CN116407625A - 一种用于增强核酸免疫效力的脂质纳米颗粒及其制备方法和用途 - Google Patents
一种用于增强核酸免疫效力的脂质纳米颗粒及其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种用于增强核酸免疫效力的脂质纳米颗粒及其制备方法和用途。
背景技术
mRNA能实现遗传信息在蛋白质上的表达,随着对mRNA的不断研究,mRNA疫苗和mRNA药物受到广泛关注。其中,mRNA疫苗是近年来兴起的一种新型疫苗,是将含有编码抗原蛋白的mRNA导入人体,直接进行翻译,形成相应的抗原蛋白,从而诱导机体产生免疫应答,达到预防免疫的作用。与传统疫苗相比,mRNA疫苗抗原选择范围广,开发速度快,疫苗制备采用全合成技术,组分明确,生产工艺简单,可以使用相似的生产工艺和相同的生产设备完成不同mRNA疫苗的生产,因此其研发周期短、成本低、便于标准化生产,适用于大流行疾病和传染病暴发流行期间的疫苗开发和生产。除针对传染病的疫苗,各类肿瘤和感染性疾病的mRNA疫苗也被不断研究。而mRNA药物是一种核酸药物,通过体外合成mRNA序列,再由递送***递送到细胞质,向患者注射mRNA可以在病人自己的细胞中启动药物的生产,补偿有缺陷的基因/蛋白质,可以mRNA平台形式转化各种蛋白质药物,如单克隆抗体、酶和细胞因子等,并用来治疗代谢疾病、心脏病和免疫肿瘤学等多种疾病。可见,mRNA具备预防和治疗多种疾病的潜力,拥有广阔的发展前景。
但是mRNA不稳定,极易被广泛存在的RNA酶降解,因此限制了其使用。为了更好的发挥mRNA的效果,mRNA递送***受到广泛的研究。其中,作为非病毒载体的脂质纳米颗粒(lipid nanoparticles,LNPs)是目前mRNA研究领域重要的递送***。mRNA分子通常被包裹在LNPs内以提高其稳定性。LNPs主要由4部分组成:可电离阳离子脂质、脂质连接的聚乙二醇(PEG化脂质)、甾醇和中性磷脂。各部分在LNPs中起的作用不同,其中负责与mRNA结合的可电离阳离子脂质为LNPs的核心组成成分,中性磷脂为促进LNPs结构稳定的辅助脂质,甾醇起到稳定LNPs和调节膜流动性的作用,PEG化脂质用于确保LNPs的体内稳定性。此外,LNPs中疏水区与亲水区的共存打开了mRNA与其它药物分子共递送的大门。同时,研究发现LNPs中的可电离阳离子脂质具有一定的佐剂效应,但是,该佐剂效应在增强抗原相关的特异性免疫上还有待加强。如何选择合适的佐剂,增强mRNA产品的效果,需要进一步研究。
Pam2Cys是由一个半胱氨酸分子、一个硫代甘油分子和两条脂肪酸链经过简单合成而形成的中性脂质氨基酸,具有简单易得和可代谢的优点。Pam2Cys及其衍生物通过TLR2/6信号通路进行信号传导,被证实能够同时激活体液免疫与细胞免疫。以往有研究将Pam2Cys与抗原表位肽偶联作为疫苗。但是并没有研究表明Pam2Cys可以在mRNA产品中发挥佐剂效应,其是否能够作为免疫佐剂,需要进一步研究。
发明内容
本发明的目的是提供一种用于增强核酸免疫效力的脂质纳米颗粒及其制备方法和用途。
本发明提供了式I所示化合物、或其盐作为增强核酸免疫效力的免疫佐剂的用途;
其中,R选自羟基、氨基、烷氧基、氨基酸、多肽、聚乙二醇。
进一步地,所述R为羟基时,式I所示化合物为Pam2Cys;
和/或,所述核酸为mRNA。
本发明还提供了一种脂质纳米颗粒,所述脂质纳米颗粒由可电离阳离子脂质、甾醇、中性磷脂、PEG化脂质和前述的式I所示化合物、或其盐为原料制备而成。
进一步地,所述原料的摩尔配比如下:
可电离阳离子脂质20~60份、甾醇30~60份、中性磷脂9~50份、PEG化脂质1~5份、前述的式I所示化合物、或其盐0.01~5份。
进一步地,所述原料的摩尔配比如下:
可电离阳离子脂质47~50份、甾醇36~39份、中性磷脂9~10份、PEG化脂质1~2份、前述的式I所示化合物、或其盐0.5~5份。
进一步地,
所述可电离阳离子脂质选自4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)、8-[(2-羟乙基)[6-氧代-6-(十一烷基氧基)己基]氨基]辛酸1-辛基壬基酯或十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯);
和/或,所述中性磷脂选自1,2-二肉豆蔻酰-SN-甘油-3-磷酰乙醇胺、1,2-二棕榈酰基-SN-丙三基-3-和磷酸乙氨醇、1,2-二硬脂酰基-SN-丙三基-3-磷脂酰乙醇胺、1,2-二油酰基-SN-丙三基-3-磷脂酰乙醇胺、二肉豆蔻酰磷脂酰胆碱、1,2-二棕榈酰-SN-甘油-3-磷酰胆碱、1,2-双硬脂酰基-SN-丙三基-3-磷酸胆碱或1,2-二油酰-SN-甘油-3-磷酰胆碱;
和/或,所述甾醇选自胆固醇、谷甾醇、麦角甾醇、菜油甾醇、豆甾醇或菜籽甾醇;
和/或,所述PEG化脂质为二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇、二硬脂酰磷脂酰乙醇胺-聚乙二醇或二肉豆蔻酰基-SN-甘油-3-甲氧基聚乙二醇;所述聚乙二醇部分的分子量为1000~5000。
本发明还提供了前述的脂质纳米颗粒的制备方法,它包括如下步骤:
(1)将可电离阳离子脂质、甾醇、中性磷脂、PEG化脂质和前述的式I所示化合物、或其盐溶于有机溶剂中,作为有机相;
(2)将有机相和水相混合,自组装形成脂质纳米颗粒。
进一步地,
步骤(1)中,所述有机溶剂为乙醇、甲醇、DMSO或四氢呋喃;
和/或,步骤(2)中,所述水相为柠檬酸缓冲液或醋酸盐缓冲液;
和/或,步骤(2)中,所述有机相和水相的体积比为1:(1~5);
和/或,步骤(2)中,所述混合使用微流控混合设备混合;
优选地,
步骤(2)中,所述柠檬酸缓冲液pH为3.0~6.0,浓度为5~20mM;
和/或,步骤(2)中,所述有机相和水相的体积比为1:3;
和/或,步骤(2)中,所述微流控混合的流速为9~20mL/min。
本发明还提供了前述的脂质纳米颗粒在制备核酸递送***的用途。
本发明还提供了一种核酸递送***,所述核酸递送***为负载一种或多种核酸的前述的脂质纳米颗粒。
进一步地,所述核酸为mRNA。
进一步地,所述mRNA为采用质粒载体转录制备而得的mRNA;所述质粒载体不含目标蛋白编码序列时的核苷酸序列如SEQ ID NO.1或SEQ IDNO.2所示。
进一步地,所述SEQ ID NO.1或SEQ ID NO.2所示的核苷酸序列顺序均为T7启动子序列、5’α-globin UTR序列、3’α-globin UTR序列和poly(A)尾序列。
进一步地,
所述质粒载体制备mRNA时,将编码目标蛋白的核苷酸序列***SEQ ID NO.1或SEQID NO.2中SEQ ID NO.3所示5’α-globin UTR序列和SEQ ID NO.4所示3’α-globin UTR序列之间的编码区克隆位点;
或者,将SEQ ID NO.6所示的核苷酸序列连接在目标蛋白编码序列5’端,将SEQ IDNO.8所示的核苷酸序列连接在目标蛋白编码序列3’端,然后将目标蛋白***SEQ ID NO.3所示5’α-globin UTR序列和SEQ ID NO.4所示3’α-globin UTR序列之间的编码区克隆位点;
或者,将SEQ ID NO.10所示的核苷酸序列连接在目标蛋白编码序列5’端,然后将目标蛋白***SEQ ID NO.3所示5’α-globin UTR序列和SEQ ID NO.4所示3’α-globin UTR序列之间的编码区克隆位点。
进一步地,所述目标蛋白为OVA;
优选地,所述mRNA为采用核苷酸序列如SEQ ID NO.11质粒载体转录制备而得的mRNA。
本发明还提供了前述的核酸递送***的制备方法,它包括如下步骤:
1)将可电离阳离子脂质、甾醇、中性磷脂、PEG化脂质和前述的式I所示化合物、或其盐溶于有机溶剂中,作为有机相;
2)将核酸溶于水溶液中,得到水相;
3)将有机相和水相混合,自组装形成脂质纳米颗粒。
进一步地,
步骤1)中,所述有机溶剂为乙醇、甲醇、DMSO或四氢呋喃;
和/或,步骤2)中,所述水相为柠檬酸缓冲液或醋酸盐缓冲液;
和/或,步骤3)中,所述有机相和水相的体积比为1:(1~5);
和/或,步骤3)中,所述有机相中的可电离阳离子脂质与水相中的核酸的氮磷摩尔比为(1~10):1;
和/或,步骤3)中,所述混合使用微流控混合设备混合;
优选地,
步骤2)中,所述柠檬酸缓冲液pH为3.0~6.0,浓度为5~20mM;
和/或,步骤3)中,所述有机相和水相的体积比为1:3;
和/或,步骤3)中,所述有机相中的可电离阳离子脂质与水相中的核酸的氮磷摩尔比为6:1;
和/或,步骤3)中,所述微流控混合的流速为9~20mL/min。
本发明还提供了前述的核酸递送***在制备核酸药物或核酸疫苗中的用途;
优选地,所述核酸药物为mRNA药物;所述核酸疫苗为mRNA疫苗。
与现有技术相比,本发明的有益效果为:
本发明发现式I所示化合物、或其盐可以作为增强核酸免疫效力的免疫佐剂,利用其制备脂质纳米颗粒,载核酸后可以增强核酸的免疫效果;同时,使用本发明脂质纳米颗粒载核酸安全性好。本发明脂质纳米颗粒尤其适用于mRNA的运载,制备得到效果更好的mRNA疫苗或mRNA药物,促使mRNA在疾病预防和治疗中发挥更好的效果,具有良好的应用前景。
本发明将Pam2Cys或其衍生物作为免疫佐剂,添加到脂质纳米颗粒中,以增强mRNA疫苗的佐剂效应,效果十分明显。与其它核酸/核苷酸或小分子佐剂所不同的是,Pam2Cys或其衍生物的亲脂性促使它进入纳米载体的脂质层,而不需要在纳米载体中取代一定比例的mRNA或者对化合物进行进一步的化学修饰,从而实现与编码抗原的mRNA的共递送。
本发明考察了Pam2Cys LNPs包载的mRNA在肿瘤预防和治疗过程中的效果,结果表明所制备的mRNA-LNPs(Pam2Cys)与mRNA-LNPs(Conventional)相比,在肿瘤预防与治疗模型中均表现出更好的效果,同时免疫后小鼠关键器官的组织切片显示mRNA-LNPs(Pam2Cys)的安全性良好。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为OVA mRNA变性琼脂糖凝胶电泳。
图2为各OVA mRNA-LNPs的粒径和电位的分布。
图3为各OVA mRNA-LNPs预防模型中的小鼠肿瘤生长曲线;其中,a为未处理组;b为常规LNPs组;c为实验组;d为无关mRNA对照组。
图4为各OVA mRNA-LNPs预防模型中的小鼠平均肿瘤体积和生存曲线;其中,a为平均肿瘤体积;b为生存曲线。
图5为各OVA mRNA-LNPs激活OVA特异性CD8+T细胞的流式分析。
图6为各OVA mRNA-LNPs治疗模型中小鼠肿瘤生长曲线;其中,a为未处理组;b为常规LNPs组;c为实验组;d为无关mRNA对照组;e为CD4基因敲除实验组;f为CD8a基因敲除实验组。
图7为各OVAmRNA-LNPs治疗模型中小鼠平均肿瘤体积和生存曲线;其中,a为平均肿瘤体积;b为生存曲线。
图8为各OVA mRNA-LNPs诱导免疫记忆模型中小鼠肿瘤生长曲线;其中,a为未处理组;b为实验组。
图9为各OVA mRNA-LNPs诱导免疫记忆模型中小鼠平均肿瘤体积和生存曲线;其中,a为平均肿瘤体积;b为生存曲线。
图10为各OVA mRNA-LNPs受试小鼠的关键器官组织切片。
具体实施方式
除另有说明外,本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。
1、DNA载体的构建和mRNA的制备
1)编码特定抗原的DNA模板载体的制备:转录模板为pT7ggAGA和pT7AGA2质粒载体。pT7ggAGA和pT7AGA2质粒载体的序列顺序均为T7启动子序列、5’α-globin UTR序列、3’α-globin UTR序列和poly(A)尾序列,但两者有8个碱基对的序列差别。克隆相应基因的编码区至pT7ggAGA或pT7AGA2质粒载体上的编码区克隆位点,即得相应基因的转录模板DNA载体。pT7ggAGA和pT7AGA2载体序列,及载体中的蛋白质编码序列如下:
不含蛋白编码序列的模板DNA载体pT7ggAGA序列(SEQ ID NO.1):
不含蛋白编码序列的模板DNA载体pT7AGA2序列(SEQ ID NO.2):
完整编码蛋白的序列由将下述编码序列按5’到3’的方向***前述两载体的ggtccccacagactcagagagaac(SEQ ID NO.3)和gctggagcc tcggtggcca tgcttcttgc(SEQ IDNO.4)两序列之间得到。
其中蛋白编码序列分为三种模式:
①只编码目标蛋白的编码序列
②依次编码额外“分泌信号肽”、目标蛋白和“跨膜和胞质结构域”的编码序列
其中:
位于目标蛋白编码序列5’端的编码的分泌信号肽(蛋白序列):
MAVMAPRTLL LLLSGALALT QTWAGS(SEQ ID NO.5)
位于目标蛋白编码序列5’端的编码的分泌信号肽(核酸序列):
atggccgtga tggctcctcg aactttgttg ctccttttga gcggagcctt ggctctcactcagacatggg cgggctct(SEQ ID NO.6)
位于目标蛋白编码序列3’端的编码的跨膜和胞质结构域(蛋白序列):
IVGIVAGLAV LAVVVIGAVV AAVMCRRKSS GGKGGSYSQA ACSDSAQGSD VSLTA(SEQ IDNO.7)
位于目标蛋白编码序列3’端的编码的跨膜和胞质结构域(核酸序列):
atcgtgggca ttgttgctgg cctggctgtc ctagcagttg tggtcatcgg agctgtggtcgctgctgtga tgtgtaggag gaagagttca ggtggaaaag gagggagcta ctctcaggct gcgtgcagcgacagtgccca gggctctgat gtgtctctca cagct(SEQ ID NO.8)
③依次编码额外“胞质和跨膜结构域”和目标蛋白的编码序列
其中:
位于目标蛋白编码序列5’端的编码的胞质和跨膜结构域(蛋白序列):
MHRRRSRSCR EDQKPVMDDQ RDLISNNEQL PMLGRRPGAP ESKCSRGALY TGFSILVTLLLAGQATTAYF LY(SEQ ID NO.9)
位于目标蛋白编码序列5’端的编码的胞质和跨膜结构域(核酸序列):
atgcacagac gacggagccg cagctgcaga gaggaccaaa aaccggtaatggacgaccaaagagacctca taagcaacaa cgagcaactc cccatgctcg gacgaaggcc cggcgcaccggaaagcaagtgcagcagagg agcgctctac acgggcttca gcatactggt gacactcctg ctggcaggccaagcgacgaccgcgtacttc ctatac(SEQ ID NO.10)
2)使用限制性内切酶BspQI在poly(A)的末尾处对质粒进行切割得到体外转录所需的线性化DNA模板。
3)向线性化DNA模板中加入T7 RNA聚合酶、核苷酸底物、RNA酶抑制剂等原料体外转录线性化DNA模板得到mRNA。随后用牛痘病毒加帽酶和二氧甲基转移酶对mRNA进行转录后加帽。最后,用oligo(dT)30磁珠纯化加帽后的mRNA,并用变性琼脂糖凝胶电泳法评估所合成mRNA的质量。
2、LNPs的制备及表征
本发明使用微流控混合设备来制备LNPs。具体制备方式为,将可电离阳离子脂质、中性磷脂、甾醇、PEG化脂质和免疫佐剂(Pam2Cys或其衍生物)溶于乙醇等溶剂内作为有机相,同时将mRNA溶于柠檬酸缓冲液中作为水相。将水相与有机相以3:1的比例在微流控混合通道内快速混合,使其自组装形成LNPs初制品。稀释LNPs初制品,以含有9%蔗糖的pH值为7.4的Tris-HCl缓冲液为透析液,透析24h置换有机溶剂,得到mRNA-LNPs终产品。
用马尔文粒度仪检测所制备mRNA-LNPs的粒径、PDI和zeta电位,用Quant-itRiboGreen RNA试剂盒检测mRNA的包封率。
3、药效学实验
以C57BL/6小鼠为实验动物,采用肌肉注射的给药方式,单次注射含20μg mRNA的mRNA-LNPs,共免疫一次,每只小鼠皮下注射5×105E.G7-OVA淋巴瘤细胞于小鼠体内构建肿瘤模型,分别设制未处理组、无关mRNA对照组(EGFP-LNPs(Pam2Cys))、常规LNPs组(OVA-LNPs(Conventional))和实验组(OVA-LNPs(Pam2Cys))。通过OVA特异性T细胞的tetramer流式细胞术检测和肿瘤生长状况来考察肿瘤预防效果。
以C57BL/6小鼠为实验动物,采用肌肉注射的给药方式,单次注射含20μg mRNA的LNPs,共免疫一次,分别设置未处理组、无关mRNA对照组(EGFP-LNPs(Pam2Cys))、常规LNPs组(OVA-LNPs(Conventional))和实验组(OVA-LNPs(Pam2Cys))。第0天时皮下注射3×105E.G7-OVA淋巴瘤细胞(实验对象还包括CD4基因敲除和CD8a基因敲除的C57BL/6小鼠),第3天时单次肌肉注射含20μg mRNA的mRNA-LNPs,随后不再干预。
检测肿瘤长度(L)和宽度(W),按照公式V=0.5×L×W2计算肿瘤体积大小。当满足以下任一条件时,对荷瘤小鼠施以安乐死:1)肿瘤体积>1000mm3,2)肿瘤溃烂或小鼠濒死。
4、安全性评价
取免疫45天后处死小鼠的关键脏器(心、肝、脾、肺、肾)进行石蜡包埋,并用苏木精-伊红染料对石蜡切片进行染色,观察潜在的组织病理学改变。
实施例1、DNA载体的构建和mRNA的制备
1)编码OVA抗原的DNA模板载体的制备:合成编码OVA的pT7ggAGA质粒载体,pT7ggAGA质粒载体的序列顺序为T7启动子序列、5’α-globin UTR序列、3’α-globin UTR序列和poly(A)尾巴序列。克隆OVA的编码区至pT7ggAGA质粒载体上的编码区克隆位点,即得OVA的转录模板DNA载体。编码OVA的pT7ggAGA载体的全序列如SEQ ID NO.11所示:
编码OVA的pT7ggAGA载体序列(SEQ ID NO.11):
2)线性化DNA模板的制备:使用限制性内切酶BspQI在poly(A)的末尾处对质粒进行切割得到体外转录所需的线性化DNA模板。
3)体外转录mRNA的合成:向线性化DNA模板中加入T7 RNA聚合酶、核苷酸底物、RNA酶抑制剂等原料体外转录线性化DNA模板得到mRNA,在反应过程中用me1Ψ-UTP代替UTP。随后用牛痘病毒加帽酶和二氧甲基转移酶对所得到的mRNA进行转录后加帽。最后,用oligo(dT)30磁珠纯化加帽后的mRNA,并用变性琼脂糖凝胶电泳法评估所合成mRNA的质量,结果见图1,左侧约1700nt的条带为OVA mRNA。
实施例2、LNPs的制备及表征
将OVA mRNA溶解于10mM pH3.0的柠檬酸缓冲液中作为水相,各脂质成分溶解于乙醇中作为有机相,各脂质成分组成如表1所示,其中ALC-0315与OVA mRNA的氮磷比为6:1(摩尔比),将水相与有机相以3:1的体积比和12mL/min的流速在微流控混合通道内快速混合,使其自组装形成mRNA-LNPs初制品。将mRNA-LNPs初制品放入截留分子量为6-8kDa的透析管内,以含有9%蔗糖的pH值为7.4的20mM Tris-HCl缓冲液为透析液,透析24h置换有机溶剂,得到mRNA-LNPs终产品。按照各脂质成分组成不同分别制备mRNA-LNPs1(脂质组成1)、mRNA-LNPs2(脂质组成2)、mRNA-LNPs3(脂质组成3)和mRNA-LNPs4(脂质组成4)。
用马尔文粒度仪检测所制备mRNA-LNPs的粒径、PDI和zeta电位,用Quant-itRiboGreen RNA试剂盒检测mRNA的包封率,结果如表2所示,粒径电位分布结果见图2。图2中Conventional LNPs为不加Pam2Cys的制备的样品。
表1.各脂质成分组成
表1中,ALC-0315:((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯);DOPE:1,2-二油酰基-SN-丙三基-3-磷脂酰乙醇胺;DSPC:1,2-双硬脂酰基-SN-丙三基-3-磷酸胆碱;DMG-PEG2000:二肉豆蔻酰基-SN-甘油-3-甲氧基聚乙二醇,聚乙二醇部分分子量为2000。
表2.各样品粒径、电位和包封率的测量结果
粒径 | PDI | Zeta电位 | 包封率 | |
mRNA-LNPs1 | 91.02nm | 0.143 | -2.227mv | 90.57% |
mRNA-LNPs2 | 77.91nm | 0.102 | -5.805mv | 92.37% |
mRNA-LNPs3 | 95.29nm | 0.104 | -6.831mv | 89.91% |
mRNA-LNPs4 | 90.78nm | 0.100 | -3.044mv | 90.13% |
以下通过具体试验例证明本发明的有益效果。
试验例1、效果试验
肿瘤预防实验
选取C57BL/6小鼠为实验动物,采用肌肉注射的给药方式,单次注射含20μg OVAmRNA的OVA mRNA-LNPs(实施例2中的mRNA-LNPs2),共免疫一次。分别设立未处理组(不注射脂质体,N.T.)、无关mRNA对照组(EGFP-LNPs(Pam2Cys))、常规LNPs组(OVA-LNPs(Conventional),即mRNA-LNPs1)和实验组(OVA-LNPs(Pam2Cys),即mRNA-LNPs2)。给药时间记为负7天,并于第0天皮下注射5×105E.G7-OVA淋巴瘤细胞。部分小鼠第0天时取外周血,用于OVA特异性T细胞的tetramer检测。
EGFP-LNPs(Pam2Cys)的制备方法参照实施例2中的mRNA-LNPs2,只是将OVA mRNA替换为EGFP mRNA。
注射淋巴瘤细胞后,第7天开始检测肿瘤大小,按照每隔4天然后隔3天的检测方法循坏检测,即按照第7、11、14、18、21、25、28、32、35、39、42、46天这样的方式检测。
结果显示(见图3、4、5),实验组(OVA-LNPs(Pam2Cys))能够使该组70%的小鼠无肿瘤形成,并且小鼠生存率高,显著优于常规LNPs组以及无关对照组。
肿瘤治疗实验
选取C57BL/6小鼠为实验动物,采用肌肉注射的给药方式,单次注射约含20μg OVAmRNA的LNPs(实施例2中的mRNA-LNPs2),共免疫一次,分别设立未处理组(不注射脂质体,N.T.)、无关mRNA对照组(EGFP-LNPs(Pam2Cys))、常规LNPs组(OVA-LNPs(Conventional),即mRNA-LNPs1)和实验组(OVA-LNPs(Pam2Cys),即mRNA-LNPs2)。第0天时皮下注射3×105E.G7-OVA淋巴瘤细胞(实验对象包括CD4基因敲除和CD8a基因敲除的C57BL/6小鼠),第3天时单次肌肉注射含20μg OVA mRNA的LNPs或者其他LNPs,随后不再干预。
EGFP-LNPs(Pam2Cys)的制备方法参照实施例2中的mRNA-LNPs2,只是将OVA mRNA替换为EGFP mRNA。
单次肌肉注射后,第7天开始检测肿瘤大小,按照每隔4天然后隔3天的检测方法循坏检测,即按照第7、11、14、18、21、25、28、32、35、39、42、46.天这样的方式检测。
实验结果见图6和图7,由该结果可知实验组(OVA-LNPs(Pam2Cys))使50%的小鼠肿瘤消退,具有更好的抑制肿瘤生长的作用,且该作用由CD4+T细胞和CD8+T细胞介导。
第60天,在因前一次mRNA疫苗接种而致E.G7-OVA肿瘤完全消退的C57BL/6小鼠上,再次皮下注射3×105E.G7-OVA淋巴瘤细胞,以验证免疫记忆反应。结果见图8和图9,表明该疫苗能够有效诱导免疫记忆。
试验例2、安全性实验
取免疫45天后的小鼠关键脏器(心、肝、脾、肺、肾)进行石蜡包埋(肿瘤预防实验中的小鼠,未接瘤),并用苏木精-伊红染料对石蜡切片进行染色,观察各个脏器潜在的组织病理学改变。
结果见图10,由该结果可知mRNA-LNPs(Pam2Cys)的安全性良好。
综上,本发明发现Pam2Cys或其衍生物可以作为增强核酸免疫效力的免疫佐剂,利用其制备脂质纳米颗粒,载核酸后可以增强核酸的免疫效果;同时,使用本发明脂质纳米颗粒载核酸安全性好。本发明脂质纳米颗粒尤其适用于mRNA的运载,制备得到效果更好的mRNA疫苗或mRNA药物,促使mRNA在疾病预防和治疗中发挥更好的效果,具有良好的应用前景。
Claims (18)
2.根据权利要求1所述的用途,其特征在于:所述R为羟基时,式I所示化合物为Pam2Cys;
和/或,所述核酸为mRNA。
3.一种脂质纳米颗粒,其特征在于:所述脂质纳米颗粒由可电离阳离子脂质、甾醇、中性磷脂、PEG化脂质和权利要求1或2所述的式I所示化合物、或其盐为原料制备而成。
4.根据权利要求3所述的脂质纳米颗粒,其特征在于:所述原料的摩尔配比如下:
可电离阳离子脂质20~60份、甾醇30~60份、中性磷脂9~50份、PEG化脂质1~5份、权利要求1或2所述的式I所示化合物、或其盐0.01~5份。
5.根据权利要求4所述的脂质纳米颗粒,其特征在于:所述原料的摩尔配比如下:
可电离阳离子脂质47~50份、甾醇36~39份、中性磷脂9~10份、PEG化脂质1~2份、权利要求1或2所述的式I所示化合物、或其盐0.5~5份。
6.根据权利要求3~5任一项所述的脂质纳米颗粒,其特征在于:
所述可电离阳离子脂质选自4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)、8-[(2-羟乙基)[6-氧代-6-(十一烷基氧基)己基]氨基]辛酸1-辛基壬基酯或十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯);
和/或,所述中性磷脂选自1,2-二肉豆蔻酰-SN-甘油-3-磷酰乙醇胺、1,2-二棕榈酰基-SN-丙三基-3-和磷酸乙氨醇、1,2-二硬脂酰基-SN-丙三基-3-磷脂酰乙醇胺、1,2-二油酰基-SN-丙三基-3-磷脂酰乙醇胺、二肉豆蔻酰磷脂酰胆碱、1,2-二棕榈酰-SN-甘油-3-磷酰胆碱、1,2-双硬脂酰基-SN-丙三基-3-磷酸胆碱或1,2-二油酰-SN-甘油-3-磷酰胆碱;
和/或,所述甾醇选自胆固醇、谷甾醇、麦角甾醇、菜油甾醇、豆甾醇或菜籽甾醇;
和/或,所述PEG化脂质为二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇、二硬脂酰磷脂酰乙醇胺-聚乙二醇或二肉豆蔻酰基-SN-甘油-3-甲氧基聚乙二醇;所述聚乙二醇部分的分子量为1000~5000。
7.权利要求3~6任一项所述的脂质纳米颗粒的制备方法,其特征在于:它包括如下步骤:
(1)将可电离阳离子脂质、甾醇、中性磷脂、PEG化脂质和权利要求1或2所述的式I所示化合物、或其盐溶于有机溶剂中,作为有机相;
(2)将有机相和水相混合,自组装形成脂质纳米颗粒。
8.根据权利要求7所述的制备方法,其特征在于:
步骤(1)中,所述有机溶剂为乙醇、甲醇、DMSO或四氢呋喃;
和/或,步骤(2)中,所述水相为柠檬酸缓冲液或醋酸盐缓冲液;
和/或,步骤(2)中,所述有机相和水相的体积比为1:(1~5);
和/或,步骤(2)中,所述混合使用微流控混合设备混合;
优选地,
步骤(2)中,所述柠檬酸缓冲液pH为3.0~6.0,浓度为5~20mM;
和/或,步骤(2)中,所述有机相和水相的体积比为1:3;
和/或,步骤(2)中,所述微流控混合的流速为9~20mL/min。
9.权利要求3~6任一项所述的脂质纳米颗粒在制备核酸递送***的用途。
10.一种核酸递送***,其特征在于:所述核酸递送***为负载一种或多种核酸的权利要求3~6任一项所述的脂质纳米颗粒。
11.根据权利要求10所述的核酸递送***,其特征在于:所述核酸为mRNA。
12.根据权利要求11所述的核酸递送***,其特征在于:所述mRNA为采用质粒载体转录制备而得的mRNA;所述质粒载体不含目标蛋白编码序列时的核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
13.根据权利要求12所述的核酸递送***,其特征在于:所述SEQ IDNO.1或SEQ IDNO.2所示的核苷酸序列顺序均为T7启动子序列、5’α-globin UTR序列、3’α-globin UTR序列和poly(A)尾序列。
14.根据权利要求13所述的核酸递送***,其特征在于:
所述质粒载体制备mRNA时,将编码目标蛋白的核苷酸序列***SEQ IDNO.1或SEQ IDNO.2中SEQ ID NO.3所示5’α-globin UTR序列和SEQ IDNO.4所示3’α-globin UTR序列之间的编码区克隆位点;
或者,将SEQ ID NO.6所示的核苷酸序列连接在目标蛋白编码序列5’端,将SEQ IDNO.8所示的核苷酸序列连接在目标蛋白编码序列3’端,然后将目标蛋白***SEQ ID NO.3所示5’α-globin UTR序列和SEQ ID NO.4所示3’α-globin UTR序列之间的编码区克隆位点;
或者,将SEQ ID NO.10所示的核苷酸序列连接在目标蛋白编码序列5’端,然后将目标蛋白***SEQ ID NO.3所示5’α-globin UTR序列和SEQ IDNO.4所示3’α-globin UTR序列之间的编码区克隆位点。
15.根据权利要求13或14所述的核酸递送***,其特征在于:所述目标蛋白为OVA;
优选地,所述mRNA为采用核苷酸序列如SEQ ID NO.11质粒载体转录制备而得的mRNA。
16.权利要求10~15任一项所述的核酸递送***的制备方法,其特征在于:它包括如下步骤:
1)将可电离阳离子脂质、甾醇、中性磷脂、PEG化脂质和权利要求1或2所述的式I所示化合物、或其盐溶于有机溶剂中,作为有机相;
2)将核酸溶于水溶液中,得到水相;
3)将有机相和水相混合,自组装形成脂质纳米颗粒。
17.根据权利要求16所述的制备方法,其特征在于:
步骤1)中,所述有机溶剂为乙醇、甲醇、DMSO或四氢呋喃;
和/或,步骤2)中,所述水相为柠檬酸缓冲液或醋酸盐缓冲液;
和/或,步骤3)中,所述有机相和水相的体积比为1:(1~5);
和/或,步骤3)中,所述有机相中的可电离阳离子脂质与水相中的核酸的氮磷摩尔比为(1~10):1;
和/或,步骤3)中,所述混合使用微流控混合设备混合;
优选地,
步骤2)中,所述柠檬酸缓冲液pH为3.0~6.0,浓度为5~20mM;
和/或,步骤3)中,所述有机相和水相的体积比为1:3;
和/或,步骤3)中,所述有机相中的可电离阳离子脂质与水相中的核酸的氮磷摩尔比为6:1;
和/或,步骤3)中,所述微流控混合的流速为9~20mL/min。
18.权利要求10~15任一项所述的核酸递送***在制备核酸药物或核酸疫苗中的用途;
优选地,所述核酸药物为mRNA药物;所述核酸疫苗为mRNA疫苗。
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