Manganese Modified Carbon Dots Enhance Photodynamic Therapy In The Treatment Of Oral Squamous Cell Carcinoma In Vitro And In Vivo

OBJECT: NCDs@MnO₂-PDA nanoparticles（NMP NPs）were prepared by loading amidation carbon quantum dots（NCDs）into hollow mesoporous manganese dioxide（Mn O2）and coating it with polydopamine（PDA）nanoparticles.Manganese doped carbon quantum dots（Mn-CDs）were prepared by doping manganese ions into carbon quantum dots（CDs）to simplify the preparation process.In order to obtain a photosensitizer with low toxicity and high efficacy of photodynamic therapy and improve the efficiency of photodynamic therapy in the treatment of oral squamous cell carcinoma,NMP NPs were verified to alleviate tumor hypoxic microenvironment and enhance the efficacy of photodynamic therapy and photothermal therapy by cell and animal experiments.METHODS: NMP NPs were prepared and their physical and chemical properties were detected by physical and chemical characterization methods to explore their micromorphology,surface functional groups,optical properties,photodynamic efficiency and photothermal effect;The cellular uptake ability and biological safety of NMP NPs were verified by cell uptake test and cytotoxicity test.Phototoxicity test,live/dead staining test and apoptosis test were used to study the anticancer effect of NMP NPs on oral squamous cell carcinoma;Oral squamous cell carcinoma cells（KB cells）were subcutaneously inoculated on the back of nude mice to establish a tumor model,and different drugs and laser irradiation were given to the tumor.The experiment was divided into 7 groups: Control group,injection of phosphate buffer salt solution（Phosphate buffer salt solution,PBS）and635 nm laser irradiation group,injection of PBS and 635 nm and 808 nm laser irradiation group,injection of NMP NPs only,injection of NMP NPs and 635 nm laser irradiation group,injection of NMP NPs and 808 nm laser irradiation group The anti-OSCC effect of NMP NPs in vivo was evaluated in the nm laser irradiation group,the injection group,and the 635 nm and 808 nm laser irradiation groups;Mn-CDs were prepared by a simple one-pot solvothermal method,and their physical and chemical properties were investigated by physical and chemical characterization methods;The uptake ability and biosafety of Mn-CDs cells were verified by cytotoxicity test and cell uptake test.Phototoxicity test,live/dead staining test and apoptosis test were used to explore the anticancer effect of Mn-CDs on oral squamous cell carcinoma.The in vivo biosafety of Mn-CDs was evaluated by toxicological test.RESULTS: Transmission electron microscopy,ultraviolet spectrophotometer and X-ray photoelectron spectroscopy showed that NMP NPs were successfully prepared,with an obvious bilayer structure and an average particle size of about 125 nm.The results of the detection of reactive oxygen species in and out of cells showed that the production of reactive oxygen species was significantly increased after 635 nm laser irradiation of NMP NPs in the presence of hydrogen peroxide.The results of extracellular photothermal detection showed that the NMP NPs had good photothermal efficiency and photothermal stability after being irradiated by 808 nm laser;CCK-8 results showed that NMP NPs had no obvious toxicity to normal oral tissue cells.The results of phototoxicity test,live/dead staining test and apoptosis test showed that NMP NPs showed obvious anticancer activity after 635 nm and 808 nm laser irradiation in the presence of hydrogen peroxide.The apoptosis rate of KB cells was 79.2%,which was significantly higher than that of other groups;The results of animal experiments showed that after injection of NMP NPs and dual irradiation with 635 nm and 808 nm laser,the tumor volume of nude mice was significantly reduced or even disappeared after 14 days,showing excellent anticancer activity compared with other groups;The results of transmission electron microscopy,high resolution transmission electron microscopy and atomic force microscopy showed that the prepared Mn-CDs and CDs had obvious graphene structure with uniform dispersion and uniform size,and the average particle size was 2.7 nm and 3.0 nm,respectively.Zeta potential,X-ray diffraction（X-ray diffraction,XRD）and Fourier infrared spectroscopy（Fourier transform infrared spectroscopy,FT-IR）results showed that Mn was successfully doped into CDs.Ultraviolet and visible spectrum（UV-Vis）absorption spectra and photoluminescence（PL）spectra showed that Mn-CDs had an obvious absorption peak at 610 nm and an optimal emission wavelength at 675 nm when the excitation wavelength was 610 nm.The production of reactive oxygen species（ROS）in the presence of hydrogen peroxide after 653 nm laser irradiation was significantly higher than that of Mn-CDs and CDs without hydrogen peroxide;CCK-8 test showed that Mn-CDs and CDs group did not show obvious toxicity to oral squamous cell carcinoma cells（OSCC-9）even at the concentration of 300 μ g/m L.However,in the presence of hydrogen peroxide and 635 nm laser,the killing effect on OSCC-9 cells could reach 96.42%,which was significantly higher than that of other groups.Live/dead staining assay and apoptosis assay showed the same results.The results of toxicological tests showed that there was no significant difference in H&E staining results of major organs,blood routine and biochemical analysis of rats injected with Mn-CDs and CDs compared with the control group（P >0.05）.CONCLUSION:1.This study successfully prepared a novel NMP NPs,which has good biocompatibility,physiological stability and tumor microenvironment responsiveness.It can alleviate the hypoxic microenvironment in the tumor and improve the efficiency of photodynamic therapy,and has good photothermal stability and photothermal conversion efficiency.In vivo and in vitro experiments showed that NMP NPs combined with photodynamic therapy and photothermal therapy were more effective than photothermal therapy or photodynamic therapy alone in the treatment of oral squamous cell carcinoma.2.This study successfully prepared a Mn-CDs with good water solubility and biocompatibility,which can react with abundant hydrogen peroxide in the tumor to generate oxygen,alleviate the hypoxic microenvironment in the tumor,and improve the efficacy of photodynamic therapy.Mn-cds provides a simple synthesis,low toxicity,and efficient photosensitizer for the treatment of oral squamous cell carcinoma.3.Two different forms of manganese modified carbon dots nanoparticles have achieved good effects on oral squamous cell carcinoma.Compared with NMP NPs,Mn-CDs can simplify the preparation process and treatment process,and only need a single laser irradiation to obtain ideal anticancer effect,which provides a new idea for the subsequent study of anticancer effect in solid tumors.