CN111253935B - 一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途 - Google Patents
一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途，可以同时在两个不同通道监测细胞线粒体中极性和粘度的实时变化，其结构式如下：
本发明双光子荧光探针分子能够利用两个不同的荧光波段同时对极性(短波，410nm)和粘度(长波，580nm)进行荧光响应。此外，细胞毒性测试表明该探针对于细胞几乎没有什么毒副作用，双光子共聚焦荧光显微成像实验表明该探针对HeLa细胞渗透性好，可以有效定位细胞中的线粒体(定位系数为0.97)，适用于细胞线粒体内极性和粘度的双通道双光子荧光成像和检测。
Description
技术领域
本发明涉及一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途，以实现 双通道双光子成像同时检测细胞线粒体内的极性和粘度变化。具有双光子吸收性能优异，高 透膜性，低细胞毒性，高光稳定性和生物相容性的优点。
背景技术
细胞和亚细胞器粘度是一种重要的微环境参数，它通过影响活细胞内生物分子和化学信 号的相互作用和传递而发挥生物学功能。细胞粘度的异常变化与糖尿病、梗死、高血压等疾 病密切相关。细胞的极性是建立和维持特定域功能的一系列复杂机制的反馈。涉及空间排列 和蛋白质组成的许多细胞过程，例如分化，局部膜生长，免疫应答激活，定向细胞迁移以及 分子跨细胞层的矢量运输，可能导致极性的改变和发展。因此，极性的异常变化与紊乱和疾 病密切相关。
线粒体作为细胞的能量来源，是细胞内重要的细胞器，在细胞代谢中起着关键的作用， 包括通过氧化磷酸化提供代谢能量，通过活性氧(ROS)产生、调节Ca2+稳态，触发细胞凋 亡的细胞信号传递平台。值得注意的是，线粒体的独特功能与维持其参数和微环境(例如pH， 粘度，极性，温度等)的稳态有关。
小分子荧光探针因其高灵敏度、实时检测、快速无损分析等优点，在荧光成像中得到了 广泛的应用。用荧光探针测量活细胞的亚细胞活性是收集特定亚细胞微环境信息的有力手段。 目前单一检测线粒体极性或者粘度的荧光探针有很多，但是能够同时检测线粒体极性和粘度 的双光子荧光探针还未见报道。
细胞凋亡是细胞程序性死亡最常见的方式，是调节细胞平衡和维持组织稳态的关键。细 胞凋亡缺陷是导致自身免疫性疾病和癌症等多种病理过程的主要原因。因此，治疗这些疾病， 特别是癌症治疗的一个有效的治疗策略是通过调节凋亡途径选择性地诱导细胞死亡。凋亡的 检测和监测不仅在许多细胞生物学和临床研究中具有重要意义，而且在凋亡靶向治疗的治疗 评估中也具有重要意义。从已有的文献报道来看，细胞凋亡会引起细胞内线粒体微环境如极 性、粘度和pH的变化。然而大部分荧光探针只能检测其中一个微环境参数的变化，这将阻 碍我们更深入地了解细胞凋亡过程中微环境之间的动态联系。因此开发性能优异的双通道检 测极性和粘度的双光子荧光探针，通过监测线粒体的粘度和极性的变化来理解线粒体相关的 细胞事件，是十分迫切的。
发明内容
本发明旨在提供一种双通道检测极性和粘度的双光子荧光探针及其制备方法和用途，所 要解决的技术问题是通过分子设计得到一种合适的荧光探针结构，具有双光子吸收性能优良， 细胞毒性低，透膜性和生物相容性好，选择性和光稳定性高等优点，以实现双通道双光子荧 光成像同时检测细胞凋亡过程中极性和粘度的实时变化和双色成像。
本发明双光子荧光探针，简记为Mito-PV，以咔唑为母体，其结构式如下：
本发明双光子荧光探针的制备方法，包括如下步骤：
步骤1：氮气气氛中，于60℃搅拌下将1-碘己烷(1.56g，7.38mmol)加入2-甲基苯并噻 唑(1.0g，6.71mmol)中，冷却至室温后得到粗产物；粗产物通过柱色谱法纯化(二氯甲烷： 甲醇＝4∶1作为洗脱剂)，得到1.50g(62％)的中间体1——3-己基-2-甲基苯并噻唑碘化物 盐；
步骤2：将化合物9-乙基-6-碘-9H-咔唑-3-甲醛(1.0g，2.86mmol)、4-氟苯乙炔(0.41g， 3.44mmol)、三苯基磷二氯化钯(9.65mg，0.014mmol)、碘化亚铜(5.24mg，0.027mmol)和 三乙胺(2ml)在30℃无水无氧条件下搅拌反应12小时；将混合物冷却至室温，沉淀过滤并 浓缩，得到粗产物；通过柱色谱法(石油/二氯甲烷＝4：1作为洗脱液)纯化粗物质，得到中 间体3——3-(4-氟苯乙炔基)-6-醛基-N-乙基咔唑，0.80g，产率为82％。
步骤3：将中间体1(0.32g，0.9mmol)和中间体3(0.3g，0.9mmol)在50mL无水乙醇中混合，在氮气保护下回流反应12小时，然后冷却，并通过旋转蒸发除去溶剂；将获得的残余物用30mL饱和盐水洗涤，然后通过二氯甲烷(3×50mL)萃取，并在真空下蒸发溶剂； 残余物通过柱色谱法纯化(二氯甲烷：甲醇＝50：1作为洗脱剂)，得到0.36g(59％)的 Mito-PV。
本发明双光子荧光探针Mito-PV的合成路线如下：
本发明双光子荧光探针的用途，是在双通道检测细胞中线粒体内的极性和粘度时作为检 测试剂使用。检测方法如下：
将本发明双光子荧光探针溶于DMSO中制得2mM的母液，各取15μL母液于3mL不同极性的溶剂中，获得探针Mito-PV在不同溶剂中的紫外谱图。在360nm波长激发下，其在410nm处的荧光强度随着测试体系极性的增大逐渐减弱。同时发现探针Mito-PV只在粘度较大的甘油溶剂中580nm处有较强的荧光发射峰。这表明Mito-PV可用于检测常见溶液的极性和粘度。为了进一步验证探针Mito-PV对极性和粘度的双响应特性，首先在具有不同比例的水和1,4-二氧六环混合溶剂中，测量Mito-PV的吸收和荧光光谱。当溶剂的极性从含10％水(极性参数Δf≈0.229)依次增大到含70％水(Δf≈0.304)时，在吸收最大值中只能看到微小的变化，与测不同溶剂极性的结果一致。相反，当溶液的极性(Δf)从0.304(70％水)降低到0.229(10％水)时，Mito-PV在410nm处的荧光强度增加了11倍。并且，Mito-PV 在410nm处的荧光强度与Δf具有良好的线性关系，证实Mito-PV对极性具有良好的荧光响应。接着在具有不同比例的甲醇和甘油混合溶剂中，测量Mito-PV的荧光发射光谱。随着混合体系中甘油含量的逐渐增加(粘度从1.0增加到300cp)，Mito-PV在580nm处的荧光强度增加了15倍，并且log I580nm与logη之间具有良好的线性关系。这表明Mito-PV对溶剂的粘度同样具有良好的荧光响应。此外，利用本探针Mito-PV测试了依托泊苷(etoposide)诱导HeLa细胞凋亡过程的极性和粘度的变化，随着细胞的凋亡，短波的蓝色通道荧光增强，说明极性减小，长波的红色通道荧光增强，说明粘度增大。
本发明的双光子荧光探针能够同时双色荧光响应极性和粘度的变化。细胞毒性实验还表 明该探针具有较低的细胞毒性，双光子共聚焦荧光显微成像实验表明该探针对HeLa细胞渗 透性好，可以有效定位细胞中的线粒体(定位系数分别为0.97)，适用于细胞线粒体内极性和 粘度的双光子荧光成像，并可以双通道检测依托泊苷诱导细胞凋亡过程中极性和粘度的实时 变化和双色成像。
附图说明
图1是10μM探针在不同极性有机溶剂中的(左)紫外吸收光谱图；(右)荧光发射光谱 图；
图2是10μM探针在不同体积比水/1,4-二氧六环混合溶剂中的(左)紫外吸收光谱图； (右)荧光发射光谱图，插图：荧光强度I410nm和Δf之间的线性关系图；
图3是10μM探针在不同体积比甲醇/甘油混合溶剂中的(左)荧光发射光谱图；(右)荧光强度I580nm和Δf之间的线性关系图；
图4是0.1mM探针在混合溶剂中的有效双光子吸收截面图(左)水/1,4-二氧六环不同体 积比的混合溶剂；(右)甲醇/甘油不同体积比的混合溶剂；
图5是在不同浓度(0μM、10μM、20μM、30μM)的探针分子的作用下的HeLa细胞存 活率图。
图6是10μM探针和1μM的Mitotracker Deep red(MTDR)同时共染HeLa细胞线粒体定位验证共聚焦荧光成像图。
图7是10μM探针在50μM依托泊苷(etoposide)诱导的HeLa细胞凋亡共聚焦荧光成像 图。
具体实施方式
下面通过实施例对本发明做进一步说明。
实施例1：荧光探针分子Mito-PV的合成
将3-己基-2-甲基苯并噻唑碘化物盐(0.32g，0.9mmol)和3-(4-氟苯乙炔基)-6-醛基-N-乙 基咔唑(0.3g，0.9mmol)在50mL无水乙醇中混合；将混合液在氮气保护下回流12小时， 然后冷却，通过旋转蒸发除去溶剂；将获得的残余物用30mL饱和盐水洗涤，然后通过二氯 甲烷(3×50mL)萃取，并在真空下蒸发溶剂，残余物通过柱色谱法纯化(二氯甲烷：甲醇＝50：1作为洗脱剂)，得到0.36g(59％)的Mito-PV。
1H NMR(400MHz,DMSO-d6,ppm):δ9.00(s,1H),8.43(dd,J＝21.2,13.9Hz,3H),8.27(t, J＝9.0Hz,2H),8.03(d,J＝15.6Hz,1H),7.87(dd,J＝8.1,4.9Hz,2H),7.81-7.72(m,3H),7.66 (dd,J＝8.1,5.7Hz,2H),7.31(t,J＝8.7Hz,2H),4.97(t,J＝7.0Hz,2H),4.56(m,J＝6.8Hz,2H), 1.88(dd,J＝15.0,7.7Hz,2H),1.46(dd,J＝14.9,7.2Hz,2H),1.40-1.24(m,7H),0.84(t,J＝7.0 Hz,3H).13C NMR(100MHz,DMSO,ppm):δ171.75,160.61,150.62,142.64,141.22,140.07, 133.49,133.41,129.95,129.34,128.79,128.12,127.78,125.69,124.29,123.98,123.81,122.48, 122.41,119.20,116.55,116.14,115.92,113.58,110.64,110.46,110.08,90.04,87.01,48.58,37.67, 30.76,28.65,25.42,21.93,13.85,13.79.
实施例2：荧光探针分子的光谱测试
将本发明双光子荧光探针溶于DMSO中制得2mM的母液，各取15μL母液于3mL不同极性的溶剂中，获得探针Mito-PV在不同溶剂中的紫外吸收谱图(图1左图)。在360nm波 长激发下，其在410nm处的荧光强度随着测试体系极性的增大逐渐减弱。探针Mito-PV只在 粘度较大的甘油溶剂中580nm处有较强的荧光峰。这表明Mito-PV可用于检测常见溶液的 极性和粘度(图1右图)。为了进一步证明探针Mito-PV的极性和粘度的双响应特性，在25℃ 下在具有不同比例的水和1,4-二氧六环混合溶剂中，测量Mito-PV的吸收和荧光光谱(图2 左图，图2右图)。当溶液的极性(Δf)从0.304(70％水)降低到0.229(10％水)时，Mito-PV 在410nm处的荧光强度增加了11倍。并且，Mito-PV在410nm处的荧光强度与Δf具有良好 的线性关系(图2右图插图)。这表明Mito-PV能够很好的响应溶剂极性的变化。接着在具 有不同比例的甲醇和甘油混合溶剂中，测量Mito-PV的荧光光谱(图2)。随着混合体系中甘 油含量的逐渐增加(粘度从1.0增加到300cp)，Mito-PV在580nm处的荧光强度增加了15 倍(图2左图)，并且log I580nm与log η之间具有良好的线性关系(图2右图)。这表明Mito-PV 对溶剂的粘度具有良好的荧光响应。以上结果表明Mito-PV可利用两个不同的荧光波段用于 检测常见溶液的极性和粘度。
实施例3：荧光探针分子的双光子性能测试
首先测试Mito-PV在不同水和1,4-二氧六环极性混合溶剂中(水的含量分别为10％，40％ 和70％)的有效双光子吸收截面。有效双光子吸收截面在720nm出现最大并随着水含量的增 加，逐渐从88GM降到29GM(图3左图)。Mito-PF在不同甲醇和甘油混合溶剂中(甘油的 含量分别为99％，80％和60％)，有效双光子吸收截面在840nm出现最大并随着甘油含量的 降低，逐渐从96GM降到48GM(图3右图)。以上证明Mito-PV有能力用于细胞内极性和 粘度的双光子生物成像。
实施例4：细胞毒性测试
我们用MTT(5-二甲基噻唑-2-基-2,5-二苯基四唑溴化物)方法进行了细胞毒性实验。 Mito-PV在活HeLa细胞中加入各种浓度(0μM，10.0μM，20.0μM，30.0μM)，24小时后测试，结果如图4所示，当探针浓度达到30μM时，细胞存活率仍然有约90％，说明了本发明 荧光探针分子对细胞无明显毒性作用，可以进行生物应用。
实施例5：细胞定位测试
为了研究Mito-V的线粒体定位性能，我们这里使用的商业染料Mitotracker Deepred (MTDR)与Mito-PV在HeLa细胞中进行共定位研究。结果表明Mito-PV的黄色通道(λem＝500-600nm，λex＝720nm)和MTDR(λem＝655-755nm,λex＝635nm)的荧光图像重叠良好， 并且Mito-PV与MTDR的Pearson共定位系数计算为0.97(图5)。这些结果表明，Mito-PF 可以很好地定位于活细胞的线粒体中。
实施例6：细胞凋亡共聚焦荧光成像
依托泊苷(etoposide)能够引起细胞凋亡，是一种公认的细胞凋亡试剂。从已有的文献 报道来看，细胞凋亡会引起细胞内线粒体微环境如极性、粘度和pH的变化。因此，我们尝 试用Mito-PV来监测在依托泊苷诱导的细胞凋亡过程中线粒体极性和粘度的实时变化。将10 μM的Mito-PV加入细胞内孵育0.5h。之后将etoposide(50μM)加入细胞内，时间每隔15分钟进行成像。通过成像可以发现在蓝色通道(Blue Channel,λem＝420-460nm)中的荧光强度逐渐增强，说明极性在逐渐减小。而红色通道(Red Channel,λem＝500-600nm，λex＝720nm) 中荧光强度也是逐渐增强，说明粘度在逐渐增大。通过以上数据的分析，我们能够清晰的发 现，随着加入依托泊苷(etoposide)的时间增长，即凋亡更深入，这时细胞内线粒体的极性 逐渐减小，粘度逐渐增大。这说明依托泊苷诱导的细胞凋亡会引起细胞内线粒体极性减小和 粘度的增大。
Claims (3)
1.一种双通道检测极性和粘度的双光子荧光探针，简记为Mito-PV，其特征在于其结构式为：
2.一种权利要求1所述的双光子荧光探针的制备方法，其特征在于包括如下步骤：
步骤1：氮气气氛中，于60℃搅拌下将1-碘己烷加入2-甲基苯并噻唑中，冷却至室温后得到粗产物；粗产物通过柱色谱法纯化，得到中间体1——3-己基-2-甲基苯并噻唑碘化物盐；
步骤2：将化合物9-乙基-6-碘-9H-咔唑-3-甲醛、4-氟苯乙炔、三苯基磷二氯化钯、碘化亚铜和三乙胺在30℃无水无氧条件下搅拌反应12小时；将混合物冷却至室温，沉淀过滤并浓缩，得到粗产物；通过柱色谱法纯化粗物质，得到中间体3——3-(4-氟苯乙炔基)-6-醛基-N-乙基咔唑；
步骤3：将中间体1和中间体3在无水乙醇中混合，在氮气保护下回流反应12小时，然后冷却，并通过旋转蒸发除去溶剂；将获得的残余物用饱和盐水洗涤，然后通过二氯甲烷萃取，并在真空下蒸发溶剂；残余物通过柱色谱法纯化，得到目标产物Mito-PV；
步骤1中，柱色谱法纯化时的洗脱剂为二氯甲烷：甲醇＝4：1，v/v；
步骤3中，柱色谱法纯化时的洗脱剂为二氯甲烷：甲醇＝50：1，v/v。
3.一种权利要求1所述的双光子荧光探针的用途，其特征在于：所述双光子荧光探针在检测细胞中线粒体内的极性和粘度时作为检测试剂使用；所述双光子荧光探针能够同时双色荧光响应极性和粘度的变化。
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