CN110507830A - 一种用于阿尔茨海默病致病蛋白的纳米探针及其制备 - Google Patents
一种用于阿尔茨海默病致病蛋白的纳米探针及其制备 Download PDFInfo
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- CN110507830A CN110507830A CN201910935823.6A CN201910935823A CN110507830A CN 110507830 A CN110507830 A CN 110507830A CN 201910935823 A CN201910935823 A CN 201910935823A CN 110507830 A CN110507830 A CN 110507830A
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Abstract
本发明属于纳米材料和生物医学分子影像技术领域,具体涉及一种用于阿尔茨海默病致病蛋白的纳米探针及其制备;本发明所述纳米探针为多模态纳米探针,由超小铁氧体纳米颗粒、聚乙二醇衍生物和吩噻嗪衍生物制得;所述纳米探针的内核为超小铁氧体纳米颗粒,外层为聚乙二醇片段偶联吩噻嗪衍生物;所述的多模态纳米探针可特异性与β‑淀粉样蛋白斑块结合,不仅具有独特的近红外荧光标记增强效应和T1‑T2磁共振影像对比增强效应;同时该探针还具有超小尺寸、良好的生物相容性、无辐射性和无潜在神经毒性等多种优点,该探针在阿尔茨海默病的早期诊断方面具有良好的应用前景。
Description
技术领域
本发明属于纳米材料和生物医学分子影像技术领域,具体涉及一种用于阿尔茨海默病致病蛋白的纳米探针及其制备。
背景技术
阿尔茨海默病(Alzheimer's disease,AD)是一种常见的痴呆病变类型,是以进行性认知功能障碍和行为损害为特征的中枢神经系统退行性病变。虽然,国内外投入了大量资金进行AD疾病的药物研发,但目前大部分研究均以失败告终5,其中最主要的原因可能是缺乏有效的早期诊断,治疗给药的时间已经处于晚期,药物治疗效果甚微。因此,目前研究人员的主要挑战就是如何利用临床有效手段进行阿尔茨海默病的病理特征分析、实时跟踪病理变化,以实现阿尔茨海默病的早期诊断,并有根据的进行预防和干预。
淀粉样蛋白级联假说是目前AD发病机制的主流学说之一,即淀粉样前体蛋白(amyloid peptide precursor,APP)的β-淀粉样多肽(β-Amyloid,Aβ),如Aβ40、Aβ42在大脑中的聚集,对阿尔茨海默病的发病具有重大的影响。而该类蛋白的聚集通常早在疾病出现临床症状前,甚至是早在20年前就已经存在了。因此,能够构建阿尔茨海默病诊断制剂,对该类致病蛋白进行分子层面的标记和跟踪,是实现早期阿尔茨海默病诊断的有效途径之一。
目前,人们在诊断制剂方面进行了一定程度的研究,比如正电子发射断层扫描(PET)成像探针11C-PIB,荧光成像分子NIAD,Methoxy-X04,AOI-987,CRANAD-2等。然而,这些标志物与放射性元素耦合、用药适用范围受限、长期跟踪监测困难以及高额的诊断费用等问题,影响了其在AD疾病的早期诊断方面的广泛应用。
发明内容
为了解决上述问题,本发明的目的在于提供一种用于阿尔茨海默病致病蛋白的纳米探针。
本发明的目的之二是提供了用于阿尔茨海默病致病蛋白的纳米探针的制备方法。
本发明是通过以下技术方案来实现的:
一种用于阿尔茨海默病致病蛋白的纳米探针,所述纳米探针为多模态纳米探针,内核为超小铁氧体纳米颗粒,外层为聚乙二醇片段偶联吩噻嗪衍生物;所述纳米探针的水动力学直径为1-30nm;所述纳米探针包含按重量份计算的以下组分:超小铁氧体纳米颗粒5-20份、聚乙二醇衍生物1-5份、吩噻嗪衍生物0.1-2份。
本发明选择超小铁氧体纳米颗粒,利用其优秀的r1弛豫率,构建具有靶向T1成像能力的探针,不仅避免了传统钆的T1对比剂带来的潜在神经损伤,而且其超小的尺寸也有利于血脑屏障的穿透。同时,吩噻嗪衍生物在探针中的偶联和引入,使探针具备了优异的Aβ绑定标记能力,以及近红外荧光特异性增强能力。聚乙二醇衍生物用于连接铁氧纳米颗粒和吩噻嗪类衍生物。
较佳地,所述的吩噻嗪衍生物结构为:
其中,m为双键的个数,包括且不限于以下数字,1、2、3等;n为烷基链个数,包括且不限于以下数字,1、2、3等;R1为连接聚乙二醇衍生物的功能基团,为甲氧基类、羧基类、氨基类、巯基类、马来酰亚胺类、叠氮类、生物素或N-琥珀酰亚胺基等分子;R2为修饰基团,为氢、羟基、甲基、乙基、卤素、羧基、伯胺基或叔胺基等分子。
较佳地,所述的聚乙二醇为双端基取代,端基为甲氧基、氨基、巯基、羧基、马来酰亚胺基或炔基。
较佳地,所述聚乙二醇衍生物结构式为:
其中,R1为连接铁氧体纳米颗粒的功能基团,为烷基、氨基、羧基、巯基、磷酸盐类、异羟肟酸类或邻苯二酚类等分子;R2为连接吩噻嗪衍生物的功能基团,为甲氧基类、羧基类、氨基类、巯基类、马来酰亚胺类、叠氮类、生物素类或N-琥珀酰亚胺基等分子;n指的是聚乙二醇的重复单元数目,根据所需要的分子量,n可以从1-200任何数字。
较佳地,所述超小铁氧体纳米颗粒的粒径为1-6nm,所述的超小铁氧体纳米颗粒的制备方法为:先将前驱体与油剂混合均匀,然后将混合物溶于溶剂中搅拌均匀,接着将混有溶剂的混合物加热至265℃±2℃,并在此温度下保持25-35min;然后将反应物冷却、乙醇洗涤、离心分离,最后得到所述超小铁氧体纳米颗粒;所述前驱体、油剂、溶剂之间的质量比为1:0.7-0.8:3-4;
其中,所述前驱体为铁芥酸络合物和/或锰油酸络合物,所述溶剂为苄醚、二苄醚或十八烯中的一种或几种混合物;所述油剂为油酸、油醇或油胺中的一种或几种组合物。
所述前驱体由如下方法制备而得:将盐、芥酸、氢氧化钠混合均匀后,溶于甲醇,于40±2℃下磁力搅拌,反应1-2小时,用去离子水和甲醇洗涤产物,所得到的在45±2℃真空中干燥12小时得到所述铁芥酸络合物;所述盐、芥酸、氢氧化钠之间的质量比为1:3-4:0.4-0.5,所述盐、酸、氢氧化钠的混合物与甲醇之间的固液比为1:10-11g/mL;所述盐为氧化铁、油酸铁、芥酸铁、油酸锰、芥酸锰中的一种或几种组合物。
用于阿尔茨海默病致病蛋白的纳米探针的制备方法,包括以下制备步骤:
(1)将超小铁氧体纳米颗粒加入至有三氯甲烷中,并加入一种或几种聚乙二醇双端基取代衍生物,加热、超声、搅拌反应,反应后除去有机溶剂;
(2)冷却至室温,加入水相溶液,超声、过滤后透析、超滤,取分散良好的水相超小铁氧体纳米颗粒溶液,冷冻干燥为粉末或放置于4℃待用;
(3)将吩噻嗪衍生物溶解至二甲基亚砜中,加入1-乙基-(3-二甲基氨基丙基)碳二亚胺,N-羟基琥珀酰亚胺进行活化反应,然后加入水相分散的步骤(2)中得到的纳米颗粒溶液,反应4小时;
(4)将步骤(3)得到的反应产物在蒸馏水中透析,取透析后的溶液,冷冻干燥,即得述用于阿尔茨海默病致病蛋白的纳米探针。
较佳地,所述步骤(1)中,反应温度为20-70℃,超声时间为10-30分钟,反应时间为2-12小时,反应后利用旋转真空去除溶剂。
较佳地,其特征在于,所述步骤(2)中,超声时间为10-30分钟,过滤的滤膜孔径为0.22-0.45微米,冷冻干燥时间为12-24小时。
较佳地,其特征在于,所述步骤(3)中,吩噻嗪衍生物与1-乙基-(3-二甲基氨基丙基)碳二亚胺和N-羟基琥珀酰亚胺的质量比为1:(2-4):(2-6);纳米颗粒溶液反应浓度为1-5mg/mL,纳米颗粒与吩噻嗪衍生物的质量比为1:(0.1-2)。较佳地,其特征在于,所述步骤(4)中,透析时所用的透析袋的截留分子量为500-14000Da,冷冻干燥时间为12-24小时。
本发明提供的靶向多模态纳米探针包括具有有效诊断能力的纳米颗粒物和可分散的生理水溶液。所述诊断物由超小铁氧体纳米颗粒、聚乙二醇衍生物包裹和偶联的吩噻嗪衍生物构成,吩噻嗪衍生物具有特定的供体受体分子结构,并具有优秀的Aβ聚集体特异性的亲和力。可实现对活体AD疾病小鼠模型的脑部Aβ斑块的靶向T1-T2MRI/NIRF三模态成像。
本发明利用超小铁氧体纳米颗粒,偶联具有Aβ特异性和敏感性的近红外荧光增强性能的吩噻嗪类衍生物,构建新型超小多模态纳米探针用于进行Aβ标记的T1-T2MRI/NIRF成像,本发明涉及的纳米探针具有超小的粒径,无钆神经毒性,生物相容性良好,属于无辐射,非入侵型探针,为早期诊断和长期示踪Aβ斑块,进行AD疾病的诊断,提供了良好的解决方案。
与现有技术相比,本发明的靶向Aβ的多模态纳米探针,将靶向致病蛋白、T1-MRI、T2-MRI和近红外荧光成像示踪为集合的整体,可实现阿尔茨海默病小鼠模型的大脑中致病蛋白斑块的定位成像,利用高空间分辨率的MRI成像,显示解剖学细节的T1-MRI成像细节,活体下定位斑块在脑中的位置,因其具有高生物相容性,体内代谢能力,不会存在多次对比剂给药导致的钆神经毒性。因此,该探针在阿尔茨海默病的早期诊断方面具有非常广泛的应用前景。
附图说明
图1为油相的超小铁氧体纳米颗粒的透射电子显微镜照片图
图2为实施例1制备的靶向多模态纳米探针的透射电子显微镜照片图
图3为实施例1制备的靶向多模态纳米探针的水动力学粒径分布图
图4为水相的超小铁氧体纳米颗粒的透射电子显微镜照片图
图5为水相的超小铁氧体纳米颗粒的水动力学粒径分布图
图6为靶向多模态纳米探针的脑切片染色NIR荧光显微图
图7为靶向多模态纳米探针的体内NIR荧光成像图
图8为靶向多模态纳米探针的体外NIR荧光成像图
图9为靶向多模态纳米探针的体外T1-T2MRI成像图
图10为靶向多模态纳米探针的体内T1-T2MRI成像图
具体的实施方式
下面结合具体实施方式对本发明作进一步的详细说明,以助于本领域技术人员理解本发明。
实施例1
一种用于阿尔茨海默病致病蛋白的纳米探针,由以下方法制备而得:
(1)称取重量组分为1份的粒径约为3nm的超小铁氧体纳米颗粒,加入5mL三氯甲烷,将混合物与重量组分为5份的邻二苯酚-聚乙二醇2000-氨基/甲氧基混合后,室温下超声10min,使纳米颗粒和高分子聚合物充分分散和溶解,混合后将烧瓶置于旋转蒸发仪上,温度为60℃,抽真空旋转蒸发,通过表面配体交换作用,在颗粒表面形成一层包裹有良好水溶性的聚乙二醇单层的分子,当有机溶剂蒸发后,超小铁氧体纳米颗粒可以成功转移至水相。
(2)待纳米颗粒冷却至室温后,用超声将纳米颗粒均匀分散在水中。经0.22微米的滤膜过滤后,再超滤离心,取分散良好的纳米胶体溶液,冷冻干燥12小时,得到粉末状的纳米颗粒,复溶至备用。
(3)将步骤(2)得到的纳米颗粒加入至1mL的水中,得到胶体溶液备用。另将0.1份的吩噻嗪衍生物溶解于甲基亚砜,加入2倍质量的1-乙基-(3-二甲基氨基丙基)碳二亚胺,2倍质量的N-羟基琥珀酰亚胺进行活化反应,反应2h,然后加入至纳米胶体溶液中,继续反应4h。
(4)将步骤(3)得到的反应混合物用10000Da的透析袋在流动的蒸馏水中透析过夜,将得到的透析液取出,超滤,并进一步冷冻干燥,得到本发明所述用于阿尔茨海默病致病蛋白的纳米探针。
用透射电子显微镜检测获得的本发明纳米探针的尺寸和形貌,结果见图2。用动态光散射仪检测本实验得到纳米探针的水动力学粒径分布,结果见图3。由图2,图3可知,本实施例获得的纳米探针的无机核心尺寸未发生明显变化,均在3nm的超小纳米粒径范围。另外,由于高分子聚合物的存在,纳米探针的水动力学直径约为20nm,说明其在水相中仍保持良好的尺寸分布,溶胶稳定性良好,分散均匀。
其中,超小铁氧体纳米颗粒的制备方法如下:
将2.7g氯化铁,10.2g芥酸,随后加入1.2g氢氧化钠,溶于150mL甲醇,40℃磁力搅拌。反应完成后,用去离子水和甲醇洗涤产物,所得到的在45℃真空中干燥12小时。2.14g铁芥酸络合物、0.62g锰油酸络合物,加入0.57g油酸,1.61g的油醇溶于10g的苄醚溶剂中。反应混合物随后加热至265℃,并在此温度下保持30min。反应后,冷却反应混合物,乙醇洗涤,离心分离,得到超小粒径的铁氧体纳米颗粒。
用透射电子显微镜检测颗粒的尺寸和形貌以及均一性,见图1。如图1所示,超小铁氧体纳米颗粒的平均粒径为3nm,粒子呈球状且具有良好的单分散性。
实施例2
本发明的靶向多模态纳米探针的一个实施例,其制备方法如下。
(1)称取重量组分为5份的粒径约为3nm的超小铁氧体纳米颗粒,加入5mL三氯甲烷,将混合物与重量组分为20份的邻二苯酚-聚乙二醇2000-氨基/甲氧基混合后,室温下超声10min,使纳米颗粒和高分子聚合物充分分散和溶解,混合后将烧瓶置于旋转蒸发仪上,温度为60℃,抽真空旋转蒸发,通过表面配体交换作用,在颗粒表面形成一层包裹有良好水溶性的聚乙二醇单层的分子,当有机溶剂蒸发后,超小铁氧体纳米颗粒可以成功转移至水相。
(2)待纳米颗粒冷却至室温后,用超声将纳米颗粒均匀分散在水中。经0.22微米的滤膜过滤后,再超滤离心,取分散良好的纳米胶体溶液,冷冻干燥12小时,得到粉末状的纳米颗粒,复溶至备用。
(3)将步骤(2)得到的纳米颗粒加入至1mL的水中,得到胶体溶液备用。另将2份的吩噻嗪衍生物溶解于甲基亚砜,加入4倍质量的1-乙基-(3-二甲基氨基丙基)碳二亚胺,6倍质量的N-羟基琥珀酰亚胺进行活化反应,反应2h,然后加入至纳米胶体溶液中,继续反应4h。
(4)将步骤(3)得到的反应混合物用10000Da的透析袋在流动的蒸馏水中透析过夜,将得到的透析液取出,超滤,并进一步冷冻干燥,得到本发明所述的靶向多模态纳米探针。
用透射电子显微镜检测获得的本实验得到纳米探针的尺寸和形貌,用动态光散射仪检测本实验得到纳米探针的水动力学粒径分布,结果与图2,图3一致。说明纳米探针的溶胶稳定性良好,分散均匀。
其中,超小铁氧体纳米颗粒的制备方法同实施例1。
实施例3
本发明的靶向多模态纳米探针的对照组的一个实施例,其制备方法如下。
(1)称取重量组分为1份的粒径约为5nm的超小铁氧体纳米颗粒,加入5mL三氯甲烷,将混合物与重量组分为5份的邻二苯酚-聚乙二醇2000-氨基/甲氧基混合后,室温下超声10min,使纳米颗粒和高分子聚合物充分分散和溶解,混合后将烧瓶置于旋转蒸发仪上,温度为60℃,抽真空旋转蒸发,通过表面配体交换作用,在颗粒表面形成一层包裹有良好水溶性的聚乙二醇单层的分子,当有机溶剂蒸发后,超小铁氧体纳米颗粒可以成功转移至水相。
(2)待纳米颗粒冷却至室温后,用超声将纳米颗粒均匀分散在水中。经0.22微米的滤膜过滤后,再超滤离心,取分散良好的纳米胶体溶液,即得对比组纳米探针。
用透射电子显微镜检测获得的本实验得到对比组纳米探针的尺寸和形貌,结果见图4。用动态光散射仪检测本实验得到纳米探针的水动力学粒径分布,结果见图5。由图4和图5可知,本实施例获得的纳米探针的无机核心尺寸均在3nm的超小纳米粒径范围。另外,由于高分子聚合物的存在,纳米探针的水动力学直径约为20nm,说明其在水相中仍保持良好的尺寸分布,溶胶稳定性良好,分散均匀。
其中,超小铁氧体纳米颗粒的制备方法同实施例1。
实施例4
一种用于阿尔茨海默病致病蛋白的纳米探针,由以下方法制备而得:
(1)称取重量组分为1份的粒径约为2nm的超小铁氧体纳米颗粒,加入5mL三氯甲烷,将混合物与重量组分为5份的邻二苯酚-聚乙二醇2000-氨基/甲氧基混合后,室温下超声20min,使纳米颗粒和高分子聚合物充分分散和溶解,混合后将烧瓶置于旋转蒸发仪上,温度为60℃,抽真空旋转蒸发,通过表面配体交换作用,在颗粒表面形成一层包裹有良好水溶性的聚乙二醇单层的分子,当有机溶剂蒸发后,超小铁氧体纳米颗粒可以成功转移至水相。
(2)待纳米颗粒冷却至室温后,用超声将纳米颗粒均匀分散在水中。经0.3微米的滤膜过滤后,再超滤离心,取分散良好的纳米胶体溶液,冷冻干燥20小时,得到粉末状的纳米颗粒,复溶至备用。
(3)将步骤(2)得到的纳米颗粒加入至1mL的水中,得到胶体溶液备用。另将0.1份的吩噻嗪衍生物溶解于甲基亚砜,加入3倍质量的1-乙基-(3-二甲基氨基丙基)碳二亚胺,4倍质量的N-羟基琥珀酰亚胺进行活化反应,反应2h,然后加入至纳米胶体溶液中,继续反应4h。
(4)将步骤(3)得到的反应混合物用8000Da的透析袋在流动的蒸馏水中透析过夜,将得到的透析液取出,超滤,并进一步冷冻干燥,得到本发明所述用于阿尔茨海默病致病蛋白的纳米探针。
用其中,超小铁氧体纳米颗粒的制备方法如下:
将2.7g油酸铁,8.1g芥酸,随后加入1.08g氢氧化钠,溶于150mL甲醇,40℃磁力搅拌。反应完成后,用去离子水和甲醇洗涤产物,所得到的在45℃真空中干燥12小时。2.14g铁芥酸络合物、0.62g锰油酸络合物,加入0.57g油酸,1.61g的油醇溶于10g的苄醚溶剂中。反应混合物随后加热至265℃,并在此温度下保持35min。反应后,冷却反应混合物,乙醇洗涤,离心分离,得到超小粒径的铁氧体纳米颗粒。
实施例5
一种用于阿尔茨海默病致病蛋白的纳米探针,由以下方法制备而得:
(1)称取重量组分为1份的粒径约为10nm的超小铁氧体纳米颗粒,加入5mL三氯甲烷,将混合物与重量组分为4份的邻二苯酚-聚乙二醇2000-氨基/甲氧基混合后,室温下超声30min,使纳米颗粒和高分子聚合物充分分散和溶解,混合后将烧瓶置于旋转蒸发仪上,温度为60℃,抽真空旋转蒸发,通过表面配体交换作用,在颗粒表面形成一层包裹有良好水溶性的聚乙二醇单层的分子,当有机溶剂蒸发后,超小铁氧体纳米颗粒可以成功转移至水相。
(2)待纳米颗粒冷却至室温后,用超声将纳米颗粒均匀分散在水中。经0.45微米的滤膜过滤后,再超滤离心,取分散良好的纳米胶体溶液,冷冻干燥24小时,得到粉末状的纳米颗粒,复溶至备用。
(3)将步骤(2)得到的纳米颗粒加入至1mL的水中,得到胶体溶液备用。另将0.1份的吩噻嗪衍生物溶解于甲基亚砜,加入4倍质量的1-乙基-(3-二甲基氨基丙基)碳二亚胺,6倍质量的N-羟基琥珀酰亚胺进行活化反应,反应2h,然后加入至纳米胶体溶液中,继续反应4h。
(4)将步骤(3)得到的反应混合物用14000Da的透析袋在流动的蒸馏水中透析过夜,将得到的透析液取出,超滤,并进一步冷冻干燥,得到本发明所述用于阿尔茨海默病致病蛋白的纳米探针。
用其中,超小铁氧体纳米颗粒的制备方法如下:
将2.7g芥酸铁,10.8g芥酸,随后加入1.35g氢氧化钠,溶于150mL甲醇,40℃磁力搅拌。反应完成后,用去离子水和甲醇洗涤产物,所得到的在45℃真空中干燥12小时。2.14g铁芥酸络合物、0.62g锰油酸络合物,加入0.77g油酸,1.48g的油醇溶于11.04g的苄醚溶剂中。反应混合物随后加热至265℃,并在此温度下保持30min。反应后,冷却反应混合物,乙醇洗涤,离心分离,得到超小粒径的铁氧体纳米颗粒。
上述实施例,只是本发明的较佳实施例,并非用来限制本发明实施范围,故凡以本发明权利要求所述的特征及原理所做的等效变化或修饰,均应包括在本发明权利要求范围之内。
本发明中聚乙二醇衍生物是合成探针中步骤所需的分子,用于连接铁氧纳米颗粒和吩噻嗪类衍生物。该类双端基取代的聚乙二醇分子可在一些商家买到。例如,如果使用聚氧乙烯二胺(CAS号24991-53-5)或聚乙二醇二羧酸(CAS号39927-08-7),可以在阿拉丁试剂公司购得。
应用本发明所得的用于阿尔茨海默病致病蛋白的纳米探针进行相应的实验,其结果详见效果例。
效果例1
本发明所述的靶向多模态纳米探针的体外NIR验证标记效果。
取12月龄的APP/PS1阳性小鼠和同年龄的野生型小鼠的大脑切片,将其与靶向多模态纳米探针进行共孵育,然后用乙醇和PBS进行洗片,再加入硫黄素进行二次染色,乙醇和PBS再次进行洗片,干燥后置于荧光显微镜下观察。结果如图6所示,靶向多模态纳米探针和硫黄素在脑片中增强显示的位置吻合良好,而野生型小鼠的大脑切片则没有明显的荧光增强发生,实验结果说明靶向纳米探针具有良好的标记能力,为之后的动物实验奠定了基础。
效果例2
本发明所述的靶向多模态纳米探针的体内NIR验证标记效果。
将12月龄的APP/PS1阳性小鼠和同年龄的野生型小鼠实施麻醉,进行NIR荧光成像,获得对照组的图像,然后对小鼠静脉注射实施例1中制备的靶向多模态纳米探针,注射后进行不同时间的脑部活体近红外荧光成像,结果见图7。从图中可以看出,纳米探针在AD阳性小鼠中存留的时间长6小时均具有很强的信号,而在野生型的小鼠的脑部,信号则随时间变化,逐渐减弱。进一步将小鼠处死取材,获得脑部切片,并进行荧光显微镜检测,见图8。检测结果显示,相较于没有明显信号变化的野生型对照小鼠,AD阳性小鼠的大脑呈现明显的斑块荧光增强效果。
效果例3
本发明所述的靶向多模态纳米探针的体外MRI验证标记效果。
取相同浓度的实施例1和实施例3的纳米探针溶液,加入等量的Aβ蛋白聚集体,孵育30分钟后离心除上清,获得Aβ蛋白聚集体混合物,用T1-T2MRI扫描检测后,得到图9。样品1、2、3分别代表Aβ蛋白聚集体加入不同制剂(PBS,实施例3对比组,实施例1实验组)后的收集结果。如图所示,相较于未加探针、和加入对照组探针的样品,加入靶向多模态探针的实验组获得了T1和T2MRI信号极大程度的增强,说明了探针对于Aβ蛋白聚集体靶向的特异性。
效果例4
本发明所述的靶向多模态纳米探针的体内MRI验证标记效果。
将12月龄的APP/PS1阳性小鼠和同年龄的野生型小鼠实施麻醉,进行T1和T2MRI成像,获得对照组的图像,然后对小鼠注射实施例1中制备的靶向多模态纳米探针,注射后进行不同时间的脑部T1和T2MRI成像,结果见图10。从图中可以看出,打药前AD阳性小鼠的大脑T1T2MRI图像中没有明显的变化和差异,而在打药后的AD阳性小鼠中,T1MRI图像中明显看到变亮的像点,T2MRI图像中同位置处也可以明显看到变暗的像点,两图像中的像点吻合良好,说明了Aβ斑块在AD小鼠脑内的存在和定位。
以上实验结果图说明本发明所述的Aβ靶向多模态纳米探针具有良好的活体Aβ斑块标记能力,且探针组成中无放射性组分、无钆离子的潜在毒性、生物相容性良好、探针在活体内的标记特异性和敏感性高,是一种在阿尔茨海默病早期诊断方面具有优秀的潜在应用价值的活体靶向标记示踪探针。
Claims (10)
1.一种用于阿尔茨海默病致病蛋白的纳米探针,其特征在于:所述纳米探针为多模态纳米探针,内核为超小铁氧体纳米颗粒,外层为聚乙二醇片段偶联吩噻嗪衍生物;所述纳米探针的水动力学直径为1-30nm;所述纳米探针包含按重量份计算的以下组分:
超小铁氧体纳米颗粒 5-20份
聚乙二醇衍生物 1-5份
吩噻嗪衍生物 0.1-2份。
2.如权利要求1所述用于阿尔茨海默病致病蛋白的纳米探针,其特征在于:所述的吩噻嗪衍生物结构为:
其中,m为双键的个数,n为烷基链个数;R1为甲氧基类、羧基类、氨基类、巯基类、马来酰亚胺类、叠氮类、生物素或N-琥珀酰亚胺基分子;R2为氢、羟基、甲基、乙基、卤素、羧基、伯胺基或叔胺基分子。
3.如权利要求1所述用于阿尔茨海默病致病蛋白的纳米探针,其特征在于:所述的聚乙二醇为双端基取代,端基为甲氧基、氨基、巯基、羧基、马来酰亚胺基或炔基。
4.如权利要求1所述用于阿尔茨海默病致病蛋白的纳米探针,其特征在于:所述聚乙二醇衍生物结构式为:
其中,R1为烷基、氨基、羧基、巯基、磷酸盐类、异羟肟酸类或邻苯二酚类分子;R2为甲氧基类、羧基类、氨基类、巯基类、马来酰亚胺类、叠氮类、生物素类或N-琥珀酰亚胺基分子;n为1-200。
5.如权利要求1所述用于阿尔茨海默病致病蛋白的纳米探针,其特征在于:所述超小铁氧体纳米颗粒的粒径为1-10nm,所述的超小铁氧体纳米颗粒的制备方法为:先将前驱体与油剂混合均匀,然后将混合物溶于溶剂中搅拌均匀,接着将混有溶剂的混合物加热至265℃±2℃,并在此温度下保持25-35min;然后将反应物冷却、乙醇洗涤、离心分离,最后得到所述超小铁氧体纳米颗粒;所述前驱体、油剂、溶剂之间的质量比为1:0.7-0.8:3-4;
其中,所述前驱体为铁芥酸络合物和/或锰油酸络合物,所述溶剂为苄醚、二苄醚或十八烯中的一种或几种混合物;所述油剂为油酸、油醇或油胺中的一种或几种组合物。
6.如权利要求5所述用于阿尔茨海默病致病蛋白的纳米探针,其特征在于:所述前驱体由如下方法制备而得:将盐、芥酸、氢氧化钠混合均匀后,溶于甲醇,于40±2℃下磁力搅拌,反应1-2小时,用去离子水和甲醇洗涤产物,所得到的在45±2℃真空中干燥12小时得到所述铁芥酸络合物;所述盐、芥酸、氢氧化钠之间的质量比为1:3-4:0.4-0.5,所述盐、酸、氢氧化钠的混合物与甲醇之间的固液比为1:10-11g/ml;所述盐为氧化铁、油酸铁、芥酸铁、油酸锰、芥酸锰中的一种或几种组合物。
7.如权利要求1-6中任一项所述用于阿尔茨海默病致病蛋白的纳米探针的制备方法,其特征在于,包括以下制备步骤:
(1)将超小铁氧体纳米颗粒加入至有三氯甲烷中,并加入一种或几种聚乙二醇双端基取代衍生物,加热、超声、搅拌反应,反应后除去有机溶剂;
(2)冷却至室温,加入水相溶液,超声、过滤后透析、超滤,取分散良好的水相超小铁氧体纳米颗粒溶液,冷冻干燥为粉末或放置于4℃待用;
(3)将吩噻嗪衍生物溶解至二甲基亚砜中,加入1-乙基-(3-二甲基氨基丙基)碳二亚胺,N-羟基琥珀酰亚胺进行活化反应,然后加入水相分散的步骤(2)中得到的纳米颗粒溶液,反应4小时;
(4)将步骤(3)得到的反应产物在蒸馏水中透析,取透析后的溶液,冷冻干燥,即得述用于阿尔茨海默病致病蛋白的纳米探针;透析时所用的透析袋的截留分子量为500-14000Da,冷冻干燥时间为12-24小时。
8.如权利要求7所述的用于阿尔茨海默病致病蛋白的纳米探针的制备方法,其特征在于,所述步骤(1)中,反应温度为20-70℃,超声时间为10-30分钟,反应时间为2-12小时,反应后利用旋转真空去除溶剂。
9.如权利要求7所述的用于阿尔茨海默病致病蛋白的纳米探针的制备方法,其特征在于,所述步骤(2)中,超声时间为10-30分钟,过滤的滤膜孔径为0.22-0.45微米,冷冻干燥时间为12-24小时。
10.如权利要求7所述的用于阿尔茨海默病致病蛋白的纳米探针的制备方法,其特征在于,所述步骤(3)中,吩噻嗪衍生物与1-乙基-(3-二甲基氨基丙基)碳二亚胺和N-羟基琥珀酰亚胺的质量比为1:2-4:2-6;纳米颗粒溶液反应浓度为1-5mg/mL,纳米颗粒与吩噻嗪衍生物的质量比为1:0.1-2。
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