Quercetin-loaded Ceria Nanocomposites Potentiate Dual-directional Immunoregulation Via Macrophage Polarization Against Periodontal Inflammation

Background:Periodontal disease was a chronic inflammatory disease that was initiated and progressed by dysbacteriosis of dental plaque and mediated by the host immune defenses.It is reported that severe periodontal disease is the 6^th most prevalent disease,with a totaled prevalence of 11.2%,therefore it made a detrimental effect on the life quality of patients.Considering the pathogenesis of periodontitis,the exposure of periodontal tissues to the microbiome would activate the host innate immune system and incur a defense response,resulting in the release of pro-inflammatory cytokines,thereby giving rise to the damage of tissues around the teeth.Mechanical debridement is the most dominant method to eliminate pathogenic microbiome and alleviate the recolonization of bacteria.However,it is only partially effective for the majority of patients suffered from periodontal disease,since the existance of bacterial was only considered as the initiating factor.Once the host immune defense starts,it is hard to control the biological response to microbial challenge causing destruction upon the periodontal tissues.Therefore,it is crucial to develop host-modulation therapies for the treatment of periodontal disease.Macrophages,as the first line of host immune-defense,play important role in the initiation and progression of periodontitis which can be classified to classically phenotype（M1 type）and alternatively phenotype（M2 type）.Classically M1macrophages mainly involve in pro-inflammatory response in periodontal disease,inducing the host defense response against pathogens via producing high level of inflammatory cytokines which subsequently stimulating periodontal tissue destruction and inducing the production of tissue-degrading proteinases.However,alternatively M2 macrophages mainly exhibits prominent anti-inflammatory and pro-healing effect.Recently,more and more evidence testified that periodontitis is closely relevant to the increased ratio of M1/M2 polarized macrophages and a decrease in the M1/M2 is essential for periodontitis treatment.Therefore,mediated macrophage polarization from M1 to M2 phenotype is the promising target of periodontitis treatment strategy.Objective:A novel nanocomposite（CeO₂@QU）which combine ceria nanoparticles（CeO₂）and quercetin was prepared.The effect of CeO₂@QU on macrophage polarization was compared with CeO₂ in order to explore its anti-inflammatory property.Methods:1.The octahedron CeO₂ was synthesized via hydrothermal method which was subsequently modified with 3-aminopropyltriethoxysilane（APTES）in order to load quercetin.2.The nanocomposite CeO₂@QU was texted via scanning electron microscopy（SEM）,transmission electron microscope（TEM）,Zeta potential,X-ray diffractometry（XRD）,Fourier transform infrared spectrometer（FTIR）and X-ray photoelectron spectroscopy（XPS）.3.The biosafety of CeO₂@QU was estimated via CCK-8 assay and real time cell viability assay（RTCA）.4.In terms of in vitro experiments,Enzyme activities of CeO₂@QU and CeO₂ were detected via total antioxidant kit in order to compare the anti-oxidant property of the new developed nanocomposite with CeO₂.In gene level,cytokines related to M1 type macrophage including tumor necrosis factor-α（TNF-α）,Interleukin-6（IL-6）and Interleukin-1β(（IL-1β）and M2 phenotype macrophage-associated cytokines such as transforming growth factor-β（TGF-β）,arginase-1（Arg-1）and Interleukin-10（IL-10）were detected via q PCR.Besides,flow cytometry and immunofluorescence tests were employed to further evaluate the effect of CeO₂@QU on macrophage differentiation and its anti-inflammatory activity in protein level.NF-κB pathway was selected to explore the anti-inflammatory mechanism of nanocomposite.5.In vivo,hematoxylin-eosin staining（H＆E）,Masson staining and immunohistochemistry（IHC）experiments were utilized in order to estimate the effect of CeO₂@QU on inflammatory inhibition via macrophage differentiation compared with CeO₂,quercetin and the mixture of CeO₂and quercetin.Result:1.The average diameter of CeO₂@QU was around 120 nm,and the well drug loading efficiency of quercetin was 7.91%.2.CeO₂@QU,which exhibited negligible toxicity with the concentration lower than 50μg/m L,possessed well biosafety.3.In terms of in vitro tests,CeO₂@QU exhibited the most prominent M1 macrophage inhibition and simultaneously,was able to make up the deficiency of single CeO₂ in promoting M2 macrophage polarization.4.In vivo experiments demonstrated compared with single CeO₂,quercetin and CeO₂+QU,CeO₂@QU possessed the most eminent anti-inflammatory and rehabilitated effect via successfully promoting macrophage polarization from M1 type to M2 type（P<0.05）.Conclusion:In this study,the chemical bonding of plants-derived quercetin with outstanding anti-oxidant activity to the surface of CeO₂ is realized through APTES.The as-prepared nanocomposite exerts an improved ROS scavenging activity and can effectively enhance the inhibition effect on M1 polarization simultaneously promote M2 type macrophage polarization due to the bonding of quercetin.Therefore,it has the capacity to potentiate dual-directional immune-regulation via shifting macrophage from M1 to M2 type against periodontitis.This new-developed nanocomposite with well-biocompatibility will become a promising candidate in periodontitis treatment due to its ability to elevate the inflammation suppression as well as promoting tissue repair and regeneration.