双载药丝蛋白水凝胶的构筑及其释药性能研究

doi:10.6023/A21050203

摘要/Abstract

可注射水凝胶因其可以长时间在肿瘤病灶部位缓慢释放药物及避免外科手术风险等而备受关注. 本工作将载有盐酸阿霉素(DOX)的介孔硅分子筛SBA-15/DOX与混有血管阻断剂康普瑞汀磷酸二钠盐(CA4P)的再生丝蛋白(RSF)水溶液均匀混合后, 根据丝蛋白能在多种促进条件下易形成β-折叠构象并成为物理交联点/区域的特性, 通过超声法制得了具有两种不同药物释放行为的(RSF/CA4P)-(SBA-15/DOX)可注射水凝胶. 其中快速释放的CA4P, 与肿瘤血管的内皮细胞中β-微管蛋白的秋水仙碱结合位点结合, 破坏已生成的肿瘤血管, 进而阻断对肿瘤细胞的氧气输送和营养输送; 而长时间持续缓慢释放的DOX, 将抑制肿瘤细胞的生长. 具有不同药物释放动力学的良好组合, 使得(RSF/CA4P)-(SBA-15/DOX)水凝胶在较长时间内对人乳腺肿瘤细胞(MDA-MB-231)的增殖有较强的抑制作用, 同时达到了降低DOX的使用剂量但仍能保持高效的抗肿瘤细胞作用的目的. 另外, 此类水凝胶具有可塑性和可注射性, 也能在暗场下发出相应的荧光, 这为其在生物医疗方面的应用带来了极大的便利.

关键词: 生物大分子, 丝蛋白, 水凝胶, 可注射性, 双药物释放行为

Injectable hydrogels, as an effective vehicle for localized drug delivery, have attracted increasing attention in recent years. With improved tumor-specific drug accumulation and lowered risk of infection, the application of in situ-forming injectable hydrogels provides an alternative to surgical implantation. In this study, based on a unique feature of regenerated silk fibroin (RSF) that it can easily form β-sheet structures, which function as physical cross-links, under various conditions, we designed an injectable silk fibroin hydrogel which encapsulated SBA-15 for the co-delivery of a vascular disrupting agent, combretastatin A4 disodium phosphate (CA4P), and doxorubicin hydrochloride (DOX). Such a CA4P and DOX co-encapsulated hydrogel ((RSF/CA4P)-(SBA-15/DOX)), simply prepared through a designed ultrasonication procedure, showed two different types of drug release behavior. CA4P was rapidly released, and it not only targeted the abnormal vasculature of tumors, causing vascular collapse, but also inhibited tumor cell proliferation by binding to tubulin and arresting mitosis. On the other hand, the sustained release of DOX can inhibit the proliferation of tumor cells for a prolonged period of time. Because of the rapid disruption of tumor vasculature and sustained inhibition of tumor cells proliferation, the (RSF/CA4P)-(SBA-15/DOX) hydrogel showed significantly enhanced cytotoxicity against human breast carcinoma cells (MDA-MB-231). By using this co-delivery system, the required dose of DOX can be lowered, and at the same time, the superior antitumor efficacy of the drugs is maintained. Our results suggest that the application of silk fibroin hydrogels to the co-delivery of a vascular disrupting agent and a chemotherapeutic agent with different drug release behaviors is a promising strategy in tumor treatment. Moreover, after sonication, the hydrogel precursor of (RSF/CA4P)-(SBA-15/DOX), can reach target irregular-shaped tumor sites via intratumor injection, and the hydrogels then form in situ, simply induced by body heat. Such silk fibroin-based hydrogels are injectable and malleable and fluorescent in dark field, which greatly facilitates their biomedical applications.

Key words: biomacromolecule, silk fibroin, hydrogel, injectable, dual-drug release behavior

引用此文

王苏杭, 孙灵娜, 曹涵, 钟一鸣, 邵正中. 双载药丝蛋白水凝胶的构筑及其释药性能研究[J]. 化学学报, 2021, 79(8): 1023-1029.

Suhang Wang, Lingna Sun, Han Cao, Yiming Zhong, Zhengzhong Shao. Development of a Dual-drug-loaded Silk Fibroin Hydrogel and Study on Its Drugs Release Behaviors[J]. Acta Chimica Sinica, 2021, 79(8): 1023-1029.

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图/表 8

图1. 双载药水凝胶按顺序释放药物的机理图

Figure 1. Schematic representation of the sequential drug release in dual drugs loaded hydrogel

图2. 冻干RSF-(SBA-15)水凝胶断面的SEM图

Figure 2. SEM images of the cross section of the lyophilized RSF-(SBA-15) hydrogel

图3. RSF和RSF-(SBA-15)(其中SBA-15的质量分数为20%)水凝胶的流变学行为. (a) 在37 ℃条件下, RSF和RSF-(SBA-15)水凝胶的储能模量(G')与频率之间的关系. (b) 在37 ℃条件下, RSF和RSF-(SBA-15)水凝胶的储能模量(G')与应变之间的关系

Figure 3. The rheology characterization of the RSF hydrogel and the RSF-(SBA-15) (w(SBA-15)=20%) hydrogel. (a) Storage modulus (G') versus frequency for the RSF hydrogel and the RSF-(SBA-15) hydrogel at 37 ℃. (b) Storage modulus (G') versus strain for the RSF hydrogel and the RSF-(SBA-15) hydrogel at 37 ℃

图4. (RSF/CA4P)-(SBA-15/DOX)水凝胶的制备图示. 该水凝胶具有可注射性, 而且在暗场下能自发荧光

Figure 4. Illustration of the preparation of the (RSF/CA4P)- (SBA-15/DOX) hydrogel. The hydrogel was injectable, and fluorescent in dark field

图5. 37 ℃时, 在0.01 mol/L, pH 7.4的PBS溶液中, (a) (RSF/CA4P)-(SBA-15/DOX)水凝胶中CA4P和DOX的体外释放情况; (b) RSF-(SBA-15/DOX)和(RSF/CA4P)-(SBA-15/DOX)水凝胶中DOX的体外释放情况和在含有5 U/mL蛋白质水解酶情况下DOX的体外释放情况

Figure 5. In vitro drug release in 0.01 mol/L, pH 7.4 PBS at 37 ℃. (a) CA4P and DOX release from the (RSF/CA4P)-(SBA-15/DOX) hydrogel; (b) DOX release from the RSF-(SBA-15/DOX) and (RSF/CA4P)- (SBA-15/DOX) hydrogels with or without 5 U/mL protease XIV

图6. HUVEC细胞的免疫荧光实验. 在37 ℃, 5% CO2的条件下, HUVEC细胞与不同水凝胶共培养12 h或24 h, 于激光共聚焦显微镜下成像, 标尺20 μm

Figure 6. Immunofluorescent staining of HUVEC cells. The HUVEC cells were imaged at 12 h or 24 h post-incubation with different hydrogels at 37 ℃ under an atmosphere of 5% CO2. Scale bar, 20 μm

图7. 通过CCK-8法测试不同水凝胶在体外的细胞毒性. 在第4天、第9天和第14天时, 将每个孔中的培养液吸弃, 分别加入1 mL含有2×104个细胞(第4天), 5×104个细胞(第9天), 1×105个细胞(第14天)的DMEM-FBS(含胎牛血清的细胞培养液)溶液.

Figure 7. In vitro cytotoxicity studies of different hydrogels were examined as a function of incubation time via CCK-8 assay. At the 4th, 9th, 14th day, the media of each well was removed and replenished with 1 mL DMEM-FBS containing 2×104 cells (at the 4th day), 5×104 cells (at the 9th day), 1×105 cells (at the 14th day)

图8. 水凝胶作用于肿瘤细胞的详细图示

Figure 8. Illustration representation of the detail cell experiment of hydrogels treated tumor cells

参考文献 37

[1]	Qian, Q.; Wang, D.; Shi, L.; Zhang, Z.; Qian, J.; Shen, J.; Yu, C.; Zhu, X. Biomaterials 2021, 265, 120403. doi: 10.1016/j.biomaterials.2020.120403
[2]	Feng, Q.; Zhang, K. Y.; Li, R.; Bian, L. M. Acta Polym. Sin. 2021, 52, 1. (in Chinese)
	( 冯茜, 张琨雨, 李睿, 边黎明高分子学报, 2021, 52, 1.)
[3]	Cui, S. Q.; Yu, L.; Ding, J. D. Acta Polym. Sin. 2018, (8), 997. (in Chinese)
	( 崔书铨, 俞麟, 丁建东, 高分子学报, 2018, (8), 997.)
[4]	Lei, N.; Gong, C. Y.; Qian, Z. Y.; Luo, F.; Wang, C.; Wang, H. L.; Wei, Y. Q. Nanoscale 2012, 4, 5686. doi: 10.1039/c2nr30731f pmid: 22875402
[5]	Li, Y.; Hou, H.; Zhang, P.; Zhang, Z. Drug Deliv. 2020, 27, 1044. doi: 10.1080/10717544.2020.1785049
[6]	Gong, H.; Chen, Y.; Yu, X.; Xiao, H.; Xiao, J.; Wang, Y.; Shuai, X. Chinese J. Polym. Sci. 2019, 37, 1224. doi: 10.1007/s10118-019-2272-6
[7]	Jiang, J.; Shen, N.; Ci, T.; Tang, Z.; Gu, Z.; Li, G.; Chen, X. Adv. Mater. 2019, 31, 1904278. doi: 10.1002/adma.v31.44
[8]	Wu, M.; Yang, W.; Chen, S.; Yao, J.; Shao, Z.; Chen, X. J. Mater. Chem. B 2018, 6, 1179. doi: 10.1039/C7TB03113K
[9]	Wei, X.; Liu, L.; Guo, X.; Wang, Y.; Zhao, J.; Zhou, S. ACS Appl. Mater. Interfaces 2018, 10, 17672. doi: 10.1021/acsami.8b04163
[10]	Zhao, G.; Sun, Y.; Dong, X. Langmuir 2020, 36, 2383. doi: 10.1021/acs.langmuir.9b03722
[11]	Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G.; Watson, N.; Kiziltepe, T.; Sasisekharan, R. Nature 2005, 436, 568. doi: 10.1038/nature03794
[12]	Aw, M. S.; Addai-Mensah, J.; Losic, D. Chem. Commun. 2012, 48, 3348. doi: 10.1039/c2cc17690d
[13]	Lian, B.; Wei, H.; Pan, R.; Sun, J.; Zhang, B.; Wu, J.; Li, X.; Tian, G. Int. J. Nanomed. 2021, 16, 457. doi: 10.2147/IJN.S283793
[14]	Yang, W. J.; Zhou, P.; Liang, L.; Cao, Y.; Qiao, J.; Li, X.; Teng, Z.; Wang, L. ACS Appl. Mater. Interfaces 2018, 10, 18560. doi: 10.1021/acsami.8b04394
[15]	Zhu, J.; Hu, M.; Qiu, L. J. Pharm. Pharmacol. 2017, 69, 844. doi: 10.1111/jphp.12725
[16]	Yuan, H.; Li, X.; Tang, J.; Zhou, M.; Liu, F. Cancer Imaging 2019, 19, 71. doi: 10.1186/s40644-019-0257-x
[17]	Zhu, J.; Xu, X.; Hu, M.; Qiu, L. J. Biomed. Nanotechnol. 2015, 11, 997. doi: 10.1166/jbn.2015.2010
[18]	Dark, G. G.; Hill, S. A.; Prise, V. E.; Tozer, G. M.; Pettit, G. R.; Chaplin, D. J. Cancer Res. 1997, 57, 1829. pmid: 9157969
[19]	Wu, L.; Qiu, L. J. Mater. Chem. B 2016, 4, 760. doi: 10.1039/C5TB02468D
[20]	Wang, Y.; Tang, Z. H. Acta Polym. Sin. 2021,doi: 10.11777/ j.issn1000-3304.2021.21024. (in Chinese) doi: 10.11777/ j.issn1000-3304.2021.21024
	( 王月, 汤朝晖, 高分子学报, 2021,doi: 10.11777/ j.issn1000-3304.2021.21024.) doi: 10.11777/ j.issn1000-3304.2021.21024
[21]	Ma, Z.; Yan, X.; Zhao, L.; Zhou, J.; Pang, W.; Kai, Z.; Wu, F. J. Agric. Food Chem. 2016, 64, 746. doi: 10.1021/acs.jafc.5b05119
[22]	Liu, L.; Mason, R. P.; Gimi, B. Cancer Lett. 2015, 356, 462. doi: 10.1016/j.canlet.2014.09.038 pmid: 25305449
[23]	Wang, Z.; Chui, W.; Ho, P. C. Pharm. Res. 2011, 28, 585. doi: 10.1007/s11095-010-0308-2
[24]	Zhang, Y.; Wang, J.; Bian, D.; Zhang, X.; Zhang, Q. Eur. J. Pharm. Biopharm. 2010, 74, 467. doi: 10.1016/j.ejpb.2010.01.002
[25]	Cao, H.; Duan, Y.; Lin, Q.; Yang, Y.; Gong, Z.; Zhong, Y.; Chen, X.; Shao, Z. Biomater. Sci. 2019, 7, 2975. doi: 10.1039/C9BM00540D
[26]	Dong, T.; Mi, R.; Wu, M.; Zhong, N.; Zhao, X.; Chen, X.; Shao, Z. J. Mater. Chem. B 2019, 7, 4328. doi: 10.1039/c9tb00783k
[27]	Seib, F. P.; Pritchard, E. M.; Kaplan, D. L. Adv. Funct. Mater. 2013, 23, 58. doi: 10.1002/adfm.201201238
[28]	Li, Z.; Zheng, Z.; Yang, Y.; Fang, G.; Yao, J.; Shao, Z.; Chen, X. ACS Sustain. Chem. Eng. 2016, 4, 1500. doi: 10.1021/acssuschemeng.5b01463
[29]	Liu, H.; Long, L.; Xu, Z.; Zheng, S. Chem. Eng. J. 2020, 400, 125987. doi: 10.1016/j.cej.2020.125987
[30]	Shen, X.; Wang, Z.; Wang, Q.; Liu, S.; Wang, G. Chinese J. Polym. Sci. 2018, 36, 1027. doi: 10.1007/s10118-018-2137-4
[31]	Cheng, S. J.; Zeng, Y.; Pei, Y.; Fan, K. N.; Qiao, M. H.; Zong, B. N. Acta Chim. Sinica 2019, 77, 1054. (in Chinese) doi: 10.6023/A19060219
	( 成诗婕, 曾杨, 裴燕, 范康年, 乔明华, 宗保宁, 化学学报, 2019, 77, 1054.) doi: 10.6023/A19060219
[32]	Qian, W. J.; Wan, M. M.; Lin, W. G.; Zhu, J. H. J. Mater. Chem. B 2014, 2, 92. doi: 10.1039/C3TB21092H
[33]	Gao, M. Y.; Jiang, D.; Sun, D. K.; Hou, B.; Li, D. B. Acta Chim. Sinica 2014, 72, 1092. (in Chinese) doi: 10.6023/A14070535
	( 高梦语, 姜东, 孙德魁, 候博, 李德宝, 化学学报, 2014, 72, 1092.) doi: 10.6023/A14070535
[34]	Yang, G. W.; Gu, K.; Shao, Z. Z. Acta Polym. Sin. 2021, 52, 16. (in Chinese)
	( 杨公雯, 顾恺, 邵正中高分子学报, 2021, 52, 16.)
[35]	Biancalana, M.; Koide, S. Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 1405. doi: 10.1016/j.bbapap.2010.04.001
[36]	Wu, C.; Wang, Z.; Lei, H.; Zhang, W.; Duan, Y. J. Am. Chem. Soc. 2007, 129, 1225. doi: 10.1021/ja0662772
[37]	Jiang, X. R.; Guan, J.; Chen, X.; Shao, Z. Z. Acta Chim. Sinica 2010, 68, 1909. (in Chinese)
	( 江霞蓉, 管娟, 陈新, 邵正中, 化学学报, 2010, 68, 1909.)