Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (6): 936-944.doi: 10.3969/j.issn.2095-4344.1549
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Wang Yang1, Nie Jinshan2, Gu Zhun1, Zhu Kai3
Received:
2018-10-16
Online:
2019-02-28
Published:
2019-02-28
Contact:
Wang Yang, Suzhou Chien-Shiung Institute of Technology, Taicang 215411, Jiangsu Province, China
About author:
Wang Yang, MD, Lecturer, Suzhou Chien-Shiung Institute of Technology, Taicang 215411, Jiangsu Province, China
Supported by:
the Natural Science Research Project of Jiangsu Provincial Universities (General Project), No. 16KJB430037 (to WY); the Three-Level Linkage Research Foundation of Suzhou Chien-Shiung Institute of Technology, No. 2017SJLD14 (to GZ); the Basic Research Project of Taicang Municipality, No. TC2016YYJC09 (to GZ); the Excellent Youth Teacher Project of Jiangsu Provincial Universities (to WY)
CLC Number:
Wang Yang, Nie Jinshan, Gu Zhun, Zhu Kai. Construction and in vitro evaluation of a biodegradable cationic gene delivery system based on hyperbranched polyamidoamine[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(6): 936-944.
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2.1 载体材料DHPAA的合成与表征 DHPAA由单体A2+B3通过迈克尔加成“一锅法”反应合成,单体A2选用含有二硫键的末端双烯基单体N,N'-双(丙烯酰)胱胺,单体B3选用多元胺1- (2-氨乙基)哌嗪,使用甲醇为溶剂,反应条件温和,操作简单。图2为聚合物DHPAA的核磁共振氢谱图,2.10 ppm处出现了来自哌嗪六元环的-CH2-特征峰,2.79-2.73 ppm处出现了来自N,N'-双(丙烯酰)胱胺 的-CH2(S-S)CH2-特征峰,2.67 ppm 处出现来自N-氨乙基中的-CH2-特征峰,2.60 ppm处出现了(-CH2CONH)的特征峰,2.80 ppm处出现了(-CH2NH2)的特征峰,3.41 ppm 处出现了(>NCH2-)的特征峰,3.34-3.23 ppm 处出现了(-CONHCH2-)的特征峰,3.15 ppm出现了(-CH2-NH-CH2-CH2N(CH2)4NH)的特征峰。DHPAA在D2O中的核磁共振氢谱图表明,所合成的聚合物符合分子设计。采用激光光散射凝胶渗透色谱系统测定聚合物DHPAA的绝对分子质量,根据GPC结果分析,数均分子质量(Mn)为4.635×104,重均分子质量(Mw)为1.372×105,重复的聚酰胺胺单元大约为119。通过酸-碱滴定评价聚合物DHPAA的质子缓冲效应。见图3,0.15 mol/L氯化钠溶液的滴定曲线接近垂直,说明其缓冲能力非常小。相反,聚合物DHPAA溶液具有很强的缓冲能力,在pH=10-3的范围内的滴定曲线非常平缓。"
2.2.3 复合物的形态、粒径及Zeta 电位 见图6。当聚合物DHPAA与DNA质粒的质量比从1∶1变化到20∶1,粒径从105.5 nm变化至59.8 nm,表明聚合物与DNA质粒质量比增加,粒径减小。此外,图6中所示的电势值表明,当质量比从1∶1变化到20∶1,电势从-9.98 mV变化至 29.1 mV(远高于纯DNA的电势-22.4 mV),当质量比大于10∶1时,复合物的电势已大于20 mV,说明聚合物已可完全包裹DNA,这与Picogreen荧光分析结果一致。图7为质量比为10∶1复合物纳米粒降解前后的透射电镜形貌,降解前复合物纳米粒呈现出规则、均匀的球形形貌,粒径约为45 nm;降解后发生粒子聚集,粒径增加。"
2.2.4 复合物的包封率测定与体外释放 通过荧光分光光度法测定不同质量比DHPAA/DNA复合物的包封率,图8A结果表明,在质量比为10∶1时已接近90%,质量比继续增加包封率不再增加。因此,选用DHPAA与DNA质量比为10∶1的复合物为研究对象,研究其在中性或者还原性介质中DNA的释放行为。图8B结果表明,在不含二硫苏糖醇的释放介质中,DNA释放百分率接近0%,说明DHPAA/ DNA复合物很稳定,无法从复合物纳米粒中释放出DNA;相反,在二硫苏糖醇浓度为10 mmol/L的释放介质中,在0-4 h 内,DNA的释放百分率从0%增加到90%,这是因为在还原性环境中,DHPAA中的二硫键被还原,聚合物发生降解促进了DNA的释放,有利于体外或体内基因的表达。"
2.3 DHPAA细胞毒性分析 基因载体的细胞毒性对DNA的转染效率有很大的影响。图9显示了使用MTT法测定不同质量浓度聚合物对HEK293、MCF-7、间充质干细胞和Hela细胞的48 h毒性。由图可见,随着质量浓度的增加,聚乙烯亚胺对所有的细胞系表现出很强的细胞毒性,尤其当质量浓度为200 mg/L时,细胞存活率为20%-30%。相比之下,聚合物DHPAA毒性较小,在整个质量浓度范围内(1-200 mg/L)几乎对细胞存活率无影响,即使在200 mg/L条件下细胞存活率仍接近90%。图10为25 mg/L不同质量比DHPAA/DNA复合物对间充质干细胞和Hela细胞的48 h毒性分析。从图中可看出,所有复合物48 h的细胞毒性均较小,当聚合物DHPAA与DNA的质量比从1∶1增加至20∶1,间充质干细胞的存活率从90.57%变化到93.02%,Hela细胞的存活率从86.96%变化到90.65%,这说明聚合物不影响细胞存活率,是一种生物相容性好的基因递送材料。"
2.4 体外基因转染实验 使用流式细胞仪和荧光共聚焦显微镜对不同质量比的HPAA/pEGFP复合物进行了间充质干细胞细胞转染实验。图11结果表明,当DHPAA与pEGFP质量比从1∶1增加至20∶1,复合物转染效率从34.9%增加至63.5%,远大于纯DNA质粒的转染效率(0.115%);当聚合物与pEGFP质量比为10∶1时,复合物的转染效率接近60%,质量比继续增加,转染效率增加缓慢。图12为不同质量比DHPAA/pEGFP复合物的荧光共聚焦图像,结果表明随着DHPAA与pEGFP质量比的增加,细胞中的绿色荧光强度增强,当质量比增加至10∶1以上,可明显地观察到视野下的绿色荧光增强,这表明较多绿色荧光蛋白被表达,这与流式细胞仪分析结果一致。"
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