Triblock Polymer Micelle Mediated Co-delivery Of Doxorubicin And P-glycoprotein Si Rna For Reversal Of Multidrug Resistance And Synergistic Tumor Therapy

Objective:To prepare a triblock copolymer micelle for codelivering doxorubicin(Dox) and si RNA-P-glycoprotern(P-gp)(si RNA-Dox-micelle). To demonstrate the effectiveness of Dox-si RNA-micelle in tumor-targeting and MDR reversing, and provide a promising strategy to develop a codelivery system with synergistic effect for combined cancer therapy.Methods:(1) Triblock copolymer micelle was prepared based on the synthetic polymer of N-succinyl chitosan- poly-L-lysine- palmitic acid(NSC-PLL-PA) to codeliver doxorubicin(Dox) and si RNA-P-glycoprotern(P-gp)(si RNA-Dox-micelle).(2) The cellular uptake and subcellular localization characteristics of si RNA-micelle、Dox-micelle and si RNA-Dox-micelle were investigated, and the intracellular drug accumulation and elimination of different Dox formulations were also quantitative evaluated.(3) The relationship between cytotoxicity of different Dox formulations and intracellular accumulation of Dox was investigated to study the effect of intracellular drug concentration on the in vitro antitumor efficacy.(4) The transfection efficiency of si RNA-micelle and the potential targeting efficacy of si RNA-Dox-micelle were detected, the relationship between P-gp transfer and the unsatisfactory therapeutic efficiency of chemotherapeutics was discussed, and the effectiveness of si RNA-Dox-micelle in synergistic tumor therapy was clarified by enhancing tumor-targeting and MDR reversing.Results:(1) NSC-PLL-PA was successfully synthesized through the condensation method,NSC as the hydrophilic shell was designed to increase the half-life of micelle in blood and decrease the toxicity of PLL, PLL as the cationic backbone was expected to electrostatic absorb the negatively charged si RNA, and PA as hydrophobic core was allowed for encapsulating Dox. The triblock copolymer tend to form the core-shell structure of micelle with PA self-assembling in the core in the water. The resulting micelle exhibited a narrow size distribution and good dispersion, accompanied with high encapsulation efficiency and drug loading for Dox, as well as efficient binding ability for si RNA. si RNA-Dox-micelle was unstable in p H 5.3 medium than in p H 7.4 medium, which corresponded with the in vitro rapid release of Dox and si RNA in acidic environments.(2) The co-localization of Dox and si RNA in the cytoplasm were indicated the simultaneous delivery of si RNA and Dox after incubated with si RNA-Dox-micelle. The fluorescent intensity of intracellular Dox and si RNA, and the proportion of both green and red positive cells increased as the incubating time prolonging. Increasing accumulation of Dox and si RNA in the cytoplasm was result in better synergistic effect of antitumor efficacy. Dox signal gradually transferred from the cytoplasm to the nucleus, while almost of si RNA still located in the cytoplasm, thus improving synergistic therapeutic efficacy in their each targeted region.(3) si RNA-Dox-micelle could improve therapeutic efficacy by bypassing the drug efflux, down-regulating the level of P-gp, increasing intracellular drug concentration and obtaining synergistic therapeutic efficacy. Morever, a further increase of intracellular drug accumulation could transfer to higher therapeutic efficacy.(4) si RNA-micelle was effective in deliving si RNA into cytoplasm and downregulating the expression of P-gp. However, P-gp could transfer between cells and retains its bioactivity, and the associated phenotype was not the concequence of mdr1 gene expression, thus limiting the desired efficiency of si RNA-micelle. si RNA-Dox-micelle showed a synergistic antitumor activity and significantly improved the survival quality of tumor-bearing mice, and enhanced the cytotoxicity of Dox through MDR reversing.Conclusion : The synthesized NSC-PLL-PA exhibited an higher encapsulation efficiency for Dox and si RNA, and effective in intracellular release in the cytoplasm. si RNA-Dox-micelle was demonstrated the effectiveness in tumor-targeting, down-regulating the expression of P-gp, overcome multidrug resistance, and synergistic therapeutic effect.