| Background:Diseases caused by bacterial infections pose a serious threat to human health and have once again been recognized as one of the most serious global threats.Its treatment mainly depends on traditional antibiotics,however,with the continuous emergence of drug-resistant strains,traditional antibiotics have been increasingly unable to meet the needs of clinical anti-infective treatment.Therefore,in order to alleviate or reverse the crisis caused by bacterial drug resistance,the development of new high-efficiency antibacterial agents has important practical significance.Nanozymes are a new type of antibacterial preparation developed in recent years.Their antibacterial mechanism lies in the activity of peroxidase and oxidase,which can catalyze hydrogen peroxide or oxygen to produce a large number of free radicals to effectively kill bacteria.in a short time.Among the common kinds of nanozymes,carbon nanozymes,which are based on carbon materials,and have attracted more and more attention in anti-infective therapy because of their multiple properties,such as mimic enzyme activity,photothermal performance,photocatalytic activity and so on.However,the low mimic activity of carbon nanozymes lead to their poor germicidal efficacy.Therefore,in order to obtain higher enzyme catalytic activity,the modification of carbon nanozymes,such as the introduction of metal active sites(Cu,Fe,Zn,etc.),is one of the effective ways to improve their antibacterial activity.However,the physical and chemical properties of metal/carbon hybrid nanozymes constructed by introducing metal active sites,such as particle size,crystal structure,element composition,content and surface charge,can affect the mimic enzyme activity,thus further affect the antibacterial activity.Therefore,the study of the relationship between the composition or state of nanomaterials and mimic enzyme activity will help us to understand the source of enzyme activity of artificial metal nanozymes and their derivatives.It also provides experimental basis and theoretical guidance for obtaining nanozymes with high catalytic activity,and then we can understand the antibacterial properties related to enzyme activityCarbon nanozymes are different from other metal and metal oxide nanozymes in that they have a large specific surface area and can be used as a carrier to encapsulate and transport drug molecules.Compared with traditional drug carriers such as phospholipids or macromolecular polymers,this carrier can not only play the pharmacological role of the drug itself,but also achieve the purpose of synergistic therapy through its own enzyme catalytic activity.For this reason,curcumin was introduced into carbon nanozymes system to construct drug-loaded carbon nanozymes.Curcumin is one of the most popular natural food pigments in the world,and it is a natural plant extract with a wide range of raw materials,low cost,non-toxic and non-pollution.Medical studies have shown that curcumin has the effects of reducing blood lipids,anti-tumor,anti-inflammation and so on,meanwhile,curcumin is also a new type of photosensitizer.Under the irradiation of blue light source(wavelength range 420 nm-480 nm),curcumin can produce singlet oxygen,which can be used in photodynamic therapy(PDT)for many diseases.Combining curcumin with carbon nanozymes as a drug carrier,on the one hand,curcumin PDT produces singlet oxygen to exert antibacterial effect,on the other hand,metal/carbon hybrid nanozymes can kill bacteria by exerting their own enzyme catalytic activity,and finally achieve the effect of synergistic therapy.Based on the above background,copper element was introduced into carbon nanozymes as metal active center.Copper/carbon hybrid nanozymes with many kinds of mimic enzyme activity were designed and synthesized,and their mimic enzyme activity was regulated by adjusting the valence of copper element(from Cu2+ to Cu0),so as to further determine the relationship between mimic enzyme activity and antibacterial activity.The morphology,particle size,structure,multi-enzyme activity,bactericidal ability and bactericidal mechanism of copper/carbon hybrid nanozymes were studied in detail,and their therapeutic effects on bacteria-induced gastroenteritis infection and wound healing were also evaluated.Finally,copper-doped carbon nanozymes were used as drug carriers to combine with curcumin to investigate the inhibition of bacteria by the composite system under blue light irradiation,so as to develop a new antibacterial agent using carbon nanozymes as drug carriers to lay the experimental foundation.Objectives:The main results are as follows:(1)Copper nanomaterials have been widely used in bacteriostasis.We prepared copper/carbon hybrid nanozymes to observe the bacteriostatic effect of nanoscale enzymes.(2)Copper has bivalent,univalent and zero valence in nature.We want to investigate the effects of the recombination of different valence copper on carbon nanozymes on mimic enzyme activity and antibacterial activity,in order to find out the relationship between valence,enzyme activity and antibacterial activity.(3)The antibacterial activity of copper/carbon hybrid nanozymes was used to treat different animal models of bacterial infection.(4)PDT with curcumin as photosensitizer has good germicidal efficacy.Copper-doped carbon nanozymes were combined with curcumin as drug carrier to investigate the inhibition of bacteria by the composite system under blue light irradiation,so as to lay an experimental foundation for the development of new antimicrobial agents using carbon nanozymes as drug carrier.Methods:Chapter 1 Study on Catalytic Activity and Antibacterial Activity of Copper/carbon Hybrid NanozymesIn this chapter,two kinds of copper/carbon hybrid nanozymes with different copper valence were synthesized by using SiO2 nanospheres as templates,one is copper-doped carbon nanozyme(Cu-HCSs),the other is copper oxide-doped carbon nanozyme(CuO-HCSs),and the undoped-copper carbon nanozyme(HCSs)is used as control.Firstly,the morphology of copper/carbon hybrid nanozymes were characterized by TEM,SEM,HRTEM and other characterization methods.In addition,their structures and compositions were analyzed by XRD,XPS and Raman spectra.Subsequently,the multi-enzyme activities of Cu-HCSs and CuO-HCSs were analyzed,including peroxidase,catalase and superoxide dismutase activity.Then,on the one hand,the antibacterial activity of CuO-HCSs was studied,on the other hand,the antibacterial effect of Cu-HCSs and H2O2 was discussed,and their antibacterial mechanism was studied respectively,including the detection of the level of reactive oxygen species,the determination of lipid oxidation level,the observation of bacterial morphology and the cleavage of nucleic acid in bacteria and so on.Finally,the biosafety of copper/carbon hybrid nanozymes were evaluated,and the models of intestinal infection caused by Salmonella typhimurium and wound infection caused by Staphylococcus aureus were established to explore the therapeutic effects of CuO-HCSs on intestinal infection and the combined use of Cu-HCSs and H2O2 on wound healing.Chapter 2 Preliminary Study on Antibacterial Effect of Curcumin-loaded Copper carbon NanozymesIn this chapter,based on the detailed determination of the properties of copper/carbon hybrid nanozymes,Cu-HCSs nanozymes with high catalytic activity were selected as the drug carrier to load curcumin,and curcumin/copper-doped carbon nanozymes were achieved(Cur/Cu-HCSs).Cur/Cu-HCSs was characterized by TEM and Fourier transform infrared spectroscopy(FTIR),and the drug loading rate was determined in detail.Finally,the antibacterial effect and the degradation of plasmids and proteins in Cur/Cu-HCSs under blue light irradiation were discussedResults:Chapter 1:The copper/carbon hybrid nanozymes prepared in this chapter have good graphitization degree,the particle size is about 100 um,the shell thickness is 5-10 nm,and the dispersion is good.The distribution of CuO particles(about 10 nm)on the carbon layer of CuO-HCSs can be observed by HRTEM,while the Cu0 in Cu-HCSs is difficult to be observed.Copper/carbon hybrid nanozymes have copper valence-dependent peroxidase,catalase and superoxide dismutase activities.In addition,their antibacterial activity mainly depends on the valence of copper,and two different antibacterial mechanisms are proposed for CuO-HCSs and Cu-HCSs:Cu2+released by CuO-HCSs,can cause cell membrane damage,lipid peroxidation and nucleic acid degradation of Gram-negative bacteria,while reactive oxygen species catalyzed by hydrogen peroxide production of Cu-HCSs,are the decisive factor against Gram-positive and Gram-negative bacteria.In order to confirm the antibacterial ability of copper/carbon hybrid nanozymes,in the mouse model of gastrointestinal infection caused by Salmonella typhimurium and wound infection caused by Staphylococcus aureus,we found that CuO-HCSs had a better therapeutic effect on gastrointestinal infection and greatly improved the survival rate of mice;meanwhile,compared with monotherapy,the combined use of Cu-HCSs and H2O2 can effectively treat wound infection and promote wound healing.Chapter 2:In this chapter,we use Cu-HCSs with high catalytic activity as drug carrier to prepare curcumin-loaded carbon nanozymes(Cur/Cu-HCSs)with a drug loading rate of 99%.The TEM diagram shows that Cur can be loaded to Cu-HCSs,and the change of the characteristic peak in the Fourier transform infrared spectrum also shows that Cur has been successfully loaded into Cu-HCSs.In the antibacterial experiment,compared with the single antibacterial therapy,Cur/Cu-HCSs have a strong antibacterial effect on all kinds of bacteria under blue light irradiation.In addition,it was also found that Cur/Cu-HCSs could accurately degrade Escherichia coli plasmid and fetal bovine serum albumin(BSA)under blue light irradiation. |
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