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浙江大学学报(医学版)  2017, Vol. 46 Issue (2): 127-133    DOI: 10.3785/j.issn.1008-9292.2017.04.03
专题报道     
普朗尼克—聚乙烯亚胺纳米胶束的制备及其细胞生物学特性
汪河滨1,2(),李洋2,刘馨刚2,周峻2,王青青2,汤谷平2,*()
1. 新疆兵团南疆化工资源利用工程实验室 塔里木大学生命科学学院, 新疆 阿拉尔 843300
2. 浙江大学化学系, 浙江 杭州 310058
Preparation, characterization and cytology study of Pluronic-PEI micelles
WANG Hebin1,2(),LI Yang2,LIU Xingang2,ZHOU Jun2,WANG Qingqing2,TANG Guping2,*()
1. Engineering Laboratory of Chemical Resources Utilization in South Xinjiang of Xinjiang Production and Construction Corps, College of Life Science, Tarim University, Xinjiang Alar 843300, China
2. Department of Chemistry, Zhejiang University, Hangzhou 310027, China
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摘要:

目的:采用普朗尼克—聚乙烯亚胺纳米胶束制备高效、低毒的药物和基因输送体系。方法:利用小分子量的聚乙烯亚胺作为交联剂制备普朗尼克—聚乙烯亚胺纳米胶束,采用动态光散射法、芘荧光探针法分别测定聚合物的粒径、表面电动电位及临界胶束浓度,考察聚合物携带基因和药物的能力,并在细胞水平上探讨普朗尼克—聚乙烯亚胺的细胞毒性、转染效率以及对细胞内ATP和P-糖蛋白水平的影响。结果:成功制备了普朗尼克—聚乙烯亚胺纳米胶束,其粒径120~180 nm,表面电位+6~+9 mV,具有良好的携载基因、药物的能力。细胞生物学研究表明,普朗尼克—聚乙烯亚胺毒性较小,其中P123-PEI600基因转染效率高,并且能降低细胞内ATP和P糖蛋白水平。结论:普朗尼克—聚乙烯亚胺是一种良好的药物和基因输送体系,P123-PEI600是理想的可用于逆转肿瘤多药耐药的载体。

关键词: 工艺学,制药聚乙烯亚胺/化学合成纳米球药物载体基因转染腺苷三磷酸P-糖蛋白抗药性,肿瘤    
Abstract:

Objective: To prepare and characterize Pluronic-PEI micelles as a drug/gene delivery system. Methods: We used the low-molecular-weight PEI as a cross-linking agent to prepare the Pluronic-PEI micelles. The particle size, zeta potential and critical micelle concentration (CMC) were measured by dynamic light scattering (DLS) and pyrene fluorescence probe. The cytotoxicity, transfection efficiency and the impact on the intracellular ATP and P-gp levels of Pluronic-PEI micelles were investigated at the cellular level. Results: Pluronic-PEI micelles were successfully prepared with a suitable particle size (120-180 nm), zeta potential (+6-+9 mv), and a good ability to carry the drug/gene. An in-vitro study showed that Pluronic-PEI had low cytotoxicity, and the P123-PEI600 possessed high gene transfection efficiency and could downregulate the intracellular ATP and P-gp levels. Conclusion: Pluronic-PEI is a good drug/gene delivery system, and P123-PEI600 is an ideal vector, which may be used in the combination therapy for reversing multidrug resistance.

Key words: Technology, pharmaceutical    Polyethyleneimine/chemical synthesis    Nanospheres    Drug carriers    Genes    Transfection    Adenosine triphosphate    P-glycoprotein    Drug resistance, neoplasm
收稿日期: 2016-11-02 出版日期: 2017-08-07
基金资助: 国家自然科学基金(51573161)
通讯作者: 汤谷平     E-mail: wanghebin329@163.com;tangguping@zju.edu.cn
作者简介: 汪河滨(1980—),男,博士,主要从事生物医用纳米材料方面的研究;E-mail:wanghebin329@163.com;http://orcid.org/0000-0003-2159-914X
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引用本文:

汪河滨,李洋,刘馨刚,周峻,王青青,汤谷平. 普朗尼克—聚乙烯亚胺纳米胶束的制备及其细胞生物学特性[J]. 浙江大学学报(医学版), 2017, 46(2): 127-133.

WANG Hebin,LI Yang,LIU Xingang,ZHOU Jun,WANG Qingqing,TANG Guping. Preparation, characterization and cytology study of Pluronic-PEI micelles. J Zhejiang Univ (Med Sci), 2017, 46(2): 127-133.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2017.04.03        http://www.zjujournals.com/xueshu/med/CN/Y2017/V46/I2/127

图 1  普朗尼克—聚乙烯亚胺的合成路线
图 2  紫杉醇吸收曲线和标准曲线
图 3  普朗尼克—聚乙烯亚胺在重水中的核磁共振氢谱图
(%)
元素 检测值 F127 F127-PEI600 F127-PEI1200 P123-PEI600 P123-PEI1200 L61-PEI600 L61-PEI1200
实测值 56.26 56.04 56.56 59.58 59.66 56.05 55.66
理论值 56.72 56.69 56.66 59.63 59.31 55.71 55.76
实测值 9.76 10.14 9.89 10.08 10.05 10.21 9.92
理论值 9.45 9.45 9.44 9.94 9.89 9.29 9.29
实测值 0.16 1.51 3.01 3.30 5.90 7.07 11.09
理论值 0.00 1.49 2.84 3.06 5.60 7.00 11.53
表 1  普朗尼克—聚乙烯亚胺元素分析结果
图 4  不同普朗尼克—聚乙烯亚胺空白胶束的透射电镜图
($\bar x \pm s$)
样品 粒径(nm) 表面电位(mV)
 F127 81.7±1.0 -0.22±0.4
 F127-PEI600 149.1±2.5 +6.16±0.9
 F127-PEI1200 181.2±8.9 +8.14±0.4
 P123 30.1±0.5 -0.17±0.4
 P123-PEI600 122.7±4.2 +6.49±1.1
 P123-PEI1200 145.9±5.2 +8.60±2.2
 L61 1698.3±745.7 -0.83±9.9
 L61-PEI600 1804.0±170.2 +7.49±7.9
 L61-PEI1200 2417.9±640.2 +9.11±5.6
表 2  普朗尼克—聚乙烯亚胺粒径和电位分析结果
图 5  普朗尼克—聚乙烯亚胺溶液浓度对数值随荧光强度比值变化曲线图
图 6  普朗尼克—聚乙烯亚胺的DNA凝胶电泳图
($\bar x \pm s$)
样品 包封率(%) 载药率(%)
  与F127比较,*P<0.05.
 F127 15.33±4.21 0.73±0.12
 F127-PEI600 16.25±3.26 0.92±0.09
 F127-PEI1200 21.21±4.07 1.01±0.23
 P123 23.73±2.28 1.13±1.22*
 P123-PEI600 26.46±3.23 1.26±0.78
 P123-PEI1200 29.40±4.18 1.40±0.19
表 3  普朗尼克—聚乙烯亚胺载药率和包封率
图 7  不同普朗尼克—聚乙烯亚胺的药物释放曲线
图 8  加入普朗尼克—聚乙烯亚胺的细胞生存活曲线
图 9  不同普朗尼克—聚乙烯亚胺和PEI25 000携带pEGFP质粒在HEK293和A549细胞上的转染结果
图 10  普朗尼克—聚乙烯亚胺对细胞内ATP和P-gp水平的影响
图 11  普朗尼克—聚乙烯亚胺对Panc-1细胞内P-gp的影响
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