CN113698425A - 一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途 - Google Patents
一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途，其中杂化材料简记为Eu(TTA)4P，结构式如下所示：
本发明铕配合物/噻吩吡啶盐杂化材料Eu(TTA)4P具有低的细胞毒性，在不同波段可以分别靶向脂滴和线粒体，通过不同窗口采集信息Eu(TTA)4P可以很好地跟踪细胞中的脂滴与线粒体，这种双靶向的荧光探针在疾病诊断方面具有重要的应用价值。
Description
技术领域
本发明涉及一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途。本发明杂化材料具有多光子效应，在不同波段可以分别靶向脂滴和线粒体。
背景技术
多光子吸收材料是一种低能量激发，高能量发射的发光材料，具有对细胞和生物组织穿透力强，光损伤小和自荧光干扰弱等优点，在生物医学领域具有重要的应用价值。其中能很好地跟踪细胞中脂滴与线粒体变化的荧光探针，在临床疾病诊断方面具有一定的应用前景。
脂滴是细胞内中性脂的主要贮存场所。最新的研究表明，脂滴并非细胞内一个简单的能量贮存器，而是一个复杂、活动旺盛、动态变化的多功能细胞器。脂滴能够沿着细胞骨架运动，并与其它细胞器相互作用，可能在脂类代谢与存储、膜转运、蛋白降解，以及信号传导过程中起着重要的作用。另外，研究还表明，多种代谢性疾病，如肥胖、脂肪肝、心血管疾病及糖尿病、中性脂贮存性疾病，往往都伴随着脂质贮存的异常。因此，关于脂滴的生物学研究日益受到人们的重视。
线粒体是细胞的“能量工厂”，它是由两层膜包裹的细胞器，是细胞有氧呼吸的重要场所。线粒体除了为细胞供能外，还参与细胞很多的新陈代谢，如细胞信息传递、免疫反应、生物合成、细胞分化和细胞凋亡等过程，并对细胞周期和细胞生长等方面具有一定的调控作用。所以通过对细胞线粒体状态的检测，可以准确对机体疾病进行诊断和治疗，研究高效低毒性的线粒体荧光探针受到了研究者的青眯。
目前常用的商业化细胞核染料DAPI、Hoechst等，大多是有机小分子，主要局限于单光子荧光成像，同时伴随着水溶性差、细胞毒性大、光损伤大、光稳定性差等缺点，不利于长时间追踪，从而限制了这些染料在生物体内的应用和作用机理探究。此外，发射能量和激发能量间的斯托克斯位移小，导致内源性荧光团的自发荧光而产生干扰。因此，开发一种亲生物、光稳定好，且长波激发的多光子荧光探针具有重要的科学意义和应用前景。
申请人对本申请的主题进行了如下的文献检索：
1、xueshu.baidu.com网检索结果：(2021/6/29)
2、中国知网检索结果：
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发明内容
本发明旨在提供一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料及其制备方法和用途。稀土离子EuIII具有显色性好，Stokes位移大，抗光漂白能力强及荧光寿命长等优异的光学性质，而噻吩吡啶盐具有良好的非线性光学性质，通过两者的组装可制备出具有两者优点的噻吩吡啶盐/铕配合物杂化材料，可望成为一类新型双靶向生物探针材料。
本发明细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料，简记为Eu(TTA)4P，结构式如下所示：
本发明细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料的合成方法，包括如下步骤：
将0.3996g(1.8mmol)HTTA和0.4mL(2.7mmol)三乙胺置于100mL圆底烧瓶中，加入15mL甲醇，常温下搅拌5min，逐滴加入15mL溶解有0.2007g(0.45mmol)Eu(NO3)3·6H2O的甲醇溶液，得到澄清溶液；将15mL已溶解0.225mmol化合物P+(二苯胺噻吩乙烯基甲基吡啶盐)的甲醇溶液缓慢加到上述澄清溶液中，回流反应12h，减压浓缩成油状，加入少量乙醇溶解，静置过夜，有固体析出，抽滤，甲醇洗涤干燥，获得配合物Eu(TTA)4P。产量0.6504g，产率：86.21％。
所述化合物P+为二苯胺噻吩乙烯基甲基吡啶盐。
合成路线如下所示：
本发明铕配合物/噻吩吡啶盐杂化材料Eu(TTA)4P的用途，是用于制备双靶向生物探针材料。所述双靶向生物探针材料在不同波段可以分别靶向脂滴和线粒体。
本发明的有益效果体现在：
本发明通过吡啶盐阳离子的组装使得Eu(TTA)4P探针具有特异性靶向线粒体。Eu(TTA)4P在近红外880nm波长处双光子荧光信号最强，在中红外波区1250nm波长处三光子荧光信号最强，是具有优良非线性光学性质的多光子荧光材料。如图1和图2所示。
本发明Eu(TTA)4P原料易得，合成简单。综合性质优于商用脂滴和线粒体染料，不存在类似功能的荧光探针，具有较强的商业价值。
本发明Eu(TTA)4P具有低的细胞毒性，通过不同窗口采集信息Eu(TTA)4P可以很好地跟踪细胞中的脂滴与线粒体，这种双靶向的荧光探针在疾病诊断方面具有重要的应用价值。
附图说明
图1(a)是杂化材料Eu(TTA)4P在DMSO溶剂中的有效双光子吸收截面图。说明：Eu(TTA)4P的双光子荧光光谱的最佳激发波长为880nm，最大吸收截面212GM。图1(b)是Eu(TTA)4P的双光子验证图。说明：输出功率(Iout)和输入功率(Iin)线性关系的斜率为1.948，接近2，Eu(TTA)4P具有双光子荧光效应。
图2(a)是杂化材料Eu(TTA)4P不同波段激发下Eu(TTA)4P的荧光光谱。说明：在1250nm波段激发的荧光最强。图2(b)是配合物Eu(TTA)4P在DMSO溶剂中的有效三光子吸收截面，其中插图是在DMSO中最佳波段1250nm激发下配合物Eu(TTA)4P的发光图。说明：Eu(TTA)4P的三光子荧光光谱的最佳激发波长为1250nm，三光子最大吸收截面为19.2GM，杂化材料具有较强的红光发射。图2(c)是Eu(TTA)4P的三光子验证图。说明：输出功率(Iout)和输入功率(Iin)线性关系的斜率为2.987，约等于3，Eu(TTA)4P具有三光子荧光效应。
图3是杂化材料Eu(TTA)4P的细胞毒性测试图。说明：Eu(TTA)4P的细胞毒性低，当Eu(TTA)4P的浓度达到25μM时，细胞成活率仍高于60％。
图4是Eu(TTA)4P在HepG2和HeLa细胞中的共聚焦显微成像。蓝色信号为采集420-489nm窗口信号，红色为600-650nm窗口信号，绿色为脂滴商染信号。其中420-489nm窗口采集的信号与脂滴商染信号完全重合，在叠加通道图(Merge)中呈现出浅蓝色，而与600-650nm窗口采集的红色信号完全不重合，说明：探针Eu(TTA)4P可以很好地区别细胞中的脂滴与线粒体，通过不同窗口采集信息可以很好地跟踪细胞中的脂滴与线粒体。相同的现象在宫颈癌细胞HeLa也得到了证实。
具体实施方式
实施例1：杂化材料Eu(TTA)4·P的合成
将0.3996g(1.8mmol)HTTA和0.4mL(2.7mmol)三乙胺置于100mL圆底烧瓶中，加入15mL甲醇，常温下搅拌5min，逐滴加入15mL溶解有0.2007g(0.45mmol)Eu(NO3)3·6H2O的甲醇溶液，得到澄清溶液；将15mL已溶解0.225mmol化合物P+(二苯胺噻吩乙烯基甲基吡啶盐)的甲醇溶液缓慢加到上述澄清溶液中，回流反应12h，减压浓缩成油状，加入少量乙醇溶解，静置过夜，有固体析出，抽滤，甲醇洗涤干燥，获得杂化材料Eu(TTA)4P。产量0.6504g，产率：86.21％。
1HNMR(CD3CN,400MHz,ppm)δ9.62(d,J＝6.6Hz,2H),8.53(d,J＝6.7Hz,2H),8.24(d,J＝15.7Hz,1H),7.45(t,J＝7.9Hz,4H),7.29(ddd,J＝23.4,12.9,4.5Hz,12H),6.85(d,J＝15.7Hz,1H),6.75(s,4H),6.69–6.56(m,4H),6.46(d,J＝4.1Hz,1H),5.13(s,3H),4.02(s,3H)；13C-NMR(CD3CN,100MHz,ppm)):δ157.09,146.45,144.01,140.95,137.87,133.41,129.84,125.95,121.97,49.81,39.98,33.91；ESI-MS m/z in positive ion mode:369.52,found:369.67,in negative ion mode:1037.81[Eu(TTA)4]-。
实施例2：目标分子的生物学研究
1、在96孔板中分别用0、5、10、15、20和25μM的Eu(TTA)4P孵育HepG2细胞24小时，观察细胞在不同浓度下的存活率均高于60％，表明Eu(TTA)4P具有较低的暗毒性。
2、对杂化材料Eu(TTA)4P进行细胞共聚焦显影实验。在1μM的浓度下细胞HepG2培养30min。选择了线粒体商染和脂滴商染进行共定位实验。蓝色信号为采集420-489nm窗口信号，红色为600-650nm窗口信号，绿色为脂滴商染信号。其中420-489nm窗口采集的信号与脂滴商染信号完全重合，在叠加通道图中呈现出浅蓝色，而与600-650nm窗口采集的红色信号完全不重合，表明探针Eu(TTA)4P可以很好地区别细胞中的脂滴与线粒体，并且通过不同窗口采集信息可以很好地跟踪细胞中的脂滴与线粒体的变化，相同的现象在宫颈癌细胞HeLa也得到了证实。
Claims (3)
1.一种细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料，其特征在于其结构式如下所示：
2.一种权利要求1所述的细胞双靶向多光子吸收铕配合物/噻吩吡啶盐杂化材料的制备方法，其特征在于包括如下步骤：
将HTTA和三乙胺置于圆底烧瓶中，加入甲醇，常温下搅拌分散均匀，逐滴加入溶解有Eu(NO3)3·6H2O的甲醇溶液，得到澄清溶液；将已溶解化合物P+——二苯胺噻吩乙烯基甲基吡啶盐的甲醇溶液缓慢加到上述澄清溶液中，回流反应12h，减压浓缩成油状，加入少量乙醇溶解，静置析出固体，抽滤，甲醇洗涤干燥，获得杂化材料Eu(TTA)4P；
合成路线如下所示：
3.一种权利要求1所述的铕配合物/噻吩吡啶盐杂化材料的用途，其特征在于：所述铕配合物/噻吩吡啶盐杂化材料用于制备双靶向生物探针材料；所述双靶向生物探针材料在不同波段可以分别靶向脂滴和线粒体。
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