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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (6): 657-667    DOI: 10.3785/j.issn.1008-9292.2019.12.11
原著     
基于聚天冬氨酸骨架的药物/基因共运输载体的制备及细胞生物学特性
沈洁1,2(),王启闻3,高东若1,吕媛媛1,汤谷平2,*()
1. 浙江大学城市学院医学院, 浙江 杭州 310015
2. 浙江大学化学系, 浙江 杭州 310028
3. 浙江大学医学院附属第一医院心血管内科, 浙江 杭州 310003
Synthesis and cell biological properties of polyaspartic acid drug/gene vector
SHEN Jie1,2(),WANG Qiwen3,GAO Dongruo1,LYU Yuanyuan1,TANG Guping2,*()
1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Chemistry, Zhejiang University, Hangzhou 310028, China
3. Department of Cardiovascular Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
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摘要：

目的: 以聚天冬氨酸作为主链，接枝胺基侧链和烷基侧链制备药物/基因共运输载体，考察不同侧链接枝率形成的纳米粒载体的载药及载基因性能，并筛选出性能较优的载体。方法: 以D，L-天冬氨酸为单体，通过聚合反应合成聚天冬氨酸，然后以不同比例的N，N-二甲基亚二丙基三胺、3，3'-二氨基二丙胺和十二胺/十六胺/十八胺进行开环接枝，得到两亲性聚天冬氨酸-3，3'-二氨基二丙胺-N，N-二甲基亚二丙基三胺-烷基胺（PEEC）材料。采用氢核磁共振确认PEEC结构，用动态光散射法测定纳米粒胶束的粒径和表面电位，用芘荧光探针法测定临界胶束浓度，用琼脂糖凝胶电泳表征RNA阻滞能力，考查载药胶束的释放行为并且在细胞层面上研究材料的细胞毒性、细胞摄取和基因沉默效率。结果: 成功合成了不同接枝率的PEEC材料，纳米粒胶束的粒径为250~350 nm，表面电位为27~45 mV，拥有较小的临界胶束浓度值，RNA阻滞的质量比约为0.8：1；PEEC胶束有一定的细胞毒性，其载药能力和基因绑定能力良好，其中PEEC16-2在细胞水平上表现出较高的基因沉默效率。结论: PEEC具有共载药物/基因的能力，其中PEEC16-2胶束具有良好的药物包载和基因运输能力。
关键词： 天冬氨酸;  药物载体;  基因治疗;  纳米粒子;  肿瘤    
Abstract:

Objective: Taking polysuccinimide as the main chain, amine side chain and alkyl side chain were grafted to prepare the drug/gene co-delivery vector. The property of the polymers with various side links were investigated to select an optimal vector. Methods: Poly-D, L-polysuccinimide was synthesized by polymerization reaction of D, L-aspartic acid as monomer. Therefore, N, N-dimethylenedipropyl-triamine and 3, 3'-diaminodipropylamine were grafted with dodecylamine/adecylamine/octadecylamine at different proportions by ring-opening reaction to obtain amphiphilic PEECs. The structure of the material was confirmed by 1H NMR; the particle size and surface potential of the micelles were measured by dynamic light scattering; the critical micelle concentration (CMC) was determined by pyrene fluorescent probe; the RNA blocking ability was characterized by agarose gel electrophoresis; the release behavior of the PEECs was examined and the cytotoxicity, cellular uptake and gene silencing efficiency of the PEECs were studied at the cellular level. Results: A series of PEECs with different grafting rates was successfully synthesized. The particle sizes and surface potential of the PEEC derived micelles were between 250 nm and 350 nm and 27 mV and 45 mV, respectively, with a small CMC value. The RNA binding ratio of PEECs was at a mass ratio of about 0.8:1. MTT assay demonstrated that PEEC micelles had certain cytotoxicity. PEECs had excellent micelle formation, drug-loading and gene binding abilities, particularly, PEEC16-2 showed high gene silencing efficiency at the cellular level. Conclusion: PEECs are able to co-delivery drug and gene, and PEEC16-2 micelles have the best ability of drug encapsulation and gene delivery.
Key words: Aspartic acid    Drug carriers    Genetic therapy    Nanoparticles    Neoplasms
收稿日期: 2019-01-30 出版日期: 2020-01-19
:  R943  
基金资助: 浙江大学城市学院教师科研基金(J16002);国家自然科学基金(81800442)
通讯作者: 汤谷平     E-mail: shenj@zucc.edu.cn;tangguping@zju.edu.cn
作者简介: 沈洁(1987-), 女, 博士, 讲师, 主要从事抗肿瘤基因药物纳米材料研究; E-mail:shenj@zucc.edu.cn; https://orcid.org/0000-0002-8116-0473
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引用本文:

沈洁,王启闻,高东若,吕媛媛,汤谷平. 基于聚天冬氨酸骨架的药物/基因共运输载体的制备及细胞生物学特性[J]. 浙江大学学报（医学版）, 2019, 48(6): 657-667.

SHEN Jie,WANG Qiwen,GAO Dongruo,LYU Yuanyuan,TANG Guping. Synthesis and cell biological properties of polyaspartic acid drug/gene vector. J Zhejiang Univ (Med Sci), 2019, 48(6): 657-667.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.12.11    或    http://www.zjujournals.com/med/CN/Y2019/V48/I6/657

样品 聚天冬氨酸(g) 烷基胺(g) N，N-二甲基亚二丙基三胺(mL) 3，3′-二氨基二丙胺(mL)
PEEC：聚天冬氨酸-3，3′-二氨基二丙胺-N，N-二甲基亚二丙基三胺-烷基胺.
PEEC12-1 1.0 0.10 1.0 1.3
PEEC12-2 1.0 0.20 1.0 1.3
PEEC16-1 1.0 0.27 1.0 1.7
PEEC16-2 1.0 0.40 1.0 1.7
PEEC18-1 1.0 0.15 1.0 1.7
PEEC18-2 1.0 0.30 1.0 1.7
表 1  合成PEEC的投料比
图 1  PEEC的合成路线
图 2  PEEC的核磁共振氢谱
样品 x y z 烷基链接枝率(%) 粒径(nm) 表面电位(mV) 临界胶束浓度(μg/mL)
x：3，3′-二氨基二丙胺；y：N，N-二甲基亚二丙基三胺；z：烷基胺；PEEC：聚天冬氨酸-3，3′-二氨基二丙胺-N，N-二甲基亚二丙基三胺-烷基胺.
PEEC12-1 0.295 0.573 0.054 5.9 308±6 32.0±0.6 43.8
PEEC12-2 0.217 0.538 0.134 15.1 279±16 29.0±1.4 21.6
PEEC16-1 0.339 0.507 0.054 6.0 342±32 37.4±2.6 45.9
PEEC16-2 0.396 0.722 0.157 12.3 302±20 31.8±2.5 4.4
PEEC18-1 0.383 0.673 0.031 2.9 341±28 42.5±2.1 42.7
PEEC18-2 0.966 1.440 0.179 6.9 326±24 37.2±1.1 28.3
表 2  PEEC的烷基链接枝率、粒径、表面电位及临界胶束浓度
图 3  不同接枝率PEEC的临界胶束浓度
图 4  不同接枝率PEEC胶束的琼脂糖凝胶电泳图
图 5  PEEC胶束对细胞存活率的影响
图 6  PEEC16-2载药胶束的体外药物释放曲线(n=3)
图 7  PEEC16-2/siRNA复合物的细胞摄取
图 8  PEEC16-2/siGFP绿色荧光蛋白沉默结果(n=3)
图 9  PEEC16-2/DOX/siRNA的细胞摄取
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