CN110974976A - 羧甲基壳聚糖修饰载药囊泡及其制备与应用 - Google Patents
羧甲基壳聚糖修饰载药囊泡及其制备与应用 Download PDFInfo
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Abstract
一种羧甲基壳聚糖修饰载药囊泡，其特征在于使用羧甲基壳聚糖对细胞外囊泡的脂质双分子层膜进行包覆或修饰。所述羧甲基壳聚糖为分子量为165至119000，脱乙酰度≥50％。所述载药囊泡包含裹的药物为双氯芬酸钠、胰岛素、紫杉醇、顺铂、阿霉素或西贝母碱中等药物的一种或多种。本发明羧甲基壳聚糖连接于细胞外囊泡的表面，羧甲基壳聚糖的修饰改变了细胞外囊泡的表面性质，并对其药物传递机制产生深远的影响，相对于普通细胞外囊泡，其在肿瘤细胞靶向。本发明所制备的制剂在肿瘤内摄取情况下细胞囊泡量更多。
Description
技术领域
本发明涉及药物技术领域，具体涉及一种羧甲基壳聚糖修饰载药囊泡及其制备与应用。
背景技术
壳聚糖是一种自然界生物所含有的碱性氨基高分子多糖，低毒性，表面带正电荷，可通过电荷之间相互作用，与带负电荷的细胞表明相结合，从而有效地摄取，并能通过打开细胞通道提高药物在细胞间的瞬间渗透能力，延长在作用部位的滞溜时间而提高药物生物利用度。但缺点是在生理条件下不溶于水。现通过对其进行修饰，从而改善水溶性，低分子量壳聚糖主要有季铵化、羧甲基化和硫酸酯化衍生物等。壳聚糖分子上存在大量的羟基和氨基，会形成分子间和分子内氢键。当壳聚糖降解后，分子量降低，分子内的氢键作用减弱，容易与溶剂分子作用，且分子构型发生变化，从而是溶解性得到显著改善。因此羧甲基壳聚糖具有良好的水溶性。由于在多种肿瘤细胞内pH值明显低于一般正常细胞的特点，利用壳聚糖与其受体的特异性结合的特点，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。
细胞外囊泡是一种新型的生物相容性药物载体，粒径30～150nm，其包裹范围非常广，可包裹水溶性物质、脂溶性物质及两性物质，能有低免疫原性、提高和延长药物疗效等优势。
发明内容
本发明的目的是针对现有技术的上述不足，提供一种羧甲基壳聚糖修饰载药囊泡及其制备与应用。
本发明的目的可通过以下技术方案实现：
一种羧甲基壳聚糖修饰载药囊泡，使用羧甲基壳聚糖对细胞外囊泡的脂质双分子层膜进行包覆或修饰。
所述羧甲基壳聚糖为分子量为165至119000，脱乙酰度≥50％。
所述细胞外囊泡来源肿瘤细胞、巨噬细胞、树突状细胞、间充质干细胞和红细胞等细胞的一种或多种。
所述细胞外囊泡的制备方法为超速离心法、蔗糖梯度离心法、尺寸排阻色谱法、超滤法、免疫亲和层析法、聚合物沉淀法、微流控技术等。
所述其中的羧甲基壳聚糖可接受叶酸、磁珠、多肽等物质再修饰。
所述载药囊泡的制备方法为共同孵育、电穿孔、超声、冻融、挤出、低渗透析等。
所述载药囊泡包含的药物为双氯芬酸钠、胰岛素、紫杉醇、顺铂、阿霉素或西贝母碱的一种或多种。
所述的一种羧甲基壳聚糖载药囊泡用于制成注射液、注射用冻干粉剂，滴眼剂、凝胶剂、混悬剂、片剂、胶囊、软胶囊、吸入制剂、透皮制剂或植入剂的一种的用途。
一种羧甲基壳聚糖修饰载药囊泡的制备方法，其特征在于包括如下步骤：
1)直接孵育；
2)然后将低分子量壳聚糖溶解于细胞外囊泡的水相中，通过静电力作用，使低分子壳聚糖连接于脂质体的表面。
一种羧甲基壳聚糖修饰载药囊泡的制备方法，其特征在于包括如下步骤：
采用直接共同孵育法：
步骤(1)膜过滤-超速离心法提取细胞外囊泡:人肝癌细胞HepG2细胞(约8x107个)用含有2％去细胞外囊泡胎牛血清(120,000xg离心过夜除去血清中的细胞外囊泡)培养48h后，收集上清并于1200xg离心10min，去除细胞或细胞碎片；再用0.45μm滤器，过滤至10KD分子量截留的超滤管中，经过4000xg离心30min，超滤浓缩；最后用0.22 μm滤器过滤后，经120,000xg超速离心90min，将沉淀溶解于PBS中得到细胞外囊泡；
步骤(2)直接共同孵育法制备羧甲基壳聚糖修饰的装载阿霉素的细胞外囊泡:取30μ g阿霉素加入到8x107个细胞提取的囊泡混悬液中用PBS溶液混匀定容至1ml，然后置于37℃恒温箱中共同孵育1h后，再添加1ml浓度为25μg/ml羧甲基壳聚糖溶液水合1h后， 10,000xg离心10min，去除残余药物及羧甲基壳聚糖；再用0.22μm滤器过滤后，经120,000xg超速离心70min，将沉淀溶解于PBS中得到羧甲基壳聚糖修饰的装载阿霉素的细胞外囊泡，即羧甲基壳聚糖修饰的载药囊泡。
有益效果：
本发明将羧甲基壳聚糖连接于细胞外囊泡的表面，羧甲基壳聚糖的修饰改变了细胞外囊泡的表面性质，并对其药物传递机制产生深远的影响，相对于普通细胞外囊泡，其在肿瘤细胞靶向。本发明所制备的制剂在肿瘤内摄取情况下细胞囊泡量更多。
附图说明
图1a为本发明按照实施例1制备的羧甲基壳聚糖修饰细胞外囊泡的透射电镜图之一。
图1b为本发明按照实施例1制备的羧甲基壳聚糖修饰细胞外囊泡的透射电镜为本发明之二。
图1c为本发明按照实施例1制备的羧甲基壳聚糖修饰细胞外囊泡的透射电镜为本发明之三。
图2a为本发明按照实施例1制备的羧甲基壳聚糖修饰细胞外囊泡的粒径分析为本发明之一。
图2b为本发明按照实施例1制备的羧甲基壳聚糖修饰细胞外囊泡的粒径分析为本发明之二。
图2c为本发明按照实施例1制备的羧甲基壳聚糖修饰细胞外囊泡的粒径分析为本发明之三。
具体实施方式
下面结合附图对本发明做进一步说明。
实施例1。
一种羧甲基壳聚糖修饰载药囊泡，其特征在于使用羧甲基壳聚糖对细胞外囊泡的脂质双分子层膜进行包覆或修饰。
所述羧甲基壳聚糖为分子量为165至119000，脱乙酰度≥50％。
所述羧甲基壳聚糖修饰载药囊泡包裹的药物为双氯芬酸钠、胰岛素、紫杉醇、顺铂、阿霉素或西贝母碱等药物的一种或多种。
一种羧甲基壳聚糖修饰载药囊泡用于制成注射液、注射用冻干粉剂，滴眼剂、凝胶剂、混悬剂、片剂、胶囊、软胶囊、吸入制剂、透皮制剂或植入剂等给药方式的一种的用途。
一种羧甲基壳聚糖修饰载药囊泡的制备方法，其特征在于包括如下步骤：
1)直接孵育；
2)然后将低分子量壳聚糖溶解于细胞外囊泡的水相中，通过静电力作用，使低分子壳聚糖连接于脂质体的表面。
初步研究表明，本发明实施例实验发现的羧甲基壳聚糖修饰的载药囊泡的性能包括：粒径大小在30到150纳米之间，粒径均匀；缓释性能明显，作为化疗药物载体时能够延长药物的作用时间，促进药物的吸收；具有pH响应性。以阿霉素为模型药物，以肝癌细胞PLC/PRF/5肝癌细胞为模型细胞，考察羧甲基壳聚糖修饰的载药囊泡对肿瘤细胞的杀伤作用。
本发明的羧甲基壳聚糖修饰的载药囊泡的制备方法，采用直接共同孵育法：
(1)膜过滤-超速离心法提取细胞外囊泡:人肝癌细胞HepG2细胞(约8x107个)用含有2％去细胞外囊泡胎牛血清(120,000xg离心过夜除去血清中的细胞外囊泡)培养48h 后，收集上清并于1200xg离心10min，去除细胞或细胞碎片；再用0.45μm滤器，过滤至10KD分子量截留的超滤管中，经过4000xg离心30min，超滤浓缩；最后用0.22μm滤器过滤后，经120,000xg超速离心90min，将沉淀溶解于PBS中得到细胞外囊泡。
(2)直接共同孵育法制备羧甲基壳聚糖修饰的装载阿霉素的细胞外囊泡:取30μg阿霉素加入到8x107个细胞提取的囊泡混悬液中用PBS溶液混匀定容至1ml，然后置于37℃恒温箱中共同孵育1h后，再添加1ml浓度为25μg/ml羧甲基壳聚糖溶液水合1h后，10,000 xg离心10min，去除残余药物及羧甲基壳聚糖；再用0.22μm滤器过滤后，经120,000 xg超速离心70min，将沉淀溶解于PBS中得到羧甲基壳聚糖修饰的装载阿霉素的细胞外囊泡，即羧甲基壳聚糖修饰的载药囊泡。
Claims (10)
1.一种羧甲基壳聚糖修饰载药囊泡，其特征在于使用羧甲基壳聚糖对细胞外囊泡的脂质双分子层膜进行包覆或修饰。
2.根据权利要求1所述的羧甲基壳聚糖修饰载药囊泡，其特征在于所述羧甲基壳聚糖为分子量为165至119000，脱乙酰度≥50％。
3.根据权利要求1所述的羧甲基壳聚糖修饰载药囊泡，其特征在于所述细胞外囊泡来源肿瘤细胞、巨噬细胞、树突状细胞、间充质干细胞和红细胞等细胞的一种或多种。
4.根据权利要求1所述的羧甲基壳聚糖修饰载药囊泡，其特征在于所述细胞外囊泡的制备方法为超速离心法、蔗糖梯度离心法、尺寸排阻色谱法、超滤法、免疫亲和层析法、聚合物沉淀法、微流控技术等。
5.根据权利要求1所述的羧甲基壳聚糖修饰载药囊泡，其特征在于所述其中的羧甲基壳聚糖可接受叶酸、磁珠、多肽等物质再修饰。
6.根据权利要求1所述的羧甲基壳聚糖修饰载药囊泡，其特征在于所述载药囊泡的制备方法为共同孵育、电穿孔、超声、冻融、挤出、低渗透析等。
7.根据权利要求1所述的羧甲基壳聚糖修饰载药囊泡，其特征在于所述载药囊泡包含的药物为双氯芬酸钠、胰岛素、紫杉醇、顺铂、阿霉素或西贝母碱的一种或多种。
8.如权利要求书1至3任意之一所述的一种羧甲基壳聚糖载药囊泡用于制成注射液、注射用冻干粉剂，滴眼剂、凝胶剂、混悬剂、片剂、胶囊、软胶囊、吸入制剂、透皮制剂或植入剂的一种的用途。
9.一种羧甲基壳聚糖修饰载药囊泡的制备方法，其特征在于包括如下步骤：
1)直接孵育；
2)然后将低分子量壳聚糖溶解于细胞外囊泡的水相中，通过静电力作用，使低分子壳聚糖连接于脂质体的表面。
10.一种羧甲基壳聚糖修饰载药囊泡的制备方法，其特征在于包括如下步骤：
采用直接共同孵育法：
步骤(1)膜过滤-超速离心法提取细胞外囊泡:人肝癌细胞HepG2细胞(约8x107个) 用含有2％去细胞外囊泡胎牛血清(120,000xg离心过夜除去血清中的细胞外囊泡)培养48h后，收集上清并于1200xg离心10min，去除细胞或细胞碎片；再用0.45μm滤器，过滤至10KD分子量截留的超滤管中，经过4000xg离心30min，超滤浓缩；最后用0.22μm滤器过滤后，经120,000xg超速离心90min，将沉淀溶解于PBS中得到细胞外囊泡；
步骤(2)直接共同孵育法制备羧甲基壳聚糖修饰的装载阿霉素的细胞外囊泡:取30μg阿霉素加入到8x107个细胞提取的囊泡混悬液中用PBS溶液混匀定容至1ml，然后置于37℃恒温箱中共同孵育1h后，再添加1ml浓度为25μg/ml羧甲基壳聚糖溶液水合1h后，10,000xg离心10min，去除残余药物及羧甲基壳聚糖；再用0.22μm滤器过滤后，经120,000xg超速离心70min，将沉淀溶解于PBS中得到羧甲基壳聚糖修饰的装载阿霉素的细胞外囊泡，即羧甲基壳聚糖修饰的载药囊泡。
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