Cellular uptake, intracellular trafficking, and antitumor efficacy of doxorubicin-loaded reduction-sensitive micelles.

Abstract Reduction-sensitive micelles were prepared from monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C 16 ), an amphiphilic poly(ethylene glycol) derivative containing a disulfide bond. The micelles were then used for the intracellular delivery of the anticancer drug doxorubicin (DOX) into tumor cells, and the cellular uptake mechanisms of the micelles were determined. To serve as a control, monomethoxy-poly(ethylene glycol)-C-C-hexadecyl (mPEG-C-C-C 16 ) with an analogous structure but without a disulfide bond was also prepared. The polymer could self-assemble into micelles in an aqueous solution and be loaded with high-content DOX. In vitro release studies revealed that DOX-loaded mPEG-S-S-C 16 micelles released DOX faster than DOX-loaded mPEG-C-C-C 16 micelles in the presence of dithiothreitol (DTT), but showed similar release rates in the absence of DTT. MTT assay demonstrated significantly enhanced cytotoxicity of DOX-loaded mPEG-S-S-C 16 micelles against the human cervical cancer cells (HeLa) compared with DOX-loaded mPEG-C-C-C 16 micelles, but there was no significant difference in the cytotoxicity between the two DOX-loaded micelles against the african green monkey SV40-transformed kidney fibroblast cells (COS-7). Confocal laser scanning microscopy observation and flow cytometry analyses indicated that DOX-loaded mPEG-S-S-C 16 micelles were efficiently internalized into HeLa cells, released DOX into the cytoplasm, and entered the nuclei. By contrast, in the case of DOX-loaded mPEG-C-C-C 16 micelles, little DOX was found in the nuclei. Endocytosis inhibition results proved that both mPEG-S-S-C 16 and mPEG-C-C-C 16 micelles entered the HeLa cells mainly through the clathrin-mediated endocytosis pathway, and caveolae-mediated endocytosis was involved to a small extent. These results indicated that 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, and the potential of this reduction-sensitive polymer for the effective intracellular delivery of anticancer drugs.