| Doxorubicin(DOX) is a kind of broad-spectrum antitumor drug with low price and good efficacy. However,the severe side-effect and drug resistance limited its clinical application. Therefore, synthesis of new DOX delivery system with tumor-targeting profile, reversing the drug-resistance and releasing free DOX in tumor cells would be potential. As it was reported that folate receptor(FR)was highly expressed on surface of many tumor cells,the folate modified system can actively target to tumor. When DOX was used to treat DOX-resistant tumor, DOX was quickly pumped out of the cells and could not accumulate in nucleus to take effect. It was proved that mitochondria are also a targeted region of DOX. So the mitochondrial targeting DOX delivery system might reverse the tumor resistance and enhance the antitumor effect. When high molecular weight drug delivery system was taken up by cells, it first distributed in acidic organelles like endolysosome. So the p H-sensitive carriers exhibited a control-released profile in tumor cells. Objective:In this paper, lipid cation(3-carboxypropyl) triphenylphosphonium bromide(TPP) was chosen as mitochondrial targeting moiety for its high affinity to mitochondria, and TPP-DOX was prepared.DOX and TPP-DOX were then co-encapsulated in folic acid(FA) modified p H-responsive micelles to enhance the antitumor activity of DOX, overcome the drug-resistance, and reduce the systemic toxicity of DOX. Methods:TPP-DOX was synthesized by connecting DOX and TPP. Palmitic acid(PA), poly aspartic acid(Pasp), FA and polyethylene glycol(polyethylene, glycol, PEG) were used to synthesize the p H-triggered materials(PA-hyd-PEG-FA and Pasp-hyd-PEG-FA) via hydrazone bond. The chemical structures were characterized by 1H NMR, MS and IR spectrum. Then DOX and TPP-DOX were co-encapsulated, while the DOX/TPP-DOX @PA-hyd-PEG-FA and DOX/TPP-DOX@Pasp-hyd-PEG-FA micelles were prepared by solvent evaporation and dialysis method, respectively. Then the micelles were characterized. The drug-loaded micelles were dispersed in phosphate buffer solution with different p H values to investigate the drug release profile in vitro. The antitumor activity of drug-loaded micelles in vitro was tested by MTT assay. The cell apoptosis was evaluated by kit method. The fluorescence microscopy and laser confocal scan microscopy were used to observe the uptake and subcellular distribution of DOX in tumor cells. The in vivo antitumor activity of drug-loaded micelles was investigated on tumor bearing nude mice models.Results:1. The spectroscopic results were consistent with the target products.2. The size of DOX/TPP-DOX@PA-hyd-PEG-FA was 237 nm, and the drug loading was 6.8% and 10.3% for DOX and TPP-DOX respectively. The particle size of micelles was stable for 4 days in p H 7.4 PBS. When the micelles incubated with phosphate buffer(p H=7.4, 6.5 or 5.0) for 72 h, the cumulative release percentage of DOX was 52%, 67% and 86%, indicating a p H-dependent profile. The blank micelles were nearly non-toxic. The drug-loaded micelles were taken up by tumor cells via folic acid receptor mediated route, and showed higher cytotoxicity than free DOX. DOX/TPP-DOX@PA-hyd-PEG-FA showed high toxicity on MCF-7/ADR cell. DOX/TPP-DOX@PA-hyd-PEG-FA could obviously increase the concentration of Caspase3. The drug-loaded micelles could deliver DOX to mitochondria and nucleus of tumor cell. The in vivo antitumor activity was evaluated by using MCF-7/ADR cell bearing nude mice. DOX mainly accumulated in tumor tissue when DOX/TPP-DOX@PA-hyd-PEG-FA was intravenously administered to tumor bearing nude mice. DOX/TPP-DOX@PA-hyd-PEG-FA inhibited the growth of tumor in a dose-dependent manner. Meanwhile, DOX/TPP-DOX@PA-hyd-PEG-FA could decrease the systemic toxicity of DOX.3. The size of DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10) was 193 nm, and the drug loading was 7.9% and 12.4% for DOX and TPP-DOX respectively. The size of micelles was stable for30 days in p H 7.4 PBS. The blank micelles were almost non-toxic. DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10) showed greater cytotoxicity than free DOX both on MCF-7/ADR cell and KB cells. DOX/TPP-DOX @Pasp-hyd-PEG-FA(Pasp:PEG=1:10) could deliver DOX to nucleus and mitochondria by the evidences of florescence microscope and laser confocal scan microscopy. The in vivo antitumor activity was investigated by using KB cells bearing nude mice. DOX mainly accumulated in tumor tissue when DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10) was intravenously administered to tumor bearing mice. DOX/TPP-DOX @Pasp-hyd-PEG-FA(Pasp:PEG=1:10) inhibited the growth of tumor in a dose-dependent manner. Meanwhile, DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10) could decrease the systemic toxicity of DOX. Conclusion:The drug release from DOX/TPP-DOX@PA-hyd-PEG-FA and DOX/TPP-DOX @Pasp-hyd-PEG-FA(Pasp:PEG=1:10) exhibited p H-dependent manner. DOX/TPP-DOX @PA-hyd-PEG-FA and DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10) could deliver DOX to nucleus and mitochondria. The two polymeric micelles showed good toxicity on DOX resistant tumor cells. The micelles reduced the accumulation of DOX in normal tissue, but increased the accumulation of DOX in tumor tissue. DOX/TPP-DOX @PA-hyd-PEG-FA and DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10) showed better antitumor activity than free DOX in vivo. Meanwhile, the systemic toxicity of DOX was reduced by using DOX/TPP-DOX@PA-hyd-PEG-FA and DOX/TPP-DOX@Pasp-hyd-PEG-FA(Pasp:PEG=1:10). |
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