Construction Of Novel Multifunctional Antibacterial Systems Based On Carbon Dots And Investigation Of Theirs Activity

Objective:Bacterial infections caused after skin trauma have become a serious public health problem worldwide.There are various clinical treatments for bacterially infected wounds,such as debridement,flushing with antiseptic water,and administration of antibiotics,but there are still cases of improper treatment that allow pathogenic bacteria to enter the blood circulation,grow and multiply in the blood,and produce toxins that cause systemic sepsis.In addition,antibiotics used to treat bacterial infections are prone to develop resistance,making the treatment less effective.A variety of new organic small molecules and other antibacterial materials have been reported to cope with this crisis,however,there are drawbacks such as complicated synthesis and expensive raw materials.Therefore,there is an urgent need to develop new materials that are simple,inexpensive and highly effective in sterilization.Carbon dots（CDs）have received a lot of attention from researchers because of their good optical properties,dispersion,stability,biocompatibility,low synthesis cost and simple synthesis methods,and have shown promising applications in metal ion sensing,bioimaging,catalysis and drug delivery.Currently,carbon dots have a single antimicrobial therapeutic modality;therefore,this paper constructs a multifunctional nanoplatform based on novel carbon dots for effective bactericidal treatment and investigates its therapeutic effects in bacterially infected wounds as follows:1.Blue fluorescent carbon dots（PDCDs）with antibacterial activity were synthesized by hydrothermal method using citric acid and 1-（2-methyl-5-nitrophenyl）guanidine nitrate as raw materials and ultrapure water as solvent.Further chelate Cu²⁺with wound healing promoting properties on PDCDs and study the in vitro killing properties of PDCDs and PDCDs@Cu against Pseudomonas aeruginosa and Staphylococcus aureus in an attempt to develop a nanomedicine with high biosafety,bactericidal and wound healing promoting properties.2.Photothermal CDs（PTCDs）with red fluorescence emission and photothermal properties were synthesized by hydrothermal method using citric acid and 1-ethyl-（3-dimethylaminopropyl）carbodiimide hydrochloride（EDC）as raw materials and formamide as solvent.The PTCDs were chelated with Ag⁺,which has antibacterial activity,and further mixed with azo diisopropylimidazoline hydrochloride（AIPH）,which generates free radicals by thermal excitation,into the hydrogel prepared from gelatin to obtain a novel multifunctional hydrogel for multi-method synergistic sterilization.Methods:1.PDCDs were synthesized by a one-step hydrothermal method using citric acid and1-（2-methyl-5-nitrophenyl）guanidine nitrate as raw materials,and their synthesis time,raw material mass ratio and synthesis temperature were optimized.The morphology and the constituent elements were characterized by TEM,AFM,XRD,XPS and FTIR;the properties of PDCDs were investigated by testing their fluorescence spectra,UV-Vis absorption spectra and photodynamic properties;and their cytotoxicity was tested by MTT method.In view of the good chelating ability of PDCDs to Cu²⁺,the metal chelating carbon dots（PDCDs@Cu）were finally prepared and their killing effects on Pseudomonas aeruginosa and Staphylococcus aureus were investigated.2.PTCDs were synthesized by a one-step solvothermal method using citric acid and EDC as raw materials and formamide as solvent,and the excellent PTCDs were prepared by optimizing the synthesis conditions;their morphological particle size and constituent elements were characterized;the optical properties of PTCDs were tested by UV-Vis spectrophotometer and fluorescence spectrophotometer;and their cytotoxicity was tested by MTT method.In view of the excellent performance of PTCDs,metal chelated carbon dots（PTCDs@Ag）were prepared,and finally they were co-loaded with AIPH in a hydrogel prepared by gelatin,and their bactericidal properties were investigated using the template bacteria Staphylococcus aureus and the bactericidal properties after biofilm formation,and finally applied in vivo to further verify their therapeutic effects on infected wounds.Results:1.TEM and AFM characterization results showed that the particle size of PDCDs was homogeneous and mainly concentrated in 2-3 nm,XRD spectra showed that they were amorphous carbon structures,XPS and FTIR spectra showed that their surfaces mainly contained hydroxyl,carboxyl and amino groups and other hydrophilic groups;fluorescence spectroscopy results showed that the fluorescence emission peak of PDCDs was located at440 nm（360 nm excitation）and the fluorescence was not affected by ion.The results of fluorescence spectroscopy showed that PDCDs fluorescence emission peak was located at445 nm（360 nm excitation）and fluorescence was not affected by ionic intensity,wide p H and storage time,and had good optical stability,and Cu²⁺had obvious fluorescence quenching phenomenon,indicating that PDCDs could chelate Cu²⁺.In addition,the killing performance of PDCDs@Cu against Pseudomonas aeruginosa and Staphylococcus aureus was investigated using coated plate assay,and the results showed that PDCDs@Cu could selectively kill Pseudomonas aeruginosa under the same conditions.2.The optimal conditions for the synthesis of PTCDs were 2 h reaction time,5:1 mass ratio of reactants（citric acid:1-ethyl-（3-dimethylaminopropyl）carbodiimide hydrochloride）,and 200℃reaction temperature.The synthesized PTCDs are small in particle size,composed of amorphous carbon,and contain hydrophilic groups on the surface,so they have good water solubility.PTCDs fluoresce red,with optimal excitation at 550 nm and optimal emission at 650 nm,and have good optical stability;Ag⁺has fluorescence quenching phenomenon for them,indicating that PTCDs can chelate Ag⁺.They have good photothermal effect and stable photothermal performance under laser irradiation at 660 nm.The temperature-sensitive hydrogel PTCDs@Ag-AIPH Hydrogel was prepared by chelating it with Ag⁺and co-loading it with AIPH in gelatin,which has a porous structure with good water absorption and is conducive to absorbing exudate from wound sites.The composite hydrogel also showed good bactericidal properties in in vivo experiments and did not cause any damage to the liver and kidney function of mice.Conclusion:In this thesis,two excellent performance carbon dots were synthesized and their synergistic bactericidal properties were explored by combining carbon dots with other antibacterial substances.Firstly,PDCDs with ROS production under xenon lamp irradiation were synthesized by hydrothermal method using citric acid and 1-（2-methyl-5-nitrophenyl）guanidine nitrate as raw materials and ultrapure water as solvent,and on the basis of which Cu²⁺,which can promote wound healing and sterilization,was connected to the composite material PDCDs@Cu,which has good sterilization performance against Pseudomonas aeruginosa in vitro.Secondly,PTCDs with red fluorescence and good photothermal properties were synthesized by the solvothermal method using citric acid and EDC as raw materials and formamide as solvent,which were chelated with Ag+and co-loaded with AIPH in the hydrogel prepared from gelatin to make a composite hydrogel with good water absorption performance and low toxicity.They were converted from solid to liquid state at ambient temperature greater than 37℃.We thermally solubilized the hydrogels by 660 nm laser irradiation of PTCDs and activated AIPH to generate alkyl radicals,which had excellent killing performance against Staphylococcus aureus and its biofilm in vitro and in vivo by heat,alkyl radicals and Ag⁺synergistic bactericide.In conclusion,we synthesized two excellent performance carbon dots and combined them with other materials to achieve good synergistic bactericidal effects.