CN116270730A - 一种用于脑部多巴供应的mRNA响应纳米催化剂及其制备方法 - Google Patents
一种用于脑部多巴供应的mRNA响应纳米催化剂及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于脑部多巴供应的mRNA响应纳米催化剂,其是以四氧化三铁纳米颗粒作为模拟TH的催化中心,酪氨酸结合序列的寡核苷酸作为捕获内源性酪氨酸的特异性结合位点,靶向序列的寡核苷酸作为血脑屏障内皮细胞结合及mRNA响应位点构建的纳米材料。其中,四氧化三铁纳米颗粒可以在脑部微环境模拟TH催化功能;酪氨酸结合序列可以捕获内源性酪氨酸,增加酪氨酸的局部浓度;靶向序列可以封闭酪氨酸结合序列的功能,并使纳米材料被血脑屏障内皮细胞主动转运进入脑部,并在脑部响应SNCA的mRNA以恢复酪氨酸结合序列的功能,因此,本发明所得催化剂在体内可以实现脑部的持续多巴生成,有望为帕金森症的治疗提供新的技术支持。
Description
技术领域
本发明属于生物医药领域,具体涉及一种用于脑部多巴供应的mRNA响应纳米催化剂及其制备方法。
背景技术
帕金森症是目前世界上第二大神经退行性疾病,体现为严重的运动障碍。帕金森症的主要病理变化是黑质多巴胺能神经元的变性和死亡,以及由此导致的纹状体多巴胺含量的显著减少。口服多巴是目前治疗帕金森症最有效的方法。多巴可以穿过血脑屏障,在神经元中转化为多巴胺。然而,由于多巴的半衰期较短,服用多巴会导致多巴的血浆水平大幅波动,从而引发多巴诱导的运动障碍。此外,多巴在外周组织中的代谢会导致全身副作用。
发明内容
本发明的目的在于提供一种用于脑部多巴供应的mRNA响应纳米催化剂及其制备方法,其采用一种简单的方法设计出了一种能经过血液循环到达脑部并响应α-突触核蛋白mRNA进行催化多巴生成的纳米材料,有望应用于帕金森症治疗药物的研究。
为实现上述目的,本发明采用如下技术方案:
一种用于脑部多巴供应的mRNA响应纳米催化剂,其包括三个组成部分:
(1)模拟酪氨酸羟化酶的催化中心;
(2)捕获内源性酪氨酸的特异性结合位点(以下称为酪氨酸结合序列);
(3)血脑屏障内皮细胞结合及mRNA响应位点(以下称为靶向序列)。
进一步地,所述模拟酪氨酸羟化酶的催化中心为四氧化三铁纳米颗粒。
进一步地,所述酪氨酸结合序列为:5’-TGTATGTGGTGTGTGAGTGCGGTGCCCAGTGTTC-3’。
进一步地,酪氨酸结合序列与四氧化三铁纳米颗粒通过共价键结合,其结合比例为每个四氧化三铁纳米颗粒上结合1~200条。
进一步地,所述靶向序列为:5’-GCGTGGTCACACGCGAGCCTACATAGAGAACACTGGGCA-3’。
进一步地,所述催化剂中酪氨酸结合序列与靶向序列通过非共价键结合,两者质量比为1:0.1~1:10。
所述用于脑部多巴供应的mRNA响应纳米催化剂是以羧基功能化的Fe3O4纳米颗粒、5’端氨基修饰的酪氨酸结合序列寡核苷酸和靶向序列寡核苷酸为原料,利用Fe3O4纳米颗粒表面的羧基和酪氨酸结合序列上修饰的氨基经酰胺缩合反应共价偶联,再利用酪氨酸结合序列和靶向序列的碱基互补配对形成双链,最终制备得到的;其制备方法包括以下步骤:
(1)Fe3O4纳米颗粒与酪氨酸结合序列的共价结合:将羧基功能化的Fe3O4纳米颗粒和酪氨酸结合序列在缓冲溶液中混合,并加入酰胺化偶联试剂,混合均匀后于4~40℃反应2~24h,反应后磁分离并收集产物,然后用二次蒸馏水洗涤数次,得到酪氨酸结合序列修饰的Fe3O4纳米颗粒;
(2)mRNA响应纳米催化剂的制备:将酪氨酸结合序列修饰的Fe3O4纳米颗粒与靶向序列在PBS缓冲溶液中混合,在4~40℃反应2~24h,反应后磁分离并收集产物,然后用二次蒸馏水洗涤数次,得到所述用于脑部多巴供应的mRNA响应纳米催化剂。
进一步地,步骤(1)中所用Fe3O4纳米颗粒与酪氨酸结合序列的质量比为1:10-3~1:1。
进一步地,步骤(1)中所用酰胺化偶联试剂与酪氨酸结合序列的摩尔比为(1~105):1;所述酰胺化偶联试剂为常规已知的酰胺化偶联试剂中的任意一种,如4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐、1,3-二环己基碳二亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐等。
除此之外,酪氨酸结合序列和四氧化三铁纳米颗粒也可通过叠氮基和炔基、马来酰亚胺和巯基等官能团修饰以产生共价键。
进一步地,步骤(1)中所用缓冲溶液为含有0.1 M 2-(N-吗啉)乙磺酸一水物的水溶液。
进一步地,步骤(2)中所用酪氨酸结合序列修饰的Fe3O4纳米颗粒与靶向序列的质量比为1:10-3~1:1。
所得用于脑部多巴供应的mRNA响应纳米催化剂能够通过在脑部催化酪氨酸产生多巴以补充多巴胺,因而可用于制备帕金森症治疗药物。
神经元的退化伴随着酪氨酸羟化酶的失活,酪氨酸羟化酶(TH)是催化儿茶酚胺神经递质合成的限速酶,负责催化酪氨酸转化为多巴。因此,用人工酶取代患病神经元中失活的酪氨酸羟化酶,是实现多巴持续稳定原位生成的理想选择。另外,核酸适配体作为底物特异性结合成分,其模拟生物酶的识别功能已得到广泛认可。核酸适配体与催化中心的结合可以大大提高细胞内的催化性能。因此,本发明以酪氨酸结合序列的寡核苷酸作为捕获内源性酪氨酸的特异性结合位点,与作为模拟酪氨酸羟化酶催化中心的Fe3O4纳米颗粒结合,实现在细胞内高效催化酪氨酸产生多巴。
另外,帕金森症的治疗需要两个先决条件,即纳米材料在脑部的富集和避免外周组织的多巴产生。因此本发明还引入了靶向序列作为第三个部分:一段结合血脑屏障内皮细胞并具有α-突触核蛋白(SNCA)mRNA响应区域的寡核苷酸。靶向序列与酪氨酸结合序列通过碱基互补配对形成双链结构,能够封闭酪氨酸结合序列的功能。该mRNA响应纳米催化剂在静脉注射后,其靶向序列与脑内皮细胞结合,通过受体介导的转运穿过血脑屏障到达脑部。在脑部帕金森症病变区域,纳米催化剂响应过度表达的SNCA,将靶向序列置换离去,然后在细胞内高效催化酪氨酸产生多巴。因此,这种用于脑部多巴供应的mRNA响应纳米催化剂,在实现脑部持续的多巴供应的同时,避免了传统多巴给药引起的多巴胺剧烈波动,对帕金森症治疗策略的发展具有重要的科学意义。
本发明的有益效果在于:
本发明提供了一种用于脑部多巴供应的mRNA响应纳米催化剂及其制备方法,该方法简单、原料易得,所得用于脑部多巴供应的mRNA响应纳米催化剂可以在静脉注射的情况下到达脑部,在帕金森症病变区域催化酪氨酸产生多巴,用于缓解帕金森症中的多巴胺缺失。
附图说明
图1为本发明所提供的用于脑部多巴供应的mRNA响应纳米催化剂的作用原理示意图。
图2为Fe3O4纳米颗粒、酪氨酸结合序列修饰的Fe3O4纳米颗粒(Apt-Fe3O4)的催化动力学曲线。
图3为用于脑部多巴供应的mRNA响应纳米催化剂(FNA-Fe3O4)在有或无mRNA情况下的催化动力学曲线。
图4为用于脑部多巴供应的mRNA响应纳米催化剂(FNA-Fe3O4)在细胞内的催化性能测试结果。
图5为用于脑部多巴供应的mRNA响应纳米催化剂(FNA-Fe3O4)在小鼠模型中的脑部催化性能测试结果。
具体实施方式
一种用于脑部多巴供应的mRNA响应纳米催化剂的制备包括以下步骤:
(1)Fe3O4纳米颗粒与酪氨酸结合序列的共价结合:将羧基功能化的Fe3O4纳米颗粒和酪氨酸结合序列在缓冲溶液中混合并加入酰胺化偶联试剂,混合均匀后于4~40℃反应2~24h,反应后磁分离并收集产物,然后用二次蒸馏水洗涤数次,得到酪氨酸结合序列修饰的Fe3O4纳米颗粒;
(2)mRNA响应纳米催化剂的制备:将酪氨酸结合序列修饰的Fe3O4纳米颗粒与靶向序列在PBS缓冲溶液中混合,在4~40℃反应2~24h,反应后磁分离并收集产物,然后用二次蒸馏水洗涤数次,得到所述用于脑部多巴供应的mRNA响应纳米催化剂;
所述酪氨酸结合序列为一段具有特定序列(5’-TGTATGTGGTGTGTGAGTGCGGTGCCCAGTGTTC-3’)的寡核苷酸,在其序列的5’端进行氨基修饰,每条酪氨酸结合序列修饰一个氨基;所述靶向序列为一段具有特定序列(5’-GCGTGGTCACACGCGAGCCTACATAGAGAACACTGGGCA-3’)的寡核苷酸。
步骤(1)中所用Fe3O4纳米颗粒与酪氨酸结合序列的质量比为1:10-3~1:1;所用酰胺化偶联试剂与酪氨酸结合序列的摩尔比为(1~105):1;所用缓冲溶液为含有0.1 M 2-(N-吗啉)乙磺酸一水物(MES)的水溶液。
步骤(2)中所用酪氨酸结合序列修饰的Fe3O4纳米颗粒与靶向序列的质量比为1:10-3~1:1。
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
所用酪氨酸结合序列及靶向序列均购自生工生物工程(上海)股份有限公司。
实施例
(1)将羧基功能化的Fe3O4纳米颗粒悬液(5 mg mL-1,40 μL)和酪氨酸结合序列溶液(100 μM,40 μL)加入到MES缓冲溶液(0.1 M,pH 5.5,100 μL)中,并加入EDC(10 mM,10 μL)和sulfo-NHS(25 mM,10 μL),混合均匀后于4 ℃反应12 h,反应后磁分离并收集产物,然后用二次蒸馏水洗涤3次,得到酪氨酸结合序列修饰的Fe3O4纳米颗粒;
(2)将酪氨酸结合序列修饰的Fe3O4纳米颗粒悬液(5 mg mL-1,40 μL)与靶向序列溶液(10 μM,400 μL)加入到PBS缓冲溶液(50 mM,pH 7.2,含有100 mM NaCl和5 mM MgCl2)中,在37 ℃反应3 h,反应后磁分离并收集产物,然后用二次蒸馏水洗涤3次,得到用于脑部多巴供应的mRNA响应纳米催化剂(FNA-Fe3O4)。
性能试验
1. 配制一系列含有:Fe3O4或Apt-Fe3O410 μg mL-1,抗坏血酸5 mM,H2O25 mM,以及酪氨酸10、25、50、100、250、500、1000、2000 mM的水溶液,放置0、1、2、3、4、5分钟时测量上清液中的多巴浓度,根据一系列酪氨酸浓度对应的反应初速度,作出催化动力学曲线。
2. 配制一系列含有:FNA-Fe3O4或FNA-Fe3O4+SNCA mRNA样品10 μg mL-1,抗坏血酸5 mM,H2O25 mM,以及酪氨酸10、25、50、100、250、500、1000、2000 mM的水溶液,放置0、1、2、3、4、5分钟时测量上清液中的多巴浓度,根据一系列酪氨酸浓度对应的反应初速度,作出催化动力学曲线。
3. 以人神经母细胞瘤细胞系SH-SY5Y为验证模型。将SH-SY5Y细胞接种在六孔培养皿中,在细胞培养箱中孵育24 h,用浓度为100 μg mL-1的FNA-Fe3O4的培养液孵育细胞。然后在不同时间点将细胞收集并裂解,通过HPLC测量细胞内的多巴胺含量。
4. 以C57BL/6J小鼠为验证模型。将浓度为1 mg mL-1的FNA-Fe3O4通过尾静脉注射入小鼠体内,在脑部纹状体区域植入微透析探针,收集纹状体微透析液,通过HPLC测量微透析液内的多巴及多巴胺含量。
图2为Fe3O4纳米颗粒、酪氨酸结合序列修饰的Fe3O4纳米颗粒(Apt-Fe3O4)的催化动力学曲线。如图所示,Fe3O4纳米颗粒的催化活性较低,而Apt-Fe3O4的催化活性更高,说明Apt-Fe3O4可以更高效的催化多巴的生成。
图3为mRNA响应纳米催化剂(FNA-Fe3O4)在有或无mRNA情况下的催化动力学曲线。如图所示,FNA-Fe3O4的催化性能较低,说明靶向序列封闭了酪氨酸结合序列的功能,而FNA-Fe3O4+SNCA mRNA的催化性能较高,说明FNA-Fe3O4在脑部可以更高效的催化多巴的生成。
图4为FNA-Fe3O4在细胞内的催化性能测试结果。如图所示,FNA-Fe3O4在正常细胞内不会催化多巴的生成,在帕金森症细胞模型中可诱导多巴胺的逐渐生成,说明FNA-Fe3O4具有mRNA响应性的催化功能。
图5为FNA-Fe3O4在小鼠模型脑部的催化性能测试结果。如图所示,FNA-Fe3O4在帕金森症小鼠模型中诱导了纹状体多巴和多巴胺的逐渐生成,说明FNA-Fe3O4能够在帕金森症小鼠模型中恢复纹状体的多巴胺含量。
由上述试验可见,本发明所提供的纳米催化剂可以在帕金森症病变区域持续催化酪氨酸产生多巴,以缓解帕金森症中的多巴胺缺失。
总之,本发明提供了一种用于脑部多巴供应的mRNA响应纳米催化剂及其制备方法,其将酪氨酸结合序列共价偶联到Fe3O4纳米颗粒上,并进一步结合了靶向序列,构建了所述的mRNA响应纳米催化剂。其方法简单,所制备的材料能够在mRNA响应的条件下催化多巴的生成,展现出在帕金森症的药物应用中的潜力。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (10)
1.一种用于脑部多巴供应的mRNA响应纳米催化剂,其特征在于,所述催化剂包括三个组成部分:
(1)模拟酪氨酸羟化酶的催化中心;
(2)捕获内源性酪氨酸的特异性结合位点;
(3)血脑屏障内皮细胞结合及mRNA响应位点;
所述模拟酪氨酸羟化酶的催化中心为四氧化三铁纳米颗粒。
2.根据权利要求1所述的用于脑部多巴供应的mRNA响应纳米催化剂,其特征在于,所述捕获内源性酪氨酸的特异性结合位点为一段具有特定序列的寡核苷酸,其序列为:5’-TGTATGTGGTGTGTGAGTGCGGTGCCCAGTGTTC-3’;其与四氧化三铁纳米颗粒通过共价键结合。
3.根据权利要求1所述的用于脑部多巴供应的mRNA响应纳米催化剂,其特征在于,所述血脑屏障内皮细胞结合及mRNA响应位点为一段具有特定序列的寡核苷酸,其序列为:5’-GCGTGGTCACACGCGAGCCTACATAGAGAACACTGGGCA-3’;其与捕获内源性酪氨酸的特异性结合位点通过非共价键结合。
4.一种如权利要求1所述的用于脑部多巴供应的mRNA响应纳米催化剂的制备方法,其特征在于,包括以下步骤:
(1)模拟酪氨酸羟化酶的催化中心与捕获内源性酪氨酸的特异性结合位点的共价结合:将四氧化三铁纳米颗粒经表面羧基功能化后与捕获内源性酪氨酸的特异性结合位点在缓冲溶液中混合,并加入酰胺化偶联试剂进行反应,磁分离并收集产物,得到特异性结合位点修饰的四氧化三铁纳米颗粒;
(2)mRNA响应纳米催化剂的制备:将特异性结合位点修饰的四氧化三铁纳米颗粒与血脑屏障内皮细胞结合及mRNA响应位点在PBS缓冲溶液中混合,经反应后磁分离并收集产物,得到所述用于脑部多巴供应的mRNA响应纳米催化剂。
5.根据权利要求4所述的用于脑部多巴供应的mRNA响应纳米催化剂的制备方法,其特征在于,步骤(1)中所用四氧化三铁纳米颗粒与捕获内源性酪氨酸的特异性结合位点的质量比为1:10-3~1:1。
6.根据权利要求4所述的用于脑部多巴供应的mRNA响应纳米催化剂的制备方法,其特征在于,步骤(1)中所用酰胺化偶联试剂与捕获内源性酪氨酸的特异性结合位点的摩尔比为(1~105):1。
7.根据权利要求4所述的用于脑部多巴供应的mRNA响应纳米催化剂的制备方法,其特征在于,步骤(1)中所用缓冲溶液为含有0.1 M 2-(N-吗啉)乙磺酸一水物的水溶液。
8.根据权利要求4所述的用于脑部多巴供应的mRNA响应纳米催化剂的制备方法,其特征在于,步骤(2)中所用酪氨酸结合序列修饰的Fe3O4纳米颗粒与血脑屏障内皮细胞结合及mRNA响应位点的质量比为1:10-3~1:1。
9.根据权利要求4所述的用于脑部多巴供应的mRNA响应纳米催化剂的制备方法,其特征在于,操作中所述反应的温度为4~40℃,时间为2~24h。
10.一种如权利要求1所述的用于脑部多巴供应的mRNA响应纳米催化剂在制备帕金森症治疗药物中的应用。
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