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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Construction and in vitro evaluation of a biodegradable cationic gene delivery system based on hyperbranched polyamidoamine
Wang Yang1, Nie Jinshan2, Gu Zhun1, Zhu Kai3
- 1Suzhou Chien-Shiung Institute of Technology, Taicang 215411, Jiangsu Province, China; 2Department of Gastroenterology, Taicang No. 1 People Hospital, the Affiliated Hospital of Soochow University, Taicang 215400, Jiangsu Province, China; 3Department of Cardiac Surgery, Zhongshan Hospital, Fudan University & Shanghai Institute of Cardiovascular Diseases, Shanghai 200032, China
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Received:2018-10-16Online:2019-02-28Published: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)
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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。"
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2.2 DHPAA/DNA复合纳米粒子的性能表征 2.2.1 凝胶阻滞分析 图4为不同质量比DHPAA/DNA质粒的电泳分析图，表明当质量比为1∶1时即可抑制DNA质粒的流动，见图4A；当DHPAA/DNA复合物分散于10 mmol/L 二硫苏糖醇时，复合物快速分解，并释放出游离DNA条带，见图4B。这些结果表明，二硫苏糖醇的存在使聚合物DHPAA中的二硫键发生断裂，引起复合物的分解。"
2.2.2 聚合物DHPAA压缩DNA形成复合物的能力 使用Picogreen试剂测定了DHPAA聚合物包裹DNA的能力，见图5所示，DHPAA/DNA复合物的荧光强度随着质量比的增加而逐渐降低，这表明当聚合物质量增加时，未包裹的游离DNA减少。在DHPAA与DNA质量比达到10∶1时，溶液的相对荧光强度不再发生变化，说明质量比10∶1时聚合物对于DNA的固缩已相对完全。当10 mmol/L二硫苏糖醇加入至复合物溶液中时，聚合物发生降解，质粒被游离出来，因此相对荧光强度几乎保持不变(90%-100%)，这些结果与电泳结果一致。"
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。"
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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。"
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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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