载奥沙利铂类弹性蛋白多肽水凝胶的制备及其对小鼠结肠癌CT26细胞的杀伤作用

doi:10.13481/j.1671-587X.20230112

摘要/Abstract

摘要：

目的制备担载奥沙利铂（OXA）的类弹性蛋白多肽（ELPs）水凝胶，研究其体外药物释放情况及对小鼠结肠癌CT26细胞的杀伤作用。方法利用大肠杆菌（E.coli）系统表达ELPs原核蛋白，利用可逆相变循环法（ITC）进行蛋白纯化，将ELPs蛋白溶液与交联剂四羟甲基氯化磷（THPC）混匀制备水凝胶，扫描电子显微镜（SEM）观察水凝胶的超微结构，CCK-8法检测不同浓度ELPs水凝胶处理后CT26细胞和小鼠成纤维NIH3T3细胞存活率，活/死细胞染色法观察30和40 g?L^－1ELPs水凝胶作用48 h后CT26细胞存活情况。制备载OXA的ELPs水凝胶，检测其在不同pH值（6.5和7.4）缓冲液中的药物释放情况，CCK-8法检测加入载不同浓度（0.25、1.00、4.00、16.00和64.00 mg?L^－1）OXA的ELPs水凝胶浸泡液后各组CT26细胞存活率和处理不同时间各组CT26细胞存活率。结果凝胶电泳分析，E.coli诱导后在预计相对分子质量38 000附近出现明显加粗的蛋白条带，ITC纯化后泳道中无明显杂蛋白存在。ELPs蛋白溶液中加入THPC后由透明液体状态转变为不透明的白色水凝胶，SEM观察显示出排列整齐的孔隙和三维结构；CCK-8法检测，经不同浓度ELPs蛋白处理后CT26细胞和NIH3T3细胞存活率仍接近100%；活/死细胞染色，ELPs水凝胶处理后的CT26细胞在荧光显微镜下呈现明亮的绿色荧光。载OXA的ELPs水凝胶能在体外持续释放OXA，在pH值6.5条件下OXA的释放速度较pH值7.4条件下稍快；CCK-8法检测，载不同浓度OXA的ELPs水凝胶浸泡液呈剂量依赖性抑制CT26细胞增殖，且随孵育时间的延长CT26细胞存活率逐渐降低。结论载OXA的ELPs水凝胶具有持续释放OXA的特性，能够高效且持续地杀伤小鼠结肠癌CT26细胞，可作为一种安全有效的药物递送载体用于抗肿瘤研究。

关键词: 类弹性蛋白多肽, 水凝胶, 奥沙利铂, 结肠肿瘤, 药物递送

Abstract:

Objective To prepare the oxaliplatin （OXA）-loaded elastin-like polypeptides （ELPs） hydrogels， and to investigate its in vitro drug release and the killing effect in the CT26 colon cancer cells. Methods The prokaryotic proteins ELPs were produced in Escherichia coli （E.coli） system and purified via the inverse transition cycling （ITC）.The ELPs hydrogel was prepared by mixing the ELPs protein solution with the cross-linking agent tetrakis hydroxymethyl phosphonium chloride（THPC）.The microstructures of hydrogels were observed by scanning electron microscope （SEM）； CCK-8 assay was used to determine the survival rates of the CT26 cells and NIH3T3 cells after hydrogel treatment and Live/Dead cell staining was used to observe the survival of the CT26 cells after treated with 30 and 40 g?L^－1 hyrogels for 48 h. The OXA-loaded ELPs hydrogels were prepared， and the drug release of OXA-loaded hydrogels was detected at different pH values （pH 6.5 and pH 7.4） in the sustained release liquid. The survival rates of CT26 cells after added with the ELPs hydrogels soak solution loading different concentrations （0.25， 1.00， 4.00， 16.00， and 64.00 mg?L^－1） of OXA were detected and the survival rates of CT26 cells after treated for different time in various groups were detected by CCK-8 assay. Results The gel electrophoresis analysis showed that an obvious bold protein band was observed in the region with expected molecular weight 38 000. After protein purification using ITC， almost no other protein was found. The ELPs protein solution was transited from transparent solution into an opaque white hydrogel after the addition of THPC. The hydrogel showed uniformed pores and three-dimensional structure under SEM.The CCK-8 assay results indicated that the survival rates of CT26 cells and NIH3T3 cells were close to 100%. The Live/Dead cell staining results showed the green fluorescence in the CT26 cells under fluorescence microscope. The OXA-loaded hydrogels showed sustained drug release profile in vitro，and a slightly faster drug release in pH 6.5 was found than that in pH 7.4.The CCK-8 assay results showed that the OXA-loaded hydrogels could significantly inhibit the proliferation of CT26 cells in a dose-dependent manner. With the prolongation of the incubation time， the survival rates of CT26 cells treated with drug-loaded hydrogel were decreased gradually. Conclusion The OXA-loaded ELPs hydrogels have sustained release profile in vitro and can kill efficiently and consistenly the colon cancer CT26 cells of the mice. ELPs hydrogels may be used as the safe and effective drug delivery system for cancer therapy.

Key words: Elastin-like polypeptides, Hydrogels, Oxaliplatin, Colon neoplasms, Drug delivery

中图分类号:

R735.35

胡楠楠,杨馥旭,穆业腾,郭冲,薛晗,范宇鑫,郭峰霖,关新刚. 载奥沙利铂类弹性蛋白多肽水凝胶的制备及其对小鼠结肠癌CT26细胞的杀伤作用[J]. 吉林大学学报(医学版), 2023, 49(1): 94-100.

Nannan HU,Fuxu YANG,Yeteng MU,Chong GUO,Han XUE,Yuxin FAN,Fenglin GUO,Xingang GUAN. Preparation of oxaliplatin-loaded elastin-like polypeptides hydrogel and its killing effect on colon cancer CT26 cells[J]. Journal of Jilin University(Medicine Edition), 2023, 49(1): 94-100.

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