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dc.contributor.advisorUlubayram, Kezban
dc.contributor.authorShahbazi, Reza
dc.date.accessioned2017-02-17T07:33:35Z
dc.date.available2017-02-17T07:33:35Z
dc.date.issued2017
dc.date.submitted2017-02-02
dc.identifier.citationReza Shahbazi, Development of a Gene-Knockdown Approach Based on siRNA/Gold Nanoparticles for Breast Cancer Therapy, Ph.D. Thesis in Nanotechnology and Nanomedicine, Hacettepe university, 2017.tr_TR
dc.identifier.urihttp://hdl.handle.net/11655/3180
dc.description.abstractBreast cancer is considered to be one of the leading causes of death in the 21st century and has claimed the lives of many people. As one of its subtypes, triple-negative breast cancer (TNBC) is a devastating disease, owing to the aggressive course, early metastasis, drug resistance, and poor clinical outcome and patient survival. Recently, different gene therapy approaches such as the use of siRNAs have brought hope for the treatment of TNBC. Although siRNA-based therapeutics offer tremendous potential as targeted therapies, their low transfection efficiency, and in vivo degradation by serum endo and exo-nucleases have prevented their delivery into tumors and made that an important obstacle for their translation into the clinic. So, the development of safe and effective delivery systems with robust target knockdown is of paramount importance. To address this problem, in this thesis it was aimed to develop a novel gold nanoparticle/siRNA based nanotherapeutic for gene knockdown approach in triple negative breast cancer. In the first and second sections of this thesis, it was aimed to develop gold nanoparticle based delivery vehicles and evaluate their cellular biodistribution. In this regard, highly stable and monodisperse polyethylenimine (PEI) functionalized gold nanoparticles were synthesized by a new method of surface modification. For the first time in literature, it was found that PEI functionalized gold nanoparticles in the size range of 45-82 nm, can enter to the cell nucleolus even in postmitotic dorsal root ganglion (DRG) neurons which are known to be very difficult cell types to transfect. It was shown that PEI functionalization increased the uptake rate of gold nanoparticles and delayed the fast excretion rate. Also, it was observed that PEI functionalization increased the emission signal intensity of gold nanoparticles. With a great potential for theranostic applications, the cellular biodistribution of nanoparticles was clearly visualized under both confocal and two-photon microscopes. In the third section, it was aimed to develop a siRNA therapeutic for TNBC therapy based on PEI-functionalized gold nanoparticles. With this purpose, eukaryotic elongation factor 2 kinase (eEF-2K) was chosen as the therapeutic target and conjugated with nanoparticles. Prepared siRNA therapeutic was highly monodisperse (PDI of 0.1) and stable. It was shown that this siRNA therapeutic was highly effective for eEF-2K gene down-regulation in vitro and in vivo and showed remarkable antitumor efficacy that was associated with eEF-2K knockdown, inhibition of Src and MAPK-ERK signaling pathways in a TNBC orthotopic tumor model. A significant tumor inhibition (90%) was achieved by only one intravenous injection of siRNA therapeutic per week at a relatively low dose of 8 µg/mouse (0.3 mg/kg), which is below the limit of any potential toxicity. Finally, siRNA therapeutic conjugated with doxorubicin was developed for combinational therapy approach which decreased the cell viability significantly in compare to sole doxorubicin chemotherapeutic drug. In conclusion, a potent theranostic gold nanoparticle based gene delivery vehicle was developed and used in the preparation of eEF-2K siRNA based therapeutics. It was concluded that prepared eEF-2K siRNA therapeutics are promising for TNBC treatment and imaging.tr_TR
dc.description.tableofcontentsABSTRACT ÖZET ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABBREVIATIONS 1. INTRODUCTION 1.1. General Information 1.2. Breast Cancer Statistics and Its Subtypes 1.2.1. Luminal Tumors 1.2.2. HER2 Over-Expression Tumors 1.2.3. Basal Tumors 1.3. Triple Negative Breast Cancer and Its Biology 1.3.1. Approved Strategies for Triple Negative Breast Cancer Therapy 1.3.2. Elongation Factor 2 Kinase as a Novel Target for TNBC Therapy 1.4. Oligonucleotide-Based Therapeutics in Gene Therapy 1.4.1. Morpholino Oligonucleotides 1.4.2. siRNAs 1.4.3. miRNAs 1.4.4. shRNAs 1.4.5. Hurdles in Front of Oligonucleotide-Based Cancer Therapeutics 1.5. Viral and Non-Viral Gene Delivery Vehicles 1.5.1. Viral Delivery Vehicles 1.5.1.1. Retroviral vectors 1.5.1.2. Adenoviral vectors 1.5.1.3. Adeno‑associated vectors 1.5.1.4. Helper‑dependent adenoviral vector 1.5.1.5. Hybrid adenoviral vectors 1.5.1.6. Herpes simplex virus 1.5.1.7. Lentiviruses 1.5.1.8. Poxvirus vectors 1.5.1.9. Epstein–Barr virus 1.5.2. Non-Viral Delivery Vehicles 1.5.2.1. Naked DNA 1.5.2.2. DNA particle bombardment by gene gun 1.5.2.3. Electroporation 1.5.2.4. Hydrodynamic 1.5.2.5. Ultrasound 1.5.2.6. Magnetofection 1.5.2.7. Cationic liposomes 1.5.2.8. Cationic polymers 1.5.2.9. Lipid–polymer systems 1.6. Nanoparticles as Robust Gene Delivery Vehicles 1.7. Gold Nanoparticles for the Delivery of Oligonucleotide Based Therapeutics 1.8. Review of the Published Literature in Cancer Gene Therapy 1.9. Aim of the Thesis 2. MATERIALS AND METHODS 2.1. Materials 2.2. Methods 2.2.1. Synthesis of Gold Nanoparticles and PEI Modification 2.2.1.1. Synthesis of seed gold nanoparticles 2.2.1.2. Growth of seed nanoparticles by seeding-growth method 2.2.1.3. Surface modification of synthesized gold nanoparticles 2.2.2. Cellular Biodistribution of Gold Nanoparticles 2.2.2.1. Isolation of DRG sensory neurons and cell culture studies 2.2.2.2. Determination of the cellular uptake by ICP-MS analysis 2.2.2.3. Imaging the cellular biodistribution of gold nanoparticles by confocal microscope 2.2.2.4. Imaging of the nanoparticle incubated cells by two-photon microscope 2.2.2.5. Toxicity studies of gold nanoparticles 2.2.2.6. Immunocytochemical studies 2.2.3. In Vitro and In Vivo Gene Delivery Studies 2.2.3.1. Determination of optimal siRNA conjugation ratio 2.2.3.2. siRNA design and in vitro gene-knockdown studies 2.2.3.3. Development of orthotopic xenograft tumor model in mice 2.2.3.4. Injection of prepared nano-formulation and tumor analysis 2.2.4. Development of Doxorubicin and eEF-2K siRNA Conjugated Gold Nanoparticles for Combinational Therapy Approach 2.2.4.1. Preparation of doxorubicin and eEF-2K siRNA conjugated gold nanoparticles 2.2.4.2. In vitro evaluation of the doxorubicin conjugated gold nanoparticles in MDA-MB-231 cells 2.2.4.3. In vitro evaluation of the doxorubicin and eEF-2K siRNA conjugated gold nanoparticles in MDA-MB-231 and MDA-MB-436 cells 2.2.5. Statistical Analysis 3. RESULTS AND DISCUSSION 3.1. Synthesis of Gold Nanoparticles 3.1.1. Determining the Optimal Synthesis Method 3.2. Surface Modification of Gold Nanoparticles with Polyethylenimine 3.2.1. Synthesis of Citrate Capped Gold Nanoparticles in Different Size Ranges 3.2.2. Polyethylenimine Modification of Gold Nanoparticles in Different Size Ranges 3.3. Cellular Biodistribution and Imaging Potential of Gold Nanoparticles 3.3.1. Effect of Gold Nanoparticles on Cell Morphology 3.3.2. Effect of Gold Nanoparticles on Cell Viability 3.3.3. Effect of Gold Nanoparticles on Neural Cell Skeleton 3.3.4. Cellular Uptake of Gold nanoparticles 3.3.5. Imaging the Cellular Biodistribution of Gold Nanoparticles by Confocal Microscope 3.3.6. Nucleolar Localization of PEI-Functionalized Gold Nanoparticles 3.3.7. Imaging Gold Nanoparticle Incubated Cells by IR Irradiation 3.3.8. Potential Mechanism for Nucleolar Localization 3.4. Gene-Knockdown Studies by siRNA/Gold Nanotherapeutics for Triple Negative Breast Cancer Therapy 3.4.1. Properties of PEI-Functionalized Gold Nanoparticles as eEF-2K siRNA Carriers 3.4.2. Conjugation of eEF-2K siRNA to PEI-Functionalized Gold Nanoparticles 3.4.3. In Vitro Down-Regulation of eEF-2K by AuNP-PEI/eEF-2K siRNA 3.4.4. Effective In Vivo Target Knockdown and Growth Inhibition in a TNBC Tumor Model 3.5. Doxorubicin Conjugated siRNA Therapeutics for Combinational Therapy 3.5.1. Characterization of Doxorubicin and eEF-2K siRNA Conjugated Gold Nanoparticles 3.5.2. Effect of Doxorubicin Conjugated Gold Nanoparticles on the Cell Viability of MDA-MB-231 Cells 3.5.3. Effect of Combinational Therapy with Doxorubicin and eEF-2K siRNA Conjugated Gold Nanoparticles on the Cell Viability of MDA-MB-231 Cells CONCLUSION REFERENCES CURRICULUM VITAE APPENDIX 1tr_TR
dc.language.isoentr_TR
dc.publisherFen Bilimleri Enstitüsütr_TR
dc.rightsinfo:eu-repo/semantics/openAccesstr_TR
dc.subjectPEI-functionalized gold nanoparticlestr_en
dc.subjectNucleolus
dc.subjecteEF-2K
dc.subjectsiRNA,
dc.subjectNanotherapeutics
dc.subjectTriple-negative breast cancer
dc.subjectTheranostics
dc.titleDevelopment of A Gene-Knockdown Approach Based on Sirna/Gold Nanopartıcles for Breast Cancer Therapytr_en
dc.typeinfo:eu-repo/semantics/doctoralThesistr_TR
dc.description.ozetMeme kanseri, 21. yüzyılda önde gelen ölüm nedenlerinden biri olarak kabul edilmekte olup birçok kişinin yaşamını yitirmesine neden olmaktadır. Meme kanserinin alt tiplerinden biri olarak bilinen üçlü negatif meme kanseri (ÜNMK), agresif seyir, erken metastaz, ilaç direnci, zayıf klinik sonuç ve düşük hayatta kalma oranı nedeniyle yıkıcı bir hastalık olarak bilinmektedir. Son zamanlarda, siRNA'ların kullanımı gibi farklı gen tedavisi yaklaşımları ÜNMK'nın tedavisi için umut vaat etmektedir. siRNA temelli terapötikler hedefe yönelik terapiler olarak büyük bir potansiyel sunmakla birlikte düşük transfeksiyon verimliliği ve in vivo şartlarında serum endo ve ekso-nükleazlar tarafından bozunmaları, siRNA’ların tümöre taşınmasını önlemekte ve kliniğe translasyonunda büyük engel teşkil etmektedir. Bu nedenle güçlü hedef gen susturmasına sahip olan güvenli ve etkin taşıma sistemlerinin geliştirilmesi büyük önem taşımaktadır. Bu sorunu çözmek için, bu tez kapsamında üçlü negatif meme kanserinde gen susturma yaklaşımı için yeni bir siRNA/altın nanopartikül bazlı nanoterapötiklerin geliştirilmesi amaçlanmıştır. Tezin, birinci ve ikinci bölümlerinde altın nanopartikül bazlı gen taşıma araçlarının geliştirilmesi ve hücresel dağılımlarının değerlendirilmesi hedeflenmiştir. Bu amaçla son derece kararlı ve monodispers polietilenimin (PEI) ile fonksiyonlaştırılmış altın nanopartiküller yeni bir yüzey modifikasyonu yöntemi ile sentezlenmiştir. Literatürde bir ilk olarak, 45-82 nm boyut aralığında PEI ile fonksiyonlaştırılmış altın nanopartiküllerin, transfeksiyonu çok zor olduğu bilinen postmitotik dorsal kök gangliyon (DKG) nöronlarında hücre çekirdekciğine girdiği açıklanmıştır. Ayrıca, PEI ile fonksiyonlaştırmanın altın nanopartiküllerin hücre alımını arttırdığı ve atılım hızını geciktirdiği gösterilmiştir. Ayrıca, PEI ile fonksiyonlaştırılan altın nanopartiküllerin emisyon sinyal yoğunluğunu artırdığı izlenmiştir. Teranostik uygulamalar için büyük potansiyele sahip olan bu nanopartiküllerin hücresel dağılımı hem konfokal hem de çift fotonlu mikroskoplar altında açıkça görüntülenmiştir. Üçüncü bölümde, ÜNMK tedavisi için PEI ile fonksiyonlaştırılmış altın nanopartikül ile siRNA terapötiklerin geliştirilmesi amaçlanmıştır. Bu amaçla, ökaryotik uzama faktörü 2 kinaz (eEF-2K) terapötik hedef olarak seçilmiş ve altın nanopartiküllerle konjuge edilmiştir. Hazırlanan siRNA terapötiklerin oldukça monodispers (PDI=0,1) ve kararlı olduğu izlenmiştir. Bu siRNA terapötiklerin, in vitro ve in vivo şartlarda gen susturma için son derece etkili olduğu ve ÜNMK ortotopik tümör modelinde eEF-2K’yı susturma, Src inhibisyonu ve MAPK-ERK sinyal yolaklarıyla ilişkili olarak dikkate değer antitümör etkinliğine sahip olduğu gösterilmiştir. Haftalık tek bir intravenöz enjeksiyonuyla fare başına 8 mikrogram (0,3 mg/kg) olan düşük bir dozda anlamlı tümör inhibisyonu (%90) sağlanmış olup, bu doz potansiyel toksisite dozunun altındadır. Son olarak doksorubisin ile konjuge edilmiş siRNA terapötikler kombinasyon tedavi yaklaşımı için geliştirilmiş ve bunların tek başına doxorubisin ile karşılaştırıldığında hücre canlılığını önemli ölçüde azalttığı gözlenmiştir. Sonuç olarak, etkili teranostik altın nanopartikül bazlı gen taşıma aracı geliştirilerek, eEF-2K siRNA terapötiklerin hazırlanmasında kullanılmıştır. Hazırlanan eEF-2K siRNA terapötiklerin ÜNMK tedavisinde ve görüntülemede umut verici olduğu sonucuna varılmıştır.tr_TR
dc.contributor.departmentNanoteknoloji ve Nanotıptr_TR


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