Glucose-Driven Transformable Complex Eliminates Biofilm And Alleviates Inflamm-aging For Diabetic Periodontitis Therapy

Periodontitis,the sixth major complication of diabetes,is closely related to tooth loss and more serious systemic diseases（including Alzheimer’s disease,atherosclerosis and various cancers）.Due to the hidden location of dental plaque biofilm in the periodontal pocket and the presence of its substances such as extracellular polysaccharides,proteases,and extracellular DNA,dental plaque biofilm has an inhibitory effect on the diffusion of foreign drugs.Therefore,current clinical surgical and drug therapy cannot completely eliminate dental plaque biofilm.In addition,the high glucose concentration in the periodontal tissue of diabetes patients will directly stimulate macrophages and fibroblasts,leading to a serious inflamm-aging state,thus affecting the efficacy of traditional therapy.Objective:We developed a glucose-driven transformable complex consisting of a rigid ZIF-8 core and an elastic calcium alginate（Ca Alg）shell.The complex was loaded with glucose oxidase/catalase（GOx/CAT）for glucose reduction and minocycline（MINO）,named Ca Alg@MINO/GOx/CAT/ZIF-8（CMGCZ）,was used for diabetic periodontitis.Upon entering the dental plaque biofilm,GOx/CAT decomposed local glucose to produce gluconic acid,which could dissolved ZIF-8 core of the complex,allowing the elastically flexible Ca Alg shell to wrap GOx/CAT and MINO.Based on the pore size of the surrounding dental plaque biofilm,the complex could transform and penetrated deeply into the biofilm.On the contrary,when exposed to the oral environment,the ZIF-8 core of the CMGCZ did not dissolve,led to no nanotoxicity and no release of MINO.The Ca Alg shell could protect GOx/CAT from degradation and adsorption by proteases and other substances within the biofilm,thus reducing local glucose concentration,while allowing the small molecule minocycline to diffuse freely out of the composite and exert its bactericidal effect within the biofilm.Methods:（1）GOx/CAT and MINO were loaded into ZIF-8 through a"one-pot"synthesis method and then cross-linked with sodium alginate after Ca²⁺adsorption to form Ca Alg@GOx/CAT/MINO/ZIF-8（CMGCZ）.（2）The structure and morphology of CMGCZ were characterized by transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,Fourier transform infrared spectroscopy（FT-IR）,X-ray diffraction（XRD）,confocal laser scanning microscopy（CLSM）and flow cytometry.（3）The morphological changes of CMGCZ at different glucose concentrations were observed using TEM,SEM,and CLSM.The enzyme activity and MINO release rate of CMGCZ at different glucose concentrations and different environments were detected using glucose content detection reagent kits,hydrogen peroxide content detection reagent kits and an enzyme-linked immunosorbent assay（ELISA）.（4）The phagocytic clearance effect of macrophages on the complex was observed using CLSM.The cytotoxicity of the complex was evaluated through CCK8 assay.The ability of CMGCZ to alleviate macrophage pyroptosis,macrophage senescence and paracrine senescence was assessed using Western blotting,Edu assays and SA-β-gal assays.（5）The therapeutic effect of CMGCZ on diabetic periodontitis in vivo was evaluated using animal modeling.The efficacy of CMGCZ and its ability to correct periodontal tissue functional disorders were evaluated using tissue slicing,micro-CT,and immunofluorescence staining methods.Results:（1）The TEM,SEM,and Mapping results of CMGCZ proved the successfully constraction of"core-shell"structure of CMGCZ.FT-IR and XRD results indicated that GOx/CAT and MINO were successfully encapsulated in the ZIF-8 core without affecting its internal structure.The results of co-location experiments also verified the exist of the"core-shell"structure of CMGCZ.（2）TEM and SEM revealed that CMGCZ could gradually transformed from a regular to an irregular shape as glucose concentration increased,indicating that the ZIF-8 core of CMGCZ degraded with increasing glucose concentration.The glucose clearance capacity of CMGCZ at different concentrations was tested,and the results showed that CMGCZ could successfully consume glucose in high glucose concentrations.In addition,CMGCZ also maintained a high glucose clearance ability in the presence of protease K（PK）,demonstrating the protective effect of the Ca Alg shell.CMGCZ released MINO most efficiently in high glucose environments also verified the glucose-driven degration of ZIF-8.（3）CLSM showed that macrophages had low phagocytic clearance of CMGCZ.The CCK8 results showed that CMGCZ had low cytotoxicity.Western blot,Edu and SA-β-gal experiments successfully demonstrated that CMGCZ effectively alleviated macrophage apoptosis,macrophage aging,and paracrine aging.（4）Animal experiments confirmed the significant therapeutic effect of CMGCZ on diabetic periodontitis in vivo.Micro-CT and immunofluorescence staining results showed that CMGCZ promoted alveolar bone regeneration and corrected the dysfunction of periodontal tissue.Conclusions:This study designed a glucose-driven transformable complex（CMGCZ）for the treatment of diabetic periodontitis.CMGCZ can consume glucose and produce glucuronic acid,leading to the degradation of the rigid ZIF-8 core.The residual Ca Alg shell allows the complex to transit from non-flexible to flexible,and penetrate into the deeplayers of the dental plaque biofilm.The remaining Ca Alg shell also protects GOx/CAT and MINO from electrostatic chelation and enzyme degradation effects within the dental plaque biofilm.During transformation,minocycline is continuously released and gradually penetrates deeply into the dental plaque for sterilization.Additionally,the glucose clearance capacity of CMGCZ also alleviate the pyroptosis and senescence of macrophage,reduce pro-inflammatory cytokine secretion and correct functional disorders of periodontal tissues.