The Study Of Hyrid Membrane-coated Bionic Nanozyme With Ultrasonic Enhanced Activity Combat Multidrug-resistant Bacteria

In recent years,multi-drug resistance（AMR）pose an urgent threat to global public health due to the misuse of antibiotics.At present,carbapenem-resistant Enterobacteriaceae（CRE）,methicillin-resistant staphylococcus aureus（MRSA）and vancomycin-resistant enterococcus（VRE）are the most threatening multi-drug resistant bacteria,can hydrolyze almost all clinically available antibiotics,triggering a significant clinical challenge.Due to the poor therapeutic effect of clinical antibiotics and the difficulty in the research and development of new antibiotics,it is particularly urgent to seek new antibacterial therapy.Reactive oxygen species（ROS）can be used as a strong oxidant,by destroying the bacterial cell membrane,inactivating proteins,breaking DNA,etc,to kill bacteria,so it has a broad-spectrum antibacterial effect.Nanozyme,as one of many nanomaterials that produce ROS,has received widespread attention with the advantages of stability,low cost,good biocompatibility and versatility.However,due to low catalytic capacity and poor in vivo selectivity of nanozyme,nanozyme-based antimicrobial therapy still faces huge challenges.Therefore,developing a novel nanozyme system that can both precisely target bacteria and efficiently kills drug-resistant bacteria is promising.Herein,a biomimetic nano enzyme system（BiPt@HM）with high ROS generation and precise pathogen targeting capacities was constructed.BiPt@HM has the following main features:（1）the BiPt nanozyme presented multiple enzymatic activities,including peroxidase activity,catalase and oxidase activity,and its enzymatic activity could be efficiently sensitized by highly penetrating ultrasonic（US）in vivo.In the assist of US,BiPt could produce large amounts of ROS explosively,which lays foundation for drug-resistant bacteria treatment;（2）a hybrid membrane of bacterial outer membrane vesicles and platelet membrane was used as the coat for BiPt nanozyme,which enables escape immune surveillance and accurately target bacterial infectious sites in vivo.In conclusion,thanks to the high catalytic activity of the enhanced nanozyme and the precise recognition ability of hybrid membrane,BiPt@HM achieved efficient clearance of NDM-1 EC1322 and M13 in vivo.Details of the study are as follows:1.Preparation and characterization of BiPt nanozymeFirst,BiPt nanozyme were prepared by one-step method,TEM,XRD,XPS,UPS and other results proved that the spherical aggregate BiPt nanozyme was successfully prepared,which has a size of about 40 nm and has good photoacoustic imaging capabilities.Then,through the determination of peroxidase reaction kinetic constants,we found that BiPt nanozyme exhibited strong H₂O₂ affinity and high peroxidase activity,and its Vmax value was about 5 times than that of Bi nanoparticles,which was 1.7 times than that of Pt nanoparticles,furthermore,its Kcat value(6.151 Min^-1)was significantly higher than that of Bi nanoparticles(0.829 Min^-1)and Pt nanoparticles(1.406 Min^-1).The results of oxygen production measurement experiments and electron spin resonance（ESR）experiments show that BiPt nanozymes also have catalase activity and oxidase activity,which can catalyze the production of·OH,O₂,·O₂^-and ¹O₂.Importantly,the enzyme activity of BiPt nanozymes is significantly enhanced after sonication.2.Preparation and characterization of BiPt@HMFirstly,platelet membrane（PMs）and bacterial outer membrane vesicles（OMVs）were prepared using the extrusion method.The results of TEM,SDS-PAGE,Western blot and FRET demonstrated HM were successfully prepared.The particle size of HM is about 35 nm.HM has the characteristic proteins CD62p,CD41,CD47 and Omp A of both OMVs and PMs.BiPt@HM was then prepared using the extrusion method.The successful preparation of BiPt@HM was demonstrated by TEM,SDS-PAGE and Western blot experiments.Results showed that BiPt@HM has a particle size of approximately 70 nm and good photoacoustic imaging capabilities,and shares the characteristic proteins of HM.BiPt@HM was later shown to be highly efficient in escaping immunosurveillance and to have excellent bacterial targeting capabilities through flow experiments,laser confocal experiments and bioscanning electron microscopy.Finally,the catalytic activity of BiPt@HM was examined and we found that BiPt@HM has excellent catalytic ability and the encapsulation of HM does not affect the catalytic activity of BiPt nanozymes.3、BiPt@HM antibacterial activity study in vitroNDM-1 E.coli EC1322 and methicillin-resistant staphylococcus aureus M13were used as models to evaluate the in vitro antibacterial activity of BiPt@HM.Firstly,the antibacterial effect of different concentrations of BiPt nanozymes on drug-resistant bacteria was investigated.Result showed that the antibacterial effect of BiPt nanozymes gradually increased as the concentration of BiPt nanozymes increased,and the addition of H₂O₂ further enhanced the antibacterial effect.Then the antibacterial effect of BiPt@HM on drug-resistant bacteria was investigated.The results showed that BiPt@HM had excellent antibacterial ability against drug-resistant bacteria with 99.999%antibacterial rate under the action of ultrasound,and the antibacterial ability was not affected by HM wrapped with BiPt nanozyme.Finally,the mechanism of bacterial killing by BiPt@HM was investigated by reactive oxygen staining experiments,and the results showed that BiPt nanozymes and BiPt@HM are efficient in treating drug-resistant bacteria by catalyzing the generation of ROS.4、Study of antibacterial activity of BiPt@HM in vivoThe M13-infected rat osteomyelitis model,the NDM-1 EC1322-infected mouse muscle model and the NDM-1 EC1322-infected mouse pneumonia model were used to evaluate the in vivo kinetic distribution and pharmacodynamic properties of the drug delivery system by observing the distribution of BiPt@HM in mice,the bacterial load at the infectious sites in mice and rats in each administration group,the blood inflammatory factor levels and the H&E staining of tissue sections.The in vivo distribution results showed that IR780@HM can effectively accumulate at the sites of bacterial infection,providing a basis for determining the time of ultrasound treatment.Photoacoustic imaging results showed that BiPt@HM has excellent targeting ability to the sites of bacterial infection and good photoacoustic imaging capability.H&E pathological slide results,blood routine results and blood biochemical results show that the prepared bionic nanozyme BiPt@HM have good biosafety in vivo.The results of the bacterial load of the infected site in the treated mice and rats showed that BiPt@HM had a good antibacterial effect in vivo under the action of ultrasound compared with other groups（Control,Control+US,BiPt,BiPt+US,HM,HM+US,BiPt@HM）,and the bacterial inhibition rate reached over 99.9%.The results of H&E staining of tissues at the site of bacterial infection showed that the BiPt@HM+US group had only a small amount of inflammatory infiltration in the infected tissues,while the other groups had obvious lesions.A bacterial outer membrane vesicle/platelet membrane hybrid membrane encapsulated bionanase system（BiPt@HM）for the treatment of multi-drug resistant bacteria was constructed.BiPt@HM has both the ability to accurately identify bacteria and the ability to efficiently produce ROS for the treatment of drug-resistant bacteria.The in vivo and in vitro results showed that bionic nanozyme system can precisely target the site of bacterial infection and efficiently treat multidrug-resistant bacteria,providing a new tool for the treatment of multidrug-resistant bacteria.