CN103143033B - 一种靶向肝脏细胞的运载体及其制备方法 - Google Patents
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Abstract
本发明提供了一种将基因靶向性导入肝脏细胞的运载体及其制备方法，所述运载体是由脂质体和载脂蛋白组成的复合物，其中脂质体为由双十二烷基二甲基溴化铵与二油酰磷脂酰乙醇胺制成，并经二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺修饰的双层包膜的磷脂质小囊，载脂蛋白为ApoE。本发明构建的靶向性基因运载体通过以配体—受体亲和性的富集作用来提高载体的靶向性，提高转染效率和肝细胞内的富集度，具有显著的肝脏细胞靶向性，可以作为各类治疗药剂的人工膜和赋形剂，以增强药物的疗效。
Description
技术领域
本发明涉及脂质体运载体，特别是涉及一种能够特异性针对肝脏细胞的脂质体运载体及其制备方法。
背景技术
肝癌是常见肿瘤之一，在全球发病率日益上升，严重危害人类的生命健康。目前治疗肝癌无外乎化疗或手术治疗，但效果都不很理想。随着基因技术的日益发展，从基因方面治疗肝癌成为了可能，且技术越来越成熟。如何实现基因治疗的靶向性，是目前研究亟待解决的问题。
应用于基因治疗的载体主要有病毒载体和非病毒载体两种。病毒载体转染效率高，但存在免疫原性高、毒性大、目的基因容量小、靶向特异性差、制备较复杂及费用较高等缺点。因此，人们愈来愈重视非病毒载体的研究。阳离子聚合物作为非病毒基因载体显示出巨大的优势和潜力，它不但可以降低药物在细胞内的蓄积和毒性，还可以通过自身降解来控制基因的释放。
阳离子脂质体作为一种高效的基因传递载体，具有下列优点：1）可防止核酸被体内物质降解，可将其特异性传递到靶细胞中；2）无毒、无免疫原性，具有生物惰性，可生物降解；3）易于制备，使用方便，可将大的DNA 片断转运到细胞中；4）基因转染率高，离体细胞可以瞬间表达外源基因。目前，阳离子脂质体介导转染技术已经在基因治疗方面取得了巨大的成果。
发明内容
本发明的目的是提供一种脂质体运载体，使其具有良好的肝细胞靶向性，从而提高所包裹药物的作用。同时，本发明还提供了该运载体的制备方法。
本发明提供的靶向肝脏细胞的运载体是一种由脂质体和载脂蛋白组成的复合物，其中，所述的脂质体为由双十二烷基二甲基溴化铵与二油酰磷脂酰乙醇胺组成，并经二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺修饰的双层包膜的磷脂质小囊，且双十二烷基二甲基溴化铵与二油酰磷脂酰乙醇胺的质量比为1：0.6-1.5，所述的载脂蛋白为ApoE，脂质体与载脂蛋白的质量比为1000-1200：1。
进一步地，所述的ApoE预先以2-亚氨基硫烷盐酸盐进行活化。
本发明还提供了上述靶向肝脏细胞的运载体的制备方法，包括以下步骤：
1）将双十二烷基二甲基溴化铵和二油酰磷脂酰乙醇胺按照1：0.6-1.5的质量比溶解在无水乙醇中，于50-70℃加入水中，分别以150-200nm、80-120nm及40-60nm的聚碳酸酯膜挤压整理，得到粒径80-110nm的脂质体；水洗除去乙醇后，稀释得到浓度13-17mg/ml的阳离子脂质体水溶液；
2）向阳离子脂质体水溶液中加入二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺，加入量4-10μg/ml，于50-70℃温育0.8-1.2h，得到活化的阳离子脂质体；
3）按照ApoE蛋白：2-亚氨基硫烷盐酸盐为1：1.5-2.5的摩尔比，将ApoE蛋白和2-亚氨基硫烷盐酸盐的PBS溶液混合，室温反应0.8-1.2h，得到活化ApoE蛋白；
4）将活化的阳离子脂质体与活化ApoE蛋白按照1000-1200：1的质量比混合，氮气保护下室温反应3-5h，得到所述靶向肝脏细胞的运载体。
本发明将得到的靶向肝脏细胞的运载体进行进一步的纯化，其方法为：将甘氨酸与2-亚氨基硫烷盐酸盐按照1.5-2.5：1的质量比混合，室温反应0.8-1.2 h后，加入到制备得到的运载体中，室温反应3-5h，微型凝胶柱离心除去未反应的小分子化合物，得到最终产品。
本发明将上述得到的最终产品的浓度调整为3.5-4.5mg/ml。
本发明将阳离子脂质体与载脂蛋白结合，共同构建成为了靶向性针对肝脏细胞的运载体。本发明所构建的靶向性基因传递载体是一种全新的高效、高靶向性、高安全性的载体体系，该运载体中含有ApoE，可以特异性识别肝细胞膜上受体，从而介导脂质体与肝细胞膜结合，通过以配体—受体亲和性的富集作用来提高载体的靶向性，提高转染效率和肝细胞内的富集度。
本发明提供的靶向肝脏细胞的运载体可以作为各类治疗药剂的人工膜和赋形剂，通过各种给药途径，使所包裹的药物具有打靶作用，准确地击中病变部位、组织和细胞，从而增强药物的疗效。
附图说明
图1为L-02肝细胞分别转染载体1和2号（100×）。
左图转染载体1号4μl+质粒pGenesil-1 8μl，右图转染载体2号4μl+质粒pGenesil-1 8μl。
图2为SMMC-7721肝癌细胞分别转染载体1和2号（100×）。
左图转染载体1号4μl+质粒pGenesil-1 8μl，右图转染载体2号4μl+质粒pGenesil-1 8μl。
图3为H9c2心肌细胞分别转染载体1和2号（100×）。
左图转染载体1号4μl+质粒pGenesil-1 8μl，右图转染载体2号4μl+质粒pGenesil-1 8μl。
具体实施方式
实施例1
靶向性导入肝脏细胞的运载体，是由脂质体和载脂蛋白ApoE组成的复合物。该复合物是一类具有细胞膜穿透作用的非病毒运载体。其脂质体是一种人工制备的携有双层包膜的磷脂质小囊，因ApoE可以特异性识别肝细胞膜上受体，从而介导脂质体与肝细胞膜结合，有利于载体的靶向性。
运载体的具体制备方法包括以下步骤：
1、阳离子脂质体的制备
称取双十二烷基二甲基溴化铵（DDAB，AVANTI POLAR LIPIDS, INC.）、二油酰磷脂酰乙醇胺（DOPE，AVANTI POLAR LIPIDS, INC.）各100mg，溶解在1.2ml无水乙醇中，于60℃下注入到12ml注射用水中。
分别用规格150-200nm、80-120nm及40-60nm的聚碳酸酯膜挤压整理，得到粒径约为80-110nm的脂质体；用8倍体积的水进行超滤，除去体系中的乙醇后，定容得到浓度为15mg/ml的普通阳离子脂质体，记作CL-1。
2、脂质体的活化
量取CL-1 5ml，加入含有25μg二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺（DSPE-PEG-MAL，AVANTI POLAR LIPIDS, INC.）的水溶液中，60℃温育1.2h，记作CL-2。
3、蛋白活化
将ApoE蛋白和2-亚氨基硫烷盐酸盐（2-IT，THERMO）分别用PBS（磷酸盐缓冲液）溶解后，按照1：2的摩尔比将两种溶液混合，室温反应1h，记作AP。
4、蛋白和脂质体连接
将CL-2和AP按照1000：1的质量比混合，氮气保护下室温反应3h，记作CL-P。
5、纯化
将甘氨酸（SIGMA）与2-IT按照2.5：1的比例混合，室温反应1.2h后，取适量加入到CL-P中，室温反应4h，以微型凝胶柱离心除去未反应的小分子化合物后，定容得到浓度为3.5-4.5mg/ml的运载体。
实施例2
靶向肝脏细胞的运载体转染肝癌细胞的效率研究
1、细胞培养
供试细胞：人肝癌细胞SMMC7721，人正常肝细胞L-02，心肌细胞H9c2。
SMMC7721体外培养于含10%胎牛血清（GIBCO）的RPMI1640中，其余细胞均培养于含10%胎牛血清的高糖型DMEM(GIBCO)中。
2、肝癌细胞系对运载体复合物的转染效率
实验设2组，样品1号为脂质体载体组，样品2号为脂质体-ApoE载体组，具体参数如表1，比较2种载体的转导效率差异。
六孔板细胞传代，密度2×105时，37℃，5%CO2隔夜培养。六孔板细胞转染，载体1和2号4μl、质粒pGenesil-1 8μl分别在250μl DMEM双无培养基孵育5分钟；然后将载体1号和2号分别与质粒混合后孵育20分钟；将六孔板中细胞用2ml DMEM双无培养基换液；载体和质粒混合物加至六孔板各孔；转染6小时后加血清培养。用荧光显微镜观察EGFP的表达来鉴定转染效果。
转染率计算：转染率%=[绿色荧光细胞数/总细胞数]×100%。
计算转染效率结果见表2。
由表2可见，在肝细胞和肝癌细胞中，带有ApoE的脂质体（2号）携带质粒的转染效率明显高于不带ApoE的脂质体（1号）的转染效率。
Claims (3)
1.一种靶向肝脏细胞的运载体的制备方法，其特征是包括以下步骤：
1）将双十二烷基二甲基溴化铵和二油酰磷脂酰乙醇胺按照1：0.6-1.5的质量比溶解在无水乙醇中，于50-70℃加入水中，分别以150-200nm、80-120nm及40-60nm的聚碳酸酯膜挤压整理，得到粒径80-110nm的脂质体；水洗除去乙醇后，稀释得到浓度13-17mg/ml的阳离子脂质体水溶液；
2）向阳离子脂质体水溶液中加入二硬脂酰磷脂酰乙醇胺-聚乙二醇-马来酰亚胺，加入量4-10μg/ml，于50-70℃温育0.8-1.2h，得到活化的阳离子脂质体；
3）按照ApoE蛋白：2-亚氨基硫烷盐酸盐为1：1.5-2.5的摩尔比，将ApoE蛋白和2-亚氨基硫烷盐酸盐的PBS溶液混合，室温反应0.8-1.2h，得到活化ApoE蛋白；
4）将活化的阳离子脂质体与活化ApoE蛋白按照1000-1200：1的质量比混合，氮气保护下室温反应3-5h，得到所述靶向肝脏细胞的运载体。
2.根据权利要求1所述的靶向肝脏细胞的运载体的制备方法，其特征是按照以下方法纯化得到的运载体：将甘氨酸与2-亚氨基硫烷盐酸盐按照1.5-2.5：1的质量比混合，室温反应0.8-1.2 h后，加入到制备得到的运载体中，室温反应3-5h，微型凝胶柱离心除去未反应的小分子化合物，得到最终产品。
3.根据权利要求2所述的靶向肝脏细胞的运载体的制备方法，其特征是所述最终产品的浓度为3.5-4.5mg/ml。
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| CN109652456B (zh) * | 2019-01-22 | 2022-04-05 | 北京大学深圳医院 | 用于细胞转染的受体转染微泡、配体转染微泡以及两段制导式细胞转染方法 |
| CN110452298A (zh) * | 2019-08-26 | 2019-11-15 | 桂林医学院 | 一种肝靶向配体及其在脂质体制剂中的应用 |
| CN111557910B (zh) * | 2020-04-08 | 2022-09-20 | 厦门诺康得生物科技有限公司 | 一种靶向肝脏的nmn脂质体纳米粒子及其制备方法和应用 |
| CN113546047B (zh) * | 2020-04-23 | 2023-01-24 | 吉林大学第一医院 | Mal官能团修饰的脂质体在靶向肝脏递送中的应用 |
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