Injectable Temperature-sensitive Hydrogel Loaded With IL-36 Receptor Antagonist For The Relief Of Osteoarthritis

Objective Osteoarthritis(OA)is an inflammatory disease accompanied by synovial joint inflammation,and IL-36 plays an important role in this process.Local application of IL-36 receptor antagonist(IL-36Ra)can effectively control the inflammatory response,thus protecting cartilage and slowing down the development of OA.However,its application is limited by the fact that it is rapidly metabolized locally.The aim of this project is to construct a temperature-sensitive hydrogel system loaded with IL-36 receptor antagonist(IL-36Ra)and evaluate the controlled release of this drug-loaded temperature-sensitive hydrogel,as well as to study its effectiveness in the treatment of osteoarthritis.Methods:First,articular cartilage tissue,synovial fluid,and subpatellar fat pad from patients with different stages of knee osteoarthritis(KOA)were collected to detect and analyze the expression levels of IL-36 and IL-36 receptor antagonist(IL-36RN)by enzyme-linked immunosorbent assay(ELISA).Next,we designed and prepared temperature-sensitive PEG-PLGA-PEG hydrogel(IL-36Ra@Gel)system carrying IL-36 Ra and evaluated its basic physicochemical characteristics.The release curves were obtained by Elisa assay at different times to evaluate the controlled release ability of the system,and also to evaluate its biodegradability.In addition,rat chondrocytes were cultured in vitro to evaluate the biocompatibility of the IL-36Ra@Gel system.Meanwhile,RT-PCR was used to detect the expression levels of various biomarkers MMP-13,ADAMTS-5,Aggrecan and Collagen X in IL-36Ra@Gel-treated chondrocytes to evaluate the effect of the drug delivery system on cartilage matrix degeneration.Finally,we induced C57/B6 mice by medial meniscal destabilization(DMM)to establish a murine knee osteoarthritis model.Groups were treated with joint cavity injection,and knee specimens were collected from each group of mice after 8 weeks,and hematoxylin-eosin staining(H&E staining)and saffranin-fast-green staining(safranin O/Fast-Green)were performed,and the degree of knee osteoarthritis was scored to evaluate the effect of IL-36Ra@Gel on delaying the progression of knee osteoarthritis.ResultsThe results of ELISA testing of articular cartilage tissue,synovial fluid,and subpatellar fat pad showed that local IL-36 and IL-36 RN levels correlated with the clinical severity of knee osteoarthritis.Specifically,IL-36 was positively correlated with the severity of knee osteoarthritis in K-L grade 0-3,while IL-36 RN was negatively correlated with the severity of knee osteoarthritis.In contrast,the joint tissues affected by severe OA,i.e.,K-L grade 4knee tissues,all exhibited a substantial decrease in IL-36 and IL-36 RN.Next,a temperature-sensitive PEG-PLGA-PEG hydrogel(IL-36Ra@Gel)system carrying IL-36 Ra was designed and prepared,and its initial gel time,initial gel temperature,and elastic rheological properties were obtained.The IL-36Ra@Gel drug release curve was also plotted,indicating that this system can release the drug slowly over a longer period of time.Also degradation experiments showed that IL-36Ra@Gel could be largely degraded from the body within one month.The biocompatibility-related results showed that IL-36Ra@Gel had no significant effect on cell proliferation compared to the control group.Also,the expression of MMP-13 and ADAMTS-5 was lower in IL-36Ra@Gel-treated chondrocytes than in the control group,while the opposite was true for Aggrecan and Collagen X.After 8weeks of treatment by joint cavity injection,HE staining and staining with saffron solid green showed that the degree of cartilage tissue destruction in the IL-36Ra@Gel-treated group was less than that in the modeling group and also better than that in the drug-only and hydrogel injection groups,and no significant inflammation was observed with intact joint structure.Meanwhile,the joints of mice in the IL-36Ra@Gel treatment group had the most intact cartilage surface,small cartilage erosion thickness,and the lowest OARSI score and Mankins score among all groups.ConclusionWe designed and prepared an IL-36Ra@Gel temperature-sensitive hydrogel system and demonstrated its excellent controlled-release ability and biocompatibility,proposing a new strategy for treating osteoarthritis through intra-articular release of therapeutic drugs.the combination of IL-36 Ra and PEG-PLGA-PEG temperature-sensitive hydrogel can greatly maximize the therapeutic effect and prolong the treatment time,thus effectively delaying the the progression of degenerative changes in osteoarthritis,providing a new feasible non-surgical treatment for osteoarthritis.