药学学报, 2018, 53(12): 2104-2112
引用本文:
王丹丹, 刘瑞, 王钰, 李芳, 陈维良, 张学农. 共载多柔比星和siRNA的还原敏感性纳米粒的体外靶向性评价[J]. 药学学报, 2018, 53(12): 2104-2112.
WANG Dan-dan, LIU Rui, WANG Yu, LI Fang, CHEN Wei-liang, ZHANG Xue-nong. In vitro targeting efficiency evaluation of reduction-responsive co-loaded doxorubicin/siRNA nanoparticles[J]. Acta Pharmaceutica Sinica, 2018, 53(12): 2104-2112.

共载多柔比星和siRNA的还原敏感性纳米粒的体外靶向性评价
王丹丹, 刘瑞, 王钰, 李芳, 陈维良, 张学农
苏州大学药学院, 江苏 苏州 215123
摘要:
本研究构建了一种透明质酸(hyaluronic acid,HA)修饰的共载多柔比星(doxorubicin,DOX)和siRNA的还原敏感性纳米粒(nanoparticles,NPs),并对其体外肺癌靶向性进行评价。经酰胺化反应合成载体材料多聚L-赖氨酸-硫辛酸聚合物(PLA)并进行核磁表征。通过透析法和静电吸附法制备共载DOX与siRNA的NPs并以HA对其修饰,得到HA-PLA/DOX-siRNA-NPs;以粒径和zeta电位为指标,考察HA-PLA/DOX-siRNA-NPs的肿瘤微环境响应性;以人源性非小细胞肺癌细胞(A549)为体外模型,通过CLSM考察HA-PLA/DOX-siRNAFAM-NPs的细胞摄取与siRNA的内涵体逃逸。1H NMR结果显示,载体材料PLA成功合成,LA的接枝率为25.1%;体外表征结果显示,HA-PLA/DOX-NPs的包封率和载药量分别为(86.93±8.91)%和(4.17±0.68)%,在载体中氮磷比(N/P)为6:1时能完全吸附siRNA;HA-PLA/DOX-siRNA-NPs的粒径为(167.3±9.9)nm,电荷为(-15.5±1.4)mV;在透明质酸酶(HAase)环境中zeta电位由负转正,而在10 mmol·L-1谷胱甘肽(GSH)环境中粒径分布变乱;体外释放结果表明,HA-PLA/DOX-NPs在pH 7.4下释药缓慢,而在10 mmol·L-1 GSH环境中能够快速释药。细胞摄取及分布实验表明,HA的包覆能够增强NPs的细胞亲和性和靶向性,且采用HAase处理后,HA-PLA/DOX-siRNAFAM-NPs组的细胞摄取增强;且摄入后能有效从内涵体逃逸,快速释放药物和siRNA至其各自的靶点。结果提示:HA-PLA/DOX-siRNAFAM-NPs对DOX和siRNA均具有较高的包载效率,能显著提高其肿瘤细胞靶向性,并具有肿瘤微环境响应特征,有作为基因与药物共递送体系的潜能。
关键词:   
In vitro targeting efficiency evaluation of reduction-responsive co-loaded doxorubicin/siRNA nanoparticles
WANG Dan-dan, LIU Rui, WANG Yu, LI Fang, CHEN Wei-liang, ZHANG Xue-nong
College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China
Abstract:
In this study a reduction-responsive nanoparticles (NPs) modified with hyaluronic acid (HA) was prepared for the co-delivery of doxorubicin (DOX) and siRNA and then evaluated as a lung cancer targeting delivery system in vitro. The amphiphilic polymer of poly-L-lysine-lipoic acid (PLA) based on poly-L-lysine (PLL) with lipoic acid (LA) was synthesized via amidation reaction and characterized by 1H NMR. The DOX loaded PLA NPs were prepared via dialysis method, and siRNA was loaded via electrostatic attraction to prepare the co-delivery NPs system (PLA/DOX-siRNA-NPs). Then PLA/DOX-siRNA-NPs were coated with HA to obtain HA-PLA/DOX-siRNA-NPs. The tumor microenvironment-responsive properties under different pH or reduction condition of HA-PLA/DOX-siRNA-NPs were evaluated by investigating the particle size and zeta potential. Cellular uptake of HA-PLA/DOX-siRNAFAM-NPs by A549 cells and endosomal escape of siRNA were studied using confocal laser scanning microscope (CLSM). 1H NMR spectrum demonstrated that PLA was successfully synthesized with LA grafting rate of 25.1%. The encapsulation efficiency (EE) and drug loading (DL) of HA-PLA/DOX-NPs was (86.93±8.91)% and (4.17±0.68)%, respectively, and siRNA was loaded at an N/P of 6:1 in carrier. HA-PLA/DOX-siRNA-NPs exhibited a suitable size of (167.3±9.9) nm and negative charge of (-15.5±1.4) mV with the optimal ratio of PLA and HA of 1:3. Additionally, the zeta potential of HA-PLA/DOX-siRNA-NPs significantly increased with charge reversal from negative to positive after the treatment with HAase, and the particle size of HA-PLA/DOX-siRNA-NPs changed significantly under the condition of 10 mmol·L-1 glutathione (GSH). The release profiles in vitro demonstrated that HA-PLA/DOX-NPs exhibited a maintained release behavior at pH 7.4 and the adding of GSH (10 mmol·L-1) led to rapid release of DOX from NPs. In vitro cellular uptake and subcellular distribution study demonstrated that themodification of HA enhanced the affinity of NPs to A549 cells and targeting ability, and the cellular uptake of HA-PLA/DOX-siRNAFAM-NPs significantly increased after the treatment with HAase. It was observed that HA-PLA/DOX-siRNAFAM-NPs could escape from endo-lysosomes followed by sharp payloads release to their relative targets. All these results demonstrated that the co-loaded NPs have a high entrapment efficiency of DOX and siRNA. And they also exhibited an active tumor targeting efficiency and tumor microenvironment-responsive properties, which were beneficial to cellular uptake and intracellular release of DOX and siRNA. In conclusion, these reduction-responsive NPs modified with HA have great potential as co-delivery systems for antitumor agents and siRNA.
Key words:   
收稿日期: 2018-06-22
DOI: 10.16438/j.0513-4870.2018-0576
基金项目: 国家自然科学基金资助项目(81571788,81773183);江苏省大学生创新创业训练项目(201610285096X).
通讯作者: 张学农,Tel/Fax:86-512-65880030,E-mail:zhangxuenong@163.com
Email: zhangxuenong@163.com
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