CN105820334A - 一种基于氨基酸的聚两性离子纳米颗粒的制备方法 - Google Patents
一种基于氨基酸的聚两性离子纳米颗粒的制备方法 Download PDFInfo
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Abstract
本发明涉及一种基于氨基酸的聚两性离子纳米颗粒的制备方法，采取两步法合成中间段疏水、两端链段含聚两性离子的三嵌段共聚物，首先将N,N‑双丙烯酰基胱胺与脂肪族伯胺溶解在去离子水与乙醇的混合溶剂中，通过亲核加成得到交替共聚物，然后在上述共聚物中添加N,N‑双丙烯酰基胱胺与赖氨酸的混合物溶液，继续进行反应，得到含N,N‑双丙烯酰基胱胺、脂肪族伯胺和赖氨酸的三嵌段共聚物，三嵌段共聚物在水溶液中自组装形成纳米颗粒，具有pH和还原敏感性，以及优异的抗蛋白质非特异吸附性能，纳米颗粒无细胞毒性，作为抗癌药物载体具有应用前景。
Description
技术领域
本发明涉及生物医用材料技术领域，尤其涉及一种基于赖氨酸的聚两性离子纳米颗粒及其制备方法。
背景技术
近年来，纳米技术在医学上的应用特别是纳米药物的发展推动了各种各样的纳米药物传输体系的发展，尤其是以脂质体和聚合物为基础的纳米颗粒。然而纳米颗粒在药物传输体系上的应用正面临着两种瓶颈，首先，纳米颗粒在肿瘤组织的累积较差，其原因是纳米颗粒易被内皮网状系统(RES)所清除且缺少肿瘤靶向的能力，这不可避免的导致不良的副作用。另一重要的问题就是以纳米颗粒为基础的药物传输体系的生物利用度。高的利用度是指一旦纳米颗粒被附着于固体肿瘤上，包裹药物的纳米颗粒就会以一定的速率释放药物；然而，低的药物利用度会导致药物利用率下降并且也会增加多药耐药性发生的可能性。
智能药物传输体系越来越引起人类的关注，它们用智能响应性原理来实现药物的传输和靶向释放，同时最大化提高了药物利用度，最小化了一些副反应。典型的刺激敏感性药物传输体系可大致分为两类，一类是来自外界的刺激，另一类是生物体本身的刺激。最具代表性的生物体内的刺激有pH的变化，氧化还原的变化，离子强度和酶的作用。所有的这些刺激中pH和还原敏感性备受世人的青睐，一方面，在生物体内不同部位pH具有明显的变化，如血液pH7.4，早期内涵体pH为6.0～6.5，晚期内涵体pH为5.0～6.0，肿瘤细胞外pH为6.5-7.2，正常组织细胞外pH为7.4；因此，智能药物载体一旦被被捕获，将经历pH从7.4到5.0甚至4.5的下降。通过利用pH的不同，药物会尽可能的释放到靶向位点。另外，智能药物载体利用pH的改变，来改变自身的表面的电荷，这样更有利于载体的利用度。因为细胞膜带负电，如果载体在此时带上正电有利于肿瘤细胞的摄取，而在血液中载体带有负电有利于载体在血液中停留，且不易被RES清除。因而制备表面电荷可转化的智能药物载体将显得尤为重要。另一方面，还原型谷胱甘肽在生物体内不同部位也有明显的变化，在细胞内的浓度为2-10mM，明显高于细胞外的浓度(2-20μM)。而且,还原型谷胱甘肽在癌细胞中的浓度是正常细胞的几倍，利用其浓度变化，可促使药物载体结构发生变化，从而将药物尽可能释放在肿瘤靶向位点。
但是目前文献报道的同时具有pH和还原敏感性的聚两性离子的文献较少，且通常所选用的原材料具有不可降解性，在体内排泄困难，不满足人体内使用的要求，因而成为此类材料实际应用的瓶颈问题。
发明内容
为解决上述技术问题，本发明的目的是提供一种基于氨基酸的聚两性离子纳米颗粒的制备方法，该纳米颗粒具有良好的生物相容性、在体内可完全降解无残留。
首先将N,N-双丙烯酰基胱胺与脂肪族伯胺混合，通过亲核加成得到交替共聚物，所用脂肪族伯胺分别为辛胺、十二胺、十四胺或十六胺，投料时N,N-双丙烯酰基胱胺与脂肪族伯胺摩尔比为1：0.8。由于在投料时将N,N-双丙烯酰基胱胺过量，则在形成交替共聚物时，聚合物末端有剩余双键；
然后在上述共聚物中添加N,N-双丙烯酰基胱胺与赖氨酸的混合物溶液，继续进行反应，得到含N,N-双丙烯酰基胱胺、脂肪族伯胺和赖氨酸的三嵌段共聚物；
合成时的溶剂选择体积比为1:2的去离子水与乙醇的混合溶剂，保证反应原料脂肪族伯胺、N,N-双丙烯酰基胱胺和赖氨酸具有良好的相容性，有利于反应进行。
共聚物合成反应完毕，向上述共聚物溶液中滴入超纯水搅拌，然后添加无水乙醚萃取共聚物溶液，除去未反应的N,N-双丙烯酰基胱胺和伯胺；
最后使用透析袋将水层溶液在纯水中透析72小时，得到含氨基酸的聚两性离子纳米颗粒分散液。透析的目的在于除去未反应的小分子和低聚物，因为分子量太低的聚合物不易形成胶束，透析袋有各种规格，选择3500的可达到目的。
本发明还提供一种基于氨基酸的聚两性离子纳米颗粒在制备化疗药物载体中的应用，用所述任何方法所制得纳米颗粒分散液，将其冷冻干燥后得到纳米颗粒粉末，称取该粉末加入到盐酸阿霉素溶液中，搅拌8小时后在超纯水中透析24小时，得到负载阿霉素的纳米颗粒。
借由上述方案，本发明至少具有以下优点：
1.所合成的三嵌段共聚物中间段含N,N-双丙烯酰基胱胺与脂肪族伯胺，呈疏水性；左右两端含N,N-双丙烯酰基胱胺和赖氨酸，其中赖氨酸同时含有氨基和羧基，因而具有两性离子性能，呈亲水性，因而三嵌段共聚物在纯水中通过自组装形成纳米颗粒。
2.纳米颗粒有灵敏的pH敏感性，在肿瘤细胞内部的弱酸性条件下，胶束结构发生变化，可促使药物释放；
3.共聚物中含有N,N-双丙烯酰基胱胺结构单元，其中的双硫键具有还原响应性，能在还原型谷胱甘肽的肿瘤环境下断裂，因而能够将药物彻底释放到肿瘤细胞靶向位点；
4.纳米颗粒外层的聚两性离子赋予其优异的抗蛋白质非特异吸附性能，因而具有特殊的抗污染性能，可保持纳米颗粒在血液中的稳定性；
5.纳米胶束无细胞毒性，满足人体使用的安全性标准；
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以本发明的较佳实施例并配合附图详细说明如下。
附图说明
图1本发明中一种基于氨基酸的聚两性离子纳米颗粒的制备反应示意图；
图2本发明中实施例2所得基于赖氨酸的聚两性离子纳米颗粒的透射电镜照片；
图3实施例2所得基于赖氨酸的聚两性离子纳米颗粒在10mM的谷胱甘肽溶液中不同时间下的粒径分布；
图4实施例2所得基于赖氨酸的聚两性离子纳米颗粒在不同蛋白质溶液中的粒径变化；
图5实施例2所得基于赖氨酸的聚两性离子纳米颗粒的细胞毒性结果。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：N,N-双丙烯酰基胱胺的合成：
将11.6g的胱胺二盐酸盐加入到250mL单口烧瓶中，然后加入50mL蒸馏水将胱胺二盐酸盐搅拌溶解。将烧瓶置于0℃的冰水混合物中；另外称取8g的氢氧化钠固体溶于20mL的蒸馏水中，将溶解好的氧氧化钠溶液一次性加入到单口烧瓶中，将预先精制好的19mL丙烯酰氯与3mL二氯甲烷混合成溶液，通过恒压滴液漏斗滴加到单口烧瓶中，在40分钟内滴加完毕后，控制反应在25℃下反应16h。产物过滤、用去离子水洗3次，最后用乙酸乙酯重结晶，在真空干燥箱中干燥24h得到产物。
实施例2：基于氨基酸的聚两性离子纳米颗粒的制备:
在25mL的三口烧瓶中，将蒸馏水1.7mL和无水乙醇3.3mL混合，将N,N-双丙烯酰基胱胺0.26g和辛胺0.103g溶解于上述溶液中，调节pH值范围为9.5～10,在氮气保护下，通过油浴锅加热反应液到40℃,恒温条件下搅拌反应48小时；
将N,N-双丙烯酰基胱胺0.26g和赖氨酸0.175g溶于蒸馏水1.7mL和无水乙醇3.3mL的混合溶剂，然后添加到上述共聚物溶液中，调节pH值为10～11，在55℃恒温条件下搅拌，继续反应72小时，得到含N,N-双丙烯酰基胱胺、辛胺和赖氨酸的三嵌段共聚物。
向上述共聚物溶液中滴入超纯水，搅拌5小时，然后用5倍于共聚物溶液体积的无水乙醚，均分三次萃取共聚物中残留的N,N-双丙烯酰基胱胺和脂肪族伯胺，分离弃去乙醚层；使用截留分子量为3500的透析袋，将水层溶液在纯水中在30℃温度下透析72小时，得到含氨基酸的聚两性离子纳米颗粒分散液。
实施例3
同实施例2，但是用十二胺0.148g代替辛胺0.103g,其余条件和操作不变，合成得到含N,N-双丙烯酰基胱胺、十二胺和赖氨酸的三嵌段共聚物，进一步合成纳米颗粒。
实施例4
同实施例2，但是用十四胺0.170g代替辛胺0.103g,其余条件和操作不变，合成得到含N,N-双丙烯酰基胱胺、十四胺和赖氨酸的三嵌段共聚物，进一步合成纳米颗粒。
实施例5
同实施例2，但是用十六胺0.193g代替辛胺0.103g,其余条件和操作不变，合成得到含N,N-双丙烯酰基胱胺、十六胺和赖氨酸的三嵌段共聚物，进一步合成纳米颗粒。
实施例6
同实施例2，但是用十六胺0.193g代替辛胺0.103g,并且改变赖氨酸的量为0.204g，其余条件和操作不变，合成得到含N,N-双丙烯酰基胱胺、十六胺和赖氨酸的三嵌段共聚物，进一步合成纳米颗粒。
表1三元共聚物合成配方一览表
实施例7
将实施例2所得纳米颗粒分散液20μL滴到铜网上，然后往铜网上滴加一滴1％磷钨酸溶液进行染色，铜网在室温下自然干燥后，用透射电子显微镜在高真空状态下观察纳米颗粒的形貌，透射电镜加速电压为120kV。图2中显示实施例2所得纳米颗粒形貌呈球形，可见粒径分布较均一。
实施例8：载药纳米颗粒的制备
用权利要求1～5所述任何一种方法所制得纳米颗粒分散液，将其冷冻干燥3天，得到纳米颗粒粉末，称取该粉末20mg,加入到5mL浓度为2mg/mL的盐酸阿霉素溶液中，搅拌8小时后在超纯水中透析24小时，得到负载阿霉素的纳米颗粒。
实施例9：纳米颗粒的抗牛血清白蛋白的非特异吸附性能
将实施例2中所得纳米颗粒分散液分别置于pH＝7.4的PBS缓冲溶液、含有浓度为45g/L的牛血清白蛋白(BSA)和10％胎牛血清(FBS)溶液中，分别孵育12h,24h和48h，利用激光光散射仪监控纳米颗粒粒径的变化，观察抗牛血清白蛋白非特异吸附性能。
实施例10：聚两性离子纳米颗粒的生物相容性
在温度为37℃的水浴锅中，迅速解冻-80℃冻存的3T3细胞和Hela细胞，将其移入到含有7mL的RPMI-1640培养液的离心管中，以800rpm速度离心，用含有10％小牛血清的RPMI-1640培养液吹打细胞制成单细胞悬液，将其移入到50mL的培养瓶中，在37℃，5％CO2孵箱中培养。采用MTT法对其形成的纳米粒子的细胞毒性进行测试，以约2.0×104/mL将小鼠成纤维细胞接种于96孔板，每孔100μL，培养24h，吸出每孔中的原培养液，每孔加入100μL的阴性对照液(pH＝7.4的10％小牛血清的RPMI-1640培养液)、阳性对照液(0.64％苯酚培养基)、样品组(样品组含pH＝7.4的10％小牛血清的RPMI-1640培养液)，继续置于37℃、5％CO2培养箱中培养48h。每组设4个平行孔。取出培养板后通过倒置显微镜观察、评价细胞生长状况。后加入MTT20μL，继续培养4h后，将培养板中的小孔内的液体吸尽后，加入二甲基亚砜，用酶标仪于570nm处测其吸光度值(A)，计算细胞存活率。3T3和Hela两种细胞在不同浓度的纳米胶束溶液中的存活率在95％以上，符合生物相容性的标准。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Claims (6)
1.一种基于氨基酸的聚两性离子纳米颗粒的制备方法，其特征是采取两步法合成三嵌段共聚物，中间段疏水，两端链段含聚两性离子，具有亲水性，三嵌段共聚物在水溶液中自组装形成纳米颗粒，制备依次包括以下步骤：
1)将N,N-双丙烯酰基胱胺与脂肪族伯胺溶解在体积比为1：2的去离子水与乙醇的混合溶剂中，浓度为6.8wt％～8.3wt％,调节pH值为9.5～10，通过亲核加成得到交替共聚物，使聚合物末端有剩余双键；
2)在上述共聚物中添加N,N-双丙烯酰基胱胺与赖氨酸的混合物溶液，继续进行反应，得到含N,N-双丙烯酰基胱胺、脂肪族伯胺和赖氨酸的三嵌段共聚物；
3)向上述共聚物溶液中滴入超纯水，搅拌5小时，然后添加无水乙醚萃取共聚物溶液3次，分离弃去乙醚层；
4)使用截留分子量为3500的透析袋，将水层溶液在纯水中在30℃温度下透析72小时，得到含赖氨酸的聚两性离子纳米颗粒分散液。
2.根据权利要求1所述一种基于氨基酸的聚两性离子纳米颗粒的制备方法，其特征是在步骤1)中，所用脂肪族伯胺分别为辛胺、十二胺、十四胺或十六胺，投料时N,N-双丙烯酰基胱胺与脂肪族伯胺摩尔比为1：0.8。
3.根据权利要求1所述一种基于氨基酸的聚两性离子纳米颗粒的制备方法，其特征是在步骤1)中，共聚反应在氮气保护下、在40℃恒温条件下搅拌反应48小时。
4.根据权利要求1所述一种基于氨基酸的聚两性离子纳米颗粒的制备方法，其特征是在步骤2)中，N,N-双丙烯酰基胱胺与赖氨酸按摩尔比为1:1.2～1:1.4，溶解在体积比为1：2的去离子水与乙醇的混合溶剂中，浓度为8.0wt％～8.5wt％,调节pH值为10～11，在55℃恒温条件下搅拌反应72小时。
5.根据权利要求1所述一种基于氨基酸的聚两性离子纳米颗粒的制备方法，其特征是在步骤3)中，用5倍于共聚物溶液体积的无水乙醚，均分三次萃取共聚物中残留的N,N-双丙烯酰基胱胺和脂肪族伯胺。
6.一种基于氨基酸的聚两性离子纳米颗粒在制备化疗药物载体中的应用，其特征是用权利要求1～5所述任何一种方法所制得纳米颗粒分散液，将其冷冻干燥3天，得到纳米颗粒粉末，称取该粉末20mg,加入到5mL浓度为2mg/mL的盐酸阿霉素溶液中，搅拌8小时后在超纯水中透析24小时，得到负载阿霉素的纳米颗粒。
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