Preparation Of Docosahexaenoic Acid Nanostructured Lipid Carriers And Experimental Study Of Their Anti-inflammatory Effects In Vitro

Background and Purpose: Peri-implantitis is a pathological condition occurring in the peri-implant tissues characterized by inflammation of the peri-implant connective tissue and progressive loss of supporting bone.The host immune response plays an important role in the development of peri-implantitis.The process involves specific recognition of lipopolysaccharide on the surface of Porphyromonas gingivalis by macrophages via Toll-like receptors,as well as the activation of macrophages into the M1 pro-inflammatory phenotype and the release of large amounts of pro-inflammatory cytokines such as Interleukin(IL)-1β,Tumor necrosis factor(TNF)-α,IL-6.This has a lytic and destructive effect on the supporting tissues around the implant,resulting in peri-implantitis.Studies have shown that Docosahexaenoic acid(DHA),an important component of cell membrane phospholipids,can bind to cell membranes and exert antibacterial,anti-inflammatory and anti-cancer effects by regulating gene expression and cell signaling.Numerous studies have demonstrated that DHA plays a positive role in oral-related inflammatory diseases.The current problems to be solved when applying DHA are: improving the stability of DHA during storage,solving the problem of easy oxidation of DHA,and making DHA more easily absorbed at the infection site.Nano-delivery systems,such as nano-emulsion,are an effective way to protect,transport and solve the problems of bioactivity and insolubility.Nanostructured lipid carriers(NLC),as the second generation of lipid nanoparticles,have the advantages of reduced drug toxicity,possess higher drug loading capacity,chemical diversity,and can be mass produced.The aim of this study is to prepare and characterize DHA-NLC using ultrasonication to investigate the biocompatibility of DHA-NLC and the modulation of macrophage inflammatory factor expression.Methods:1.NLC was synthesized from squalene,glycerol monostearate,Tween80 and propanetriol by ultrasonication.DHA-NLC was synthesized from DHA and NLC.Transmission electron microscopy and nanoparticle size potentiometry were applied to observe and measure the size,morphology,polydispersity index,zeta potential and stability of DHA-NLC.The infrared spectra of DHA-NLC were obtained by Fourier transform infrared spectroscopy.The encapsulation rate,drug loading and free radical scavenging rate of DHA-NLC were measured by UV-Vis spectrophotometer.2.RAW264.7 cells were pretreated with 25 μmol/L,50 μmol/L and 100 μmol/L of DHA-NLC medium solution for 2 h,and replaced with 1 mg/L of P.gingivalis-LPS to stimulate RAW264.7 cells for 24 h.The cytotoxicity of the drug was analyzed by CCK-8 assay.3.RAW264.7 cells were pretreated with 50 μmol/L DHA-NLC medium solution for 2 h,replaced with 1 mg/L P.gingivalis-LPS to stimulate RAW264.7 cells for 24 h.The cytotoxicity of IL-1β,IL-6 and the gene expression levels of TNF-αpro-inflammatory factors were determined by quantitative real-time PCR(q PCR).Results: 1.The average particle size of DHA-NLC was about 170.7±0.53 nm,the polydispersity index was 0.130±0.024,the zeta potential was-40.73±0.68 m V,the encapsulation rate was 78±2%,and the drug loading was 28±0.5%.Transmission electron microscopy showed sphere-like shape and Fourier transform infrared spectroscopy confirmed that DHA was successfully encapsulated in NLC.DHA-NLC has better antioxidant properties than DHA.DHA-NLC can be stored stably at 4 °C for 4 weeks(28 days).2.When the concentration of DHA-NLC was ≤100 μmol/L for 1 mg/L P.gingivalis-LPS stimulated RAW264.7 cells,no significant cytotoxicity was shown and the cell activity reached more than 90%.3.In the inflammation model of RAW264.7 cells stimulated by 1 mg/L P.gingivalis-LPS,50 μmol/L DHA-NLC could down-regulate the gene expression of inflammatory factors such as IL-1β,IL-6 and TNF-α,and the degree of down-regulation was better than that of DHA.Conclusions: 1.DHA-NLC with small particle size,good dispersion and high encapsulation rate was successfully synthesized and improved DHA easy oxidation characteristics.2.DHA-NLC has no toxic effects at concentrations ≤100 μmol/L.3.DHA-NLC has better inhibitory effect on the release of inflammatory factors compared with DHA.