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Nanoparticles with siRNA targeted to Hsp70 mRNA effectively silence Hsp70 and promote apoptosis

Purpose: RNA interference (RNAi) is an endogenously present regulatory mechanism in eukaryote cells. It involves the destruction of messenger RNA (mRNA) upon interaction with homologous double stranded RNA (dsRNA), known as small interfering RNA (siRNA). Apoptosis resistance of tumor cells is associated with increased expression of inducible heat shock proteins (Hsp). SiRNAs targeted to Hsp gene family can be introduced into tumor cell in order to accomplish greater sensitivity of tumor cells to apoptosis. Methods: In our study, aqueous solutions of chitosan, in the form of glutamate salt (molecular weight <200kDa ; deacetylation 75-90%), sodium tripolyphosphate and siRNA were used for nanoparticles preparation (siRNA-NP) with N:P ratio of 100:1. The average particle size and size distribution (polydispersity index ; PDI) was determined by photon correlation spectroscopy. The zeta potential was obtained by laser Doppler anemometry using a Zetasizer 3000 HS. Colorimetric MTT assay was performed to assess cell viability after 24h incubation with different volumes of siRNA-NPs. Quantitative RT-PCR was used for evaluation of siRNA(targeted against Hsp70)-NPs knockdown efficiency. Staurosporine was used as a model chemotherapeutic to study synergistic effect with Hsp70 gene silencing. The effect of siRNA-NPs on staurosporine induced apoptosis was measured with a quantitative immunoassay using mouse monoclonal antibodies directed against DNA (mono- and oligonucleosomes) and histones, that characterize apoptotic cell death. Results: Mean particle size of prepared siRNA-NPs was in the nano-range (~170-220 nm) when incubated in serum free medium. Surface charge of prepared siRNA-NP was ~ +30 mV but decreased to +20 mV (± 2, 2) when NPs were incubated in serum free medium, showing minor loss of chitosan positive charge in pH approaching pKa of chitosan. Cell viability was reduced to 80% (SD 5-10%) when different cell lines were incubated with 40 nM and 80 nM of siRNA entrapped into NPs. Hsp70 mRNA expression was significantly decreased after 24h incubation of cells with siRNA-NP in serum free medium. Expression levels of Hsp70 mRNA in different cell lines were approximately 56% of Hsp70 mRNA in negative control. Apoptosis was increased in the cells with posttranscriptionaly silenced Hsp70 gene. Conclusion: Chitosan-based NP containing HSP70 SiRNA were successfully delivered into different cell lines. Hsp70 mRNA expression was significantly decreased (for 44%), and apoptosis was enhanced in cells exposed to siRNA-NP. These results indicate that siRNA70-NP could be effective in reducing the tumor growth in vivo

RNA interference; Heat shock proteins; apoptosis; tumor

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