Fundamental Study On The Construction Of Novel Polysaccharide Hydrogel Based On Macrophage Immune Response Mechanism For Diabetic Ulcer

ObjectiveWith the increasing prevalence of diabetes and its complications of diabetic foot ulcer,to improve the economic loss,amputation and other consequences,this study aims to synthesizing a kind of new functional hydrogel material,which similar to the extracellular matrix.Immune regulation of macrophage by interleukin 4(IL-4)-loaded hydrogel to induce the macrophage differentiate into selectively activated macrophages(M2 subtype),and M2 type macrophage secrete anti-inflammatory cytokines to play an anti-inflammatory effect to promote the rapid wound healing of diabetic foot ulcer.MethodWe designed and synthesized thiolated chitosan and maleic acid-grafted dextran with chitosan and dextran as the main raw material,and then obtained the novel functionalized composite hydrogel(CSDM hydrogel)by the Michael addition reaction.A series of hydrogels with different concentrations and different proportions were prepared by adjusting the content of mercapto groups and maleic acid.We used the nuclear magnetic resonance spectrometer and Fourier infrared spectrometer to analysis hydrogel basic structure.The microstructure of the hydrogels was observed by scanning electron microscope(SEM),and the swelling and rheological properties of the hydrogels were measured and analyzed.Meanwhile the biocompatibility of the hydrogels was evaluated by MTT assay.Based on the above-mentioned basic performance evaluation of hydrogels,the hydrogel was loaded with IL-4 drug,and the mouse mononuclear macrophage leukemia cells(RAW264.7)were intervened using IL-4-loaded hydrogel.We established five groups,namely normal control(NC)group,LPS group,IL-4 group,CSDM hydrogel group,and CSDM hydrogel+IL-4 group,respectively.Then the morphology of RAW264.7 cells of each group was observed and photographed records under the microscope.The total nitric oxide test kit was applied to detect the NO content in the supernatant of each group.The enzyme-linked immunosorbent assay(ELISA)method was used to detect the secretion of Tumor necrosis factor a(TNF-a)and interleukin-10(IL-10)in each group cells.And the expression of inducible nitric oxide synthase(iNOS)and Arginase-1(Arg-1)in each group were measured by Real-time quantitative polymerase chain reaction(qPCR).ResultWe achieved 5 kinds of proportion of hydrogels by adjusted the content of mercapto groups and maleic acid,the molar ratio of mercapto group and double bond was 1:4,1:2,1:1,2:1 and 4:1,respectively marked CS1DM4 CS1DM2 CS1DM1 CS2DM1 and CS4DM1.The results of nuclear magnetic and infrared test proved that the hydrogel was successfully synthesized.The hydrogel was a three-dimensional porous structure with a pore size about 5?m by SEM,this structure of the hydrogel is the basic condition to play its function.At the same time this kind of porous structure made the swelling ratio of hydrogel rapidly rising in 60 min and achieved the swelling balance,which biggest swelling rate was 400%.The hydrogel swelling rates increased to 700% over the time,which proved the hydrogel has good hydroscopicity.Hydrogel rheological results showed that,the five hydrogels G' were greater than G" with the growing angular frequency(0.1~100rad/s),which indicating the stable formation of hydrogel.Importantly,the viability of NIH3T3 cells and RAW264.7 macrophages were over 80% after treatment with hydrogel(<4mg/mL)for 24 h and 48 h detected by MTT,indicating that the biocompatibility was well and the cytotoxicity was small.After the cells were intervened,the cell morphology of NC group was small and round,but the LPS group was abnormal with polygon and extended pseudopodia.IL-4 group,CSDM group and CSDM+IL-4 group had no obvious change of cell morphology.The NO test results showed that NO emission of CSDM+IL-4 group was significantly lower than that in CSDM group.ELISA assay indicated the secretion of TNF-a in CSDM+IL-4 group was significantly decreased than that in CSDM group;the secretion of IL-10 of CSDM+IL-4 group was obviously higher than that of CSDM group.The qPCR results showed that the relative expression of iNOS in CSDM+IL-4 group was significantly lower than CSDM group,while the expression of Arg-1 in CSDM + IL – 4 group significantly increased than that in CSDM group.ConclusionCSDM composite hydrogel has the basic conditions and good biocompatibility as the wound dressing,it not only provides an excellent moist environment for wound tissue,but also can absorbs inflammatory exudates.The IL-4-loaded hydrogel can successfully induced the macrophage differentiating into M2 macrophages,so that the secretion of anti-inflammatory cytokines IL-10 significantly increased,and the mRNA expression of Arg-1 also significantly increased,which all play the role of anti-inflammatory to promote the rapid healing of diabetic foot ulcers.