| ObjectiveBreast cancer is one of common cancer in women worldwide.It seriously harms the physical and mental health of women,and even risks their lifes.Comprehensive therapy of surgical resection,chemotherapy,radio therapy,endocrine therapy,and immunotherapy has been considered as an optimal strategy for breast cancer treatment in clinic.Chemotherapy plays an irreplaceable role in comprehensive treatment of breast cancer,but long-term chemotherapy may produce drug resistance,and thus causing chemotherapy failure.Sonodynamic therapy(SDT)is an emerging method for cancer treatment.SDT can use ultrasound to activate sonosensitizers in tumor tissues,trigger the generation of reactive oxygen species(ROS)and thus exert anti-tumor effects.The recent investigations have shown that SDT can reverse cancer drug resistance by down-regulating the expression of P-glycoprotein(P-gp)and activating the mitochondrial apoptosis in cancer cells.In this study,we designed a nanomicelle system containing both chemotherapeutic drug and sonosensitizer(HPDF nanomicelles),which had a simple structure,but exhibited a high reversl efficiency for breast cancer resistance.HPDF nanomocelles had a classic core-shell structure,in which anthracycline antitumor drug doxorubicin(DOX)and sonosensitizer hematoporphyrin(HP)were complexed to form hydrophobic cores,and meanwhile polymeric surfactant Pluronic F68 was coated on the surfaces to form hydrophilic shells.Here,we hope that HPDF nanomicells will combine SDT and chemotherapy to reverse drug resistance,and therefore achieving synergistic effects against dru-resistant breast cancer.ContentsThis study manily contains two sections.The first section involves the preparation,characterization,drug-carrying capability of HPDF nanomicelles.The second part involves the in vitro evaluation of drug-resistance reversal efficiency of HPDF nanomicelles with ultrasonic irradiation in drug-resistant breast cancer MCF-7/ADR cells and the preliminary discussion of their reversal effect mechanisms by combining SDT and chemotherapy.Methods1.Preparation and characterization of HPDF nanomicelles as well as drug-loading capability and drug release characteristics.HPDF nanomicelles were prepared by the thin film hydration method.The morphology,particle size and size distribution of HPDF nanomicelles were characterized by the transmission electron microscope(TEM)and the particle size & zeta potential analyzer.The loading contents and encapsulation efficiencies of DOX and HP in HPDF nanomicelles were detected using the Ultra performance liquid chromatograph(UPLC)method.2.In vitro evaluation of the reversal efficiency of HPDF nanomicelles against drug resistant breast cancer MCF-7/ADR cells.CCK-8 assay was used to evaluate the cytotoxicities of free DOX and HPDF nanomicelles with ultrasonic irradiation in drug resistant breast cancer MCF-7/ADR cells,and the reversal efficiency of HPDF nanomicelles with ultrasonic irradiation was obtained by comparing their half drug inhibitory concentration(IC50)with free DOX.Flow cytometry was applied to investigate the effects of cell apoptosis induction and cell cycle arrest of HPDF nanomicelles with ultrasound irradiation in MCF-7/ADR cells.3.The reversal mechanism of HPDF nanomicelles with ultrasonic irradiation against drug resistance.The cellular uptake and intracellular location of DOX loaded by HPDF nanomicelles were analyzed in MCF-7/ADR cells using the flow cytometry and confocal laser scanning microscopy,thus to preliminarily evaluate the inhibitory effect of HPDF nanomicelles on the drug-efflux function of P-gp.After treatment of HPDF nanomicelles and ultrasonic irradiation,the intracellular generation of ROS in MCF-7/ADR cells was monitored using the fluorescence probe method and the subcellular location of Cyt c was observed using the laser confocal microscopy,thus to evaluate the activation of mitochondrial apoptotic pathway.Results1.HPDF nanomicelles containing both HP and DOX were successfully prepared.HPDF nanomicelles had a classic “core-shell” structure and a regular spherical shape.The mean diameter of HPDF nanomicelles was 77.8±17.5 nm and showed a relatively uniform distribution.In HPDF nanomicelles,the loading contents of HP and DOX were about 12.6% and 3.0%,and correspondingly their encapsulation efficiencies were 86.7% and 69.2%,respectively.2.HPDF nanomicelles combined with ultrasonic irradiation significantly reversed drug resistance in MCF-7/ADR cells.As compared to free DOX,they exhibited a much higher cytotoxicity,stronger induction effect on the cell apoptosis and more potent efficacy on the cell cycle arrest at S-phase.3.HPDF nanomicelles notably inhibited the drug-efflux function of P-gp and efficiently improved the cellular uptake of DOX in MCF-7/ADR cells,which would be beneficial for exerting the cytotoxic effect of DOX and reversing the drug resistance of breast cancer.HPDF nanomicelles with ultrasonic irradiation significantly triggered the generation of a large amount of ROS in MCF-7/ADR cells,damaged the mitochondrial membrane,and further caused the release of Cyt c from the mitochondria to the cytoplasm,which subsequently resulting in the activation of mitochondrial apoptotic pathway.ConclusionsIn this research,we successfully prepared HPDF nanomicelles containing both HP and DOX.HPDF nanomicelles had a classic “core-shell” structure and a very small below 100 nm with a uniform distribution.HPDF nanomicelles showed very high reversal efficiency for drug resistance in MCF-7/ADR cells through promoting the cellular uptake of DOX.In MCF-7/ADR cells,HPDF nanomicelles with ultrasonic irradiation significantly inhibited the cell proliferation,induced the cell apoptosis and cell cycle arrest at S-phase,and furthermore notably activated the mitochondrial apoptotic pathway.Thus it can be seen that HPDF nanomicelles can efficiently combine SDT and chemotherapy to exert synergistic therapeutic effects against drug resistant breast cancer. |
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