CN113717164B - 一种红色荧光探针及制备与其在细胞成像中的应用 - Google Patents
一种红色荧光探针及制备与其在细胞成像中的应用 Download PDFInfo
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Abstract
本发明提供了一种红色的荧光探针并将其用于癌细胞膜的高保真时空成像，该探针以香豆素为给电子基团(D)，C＝C为π桥，吡啶盐的正电荷部分为吸电子基团(A)，通过引入柔性的醚氧链，制备了具有红色发射的两亲性双香豆素、双吡啶盐目标产物MO。细胞显影实验表明，该探针能够均匀地点亮癌细胞的细胞膜，在黑暗条件下能保留6h而不发生生易位情况，实时高保真成像。同时，开发的探针具有经济性，光稳定性好，细胞暗毒性低和适用范围广等特点，预示该红色荧光探针作为膜荧光标记物可成为潜在的癌细胞膜商染。
Description
技术领域
本发明涉及一种荧光成像荧光探针及其制备方法，具体地说是一种红色的荧光探针并将其用于癌细胞膜的高保真时空成像。
背景技术
生物成像是表征生物体组织结构、了解其生理功能的重要研究工具，通常是利用光学或电子显微镜观察生物细胞和组织的微观结构，解析生物细胞的各种生理过程。与传统的检测方法相比，生物发光成像具有灵敏度高、创伤性低、选择性高、实时成像等优点，成为生命系统中监测生物分子的有力工具。荧光成像技术因其操作简单灵活，信噪比高，成本低、无电离辐射等优势，已广泛应用于监测药物分子的药代动力学，对研究机体疾病的发生和发展具有重要的作用。
细胞膜作为生物系统的边界，主要由脂质和蛋白质组成。磷脂分子是细胞膜的主要脂质组分，具有疏水的烷基链和亲水的极性基团；胆固醇分散在甘油磷脂双分子层中，是调节细胞膜硬度的重要组成部分。除脂质外，细胞膜上还负载蛋白质，占大多数细胞膜的质量比约为50％。细胞膜以脂质和蛋白质为骨架，因在活细胞中起着屏障和把关的作用而扮演着重要的角色，如将细胞内部与环境隔开，参与细胞信号传导和溶质运输。因此，揭示细胞膜的生物学功能对于药物筛选、疾病早期诊断和治疗、信号传导等各个方面的应用都至关重要。
细胞膜标记的荧光探针通常是将细胞膜靶向单元与荧光团结合而设计的。选择有效的靶向单元是膜荧光探针发展的重要步骤之一。传统的膜探针大多数面临着脱靶或失真的现象，从而不能真实地反馈细胞膜荧光标记情况。基于此，迫切需要开发长时间对细胞膜进行高保真时空成像的荧光标记物来填补膜染料方面的空缺，不断推动商业染料的发展。
发明内容
本发明旨在提供一种红色的荧光探针用于癌细胞膜的高保真时空成像，所合成的荧光分子以香豆素为基本骨架形成双香豆素双吡啶盐类衍生物。通过引入合适的亲脂性和亲水性基团赋予探针长时间特异性靶向癌细胞膜的特性，通过构筑D-π-A结构，增强分子内电子流动，使发射波长红移，产生红色荧光，实现超长时间(360min以上)高保真成像细胞膜的生命状态，有望填补红色细胞膜染料方面的空缺。
本发明涉及的一种用于癌细胞膜高保真时空成像的红色荧光探针，结构式如下：
本发明同时请求保护上述红色荧光探针MO的制备方法，包括如下步骤：
A、中间体M1的制备
将1，2-双(2-碘乙氧基)乙烷，4-甲基吡啶和乙醇混合，60-70℃条件下回流反应20-30h，TLC跟踪，反应结束后，柱层析分离得白色晶状物M1，分子式C18H26I2N4O2。
B、MO的制备
将M1和7-(二乙氨基)-2-氧代-2H-香豆素-3-甲醛溶于乙醇中，加入引发剂，60-80℃回流反应20-30h后，TLC跟踪，反应结束，冷却到室温后，析出固体，抽滤，干燥得红色固体MO，分子式C47H54I2N4O4；
其中，M1命名为：(N,N'-(2,2'-(乙基-1,2-双乙氧基)-双-(4-甲基-吡啶碘盐)，MO命名为：(N,N'-(2,2'-(乙基-1,2-双乙氧基))-双-4-((E)-2-(7-(二乙氨基)-2-氧代-2H-香豆素-3-基)乙烯基)吡啶碘盐)，
制备过程反应如下：
上述制备方法中，作为优选，步骤A中，1，2-双(2-碘乙氧基)乙烷和4-甲基吡啶的摩尔用量比为1：2.5-3.5；步骤B中，M1和7-(二乙氨基)-2-氧代-2H-香豆素-3-甲醛摩尔用量比为1：2.0-3.0为佳；所述引发剂可以为一些有机碱性物质如有机胺包括但不限于哌啶、三乙胺、三甲胺，其用量一般较小不做特殊要求；所述乙醇浓度97％(v/v)以上，无水乙醇为佳；TLC薄层色谱溶剂可选用二氯甲烷：甲醇＝4-6：1(v/v)。
本发明所述的红色荧光探针可作为膜荧光标记物或细胞膜染料在细胞成像中应用；更具体的，所述细胞成像为癌细胞膜的高保真时空成像；所述癌细胞包括但不限于宫颈癌细胞、肝癌细胞、肺癌细胞、乳腺癌细胞等。
细胞显影实验表明，该探针能够均匀地点亮癌细胞的细胞膜，在黑暗条件下能保留6h而不发生易位情况，实时高保真成像。同时，开发的探针具有经济性，光稳定性好，细胞暗毒性低和适用范围广等特点，预示该红色荧光探针作为膜荧光标记物可成为潜在的癌细胞膜商染。
与已有技术相比，本发明的有益效果体现在：
1、本发明以刚性平面结构香豆素为给电子基团(D)，C＝C为π桥，吡啶盐基团作为吸电子基团(A)，通过亲核取代及缩合反应合成目标产物MO。探针MO具有优良的红光发射，并且因为双亲结构能够很好地靶向癌细胞的细胞膜，实现长时间高保真3D时空成像细胞膜结构。这一研究成果对于发展红色荧光标记物特异性点亮膜具有极大的参考价值。
2、本发明两亲性红色荧光材料是一种水溶性较好的光学材料，暗毒性低，生物相容性好，对癌细胞如宫颈癌细胞、肺癌细胞、肝癌细胞、乳腺癌细胞具有较强的亲和性，能特异性追踪癌细胞膜变化，具有明显的应用价值。
3、本发明制备方法原料易得，成本低，合成步骤简单，易于操作。
附图说明
图1为本发明红色荧光探针的设计原理及作用机制图。
图2是MO在HeLa细胞中的共定位图。
图3是不同细胞系对MO的摄取情况及细胞毒性测试图。
图4是目标产物MO与商业膜染料的深度扫描对比图。
图5目标产物MO和商业膜染料在不同时间的细胞摄取图。
具体实施方式
下述实施例是对于本发明内容的进一步说明以作为对本发明技术内容的阐释，但本发明的实质内容并不仅限于下述实施例所述，本领域的普通技术人员可以且应当知晓任何基于本发明实质精神的简单变化或替换均应属于本发明所要求的保护范围。
实施例1
一种癌细胞膜高保真时空成像的红色荧光探针的制备方法，包括以下步骤
A、中间体M1的制备
将1，2-双(2-碘乙氧基)乙烷(1.0g,2.7mmol,sigma-aldrich)，4-甲基吡啶(0.76g,8.1mmol)和无水乙醇混合，65-67℃条件下回流反应24h，TLC跟踪，反应结束后，进行硅胶柱层析分离，得白色油状物M1，产率为48.3％。1H NMR(400MHz,DMSO-d6),δ(ppm):8.93-8.92(d,J＝6.48MHz,4H),8.06-8.05(d,J＝6.32MHz,4H),4.78-4.76(t,J＝4.76MHz,4H),3.88-3.86(d,J＝4.84MHz,4H),3.52(s,4H),2.66(s,6H).13C NMR(100MHz,DMSO-d6),δ(ppm):159.08,144.01,128.04,69.46,68.59,59.43,21.56；MS(ESI):calcd for:C18H26N2O2 2+[M/2],151.10；found,151.0982.B、MO的制备
将M1(0.2g,0.36mmol)和7-(二乙氨基)-2-氧代-2H-香豆素-3-甲醛(0.22g,0.90mmol)溶于无水乙醇中，加入0.05mL哌啶，68-70℃回流反应24h后，TLC跟踪，反应结束，冷却到室温后，析出固体，抽滤，干燥得红色固体MO，产率为85.7％。1H NMR(400MHz,DMSO-d6),δ(ppm):8.76-8.75(d,J＝6.76MHz,4H),8.13(s,2H)8.11-8.09(d,J＝6.76MHz,4H),7.77-7.73(d,J＝16.0MHz,2H),7.61-5.57(d,J＝16.0MHz,2H),7.47-7.45(d,J＝9.0MHz,2H),6.74-6.71(m,2H),6.52-6.51(d,J＝2.04MHz,2H),4.65-4.63(t,J＝4.40MHz,4H),3.89-3.86(t,J＝4.52MHz,4H),3.57(s,4H),3.48-3.43(m,8H),1.16-1.12(t,J＝7.00MHz,12H).13C NMR(100MHz,DMSO-d6),δ(ppm):159.48,156.24,153.48,151.94,145.35,144.25,137.08,130.66,122.87,122.39,113.54,109.92,108.34,96.12,69.36,68.61,58.77,44.33,12.35.MS(ESI):calcd for:C46H52N4O6 2+[M/2]2+,378.1938；found,378.1950.
实施例2
目标产物MO在HeLa细胞中的共定位图
探针MO因优良的光学性质促使我们进一步探索其在细胞水平上的运用。首先，HeLa细胞(北纳生物，BeNa culture collection)被选择用来评估MO的细胞摄取能力，如图2所示，将MO与HeLa共孵育20min，采用488nm激发光激发，显影结果表明目标分子MO能迅速、均匀地点亮HeLa细胞的细胞膜(细胞膜产生明显的红色荧光，细胞核内几没有荧光)，这与明场细胞的轮廓基本吻合。为了进一步探索MO在细胞中的分布情况，商业化染料cell maskgreen(赛默飞，Thermo Fisher)被使用。我们将cell mask green加入到已经孵育MO的细胞中继续孵育15min,培养结束后用PBS清洗3次进行共聚焦成像。实验结果表明，目标产物MO明亮的红色荧光与商业染料的绿色荧光(cell mask green)有很好的重叠(细胞膜产生明显的黄色荧光且细胞核内有大量绿色荧光点)，其相关系数Pr为0.87，说明两亲性MO具有显著的细胞膜靶向能力。
实施例3
不同细胞系对MO的摄取情况及细胞毒性测试图
考虑到癌细胞表面通常比正常细胞带有更多的负电荷，本发明制备的带正电荷吡啶盐探针理论上对癌细胞具有更高的亲和力。基于此，我们筛选了几种常见的细胞系，包括四种癌细胞(HeLa宫颈癌细胞,A549肺癌细胞,Hep G2肝癌细胞和MCF-7乳腺癌细胞)和三种正常细胞(293T人胚肾细胞,HL-7702人肝细胞和HK2人肾小管上皮细胞)，其中，HeLa和HepG2购买于北纳生物，BeNa culture collection；其他细胞来自美国菌种保藏中心ATCC，American type culture collection。研究了MO在不同细胞系的染色情况。有趣的是，正如预期地那样，只有癌细胞对MO表现出较强的摄取能力，且细胞膜可以被目标产物MO特异性点亮。相比之下，正常细胞对MO的摄取能力较弱甚至不摄取。如图3b的上图仅显示细胞膜部的红色荧光，无其他荧光；图3b的中图显示了细胞膜部的红色荧光以及细胞核内的蓝色荧光；图3b的下图仅显示细胞核内的蓝色荧光，无红色荧光。表明MO使四种癌细胞膜均可发出红色荧光，所有细胞的细胞核借助商染DAPI均发射蓝色荧光。随后，为了定量地评估目标产物的细胞毒性，本发明借助标准的3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(MTT)对7种细胞株进行毒性测定(图3a显示MO对细胞的暗毒性较低)，实验结果表明MO具有良好的生物相容性和安全性，可进一步应用于生物成像。
实施例4
目标产物MO与商业膜染料DiO的深度扫描对比图
鉴于本发明制备的探针对细胞膜表现出高的亲和力，我们进一步研究MO和商业染料分别标记细胞膜的深度扫描。具体实施方法如下，分别将MO和DiO与HeLa细胞在37℃条件下共同孵育20min，培养完成后用PBS清洗3次，置于共聚焦显微镜下采集细胞膜的三维(3D)结构信息。如图4上面两组图片显示细胞膜被MO标记为红色荧光，下面两组图片显示细胞膜被DiO标记为绿色荧光。通过对细胞进行不同深度的扫描，我们记录了一系列共聚焦图像，发现HeLa细胞的细胞膜被成功地标记上红色荧光，且在深度为1μm-9μm之间均可以观察到膜均匀地分布在周围，这个现象不仅有助于观察细胞之间的相互作用，而且可以实时可视化细胞之间的信息交换过程。作为对比，商业的染料DiO虽然能清晰地标记出细胞膜的轮廓，但是染色不够均匀，缺乏高的信噪比。综上所述,与细胞膜商染相比，我们的设计的两亲性活性探针MO可以实现高保真的二维和三维细胞膜成像以及更高的信噪比。
实施例5
目标产物MO和商业膜染料DiO在不同时间的细胞摄取图
为了进一步证实MO的两亲性可以避免不良聚集，适当的亲水性和疏水性可以确保对磷脂双分子层的强锚定能力，我们比较了目标分子与商业染料的细胞膜成像性能。首先，用探针MO孵育HeLa细胞6小时，并在孵育过程中实时进行细胞显影，分别间隔10,30,60,360min对细胞膜的成像情况进行捕捉。如图5所示，DiO仅能对细胞膜进行染色约60min，然后逐渐进入细胞质展现出不希望出现的假染色。360min后，膜上的荧光强度仅为总荧光强度的63.7％，这可能与DiO与细胞膜的结合亲和力较弱有关。这些结果明显表明DiO不能长时间跟踪细胞膜。相比之下，MO对细胞膜的染色较好，即使在360min后，膜上的荧光强度仍占总强度的100％，说明合适的脂性和亲水性基团有助于目标分子长时间高保真锚定于细胞膜上，这对长期监测细胞膜的动态变化等相关生物过程具有重要意义。
应当说明的是，本发明的上述所述之技术内容仅为使本领域技术人员能够获知本发明技术实质而进行的解释与阐明，故所述之技术内容并非用以限制本发明的实质保护范围。本发明的实质保护范围应以权利要求书所述之为准。本领域技术人员应当知晓，凡基于本发明的实质精神所作出的任何修改、等同替换和改进等，均应在本发明的实质保护范围之内。
Claims (10)
1.一种红色荧光探针，命名：MO，结构式如下：
2.权利要求1所述的红色荧光探针的制备方法，其特征在于，以香豆素为给电子基团，C＝C为π桥，吡啶盐的正电荷部分为吸电子基团，通过引入醚氧链，制备了具有红色发射的两亲性双香豆素双吡啶盐目标产物MO。
3.如权利要求2所述的制备方法，包括下述步骤：
将1,2-双(2-碘乙氧基)乙烷、4-甲基吡啶和乙醇混合，60-70℃条件下回流反应20-30h，TLC跟踪，反应结束后，柱层析分离得白色晶状物C18H26I2N2O2即M1；
将M1和7-(二乙氨基)-2-氧代-2H-香豆素-3-甲醛溶于乙醇中，加入引发剂，60-80℃回流反应20-30h后，TLC跟踪，反应结束，冷却到室温后，析出固体，抽滤，干燥得红色固体C46H52I2N4O6即MO。
4.如权利要求3所述的制备方法，其特征在于，所述1，2-双(2-碘乙氧基)乙烷、4-甲基吡啶的摩尔用量比为1∶2.5-3.5。
5.如权利要求3所述的制备方法，其特征在于，所述M1，7-(二乙氨基)-2-氧代-2H-香豆素-3-甲醛摩尔用量比为1∶2.0-3.0。
6.权利要求1所述的或者由权利要求2-5任一项所述制备方法得到的红色荧光探针在制备用于细胞成像标记物中的应用。
7.如权利要求6所述应用，其特征在于，所述细胞成像为癌细胞膜的高保真时空成像。
8.如权利要求7所述应用，其特征在于，所述癌细胞为宫颈癌细胞或肝癌细胞。
9.如权利要求6-8任一项所述应用，其特征在于，所述红色荧光探针作为膜荧光标记物的应用。
10.如权利要求6-8任一项所述应用，其特征在于，所述红色荧光探针作为细胞膜染料的应用。
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