Preparation Of Enzyme-responsive Antibacterial Nanoprobes And Its Antibacterial Research

Nowadays,we are facing with more and more serious clinical bacterial infections.In recent years,a large number of drug-resistant bacteria and multi-drug-resistant bacteria continue to appear.The spontaneous drug-resistant mutation of bacteria,the screening of antibiotics and the adaptability of pathogenic bacteria to the environment are the basis of the generation of clinical drug-resistant bacteria and multidrug-resistant bacteria.In fact,it is wound tissue that is prone to bacterial infection.Human skin is the first barrier for humans to prevent bacterial invasion.As the integrity of the skin is destroyed,trauma patients lose their function as a barrier and cannot effectively isolate bacteria.Cause bacteria to gain a quick way to invade the human body.At the same time,the wound is infected by bacteria,and the healing speed of the wound is first affected.In severe cases,bacteria invade the blood circulation and cause acute systemic infections that endanger the lives of patients.Therefore,how to quickly identify and eliminate wound bacterial infections and reduce the risk of trauma patients exposed to the bacterial environment is of great research significance.This project used gold nanostar(AuNS)as a carrier,introduced a gelatinase cleavage site(PLGVR)at the C-terminus of the GKRWWKWWRR antimicrobial peptide,and introduced a S.aureus targeting recognition peptide(GLFVD)at the N-terminus,and synthesized a peptide sequence and the sequence of the polypeptide GLFVDK(-Cy7)GKRWWKWWRRGPLGVRGC is digested,and the polypeptide was coupled with the gold nanostar through a gold-sulfur bond to prepare AuNS-PEG-AMP.Generally,the nanomaterial did not produce fluorescence because there is fluorescence resonance energy transfer(FRET)between the Cy7 dye and the AuNS core.However,in the pathological enzyme microenvironment of S.aureus infection,the PLGVR sequence between AuNS and Cy7 would be cleaved by overexpressed gelatinase to restore the fluorescence of Cy7 and realize the in situ NIR fluorescence of S.aureus infection imaging.In addition,GLFVD can significantly enhance the targeted accumulation of nanomaterials at the site of S.aureus infection,and avoid thermal damage to surrounding healthy tissues,thereby nanomaterials can be enhanced for more specific and localized photothermal treatment of S.aureus infected wounds.Results:(1)The material characterization results show that the AuNSPEG-AMP nanomaterial has good dispersibility in aqueous solution,and its hydrodynamic particle size is 201.3 ± 3.7 nm.In addition,nanomaterials have excellent particle size stability,photostability,photothermal effect and antibacterial ability;(2)In vitro experimental results show that there is fluorescence resonance energy transfer between the Cy7 dye(FRET)on the targeted antibacterial enzyme cleaved peptide and the AuNS core,Cy7 fluorescence quenching.In the microenvironment of the pathological enzymes of S.aureus infection,the over-expressed gelatinase will cleave the PLGVR sequence,restore the fluorescence of Cy7,and realize the in situ NIR fluorescence imaging of S.aureus infection.In addition,bacterial growth curve experiments,bacterial biofilm inhibition/destruction experiments,and bacterial live/dead staining experiments confirmed that AuNS-PEG-AMP nanomaterials can effectively inhibit the growth of bacteria.Bacterial coating experiments on agar plates confirmed that AuNS-PEG-AMP nanomaterials can specifically kill S.aureus in the microenvironment where mixed strains exist.In vitro cytotoxicity experiments show that AuNS-PEG-AMP nanomaterials have excellent biocompatibility;(3)In vivo experimental results show that AuNS-PEGAMP nanomaterials are used for wound treatment of S.aureus/E.coli infected mice.Compared with the non-irradiation group,the wound area of mice in the AuNS-PEG-AMP illuminated group was significantly reduced.It is worth noting that the targeting properties of AuNS-PEGAMP nanomaterials prove that its effect in the treatment of S.aureus wounds is significantly better than that of E.coli wound infections.