CN111450261A - 一种用于增强siRNA递送的多功能脂质体的制备方法 - Google Patents
一种用于增强siRNA递送的多功能脂质体的制备方法 Download PDFInfo
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Abstract
本发明提供一种用于增强siRNA递送的多功能脂质体的制备方法，采用固相肽合成的方法，利用酰胺键将R8与不同疏水性的脂肪酸进行共价连接，制成三种两亲性的穿膜肽；采用多聚阳离子鱼精蛋白将siRNA压缩，保护了siRNA免受核酸酶的降解，提高siRNA的包封率；然后通过乙醇稀释法将内核包裹进入脂质体中间层，中间层的改性穿膜肽能够帮助脂质体穿过细胞膜，而条件性电离的阳离子磷脂能够帮助内涵体逃逸；最后对脂质体进行转铁蛋白和聚乙二醇的修饰，使脂质体能够靶向肿瘤的同时延长在血液中的循环时间。这种新型的多功能脂质体综合了各种载体系统所具有的优点，能够在体内递送siRNA到达肿瘤部位，发挥高效的RNA干扰作用。
Description
技术领域
本发明公开一种用于增强siRNA递送的多功能脂质体的制备方法，为一种多功能脂质体，属于医药生产技术领域。
背景技术
随着siRNA研究的兴起, 以特定的基因作为靶位的治疗策略成了国际制药领域的一大研究和开发的热点。在实际应用过程中，基因治疗存在的最大障碍是缺少安全、高效的载体系统。目前所采用的载体主要分为两大类，即病毒载体和非病毒载体。病毒载体因具有转导效率及表达效率高的特点而得到广泛应用,但其本身存在不可避免的安全性问题，如表达外源基因时间短，免疫原性强，易引发强烈的炎症反应和免疫反应，毒性较大等。相比之下，非病毒载体具有安全性好、重复性高、制备简单等多项优点。脂质体作为常用的非病毒性载体容易制备、安全性高，却难达到质控要求，体内基因导入效率低，且无靶向性，使得其应用受到限制。因此，提供一种安全性高，具有靶向性，转染效率好的脂质体是本发明要完成的任务。
细胞穿膜肽是一类能够携带大分子物质进入细胞的短肽，可用于提高体内基因载体的递送效率，例如八聚精氨酸（R8）。然而由于 R8 极性较强，自身的穿膜效果并不理想。因此对R8进行脂肪酸修饰，能够使其具有两亲性。此外，将改性穿膜肽与靶向配体联合应用，既能克服改性穿膜肽特异性差的缺点，又能改善靶向配体穿膜效率低的不足，有效地提高 siRNA 进入细胞的效率。
常规的脂质体采用的磷脂，胆固醇作为脂相来制备。当用于递送siRNA时，选择阳离子磷脂，如DODMA，DOTMA和DOTAP等。聚乙二醇的加入能够延长脂质体在血液中的循环时间。为了增强脂质体对细胞的靶向性和穿透能力，使用改性细胞穿膜肽和肿瘤特异性配体转铁蛋白共同修饰脂质体，以达到目的。
发明内容
本发明提供一种用于增强siRNA递送的多功能脂质体的制备方法，采用固相肽合成的方法，利用酰胺键将R8与不同疏水性的脂肪酸进行共价连接，包括油酸（OA）亚油酸（LA）和硬脂酸（StA），制成三种两亲性的穿膜肽（OA-R8, LA-R8, StA-R8）。
本发明利用改性穿膜肽和靶向配体制备一种多功能脂质体，细胞毒性低，对siRNA的转染效率高，靶向性强。
本发明进一步提供了一种多功能脂质体的制备工艺，粒径低于200nm，粒度均一，分散性好，呈正电性。
本发明提供一种多功能脂质体，将鱼精蛋白结合siRNA包裹在核心防止其降解，其特征在于由改性穿膜肽与肿瘤细胞特异性配体转铁蛋白共修饰，是一种新型的基因传递系统。
本发明所述的一种多功能脂质体制备方法，包括以下步骤：
1、采用乙醇稀释法制备多功能脂质体。将卵磷脂和胆固醇作为辅助脂质掺入脂质体中，并使用阳离子脂质与改性穿膜肽混合制备脂质体。制备处方为阳离子磷脂20%~30%，改性穿膜肽15%~25%，卵磷脂 18%，胆固醇 35%，聚乙二醇2%，在乙醇中溶解后按照比例混合均匀后，采用透析法除去乙醇，透析液为pH 7.2~7.4缓冲液，室温透析2~3小时后，取出脂质体。在马尔文激光粒度分析仪上测定粒径和Zeta电位。
2、按照多功能脂质体的制备处方进行siRNA和转铁蛋白的加载。鱼精蛋白和siRNA分别用柠檬酸盐缓冲液溶解；按照优化后的摩尔比在乙醇溶液加入阳离子磷脂，改性穿膜肽，卵磷脂，胆固醇和聚乙二醇，混合均匀后，将混合液与鱼精蛋白溶液混合，涡旋30~50秒；然后将 siRNA 溶液滴加至上述混合液中，涡旋20~30秒。采用透析法除去乙醇和游离的siRNA，室温透析2~3小时后，取出脂质体。在上述脂质体中加入转铁蛋白-胆固醇，37摄氏度孵育1小时，制备载siRNA的改性穿膜肽与转铁蛋白双修饰的多功能脂质体。
本发明的积极效果在于：
多功能脂质体在改性穿膜肽的基础上设计了一种新型的基因传递系统：鱼精蛋白压缩siRNA 作为核心，阳离子磷脂和改性穿膜肽作为中间层，靶向配体转铁蛋白和聚乙二醇作为外壳。首先，采用多聚阳离子鱼精蛋白将siRNA压缩，这样可以保护siRNA 免受核酸酶的降解，提高siRNA的包封率；然后通过乙醇稀释法将内核包裹进入脂质体中间层，中间层的改性穿膜肽能够帮助脂质体穿过细胞膜，而条件性电离的阳离子磷脂能够帮助内涵体逃逸；最后对脂质体进行转铁蛋白和聚乙二醇的修饰，使脂质体能够靶向肿瘤的同时延长在血液中的循环时间。这种新型的多功能脂质体综合了各种载体系统所具有的优点，能够在体内递送siRNA到达肿瘤部位，发挥高效的RNA干扰作用。
具体实施方式
通过以下实施例进一步举例描述本发明，并不以任何方式限制本发明，在不背离本发明的技术解决方案的前提下，对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
1、阳离子脂质体制备
在乙醇溶解的磷脂溶液加入DODMA，OA-R8，Chol和DSPE-PEG2000，使其摩尔比为20/25/35/2，其中卵磷脂摩尔比为18，混合均匀后，采用pH为7.2~7.4的HEPES缓冲液透析除去乙醇，室温透析2~3小时后，取出脂质体。测得的平均粒径为98.8nm，多分散系数平均为0.107，Zeta电位是38.5 mV。
2、多功能脂质体载siRNA传递系统制备
鱼精蛋白和siRNA分别用柠檬酸盐缓冲液（20 mM，pH 4）溶解，将步骤1中的脂质混合液与鱼精蛋白溶液混合，涡旋 30~50秒；然后将siRNA溶液滴加至上述混合液中，涡旋20~30秒。采用透析法除去乙醇和游离的siRNA，室温透析2~3小时后，取出脂质体。转铁蛋白-胆固醇与磷脂按照摩尔比1:100，加入到制备好的脂质体中，37摄氏度孵育1小时，得到载siRNA的改性穿膜肽与转铁蛋白双修饰的多功能脂质体（sTOLP）。测得平均粒径为150.5 nm，Zeta电位是3.4 mV。
实施例2
1、阳离子脂质体制备
在乙醇溶解的磷脂溶液加入DODMA，LA-R8，Chol和DSPE-PEG2000，使其摩尔比为20/25/35/2，其中卵磷脂摩尔比为18，混合均匀后，采用pH为7.2~7.4的HEPES缓冲液透析除去乙醇，室温透析2~3小时后，取出脂质体。测得的平均粒径为105.3 nm，多分散系数平均为0.159，Zeta电位是33.4 mV。
2、多功能脂质体载siRNA传递系统制备
鱼精蛋白和siRNA分别用柠檬酸盐缓冲液（20 mM，pH 4）溶解，将步骤1中的脂质混合液与鱼精蛋白溶液混合，涡旋30~50s；然后将siRNA溶液滴加至上述混合液中，涡旋20~30秒。采用透析法除去乙醇和游离的siRNA，室温透析2~3小时后，取出脂质体。转铁蛋白-胆固醇与磷脂按照摩尔比1:100，加入到制备好的脂质体中，37摄氏度孵育1小时，得到载siRNA的改性穿膜肽与转铁蛋白双修饰的多功能脂质体（sTOLP）。测得平均粒径为165.1 nm，Zeta电位是5.7 mV。
实施例3
1、制备多功能脂质体以及对照脂质体
采用乙醇稀释法制备四组脂质体，分别是（1）改性穿膜肽与转铁蛋白双修饰的多功能脂质体（TOLP）：按照DODMA/ OA-R8/Egg-PC/ Chol/ DSPE-PEG 2000= 20/25/18/35/2摩尔比制备乙醇溶液，与HEPES缓冲液混合均匀后，透析除去乙醇。室温透析2~3小时后，将转铁蛋白-胆固醇与磷脂按照摩尔比1:100，加入到制备好的脂质体中，37摄氏度孵育1小时，得到TOLP；（2）未经修饰的脂质体（LP）：按照Egg-PC/ Chol=1/3制备乙醇溶液，注入HEPES缓冲中液后涡旋30~50秒，得到LP；（3）只有改性穿膜肽OA-R8修饰的脂质体（OLP）：按照OA-R8/ePC/Chol = 45/18/35制备乙醇溶液，注入HEPES缓冲中液后涡旋30~50秒，得到OLP；（4）只有转铁蛋白修饰的脂质体（TLP）：按照Egg-PC/ Chol=1/3制备乙醇溶液，注入HEPES缓冲中液后涡旋30~50秒，将转铁蛋白-胆固醇与磷脂按照摩尔比1:100，加入到制备好的脂质体中，37摄氏度孵育1小时，得到TLP。
2、脂质体细胞毒性鉴定
收集处于对数生长期的肝癌细胞HepG2，胰酶消化计数后以5×103~8×103个/孔的密度接种于96孔板中，培养过夜后，吸除培养基，加入100微升含有四组步骤1中制备的DMEM完全培养基稀释的脂质体，同时设定空白对照和背景孔，每组设6个平行孔，培养24~48小时后，每孔加入浓度5mg/ml MTT溶液15~20微升，置于CO2培养箱中继续培养4小时，轻轻吸除上清液后加入100~150微升的DMSO溶解甲臜颗粒，酶标仪于490 nm处测定OD值，计算细胞活力。其中，TOLP的细胞活力为93.33%，TLP组的细胞活力为99.83%，OLP组的细胞活力为97.07%，LP组细胞活力为102.33%，四组脂质体与空白对照组之间均无显著差异。
以下实验表明本发明和常规的阳离子脂质体制备处方对比（详见表1）
表1 .本发明和常规的阳离子脂质体制备处方对比
| 项目 | 本发明 | 常规脂质体1 | 常规脂质体2 |
| 组分 | 阳离子磷脂/ 改性穿膜肽/卵磷脂/ 胆固醇/ 聚乙二醇2000 | 阳离子磷脂DODMA/卵磷脂/ 胆固醇 | 阳离子磷脂DOTMA/卵磷脂/ 胆固醇 |
| 比例范围 | 20~30/15~25/18/35/2 | 45/18/35 | 45/18/35 |
| 粒径（nm） | 98.8~105.3 | 107.8~117.8 | 120.3~131.7 |
| 多分散系数PDI | 0.107~0.159 | 0.265 | 0.215 |
| Zeta电位（mV） | 33.4~38.5 | 8.4~9.2 | 35.7 ± 37.5 |
结论：
本发明制备的多功能脂质体粒径适宜，粒度均一，具有正电性，并且无明显细胞毒性，有利于siRNA的装载与递送。采用改性穿膜肽与肿瘤特异性配体转铁蛋白共同修饰脂质体，能够增强siRNA的转染。多功能脂质体制备方法简单、操作方便，是一种适用于siRNA递送的安全有效的传递系统。
Claims (1)
1.一种用于增强siRNA递送的多功能脂质体的制备方法，包括以下步骤：
1）采用乙醇稀释法制备多功能脂质体
将卵磷脂和胆固醇作为辅助脂质掺入脂质体中，并使用阳离子脂质与改性穿膜肽混合制备脂质体；
制备处方为阳离子磷脂20%~30%，改性穿膜肽15%~25%，卵磷脂 18%，胆固醇 35%，聚乙二醇2%，在乙醇中溶解后按照比例混合均匀后，采用透析法除去乙醇，透析液为pH 7.2~7.4缓冲液，室温透析2~3小时后，取出脂质体；
在马尔文激光粒度分析仪上测定粒径和Zeta电位；
2）按照多功能脂质体的制备处方进行siRNA和转铁蛋白的加载
鱼精蛋白和siRNA分别用柠檬酸盐缓冲液溶解；按照优化后的摩尔比在乙醇溶液加入阳离子磷脂，改性穿膜肽，卵磷脂，胆固醇和聚乙二醇，混合均匀后，将混合液与鱼精蛋白溶液混合，涡旋30~50秒；然后将 siRNA 溶液滴加至上述混合液中，涡旋20~30秒；
采用透析法除去乙醇和游离的siRNA，室温透析2~3小时后，取出脂质体；
在上述脂质体中加入转铁蛋白-胆固醇，37摄氏度孵育1小时，制备载siRNA的改性穿膜肽与转铁蛋白双修饰的多功能脂质体。
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| CN114432328A (zh) * | 2020-11-05 | 2022-05-06 | 广州中医药大学第一附属医院 | 一种用于治疗肝细胞癌的药物组合物及其应用 |
| CN114832117A (zh) * | 2022-05-20 | 2022-08-02 | 天津科技大学 | 一种细胞穿透肽修饰的核酸-阳离子热敏脂质体及其制备方法 |
| CN117025592A (zh) * | 2023-07-05 | 2023-11-10 | 北京大学 | 一种靶向jfk的小干扰rna及其用途 |
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| CN114432328A (zh) * | 2020-11-05 | 2022-05-06 | 广州中医药大学第一附属医院 | 一种用于治疗肝细胞癌的药物组合物及其应用 |
| CN114832117A (zh) * | 2022-05-20 | 2022-08-02 | 天津科技大学 | 一种细胞穿透肽修饰的核酸-阳离子热敏脂质体及其制备方法 |
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