| Periprosthetic infection(PJI)is one of the major causes of joint replacement failure and is difficult to treat with a high recurrence rate,especially when made more difficult by methicillin-resistant Staphylococcus aureus(MRSA)infection.Treatment with vancomycin usually leads to extremely toxic side effects and low efficacy due to strong drug toxicity and weak targeting.Therefore,there is an urgent need to develop a novel antimicrobial agent and targeted drug delivery system.The depolymerized Fucoidan(low-molecular-weight Fucoidan,LMWF)can effectively kill MRSA strains and inhibit osteoclast activation while killing the bacteria,which can be an ideal drug for application in periprosthetic infections.However,LMWF is easily degraded in vivo,so it is necessary to construct a drug delivery system that can effectively load LMWF and have the ability to target the infection site,so that LMWF can be efficiently released at the infection site to help the treatment of periprosthetic infections.In this experiment,we demonstrated that LMWF has ideal anti-MRSA effect and good biosafety.In order to improve the slow release efficiency of LMWF and maintain the effective local drug concentration,we prepared an anti-infection bionanoparticle(LMWF@PLGA@NM NPs,LMM NPs)loaded with LMWF and wrapped with neutrophil membrane with autonomous targeting ability,and investigated its therapeutic effect on PJI of MRSA infection.Part Ⅰ.Ex vivo and in vivo studies on the anti-MRSA effect of LMWFObjective:To explore the antibacterial effect of low molecular weight fucoidan(LMWF)on MRSA and to explore the feasibility of LMWF as a new antibacterial agent.Methods : LMWF was obtained by depolymerization of Fucoidan and co-incubated with MRSA to detect the survival rate of MRSA after LMWF treatment;the damage of LMWF on MRSA bacterial cell membrane was examined.The in vivo antibacterial effect of LMWF was investigated using a mouse MRSA-infected skin defect model.Results : 1.The depolymerization product of Fucoidan(LMWF)was successfully obtained,and its molecular weight was in the range of 10-30 k Da.FTIR results showed that the main characteristic peaks were retained.2.The results of bacterial smear plate showed that the number of bacteria was negatively correlated with the concentration of LMWF,and the number of bacteria decreased significantly after 1.25 mg/m L LMWF treatment for 2 hours,and no statistical difference with vancomycin treatment group;the results of ZOI showed that the size of inhibition ring was negatively correlated with the concentration of LMWF,and the ZOI of 5 mg/m L LMWF was not statistically different with vancomycin;the results of live-dead fluorescence of bacteria showed that the same After treatment with the same concentration,the bacterial death increased in the LMWF group compared with the Fucoidan group.3.The integrity of the cell membrane of MRSA bacteria treated with LMWF was damaged;the protein concentration in the supernatant increased and the bacterial cell contents leaked out;the bacteria were crumpled,depressed and died.4.In the mouse MRSA infected skin defect model,the size of wound area in the LMWF-treated group was comparable to that in the vancomycin-treated group,and HE staining showed wound healing;Giemsa staining showed that the number of wound bacteria was significantly reduced;blood tests in mice showed that the white blood cell count and neutrophil ratio in the LMWF-treated group were not statistically different from those in normal mice.Conclusions: The depolymerization treatment does not change the main functional structure of Fucoidan;LMWF has ideal anti-MRSA effect both in vivo and in vitro,and LMWF is feasible as a new antibacterial agent.Part Ⅱ.Preparation of LMWF-loaded neutrophil membrane coated nanoparticles and evaluation of their propertiesObjective:To prepare the LMWF-loaded PLGA nanoparticles wrapped with neutrophil membranes(LMWF@PLGA@NM nanoparticles,LMM NPs)and evaluate their properties.Methods:To prepare the LMWF-loaded neutrophil membrane-mimetic nanoparticles LMM NPs were and analyze using transmission electron microscopy(TEM)and DLS;to detect the adhesion of nanoparticles to MRSA bacteria;to detect the targeting effect of nanoparticles on infection sites in vivo by in vivo fluorescence in rat;to collect venous blood to detect the biometabolism of nanoparticles in vivo.Results : 1.Neutrophil membranes were successfully extracted and obtained,and wrapped on the surface of LMWF-loaded PLGA nanoparticles to produce neutrophil membrane-mimetic nanoparticles LMM NPs.LMWF can continuously release from LMM NPs.2.The results of CCK-8 showed that LMM NPs had no significant cytotoxic.3.The antibacterial effect of LMM NPs on MRSA gradually increased with the increase of nanoparticle concentration;after wrapping the neutrophil membrane,the phagocytosis of nanoparticles by macrophages decreased;after wrapping the neutrophil membrane,the adhesion of nanoparticles to bacteria increased.4.In the rat PJI model,after LMM NPs were injected via tail vein,the in vivo fluorescence results showed that nanoparticles aggregated at the infection site;after wrapping neutrophil membranes,the blood circulation time of LMM NPs in vivo was prolonged.Conclusions : The LMWF-loaded neutrophil membrane-coated nanoparticles(LMM NPs)were successfully prepared with uniform size and stable properties;the LMM NPs can evade phagocytosis by macrophages and can target and accumulate at the site of infection in vivo;the LMM NPs have the ability of drug slow release;the LMM NPs are feasible for the treatment of periprosthetic infections.Part Ⅲ.Experimental study of LMWF-loaded neutrophil membrane-coated nanoparticles for the treatment of periprosthetic infectionsObjective:To investigate the therapeutic efficacy of LMM NPs in the treatment of PJI.Methods:We used an animal model of PJI in rats to test the therapeutic effect of LMM NPs.We measured the changes of body weight and leg circumference on the molded side of the rats to assess the progress of infection;detected blood leukocyte count and neutrophil ratio to assess the treatment of infection;removed the prosthesis to coat the LB plate to detect the bacterial count on the surface of the prosthesis;micro-CT,H&E staining and Trap staining to assess bone destruction and bone repair;blood,liver and kidney function and organ H&E sections to assess the biosafety of nanoparticles;in vitro osteoclast Osteoclast induction intervention was performed to assess the inhibitory effect of nanoparticles on osteoclast activation.Results:1.After treatment with LMM NPs injection,the joint swelling,blood leukocyte count and neutrophil ratio of the modeled side of the rats were not significantly different from those of normal rats;micro-CT showed that the bone joint surface of the LMM NPs treated group was smoother and the bone volume was not statistically different from that of normal rats;H&E staining showed that the joint surface defects in the LMM NPs treatment group had been repaired;Trap staining showed that the number of Trap-positive cells decreased after LMM NPs treatment.2.The results of blood liver and kidney function tests showed that there were no significant abnormalities in ALT,AST,BUN and CRE;H&E sections showed that there was no significant damage to the major organs(heart,lung,liver,spleen and kidney).3.The number of osteoclast induction in vitro decreased with increasing nanoparticle concentration;the expression of IκBα increased,and the expression of p-IκBα,c-Fos and NFATc1 decreased.Conclusions: LMM NPs can treat PJI caused by MRSA,and can inhibit osteoclast activation and osteolysis while being effective antibacterial;LMWF may inhibit osteoclast activation by inhibiting NF-κB pathway activation. |
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