The Construction Of Tumor Cell Mitochondrial Targeting Drug Delivery Systems And Its Anti-tumor Activity

Doxorubicin(DOX) is one of most commomly used broad-spectrum anti-tumor drugs because of its exact curative effect and the low price. However, the cardiac toxicity and high resistance rate severely limit its clinic use. Studies have found that DOX can interact with DNA in nucleus, and mitochondrion is also its important targeting site. If DOX is specifically delivered to nucleus and mitochondria in the tumor cells, its efficacy can be greatly improved, and its systemic toxicity can be significantly reduced. A large numbers of human cancers such as melanoma, non-small cell lung cancer, breast cancer and prostate cancer overexpress sigma receptor. Fennel amide(AA) is a selective receptor ligand of sigma receptor. AA modified drug delivery system shows tumor active targeting characteristics.Objective:In order to overcome the drug-resistance, enhance the anti-tumor activity, and reduce the systemic toxicity of DOX, a p H-sensitive micelle based on DSPE, and a modified p H-sensitive nanoparticle based on PLGA drug delivery system were set up to specifically deliver DOX to mitochondria of the tumor cell by using AA as the tumor cell specific targeting ligand. Methods:Mitochondrial targeting conjugate DQA-DOX, fennel amide-polyethylene glycol-DSPE(AA-PEG-hyd-DSPE), fennel amide-polyethylene glycol-cholesterol(AA-PEG-hyd-CHOL) and triphenylphosphonium-cholesterol(TPP-CHOL) were synthesized. The structure of conjugates was identified by MS, IR and 1H NMR. The DOX and DQA-DOX loaded micelle(DOX/DQA-DOX@DSPE-hyd-PEG-AA), DOX loaded micelle(DOX@DSPE-hyd-PEG-AA) and the DOX loaded PLGA nanoparticle modificated with AA-PEG-hyd-CHOL and TPP-CHOL(LNPs) were prepared by emulsion-solvent evaporation method. The delivery systems were characterized. The anti-tumor activity of DOX/DQA-DOX@DSPE-hyd-PEG-AA, DOX@DSPE-hyd-PEGAA and LNPs was tested in vitro by the MTT method. The apoptosis induced by DOX/DQA-DOX@DSPE-hyd-PEG-AA, DOX@DSPE- hyd-PEG-AA and LNPs in MDA-MB-231/ADR cells and A549 cells were tested by kit method. Laser confocal scan microscopy was used to observe the cellular uptake of DOX/DQA-DOX@DSPE-hydPEG-AA and LNPs and the sub-cellullar distribution of DOX in tumor cell. The anti-tumor activity of DOX/DQA-DOX@ DSPE-hyd-PEG-AA and LNPs in nude mice was observed on subcutaneous transplantation tumor(MDA-MB-231/ADR cells and A549 cells) model in nude mice. The distribution of DOX in tumor tissue and in normal organs was detected by the Caliper IVIS Lumina Ⅱ in vivo image after DOX/DQA-DOX@DSPE-hyd-PEG-AA and LNPs were injected via the tail vein to the tumor-bearing nude mice. Results:1. The results of spectroscopy identification showed that the synthesized materials are the target products.2. The particle size of DOX/DQA-DOX@DSPE-hyd-PEG-AA were 224 nm, and the drug loading of DOX and DQA-DOX were 6.2% and 6.5%, respectively. Drug release of DOX/DQA-DOX@DSPE-hyd-PEG-AA showed p H-dependent manner. In p H 5.0, the cumulative release percentage of DOX and DQA-DOX from DOX/DQA-DOX@DSPE- hyd-PEG-AA was 90% and 80% in 96 h. The results of anti-tumor activity in vitro showed that DOX/DQA-DOX@DSPE-hyd-PEG-AA could inhibit the tumor cell growth in concentration-dependent manner. When DOX concentration was 50 μmol/L, the cell survival rate of DOX/DQA-DOX@DSPE-hyd-PEG-AA treated A549 cell and MDA-MB-231/ADR cell was 24% and 34% respectively. The results of laser confocal scan microscopy indicated that the cellular uptake of DOX/DQA-DOX@DSPEhyd-PEG-AA was mediated by AA receptor. When tumor cell was cultured with DOX/DQA-DOX@DSPE-hyd-PEG-AA, DOX firstly localized in lysosomes, then distributed in mitochondria. Under the same concentration of DOX, DOX/DQA-DOX @DSPE-hyd-PEG-AA could deliver 80% of DOX to mitochondria of A549 cell and 75% of DOX to mitochondria of MDA-MB-231/ADR cell. When the dose of DOX was 10 μmol/kg, the tumor inhibition rate of free DOX and DOX/DQA-DOX@DSPEhyd-PEG-AA were respectively 22% and 77% in MDA-MB-231/ADR cell planted model. Compared with free DOX treated mice, after DOX/DQA-DOX@DSPE-hyd-PEG-AA was injected via the tail vein to the tumor-bearing nude mice, most of the DOX was specifically accumulated in tumor tissues, and little amount of DOX was accumulated in liver and heart. HE staining results showed that morphology of cardiac tissue in free DOX treated mice was changed significantly. However, morphology of cardiac tissue in DOX/DQA-DOX@DSPE-hyd-PEG-AA treated mice did not change.3. The particle size and drug loading of LNPs were 5.4% and 188 nm, respectively. The in vitro drug release of LNPs had the same p H-dependent manner. The results of anti-tumor activity in vitro showed that LNPs could inhibit the tumor cell growth in concentration-dependent manner. When DOX concentration was 50 μmol/L, the cell survival rate of LNPs treated A549 cell and MDA-MB-231/ADR cell was 19% and 29% respectively. The results of laser confocal scan microscopy indicated that the cellular uptake of LNPs was mediated by AA receptor. When tumor cell was cultured with LNPs, DOX firstly localized in lysosomes, then distributed in mitochondria. Under the same concentration of DOX, LNPs could deliver 81% of DOX to mitochondria of A549 cell and 87% of DOX to mitochondria of MDA-MB-231/ADR cell. When the dose of DOX was 10 μmol/kg, the tumor inhibition rate of free DOX and LNPs were respectively 53% and 80% in A549 cell planted model. Compared with free DOX treated mice, after LNPs were injected via the tail vein to the tumor-bearing nude mice, most of the DOX was specifically accumulated in tumor tissues, and little amount of DOX was accumulated in kidney. HE staining results showed that morphology of cardiac tissue in free DOX treated mice was changed significantly. However, morphology of cardiac tissue in LNPs treated mice did not change. Conclusion:Drug release from DOX/DQA-DOX@DSPE-hyd-PEG-AA and LNPs showed p H-dependent manner. Both of LNPs and DOX/DQA-DOX@DSPE-hyd-PEG-AA could deliver DOX to mitochondria; DOX/DQA- DOX@DSPE-hyd-PEG-AA and LNPs could increase the accumulation of DOX in tumor tissues, enhance the anti-tumor activity, overcome the drug-resistance, and reduce the systemic toxicity of DOX.