CN110038138B - 一种靶向Aβ老年斑的普鲁士蓝纳米颗粒及其制备方法 - Google Patents
一种靶向Aβ老年斑的普鲁士蓝纳米颗粒及其制备方法 Download PDFInfo
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Abstract
本发明涉及生物医学领域的纳米材料,特别涉及一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法。通过对普鲁士蓝纳米颗粒进行功能化修饰,可以负载具有穿过血脑屏障功能的转铁蛋白和靶向Aβ老年斑的刚果红分子,所制备的纳米颗粒具有良好的r1纵向弛豫率和r2横向弛豫率,可以实现对Aβ老年斑的MRI(T1/T2)同时成像。
Description
技术领域
本发明涉及生物医学领域的纳米材料,特别涉及一种靶向Aβ老年斑的普鲁士蓝纳米颗粒及其制备方法。
背景技术
阿尔茨海默症(Alzheimer's disease,AD)是一种神经退行性疾病,主要临床表现为记忆力和认知能力下降、日常生活能力下降等,典型的病理特征是β淀粉样蛋白(amyloidβpeptide,Aβ)在细胞外沉积、细胞内tau蛋白过度磷酸化形成神经纤维缠结和神经元缺失。其中Aβ沉积造成的老年斑会引起脑组织中的一系列病变,如炎症、氧化应激、线粒体功能障碍等,在AD病程中发挥着重要作用,被普遍认为是AD诊断的理想靶点。MRI具有无放射性、空间分辨率高、适用范围广等优点,但现有MRI示踪剂对Aβ老年斑缺乏特异性和靶向性,限制了AD的MRI临床诊断。
普鲁士蓝是一种古老的蓝色染料,具有良好的生物相容性及生物安全性,已被美国食品药品监督局认证临床治疗铊等放射性元素中毒的解毒剂。普鲁士蓝制备过程简单、反应条件温和且易于表面修饰,由于本身独特的电子自旋特性具有MRI的T1成像和T2成像能力,近年来其在生物医学诊断领域的应用已成为研究热点。但是,普鲁士蓝纳米颗粒很难进行靶向性修饰,难以精确聚集到病变部位,且目前大部分合成的颗粒多用于肿瘤诊断。因此,制备一种具有靶向Aβ老年斑功能的普鲁士蓝纳米颗粒对于AD精准诊断具有重要意义。
据报道,转铁蛋白受体在血脑屏障中呈高表达,以转铁蛋白为主动靶向分子特异性结合转铁蛋白受体,能够使纳米材料有效穿过血脑屏障,可作为跨血脑屏障的有效靶向剂。刚果红是一种长线状酸性偶氮染色剂,以氢键的方式将其氨基和淀粉样蛋白的羟基结合,从而嵌入两个毗邻的淀粉样纤维间,平行地附着到淀粉样纤维上,因此,刚果红与淀粉样纤维具有很强的亲和力,可作为Aβ老年斑的靶向剂。
发明内容
本发明的目的旨在提供一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,该纳米颗粒可主动穿过血脑屏障并靶向Aβ沉积的老年斑,从而实现AD的精确诊断。
本发明的技术方案是一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,包括如下步骤:
1)将亚铁氰化钾与羧基化聚乙二醇分别溶于去离子水中,充分混匀得到澄清溶液A;
将氯化铁溶于去离子水中,充分溶解得到澄清溶液B;
将溶液B逐滴加入溶液A中,亚铁氰化钾与氯化铁摩尔比为1:1,40~80℃下反应0.5~2h;
2)待反应体系降至室温,反应0.5~2h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于去离子水中,室温反应10~30min,得到溶液D;
4)将N-羟基琥珀酰亚胺、转铁蛋白、刚果红按照一定浓度加入到溶液D中,N-羟基琥珀酰亚胺溶液与所述步骤3)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液的摩尔比为1:(1~10),转铁蛋白溶液浓度为0.5~2.5μg/mL,刚果红溶液浓度为0.004~0.06mmol/L室温反应12~36h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所述步骤1)中亚铁氰化钾水溶液浓度为1~5mmol/L。
所述步骤1)中氯化铁水溶液浓度为1~5mmol/L。
所述步骤1)中羧基化聚乙二醇包括单羧基聚乙二醇(mPEG-COOH)、二羧基聚乙二醇(COOH-PEG-COOH)、多臂聚乙二醇羧基(nARM-PEG-COOH,n=3,4,5……);所述的羧基化聚乙二醇水溶液浓度为0.1~2mmol/L。
所述步骤3)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为1~20mmol/L。
所述步骤4)中N-羟基琥珀酰亚胺溶液浓度为1~20mmol/L。
本发明的第二个技术方案是在普鲁士蓝纳米颗粒的基础上对其进行功能化修饰,利用合成的普鲁士蓝纳米颗粒上的羧基基团与转铁蛋白和刚果红分子上的氨基相结合,制备出一种靶向Aβ老年斑的普鲁士蓝纳米颗粒,粒径在150~250nm,紫外吸收光谱在260~300nm、450~530nm、650~800nm具有最大紫外吸收峰。
本发明与现有技术相比优点如下:
1.制备的纳米颗粒具有良好的生物相容性及生物安全性,稳定性好;
2.通过对普鲁士蓝纳米颗粒进行功能化修饰,可以负载具有穿过血脑屏障功能的转铁蛋白和靶向Aβ老年斑的刚果红分子,所制备的纳米颗粒具有良好的r1纵向弛豫率和r2横向弛豫率,可以实现对Aβ老年斑的MRI(T1/T2)同时成像。
3.提供了一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,该方法可重复性高,操作简单,绿色环保。
附图说明
图1:实施例1中制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的粒径分布图。
图2:实施例1中制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的透射电镜照片(形貌分析)。
图3:实施例1中制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的紫外可见吸收光谱图。
图4:实施例2中制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的核磁纵向弛豫率图(MRI造影剂成像效果分析)。
图5:实施例2中制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的核磁横向弛豫率图(MRI造影剂成像效果分析)。
具体实施方式
下面的实施例中将对本发明做进一步的阐述,但本发明范围不限于此。
实施例1:
1)将0.01毫摩尔亚铁氰化钾与0.001毫摩尔mPEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.01毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,60℃下反应0.5h;
2)待反应体系降至室温,反应0.5h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.1毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应15min,得到溶液D;
4)将0.1毫摩尔N-羟基琥珀酰亚胺、0.01毫克转铁蛋白、0.4微摩尔刚果红加入到溶液D中,室温反应24h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为180nm,如图1所示为制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的粒径分布图;如图2所示为制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的透射电镜图;如图3紫外吸收图谱所示,所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒在288nm、495nm、710nm处显示出最大紫外吸收峰,说明转铁蛋白和刚果红成功连接到普鲁士蓝纳米颗粒上。
实施例2:
1)将0.005毫摩尔亚铁氰化钾与0.005毫摩尔mPEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.005毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,55℃下反应0.5h;
2)待反应体系降至室温,反应1h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.05毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应10min,得到溶液D;
4)将0.04毫摩尔N-羟基琥珀酰亚胺、0.005毫克转铁蛋白、0.48微摩尔刚果红加入到溶液D中,室温反应24h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为210nm,紫外吸收光谱在285.5nm、500nm、715nm具有最大紫外吸收峰。如图4核磁纵向驰豫率测试结果所示,所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒的纵向弛豫率r1为6.84mM-1·s-1;如图5核磁横向弛豫率测试结果所示,横向弛豫率r2为5.93mM-1·s-1,说明此纳米颗粒具有良好的MRI(T1/T2)成像能力。
实施例3:
1)将0.0195毫摩尔亚铁氰化钾与0.003毫摩尔COOH-PEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.0195毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,60℃下反应1h;
2)待反应体系降至室温,反应0.5h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.12毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应25min,得到溶液D;
4)将0.02毫摩尔N-羟基琥珀酰亚胺、0.02毫克转铁蛋白、0.6微摩尔刚果红加入到溶液D中,室温反应12h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为245nm,紫外吸收光谱在292nm、507nm、692.5nm具有最大紫外吸收峰。
实施例4:
1)将0.013毫摩尔亚铁氰化钾与0.004毫摩尔4ARM-PEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.013毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,70℃下反应2h;
2)待反应体系降至室温,反应1h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.18毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应10min,得到溶液D;
4)将0.06毫摩尔N-羟基琥珀酰亚胺、0.023毫克转铁蛋白、0.2微摩尔刚果红加入到溶液D中,室温反应12h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为195nm,紫外吸收光谱在280.5nm、483nm、720nm具有最大紫外吸收峰。
实施例5:
1)将0.022毫摩尔亚铁氰化钾与0.007毫摩尔mPEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.022毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,65℃下反应1h;
2)待反应体系降至室温,反应1h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.08毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应15min,得到溶液D;
4)将0.08毫摩尔N-羟基琥珀酰亚胺、0.019毫克转铁蛋白、0.32微摩尔刚果红加入到溶液D中,室温反应18h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为160nm,紫外吸收光谱在283nm、493.5nm、717nm具有最大紫外吸收峰。
实施例6:
1)将0.0095毫摩尔亚铁氰化钾与0.0095毫摩尔COOH-PEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.0095毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,60℃下反应1h;
2)待反应体系降至室温,反应1.5h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.13毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应15min,得到溶液D;
4)将0.1毫摩尔N-羟基琥珀酰亚胺、0.009毫克转铁蛋白、0.52微摩尔刚果红加入到溶液D中,室温反应24h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为170nm,紫外吸收光谱在278nm、502nm、687.5nm具有最大紫外吸收峰。
实施例7:
1)将0.0175毫摩尔亚铁氰化钾与0.0025毫摩尔8ARM-PEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.0175毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,60℃下反应0.5h;
2)待反应体系降至室温,反应2h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.2毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应20min,得到溶液D;
4)将0.1毫摩尔N-羟基琥珀酰亚胺、0.015毫克转铁蛋白、0.24微摩尔刚果红加入到溶液D中,室温反应36h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为225nm,紫外吸收光谱在276.5nm、508.5nm、705.5nm具有最大紫外吸收峰。
实施例8:
1)将0.014毫摩尔亚铁氰化钾与0.008毫摩尔mPEG-COOH分别溶于5毫升去离子水中,充分混匀得到澄清溶液A;将0.014毫摩尔氯化铁溶于5毫升去离子水中,充分溶解得到澄清溶液B;将溶液B逐滴加入溶液A中,60℃下反应1.5h;
2)待反应体系降至室温,反应1h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C、0.15毫摩尔1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于10毫升去离子水中,室温反应10min,得到溶液D;
4)将0.03毫摩尔N-羟基琥珀酰亚胺、0.012毫克转铁蛋白、0.08微摩尔刚果红加入到溶液D中,室温反应24h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
所制备的靶向Aβ老年斑的普鲁士蓝纳米颗粒其粒径约为175nm,紫外吸收光谱在284nm、504nm、709nm具有最大紫外吸收峰。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,其特征在于,包括如下步骤:
1)将亚铁氰化钾与羧基化聚乙二醇分别溶于去离子水中,充分混匀得到澄清溶液A;
将氯化铁溶于去离子水中,充分溶解得到澄清溶液B;
将溶液B逐滴加入溶液A中,亚铁氰化钾与氯化铁摩尔比为1:1,40~80℃下反应0.5~2h;
2)待反应体系降至室温,反应0.5~2h,离心洗涤,得到含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C;
3)将含有羧基的聚乙二醇修饰的普鲁士蓝纳米颗粒的沉淀C与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于去离子水中,室温反应10~30min,得到溶液D;
4)将N-羟基琥珀酰亚胺、转铁蛋白、刚果红按照一定浓度加入到溶液D中,N-羟基琥珀酰亚胺溶液与所述步骤3)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液的摩尔比为1:(1~10),转铁蛋白溶液浓度为0.5~2.5μg/mL,刚果红溶液浓度为0.004~0.06mmol/L,室温反应12~36h,离心洗涤,所得沉淀用去离子水重悬,得到靶向Aβ老年斑的普鲁士蓝纳米颗粒。
2.根据权利要求1所述的一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,其特征在于,所述步骤1)中亚铁氰化钾水溶液浓度为1~5mmol/L。
3.根据权利要求1所述的一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,其特征在于,所述步骤1)中氯化铁水溶液浓度为1~5mmol/L。
4.根据权利要求1所述的一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,其特征在于,所述步骤1)中羧基化聚乙二醇包括单羧基聚乙二醇、二羧基聚乙二醇、多臂聚乙二醇羧基;所述的羧基化聚乙二醇水溶液浓度为0.1~2mmol/L。
5.根据权利要求1所述的一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,其特征在于,所述步骤3)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液浓度为1~20mmol/L。
6.根据权利要求1所述的一种靶向Aβ老年斑的普鲁士蓝纳米颗粒的制备方法,其特征在于,所述步骤4)中N-羟基琥珀酰亚胺溶液浓度为1~20mmol/L。
7.一种靶向Aβ老年斑的普鲁士蓝纳米颗粒,采用权利要求1至6中任一项所述的制备方法制备得到,其特征在于,粒径在150~250nm,紫外吸收光谱在260~300nm、450~530nm、650~800nm具有最大紫外吸收峰。
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