The Development Of Functional Carbon Dots In Antiviral And Adjuvant Agents

Infectious diseases caused by viruses account for more than three-quarters of global infectious diseases,which threaten the health of human beings and animals.Therefore,antiviral drugs are very important in the treatment of viral infectious diseases.However,there are many types of viruses,and antiviral drugs currently available for clinical application are only effective against nine viruses(HIV,HCV,HBV,HCMV,HSV,HPV,IFV,RSV and VZV).Despite large number of antiviral agents were researched,there are some deficiencies in drug toxicity,drug resistance and low pharmacodynamic effect.Thereby,it is urgent to find novel antiviral materials to deal with viral infections.Vaccine is the most effective tool in the prevention and control of viral infectious.Due to the long period and biosafety risks for the development of attenuated vaccines,inactivated and subunit vaccines are hotspots researches.However,specific adjuvants need to be added to enhance the immune effect of these vaccines because of their poor immunogenicity.Nevertheless,available adjuvants in the market are limited and they are not suitable for all vaccines.Thus,it is necessary to develop new adjuvants.At present,nanotechnology has shown notable advantages in biomedicine.Novel antiviral drugs and adjuvants can be achieved by nanotechnology.However,finding nanomaterials that are economical,effective and suitable for large-scale production is a major challenge in clinical application.Carbon dots are new kinds of carbon nanomaterials with sizes below 10 nm.This materials have advantages of abundant raw materials,simple operation,low toxicity,environmental friendliness,various functions and a wide range of biological application potential.Moreover,some carbon dots recently have exhibited antiviral and adjuvant abilities in vitro.Therefore,based on the biological application of carbon dots,our study aim to explore the broad-spectrum antiviral effect of novel carbon dots,and develop carbon dots with good immunoadjuvant activity for the prevention and treatment of viral infectious.The detail researches and results are shown as follows:1.Development of carbon dots derived from benzoxazine with broad-spectrum antiviral capability1.1 Synthesis,characterization and cytotoxicity analysis of carbon dotsIn this study,benzoxazine monomer-derived carbon dots(BZM-CDs)were prepared by hydrothermal synthesis.Carbon dots were observed to be a dispersed spherical shape with 4.4±0.6 nm under transmission electron microscopy(TEM).The zeta potential of BZM-CDs was 0.124±0.026 eV in pH 7.4 aqueous solution.Amino and carboxyl groups are rich in the surface of BZM-CDs by XPS and FT-IR analysis,and green fluorescence was emitted under excitation of 365 nm ultraviolet light for BZM-CDs,which quantum yield was 13.1%.When BHK-21 and Vero cells were incubated with BZM-CDs for 72 h at a concentration of 75μg/mL,all the cell viability were more than 90%.1.2 BZM-CDs inhibited the infection of enveloped and non-enveloped viruses in vitroEnveloped viruses(JEV,ZIKV,DENV2)and non-enveloped viruses(AAV and PPV)were used as viral models to evaluate the antiviral function of BZM-CDs.In vitro,the infection of enveloped viruses(JEV,ZIKV and DENV2)were inhibited by BZM-CDs through plaque forming assay,immunofluorescence assay and western blot.Antiviral ability of BZM-CDs was in a dose-and time-dependence manner with EC50 of 18.63μg/mL(JEV),3.715μg/mL(ZIKV),37.49μg/mL(DENV2).In addition,non-enveloped viruses(AAV,PPV)were also blocked by BZM-CDs,which EC50 were 40.25μg/mL(AAV)and 45.51μg/mL(PPV),respectively.1.3 Entry period was blocked by BZM-CDs through adsorbing on the viral surfaceBy comparing the inhibitory effects of BZM-CDs on the treatment of cells or viruses,it was found that the antiviral function of BZM-CDs mainly acted on the virus.Furthermore,TEM images were displayed that viral surface was adsorbed by BZM-CDs.The results of cell binding assay were shown that BZM-CDs inhibited the binding of virus to host cells.Therefore,it is suggested that the antiviral effect of BZM-CDs was through blocking viral entry process.1.4 Distribution of surface functional groups of BZM-CDs affects its antiviral activityAmmonium hydroxide and hydrochloric acid were respectively used to obtain two kinds of carbon dots(Ami-CDs and Car-CDs)by hydrothermal synthesis.Compared with BZM-CDs,amino and carboxyl groups were more in Ami-CDs and Car-CDs,respectively.At the same concentration,antiviral ability of Ami-CDs was stronger than BZM-CDs,while the antiviral function of Car-CDs was weaker.Therefore,amino groups may enhance the antiviral effect of carbon dots,while carboxyl groups may weaken them.In summary,BZM-CDs can be used as a new candidate in the research of antiviral drugs in the future.2.Research of cation carbon dots derived from biquaternary ammonium salts in adjuvant activity and its mechanism2.1 Synthesis and characterization of cationic carbon dots derived from biquaternary ammonium saltsIn this study,a new kind of cationic carbon dots(BQAS-CDs)was derived from biquaternary ammonium salts.The carbon dots are well-dispersed spherical particles under TEM,and the particle size distribution was 3.74±0.63 nm.The analysis of FT-IR and XPS showed that the surface of BQAS-CDs were rich in quaternary ammonium groups.Blue fluorescence was emitted under 365 nm ultraviolet excitation for the materials,which quantum yield of BQAS-CDs was 7.8%.Moreover,stability of BQAS-CDs was detected under different pH aqueous solution,temperature,ionic force and different time exposed under UV.It was found that pH had a great influence on the stability of BQAS-CDs,while the ionic force and temperature had weak impact on it.After exposure under UV for 6 h,the fluorescence intensity of BQAS-CDs remained at89.4±0.2%.Thereby,it was indicated that BQAS-CDs got a good stability.2.2 Self-emulsification occured after proteins mixed with BQAS-CDsOvalbumin(OVA)was used as a model protein,and self-emulsification occurs when mixed with BQAS-CDs.The observations by scanning electron microscopy(SEM)and TEM were showed that the size of different aggregation for protein range from 50 nm to405 nm(1:1,w/w),130.8 nm(1:2,w/w)and 50.29 nm(1:3,w/w).The zeta potential of the protein solution varied from-5.99±0.21 to-2.49±0.18(1:1,w/w),-3.81±0.16(1:2,w/w)and-4.82±0.44(1:3,w/w).Furthermore,PL of BQAS-CDs was showed a red shift,and UV-vis analysis of the protein became wider after mixing with BQAS-CDs.Thus,interaction between BQAS-CDs and OVA led to aggregation of protein to fuse into large particles by electrostatically.2.3 Immune responses against antigen were enhanced by BQAS-CDsAfter injecting mice muscle with nanocomplexes formed by BQAS-CDs and OVA,the antibody levels were 60-fold higher than those of the naked-protein group and maintain for a longer period of time.Compared with the commercial adjuvant Alum,higher production of IgG2a and IgG2b antibodies were induced by BQAS-CDs,which were 3 and 5 fold higher respectively.It was suggested that BQAS-CDs were on a tendency to be a Th1 type immunized.Additionally,stronger proliferation of OVA-specific CD4~+T and CD8~+CTL cells were stimulated by BQAS-CDs,which suggested that cellular immunity was improved.2.4 Antigen uptake is promoted by BQAS-CDs in APCs through endocytosisAfter BQAS-CDs and OVA-FITC complexes were incubated with BMDCs and RAW264.7 cells,antigen uptake was significantly promoted by BQAS-CDs through confocal fluorescence microscopy and flow cytometry analysis.When the complex was incubated at low temperature,antigen uptake was not improved significantly.Therefore,it was suggested that antigen uptake of the complex mainly through endocytosis in APCs.Furthermore,the ratio of CD40 and CD80 of BMDCs were not improved by BQAS-CDs,which indicated that maturation of DCs was not directly affected by BQAS-CDs.2.5 Good biocompatibility of BQAS-CDs in vivoBiocompatibility analysis in vivo showed that the body weight of mice was not affected by BQAS-CDs after immunization.Hematological changes were not significant in all mice.Additionally,there were no obvious changes in histology in the mice.It was indicated that BQAS-CDs had good biocompatibility in vivo.Collectively,BQAS-CDs may have potential for the development of new adjuvants.