Docetaxel-loaded Polymeric Micelles For Overcoming Multidrug Resistance

Polymeric micelle is a novel drug delivery vehicle in nanoparticles drug delivery field in recent years. Amphiphilic copolymers could form spontaneously into polymeric micelles with core-shell structure in aqueous solution. Its hydrophobic cores are able to be drug reservoirs, and its shell is hydrophilic. It includes two types:block copolymer micelles and grafted copolymer micelles (self-assembled nanoparticles) in broad definition. Multidrug resistance (MDR) is one of the main obstacles to the treatment of cancer in chemotherapy. In this study, a novel mixed micelle that comprised of MPEG-PLA (MPP) and Pluronic(?) copolymers was developed for enhanced bioavailability and to overcome multidrug resistance of docetaxel in cancer therapy. In the mean time, cholesterol-modified chitosan self-assembled nanoparticles (CHS-CS NPs) with a relatively high degree of substitution (15.3) were prepared by probe sonication for overcoming the multidrug resistance and thereby enhancing the intracellular uptake of docetaxel in cancer therapy.A series of methoxy poly (ethylene glycol)-b-poly (L-lactic acid) diblock copolymers (MPEG-PLA) with different molecular weight were synthesized by a ring-opening polymerization of L-lactide using MPEG as a maroinitiator with tin octoate as catalyst. Cholesterol-modified chitosan was synthesized with cholesteryl hemisuccinate and chitosan oligosaccharide by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) mediated coupling reaction. The degree of amino substitution was 15.3 determined by 2,4,6-trimitrobenzene sulfonic acid (TNBS) method.Polymeric micelle was prepared by co-solvent evaporation method. The optimized concentration of polymer in acetone was 150 mg/mL, and the ratio of acetone to water was 1:20 according to the investigation of micellar size. The prepared micellar size of plain MPEG-PLA micelles was between 20 and 50 nm. According to the drug loading content of docetaxel in different MPEG-PLA micelles, MPEG2-PLA2.5 was selected as the best block copolymer for further study. Three different Pluronic copolymers-L61, L62 and P85 were employed to investigate the influence of HLB and their ratios to MPEG-PLA on drug loading content and entrapment efficiency of docetaxel in MPEG-PLA/Pluronic mixed micelles (MPP/Pluronic MM). The optimized ratio of MPEG-PLA and Pluronic copolymer was 6:1 (w/w), and the final loading content of docetaxel in mixed micelles was above 3%. MPP/Pluronic MM solution was clear. The mean diameter of micellar size was 20～30 nm with narrow distribution. The micelles were well spherical particles observed by TEM. The release percent of docetaxel from MPP/Pluronic MM was below 60% in pH 7.4 buffer solution at 72 h by dialysis method. The release rate was also related to constitute of mixed micelles. The critical micellar concentration of MPEG-PLA/Pluronic mixed copolymer was around 4 mg/L detected by pyrene fluorescent probe method, which indicated micelles cannot be dissociated in diluted condition. The docetaxel loaded MPP/Pluronic MM showed good stability in diluted condition in virto.Compared to MPEG-PLA micelles (MPPM) and Taxotere(?), MPP/Pluronic MM could greatly increase the cytotoxicity of docetaxel against MCF-7/ADR and KBv mulidrug resistance tumor cells. It was also demonstrated that MPP/Pluronic MM could improve docetaxel absorption in KBv cells compared to the control group. Clear differences in the intracellular uptake of Coumarin-6 between MPPM and mixed micelles were observed using confocal laser scanning microscopy in KBv cells at 4 h.An effective HPLC method was developed for the determination of docetaxel in rat plasma samples. In vivo pharmacokinetics study in rats showed that the mixed micelles significantly enhanced the bioavailability of docetaxel (3.6 fold) than Taxotere(?), and it also prolonged the circulation time of docetaxel in vivo (2.05 h v.s.1.04 h). Moreover, antitumor activity assessed in KBv cancer xenograft BALB/C nude mice models showed that the mixed micelles significantly reduced the tumor size than the control (Taxotere(?)). The systemic toxicity of MPP/P85 MM also was lower than Taxotere(?) by determination of body weight change after i.v. administration.Plain CHS-CS NPs was prepared by probe sonication method. The mean diameter of nanoparticles was 304 nm with spherical appearance by TEM, itsζpotential was 21.3 mV. The loading content of docetaxel in CHS-CS NPs was around 2% by dialysis method for drug loading. The critical aggregation concentration (CAC) of CHS-CS copolymer was 0.0079 mg/mL, which could make its NPs to be stable in diluted condition. The in vitro release profile of the optimized CHS-CS NPs in different pH media revealed the nature of pH sensitive drug release profile from the prepared NPs. The release rate of docetaxel from CHS-CS NPs in pH 6.0 buffer solution was faster than that in pH 7.4 buffer solution.The in vitro cytotoxicity studies revealed the superiority of CHS-CS NPs over the control formulation in KBv cells. In vitro cellular uptake studies in sensitive KB and its multidrug resistance KBv cells showed that CHS-CS NPs significantly enhanced the cellular uptake of docetaxel than the control and these results further confirmed by FITC-labeled NPs. Moreover, clear differences in the intracellular uptake of docetaxel between chitosan and CHS-CS NPs were observed using confocal laser scanning microscopy. Our results suggested that CHS-CS NPs could be a potential carrier of docetaxel to overcome MDR effect of cancer cells.