CN110575544A - 一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法 - Google Patents

一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法 Download PDF

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CN110575544A
CN110575544A CN201910929623.XA CN201910929623A CN110575544A CN 110575544 A CN110575544 A CN 110575544A CN 201910929623 A CN201910929623 A CN 201910929623A CN 110575544 A CN110575544 A CN 110575544A
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folic acid
solution
chitosan
adriamycin
dissolving
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王秉
徐城锋
曹澳
陈碧玲
万军民
彭志勤
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Zhejiang University of Technology ZJUT
Zhejiang Sci Tech University ZSTU
Zhejiang University of Science and Technology ZUST
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Abstract

本发明涉及医用材料领域，公开了一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法，本发明先制备得到叶酸活性酯，并通过酰胺反应得到了叶酸修饰壳聚糖；向叶酸修饰壳聚糖溶液中加入配制好的阿霉素溶液和聚多巴胺溶液，得到阿霉素‑叶酸‑壳聚糖；最后将核酸适配子AS1411与阿霉素‑叶酸‑壳聚糖混合，离心，冷冻，干燥，得到以叶酸修饰壳聚糖为载体的阿霉素纳米微粒。发明通过制备一种包埋有抗癌药物的聚合物纳米微粒，可实现高效的肿瘤热疗和靶向为一体的治疗手段，增加药物的稳定性，实现主动靶向性，增加与受体的结合能力，从而大大提升对癌细胞的治疗效果。

Description

一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法
技术领域
本发明涉及医用材料理应，尤其涉及一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法。
背景技术
尽管基因治疗在基础研究取得很多成绩，但临床试验研究的结果尚不令人满意，存在的问题主要有基因转染效率低、载体安全性差和基因治疗缺乏靶向性等。因此，研制具有高效转染、安全低毒和器官或细胞特异性的基因载体已成为制约基因治疗发展的瓶颈。
壳聚糖能被人体吸收利用，与人体的组织器官及细胞有良好的生物相容性，无毒，具有生物降解性，降解过程中产生的壳寡糖在体内不积累，几乎无免疫原性，同时具有多种生物活性。壳聚糖对DNA有一定的保护作用，使其免受核酸酶的降解，提高DNA半衰期，延长基因表达时间。用壳聚糖作基因载体，能转载多种基因药物，具有靶向、降低毒副作用、自身安全无毒等优点。
随着聚合物纳米粒子作为药物载体在生物医药方面的广泛应用，如何让聚合物纳米粒子同时兼具靶向性、光热效应，改善其在药物靶向和代谢稳定性方面的不足等问题已成为关注的焦点。与此同时，各种多功能聚合物纳米粒子作为一种药物输送载体，不仅要使药物的毒性最小化，还要改善其稳定性以及在所需部位的保留时间。因此，研究一种同时兼具靶向性、肿瘤热疗为一体的聚合物纳米粒子迫在眉睫。
发明内容
为了解决上述技术问题，本发明提供了一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法，本发明首先制备得到了叶酸活性酯，并通过酰胺反应得到了叶酸修饰壳聚糖；向叶酸修饰壳聚糖溶液中加入配制好的阿霉素溶液和聚多巴胺溶液，得到阿霉素-叶酸-壳聚糖；最后最后将核酸适配子AS1411与阿霉素-叶酸-壳聚糖混合，离心，冷冻，干燥，得到阿霉素纳米微粒。
本发明的具体技术方案为：一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法，包括以下步骤：
1)将阿霉素溶于水中，配得浓度为4-6wt％的阿霉素溶液；将聚多巴胺溶于水中，配得浓度为2-4wt％的聚多巴胺溶液；将溶液预冷，备用；
2)称取1-1.2g叶酸置于容器中，将其溶解于无水DMSO，然后加入0.67-0.77g EDC和0.4-0.5g NHS，在室温下于暗室搅拌1-3h使叶酸充分溶解；
在步骤2)中，加入EDC和NHS的作用是活化叶酸表面的羧基。
3)得到了叶酸活性酯的DMSO溶液，溶液为酒红色；
步骤3)中，本发明得到叶酸活化酯，方便进一步与壳聚糖偶联。
4)称取1-1.4g壳聚糖粉末，溶解于1.5-2.5％乙酸溶液中混合均匀，并调节pH＝5.5—6.5，超声后得到壳聚糖溶液；
在步骤4)中，加入带正电及生物相容性好的壳聚糖对带负电荷的肿瘤细胞有好的粘附性能。
5)在暗室中，搅拌条件下将壳聚糖溶液缓慢加入40-60mL含叶酸活性酯的二甲基亚砜溶液，室温下避光反应12-18h；
6)然后用1mL注射器逐滴加入NaOH溶液(0.1M)，将其pH调至9.0，有黄色沉淀析出；
7)将步骤6)所得反应中产物装入透析袋分别用磷酸缓冲液和去离子水透析48-60h，冷冻干燥后得到叶酸-壳聚糖粉末；
在步骤7)中，本发明成功将叶酸修饰到壳聚糖表面。
8)将所得叶酸-壳聚糖粉末溶于水中配成1.5-2.5mg/mL的叶酸-壳聚糖溶液并调节pH至7-7.5，取3—5mL叶酸-壳聚糖溶液，磁搅拌下将步骤1)所得阿霉素溶液600-700μL滴加到叶酸-壳聚糖溶液中并超声；
9)将步骤8)所得溶液加入到6-10mL步骤1)所得聚多巴胺溶液中，超声混合均匀；
在步骤9)中，聚多巴胺溶液作为分散剂，聚多巴胺具有良好的亲水性、稳定性和生物相容性，可用于聚合物粒子在溶液中的分散。
10)取1OD的核酸适配子AS1411溶于5—8mL水中，然后加入8-12mM三(2-羧乙基)膦反应1-2h，再与步骤9)所得产物混合搅拌过夜，最后经离心、洗涤、冷冻干燥处理，制得阿霉素纳米微粒。
在步骤10)中，将核酸适配子AS1411连接在纳米粒子上，使其能够与多数肿瘤细胞表面过表达的核仁素特异性结合，从而实现主动靶向，并介导纳米粒子靶向进胞。
作为优选，步骤2)中，搅拌速度为200-400r/min。
作为优选，步骤5)中，搅拌速度300r/min下进行避光反应。
作为优选，步骤7)中，所述透析袋的截留分子量为8000-14000Da。
作为优选，步骤10)中，离心转速为16000-18000r/min，离心时间为15-20min。
与现有技术对比，本发明的有益效果是：
1.在步骤1)中，通过将抗癌药阿霉素溶解于去水中并充分搅拌使其分散均匀，增强其在后期的包封率以及稳定性。
2.在步骤4)中，壳聚糖能被人体吸收利用，与人体的组织器官及细胞有良好的生物相容性，无毒，具有生物降解性，降解过程中产生的壳寡糖在体内不积累，几乎无免疫原性，保持壳聚糖带正电及生物相容性好的特点，同时具有较好的水溶性，通过壳聚糖修饰改变纳米载体的表面电性，增加载体对肿瘤细胞的粘附性。
3.在步骤9)中，聚多巴胺具有良好的亲水性、稳定性和生物相容性，可用于聚合物粒子在溶液中的分散。
4.在步骤10)中，以适配子AS1411为靶向配体，它能够与大多数肿瘤细胞表面过表达的核仁素特异性结合，介导纳米粒子入胞，实现纳米粒子的主动靶向。
5.发明通过制备一种包埋有抗癌药物的聚合物纳米微粒，可实现高效的肿瘤热疗和靶向为一体的治疗手段，增加药物的稳定性，实现主动靶向性，增加与受体的结合能力，从而大大提升对癌细胞的治疗效果。
6.发明通过构建一种安全，无毒性，并且能够高效运输光敏剂的药物载体，能够提高药物的稳定性，便于贮存。
具体实施方式
下面结合实施例对本发明作进一步的描述。
实施例1
1)将阿霉素溶于水中，配得浓度为4wt％的阿霉素溶液；将聚多巴胺溶于水中，配得浓度为2wt％的聚多巴胺溶液；将溶液预冷，备用；
2)称取1g叶酸置于容器中，将其溶解于无水DMSO，然后加入0.67g EDC和0.4gNHS，在室温下于暗室搅拌1h使叶酸充分溶解；
3)得到了叶酸活性酯的DMSO溶液，溶液为酒红色；
4)称取1g壳聚糖粉末，溶解于1.5％乙酸溶液中混合均匀，并调节pH＝5.5，超声后得到壳聚糖溶液；
5)在暗室中，搅拌条件下将壳聚糖溶液缓慢加入40mL含叶酸活性酯的二甲基亚砜溶液，室温下避光反应12h；
6)然后用1mL注射器逐滴加入NaOH溶液(0.1M)，将其pH调至9.0，有黄色沉淀析出；
7)将步骤6)所得反应中产物装入透析袋分别用磷酸缓冲液和去离子水透析48h，冷冻干燥后得到叶酸-壳聚糖粉末；
8)将所得叶酸-壳聚糖粉末溶于水中配成1.5mg/mL的叶酸-壳聚糖溶液并调节pH至7，取3mL叶酸-壳聚糖溶液，磁搅拌下将步骤1)所得阿霉素溶液600μL滴加到叶酸-壳聚糖溶液中并超声；
9)将步骤8)所得溶液加入到6mL步骤1)所得聚多巴胺溶液中，超声混合均匀；
10)取1OD的核酸适配子AS1411溶于5mL水中，然后加入8mM三(2-羧乙基)膦反应1h，再与步骤9)所得产物混合搅拌过夜，最后经离心、洗涤、冷冻干燥处理，制得阿霉素纳米微粒。
对叶酸进行红外检测，可从检测中看到叶酸具有3个强吸收峰，它们分别是1694cm-1处叶酸分子中标志性的C＝O结构，即-COOH的吸收峰，1606cm-1处叶酸分子中喋呤环上的-NH2基团，以及1484cm-1处叶酸分子中的苯环。对壳聚糖进行红外检测，壳聚糖的红外光谱中，由于N-H和-OH两个基团的伸缩振动，造成两个吸收峰相叠加，显示在3448cm-1处具有较宽的一段吸收。由于C-O-C造成的，壳聚糖在1155cm-1和1072cm-1处的吸收峰。另外，壳聚糖的两个氨基基团特征峰在1642cm-1和1523cm-1处。
对本实施例所得产物进行红外检测，发现其与叶酸和壳聚糖二者的红外吸收峰显示明显的变化，提示并不是简单的物理混合。由于叶酸、壳聚糖发生缩合反应，壳聚糖的部分氨基被叶酸取代，叶酸-壳聚糖的吸收峰减弱，主要显示在1646cm-1和1514cm-1处。此外，叶酸-壳聚糖中有叶酸分子中喋呤环的吸收峰，主要显示在1606cm-1处。以上特征吸收峰的变化证明：壳聚糖上的氨基基团与叶酸的羧基基团发生缩合反应，生成新的化合物叶酸-壳聚糖。
实施例2
1)将阿霉素溶于水中，配得浓度为5wt％的阿霉素溶液；将聚多巴胺溶于水中，配得浓度为3wt％的聚多巴胺溶液；将溶液预冷，备用；
2)称取1.1g叶酸置于容器中，将其溶解于无水DMSO，然后加入0.72g EDC和0.45gNHS，在室温下于暗室搅拌2h使叶酸充分溶解；
3)得到了叶酸活性酯的DMSO溶液，溶液为酒红色；
4)称取1.2g壳聚糖粉末，溶解于2％乙酸溶液中混合均匀，并调节pH＝6.0，超声后得到壳聚糖溶液；
5)在暗室中，搅拌条件下将壳聚糖溶液缓慢加入50mL含叶酸活性酯的二甲基亚砜溶液，室温下避光反应15h；
6)然后用1mL注射器逐滴加入NaOH溶液(0.1M)，将其pH调至9.0，有黄色沉淀析出；
7)将步骤6)所得反应中产物装入透析袋分别用磷酸缓冲液和去离子水透析54h，冷冻干燥后得到叶酸-壳聚糖粉末；
8)将所得叶酸-壳聚糖粉末溶于水中配成2mg/mL的叶酸-壳聚糖溶液并调节pH至7.3，取4mL叶酸-壳聚糖溶液，磁搅拌下将步骤1)所得阿霉素溶液650μL滴加到叶酸-壳聚糖溶液中并超声；
9)将步骤8)所得溶液加入到8mL步骤1)所得聚多巴胺溶液中，超声混合均匀；
10)取1OD的核酸适配子AS1411溶于6.5mL水中，然后加入10mM三(2-羧乙基)膦反应1.5h，再与步骤9)所得产物混合搅拌过夜，最后经离心、洗涤、冷冻干燥处理，制得阿霉素纳米微粒。
实施例3
1)将阿霉素溶于水中，配得浓度为6wt％的阿霉素溶液；将聚多巴胺溶于水中，配得浓度为4wt％的聚多巴胺溶液；将溶液预冷，备用；
2)称取1.2g叶酸置于容器中，将其溶解于无水DMSO，然后加入0.77g EDC和0.5gNHS，在室温下于暗室搅拌3h使叶酸充分溶解；
3)得到了叶酸活性酯的DMSO溶液，溶液为酒红色；
4)称取1.4g壳聚糖粉末，溶解于2.5％乙酸溶液中混合均匀，并调节pH＝6.5，超声后得到壳聚糖溶液；
5)在暗室中，搅拌条件下将壳聚糖溶液缓慢加入60mL含叶酸活性酯的二甲基亚砜溶液，室温下避光反应18h；
6)然后用1mL注射器逐滴加入NaOH溶液(0.1M)，将其pH调至9.0，有黄色沉淀析出；
7)将步骤6)所得反应中产物装入透析袋分别用磷酸缓冲液和去离子水透析60h，冷冻干燥后得到叶酸-壳聚糖粉末；
8)将所得叶酸-壳聚糖粉末溶于水中配成2.5mg/mL的叶酸-壳聚糖溶液并调节pH至7.5，取5mL叶酸-壳聚糖溶液，磁搅拌下将步骤1)所得阿霉素溶液700μL滴加到叶酸-壳聚糖溶液中并超声；
9)将步骤8)所得溶液加入到10mL步骤1)所得聚多巴胺溶液中，超声混合均匀；
10)取1OD的核酸适配子AS1411溶于8mL水中，然后加入12mM三(2-羧乙基)膦反应2h，再与步骤9)所得产物混合搅拌过夜，最后经离心、洗涤、冷冻干燥处理，制得阿霉素纳米微粒。
本发明中所用原料、设备，若无特别说明，均为本领域的常用原料、设备；本发明中所用方法，若无特别说明，均为本领域的常规方法。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制，凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换，均仍属于本发明技术方案的保护范围。

Claims (5)

1.一种以叶酸修饰壳聚糖为载体的阿霉素纳米微粒的制备方法，其特征在于，以g和μL、mL计，包括以下步骤：
1)将阿霉素溶于水中，配得浓度为4-6wt％的阿霉素溶液；将聚多巴胺溶于水中，配得浓度为2-4wt％的聚多巴胺溶液；将溶液预冷，备用；
2)称取1-1.2g叶酸置于容器中，将其溶解于无水DMSO，然后加入0.67-0.77g EDC和0.4-0.5g NHS，在室温下于暗室搅拌1-3h使叶酸充分溶解；
3)得到了叶酸活性酯的DMSO溶液，溶液为酒红色；
4)称取1-1.4g壳聚糖粉末，溶解于1.5-2.5％乙酸溶液中混合均匀，并调节pH＝5.5-6.5，超声后得到壳聚糖溶液；
5)在暗室中，搅拌条件下将壳聚糖溶液缓慢加入40-60mL含叶酸活性酯的二甲基亚砜溶液，室温下避光反应12-18h；
6)然后用1mL注射器逐滴加入NaOH溶液(0.1M)，将其pH调至9.0，有黄色沉淀析出；
7)将步骤6)所得反应中产物装入透析袋分别用磷酸缓冲液和去离子水透析48-60h，冷冻干燥后得到叶酸-壳聚糖粉末；
8)将所得叶酸-壳聚糖粉末溶于水中配成1.5-2.5mg/mL的叶酸-壳聚糖溶液并调节pH至7-7.5，取3-5mL叶酸-壳聚糖溶液，磁搅拌下将步骤1)所得阿霉素溶液600-700μL滴加到叶酸-壳聚糖溶液中并超声；
9)将步骤8)所得溶液加入到6-10mL步骤1)所得聚多巴胺溶液中，超声混合均匀；
10)取1 OD的核酸适配子AS1411溶于5-8mL水中，然后加入8-12mM三(2-羧乙基)膦反应1-2h，再与步骤9)所得产物混合搅拌过夜，最后经离心、洗涤、冷冻干燥处理，制得阿霉素纳米微粒。
2.如权利要求1所述的制备方法，其特征在于，步骤2)中，搅拌速度为200-400r/min。
3.如权利要求1所述的制备方法，其特征在于，步骤5)中，搅拌速度300r/min下进行避光反应。
4.如权利要求1所述的制备方法，其特征在于，步骤7)中，所述透析袋的截留分子量为8000-14000Da。
5.如权利要求1所述的制备方法，其特征在于，步骤10)中，离心转速为16000-18000r/min，离心时间为15-20min。
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