CN113384713A - 酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系及其制备方法 - Google Patents
酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系及其制备方法 Download PDFInfo
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Abstract
酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系及其制备方法，其中咪唑环糊精作为主体，透明质酸作为靶向剂，其构筑单元的化学结构式如下：
本发明的优点是：该纳米粒子可以在透明质酸酶(HAase)的作用下特异性水解超分子组装体中的透明质酸，从而实现抗癌药物阿霉素(DOX)的可控释放，该体系制备简单，与普通的药物释放相比具有药物缓释的效果，使服药次数减少，降低了药物的毒副作用。此外，这种具有酶响应性、生物相容性和抗癌活性的超分子组装体可以对抗癌药物DOX缓释的策略为在生物医学上进行癌症治疗提供了一种新的思路，具有良好的潜在应用前景。
Description
技术领域
本发明属于超分子靶向药物传递技术领域，特别是酶响应的超分子纳米粒子用于可控释放抗癌药物阿霉素体系。
背景技术
癌症是令世界人民都非常头痛的问题，每年许多科学家都会做大量研究用以解决癌症问题。因此许多抗癌药物也陆陆续续被研究出来用以癌症的治疗。阿霉素(DOX)就是比较常见的抗癌药物，可以治疗多种癌症。现如今，通过超分子化学进行靶向传递抗癌药物已经得到了许多科研学者的关注和研究。在超分子大环中，环糊精由于其价格低廉、无毒、生物相容性和水溶性好等优点，此外，其疏水性空腔可以负载许多具有特定功能结构的客体分子，使得其在酶响应的超分子生物材料领域得到广泛研究。
发明内容
本发明利用生物相容性的咪唑环糊精(Im-CD)和具有靶向性的透明质酸(HA)通过静电相互作用构筑了超分子组装体(Im-CD/HA)，Im-CD/HA负载了抗癌药物阿霉素(DOX)形成新的超分子组装体DOX@Im-CD/HA。通过紫外分光光度计、透射电镜(TEM)、动态光散射(DLS)以及细胞实验等对超分子组装体进行了研究，可以看出超分子组装体是具有球形结构的纳米颗粒，在加入透明质酸酶(HAase)3h之后可以使组装体中的透明质酸(HA)被特异性水解，从而使超分子组装体解组装释放出抗癌药物阿霉素(DOX)，由于DOX在此过程中释放缓慢，所以可以起到一个缓释的作用，使服药次数减少，降低了药物的毒副作用。此外，该体系制备简单，与普通的药物释放相比更好的效果。总之，这种具有酶响应性、生物相容性和抗癌活性的超分子组装体可以对抗癌药物DOX缓释的策略为在生物医学上进行癌症治疗提供了一种新的思路，具有良好的潜在应用前景。
本发明的技术方案：
酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系，其中咪唑环糊精作为主体，透明质酸作为靶向剂，其构筑单元的化学结构式如下：
酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系的制备方法，包括以下步骤：
步骤1、抗癌药物阿霉素(DOX)溶液的制备；
步骤2、咪唑环糊精-透明质酸二元超分子纳米粒子溶液的制备；
步骤3、将步骤2所得二元超分子纳米粒子溶液加入步骤1制得的DOX溶液中搅拌后进行透析；
其中咪唑环糊精是环糊精上的6位羟基被甲基咪唑全取代的大环主体，透明质酸是在网上商城购买所得，通过静电相互作用构筑形成超分子组装体，再通过环糊精空腔对DOX的负载作用构筑成超分子纳米粒子，然后用透射电镜以及紫外可见光谱和荧光发射光谱来证明超分子纳米粒子的形成，然后用多种仪器监测其加入透明质酸酶之后的变化，并对其进行细胞实验测试。
进一步的，其中步骤2中咪唑环糊精的制备方法如下：
1)将三苯基膦(20.2g，77.0mmol)溶于无水DMF(80mL)中，在氮气保护下10-15分钟内缓慢地加入碘(20.2g，77.2mmol)。然后将干燥的β-环糊精(5g，4.4mmol)加入到上述的深棕色溶液中，将其在70℃和氮气氛围下充分搅拌18小时。之后将反应液在减压的条件下蒸出一半的溶液。在冰浴条件下，在搅拌的情况下向其中加入甲醇钠的甲醇溶液，后将溶液的pH调到9-10。在室温下，将混合液搅拌30分钟，随后在剧烈搅拌下将混合液加入到冷甲醇中(400mL)，将析出的不溶物过滤并收集，用甲醇洗涤后，并用甲醇对得到的固体进行索氏提取，直至溶剂不变色，最终得到目标产物为白色粉末(7.70g，产率为92％)。
2)在氮气氛围下，将1)中制得的产物(1g，0.52mmol)，使之与1-甲基咪唑(6.0ml，90mmol)在氩气氛围保护和80℃条件下加热搅拌48h。反应结束后，将反应瓶溶液转移至200ml丙酮中，会有大量白色沉淀析出。减压抽滤后用丙酮洗涤沉淀，然后将得到的白色沉淀用少量二次水进行重结晶。将以上过程重复三次，得到物质即为目标产物(715.2mg，产率56％)。1HNMR(D2O，400MHz)：δ(ppm)＝3.30-3.37(t，1H)，3.50-3.55(d，1H)，3.75-3.83(s，3H)，3.95-4.01(t，1H)，4.08-4.18(m，1H)，4.45-4.54(m，2H)，5.01-5.08(s，1H)，7.40-7.45(s，1H)7.49-7.54(s，1H)。
进一步的，步骤2中咪唑环糊精-透明质酸二元超分子纳米粒子溶液的制备，步骤如下：
咪唑环糊精-透明质酸二元超分子纳米粒子是以甲基咪唑取代的β-环糊精为主体，以具有靶向性的透明质酸为客体，通过静电相互作用构筑了超分子纳米粒子；将甲基咪唑取代的β-环糊精和靶向性的透明质酸按照0.05mM咪唑环糊精和0.5mM浓度透明质酸的比例溶解于水中，均匀混合后得到二元超分子纳米粒子溶液。
进一步的，步骤3中透析的步骤如下：
将制备好的咪唑环糊精-透明质酸二元超分子纳米粒子溶液加入到阿霉素溶液中后放置在截留量为3500的透析袋中搅拌24h透析，直至透析液的颜色不发生变化为止。
本发明的优点是：
利用具有水溶性以及生物相容性的C6位被甲基咪唑全部修饰的β-环糊精(Im-CD)和具有靶向性的透明质酸(HA)通过静电相互作用进行自组装形成超分子组装体，超分子组装体可以负载抗癌药物阿霉素(DOX)，形成新的超分子纳米粒子，该纳米粒子可以在透明质酸酶(HAase)的作用下特异性水解超分子组装体中的透明质酸，从而实现抗癌药物阿霉素(DOX)的可控释放，该体系制备简单，与普通的药物释放相比具有药物缓释的效果，使服药次数减少，降低了药物的毒副作用。此外，这种具有酶响应性、生物相容性和抗癌活性的超分子组装体可以对抗癌药物DOX缓释的策略为在生物医学上进行癌症治疗提供了一种新的思路，具有良好的潜在应用前景。
附图说明
图1为咪唑环糊精合成方法示意图。
图2为咪唑环糊精氢谱谱图。
图3为客体透明质酸的临界聚集浓度测试谱图。
图4为固定主体浓度改变客体浓度以及固定客体浓度改变主体浓度的临界聚集浓度测试谱图。
图5为固定阿霉素浓度改变透明质酸浓度的临界聚集浓度测试谱图。
图6为单独大环主体及组装体的紫外透过率测试谱图。
图7为DOX的药物标准曲线测试谱图。
图8为DOX药物负载的荧光发射及负载率的测试谱图。
图9为超分子组装体的动态光散射(DLS)测试谱图。
图10为客体的透射电镜(TEM)测试谱图。
图11为超分子组装体的透射电镜(TEM)测试谱图。
图12为超分子组装体在PBS缓冲溶液中的稳定性测试谱图。
图13为超分子组装体的酶响应在荧光发射及紫外可见光谱仪器上的测试谱图。
图14为超分子组装体的细胞实验测试谱图。
具体实施方式
实施例：
酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系，其中咪唑环糊精作为主体，透明质酸作为靶向剂，其构筑单元的化学结构式如下：
本发明提供的酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系制备方法，包括以下步骤：
步骤1、抗癌药物阿霉素(DOX)溶液的制备；
步骤2、咪唑环糊精-透明质酸二元超分子纳米粒子溶液的制备；
步骤3、将步骤2所得二元超分子纳米粒子溶液加入步骤1制得的DOX溶液中搅拌后进行透析；
参见附图1，以上制备方法中，步骤2中咪唑环糊精的制备方法如下：
1)将三苯基膦(20.2g，77.0mmol)溶于无水DMF(80mL)中，在氮气保护下10-15分钟内缓慢地加入碘(20.2g，77.2mmol)。然后将干燥的β-环糊精(5g，4.4mmol)加入到上述的深棕色溶液中，将其在70℃和氮气氛围下充分搅拌18小时。之后将反应液在减压的条件下蒸出一半的溶液。在冰浴条件下，在搅拌的情况下向其中加入甲醇钠的甲醇溶液，后将溶液的pH调到9-10。在室温下，将混合液搅拌30分钟，随后在剧烈搅拌下将混合液加入到冷甲醇中(400mL)，将析出的不溶物过滤并收集，用甲醇洗涤后，并用甲醇对得到的固体进行索氏提取，直至溶剂不变色，最终得到目标产物为白色粉末(7.70g，产率为92％)。
2)在氮气氛围下，将1)中制得的产物(1g，0.52mmol)，使之与1-甲基咪唑(6.0ml，90mmol)在氩气氛围保护和80℃条件下加热搅拌48h。反应结束后，将反应瓶溶液转移至200ml丙酮中，会有大量白色沉淀析出。减压抽滤后用丙酮洗涤沉淀，然后将得到的白色沉淀用少量二次水进行重结晶。将以上过程重复三次，得到物质即为目标产物(715.2mg，产率56％)。1HNMR(D2O，400MHz)：δ(ppm)＝3.30-3.37(t，1H)，3.50-3.55(d，1H)，3.75-3.83(s，3H)，3.95-4.01(t，1H)，4.08-4.18(m，1H)，4.45-4.54(m，2H)，5.01-5.08(s，1H)，7.40-7.45(s，1H)7.49-7.54(s，1H)。
图2为咪唑环糊精氢谱谱图。图中表明：合成的咪唑环糊精的结构正确。
图3为客体透明质酸的临界聚集浓度测试谱图。图中表明：在1mM浓度范围内，透明质酸不能自聚集形成纳米粒子。
图4为固定主体浓度改变客体浓度以及固定客体浓度改变主体浓度的临界聚集浓度测试谱图。图中表明：在加入主体后有明显的透过率，可以证明两者形成了组装体。
本发明制备方法的步骤2中，咪唑环糊精-透明质酸二元超分子纳米粒子溶液的制备，步骤如下：
咪唑环糊精-透明质酸二元超分子纳米粒子是以甲基咪唑取代的β-环糊精为主体，以具有靶向性的透明质酸为客体，通过静电相互作用构筑了超分子纳米粒子。将甲基咪唑取代的β-环糊精和靶向性的透明质酸按照0.05mM咪唑环糊精和0.5mM浓度透明质酸的比例溶解于水中，均匀混合后得到二元超分子纳米粒子溶液。
本发明步骤3中透析的步骤如下：
将制备好的咪唑环糊精-透明质酸二元超分子纳米粒子溶液加入到阿霉素溶液中后放置在截留量为3500的透析袋中搅拌24h透析，直至透析液的颜色不发生变化为止。
图5为固定阿霉素浓度改变透明质酸浓度的临界聚集浓度测试谱图。图中表明：阿霉素与透明质酸在此浓度范围内无法形成纳米粒子。
图6为单独大环主体及组装体的紫外透过率测试谱图。图中表明：在只有加入咪唑环糊精后才能形成超分子组装体。
图7为DOX的药物标准曲线测试谱图。图中表明：阿霉素的药物标准曲线是线性关系。
图8为DOX药物负载的荧光发射及负载率的测试谱图。图中表明：阿霉素的确是被负载进了超分子组装体中。
图9为超分子组装体的动态光散射(DLS)测试谱图。图中表明：超分子组装体的粒径尺寸是100nm左右。
图10为客体的透射电镜(TEM)测试谱图。图中表明：客体的形貌结构为线型结构。
图11为超分子组装体的透射电镜(TEM)测试谱图。图中表明：超分子组装体形成了球型纳米粒子结构。
图12为超分子组装体在PBS缓冲溶液中的稳定性测试谱图。图中表明：超分子组装体在PBS缓冲溶液中6h范围内能保持良好的稳定性。
图13为超分子组装体的酶响应在荧光发射及紫外可见光谱仪器上的测试谱图。图中表明：加入透明质酸酶3h后组装体解组装释放出抗癌药物阿霉素(DOX)。
图14为超分子组装体的细胞实验测试谱图。图中表明：在加入透明质酸酶3h后组装体具有杀伤A549癌细胞的能力。
Claims (5)
1.酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系，其特征在于：该体系是以咪唑环糊精作为主体，透明质酸作为靶向剂，其中构筑单元咪唑环糊精的化学结构式如下：
2.一种权利要求1所述的酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系的制备方法，包括以下步骤：
步骤1、抗癌药物阿霉素DOX溶液的制备；
步骤2、咪唑环糊精-透明质酸二元超分子纳米粒子溶液的制备；
步骤3、将步骤2所得二元超分子纳米粒子溶液加入步骤1制得的DOX溶液中搅拌后进行透析。
3.一种如权利要求2所述酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系的制备方法，其特征在于：步骤2中咪唑环糊精的制备方法如下：
1)将20.2g、77.0mmol的三苯基膦溶于80mL无水DMF中，在氮气保护下10-15分钟内缓慢地加入20.2g、77.2mmol的碘；然后将5g、4.4mmol的干燥的β-环糊精加入到上述的深棕色溶液中，将其在70℃和氮气氛围下充分搅拌18小时；之后将反应液在减压的条件下蒸出一半的溶液；在冰浴条件下，在搅拌的情况下向其中加入甲醇钠的甲醇溶液，后将溶液的pH调到9-10；在室温下，将混合液搅拌30分钟，随后在剧烈搅拌下将混合液加入到400mL冷甲醇中，将析出的不溶物过滤并收集，用甲醇洗涤后，并用甲醇对得到的固体进行索氏提取，直至溶剂不变色，最终得到目标产物为白色粉末7.70g，产率为92％；
2)在氮气氛围下，将1)中制得的产物1g、0.52mmol，使之与6.0ml、90mmol的1-甲基咪唑在氩气氛围保护和80℃条件下加热搅拌48h；反应结束后，将反应瓶溶液转移至200ml丙酮中，会有大量白色沉淀析出；减压抽滤后用丙酮洗涤沉淀，然后将得到的白色沉淀用少量二次水进行重结晶；将以上过程重复三次，得到物质即为目标产物715.2mg，产率56％；核磁表征产物结构为：1H NMR(D2O，400MHz)：δ(ppm)＝3.30-3.37(t，1H)，3.50-3.55(d，1H)，3.75-3.83(s，3H)，3.95-4.01(t，1H)，4.08-4.18(m，1H)，4.45-4.54(m，2H)，5.01-5.08(s，1H)，7.40-7.45(s，1H)7.49-7.54(s，1H)。
4.一种如权利要求2所述酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系的制备方法，其特征在于：步骤2中咪唑环糊精-透明质酸二元超分子纳米粒子溶液的制备，步骤如下：
咪唑环糊精-透明质酸二元超分子纳米粒子是以甲基咪唑取代的β-环糊精为主体，以具有靶向性的透明质酸为客体，通过静电相互作用构筑了超分子纳米粒子；将甲基咪唑取代的β-环糊精和靶向性的透明质酸按照0.05mM咪唑环糊精和0.5mM浓度透明质酸的比例溶解于水中，均匀混合后得到二元超分子纳米粒子溶液。
5.一种如权利要求2所述酶响应的超分子纳米粒子可控释放抗癌药物阿霉素体系的制备方法，其特征在于：步骤3中透析的步骤如下：
将制备好的咪唑环糊精-透明质酸二元超分子纳米粒子溶液加入到阿霉素溶液中后放置在截留量为3500的透析袋中搅拌24h透析，直至透析液的颜色不发生变化为止。
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