CN114874353B - 基于壳寡糖的非病毒基因药物载体及其制备方法 - Google Patents
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Abstract
本发明属于药物制剂技术领域，具体涉及一种基于壳寡糖的非病毒基因药物载体及其制备方法。该载体由壳寡糖(CSO)和胱胺二丙烯酰胺(CBA)通过Michael加成制备；CBA是一种交联剂，具有还原响应性，可将多个CSO单体通过双键加成组装在一起；CSO‑CBA通过静电作用力与pDNA吸附形成复合物进入细胞，该复合物生物相容性好，在多种细胞中均有很好的转染效率。
Description
技术领域
本发明属于药物制剂技术领域，具体涉及一种基于壳寡糖的非病毒基因药物载体及其制备方法。
背景技术
基因治疗通过纠正或补偿遗传缺陷的方法来改善乃至治疗某些疾病，比如取代病变细胞中的突变基因来治疗血友病、大疱性表皮松解症、囊性纤维化等诸如此类的遗传性疾病，或者向靶细胞递送遗传物质来治疗遗传病。研究发现，基因药物自身在体内很容易降解，所以为了有效地将基因药物递送至靶细胞，开发安全高效的载体体系是十分必要的。目前基因递送载体体系主要分为病毒载体和非病毒载体两类，非病毒载体以成本低、制备简单、生物相容性好的优点被广泛应用，非病毒载体通过增强核酸物质的容纳性，延长体内的循环周期，从而提高治疗效果。然而仅靠单纯的非病毒载体，负载货物的能力有限，靶向性也不足，需要引入新的配体与之进行修饰，改善递送系统的持续释放和靶向性能。
壳聚糖是一类由氨基葡萄糖组成的阳离子聚合物，具有很好的生物相容性和生物降解性。壳聚糖对生物大分子类药物和核酸类物质具有保护作用，然而众所周知，作为共聚体的亲水片段，壳聚糖主要存在两个缺陷：一个是阳离子聚合物自身毒性较大，另一个是在生理pH 7.4时的水溶性差。而低分子量壳聚糖，也就是壳寡糖既能有壳聚糖的优势，又很好的规避潜在的劣势。
发明内容
目的：为了克服现有技术中存在的不足，本发明提供一种基于壳寡糖的非病毒基因药物载体及其制备方法，该载体是可用于细胞中基因药物输送的非病毒载体。本发明的自组装纳米递送系统通过将重复单元1-20的壳寡糖通过交联剂胱胺二丙烯酰胺连接形成聚合物，提高基因药物进入靶细胞的效率。
技术方案：为解决上述技术问题，本发明采用的技术方案为：
一种基于壳寡糖的非病毒基因药物载体(CSO-CBA)，其化学结构式如下：
其中，m为1-20的正整数，x为正整数。
本发明还提供了上述基于壳寡糖的非病毒载体的制备方法，包括如下步骤：由壳寡糖和胱胺二丙酰胺通过迈克尔加成方法获得，壳寡糖和胱胺二丙酰胺的摩尔比为1:0.2-1:1.2，壳寡糖重复单元数为1-20。
壳寡糖表面有丰富的功能基团，可以包裹或吸附目的基因，它的修饰位点主要是伯胺和羟基，可以通过酰胺化或酯化、环氧-胺/羟基偶联、席夫碱形成和迈克尔加成等方法对其进行化学改性，在这里选择了迈克尔加成法进行伯胺修饰。修饰后的壳寡糖可与基因有更好的结合，交联后的壳寡糖/基因复合物经内吞进入细胞，可在细胞还原环境下响应性降解，能更好地释放基因。同时与交联剂胱胺二丙烯酰胺组装，能增加链长，而且经过修饰的壳寡糖与pDNA结合稳定性提高，从而提高转染效率，在L929、A549、4T1和NIH/3T3细胞转染中均得到很好地验证。
具体的，包括以下步骤：
(1)壳寡糖(CSO)，用无水DMSO室温下溶解，加入三乙胺，搅拌均匀，得壳寡糖的DMSO溶液；(三乙胺是为了去掉壳寡糖中的酸根离子，优选的，CSO和三乙胺的摩尔比为1：3)
(2)将胱胺二丙烯酰胺，用无水DMSO室温下溶解，得胱胺二丙酰胺的DMSO溶液；
(3)将步骤(1)的壳寡糖的DMSO溶液和步骤(2)的胱胺二丙酰胺的DMSO溶液室温下混合均匀，转移至耐压瓶中，在反应温度下进行迈克尔加成反应(反应条件优选60℃油浴24小时)，反应结束后调节PH去掉多余的铵根离子(PH优选为4)，接着用分子量截留值3500的透析袋纯水透析，冻干后得成品CSO-CBA。
反应式如下：
上述胱胺二丙烯酰胺(CBA)的合成：胱胺二盐酸盐的水溶液、丙烯酰氯的二氯甲烷溶液和氢氧化钠在冰浴下滴定混匀，接着室温搅拌6小时得到胱胺二丙烯酰胺粗品，再通过萃取、旋蒸和干燥等一系列方式得到纯品。
有益效果：本发明以壳寡糖(重复单元1-20)为基础，使用交联剂胱胺二丙烯酰胺通过Michael加成连接形成复合物后(壳寡糖和胱胺二丙酰胺的摩尔比为1:0.2-1:1.2)，可与质粒pDNA在静电力的作用下组装。该基于壳寡糖的基因载体在L929、A549、4T1和NIH/3T3细胞中均能将核酸物质递送至胞内，转染效果明显。
附图说明
图1是本发明依照实施方式例2合成的壳寡糖载体CSO-CBA的傅里叶红外光谱图。
图2是本发明依照实施方式例4制备CSO-CBA载体与pDNA自组装纳米粒结果图；
图3是本发明依照实施方式例5的方法做出的非病毒载体CSO-CBA生物相容性的表征：(a)CSO-CBA的细胞毒性测试。(b)CSO-CBA的溶血实验；
图4是本发明按照实施方式例6针对L929、A549、4T1和NIH/3T3细胞的转染效果的表达。
具体实施方式
本发明不局限于下列具体实施方式，本领域一般技术人员根据本发明公开的内容，可以采用其他多种具体实施方式实施本发明的，或者凡是采用本发明的设计结构和思路，做简单变化或更改的，都落入本发明的保护范围。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
本发明下面结合实施例作进一步详述：
基于壳寡糖的非病毒载体CSO-CBA的制备方法如下：将胱胺二盐酸盐和丙烯酰氯合成得到胱胺二丙酰胺，接着与壳寡糖通过迈克尔加成反应连接，再通过调节PH，透析冻干后得到纯品CSO-CBA载体。
自组装纳米共递送系统均是新鲜制备，制备方案具体如下：将pDNA溶液在涡旋状态下逐滴加入等体积的CSO-CBA载体溶液中，涡旋混匀，室温放置20min，便得到CSO-CBA/pDNA。
上述非病毒纳米载体可用于细胞中基因输送。
实施例1
CSO-CBA的合成：
取CSO粉末160mg(分子量970Da)，加入0.5mL无水DMSO和413mL三乙胺溶解(CSO和三乙胺的摩尔比为1：3)。取CBA粉末52mg，加入适量1mLDMSO溶解，缓慢加入CSO溶液中(CSO和CBA的摩尔比为1:1.2)，所有操作均在25℃环境下进行。接着将溶液PH调为4，放入耐压瓶中60℃油浴24小时。使用分子量截留值为3500的透析袋进行透析，真空冷冻干燥后得到纯品CSO-CBA载体，收率为18％。
实施例2
CSO-CBA聚合物的结构鉴定。
CSO-CBA聚合物通过傅里叶红外光谱来鉴定结构。图1显示，CSO-CBA中属于CBA的碳碳双键特征峰消失，说明CSO-CBA的成功合成。
实施例3
壳寡糖载体与pDNA自组装纳米粒的制备。
制备方案如下所述：pDNA溶液处于涡旋状态，将CSO-CBA溶液缓慢逐滴加入，再一次涡旋将其混匀，室温放置20min即可。
实施例4
自组装纳米粒的成纳米结果表征。
CSO-CBA/pDNA的成纳米结果通过琼脂糖凝胶电泳来表征。采用1％的琼脂粉制备的凝胶，电解质选用TAE缓冲液，将CSO-CBA与pDNA以五种不同质量比结合，添加烯释液和上样显色剂，最终总体积统一为12uL。上样，通电，110v，30min。图2显示不同质量比的CSO-CBA均与pDNA成功结合在上样孔处，说明自组装纳米粒的成功合成。
实施例5
CSO-CBA自组装纳米递送系统的生物相容性表征。
将正常培养的NIH/3T3细胞用PBS清洗、胰酶消化后，1500rpm离心3min沉淀细胞，之后使用细胞计数板计数，将细胞以8000个/mL的密度接种于96孔板中，37℃、4％CO2，孵育12小时。弃去旧培养基，加入培养基烯释的不同浓度的CSO-CBA样品(0、10、20、30、50、100μg/mL)，以1％的曲拉通作为阳性对照，每个样设置4个复孔。相同孵育条件下再次培养24小时。每孔加入20μL的MTT溶液，4h培养后弃去孔内液体，加入无水DMSO，摇床培养5min后，使用酶标仪测定OD570nm。图3(A)中NIH/3T3细胞形态没有发生改变，数量没有发生减少，数据显示CSO-CBA对NIH/3T3细胞的毒性很低，样品在低浓度时也有细胞增殖迹象。
从正常小鼠的眼眶静脉中取出新鲜血液，用抗凝管收集。冷冻离心机2000rpm，4℃条件下离心10min，收集红细胞。取300μL预冷的PH 7.4的PBS清洗红细胞3次，4℃，2000rpm离心5min，再次弃上清。然后往处理好的红细胞加入PH 7.4的PBS，配置成20％的红细胞悬液。1％的曲拉通和PH 7.4的PBS分别作为阳性对照和阴性对照。CSO-CBA用PH 7.4的PBS烯释成浓度为20、50、100、200、400、800μg/mL的样品。往准备好的1mL的阴性对照、阳性对照、6个浓度梯度的样品中各加入20μL 20％的红细胞悬液。之后在37℃条件下孵育2小时。最后将这些样品4℃，2000rpm离心10min并拍照。图3(B)显示CSO-CBA的溶血率均低于5％，说明红细胞没有发生破裂溶解现象，该载体生物相容性好。
实施例6
CSO-CBA/pDNA对L929、A549、4T1和NIH/3T3细胞的转染效率研究。
将正常培养的L929细胞用PBS清洗、胰酶消化后，1500rpm离心3min沉淀细胞，之后使用细胞计数板计数，将细胞以106个/mL的密度接种于24孔板中，37℃、4％CO2，孵育16小时左右。将pDNA在涡旋状态下加入等量的CSO-CBA溶液中，涡旋混匀，放置20min后替换24孔板中原先培养液，继续孵育4小时后再替换成新鲜的细胞培养基。24小时后对每个孔采用倒置荧光显微镜进行拍照。A549、4T1和NIH/3T3细胞采用相同方法操作。图4显示CSO-CBA载体在L929、A549、4T1和NIH/3T3细胞的转染效果比单纯的CSO效果好。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种基于壳寡糖的非病毒载体，其特征在于：其化学结构式如下：
其中，m为1-20的正整数，x为正整数；
由壳寡糖和胱胺二丙烯酰胺通过Michael加成获得，壳寡糖和胱胺二丙酰胺的摩尔比为1:0.2-1:1.2，壳寡糖重复单元数为1-20。
2.根据权利要求1所述的基于壳寡糖的非病毒载体的制备方法，其特征在于，还包括以下步骤：（1）壳寡糖用无水DMSO室温下溶解，加入三乙胺，搅拌均匀，得壳寡糖的DMSO溶液；
（2）将胱胺二丙烯酰胺，用无水DMSO室温下溶解，得胱胺二丙酰胺的DMSO溶液；
（3）将步骤（1）的壳寡糖的DMSO溶液和步骤（2）的胱胺二丙酰胺的DMSO溶液室温下混合均匀，转移至耐压瓶中，在反应温度下进行迈克尔加成反应，反应结束后调节PH去掉多余的铵根离子，接着用分子量截留值3500的透析袋纯水透析，冻干后得成品CSO-CBA。
3.根据权利要求2所述的基于壳寡糖的非病毒载体制备方法，其特征在于，步骤（1）中壳寡糖和三乙胺的摩尔比为1:3。
4.根据权利要求2所述的基于壳寡糖的非病毒载体制备方法，其特征在于，步骤（3）中反应条件为60℃油浴24小时。
5.根据权利要求2所述的基于壳寡糖的非病毒载体制备方法，其特征在于，步骤（3）中PH调节为4。
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