CN117088820A - 一种新型阳离子lnp递送系统及制备方法 - Google Patents
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Abstract
本发明提供一种新型阳离子LNP递送系统及制备方法,属于医药技术领域。本发明提供了一种阳离子脂质,为式I或其盐或异构体;其中R为含阳离子性杂原子的杂环基团。本发明的阳离子脂质构建的递送系统较现有递送系统,有更好的递送效率。
Description
技术领域
本发明属于医药技术领域,具体涉及一种新型阳离子LNP递送系统及制备方法。
背景技术
核酸治疗在遗传紊乱到获得性病症的疾病中均具有应用前景,具体包括癌症、感染性紊乱(AIDS)、心脏病、关节炎、和神经变性疾病(如帕金森病和阿尔茨海默病)等等。这种新型疗法不仅可递送功能基因来修复遗传缺陷或诱导外源基因产物表达,而且还可递送核酸来抑制内源基因表达以提供治疗效果。基因表达的抑制可通过例如反义寡核苷酸、双链RNA(如siRNAs、miRNAs)或核酶来介导,此种疗法也称为RNA疗法。
RNA疗法(mRNA,siRNA,saRNA,RNAi,circRNA)正在作为一种新型技术,用于多种疾病的预防和治疗。RNA药物发挥作用需要将带负电荷的大RNA分子穿过细胞膜,但裸露RNA(裸RNA)一旦进入细胞,即面临被核糖核酸酶降解的风险。除此之外裸RNA还会引起过高的免疫反应产生副作用,因此,有效的递送系统对于实现良好的治疗至关重要。
当前应用最为广泛的是基于脂质材料的递送系统,这类材料被称为脂质纳米颗粒(Lipid Nanoparticle,LNP)。该技术是由磷脂组成的脂质纳米颗粒,将mRNA包载其中,可保护mRNA免于递送过程的酶降解和免疫系统的清除,促进其跨膜转运且在细胞质中释放mRNA用于翻译蛋白,中和抗体实现机体免疫。
LNP主要由4种物质组成:可电离脂质、中性辅助脂质、胆固醇、聚乙二醇(PEG)化脂质。具体可由pH依赖性可电离脂质、中性辅助磷脂、胆固醇、聚乙二醇(PEG)化脂质组成。其中:
pH敏感性的可电离脂质,在包载过程中提供正电荷与带负电荷的核酸结合,同时在内涵体中再次转为正电荷有助于内涵体逃逸,提高mRNA体内转染效率。因此可电离脂质被设计为在pH值下降时获得正电荷,促进脂质体的内体摄取,同时保留带负电荷的mRNA分子的封装。氨基脂质成分的结构在递送效率、耐受性和组织清除中起关键作用,是递送核酸和穿过细胞膜的关键性物质,也是技术围绕的重点;
中性辅助脂质(一般为饱和磷脂)用于支撑脂质双层结构的形成并稳定其结构排列;
具有膜融合性的胆固醇调整脂质膜的完整性和硬度,增强LNP的稳定性;
能改善亲水性的PEG化脂质位于LNP表面,既可以避免粒子被免疫系统快速清除以延长循环时间,又可以防止颗粒聚集以增加稳定性,PEG在防止巨噬细胞介导的降解方面发挥着重要作用。
专利US8058069B2公开了:一种或多种活性剂或治疗剂的新颖、稳定的脂质颗粒、制备脂质颗粒的方法以及递送和/或施用脂质颗粒的方法。更具体地,提供包含核酸(例如一个或多个干扰RNA)的稳定核酸脂质颗粒(SNALP)、制备SNALP的方法以及递送和/或施用SNALP的方法。目前上市三种LNP递送系统,可电离脂质分别使用Dlin-MC3-DMA,SM-102和ALC 0315。以上递送系统的递送效率仍存在提高空间。
发明内容
为了解决上述问题,本发明通过新型可电离阳离子脂的开发,并将其用于LNP递送系统,寻找当前LNP递送系统替代方法,以期提高包封效果和递送效率,带来新的递送思路。
本发明基于现有技术中SM-102的结构进行改造,调整了氨基结构位置,以期达到更好的效果。
SM-102结构为:
一方面,本发明提供了一种阳离子脂质。
所述的阳离子脂质为式Ⅰ或其盐或异构体:
以上通式中,R为可质子化的杂环基团,可以是含阳离子性杂原子的杂环基团。
所述的阳离子性杂原子可以是O、N、S中的任意一种或多种,所述的阳离子性杂原子的数量可以是任意一个或多个。
所述的杂环基团可以是饱和杂环基团或不饱和杂环基团,优选为饱和杂环基团。
所述的杂环基团可以是螺环杂环基团或稠合杂环基团。
优选地,所述的R可以是含N杂环基团。
所述的含N杂环为烷基N杂环,优选为辛烷基氮杂环。
优选地,所述的含N杂环基团包括至少1个N,具体地,可以包括1个或多个N,也可以包括1个N和一个或多个其他阳离子性杂原子,优选地,包括2个N。
所述的含N杂环基团的N中,至少有1个N含有至少1个氢,具体地,可以有1个N上含有1个氢,也可以有2个N上各含有1个氢,还可以有1个N上含有2个氢,还可以有2个N上各含有2个氢。
所述的R可以是杂双环辛烷基,优选为含N的杂双环辛烷基,进一步优选为二氮杂双环辛烷基。
优选地,所述的R可以是1,4-二氮杂螺[2.3]辛烷基或1,5-二氮杂二环[3.3.0]辛烷基。
优选地,R的连接方式可以是C-N。
优选地,所述的阳离子脂质选自BJL-ALC-6或BJL-ALC-9:
另一方面,本发明提供了前述的阳离子脂质在制备脂质纳米颗粒中的应用。
一般来说,所述的阳离子脂质可以与胆固醇、聚乙二醇或中性辅助磷脂联用。
本发明同时提供了包含前述的阳离子脂质的脂质纳米颗粒。
所述的脂质纳米颗粒还包括胆固醇、聚乙二醇或中性辅助磷脂。
又一方面,本发明提供了前述的阳离子脂质或脂质纳米颗粒在制备RNA药物中的应用。
具体地,前述的阳离子脂质或脂质纳米颗粒通过递送RNA实现应用。
本发明同时提供了包含前述的阳离子脂质或脂质纳米颗粒的RNA药物。
所述的RNA药物中可以包括:mRNA,siRNA,saRNA,RNAi,circRNA中的一种或多种。
所述的药物中还可以包括其他药学上可接受的载体或赋形剂。
本发明的有益效果:
可电离阳离子脂在酸性环境下,氨基带正电荷,可以与带负电的RNA的磷酸基团结合。相较于已经上市的SM102,本发明的BJL-ALC-6、BJL-ALC-9将氨基结构从主链移到支链,更方便与RNA相结合,从而提高递送效率。
本发明基于BJL-ALC-6、BJL-ALC-9构建的递送系统较现有的MC3-LNP及SM102-LNP递送系统,有更好的递送效率。
附图说明
图1为4种阳离子脂质制备的药物包封率和粒径测定结果。
图2为给药后小鼠活体成像图。
图3为给药后小鼠活体成像荧光强度。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例中BJL-ALC-6和BJL-ALC-9均委托南京博济达医药科技有限公司进行制备。
实施例1RNA药物的制备方法
基础实施例制备得到的两种阳离子脂质BJL-ALC-6、BJL-ALC-9,分别与胆固醇,聚乙二醇(PEG)化脂质,中性辅助磷脂DSPC共同制成脂相溶液,然后与水相mRNA药物进行LNP包封。获得的包封产品。将SM-102和另一种常见的已上市阳离子脂Dlin-MC3-DMA(以下简称为MC3)作为阳性对照对比。
试剂材料 | 品牌 | 货号 |
DSPC | 艾伟拓 | S01005 |
Cholestrol | 艾伟拓 | O01001 |
PEG2000 | 艾伟拓 | O02005 |
药物具体制备步骤包括:
1.逐一称量并在无水乙醇中溶解上述脂相物料。
2.冰水浴融化mRNA原液,加入枸橼酸缓冲液中按RNA浓度进行稀释。
3.使用微流控设备按脂相:水相比例1:3的流速比,进行脂质纳米颗粒制备。
4.对包封后的样品进行稀释,并使用超滤管进行超滤浓缩,然后进行无菌过滤。
5.检测样品包封率和粒径指标。
包封率测量方式:
包封率测定的关键是将包封药物和未包封的游离药物分离,再利用光谱分析手段检测包封药物或游离药物的浓度。首先检测LNP-RNA溶液中游离在LNP颗粒外的RNA数量,接着用Triton-100破坏LNP结构,使得RNA释放到外部溶液中,检测出溶液中全部的RNA数量。两者的差值就是被包封在LNP颗粒内部的RNA数量,从而获得包封率。
使用赛默飞的RiboGreen试剂盒,TR值为含浓度为2%的Triton-100的TE缓冲缓冲溶液破坏LNP结构后的酶标仪荧光示数,TE值为纯TE缓冲溶液中LNP的荧光示数。
包封率(%)=[(TR值-TE值)/TR值]×100%
粒径测量方式:
使用马尔文粒度分析仪,将LNP产品用PBS稀释20倍后测量。均按照以下体系制备药物:
油相总浓度12.5mmol/L,其中四个成分的浓度占比(物质的量浓度占比)为阳离子脂质50%,DSPC 10%,Cho 38.5%,PEG 1.5%,NP比3.4,RNA种类为自复制荧光素酶RNA,RNA浓度0.2ug/uL,pH为4,体积共计4mL。
自复制荧光素酶RNA制备方法:
将线性化质粒DNA(一万bp,购买自金斯瑞生物科技有限公司)利用IVT试剂盒(南京诺唯赞生物科技股份有限公司)进行体外转录,RNA和LiCl混合溶液静置离心后,加入70%乙醇溶液,离心并去掉上清溶液,在生物安全柜干燥后,用RNase-free H2O溶解RNA沉淀,得到纯化后的RNA溶液。
4种阳离子脂质制备的药物包封率和粒径测定结果见表1和图1。
G1 MC3 | G2 SM102 | G3 BJL-ALC-6 | G4 BJL-ALC-9 | |
包封率/% | 91.10 | 93.65 | 92.60 | 94.49 |
粒径/nm | 84.98 | 86.76 | 85.33 | 85.58 |
结果表明,本发明制备的LNP包封率稳定在90%以上,粒径稳定在70-90nm间。
实施例2动物实验
根据实施例1种制备的4种药物设置动物实验组别,每组小鼠数量3只,在小鼠身上进行左右腿肌肉注射,注射量以自复制荧光素酶RNA为2μg为准,实际注射量为10μL药物,给药当天记为Day0。Day3活体成像观察注射后效果,评估新型阳离子脂质的递送效果。
小鼠信息:
品种 | 小鼠Balb/c |
周龄 | 6-8 |
性别 | 雌 |
体重 | 18-25g |
供应商 | 浙江维通利华实验动物有限公司 |
活体成像:使用IVIS Lumina XRMS Series III小动物活体成像系统检测后,用配套的Living Image软件进行数据分析。
结果如图2-3所示。
从活体成像荧光强度可以看出,新合成的阳离子脂质体BJL-ALC-6和BJL-ALC-9替代市面上现有的阳离子MC3和SM102之后,在小鼠体内表达的荧光素酶更多,说明递送效率更好。图3对应的数据如下:
Claims (16)
1.一种阳离子脂质,其特征在于,为式I或其盐或异构体:
其中R为可质子化的杂环基团。
2.根据权利要求1所述的阳离子脂质,其特征在于,所述的杂环基团为饱和杂环基团。
3.根据权利要求1所述的阳离子脂质,其特征在于,所述的杂环基团为螺环杂环基团或稠合杂环基团。
4.根据权利要求1所述的阳离子脂质,其特征在于,R为含N杂环基团。
5.根据权利要求4所述的阳离子脂质,其特征在于,R为烷基N杂环,优选为辛烷基N杂环。
6.根据权利要求4所述的阳离子脂质,其特征在于,所述的含N杂环基团包括至少1个N;所述的含N杂环基团的N中,至少有1个N含有至少1个氢。
7.根据权利要求6所述的阳离子脂质,其特征在于,R的连接方式为C-N。
8.根据权利要求5所述的阳离子脂质,其特征在于,R为杂双环辛烷基。
9.根据权利要求8所述的阳离子脂质,其特征在于,R为二氮杂双环辛烷基。
10.根据权利要求9所述的阳离子脂质,其特征在于,R为1,4-二氮杂螺[2.3]辛烷基或1,5-二氮杂二环[3.3.0]辛烷基。
11.根据权利要求1所述的阳离子脂质,其特征在于,选自BJL-ALC-6或BJL-ALC-9:
12.权利要求1-11任一项所述的阳离子脂质在制备脂质纳米颗粒中的应用。
13.包括权利要求1-11任一项所述的阳离子脂质的脂质纳米颗粒。
14.根据权利要求13所述的脂质纳米颗粒,其特征在于,还包括胆固醇、聚乙二醇或中性辅助磷脂。
15.权利要求1-11任一项所述的阳离子脂质或权利要求13-14任一项所述的脂质纳米颗粒在制备RNA药物中的应用。
16.包括权利要求1-11任一项所述的阳离子脂质或权利要求13-14任一项所述的脂质纳米颗粒的RNA药物。
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