Preparation Of Tumor Microenvironment-responsive Multifunctional Nanomaterials And Their Application In Tumor Combination Therapy

Objective:1.PtTeCu@RM nanorods with good photothermal/thermoelectric/chemodynamic properties and good biocompatibility was prepared to investigate the combined effects of photothermal therapy（PTT）/pyroelectric dynamic therapy（PEDT）/chemodynamic therapy（CDT）in killing Hep G-2 cells under the internal biochemical signals and external near-infrared light stimulation of the tumor microenvironment in vitro.2.In₂S₃-CIS/RM hollow nanotubes with good photothermal/chemodynamic properties and good biocompatibility was prepared to construct a combined photothermal/chemodynamic In₂S₃-CIS/RM nanotherapeutic system.To explore its PTT/CDT dual-mode combined killing effect on Hep G-2 cells mediated by NIR light and H₂O₂.3.Injectable multifunctional hydrogel CuIn-MOF/GOx/ALG nanocones with good chemokinetic properties was prepared for starvation therapy ST/CDT combination for local killing of Hep G-2 cells.Methods:1.First,PtTeCunanorods were prepared by solvothermal reaction,and the erythrocyte membrane was wrapped around the nanorods by ultrasonic extrusion to synthesize PtTeCu@RM nanorods,and then characterized by transmission electron microscope（TEM）analysis,X-ray diffraction（XRD）,X-ray photoelectron spectroscopy（XPS）,particle size and zeta potential.Secondly,the photothermal conversion capability of PtTeCu@RM was investigated by photothermal conversion experiments,while its thermoelectric conversion performance was inspected by measuring the electrochemical impedance,and its chemodynamic performance was examined by measuring peroxidase-like activity,detecting the production of singlet oxygen and consuming glutathione.Finally,human hepatocellular carcinoma cells（Hep G-2）were used as the research object.2’,7’-dichlorodihydrofluorescein diacetate（DCFH-DA）probe method was used to detect reactive oxygen species（ROS）in Hep G-2 cells,and Calcein-AM/PI live-dead cell staining method was used to furtherly investigate the multimodal synergistic killing effect of PtTeCu@RM on Hep G-2 cells in vitro.2.Firstly,In₂S₃-CIS hollow nanotubes were prepared by two steps of liquid-phase vulcanation and water-thermal ion exchange,and the red blood cell membrane was coated on the nanotubes by ultrasonic extrusion to synthesize In₂S₃-CIS/RM hollow nanotubes.Secondly,various characterization methods such as TEM,EDS,XRD,and XPS were used for characterization,and their photothermal conversion capacity was investigated by photothermal conversion experiments,as well as their chemodynamic performance was inspected by peroxidase-like activity assay,detection of singlet oxygen production and glutathione depletion experiments.Finally,Hep G-2 was used as the research object,and the DCFH-DA probe method was used to detect intracellular ROS in Hep G-2 cells,and Calcein-AM/PI live-dead cell staining method was used to furtherly investigate the combined PTT/CDT killing effect of In₂S₃-CIS/RM on Hep G-2 cells.3.Firstly,CuIn-MOF was synthesized using In（NO₃）₃·4H₂O and Cu（NO₃）₂·3H₂O as metal precursors,terephthalic acid as organic ligand and DMF as solvent,and GOx was coupled to CuIn-MOF,and then the synthesized CuIn-MOF/GOx was compounded with ALG to form CuIn-MOF/GOx/ALG nanocones.Secondly,it was characterized by various characterization methods such as TEM,XRD and XPS,its chemodynamic property was inspected by peroxidase-like activity assay and glutathione depletion experiments.The solution was converted into hydrogel by the complex reaction between the solution and Ca²⁺,and its gel property and chemodynamic property were examined.Finally,Hep G-2cells were used as the research object,and the combined ST/CDT local killing effect of CuIn-MOF/GOx@RM on cancer cells was furtherly investigated by using DCFH-DA probe assay to detect ROS in Hep G-2 cells and Calcein-AM/PI live-dead cell staining in vitro.Results:1.The TEM image showed that the PtTeCunanorods exhibited one-dimensional rod-like morphology with uniform dispersion and were wrapped by an erythrocyte membrane,indicating that PtTeCu@RM nanorods had been successfully prepared.The XRD map results revealed that PtTeCumatches the crystal structure of Pt Te₂in the standard crystal structure database.The XPS full spectra furtherly indicated that the PtTeCunanorods are composed of three elements,Pt,Te and Cu.The particle size,potential diagrams and FTIR spectrogram demonstrated that the erythrocyte membrane was successfully wrapped on the PtTeCunanorods.The results of photothermal conversion experiments suggested that PtTeCu@RM had good photothermal performances and stabilities under 808 nm NIR light(light intensity 1 W·cm^-2).The electrochemical impedance spectra showed that PtTeCu@RM had good charge transfer ability under 808nm NIR light irradiation and it is a better pyroelectric material.The results of the enzyme kinetic assay cleared that PtTeCu@RM nanorods had excellent peroxidase-like activity and were capable of generating reactive oxygen species such as singlet oxygen in vitro.Glutathione depletion experiments implied that PtTeCu@RM nanorods could deplete GSH to a greater extent,thereby increasing their killing cancer effect.In vitro cellular experiments showed that PtTeCu@RM nanorods have good biosafety and had a significant effect on killing Hep G-2 cells under 808 nm NIR light.2.The TEM image showed the tubular morphology of In₂S₃-CIS nanotubes with homogeneous dispersion and a red cell membrane wrapped around the outer layer,indicating the successful preparation of In₂S₃-CIS/RM hollow nanotubes.The EDS pattern confirmed that In₂S₃-CIS nanotubes were composed of three elements,In,Cuand S;The XRD pattern results indicated that In₂S₃-CIS matches the crystal structure of In₂S₃in the standard crystal structure database.The XPS full spectra furtherly stated that the In₂S₃-CIS hollow nanotubes were composed of Cu,In and S.Particle size,potential diagrams and FTIR spectrogramdemonstrated that the red blood cell membrane was successfully encapsulated on the In₂S₃-CIS hollow nanotubes.The results of photothermal conversion and stability experiments suggested that In₂S₃-CIS/RM hollow nanotubes exhibited good photothermal performances and stabilities under 808 nm NIR light(light intensity 1W·cm^-2).In vitro cellular assays implied that the In₂S₃-CIS/RM hollow nanotubes were biologically safe and exhibited significant photothermal combined chemodynamic effects on Hep G-2 cells under 808 nm NIR light.3.The TEM image showed that CuIn-MOF was cone-shaped and uniformly dispersed.The XRD results confirmed that the spectra match the diffraction peak positions of the fitted MIL-68（In）pattern,indicating that CuIn-MOF had the same crystal structure as MIL-68（In）.The XPS full spectra furtherly stated that the CuIn-MOF nanocone is composed of Cu,In and O.Zeta diagrams demonstrated that the zeta potential gradually decreased as GOx and ALG were loaded onto CuIn-MOF,indicating that CuIn-MOF/GOx/ALG was successfully synthesized.FTIR spectrograms further supported the successful synthesis of CuIn-MOF/GOx/ALG.After adding CuIn-MOF/GOx/ALG to the Ca²⁺solution,the solution was transformed into a hydrogel,indicating that CuIn-MOF/GOx/ALG was converted into a hydrogel by complexation with the Ca²⁺inter.The gluconic acid detection experiments suggested that CuIn-MOF/GOx/ALG could catalyse the oxidation of glucose to gluconic acid,thus achieving the anti-tumour effect of starvation therapy.The effects of different p H and different concentration of H₂O₂and CuIn-MOF/GOx/ALG on the chemodynamic properties of CuIn-MOF/GOx/ALG nanocones injectable multifunctional hydrogels were studied,and the results implied that the catalytic activity of peroxidases of CuIn-MOF/GOx/ALG nanocones in acid environment was good,and the peroxidase-like activity of CuIn-MOF/GOx/ALG was better with increasing concentrations of H₂O₂and CuIn-MOF/GOx/ALG.Enzyme kinetic assay and glutathione depletion assay further demonstrated that CuIn-MOF/GOx/ALG nanocones had excellent peroxidase-like activity.In vitro cellular experiments had shown that CuIn-MOF/GOx/ALG converts the injected solution into a hydrogel by complexation with Ca²⁺in tumour cells,and acts precisely and continuously at the tumour site,achieving a synergistic effect of starvation therapy and chemodynamic therapy in killing cancer cells.Conclusion:1.The PtTeCu@RM nanorods were successfully synthesized;PtTeCu@RM nanorods had good photothermal,thermoelectric and chemodynamic properties,good biological safety,and significant photothermal/thermoelectric/chemodynamic synergistic killing effect on Hep G-2 cells under 808 nm NIR light.2.The In₂S₃-CIS/RM hollow nanotubes were successfully prepared by the two-step method and ultrasonic extrusion.In₂S₃-CIS/RM hollow nanotubes had good photothermal and chemodynamic properties and good biosafety,and had a significant combined photothermal chemodynamic killing effect on hepatocellular carcinoma Hep G-2 cells under 808 nm near infrared light.3.The injectable multifunctional CuIn-MOF/GOx/ALG nanocone was successfully prepared,it acted precisely and continuously at the tumor site,exerting a good effect of starvation therapy combined with chemodynamic therapy for local killing of hepatocellular carcinoma Hep G-2 cells.