Non-viral siRNA and shRNA Delivery Systems in Cancer Therapy

RNA interference represents a promising therapeutic strategy for the silencing of specific target genes in cancer therapy. Small interfering RNAs and DNA-based vectors encoding short hairpin RNAs provide sequence-specific post-transcriptional gene silencing by binding to its complementary RNA. For the therapeutic use of siRNA in cancer, efficient intracellular delivery is necessary. The efficient cancer therapy with RNAi is not still accomplished because of internalization and intracellular trafficking problems such as low transfection efficiency, enzyme degradation, inappropriate subcellular localization, and endosomal trapping of siRNAs in cells. Cancer is a complex disease including multiple genes and pathways. The most important benefits of siRNA therapy are high target specificity and non-toxicity compared with chemotherapy. The uptake of siRNA by cells without a carrier system is possible, but naked siRNA is mostly degraded with nucleases and activates the immune responses. Development of appropriate delivery systems is an important issue in the use of siRNA-based therapeutics. Non-viral delivery systems have great potential for safe and effective delivery of siRNA therapeutics to tumor cells. Nanocarriers such as nanoplexes, lipoplexes, nanoparticles, and liposomes have been commonly used for siRNA delivery. This chapter highlights the importance of non-viral delivery systems in vitro and in vivo cancer therapy

Evaluation of the effect of honey-containing chitosan/hyaluronic acid hydrogels on wound healing

The 3D polymeric network structure of hydrogels imitates the extracellular matrix, thereby facilitating cell growth and differentiation. In the current study, chitosan/hyaluronic acid/honey coacervate hydrogels were produced without any chemicals or crosslinking agents and investigated for their wound-healing abilities. Chitosan/hyaluronic acid/honey hydrogels were characterized by FTIR, SEM, and rheology analysis. Moreover, their water content, water uptake capacities, and porosity were investigated. In FT-IR spectra, it was discovered that the characteristic band placement of chitosan with hyaluronic acid changed upon interacting with honey. The porosity of the honey-containing hydrogels (12%) decreased compared to those without honey (17%). Additionally, the water-uptake capacity of honey-containing hydrogels slightly decreased. Also, it was observed that hydrogels’ viscosity increased with the increased hyaluronic acid amount and decreased with the amount of honey. The adhesion and proliferation of fibroblast cells on the surface of hydrogel formulations were highest in honey-containing hydrogels (144%). In in vivo studies, wound healing was accelerated by honey addition. It has been demonstrated for the first time that honey-loaded chitosan-hyaluronic acid hydrogels, prepared without the use of toxic covalent crosslinkers, have potential for use in wound healing applications

Investigation of therapeutic effects in the wound healing of chitosan/pGM-CSF complexes

Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to promote the growth, proliferation, and migration of endothelial and keratinocyte cells. Chitosan has been widely used as a biopolymer in wound-healing studies. The aim of this study was to investigate the in vitro proliferative effects of chitosan/pGM-CSF complexes as well as the therapeutic role of the complexes in an in vivo rat wound model. The effect of complexes on cell proliferation and migration was examined. Wounds were made in Wistar-albino rats, and examined histopathologically. The cell proliferation and migration were increased weight ratio- and time-dependently in HaCaT and NIH-3T3 cell lines. Wound healing was significantly accelerated in rats treated with the complexes. These results showed that the delivery of pGM-CSF using chitosan complexes could play an accelerating role in the cell proliferation, migration, and wound-healing process

Synthesis and cytotoxicity studies on new pyrazolecontaining oxime ester derivatives

Purpose: To synthesize a series of new 1-(2-naphthyl)-2-(1H-pyrazol-1-yl)ethanone oxime ester derivatives (5-12) with potential anticancer properties, and to determine their cytotoxic effects in mouse fibroblast and human neuroblastoma cell lines. Methods: The title compounds were obtained through sodium salt reaction of 1-(naphthalene-2-yl)-2- (1H-pyrazol-1-yl)etanone oxime (4) with various acyl chlorides. The cytotoxic effects were evaluated by MTS colorimetric assay, while physicochemical descriptors were calculated using QikProp software. Results: Most of the compounds showed approximately 50 – 60 % inhibition against SH-SY5Y neuroblastoma cells at 100 μM. Of these, compound 7a was the most active combination with an IC50 value of 85.94 µM. The toxic effect of the compounds on mouse fibroblast cell line was insignificant (p < 0.05) even when the dose was increased. The calculated physicochemical properties of the compounds were within drug-like chemical space. Conclusion: The synthesized oxime ester derivatives with pyrazole ring exhibit selective toxicity to neuroblastoma cells without affecting healthy mouse fibroblast cells. The compounds proved to be druglike while their pharmacokinetic features were also encouraging, and were in line with in silico predictions

siRNA’nın biyodağılımına kitozan komplekslerinin etkisi

Amaç: RNAi kanser dahil olmak üzere birçok hastalığın moleküler mekanizmasının analizinde ve gen susturulmasında hücresel proseslerin kontrolü için önemli bir araçtır. VEGF sinyali meme kanserinde siRNA taşınmasında önemli bir hedeftir. siRNA farklı hastalıklar için potansiyel bir ajan olmasına rağmen, siRNA’nın intrasellüler taşınması, terapötik olarak aktif bir moleküle dönüşmesindeki önemli engellerden biridir. Bugüne kadar birçok transfeksiyon yöntemi ve taşıyıcı sistem geliştirilmiştir. Bunlar arasında kitozan, biyouyumlu, biyoparçalanabilir olması, toksik ve immunojenik olmaması gibi özellikleri nedeniyle önemli bir gen taşıyıcısıdır. Bu çalışmanın amacı, meme kanserinde kitozan/VEGF-siRNA komplekslerinin tümör lokalizasyonunu ve biyodağılımını araştırmaktır. Yöntem: Çalışmamızda meme tümörü taşıyan sıçanlara serbest FITC-işaretli siVEGF (40 µg/sıçan) ve kitozan/ FITC-işaretli siVEGF (40 µg/sıçan) kompleksleri intravenöz olarak enjekte edildi. Bulgular: Kitozan/siVEGF komplekslerinin beyin ve kalbe biyodağılımı, serbest siVEGF ile hemen hemen benzerken, dalak, karaciğer, akciğer ve kasta biraz daha düşük ve böbrekte ise biraz daha yüksektir. Meme tümör dokusunda, kompleksler enjeksiyon sonrası 15 dakikada tümörde lokalize iken, serbest FITC-siVEGF tümör dokusunda lokalize değildir. Sonuç: Bu ön çalışmada, biz biyodağılım için VEGF siRNA taşıyıcı sistem olarak kitozanın umut verici olduğunu gösterdik

Sirna için uygun taşıyıcı sistem geliştitilmesi; İn Vitro ve İn Vivo çalışmalar

ÖZETRNAi (RNA interferans), son zamanlarda başta kanser olmak üzere diğer önemli hastalıkların tedavisinde diğer geleneksel tedavilere alternatif olarak araştırılmaktadır. Ancak siRNA ile tedavide, uygun taşıyıcı sistemlerin geliştirilmesi önemli bir sorundur. Çalışmamızda biyogeçimli, toksik ve immunojenik olmayan bir polimer olan kitozan ile VEGF siRNA ve shRNA ile oluşturulan kompleksleri kullanarak deneysel meme tümör modelinde VEGF genini baskılamayı ve tümörde anjiogenezi durdurmayı amaçladık. Bu çalışmada katyonik bir polimer olan kitozan ile sentetik siRNA ve plazmid siRNA’ların kompleks ve nanopartikül formları hazırlanarak karakterizasyonu ve transfeksiyon etkinliği araştırılmıştır. Uygun formülasyonların in vitro HEK293, HeLa, MCF-7 ve MDA-MB-435 hücre hatlarında antisens etkinlikleri ve ELISA yöntemi ile de VEGF protein ekspressiyonu incelenmiştir. In vivo deneysel tümör modelinde de (Sprague-Dawley sıçanlarda) siVEGF ve shVEGF içeren kitozan komplekslerin VEGF mRNA’sı ve proteinini inhibe etme özellikleri, tümör hacminin ölçülmesi, immunohistokimya, kantitatif Real-Time PCR ve Western blot gibi değişik yöntemlerle araştırılmıştır. Sonuç olarak, 280 nm boyutundaki kompleks ve 246 nm boyutundaki nanopartikül formlarında kitozanın, VEGF siRNA’ları ekstrasellüler enzim ve serum parçalanmasına karşı koruduğu saptanmıştır. Farklı kompleks ve nanopartikül formlarının, in vitro transfeksiyon etkisini arttırarak gen susturma etkinliğini arttırdığı saptanmıştır. In vivo çalışmada ise, her iki tip siRNA’yı içeren kompleks formlarının meme tümöründe VEGF ekspressiyonunu hem mRNA, hem de protein seviyesinde azalttığı saptanmıştır. Özellikle tümör hacmi ve damar yoğunluğunda en fazla azalma, kitozan/siVEGF kompleksinin i.t uygulandığı grupta gözlenmiştir. shVEGF ile elde edilen sonuçlar daha düşük olmakla beraber i.t uygulanan grupta susturma i.p uygulanan gruplara göre daha yüksek bulunmuştur. Kitozanın VEGF siRNA için etkin ve güvenilir bir taşıyıcı sistem olarak kullanılabileceği görülmüştür. Bu çalışma gelecekte kitozan polimeri ile yapılacak siRNA taşıyıcı sistem araştırmaları için temel oluşturmaktadır. Anahtar Sözcükler: kitozan, kompleks, nanopartikül, shRNA, siRNA SUMMARYTHE DEVELOPMENT OF APPROPRIATE CARRIER SYSTEM for siRNA; In Vitro and In Vivo StudiesRecently, RNAi is frequently used in treatmet of the primarily to cancer, infectious, inflammation, apoptosis, oculer and respiratory tract diseases. However, treatment with siRNA, the development of appropriate systems are important problem. The aim of this study, is to VEGF gene silencing and angiogenesis inhibition in the experimental mammary tumor model using plasmid siRNA and synthetic siRNA to specific VEGF gene with chitosan as non viral vector for transfection because of several advantages such as biocompatibility, biodegredability, low immune response and non toxic. In this thesis, the complex and nanoparticle forms of siRNAs were prepared by using a cationic polymer called chitosan. Afterwards, the physicochemical properties such as zeta-potential, particle size, serum and enzyme stability and morphologic features of these formulations were measured and ELISA method with VEGF protein expression and the antisense activity in the different cell lines of the appropriate formulations were investigated in vitro. Measuring of the tumor volume, immunohistochemistry, Real-Time PCR and Western Blot methods inhibition to VEGF mRNA and protein of the complexes were investigated in vivo experimental mammary tumor model. Eventually, both forms protected siRNAs from potential enzymatic degredations. We were established to increased gene silencing efficiency to increase in vitro transfection efficiency of the different complex and nanoparticle forms. In vivo study was to performed determine decreased in the VEGF mRNA and protein expression in the mammary tumor of the complex forms containing siVEGF and psiVEGF. As a result, chitosan can be considered as a suitable carrier for siRNAs. This study can form the basis for the forthcoming studies related with carrier systems of siRNAs that will be done with chitosan polymer.Key Words: chitosan, complex, nanoparticle, shRNA, siRN