The Construction And Performance Of Smart ROS Controllable Ceria-based Nanocomposites

Periodontal disease was defined as a complex body’s inflammation response to bacterial plaque frequently causing the immunosuppression.The accumulation of plaque caused the endogenous immune response and secreted inflammatory factors,which led irreversible damage to periodontal tissues,even the teeth loss.Traditional scaling treatment with local antibiotics treatment would cause some problems,such as gum trauma and drug resistant bacteria,which limited the application.Nowadays,the antimicrobial photodynamic therapy（aPDT）was of great concern by the powerful antibacterial ability.In the aPDT progress,photo-activatable photosensitizers（PS）are able to instantaneously and abundantly generate reactive oxygen species（ROS）by photocatalysis under the irradiation of the excitation light and lead to the favorable therapeutic effect with a reduced potential to develop drug-resistant bacteria.The antibacterial effect of aPDT depended on the great amount of instantaneous ROS generation.However,the residual ROS would induce the high level of intracellular ROS,cause the accumulation of pro-inflammatory cytokines and subsequently lead the damage to proteins,nucleic acids,lipids,and ultimately triggering cell death and tissue damage.Therefore,during the aPDT treatment,after inactivating the bacteria by excess ROS,the local imbalance of redox homeostasis would be aggravated in the periodontal pocket,inflammatory cells would be recruited,and the macrophages would be changed to M1 polarization,which could cause the irreversible damage to the periodontal tissue.Correspondingly,ROS can not only kill bacteria but also cause inflammation,which is contradictory and seriously hinder the progress of aPDT clinical application.How to not only retain the bactericidal ability of aPDT,but also reduce the inflammation is of great significance to enhance the periodontitis treatment via aPDT.Analyzing the generation and mechanism of ROS from the perspective of time and space is the key to solving this contradiction.In this regard,a simple and“all in one”strategy was proposed in this work by involving superoxide dismutase（SOD）and catalase（CAT）function in aPDT system to deal with the problem of local massive ROS accumulation,that is introduced cerium oxide nanoparticles（CeO₂）in the plan.CeO₂NPs with different morphologies can exhibit dissimilar nano-enzymatic activity due to the proportion of Ce³⁺/Ce⁴⁺,and simulate the SOD and CAT activities by the transition of oxygen vacancies.In summary,this research designed a new type of multi-functional CeO2@Ce6NPs,which could achieve the antibacteria and anti-inflammation simultaneously.This nano-based platform could utilize the aPDT for antibacterial purpose in the first stage with red-light irradiation,and subsequently scavenge the residual ROS via nanoceria to modulate host immunity by down-regulating the M1 polarization（pro-inflammatory）of macrophages and up-regulating the M2 polarization（anti-inflammatory and regenerative）of macrophages.Moreover,the local ROS level induced by activated inflammation pathway could reduce the local oxidative stress and inflammatory response by aPDT,which could regulate the inflammatory microenvironment,protect the normal cells,and restore the regenerative potential of inflammatory surrounding tissues.In Chapter one,CeO₂ was aminated via 3-aminopropyltriethoxysilane（APTES）.The amino group was combined with the carbonyl group of Chlorin e6（Ce6）.Prepared different ratios of CeO₂ and Ce6.And then by comparing the basic morphology and ROS balance ability of CeO₂@Ce6 with different ratios,the ratio which met the requirements were screened out.The results proved that CeO₂@Ce6with medium proportion of Ce6（3 mg Ce6）could be accorded with the requirements of time-sequential antibacterial/anti-inflammatory effect,yield ROS production in the first 180 s,and ROS clearance of CeO₂ takes places after the nearly exhaustion of Ce6 in CeO₂@Ce6 nanocomposite,which can effectively avoid inflammation.In Chapter two,the physical and chemical properties of nanoparticles,evaluation of ROS regulation ability and biological safety were characterized.The results indicated that CeO₂@Ce6NPs took the stable octahedral CeO₂ as the core,and Ce6 covered the surface of CeO₂ via chemical conjugation.CeO₂@Ce6 NPs exhibited good chemical stability,SOD and CAT activities and biocompatibility.In Chapter three,the antibacterial properties of CeO₂@Ce6 nanoparticles against single-species biofilm and multi-species biofilm were tested,and it was proved that the aPDT of CeO₂@Ce6 nano-particles could be effective against the single species biofilm and multi-strain biofilms which were formed by periodontitis-related bacteria Streptococcus gordonii,（S.gordonii）,Porphyromonas gingivalis（P.gingivalis）,Fusobacterium nucleatum（F.nucleatum）.The results indicated that the nanoparticles had inherited the great antibacterial effect of aPDT and also can inhibit the expression of the virulence factor gene of the main pathogenic bacteriaP.gingivalis,effectively hindered bacterial colonization,and intervened the interaction between bacteria and the host.It has great prospects in the treatment of periodontitis.In Chapter four,according to the anti-inflammatory test of CeO₂@Ce6,it has been proved that CeO₂@Ce6 could inhibit the M1 polarization of macrophages and promote the M2 polarization,and inhibit the activation of the pro-inflammatory signaling pathway NF-κB signal.And by further formulating the stimulation condition medium to simulate the inflammation microenvironment,the effect of CeO₂@Ce6 on the osteogenic differentiation ability of MC3T3 cells under inflammation was tested,and the results proved that CeO₂@Ce6 could restore the potential of tissue regeneration.In Chapter five,according to the establishment of rat periodontitis model and mouse back wound inflammation model,we explored the effect of CeO₂@Ce6 in vivo,the results showed that CeO₂@Ce6 could play the time-sequential function of antibacteria and anti-inflammation to relieve the gingival inflammation.The positive expression of TNF-α,IL-1βand IL-6 in the CeO₂@Ce6 group were restrained significantly.The gene expression of M1-related pro-inflammatory factors was down-regulated,and the M2-related anti-inflammatory factors was up-regulated.And CeO₂@Ce6 also could accelerate the healing of inflammatory wounds.In summary,we successfully combined Ce6 molecule with aPDT function and nanoceria via chemical conjugation to form CeO₂@Ce6.Under the excitation of red light（630 nm）,the aPDT sterilization function is realized with instantaneously increased ROS.After the aPDT,the inner nano-octa-CeO₂NPs play the superior ROS-scavenging capacity to prevent the oxidative stress damage.Simultaneously,CeO₂@Ce6 could reverse the macrophage polarization from M1 to M2,change the microenvironment and recover the regeneration of surrounding tissues including alveolar bone.This simple nanocomposite overcomes the shortcomings of aPDT and paves the way for the clinical treatment of periodontal diseases in the future.