CN114181253A - 一种基于氟硼二吡咯的线粒体靶向型光敏剂的制备及应用 - Google Patents
一种基于氟硼二吡咯的线粒体靶向型光敏剂的制备及应用 Download PDFInfo
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Abstract
本发明提供了一种基于氟硼二吡咯的线粒体靶向型光敏剂的制备及应用。基于重原子引入具有增强光敏性的BODIPY染料(BD)，通过酯化反应将广泛认可的具有线粒体靶向的三苯基膦阳离子(TPP)衍生在BODIPY核的头部得到mitoBD。利用TPP和线粒体膜电位的静电吸附作用以及脂溶性，mitoBD可以选择性精准地靶向到细胞器‑线粒体，进而增强对肿瘤细胞的光动力治疗效果，同时具有荧光成像的应用潜力。
Description
技术领域
本发明属于化学生物学领域，研究内容是基于三苯基膦共价修饰的氟硼二吡咯(BODIPY)染料分子，通过靶向细胞器-线粒体增强对肿瘤细胞的光动力治疗效果，同时具有荧光成像的应用潜力。
背景技术
光动力治疗(PDT)是一种众所周知的广泛用于治疗各种癌症的非侵入性疗法，尤其是对膀胱癌、泌尿道癌、肺癌和食管癌。PDT的作用机制是通过光敏剂、光和氧气相互作用引发光化学反应产生活性氧(ROS)，进而触发一系列反应杀伤癌细胞：(1)活性氧能够氧化细胞内的生物大分子直接引起细胞凋亡或者坏死；(2)它还能诱导局部的炎症反应，激活靶细胞的免疫反应从而对微脉管系统产生损伤，间接杀伤靶细胞。然而，1O2的寿命很短(≈3.0μs)并且扩散半径有限(0.02μm)，只会对其附近的细胞构造产生光毒性。因此，如何将光敏剂精确地运输到重要或者较为脆弱细胞器对于提高光动力的治疗效果尤其重要。
随着对光动力机制认识的深入，人们逐渐开发靶向特定细胞器(细胞膜、细胞核、内质网、溶酶体和线粒体)的光敏剂用于对抗肿瘤。目前，细胞核靶向的光动力治疗虽然对癌细胞有很大的杀伤性，但同时伴有遗传变异的风险。考虑到光敏剂小分子脂溶性以及细胞膜不可避免地快速摄取特性，实现长时间的锚定在细胞膜上仍然是一个挑战。溶酶体靶向的光动力疗法并没有像其它靶向疗法那样引起人们的兴趣，主要是因为含有蛋白质和多肽的治疗剂对溶酶体微环境不具有耐受性。内质网靶向的光敏剂目前研究较少，相关的靶向作用机制尚不清楚，需要进一步探索。相比于前面那些缺陷，线粒体因其独特结构特点以及在细胞中重要功能使其在细胞器靶向的PDT中脱颖而出，进而受到了科学家们越来越多的关注。
线粒体是在大多数细胞中含有的由两层膜包被的细胞器，它负责有氧呼吸的第二和第三阶段，即三羧酸循环与氧化磷酸化，是细胞进行产生ATP的主要场所，因而被称为“power house”。同时，线粒体是大部分ROS产生的主要场所，它拥有一系列维持癌细胞稳态的调节系统，在调节氧化还原信号、细胞凋亡以及坏死途径中起重要作用。当线粒体中ROS过表达时，表现为氧化应激增加，进而发生线粒体膜去极化以及释放细胞色素c和激活半胱天冬酶等细胞凋亡过程。此外，线粒体自身结构特点较为突出：外膜具有高渗透性，而内膜是不可渗透的。内膜上分布的电子传递链在产生能量时会将电化学势能存储，在内膜两侧造成质子及其他离子浓度的不对称分布而形成远远高于其他细胞器的线粒体膜电位(MMP，-180mV)。因此，线粒体是PDT的理想靶标细胞器，它可以切断细胞的能量供应并导致线粒体功能障碍引发细胞凋亡。
发明内容
本发明提供了一种基于氟硼二吡咯的线粒体靶向型光敏剂的制备及应用。基于重原子引入具有增强光敏性的BODIPY染料(BD)，通过酯化反应将广泛认可的具有线粒体靶向的三苯基膦阳离子(TPP)衍生在BODIPY核的头部得到mitoBD。利用TPP和线粒体膜电位的静电吸附作用以及脂溶性，mitoBD可以选择性精准地靶向到细胞器-线粒体，进而增强对肿瘤细胞的光动力治疗效果，同时具有荧光成像的应用潜力。
本发明提供的mitoBD分子，其结构式如图1所示
本发明所提供合成方法如图2所示，通过酯化反应，以二氯甲烷作为反应溶剂，在1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI，0.10mmol)和4-二甲基氨基吡啶(DMAP，0.10mmol)催化下，分别加入BD和三苯基膦(0.95g，0.42g)于圆底烧瓶中，常温搅拌反应6小时，即得mitoBD。
本发明所提供了mitoBD分子的紫外-可见光吸收光谱，如图3所示：当mitoBD处于有机溶液中以单分子形式存在时，其最大吸收峰在678nm。
本发明所提供了mitoBD分子荧光性质的考察结果，如图4所示：当mitoBD处于有机溶液中以单分子形式存在时，其可以发射红色荧光，最大发射峰在723nm。
本发明所提供了mitoBD分子光敏性的考察结果，如图5(A)和5(B)所示：使用DPBF作为检测ROS的探针，评价mitoBD分子在黑暗以及655nm激光下照射90秒活性氧的产生，每间隔15秒利用紫外分光光度计扫描记录探针吸收变化。在90秒内，DPBF在413nm的吸收从1.0降至0.2，表明mitoBD分子具有作为光敏剂的潜能。
选用HeLa细胞，利用Leica TCS SP8共焦激光扫描仪对mitoBD分子在细胞内的荧光以及共定位进行了考察，结果如图6所示：mitoBD分子可以发射明亮的红色荧光，具有成像诊断的应用潜力。同时选用商用的线粒体探针
Green FM根据相关操作进行了共定位实验，mitoBD分子与线粒体皮尔森相关系数达到0.91，即实现了很好的共定位。
选用HeLa细胞，利用DCFH-DA探针和MTT法分别检测mitoBD分子在细胞内ROS产生以及抑制癌细胞生长的情况，结果如图7(A)和7(B)所示：同黑暗条件相比，光照组mitoBD分子在655nm激光下可以产生明显的ROS，进而产生光毒性杀死癌细胞。
附图说明
图1为本发明中所合成的化合物分子结构式。
图2为本发明中所设计分子的合成路线。
图3为本发明中mitoBD分子在DMSO中的紫外可见吸收光谱。
图4为本发明中mitoBD分子在DMSO中的荧光发射图谱。
图5为本发明中mitoBD分子在黑暗以及光照下活性氧的产生情况。
图6为本发明中mitoBD分子在细胞中荧光以及与线粒体共定位的情况。
图7为本发明中mitoBD分子在细胞中产生活性氧情况以及MTT实验的结果。
具体实施方式
下面结合具体实施例对本发明做出进一步的解释和说明，基于本发明中的实施例，本领域的技术人员，在没有做出创造性的劳动成果下所获得的其他实施例，都属于本发明的保护范围。
实施例1。光敏剂氟硼二吡咯-三苯基膦(mitoBD)分子的合成，合成路线见图2：向化合物BD(0.95g，1.0mmol)和三苯基膦溴丁酸(0.42g，1.0mmol)的二氯甲烷混合溶液中加入1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI，0.10mmol)和4-二甲基氨基吡啶(DMAP，0.10mmol)，将混合物在室温避光条件下搅拌过夜。待反应完全后停止反应，将所得的混合物用饱和盐水洗涤三次，无水硫酸钠干燥，减压下旋蒸除去溶剂。将粗产物在硅胶柱上进行纯化分离，以二氯甲烷/甲醇＝100/4作为洗脱剂，得到深绿色固体0.54g，命名为mitoBD，产率为40.5％。1H NMR(400MHz,CDCl3)δ(ppm)8.08(d,J＝16.5Hz,2H),7.93–7.84(m,6H),7.79(m,3H),7.70(m,6H),7.60(d,J＝16.5Hz,2H),7.24(d,J＝8.4Hz,2H),7.16(s,2H),6.91(d,J＝8.4Hz,2H),6.76(s,2H),4.23(m,2H),4.17(m,2H),4.07(m,2H),3.97(s,6H),3.93(s,6H),3.73(m,6H),2.96(m,2H),2.09(s,6H),1.93(m,4H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ(ppm)173.16,150.45,149.20,148.17,140.33,138.86,136.98,135.00,134.97,133.83,133.73,131.63,130.50,130.38,130.16,121.64,118.63,117.77,116.41,114.48,111.23,109.86,70.81,70.65,69.76,68.95,67.35,63.93,56.03,55.98,20.03,18.03,12.65.HRMS(ESI):m/z[M+NH4]+calcd.for C67H69BBr2F2N2O9P 1285.3148；found1285.3155.
实施例2。对mitoBD分子吸收以及荧光性质的研究：通过紫外-可见吸收分光光度计和荧光分光光度计(λEx＝676nm)测量mitoBD分子在DMSO中的荧光光谱。结果如图3和图4所示。
实施例3。对mitoBD分子光敏性的研究：通常，将DPBF探针溶液稀释添加到2mLmitoBD/DMSO溶液中并置于比色皿，同时确保探针在412nm处的吸收为1.0，然后用655nm激光照射90秒。每隔15秒记录探针在412nm处吸光度的变化。mitoBD分子在黑暗以及光照下产生1O2结果如图5(A)和5(B)所示。
实施例4。对mitoBD分子在细胞内荧光成像以及共定位进行研究。将HeLa细胞(密度约1×105)接种到规格为20mm的共聚焦细胞培养皿中，置于细胞培养箱(5％CO2，37℃)孵育过夜。待细胞完全贴壁除去旧培养液，然后用药物继续孵育。随后在避光的环境下，加入1mLMito-tracker探针溶液(浓度为200nM)，置于培养箱染色30分钟。然后将细胞用PBS(10mM，pH 7.4)洗涤几次，通过Leica TCS SP8共焦激光扫描仪拍摄(mitoBD：λEx＝633nm，λEm＝675-735nm，Tracker：λEx＝488nm，λEm＝500-580nm)，实验结果如图6所示。
实施例5。为了测定细胞内活性氧产生以及对癌细胞的抑制情况。首先选用DCFH-DA探针进行ROS测定。铺板以及给药孵育时间同上，随后，在避光的环境下加入新鲜配置的DCFH-DA探针溶液(1mL，浓度为20μM)，置于培养箱共同孵育30分钟。洗涤几次并置于黑暗或者655nm激光下照射1分钟。最后,通过在倒置荧光显微分别在明场和荧光场下成像，实验结果如图7(A)所示。其次，利用MTT法进行光毒性测定。将HeLa细胞(密度约5×103)接种到96孔板中，置于细胞培养箱(5％CO2，37℃)孵育过夜。待细胞完全贴壁除去旧培养液，然后用不同浓度的药物继续孵育。最后，洗涤几次并置于黑暗或者655nm激光下照射1分钟，37℃继续孵育，随后加入MTT，37℃孵育4小时后加入DMSO，用酶标仪测试96孔板的吸收值。实验结果如图7(B)所示。
以上所述仅是本发明的优选实施方式，应当指出，对于使用本技术领域的研究人员，在不脱离本发明技术原理的前提下，对这些实施例进行的多种修改，这些修改应该视为本发明的保护范围。
Claims (8)
1.靶向线粒体的光敏剂mitoBD分子结构。
2.根据权利要求1所述的光敏剂结构，在溶液和细胞内均具有优异的光敏性和荧光，并且能够精准地靶向线粒体，可用于癌细胞的光动力治疗及成像。
3.根据权利要求1所述光敏剂的制备方法，其特征在于以干燥的二氯甲烷作为反应溶剂，DMAP为催化剂，常温反应6小时。
4.根据权利要求1所述的光敏剂，提供了利用DPBF探针检测光敏剂在溶液中产生ROS的能力。
5.根据权利要求1所述的光敏剂，我们提供了以HeLa细胞为例的mitoBD分子在细胞内荧光成像能力以及靶向线粒体的能力。
6.根据权利要求1所述的光敏剂，我们提供了HeLa细胞为例的mitoBD分子在细胞内活性氧进而产生光毒性杀死癌细胞的能力。
7.根据权利要求1所述的线粒体靶向光敏剂mitoBD分子结构以及制备方法，均在本发明的保护范围之内。
8.根据权利要求1所述的线粒体靶向光敏剂mitoBD分子的光敏性、线粒体靶向性以及荧光成像能力，凡使用本分子对癌细胞或者活体进行PDT以及成像的应用，均在本发明的保护范围之内。
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