Reduction-sensitive Polymeric Nanoparticles For Synergistic Drug Delivery

Nanoscaled drug delivery systems based on biodegradable amphiphilic copolymers have many merits, such as excellent biocompatibility, favorable size distribution, reduced drug side effects, improved drug tolerance, delivering multiple drugs at the same time and enhanced permeation retention (EPR) effect. However, the concentration of drugs in the cancer cells is often insufficient for killing cancer cells due to the low dose of drug released or the slow release of drug from the conventional drug carriers. Stimuli-responsive nanoparticles have been designed to control drug release temporally and spatially upon changes in tumor tissue environments, such as redox potential, owing to the markedly different glutathione (GSH) concentration between intracellular (2-10 x 10-3 M) and extracellular environment (2-20 ?M) for tumor cells. In addition,the anti-tumor effect can be further improved by the introduction of targeting ligand, such as folic acid (FA) that exhibits superior targeting ability to folate receptor (FR) on many tumor cells, including ovarian and breast cancers. Moreover, the drugs encapsulated in polymeric nanoparticles enter into drug-resistant tumor cells via endocytotic internalization pathway, which is different from free drugs and avoids the P-gp-mediated drug efflux in drug-resistant cells, resulting in effective drug accumulation in MCF-7/ADR cells. In this work, our studies are focused on a series of reduction-sensitive drug carriers for synergistic drug delivery and overcoming multidrug resistance.In chapter 1, developments and applications of smart drug delivery systems were briefly reviewed, with the emphasis on the delivery systems for synergistic delivery of multiple drugs, the mechanisms of tumor multidrug resistance and the strategies to reverse multidrug resistance.In chapter 2, the crosslinked reduction-sensitive copolymer PEG-P(TMC-co-TDCSS) was obtained via a facile one-step procedure for efficient delivery of doxorubicin (DOX) into cancer cells. To serve as a control, PEG-P(TMC-co-TDCCC) that has an analogous structure but without disulfide bond and a linear polymer PEG-PTMC were also prepared. The copolymers could self-assemble to form nano-sized micelles in aqueous solution. As compared to PEG-PTMC, crosslinked PEG-P(TMC-co-TDCSS) and PEG-P(TMC-co-TDCCC) showed lower CMC values and thus induced a much better micelle-forming ability. In vitro release studies revealed that the drug release behavior of DOX-loadedPEG-P(TMC-co-TDCSS) micelles which could be accelerated in the presence of 10 mM dithiothreitol (DTT). Therefore, although DOX-loaded PEG-P(TMC-co-TDCSS) micelles showed lower cytotoxicity in vitro than free DOX, it still could deliver DOX efficiently to the cell nuclei, displaying higher cytotoxicity against HeLa cells than PEG-PTMC and PEG-P(TMC-co-TDCCC) micelles. Confocal laser scanning microscopy (CLSM) indicated that DOX-loaded PEG-P(TMC-co-TDCSS) micelles were efficiently internalized into Hela cells, released DOX into the cytoplasm and the drug finally entered the nuclei.In chapter 3, we first synthesized an amphiphilic block copolymer PEG-P(TMBPEC-co-MPMC), with pendent reactive alkynyl groups as well as pH-sensitive acetal groups. Next, core crosslinked (CCL) micelles were prepared by the introduction of 1,6-diazidohexane and bis(azidoethyl) disulfide into micelles via azide-alkyne click chemistry, which were denoted as CCL/CC and CCL/SS, respectively. The CCL micelles had superior stability and drug loading efficiency to the uncrosslinked (UCL) micelles. In comparison with free DOX, DOX-loaded CCL micelles exhibited lower cell viability in MCF-7/ADR cells due to their "stealth" endocytosis effect that might be beneficial for overcoming delivery barriers of drug resistance. More interestingly, as compared with DOX-loaded CCL/CC micelles, DOX-loaded CCL/SS micelles were found to further enhance cytotoxicity in MCF-7/ADR cells because of their better on-demand drug release capability of pH and redox dual-sensitive CCL/SS micelles. These results suggest the self-assembled pH and redox dual-sensitive CCL/SS micelle could inhibit the growth of normal tumor cells and overcome multi-drug resistance of MCF-7/ADR cells.In chapter 4, reduction-sensitive PTX-conjugated amphiphilic polycarbonate prodrugs were synthesized and used to encapsulate another chemotherapeutic agent for enhancing chemotherapy efficacy. By adjusting the grafted ratio of PTX, amphiphilic polycarbonate prodrugs could easily self-assemble into polymersomes or micelles to load hydrophilic DOX·HCl or hydrophobic DOX, respectively. In order to further improve the stability and drug loading efficiency of nanocarriers, the reversible CL nanoparticles were prepared. The drug release from the carriers was suppressed in normal physiological conditions, whereas markedly accelerated under 10 mM dithiothreitol (DTT) conditions. The dual drug-loaded nanoparticles exhibited significant growth-inhibition for HeLa cells and drug-resistant MCF-7/ADR cells as compared with the single drug-loaded nanoparticles, especially for drug resistant tumor cell. More importantly, DOX·HCl-loaded polymersomes of polymeric prodrug exhibited synergistic effect of cell-growth inhibition on HeLa and MCF-7/ADR cells.In chapter 5, the folate-targeting core crosslinked polymeric micelles (CCL/FA) containing multiple disulfide bonds located on the interface and core of the micelles to co-deliver doxorubicin (DOX) and P-glycoprotein (P-gp) inhibitor tariquidar (TQR) for reversing drug resistance was prepared. As expect, the hydrophobic middle block of FA-PEG-PPDD could be degraded into small fragments under reducing conditions. On the one hand, the micelles possessed good stability that led to the suppression of drug release from the CCL micelles in physiological environment. On the other hand, under reductive conditions, the CCL micelles could collapse rapidly and accelerate the drug release markedly. In vitro cytotoxicity measurements, combined with confocal laser scanning microscopy (CLSM) and flow cytometry, confirmed that the dual-drug loaded micelles exhibited obviously higher cytotoxicity to MCF-7/ADR resistant cells than free DOX·HCl, single-drug loaded CCL micelles and non-targeted CCL micelles. The results imply that co-delivering DOX and TQR by CCL/FA micelles may be promising to overcome multi-drug resistance in tumor treatments.