engleski

Chitosan-TPP/siRNA mediated Hsp70 knockdown: potential cancer therapeutic

Apoptosis resistance of tumor cells is associated with increased expression of heat shock proteins (Hsp). RNA interference nanotechnology can be used for Hsp gene silencing, in order to accomplish greater sensitivity of tumor cells against chemotherapeutics, which are based on apoptosis promotion. Solution of chitosan, in the form of glutamate salt (<200kDa), in acetate buffer (pH 4, 5), pentasodium tripolyphosphate (TPP) aqueous solution and siRNA (targeted against Hsp70) aqueous solution were used for chitosan-TPP and chitosan-TPP-siRNA nanoparticles (NPs) preparation. RPMI medium supplemented with 2mM L-Glutamine, 1% Antibiotic-Antimycotic solution, with or without 10% foetal bovine serum (FBS), was used for Jurkat cell line growth and treatment with NPs. Mean particle size of NPs obtained was 194, 95 ± 24, 05 nm for chitosan-TPP NPs and 197, 8 ± 3, 9 nm for chitosan-TPP-siRNA NPs. Greater size of chitosan-TPP NPs was observed when NPs were incubated in serum free medium (from 240, 9 nm to 852, 0 nm) contrary to the size of chitosan- TPP-siRNA NPs which showed no significant change (214, 8 ± 4, 3 nm). Surface charge for all NPs obtained was ~ +30 mV, but decreased to values around +20 mV when NPs were incubated in the serum free RPMI medium. The changes in size and surface charge were ascribed to the change in pH from 4, 5 (acetate buffer) to 6, 5 (growth medium when mixed with NPs). Chitosan-TPP-siRNA NPs loading efficiency was measured to be ~ 75-85%. Binding of siRNA with chitosan determined by agarose gel electrophoresis, showed retarded movement of siRNA loaded into NPs compared to naked siRNA. Viability of Jurkat cells decreased to ~ 80% after 24 h of incubation with chitosan-TPP-siRNA NPs in serum free medium. Only ~ 40% (p < 0, 01) of the amount of Hsp70 gene transcript was detected with real-time PCR analysis after this treatment (final concentration of siRNA was 50 nM). This work demonstrates the efficiency of chitosan-TPP-siRNA NPs as a gene silencing tool.

RNA interference; Heat shock proteins; apoptosis; tumor

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