CN117384052A - 一种线粒体靶向的氟化可电离脂质及其作为线粒体基因递送载体的用途 - Google Patents
一种线粒体靶向的氟化可电离脂质及其作为线粒体基因递送载体的用途 Download PDFInfo
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- CN117384052A CN117384052A CN202310980694.9A CN202310980694A CN117384052A CN 117384052 A CN117384052 A CN 117384052A CN 202310980694 A CN202310980694 A CN 202310980694A CN 117384052 A CN117384052 A CN 117384052A
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Abstract
本发明公开了一种线粒体靶向的氟化可电离脂质及其作为线粒体基因递送载体的用途。一种线粒体靶向的氟化可电离脂质,其为式(I)化合物,或其药学上可接受的盐、立体异构体、互变异构体,其中:a=2~8的整数,b=4~8的整数;R1为可电离脂质头部基团,含有伯胺或叔胺结构的一元或多元胺。一种线粒体靶向的脂质载体,所述载体包括本发明所述氟化可电离脂质、阳离子脂质、辅助脂质、结构性脂质、含线粒体靶向基团修饰的功能性聚合物共轭脂质。本发明提供的具有线粒体靶向能力的氟化可电离脂质基因递送载体生物相容性良好,能够同时增加基因药物的细胞摄取及线粒体蓄积,提高线粒体基因转染效率,实现基因药物线粒体基质的高效递送。
Description
技术领域
本发明属于生物制药技术领域,具体涉及一种线粒体靶向的氟化可电离脂质,及其作为线粒体基因递送载体的应用。
背景技术
针对Leber遗传性视神经病变(LHON)等线粒体基因突变疾病的基因疗法主要分为基于病毒载体的异位表达和基于非病毒载体的原位线粒体基因治疗。目前病毒载体介导的基因治疗在线粒体相关疾病临床研究中已取得一定进展,但其存在的潜在安全问题和免疫原性风险限制了病毒载体介导的基因治疗在该领域的广泛应用。原位线粒体基因治疗可将治疗基因直接递送至线粒体基质,因此,非病毒载体介导的原位线粒体基因治疗可能是克服现有LHON基因治疗瓶颈的一种有效策略。然而,复杂的线粒体双层膜结构增加了原位基因递送的难度,导致现有的线粒体基因递送体系普遍存在基因转染效率有限的问题。因此,为了实现高效低毒的线粒体基因治疗,开发一种新型非病毒线粒体基因递送载体用于LHON等线粒体基因突变相关疾病的治疗是至关重要的。
发明内容
本发明提供一种具有线粒体靶向能力的氟化可电离脂质及其作为线粒体基因递送载体用于治疗由线粒体基因突变引起的疾病的用途。
为了解决上述技术问题,本发明采用的技术方案为:
一种线粒体靶向的氟化可电离脂质,其为式(I)化合物或其药学上可接受的盐、立体异构体、互变异构体,其中:a=2~8的整数,b=4~8的整数;R1为可电离脂质头部基团,含有伯胺或叔胺结构的一元或多元胺。
优选地,所述化合物具有以下式(I-1)、式(I-2)、式(I-3)所示结构中的一种:
R1选自以下结构式中的任意一种,其中波浪线表示与相邻结构共价连接:
在一种实施方案中,R1为
一种线粒体靶向的氟化可电离脂质或其药学上可接受的盐、立体异构体、互变异构体,选自以下化合物:
本发明又一方面提供一种线粒体靶向的脂质载体,所述载体包括本发明所述氟化可电离脂质、阳离子脂质、辅助脂质、结构性脂质、含线粒体靶向基团修饰的功能性聚合物共轭脂质。
在一种实施方案中,所述氟化可电离脂质占载体的摩尔比为12.5%~50%。
在一种实施方案中,所述阳离子脂质选自以下中的一种或多种:氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)、溴化三甲基-2,3-二油酰氧基丙基铵(DOTAP)、O-[(N,N-二甲基氨基乙基)-氨基甲酰基]胆固醇盐酸盐(DC-Chol)、4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯(DLin-MC3-DMA)、十七烷-9-基-8-((2-羟乙基)(6-氧代-6-((癸氧基)己基)氨基)辛酸酯)(SM-102)、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)(ALC-0315)。在一种实施方案中,所述阳离子脂质为ALC-0315,所述阳离子脂质占载体的摩尔比为12.5%~50%。
在一种实施方案中,所述辅助脂质选自以下中的一种或多种:二油酰磷脂酰乙醇胺(DOPE)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、磷脂酸(PA)、磷脂酰丝氨酸(PS)、鞘磷脂(SM)。在一种实施方案中,所述辅助脂质为DSPC。
在一种实施方案中,所述结构性脂质选自以下中的一种或多种:胆固醇、非甾醇、谷固醇、麦角固醇、α-生育酚、皮质类固醇。在一种实施方案中,所述结构性脂质是胆固醇。
在一种实施方案中,所述载体还包含含线粒体靶向基团的聚合物共轭脂质。所述聚合物共轭脂质选自以下中的一种或多种:二硬脂酰基磷脂酰乙醇胺聚乙二醇2000-马来酰亚胺(DSPE-PEG2000-MAL)、二硬脂酰基磷脂酰乙醇胺聚乙二醇2000(DSPE-PEG2000)、二肉豆蔻酰甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)和甲氧基聚乙二醇双十四烷基乙酰胺(ALC-0159);所述线粒体靶向基团选自线粒体靶向序列(MTS)MLSLRQSIRFFKC、MLRAALSTARRGPRLSRLLC、MVSGSSGLAAARL LSRTFLLQQNGIRHGSYC中的一种。在一种实施方案中,所述MTS为MLSLRQSIRFFK C,其通过末端巯基与聚合物共轭脂质马来酰亚胺基团反应接枝在载体表面;所述含线粒体靶向基团的聚合物共轭脂质占载体的摩尔比为0.5%~1.5%。
作为本发明的一种优选实施方式,在所述脂质载体中,所述氟化可电离脂质、阳离子脂质、辅助脂质、结构性脂质、含线粒体靶向基团修饰的功能性聚合物共轭脂质的摩尔比为(10~50):(10~40):(5~25):(10~55):(0.3~5)。
本发明又一方面提供一种用于线粒体基因递送的组合物,所述组合物包括本发明所述的氟化可电离脂质化合物或其药学上可接受的盐、立体异构体、互变异构体或本发明所述的脂质载体,以及功能性基因。
在一种实施方案中,所述线粒体基因递送组合物中功能基因选自以下中的一种或多种:人NADH泛醌氧化还原酶亚基4(ND4)、人NADH泛醌氧化还原酶亚基6(ND6)和人NADH泛醌氧化还原酶亚基1(ND1)。在一种实施方案中,所述功能基因为ND4。
在一种实施方案中,所述功能性基因位于质粒DNA载体中,除编码功能基因的核苷酸序列外,还编码Flag表位标签序列和荧光素酶序列,用于检测线粒体转染效率。
在一种实施方案中,所述线粒体基因递送组合物为纳米颗粒制剂,所述纳米颗粒制剂的平均粒径为50nm~500nm,优选100nm~200nm;所述纳米颗粒制剂的多分散系数(PDI)小于50%,优选小于30%。
本发明又一方面提供上述线粒体基因递送组合物用于治疗由线粒体基因突变引起的疾病。
例如,所述疾病选自以下中的一种或多种:LHON、线粒体脑肌病伴乳酸酸中毒及卒中样发作综合征(MELAS)、肌阵挛性癫痫和不规整红纤维病(MERRF)。优选LHON。
本发明的有益效果:
本发明提供的具有线粒体靶向能力的氟化可电离脂质基因递送载体生物相容性良好,并且载体中功能性氟化可电离脂质和MTS能够同时增加基因药物的细胞摄取及线粒体蓄积,提高线粒体基因转染效率,实现基因药物线粒体基质的高效递送。通过负载功能性基因,可以用于治疗由不同线粒体基因突变引起的疾病,如LHON。
附图说明
图1为本发明实施例8中不同氟化载体不同浓度下细胞存活情况示意图。
图2为本发明实施例9中不同氟化载体体外细胞摄取情况示意图。
图3为本发明实施例10中不同氟化载体体外线粒体转染情况示意图。
图4为本发明实施例11中不同载体体外线粒体摄取情况示意图。
图5为本发明实施例11中不同载体与线粒体共定位系数图。
图6为本发明实施例12中不同载体体外目的蛋白表达情况示意图。
图7为本发明实施例13中线粒体靶向最佳氟化载体制剂体内目的蛋白表达情况示意图。
具体实施方式
下面结合附图和具体实施例对本发明作更进一步的说明。
实施例1:氟化可电离脂质化合物1(4F)的合成
合成路线如下:
化合物1a的合成:将连二亚硫酸钠(3.14g,17.8mmol)缓慢加入到八氟-1,4-二碘丁烷(4.10g,9.0mmol)、苯甲酸乙烯酯(1.25mL,9.0mmol)、1-己烯(1.41mL,9.0mmol)和NaHCO3(1.91g,18.0mmol)的CH3CN/H2O(8:7,v/v,18.4mL)的混合物中。反应液于0℃下搅拌50min,反应结束后,用HCl 3N将pH调至5~7,继续于室温搅拌20min。经CH2Cl2萃取后有机相用MgSO4干燥。采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(95:5,v/v),得到化合物1a(1.42g,22%),为微黄色蜡状固体。
化合物1b的合成:将化合物1a(1.42g,2.0mmol)和AIBN(34.0mg,0.20mmol)溶于乙醚(50mL)中,加入三丁基氢化锡(2.65mL,10.0mmol),将反应混合物在卤素灯下回流15h。减压除去溶剂,采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(90:10,v/v),得到化合物1b(0.74g,80%),为白色蜡状固体。
化合物1c的合成:将化合物1b(0.69g,1.5mmol)在1M LiOH(4mL)甲醇中室温搅拌3h。真空干燥后将产物溶解于CH2Cl2中,分别用水和生理盐水洗涤,再将有机相用MgSO4干燥。采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(50:50,v/v),得到化合物1c(0.40g,75%),为白色蜡状固体。
化合物1d的合成:将化合物1c(0.39g,1.1mmol)溶于丙酮(40mL)和水(0.5mL)中。在室温下滴加琼斯试剂,直至得到持久的红棕色溶液。反应混合物搅拌1h后加入i-PrOH(2mL),继续搅拌15min。减压除去溶剂,加入水(5mL),用乙醚萃取。有机相分别用水和生理盐水洗涤,最后经MgSO4干燥。过滤,真空干燥得到化合物1d(0.37g,91%),为白色蜡状固体。
化合物1的合成:将化合物1d(0.37g,1.0mmol)、3-(二丙胺)丙烷-1,2-二醇(96mg,0.5mmol)、DMAP(13mg,0.1mmol)和DIC(126.2mg,1.0mmol)溶于CH2Cl2(150mL)中。室温搅拌24h后,过滤除沉淀,滤液经旋转蒸发干燥。采用硅胶柱层析法纯化,洗脱剂为石油醚/乙酸乙酯(3:1,v/v),得到化合物1(0.31g,71%)。1H NMR(600MHz,Chloroform-d):δ5.22(m,1H),4.48(m,1H),4.29(m,1H),3.14(m,2H),2.60(m,2H),2.39(m,4H),2.03(m,4H),1.31(m,30H),0.86(m,12H)。19F NMR(565MHz,Chloroform-d):δ-113.8(m,4F),-114.5(m,8F),-123.9(m,4F)。
实施例2:氟化可电离脂质化合物2(6F)的合成
合成路线如下:
化合物2a的合成:将连二亚硫酸钠(3.14g,17.8mmol)缓慢加入到十二氟-1,6-二碘己烷(5.00g,9.0mmol)、苯甲酸乙烯酯(1.25mL,9.0mmol)、1-己烯(1.12mL,9.0mmol)和NaHCO3(1.91g,18.0mmol)的CH3CN/H2O(8:7,v/v,18.4mL)的混合物中。反应液于0℃下搅拌50min,反应结束后,用HCl 3N将pH调至5~7,继续于室温搅拌20min。经CH2Cl2萃取后有机相用MgSO4干燥。采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(95:5,v/v),得到化合物2a(1.51g,20%),为微黄色蜡状固体。
化合物2b的合成:将化合物2a(1.5g,1.9mmol)和AIBN(31mg,0.19mmol)溶于乙醚(50mL)中,加入三丁基氢化锡(2.56mL,9.5mmol),将反应混合物在卤素灯下回流15h。减压除去溶剂,采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(90:10,v/v),得到化合物2b(0.84g,83%),为白色蜡状固体。
化合物2c的合成:将化合物2b(0.80g,1.5mmol)在1M LiOH(4mL)甲醇中室温搅拌3h。真空干燥后将产物溶解于CH2Cl2中,分别用水和生理盐水洗涤,再将有机相用MgSO4干燥。采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(50:50,v/v),得到化合物2c(0.52g,80%),为白色蜡状固体。
化合物2d的合成:将化合物2c(0.52g,1.2mmol)溶于丙酮(40mL)和水(0.5mL)中。在室温下滴加琼斯试剂,直至得到持久的红棕色溶液。反应混合物搅拌1h后加入i-PrOH(2mL),继续搅拌15min。减压除去溶剂,加入水(5mL),用乙醚萃取。有机相分别用水和生理盐水洗涤,最后经MgSO4干燥。过滤,真空干燥得到化合物2d(0.45g,85%),为白色蜡状固体。
化合物2的合成:将化合物2d(0.44g,1.0mmol)、3-(二丙胺)丙烷-1,2-二醇(70mg,0.4mmol)、DMAP(10mg,0.08mmol)和DIC(2.9g,23mm ol)溶于CH2Cl2(150mL)中。室温搅拌24h后,过滤除沉淀,滤液经旋转蒸发干燥。采用硅胶柱层析法纯化,洗脱剂为石油醚/乙酸乙酯(3:1,v/v),得到化合物2(0.36g,35%)。1H NMR(600MHz,Chloroform-d):δ5.22(m,1H),4.56(m,1H),4.31(m,1H),3.15(m,2H),2.61(m,2H),2.40(m,4H),2.03(m,4H),1.40(m,22H),0.87(m,12H)。19F NMR(565MHz,Chloroform-d):δ-111.6(m,4F),-114.5(m,4F),-118.8(m,8F),-121.9(m,4F),-123.7(m,4F)。
实施例3:氟化可电离脂质化合物3(8F)的合成
合成路线如下:
化合物3a的合成:将连二亚硫酸钠(1.76g,10.0mmol)缓慢加入到十六氟-1,8-二碘辛烷(3.3g,5.0mmol)、苯甲酸乙烯酯(0.7mL,5.0mmol)、1-溴丁烯(0.68g,5.0mmol)和NaHCO3(0.84g,10.0mmol)的CH3CN/H2O(8:7,v/v,18.4mL)的混合物中。反应液于0℃下搅拌50min,反应结束后,用HCl 3N将pH调至5~7,继续于室温搅拌20min。经CH2Cl2萃取后有机相用MgSO4干燥。采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(95:5,v/v),得到化合物3a(0.94g,20%),为微黄色蜡状固体。
化合物3b的合成:将化合物3a(0.94g,1.0mmol)和AIBN(34.0mg,0.20mmol)溶于甲苯(50mL)中,加入三丁基氢化锡(2.65mL,10.0mmol),将反应混合物在卤素灯下回流15h。减压除去溶剂,采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(90:10,v/v),得到化合物3b(0.32g,53%),为白色蜡状固体。
化合物3c的合成:将化合物3b(0.32g,0.53mmol)在1M LiOH(3mL)甲醇中室温搅拌3h。真空干燥后将产物溶解于CH2Cl2中,分别用水和生理盐水洗涤,再将有机相用MgSO4干燥。采用硅胶柱层析法纯化,洗脱剂为C6H14/CH2Cl2(50:50,v/v),得到化合物3c(0.19g,72%),为白色蜡状固体。
化合物3d的合成:将化合物3c(0.19g,0.38mmol)溶于丙酮(20mL)和水(0.2mL)中。在室温下滴加琼斯试剂,直至得到持久的红棕色溶液。反应混合物搅拌1h后加入i-PrOH(2mL),继续搅拌15min。减压除去溶剂,加入水(5mL),用乙醚萃取。有机相分别用水和生理盐水洗涤,最后经MgSO4干燥。过滤,真空干燥得到化合物3d(0.16g,81%),为白色蜡状固体。
化合物3的合成:将化合物3d(0.16g,0.3mmol)、3-(二丙胺)丙烷-1,2-二醇(18mg,0.1mmol)、DMAP(13mg,0.1mmol)和DIC(38.2mg,0.3mmol)溶于CH2Cl2(50mL)中。室温搅拌24h后,过滤除沉淀,滤液经旋转蒸发干燥。采用硅胶柱层析法纯化,洗脱剂为石油醚/乙酸乙酯(3:1,v/v),得到化合物3(65.6mg,56%)。1H NMR(600MHz,Chloroform-d):δ5.17(m,1H),4.54(m,1H),4.23(m,1H),3.15(m,2H),2.67(t,4H),2.56(t,2H),2.38(m,4H),1.38(m,14H),0.87(m,12H)。19F NMR(565MHz,Chloroform-d):δ-111.8(m,4F),-114.1(m,4F),-114.5(m,16F),-123.5(m,4F),-124.8(m,4F)。
实施例4:非氟化可电离脂质化合物(0F)的合成
合成路线如下:
化合物4的合成:将化合物4a(0.45g,0.3mmol)、3-(二丙胺)丙烷-1,2-二醇(54mg,0.3mmol)、DMAP(38mg,0.3mmol)和DIC(95.5mg,0.75mmol)溶于CH2Cl2(50mL)中。室温搅拌24h后,过滤除沉淀,滤液经旋转蒸发干燥。采用硅胶柱层析法纯化,洗脱剂为石油醚/乙酸乙酯(5:1,v/v),得到化合物4(0.13g,70%)。1H NMR(600MHz,Chloroform-d):δ5.10(m,1H),4.35(dd,1H),4.12(dd,1H),2.52(m,2H),2.37(m,4H),2.28(m,4H),1.59(m,4H),1.40(m,4H),1.26(m,40H),0.86(m,12H)。
实施例5:线粒体靶向氟化载体制剂的制备
将实施例1~3中的氟化可电离脂质化合物1~3(4F、6F、8F)分别与现有技术代表性阳离子脂质ALC-0315、DSPC、胆固醇、DSPE-PEG2000以表1中的摩尔比溶解于第一溶液中,配制成总脂质浓度为1.28mg/mL的溶液,备用。取质粒DNA溶液稀释于第二溶液中,配制成质粒DNA浓度为0.013mg/mL的稀释液,备用。其中,第一溶液为乙醇溶液,第二溶液为pH 4.0的10mM柠檬酸缓冲盐水溶液,二者体积比1:3,使用微流控芯片将两相快速混合,超滤除去体系中乙醇并置换成NaCl溶液,得到一系列载基因的氟化制剂4F-LNP@DNA、6F-LNP@DNA、8F-LNP@DNA。同时以实施例4中非氟化可电离脂质(0F)按同法制备得到的载基因非氟化制剂0F-LNP@DNA以及单独ALC-0315阳离子脂质按同法制备得到的载基因的非氟化制剂LNP@DNA作为对照组。表1组别中百分比表示氟化可电离脂质占总阳离子脂质的摩尔百分比。
表1氟化/非氟化可电离脂质与其他脂质在第一溶液中的摩尔比例
实施例6:线粒体靶向最佳氟化载体制剂的制备
将实施例2中的氟化可电离脂质化合物2(6F)分别与ALC-0315、DSPC、胆固醇、DSPE-PEG2000、DSPE-PEG2000-MAL以表2中的摩尔比溶解于第一溶液中,配制成总脂质浓度为1.28mg/mL的溶液,备用。取质粒DNA溶液稀释于第二溶液中,配制成质粒DNA浓度为0.013mg/mL的稀释液,备用。其中,第一溶液为乙醇溶液,第二溶液为pH 4.0的10mM柠檬酸缓冲盐水溶液,二者体积比1:3,使用微流控芯片将两相快速混合,超滤除去体系中乙醇并置换成NaCl溶液。向溶液中加入适量MTS溶液,4℃过夜反应,通过MTS末端巯基与载体外侧马来酰亚胺基团反应得到载基因的含MTS的氟化制剂6F-M-LNP@DNA。同时以实施例4中非氟化可电离脂质(0F)按同法制备载基因的含MTS非氟化制剂0F-M-LNP@DNA作为对照。
表2氟化/非氟化可电离脂质与其他脂质在第一溶液中的摩尔比例
实施例7:不同载体制剂的理化表征
采用马尔文激光粒度仪测定实施例5和6中载体制剂@DNA的粒径、多分散系数(PDI)以及Zeta电位。取各载体制剂@DNA溶液40μL,用去离子水稀释至1mL,加入样品池,每个样品重复测定3次。检测结果如表3所示,实施例5和6中制备的脂质纳米粒粒径均在100nm~200nm之间,PDI在0.1~0.4之间,Zeta电位在-20~0mV之间。
表3各载体制剂@DNA的粒径、PDI及电位表征
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实施例8:不同氟化载体体外细胞毒性情况
将L02细胞接种于96孔板中(1×104个细胞/孔),在细胞培养箱中孵育过夜,用实施例5中不同氟化载体(0F、4F、6F、8F)对细胞进行处理,并继续于培养箱中孵育72h。随后加入20μL噻唑蓝(MTT)(5mg/mL)溶液孵育4h,吸走上清,加入150μL DMSO溶解96孔板中沉淀,并在490nm下测定其紫外吸收,计算不同氟化载体不同氟化脂质浓度下细胞存活情况,结果如图1所示。MTT实验结果表明,不同氟化载体在氟化脂质浓度10~60μg/mL浓度范围内细胞存活率均保持在90%以上,表现出较好的体外安全性。
实施例9:不同氟化载体体外细胞摄取情况
将L02细胞接种于24孔板中(8×104个细胞/孔),在细胞培养箱中孵育过夜,用实施例5中不同载体@Cy5-DNA复合物纳米粒(含1μg Cy5-DNA)对细胞进行处理,并继续于培养箱中孵育24h。吸走上清,用PBS清洗一遍,加入0.2mL胰酶消化细胞,并利用新鲜培养基将消化好的细胞收集至离心管中。最后加入0.2mL PBS重悬并转移至流式管内备用,经流式细胞仪上机定量检测Cy5荧光强度,考察细胞摄取纳米粒的情况。结果如图2所示,氟化制剂组普遍表现出较非氟化制剂组更高的细胞摄取水平,且不同氟化可电离脂质浓度的不同氟化载体组间荧光强度差异明显,其中50%-6F-LNP和25%-8F-LNP具有最佳的细胞摄取效果。
实施例10:不同氟化载体体外线粒体转染情况
将L02细胞接种于6孔板中(2×105个细胞/孔),在细胞培养箱中孵育过夜,用实施例5中不同载体@DNA复合物纳米粒(含2μg DNA)对细胞进行处理,孵育24h后换液,继续培养48h。吸走上清,用PBS清洗一遍,加入0.3mL胰酶消化细胞,并利用新鲜培养基将消化好的细胞收集至离心管中。细胞经固定透化处理后,加入Flag一抗室温孵育30min。随后,吸走上清,用PBS清洗一遍,加入对应Alexa Fluor 647荧光二抗,室温继续孵育30min。最后吸走上清,用PBS清洗一遍后加入0.2mL PBS重悬,转移至流式管内备用,经流式细胞仪上机定量检测Alexa Fluor 647荧光强度,考察不同制剂组体外线粒体转染情况。结果如图3所示,非氟化制剂组荧光强度较低,不同氟化可电离脂质浓度的不同氟化载体组荧光强度差异明显,代表不同氟化载体组具有不同的线粒体转染能力,其中50%-6F-LNP线粒体转染能力最佳。另外,对相同氟化/非氟化可电离脂质摩尔比例制备的不同载体@DNA复合物纳米粒(50%-0F-LNP、50%-4F-LNP、50%-6F-LNP、50%-8F-LNP)转染能力进行比较,结果如表4所示,50%-6F-LNP组的荧光强度是50%-0F-LNP组的2.37倍,是50%-4F-LNP组的2.21倍,是50%-8F-LNP组的1.42倍。经单因素方差分析(ANOVA)检验分析,50%-6F-LNP组与其他组相比均具有高度显著性差异(p<0.001),线粒体转染能力显著提高。
表4不同载体@DNA复合物纳米粒转染后荧光强度检测结果
组别 | 平均荧光强度(MFI) |
50%-0F-LNP | 76.5±1.5 |
50%-4F-LNP | 82.1±1.7 |
50%-6F-LNP | 181.3±1.2 |
50%-8F-LNP | 127.7±1.5 |
实施例11:不同载体体外线粒体摄取情况
将L02细胞接种于35mm玻璃培养皿中(5×104个细胞/皿),在细胞培养箱中孵育过夜,利用不同载体LNP、0F-LNP(即50%-0F-LNP)、0F-M-LNP、6F-LNP(即50%-6F-LNP)、6F-M-LNP@Cy5-DNA复合物纳米粒(含1μg DNA)进行处理并孵育24h。其中,现有技术中代表性阳离子脂质ALC-0315单独制备得到的LNP组作为阳性对照组。随后,吸走上清,用PBS清洗一遍,加入1mL线粒体绿色荧光探针于37℃染色30min。PBS清洗后在激光共聚焦显微镜下进行观察,并通过ImageJ软件对CLSM图像进行线粒体共定位分析。线粒体经绿色荧光染料染色后呈绿色荧光,Cy5-DNA呈红色荧光,黄色表示载体和线粒体的共定位情况。结果如图4所示,氟化制剂组6F-LNP和6F-M-LNP显示出较其他组更强的黄色荧光。采用ANOVA检验进行多组间共定位系数均值比较,结果如图5所示,图中ns表示p>0.05,不具有显著性差异;*表示p<0.05,具有显著性差异;**表示p<0.01,具有中度显著性差异;***表示p<0.001,具有高度显著性差异。可以看出,最佳制剂6F-M-LNP组线粒体共定位系数最高,说明其具有最佳的线粒体靶向效率。
实施例12:不同载体体外目的蛋白表达情况
将L02细胞接种于10cm细胞培养皿中(1.5×106个细胞/皿),在细胞培养箱中孵育过夜,利用不同载体LNP、0F-LNP、0F-M-LNP、6F-LNP、6F-M-LNP@DNA复合物纳米粒(含10μgDNA)进行处理,孵育24h后换液,继续培养48h。收集各制剂组处理的细胞,提取线粒体蛋白,上样进行SDS-PAGE凝胶电泳及Western Blot,利用ND4目的蛋白一抗和线粒体内参COX IV蛋白一抗以及对应二抗进行孵育并显影,通过ImageJ软件对蛋白条带进行定量分析,考察各制剂组线粒体ND4蛋白表达情况。其中,现有技术中代表性阳离子脂质ALC-0315单独制备得到的LNP组作为阳性对照组。采用ANOVA检验进行多组间均值比较,结果如图6所示,图中*表示p<0.05,具有显著性差异;***表示p<0.001,具有高度显著性差异。可以看出,氟化制剂组6F-LNP和6F-M-LNP较非氟化制剂组ND4蛋白表达量有显著性差异,且6F-M-LNP组ND4蛋白表达水平最高,说明最佳制剂6F-M-LNP组可以实现更高的线粒体基因转染以及目的蛋白的表达。
实施例13:线粒体靶向氟化载体最佳制剂体内目的蛋白表达情况
通过玻璃体内给药的方式将实施例6中的最佳制剂6F-M-LNP@DNA复合物纳米粒注射进小鼠眼内(1μg DNA/眼),分别于给药3,7,14,30天后,收集小鼠眼球,提取线粒体蛋白,上样进行SDS-PAGE凝胶电泳及Western Blot,利用ND4目的蛋白一抗和线粒体内参COX IV蛋白一抗及对应二抗进行孵育并显影,通过ImageJ软件对蛋白条带进行定量分析,考察最佳制剂F-M-LNP递送的ND4基因在小鼠体内的蛋白质表达水平和持续时间。采用ANOVA检验进行多组间均值比较,结果如图7所示,图中ns表示p>0.05,不具有显著性差异;***表示p<0.001,具有高度显著性差异。可以看出,6F-M-LNP递送载体能将ND4基因有效地递送至小鼠眼球内,实现线粒体基因转染并持续表达30天。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种线粒体靶向的氟化可电离脂质或其药学上可接受的盐、立体异构体、互变异构体,其特征在于所述氟化可电离脂质的结构如式(I)所示:
其中,a=2~8的整数,b=4~8的整数;R1为可电离脂质头部基团,含有伯胺或叔胺结构的一元或多元胺。
2.根据权利要求1所述的式(I)化合物或其药学上可接受的盐、立体异构体、互变异构体,其特征在于,所述式(I)化合物具有以下结构中的一种:
其中,R1选自以下结构式中的任意一种,波浪线表示与相邻结构共价连接:
3.一种线粒体靶向的氟化可电离脂质或其药学上可接受的盐、立体异构体、互变异构体,其特征在于选自以下化合物:
4.一种线粒体靶向的脂质载体,其特征在于,所述载体包括权利要求1~3中任一项所述的氟化可电离脂质、阳离子脂质、辅助脂质、结构性脂质、含线粒体靶向基团修饰的功能性聚合物共轭脂质。
5.根据权利要求4所述的脂质载体,其特征在于,所述阳离子脂质选自以下中的一种或多种:DOTMA、DOTAP、DC-Chol、DLin-MC3-DMA、SM-102、ALC-0315,优选ALC-0315;所述辅助脂质选自以下中的一种或多种:DOPE、DOPC、DSPC、PA、PS、SM,优选DSPC;所述结构性脂质选自以下中的一种或多种:胆固醇、非甾醇、谷固醇、麦角固醇、α-生育酚、皮质类固醇,优选胆固醇;所述聚合物共轭脂质选自以下中的一种或多种:DSPE-PEG2000-MAL、DSPE-PEG2000、DMG-PEG2000、ALC-0159,优选DSPE-PEG2000-MAL和/或DSPE-PEG2000;所述线粒体靶向基团选自线粒体靶向序列:MLSLRQSIRFFKC、MLRAALSTARRGPRLSRLLC、MVSGSSGLAAARLLSRTFLLQQNGIRHGSYC中的任意一种,优选MLSLRQSIRFFKC;在所述脂质载体中,所述氟化可电离脂质、阳离子脂质、辅助脂质、结构性脂质、含线粒体靶向基团修饰的功能性聚合物共轭脂质的摩尔比为(10~50):(10~40):(5~25):(10~55):(0.3~5)。
6.一种用于线粒体基因递送的组合物,其特征在于,所述组合物包括权利要求1~3中任一项所述的氟化可电离脂质化合物或其药学上可接受的盐、立体异构体、互变异构体或权利要求4~5中任一项所述的脂质载体,以及功能性基因。
7.根据权利要求6所述的组合物,其特征在于,所述组合物为纳米颗粒制剂,所述纳米颗粒制剂的平均粒径在50nm~500nm之间;所述纳米颗粒制剂的多分散系数PDI小于50%。
8.根据权利要求6所述的组合物,其特征在于,所述的功能性基因为含有目的基因的质粒DNA载体。
9.权利要求1~3中任一项所述的氟化可电离脂质化合物或其药学上可接受的盐,或者权利要求4~5中任一项所述的脂质载体,或者权利要求6所述的组合物在制备用于治疗由线粒体基因突变引起的疾病的药物中的应用,所述疾病优选Leber遗传性视神经病变。
10.根据权利要求9所述的应用,其特征在于,所述组合物中功能基因选自人NADH泛醌氧化还原酶亚基4、人NADH泛醌氧化还原酶亚基6和人NADH泛醌氧化还原酶亚基1中的一种。
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