Polylysine-derived Carbon Quantum Dots Modulate T Lymphocyte Responses For Periodontitis Treatment

Objective Polylysine Carbon Quantum Dots(PL-CQDs)derived from Polylysine(PL)were synthesized,their physicochemical properties were characterized,and the effects of different concentrations of PL-CQDs on the viability and proliferation of mouse T cells were investigated.PL-CQDs were co-cultured with mouse primary T cells and then with mouse embryonic osteoblast precursor cells(MC3T3-E1)to detect the effects on their osteogenic differentiation.The therapeutic effects of PL-CQDs were then examined in an animal model of periodontitis in rats,and PL-derived PL-CQDs were initially investigated to regulate T lymphocyte responses for periodontitis treatment.Methods PL-CQDs containing amino groups were synthesized by hydrothermal method.The particle size and morphology were examined by high-resolution transmission electron microscopy(HRTEM).The surface charge and surface functional groups were analyzed by Zeta potential and Fourier Transform Infrared Spectroscopy(FT-IR).The fluorescence properties of PL-CQDs were detected by ultraviolet and fluorescence spectrophotometer.T cells were co-cultured with PL-CQDs at different concentrations(0μg/m L,50μg/m L,200μg/m L,and 800μg/m L),and their viability was measured by CCK-8 method,and the appropriate concentration was selected for subsequent experiments.The effect of PL-CQDs on the differentiation of T cells in the spleen of mice and their spleen was detected by flow cytometry 1 day after tail vein injection of PL-CQDs.PL-CQDs were co-cultured with T cells and then co-cultured with MC3T3-E1 cells,and their effect on the osteogenic differentiation ability of MC3T3-E1 cells was assessed by alkaline phosphatase activity assay,alkaline phosphatase staining,and alizarin red staining.A rat model of periodontitis was established by ligating the maxillary second molar of rats for 14 days and feeding them with high glucose.And its in vivo therapeutic effect in rat periodontitis animals was examined by using Micro-CT,Gingival bleeding index(GBI),Masson staining,osteoclast staining,and immunohistochemical staining.Results HRTEM showed that PL-CQDs were spherical,uniformly dispersed,and had no obvious aggregation.The average particle size was 2.31±0.70 nm,and a lattice stripe with a lattice spacing of 0.21 nm was also observed.Zeta potential found that the surface charge of PL-CQDs and PL was positive and there was no statistically significant difference.FT-IR analysis found that both PL and PL-CQDs had amino functional groups,indicating that PL-CQDs inherited the properties of precursor PL.It showed blue fluorescence under ultraviolet light with a wavelength of 365 nm and transparent pale yellow under visible light.UV absorption spectra showed that PL-CQDs had two obvious absorption peaks at 216 nm and 300 nm.The photoluminescence of PL-CQDs was related to the excitation wavelength.When the excitation wavelength was 380 nm,the photoluminescence intensity of the dispersive emission of PL-CQDs was the highest at 480 nm.T cells were co-cultured with PL-CQDs at different concentrations.CCK-8 method showed that the cell viability of PL-CQDs was significantly higher than that of other groups at 200 and 800μg/m L.As the culture time increased from 1 to 5 days,all but 50μg/m L concentrations of carbon quantum dot T cells showed good proliferative viability.The effect of PL-CQDs on T cell differentiation was evaluated by flow cytometry,indicating that PL-CQDs could affect the differentiation of T cells and reduce the proportion of CD4~+T cells.The T cells+PL-CQDs group significantly affected the osteogenic differentiation ability of MC3T3-E1 cells and significantly promoted the alkaline phosphatase expression of MC3T3-E1 cells.In addition,the results of alizarin red quantification showed that the values of the T cells+PL-CQDs group were significantly higher than the other groups,indicating that T cells+PL-CQDs promoted the formation of calcium nodules.After the establishment of a rat periodontitis model,treatment was performed by injecting the clinical drug minocycline hydrochloride and PL-CQDs into the periodontal pockets.The results of GBI,Micro-CT,Masson staining,osteoclast staining,and immunohistochemical staining demonstrated that PL-CQDs could reduce the GBI index,promote alveolar bone regeneration,and collagen deposition,inhibit osteoclast activity and reduce the level of periodontal inflammation,thus producing good therapeutic effects in vivo.Conclusion Positively charged PL-CQDs with uniform particle size and good dispersion were successfully prepared by the hydrothermal method.PL-CQDs with good biocompatibility enhanced T-cell viability with increasing concentration.In addition,PL-CQDs inhibited the differentiation of T cells to CD4~+T cells in mice and promoted the expression of alkaline phosphatase and the formation of calcium nodules in MC3T3-E1 cells together with T cells.In an animal model of periodontitis,PL-CQDs were able to improve the periodontal inflammatory environment and promote alveolar bone regeneration for the treatment of periodontitis.Therefore,PL-CQDs have great potential for application in periodontitis treatment and provide new ideas for their mediated T cell-osteoblast interactions.