Synthesis Of Minocycline Carbon Dots And Preliminary Study On Their Antibacterial And Osteogenic Effects

Background and Objective:The 21st century is a period of rapid development of nanotechnology.Nanoparticles have been widely used in many fields,and more and more nanoparticles have been invented.Carbon dots are a class of spherical nanoparticles with a particle size of 1-10 nm,generally using sp~2 or sp~3 hybrid carbon as their structural unit.Due to its good biocompatibility,excellent water solubility and stability,it has been widely used in the fields of drug delivery,bioimaging,biosensing,and free radical scavenging in recent years.Minocycline is currently approved for clinical use in tetracycline antibiotics with the strongest antibacterial effect,it was approved for clinical use in 1971,belongs to the second generation of semi-synthetic tetracycline antibiotics,with high efficiency and long-acting characteristics.Minocycline is widely used in the treatment of chronic periodontitis and dental pulp revascularization due to its good antibacterial properties,as well as anti-inflammatory,anti-apoptotic,osteogenesis-promoting,and proteolytic-inhibiting effects.However,due to the poor biocompatibility of minocycline,excessive use can inhibit tissue regeneration and new bone formation,limiting its topical application in the oral cavity.The preparation of minocycline into carbon dots and the exploration of its therapeutic effect on infected pulp and periodontal tissue can open up new methods for the application of minocycline.In this experiment,minocycline carbon dots were prepared by hydrothermal method,the physicochemical properties of minocycline carbon dots were characterized,the cytotoxicity of minocycline carbon dots was studied,and the cytotoxicity of minocycline carbon dots was further explored.Antibacterial properties and osteogenic differentiation ability,in order to provide a theoretical basis for the application of minocycline carbon dots in the oral cavity.Methods:1.Synthesis of minocycline C-dots:Minocycline C-dots were synthesized by one-step hydrothermal method using minocycline and ethylenediamine as raw materials.2.Characterization of minocycline carbon dots:The size,morphology,and dispersion of minocycline carbon dots were observed by transmission electron microscopy(TEM);the content of each element and surface chemical properties of minocycline carbon dots were obtained by X-ray photoelectron spectroscopy(XPS);fourier transform infrared spectroscopy(FT-IR)was used to study the molecular group changes of minocycline carbon dots;fluorescence spectrometer was used to study the fluorescence properties of minocycline carbon dots.3.Detection of cytotoxicity of minocycline carbon dots:MC3T3-E1 cells were co-cultured with different concentrations of carbon dots,and the effect of carbon dots on the viability of MC3T3-E1 cells was determined by the absorbance value of CCK-8method at 450 nm wavelength.4.Detection of antibacterial properties of minocycline carbon dots:Streptococcus mutans were co-cultured with carbon dots of different concentrations,and the minimum inhibitory concentration and minimum bactericidal concentration of carbon dots were determined;the morphological changes of bacteria were observed by scanning electron microscope(SEM);the ability to inhibit bacterial biofilm formation was determined by crystal violet staining;the destruction ability of mature bacterial biofilm was detected by MTT method.5.Preliminary study on the osteogenic differentiation effect of minocycline carbon dots:MC3T3-E1 cells were co-cultured with carbon dots of different concentrations,and the expression of osteogenesis-related gene Runx2,Col I and OCN were determined by real-time quantitative PCR(Real-time PCR);the effect of carbon dots on the osteogenic differentiation of cells was detected by alkaline phosphatase(ALP)staining.Results:1.Synthesis and characterization of minocycline carbon dots:TEM results showed that minocycline carbon dots had an average particle size of 3.57 nm and a lattice size of 0.22 nm;XPS and FT-IR results showed that minocycline carbon dots It is rich in carbonyl,amino,and other groups;the fluorescence spectrum shows that the excitation and emission spectra of carbon dots are wide,and the optimal emission wavelength is460 nm when the optimal excitation wavelength is 377 nm.2.Cytotoxicity of minocycline carbon dots:CCK-8 showed minocycline carbon dots have good biocompatibility with MC3T3-E1 even when the concentration of carbon dots reached 200?g/m L,and Carbon dots can promote cell proliferation in MC3T3-E1 cells at a concentration of 50?g/m L.3.Research on the antibacterial properties of minocycline carbon dots:the minimum inhibitory concentration of carbon dots against Streptococcus mutans is 1?g/m L,and the minimum bactericidal concentration against Streptococcus mutans is 4?g/m L;SEM shows that it can rupture the bacterial envelope;The crystal violet staining showed that the carbon dots had a good inhibitory effect on the formation of bacterial biofilm;MTT results showed that the carbon dots had a good destruction effect on the mature biofilm.4.Preliminary study on the effect of minocycline carbon dots on promoting osteogenic differentiation:Real-time PCR and ALP results showed that in the range of1-200?g/m L,minocycline carbon dots could promote the expression of osteogenesis-related genes in MC3T3-E1 cells and also promote the expression of alkaline phosphatase;100?g/m L minocycline carbon dots have the best pro-osteogenic effect on MC3T3-E1 cells.Conclusion:1.One-step hydrothermal method can be used to synthesize minocycline carbon dots.The synthesized minocycline carbon dots have a uniform particle size,and are spherical-like carbon nanoparticles with excitation wavelength-dependent characteristics and good dispersibility.2.Minocycline carbon dots have good biocompatibility and antibacterial ability;they can inhibit the formation of biofilms and destroy mature biofilms,and can promote the osteogenic differentiation of MC3T3-E1 cells in a concentration-dependent manner.