Near‐Infrared Light Triggered Nanocomposites With Nitric Oxide Releasing For Photodynamic/Photothermal Complementary Therapy Against Periodontal Biofilm

Objective:Periodontal disease is an oral disease caused by plaque biofilm infection.It affects10%of the global population and is the leading cause of tooth loss in humans today.However,due to the complexity of root anatomy,natural resistance and antibiotic resistance of biofilm,traditional mechanical debridement and antibiotic removal methods of biofilm are not ideal.Gas therapy,as a new antibacterial therapy,has come into view.Nitric oxide（NO）gas molecule is also a hot research topic for its high bactericidal and dispersive biofilm effects.NO gas therapy and its multifunctional therapy are effective methods for removing biofilms.However,large-scale and controlled molecular transport of NO gas is currently a great challenge.In this study,we designed and synthesized nanocomposites for NO-assisted photothermal and photodynamic therapy under the control of 808 nm near-infrared light,aiming to solve the precise and controllable release of NO,and enhance the effect of photothermal and photodynamic therapy on anti-periodontal plaque biofilm and alleviate periodontal inflammation.Methods:The core-shell structure of Ag₂S@ZIF-90/Arg/ICG has been successfully developed and characterized by transmission electron microscopy（TEM）,Ultraviolet–visible spectroscopy（UV-vis）absorption spectrum,X-ray diffraction analysis（XRD）,Zeta potential,Fourier transform infrared spectroscopy（FT-IR）,N₂adsorption and X-ray photoelectron spectroscopy（XPS）.The ability of Ag₂S@ZIF-90/Arg/ICG to produce heat,reactive oxygen species（ROS）and NO at 808 nm near-infrared excitation was determined by infrared photothermal camera,1,3-diphenyl isobenofuran（DPBF）probe,9,10-anthracene diphenyl-bis（methylene）dimalonic acid（ABDA）probe and Griess method.Colony forming unit（CFU）count,live/dead staining and biofilm metabolic activity（MTT）were used to evaluate the antibiofilm effect in vitro.Real-time polymerase chain reaction（RT-PCR）was used to evaluate the effects of Ag₂S@ZIF-90/Arg/ICG on virulence factors of porphyromonas gingivalis and genes encoding cyclic diadenosine 3,5-monophosphate（c-di-AMP）synthetase and decomposase.The treatment effect of Ag₂S@ZIF-90/Arg/ICG was analyzed by Hematoxylin-Eosin（H&E）staining,Masson staining and immunofluorescence staining in animal models of biofilm infection of periodontium pathogens.In addition,near infrared imaging technology was used to verify that Ag₂S@ZIF-90/Arg/ICG can be used for oral administration of near infrared region II imaging.Results:1.Ag₂S@ZIF-90/Arg/ICG is a core-shell structure with a diameter of about 50 nm.It is not only successfully loaded with excellent biosafe NO donor L-arginine,but also successfully loaded with ICG,which will achieve the controlled generation of NO,a PTT,and a PDT achieved by a single 808 nm NIR excitation.2.Ag₂S@ZIF-90/Arg/ICG is capable of generating heat,reactive oxygen species（ROS）and NO under the control of 808 nm near-infrared light.It has good biosafety when the concentration of Ag₂S@ZIF-90/Arg/ICG is not higher than 50μg m L^-1.3.In vitro,CFU counting,live/dead bacterial staining and MTT demonstrated that Ag₂S@ZIF-90/Arg/ICG had excellent anti-biofilm effects.Further investigation by RT-PCR confirmed that NO produced by Ag₂S@ZIF-90/Arg/ICG under 808 nm NIR could activate the decomposing enzyme of c-di-AMP,which triggered the decomposition of biofilms and enhanced the bactericidal effect of ROS and heat.4.In the animal model of periodontal pathogen biofilm infection,Ag₂S@ZIF-90/Arg/ICG had the best anti-biofilm effect with CFU decreased by 3-log,and pro-inflammatory factor IL-1βwas most significantly down-regulated while anti-inflammatory factor Arg-1 was most significantly up-regulated.This proved that Ag₂S@ZIF-90/Arg/ICG also had good anti-biofilm and anti-inflammatory effects in vivo.Conclusion:We have successfully prepared a novel nanocomposite with NO synergistic a PTT and a PDT.It had an outstanding therapeutic effect in treating deep tissue biofilm infection.This study not only enriches the research of compound therapy with NO gas therapy,but also provides a new solution for other biofilm infection diseases.