Functionalized Gold Nanomaterials For Bacterial Detection And Multidrug Resistance Infection Therapy

Multidrug-resistant bacterial infection represented by ESKAPE has become one of the most serious threats to public health worldwide.At present,the development of traditional small-molecule antibiotics has been unable to cope with the increasingly severe multidrug-resistant infection.With the development of nanomedicine,a variety of antimicrobial drugs based on nanomaterials have been widely developed and used for the treatment of multidrug resistance infections.Gold nanomaterials are widely used in biomedical fields such as biological analysis,cancer treatment,drug loading,and disease diagnosis due to their unique physical and chemical properties as well as excellent biocompatibility.The latest research shows that in addition to antimicrobial drug loading,gold nanomaterials can also be used for bacterial infection treatment by surface ligand or structural control to obtain antibacterial activity.Moreover,due to the unique optical properties,gold nanomaterials?such as fluorescent gold nanoclusters?have also been applied to the bacteria detection.At present,the development of antibacterial substances based on gold nanostructures and nano-diagnostic probes has become one of the most popular directions for biomedical applications of gold nanomaterials.Therefore,it is of great significance to further develop new gold nanomaterials for the diagnosis and treatment of bacterial infections,especially multidrug-resistant infections.This thesis is dedicated to the applications of gold nanomaterials in the bacterial detection and treatment of multidrug resistance infection.The effects of surface ligands,sizes,crystal facets and antimicrobial peptides binding on the antibacterial properties of gold nanomaterials were highlighted.The label-free fluorescence detection of Acinetobacter baumannii was achieved by using the aggregation-induced emission?AIE?enhancement effect of gold nanoclusters to obtain a fluorescent probe with strong fluorescence.A series of structurally similar mercaptopyrimidines were used as surface ligands to synthesize ultrasmall gold nanoclusters by hydrothermal reaction,and a gold nanoantibiotic with strong antibacterial activity against multidrug resistant bacteria was obtained.The effects of sizes and antimicrobial peptides loading on its antibacterial properties and its mechanism of action were further studied.In addition,the effects of crystal facets on the antibacterial activity were further investigated by synthesizing gold nanocrystals with different crystal facets.The main contents are as follows:?1?The weakly fluorescently emitted gold nanoclusters were synthesized by changing the ratio of HAuCl₄ to the thiol ligands and the reaction conditions.By introducing a certain concentration of Ag?I?as linkers to connect the Au?I?-thiol motifs forming Au?I?/Ag?I?-thiol motifs on the tailored thiolated gold nanoclusters,resulting in a significant fluorescence enhancement.The strong fluorescence of the as-synthesized AuAg bimetallic nanoclusters can be rapidly and selectively quenched by A.baumannii due to its induced agglomeration of AuAg bimetallic nanoclusters,which allows this fluorescent material to act as a label-free A.baumannii sensor in aqueous solution.A linear relationship was demonstrated between the fluorescence intensity of the thiolated AuAg bimetallic nanoclusters and the concentration of A.baumannii,in the range of 1×10⁴-5×10⁷ colony forming unit?CFU?/mL with a limit of detection of 2.3×10³ CFU/mL.The A.baumannii content in sputum was also analyzed.As far as we know,this is the first report of a fluorescent sensor for the detection of A.baumannii.This approach represents a rapidly alternative method for the analysis of sputum A.baumannii in clinical diagnosis.?2?Four mercaptopyrimidine analogues,namely,4-amino-2-mercaptopyrimidine?AMP?,4,6-diamino-2-mercaptopyrimidine?DAMP?,4-amino-6-hydroxyl-2-mercaptopyrimidine?AHMP?,and 4,6-dihydroxyl-2-mercaptopyrimidine?DHMP?,were used as ligands for the synthesis of Au NCs.We assess the minimal inhibitory concentration?MIC?of these mercaptopyrimidine-conjugated Au NCs against clinically isolated ESKAPE strains,i.e.,multidrug resistant Escherichia coli,A.baumannii,Pseudomonas aeruginosa,Klebsiella pneumoniae,methicillin-resistant Staphylococcus aureus?MRSA?,and vancomycin-resistant Enterococcus faecium?VRE?).These superbugs are isolated from the sputum samples of clinical patients with respiratory tract infections.As the best antibacterial activity gold nanoclusters,AuDAMP can inhibit the multiplication of multidrug resistant E.coli,A.baumannii,P.aeruginosa,K.pneumoniae,MRSA,and VRE,with a MIC of 4?g/mL,2?g/mL,4?g/mL,2?g/mL,2?g/mL,and 8?g/mL,respectively.Drug resistance study showed that no resistance development towards the AuDAMP was demonstrated even after continuous passages for 30 days.The AuDAMP kill these superbugs through a combined mechanism including cell membrane destruction,DNA damage,and reactive oxygen species?ROS?generation,and exhibit excellent treatment effects in both macrophages and animal infection models induced by MRSA as representative.Moreover,the induction of intracellular ROS production in bacterial cells mainly attributed to the AuDAMP?intrinsic oxidase-and peroxidase-like catalytic activities has been demonstrated for the first time.Size effect studies have found that DAMP-mediated large-size gold nanoparticles have significantly lower antibacterial activity than AuDAMP.Cytotoxicity evaluation showed that AuDAMP has excellent biocompatibility for mammalian cells,and the cell activity is still as high as 85%or more at a concentration of 256?g/mL.Both macrophages infection model and mouse infection model showed that AuDAMP has better in vivo treatment capability and potential application in the treatment of multidrug resistance infection.?3?Based on AuDAMP,we demonstrated an effective antibacterial hybrid formed by covalently conjugating antibacterial gold nanoclusters?AuDAMP?and daptomycin?Dap,a cyclic lipopeptide antimicrobial peptide?.The as-synthesized hybrid structure?Dap-AuDAMP?not only inherits the intrinsic properties from both agents but also renders an enhanced synergistic effect.Compared with the physically mixed AuDAMP and daptomycin?Dap+AuDAMP?,the Dap-AuDAMP hybrid structure has a stronger bactericidal effect toward MRSA,a representative of multidrug-resistant bacteria.Dap-AuDAMP could effectively disrupt bacterial membranes by creating more and/or larger holes in the membranes due to the localized daptomycin within the conjugated structure.These larger?and possibly more?holes motivate the entry of Dap-AuDAMP into bacterial cells and lead to more serious damage of the bacteria at subcellular levels.Moreover,bacterial genomic DNA fragmentation was further quantified to show that Dap-AuDAMP may induce severe DNA breaks.The strong DNA destruction benefited from localized high concentrations of ROS induced by the localization of AuDAMP in the antimicrobial conjugation.The conjugated AuDAMP could serve as a critical free radical generator to continuously produce ROS within the bacteria.The continuous ROS bombings also limit the capacity of the bacteria to develop drug resistance.In addition,a significant fluorescence enhancement of the hybrid structure was observed due to a novel AIE pattern caused by the AuDAMP and daptomycin conjugation.This conjugation strategy provides a new perspective for the synthesis of new antimicrobial agents as well as AIE-type fluorescence materials.?4?In this section,the antibacterial toxicity behavior of well-defined crystallographic facets of a series of Au nanocrystals,including{100}-facet cubes,{110}-facet rhombic dodecahedra,{111}-facet octahedra,{221}-facet trisoctahedra and{720}-facet concave cubes,was investigated in the model bacteria S.aureus by experimental approach.We find that Au nanocrystals display substantial facet-dependent antibacterial activities.The low-index facets of cubes,octahedra,and rhombic dodecahedra showed a certain degree of antibacterial activity,whereas the high-index facets of trisoctahedra and concave cubes remained inert under biological conditions.The antibacterial toxicity mechanism studies have shown that the facet-dependent antibacterial behaviors of Au nanocrystals mainly caused by differential bacterial membrane damage as well as inhibition of protease activity and energy metabolism.These faceted Au nanocrystals are unique in that they do not produce reactive oxygen species as most antibiotics as well as antimicrobial nanostructures do.Our findings provide a deeper understanding of facet-dependent toxicological responses and might advance the development of gold-based nanomaterials with enhanced antibacterial activity.