Preparation Of Multifunctional Reduced Graphene Oxide Nanocomposites And Its Applications In Breast Cancer Detection And PTT/PDT

Objective:To prepare a reduced graphene oxide-ferrocene?RGO-Fc?composite with low toxicity,good dispersion,high stability and high electrochemical performance and be used to construct a folate-functionalized cytosensor in the first part.To explore a cytosensor with simple operation,high electrochemical performance,rapid and accurate detection of breast cancer cell MCF-7.To prepare a multifunctional reduced graphene oxide-manganese dioxide-copper phthalocyanine?RGO-MnO₂-CuPc?composite with good dispersion,high biological safety,excellent photothermal conversion capability and high singlet oxygen production yield.It is applied to study of killing breast cancer MCF-7 cells by combination of photothermal therapy and photodynamic therapy?PTT/PDT?in the second part.Methods:In the first part,a RGO-Fc composite was firstly prepared by?-?stacking and characterized by ultraviolet-visible?UV-Vis?spectroscopy,fourier transform infrared?FT-IR?spectroscopy and transmission electron microscope?TEM?.Secondly,folate molecules were adhered to MCF-7 cells and fixed on the surface of RGO-Fc modified glassy carbon electrode by drop coating and covalent cross-linking method.Breast cancer cells?MCF-7?were used as research objects,electrochemical impedance spectroscopy and square wave voltammetry were used to study the electrochemical behavior of MCF-7 cells on modified electrodes.In the second part,a RGO-MnO₂-CuPc composite was prepared by self-assembly method and characterized by ultraviolet-visible?UV-Vis?spectroscopy,fourier transform infrared?FT-IR?spectroscopy and X-ray diffraction?XRD?.Its photothermal conversion capability was detected by photothermal conversion test.The degradation behavior of MnO₂ in different media of RGO-MnO₂-CuPc was investigated by UV-Vis spectrophotometry.Magnetic resonance imaging?MRI?technology was used to investigate T1 enhanced signal capability of RGO-MnO₂-CuPc.The cytotoxicity of RGO-MnO₂-CuPc composite was examined using normal hepatocytes?HL-7702?.Using human breast cancer cells?MCF-7?as research objects,PTT,PDT and PTT/PDT effects of RGO-MnO₂-CuPc composite were studied in vitro.The survival rate of MCF-7 cells was measured by modified MTT assay,and the killing effect of PTT/PDT and single PTT and PDT on MCF-7 cells were compared.And,the effect of ¹O₂ on PDT killing effect was studied by ¹O₂ quencher experiment in vitro.The effect of RGO-MnO₂-CuPc composite on the migration of MCF-7 cells in vitro was evaluated by cell scratch test.The apoptosis-inducing effect of PTT/PDT of RGO-MnO₂-CuPc composite on MCF-7 cells was inspected by flow cytometry and its mechanism of killing MCF-7 cells was initially investigated.Results:In the first part,the UV-Vis spectrum of RGO-Fc composite demonstrated that the absorption peaks of RGO-Fc composite at 375 nm and 498 nm were red-shifted,and the intensity of the absorption peak at 498 nm was significantly reduced,indicating that RGO and Fc successfully combined by?-?stacking.The results of FT-IR demonstrated that the C=O stretching vibration in the carboxyl functional group of the RGO-Fc composite was red-shifted from 1463 cm^-1 to 1457 cm^-1,and there were still some characteristic absorption peaks of ferrocene at 1089,1048,881 cm^-1,respectively.It indicated that RGO combined with Fc by?-?stacking,and the preparation of the RGO-Fc composite had been successful prepared.The TEM results illustrated that the Fc nanoparticles were uniformly distributed on the RGO sheet,and the composite was still in the shape of a nanosheet.The results of electrochemical impedance spectroscopy and square wave voltammetry indicated that RGO-Fc had excellent electron transport ability,and the electrochemical performance of its modified glassy carbon electrode?RGO-Fc/GCE?was significantly enhanced.After FA was assembled on the surface of RGO-Fc/GCE,the electrochemical signal of RGO-Fc/GCE changed significantly.It showed that FA had been immobilized on the surface of RGO-Fc/GCE by EDC/NHS reaction,and folate-functional electrode had been successfully prepared.The optimization of experimental conditions revealed that the incubation time of cells and antibodies had a significant effect on the sensitivity of the electrodes,and the optimal incubation time was 60 min.The results of square wave voltammetry showed that a good linear relationship between the peak current and the logarithm of MCF-7 cell concentration.The linear range was 1×10²-1×10^5.8 cells·mL^-1,and the detection limit was 53 cells·mL^-1.In the second part,the results of XRD of RGO-MnO₂ composite showed that only the characteristic diffraction peaks of?-MnO₂ appeared in the spectrum,indicating that the prepared MnO₂ crystal form was?-type.The results of UV-Vis revealed that the characteristic absorption peak of MnO₂ appeared in the range of 350-440 nm,while the characteristic absorption peak of GO disappeared in the range of 200-300 nm.It indicated that the RGO-MnO₂ composite had been successfully prepared.The results of FT-IR demonstrated that the C=O vibrational absorption peak of the RGO-MnO₂composite at 1754 cm^-1 disappeared,the other absorption peaks weakened,and the Mn-O stretching vibrational absorption peak of MnO₂ appeared at 525 cm^-1.It was further confirmed that the RGO-MnO₂ composite was successfully prepared.After CuPc was loaded on surface of the RGO-MnO₂ composite,the vibrational absorption peak of the O-H bond of RGO-MnO₂ was red-shifted by 101 cm^-1,the absorption peak was broadened,and the absorption intensity was enhanced.It indicated that RGO-MnO₂ and CuPc had been successfully compounded.The results of TEM showed that the prepared RGO-MnO₂-CuPc nanocomposite had a sheet-like structure.The results of dynamic light scattering demonstrated that the average hydrated particle size of RGO-MnO₂-CuPc composite was 215.6 nm,and it had good dispersion stability.The results of photothermal conversion experiments showed that RGO-MnO₂-CuPc composite had excellent photothermal conversion capability under 808 nm NIR irradiation,and its photothermal effect was time-dependent.The results of UV-Vis showed that MnO₂ in RGO-MnO₂-CuPc composite could react with H₂O₂ to form O₂ at pH 6.50.The results of MRI showed that the T1 weighted imaging of RGO-MnO₂-CuPc composite became brighter with the increase of concentration,and its enhancement effect was concentration dependent.It indicated that it could be used as a T1 contrast agent for magnetic resonance imaging.The results of cytotoxicity test in vitro showed that RGO-MnO₂-CuPc composite had no obvious toxicity to HL-7702 cells in the range of 0²00?g·mL^-1,and the survival rate of HL-7702 cells was not less than 75%after 48 h incubation,indicating that it had good biosafety.The survival rate of HL-7702 cells exposed to light alone was higher than 90%,indicating that light alone had little damage to HL-7702 cells.When the concentration of RGO-MnO₂-CuPc composite was between 0 and 200?g·mL^-1,its dark toxicity to MCF-7 cells was lower.The IC₅₀ value of RGO-MnO₂-CuPc composite was significantly lower than that of a single nanomaterial,which was25.96?g·mL^-1.RGO-MnO₂ composite had dual photothermal killing effect,and the killing effect of RGO-MnO₂-CuPc composite on MCF-7 cells in PTT/PDT was stronger than that of single PTT and PDT.The results of ¹O₂ quencher experiments in vitro showed that RGO-MnO₂-CuPc could produce ¹O₂ and killed MCF-7 cells during PDT.The results of flow cytometry demonstrated that the killing effect of PTT/PDT of RGO-MnO₂-CuPc composite on MCF-7 cells was mainly achieved by inducing apoptosis.The results of cell scratch experiments showed that RGO-MnO₂-CuPc composite could inhibit the migration ability of MCF-7 cells in vitro during the PTT/PDT process.Conclusion:In the first part,RGO-Fc composite with low toxicity,good dispersion,high stability and high electrochemical performance was successfully prepared,and a folate-functionalized cytosensor was successfully constructed.It could selectively detect breast cancer MCF-7 cells and had good stability.Folate-functional cytosensor had great potential in the early diagnosis of breast cancer.In the second part,RGO-MnO₂ and RGO-MnO₂-CuPc composite were successfully prepared by hydrothermal method and self-assembly method.This method was simple and low cost.The prepared RGO-MnO₂-CuPc composite had excellent photothermal conversion capability,could effectively generate ¹O₂,and could be used as a T1 contrast agent for MRI.Compared with PTT or PDT alone,the killing effect of PTT/PDT of RGO-MnO₂-CuPc composite on MCF-7 cells was more significant,and it killed MCF-7 cells mainly by inducing apoptosis.And it could inhibit the migration ability of MCF-7 cells in vitro.The RGO-MnO₂-CuPc composite showed potential application prospects in the combined treatment of breast cancer.