CN115073433A - 具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针 - Google Patents
具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针 Download PDFInfo
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Abstract
本发明公开一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针，属于荧光探针技术领域。本发明提供的一种线粒体靶向近红外粘度荧光探针具有聚集诱导发光特性，可作为检测试剂在脂肪肝、炎症和肿瘤模型中检测粘度变化，而且该探针具有良好的活性氧产生能力，可用作光敏剂对癌症进行光动力治疗，检测和治疗的手段简单灵敏。
Description
技术领域
本发明属于粘度荧光探针技术领域，具体涉及一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针。
背景技术
粘度作为细胞微环境的重要参数，在物质扩散和信号传导等方面发挥重要作用。线粒体作为真核细胞的一种重要细胞器，被认为是细胞的主要动力库，其微环境稳态对正常生命活动至关重要，线粒体的粘度水平在调节其生理过程中起着重要的作用，其粘度的异常变化往往会导致炎症、神经退行性疾病甚至癌症等多种疾病。因此，开发监测线粒体粘度的荧光探针将有助于深刻理解与线粒体粘度相关的生理和病理过程。现有粘度敏感探针大多是基于聚集导致淬灭发光机理，即低浓度发光，高浓度或聚集状态时会发生荧光淬灭，严重影响其作为发光材料的有效性。而具有聚集诱导发光(AIE)特性的分子在溶解时，由于分子内自由旋转不发光或发光很弱；然而，一旦聚集或限制在刚性环境中，分子内运动的受限就会发出强烈的荧光。因此，开发具有聚集诱导发光特性且可监测线粒体粘度发生过程的高灵敏度荧光粘度探针是非常有必要的。
光动力治疗(PDT)由于其微创性、高选择性、毒副作用小和良好的靶向治疗效果的优点，已成为临床认可的一种很有潜力的癌症治疗方式。PDT是在光照射下激活光敏剂(PS)产生ROS，特别是单线态氧(1O2)，导致癌细胞坏死或凋亡。由于ROS具有反应活性高和扩散距离短的特点，因此合理设计具有细胞器靶向的PSs可以显著提高PDT的疗效。考虑到线粒体在细胞呼吸、分裂和凋亡中的重要作用，及其对ROS和药物刺激的高度敏感性，将PSs直接靶向到线粒体，理论上可以最大限度地提高PDT的效率[130]。因此，线粒体靶向PSs的开发近年来受到广泛关注，并成为传统细胞核靶向药物的替代品，用于杀伤或抑制癌细胞和肿瘤发生。
目前具有聚集诱导发光特性，能够用于多种疾病模型(脂肪肝、炎症甚至癌症)的可视化诊断，并且能够进行癌症的光动力治疗，以及通过线粒体粘度的变化对治疗效果进行即时评估的荧光探针，鲜有报道。因此，开发具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针用于多种疾病模型的可视化诊断和癌症的光动力治疗，进而实现诊疗一体化是非常有必要的。
发明内容
针对现有技术中存在的缺陷，本发明提供一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针(POTA-OH)，利用其正电荷结构与线粒体负性膜电位的静电作用相结合，使探针靶向聚集于线粒体中，同时利用AIE分子运动受限和多个可自由旋转的苯基及单键自由旋转受到限制使分子的有效共轭，从而实现对线粒体粘度的高灵敏识别。同时，具有粘度依赖性的POTA-OH能够在体外PDT过程的同时进行治疗效果的即时评估。此外，POTA-OH高效的PDT在体内抗肿瘤方面也取得了成功，为肿瘤治疗及其疗效即时评估提供了有效的策略。
为达到上述目的，本发明采用了以下技术方案：
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针，结构式为：
本发明提供的荧光粘度探针具有聚集诱导发光的特性，探针的近红外荧光强度随环境粘度的增加而逐渐增强。
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备监测活细胞线粒体内粘度变化试剂中的应用。
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在检测药物诱导活细胞中粘度变化的应用。
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备检测炎症模型粘度变化试剂中的应用。
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备检测脂肪肝模型粘度变化试剂中的应用。
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备检测肿瘤模型粘度变化试剂中的应用。
一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备光动力治疗试剂中的应用。
与现有技术相比，本发明具有以下有益效果：
(1)本发明所述的检测线粒体粘度的荧光探针以对羟基二苯胺取代氧杂蒽基团作为聚集诱导荧光核和电子供体(D)，吡啶阳离子作为电子受体(A)并兼具线粒体靶向基团。该分子结构中存在强大的推拉电子效应和扭曲的D-A效应；在二甲基亚砜/甲苯混合溶剂中随着甲苯含量的增加，探针荧光逐渐增强，具有典型的聚集诱导荧光发射特性。
(2)所述探针对粘度变化的响应原理：由于多个可自由旋转的苯基和乙烯基单键，使分子在粘度较低的介质中可自由旋转，且处于扭曲态分子内电荷转移状态，导致荧光猝灭；当处于粘度较高的体系时，自由旋转受到限制，扭曲态分子内电荷转移效应被抑制，使得整个分子共平面，探针就会发出很强的近红外荧光。
(3)所述探针在水-甘油体系中，当粘度η从0.89cP增加到945cP时，该探针在720nm处的近红外荧光强度逐渐增强，且增强倍数超过157倍。此外，logI720与logη存在良好的线性关系(R2＝0.9896)，斜率为0.725，具有高灵敏定量检测环境粘度的特性。
(4)所述探针对环境粘度的检测具有高选择性和高灵敏性，且不受生物体系及环境中其他物质、极性和pH的干扰。
(5)所述探针利用分子中吡啶正电荷结构与线粒体负性膜电位的静电作用相结合，使探针靶向聚集于线粒体中；应用于线粒体粘度细胞模型中对粘度变化的实时原位监测时，并在活体粘度和炎症小鼠体内也表现出高灵敏响应。此外，还应用于脂肪肝组织切片及活体小鼠肿瘤和临床癌症患者病理样本的可视化识别，具有高灵敏、可靠性、可视化、快速便捷等特点，预示该探针在线粒体相关生物研究与医疗诊断方面具有良好的应用前景。
(6)所述探针具有良好的活性氧产生能力，该探针能够同时进行体外光动力治疗和即时评估治疗效果，实现了对体内肿瘤高效的光动力治疗，在生物体系中的可视化检测及疾病诊断和治疗方面具有潜在的应用价值，并且为实现癌症的诊疗一体化提供了一种非常有前景的荧光试剂。
(7)所述的检测手段简单，只需包括荧光分光光度计、激光共聚焦显微镜、多模式活体成像仪。
附图说明
图1为本发明探针POTA-OH在二甲基亚砜-甲苯混合溶剂中随含甲苯体积含量变化的荧光发射光谱图；
图2为本发明探针POTA-OH的相对荧光强度(I/I0)在二甲基亚砜-甲苯混合体系中随体积含量变化的曲线；
图3为本发明探针POTA-OH在水-甘油混合溶剂中随甘油体积含量变化的荧光发射光谱图；
图4为本发明探针POTA-OH的log I在水/甘油混合体系中随logη变化的曲线；
图5为本发明探针POTA-OH在不同极性溶液中的荧光发射光谱图；
图6为本发明探针POTA-OH在不同甘油体积含量的PBS-甘油(0％和95％)混合溶剂中，随pH由3.0变化到10.0时的荧光发射光谱图；
图7为本发明探针POTA-OH在常见金属离子、阴离子和生物活性小分子存在下，对甘油的荧光光谱的选择性柱状图；
图8为本发明探针POTA-OH与市售线粒体特异性染料(MTG)共染活细胞，二者的荧光共定位成像图；
图9为本发明探针POTA-OH在制霉菌素刺激的HeLa细胞粘度变化的实时荧光成像图；
图10为本发明探针POTA-OH在制霉菌素、脂多糖分别刺激的活体小鼠中粘度变化的荧光成像图；
图11为本发明探针POTA-OH在肿瘤小鼠模型中左(肿瘤)和右(正常)腋下组织内粘度变化的荧光成像图；
图12为本发明探针POTA-OH在临床癌症患者病理组织切片中粘度变化的荧光成像图；
图13为本发明探针POTA-OH在脂肪肝组织切片中粘度变化的荧光成像变化图；
图14为本发明探针POTA-OH在黑暗或激光照射时HeLa细胞的细胞成活率图；
图15为本发明探针POTA-OH在激光照射下有无指示剂——2’7’-二氯荧光素二乙酸酯(DCFH-DA)存在或DCFH-DA单独存在时HeLa细胞的荧光成像变化图；
图16为本发明荧光探针POTA-OH分别在活HeLa细胞、激光照射活HeLa细胞、固定HeLa细胞中荧光成像变化图；
图17为本发明荧光探针POTA-OH对荷瘤小鼠进行体内光动力治疗，21天后肿瘤体积变化图；
图18为本发明荧光探针POTA-OH对荷瘤小鼠进行体内光动力治疗，21天后小鼠体重变化图；
图19为本发明荧光探针POTA-OH对荷瘤小鼠进行体内光动力治疗，21天后肿瘤组织切片免疫组化染色图。
具体实施方式
实施例1
将荧光探针POTA-OH在二甲基亚砜-甲苯混合溶剂中的聚集诱导荧光发射特性。用二甲基亚砜-甲苯混合溶剂将实施例1中的荧光探针稀释至终浓度为10μmol/L，固定激发波长为565nm，记录探针随甲苯体积含量变化的荧光发射光谱(图1)，并绘制探针相对荧光强度(I/I0)在二甲基亚砜-甲苯混合体系中随体积含量变化的曲线(图2)。随着甲苯体积比由0％增加到100％，730nm的荧光强度逐渐增强并蓝移至714nm，且在甲苯体积为95％时达到最大值，说明探针具有典型的聚集诱导发光特性。
实施例2
将实施例1中的荧光探针POTA-OH用水和甘油混合溶剂稀释至终浓度为10μmol/L，固定激发波长为565nm，记录探针随含甘油体积含量变化(或粘度系数η)的荧光发射光谱(图3)，并绘制探针在720nm处的荧光强度数值(log I720)，在水和甘油混合体系中随粘度系数(logη)变化的线性相关曲线(图4)。随着甘油体积比由0％(0.89cP)增加到99％(945cP)，720nm的荧光强度逐渐增加，且在甘油体积为99％时达到最大值，说明探针的相对荧光强度随着环境粘度的增加显著增强。
实施例3
将实施例1中的荧光探针POTA-OH浓度保持在10μmol/L，考察该探针在不同极性溶液中的荧光发射光谱。如图5所示，探针在甘油中具有显著荧光增强，而在其他不同极性溶剂中，荧光强度相对变化不大，说明探针对粘度的响应基本不受溶剂极性的影响。
实施例4
将实施例1中的荧光探针POTA-OH浓度保持在10μmol/L，考察该探针分别在含有0％和95％甘油的PBS/甘油体系中，随pH变化的荧光光谱。如图6所示，随着pH从3.0增加到10.0，探针的荧光强度基本保持稳定，说明探针对粘度的响应不受pH变化的影响。
实施例5
将实施例1中的荧光探针POTA-OH浓度保持在10μmol/L，分别考察该探针在常见离子及生物活性小分子存在下，对其荧光光谱的选择性。如图7所示，在磷酸盐缓冲液PBS(2mL，pH 7.4)体系中时，分别加入下述物质(100μmol/L)对探针的荧光强度几乎没有干扰。图10中各物质依次为：1.空白；2.99％Glycerol；3.Trp；4.Cys；5.GSH；6.Hcy；7.Pro；8.Ser；9.Ala；10.Zn2+；11.Cu2+；12.Pb2+；13.K+；14Mg2+；15Na+；16.Ba2+；17.Ca2+；18.Cd2+；19.Fe2+；20.Fe3+；21.Br-；22.Cl-；23.CN-；24.CO3 2-；25.H2PO4 -；26.HSO3 -；27.HSO4 -；28.NO2 -；29.NO3 -；30.S2-；31.S2O3 2-；32.SO4 2-。
实施例6
为了观察探针POTA-OH是否能够靶向聚集于活细胞线粒体中，进行了探针与市售线粒体特异性染料MitoTracker Green(MTG)的共定位实验。将贴壁的HeLa细胞与MTG(终浓度1.0μmol/L)在pH 7.4的条件下，于37℃、5％CO2的孵育箱中孵育30min后，用PBS缓冲液(pH 7.4)洗涤3次，除去多余的染料。然后加入探针POTA-OH(终浓度5μmol/L)继续共同孵育30min，在激光共聚焦显微镜下观察二者的共定位情况。探针POTA-OH固定激发波长为561nm，选择红色荧光成像，收集红色通道范围650-754nm；MTG的固定激发波长为488nm，收集红色通道范围490-530nm。由图8可知，荧光探针POTA-OH呈典型的红色棒状线粒体形态，且与MTG能够很好地重叠，得到黄色重叠荧光，且二者皮尔森共定位系数PC为0.91。说明荧光探针POTA-OH与MTG具有显著的共定位成像，能够靶向定位于线粒体中。
实施例7
将贴壁的HeLa细胞与实施例1中的荧光探针POTA-OH(终浓度5μmol/L)，于37℃、5％CO2的孵育箱中孵育30min后，无需洗涤，在激光共聚焦显微镜下观察探针的荧光成像。如图9所示，探针本身在细胞中发射较弱的荧光，然后加入5μmol/L制霉菌素，刺激细胞30min，可原位观察到细胞内荧光发射逐渐增强；同时，线粒体形态由棒状转变为球状，说明制霉菌素会导致线粒体内粘度的升高以及线粒体形态的变化，而且探针POTA-OH能够高灵敏监测线粒体内粘度的变化。
实施例8
将实施例1中的荧光探针POTA-OH(100μL，200μM)腹腔注射加入正常小鼠、经制霉菌素处理的小鼠(粘度小鼠模型)和经脂多糖处理的小鼠(炎症小鼠模型)体内，如图10所示，在活体成像仪下观察到正常小鼠体内非常微弱的红色荧光，经制霉菌素和脂多糖处理的小鼠体内有明显的红色荧光。由此说明在腹腔注射制霉菌素和脂多糖均可使小鼠体内粘度增加，表明该探针可用于监测粘度和炎症活体小鼠模型体内的粘度的实时变化。
实施例9
将实施例1中的荧光探针POTA-OH((50μL，200μmol/L)分别注射到荷瘤小鼠左(肿瘤)和右(正常)腋下，在活体成像仪下观察探针的荧光成像，如图11所示，探针在左腋下肿瘤中表现出明亮的红色荧光，而在右腋下正常组织中的荧光可忽略不计，说明肿瘤组织内粘度水平明显高于正常组织，而且探针POTA-OH能够实现对肿瘤进行高灵敏可视化识别。
实施例10
将实施例1中的荧光探针POTA-OH(终浓度20μmol/L)分别孵育到人体良性(乳腺和甲状腺)和恶性癌症(乳腺和甲状腺)组织切片中，在激光共聚焦显微镜下探针的荧光成像，其中，恶性癌症组织中观察到显著的红色荧光增强信号，而在良性组织切片中的荧光信号可忽略不计(图12)，表明恶性癌症组织中的粘度显著高于良性组织，而且探针POTA-OH具有良好的癌症可视化识别能力。
实施例11
将实施例1中的荧光探针POTA-OH(终浓度20μmol/L)分别孵育到正常肝组织和脂肪肝组织中，在激光共聚焦显微镜下观察探针的荧光成像，探针在脂肪肝切片中表现出明亮的红色荧光，而在正常肝组织切片中的荧光可忽略不计(图13)，说明脂肪肝组织内粘度水平明显高于正常肝组织，而且探针POTA-OH能够实现对脂肪肝组织的高效可视化识别。
实施例12
将实施例1中不同浓度的荧光探针POTA-OH(0、0.5、1.0、5.0、10.0、15.0μmol/L)在黑暗中或在635nm激光(50mW·cm-2，20min)照射，如图14所示，POTA-OH在黑暗中表现出较好的生物相容性，而在激光照射后，表现出探针剂量依赖性的细胞毒性，表明POTA-OH对肿瘤细胞良好的光动力治疗作用。
实施例13
将实施例1中的探针POTA-OH在635nm激光(100mW·cm-2)照射下有无指示剂——2’7’-二氯荧光素二乙酸酯(DCFH-DA)存在或DCFH-DA单独存在时HeLa细胞的实时原位荧光成像。如图15所示，在探针和DCFH-DA共同存在时，细胞内荧光强度显著增强，而DCFH-DA或探针分别单独存在时，细胞内荧光无明显变化，表明该探针在细胞内有效产生活性氧。
实施例14
将实施例1中的探针POTA-OH在活HeLa细胞、635nm激光(100mW·cm-2)照射的活HeLa细胞以及固定HeLa细胞(通过多氯甲醛固定，使细胞收缩，导致固定细胞的粘度增大)分别共孵育。如图16所示，随激光照射时间的增加，探针在活细胞中产生光动力治疗作用，诱导细胞逐渐凋亡，从而导致细胞内粘度明显增大，表现出荧光强度的增强，这与固定细胞内的荧光现象相一致，进一步证明了该探针具有良好的体外癌细胞光动力治疗能力，且能够通过细胞粘度变化实现对治疗效果的即时评估。
实施例15
将实施例1中的探针POTA-OH(3mM，100μL/200mm3肿瘤)通过瘤内注射方式加入小鼠肿瘤中2h后，用635nm激光(100mW·cm-2，30min)照射肿瘤部位，每3天测量一次小鼠的体重和肿瘤体积。把小鼠注射PBS后相同条件光照(PBS+Laser)，以及注射POTA-OH但不光照作为平行对照组。21天后，如图17所示，PBS+Laser组和POTA-OH组的平均肿瘤体积迅速增大，仅POTA-OH+Laser(635nm，100mW·cm-2)实验组，肿瘤体积逐渐缩小，表明肿瘤得到抑制，进而证实POTA-OH在体内表现出优异的PDT效应。如图18所示，对照组和实验组中小鼠体重几乎不变，表明该探针对肿瘤的光动力治疗几乎没有副作用。更重要的是，21天后，将所有实验小鼠解剖分离出肿瘤组织，使用Ki67对肿瘤组织切片进行免疫组化染色，与对照组相比，POTA-OH+Laser组癌细胞几乎完全丧失增殖能力(图19)，进一步证实了POTA-OH的治疗作用,可有效抑制肿瘤的生长。
Claims (7)
1.一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针，其特征在于，结构式为：
2.一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备监测活细胞线粒体内粘度变化试剂中的应用。
3.如权利要求1所述的一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在检测药物诱导活细胞中粘度变化的应用。
4.如权利要求1所述的一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备检测炎症模型粘度变化试剂中的应用。
5.如权利要求1所述的一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备检测脂肪肝模型粘度变化试剂中的应用。
6.如权利要求1所述的一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备检测肿瘤模型试粘度变化剂中的应用。
7.如权利要求1所述的一种具有聚集诱导发光特性的线粒体靶向近红外粘度荧光探针在制备光动力治疗试剂中的应用。
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| CN112779001A (zh) * | 2019-11-02 | 2021-05-11 | 复旦大学 | 一种近红外粘度荧光探针的制备及其应用 |
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| CN112321492A (zh) * | 2020-10-23 | 2021-02-05 | 山西大学 | 监测线粒体自噬的荧光粘度探针及制备和应用 |
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