Novel Twin-crystal Nanosheets With MnO₂ Modification To Combat Bacterial Biofilm Against Periodontal Infections Via Multipattern Strategies

Background:Periodontal disease is one of the most common chronic oral diseases,which is caused by plaque attached to the surface of teeth and regulated by the host itself,resulting in oral connective tissue destruction,bone tissue absorption,and tooth loss in severe cases.As plaque is the initial factor in the occurrence and development of periodontal disease,the primary goal of periodontal disease prevention is to inhibit the accumulation and retention of periodontal disease-related bacterial biofilm on the tooth surface.However,as a complex multi-bacterial symbiotic environment,own characteristics of oral cavity make it difficult to achieve this goal.For stubborn bacterial biofilm,the traditional treatment is the use of antibiotics and mechanical debridement.Due to the special physiological structure,such as deep periodontal pocket or uneven root structure,the effect of mechanical debridement is not as expected.Meanwhile,due to the abuse of antibiotics and the enhancement of bacterial drug resistance,it is difficult to treat periodontal disease.Therefore,the emergence of nano-sized drugs and new antibacterial strategies have greatly promoted the prevention and treatment of periodontal disease.However,single-pattern antibacterial strategies such as photodynamic therapy and photothermal therapy show unsatisfactory results in the fight against periodontal bacterial biofilm.For example,a single photodynamic therapy can produce a variety of ROS,showing a great bactericidal effect,but for mature bacterial biofilm,ROS is difficult to penetrate into its inner cloth to play a bactericidal role.Meanwhile,a single chemodynamical therapy is also limited in dealing with stubborn bacterial infections.For example,the concentration of H₂O₂ in the inflammatory environment is low,which cannot effectively catalyze the production of hydroxyl radicals,seriously hindering its therapeutic effect on bacterial infections.Therefore,there is an urgent need to develop a nano-drug that integrates multi-mode antibacterial strategies to achieve the effect of efficient anti-biofilm and free bacteria,so as to treat stubborn periodontal diseases more effectively.Objective:A kind of composite nanoparticles（Cu S/Mn S@Mn O₂）was prepared,which consists of copper sulfide（Cu S）and manganese sulfide（Mn S）twin crystal（Cu S/Mn S）as the core and manganese dioxide（Mn O₂）as the core.The effect of anti-biofilm and killing free bacteria in the treatment of oral periodontal bacterial infection was studied in vivo and in vitro.Methods:1.Hexagonal sheet twin crystals（Cu S/Mn S）were synthesized by an improved one-step hydrothermal method and modified by redox reaction to encapsulate manganese dioxide（Mn O₂）.2.The nanocomposites were characterized by scanning electron microscope（SEM）,transmission electron microscope（TEM）,ultraviolet spectrum,an X-ray diffraction pattern（XRD）,X-ray photoelectron spectroscopy（XPS）,ultraviolet-visible spectrophotometer（UV-vis）and so on.3.The material properties of Cu S/Mn S@Mn O₂ were evaluated by in vitro experiments(infrared photothermal camera,inductively coupled plasma emission spectroscopy（ICP-AES）,fluorescence staining,and dissolved oxygen meter,including photothermal conversion ability,·OH production ability,and oxygen production capacity.4.The biosafety and cytotoxicity of Cu S/Mn S@Mn O₂ were evaluated by in vitro experiments（CCK-8,cell staining,and RTCA）,in vivo experiments（Hype staining of important tissues and organs）,and hemolysis test.5.The in vitro experiments（live/dead staining,colony forming unit（CFU）count,biofilm metabolic activity,and FISH）were used to explore the killing effect of Cu S/Mn S@Mn O₂ on multi-species biofilm and internal bacteria formed by pathogenic bacteria（Porphyromonas gingivalis（P.gingivalis）,Fusobacterium nucleatum（F.nucleatum）and Streptococcus gordonii（S.gordonii））.The scavenging effect of Cu S/Mn S@Mn O₂ on the extracellular DNA（e DNA）of bacteria was evaluated by fluorescence staining experiment.Evaluation of the regulatory effect of Cu S/Mn S@Mn O₂ on bacterial virulence factors by polymerase chain reaction（Polymerase Chain Reaction,PCR）.6.The therapeutic effect of Cu S/Mn S@Mn O₂ on periodontal disease and the changes of inflammatory factors caused by sterilization were evaluated by in vivo experiments（gingival tissue hematoxylin-eosin staining,Masson staining,immunofluorescence staining）.Results:1.Copper and manganese in the Cu S/Mn S twin are uniformly distributed in the hexagonal frame,and manganese dioxide（Mn O₂）is uniformly wrapped in the outer layer of the hexagonal twin-crystal,forming a stable composite nanostructure.2.Cu S/Mn S@Mn O₂ has excellent photothermal performance,and the photothermal conversion rate is as high as 37.4%.Also,the CDT process can be triggered by Mn²⁺to generate a high level of·OH.Importantly,photothermal interaction demonstrations have an excellent promoting effect on the CDT process.Meanwhile,a large amount of oxygen is released through the Mn O₂ layer under physiological conditions.3.Cu S/Mn S@Mn O₂ has excellent biocompatibility and has no obvious cytotoxicity when the concentration of the drug is less than 15μg m L^-1.4.In the in vitro antibacterial experiment,the CFU of the Cu S/Mn S@Mn O₂experimental group decreased by about four orders of magnitude.At the same time,it showed an excellent scavenging effect on multi-strain biofilm.5.In the periodontal disease model experiment in vivo,Cu S/Mn S@Mn O₂nanocomposites can effectively kill bacterial pathogens,promote host autoimmune regulation,and alleviate local inflammation.Conclusions:In this study,a multi-functional collaborative integrated nano platform（Cu S/Mn S@Mn O₂）is developed.Core Cu S/Mn S twins crystal can effectively realize photothermal conversion and in situ heat transfer.The photothermal effect not only dissipated the bacterial biofilm but also enhanced the Mn²⁺-mediated CDT process.Meanwhile,·OH can destroy the important component of biofilm（e DNA）and form an effective synergistic effect with PTT to eliminate stubborn bacterial biofilm.Meanwhile,the shell Mn O₂ layer can alleviate the local anoxic microenvironment and regulate the species changes of oral microorganisms by producing a large amount of O₂.This work provides a new strategy for PTT/CDT/O₂ in the treatment of periodontitis and may show excellent potential in other infectious diseases caused by anaerobes.