CN116514672A - 一种用于核酸递送的新型可电离脂质及其lnp组合物和疫苗 - Google Patents
一种用于核酸递送的新型可电离脂质及其lnp组合物和疫苗 Download PDFInfo
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- CN116514672A CN116514672A CN202310045868.2A CN202310045868A CN116514672A CN 116514672 A CN116514672 A CN 116514672A CN 202310045868 A CN202310045868 A CN 202310045868A CN 116514672 A CN116514672 A CN 116514672A
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Abstract
本发明提供了一种新型阳离子脂质和脂质纳米颗粒及核酸疫苗。本发明选择特定的阳离子脂质制备的脂质纳米颗粒mRNA疫苗,发现其较现有技术中的阳离子脂质制备的LNP具有更好的体外稳定性并能激发更强的免疫反应。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种用于核酸递送的新型可电离脂质及其LNP组合物和疫苗。
背景技术
目前递送mRNA在临床上经过验证的系统为脂质纳米粒(Lipid Nanoparticle,LNP),属于脂质形成的纳米微粒,其中原理包括阳离子脂质,而现有技术研究表明,mRNA递送至细胞内后,mRNA表达率偏低,例如Dlin-MC3-DMA作为阳离子脂质构建LNP,mRNA的表达量为0.63%(Maugeri,Marco et al.“Linkage between endosomal escape of LNP-mRNAand loading into EVs for transport to other cells.”Nature Communications,2019),因此,阳离子脂质的结构是影响mRNA表达量的关键因素,对于阳离子脂质的结构需要进一步的优化。
水痘-水痘-带状疱疹病毒(varicella-zoster virus,VZV)会导致两种不同的疾病:水痘和带状疱疹。带状疱疹疫苗针对已经感染过VZV病毒、对水痘具有免疫力,但体内潜伏着的VZV病毒的人群。因此,相比于针对VZV病毒易感人群的水痘疫苗,带状疱疹疫苗的功能类似于治疗性疫苗,需要引起更强的免疫反应以防止潜伏VZV病毒的重新激活。
目前上市的两种带状疱疹疫苗为减毒活疫苗ZOSTAVAX和亚单位疫苗SHINGRIX。ZOSTAVAX为通过低温传代获得的减毒Oka株,冻干形式在-15℃到-50℃保存;FDA批准的目标人群为50岁及以上的成年人,但美国免疫接种咨询委员会推荐60岁及以上的人群使用,因为该疫苗保护力最多持续8年。SHINGRIX的组分包括VZV病毒的GE蛋白胞外区和AS01B佐剂,以GE蛋白冻干、佐剂液体的形式在2℃到8℃保存;FDA批准的目标人群为50岁及以上的成年人和免疫缺陷、抑制的18岁以上的成年人,但美国免疫接种咨询委员会推荐50岁及以上的人群和ZOSTAVAX免疫8周及以上的人群使用;该疫苗保护力可以持续10年或以上(目前研究到10年)。除上述两款疫苗外,目前国内的带疱疫苗临床研发进展如下表所示。
表1国内带状疱疹疫苗临床研发进展表
虽然VZV病毒重新激活的原理现在并不清楚,但已知VZV特异性细胞免疫是限制病毒重新激活和复制的关键。分泌IFNγ的T细胞频数目前被认为是考察带状疱疹疫苗保护作用的最佳替代性指标,而关于特异性抗体应答水平与保护作用相关性的争议性则较大。
GE蛋白是VZV病毒当中可引起CD4+T细胞反应的主要蛋白;而AS01B佐剂当中的QS-21组分也是一种促进CD4+T细胞反应的天然皂苷。但是佐剂本身具有一定毒性。
发明内容
本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。
本发明术语“聚乙二醇(PEG)-脂质缀合物”是指包含脂质部分和聚乙二醇部分的分子。
本发明术语“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒,其包含至少一种脂质。
本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物,在施用后诱导免疫应答,其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。
本发明术语“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。
本发明术语,N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。
本发明术语“烃基”是指相应的烃失去一个氢原子后剩余的基团,在本发明中特别指脂烃基,例如烷基、烯基、炔基,特别是烷基。
本发明涉及一种阳离子脂质具有如下式I结构:
其中:
L1和L2至少一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-或-NRa-,
并且,
L1或L2中的另一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-、-NRa-、-O(C=O)-、-(C
=O)O-、-C(=O)-、-S(O)x-、-S-S-、-C(=O)S-、-SC(=O)-、-NRaC(=O)NRa-、-OC(=O)NRa-或-NRaC(=O)O-;
G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基;
G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基;
Ra为H或C1-C12烃基;
R1和R2各自独立地为C6-C24烷基或C6-C24烯基;
R3为H、OH、OR4、CN、-C(=O)OR4、-OC(=O)R4或–NR5C(=O)R4;
R4为C1-C12烃基;
R5为H或C1-C6烃基;
x为0、1或2。
具体地,其中的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C=O)O-、-(C=O)NH-、-NH(C=O)-和-NH-。
具体地,其中的阳离子脂质式I结构中,L1和L2均为-O-,或者,L1和L2均为-O(C=O)O-,或者,L1和L2均为-NH-,或者,L1为-NH(C=O)-,L2为-(C=O)NH-。
具体地,其中的阳离子脂质其有以下结构(IA):
其中:
R6在每次出现时独立地为H、OH或C1-C24烃基;
n为1至15的整数。
具体地,其中的阳离子脂质其有以下结构(IB):
其中y和z各自独立地为1至12的整数。
具体地,其中的阳离子脂质结构中n为2至12的整数,优选的,n为2、3、4、5或6;其中y和z各自独立地为2至10的整数,优选的,为4至9的整数。
具体地,其中的阳离子脂质结构中R1和R2各自独立地具有以下结构:
其中:
R7a和R7b在每次出现时独立地为H或C1-C12烃基;并且a为2至12的整数,优选的,a为8至12的整数;
其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。
具体地,其中的阳离子脂质结构中至少一次出现的R7a为H,优选的,R7a在每次出现时为H。
具体地,其中的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基;优选的,其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。
具体地,其中的阳离子脂质结构中R1或R2或两者具有以下结构之一:
具体地,其中的阳离子脂质化合物下结构如下:
本发明提供一种脂质纳米颗粒,包含:上述的阳离子脂质、非-阳离子脂质和/或聚乙二醇(PEG)-脂质缀合物,优选地,包含:阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。
具体地,所述的聚乙二醇(PEG)-脂质缀合物选自:2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合,优选的为DMG-PEG2000。
具体地,所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合,优选的为DSPC。
具体地,所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇;羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合,优选的为胆固醇。
具体地,所述的阳离子脂质在脂质组分中的摩尔百分含量为20~60%、中性磷脂在脂质组分中的摩尔百分含量为5%~25%、甾族脂质在脂质组分中的摩尔百分含量为25%~55%;聚乙二醇(PEG)-脂质缀合物在脂质组分中的摩尔百分含量为0.5%~15%。
具体地,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10,优选的,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为40-60:10-20:30-50:1-5,更优选的,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为45:10:43:2或40:10:48:2。
具体地,所述疫苗中还包含其他辅料,所述辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。
具体地,所述纳米颗粒的平均粒径为50~200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。
本发明提供一种脂质纳米颗粒的制备方法,包括将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合的步骤。
具体地,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得;优选的,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得;优选的,所述的超滤方式为切向流过滤;更优选的,所述的混合方式可为湍流混合、层流混合或微流体混合。
具体地,稀释液为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。
具体地,所述缓冲液pH为3~6,浓度为6.25~200mM。
具体地,将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1~5:1。
具体地,采用脂质包封mRNA时的N/P为2-10,优选的N/P为3-8,更优选的,N/P为3、4、5、6、7、8,所述N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。
具体地,所述的超滤液选自由以下组成的组:钠盐和三(羟甲基)氨基甲烷(Tris)盐,优选的,超滤液pH为6.5~8.5。
具体地,疫苗的剂型为口服制剂、肌肉注射制剂、静脉注射制剂、吸入制剂、液体制剂、冻干粉剂、雾化吸入剂或干粉吸入剂。
本发明提供一种水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗,包含:编码水痘-带状疱疹病毒GE蛋白的mRNA;所述mRNA被所述的脂质纳米颗粒包裹。
具体的,其中所述mRNA编码GE蛋白的氨基酸序列为SEQ ID NO:1所示序列,或者与SEQ ID NO:1所示序列具有80%或以上同一性的氨基酸序列,优选具有85%、90%、95%、96%、97%、98%、99%以上或100%同一性的氨基酸序列。
本发明提供一种水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗在制备用于预防水痘-带状疱疹病毒感染的预防性药物中的应用。
本发明的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗包含编码水痘-带状疱疹病毒GE蛋白的mRNA、阳离子脂质、非-阳离子脂质及聚乙二醇(PEG)-脂质缀合物。本发明选择特定的阳离子脂质并与非-阳离子脂质及聚乙二醇(PEG)-脂质组合制备脂质纳米颗粒,实验发现具有良好的体外稳定性并能激发更强的免疫反应。
本发明相比现有技术的有益效果为:
1、采用本发明所述的阳离子脂质制备的脂质纳米颗粒,其包封率显著优于已上市的阳离子脂质;
2、采用本发明所述的脂质纳米颗粒制得的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗,其引起的体液免疫反应及细胞免疫反应显著优于已上市的阳离子脂质;
3、本发明所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗可有效促进抗原提呈细胞吞噬和高效递送抗原,并实现疫苗的缓释持续刺激机体产生针对VZV-gE特异的细胞免疫应;
4、本发明所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗与已上市的带状疱疹疫苗SHINGRIX相比,除了可诱导CD8+4细胞反应外,还可显著诱导CD8+T细胞反应。
附图说明
图1所示为BALB/c小鼠免疫程序。
图2不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗检测结果。
图3血清IgG抗体滴度(Log值)。
图4所示为BALB/c小鼠模型上ICS法检测IFNγ分泌T细胞频数。
图5所示为BALB/c小鼠模型上ELISPOT法检测IFNγ分泌T细胞频数。
图6所示为C57BL/6小鼠免疫程序。
图7所示为C57BL/6小鼠模型上ELISA法检测gE特异性IgG滴度。
图8所示为C57BL/6小鼠模型上ICS法检测IFNγ分泌T细胞频数。
图9所示为C57BL/6小鼠模型上ELISPOT法检测IFNγ分泌T细胞频数。
图10所示为C57BL/6小鼠模型上ICS法检测特异性分泌TNFα、IFNγ、IL-2、IL-4和IL-5的CD4+T细胞频数。
图11所示为C57BL/6小鼠模型上ICS法检测特异性分泌TNFα、IFNγ、IL-2、IL-4和IL-5的CD8+T细胞频数。
具体实施方式
下面将结合本发明的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
化合物1的合成
6-溴己基(2-己基癸基)碳酸酯(1a)的合成
将6-溴正己醇(0.91g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(0.90g,7.5mmol),再分批次加入对硝基氯甲酸苯酯(1.20g,6.0mmol),反应室温搅拌3h,在此反应液中加入2-己基癸醇(1.36g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到6-溴己基(2-己基癸基)碳酸酯1a(1.53g,淡黄色油状物),产率68%。
MS m/z(ESI):449.3[M+1]
化合物1的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物1(454mg,淡黄色油状物),产率55%。
MS m/z(ESI):826.9[M+1]
1H NMR(300MHz,CDCl3):δ4.13(t,4H,J=6.6Hz),4.05(d,4H,J=5.7Hz),3.56-3.55(m,2H),2.47-2.42(m,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J=6.2Hz)
实施例2
化合物2的合成
7-溴庚基十七烷-9-基碳酸酯(2a)的合成
将7-溴庚醇(0.98g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(1.22g,10mmol),再分批次加入对硝基氯甲酸苯酯(1.11g,5.5mmol),反应室温搅拌3h,在此反应液中加入9-羟基十七醇(1.44g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到7-溴庚基十七烷-9-基碳酸酯2a(1.50g,淡黄色油状物),产率65%。
MS m/z(ESI):477.3[M+1]
十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(2b)的合成
室温条件下,将7-溴庚基十七烷-9-基碳酸酯(2a)(1.38g,3mmol)溶于20mL乙醇中,加入乙醇胺(2.75g,45mmol),升温至50℃,搅拌8h,监控反应进程,原料消耗完全后降温至45℃旋干除去乙醇,用二氯甲烷溶解粗品,用饱和食盐水洗涤三次,有机相用无水硫酸钠干燥,浓缩得到产品十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯2b(1.35g,淡黄色油状物)。
MS m/z(ESI):458.4[M+1]
5-溴戊基十一烷基碳酸酯(2c)的合成
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将5-溴戊醇(0.84g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(1.22g,10mmol),再分批次加入对硝基氯甲酸苯酯(1.11g,5.5mmol),反应室温搅拌3h,在此反应液中加入十一醇(0.97g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到5-溴戊基十一烷基碳酸酯2c(1.20g,淡黄色油状物),产率66%。
MS m/z(ESI):365.2[M+1]
化合物2的合成
将十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(457mg,1.0mmol)溶于四氢呋喃中,加入乙腈,5-溴戊基十一烷基碳酸酯(437mg,1.2mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物2(440mg,淡黄色油状物),产率57%。
MS m/z(ESI):742.8[M+1]
1H NMR(300MHz,CDCl3):δ4.71-4.68(m,1H),4.15-4.10(m,6H),3.53(t,2H,J=5.4Hz),2.94(br,1H),2.58(t,2H,J=5.4Hz),2.45(t,4H,J=5.7Hz),1.75-1.34(m,62H),0.90(t,9H,J=6.3Hz)
实施例3
化合物3的合成
6-溴己基十一烷基碳酸酯(3a)的合成
将6-溴正己醇(0.91g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(0.90g,7.5mmol),再分批次加入对硝基氯甲酸苯酯(1.20g,6.0mmol),反应室温搅拌3h,在此反应液中加入十一醇(0.97g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到6-溴己基十一烷基碳酸酯3a(1.25g,淡黄色油状物),产率66%。
MS m/z(ESI):379.2[M+1]
化合物3的合成
将6-溴己基十一烷基碳酸酯(948mg,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物3(412mg,淡黄色油状物),产率60%。
MS m/z(ESI):686.8[M+1]
1H NMR(300MHz,CDCl3):δ4.13(t,8H,J=6.6Hz),3.58(t,2H,J=5.7Hz),2.52(t,6H,J=8.4Hz),1.74-1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J=6.2Hz)
实施例4
化合物4的合成
6-溴己基十七烷-9-基碳酸酯(4a)的合成
将6-溴正己醇(0.91g,5.0mmol)溶于30mL二氯甲烷中,加入4-二甲氨基吡啶(0.90g,7.5mmol),再分批次加入对硝基氯甲酸苯酯(1.20g,6.0mmol),反应室温搅拌3h,在此反应液中加入9-十七醇(1.44g,5.6mmol),混合物在室温下搅拌过夜,TLC显示反应完成后,加入20mL二氯甲烷稀释,然后用30mL饱和食盐水洗涤,有机相用无水硫酸钠干燥,过滤并浓缩,柱层析分离得到6-溴己基十七烷-9-基碳酸酯4a(1.53g,淡黄色油状物),产率66%。
MS m/z(ESI):464.3[M+1]
化合物4的合成
将6-溴己基十七烷-9-基碳酸酯(1.16g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物4(502mg,淡黄色油状物),产率59%。
MS m/z(ESI):855.4[M+1]
1H NMR(300MHz,CDCl3):δ4.71-4.68(m,2H),4.13(t,4H,J=6.6Hz),3.57(t,2H,J=5.4Hz),2.49-2.44(m,6H),1.74-1.28(m,76H),0.90(t,12H,J=6.3Hz)
实施例5
化合物5的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,乙醇胺(61.0mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物5(487mg,淡黄色油状物),产率61%。
MS m/z(ESI):798.9[M+1]
1H NMR(300MHz,CDCl3):δ4.14(t,4H,J=6.6Hz),4.04(d,4H,J=5.7Hz),3.54(t,2H,J=5.4Hz),2.58(t,2H,J=5.4Hz),2.46(t,4H,J=7.2Hz),1.72-1.65(m,6H),1.49-1.28(m,61H),0.69(t,12H,J=6.2Hz)
实施例6
化合物6的合成
将5-溴戊基十一烷基碳酸酯(910mg,2.5mmol)溶于四氢呋喃中,加入乙腈,乙醇胺(61.0mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物6(410mg,淡黄色油状物),产率65%。
MS m/z(ESI):630.7[M+1]
1H NMR(300MHz,CDCl3):δ4.10(t,8H,J=6.6Hz),3.52(d,2H,J=5.4Hz),2.83(br,1H),2.57(t,2H,J=5.4Hz),2.45(t,4H,J=7.2Hz),1.73-1.62(m,8H),1.52-1.39(m,40H),0.69(t,6H,J=6.2Hz)
实施例7
化合物7的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,3-甲氧基丙胺(89mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物7(495mg,淡黄色油状物),产率60%。
MS m/z(ESI):826.7[M+1]
实施例8
化合物8的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,3-氨基丙腈(70mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物8(469mg,淡黄色油状物),产率58%。
MS m/z(ESI):807.7[M+1]
实施例9
化合物9的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基丁酸乙酯盐酸盐(167mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物9(546mg,淡黄色油状物),产率63%。
MS m/z(ESI):868.8[M+1]
实施例10
化合物10的合成
将6-溴己基(2-己基癸基)碳酸酯(1.12g,2.5mmol)溶于四氢呋喃中,加入乙腈,N-(4-氨基丁基)-乙酰胺盐酸盐(167mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物10(560mg,淡黄色油状物),产率69%。
MS m/z(ESI):867.8[M+1]
实施例11
化合物11的合成
8-溴-N-(十七烷-9-基)辛酰胺(11a)的合成
将8-溴辛酸(1.12g,5.0mmol)溶于50mL二氯甲烷中,在0℃下分批加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(1.05g,5.5mmol),搅拌30min后,在反应液中逐滴加入9-氨基十七烷(1.28g,5.0mmol),滴加完毕后,混合物在室温下搅拌过夜,TLC显示反应完成后,用100ml水洗涤2次,有机相用无水硫酸钠干燥,过滤并浓缩,得到化合物11a(1.95g,黄色油状物),产率82%。
MS m/z(ESI):461.3[M+1]。
化合物11b的合成
将8-溴-N-(十七烷-9-基)辛酰胺(1.15g,2.5mmol)溶于四氢呋喃中,加入乙腈,4-氨基-1-丁醇(89.2mg,1.0mmol),碳酸钾(550mg,4.0mmol),碘化钾(332mg,2.0mmol),在83℃下搅拌16-20h。冷却至室温,过滤,滤渣用二氯甲烷洗涤,得到的滤液中加入饱和碳酸氢钠溶液,用二氯甲烷萃取2次,合并有机相,经无水硫酸钠干燥,过滤并浓缩,柱层析分离,得到产物11b(534mg,淡黄色油状物),产率63%。
MS m/z(ESI):848.8[M+1];
1H NMR(300MHz,CDCl3):δ8.10(s,2H),4.21(s,1H),3.46-3.4(m,4H),3.02(t,6H,J=6.2Hz),2.14(t,4H,J=4.8Hz),1.57-1.47(t,14H,J=6.3Hz),1.36-1.26(m,66H),0.90(t,12H,J=6.3Hz)。
化合物11的合成
在0℃下,将化合物11b(1.70g,2mmol)缓慢加入四氢铝锂(379mg,10mmol)的无水四氢呋喃(10ml)溶液中,混合物加热回流5小时。反应完全后,降温,在体系中加入水使过量的还原剂完全分解。过滤,滤渣用乙酸乙酯洗涤,得到的滤液用水洗涤,经无水硫酸钠干燥,过滤并浓缩,得到化合物11(1.45g,黄色油状物),产率90%。
MS m/z(ESI):820.8[M+1];
1H NMR(300MHz,CDCl3):δ4.11(s,1H),3.44(t,2H,J=4.8Hz),3.32(s,2H),3.00(t,6H,J=6.3Hz),2.52(t,4H,J=6.3Hz),2.48-2.43(m,2H),1.61-1.56(m,2H),1.36-1.26(m,82H),0.86(t,12H,J=4.8Hz)。
实施例12脂质纳米颗粒包裹mRNA抗原
本发明分别使用阳离子脂质I~ⅩⅣ制备脂质纳米颗粒核酸疫苗,14种阳离子脂质结构如下表。
表2阳离子脂质结构式
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100mM醋酸钠缓冲液(pH 4.0)稀释水痘-带状疱疹病毒mRNA疫苗原液至浓度为150μg/ml,该疫苗原液含有编码水痘-带状疱疹病毒GE蛋白的mRNA抗原的氨基酸序列,抗原序列如SEQID NO:1。按照阳离子脂质:DSPC:胆固醇:DMG-PEG2000摩尔比为45:10:43:2配制脂质混合溶液;设定纳米药物制造设备总流速12ml/min、mRNA溶液与脂质混合溶液流速比3:1并开始包封,包封完成后,切向流过滤系统超滤换液收集样品,并加入蔗糖溶液。在N/P(可电离的阳离子脂质与核苷酸磷酸盐)摩尔比不同条件下进行试验(N/P摩尔比分别为3、6、9)。取样检测包封率(图2)、平均粒径、PDI及Zeta电位,结果如下表3。
表3不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗检测结果
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由以上结果可以看出,在相同N/P条件下,阳离子脂质I、II、VI-XIV所制得的脂质纳米颗粒mRNA疫苗的包封率均高于阳离子脂质III、IV、V。阳离子脂质III的包封率略高于IV、V。
实施例13带状疱疹脂质纳米颗粒mRNA疫苗体液免疫评价
将实施例12制备的样品1~14(A、B、C)分别在BALB/c小鼠模型上的进行体液免疫的评价,设置不同N/P(3、6、9)对脂质纳米颗粒mRNA疫苗的免疫原性影响。
如图1所示,BALB/c小鼠在第0天和14天免疫5μg的mRNA-LNP。在第28天采血进行抗体滴度检测,检测结果如下表4及图3所示。
表4不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗抗体滴度
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由上表的抗体滴度检测结果可以看出,阳离子脂质I、II、VI-XIV所制得的脂质纳米颗粒mRNA疫苗的滴度高于阳离子脂质III、IV、V。阳离子脂质III略高于IV、V。
实施例14带状疱疹脂质纳米颗粒mRNA疫苗小鼠免疫与检测
1、在BALB/c小鼠模型上的细胞免疫反应评价
将实施例12制备的样品B-1、B-2、B-3、B-4(编号为mRNA-LNP1、mRNA-LNP2、mRNA-LNP3、mRNA-LNP4)分别在BALB/c小鼠模型上的进行细胞免疫反应的评价。
如图1所示,BALB/c小鼠在第0天和14天免疫5μg的mRNA-LNP。在第28天,处死小鼠并收获脾细胞并使用VZV gE抗原的重叠肽库进行刺激。通过细胞内细胞因子染色流式细胞术(ICS)方法和酶联免疫斑点(ELISpot)方法测量产生IFN的细胞。
IFN分泌T细胞频数是目前公认的带状疱疹疫苗保护作用的最佳替代性指标。如图4和图5所示,两种检测方法的结果一致,使用本专利配方的mRNA疫苗引发的细胞免疫反应可以产生较高的IFN分泌T细胞频数。
综上,本发明制备的mRNA疫苗显示出较好的用于预防带状疱疹的潜力,且阳离子脂质I、II所制得的脂质纳米颗粒mRNA疫苗的细胞免疫反应较阳离子脂质III、IV更好。
2、在C57BL/6小鼠模型上对比阳性疫苗的免疫反应评价
mRNA-LNP1为用含阳离子脂质I的配方(B-1)制备的mRNA疫苗;mRNA-LNP2为用含阳离子脂质II的配方(B-2)制备的mRNA疫苗;SHINGRIX为阳性市售亚单位疫苗(水痘-带状疱疹病毒糖蛋白E及AS01B佐剂)。如图6所示,C57BL/6小鼠在第0天和第30天免疫5免疫的mRNA-LNP或5RN的SHINGRIX。在第44天,处死小鼠并收获脾细胞用于通过ICS方法和ELISpot方法评估细胞免疫反应。在第30天和第44天收集血清用于检测gE特异性IgG抗体滴度。
如图7所示,在加强注射后,mRNA疫苗和SHINGRIX之间的gE特异性IgG滴度具有可比性。如图8和图9所示,两种检测方法的结果一致,mRNA疫苗诱导的产生IFN导的细胞的百分比显着高于SHINGRIX诱导的细胞百分比。如图10和图11所示,mRNA疫苗和SHINGRIX都诱导了Th1偏向反应。SHINGRIX如公开数据显示,只能激活CD4+T细胞;而mRNA疫苗除可以激活CD4+T细胞外,还可以诱导CD8+T细胞反应。
AS01佐剂是一种包含免疫刺激剂单磷酸酰脂质A(MPL)和皂树皂苷QS-21的脂质体佐剂,能够激发细胞免疫和体液免疫。Shingrix高保护率得益于添加了AS01佐剂,AS01佐剂虽然很大程度提升了疫苗的有效性,但同时也增加了疫苗不良反应比例。本发明mRNA疫苗不含佐剂,具有显著优势。
综上,本发明制备的mRNA疫苗显示出比市售阳性疫苗更好的用于预防带状疱疹的潜力。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (29)
1.一种阳离子脂质,其特征在于,所述的阳离子脂质具有如下式I结构:
其中:
L1和L2至少一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-或-NRa-,
并且,
L1或L2中的另一个为-O-、-O(C=O)O-、-(C=O)NRa-、-NRa(C=O)-、-NRa-、-O(C=O)-、-(C=O)O-、-C(=O)-、-S(O)x-、-S-S-、-C(=O)S-、-SC(=O)-、-NRaC(=O)NRa-、-OC(=O)NRa-或-NRaC(=O)O-;
G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基;
G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基;
Ra为H或C1-C12烃基;
R1和R2各自独立地为C6-C24烷基或C6-C24烯基;
R3为H、OH、OR4、CN、-C(=O)OR4、-OC(=O)R4或–NR5C(=O)R4;
R4为C1-C12烃基;
R5为H或C1-C6烃基;
x为0、1或2。
2.根据权利要求1所述的阳离子脂质,其特征在于,所述的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C=O)O-、-(C=O)NH-、-NH(C=O)-和-NH-。
3.根据权利要求1-2任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质式I结构中所述L1和L2均为-O-,或者,L1和L2均为-O(C=O)O-,或者,L1和L2均为-NH-,或者,L1为-NH(C=O)-,L2为-(C=O)NH-。
4.根据权利要求1-3任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质其有以下结构(IA):
其中:
R6在每次出现时独立地为H、OH或C1-C24烃基;
n为1至15的整数。
5.根据权利要求1-4任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质其有以下结构(IB):
其中y和z各自独立地为1至12的整数。
6.根据权利要求1-5任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中n为2至12的整数,优选的,n为2、3、4、5或6;其中y和z各自独立地为2至10的整数,优选的,为4至9的整数。
7.根据权利要求1-6任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中R1和R2各自独立地具有以下结构:
其中:
R7a和R7b在每次出现时独立地为H或C1-C12烃基;并且a为2至12的整数,优选的,a为8至12的整数;
其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。
8.根据权利要求1-7任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中至少一次出现的R7a为H,优选的,R7a在每次出现时为H。
9.根据权利要求1-8任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基;优选的,其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。
10.根据权利要求1-9任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质结构中R1或R2或两者具有以下结构之一:
11.根据权利要求1-10任一项所述的阳离子脂质,其特征在于,所述的阳离子脂质化合物下结构如下:
12.一种脂质纳米颗粒,其特征在于,包含:如权利要求1-11任一项所述的阳离子脂质、非-阳离子脂质和/或聚乙二醇(PEG)-脂质缀合物,优选地,包含:阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。
13.根据权利要求12所述的脂质纳米颗粒,其特征在于,所述的聚乙二醇(PEG)-脂质缀合物选自:2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合,优选的为DMG-PEG2000。
14.根据权利要求12-13任一项所述的脂质纳米颗粒,其特征在于,所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合,优选的为DSPC。
15.根据权利要求12-14任一项所述的脂质纳米颗粒,其特征在于,所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇;羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合,优选的为胆固醇。
16.根据权利要求12-15任一项所述的脂质纳米颗粒,其特征在于,所述的阳离子脂质在脂质组分中的摩尔百分含量为20~60%、中性磷脂在脂质组分中的摩尔百分含量为5%~25%、甾族脂质在脂质组分中的摩尔百分含量为25%~55%;聚乙二醇(PEG)-脂质缀合物在脂质组分中的摩尔百分含量为0.5%~15%。
17.根据权利要求12-16任一项所述的脂质纳米颗粒,其特征在于,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10,优选的,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为40-60:10-20:30-50:1-5,更优选的,所述阳离子脂质:中性磷脂:甾族脂质:聚乙二醇(PEG)-脂质缀合物摩尔比为45:10:43:2或40:10:48:2。
18.根据权利要求12-17任一项所述的脂质纳米颗粒,其特征在于,所述疫苗中还包含其他辅料,所述辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。
19.根据权利要求12-18任一项所述的脂质纳米颗粒,其特征在于,所述纳米颗粒的平均粒径为50~200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。
20.一种根据权利要求1-19任一项所述的脂质纳米颗粒的制备方法,其特征在于,包括将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合的步骤。
21.根据权利要求20所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗的制备方法,其特征在于,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得;优选的,将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得;优选的,所述的超滤方式为切向流过滤;更优选的,所述的混合方式可为湍流混合、层流混合或微流体混合。
22.根据权利要求20-21任一项所述的脂质纳米颗粒的制备方法,其特征在于,稀释液为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。
23.根据权利要求20-22任一项所述的脂质纳米颗粒的制备方法,其特征在于,所述缓冲液pH为3~6,浓度为6.25~200mM。
24.根据权利要求20-23任一项所述的脂质纳米颗粒的制备方法,其特征在于,将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1~5:1。
25.根据权利要求20-24任一项所述的脂质纳米颗粒的制备方法,其特征在于,采用脂质包封mRNA时的N/P为2-10,优选的N/P为3-8,更优选的,N/P为3、4、5、6、7、8,所述N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。
26.根据权利要求20-25任一项所述的脂质纳米颗粒的制备方法,其特征在于,所述的超滤液选自由以下组成的组:钠盐和三(羟甲基)氨基甲烷(Tris)盐,优选的,超滤液pH为6.5~8.5。
27.根据权利要求20-26任一项所述的脂质纳米颗粒的制备方法,其特征在于,疫苗的剂型为口服制剂、肌肉注射制剂、静脉注射制剂、吸入制剂、液体制剂、冻干粉剂、雾化吸入剂或干粉吸入剂。
28.一种水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗,其特征在于,包含:编码水痘-带状疱疹病毒GE蛋白的mRNA;所述mRNA被权利要求12-27任一项所述的脂质纳米颗粒包裹或被ALC-0315制备的脂质纳米颗粒包裹。
29.一种权利要求1-19、28任一项所述的水痘-带状疱疹病毒脂质纳米颗粒mRNA疫苗在制备用于预防水痘-带状疱疹病毒感染的预防性药物中的应用。
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