CN114105799B - 一种氨基脂质及其制备方法和应用 - Google Patents

一种氨基脂质及其制备方法和应用 Download PDF

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CN114105799B
CN114105799B CN202210082815.3A CN202210082815A CN114105799B CN 114105799 B CN114105799 B CN 114105799B CN 202210082815 A CN202210082815 A CN 202210082815A CN 114105799 B CN114105799 B CN 114105799B
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cancer
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lipid
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CN114105799A (zh
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查高峰
潘逸航
胡月潇
彭星华
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SHENZHEN HONGXIN BIOTECHNOLOGY CO Ltd
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Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
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Priority to PCT/CN2022/131322 priority patent/WO2023142600A1/zh
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Abstract

本发明属于医药化学技术领域，特别涉及一种氨基脂质及其制备方法和应用。本发明提出通式如式（I）所示的可离子化氨基脂质，或其药物可用的盐。本发明的氨基脂质可用于递送核酸及小分子药物。上述氨基脂质化合物具备两个酯键，显著增强了可离子化氨基脂质的溶酶体逃逸能力，有助于目的药物或基因等递送目标的释放，进而提高递送效率，在体外、体内的递送研究中，显示了优良的递送核酸至细胞中的能力。

Description

一种氨基脂质及其制备方法和应用
技术领域
本发明属于医药化学技术领域，特别涉及一种氨基脂质及其制备方法应用。
背景技术
核酸药物在癌症、传染性疾病、遗传性疾病和心血管疾病的预防与治疗方面具有极为广阔的应用前景。然而，由于RNA、DNA和siRNA等体内极易被降解，直接通过口服或静脉注射给药，生物利用度极低，因此需要载体的递送。
常用的核酸载体包括病毒载体和非病毒载体，病毒载体具有较高的转染效率，但是病毒载体缺乏靶向性，存在较大的安全顾虑，且载体容量小，生产成本较高。非病毒载体具有安全性较高，载体分子容易修饰，适合大量生产等优点，具有广阔的应用前景。其中基于脂质的纳米颗粒递送系统（Lipid Nanoparticles, LNP）的应用走在了前列，LNP一般由可离子化或阳离子脂质、膦脂、胆固醇和聚乙二醇化的脂质组成。结构上均为具有自组装性能的两亲性分子，LNP各成分结构确定，重现性好，利于质量监管，同时具有较长的体内循环时间、良好的生物相容性等优点而受到广泛关注。纳米颗粒在进入细胞后，需逃出内涵体/溶酶体，才能在细胞质中释放RNA，使其得以表达生成目的蛋白。但目前LNP的内涵体/溶酶体逃逸率普遍较低，尽管DLin-MC3-DMA作为氨基脂质体内评价的“金标准”，是目前最高效的氨基脂质，并被FDA批准用于首个siRNA治疗性药物Onpattro（patisiran），也仅有1% -4%的RNA逃出内涵体/溶酶体，内涵体/溶酶体逃逸已成为影响核酸递送的关键步骤，因此设计具有良好包载核酸能力，同时又具有较高的内涵体/溶酶体逃逸能力的氨基脂质，以解决核酸递送问题，具有重大的研究意义和现实需求。
发明内容
针对现有技术中阳离子脂质体的转染的效率低、正电荷所导致的细胞毒性等技术问题，本发明提供了一种氨基脂质及其应用。
本发明的目的通过以下技术方案予以实现：
本发明的第一个方面，提供：
一种氨基脂质，其结构如式（I）所示：
Figure 229645DEST_PATH_IMAGE001
其中，L为C1-C6亚烷基、C1-C6亚烯基、C1-C6亚炔基、C3-C6亚环烷基、C3-C6亚环烯基；
R1和R2相同或不同，并且各自独立地选自取代或未取代的C1-C20烷基、C1-C20烯基、C1-C20炔基、C1-C20环烷基、C1-C20环烯基、C1-C20环炔基，所述C1-C20烷基、C1-C20烯基、C1-C20炔基、C1-C20环烷基、C1-C20环烯基、C1-C20环炔基的取代基选自C1-C6烃基、氟；
R3和R4彼此相同或不同，并且各自独立地选自H，取代或未取代的C1-C10烷基、C2-C10烯基、C2-C10炔基，所述C1-C10烷基、C2-C10烯基、C2-C10炔基的取代基为C1-C6烃基；
或R3和R4相连接形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。
优选地，所述R1选自取代或未取代的C4-C17烷基、C4-C17烯基、C4-C17炔基、C4-C17环烷基、C4-C17环烯基、C4-C17环炔基，所述C4-C17烷基、C4-C17烯基、C4-C17炔基、C4-C17环烷基、C4-C17环烯基、C4-C17环炔基的取代基选自C1-C6烃基、氟。
优选地，R1选自E1、E2、E3、E4、E5、E6、E7、E8、E9、E10、E11、E12、E13、E14、E15、E16、E17、E18、E19、E20、E21、E22、E23、E24、E25中的一种：
Figure 956293DEST_PATH_IMAGE002
。
更优选地，R1选自E3、E4、E5、E6、E7、E8、E9、E10、E11、E12、E13、E15、E17、E18、E20、E21、E24中的一个。
优选地，所述R2选自取代或未取代的C5-C17烷基、C5-C17烯基、C5-C17炔基、C5-C17环烷基、C5-C17环烯基、C5-C17环炔基，所述C5-C17烷基、C5-C17烯基、C5-C17炔基、C5-C17环烷基、C5-C17环烯基、C5-C17环炔基的取代基选自C1-C6烃基、氟。
优选地，R2选自C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30、C31、C32、C33、C34、C35、C36、C37、C38、C39、C40、C41、C42、C43、C44、C45、C46、C47、C48、C49、C50、C51、C52、C53、C54、C55、C56、C57、C58、C59、C60、C61、C62、C63、C64、C65、C66、C67、C68、C69、C70、C71、C72、C73、C74、C75、C76、C77、C78、C79、C80、C81、C82、C83、C84、C85、C86、C87、C88、C89、C90、C91、C92、C93、C94、C95、C96、C97、C98、C99、C100、C101中的一种：
Figure 854979DEST_PATH_IMAGE003
Figure 267243DEST_PATH_IMAGE004
。
更优选地，R2选自C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C56、C57、C58、C60、C62、C63、C64、C66、C67、C71、C72、C74、C79、C82、C83中的一个。
优选地，R3、R4、L形成R3R4-N-L的含胺的羧酸结构为：
Figure 276788DEST_PATH_IMAGE005
和/或R3和R4相连接 形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。
优选地，
Figure 389100DEST_PATH_IMAGE006
选自：A1、A2、A3、A4、A5、A6、A7、A8、A9、A10、A11、A12、A13、A14、 A15、A16、A17、A18、A19、A20、A21、A22、A23、A24、A25、A26、A27、A28、A29、A30、A31、A32、A33、 A34、A35、A36、A37、A38、A39、A40中的一种：
Figure 396371DEST_PATH_IMAGE007
更优选地，
Figure 594134DEST_PATH_IMAGE006
选自A1、A7、A8、A9、A10、A11、A12、A14、A15、A16、A23、A24、 A28、A33、A37中的一个。
优选的，所述氨基脂质选自如下结构所示化合物的一种：
Figure 407369DEST_PATH_IMAGE008
。
本发明的第二个方面，提供：
本发明第一个方面所述氨基脂质的制备方法，包括以下步骤：
S1. 化合物R2COOH与环氧类化合物在催化剂FeCl3/Py的催化下无溶剂搅拌反应；
S2. 在步骤S1反应体系中加入R3R4NLCOOH，并在缩合剂存在的条件下反应即得。
反应的流程如下：
Figure 75985DEST_PATH_IMAGE009
优选地，所述方法包括以下步骤：
（1）常温下，环氧类化合物与由R2COOH表示的化合物在催化剂FeCl3/Py的催化下进行第一反应，得到第一中间体；
（2）分离第一中间体，在缩合剂的作用下，加入催化量的DMAP，使所述第一中间体与R3R4NLCOOH的羧酸在常温下进行第二反应，得到所述式I的氨基脂质化合物。上述制备方法中使用的缩合剂为EDC·HCl、DCC等。
本发明的第三个方面，提供：
本发明第一个方面所述氨基脂质或其药物可接受的盐、前药或立体异构体在制备用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物中的应用。
优选地，所述药物用于癌症或遗传疾病的治疗。
优选地，所述肿瘤包括但不限于肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌或、膀胱癌、前列腺癌，所述遗传疾病包括但不限于血友病，地中海贫血或高雪氏病中。
优选地，所述药物用于治疗癌症、过敏、毒性和病原体感染。
优选地，上述应用为在制备用于核酸转移的药物中的应用。
优选地，所述核酸为RNA，包括但不限于信使RNA (mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA（rRNA）、微RNA（miRNA）、转移RNA（tRNA）、小干扰RNA（siRNA）和小核RNA（snRNA）。
本发明的第四个方面，提供：
一种纳米颗粒递送系统，其原料本发明第一个方面所述的氨基脂质。
与现有技术相比本发明具有以下技术效果：
本发明公开的通式(Ⅰ)
Figure 785315DEST_PATH_IMAGE010
所示的可离子化氨基脂质或其药物可 用的盐，在构建氨基脂质的过程中反应条件温和，不需要保护和脱保护，原子经济性高。在 体外、体内的递送研究中，显示了优良的递送核酸至细胞中的能力。上述氨基脂质化合物具 备两个酯键，该基团的引入显著增强了阳离子聚合物的降解能力，在大幅度降低细胞毒性 的同时，还能有助于目的药物或基因等递送目标的释放，进而提高递送效率。所述氨基脂质 化合物的制备方法具有原料易得、反应条件温和、反应选择性好、反应产率高、仪器设备要 求低和操作简单的优点。
附图说明
图1为实施例5中E7C71A9的1H-NMR图谱；
图2为实施例5中E7C71A9的13C-NMR图谱；
图3为实施例15中肌肉注射OVA mRNA疫苗后的荷瘤小鼠的肿瘤生长曲线图；
图4为实施例15中肌肉注射OVA mRNA疫苗后的荷瘤小鼠生存曲线图。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
本发明所用的术语“任选地取代的”意指与原子或基团连接的一个或多个氢原子独立地未被取代，或被一个或多个例如一、二、三或四个取代基取代。当一个原子或基团被多个取代基取代时，所述多个取代基可以相同或不同。
文中的缩写：
RNA 核糖核酸
DSPC 二硬脂酰磷脂酰胆碱
DOPE二油酰基磷脂酰乙醇胺
DOPC二油酰磷脂酰胆碱
DSPE二硬脂酰磷脂酰乙醇胺
PEG2000-DMG (1- (单甲氧基聚乙二醇) -2, 3 二肉豆寇酰基甘油
kD千道尔顿
PBS 磷酸盐缓冲溶液。
以下实施例中如无特别说明，氨基脂质结构通式如式(Ⅰ)所示
Figure 470375DEST_PATH_IMAGE010
，编号指代的氨基脂质结构中，E1～E25为上述定义的R1取代基、 C1～C101为上述定义的R2取代基、A1～A40为上述定义的
Figure 24984DEST_PATH_IMAGE006
基团。如E7C71A1的结 构式为
Figure 846309DEST_PATH_IMAGE011
。
实施例1：E7C71Ay系列氨基脂质化合物库的平行合成与表征
Figure 523278DEST_PATH_IMAGE012
在25mL的反应管中依次加入FeCl3（4mg，0.005mmol）、Py（1μL，0.0025mmol）、2-己基癸酸（0.3 mL, 1 mmol）和1,2-环氧十二烷（0.27 mL, 1.2mmol），室温下搅拌反应过夜，得 Step I（1mmol），加入10mL的DCM，配成0.1 M的Step I溶液。
用移液枪将Step I溶液分别转移至1.5 mL的96孔板中（每个0.1 mL，0.01mmol），每孔中各加入带三级胺的羧酸的DCM溶液（0.1 mL，0.02 mmol，0.2M）、DIPEA和EDC·HCl的DCM溶液（0.2 mL，0.04 mmol，0.2 M），DMAP的DCM溶液（0.1 mL，0.005 mmol，0.05 M）常温搅拌6h，TLC检测无Step I 原料。反应结束后，常温挥法至干，即得到15个氨基脂质化合物E7C71Ay。进行质谱检测，结果见下面的表1。
表1：E7C71Ay系列氨基脂质化合物库的MW/z值
Figure 934449DEST_PATH_IMAGE013
Figure 27170DEST_PATH_IMAGE014
。
实施例2：2-羟基十六烷基十二烷酸酯
Figure 968581DEST_PATH_IMAGE015
在25mL的反应管中依次加入FeCl3（20mg，0.025mmol）、Py（5μL，0.0125 mmol）、月桂酸（1g，5 mmol）和1,2-环氧十六烷（1.7 mL, 6 mmol），室温下搅拌反应过夜，使用柱层析纯化（hexane：EA=20：1至5：1）得2-羟基十六烷基十二烷酸酯（2.0g，90%）。1H NMR (400MHz，CDCl3)：δ 0.88（t，6H），1.26-1.45（m，40H），1.47（m，2H），1.63（m，2H），2.02（m，1H），2.34（t，2H），3.82（m，1H），3.95（m，1H），4.13（m，1H）。ESI-MS calculated for C28H57O3 + [M+H]+441.4, found 441.6。
实施例3：2-((4-(二甲氨基)丁酰基)氧基)十二烷酸十六烷酯
Figure 816451DEST_PATH_IMAGE016
在10 mL的反应管中依次加入EDC·HCl（192mg，1 mmol），DIPEA（174μL，1 mmol），DMAP （3.0 mg，0.025 mmol），4-(二甲氨基)丁酸（101mg，0.6 mmol），2-羟基十六烷十二烷酸酯（220mg，0.5mmol），4 mL DCM。室温下搅拌3 h得到化合物E11C7A9（235 mg,，85%）。1HNMR (400 MHz，CDCl3)：δ 0.88 （t，6H），1.25-1.45（m，40H），1.58（m，4H），1.78（m，2H），2.23（s，6H），2.30（m，6H），4.01（m，1H），4.21（m，1H），5.08（m，1H）。13C NMR (400 MHz，CDCl3)：δ14.03，14.08，22.59，22.64，23.35，25.11，27.39，27.43，29.23，29.27，29.29，29.45，29.53，29.59，30.80，31.65，31.85，31.89，32.16，32.39， 47.39，47.69，58.86，64.49，71.53，171.87，173.43。ESI-MS calculated for C34H68NO4 + [M+H]+ 554.5, found 554.7。
实施例4：2-羟基十二烷基2-己基癸酸酯
Figure 413786DEST_PATH_IMAGE017
在25mL的反应管中依次加入FeCl3（20mg，0.025mmol）、Py（5μL，0.0125 mmol）、2-己基癸酸（1.3g，5 mmol）和1,2-环氧十二烷（1.3 mL, 6 mmol），室温下搅拌反应过夜，使用柱层析纯化（hexane：EA=20：1至5：1）得2-羟基十二烷基2-己基癸酸酯（1.9 g，85%）。1H NMR(400 MHz，CDCl3)：δ 0.88 （t，9H），1.26-1.45 （m，36H），1.47（m，2H），1.63（m，2H），2.02（m，1H），2.34（t，2H），3.82（m，1H）, 3.95（m，1H），4.13（m，1H）。ESI-MS calculated for C28H56O3 + [M+H]+ 441.4, found 441.5。
实施例5：2-((4-(二甲氨基)丁酰基)氧基)十二烷基2-己基癸酸酯
Figure 106936DEST_PATH_IMAGE018
在10 mL的反应管中依次加入EDC·HCl（192 mg，1 mmol），DIPEA（174μL，1 mmol），DMAP （3.0 mg，0.025 mmol），4-(二甲氨基)丁酸（101 mg，0.6 mmol），2-羟基十二烷基2-己基癸酸酯（220 mg，0.5mmol），4 mL DCM。室温下搅拌3 h得到化合物E7C71A9（235 mg,，85%）。1H NMR (400 MHz，CDCl3)：δ 0.88 （t，9H），1.25-1.45（m，38H），1.58（m，4H），1.79（m，2H），2.12-2.30（m，11H），4.01（m，1H），4.22（m，1H），5.07（m，1H）（图1）。13C NMR (400 MHz，CDCl3)：δ14.04，14.07，22.59，22.64，22.95，25.11，27.39，27.43，29.23，29.27，29.29，29.45，29.53，29.59,30.79，31.68，31.85，31.87，32.18，32.37, 45.39，45.69，58.84，64.48，71.51，172.98，176.23（图2）。ESI-MS calculated for C34H67NO4 + [M+H]+ 554.5,found 554.6。
实施例6：2-羟基癸基-十八烷-9-烯酸酯
Figure 401388DEST_PATH_IMAGE019
在25mL的反应管中依次加入FeCl3（20mg，0.025mmol）、Py（5μL，0.0125 mmol）、油酸（1.6 mL，5 mmol）和1,2-环氧癸烷（1.1mL, 6 mmol），室温下搅拌反应过夜，使用柱层析纯化（hexane：EA=20：1至5：1）得2-羟基癸基-十八烷-9-烯酸酯（1.9g，85%）。1H NMR (400MHz，CDCl3)：δ 0.88 （t，6H），1.26-1.45（m，34H），1.63（m，2H），2.17（m，4H），2.33（m，2H），4.09-4.35（m，3H），5.35-5.43（m，2H）。 ESI-MS calculated for C28H55O3 + [M+H]+439.4,found 439.6。
实施例7：1-(油酰氧基)癸烷-2-基1-甲基哌啶-4-羧酸酯
Figure 154581DEST_PATH_IMAGE020
在10 mL的反应管中依次加入EDC·HCl（192 mg，1 mmol），DIPEA（174μL，1 mmol），DMAP （3.0 mg，0.025 mmol），1-甲基哌啶-4-羧酸（86 mg，0.6 mmol），2-羟基癸基-十八烷-9-烯酸酯（219 mg，0.5mmol），4 mL DCM。室温下搅拌3 h得到化合物E5C82A23（226 mg，80%）。1H NMR (400 MHz，CDCl3)：δ 0.88 （t，6H），1.23-1.46（m，32H），1.49（m，2H），1.66（m，2H），1.73-2.03（m，4H），2.11-2.20（m，7H），2.33-2.51（m，7 H），4.03（m，1H），4.24（m，1H），5.07（m，1H），5.43（m，2H）。13C NMR (400 MHz，CDCl3)：δ14.04，14.06，22.59，22.64，25.01，25.26，29.23，29.25，29.27，29.29，29.45，29.53，29.59，30.80，31.65，31.85，31.90，32.16，32.39，47.39，47.69，58.86，64.49，71.53，130.57，130.63，171.97，173.73。ESI-MScalculated for C35H66NO4 + [M+H]+ 564.5, found 564.6。
实施例8：3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟-1-羟基辛基十四烷酸酯
Figure 239211DEST_PATH_IMAGE021
在25mL的反应管中依次加入FeCl3（20mg，0.025mmol）、Py（5μL，0.0125 mmol）、豆蔻酸（1.1 g，5 mmol）和3-(全氟正己基)环氧丙烷（1.4 mL, 6 mmol），室温下搅拌反应过夜，使用柱层析纯化（hexane：EA=20：1至5：1）得3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟-1-羟基辛基十四烷酸酯（2.7g，90%）。1H NMR (400 MHz，CDCl3)：δ 0.88 （t，3H），1.26-1.45（m，20H），1.63（m，2H），2.03（m，2H），2.36（t，2H），6.68（t，1H）。ESI-MS calculated forC22H32F13O3 + [M+H]+591.2，found 591.3。
实施例9：1-((4-(二甲氨基)丁酰基)氧基)-3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟辛基十四烷酸酯
Figure 736052DEST_PATH_IMAGE022
在10 mL的反应管中依次加入EDC·HCl（192mg，1 mmol），DIPEA（174μL，1 mmol），DMAP （3.0 mg，0.025 mmol），4-(二甲氨基)丁酸（101mg，0.6mmol），3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟-1-羟基辛基十四烷酸酯（295mg，0.5mmol），4 mL DCM。室温下搅拌3 h得到化合物E24C9A9（263.8 mg，75%）。1H NMR (400 MHz，CDCl3)：δ 0.88（t，3H），1.23-1.60（m，20H），1.66（m，2H），1.88（m，2H），2.05-2.16（m，8H），2.36（m，4H），3.10（t，2H），7.46（t，1H）。13C NMR (400 MHz，CDCl3)：δ 14.07，22.69，22.95，25.06，27.39，29.25，29.41，29.57，29.59，30.79，31.85，31.87，32.18，32.37，46.39，46.69，59.84，88.37，109.05，110.09，111.89，112.37，118.49，173.67，176.23。ESI-MScalculated for C28H43F13NO4 + [M+H]+704.3，found 704.5。
实施例10：8-乙基-2-羟基癸基棕榈酸酯
Figure 652055DEST_PATH_IMAGE023
在25mL的反应管中依次加入FeCl3（20mg，0.025mmol）、Py（5μL，0.0125 mmol）、2-己基癸酸（1.3 g，5 mmol）和2-(6-乙基辛基)环氧乙烷（1.3 mL, 6 mmol），室温下搅拌反应过夜，使用柱层析纯化（hexane：EA=20：1至5：1）得8-乙基-2-羟基癸基棕榈酸酯（2.0 g，90%）。1H NMR (400 MHz，CDCl3)：δ 0.88 （t，9H），1.26-1.45 （m，37H），1.47（m，2H），1.63（m，2H），2.02（m，1H），2.34（t，2H），3.82（m，1H），3.95（m，1H），4.13（m，1H）。ESI-MS calculatedfor C28H57O3 + [M+H]+ 441.4, found 441.6。
实施例11：2-((4-(二甲氨基)丁酰基)氧基)-8-乙基癸基棕榈酸酯
Figure 310570DEST_PATH_IMAGE024
在10 mL的反应管中依次加入EDC·HCl（192 mg，1 mmol），DIPEA（174μL，1 mmol），DMAP （3.0 mg，0.025 mmol），4-(二甲氨基)丁酸（101 mg，0.6 mmol），8-乙基-2-羟基癸基棕榈酸酯（220mg，0.5mmol），4 mL DCM。室温下搅拌3 h得到化合物E20C11A9（221 mg,，80%）。1H NMR (400 MHz，CDCl3)：δ 0.88 （t，9H），1.25-1.45（m，37H），1.58（m，4H），1.78（m，2H），2.23（s，6H），2.30（m，6H），4.01（m，1H），4.21（m，1H），5.08（m，1H）。13C NMR (400 MHz，CDCl3)：δ12.01，12.08，14.09，22.59，22.64，22.95，25.11，27.39，27.43，29.23，29.25，29.31,29.44,29.56,29.59,30.80, 31.64，31.83，31.87，32.21，32.33，46.48，46.89，59.45，65.36，71.70，173.48, 176.73。ESI-MS calculated for C34H68NO4 + [M+H]+ 554.5,found 554.7。
实施例12：氨基脂质化合物作为mRNA载体的体外评价
细胞系：HeLa细胞系 (ATCC)
培养基：补充了10%胎牛血清的1640 (Invitrogen)
筛选形式：96孔板细胞转染
检测：使用多功能酶标仪检测荧光强度。根据制造商的说明，Lipofectamine3000(Invitrogen) 用作阳性对照组。
方法：使用8通道移液管加样。所示的含量为96孔平板的单孔。
1. 参考实施例1中所述中路线合成得到一系列氨基脂质化合物，将其与二硬脂酰磷脂酰胆碱（DSPC）, 胆固醇， PEG2000-DMG按摩尔比为50：10：38.5：1.5的比例混合溶解在无水乙醇中；Luc-mRNA (TriLink)溶解在醋酸钠溶液（25 nM，pH = 5.0）中，使用排枪取出上述的混合脂质溶液，加入至Luc-mRNA溶液中，充分混合，控制其配比为乙醇溶液与醋酸钠溶液（25nM， pH = 5.0）的比例为1:3，制得脂质纳米颗粒溶液。氨基脂质化合物与荧光素酶mRNA(Luc mRNA)的质量比约为10:1，每孔mRNA的用量为100 ng。
2. 脂质纳米颗粒溶液在室温下孵育30min后，于96孔全白酶标板中每孔加入100μL新鲜重悬浮的HeLa细胞（1×104细胞），再用移液管将脂质纳米颗粒溶液加入到96孔板中（每孔10 μL）。置于37℃的含有5% CO2的培养箱中孵育。
3. 细胞初始转染后16至20小时，将底物ONE-GloTMLuciferase以100 μL/孔加入细胞中，2 min后用多功能酶标仪（Biorek SynergyH1）进行检测。
4.相对转染效率如下计算：
Figure 381032DEST_PATH_IMAGE025
结果：部分化合物对HeLa细胞的Luc-mRNA的转染效率示于表2中。
表2：4345种化合物对HeLa细胞的mRNA相对转染效率
Figure 681563DEST_PATH_IMAGE026
Figure 452073DEST_PATH_IMAGE027
Figure 484751DEST_PATH_IMAGE028
Figure 340711DEST_PATH_IMAGE029
Figure 444934DEST_PATH_IMAGE030
Figure 506168DEST_PATH_IMAGE031
Figure 772065DEST_PATH_IMAGE032
Figure 115321DEST_PATH_IMAGE033
Figure 757655DEST_PATH_IMAGE034
Figure 440440DEST_PATH_IMAGE035
Figure 877238DEST_PATH_IMAGE036
实施例13：氨基脂质化合物制备的脂质纳米颗粒在BMDC原代细胞上的转染
制剂方法：同实施例7。
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20 g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
细胞获取：把C57BL/6小鼠进行脱颈臼处死，并置于75%酒精中浸泡5分钟进行消毒，解剖获取小鼠大腿及小腿胫骨，并把附着的肌肉剔除露出骨质，然后用1ml吸有PBS的注射器把胫骨中的骨髓吹出，把所得骨髓吹散后通过50um滤网滤去杂质，向所得过滤物中加入红细胞裂解液（3~4 mL）后放置5分钟后进行800g、5分钟的离心除去上清液，将所得细胞置于1640培养基（含10%胎牛血清、20ng/ml GMCSF、10ng/ml IL4）中重悬，并接种于6孔板中，接种密度为100000个细胞/毫升培养基，放置于37℃、5%CO2细胞培养箱中，每2天进行半换液一次，于第七天收集悬浮细胞和松散贴壁的细胞，并接种到96孔全白酶标板接种密度为每孔20000个细胞，培养基体积为100uL。
细胞转染：向铺有原代细胞的96孔全白酶标板中加入包裹萤光素酶mRNA的脂质纳米颗粒，控制每孔中的mRNA脂质纳米颗粒加入体积为10μL。随后放置在37℃、5%CO2浓度的培养箱中16小时。
转染效率检测：往96孔全白酶标板中每孔加入20 uL底物ONE-GloTMLuciferase，1min后用多功能酶标仪（Biorek SynergyH1）进行检测。代表性氨基脂质化合物在BMDC上转染LucmRNA的表达强度见表3。DLin-MC3作为对照,所述的氨基脂质多个与MC3表达强度相似，并有多个显著优于阳性对照。
表3：98个氨基脂质化合物在BMDC上的转染的表达强度
Figure 707791DEST_PATH_IMAGE037
Figure 380912DEST_PATH_IMAGE038
实施例14：氨基脂质化合物制备的脂质纳米颗粒的萤光素酶mRNA体内递送性能评价
1. 脂质纳米颗粒的制备
将本发明的氨基脂质化合物与中性脂质（如：DSPC、DOPE、胆固醇）、聚乙二醇化脂质（如：PEG2000-DMG、PEG2000-DSPE）按优化后的摩尔比混合并溶解在无水乙醇中。使用微流控制备系统使所得的乙醇溶液和溶解有Luc-mRNA (TriLink)的醋酸钠溶液（25mM, pH =5.0）以1:3的体积比在微流控芯片中混合以制得脂质纳米颗粒的粗溶液，然后将其用透析盒（Fisher，MWCO 20,000）在1 X PBS、控温4 ℃下透析6h，在使用前通过0.22 μm的微孔滤膜过滤。氨基脂质化合物与萤光素酶mRNA (Luc mRNA)的质量比约为10 : 1。
脂质纳米颗粒的表征：
粒径的表征：所制备的脂质纳米颗粒的粒径和PDI通过Nano-ZSZEN3600(Malvern)测定。取LNP溶液20uL进行粒径测量，循环三次，每次循环30s。
包封率测定：参照Quant-iT RiboGreen RNA试剂盒标准规程进行测定。
表4：使用代表性氨基脂质化合物制备的LNP的表征数据
Figure 918204DEST_PATH_IMAGE039
Figure 260323DEST_PATH_IMAGE040
注：上表中：
LNP-1～LNP-36的脂质配方为：氨基脂质:DSPC:胆固醇:PEG2000-DMG= 50:10:38.5:1.5；
LNP-37～LNP-39的脂质配方为：氨基脂质:DOPE:胆固醇:PEG2000-DMG= 45:10:42.5:1.5；
LNP-40和LNP-41的脂质配方为：氨基脂质:DOPC: 胆固醇:PEG2000-DMG= 55:5:38.5:1.5；
LNP-42和LNP-43的脂质配方为：氨基脂质: 胆固醇:PEG2000-DSPE= 60:35.5:4.5；
LNP-44和LNP-45的脂质配方为：氨基脂质:DSPC:DOPC: 胆固醇:PEG2000-DMG=45:10:5:38.0:2.0；
LNP-46和LNP-47的脂质配方为：氨基脂质:DSPC:DOPE: 胆固醇:PEG2000-DSPE=50:10:5:33.5:1.5。
动物实验
动物准备：选取体重约20 g的6周龄的雌性C57BL/6小鼠，于SPF级的饲养室中饲养。动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：每组随机选取9只C57BL/6小鼠，按0.5 mg/kg mRNA的用量，分别使用皮下、肌肉和尾静脉注射三种给药方式注射脂质纳米颗粒溶液（每种给药方式3只小鼠）。12小时后，往每只小鼠体内通过尾静脉注射200 μL 10 mg/mL的D-萤光素钾盐，10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，并拍照记录下来。代表性氨基脂质化合物通过3种给药方式递送的Fluc mRNA的表达强度见表5-7。DLin-MC3作为对照。
表5：代表性氨基脂质化合物LNP皮下给药递送的Luc mRNA的表达强度
Figure 515855DEST_PATH_IMAGE041
表6：代表性氨基脂质化合物LNP肌注给药递送的Luc mRNA的表达强度
Figure 234413DEST_PATH_IMAGE042
表7：代表性氨基脂质化合物LNP尾静脉给药递送的Luc mRNA的表达强度
Figure 452642DEST_PATH_IMAGE043
实施例15：氨基脂质化合物制备的脂质纳米颗粒的体内免疫和肿瘤治疗效果评价
制剂方法：将本发明中所述的氨基脂质化合物与DSPC，胆固醇，PEG2000-DMG的摩尔比为50：10：38.5：1.5的比例混合溶解在无水乙醇中。卵清蛋白mRNA(OVA mRNA)溶解在醋酸钠溶液（50 mM, pH = 4.0）中。使用微流控制备系统使所得的乙醇溶液和溶解有Luc-mRNA (TriLink)的醋酸钠溶液（25mM, pH = 5.0）以1:3的体积比在微流控芯片中混合制得脂质纳米颗粒，再使用透析盒（Fisher，MWCO 20,000）在1X PBS、控温4 ℃下透析6h，使用前用0.22 μm的微孔滤膜过滤。氨基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为10:1。
动物准备：选取5-6周龄的雌性C57BL/6小鼠，体重在18-20 g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：将 B16-OVA 黑色素瘤细胞 (1.5 × 105 ) 皮下注射到小鼠大腿外侧。当肿瘤大小等于 50 mm3时（约在肿瘤接种后第6天或第7天）开始接种疫苗。通过肌肉注射含有1 µg OVA-mRNA 的LNP 制剂对动物进行两次免疫，第二针间隔7天。使用数显卡尺每周测量肿瘤生长 3 次，计算公式为 0.5 × 长度 × 宽度。当肿瘤体积达到1500 mm3时对小鼠实施安乐死。E7C71A9和E6C71A12的肿瘤生长速度显著慢于MC3组（如图3所示），且分别有60%（E7C71A9组）和40%（E6C71A12组）的小鼠达到了完全缓解，显著好于MC3组（如图4所示）。
以上所述仅为本发明专利的较佳实施例而已，并不用以限制本发明专利，凡在本发明专利的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明专利的保护范围之内。

Claims (6)

1.一种氨基脂质，其特征在于，其结构如式（I）所示：
Figure DEST_PATH_IMAGE002
其中，R1选自E6、E7、E8、E9、E10、E11、E13、E17、E18、E20、E21、E24：
Figure DEST_PATH_IMAGE004
所述R2选自C6、C7、C8、C10、C11、C12、C13、C14、C56、C57、C64、C71、C72、C79、C82、C83：
Figure DEST_PATH_IMAGE006
Figure DEST_PATH_IMAGE008
选自A9、A10、A11、A12、A16、A23、A28：
Figure DEST_PATH_IMAGE010
其中，当R1为E24，R2为C57时，
Figure DEST_PATH_IMAGE011
不为A16。
2.一种氨基脂质，其特征在于，其结构如式（I）：
Figure DEST_PATH_IMAGE012
其中，R1、R2、R3、R4及L选择不同的取代基以后形成如下表的化合物：
编号 氨基脂质化合物的结构式 E3C79A10
Figure DEST_PATH_IMAGE014
 E3C79A11
Figure DEST_PATH_IMAGE016
 E3C79A12
Figure DEST_PATH_IMAGE018
 E4C72A11
Figure DEST_PATH_IMAGE020
 E4C74A9
Figure DEST_PATH_IMAGE022
 E4C74A10
Figure DEST_PATH_IMAGE024
 E4C74A11
Figure DEST_PATH_IMAGE026
 E4C79A9
Figure DEST_PATH_IMAGE028
 E4C79A10
Figure DEST_PATH_IMAGE030
 E4C79A11
Figure DEST_PATH_IMAGE032
 E4C79A12
Figure DEST_PATH_IMAGE034
 E5C13A12
Figure DEST_PATH_IMAGE036
 E5C71A11
Figure DEST_PATH_IMAGE038
 E5C74A10
Figure DEST_PATH_IMAGE040
 E5C79A11
Figure DEST_PATH_IMAGE042
 E5C82A23
Figure DEST_PATH_IMAGE044
 E6C74A10
Figure DEST_PATH_IMAGE046
 E6C74A11
Figure DEST_PATH_IMAGE048
 E7C74A9
Figure DEST_PATH_IMAGE050
 E7C74A10
Figure DEST_PATH_IMAGE052
 E7C74A11
Figure DEST_PATH_IMAGE054
 E8C74A9
Figure DEST_PATH_IMAGE056
 E8C74A11
Figure DEST_PATH_IMAGE058
 E8C74A23
Figure DEST_PATH_IMAGE060
 E9C58A11
Figure DEST_PATH_IMAGE062
 E9C60A23
Figure DEST_PATH_IMAGE064
 E9C62A10
Figure DEST_PATH_IMAGE066
 E9C63A10
Figure DEST_PATH_IMAGE068
 E9C66A9
Figure DEST_PATH_IMAGE070
 E9C67A10
Figure DEST_PATH_IMAGE072
 E10C74A9
Figure DEST_PATH_IMAGE074
 E10C74A23
Figure DEST_PATH_IMAGE076
 E11C58A11
Figure DEST_PATH_IMAGE078
 E11C60A11
Figure DEST_PATH_IMAGE080
 E11C62A11
Figure DEST_PATH_IMAGE082
 E12C6A11
Figure DEST_PATH_IMAGE084
 E12C64A23
Figure DEST_PATH_IMAGE086
 E12C74A11
Figure DEST_PATH_IMAGE088
 E15C79A10
Figure DEST_PATH_IMAGE090
 E15C79A11
Figure DEST_PATH_IMAGE092
 E15C79A12
Figure DEST_PATH_IMAGE094
 E20C9A10
Figure DEST_PATH_IMAGE096
 E20C9A11
Figure DEST_PATH_IMAGE098
 E20C66A9
Figure DEST_PATH_IMAGE100
 E21C67A9
Figure DEST_PATH_IMAGE102
 E24C9A9
Figure DEST_PATH_IMAGE104
。
3.根据权利要求1～2任一项所述氨基脂质的制备方法，其特征在于，包括以下步骤：
S1. 化合物R2COOH与环氧类化合物在催化剂FeCl3/Py的催化下无溶剂搅拌反应；
其中，环氧类化合物的结构为：
Figure DEST_PATH_IMAGE106
；
S2. 在步骤S1反应体系中加入R3R4NLCOOH，并在缩合剂存在的条件下反应即得；
其中，所述R1、R2、R3、R4、L选择的取代基与权利要求1或2相同。
4.权利要求1或2所述氨基脂质及其药物可接受的盐在制备用于基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物中的应用。
5.根据权利要求4所述应用，其特征在于，在制备用于癌症和病原体感染疾病的药物中的应用；所述癌症为肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌、前列腺癌或黑色素瘤。
6.根据权利要求1或2所述氨基脂质及其药物可接受的盐在制备用于核酸转移的药物中的应用。
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