Preparation Of FA/rGO/MSN-IR820 Nanocomposite And Its Application In Cancer Detection And PTT/PDT

Objective:To prepare a folic acid-targeted nanocomposite folic acid/reduced graphene oxide/mesoporous silica-new indocyanine green（FA/rGO/MSN-IR820）with high biosafety,excellent electrochemical performance and good photothermal conversion ability and to explore the construction of an electrochemical cell sensor with simple operation and rapid detection of liver cancer cells HepG2.The photothermal therapy combined photodynamic therapy killing effect of FA/rGO/MSN-IR820 nanocomposite on HepG2 cells was also investigated.Methods:First,mesoporous silica nanoparticles were prepared by phase transition method and encapsulated with new indocyanine green;secondly,rGO was loaded by self-assembly method;finally,FA/rGO/MSN-IR820 composite was prepared by amidation reaction and characterized by Fourier transform infrared（FTIR）spectroscopy,ultraviolet visible（UV-Vis）spectroscopy,hydrated particle size,zeta potential,thermogravimetric analysis,nitrogen adsorption-desorption（BET）measurement and transmission electron microscope（TEM）.Meanwhile,the photothermal conversion ability of FA/rGO/MSN-IR820 composite was investigated by photothermal conversion test.The synthesized nanocomposites were modified on the surface of glassy carbon electrode by drop coating method,and electrochemical characterization of different modified electrodes was carried out.HepG2 cells were immobilized on the surface of the modified glassy carbon electrode,and the experimental conditions were optimized to obtain the best incubation time.Under the optimal incubation time,the current responses of HepG2 cells with different concentrations were measured by differential pulse voltammetry,and their selectivity and stability were investigated.The drug release studies of FA/rGO/MSN-IR820 composite at different pH values were conducted in vitro.The toxicity of FA/rGO/MSN-IR820 composite to human normal hepatocyte L02 cells was investigated.The survival rate of human hepatoma HepG2 cells was detected by the modified methyl thiazol tetrazolium method,and the combined photothermal/photodynamic killing effect of FA/rGO/MSN-IR820composite on HepG2 cells was investigated in vitro.The apoptosis-inducing effect of FA/rGO/MSN-IR820 composite on HepG2 cells was investigated by Hoechst 33342fluorescence staining and Annexin V-FITC/PI double staining and the mechanism of FA/rGO/MSN-IR820 composite killing HepG2 cells was preliminarily explored.Results:FT-IR results showed that the stretching vibration peaks of Si-O-Si appeared at1087 cm^-1 and 800 cm^-1 in MSN,respectively,and the characteristic absorption peaks of carbonyl group and amide group appeared at 1737 cm^-1 and 1556 cm^-1,indicating that FA/rGO/MSN-IR820 was successfully synthesized.UV-vis results indicated that MSN had no obvious absorption peak.After being loaded with IR820,strong absorption peaks appeared at 405 nm,750 nm and 845 nm,respectively.rGO had a characteristic absorption peak at 251 nm,which is red-shifted to 261 nm after complexing with MSN-IR820.The new peak at 285 nm of the FA/rGO/MSN-IR820 composite was attributed to the characteristic peak of grafted folic acid FA.The characterization result of hydrated particle size showed that the particle size gradually increased after each step of loading the material.The zeta potential results revealed that the FA/rGO/MSN-IR820composite was negatively charged with a potential value of-21.5 mV.TEM results showed that the rGO/MSN-IR820 composite had a wrinkled sheet structure with spherical MSN distributed.The thermogravimetric results indicated that the weight loss rate of FA/rGO/MSN-IR820 was 80.79%.The nitrogen adsorption-desorption results demonstrated that the specific surface area and pore size of MSN-IR820 nanoparticles decreased significantly after MSN was loaded with IR820.The results of photothermal conversion experiments revealed that the FA/rGO/MSN-IR820 composite had fine photothermal functions under the irradiation of 808 nm near-infrared light（0.5 W·cm^-2）.Different modified electrodes were detected by electrochemical impedance spectroscopy and differential pulse voltammetry.The results showed that compared with MSN,the current of MSN-IR820 decreased and the resistance increased.After rGO was loaded,the current increased and the resistance decreased.After the modification of FA,the current decreased and the resistance increased.After further capturing HepG2 cells,the current peak continued to decrease and the resistance value further increased,indicating that the electrochemical cell sensor was successfully prepared.The optimization results of the experimental conditions demonstrated that the incubation time of cells with materials had a significant impact on the current signal,and 20 min was the optimal incubation time.The peak current measured by differential pulse voltammetry showed a good linear relationship with the logarithm of HepG2 cell concentration,its linear range was 50-5×10⁵ cells·mL^-1,the correlation coefficient R²was 0.9981,and the cell sensor had good selectivity and stability.The results of drug release study in vitro of FA/rGO/MSN-IR820 composite at different pH values indicated that IR820 was more easily released from FA/rGO/MSN-IR820 composite at weakly acidic pH 5.50.The cytotoxicity experiments in vitro showed that when the concentration of FA/rGO/MSN-IR820 nanocomposite was between 0-20μg·mL^-1,it was less toxic to normal hepatocyte L02 cells,and it had good biological safety.The survival rate of HepG2 cells was still higher than 90%after only808 nm near-infrared irradiation for 20 min,and the damage to HepG2 cells was weak.In vitro,the combined photothermal/photodynamic killing effect of FA/rGO/MSN-IR820 composite on HepG2 cells showed that the killing effect of FA/rGO/MSN-IR820composite on HepG2 cells was stronger than that of photothermal agent rGO and also stronger than that of photosensitizer IR820.Hoechst 33342 fluorescence staining showed that the FA/rGO/MSN-IR820 composite had a strong apoptosis-inducing effect on HepG2 cells.Annexin V-FITC/PI double staining flow cytometry results further confirmed that the killing effect of FA/rGO/MSN-IR820 composite on HepG2 cells was mainly achieved by inducing cell apoptosis.Conclusion:The FA/rGO/MSN-IR820 nanocomposite was successfully prepared,and its preparation method was simple,low cost and easy to operate.The electrochemical cell sensor was successfully constructed.The FA/rGO/MSN-IR820 composite had good tumor cell targeting and stability,and could selectively detect liver cancer cells.Moreover,the FA/rGO/MSN-IR820 nanocomposite had good photothermal conversion ability,and had both photothermal and photodynamic killing effects.The killing effect of FA/rGO/MSN-IR820 nanocomposite on HepG2 cells was stronger than that of photothermal or photodynamic killing alone.It killed HepG2 cells by inducing early and late apoptosis at the same time.The results of the study provide new ideas for the diagnosis and treatment of cancer.