| Objective:Doxorubicin(DOX), with the advantages of exact curative effects and low price, was used as the first-line chemotherapeutic drug to treat many cancers for a long time. But the obvious toxicity to normal tissues and the development of DOX resistance in tumor cells limit the clinical application of DOX.In this study, DOX and resveratrol(RES) was co-encapsulated in poly(lactic-coglycolic acid)(PLGA) based nanoparticles. And polymer cholesterol-hydrazone-polyethylene glycol-anisamide(AA-PEG-hyd-CHOL) was coated on the surface of PLGA nanoparticle to get DOX and RES co-delivery PLGA nanoparticles(NPS) to prolong the half-life of DOX and RES and increase the anti-tumor activity of NPS on DOX-resistant tumor. The molecular mechanism of anti-tumor activity of NPS was deeply investigated in vitro and in vivo. Methods:1. The solvent evaporation method was used to prepare DOX/RES-loaded NPSwhich surface was coated with AA-PEG-hyd-CHOL. The DOX/RES-loaded NPSwas characterized by zeta-potential & particle size analyzer(DLS) and transmission electron microscopy(TEM).2. The DOX and RES release from DOX/RES-loaded NPS in vitro were investigated by detecting the fluorescent intensity of DOX and RES in release medium(pH5.0, pH6.5 and pH7.4) by using 970 CRT fluorescence spectrophotometer.3. The cytotoxicity of NPS, free DOX, free RES and MIX(mixture of free DOX and free RES) on MDA-MB-231/ADR cells and MCF-7/ADR cells was tested by MTT assay. The Caspase-3 level induced by NPS, DOX, RES and MIXin MDA-MB-231/ADR cells and MCF-7/ADR cells was measured by Caspase-3 Activity Assay Kit.4. Confocal laser scanning microscopy(CLSM) was used to evaluate the cellular uptake and intracellular localization of DOX and RES delivered by NPS in MDA-MB-231/ADR cells and MCF-7/ADR cells.5. The effect of NPS, DOX, RES and MIX on cell cycle in MDA-MB-231/ADR cells and MCF-7/ADR cells were tested by flow cytometry, and the cell cycle related proteinswere measured by Western blot.6. The effect of NPS, DOX, RES and MIX on drug-resistance related proteins and apoptotic related proteins in MDA-MB-231/ADR cells and MCF-7/ADR cells were measured by Western blot.7. The cytotoxicity of NPS, free DOX, free RES and MIX on A549 cells was tested by MTT assay. The effect of NPS, DOX, RES and MIX on cell cycle related proteins, drug-resistance related proteins and apoptotic related proteins in A549 cells were measured by Western blot.8. MDA-MB-231/ADR cells were grafted on nude mice. The mice were observed and weighted every day after being intravenously injected NPS, DOXand RES. The tumor volume was measured as an indicator of in vivo antitumor activity of NPS.Caliper IVIS Lumina II in-Vivo image system was used to evaluate the DOX and RES distribution delivered by NPS in tumor tissues and various organs. The fluorescence microscopywas used to observe RES and DOXdelivered by NPS in the section of tumor tissues and organs.The H&E(hematoxylin and eosin) staining sections were used to observe the morphological changes of major organs and tumor tissue. The proteins in the tumor tissues were extracted, and the level of drug-resistance related proteins and apoptotic related proteins were measured by Western blot.9. A549 cells were grafted on nude mice. The mice were observed and weighted every day after being intravenously injected NPS, DOX and RES. The tumor volume was measured.Caliper IVIS Lumina II in-Vivo image system was used to evaluate the DOX and RES distribution delivered by NPS in tumor tissues and various organs. And the fluorescence microscopy was used to observe RES and DOX delivered by NPS in the section of tumor tissues and organs. The H&E(hematoxylin and eosin) staining sections were used to morphological changes of major organs and tumor tissue. Results:1. The particle size of NPS was(204±27) nm and the zeta potential was(-15.8±0.8) mV. The drug loading of NPS was(4.6±1.4)% for DOX and(15.5±5.3)% for RES.2. The release rate of DOX and RES from the NPS was dependent on the pH of the medium. In pH5.0 medium, NPS released out 67.7% of loaded DOX and 83.4% of loaded RES in 24 h, respectively. However, NPS released out 43.7% of loaded DOX and 43.6% of loaded RES in 24 h in pH7.4 medium.3. The cytotoxicity of NPS on MDA-MB-231/ADR cells and MCF-7/ADR cells was exhibited a dose-depended manner. And the Caspase-3 levels induced by NPS were also exhibited a dose-depended manner.4. The DOX and RES delivered by NPS could accumulate in the nucleus of MDA-MB-231/ADR cells and MCF-7/ADR cells in a time dependent manner.5. NPS could arrest the cell cycle in G1 phase, subsequently induced the apoptosis of DOX-resistant tumor cell and inhibited the proliferation of the DOX-resistant tumor cells.6. NPS significantly reduced the expression of Cyclin A, Cyclin D1, CDK2 and CDK4 in MDA-MB-231/ADR cells and MCF-7/ADR cells, and down-regulated the expression of MRP-1, P-gp and BCRP. NPS also significantly inhibited the expression of NF-?B and BCL-2, and increased the expression of Bax in MDA-MB-231/ADR cells and MCF-7/ADR cells.7. The cytotoxicity of NPS on A549 cells was exhibiteddose-depended manner. The results of Western blot indicated that NPS could significantly reduce the expression of Cyclin A, Cyclin D1, CDK2 and CDK4 in A549 cells. NPS also significantly inhibited the expression of NF-?B and BCL-2, and increased the expression of Bax in A549 cells.8. NPS inhibited tumor volume in a dose-depended manner in MDA-MB-231/ADR cell tumor-bearing mice. The distribution of DOX and RES in normal tissue was reduced. NPS significantly attenuated the toxicity of DOX to normal organ.Compared with the tumor tissue from normal saline treated mice, the expression of Bax and Caspase-3 were up-regulated, but the expression of P-gp, MRP-1, BCRP, NF-?B and BCL-2 was down-regulated in the tumor tissue from NPS treated tumor-bearing mice.9. NPS inhibited the tumor volume in a dose-depended manner in A549 cell tumor-bearing mice. NPS simultaneously delivered DOX and RES to tumor tissue, and the distribution of DOX and RES in normal tissue was reduced. The H&E staining sections indicated that NPS significantly increase the anti-tumor activities, and attenuated the toxicity of DOX to normal organs. Conclusion:NPS could co-deliver DOX and RES to tumor tissue in tumor-bearing mice, and increase the accumulation of DOX and RES in tumor cells through down-regulating the expression of drug-resistancer related proteins(P-gp, MRP-1 and BCRP). Consequently, NPS could overcome the DOX-resistance and enhanced the antitumor activity by decreasing the expression of NF-?B and BCL-2 and increasing the expression of Bax and Caspase-3 in DOX-resistance breast cancer cells and lung cancer cells. In addition, NPS significantly decreased the systemic toxicity of DOX. |
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