Study On The Antibacterial Properties Of Carbon Dot-based Nanozymes And Their Application In The Treatment Of Vaginal Infectious Disease

Vulvovaginal candidiasis（VVC）and aerobic vaginitis（AV）are common vaginal infectious diseases,which seriously endanger the health of women.At present,the drug resistance of pathogens has brought great difficulties to the treatment of these infectious diseases.The development of new antibacterial agents based on nanozyme provides a new idea to solve the above problems.The antibacterial mechanism of nanozymes is mainly due to their enzyme-like activity to catalyze the corresponding substrate to produce reactive oxygen species（ROS）.Carbon dots（CDs）are a class of nanozymes with ultra-small size.There are a large number of active sites,and beneficial to modify functional groups in the CDs.The above characteristics make CDs have excellent optical and catalytic properties,as well as good biocompatibility.There was great potential of the CDs in antibacterial therapy.Improving catalytic efficiency and pathogen binding ability of the CDs are the key to fight pathogenic microorganisms.Atomic doping is widely used to improve the catalytic properties of CDs.This research proposes the doping strategy of CDs based nanozymes.It constructs a new high-efficiency nanozymes antibacterial system.The research analyzed the structural characterization,catalytic performance and antibacterial mechanism of the CDs.The CDs was applied to the clinical samples at the same time.The main content includes the following four parts:In the first part of this study,49064 cases of vaginal microecology results of patients with vaginitis in the gynecological clinic of our hospital from January 2015 to December 2018 were analyzed.It was compared with domestic and foreign literature to explore the changes of vaginal microecology in patients with vaginitis.In this study,vaginal p H was higher than 4.5 in 4280 VVC patients,and the measured p H ranged from 4.6 to 5.4.They accounted for 76.94%of VVC patients.There were 1514 cases of vaginal p H>4.5,accounting for 97.68%of the total AV patients.The H₂O₂ produced by Lactobacillus in the vagina and the range of p H which can provide natural conditions for the catalytic activity of CDs.The scientific idea of treating vaginitis with CDs was proposed.In the second part of the thesis,iodine,nitrogen-doped carbon dots（I-CDs）with high peroxidase activity were synthesized.When I-CDs-3（2.72 mg/m L）was combined with exogenous H₂O₂（0.5 m M）,which was lower than the physiological concentration in the vagina,and treated with visible light（2 W/cm²）for 120 min,the antibacterial efficiency of the system against Candida albicans was more than 90%.The antifungal effect of the synergistic system of I-CDs and H₂O₂ on C.albicans may be achieved by the combined effect of simulated POD activity and photocatalytic activity.In addition,the simulated POD activity and photocatalytic activity of I-CDs were closely related to iodine content.The higher iodine content in CDs,the higher simulated POD activity and photocatalytic activity.These results indicate that the prepared I-CDs have the potential to be used as antifungal agents for the treatment of VVC.In the third part of this thesis,Cu/I-CDs was synthesized by adding Cu to iodine to improve the catalysis efficiency of CDs-based nanozymes.The synthesized Cu/I-CDs had a high activity of mimetic POD nanozymes,and the inhibition rate of C.albicans was more than 95%at the concentration of 0.585 mg/m L combined with 0.5m M H₂O₂.The synthesized Cu/I-CDs nanozymes showed strong antifungal efficiency on the biofilm formation and maturation of C.albicans.In order to verify the antifungal effect of Cu/I-CDs system in vivo,a mouse VVC model was established.Experimental results showed that Cu/I-CDS could inhibit C.albicans in the mouse VVC model.A series of experiments were also performed to verify the intracellular ROS production.The low toxicity and high biocompatibility of Cu/I-CDs were also verified.In the fourth part of this thesis,Cu,Mo-CDs nanozymes with both abundant oxygen vacancies and Cu substitutions were generated by designing substitution defects.The high POD activity of Cu,Mo-CDs nanozymes was verified.The Ampicillin-resistant Escherichia coli（AREC）and Methicillin-resistant Staphylococcus aureus（MRSA）were selected to showed high antibacterial activity of the Cu,Mo-CDs nanozymes against drug-resistant Gram-positive and Gram-negative bacteria.Cu,Mo-CDs nanozymes showed high antibacterial activity against MRSA（97.8%）and AREC（96.3%）under the condition of H₂O₂ lower than the physiological concentration in the vagina.Concentration of H₂O₂ required was lower than the physiological concentration of H₂O₂ in the vagina.A series of experiments were carried out to study the antibacterial mechanism of the antibacterial agent.Including free radical capture test,SEM analysis of Cu and Mo-CDs treated bacteria,dead or alive staining analysis under laser confocal scanning microscope（CLSM）,and biofilm removal with crystal violet（CV）staining were carried out.The results showed that Cu,Mo-CDs nanozymes had a high antibacterial activity against drug-resistant bacteria and could clear the biofilm of drug-resistant bacteria.In the fifth part of this thesis,vaginal secretions from patients with typical clinical symptoms of VVC and AV were collected.The I-CDs,Cu/I-CDs,Cu,Mo-CDs nanozymes synthesized in this study were used in vaginal secretions of clinical patients.The clinical exudates of VVC patients were identified and cultured by isolation and purification fungal culture.The antifungal experiments with I-CDs and Cu/I-CDs showed good antifungal effect.The secretion of clinical AV patients was cultured for Gram-positive bacteria and Gram-negative bacteria,and Cu,Mo-CDs was used for antibacterial test.The results confirmed that the system with carbon dot nanozyme had satisfactory antibacterial effect on the samples of clinical vaginitis patients.In summary,the synthesized CDs based nanozymes exhibited high peroxidase activity and good biocompatibility.The CDs exhibited anti-biofilm activity against common vaginitis fungal pathogens and drug-resistant bacteria.It has potential medical value and broad application prospect in the treatment of vaginal infectious diseases,and provides important data and ideas for the application of doped carbon dot based nanozymes in many fields.