CN116574046A - 用于核酸递送的新型阳离子脂质化合物及其制备方法和应用 - Google Patents
用于核酸递送的新型阳离子脂质化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了用于核酸递送的新型阳离子脂质化合物及其制备方法和应用,使用本发明提供的式(Ⅰ)~(Ⅵ)所示的新型阳离子脂质化合物制备的脂质纳米颗粒,粒径更均匀,分散性良好;与核酸结合性更高,可以将核酸药物递送至靶细胞并产生抗原蛋白,提高了核酸药物递送的有效性;且在体内更容易降解代谢,毒性更低,应用前景广阔。
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阳离子脂质化合物,还涉及其制备方法和应用。
背景技术
核酸药物可以通过将外源基因引入靶细胞来替代或修正特定基因,以达到预防和治疗疾病的目的。核酸药物的研发生产流程相对简单,并且具有研发周期短、临床开发成功率高等优点。
然而,裸露的mRNA在体内极易被降解,并且难以进入靶细胞发挥作用。因此,提高mRNA的体内递送效率,是提高核酸药物有效治疗作用的关键途径。
目前,脂质纳米颗粒是有效递送核酸药物的主要载体之一,可以保护核酸药物在体内不被快速降解,延长其体内循环时间,且能够更有效的递送核酸药物进入靶细胞。脂质纳米颗粒与细胞膜的组成成分相似,均由脂质分子构成,它由4个脂质组分构成,其中包括阳离子脂质、胆固醇、辅助型脂质和PEG脂质。其中,阳离子脂质在核酸药物的包封和释放中扮演着关键作用,因此,研究开发新型阳离子脂质化合物具有非常重要的意义。
发明内容
有鉴于此,本发明的目的在于提供一种用于递送核酸药物的新型阳离子脂质化合物;本发明的目的之二在于提供一种制备所述用于递送核酸药物的新型阳离子脂质化合物的方法;本发明的目的之三在于提供所述新型阳离子脂质化合物在制备脂质纳米颗粒制剂中的应用。
为达到上述目的,本发明提供如下技术方案:
1、一种用于递送核酸药物的新型阳离子脂质化合物,具有以下通式结构:
或为其可接受的盐、互变异构体或立体异构体,其中,n为1或2;R1选自以下基团中的任意一种:
R2及R3选自以下基团中任意一种,R2、R3可以相同:
本发明优选的,所述新型阳离子脂质化合物具有如下式(Ⅰ)所示的结构:
本发明优选的,所述新型阳离子脂质化合物具有如下式(Ⅱ)所示的结构:
本发明优选的,所述新型阳离子脂质化合物具有如下式(Ⅲ)或(Ⅳ)或(Ⅴ)或(Ⅵ)所示的结构:
2、一种制备权利要求1所述的用于递送核酸药物的新型阳离子脂质化合物的方法,包含如下步骤:
步骤1:
步骤2:
步骤3:
3、权利要求1或2任一项所述的新型阳离子脂质化合物在制备脂质纳米颗粒制剂中的应用。
本发明优选的,所述脂质纳米颗粒制剂还所述脂质纳米颗粒制剂还包含一种或多种其它脂质成分,包括结构脂质、类固醇和聚合物缀合的脂质。
本发明优选的,所述结构脂质为DSPC或DOPC或DOPE;所述结构脂质和阳离子化合物的摩尔比为(0~0.5):1。
本发明优选的,所述类固醇为胆固醇,所述胆固醇和阳离子化合物的摩尔比为(0~1.5):1。
本发明优选的,所述聚合物缀合的脂质为DSPE-mPEG或DMG-mPEG;所述聚合物缀合的脂质和阳离子化合物的摩尔比为(0.01~0.05):1。
本发明的有益效果在于:本发明的新型阳离子脂质化合物具有形成的脂质纳米颗粒粒径更均匀,分散性良好;与核酸结合性更高,递送效果更好;且在体内更容易降解代谢,毒性更低。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为实施例3由化合物1制备所得脂质纳米颗粒的粒度分布结果;;
图2为实施例4由化合物1制备所得脂质纳米颗粒的透射电镜结果;
图3为实施例5由化合物1~6制备所得脂质纳米颗粒细胞毒性实验结果;
图4为实施例6由化合物1制备所得脂质纳米颗粒的体外转染效果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
本发明提供了上述含阳离子脂质分子的制备方法,包括以下反应步骤:
步骤1:
步骤2:
步骤3:
上述R1选自以下任意基团:
R2和R3选自以下任意基团(R2和R3可以相同):
本发明优选的,所述新型阳离子脂质化合物具有如下式(Ⅰ)所示的结构:
本发明优选的,所述新型阳离子脂质化合物具有如下式(Ⅱ)所示的结构:
本发明优选的,所述新型阳离子脂质化合物具有如下式(Ⅲ)或(Ⅳ)或(Ⅴ)或(Ⅵ)所示的结构:
实施例1新型阳离子脂质化合物1的合成
新型阳离子脂质化合物1的结构式如下:
步骤1:
N2保护下向一个两口圆底烧瓶中注射加入15moL 2-(吡啶-2-基)乙烷-1-醇的和60moL碘甲烷,随后加入50mL乙腈作为溶剂,80℃反应16小时,随后降至室温,减压条件下除去多余的碘甲烷以及溶剂,得到粗产品,粗产品用乙腈和乙酸乙酯混合溶剂重结晶得到2-(2-羟乙基)-1-甲基吡啶-1-鎓,备用。
步骤2:
将0.25moL 2-(2-羟乙基)-1-甲基吡啶-1-鎓、0.2moL碘单质、1moL碳酸钾溶于20mL水中,在80℃下搅拌24小时,反应结束后,冷却至室温,后旋转蒸馏除去溶剂后用硅胶柱层析法分离纯化得到5-(2-羟乙基)-1-甲基-1H-吡咯-2-甲醛,备用。
步骤3:
在氩气保护下,将0.1moL 8-((E)-十二碳-3-烯-1-基)-1-((E)-十三碳-3-烯-1-基)-4-(甲基氨基)甲基)辛二酸酯溶解在100mL二氯甲烷中,加入0.1moL NaBH(OAc)3并搅拌20分钟。之后将溶于100mL二氯甲烷中的0.1moL 5-(2-羟乙基)-1-甲基-1H-吡咯-2-甲醛缓慢地加入到反应物中。添加完成后,让反应物在室温搅拌15-20分钟。反应物用饱和NaHCO3(2x500 mL)洗涤。水层用二氯甲烷重新萃取。合并的有机层用盐水洗涤。有机层在Na2SO4上干燥,过滤并浓缩。得到的粗品通过硅胶柱层析法分离纯化。得到目的产物,产率46%。
产物的表征数据:
1H NMR(400MHz,Chloroform-d)δ5.83(d,J=6.8Hz,1H),5.58(dtt,J=15.1,5.7,1.0Hz,2H),5.43(dtt,J=15.0,5.5,1.0Hz,2H),4.21(t,J=6.2Hz,4H),3.84(qd,J=5.2,1.0Hz,2H),3.70–3.60(m,2H),3.46(s,3H),3.31(t,J=5.5Hz,1H),2.80–2.69(m,2H),2.48(dd,J=11.3,5.7Hz,1H),2.37–2.22(m,10H),2.01(tdt,J=7.8,5.4,1.1Hz,4H),1.81–1.71(m,1H),1.67–1.54(m,4H),1.40(tdd,J=8.6,7.3,2.0Hz,2H),1.33–1.25(m,26H),0.91–0.87(m,6H).
实施例2新型阳离子脂质化合物2的合成
新型阳离子脂质化合物2的结构式如下:
步骤1:
N2保护下向一个两口圆底烧瓶中注射加入15moL 2-(吡啶-2-基)乙烷-1-胺的和60moL的碘甲烷,随后加入50mL的乙腈作为溶剂,80℃反应16小时,随后降至室温,减压条件下除去多余的碘甲烷以及溶剂,得到粗产品,粗产品用乙腈和乙酸乙酯混合溶剂重结晶得到2-(2-氨基乙基)-1-甲基吡啶-1-鎓,备用。
步骤2:
将0.25moL 2-(2-氨基乙基)-1-甲基吡啶-1-鎓、0.2moL碘单质、1moL碳酸钾溶于20mL水中,在80℃下搅拌24小时,反应结束后,冷却至室温,后旋转蒸馏除去溶剂后用硅胶柱层析法分离纯化得到5-(2-氨基乙基)-1-甲基-1H-吡咯-2-甲醛,备用。
步骤3:
在氩气保护下,将0.1moL 2-((7,10-十六烯-7,10-二烯-1-yl)-N-甲基十八烯-8,11-二烯-1-胺溶解在100mL二氯甲烷中,加入0.1moL NaBH(OAc)3并搅拌20分钟。之后将溶于100mL二氯甲烷中的0.1moL 5-(2-氨基乙基)-1-甲基-1H-吡咯-2-甲醛缓慢地加入到反应物中。添加完成后,让反应物在室温搅拌15-20分钟。反应物用饱和NaHCO3(2x500 mL)洗涤。水层用二氯甲烷重新萃取。合并的有机层用盐水洗涤。有机层在Na2SO4上干燥,过滤并浓缩。得到的粗品通过硅胶柱层析法分离纯化。得到目的产物,产率57%。
产物的表征数据:
1H NMR(400MHz,Chloroform-d)δ6.07(d,J=6.8Hz,1H),5.83(d,J=7.0Hz,1H),5.56–5.21(m,11H),3.70–3.59(m,2H),3.43(s,3H),2.99(tt,J=6.0,4.5Hz,3H),2.84(td,J=4.5,2.7Hz,3H),2.53(ddt,J=4.9,3.9,1.1Hz,5H),2.46(dd,J=11.4,5.7Hz,1H),2.06–1.98(m,10H),1.62(pt,J=7.0,5.7Hz,1H),1.44(t,J=6.1Hz,3H),1.42–1.33(m,8H),1.33–1.25(m,30H),0.91–0.87(m,7H).
实施例3脂质纳米颗粒制备以及性状表征
将EGFP-mRNA稀释于10mM~50mM柠檬酸盐缓冲液(pH=4.0)中;将各脂质组分(本发明制备的新型阳离子脂质化合物:DSPC:胆固醇:PEG脂质)按总浓度10mg/mL~30mg/mL溶解于无水乙醇中,使用注射器将脂质溶液与mRNA缓冲液以1:5至1:2(v/v)的比例,通过微流控装置,设定总流速为10mL/min~20mL/min,制备脂质纳米颗粒,将收集到的样品通过透析去除乙醇,再将脂质纳米颗粒通过0.22μm无菌过滤器过滤,保存于4℃。
粒径大小、PDI以及Zeta电位的测定:取制备好的脂质纳米颗粒溶液100μL,用磷酸盐缓冲液稀释至1mL,混匀后置于马尔文激光粒度仪上测定粒径大小、粒径分布以及Zeta电位。测定温度恒定为25℃,测定前将样品放置于仪器内平衡2min,样品平行测定3次。由化合物1制备所得的脂质纳米颗粒测定结果如图1所示,结果表明,脂质纳米颗粒粒径在70nm到100nm左右,大小均一,分散性良好,媲美目前市面的SM-102等。
包封率的测定:按照Ribogreen试剂盒说明书,测定上述脂质纳米颗粒的包封率。具体操作如下:依次配置2% Triton X-100溶液,20×TE buffer溶液,200×RiboGreenRNA染色剂溶液;吸取适量脂质纳米颗粒溶液,加入20×TE buffer溶液稀释至适当倍数,混匀后加入相同体积2% Triton X-100溶液,混匀,室温条件下放置,裂解脂质纳米颗粒1h;吸取裂解后溶液100μL,加入100μL 200×RiboGreen RNA染色剂溶液,混匀,避光条件下孵育10min;酶标仪测定荧光强度,仪器设定激发波长为480nm,发射波长为520nm;代入标准曲线计算脂质纳米颗粒溶液中mRNA的含量。包封率(%)计算公式如下:
表1脂质纳米颗粒表征实验结果
*SM-102为商业核酸递送系统Spikevax的阳离子脂质。
**ALC-0315为商业核酸递送系统Comirnaty的阳离子脂质。
本实施例所制备的负载mRNA的脂质纳米颗粒的包封率、粒径、PDI和Zeta电位的检测结果如表1、图1所示。结果表明,该配方下的脂质化合物和mRNA形成的脂质纳米颗粒包封率较高,粒径均一,大约为80nm,符合核酸药物递送载体的基本特征。同时,通过与商业核酸递送系统Spikevax以及Comirnaty的阳离子脂质化合物的对比,本发明中含有吡咯环的新型阳离子脂质化合物制备而成的脂质纳米颗粒具有更高的核酸包封率,且PDI更小,脂质纳米颗粒分散性更好。
实施例4脂质纳米颗粒的透射电镜结构测定
将由实施例3中化合物1制备所得的脂质纳米颗粒通过透射电镜观察脂质纳米颗粒形态,具体操作如下:通过透射电子显微镜(TEM)观察脂质纳米颗粒的形态。将脂质纳米颗粒溶液用磷酸盐缓冲液稀释,滴至专用铜网上,静置风干后,用2%(w/v)的磷钨酸溶液染色30s。用一张滤纸吸去多余的液体,然后将样品在室温下干燥,用电镜观察。
观察结果如图2所示,结果表明,由实施例3中化合物1制备所得的脂质纳米颗粒呈球形,表面光滑且分布均匀。
实施例5脂质纳米颗粒的细胞毒性测定
将由实施例3中化合物1~6制备所得的脂质纳米颗粒通过MTT实验测定细胞毒性,具体操作如下:取处于对数生长期的人胚胎肾细胞HEK-293T细胞,分别进行消化、离心,离心获得的细胞悬液弃掉上清液后,用DMEM含血清培养基重悬,取20μL细胞悬液用PBS溶液稀释10倍,用流式细胞仪计数,根据计数结果将细胞悬液稀释到5×104个/mL,按每孔100μL细胞悬液均匀接种在96孔板上。将接种好细胞的96孔板做好标记后,放置于二氧化碳培养箱中过夜培养。
待细胞完全贴壁后,分别取由实施例3中化合物1~6制备所得的脂质纳米颗粒以及对照试剂Lipofectamine 2000 5μL,加入到细胞中,给药后继续放置于二氧化碳培养箱中,药物作用24h。
药物作用24h后,每孔加入20μL MTT溶液,振荡30s后转入二氧化碳培养箱,4h后吸出上清液,每孔加入150μL DMSO,避光振荡10min后放入酶标仪中,设置波长为490nm,测定OD值。根据以下公式计算细胞存活率:
OD(Experimental)为供试药液组测得的OD值;OD(Blank)为空白组测得的OD值;OD(Control)为阴性对照组测得的OD值。
细胞毒性结果如图3所示,结果表明,本发明制备的脂质纳米颗粒对于正常细胞无明显毒性,是一种具有良好生物相容性和安全性的载体,适合用于递送核酸药物。
实施例6脂质纳米颗粒的转染效果测定
将由实施例3中化合物1制备所得的脂质纳米颗粒通过转染实验测定转染效果,具体操作如下:取处于对数生长期的人胚胎肾细胞HEK-293T细胞,分别进行消化、离心,离心获得的细胞悬液弃掉上清液后,用DMEM含血清培养基重悬,取20μL细胞悬液用PBS溶液稀释10倍,用流式细胞仪计数,根据计数结果将细胞悬液稀释到2×105个/mL,按每孔1mL细胞悬液均匀接种在12孔板上。将接种好细胞的12孔板做好标记后,放置于二氧化碳培养箱中过夜培养。
待细胞密度达到70%~80%左右后,分别取由实施例3中化合物1、商业核酸递送系统Spikevax的阳离子脂质化合物SM-102和商业核酸递送系统Comirnaty的阳离子脂质化合物ALC-0315制备所得的脂质纳米颗粒以及转染试剂Lipofectamine 2000,按1~5μg/mL的EGFP-mRNA浓度给细胞加药,轻柔摇晃12孔板使药物充分扩散到整个细胞生长面,放置在二氧化碳培养箱内孵育24h。
药物作用24h后,在倒置荧光显微镜下观察细胞绿色荧光蛋白表达情况并拍照。结果如图4所示,表明采用本发明实施例3中化合物1制备所得的脂质纳米颗粒,其转染能力强,与商品化Lipofectamine 2000转染试剂相同或略高。同时,通过与商业核酸递送系统Spikevax(对应图4中的SM-102)以及Comirnaty(对应图4中的ALC-0315)的阳离子脂质化合物的对比,本发明中含有吡咯环的新型阳离子脂质化合物制备而成的脂质纳米颗粒转染能力更强。
综上,本发明提供了一种新型阳离子脂质化合物的合成方法以及一种包含上述新型阳离子脂质化合物的脂质纳米颗粒的制备方法。以上实验结果表明,本发明提供的新型阳离子脂质所制备的脂质纳米颗粒,可以将核酸药物有效递送至靶细胞产生抗原蛋白,提高了核酸药物递送的效率,应用前景广阔。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种用于递送核酸药物的新型阳离子脂质化合物,其特征在于,具有以下通式结构:
或为其可接受的盐、互变异构体或立体异构体,其中,n的数目为1或2,R1选自以下基团中的任意一种:
R2及R3选自以下基团中任意一种,R2、R3可以相同:
2.根据权利要求1所述的用于递送核酸药物的新型阳离子脂质化合物,其特征在于,具有如下式(Ⅰ)所示的结构:
3.根据权利要求1所述的用于递送核酸药物的新型阳离子脂质化合物,其特征在于,具有如下式(Ⅱ)所示的结构:
4.根据权利要求1所述的用于递送核酸药物的新型阳离子脂质化合物,其特征在于,具有如下式(Ⅲ)或(Ⅳ)或(Ⅴ)或(Ⅵ)所示的结构:
5.一种制备权利要求1所述的用于递送核酸药物的新型阳离子脂质化合物的方法,其特征在于,包含如下步骤:
步骤1:
步骤2:
步骤3:
6.权利要求1或2任一项所述的新型阳离子脂质化合物在制备脂质纳米颗粒制剂中的应用。
7.根据权利要求4所述的应用,其特征在于,所述脂质纳米颗粒制剂还所述脂质纳米颗粒制剂还包含一种或多种其它脂质成分,包括结构脂质、类固醇和聚合物缀合的脂质。
8.根据权利要求5所述的应用,其特征在于,所述结构脂质为DSPC或DOPC或DOPE;所述结构脂质和阳离子化合物的摩尔比为(0~0.5):1。
9.根据权利要求5所述的应用,其特征在于,所述类固醇为胆固醇,所述胆固醇和阳离子化合物的摩尔比为(0~1.5):1。
10.根据权利要求5所述的应用,其特征在于,所述聚合物缀合的脂质为DSPE-mPEG或DMG-mPEG;所述聚合物缀合的脂质和阳离子化合物的摩尔比为(0.01~0.05):1。
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