CN115417815B - 一种靶向线粒体检测粘度的荧光探针及其应用 - Google Patents
一种靶向线粒体检测粘度的荧光探针及其应用 Download PDFInfo
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Abstract
本发明提供了一种新型的线粒体靶向近红外发射粘度荧光探针Mito‑ND，其化学结构式为。该探针合成简单，细胞毒性低，具有良好的化学稳定性和光稳定性；能定位线粒体用于监测线粒体内粘度的变化。体内成像实验表明该探针还可以有效检测损伤条件下肝、肾组织的粘度变化。
Description
技术领域
本发明属于分析化学技术领域，具体涉及一种靶向线粒体检测粘度的荧光探针及其应用。
背景技术
肾、肝作为人体的重要器官，在机体的正常运作中起着不容忽视的作用。肾脏具有将代谢产物和某些废物及毒素排出体外的功能，同时保留水分和电解质，调节水和电解质的平衡，维持酸碱平衡。肾脏具有内分泌功能，可分泌肾上腺素、促红细胞生成素、前列腺素、激肽等。肾脏的这些功能保证了体内环境的稳定，保持人体的正常代谢。肝脏就像是脊椎动物体内的一种工具，主要发挥代谢功能。肝脏是许多体外非营养性物质和体内特定代谢物的生物转化器，如各种药物、激素、维生素等，这些物质被完全新陈代谢分解并从体内排出，这一作用也被称为“肝的解毒功能”。当出现肝功能障碍或肾功能障碍的情况下，有可能存在药物或毒物摄入体内后无法正常排出而产生不良反应的风险，因此检查肝、肾至关重要。
粘度作为生物微环境的典型代表，在控制扩散速率、信号转导和产物代谢的转运等生理过程中起着不可或缺的作用。研究发现生物体中粘度的异常表达与恶性肿瘤、动脉粥样硬化、糖尿病、炎症、阿尔茨海默病等多种疾病相关。由于这些临床疾病与生物粘度之间有相互关系，因此发展无创生物粘度检测技术可以更直接、有效地研究相关疾病的发病机制，为与粘度相关疾病的早期诊断提供可靠的理论依据。
许多传统的粘度分析方法，如毛细管粘度计，落球粘度计，转子粘度计等，由于实际因素，不能用于活细胞或特定细胞器的粘度测量。为了解决这一问题，荧光探针技术以其无创、操作简单、选择性高、实时监测等优点，逐渐被广泛应用于细胞或细胞器粘度的检测，可对细胞微环境粘度的变化产生荧光响应。在低粘度溶液中，探针分子可以自由旋转，形成扭曲的分子内电荷转移态(TICT态)，这种状态允许电子通过非辐射过程进行弛豫，从而抑制荧光发射。然而，随着体系粘度的增加，对分子自由旋转的抑制程度逐渐增强，阻碍了TICT态的内部跃迁，荧光强度也会增加。
现有的荧光探针属于近红外区的较少，且难以实现原位检测，本探针是一个近红外荧光探针，且实现了疾病原位检测，并首次应用于肝损伤和肾损伤的粘度检测。
发明内容
针对现有技术中的问题，本发明提供一种能够定位线粒体检测粘度的荧光探针，响应倍数高、化学和光稳定性好。
本发明的另一目的是提供一种上述荧光探针在检测溶液中或生物细胞内粘度的应用。
为实现上述目的，本发明采用如下技术方案。
一种靶向线粒体检测粘度的荧光探针，简称Mito-ND，其化学结构式如式（I）所示：
式（I）。
上述荧光探针的制备方法，包括以下步骤：
（1）在氮气保护下，4-甲基喹啉与溴化苄于甲苯中加热反应，反应后冷却至室温，固液分离后固体用石油醚洗涤，得到化合物1：
；
（2）将化合物1与4-二甲氨基苯甲醛于无水乙醇中，在哌啶催化下，加热回流反应，冷却后分离提纯，得探针：
。
步骤（1）中，所述4-甲基喹啉与溴化苄物质的量比为20:21。
步骤（1）中，所述加热反应条件为：115 ℃回流搅拌8 h。
步骤（2）中，所述化合物1与4-二甲氨基苯甲醛的物质的量比为1:1。
步骤（2）中，所述反应温度为90℃。
步骤（2）中，所述分离提纯条件为：将反应液旋蒸除去溶剂，进行柱层析；所述层析液为体积比20:1的二氯甲烷:甲醇溶液。
一种上述荧光探针在检测溶液、细胞、组织或生物体中粘度的应用。
本发明的机理如下：
本发明的探针分子通过扭曲分子内电荷转移（Twisted Intramolecular ChargeTransfer, TICT）机制实现对溶液粘度的识别。该探针分子中，4-甲基喹啉季铵盐作电子受体基团，此外，4-甲基喹啉季铵盐携带正电荷，通过静电吸附与线粒体外膜的负电荷相互作用，导致荧光化合物在细胞线粒体内快速富集；4-二甲氨基苯基具有强给电子能力，作为探针分子中的给电子基团。由于探针分子中的给电子基团在低粘度溶剂中可以自由旋转，表现出微弱的荧光信号。然而，随着溶剂粘度的增加，分子内的旋转能力被抑制，从而在近红外发射区观察到更强的荧光信号。
本发明具有以下优点：
本发明提供了一种以线粒体为靶向的近红外荧光粘度探针Mito-ND，对活细胞具有低的细胞毒性，具有良好的化学稳定性和光稳定性；细胞成像结果表明，探针在线粒体处具有明显的富集特性，可用于监测线粒体内粘度的变化。此外，体内成像实验表明，该探针可以有效检测损伤条件下肝、肾组织的粘度变化。
附图说明
图1是荧光探针Mito-ND的1H NMR图谱；
图2是荧光探针Mito-ND的13C NMR图谱；
图3是荧光探针Mito-ND的质谱图谱；
图4是荧光探针Mito-ND在不同粘度下荧光实验；其中激发波长为560 nm；探针的浓度：10 µM；
图5是荧光探针Mito-ND在PBS和70%甘油中对不同物质的选择性；其中激发波长为560 nm，探针的浓度：10 µM，选择性离子(或氨基酸)的浓度为100 µM；
图6是荧光探针Mito-ND在细胞中线粒体靶向；激发波长：560 nm，发射波段：620-720 nm；
图7是荧光探针Mito-ND的细胞毒性；
图8是探针Mito-ND的不同pH下的检测实验；其中激发波长为560 nm；探针的浓度：10 µM；
图9是探针Mito-ND光稳定性实验；其中激发波长为560 nm；探针的浓度：10 µM。测试时间：60 min；
图10是荧光探针Mito-ND斑马鱼成像应用，激发波长：560 nm，发射波段：620-720nm，A为斑马鱼只与10 µM的Mito-ND孵育，或制霉菌素、莫能菌素、脂多糖预处理后与Mito-ND孵育的荧光共聚焦图像，其中a1，b1，c1，d1为明场图像，a2，b2，c2，d2为荧光场图像，a3，b3，c3，d3为叠加场图像；B为荧光强度；
图11是荧光探针Mito-ND肾损伤的粘度成像，激发波长：561 nm，发射波段：670nm， 其中，A为荧光探针Mito-ND肾损伤的粘度成像，左侧为正常肾，右侧为肾损伤；B为荧光强度；
图12是荧光探针Mito-ND肝损伤的粘度成像；激发波长：561 nm，发射波段：670nm，其中，A为荧光探针Mito-ND肝损伤的粘度成像，左侧为正常肝，右侧为肝损伤；B为荧光强度。
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例1 荧光探针Mito-ND的合成
（1）化合物1的合成
将4-甲基喹啉(2.86 g,20 mmol)和溴化苄(3.60 g,21 mmol)加入到250 mL圆底烧瓶中，倒入50 mL甲苯作溶剂。在氮气保护下，升温至115 ℃，回流搅拌8 h，待反应完全后冷却至室温，抽滤，固体用石油醚洗涤三次，得到白色固体，产率为93%。化合物1未经纯化直接进入下一个反应步骤。
（2）Mito-ND的合成
将化合物1(3.13 g,10 mmol)和4-二甲氨基苯甲醛(1.49 g,10 mmol)溶于60 mL无水乙醇后，加入三滴哌啶，并保持在90 ℃回流过夜。待溶液冷却至室温后，通过旋转蒸发除去溶剂，用二氯甲烷:甲醇（20:1v/v）柱层析得到蓝紫色固体Mito-ND(产率为87%)。
其1H NMR图谱如图1：1H NMR (500 MHz, DMSO-d 6 ) δ 9.37 (d, J = 10 Hz, 1H),9.06 (d, J = 10 Hz, 1H), 8.47 (d, J = 10 Hz, 1H), 8.29 (dd, J = 20 Hz, J = 5Hz, 2H), 8.09 (m, 1H), 8.06 (d, J = 15 Hz, 1H), 7.92 (d, J = 5 Hz, 2H), 7.37(m, 6H), 6.84 (d, J = 10 Hz, 2H), 6.18 (s, 2H), 3.09 (s, 6H).
其13C NMR图谱如图2：13C NMR (125 MHz, DMSO-d 6 ) δ 154.49, 152.98, 147.26,146.23, 138.45, 135.23, 135.19, 132.16, 129.50, 128.91, 127.35, 127.27,126.73, 123.59, 119.68, 114.64, 113.58, 112.41, 58.88, 40.22.
其质谱图谱如图3：HR-MS (ESI) calcd. for C26H25N2 + . [M+H]+ m/z 365.2012,found 365.2015.
荧光探针Mito-ND的荧光量子产率为7.92%。
实施例2 荧光探针对不同粘度体系的响应
配制实施例1制备的探针溶液，终浓度为10 µM，分别与不同粘度的体系（0%，10%，20%，30%，40%，50%，60%，70%，80%，90%，100%甘油）相互作用，设置激发波长为560 nm，在580-800 nm波段进行荧光检测。如图4所示，反应体系荧光强度随粘度增加而逐渐增强。
实施例3 荧光探针Mito-ND对不同物质的选择性
配制实施例1制备的探针Mito-ND溶液，终浓度为10 µM。将探针Mito-ND溶液与相关生物活性分子(1-22分别为空白对照、L-Cys、L-Ala、NaClO、L-Met、KI、NaF、CaCl2、Gly、DL-Hcy、GSH、H2O2、L-Arg、Na2S、Na2SO4、MgCl2、MnCl2、•OH（H2O2/FeCl3 =1:1，V/V）、CuSO4、L-Val、ZnCl2、Na2SO3)。在两种测试体系(纯PBS缓冲液pH=7.4和70%甘油溶液)中进行选择性实验。设置激发波长为560 nm、检测波长为676 nm进行荧光检测，结果如图5所示，在两种溶液中分别加入100 µM浓度的代表性阴离子、金属离子、活性氧和还原剂后，探针Mito-ND的荧光强度基本保持不变，说明生物样品中化合物的复杂组成不会干扰Mito-ND探针对粘度的检测。
实施例4 荧光探针在细胞中线粒体靶向应用
为了确定探针Mito-ND是否有线粒体靶向，我们用探针Mito-ND(终浓度10 µM)和线粒体橙色定位染料(Mito Tracker Orange, MTO, 200 nM)进行线粒体共定位成像实验。HeLa细胞预先用商业化线粒体荧光染料(Mito tracker Orange)孵育30 min，再用Mito-ND(10 μM)处理30 min。随后，使用共聚焦激光显微镜进行共定位成像实验。绿色通道激发波长为560 nm，在570-620 nm处收集荧光；红色通道激发波长为560 nm，在620-670 nm处收集荧光。结果如图6所示，红色荧光通道(b)显示的是探针Mito-ND和LPS的荧光信号，橙色荧光通道(c)显示的是MTO的荧光信号。在HeLa细胞中，红色荧光信号与橙色荧光信号(d)能够很好的重叠，皮尔逊相关系数为0.83，说明探针Mito-ND具有明显的线粒体靶向性。
实施例5 荧光探针对细胞的毒性
将HeLa细胞株接种到96孔板上，置于培养箱(37 °C,5% CO2)中培养，直至细胞覆盖培养皿底部的80-90%。将不同浓度(0、2、5、10、20、30 μM)的Mito-ND添加到96孔板中，继续培养24 h。接着每个孔添加10 μL的MTT孵育4 h。最后，96孔板放在摇床上摇晃10 min，并分析每一个孔的读数。在492 nm处检测吸光度，如图7所示，探针Mito-ND对活细胞的细胞毒性低，安全性高。
实施例6 荧光探针的化学稳定性和光稳定性
研究了探针Mito-ND(10 µM)在两种测试溶剂(纯PBS缓冲液pH=7.4和70%Gly溶液)中不同pH值(4.0、4.5、5、5.5、6、6.5、7、7.5、8、8.5)下荧光强度的变化，结果如图8所示。在两种溶液中，pH不同，探针Mito-ND的荧光强度变化不显著，说明该探针不受生物样品pH变化的干扰，能够有效跟踪生物样品的粘度变化。
在两种测试溶剂(纯PBS缓冲液pH=7.4和70%Gly溶液)中，研究了探针Mito-ND(10µM)的光稳定性。进行荧光检测，每隔5 min测试一次，测试60 min，计算各体系中随时间变化的荧光强度，建立荧光强度与作用时间标准曲线，如图9所示。在676 nm处，探针Mito-ND的荧光发射强度随光照时间(0-60 min)变化不显著。
应用例1 荧光探针对斑马鱼内粘度的成像应用
以斑马鱼为模型，进行共聚焦荧光成像实验。将相同生长条件的斑马鱼分为实验组和对照组进行成像实验。实验组的斑马鱼用刺激物制霉菌素LPS、莫能菌素Mon、脂多糖Nys分别刺激，再加探针Mito-ND孵育60 min，随后进行荧光成像实验。作为对照组，将未被刺激的斑马鱼用探针Mito-ND孵育20 min，然后进行荧光成像实验。设置激发波长为560 nm并在620-670 nm处收集荧光信号。结果如图10A所示，未被药物刺激的斑马鱼在加入探针Mito-ND(10 µM)后显示微弱的荧光信号。然而，制霉菌素、莫能菌素、脂多糖三种药物刺激的斑马鱼在探针加入后分别表现出明显的荧光信号，荧光强度分别比正常斑马鱼高3.04、2.73和2.69倍(图10B)。
应用例2 荧光探针对小鼠肾损伤和肝损伤的粘度成像
采用肝损伤和肾损伤两种小鼠组织模型进行成像实验。将肝损伤、肾损伤和正常的肝、肾分别在10 µM探针Mito-ND溶液中孵育6 h，然后用活体成像仪进行组织成像实验。成像结果如图11、12所示，正常肝脏和肾脏荧光较弱，肝损伤和肾损伤模型组表现出较强的荧光信号，荧光强度分别增加了2.29倍和2.93倍。实验结果表明，该探针在肝脏和肾脏疾病的诊断中具有潜在的应用价值。
Claims (1)
1.一种如式（I）所述的荧光探针在制备检测溶液、细胞、组织或生物体粘度的试剂中的应用：
式（I）。
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