Preparation Of Graphitic-phase Nitride Carbon Dots And Its Application In Biological Imaging And Drug Delivery

Graphitic-phase nitride carbon dots?g-CNQDs?as a kind of new non-metallic nanomaterial have the advantages of strong fluorescence,good stability,good water solubility,good biocompatibility and low toxicity,which can be used in biomedical field instead of traditional quantum dots.However,the main bottleneck of the development of g-CNQDs is the lack of effective synthesis methods and low quantum yield.Therefore,it is very important to develop a new method for the preparation of g-CNQDs.The preparation and fluorescence properties of g-CNQDs and g-CNQDs-PEG passivated by polyethylene glycol?PEG?were studied systematically in this paper.The applications of g-CNQDs and g-CNQDs-PEG in biological imaging and drug delivery were discussed.The main content of this paper is divided into the following four parts:1.Graphitic carbon nitride?g-C₃N₄?was prepared by calcining urea at high temperature.g-CNQDs with blue fluorescence were obtained by refluxing bulk g-C₃N₄ in HNO₃ followed by a direct hydrothermal treatment.The structure,morphology and optical properties of the g-CNQDs were characterized systematically.The results show that the g-CNQDs prepared by this method are monodispersed with uniform size and their average diameter is 4.1 nm.g-CNQDs exhibit good fluorescence stability at different pH values from 3 to 11,and under the optimal excitation of 300 nm,the fluorescence quantum yield is 7.5%with quinine sulfate as reference.2.The drug delivery system?g-CNQDs-DOX?was constructed for the first time using g-CNQDs as a drug carrier,using hydrogen bonding,?-?conjugation and electrostatic interaction to load anti-cancer drug doxorubicin?DOX?.The feasibility of g-CNQDs as drug carrier was studied by cytotoxicity test,drug release test in vitro and cell imaging test.The results show that the drug loading of g-CNQDs-DOX is 69.6%.The cytotoxicity of g-CNQDs-DOX is significantly higher than that of free DOX.The release rate of DOX in acidic environment is significantly higher than that in pH=7.4.This property of the drug delivery system allows it to improve therapeutic efficiency without increasing the dose of DOX while reducing adverse reactions of anticancer drugs to normal tissues.The inherent fluorescence of g-CNQDs and DOX provide g-CNQDs-DOX with dual-color imaging for revealing the intracellular localization of g-CNQDs and DOX release.After 16 h incubation of U251 human glioma cells with g-CNQDs-DOX,the released DOX and partially g-CNQDs entered the nucleus,which may be the reason why g-CNQDs-DOX is more toxic than free DOX.The results indicated that g-CNQDs,as a traceable and pH-responsive drug carrier,have a promising application prospect in the field of drug delivery.3.Fluorescent PEGylated g-CNQDs?g-CNQDs-PEG?by refluxing g-C₃N₄ in HNO₃followed by a direct hydrothermal treatment using diamine-terminated oligomeric poly?ethylene glycol??PEG?as a surface passivation agent.The structure,morphology and optical properties of tg-CNQDs-PEG were characterized systematically.The results show that g-CNQDs-PEG is composed of regular nanospheres with an average particle size of57 nm.These nanospheres consist of smaller-sized individual nanoparticles?⁶ nm?embedded in the matrix.g-CNQDs-PEG also have stable fluorescence in the pH range of3-11.The quantum yield of g-CNQDs-PEG is 9.3%,which is significantly higher than that of g-CNQDs.The stability of g-CNQDs-PEG in physiological environment is obviously better than that of g-CNQDs,and it is more suitable for biomedical field.4.The drug delivery system?g-CNQDs-PEG-DOX?was constructed by loading DOX on g-CNQDs-PEG.The results show that the toxicity of g-CNQDs-PEG is signif-icantly lower than that of g-CNQDs.When the concentration of g-CNQDs-PEG is as high as 1.5mg/ml,the survival rate of U251 human glioma cells is still above 85%.The drug loading of g-CNQDs-PEG-DOX is 56.6%.The cytotoxicity of g-CNQDs-PEG-DOX is lower than that of free DOX,and it shows obvious pH-dependent drug release behavior.The total amounts of DOX released after 72 h are 83.1%,73.2%,48.0%,and 19.5%at pH values of 4.0,5.0,6.0,and 7.4,respectively,which was significantly higher than that of g-CNQDs-DOX at the same pH.Based on the intrinsic fluorescence of g-CNQDs-PEG and DOX,g-CNQDs-PEG-DOX also can dual-color imaging for revealing the intracellular localization of g-CNQDs-PEG and DOX release.After 16 h of incubation of U251 human glioma cells with g-CNQDs-PEG-DOX,most DOX is distributed in the cell nuclei,while g-CNQDs-PEG remains in the cytoplasm.The results indicate that g-CNQDs-PEG is expected to be used as a pH responsive fluorescent drug carrier,which have better biocompatibility and physiological stability than g-CNQDs,in the field of cell imaging and drug delivery.