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

doi:10.3969/j.issn.1674-8115.2021.02.003

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (2): 140-146.doi: 10.3969/j.issn.1674-8115.2021.02.003

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

蔡传栋¹(), 王非², 崔文国², 范存义¹, 刘珅¹()

^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

收稿日期:2020-06-01 出版日期:2021-02-28 发布日期:2021-02-28
通讯作者: 刘珅 E-mail:caicd123@sina.com;liushensjtu@sjtu.edu.cn
作者简介:蔡传栋（1995—），男，硕士生；电子信箱：caicd123@sina.com。
基金资助:
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)

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

Chuan-dong CAI¹(), Fei WANG², Wen-guo CUI², Cun-yi FAN¹, Shen LIU¹()

^1.Department of Orthopedics, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China
^2.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

Received:2020-06-01 Online:2021-02-28 Published:2021-02-28
Contact: Shen LIU E-mail:caicd123@sina.com;liushensjtu@sjtu.edu.cn
Supported by:
National Natural Science Foundation of China(81772314);Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(20191829)

摘要/Abstract

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300 μg/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100 μg/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 PAMAM-IBU/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胞增殖。

关键词: 水凝胶, 第4代聚酰胺-胺, 布洛芬, 肌腱粘连

Abstract:

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 300 μg/mL to inhibit the proliferation of fibroblasts (P=0.023), while the concentration of IBU in G4 PAMAM-IBU was 100 μg/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 vitro results showed that compared with the IBU/GelMA hydrogel, there were less live cells and more dead cells in the G4 PAMAM-IBU/GelMA hydrogel-treated fibroblasts. G4 PAMAM-IBU/GelMA hydrogel had a greater proliferation inhibitory effect than the IBU/GelMA hydrogel. And the addition of MMP further enhanced the inhibitory effect on fibroblasts.

Conclusion·G4 PAMAM can significantly increase the solubility of IBU and improve its drug effect; the G4 PAMAM-IBU/GelMA hydrogel has a sustained drug release behavior responding to MMP and can inhibit the proliferation of fibroblasts sustainably.

Key words: hydrogel, generation 4 polyamindoamine (G4 PAMAM), ibuprofen (IBU), peritendinous adhesion

中图分类号:

R686.1

蔡传栋, 王非, 崔文国, 范存义, 刘珅. 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[J]. 上海交通大学学报(医学版), 2021, 41(2): 140-146.

Chuan-dong CAI, Fei WANG, Wen-guo CUI, Cun-yi FAN, Shen LIU. Fabrication and characterization of matrix metalloproteinase-responsive G4 PAMAM-IBU/GelMA hydrogel[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(2): 140-146.

图/表 4

图1 IBU与G4 PAMAM的结合与解离表征Note:A. Schematic representation of the combination of G4 PAMAM and IBU. B. Solubility increasement in water of IBU assisted by G4 PAMAM. C. The dissociation of G4 PAMAM-IBU polyplexes and the release of free IBU.

Fig 1 Characterization of combination and dissociation of G4 PAMAM-IBU

图2 MMP所致GelMA水凝胶的降解及G4 PAMAM-IBU/GelMA水凝胶中IBU的响应性释放Note:A. Schematic representation of G4 PAMAM-IBU/GelMA hydrogel formation and degradation under MMP. B. Microstructures of blank GelMA and G4 PAMAM-IBU/GelMA hydrogel with or without MMP treatment. C. Responsive release of IBU from G4 PAMAM-IBU/GelMA hydrogel with or without MMP treatment.

Fig 2 MMP-induced GelMA hydrogel degradation and MMP-responsive IBU release from G4 PAMAM-IBU/GelMA hydrogel

图3 G4 PAMAM与G4 PAMAM-IBU对成纤维细胞增殖的抑制作用Note:A. Inhibitory effect of G4 PAMAM on cell proliferation. ①P=0.023, ②P=0.004, ③P=0.001, compared with G4 PAMAM 0 μg·mL-1. B. Inhibitory effects of IBU and G4 PAMAM-IBU on cell proliferation.④P=0.023, ⑤P=0.000, compared with IBU 0 μg·mL-1.

Fig 3 Inhibitory effects of G4 PAMAM and G4 PAMAM-IBU on cell proliferation

图4 MMP响应性G4 PAMAM-IBU/GelMA水凝胶对成纤维细胞增殖的抑制作用Note:A. Live/dead cell staining of rat fibroblasts in different groups. B. CCK-8 analysis of rat fibroblast in different groups. ①P=0.000, compared with the IBU/GelMA group; ②P=0.001, ③P=0.000, compared with the IBU/GelMA+MMP group; ④P=0.000, compared with the G4 PAMAM-IBU/GelMA group.

Fig 4 Inhibitory effect of MMP-responsive G4 PAMAM-IBU/GelMA hydrogel on cell proliferation

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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

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