Upconversion Nanoparticle-Based Carvacrol Delivery System For The Treatment Of Periodontitis

Background:Periodontitis is the sixth largest chronic bacterial infectious disease in the world,affecting people of all ages,and the prevalence rate gradually increases with age.Periodontitis eventually leads to inflammatory destruction of periodontal support tissue and even tooth loss.Bacterial infection,biofilm formation,and immune dysfunction are common triggers and aggravator of periodontitis.At present,mechanical debridement combined with antibiotics is the preferred treatment,but it has disadvantages such as difficulty in effectively removing subgingival plaque,tissue damage,risk of postoperative complications,drug resistance and gastrointestinal reactions.Moreover,the complex tooth anatomy and deep periodontal pocket make antibiotics difficult to achieve satisfactory results.Carvacrol（CA）,a monomer of traditional Chinese medicine,dominates antibacterial,anti-inflammatory and immunomodulatory activities.Importantly,under blue light（BL）irradiation,CA can achieve specific anti-bacteria.However,CA has poor water solubility.In addition,BL has shallow tissue penetration and is cytotoxic after long-term irradiation.In contrast,near-infrared（NIR）light is an ideal light source with deep tissue penetration,low light absorption and good biological safety.Therefore,improving the water solubility of CA and using NIR as the light source to emit BL in the deeper tissue are two major difficulties in the study.Objective:Based on the optical characteristics of upconversion nanoparticles（UCNPs）that can upconvert NIR to emit BL,we wrapped the drug carrier mesoporous silica（mSiO₂,with good water solubility and large specific surface area）on the surface of UCNPs,loading CA to mSiO₂by electrostatic adsorption.And thus CA-based NIR light-responsive nano-drug delivery system（UCNP@mSiO₂-CA,UCMCs）was prepared for the first time.This system focused on solving the two difficulties mentioned above.Besides,we also explored the specific anti-bacteria,anti-inflammation,immunoregulation,periodontal restoration ability and biological safety,proving the feasibility of UCMCs with 808 nm NIR irradiation for the systemic treatment of periodontitis.Methods:1.The UCNPs with core–shell–shell structures were synthesized by the thermal decomposition method.Then the hydrophilic drug carrier UCNP@mSiO₂（UCMs）was synthesized.Under dark conditions,UCMs were further stirred in an ethanol solution containing CA,and UCMCs were obtained by electrostatic adsorption.The performance and biological safety of UCMCs were verified by material characterization,drug loading rate,drug release,cell compatibility in vitro,metabolic distribution in vivo and H&E staining of main organs.2.To screen the concentration of UCMCs and the irradiation parameters of NIR in vitro,the effects of UCMCs with NIR irradiation were explored as follows:（1）Antibacterial activity against the planktonic bacterial and biofilm of Porphyromonas gingivalis（the main periodontal pathogen,anaerobes）,Staphylococcus aureus and Escherichia coli.（2）The ability to regulate the phenotype of macrophages（RAW264.7 cells）,exerting anti-inflammatory and immunoregulatory functions,and the molecular mechanism of immune regulation was explored by RNA-seq and verified by the Western blot（WB）assay.3.The Sprague-Dawley（SD）rat model of periodontitis was constructed.Local injection of UCMCs followed by NIR irradiation was used as experimental treatment and compared with minocycline cream（a first-line clinical drug）.The systemic therapeutic effects of UCMCs with NIR irradiation on periodontitis were verified by clinical index evaluations,plate counts of periodontal bacteria,Micro-CT scanning analyses,histopathological staining,blood routine tests,blood biochemical tests and other experiments.Results:1.The results of SEM and TEM showed that the UCNPs with the core–shell–shell structures were monodisperse hexagonal with an average diameter of about 32 nm.The fluorescence emission spectrum displayed that UCMs could upconvert NIR（808 nm）to emit BL.When the concentration of CA was 300.0μg mL^–1,and the reaction was stirred for 6 h,UCMs could obtain a drug loading rate of about 14.50%（w/w）.In various media,UCMCs rapidly released about 80.00%of CA in the first 6 h.What’s more,UCMCs possessed good cell compatibility.And in vivo fluorescence imaging of Kunming mice showed that UCMCs were mainly metabolized by the liver and kidney.The results of H&E staining also showed no obvious organ toxicity,which supported the good biocompatibility of UCMCs in vivo.2.The treatment parameters of three kinds of bacteria and RAW 264.7 cells were obtained in vitro.Moreover,（1）according to the results of anti-planktonic bacteria and anti-biofilm experiments,UCMCs with NIR irradiation destroyed the integrity of bacterial membrane and biofilm,increased production of reactive oxygen species and aggravated bacterial protein leakage.But it was noteworthy that there was no obvious toxic effect on human gingival epithelial（HGE）cells.These results suggested that UCMCs with NIR irradiation had highly effective and specific antibacterial effects.（2）In the inflammatory cell model,UCMCs with NIR irradiation could reduce the expression of pro-inflammatory factors from the level of the mRNA and protein,promoting the transformation to M2 type RAW 264.7 cells,and thus playing the role of anti-inflammatory and immune regulation.The RNA-seq results showed that many inflammatory immune-related signal pathways,such as the MAPK signal pathway,the TNF signal pathway and the IL-17 signal pathway,were enriched to reshape the immune microenvironment.And the crucial role of the MAPK signal pathway was also verified by the WB assay.3.The periodontitis model of SD rats was successfully constructed.UCMCs with NIR irradiation had the properties of lower the clinical index score,decreasing the content of periodontal plaque,alleviating the loss of alveolar bone,easing the periodontal inflammation,preventing the progression of the periodontitis,promoting the reattachment of the periodontal ligament,and thus facilitating the tissue repair of periodontitis,which was similar with the therapeutic effect of minocycline cream.Moreover,blood routine,blood biochemistry and H&E staining of main organs showed no abnormality.Conclusions:1.UCMCs have been successfully prepared with adequate performance,good cell compatibility and biosafety in vivo.2.UCMCs with NIR irradiation have the characteristics of specific anti-bacteria（including anaerobic bacteria）,anti-inflammation and immunomodulation.And the MAPK signal pathway is the main anti-inflammation and immune regulation mechanism.3.From the animal level,the periodontitis model of SD rats verifies that UCMCs with NIR irradiation can achieve the systemic therapeutic effect of anti-bacteria,anti-inflammation,immunomodulation and tissue repair in periodontitis.And UCMCs with NIR irradiation also have the advantages of high efficiency and safety,which provides an effective and reliable strategy for the various potential application of deep tissue diseases.