CN115433099A - 可离子化的阳离子脂c6及由其组成的纳米脂质体颗粒 - Google Patents
可离子化的阳离子脂c6及由其组成的纳米脂质体颗粒 Download PDFInfo
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- CN115433099A CN115433099A CN202211370059.0A CN202211370059A CN115433099A CN 115433099 A CN115433099 A CN 115433099A CN 202211370059 A CN202211370059 A CN 202211370059A CN 115433099 A CN115433099 A CN 115433099A
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- glycero
- lipid
- phosphocholine
- ionizable cationic
- nanoliposome
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- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
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Abstract
本发明提供了一种可离子化的阳离子脂C6及由其组成的纳米脂质体颗粒,所述纳米脂质体颗粒由所述可离子化的阳离子脂C6,辅助脂如DSPC,结构脂,PEG化脂组成。所述纳米脂质体颗粒可用于递送DNA,RNA或小分子药物至哺乳动物的细胞或器官,发挥预防或者治疗的效果。
Description
技术领域
本发明公开了一种可离子化的阳离子脂以及包含此类化合物的纳米脂质体颗粒载体。此纳米脂质体颗粒还包括特定比例的不饱和脂质在内的磷脂,PEG脂质,结构脂质和治疗剂或预防剂。这种纳米脂质体颗粒载体可以递送一种或多种预防剂或治疗剂到哺乳动物的细胞或器官。
背景技术
核酸疫苗特别是mRNA疫苗具有研发周期短,灵活性强,安全性高等优势,在应对突发疾病如新冠时具有显著的优势。然而核酸药物在成药过程中具有很多的挑战,特别是mRNA作为一种生物大分子不稳定,易被无处不在的核酶降解。同时mRNA富含磷酸基团呈负电,而组成细胞膜的磷脂同样呈负电状态,因此裸mRNA与细胞膜相互排斥,难以进入细胞内。而核酸药物需要进入细胞内才能发挥相应的生物学功能,因此对于核酸药物需要相应的载体,一方面将核酸递送至细胞内,另一方面保护核酸药物不被机体迅速降解。目前常见的核酸分为两类:病毒载体和非病毒载体,病毒载体如病毒样颗粒常常面临免疫原性高,副反应大等问题,临床上使用的不多;非病毒载体如聚合物、纳米乳、脂质体等,目前临床上最成熟,使用最多的是纳米脂质体颗粒(LNP)。纳米脂质体颗粒载体有递送效率高,安全性好等优势,目前已上市的两款新冠mRNA疫苗所用的递送载体就是纳米脂质体颗粒。这种高效安全的递送载体也被广泛研究,特别是在提升纳米脂质体颗粒载体递送效率,安全性方面是目前相关研究的重点。
虽然纳米脂质体颗粒载体成功应用于临床,相较于其它载体具有显著的优势,但是具有很强的专利壁垒,技术效果也有待于提升,设计新的纳米脂质体载体规避专利限制并提升技术效果,以适应于不断增加的应用需求,是目前纳米脂质体载体研究的重要课题。常见的纳米脂质体颗粒由四种组分组成,可离子化的阳离子脂,磷脂,结构脂和PEG化脂。四种脂中可离子化的阳离子脂的化学结构对纳米脂质体颗粒载体的递送效率以及安全性具有重要影响。设计并筛选高效,安全的可离子化阳离子脂成为纳米脂质体颗粒载体的焦点问题。本发明的目的就是提供一种新的可离子化阳离子脂,进而提供一种由该可离子化阳离子脂参与构成的纳米脂质体颗粒载体,以解决现有技术存在的问题。
发明内容
基于上述目的,本发明首先提供了一种可离子化的阳离子脂,所述可离子化的阳离子脂为3-[3-[3-[(2-羟基-16-甲基-十七烷基)-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙氨基]丙酸3,7,11,15-四甲基十六烷基酯,所述可离子化的阳离子脂的化学结构式如式(Ⅰ)所示:
在本发明中,所述可离子化的阳离子脂被命名为C6。
在一个优选的实施方案中,所述可离子化的阳离子脂具有如图1所示的核磁谱图(1H NMR: ( 400 MHz CDCl3)),C6的特征为:δ4.12 (tt, J = 6.93, 3.46 Hz, 4 H),3.54 - 3.65 (m, 1 H), 2.85 - 2.99 (m, 3 H), 2.65 - 2.82 (m, 4 H), 2.53 - 2.64(m, 3 H), 2.33 - 2.51 (m, 8 H), 2.17 - 2.27 (m, 3 H), 1.60 - 1.82 (m, 6 H),1.56 (br s, 72 H), 0.80 - 0.95 (m, 36 H),因此C6含有-CH3, -CH2-, -RH2-C00-RH2-,-RH(CH3)-, -CH(OH)-等特征基团。
其次,本发明提供了一种纳米脂质体颗粒载体,所述纳米脂质体颗粒载体为含有上述的可离子化的阳离子脂和磷脂,结构脂,PEG化脂的组合物。
在一个优选的实施方案中,所述可离子化的阳离子脂和磷脂,结构脂,PEG化脂以(40-65%):(5-25%):(25-50%):(0.5-15%)的摩尔配比混合制备而成,制备时可离子化阳离子脂质中的氮原子与作为药物分子的RNA或DNA中的磷酸基团的摩尔比,即,氮磷比为6:1(N:P=6:1),所述可离子化的阳离子脂和磷脂,结构脂,PEG化脂的总脂浓度为6mM。
在一个更为优选的实施方案中,所述可离子化的阳离子脂和磷脂,结构脂,PEG化脂以50:10:38.5:1.5的摩尔配比混合制备而成。
本发明所述的磷脂是指一种两性脂,含亲水的头部和疏水的尾部,是纳米脂质体颗粒球形外层的重要组成部分,能够促进纳米脂质体颗粒与细胞的融合。在一个优选的实施方案中,其中所述的磷脂选自由以下组成的组中的一种或多种磷脂化合物:1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二(十一烷酰基)-sn-甘油-磷酸胆碱(DUPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八烯基-sn-甘油-3-磷酸胆碱(18:0DietherPC)、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基-sn-甘油-3-磷酸胆碱(C16LysoPC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-二(二十二碳六烯酰基)-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二(二十二碳六烯酰基)-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酸-外消旋-(1-甘油)钠盐(DOPG)、鞘磷脂。
本发明所述的结构脂质是指可以增加纳米脂质体颗粒的稳定性,减少纳米脂质体颗粒中磷脂的流动性,减少纳米脂质体颗粒包裹核酸的泄露,同时增加纳米脂质体颗粒的内涵体逃逸效率的脂质,在本发明的一个优选的实施方案中,结构脂质选自以下物质的任一种或多种:胆固醇、粪甾醇、谷甾醇、麦角甾醇、菜油甾醇、豆甾醇、菜籽甾醇、番茄碱、番茄苷、熊果酸、α-生育酚。在另一个优选的实施方案中,所述结构脂选自由以下组成的组中的任一结构脂化合物:胆固醇、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、熊果酸、α-生育酚、皮质类固醇。
在另一个优选的实施方案中,所述的PEG化脂选自由以下组成的组中的任一PEG化脂化合物:经PEG修饰的磷脂酰乙醇胺、经PEG修饰的磷脂酸、经PEG修饰的神经酰胺、经PEG修饰的二烷基胺、经PEG修饰的二酰基甘油、经PEG修饰的二烷基甘油。
在本发明的一个具体实施方案中,所述磷脂,结构脂,PEG化脂分别为1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱、胆固醇、PEG2000-DMG。
最后,本发明还提供了所述的纳米脂质体颗粒载体在制备核酸递送药物中的应用。
在一个优选的实施方案中,所述核酸为RNA或DNA。
在一个优选的实施方案中,所述核酸递送药物的制备方法包括以下步骤:
(1)将作为药物分子的RNA或DNA溶解于醋酸钠缓冲液中形成水相;
(2)以可离子化阳离子脂质中的氮原子与作为药物分子的RNA或DNA中的磷酸基团的摩尔比(N∶P)为(2-30)∶1确定可离子化阳离子脂质的摩尔数,再按照可离子化阳离子脂质, 1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱, 胆固醇, PEG2000-DMG的摩尔比为50:10:38.5:1.5的比例将四种组分混合均匀,形成均一的有机相;
(3)将步骤(1)制备的水相和步骤(2)制备的有机相以微流控混合的形式制备成装载mRNA或DNA的纳米脂质体颗粒,其中,核酸水相的体积是有机相的3倍,所述纳米脂质体颗粒载体中的脂质和核酸的质量比wt/wt比率为3∶1至100∶1。
优选的,所述N∶P的比率为5-20∶1。
更为优选的,所述N∶P的比率为8∶1。
尤为优选的,所述N∶P的比率为6∶1。
优选的,所述脂质和核酸(mRNA/DNA)的质量比wt/wt比率为约20∶1。
更为优选的,所述脂质和核酸(mRNA/DNA)的质量比wt/wt比率为约6∶1。
在一个更为优选的实施方案中,所述载体与所述核酸的质量比(5~ 50):1。
更为优选地,所述RNA为短干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、RNA干扰(RNAi)分子、微小RNA(miRNA)、反义RNA (antagomir)、小发夹RNA(shRNA)或信使RNA(mRNA)。
本发明中可离子化的阳离子脂具有可电离的胺基头部,在酸性环境下可结合氢离子带正电,从而耦合带负电的RNA分子,将mRNA分子包裹在可电离阳离子脂形成的小球中形成纳米脂质体颗粒。所述纳米脂质体颗粒的平均直径为50-150nm。该可电离阳离子脂头部多个氨基,可带多个正电能够更好的耦合mRNA分子,达到较高的包封效率;该脂还有尾部的分支结构和酯键,大大增加其可降解性,减少脂在机体内的保留时间,减少副反应的概率,增加安全性和耐受性。1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)和胆固醇作为辅助脂,有助于提高纳米脂质体颗粒的膜融合性,维持其结构和稳定性,增加纳米脂质体颗粒的内涵体逃逸效率,从而提升递送效率,1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(PEG2000-DMG)可使纳米脂质体颗粒形成致密的球形结构,延长纳米脂质体颗粒在体内的循环时间,减少纳米脂质体颗粒的聚集,增加其稳定性。
附图说明
图1. 可离子化的阳离子脂化合物C6的核磁共振图谱;
图2. 纳米脂质体颗粒递送Luc-mRNA转染效率检测;
图3. 纳米脂质体颗粒递送eGFP-mRNA荧光显微镜观察图;
图4. 纳米脂质体颗粒递送Luc-DNA转染效率检测;
图5. 纳米脂质体颗粒递送eGFP-DNA荧光显微镜观察图。
具体实施方式
以下将参照附图,对本发明的优选实施案例进行详细描述。除非另外确切定义,本文所使用的所有技术和科学术语具有与本披露所属领域的技术人员通常所理解的相同的意义。除非另有说明,本文采用或考虑的技术是本领域普通技术人员熟知的标准方法、或按照制造厂商所建议的条件进行。除非另外说明,本披露的实践将使用本领域技术范围内的微生物学、组织培养、分子生物学、化学、生物化学以及重组DNA技术的常规技术。这些材料、方法、和实例仅是说明性的并且不是限制性的。以下通过说明的方式呈现,并非旨在限制本披露的范围。
在一些实施例中,在说明书中阐述的数字参数是近似值,其可以根据由特定实施例寻求获得的期望性质而变化。在一些实施例中,这些数值参数应该按照报告的有效数字的数量以及通过应用普通的舍入技术来解释。虽然阐述本披露的一些实施例的广泛范围的数字范围和参数是近似值,但是在具体实例中阐述的数值被尽可能地精确地报道。在本披露的一些实施例中呈现的数值可含有由其各自测试测量中发现的标准偏差必然导致的某些误差。本文中对数值范围的描述仅旨在用作逐个提及落入所述范围内的各个独立值的一种简化方法。除非在本文中另有说明,否则将各个独立的值并入说明书中,就如同在本文中对它进行了逐个描述一样。
为方便,这里收集了在整个申请(包括本说明书、实例和所附权利要求书)中使用的某些术语。除非另外定义,本文所使用的所有技术和科学术语具有与本披露所属领域的技术人员通常所理解的相同的意义。
在一些实施例中,用于描述和要求保护本披露的某些实施例的表达成分的数量、性质(例如分子量)、反应条件和结果等的数字应理解为在某些情况下被术语“约”修饰。本领域普通技术人员将在限定值的上下文中理解术语“约”的含义。在一些实施例中,术语“约”用于表示一个值包括用于确定所述值的设备或方法的平均值的标准偏差。在一些实施例中,在说明书中阐述的数字参数是近似值,其可以根据由特定实施例寻求获得的期望性质而变化。在一些实施例中,这些数值参数应该按照报告的有效数字的数量以及通过应用普通的舍入技术来解释。虽然阐述本披露的一些实施例的广泛范围的数字范围和参数是近似值,但是在具体实例中阐述的数值被尽可能地精确地报道。在本披露的一些实施例中呈现的数值可含有由其各自测试测量中发现的标准偏差必然导致的某些误差。
术语“化合物”包括所示结构的所有同位素和异构体。“同位素”是指具有相同原子序数但质量数不同的原子,其由核中不同数目的中子产生。例如,氢的同位素包括氚和氘。此外,本披露的化合物、盐或复合物可以与溶剂或水分子组合制备,以通过常规方法形成溶剂化物或水合物。“异构体”意指化合物的任何几何异构体、互变异构体、两性离子、立体异构体、对映异构体或非对映异构体。化合物可以包括一个或多个手性中心和/或双键,因此可以立体异构体(例如双键异构体或非对映异构体)存在。本披露涵盖本文所述化合物的任何和所有异构体,所述异构体包括立体异构纯形式以及对映体和立体异构体混合物,例如外消旋体。化合物的对映体和立体异构体混合物以及将它们分解成其组分对映体或立体异构体的方法是本领域熟知的。
术语“包含”、“具有”和“包括”是开放式连接动词。这些动词中的一个或多个的任何形式或时态例如“包含”、“具有”、“包括”也是开放式的。例如,“包含”、“具有”或“包括”一个或多个步骤的任何方法不限于仅具有那一个或多个步骤,并且还可以涵盖其他未列出的步骤。类似地,“包含”、“具有”或“包括”一个或多个特征的任何组合物不限于仅具有那一个或多个特征并且可以涵盖其他未列出的特征。本文关于某些实施例提供的任何和所有实例或示例性语言(例如“例如”)的应用仅旨在更好地说明本披露,而不对另外要求保护的本披露范围做出限制。说明书中的语言不应当被解释为指示任何未要求保护的要素为实践本披露所必需的。
术语“由......组成”是指如本文所述的组合物、方法、及其对应的组分,其排除在该实施例的这个说明中没有描述的任何要素。
如本文所用,术语“递送”意指向目的地提供实体。例如,向细胞递送治疗剂可涉及向细胞施用包含至少一种包含mRNA的纳米颗粒的药物组合物。
如本文所用,核酸序列的“表达”是指一个或多个以下事件:(1) 从DNA序列产生RNA模板(例如通过转录);(2) RNA转录物的加工(例如通过剪接、编辑、5′帽形成和/或3′末端加工);(3)将RNA翻译成多肽或蛋白质;和(4)多肽或蛋白质的翻译后修饰。
如本文所用,术语“脂质组分”是包括一种或多种脂质的纳米颗粒的组分。例如,脂质组分可以包括一种或多种可离子化阳离子脂质、磷脂、结构脂、PEG脂。
如本文所用,“纳米颗粒”是包含一种或多种脂质和一种或多种治疗剂的颗粒。纳米颗粒的尺寸量级通常为微米或更小,并且可包括脂质双层。在一些实施例中,通过DLS(动态光散射)纳米颗粒的平均直径 (例如,等效直径) 为约70 nm与约150 nm之间,例如直径为约80 nm与约120 nm之间、90 nm与110 nm之间。在一些实施例中,纳米颗粒的平均动力学直径为约90 nm、91 nm、92 nm、93 nm、94 nm、95 nm、96 nm、97 nm、98 nm、99 nm、100 nm、101 nm、102 nm、103 nm、104 nm、105 nm、106 nm、107 nm、108 nm、109 nm或110nm。在一些实施例中,治疗剂是mRNA或DNA。
如本文所用,“多分散指数(PDI)”是纳米颗粒样品中纳米颗粒尺寸分布的量度。在一些实施例中,多分散指数为约0.10与0.20之间,例如约0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19或0.20。
如本文所用,“总脂”、“总脂质”是纳米共沉淀法合成脂质体纳米颗粒时,先期溶解并混合在乙醇中的所有脂质,包括可离子化阳离子脂质、磷脂、结构脂、辅助聚合物及PEG脂。在一个实施案例中,总脂包括所述可离子化的阳离子脂为3-[3-[3-[(2-羟基-16-甲基-十七烷基)-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙氨基]丙酸3,7,11,15-四甲基十六烷基酯,所述可离子化的阳离子脂的化学结构式如式(Ⅰ)所示:
在本发明中,所述可离子化的阳离子脂被命名为C6。
1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC),分子式如式(II)所示:
胆固醇,分子式如式(III)所示:
DMG-PEG2000(1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000),分子式如式(IV)所示:
其中式(IV)中的数字44是指:括号内乙二醇的重复单元数为44个。
如本文所用,N∶P(N/P比)是可离子化阳离子脂质中的氮原子与治疗剂mRNA或DNA中的磷酸基团的摩尔比。
如本文所用,“mRNA:总脂体积比”、“mRNA:总脂质体积比”是脂质合成时,mRNA与脂质组分(包括可离子化阳离子脂质、结构脂、辅助脂、PEG脂)混合时,二者的体积比。在一个实施案例中mRNA:总脂体积比为3:1。
如本文所用,“mRNA:总脂质流速比”、“总流速”、“Start waste”、“End waste”是在Precision Nanosystems Inc微流控纳米颗粒制备系统Ignite上制备纳米脂质体颗粒时,仪器的设定参数,其定义符合Precision Nanosystems对的其基本定义,其结果为实验调整所得。在一个实施案例中mRNA:总脂质流速比为3,总流速为12 mL/min,Start waste为0.15mL,End waste为0.05 mL。
如本文所用如本文使用,术语“核酸”在其最广泛的意义上包括包含通过磷酸二酯键连接的核苷酸聚合物的任何化合物和/或物质。这些聚合物通常称为寡核苷酸或多核苷酸,这取决于大小。
如本文使用,“蛋白质”是基本上由20个氨基酸中的任何氨基酸组成的聚合物。虽然“多肽”通常用于提及相对较大的多肽,而“肽”通常用于提及小多肽,但本领域中这些术语的使用重叠并且是变化的。本文中术语“肽”、“蛋白质”和“多肽”有时可以互换使用。
本发明的实施案例中,可离子化的阳离子脂,DSPC, 胆固醇,PEG溶解在无水乙醇中,四种组分的摩尔比分别为50%,10%,38.5%,1.5%;有机相的总脂浓度为6mM。为了形成均一的有机相,所有溶液充分涡旋混匀,产生良好的包封效果。其中,在制备纳米脂质体颗粒包裹萤火虫荧光素酶(luciferase)和绿色荧光蛋白(eGFP)报告基因的mRNA和DNA时,使用的N:P=6:1。核酸(mRNA和DNA)溶解在25mM的醋酸钠溶液中(pH 5.2),其中脂混合液组成的有机相和mRNA醋酸钠缓冲液组成的水相的体积比为1:3。
采用纳米共沉淀法的原理,在Ignite (Precision Nanosystems Inc)纳米脂质体颗粒制备系统上制备LNPs溶液,总流速为12 mL/min,Start waste及End waste分别为0.15mL和0.05 mL,实施方案示于表1。
表1制备可离子化阳离子脂质的技术参数
制备完成后迅速将LNPs溶液转移到50ml超滤管(Millipore,100KD)并用1×PBS10倍稀释LNPs溶液,在4度条件下,1300g(Thermo)离心30分钟,最后收取超滤后的LNPs溶液,用1×PBS定容至浓度为150ug/mL。
制备完成的LNPs溶液稀释后,使用NanoBrook Omni(Brookhaven)多角度粒度与高灵敏度Zeta电位分析仪测定其有效粒径、多分散指数(PDI)及Zeta电位,同时使用Quant-iT™ RiboGreen™ RNA定量检测试剂盒(Life Technology)精确测量LNPs的包封率和LNPs里核酸的浓度。
实施例1. 可离子化阳离子脂质C6的合成路线
可离子化阳离子脂质C6的合成原理为首先将烯醇和氢气反应加成生成醇,然后和酰氯发生取代反应生成丙烯酸酯,丙烯酸酯和二胺进行加成反应,然后脱去苄基,最后和烷基环氧乙烷进行加成反应产生目标产物。合成路线为由化合物C6-1出发合成化合物C6-2,由化合物C6-2出发合成化合物C6-3;另外由化合物1出发合成化合物2,再由化合物2出发合成化合物3;再由化合物3与化合物C6-3合成化合物C6-4,再由化合物C6-4出发合成化合物C6-5;同时由化合物A-1合成化合物A-2,由化合物A-2合成化合物A-3,由化合物A-3合成化合物A-4,由化合物A-4合成化合物A-5,最后,由化合物C6-5与化合物A-5合成目的产物可离子化阳离子脂质C6。具体步骤如下。
向化合物C6-1 (3,7,11,15-四甲基-2-十六碳烯-1-醇,50.0 g, 169 mmol, 1.00eq,Sigma-Aldrich)的四氢呋喃(THF, 400mL, Sigma-Aldrich)溶液中加入钯碳催化剂(Pd/C , 4.00 g, 337 uL,10%),悬浮液脱气,氢气净化3次。混合物在25℃的氢气 (50Psi)下搅拌12小时至核磁表明反应物C6-1被完全消耗。反应物经过过滤,在减压下浓缩,形成的残渣通过柱层析(二氧化硅,石油醚/乙酸乙酯=1/0 ~ 0/1)得到黄色油状化合物C6-2(3,7,11,15-四甲基十六烷醇35.0 g, 117 mmol,69.5%)。
在0℃下, 向化合物C6-2 (3,7,11,15-四甲基十六烷醇,35.0 g, 117 mmol,1.00 eq)的二氯甲烷(DCM, 240 mL, Sigma-Aldrich)溶液中加入丙烯酰氯(11.7 g, 129mmol, 10.5 mL, 1.10 eq,Aladdin)和三乙胺(TEA, 23.7 g, 234 mmol, 32.6 mL, 2.00eq, Sigma-Aldrich)。混合物在25°C下搅拌3小时至薄层色谱(石油醚:乙酸乙酯=5;1)显示反应物C6-2 (3,7,11,15-四甲基十六烷醇)完全消耗,反应完全。将反应混合物倒入水中(200 mL),用乙酸乙酯(300 mL, 200 mL,Sigma-Aldrich)萃取的有机层用200 mL盐水洗涤,经硫酸钠干燥,过滤,减压浓缩,得到的残渣通过柱层析(二氧化硅,石油醚/乙酸乙酯=1/0 ~ 0/1)得到无色油状的化合物C6-3 (3、7、11、15-四甲基十六烷基丙烯酸酯,36.0 g,102 mmol,87.1%)。
在0℃条件下,向化合物1 (N,N-双(3-氨丙基)甲胺, 20.0 g, 138 mmol, 1.00eq,Sigma-Aldrich)的二氯甲烷 (DCM, 140 mL,Sigma-Aldrich)溶液中加入三乙胺 (TEA,41.8 g, 413 mmol, 57.5 mL, 3.00 eq,Sigma-Aldrich)和苯甲酰氯(42.6 g, 303 mmol,35.2 mL, 2.20 eq,Sigma-Aldrich),混合物在25℃下搅拌3小时至反应完全。将反应混合物倒入水中(50 mL),用乙酸乙酯(100 mL, 80 mL,Sigma-Aldrich)提取,提取的有机层用盐水20 mL洗涤,后用硫酸钠干燥,过滤,减压浓缩,得到白色固体状的化合物2(N-[3-[3-苯甲酰胺丙基(甲基)氨基]丙基]苯甲酰胺50.0 g)。
在25℃,将四氢铝锂 (LAH, 6.44 g, 170 mmol, 6.00 eq,Sigma-Aldrich)加入到四氢呋喃 (THF, 200 mL,Sigma-Aldrich)中混合均匀,然后加入化合物2 (N-[3-[3-苯甲酰胺丙基(甲基)氨基]丙基]苯甲酰胺, 10.0 g, 28.3 mmol, 1.00 eq)的四氢呋喃(THF,50 mL,Sigma-Aldrich)溶液。混合物在80℃下搅拌48小时,液质联用(Rt=0.487, MS+1 = 326)显示化合物2被完全消耗,并检出一个符合要求质量的主峰。在0℃条件下,向反应混合物中缓慢的加入水(6.44mL)、15% 氢氧化钠(6.44mL,Sigma-Aldrich)、水(19.4 mL)和硫酸钠然后在20℃搅拌20 min后,对混合物进行过滤和减压浓缩,得到黄色油状的化合物3(N-苄基-N'-[3-(苄氨基)丙基]-N'-甲基-丙烷-1,3-二胺,37.0 g)。
向化合物3(N-苯甲基-N’-3-苄氨基1-丙醇-N’-甲基-丙烷-1,3-二胺 5.00 g,15.4 mmol, 1.00 eq)的乙醇(EtOH, 50.0 mL,Sigma-Aldrich)溶液中加入C6-3(3,7,11,15-四甲基十六烷基丙烯酸酯,11.9 g, 33.8 mmol, 2.20 eq)。混合物在70°C下搅拌12小时至反应物C6-3(3,7,11,15-四甲基十六烷基丙烯酸酯)被完全消耗,并检测出一个符合要求质量的主峰。将反应混合物倒入水中(10 mL),用乙酸乙酯(30 mL, 20 mL,Sigma-Aldrich)提取,结合的有机层用(10 mL)盐水洗涤,用硫酸钠干燥,过滤,减压浓缩,得到残渣并通过柱层析(二氧化硅,石油醚/乙酸乙酯制二氯甲烷:甲醇= 50/1 ~ 10/1)得到黄油状的化合物C6-4 ( 3-[苯甲基-[3-[3-[苯甲基-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙基]氨基]丙酸3,7,11,15-四甲基十六烷基酯 37.0 g,35.9 mmol,77.9%)。
向化合物C6-4 (3-[苯甲基-[3-[3-[苯甲基-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙基]氨基]丙酸3,7,11,15-四甲基十六烷基酯,18.5 g, 17.9 mmol, 1.00 eq)的四氢呋喃 (THF,140 mL,Sigma-Aldrich)溶液中加入Pd(OH)2 (氢氧化钯,3.70 g, 5.27 mmol, 20%),悬浮液脱气,氢气净化3次。混合物在25℃氢气 (50 Psi)条件下搅拌12小时,薄层色谱(二氯甲烷:甲醇= 5:1)显示反应物C6-4(3-[苯甲基-[3-[3-[苯甲基-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙基]氨基]丙酸3,7,11,15-四甲基十六烷基酯)消耗完全,产物经过过滤,减压浓缩,得到黑棕色油状的化合物C6-5(3-[3-[甲基-3-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基]氨基]丙氨基]丙酸3,7,11,15-四甲基十六烷基酯23g)。
在30℃条件下,向镁(9.48 g, 390 mmol, 5.17 eq,Sigma-Aldrich)和碘(191mg, 754umol, 151uL, 0.01eq, Sigma-Aldrich)的四氢呋喃(THF, 100 mL,Sigma-Aldrich)中加入化合物A1(1-溴-3-甲基丁烷, 56.9 g, 377 mmol, 47.4 mL, 5.00 eq,Sigma-Aldrich)的四氢呋喃(THF 50 mL, Sigma-Aldrich)溶液后,混合物在45℃下搅拌2小时。将混合物加入到化合物A-1 (12-溴-1-十二烷醇,20 g, 75.4 mmol, 1.00 eq,Sigma-Aldrich)和四氯合铜酸二锂(Ⅱ)(0.1 M, 45.2 mL, 0.06 eq,Sigma-Aldrich)的四氢呋喃(THF, 50 mL, Sigma-Aldrich)溶液中。混合物在氮气条件下下,室温过夜至薄层色谱显示反应物A-1完全消耗。反应完全后,向反应液中加入稀盐酸(1M,500mL,Sigma-Aldric),加水(300mL)稀释,用乙酸乙酯(EtOAc, 300 mL, 200 mL, Sigma-Aldric) 萃取,结合的有机层用盐水(300 mL)洗涤,用硫酸钠(Na2SO4, Sigma-Aldric)干燥,过滤,减压浓缩,然后通过柱层析(SiO2,石油醚/乙酸乙酯=0 ~ 1)得到白色固体状的化合物A-2 (15-甲基-十六烷醇15.7 g, 61.2 mmol, 81.1%)。
向化合物A-2(15-甲基-十六烷醇, 15.7 g, 61.2 mmol, 1.00 eq)的乙腈(ACN,310mL, Sigma-Aldric)溶液中加入2-碘酰基苯甲酸(IBX, 25.7 g, 91.8 mmol, 1.50 eq,Sigma-Aldric), 混合物在85℃搅拌2小时至薄层色谱显示反应完全。将反应混合物过滤,滤液减压浓缩,得到白色固体状的化合物A-3 (15-甲基-16烷醛, 15 g)。
在-20℃条件下,向甲基三苯基溴化磷(109 g, 305 mmol, 5.18 eq,Sigma-Aldrich)的四氢呋喃溶液(THF, 300ml, Sigma-Aldrich)中加入叔丁醇钾(t-BuOK, 20.5g, 183 mmol, 3.10 eq, Sigma-Aldrich),混合物在25℃下搅拌1 h,在-20℃加入化合物A-3 (15-甲基-16烷醛, 15.0 g, 58.9 mmol, 1.00 eq)的四氢呋喃(THF, 30 mL, Sigma-Aldrich)溶液。混合物在氮气条件下,25℃搅拌2小时至薄层色谱显示反应干净。将反应混合物倒入氯化铵(NH4Cl, 200 mL, Sigma-Aldrich)中,用乙酸乙酯(500 mL, 300 mL,Sigma-Aldrich)提取,结合的有机层用150 mL盐水洗涤,用硫酸钠干燥,过滤,减压浓缩后,通过柱层析(SiO2,石油醚/乙酸乙酯= 1/0 ~ 50/1)得到无色油状的化合物A-4(16-甲基-17-烷基-1-烯, 14 g, 55.45 mmol, 94%)。
在0℃下, 向化合物A-4 (16-甲基-17-烷基-1-烯,14.0 g, 55.4 mmol, 1.00eq)的二氯甲烷 (DCM, 98 mL,Sigma-Aldrich)溶液中,加入3-氯过氧苯甲酸(m-CPBA,17.9 g, 83.2 mmol, 80%,1.50 eq)。混合物在25°C下搅拌3小时至薄层色谱显示反应完全。反应混合物在0℃下加入硫代硫酸钠(Na2S2O3 30 mL, Sigma-Aldrich)淬火,然后用水(20 mL)稀释,用乙酸乙酯(50 mL, 30 mL,Sigma-Aldrich )提取后有机层用盐水(8mL)洗涤,在硫酸钠上干燥,过滤,减压浓缩,然后通过柱层析(SiO2,石油醚/乙酸乙酯=1/0 ~ 0/1),得到无色油状的化合物A-5(2-(14-甲基-十五烷基)环氧乙烷, 13.3 g, 49.5 mmol,89.3%)。
向化合物C6-5 的叔丁醇 (t-BuOH,10 mL)溶液中加入化合物A-5(2-(14-甲基十五烷基)环氧乙烷, 316 mg, 1.18 mmol, 1.00 eq),混合物在90℃下搅拌48小时。产物通过柱层析(SiO2,石油醚/乙酸乙酯到二氯/甲醇= 50/1至1/1)得到化合物C6(3-[3-[3-[(2-羟基-16-甲基-十七烷基)-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙氨基]丙酸3,7,11,15-四甲基十六烷基酯 0.7 g, 626 umol)。
C6的核磁图谱如图1所示。由图1所示的核磁谱图(1H NMR: ( 400 MHz CDCl3))可知,C6的特征为:δ4.12 (tt, J = 6.93, 3.46 Hz, 4 H), 3.54 - 3.65 (m, 1 H), 2.85- 2.99 (m, 3 H), 2.65 - 2.82 (m, 4 H), 2.53 - 2.64 (m, 3 H), 2.33 - 2.51 (m,8 H), 2.17 - 2.27 (m, 3 H), 1.60 - 1.82 (m, 6 H), 1.56 (br s, 72 H), 0.80 -0.95 (m, 36 H),因此C6含有-CH3, -CH2-, -RH2-C00-RH2-, -RH(CH3)-, -CH(OH)-等特征基团。
此可离子化的阳离子脂C6(3-[3-[3-[(2-羟基-16-甲基-十七烷基)-[3-氧代-3-(3,7,11,15-四甲基十六烷氧基)丙基]氨基]丙基-甲基-氨基]丙氨基]丙酸3,7,11,15-四甲基十六烷基酯)是一种多氮的结构,在酸性条件下能够结合氢离子从而带正电,与呈负电的mRNA静电结合,从而很好的包裹mRNA,因此由该可离子化的阳离子脂组成的纳米脂质体颗粒对mRNA有良好的包封率,达到百分之九十以上。另一方面,此化合物含有酯键,能够在体内相关酶的作用下水解,从而加速化合物在体内的降解,减少其在体内的半衰期,从而减少化合物在体内可能诱导的副反应。此化合物还是一个多支链的结构,这些支链使化合物组成的纳米脂质体颗粒具有较低的相变温度,使纳米脂质体颗粒易于形成锥型结构,从而破坏内涵体的膜以及自身的结构从而释放包裹的mRNA。从整体结构来看,化合物的多氮,酯键,支链等结构一方面有助于其在酸性环境下很好的耦合mRNA,使其组成的纳米脂质体颗粒能够很好的包裹mRNA,达到较高的包封率;另一方面促进纳米脂质体颗粒在细胞内发生构象的改变,破环自己和内涵体的结构完整性,从而促进mRNA释放到胞质中,提高翻译效率。
实施例2. 纳米脂质体颗粒的制备并包裹mRNA
可离子化的阳离子脂C6由药明康德合成,按照实施案例1的合成路线合成, DSPC(二硬脂酰磷脂酰胆碱), 胆固醇,DMG-PEG 2000(1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000)三种脂均来自Avanti Polar Lipids(阿万蒂极性脂质公司)。将这四种脂溶解在无水乙醇(纯度>99.9%, Sigma)中配制成10mg/mL的储备溶液。四种脂的按照摩尔比为50:10:38.5:1.5的比例混合,四种脂的总脂浓度为6mM,制备时氮磷比为6:1(N:P=6:1)。
mRNA溶解在25mM的醋酸钠缓冲液(pH5.2)中,水相和有机相的体积比为3:1,使用Ignite(Precision Nanosystem Inc)纳米制造系统制备LNPs溶液,制备参数为水相和有机相的流速比为3:1,总流速为12mL/min,前废液(start waste)为0.15mL, 后废液(endwaste)为0.05mL。
根据上述制备参数使用Ignite制备纳米脂质体颗粒并包裹Luc-mRNA或者eGFP-mRNA得到相应的LNPs-Luc-mRNA或LNPs-eGFP-mRNA溶液(Luc-mRNA为萤火虫荧光素酶mRNA,长度为1929个核苷酸,eGFP-mRNA是绿色荧光蛋白,能在荧光激发下发出绿光,是一种常见的报告基因,长度为996个核苷酸,Luc-mRNA和eGFP-mRNA均由TriLink BioTechnologies公司提供)。迅速将所得的LNPs-mRNA溶液转移到50ml超滤管(Millipore,100KD)并用1×PBS10倍稀释LNPs溶液,在4度条件下,1300rpf(Thermo)离心30分钟,最后收取超滤后的LNPs溶液,用1×PBS定容至浓度为150ug/mL。将最终的LNPs-Luc-mRNA溶液使用纳米粒径分析仪(NanoBrook Omni,Brookhaven)测定LNPs-Luc-mRNA的理化参数包括粒径,多分散系数(PDI),Zeta电位,使用Quant-iT™ RiboGreen™ RNA定量检测试剂盒(Life Technology)测定包封率如下:
表2. LNPs-mRNA各组的理化参数和包封率
从理化参数来看,可离子化的阳离子脂化合物C6和另外的三种脂可以形成良好的纳米脂质体颗粒并包裹mRNA。因此,进一步在细胞模型上进行体外递送效率的检测。使用HEK293细胞,以每孔20000个细胞的密度铺96孔板,过夜后,以500ng的LNPs-mRNA的转染量,每组五个复孔进行转染实验,24小时后使用萤火虫荧光素酶检测试剂盒(Promega)检测转染效率(参见图2)。
由体外转染结果显示,此可离子化的阳离子脂C6组成的纳米脂质体颗粒载体对萤火虫荧光素酶mRNA具有良好的递送效率,其递送效率是已上市药物Onpattro中所用的可离子化阳离子脂MC3(CN102625696B)的近1.5倍。进一步使用此纳米脂质体颗粒载体递送eGFP-mRNA,通过绿色荧光蛋白(eGFP)的表达量来检测纳米脂质体颗粒的递送效率。使用HEK293细胞,以每孔1×105个细胞的密度铺24孔板,过夜后,以500ng的LNPs-mRNA的转染量,每组3个复孔进行转染实验,24小时后使用荧光显微镜观察绿色荧光蛋白的表达量,可离子化的阳离子脂化合物C6的表达量同样显著高于MC3(参见图3)。
由可离子化的阳离子脂C6组成的纳米脂质体颗粒载体递送萤火虫荧光素酶mRNA(luciferase-mRNA)和绿色荧光蛋白mRNA(eGFP-mRNA)在细胞模型上不仅有良好的理化参数,能够很好的包裹mRNA,而且呈现很好的递送效率,显著高于MC3。
实施例3:纳米脂质体颗粒的制备并递送DNA
同样将上文提到的可离子化的阳离子脂C6,DSPC(二硬脂酰磷脂酰胆碱), 胆固醇,DMG-PEG 2000(1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000)按照摩尔比为50:10:38.5:1.5的比例混合。四种脂的总脂浓度为6mM,制备时氮磷比为6:1(N:P=6:1)。DNA溶解在25mM pH5.2的醋酸钠缓冲液中,水相和有机相的体积比为3:1,使用Ignite(PrecisionNanosystem Inc)纳米制造系统制备LNPs溶液,制备参数为水相和有机相的流速比为3:1,总流速为12mL/min,前废液(start waste)为0.15mL, 后废液(end waste)为0.05mL同样的参数包裹Luc-DNA和eGFP-DNA。将制备好的纳米脂质体颗粒使用纳米粒径分析仪(NanoBrook Omni,Brookhaven)测定LNPs-Luc-mRNA的理化参数包括粒径,多分散系数(PDI),Zeta电位,使用Quant-iT™ RiboGreen™ RNA定量检测试剂盒(Life Technology)测定包封率如下:
表3. LNPs-DNA各组的理化参数和包封率
从理化参数可知:可离子化的阳离子脂C6组成的纳米脂质体颗粒可以很好的包裹萤火虫荧光素酶DNA(luciferase-DNA)和绿色荧光蛋白DNA(eGFP-DNA), 粒径和多分散性(PDI)良好,包封率接近百分之九十。
进一步在细胞模型上进行递送效率的检测:使用HEK293细胞,以每孔20000个细胞的密度铺96孔板,过夜后,以500ng的LNPs-DNA的转染量,每组五个复孔进行转染实验,24小时后使用萤火虫荧光素酶检测试剂盒(Promega)检测纳米脂质体颗粒的体外转染效率。
萤火虫荧光素酶DNA的体外递送结果(参见图4)可知,由可离子化的阳离子脂C6组成的纳米脂质体颗粒载体对DNA同样有很好的递送效率,递送效率高于已上市的可离子化的阳离子脂MC3,C6的递送效率是MC3的两倍。进一步使用此载体递送绿色荧光蛋白DNA同样用HEK293细胞,以每孔1×105个细胞的密度铺24孔板,过夜后,以500ng的LNPs-mRNA的转染量,每组3个复孔进行转染实验,24小时后使用荧光显微镜观察绿色荧光蛋白的表达量,荧光图片显示C6的递送效率同样显著高于MC3(如图5所示)。
由可离子化的阳离子脂C6组成的纳米脂质体颗粒载体同样能够很好的递送绿色荧光蛋白DNA(eGFP-DNA),细胞模型上转染效率很高。从细胞模型上的转染效率可知:由C6组成的纳米脂质体颗粒载体能够很好的包裹mRNA和DNA,理化参数良好,包封率很高,同时递送效率良好,因此是很好的核酸递送载体,在核酸药物递送方面具有巨大的前景。
Claims (12)
2.根据权利要求1所述的可离子化的阳离子脂,其特征在于,所述可离子化的阳离子脂具有δ4.12 (tt, J = 6.93, 3.46 Hz, 4 H), 3.54 - 3.65 (m, 1 H), 2.85 - 2.99 (m,3 H), 2.65 - 2.82 (m, 4 H), 2.53 - 2.64 (m, 3 H), 2.33 - 2.51 (m, 8 H), 2.17- 2.27 (m, 3 H), 1.60 - 1.82 (m, 6 H), 1.56 (br s, 72 H), 0.80 - 0.95 (m, 36H)的核磁谱图。
3.一种纳米脂质体颗粒载体,其特征在于,所述纳米脂质体颗粒载体为含有权利要求2所述的可离子化的阳离子脂和磷脂,结构脂,PEG化脂的组合物。
4.根据权利要求3所述的纳米脂质体颗粒载体,其特征在于,所述可离子化的阳离子脂和磷脂,结构脂,PEG化脂以(40-65%):(5-25%):(25-50%):(0.5-15%)的摩尔配比混合制备而成,制备时可离子化阳离子脂质中的氮原子与作为药物分子的RNA或DNA中的磷酸基团的摩尔比=6:1,所述可离子化的阳离子脂和磷脂,结构脂,PEG化脂的总脂浓度为6mM。
5.根据权利要求4所述的纳米脂质体颗粒载体,其特征在于,所述可离子化的阳离子脂和磷脂,结构脂,PEG化脂以50:10:38.5:1.5的摩尔配比混合制备而成。
6. 根据权利要求5所述的纳米脂质体颗粒载体,其特征在于,其中所述的磷脂选自由以下组成的组中的一种或多种磷脂化合物:1,2-二亚油酰基-sn-甘油-3-磷酸胆碱、1,2-二肉豆蔻酰基-sn-甘油-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸胆碱、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱、1,2-二(十一烷酰基)-sn-甘油-磷酸胆碱、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱、1 ,2-二-O-十八烯基-sn-甘油-3-磷酸胆碱、1-油酰基-2-胆固醇基半琥珀酰基-sn-甘油-3-磷酸胆碱、1-十六烷基-sn-甘油-3-磷酸胆碱、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-二(二十二碳六烯酰基)-sn-甘油-3-磷酸胆碱、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二(二十二碳六烯酰基)-sn-甘油-3-磷酸乙醇胺、1 ,2-二油酰基-sn-甘油-3-磷酸-外消旋-(1-甘油)钠盐、鞘磷脂。
7.根据权利要求5所述的纳米脂质体颗粒载体,其特征在于,其中所述的结构脂选自由以下组成的组中的任一结构脂化合物:胆固醇、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、熊果酸、α-生育酚、皮质类固醇。
8.根据权利要求5所述的纳米脂质体颗粒载体,其特征在于,其中所述的PEG化脂选自由以下组成的组中的任一PEG化脂化合物:经PEG修饰的磷脂酰乙醇胺、经PEG修饰的磷脂酸、经PEG修饰的神经酰胺、经PEG修饰的二烷基胺、经PEG修饰的二酰基甘油、经PEG修饰的二烷基甘油。
9.根据权利要求3-8任一所述的纳米脂质体颗粒载体,其特征在于,所述磷脂,结构脂,PEG化脂分别为1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱、胆固醇、PEG2000-DMG。
10.权利要求3-8任一所述的纳米脂质体颗粒载体在制备RNA或DNA递送药物中的应用。
11.根据权利要求10所述的应用,其特征在于,所述RNA或DNA递送药物的制备方法包括以下步骤:
(1)将作为药物分子的RNA或DNA溶解于醋酸钠缓冲液中形成水相;
(2)以可离子化阳离子脂质中的氮原子与作为药物分子的RNA或DNA中的磷酸基团的摩尔比为(2-30)∶1确定可离子化阳离子脂质的摩尔数,再按照可离子化阳离子脂质, 1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱, 胆固醇, PEG2000-DMG的摩尔比为50:10:38.5:1.5的比例将四种组分混合均匀,形成均一的有机相;
(3)将步骤(1)制备的水相和步骤(2)制备的有机相以微流控混合的形式制备成装载mRNA或DNA的纳米脂质体颗粒,其中,核酸水相的体积是有机相的3倍,所述纳米脂质体颗粒载体中的脂质和核酸的质量比wt/wt比率为3∶1至100∶1。
12.根据权利要求10所述的应用,其特征在于,所述RNA为短干扰RNA、不对称干扰RNA、RNA干扰分子、微小RNA、反义RNA、小发夹RNA或信使RNA。
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