CN102350278B - 可载药壳聚糖微球的制备 - Google Patents

可载药壳聚糖微球的制备 Download PDF

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CN102350278B
CN102350278B CN201110168817.6A CN201110168817A CN102350278B CN 102350278 B CN102350278 B CN 102350278B CN 201110168817 A CN201110168817 A CN 201110168817A CN 102350278 B CN102350278 B CN 102350278B
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CN102350278A (zh
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聂俊
朱晓丹
马贵平
徐娟
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Beijing Do&done Establish A Business Technology Co ltd
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Changzhou Institute for Advanced Materials Beijing University of Chemical Technology
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Abstract

本发明涉及可用作药物载体的壳聚糖微球的制备。本发明采用自组装法制备壳聚糖微球。首先用10-溴癸酸甲酯对羟甲香豆素进行改性,制备含有羧基的羟甲香豆素,接着将其加入壳聚糖的乙酸溶液进行反应,用NaOH调节反应体系的pH为7~9,在丙酮中沉淀出产品。取20~30mg的产品溶于50mL的pH为6~9的PBS缓冲液中,用探针式超声均化仪均化,过滤膜过滤,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。本方法制备工艺简单,成本较低,且制备的纳米粒子无细胞毒性,可用作药物载体。

Description

可载药壳聚糖微球的制备
技术领域
本发明涉及可载药壳聚糖微球的制备 
背景技术
甲壳素是地球上储量仅次于纤维素的天然的可再生资源,壳聚糖是其N-乙酰基脱去55%以上而得到的一种碱性多糖。由于壳聚糖具有良好的生物相容性、生物可降解性、抗菌性、防腐性以及止血和促进伤口愈合等诸多特性,因此壳聚糖在伤口敷料、药物控释系统和组织工程等方面有着良好的应用前景。 
壳聚糖微球由高分子材料形成的外壳和水或油状内核构成,而药物通常就被聚合物膜包封在此内核层。理想的微球载体可生物降解并且无毒,所具有的特异靶向性使药物被定向释放出来,载体则被生物降解,避免转运过程中药物在其它组织释放而产生副作用或过早被灭活。由于壳聚糖具有可被体内多种酶生物降解,降解产物无毒且能被生物体完全吸收等优良特性,是药物缓释的理想载体,因此其作为药物载体材料的研究受到了极大的重视。 
制备壳聚糖微球的方法主要有乳化交联法、离子凝胶化法、分子自组装法等。有文献报道以壳聚糖为载体,戊二醛为交联剂,以Span-80为乳化剂,环己烷为油相,通过乳化交联法制备出分散性及圆整度良好的5-氟尿嘧啶壳聚糖微球。还有文献采取离子交联法利用壳聚糖的游离氨基与多聚磷酸钠阴离子发生分子间或分子内交联反应,制备平均直径为100纳米左右的壳聚糖球状凝胶。 
乳化交联法制备的纳米粒子粒径较小、球形度好,用作药物载体药物释放周期较长,但此方法常用戊二醛作交联剂,戊二醛具有抗基因毒性,而且蛋白质或多肽也与戊二醛反应,因此这种制备方法对于蛋白类和肽类药物方面的应用很受限制。离子交联法的机理主要是壳聚糖与带负电荷的聚阴离子电解质通过聚电解质大分子的分子内和分子间的静电相互作用而交联形成纳米粒子,这种反应条件温和,易于得到均一粒径的纳米粒且粒径的范围可调整,但由于较弱的离子键作用,使得形成的粒子骨架密度低,用作药物载体的药物主要分布在粒子的表面,药物突释效应严重。 
分子自组装是指在一定条件下,通过非化学键的相互作用,分子间自发组合,形成一类具有明确、稳定结构的且有某种特定性能的分子聚集体。分子自组装法在制备过程中不需要添加乳化剂、表面活性剂等有机溶剂,可减少载体的毒性;此外该方法工艺简单、成本低,是环境友好型的绿色方法,具有很好的产品开发前景。 
发明内容
针对离子交联法和乳化交联法的限制,本发明的目的在于用自组装法制备可载药壳聚糖微球 
本发明的原理及方法:本发明以壳聚糖为基体,向壳聚糖链引入疏水性基团,加上壳聚糖链本身具有亲水基团,采用自组装法制备壳聚糖微球。 
制备本发明所述的壳聚糖微球的方法包括如下步骤: 
(1)称取一定量的10-溴癸酸甲酯,羟甲香豆素和碳酸钾,将其溶于丙酮中,40~70℃下搅拌反应5~10h。 
(2)将步骤(1)中所得产物与5%NaOH反应,再用HCl酸化,反复用水洗。 
(3)称取一定量的上述产物,溶于乙醇中,加入EDC和NHS活化羧基0.5h~2h后,慢慢滴加至溶于2%乙酸的壳聚糖水溶液中,CS∶EDC∶NHS=1∶1.2∶1.2~1∶2∶2,室温下避光反应。 
(4)用1mol/L的NaOH溶液调反应体系的pH值为7~9,于丙酮中沉淀出产品。 
(5)取20~30mg的产品溶于50mLpH为6~9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
本发明的优点: 
1.本发明提供的制备方法中,不需要加任何交联剂,避免了交联剂潜在的细胞毒性。 
2.本发明提供的制备方法工艺简单,成本较低。 
附图说明
图1为可载药壳聚糖微球的扫描电镜图 
具体实施方式
实施例1 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,40℃下搅拌反应5h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为3.5,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.18gEDC和0.11gNHS,磁力搅拌0.5h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应24h。用1mol/L的NaOH溶液调反应体系的pH值为7,于丙酮中沉淀出产品。取20mg的产品溶于50mLpH为6的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例2 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,40℃下搅拌反应10h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4.5,反复用水洗。称取上述产物0.66g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌2h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应48h。用1mol/L的NaOH溶液调反应体系的PH值为7,于丙酮中沉淀出产品。取20mg的产品溶于50mLpH为6的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯 净的壳聚糖微球。 
实施例3 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,40℃下搅拌反应5h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为3.5,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.18gEDC和0.11gNHS,磁力搅拌0.5h,活化羧基。称取壳聚糖0.13g溶于1%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应24h。用1mol/L的NaOH溶液调反应体系的PH值为9,于丙酮中沉淀出产品。取30mg的产品溶于50mLpH为9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例4 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,50℃下搅拌反应5h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌0.5h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应24h。用1mol/L的NaOH溶液调反应体系的PH值为7,于丙酮中沉淀出产品。取20mg的产品溶于50mLpH为8的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例5 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,50℃下搅拌反应5h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌0.5h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应24h。用1mol/L的NaOH溶液调反应体系的pH值为7,于丙酮中沉淀出产品。取20mg的产品溶于50mLpH为9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例6 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,50℃下搅拌反应5h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌0.5h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应24h。用1mol/L的NaOH溶液调反应体系的pH值为7,于丙酮中沉淀出产品。取30mg的产品溶于50mLpH为9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例7 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,60℃下搅拌反应5h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4.5,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌1h,活化羧基。称取壳聚糖0.13g溶于1%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应48h。用1mol/L的NaOH溶液调反应体系的pH值为7,于丙酮中沉淀出产品。取30mg的产品溶于50mLpH为9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例8 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,60℃下搅拌反应8h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌1h,活化羧基。称取壳聚糖0.13g溶于1%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应48h。用1mol/L的NaOH溶液调反应体系的pH值为7,于丙酮中沉淀出产品。取30mg的产品溶于50mLpH为9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例9 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,60℃下搅拌反应8h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.33g,溶于乙醇中,加入0.3gEDC和0.18gNHS,磁力搅拌1h,活化 羧基。称取壳聚糖0.13g溶于1%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应48h。用1mol/L的NaOH溶液调反应体系的pH值为8,于丙酮中沉淀出产品。取30mg的产品溶于50mLpH为7的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例10 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,70℃下搅拌反应9h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.66g,溶于乙醇中,加入0.18gEDC和0.11gNHS,磁力搅拌2h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应48h。用1mol/L的NaOH溶液调反应体系的pH值为8,于丙酮中沉淀出产品。取20mg的产品溶于50mLpH为6的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 
实施例11 
称取1.5g的10-溴癸酸甲酯,1g羟甲香豆素,和0.8g碳酸钾于25mL丙酮中,70℃下搅拌反应9h。抽滤,旋蒸出产品,再将产品与5%NaOH反应,用HCl酸化至体系pH值为4,反复用水洗。称取上述产物0.66g,溶于乙醇中,加入0.18gEDC和0.11gNHS,磁力搅拌2h,活化羧基。称取壳聚糖0.13g溶于2%的乙酸溶液,将活化好的溶液滴到壳聚糖溶液中。室温下避光反应48h。用1mol/L的NaOH溶液调反应体系的pH值为8,于丙酮中沉淀出产品。取25mg的产品溶于50mLpH为8的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析一冻干纯化后,得到较为纯净的壳聚糖微球。 

Claims (2)

1.可载药壳聚糖微球的制备方法,其特征在于包括以下步骤:
(1)按摩尔比为1∶1∶1称取一定量的10-溴癸酸甲酯,羟甲香豆素和碳酸钾,将其溶于25mL丙酮中,40~70℃下搅拌反应5~10h;
(2)将步骤(1)中所得产物与5%NaOH反应,再用HCl酸化至pH为3.5~4.5,反复用水洗;
(3)按摩尔比为1∶1~1∶2称取一定量的壳聚糖和上述产物,将产物溶于乙醇中,加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)活化羧基0.5h~2h后,慢慢滴加至1%~2%乙酸的壳聚糖水溶液中,室温下避光反应1~2天;
(4)用1mol/L的NaOH溶液调反应体系的pH值为7~9,于丙酮中沉淀出产品;
(5)取20~30mg的产品溶于50mLpH为6~9的PBS缓冲液中,将所得溶液用探针式超声均化仪均化,然后通过过滤膜过滤掉沉淀物质,将所得样品进行冻干,反复透析-冻干纯化后,得到较为纯净的壳聚糖微球。
2.如权利要求1所述的制备方法,其特征在于步骤(3)中壳聚糖与EDC、NHS的摩尔比为1∶1.2∶1.2~1∶2∶2。
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