| Background:Periodontitis is a highly prevalent inflammatory oral disease caused by subgingival plaque and mediated by the host.Periodontitis is a serious threat to human physical and psychological health.Plaque biofilm is the initiating factor of periodontitis.Pathogenic plaque biofilm can lead to a significant destruction of periodontal tissue,bone resorption acceleration,and inhibition of host protective mechanisms.Etiologically,periodontal pathogenic bacteria stimulate immune cells to form hypersensitivity reactions and secrete many inflammatory factors to induce host reactions.Consequently,it caused the destruction of periodontal tissue and led to the occurrence of periodontitis.Therefore,there are two vexing issues in treating periodontitis:One is that the pathogenic plaque biofilm formed by periodontal pathogens is difficult to remove,and the other is that a large number of inflammatory factors produced are difficult to control.Mechanical debridement and antibiotic-assisted treatment are currently commonly used in clinical practice.However,mechanical debridement is mostly able to remove large pieces of inflammatory granulation tissue,and is difficult to thoroughly remove the plaque biofilm from complex periodontal pockets,especially deeper ones.In addition,long-term antibiotic treatment can lead to antibiotic resistance.Recently,antimicrobial photodynamic therapy（a PDT）and photothermal therapy（PTT）have attracted a great deal of attention because of their non-invasive,rapid bactericidal,and non-bacterial resistance advantages.The combination of a PDT/PTT has been used with good results in the antimicrobial field.However,there are still some problems with the combined use of a PDT/PTT:On the one hand,the dense structure that the plaque biofilm formed can shield the bacteria,and its component extracellular polymeric substance can greatly limit the diffusion depth of reactive oxygen species（ROS）and heat,resulting in the incomplete removal of plaque biofilms.On the other hand,the problem of immune hyperactivity caused by microorganisms in periodontitis remains unsolved.Gas molecules,as second messengers of inflammatory immunity,have the advantage of controlled release and efficient regulation and are a new tool for immune modulation.For example,nitric oxide（NO）not only has antibacterial and biofilm dispersing effects but can also modulate the over-activated NLRP3inflammasome in periodontal disease by affecting key proteins of NLRP3inflammasome assembly such as NLRP3 and caspase-1 through thiol nitrosylation.Objectives:In this study,a multimodal nanotherapeutic platform combining a PDT/PTT/NO release triggered by Near infrared light（NIR）was designed to address the challenge of treating deep infections in periodontal tissues.On the one hand,the multifunctional nanoplatforms have bacterial killing effects,prevent biofilm forming and promote biofilm dispersal;on the other hand,it exhibits anti-inflammatory and immunomodulatory capabilities.Furthermore,the potential mechanisms of antibacterial and anti-biofilm properties and the immunomodulatory pathways of the multifunctional nanoplatforms involved in periodontal inflammation were deeply investigated.Methods:In Chapter 2,GNRs@m Si O2 photothermal carriers were first prepared,and the NO donor S-nitrosothiol（SNO）was modified on their surface.Finally,indocyanine green（ICG）was uploaded to construct GNRs@m Si O2-SNO/ICG nanoplatforms with integrated a PDT/PTT/NO release multifunction.The characterizations of multifunctional nanoplatforms were confirmed by transmission electron microscopy（TEM）,energy-dispersive X-ray mapping,Fourier transform infrared spectroscopy,and UV-vis absorption spectroscopy.After that,in vitro photothermal properties,photodynamic properties,and NO release properties were tested and the stability of the nanoplatforms in a simulated microenvironment was then assessed.Finally,the biosafety of the nanoplatforms was tested.In Chapter 3,the effect of each treatment group on bacterial membrane permeability was first investigated by detecting nucleic acid leakage at OD260.The inhibitory effect of each treatment group on the biofilm formation of single species and the eradication effect on mature biofilms of single species were then explored by live/dead staining and colony-forming unit（CFU）counts.Next,the mature biofilm eradication of multiple species was investigated by fluorescence in situ hybridization,CFU counts,TEM,and scanning electron microscopy.Finally,the potential mechanisms of antibacterial and anti-biofilm effects of the nanoplatforms were explored at the genetic level by quantitative real-time polymerase chain reaction（q PCR）.In Chapter 4,the anti-inflammatory and immunomodulatory properties of the GNRs@m Si O2-SNO/ICG NPs multifunctional nanoplatforms were investigated at the molecular and protein levels by q PCR,immunofluorescence（IF）staining,and Western Blot,respectively.In Chapter 5,the photothermal properties in vivo were first evaluated.Then the in vivo antibacterial properties of the multifunctional nanoplatforms were evaluated by CFU counts.Finally,the in vivo anti-inflammatory properties of the nanoplatforms were verified by small animal fluorescence imaging,H&E staining,Masson staining,immunohistochemical staining（IHC）,and q PCR.Results:（1）GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms with good dispersion,stability,and biocompatibility were successfully constructed.After a 5 min NIR irradiation,the nanoplatforms temperature could reach 42.5 oC and the photothermal conversion efficiency was 52.76%.GNRs@m Si O2-SNO/ICG could reduce the absorption peak intensity of DPBF probes by about 60%,demonstrating ROS production.（2）GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms could alter bacterial membrane permeability to kill bacteria.A significant biofilm eradication effect of the multifunctional nanoplatforms was observed in live/dead bacterial staining images.In addition,the multifunctional nanoplatforms significantly down-regulated all tested adhesion molecule-related genes fim A,Hag A,Hag B,and virulence factor-related genes PPAD,Kgp,Rgp A,Rgp B,compared to the Control group（P<0.0001）.（3）q PCR,IF staining,and Western Blot results showed that GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms affected NLRP3 inflammasome assembly by inhibiting the expression of NLRP3 and caspase-1.In addition,NF-κB/p65 subunit nuclear translocation experiments showed that the multifunctional nanoplatforms could also indirectly affect pro-inflammatory factor expression and NLRP3 inflammasome activation by inhibiting NF-κB activation.（4）GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms could reduce bacterial CFU by approx.1.5 log in vivo experiments.Small animal fluorescence imaging and H&E staining showed reduced local inflammation and less inflammatory cell infiltration in the multifunctional nanoplatforms group.Masson staining showed no significant difference in collagen degradation in the multifunctional nanoplatforms group compared to the control group.Finally,IHC staining and q PCR findings revealed that the expression of inflammatory factors was down-regulated in the multifunctional nanoplatforms group compared to the control group.Conclusions:（1）The GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms with integrated a PDT/PTT/NO release had good photothermal,photodynamic,and controlled NO release properties.（2）GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms had excellent functions of bacterial killing and biofilm formation inhibition.GNRs@m Si O2-SNO/ICG NPs inhibited biofilm formation by down-regulating the expression of adhesion molecule-related genes and virulence factor-related genes,as well as reducing the antigenicity of bacteria.（3）GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms affected the assembly of NLRP3 inflammasome by inhibiting the expression of NLRP3 and caspase-1,and indirectly affected the expression of pro-inflammatory factors by inhibiting the activation of NF-κB,thus acting as an anti-inflammatory and immunomodulatory role.（4）GNRs@m Si O2-SNO/ICG multifunctional nanoplatforms had good in vivo bactericidal effects and reduced periodontal inflammation by down-regulating the expression of inflammatory factors as well as reducing collagen degradation. |
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