| Multi-drug resistance (MDR) is one of the major obstacles in cancer chemotherapy. P-glycoprotein (P-gp), overexpressed in drug-resistant tumor cells, is an energy-dependent efflux transporter and plays an important role in decreased intracellular accumulation and cytotoxic effect of anticancer drugs. In the recent years, small interference RNA (siRNA) is used to silence the expression of P-gp, so that the cells can restore chemosensitivity to anticancer drug such as doxorubicin. In this study, a strategy of sequential treatment with redox-responsive glucolipid conjugate micelles (CSO-ss-SA) loading MDR1 targeting siRNA and doxorubicin was applied to achieve optimal efficacy by administrated with anticancer drugs when the concentration of P-gp was the lowest.The selective redox-response nano carrier (CSO-ss-SA) was prepared in this study. The CSO-ss-SA could self-assemble into micelles in aqueous medium, which had a low critical micelle concentration (CMC) of 38.9μg/mL and showed a uniform spherical shape. The particle size and zeta potential were 79.9±3.7nm and 23.3±1.7mV, respectively.CSO-ss-SA could compact siRNA effectively to form nanocomplexes due to its cationic characteristics. The particle size and zeta potential of CSO-ss-SA/siRNA (N/P=150) were 39.6±3.9nm and 10.5±0.9mV. The gel retardation assay illustrated that the nanocarrier had the ability to condense siRNA and the RNase protection assay showed the CSO-ss-SA could protect siRNA from degradation by nucleases. The results of in vitro siRNA release indicated that the complexes could response to reducing environment of lOmM GSH and the cumulative siRNA release showed a decrease at the medium without GSH.The model drug doxorubicin (DOX) could be encapsulated into the hydrophobic core of CSO-ss-SA by the dialysis method to form CSO-ss-SA/DOX. The particle size and zeta potential of CSO-ss-SA/DOX were 65.7±2.2nm and 13.9±0.8mV. The encapsulation efficiency and drug loading were 67.4±3.0% and 6.39±0.26%, respectively. The result of in vitro drug release showed that the CSO-ss-SA could degrade fast and release drug in the medium of 10mM GSH.Take the MCF-7/ADR cells as model. The results of cellular uptake revealed that the CSO-ss-SA/siRNA had a time-dependent increase in internalization which was close to positive control. Intracellular trafficking observation indicated the complexes could escape from lysosomes effectively and widely distributed in the cytosol. Real Time-PCR assays showed that the CSO-ss-SA/siRNA could down-regulate MDR1 gene expression to 40% after incubation with MCF-7/ADR cells for 48h. The P-gp expression in the cells was decreased gradually after incubation with the CSO-ss-SA/siRNA for 1 to 3d since the down-regulation of MDR1 gene induced by siRNA was a time-dependent process; however, the expression was increased gradually after incubation for 4 to 7d due to the metabolism of siRNA.The internalization of CSO-ss-SA/DOX indicated that the drug delivery system could avoid the DOX molecular recognition by P-gp. After incubation with CSO-ss-siRNA for 0 to 7d, the MCF-7/ADR cells were sequentially incubated with CSO-ss-SA/DOX for 4h. The results showed the cells incubated with CSO-ss-SA/siRNA for 3d had the strongest ability to uptake CSO-ss-SA/DOX. This was the point when the P-gp expression was the lowest. The sequential treatment could attain optimal efficacy at this moment. Evaluate the cell viability by MTT method. The results confirmed that CSO-ss-SA and CSO-ss-SA/siRNA were both low cytotoxic. The sequential treatment with CSO-ss-SA/siRNA and CSO-ss-SA/DOX had the optimal cytotoxic which was 2.3 fold higher compared with the chemotherapy of CSO-ss-SA/DOX. The results of ATP assay revealed that the process of CSO-ss-SA/DOX endocytosis was ATP-consuming which consequently could counteract the activity of P-gp. In conclusion, the nano drug delivery systems had synergistic effect to reverse multidrug resistance by down-regulating the concentration and activity of P-gp. |
