Study On Co-delivery Of Doxorubicin And Breast Cancer MDR Reversal Inhibitor Elacridar Using A Redox And PH Dual Responsive PAMAM Nanoparticle

Objective: To prepare a redox and pH dual sensitive nano-carrier based on PAMAM in order to co-deliver chemotherapeutic agent doxorubicin and breast cancer multidrug resistance reversal agent elacridar, and study their in vitro reversal effect.Methods: The 1H NMR and FTIR were used to characterize the carrier PSSP.PSSP/DOX/ELC nanoparticles were prepared to co-delivery DOX and ELC by hydrophobic effect. The nanoparticles were charactered using particle size, Zeta potential,and the drug loading content and encapsulation efficiency were also determined. Dialysis bag method was applied to evaluate in vitro drug release behavior. MCF-7(sensitive to DOX) and MCF-7/ADR(nonsensitive to DOX) cells were chose as the model cells. The cytotoxicity of blank-carrier, free ELC, PSSP/DOX and PSSP/DOX/ELC nanoparticles, as well as IC50 values, resistant index and reversal factor were evaluated by MTT assay.Confocal, flow cytometry and acidified isopropanol extraction methods were used to qualitatively and quantitatively analyse cellular uptake. Cellular uptake mechanism was investigated by representative inhibitors, and intracellular triggered drug release property was evaluated by redox and acid inhibitors. Rhodamine 123, as one of the natural P-gp substrate, was used to determine the intracellular efficient concentration and efflux inhibition effect of ELC by flow cytometry. The ATP level and P-gp expression were also analyzed to reveal function mechanism of ELC. In vivo antitumor experiments were performed on MCF-7 tumor-bearing nude mice with different therapy strategies. The pathological change of cancer and major organs was investigated using hematoxylin and eosin(HE) staining.Results: The pH and redox dually sensitive carrier PSSP were successfullysynthesized. The particle size of nanoparticle entraping DOX and ELC with different molar ratios ranged from 10 to 20 nm, and Zeta potential were between +2 to 5 mV. The results suggested that PSSP had high encapsulation efficiency, and the drug loading content of DOX and ELC were about 10% and 2%, respectively. Obvious redox and pH sensibility of PSSP was investigated by in vitro release experiment. PSSP/DOX/ELC nanoparticle exhibited higher cytotoxicity and enhanced reversal effect of MDR compared to PSSP/DOX nanoparticle. Cellular uptake studies revealed that the uptake of PSSP/DOX/ELC were superior to that of PSSP/DOX nanoparticle. The main cell endocytosis way of nanoparticles was proved to be mainly mediated by clathrin, and drug intracellular release was triggered by lysosomal acid environment and high extranuclear reductive environment. What’s more, inhibition of DOX efflux by P-gp, increased cellular ATP content caused ELC treatment was investigated at a low concentration, which resulted to the increased cellular uptake. In vivo anticancer study, indicated that PSSP/DOX/ELC nanoparticle could obviously inhibit the growth of tumor, at same time, significantly reduce the side effects of DOX. Moreover, PSSP/DOX/ELC nanoparticle could inhibit drug efflux stress reaction at early stage of chemotherapy and reverse MDR, which would greatly reduce the difficulty of tumor therapy.Conclusions: In this study, we have successfully constructed a redox and pH dual sensitive nano-carrier(PSSP) based on PAMAM to co-deliver doxorubicin(DOX) and ELC. The prepared nanoparticles with uniformed size had high encapsulation efficiency and drug loading content, and presented well redox and pH dual sensibility in vitro. Both in vitro and in vivo anticancer studies revealed that PSSP/DOX/ELC nanoparticle could effectively reverse MDR of breast cancer and enhance the effect of tumor chemotherapy.Besides,PSSP/DOX/ELC treatment in early stage of tumor therapy can reduce drug efflux stress reaction and reverse MDR. All of these suggested PSSP/DOX/ELC would have a broad application prospect in the field of the antitumor drug delivery system.