Construction Of Poly(L-histidine)and TPGS Based Nano Drug Delivery Systems For Reversal Of Multidrug Resistance

Currently,chemotherapy is one of the main strategies for cancer treatment.However,the emergence of multidrag resistance(MDR)renders cancer cells immune to cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents thus becomes a major obstacle in cancer chemotherapy.Therefore,it is important and challenging to develop safe and efficient drug delivery systems to reverse MDR in cancer cells.The main objectives of the proposal are to design and study two functional nano drug delivery systems with triggered release property for intracellular delivery of doxorubicin(DOX)and overcoming MDR.The main contents of this study include the synthesis and identification of the polymers and the evaluation of the in vitro and in vivo antitumor activity of DOX-loaded nanocarriers on MCF-7/ADR cells.Meanwhile,the corresponding cell uptake mechanisms and reversing MDR mechanisms were also preliminarily investigated.A novel pH-sensitive polymer,poly(L-histidine)-poly(lactide-co-glycolide)-tocopheryl polyethylene glycol succinate(PLH-PLGA-TPGS),was first synthesized to design a biocompatible drug delivery system for cancer chemotherapy.The structure of the PLH-PLGA-TPGS copolymer was confirmed by FT-IR,1H-NMR and GPC.The apparent pKa of the PLH-PLGA-TPGS copolymer was calculated to be 6.33 according to the acid-base titration curve.The doxorubicin(DOX)-loaded nanoparticles(PLH-PLGA-TPGS nanoparticles and PLGA-TPGS nanoparticles)and corresponding blank nanoparticles were prepared by a co-solvent evaporation method.The blank PLH-PLGA-TPGS nanoparticles showed an acidic pH-induced increase in particle size.The DOX-loaded nanoparticles based on PLH-PLGA-TPGS showed a pH-triggered drug-release behavior under acidic conditions.Facilitated by the cytotoxicity and uncompetitive P-gp ATPase inhibition by PLH-PLGA-TPGS,the results of in vitro cytotoxicity experiment on MCF-7 and MCF-7/ADR cells showed that the DOX-loaded PLH-PLGA-TPGS nanoparticles resulted in lower cell viability versus the PLGA-TPGS nanoparticles and free DOX solution.Confocal laser scanning microscopy images showed that DOX-loaded PLH-PLGA-TPGS nanoparticles were internalized by MCF-7/ADR cells after 1 and 4 h incubation and most of them accumulated in lysosomes to accelerate DOX release under acidic conditions.In order to design an intelligent drug delivery system with prolonged circulation time and active tumor targeting characteristics,two novel functional copolymers(methoxy poly(ethyleneglycol)-poly(L-histidine)-d-a-Vitamin E Succinate(MPEG-PLH-VES)and TPGS 3400-Biotin)were then synthesized.The structures of the two copolymers were confirmed by 1H-NMR,FT-IR and GPC.Meanwhile,the pH-sensitve characteristics of MPEG-PLH-VES were also investigated by acid-base titration method and laser light scattering technique.The DOX-loaded functional micelles(with or without Biotin decoration)showed a pH-triggered drug-release behavior under acidic conditions.The resistant MDR effect of MPEG-PLH-VES micelles(with or without Biotin decoration)and relevant reversal mechanism were investigated in DOX resistant MCF-7/ADR cells.MDR reversal mechanism experiments showed that the blank MPEG-PLH-VES micelles(with or without Biotin decoration)inhibited P-gp activity by reducing mitochondrial membrane potential and depletion of ATP.Furthermore,MPEG-PLH-VES copolymers were able to inhibit the Verapamil-induced ATPase activity of P-gp without altering P-gp expression.The Cellular uptake tests showed that the MPEG-PLH-VES/B micelles resulted in higher cellular uptake than the MPEG-PLH-VES micelles and free DOX solution.The uptake of MPEG-PLH-VES/B micelles were mainly through SMVT receptor mediated endocytosis.The main endocytic pathway was clathrin-mediated endocytosis.The results of in vitro cytotoxicity experiments on MCF-7 and MCF-7/ADR cells showed that DOX-loaded micelle formulations exhibited better cytotoxic effect against MCF-7/ADR cells than free DOX solution.CLSM images showed that DOX-loaded MPEG-PLH-VES micelles(with or without Biotin decoration)were internalized by MCF-7/ADR cells after 1 and 4 h incubation and most of them accumulated in lysosomes to accelerate DOX release under acidic conditions.For DOX-loaded micelles,obvious DOX endo-lysosomal escape was observed by CLSM at 4 h of incubation.The time-dependent biodistribution and in vivo anti-tumor efficacy of the DOX-loaded MPEG-PLH-VES micelles(with or without Biotin decoration)were evaluated in nude mice bearing MCF-7/ADR tumor.In vivo fluorescence imaging test showed DIR/MPEG-PLH-VES/B micelles retained in the body for a longer time and displayed higher fluorescence accumulation in tumor site compared with that of DIR/MPEG-PLH-VES micelles.To evaluate the in vivo antitumor efficacy,the saline,DOX solution and DOX-loaded micelles were intravenously injected to the nude mice bearing MCF-7/ADR tumor,respectively.MPEG-PLH-VES/B micelles showed more significant inhibition of tumor growth as compared to MPEG-PLH-VES micelles(P<0.05)and DOX solution(P<0.01).Meanwhile,no significant change in body weight of mice was observed during treatment with DOX-loaded micelles.This result indicates that the DOX-loaded micelles could effectively inhibit MCF-7/ADR tumor growth with decreased systemic toxicity.