CN113181371A - 一种pH/ROS响应型纳米药物递送系统及制备方法 - Google Patents
一种pH/ROS响应型纳米药物递送系统及制备方法 Download PDFInfo
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Abstract
本发明公开了一种pH/ROS响应型纳米药物递送系统及制备方法：主要由mPEG‑NH2、Cbz‑Lys‑NCA为原料聚合形成前药骨架，再以α‑TOS和D(Boc)作为载体等为原料合成聚合物，并将DMA与骨架共轭，再以pHLIP修饰聚合物骨架，同时包载DOX，其上修饰有pHLIP，其内包裹抗癌药物。本发明通过集合DMA和pHLIP实现对肿瘤微环境的pH响应性和电荷反转，促进细胞摄取；通过将TOS结合于聚合物上，阻断线粒体电子传递链，实现ROS快速生成，促进药物及更多TOS的释放，形成药物正反馈释放；通过释放包裹的抗癌药物来实现对肿瘤的杀伤作用；通过独具的pH/ROS双响应型特性能在血液循环环境中保持稳定而在肿瘤微环境中增加药物释放，实现靶向性释药，并能够提高肿瘤细胞对药物的靶向摄取，实现更高效的肿瘤杀伤作用。
Description
技术领域
本发明属于药物制剂领域，涉及药物载体、胶束及其制备方法和应用，具体涉及一种pH/ROS响应型纳米药物递送系统及制备方法。
背景技术
恶性肿瘤是一类严重威胁人类生命健康的常见病，药物治疗作为抗肿瘤治疗的常用手段之一，应用的药物种类及给药途径始终在不断更新。近年来随着纳米技术的兴起和发展，纳米粒子(NP)介导的药物递送已被广泛追求，以开发更安全、更有效的治疗方式来治疗多种重要疾病，尤其在纳米抗癌药物研发领域发挥着巨大作用。
然而，迄今为止，大多数纳米药物仍面临许多挑战，难以实现令人满意的治疗效果。例如，靶向性低、肿瘤渗透性差、药物释放位置无选择性、释放速度不受控制等，阻碍了纳米载体在抗肿瘤领域中的进一步发展和应用。
因此，需构建一种具有高载药量和肿瘤靶向效果及多种治疗模式相结合的纳米胶粒递送系统，来解决上述弊端。构建具有良好靶向性、生物相容性及生物可降解性的纳米胶粒递送系统，将是纳米胶粒聚合物在肿瘤治疗领域方面新的突破。
通过利用智能纳米材料的刺激反应性，可以实现药物的受控释放，从而提高了生物利用度并减少了副作用。实现按需药物释放的一种策略是使用刺激响应性纳米载体来封装药物。然而，它们中的大多数仍然遭受血液循环中药物载量低和药物泄漏的困扰，而使用刺激响应性键将药物偶联到载体材料上提供了另一种策略。
发明内容
针对背景技术中所提到的问题，本发明研究了一种pH/ROS响应型纳米药物递送系统，该载药纳米递送系统对肿瘤微环境的pH值范围敏感，触发电荷反转并可通过ROS自我生成和局部放大，达到加速载体解体，增强药物释放。首先以mPEG-NH2、Cbz-Lys-NCA为原料聚合形成前药骨架，再以α-TOS(α-维生素E琥珀酸酯)和D(Boc)作为载体等为原料合成聚合物骨架，并将DMA与骨架共轭，再以pH敏感肽(pHLIP)修饰聚合物骨架，同时包载阿霉素(DOX)。当处于肿瘤所在的相对酸性环境(pH＝6.2-6.9)时，pHLIP响应pH，并触发DMA电荷反转，从负电荷转为正电荷，两者促进细胞摄取；α-TOS促进ROS自我生成和局部放大一方面使聚合物骨架上的甲硫氨酸基团转变为亲水基团，加速载体解体和增强药物释放，另一方面，对肿瘤细胞有特异杀伤作用。
本发明提供的pH/ROS响应型纳米药物递送系统具有良好的生物降解性、生物相容性、pH响应性和ROS响应性，具体是通过如下技术方案实现的：
一种pH/ROS响应型纳米药物递送系统及制备方法，包括以下步骤：
1)将mPEG-NH2和Cbz-Lys-NCA混合后加入DMF溶解，25℃条件下搅拌后将反应体系倒入冰乙醚得到纯mPEG-b-PLL(Z)沉淀；
2)将步骤1)得到的mPEG-b-PLL(Z)加入到TFA与HBr/HOAc的混合溶液中，冰浴搅拌将反应体系倒入冰乙醚并对蒸馏水透析，将透析产物冷冻干燥得到mPEG-b-PLL；
3)将步骤2)得到的mPEG-b-PLL与D(Boc)、NHS、DCC与氮气环境下溶解于DMSO中，25℃条件下搅拌后过滤，用DMSO透析，再用蒸馏水透析，将透析产物冷冻得到PPD(Boc)；
4)将步骤3)得到的PPD(Boc)与α-TOS、NHS、DCC于氮气环境下溶解于DMSO中，25℃条件下搅拌后对DMSO和蒸馏水透析，将透析产物冷冻干燥得到PPT/D(Boc)；
5)将步骤4)得到的PPT/D(Boc)溶解于DCM中，将体系冰浴并滴加TFA搅拌，过滤将滤液加入冰乙醚中，对蒸馏水透析，将透析产物冷冻干燥得到PPT/D；
6)将步骤5)得到的PPT/D与DMA溶解于DMSO中，加入TEA和吡啶室温下搅拌过夜，对DMSO透析以纯化混合物，再经过二次透析去除DMSO，将透析产物冷冻干燥得到PPT/D(DMA)。
7)将步骤6)得到的PPT/D(DMA)溶解于DMF中并与DOX混合，避光搅拌，随后在反应体系滴入PBS搅拌，然后对水透析，得到的溶液温和离心弃沉淀得到PPT/D(DMA)@DOX纳米胶粒；
8)将步骤7)得到的PPT/D(DMA)@DOX纳米胶粒与pH敏感肽混合溶解于DMSO中，得到的均匀溶液逐滴加入到高速搅拌的水中自组装，然后用PBS透析，滤膜过滤，冷冻干燥得到PPT/D(DMA)@DOX-pHLIP自组装载药纳米粒子。
进一步地，步骤1)所述mPEG-NH2和Cbz-Lys-NCA混合的摩尔比为1：30。
进一步地，步骤3)所述mPEG-b-PLL与D(Boc)、NHS、DCC混合的摩尔比为1：10：135：135。
进一步地，步骤4)所述PPD(Boc)与α-TOS、NHS、DCC混合的摩尔比为4：33：66：66。
进一步地，步骤6)所述PPT/D与DMA混合的摩尔比为1：2。
进一步地，步骤7)所述PPT/D(DMA)溶解于DMF中并与DOX混合具体为将DOX·HCl溶解于含TEA的DMF中搅拌，再将PPT/D(DMA)溶解于DMF并与上述体系混合；其中，PPT/D(DMA)、DOX·HCl、TEA的摩尔比为2：1：3。
进一步地，空微粒的平均直径为98.1±4.5nm，以10％的比例载DOX后其平均直径增至84.3±3.6nm，颗粒尺寸分布为PDI＝0.108；装载量(LC)和封装效率(EE)分别为9.64％和96.4％。
进一步地，步骤8)所述pH敏感肽来源于细菌视紫红质的跨膜螺旋蛋白C，溶于水，是一条中等疏水性多肽；所述pH敏感肽能够在酸性条件下折叠形成稳定的TM螺旋结构，嵌入并跨越细胞膜进入细胞，在中性条件下，pH敏感肽的变化处于一种平衡过程。
进一步地，步骤8)所述PPT/D(DMA)@DOX-pHLIP自组装载药纳米粒子其内包裹抗癌药物，所述抗癌药物为具有芳香结构的抗肿瘤药物。
一种pH/ROS响应型纳米药物递送系统用于肿瘤治疗药物中的应用。
进一步地，所述抗肿瘤纳米药物递送系统在pH，ROS和pHLIP三重响应刺激条件下，PPT/D(DMA)@DOX纳米胶粒聚合物结构发生改变，促进包载的药物快速释放。
与现有技术相比，本发明的有益效果是：
本发明通过集合DMA和pHLIP实现对肿瘤微环境的pH响应性和电荷反转，促进细胞摄取；通过将TOS结合于聚合物上，以阻断线粒体电子传递链，实现ROS的快速生成，促进药物及更多TOS的释放，形成药物的正反馈释放；通过释放包裹的抗癌药物来实现对肿瘤的杀伤作用；通过独具的pH/ROS双响应型特性能在血液循环环境中保持稳定而在肿瘤微环境中增加药物释放，实现靶向性释药，并能够提高肿瘤细胞对药物的靶向摄取，实现更高效的肿瘤杀伤作用。
附图说明
图1为PPT/D(DMA)聚合物的合成工艺流程图。
图2为PPT/D(DMA)微粒聚合物的核磁示意图。
图3为PPT/D(DMA)@DOX胶束的大小和表面形态特征示意图。
图4为PPT/D(DMA)@DOX胶束在不同pH下和ROS反应特性的药物释放行为示意图。
具体实施方式
下面结合具体实施例对本发明作进一步的说明，但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法，所述原材料如无特别说明均能从公开商业途径获得。
实施例1
(1)首先，溶解在DMF中的Lys(Z)-NCA以mPEG-NH2为引发剂以开环聚合法对苄基进行了脱保护。将干燥的mPEG-NH2(1.0g，0.2mmol)和Cbz-Lys-NCA(1.8g，6.0mmol)放入干燥的50ml玻璃反应器中，并加入30mL DMF。25℃条件下搅拌3天后将反应体系倒入150.0mL冰乙醚中三次以得到纯mPEG-b-PLL(Z)沉淀物(产率为88.6％)。1H-NMR测得聚合度为30，mPEG-b-PBLG得平均摩尔质量为12870(mPEG5000-b-PLL(Z)7870)。取1.6g得到的mPEG-b-PLL(Z)加入10.0mL TFA和0.8mL HBr/HOAc溶液中以去除其Cbz基团。冰浴中搅拌1小时后将体系倒入150.0mL冰乙醚并对蒸馏水透析。冷冻干燥透析产物得mPEG-b-PLL产品。产品纯化后于室温下真空干燥。
(2)将干燥的mPEG-b-PLL(1.0g，0.11mmol)、D(Boc)(0.74g，3.30mmol)、N-羟基琥珀酰亚胺(NHS)(1.71g，14.85mmol)和二环己基碳二亚胺(DCC)(3.06g，14.85mmol)于氮气环境下溶解于30.0mL DMSO中。25℃条件下搅拌24h后先过滤，用二甲亚砜(DMSO)透析，再用蒸馏水透析，冷冻得到甲氧基聚(乙二醇)-聚块聚(l-赖氨酸)-接枝蛋氨酸(Boc)(PPT/D(Boc))产物(PPD(Boc))。1H-NMR测得移植来的D(Boc)数为24。
(3)再将PPD(Boc)(1.15g，0.08mmol)、α-TOS(0.35g，0.66mmol)、NHS(0.15g，1.32mmol)和DCC(0.27g，1.32mmol)于氮气环境中溶解于30.0mL DMSO中。25℃条件下搅拌48h后先后对DMSO和蒸馏水透析，冷冻干燥得PPT/D(Boc)产品。
(4)然后将0.8g PPT/D(Boc)产品溶解于5.0mL DCM中，将体系置于冰浴中并滴加5.0mL TFA，搅拌1h。过滤沉淀后加入100.0mL冰乙醚中，对蒸馏水透析，冷冻干燥得PPT/D产品。
(5)最后将得到的PPT/D与DMA反应制备壳，将PPT/D和双倍的DMA溶解于DMSO中，加入TEA和吡啶室温下搅拌过夜。对DMSO透析24h(MWCO 3500Da)以纯化混合物，再置于透析袋中(MWCO 10,000Da)透析24h以去除DMSO，冻干得PPT/D(DMA)壳。对比验证此壳的pH响应电荷反转性质，以同样的方法制备无电荷敏感性及诱发电荷反转性质的丁二酸(SA)改良壳作为对比，标记为PPT/D(SA)。为验证ROS反应性，甲硫氨酸与H2O2在D2O中分别共孵育0，4，12h。使用1H-NMR记录化学变化，可见甲硫氨酸的氧化产物在共孵育4h后被检测到，12h后甲硫氨酸被完全氧化。
(6)制备纳米胶粒。将DOX·HCl(5.0mg)溶解于含2.6mgTEA的DMF中搅拌2h以去除其上的HCl。一定数量的PPT/D(DMA)溶解于DMF中并与上述体系混合。避光搅拌2h后反应体系以2mL/h的速度滴入5mL PBS(pH5.5)中，搅拌3h。之后将反应体系置于MWCO 3500Da透析袋中对pH8.5水透析24h，得到的溶液温和离心，弃沉淀，即得到PPT/D(DMA)@DOX纳米胶粒。
(7)PPT/D(DMA)@DOX纳米胶粒与pH敏感肽混合，溶解在1mL DMSO中，以获得聚合物浓度为10mg/mL的均匀溶液。然后将上述混合溶液逐滴加入到高速搅拌的水中自组装10分钟，然后用PBS透析24小时，滤膜过滤，冷冻干燥后得到PPT/D(DMA)@DOX-pHLIP自组装载药纳米粒子。
采用透析法监控PPT/D(DMA)@DOX在pH为5.5时释放DOX的情况，测得累积DOX释放为73.9％。
最后应当说明的是：以上实施例仅用以说明本申请的技术方案而非对其限制；尽管参照较佳实施例对本申请进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本申请的具体实施方式进行修改或者对部分技术特征进行等同替换，其均应涵盖在本申请请求保护的技术方案范围当中。
Claims (10)
1.一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，包括以下步骤：
1)将mPEG-NH2和Cbz-Lys-NCA混合后加入DMF溶解，25℃条件下搅拌后将反应体系倒入冰乙醚得到纯mPEG-b-PLL(Z)沉淀；
2)将步骤1)得到的mPEG-b-PLL(Z)加入到TFA与HBr/HOAc的混合溶液中，冰浴搅拌将反应体系倒入冰乙醚并对蒸馏水透析，将透析产物冷冻干燥得到mPEG-b-PLL；
3)将步骤2)得到的mPEG-b-PLL与D(Boc)、NHS、DCC与氮气环境下溶解于DMSO中，25℃条件下搅拌后过滤，用DMSO透析，再用蒸馏水透析，将透析产物冷冻得到PPD(Boc)；
4)将步骤3)得到的PPD(Boc)与α-TOS、NHS、DCC于氮气环境下溶解于DMSO中，25℃条件下搅拌后对DMSO和蒸馏水透析，将透析产物冷冻干燥得到PPT/D(Boc)；
5)将步骤4)得到的PPT/D(Boc)溶解于DCM中，将体系冰浴并滴加TFA搅拌，过滤将滤液加入冰乙醚中，对蒸馏水透析，将透析产物冷冻干燥得到PPT/D；
6)将步骤5)得到的PPT/D与DMA溶解于DMSO中，加入TEA和吡啶室温下搅拌过夜，对DMSO透析以纯化混合物，再经过二次透析去除DMSO，将透析产物冷冻干燥得到PPT/D(DMA)。
7)将步骤6)得到的PPT/D(DMA)溶解于DMF中并与DOX混合，避光搅拌，随后在反应体系滴入PBS搅拌，然后对水透析，得到的溶液温和离心弃沉淀得到PPT/D(DMA)@DOX纳米胶粒；
8)将步骤7)得到的PPT/D(DMA)@DOX纳米胶粒与pH敏感肽混合溶解于DMSO中，得到的均匀溶液逐滴加入到高速搅拌的水中自组装，然后用PBS透析，滤膜过滤，冷冻干燥得到PPT/D(DMA)@DOX-pHLIP自组装载药纳米粒子。
2.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤1)所述mPEG-NH2和Cbz-Lys-NCA混合的摩尔比为1：30。
3.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤3)所述mPEG-b-PLL与D(Boc)、NHS、DCC混合的摩尔比为1：10：135：135。
4.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤4)所述PPD(Boc)与α-TOS、NHS、DCC混合的摩尔比为4：33：66：66。
5.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤6)所述PPT/D与DMA混合的摩尔比为1：2。
6.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤7)所述PPT/D(DMA)溶解于DMF中并与DOX混合具体为将DOX·HCl溶解于含TEA的DMF中搅拌，再将PPT/D(DMA)溶解于DMF并与上述体系混合；其中，PPT/D(DMA)、DOX·HCl、TEA的摩尔比为2：1：3。
7.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤8)所述pH敏感肽来源于细菌视紫红质的跨膜螺旋蛋白C，溶于水，是一条中等疏水性多肽；所述pH敏感肽能够在酸性条件下折叠形成稳定的TM螺旋结构，嵌入并跨越细胞膜进入细胞，在中性条件下，pH敏感肽的变化处于一种平衡过程。
8.根据权利要求1所述一种pH/ROS响应型纳米药物递送系统及制备方法，其特征在于，步骤8)所述PPT/D(DMA)@DOX-pHLIP自组装载药纳米粒子其内包裹抗癌药物，所述抗癌药物为具有芳香结构的抗肿瘤药物。
9.权利要求1-8任一项所述的pH/ROS响应型纳米药物递送系统用于肿瘤治疗药物中的应用。
10.根据权利要求9所述的pH/ROS响应型纳米药物递送系统用于肿瘤治疗药物中的应用，其特征在于，所述抗肿瘤纳米药物递送系统在pH，ROS和pHLIP三重响应刺激条件下，PPT/D(DMA)@DOX纳米胶粒聚合物结构发生改变，促进包载的药物快速释放。
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