AMPHIPATHIC CHITOSANS AS VECTORS FOR SIRNA DELIVERY

Abstract

Background Polycations are promising candidates for siRNA delivery due to their ability to complex siRNA molecules into small nanoparticles (NPs)[1,2]. In this study, we have synthesized and evaluated the potential of amphipathic chitosans as siRNA carriers for the intracellular delivery of siRNA molecules. Methods Chitosans with varying degrees of acetylation (DA) and increasing proportions of diethylaminoethyl groups (DEAE) were synthesized and characterized. siRNA nanoparticles were prepared and their physicochemical properties and transfection efficiency evaluated on Raw 264.7 macrophages and HeLa-GFP cell lines. Results The results showed that the adjustment of DA and DEAE groups decreased the interparticle interactions allowing colloidal nanoparticles 150 nm in size, low polydispersity indices (0.15 - 0.2) and positive Zeta potentials (+4 mV to +12 mV) to be formed. The resistance of nanoparticles to aggregation was provided by changes in the surface of nanoparticles and their pegylation reduced the nanoparticle sizes to around 100 nm at pH 6.3. Real-time confocal microscopy measurements revealed a fast release of siRNA-FAM in the intracellular environment. In vitro transfection studies with pegylated NPs were more efficient than lipofectamine in the silencing of TNF-alpha in macrophages by 63% and 48%, respectively. Similarly, transfection studies on epithelial cells of cervical carcinoma (HeLa-GFP) also showed that derivatives modified with DEAE and PEG formed NPs that enabled an efficient GFP knockdown. Conclusion The results have provided a clear approach to overcome the limited stability of chitosan nanoparticles under physiological conditions and confer potential to these vectors for in vivo studies. References [1]Yin, H., Kanasty, R. L., Eltoukhy, A. A., Vegas, A. J., Dorkin, J. R., & Anderson, D. G. (2014). Non-viral vectors for gene-based therapy. Nature Reviews Genetics, 15(8), 541. [2] MJ Tiera, Q Shi, HFG Barbosa, JC Fernandes (2013) . Polymeric Systems as Nanodevices for siRNA Delivery. Current Gene Therapy, 13 (5) 358. The Authors thank FAPESP (Grants No. 2017/10331-5; 2015/05148-1 and 2015/20206-8; 2012/24259-0). CAPES (Grants PNPD 1267244 and 1743469). The authors report no conflicts of interest in this work. The authors would like to thank L. R. F. Junior and L. F. M. Ferraz (Embrapa Instrumentacao-Sao Carlos) for help with the Scanning Electron Microscopy, and C. R. Bonini Domingos for access to instrumentation.