CN115054699A - 一种肝靶向递送miR-26a类似物的纳米药物载体及其制备方法 - Google Patents

一种肝靶向递送miR-26a类似物的纳米药物载体及其制备方法 Download PDF

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CN115054699A
CN115054699A CN202210545823.7A CN202210545823A CN115054699A CN 115054699 A CN115054699 A CN 115054699A CN 202210545823 A CN202210545823 A CN 202210545823A CN 115054699 A CN115054699 A CN 115054699A
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傅湘辉
田艳
简月
邹赛兰
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Abstract

本发明公开了一种肝靶向递送miR‑26a类似物的纳米药物载体及其制备方法，采用三次酰胺反应，可将不同分子量的PLGA、PEG、HA接枝到阳离子聚合物支化PEI上，通过透析、过滤、冻干等方法得到用于miRNA体外及体内递送的载体PLGA‑PEI‑PEG‑HA。递送载体在去离子水中水化后，自组装形成球型的核壳结构，表面电荷为40～60mV，能以N/P比为20:1完全有效地包载miR‑26amimics，可用于体外和体内低毒高效地主动递送至肝癌细胞。

Description

一种肝靶向递送miR-26a类似物的纳米药物载体及其制备 方法
技术领域
本发明属于药物载体技术领域，具体涉及到一种肝靶向递送miR-26a类似物的纳米药物载体及其制备方法。
背景技术
近年来，在大量临床样本检测中发现miR-26a的表达水平变化与肝炎、肝硬化、肝癌呈显著相关，证实miR-26a可作为临床早期鉴定及肝癌分期的生物标志物。同时，临床数据分析显示，与miR-26a表达水平较低的患者相比，miR-26a表达水平高的患者总生存期和复发时间更长。针对临床肝细胞癌(HCC)细胞显著低表达的miR-26a，许多研究者探索了miR-26a与HCC发生发展的分子机制，证明了miR-26a通过介导肝癌发生发展各通路的关键因子的表达，如细胞周期蛋白家族、IL-6/Stat3、生长因子等，来抑制肝癌细胞的增殖、迁移、血管生成等，并且miR-26可通过抑制细胞自噬，增强化学敏感性，促进肝癌细胞凋亡。鉴于miR-26a对肝癌发展的抑制作用，外源补充miR-26a，使肝癌细胞恢复miR-26a水平，从而抑制癌细胞生长，成为了一种前景良好的肝癌治疗手段。
近年来随着高分子材料领域的不断发展，越来越多的聚合物分子被证明具有良好的基因递送潜力。聚乙烯亚胺(PEI)是基础研究中被广泛用于纳米药物载体构建的聚合物。PEI是一种水溶性高分子聚合物，具有质子海绵效应，即进入细胞后引起溶酶体破裂从而导致线粒体损伤和细胞凋亡，被广泛用于基因转染和药物递送中。该聚合物具有超支化结构，并带有许多伯胺基团，这些伯胺基团在生理条件下被质子化。PEI的高正电荷特性可使该聚合物与带负电的细胞膜发生静电相互作用，从而促进细胞摄取。
聚乳酸-羟基乙酸共聚物(PLGA)是美国FDA批准的药用辅料，在体内可经过三羧酸循环最后降解为水和二氧化碳，具有良好的生物相容性和生物降解性，此外，PLGA还具有良好的成囊成膜的性能，广泛用于制药、医用工程材料等。研究表明，经PLGA修饰后的PEI(PLGA-PEI)纳米粒的基因转染效果优于PEI纳米粒，可以显著降低PEI的细胞毒性。
聚乙二醇(PEG)是最常用亲水性修饰材料，也是药物载体系统想载体材料之一，其免疫原性及抗原性低，能有效屏蔽阳离子聚合物纳米粒的部分正电荷，降低聚阳离子的细胞毒性，同时降低网状内皮系统的识别和吞噬。PEG的亲水性有利于延长载体在体内的循环时间，增强载体在血液中的稳定性，并有利于通过“增强渗透与滞留”(EPR)效应增加肿瘤组织的蓄积量。对于载生物药物的纳米粒，PEG可以屏蔽生物药物的免疫原性，减少外界条件(酸、碱、酶、水和热等)对生物药物活性的影响，延长生物药物在体内的半衰期。
透明质酸(HA)是一种酸性粘多糖，能识别肿瘤细胞表面高表达的CD44受体，与之结合使药物入胞，成为介导药物细胞膜内转运的非常有潜力的物质。已有研究表明，HA修饰的PEI阳离子纳米粒能靶向肿瘤细胞，显著提高肿瘤药物的细胞摄取，提高药物疗效。
不断深入的研究发现，miRNA药物的体内递送面临许多挑战。例如，要实现组织特异性递送、靶细胞的有效摄取；要避免核酸酶的降解、脱靶效应、免疫系统激活；要降低递送材料的毒副作用等。
发明内容
本发明的目的是提供一种肝靶向递送miR-26a类似物的纳米药物载体及其制备方法，可以利用其透明质酸靶向于肝脏肿瘤部位，将基因药物miR-26a类似物(miR-26amimics)递送至肝癌细胞，实现靶点可控定位释药，从而恢复肝癌细胞miR-26a水平，达到抑制肝癌发展的治疗作用。
为达上述目的，本发明提供了一种肝靶向递送miR-26a类似物的纳米药物载体的制备方法，包括以下步骤：
(1)将b-PEI溶液滴加于活化后的聚乳酸-羟基乙酸共聚物溶液中，恒温搅拌后经透析、过滤和冷冻干燥，制得PLGA-PEI；
(2)将步骤(1)制得的PLGA-PEI与聚乙二醇共溶于二甲基亚砜中，加入活化剂，恒温搅拌后经透析、过滤和冷冻干燥，制得PLGA-PEI-PEG；
(3)将透明质酸溶于二甲基亚砜中，加入活化剂，室温搅拌后制得活化的透明质酸溶液；
(4)将步骤(2)制得的PLGA-PEI-PEG与步骤(3)制得的透明质酸溶液混合后，恒温搅拌后经透析、冷冻干燥后制得，命名为PLGA-PEI-PEG-HA。
进一步地，步骤(1)中的活化后的聚乳酸-羟基乙酸共聚物溶液通过以下方法制备得到：将聚乳酸-羟基乙酸共聚物溶于二甲基亚砜中，加入活化剂后室温搅拌3-5h，制得；聚乳酸-羟基乙酸共聚物与活化剂的质量比为3.5-4.0:0.85。
进一步地，b-PEI(枝状聚乙烯亚胺Branched polyethylenimine，b-PEI)溶液通过以下方法制备得到：将b-PEI溶于二甲基亚砜溶液中，于室温下搅拌3-5h，制得；b-PEI溶解后的浓度为0.08-0.1g/mL。
进一步地，步骤(1)、步骤(2)和步骤(4)中恒温的温度为30-40℃，搅拌的时间为20-25h，透析的时间为45-50h，冷冻干燥的温度为-80℃，冷冻干燥的时间为2h以上。
进一步地，活化剂为1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺按照质量比8-250:5-150的混合物。
进一步地，步骤(2)中的PLGA-PEI与聚乙二醇的质量比为3.3:0.15-0.17，所述聚乙二醇溶于二甲基亚砜后的浓度为2.0*10-3-2.1*10-3g/mL。
进一步地，步骤(3)中的透明质酸通过以下方法制备得到：将透明质酸钠盐溶解于去离子水中，以pH为3.5的盐酸溶液为透析液，透析10-14h后，取透析袋内混合液于-80℃冷冻2h以上，制得；
溶解于去离子水中的透明质酸钠盐浓度为0.01-0.03g/mL。
进一步地，步骤(4)中PLGA-PEI-PEG与透明质酸溶液中透明质酸的质量比为1.5-2.0:0.07。
进一步地，聚乳酸-羟基乙酸共聚物溶液的分子量为6-36kDa。
本发明还提供了一种肝靶向递送miR-26a类似物的纳米药物载体的制备方法制备得到的PLGA-PEI-PEG-HA。
采用上述方案的有益效果是：该纳米药物载体可以递送miR-26a类似物，并且具有肝靶向作用。
综上所述，本发明具有以下优点：
1、本发明所用的纳米复合物形成的原料生物安全性均良好，且具有生物可降解性，已被广泛应用于生物医药领域。
2、通过本发明制备的肝靶向递送miR-26a类似物的纳米药物载体为具有包载肝癌抑制功能的miRNA的纳米复合物，制备过程简便温和，制备原料廉价易得，载体粒径范围适宜。
3、本发明制备的纳米药物载体在小鼠肝癌模型上应用时，可采用尾静脉注射的方式，给药后纳米复合物通过肝癌的高渗透性和滞留效应被动蓄积到肿瘤部位。载体表面的HA可主动靶向肝癌细胞，使载体被靶细胞摄取，并且基于合成的递送载体的高入胞和内涵体/溶酶体逃逸能力可实现目标miRNA(miR-26a mimics)的高效递送，在miRNA的肝癌治疗方面有良好的应用前景。
4、本发明采用三次酰胺反应，可将不同分子量的PLGA、PEG、HA接枝到阳离子聚合物支化PEI上，通过透析、过滤、冻干等方法得到用于miRNA体外及体内递送的载体PLGA-PEI-PEG-HA。
递送载体在去离子水中水化后，自组装形成球型的核壳结构，表面电荷为40～60mV，能以N/P比为20:1完全有效地包载miR-26a mimics，可用于体外和体内低毒高效地主动递送至肝癌细胞。
附图说明
图1为PLGA-PEI、PLGA-PEI-PEG、PLGA-PEI-PEG-HA纳米载体的透射电镜图；
其中A为PLGA-PEI；B为PLGA-PEI-PEG，C为PLGA-PEI-PEG-HA；
图2为PLGA-PEI的核磁共振氢谱；
图3为PLGA-PEI-PEG的核磁共振氢谱；
图4为PLGA-PEI-PEG-HA的核磁共振氢谱；
图5为PLGA-PEI、PLGA-PEI-PEG、PLGA-PEI-PEG-HA纳米载体的傅里叶变换红外光谱图；
图6为PLGA-PEI、PLGA-PEI-PEG、PLGA-PEI-PEG-HA纳米载体的粒径和电位示意图；
图7为PLGA-PEI、PLGA-PEI-PEG、PLGA-PEI-PEG-HA纳米载体的PEI结枝率；
其中由上至下依次为结合HA(％)、结合PEG(％)、结合PLGA(％)和游离PEI(％)；
图8为PLGA-PEI-PEG、PLGA-PEI-PEG-HA纳米载体的琼脂糖凝胶电泳图像：
图9和图10为PLGA-PEI-PEG、PLGA-PEI-PEG-HA纳米载体的细胞毒性的实验结果；
图11为PLGA-PEI-PEG、PLGA-PEI-PEG-HA载体的琼脂糖凝胶电泳图像；
图12为PLGA-PEI-PEG、PLGA-PEI-PEG-HA载体的细胞毒性实验结果；
图13为实施例4中PLGA24kDa-PEI-PEG、PLGA24kDa-PEI-PEG-HA载体包载si-CHMP2B转染SNU449细胞后(载体包载si-DHX36转染Huh7细胞后)，基因CHMP2B(DHX36)的蛋白表达情况。
具体实施方式
以下结合实施例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
本实施例提供了PLGA-PEI的制备方法，包括以下步骤：
(1)将0.3840g PLGA(Mw＝24kDa)溶于5mL DMSO，加入0.0530gEDC·HCl和0.0320gNHS，于室温下搅拌4h，活化PLGA的羧基基团，制得PLGA反应液。
(2)将0.400g b-PEI溶于另一5mL的DMSO溶液中，于室温下搅拌4h后于搅拌下缓慢滴入PLGA反应液中，恒温35℃搅拌反应24h。
(3)将步骤(2)的反应液转至去离子水中透析(MWCO＝25kDa)48h，期间多次更换透析液。
(4)透析结束后，将透析袋内混合液过滤(0.45μm)，收集滤液置于-80℃冷冻2h以上，经冷冻干燥获得PLGA-PEI。
实施例2
本实施例提供了PLGA-PEI-PEG的制备方法，包括以下步骤：
(1)将0.3300g PLGA-PEI、0.0165g PEG(2kDa)溶于8mL DMSO，0.2190g EDC·HCl和0.1320g NHS，于恒温35℃搅拌反应24h。
(2)反应结束后，将反应液转至去离子水中透析(MWCO＝2kDa)48h，期间多次更换透析液。
(3)透析结束后，将透析袋内混合液过滤(0.45μm)，收集滤液置于-80℃冷冻2h以上，再经冷冻干燥获得PLGA-PEI-PEG。
实施例3
本实施例提供了PLGA-PEI-PEG-HA的制备方法，包括以下步骤：
(1)将0.100g HA钠盐溶解在5mL去离子水中，并使用MWCO＝2kDa透析袋对稀HCl溶液(pH＝3.5)透析12h，期间多次更换透析液。
(2)透析结束后，将透析袋内混合液置于-80℃冷冻2h以上，经冷冻干燥获得HA。
(3)将冻干所得的HA取0.0070g溶于5mL DMSO中，加入0.0093g EDC·HC、0.0056gNHS，室温搅拌2h，制得活化的HA。
(4)将冻干所得的PLGA-PEI-PEG取0.1785g加入活化的HA中，于恒温35℃搅拌反应24h。
(5)反应结束后，将反应液转至去离子水中透析(MWCO＝2kDa)，期间多次更换透析液。
(6)透析结束后，将透析袋内混合液置于-80℃冷冻2h以上，经冷冻干燥获得PLGA-PEI-PEG-HA。
本实施例中的PLGA的分子量可按反应比例做出相应调整。例如，使用6kDa、12kDa、24kDa、36kDa的PLGA均可通过上述制备步骤获得PLGA-PEI-PEG-HA载体(如图11)。
试验例1
PLGA-PEI-PEG-HA包载miR-26a mimics对肝癌细胞的体外和体内递送检测试验
S1：分别对实施例1～3制备得到的纳米载体进行透射电子显微镜检测(如图1)，傅里叶变换红外光谱分析(如图5)，核磁共振氢谱分析(如图2-4)，粒径与电位测定(如图6)。
S2：分别对实施例1～3制备得到的纳米药物载体进行PEI结枝率测定(如图7)、miRNA包载效率测定(如图8)、细胞毒性测试(如图9-10)、转染效率测定(如图13)测试，筛选得到转染效率最高且细胞毒性最低的递送载体，即可用于体外递送miR-26a mimics以达到抑制肝癌细胞增殖和迁移的作用；用于体内递送mIR-26a mimics以发挥肝癌的治疗作用。
从图1-7可以看出，PLGA、PEG、HA三部分成功接枝到阳离子聚合物PEI上，合成的递送载体具有明显且规整的球形的核壳结构，粒径约为150～250nm，表面Zeta电位约为40～60mV，有利于核酸片段的包载。
从图8中可以看出，PLGA-PEI-PEG、PLGA-PEI-PEG-HA载体与包载的核酸片段的N/P比在10:1及以上时，核酸片段可被完全有效地包载。
从图11-12可以看出，当PLGA分子量为24kDa时，PLGA-PEI-PEG-HA载体以N/P＝20:1包载核酸片段，载体的毒性最低。其中PPP24载体即为PLGA(24kDa)-PEI-PEG，HA-PPP载体即为PLGA(24kDa)-PEI-PEG-HA载体。
试验例2
PLGA-PEI-PEG-HA载体包载核酸片段si-CHMP2B对肝癌细胞SNU499、包载核酸片段si-DHX36对肝癌细胞Huh7的递送效果的验证
S1：按照实施例1～3的制备方法合成PLGA(24kDa)-PEI-PEG、PLGA(24kDa)-PEI-PEG-HA载体，分别将其分散在去离子水中，形成一定浓度(1mg/mL或10mg/mL)的分散液。将合成载体以不同的N/P比与一定量的核酸片段si-CHMP2B(或si-DHX36)混合后常温静置孵育30min，使合成载体通过静电吸附作用包载核酸片段。
S2：将处于对数生长期且生长状态良好的肝癌细胞SNU449(Huh7)，加入胰蛋白酶消化，RPMI 1640(DMEM)完全培养基终止消化后，将细胞悬液离心，弃去上清，用RPMI 1640(DMEM)完全培养基重悬后接种于24孔板中。
S3：待细胞贴壁后，弃去培养液，分别加入用RPMI 1640(DMEM)培养基稀释纳米复合物si-CHMP2B@PLGA-PEI-PEG、si-DHX36@PLGA-PEI-PEG-HA，孵育36h后，收取细胞蛋白，通过检测基因CHMP2B(或DHX36)的表达来反映出合成载体对肝癌细胞SNU449(或Huh7)的递送效果。其中，N/P比为PEI游离氨基的摩尔数与核酸片段磷酸基团的摩尔数之比。
实验结果：如图13所示，在N/P比为20:1～30:1时，PLGA-PEI-PEG、PLGA-PEI-PEG-HA载体均表现出对肝癌细胞SNU449(或Huh7)良好的递送效果。
虽然对本发明的具体实施方式进行了详细地描述，但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内，本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。

Claims (10)

1.一种肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，包括以下步骤：
(1)将b-PEI溶液滴加于活化后的聚乳酸-羟基乙酸共聚物溶液中，恒温搅拌后经透析、过滤和冷冻干燥，制得PLGA-PEI；
(2)将步骤(1)制得的PLGA-PEI与聚乙二醇共溶于二甲基亚砜中，加入活化剂，恒温搅拌后经透析、过滤和冷冻干燥，制得PLGA-PEI-PEG；
(3)将透明质酸溶于二甲基亚砜中，加入活化剂，室温搅拌后制得活化的透明质酸溶液；
(4)将步骤(2)制得的PLGA-PEI-PEG与步骤(3)制得的透明质酸溶液混合后，恒温搅拌后经透析、冷冻干燥后制得。
2.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述步骤(1)中的活化后的聚乳酸-羟基乙酸共聚物溶液通过以下方法制备得到：将聚乳酸-羟基乙酸共聚物溶于二甲基亚砜中，加入活化剂后室温搅拌3-5h，制得；所述聚乳酸-羟基乙酸共聚物与活化剂的质量比为3.5-4.0:0.85。
3.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述b-PEI溶液通过以下方法制备得到：将b-PEI溶于二甲基亚砜溶液中，于室温下搅拌3-5h，制得；所述b-PEI溶解后的浓度为0.08-0.1g/mL。
4.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述步骤(1)、步骤(2)和步骤(4)中恒温的温度为30-40℃，搅拌的时间为20-25h，透析的时间为45-50h，冷冻干燥的温度为-80℃，冷冻干燥的时间为2h以上。
5.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述活化剂为1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺按照质量比8-250:5-150的混合物。
6.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述步骤(2)中的PLGA-PEI与聚乙二醇的质量比为3.3:0.15-0.17，所述聚乙二醇溶于所述二甲基亚砜后的浓度为2.0*10-3-2.1*10-3g/mL。
7.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述步骤(3)中的透明质酸通过以下方法制备得到：将透明质酸钠盐溶解于去离子水中，以pH为3.5的盐酸溶液为透析液，透析10-14h后，取透析袋内混合液于-80℃冷冻2h以上，制得；
所述溶解于去离子水中的透明质酸钠盐浓度为0.01-0.03g/mL。
8.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述步骤(4)中PLGA-PEI-PEG与透明质酸溶液中透明质酸的质量比为1.5-2.0:0.07。
9.如权利要求1所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法，其特征在于，所述聚乳酸-羟基乙酸共聚物溶液的分子量为6-36kDa。
10.采用权利要求1-9任一项所述的肝靶向递送miR-26a类似物的纳米药物载体的制备方法制备得到的肝靶向递送miR-26a类似物的纳米药物载体。
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