鱼精蛋白-siRNA不同形貌复合体的制备、 结构及药效学分析

doi:10.7503/cjcu20180833

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (6): 1164.doi: 10.7503/cjcu20180833

鱼精蛋白-siRNA不同形貌复合体的制备、结构及药效学分析

史迈¹, 江芮², 崔新霞¹, 张欣², 申世刚¹, 丁良²(), 潘学峰^1,^2,³()

1. 河北大学化学与环境科学学院, 保定 071002
2. 河北大学医学院, 保定 071000
3. 北京理工大学生命学院, 北京 100081

收稿日期:2018-12-13 出版日期:2019-06-10 发布日期:2019-04-18
作者简介:
联系人简介: 潘学峰, 男, 博士, 教授, 博士生导师, 主要从事生物化学、分子遗传学及分子诊断方面的研究.E-mail: xuefengpancam@aliyun.com ;丁良, 女, 博士, 教授, 主要从事分析化学方面的研究. E-mail: 345823685@qq.com
基金资助:
河北省医学科学研究重点课题(批准号: 20160051)、京津冀新药研制协同创新计划项目和保定市莲池区科技局重点项目(批准号:15J04)资助.

Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes^†

SHI Mai¹, JIANG Rui², CUI Xinxia¹, ZHANG Xin², SHEN Shigang¹, DING Liang²(), PAN Xuefeng^1,^2,³()

1. School of Chemistry and Environment, Hebei University, Baoding 071002, China
2. Medical School, Hebei University, Baoding 071000, china
3. School of Life Science, Beijing Institute of Technology, Beijing 100081, China

Received:2018-12-13 Online:2019-06-10 Published:2019-04-18
Supported by:
†Supported by the Hebei Provincial Key Research Programs for Medical Science, China(No.20160051), the Collaborative Innovation Project for New Drug Development between Beijing, Tianjin and Hebei Province, China and the Key Project of Baoding Lianchi District Science and Technology Bureau, China(No.15J04).

摘要/Abstract

摘要：

利用涡旋混匀及室温孵育的方法制备了不同形貌的鱼精蛋白-siRNA复合体, 并利用聚丙烯酰胺凝胶电泳结合银染、原子力显微镜和透射电子显微镜等手段进行了结构表征. 结果表明, 鱼精蛋白可以和不同的siRNA分子以不同的质量比形成球形和纤维状鱼精蛋白-siRNA复合物. 在此基础上, 利用Mg²⁺等金属离子探针, 确认鱼精蛋白与siRNA的磷酸-戊糖骨架之间通过静电力发生作用. 利用原子力和透射电子显微镜表征了鱼精蛋白-siRNA复合体的形貌和结构特征; 利用激光纳米粒度仪测量了该复合体的粒径; 并进一步比较了黑色素瘤细胞对不同形貌复合体的吞噬及药效学特征.

关键词: 鱼精蛋白, siRNA, 黑色素瘤, 药物递送

Abstract:

Protamine-siRNA complexes of different morphologies were prepared by vortex mixing different mass ratios of protamine and siRNA at room temperature, which could be resolved by polyacrylamide gel electrophoresis with silver staining. The protamine-siRNA complexes were observed under atomic force and transmission electron microscopy, respectively. It was found that similarly shaped protamine-siRNA complexes could be formed by protamine with different siRNA molecules, indicated that the interaction between protamine and siRNA was independent on the base composition of siRNA. Moreover, the interactions between the protamine and the siRNA molecules were characterized by ionic probes, including Mg²⁺ etc. And the morphologies, the particle sizes as well as the structural properties of the protamine-siRNA complexes were further characterized using atomic force microscope, dynamic light scattering and transmission electron microscope, respectively. The results show that protamine formed toroid complexes and fibrous composites with siRNA molecules respectively in the light of the different mass ratios between protamine and siRNA. In the meantime, the phagocytosis and the pharmacodynamics of protamine-siRNA complexes of different morphologies were compared and measured using melanoma cells.

Key words: Protamine, siRNA, Melanoma, Drug delivery

中图分类号:

O629.74

TrendMD:

史迈, 江芮, 崔新霞, 张欣, 申世刚, 丁良, 潘学峰. 鱼精蛋白-siRNA不同形貌复合体的制备、结构及药效学分析. 高等学校化学学报, 2019, 40(6): 1164.

SHI Mai,JIANG Rui,CUI Xinxia,ZHANG Xin,SHEN Shigang,DING Liang,PAN Xuefeng. Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes^†. Chem. J. Chinese Universities, 2019, 40(6): 1164.

图/表 10

Table 1 Preparation of protamine-siRNA-1 complex with a mass ratio of 20∶1

V(Total)/μL	V(siRNA)/μL	V(Protamine)/μL	V(HEPES)/μL
50.0	2.5	2.5	45.0
50.0	5.0	5.0	40.0
50.0	7.5	7.5	35.0
50.0	10.0	10.0	30.0
50.0	12.5	12.5	25.0
50.0	15.0	15.0	20.0

Fig.1 PAGE analysis on the formations of protamine-siRNA complexes Protamine-siRNA-1 complexes in (A) or protamine-siRNA-2 complexes in (B) were formed by protamine and siRNA by mass ratios of 15∶1, 20∶1, 25∶1, 30∶1, 35∶1, 40∶1, respectively. Lane 1: siRNA-1(A) or siRNA-2(B); lanes 2—7: protamine-siRNA complexes formed by mass ratios of protamine to siRNA-1(A) or siRNA-2(B) by 15∶1, 20∶1, 25∶ 1, 30∶ 1, 35∶ 1, 40∶ 1, respectively. Protamine-siRNA-1 complexes formed by a mass ratio of protamine to siRNA-1 by 20∶1(C). Lane 1: siRNA-1, lanes 2—7: protamine-siRNA-1 complexes formed by simultaneously increasing the protamine and siRNA-1 by 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 μL, respectively.

Fig.2 PAGE analysis on the effects of different metal ions on the stabilities of protamine-siRNA complexes

Fig.3 TEM images of protamine-siRNA complexes formed by protamine and siRNA with the mass ratios of 20∶1(A) and 40∶1(B)

Fig.4 AFM visualizations of protamine-siRNA complexes with the mass ratios of protamine to siRNA 20∶1(A, toroid) and 40∶1(B, filamentous) and protamine proteins(white dots) in the filamentous protamine-siRNA complexes(C)

Scheme 1 Schematic illustrations for the interactions between protamine and siRNA in protamine-siRNA complexes of different morphologies

Fig.5 PAGE analysis on the formation of disulfide bonds in the protamine-siRNA complexes Lane 1: siRNA, lanes 2—7: protamine-siRNA complexes of filament morphologies treated using β-mercaptoethanol with a final concentration of 0, 5, 10, 15, 20 and 40 mmol/L, respectively.

Table 2 Particle sizes of the protamine-siRNA complexes

Mass ratio of protamine to siRNA	d(90)/nm	Mass ratio of protamine-siRNA	d(90)/nm
15∶1	217.5±6.7	30∶1	325.8±7.6
20∶1	220.0±7.2	35∶1	384.2±6.2
25∶1	275.5±5.0	40∶1	405.0±4.3

Fig.6 Phagocytosis of A375 melanoma cells on toroid(A) and fibrous(B) protamine-siRNA complexes under confocal fluorescence microscopy

Fig.7 Effects of protamine-siRNA complexes on the proliferation of A375 cells a. Control; b. lipoHigh; c. phospholipid vesicles; d. siRNA; e. spherical complexes; f. filamentous complexes.

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鱼精蛋白-siRNA不同形貌复合体的制备、结构及药效学分析

Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes^†

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鱼精蛋白-siRNA不同形貌复合体的制备、 结构及药效学分析

Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes†

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鱼精蛋白-siRNA不同形貌复合体的制备、结构及药效学分析

Preparation, Structure and Pharmaceutical Analysis of Protamine-siRNA Complexes^†