Novel N-Acetyl-L-Cysteine Carbonyl Polymer Dots Regulate Tissue Homeostasis In Periodontitis Via The Keap1/Nrf2 Axis

As the sixth largest public health problem in the world,periodontitis is a chronic inflammatory disease characterized by the destruction of periodontal connective tissue and gradual resorption of alveolar bone.As the major cause of tooth loss in adults,periodontitis not only affects the local health of the oral cavity but is also closely related to many systemic diseases.Therefore,active treatment of periodontitis is important to maintain oral and general health.Conventional treatment of periodontitis consists mainly of mechanical treatment as well as tissue regeneration surgery for severe periodontitis,supplemented by medication.Although the treatments mentioned above alleviate the clinical symptoms of periodontitis to varying degrees,due to the limitations of the different therapies,there is an urgent need for scholars to develop a safe and effective treatment to overcome the shortcomings of traditional periodontitis treatments.Numerous studies have confirmed that oxidative stress plays an important role in the pathogenesis of periodontitis.In the microenvironment of chronic periodontitis,the local accumulation of excess reactive oxygen species（ROS）not only causes direct damage through lipid peroxidation,protein denaturation,and DNA damage but also indirect damage to periodontal tissues through the signaling pathways and transcription factors that regulate the immune-inflammatory response.Removal of excess ROS and restoration of redox balance in periodontal tissues is considered to be an effective means of treating periodontitis,so scholars have identified the removal of excess ROS as a new target for periodontitis treatment.Carbon dots（CDs）have become the preferred nanomaterials for biomedical applications due to their small particle size,good water solubility,chemical inertness,good biocompatibility,simple synthesis method,and easy modification.However,carbon dots with ROS scavenging effect have been rarely studied in periodontitis,and even less explored in terms of their mechanism.We designed and synthesized carbonized polymer dots（CPDs）with ROS scavenging effect to treat periodontitis and to investigate the regulation of oxidative stress-related pathways.In the first part of the experiment,NAC-CPDs were prepared by solvothermal method using citric acid,N-Acetyl-L-Cysteine（NAC）,and formamide as raw materials.The prepared NAC-CPDs were characterized for their physicochemical properties.Transmission electron microscopy observed that the NAC-CPDs were uniformly distributed in size and nearly spherical in morphology,with an average diameter of 3.8 nm,and excellent water solubility.Fluorescence spectroscopy analysis showed that NAC-CPDs have red light characteristics and possess in vivo fluorescent tracer properties.Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy showed that the NAC-CPDs consisted of the elements C,N,O,and S,with abundant oxygen-containing and doped sulfur functional groups.The total antioxidant capacity assay revealed that NAC-CPDs exhibited excellent antioxidant properties.The kinetic assay for H₂O₂scavenging revealed that NAC-CPDs did not possess catalase-like activity,whereas the electron spin resonance spectroscopy assay revealed that NAC-CPDs had excellent·OH scavenging ability.Therefore,NAC-CPDs function mainly as antioxidants.In the second part of the experiment,NAC-CPDs biosafety and imaging properties were assessed by in vitro cellular assays and in vivo animal experiments.CCK-8 evaluated the toxicity of NAC-CPDs on human periodontal ligament cells（h PDLCs）,and the results preliminarily indicated that NAC-CPDs have good biological safety.In vivo,animal experiments demonstrated that NAC-CPDs have good biosafety by evaluating body weight,liver function indexes,blood routine indexes,and H&E staining of important organs.The imaging of NAC-CPDs within h PDLCs was observed by laser confocal microscopy,and the results showed that NAC-CPDs were able to enter the cells and distribute in the cytoplasm,and their red fluorescence was consistent with the results of fluorescence spectroscopy.Meanwhile,the red fluorescence of NAC-CPDs overlapped with the green fluorescence of the mitochondria-targeting dye Mito-green,which proved that NAC-CPDs could reach the mitochondria to exert their antioxidant function.Finally,the imaging characteristics and metabolism of NAC-CPDs in mice were evaluated by in vivo imaging in small animals.For the first time,it was found that NAC-CPDs could be enriched in the maxilla,which was more beneficial for the treatment of periodontitis.NAC-CPDs were mainly metabolized by the liver and kidney and excreted from the body through feces and urine.The above results indicate that NAC-CPDs have good biological safety and imaging function.In the third part of the experiment,the antioxidant,anti-inflammatory and bone-enhancing abilities of NAC-CPDs were evaluated in vitro by h PDLCs.Firstly,it was experimentally found that NAC-CPDs could effectively restore the H₂O₂-induced deformation of h PDLCs and the decrease in cell viability.Secondly,H₂O₂and E.coli endotoxin stimulated h PDLCs as exogenous and endogenous ROS,respectively.DCFH-DA results showed that NAC-CPDs were able to effectively scavenge ROS from different sources.Immunofluorescence assay revealed that NAC-CPDs effectively reduced the fluorescent high expression of inflammatory factors NF-κB and TLR4,and demonstrated good antioxidant and anti-inflammatory effects.Finally,the effects of NAC-CPDs on the osteogenic differentiation of h PDLCs were evaluated by ALP and ARS,and the results showed that NAC-CPDs had a good osteogenesis effect on h PDLCs under oxidative stress,but had no significant effect on the osteogenesis of h PDLCs under physiological state.In the fourth part of the experiment,the mouse periodontitis model was constructed by ligation to evaluate the therapeutic effect of NAC-CPDs on periodontitis in mice and to explore the mechanism.The results of Micro-CT showed that NAC-CPDs could effectively alleviate the resorption of alveolar bone in periodontitis mice and improve the indicators of bone parameters.The results of H&E staining,MT staining,and TRAP staining showed that NAC-CPDs could significantly improve the local inflammatory state of periodontal tissue and collagen fiber formation as well as the resorption of alveolar bone in periodontitis mice.The results of H&E staining and TRAP staining showed that NAC-CPDs could significantly improve local inflammation,collagen fiber formation,and alveolar bone resorption in periodontitis mice.The effect of NAC-CPDs on osteogenesis-related proteins was evaluated by IHC,and the results showed that NAC-CPDs promoted the expression of osteogenesis-related proteins OPN and RUNX2 in periodontitis mice.Next,the effect of NAC-CPDs on inflammatory indicators was assessed by IHC and ELISA.IHC results showed that NAC-CPDs could reduce the expression of TNF-αin periodontal tissues of periodontitis mice,and ELISA results revealed that NAC-CPDs could effectively reduce the levels of LPS and inflammatory factors TNF-α,IL-6,and IL-1βin the blood.The blood routine results showed that NAC-CPDs reduced the level of inflammatory cells in the blood.These results suggest that NAC-CPDs can effectively alleviate the local and systemic inflammatory state of periodontal tissues.Finally,to elucidate the antioxidant therapeutic mechanism of NAC-CPDs in periodontitis,the present study examined Keap1 and Nrf2,important regulators of oxidative stress,and the IHC results showed that NAC-CPDs effectively increased the expression of Nrf2and suppressed the expression of Keap1 in periodontal tissues under the inflammatory state.Meanwhile,NAC-CPDs effectively reversed the expression of oxidative stress-related indexes in serum,and NAC-CPDs played an antioxidant role by regulating the Keap1/Nrf2 axis,which not only alleviated local inflammation and alveolar bone resorption in periodontal tissues but also ameliorated the systemic oxidative stress and inflammation status.To summarize,in this study,novel red light NAC-CPDs with ROS scavenging efficacy were successfully prepared with good ex vivo biosafety and imaging characteristics,which could effectively improve the local and systemic oxidative stress and inflammatory state of periodontal tissues in periodontitis mice via the Keap1/Nrf2 axis.