CN112079771B - 一种水溶性红色荧光的线粒体靶向探针及应用 - Google Patents

一种水溶性红色荧光的线粒体靶向探针及应用 Download PDF

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CN112079771B
CN112079771B CN202011009611.4A CN202011009611A CN112079771B CN 112079771 B CN112079771 B CN 112079771B CN 202011009611 A CN202011009611 A CN 202011009611A CN 112079771 B CN112079771 B CN 112079771B
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李冲
田蕊
朱明强
王亚龙
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Huazhong University of Science and Technology
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Abstract

本发明涉及一种水溶性红色荧光的线粒体靶向探针及应用，属于新材料领域。本发明公开了一种水溶性红色荧光的线粒体靶向探针，结构通式为
Figure DDA0002697131370000011
其中M为芳环，N为亲水基团。本发明中的靶向探针拥有相对其他商用线粒体荧光探针而言比较良好的水溶性。使用本发明所述的荧光探针对细胞染色，可以靶向线粒体；具有更好生物相容性，几乎没有背景荧光，生物样品在红色荧光波段处的散射光和自发荧光较弱有利于降低背景信号，因此具有更好的信噪比。此外红色荧光具有更好的穿透深度，用于细胞的线粒体成像具有更加突出的优势，可在免漂洗的情况下，用于原位观察细胞线粒体成像过程，并长时间追踪细胞线粒体的状态。

Description

一种水溶性红色荧光的线粒体靶向探针及应用
技术领域
本发明属于新材料领域，更具体地，涉及一种水溶性红色荧光的线粒体靶向探针及应用，尤其涉及一种基于聚集诱导发光性质的水溶性荧光探针新材料与其在生物成像中可以靶向定位染色线粒体的应用。
背景技术
细胞是人体结构中重要的组成单元，不同种类的细胞具有不同的功能，它们共同维持着生命活动的正常运行。人体细胞具有精密复杂的结构，包括细胞器、细胞膜、细胞质、细胞核和细胞质基质。其中细胞器包括线粒体、内质网、溶酶体、脂质小滴等。不同的细胞器具有不同的功能，共同参与细胞的整个代谢过程，细胞内各个结构之间紧密协调、分工合作、缺一不可，使得生命活动能够有序进行。线粒体是细胞进行有氧呼吸的场所，被视为细胞的能量工厂，为细胞的代谢过程、以及其他生命活动提供所需能量。对着科学技术的不断进步与发展，越来越多的研究表明线粒体参与众多的生理过程，比如细胞传递、细胞分化、细胞凋亡等，还可以调控细胞生长和细胞周期。因此细胞的线粒体功能障碍，可能导致许多严重的疾病甚至癌症。
由此可知，线粒体等细胞器的研究，对于揭开生命的奥秘、研究疾病起因和治疗疾病具有重要的意义。细胞荧光成像已经成为现代细胞生物学研究的重要手段。荧光成像技术的发展很大程度上依赖于荧光材料的性能，AIEgens由于其高亮度，大的斯托克斯位移和良好的光稳定性，以及出色的生物相容性，被广泛应用于细胞成像的研究中，但是成像效果的清晰度以及操作步骤的简洁度有待提高。
发明内容
本发明提供了结构通式为
Figure BDA0002697131350000021
的水溶性红色荧光的线粒体靶向探针，该探针能够单独靶向定位线粒体并进行染色，在不需要二次漂洗的情况下，形成清晰度、对比度、信噪比均较高的线粒体荧光成像。
按照本发明的第一方面，提供了一种水溶性红色荧光的线粒体靶向探针，所述靶向探针具有如式(一)所示的结构通式：
Figure BDA0002697131350000022
其中M为芳环，N为亲水基团。
优选地，所述M为疏水芳环。
优选地，所述M为不含取代基或带有取代基苯环、萘环、蒽环、噻吩、噻唑、咪唑、吲哚、苯胺、二苯胺、三苯胺、咔唑或芴。
优选地，所述M为
Figure BDA0002697131350000023
Figure BDA0002697131350000031
其中R1和R2各自独立地为氢、氟、氯、溴、碘、硝基、氰基、羧基、羟基、酯基、烷基、端胺基烷基或端羟烷基。
优选地，所述N为亲水性的芳杂环阳离子。
优选地，所述N为吡啶类阳离子、喹啉类阳离子或嘧啶阳离子。
优选地，所述N为
Figure BDA0002697131350000032
其中R3为水溶性基团，R4为氢、氟、氯、溴、碘、硝基、氰基、羧基、羟基、酯基、烷基、端胺基烷基或端羟烷基；
优选地，所述R3为
Figure BDA0002697131350000033
按照本发明的另一方面，提供了任一所述的水溶性红色荧光的线粒体靶向探针在靶向细胞线粒体荧光成像中的应用，将含有所述靶向探针的溶液浸染细胞，所述靶向探针能够靶向染色定位细胞中的线粒体。
优选地，含有所述靶向探针的溶液为水溶液或有机溶液。
优选地，含有所述靶向探针的溶液中所述靶向探针的浓度为0.1μmol/L-2mol/L；
优选地，含有所述靶向探针的溶液中所述靶向探针的浓度为5μmol/L-50μmol/L。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得下列有益效果：
(1)本发明中的靶向探针其结构的特殊性，使其拥有相对其他商用线粒体荧光探针而言比较良好的水溶性。使用本发明所述的荧光探针对细胞染色，可以靶向线粒体。本发明中所述的荧光探针为两亲/水溶性，具有更好生物相容性，几乎没有背景荧光，生物样品在红色荧光波段处的散射光和自发荧光较弱有利于降低背景信号，因此具有更好的信噪比。此外红色荧光具有更好的穿透深度，用于细胞的线粒体成像具有更加突出的优势，可在免漂洗的情况下，用于原位观察细胞线粒体成像过程，并长时间追踪细胞线粒体的状态。
(2)本发明所述的荧光探针在生理状态下，可以溶解，考虑到细胞代谢是在生理环境中进行的，基于其聚集诱导发光的原理，在溶液中的荧光可以忽略不计，只有与靶向目标结合后才会发射出很强的荧光，基于此，可以实现对细胞线粒体的靶向成像。
(3)本发明所制备的聚集诱导荧光探针，其溶解特性具有两亲性，既可以在水性环境中溶解，同时在油性环境中也具有一定的溶解性。这为其在多种环境中的应用打好了基础，使其具有更好的普适性。
(4)本发明所述的荧光探针用于靶向线粒体的细胞成像，具有良好的生物相容性、极低的背景荧光，免于二次漂洗，且成像清晰，操作简单，在长期监测与跟踪细胞的生态过程中具有不可忽略的优势。
附图说明
图1为本发明实施例1中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像的红色通道图。
图2为本发明实施例1中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像的绿色通道图。
图3为本发明实施例1中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像图。
图4为本发明实施例2中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像的红色通道图。
图5为本发明实施例2中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像的绿色通道图。
图6为本发明实施例2中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像图。
图7为本发明实施例3中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像的红色通道图。
图8为本发明实施例3中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像的绿色通道图。
图9为本发明实施例3中化合物TPA-OH与线粒体绿色荧光探针(Mit o-trackerGreen)共同染色Hela细胞的共聚焦成像图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
一种水溶性红色荧光的线粒体靶向探针，所述荧光探针具有如式(一)所示的结构通式：
Figure BDA0002697131350000061
其中M和N为任意位置取代基，且M为芳环，N为吡啶或哌嗪等亲水基团。
通式中M为疏水芳环，如不含取代基或带有取代基苯环，萘环，蒽环，噻吩，噻唑，咪唑，吲哚，苯胺，二苯胺，三苯胺，咔唑，芴等及其衍生物，如：
Figure BDA0002697131350000062
其中R1和R2为氢、氟、氯、溴、碘、硝基、氰基、羧基、羟基、酯基、烷基、端胺基烷基或端羟烷基；
N为亲水性的芳杂环阳离子，如吡啶类阳离子、喹啉类阳离子、嘧啶阳离子等及其衍生物，如：
Figure BDA0002697131350000071
其中R3为水溶性基团，如：
Figure BDA0002697131350000072
R4为氢、氟、氯、溴、碘、硝基、氰基、羧基、羟基、酯基、烷基、端胺基烷基或端羟烷基等。
本发明中的靶向探针以供电子基，通过简单的Knoevenagel缩合反应，制备单取代乙烯型聚集诱导发光分子。
本发明所述荧光探针在靶向细胞线粒体荧光成像中的应用，其具体包括如下步骤：
使含有所述荧光探针的溶液浸染细胞，所述荧光探针能够靶向染色定位细胞中的线粒体，不需要二次漂洗，在红色通道下，形成清晰度、对比度、信噪比均较高的线粒体荧光成像。
本发明所述荧光探针的溶液为将所述荧光探针溶解于溶剂中获得的溶液，一些实施例中，所述溶剂为去离子水或有机溶剂。
一些实施例中，所述荧光探针的溶液为荧光探针的水溶液。
一些实施例中，所述荧光探针溶液中荧光探针的浓度为0.1μmol/L至2mol/L，优选为5μmol/L至50μmol/L。
本发明荧光探针由于其结构的特殊性，其具有两亲性/水溶性，因此在靶向线粒体染色的荧光成像中相比于脂溶性的化合物具有很多突出的优势。
本发明所述的基于聚集诱导发光的两亲/水溶性荧光探针，由于其特殊的分子结构，具有聚集诱导发光的良好性质。
本发明提供的如式(一)所示的AIE分子，其为两亲/水溶性分子。由于其具有水溶性，在侵染细胞时，不会因为不溶于细胞的生理环境而在其中聚集，而产生比较强烈的背景荧光，即本发明荧光探针成像没有背景荧光，不需要二次清洗。
本发明利用荧光探针靶向细胞线粒体成像的操作非常简单；对细胞线粒体成像清晰，并且没有背景荧光，不需要二次洗涤。
实施例1
一种结构如
Figure BDA0002697131350000081
所示的荧光探针，其名称缩写为TPA-OH。通过将TPA-OH与线粒体绿色荧光探针(Mito-tracker Green)共同染色Hela细胞。其中TPA-OH的浓度为2μM，线粒体绿色荧光探针浓度为100nM。通过共聚焦显微镜进行荧光成像。最终得到多通道的荧光图像。
TPA-OH的红色通道和线粒体绿色荧光探针(Mito-tracker Green)都可以看到明显的细胞轮廓，将两个通道成像图进行图像叠加，如图1共聚焦显微镜荧光成像的绿色通道图中绿色部分和图2共聚焦显微镜荧光成像的红色通道图中的红色部分重叠变为黄色，如图3所示，证明TPA-OH和线粒体绿色荧光探针(Mito-tracker Green)靶向的是同一个细胞器，即为线粒体。
实施例2
一种结构如
Figure BDA0002697131350000082
所示的2号荧光探针。通过将2号荧光探针与线粒体绿色荧光探针(Mito-tracker Green)共同染色Hela细胞。其中2号荧光探针的浓度为2μM，线粒体绿色荧光探针浓度为100nM。通过共聚焦显微镜进行荧光成像。最终得到多通道的荧光图像。
2号荧光探针的红色通道和线粒体绿色荧光探针(Mito-tracker Green)都可以看到明显的细胞轮廓，将两个通道成像图进行图像叠加，如图4共聚焦显微镜荧光成像的绿色通道图中绿色部分和图5共聚焦显微镜荧光成像的红色通道图中的红色部分重叠变为黄色，如图6所示，证明TPA-OH和线粒体绿色荧光探针(Mito-tracker Green)靶向的是同一个细胞器，即为线粒体。
实施例3
一种结构如
Figure BDA0002697131350000091
所示的3号荧光探针。通过将3号荧光探针与线粒体绿色荧光探针(Mito-tracker Green)共同染色Hela细胞。其中3号荧光探针的浓度为2μM，线粒体绿色荧光探针浓度为100nM。通过共聚焦显微镜进行荧光成像。最终得到多通道的荧光图像。
3号荧光探针的红色通道和线粒体绿色荧光探针(Mito-tracker Green)都可以看到明显的细胞轮廓，将两个通道成像图进行图像叠加，如图7共聚焦显微镜荧光成像的绿色通道图中绿色部分和图8共聚焦显微镜荧光成像的红色通道图中的红色部分重叠变为黄色，如图9所示，证明TPA-OH和线粒体绿色荧光探针(Mito-tracker Green)靶向的是同一个细胞器，即为线粒体。
结果分析：
由实施例1、2、3可知，结构通式为
Figure BDA0002697131350000092
的基于聚集诱导发光性质的两亲/水溶性荧光探针的确具有靶向线粒体染色定位，并在不需要二次漂洗的情况下由共聚焦显微镜得到清晰的、对比度较高的荧光成像。
由图1-9可知，将实施例1、2、3中合成的荧光分子探针与线粒体绿色荧光探针(Mito-tracker Green)对Hale细胞进行共同染色后的荧光成像，红色通道荧光成像清晰，绿色通道荧光成像清晰，将两个通道重叠之后形成红色部分与绿色部分转变为黄色的荧光成像，由此可以证明，实施例1、2、3中合成的基于聚集诱导发光性质的两亲/水溶性荧光探针与线粒体绿色荧光探针(Mito-tracker Green)共同具有特异性靶向线粒体，并且对其进行定位染色的性能。由于其特殊的溶解性质，在多种环境中都具有同样优良的性质的。同时，由于细胞的生理环境更接近水性环境，两亲/水溶性的荧光探针分子在环境中的溶解性更好，因此没有背景荧光，不需要进行二次漂洗。
实施例1、2、3中合成的三种基于聚集诱导发光性质的两亲/水溶性荧光探针相比市面上的商用线粒体绿色荧光探针(Mito-tracker Green)具有更加优良的溶解性质，以及清晰度、穿透度、对比度、信噪比更高的成像性能。除此之外，有希望用于原位观察细胞成像过程，并长时间追踪细胞状态。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (5)

1.一种水溶性红色荧光的线粒体靶向探针，其特征在于，所述靶向探针的结构式为
Figure FDA0003469393240000011
2.如权利要求1所述的水溶性红色荧光的线粒体靶向探针用于制备靶向细胞线粒体荧光成像试剂的应用，其特征在于，将含有所述靶向探针的溶液浸染细胞，所述靶向探针能够靶向染色定位细胞中的线粒体。
3.如权利要求2所述的应用，其特征在于，含有所述靶向探针的溶液为水溶液或有机溶液。
4.如权利要求2所述的应用，其特征在于，含有所述靶向探针的溶液中所述靶向探针的浓度为0.1μmol/L-2mol/L。
5.如权利要求4所述的应用，其特征在于，含有所述靶向探针的溶液中所述靶向探针的浓度为5μmol/L-50μmol/L。
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