CN117603078A - 新型含氟可离子化脂质、制备方法及在递送体系中的应用 - Google Patents
新型含氟可离子化脂质、制备方法及在递送体系中的应用 Download PDFInfo
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Abstract
本发明涉及mRNA递送技术领域,具体涉及一种新型含氟可离子化脂质、制备方法及在递送体系中的应用,所述含氟可离子化脂质的结构式如式I所示:式I,化学式为C47H88F3NO3,分子量为772.22,稳定性更好,递送效率也更高。
Description
技术领域
本发明涉及mRNA递送技术领域,具体涉及一种新型含氟可离子化脂质、制备方法及在递送体系中的应用。
背景技术
核酸分子做为天然的高负电荷分子,其难以直接穿过负电荷的细胞膜进入细胞而发挥治疗作用,因此通常需要递送载体来协助核酸进入目标细胞内。而且,裸露的mRNA还存在进一步的问题:第一,为保护的mRNA会被体内无处不在的RNA酶降解;第二,裸露的mRNA可能会增加其对免疫系统的激活,影响最终的蛋白翻译。在现有的非病毒类载体中,脂质纳米粒(LNP)是经过广泛真实世界验证的递送工具,已经至少在三款经FDA批准上市的核酸药物中采用,其中包括了一款siRNA药物与两款新冠mRNA疫苗。
尽管已经上市的几款LNP递送工具能在一定程度上实现核酸——特别是mRNA分子在体内的翻译表达,但是它们的递送效率仍有很大的提升空间。在mRNA疫苗领域,增加对目标mRNA的翻译表达效率能直接增加体内的目标抗原分子数量,从而增加最终特异性抗体的增加。LNP的核酸递送效率主要依赖于其中可离子化脂实现,因此可离子化脂结构的设计是其中最为关键的因素。
而现有的可离子化脂质参与形成的LNP的稳定时间不够持久,对mRNA的递送效率以及转染效率还不够高。
发明内容
本发明的目的是提供一种新型含氟可离子化脂质、制备方法及在递送体系中的应用。
本发明中的含氟可离子化脂质具有一种特定的脂质疏水链组合,其中一条疏水脂质链为包含2个不饱和双键的碳十八链,另一条为支化的可降解疏水链;进一步的,可降解疏水链包含一个9位羟基取代的碳十八链,并通过一个酯键以及七亚甲基疏水连接子与可离子化中心连接。含三氟取代基的脂质头能够与膜具有更好的亲和性,同时,特定的脂质疏水链组合能很好参与形成非常稳定的LNP,同时极大促进对所包封mRNA的递送能力。
本发明的上述目的是通过以下技术方案得以实现的:一种新型含氟可离子化脂质,所述含氟可离子化脂质的结构式如式I所示:
式I,化学式为C47H88F3NO3,分子量为772.22。
一种新型含氟可离子化脂质的制备方法,所述制备方法包括如下步骤:
(a)将9-十八醇和8-溴辛酸进行酯化反应,得到中间体1;
(b)将中间体1和3-氨基-1,1,1-三氟丙烷-2-醇进行缩合反应,得到中间体2;
(c)将中间体2和6,9二烯十八溴进行缩合反应,得到所述可离子化脂质。
作为本发明的优选,在步骤(a)中,9-十八醇的摩尔量为9-11mmol,8-溴辛酸的摩尔量为9-11mmol。
作为本发明的优选,在步骤(b)中,中间体1的摩尔量为0.4-0.6mmol。
作为本发明的优选,在步骤(b)中,中间体1和3-氨基-1,1,1-三氟丙烷-2-醇在异丙醇中搅拌得到的粗品通过硅胶柱层析得到中间体2。
作为本发明的优选,在步骤(c)中,中间体2的摩尔量为1mmol,6,9二烯十八溴的摩尔量为1mmol。
前述含氟可离子化脂质在制备mRNA递送体系中的应用。
一种mRNA递送体系,包括前述的含氟可离子化脂质、磷脂、PEG脂质和结构性脂质和mRNA。
作为本发明的优选,含氟可离子化脂质与mRNA的N/P为6∶1。
前述一种mRNA递送体系的制备方法,包括如下步骤:(a)将可离子化脂质、结构性脂质、磷脂和PEG脂质按照摩尔比为50∶38.5∶10∶1.5的比例溶于乙醇,得到脂质溶液;(b)在微流控系统中,将脂质溶液以10mL/min的流速注入mRNA溶液中,得到纳米脂质分散液,经无菌过滤器过滤后,得到mRNA递送体系。
本发明的有益效果:本发明上述可离子化脂质参与形成的LNP的稳定性更好,能在4oC条件下稳定长达至少24个月,在37oC条件稳定至少1个月时间;
本发明上述可离子化脂质参与形成的LNP对mRNA的递送效率更高,与市售脂质MC3相比,转染效率在同等条件下能提升至少两倍。
附图说明
图1是实施例1中的脂质1的合成过程示意图;
图2是实施例1的可离子化脂质在应用中经肌肉注射后LNP递送Fluc-mRNA在小鼠中的IVIS成像图;
图3是实施例1的可离子化脂质在应用中经尾静脉注射后LNP递送Fluc-mRNA在小鼠中的IVIS成像图;
图4是实施例1的可离子化脂质在应用中LNP的理化性质与递送效率数据。
具体实施方式
以下结合附图对本发明作进一步的详细说明。
本具体实施例仅仅是对本发明的解释,其并不是对本发明的限制,本领域技术人员在阅读完本说明书后,可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本发明的权利要求范围内都受到专利法的保护。
实施例1,本实施例为一种可离子化脂质的制备方法,该可离子化脂质的合成工艺如图1所示,该制备方法包括如下步骤:
(a)合成中间体1:在0℃,氮气保护的条件下,向9-十八醇(2.7g,9-11mmol)、8-溴辛酸(2.22g,9-11mmol)和DMAP(0.33g,2.64mmol)的二氯甲烷中溶液中添加EDCI(3.79g,19.8mmol)粉末,室温搅拌反应过夜,TLC显示反应结束,用2M盐酸酸化,用乙腈:正己烷=1:1的萃取液进行萃取,干燥脱溶得到中间体1,计算收率为95%,不进行纯化直接用于下一步反应,其中,9-十八醇进一步优选采用10mmol,8-溴辛酸一步优选采用10mmol;
(b)合成中间体2:
在室温下,将中间体1(0.24g,0.4-0.6mmol)和3-氨基-1,1,1-三氟丙烷-2-醇(0.066g,0.75mmol)在异丙醇中搅拌过夜,反应温度70℃,TLC显示反应结束,粗品通过硅胶柱层析中间体2,纯化梯度:DCM/MeOH=10:0~10:1,计算收率为62%,其中,中间体1进一步优选采用0.5mmol;
(c)合成可离子化脂质,简称为(脂质1):
在氮气保护的条件下,向中间体2(0.52g,1.0mmol)在DMF中的溶液中加入6,9二烯十八溴(0.33g,1.0mmol)、碳酸钾(0.54g,3.93mmol)、碘化钾(0.002g,0.013mmol);氮气保护80℃反应16h,TLC监测反应进程反应结束后,用水稀释有机相,用乙酸乙酯萃取,干燥脱溶,粗品通过色谱纯化得到可离子化脂(脂质1),纯化梯度:DCM/MeOH=10:0~10:2,计算收率为67.6%;
对脂质1进行氢谱分析,得到脂质1的HNMR数据如下所示:
1HNMR(300MHz,CDCl3)δ5.66(m,1H),5.38(m,4H),4.10-3.97(m,1H),2.98(dd,2H),2.81(dd,2H),2.75-2.59(m,4H),2.34-2.21(m,2H),1.60-1.12(m,54H),0.93-0.77(m,9H)。
实施例2,本实施例为一种mRNA递送体系的制备方法,该制备方法包括如下步骤:(a)按照摩尔比为50∶38.5∶10∶1.5,将可离子化脂质、结构性脂质、磷脂和PEG脂质溶于乙醇,得到脂质溶液;
(b)在微流控系统中,将脂质溶液以10mL/min的流速注入mRNA溶液中,得到纳米脂质分散液,经0.22μm无菌过滤器过滤后,得到mRNA递送体系,其中,mRNA溶液的浓度为0.1mg/mL,溶剂为pH值为3、50mM的柠檬酸钠缓冲液,mRNA溶液和脂质溶液的体积比为3∶1,可离子化脂质与mRNA的N/P(氮磷比)为6∶1;mRNA为trilink公司商品化的CleanCap mRNA。
使用Zetasizer Nano ZS(Malvern Instru ments Ltd,Malvern,Worcestershire,UK)测定上述制备得到的mRNA递送体系的粒度、多分散指数(PDI)和zeta电势,粒度是在1×PBS中测定且zeta电势是在15mM PBS中测定的;其中,粒径和电位测试结果如表1所示,本发明递送体系的粒径为85.2nm,Zeta电位为-5.1mV。
对于包含mRNA的递送体系,可以使用QUANT-ITTM RNA测定(InvitrogenCorporation Carlsbad,CA)评价递送体系对RNA的包封情况。在1×TE缓冲溶液中将样品稀释至约5μg/mL的浓度。将50μL稀释过的样品转移至96孔板上并向各孔中添加50μL TE缓冲液或50μL 5%TritonX-100溶液。在37℃温度下孵育板15分钟。将试剂以1:100稀释于TE缓冲液中,并向各孔中添加100μL该溶液。根据试剂盒提供的参数使用酶标仪检测相应的荧光值,并换算游离的与总的RNA量。
包封率计算公式为:包封率(EE%)=(总RNA浓度-游离mRNA浓度)/总RNA浓度,测得的本发明递送体系的包封率为94%。
实施例3,在实施例2制备得到的递送体系的荧光素酶mRNA体内递送性能的评价:
肌肉注射:按1ug mRNA每只的用量,使用肌肉注射实施例2制备的递送体系,以MC3脂质体纳米颗粒分别作为对照。6小时后,分别往每只小鼠体内通过腹腔注射200μL 10mg/mL的D-荧光素钾盐,15分钟后,将小鼠放置于活体成像系统(IVIS-200,Xenogen)下,观察每只小鼠总的萤光强度,通过软件读取信号。Fluc mRNA的表达强度记录在表1中。代表性小鼠整体成像结果如图2所示。所述的递送体系与MC3控制组均在肌肉中检测到明显的阳性表达,所述递送系统的表达效率显著优于控制组对照。
尾静脉注射给药:按0.25mg/kg的用量,使用尾静脉注射实施例2制备的递送体系,以MC3脂质体纳米颗粒分别作为控制组对照。
6小时后,分别往每只小鼠体内通过腹腔注射200μL 10mg/mL的D-荧光素钾盐,15分钟后,将小鼠放置于活体成像系统(IVIS-200,Xenogen)下,观察每只小鼠总的萤光强度(表1)。成像结果如图3所示。所述的递送体系与MC3控制组均在肝脏中检测到明显的阳性表达,所述递送系统的表达效率显著优于控制组对照。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到各种等效的修改或替换,这些修改或替换都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以权利要求的保护范围为准。
Claims (10)
1.一种新型含氟可离子化脂质,其特征在于,所述含氟可离子化脂质的结构式如式I所示:
式I,化学式为C47H88F3NO3,分子量为772.22。
2.根据权利要求1所述的一种新型含氟可离子化脂质的制备方法,其特征在于,所述制备方法包括如下步骤:
(a)将9-十八醇和8-溴辛酸进行酯化反应,得到中间体1;
(b)将中间体1和3-氨基-1,1,1-三氟丙烷-2-醇进行缩合反应,得到中间体2;
(c)将中间体2和6,9二烯十八溴进行缩合反应,得到所述可离子化脂质。
3.根据权利要求2所述的一种新型含氟可离子化脂质的制备方法,其特征在于,在步骤(a)中,9-十八醇的摩尔量为9-11mmol,8-溴辛酸的摩尔量为9-11mmol。
4.根据权利要求2所述的一种新型含氟可离子化脂质的制备方法,其特征在于,在步骤(b)中,中间体1的摩尔量为0.4-0.6mmol。
5.根据权利要求2所述的一种新型含氟可离子化脂质的制备方法,其特征在于,在步骤(b)中,中间体1和3-氨基-1,1,1-三氟丙烷-2-醇在异丙醇中搅拌得到的粗品通过硅胶柱层析得到中间体2。
6.根据权利要求2所述的一种新型含氟可离子化脂质的制备方法,其特征在于,在步骤(c)中,中间体2的摩尔量为1mmol,6,9二烯十八溴的摩尔量为1mmol。
7.权利要求1所述的含氟可离子化脂质在制备mRNA递送体系中的应用。
8.一种mRNA递送体系,其特征在于,包括权利要求1所述的含氟可离子化脂质、磷脂、PEG脂质和结构性脂质和mRNA。
9.根据权利要求8所述的一种mRNA递送体系,其特征在于,含氟可离子化脂质与mRNA的N/P为6∶1。
10.根据权利要求8或9所述的一种mRNA递送体系的制备方法,其特征在于,包括如下步骤:(a)将可离子化脂质、结构性脂质、磷脂和PEG脂质按照摩尔比为50∶38.5∶10∶1.5的比例溶于乙醇,得到脂质溶液;(b)在微流控系统中,将脂质溶液以10mL/min的流速注入mRNA溶液中,得到纳米脂质分散液,经无菌过滤器过滤后,得到mRNA递送体系。
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