CN113200940B - 一种Aβ斑块响应型荧光探针及其制备和应用 - Google Patents
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Abstract
本发明提供一种Aβ斑块响应型荧光探针及其制备和应用,涉及响应型荧光探针设计合成与应用技术领域。所述Aβ斑块响应型荧光探针主要采用甲基苯并噻唑与碘甲烷发生甲基化反应后产物与4‑羟基‑2‑甲氧基苯甲醛发生缩合反应来制成。本发明克服了现有技术的不足,探针的合成路线简单,可操作性强,且目标探针具有良好的水溶性和生物相容性,为Aβ蛋白异常相关疾病尤其是AD的诊断和治疗提供潜在的应用价值。
Description
技术领域
本发明涉及响应型荧光探针设计合成与应用技术领域,具体涉及一种Aβ斑块响应型荧光探针及其制备和应用。
背景技术
目前,随着全球老龄化加剧,阿尔兹海默氏症(Alzheimer’s Disease,AD)发病率呈现逐年上升的趋势,已经严重威胁到人类健康和经济社会发展。在我国,60岁以上人群中患阿尔兹海默氏症达3%左右,确诊患者通常在诊断后7-10年内死亡,只有少数患者在发病后可以生存10年或更长,给患者家庭和社会造成了沉重的负担。
尽管AD的发病机制仍不明确,但是β-淀粉样蛋白(β-amyloid,Aβ蛋白)在神经元细胞外周沉积聚集形成的β-淀粉样斑块(Aβ-plaque,Aβ斑块)被认为是AD最重要的病理标志物之一。在研究和治疗阿尔兹海默症的方法中,利用功能性基团特异性识别Aβ蛋白并激活荧光信号,是近年来研究的热点。主要策略是:设计特异性靶向Aβ蛋白的小分子荧光探针,优化结构使其能够穿过血脑屏障,最终达到对阿尔兹海默症的早期诊断和有效治疗。
然而,目前报道的Aβ荧光探针仍旧存在难以穿过血脑屏障,生理毒性较大,对深组织的Aβ蛋白的成像效果较差等问题,难以进行后续的生物学应用。因此,设计合成新型小分子荧光探针检测异常的Aβ斑块水平,具有重要的科研及临床价值。
发明内容
针对现有技术不足,本发明提供一种Aβ斑块响应型荧光探针及其制备和应用,通过新设计构建的Aβ斑块响应型荧光探,在体外条件下,Aβ斑块能够有效激活探针的荧光信号,且不受BSA干扰,实现荧光信号从“OFF”到“ON”的转变,同时毒性低,方便后续生物学应用。
为实现以上目的,本发明的技术方案通过以下技术方案予以实现:
一种Aβ斑块响应型荧光探针,所述Aβ斑块响应型荧光探针的化学结构式如下所示:
所述Aβ斑块响应型荧光探针的制备方法包括以下步骤:
(1)将甲基苯并噻唑与碘甲烷发生甲基化反应,得到化合物M1;
(2)将化合物M1与4-羟基-2-甲氧基苯甲醛在弱碱存在下发生缩合反应,得到目标探针,称为探针BP。
优选的,所述步骤(1)中甲基苯并噻唑与碘甲烷发生甲基化反应在丙酮中进行,且甲基苯并噻唑与碘甲烷的摩尔比为1∶3。
优选的,所述步骤(2)中M1与4-羟基-2-甲氧基苯甲醛反应在乙腈/甲醇的混合溶剂中进行,且添加哌啶作为催化剂,所述M1与4-羟基-2-甲氧基苯甲醛的摩尔比为1:1。
优选的,所述甲基化反应和缩合反应均在氮气保护下进行,且甲基化反应为回流反应6h,缩合反应为回流反应12h。
所述Aβ斑块响应型荧光探针的应用包括在体内外检测Aβ斑块水平中的应用;或者在制备Aβ斑块检测试剂盒中的应用;或者在细胞共聚焦荧光成像中的应用;或者在小动物活体检测Aβ斑块中的应用。
优选的,所述Aβ斑块响应型荧光探针在细胞共聚焦荧光成像中的应用方式包括以下步骤:
(1)将Aβ斑块响应型荧光探针溶液加入细胞中,培养孵化后吸去培养液;
(2)将上述培养孵化后的细胞加入加入缓冲液,进行荧光检测;
(3)完成细胞成像。
优选的,所述Aβ斑块响应型荧光探针溶液为含1%的DMSO水溶液,细胞为人源肝癌HepG2细胞
本发明提供一种Aβ斑块响应型荧光探针及其制备和应用,与现有技术相比优点在于:
(1)本发明采用甲基化反应和缩合反应两步设计合成一种新型响应型小分子荧光探针BP,合成方法简便易行;
(2)本发明中所设计的新型目标探针BP在被Aβ斑块识别后,荧光信号实现“OFF”到“ON”的转变,具有激活型成像的特点,能够进行细胞内共聚焦荧光成像,便于探索Aβ斑块形成与AD发生之间的作用机理;
(3)本发明中目标探针BP对Aβ斑块的响应是特异性的识别,不受BSA的干扰;
(4)本发明中目标探针BP是离子型小分子探针,具有良好的水溶性和生物相容性,有利于进行后续生物学应用。
附图说明
图1为本发明实施例1中Aβ斑块响应型荧光探针BP的合成示意图;
图2为本发明实施例2中探针BP在不同溶剂中的紫外可见吸收光谱;
图3为本发明实施例3中探针BP在相同当量Aβ1-42聚集体和BSA存在下的荧光发射光谱;
图4为本发明实施例4中探针BP的细胞毒性测试;
图5为本发明实施例5中探针BP的活细胞共聚焦荧光成像。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面结合本发明实施例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作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:
一种Aβ斑块响应型荧光探针BR的构建:
(1)氮气保护下,50mL圆底烧瓶中加入甲基苯并噻唑0.74g(149.21g/mol,5.0mmol)、碘甲烷2.12g(141.94g/mol,15.0mmol)以及25mL丙酮作为溶剂,混合液磁力搅拌并回流6h;反应完成,冷却至室温,析出固体,抽滤,适量正己烷洗三遍,真空干燥,产物为M1(1.13g,产率:78%)。
(2)氮气保护下,100mL圆底烧瓶中加入M1(1.45g,5.0mmol)、4-羟基-2-甲氧基苯甲醛(0.76g,5.0mmol)以及60mL乙腈和甲醇的混合溶剂(体积比1:1),并加入适量哌啶作为催化剂,混合液磁力搅拌并回流12h;反应完成,旋干溶剂,粗产物柱层析分离纯化(二氯甲烷:甲醇=10:1,v/v),产物为浅红色固体(1.33g,产率:63%)。
实施例2:
Aβ斑块响应型荧光探针的溶剂化紫外可见吸收光谱的建立:
将上述获得的目标探针BP溶于DMSO溶液中,配置成浓度为1×10-3mol/L的母液;取50μL母液分别加入4500μL的色谱纯溶剂中,按溶剂极性由低到高的顺序为:二氯甲烷,四氢呋喃,乙酸乙酯,乙腈,二甲基亚砜和PBS;取适量上述样品于1cm的石英比色皿进行紫外可见吸收光谱测试,测试仪器使用SPECORD S600分光光度计(如图2所示)。
结果表明,目标探针BP在不同极性溶剂中的最大吸收波长均在450~550nm,且随着溶剂极性大的增加,最佳吸收波长显著红移,呈现明显的溶剂化效应;这是由于:处于激发态分子的极性大于基态,所以极性溶剂虽然使激发态和基态的能量都有所降低,但是激发态降低的更多,因而分子的电子吸收能量较非极性溶剂中减小,故吸收带发生红移。值得注意的是,探针BP在PBS溶液中的最佳吸收波长为505nm,吸光度与在有机溶剂中相当,表明探针在缓冲溶液中具有良好的光物理性质。
实施例3:
Aβ斑块响应型荧光探针在相同当量Aβ1-42聚集体和BSA存在下的荧光发射光谱
Aβ1-42聚集体的制备:将购买的Aβ1-42粉末溶解在含1%氨水的PBS溶液中并稀释至200μM,37℃恒温震荡3天,即得Aβ1-42聚集体。
将上述获得的目标探针BP溶于DMSO溶液中,配置成浓度为1×10-4mol/L的母液。取10μL母液分别加入970μL的PBS溶液(10mM,pH=7.4)中,并加入浓度为200μM的Aβ1-42聚集体溶液20μL,并以相同当量的BSA作为对照,最终使探针的浓度为1μM,Aβ1-42聚集体或者BSA的浓度为4μM。37℃恒温震荡1h后,取适量上述样品于1cm的石英比色皿进行荧光发射光谱测试,测试仪器使用日立F-7000荧光分光光度计。
如图3所示,空白探针溶液的荧光发射波长为552nm,且发射强度很弱;当加入Aβ1-42聚集体后,荧光强度显著增强,约是空白探针溶液的5.5倍,最佳发射波长无明显改变;而加入相同当量的BSA溶液,荧光强度无明显改变。
上述结果表明探针BP仅在Aβ1-42聚集体存在下荧光增强,是对Aβ1-42聚集体具有单一性和敏感性响应的小分子荧光探针。
实施例4:
Aβ斑块响应型荧光探针的细胞毒性测试
在细胞毒性实验中:
(1)将对数期生长期的HepG2细胞接种到平底的96孔板中,在含5%CO2的37℃恒温培养箱中孵育24h;
(2)使用新鲜DMEM培养基将探针BP稀释至所需浓度(0、10、20、40、80和160μM),吸去96孔板中原有培养基,分别加入100μL不同浓度培养基稀释的探针溶液,继续37℃孵育24h;
(3)探针孵育完成,吸去原有培养基,每孔中使用100μL冷的PBS洗涤两遍,再分别加入100μL培养基稀释的MTT溶液(0.5mg/mL),在5%CO2的37℃恒温培养箱中孵育4h;
(4)孵育完成,小心吸去96孔板中的溶液,并加入100μL的二甲基亚砜溶液,常温震荡5min后使用酶标仪进行测定;
(5)以同一实验条件下未经探针处理的细胞群体的吸光度作为参比,利用公式计算细胞成活率;实验重复测定三次。
如图4,结果显示:在探针浓度为80μM的条件下,HepG2细胞的存活率均达到85%以上,这种较低的毒性可以用于后续的细胞共聚焦荧光成像。
实施例5:
将上述实施例1获得的目标探针BP溶于含1%的DMSO的水中,加入到细胞富集程度达60%的HepG2细胞培养皿中(5μM),放置在恒温培养箱孵化30min,随后吸去培养液,并用PBS缓冲液洗两遍(2×1mL),最后将每个孔注入1mL的PBS缓冲液进行共聚焦荧光成像;对照组实验为不加入探针溶液的相同条件下的HepG2细胞。
用Olympus共聚焦荧光显微镜进行观察,实验结果显示:与没有添加探针的对照组相比,实验组具有显著增强的细胞内荧光信号,且信号区域集中在胞质内,表明探针BP能够有效的穿透细胞膜并对胞质进行荧光成像,这对后续开展的细胞内转染Aβ蛋白和小动物活体实验奠定了基础。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (5)
1.一种Aβ斑块响应型荧光探针,其特征在于,所述Aβ斑块响应型荧光探针的化学结构式如下所示:
。
2.一种如权利要求1所述的Aβ斑块响应型荧光探针的制备方法,其特征在于,所述Aβ斑块响应型荧光探针的制备方法包括以下步骤:
(1)将甲基苯并噻唑与碘甲烷发生甲基化反应,得到化合物M1;
(2)将化合物M1与4-羟基-2-甲氧基苯甲醛在弱碱存在下发生缩合反应,得到目标探针。
3.根据权利要求2所述的一种如权利要求1所述的Aβ斑块响应型荧光探针的制备方法,其特征在于:所述步骤(1)中甲基苯并噻唑与碘甲烷发生甲基化反应在丙酮中进行,且甲基苯并噻唑与碘甲烷的摩尔比为1∶3。
4.根据权利要求2所述的一种如权利要求1所述的Aβ斑块响应型荧光探针的制备方法,其特征在于:所述步骤(2)中M1与4-羟基-2-甲氧基苯甲醛反应在乙腈/甲醇的混合溶剂中进行,且添加哌啶作为催化剂,所述M1与4-羟基-2-甲氧基苯甲醛的摩尔比为1:1。
5.根据权利要求2所述的一种如权利要求1所述的Aβ斑块响应型荧光探针的制备方法,其特征在于:所述甲基化反应和缩合反应均在氮气保护下进行,且甲基化反应为回流反应6h,缩合反应为回流反应12 h。
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