CN101235102B - 一种叶酸偶联羟丙基壳聚糖纳米微粒的制备方法 - Google Patents
一种叶酸偶联羟丙基壳聚糖纳米微粒的制备方法 Download PDFInfo
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Abstract
一种叶酸偶联羟丙基壳聚糖纳米微粒的制备方法，其制备方法为：将羟丙基壳聚糖水溶液与等体积叶酸溶液混合，70℃下避光充分反应；产物透析24小时，透析后产物冷冻干燥即得到叶酸偶联羟丙基壳聚糖淡黄色固体；将叶酸偶联羟丙基壳聚糖水溶液的pH调节至5-6，滴加多聚磷酸钠溶液，待溶液黄色消退微显乳光时停止滴加，即得到纳米微粒。制备的叶酸偶联羟丙基壳聚糖纳米微粒对肿瘤细胞具有明显的靶向作用，且粒径可控、可生物降解，无毒无害，方法温和，可用于作为蛋白质类药物的靶向缓释载体。
Description
技术领域
本发明涉及一种对肿瘤细胞具有明显靶向作用，粒径可控、稳定性较好的、可用作靶向药物载体的叶酸偶联羟丙基壳聚糖纳米微粒的制备方法。
背景技术
抗肿瘤药物往往选择性不高，在杀伤肿瘤细胞的同时也可能杀死机体正常细胞，毒副作用较大。叶酸受体是肿瘤选择性药物输送的特异性靶点，在多种肿瘤细胞(如卵巢癌、结肠直肠癌、肺癌、肾细胞癌等)膜表面的活性和数量明显高于一般正常细胞。由于受体与其配体的结合具有特异性、选择性、亲合力强等特点，利用配体作为药物载体或与载体偶联，通过受体介导作用，可增加药物在病灶局部的浓度，提高疗效，降低毒副作用，达到靶向治疗的目的。因此，作为叶酸受体的配体，叶酸在肿瘤治疗及预防中越来越显示出其应用价值。
壳聚糖是一种带正电的天然多糖，本身具有无毒、无刺激性、无致敏性、无致突变的性质，且有良好的生物相容性和生物降解性。将壳聚糖及其衍生物制备成纳米微球或微粒，作为药物传递和控释载体，具有药物包封率高、稳定性强和释药时间长等优点，可有效避免药物浓度的剧烈变化引起的副作用和治疗效果不理想等问题，近年来在缓释给药系统中得到了越来越广泛的应用。
目前，将叶酸分子偶联到药物载体上的方法，多是将叶酸制备成叶酸活性酯或者使用大量有机溶剂如二甲基亚砜作为液相，来进行反应。有机溶剂在人体内不能正常代谢，有一定毒害作用。通过将壳聚糖亲水改性，直接在水溶液中与叶酸偶联即可避免有机溶剂的使用。
发明内容
本发明的目的是提供一种叶酸偶联羟丙基壳聚糖纳米微粒的制备方法，叶酸偶联羟丙基壳聚糖纳米微粒可用作包裹蛋白类药物的肿瘤靶向载体。
本发明是这样来实现的，其特征在于：将羟丙基壳聚糖配制成浓度为2.0mg/ml的水溶液，以0.22μm滤纸过滤，加入等体积120μg/ml的叶酸溶液，持续搅拌条件下加热至70℃，避光反应70分钟，产物冷却至室温，透析24小时，透析过程中每隔2小时取透析液，于紫外分光光度计上测定游离叶酸的含量，监测透析效果，透析后产物冷冻干燥，得到淡黄色叶酸偶联羟丙基壳聚糖固体。将叶酸偶联羟丙基壳聚糖配制成浓度分别为0.8、1.0、1.5、2.0mg/ml的水溶液，调节pH于5-6之间，将多聚磷酸钠配制成浓度分别为0.4、0.8、1.0mg/ml的水溶液，在持续磁力搅拌下，向叶酸偶联羟丙基壳聚糖水溶液中缓慢滴加多聚磷酸钠溶液，待反应液黄色消退微显乳光为止。
本发明的技术效果是：反应条件温和，生产工艺简单，易于工业化生产，得到的叶酸偶联羟丙基壳聚糖纳米微粒对肿瘤细胞具有明显靶向作用，粒径可控、可生物降解，无毒无害，可用于作为蛋白质类药物的靶向缓释载体。
附图说明
图1为实施例1制备的叶酸偶联羟丙基壳聚糖纳米微粒的透射电镜图；
图2为实施例2制备的叶酸偶联羟丙基壳聚糖纳米微粒的透射电镜图。
具体实施方式
实施例1
本发明以脱乙酰度大于90％的壳聚糖为原料，在碱性条件下使其与过量环氧丙烷发生反应，生成水溶性的羟丙基壳聚糖。将羟丙基壳聚糖配制成浓度为2.0mg/ml的水溶液，以0.22μm滤纸过滤，加入等体积120μg/ml的叶酸溶液，持续搅拌条件下加热至70℃，避光反应70分钟，产物冷却至室温，透析24小时，透析过程中每隔2小时取透析液，于紫外分光光度计上测定游离叶酸的含量，监测透析效果，透析后产物冷冻干燥，得到淡黄色叶酸偶联羟丙基壳聚糖。量取5ml的1.0mg/ml叶酸偶联羟丙基壳聚糖水溶液于三口烧瓶中，调节pH于5-6之间，在持续磁力搅拌条件下，缓慢滴加0.8mg/ml的多聚磷酸钠溶液，滴加速度为20-40滴/min，待溶液黄色消退微显乳光时停止滴加。
由图1的透射电镜照片可以看出，该叶酸偶联羟丙基壳聚糖纳米微粒形状呈现较规则的球形，且粒径在140nm左右。
实施例2
叶酸偶联羟丙基壳聚糖的制备同实施例1。量取5ml的2.0mg/ml叶酸偶联羟丙基壳聚糖水溶液于三口烧瓶中，调节pH于5-6之间，在持续磁力搅拌条件下，缓慢滴加0.8mg/ml的多聚磷酸钠溶液，滴加速度为20-40滴/min，待溶液黄色消退微显乳光时停止滴加。
由图2的透射电镜照片可以看出，该叶酸偶联羟丙基壳聚糖纳米微粒呈现较规则的球形，且粒径在360nm左右。实施例2中微粒粒径比实施例1中的粒径有所增大，这主要是由于反应物叶酸偶联羟丙基壳聚糖浓度增大造成的。
Claims (1)
1.一种叶酸偶联羟丙基壳聚糖纳米微粒的制备方法，其特征在于：将羟丙基壳聚糖配制成浓度为2.0mg/ml的水溶液，以0.22μm滤纸过滤，加入等体积120μg/ml的叶酸溶液，持续搅拌条件下加热至70℃，避光反应70分钟，产物冷却至室温，透析24小时，透析过程中每隔2小时取透析液，于紫外分光光度计上测定游离叶酸的含量，监测透析效果，透析后产物冷冻干燥，得到淡黄色叶酸偶联羟丙基壳聚糖，量取5ml的1.0mg/ml叶酸偶联羟丙基壳聚糖水溶液于三口烧瓶中，调节pH于5-6之间，在持续磁力搅拌条件下，缓慢滴加0.8mg/ml的多聚磷酸钠溶液，滴加速度为20-40滴/min，待溶液黄色消退微显乳光时停止滴加。
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| EP0579996A1 (de) * | 1992-07-13 | 1994-01-26 | EPROVA Aktiengesellschaft | 5,10-Methylentetrahydrofolsäure-Cyclodextrin-Einschlussverbindungen |
| CN1287127A (zh) * | 2000-04-17 | 2001-03-14 | 上海医科大学 | 叶酸－多聚糖复合物,其制备方法和以该复合物为活性成分的药物组合物 |
| CN1472235A (zh) * | 2003-06-26 | 2004-02-04 | 上海复康医药科技发展有限公司 | 叶酸-聚二醇类化合物及其药物复合物 |
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| EP0579996A1 (de) * | 1992-07-13 | 1994-01-26 | EPROVA Aktiengesellschaft | 5,10-Methylentetrahydrofolsäure-Cyclodextrin-Einschlussverbindungen |
| CN1287127A (zh) * | 2000-04-17 | 2001-03-14 | 上海医科大学 | 叶酸－多聚糖复合物,其制备方法和以该复合物为活性成分的药物组合物 |
| CN1472235A (zh) * | 2003-06-26 | 2004-02-04 | 上海复康医药科技发展有限公司 | 叶酸-聚二醇类化合物及其药物复合物 |
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| Title |
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| 张良珂.叶酸偶联白蛋白纳米粒肿瘤细胞靶向性研究.四川大学学报35 2.2004,35(2),165-168. |
| 张良珂.叶酸偶联白蛋白纳米粒肿瘤细胞靶向性研究.四川大学学报35 2.2004,35(2),165-168. * |
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