Investigation Of Symbiotic Biofilm Clearance By Self-Supplying H₂O₂ Nanocomposites Loaded In Bacterial Membrane Vesicles

The unique structure of the oral cavity allows microorganisms to colonize in the dental space and gingival crevice,forming symbiotic biofilms(dental plaque).It involves the participation of a variety of bacteria in the oral cavity in a certain sequence.For example,the initial colonizing bacteria accumulate in with the assistance of saliva and gingival crevicular fluids.Subsequently,the initial colonizing bacteria and subsequent bacteria interact with each other through nutrient transfer and/or with assistance of adhesins for mediation of the secondary colonization.Streptococcus gordonii is a typical initial colonizing bacterium.It forms a biofilm on the tooth surface and then provides adhesion sites and nutrients for the secondary colonization of Porphyromonas gingivalis(one of the main pathogens of periodontitis).However,physical clearance and systemic antibiotic therapies the commonly-used treatments against periodontitis suffer from low efficacy,risks of antibiotic resistance,and incomplete clearance.The chemodynamic therapies based on functional nanomaterials typically rely on nano-catalyst assisted Fenton or Fenton-like reactions to generate highly toxic hydroxyl radical(·OH)under acidic conditions for bacterial ablation.Inspired by the special colonization sequence of oral mixed biofilms,I developed a new strategy to clear periodontitis pathogens by preparing bacterial membrane vesicle loaded with selfsupplying H2O2 nanocomposites.In my thesis,the research contents include the following three parts:In the first part,ZnO2-Fe3O4 NPs nanoparticles were assembled with membrane vesicles of Streptococcus gordonii with the aqueous-phase extruding method,collecting the final product of ZnO2-Fe3O4@MV composite nanoparticles.The structure of ZnO2Fe3O4@MV was characterized by EDS mapping,XRD,XPS and FT-IR.The results of TMB chromogenic reactions verified generation of ·OH by the Fenton reaction with ZnO2-Fe3O4@MV NPs as low as 20 μg/mL(pH 5.0).The data of CFU counting and cytotoxicity experiments suggested that ZnO2-Fe3O4@MV NPs(100 μg/mL)can effectively inhibit proliferation of planktonic S.gordonii,while inducing minimal cytotoxicity on normal mammalian cells.In the second part,the data of confocal microscopy and flow cytometry validated an enhanced internalization of bacterial membrane vesicles in comparison to traditional liposomes.Selective targeting the parental bacteria by bacterial membrane vesicles was further demonstrated in my experiments.Effective ablation of planktonic S.gordonii or biofilms can be induced by ZnO2-Fe3O4@MV NPs.In addition,an ex vivo mixed biofilms of Streptococcus gordonii and Porphyromonas gingivalis were developed on the basis of mouse gingival tissue slices.In this new model,gingival tissue slices can provide not only nutrients for bacterial colonization,but also quasi-3D frameworks for the culture of mixed biofilms,thus serving a mimicking microenvironment for periodontitis pathogens better than bare glass substrates.ZnO2-Fe3O4@MV NPs were shown to enhance the elimination of mixed biofilms,in comparison to the control treatment of ZnO2-Fe3O4 NPs or tetracycline as a commonly-used antibiotic against periodontitis.In the third part,the effects of S.gordonii MVs and ZnO2-Fe3O4@MV NPs on macrophage RAW 264.7 were investigated,including their internalization patterns,cell polarization,proliferation,cytotoxicities,and antibacterial behaviors.The results revealed a remarkable M1-type polarization of RAW 264.7 cells induced by S.gordonii MVs or ZnO2-Fe3O4@MV NPs.When the concentration of ZnO2-Fe3O4@MV NPs was further increased to 40 μg/mL,the macrophages presented an elevated tendency for M2type polarization.Under the stimulation of S.gordonii MVs,macrophages were promoted for higher proliferation rates and enhanced antibacterial activities.These preliminary results revealed the impacts of bacterial membrane vesicles on the host immune cells,providing useful clues for optimization of nanocomposites against pathogenic biofilms.In summary,a new strategy of bacterial elimination was developed,inspired by the unique co-colonization of pathogens in the oral cavity.It features membrane vesicles of S.gordonii loaded with ZnO2-Fe3O4 NPs for highly effective destruction of oral mixed biofilms in a "Jenga" style,as evidenced by both in vitro and ex vivo experiments.The impacts of the nanocomposites on host immune cells were preliminarily investigated by detecting macrophages of RAW 264.7.It will foster advanced development of new designs and optimization of composite nanomaterials loaded in bacterial membrane vesicles in the fight against pathogenic biofilms.