Bildiriler ve Tam Metinler.

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1 Bildiriler ve Tam Metinler ivekvakfi ivek_vakfi ivekvakfi

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3 Uluslararası İVEK BİO Kongresi Kasım Osmanlı Arşivi Kongre Merkezi 2018 Kağıthane, Istanbul İÇİNDEKİLER / INDEX Önsöz Foreword Bilimsel Program Program Poster Program Poster Program Davetli Konuşmacılar Invited Lectures Sözlü Bildiriler ve Tam Metinler Oral Abstracts and Full Texts Poster Bildiriler ve Tam Metinler Poster Abstracts and Full Texts Yazar Dizini Author Index

4 Değerli Akademisyenler, Sayın Eczacı Meslektaşlarım, Bürokratlarımız, İlaç Endüstrimizin Çok Değerli Temsilcileri ve Sevgili Öğrenciler, Uluslararası İVEK Biyoteknoloji Kongresi (İVEK BİO 2018) Kasım 2018 tarihlerinde İstanbul da, Osmanlı Arşivleri toplantı salonlarında sizlerin katılımı ve katkılarıyla gerçekleştirilecektir. Sağlık alanına yönelik biyoteknolojik çalışmaların artmasıyla, biyoteknolojik ilaç üretimi gün geçtikçe önem kazanmaktadır. Biyoteknolojik ürünlerin tedavide kullanılması ve ekonomide büyük pay almasıyla Farmasötik Biyoteknoloji alanında yapılan çalışmalar ve geliştirilen ürünlerin sayısı da oldukça artmıştır yılında en çok pazar payı bulunan ilaçların kanser, diyabet, otoimmün bozukluklar ve viral hastalıklara karşı kullanılan, biyoteknolojik yöntemlerle üretilen ilaçlar olduğu görülmektedir. Bu nedenle, gerçekleştirmeyi planladığımız kongrede sağlıkta biyoteknoloji alanında bilimsel katkıda bulunan, çalışan, biyoteknolojik ilaç üreten farklı platformları biraraya getirerek bu konuda güncel bilgileri aktarmayı ve paylaşmayı amaçladık. Ayrıca, ülkemizdeki Teknokentler ve Girişim (Startup) firmalarının da katılımıyla biyoteknolojik ilaç ve ürünlerdeki gelişmelerin ve yeni buluşların da duyurulmasını hedefledik. Aşağıda konu başlıkları belirtilen toplantıya sizleri davet etmekten büyük onur ve mutluluk duymaktayız. Nanobiyoteknoloji Gen Tedavisi ve Gen Taşıyıcı Sistemler Sağlık Alanında Biyoteknolojik Ürünler Biyoteknolojik İlaçların Formülasyonu Biyoteknolojik İlaçların Üretimi Biyoteknolojik İlaçlarda Preklinik ve Klinik Araştırmalar Biyoteknolojik Aşılar Biyoteknolojik İlaçlarda Ruhsat ve Patent Biyoteknolojide Medikal İnformatik İVEK BİO 2018 Kongresi nin dili Türkçe ve İngilizce olacaktır. İngilizce hazırlanmış Bildiri Özetleri değerlendirilerek, kabul edilenler bildiri kitapçığında basılacaktır. Sevgi ve Saygılarımızla, Kongre Başkanı Prof. Dr. Ayşe Gülten KANTARCI Kongre Genel Sekreteri Prof. Dr. Ahmet HACIMÜFTÜOĞLU 2

5 BİLİMSEL PROGRAM SCIENTIFIC PROGRAM

6 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM 26 KASIM 2018, Pazartesi Açılış Konuşmaları Kahve Molası Biotechnology : Scope and its Workforce Oturum Başkanı: Prof.Dr. Ahmet Hacımüftüoğlu (Atatürk Üniversitesi) Davetli Konuşmacı(DK-01): Prof. Hisham Ibrahim (Kagoşima Üniversitesi, Japonya) Öğle Yemeği Panel Konusu: Türkiye de Biyoteknolojik İlaçların Bugünü ve Geleceği Moderatör: Doç. Dr. Ali Demir Sezer Panelistler: - "Dünyada ve Türkiye'de biyoteknolojik ilaçlarda gelişmeler" - Uzm. Ecz. Vedat Eğilmez (İEİS), - Biyoteknoloji Ekosistemi için doğru politikanın belirlenmesi - Dr. Ümit Dereli (AİFD) - "İlaç Biyoteknolojisinde Güncel Gelişmeler" - Zeynep Atabay (TİSD) - Sektöre Kısa Bir Bakış - Gürsel Bayat (SÜRDER) - "Türkiye de Biyoteknolojik İlaçların Bugünü ve Geleceği" - Dr. Ali Alkan (TİTCK Başkan Yardımcısı) Kahve Molası Oturum Başkanı: Dr. Mahmut Tokaç (İVEK Yönetim Kurulu Başkanı) Biyoteknolojik İlaçlarda Ruhsat Ecz. Hacer Coşkun (TİTCK) Biyoteknolojik İlaçlarda Fikri Mülkiyet Salih Bektaş (Türk Patent Enstitüsü) 27 KASIM 2018, Salı 1. Salon Biyoteknolojik İlaçların Üretimi Oturum Başkanları: Prof. Dr. Türkan Eldem (Hacettepe Üniversitesi), Prof.Dr. Zeki Topçu (Ege Üniversitesi) Biyoteknolojik İlaç Üretiminde Hücre Bankaları Davetli Konuşmacı (DK-02): Doç. Dr. Emine Şalva (İnönü Üniversitesi) Sürdürülebilir Biyofarmasötik Üretim Sağlamak İçin Platform Yaklaşımları Davetli Konuşmacı (DK-03): Dr. Aziz Çaylı (Florabio A.Ş.) Maya Hücrelerinin Farmasötik Önemi Davetli Konuşmacı (DK-04): Prof. Dr. Zeki Topçu (Ege Üniversitesi) Kahve Molası 4

7 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Oturum Başkanı: Prof. Dr. Şaban Tekin (Tubitak MAM) Farmasötik biyoteknolojide mikrobiyolojik üretimde üst akış işlemleri Davetli Konuşmacı (DK-05) : Dr. Turgay Kaçar (Arven İlaç) Farmasötik biyoteknolojide memeli hücreleri ile üretimde üst akış işlemleri Davetli Konuşmacı (DK-06) : Dr. Ramazan Karaduman (Abdi İbrahim İlaç) Biyoteknolojik İlaç Üretiminde Alt Akış İşlemleri Davetli Konuşmacı (DK-07) : Uzm. Özgür KARBAN (Centurion Pharma İlaç) 12:30-13:30 Öğle Yemeği Oturum Başkanı: Prof. Dr. Sadrettin Pençe (Sağlık Bilimleri Üniversitesi) Monoklonal Antikorların Üretiminde Üst Akış İşlemleri Davetli Konuşmacı (DK-08) : Prof. Dr. Serdar Alpan (Turgut İlaç) Monoklonal Antikorların Üretiminde Alt Akış İşlemleri Davetli Konuşmacı (DK-09) : Uzm. Mehran Montajabi Niyat (CinnaGen İlaç) Biyoteknolojik Ürünlerde GMP Kuralları Davetli Konuşmacı (DK-10) : Catherine Oakes (Oakes Consulting LLC, USA) Biyoteknolojik İlaçlarda Pre-Klinik ve Klinik Çalışmalar Davetli Konuşmacı (DK-11) :Prof. Dr. Suliman AlFayoumi (Principal Consultant, Al-fayoumi Consulting, Jordon) Kahve Molası Oturum Başkanları: Prof.Dr. Zeki Topçu, Prof. Dr. Türkan Eldem (Hacettepe Üniversitesi) Biyoteknolojik ilaç geliştirme sürecinde TUBİTAK GMBE nin mevcut konumu Davetli Konuşmacı (DK-12) : Prof. Dr. Berrin Erdağ (TÜBİTAK-MAM) Biyobenzer İlaçlarda Kalite ve Farmasötik Kalite Güvencesi Davetli Konuşmacı (DK-13) : Prof. Dr. Türkan Eldem (Hacettepe Üniversitesi) Üretimden Bitmiş Ürüne Biyoteknolojik Ürünlerde İmmunojenisite Davetli Konuşmacı (DK-14) : Dr. Öğr. Üyesi Devrim Demir Dora (Akdeniz Üniversitesi) 2. Salon Gen Tedavisi ve Gen Taşıyıcı Sistemler Oturum Başkanı: Doç. Dr. Gülay Büyükköroğlu (Anadolu Üniversitesi) Rekombinant Proteinlerin ve nükleik asitlerin aktarımında Nanopartikül sistemler Davetli konuşmacı (DK-15) : Prof. Dr. Ayşe Gülten Kantarcı (Ege Üniversitesi) Nanocomplexes for gene therapy of respiratory diseases: Targeting and overcoming the mucus barrier. Davetli Konuşmacı (DK-16) : Prof. Massimo Conese ( Universita Degli Studi di Foggia, Italy) 5

8 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Sözlü Sunum Solid Lipid Nanoparticle complexes with plasmid or oligonucleotide for mir- 34a delivery Mustafa Kotmakçı, Vildan Bozok Çetintaş, Zekeriya Düzgün, Uğur Karagöz, Ayşe Gülten Kantarcı Kahve Molası Oturum Başkanı: Prof.Dr. A. M. Abd El-Aty (Atatürk Üniversitesi) Taşıyıcı sistemler olarak Mikrobial Polihidroksialkanoatların (PHA) Üretimi ve Uygulamaları Davetli Konuşmacı (DK-17) : Doç. Dr. Gülay Büyükköroğlu (Anadolu Üniversitesi) The development of novel drug-delivery systems for specific targeting of drugs into their intracellular targets Davetli Konuşmacı (DK-18) : Prof. Hisham Ibrahim (Kagoşima Üniversitesi, Japonya) Öğle Yemeği Oturum Başkanı: Prof. Dr. Filiz Öner (Hacettepe Üniversitesi) Akciğer Kanseri Tedavisinde Gen Taşıyıcı Nanobaloncuklar Davetli Konuşmacı (DK-19) : Dr. Öğr. Üyesi Yücel Başpınar (Ege Üniversitesi) The role of Surface Plasmon Resonance in supporting Drug Development & Drug submission Dr. Fredrik Sundberg (GE Healthcare, Sweden) Sözlü Sunum An alternative treatment approach to anti-androgenic drugs with high side effects; Development and in vitro evaluation of solid lipid nanoparticles for gene silencing Gülşah Erel Akbaba, Ayşe Gülten Kantarcı Kahve Molası Oturum Başkanı: Dr. Öğr. Üyesi Yücel Başpınar (Ege Üniversitesi) Sözlü Sunum Effectiveness of Combined sirna and Docetaxel-Loaded SLNs on Different Breast Cancer Cell Lines Behiye Şenel, Gülay Büyükköroğlu Sözlü Sunum Superparamagnetic Iron Oxide Nanoparticle-Based Targeted Gene Delivery Systems for Cancer Therapy Kurtulus Gokduman 6

9 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Sözlü Sunum Technetium-99m radiolabeled glucagon-like peptide analog Exendin-4 for cancer diagnosis Emre Özgenç, Meliha Ekinc, Derya Ilem Özdemir, Evren Gündoğdu, Makbule Aşıkoğlu Sözlü Sunum Preparation, optimization and in vitro evaluation of novel self emulsifying drug delivery systems for oral administration Neslihan Üstündağ Okur, Emre Şefik Çağlar, Mine Diril, Hatice Yeşim Karasulu Sözlü Sunum Antibacterial Activity and Cytocompatibility of Silk Sericin-Capped Gold Nanoparticles Synthesized Under UVC Radiation Ömer Aktürk, Zehra Gün Gök, Memik Taylan Daş, Özge Erdemli, Mustafa Yiğitoğlu Sözlü Sunum Progesteron hormonu ve glial beyin tumorlerinde etkileri Adil Meriç Altınöz 3. Salon Biyoteknolojik Aşılar Oturum Başkanları: Prof. Dr. Filiz Öner (Hacettepe Üniversitesi), Doç. Dr. Mert Döşkaya (Ege Üniversitesi) Rekombinant DNA Aşılar Davetli Konuşmacı (DK-20) : Prof. Dr. Filiz Öner (Hacettepe Üniversitesi) Erişkinlerde Aşılama Davetli Konuşmacı (DK-21) : Prof. Dr. Serhat Ünal (Hacettepe Üniversitesi Aşı Enstitüsü) Kırım Kongo Aşısı ve Faz I Çalışmaları Davetli Konuşmacı (DK-22) : Prof. Dr. Aykut Özdarendeli (Erciyes Üniversitesi) Kahve Molası Oturum Başkanları: Prof. Dr. Yücel Kadıoğlu (Atatürk Üniversitesi), Doç. Dr. Emine Şalva (İnönü Üniversitesi) DNA aşıları ve Rekombinant protein aşılarının geliştirilmesinde antijen keşfi yaklaşımının önemi Davetli Konuşmacı (DK-23) : Doç. Dr. Mert Döşkaya (Ege Üniversitesi) Sözlü Sunum Understanding the mechanisms that regulate cutaneous resident memory T cells on autoimmune skin diseases and preclinical studies in terms of vitiligo Hasan Akbaba, Shannon K Bromley 7

10 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Öğle yemeği Oturum Başkanı: Prof.Dr. Makbule Aşıkoğlu (Ege Üniversitesi) Amerikadaki start-up ların durumu ve örnek olabilecek hususlar Davetli Konuşmacı (DK-24) : Mehmed Bulend Ugur (TAEB) Sözlü Sunum A Novel Diagnosis Approach on Paraffin Embedded Gastric Tissue Samples via Chemometrics Assisted Raman Spectroscopy Yücel Kadoğlu, Onur Şenol Sözlü Sunum Comparision of Invasive duktal carsinoma (IDC) and Invasive lobular carcinoma (ILC) for Identification of Novel Biomarkers Umut Ağyüz, Yasemin Müşteri Oltulu, Tuba Saraç, Tuğba Elgün Sözlü Sunum Targeted Cancer Therapy Using Nano Drug Delivery Systems, Struggles, Strategies and Prospects Fatemeh Bahadori Kahve Molası Oturum Başkanı: Dr. Öğretim Üyesi Bilge Debeleç Bütüner (Ege Üniversitesi) Sözlü Sunum Possible toxic effects of biotechnological vaccines and the safety concerns Pınar Erkekoğlu Sözlü Sunum Development, Characterization and "In Vitro"-"In Vivo" Evaluation of "In Situ" Gel Systems for Improving Therapeutic Efficacy of Ocular Drug Delivery Neslihan Üstündağ Okur, Vildan Yozgatli, Mehmet Evren Okur, Panoraia Siafaka Sözlü Sunum Gene drives with CRISPR-CAS9 technology in agriculture and ethical issues Mehtap Kara, Özlem Akbal Dağıstan, Özlem Nazan Erdoğan Sözlü Sunum Chemical Characterization And Biological Study Of The Species Senecio cineraria Ababsa Zine El Abidine, Kara Ali. Wahiba, Medjroub Kamel, Akkal Salah, Abidli Nacira Sözlü Sunum Production of new generation, most advanced Anthrax and Malaria vaccines in green plant leaves for human and animal using transient expression technology Prof.Dr.Tarlan Mammedov (Akdeniz Üniversitesi) 8

11 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM 28 KASIM 2018, Çarşamba 1. Salon Teşhis ve Tedavide Biyoteknolojik Yöntemler Oturum Başkanları: Prof.Dr. A. M. Abd El-Aty (Atatürk Üniversitesi), Prof. Dr. Mehmet Öztürk (IBG Izmir Biyotıp ve Genom Merkezi) In vivo Sensörler ve Biyoteknolojik Sinir Bilim Uygulamaları Davetli konuşmacı (DK-25) : Prof. Dr. Ahmet Hacımüftüoğlu (Atatürk Üniversitesi) Kantitatif DNA Hasarı Ölçümlerinin Tanı ve Tedavide Kullanımı Davetli Konuşmacı (DK-26) : Dr. Öğretim Üyesi Bilge Debeleç Bütüner (Ege Üniversitesi) Kanserde Teranostik ve Kişiye Özgün Tıp Davetli Konuşmacı (DK-27) : Prof. Dr. Mehmet Öztürk (IBG Izmir Biyotıp ve Genom Merkezi) Kahve Molası Eğitim Oturumu Oturum Başkanı: Prof.Dr. Julide Akbuğa (Altınbaş Üniversitesi) Challenges in Scientific Publication: Editor, reviewer, and author perspectives Davetli Konuşmacı (DK-28) : Prof.Dr. A. M. Abd El-Aty (Atatürk Üniversitesi) Editor, Journal of Advanced Research Sözlü Sunum Farmasötik Biyoteknoloji de lisans ve lisansüstü eğitim Prof. Dr. Türkan Eldem, Prof.Dr. Filiz Öner, Özgün Fırat Düzenli 12:20-13:30 Öğle yemeği Teknoparklar Kapsamında İlaç Biyoteknolojisi ile İlgili Yeni Gelişmeler Biyoteknolojide Güncel Stratejiler Oturum Başkanı: Dr. Mahmut Tokaç (İVEK Yönetim Kurulu Başkanı) Biyoteknolojide Medikal İnformatik Davetli konuşmacı (DK-29) : Bakan Yardımcısı Dr. Şuayip Birinci Biyomedikal Inovasyonda Güncel Stratejiler ve Yol Haritaları Davetli Konuşmacı (DK-30) : Prof. Dr. Murat Özgören Sözlü sunum (Dr.Ferhat Farshi) Dünya da ve Türkiye de Biyoteknolojik İlaçlara Hızlı Bir Bakış Kahve Molası Sağlık Teknopark/kent Sunumları Moderatör: Prof.Dr. Murat Özgören ATA Teknokentte biyoteknoloji çalışmaları ve Potansiyeli Davetli Konuşmacı (DK-31) : Doç.Dr. Ersin Karaman (Atatürk Üniversitesi) 9

12 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Türkiye ve Dünyada Biyoteknoloji ve Sentetik Biyoloji Devrimi Davetli Konuşmacı (DK-32) :Prof.Dr. Işıl Kurnaz (Gebze Teknik Üniversitesi) İzmir Biyotıp ve Genom Merkezi (IBG) "Yeni nesil biyoteknolojik ilaçlar, mab'lardan sonra ne gelecek" Davetli Konuşmacı (DK-33) :Prof.Dr. Hülya Ayar Kayalı Tübitak MAM Gen Mühendisliği ve Biyoteknoloji Enstitüsünde Yapılan Çalışmalar Davetli Konuşmacı (DK-34) :Prof.Dr. Şaban Tekin (Tübitak MAM) Kapanış Konuşması Dr. Nureddin NEBATİ T.C. Hazine ve Maliye Bakan Yardımcısı Ödül* Töreni ve Kapanış *2019 Bio International Convention Katılımı 2. Salon Girişimciler ve yenilikçi çalışmalar Oturum Başkanı: Ecz. İbrahim Yavuz (İVEK Vakfı) Biyoteknolojik İlaçların Test Kitlerini Üreten Sürdürülebilir Bir Biyoteknoloji Şirketi Olmaya Giden Yol Davetli Konuşmacı (DK-35) : Prof.Dr. Haluk Ataoğlu (Matriks Biotek) Kahve Molası TİTCK SUNUMU TITCK Startup Online Platformu Oturum Başkanı: Prof.Dr. Aydın Erenmemişoğlu (Farmagen Klinik Araştırma Merkezi) Doç.Dr. İsmail Mert Vural (TİTCK Başkan Yardımcısı) Recep Uslu (TİTCK Başkan Yardımcısı) Dr. Sinan Tandoğan (TÜBİTAK, Girişimcilik Destekleme Grubu) Öğle Yemeği Oturum Başkanı : Prof.Dr. Hülya Ayar Kayalı (IBG) Biyogirişimcilik ve Ticarileşme Davetli Konuşmacı (DK-36) : Prof. Dr. Nezih Hekim (Biruni Üniversitesi) Panel: Yatırım almanın ipuçları / Risk Sermayesi Oturum Başkanı: Prof.Dr. Cengizhan Öztürk - Selin Arslanhan Memiş (Kurucu, ReDis Innovation - İnovasyon Arayüzü) - Umut Ağyüz (Start-up) - Sena Nomak (Kurucu Ortak, RS Research) - Utku Gökkaya (Vestel Ventures) - Akın Kozanoglu (Şirketortağım modeli) - Gökhan Güner/Okan Kara (Fon yönetimi) - Prof. Cengizhan Öztürk (Boğaziçi Üniversitesi) 10

13 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Video Konferanslar Oturum Başkanı: Umut Ağyüz "Biyoteknolojik Ürün Ticarileşmesi" Dr. Barry Horwitz (Boston University, USA, Strategy and Innovation) Dr. Jean Jacques Yarmoff (International strategist, Biolabs, Cambridge, MA) Joseph Damond, (Executive Vice President for International Affairs at the Biotechnology Innovation Organization-BIO) 3. Salon Oturum Başkanı: Dr. Öğr. Üyesi Devrim Demir Dora (Akdeniz Üniversitesi) Sözlü Sunum Social and Ethical Aspects of Biotechnology Merve Memisoglu Sözlü Sunum Ethical Issues Related to Gene Editing Using CRISPR Zehra Şeker, Özlem Bildik-Şanlı Sözlü Sunum The Use of L-Ascorbic Acid Containing Dextran Coated Silica Aerogel in Controlled Drug Targeting Systems Cem Özel, Ceren Keçeciler, Ali Değirmenci, Ecem Tiryaki, Yeliz Başaran Elalmış, Burcu Karakuzu Ikizler, Sevi Yücel Sözlü Sunum Cytotoxic effect of boric acid-glycerin on SH-SY5Y cell line Ayça Taş, Neşe Kekli kçiȯğlu Çakmak, Yavuz Si li ğ Kahve Molası Oturum Başkanı: Prof.Dr. Berrin Erdağ (Tübitak MAM) Sözlü Sunum A pilot scale production of L-asparaginase, L-glutaminase and Taxol from endophytic fungi isolated from Punica granatum Ibrahim Karidio Diori, Şenay Hamarat Şanlier Sözlü Sunum Electrochemical Biosensor for Monitoring of Interaction Between Anticancer Drug Dacarbazine and Nucleic Acids Ece Eksin, Arzum Erdem Sözlü Sunum The Design, Synthesis, NMR Analysis and Cytotoxic Activities of 6-(3-Aryl-2- Propenoyl)-2(3H)-Benzoxazolones Sinan Bilginer, Halise Inci Gül 11

14 BİLİMSEL PROGRAM / SCIENTIFIC PROGRAM Sözlü Sunum A Comparative Docking Study of Psoriasis Disease Proteins with Psoriasis Drugs versus Plant Extracts Used in Phytotherapy Büşra Çetin, Vildan Enisoğlu Atalay, Tuba Sevimoğlu Sözlü Sunum Formulation and Evaluation of Nano-Niosomes as Gene Delivery Systems Devrim Demir Dora, Büşra Cesur, Feride Öner 4. Salon Moderatör: Prof.Dr. Erdal Cevher (İstanbul Üniversitesi) Yeni Fikirler sunumlar Start-up Sunumları 8-10 adet (15 Dak) CRISPR teknikleri ve biyoteknoloji alanında uygulamaları Panel Başkanı: Prof Dr. Nezih Hekim (Sivas Cumhuriyet Üniversitesi) Panelistler: Dr. Öğr. Üyesi Oktay İsmail Kaplan (Kayseri Abdullah Gül Üniversitesi) Dr. Cihan Taştan (Acıbadem LabCell Laboratuvarı) Dr. Öğr. Üyesi Cihan Aydın (İstanbul Medeniyet Üniversitesi) Dr. Öğr. Üyesi Kaan Yılancıoğlu (İstanbul Üsküdar Üniversitesi) Ar. Gör. Burak Berber (Eskişehir Teknik Üniversitesi) :30 Birebir görüşmeler 12

15 POSTER PROGRAM POSTER PROGRAM

16 POSTER PROGRAMI / POSTER PROGRAM P-003 Economic analysis of biosimilar drugs approved in Turkey: a study covering the period Sule Apikoglu Rabus, Baran Arslan P-004 HPLC-DAD analysis of best anti-ages extracts from Rosmarinus tournefortii leaves Salah Akkal, Amel Achouri, Séverine Derbré P-007 Chemical composition of organic volatile compounds of an endemic and rare Apiaceae of Algeria, Bunium crassifolium Batt Djarri Lakhdar, Souileh Nabila, Medjroubi Kamel, Hamel Tarek, Demirtas Ibrahim P-008 Taxol -producing endophytic fungi: a promising red biotechnology Radia Ayad, Kamel Medjroubi, Salah Akkal P-011 Quantification and assessment of the Anti-proliferative activity of essential oils of Rosmarinus Officinalis Collected from Three climatically dissimilar locations in Algeria Ouroud Fellah, Samir Hameurlaine, Noureddine Gherraf, Amar Zellagui, Muhammed Altun, Ibrahim Demirtas, Ayse Sahin Yaglioglu P-014 Anti-Proliferative and Antioxidant Activities of Chloroformic Extract from Two Euphorbia Species Growing in Algeria Amar Zellagui, Agena Ghout, Noureddine Gherraf, Ibrahim Demirtas P-022 In vitro studies on inhibition of mammaglobin gene expression with chitosan/shrna nanoplexes in breast cancer cell lines Aylin Ozkan, Ceyda Ekentok, Emine Şalva, Suna Ozbas Turan P-023 In Vitro Studies on Inhibition of Chitosan / sirna Nanoplexes and Mammaglobin Gene Expression in Breast Cancer Cell Lines Sadiye Burcu Ozkavak, Ceyda Ekentok, Emine Şalva, Suna Ozbas Turan P-024 In vitro antioxidant and photoprotective activities of ethyl acetate fraction of hydroalcoholic extract of the aerial parts of Algerian Plumbaginaceae Limonium thouinii (viv.) Kuntze Mostefa Lefahal, El Hani Makhloufi, Nabila Zaabat, Salah Akkal, Kamal Medjroubi, Merzoug Benahmed, Houcine Laouer P-025 Design and Development of GO/PLA Pharmaceutical System for the Treatment of Diabetic Injuries Sümeyra Ayan, Ebru Demir, Cem Bülent Üstündağ P-026 Graphen Oxide / PLGA Scaffold Design for Tissue Engineering Applications Ebru Demir, Nuran Çalımlı, Sümeyra Ayan, Cem Bülent Üstündağ P-027 Production of Biotechnological Drugs and Recent Developments Makbule Ayaydın, Şeyma Hande Tekarslan Şahin P-028 Applications of DNA-loaded Nanoparticles in Brain Tumors Nahide Zeren Arda, Özgen Özer P-029 Biotechnological Drugs on Treatment of Neurodegenerative Diseases Hazal Eken, Rana Arslan, Nurcan Bektaş Türkmen P-030 Propolis as a natural source for drug development Segueni Narimane, Akkal Salah, Rhouati Salah 14

17 POSTER PROGRAMI / POSTER PROGRAM P-031 Developments in treatment of autoimmune and inflammatory diseases with monoclonal antibodies Yağmur Okçay, Rana Arslan, Nurcan Bektaş Türkmen P-032 Biotechnological applications of heat shock proteins Mevcude Çam, Rana Arslan, Nurcan Bektaş Türkmen P-033 Graphene Based Nano Drug Design for the Treatment of Breast Cancer Elif Akyar, Fadime Çetin P-035 Biological applications as an anti-microbial, DNA binding, and DNA cleavage of trifloromethanesulfonamide functionalized graphene quantum dots Elif Ünaldi, Neslihan Demir, Mustafa Yıldız P-036 Anti-QS and Anti-biofilm Activities of Cucurbita pepo Leaf Extracts against Pseudomonas aeruginosa Arhun Ali Balkan, Ayla Yıldız, Ayten Şen, Aleyna Topaç, Barış Gökalsın, Nüzhet Cenk Sesal P-037 Determining the Effects of Lichen and Endolichenic Bacteria Extracts on Pseudomonas aeruginosa Quorum Sensing and Biofilm Form Tunahan Irmak Başaran, Barış Gökalsın, Nüzhet Cenk Sesal P-039 Pseudomonas aeruginosa Quorum Sensing Inhibition by Cherry Stalk Extracts Ayla Yıldız, Arhun Ali Balkan, Deniz Ezgi Budak, Ceren Özbek, Barış Gökalsın, Nüzhet Cenk Sesal P-040 Quorum Sensing Inhibitory Potential of Halomonas campaniensis on Pseudomonas aeruginosa Tunahan Irmak Başaran, Didem Berber, Barış Gökalsın, Abbamondi Gennaro Roberto, Giuseppina Tommonaro, Nüzhet Cenk Sesal P-041 Quorum Sensing and Biofilm Inhibition Properties of Ethyl Acetate Extracts of Sunflower (Helianthus annuus) Leaves against Pseudomonas aeruginosa Arhun Ali Balkan, Ayla Yıldız, Ayten Şen, Ezgi Ay, Barış Gökalsın, Göksel Evci, Nüzhet Cenk Sesal P-043 Quorum Sensing Inhibitory Potential of Natrinema versiforme on Pseudomonas aeruginosa Tunahan Irmak Başaran, Didem Berber, Barış Gökalsın, Iodice Carmine, Giuseppina Tommonaro, Nüzhet Cenk Sesal P-044 A simple silica based method for cell free DNA isolation from blood plasma Burhanettin Yalcinkaya, Kubra Coskun, Engiṅ Aydiṅ, Muslum Akgoz, Sadrettiṅ Pence P-045 Optimization of primers and probes that can be used in the diagnosis of mitochondrial diseases Burhanettin Yalcinkaya, Sadrettin Pence P-049 Biotechnological Products Used in the Treatment of Rheumatoid Artritis Elif Taşdemir, Rana Arslan P-050 Many Algerian Plants with thymol as major or active compound Hocine Laouer, Salah Akkal P-053 Polyphenols and phytochemical screening of asphodelus Racha Lydia Bouchouka 15

18 POSTER PROGRAMI / POSTER PROGRAM P-056 Emicizumab and Recombinant Products Used in the Treatment of Hemophilia A Elif Taşdemir P-058 Assesment of Quality Control ın Biotechnological Drugs Evren Alğın Yapar, Burçin Çağan P-059 International Standards for Biotechnological Drugs Evren Alğın Yapar, Burçin Çağan P-060 The Role of Antibody-Dependent Cell-Mediated Cytotoxicity Mechanism ın Cancer Treatment Burçin Çağan, Evren Alğın Yapar P-061 Biotechnological Drugs for Cancer Treatment and Clinical Trials Burçin Çağan, Evren Alğın Yapar P-063 Biotechnology and Pharmacognosy Timur Hakan Barak P-066 Development of a Novel Gene Specific Silencing Method by RNAi in Targeted Cells Using PLGA Nano-Particles Şeyma Ceylan, Fahri Akbaş, Fatemeh Bahadori P-072 MicroRNA in Hepatitis B and Hepatitis C associated Hepatocellular Carcinoma and Cirrhosis Gokhan Kucukkara, Ferhat Gurkan Aslan, Bilal Toka, Mehmet Koroglu, Mustafa Altindis P-078 Monoclonal Antibodies for Cancer Treatment Burçin Çağan, Evren Alğın Yapar P-083 Determination of Bioactive Goat Milk Peptide Structures by LC-QTof-MS System Bilal Cakir, Tuğba Tunali Akbay P-088 Synergy Study, Plant Extracts and Antibiotics Against Bacteria Mohamed Mihoub Zerroug, Nora Haichour, Samia Mezaache Aichour P-089 Investigation of the relationship between influenza A virus RNA polymerase and human actin beta protein Nazife Gelmez, Erkan Rayaman, Atsushi Kawaguchi, Kadir Turan P-090 Mechanisms or Instability for Protein Based Pharmaceuticals Timur Hakan Barak P-091 Antioxidant activities of Santolina chamaecyparissus aqueous extract Abderrahmane Senator, Bouriche Hammama P-093 Antimicrobial activity of a fungal microorganism Agit Çetinkaya, Ragıp Soner Silme, Ömür Baysal P-095 In vitro culture of tumor infiltrating lymphocytes for adoptive immunotherapy in breast cancer patients Erdoğan Selçuk Şeber, Tarkan Yetişyiğit, Hülya Dönmez, Sinem Buluş, Sibel Özkan Gürdal, Meltem Öznur, Burhan Turgut P-096 Preparation of drug loaded polymeric nanoparticles as ceramidase inhibitor Ferdane Danışman, Hüsniye Birman, Merve Ilgar, Ezgi Tan, Selcan Karakuş, Ayben Kilislioğlu, Serap Kuruca 16

19 POSTER PROGRAMI / POSTER PROGRAM P-098 Synthesis and Herbicidal Activities of New Cu(II) Complexs of Acylthiourea Derivatives Turkan Kiral, Mehmet Yakan, Irfan Koca, Serhat Mamaş, Nurcan Karacan, Ahmet Tansel Serim P-099 In Silico Analysis of Goat Milk Derived Bioactive Peptides Kıymet Özlem Şahna P-100 Isolation and screening method for antibiotic-producing fungi Ragıp Soner Silme, Gülperi Kayran, Ömür Baysal P-101 Perspectives of Medical Students about the Biotechnological Drugs Zinnet Şevval Aksoyalp, Devrim Demir Dora P-102 Monoclonal antibodies which licenced in Turkey Timur Hakan Barak P-106 Curcumin increase antitumor effect of CDs+5-Fluorouracil against prostate cancer Yeşim Yeni, Ali Taghizadehghalehjoughi, Ismail Çagrı Aydın, Sidika Genç, Aysegul Yılmaz, Fatma Yesilyurt, Selma Sezen, Ahmet Hacımuftuoglu P-107 Evaluation neuroprotective effects of Origanum majorana extract on Fe3O4@SiO2 NPs induced toxicity in cortex culture Selma Sezen, Ali Taghizadehghalehjoughi, Yeşim Yeni, Sidika Genç, Medine Güllüce, Ahmet Hacımuftuoglu, Ahmet Mavi, Kübra Solak P-108 Comparing Our New Electrodes with Pt-Iridyum Commercial Electrodes Fatma Yesilyurt, M.sait Ertugrul, Aysegul Yilmaz, Ufuk Okkay, Ali Taghizadehghalehjoughi, Ahmet Hacimuftuoglu P-110 Temozolomide Cds NPs combination shows more effectivity in compare pure drug against T98G cells Ayşegül Yılmaz, Ali Taghizadehghalehjoughi, Selma Sezen, Fatma Yesilyurt, Yesim Yeni, Sidika Genc, Atefeh Varmazyari, Ahmet Hacimuftuoglu P-111 An anticancer agent based on ehtylenediamine against breast cancer cells, MDA-MB-231 and MCF-7 Elif Avcu Altıparmak, Gökçe Erdemir, Serap Erdem Kuruca, Bahri Ülküseven, Tülay Bal Demirci P-112 DNA vaccines: from bench to bedside Genada Sinani, Melike Sessevmez P-113 Recombinant Protein-Based Malaria Vaccines Melike Sessevmez, Genada Sinani P-115 Vitex Agnus Castus Combination Cds and 5-Fluorouracil Effectively Kill Prostate Cancer: İn Vitro Sıdıka Genç, Ali Taghizadehghalehjoughi, Cemil Bayram, Yeşim Yeni, Fatma Yesilyurt, Aysegul Yılmaz, Selma Sezen, Ahmet Hacımuftuoglu P-117 Impurities in Biologicals, Their Safety/Toxicity and Analytical Methods Pınar Erkekoğlu, Emirhan Nemutlu P-119 3D Tissue Models for Nanotoxicology Applications Suna Sabuncuoğlu, Pınar Erkekoğlu 17

20 POSTER PROGRAMI / POSTER PROGRAM P-120 Perspectives of Medical Students about Biotechnological Drugs Zinnet Şevval Aksoyalp, Devrim Demir Dora P-121 Biosimilar Safety Suna Sabuncuoğlu, Gözde Girgin P-124 Effect of Niosome Preparation Techniques on Physicochemical Properties of Niosome/ pdna Complexes (Nioplexes) Devrim Demir Dora, Büsra Cesur, Feride Öner P-138 Analysis of Gene Regulation and Relationships of Breast Carcinoma due to Epithelial or Stromal Origin Tuğba Elgün, Tuba Saraç, Lokman Tunca, Asiye Gök Yurttaş, Umut Ağyüz P-145 A Novel Nano Formulation for Targeting Breast Cancer Zahra Eskandari, Fatemeh Bahadori, Melda Altikatoglu, Vildan Betul Yenigun, Abdurrahim Kocyigit, Hayat Onyuksel P-148 Chitosan-Based Nanoparticles as Bioadhesive Drug Delivery System for Buccal Route Muhammet Davut Arpa 18

21 DAVETLİ KONUŞMACILAR INVITED LECTURES

22 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-02 Cell Banks in Biotechnological Drug Manufacturing Emine Şalva Inonu University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, Malatya Biotechnologic based drugs, known as biologics or biopharmaceuticals are produced by the genetic manipulation of living cells or organisms. Biologics are widely used to teratment of many diseases such as cancer, inflammatory or neuropathologic diseases. Many biotechnologic drugs including recombinant proteins produced by recombinant DNA technology, monoclonal antibodies produced by hybridoma technology, gene therapy products and cell based products represent more specific and efficient therapy. Production of recombinant proteins begins with selecting the appropriate cell line. The choice of expression system and expression platform generally depend on the nature of the protein to be expressed, the amount needed, and the continuity of production. For the production of small amounts of biologics in a short period of time, transient expression systems are preferred. For clinical and commercial production, stable expression systems are preferred for continuous production of the product over long periods (months). Prokaryotic cells, yeasts, fungi, insect cells, transgenic animals and plants and mammalian cells are widely used in the production of biotechnologic drugs. In the selection of the expression system for high-level recombinant protein; cell growth characteristics, expression levels, intracellular and extracellular production, biological activity of the target protein are important. In addition, the cost and the design of each expression system is important. Cells used for commercial production; 1) support high-level product expression in culture for several months, 2) be able to scale at wide volumes (100-10,000 L), 3) achieve and maintain a high live cell density in the acceptable production process (>5x10 6 cells / ml in batch culture, >10 8 cells/ml in perfusion), 4) provide appropriate posttranslational process capability, 5) be able to characterize to allow the separation of cells from adventitious agents such as viruses. During the initial selection process and in the final cycle of subsequent amplification, individual clones are isolated from the cell pool and screened for desired specific clones with high specific production (pg/cell/day), good growth rate and appropriate biochemical product quality. The first step of screening is to evaluate a large number of cells, with a minimum number of cells and the highest producer cell for evaluation. The clones collected from the transfection and amplification steps are isolated by limited dilution, while the well-isolated colonies at low-density plates are evaluated by cell culture methods. Specific productivity, quantity of product produced in each cell at each unit time (pg/cell/day). Volumetric productivity is the product of cumulative live cell mass and specific productivity in the long-term culture. Volumetric productivity or production of cell culture at the end of the titer (mg / L) is important. The growth characteristics of the cells are different. The cells are adapted to grow in suspension or serum free medium. ELISA and bioassays are used to measure the protein product accumulated in the cell culture or mrna expression is examined. Cell culture is the formation of cells isolated from multicellular organisms by production in vitro. Cell bank system is a series of products produced in culture by cells in the same host cell bank, which have been completely characterized for identification. A cell bank system is validated in terms of the level of passages or duplications that occur during and after routine production. After cell line development through transfection, amplification, and final clone selection, a master cell bank and a working cell bank are created. Master cell bank (MCB) is a fully characterized cell culture which is distributed to its containers with a single pro- 20

23 DAVETLİ KONUŞMACILAR / INVITED LECTURES cess and processed so as to ensure uniformity and stored to ensure stability. Working cell bank (WCB) is a cell culture which is produced from culture in master cell bank and used in production cell cultures. For each production, a new vial from WCB is taken, heated and characterized. It is started production and inoculated by expanding to the production reactor. At the end of production, the cell sample is taken from the reactor (end of production cells, EOPC). Both cultures should be re-characterized and retained as Extended Cell Banks (ECB) for future references. MCB and WCB should be evaluated in terms of identification of cell line, removal of adventitous agents, accuracy of gene product, adaptation to growth and productivity, and product quality and potency. MCB and WCB are produced and characterized under good manufacturing practice (GMP) conditions. The characterization of cell banks consists of identity, purity and suitability. Timing and extent of MCB and WCB testing and characterization should be determined with risk-based approaches and legal authorities. For the production of recombinant protein, the cell line must first be defined by karyology, isoenzyme analysis, whole genome sequencing, SNP and STR (origin of cell, morphology and doubling time, genotypic features, integration region number, properties of expression vector). Cell line stability can be evaluated by considering phenotypic, genotypic and product quality changes. In phenotypic stability, live cell density, titre and specific productivity are evaluated. In genotypic stability, gene copy number (PCR), integration region (Southern Blot) and accuracy of gene transcript (cdna sequencing) are determined. Changes in product quality during the cell culture process, protein amino acid sequence change and whether protein sequence variants are determined by LC/MS/MS and peptide mapping. In purity studies of cell banks, sterility tests, mycoplasma test, in vitro and in vivo tests, tests for retroviruses and antibody production tests should be made. 21

24 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-05 Microbial Upstream Process in Pharmaceutical Biotechnology Turgay Kaçar Arven Pharma, R&D Center, İstanbul, Turkey In recent years, progress in the field of both pharmaceutical biotechnology and molecular biology (particularly in recombinant DNA technology) allow us to manufacture biotechnology drug products as many as to reach a major share of prescribed pharmaceuticals (1, 2). These products, called biopharmaceuticals, mostly include peptides and proteins, including monoclonal antibodies, hormones etc., as well as nucleotide based biomacromolecules (3). For instance, monoclonal antibodies have more than 50 billion dollars in sales volume globally (2). The biopharmaceuticals are manufactured by biotechnological processes which can be divided into upstream (USP) and downstream processes. USP is defined as in which the living organisms are utilized to obtain mass amount of bioproducts and involves a series of stages including the selection of host cell line, culture media/feed, growth parameters, and process optimization to achieve optimal conditions for cell growth and biopharmaceutical production. Not only prokaryotic cells like bacteria (e.g. E.coli) are used, but also eukaryotic cells (e.g. mammalian cells) are utilized (1). During USP, an efficient transformation of substrates into the desired metabolic products are required (4). Here, main goal is to utilize the bacteria as a factory to reach high accumulation of the product. Several factors should be taken into consideration such as the type of process, temperature, ph, aeration, agitation, equipment employed, and maintenance of the environment to ensure that it is free of contaminating microorganisms. The product itself can be part of the host cell s natural metabolism, or it can be genetically engineered protein. If it is protein, it can be accumulated in soluble form or in cytoplasmic aggregates as inclusion bodies. Whatever form is selected, the optimized USP should yield an appropriate intermediate product for the downstream part to reach the final product with desired quality profile. References: 1. Crommelin, D. J. A., et al. Pharmaceutical Biotechnology: Fundamentals and Applications. Informa Healthcare, Udpa, N., and Ryan P. M., Nature Reviews Drug Discovery, 2015:15(1):13 14., 3. Crommelin D.J., Storm G., Verrijk R., De Leede L., Jiskoot W., Hennink W.E., Int J Pharm., 2003;266(1 2): Gronemeyer P., Ditz R., Strube J., Bioengineering. 2014;1(4): Keywords: upstream process, bacteria, therapeutic protein, fermentation 22

25 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-12 Current Position of TUBITAK MRC GEBI in the Process of Biotechnological Drug Development Berrin Erdağ TÜBİTAK Marmara Araştırma Merkezi Gen Mühendisliği ve Biyoteknoloji Enstitüsü The global market for pharmaceuticals in 2018 reached $1.2 trillion, The share of biotechnological drugs in the market is growing rapidly. Ever since their market approval in the 1980s, biotechnological drugs have contributed greatly to the treatment of many life threatening diseases including cancer, alzheimer, heart diseases, diabetes and rheumatoid arthritis. The market share, which was 18 percent in 2010, reached 27 percent in Biotechnological drugs contain larger molecules than conventional drugs While a small molecule drug, produced by the conventional method such as aspirin, can consist of as few as 21 atoms, biotechnological pharmaceutical can be made of over 25,000 atoms. They are produced by using living organisms such as a microorganism, plant cell, or animal cell. The production process is complex and requires multistep advanced technology since the biologically active substance which is the final product inside the living cell or organisms must be separated and purified from thousands of other molecules. It is impossible to guarantee that each batch of a biologic will be identical to the last, which means every dose of a biologic has slight variations. This variability can be the type and length of any sugar or carbohydrate that may be molecularly added by the shape of the molecule. However, because of their harder and multistep complex production technology compared to that of conventional drugs, biotechnological drugs are priced beyond the reach of many patients. Therefore, reduction of cost of biopharmaceuticals will increase not only access to drugs but also efficiency in public health spending. To this end, it is of critical importance to develop the similar biological drugs, called biosimilars, with expired patent protection. Biosimilars are highly similar versions of approved branded biologics in terms of their quality, efficacy and safety.. Unlike generics, they are not exact replicas of reference products. Minor differences between biosimilars and reference products in some aspects are expected; likewise, biosimilar products will differ from each other. This is why the term biosimilar is used instead of biogeneric. The development of a biosimilar product involves a stepwise approach of optimising the production physicochemical and biological characterisation process, of the product followed by pre-clinical and/or clinical studies. It may be noted that demonstration of similarity for a biosimilar product to the innovator product is a pre-requisite for the reduction of requirements for pre-clinical and clinical studies before gaining final marketing approval through an abbreviated regulatory process in most countries. Identification of any significant differences in quality, safety and efficacy studies would mean the need for a more extensive pre-clinical and clinical evaluation and the product will not qualify as a biosimilar. The European Medicines Agency (EMA) is the body responsible for approval of biosimilars within the European Union (EU). A legal framework for approving biosimilars was established in All biopharmaceuticals for human and animal use must be approved via the centralized EMA procedure. EMA has both, product specific, including monoclonal antibody, and general biosimilar guidelines. EMA approved the first biosimilar in Turkish Medicines And Medical Devices Agency (TMMDA) approved the first biosimilar in The biosimilar market in the USA is relatively new, FDA approved the first biosimilar in According to our knowledge, there are 45 biosimilars in EU; 14 biosimilars in USA and 57 biosimilars ( with all forms) in Turkey. Turkey's pharmaceutical market in 2017 reached 25 billiontl and it is ranked 16th in the world. Biotechnological drugs reached 5.0 billion TL in Turkey imports almost all of biotechnological drugs. Turkey pays at least, 1 23

26 DAVETLİ KONUŞMACILAR / INVITED LECTURES million dollars per kilogram of biotechnological drugs. Import-dependent biotechnological drug supply model is not sustainable for Turkey, and it is clear that domestic production of biopharmaceuticals has strategic importance. Even if Turkey have a relatively strong background and a growing market in the pharmaceuticals industry. The government support regarding R&D investment in biopharmaceutical drugs development has just been initiated recently. The Genetic Engineering and Biotechnology Institute (GEBI) is the first National constitution focused on the research and development of cancer related original and biosimilar biopharmaceutical drugs. In terms of biosimilar drugs, Biosimilar Drug Development and Production Project (BIOSIM-1) is supported by TUBITAK TARAL 1007 programme in December The biotechnological anti-cancer drug, funded by National capital will be produced for the treatment of cancer patients, with the collaboration of the Governmental Research Center and a National Pharmaceutical Company. For this purpose, a recombinant cell bank producing the Biosimilar molecule will be developed at GEBI, the Governmental Research Center. The final product will be manufactured at the Pharmaceutical Company s pilot facility. The phase I trial assays will be funded by the company itself. TÜBİTAK Marmara Research Center will ensure the sustainability of the knowledge and acquisitions obtained during the project. At the end of this collaboration, a significant Biosimilar manufacturing infrastructure and technological know-how at worldwide standard will be acquired in Turkey. The transfer technology will lead to the development of new innovative and competitive biopharmaceutical products contributing to the National Economy. On the other hand, GEBI is Turkey's first institute to develop recombinant antibodies by using phage display technology and has an experience in the field of antibody engineering since Two novel anti-angiogenic recombinant antibodies were selected from a single chain variable fragment antibody library generated from mice immune repertoire. Then, their anti-angiogenic properties were demonstrated in in-vivo rat cornea angiogenesis and mice tumor models. These national anti-angiogenic recombinant antibodies were patented in USA and China. With the DAKINAT project these two anti-angiogenic recombinant antibodies were humanized. The market share of anti-angiogenic drug in Turkey is 100 million dollars since year 2017 and the market share is growing every year. So these anti-angiogenic recombinant antibodies have a great potential. But these molecules are pending on theway to be original biopharmaceuticals developed in Turkey, because of budget requirement The infrastructure project called MedBiyo was funded in 2016 by the Ministry of Development of Turkey for the development and production of bio-therapeutics in Turkey. It is aimed to realize physicochemical and biological activity characterizations under GLP conditions.indispensable for the biotechnological drug development process. As summarized above, GEBI has accumulated knowledge to manage research projects on designing new drugs and production. Thus, GEBI has aimed to set up a bridge through strong collaboration in between research and pharmaceutical companies. This overarching goal is compatible with 2023 Turkey`s National Strategic Plan. Presentation in this session will focus on engineered antibody landscape, beginning with a historical perspective of technology development, and finally selected projects such as BIOSIM, DAKINAT and MEDIBIYO about the development of biopharmaceuticals in TUBİTAK Marmara Research Center Genetic Engineering and Biotechnology Institute. 24

27 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-13 Quality and Pharmaceutical Quality Assurance in Biosimilar Drugs Türkan Eldem Hacettepe University, Faulty of Pharmacy, Pharmaceutical Biotechnology Department, Ankara, Turkey There has been a rapid increase in the number of biosimilar drugs in our country and worldwide. When a medicinal product having a recombinant protein as the active substance is produced by another company at the end of the patent expiry, it is necessary to perform comparability studies together with the reference medicinal product in terms of quality, safety and efficacy and to prove its biosimilarity. During comparability studies the quality of the medicinal product should be ensured before starting safety and efficacy studies The aim of this presentation is to provide a brief perspective on the national legislation related to the biosimilar medicinal products, their quality, and the pharmaceutical quality assurance, especially in upstream and downstream processes during the manufacturing of biosimilar medicinal products. 25

28 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-14 Immunogenicity of Biotechnological Medicinal Products: From Cell Line to Finished Product Devrim Demir Dora Department of Medical Pharmacology, Faculty of Medicine, Akdeniz University, Antalya,Turkey Introduction Therapeutic protein products provide effective treatments for several human diseases and the number of proteins used as therapeutic agents is increasing in recent years. Therapeutic proteins are >40 aa polypeptides whose active components are derived from a biological source by being produced in microorganisms and human or animal cells using biotechnology (1). They are not chemically synthesized and when compared with chemically synthesized small molecule drugs, the manufacturing process and molecular structure of biopharmaceuticals are more complex (2). As they are produced mainly in a genetically modified living systems, their synthesis is influenced by multiple parameters during the manufacturing process. Three-dimensional structure of the biopharmaceutical could be changed by manufacturing process and it should be controlled to ensure patient safety. Quality, safety, and efficacy of the therapeutic recombinat protein depends on the structure and post translational modifications of the protein which is directly related with manufacturing process (3). Factors Affecting Immunogenicity of Biotechnological Medicinal Products The most important safety issue for biopharmaceuticals is immunogenicity. Therapeutic proteins are recognized by the human immune system and followed by an unwanted immune response. The immune response against therapeutic proteins differs not only between products and product categories but also between individuals and patient groups. Factors that may affect the development of an immune response against a therapeutic protein can be either patient- and disease-related or product related (4,5). Patient- and disease-related factors Patient s genetic factors, age, other diseases, concomitant treatment and treatment related factors can affect immune response. Length of treatment, dosage schedule and route of administration are also factors in antibody formation. Higher doses or prolonged duration of treatment increase exposure and thereby heighten the risk of developing immunogenicity. Immunogenicity appears to be greater if the biopharmaceutical is administered subcutaneously (sc) or intramuscularly (ım) and has decreasing severity with intravenous (ıv) administration. Inhalational, intradermal, as well as ocular administration may also enhance immune responses towards the therapeutic protein. Product-related factors Product-related factors that can influence the immune response include the manufacturing process, formulation, stability characteristics, the interactions between the drug and/or formulation with the primary packaging material (container closure system) and the level of impurity or presence of contaminants. Manufacturing of Recombinant Proteins The protein production process begins with the isolation and cloning of the required gene into a vector and transfer into a host cell. Prokaryotic systems (Escherichia coli), yeast (S.cerevisiae), mammalian cells (CHO), insect cells, 26

29 DAVETLİ KONUŞMACILAR / INVITED LECTURES transgenic animals and transgenic plants can be used as expression sytems. Any change in the vector, cell line, growth medium, upstream and down stream processes, filtration or chromatography reagents can change immunogenicity of the product. Each expression system has different impurity profile. Product related or manufacturing process related impurities or post-translational protein modifications can cause immunogenicity (5). Formulation of Drug Substance The formulation of a biopharmaceutical is as important as the active ingredient. Proteins usually aggregate from partially unfolded molecules. Excipients (e.g. Human serum albumin (HSA), polysorbates, glycine and amino acids) are routinely used to stabilize protein drugs. However, the excipients may also influence a drug s safety profile. Although formulations are developed to make final protein product stable, dilution with infusion solutions, exposure to interfaces, container-closure systems, filling pumps, light, temperature fluctuations or minor impurities can induce aggregation and cause immunogenicity (5). Purity and Impurities of Finished Product Purity is an important critical quality attribute for a biological/biotechnological product. The determination of purity depends on an analytical technique and is assessed by a combination of analytical procedures. The results are method dependent (6). The impurities observed in Drug Substance and Drug Product are classified as process-related impurities or product related impurities. Impurities should be controlled by in-process acceptance criteria or action limit on active ingredients or products. New impurities should be evaluated regarding its impact on quality, safety, and efficacy. Safety is ensured by the specified limits for bioburden and endotoxin and process-related contaminants (7). Process-related impurities include host cell DNA, host cell protein, cell culture components and/or antibiotics, leachates and Protein A. Product-related impurities include structural properties, such as protein sequence, molecular variants, the presence of exogenous or endogenous epitopes and the degree of glycosylation influencing protein degradation, exposure of antigenic sites and solubility. Conclusion Nearly all protein drugs induce antibodies in humans and responses are individual. Individual characteristics such as genetic background and disease are important parameters. Immune responses against non-vaccine biologics can affect their efficacy and safety, resulting in adverse events that could include administration reactions, hypersensitivity, deficiency syndromes and lack of a clinical response in treated patients. An unwanted immune response to a therapeutic protein may lead to a loss of efficacy and/or to severe side effects. Neutralizing antibodies can ocur during the treatment which can interfere with the efficacy of the therapeutic protein. Immunogenicity can not be predicted completely from physicochemical characterization, epitope analysis or animal studies. References [1.] Immunogenicity Assessment for Therapeutic Protein Products, U.S. Department of Health and Human Services Food and Drug Administration, August [2.] Devrim Demir-Dora, Biyofarmasötik Ürünlerin Geliştirilmesinde Biyobelirteçler, Turkiye Klinikleri J Pharmacol-Special Topics, 5(2):75-83, [3.] K. Anshu, C. Narendra, and N. Pradip, Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications, Journal of Immunology Research, June 2016, Vol. 2016, [4.] Guideline on Immunogenicity assessment of therapeutic proteins, 18 May 2017 EMEA/CHMP/ BMWP/14327/2006 Rev 1. [5.] Sorularla Biyoteknolojik ve Biyobenzer İlaçlar. Sadi Özdem, İrfan Çiçin, Devrim Demir Dora, Ceyda Korucu 27

30 DAVETLİ KONUŞMACILAR / INVITED LECTURES Nazlı, Ed: İrfan Çiçin, Güneş Tıp Kitapevleri ISBN: [6.] Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products, U.S. Department of Health and Human Services Food and Drug Administration, April 2016 Pharmaceutical Quality/ CMC Revision 1. [7.] S. Zuben, L. Daniel, P.V. Joao, G. Basil, and R.S. Amy, Evaluating and Mitigating the Immunogenicityof Therapeutic Proteins, Trends in Biotechnology, October 2018, Vol. 36, No. 10, Keywords: biotechnological medicinal products, immunogenicity, manufacturing, process-related factors, produc-related factors 28

31 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-15 Nanoparticle Systems for the Delivery of Recombinant Proteins and Nucleic Acids Ayşe Gülten Kantarcı Department of Pharmaceutical Biotechnology,Faculty of Pharmacy, University of Ege, İzmir, Turkey Abstract: Many viral and nonviral systems have been developed for the delivery of biologically active molecules into cells such as recombinant proteins and nucleic acids. Among these, nanoparticle systems have received increasing attention due to their physiologically well-tolerated ingredients, availability to produced in large scale, having good storage capabilities and being low cytotoxic. Here, we have reviewed our studies on gene and protein delivery by developed nanoparticle systems in our laboratories. Keywords: gene delivery, antisense gene therapy, solid lipid nanoparticle, magnetic nanoparticle, oral insulin delivery Introduction Solid lipid nanoparticles (SLNs) are one of the most promising nanoparticulate systems that are being used for drug and gene delivery. SLNs usually consist of physiologically well-tolerated ingredients already approved for pharmaceutical applications in humans. They have many advantages as carrier systems such as; They can be produced in large scale, have good storage capabilities including freeze-drying, can be sterilized, have low cytotoxicity. In addition, the charge of the particles can be modulated via the composition, thus allowing binding of oppositely charged nucleic acid molecules via electrostatic interactions. Using SLNs as protein and gene delivery systems, many studies were carried out in Pharmaceutical Biotechnology Department, Pharmacy Faculty, Ege University. Results and Discussion a. Optimization of SLN production by the hot microemulsion method We aimed to investigate the microemulsion formation area with solid lipids using hot ternary phase diagrams at elevated temperatures and to use selected microemulsions for SLN production. Also, we aimed to characterize obtained SLNs in terms of physicochemical properties, in vitro cell toxicity, and hemocompatibility. Phase diagrams of solid lipids were screened at elevated temperatures and oil-in-water microemulsion regions were determined. Microemulsions were selected, and SLNs were produced by modification of the microemulsion dilution method and characterized in terms of visual appearance, turbidity, particle size, and zeta potential. Nanoparticles prepared by the new method exhibit lower toxicity on L929 cells and have lower hemolytic potential than the formulations prepared by direct mixing of the components [1]. The method can be used to prepare SLNs with controllable composition and small particle size below 100 nm. These SLNs are low toxic and are used for gene delivery purposes for STAT3 silencing in lung cancer cells [2]. b. SLN Mediated Gene Silencing of 5 Αlpha-Reductase-2 For The Treatment of Androgenic Alopecia In another study, in order to treat androgenic alopecia (AA), we aimed to develop a non-viral gene delivery system for topical delivery of 5α-reductase-2 short hairpin RNAs (5-αR2 shrnas) plasmid. AA is the most common form 29

32 DAVETLİ KONUŞMACILAR / INVITED LECTURES of hair loss. The responsible androgen of AA is dihydrotestosteron (DHT). The conversion of testosterone to DHT is catalyzed by 5-αR2. Then, DHT binds the androgen receptors with an avidity five-fold higher than testosterone and leads to AA development. Therapeutics based on shrnas, which act by inhibiting the expression of target transcripts, represent a new class of highly specific treatments [3]. Within this study, SLNs were developed by modified microemulsion dilution technique using compritol as matrix lipid, Tween 80 as surfactant, ethanol as co-surfactant, and distilled water as dispersant. EQ 1 and DDAB were incorporated into the formulation as charge carrier [4]. Nanoparticles were evaluated for complexation with shrna encoding plasmid DNA using agarose gel retardation assay. The obtained formulation was evaluated for in vitro DNA release and DNase I protection ability. Cytotoxicity of complexes was determined on mammalian cell lines. According to the results, developed formulation loaded with 5-αR2 shrna-encoding plasmid has promising potential as RNAi therapeutic for treatment of androgenic alopecia. c. A Novel Synthesis Method of Cationic Lipid Coated Magnetic Nanoparticles (MNP) For targeting with magnetic SLNs, a novel iron oxide nanoparticle synthesis method with in-situ surface coating was developed in our department. Such delivery systems need to be biocompatible and non-toxic. In addition, surface coating is required for loading drugs or to form complexes with nucleic acids. With this method multiple emulsions were used as microreactors and magnetic iron oxide particles synthesized in the core of cationic solid lipid nanoparticles for the first time. 1M Fe +2 and 2M Fe +3 solutions were incorporated into the inner water phase of multiple emulsion (w 1 /o/w 2 ) and 2N NH 4 OH was added in the outer phase of the multiple emulsion (w 1 /o/w 2 ) [5]. [OH - ] ions leaked to the interior water phase of the multiple emulsion and reacted with Fe solutions. Therefore, magnetic iron oxide particles formed in the core of cationic solid lipid nanoparticles. Obtained MNPs are nano-sized with narrow particle size distribution, and superparamagnetic [6]. They are positively charged due to cationic lipid coating during synthesis process. MNPs were than complexed with green fluorescent encoding plasmid DNA (pegfp-c1). Cytotoxicity and transfection studies were carried out in mammalian cell culture. Consequently, a well-defined targetable MNPs with highly effective magnetofection ability were developed. d. A novel nanoencapsulated system for oral protein delivery In this study, we aimed to develop a novel protein - nanoencapsulated system for oral administration. For this purpose, insulin was selected as the model drug. Insulin loaded chitosan nanoparticles (INS-CS-NPs) were obtained by ionic gelation between chitosan (CS) and sodium tripolyphosphate (TPP) [7]. Afterwards, as a novel strategy the nanoparticles were loaded into the inner phase of prepared water in oil microemulsion to provide sustained released, increased in vivo stability and enhanced drug absorption in the gastrointestinal tract [8]. By this way, INS- CS-NPs encapsulated in microemulsion (INS-CS-NP-ME) was formed. In vitro release study in ph 2.5 revealed that insulin release was significantly low under higher CS ratios (p< 0.05). Circular dichroism analyses showed that the conformational stability of insulin was not affected from preparation process. Furthermore, in vivo experiments in Wistar Albino rat model demonstrate that INS-CS-NP-ME effectively reduced blood glucose levels over a period of 8 h after oral administration. In conclusion, insulin loaded chitosan nanoparticles encapsulated in microemulsion system provides a protective and prolonged effect for oral delivery, which may reduce the dosing interval in insulin dependent diabetes mellitus. Moreover, as it provides protection in the gastric conditions, it could also be useful for local delivery of protein therapeutics for intestinal diseases, such as intestinal cancer and Crohn's disease. Conclusions All studies in our laboratories have shown that developed nanoparticle systems may be used as safe, non-toxic, targetable delivery systems for recombinant proteins and nucleic acids. 30

33 DAVETLİ KONUŞMACILAR / INVITED LECTURES References [1.] M. Kotmakçı, H. Akbaba, G. Erel, G. Ertan, G. Kantarcı, Improved method for solid lipid nanoparticle preparation based on hot microemulsions: preparation, characterization, cytotoxicity, and hemocompatibility evaluation, AAPS PharmSciTech(2016), [2.] M. Kotmakçı, V.B. Çetintaş, A.G. Kantarcı, Preparation and characterization of lipid nanoparticle/pdna complexes for STAT3 downregulation and overcoming chemotherapy resistance in lung cancer cells, Int. J. Pharm. (2017) [3.] Sincalair RD. Male androgenic alopecia, Journal of Men s Health and Gender 1: , [4.] del Pozo-Rodriguez A, Delgado D, Solinis MA, Gascon AR, Pedraz JL. Solid lipid nanoparticles: formulation factors affecting cell transfection capacity. Int J Pharm 18;339(1-2): , [5.] Schillinger U, Brill T, Rudolph C, Huth S, Gersting S, Krötz F, et al., J Magn Magn Mater, 293, 501 8, [6.] Kumar A, Jena PK, Behera S, Lockey RF, Mohapatra S, Mohapatra S., Nanomedicine Nanotechnology, Biol Med, 6, 64 9 [7.] Martinez L.; Agnely F.; Leclerc B.; Siepmann J.; Cotte M.; Geiger S, et al. Cross-linking of chitosan and chitosan/poly(ethylene oxide) beads: A theoretical treatment. Eur J Pharm Biopharm., 67, (2007). [8.] Soudry-Kochavi L.; Naraykin N.; Nassar T and Benita S. Improved oral absorption of exenatide using an original nanoencapsulation and microencapsulation approach. J Control Release, 217, (2015). 31

34 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-16 Nanocomplexes Targeting the Mucus Barrier in Chronic Respiratory Diseases Massimo Conese, Stefano Castellani, Sante Di Gioia Laboratory of Experimental and Regenerative Medicine, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy Gene delivery to the respiratory tract offers the development of innovative therapies having the ability to target local disorders and to reduce at the same time systemic dose minimizing side effects. Chronic obstructive diseases can be either derived from inheritance of mutated genes, such as cystic fibrosis (CF), or can be acquired (smoke, pollutants), such as chronic obstructive pulmonary disease (COPD) and asthma, whose pathogenesis include genetic susceptibility [1]. CF, COPD and asthma are characterized by hypersecretion of respiratory mucus, whose composition and biophysical properties are changed by inflammation affecting the respiratory tract in these diseases [2]. The expectorated excessive secretions are referred to as sputum. Inflammation is also responsible for ciliary dysfunction that, in combination with a thick and more than normal viscolestatic sputum, impairs muco-ciliary clearance. Mucus is composed primarily of crosslinked and entangled mucin fibers, that are formed by the linking of numerous mucin monomers, and are coated with a complex and highly diverse array of proteoglycans. In sputum, necrotic neutrophils release deoxyribonucleic acid (DNA) and filamentous actin (F-actin) from the cytoskeleton. DNA and F-actin copolymerize to form a second rigid network within airway secretions [2, 3]. Entanglement of mucin results in formation of dense fiber mesh in mucus gel layer structure. Interaction of drug and delivery systems with mucus components is due to ionic interactions, hydrogen bonds and hydrophobic interactions. Furthermore, enzymes derived from bacteria and fungi colonizing the airways may degrade gene delivery vectors [4]. Thus, in order to penetrate mucus or sputum, synthetic nanoparticles must avoid adhesion to mucus components and be small enough to avoid significant steric inhibition by the dense fiber mesh [5, 6]. The use of mucoadhesive gene delivery vectors is one of the drawbacks that have not allowed to fully realize the promise of gene therapy in the clinical setting, although positive encouraging results had been obtained in preclinical animal models. For example, more than 25 clinical trials have been performed in CF, using both viral and nonviral delivery methods, but without the emergence of a clinical therapy [7]. Although initial studies found that cellular receptors and intracellular barriers (e.g. endosomes) could be a detrimental factor for efficient gene delivery to airway epithelial cells [8], most recently the extracellular barriers, such as the mucus and glycocalyx, have been recognized as the most important setbacks. Thereby, different strategies have been set up to accomplish the efficient crossing of mucus and/or sputum. Trapping mechanisms in the airway mucus can be divided into adhesive trapping and steric trapping [9, 10]. The adhesive trapping is due to the negatively-charged glycosylated domains of mucins that likely pose a significant hindrance to the penetration of positively-charged particulates. The same holds for F-actin and DNA. Most nonviral gene vectors are formulated with positively charged carrier materials, including cationic polymers and lipids that condense nucleic acids into NP with net positively-chargedsurfaces. To avoid these interactions, nonviral gene carriers (i.e. cationic liposomes and polymers) have been formulated by exposing on their surface either poloxamers or hydrophilic, neutrally-charged polyethylene glycol (PEG) molecules [11]. While the use of poloxamers have been limited in the respiratory field, much attention has been paid to PEG. To facilitate mucopenetration, proper configuration and molecular weight of PEG are needed. Various types of nanoparticles coated thoroughly as dense- 32

35 DAVETLİ KONUŞMACILAR / INVITED LECTURES brush with PEG (5 kda) exhibited the highest mucus penetrating ability compared to loosely as brush coated particles [12]. Dense PEGylation of cationic polymer-based gene vectors also greatly improved mucopenetration as well as the following after inhalation into the lungs of mice: (i) gene vector distribution uniformity within the airways, thereby allowing access of gene vectors to a much higher percentage of the underlying epithelium, (ii) gene vector retention within the airways owing to the deep penetration of the particles through the mucus gel layer and into the PCL, and (iii) transgene expression [13]. Another approached pursued by our group is to shield the dense cationic charges of polyethylenimine (PEI), a polymer which condenses tightly cdna, with albumin with the aim to avoid electrostatic interaction between PEI and mucus components. The ternary PEI-albumin-DNA complexes not only allowed to increase gene transfer into airway epithelial cells but did also in the presence of soluble phase of CF sputum [14]. Formulation of PEI polyplexes in the presence of albumin resulted in vivo in a 2-fold increase in luciferase expression and nuclear b-gal localization in bronchiolar epithelium with a 4-fold increase in the number of transfected cells [15]. Specific adhesive interactions between gene vectors and mucus in the gel layer may also reduce viral gene vector penetration, distribution uniformity, and transfection efficiency. Specific sugars on cell surfaces are used by viruses in order to enter the cell, but soluble mucins found in abundance in the mucus gel layer may possess the same sugars. Indeed, adenovirus serotype 5 andadenoassociated virus (AAV) serotype 1, 2, and 5 all strongly adhere to the CF mucus gel [16]. AAV2 adhesion to CF mucus may be partially mediated by specific binding to heparan sulfate on airway mucins, whereas a mutated AAV2 wherein specific binding to heparan sulfate was abolished, lead to a twofold increase in transport in CF mucus [17]. Recently, AAV6 diffusion in CF sputum was enhanced by 3- to 10-fold in comparison to AAV1, while the diffusion of a AAV6 mutant, AAV6-K531E, which has been previously shown to confer AAV6 with an AAV1-like binding affinity to glycans, was significantly lower than those of wild-type [18]. Steric (physical) trapping is determined by the pore size within airway mucus and sputum [9]. Pores within airway mucus gels from human without lung disease were estimated to have size ranging from nm. In one study, PEG nanoparticles as large as 200 nm were shown to efficiently penetrate CF airway sputum, whereas the transport of 500 nm PEG-nanoparticles was strongly hindered by steric obstruction [19]. Based on this and on other studies [17, 20], it is now recognized that inhaled gene vectors with sizes significantly above 100 nm are unlikely to efficiently penetrate the mucus gel layer in CF patients due to steric trapping. To study physical and steric trapping in the same delivery systems, liposomes with size < 100 nm and coated with either thepoloxamerpluronic F-127 (a triblock copolymer of poly (ethylene glycol)-poly (propylene oxide)-poly (ethylene glycol) (PEG-PPO-PEG) or PEG were allowed to migrate through sputum collected from COPD patients [21]. It was demonstrated that PEGliposomes outperformed plain and PF-liposomes in crossing COPD patients but only at a time as long as 27 hours, indicating that a combination of small size and avoiding of hydrophobic interactions is propedeutic for penetrating pathological mucus. Mucopenetrating particles can be used in combination with mucolytic agents in order to cleave certain substructures within the three dimensional network of the mucus, including disulfide breaking agents such as N-acetyl-cysteine (NAC), or DNase to break DNA strands superimposed onto the mucin mesh. Pre-treatment with NAC led to more rapid diffusion of AAV1 [17] and a pegylated poly-lysine (CK 30 PEG 10k )-based system [22], through CF mucus. Intranasal dosing of NAC prior to CK 30 PEG 10k /DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, indicating that NAC may be used as an adjuvant in gene therapy applied to lung diseases. On the other hand, recombinant human (rh)dnase was shown to exhibit only minimal to no effect on the diffusion of latex nanoparticles and lipid-based gene carriers in CF sputum [23, 24]. However, pegylated polymers based on polyaspartamide (PHEA) were enhanced in their gene transfer efficiency in the presence of CF sputum sol phase but not significantly, again indicating that the coverage of PEG was not sufficient to overcome the hindrance of polyplexes by sputum components also in the presence of DNA disrupting agent (Di Gioia S., Licciardi M., Cavallaro G., Conese M., unpublished results). Overall, these findings underscore the importance of rapid penetration of the airway mucus barrier to achieve high 33

36 DAVETLİ KONUŞMACILAR / INVITED LECTURES levels of airway gene transfer in vivo. In order to achieve this goal, magnetofection (i.e., the nucleic acid delivery under the influence of a magnetic field acting on nucleic acid vectors that are associated with magnetic nanoparticles) can be used to improve the kinetics of the delivery process, as well as to localize nucleic acid delivery to an area which is under magnetic field influence [25]. We have shown that magnetofection can reduce the time of contact between lentiviral vectors (LV) and airway epithelial cells to obtain efficient transduction [26]. With only 15 min of incubation, the LV mediated transduction was increased by 81 fold by paramagnetic LV vectors. Importantly, magnetofection enhanced LV-mediated transduction through CF sputum sol phase. This method should be also useful in the context of nonviral vectors. Our preliminary experience with unmodified solid lipid nanoparticles (SLN) shows that magnetofection enhances penetration of paramagnetic SLN through COPD sputum as compared with nonmagnetic SLN (Castellani S., Trapani A., Di Gioia S., Conese M., unpublished results). We have previously shown that these SLN are taken up efficiently by airway epithelial cells and exert anti-oxidant and anti-inflammatory effects [27]. In conclusion, for efficient gene transfer to the airway epithelium in vivo, inhaled gene vectors must be small enough to diffuse through the mucus mesh while possessing a muco-inert surface to avoid adhesion to mucus constituents. Simultaneous modulation of gene vectors and the mucus barrier may synergistically improve mucus penetration and thus efficacy of inhaled gene therapy. Magnetofection may further increase speed of mucopenetrating gene therapy vectors and increased precision. S.C. has been funded by Intervento Cofinanziato dal Fondo di Sviluppo e Coesione APQ Ricerca Regione Puglia Programma regionale a sostegno della specializzazione intelligente e delle sostenibilità sociale ed ambientali Future In Research. REFERENCES [1.] Di Gioia, S., et al., From Genesis To Revelation: The Role Of Inflammatory Mediators In Chronic Respiratory Diseases And Their Control By Nucleic Acid-Based Drugs. Curr Drug Deliv, 2017;14(2): [2.] Rogers, D.F., Mucoactive agents for airway mucus hypersecretory diseases. Respir Care, 2007; 52(9): [3.] Sanders, N., et al., Extracellular barriers in respiratory gene therapy. Adv Drug Deliv Rev, 2009; 61(2): [4.] Mottais, A., et al., Enhancement of lung gene delivery after aerosol: a new strategy using non-viral complexes with antibacterial properties. Biosci Rep, 2017; 37(6). pii: BSR [5.] Lai, S.K., Y.Y. Wang, and J. Hanes, Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv Drug Deliv Rev, 2009; 61(2): [6.] Di Gioia, S., et al., Nanocomplexes for gene therapy of respiratory diseases: Targeting and overcoming the mucus barrier. Pulm Pharmacol Ther, 2015; 34: [7.] Hart, S.L. and P.T. Harrison, Genetic therapies for cystic fibrosis lung disease. Curr Opin Pharmacol, 2017; 34: [8.] Gill, D.R. and S.C. Hyde, Delivery of genes into the CF airway. Thorax, (10): [9.] Duncan, G.A., et al., The Mucus Barrier to Inhaled Gene Therapy. Mol Ther, 2016; 24(12): [10.] Kim, N., et al., Barriers to inhaled gene therapy of obstructive lung diseases: A review. J Control Release, 2016; 240: [11.] Netsomboon, K. and A. Bernkop-Schnurch, Mucoadhesive vs. mucopenetrating particulate drug delivery. Eur J Pharm Biopharm, 2016; 98: [12.] Suk, J.S., et al., PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev, 2016; 99(Pt A): [13.] Suk, J.S., et al., Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier. J Control Release, 2014; 178: [14.] Carrabino, S., et al., Serum albumin enhances polyethylenimine-mediated gene delivery to human respiratory epithelial cells. J Gene Med, 2005; 7(12): [15.] Di Gioia, S., et al., Role of biophysical parameters on ex vivo and in vivo gene transfer to the airway epithelium by 34

37 DAVETLİ KONUŞMACILAR / INVITED LECTURES polyethylenimine/albumin complexes. Biomacromolecules, 2008; 9(3): [16.] Hida, K., et al., Common gene therapy viral vectors do not efficiently penetrate sputum from cystic fibrosis patients. PLoS One, 2011; 6(5): e [17.] Schuster, B.S., et al., Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors. Mol Ther, 2014; 22(8): [18.] Duncan, G.A., et al., An Adeno-Associated Viral Vector Capable of Penetrating the Mucus Barrier to Inhaled Gene Therapy. Mol Ther Methods Clin Dev, 2018; 9: [19.] Suk, J.S., et al., The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles. Biomaterials, 2009; 30(13): [20.] Schuster, B.S., et al., Nanoparticle diffusion in respiratory mucus from humans without lung disease. Biomaterials, 2013; 34(13): [21.] De Leo, V., et al., Preparation of drug-loaded small unilamellar liposomes and evaluation of their potential for the treatment of chronic respiratory diseases. Int J Pharm, 2018; 545(1-2): [22.] Suk, J.S., et al., N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles. Mol Ther, 2011; 19(11): [23.] Dawson, M., D. Wirtz, and J. Hanes, Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport. J Biol Chem, 2003; 278(50): [24.] Sanders, N.N., et al., On the transport of lipoplexes through cystic fibrosis sputum. Pharm Res, 2002; 19(4): [25.] Plank, C., O. Zelphati, and O. Mykhaylyk, Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Adv Drug Deliv Rev, 2011; 63(14-15): [26.] Castellani, S., et al., Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers. Genes (Basel), 2016; 7(11). pii: E103. [27.] Castellani, S., et al., Nanoparticle delivery of grape seed-derived proanthocyanidins to airway epithelial cells dampens oxidative stress and inflammation. J Transl Med, (1):

38 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-17 Production and Applications of Mıcrobial Polyhydroxyalkanoates (PHAs) as Drug Delivery Systems Gülay Büyükköroğlu, Behiye Şenel Anadolu University, Faculty of Pharmacy Department of Pharmaceutical Biotechnology, Türkiye Abstract Poly(hydroxyalkanoates) (PHA) are FDA approved, non-cytotoxic, highly biocompatible and biodegradable polymers. PHAs which are the polyesters of various hydroxyalkanoate monomers which accumulate as energy/carbon storage materials in the form of granular inclusions in the cytoplasm of various bacterial cells, usually under unbalanced growth conditions. These polymers are potential candidates as carriers in drug delivery systems due to their unique and interesting features. Surface erosion of the polymers during degradation which leads to controlled release of the active ingredient rather than burst release. The physicochemical characterizations of Venetoclax (VEN) loaded nanoparticles the such as particle size and shape, zeta potential were examined. Cell culture studies were done via MTT assays on MCF-7 (human breast cancer) and A549 (lung carcinoma) cell lines. Results showed that particle sizes of complexes were below 300 nm, with a negative zeta potential.venetoclax (VEN) loaded nanoparticles were more toxic to MCF-7 cell line when compared to A549. Keywords Poly(hydroxyalkanoates) (PHA), drug delivery, Venetoclax (VEN), Poly(3-hydroxyalkanoates) P(3HB) Introduction: Polyhydroxyalkanoates (PHA) are biosynthetic and biodegradable and fully recyclable materials known as a group of microbial poly (oxoesters) and prokaryotic storage materials. Poly(3-hydroxyalkanoates) P(3HB) is a type of PHA, which are a class of versatile biodegradable and biocompatible materials that have attracted much attention from scientists in biomedical fields. P(3HB) has been widely used in bone tissue engineering applications (1). These polymers are also potential candidates as carriers in drug delivery systems due to their unique and interesting features including surface degradation properties, leading to controlled release of the encapsulated active ingredient as opposed to bulk degradation observed for PLA/PLLA/PLGA; the degradation products are less acidic leading to lower inflammation; the degradation rate can be tailored depending on the type of PHA produced and the rate of degradation is relatively slower leading to long term delivery (2). PHAs are the polyesters of various hydroxyalkanoate monomers which accumulate as energy/carbon storage materials in the form of granular inclusions in the cytoplasm of various bacterial cells, usually under nutrient limiting conditions (3). Biodegradable polymers containing an entrapped drug can be placed in the body, and they are used for localized drug delivery accompanied with the controlled release of a drug over a period of months (4). PHAs are biocompatible and hydrophobic; they can also be turned into films, porous matrices, microcapsules, microspheres, and nanoparticles. Drugs can be entrapped or microencapsulated in a PHA homopolymer or copolymer. Microsphere or microcapsule based delivery systems have been extensively used for the delivery of a number of drugs such as anaesthetics, antibiotics, anti-inflammatory agents, anticancer agents, hormones, steroids, and vaccines (3, 5). Cancer leads to the formation of tumors which are characterized by uncontrolled cell proliferation. The uncontrolled proliferation that occurs as a result of mutations in DNA hinders the apoptosis of the cell. Apoptosis is a crucial physiological mechanism that controls the development and elimination of unwanted and malignant cells. The BCL-2 protein is encoded by the BCL-2 gene and plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (6). Venetoclax (VEN) is the first FDA-approved (April 2016) drug which targets BCL-2 protein in cancer cells and is being used for the treatment of patients with chronic 36

39 DAVETLİ KONUŞMACILAR / INVITED LECTURES lymphocytic leukemia (CLL). VEN (ABT-199, GDC-0199) blocks an important pathway that promotes cell survival in tumour cells with overexpressed BCL-2, so VEN causes tumour cells to die (pro-apoptotic). BCL2, which is part of the p53 signaling pathway and regulates a form of cell death called apoptosis, is overexpressed in many patients with chronic lymphocytic leukemia (CLL) (7). The aim of the project is formulating and evaluating of Venetoclax-loaded nanoparticles using Poly(3-hydroxyalkanoates), aiming for their use cancer treatment. Method Preparation of formulation: P(3HB) nanoparticles (NP) were prepared by solvent emulsification/evaporation method according to the procedure described by Şenel, et al., (2015) with some modifications. Briefly, 10% (w/v) P(3HB) was dissolved in dichloromethane (DCM) and the surfactant 1% (w/v) (Tween 80) was dissolved in distilled water (Millipore Milli-Q A10, ph 6.8) and autoclaved (121 C, 20 min). The aqueous phase was added to the oily phase in a round bottom 10 ml glass flask and the mixture was sonicated using a sonicator in a temperature-controlled (2 C) water bath. The mixture was evaporated under 20 rpm stirring and at about 150 MBar for min in a water bath of 33 C to remove the residual organic solvent using a rotary evaporator. For the preparation of VEN-loaded NPs (V-NP), VEN, was dissolved in DCM to form the oily phase and the both the methods explained above was used. Morphology of nanoparticles: Surface morphology of the NPs was investigated using Carl Zeiss scanning electron microscope (SEM). Prior to analysis, NPs were dropped on double-sided carbon tape pre-affixed on a specimen stub and were dried at room-temperature. Samples were coated with a thin layer of gold (100 Å) by a sputter coater before observed under SEM (9). Determination of particle size and zeta potential: NPs were characterized using a particle size analyzer (Zetasizer NanoZS, Malvern Instruments, UK) to deter-mine average diameter and size distribution represented by the polydispersity index (PDI). The ph of water was adjusted to ph 7.4 prior to analyses (10). The Malvern Zeta capillary cuvettes were used for the zeta potential analyses and all analyses were repeated in triplicate at room temperature. Cell culture studies: MCF-7 (human breast cancer) and A549 (lung carcinoma) cell lines were used for in vitro cell cultures studies. The colorimetric 3-(4,5-dimethylthia zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used for the quantitative determination of cell cytotoxicity (11). The absorbances of the plates were measured at 570 nm using a spectrometric microplate reader. Results were expressed as the percentage of absorbance of control cells. Results Size and Morphology of NPs: Particle sizes of the NPs and V-NPs were in the range of ± 8.22 and ± 4.23 nm. These results were SEM images showed that the particles were spherical in shape, with sizes also in the nanometer range (Fig. 1). The PDI value of SLN was ranged between ± 0.05 and ± 0.08 indicating that the formulations are polydisperse systems with relatively homogenous size distributions. Zeta potential values of the formulation were found to be between ± 0.44 and ± 0.95 at ph

40 DAVETLİ KONUŞMACILAR / INVITED LECTURES Figure 1: SEM images of NP (A) and V-NP (B) Cytotoxicity of formulations: Toxicities of VEN and formulations on MCF-7 and A549 cell lines were determined after 48 h. Results obtained are presented in Figure 2. Figure 2: Viability of MCF-7 and A549 cells after being treated with VEN and formulations for 48 h. Discussion The literature also states that while complexes with particle sizes less than 300 nm were uptaken by clathrin mediated endocytosis, complexes with sizes above 500 nm are uptaken by caveolar endocytosis (Rejman et al. 2004). In this study the particle sizes of the NPs were found below 300 nm with relatively homogenous size distributions. These results indicate that NPs can be up taken by cell. Zeta potential (ZP) value associated with short/long term stability of polymeric nanoparticles demonstrating high positive or low negative (+30/-30 mv) values lead to high stability (12). The ZP of NPs were measured below -20 mv, proving the high stability of the NPs suspended in aqueous medium. According to the 48 h cytotoxicity test results, free VEN and NP showed cell viability over 60% even at the highest doses. Free VEN showed less toxicity on A549 cell lines compared to MCF-7 cell lines with cell viability of ± 1.54% and ± 2.01% at the highest doses, respectively. Dose dependent cytotoxicity was found in cytotoxicity studies for V-NP on MCF-7 cell line. These results indicate that MCF-7 cells overexpressed the BCL-2 protein than A549 cells, and the bcl-2 gene blocked by VEN in MCF-7 cell. 38

41 DAVETLİ KONUŞMACILAR / INVITED LECTURES Conclusions These results show that P(3HB) can be used as a drug delivery system without toxicity and that VEN can be loaded to P(3HB) and it can be effective in different cancer cells with this carrier system. References [1.] Bugnicourt, E., et al "Polyhydroxyalkanoate (PHA): Review of synthesis, characteristics, processing and potential applications in packaging", EXPRESS Polymer Letters, 8 (11), [2.] Nigmatullin, R., et al Polyhydroxyalkanoates and their applications in Drug Delivery, J. Chem. Technol. Biotechnol. 90(7) [3.] Shrivastav, A., et al Advances in the Applications of Polyhydroxyalkanoate Nanoparticles for Novel Drug Delivery System, Biomed Res Int. 2013: [4.] Lenz, R.W. and Marchessault, RH, "Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology." Biomacromolecules, 6.1, 1-8. [5.] Nobes, GAR., et al "Polyhydroxyalkanoates: materials for delivery systems." Drug Delivery 5.3, [6.] Bruckheimer, EM. Et al The Bcl-2 gene family and apoptosis, Adv Biochem Eng Biotechnol. 62: [7.] Roberts AW, et al., Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia. N Engl J Med. 374(4): [8.] Şenel, B., et al Solid lipid and chitosan particulate systems for delivery of sirna, Pharmazie, 70, [9.] Büyükköroğlu, G., et al Preparation and in vitro evaluation of vaginal formulations including sirna and paclitaxel-loaded SLNs for cervical cancer Eur J Pharm Biopharm,109, [10.] Büyükköroğlu, G., et al Preparation and physicochemical characterizations of solid lipid nanoparticles containing DOTAP for DNA delivery, Turk J Chem, 39, [11.] Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity tests, J Immunol Methods, 63, [12.] Martín-Banderas, L., et al Cannabinoid derivate-loaded PLGA nanocarriers for oral administration: formulation, characterization, and cytotoxicity studies, Int. J. Nanomed. 7:

42 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-18 Development of Novel Drug-Delivery Systems For Specific Targeting of Drugs into Their Intracellular Targets Hisham R. Ibrahim Department of Biochemistry & Biotechnology, Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima , Japan Drug resistance is a serious problem that continues to challenge the healthcare sectors and has become increasingly alarming in the past few years. To face this emerging global crisis, there is a need to find a new class of drugs that act on new targets or harness existing drugs by developing new drug-targeting strategies. Substantial discoveries of new drugs have been made in the last decades, but they are bearing considerable drawbacks due to poor solubility, allergic reactions and systemic toxicity and/or poor cellular transport properties, hence the drug concentrations inside the cells are below the minimum inhibitory concentrations. One novel strategy to overcome this problem is to load efficient drugs into a safe protein carrier that can specifically recognize and then deliver the drug into the target cells. Egg proteins provide endless opportunities in the area of drug delivery due to their biological activities, less toxicity, ease of functionalization, and number of them are being recognized by diseased cellular receptors involved in various uptake events. In this study, I will introduce our recent approaches in which ovotransferrin (OTf) from egg albumen, was loaded with hydrophobic drugs for direct delivery and improved permeability of therapeutics or as targeting ligand for drug-loaded gold nanoparticles. Ovotransferrin is a member of iron-binding proteins called transferrin family. Transferrins are recognized by the transferrin receptor (Tf R) on the surface of mammalian cells and pathogens to uptake the iron necessary for cell growth. Cancer cells require more iron than normal cells, and the majority of cancer cells overexpress Tf R. It is thus, molecules of transferrin family have unique features worth using to target drugs specifically to cancer cells as well as to intracellular infections. Therefore, this study is aimed to develop a drug targeting delivery system to cancer cells by loading OTf with anticancer drugs. Antifolate and platinum-based drugs was conjugated with OTf through non-covalent interaction at various molar ratios. In another set OTf-drug complexes were coupled to gold nanoparticles to increase drug load. The complex formation between OTf and drugs was confirmed using fluorescence analysis and ANS binding assay. The binding and uptake of the complexes by human colon adenocarcinoma cells (HCT-116) was assessed by using anti OTf antibody in receptor binding assay. Cytotoxicity was measured using Acridine Orange/Propidium Iodide Assay, and Proliferation was measured using CyQuant Direct Assay. The binding assay revealed that drugs are bound to the hydrophilic and acidic residues of OTf and all complexes were recognized by Tf R present on the surface of cancer cells. OTf-drug complexes showed stronger anti-cancer and anti-infective actions than free drugs. Further, OTf displayed unique features as it rendered the hydrophobic drugs water-soluble and exhibited specific drug targeting to human intracellular infections as well as to human colon cancer cells. The strategy offers tremendous opportunities for hen egg OTf potential utility as novel drug-targeting molecule in therapy for the treatment of cancers and the emerging infectious diseases. 40

43 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-19 Nanobubbles as Gene Carriers for the Therapy of Lung Cancer Yücel Başpınar 1, Gülşah Erel 2, Mustafa Kotmakçı 1, Hasan Akbaba 1 1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Ege, 35100, İzmir, Turkey; 2 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Izmir Katip Çelebi, 35620, İzmir, Turkey Abstract The worldwide cancer-related death rate for lung cancer is about 23 %, more than the sum of breast, colon and prostate cancer. Chemotherapy with the first-line drug paclitaxel is an often used option. An overexpression of the apoptosis inhibitor survivin is a similarity of almost each cancer type, including lung cancer. Thus, Sur is a target for cancer therapy, lead to synthesis of the Sur inhibitor sepantronium bromide (YM155) Due to the low aqueous solubility of paclitaxel and serious side effects like life-threatening hypersensitivity reactions and hemo-toxicity of approved products, alternative formulations of paclitaxel are required. A promising approach to improve the poor solubility of paclitaxel and reduce the side effects is the use of nanoparticles like nanobubbles, which are promising carrier for gene and drug delivery. Due to their potential to be activated in the presence of ultrasound and mediate the delivery of drugs or genes to specific cells, like tumour cells, they can be used for drug targeting. Nanobubbles (1 500 nm) are spherical vesicles with a gas core and a shell. The shell can be composed of phospholipids, polymers or proteins and perfluorocarbons (perfluoropentane, perfluoropropane, perfluorobutane etc.), sulphur hexafluorid, air and CO 2 can be used as the core material. After application, nanobubbles have to penetrate through the epithelial cells of the vasculature to the target tissue. For that purpose, the particle size of nanobubbles (1-500 nm) is very promising for extravasation. The aim of this study is to develop nanobubble formulations containing paclitaxel, survivin inhibitor YM155 and sirna against the lung cancer cells A549. For that purpose nanobubbles will be prepared and characterized in-vitro, in terms of particle size, size distribution (polydispersity index), zeta potential and cytotoxicity. Keywords Lung cancer, paclitaxel, YM155, nanobubbles, sirna 1. Introduction 23 % of the cancer-related deaths worldwide are caused by lung cancer, more than the sum of breast, colon and prostate cancer ( Jemal et al., 2011). The two main types of lung cancer are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). A first-line drug for the chemotherapy of NSCLC is paclitaxel (PTX). A similarity of almost each cancer type, including lung cancer, is an overexpression of the apoptosis inhibitor survivin (Sur), while it is not detectable or only in traces found in normal lung tissues (Sun et al., 2010). The over-expression of Sur in lung cancer cells (Altieri 2002; Adida et al., 1998), makes Sur to a target for cancer therapy (Muzio et al., 2003). 2007, the Sur inhibitor sepantronium bromide (YM155) (Nakahara et al., 2007) was synthesized. The low aqueous solubility of PTX is a serious limiting factor for its application, so using surfactants and cosolvents Taxol was approved by FDA Unfortunately, serious side effects like life-threatening hypersensitivity reactions and hemo-toxicity are limiting the use of Taxol. Because of this alternative formulations of PTX are required. An important approach to improve the poor solubility is to prepare nanoparticles (NPs) (Ruenraroengsak 2010). Although several methods has been described for NPs, nanobubbles (NBs) are promising carrier for gene delivery (Cavalli et al., 2012). Due to their potential to be activated in the presence of ultrasound ( US) and mediate the delivery of drugs or genes to specific cells, like tumour cells, they can be used for drug targeting. NBs (1 500 nm) are spherical vesicles with a gas core and a shell. The shell can be composed of phospholipids, polymers or proteins and perfluo- 41

44 DAVETLİ KONUŞMACILAR / INVITED LECTURES rocarbons (PFCs; perfluoropentane, perfluoropropane, perfluorobutane etc.), sulphur hexafluorid, air and CO 2 can be used as the core material. After application, NBs have to penetrate through the epithelial cells of the vasculature to the target tissue. For that purpose, the particle size of NBs (1-500 nm) is very promising for extravasation. The aim of this study is to develop nanobubble (NB) formulations containing PTX, Sur inhibitor YM155 and sirna against the lung cancer cells A549. For that purpose NBs will be prepared and characterized in-vitro, in terms of particle size, size distribution (polydispersity index, PDI), zeta potential (ZP) and cytotoxicity. 2. Materials and Methods 2.1 Materials Ethanol, lecithin, medium molecular weight (MMW) chitosan, perfluoropentan, pluronic F68, PTX, YM155, filter, US, DDAB, A549 cells, DMEM, penicillin, streptomycin, L-glutamin, fetal bovine serum, wate Perfluoropentane (2H,3H-Decafluoropantane) and DDAB were purchased from TCI, Tokyo Chemical Industry, Tokyo, Japan. Lecithin was obtaind from AppliChem, Darmstadt, Germany. Paclitaxel (Alfa Aesar, Kandel, Germany), YM155 (Cayman Chemicals, Ann Arbor, USA), low molecular weight (LMW) (Acros, Geel, Belgium), medium molecular weight (MMW) (Aldrich, Steinheim, Germany) and Minisart filter (Sartorius, Göttingen, Germany) were all used as received. Pluronic F68 was a gift from BASF, Ludwigshafen, Germany. A549 cells were obtained from ATCC. DMEM, penicillin, streptomycin, L-glutamin and fetal bovine serum were purchased from Gibco, Auckland, New Zealand. Ethanol (Merck, Darmstadt, Germany) was of analytical grade. 2.2 Methods Development of the nanobubbles For developing the NBs four different NB formulations were prepared: a) blank NBs (NB), b) 1 mg/ml PTX containing NBs (NB-PTX), c) 1 mg/ml PTX and 0.2 mg/ml YM155 containing NBs (NB-PTX-YM) and d) 1 mg/ml PTX and 5 µm sirna containing NBs (PTX:siRNA 3:1, v:v) The development of blank NBs comprised of 6 steps. First, the lecithin concentration was optimized (1.0, 2.0 and 3.0%, w/w). Then, to determine the effect of filtration on the particle size, filtration through 0.4 µm filters were performed. Next, the effect of chitosan molecular weight was assessed by using LMW and MMW chitosans after which the chitosan concentration was optimized (2.7, 1.0 and 0.25%, w/w). To determine the impact of a stabilizing agent, 0.01% Pluronic F 68 was tested. Finally, to investigate the physical conditions of preparation a high shear mixer (Ultra Turrax) and ultrasound processor were separately tested for preparing NBs. For determining the optimized formulation, blank NBs were characterized. After these formulation parameter and the preparation method were optimized, the preparation temperature was investigated and PTX, YM155 and sirna, respectively, were loaded into the NBs Cytotoxicity studies Human NSCLC adenocarcinoma A549 cells were cultured in DMEM medium, supplemented with 1 % penicillin-streptomycin, 1 % L-glutamine, 10 % fetal bovine serum. The cells were checked daily with an inverted microscope and passaged freshly after reaching a confluency of 90 %. A549 cell line was grown in a highly humidified atmosphere of 5% CO 2 in air at 37 C. A549 cells were seeded on wells and incubated for 24 h in a 37 C incubator under 5 % CO 2. The next day, the culture medium was removed and the different NB formulations were applied after sterilization by filtration through a 0,22 µm filter. PBS (control), PTX loaded NBs, PTX and YM155 loaded NBs and PTX and sirna loaded NBs were applied. 42

45 DAVETLİ KONUŞMACILAR / INVITED LECTURES After application of the NBs, the cells were incubated for 48 hours, respectively, in an incubator at 37 C and 5 % CO 2. Following the incubation, 50 µl XTT was applied to each well and incubated for 2 hours. The orange color was measured at defined wavelength colorimetric. The results were compared to the control group and defined as % viability. The viability of the control group was taken as 100 %. 3. Results and Discussion The blank NB formulation after the optimization process had a particle size of 178 nm, a PDI of 0.41 and a ZP of 38 mv. The particle size (308 nm), PDI (0.44) and ZP (53 mv) of the NBs prepared with DDAB at 25 C were increased. After adding PTX to the NBs, the particle size (335 nm) and the PDI (0.57) futher increased, while the ZP (34 mv) decreased. The PTX and YM155 loaded NBs had compared to PTX loaded NBs had decreased particle size (76 nm) and PDI (0.24), and increased ZP (50 mv). Comparing these results obtained after preparation at 25 C with a preparation temperature of 4 C revealed that the particle sizes of all NBs were decreased. The PDI of the blank and PTX + YM155 loaded NBs increased, while the PDI of the PTX loaded NB decreased. The ZPs of all NBs prepared at 25 C and 4 C showed no significant differences. Due to stability issues of sirna the PTX + sirna loaded NBs were prepared only at 4 C with a particle size of 252 nm, a PDI of 0.64 and a ZP of 27 mv. The particle size and PDI can be explained with adhesion of sirna on the NBs. The decreased ZP was caused by the negative charge of sirna. Even the application of low concentration (4 µl/well) of the PTX and YM155 loaded NBs resulted in significant decrease of the viability of A549 lung cancer cells. 4. Conclusions For preparing suitable NBs with a small particle size and a high ZP, 2 % lecithin, 0.25 % medium molecular weight chitosan, filtration through a 0.4 µm filter, 0.01 % of the stabilizing agent Pluronic F 68 and DDAB were necessary. Furthermore, these NBs revealed an enhanced cytotoxic effect on A549 lung cancer cells. Acknowledgement: This study has been financially supported by TUBITAK under grant code 116S213. References [1.] Adida, C., Crotty, P.L., McGrath, J., vd Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. The American J. Path. 152(1), 43. [2.] Altieri, D.C., Survivin in apoptosis control and cell cycle regulation in cancer. Progress Cell Cycle Res. 5, [3.] Cavalli, R., Bisazza, A., Trotta, M., Argenziano, M., Civra, A., Donalisio, M., Lembo, D., New chitosan nanobubbles for ultrasound-mediated gene delivery:preparation and in vitro characterization. Int. J. Nanomed. 7, [4.] Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., Global cancer statistics. CA Cancer J. Clin. 61(2), [5.] Muzio, L.L., Pannone, G., Staibano, S., vd Survivin expression in oral squamous cell carcinoma. Br. J. Cancer 89(12), [6.] Nakahara, T., Takeuchi, M., Kinoyama, I. vd YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts. Cancer Res. 67, [7.] Ruenraroengsak, P., Cook, J.M., Florence A.T., Nanosystem drug targeting: facing up to complex realities. J. Control. Release 141, [8.] Sun, M., Lou, W., Chun, J.Y.vd Sanguinarine suppresses prostate tumor growth and inhibits survivin expression, Genes Cancer, 1(3),

46 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-20 DNA Vaccines Filiz Öner Hacettepe University, Faculty of Pharmacy, Pharmaceutical Biotechnology Department 06100, Ankara Immunization is the process whereby a person is made immune or resistant to an infectious disease, by the administration of a vaccine. Vaccines are biological pharmaceutical products that stimulate the body's own immune system to protect the person against subsequent infection or disease (1). Main types of vaccines are classified as traditional vaccines (live attenuated vaccines inactivated vaccines, toxoid vaccines) and new vaccines (subunit, recombinant, polysaccharide, conjugate). In recent years most studied new generation vaccine types are recombinant DNA vaccines and DNA vaccines. Action mechanism of recombinant DNA vaccines and DNA vaccines differ from each other. The immunogenic antigen expressed with a DNA vaccine is generated by the host cells while Recombinant DNA vaccines are not made of DNA, they made of a protein antigen produced by using recombinant DNA technology. Expressed antigen is injected into the host recipient in a parenteral dosage form. A DNA vaccine is composed of a bacterial plasmid that encodes an antigen (a protein) under a mammalian promoter enabling it to function in the transfected mammalian cells. DNA vaccination could trigger immune responses through the antigen or plasmid DNA itself which has a possible unwanted effect or an adjuvant activity. First DNA vaccine developed in 1990 s with limited knowledge. DNA vaccination involves the direct introduction of plasmid DNA containing the antigen encoding genes into appropriate host tissues and the in situ production of the desired antigens (2). This type of vaccines have potentially important advantages over the direct application of the antigen itself, including long-lasting immunogenicity, no risk of infection (safe), easy production, improved stability, stimulation of both humoral and cellular immune responses, administration of mixture of plasmids or antigen coding genes and low cost. But DNA vaccines also have disadvantages e.g. they are not applicable for non-protein based antigens (bacterial polysaccharides), they can elicit anti-dna antibodies, expressed antigens can induce immunologic tolerance, they have relatively poor immunogenicity and foreign DNA can be inserted into the host genome causing possible oncogenic effects (2-4). Manufacturing requirements in the rules of good manufacturing practices (GMP) for pharmaceutical and biological products are necessary also for plasmid DNA vaccines (3,4). Quality issues of biological products, such as potency, impurities (endotoxin), stability, toxicity and sterility are important for also DNA vaccines. Among the methods to enhance immune response of DNA vaccines; stability of expression, modulation of the immune response, inclusion of molecular adjuvants or co plasmids and formulating the DNA within new delivery systems (microparticles, liposomes) can be counted. As an example, PLGA microsphere vaccine formulations containing pdna coexpressing Hepatitis B surface antigen and Interleukin 2 within or outher surface of the particles are seen in Figure 1. (5) Based on the results from in vivo experiments in mice, microsphere formulations were able to induce immune response upon delivery of pdna. 44

47 DAVETLİ KONUŞMACILAR / INVITED LECTURES Figure 1. pdna encapsulated within the microspheres in A2 and B2 formulations, while B2a contained pdna adsorbed onto the microspheres. A1 and B1 are empty particles. pdna loading capacities of A2, B2 and B2a were 15%, 25% and 45%, respectively. Protective immune responses observed against many infectious agents in fish and farm animals. Five veterinary vaccines manufactured by using plasmid DNA as an active substance have been authorized by different authorities but there is not an authorized human DNA vaccine yet. However clinical phase studies are ongoing on some DNA vaccines against malaria, HIV, influenza, tuberculosis (TB) and Ebola (6). Veterinary vaccines include one against West Nile virus in horses, one against infectious hematopoietic necrosis virus in salmon fish, one for treatment of melanoma in dogs, one for against salmon alphavirus subtype 3 and recent one for influenza in chickens. Manufacturing methods, quality controls and nonclinical safety studies (biodistribution, plasmid persistence and chromosomal integration) should be specifically designed for these vaccines. (7) The final composition of the vaccine, manufacturing steps from the bulk purified plasmid to the final vaccine, identification of materials, equipment and in-process controls should be well documented (2). In this presentation pdna vaccines and scientific issues associated with their development will be reviewed and presented. References [1.] [2.] WHO Technical Report Series No 941, 2007, Annex 1 Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines [3.] FDA (CBER), Plasmid DNA vaccines for Preventative Infectious Disease Indications, DECEMBER 1996 [4.] EMA (2012b) Concept paper on guidance for DNA vaccines, Scientific guideline/2012/03/ [5.] Eratalar A, Coşkun-Arı FF, Öner F, Öz cengiz E., Jurnal of Microencapsulation, 2010, 27(1): [6.] Concept paper on DNA vaccines non-amplifiable in eukaryotic cells for veterinary use EMA/CVMP/ IWP/867401/

48 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-24 Star-T UP in America Mehmed Ugur Turkish American Pharmacists Association, NJ, USA Kapitalizmle Bilimin Buluşması Kapitalizmin olaylara bakış açısı ben bu işten para yapar mıyım? sorusuna cevap şeklindedir. Yani sosyal bir devlet mantığı yoktur. Para yapabileceklerine inanmıyorlarsa, dünyayı kurtarsanız bile yatırımcılardan bir kuruş alamazsınız. Buluşun sahibi ve parayı yatıran aynı kişi olmak zorunda değildir. Ironi burada başlar; zıt gibi görünseler de bir araya gelebilirler. Önemli olan; elindeki fikri paraya çevirecek yatırımcı bulmak ve bu yatırımcıyı ikna edip, sonuç almak için araştırma ve çalışmalar yapmaktır. Yatırımcı için yeni fikirler çok önemli, çünkü yeni bir fikir yoksa daha fazla para kazanma şansı da yoktur. Fikir sahibi için de yatırımcı çok önemli bir unsurdur. Eğer para yoksa, buluşun ekonomiye kazanımı da yok. Bu da yatırımcıyı Ar-Ge ye ve yeniliklerin yapılmasına yönlendiriyor. Amerika neden Amerika olmuş sorusunun cevabı, en çok Ar-Ge nin yapıldığı ülke olmasında yatıyor. Sistem buna göre kurulmuş ve kurgulanmış. Her yerde irili ufaklı Ar-Ge firması bulmak mümkün. Sadece New Jersey eyaletinde yüzlerce ilaç Ar-Ge firmaları ve laboratuvarları var. Bugün dünyanın en önde gelen ilaç firmaları bu Ar-Ge laboratuvarlarına belli koşullarda ortaklar. Yani finans sağlıyorlar ama laboratuvarlar bağımsız olarak çalışıyorlar. İlaç firması parayı en kolay ya da en çok gelecek vaat eden fikirlere yatırıyor. Bir iki ürün yerine birçok yeni ürün üzerine çalışmalar yapma fırsatını yakalamış oluyorlar. Genelde bu yapılar yarı özerktirler. Parasal yardımlar, banka kredileri ve yatırımcılarla çalışmalarını sürdürürler. Ama her alınan paranın nereye nasıl harcandığı ve projede gelinen noktaları, sürekli raporlarla gerekli yerlere bildirirler. Bilim insanları işini yapar, yatırımcı parasını kazanır. Herkes kazanır. Bunun devletçi yaklaşımdan farkı parayı veren sonuç görmek ister, belli bir ilerleme yoksa yatırım durur. Yapılan her araştırma bir şekilde ekonomiye kazandırılır. Ya tecrübe olmuş olur ki bu tecrübeyi üniversitelerde okuturlar ya da ürün olarak raflarda yerini alır. Yani yatırılan para bir şekilde ekonomiye döner. Daha agresiftirler, çünkü herkes sonuç görmek ister. Sadece Ar-ge departmanları açmak yerine, bugün buluşu olan insanlara kendilerini ispat etmeleri için bu departmanların kapılarını açarak fırsat vermek daha doğru olur. Böylece bilim insanlarının pastadan alacakları payda artmış olacaktır. Star-Türkiye UP Ülkemizi daha ileriye taşımak için yapılabileceklerden bir tanesi de olaylara bakış açımızı değiştirmektir. Esneklik kazanıp, hızlı hareket etme kabiliyetine ulaşmak çok önemli. Amerika da yaşayan birçok Türk kökenli bilim insanı var. Bu bilim insanlarının ülkemize geri dönüp, bilgi ve kazanımlarını bize aktarması çok güzel olurdu. Ama maalesef teknik, ailevi, iş yoğunluğu, zaman ve daha birçok sebeplerden dolayı bu pek de mümkün olmuyor, olamıyor. Bu durumda bizlerin onların ayağına gitmesi daha güzel olmaz mı? Örneğin; Amerika' da bir Ar-Ge merkezi kurulsa ve bu merkezde Türk kökenli bilim insanlarına, hatta her başarılı bilim insanına kendi alanlarında çalışmalar yapmalarına imkân verilse, aynı zaman Türkiye den de asistan seklinde insan kaynağı ve çalışmalar için gerekli ekipman vs. karşılanırsa işte o zaman Star-Türkiye UP başlamış olur. Önce kendi bilim insanlarımızı geri kazanmış oluruz. Aynı zamanda yapılan bilimsel çalışmalar da dünyada daha hızlı kabul görür. Amerika ile Türkiye arasında oluşacak bu bilgi ve insan trafiğiyle, Know-How kazanımı artmış ve Türkiye deki eğitim ve standartlarının artmasının yanı sıra kısa ve uzun vade de daha birçok kazanımları olacaktır. Türkiye bugün Antartika da bir laboratuvar açmayı düşünüyor da bu neden Amerika da da olmasın. Konu; bioteknoloji ve bioteknolojiye ulaşmak ise bunun birçok yöntemi var. Direkt veya dolaylı bir şekilde ulaşılabilinir. İlaç ve eczacılık konusuna gelince, ürünler kadar onların uygulama yöntemleri, yardımcı maddeler, hasta odaklı yaklaşımlar ve alternatif olabilecek ürünler üzerinde de çalışılmalı. Örneğin, sadece insülini veya grip aşısını üretmek değil onu daha kolay uygulanır hale getirmek, bunun üzerine çalışmalar yapıp basınçlı enjektörleri geliştirmek. Hastalara ilaçlarını daha kolay kullanabileceği, (smart box) akıllı kutu ile vermek. Akıllı telefon uygulamalarıy- 46

49 DAVETLİ KONUŞMACILAR / INVITED LECTURES la hastanın ilacı doğru ve zamanında kullanmasını sağlamak gibi... Global dünyada olduğu gibi ülkemizde de eczacılıkta branşlaşma gerekmektedir. Konusunda branşlaşmış eczacıların daha detaylı ve derinlemesine bilgi birikimi ve aktarımını sağlamış oluruz. Bu da eczacıların daha çok yeni fikirler geliştirmesini, kendilerine ve ülkemize daha fazla katkı yapmalarını sağlayacaktır. Bu branşlar, klinik eczacılık, majistral(compounding), fitoterapi, homeopathy, dermokozmetik, ilaç sanayi seklinde olabilir. Dermokozmetik uzmanı bir eczacı, majistral uzmanı bir eczacıyla ortaklaşa çalışarak, oldukça faydalı kozmetik veya ilaç kategorisinde ürünler geliştirebilirler. Bugün dünyanın birçok ülkesindeki eczacılar başta Amerika olmak üzere Türkiye ve dünyanın her yerinde iş imkânlarını ararken, neden Türk eczacılarımız, gençlerimiz kendilerini Anadolu topraklarıyla sınırlıyorlar. Bizler genç ve yeni nesil eczacılara yol göstermeliyiz, hatta onların ellerinden tutup onlara imkanlar sunarak, Türkiye nin global eczacılıktaki yerini almasını sağlamalıyız. Bu bir stratejidir, vizyondur, Türkiye yi ve Türk eczacılığını ileriye taşımaktır. Dünya her geçen gün küçülürken, kapalı kapılar arkasında kendimizi saklayamayız ve saklamamalıyız. Star-T UP in America Pharm. Mehmed Ugur President of Turkish American Pharmacists Association The Meeting of Capitalism and Science Capitalism's perspective on events do I make money from this? The answer to the question is. So there is no logic of a social state. If they don't believe they can make money, you can't buy a penny from investors even if you save the world. The owner of the invention does not have to be the same person who makes the money. they may appear together, but they may come together. The important thing is; to invest in research and studies to find an investor and to convince this investor. New ideas are very important for the investor because if there is no new idea, there is no chance of making more money. The investor is a very important element for the owner of the idea. This leads the investor to R & D and innovation. The answer to the question of why America is America is the fact that it is the country where the R & D is made. The system was installed and constructed accordingly. It is possible to find R & D companies of all sizes. Only in the state of New Jersey there are hundreds of pharmaceutical R & D companies and laboratories. Today, the world's leading pharmaceutical companies are partners in these R & D laboratories under certain conditions. So they provide finance, but laboratories work independently. The pharmaceutical company invests money in the easiest or most promising ideas. Instead of one or two products, they have the opportunity to work on many new products. In general, these structures are semi-autonomous. They continue to work with financial aids, bank loans and investors. But every time the money is spent and the points reached in the project, continuous reports to report where necessary. Scientists do their job, the investor earns his money. Everybody wins. The difference from the statist approach to this is whether the result of the money to see whether the investment stops. Each research is somehow brought into the economy. Either it has been experienced and these experiences teach in the universities, or as a product will take place on the shelves. So money invested somehow returns to the economy. They are more aggres- 47

50 DAVETLİ KONUŞMACILAR / INVITED LECTURES sive because everyone wants to see results. Instead of simply opening R & D departments, it is better to give the opportunity to people who have the invention today by opening the doors of these departments to prove themselves. Thus, the share of scientists will be increased from the pie. Star-Turkey UP One of the things that can be done to move our country forward is to change our perspective on events. Gaining flexibility and ability to move fast is very important. There are many scientists of Turkish origin living in America. It would be nice for these scientists to go back to our country and pass on their knowledge and gains to us. But unfortunately, technical, family, work intensity, time and for many other reasons, this is not possible, can not be. Wouldn't it be better for us to go to their feet? For example; in America an R & D center is founded and the centers of Turkish origin scientists, even every successful scientist to enable them to work in their fields until given. At the same time, the necessary equipment etc. and for human resources as assistant provides from Turkey, then it would have started Star-Turkey UP. First, we'll have our own scientists back. At the same time scientific studies are accepted faster in the World literature. With this information with the United States will occur between Turkey and human traffic, Know-How and enhanced recovery as well as the increase of education and standards in Turkey will be both short and long term gains many more. Today, Turkey is considering whether to open a laboratory in Antarctica in this cause why not in America. If the topic; Biotechnology and Access to biotechnology there are many methods. It can be accessed directly or indirectly. When it comes to pharmaceuticals and pharmaceuticals, products should be worked on as well as their application methods, excipients, patient-oriented approaches and alternative products. For example, it is not only to produce insulin or influenza vaccine, but to make it easier to apply, to do pressurized injectors. Give patients a smart box, which they can use more easily. Smart phone applications to ensure the patient to use the drug correctly and timely... As in the global world, there is a need for branching in pharmacy. We provide more detailed and in-depth knowledge and transfer of specialized pharmacists. This will allow pharmacists to develop more new ideas and make more contributions to themselves and our country. These branches can be clinical pharmacy, compounding, phytotherapy, homeopathy, dermocosmetic, pharmaceutical industry. Dermocosmetic specialty pharmacist, working together with a compounding expert pharmacist, can develop products in the highly beneficial cosmetic or drug category. Today many pharmacists around the world, including Turkey and particularly in America when looking for jobs all over the world, why our Turkish pharmacists, our young people are limiting themselves to Anatolia. We must show the way for a new generation of young and pharmacists, and even hold them in their hands by providing opportunities, we must ensure Turkey to take its place in the global pharmaceuticals. This is a strategy, and vision to move forward on Turkey and the Turkish pharmacy. While the world is getting smaller every day, we cannot hide and hide ourselves behind closed doors. Keywords: America, Start up, biotechnology 48

51 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-25 In vivo Sensors and Biotechnological Neuroscience Implementations Ahmet Hacimuftuoglu 1, Bulent Cavusoglu 2, Yucel Ozbek 2, Mehmet Ertugrul 2, Ufuk Okkay 1, Ali Tagizadehghalehjoughi 3 1 Ataturk University, Medical Faculty, Medical Pharmacology, Erzurum, Turkiye 2 Ataturk Univ. Engineering Faculty, Dep. of Electric-Electronic, Erzurum, Turkiye 3 Ataturk Univ. Veterinary Faculty, Dep. Of Pharmacology, Erzurum, Turkiye Neurotransmitters mediate communication between neurons and/or non-neuronal cells. Changings in neurotransmitter levels in the synaptic area can cause or aggravate central nervous system (CNS) disorders. In the past, detecting directly of these neurotransmitter levels has been limited in their temporal and spatial resolution because of their nature of chemical signaling and their structures (1,2). Minimally invasive techniques for monitoring brain chemistry in vivo provided better understanding of neuropharmacology of CNS disorders. For monitoring and sampling brain chemistry; voltammetric electrodes, microdialysis and related analytical techniques had been used (3,4,5). Microdialysis, compared to voltammetry, offers lower temporal and spatial resolution. What we did new with our projects? New biocompatable microelectrodes New biocompatable barriers Appropriate wireless system Smallest device The fabrication of our biocompatible sensors, which can detect brain neurotransmitter concentrations, consists of three steps. These are production of the microelectrodes by photolithographic methods, packaging and coating with selective chemical barriers respectively. The produced sensors are cost efficient and biocompatible. Applied new barriers have increased the selectivity and sensitivity values. A mojor problem of in-vivo monitoring of neurotransmitters in the brain tissues is interference effect of other co-existing oxidizable compounds, particulary ascorbic acid. Cation-exchange polymers, such as Nafion, have been particulary attractive in connection with neurochemical studies of neurotransmitter release. However, nafion is not member of bio-compatible polymer groups are allowed to using in vivo measurement of human tissues. The aim was finding out a novel electropolymerization system which is selective and sensitive. It is thought that the majority of brain diseases are caused by changes in the basal levels of neurotransmitters which are acting at synaptic space in brain or removal parameters of them from synaptic cleft. In our studies, a biocompatible sensor, a wireless neurotransmitter detection device and a new software have been produced for the diagnosis, treatment and follow-up of the brain diseases. 49

52 DAVETLİ KONUŞMACILAR / INVITED LECTURES The device used to process the electrical current information obtained from the sensors is produced within the scope of these projects. This device amplifies the na level currents which stem from neurotransmitter concentration by factor of 107. The device is designed to be small and light enough to be carried on the subjects. This device, together with the battery, weighs a total of 11.8 g and measures 2.9 cm x 2.4 cm. Also the device developed within the scope of this project can display the necessary calculations during the experiment and follow up. This information is transmitted via the wireless communication unit to the Labview based software. In addition, the noise performance of the system has been improved thanks to filtering operations. This system which we developed has been experimentally tested in animal models of depression, chronic pain, hepatic encephalopathy, epilepsy and Alzheimer's disease, and measurements of glutamate and other neurotransmitters were performed by in-vivo voltammetry. The drugs were selected according to the type of neurotransmitters in which the change were detected and the differences were tried to be removed. Numerous drug trials were performed. Results show that a large proportion of brain diseases may be associated with neurotransmitter level changes. Treatments focused on the changes in neurotransmitters levels have also been shown to improve the clinical course of the disease. We have performed statistical analysis of wavelet coefficients and determined that the feature set of Fx which includes only 5 statistics can be used instead of Flit which is generally used in literature and includes 8 statistics. Fx has lower dimension. Moreover, through statistical analysis we showed that it is necessary to include higher order moments for CA coefficients. We validated that our effective power measurement helpful in determining the effective features. In the scope of this activities, a great deal of data on the etiology of brain diseases is presented and even the treatment can be shed. The device, software and sensors are patented. In addition, within the scope of this work, 4 Ph.D. and 5 M.Sc. thesis were completed. 2 Ph.D., 3 M.Sc. thesis are in completion stage. This studies are supported by Tubitak (The Scientific and Technological Research Council of Turkey) projects # 107S067, 113S083 and Ataturk University BAP project # 2008/126. References: 1. Ahmet, H., S. Fatih, S. Halis, C. Damla, H. Zekai and G. Fatma (2010). "Paclitaxel-Evoked Pain And Its Theraphy; In Vivo Voltammetric Study." WorldPharma: Headley PM, Grillner S. Excitatory amino acids and synaptic transmission: the evidence for a physiological function. Trends Pharmacol Sci, 1990, 11: Rawls, S. M., M. Zielinski, H. Patel, S. Sacavage, D. A. Baron and D. Patel (2010). "Beta-lactam antibiotic reduces morphine analgesic tolerance in rats through GLT-1 transporter activation." Drug and Alcohol Dependence 107(2-3): Silpa, N. (2007). Nanostructured Sensors for In Vivo Neurochamical Recording Master s Theses, University of Kentucky. 5. Liu, P., Y. S. Zhu, S. H. Lee and M. Yun (2016). "Two-dimensional polyaniline nanostructure to the development of microfluidic integrated flexible biosensors for biomarker detection." Biomedical Microdevices 18(6). 50

53 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-26 Quantitative Measurements of DNA Damage in Diagnosis and Treatment Bilge Debeleç Bütüner Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Izmir, Turkey Abstract Recent studies showed that 8-OHdG is biologically significant for measuring oxidative DNA damage as a leading factor of initiation and promotion of several pathologies. This alteration has been used to assess DNA damage in humans after exposure to various oxidant chemicals, pathological conditions, and heavy physical exercise. In addition, changes in 8-OHdG levels have been approved in many studies as a biomarker indicating the risk for development of many cancers, degenerative diseases and aging. Detection of 8-OHdG-base damage was a big challenge for decades, though different analytical methods were developed. Automated damage detection from a single cell, which has been previously optimized for γh2ax detection, which indicates DNA double strand breaks, has successfully applied to quantitation of 8-OHdG damages by using different types of cells allowing this method to be used in diagnosis and treatment of the related diseases. Keywords 8-OHdG; DNA damage, γh2ax; Aklides; Biomarker Introduction 8-hydroxy-2'-deoxyguanosine (8-OHdG) or 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG) are the most commonly observed single nucleotide-base lesions in nuclear and mitochondrial DNA. Further, these base lesions are induced by free radicals and recognized as potential biomarkers of oxidative DNA damage [1, 2]. Recent studies have confirmed the role of ROS accumulation and subsequent 8-OHdG oxidative DNA damage in carcinogenesis [3], a wide range of inflammatory diseases [4, 5], aging [6, 7], and exercise were also reported [8-11]. In mammalian cells, 8-OHdG is repaired by 8-oxoguanine DNA glycosylase-1 (OGG1), which is one of the major players of base excision repair (BER) [12, 13]. Quantitation of 8-OHdG currently performed by a wide range of methodologies. LC-MS/MS, which is commonly used, produces highly precise and sensitive data from tissue, plasma and urine samples [5-7, 14]. There are also antibody labeling-based techniques such as immunohistochemistry (IHC) [3, 4], immunocytochemistry (ICC) [15] and immunofluorescence (IF) allowing detection from plasma and tissue [16-19]. Potential use of the 8-OHdG damages quantitation is required to be performed by choosing the most efficient method with the appropriate sample material. These assays should be evaluated according to their precision, reliability, sensitivity, and time. Materials and Methods Cell Culture and Treatments LNCaP, PC3, Du145, U937 and Jurkat cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and propagated as recommended. Antibodies The following antibodies were purchased from manufacturers: OGG1 (ThermoScientific, Germany), 8-OHdG (Santa Cruz, Germany). Foci analysis using Aklides.Nuk platform (Medipan, Germany) Aklides.Nuk is an automated immunofluores- 51

54 DAVETLİ KONUŞMACILAR / INVITED LECTURES cence-based system that consists of a high-resolution semi-confocal immunofluorescence microscopy and image analysis software. Experimentally, immunofluorescence labeled cells were fixed onto the coverslips and placed to Aklides.Nuk system. The parameters (nucleus diameter, nucleus height/width ratio, foci diameter, foci intensity, foci convexity) of the software were adjusted to pre-optimized values for each cell type and foci were defined in 5 focal planes and counted by the software. Results Quantitation of 8-OHdG damages by Aklides.Nuk system has been performed and optimized for different cell types including adherent and suspension cells allowing this method to be used for different approaches. Damage quantitation in lymphocytes and monocytes is an effective method to detect chemotherapeutic drug sensitivity and exercise-induced DNA damage for personalized therapy approaches. In addition, optimization of the method for a wide range of normal and cancer cells leads the system to detect damage heterogeneity among cells to better understand the role of DNA damage and repair in carcinogenesis. Further, detection of OGG1 foci, indicating 8-OHdG damage repairs has been also achieved by this method enabling the system to detect DNA damage and simultaneous repair at a single cell. Discussion The Aklides system determines the shape as well as size of the cell nucleus and the damage foci enabling the quantitation of their fluorescent signals. Indeed, this foci determination system was optimized for quantitative analysis of DNA double strand break γ-h2ax (S139) foci analysis originally [20, 21] allowing to measure DNA damage levels in each cell at single focus level. Recent studies have been shown that it can be utilized for clinical purposes to designate drug resistance and radiotherapy sensitivity in patients [22-24]. Quantitation of 8-OHdG foci by IF-based automated system has been optimized in our studies and revealed the advantages as well as limitations. Conclusions The biological significance of alterations in 8-OHdG levels has been revealed in cancer, inflammatory diseases, aging, and severe exercise-induced oxidative damage. 8-OHdG oxidative damages could be recognized as a biomarker for a wide range of DNA/protein-related pathologies and quantitation of 8-OHdG foci could be achieved by an IF-based automated system with plasma as sample material leading fast and reliable data acquisition. The parameters of Aklides system need to be adjusted for each cell type and predicted damage levels for different pathologies. References [1.] Kryston TB, Georgiev AB, Pissis P, Georgakilas AG. Role of oxidative stress and DNA damage in human carcinogenesis. Mutation research. 2011;711(1-2): doi: /j.mrfmmm PubMed PMID: [2.] Cadet J, Loft S, Olinski R, Evans MD, Bialkowski K, Richard Wagner J, et al. Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids. Free Radic Res. 2012;46(4): doi: / PubMed PMID: ; PubMed Central PMCID: PMC [3.] Murata M, Thanan R, Ma N, Kawanishi S. Role of nitrative and oxidative DNA damage in inflammation-related carcinogenesis. Journal of biomedicine & biotechnology. 2012;2012: doi: /2012/ PubMed PMID: ; PubMed Central PMCID: PMC [4.] Ding X, Hiraku Y, Ma N, Kato T, Saito K, Nagahama M, et al. Inducible nitric oxide synthase-dependent DNA 52

55 DAVETLİ KONUŞMACILAR / INVITED LECTURES damage in mouse model of inflammatory bowel disease. Cancer Sci. 2005;96(3): doi: /j x. PubMed PMID: [5.] Kurgan S, Onder C, Altingoz SM, Bagis N, Uyanik M, Serdar MA, et al. High sensitivity detection of salivary 8-hydroxy deoxyguanosine levels in patients with chronic periodontitis. Journal of periodontal research. 2015;50(6): doi: /jre PubMed PMID: [6.] Gan W, Nie B, Shi F, Xu XM, Qian JC, Takagi Y, et al. Age-dependent increases in the oxidative damage of DNA, RNA, and their metabolites in normal and senescence-accelerated mice analyzed by LC-MS/MS: urinary 8-oxoguanosine as a novel biomarker of aging. Free Radic Biol Med. 2012;52(9): doi: /j. freeradbiomed PubMed PMID: [7.] Nie B, Gan W, Shi F, Hu GX, Chen LG, Hayakawa H, et al. Age-dependent accumulation of 8-oxoguanine in the DNA and RNA in various rat tissues. Oxidative medicine and cellular longevity. 2013;2013: doi: /2013/ PubMed PMID: ; PubMed Central PMCID: PMC [8.] Fogarty MC, Devito G, Hughes CM, Burke G, Brown JC, McEneny J, et al. Effects of alpha-lipoic acid on mtdna damage after isolated muscle contractions. Medicine and science in sports and exercise. 2013;45(8): doi: /MSS.0b013e31828bf31e. PubMed PMID: [9.] Soares JP, Silva AM, Oliveira MM, Peixoto F, Gaivao I, Mota MP. Effects of combined physical exercise training on DNA damage and repair capacity: role of oxidative stress changes. Age. 2015;37(3):9799. doi: / s PubMed PMID: ; PubMed Central PMCID: PMC [10.] Villano D, Vilaplana C, Medina S, Cejuela-Anta R, Martinez-Sanz JM, Gil P, et al. Effect of elite physical exercise by triathletes on seven catabolites of DNA oxidation. Free Radic Res. 2015;49(8): doi: / PubMed PMID: [11.] Yasuda N, Bolin C, Cardozo-Pelaez F, Ruby BC. Effects of repeated bouts of long-duration endurance exercise on muscle and urinary levels of 8-hydroxy-2'-deoxyguanosine in moderately trained cyclists. Journal of sports sciences. 2015;33(16): doi: / PubMed PMID: ; PubMed Central PMCID: PMC [12.] Ba X, Aguilera-Aguirre L, Rashid QT, Bacsi A, Radak Z, Sur S, et al. The role of 8-oxoguanine DNA glycosylase-1 in inflammation. International journal of molecular sciences. 2014;15(9): doi: / ijms PubMed PMID: ; PubMed Central PMCID: PMC [13.] Paz-Elizur T, Sevilya Z, Leitner-Dagan Y, Elinger D, Roisman LC, Livneh Z. DNA repair of oxidative DNA damage in human carcinogenesis: potential application for cancer risk assessment and prevention. Cancer Lett. 2008;266(1): doi: /j.canlet PubMed PMID: ; PubMed Central PMCID: PMC [14.] Dizdaroglu M, Jaruga P, Rodriguez H. Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry. Nucleic Acids Res. 2001;29(3):E12. PubMed PMID: ; PubMed Central PMCID: PMC [15.] Thompson CM, Fedorov Y, Brown DD, Suh M, Proctor DM, Kuriakose L, et al. Assessment of Cr(VI)-induced cytotoxicity and genotoxicity using high content analysis. PloS one. 2012;7(8):e doi: / journal.pone PubMed PMID: ; PubMed Central PMCID: PMC [16.] Kim J, Kim NH, Sohn E, Kim CS, Kim JS. Methylglyoxal induces cellular damage by increasing argpyrimidine accumulation and oxidative DNA damage in human lens epithelial cells. Biochem Biophys Res Commun. 2010;391(1): doi: /j.bbrc PubMed PMID: [17.] Cafueri G, Parodi F, Pistorio A, Bertolotto M, Ventura F, Gambini C, et al. Endothelial and smooth muscle cells from abdominal aortic aneurysm have increased oxidative stress and telomere attrition. PloS one. 2012;7(4):e doi: /journal.pone PubMed PMID: ; PubMed Central PM- CID: PMC [18.] Puente BN, Kimura W, Muralidhar SA, Moon J, Amatruda JF, Phelps KL, et al. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell. 2014;157(3):565-53

56 DAVETLİ KONUŞMACILAR / INVITED LECTURES 79. doi: /j.cell PubMed PMID: ; PubMed Central PMCID: PMC [19.] Tsai-Turton M, Luong BT, Tan Y, Luderer U. Cyclophosphamide-induced apoptosis in COV434 human granulosa cells involves oxidative stress and glutathione depletion. Toxicol Sci. 2007;98(1): doi: / toxsci/kfm087. PubMed PMID: [20.] Runge R, Hiemann R, Wendisch M, Kasten-Pisula U, Storch K, Zophel K, et al. Fully automated interpretation of ionizing radiation-induced gammah2ax foci by the novel pattern recognition system AKLIDES(R). International journal of radiation biology. 2012;88(5): Epub 2012/01/28. doi: / PubMed PMID: [21.] Willitzki A, Lorenz S, Hiemann R, Guttek K, Goihl A, Hartig R, et al. Fully automated analysis of chemically induced gammah2ax foci in human peripheral blood mononuclear cells by indirect immunofluorescence. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2013;83(11): Epub 2013/09/07. doi: /cyto.a PubMed PMID: [22.] Leifert WR, Siddiqui SM. gammah2ax is a biomarker of modulated cytostatic drug resistance. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2015;87(8): doi: /cyto.a PubMed PMID: [23.] Menegakis A, von Neubeck C, Yaromina A, Thames H, Hering S, Hennenlotter J, et al. gammah2ax assay in ex vivo irradiated tumour specimens: A novel method to determine tumour radiation sensitivity in patient-derived material. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology. 2015;116(3): doi: /j.radonc PubMed PMID: [24.] Reddig A, Lorenz S, Hiemann R, Guttek K, Hartig R, Heiserich L, et al. Assessment of modulated cytostatic drug resistance by automated gammah2ax analysis. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2015;87(8): doi: /cyto.a PubMed PMID:

57 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-32 Biotechnology and Synthetic Biology Revolution in the World and Turkey Işıl Aksan Kurnaz 1 1 Gebze Technical University, Institute of Biotechnology, Gebze Kocaeli, TURKEY 2 Original Bioeconomy Resources (OBEK) Center of Excellence, Gebze Technical Education and Research Foundation, Gebze Kocaeli, TURKEY Absract Biotechnology is no longer tomorrow s technology it has been here for nearly half a century, and together with novel tools and technologies it is at a new stage of evolution. The awarding of this year s Nobel Prize for Chemistry partly for Prof. Frances Arnold for her work on directed enzyme evolution was a sign in that direction Prof. Arnold, in addition to being a Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry at Caltech, is also the founder of a synthetic biology company, Gevo. Synthetic biology itself is not a new concept it originated in 1990s, hand-in-hand with the genomics revolution and the rise of systems biology, and was launched primarily by engineers who aimed to merge engineering principles with the concept of microbial cell factories, modifying and evolving not just new enzymes or proteins, but entire new metabolic pathways. The recent emergence of genome editing tools has further enabled and expanded the use of synthetic biology in a wide range of applications, and with the possibility of using expanded genetic codes the protein engineering landscape is now even greater than before. Quite a number of funds and investment opportunities are currently focusing on yesterday s technology, ie traditional biotechnology and bioengineering, whereas it is now the time to focus on tomorrow s technology, synthetic biology, before it is too late. GTU Institute of Biotechnology was established in 2014 and actively started graduate education and research activities in the Academic Year. With its internationally-renowned Scientific Advisory Board, its main vision has been to educate tomorrow s bioentrepreneurs and biotechnology leaders, in close association with the biotechnology industry. Keywords biotechnology; bioengineering; synthetic biology Biotechnology Past, Present and Future Biotechnology is a coin termed in the early 1900s to describe the technology to convert raw materials to more useful products with the help of organisms (Swarup et al, 2011). Towards 1990s it had already evolved from simple fermentation to genetically modifying microorganisms using recombinant DNA technologies. This period had also seen the rise of many biotechnology companies, such as Genentech that commercialized recombinant human growth hormone. Now, biotechnology is everywhere, from medical and pharmaceutical industry to agricultural and environmental industries, with global biotechnology crop area up to 4.7 million hectares in 2017 and providing USD billion in economic gain worldwide between (ISAAA Brief 53:2017). With the use of site-directed mutagenesis technology in 1980s and the ability to design proteins as required, came the then-novel technology of protein engineering, soon followed by enzyme engineering and antibody engineering, which enabled researchers to improve either the proteins themselves, or the production process (Brannigan and 55

58 DAVETLİ KONUŞMACILAR / INVITED LECTURES Wilkinson, 2002). Synthetic biology field was founded in late 1990s, with the idea that engineering concepts could be applied to biological systems, with cells, proteins, genes and promoters treated as synthetic parts and devices to design and engineer completely novel circuits and systems not found in nature (Cameron et al, 2014). The standardized parts have made synthetic biology available to everyone (also promoting Do-It-Yourself technologies), but more importantly synthetic biology enabled researchers to design improved products or processes. When one considers that total domestic revenues in the US from biotechnology products of all sub-domains totalled almost USD 325 billion, one can appreciate the strong interest of governments as well as private funds (see report Preparing for Future Products of Biotechnology). Today, with the societial and economical pressures and challenges, sustainability and circular economy (indlucing circular bioeconomy and bio-based industries) have become new arenas where researchers and policy-makers alike try to push boundaries and find innovative solutions, which is driving the transformation of biotechnology. Bioeconomy and sustainable manufacturing technologies Since the beginning of the new millenium, and with the increasing demand for biotechnology products, new trends in biotechnology have been biobased chemicals, biofuels and new and more sustainable methods of manufacturing, exemplified by the National Bioeconomy Blueprint of Obama Administration in The European Commission defined the circular economy as one where the value of products, materials and resources is maintained in the economy for as long as possible, with the minimum amount of waste generated, and the intersection of circular economy with bioeconomy was redefined as circular bioeconomy (Carus and Dammer, 2018). As with circular economy, the main concepts of circular bioeconomy aim sustainability and include bio-based products, a framework for sharing/reusing/remanufacturing/recycling, utilization of organic wastes, resource-efficient chains, and nutrient recycling. Synthetic biology is currently in the driver seat for bioeconomy, and is revolutionizing the biotechnology industry (Bueso and Tangney, 2017). In September 2018, a new $ 4.5 million Synthetic Biology Initiative was announced as part of the Australian Industrialized Biotechnology Alliance Initiative (which itself reportedly has a total investment of $ 20 million) in order to help drive sustainable, export-oriented biotechnologies ( article/2018/09/new-synthetic-biology-initiative-boost-bio-economy). Future trends As discussed above, global challenges such as food security and climate change bring about the search for sustainable and circular bioeconomy solutions a major driver of new biotechnology trends. Among some innovative solutions startup companies are working on include lab-grown synthetic meat and to come up with these solutions, the biotechnology accelerator IndieBio gives USD and four months access to a fully functional biotech laboratory the new Bio-Silicon Valley. GTU Institute of Biotechnology was established in 2014 and actively started graduate education and research activities in the Academic Year. With its internationally-renowned Scientific Advisory Board, its main vision has been to educate tomorrow s bioentrepreneurs and biotechnology leaders, in close association with the biotechnology industry. To that end, in addition to Medical, Agricultural and Industrial Biotechnology tracks in Biotechnology Graduate Programs, our Institute also offers graduate degree in Synthetic and Systems Biotechnology track option. We aim to integrate and align our research with the needs of not only the newly blossoming Turkish biotechnology industry, but also the world trends in the field. With this in mind, we have coordinated a national consortium to structure the first bioeconomy platform of Turkey, namely Original Bio-Economy Resources (OBEK) Center of Excellence within Gebze Technical Education and Research Foundation. This is a platform of collaboration, where top-notch researchers from various universities as well as industrial partners come together 56

59 DAVETLİ KONUŞMACILAR / INVITED LECTURES to devise new strategies towards sustainable bioeconomy solutions. Recently, quite a number of funds and investment opportunities in Turkey are focused on biotechnology, mainly in biopharmaceuticals and biomedical devices, whereas it is now the time to focus on tomorrow s technology before it is too late. It is not the time to make redundant infrastructure investments, neither at public nor at private end, but rather to devise innovative accelerator or biotechnology collaboration hubs or platforms to mobilize existing infrastructure to dream and realize cutting-edge technologies. References [1.] Preparing for Future Products of Biotechnology. National Academy of Sciences, Engineering and Medicine; Division on Earth and Life Studies; Board on Chemical Sciences and Technology; Board on Agriculture and Natural Resources; Board on Life Sciences; Committee on Future Biotechnology Products and Opportunities to Enhance Capabilities of the Biotechnology Regulatory System. Washington DC: National Academies Press (US); 2017 ( [2.] Swarup Verma A, Agrahari S, Rastogi S, Singh A (2011). Biotechnology in the realm of history. J Pharm Bioallied Sci 3(3): [3.] Cameron DE, Bashor CJ, Collins JJ (2014). A brief history of synthetic biology. Nature Rev Microbiol 12: 381 [4.] Brown KV (2018). Silicon valley wants to disrupt biology. Bloomberg, Nov 20 ( com/news/articles/ /biology-is-silicon-valley-s-next-big-thing ). [5.] ISAAA Brief Global Statu of Commercialized Biotech/GM Crops: Biotech Crop Adoption Leads to Greater Sustainability and Socioeconomic Opportunities for Global Farmers and Citizens. [6.] Brannigan JA and Wilkinson AJ (2002). Protein engineering 20 years on. Nature Rev Mol Cell Biol 3: [7.] Buesco YF and Tangney M (2017). Synthetic biology in the driving seat of the bioeconomy. Trends in Biotech 35(5): [8.] Carus M, Dammer L (2018). The Circular Bioeconomy concepts, opportunities and limitations. Nova paper #9 on bio-based economy ( ). 57

60 DAVETLİ KONUŞMACILAR / INVITED LECTURES DK-36 Biyogirişimcilik ve Ticarileşme Nezih Hekim T.C. Cumhuriyet Üniversitesi Klinik Biyokimya Ana Bilim Dalı Öğretim Üyesi Girişimcilik ve inovasyon konusunda şüphesizdir ki herkesin bir fikri vardır. Ancak iş tanımlamaya geldiği zaman herkesin girişimcilik ve inovasyon konusunda aynı şeyleri mi düşünmediği ortaya çıkıyor. İnovasyon sözcüğü kendi dilimize yenilik, yenilikçilik olarak tercüme edilmiştir. OECD, Oslo kılavuzunda inovasyon konusunda tanım ve uygulamalarına ilişkin detaylı bilgiler mevcuttur (1). Oslo kılavuzunda bir ürünün tek başına yeni olmasının inovasyon için yeterli olmadığı belirtilmektedir. Bu kılavuza göre; inovasyon için ürün eskisinden daha değerli olmalıdır. İnovatif ürün, evet, yeni olmalı, ancak inovasyonu ortaya çıkardığı için kuruma ticari bir değer katacağı kadar, ülkeye ve hatta tüm insanlığa ekonomik ve sosyal değerler de katmalıdır. Unutulmamalıdır ki, inovasyon her zaman radikal olmayabilir. Artımsal da olabilir. Telefonun icadı radikal bir inovasyondır. Bu sayede haberleşme kolaylaşmış ve sadece telefon şirketine ticari bir değer katmamış, tüm ülkelerin ekonomik kalkınmalarına ve hatta tüm insanlığın refahına yol açmıştır. Cep telefonları ise artımsal bir inovasyondır. Dünyayı değiştirmemiştir, ancak ürün mükemelleşmiş ve üretici firmayı en çok kazanan firmalardan biri haline getirmiştir. Oslo kılavuzuna göre; bir süreç, bir organizasyon, bir pazarlama yöntemi de firma için ilk, pazar için ilk ve hatta o ülke için bir ilkse, bir inovasyondur. Bu yer kürede girişimcilik ve inovasyonu ilk kez konuşan ekonomistlerden biri Joseph Alois Schumpeter dir. Bugün onu anmadan geçmememiz gereklidir. Schumpeter, tekstil üreticisinin oğlu olarak 1883 yılında Triesch, Moravia da doğmuştur. Aristrokrat bir ailede yetişen Schumpeter, Hukuk okumuş ve insana değer veren ekonomik gelişmelerin gerekliliğini ortaya koyarak tüm insanlığa örnek olmuştur. 58

61 DAVETLİ KONUŞMACILAR / INVITED LECTURES Bilindiği gibi, inovasyon üniversitelerdeki bilim adamlarının veya mucitlerin keşifleri ile ilgili bir kavram değildir. Yeni bir tekniğin veya yeni bir organizasyonun pazara girmesi ile ilintili bir kavramdır. Şurası çok nettir ki, yoğun rekabet ekonomiye yön veren önemli bir etmendir. Schumpeter, şirketlerin gelişmesinde kendi kendini tekrar eden değişmeyen (statik) bir model yerine, dinamik ve sürekli bir gelişme modeli önermiştir. Schumpeter e göre, girişim; dengeye ulaşmış ve statik hale gelmiş mevcut sistemi değiştirmek için eski modeli yıkıp, yeni bir modelle, yeni bir dengeye koşmaktır. Aslında bu fiziğin evrensel bir kuramı olan termodinamiğin 2. kuralıdır. Sistem dengeye ulaşmışsa (k=1), iş yapmaz (W=0). Üretim tekniğine ve içeriğine hemen uygulanabilecek olan inovasyonlar, u retim faktörlerinin bileşimine değişiklik getiren ve bu sayede girişimci kârını arttıran faaliyetler olarak tanımlanır. Bu nedenle, biraz önce de değinildiği gibi, inovasyon endüstrideki oyuncuların terminolojisidir. Amaç; eskiyi yıkan yeniliklerle daha yüksek kazançlar elde etmektir. Örneğin, kömürlü lokomotifler artık yok. Dizel lokomotifler de yavaş yavaş yok oluyor. Artık elektrikli ve yüksek hızlı trenler var. Manyetolu telefonlar, telgraf direkleri de yok olup gitti. Artık uydu haberleşmeler var. Bu gelişmelerin arka planı; buharlı lokomotifler, manyetolu telefonlar gibi yok olmaya yüz tutmuş, eskiyen ürünlerin üretim hatlarını kapatıp, yeninin üretim hatlarını açmak, yeniyi daha da geliştirmek ve geliştirdiğinle daha çok kazanmaktır. İnovasyonun bir diğer kahramanı, inovasyonun algoritması olarak kabul edilen TRIZ in bulucusu Altschuler dir. Genrich Saulovich Altschuler ( ), Rusya da yaşamış bir bilim adamı ve mühendistir. Altschuler e göre inovasyon, yaşamda kalmak için her gün daha iyisini yapmak zorunda olduğumuz ardı arkası kesilmeyen faaliyetlerdir. Girişimcilik aynı ürünü herkesten farklı bir yoldan giderek geliştirmek, ucuzlaştırarak daha verimli üretmek ve hayatta kalmak için daha fazla kazanmaktır. Girişimci mevcut başarızılıkları başarıya döndermek için daima daha farklı yolları test eder. Kanserin tedavisinde gidilen yolun sonu her defasında aynı başarısızlık çıkmazında kilitleniyorsa, genç girişimciler hiç çekinmeden farklı bir yol arayacaktır. Benim rast geldiğim üniversitelerin Kimya Mühendisliği, Kimya, Eczacılık Fakülteleri ve Temel Tıp Bölümleri, sanki hep birlikte sözleşmiş gibi hücre kültürü ile kanser ilacı bulmaya çalışıyor. Bu artık bulaşıcı bir hastalık gibi yayılmış. Oysa, bugün büyük ilaç fabrikaları immunoterapi (CAR-T, Mabs...), Peptitlerle hedef hücreye saldırı, teranostiks, arızalı geni saptayıp hedefe yönelik tedavi, bireyin kalıtsal özelliklerine özgü kişiselleştirilmiş tedavi gibi yeni alanlara ucu açık yatırımlar yapıyor (2-5). İşte IVEK-Bio 2018 toplantısında inovasyon ve girişimden söz açılmasının nedeni budur (2-5). Jenerik ilaç üretmenin, ambalajlamanın, dünyanın iyi bir pazarı olmamızın dışında, ülkemizi bir yere getirmediği çok açıktır. 59

62 DAVETLİ KONUŞMACILAR / INVITED LECTURES Hindistan ve Çin in ilaç endüstrisindeki başarısının arkasında eğitimde yaptıkları büyük değişiklikler yatmaktadır. Translasyonel bilim denilen, nasıl bir ürüne dönüşeceği önceden belirlenmiş projelere yatırım yapılmadığı sürece, ülkenin gelişmek için bilimsel araştırmaya yatıracağı paralar geriye dönüşümü olmayacak yatırımlar olarak heba olacaktır (6 ve 7). Girişimcinin yatırım için aradığı gerçek bazen çok basit ve öteden beri gözünün önünde oluyor olabilir. Basiti yakalamak için kullanıcılar ile iletişim iyi bir yol göstericidir. Buna; Kullanıcıdan Gelen İnovasyon Süreci de denilmektedir. Bir yatırımcıyı; işini sürdürebilmek, krizlere karşı hazırlıklı olmak, değişen teknolojileri bırakıp daha verimli yöntemler bulmak, daha çok kazanmak gibi inovasyon yapmaya iten nedenler vardır. Bunlara inovasyona iten faktörler de denilmektedir. Elbette ki bu tür itici faktörler firmaların yaşaması için gerelidir. Ancak daha da önemlisi, yapılacak inovasyonun o alanda çalışanların şiddetle duyduğu ihtiyaçlarla kesişmesidir. Bunlara da çekici faktörler denilmektedir. İşe başlarken basitçe sahada bu işi yapan kullanıcılarla görüşmek ve onları dinlemek yaşamsaldır. Örneğin kanser tedavisi için gerçekten eski ilaçları taşıyacak yeni ilaç taşıyıcılarına ihtiyaç var mıdır? Araştırıcı hangi yatırımcıyı yanına aldı, hangi ilaç üreticisi ile konuştu, hangi üretici onlara ilaç taşıyıcılarına ihtiyaçları olduğunu belirtti ve hatta sipariş vermek için niyet mektubu verdi? Bu basit bir pazar araştırmasıdır. İnovasyon için tek başına hayal etmek yetmiyor. Edison un dediği gibi başarının %5 i hayal gücü, %95 i çalışmak ve ter dökmektir. Tıp alanında inovasyon yapmak için sadece kanser ilaçlarına odaklanmak da şart değildir. Koruyucu ve Sosyal Tıp parlayan bir alandır. Kanseri önlemek, tedavi etmekten daha kolaydır. Hasta bakımı, hastane yönetim organizasyonu ve/veya wellness klinikleri Organizasyonel innovasyonları gerçekleştiren hastanelerin daha iyi geliştikleri ortadadır. Yeni fırsatlar ve teknolojik imkanlardaki gelişmeler, daha etkin organizasyonel innovasyonların önünü açmaktadır. Tıp biliminde sınırlar zorlanabilir. Tanısı zor hastalıklarda genomiks, proteomiks, metabolomiks gibi sistem temelli yaklaşımlar klinik pratikte yerini bulabilir. Bulmalıdır da. Yeni proteomik teknolojiler gelişti. MALDI-TOF mikrobiyoloji laboratuvarlarının vaz geçilmez aygıtları oldu. Post-genomik çağda oluşumuz önemli bir fırsattır yılında, laboratuvarda çalışan moleküler biyologlar ilaç teknolojisini zorladılar Asilomar Konferansı (USA), dünyanın bir ucunda akla hayale gelmeyecek bir yerde gerçekleşti. Paul Berg, rekombinant teknolojisi ile ilaç üretimini anlatıyor ve takım lideri olarak ABD nin bu işe girmesi gerektiğini savunuyordu. Bu sunum sadece, 1980, Walter Gilbert ve Frederick Sanger ile ortaklaştığı Nobel Kimya Ödülünü getirmedi. Rekombinant ilaç üretimi, İnsülin, Büyüme Hormonu, Somatostatin. gibi biyolojik ilaçların standart üretim 60

63 DAVETLİ KONUŞMACILAR / INVITED LECTURES yöntemi haline geldi. Bugünün hayali; OMICs i teşhis, tedavi ve ilaç seçiminde kullanmak olabilir. OMICs, Latince herşeyi içine alan, demetleyen demektir. Kısaca, sistem temelli yaklaşım da denilmektedir. Genomiks, Proteomiks, Metabolomiks, Vaksinomiks, Agrigenomiks, Pharmakomicrobiomiks ve bizim kendi geliştirdiğimiz Iatromics gibi (8). Sistem temelli tıp, tek bir ağaca bakmak yerine ormana bakmaktır. Bazen, asıl sorun baktığınız ağaçta değil, ormanın bir başka yerindeki ağaçlarda olabilir. BiyoYatırımcı değişen paradigmaları içselleştirmelidir. Bundan Doksan yıl önce Toplum bilimin takipçisidir deniliyordu (9). Bugün Bilimin toplumun takipçisi olduğunu konuşuyoruz. Yönetişim Bilimini, yönetişimi (Governance) konuşuyoruz. Devlet adamlarını, teknokratları, bürokratları, muhalefet bile olsa siyaset adamlarını içerisine almayan inovatif yaklaşımlar başarısız olup sönüyor. Ülkenin yasalarını, yönetmeliklerini değiştirmek kolay değildir. Bu devlet adamlarının konuya ne kadar vakıf olduklarına ve bu yeniliği ne kadar benimsediklerine bağlıdır. Girişim, inovasyon ve yönetişim (Governance) artık birbirlerinden ayrılmaz üçlü bir sacayağıdır (2-3). Networking (doğru iletişim ağları kurmak) başarının kaçınılmaz bir parçasıdır (10). Avrupa ile Asya, Çin ve Afrika nın kesiştiği irtibat noktası ülkemizdir. İpek yolunun ticareti yeniden bizim üzerimizden Avrupa ve Amerika kıtasına açılabilir. Bu bizim ağ kurmadaki başarımıza bağlıdır. İtibar Yönetimi, İnternet-Ağ Yönetimi, Yapay zeka, Derin-öğrenme, Nöromorfik öğrenme ülkemiz gençlerinin çok başarılı olacağı alanlardır. Şu anda geç kalıyoruz. Genç yetenekleri yaşatmak ve gün yüzüne çıkarmak için ciddi bir yetenek yönetimine ihtiyaç vardır. Yetenek yönetimi (Talent Management) günümüzde başlı başına sosyal bir bilimdir. Bilim Barış Elçilerine olan ihtiyacı çok büyük bir bilim adamı grubu ile dile getirdik (6). Genç bilim insanı adaylarını, henüz öğrenciyken dünyayı tanımaları için desteklemiliyiz. Çin, Kazakistan, Özbekistan gibi Türk Devletleri, Afrika Ülkelerine birer Bilim ve Barış Elçileri olarak göndermemiz gereklidir (6). Yüzyılımızda 4.8 Milyar insan gelişmekte olan ülkelerde yaşadığını ve 2.7 milyar insan günde gayri safi milli gelir olarak 2 Amerikan Dolarının altında bir gelirle yaşadığını hesaba katarak kapsayıcı yeni bir inovasyon ve girişim modeli üzerinde çalışmalıyız (6). Unutulmamalıdır ki, BiyoGirişimcinin yatırım alanı, mutlaka kanser ilacı bulmak veya genetikle uğraşmak değildir. Kanserden koruyucu hekimlikte atılabilecek yeni adımlar ve wellness merkezleri Tohum. Çevre. İçme suları. Atıkların temizlenmesi. Toksikoloji. Yeni biyomalzemeler, protezler. Protezlerin peptitle kaplanması. Fotodinamik tedaviyi radyoterapi ile bütünleştirme. Non-invazif tanı yöntemleri, Manyetik levitasyonla flow-sitometri genç Türk girişimcilerinin hali hazırda gerçekleştirdiği buluş ve yatırımlardır. 61

64 DAVETLİ KONUŞMACILAR / INVITED LECTURES Ayhan Doyuk-Türk yatırımcılarla (Dezenkon, Solars, Anti-Mikrop laboratuvarları) çevre. içme suları. atıkların temizlenmesi. Toksikoloji alanlarında İspanya ve Ankara merkezli inovatif ürünlerle dünyayı değiştirmeye hazırlanıyor. Sloganları Ekoloji den Ekonomi ye. Utkan Demirci-USA, Manyetik levitasyonla flow-sitometri, mikro kanalların tıbbi uygulamaları ile tüm dünyada tanındı ve ciddi bir üretici oldu. Aydoğan Özcan-USA, cep-telefonuna bağlanan cep-mikroskobu ile Afrika ve tüm dünyada AIDS hastalarının bağışık hücre sayılarını takip ediyor. Mehmet Sarıkaya-USA, metal ve plastik yüzeylerini çok sağlam ve kalıcı olarak peptitle kaplıyor ve şimdiden yüzlerce patent aldı. Banu Onaral-USA, beyin kanamlarını acil paramediklerin ambulansta veya askerlerin cephede teşhis edeceği bir alet geliştirdi. Üretimi ile tüm dünyaya satışını gerçekleştirdi. INOVITA, genç bilim adamları ve hekimler kendi alanlarında yenilikler yapsın, buluşlarının patentini alsın, kendi işlerini kursunlar diye kuruldu. Türkiye Cumhuriyeti Kalkınma Bakanlığı ve İstanbul Kalkınma Ajansı destekledi. Üç üniversiteyi biraraya getirdi. Genç bilim adamlarını spin-off şirket kurma, start-up şirket başarısı, fikri haklar konusunda eğitiyor ve ISEK kapsamında onlara yer veryor. SEM firması ilk kez İnovita da biraraya geldi ve JASEM kuruldu. Tüm dünyaya kütle spektroskopi kitlerini satıyor. Hemen hemen rakipsizler. Prof. Vural Özdemir ile birlikte ülkemizin kendi ihtiyaçlarına uygun bir bilim politikası üzerinde düşünüyor ve çalışıyoruz (11 ve 12). Vural Özdemir OMICS dergisinin editörüdür (13) ve onun sayesinde ülkemiz çok saygın bir düzeyde temsil ediliyor. Ülkemizin geleceği ve bilim politikalarının üretilmesi için bu ülkede herkese büyük görevler düşmektedir. Ekosistemin korunması, ülkelerin bilim politikaları, sosyal ıstıraplar. Şimdi bilimin sadece tedaviye değil, bilimin topyekün barışa ve kalkınmaya dönüşümünü konuşma zamanıdır. Biz çalışmalarımızı daha verimli, etik ve küresel kalkınmaya yönelik planlıyorsak devlet adamları ile daha sıkı işbirlikleri geliştirmeliyiz. Onları bilgilendirmek için yeni yönetişim araçlarının geliştirilmesi, inovasyonun yönetilmesi ihtiyacımız olan konulardır. Bu kongrede kesinlikle şunu gördüm. Tüm genç Türk bilim adamları, girişimci ve yatırımcılar, bilimini ve Teknolojisini geliştirmiş üreten bir Türkiye için canla başla çalışıyor. Devamını diliyor ve teşekkürlerimi sunuyorum. Bu konuşmanın eksik kalan Ticarileşme boyutunu biraz sonra Prof. Cengizhan Öztürk Hoca ve panelistler tüm detayları ile ortaya koyacaktır. 62

65 DAVETLİ KONUŞMACILAR / INVITED LECTURES Kaynaklar [1.] OECD, Oslo Kılavuzu. TÜBİTAK Avrupa Komisyonu 3.Baskı [2.] Özdemir V, Hekim N.: Innovation Management? Orienting Sepsis R&D and Technology Transfer Towards Stratified Medicine.: EBioMedicine Apr;6:8-9. [3.] Dandara C. Endrenyi L. Kolker E. Hekim N. Steuten LM.G. Güngör K. Aklillu E. Özdemir V. Precision Medicine 2.0: The Rise of Glocal Innovation, Superconnectors, and Design Thinking. OMICS A Journal of Integrative Biology 2016; 20(9): [4.] Saygılı Eİ, Abou-Zeid AH, Akkın SM, Aklillu E, Barlas İÖ, Borda-Rodriguez A, Boschele FA, Çetin Z, Coşkun E,Coşkun Y, Dağlı G, Dai TU, Dandara C, Dereli T, Elbeyli L, Endrenyi L, Eyigün CP, Georgakilas A, Günbulut B, Güngör K, Güzelbey A, Hekim C, Huzair F, Kimyon S, Karakaş Ü, Lin B, LLerena A, Masimirembwa C,McNally R, Mete A, Sancar P, Srivastava S, Steuten LM, Tanrıöver O, Tyfield D, Töre Vİ, Vuruşkan D,Wang W, Warnich L, Wonkam A, Yıldırım YZ, Yılmaz İ, Sınav A, Hekim N: An Open Letter in Support of Transformative Biotechnology and Social Innovation: SANKO University Innovation Summit in Medicine and Integrative Biology, Gaziantep, Turkey, May 5-7, OMICS Apr;20(4): doi: /omi [5.] Edward S. Dove, İ. Ömer Barlas, Kean Birch, Patricia A. Broderick, William M. Byne, Yavuz Coşkun, Marja-Liisa Dahl, Türkay Dereli, Shyam Diwakar, Levent Elbeyli, Laszlo Endrenyi, Lynnette R. Ferguson, Kıvanç Güngör, Ulvi Gürsoy, Just Haffeld, Nezih Hekim, Farah Huzair, Kabeer Kaushik, Olcay Kıroğlu, Ilinoa Kickbusch, Eija Könönen, Biaoyang Lin, Adrian LLerena, Faruk Malhan, Bipin Nair, Bülent Özkan, George Patrinos, Semra Şardaş, Sanjeeva Srivastava, Lotte M.G. Steuten, Cengiz Toraman, Effy Vayena, and Vural Özdemir. An Appeal to the Global Health Community for a Tripartite Policy Innovation: An Essential Diagnostics List, Health in All Policies and See-Through 21 st Century Science and Ethics. Journal Accepted. OMICS [6.] Nezih Hekim, Yavuz Coşkun, Ahmet Sınav, Alaa H Abou-Zeid, Mehmet Ağırbaşlı,Simisola O Akintola, Şükrü Aynacıoğlu, Mustafa Bayram, Nicola Luigi Bragazzi,Collet Dandara, Türkay Dereli, Edward S Dove, Levent Elbeyli, Laszlo Endrenyi,Kamile Erciyas, Jack Faris, Lynnette R Ferguson, Fahrettin Göğüş, Kıvanç Güngör,Mervi Gürsoy, Ulvi K Gürsoy, M Asım Karaömerlioğlu, Ilona Kickbusch, Türker Kılıç, Metin Kılınç, Tanıl Kocagöz, Biaoyang Lin, Adrián LLerena, Vangelis G Manolopoulos, Bipin Nair, Bülent Özkan, Tikki Pang, Şemra Sardaş, Sanjeeva Srivastava, Cengiz Toraman, Kemal Üstün, Louise Warnich, Ambroise Wonkam,Mustafa Cengiz Yakıcıer, Ümit Yaşar, Vural Özdemir. Translating biotechnology to knowledge-based innovation, peace, and development? Deploy a Science Peace Corps--an open letter to world leaders. OMICS 2014 Jul 23;18(7): Epub 2014 Jun 23. [7.] Özdemir, V., Dandara, C., Hekim, N., Birch, K., Springer, S., Kunej, T., & Endrenyi, L. (2017). Stop the Spam! Conference Ethics and Decoding the Subtext in Post-Truth Science. What Would Denis Diderot Say? OMICS: A Journal of Integrative Biology, 21(11), doi: /omi [8.] Nezih Hekim, Eugene Kolker and Vural Özdemir. A General Theory for Post Systems Biology: Iatromics and the Environtome. OMICS May;20(5):325-7 [9.] William Ogburn, (1922). Sosyal Değişim. New York: Dell. [10.] Kickbusch, I., Bayram, M., Bayram, Ö., Hekim, N., Kayalı, B., & Özdemir, V. (2018). Interview: The New Silk Road Health as Soft Power. OMICS: A Journal of Integrative Biology, 22(6), doi: / omi [11.] Vural Özdemir ve Nezih Hekim. Birth of Industry 5.0: Making Sense of Big Data with Artificial Intelligence, The Internet of Things and Next-Generation Technology Policy. OMICS. a journal of integrative biology 2018 Jan;22(1): [12.] Özdemir, V., Endrenyi, L., Hekim, N., Kunej, T., Steuten, L. M., Springer, S., Bayram, M. (2018). To Genotype or Phenotype for Drug and Food Safety? Exiting the Technology Echo Chambers. OMICS: A Journal of Integrative Biology, 22(8), doi: /omi [13.] OMICS, A Journal of Integrative Biology endorsed in January 2013 a new editorial vision entitled OMICS

66 SÖZLÜ BİLDİRİLER ve TAM METİNLER ORAL ABSTRACTS and FULL TEXTS

67 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-005 A Pilot Scale Production of L-asparaginase, L-glutaminase and Taxol from Endophytic Fungi Isolated from Punica Granatum Ibrahim Karidio Diori, Şenay Hamarat Şanlier Department of Biochemistry, Ege University, İzmir, Turkey Endophytes encompass the ubiquitous diversity of endosymbionts taxa that succeeded in escaping to plants defense barriers, and settled house within them asymptomically. Therefrom, they entertain interrelationship with their respective hosts that ranges from commensalism to mutualism. These microbial inhabitants of plants are believed to be sources of secondary metabolites to their hosts, thereby assisting them with growth promoting biomolecules and/or metabolites that promote their resistance against biotic/ abiotic stresses. Some of these biomolecules are novel, chemically, and biologically valuable secondary metabolites that are sometimes extracted from the plants for pharmaceutical or industrial purposes. The Taxol production reported by Strobel et al. (1993), is an illustrative example, which pioneered new research area with a growing interest toward these microbiota for pharmaceutical purposes. Herein, we report the isolation of endophytic fungi from flowers, leaves, twigs, and roots of Punica granatum. In a pilot screening study, we investigated six fungal species isolated from roots and twigs for the production of Taxol, and amidohydrolases i.e. L-asparaginase (E.C ) and L-glutaminase (E.C ), with antineoplastic activities. Many scientific reports support that L-asparaginase could have L-glutaminase co-activity. Clinically, L-asparaginase is used to lower the serum's content of L-asparagine, an amino acid crucial for the tumor's growth. It has a wide use in therapeutics as antineoplastic drug, especially in the treatment of acute lymphoblastic leukemia, but also in lymphosarcoma and melanosarcoma. Clinically, Taxol is used for its antineoplastic properties, especially to treat breast cancer. Taxol acts by freezing the cytoskeleton, thereby exhibits an antimitotic activity. After the fermentation process for the production of Taxol (10 days), the culture broth was filtered to separate fungal mycelia from the filtrate. The latter was exhaustively extracted with EtOAc and n-hexane respectively. The absorption profiles of both organic extracts were determined. The EtOAc extract exhibited an absorption profile that included the absorption range of previously reported Taxol i.e nm. The amidohydrolases production potentials were tested on solid culture media with L-asparagine / L-glutamine as a sole nitrogen source supplemented with phenol red to monitor ph change. Subsequently, Nesslerization reaction served to monitor the ammonium content of the culture broth in a pilot fermentation. Keywords: endophytes, pomegranate, asparaginase, glutaminase,taxol Introduction Endophyte etymologically means inside plant as it is derived from the Greek words endo meaning inside and phyto meaning plant. The term was first coined by De Bary in The nowadays meaning was given by Bacon & White (2009). This encompasses a broad scope, but yet unknown Taxa of microbes including bacteria, fungus, and algae. Every plant living in a natural ecosystem is believed to host at least one kind of endophytic species either 65

68 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS or both in its rhizosphere and phyllosphere. These microbial inhabitants of plants are believed to be a source of secondary metabolites to their hosts, thereby assisting them with growth-promoting biomolecules and/ or metabolites that promote their resistance against biotic and / abiotic stresses. With the Taxol production reported by Strobel et al. (1993) researchers manifest a growing interest toward these microbiota for pharmaceutical, industrial and also for agricultural purposes. However, a lot remains to do. Herein, we do report a preliminary pilot study of a thesis research in which we aim to isolate an array of endophytic species from a Punica granatum (pomegranate), screen them for bioactive metabolites and eventually optimize the most promising bioactive compounds produced in the lab. The present report, especially focuses on the potential production of fungal species isolated from roots and twigs of Punica granatum for the production of L-asparaginase, L-glutamine, and Taxol. Material and method Fresh and apparently healthy flower, fruit, leave, twig and root samples were collected from a Punica granatum tree at the Faculty of Science of Ege University (38 27'34.2"N 27 13'51.8"E), and transported in sterile plastic bags to the laboratory of Biotechnology of the Department of Biochemistry, where they were immediately washed under running tap water before submitting them to surface sterilization procedure. The roots and twigs were cut into small fragment by means of sterile bistoury. Depending on the sample types the immersion time during the surface sterilization process varied. Fruits, flowers, and leaves were immersed for 30s in 70% EtOH, then at 2% NaOCl for 10-15s, again in 70% EtOH for 30s and rinsed twice in sterile distilled water. Throughout the process, each step was carried out in a different beaker. Root and twig samples were surface-sterilized through a similar procedure, but with longer immersion periods. First, immersion was at 70% EtOH for 3 min, followed by 1 min at 2% NaOCl, 1 min at 70% EtOH and 1 min in sterile-distilled water twice. Following surface sterilization, samples were placed into the folds of sterile tissue papers to dry. The samples were then cut into small fragments (twigs and roots were also cut longitudinally), and transferred onto freshly prepared artificial microbiological culture media so that the newly cut surface came into contact with the culture media. As a control for the efficiency of the sterilization process, 0.1 ml of the last rinsing water was inoculated onto a separate culture medium and incubated the samples under the same conditions. The used culture media included LB-agar, PDA + chloramphenicol. Culture medium for L-glutaminase production KCl-0.5g; MgSO 4-0.5g; KH 2 PO 4-1.0; FeSO 4-0.1g; ZnSO 4-0.1g; NaCl-0.5g; Agar-20g; L-Glutamate-10.0g; phenol red-0.012g; ph 6.8; temperature 37ºC. Culture medium for L-Asparaginase production Glucose-2.076g; L-Asparagine-5.202; Na 2 HPO 2 O-6.0g; Agar-20g; KH 2 PO g; NaCl-0.5g; Mg- SO 4.7H 2 O-0.373g; yeast extract-1.0g; peptone-1.0g; Temperature 37ºC; In the biotechnological production process, the screening was first done on solid artificial microbiological culture media with sole or alternative nitrogen-source L-glutamine/ L-asparagine, and phenol-red to monitor any eventual ph change that would testify of a positive result if the color changes to pink. The hydrolysis of L-glutamine/ L-asparagine would release L-glutamic acid/ L-aspartic acid and ammonium into the medium. The ammonium would cause the color change to pink. Culture medium for Taxol production Yeast extract-1%; peptone-2%; Glucose-2%; temperature 30ºC; 80rpm; time 7days; The culture was brought onto an end by adding EtOAc into each of the Erlenmeyer flasks containing the culture and incubating the mixture at 40 ºC; 150 rpm overnight. Then, the polar and apolar phases of the culture were extracted using EtOAc: n-hexane system in a liquid-liquid extraction. The absorption profile of each of the phases was determined by means of spectroscopic absorption wavelength scanning procedure. 66

69 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Results and discussion Many fungal species grew from the plant s tissue samples. Although not yet molecularly characterized, considering the homogeneity of their appearance, morphology and upside-down culture image, we report the isolation of many apparently pure fungal species from the plant tissue samples., A developing pink color was observed around the growing cultures, reflecting positive a result for each of the tested organisms, but with distinct color intensities. Thus, these organisms could potentially be producers of L-Asparaginase and L-glutaminase. In the second step, Nesslerization reaction was used to monitor the qualitative and quantitative enzyme content of the broth culture media throughout the conducted pilot study that lasted ten (10) days. There was observed an increasing ammonium content of the cultures broth separated from the mycelia prior to the test. In pharmaceutical industries, the most common sources of L-asparaginase / L-glutaminase has been E. coli, Erwinia chrysanthemi, Streptomyce canarius, and plants. However, side effects and / or yield considerations are directing scientists towards new sources. The major advantages of fungal L-asparaginase and L-glutaminase are: (i) that they have lower risks to generate side effects in patients such as allergy compared to the bacterial sourced ones; (ii) Fungi mostly produce L-asparginase and L-glutaminase as exozymes, making isolation, purification, and yield higher compared to bacterial production; (iii) Fungi, being eukaryotic organisms are closer to human beings than bacteria, thus provoke less immunological response. The organisms were also screened for the production of Taxol. After the fermentation and extraction of the culture broths, the absorption profile of each of the polar and apolar phases were determined. The broths polar extracts showed absorbance in the characteristic absorption range of Taxol i.e nm; while the apolar phases showed absorption range between nm. The investigated endophytic fungal species showed promising results, some being better than others. However, an optimization of individual fermentation parameters, purification, and anticancer activities of the eventually produced biomolecules would be necessary to know if they could be alternative sources of these molecules for pharmaceutical purposes. Conclusion The endophytic fungi screened for the production of amidohydrolases i.e. L-glutaminase, L-asparaginase and the production of Taxol showed promising results. These organisms are potential producers. However, optimization of the fermentation process, purification and test for the biological activity would be necessary to confirm that they would be alternative sources for the anticancer biomolecules for pharmaceutical industries. 67

70 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-010 Possible Toxic Effects of Biotechnological Vaccines and the Safety Concerns Pınar Erkekoğlu Hacettepe University Faculty of Pharmacy Department of Toxicology Abstract Vaccines are crucial products for the well-being of populations. Toxic effects of vaccine preservatives and adjuvants have been subject of several research for many years. Today, new techniques of molecular biology and complete chemical synthesis of genes can provide genetic engineering of microorganisms and novel biotechnological/biological-derived vaccines are now generated. However, due to their being new on market, there are potential safety concerns that arise from the novel manufacturing processes and complex structural and biological characteristics of these products. These vaccines may also contain the same preservatives and adjuvants that are used in classical vaccines. Immunogenicity is the primary toxic effects of vaccines, including biotechnological vaccines. The risk associated their immunotoxic effects is linked to purification processes as antigenic fractions can be polluted by vectors which were not totally eliminated in the finished product. Other than classical toxic effects of their ingredients and immunotoxicity, reduction of efficacy can also be considered as an unwanted effect for biotechnological vaccines. It can clearly be stated that evaluation of the quality, toxicity/safety, efficacy and stability of biotechnological vaccines need complex analytical methods and appropriate physicochemical, biochemical and immunochemical analyses. Regulatory authorities should develop new approaches to control their safety and new toxicity testing strategies should be implanted. Keywords vaccine, biotechnological vaccine, immunogenicity, immunotoxicity, safety, biotechnology, toxicity Introduction Vaccines are crucial products for the well-being of populations. Human vaccines are being produced using classical manufacturing methods (culture, various concentration, inactivation, detoxification and conjugation production processes) for decades. Main toxic effect of classical vaccines is their immunogenicity. On the other hand, preservatives (e.g. thiomersal, formaldehyde) and adjuvants (e.g. aluminum) in classical vaccines have been subject of several research for many years. Today, new techniques of molecular biology and complete chemical synthesis of genes can provide genetic engineering of microorganisms and novel biotechnological/biological-derived vaccines are now generated. However, due to their being new on market, there are potential safety concerns that arise from the novel manufacturing processes and complex structural and biological characteristics of these products. These vaccines may also contain the same preservatives and adjuvants that are used in classical vaccines. However, the main concern is their immunogenicity. Their immunotoxic effects can occur as a result of multiple factors. On the other hand, reduction of efficacy can also be considered as an unwanted effect for biotechnological vaccines. Other than toxicity of the preservatives/adjuvants, immunogenicity and low efficacy; skin reactions, several other possible toxic effects were also reported as adverse drug reaction toward biotechnological vaccines. Therefore, our 68

71 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS aim was to consider the possible toxic effects of biotechnological vaccines and their safety from the point of view of toxicologist. Materials and Methods Limited amount of literature on the toxicity and safety of biotechnological vaccines was searched on PubMed and Scopus. Moreover, the toxic effects of preservatives and adjuvants in biotechnological vaccines and related studies were also searched. Results Limited amount of literature indicated that the toxic effects of biotechnological vaccines and their safety profile should be discussed under four sub-titles: Toxic effects of preservatives and adjuvants in biotechnological vaccines Thiomersal (sodium ethylmercury thiosalicylate) is used as an antibacterial and antifungal vaccine preservative. This compound is mainly and it contains almost 49% mercury within the structure. Thiomersal increases the shelflife of the vaccines. In 1999, concerns were raised in the USA about exposure to mercury (i.e. thiomersal) in vaccines. This was based on the realization that the cumulative amount of mercury in the infant immunization schedule potentially exceeded the recommended threshold set by the United States government for methyl mercury. However, thiomersal, the preservative in some vaccines, contains ethyl mercury not methyl mercury. Thiomersal is now associated with autism and it is now not used in vaccines applied to children. Formaldehyde is another vaccine preservative and a nearly colorless gas with a pungent, irritating odor even at very low concentrations (below 1 ppm). Because the pure gas tends to polymerize, it is commonly used and stored in solution. Formalin, the aqueous solution of formaldehyde (30% to 50% formaldehyde), typically contains up to 15% methanol as a stabilizer. The systemic effects of formaldehyde are due primarily to its metabolic conversion to formate, and may include metabolic acidosis, circulatory shock, respiratory insufficiency, and acute renal failure. Formaldehyde is classified as Group I carcinogen by International Agency for Research on Cancer (IARC) and it mainly causes cancers of nasal cavity. Aluminum is a vaccine adjuvant that causes an oxidative stress within brain tissue. Since the elimination half-life of aluminum from the human brain is 7 years, this can result in cumulative damage via the element's interference with neurofilament axonal transport and neurofilament assembly. A possible etiologic link between aluminum exposure and Alzheimer s disease emerged from a 1965 study showing that aluminum causes neurofibrillary tangles in the brains of rabbits. Subsequent research has largely failed to support this hypothesis, however. For example, the clinical manifestations and underlying neuropathology of aluminum-induced encephalopathy in dialysis patients bear no resemblance to those of Alzheimer disease. It was suggested that the heterogeneous symptoms of autism spectrum disorders have a connection with dysregulation of glutamatergic neurotransmission in the brain, along with enhancement of excitatory receptor function by pro-inflammatory immune cytokines, as the underlying pathophysiological process. It was also suggested that aluminum and other environmental and dietary excitotoxins (eg, mercury, fluoride) can exacerbate the pathological and clinical problems by worsening excitotoxicity and by microglial priming. Aluminum is also associated with kidney toxicity in later life. Exposure to aluminum is stated as Group I carcinogen by IARC. Immunotoxicity of biotechnological vaccines Immunogenicity is the main problem associated with both classical and biotechnological vaccines. The strain of the microorganism used in the vaccines, storage conditions, purification, or formulation of the product or most importantly the presence of impurities can trigger an immunogenic response, which can be life-threatening. Inert recombinant vaccines contain proteins, usually expressed in a bacterium or yeast. The risk associated with 69

72 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS use of these vaccines is linked to purification processes as antigenic fractions can be polluted by vectors which were not totally eliminated in the finished product. Risk of using antigens expressed in a replicative vector is associated with the vector used, and is therefore dependent on the biological properties of this vector in the target host and potential non-target hosts. Reduction of efficacy for biotechnological vaccines Reduction in the efficacy of vaccines a major problem for biotechnological vaccine producers and receivers. For instance, synthetic biotechnological vaccines may sometimes have limited efficacy. Other toxic effects of biotechnological vaccines Both classical and biotechnological vaccines were suggested to show several other toxic effects, including asthma, digestive problems, increased risk of food allergies, liver and kidney problems, skin reactions (like eczema, acne, soreness on the application site), attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD). Discussion It is evident that biotechnological vaccines may exert the same risks concerning their ingredients. However, their immunogenic properties may differ from classical vaccines and their safety evaluation should be performed by taking into account of their complex nature. Mechanistic approach is needed to evaluate their toxicological profiles and each product should be evaluated separately for their safety. This evaluation should be done by scientists who have high and sophisticated knowledge in this field. It can clearly be stated that determination of the quality, toxicity/safety, efficacy and stability of biotechnological vaccines need complex analytical methods and appropriate physicochemical, biochemical and immunochemical analyses. Moreover, Biotechnological vaccines have distinguishing characteristics to which consideration should be given in well-defined quality control and toxicity/safety testing programs. Regulatory authorities should develop new approaches to control their safety and new toxicity testing strategies should be implanted. Conclusions In conclusion, we can state that vaccination should be encouraged as it is the most important way for providing public health throughout the generations. Biotechnological vaccines are new on market and may have safety issues. Before marketing biotechnological vaccines, particularly for vaccines in children use, the production companies should perform risk assessment for each ingredient as well as for the whole vaccine. Their safety, efficacy and quality should be considered and their toxicological profile should be evaluated thoroughly. 70

73 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-015 Chemical Characterization and Biological Study of the Species Senecio cineraria Ababsa Zine El Abidine 1, Kara Ali. Wahiba 2, Medjroub Kamel 3, Akkal Salah 4, Abidli Nacira 5 1 Département des Sciences de la matière, Faculté des sciences exactes et de la nature et vie, Université Larbi BEN M HIDI, Oum El Bouaghi- Algérie 2 Laboratoire de Biologie et de l Environnement, Faculté des Sciences de la Nature et Vie, Université Mentouri -Constantine1- Algérie 3 Unité de Valorisation des ressources naturelles, Molécules Bioactives et Analyses Physico-Chimiques et biologiques, Faculté des Sciences Exactes, Département de Chimie, Université Mentouri de Constantine 1- Algérie 4 Unité de Valorisation des ressources naturelles, Molécules Bioactives et Analyses Physico-Chimiques et biologiques, Faculté des Sciences Exactes, Département de Chimie, Université Mentouri de Constantine 1- Algérie 5 Département de Biologie, école nationale supérieure des enseignants ENS, Kouba, Alger- Algérie Abstract Senecio cineraria is a perennial shrub of Mediterranean origin, belongs to the family of Asteraceae, it is used in pharmaceutical preparations and in homeopathy. This study is a scientific contribution to the determination of certain phytochemicals, as well as the study of some in vitro biological activities of the methanolic extract of the plant. The analysis of the extract by colorimetric tests was revealed the presence of flavonoids, alkaloids and tannins. Qualitatively, the TLC analysis of the extract showed the presence of a multitude of flavonoid varieties. The flavonoid assay showed a significant content on the order of mg EQ / g E. The study of the antioxidant power by the DPPH method has shown that the concentration which traps 50% of the DPPH (IC50) radical is 0.35 mg / ml. EMSC revealed a significant anti-hemolytic effect compared to the positive control. This is proportional to the concentration of the extract used during the test. The antibacterial potential of the extract was confirmed on strains: Staphylococcus aureus, Listeria monocytogenes, Klebsielaoxytoca, with MICs of 10 mg / ml, 20 mg / ml and 2.5 mg / ml respectively. Chronometric coagulation tests (TCK and TQ) have shown that the extract has significant anticoagulant activity. Keywords: Senecio cineraria, flavonoids, antioxidant power, The antibacterial potential, Chronometric coagulation tests. 71

74 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-019 Gene Drives with CRISPR-CAS9 Technology in Agriculture and Ethical Issues Mehtap Kara 1, Özlem Akbal Dağıstan 2, Özlem Nazan Erdoğan 3 1 Istanbul University Faculty of Pharmacy Department of Pharmaceutical Toxicology 2 Istanbul University Faculty of Pharmacy Department of Pharmaceutical Technology 3 Istanbul University Faculty of Pharmacy Department of Pharmacy Management Abstract For increasing crop productivity, several types of pesticides using in modern agricultural applications. Thus, these chemicals cause different serious toxic effects through accumulating in the environment. Most of these chemicals have effects on non-target and non-harmed insect species, fishes and animals. And also these chemicals may revealed resistance of pests via genetic modification mechanisms. As a fast growing technology CRISPR-Cas9 gene editing system may play key role in improving environmental and public health. Because of its key role on cutting DNA, low cost and application in several areas as analyzing gene functions, genomic rearragements, cancer progression, correcting genetic mutations, observing diseases survey, agricultural practices etc. CRISPR-Cas9 biotechnology draw attention by different area scientists and preffered method. This technology based from bacterial immune system which recognize short DNA sequences, cut these sequences and insert new one that area CRISPR-Cas9 technology make easy to insert new genetic sequences into the genome. Main three element of CRISPR-Cas9 technology are; Cas9 protein encoding gene, guide RNA coding gene and flanking sequences. Cas9 enzyme generate breaks in target double stranded DNA. RNA guided endonucleases induce site specific cleavage, use a short guide RNA (grna) which recognize target DNA. This derived from the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) found in bacteria that play role as immune system to destroy foreign DNA. Gene sequence insertion with gene drive may give several advantages in pest control as low cost, more precision, direct manipulation of pest species compared to other techniques. This technology give opportunity to easily eradication of species. Although these features make this technology attractive, regulative and ethical issues primarily are on the front burner. In this review we discussed one of important application area of CRISPR based gene drive that aiming increase crop production with pest control and ethical issues. Keywords CRISPR-Cas9, Agriculture, Ethic, Gene Modification, Toxicity Introduction CRISPR-Cas9 is recently developed gene technology and have applications in biomedical and drug delivery researches. While it is predicted potentially valuable technology, researches have wonder about future mutational effects on species and this brign into questions about ethical issues. CRISPR-Cas9 gene technology has advantages as cheap, easy, precise, and efficient genetic modification. This technology make easy to create new mutations, gene insetrion and deletions on genome with diverse effects. Due to make this technology possible to genome changes, ethical values and debates draw attentions (Scherz ). As same eukaryote species prokaryotes have different defence mechanisms to protection from different type infections. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) one of the main bacterial defence mechanism with spacers invading genomes as short sequences to virus infections 72

75 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS and develop immune memory. Spacers include repeats that express small guide CRISPR RNAs (crrnas) (Hille F, Charpentier E,. 2016) CRISPR-Cas9 mechanism is based on bacteria immune system which protects bacteria genome from viral infections. Bacteria have guide sequences include approximately nucleotides which match with virus genome. These guide sequences play role as guide RNA and then interact to Cas (CRISPR-associated proteins). This bacterial RNA-protein complex have ability to rapidly cut viral DNA and finish viral infections in bacteria (Figure 1) (Wright, 2016; Scherz ;Martin Jinek et al., 2012) This gene editing technology have advantages as; significantly reduces the time required to experiments which have previously taken years, simplicity and efficiency. The important disadvantage is unwanted mutational results during applications. (Mulvihill et al. 2017) Figure 1. Basic mechanism of CRISPR-Cas9 gene editing technology (Figure from: spring-2016-opening-pandoras-box-the-future-of-genetic-engineering/ ) In this review article we aimed to discuss CRISPR-Cas9 gene drive technology in agricultural area and important ethical issues. CRISPR-Cas9 Gene Technology In Agricultural Applications Modern agriculture improvements with research and technology permit increasing of crop yield and quality in recent years. CRISPR-Cas9 gene technology could be an effective tool for crop improvement and raises specific issue. This technology could be use for increase active ingredients, defense mechanisms and yield of crops. Give opportunity to grow pesticide and herbicide tolerent crops, however Gene-edited organisms could be express a novel protein which is uncertain about its safety. CRISPR-Cas9 gene editing technology future evolving in agricultural applications has not yet been clear (Gao, 2018; Courtier-Orgogozo, et al.,2017) Ethical Issues The most important biotechnological discovery of the century, CRISPR technology give amazing resource for biomedical, drug delivery, agircultural and environmental reserach areas. Due to its diverse application areas and giving advantes to genome modifications, this technology brings out ethical questions with itself. Speculations about growing CRISPR technology could give rise to solidarity on this conceptualizing. Main question is how this technology regulates by governments to prohibit misused by war industry. Some of important questions about this genetic technology are listed below; How this technology affect next generations due to its genome modification advantage? How it will control by authorities, if its applicate for create more excellent generations? 73

76 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS In drug delivery, is it possible to develop radical therapy for several diseases, who will fund this or this medical applications create more dangerous diseases? Will this technology use for solve scarcity problem with effectice agricultural applications or will this in turn an environmental disaster? (Scherz 2017 ; Mulvihill et al., 2017; Zimmer. 2015; Deborah M. Thurtle-Schmidt,- Te-Wen Lo. 2018) In conclusion, CRISPR/Cas9 genome engineering technology provides accelerated and valuable methods for scientisct in several applications areas, however this technology should be handled by authorities in accordance with scientists carefully about its misuse. References [1.] Courtier-Orgogozo, V., Morizot, B. & Boëte, C Agricultural pest control with CRISPR based gene drive: time for public debate. EMBO Reports 18: [2.] Deborah M. Thurtle-Schmidt,Te-Wen Lo Molecular Biology at the Cutting Edge: A Review on CRISPR/CAS9 Gene Editing for Undergraduates. Topical Review. Biochemistry and Molecular Biology Education. Volume 46, Number 2, March/April 2018, Pages [3.] Gao C The future of CRISPR Technologies in agriculture Nature Reviews,Molecular Cell Biology Volume 19; 1-2. [4.] Hille F, Charpentier E CRISPR-Cas: biology, mechanisms and relevance. Phil. Trans. R. Soc. B 371: [5.] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E A programmable dual-rna-guided DNA endonuclease in adaptive bacterial immunity. Science. 337(6096): [6.] Mulvihill JJ, Capps B, Joly Y, Lysaght T, Zwart HAE, Chadwick R; International Human Genome Organisation (HUGO) Committee of Ethics, Law, and Society (CELS) Ethical issues of CRISPR technology and gene editing through the lens of solidarity. Br Med Bull.122(1):17-29 [7.] Scherz P The Mechanism and Applications of CRISPR-Cas9. National Catholic Bioethics Quarterly 17.1: [8.] Wright AV, Nuñez JK, and Jennifer A. Doudna Biology and Applications of CRISPR Systems: Harnessing Nature s Toolbox for Genome Engineering. Cell 164.1: [9.] Zimmer C. Breakthrough DNA editing born of bacteria. Quanta Magazine

77 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-047 Superparamagnetic Iron Oxide Nanoparticle-Based Targeted Gene Delivery Systems for Cancer Therapy Kurtulus Gokduman Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA Abstract Superparamagnetic iron oxide nanoparticles (SPION), which possess unique physicochemical features such as ultra-fine sizes and a large surface area to mass ratio, are currently the only clinically approved metal oxide nanoparticles and the most commonly used superparamagnetic nanoparticles. Among vast variety of biomedical applications, SPION are of great interest in terms of cancer gene therapy due to the enabling possibilities like targeted control, image-guided therapy and hyperthermia. In parallel to their increasing popularity in cancer gene therapy, safety concerns about SPION have increased and to date, there is neither adequate information regarding their safety nor standardized method to establish such parameters. Herein, a methodology for the design and cancer gene therapy applications of SPION with a focus on the obtained nanotoxicology data will be discussed. Keywords Superparamagnetic iron oxide nanoparticles (SPION), targeted gene delivery, cancer therapy, hepatotoxicity, reactive oxygen species (ROS) Full Text Overcoming multi drug resistance (MDR) in cancer cells using gene therapy offers an interesting therapeutic window. Although the first trials to overcome the MDR are quite successful, they have chronic toxicity and side effects (e.g., despite successes of first trials containing P-gp inhibitors such as verapamil and cyclosporin A, generally they cause chronic toxicity and systemic side effects such as bradycardia, suppression of the immune system, etc.) [1, 2]. On the other hand, silencing of sequence specific genes (resistance genes) using double-stranded, non-coding small interfering RNA (sirna) with nucleotide has become increasingly important approach in recent years, as it offers a highly secure method of inhibiting the expression of proteins involved in MDR without side effects [1-3]. Although sirna is a very promising approach for cancer treatment, it has some limitations: I- Insufficient membrane permeability limits the cellular uptake of sirna; II- Most of the substances (e.g. Lipofectamine) used for the release of sirna are toxic; III- sirna is unstable and is rapidly degraded by nucleases [3]. Thus, an ideal carrier system design has a key role in sirna activity in cancer therapy; carrier systems can be categorized in two main groups as viral and non-viral systems. Although viral vectors have high sirna transfer efficiency, they have serious disadvantages in terms of toxicity (cytotoxicity, immunogenicity, mutagenicity, carcinogenicity etc.) and limitation of the size of the added genetic material [4, 5]. On the other hand, nanotechnology provides highly effective solutions to overcome the above mentioned limitations of sirna by using bio-compatible nanoparticles as sirna delivery systems without causing problems induced by viral vectors [3, 5]. SPION with unique physicochemical features such as ultra-fine sizes and a large surface area to mass ratio are currently the only clinically approved metal oxide nanoparticles and the most commonly used superparamagnetic nanoparticles [6, 7]. Among vast variety of biomedical applications, SPION are of great interest in terms of cancer gene therapy due to the enabling possibilities like targeted control, image-guided therapy and hyperthermia [8-10]. In parallel to their increasing popularity in cancer gene therapy, safety concerns about SPION have increased and to date, there is neither adequate information regarding their safety nor standardized method to establish such param- 75

78 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS eters. Hepatotoxicity is a major factor for biosafety of any formulation before its clinical translation; more than 900 drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market [11]. Therefore, to establish a method that can be used towards identifying toxicity, adverse effects, and mechanism of action of SPION on the liver and its parenchymal cells, hepatocytes, we have investigated the concentration-dependent (0-400 μg/ml) and exposure-dependent (single dosing vs. cumulative dosing) effects of SPI- ON (d=10 nm) on primary rat hepatocytes in a time-dependent manner. To this end, we have used a sandwich-cultured hepatocyte model and evaluated the temporal effects of SPION on the viability and hepatocyte specific functions (the secretion of albumin and urea). In addition, to investigate the role of reactive oxygen species (ROS) in SPION induced hepatotoxicity, we also studied the time-dependent ROS production upon treatment of primary rat hepatocytes with SPION in a concentration-dependent manner [12]. Both in single dosing and cumulative dosing groups, statistically significant effects on the viability of the primary rat hepatocytes were observed upon treatment with SPION with 200 μg/ml and 400 μg/ml at 24th hour (p<0.05 for 200 μg/ml, p<0.01 for 400 μg/ml) and 48th hour (p<0.01 for 200 μg/ml and 400 μg/ml) after the start of treatment. Live/dead assay carried out at 7th day after the start of treatment illustrated that damaging effect of SPION on the viability of the primary rat hepatocytes was progressive with time. The results also revealed the loss of hepatocyte specific functions, albumin and urea synthesis, upon treatment with SPION in a dose and time dependent manner; significantly (p<0.05) more deleterious effects on the functions were observed at 48th hour after the start of treatment in the cumulative dosing group. All concentrations of SPION induced highly significant (p<0.01) production of ROS at 30th minute; and then, statistically significant differentiation (p<0.05) among concentration groups took place at certain time points within the 18-hour time-lapse experiment. Thus, the study reveal that a combination of various biomarkers should be employed for the evaluation of the effect of SPION on liver, and, each biomarker should be analyzed in a time and exposure dependent manner. In addition, cancer gene therapy and other biomedical applications of SPION should be considered in the light of accumulating evidence of their nanotoxicology profiles. References [1.] Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 5(3): [2.] Yhee JY, Song S, Lee SJ, Park SG, Kim KS, Kim MG, Son S, Koo H, Kwon IC, Jeong JH, Jeong SY, Kim SH, Kim K Cancer-targeted MDR-1 sirna delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance. J. Control. Release 198: 1-9. [3.] Susa M, Iyer AK, Ryu K, Choy E, Hornicek FJ, Mankin H, Milane L, Amiji MM, Duan Z Inhibition of ABCB1 (MDR1) expression by an sirna nanoparticulate delivery system to overcome drug resistance in osteosarcoma. PLoS One 5(5): e [4.] Chen Y, Wang W, Lian G, Qian C, Wang L, Zeng L, Liao C, Liang B, Huang B, Huang K, Shuai X Development of an MRI-visible nonviral vector for sirna delivery targeting gastric cancer. Int. J. Nanomedicine 7: [5.] Grumezescu AM Nanobiomaterials in drug delivery (Vol 9). Oxford, UK: Elsevier Inc. [6.] Gokduman K Strategies targeting DNA topoisomerase I in cancer chemotherapy: Camptothecins, nanocarriers for camptothecins, organic non-camptothecin compounds and metal complexes. Curr. Drug. Targets 17(16): [7.] Singh N, Jenkins GJS, Asadi R, Doak SH Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION). Nano Rev. 1: [8.] Dey S, Maiti TK Superparamagnetic nanoparticles and RNAi-mediated gene silencing: Evolving class of cancer diagnostics and therapeutics. J. Nanomater [9.] Wahajuddin AS Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. 76

79 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Int. J. Nanomedicine 7: [10.] Gokduman K, Demir AS Camptothecin anticancer drug loaded iron oxide micro and nanoparticles: Tuning targeted and sustained release of the drug in bioactive form. Curr. Nanosci. 9(6): [11.] Rajan B, Sathish S, Balakumar S, Devaki T Synthesis and dose interval dependent hepatotoxicity evaluation of intravenously administered polyethylene glycol-8000 coated ultra-small superparamagnetic iron oxide nanoparticle on Wistar rats. Environ. Toxicol. Pharmacol. 39(2): [12.] Gokduman K, Bestepe F, Li L, Yarmush ML, Usta OB Dose-, treatment- and time-dependent toxicity of superparamagnetic iron oxide nanoparticles on primary rat hepatocytes. Nanomedicine 13(11):

80 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-064 Antibacterial Activity and Cytocompatibility of Silk Sericin- Capped Gold Nanoparticles Synthesized Under UVC Radiation Ömer Aktürk 1, Zehra Gün Gök 2, Memik Taylan Daş 3, Özge Erdemli 4, Mustafa Yiğitoğlu 1 1 Department of Bioengineering, Kirikkale University, Kirikkale, Turkey 2 Institute of Science, Department of Bioengineering, Hacettepe University, Ankara, Turkey 3 Department of Mechanical Engineering, Kirikkale University, Kirikkale, Turkey 4 Department of Molecular Biology and Genetic, Baskent University, Ankara, Turkey Abstract Biosynthesis of metal nanoparticles is gaining importance in view of their biocompatibility, good colloidal stability and eco-friendly features. In our previous work, sericin-capped gold nanoparticles (S-AuNPs) were synthesized by an easy one-pot synthesis method involving only gold salt and sericin extracted from silk worm cocoon in the presence of UVC light at different sericin solutions (0.25, 0.5, 1% w/v). According to the physicochemical test results, S-AuNP0.5 group was selected as the most suitable group and in this work, the colloidal stability, antibacterial and cytotoxicity effects of S-AuNP0.5 were investigated. The colloidal stability of S-AuNPs was determined in both high ionic strength solution and cell culture medium for either short and long-term incubation periods. Characterization results from UV-Vis spectroscopy showed that the synthesized AuNPs were stable in both at high salt concentration or in cell culture medium. The antibacterial properties of S-AuNPs and mixtures of S-AuNPs and streptomycin antibiotic were determined by minimal inhibitory concentration (MIC) test on Bacillus subtilis bacteria. According to the MIC tests performed, the synthesized S-AuNP0.5 group had no antibacterial effect on B. subtilis when used alone but, when it was used with streptomycin, it decreased the MIC value of the antibiotics for B. subtilis. In addition, in-vitro biocompatibility of S-AuNPs were assessed with WST-1 assay, proliferation (xcelligence microelectronic system) and apoptosis/necrosis observation. Cytotoxicity assays carried out with L929 cells demonstrated the non-toxic nature of S-AuNPs. As a whole, the synthesized S-AuNPs could be proposed as potential candidates for many biological applications due to their good colloidal stability, ability to act as reinforcing agents for the streptomycin antibiotic and biocompatible features. Keywords Bombyx mori silk sericin; Gold nanoparticles; Aqueous stability; Biocompatibility; Antibacterial activity 1. Introduction Gold nanoparticles (AuNPs) of different sizes and shapes are nowadays have many different application such as biosensors, drug delivery, and diagnostic assays [1, 2]. The synthesis of AuNPs is easily done by direct reduction of the gold salt solution (HAuCl 4 ), with common reduction agent such as citric acid [3, 4]. Lately, different methods have been suggested to produce biocompatible, stable and antibacterial AuNPs in a more environmentally friendly manner [4]. For example, biocompatible AuNPs were synthesized using gelatin [5], gallic acid [6], chitosan [7], silk fibroin [2]. In this work, we examined the stability, antibacterial activity and cytotoxicity properties of silk sericin capped gold nanoparticles which were synthesized with a simple and green synthesis method with the assistance UVC light in our previous work. 2. Material and Methods 78

81 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 2.1. Colloidal stability The stabilities of S-AuNPs at different salt concentrations (10, 5, 2.5, 1.25, %) and after different time incubation periods were measured with UV-Vis spectroscopy in the wavelength range of nm. Similarly, the stabilities of S-AuNPs incubated in cell culture medium (DMEM w/o phenol supplemented with 10% FBS) at different time periods were investigated Antibacterial test To evaluate antibacterial activity of the synthesized S-AuNPs, Bacillus subtilis RSHI (pathogen isolate taken from Refik Saydam Hifzissihha Institute, Ankara, Turkey) was used. The antibacterial activity of S-AuNPs0.5 group was evaluated using the standard broth macrodilution assay. In this assay, the bacterial cells were incubated with different concentration of a mixture of streptomycin antibiotic solution and S-AuNPs. Turbidity of these diluted test groups were determined by measurement of optical density at 600 nm. Clear suspension from the test groups were also selected and cultured on agar petri plates to visualize the CFUs on the agar surface. SEM micrographs were also utilized to image the cell number and morphology seeded on smooth mica surfaces In vitro cytotoxicity assay and cell morphology analysis The cytotoxicity effects of S-AuNPs0.5 solutions at different concentrations (0.26, 0.13 and mg/ml) on L929 cell line were examined with WST-1 assay using 96-well plates. Cell proliferation, viability and cytotoxicity tests of S-AuNPs were also performed with the cell-based xcelligence microelectronic system. For apoptosis and necrosis assessment, double flourescence stain was performed. 3. Result and Discussion Long term stability of nanoparticles is an important feature for their biomedical application [8].For this reason, in this work, the colloidal stability of synthesized S-AuNPs groups were determined in different salt concentration and in cell culture medium. Figure 1 shows the UV Vis absorption spectra of S-AuNPs in different NaCI concentration and DMEM at different time. The spectra of S-AuNPs showed no distinguishable changes in the intensity or position of the absorbance peak either at different salt concentration or in DMEM. The maintenance of peak positions of AuNPs during incubation periods indicated extra stability of S-AuNPs as stated elsewhere [9]. Absorbance (a.u.) 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0, Wavelength (nm) (A1) Absorbance (a.u.) 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0, Wavelength (nm) (A2) Day 0 % 0 NaCl % NaCl % 1.25 NaCl % 2.5 NaCl % 5 NaCl % 10 NaCl Day 3 % 0 NaCl % NaCl % 1.25 NaCl % 2.5 NaCl % 5 NaCl % 10 NaCl Absorbance (a.u.) Absorbance (a.u.) 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0, Wavelength (nm) (A3) 0,23 0,18 0,13 0, Wavelength (nm) Day 7 % 0 NaCl % NaCl % 1.25 NaCl % 2.5 NaCl % 5 NaCl % 10 NaCl Day 0 (before incubation) Day 1 Figure 1. UV-Vis spectra of S-AuNP0.5 (A1-A3) in different concentrations of NaCl and UV-Vis spectra of S-AuNPs0.5 (A4) in DMEM (w/o phenol red, w 10% FBS) (A4) Day 3 Day 5 79

82 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS The in vitro antibacterial activity was demonstrated by a broth macrodilution assay in which the bacterial cells were incubated at different concentrations of streptomycin antibiotic, gold nanoparticle solutions and a mixture of antibiotics and nanoparticles. The measured optical density values of different serial dilutions of streptomycin (0.1, 0.075, 0.05 and mg/ml), S-AuNP0.5 (0.26, 0.195, 0.13 and mg/ml), mixtures of streptomycin and S-AuNP0.5 (S1Au1: 0.05 mg/ml streptomycin and 0.13 mg/ml S-AuNP0.5), and negative control (only LB broth) were given in Figure 2. For the dilution ratios used here, in contrast to expectations, S-AuNP0.5 fostered the growth of bacteria to a large extent. However, when S-AuNP0.5 and streptomycin was combined, the OD value of groups containing the same amount of antibiotic was significantly lowered. Figure 2. Antibacterial activity assessment of different serial dilutions of streptomycin, mixtures of streptomycin and S-AuNP0.5 and negative control on B. Subtilis (Note: Clarity and turbidity were indicated with the symbols * and #, respectively.) Biomedical application of nanoparticles demands their biocompatibility that is the important criteria to be evaluated [10]. On this basis, the cytotoxicity effects of S-AuNPs0.5 were investigated with different tests. Different concentrations of S-AuNP0.5 group showed no cytotoxicity toward L-929 cells during 1 and 3 days of incubation period as seen in Figure 3A. These findings were supplied with the real-time cell monitoring results (Figure 3B). S-AuNP0,5 group was shown to have no any apoptotic-positive effect on L-929 cells (Figure 4 C1-F1). A slight necrosis was observed for control and Au1 groups only (Figure 4 C2-F2). Allh of these results show that the synthesized S-AuNPs have no cytotoxicity effects on L-929 cells. Similarly, in other works, it was shown that the green synthesized of gold nanoparticles with gelatin [5] and gallic acid [6] have no cytotoxicity on osteoblast cells and mouse embryonic fibroblast cells. 80

83 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS (C1) Hoechst (C2) PI Cell viability (%) Au1 Au1/2 Au1/4 Control (D1) (D2) (A) Time (Day) (E1) (E2) 2 Cell Index 1 Au1 Au1/2 Au1/4 Control (F1) (F2) (B) Time (Hour) Figure 3. In vitro cytotoxicity assays and morphology analysis: (A) Cell viability measurement results of S-AuNP0.5 group during 1 day and 3 days of incubation period with WST-1 assay and (B) real-time monitoring of L-929 cell index with xcelligence system for S-AuNP0.5 group. Representative fluorescence microscopy images of L-929 cell lines incubated for 1 day in complete cell culture media containing S-AuNP0.5 in different concentrations (C1, C2) Au1 (0.26 mg/ml), (D1, D2) Au1/2 (0.13 mg/ml), (E1, E2) Au1/4 (0.065 mg/ml) and (F1, F2) control group (complete cell culture medium only) (Note: Blue (Hoechst 33332) and red (Propidium iodide) fluorescence stains indicate apoptotic and necrotic cells, respectively.) Conclusion The findings showed that the synthesized S-AuNPs with a single step green process were stable in both saline and cell culture medium, had no cellular toxicity on L-929 fibroblast cell lines and acted as an effective strengthening agent to increase the antibacterial effect of a well-known antibiotic streptomycin. Consequently, sericin-capped AuNPs synthesized under UVC light can be used for various biomedical applications. Acknowledgments This project was financially supported by Kirikkale University Scientific Research Projects (BAP) Coordination Unit via project No: 2016/141. References [1.] Daniel, M.C., Astruc, D., Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chemical Review 104, , [2.] Jia, L., Guo, L., Zhu, J., Ma, Y., Stability and cytocompatibility of silk fibroin-capped gold nanoparticles. Materials Science and Engineering C 43, , [3.] Frens, G., Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions, Nature Physical Science 241 (105), 20-22, [4.] Teixeira, P.R., Santos, M.S.C., Silva, A.L.G., Báo, S.N., Azevedo, R.B., Sales, M.J.A., Paterno, L.G., Photochemically-assisted synthesis of non-toxic and biocompatiblegold nanoparticles. Colloids and Surfaces B: Biointerfaces 148, , [5.] Suarasan, S., Focsan, M., Soritau, O., Maniu, D., Astilean, S., One-pot, green synthesis of gold nanoparticles 81

84 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS by gelatin and investigation of their biological effects on Osteoblast cells. Colloids and Surfaces B: Biointerfaces 132, , [6.] Kim, D.Y., Kim, M., Shinde, S., Sung, J.S., Ghodake, G., Cytotoxicity and antibacterial assessment of gallic acid capped goldnanoparticles. Colloids and Surfaces B: Biointerfaces 149, , [7.] Yang, N., Li, W.H., Preparation of gold nanoparticles using chitosan oligosaccharide as a reducing and capping reagent and their in vitro cytotoxic effect on Human fibroblasts cells. Materials Letters 138, , [8.] Kumar, C.G., Poornachandra, Y., Mamidyala, S.K., Green synthesis of bacterial gold nanoparticles conjugated toresveratrol as delivery vehicles, Colloids and Surfaces B: Biointerfaces 123, , [9.] Leonard, K., Ahmmad, B., Okamura, H., Kurawaki, J., In situ green synthesis of biocompatible ginseng capped gold nanoparticles with remarkable stability. Colloids and Surfaces B: Biointerfaces 82, , [10.] Vinodhini, A., Govindaraju, K., Singaravelu, G., Mohamed Sadiq, A., Kumar, V.A., Cardioprotective potential of biobased gold nanoparticles, Colloids and Surfaces B: Biointerfaces 117, ,

85 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-065 Solid Lipid Nanoparticle Complexes with Plasmid or Oligonucleotide for mir-34a Delivery Mustafa Kotmakçı 1, Vildan Bozok Çetintaş 2, Zekeriya Düzgün 2, Uğur Karagöz 1, Ayşe Gülten Kantarcı 1 1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Bornova Izmir, Turkey 2 Department of Medical Biology, Faculty of Medicine, Ege University, Bornova Izmir, Turkey Abstract Recognition of micrornas (mirnas) as potential diagnostic tools started to gain increasing attention in the last years. Moreover, focusing on their functions in different disease conditions revealed them to be also good therapeutic targets. Their therapeutic use can be achieved either by using mirna-mimicks in cases where downregulation of certain mirnas lead to disease development, or by antagonizing their function in cases where their overexpression contribute to the disease development. mir-34a is a tumor suppressor mirna which suppresses cellular proliferation, survival and plasticity in cancer cells. Its expression is decreased in many cancer. However its use as a therapeutic agent is challenged by lack of effective, specific and safe delivery system. The aim of this study was to develop solid lipid nanoparticle (SLN) system and to modify their surface with monoclonal antibody fragments in order to obtain immuno SLNs (isln) and provide specific targeting of mir-34a to epidermal growth factor receptors (EGFR) on cancer cells. After preparation of islns, these were complexed with mir-34a oligonucleotide or mir-34a-encoding plasmid DNA (pmir-34a) at different ratios and characterized. Cell culture experiments were performed on Calu-1, HCC827, Calu-3 and H1299 non small cell lung cancer (NSCLC) sell lines which have distinct mir-34a and EGFR expression levels. It has been shown that, in the case of the SLNs functionalized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG- MAL), the zeta potential of the islns obtained after the monoclonal antibody F(ab ) fragment konjugation decreased to neutral or negative values preventing complexation with the nucleic acids. Therefore, it was assumed that addition of the F(ab ) fragment separately conjugated to DSPE-PEG-MAL to pre-formed csln:nucleic acid complexes will be of beneft. The mirna-containing complex had a particle size of about 66 nm and a zeta potential value of mv, while pdna-containing complex had a particle size of approx. 114 nm and a zeta potential value of +3.9 mv. In in vitro activity studies, complexes obtained without F(ab ) fragments were found more effective in increasing mir-34a levels in the cancer cells. Keywords gene delivery, active grug targeting, solid lipid nanoparticles, mir-34a, lung cancer Introduction The inadequacy and the extent of side effects of conventional cancer chemotherapy neccessitates the determination of new targets and development of new treatment modalities for cancer eradication. Gene therapy is one of these modalities. mirnas play important roles in the cells by regulating the expression of several proteins which contribute to the wellbeing and survival of the cell. With regard to their association with cancer development, there are two types of mirnas. The first type mirnas act like procarcinogenic regulators and their overexpression lead to increased cell motility, proliferation, invasiveness, immortalization and induced vascularization which are among the main hallmarks of cancer development (Hanahan & Weinberg, 2011). The second group of mirnas act like tumor suppressors and reverse the development of cancer by upregulation of proapoptotic molecules, 83

86 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS downregulation of antiapoptotic proteins and reducing angiogenesis. mir-34a is a tumor suppressor mirna adjusting cell proliferation, survival and plasticity in both in normal and cancer cells. Its expression is lost or decreased in many cancers including lung, liver, colon, prostate, breast, ovary, glioblasytoma, myeloid leukemia, bladder and gastric cancers (Slabáková vd., 2017). When applied naked, nucleic acids suffer from stability problems until reaching their target. Therefore, they are usually delivered by a suitable vector (either viral or non-viral vectors) for gene therapy. Viral vectors, although efficien, could cause damage to normal cells and stimulate immune response thus, non-viral vectors are being developed as substituents for vial vectors (Zhang vd., 2013). Loading nucleic acids to pharmaceutical formulations that provide specific targeting of the cancerous tissue minimizes their side effects, provides more stable formulations and enhances their therapeutic efficacy. Solid lipid nanoparticles (SLNs) represent an example of such delivery systems. SLNs are prepared from lipids that are solid at room temperature and are generally recognised as safe. They can be easily prepared by the microemulsion method (Kotmakçı vd., 2017). By incorporating cationic lipids to their structure these nanoparticles are used as DNA delivery systems for gene therapy (Akbaba vd., 2018; Karagöz vd., 2018). The aim of this study was to prepare SLN s with suitable properties for mirna and pdna delivery, and to investigate the possibility of targeting these systems by attaching antibody fragments to their surface. Materials and Methods SLNs were prepared by hot microemulsion method. Ternary phase diagrams were screened with various solid lipids and surfactant/ cosurfactant mixtures (S/CoS) in order to determine the microemulsion formation area. Subsequently, microemulsions were selected for SLN generation. Briefly, the microemlsion was prepared and ultrapure water, preheated to the same temperature was added (1:4 microemulsion-to-water v/v) and the mixture was left to cool to room temperture by stirring at 1500 rpm on a magnetic stirrer. Cationic SLNs (csln) were prepared by adding Esterquat 1, CTAB or DDAB to the composition of the microemulsions. To obtain maleimidefıunctionalized cslns (fcsln) for anibody fragment attachment, DSPE-PEG-MAL was added to the csln composition after dilution of the microemulsion with ultrapure water. Complexes of either fcsln or csln with pdna or mir-34a oligonucleotide were prepared by combining selected formulations with predetermined amount of the respective nucleic acid and incubation at 25 C for 30 min. Complex formation was observed by 0.8% agarose gel electrophoresis followed by ethidium bromide staining. Cetuximab, a monoclonal antibody targeting the EGF receptor of cancer cells was used to obtain isln s. For this purpose, the antibody was first subjected to pepsin-digestion in order to remove the Fc fragment and to obtain the F(ab )2 fragment. Protein concentration was measured by Bradford assay. Cleavage of the antibody was confirmed by SDS-PAGE with silver staining. 300 ng of the F(ab )2 fragment were further processed with varying concentrations of tris(2-carboxiethy)phosphine (TCEP) for 30 min at room temperature in the dark in order to reduce the interchain disulfite bonds and to obtain free thiol containing F(ab ) fragments. Free thiol content was assessed by Ellman s reagent. The obtained F(ab ) fragment was used for isln formation using two methods. 1) F(ab ) was combined with pre-formed fcsln:pmir-34a complexes and incubated overnight at 4 C (Figure 1A). 2) csln:pmir-34a was prepared first; separately F(ab ) was conjugated to DSPE-PEG-MAL forming DSPE-PEG- MAL-Fab micelles. Then, pre-formed complexes were combined with the DSPE-PEG-MAL-Fab micelles and either incubated at 50 C or sonicated for 2 min at room temperature (Figure 1B). mir-34a oligonucleotide containing complexes were prepared using the second method. 84

87 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Figure 1. Schematic representation of the two strategies followed to incorporate targeting antibody F(ab ) fragments onto the surface of developed fcsln or csln nanoparticles. A: conjygation of F(ab ) to pre-formed fcsln:pdna complex; B: post-insertion of the DSPE-PEG-MAL-Fab to pre-formed csln:pdna complex. Results A representative ternary phase diagram obtained with PATO5 as the solid lipid and S/CoS mixture of Kolliphor HS15/propyleneglicol (1:1, w/w) is presented in Figure 2A. In Figure 2B, results for complex formation of fcslns containing 10 % lipid with pdna are presented. Results for complexation of fcslns containing 5% lipid are presented in Figure 3C. Results of RT-PCR experiments after transfection of non-small cell lung cancer (NSCLC) cells with the developed formulations are presented in Figure 3. Figure 2. A: representative ternary phase diagrams obtained with the solid lipid Precirol ATO5 and S/CoS mixture Kolliphor HS15/propyleneglicol (1:1, w/w) at 66 C. B: fcsln:pdna complexes with SLN s contaning 10 % lipid. C: upper panel- fcsln:pdna complexes with fcsln s contaning 5 % lipid; middle panel- complexes with fcsln s contaning 5 % lipid and diluted 1:10 v/v; lower panel shows fcsln:pdna complexes with high pdna content and corresponding particle characteristics (D-particle size, PDI- polydispersity index, ZP- zeta potential) 85

88 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Figure 3. Relative mir-34a expression in three NSCLC cell lines (Calu1, H1299 and HCC827) after transfection with the developed formulations. Discussion After trying to obtain islns with fcslns and have observed that the addition of targeting ligand decreases the zeta potential, we decided to prepare isln s by pre-complexation of csln and pdna and subsequently incubating these complexes with pre-formed DSPE-PEG-MAL-Fab. Incubation at high temperature leads to increase in particle size, therefore we continued with the ultrasound-mediated incorporaiton for further stugies. Similar procedure was followed for the preparation of isln:mirna complexes. The pdna-containing complex consisted of cationic SLN:pDNA at a ratio of 1:3 (v/v). After adding DSPE-PEG-MAL-Fab by ultrasonication, an isln:pdna complex with a size of approx. 114 nm and a zeta potential value of +3.9 mv was obtained. The mirna-containing complex consisted of cationic SLN:miRNA at a ratio of 3: 2 (v/v), and after addition of 1 µl DSPE-PEG-MAL-Fab by 2 min ultrasonication, the isln:mirna complex with a size of about 66 nm and a zeta potential value of mv was obtained. Both complexes had suitable properties for transfection studies. The addition of Fab fragment to the surface of the complexes reduced their efficiency in increasing the intracellular mir-34a expression. However, the non-targeted particles were quite effective in increasing the mir-34a level in the cancer cells. Conclusions In this study, csln:pdna and csln:mirna complexes that effectively deliver mir-34a to cancer cells were developed. The efficacy of these systems should be tested in in vivo disease models in order to evaluate the specificity of the targeting. Acknowlwdgement We greatly appreciate the financial support provided by the Scientific and Technological Research Council of Turkey - TUBİTAK (grant no: 115S566). References [1.] Akbaba, H., Erel Akbaba, G., & Kantarcı, A. G Development and evaluation of antisense shrnaencoding plasmid loaded solid lipid nanoparticles against 5-α reductase activity, J. Drug Delivery Sci. Technol., 44, [2.] Hanahan, D., & Weinberg, R. A Hallmarks of cancer: the next generation., Cell, 144(5), [3.] Karagöz, U., Kotmakçı, M., Akbaba, H., Çetintaş, V. B., & Kantarcı, G Preparation and characterization of non-viral gene delivery systems with pegfp-c1 Plasmid DNA, Braz. J. Pharm. Sci., 54(1), e [4.] Kotmakçı, M., Akbaba, H., Erel, G., Ertan, G., & Kantarcı, G Improved Method for Solid Lipid Nanoparticle Preparation Based on Hot Microemulsions: Preparation, Characterization, Cytotoxicity, and Hemocompatibility Evaluation, AAPS PharmSciTech, 18(4), [5.] Slabáková, E., Culig, Z., Remšík, J., & Souček, K Alternative mechanisms of mir-34a regulation in cancer, Cell Death Disease, 8(10), e3100. [6.] Zhang, Y., Wang, Z., & Gemeinhart, R. a Progress in microrna delivery, J. Control. Release, 172(3),

89 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-067 The Design, Synthesis, NMR Analysis and Cytotoxic Activities of 6-(3-Aryl-2-Propenoyl)-2(3H)-Benzoxazolones Sinan Bilginer, Halise Inci Gül Department of Phameceutical Chemistry Faculty of Pharmacy, Atatürk University, Erzurum, Turkey Abstract BACKGROUND: According to the WHO, 13.1 million people will die from cancer by Drugs used for the treatment of cancer have some problems such as various side effects, gained resistance and low selectivity. So, there is a necessity for new drugs. Chalcones have a wide range of biological activities. Benzoxazolones are known with several bioactivities such as anticancer. There is a limited number of studies reporting the anticancer activities of benzoxazolone derived compounds. So, we aimed to synthesize new chalcone compounds of benzoxazolone (Compounds S1-10), to evaluate their cytotoxic/anticancer activities to find out new possible drug candidate. METHODS: First, 2(3H)-benzoxazolone was acylated by Friedel-Craft acylation reaction to use in synthesizing our chalcones. The compounds S1, S2,S3, S4, S6, S9 and S10 were synthesized by Claisen-Schmidt Condensation in basic condition. On the other hand, the compounds S5, S7 and S8 were synthesized by microwave irradiation (MW) method. The chemical structure of the compounds were confirmed by 1H-NMR spectra. Cytotoxicities of the compounds were evaluated against human oral squamous cell carcinoma (OSCC) cell line (HSC-2) and two human normal oral cells (HGF and HPLF) by MTT test. RESULTS: The ten compounds designed were successfully synthesized and their chemical structures were confirmed by 1H-NMR spectra. The compounds showed relatively high cytotoxicity against HSC-2 cell line over two cells (HGF, HPLF) (Table 1). The CC50 values of the compounds are in the range of µm towards HSC-2 cell line. The reference drugs doxorubicin (CC50 value 0,5 µm) and 5-fluorouracil (5-FU) (CC50 value µm) were also tested. The conclusion to be drawn is that chalcones synthesize had remerquable antineoplastic potencies. CONCLUSIONS: Newly synthesized benzooxazolone bearing chalcones, 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones S1-10, were reported here with their cytotoxicities against HSC-2 cell. The compound S5, 6-[3-(4-Bromophenyl)-2-propenoyl]-3H-benzooxazol-2-one, was found to be the most selective cytotoxic compound with the highest tumour selectivity (TS:13.3). On the other hand, the compound S4 was found most cytotoxic compound with the highest CC50 (4.0 µm) and PSE (159) values among the compounds studied. Therefore, S4 can be considered as a lead compound of the study for further modifications in designing new anticancer drug candidate compound. Keywords 2(3H)-benzoxazolone, chalcone, cytotoxicity, 1H-NMR spectra Background Cancer is the second cause of death in the world after the cardiovascular system diseases (Mahapatra et al., 2015). According to the WHO, 13.1 million people will die from cancer by 2030 (WHO. World Health Organization). Surgery, radiation and chemoteraphy are used for the treatment of cancer. The most important one is chemoteraphy 87

90 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS in terms of Medicinal Chemistry. Drugs used for the treatment of cancer have some problems such as various side effects, gained resistance and low selectivity. So, there is a necessity for the drugs whose toxicity are low and selectivity are high. Chalcones are 1,3-diaryl-2-propene-1-ones. They have a wide range of biological activities, antiinflamatuar, antimicrobial, antioxidant, cytotoxic, anticancer activities (Singh P, 2014). Benzoxazolones are known with several bioactivities such as anti-hiv (Deng et al., 2006), anticancer (Ivanova et al., 2007), analgesic (Unlu et al., 2003) antiinflamatuar, antimicrobial activities (Koksal et al., 2008). There is a limited number of studies reporting the anticancer activities of benzoxazolone derived compounds. So, we aimed to synthesize new chalcone compounds, 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolone (Compounds S1-10), to evaluate their cytotoxic/anticancer activities to find out new possible drug candidate. Methods First, 2(3H)-benzoxazolone was acylated by Friedel-Craft acylation reaction to use in synthesizing our chalcones. The compounds S1, S2,S3, S4, S6, S9 and S10 were synthesized by Claisen-Schmidt Condensation in basic condition. For this, aqueous solution of KOH ( 10%, 5ml) was added to the mixture of 6-acetyl-2(3H)-benzoxazolone and a suitable benzaldehyde in 1:1 mol ratios in ethanol (5ml). Reaction content was stirred at room temperature for 24 hours. Reactions were followed by TLC. On the other hand, the compounds S5, S7 and S8 were synthesized by microwave irradiation (MW) method. For the compounds at issue, to mixture of 6-acetyl-2(3H)-benzoxazolone and a suitable benzaldehyde in 1:1 mol ratios in ethanol (2ml); aqueous solution of KOH ( 10%, 2ml) was added. The mixture was irradiated in microwave oven for several minutes (25-60 W, C). When the reactions finished, the content of the reaction flask was poured on ice-water and neutralized by HCl (%37). The solid precipitaced was filtered and washed with cold water. The crude compounds were purified by crystalization. The chemical structure of the compounds were confirmed by 1 H-NMR spectra. Synthetic pathway was summarized in Scheme 1. Cytotoxicities of the compounds were evaluated against human oral squamous cell carcinoma (OSCC) cell line (HSC-2) and two human normal oral cells (HGF and HPLF) by MTT test. Scheme 1: Synthetic pathway of ten compounds of 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolone derivaties. O O N H O Ar O H EtOH KOH Ar: Ar O O N H O H 3 C S1: S4: F 3 C S7: H 3 C N S2: H 3 C S5: Br S8: H 2 CO H 3 C S3: H 3 CO S6: H 3 C CH S9: O S10: S Results The ten compounds designed were successfully synthesized and their chemical structures were confirmed by 1 H-NMR spectra. The compounds showed relatively high cytotoxicity against HSC-2 cell line over two human 88

91 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS normal oral cells (HGF, HPLF) (Table 1). The CC 50 values of the compounds are in the range of µm towards HSC-2 cell line. The reference drugs doxorubicin (CC 50 value 0,5 µm) and 5-fluorouracil (5-FU) (CC 50 value µm) were also tested. The conclusion to be drawn is that chalcones synthesize had remerquable antineoplastic potencies. Table 1. Cytotoxicity results of the compounds S1-10 CC 50 (μm) OSCC Human normal oral cells HSC-2 SD HGF SD HPLF SD mean TS PSE Compound (A) (B) (B/A) (B/A 2 ) 100 S1 22,1 1,2 71,0 9,5 83,3 14,0 77,2 3,5 16 S2 10,1 0,4 87,7 18,5 105,3 3,1 96,5 9,5 94 S3 14,5 2,9 88,3 17,9 84,0 0,0 86,2 5,9 41 S4 4,0 0,6 24,7 0,6 26,3 0,5 25,5 6,4 159 S5 14,7 4,5 351,3 84,3 40,0 0,0 195,7 13,3 90 S6 10,1 0,5 53,0 5,3 76,0 7,0 64,5 6,4 64 S7 30,2 3,2 76,3 9,3 70,0 2,0 73,2 2,4 8 S8 11,9 0,2 98,0 50,2 32,7 1,5 65,3 5,5 46 S9 63,7 3,1 54,7 23,3 81,0 3,0 67,8 1,1 2 S10 32,0 1,7 24,5 0,5 127,0 0,0 75,8 2,4 7 5-FU > >1000 0,0 >1000 > DXR 0,5 0,1 0,7 0,0 >10 0,0 >5.3 >10.4 >2030 Conclusions Newly synthesized benzooxazolone bearing chalcones, 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones S1-10, were reported here with their cytotoxicities against HSC-2 cell. The compound S5, 6-[3-(4-Bromophenyl)-2-propenoyl]-3H-benzooxazol-2-one, was found to be the most selective cytotoxic compound with the highest tumour selectivity (TS:13.3). On the other hand, the compound S4 was found most cytotoxic compound with the highest CC 50 (4.0 µm) and PSE (159) values among the compounds studied. Therefore, S4 can be considered as a lead compound of the study for further modifications in designing new anticancer drug candidate compound. References [1.] Deng T, Sharps J L, Brownlee G G. Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication. (2006) Journal of general virology; 87(11): [2.] Ivanova Y, Momekov G, Petrov O, Karaivanova M, Kalcheva V. Cytotoxic Mannich bases of 6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones. (2007) European Journal of Medicinal Chemistry; 42(11): [3.] Katsori A M, Hadjipavlou-Litina D. Chalcones in cancer: understanding their role in terms of QSAR. (2009) Current medicinal chemistry; 16(9): [4.] Koksal M, Kelekci N G, Mercanoglu G O, Erdogan H. Synthesis and evaluation of analgesic, anti-inflammatory and antioxidant activities of new 6-acyl-3-alkyl-5-methyl-2 (3H)-benzoxazolones. (2008) Arzneimittelforschung; 58(08):

92 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-069 Cytotoxic effect of boric acid-glycerin on SH-SY5Y cell line Ayça Taş 1, Neşe Kekli kçiȯğlu Çakmak 2, Yavuz Si li ğ 3 1 Faculty of Health Sciences, Department of Nutrition and Dietetics, Cumhuriyet University, Sivas, Turkey 2 Engineering Faculty, Chemical Engineering, Cumhuriyet University,Sivas, Turkey 3 Faculty of Medicine, Department of Biochemistry, Cumhuriyet University, Sivas, Turkey Abstract Objective: Naturally occurring compounds are a potentially source of new anticancer agents. As an anticancer agent boron and its derivatives are promising. Boric acid has a number of different properties to make it a promising pharmaceutical agent for cancer treatment. The aim of this study is to prepare boric acid in glycerin, an organic and biocompatible solvent, and to determine the anticancer activity on neuroblastoma SH-SY5Y cell lines. Materials&- Methods: Boric acid used in this study is commercially purchased. The boric acid was dissolved in glycerin by probe sonicator under certain conditions and characterization analyses were carried out. The synthesized boric acid-glycerin was applied to the neuroblastoma SH-SY5Y cell line and the cytotoxic effect of these drugs were determined by using MTT method. SH-SY5Y cells were treated with different concentrations of boric acid-glycerin ( μg/ml) for 24, 48 and 72 hours. Results: In this study, the characterization analyzes of the boric acid-glycerin system were performed. The effects of boric acid-glycerin on the SH-SY5Y cell line were compared to the control group and IC 50 values were found for 24, 48 and 72 hours. Conclusion: In this study, it was shown that the effect of boric acid-glycerin system on SH-SY5Y cell was inhibitory to growth in cancer cells when compared with control group. However, there is no study on boric acid-glycerin so far. This study will provide new information in that regard. Keywords Neuroblastoma, SH-SY5Y, Boric acid, Glycerin, MTT 1. Introduction Neuroblastoma (NB),1 the most common malignant sympathetic nervous system tumor of childhood, arises from the neural crest [1,2]. Despite the array of chemotherapeutic agents presently available and current strategies employing intensive myeloablative chemotherapy with autologous bone marrow transplantation, most patients with high risk NB die of their disease [3,4]. To improve this situation, the basis for treatment failures as well as the mechanisms that underlie successful responses to chemotherapy in NB cells must be understood. The molecular response of the tumor cells to cytotoxic agents has become the focus of these efforts because it is clear that these pathways can lead to tumor cell death, whereas their absence or failure leads to resistant disease [5]. In the current report, several studies examined the effects of pharmacological concentrations of boric asid (BA) on cell morphology and molecular markers of proliferation, senescence, metastasis and motility [6]. Boron compounds are very effective against leukemia cells, breast cancer cells, lung cancer cells, prostate cancer cells and ovarian cancer cells. As such, interest stems from the tremendous importance of BA in the synthesis of biologically active compounds and the use of BA itself as pharmaceutical agent [7]. The incorporation of boron in some boron compounds imparts antitumor properties to different cancer cell lines. There are experiments in which boron compounds work as a proteasome inhibitor in cancer cells [8]. The mechanism of its lethal effects did not involve apoptosis, but it is suspected to be through histone deacetylase inhibition [8-10]. Glycerine (glycerol, 1,2,3-propanetriol), which is a typical polyol, is conveniently used for the preparation of a 90

93 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS condensed product with H 3 BO 3 and it has been used for many years as a solvent for boric acid. Furthermore, glycerol is an organic solvent of low molecular weight, thus, homogeneous and easy blending is possible with an organic compound and it is one of the most extensively studied hydrogen-bonded systems. The extremely wide range of uses for glycerol is due in large measure not to a single property, but to its unique combination of properties. Nature made glycerol the most widely distributed of the polyhydric alcohols, as combined in fats and other lipids essential to life processes. Derivatives dependent on its chemical structure have now been extended to virtually every field of research and technology, from explosives to emulsifiers. In order to be able to use the boric acid-glycerol solution as a medicine in medical applications, its characteristics must be well known [11]. In this study, boric acid and glycerin molecules were activated for a new cancer treatment strategy and anticancer activity was assessed in SH-SY5Y neuroblastoma cells. 2. Materials and Methods 2.1 Materials Boric acid (BA) and glycerine ( 99.5%) were obtained commercially from Sigma Aldrich and were used as purchased. No dispersant/surfactant was used in the suspension. 2.2 Dispersion of boric acid The commercially purchased boric acid was dispersed in 50 ml glycerine (99.5 wt.%) with different mass fractions with the help of a sonicator (Sonics & amp; materials INC, USA). The reaction was then sonicated for 30 min. The fluid of boric acid in glycerine were stable for longer than three months. 2.3 Characterization The morphologies of boric acid was measured by scanning electron microscope (TESCAN MIRA3 XMU). UV-Vis spectrophotometer (UV-1280, Shimadzu, Japan) was utilized to record the spectra of prepared samples range from 200 to 800 nm. Finally, in order to homogeneously disperse the boric acid in glycerine, a probe sonicator (Sonics & amp; materials INC, USA) at 750W power was used. 2.4 Cell Culture Cell lines including SH-SY5Y cells were maintained in DMEM medium, containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 mg/l). Cells were grown in at 37 C, 5% CO2 and 95 % air in a humidified incubator. For each cell line, 70-80% confluent cell culture flask was trypsinized and cells were seeded in 96 well plates. 2.5 Cytotoxic effect of BA-glycerin on SH-SY5Y cells The in vitro cytotoxicity of the BA-glycerin against SH-SY5Y cell lines was performed with the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the Skehan s method [12]. 1 μl of test substance at concentrations ranging between µg/mL were added into each well containing the cells. After mixing with a mechanical plate mixer for 15min, the absorbance of plates were recorded at 570 nm on a microplate reader (Bio-Tek, USA). All drug doses were parallel tested in triplicate and were performed at least 3 times; control samples were run with 1% sterilized water. 3. Results and Discussion 3.1. Structures and properties of boric acid-glycerin solution The surface morphology of boric acid characterized by SEM analysis as shown in Fig. 1. As can be seen from the SEM micrographs, these particles have a spherical shape. From SEM images, the average particle size was evaluated to be around 250 µm for particles. 91

94 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Figure 1. SEM micrographs of boric acid. The UV-Vis spectra of boric acid-glycerin solution are shown in Fig. 2. As we can see a clearspectra without any crowding of signals in the spectrum. This shows that best solvent for the boric acid is glycerin. Figure 2. UV-Vis absorption spectra of 0.5% boric acid in glycerin 3.2 Cytotoxicity activities of BA-glycerin on SH-SY5Y cells In order to determine whether there were cytotoxic effects associated with the presence of intracellular BA-glycerin, SH-SY5Y cells were exposed to a range of concentrations of these drugs rate was examined by MTT (Figure 1). Figure 1 shows changes in cell inhibition for 24, 48 and 72 hours versus increasing concentrations of SH-SY5Y cell lines. x-axis shows cell types and varying time points, while the y-axis shows the inhibition rates of cancer cells relative to the control. Compared to the control group, BA-glycerin treated human SH-SY5Y neuroblastoma cells showed significantly decreased tumor survival rate after 24h, 48h and 72h of incubation. Compared to the control group, the BA-glycerin group had significantly reduced survival rate after 24h, 48h and 72 h of incubation. Cell survival rates in all groups after 24h, 48h and 72 h of incubation were significantly decreased than those in the control group. With elongated treatment time, the survival rate of tumor cells was significantly reduced. BA-glycerin on SH-SY5Y cells was the most active for 72 h of incubation. In addition, BA-glycerin IC 50 values for 24, 48 and 72 hours were 27,10 μg/ml, 26, 32μg/ml and 18,47 μg/ml respectively (Table 1). 92

95 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Table 1 Comparison of IC 50 values between boric acid-glycerin on SH-SY5Y after 24 h, 48 h and 72 h of incubation Time Boric acid-glycerin IC 50 (μg/ml) 24h 27,10 48h 26,32 72h 18,47 SH-SY5Y R e la tiv e C e ll V ia b ility (% ) h 48h 72h Control 24h 48h 72h Control Boric Asid-G lycerine Concentration (µg/m l) 4. Conclusions Figure 3. Cytotoxity activities of of BA-glycerin on SH-SY5Y cell line In summary, the present study shows that BA-glycerin has a cytotoxic effect on SH-SY5Y cells compared with control group. We conclude that high concentrations of BA-glycerin affect -SY5Y cells in 24,48 and 72h times. Not enough data is available about BA toxicity, so BA use as cancer treatment can be possible if new toxicity studies are performed. Our study is essential for the use of new treatment options, those deploying BA-glycerin, for the treatment of neuroblastoma. Further studies on different neuroblastoma cell lines are necessary. 5. Acknowledgements This study was carried out at Cumhuriyet University s Advanced Technology Application and Research Center (CUTAM). REFERENCES [1.] Saxen, L., And Saxen, E. (1960) Cancer Gene Ther. 13, [2.] Machlin, G. A. (1982) in Neuroblastoma Clinical and Biological Manifestations (Pochedly, C., ed) pp , Elsevier Biomedical, New York [3.] Frappaz, D., Michon, J., Coze, C., Berger, C., Plouvier, E., Lasset, C., Bernard, J. L., Stephan, J. L., Bouffet, E., Buclon, M., Combaret, V., Fourquet, A., Philip, T., and Zucker, J. M. (2000) J. Clin. Oncol. 18, [4.] Schmidt, M. L., Lukens, J. N., Seeger, R. C., Brodeur, G. M., Shimada, H., Gerbing, R. B., Stram, D. O., Perez, C., Haase, G. M., and Matthay, K. K. (2000) J. Clin. Oncol. 18, [5.] Kaufmann, S. H., and Earnshaw, W. C. (2000) Exp. Cell Res. 256, [6.] B, WT and E, CD. Cellular changes in boric acid-treated DU-145 prostate cancer cells. British Journal of Cancer (2006) 94, [7.] Chapin RE, Ku WW (1994) The productive toxicity of Boric Acid. Environ Health Perspect 102:87 91 [8.] Bone R, Ashok B (1987) Serine protease mechanism: structure of an inhibitory complex of alfa-lytic protease and a tightly bound peptide boronic acid. Biochemistry 26:

96 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS [9.] R. Scorei, R. Ciubar, C. M. Ciofrangeanu, V. Mitran, A. Cimpean and D. Iordachescu,"Comparative Effects of Boric Acid and Calcium Fructoborate on Breast Cancer Cells,"Biological Trace Element Research, vol. 122, no. 3, pp , [10.] F. Di Renzo, G. Cappelletti, M. L. Broccia, E. Giavini and E. Menegola, "Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity," Toxicology and Applied Pharmacology, vol. 220, p , [11.] SS. Claude, Lipid Fett, 1999, 101, [12.] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cytotoxicity assay for anticancer-drug screening. (1990) J Natl Cancer Inst.; 82(13):

97 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-071 Social and Ethical Aspects of Biotechnology Merve Memisoglu Department of Pharmacy Management and Social Pharmacy, Biruni University, Istanbul, Turkey Abstract The aim of the study is to present and interpret implications of advanced technology within pharma and medical fields in terms of social and ethical aspects. For this purpose, related literature screening was needed to obtain the extent of exposure to technological and scientific innovation in Turkey. Keywords Biotechnology; Nanobiotechnology; Biopharmaceuticals; Ethics; Social Aspects Main Text -"Technological progress is like an axe in the hands of a pathological criminal."- (A. Einstein) Neil Postman, the author of Technopoly: The Surrender of Culture to Technology a review of technology and culture in our modern world underlines the fact that technology is both the friend and enemy to humans. Postman underlines that technology, more specifically, medical and pharmaceutical advancements makes life easier, cleaner, longer and goes even further by stating that technology improves health by increasing life expectancy. On the other hand, Postman emphasizes that companies in this process make money regardless of moral responsibility [1]. The advancements in technology, and the nature of technological change is a key component of social transformation which also impacts biotechnology and the biotech society. Evolution of biotechnology, from the ancient Sumerians in Mesopotamia to Pasteur s time, includes use of micro-organisms for fermentation and vaccine development. Today, biotechnology with experiments in gene cloning and recombinant DNA technology has become one of the mainstream technologies. In general, biotechnology is a technology by which manipulated living organisms are utilized to generate useful products such as biopharmaceuticals. Biotechnology products which are protein- peptide- or gene-based drugs, they involve using biological materials to create safer and more effective versions of conventionally produced pharmaceuticals. For example, recombinant human insulin is at least as effective as insulin of animal origin, is safer than animal-source insulin. Biotechnology in the pharmaceutical industry also show cost-effectiveness in comparison to conventional alternatives. Biotechnology is one of the world's fastest growing technologies. The worldwide use of biotechnological products has exceeded 20% and is expected to rise further. In Turkey, according to IEIS Report (2017), there are 208 originator biotechnological products under 93 brands and 46 biosimilar products under 17 brands in pharmaceutical industry [2]. Despite the enormous developments in the field of biotechnology, the social and ethical issues should also be discussed, in order to balance benefits and risks as the Biotechnology industry hides unsurfaced social and ethical issues like; morality, safety, environmental concerns. This study is planned to examine these dimensions of biotechnology. 95

98 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS - Safety first! - Biopharmaceuticals have a complex three-dimensional structure, which makes production of an exact copy difficult. Large scale production process of biopharmaceuticals has some variations from batch to batch and can induce an unwanted immune response. Many factors which are patient, disease or product-related, may influence the immunogenicity of therapeutic proteins. These are the priority issues that need to be examined regarding the safety and efficacy of biosimilar and biological reference products. The same criticism is prevalent even in new discoveries like the developments in nanobiotechnology which has introduced several problems such as toxicity and environmental risks of nanomaterials at the outset, alongside its unique and gound-breaking properties. Nanomaterials, which can be found in diesel exhaust, smoke, and some viruses can be actually activate immune system pathway that cause serious toxicity problems. Nanoparticles consisting of heavy metals may cross cell membranes and the blood-brain barrier. Furthermore, inhaled nanomaterials can enter the capillaries, in the circulatory system. Nanoscale materials may also accumulate in parts of the body and produce adverse effects. Those nanoparticles may also be released into the environment. Long-term effects on human health and environment are ambiguous. For this reason, researchers who are exposed to these nanoparticles can be at risk. This necessitates the examination of toxicity and ecotoxicity of nanoparticles [3-4]. - Without trust, there can no true growth! - Some critics have no confidence in global pharmaceutical industry known as Big Pharma, and therefore resist innovation in the discipline that includes biotechnology, nanotechnology. Human attitudes towards technology can be positive or negative. Attitudes related to technologies are different, such as; technophobia, technophilia, technoprogressivism, and bioconservatism. Nowadays, people can also become addicted and reliant on technology and use it as their main form for creating social relations. This newly emerged discipline is developing so fast that there is a regulatory lag in the process. The regulators are slow in keeping up with the pace of technological advancement. This lack of or slow pace of - regulatory environment brings another ethical and legal dimension, like heterogenicity in the patent law creating potential risks in terms of legal and ethical aspects. The products created, often for global consumption also face national boundaries as regulations and guidelines of biotechnology products especially biosimilars are different from country to country [5]. There is also the cost of technological and scientific evolution. Nanotechnology and biotechnology-based products are very expensive when they first enter the market, and which implicitly creates national and international health inequalities. What are the costs of biotechnology and nanotechnology-based drugs? Who has access to them? Can emerging and developing world have equal access to these advanced technologies? Can they be subsidized? Nano-divide is the potential for nanotechnologies to increase the gap between the rich and the poor countries and reinforce global inequalities. These issues and concerns related to social and global justice should be discussed in-depth. Technology also raises privacy concerns. Search into gene-related areas like genetic engineering creates concerns as to who gets to see the findings and whether the results should be disclosed to third parties? Yet again, this issue creates another problem ethics in the development of technology. For example, it may become possible to know that a 5-year-old is going to develop serious heart disease later in life, but does a prospective employer have the right to know that? How will this knowledge affect the individual s ability to obtain a job, or insurance? [6]. 96

99 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS At present, another ethical issue is related to risk assessment, risk management, and risk communication in clinical trials, like clinical trials on animals and humans. Here do stakeholders allow enough information to be disclosed to the subjects? Usually, patients with terminal illnesses are willing to try new developmental medicines as they see a potential cure - even if side-effects are not known for biotech products. Autonomy is the right of the patient to determine his/her own health issues. Informed consent is another patient right which means receive information about his/her treatments. It s necessary for patient s freedom, privacy and safety according to bioethical principle in health care. It is also essential for building trust between healthcare professionals and patients. Informed consent is the only model in which communication is directed at individuals, otherwise most people receive news primarily from the media. Therefore, the Impact of mass and social media on society cannot be ignored. In US, for example, direct to consumer advertising is free for prescription drugs and medical devices which involve nanobiotechnology products. Within communication strategies, effective risk communication should also be used by pharmaceutical manufacturers. However, when they plan communication strategies, technology producers shouldn't ignore the role of individual and social values in making decisions about risky technologies. In practice, it means value-based approach to risk management [7]. Communication strategies should cover all stakeholders; Manufacturers, researchers, and governments should educate and inform the public about the benefits and the risks of advanced technology products. Communication between healthcare professionals and patients should also be analyzed. Both sides should not only seek more information about nanotechnology and biotechnology but must find more effective ways of communication. Now we can ask; is the pharmacist and physician ready for the new technologies? First, healthcare professionals biotechnology and nanotechnology knowledge should be investigated. They could start with biosimilars where the key issues are interchangeability, immunogenicity risk management, and differences between generic drugs and biosimilars. Most people are likely to remain uninformed and disregardful and unaware about new technologies. At this point, it is essential that health professionals lead and guide the society. For this, it is of utmost importance for their adaptation to these emerging technologies. In our further research, we are planning to examine the responses of the health professionals about those questions. References [1.] Rosenberg, R. The social impact of computers, London, Elsevier, [2.] Turkish Pharmaceutical Market 2017, İlaç Endüstrisi İşverenleri Sendikası IEIS Rapor, [3.] Oberdörster, G.; Oberdörster, E.; Oberdörster, J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environmental health perspectives 2005, 113(7), [4.] Hoet, P. H.; Brüske-Hohlfeld, I.; Salata, O. V. Nanoparticles known and unknown health risks. Journal of nanobiotechnology 2004, 2(1), 12. [5.] Burk, D. L.; Lemley, M. A. Biotechnology's Uncertainty Principle, [6.] Silverman, E. The 5 most pressing ethical issues in biotech medicine. Biotechnology healthcare 2004, 1(6), 41. [7.] Priest, S. H. Risk communication for nanobiotechnology: To whom, about what, and why? The Journal of Law, Medicine & Ethics 2009, 37(4),

100 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-074 Immobilization of Sericin Capped Silver Nanoparticles to Amine-functionalized Methacrylic Acid-g-poly(ethylene terephthalate) Fiber and It s Antibacterial Applications Zehra Gün Gök 1, Kübra Günay 2, Metin Arslan 3, Mustafa Yiğitoğlu 4, Ibrahim Vargel 5 1 Institute of Science, Department of Bioengineering, Hacettepe University, Ankara, Turkey 2 Graduate School of Natural and Applied Sciences, Kırıkkale University, Yahsihan, Kirikkale, Turkey 3 Department of Chemistry and Chemical Processing Technologies, Kırıkkale Vocational High School, Kırıkkale University, Yahsihan, Kirikkale, Turkey 4 Department of Bioengineering, Kirikkale University, Kirikkale, Turkey 5 Department of Plastic and Reconstructive Surgery, Hacettepe University Hospitals, Ankara, Turkey Abstract Polyethylene terephthalate (PET) is one of the most widely and commonly used polymeric materials. However, because of the its lack of antimicrobial properties, PET fabrics don t prevent the growth of microorganisms. For this reason, the studies made to add antimicrobial properties to PET fibers have gained industrial importance. In the present work, a kind of amine-type fibers were synthesized by reacting hexamethylenediamine (HMDA) with methacrylic acid-g-poly(ethylene terephthalate) (PET-g-MAA) fiber for the adsorption of silk sericin capped silver nanoparticles (S-AgNPs) from an aqueous solution to produce an antimicrobial PET fibers. Firstly, PET fibers were grafted MAA by using benzoyl peroxide (Bz2O2) as initiator. Then, HMDA was then covalently attached to this MAA grafted PET fibers. The amine modified PET fibers was then coated with S-AgNPs solution to obtain an antimicrobial surface. The S-AgNPs coated modified PET fibers were characterized by Fourier Transformed Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). The antimicrobial activities of the obtained PET fibers were investigated on Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) bacteria by using agar diffusion test. It was found that the S-AgNPs coated modified PET fibers exhibited antimicrobial activities toward both gram-positive and gram negative bacteria. The obtained polymeric PET fibers containing nano silver at different concentration can be used as an antimicrobial surface for many applications such as wound dressing. Keywords PET; Grafting copolymerization; Silver nanoparticles; Silk sericin; Antimicrobial application 1. Introduction Immobilization of nanoparticles onto the surface of polymers provides amazing opportunities for the making of smart composite materials [1]. PET is a type of well-known polymeric materials and its application field has been branched widely in clothing, cosmetics and biomaterials because of its good mechanical properties, high stability in the presence of body fluids and high radiation resistance [2]. PET has no antimicrobial effect despite these good properties, particularly for the use in medical field (for example, antibacterial PET fabric), adding of antibacterial and antifungal agents such as AgNPs in the PET structure is desirable [3]. In this work, PET as functionalized with graft copolymerization and by coating of PET fibers with S-AgNPs, the antibacterial property was gained to PET fibers. 98

101 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 2. Material and Methods 2.1. Grafting of MAA to PET fibers Firstly, the PET fibers were swollen in 1,2-dichloroethane for 2 hours at 90 ºC and the excess solvent was removed by filter paper. The swelled PET fibers were placed in polymerization tubes which contained 0.3 M MAA in 18 ml water. Then Bz 2 O 2 dissolved in 2 ml acetone was added to the polymerization tube as a radical initiator and the tubes were incubated at 85 ºC for two hours under reflux [4]. After 2 hours, the fibers were taken and washed with methanol for 8 hours to remove homopolymers. The obtained copolymers were dried at 37 ºC and by calculating weight increasing, the grafting yield of MAA was calculated Coating of PET-g-MAA Before coating of PET-g-MAA fibers with the synthesized S-AgNPs (in our previous work), HMDA was covalently attached to PET-g-MAA fibers by placing of the grafted PET fibers in a 30 ml of 50% HMDA (dissolved in ethanol) at 30 C for 90 minutes [4]. The modified PET fibers with HMDA (PET-g-MAA-HMDA) were put into a buffer solution at ph 3 for 90 min and then, the activated fibers were placed into 20 ml S-AgNPs (5 mm and 10 mm) solution and incubated for 24 h at 30 C. After 24 hours, the fibers were removed from S-AgNPs solution and the absorbance spectra of the remained S-AgNPs solution was measured with a UV-Vis spectrophotometer (BioTek, USA) between nm. The amounts of nanoparticles adsorbed on PET-g-MAA-HMDA fibers were calculated by the following equation: q = (C o -C)V/m where q is the amount of nanoparticles concentration adsorbed by one gram of adsorbent (mm/g), C o is initial concentration of S-AgNPs solution (mm/l), C is equilibrium concentration of solution of S-AgNPs (mm/l), V is volume (L) of S-AgNPs solution and m is the amount of adsorbent (g) Characterization of modified PET fibers The chemical structure of the modified and un-modified PET fibers were analyzed by FTIR (Bruker Vertex 70V) spectrum analysis. The morphologies of the modified and un-modified PET fibers were analyzed SEM ( JEOL Model JSM 5600) Antibacterial studies The antibacterial properties of modified and original PET fibers were investigated with disk diffusion test method. Bacterial cultures of E. coli and S. aureus with McFarland standard were incoluated on Mueller-Hinton agar surface. Antibiotic disk, unmodified PET fibers and modified PET fibers (shaped into disc shape and sterilized with UV) were put in the bacterial culture inoculated petri dishes and the petri dishes were incubated at 37 C incubator for h. Zone diameters were measured using a ruler. 3. Result and Discussion In this study, PET fibers were modified to adsorb AgNPs in three steps. In the first step, the PET fibers were grafted with MAA by using free radical polymerization technique and PET was functionalized by the addition of COOH groups. In the second step, HMDA was covalently attached to the copolymer via COOH groups of MAA. In the third step, NH 2 groups of HMDA were used to sorption of S-AgNPs by the PET fibers, and then the polymer metal hybrid materials were developed. The S-AgNPs sorption process of modified PET fibers was shown in Fig. 1. This kind of adsorption strategy shows some advantages. For example, the amount of immobilized AgNPs can be controlled by grafting amount of carboxylic groups. 99

102 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS PET PET CH 2 O CH 2 O H 3C C C OH NH 2 (CH 2 ) 6 NH 2 H 3C C C NH(CH 2) 6NH 2 PET-g-MAA PET-g-MAA-HMDA PET PET H 3C CH 2 C O C NH(CH 2) 6NH 2 H + H 3C CH 2 C O C NH(CH 2) 6NH 3 + PET-g-MAA-HMDA PET-g-MAA-HMDA PET PET CH 2 O H 3C C C + NH(CH 2) 6NH 3 PET-g-MAA-HMDA CH 2 O S - -AgNPs H 3C C C PET-g-MAA-HMDA NH(CH 2) 6NH 3 + S - -AgNPs Fig. 1. Sorption mechanism of S-AgNPs by the modified PET fibers The FT-IR spectra is known to be convenient to guarantee strong proof for the grafting [4]. Thus, FT-IR spectra of original and modified PET fibers have been analyzed and were displayed in Fig. 2. The FT-IR spectra of original PET fibers had peaks owing to C=O (at 1712 cm -1 ), C=C and aliphatic C H (at 1411 and 1578 cm -1 ) of PET fibers and in the spectra of the PET-g-MAA fibers, the peaks at 1712 cm -1 were expanded due to C=O groups of MAA [5]. In the spectra of the HMDA attached PET fibers, a new peak was found at 1627 cm-1 due to C=O groups of amide and this new peak observation represented that HMDA was covalently attached to PET-g-MAA fibers as reported in previous works [4, 6, 7]. Fig. 2. FTIR spectrum of the original and modified PET fibers The morphologies of the modified and unmodified PET fibers were analyzed by SEM (Fig. 3). As shown in the SEM images of original PET fibers, the surface was smooth and homogeneous. SEM photograph of PET-g-MAA fibers, however, rough and heterogeneous surface due to grafting of MAA. In the SEM images of S-AgNPs coated PET fibers, presence of AgNPs was clearly seen like in other works [3]. 100

103 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Fig. 3. SEM images of (A) original, (B) MAA grafted PET fibers (100 % grafting yield), (C) PET/AgNPs obtained by coating with 5 mm S-AgNPs and (D) PET/AgNPs obtained by coating with 10 mm S-AgNPs. The antibacterial activities of the modified and original PET fibers were given in Table 1 and the agar images of the study were given in Fig. 4. The original PET fibers did not have antibacterial activity, however, when the PET fibers were coated with S-AgNPs, the constructed materials had antibacterial activity on both gram negative and gram positive bacteria. Table 1. The inhibition zones formed around the antibiotic disk, original and mofidfied PET fibers Polymers E. coli zone diameters (mm) S.aureus zone diameters (mm) Gentamicin (10 µg) Unmodified PET fibers 0 0 S-AgNPs-PET-g-MAA-HMDA fibers (5 mm) S-AgNPs-PET-g-MAA-HMDA fibers (10 mm) Fig. 4. Antimicrobial activities of the modified and unmodified PET fibers. Gentamicin (A, E), Unmodified PET fiber (B, F), PET-AgNPs (containing 1,4 mm S-AgNPs) produced by coating with 5 mm S-AgNPs (C, G) and PET-AgNPs (containing 2,1 mm S-AgNPs) produced by coating with 10 mm S-AgNPs (D, H). 101

104 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Conclusion The findings showed that the synthesized S-AgNPs coated PET fibers have antibacterial activity and they can be used as a amnimicrobial surface im many applications in which PET is used.. References [1.] Reznickova, A., Novotna, Z., Kolska, Z., Svorcik, V. Immobilization of silver nanoparticles on polyethylene terephthalate. Nanoscale Research Letters, 9:305, [2.] Ping, X., Wang, M., Ge, X. Surface modification of poly(ethyleneterephthalate) (PET) film by gamma-ray induced grafting of poly(acrylicacid) and its application in antibacterial hybrid film. Radiational Physical Chemistry, 80: , [3.] Fraga,l V.H., Cellet, T.S.P., Pereira, G.M., Fragal, E.H., Costa, M.A., Nakamura, C.V., Asefa, T., Rubira, A.F., Silva, R. Covalently-layers of PVA and PAA and in situ formed Ag nanoparticles as versatile antimicrobial surfaces. International Journal of Biological Macromolecules, 91: , [4.] Arslan, M., Günay, K. Synthesis of amine-functionalized methacrylic acid-g-poly(ethylene terephthalate) fiber and its Congo red removal ability. Polymer Bulletin, [5.] Abdolahifard, M., HajirBahrami, S., Malek, R.M.A. Surface Modification of PET Fabric by Graft Copolymerization with Acrylic Acid and Its Antibacterial Properties. ISRN Organic Chemistry, /2011/265415, 1-8, [6.] Liu, C., Bai, R., Quan San, L. Selective removal of copper and lead ions by diethylenetriaminefunctionalized adsorbent: behaviors and mechanisms. Water Research, 42: , [7.] Hu, X.P., Li, W.Y., Wang, Y.Z. Synthesis and characterization of a novel nitrogen-containing flame retardant. Journal of Applied Polymer Science, 94(4): ,

105 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-077 Electrochemical Biosensor for Monitoring of Interaction Between Anticancer Drug Dacarbazine and Nucleic Acids Ece Eksin, Arzum Erdem Ege University, Faculty of Pharmacy, Analytical Chemistry Department, 35100, Izmir, Turkey Abstract Dacarbazine (DCB) is a chemotherapy drug that is used for the treatment of Hodgkin lymphoma, melanoma and soft tissue sarcoma. In our study, the biomolecular interaction between DCB and DNA was investigated electrochemically using single-use pencil graphite electrodes (PGEs). The oxidation signals of DCB and guanine were measured voltammetrically before and after interaction process. The interaction of DCB and nucleic acids was also examined. Furthermore, EIS technique was utilized for detection of the interaction between DCB and DNA. Keywords Dacarbazine, nucleic acid, electrochemical detection, graphite electrode, drug-dna interaction Introduction Exploration of interaction between drugs and DNA is one of the most important subjects for drug discovery, potential chemotherapeutic applications and the association between genetics and drug response. Anticancer drugs interact with nucleic acids in many different ways including intercalation between base pairs, non-covalent minor and major groove binding, covalent binding/cross-linking, DNA cleavage and nucleoside-analog incorporation [1]. Dacarbazine [5-(3, 3-dimethy-1-triazenyl) imidazole-4-carboxamide; DCB) is one of the most important anticancer therapeutic agents used in the clinical treatment of malignant melanoma [2], Hodgkin s lymphoma [3], soft-tissue sarcomas [3,4], and childhood solid tumors [5,6]. Electrochemical DNA biosensors play an important role in many areas such as clinical diagnosis, environmental monitoring, forensic applications, and pharmaceutical investigation, because they provide rapid, simple, high sensitivity and low-cost detection of specific nucleic acid sequences [7-9]. The pencil graphite electrodes have a special compatibility to microfabrication technology to detect drugs, and drug DNA interactions [10,11]. In our study, the interaction between DCB and DNA was investigated at the surface of disposable pencil graphite electrodes (PGEs). Before and after interaction process, the oxidation signals of DCB and guanine were measured by using differential pulse voltammetry (DPV) and the changes at the oxidation signals were evaluated. The electrochemical detection of surface-confined interaction between DCB and DNA was explored by using electrochemical impedance spectroscopy (EIS) techniques. Materials and Methods Procedure: The experimental procedure mainly included four steps; (i) DCB immobilization and its electrochemical detection at the surface of PGEs, (ii) the immobilization of DNA onto the surface of electrode, (iii) electrochemical detection of interaction between DCB and DNA. Voltammetric measurements: DPV measurements were performed in buffer to measure the signals of drug and guanine before and after interaction process. Impedimetric measurements: EIS measurements were performed with frequency range from 100 mhz to 1 khz in 103

106 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS the presence of redox probe solution containing 0.1 M KCl. Results and Discussion The interaction process was evaluated in the presence of ssdna or dsdna (Fig. 1). The oxidation signals of DCB and guanine were measured before and after interaction process at the same voltammetric scale using DPV, then the changes at the signals were evaluated in terms of interaction process. Figure 1. (A) DPVs representing the oxidation signal of DCB and guanine before and after interaction process. The guanine oxidation signal of ssdna before (a) and after (a ) interaction. DCB oxidation signal before (b) and after (b ) interaction. (B) DPVs representing the guanine oxidation signal of dsdna before (a) and after (a ) interaction. DCB oxidation signal before (b) and after (b ) interaction (n=3). The surface confined interaction between 12 µm DCB and 20 µg/ml ss/dsdna onto the PGE surface were also examined using EIS technique (Fig. 2). The changes at charge transfer resistance (R ct ) value was evaluated before and after interaction process. Figure 2. Nyquist diagrams representing the average R ct values recorded before and after interaction of 12 µm DCB with 20 µg/ml ssdna or dsdna during 10 min. (a) PGE, (b) 12 µm DCB immobilized PGE, (c) 20 µg/ ml ssdna immobilized PGE, (d) 20 µg/ml dsdna immobilized PGE, (e) after interaction of DCB with ssd- NA, (f) after interaction of DCB with dsdna onto PGE. Inset is equivalent circuit model used to fit the impedance data, the parameters. Conclusions The single-use pencil graphite electrodes developed herein for monitoring biorecognition processes have presented many advantages like cost effectiveness, rapid and sensitive detection with good repeatability of drug-dna interaction in comparison to other electrodes, such as carbon paste electrode, glassy carbon electrode and gold electrode [12,13]. The electrochemical monitoring of the surface-confined interaction between DNA and DNA-targeted mo- 104

107 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS lecules would be valuable in the rational design of the molecule-specific electrochemical biosensor, and in the further development of novel drugs for the chemotherapy. Acknowledgements A.E would like to express her gratitude to the Turkish Academy of Sciences (TUBA) as a Principal member of TUBA for its partial support. References [1.] G.M. Bleckburn, M.N. Gait, Nucleic Acids in Chemistry and Biology, IRL Press, New York, 1990, [2.] J.K. Luce, W.G. Thurman, B.L. Isaacs, R.W. Talley, CancerChemother. Rep. Part 1 (1970) [3.] M. Pfreundschuh, W. Schoppe, R. Fuchs, K. Pfluger, M. Loeffler, V. Diehl, CancerTreat. Rep. 71 (1987) [4.] G. Beretta, P. Fraschini, L. Tedeschi, Oncology 37 (1980) [5.] D.M. Thomas, B. O Sullivan, A. Gronchi, Expert Rev. Anticancer Therapy 9 (2009) [6.] M. Slavik, Cancer Treat. Rep. 60 (1976) [7.] F. Jelen, A. Erdem,E. Palecek, Bioelectrochem. 2002, 55, [8.] A. Erdem, Talanta, 2007, 71, [9.] A. Erdem, E. Eksin, E. Kesici, Biosensors and Nanotechnology: Applications in Health Care Diagnostics, Chapter 15: Biosensors for Detection of Anticancer Drug DNA Interactions, John Wiley & Sons, Inc. (2018) [10.] E. Kanat, E. Eksin, B. Karacicek, Y. Eraç, A. Erdem, Electroanalysis, 2018, 30, [11.] E. Eksin, E. Zor, A. Erdem, H. Bingol, Biosens. Bioelectron. 2017, 92, [12.] M. Song, R.Y. Zhang, X.M. Wang, Mater. Lett. 60 (2006) [13.] Q. Shen, X. Wang, D. Fu. Applied Surface Science 255 (2008)

108 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-082 A Comparative Docking Study of Psoriasis Disease Proteins with Psoriasis Drugs versus Plant Extracts Used in Phytotherapy Büşra Çetin 1, Vildan Enisoğlu Atalay 2, Tuba Sevimoğlu 2 1 Department of Pharmacology, Marmara University, İstanbul, Turkey 2 Department of Bioengineering, Üsküdar University, İstanbul, Turkey Abstract Psoriasis is a chronic, immune-related and a multisystemic disease that affects both skin and joints. It is characterized with abnormal keratinocyte proliferation in dermis and epidermis. Though there is still no cure for this complex disease, lesions caused by psora can be inhibited with topical and systemic treatments, phototherapies and herbals. In this comparative study, drug active ingredients and the effective molecules of herbal plants used in phytotherapy are docked to selected disease proteins to understand which molecule type (drug active ingredient or plant effective molecule) is more efficient as per binding energy and interaction. AutoDock 4.2 program was used to calculate the binding energies and the interactions are defined through AutoDock Vina program between protein and drug agent/effective plant molecules. The highest affinity of the drug agents and proteins for binding were found between the anthralin drug molecule and the MMP12 protein (K i =-11.1 Kjmol -1 ). On the other hand, for plant effective molecule interaction with proteins, the highest affinity was found between cassia tora and the protein (MMP12) with K i =-11.5 Kjmol -1. Keywords Psoriasis proteins, phytotherapy, effective molecules, affinity, drug agents, Autodock 4.2 Introduction Psoriasis is a chronic and immune-related disease that affects skin and joints. It has significant negative impact on a patients s quality of life. Statically obtained by traditional gene-related analysis methods, 10 chromosomal area (PSORS1 PSOR10) and multiple genes including MMP12 and CDK1 gene groups are related to this disease. The disease is characterized with abnormal keratinocyte proliferation in dermis and epidermis which has more significant roles for dendritic and T cells than other cells. Although there is no real solution for Psoriasis, lesions caused by psora can be inhibited with topical and systemic treatments, phototherapies and herbals. Systemic drugs used in the treatment of psoriasis include methotrexate, cyclosporin, acitretin, and retinoids. Materials and Methods Each molecule (drug active ingredients and effective molecules of plants) is examined with Spartan 14 and optimized structures are used for docking to selected Psoriasis protein structures via AutoDock 4.2 and AutoDock Vina programs. Results The aim of this study was to compare the drug molecules and plant active ingredients in terms of binding energy to Psoriasis disease proteins. The highest affinity for binding were found between the anthralin drug molecule and the MMP12 protein (K i =-11.1 Kjmol -1 ). The highest affinity binding value (K i =-11.8 Kjmol -1 ) between plant activators 106

109 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS and proteins was found between astragalus membranaceus and MMP12 protein. The second highest affinity binding was for cassia tora with K i =-11.5 Kjmol -1, and as a prominent result this plant has showed high affinity with MMP12. Discussion and Conclusion This study has been an important key to compare between synthetic drug and natural plant molecules in the Psoriasis disease. The effective result is observed that the herbal plant molecule is satisfied according to the inhibition value (K i ). Cassia tora plant molecule (with the K i =-11.5 Kjmol -1 value) is more effective than the anthralin drug molecule (K i =-11.1 Kjmol -1 ). These results point out that the herbal plant molecules can be important candidate molecules for treatment of Psoriasis. This study can also be extended for other diseases. Potential phytotherapy treatments might be a better treatment option for such diseases as drugs have many side effects. 107

110 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-084 Technetium-99m Radiolabeled Glucagon-like Peptide Analog Exendin-4 for Cancer Diagnosis Emre Özgenç, Meliha Ekinc, Derya Ilem Özdemir, Evren Gündoğdu, Makbule Aşıkoğlu Department of Radiopharmacy, Faculty of Pharmacy, Ege University, Izmir, Turkey Abstract Cancer is one of the leading causes of mortality worldwide. Usually, the diagnosis of cancer at an early stage is important to facilitate proper treatment and survival (1). Nuclear medicine has been successfully and widely used in the diagnosis, staging, therapy, and monitoring of cancers by allowing scientists and physicians to see what is happening in the body at a cellular level (2). Radiopharmaceuticals are radioactive drugs, which consist of a pharmaceutical compound and a radionuclide part (3). In accordance with the aim, it is important to choose the target specific pharmaceutical compound for radiolabeling. Overexpression of glucagon-like peptide 1 (GLP-1) receptors in many cancer may be useful for diagnosis of the disease (4). Exendin-4 is a peptide agonist of the GLP receptor that promotes insulin secretion (5). In the present study, exendin-4 was radiolabeled with Technetium-99m ( 99m Tc). To optimize the labeling conditions, reducing agent concentration, ph, specific activity and incubation time effect on radiolabeling yield and stability was performed. Binding affinity to cancer cells were evaluated by in vitro cell culture incorporation studies. In accordance with the performed study, exendin-4 was labeled with 99m Tc with high radiolabeling yield (>95%). The resulting complex was quite stable and labeling efficiency was maintained for up to 6 h. The maximum radiolabeling yield was obtained with 50 μg stannous chloride and 37 MBq TcO4 - including formulations at ph 6.6. According to the cell culture studies, further studies with 99m Tc-exendin-4 should be done for cancer diagnosis. Keywords Exendin-4; Tc-99m; Radiolabeled Studies; Cancer Diagnosis; Cell Culture Studies Introduction Exendin-4 is a peptide of 39 amino acids that has been isolated from the venom of the lizard Heloderma suspectum (Gila monster) (6). It is currently approved by the FDA (Food and Drug Administration) for clinical use as an antidiabetic drug for type 2 diabetes treatment, and its safety and tolerance in humans have been demonstrated. Several Exendin-4 derivatives have been radiolabeled and evaluated for nuclear imaging and a small number of clinical studies were performed (7, 8). Radiopharmaceuticals are consist of pharmaceutical and radionuclide parts. Choice of a suitable radionuclide for radiolabeling studies can be ascertained by considering factors such as the radiation type and dose, cost and availability. Recently, 99m Tc is the most popular radionuclide for labeling studies (9). The use of 99m Tc may improve the quality of images and radiation safety for patients and the staff by many procedural advantages related to the physical properties of this isotope (10). Because, 99m Tc radionuclide has some ideal properties such as; 140 KeV monoenergetic gamma rays, 6 h half-life and versatile chemistry to make complexes (11). Although experimental animal models are substantial for cancer diagnosis, also in vitro cell culture studies were performed to evaluate the cell binding affinities. (12-15). In this method, newly improved radiopharmaceutical is incubated with the different types of cells and, the radioactivity in cells are measured after administration of labeled 108

111 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS formulations to the cells at different time intervals. The aim of this study was to prepare a new radiopharmaceutical to use in cancer diagnosis and evaluate the uptake ratios to the different of cell lines. For this purpose, exendin-4 was radiolabeled with 99m Tc. Labeling efficiency and stability of the compound were investigated. Binding affinity of the newly developed radiopharmaceutical to cancer cells were evaluated by cell culture incorporation studies. As part of the cell culture studies, incorporation of 99m Tc-exendin-4 to cancer and normal cell lines was evaluated in cancer cell line (PCS, Calu) and healthy cell line (CRL, HaCat). Materials and Methods The optimum conditions were determined by changing the radiolabeling conditions of Exendin-4 with 99m Tc. In order to determine the effect of reducing agent concentraiton, incubation time, radioactivity dose and ph, radiolabeling studies were performed. Radiochemical purity (RP) was determined with radio thin layer chromatography (RTLC) analysis. Cell uptake of 99m Tc-Exendin-4 to cancer cell line (PCS, Calu) and healthy cell line (CRL, HaCat) were investigated. For this purpose, 3.7 MBq 99m Tc-Exendin-4 was incubated with cells for 60 and 120 min at 37 C. After incubation period, the samples were taken from cell medium to eppendorf tubes. Also, adherent cells were trypsinised and taken to another eppendorf tubes. The amount of radioactivity in the cell medium and cells were counted in the gamma counter. The cellular uptake was calculated as the percentage of the activity counted in the cells relative to the total activity counted. The percentage radioactivity of cells was calculated from the following equation. % Radioactivity of cells = (Radioactivity of cells / Total radioactivity) x 100 Results A new, simple, easy and efficient direct method for labeling of Exendin-4 with 99m Tc was developed by our research group. Labeling efficiency of the 99m Tc-Exendin-4 was assessed with RTLC studies. The radiochemical purity of 99m Tc-Exendin-4 was >95 %, acquired via RTLC. In this study, the incorporation percentage of 99m Tc-Exendin-4 to PCS, Calu, CRL and HaCat cell lines was evaluated. According to the cell culture studies, the highest incorporation percentage was observed with Calu cell line at 120 min. Discussion Optimal radiolabeling conditions to 99m Tc-Exendin-4 were determined and radiopharmaceutical with high radiochemical purity was prepared. Cell culture studies were performed with radiopharmaceutical that were radiolabeled under optimum conditions. In these studies, Calu, which is an adenocarcinoma cell line, had a higher incidence than the health cells lines. Thus, it was shown that 99m Tc-Exendin-4 radiopharmaceutical could be used in the diagnosis of cancer cells in the future. Conclusions In this work, we have shown that Exendin-4 can be labeled with 99m Tc with high radiochemical purities (>95 %) with a simple method assessing with RTLC. The resulting complex was found quite stable and labeling efficiency was maintained for up to 6 h. Maximum radiochemical purity was obtained with 50 μg stannous chloride and 37 MBq TcO4 - including formulations at ph 6.6. According to the cell culture studies results, 99m Tc-Exendin-4 proved a useful tool for assessing the in vitro affinity of the cancer and normal cell binding. 99m Tc-Exendin-4 uptake to cancer cell line was found higher than healthy cell line. Consequently, according to the study results show that 99m Tc-Exendin-4 can be a candidate for cancer diagnosis. Also, further studies with 99m Tc-Exendin-4 should be done for cancer diagnosis. 109

112 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS References [1.] Fass L. Imaging and cancer: A review. Molecular Oncology vol: 2 (2) pp: [2.] Abedi S., Mardanshahi A., Shahhosseini R., Hosseinimehr S. Nuclear medicine for imaging of epithelial ovarian cancer. Future Oncology vol: 12 (9) pp: [3.] Kydd J Jadia R Velpurisiva P Gad A Paliwal S. et. al. Targeting Strategies for the Combination Treatment of Cancer Using Drug Delivery Systems. Pharmaceutics vol: 9 (4) pp: 46 [4.] Tomaszuk M., Sowa-Staszczak A., Lenda-Tracz W., Glowa B., Pach D. et. al. Dosimetry of exendin-4 based radiotracer for glucagonlike peptide-1 receptor imaging: an initial report. J. Phys.: Conf. Ser. 2011; 317: 01. [5.] Ding X., Saxena N., Lin S., Gupta NA., Anania FA. Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis inob/ob mice. Hepatolog vol: 43 (1) pp: [6.] Eng J, Kleinman WA, Singh L, Singh G, Raufman JP (1992) Isolation and characterization of exendin-4, an exendin-3 analogue from Heloderma suspectum venom. J Biol Chem. 267(11): [7.] Baggio LL, Drucker DJ (2007) Biology of incretins: GLP-1 and GIP. Gastroenterology. 132(6): [8.] Selvaraju RK, Velikyan I, Asplund V, Johansson L, Wu Z, Todorov I, Shively J, Kandeel F, Eriksson B, Korsgren O, Eriksson O (2014) Pre-clinical evaluation of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 for imaging of insulinoma. Nucl Med Biol. 41(6): [9.] Häfeli U (2002) Radioactive microspheres for medical applications. In: Cuyper MD, Bulte JWM (eds) Physics and chemistry basis of biotechnology. Springer, Netherlands. 7: [10.] Sowa-Staszczak A, Pach D, Mikołajczak R, Mäcke H, Jabrocka-Hybel A, Stefańska A, Tomaszuk M, Janota B, Gilis-Januszewska A, Małecki M, Kamiński G, Kowalska A, Kulig J, Matyja A, Osuch C, Hubalewska-Dydejczyk A (2013) Glucagon-like peptide-1 receptor imaging with [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 for the detection of insulinoma. Eur J Nucl Med Mol Imaging. 40(4): [11.] Ting G, Chang CH, Wang HE (2009) Cancer nanotargeted radiopharmaceuticals for tumor imaging and therapy. Anticancer Res. 29: [12.] Gundogdu E, Ilem-Ozdemir D, Ekinci M, Ozgenc E, Asikoglu M (2015) Radiolabeling efficiency and cell incorporation of chitosan nanoparticles. J Drug Deliv Sci Technol. 29:84-89 [13.] Ekinci M, Ilem-Ozdemir D, Gundogdu E, Asikoglu M (2015) Methotrexate loaded chitosan nanoparticles: Preparation, radiolabeling and in vitro evaluation for breast cancer diagnosis. J Drug Deliv Sci Technol. 30: [14.] Ozgenc E, Ekinci M, Ilem-Ozdemir D, Gundogdu E, Asikoglu M (2016) Radiolabeling and in vitro evaluation of 99mTc-methotrexate on breast cancer cell line. J Radioanal Nucl Chem. 307(1): [15.] Ilem-Ozdemir D, Karavana SY, Ay-Senyigit Z, Caliskan C, Ekinci M, Asikoglu M, Baloglu E (2016) Radiolabeling and cell incorporation studies of gemcitabine HCl microspheres on bladder cancer and papilloma cell line. J Radioanal Nucl Chem. 310(2):

113 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-092 Understanding the Mechanisms that Regulate Cutaneous Resident Memory T Cells on Autoimmune Skin Diseases and Preclinical Studies in Terms of Vitiligo Hasan Akbaba 1, Shannon K Bromley 2 1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, 35100, Izmir, Turkey 2 Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA Abstract The immune system must defend the host at the most likely sites of pathogen encounter. Recent data demonstrate that a population of memory T cells (TRM) resides long-term within peripheral tissues and provides the first line of host defense from pathogens that cause a local infection. However, the local persistence of memory T cells that provide robust response may be harmful in some cases. TRM are thought to play a key role in local, recurring inflammatory skin diseases such as psoriasis and vitiligo. Defining mechanisms that control TRM precursor localization and persistence within skin will be important for optimizing vaccines to provide local protection as well as therapies to prevent unwanted inflammation. In this study, we investigated the mechanisms that direct the interstitial migration, persistence and response of cutaneous TRM. While the local cytokine microenvironment is known to direct TRM formation, factors that inhibit TRM differentiation/persistence are unknown. We also investigated the promotion or inhibition of TRM formation depending on the local cytokine microenvironment. Lastly, although TRM have been identified at sites of cutaneous inflammatory disease, targeting TRM is an untested therapeutic approach. We used a mouse model of vitiligo with preclinical application to investigate that autoreactive TRM maintain depigmentation, and that depletion of autoreactive TRM will prevent and/or ameliorate disease. Keywords Resident memory T cell, vitiligo, CD49a, cytokine microenvironment, autoimmune skin disease Introduction Before the investigation of TRM, it was thought that T cells in the tissues were considered to remain in the circulation until recruitment of the inflammation and there is only a few T cells remain in the peripheral tissues without inflammation. T cells that are taken into the tissues during inflammation were thought to have undergone apoptosis after infection. However, studies have found that a group of T cells settled in the tissues and remained there for a long time (Hill, 2015). Those cells named as tissue resident memory T cells (TRM). TRM cells are transcriptionally, phenotypically and functionally distinct from TCM and TEM T cells which recirculate between blood, secondary lymphoid organs, and non-lymphoid tissues. They undergo a distinct proliferation that discriminates them from circulating T cells (Carbone et al., 2013). Their main cell surface markers are CD69, CD103, and CD49a. CD69 marker has a key role in distinguishing T cells in tissues from circulating T cells and it is an activation marker (Topham and Reilly, 2018). Integrin, alpha E (ITGAE) also known as CD103 is an integrin protein that binds E-cadherin, which is highly expressed on epithelial cells (Cheuk et al., 2017). Another marker that can be used to separate the TRM is CD49a. CD49a, or integrin α1, is an integrin protein and take part within VLA1 complex. VLA-1 is a collagen-binding integrin, and it has a direct role in attaching to epithelial cells (Ray et al., 2004). However, expression levels of markers can differ between T cells in different tissues. CD8 + CD49a + TRM cells produce perforin and IFN-gamma, which are key cytokines in clearing virus infections. They also differentiate form TEM by their expression of inflammatory chemokine receptors and a lack of lymphoid homing molecules, specifically CD62L and CCR7 (Mackay and Kallies, 2017). 111

114 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Skin is one of the main barriers to the external environment, which is constantly exposed to colonizing microbiota, invasive pathogens, and allergens. These encounters provide differentiation and colonization of TRM. While such local immune responses contribute to immunization, abnormal activation may cause diseases in some cases. The formation of various T-cell mediated diseases, such as psoriasis and vitiligo, is an immunopathological symptom of T cells located in the tissue rather than in the circulating cells (Cheuk et al., 2017). Here, we determined the anatomical localization, transcriptional profiles, and functional properties of TRM cell subsets in the skin with cutaneous HSV mouse model in respect to CD49a, CD69, and CD103 markers. We also used the vitiligo mouse model for the new treatment approaches depending on CD49a. Materials and Methods We used wild type (WT) and CD49a knockout (KO) mouse models of infection (herpes simplex virus) to study the migration, differentiation, persistence, and response of memory T cells within the skin. In addition, we investigated the in vitro CD8 + T cell proliferation profiles in the presence of cytokines in order to investigate TRM formation and inhibition in terms of local microenvironment. Furthermore, we investigated the formation of Melanocyte-Specific TRM in a mouse model of vitiligo to guide the autoimmunity for future studies. Results and Discussion Lymphocyte recirculation is a dynamic and tightly regulated process necessary for delivery of effective immune responses. While many memory T cells circulate through blood, a subset of memory T cells persists long-term within peripheral tissues. We demonstrated the increase in CD8 + HSV-1 gb / Kb + T cells in the skin, spleen and lymph nodes with cutaneous HSV mouse model buy using CD103, CD69 and CD49a as a marker of TRM formation (Figure 1a). We demonstrated the in vitro TRM formation and amount by performing various cytokine induction (Figure 1b). For the detection of genes required for in vitro TRM formation, naïve T cells were isolated from the spleen and followed by cytokine induction. It has been shown that CD49a has an important role in the localization of TRM cells in tissue as well as CD103. Upregulation of Itga1 (integrinα1), Itgae (integrinαe), IL4Ra, TGFBRII genes was determined quantitatively using qpcr technique (Data not shown). Figure 1. a. HSV-1 specific T cell percentage in the skin, spleen and DLN, b. Cytokine induction for in vitro TRM formation, c. Table of vitiligo score and the percentages TRM markers in vitiligo mouse model. d.e. CD49a expression on Epidermal and DLN CD44 hi CD69 + CD103 + PMEL CD8 + T Cells We used a mouse model of vitiligo with preclinical application to investigate that autoreactive TRM maintain depig- 112

115 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS mentation, and that the role of CD49a. We scored mice that showed symptoms of vitiligo and determined the percentages of CD69 and CD103 as TRM markers. We found a correlation between vitiligo scores and these percentages (Figure1c). We also examined the amount of CD49a in the epidermis and draining lymph node (DLN) in the mouse model of vitiligo, results have shown that CD49a CD8 + T cells were localized in the skin at a high rate (Figure1d,e). As a result, it can be said that vitiligo is characterized by the accumulation of a CD49a + T cells which recognize and attack pigment cells. Therefore, suppression of the CD49a + T cells in the skin could be a promising treatment approach for vitiligo. Conclusions Up-regulating pathways that promote the persistence of T cells in peripheral tissues is a critical focus of current vaccine design; it may be equally important to create approaches that reverse these pathways to prevent unwanted TRM accumulation that causes vitiligo. CD49a requirement for vitiligo development, and can vitiligo be reversed by CD49a neutralization is further investigated. Acknowledgments This work was supported by National Institutes of Health under grant code: [NIH-R01-AI A1]. References [1.] Carbone, F.R., Mackay, L.K., Heath, W.R., Gebhardt, T., Distinct resident and recirculating memory T cell subsets in non-lymphoid tissues. Curr. Opin. Immunol. 25, [2.] Cheuk, S., Schlums, H., Gallais Sérézal, I., Martini, E., Chiang, S.C., Marquardt, N., Gibbs, A., Detlofsson, E., Introini, A., Forkel, M., Höög, C., Tjernlund, A., Michaëlsson, J., Folkersen, L., Mjösberg, J., Blomqvist, L., Ehrström, M., Ståhle, M., Bryceson, Y.T., Eidsmo, L., CD49a Expression Defines Tissue-Resident CD8+T Cells Poised for Cytotoxic Function in Human Skin. Immunity 46, [3.] Hill, C., Resident cells in human disease. Sci. Transl. Med. 73, [4.] Mackay, L.K., Kallies, A., Transcriptional Regulation of Tissue-Resident Lymphocytes. Trends Immunol. 38, [5.] Ray, S.J., Franki, S.N., Pierce, R.H., Dimitrova, S., Koteliansky, V., Sprague, A.G., Doherty, P.C., De Fougerolles, A.R., Topham, D.J., The Collagen Binding α1β1 Integrin VLA-1 Regulates CD8 T Cell-Mediated Immune Protection against Heterologous Influenza Infection. Immunity 20, [6.] Topham, D.J., Reilly, E.C., Tissue-resident memory CD8+T cells: From phenotype to function. Front. Immunol

116 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-094 An Alternative Treatment Approach to Anti-androgenic Drugs with High Side Effects; Development and In Vitro Evaluation of Solid Lipid Nanoparticles for Gene Silencing Gülşah Erel Akbaba 1,2, Ayşe Gülten Kantarcı 2 1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Izmir Katip Celebi University, 35620, Izmir, Turkey 2 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, 35100, Izmir, Turkey Abstract In general, androgens are hormones that regulate the development of sex characteristics. In diseases such as benign prostatic hyperplasia, prostate cancer or androgenic alopecia, local androgenic activity increases undesirably. Anti-androgenic drugs work by blocking the effects of androgens, such as testosterone. They do this by binding to proteins called androgen receptors. However, this binding brings along undesirable side effects. In this study, we aimed to develop a novel system for nucleic acid delivery to reduce local androgenic activity. For this purpose, 5α-reductase which is responsible for the conversion of testosterone to five-times more potent dihydrotesterone was chosen as a target protein for gene silencing. In order to achieve anti-androgenic activity, shrna-encoding plasmid against 5-α reductase (p5α-red) was loaded to a newly developed delivery vector. To this end, solid lipid nanoparticles (SLNs) were produced by the melt-emulsification process by using Compritol ATO 888 as internal oil phase; Tween 80 as a surfactant; ethanol as co-surfactant and ultra-pure water as the continuous water phase. The formulated nanoparticles were electrostatically bound to p5α-red to form SLN:p5α-Red vectors. The SL- N:p5α-Red vectors have particle sizes of nm, and zeta potential values of 12.9 mv, able to protect the p5α-red from degradation and showed no cytotoxicity in the concentration range of µg/well on DU-145 cell line. Furthermore, in vitro gene silencing experiment demonstrated that SLN:p5α-Red vector effectively reduced 5α-Red enzyme level in 48h. Considering the role of 5-α reductase in androgenic diseases, the developed SL- N:p5α-Red vector system may be promising for future therapies. Keywords Antisense gene therapy, 5-α reductase, solid lipid nanoparticle, shrna, androgenic disease Introduction RNA interference is a specific post-transcriptional gene silencing mechanism within a cell via the transfection of micrornas (mirna), exogenous small interfering RNAs (sirna) or small hairpin RNAs (shrna) [1]and although current treatments have efficacy in treating primary prostate cancer, they are associated with a decreased quality of life and are ineffective in treating the metastatic disease. The identification of oncogenes associated with the formation, proliferation, and metastasis of prostate cancer has presented promising targets for RNA interference (RNAi. Transfection of shrna can be done by shrna-encoding expression plasmids. These vectors have several advantages. They ensure sustained silencing of the targeted genes via prolonged expression of shrnas. On the contrary, shrna-encoding expression plasmids are larger than shrna because of including elements for plasmid formation like promoters and antibiotic resistance sequences. To obtain high transfection efficiencies a convenient plasmid delivery system is required. Therefore, we aimed to develop a novel solid lipid nanoparticle system for local delivery of shrna-encoding plasmid against 5-α reductase (SLN:p5α-Red) in order to silence the expression of 5α-reductase enzyme transcripts. 114

117 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 5α-reductase were chosen as a target protein in this study for gene silencing due to its important role in steroid metabolism [2]. The conversion of testosterone to dihydrotesterone (DHT) is catalyzed by 5α-reductase and 5α-reductase inhibitor drugs such as finasteride, dutasteride, and turosteride are mainly used in benign prostatic hyperplasia (BPH) and androgenic alopecia (AA). Although, 5-α reductase inhibitors are primarily used to treat BPH and AA, these inhibitors are under investigation to be used against prostate cancer, as well [2 3]. Their efficacy against prostate cancer has been evaluated in recent years and there are some 5α-reductase inhibitors that have ongoing phase II and phase III trials for the markets. Beside their promising uses in different disease, gynecomastia, erectile dysfunction, fatigue, hypoglycemia, and depression, are only a few of the possible side-effects of conventional 5α-reductase inhibitors. These effects reduce their systemic usage especially for AA indication [4]. Due to all these side effects, topical administration of nucleic acid-based therapeutics against 5α-reductase activity may be an alternative and effective delivery route without systemic side effects. In this study, we aimed to develop SLN:pDNA vector system for delivery of shrna-encoding plasmid against 5-α reductase in order to silence the expression of 5α-reductase enzyme transcripts. DU-145 cell line was chosen for its well-known 5-α reductase activity and to test the silencing effects of developed vector system [5]. Permeation ability of vector system was also evaluated for its possible use in topical therapy in the future for some diseases such as AA. Obtained data in this study may help to develop drugs or vectors with less side effects against BPH, prostate cancer or AA for further studies. Materials and Methods SLNs as gene delivery vectors were prepared by the melt-emulsification process. Firstly, oil in water (o/w) microemulsion system was formed with Compritol ATO 888, DDAB, Tween 80, ethanol and water. Following microemulsion formation, obtained o/w microemulsion was dispersed in cold ultra-pure distilled water and SLNs were formed when hot microemulsion droplets were met with cold water [6]. Gel retardation assay was performed to evaluate the complex formation ability and protection capability of SLNs against serum nucleases. Particle size, polydispersity index (PDI), and zeta potential of SLN formulations and SLN: p5α-red vectors were measured on Zetasizer Nano ZS instrument (Malvern, UK) [7]. Dialysis membrane method was used for the in vitro penetration study of SLN:p5α-Red (3:1, v/v) vectors. The viability of cells was determined by colorimetric XTT cell proliferation assay prior to the transfection studies. Gene silencing efficiency was evaluated by western blot analysis after optimizing the transfection protocol of the obtained SLN: p5α-red vector system. Results and Discussion Agarose gel electrophoresis image in Figure 1a shows the capability of the increasing amount of SLNs to protect p5α-red against the nucleases. SLN:p5α-Red vectors were prepared and incubated with and without DNase I (0.4 IU DNase I/1 µg pdna) enzyme to determine the optimal complex formation ratio and we observed that SL- N:p5α-Red vector with the ratio of 3:1 (v/v) was able to protect p5α-red (Figure 1a-Lane 5). Once the optimal SLN and SLN:p5α-Red vectors were determined, we proceed to the physiochemical characterization. DLS measurements were performed in order to investigate the particle size, PDI and zeta potential of SLNs and SLN:p5α-Red (3:1, v/v) vectors. Particle size was measured as nm for SLNs and nm for SL- N:p5α-Red (3:1, v/v) vectors (Table 1). Furthermore, SLN formulations have the positive electrical charge in order to be used for pdna complexation and by addition of p5α-red, the surface charge values changed into negative direction due to the negative charge of plasmid, as expected. Table 1. Physicochemical properties of the SLNs and SLN:p5α-Red vectors (3:1, v/v) Particle diameter (nm ±SD) PDI (±SD) Zeta Potantial (mv±sd) SLNs ± ± ±1.11 SLNs: p5α-red ± ± ±

118 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS The permeation study revealed that SLN:p5α-Red vectors are able to pass the cellulose membrane and 80.2 % of the SLN:p5α-Red vectors were dialyzed after 6 hours. Prior to transfection, the cytotoxicity assay was examined to determine the viability of cells. As seen in Figure 1b, cell viability is reduced in relative order against increasing SLNs and SLN:p5α-Red (3:1, v/v) vector doses. One of the main causes of toxicity in cationic SLN formulations is zeta potential. SLNs have higher zeta potential than the SLN:p5α-Red (3:1, v/v) vectors and show relatively higher cytotoxicity in parallel. SLN:p5α-Red (3:1, v/v) vector doses showing cell viability above 70% were accepted as viable reference doses for transfection studies. Figure 1: a) Agarose gel electrophoresis image of SLN: p5α-red vectors showing SDS-induced release profile of vectors and protection capability against DNase I enzyme, b) Viability percentages of DU-145 cells treated with increasing doses of SLNs and SLN:p5α-Red (3:1, v/v) vectors, c). Images of GFP-positive cells obtained with SL- N:pEGFP-C1 (v/v) (3:1) vector observed by inverted fluorescent microscopy after 24 h and 48 h of transfection. d) Detection of p5α-red protein expression levels by Western Blot in DU-145 cell line using SLN: p5α-red (3:1, v/v) vectors. Calculated percentages of band densities were mentioned on blot image for each band. One of the easiest and most convenient methods of monitoring transfection is the expression of genes encoding reporter proteins like fluorescent biomarkers. To determine the transfection ability before gene silencing assay, transfection was performed using the pegfp vector as well. EGFP expression was observed under fluorescence microscope. As can be seen in Figure 1c, it was observed that the developed SLN: pegfp-c1 vectors (3:1, v/v) have the ability to transfect DU-145 cells. After the optimization of transfection protocol, transfection was then repeated using the SLN:p5α-Red vectors (3:1, v/v). Forty-eight hours after transfection, the amount of p5α-red enzyme was analyzed by western blot method. Figure 1d, shows the membrane image obtained after western blot analysis and we observed that the western blot band density of 5 α-red enzyme reduced about 65.1% compared to the control. Gene silencing was showed that SLN:p5α-Red (3:1, v/v) vectors were able to silence p5α-red enzyme synthesis in DU-145 cell line. Conclusions The results of gene suppression study revealed that antisense shrna-encoding plasmid loaded solid lipid nanoparticles are promising and encouraging for local delivery against 5-α reductase activity. This newly developed approach may become a novel treatment strategy in related androgenic diseases such as benign prostatic hyperplasia, androgenic alopecia and prostate cancer. 116

119 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Acknowledgments This work was supported by Ege University Research Fund [BAP, 14-ECZ-030, 2016]. References [1.] Guo, J.; Evans, J.C. and O Driscoll, C.M. Delivering RNAi therapeutics with non-viral technology: A promising strategy for prostate cancer?. Trends Mol. Med., 19, (2013). [2.] Ellis, J. A. and Sinclair, R.D. Male pattern baldness: current treatments, future prospects. Drug Discov. Today. 13, (2008). [3.] L.J. Schmidt, D.J. Tindall, Steroid 5 α-reductase inhibitors targeting BPH and prostate cancer, J. Steroid Biochem. Mol. Biol. 125, (2011). [4.] J.A.R. Salvador, R.M.A. Pinto, S.M. Silvestre, Steroidal 5alfa-reductase and 17alfa-hydroxylase/17,20-lyase (CYP17) inhibitors useful in the treatment of prostatic diseases, J. Steroid Biochem. Mol. Biol. 137, (2013). [5.] C. Iehlé, S. Délos, O. Guirou, R. Tate, J.P. Raynaud, P.M. Martin, Human prostatic steroid 5 alpha-reductase isoforms--a comparative study of selective inhibitors., J. Steroid Biochem. Mol. Biol. 54, 273 9(1995). [6.] Müller, R.H.; Mäder, K. and Gohla, S. Solid lipid nanoparticles (SLN) for controlled drug delivery - A review of the state of the art. Eur. J. Pharm. Biopharm., 50, (2000). [7.] A. del Pozo-Rodríguez, S. Pujals, D. Delgado, M.A. Solinís, A.R. Gascón, E. Giralt, J.L. Pedraz, A proline-rich peptide improves cell transfection of solid lipid nanoparticle-based non-viral vectors, J. Control. Release. 133, 52 59(2009). 117

120 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-103 Effectiveness of Combined sirna and Docetaxel-Loaded SLNs on Different Breast Cancer Cell Lines Behiye Şenel, Gülay Büyükköroğlu Department of Pharmaceutical Biotechnology, Anadolu University, Eskişehir, Turkey Abstract Nowadays, short interfering RNA (sirna) therapeutics has brought a great hope for treatment of various diseases, including genetic diseases, cancer, and resistant viral infections. In this study, it was planned to investigate the cytotoxicity and transfection effects of the previously prepared DTX and sirna loaded cationic lipid nanoparticles on three different breast cancer cells. When the results were analysed, the formulations were found to be suitable for carrying a gene and chemotherapeutic agent together. The cell types were also found to be important on the efficacy of formulations Keywords Solid lipid nanoparticle, sirna, BCL-2, Breast cancer Introduction With the discovery of RNAi, new approaches to diseases have been introduced. The therapeutic advantage of SiR- NA is that it can target many different genes [1]. sirna is a promising therapeutic solution as a post-transcription gene regulation process for various pathological conditions such as viral infections, cancer, genetic disorders and autoimmune disorders. This therapeutic method is currently actively involved in cancer therapy [2]. However, the naked sirna is unstable in the bloodstream and cannot effectively pass cell membranes alone. Therefore, there is a need for the design of various carrier systems for efficient use of sirnas [3]. Solid lipid nanoparticles are widely used for the delivery of poorly soluble drugs. To date, the administration of solid lipid nanoparticles for sirna administration has been performed in a limited. The solid lipid nanoparticles have been shown to have advantageous properties over other carriers, such as high stability in body fluids and tissues, the ability of the nanoparticle matrix to penetrate into the cell easily due to the lipid component, biodegradability, ease of production, and the ability to scale to industrial production levels at relatively at low cost. However, in studies conducted was insufficient, the use of solid lipid nanoparticles for anticancer chemotherapeutics / sirna co-delivery systems [4]. In this study, the combined effect of docetaxel, as a chemotherapeutic agent, and BCL-2 sirna were investigated on breast cancer cell lines. Materials Gelucire 50/13 was used as the solid lipid and was purchased from Gattefosse (Cedex, France). sirna Bcl-2 and FITC (fluorescein isothiocyanate) conjugated sirna were provided by Santa Cruz Biotechnology (Heidelberg, Germany). Docetaxel (DTX) was obtained from Sigma-Aldrich (Hong Kong, China), the cationic agent Octadecylamine were from Sigma-Aldrich (Steinheim, Germany). Methods Preparation of SLN, DTX/SLN and DTX/siRNA/SLN SLNs were prepared by solvent emulsification/evaporation method according to the procedure described before 118

121 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS with changing the cationic agent [23]. Briefly, Gelucire 50/13 and Octadecylamine (as a cationic agent) were dissolved in 1 ml dichloromethane (DCM) and the surfactants were dissolved in distilled water. The aqueous phase was added to the oily phase in a round bottom glass flask and the mixture was sonicated for 1 min at 20% power using a sonicator (Sonics & Materials Inc, USA). The mixture was evaporated to remove the residual organic solvent using a rotavapor (Buchi, Switzerland). To reduce the particle size and also to obtain a homogeneous dispersion, formulations was homogenized using a nano-homogenizer (Microfluidic LV1, USA). For the preparation of DTX-loaded, sirna-encapsulated and DTX and sirna combination included SLNs were proceeded as explained before [23] with changing the cationic agent. Homogenization was performed following the organic solvent evaporation. All homogenized dispersions were filtered through a 0.2 µm non-pyrogenic filter. Characterisation studies Mean diameter of the bulk population, particle size distribution via polydispersity index (PI) and zeta potential of freshly prepared samples were determined using a Zetasizer NanoZS (Malvern Instruments, UK). Surface morphology of the SLNs prepared was investigated using Carl Zeiss scanning electron microscope (SEM) (SUPRA 50VP model, Oberkochen, Germany). Determination of encapsulated sirna and serum stability analyses Gel retardation studies were used to determine sirna encapsulation efficiency of formulations, to evaluate the possible degradation of sirna encapsulated in serum. Cell viability and transfection studies The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used for the quantitative determination of cell viability. In order to determine the cell viability effects of SLN and loaded formulations, MDA-MB-453, MDA-MB-231 and MCF-7 cell lines were used. Cell viability was determined after 24 and 48 hr. For the transfection study, the same procedure was used but FITC-conjugated control sirna used as a genetic material. Results Particle sizes of SLN formulations prepared in this study were between ± 4.25 and ± 4.26 nm. SEM images showed that the particles were spherical in shape, with sizes also in the nanometer range These formulations have high transfection efficiencies and serum stability. The cell viability results of MDA-MB453, MDA-MB231 and MCF-7 cell line was shown in Figure

122 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Figure 1: Cell viability results for 24 and 48 h. According to cell viability results, prepared formulations was most effective on MCF-7 and MDA-MB-231 compared to MDA-MB-453. It was found that combined formulations started to show their effectiveness even at the smallest dose. Discussion and Conclusions Cell viability is crucial parameter for evaluation of a therapeutic agent prior to in vivo and also clinical use. Compared with individual effects, when combined with sirna and chemotherapeutic drugs, synergistic effects between them can increase the potential of therapeutic effect [5]. Our study offers new insights for future optimization of sirna-based drug delivery systems for breast cancer therapy. Our formulations demonstrated substantial cellular internalization. Targeted Bcl2 and hence its mediators on other pathways have been shown to inhibit cell proliferation, and this inhibition varies according to the difference between cells. We demonstrated that the importance of using combined Docetaxel and sirna-bcl-2 loaded cationic lipid nanoparticles order to inhibit cancer progression in vitro on different breast cancer cell lines. We believe that these formulation strategies developed in this study may be applicable to knockdown of Bcl2-mRNA and other gene targets in vivo, and will be an excellent platform to apply in future anti-cancer therapy. References [1] Draz, M. S., Fang, B. A., Zhang, P., Hu, Z., Gu, S., Weng, K. C., Gray, J. W., Chen, F. F. (2014). Nanoparticle-mediated systemic delivery of sirna for treatment of cancers and viral infections. Theranostics, 4(9), [2] Tatiparti, K., Sau, S., Kashaw, S.K., & Iyer, A.K. (2017). sirna Delivery Strategies: A Comprehensive Review of Recent Developments. Nanomaterials. [3] Wang, J., Mi, P., Lin, G., Wáng, Y.X.J., Liu, G.; Chen, X. (2015). Imaging-guided delivery of RNAi for anticancer treatment. Adv. Drug Deliv. Rev., 104, [4] Yu, Y.H., Kim, E., Park, D.E., Shim, G., Lee, S., Kim, Y.B., Kim, C.W., Oh, Y.K. (2012). Cationic solid lipid nanoparticles for co-delivery of paclitaxel and sirna. Eur J Pharm Biopharm. 80(2), [5]Young, S.W., Stenzel, M.H., & Yang, J.L. (2016). Nanoparticle-siRNA: A potential cancer therapy? Critical reviews in oncology/hematology, 98,

123 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-104 The Use of L-Ascorbic Acid Containing Dextran Coated Silica Aerogel in Controlled Drug Targeting Systems Cem Özel, Ceren Keçeciler, Ali Değirmenci, Ecem Tiryaki, Yeliz Başaran Elalmış, Burcu Karakuzu Ikizler, Sevi Yücel Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Bioengineering Department Abstract Dextranase is an enzyme found in the large intestine but not in the gastric in the human digestive system. Thus, model drug would be transferred to large intestine without release. In this research, dextran cross-linked with glutaraldehyde was used as a coating material for silica aerogel powders. L-Ascorbic acid was used as a model drug. Dextran concentration in coating medium was used as 5% and 10% with 5% glutaraldehyde crosslinker. 10% dextran coated silica aerogel showed only 1.36% drug release in the gastric fluid in 120 min. The results are promising for colon targeted drug delivery. Keywords Silica aerogel, L-Ascorbic acid, controlled drug delivery system, dextran coating, glutaraldehyde. Introduction Controlled drug delivery system (CDDS) attracts attention in pharmaceutical technology for years. Pure drugs using in the body may introduce side effect, some toxic effect, high dosage, low effective biodistribution, dissolve rapidly, affecting a non-target cell. CDDS try to prevent these undesired effects. Controlled drug delivery carriers provide the effectiveness of the model drug, targeted delivery, enhancing the solubility of the drug, prolonged releasing, and reduction of using a dose [1, 2]. In recent years, aerogels have gained increasing interest within biomedical sciences and across various scientific disciplines including CDDS [3]. Silica aerogels, are one of them, gain a tremendous attention in CDDS last 30 years thanks to its biocompatibility, non-toxic properties, high porosity with open interconnected porous, suitable pore-size distribution, and large surface area [4, 5]. Dextran is a water-soluble polysaccharide which consists mainly of α-1,6 linked D-glucopyranose residues with a low percentage of α-1,2, α-1,3 and α-1,4 linked side chains. There are several applications of dextran in market. One of them is plasma volume expender as dextran70 which uses for treatment of shock or hemorrhage, burns, surgery or trauma. Also, dextran is used in food or pharmaceutical industry as component of packaging substances. Dextran is used for shielding material as a polysaccharide in CDDS. Vitamin C (L-Ascorbic Acid) functions as an antioxidant and is cofactor for a lot of enzymes. In some situations, however, it can act as a prooxidant, where it accepts a hydrogen atom instead of giving one. Whether vitamin C acts as an antioxidant or a prooxidant depends on its oxidative state. Body cell L-Ascorbic acid absorption get decreased on high concentration while at low concentration, absorption of the ascorbic acid increased. Its excretion from body lasts 30 minutes [6]. In this report, we make dextran coated silica aerogels for releasing mostly in human intestine ascorbic acid thanks to dextranase enzyme. Materials and Methods Tetraethyl orthosilicate (TEOS, Merck), HCl and NH 4 OH (Merck), dextran (Sigma-Aldrich), L-Ascorbic acid (AA) and Glutaraldehyde (Sigma-Aldrich) used were purchased commercially. Silica aerogels synthesis occurs in 121

124 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS two steps sol-gel reactions; (1) obtaining alcogel by two-step sol-gel method with hydrolysis and condensation reaction and aging, (2) drying with an air dryer. In order to prepare silica aerogels samples mixture of TEOS:EtOH:H 2 O:HCl at 1:3.9:1:7.8x10-4 molar ratios were prepared. Hydrolysis reaction s (ph 4.0), condensation reaction s (ph 8.0 with NH 4 OH) and aging (at 50 C) were performed, respectively. The aged gel was washed with ethanol and then was placed in an incubator at 50 C for 24 h. Three different formulation (S1, S2, S3) were prepared by 0.1g silica aerogel and ascorbic acid solution (3 mg/ml). Silica aerogel were loaded with ascorbic acid (S1), %5(w/v) dextran and %5(v/v) glutaraldehyde coating (S2) and %10(w/v) dextran and %5(v/v) glutaraldehyde coating (S3). The silica aerogels were characterized using FT-IR (SHIMADZU CORP, IRPrestige21) to determine functional groups. Ascorbic acid loaded silica aerogel powders were placed into a dialysis bag for drug release experiment in gastric fluid (ph 1.2) for 120 min and continued with intestinal fluid (ph 6.8), respectively. Drug concentrations were determined via UV-visible spectrophotometer (Scinco, S-3100) at 265 nm for loading and release studies. Results Ascorbic acid loaded, and unloaded silica aerogels were characterized via FT-IR (Figure 1). Band present in the FT- IR spectrum of unloaded and uncoated silica aerogel samples at 3334, 1630, 1055 and 800 cm -1 represents OH bond on silica surface (Si-OH), the adsorbed H 2 O and Si-O-Si (siloxane) asymmetric stretching, respectively (Figure 1a). FT-IR spectrum of dextran coated, and AA loaded silica aerogel samples shows a band at 1714 cm -1 due to the C=O group of glutaraldehyde cross-linker and AA (Figure 1b-c). The band at 3379 cm 1 could be due to the OH groups of dextran, AA and silanol. Band present at 2927 cm 1 could come from the CH 2 asymmetric stretching vibration of dextran and ascorbic acid. Figure 1. FT-IR spectra of the (a) unloaded-uncoated, (b) L-Ascorbic acid loaded % 10 dextran coated (c) Ascorbic acid loaded %5 dextran coated silica aerogel. Ascorbic acid loading efficiencies for S1, S2 and S3 were calculated as 5.57%, 23.40% and 49.90%, respectively. The results of ascorbic acid release in gastric buffer (ph 1.2) and intestine buffer (ph 6.8) for 240 min for S1, S2 and S3 were presented in Figure 2. Releasing of the ascorbic acid from the coated-loaded silica aerogels was investigated in (Figure 2). 122

125 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS (a) (b) Discussion (c) Figure 2. L-Ascorbic acid release from (a) unmodified silica aerogel, (b) %5 dextran coated silica aerogel, (c) %10 dextran coated silica aerogel. In this work, dextran coating was applied to silica aerogel particles prepared with glutaraldehyde cross-linker. As the concentration of dextran increased, the loading rate increased and the release of the L-Ascorbic acid in gastric fluid buffer decreased. FT-IR results confirmed dextran coating and ascorbic acid loading of the silica aerogel powders. Drug release in human gastric fluid was a crucial step in this experiment. When we compared drug release at 120 min, silica aerogel coated with 10% dextran shows only 1.36% drug release while 5% coated shows 8% drug release. This is a strong indication that when usage of optimal amounts of dextran with glutaraldehyde, coating becomes more rigid and limits the drug release. Crosslinked dextran is reported to be stable in the stomach and small intestine but degradable in the large intestine [8]. Our drug release results are in agreement with Hovgaard and Brøndsted s results which suggest that the dextran hydrogels are promising as drug carriers for colon specific drug delivers [9]. Conclusions In this study, firstly silica aerogel was produced by sol-gel method. Ascorbic acid was loaded to produced silica aerogel in the presence and absence of dextran and glutaraldehyde. Applied dextran concentrations were 5% and 10%. Minimum release was achieved with 10% dextran coated silica aerogels. In conclusion, our results show that dextran concentration in coating is important for drug delivery and release systems for colon targeted drugs. 123

126 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS References [1.] L. Yan, J. Zhang, C.-S. Lee, and X. Chen, Micro- and Nanotechnologies for Intracellular Delivery, Small, vol. 10, no. 22, pp , Nov [2.] V. Mamaeva, C. Sahlgren, and M. Lindén, Mesoporous silica nanoparticles in medicine Recent advances, Advanced Drug Delivery Reviews, vol. 65, no. 5, pp , May [3.] G. J. Owens et al., Sol gel based materials for biomedical applications, Progress in Materials Science, vol. 77, pp. 1 79, Apr [4.] J. L. Gurav, I.-K. Jung, H.-H. Park, E. S. Kang, and D. Y. Nadargi, Silica Aerogel: Synthesis and Applications, Journal of Nanomaterials, vol. 2010, p. e409310, Aug [5.] P. B. Sarawade, G. N. Shao, D. V. Quang, and H. T. Kim, Effect of various structure directing agents on the physicochemical properties of the silica aerogels prepared at an ambient pressure, Applied Surface Science, vol. 287, pp , Dec [6.] M. Levine et al., Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance., Proc Natl Acad Sci U S A, vol. 93, no. 8, pp , Apr [7.] J. Kim et al., Enhanced antitumor activity of vitamin C via p53 in Cancer cells, Free Radical Biology and Medicine, vol. 53, no. 8, pp , Oct [8.] Brøndsted, H., Andersen, C., & Hovgaard, L. (1998). Crosslinked dextran a new capsule material for colon targeting of drugs. Journal of controlled Release, 53(1-3), [9.] Hovgaard, L., & Brøndsted, H. (1995). Dextran hydrogels for colon-specific drug delivery. Journal of Controlled Release, 36(1-2),

127 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-114 Ethical Issues in Gene Editing Tool CRISPR Zehra Şeker 1, Özlem Bildik Şanlı 2 1 Clinical Pharmacy, Marmara University, Istanbul, Turkey 2 Medical Ethics and History of Medicine, Bezmialem University, Istanbul, Turkey Abstract CRISPR is a new gene editing tool that get inspired from fighting of bacteria against viruses. It is widely used in genetic technology since it s ease of use, lower cost, efficient and specificity characteristics and it will be full of hope for many genetic diseases. However, the problem arises with the germline gene editing that include manipulation of embryo genome. Germ line gene editing brings many ethical concerns among public, scientists and bioethicist. There are different approaches argue whether applying these techniques to germ line is moral or not. The questions are that will designer babies turn to a horror story? Will eugenism come back into society via this new gene editing tool? How could we preclude non-therapeutic applications such as human enhancement? In this presentation, we will illustrate the debates and related ethical issues in the recent literature. Keywords Genome editing, ethics of genomics, ethics 125

128 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-123 Ethical Issues Related to Gene Editing Using CRISPR Zehra Şeker 1, Özlem Bildik-Şanlı 2 1 Clinical Pharmacy, Marmara University, Istanbul, Turkey 2 Medical Ethics and History of Medicine, Bezmialem University, Istanbul, Turkey Abstract CRISPR is a new gene editing tool that get inspired from fighting of bacteria against viruses. It has been widely used in genetic technology because of it s ease of use, lower cost, high efficiency and selection of specific characteristics. Germline gene editing brings many ethical concerns among public, scientists and bioethicist. Numerous studies has come to exist in the literature and there are different approaches argue whether applying these techniques to germ line is moral or not. The questions are that will designer babies turn to a horror story? Will eugenism come back into society via this new gene editing tool? How could we preclude non-therapeutic applications such as human enhancement? In this presentation, we will illustrate the debates and related ethical issues in the recent literature. Keywords Genome Editing, CRISPR, Genetic Engineering, Ethics Full Text CRISPR is a new gene editing tool that get inspired from fighting of bacteria against viruses. Greater part of bacteria and archaea have their own immune system that enable them to fight against viruses. The acquired immunity system CRISPR- Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated proteins ), is a new gene editing tool that get inspired from fighting of bacteria against viruses. The technique have two essential components; guide RNA which is designed to make Watson-Crick base pairing with targeted DNA sequence and Cas endonuclease which provides the double strand break of DNA. It is widely used in genetic technology since it s ease of use, lower cost, efficient and specificity characteristics and it will be full of hope for many genetic diseases. However, the problem arises with the germline gene editing that include manipulation of embryo genome. Although the RNA guided CRISPR-Cas system has many advantages, it also has number of technical challenges such as mosaicism and off-target effects that can cause unpredictable effects on future generations when it is applied to human embryos and germ cells. Germ line gene editing brings many ethical concerns among public, scientists and bioethicist. Two different approaches argue whether applying these techniques to germ line is moral or not and this gene editing tool has any superiority to IVF PGD. The questions are that will designer babies turn to a horror story? Will eugenism come back into society via this new gene editing tool? How could we preclude non-therapeutic applications such as human enhancement? First discovery of CRISPR system is based upon the study of Yoshizumi Ishino and colleagues in In this study, the scientists hadn t know the function of CRISPR system yet but they initiated a new adventure in gene editing by finding out the presence of repeated DNA sequences in bacteria s CRISPR loci. After 20 years, the actual function of these repeated sequences are figured out via the yoghurt fermentation process. Since the bacteria that are used in dairy industry are very sensitive to invasion via viruses, a Danish food company Danisco published a study. In this study, they show that the CRISPR system is an acquired immune system of prokaryotes. Scientists of Danisco Food Company helped to reveal the mechanism of action of this acquired immune system. 126

129 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS CRISPR system has a main pattern with palindromic repeated sequences that are separated by non-repeated sequences called as spacers. Thanks to human genome project, genomes of many organisms including bacteriophages were sequenced and these developments offer an insight into understand the meaning of these spacers. Once the viruses invade bacteria, they first leave their genetic material into bacteria. The CRISPR system keeps the viral DNA in the memory by inserting short viral DNA sequences into spacer sequences. So, when the bacteria meet with these phages again, the system step in to inactivate the virus. The CRISPR acquired immunity system occurs in three steps. The first one is the insertion of the brief foreign DNA into spacer sequence. The second step is the transcription of CRISPR array to generate a crrna/guiderna which makes the Watson-Crick base pairing with the invading DNA targeted sequence. Guide RNA binds and forms a complex with the CRISPR associated genes (Cas). In the last step, Cas enzymes cleave the foreign DNA by their nuclease activity which leads to inactivation of viruses. After clearing the function and mechanism of RNA guided CRISPR-Cas system in bacteria, scientists try to adaptate this technique to manipulate the human genome. First in vitro application of this technique as a gene editing tool in human genome was occured in 2012 and after a year, an in vivo study was published which shows the therapeutic application of CRISPR technique in human genome. Thereafter, numerous studies has come to exist in the literature. It has been widely used in genetic technology because of it s ease of use, lower cost, high efficacy and selection of specific characteristics. It also hold promise for many genetic diseases such as Huntington disease, duchenne muscular dystrophy, cystic fibrosis, AIDS, beta thalassemia etc. So, the necessary components are just a designed guide RNA to make Watson-Crick base pairing with targeted sequence which identify the specificity of the method and Cas9 nuclease to make double strand DNA break. Cold war in the science world has begun with the publication of a study which showed that CRISPR-Cas9 system is applicable for gene editing in human germ line in Until that time gene editing was only used in somatic cells that couldn t cause to be inherited the next generations. Chinese scientists Liang and co-workers used tripronuclear zygotes which have two sperm nucleus and one oocyte nucleus. So, even if these zygotes don t have the chance to lead healthy pregnancy and birth, many ethical concerns come up about manipulation of embryo genome. Besides that, the researchers observed off-target effects and mosaicism also. Large genomes can involve similar or same arrays with the targeted DNA sequences. This can lead to cleavage of non-specific, off-target DNA sequences by Cas enzyme and mutations. Mosaicism is the alteration and differentiation of only some of genes rather than all of them. The manipulation of embryo genome, off-target and mosaicism challenges that can be inherited from individual to next generations, editing genes of embryos without consent are the main topics about ethical debates. 127

130 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-128 Formulation and Evaluation of Nano-Niosomes as Gene Delivery Systems Devrim Demir Dora, Büşra Cesur, Feride Öner Akdeniz University, Faculty of Medine, Department of Medical Pharmacology, Antalya Abstract Gene therapy aims to cure or treat diseases by delivering DNA into the cell nucleus to produce or regulate defective genes. Gene therapy success depends on the efficient delivery of therapeutic genes into the target cells (1). Genes can be delivered by viral or non-viral vectors and appropiate gene carriers should be able to keep the integrity of the genetic material to transport the DNA to the cell nucleus (2). To deliver genes efficiently into the nucleus, vectors must circumvent extracellular and intracellular barriers. Extracellular barriers are the first barriers that protect the integrity of the cells from foreign agents. Enzymes, serum proteins and blood cells are responsible for the degradation or aggregation of the DNA and delivery systems in the circulatory system. Vesicular systems, such as liposomes and niosomes are widely used as carriers for delivery of nucleic acids (3). Niosomes are non-ionic surfactant vesicles (NSVs) that are more stable and less toxic than liposomes. Nioplexes are formed by complexation and condensation of negatively charged nucleic acids by positively charged niosomes (4,5). In this study it was aimed to investigate the effect of different non ionic surfactants on physical properties and serum stability of nioplexes. INTRODUCTION Gene therapy aims to cure or treat diseases by delivering DNA into the cell nucleus to produce or regulate defective genes. Gene therapy success depends on the efficient delivery of therapeutic genes into the target cells (1). Genes can be delivered by viral or non-viral vectors and appropiate gene carriers should be able to keep the integrity of the genetic material to transport the DNA to the cell nucleus (2). To deliver genes efficiently into the nucleus, vectors must circumvent extracellular and intracellular barriers. Extracellular barriers are the first barriers that protect the integrity of the cells from foreign agents. Enzymes, serum proteins and blood cells are responsible for the degradation or aggregation of the DNA and delivery systems in the circulatory system. Vesicular systems, such as liposomes and niosomes are widely used as carriers for delivery of nucleic acids (3). Niosomes are non-ionic surfactant vesicles (NSVs) that are more stable and less toxic than liposomes. Nioplexes are formed by complexation and condensation of negatively charged nucleic acids by positively charged niosomes (4,5). In this study it was aimed to investigate the effect of different non ionic surfactants on physical properties and serum stability of nioplexes. 128

131 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS MATERIALS AND METHODS Materials Non ionic surfactants Span 60, Span 80 and Brij 72 ; cholesterol and cationic agent (CTAB) was purchased from Sigma&Aldrich. LV-RFP pdna was purchased from Addgene. All other chemicals used were of analytical grade and were obtained from Sigma & Aldrich. Plasmid DNA Isolation plv-rfp pdna was amplified using E. coli DH5α strain and purified by using QIAGEN Plasmid Maxi Kit. The purity of pdna was checked by 1% agarose gel electrophoresis and concentration of DNA was determined by measuring UV absorbance at 260 and 280 nm. Preparation of Niosomes Cationic niosomes were prepared by thin film hydration technique using non ionic surfactant, cholesterol and cationic agent (CTAB) at molar ratios of 72Mm:18Mm:10Mm, respectively. Span 60, Span 80 and Brij 72 were used as non ionic surfactants which have HLB values of 4.7, 4.3 and 4.9, respectively. Cholesterol, surfactant and cationic agent were dissolved in 1ml chloroform. The organic solvent was then removed above the lipid transition temperature by using a rotary evaporator at 60 C, 20 kpa, leaving a thin layer of solid mixture deposited on the flask. The dried surfactant film was hydrated with 1 ml deionized water at 60 C. Particle size was reduced by sonication for 10 minutes. Span 60, Span 80 and Brij 72 niosomes were coded as N1, N2 and N3 respectively. Niosome formulations were characterized with respect to shape, particle size distribution, zeta potential and poly dispersity index (PDI). Morphology of niosome formulations was determined by optical microscopy. Preparation of Niosome/pDNA Complexes (Nioplexes) Nioplexes were prepared by mixing the cationic niosomes with the anionic plv-rfp plasmid DNA at 1:1w/w ratio. The mixtures were incubated for 30 minutes at 37 C for electrostatic interaction and nioplex formation and DNA binding was checked by 1% agarose gel electrophoresis. Nioplexes were characterized in terms of size, surface charge, polydispersity index and ability for DNA condensation and protection. Zeta Potential and Vesicle Size Analysis of Nioplexes The hydrodynamic diameter of the niosomes and nioplexes were determined by Dynamic Light Scattering (DLS) using a Zetasizer (Malvern Zetasizer ZEN3600). All niosomes and nioplex formulations were measured at room temperature in triplicate for vesicle size and zeta potential analysis. Serum Stability of Nioplexes The prepared nioplexes were incubated in the DMEM with 10 % FBS for 4h. At the end of the incubation, the reaction was terminated with 1% sodium dodecyl sulphate (SDS) and stability in the serum was evaluated by 1% agarose gel electrophoresis. 129

132 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS RESULTS AND DISCUSSION Morphology of the Niosomes Morphologies of the niosomes checked by optical microscopy (40Xmagnification) are shown in Figures 1-3. Figure 1. Optical microscopy image of Span 60 niosomes Figure 2. Optical microscopy image of Span 80 niosomes Figure 3. Optical microscopy image of Brij 72 niosomes Preparation of Nioplexes Complexation by Span 60, Span 80 and Brij 72 niosomes retarded pdna ( Figure 4). Figure 4: Agarose gel electrophoresis photograph of Span 60, Span 80 and Brij 72 nioplexes (1:DNA mw st., 2:Naked pdna, 3: Span60/pDNA 1:1 (w/w), 4:Span 80/pDNA 1:1 (w/w), 5:Brij 72/pDNA 1:1 (w/w)). 130

133 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Zeta Potential and Vesicle Size Analysis of Nioplexes Vesicle size, zeta potential and PDI values of Span 60, span 80 and Brij 72 niosomes and nioplexes are seen in Table 1. Zeta potential and size of the niosomes were effected by type of non ionic surfactants. Table 1. Particle size, zeta potential and PDI values of niosomes and nioplexes Niosomes and Nioplexes Zeta potential (mv) Vesicle size (nm) PDI N1 +81 ±1.52 mv 269 ± N ± 2.20 mv ± N3 +54 ± 1.74 mv ± N1P ± 1.72 mv 498 ± N2P +41 ± 2.40 mv 370 ± N3P +24 ± 1.96 mv 484 ± Serum Stability of Nioplexes pdna was protected in the serum for 4 hours by niosomes as seen in Figure 5. Conformation of the naked plasmid DNA was changed after incubation with serum. Plasmid DNA conformation can effect stability and transfection efficiency. Figure 5: Electrophoretic mobility of plasmid DNA on agarose gel after 4 h incubation in DMEM with serum (1:DNA mw st., 2:Naked pdna with serum, 3:Naked pdna, 4:N1P, 5:N2P, 6:N3P) CONCLUSION Extracellular barriers such as serum proteins are responsible for the degradation or aggregation of the DNA and delivery systems in the circulatory system. Niosomes can remain in the bloodstream for a reasonable time. Plasmid DNA could be protected by niosomes for 4 hours in serum containing media. Cationic niosomes can keep the integrity of the genetic material to transport the DNA to the cell nucleus and can be used as gene delivery systems. The average niosome size varied between and nm and increased as the HLB value of surfactants increased. These results are compatible with the findings in the literature. However, positively charged niosomes have provided electrostatic interaction with negatively charged pdna, so that the vesicle size of niosomes was increased. The zeta potential and vesicle size of niosomes are important in the interaction with the cell membrane. Niosomes are more economic and chemically stable alternatives compared to liposomes.we suggest that our niosomes could be used for delivery of nucleic acids to various cells. 131

134 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS REFERENCES [1.] Heilbronn, R., Weger S., Viral vectors for gene transfer: current status of gene therapeutics, in: M. Schäfer- Korting (Ed.), Drug Delivery, Vol. 197 of Handbook of Experimental Pharmacology, Springer, Berlin, Heidelberg, 2010; [2.] Wang, W., Li, W., Ma, N., Steinhoff G., Non-viral gene delivery methods, Curr. Pharm. Biotechnol. 2013; 14: [3.] N.B.Mahale, P.D.Thakkar, R.G.Mali, D.R.Walunj, S.R.Chaudhari, Niosomes: Novel Sustained Release Nonionic Stable Vesicular Systems An Overview, Advances In Colloid And Interface Science, 2012; : [4.] M. H. Nematollahi, A. Pardakhty, M.Torkzadeh-Mahanai, M. Mehrabani, G. Asadikaram; Changes in Physical and Chemical Properties of Niosome Membrane Induced by Cholesterol: a Promising Approach for Niosome Bilayer Intervention; Royal Society of Chemistry, 2017; 7; [5.] Huang Y, Chen J, Chen X, Gao J, Liang W. PEGylated synthetic surfactant vesicles (Niosomes): novel carriers for oligonucleotides, J Mater Sci Mater Med. 2008; 19:

135 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-129 Immunogenicity of Biotechnological Medicinal Products: From Cell Line to Finished Product Devrim Demir-Dora Akdeniz University, Faculty of Medicine, Department of Medical Pharmacology, Antalya, Turkey. Introduction Therapeutic protein products provide effective treatments for several human diseases and the number of proteins used as therapeutic agents is increasing in recent years. Therapeutic proteins are >40 aa polypeptides whose active components are derived from a biological source by being produced in microorganisms and human or animal cells using biotechnology (1). They are not chemically synthesized and when compared with chemically synthesized small molecule drugs, the manufacturing process and molecular structure of biopharmaceuticals are more complex (2). As they are produced mainly in a genetically modified living systems, their synthesis is influenced by multiple parameters during the manufacturing process. Three-dimensional structure of the biopharmaceutical could be changed by manufacturing process and it should be controlled to ensure patient safety. Quality, safety, and efficacy of the therapeutic recombinat protein depends on the structure and post translational modifications of the protein which is directly related with manufacturing process (3). Factors Affecting Immunogenicity of Biotechnological Medicinal Products The most important safety issue for biopharmaceuticals is immunogenicity. Therapeutic proteins are recognized by the human immune system and followed by an unwanted immune response. The immune response against therapeutic proteins differs not only between products and product categories but also between individuals and patient groups. Factors that may affect the development of an immune response against a therapeutic protein can be either patient- and disease-related or product related (4,5). Patient- and disease-related factors Patient s genetic factors, age, other diseases, concomitant treatment and treatment related factors can affect immune response. Length of treatment, dosage schedule and route of administration are also factors in antibody formation. Higher doses or prolonged duration of treatment increase exposure and thereby heighten the risk of developing immunogenicity. Immunogenicity appears to be greater if the biopharmaceutical is administered subcutaneously (sc) or intramuscularly (ım) and has decreasing severity with intravenous (ıv) administration. Inhalational, intradermal, as well as ocular administration may also enhance immune responses towards the therapeutic protein. Product-related factors Product-related factors that can influence the immune response include the manufacturing process, formulation, stability characteristics, the interactions between the drug and/or formulation with the primary packaging material (container closure system) and the level of impurity or presence of contaminants. Manufacturing of Recombinant Proteins 133

136 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS The protein production process begins with the isolation and cloning of the required gene into a vector and transfer into a host cell. Prokaryotic systems (Escherichia coli), yeast (S.cerevisiae), mammalian cells (CHO), insect cells, transgenic animals and transgenic plants can be used as expression sytems. Any change in the vector, cell line, growth medium, upstream and down stream processes, filtration or chromatography reagents can change immunogenicity of the product. Each expression system has different impurity profile. Product related or manufacturing process related impurities or post-translational protein modifications can cause immunogenicity (5). Formulation of Drug Substance The formulation of a biopharmaceutical is as important as the active ingredient. Proteins usually aggregate from partially unfolded molecules. Excipients (e.g. Human serum albumin (HSA), polysorbates, glycine and amino acids) are routinely used to stabilize protein drugs. However, the excipients may also influence a drug s safety profile. Although formulations are developed to make final protein product stable, dilution with infusion solutions, exposure to interfaces, container-closure systems, filling pumps, light, temperature fluctuations or minor impurities can induce aggregation and cause immunogenicity (5). Purity and Impurities of Finished Product Purity is an important critical quality attribute for a biological/biotechnological product. The determination of purity depends on an analytical technique and is assessed by a combination of analytical procedures. The results are method dependent (6). The impurities observed in Drug Substance and Drug Product are classified as process-related impurities or product related impurities. Impurities should be controlled by in-process acceptance criteria or action limit on active ingredients or products. New impurities should be evaluated regarding its impact on quality, safety, and efficacy. Safety is ensured by the specified limits for bioburden and endotoxin and process-related contaminants (7). Process-related impurities include host cell DNA, host cell protein, cell culture components and/or antibiotics, leachates and Protein A. Product-related impurities include structural properties, such as protein sequence, molecular variants, the presence of exogenous or endogenous epitopes and the degree of glycosylation influencing protein degradation, exposure of antigenic sites and solubility. Conclusion Nearly all protein drugs induce antibodies in humans and responses are individual. Individual characteristics such as genetic background and disease are important parameters. Immune responses against non-vaccine biologics can affect their efficacy and safety, resulting in adverse events that could include administration reactions, hypersensitivity, deficiency syndromes and lack of a clinical response in treated patients. An unwanted immune response to a therapeutic protein may lead to a loss of efficacy and/or to severe side effects. Neutralizing antibodies can ocur during the treatment which can interfere with the efficacy of the therapeutic protein. Immunogenicity can not be predicted completely from physicochemical characterization, epitope analysis or animal studies. References [1.] Immunogenicity Assessment for Therapeutic Protein Products, U.S. Department of Health and Human Services Food and Drug Administration, August [2.] Devrim Demir-Dora, Biyofarmasötik Ürünlerin Geliştirilmesinde Biyobelirteçler, Turkiye Klinikleri J Pharmacol-Special Topics, 5(2):75-83, [3.] K. Anshu, C. Narendra, and N. Pradip, Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications, Journal of Immunology Research, June 2016, Vol. 2016, [4.] Guideline on Immunogenicity assessment of therapeutic proteins, 18 May 2017 EMEA/CHMP/ 134

137 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS BMWP/14327/2006 Rev 1. [5.] Sorularla Biyoteknolojik ve Biyobenzer İlaçlar. Sadi Özdem, İrfan Çiçin, Devrim Demir Dora, Ceyda Korucu Nazlı, Ed: İrfan Çiçin, Güneş Tıp Kitapevleri ISBN: [6.] Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products, U.S. Department of Health and Human Services Food and Drug Administration, April 2016 Pharmaceutical Quality/ CMC Revision 1. [7.] S. Zuben, L. Daniel, P.V. Joao, G. Basil, and R.S. Amy, Evaluating and Mitigating the Immunogenicityof Therapeutic Proteins, Trends in Biotechnology, October 2018, Vol. 36, No. 10,

138 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-136 Undergraduate and Graduate Education in Pharmaceutical Biotechnology Ozgun Firat Duzenli, Ayse Filiz Oner, Turkan Eldem Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Hacettepe University, Ankara Abstract In this presentation, undergraduate and graduate courses given by the Departments of Pharmaceutical Biotechnology in Pharmacy Faculties in Turkey was summarized. In addition Graduate Programmes of Pharmaceutical Biotechnology in Turkey were examined and Pharmaceutical Biotechnology Master s and Doctoral programs offered by the Department of Pharmaceutical Biotechnology, Graduate School of Health Sciences, Hacettepe University was outlined as an example. Keywords Pharmaceutical Biotechonology, Undergraduate Education in Pharmaceutical Biotechnology, Master and Doctoral Education in Pharmaceutical Biotechnology. Aim Aim of this presentation is to give information about undergraduate education given by the Pharmaceutical Biotechnology Departments in Pharmacy Faculties and graduate education programs of Pharmaceutical Biotechnology in Hacettepe University, Graduate School of Health Sciences. Method Yöntem: Investigation method for this study is searching websites of the Council of Higher Education (CoHE) and CoHE registered universities having a Pharmaceutical Biotechnology Department that provide education within the Faculty of Pharmacies. Results: In 1981, with the Law of Higher Education, all higher education institutions in Turkey have gathered under the roof of the Council of Higher Education (CoHE, YÖK) (1). The universities offering pharmacy degree program registered to the YÖK are shown in Table 1 (2). Table 1. Universities having Pharmacy Undergraduate Education Program No Name of the University No Name of the University 1 Adıyaman 19 Mersin 2 Agrı Ibrahim Cecen 20 Saglık Bilimleri 3 Anadolu 21 Selcuk 4 Ankara 22 Trakya 5 Ataturk 23 Van Yüzüncü Yıl 6 Bulent Ecevit 24* Acıbadem Mehmet Ali Aydınlar 7 Cukurova 25* Altınbaş 8 Cumhuriyet 26* Bezm-i Âlem Vakıf 136

139 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 9 Dicle 27* Biruni 10 Ege University 28* İstanbul Medipol 11 Erciyes University 29* İstanbul Yeni Yüzyıl 12 Erzincan Binali Yıldırım 30* İstinye 13 Gazi 31* Yeditepe 14 Hacettepe 32* Eastern Mediterranean 15 Inonu 33* Girne American 16 Istanbul 34* Cyprus International 17 Karadeniz Technical 35* Near East 18 Marmara Ranking was same as in the reference (2). *Private universities registered to the YÖK. Minimum issues which should be included by all faculties in their Core Education Program (National-CEP) were described by Regulation on the Determination of the Minimum Education Conditions for Medicine, Nursing, Obstetrics, Dentistry, Veterinary Medicine, Pharmacy and Architecture Education Programs which had been announced in the Official Gazette dated 2/2/2008 (Nr: 26775), and updated in the Official Gazette dated 31/12/2009 (Nr 27449) by CoHE (3). National-CEP is only an advice for pharmacy faculties creating their own education program not a general education program. It reflects approximately 70-80% of courses and course hours in pharmacy education. Division of Pharmaceutical Professional Sciences, Division of Pharmaceutical Technology and Division of Pharmaceutical Basic Sciences are the main fields in the National-CEP. The Department of Pharmaceutical Biotechnology has been shown under the Division of Pharmaceutical Technology in accordance with its establishment by YÖK (4). Pharmacy faculties having Pharmaceutical Biotechnology Departments and undergraduate courses are shown in Table 2 (5-17). Table 2. Pharmacy Faculties having Pharmaceutical Biotechnology Departments and Compulsory Courses in Undergraduate Education. No University Name Course Code and Name ECTS S Ref 1 Adıyaman NIA NIA NIA 5 2 Anadolu ECZ358 Pharmaceutical Biotechnology 2 VI 6 3 Bülent Ecevit ECZ416 Pharmaceutical Biotechnology 3 VIII 7 4 Cumhuriyet ECZ4010 Pharmaceutical Biotechnology 4 VIII 8 5 Ege Pharmaceutical Biotechnology 3 VII Introduction to Molecular Biology 3 III 6 Erciyes BTG202 Introduction to Biotechnology 2 IV 10 BTG204 Introduction to Biotechnology Laboratory 2 IV FBT302 Pharmaceutical Biotechnology 4 V 7 Hacettepe ECF 448 Pharmaceutical Biotechnology 4 VII 11 8 Inönü Pharmaceutical Biotechnology 2 VII 12 9 Marmara ECZ453 Pharmaceutical Biotechnology 4 VII 13 ECZ312 Genetics 2 VI 10 Mersin ECZ405 Pharmaceutical Biotechnology NIA NIA Sağlık Bilimleri NIA NIA NIA Trakya ECZ406 Pharmaceutical Biotechnology 2 VIII Van Yüzüncü Yıl NIA NIA NIA 17 Abbr: ECTS, European Credit Transfer System; S, Semester; Ref, Reference; U, University; NIA, No Information Available. 137

140 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Core Education Program of Hacettepe University Faculty of Pharmacy (Hacettepe-CEP) was excluded from the content of the National-CEP. So there is no detailed information about the graduate course titled Pharmaceutical Biotechnology in National-CEP. Therefore the details of the course titled Pharmaceutical Biotechnology defined in the CEP of Hacettepe University, Faculty of Pharmacy will be discussed (18). 28 hours (14 week x 2 hours) were accepted as minimum course hour. Contents and aim recommended for the course titled Pharmaceutical Biotechnolog in Hacettepe-CEP are to provide information about pharmaceutical biotechnology based biological medicinal products, biosimilars drugs, advanced therapy medicinal products ((ATMP) somatic cell therapy medicinal products, gene therapy medicinal products, tissue engineered products, combined advanced therapy medicinal products), stem cell based drugs, personalized medicines, their characteristics, manufacturing technologies, quality assurance, comparability exercises and risk-based approach in their regulatory affairs, distribution, tracing and clinical applications. In addition, another goal of the course is to describe roles and responsibilities of pharmacist, who has knowledge in the field of pharmaceutical biotechnology based drugs, biological medicinal products, medical device combined products and follows recent improvements, in the health system during the life cycles of these drugs and products including their clinical trials (18). The most important objectives of the Pharmaceutical Biotechnology graduate programs are to provide the necessary knowledge and experience at the expert level required for research, development, design, manufacturing, quality assurance, quality control, regulatory affairs, clinical investigations, therapeutic applications of Pharmaceutical biotechnology derived biological drugs (medicinal products), combination products and advanced technologies related to them (19). All higher education institutions offering graduate degree in the field of pharmaceutical biotechnology are listed in Table 3 (20-22). Table 3. Pharmaceutical Biotechnology Graduates Programmes given by Pharmaceutical Biotechnology Departments of Higher Education Institutes. No University Name Institute/Graduate School Master's Program Doctoral Program 1 Ege Institute of Health Science YES YES* 20 2 Hacettepe Graduate School of Health Science YES YES 21 3 Marmara Institute of Health Science YES YES* 22 *These two Higher Education Institute have a joint doctoral program. Abbr: Ref, Reference. To explain the structure of Pharmaceutical Biotechnology Master s and Doctoral programs in Turkey the Department of Pharmaceutical Biotechnology, Graduate School of Health Sciences, Hacettepe University was chosen. In Master s program the student must earn a minimum of 60 ECTS credits (20% from compulsory courses and 80% from elective courses) from the courses and 60 ECTS credits from the thesis and must be successful in related courses to complete the program. In the context of elective courses, student can take courses from other programs. The Selection of elective courses must take into consideration the learning outputs in Pharmaceutical Biotechnology Master s Degree Program. The courses in Hacettepe University Graduate School of Health Science Department of Pharmaceutical Biotechnology Master s Program are shown in Table 4 (19,23). Ref 138

141 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Table 4. Hacettepe University Graduate School of Pharmacy Pharmaceutical Biotechnology Master s Program Courses Course Code Course Title M/E T P C ECTS EFB641 Targeting Techniques in Pharmaceutical E Biotechnology EFB650 Protein and Peptides Dosage Form Design I M EFB658 Fundamentals of Pharmaceutical Biotechnology E EFB685 Gene, Cell and Tissue Engineering in Advanced E Therapy Medicinal Products (ATMP): Quality Assurance and Good Manufacturing Practises EFB Special Subjects-1 M EFB Special Subjects-2 M EFB610 Seminar M EFB612 Immunopharmaceuticals E EFB620 Molecular Biotechnology E EFB680 Medical Devices, Combination Products and Combined ATMP in Pharmaceutical Biotechnology: Good Practices, Ethics and Regulations E Abbr: M, Must course; E, Elective course; P, Practical; T, Theoretic; C, Credit, ECTS, European Credit Transfer System. In Doctoral program the student must earn 120 ECTS credits (42 ECTS credits (35%) from compulsory courses and 78 ECTS credits (%65) from elective courses) from the courses and 120 ECTS credits from the thesis and must be successful in related courses to complete the program. In the context of elective courses, the student can take courses from other programs. The Selection of elective courses must take into consideration the learning outputs in Pharmaceutical Biotechnology Doctoral Program. The courses in Hacettepe University Graduate School of Health Sciences Department of Pharmaceutical Biotechnology Doctoral Program are shown in Table 5 (19,24). Recently in April 16, 2018 the doctoral programs of Hacettepe University Institute of Health Sciences including Pharmaceutical Biotechnology Doctoral Program are accredited with the ORPHEUS Label (25). Table 5. Hacettepe University Graduate School of Pharmacy Pharmaceutical Biotechnology Doctoral Program Courses Course Code Course Title M/E T P C ECTS EFB Special Subjects-1 M EFB Special Subjects-2 M EFB Special Subjects-3 M EFB Special Subjects-4 M EFB720 Pharmaceutical Biotechnology Based Biological Medicinal M products: Quality Assurance, Good Manufactu- ring Practices and Registration EFB753 Legislations and Quality Assurance on Cell and Gene E Therapy Products, Advanced Therapy Medicinal Products (ATMP) EFB711 Seminar M

142 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS EFB730 Pharmaceutical Biotechnology Research Techniques E and Laboratory Methods EFB754 Biosimilar Medicinal Products: Legislations on Comparability, E Quality Assurance, Efficacy and Safety EFB755 Cell and Tissue Culture: Laboratory Techniques in E Pharmaceutical Biotechnology and Good Cell Culture Practices EFB760 Protein and Peptides Dosage Form Design II E EFB770 Lipids, Phospholipids and Pharmaceutical Biotechnology E Application Fields EFB785 Pharmaceutical Bionanotechnology: Supramolecular E Structures in Drug, Gene and cellular Delivery System Design EFB799 Preparation to Doctoral Proficiency Exam M EFB800 Research Studies on Thesis I E EFB801 Research Studies on Thesis II E EFB802 Research Studies on Thesis III E Abbr: M, Must course; E, Elective course; P, Practical; T, Theoretic; C, Credit, ECTS, European Credit Transfer System Courses in Master s and Doctoral Programs are preferred by foreign students came Turkey under Erasmus program, pharmacists working at Republic of Turkey Ministry of Health Turkish Medicines and Medical Devices Agency (TMMB) and private sector employees. A significant contribution is also provided to Registration of Medicinal Products (Non-Thesis) Master s Program and Stem Cell Doctoral Program by the the Department of Pharmaceutical Biotechnology (19). Conclusion The pharmaceutical biotechnology educaiton programs in the faculties of pharmacy in Turkey have similar structure and the courses in this field are highly qualified to contribute to innovative and competitve profile of the researchers in pharmaceutical biotechnology. Since there is great demand for specialized and trained human resources in the field of Pharmaceutical Biotechnology in Turkey, it is important that other pharmacy faculties begin undergraduate and graduate educations with the departments of pharmaceutical biotechnology. References [1.] 16 November 2018 [2.] Accessed 16 November 2018 [3.] Y- Rx/10279/18093 Accessed 16 November 2018 [4.] Mezuniyet Öncesi Eczacılık Eğitimi, Ulusal Çekirdek Eğitim Programı, EczÇEP, [5.] Accessed 16 November 2018 [6.] Accessed 16 November 2018 [7.] Accessed 16 November 2018 [8.] Accessed 16 November 2018 [9.] tr/tr-4886/lisansustu_programlarimiz.html Accessed 16 November

143 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS [10.] Accessed 16 November 2018 [11.] Accessed 16 November 2018 [12.] Accessed 16 November 2018 [13.] Accessed 16 November 2018 [14.] Accessed 16 November 2018 [15.] Accessed 16 November 2018 [16.] Accessed 16 November 2018 [17.] Accessed 16 November 2018 [18.] Mezuniyet Öncesi Eczacılık Eğitimi, Çekirdek Eğitim Programı, Hacettepe-ÇEP, [19.] I. Bologna Süreci Araştırmaları Kongresi, s , 2015, Hacettepe Üniversitesi, Beytepe, Ankara. [20.] Accessed 16 November 2018 [21.] Accessed 16 November 2018 [22.] Accessed 16 November 2018 [23.] kod=2070&submenuheader=2&prg_kod=20701&programduzey=3&dil_kod=1 Accessed 16 November 2018 [24.] kod=2070&submenuheader=2&prg_kod=20702&programduzey=5&dil_kod=1 Accessed 16 November 2018 [25.] Accessed 16 November

144 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-137 Targeted Cancer Therapy Using Nano Drug Delivery Systems, Struggles, Strategies and Prospects Fatemeh Bahadori Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, Istanbul Turkey Abstract Cancer, one of the leading causes of death in the last decades, is hard to be completely treated due to the many reasons including insufficient efficiency of chemotherapeutic drugs etc. Nano-technology, is a promising area in cancer treatment along with other bio-medical applications. Damaged vein structure of cancer tumors provides facility of accumulating nan-drug delivery systems (NDDS) at this side. While the gap between the endothelial cells of the vein surface are between nm in healthy tissue, this gap reaches up to 200 nm in cancer tumors due to inflammation. Encapsulation of chemotherapeutic drugs inside of the NDDS with size less than 200 nm, enhances their accumulation at the tumor side. The so called Enhanced and Permeability Retention (EPR) effect constitutes the basics of passive targeted cancer therapy. Nevertheless, the lack of lymphatic system and drainage at tumor side causes a high pressure, by which the accumulated NDDS are pushed back to the veins. This is where decoration of the surface of NDDS with the ligands of the over expressed receptors on cancer cell (active targeting) takes a very important place in treating cancer in a more efficient way. Scientific research on targeted cancer therapy using NDDS, their pre-clinical and clinical applications and commercially available liposomal formulations have shown their success in enhancement of activity and reduction of side effects of chemotherapeutic agents. On the other hand, while optimization of size and stability of NDDS are of great importance for achieving targeting purpose, a small number of NDDS are capable of production in the industrial scale. Thus, the process of NDDS production from research to industry remains fruitless. Although normalization of vein structure is one of the most important strategies in cancer treatment, this attempt could reduce the efficacy of EPR effect, thus, new approaches to this effect suggest using NDDS with size less than 40 nm to provide possibility of using antiangiogenetic therapeutics. The samples of struggling and prospects of targeting cancer therapy mentioned above could be extended to pages, however, with no doubt NDDS will be the most promising vehicles in treating cancer in near future. Keywords Nanotechnology, Targeted Cancer Therapy, EPR Effect, Active Targeting, Industrial Production 142

145 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-141 Preparation, Optimization and In Vitro Evaluation of Novel Self Emulsifying Drug Delivery Systems for Oral Administration Neslihan Üstündağ Okur 1, Emre Şefik Çağlar 2, Mine Diril 3, Hatice Yeşim Karasulu 3 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Health Sciences, İstanbul, Turkey 2 Department of Pharmaceutical Technology, School of Pharmacy, Istanbul Medipol University, İstanbul, Turkey 3 Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege University, İzmir, Turkey Abstract Oral Drug Delivery (ODD) is one of the most preferred drug administration routes due to various advantageous. Bioavailability which is effected by the solubility of drug molecule is the key factor of therapeutic effectiveness. In this study, the SEDDS for oral delivery of Domperidone maleate (DMP) were developed through the construction of pseudo-ternary phase diagram and optimized with a simple method. The developed DMP loaded SEDDS achieved good stability, narrow particle size distribution, low PDI. In addition, all formulations had suitable ph and conductivity for SEDDS. The prepared formulations especially S3-DMP are promising for oral delivery of BCS class II drugs. Keywords Self-emulsifying drug delivery; oral administration; bioavailability 1. Introduction ODD is one of the most preferred drug administration routes due to various advantageous features such as safety, patient convenience and compliance, cost effectiveness, least sterility constraints, flexibility in formulation design and ease of manufacturing [1 4]. Drug candidates which have low solubility and high permeability [Biopharmaceutical Classification System (BCS) Class II drugs] are being more prevelant in pharmaceutical industry. At this point, the low solubility of the active pharmaceutical ingredient is affecting the drug delivery and concentration in the blood circulation [4,5]. DMP is lipophilic and BCS Class II drug, is poor water-soluble and it is rapidly absorbed from GIT. Self-Emulsifying Drug Delivery System (SEDDS) is isotropic mixtures of drug, lipids and surfactants, usually with one or more hydrophilic cosolvents or coemulsifiers [6]. The objective of this study was to develop and characterize new domperidone loaded SEDDS as an alternative formulation and to evaluate the permeability of domperidone SEDDS by using Caco-2 cells. 2. Material and Methods 2.1. Materials DMP was gifted by Deva Holding, Turkey. Oleic acid, PEG 400, ethanol, Span 80, Tween 20 and Tween 80 were purchased from Merck, Germany. Cremophor EL was purchased from Sigma-Aldrich, Germany. Labrasol, Capyrol 90, Labrafac PG, and Transcutol HP were gifted by Gattefosse, France. HPLC grade acetonitrile and methanol was obtained from Sigma (Germany). Intestinal medium tablets were purchased from Merck, Germany. Distilled water was used throughout the study. 143

146 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 2.2. Methods Screening of excipients An excess amount of DMP was added into 2mL solvent and then allowed to equilibrate under continuous mixing for 72h at 25±2 o C. Drug concentration was determined with HPLC HPLC analysis For cell culture studies, the modified method was used. The samples were analyzed at 230nm with 2mL/min at 27±2 o C flow rate. Injection volume of the samples was 5 µl. The mobile phase used was phosphate buffer (50mM, ph 3.5) and acetonitrile (80:20, v/v). Retention time of the drug was 15 min. Both methods were validated partially linearity, limit of detection (LOD) and limit of quantitation (LOQ), precision, accuracy and specificity, selectivity and stability Preparation of DMP loaded Self-Emulsifying Drug Delivery Systems The formulations were prepared using oleic acid as oil, Labrasol, PEG 400 and Tween 80 as surfactant, and Transcutol HP as a co-surfactant. After the identification of SEDDS region in the phase diagrams, the formulations were selected at desired component ratios. 10mg DMP was added into the each optimum 5g SEDDSs Characterization of DMP Loaded Self-emulsifying Drug Delivery System The average droplet size, conductivity, viscosity, robustness to dilution and ph of formulations were measured for characterization of the formulations In vitro Drug Release Studies Dissolution vessels were filled with 900mL simulated intestinal medium (ph 6.8) and set 37 C and stirred at 50rpm. Samples (1mL) were withdrawn from release medium at predetermined time interval Permeability studies in Caco-2 Cell Monolayer Caco-2 Cell Culture Caco-2 were cultured in Dulbecco s modified Eagle medium (DMEM). The medium was composed of 50mL foetal bovine serum, 5mL of penicillin (100IU/mL), 5mL of alanine and 5mL of Hank s Balanced Salt Solution (HBSS) supplemented with 450mL L-glutamine (1%, v/v, 2 mm). The Caco-2 cells were cultured from passage number 21 to 55. Cell monolayers were prepared by seeding 4x105 cells/4.67 cm 2 on six transwell insert filter for permeability Permeability Studies The in vitro permeability study was carried out on Caco-2 cell monolayers. The cells were maintained at 37 C in an atmosphere as described above. The medium was replaced every second day for 3 weeks. 3. Results It was seen that the highest solubility of DMP was in PEG 400 (8.622±0.009 mg/ml), Tween 20 (5.898±0.009mg/ ml), Tween 80 (5.038±0.006mg/mL), Cremophor EL (4.718±0.053mg/mL), Oleic Acid (2.981±0.033mg/mL), Transcutol HP (2.630±0.027mg/mL), and Labrosol (2.400±0.009mg/mL), respectively. Figure 1 shows the pseudo ternary phase diagrams for drug free SEDDS formulations. Table 1 shows the concentration of each component for preparation of optimum formulations. 144

147 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Table 1: The amount of components of SEDDSs Components (%) S1 S2 S3 Oleic acid Tween Labrasol PEG Trasncutol HP HLB Viscosity values of the formulations were found between ± 0.049cP. In addition, ph values of formulations were measured in between 6.46 ± ± For robustness to dilution studies, formulations showed no signs of phase separation, cloudiness, and precipitation of the drug itself that ensured the stability of the reconstituted formulations. Each of the formulations in all dilutions and media (Table 2) showed similar droplet size. Table 2: The effect of various dispersion medium and volume on droplet size (nm) of formulations ph 1.2 Time/dilution ratio 1:10 1:100 1:200 1: h 331.1± ± ±7 367± h 345± ± ± ±6 S1-DMP 12. h 352.6±9 365±10 376± ±8.5 ph 6.8 Time/dilution ratio 1:10 1:100 1:200 1: h 320± ± ± ±11 4. h 332± ± ± ± h 344± ± ± ±9.8 ph 1.2 Time/dilution ratio 1:10 1:100 1:200 1: h 251.8± ± ± ± h 268.8± ± ± ±.9.6 S2-DMP 12. h 271±12 283± ± ±8.8 ph 6.8 Time/dilution ratio 1:10 1:100 1:200 1: h 242.1± ± ± ± h 250± ±12 260± ± h 256± ± ±11 273±4.7 ph 1.2 Time/dilution ratio 1:10 1:100 1:200 1: h 553.6± ± ± ±11 4. h 561± ± ± ±13.5 S3-DMP 12. h 574± ± ± ±15 ph 6.8 Time/dilution ratio 1:10 1:100 1:200 1: h 482.5± ±10 499± ± h 487± ± ± ± h 498± ± ± ±2.5 In addition, PDI values for all formulations were found smaller than 0.5. At the end of heating and cooling cycle test, S1-DMP, S2-DMP and S3-DMP passed the test. Phase separation signs were not observed for all formulations. The cumulative percentage drug release from S1-DMP and S3-DMP formulations were found to be 100 ± 4.176% and ± 3.250%, respectively, for 4 h which was significantly higher than the marketed formulation (52.58 ± 145

148 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 2.866%). Table 3 shows the permeability coefficent (Papp) values [Papp (A B) and Papp (B A)] of DMP loaded formulations S1-DMP and S3-DMP, solution and commercial tablet. In addition, Figure 2 shows the permeated concentration of DMP from SEDDS, solution and marketed product across the Caco-2 cell monolayer. Table 3: The permeability values of DMP loaded S1-DMP and S3-DMP formulations, commercial tablet and DMP solution with SD. Papp (A B), permeability value for A B direction. Papp (B A), permeability value for B A direction Formulation/ Papp Papp (A B) Papp (B A) Papp(B A) Papp(A B) Ratio SOL S1-DMP S3-DMP C 1.56x10-4 ±1.05x x10-4 ± 8.29x x10-4 ±9.7 x x10-6 ± 2.65x x10-3 ±9.31x x10-4 ±7.8x x10-4 ±3.4x x10-4 ±1.17x10-4 The TEER values of formulations were found between ± and ± (ohms/cm) before the experiment. After the experiment, TEER measurements showed that the TEER values were in between ± and ± (ohms/cm). 4. Discussion Surfactants which have high HLB values have good efficiency of self-emulsification [7]. Formulations S1 and S3 showed increased self-emulsification attitude. Each of the formulations in all dilutions and media showed similar droplet size and the droplet size was found in the self-emulsifying region. The data obtained from the in vitro drug release studies indicate that the release rate of DMP from SEDDS formulations was considerably faster than the marketed drug formulation. Thus, it can be evaluated as that the prepared SEDDS formulations showed improved solubility of DMP. The results of permeability study indicated that permeability values of DMP from SEDDS formulations were greater than that of commercial tablet and solution. In this study, prepared formulations had efflux ratios less than 2. This can be evaluated as that the transport mechanism of SEDDS formulations in this study is passive. Even though TEER changes were observed, cell monolayer was not damaged because the variation of TEER values was not higher than 40%. This demonstrated that Caco-2 cells were still viable after the completion of all permeability studies. 5. Conclusion In this study, the SEDDS for oral delivery of DMP were developed through the construction of pseudo-ternary phase diagram and optimized with a simple method. The developed DMP loaded SEDDS achieved good stability, narrow particle size distribution, low PDI. The prepared formulations especially S3-DMP formulations are promising for oral delivery of BCS class II drugs. Furthermore, the bioavailability of insulin was improved by using SED- DSs [8]. In this regard, our formulations are promising way of drug delivery for biotechnologic compounds. References [1.] Thanki K, Gangwal RP, Sangamwar AT, Jain S. Oral delivery of anticancer drugs: Challenges and opportunities. J Control Release [Internet]. Elsevier B.V.; 2013;170: Available from: jconrel [2.] Viswanathan P, Muralidaran Y, Ragavan G. Challenges in oral drug delivery: a nano-based strategy to overcome. Nanostructures Oral Med [Internet]. Elsevier; 2017 [cited 2018 Feb 10]. p Available from: 146

149 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS [3.] Prabhu S, Ortega M, Ma C. Novel lipid-based formulations enhancing the in vitro dissolution and permeability characteristics of a poorly water-soluble model drug, piroxicam. Int J Pharm [Internet]. Elsevier; 2005 [cited 2018 Feb 8];301: Available from: S ?via%3Dihub [4.] Kansara H, Panola R, Mishra A. International journal of drug development & research. [Internet]. Int. J. Drug Dev. Res. International Journal of Drug Development & Research; 2009 [cited 2018 Feb 10]. Available from: [5.] Pouton CW. Formulation of poorly water-soluble drugs for oral administration: Physicochemical and physiological issues and the lipid formulation classification system. Eur J Pharm Sci. 2006;29: [6.] Neslihan Gursoy R, Benita S. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs. Biomed Pharmacother [Internet]. Elsevier Masson; 2004 [cited 2018 Feb 8];58: Available from: [7.] Shen H, Zhong M. Preparation and evaluation of self-microemulsifying drug delivery systems (SMEDDS) containing atorvastatin. J Pharm Pharmacol [Internet]. Blackwell Publishing Ltd; 2006 [cited 2018 Feb 19];58: Available from: [8.] Li P, Tan A, Prestidge CA, Nielsen HM, Müllertz A. Self-nanoemulsifying drug delivery systems for oral insulin delivery: In vitro and in vivo evaluations of enteric coating and drug loading. Int J Pharm [Internet] [cited 2018 Nov 16];477: Available from: 147

150 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-142 Development, Characterization and In Vitro-In Vivo Evaluation of In Situ Gel Systems for Improving Therapeutic Efficacy of Ocular Drug Delivery Neslihan Üstündağ Okur 1, Vildan Yozgatli 2, Mehmet Evren Okur 3, Panoraia I. Siafaka 4 1 University of Health Sciences, Faculty of Pharmacy, Department of Pharmaceutical Technology, Üsküdar, 34668, İstanbul, Turkey 2 Istanbul Medipol University, School of Pharmacy, Department of Pharmaceutical Technology, Beykoz, 34810, Istanbul, Turkey 3 University of Health Sciences, Faculty of Pharmacy, Department of Pharmacology, Üsküdar, 34668, İstanbul, Turkey 4 Aristotle University of Thessaloniki, Department of Chemistry, 54124, Thessaloniki, Greece Abstract: The in situ gelling system was applied to increase the residence time and thus the bioavailability of voriconazole (VCZ) in the ocular mucosa. Temperature triggered in situ gels were prepared by cold method using poloxamer 188, poloxamer 407. These formulations were evaluated for clarity, sol-gel transition temperature, gelling capacity, ph, viscosity, FTIR and drug content for ocular administration. Furthermore, sterility, antifungal activity, stability, in vitro drug release, ex vivo permeation and penetration and in vivo study of these formulations were also examined. In conclusion, VCZ loaded in situ gels could be offered as a promising strategy for ocular drug delivery. Keywords: thermo-sensitive in situ gel; ocular drug delivery; in vivo; rabbit. 1. Introduction The anatomy, physiology, and biochemistry of the eye render this organ exquisitely impervious to foreign substances such as active ingredients [1]. The meager bioavailability (<5%) of conventional eye drops (suspensions, solutions, etc.) is one of the major concern for pharmaceutical technologists and companies [2]. In situ gels, in contrast to already gelled formulations, are advantageous formulations considering that can be accurately administered in reproducible quantities, promoting precorneal retention [3]. Fungal keratitis is a leading cause of serious ocular morbidity and blindness. Although, fungal infections are distributed worldwide, are found more frequent in the tropics and subtropical regions. Voriconazole (VCZ) is a potent new triazole derivative with a broad spectrum of antifungal activity against many opportunistic fungal pathogens including Candida, Cryptococcus, and Asprgillus species [4,5]. The aim of this research was to develop new ocular in situ gel formulations containing VCZ for the treatment of fungal keratitis by topical use while these ocular in situ gels were evaluated for effective topical ocular delivery. 2. Materials and Methods 2.1. Materials VCZ was purchased from Sigma, Germany. Poloxamer 407(P407) and poloxamer 188(P188) were kind gift from BASF, Turkey. Carboxymethyl cellulose (CMC) was purchased from ZAG, Turkey. High pressure liquid chromatography (HPLC) grade acetonitrile (Sigma, Germany) was used for HPLC studies. For microbiological studies Fluid thioglycollate medium purchased from Merck Millipore Corporation, Darmstadt Germany, soya-bean casein digest medium (CM0129) purchased from Oxoid, Thermo Fisher Scientific Inc. was used. 148

151 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS 2.2. Preparation of In Situ Gel Formulations For the preparation of an ocular in situ gel; CMC, VCZ, BZC as well as sodium chloride were incorporated in aqueous solutions containing P407, P188 and distilled water. BZC (0.02% w/w) was added as a preservative to the solutions. Sufficient amount of sodium chloride (0.9% w/w) was added to adjust the isotony. In situ gel formulations were selected according to ph values and gelling temperatures results of formulations Characterization and Stability of In Situ Gels For the characterization studies of in situ gels, clarity, sol-gel transition temperature, gelling capacity, ph, viscosity, FTIR and drug content parameters of formulations were measured. In physical stability studies, VCZ loaded in situ gels were stored at 4±1 C in the refrigerator for 3 months in the stability cabinets and ph and VCZ content of gels were investigated In Vitro Drug Release Studies In vitro release study of in situ gel solution was carried out in simulated tear fluid as a medium, at 32±0.5 C. 5g of formulations was separated from release media by means of dialysis membrane (Spectra/por, MW of kda) and capped with closures. The samples were analyzed with HPLC Microbiological Studies of In Situ Gel To check the sterility of the prepared with or without VCZ ocular formulations, sterility control testing were performed and it was carried out under aseptic conditions according to the international pharmacopoeia. Disk diffusion testing was performed according to CLSI standard M44-A2 for yeasts and M51-A for filamentous fungi. C. albicans ATCC and C. tropicalis RSKK 2412, A. fumigatus ATCC A. niger ATCC 10864, A. terreus ATCC and A. flavus ATCC were used Ex Vivo Studies (Permeation and Penetration studies) The corneal permeation and penetration experiments were carried out using diffusion cells. The excised goat corneas were clamped between the donor and the receptor chamber of vertical diffusion cells. Penetrated and permeated drug were assayed by means of HPLC In Vivo Studies Determination of the VCZ in tear New Zealand albino rabbits ranging in weight from 2.5 to 3.5 kg were used. The in vivo experimental protocol was approved by the Ethical Committee of Bezmialem Vakıf University (No. 2018/118). Six rabbits were used in the study and during the experiments, the rabbits were allowed to move their heads freely, and their eye movements were not restricted. Schirmer Tear Test Strip (ERC ) was used for the take tears. After the formulations were applied to eye, tears were collected at 0.5, 1, 2, 3, 4, 6, 8, and 24hr using tear strip. Statistical data analysis was performed using the student's t-test with P < 0.05 as the minimal level of significance Ocular irritation test The possible ocular irritancy and/or damaging effects of VCZ loaded in situ gels were evaluated according to modified Draize test. 3. Results The components of ocular in situ gels are shown in Table

152 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Table 1. Formulation codes (FC) and components FC P407 P188 CMC VCZ BZC Physiological Saline (q.s) IS-1-VCZ IS-2-VCZ IS-3-VCZ IS-4-VCZ IS-5-VCZ IS-6-VCZ IS-7-VCZ IS-8-VCZ The physicochemical parameters of VCZ in situ gels are reported in Table 2. Table 2. Physicochemical properties of VCZ loaded in situ gels FC IS-1- VCZ IS-2- VCZ IS-3- VCZ IS-4- VCZ IS-5- VCZ IS-6- VCZ IS-7- VCZ IS-8- VCZ Gelling Temperature ph Clarity Gelling Capacity (sec) Spreadability (cm) Viscosity (cp) Drug content ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.219 For stability studies the samples were analyzed periodically on every month, and found that there are no changes in visual appearance, clarity, and gelation time. FT-IR spectroscopy belongs to the most useful techniques of pharmaceutical formulations given that can provide information concerning interactions between carriers and active molecules as well as compatibility of drug and excipients. VCZ unloaded and loaded in situ gels FT-IR peaks are shown in Figure 1-a),b). In vitro drug release results of VCZ loaded in situ gels are shown Figure 2. The prepared in situ gels containing VCZ were tested in terms of their antifungal ability. The developed in situ gels were demonstrated anti fungal activity against C. albicans, C. tropicalis, A. fumigatus and A. flavus. In order to check the sterility of the formulations sterility control testing in fluid thioglycollate medium for anaerobic and soya-bean casein digest medium for fungi and aerobic bacteria were used and after incubation period for 14 days no growth of any microorganism was seen. Ex vivo permeation and penetration studies results of in situ gels, after 24 h of the contact period, the amount of VCZ 150

153 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS permeated thorough the cornea from IS-1 and IS-5 formulations was determined ±6.81%, ±2.546%, respectively. The penetration to the corneal tissue was examined it was seen that results for IS-1-VCZ and IS-5-VCZ formulations were obtained 4.992±1.056% and 7.569±0.257%, respectively. Figure 3 depicts VCZ concentration in the tear fluid as in vivo studies demonstrated. Maximum concentration of VCZ obtained from in situ gel (IS-1-VCZ) found to be increased in comparison to VCZ solution. To evaluate the ocular irritation of ideal formulations Draize Rabbit Eye Test was used. Only grades 0 and occasionally 1 were recorded according to modified Draize test. No difference between control and treated eyes for each group of rabbits were observed (Figure 4). 4. Discussion Temperature sensitive in situ gels were successfully prepared by cold method using P407, P188. Psysicochemical characterizations of in situ gel formulations are important issues to be considered in the formulation stage, especially those intended for ocular administration. Physicochemical properties of IS-1 and IS-5 formulations are within the desired range, so the above mentioned gels could be promising vehicle for opthalmic drug delivery and all the formulations showed sustained drug release for a period of 8 h, which is satisfying in order to treat ocular diseases. IS-1VCZ formulation was selected for in vivo studies according to the characterization, in vitro release, ex vivo permeation and penetration studies results. 5. Conclusions In the present study, the potential of VCZ loaded in situ gels as drug carriers for ocular delivery was investigated. The clarity, ph, gellation time and drug content of all formulations was found to be satisfactory. According to characteristics, in vitro, ex vivo and in vivo studies of formulations, IS-1 found to be suitable for application to the eye. In conclusion, this research can open up a window on opthalmic field since in situ gels loaded with API, found to be better alternative to conventional eye drops in the treatment of ocular disease. Furthermore, in situ gelling systems could be useful with biotechnological materials treatment of glaucoma [6]. In this regard, our formulations are promising way of drug delivery for biotechnologic compounds. 6. References 1. Aldrich DS, Bach CM, Brown W, Chambers W, Fleitman J, Hunt D, et al. Ophthalmic Preparations. Stimuli to Revis Process - US Pharmacopeial Conv. 2013;39: Kumar R, Sinha VR. Colloids and Surfaces B : Biointerfaces Preparation and optimization of voriconazole microemulsion for ocular delivery. Colloids Surfaces B Biointerfaces [Internet]. Elsevier B.V.; 2014;117:82 8. Available from: 3. Üstündag-Okur N, Yoltas A, Yozgatli V. Development and Characterization of Voriconazole Loaded In Situ Gel Formulations for Ophthalmic Application. Turk J Pharm Sci. 2016;13: Siafaka P, Üstündağ Okur N, Mone M, Giannakopoulou S, Er S, Pavlidou E, et al. Two Different Approaches for Oral Administration of Voriconazole Loaded Formulations: Electrospun Fibers versus β-cyclodextrin Complexes. Int J Mol Sci. 2016;17: Üstündağ Okur N, Çağlar EŞ, Yozgatli V. Development and Validation of an Hplc Method for Voriconazole Active Substance in Bulk and its Pharmaceutical Formulation. MARMARA Pharm J. 2016;20: Mittal N, Kaur G. In situ gelling ophthalmic drug delivery system: Formulation and evaluation. J Appl Polym Sci. 2014;131:

154 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-143 Comparision of Invasive duktal carsinoma (IDC) and Invasive lobular carcinoma (ILC) for Identification of Novel Biomarkers Umut Ağyüz 1, Yasemin Müşteri Oltulu 2, Tuba Saraç 3, Tuğba Elgül 4 1 Umut Ağyüz 2 Yasemin Müşteri Oltulu 3 Tuğba Elgül 4 Tuğba Saraç Abstract Purpose: The purpose of this study is to compare two main subtypes of breast cancer; invasive ductal carcinome (IDC) and invasive lobular carcinome (ILC). Introduction: Invasive Ductal means that the cancer has invaded or spread to the surrounding breast tissues from the ductal wall. In this type, the cancer is surrounding milk duct, but their shapes are not deformed. This is why it is very difficult to identify in mamography. Methodology: 28 ILC and 5 IDC patients data was gathered from online databases. GEO, Arrayexpress, SRA. We developed a software using R bioconductor for differential expression analysis of the data. Our analysis picked TFF3, MMP9, DUSP1, SCGB2A2, CTHRC1, APOD, TGFB3, NMU, IGFBP1, CD34 genes as differentially expressed. The relations among genes are demonstrated using online STRING tool. 3 genes were strongly linked, MM9, CD34 and CTHRC1. Result: CD34 is up regulated in ILC ( logfc 1.693, p <0.05) and down regulated in IDC ( logfc -0.9, p <0.05). CD34 is a transmembrane phosphoglycoprotein protein encoded by the CD34 gene in humans. Lack of CD34 gene expression in the cell, causes myofibrosarcoma in breast Discussion: CD34 is glikoprotein playying role in cell adhession and signal transduction. It is expressed by mesenchymal cells. Breast invasive neoplasm includes lots of CD34+ stromal cells in stroma. Matrix metalloproteinase (MMP) is a member of endopettidase protein family digesting extracellular matrix (ECM). These proteases cause cell migration and malignancy with seperating collogen from epitel and vascular main membrane. Resources - Romualdo Barroso-Sousa and Otto Metzger-Filho, Differences between invasive lobular and invasive ductal carcinoma of the breast: results and therapeutic implications; Ther Adv Med Oncol Jul; 8(4): Oh CD, Maity SN, Lu JF, Zhang J, Liang S, Coustry F, de Crombrugghe B, Yasuda H PLoS One. Identification of SOX9 interaction sites in the genome of chondrocytes 5(4):e10113 (2010). - KAÇAR A, PAKER İ, AKBIYIK F, ARIKÖK AT, MAMBET A; Cd117 and Cd34 Staining Patterns in Childhood Benign Mammary Lesions; Cilt/Vol. 28, No. 1, 2012; Sayfa/Page 31-37; doi: /tjpath Keywords Breast cancer, biomarker, ductal carcinoma, lobular carcinoma, gene expression. 152

155 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS S-149 A Novel Diagnosis Approach on Paraffin Embedded Gastric Tissue Samples via Chemometrics Assisted Raman Spectroscopy Yücel Kadoğlu, Onur Şenol Ataturk University, Faculty of Pharmacy, Department of Analytical Chemistry, 25240, Erzurum, Turkey Abstract Gastric cancer is mortal and several different methods are applied related to the technological developments to understand its ethiopatogenesis and diagnosis. In these days, raman strategies are accepted to be one of the most encountered analytical methods. Identifying and following of proteins, metabolites and bioelements is crucial in protecting, diagnosing and treatment of gastric and other cancer species. The concentration of these compounds may cause several pathological changes (1). Differences in concentration of protein and metabolite or bioelement level may cause toxicologic effect in organism and may even lead to death in next stages. In health sciences, biomarkers are frequently used in early stage-diagnosis and treatment. It is strongly suggested by the scientific groups that simultaneous observation and analysis of different unrelated potential biomarkers are preffered to usage of single biomarkers due to lack of sensitivity and specificity. Medical photonics term described as using optical technology and instruments for diagnosis, therapeutic and basic science application in medicine. Nowadays, Medical photonics methods such as FTIR and Raman spectroscopy assisted with chemometrics became popular for diagnosis of gastric cancer as it is in other types of cancer. It can be perform chemical analysis with these non-invasive analytical methods by providing spectral differences observed as a function of tissue physiology or pathology. The obtained data can be analyzed with chemometrics methods or different statistical methods and diagnostic algorithms gives knowledge of cancer or not. The important advantage of such a diagnosis is automation, which allows objective and real-time diagnosis of pathologies (2). Keywords 1. Garattini SK, Basile D, Cattaneo M, Fanotto V, Ongaro E, Bonotto M, Negri FV, Berenato R, Ermacora P, Cardellino GG, Givannoni M, Pella N, Scartozzi M, Antonuzzo L, Silvestris N, Fasola G, Aprille G, Molecular classification of gastric cancers: No Material and Method A WITec Alpha 300R Micro Confocal Raman Spectroscope including two laser wavelengths (532 and 758 nm) by filtering the Raleigh scattering was used for analyze carcinoma and healthy tissue samples. The grating was 600 g/ mm (blaze value of 750 nm). The analyzed spectral range was recorded in the range of cm -1 to give a resolution of 0.5 µm. Each Raman spectrum was recorded on the spectrometer using an acquisition period of 0.5 s and the width and height of the pixels of spectrums were set to be 1024 and 128, respectively. Background spectrum was obtained and removed from sample spectrum automatically. Every line 20 points were recorded and 50 images were taken for each line which included 20 points Extraction of Parafin Embedded Tissues Extraction of paraffin embedded tissues without lose of significant biological components in a shorter time is a big 153

156 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS concern in raman spectroscopy measurements. Because most of the tissue samples were present in paraffin films and in order to perform a widespread study it is impossible to neglect these samples. Extraction of sample is crucial part due to interferences resulting from wax. Recent methods generally suggest 20 hour or more time for extraction. In our proposed method, extraction were maintained via hexane:mtbe mixture (v/v) keeping much less time for wax removing process (around 12 hour). Results 8 healthy and cancer tissues were taken into account for raman analysis after paraffin extraction process. OPLS-DA characterization were performed to separate each tissue in accordance with the alteration in biological structure. Preprocessing were maintained via Orthogonal Signal orrection method. Preporcessed data has been plotted in figure 1. Figure 1 Preprocessed Data for healthy and cancer tissue via OSC. Score plot regarding Latent Varible 1 & 2 has been monitored in Figure 2. Figure 2. Score Plot for LV1 vs LV2 Specificity and sensitivity of the proposed method was quite high that prove the goodness of fit and found to be 0.92 and 0.96, respectively. 154

157 SÖZLÜ BİLDİRİLER ve TAM METİNLER / ORAL ABSTRACTS and FULL TEXTS Conclusion A novel extraction method for prafin embedded tissue samples were developed for gatric cancer tissue samples. Developed method has been applied onto raman spectroscopy and encouraging results has been obtained. Simple fast, accurate, sensitive chemometrics assisted raman spectroscopy method has been successfully applied onto tissue samples for diagnosis of gastric cancer. References [1.] Garattini SK, Basile D, Cattaneo M, Fanotto V, Ongaro E, Bonotto M, Negri FV, Berenato R, Ermacora P, Cardellino GG, Givannoni M, Pella N, Scartozzi M, Antonuzzo L, Silvestris N, Fasola G, Aprille G, Molecular classification of gastric cancers: Novel insights and possible future applications, World J Gastrointest Oncol (2017) 15, [2.] Pence I, Mahadevan-Jansen A, Clinical instrumentation and applications of Raman spectroscopy, Chem Soc Rev (2016) 45,

158 POSTER BİLDİRİLER ve TAM METİNLERİ POSTER ABSTRACTS AND FULL TEXTS

159 POSTERLER / POSTERS P-003 Economic Analysis of Biosimilar Drugs Approved in Turkey: A Study Covering the Period Sule Apikoglu Rabus 1, Baran Arslan 2 1 Clinical Pharmacy Department, Faculty of Pharmacy, Marmara University, Istanbul, Turkey 2 Faculty of Pharmacy, Marmara University, Istanbul, Turkey Abstract There has been an exponential growth in development of biologic agents. This tremendous growth is life saving on one hand, causing a serious financial threat to the health care system on the other. Manufacturing of biosimilar products help overcome this financial concern. This study aimed to make an economic analysis of a potential therapeutic interchange scenario regarding biosimilar products and their reference products marketed in Turkey. The study was conducted using the data regarding the biosimilar products (ie. insulin glargine, enoxaparin, somatropin, infliximab, filgrastim, epoetin alfa) available in Turkey between 2014 and IMS sales data was obtained from the Turkish Pharmacists Association. Only biosimilar products with the potencies comparable to their reference biologics were taken into analysis. The amount of money that would have been saved (and the number of extra doses that could have been bought with this saved amount) was calculated for a scenario of therapeutic interchange regarding biosimilar products and their reference biologics. Our results showed that starting the treatment with a biosimilar product in treatment-naïve patients or a therapeutic interchange with biosimilar products would help thousands of more patients receive treatment with the money spent on reference biologics. Establishment of a rational policy on biosimilar substitution on institutional as well as national levels is recommended. Keywords Biotechnology, economic analysis, therapeutic interchange, biologicals 157

160 POSTERLER / POSTERS P-004 HPLC-DAD Analysis of Best Anti-AGEs Extracts from Rosmarinus Tournefortii Leaves Salah Akkal 1, Amel Achouri 1, Séverine Derbré 2 1 Varenbiomol Unit, Department of chemistrty, University Mentouri Constantine, Constantine, 25000, Algeria. 2 Université d'angers, SFR QUASAV 4207, EA 921 SONAS, UFR SPIS, Angers, France Abstract During this study, we valorized Rosmarinus tournefortii leaves (Lamiacées) extract selected for its best activity of Advanced Glycation End-products (AGEs). It appeared that several extracts, particularly rich in phenolic acids significantly decreased Advanced glycation endproducts formation. In this context, a phytochemical investigation using dereplication (LC-DAD-MSn) and/or bioguided fractionation was undertaken to identify potent anti-age natural products. These results will be presented. Except for essentiel oil analysis, no phytochemical study was reported for this plant. A local valorization of these extracts either as food supplements for diabetics or as cosmetic active ingredients could be investigated. Keywords Lamiaceae, Rosmarinus tournefortii, Phenolic, LC-DAD-MS, Anti-AGEs 158

161 POSTERLER / POSTERS P-007 Chemical Composition of Organic Volatile Compounds of an Endemic and Rare Apiaceae of Algeria, Bunium Crassifolium Batt Djarri Lakhdar 1, Souileh Nabila 1, Medjroubi Kamel 1, Hamel Tarek 2, Demirtas Ibrahim 3 1 Phytochemistry and Physico-Chemical and Biological Analyzes Laboratory, Faculty of Exact Sciences, Constantine-1 University, Aïn El Bey Road, Constantine, Algeria. 2 Plant Biology and Environment Laboratory. Badji Mokhtar University, 23000, Annaba, Algeria. 3 University of Çankırı Karatekin, Faculty of Science, Department of Chemistry, Ballica Campus, Çankırı, Turkey Abstract The Bunium genus belongs to the Apiaceae family. This genus contains about fifty species distributed in Europe and North Africa, to the southwest and central Asia. It contains aromatic and medicinal plants such as Bunium bulbocastanum and Bunium persicum. In Algeria, the Bunium genus is represented by seven species of which four are endemic. Bunium crassifolium Batt. from the North East of Algeria is the subject of our investigation it s an extremely rare endemic species of the genus. As there was no attempt to study these species until now, we were invited to study the chemical composition of volatile organic compounds (VOC) not essential oils (EO), view the extreme rarity and endemism of the species. The analysis of volatile organic compounds (VOC) of aerial parts were performed using an Agilent G1888 network headspace sampler coupled with an Agilent7890 GC system. GC-MS and GC-FID chromatography revealed the presence of 22 products, 20 of which were identified representing 97.48% of the total composition, the major components are β-cubebene (44.67%), β-caryophyllene (8.82%), γ-elemene (7.04%), δ-cadinene (4.70%), γ-cadinene (4.11%), Ascaridole (3.77%) and β-elemene (3.33%), along with other constituents at relatively low amount. Keywords Apiaceae, {Bunium crassifolium}, volatile organic compounds (VOC), network headspace sampler, GC-MS and GC-FID. 159

162 POSTERLER / POSTERS P-008 Taxol -Producing Endophytic Fungi: A Promising Red Biotechnology Radia Ayad, Kamel Medjroubi, Salah Akkal VARENBIOMOL, Department of Chemistry, Mentouri Constantine 1 University, Constantine, Algeria Abstract Taxol (generic name paclitaxel) represents one of the most clinically valuable natural products known to man- kind in the recent past. The name taxol is derived from the generic name of the producer plant, Taxus (yew). Taxol was first isolated in 1963 from the bark of Taxus brevifolia belonging to family Taxaceae. While,the original source of natural taxol was bark of the Pacific yew, where it is found in minute concentrations of 0.01%-0.05%. Taxol could also be produced by an endophytic fungus, Taxomyces andreanae. This fungus was isolated from a taxol producing plant, Taxus brevifolia. This discovery was projected as the dawn of a new era of endophyte biotechnology with billions of dollars worth of global market for Taxol already in place, and agreements were immediately underway among leading pharmaceutical companies to explore the possibility of fungal Taxol production through industrial fermentation. At the present time, around 200 endophytic fungi belonging to more than 40 fungal genera from several different orders representing mostly Ascomycota and Deuteromycota, with only a few from Basidiomycota and Zygomycota, have been reported to produce Taxol. In the present work, we embark on the current state of the potential use of endophytic micro- organisms (or endophytes ) using red biotechnology, for the commercial production of taxol. Keywords Taxol, {Taxus brevifolia},endophytic fungi, red biotechnology 160

163 POSTERLER / POSTERS P-011 Quantification and Assessment of the Anti-proliferative Activity of Essential Oils of Rosmarinus Officinalis Collected from Three Climatically Dissimilar Locations in Algeria Ouroud Fellah 1, Samir Hameurlaine 2, Noureddine Gherraf 2, Amar Zellagui 3, Muhammed Altun 4, Ibrahim Demirtas 4, Ayse Sahin Yaglioglu 4 1 Faculty of Sciences, Department of Natural and Life Sciences, University of Badji Mokhtar Annaba, 23000, Algeria:: fellah_wouroud@yahoo.fr 2 Laboratory of Natural Resources and Management of Sensitive Environments, Larbi Ben M'hidiniversity, Oum El Bouaghi, Algeria, Ngherraf@Yahoo.Com, Ham_Sar18@Yahoo.Fr 3 Laboratory of Biomolecules and Plant Breeding, Life Science and Nature Department, Faculty of Exact Science and Life Science and Nature, University of Larbi Ben Mhidioum El Bouaghi, Algeria. Zellaguia@Yahoo.Com 4 Laboratory of Plant Research, Department of Chemistry, Faculty Of Science, Uluyazi Campus, Cankirikaratekin University, Cankiri, Turkeymaltun80@Gmail.Com, Ibdemirtas@Gmail.Com, Aysesahin1@Gmail.Com Abstract The present work is aimed mainly to appraise the effect of the climate factors on the quality, quantity and antiproliferative effect of essential oils of the aerial parts of Rosmarinus officinalis collected from three geographic localities in Algeria (humid, semi arid and arid). The Gas-Chromatography/Mass-Spectroscopy analysis revealed important disparities both quantitatively and qualitatively. α-pinene and camphor as major components were found to range from to 40.95% and from to 36.72% respectively. Moreover, The crude oils were subjected to antiproliferative essays against two tumor cell lines, rat brain tumor (C6) and human cervix carcinoma (HeLa) using BrdU (bromodeoxyuridine) ELISA (Enzyme-linked immunosorbent assay) and xcelligence assays. Important findings have been recorded. Keywords Rosmarinus Officinalis, essential oils, climate factors, GCMS, antiproliferative effect 161

164 POSTERLER / POSTERS P-014 Anti-Proliferative and Antioxidant Activities of Chloroformic Extract from Two Euphorbia Species Growing in Algeria Amar Zellagui 1, Agena Ghout 1, Noureddine Gherraf 2, Ibrahim Demirtas 3 1 Laboratory of Biomolecules and Plant Breeding, Life Science and Nature Department, Faculty of Exact Science and Life Science and Nature, University of Larbi Ben Mhidioum El Bouaghi, Algeria. Zellaguia@Yahoo.Com 2 Laboratory of Natural Resources and Management of Sensitive Environments, Larbi Ben M'hidiniversity, Oum El Bouaghi, Algeria, Ngherraf@Yahoo.Com 3 Laboratory of Plant Research, Department of Chemistry, Faculty Of Science, Uluyazi Campus,Cankirikaratekin University, Cankiri, Turkey Ibdemirtas@Gmail.Com Abstract The purpose the present study was to identify the bioactive compounds from two extracts of Euphorbia sp. collected in Algeria by HPLC-TOF-MS technique. the total phenolic and flavonoids contents extracted by chloroform were measured using folin-ciocalteu procedure and flavonoid-aluminium method, The HPLC analysis revealed the presence of 13 compounds in E. dindroide including gallic and chlorogenic acids as major components and 6 compounds in E. cornuta respectively. The growth inhibitory effects of extracts on two different cancer cell lines namely C6 (rat brain tumor) cells, and Hela (human uterus carcinoma) cell lines and various antioxidant assays including scavenging effect against DPPH, reducing power and inhibition of lipid peroxidation were comparatively studied and were compared to various standard such as quercetin, gallic acid and ascorbic acid etc, the Euphorbia cornuta extract with the highest phenolic and flavonoids (43.07 mg EQ/g extracts ) than E. dindroides 7.83±0.12 mg GAE/g extracts contents, showed excellent activity by the DPPH assay and good lipid peroxidation inhibition activity. The chloroformic extract of the two species showed the highest antiproliferative properties than 5-FU against C6 cells and better than those of other polar extract at 250 µg/ml, the results indicate that the pourcentage of inhibition of all extracts are dose dependent and can be attributed to its content phenolic compounds Keywords Euphorbia cornuta, Euphorbia dindroide, HPLC, anti-proliferative activity, lipid peroxidation 162

165 POSTERLER / POSTERS P-022 In Vitro Studies on Inhibition of Mammaglobin Gene Expression with Cchitosan/shRNA Nanoplexes in Breast Cancer Cell lines Aylin Ozkan 1, Ceyda Ekentok 1, Emine Şalva 2, Suna Ozbas Turan 1 1 Department of Pharmaceutical Biotechnology, Marmara University, Istanbul, Turkey 2 Department of Pharmaceutical Technology, Inonu University, Malatya, Turkey Abstract Cancer deaths are in second place in developed societies due to the complex nature of cancer despite of the new treatment approaches. Breast cancer is one of the most common malignant tumors in the world. Human mammaglobin (hmam) is associated with the family of epithelial secretory proteins, limited to mammary gland and breast tumor cell lines. It is shown that hmam mrna is frequently upregulated in the peripheral blood of breast cancer patients. Inhibition of gene expression can be accomplished by RNA interference (RNAi) technology which provides sequence-specific post-translational gene inhibition. Short hairpin RNA (shrna) selected in this study as gene silencing agent due to its longer suppression property. This target-specific method provided a new perspective for gene therapy. Delivery system is also important for successful gene therapy. Chitosan is one of most popular delivery system due to its biodegradable, non-toxic and cationic properties. In this study, chitosan/mammaglobin shrna nanoplexes were prepared to silence hmam gene. For this purpose, plasmid containing hmam shrna was transformed to competent Escherichia coli (GT116 strain) made by CaCl2 method. Thereafter, plasmid DNA was isolated from selected single colony and then spectrophotometric and electrophoretic controls were carried out. The nanoplexes were prepared via the electrostatic interaction of the plasmid and chitosan with different chitosan/shrna ratios (w/w) (0.5/1, 1/1, 2/1, 3/1, 4/1, 5/1). Fully nanoplexation at all ratios was observed according to electrophoresis result. Particle size, zeta potential, gel electrophoresis and serum stability analyses of these nanoplexes were performed. The average size of the nanoplexes were between nm and nm depending on the N/P ratio. The surface charge of nanoplexes was positive with zeta potential of +0.6 to mv. According to serum stability studies results, nanoplexes protect the shrna during 72 hours. Cytotoxicity of nanoplexes is investigated with MTT and BrdU, and no cytotoxic effect was observed. The transfection efficiency and silencing effect of these nanoplexes on mammaglobin gene expression will be investigated in breast cancer cell lines. These studies can benefit that delivery of shrna targeted to hmam using chitosan nanoplexes are promising for the therapy of breast cancer. Keywords RNAi, shrna, mammaglobin, chitosan, breast cancer 163

166 POSTERLER / POSTERS P-023 In Vitro Studies on Inhibition of Chitosan / sirna Nanoplexes and Mammaglobin Gene Expression in Breast Cancer Cell Lines Sadiye Burcu Ozkavak 1, Ceyda Ekentok 1, Emine Şalva 2, Suna Ozbas Turan 1 1 Department of Pharmaceutical Biotechnology, Marmara University, Istanbul, Turkey 2 Department of Pharmaceutical Technology, Inonu University, Malatya, Turkey Abstract Cancer is one of the leading health problems in the world in terms of mortality and morbidity rates. Breast cancer is one of the leading causes of cancer death in women. At the present time, surgery, radiotherapy, chemotherapy and hormonal treatment methods are frequently used in cancer treatment. New methods such as immunotherapy and gene therapy have been developed for the treatment of cancer in recent years. In particular, gene therapy is considered to be the most promising treatment in the future. The RNAi pathway has been one of the remarkable strategy for gene therapy over the last decade. In our study it was aimed to inhibit mamoglobin expression in different breast cancer cells using synthetic sirna targeted to hmam gene. The hmam gene, a 10 kda glycoprotein, is localized to the 11q13 side of the chromosome. In the breast tumor biopsy analysis, hmam mrna levels increased in 23% of cases. Although the previous studies showed that hmam mrna is the most effective marker in breast cancer, the literatures about mammaglobin are limited availability. Cellular uptake and stability of sirna is poor, therefore carrier systems are used to achieve delivery of sirna. Accordingly, we preferred a cationic, biocompatible and nontoxic biopolymer chitosan as the carrier system. In this study, we prepared chitosan/ mammaglobin sirna nanoplexes to silence hmam gene. The effect of these nanoplexes on the suppression of mammaglobin gene expression was investigated in different breast cancer cells in vitro. For this purpose, controls of hmam sirna was researched. Nanoplexes were prepared at different the ratios (20/1, 25/1) of chitosan/sirna. The average size of the complexes was between 177 and 320 nm and zeta potential was between +14 and 23 mv. Nanoplexes need to be protected sirna against enzymatic degradation for activity, therefore serum stability studies was performed. The cytotoxicity and the gene silencing efficiency of the prepared nanoplexes will be investigated. These study can avail that delivery of sirna targeted to hmam using chitosan nanoplexes may be effective for the therapy of breast cancer. Keywords sirna, breast cancer, mammaglobin, chitosan, nanoplexe 164

167 POSTERLER / POSTERS P-024 In Vitro Antioxidant and Photoprotective Activities of Ethyl Acetate Fraction of Hydroalcoholic Extract of the Aerial Parts of Algerian Plumbaginaceae Limonium Thouinii (viv.) Kuntze Mostefa Lefahal 1, El Hani Makhloufi 1, Nabila Zaabat 1, Salah Akkal 1, Kamal Medjroubi 1, Merzoug Benahmed 2, Houcine Laouer 3 1 Department of Chemistry,Constantine University, Constantine, Algeria 2 Department of Chemistry, Tebessa University, Tebessa, Algeria 3 Department of Biology and Plant Ecology, Setif University, Setif, Algeria Abstract The genus Limonium belongs to the Plumbaginaceae family is represented by 350 species that are growing throughout the world. The Algerian flora contains 20 species of Limonium among them, 8 are endemic. Several members of this genus were well known in folk medicine for their cure proprieties.this study was aimed to evaluate the antioxidant and photoprotective capacities of ethyl acetate (EtOAc) fraction of hydroalcoholic extract of the aerial parts of Algerian Plumbaginaceae Limonium thouinii (viv.) Kuntze. The in vitro antioxidant activity was evaluated through two established in vitro METHODS: DPPH radical scavenging and total antioxidant capacity by phosphomolybdenum method (TAC).The in vitro photoprotective effect against UV-B radiations was performed according to the parameter SPF (Sun Protection Factor) by using UV spectroscopic technic at nm and Mansur equation. Estimation of total phenolic and flavonoid contents were also done. Our results revealed that the EtOAc fraction has a significant amount of phenolics and flavonoids (136,62±3,57 mg GAE/g, 27,92± 5,78 mg QE/g, respectively) and exhibited good antioxidant activity in the two systems, (IC50=0,134±0,004 mg/ml) in DPPH test and (82,62±6,01 mg) AAE/g in TAC assay. Furthermore the EtOAc fraction showed high photoprotective activity with the sun protection factor (SPF) value=36,02 ± 0,14. The findings of this study revealed that the EtOAc fraction was found to have a high phenolic and flavonoid contents as well as antioxidant and photoprotective activities.therefore, it appears to be used a sunscreen in pharmaceutical or cosmetic preparations and as a natural source of antioxidant. Keywords Limonium thouinii, Plumbaginaceae, Antioxidant activity, SPF, EtOAc 165

168 POSTERLER / POSTERS P-025 Design and Development of GO/PLA Pharmaceutical System for the Treatment of Diabetic Injuries Sümeyra Ayan, Ebru Demir, Cem Bülent Üstündağ Department of Bioengineering, Yıldız Technical University, Istanbul, Turkey Abstract The healing process of wounds in diabetic patients is more complicated than others. In diabetic patients, the wounds are chronic and the time that is expected to take place to restore the tissues in the skin is much longer. Systems are developed by using tissue engineering information to accelerate the healing process of the tissues that have lost their function. Scaffold preparation is one of the most significant method that can be used for the formation of new tissues to improve chronic wounds in patients with diabetes. In this study, graphene oxide (GO) is designed by modified Hummers method. After that, according to our designed system, PLA is combined with GO. Subsequently, TGF-β and macrolide are added into the optimized GO/PLA dressing. Macrolide is effective antimicrobial agent in order to reduce the risk of hypoglycemia and/or hyperglycemia in patients with diabetic. Hence, we assume that the designed system is promising solution so as to heal wounds on diabetic patients. Keywords PLA, Graphene oxide, TGF-β, Macrolide, Diabetic wounds 166

169 POSTERLER / POSTERS P-026 Graphen Oxide / PLGA Scaffold Design for Tissue Engineering Applications Ebru Demir, Nuran Çalımlı, Sümeyra Ayan, Cem Bülent Üstündağ Department of Bioengineering, Yıldız Technical University, Istanbul, Turkey Abstract Tissue engineering has emerged as a promising approach to treating a lost or defective wound site and thereby improving the wound healing process. Such an approach involves scaffolds, cells, alone or in combination biological factors. There are many studies on the use of electrospinning in scaffolding construction because the mechanical, biological and physicochemical properties of the scaffold can easily be adjusted by changing the composition of the polymer solution and the process parameters. Furthermore, the electrospinning method has the ability to produce a non-woven nano- / micro-fibrous structure with morphological and architectural similarity to the extracellular matrix (ECM) of natural tissue. Poly (D, L-lactic-co-glycolic acid) (PLGA) is a biocompatible and biodegradable copolymer. Due to this feature, there is a wide area of use three-dimensional (3D) scaffolds in tissue engineering applications. PLGA is one of the most successfully used biodegradable polymers because its hydrolysis leads to metabolite monomers, lactic acid and glycolic acid. Additionally, PLGA can be readily electrospun into micro-fibrous or nanofibrous structures. It also supports the attachment, proliferation, and propagation of cells, but conductivity is an important problem in spinal cord injury. In this study, PLGA decorated with graphene oxide (GO) as a scaffold structure was designed for the treatment of spinal cord injuries. Graphene among the most popular nanomaterials thanks to its excellent physical and chemical properties including high electrical and thermal conductivities, physical and chemical stability, exceptional mechanical strength and stiffness. It is aimed to increase the conductivity and fiber structure of the scaffold by modifying PLGA with GO. The addition of GO into a PLGA matrix to obtain better neural tissue regeneration. The biological properties of the PLGA/GO composite material will also be discussed. Keywords PLGA, extracellular matrix, electrospinning, graphene oxide 167

170 POSTERLER / POSTERS P-027 Production of Biotechnological Drugs and Recent Developments Makbule Ayaydın, Şeyma Hande Tekarslan Şahin Department of Pharmaceutical Technology, Istanbul University, Istanbul, Turkey Abstract Biotechnology has a wide range of applications and plays an important role in the pharmaceutical industry. Biotechnological drugs, produced by recombinant DNA technology, are focused on increasing the production of restricted substances and developing new treatments. Medical needs and pharmaceutical production are met by recombinant DNA technology based on controlling target genes. In this process, biotechnological drugs are produced using various enzymes, vectors, host cells. Monoclonal antibodies, vaccines, hormones and biosimilars are products produced by recombinant DNA technology. They improve the standard of living by enabling the treatment of chronic diseases. In this context, new technologies are needed to meet the increasing need for treatment. The manufacturing process comprises obtaining a biopharmaceutical product after a series of treatments, including successive upstream and downstream processes. Advances in the elucidation of the DNA structure suggest that drugs produced by recombinant DNA technology cause an increase in treatment scale. These biopharmaceuticals increase the quality of life as well as hope for other life-threatening diseases that cause a serious number of deaths. Keywords Biotechnology, Upstream Process, Downstream Process, Recombinant DNA Technology, Biopharmaceuticals 168

171 POSTERLER / POSTERS P-028 Applications of DNA-loaded Nanoparticles in Brain Tumors Nahide Zeren Arda, Özgen Özer Pharmaceutical Technology Department, Ege University Abstract One of the methods used in the treatment of brain tumors is gene therapy. Generally, alone active substances cannot cross the blood-brain barrier, so they need a vector to reach the brain. Viruses are one of the most commonly used vectors, but they have safety problems. In this study, studies on the use of modified by various active substances (Chlorotoxin, Lactoferrin, Angiopep-2) DNA-loaded nanoparticles in brain tumors have been reviewed. The capacity of nanoparticular systems to be used as vectors instead of viruses and the results of various active substances when loaded on these systems were evaluated as holistic. Keywords Glioma, DNA-loaded Nanoparticles, Nanobiotechnology, Brain Tumors Introduction Gene therapy has a great potential for brain tumors but therapeutic genes can not arrive to tumor cells automatically. Brain capillary endothelium forms a barrier to the entry of drugs. Thus, ligand-mediated brain-targeting drug delivery has become one of the increasingly important technologies. Co-administration of drugs and DNA has been proposed to enhance gene expression or to provide a synergistic / combined effect of drug and gene therapy. In addition, the use of non-viral gene-delivery systems are more secure and easier than viral vectors. For these reasons, the use of active substance-modified DNA-loaded nanoparticles have been one of the promising approaches for the treatment of brain tumors. Materials and Methods In 3 different studies, the use of chlorotoxin (CTX), lactoferrin (Lf) and angiopep-modified DNA-loaded nanoparticles (NPs) have been studied as a brain-targeting ligand. Vectors are formed by synthesizing conjugates in various ratios of Polyamidoamine (PAMAM), polyethyleneglycol (PEG) and active substances. The DNA and prepared vector solution were added to 50mM / L sodium sulfate solution and vortexed for 30 seconds. In this way, chlorotoxin-modified DNA-loaded NPs, lactoferrin-modified DNA-loaded NPs and angiopep-modified DNA-loaded NPs were prepared. Results CTX modified NPs observed with transmission electron microscopy are in spherical shape. Particle sizes are around 200 nm and the zeta potential is 7.47 ± 0.35 mv. When used alone CTX was administered at concentrations ranging from 1 µg / ml to 10µg / ml, the passage of CTX to C6 glioma cells increased as concentration increased, but the passage to 293 cells remained low. The cellular uptake of PAMAM-PEG-CTX was significantly increased on C6 glioma cells compared to CTX alone, but cellular uptake was reduced when run at low temperature. Cellular uptake in the 293 cells was not altered by modification. Gene expression for PAMAM-PEG-CTX / DNA NPs was observed primarily in glioma 14 days after brain glioma implantation (48 hours after NPs administration) and NPs showed high efficacy. The PAMAM-PEG-Lf / DNA NPs observed with transmission electron microscopy were spherical and the particle size was 211 ± 15.3nm while the zeta potential value was ± 2.12mV. In addition, cellular uptake at different 169

172 POSTERLER / POSTERS temperatures was investigated. The cellular uptake of Lf-modified vectors at 37 C was much higher than at 4 C. When incubated with excess Lf, the intracellular uptake of the vectors is markedly reduced. In Confocal Microscopy Studies, Lf-modified NPs have been shown to enter acidic endolysosomal compartments within 5 minutes and moves partially out after 30mins of incubation. This suggests that the endocytic process may be the main pathway for the removal of Lf-modified NPs. Furthermore, some Lf-modified NPs remained in the acid compartments, indicating the likelihood of reaching BBB (Blood-Brain Barrier). With in vitro studies Lf-modified NPs were observed to be transported across the BBB monolayer. With in vivo studies the exogenous gene expression using Lf-modified NPs was observed in both the BBB and the parenchym. PAMAM-PEG-Angiopep / DNA NPs observed by transmission electron microscopy are in spherical in shape. The particle sizes are around 115 nm and the zeta potential is 16.3 ± 1.6 mv. The cytotoxicity of PAMAM-PEG-Angiopep / DNA NPs at a range of concentrations was evaluated in C6 glioma cells by MTT analysis. After 2 hours of incubation with NPs, the cytotoxicity of NPs is relatively low. When the PAMAM concentration reached 300 mg / ml, the cell viability was about 50%. After 6 hr of incubation, cytotoxicity was relatively increased compared to 2 hr. Conclusions It has been proven that CTX can be used as a specific glioma-targeting ligand. Furthermore, PAMAM-PEG-CTX / DNA NPs are a potential non-viral delivery system for gene therapy of glioma via intravenous administration. In further studies, PAMAM-PEG-CTX can be studied as the delivery vector of lipophilic small molecule drugs such as doxorubicin for glioma. The results of Lf-modified DNA-loaded NPs have shown that better brain targeting can be achieved by the synergistic effects of macromolecular polymers and ligand. Lactoferrin can be used as a brain-targeting ligand to generate drug delivery systems that efficiently target the brain and transcend the BBB. Present findings from studies for PAMAM-PEG-Angiopep / DNA NPs promote further study of the application of non-viral vectors for non-invasive gene therapy of malignant glioma. Based on this review, it can be used as a transmission vector of PAMAM-PEG-active substance nanoparticles prepared using other active agents in gene therapy for brain tumors in future studies. 170

173 POSTERLER / POSTERS P-029 Biotechnological Drugs on Treatment of Neurodegenerative Diseases Hazal Eken, Rana Arslan, Nurcan Bektaş Türkmen Anadolu University Faculty of Pharmacy Department of Pharmacology, Eskişehir, Turkey Abstract Neurodegenerative diseases are a group of diseases that develop with the progressive loss of nerve cells and weakens the functions of the nervous system. Alzheimer's disease, Parkinson's disease, and Huntington's disease, are the most common neurodegenerative diseases (1). The incidence of neurodegenerative diseases increases due to the average life expectancy. There is no effective and satisfactory treatment for neurodegenerative diseases yet. The one of the major obstacle for the successful treatment of neurodegenerative diseases is that drug molecules cannot cross the blood-brain-barrier (BBB) (2). However, recent developments in nanotechnology are promising for the solution of this problem. It is expected that drug-carrying nanostructures will increase the selectivity and blood permeability of the BBB and reduce the risk of neurotoxicity (3). The aim of this study was to investigate the current biotechnological developments in the treatment of neurodegenerative diseases. In recent years, nanotechnology based drug delivery systems, such as polymeric nanoparticles, liposomes, micelles are developed. Nowadays, it is possible to target the active substance to the brain by nanotechnology products. They also ensure the desired drug concentration in the brain by providing sustained release of active agent (1). Meanwhile, studies on nanotechnology products that completely bypass the BBB using the anatomical connection between the nasal cavity and the brain have recently come to the forefront (3, 4). It was demonstrated that the nasal bioavailability of liposomal formulation of donepezil, recently developed nanotechnology product, was significantly higher than traditional donezepil formulation. In addition, therapeutic ways to produce an immune response to amyloid for Alzheimer's are being investigated. Active and passive vaccines are used to prevent amyloid plaques accumulation in the brain prior to functional damage (3). Moreover, oral sustained-release or gastro-retentive formulations, subcutaneous levodopa infusion, and intestinal levodopa/entacapone/carbidopa infusion are being developed for Parkinson s disease treatment (5). Nanotechnological developments show satisfactory results in preclinical studies. In the future, it is thought that nanotechnology based drug delivery systems will improve the clinical response and improve the quality of life of patients with neurodegenerative diseases (4). Keywords Biotechnological drugs, Neurodegenerative diseases, Blood- Brain-Barrier 171

174 POSTERLER / POSTERS P-030 Propolis as a Natural Source for Drug Development Segueni Narimane 1, Akkal Salah 2, Rhouati Salah 2 1 Laboratoire des produits naturels d origine végétale et de synthèse organique. Département de chimie, Université Constantine 1. Constantine. Algérie 2 Laboratoire de Phytochimie et Analyses Physico-chimiques et Biologiques, Département de chimie, Université Constantine 1. Constantine. Algérie Abstract Natural products have become the subject of increasing interest of health industry. The large diversity in their structure and the synergetic effect of their combination are well documented. Propolis, a natural hive product is collected from several plants and mixed with salivary enzymes and beewax. Since ancient time propolis was used as a natural remedy and a food preservative. Propolis was used as a wound healing and cicatrizing substance by the Greek and the Roman. Matrix metalloproteinze-3 (stromelysin) occupies an important role in collagenolytic and ellastolytic cascade leading to skin aging. The objective of the present study is the isolation of a selective inhibitor of this enzyme from Algerian propolis. A bioguided isolation of propolis extract lead to the isolation and the identification of five caffeic acid derivatives. Butanolic extract was found to selectively inhibit MMP-3 activity. This fraction also inhibited plasmin amidolytic activity. Chicoric acid was the most actif compound. This compound could be used as a molecular basis for the development of selective MMP-3 inhibitors. Keywords propolis, skin aging, selective inhibitor 172

175 POSTERLER / POSTERS P-031 Developments in Treatment of Autoimmune and Inflammatory Diseases with Monoclonal Antibodies Yağmur Okçay, Rana Arslan, Nurcan Bektaş Türkmen Anadolu University Faculty of Pharmacy, Department of Pharmacology, Eskişehir, Turkey Abstract Monoclonal antibodies (mabs) are a group of complex and effective biologic agents used in the treatment of immune-mediated inflammatory diseases such as rheumatoid arthritis (RA). This study focuses on the biotechnologic developments in treatment of autoimmune and inflammatory diseases; a review of current literature and a brief overview of results of recent clinical trials are presented. Monoclonal antibodies have exquisite target selectivity and less toxicity as a result of binding specific targets. The clinical value of both mabs and ligand traps has been proven. Despite the therapeutic value of current RA treatments, agents with alternative modes of action are under investigation. Over the last decade, directed against a number of different target molecules, mabs have received US Food and Drug Administration (FDA) approval for the treatment of RA and other rheumatic diseases. These mabs are mainly directed against tumor necrosis factor-α, CD20-positive B cells, interleukin-1 and interleukin-6. In recent studies, new targets such as fractalkine (FKN)-a chemokine that regulates chemotaxis and adhesion of CX3C chemokine receptor 1 (CX3CR1)-expressing inflammatory cells, are being directed with mabs and effective results are acquired in clinical trials. E6011 as a biologic disease modifying anti-rheumatic drug targets FKN/CX3CR1 interaction and has been found to be well tolerated in patients with RA. RA disease activity significantly decreased in a study performed with mavrilimumab, a fully human mab targeting the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor-α, and clinically meaningful responses are observed which represents a novel mechanism of action with persuasive therapeutic potential. Secukinumab, the first in its class to receive FDA approval, is a fully human monoclonal antibody that selectively binds to IL-17A. It prevents IL-17A from binding to its receptor and inhibits its ability to trigger inflammatory responses that play a role in the development of various autoimmune diseases. Monoclonal antibodies have now being designed to target two or more targets simultaneously, augmenting their therapeutic potential. A fully in-human study of bimekizumab, selective dual inhibitor of interleukin-17a and interleukin-17b, showed fast onset of clinically meaningful efficacy. Above mentioned scientific developments point out the increase in selectivity of mabs, thereby an increase in benefit. Keywords monoclonal antibodies, autoimmune and inflammatory diseases, rheumatoid arthritis 173

176 POSTERLER / POSTERS P-032 Biotechnological Aapplications of Heat Shock Proteins Mevcude Çam, Rana Arslan, Nurcan Bektaş Türkmen Anadolu University Faculty of Pharmacy Department of Pharmacology, Eskişehir, Turkey Abstract Heat shock proteins (HSPs) are involved in all living cells and their expression increase when cells are exposed to stressors. HSPs are very important in the control of cell metabolism, cell growth, differentiation, division and apoptosis, and also in development and metastasis of cancer by shortening of cell turnover, inhibition of apoptosis, stabilization of mutant genes and induction of immunostaining[1]. Heat shock proteins are classed according to molecule weight as HSP 90, HSP 70 HSP 60 and small heat shock proteins (shsps) family[2]. In this study, we aim to evaluate the role of therapeutic and prophylactic potential of HSPs in biotechnological studies. Recent studies it has been seen that shsps have important biological functions in thermostability, disaggregation, and proteolysis inhibition. These functions can be used to develop new techniques and show promise for various applications including proteomics, nanobiotechnology, bioproduction, bioseparation, and other processes[3]. In additionally, shsps can be applied for the development of protein and peptide systems including chip-based and diagnostic immunological assays. Ehrnsperger et al. developed an approach using murine Hsp25 to stabilize the enzymatic activity and antigenicity of the proteins used in immunogenic detection[4]. New biotechnological products such as HSP-based vaccines have been investigated for therapeutic and prophylactic purposes in cancer and infectious diseases treatment. The action mechanisms of these vaccines are the activation of tumor specific immunity, increase of cell proliferation and cytotoxic properties of cancer-specific CD8+ T-cells, and prevention of tumor growth and autoimmune disease[5]. In recent study, it has been claimed that DNA vaccine, including recombinant HSP 90 or active fragments, treat inflammatory autoimmune diseases[6]. In another study, it has been observed that the vaccine, purified recombinant HSP 65 from M. Bovis, inhibits the tumor growth and prolongs mice survival[7]. Biotechnological HSP based vaccines may be as promising therapeutic options in the treatment of cancer and autoimmune diseases since they target specific proteins. Keywords Heat shock proteins, Recombinant DNA vaccine, immunity, cancer, inflammatory autoimmune disease. 174

177 POSTERLER / POSTERS P-033 Graphene Based Nano Drug Design for the Treatment of Breast Cancer Elif Akyar 1, Fadime Çetin 2 1 Department of bioengineering, Yıldız Technical University, Istanbul, Turkey 2 Department of bioengineering, Yıldız Technical University, Istanbul, Turkey Abstract Breast cancer is the most common type of cancer in women and begins in breast cells, and caused by an uncontrolled growth of cells in the breast tissues. Surgical procedures and chemotherapeutic drugs are used in the treatment. The main disadvantages of classical therapies are the inability to completely clear the tumor in surgical procedures and the damage to the healthy cells with tumor cells in chemotherapy. In this study, it has been proposed to use nanoscale graphene oxide as the carrier material. GO has a large surface area that targets therapeutic agents. Single atom size graphene is the best nano-platform for targeted drug systems because of its efficient molecular loading and bioconjugation, mechanical and chemical stability, flexible physicochemical properties, excellent drug bio-molecule conjugation, good biocompatibility and low toxicity. In order to bind to graphene, FDA-approved chemotherapeutic drug doxorubicin, photodynamic therapy to eliminate tumor by reactive oxygen, HER2 receptor to target cancer cells, hormonal drug trastuzumab to direct the drug system to the receptor, and observe the binding of the ligand to the receptor, indocyanine green, which provides fluorescence imaging were selected. In the literature researches, it is considered that the system will be successful because the designed system elements do not have any negative effects on cancer treatments. Keywords Breast cancer, graphene, photodynamic therapy, targeted drug, fluorescence imaging 175

178 POSTERLER / POSTERS P-035 Biological Applications as an Anti-microbial, DNA Binding, and DNA Cleavage of Trifloromethanesulfonamide Functionalized Graphene Quantum Dots Elif Ünaldi 1, Neslihan Demir 2, Mustafa Yıldız 3 1 Graduate School of Natural and Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey 2 Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey 3 Department of Chemistry, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey Abstract Graphene quantum dots (GQDs) display desirable properties for biomedical applications, including low toxicity, excellent photoluminescence, and good biocompatibility. They have been widely studied during the past years due to their optoelectronic properties and their potential applications. Trifloromethanesulfonamide functionalized luminescent graphene quantum dots (GQDs) was synthesized using a hydrothermal reaction between citric acid and trifloromethanesulfonamide. Graphene quantum dots have been characterized by UV-Vis, FT-IR spectroscopy, TEM, EDX spectroscopy and DLS. The antimicrobial activity of the compound was investigated for its minimum inhibitory concentration (MIC) to bacteria and yeast cultures. UV-Vis spectroscopy studies of the interactions between the GQDs and calf thymus DNA (CT-DNA) showed that the compound interacts with CT-DNA via electrostatic binding. DNA cleavage study showed that the GQDs cleaved DNA without any external agents. Keywords graphene quantum dots, anti-microbial, DNA binding, DNA cleavage 176

179 POSTERLER / POSTERS P-036 Anti-QS and Anti-biofilm Activities of Cucurbita pepo Leaf Extracts against Pseudomonas aeruginosa Arhun Ali Balkan 1, Ayla Yıldız 1, Ayten Şen 2, Aleyna Topaç 2, Barış Gökalsın 1, Nüzhet Cenk Sesal 2 1 Department of Biology, Marmara University, Institute of and Applied Sciences, Istanbul, Turkey 2 Department of Biology, Marmara University, Faculty of Arts and Sciences, Istanbul, Turkey Abstract In recent years, antibiotic resistant strains have become a global healthcare problem due to misuse or overuse of these drugs. Pseudomonas aeruginosa is an opportunistic pathogen which may have multiple antibiotic resistance and may cause various difficulties in treatment of several diseases. P. aeruginosa is frequently encountered in the airways and hospital infections especially in patients with cystic fibrosis.the importance of P. aeruginosa pathogenesis is attributed to its virulence factors and its potential for biofilm formation. These characteristics are regulated by quorum sensing (QS) mechanism. The urgent need for alternative treatment strategies can be met by inhibiting this mechanism through natural compounds. It is well known that Cucurbita pepo has several biological activities such as antimicrobial. Therefore, we aimed to evaluate potential inhibitory effects of C. pepo on P. aeruginosa QS systems and biofilm formation. Ethyl acetate extracts were obtained from C. pepo leaf samples that were decocted and infused. The extracts were applied at a certain concentrations of 240, 120 and 60 μg/ml. lasb-gfp, rhla-gfp and pqsa-gfp biosensor strains of P. aeruginosa were used to monitor QS inhibition and measurements were performed on Cytation 3 multimode microplate reader (Biotek). Results showed that decocted ethyl acetate extracts of C. pepo leaf samples have inhibitory effects on three QS systems: 48.57% for las; 30.90% for rhl; 53.10%, for pqs at 240 μg/ml. Also, highest biofilm inhibition rate was determined as 37.42% ( 16.1). The infused ethyl acetate extracts have also shown inhibitory effects on QS systems and biofilm formation at 240 μg/ml (las: 67.28% rhl: 33.32%; pqs: 49.87%; Biofilm: 32.73% 12.9). According to the results, infused extracts was more effective on las system compared to decocted samples. There were similar findings for rhl system with infused and decocted extracts. Furthermore, the decocted extracts had more inhibitory effects on pqs system and biofilm formation compared to infused extracts. Therefore, decocted extracts of C. pepomay have more potential as QS inhibitors against P. aeruginosa. Keywords Pseudomonas aeruginosa, quorum sensing inhibition, biofilm, Cucurbita pepo 177

180 POSTERLER / POSTERS P-037 Determining the Effects of Lichen and Endolichenic Bacteria Extracts on Pseudomonas aeruginosa Quorum Sensing and Biofilm Form Tunahan Irmak Başaran 1, Barış Gökalsın 1, Nüzhet Cenk Sesal 2 1 Department of Biology, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey 2 Department of Biology, Faculty of Arts and Sciences, Marmara University, Istanbul, Turkey Abstract Multidrug resistance in Pseudomonas aeruginosa is constantly increasing and poses a significant risk to public health. P. aeruginosa is capable of developing into biofilm forms in which they show increase resistance against antibiotics. Biofilm formation and virulence factors are linked to the mechanism known as Quorum sensing (QS). QS related genes are regulated by the release of signal molecules and detection of these molecules at the group level due to bacterial population density. It is thought that QS systems can be inhibited via molecules defined as QS inhibitors (QSIs). For this purpose, discovering novel natural QSIs is deemed necessary. QSIs can be obtained from natural sources such as lichens, plants, fungi, bacteria etc. It is well known that lichens produce unique secondary metabolites and some of them have antimicrobial activities. Lichens, which have symbiotic relations with fungi, cyanobacteria or algae, also provide special habitats to various bacterial groups. Moreover, these bacteria, called endolichenic bacteria (ELB) live on lichen s surface and partly within thalli in a competitive environment. Therefore, ELB should make use of antimicrobial compounds including potential QSIs. To determine QSI potential of ELB, we isolated ELB species coded TB49 from Plasmatia glauca and we extracted ethyl acetate cell free supernatant extracts (CFSE). Dosages were adjusted to 240, 120 and 60 μg/ml. The extracts were applied on P. aeruginosa lasb-gfp and rhla-gfp biosensor strains to observe QS inhibition. P.aeruginosa PAO1 (wild type) strain was used to determine biofilm inhibition. QS and biofilm inhibition tests were monitored with Cytation 3-multimode microplate reader (Biotek). For 240 μg/ml CFSE, inhibition ratio was calculated as 66,19% for lasb-gfp and 27,94% for rhla-gfp. Biofilm inhibition ratio of CFSE was observed as 55,75 % (±2,5). As a result, CFSE of TB49 has been observed to be more effective on the las system than the rhl system. We concluded that, if the concentrations are higher, inhibition rates may increase. Also we understand that, more effective results can be observed by inhibiting las and rhl inhibition along with other pathways. Further studies should be considered including determination of the active metabolite and its effects on human cells. Keywords Pseudomonas aeruginosa, lichen, endolichenic bacteria, quorum sensing, biofilm Acknowledgement This study was funded by BAPKO Project No: FEN-C-YLP We thank TÜBİTAK-BİDEB 2210/C scholarship program. 178

181 POSTERLER / POSTERS P-039 Pseudomonas aeruginosa Quorum Sensing Inhibition by Cherry Stalk Extracts Ayla Yıldız 1, Arhun Ali Balkan 1, Deniz Ezgi Budak 2, Ceren Özbek 2, Barış Gökalsın 1, Nüzhet Cenk Sesal 2 1 Biology Department, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey 2 Biology Department, Faculty of Arts and Sciences, Marmara University, Istanbul, Turkey Abstract Antibiotic resistance problem is a global healthcare problem arising from misuse or overuse of these drugs. Pseudomonas aeruginosa is responsible for approximately 10% of all nosocomial infections. Furthermore, high morbidity and mortality rates in Cystic Fibrosis (CF) patients due to biofilm formation are reported. Most virulence factors and bacterial behaviors such as biofilm are controlled by Quorum Sensing (QS) systems. It is believed that inhibition of QS systems by natural or synthetic inhibitor molecules may be an alternative approach without killing bacteria compared to conventional antibiotics. It has been reported that most inhibitor molecules from natural sources such as plant, bacteria or fungi etc. have potential quroum sensing inhibitor (QSI) activities. In this study, we evaluated QSI and anti-biofilm potential of cherry (Prunus avium) stalk extracts against P. aeruginosa. The methanol and acetone were used as solvents for extraction from cherry stalk. The extracts were applied at certain dosages of 240, 120 and 60 μg/ml. lasb-gfp, rhla-gfp and pqsa biosensor strains of P. aeruginosa were used to monitor QS inhibition and fluorescence and absorbance measurements were performed on Cytation 3 multimode microplate reader (Biotek). According to our results, the first applied dosages of all extracts showed maximum inhibition on tested parameters. We determined that QS inhibition ratios on las, rhl and pqs systems and biofilm of acetone extracts were 54.54% 32.43%, 53.54%, 45.05% (±0.05), respectively. The inhibition ratios of methanol extract values on QS and biofilm formation were 48.84%, 22.63%, 45.39%, and 13.80% (±12) respectively. As a result, when acetone and methanol extracts obtained from the cherry stalk are compared, inhibition properties of the acetone extract was better than that of the methanol extract. Further investigations are needed to discover inhibitor compounds and their effects on human cells so that these compounds may be used in new drug discoveries. Keywords Psudomonas aeruginosa, antibiotic resistance, quorum sensing inhibition, biofilm 179

182 POSTERLER / POSTERS P-040 Quorum Sensing Inhibitory Potential of Halomonas campaniensis on Pseudomonas aeruginosa Tunahan Irmak Başaran 1, Didem Berber 1, Barış Gökalsın 1, Abbamondi Gennaro Roberto 3, Giuseppina Tommonaro 3, Nüzhet Cenk Sesal 2 1 Department of Biology, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey 2 Department of Biology, Faculty of Arts and Sciences, Marmara University, Istanbul, Turkey 3 Institute of Biomolecular Chemistry-CNR, Via Campi Flegrei, 34, Pozzuoli (NA), Italy Abstract Increased drug resistance of Pseudomonas aeruginosa against conventional antibiotics is a major health problem all over the world. It is well documented that biofilm colonization has an important role in the bacterial pathogenesis during chronic infections. Biofilm formation is regulated by Quorum Sensing (QS) system which is defined as a bacterial communication system that coordinates bacterial group behaviors based on the cell density. Quorum sensing inhibitors (QSIs) against drug-resistant bacteria seems to be fairly promising improvement to overcome the resistance problem. It is believed that diverse QSIs are secreted especially in extreme conditions by natural sources such as plants, animals, fungi, bacteria etc. As known, halophiles and thermophiles grow in extreme conditions such as hypersalinity or high temperature. For this purpose, we studied QSI potentials of ethyl acetate cell and supernatant extracts of extremophilic Halomonas campaniensis against P.aeruginosa. Three dosages of 240, 120 and 60 μg/ml of each extracts were tested. To evaluate QS inhibition, biosensor strains of P. aeruginosa, lasb-gfp and rhla-gfp were used. QS and biofilm inhibition tests were performed by 96-well microplates and monitored on Cytation 3 multimode microplate reader (Biotek). According to the results, cell free supernatant extract is capable of inhibiting approximately 67,15% of GFP expression for lasb-gfp and 51,49% for rhla-gfp at 240 μg/ml. For 240 μg/ml cell extract, inhibition ratio is calculated as 67,55% for lasb-gfp and 51,61% for rhla-gfp. In addition, biofilm inhibition ratio of cell free supernatant extract is observed as 35,7%(±0,6) and biofilm inhibition ratio of cell extract is observed as 40,24%(±8,1). Results indicate that bacterial cell extracts and supernatant extracts have nearly same inhibition ratios on QS and biofilm inhibition. We concluded that, both cell free supernatant extract and cell extract might have same potential QSIs. Moreover, comparing QS and biofilm inhibition rates, it can be seen that biofilm formation is not only regulated by las and rhl systems, and therefore other pathways should also be targeted. Acknowledgement This study was funded by TÜBİTAK Project No: 315S092. Keywords Pseudomoas aeruginosa, Halomonas campaniensis, quorum sensing, QSI 180

183 POSTERLER / POSTERS P-041 Quorum Sensing and Biofilm Inhibition Properties of Ethyl Acetate Extracts of Sunflower (Helianthus annuus) Leaves against Pseudomonas aeruginosa Arhun Ali Balkan 1, Ayla Yıldız 1, Ayten Şen 2, Ezgi Ay 2, Barış Gökalsın 1, Göksel Evci 3, Nüzhet Cenk Sesal 2 1 Biology Department, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey 2 Biology Department, Faculty of Arts and Sciences, Marmara University, Istanbul, Turkey 3 Trakya Agricultural Research Institute, Edirne, Turkey Abstract Nowadays, conventional antibiotics are ineffective and drug resistance is a major problem in the treatment of several diseases such as cystic fibrosis. Therefore, the need for alternative treatment approaches have arisen. Most researchers have alternatively focused on inhibition of bacterial communication systems called quorum sensing (QS). In QS, signal molecules are secreted in the surrounding milleu and detected when bacteria reach a certain density. Pseudomonas aeruginosa is one of the drug resistant opportunistic pathogens. This bacterium may form biofilm via QS regulation and affect the course of the disease by secreting a number of virulence factors and biofilm formation. In recent years, one of the antivirulence approaches in treatment is the inhibition of its QS systems by natural or synthetic molecules. It was reported that sunflower (Helianthus annuus), which has 3000 years of ethnobotany value, has several biological activities. For this purpose, anti-qs activities of H. annuus leaf extracts from Thrace region were tested on biosensor strains of P. aeruginosa, lasb-gfp, rhla-gfp and pqsa-gfp as well as anti-biofilm activities on PAO1 wild type strain. The collected samples of H. annuus were divided into two groups, and infusion or decoction method was applied and extracted with ethyl acetate. The extracts dissolved in DMSO were tested at concentrations of 240, 120, 60 μg/ml in 96-well microplates. GFP fluorescence and absorbance were measured in Citation 3 multimode microplate reader (Biotek). According to our results, first applied dosages of all extracts showed maximum inhibition on tested parameters. Infused extracts showed inhibitory effects on las, rhl, and pqs system in varying percentages (47.59%, 20.63%, 51.91%, respectively). In addition, the anti-biofilm activity against PAO1 was determined as 48.57% (±7.7). Anti-QS potentials of the decocted extracts were detected as 48.26% on las, 40.3% on rhl and 53.88% in pqs. Futhermore, the anti-biofilm activity was 43.98% (±11). As a result, QS inhibition was more successful in infused H.annus leaf extracts. On the other hand, we observed that anti-biofilm activity of decocted H. annus leaf extracts was considerably high compared to infused samples. Acknowledgement We thank Malatya Pazarı for their support. Keywords Pseudomonas aeruginosa, quorum sensing inhibition, biofilm, Helianthus annuus 181

184 POSTERLER / POSTERS P-043 Quorum Sensing Inhibitory Potential of Natrinema versiforme on Pseudomonas aeruginosa Tunahan Irmak Başaran 1, Didem Berber 1, Barış Gökalsın 1, Iodice Carmine 3, Giuseppina Tommonaro 3, Nüzhet Cenk Sesal 2 1 Department of Biology, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey 2 Department of Biology, Faculty of Arts and Sciences, Marmara University, Istanbul, Turkey 3 Institute of Biomolecular Chemistry-CNR, Via Campi Flegrei, 34, Pozzuoli (NA), Italy Abstract Pseudomonas aeruginosa is an oppurtunistic pathogen that can be found in implamented medical devices such as stents or intubation tubes. It causes high mortality rates due to ability of forming biofilm. Biofilm formation provides up to 1000 times more resistance against drugs such as antibiotics which is related with Quorum sensing (QS) mechanism. QS related genes are regulated due to bacterial population density by using signal molecules. Also, compounds called Quorum sensing inhibitors (QSIs) which can be obtained from natural sources such as fungi, bacteria, archea etc. In addition, especially extracts from natural sources in extreme environments inhibit QS system. Therefore, extremely halophilic archaea that live in extreme conditions such as high salinity are thought to produce QSI compounds. For this purpose, we used Natrinema versiforme ethyl acetate cell free supernatant (CFSE) and cell extracts (CE). The concentrations of extracts were adjusted to 240, 120 and 60 μg/ml. To observe QS inhibition, extracts were applied on P. aeruginosa lasb-gfp and rhla-gfp biosensor strains. As a result N. versiforme CFSE is capable of inhibiting approximately 70,17%of GFP expression for lasb-gfpand 40,09% for rhla-gfp at 240 μg/ml. For 240 μg/ml CE, inhibition ratio is calculated as 63,75% for lasb-gfp and 54,81% for rhla-gfp. On the other hand, to observe biofilm inhibition P.aeruginosa PAO1 strain (wild type) was used. Biofilm inhibition ratio of cell free supernatnat extract is observed as 40,72%( ±4,1) and biofilm inhibition ratio of cell extract is observed as 32,21% (±4,2). Both tests were performed by 96-well microplates and monitored on Cytation 3 multimode microplate reader (Biotek). As a result, to QS inhibition CFSE was more effective than the CE on las system. On the other hand, CE was more effective than CFSE on rhl system. In addition, cell free supernatant extracts inhibition capability was higher than cell extracts inhibition capability. Moreover, comparing QS and biofilm inhibition rates, it can be seen that biofilm formation is not only regulated by las and rhl systems, and therefore other pathways should also be targeted. Acknowledgement This study was funded by TÜBİTAK Project No: 315S092. Keywords Pseudomonas aeruginosa, Natrinema versiforme, biofilm, quorum sensing, QSIs 182

185 POSTERLER / POSTERS P-044 A Simple Silica Based Method for Cell Free DNA Isolation from Blood Plasma Burhanettin Yalcinkaya 1, Kubra Coskun 2, Engiṅ Aydiṅ 4, Muslum Akgoz 2, Sadrettin Pence 3 1 Department of Molecular Medicine, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey 2 Bioanalysis Laboratory, TUBITAK National Metrology Institute (UME), Gebze, Kocaeli, Turkey 3 University of Health Sciences, Medical School, Istanbul, Turkey 4 Department of Pediatrics, Sakarya University School of Medicine, Sakarya, Turkey Abstract Circulating free DNA (cfdna) defined as DNA fragments in double-stranded structure are released into the circulation due to apoptosis, cell necrosis, or direct DNA release from the cell. The cf DNA concentration of only tumor cells increases in the blood stream. Thus, it becomes a valuable tool to determine the type of cancer type with a non-invasive procedure avoiding conventional biopsy procedures. cf DNA analyzes are mostly conducted by polymerase chain reaction (PCR). The success of PCR depends on the amount and purity of DNA extract of plasma. For PCR applications, several commercial kits have been developed so far extraction of cf DNA from blood serum and plasma samples, but, the DNA yield is low and they are quite expensive. Therefore, it is very important to develop new in-house methods with high yield at low cost to combat cancer in developing countries. In this study two commercial kits (Quick-cf DNA Serum & Plasma Kit- Zymo Research, Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit-Norgen Biotek) and seven modified in-house extraction methods (Silica gel method-1, Sodium acetate method, Phenol chloroform method, Ammonium acetate method, Sodium N-lauryl sarcosine method, sodium acetate method-2 and Silica gel method-2,) were compared for DNA yield and purity of the extracted DNA. The DNA concentration of extracts were compared by qpcr with using EGFR (nuclear gene) and mtdna (Mitochondrial genom; 7S region) primers and probes. For inhibition control of the extracted DNA, we also included 10 fold dilutions in PCR. At the same time, the purity of the extracted DNA were determined by NanoDrop 1000 spectrophotometer (Thermo Fisher Sci. Inc, USA) as well as spectrophotometric methods. The results show that the commercial kits and Silica gel method-1 are more effective method in terms of DNA yield and purity than others. Since, the Silica gel method-1 is very simple, inexpensive and environmentally friendly method, we would suggest this method as an alternative method for cfdna extraction from blood plasma samples. Keywords cell free DNA, cf DNA extraction method, qpcr, Biometrology 183

186 POSTERLER / POSTERS P-045 Optimization of Primers and Probes that can be used in the Diagnosis of Mitochondrial Diseases Burhanettin Yalcinkaya 1, Sadrettin Pence 2 1 Department of Molecular Medicine, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; Bioanalysis Laboratory, TUBITAK National Metrology Institute (UME), Gebze, Kocaeli, Turkey 2 Department of Molecular Medicine, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey; University of Health Sciences, Medical School, Istanbul, Turkey Abstract Mitochondria, one of the cell organelles are responsible for the production of ATP by the citric acid cycle and oxidative phosphorylation and is the production center of cellular energy. mtdna is hypersensitive to oxidative stress and other genotoxic insults because it does not have complex DNA repair mechanisms. Following metabolic events in the mitochondria, high levels of reactive oxygen species occur around mtdnas making it highly susceptible to mutagenesis. In this case, mitochondria, as defense mechanism, increases the number of DNA copies in order to alleviate the negative effects that may arise from the high mutation rates in their genomes. As a result of changes in point mutation, deletion, and copy number in mitochondrial DNA, many organs and cell types of human body are affected by dysfunction of mitochondrial functions. Thus interaction, many clinical, histological, genetic and biochemical diseases have been associated with mtdna damage, irrespective of nuclear DNA. The most prominent feature of these diseases is maternal inheritance, which is the transition from the mother. In this study, primer and probe conditions that can be used to determine the relative amount of mtdna copy number has been optimized. Firstly annealing temperature of the primers and probes were tested from 60 to 68 C by gradient PCR and confirmed by agarose gel electrophoresis. The optimum annealing temperature of the mtdna and beta actin gene s primers and probes were determined to be 68 C and 60 C respectively. The specificity analysis, final primer concentration analysis and the optimized fluorescence value determination of probes were performed with qpcr and checked by agarose gel electrophoresis. For this purpose, nanomoles, nanomoles, nanomoles, and nanomoles forward and reverse primer concentration combinations per reaction were tested. At the same time, the probe concentrations were tested separately with all primer combinations; 250 nanomoles and 150 nanomoles. This procedure was performed separately for primer probe sequences targeting both the mitochondrial genome and nuclear DNA. From the combined evaluation of threshold value (Cq) and agarose gel images the optimum primer and probe concentrations were found as nanomolar and nanomolar for mtdna and Beta actin, respectively. Keywords mitochondrial diseases, diagnosis, mtdna, qpcr optimization 184

187 POSTERLER / POSTERS P-049 Biotechnological Products Used in the Treatment of Rheumatoid Artritis Elif Taşdemir, Rana Arslan Department of Pharmacology, Anadolu University, Eskişehir, Turkey Abstract Rheumatoid arthritis (RA) is one of the most common systematic inflammatory autoimmune disease. According to WHO data, the prevalence varies of rheumatoid arthritis is 0,3-1 % in developed countries. RA affects joints, connective tissues, muscle, tendons, and fibrous tissue. The etiology of rheumatoid arthritis has not been fully explained yet. Treatment of this disease is inadequate and it can lead to permanent joint damage and disability. It was known that cytokines including tumor necrosis factor alpha, interleukin (IL)-1β and IL-6 play important role in the RA pathogenesis. Thus, targeting therapies have come into use taking into account these factors in the last 10 years and various biotechnological products have been developed to antagonize especially proinflammatory cytokines in treatment of the RA. Although we have limited knowledge about these products, the symptoms of disease and radioactively measurable joint damage were reduced with these agents. Monoclonal antibodies such as infliximab, golimumab, tocilizumab, rituximab and adalimumab and drugs produced by recombinant DNA technology like as etanercept, abatasept was licensed for the treatment of RA by ministry of health in Turkey. The clinical uses and possible side effects of biological agents continue to be investigated. This study summarizes recently published data on several biotechnological products treatment of the RA. Keywords Romatoid arthritis, monoclonal antibodies, biolotechnological products 185

188 POSTERLER / POSTERS P-050 Many Algerian Plants with Thymol as Major or Active Compound Hocine Laouer 1, Salah Akkal 2 1 Laboratoire de Valorisation des Ressources Naturelles Biologiques, Département de Biologie et d écologie végétale. Université Ferhat Abbas de Sétif, Algérie 2 Laboratoire de Phytochimie et Analyses physicochimiques et Biologiques, Département de Chimie, Faculté de Sciences exactes, Université Mentouri Constantine, Algérie Abstract Thymol is the active or the main monoterpene phenol found in different essential oil plants. This compound has revealed several biological properties, including antibacterial, and antioxidant activities. In this work, a comparison was made between the oil chemical compositions. The GC/MS analysis of four essential oils revealed the presence of thymol in Thymus numidicus Poiret., Ammoides pusilla (Brot.) Breistr., Ammoides atlantica (coss. Et Dur.) wolf. and Soccocalyx satureoides Coss. et Durieu. Thymol dominated in practically all Ammoides sp (44.5%- 53.2%) and T. numidicus (59.0%, 68.0%) but (+)-α-terpineol (35.9%), thymol (15.6%) and borneol (12.4%). were the main constituents. The in vitro antibacterial and antifungal activities of the essential oil were assessed by the disc diffusion method, and were significant on the different microorganisms tested. Keywords Essential oil, GC/MS, Thymol 186

189 POSTERLER / POSTERS P-053 Polyphenols and Phytochemical Screening of Asphodelus Species Bouchouka Racha Lydia 1, Tebibel Soraya 1, Kabouchezahia 2 1 Université des frères Mentouri-Constantine, Departement de chimie, Laboratoire d obtention de substances thérapeutiques (L.O.S.T ), Constantine, Algeria. 2 Université des frères Mentouri-Constantine, Departement de biologie animale, Laboratory of EthnobotanyPalynology and Ethnopharmacology and toxicology (E.P.E.T), Constantine, Algeria. Absract Asphodelus tenuifolius is a perennial plant with an average height of one meter, found throughout the Mediterranean region and in India. The aerial part of the plant is used for nutritional and medicinal purposes, particularly in cardiovascular; dermatological and gastrointestinal diseases. This study is designed to assess the phytochemical screening of Asphodelus tenuifolius collected from Ghardaïa, Algeria, and to analyze its total phenolic contents by the Folin Ciocalteure active. The results showed that the phytochemical screening of the aerial parts of Asphodelus tenuifolius revealed the presence of flavonoids and tannins, and the absence of the alkaloids and triterpenoids. Additionally, the extracts of this plant contain relatively high concentrations of polyphenols ( 315,14 ± 3,37 mg/g). Keywords Asphodelus, screening, polyphénols,phytochemical,medicinal 187

190 POSTERLER / POSTERS P-056 Emicizumab and Recombinant Products Used in the Treatment of Hemophilia A Elif Taşdemir Department of Pharmacology, Anadolu University, Eskişehir, Turkey Absract Hemophilia is a serious blood disorder depending on X-linked hereditary factors. The disease, caused by a lack of coagulation factor VIII and IX. Hemophilia A (factor VIII deficiency) is 80% and hemophilia B (factor IX deficiency) is 15% of patients with hemophilia. The incidence of men is higher than women. Typical features of the disease are hematoma and hemarthroses. The most common bleeding joints are knee, elbow and ankle in hemophilia, but every joint can bleed. The use of coagülation factor concentrate is the main component of treatment of hemophilia. If hemophilia patients don t use enough coagulation factor for a long time, this situation can cause permanent joint disabilities. Recently, monoclonal antibody (emicizumab) and drugs produced by recombinant DNA technology, each with ascending minimal risks of viral transmission have remarked. In connection with this, lonoctocog, efraloctocog, moroctocog, octocog, eptacog, simoctocog, susoctocog, turoctocog alfa are approved for the prophylaxis and/or treatment of bleeding in patients with haemophilia A in several countries worldwide. The biological agents go on to be searched. This study sumps up recently in the light of published datas on many biotechnological products prophylaxis and/or treatment of the hemophilia A. Keywords Hemophilia A, monoclonal antibody, recombinant DNA technology. 188

191 POSTERLER / POSTERS P-058 Assesment of Quality Control in Biotechnological Drugs Evren Alğın Yapar 1, Burçin Çağan 2 1 Republic of Turkey Ministry of Health, Turkish Medicines And Medical Devices Agency, Vice Presidency of Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey 2 Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey Absract The quality control of biotechnological drugs has a great prospect in terms of the efficacy and safety of the products. The quality of the biotechnological drugs must comply with relevant internationally accepted criteria. In this context, the quality of a biotechnological pharmaceutical product refers to a concept that expresses its conformity with good manufacturing standards including formulation, production, specifications and control.¹ Biotechnological drugs are subject to quality control criteria and analysis within the scope of the international standards and guidelines specified or guided in accordance with their individual characteristics and differences, in particular the formulations and the application routes.² General quality control tests that can be carried on the biotechnological drugs are; Biological activity by cell culture method, Diagnosis and quantification by ELISA and HPLC methods, Total and free PRP quantification by IEF, SDS PAGE, Western Blotting methods, In vivo potency testing, Physical tests (physical examination, ph determination, total and bound protein, moisture, residual moisture, volume etc.), Sterility test, LAL test and Pyrogen test.³ In this study, quality control tests and assessments will be given for biotechnological drugs. Keywords Biotechnological drugs, Quality control, GMP, Standards 189

192 POSTERLER / POSTERS P-059 International Standards for Biotechnological Drugs Evren Alğın Yapar 1, Burçin Çağan 2 1 Republic of Turkey Ministry of Health, Turkish Medicines And Medical Devices Agency, Vice Presidency of Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey 2 Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey Absract The criteria for the acceptance of a biotechnological product as a medicine constitute the specifications of the product. The specifications are critical quality standards recommended and proposed by the manufacturer and approved by the regulatory authorities. Specifications are part of the entire control strategy designed to ensure product quality and consistency. However, biotechnological drugs, the active substance of which are proteins or polypeptides, are products which are difficult to establish quality standards due to the complexity of their structures, production conditions and determination of their specifications.¹ The European Pharmacopoeia has been engaged in the development of monographs for biotechnology products for many years. The monographs, which have been started to be created for biotechnological drugs, have begun to take place in the European Pharmacopoeia since the 1990s.² Also, in 1999, International Conference on Harmonization (ICH) published a guideline describing the Biotechnological / Biological Products Testing Procedures and Acceptance Criteria. The key to identifying the specifications in the ICH manual is to focus on molecular and biological properties that are known to be useful in ensuring the safety and efficacy of the active substance and the finished product, rather than providing full characterization. In this context, the characterization of biotechnological products includes the determination of physicochemical properties, biological activity, immunochemical properties, purity and impurities.³ Within the scope of this study, the standards and contents prepared for biotechnological drugs will be discussed and explained in detail.¹ ⁵ Keywords Biotechnological drugs, ICH, Pharmacopeia, ISO, Standards 190

193 POSTERLER / POSTERS P-060 The Role of Antibody-Dependent Cell-Mediated Cytotoxicity Mechanism in Cancer Treatment Burçin Çağan 1, Evren Alğın Yapar 2 1 Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey 2 Republic of Turkey Ministry of Health, Turkish Medicines And Medical Devices Agency, Vice Presidency of Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey Absract Monoclonal antibodies used in cancer treatment show anti-tumor effects with many different mechanisms. ADCC, which is one of these mechanisms, is the main mechanism of action of monoclonal antibody. In fact, ADCC functions as the first line of defense of the body against pathogens by creating an important cell-mediated innate immune response.¹ ADCC consists of three basic components. These are the expression of the target antigen on cancer cells, the presence of antigen specific antibodies of the appropriate isotype, and effector cells carrying Fc receptors. Binding of the antibodies to the antigens on the target cell surface cross-link the Fc receptors on effector cells. As a result of this binding, the target cells are activated, triggering functions such as NK cells killing cancer cells as well as chemokine and cytokine release. The effector cells that can mediate ADCC include natural killer (NK) cells, monocyte-macrophages, polymorphonuclear leukocytes and neutrophils. NK cells form the major ADCC effector cells. On the surface of these cells are low affinity type II (FcyRIIc; CD32c) and type IIIA (FcyRIIIa; CD16a) Fc receptors. NK cells kill target cells by secreting cytotoxic granules such as perforin, granules and granzymes.² For a long time, monoclonal antibodies have been used to treat cancer, but the antibodies that modulate the immune system and induce ADCC have been used much more recently in the treatment of various types of cancer.³ In this study monoclonal antibodies that are used in the cancer treatment will be discussed by means of their mechanisms of action. Keywords Biotechnological drugs, ADCC, Cancer, Monoclonal antibody 191

194 POSTERLER / POSTERS P-061 Biotechnological Drugs for Cancer Treatment and Clinical Trials Burçin Çağan 1, Evren Alğın Yapar 2 1 Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey 2 Republic of Turkey Ministry of Health, Turkish Medicines And Medical Devices Agency, Vice Presidency of Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories, Ankara, Turkey Absract Biotechnological drugs are complex molecules produced in viable biological systems such as bacteria or mammalian cells using recombinant DNA technology. These drugs are used in the treatment of many diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriasis, ulcerative colitis and cancer. Many biological therapies, especially immunotherapy, are used in cancer treatment.¹ These therapies are fighting against cancer cells using different mechanisms. These mechanisms are as follows: Immune Control Point Inhibitors (pembrolizumab, nivolumab, avelumab, durvalumab and atzolizumab), The immune cell therapy(tisagenlecleucel and axicabtagen), Therapeutic antibodies (therapeutic antibodies work in many ways), Therapeutic vaccines (prostatic acid phosphatase-granulocyte macrophage colony stimulating factor). Immune regulating agents are ones strengthening an individual s immune system against cancer. These are cytokines (interferons and interleukins) and immunomodulatory drugs (lenalidomide, pomalidomide, imiquimod).² ³ In this study, biotechnological drugs and clinical trials will be discussed.⁴ ⁵ Keywords Biotechnological drugs, Cancer, Monoclonal antibody, Clinical trials 192

195 POSTERLER / POSTERS P-063 Biotechnology and Pharmacognosy Timur Hakan Barak Department of Pharmacognosy, Yeditepe University, İstanbul, Turkey Absract American Society of Pharmacognosy defines Pharmacognosy as the study of physical, chemical, biochemical and biological properties of drugs, drug substances, or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources (ASP). Briefly, Pharmacognosy is to acquire knowledge of drugs. Earlier, most of the drugs were derived from herbs and conventional pharmacognostical studies were focused on the systematic study of medicinal plants and medicines derived from plants. In years, Pharmacognosy has become a multidisciplinary science of natural drugs and drug substances and it deals with medicinal plant cultivation, crude drug production, chemical; biological; pharmacological and molecular analysis of crude drugs and drug substances to assure their production, potency, purity and safety as well as to assist new drug discoveries (Dhami, 2013). When plant cell biotechnology emerged as a new possibility for the production of plant secondary metabolites pharmacognosy altered its route to this field. The aim of biotechnology in pharmacognosy was the production of known pharmaceuticals by means of plant cell cultures in higher amounts than isolation from plants. Plant cell cultures play a crucial role in the development of new drugs from plants. Plant cells may provide the required amount of a compound during its development, when an agricultural or horticultural production is not yet available. In those cases in which agriculture does not work, plant cell biotechnology might be the final production method (Verporte, 2000). The Catharanthus (or Vinca) alkaloids are the examples of biotechnologically produced pharmacognostical compounds. Vinblastine is marketed for more than 40 years as an anticancer drug and became a true lead compound for drug development. Due to the pharmaceutical importance and the low content of vinblastine and vincristine in crude plant, biotechnologically those alkaloids are produced by in vitro cell culture of C. roseus cells (Heijden et al. 2004). Application of biotechnological processes in pharmacognosy has been risen recently and the examples might be augmented. Keywords pharmacognosy, biotechnology, cell culture 193

196 POSTERLER / POSTERS P-066 Development of a Novel Gene Specific Silencing Method by RNAi in Targeted Cells Using PLGA Nano-Particles Şeyma Ceylan 1, Fahri Akbaş 2, Fatemeh Bahadori 3 1 Department of Biotechnology, Bezmi Alem Vakıf Univesity, Istanbul, Turkey 2 Department of Medicinal Biology, Bezmi Alem Vakıf Univesity, Istanbul, Turkey 3 Department of Pharmaceutical Biotechnology, Bezmi Alem Vakıf Univesity, Istanbul, Turkey Absract Pancreatic cancer is considered among the types of cancer with the highest incidence mortality rate both in the world and in Turkey. This truth shows the insufficiency of existing treatment strategies. In addition, chemotherapeutic drugs show numerous side effects on healthy tissue because of their inability in targeting tumor side and methods such as radiotherapy cause loss of function in the healthy tissue exposed to radiation. Traditional cancer therapy methods are replaced by the new targeted therapies which are of great interest as they do not harm healthy cells and show high selectivity. RNA interference (RNAi) is the best experimental method for effectively silencing gene expression to study the function of proteins in various cell types. Nanoparticles consisting of poly (lactic-co-glycolic acid) (PLGA) are used in cancer diagnosis and treatment because of their unique properties such as biodegradability, high biocompatibility and low toxicity. The aim of this study was to suppress the expression of the GPR87 gene, which is overexpressed in pancreatic cancer, by sirna technique using PLGA nanoparticles. For this purpose, the GPR87 gene was amplified by PCR and cloned into the psicheck -2 vector. The luciferin expression of the recombinant plasmid transferred to the cell line is measured and the chimeric GPR87 expression of the cell is shown. The dsrna (double-strand RNA) consisting of 21-base oligonucleotides molecules were then prepared by targeting the GPR87 mrna. The prepared sirnas were loaded onto PLGA nanoparticles and transfected into the pancreatic cancer cell line showing luciferin expression. Gene silencing has been demonstrated by a reduction in Luciferase radiation. The silence rate of Luciferase expression in PLGA nanoparticles prepared by different methods was determined and the optimum effective dose was determined in cell culture As a result, it was determined that the mirnas passed through the targeted cell membrane via PLGA nanoparticles and the overexpressed gene in pancreatic cancer was successfully silenced by the RNAi mechanism in the cell. Keywords sirna, PLGA, Pancreatic Cancer, Gene Delivery 194

197 POSTERLER / POSTERS P-072 MicroRNA in Hepatitis B and Hepatitis C associated Hepatocellular Carcinoma and Cirrhosis Gokhan Kucukkara 1, Ferhat Gurkan Aslan 1, Bilal Toka 2, Mehmet Koroglu 1, Mustafa Altindis 1 1 Sakarya Ünv School Of Medicine Dept Of Medical Microbiology, Sakarya, Türkey 2 Sakarya Ünv School Of Medicine, Dept Of Gastroenterology, Sakarya, Türkey Absract Introduction and AIM: MicroRNAs (mirnas) are small, uncoded RNA molecules that regulate post-transcriptional gene expression through base-to-mrna binding. Recent studies have revealed the role of mirnas in the pathogenesis of many human diseases, particularly liver diseases. Analysis of circulating mirnas is used to diagnose hepatocellular carcinoma (HCC) and liver cirrhosis (LC), or to determine the progression of liver disease. In this study, HSA-miRNA-21-3p, hsa-mirna-29a-3p, hsa-mirna-122-3p, HsA-miRNA-192-5p, Hepatitis B (HBV) and Hepatitis C The aim of this study was to determine the biomarker potentials. MATERIAL-METHOD: Sixty patients and 26 healthy volunteers were included in the study and the patients were grouped as HBV-HCC (n = 18), HCV-HCC (n = 8), HBV-LC (n = 15) and HCV-LC (n = 19). 5 ml blood samples taken from the participants with gelled dry tubes were centrifuged at 4000 rpm for 10 minutes and the sera separated and stored at -80 C until total RNA isolation. Total RNA isolation was performed with the Direct-zolTM RNA MiniPrep (Zymo Research Corp., USA) commercial kit followed by cdna synthesis and real-time PCR amplification with EPIK mirna Select Hi / Lo-ROX (Bioline Reagents Ltd., USA). For amplification and analysis, Rotor-Gene Q (QIAGEN, Germany) instrument was used and statistical analyzes were performed with SPSS 21 (IBM, USA) program. RESULTS: Hsa-miRNA-21-3p and hsa-mirna-122-3p levels increased 3-4 fold in patients other than HCV-LC and significantly decreased in hsa-mirna-29a-3p expression in HCV infected patients (p <0,05 ). hsa-mirna p showed a 3-fold increase in HBV-LC group (p <0.05) but not in other groups. The hsa-mirna-122-3p value, which is known to be specific for liver, is increased in HCV-LC patients. Decrease in hsa-mirna-29a-3p expression was detected in patients with HCV infection (p <0.05). CONCLUSION: In our study, as non-invasive diagnostic markers; MiRNA-21-3p and hsa-mirna-122 for HBV- HCC and HCV-HCC diseases, hsa-mirna-21-3p, hsa-mirna-122 and hsa-mirna-192-5p for HBV- MiRNA- 29a-3p test for LC could be used. Keywords HBV, HCV, Hepatocellular Carcinoma, Cirrhosis, micro RNA 195

198 POSTERLER / POSTERS P-078 Monoclonal Antibodies for Cancer Treatment Burçin Çağan 1, Evren Alğın Yapar 2 1 Republic of Turkey Ministry of Health, Turkish Medicines and Medical Devices Agency, Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories 2 Republic of Turkey Ministry of Health, Turkish Medicines And Medical Devices Agency, Vice Presidency of Economic Assessments and Laboratory Services, Department of Analysis and Control Laboratories Absract Millions of people die every year due to cancer. Although many treatments have been used in the treatment of cancer, such as surgery, radiation, chemotherapy, hormone therapy, biological and targeted therapies, yet no method is satisfactory.¹ Immunotherapy, which is one of these methods, is targeted to the immune system cells of the individual and thus, the formation and elimination of cancer can be achieved without damaging the healthy cells. Among all immunotherapeutic methods for cancer treatment, monoclonal antibodies are the most commonly studied and approved clinical trials. Depending on their mechanism of action, monoclonal antibodies alone can induce target cell death or increase target cell sensitivity to chemotherapeutics or radiotherapeutics by modulating anti-apoptotic pathways.² Many monoclonal antibodies are approved by authorities such as Turkish Medicines and Medical Devices Agency (TMMDA), European Medicines Agency (EMA) and Food and Drug Administration (FDA) every year. Within the scope of this study, monoclonal antibodies, their targets and indications approved by TMMDA, EMA and FDA for cancer treatment will be described.³ ⁵ Keywords Cancer, Monoclonal antibody, TMMDA, EMA, FDA 196

199 POSTERLER / POSTERS P-083 Determination of Bioactive Goat Milk Peptide Structures by LC-QTof-MS System Bilal Cakir, Tuğba Tunali Akbay Marmara University, Faculty of Dentistry, Department of Basic Medical Sciences, Başıbüyük, Maltepe, İstanbul. Absract Bioactive peptides are widely used in biotechnology such as therapeutic proteins and enzymes, recombinant cultures for the production of therapeutic drugs and vaccine, animal cell cultures for the production of monoclonal antibodies. Although some bioactive peptides exist free in its natural source, the vast majority of known bioactive peptides are encrypted in the structure of the parent proteins and are released mainly by enzymatic processes. Milk is the primary source of bioactive peptides that have a wide range of biological functions when hydrolyzed with different enzymes. Goat milk is a natural functional food that becomes a particular interest with its various biochemical properties and with its similarities to the breast milk. The bioactive peptides of goat milk exhibites advantages for future drug researches as. they are structurally diverse, have wide spectrum of therapeutic action, low biodeposition in body tissues and high biospecificity to targets. In the present study, fresh goat milk samples were obtained from Saanen-Maltız crossbreed goats that were grown in the Thrace region of Turkey. Total protein, fat and carbohydrate levels were determined in milk samples. After the biochemical milk composition determination, milk casein was precipitated in ph 4.6 and whey protein fraction was seperated. Both casein and whey fractions were hydrolyzed with trypsin and papain enzymes under specified conditions. The peptide structure of hydrolysed milk proteins were characterized by LC-QTof-MS system. After characterization, the peptide structures were identified in terms of their bioactivity by using different protein and peptide databases. According to the in silico analyze results, bioactive peptides of goat milk has been found to have some therapeutic effects in cancer and diabetes. As a conclusion, the utilization of goat milk as a source of bioactive peptides may present unique structures and biofunctionalities that can be exploited in the pharmaceutical industry. Keywords Bioactive peptides, In silico analysis, Goat milk, Protein, Pharmaceutical industry. 197

200 POSTERLER / POSTERS P-088 Synergy Study, Plant Extracts and Antibiotics Against Bacteria Mohamed Mihoub Zerroug, Nora Haichour, Samia Mezaache Aichour Laboratoire de Microbiologie appliqué, Faculté des Sciences de la Nature et de la Vie, Université Ferhat Abbas Sétif 1, Setif Algeria Absract Antimicrobial drugs effective for treatment of pathogenic bacterial infections are limited, thus it is important and valuable to find compounds that potentiate antimicrobial activity of antibiotics against this bacteria. So, our study aims to evaluate the synergistic effect between antibiotics and extracts of pine resin and propolis against five strains of bacteria, using the diffusion method disks. This study constitute a confirmation of the synergistic effect of the two extracts with antibiotics especially against Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa (with 10-30mm diameters of inhibition zones) which were resistant to several antimicrobial agents and antibiotics. Our results seem to validate the traditional use of the resin and propolis in modern medicine. Keywords Pinus halepensis, resin, propolis, antibacterial activity, synergistic effect 198

201 POSTERLER / POSTERS P-089 Investigation of the Relationship Between Influenza A Virus RNA Polymerase and Human Actin Beta Protein Nazife Gelmez 1, Erkan Rayaman 1, Atsushi Kawaguchi 2, Kadir Turan 1 1 Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Marmara University, Istanbul-Turkey 2 Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan Absract Influenza A viruses have segmented RNA genome composed of eight single stranded negative sense RNA molecules. Replication and transcription ofviral RNAs are entirely dependent on the viral RNA polymerase. Viral RNA polymerase is a trimeric structure including of PB1, PB2 and PA subunits. These proteins are more conserved than other viral proteins in different influenza virus subtypes. This makes the viral RNA polymerase an ideal target for development of anti-influenza virus drugs. Therefore, elucidation of the relationship between viral RNA polymerase and host proteins have a vital importance. In this work, we screened the potential human interactor proteins for influenza viral RNA polymerase PA subunit with yeast two-hybrid method. More than 15 host proteins, including actin beta were found related with PA protein in yeast cells. The interaction between human actin and viral PA protein was also investigated in human cells. Human actin beta complete cdna was cloned into pcha mammalian expression vector and obtained pcha-actb plasmid. A serial plasmid vectors encoding beta actins deleted some exonic domains were derived with invers PCR from pcha-actb. The expression of actin genes and the localization of native and deleted actin proteins in transiently transfected HEK293 and/or HeLa cells were defined with western blotting and immunofluorescence techniques. The interaction between PA and actin proteins were investigated with co-immunoprecipitation assays and co-localization analysis of the proteins in the cells. Co-localization analysis of viral and host proteins was carried out with immunofluorescent staining and proximity ligation assay. Immunoprecipitation and western blot analyses revealed interactions between virus PA protein and actin proteins in mammalian cells. It was observed that the N-terminal domains of actin and C-terminal half of PA have a role in interaction. This interaction was supported with confocal microscopic examinations. Actin beta proteins are essential components of actin filaments and highly conserved between species. The results obtained in this work suggest that actin filaments may have a role in intracellular traffic of influenza vrnps consisting of the viral RNA, RNA polymerase subunits and NP in human cells. *This work was supported by The Scientific and Technological Research Council of Turkey (grant number 112S518). Keywords Influenza A viruses, viral RNA polymerase, PA protein, human actin beta 199

202 POSTERLER / POSTERS P-090 Mechanisms or Instability for Protein Based Pharmaceuticals Timur Hakan Barak Department of Pharmacognosy, Yeditepe University, İstanbul, Turkey Absract Physical and chemical stability of proteins peptides and other macromolecules indicate in altering grades. Stability ratio of these products effects the route of production, distribution, and administration. There are two main classes for defining the stability of biotechnological products: physical stability and chemical stability (Ho 2013). Chemical stability can be defined as active molecules to be ready for undergoing chemical reactions which desired to occur. Meanwhile, physical stability can be defined how readily the molecule loses its tertiary or secondary formation, aggregates with itself, and precipitates from solution. Deamidation, racemization, oxidation, beta-elimination and disulfide exchange are some mechanisms of chemical instability (Li et al. 1995). Denaturation, aggregation, precipitation and adsorption are some examples of physical instability (Fast et al. 2009). This study was designed to compile instability mechanisms and examples of biotechnological protein based medicine. Keywords Biotehnological medicine, chemical stability, physical stability 200

203 POSTERLER / POSTERS P-091 Antioxidant Activities of Santolina Chamaecyparissus Aqueous Extract Abderrahmane Senator 1, Bouriche Hammama University Batna2, Faculty SNV, Algeria 2 1. Laboratory of Applied Biochemistry, University Setif 1, Algeria Absract This study aims to detect the in-vitro antioxidant capacities of the aqueous extract of the areal part of Santolinachamaecyparissus, a plant largely used in Algerian folkloric medicine and to estimate its phenolic and flavonoid contents.total phenolic and flavonoid content was estimated by Folin-Ciocalteau s reagent and Aluminum chloride colorimetric method, respectively.the antioxidant activities were determined by using free radical scavenging and reducing power assays.results showed that polyphenols and flavonoids are quantitatively important in the aqueousextract.compared to the quercetin and rutin, used as standard antioxidant, aqueous extract exhibited a goodscavenging effect against hydrogen peroxide. The inhibition rates are similar, quercetin(93%), rutinvalue (98%) and extract (91%). The concentration of standards is 80µg/ml but that of the extract is 200µg/ml. The IC50value obtained are 101.3± 0.68 for extract and 37,4± 0.15 for quercetin and 27.4 ± 0.11 for rutin. This activity increased with the increasing concentration. Moreover, the Santolina chamaecyparissus extract exerted a good concentration-dependent reducing power, with a maximum effect at the concentration of 400µg/ml. The corresponding IC50 is ± 8.33 µg/ml. This value is lower than that of BHT, used as standard antioxidant, which gives the maximum effect at 50µg/ml. These findings suggest that aqueous extract of Santolina chamaecyparissus possess phenolic and flavonoid constituents that are responsible for antioxidant activities and can be exploited as a natural antioxidant in food and medicinal uses. Keywords antioxidant, Santolina chamaecyparissus, aqueous extract 201

204 POSTERLER / POSTERS P-093 Antimicrobial Activity of a Fungal Microorganism Agit Çetinkaya 1, Ragıp Soner Silme 2, Ömür Baysal 1 1 Department of Molecular Biology and Genetics, Faculty of Science, Muğla Sıtkı Koçman University, Muğla, Turkey 2 Technology Transfer Office, Istanbul University, Istanbul, Turkey Absract Biological property provides advantages and control on disease caused by pathogens that are beneficial for protection of humans in medicine. In purpose of this aim to find and select the effective agents showing rapidly secretion of inhibitory metabolics of importance with successful inhibition on pathogens. Therefore, inhibitory properties should be screened using biological and physiological parameters relied on confirmation by molecular tools. We have isolated a new fungus which has secondary metabolics showing antibiotic properties. It has been identified using ITS analysis as Phomopsis viticola. Although this is a plant pathogen fungus, antibiotic production have been found in in vitro condition, which is required activity for effective control of human pathogen microorganisms. Moreover, experiments carried out under controlled condition showed remarkable inhibitory effect on Citrobacter freundii, Escherichia coli and Staphylococcus aureus compared to control groups. These preliminary data showed our isolate is a new antibiotic producing candidate for controlling of pathogenic microorganisms. Our further studies will be carried out on characterization of the antibiotics produced by fungus by using X-ray crystallography and NMR. Keywords Phomopsis viticola, Antibiotics, Citrobacter freundii, Escherichia coli, Staphylococcus aureus 202

205 POSTERLER / POSTERS P-095 In Vitro Culture of Tumor Infiltrating Lymphocytes for Adoptive Immunotherapy in Breast Cancer Patients Erdoğan Selçuk Şeber 1, Tarkan Yetişyiğit 1, Hülya Dönmez 5, Sinem Buluş 5, Sibel Özkan Gürdal 3, Meltem Öznur 4, Burhan Turgut 2 1 Department of Medical Oncology, Tekirdağ Namık Kemal University, Tekirdağ, Turkey 2 Department of Hematology, Tekirdağ Namık Kemal University, Tekirdağ, Turkey 3 Department of General Surgery Tekirdağ Namık Kemal University, Tekirdağ, Turkey 4 Department of Pathology Tekirdağ Namık Kemal University, Tekirdağ, Turkey 5 Hematology Laboratory, Tekirdağ Namık Kemal University, Tekirdağ, Turkey Absract BACKGROUND: Tumor infiltrating lymphocytes (TIL) have a key role in adoptive immunotherapy and have been shown to be effective in various types of cancer patients. However, formation of in vitro TIL cultures is a labor intensive and time taking process and breast cancer (BC) has been regarded as a poor target for TIL therapy. We therefore investigated whether in vitro patient specific TIL cultures can be established from breast cancer patients and also evaluated their phenotypes. MATERIALS-METHODS: After surgical resection of breast cancer tissue and pathologic examination, tumor tissue was sliced into small pieces, and physically disaggregated. The resulting single cell suspension was layered with Ficoll gradient and TIL enriched fraction was plated in 24-well plates with complete medium and 6000 IU/mL rhil-2 and maintained at a concentration of 0.5-2X106TIL/mL. Short-term cultured TIL were then immediately cryopreserved for future evaluation. RESULTS: A total of 12 patients were included in the study group. Before plating, flow cytometric analysis was performed for each specimen to determine lymphocytic content. Seven specimens with no or very little lymphocyte presence were excluded from the study. All of the tumors that were devoid of lymphocyte presence were estrogen and progesterone receptor positive. The remaining 5 specimens were successfully cultured for ex vivo expansion of tumor derived TIL and flow cytometric analysis was done for determination of differentiation and activation markers. CONCLUSIONS: We have shown that ex vivo tumor derived TIL expansion is possible in patients with breast cancer. Especially triple negative and poorly differentiated subtypes of BC seem to be more suitable for this process. Further work should be done to elucidate whether selection of certain cytotoxic T cell subtypes would be more effective in adaptive immunotherapy applications Keywords tumor infiltrating lymphocytes, breast cancer, adoptive immunotherapy Flow cytometric analysis of TIL Patient /receptor status CD3+ CD4+ CD3+ CD8+ CD8+ CD57+ CD8+ CD137+ CD8+ CD28+ TIL2A/triple neg ,5 18,5 3 TIL3C/triple neg. 37,3 47,2 9,7 1,2 0,6 TIL4A/er + pr+ 30,4 40,7 65,4 42,3 6,2 TIL7A/er+ pr- 59,2 33,8 4,3 85,5 47,3 TIL10A/triple neg. 64,1 30, ,3 0,4 203

206 POSTERLER / POSTERS P-096 Preparation of Drug Loaded Polymeric Nanoparticles as Ceramidase Inhibitor Ferdane Danışman 1, Hüsniye Birman 1, Merve Ilgar 2, Ezgi Tan 2, Selcan Karakuş 2, Ayben Kilislioğlu 2, Serap Kuruca 1 1 Istanbul University, Faculty of Medicine, Department of Physiology 2 Istanbul University-Cerrahpasa, Faculty of Engineering, Department of Chemistry Absract Introduction: Nano-drug delivery systems have been developed to reduce the side effects of classical chemotherapy and increase the effectiveness of prolonged release of drugs. Ceramidases catalyze the degradation of ceramide to fatty acids and sphingosine. Ceramide is not only a key molecule of sphingolipid biosynthesis and degradation, it is also in sphingolipid signaling, promoting differentiation or apoptosis. Several studies have shown that acid ceramidase inhibitors are potential anti-proliferative drugs for cancer chemotherapy. Fluorouracil (5FU), a ceramidease inhibitor, is a chemotherapeutic drug used to treat various cancers parenterally. 1-hexylcarbamoyl-5-fluorouracil (carmofur), is a derivative of 5FU, being a lypophilic-masked analog of 5-FU that can be administered orally and is reported to be converted, enzymatically or nonenzymatically, into 5-FU. At the same time, carmofur is more cytotoxic than 5-fluorouracil against human tumors in vitro. Although there are studies about the nanoforms of 5FU, the nano structures related to carmofur are almost non-existent. For purpose, we have designed nanoparticles of 5FU and its derivative carmofur that is ceramidease inhibitor and used commonly in chemotherapy. Materials and Methods : Nanoparticles were prepared by ultrasound assisted solvothermal method. Polymeric nanoparticles were based on rosin ester and dispersed in Polyethylene glycol (PEG). Characterization of nanoparticles were done by Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS) and Fourier transform infrared (FTIR) spectroscopy. Results: The physicochemical properties of the nanostructure were evaluated by using SEM, DLS and FTIR spectroscopy. It is important to understand the size of nanoparticles in liquid since they are going to be used in biological fluids. In this study the average hydrodynamic diameters of the polymeric nanoparticles were measured by DLS. Average nanoparticle diameter was found approximately 210 nm. According to SEM images the diameter of the polymeric nanoparticles were found approximately 100 nm which were in agreement with hydrodynamic sizes. Chemical structure of the nanostructure and drug loaded nanostructure FTIR spectra were analyzed on an attenuated total reflection Fourier transform infrared (ATR-FTIR) instrument in wavelength range of cm 1 with a resolution accuracy of 4 cm 1. Keywords Nanodrug, ceramidase inhibitors,carmofur, 5FU. 204

207 POSTERLER / POSTERS P-098 Synthesis and Herbicidal Activities of New Cu(II) Complexs of Acylthiourea Derivatives Turkan Kiral 1, Mehmet Yakan 2, Irfan Koca 2, Serhat Mamaş 1, Nurcan Karacan 1, Ahmet Tansel Serim 3 1 Gazi University, Science Faculty, Chemistry Department, Ankara 2 Bozok University, Science and Art Faculty, Chemistry Department, Yozgat 3 Plant Protection Central Research Institute, Yenimahalle, Ankara Absract In order to find novel herbicidal active compounds, a series of acylthiourea derivatives and their copper(ii) complexes were synthesized and evaluated for their herbicidal activity. All target compounds were characterized by elemental analysis, FT-IR, 1H-NMR, 13C-NMR and ESI-MS methods. Herbicidal activities against various weeds of N-(diethylcarbamoyl)-1-ethyl-2-oxo-4-phenyl-1,2-dihydropyrimidine-5-carboxamide and its copper(ii) complex were evaluated. Efficacy of the compounds on the weeds and crop plants was determined using bioassay in the growth chamber (22-25oC: 16/8 h light/dark photoperiod). The compounds were applied to the test plants at 2-4 true leaf stage with an application volume of 195 l/ha water included non-ionic surfactant at 0.1%, v/v. Herbicidal effects of these compounds were visually evaluated 30 days after treatment. The results showed that the title compounds had moderate herbicidal activity against wild mustard, wild oat, common cocklebur and cheatgrass. They also had non-selective activity on the crop plant tested wheat, safflower, maize and sunflower. Acyl thiourea derivatives showed more herbicidal activities than copper(ii) complexes. Keywords acyl thiourea, copper(ii) complex, herbicidal activity 205

208 POSTERLER / POSTERS P-099 In Silico Analysis of Goat Milk Derived Bioactive Peptides Kıymet Özlem Şahna, Bilal Çakır, Tuğba Tunalı Akbay Marmara University, Faculty of Dentistry, Department of Basic Medical Sciences, Başıbüyük, Maltepe, İstanbul Absract Goat milk is an ideal food for children and adults, and is traditionally used as a supplement for the treatment of asthma, eczema, migraine, frequent vomiting and weight loss. These beneficial effects of goat milk are thought to be due to its bioactive peptides. Some bioactive peptides have already been approved by the FDA for commercialization as therapeutic peptides. Therefore, the specific objectives of this study were to isolate the proteins from goat milk by extraction and precipitation techniques, to digest the goat milk with pepsin, to identify the bioactive goat milk peptides with proteomic techniques and to use an in silico analysis for the prediction of the potential role of goat milk based bioactive peptides. Goat milk samples were collected from the goats on the same farm being managed and fed in the same way. Goat milk casein was precipitated in ph 4.6 and whey protein fraction was seperated. Both casein and whey fractions were hydrolyzed with pepsin enzyme under specified conditions. Peptides of goat milk were identified by mass-chromatography systems. The bioactive role of these peptides were evaluated with in silico analysis in BIOPEP database system. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of peptides. According to the results of BIOPEP database, goat milk bioactive peptides were found to have variety of beneficial functions in the body. In conclusion, bioactive peptides of goat milk can be used as functional ingredients in the formulation of health-enhancing nutraceuticals, and as potent drugs with positive impact on body functions. Keywords Bioactive Peptide, Goat Milk, In silico analysis, Casein, Whey proteins 206

209 POSTERLER / POSTERS P-100 Isolation and Screening Method for Antibiotic-Producing Fungi Ragıp Soner Silme 1, Gülperi Kayran 2, Ömür Baysal 2 1 Technology Transfer Office, Istanbul University, Istanbul, Turkey 2 Department of Molecular Biology and Genetics, Faculty of Science, Muğla Sıtkı Koçman University, Muğla, Turkey Absract A fast screening method was established in order to isolate antifungal antibiotic producing fungi from soil samples. In this method, soil samples were diluted and directly plated in agar medium by the standard fungi isolation method, and the plates were cultured at 27 C for 2-3 days to permit the growth of fungal colonies. Then, the suspension of Fusarium oxysporum (50 μl, ⁵ CFU ml ¹) was overlaid by spraying on the plates under controlled conditions. After 1-2 day incubation, fungal colonies showing an antagonistic effect with the inhibition zone against sprayed F. oxysporum were selected. Among 183 isolates, 52 strains were found producing antibiotics in liquid medium. The results indicated that this method has a higher selection rate (22.2%) than the traditional method (4.5%). This new method can be applied for isolation and screening of microorganisms that produce antibiotics against pathogenic microorganisms. Keywords Antibiotics, screening, isolation, Fungus, Fusarium oxysporum 207

210 POSTERLER / POSTERS P-101 Perspectives of Medical Students about the Biotechnological Drugs Zinnet Şevval Aksoyalp, Devrim Demir Dora Department of Medical Pharmacology, Akdeniz University, Antalya, Turkey Absract BACKGROUND: Biotechnological drugs are produced by using biotechnological methods of biological products similar to natural substances in the human body. Biosimilars are the similar version of the patent expired original biotechnological products. Currently, biotechnological drugs and biosimilars are produced in Turkey and are prescribed by a doctor. About the biotechnological drugs, the opinions and knowledge level of medical students who will prescribe the this drugs in the future are an important issue. For this reason, the perspectives of students of the Akdeniz University Medical Faculty on biotechnological drugs and biosimilars were evaluated. METHODS: This is a cross-sectional survey conducted at the Faculty of Medicine of Akdeniz University between September 2018 and October This study was conducted over the internet by using a questionnaire consisting of 9 questions addressing students of the Akdeniz University Faculty of Medicine. RESULTS: Ninety-seven students participated in the survey. Forty-three percent of the students who participated in the survey is the second year students and 62% of them are women. Seventy percent of the students did not know about biotechnological drugs. Students stated that they had textbooks/notes (% 21) as the source of their knowledge about biotechnological drugs. More than half of the students know that biotechnological drugs are obtained from living cells and the cost of these drugs is high. When the drugs in the biological/biotechnological classifications are evaluated, more than half of the students knew that monoclonal antibodies are included in the biological/ biotechnological group. Ninety-one percent of the students stated that they do not know about biosimilars. Furthermore, they do not know that these products are produced/marketed in Turkey. Ninety-four percent of the students think that a lecture on biotechnological drugs should be in the curriculum of medical education. CONCLUSIONS: As a result, it is seen that medical students' level of knowledge about biotechnological drugs is not enough. Lecture of biotechnological drugs should be included in the medical education curriculum. Keywords Biotechnological drugs, biosimilars, medical education, insulin, erythropoietin. 208

211 POSTERLER / POSTERS P-102 Monoclonal Antibodies Which Licenced in Turkey Timur Hakan Barak Department of Pharmacognosy, Yeditepe University, İstanbul, Turkey Absract Biotechnological drugs are of key importance in the advancement of the pharmaceutical industry and in the development of human health. Only ten thousand of the thirty thousand diseases entered into the literature are in the class of treatable maladies. In recent years, a significant part of the innovations in the pharmaceutical industry has been shaped around biotechnological drugs. Biotechnological drugs in the pharmaceutical industry are developing much faster than chemical and herbal medicines. Drugs produced with monoclonal antibody technology are the most important ones among the biotechnological drugs. This study was conducted to investigate licensed drugs contain monoclonal antibodies in Turkey The aim of this study was; monoclonal antibodies, which is a class of biotechnological drugs that are increasing every year, is the compilation and classification of our country in order to better understand the position of our country in the pharmaceutical sector. A scientific databases containing monoclonal antibodies have been identified worldwide licensed drugs and conditions of the license Turkey was determined using the pharmaceutical care assistant: TEBRP. The number of monoclonal antibodies have been licensed in Turkey is seen to increase with each passing year. Since these drugs are innovative products produced with the latest technology, they are found to have a high economic value. It was determined that, in case of biosimilars of biotechnological drugs can be produced in Turkey will create significant economic value. Keywords Monoclonal antibodies, biotechnological drugs, Turkey 209

212 POSTERLER / POSTERS P-106 Curcumin Increase Antitumor Effect of CDs+5-Fluorouracil Against Prostate Cancer Yesim Yeni 1, Ali Taghizadehghalehjoughi 1,2, İsmail Çagrı Aydın 1, Sidika Genç 1, Aysegul Yılmaz 1, Fatma Yesilyurt 1, Selma Sezen 1, Ahmet Hacımuftuoglu 1 1 Ataturk University, Faculty of Medicine, Department of Medical Pharmacology, 25240, Erzurum, Turkey 2 Ataturk University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Erzurum, Turkey Absract Introduction: Nanotechnology creates many exciting new tools for diagnosis and treatment of diseases. Metalnanoparticles are widely use as diagnostic and therapeutic agents for cancers. In recent studies it was shown that plants increase the effects of anticancer drugs. Curcumin is known to have cytotoxic and anticancer effects on prostate cancer (PC). Spheroid culture models is a valuable method for evaluation of drug and cancer cell interaction. Based on these data, we aimed to investigate the antiproliferative and cytotoxic effect on spheroid PC culture line with 5-Fluorouracil(5-FU), Quantum dot (CdS) nanoparticle (NPs) and curcumin. Materials and Methods: The 2D cultures of prostate carcinoma cells was grown in normal cell culture medium. 3D cultures of PC cells were then prepared. Curcumin essential oil were obtaining by using Clevenger distillated device. The centrifuged QDs precipitate was washed with n-hexane, methanol and dh 2 O, respectively. The Cds cultures were grown over night in an incubator inoculated with 100 ml Luria Bertani broth medium and left to incubate. QDs NPs were characterized by XRD, SEM. Different dose of QDs NPs (0.01 mg/μl), 5 FU (25mM), QDs NPs +5 FU +Curcumin (10-3,10-4, 10-5, 10-6,10-7 and 10-8 μl) was applied on PC cell lines for 24 hours. The viability, antioxidant and antioxidant level were determined by using MTT kit, TAC and TOS test. Results and Discussion: As a result of the MTT test, it was determined that the combination of QDs NPs +5 FU +Curcumin (10-3 and 10-4 μl) reduced cell proliferation by approximately 50% and 49%, respectively, when compared with control group. It appears that the combination of QDs NPs +5 FU +Curcumin (10-3 and 10-4 μl) does make a sense in compare to pure 5-FU and QDs NPs (P<0.05). In addition, antioxidant capacity of both group are near to control group and shows highest antioxidant capacity (P<0.05). Oxidant status have correlation with MTT result and lowest ratio were seen in QDs curcumin combination QDs NPs +5 FU +Curcumin (10-3 and 10-4 μl). J.Ravindran and colleagues shown curcumin have great antiproliferative and anticancer effect this data have correlation with our data. Keywords QDs NPs, 3D PC, curcumin 210

213 POSTERLER / POSTERS P-107 Evaluation Neuroprotective Effects of Origanum Majorana Extract on Fe3O4@SiO2 NPs Induced Toxicity in Cortex Culture Selma Sezen 1, Ali Taghizadehghalehjoughi 2, Yeşim Yeni 1, Sidika Genç 1, Medine Güllüce 3, Ahmet Hacımuftuoglu 1, Ahmet Mavi 4, Kübra Solak 4 1 Ataturk University, Faculty of Medicine, Department of Medical Pharmacology, 25240, Erzurum, Turkey 2 Ataturk University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Erzurum, Turkey 3 Atatürk University, Faculty of Science, Department of Biology, Erzurum, Turkey 4 Department of Nanoscience and Nanoengineering, Institute of Naturel and Applied Sciences, Ataturk University, 25240, Erzurum, Turkey Absract Nanoparticles (NPs), a particle size of at least one dimension in the range of nm, have some unique physical and chemical properties such as small size effect, high reactivity, surface effect, quantum size effect, and dielectric confinement effects. Since exposure to NPs has increased in human life, its impact on human health has gradually attracted attention. Studies on toxicity on NPs have shown that NPs have a variety of toxicities such as neurotoxicity, genotoxicity, immunotoxicity, teratogenicity, and reproductive toxicity. Origanum majora extracts are known to have compounds with high antioxidant and neuroprotective properties. However, the neuroprotective effect of the essential oil of Origanum majorana is unknown. The aim of this study was to investigate the neuroprotective effect of Origanum majorana essential oil in combination with a neurotoxic nanoparticle known to be used in drug targeting on primer neuron cell culture. The oleylamine stabilized monodisperse magnetite nanoparticles (Fe3O4) was prepared by thermal decomposition method. The core/shell Fe3O4@SiO2 NPs were obtained by hydrolysis of tetraethyl orthosilicate (TEOS) in the presence of ammonia solution. After the precipitate was collected, the organic materials were removed by the calcination at 550 ºC to produce magnetite nanoparticles embedded in mesoporous silica spheres. The bio synthesized Fe3O4@SiO2 were characterized by XRD, SEM. Different dose of Origanum majorana (10-3,10-4, 10-5, 10-6,10-7and 10-8 μl) was applied on neuron cell lines for 24 hours. MTT cell As a result of the MTT test, it was determined that the combination of Fe3O4@SiO2 (0.1 mg/μl) + Origanum majorana (10-3 and 10-4 μl) protect neuron cells by approximately 87% and 81% from Fe3O4@SiO2 NPs, respectively, when compared with group of Fe3O4@SiO2. It appears that the combination of Fe3O4@SiO2 NPs (0.1 mg/μl) + Origanum majorana (10-3 and 10-4 μl) reduced neurotoxic effect of NPs (P<0.05). In addition, antioxidant capacity of both group are near to control group and shows highest antioxidant capacity (P<0.05). Yusra Al Dhaheri and colleagues were shown oregano majorana have Anti-Metastatic and Anti-Tumor Growth Effects on Highly Metastatic Human Breast Cancer Cells. This data has correlation with our data. Keywords Oreganom majorana, Fe3O4@SiO2 NPs, Neuron, TAC 211

214 POSTERLER / POSTERS P-108 Comparing Our New Electrodes with Pt-Iridyum Commercial Electrodes Fatma Yesilyurt 1, M.sait Ertugrul 1, Aysegul Yilmaz 1, Ufuk Okkay 1, Ali Taghizadehghalehjoughi 2, Ahmet Hacimuftuoglu 1 1 Department of Medical Pharmacology, Faculty of Medicine, Ataturk University, Faculty of Medicine, Erzurum, Turkey 2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey Absract Introduction: The use of electrodes in the measurement of neurotransmitters and neurochemicals was initiated in the early 1970s. Voltammetry recordings are performed using a precision potentiostat and a recording method connected to the microelectrodes to measure neurotransmitters. The primary tools used to perform voltammetry recordings are microelectrodes implanted in the central nervous system (CNS) tissue or placed too close to the cells in the case of single cell recordings. In order to increase the selectivity of voltammetry methods, microelectrode surfaces were modified and different recording methods were used. Platinum (Pt) or platinum-iridium(pt-ir), usually glass-coated wires, are the basis for an electrode array being developed to measure neurochemical and other neurotransmitter surfaces using electrode-dependent oxidative enzymes. The working or recording electrode used for in vivo voltammetry measurements in CNS tissues is generally produced to obtain an end diameter of µm depending on the application. The most common reference electrode used for voltammetry recordings in CNS tissues is the Miniature Ag / AgCl electrode. Material and Method: The method is based on the potential difference in the working electrode to record the oxidation or reduction of the analytes. In amperometry, which is used as a recording method, the current can be monitored continuously, so this technique has the ability to record very fast events ( Hz). Aluminum oxide (Al 2 O 3 ) was covered with platinum on ceramic substrates. The platinum electrodes and platinum pathways were formed by photolithography. Then electrodes were formed by slicing Al 2 O 3 with slicing device. Al 2 O 3 electrode ends are bonded to the PCB with adhesive. Then the electrodes on the PCB were welded by copper wire to the platinum electrodes by wire connecting device. Result and discussion: Commercial electrodes had better results but our results are also not bad. We will work to do thinner studies for decreasing the LOD levels less than 1 and less than the commercial electrodes. 212

215 POSTERLER / POSTERS Figure 1. Pt+Irıdyum Figure 2. Electrodes which was built from platin+aluminum oxide+ copper wire Keywords: Electrodes, voltammetry, neurotransmitter 213

216 POSTERLER / POSTERS P-110 Temozolomide Cds NPs Combination Shows More Effectivity in Compare Pure Drug Against T98G Cells Aysegul Yılmaz 1, Ali Taghizadehghalehjoughi 1,2, Selma Sezen 1 Fatma Yesilyurt 1, Yeşim Yeni 1, Sidika Genç 1, Atefeh Varmazyari 3 Ahmet Hacımuftuoglu 1 1 Ataturk University, Faculty of Medicine, Department of Medical Pharmacology, 25240, Erzurum, Turkey 2 Ataturk University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Erzurum, Turkey 3 Ataturk University, Department of Nanoscience and Nanoengineering, Institute of Naturel and Applied Sciences, 25240, Erzurum, Turkey Absract Introduction: Glioblastoma multiforme are among the most aggressive and challenging cancers type. Temozolomide is the current standard-of-care chemotherapy for glioblastoma. The Origanum genus belongs to the Lamiaceae family and 14 of which are endemic in Turkey. Although reports on the essential oil composition and bio-logical activity of O. majorana are rare. In addition, although the anti-proliferative effects of Origanum species are known, the effects of O. majorana are unknown. Quantum dots (QDs) and polymeric nanoparticles (NPs) are considered good binomials for the development of multifunctional nanomedicines for multimodal imaging. In this study, it was aimed to determine the effects of temozolomide, anti-cancer chemotherapy drug, Origanum majorana, and Cds- NPs on T98G glioblastoma multiforme cell line by using different doses and the combination of all. Material and Method: The Cds cultures were grown over night in an incubator (120 rpm/m, 32 C) inoculated with 100 ml LB (Luria Bertani) broth medium and left to incubate. The culture on LB broth medium was centrifuged at 6000 rpm at 20 C for 10 m. After 24 h at room temperature, the synthesis solution was transferred to a 50 ml tube and centrifuged at 10,000 rpm at 20 C for 10 m. The precipitate containing Cds was washed with n-hexane, methanol and ddh 2 O, respectively. The resulting precipitate was dried for 24 h at 60 C prior to the characterization process. The biosynthesized Cds-NPs were characterized by XRD, SEM. Origanum majorana was collected from Alanya and was diagnosed at the Biology department of Erzurum Atatürk University and dried and stored in a dark place at room temperature. The plants were then ground to obtain the essential oil using the clover device. The cytotoxicity assays of the study were performed by using MTT, TAC and TOS respectively. Results and Discussion: When the results were examined, CdS, temozolomide and Origanum majorana 10-3 combination values were found to be significant (P<0.05). According to our study TAC capacity only in Cds + temozolomide + Origanum majorana 10-3 combination group shows same capacity with control group. TOS status were highest in Cds + temozolomide + Origanum majorana 10-8 combination group. Keywords Temozolomide, CdS NPs, Origanum majorana 214

217 POSTERLER / POSTERS P-111 An Anticancer Agent Based on Ehtylenediamine Against Breast Cancer Cells, MDA-MB-231 and MCF-7 Elif Avcu Altıparmak 1, Gökçe Erdemir 2, Serap Erdem Kuruca 2, Bahri Ülküseven 1, Tülay Bal Demirci 1 1 Department of Chemistry, Istanbul University-Cerrahpasa, Istanbul, Turkey 2 Department of Molecular Medicine, Istanbul University, Istanbul, Turkey Absract Breast cancer is the most common type of cancer in women and the rate of causing death is even higher than lung cancer. The incidence of breast cancer is dramatically rising in recent years. By 2020, the number of women with breast cancer will be increased 1.7 million annually [1]. The use of novel ligands and complexes including nitrogen atoms in cancer treatment has become important in recent years because of biological activity. N,N-bis(salicylidene)ethylenediamine and its derivatives are known as salens. Salen compounds are well-known ligand species in organometallic chemistry and coordination chemistry. These compounds are derivates of schiff bases and they have potential biological activity, such as anti-inflammatory, antifungal, antimicrobial, antiviral, anticancer and antioxidant [2]. In this study, a salen complex was synthesized and investigated the cytotoxic activity on breast cancer cells, MDA- MB-231 and MCF-7. The complex was obtained by the reaction of 2-hydroxybenzaldehyde thiosemicarbazone with ethylenediamine in the presence of Cu(II) ion. Complex was characterized by elemental analysis, IR, ESI-MS and single crystal X-ray diffraction. Cytotoxic effects of the complex was evaluated by MTT test for MDA-MB-231 ve MCF-7 cell line and compound has shown that cytotoxic effects by reducing cell viability. According to the results of MTT toxicity test performed with MCF-7 breast cancer cell line; complex has a 10% lethal effect on cells at low doses (1µg/mL), but it has been found to decrease cell viability at doses of 5 µg/ml and over. According to the results of MTT toxicity test performed with MDA-MB-231 breast cancer cell line; complex no lethal effect on cells at low dose (1 µg/ml). It has been determined that it decreases cell viability by 10-20% at doses of 5 µg/ml and above. The effect in the MCF-7 cell line studies was not high in the MDA-MB-231 cell line because MDA-MB-231 is a more aggressive cell line than MCF Z. Ma, X. Qiao, C. Xie, J. Shao, J. Xu, Z. Qiang, J. Lou, Journal of Inorganic Biochemistry, 2012, 117, P.G. Cozzi, Chemical Society Reviews, 2004, 33, Keywords Breast Cancer, Salen Complexes, Anticancer Activity 215

218 POSTERLER / POSTERS Ortep Diagram of the Complex Results of MTT toxicity test performed with MCF-7 breast cancer cells Results of MTT toxicity test performed with MDA-MB-231 breast cancer cells 216

219 POSTERLER / POSTERS P-112 DNA Vaccines: From Bench to Bedside Genada Sinani 1, Melike Sessevmez 2 1 Department of Pharmaceutical Technology, Altinbas University, Istanbul, Turkey 2 Department of Pharmaceutical Technology, Istanbul University, Istanbul, Turkey Absract The DNA vaccines contain a gene encoding a specific antigen that lead to the stimulation of immune responses after administration. DNA vaccination offers several opportunities compared to traditional approaches. Plasmid DNA can be produced easier in large scale and the process is relatively cheaper compared to recombinant protein antigens. DNA vaccination induces both antibody and cell-mediated immunity. The latter is particularly important to ensure protection against intracellular pathogens. Moreover, DNA vaccines show potential for vaccination of broad population. Following the demonstration of plasmid DNA to induce immune responses after in vivo injection in the early 1990 s, DNA vaccines have been consider as a potential tool for both prophylactic against viral, bacterial, or parasitic diseases and therapeutic vaccination for different diseases. However, all currently licensed DNA vaccines are approved only for animal use. Many experimental DNA vaccines for human are still in clinical trials to assess their efficacy and safety. The weak immunogenicity of these vaccines in humans remains a challenge. Although extensive research has been carried and several adjuvants and methods of administration have been developed and tested, improvements still need to be done to have human DNA vaccines into clinic. Additionally, safety concerns related to the probability of integration of DNA into the chromosome of the host need to be carefully examined. This study aims to provide a brief summary of DNA vaccines, including current knowledge on the vectors, immunological mechanisms and generated immune responses, and discuss both their safety and risk assessment. The development of useful DNA vaccines may enhance prophylactic vaccination and immunotherapy. Keywords DNA vaccines, gene, safety, infectious diseases, clinical efficacy 217

220 POSTERLER / POSTERS P-113 Recombinant Protein-Based Malaria Vaccines Melike Sessevmez 1, Genada Sinani 2 1 Department of Pharmaceutical Technology, Istanbul University, Istanbul, Turkey 2 Department of Pharmaceutical Technology, Altinbas University, Istanbul, Turkey Absract According to World Health Organization (WHO), malaria caused 445,000 deaths and 216 million clinical cases worldwide in There are five types of parasite species responsible for malaria in humans, of which Plasmodium falciparum is the most dangerous one and responsible for more than 98% of malaria mortality. Plasmodium parasites could gain resistance to antimalarial drugs. This has raised the need to develop highly protective vaccine. Despite the enormous efforts for developing a vaccine against malaria there is still no commercially available malaria vaccine. Fortunately, the use of recombinant technology has shown great opportunity to develop new effective and safe vaccines against challenging infectious diseases. Recently, The European Medicines Agency s (EMA) Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion to Mosquirix vaccine a recombinant protein based malaria vaccine- against malaria caused by Plasmodium falciparum for people living outside the European Union. Mosquirix is composed of Plasmodium falciparum circumsporozoite protein fused with hepatitis B surface antigen, combined with hepatitis B surface antigen(s) in the form of non-infectious virus-like particles (VLP) and is produced in yeast cells (Saccharomyces cerevisiae) by recombinant DNA technology. The clinical data has indicated that Mosquirix can protect children from 6 weeks to 17 months, while effectiveness of the vaccine has reduced with time which has caused the need of repeated injections. WHO has reported that there are more than 20 new vaccine candidates in clinical trials or advanced preclinical development against malaria. In the near future, recombinant protein based malaria vaccines will be more efficient and provide long term protection in humans through progression in new antigen discoveries and antigen combinations, protein expression platforms, adjuvants and protein vaccine delivery platforms according to current good manufacturing practice (cgmp) standards. Arama, C., & Troye Blomberg, M. (2014). The path of malaria vaccine development: challenges and perspectives. Journal of internal medicine, 275(5), Draper, S. J., Angov, E., Horii, T., Miller, L. H., Srinivasan, P., Theisen, M., & Biswas, S. (2015). Recent advances in recombinant protein-based malaria vaccines. Vaccine, 33(52), World Health Organization (November 2017). World Malaria Report Retrieved from malaria/publications/world-malaria-report-2017/report/en/ Keywords recombinant protein, vaccine, malaria, plasmodium falciparum 218

221 POSTERLER / POSTERS P-115 Vitex Agnus Castus Combination Cds and 5-Fluorouracil Effectively Kill Prostate Cancer: In Vitro Sidika Genç 1, Ali Taghizadehghalehjoughi 1,2, Cemil Bayram 1, Yeşim Yeni 1, Fatma Yesilyurt 1, Aysegul Yılmaz 1, Selma Sezen 1, Ahmet Hacımuftuoglu 1 1 Ataturk University, Faculty of Medicine, Department of Medical Pharmacology, 25240, Erzurum, Turkey 2 Ataturk University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Erzurum, Turkey Abstract Introduction: 5-Fluorouracil (5-FU) inhibits cell division by interfering with DNA and RNA synthesis by blocking the conversion of deoxyuridic acid to timidic acid via the timidylat synthetase enzyme. Vitex agnus-castus (VAC) is using in the treatment of several cancer type because of anticancer effects by inhibiting DNA and RNA synthesis. Targeted delivery systems, likes Quantum dot (CdS) nanoparticle (NPs) have been used along with combinatorial therapy. 3D culture is an effective strategy for enriching/culturing Cancer stem-like cells (CSCs) in vitro to facilitate cancer research and therapy development. For this reason, 3D in vitro cell culture was used for Prostate Cancer (PC) research. In this study, Cds Np and VAC was used to increase the anti-cancer effect 5 florouracil. Material and Method: The 2D cultures of prostate carcinoma cells were grown in normal cell culture medium. Then, 3D cultures of prostate carcinoma cells were prepared. VAC essential oil were obtaining by using Clevenger distillated device. The Cds cultures were grown over night in an incubator (120 rpm/m, 32 C) inoculated with 100 ml LB (Luria Bertani) broth medium and left to incubate. The culture on LB broth medium was centrifuged at 6000 rpm at 20 C for 10 m. To isolation Cds NPs centrifuged at 10,000 rpm at 20 C for 10 m. The precipitate containing Cds was washed with n-hexane, methanol, and dh 2 O, respectively. The Cds nanoparticles were characterized by XRD, SEM. Cds (0.01 mg/μl), 5-FU (25mM), Cds (0.01 mg/μl) +5-FU (25mM) + different dose of VAC (10-3,10-4, 10-5, 10-6,10-7 and 10-8 μl) was applied on PC cell lines for 24 hours. MTT cell viability test, TAS and TOS were performed after the application. Result and Discussion: According to our results, the most death was in the cell line where Cds (0.01 mg/μl) + 5-FU (25mM) +VAC (10-3 μl) combination (P<0.001). Antioxidant and oxidant status show correlation with MTT results. It was observed that Cds + 5-FU + VAC combination therapy is more effective than single drug administration. Our result shows correlation with K Ohyama (2003) study. The authors show VAC have apoptotic and cytotoxic effect on breast cancer. Keywords 5-Fluorouracil, Cadmium nanoparticle, Pancreas cancer, MTT, Vitex agnus-castus. 219

222 POSTERLER / POSTERS P-117 Impurities in Biologicals, Their Safety/Toxicity and Analytical Methods Pınar Erkekoğlu 1, Emirhan Nemutlu 2 1 Department of Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 2 Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey Absract The entire manufacturing process determines the quality of a biotechnological medicinal product. Biological medicine manufacturing is more complex and minor changes may affect quality, safety and efficacy of these products, including vaccines. Safety of these products should be of major concern both for the companies and for regulatory authorities. Characterization of process- and product-related impurities is required and sophisticated analytical methods and specific controls are required to demonstrate identity quality, potency and purity. Impurities in biologicals include, but are not limited to, product-related impurities, process-related (fermentation, purification, formulation, filling) impurities, degradation products and contaminants. Process related impurities are molecular variants arising during manufacture and/or storage, which do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety. Product related impurities are aggregates, breakdown products and product variants (i.e. aberrant glycosylation, oxidation, deamidation, denaturation, etc.). Gel permeation (GP) / Size exclusion (SE) high pressure liquid chromatography (HPLC), ion exchange HPLC, reversed phase HPLC, peptide mapping/ mass spectroscopy (MS) analysis, carbohydrate analysis and gel electrophoresis can be used to determine the process- and product-related impurities. However, new methods should be in use in order to identify smaller amounts of impurities in biologicals and scientists should develop more sensitive methods for the determination of different impurities arising from each step of the production and process of biological products. Keywords biological, product-related impurities, process-related impurity, toxicity, analytical method 220

223 POSTERLER / POSTERS P-119 3D Tissue Models for Nanotoxicology Applications Suna Sabuncuoğlu, Pınar Erkekoğlu Hacettepe University, Faculty of Pharmacy, Department of Toxicology Absract Nanotechnology mainly deals with the nano-sized materials. Nanomaterials (nanoparticles, nanotubes, quantum dots, single-walled carbon nanotubes, nanorods, fullerenes] have a diameter of nm and they can be used in different fields, including electronics, engineering, textiles, aerospace, machinery and pharmaceutical industry. Nanotoxicology, a branch of toxicology, studies the toxicity of nanomaterials, including those used for medical purposes. There are many concerns about the potential toxicity of nanomaterials and new toxicological approaches are needed in order to determine the safety of these products. Thus, we have very little information regarding the toxicity of nanomaterials. Potential adverse effects of nanoparticles include cytotoxicity, immunotoxicity and genotoxicity. There are not accurate in vitro tools to predict or evaluate the risk of nanomaterials. Although there are not guidelines for the determination of toxicity of the nanomaterials (including nanopharmaceuticals) yet, basic in vitro and in vivo toxicological studies as well as basic toxicity testing guidelines can be used to evaluate their toxic effects. Cell cultures are the first step to test the toxicity of several chemicals, including nanomaterials. However, existing in vitro cell culture methods provide little correlation with in vivo system. Rather than cell cultures, tissue models carrying close characteristics to human skin are now being preferred in the toxicity assessment of nanomaterials (e.g. for zinc or silver nanoparticles) that can be used by dermal route. In vitro 3-D reconstructed human epidermis (RhE) tissue models are in vitro reconstructed human epidermis from normal human keratinocytes cultured on a collagen matrix at the air-liquid interface. These tissue models are now being used to evaluate the cytotoxicity, immunotoxicity, genotoxicity, skin irritation, skin corrosion and other toxicological effects of chemical products, including nanomaterials. These new 3D models are suggested to provide an accurate prediction for toxicity of nanomaterials. However, further studies are still necessary to show whether these models can replace in vivo research and whether they can accurately determine toxicological potential of nanomaterials. Keywords 3D Tissue Models, Nanomaterials, nanotoxicology 221

224 POSTERLER / POSTERS P-120 Perspectives of Medical Students about Biotechnological Drugs Zinnet Şevval Aksoyalp, Devrim Demir Dora Department of Medical Pharmacology, Akdeniz University, Antalya, Turkey Absract BACKGROUND: Biotechnological drugs are produced by using biotechnological methods of biological products similar to natural substances in the human body 1. Biosimilars are the similar version of the patent expired original biotechnological products 2. Currently, biotechnological drugs and biosimilars are produced and prescribed in Turkey. The opinion and knowledge level about biotechnological drugs of medical students who will prescribe these drugs in the future is an important issue. For this reason, the perspectives of students of the Akdeniz University Medical Faculty on biotechnological drugs and biosimilars were evaluated. METHODS: This was a cross-sectional survey conducted at the Akdeniz University Faculty of Medicine between September 2018 and October This study was conducted over the internet by using a questionnaire consisting of 9 questions addressing students of the Akdeniz University Faculty of Medicine. RESULTS: Ninety-seven students participated in the survey. Forty-three percent of the students who participated in the survey were second year students and 62% of them were women. Seventy percent of the students did not know about biotechnological drugs. Students stated that they had textbooks/notes (21%) as the source of their knowledge about biotechnological drugs. More than half of the students knew that biotechnological drugs were obtained from living cells and the cost of these drugs was high. When drugs in the biological/biotechnological classification were evaluated, more than half of the students knew that monoclonal antibodies were included as biological/biotechnological drugs. Ninety-one percent of the students stated that they did not know about biosimilars. Furthermore, they did not know that these products were produced/marketed in Turkey. Ninety-four percent of the students thought that a lecture on biotechnological drugs should be included in the curriculum of medical education. CONCLUSIONS: As a result, it is seen that medical students' level of knowledge about biotechnological drugs is not enough. Lectures of biotechnological drugs should be included in the medical education curriculum. REFERENCES 1. Kresse GB. Biosimilars-science, status, and strategic perspective. Eur J Pharm Biopharm. 2009;72(3): World Health Organization. (2015). Expert Committee on Biological Standardization. Guidelines on evaluation of similar biotherapeutic products (SBPs) Keywords Biotechnological drugs, biosimilars, medical education, questionnaire 222

225 POSTERLER / POSTERS P-121 Biosimilar Safety Suna Sabuncuoğlu, Gözde Girgin Hacettepe University, Faculty of Pharmacy, Department of Toxicology, Sihhiye, Ankara-Turkey Absract Biosimilar products are biological drugs that are very similar to a previously licensed biopharmaceutical product. In 2010, biosimilar drugs were almost absent among biotechnological drugs, while in 2016, their market share rised to 13.4%. Biosimilars can be authorized only if they are demonstrated to be highly similar to the original drug from an analytical and clinical perspective. Hence, they cannot be considered as the generic versions of original biological drugs. In comparison with traditional drugs, biosimilars have unique safety considerations. One of the most significant safety concerns with biosimilars is the potential risk of adverse immune reactions. Because of their molecular size, biologics can directly induce antibodies which may have significant consequences for both safety and efficacy. There is stil potential uncertainty on the biosimilar-induced immunogenic alterations. Because of the diversity in their structural complexity and indications, safety assessments have to be done on a drug basis. Early experience indicates that once biosimilars become available, initial safety concerns will decrease. Moreover, it can also be predicted that adverse event risks would be lower for a biosimilar drug compared to a new active substance. Regulatory guidelines mostly focus on biosimilars because of their safety concerns and interchangeability, automatic substitution, and nomenclature are the topics still being debated. Appropriate pharmacovigilance studies are very important and necessary for the safety considerations of biosimilars. Keywords biosimilar, safety, biologic drug 223

226 POSTERLER / POSTERS P-124 Effect of Niosome Preparation Techniques on Physicochemical Properties of Niosome/pDNA Complexes (Nioplexes) Devrim Demir Dora, Büsra Cesur, Feride Öner Department of Medical Pharmacology, Faculty of Medicine, Akdeniz University, Antalya,Turkey Absract Vesicular delivery systems such as liposomes and niosomes are widely used as non viral gene delivery systems. Niosomes are formed by self-association of nonionic surfactants and cholesterol in an aqueous phase. Physicochemical characterization of niosomes is essential before transfection studies. In this study we have evaluated the effect of niosome preparation techniques on physicochemical properties such as morphology, vesicle size, polydispersity index (PDI), zeta potential and stability of niosomes in the serum. Cationic niosomes were prepared by two different methods such as thin film hydration and proniosome technology. Span 60 and Brij 72 were used as surfactants for both methods at molar ratios of surfactant:cholesterol:cationic agent (CTAB) 60mM:15mM:25mM for Span 60 (S1, S2) and 72mM:18mM:10mM Brij 72 (B1, B2), respectively. In film formation method, nonionic surfactant, cholesterol and cationic agent were dissolved in 1 ml chloroform. Resolved mixture was evaporated in a rotary evaporator and the film was hydrated with 1 ml dh2o. The other niosome was prepared as proniosomes and resolved mixture was hydrated directly with 1 ml dh2o. LV-RFP plasmid was used for serum stability assay. Nioplexes were prepared at 1:1 (w/w) and coded as S1P, S2P, B1P and B2P. Then, serum stability was performed in DMEM containing 10% FBS. Nioplexes were incubated at 37ºC for 4 h. After 4 hours, the experiment was terminated with 1% SDS. The zeta potential results of S1P, S2P, B1P and B2P nioplexes were measured +43.7mV, mv, mv and +17 mv; the particle size of nioplexes were measured 498 nm, 654 nm, 484 nm and 466 nm; and the PDI of nioplexes were measured 0.70, 0.69, 0.68 and 0.66, respectively. When the serum stability of S1P, S2P, B1P and B2P nioplexes were compared to naked plasmid DNA, S1, S2, B1 and B2 niosomes prevented degradation of naked plasmid DNA against DMEM containing of 10 % FBS. Although niosome preparation methods have effect on charge and vesicle size of nioplexes they have no effect on serum stability. Thin film hydration method and proniosome technology can be used safely as DNA delivery systems. Keywords Niosome, Span 60, Brij 72, film hydration technique, proniosome 224

227 POSTERLER / POSTERS P-138 Analysis of Gene Regulation and Relationships of Breast Carcinoma due to Epithelial or Stromal Origin Tuğba Elgün 1, Tuba Saraç 2, Lokman Tunca 2, Asiye Gök Yurttaş 3, Umut Ağyüz 4 1 Biruni Üniversitesi, Tıp Fakültesi, Histoloji ve Embriyoloji Anabilim Dalı, İstanbul, Türkiye 2 Biruni Üniversitesi, İstanbul, Türkiye 3 Biruni Üniversitesi, Mühendislik ve Doğa Bilimleri Fakültesi, İstanbul, Türkiye 4 Genz Biotechnology, Ankara, Türkiye Absract Aim: In our study, it was aimed to interpret the related gene regulation and relationships with bioinformatics analysis in order to manage the early diagnosis and treatment process in tumor formation or cancer according to the epithelial or stromal origin in breast carcinoma. Introduction: Breast cancer is the most common type of cancer in women. Fibrocystic changes are the most common benign lesions of the breast. Benign (poor) histological changes of the breast are important for the development of invasive cancer in the future. Dysplasia, also known as non-invasive carcinoma, is an early stage cancer symptom. The main goal is to catch cancer at this stage. Dysplasia refers to an anomaly in the differentiation and maturation of cells. If there is a change in epithelium of the breast tissue, it is a dysplasia, but if it crosses the epithelium and passes to the stroma, it becomes an invasive cancer. Epithelial or stroma is of great importance in diagnosis and treatment. The histopathological classification accepted today is based on tumor characteristics and source cells. Method: In our study, the comparison of epithelial and stromal-induced breast cancers (carcinoma) in terms of KIT, EGF,MHGE8,SIK1,JUN, NTRK2,PAD12,MYH11,CX3CL1,ANXA1,OXTR,COL1A2,FN1 genes were performed using GenBank Overview-NCBI-NIH database. The relationship between genes has been evaluated by STRING: functional protein association networks, and direct or indirect effects have been tried to be determined according to literature and expression data (Figure 1). Logfold change (LogFC) values are taken into account when determining expression levels. LogFC <;0.9, p <0.05 in determining low expression levels; In the determination of high expression levels, logfc> 0.9, p<0.05 values were taken as criteria. 225

228 POSTERLER / POSTERS Result: Figure 1: Relations between Genes In terms of expression levels, no significant difference was found for the two breast cancer origins. In both of the origins, the expression level was increased in KIT, EGF, MHGE8, SIK1, JUN, NTRK2, PAD12, MYH11, CX3CL1, ANXA1, OXTR genes; The expression levels of the COL1A2, FN1 genes were decreased (Table 1). When the inter-gene interaction was examined, it was found that the maximum number of linkages was in the EGF gene (Chart 2). Table 1: Evaluation of gene regulation in carcinomas of stromal and epithelial origin 226

229 POSTERLER / POSTERS Figure 2: Evaluation of inter-gene relationships Reference: Koledova Z et al.spry1 regulates mammary epithelial morphogenesis by modulating EGFR-dependent stromal paracrine signaling and ECM remodeling. ProcNatlAcad Sci USA.2016Sep27;113(39):E doi: / pnas