聚乙烯亚胺改性的双介孔氧化硅基因载体构建

doi:10.15541/jim20190099

无机材料学报 ›› 2020, Vol. 35 ›› Issue (2): 187-192.DOI: 10.15541/jim20190099 CSTR: 32189.14.10.15541/jim20190099

所属专题：生物材料论文精选(2020)

聚乙烯亚胺改性的双介孔氧化硅基因载体构建

方美蓉¹,秦利梅²,贾晓博²,李永生²,牛德超²(),胡泽岚¹()

1. 华东理工大学药学院, 上海新药设计重点实验室, 上海 200237
2. 华东理工大学材料科学与工程学院, 上海 200237

收稿日期:2019-03-05 修回日期:2019-04-01 出版日期:2020-02-20 网络出版日期:2019-05-29
作者简介:方美蓉(1993-), 女, 硕士研究生. E-mail: meirongfang@163.com
基金资助:
国家自然科学基金(51572083);国家自然科学基金(81772689);上海市科技启明星计划(16QA1401300)

Construction of Polyethyleneimine-modified Dual-mesoporous Silica Gene Carrier

FANG Mei-Rong¹,QIN Li-Mei²,JIA Xiao-Bo²,LI Yong-Sheng²,NIU De-Chao²(),HU Ze-Lan¹()

1. Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
2. School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China

Received:2019-03-05 Revised:2019-04-01 Published:2020-02-20 Online:2019-05-29
Supported by:
National Natural Science Foundation of China(51572083);National Natural Science Foundation of China(81772689);Shanghai Rising-Star Program(16QA1401300)

摘要/Abstract

摘要：

为了开发一种新型的非病毒无机基因载体, 采用3-氨丙基三乙氧基硅烷(APTMS)和聚乙烯亚胺(PEI)对嵌入型双介孔氧化硅球(EDMSNs)进行改性, 分别得到EDMSNs-NH₂和EDMSNs-PEI, 并比较了两种载体结合和保护pCMV-EGFP-N1质粒(pDNA)的能力及细胞转染性能。利用透射电镜、动态光散射及氮气吸附-脱附实验对材料的颗粒形态, 动力学粒径, Zeta电位及孔结构参数进行表征。结果显示, EDMSNs-NH₂和EDMSNs-PEI均表现出明显的双介孔结构, 形貌为规整的球形且平均动力学粒径分别为343.2和338.9 nm, 表面电位分别为+18和+43 mV。琼脂糖凝胶电泳、CCK-8法及荧光显微镜结果表明, EDMSNs-PEI对pDNA的担载量为8%, 远高于EDMSNs- NH₂(1%)。与PEI和lipofectamine2000相比, EDMSNs-PEI载体展示出更低的细胞毒性。EDMSNs-PEI/pDNA质量比为33 : 1时, EDMSNs-PEI/pDNA对293T细胞的转染效率在72 h达到最大值。因此, 与EDMSNs-NH₂相比, EDMSNs-PEI具有更高的正电位及pDNA担载量, 可作为一类有前景的非病毒无机类基因载体用于重大疾病如恶性胶质瘤的治疗。

关键词: 双介孔氧化硅球, 聚乙烯亚胺, 基因治疗, 恶性胶质瘤

Abstract:

To develop a new kind of non-viral inorganic gene vector, embedded dual-mesoporous silica spheres (EDMSNs) was modified with polyethyleneimine (PEI) or 3-aminopropyltriethoxysilane (APTMS). Then gene loading and transfection ability of the obtained EDMSNs-PEI and EDMSNs-NH₂ was tested by choosing the pCMV-EGFP-N1 plasmids (pDNA) as a gene model. Particle morphologies, particle sizes, zeta potentials, and pore structure parameters of EDMSNs-NH₂ and EDMSNs-PEI were characterized by transmission electron microscopy, dynamic light scattering and nitrogen adsorption-desorption. Both EDMSNs-NH₂ and EDMSNs-PEI display obvious dual-mesoporous structure and well-defined spherical morphology. Average dynamic sizes of EDMSNs-NH₂ and EDMSNs-PEI are 343.2 and 338.9 nm, while zeta potentials of EDMSNs-NH₂ and EDMSNs-PEI are +18 and +43 mV, respectively. The results of gel electrophoresis, CCK-8 assay, and fluorescence microscope show that the pDNA loading amount of EDMSNs-PEI is 8%, much higher than that (1%) of EDMSNs-NH₂. In addition, EDMSNs-PEI exhibits lower cytotoxicity than that of pure PEI polymers and lipofectamine2000. When the mass ratio of EDMSNs- PEI/pDNA is 33 : 1, the transfection rate of the 293T cells by EDMSNs-PEI/pDNA reaches a maximum at 72 h. In conclusion, EDMSNs-PEI, compared with EDMSNs-NH2, has a higher positive potential and pDNA loading capacity, which can be used as a promising non-viral inorganic gene vector for the treatment of diseases such as malignant glioma.

Key words: embedded dual-mesoporous silica spheres, polyethyleneimine, gene therapy, malignant glioma

中图分类号:

TQ174

方美蓉,秦利梅,贾晓博,李永生,牛德超,胡泽岚. 聚乙烯亚胺改性的双介孔氧化硅基因载体构建[J]. 无机材料学报, 2020, 35(2): 187-192.

FANG Mei-Rong,QIN Li-Mei,JIA Xiao-Bo,LI Yong-Sheng,NIU De-Chao,HU Ze-Lan. Construction of Polyethyleneimine-modified Dual-mesoporous Silica Gene Carrier[J]. Journal of Inorganic Materials, 2020, 35(2): 187-192.

图/表 6

图1 EDMSNs-PEI(a)和EDMSNs-NH2(b)的透射电镜照片

Fig. 1 TEM images of EDMSNs-PEI (a) and EDMSNs- NH2 (b)

图2 EDMSNs, EDMSNs-PEI和EDMSNs-NH2的Zeta电位(a)和动力学粒径分布(b)

Fig. 2 Zeta potentials (a) and particle size distributions (b) of EDMSNs, EDMSNs-PEI and EDMSNs-NH2

图3 EDMSNs-PEI (a~b)和EDMSNs-NH2 (c~d)的氮气吸附-脱附曲线及相应的DFT孔径分布

Fig. 3 N2 adsorption-desorption isotherms and the corresponding DFT pore size distributions of EDMSNs-PEI (a-b) and EDMSNs-NH2 (c-d)

图4 EDMSNs-PEI/pDNA和EDMSNs-NH2/pDNA纳米复合物在不同质量比下的电泳照片(a), EDMSNs-PEI/pDNA DNaseI酶切电泳照片(b)和Zeta电位(c)

Fig. 4 Electrophoresis diagram of EDMSNs-PEI/pDNA and EDMSNs-NH2/pDNA nanoparticles at different weight ratios (a), DNaseI digestion electrophoresis diagram (b) and zeta potentials (c) of EDMSNs-PEI/pDNA with M indicating DNA marker and C indicating naked pDNA Weight ratio=weight ratios of EDMSNs-PEI and pDNA.1, 2, 3, 4, 5, 6, 7: weight ratios of 5 : 1, 12.5 : 1, 25 : 1, 33 : 1, 50 : 1, 100 : 1, 200 : 1; 8, 9, 10, 11, 12, 13: weight ratios of 200 : 1 ,100 : 1, 50 : 1, 33 : 1, 25 : 1, 12.5 : 1 for EDMSNs-PEI/pDNA nanocomplexes treated with DnaseI

图5 PEI 25kD、LIP2K和EDMSNs-PEI在293T(a)和u87(b)上的细胞毒性

Fig. 5 Cytotoxicity assay of PEI 25kD, LIP2K and EDMSNs-PEI in 293T (a) and u87(b). * p<0.01, EDMSNs-PEI

图6 EDMSNs-PEI/pDNA纳米复合物在293T细胞内的转染效果

Fig. 6 Transfection efficacy of EDMSNs-PEI/pDNA nanoparticle in 293T cell lines.

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聚乙烯亚胺改性的双介孔氧化硅基因载体构建

Construction of Polyethyleneimine-modified Dual-mesoporous Silica Gene Carrier

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