CN114940756B - 一种聚(2-噁唑啉)脂质与脂质纳米颗粒及应用 - Google Patents
一种聚(2-噁唑啉)脂质与脂质纳米颗粒及应用 Download PDFInfo
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- CN114940756B CN114940756B CN202210639673.6A CN202210639673A CN114940756B CN 114940756 B CN114940756 B CN 114940756B CN 202210639673 A CN202210639673 A CN 202210639673A CN 114940756 B CN114940756 B CN 114940756B
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Abstract
本发明属于生物医药技术领域,具体公开了一种聚(2‑噁唑啉)脂质与脂质纳米颗粒及应用。本发明公开了一种聚(2‑噁唑啉)脂质,并通过该聚(2‑噁唑啉)脂质与可离子化脂质、甾醇类化合物、磷脂按特定比例混合得到了一种用于核酸递送的脂质纳米粒。该类聚(2‑噁唑啉)脂质可替代现有的PEG化脂质实现核酸药物的稳定包裹、粒径控制和有效递送,同时避免了见诸报道的PEG化脂质诱发的免疫反应;同时,聚(2‑噁唑啉)脂质本身的pH值响应特点可进一步增强核酸药物LNP制剂的环境适应性靶向摄取及内涵体逃逸,更加适用于相关核酸药物的重复给药和转染效率。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种聚(2-噁唑啉)脂质与脂质纳米颗粒及应用。
背景技术
核酸药物是生物医药发展的前沿领域,包括反义核酸(ASO)、小干扰RNA(siRNA)、小向导RNA(sgRNA)、微小RNA(miRNA)、小激活RNA(saRNA)、信使RNA(mRNA)等,核酸药物是基因治疗的一种形式,也是继小分子药物、蛋白药物、抗体药物之后的新一代制药技术。核酸药物能够直接作用于致病靶基因或者靶mRNA,在基因水平上发挥治疗疾病的作用,核酸药物从转录后水平进行基因沉默或激活治疗,相比传统蛋白水平发挥作用的药物具有高特异性、高效性、长效性等明显优势。
在过去的30年里,核酸药物发展曲折,核酸药物想要进入体内主要面临3大个难关:1)核酸的分子量和负电荷使其不能自由通过生物膜;2)RNA容易被血浆和组织中RNase酶降解,被肝脏和肾脏快速清除和被免疫系统识别;3)进入细胞后“卡”在内吞小体中无法发挥功能。药物递送系统是克服核酸类药物发展面临的技术障碍的关键,目前,解决递送问题主要有两个方法:一个是改造核酸分子,让其稳定并躲避免疫系统的识别;另外一个就是利用药物传输系统,比如说脂质纳米颗粒(LNP)和GalNAc(N-乙酰化的半乳糖胺)偶联技术。
LNP是研究最久最成熟的核酸类药物递送系统,目前已上市的第一个核酸干扰药物Alnylam的Onpattro、辉瑞与Moderna的mRNA新冠疫苗以及国内艾博生物mRNA新冠疫苗等均采用了LNP递送系统。可离子化脂质是LNP递送系统的关键,pH=4时,脂质分子中的氮会完全带正电荷。生理条件pH=7.4,脂质基本不带电,在保持一定的细胞结合能力的情况下减弱细胞毒性。进入细胞之后,脂质体形成内涵体pH达到5,由于其带正电,与内涵体膜上的负电荷脂质结合使内涵体破裂,将mRNA释放出来避免最后被溶酶体降解。所以可电离阳离子脂质最大的优点就是有效降低LNP细胞毒性,提高mRNA的体内稳定性,并帮助mRNA逃避溶酶体的降解。专利201680063235.2、200980154346.4及200980122413.4等公开了新型可离子化脂质在递送核酸类药物中的应用,可以有效的将核酸类药物递送转染细胞发挥其药效。
可离子化脂质固然重要,但是PEG化脂质对RNA类药物递送也非常关键。PEG化脂质虽然用于控制粒径并充当空间屏障起稳定作用,防止储存过程LNP微粒聚集,延长循环时间,但是,PEG化脂质降低了细胞对LNP的摄取,阻碍了LNP的内涵体逃逸,从而降低RNA转染效率。另外多次注射PEG化脂质体可诱发免疫反应,引起加速血液清除(ABC)现象。
发明内容
有鉴于此,本发明提供了一种聚(2-噁唑啉)脂质与脂质纳米颗粒及应用,该类聚(2-噁唑啉)脂质可替代现有的PEG化脂质实现核酸药物的稳定包裹、粒径控制和有效递送,同时避免了见诸报道的PEG化脂质诱发的免疫反应;同时,聚(2-噁唑啉)脂质本身的pH值响应特点可进一步增强核酸药物LNP制剂的环境适应性靶向摄取及内涵体逃逸,更加适用于相关核酸药物的多次给药和转染效率。
为了达到上述目的,本发明采用如下技术方案:
一种聚(2-噁唑啉)脂质,所述聚(2-噁唑啉)脂质的结构式为式1:
式1聚合度n独立的为10~100的任一整数;
所述L1为C1-C5烷基;
所述的R为H、C1-C18烷基、
所述的R1独立为H、
所述R2为C1-C5烷基、
所述R3为
所述R4为
优选的,所述聚合度n独立的为30~70的任一整数。
优选的,所述L1为CH2、C2H4、或C3H6;
所述的R独立为H、CH3、C2H5、
所述的R1独立为H、
所述R2为CH3、
所述R3为
所述R4为
优选的,所述聚(2-噁唑啉)脂质的结构式为:
本发明的另一个目的是提供一种包含聚(2-噁唑啉)脂质的脂质纳米粒,包含可离子化脂质、甾醇类化合物、磷脂和聚(2-噁唑啉)脂质;所述可离子化脂质、甾醇类化合物、磷脂和聚(2-噁唑啉)脂质的摩尔比为40~60∶25~40∶5~20∶0.5~5。
优选的,所述可离子化脂质的结构通式为
其中,X为C2~C5直链烷基或CH2CH2OCH2CH2;
Y为(C=O)O;
R5独立的为C10~C20直链烷基、C10~C20直链烯基或C10~C24酯基;
R6独立的为C5~C10直链烷基;
R7独立的为C10~C24支链烷基。
优选的,所述甾醇类化合物为胆甾醇、二氢胆甾醇、豆甾醇、二氢豆甾醇、菜油甾醇、麦角甾醇、豆甾醇或二氢谷甾醇。
优选的,所述磷脂为二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰磷脂酰乙醇胺、二油酰磷脂酰胆碱、二硬脂酰磷脂酰甘油、二棕榈酰磷脂酰甘油、二肉豆蔻酰磷脂酰甘油、二硬脂酰磷脂酸和二棕榈酰磷脂酸中的一种或几种。
本发明的再一目的是提供一种脂质纳米粒在制备核酸药物中的应用,其特征在于,所述核酸药物包含脂质纳米粒和核酸分子,所述脂质纳米粒中可离子化脂质的氮原子与核酸分子中的磷酸基团的摩尔比为1.5~12∶1。
优选的,所述核酸药物还包括pH调节剂和保护剂;
所述pH调节剂为药学接受的pH调节剂;
所述保护剂为甘油、蔗糖、海藻糖、葡萄糖、甘油葡萄糖苷和四氢嘧啶中的一种。
经由上述的技术方案可知,与现有技术相比,本发明具有以下有益效果:
PEG化脂质是现有技术中制备核酸药物-LNP制剂的重要辅料成分,但由于PEG成分在食品、药品、化妆品领域中的广泛应用,见诸报道的PEG成分预存免疫已屡见不鲜,一定程度上限制了PEG化核酸药物-LNP制剂的人群适用性,尤其是在多次给药的情景下,会发生PEG成分诱发免疫反应,引起加速血液清除(ABC)现象的风险。本发明中的聚(2-噁唑啉)脂质可以替代现有的PEG化脂质用于制备核酸药物-LNP制剂,是现有技术中PEG化脂质的一种替代策略,可实现核酸药物的稳定包裹、粒径控制和有效递送的同时避免了PEG成分的免疫反应风险,提高了核酸药物-LNP制剂的人群适用性。
聚(2-噁唑啉)脂质本身具有一定的pH响应性特点,在低pH环境,如肿瘤病灶部位、细胞内涵体等会提高LNP制剂的摄取与内吞逃逸效率,相比于PEG化脂质能增强核酸药物-LNP制剂的靶向摄取和转染效能。同时,聚(2-噁唑啉)脂质比PEG化脂质合成更加容易,成本更低,分子量更可精确控制,而且基团侧链具有易修饰性特点,可以使得后续处方产品开发中能针对不同的处方特点和靶向需求较便捷的进行修饰筛选,能进一步拓展核酸药物-LNP制剂技术的应用领域。
具体实施方式
本发明提供了一种聚(2-噁唑啉)脂质,所述聚(2-噁唑啉)脂质的结构式为式1:
式1聚合度n独立的为10~100的任一整数,优选为20-70的任一整数,进一步优选为30-50的任一整数;
所述L1为C1-C5烷基,选优L1为CH2、C2H4、或C3H6;再优选L1为CH2、C2H4、;
所述的R为H、C1-C18烷基、优选R为H、CH3、C2H5、/> 再优选R为CH3、
所述的R1独立为H、优选R1独立为/>
所述R2独立为C1-C5烷基、优选R2为CH3、
所述R3独立为优选R3为/>
所述R4独立为优选R4为/>
在本发明中,所述聚(2-噁唑啉)脂质优选如下结构:
本发明还提供了一种由聚(2-噁唑啉)脂质构成的脂质纳米粒,包含可离子化脂质、甾醇类化合物、磷脂和聚(2-噁唑啉)脂质;所述可离子化脂质、甾醇类化合物、磷脂和聚(2-噁唑啉)脂质的摩尔比为40~60∶25~40∶5~20∶0.5~5;优选为45~55∶30~40∶8~12∶1~3;进一步优选为50∶38.5∶10∶1.5。
在本发明中,所述可离子化脂质的结构通式为
其中,X为C2~C5直链烷基或CH2CH2OCH2CH2;
Y为(C=O)O;
R5独立的为C10~C20直链烷基、C10~C20直链烯基或C10~C24酯基;
R6独立的为C5~C10直链烷基;
R7独立的为C10~~C24支链烷基。
在本发明中,所述可离子化脂质优选如下结构:
在本发明中,所述甾醇类化合物为胆甾醇、二氢胆甾醇、豆甾醇、二氢豆甾醇、菜油甾醇、麦角甾醇、豆甾醇或二氢谷甾醇。
在本发明中,所述磷脂为二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰磷脂酰乙醇胺、二油酰磷脂酰胆碱、二硬脂酰磷脂酰甘油、二棕榈酰磷脂酰甘油、二肉豆蔻酰磷脂酰甘油、二硬脂酰磷脂酸和二棕榈酰磷脂酸中的一种或几种。
在本发明中,所述脂质纳米粒的粒径为50~200nm,优选为60~170nm,进一步优选为70~150nm,再一步优选为90nm;所述脂质纳米粒的多分散性指数为0.05-0.2,优选为0.08-0.18。
本发明还提供了一种脂质纳米粒在制备核酸药物中的应用,所述核酸药物包含脂质纳米粒和核酸分子,所述脂质纳米粒中的可离子化脂质的氮原子与核酸分子中的磷酸基团的摩尔比为1.5~12∶1,优选为3~8。
在本发明中,所述核酸分子包括任何形式的核酸分子,优选为DNA、小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、microRNA(miRNA)、Dicer-substrate RNA(dsRNA)、小向导RNA(sgRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)。
在本发明中,所述核酸药物还包括pH调节剂和保护剂;
pH调节剂为药学接受的pH调节剂,优选为乙酸、乙酸钠、柠檬酸、柠檬酸钠、磷酸、磷酸氢二钠、磷酸二氢钠、磷酸二氢钾、三羟甲基氨基甲烷盐酸盐磷酸氢二钾中的一种或几种;
所述保护剂为甘油、蔗糖、海藻糖、葡萄糖、甘油葡萄糖苷和四氢嘧啶中的一种。
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。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
将2-甲基-2-噁唑啉(45mmol,3.83g)、对甲苯磺酸甲酯(Me-OTs,1mmol,0.19g)溶解于乙腈(ACN,50mL)中,加入烧瓶中,通入氮气,密封,在油浴中70℃搅拌24h,恢复室温,加入终止剂3-氨基-1,2-十四烷基丙二醚(1.5mmol,0.72g)继续搅拌6h,加入250ml无水乙醚,搅拌30min,过滤滤液得沉淀,该沉淀通过G25凝胶柱去离子水洗脱纯化得化合物1洗脱液,冷冻干燥得粉末化合物1(2.56g,59.1%)。1HNMR(CDCl3,400MHz)δ:3.75(s,1H),3.52-3.42(m,176H),3.38-3.21(m,9H),3.02-2.93(m,3H),2.27-2.14(m,135H),1.33-1.22(m,48H),0.87(t,6H)。
实施例2化合物2的合成
将2-甲基-2-噁唑啉(30mmol,2.55g)、对甲苯磺酸甲酯(Me-OTs,1mmol,0.19g)溶解于乙腈(ACN,50mL)中,加入烧瓶中,通入氮气,密封,在油浴中70℃搅拌24h,恢复室温,加入终止剂3-氨基-1,2-十四烷基丙二醚(1.5mmol,0.72g)继续搅拌6h,旋蒸去除溶剂,将获得的固形物加入30ml水中,透析24h,冷冻干燥得化合物2-1,将化合物2-1(0.5mmol,1.53g)、乙酰氯(0.6mmol,0.04g)、无水吡啶(Pyridine,50ml)加入二氯甲烷(DCM,100ml)中,室温搅拌6h,旋转蒸发去除溶剂等挥发物,沉淀采用无水乙醚漂洗,过滤滤液得沉淀,该沉淀通过G25凝胶柱去离子水洗脱纯化得化合物2洗脱液,冷冻干燥得得到化合物2(1.21g,78%)。1HNMR(CDCl3,400MHz)δ:4.18(s,1H),3.52-3.41(m,122H),3.38-3.25(m,6H),3.02-2.93(m,3H),2.27-2.14(m,93H),1.33-1.22(m,48H),0.87(t,6H)。
实施例3化合物3的合成
将2-甲基-2-噁唑啉(60mmol,5.11g)、乙酸-2-溴乙醇酯(1mmol,0.17g)溶解于乙腈(ACN,80mL)中,加入烧瓶中,通入氮气,密封,在油浴中70℃搅拌24h,恢复室温,加入终止剂3-氨基-1,2-十四烷基丙二醚(1.5mmol,0.72g)继续搅拌6h,加入250ml无水乙醚,搅拌30min,过滤滤液得沉淀,将所得沉淀加入甲醇(50ml),同时加入水(30ml)及碳酸钾(10mmol,1.38g),室温搅拌6h,旋蒸去除溶剂,将获得的固形物加入二氯甲烷(30ml),过滤滤液旋蒸,沉淀通过G25凝胶柱去离子水洗脱纯化得并得到化合物3(2.42g,43%)。1HNMR(CDCl3,400MHz)δ:4.84(s,1H),3.52-3.42(m,238H),3.38-3.21(m,8H),3.02-2.93(m,3H),2.27-2.14(m,180H),1.33-1.22(m,48H),0.87(t,6H)。
实施例4化合物4的合成
将2-乙基-2-噁唑啉(40mmol,3.97g)、对甲苯磺酸甲酯(Me-OTs,1mmol,0.19g)溶解于乙腈(ACN,50mL)中,加入烧瓶中,通入氮气,密封,在油浴中70℃搅拌24h,恢复室温,加入终止剂3-甲氨基-1,2-十八烷酰基丙二酯(1.5mmol,0.96g)继续搅拌6h,加入250ml无水乙醚,搅拌30min,过滤滤液得沉淀,该沉淀通过G25凝胶柱去离子水洗脱纯化得化合物4洗脱液,冷冻干燥得粉末化合物得到化合物4(2.59g,56%)。1HNMR(CDCl3,400MHz)δ:5.23(s,1H),4.44-4.18(m,2H),3.52-3.42(m,156H),3.02-2.93(m,3H),2.38-2.25(m,84H),1.33-1.22(m,56H),1.11-1.01(m,120),0.87(t,6H)。
实施例5化合物5的合成
将2-甲基-2-噁唑啉(45mmol,3.83g)、对甲苯磺酸甲酯(Me-OTs,1mmol,0.19g)溶解于乙腈(ACN,50mL)中,加入烧瓶中,通入氮气,密封,在油浴中70℃搅拌24h,恢复室温,加入终止剂4-氨基-1,2-十六烷基丁二醚(1.5mmol,0.83g)继续搅拌6h,加入250ml无水乙醚,搅拌30min,过滤滤液得沉淀,沉淀通过G25凝胶柱去离子水洗脱纯化得化合物5洗脱液,冷冻干燥得粉末化合物5(2.02g,46%)。1HNMR(CDCl3,400MHz)δ:3.52-3.42(m,176H),3.38-3.21(m,8H),3.02-2.93(m,3H),2.27-2.14(m,135H),1.33-1.22(m,52H),0.87(t,6H)。
实施例6化合物6的合成
将2-乙基-2-噁唑啉(45mmol,4.47g)、TBS-溴乙酸酯(1mmol,0.25g)溶解于乙腈(ACN,80mL)中,加入烧瓶中,通入氮气,密封,在油浴中70℃搅拌24h,恢复室温,加入终止剂4-氨基-1,2-十六烷基丁二醚(1.5mmol,0.83g)继续搅拌6h,加入250ml无水乙醚,搅拌30min,过滤滤液得沉淀,将所得沉淀加入二氯甲烷(DCM,100ml)中,并依次加入丙酰氯(1mmol,0.09g)及无水吡啶(Pyridine,50ml),室温搅拌6h,旋转蒸发去除溶剂等挥发物,沉淀采用无水乙醚漂洗,将漂洗后所得沉淀加入甲醇(50ml),同时加入水(30ml)及碳酸钾(15mmol,2.07g),室温搅拌6h,旋蒸去除溶剂、将获得的沉淀加入二氯甲烷(30ml),过滤滤液旋蒸,所得固形物通过G25凝胶柱去离子水洗脱纯化得化合物6洗脱液,冷冻干燥得粉末化合物6(2.74g,54%)。1HNMR(CDCl3,400MHz)δ:4.84(s,1H),3.52-3.42(m,238H),3.38-3.21(m,8H),3.02-2.93(m,3H),2.27-2.14(m,180H),1.33-1.22(m,48H),0.87(t,6H)。
实施例7人促红细胞生成素(hEPO)mRNA脂质纳米粒(hEPO-mRNA LNP)的制备与检测
将阳离子脂质(SM-102)(艾伟拓(上海)医药科技有限公司)、DSPC(磷脂)(艾伟拓(上海)医药科技有限公司)、胆甾醇(艾伟拓(上海)医药科技有限公司)和聚(2-噁唑啉)脂质(实施例1~实施例6)以50∶10∶38.5∶1.5的摩尔比溶于乙醇中制备乙醇脂质溶液,并将hEPO-mRNA用25mM柠檬酸盐缓冲液(pH=4)中配制mRNA水溶液。使用微流控设备以1∶3的速度比混合乙醇脂质溶液和mRNA水溶液,SM-102与mRNA的N/P比为6∶1制备脂质纳米粒。经微流控设备制备的hEPO-mRNALNP混悬液在4℃温度下透析24h除去乙醇并调整pH值为中性(透析液为pH7.4磷酸氢二钠/磷酸二氢钠缓冲液、透析膜截留分子量为8-14kD),并定容至100μg/mL。最后,hEPO-mRNA LNP混悬液通过0.2μm无菌过滤器过滤,得到可使用hEPO-mRNA LNP终制剂。使用BeNano 180纳米粒度仪(丹东百特仪器有限公司),以173°反向散射检测模式通过动态光散射测定脂质纳米颗粒的大小及多分散指数PDI,测试结果见表1。使用Quant-iT Ribogreen RNA定量测定试剂盒(Thermo Fisher)确定脂质纳米颗粒的包封效率,测试结果见表1。
表1
从表1可以看出本发明制备的核酸药物粒径较小,包封率高。
实施例8 hEPO-mRNA LNP动物在体测试
按照0.5mg/kg的剂量对4~6周龄雌性ICR小鼠通过尾静脉注射施用部分实施例7制备的hEPO-mRNA LNP,并在给药后6h采集小鼠血液,采集完血液样品后,使用二氧化碳将小鼠安乐处死。在4℃下以4800g离心15分钟从全血样本中分离血清,采集血清样本液氮速冻并在-80℃下储存以用于分析。使用人促红细胞生成素(hEPO)Quantikine IVD ELISA试剂盒对采集的血清样本进行ELSA分析,检测hEPO表达水平(ng/mL)详见表2。
表2检测结果
通过动物在体测试,本发明聚(2-噁唑啉)脂质形成的LNP能够递送核酸药物,将核酸分子成功转运至细胞中并进行表达。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种聚(2-噁唑啉)脂质,其特征在于,所述聚(2-噁唑啉)脂质的结构式为
式1聚合度n独立的为10~100的任一整数;
所述L1为C1-C5烷基;
所述的R为H、C1-C18烷基、
所述的R1为H、
所述R2为C1-C5烷基、
所述R3为
所述R4为
2.根据权利要求1所述的一种聚(2-噁唑啉)脂质,其特征在于,所述聚合度n独立的为30~70的任一整数。
3.根据权利要求2所述的一种聚(2-噁唑啉)脂质,其特征在于,所述L1为CH2、C2H4、或C3H6;
所述的R独立为H、CH3、C2H5、
所述的R1独立为H、
所述R2为CH3、
所述R3为
所述R4为
4.根据权利要求1~2任一项所述的一种聚(2-噁唑啉)脂质,其特征在于,所述聚(2-噁唑啉)脂质的结构式为:
5.一种包含聚(2-噁唑啉)脂质的脂质纳米粒,其特征在于,包含可离子化脂质、甾醇类化合物、磷脂和聚(2-噁唑啉)脂质;所述可离子化脂质、甾醇类化合物、磷脂和聚(2-噁唑啉)脂质的摩尔比为40~60∶25~40∶5~20∶0.5~5;所述聚(2-噁唑啉)脂质为权利要求1~4任一项所述的聚(2-噁唑啉)脂质。
6.根据权利要求5所述的脂质纳米粒,其特征在于,所述可离子化脂质的结构通式为
其中,X为C2~C5直链烷基或CH2CH2OCH2CH2;
Y为(C=O)O;
R5独立的为C10~C20直链烷基、C10~C20直链烯基或C10~C24酯基;
R6独立的为C5~C10直链烷基;
R7独立的为C10~C24支链烷基。
7.根据权利要求6所述的脂质纳米粒,其特征在于,所述甾醇类化合物为胆甾醇、二氢胆甾醇、豆甾醇、二氢豆甾醇、菜油甾醇、麦角甾醇或二氢谷甾醇。
8.根据权利要求7所述的脂质纳米粒,其特征在于,所述磷脂为二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二油酰磷脂酰乙醇胺、二油酰磷脂酰胆碱、二硬脂酰磷脂酰甘油、二棕榈酰磷脂酰甘油、二肉豆蔻酰磷脂酰甘油、二硬脂酰磷脂酸和二棕榈酰磷脂酸中的一种或几种。
9.权利要求5~8任一项所述的脂质纳米粒在制备核酸药物中的应用,其特征在于,所述核酸药物包含脂质纳米粒和核酸分子,所述脂质纳米粒中可离子化脂质的氮原子与核酸分子中的磷酸基团的摩尔比为1.5~12∶1。
10.根据权利要求9所述的应用,其特征在于,所述核酸药物还包括pH调节剂和保护剂;
所述pH调节剂为药学接受的pH调节剂;
所述保护剂为甘油、蔗糖、海藻糖、葡萄糖、甘油葡萄糖苷和四氢嘧啶中的一种。
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