| Multidrug resistance(MDR)is caused by drug resistance of tumor cells.MDR produces a variety of mechanisms:inhibition of drug-induced apoptosis,activation of DNA damage repair mechanisms,and increased efflux pump identification.And quickly and efficiently pump chemotherapeutic agents from cancer cells.Currently,one of the strategies to overcome MDR is to use a drug delivery system based on nanomedicine technology.Since the delivery system is nano-sized,it enters the cells mainly through endocytosis,thereby effectively bypassing the action of the drug efflux pump responsible for MDR,and at the same time,different modifications can be made on the surface to enhance the anti-tumor effect.To date,a variety of nanomaterials have been developed for therapeutic agents for reversing tumor MDR,such as micelles,liposomes,and dendrimers.In this study,the liposoluble acoustic sensitizer chlorin(Ce6)was covalently linked with the surfactant material PF127 to synthesize the polymer PF127-Ce6(PFC)with acoustic sensitivity activity,followed by the chemotherapy drug doxorubicin(DOX)was carried in the polymer core formed by PF127-Ce6 and P123,and finally the nano-mixed micelle(PFC@DOX)co-loaded with the acoustic sensitizer Ce6 and DOX was prepared.In this study,the physical properties of nano-formulations,such as morphology,stability and spectroscopy,were characterized.The prepared PFC@DOX nano-mixed micelles were combined by sono-chemotherapy to inhibit tumor tissue/cell growth and reverse tumors in vitro and in vivo.Content(1)Preparation and characterization of PFC@DOX nano preparationsIn this study,PF127 and Ce6 were covalently linked in DMSO,and the connection between PF127 and Ce6 was detected by infrared spectroscopy and nuclear magnetic resonance spectroscopy.The spectral properties of PF127-Ce6 were analyzed to confirm the synthesized sound-sensitive polymerization.The PF127-Ce6 did not change the optical properties of Ce6,and the connection ratio was 94.45%.The DOX was loaded into the polymer core by self-assembly of PF127-Ce6 and P123,and the morphology,particle potential and stability of the prepared PFC@DOX nano-mixed micelles were examined,showing the O used in this study.The drug-loaded nanocomposites with a particle size of 82.82 ± 4.62 nm and a dispersion coefficient of 0.131 ± 0.021 were successfully prepared by the single-milk method.Further,the pharmacokinetic properties and stability of the nano-mixed micelles were analyzed.The results showed that DOX was successfully packaged.Loaded in the nanocore,the drug loading is 6.57%.Under ultrasound,DOX is released from the polymer micelles.(2)Acoustic dynamic effect detection of PF127-Ce6(PFC)nano preparationsBy detecting the intracellular ROS production,the experimental results show that PFC nano-mixed micelles can produce a large amount of ROS under the action of ultrasound,suggesting that the anti-tumor effect may be related to the ROS production.Further,the membrane potential changes of mitochondria,the total amount of NADH in the respiratory chain,the oxidative phosphorylation level of cells,and the intracellular ATP content were detected.The results showed that MCF-7 cells and MCF-7/ADR cells were treated with PFC+US.After that,the membrane potential of the mitochondria changed,the content of NADH decreased,the level of oxidative phosphorylation of the cells decreased,and the intracellular ATP level decreased to 49%of the normal level.(3)PFC@DOX nano preparations for tumor killing abilityThe MTT method was used to analyze the killing effect of PFC@DOX nano-preparation on tumor cells under ultrasonication.The results showed that nano-mixed micelles can effectively kill MCF-7 and MCF-7/ADR cells through the synergistic effect of sono-chemotherapy.And as the concentration of the drug increases,the killing effect on the cells is also enhanced.For MCF-7/ADR,when PFC@DOX combined with ultrasound,the cell survival rate decreased to 30.94%,indicating that PFC@DOX+US can be combined by sono-chemotherapy to effectively reverse tumor multidrug resistance.The IC50 values of-DOX in DOX and micelles were 48.6± 0.14 mg/mL and 5.5 ± 0.14 mg/mL,respectively.The apoptosis of tumor cells was detected by flow cytometry combined with Annexin V/PI.The results showed that the cells were treated with PFC@DOX+US and compared with Control group,MCF-7/ADR(MCF-7)cells were apoptosis.The rate increased to 57.36%(87.84%).(4)Analysis of MDR mechanism by ultrasound combined with PFC@DOX nano-preparationFirst,the enrichment of DOX in tumor cells was detected by fluorescence microscopy.The experimental results confirmed that PFC@DOX+US can be enriched in both tumor cells.However,for MCF-7/ADR tumor cells,the free DOX can not effectively enter the tumor cells.When PFC@DOX+US treats the drug-resistant cells,the fluorescence intensity of DOX in the cells is stronger.By detecting the cell cycle of MCF-7/ADR cells,cells in the PFC@DOX+US group were mainly retained in the G2/M phase compared with the Control group.At the same time,the expression of P-gp and p-H2A.X proteins in drug-resistant cells was detected by Westerblot.The results showed that the ATP-dependent drug efflux pump P-gp was expressed in the PFC@DOX+US group.Compared with the Control group,the expression of P-gp in MCF-7/ADR cells was down-regulated.When the MDR tumor was co-incubated with PFC@DOX and exposed to ultrasound,the expression level of the labeled p-H2A.X protein of DN A double-strand break was increased.(5)Research and safety evaluation of PFC@DOX nano-preparation overcoming tumor multidrug resistance at body levelThe reversal effect of PFC@DOX nano-preparation on multidrug resistance of tumor under ultrasound was explored by establishing a subcutaneous xenograft model of MCF-7/ADR nude mice.The results of the study showed that SDT-mediated PFC@DOX therapy inhibited tumors by 85.49%.HE staining of tumor tissues confirmed that PFC@DOX+US can kill drug-resistant cells through sonic-chemotherapy synergy.Real-time measurement of mouse body weight and detection of various inflammatory factors in mouse serum and histomorphological observation of heart,liver,spleen and lung of each group of mice confirmed that PFC@DOX nano preparation has tumor-bearing mice.ConclusionIn summary,this study successfully prepared a new nano-formulation PFC@DOX with sonotherapy effect.The nano-preparation has the characteristics of moderate particle size,uniform structure and good stability.Under the action of ultrasound,the sound-controlled release of the drug can be achieved;both in vitro and in vivo experiments confirm that the PFC@DOX+US has a killing effect on the tumor tissue/cell.By reversing the mechanism of tumor MDR by PFC@DOX+US,the experimental results confirmed that PFC@DOX enhanced the ATP production and decreased the expression of P-gp under ultrasound,increasing the accumulation of DOX in the cells and enhancing the accumulation of DOX.The killing effect of chemotherapy drugs on tumor cells. |
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