基质金属蛋白酶响应性G4 PAMAM-IBU/GelMA水凝胶的构建及其特征研究  

Fabrication and characterization of matrix metalloproteinase-responsive G4 PAMAM-IBU/GelMA hydrogel

作　　者：蔡传栋 王非 崔文国 范存义[1] 刘珅[1] CAI Chuan-dong;WANG Fei;CUI Wen-guo;FAN Cun-yi;LIU Shen(Department of Orthopedics,Shanghai Sixth People's Hospital,Shanghai Jiao Tong University,Shanghai 200233,China;Department of Orthopedics,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai Institute of Traumatology and Orthopedics,Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases,Shanghai 200025,China)

机构地区：[1]上海交通大学附属第六人民医院骨科,上海200233 [2]上海交通大学医学院附属瑞金医院骨科,上海市伤骨科研究所,上海市中西医结合防治骨与关节病损重点实验室,上海200025

出　　处：《上海交通大学学报（医学版）》2021年第2期140-146,共7页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金面上项目(81772314);上海市教育委员会高峰高原学科建设计划(20191829)。

摘　　要：目的·构建负载第4代树枝状大分子聚酰胺-胺(generation 4 polyamindoamine,G4 PAMAM)/布洛芬(ibuprofen,IBU)复合物(G4 PAMAM-IBU)的基质金属蛋白酶(matrix metalloproteinase,MMP)响应性明胶(gelatin,Gel)水凝胶,并探究其体外特征。方法·将IBU粉末加入G4 PAMAM溶液中,经超声振荡得到G4 PAMAM-IBU复合物,利用紫外分光光度计检测饱和G4 PAMAM-IBU溶液中IBU的浓度,研究G4 PAMAM对IBU的增溶能力和G4 PAMAM-IBU复合物中IBU的释放。采用CCK-8试剂盒评估IBU与G4 PAMAM-IBU对大鼠成纤维细胞增殖的抑制能力。同时,为实现G4 PAMAM-IBU的按需释放,利用Gel与甲基丙烯酸酐的加成反应合成甲基丙烯酸酯明胶(methacrylate gelatin,GelMA),并在365 nm光交联下形成MMP响应性水凝胶,并将G4 PAMAM-IBU复合物包载于该水凝胶之中。扫描电子显微镜观察水凝胶的微观形态,紫外分光光度计测定水凝胶中药物的释放量。之后,将载药GelMA水凝胶与成纤维细胞共培养,采用CCK-8试剂盒与活/死细胞染色法评估水凝胶对成纤维细胞增殖的影响。采用单因素方差分析进行组间定量资料的比较。结果·IBU与G4 PAMAM成功组成复合物,并使IBU在水中的溶解度显著提高,且IBU在12 h内与G4 PAMAM逐渐解离并释放。与空白对照组相比,单纯的IBU浓度需达到300μg/mL才具有抑制成纤维细胞增殖的能力(P=0.023),而G4 PAMAM-IBU中IBU的浓度为100μg/mL时即可显著抑制成纤维细胞增殖(P=0.000)。所制备的G4 PAMAM-IBU/GelMA水凝胶经MMP处理后,内部孔隙增大,药物释放加快。同时,体外实验发现,与载IBU的GelMA水凝胶相比,G4 PAMAMIBU/GelMA水凝胶处理后的成纤维细胞活细胞减少,死细胞增多,对细胞增殖的抑制作用显著增强(均P=0.000);且加入MMP后,抑制作用进一步增强。结论·G4 PAMAM对IBU具有显著的增溶作用并可增强其药物效应;G4 PAMAM-IBU/GelMA水凝胶具有MMP响应性的药物缓释行为,并能够持续抑制成纤维细�Objective·To fabricate matrix metalloproteinase(MMP)-responsive gelatin(Gel)hydrogel loaded with generation 4 polyamindoamine(G4 PAMAM)/ibuprofen(IBU)polyplexes(G4 PAMAM-IBU),and investigate its characteristics in vitro.Methods·The G4 PAMAM was mixed with IBU powder to form G4 PAMAM-IBU polyplexes under ultrasound vortex.The concentration of IBU in the saturated G4 PAMAM-IBU solution was determined by ultraviolet spectrophotometer.The solubilization ability of G4 PAMAM for IBU and the release of IBU in G4 PAMAM-IBU were assessed.And the ability of IBU and G4 PAMAM-IBU to inhibit the proliferation of rat fibroblasts was verified by CCK-8 assay.Meanwhile,to achieve the on-demand release of G4 PAMAM-IBU,Gel and methacrylic anhydride were used to synthesize methacrylate modified gelatin(GelMA)through an addition reaction,MMP-responsive hydrogel was formed under 365 nm light,and the G4 PAMAM-IBU polyplexes were embedded in the hydrogel.The microscopic morphology of the hydrogel was observed by scanning electron microscope(SEM)and the amount of released IBU was measured by ultraviolet spectrophotometer.Then,drug-loaded GelMA hydrogel was co-cultured with fibroblasts,and the effect of hydrogel on proliferation of fibroblasts was evaluated by CCK-8 assay and live/dead cell staining.The differences in the quantitative data among the groups were analyzed by using one-way ANOVA.Results·G4 PAMAM-IBU was formed successfully,the solubility of IBU in water increased obviously,and G4 PAMAM-IBU could gradually dissociate within 12 h and thereby release IBU as well.Compared with the blank control group,the concentration of IBU alone needed to reach 300μg/mL to inhibit the proliferation of fibroblasts(P=0.023),while the concentration of IBU in G4 PAMAM-IBU was 100μg/mL to significantly inhibit the proliferation of fibroblasts(P=0.000).The SEM images and released IBU detection results showed that the internal porosity of the prepared G4 PAMAM-IBU/GelMA hydrogel increased and the release of the drug accelerated.Besides,the in vi

关 键 词：水凝胶 第4代聚酰胺-胺 布洛芬 肌腱粘连 

分 类 号：R686.1[医药卫生—骨科学]