Application Of Self-adaptive Theranostics Nanosystem In The Prevention And Treatment Of Implant-related Infections

As medical technology and biomaterials continue to advance,more and more medical implants are being used in surgical procedures.However,the number of surgical complications is increasing every year,and one of the most common and serious complications after biomaterial implantation is implant-related infections(IRIs),which can lead to implant failure and even disability and death.Staphylococcus aureus(S.aureus)is the most common causative agent of IRIs.As a bacterium widely present in human skin and hospital environments,S.aureus can enter the surgical site during the perioperative period to colonize the surgical site and cause early infection around the implant,or it can cause delayed infection from other sites through hematogenous spread to the implant area.In addition,lactate produced by S.aureus biofilms can inhibit histone deacetylase 11(HDAC11)activity in macrophages,thereby increasing the transcriptional levels of anti-inflammatory cytokines such as interleukin-10(IL-10).These cytokines contribute to a shift in the polarization state of macrophages from an M1 pro-inflammatory phenotype to an M2anti-inflammatory phenotype,which favors the survival of pathogenic bacteria and predisposes to the recurrence of infection.Therefore,the development of novel strategies for complete biofilm clearance and reversal of immunosuppression is an urgent and important clinical problem for the prevention and treatment of IRIs.Objective: This experiment aims to design a phase change material-based self-adaptive theranostics nanosystem Ca Alg/LOD/POD@MNO/FAs(CLPM)and investigate its characterization,temperature-controlled drug release performance,enzymatic response performance,cytocompatibility,in vitro antibacterial performance,effect on macrophage polarization and mechanism,and in vivo use for treatment and prevention of implant-related infections.Methods:(1)The self-adaptive theranostics nanosystem CLPM was synthesized by mechanical stirring and ionic cross-linking,and the structure and morphology of CLPM were characterized by laser particle sizer analysis,transmission electron microscopy(TEM)observation,Fourier transform infrared spectroscopy(FTIR)detection,fluorescence labeling and confocal laser scanning microscopy(CLSM)observation.(2)The stability of CLPM was verified by observing TEM images after multiple phase change cycles,detecting particle size changes in serum-containing medium for7 days,and measuring zeta potential.The temperature responsiveness of CLPM was verified by in vitro temperature-controlled drug release assay.The degradation ability of CLPM to lactic acid was verified by enzymatic reaction assay,and the cytocompatibility of CLPM was demonstrated by cell morphology observation and CCK8 assay.(3)The antibacterial effects of CLPM on on the planktonic and biofilm states of S.aureus at 37°C and 39°C were investigated by bacterial live/dead staining,spread plate method,microstructure observation,and rheological performance assay using flow cytometry,scanning electron microscope(SEM),TEM,CLSM,and rheometer,respectively.(4)By using different treated S.aureus biofilm supernatants co-cultured with macrophages,markers of different phenotypes of macrophages(i NOS: M1;Arg-1:M2)were observed under CLSM using immunofluorescence staining method,while different cytokines(IL-6,TNF-α,IL-10 and TGF-β)in macrophage supernatants were measured by enzyme-linked immunosorbent assay(ELISA)to determine the effect of CLPM on macrophage polarization in vitro.In addition,a mouse subcutaneous implant-related infection model was established,and immunofluorescence staining was performed on the peri-implant tissues of mice after different treatments to determine the effect of CLPM on macrophage polarization in vivo.(5)A mouse subcutaneous implant-related infection model and a rat intra-tibial implant-related infection model were established to study the therapeutic effect of CLPM on IRIs in vivo.(6)A mouse subcutaneous implant-related infection recurrence model was established to study the preventive effect of CLPM on infection recurrence in vivo and the effects on the ratio of memory B cells,effector B cells,serum Ig G and IL-1βlevels.Transcriptomic analysis of CLPM-treated macrophages was performed to investigate the mechanism of CLPM in promoting immune memory.Total protein extraction was performed on peri-implant tissues of the CLPM-treated groups in a mouse subcutaneous implant infection model to study the expression of proteins related to key signaling pathways by Western blot.Results: In this experiment,we have completed the synthesis of CLPM,a self-adaptive theranostics nanosystem,and verified that it has an excellent core-shell structure with good stability,dispersion,temperature responsiveness,lactate degradation ability,and cytocompatibility.CLPM has excellent in vitro anti-planktonic bacterial and anti-biofilm properties,and can reverse the polarization direction of macrophages in vivo and in vitro to convert them to the M1 phenotype.Animal studies have demonstrated that CLPM can effectively treat implant infections and prevent the recurrence of implant infections.Transcriptomic studies demonstrated that the promotion of immune memory by CLPM may originate from the upregulation of co-stimulatory molecules and pro-inflammatory cytokine expression,and several signaling pathways involved in antigen presentation and processing,such as NOD-like receptor signaling pathway,TNF signaling pathway and MAPK signaling pathway,were activated.Protein immunoblot analysis of the titanium periplasmic tissues from CLPM-treated mice showed increased levels of JNK and p38 phosphorylation and expression of pro-inflammatory cytokines IL-6,IL-1β and TNF-α,further verifying the promoting effect of CLPM on related signaling pathways in vivo.Conclusion: CLPM can effectively clear biofilms,reverse immunosuppression and promote immune memory both in vitro and in vivo,showing great potential for application in the prevention and treatment of implant-related infections.