Preparation Of Mitochondrial Targeting MoS₂/PDA-TPP-DOX Composite And Its Application In Cancer Photothermal/Chemotherapy

Objective:To prepare a mitochondrial targeting nanocomposite molybdenum disulfide/polydopamine-triphenylphosphine（MoS₂/PDA-TPP）with high biosafety,good biocompatibility and excellent photothermal conversion capability.Its photothermal killing effect on lung cancer A549 cells was investigated.The chemotherapeutic drug doxorubicin（DOX）was loaded to prepare molybdenum disulfide/polydopamine-triphenylphosphine-doxorubicin（MoS₂/PDA-TPP-DOX）composite,which was applied to the study of killing A549 cells by photothermal therapy combined with chemotherapy.Methods:First,a one-pot hydrothermal method was used to prepare MoS₂ nanosheet.Secondly,the dopamine layer was wrapped by self-polymerization to form MoS₂/PDA.Finally,the MoS₂/PDA-TPP composite were prepared by amidation reaction.The MoS₂/PDA-TPP composite was characterized by fourier transform infrared（FT-IR）spectroscopy,ultraviolet-visible（UV-vis）spectroscopy,hydrodynamic diameter,Zeta potential and transmission electron microscope（TEM）.And the photothermal conversion ability of the MoS₂/PDA-TPP composite was investigated through photothermal conversion test at the same time.The cytotoxicity of the MoS₂/PDA-TPP composite was examined using normal hepatocytes（L02）.The mitochondrial targeting ability was studied by detecting mitochondrial membrane potential.The tetramethylazolyl blue（MTT）method was used to determine the survival rate of human lung cancer A549 cells,and the photothermal killing effect of the MoS₂/PDA-TPP composite on A549 cells was investigated.The apoptosis-inducing effect of the MoS₂/PDA-TPP composite on A549cells was inspected by Hoechst 33342 fluorescence staining method and Annexin V-FITC/PI double staining.The content of ROS in A549 cells treated with MoS₂/PDA-TPP composite was detected by flow cytometry,and the mechanism of the MoS₂/PDA-TPP composite killing A549 cells was preliminarily explored.The chemotherapeutic drug DOX was loaded on the MoS₂/PDA-TPP composite to prepare the MoS₂/PDA-TPP-DOX composite with chemotherapy and photothermal effect.The drug release studies at different pH values were conducted in vitro.The uptake ability of lung cancer A549 cells to the MoS₂/PDA-TPP-DOX composite were investigated,and the killing effect of MoS₂/PDA-TPP-DOX composite on A549 cells by photothermal/chemotherapy in vitro were evaluated.Results:The results of FT-IR showed that the MoS₂/PDA-TPP composite presented a broadened new characteristic peak at 1231.33 cm^-1,and the C=O stretching vibration peak and C-N stretching vibration peak in the amide bond appeared at 1571.22 cm^-1 and1428.51 cm^-1,indicating that TPP and PDA were combined successfully.The results of UV-vis indicated that the MoS₂/PDA composite had a characteristic absorption peak at278.98 nm,and it disappeared after the formation of the MoS₂/PDA-TPP composite.The characterization results of hydrated particle size was that the average hydrated particle size of the MoS₂/PDA-TPP composite was 187.8 nm,which had good dispersion stability.The zeta potential result showed that MoS₂/PDA-TPP composite was negatively charged,and the potential value was-20 m V.The results of TEM demonstrated that the MoS₂/PDA-TPP composite was in the form of flakes,and the agglomeration phenomenon was improved compared with pure MoS₂.The results of photothermal conversion experiment revealed that the MoS₂/PDA-TPP composite had good photothermal performance under the irradiation of 808 nm near-infrared light irradiation(0.5 W·cm^-2).The results of cytotoxicity experiment in vitro showed that the MoS₂/PDA-TPP composite was less toxicity to normal hepatocytes L02 and had good biosafety when its concentration was between 0-100μg·m L^-1.The survival rate of A549 cells was higher than 90%,and the killing effect on A549 cells was small under simple 808 nm near-infrared light for 0 to 20 min.The results of JC-1 dye detection of mitochondrial membrane potential（MMP）showed that the MoS₂/PDA-TPP composite had good mitochondrial targeting to tumor cells.After 808 nm near-infrared light(0.5 W·cm^-2)irradiation for 10 min,the survival rate of A549 cells in the MoS₂/PDA-TPP group was57.2%,which was decreased 25.5%and 11.8%lower than those in the MoS₂ group and MoS₂/PDA group,respectively.It had a strong photothermal killing effect on A549 cells.The detection of intracellular ROS content by the dihydroethidium probe method showed that the cells treated with the MoS₂/PDA-TPP composite had higher intracellular ROS content.The result of Hoechst 33342 fluorescence staining method showed that MoS₂/PDA-TPP composite had a strong apoptosis effect on A549 cells.The result of Annexin V-FITC/PI double staining flow cytometry further showed that the killing effect of the MoS₂/PDA-TPP composite on A549 cells was mainly achieved by inducing early cell apoptosis.The results of drug release study in vitro of MoS₂/PDA-TPP-DOX composite at different pH values indicated that DOX was more likely to be released from the MoS₂/PDA-TPP-DOX composite at pH 5.50.The uptake ability of lung cancer A549cells to the MoS₂/PDA-TPP-DOX composite results revealed that the uptake of MoS₂/PDA-TPP-DOX composite by A549 cells increased under 808 nm near-infrared light(0.5 W·cm^-2)irradiation.The results of photothermal/chemotherapy combined killing effect of MoS₂/PDA-TPP-DOX composite on A549 cells in vitro demonstrated that the killing effect of MoS₂/PDA-TPP-DOX composite on A549 cells was stronger than the photothermal killing effect of the MoS₂/PDA-TPP composite on A549 cells,and it is also stronger than the killing effect of the chemotherapy drug DOX on A549 cells.Combination therapy was beneficial to improve the effect of cancer efficacy.Conclusion:The MoS₂/PDA-TPP composite and MoS₂/PDA-TPP-DOX composite were successfully prepared.Their preparation method was simple and easy to operate.The dispersion of MoS₂/PDA-TPP composite was better than MoS₂,and it had excellent mitochondrial targeting to tumor cells and good photothermal conversion ability.It had a strong killing effect on A549,and killed A549 cells by inducing early apoptosis,leading to an increase of ROS content in A549 cells.The MoS₂/PDA-TPP-DOX composite had both chemotherapy and photothermal effects,and its killing effect on A549 cells was stronger than that of chemotherapy or photothermal alone.The research results provided a new idea for cancer treatment and theoretical basis and guidance for its clinical application.