Bacterial Toxin-responsive Nanoparticles For Detection And Synergistic Therapy Of Intracellular Bacteria

Bacterial infectious diseases seriously threaten human life and health,causing huge economic losses.Antibacterial drugs can control infection,but drug resistance increases and bacteria can invade host cells to form intracellular bacteria（ICB）,causing recurrent or chronic infections.Antibiotic therapy,as the most commonly used anti-ICB method,are currently limited due to the low bioavailability,high toxic side effect and bacterial resistance.Therefore,the design of non-antibiotic methods to treat ICB infections is of great significance in clinical research and applications.The diagnosis of bacterial infection is a prerequisite for ICB treatment.Sensitive,accurate and convenient diagnosis can realize on-demand treatment,improve the therapeutic effect and reduce bacterial resistance.Fluorescence imaging is widely used for bacterial detection due to its high selectivity,high sensitivity,low cost and simplicity.The design of nanoparticles（NPs）to achieve selective imaging and on-demand treatment has important scientific significance and practical value for ICB theranostics.Phenyl boronate modified-methylene blue probes（MP）and mannose-modified phosphatidylethanolamine（DOPE-PEG-Man）were synthesized.L-arginine（LA）was loaded into calcium peroxide（Ca O₂）NPs to obtain LA-Ca NPs.These NPs and MP-loaded liposomes were mixed to prepare LA-Ca@mLip-MP composite NPs by thin film extrusion for theranostics of ICB.The NPs could actively target macrophages through mannose mediation and were internalized into infected macrophages through endocytosis.Under the action of toxin secreted by bacteria in the infected cells,LA-Ca@mLip-MP NPs were broken down to rapidly release Ca O₂,MP and LA.The bare Ca O₂NPs could react with H₂O to generate H₂O₂and O₂,and MP was decomposed and activated by H₂O₂to generate methylene blue（MB）as a photosensitizer,which restored its fluorescence emission and photosensitivity properties for fluorescence imaging and cytotoxic singlet oxygen（¹O₂）generation.The generated O₂could also alleviate MB-mediated PDT-induced hypoxic conditions.Meanwhile,H₂O₂could induce the guanidine oxidation of LA to release NO to synergistically destruct ICB.By examining the antibacterial effects of different LA/MP molar concentration ratios,the results showed that when the LA/MP ratio was 1/2,the optimal synergistic antibacterial effect was achieved.The in vitro ICB model was established with S.aureus-infected macrophages,and the in vitro diagnosis and antibacterial effect showed that LA-Ca@mLip-MP NPs had good fluorescence imaging ability and antibacterial effect.The in vivo results showed that LA-Ca@mLip-MP could specifically perform fluorescence imaging in response to the toxin microenvironment of ICB infection,and could effectively remove ICB,reduce the inflammation level,and cause minimal side effects to blood,liver and kidney.These results prove the effectiveness and theranostic potential of LA-Ca@mLip-MP NPs in the precise imaging and therapy of ICB.