CN106977585B - 一种线粒体定位用于光动力疗法的双光子荧光探针库及其应用 - Google Patents
一种线粒体定位用于光动力疗法的双光子荧光探针库及其应用 Download PDFInfo
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Abstract
本发明涉及一种线粒体定位用于光动力疗法的双光子荧光探针库及其应用，属于有机荧光探针领域。本发明开发了一系列原料廉价易得，操作简便，反应条件温和，生产成本低，便于产业化的一种具有光动力疗法的线粒体靶向的双光子荧光探针，其化学结构特点主要是具有线粒体靶向基团和光学调控基团链接组成。此类探针可应用于人类健康/疾病的检测和诊断，例如，利用此类探针可以准确靶向线粒体，从而可以达到对疾病的早期预警。
Description
技术领域
本发明涉及一种线粒体定位用于光动力疗法的双光子荧光探针库及其应用，具体涉及一种线粒体定位用于光动力疗法的双光子荧光探针库的构建及其在人类健康疾病检测领域的应用，属于有机荧光探针领域。
背景技术
荧光探针是化学传感技术领域在上个世纪八十年代的一项重大发现，目前己有愈来愈多的荧光探针应用于分子水平上进行实时检测。荧光检测技术由于灵敏度高，操作简便，可视性强，且对细胞、生物体的损伤小，成为了用于临床分析、环境监测、生物分析及生命科学等领域不可缺少的检测工具分子荧光探针的检测对象包括各种离子，小分子，自由基，多肽，酶，甚至还包括温度，极性，粘度等。低分子量的探针与靶标分子相互作用时往往通过化学反应，静电作用及间接作用力等方式来达到检测靶标的目的。人们可以使用荧光显微镜，荧光光谱仪，流式细胞仪，荧光活体成像系统等仪器获取荧光探针检测的相关信息，借助荧光成像技术我们能够实时检测活细胞内分子或离子的浓度以及生物大分子结构的变化过程，也可以获得关于生物组织生理代谢过程的相关信息，还可以实现生物活体的荧光成像。
无论是各种疾病的诊断和治疗，还是对疾病发病机理的研究，都必须要借助于灵敏度高特异性强的检测手段。荧光检测特别是有机小分子荧光探针一方面具有灵敏度高，操作简便的优点，另一方面研究者们能够根据需要设计合成出满足特定要求的探针分子，基于此，荧光探针和荧光检测技术在生命科学的发展中起到举足轻重的。
近年来，生命科学，环境科学，材料科学，生命医学等学科的发展对荧光探针的性能提出了更高的要求。更高的灵敏度和选择性，更低的检测限，更高的准确度和精密度，更完善可信的形态分析，更快的分析速度和自动化程度，更小的样品量，更好的生物相容性，微损或无损分析，活体，实时分析，分析器件小型化，微型化和智能化等。因此，开发出性能更为优良的荧光探针以便用于生物化学，分子生物学和细胞生物学的研究是一项紧迫而意义重大的工作。
发明内容
本发明的目的提出一种灵敏度高，操作简便，可视性强，且对细胞、生物体的损伤小的线粒体定位用于光动力疗法的双光子荧光探针库及其应用。其具有高的线粒体靶向性，双光子性能突出。该类荧光探针可以应用于人类健康疾病的检测中，可获得高效的荧光成像图。
为了解决上述技术问题，本发明提出的技术方案是：一种线粒体定位用于光动力疗法的双光子荧光探针库，以荧光素衍生化合物为主体结构，以多肽链基团或三苯基膦基团为线粒体靶向基团，得到线粒体定位的双光子荧光探针，具体为如下结构中的一种：
为了解决上述技术问题，本发明提出的另一技术方案是：所述双光子荧光探针库的探针在波长为800nm的光照射有很强的双光子信号，在375nm激发下检测到单线态氧浓度增强的信号。
优选的，线粒体定位的双光子荧光探针的单光子激发波长为G1/G2 450nm，R1/R2560nm，Y2 380nm。
优选的，所述的荧光探针库可应用于靶向线粒体，并具有良好的双光子性能，通过光动力疗法使特定的线粒体受损。
有益效果：
线粒体是细胞的能量工厂，参与众多生命活动。其中双光子荧光显微镜在活细胞方面具有成像深度深、背景小和样品光损伤小等优势。设计新型的双光子荧光线粒体示踪剂，并通过单双光子荧光活细胞成像，研究了示踪剂的活细胞线粒体成像效果，为进一步开发新一代的双光子荧光线粒体示踪剂提供了科学参考。在激光激发下检测到单线态氧的生成，可用于光动力疗法。
本发明的荧光探针具有较好的线粒体靶向性、化学稳定性和生物兼容性。激光共聚焦成像实验表明该探针具有较好的细胞通透性，对细胞和生物体无毒副作用。
本发明的荧光示踪剂在可应用于细胞体系精准靶向线粒体且双光子性能优越，主要用的活细胞为HepG2细胞株。
激光共聚焦成像实验表明该探针具有较好的细胞通透性，对细胞和生物体无毒副作用,可以实现亚细胞水平活性氧水平的检测，并进一步应用于神经退行性疾病及癌症的研究。
附图说明
下面结合附图对本发明的作进一步说明。
图1为化合物G1,G2,R1,R2，Y2在室温下随着探针浓度增加的荧光光谱。
图2为化合物G1,G2,R1,R2，Y2在HepG2细胞中成像图。
图3是化合物质谱图(图中a是化合物G1,G2的质谱图，b是化合物R1,R2的质谱图)。
图4是化合物G2,Y2的光动力疗法细胞成像图。
具体实施方式
为了更好地理解本发明，下面通过具体的实施例来具体说明本发明的技术方案。
实施例1:特异性靶向线粒体荧光探针。记为化合物G1，结构式如下：
在25ml的圆底烧瓶中加入化合物4,4'-((1E，1'E)-(2-(丙-2-炔-1-基氧基)嘧啶-4,6-二基)双(乙烯-2,1-二基))双，N-二乙基苯胺)(30μl,40mM)和叠氮化合物(R)-6-氨基-2-((R)-2-((S)-2-((R)-2-(4-叠氮基丁酰氨基)-3-环己基丙酰胺基)-5-胍基戊酰氨基)-3-环己基丙酰胺基)己酰胺(10μl,40mM)，将8μlCuSO4·5H2O(32mM储备液)和40μL三-[苄基三唑基甲基]-胺(TBTA)(10mM储备液)加入48μLDMSO/H2O(1:1)中。将所得溶液混合，将管加盖并在室温下摇动2天。
实施例2：特异性靶向线粒体荧光探针。记为化合物R1，结构式如下：
在25ml的圆底烧瓶中加入荧光素衍生化合物4,4'-((1E，1'E-(2-(丙-2-炔-1-基氧基)嘧啶-4,6-二基)双(乙烯-2,1-二基))双，N-二乙基苯胺)(10μl,10mmol)和叠氮化合物(R)-6-氨基-2-((R)-2-((S)-2-((R)-2-(4-叠氮基丁酰氨基)-3-环己基丙酰胺基)-5-胍基戊酰氨基)-3-环己基丙酰胺基)己酰胺(10μl,40mM)，将8μlCuSO4·5H2O(4mM储备液)和40μL三-[苄基三唑基甲基]-胺(TBTA)(10mM储备液)加入48μLDMSO/H2O(1:1)中。将所得溶液混合，将管加盖并在室温下摇动2天。
实施例3:特异性靶向线粒体荧光探针。记为化合物G2，结构式如下：
将荧光素衍生化合物(16μl,20mM)和4-叠氮丁基三苯基膦(8μl,20mM)依次加入到3ml的乙腈中，常温搅拌8小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物。
实施例4:特异性靶向线粒体荧光探针。记为化合物R2，结构式如下：
将荧光素衍生化合物(20μl,10mM)和4-叠氮丁基三苯基膦(10μl,40mM)依次加入到3ml的乙腈中，常温搅拌8小时，冷却抽滤，除去溶剂，经硅胶柱层析提纯得到目标产物。
实施例5：特异性靶向线粒体荧光探针。记为化合物Y2，结构式如下：
加入6-(二甲基氨基)萘并[2,3-c]呋喃-1,3-二酮100mg，溴丙胺200mg，4ml醋酸，在油浴120℃下回流10h。用硅胶柱进行提纯(洗脱剂为石油醚：乙酸乙酯＝5:1)，得到(3-溴丙基)-6-(二甲基氨基)-1H-苯并[f]异吲哚-1,3(2H)-二酮。1H-NMR(300MHz,(CD3)2SO):δ＝8.222(1H,s),8.124(1H,s),7.991(1H,d,J＝9.3Hz),7.359(1H,m),7.238(1H,s),3.718(2H,m),3.567(2H,m),3.088(6H,s),2.140(2H,m)；13C-NMR(75MHz,(CD3)2SO):δ＝167.566,167.463,150.144,136.882,127.837,126.161,123.903,121.932,121.636,117.580,107.287,35.888,31.601,30.872,29.513,28.641.
分批加入三苯基膦，保证其过量；油浴120℃回流过夜。若反应结束后，三苯基膦固体依旧很多，用甲醇洗涤，收集溶液。粗产物用硅胶柱进行分离提纯(洗脱剂为乙酸乙酯:甲醇＝5:1)。由于化合物Y1003极性十分大，所以选用短硅胶柱过柱，硅胶选用细硅胶可以提高纯度。1H-NMR(300MHz,(CD3)2SO):δ＝8.207(1H,s),8.114(1H,s),7.989(1H,d,J＝9.3Hz),7.877(3H,m),7.342(1H,d,J＝11.1Hz),7.236(1H,s),3.761(4H,m),3.079(6H,s),1.950(2H,m)；13C-NMR(75MHz,(CD3)2SO):δ＝167.616,167.512,150.021,133.382,129.829,127.933,126.328,123.892,122.054,121.634,118.479,117.563,117.340,107.227,37.371,37.098,20.747,18.434,17.746.
实施例7：
1.化合物G1,G2,R1,R2，Y2在室温下随着探针浓度增加的荧光光谱(图1)：
用缓冲溶液配制1-10uM的探针，用荧光分光光度法分别测试，并绘制G1,G2,R1,R2，Y2不同浓度的荧光光谱图。结果描述：探针的荧光强度随着浓度的增加而增加，并不会因为探针浓度增加发生淬灭。
2.化合物G1,G2,R1,R2，Y2在HepG2细胞中成像图(图2)：
向含有HepG2细胞的培养皿中加含有探针的细胞培养液，探针在培养液中的浓度为1uM，与细胞培养液混合均匀后，染色2个小时后，用pH＝7.35的缓冲溶液冲洗3次，将该培养皿在共聚焦显微镜下成像。结果描述：(1)/(7)是有细胞的明场图，(2)/(8)是有细胞的加探针的单光子照射的暗场图，(3)/(9)是有细胞的加探针的双光子照射的暗场图，(4)/(10)是有细胞的加商业线粒体探针的暗场图，(5)/(11)是明场图和单光子暗场图的拟合重叠图，从图中可以发现探针成功进入了细胞，(6)/(12)是双光子暗场图和商业线粒体探针暗场图的拟合重叠图，从图中我们可以发现所设计的探针成功地靶向了线粒体，并具有优秀的双光子性能。
3.化合物G1,G2,R1,R2的质谱图(图3)
4.化合物G2,Y2的光动力疗法细胞成像图(图4)：
向含有HepG2细胞的培养皿中加含有探针的细胞培养液，探针在培养液中的浓度为1uM，与细胞培养液混合均匀后，染色2个小时后，用pH＝7.35的缓冲溶液冲洗3次，将该培养皿在共聚焦显微镜下成像。结果描述：图(1)(3)(5)分别是在800nm照射前加了化合物G2，Y2和DMSO(参比)的明场图，图中右下角是同种情况下的单光子的暗场成像图；图(2)(4)(6)分别是在800nm照射后对应的明场和暗场图，可以看出加了化合物G2，Y2的细胞在双光子照射一段时间后的区域，呈现出很大程度的损伤，而未加示踪剂的参比中的细胞完好无损。说明了示踪剂G2，Y2具有应用于光动力疗法的潜力。
本发明的不局限于上述实施例所述的具体技术方案，凡采用等同替换形成的技术方案均为本发明要求的保护范围。
Claims (4)
1.一种线粒体定位用于光动力疗法的双光子荧光探针库，其特征在于：以荧光素衍生化合物为主体结构，以三苯基膦基团为线粒体靶向基团，得到线粒体定位的双光子荧光探针，具体为如下结构中的一种：
2.根据权利要求1所述线粒体定位用于光动力疗法的双光子荧光探针库在制备检测试剂盒的应用，其特征在于：所述双光子荧光探针库的探针在波长为800nm的光照射有很强的双光子信号，在375nm激发下检测到单线态氧浓度增强的信号。
3.根据权利要求1所述的线粒体定位用于光动力疗法的双光子荧光探针库在制备检测试剂盒的应用，其特征在于：线粒体定位的双光子荧光探针的单光子激发波长为G2 450nm，Y2 380nm。
4.根据权利要求1所述的线粒体定位用于光动力疗法的双光子荧光探针库在制备应用于神经退行性疾病及癌症药物的应用，其特征在于：所述的荧光探针库可应用于靶向线粒体，并具有良好的双光子性能，通过光动力疗法使特定的线粒体受损。
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