CN110917359A - 离子键自组装制备PEG-P(Asp-AP)-ANTAGOMIR-RNA微球 - Google Patents
离子键自组装制备PEG-P(Asp-AP)-ANTAGOMIR-RNA微球 Download PDFInfo
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Abstract
本发明属于生物医用领域,具体涉及通过离子键自组装制备聚(乙二醇)‑b‑聚[N‑(5‑氨基戊基)‑α,β‑天冬酰胺]‑ANTAGOMIR‑RNA微球及其制备方法。本发明所述制备方法能够通过离子键作用进行自组装制备得到PEG‑P(Asp‑AP)‑ANTAGOMIR‑RNA微球,操作步骤简单,无设备要求,成本低,操作简单;本发明方法将ANTAGOMIR‑RNA通过离子键结合装入微球,避免了ANTAGOMIR‑RNA因为外界因素而失活或变性;所制备的微球能够通过离子键的结合与断键的原理,由于病灶部位病理微环境与正常组织存在差异,从而有望实现特异性刺激相应,在靶向治疗中有独特的优势和应用前景。
Description
技术领域
本发明属于生物医用领域,具体涉及通过离子键自组装制备聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球及其制备方法。
背景技术
生物活性大分子通常具有丰富的生命功能,但是它因易被体内环境广泛的RNA酶、DNA酶、蛋白酶等酶解、变性和氧化,限制了其在医用领域上的应用。MicroRNA是微小RNA的简称,是一类长度在19~25核苷酸的RNA,在人体的基因中大部分的表达都受到MicroRNA的调控。MicroRNA能够对细胞的分化、增殖、凋亡进行多种机制的调控,而在许多癌细胞中,已有文献证明MicroRNA处于正常表达之上,故认其为癌细胞的特征表达片段之一。
MicroRNA在抗癌领域方面,根据不同癌细胞的不同表达程度,被确认为可控阻碍片段,并通过实验证明使MicroRNA表达下降时,出现明显的抗癌效果。所以现在不少MicroRNA阻断剂都确认为有效特异性抗癌药物。
ANTAGOMIR-RNA是根据microRNA成熟体序列设计,经过特殊标记与化学修饰的单链小RNA,是专门用于抑制内源性microRNA的高效阻断剂,具有比普通MicroRNA阻断剂更加优异的亲细胞膜性能,而且可富集于靶向细胞,表现出高效的特异性稳定干扰,并拥有着长效性,效果最长可以持续五周左右。但是由于环境条件限制,该类阻断剂只能置于低温长期储存。
聚合物微球具有可调节核-壳结构,产生与外部环境隔离的特定空间或内核,该环境为保护生物活性大分子的活性,避免被酶解、被氧化等方面提供了条件。聚合物微球已经被设计用于细胞或细胞器模拟物,纳米/微反应器和药物的递送载体,除了简单的保护屏障之外,微球壳还具有作为通用储库的功能。两亲性嵌段共聚物微球,即聚合物微球,可以在其疏水膜中包含疏水性低分子量药物。然而,有效且稳定地嵌入生物活性大分子,特别是强亲水性或带电荷的生物活性大分子,仍是一个需要解决的难题,因为由于负载时所使用的物理或者化学方法使生物大分子易被氧化、被分解和变性等特征上限制了生物活性大分子能够嵌入共聚物中的种类,并且嵌入的生物活性大分子的量的增加更可能损害微球外壳的完整性,使微球的构成不稳定。
发明内容
本发明针对现有技术的不足,目的在于提供通过离子键自组装制备聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球及其制备方法。
为实现上述发明目的,本发明所采用的技术方案为:
聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球的制备方法,包括如下步骤:
(1)将一定量的聚(乙二醇)-b-聚(β-苄基-L-天冬氨酸)(PEG-b-PBLA)溶解在N-甲基吡咯烷酮(NMP)中得到PEG-b-PBLA溶液;
(2)将PEG-b-PBLA溶液逐滴添加到NMP与1,5-二氨基戊烷互溶溶液中,5℃条件下剧烈搅拌1~2小时;随后冰浴条件下使用乙酸中和反应所得混合物,再置于4℃条件下依次用HCl溶液、去离子水透析产物后,冻干,得到PEG-b-P(Asp-AP);
(3)将PEG-b-P(Asp-AP)和ANTAGOMIR-RNA分别溶解于羟乙基哌嗪乙硫磺酸(HEPES)缓冲液中;通过涡旋混合将PEG-b-P(Asp-AP)溶液和ANTAGOMIR-RNA溶液混合均匀,得到前驱基底材料;
(4)将前驱基底材料与溶解在HEPES缓冲液的戊二醛(GA)溶液混合,置于25℃温育25分钟后,用甘氨酸淬灭过量的GA,得到PEG-b-PBLA-ANTAGOMIR-RNA微球。
上述方案中,所述聚(乙二醇)-b-聚(β-苄基-L-天冬氨酸)中,PEG的分子量Mn为2000~5000g/mol,PBLA的聚合度DP=70~100。
上述方案中,所述PEG-b-PBLA与1,5-二氨基戊烷的质量体积比为100mg:2.7mL。
上述方案中,所述HCl溶液的浓度为0.01M~0.1M,透析袋截留分子量Mw=3500~4000。
上述方案中,所述羟乙基哌嗪乙硫磺酸(HEPES)缓冲液的pH值为7.3~7.8之间。
上述方案中,所述PEG-b-P(Asp-AP)嵌段共聚物中的氨基与所述ANTAGOMIR-RNA中的磷酸基团的摩尔比为7:5~7:10。
上述方案中,所述前驱基底材料中未反应氨基的量与戊二醛的摩尔比为5:2~5:4,其中剩余氨基是通过第(4)步中的投料比在理论中未反应的氨基来确定。
上述方案中,步骤(5)所述戊二醛(GA)充当交联剂的作用,溶液的浓度为10~15mM,所述甘氨酸的摩尔当量浓度为戊二醛(GA)溶液浓度的10倍。
本发明的有益效果:本发明所述制备方法能够通过离子键作用进行自组装制备得到PEG-P(Asp-AP)-ANTAGOMIR-RNA微球,操作步骤简单,无设备要求,成本低,操作简单;本发明方法将ANTAGOMIR-RNA通过离子键结合装入微球,避免了ANTAGOMIR-RNA因为外界因素而失活或变性;所制备的微球能够通过离子键的结合与断键的原理,由于病灶部位病理微环境与正常组织存在差异,从而有望实现特异性刺激相应,在靶向治疗中有独特的优势和应用前景。
附图说明
图1为本发明方法通过离子键结合制备PEG-P(Asp-AP)-ANTAGOMIR-RNA微球的原理示意图。
图2为本发明实施例1所制备的PEG-P(Asp-AP)-ANTAGOMIR-RNA微球80kv下透射电子显微镜(TEM)照片。
图3为本发明合成前驱体的化学式过程。
具体实施方式
为了更好地理解本发明,下面结合实施例进一步阐明本发明的内容,但本发明的内容不仅仅局限于下面的实施例。
实施例1
聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球的制备方法,包括如下步骤:
(1)将100mg冻干的PEG-b-PBLA溶解在5mL N-甲基吡咯烷酮(NMP)中得到PEG-b-PBLA溶液;将PEG-b-PBLA溶液逐滴添加到2.5mL NMP与2.7mL 1,5-二氨基戊烷互溶溶液中,5℃条件下剧烈搅拌1小时;完成搅拌后,在冰浴条件下使用14mL、20%(v/v)乙酸中和反应所得混合物,再置于4℃条件下依次用0.01M HCl溶液透析产物(透析袋的截留分子量Mw为3500,透析36h)、用去离子水透析产物(透析袋的截留分子量Mw为3500,透析12h)后,冻干,得到PEG-b-P(Asp-AP);
(2)将PEG-b-P(Asp-AP)和ANTAGOMIR-RNA分别以0.25~1.0mg/mL溶解于10mM羟乙基哌嗪乙硫磺酸(HEPES)缓冲液(pH7.3)中,并通过0.22μm膜滤器过滤;通过涡旋混合将PEG-b-P(Asp-AP)嵌段共聚物溶液中的氨基和ANTAGOMIR-RNA中的磷酸基团以7:5的比例混合均匀,得到前驱基底材料;
(3)将前驱基底材料与溶解在10mM HEPES缓冲液(pH7.3)的戊二醛(GA)溶液混合,其中前驱基底材料中未反应氨基的量与戊二醛的摩尔比为5:2,置于25℃温育25分钟后,随后用10摩尔当量于GA的甘氨酸在25℃处理24小时以淬灭过量的GA,得到PEG-b-PBLA-ANTAGOMIR-RNA微球。
实施例2
聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球的制备方法,包括如下步骤:
(1)将100mg冻干的PEG-b-PBLA溶解在5mL N-甲基吡咯烷酮(NMP)中得到PEG-b-PBLA溶液;将PEG-b-PBLA溶液逐滴添加到2.5mL NMP与2.7mL 1,5-二氨基戊烷互溶溶液中,5℃条件下剧烈搅拌1小时;完成搅拌后,在冰浴条件下使用14mL、20%(v/v)乙酸中和反应所得混合物,再置于4℃条件下依次用0.01M HCl溶液透析产物(透析袋的截留分子量Mw为3500,透析36h)、用去离子水透析产物(透析袋的截留分子量Mw为3500,透析12h)后,冻干,得到PEG-b-P(Asp-AP);
(2)将PEG-b-P(Asp-AP)和ANTAGOMIR-RNA分别以0.25~1.0mg/mL溶解于10mM羟乙基哌嗪乙硫磺酸(HEPES)缓冲液(pH7.3)中,并通过0.22μm膜滤器过滤;通过涡旋混合将PEG-b-P(Asp-AP)嵌段共聚物溶液中的氨基和ANTAGOMIR-RNA中的磷酸基团以7:5的比例混合均匀,得到前驱基底材料;
(3)将前驱基底材料与溶解在10mM HEPES缓冲液(pH7.3)的戊二醛(GA)溶液混合,其中前驱基底材料中未反应氨基的量与戊二醛的摩尔比为5:4,置于25℃温育25分钟后,随后用10摩尔当量于GA的甘氨酸在25℃处理24小时以淬灭过量的GA,得到PEG-b-PBLA-ANTAGOMIR-RNA微球。
实施例3
聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球的制备方法,包括如下步骤:
(1)将100mg冻干的PEG-b-PBLA溶解在5mL N-甲基吡咯烷酮(NMP)中得到PEG-b-PBLA溶液;将PEG-b-PBLA溶液逐滴添加到2.5mL NMP与2.7mL 1,5-二氨基戊烷互溶溶液中,5℃条件下剧烈搅拌1小时;完成搅拌后,在冰浴条件下使用14mL、20%(v/v)乙酸中和反应所得混合物,再置于4℃条件下依次用0.01M HCl溶液透析产物(透析袋的截留分子量Mw为3500,透析36h)、用去离子水透析产物(透析袋的截留分子量Mw为3500,透析12h)后,冻干,得到PEG-b-P(Asp-AP);
(2)将PEG-b-P(Asp-AP)和ANTAGOMIR-RNA分别以0.25~1.0mg/mL溶解于10mM羟乙基哌嗪乙硫磺酸(HEPES)缓冲液(pH7.3)中,并通过0.22μm膜滤器过滤;通过涡旋混合将PEG-b-P(Asp-AP)嵌段共聚物溶液中的氨基和ANTAGOMIR-RNA中的磷酸基团以7:10的比例混合均匀,得到前驱基底材料;
(3)将前驱基底材料与溶解在10mM HEPES缓冲液(pH7.3)的戊二醛(GA)溶液混合,其中前驱基底材料中未反应氨基的量与戊二醛的摩尔比5:2,置于25℃温育25分钟后,随后用10摩尔当量于GA的甘氨酸在25℃处理24小时以淬灭过量的GA,得到PEG-b-PBLA-ANTAGOMIR-RNA微球。
实施例4
聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球的制备方法,包括如下步骤:
(1)将100mg冻干的PEG-b-PBLA溶解在5mL N-甲基吡咯烷酮(NMP)中得到PEG-b-PBLA溶液;将PEG-b-PBLA溶液逐滴添加到2.5mL NMP与2.7mL 1,5-二氨基戊烷互溶溶液中,5℃条件下剧烈搅拌1小时;完成搅拌后,在冰浴条件下使用14mL、20%(v/v)乙酸中和反应所得混合物,再置于4℃条件下依次用0.01M HCl溶液透析产物(透析袋的透析分子量Mw为3500,透析36h)、用去离子水透析产物(透析袋的透析分子量Mw为3500,透析12h)后,冻干,得到PEG-b-P(Asp-AP);
(2)将PEG-b-P(Asp-AP)和ANTAGOMIR-RNA分别以0.25~1.0mg/mL溶解于10mM羟乙基哌嗪乙硫磺酸(HEPES)缓冲液(pH7.3)中,并通过0.22μm膜滤器过滤;通过涡旋混合将PEG-b-P(Asp-AP)嵌段共聚物溶液中的氨基和ANTAGOMIR-RNA中的磷酸基团以7:10比例混合均匀,得到前驱基底材料;
(3)将前驱基底材料与溶解在10mM HEPES缓冲液(pH7.3)的戊二醛(GA)溶液混合,其中前驱基底材料中未反应氨基的量与戊二醛的摩尔比为5:4,置于25℃温育25分钟后,随后用10摩尔当量于GA的甘氨酸在25℃处理24小时以淬灭过量的GA,得到PEG-b-PBLA-ANTAGOMIR-RNA微球。
显然,上述实施例仅仅是为清楚地说明所作的实例,而并非对实施方式的限制。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。因此所引申的显而易见的变化或变动仍处于本发明创造的保护范围之内。
Claims (8)
1.聚(乙二醇)-b-聚[N-(5-氨基戊基)-α,β-天冬酰胺]-ANTAGOMIR-RNA微球的制备方法,其特征在于,包括如下步骤:
(1)将一定量的聚(乙二醇)-b-聚(β-苄基-L-天冬氨酸)(PEG-b-PBLA)溶解在N-甲基吡咯烷酮(NMP)中得到PEG-b-PBLA溶液;
(2)将PEG-b-PBLA溶液逐滴添加到NMP与1,5-二氨基戊烷互溶溶液中,5℃条件下剧烈搅拌1~2小时;随后冰浴条件下使用乙酸中和反应所得混合物,再置于4℃条件下依次用HCl溶液、去离子水透析产物后,冻干,得到PEG-b-P(Asp-AP);
(3)将PEG-b-P(Asp-AP)和ANTAGOMIR-RNA分别溶解于羟乙基哌嗪乙硫磺酸(HEPES)缓冲液中;通过涡旋混合将PEG-b-P(Asp-AP)溶液和ANTAGOMIR-RNA溶液混合均匀,得到前驱基底材料;
(4)将前驱基底材料与溶解在HEPES缓冲液的戊二醛(GA)溶液混合,置于25℃温育25分钟后,用甘氨酸淬灭过量的GA,得到PEG-b-PBLA-ANTAGOMIR-RNA微球。
2.根据权利要求1所述的制备方法,其特征在于,所述聚(乙二醇)-b-聚(β-苄基-L-天冬氨酸)中PEG的分子量Mn为2000~5000g/mol,PBLA的聚合度DP=70~100。
3.根据权利要求1所述的制备方法,其特征在于,所述PEG-b-PBLA与1,5-二氨基戊烷的质量体积比为100mg:2.7mL。
4.根据权利要求1所述的制备方法,其特征在于,所述HCl溶液的浓度为0.01M~0.1M,透析袋截留分子量Mw=3500~4000。
5.根据权利要求1所述的制备方法,其特征在于,所述羟乙基哌嗪乙硫磺酸(HEPES)缓冲液的pH值为7.3~7.8之间。
6.根据权利要求1所述的制备方法,其特征在于,所述PEG-b-P(Asp-AP)嵌段共聚物中的氨基与所述ANTAGOMIR-RNA中的磷酸基团的摩尔比为7:5~7:10。
7.根据权利要求1所述的制备方法,其特征在于,所述前驱基底材料中未反应氨基的量与戊二醛的摩尔比为5:2~5:4,其中剩余氨基是通过第(4)步中的投料比在理论中未反应的氨基来确定。
8.根据权利要求1所述的制备方法,其特征在于,步骤(5)所述戊二醛(GA)充当交联剂的作用,溶液的浓度为10~15mM,所述甘氨酸的摩尔当量浓度为戊二醛(GA)溶液浓度的10倍。
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