Reduction-Sensitive Nanoparticles As Anti-Cancer Drug Carriers

Nanocarriers for drug delivery possess several advantages, such as largely enhanced drug solubility in water, delivering multiple drugs at the same time, elongated blood circulation time, avoided degradation of the drugs, improved drug bioavailability, and reduced side effects. However, the concentration of anticancer drugs in the intracellular compartments of cancer cells is often insufficient for killing cancer cells because of the low dose of anticancer drug released or the slow release of drug from the conventional drug carriers. Therefore, stimuli-triggered drug carriers that respond to minute environmental changes with massive changes in their physicochemical properties have been explored for drug encapsulation to protect the drug during circulation in vivo and enhance drug effects via specifical and rapid release of drug into the cellular compartments. Among them, reduction-sensitive drug carriers containing disulfide bonds have been extensively studied due to the existence of a high difference in the redox potential between the mildly oxidizing extracellular milieu and the reducing intracellular fluids. Upon reaching the target cells, reduction-sensitive drug carriers containing disulfide bonds tend to burst release encapsulated drugs because of the rapid degradation of the carriers caused by the cleavage of disulfide bonds under the reductive environment present in intracellular fluids of cells. This intracellular fast release of drugs enhances therapeutic efficacy. We have designed and prepared a series of reduction-sensitive drug carriers for delivering anticancer drugs into tumor cells in this dissertation, which can be further categorized into six parts as described below:1. Reduction-sensitive amphiphilic graft polymer PHEA-S-S-C16with PHEA as a backbone and a disulfide-containing alkyl as a side chain was synthesized and evaluated as an anticancer drug carrier in this work. Reduction-insensitive PHEA-C-C-C16with an analogous structure but without disulfide bonds was also prepared as a control. PHEA-S-S-C16could self-assemble into micelles in aqueous media and load DOX with high content. Studies on the in vitro release revealed that the presence of DTT accelerated the release of DOX from DOX-loaded PHEA-S-S-C16micelles. Cell experiments demonstrated that DOX-loaded PHEA-S-S-C16micelles entered cells via caveolae-and clathrin-mediated endocytosis, however, the most prominent pathway is clathrin-mediated endocytosis. Once internalized in the cells, DOX-loaded PHEA-S-S-C16micelles could accomplish reduction-sensitive release and show enhanced inhibition of HeLa cell proliferation.2. Reduction-sensitive micelles were prepared from mPEG-S-S-C16, a simple amphiphilic polymer containing a disulfide bond. The micelles were then used for the intracellular delivery of the anticancer drug DOX into tumor cells, and the cellular uptake mechanisms of the micelles were determined. To serve as a control, mPEG-C-C-C16with an analogous structure but without a disulfide bond was also prepared. The results of MTT assay, CLSM observation and FCM analyses indicated that reduction-sensitive micelles mPEG-S-S-C16could achieve rapid drug release in HeLa cells. Endocytosis inhibition results indicated that both micelles entered cells mainly through the clathrin-mediated endocytosis pathway, and the different behaviors of cell uptake between reduction-sensitive and insensitive micelles may occur after the micelles were internalized into the cells, but not during endocytosis.3. A novel "Y" shaped amphiphilic polymer (mPEG-S-S-(PCL)2) with disulfide linkages between the hydrophobic polyester and hydrophilic PEG was synthesized for the intracellular delivery of the anticancer drug DOX. The polymer self-assembled into micellar aggregates in aqueous solution, and loaded DOX with high DLC. The PEG shell of the micelles could be shed in the presence of reducing agent DTT, which resulted in size change of the micelles and the rapid release of DOX. Cell experiments showed that DOX-loaded mPEG-S-S-(PCL)2micelles efficiently delivered DOX to the cells, displaying higher cytotoxicity against GSH-OEt pretreated HeLa cells compared with nonpretreated cells. Endocytosis inhibition results indicated that DOX-loaded mPEG-S-S-(PCL)2micelles entered cells mainly through the clathrin-mediated endocytosis pathway.4. On the basis of our previous work, we prepared the biotin targeted micelles, which could recognize the receptor on MCF-7cells and achieve active targeting. We prepared Biotin-PHEA-S-S-C16by connecting of biotin with PHEA-S-S-C16, and we prepared PHEA-S-S-C16as a control. In vitro release of DOX revealed that DOX-loaded Biotin-PHEA-S-S-C16micelles released DOX faster in the presence of DTT. The results of MTT assay, CLSM observation and FCM analyses implied that Biotin-PHEA-S-S-C16micelles could achieve active targeting and reduction sensitivity at the same time. Endocytosis inhibition results indicated that both micelles entered cells mainly through the caveolin-mediated endocytosis pathway, and the different behaviors of cell uptake between the two micelles may be due to the active targeting of Biotin-PHEA-S-S-C16micelles, but not the endocytosis. 5. Reduction-sensitive polymer mPEG-S-S-C16, prepared in part two, was used for encapsulation of SPION to form SPION-mPEG-S-S-C16micelles with SPION in their hydrophobic core during self-assembly process. We prepared reduction-insensitive SPION-mPEG-C-C-C16micelles as a control. Further evidences from in vitro drug release, MTT assay, Prussian blue staining, TEM, CLSM observation and FCM analyses demonstrated that more SPION-mPEG-S-S-C16micelles could be taken up by the cells when a magnet field was applied. Once internalized in the cells, the micelles were damaged by the higher concentration of GSH, accelerating the release of DOX. Reduction-sensitive SPION-mPEG-S-S-C16micelles could achieve magnetic targeting and reduction sensitivity at the same time.6. The redox-sensitive LPNPs were prepared by self-assembly through a modified nanoprecipitation method in this study. LPNPs were composed of (1) a PLGA hydrophobic core,(2) a soybean lecithin monolayer, and (3) an amphiphilic redox-sensitive polymer mPEG-S-S-C16. The LPNPs were prepared and evaluated for anticancer drug delivery. Redox-insensitive LPNPs with analogous structures but without the disulfide bond were prepared as controls. The PEG shell of redox-sensitive LPNPs could be shed in the presence of reducing agent DTT, which resulted in aggregation of the nanoparticles and the rapid release of DOX. Cell experiments showed that DOX-loaded redox-sensitive LPNPs entered cells mainly through the clathrin-mediated endocytosis pathway. Once internalized in the cells, redox-sensitive LPNPs efficiently delivered DOX to the cell nuclei, displaying higher cytotoxicity. LPNPs combine the merits of polymeric micelles and liposomes, demonstrating the potential of redox-sensitive PLNPs for the effective intracellular delivery of anticancer drugs.