Preparation And Evaluation Of IL-1ra-loaded Microsphere For Inhibiting Periodontal Inflammation In Vitro

BackgroundPeriodontitis is a common oral disease caused by the loss of homeostasis between the resident symbiotic flora and the host.It is the main cause of tooth loosening,displacement and even loss,and it has a great impact on chewing,digestion and other systemic inflammatory diseases.Currently,effective treatments include scaling and root planning,flap surgery,guided bone regeneration and guided tissue regeneration.The reason for failure of periodontal treatment in clinical practice is often because of the uncontrollable inflammation.If the strong inflammatory response is gradually amplified,periodontal tissue will be further damaged.In the host-mediated immune inflammatory response,interleukin-1?(IL-1?)plays a key role.Interleukin-1 receptor antagonist(IL-1RA)blocks the biological activity of IL-1? by binding to the IL-1 receptor,without stimulating host cells or triggering any intracellular signaling,which is an excellent candidate for inhibiting periodontal inflammation.Studies have confirmed that intra-articular injection of water-soluble IL-IRA can effectively inhibit arthritis,but its short half-life limits it's clinical treatment effect.Therefore,searching a method to extend the biological activity of IL-1RA and to improve clinical efficacy has become urgent.In this study,IL-1RA microspheres were prepared using water-in-oil-in-water(W/O/W)and oil-in-water-in-water(S/O/W)methods respectively.We evaluated their physical and chemical properties and the ability to resist periodontal inflammation in vitro,expecting to provide a theoretical basis for the treatment of periodontitisMethod1.The dextran/PLGA&IL-IRA microspheres were prepared by S/O/W method,and PLGA&IL-1RA microsphere were prepared by W/O/W method as a control of traditional methods2.Observe the morphology and particle size of the microspheres by an inverted fluorescence microscope and a scanning electron microscope,and use the IL-1RA Elisa kit to detect the drug loading rate,encapsulation efficiency and release characteristics of the microspheres3.Take out the gum tissue that needs to be trimmed when the impacted tooth is clinically removed,aseptically extract human gingival fibroblast(HGFs),subculture,and identify by Immunohistochemistry4.The cytotoxicity of microsphere assays were conducted by using Cell Counting Kit-8.The HGFs were incubated with different formulations and various concentrations(0.1?1?10?100?1000?10000 ?g/mL)of drug-loaded PLGA microspheres for 24 h.The effect of microspheres on the proliferation of HGF cells was detected by microplate reader after 24 hours5.Detecte anti-inflammatory activity of microspheres in vitro:we used IL-1? to stimulate human gingival fibroblasts to establish an in vitro model of periodontitis Firstly,HGFs were seeded with different microspheres with or without IL-1RA for 3h and stimulated by IL-1? for 24 h.The target genes of IL-6,MMP-1 and Tumor Necrosis Factor(TNF-?)was determined by RT-qPCR.To explore the loog-term anti-inflammatory property of dextran/PLGA&IL-IRA microspheres in activated human gingival fibroblasts in vitro,IL-1RA conditioned media in different release intervals(0 to 3,3 to 7,7 to 10,10 to 14,14 to 17,and 17 to 21 days)were co-cultured with HGF cells with the stimulation of IL-1?.mRNA expression levels of IL-6,TNF-? and MMP-1 were measured using RT-qPCR.Result1.The dextran/PLGA&IL-IRA microspheres prepared by the S/O/W method were round with a smooth surface and a uniform size.The average particle diameter is 12.76±4.89 ?m,the encapsulation rate is about 67.02%.The dextran/PLGA&IL-IRA microspheres showed a nearly zero order release profile up to 50 days.They are better than PLGA&IL-1RA microspheres prepared by the W/O/W method,whose surface covered pores and the encapsulation rate and drug loading rate are low,with a burst release effect2.HGFs are long spindle-shaped,a few are angular or star-shaped.The nucleus is centrally located,and the growth curve is roughly an inverted "S" shape.The results of Immunohistochemistry staining were consistent with the characteristics of mesoderm-derived fibroblasts3.The microspheres prepared by two methods have no obvious cytotoxicity even at high concentrations of 10,000 ?g/mL(P<0.05)4.Co-culture of dextran/PLGA&IL-IRA microspheres or PLGA&IL-IRA microspheres with and HGFs resulted in a significant decrease in mRNA levels of IL-6,MMP-1,and TNF-?(P<0.05).When IL-1RA conditioned media collected in different release interval and IL-1? were co-cultured with HGFs,and the expression levels of inflammation-related genes were significantly lower than those in the IL-1?-stimulated group(P<0.01)Conclusion1.The dextran/PLGA&IL-IRA microspheres prepared by the S/O/W method have good sphericity,smooth surface,uniform size,high encapsulation efficiency and almost zero-order release release property.They are better than microspheres prepared by traditional W/O/W method2.The dextran/PLGA&IL-IRA microspheres have good biocompatibility3.The dextran/PLGA&IL-IRA microspheres can effectively inhibit the IL-1?-induced inflammatory response of HGF cells,and with the slow and long-term release of the drug,the anti-inflammatory effect is durable.