CN114195774A - 一种具有次氯酸激活荧光和线粒体靶向功能的光敏剂及其制备方法和应用 - Google Patents
一种具有次氯酸激活荧光和线粒体靶向功能的光敏剂及其制备方法和应用 Download PDFInfo
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- CN114195774A CN114195774A CN202111299589.6A CN202111299589A CN114195774A CN 114195774 A CN114195774 A CN 114195774A CN 202111299589 A CN202111299589 A CN 202111299589A CN 114195774 A CN114195774 A CN 114195774A
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- photosensitizer
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- hypochlorous acid
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- C—CHEMISTRY; METALLURGY
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
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- C—CHEMISTRY; METALLURGY
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
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- C09K2211/1025—Heterocyclic compounds characterised by ligands
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
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- C—CHEMISTRY; METALLURGY
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Abstract
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种具有次氯酸激活荧光和线粒体靶向功能的光敏剂及其制备方法和应用。
背景技术
随着医疗技术的发展,人们对医疗质量的要求也越来越高,对于肿瘤组织的精准定位和精确治疗是未来医疗的必然研究方向。近年来,光动力疗法(PDT)成为一种有吸引力的肿瘤治疗技术,这主要是因为其在非侵入性治疗和精确可控性方面具有显著优势。原则上,PDT使用可忽略不计细胞毒性的光敏剂(PS)与光的组合,将肿瘤细胞中的分子氧转化为一系列反应性极强的活性氧(ROS),特别是单线态氧(1O2),诱发细胞死亡。得益于PS固有的荧光,PDT为荧光影像引导肿瘤治疗提供了令人兴奋的前景。
不幸的是,传统光敏剂由于分子间π-π堆叠而遭受聚集引起淬灭(ACQ)效果,导致成像质量和ROS产氧量低下。新型聚集诱导发光(AIE)光敏剂相比于传统的ACQ类光敏剂,具有更好的光稳定性,保证了成像质量和光动力学治疗效果。虽然AIE光敏剂具备诸多优点,但仍存在一些问题亟待解决。第一,如何实现更有效的系间窜越(ISC)过程以达到更高效的ROS产生效率;第二,如何实时监测基于AIE光敏剂的PDT过程,以实现更有效的细胞信息传递;第三,如何将高效发光的AIE光敏剂在实际应用中靶向肿瘤,以实现精准影像诊断并引导光动力治疗。显然,目前迫切需要开发一种高性能AIE光敏剂,能够准确地区分正常组织和肿瘤组织,从而为临床提供准确的诊断和治疗。
发明内容
本发明的目的是在现有技术的基础上,提供一种具有次氯酸激活荧光和线粒体靶向功能的光敏剂PTZSPY,具有线粒体靶向能力、高识别特异性、聚集诱导发光特性、ClO-激活荧光以及伴随的1O2原位产生用于PDT,可用于选择性监测ClO-以区分癌细胞和PDT诱导的癌细胞消融。
本发明的另一目的是提供一种上述光敏剂PTZSPY的制备方法。
本发明的又一发明目的是提供上述光敏剂,具有靶向线粒体进行体内外荧光成像诊断、发挥光动力治疗效果的医药用途,特别是体内外肿瘤细胞和组织的荧光成像诊断和/或治疗。
本发明的技术方案如下:
一种具有次氯酸激活荧光和线粒体靶向功能的光敏剂PTZSPY,具体结构式如下所示:
在光照射下,光敏剂可以被激发到单线激发态,光敏剂可以从该激发态通过辐射途径发出荧光,或进行系统间交叉(ISC)成为三线态后将能量给氧气形成ROS。不幸的是,这两者是竞争关系的,因为光敏剂(PSs)在PDT过程中不可避免地会以荧光的形式释放大量能量。许多报告表明,I-作为抗衡阴离子在其他盐中表现出最高的光敏性能。然而,I-的存在也使得PS的发射性较弱。本发明基于这一原理并利用肿瘤微环境中高表达的次氯酸刺激选择性地激活荧光。因此,即使在聚集状态下开发“开启”荧光和高ROS生产能力对于高效的荧光引导光动力治疗也具有重要意义。
在本发明中,在制备用于ClO-线粒体靶向可视化和癌细胞消融的聚集诱导发射荧光探针PTZSPY作为光敏剂时,首先,利用吩噻嗪作为强电子供体和ClO-荧光检测基团,开发了基于N-取代的AIE构建块以克服聚集引起的猝灭。随后,引入了带正电荷的吡啶鎓盐通过静电作用靶向线粒体。最后,噻吩桥接单元扩展了π共轭骨架以进行红色发射,并进一步提高分子内D-A强度。
本发明提供的用于ClO-激活荧光的线粒体靶向和可视化肿瘤细胞消融的聚集诱导发射荧光探针PTZSPY作为光敏剂时,具有以下几点优势:(1)引入配位的碘负离子,通过碘重原子效应使其达到更高效的ROS产生效率;(2)通过吡啶鎓盐的阳离子靶向肿瘤细胞中膜负电位较高的线粒体,实现了动态监测肿瘤细胞光动力消融;(3)肿瘤细胞中ClO-含量高于正常细胞,利用肿瘤细胞中高表达的ClO-准确开启荧光,与ClO-响应之后的PTZSPY=O同时具备AIE光学性质和PDT功能,从而达到精准诊疗的目的。
在本发明中,具有次氯酸激活荧光和线粒体靶向功能的光敏剂PTZSPY的制备方法,其特征在于,它包括以下步骤:
进一步地,具有次氯酸激活荧光和线粒体靶向功能的光敏剂PTZSPY的制备方法,它包括以下更详细的步骤:
(1)10H-吩噻嗪、N-溴代琥珀酰亚胺与THF混合均匀进行化学反应制备3-溴-10H-吩噻嗪;
(2)在PdCl2(dppf)和K2CO3存在的条件下,3-溴-10H-吩噻嗪与(5-甲酰基噻吩-2-基)硼酸进行化学反应制备化合物3;
(3)在Pd2(dba)3、t-Bu3P和t-BuONa存在的条件下,化合物3与溴苯进行化学反应制备化合物4;
(4)在哌啶存在的条件下,化合物1、化合物4与无水乙醇混合均匀,加热至回流温度进行化学反应制备荧光探针PTZSPY。
对于本发明而言,在步骤(1)中,10H-吩噻嗪与N-溴代琥珀酰亚胺的摩尔比为1:0.8~1.5,可以但不局限于1:0.8、1:0.9、1:0.95、1:1、1:1.05、1:1.2、1:1.3或1:1.5,为了获得更好的效果,10H-吩噻嗪与N-溴代琥珀酰亚胺的摩尔比为1:1。
在步骤(2)中,3-溴-10H-吩噻嗪与(5-甲酰基噻吩-2-基)硼酸的摩尔比为1:1~2,可以但不局限于1:1、1:2、1:25、1:1.3、1:1.35、1:1.4、1:1.6、1:1.8或1:2,为了获得更好的效果,3-溴-10H-吩噻嗪与(5-甲酰基噻吩-2-基)硼酸的摩尔比为1:1.3。
进一步地,3-溴-10H-吩噻嗪与PdCl2(dppf)的摩尔比为1:0.03~0.1,可以但不局限于1:0.03、1:0.04、1:0.05、1:0.06、1:0.07、1:0.08、1:0.09或1:0.1,为了获得更好的效果,3-溴-10H-吩噻嗪与PdCl2(dppf)的摩尔比为1:0.07。
进一步地,3-溴-10H-吩噻嗪与K2CO3的摩尔比为1:2~3,可以但不局限于1:2、1:2.2、1:2.3、1:2.5、1:2.6、1:2.65、1:2.7、1:2.75、1:2.8或1:3,为了获得更好的效果,3-溴-10H-吩噻嗪与K2CO3的摩尔比为1:2.7。
在步骤(3)中,化合物3与溴苯的摩尔比为1:0.8~1.5,可以但不局限于1:0.8、1:0.9、1:0.95、1:1、1:2、1:25、1:1.3、1:1.35、1:1.4或1:5,为了获得更好的效果,化合物3与溴苯的摩尔比为1:1。
进一步地,化合物3与Pd2(dba)3的摩尔比为1:0.03~0.1,可以但不局限于1:0.03、1:0.04、1:0.05、1:0.06、1:0.07、1:0.08、1:0.09或1:0.1,为了获得更好的效果,化合物3与Pd2(dba)3的摩尔比为1:0.06。
进一步地,化合物3与t-Bu3P的摩尔比为1:0.03~0.1,可以但不局限于1:0.03、1:0.04、1:0.05、1:0.06、1:0.07、1:0.08、1:0.09或1:0.1,为了获得更好的效果,化合物3与t-Bu3P的摩尔比为1:0.06。
进一步地,化合物3与t-BuONa的摩尔比为1:0.8~1.5,可以但不局限于1:0.8、1:0.9、1:0.95、1:1、1:2、1:25、1:1.3、1:1.35、1:1.4或1:5,为了获得更好的效果,化合物3与t-BuONa的摩尔比为1:1。
在步骤(4)中,化合物1与化合物4的摩尔比为1:0.8~1.5,可以但不局限于1:0.8、1:0.9、1:0.95、1:1、1:2、1:25、1:1.3、1:1.35、1:1.4或1:5,为了获得更好的效果,化合物1与化合物4的摩尔比为1:1。
本发明提供的荧光探针作为光敏剂可用于在制备靶向肿瘤细胞线粒体的荧光成像诊断药物中,具有靶向肿瘤细胞线粒体的荧光特性,可实现体内外的肿瘤组织或细胞的荧光成像,特别地,可用于在制备靶向肿瘤细胞次氯酸荧光成像诊断药物中,具有次氯酸激活荧光的特性,可区分正常细胞与肿瘤细胞。
本发明提供的荧光探针作为光敏剂可用于在制备具有光动力肿瘤治疗药物中,可实现体内外的肿瘤组织或细胞的荧光成像和/或治疗,经过激发光照射后产生单线态氧杀伤肿瘤细胞。
采用本发明的技术方案,优势如下:
本发明提供的荧光探针PTZSPY是一种多功能的线粒体靶向探针,作为光敏剂时具有线粒体靶向能力、高识别特异性、ClO-激活荧光以及伴随的1O2原位产生用于PDT,可用于选择性监测ClO-以区分癌细胞和PDT诱导的癌细胞消融。线粒体靶向和ClO-可激活的AIE光敏剂PTZSPY的设计为准确的荧光引导成像提供了新的见解。
附图说明
图1是化合物1的1H NMR图;
图2是化合物3的13C NMR图;
图3是化合物3的HR-MS;
图4是化合物4的13C NMR图;
图5是化合物4的HR-MS;
图6是荧光探针PTZSPY的1H NMR图;
图7是荧光探针PTZSPY的13C NMR图;
图8是荧光探针PTZSPY与ClO–反应后的HR-MS;
图9是荧光探针PTZSPY在ClO-激活前后的荧光光谱;
图10是荧光探针PTZSPY在具有不同乙腈体积分数的乙腈-水混合物中的发射强度,λem=610nm;
图11是荧光探针PTZSPY在添加NaClO(50μM)后的荧光强度随时间的变化,λex=450nm;λem=610nm;
图12是荧光探针PTZSPY荧光光谱;(A)荧光探针PTZSPY的激发和发射荧光光谱,其中,左边的曲线为荧光激发光谱,右边的曲线为荧光发射光谱;(B)PTZSPY在添加ClO–(10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160μM)后的荧光滴定光谱,在PBS(pH 7.26,10mM,含有0.05%DMSO)中在450nm处激发;(C)添加ClO–(0-180μM)后PTZSPY,在625nm处的荧光强度与ClO–(0-100μm)的函数关系;LOD=16.9nM;
图13是在PBS(pH 7.3,10mM,含0.5%DMSO)中加入各种ROS前后,PTZSPY(5μM)在610nm处的荧光强度;1.H2O2 2.t-BuOOH 3.O2 -·4.·OH 5.1O2 6.ONOO-7.GSH 8.L(+)-Cysteine 9.Fe3+10.Ca2+11.NaClO;
图14是在不存在或存在ClO–(100μM)的情况下,pH值对PTZSPY=O荧光强度的影响;λex=450nm;λem=610nm;
图15是添加ClO–(50μM)后5μM PTZSPY的抗光漂白性;λex=450nm;λem=610nm;
图16是ABDA在Rose Bengal、PTZSPY、PTZSPY=O白光(100mW cm-2)下的吸收光谱辐照;在光照射下不同PSs存在下ABDA的分解率,其中A0和A是ABDA在378nm处的吸光度;ABDA在不同PSs存在下光照射下的分解速率,其中A0和A是ABDA在378nm处的吸光度;PSs的吸收光谱范围为400-700nm;
图17是在不存在或存在白光照射的情况下,用不同浓度的PTZSPY染色的MCF-7癌细胞和HUVEC正常细胞的细胞活力;
图18是使用PTZSPY在活的MCF-7细胞中检测ClO-的共聚焦荧光成像;(A-D)MCF-7细胞的荧光图像与PTZSPY(1μM)和DAPI孵育30分钟(对照);(E-H)MCF-7细胞的荧光图像与PTZSPY(1μM)和DAPI孵育30分钟,然后与NaClO(10μM)孵育30分钟;(I-L)PTZSPY(1μM)加载细胞的荧光图像用NAC(10μM)预处理30分钟。激发波长=405nm;发射范围:(A、E和I)450-500nm和(B、F和J)600-650nm;
图19是正常细胞和癌细胞共培养并用PTZSPY(5μM)染色30分钟;(A)共培养癌症(MCF-7)和正常细胞(HUVEC)的明场;(B)共培养的癌症(MCF-7)和正常细胞(HUVEC)的荧光图像。(C)和明场图像的合并图像;
图20是共聚焦荧光图像,其中,(A)将细胞与PTZSPY(5μM)和Mito Tracker Green(100nM)孵育60分钟;(B)红色通道发射λex=488nm,λem=580-650nm);(C)明场图像;(D)和明场图像的合并图像;(E)MCF-7细胞中ROI的强度分布;红线代表探针PTZSPY的强度,绿线代表Mito Tracker Green的强度;(F)Mito Tracker Green和PTZSPY强度的相关图;Rr=0.91;
图21是PTZSPY(5μM)处理的MCF-7细胞的活/死染色(A-C)0分钟、(D-F)5分钟、(G-I)10分钟、(J-L)15分钟和(M-O)无PTZSPY处理的MCF-7个细胞,光照15分钟。活细胞被AM(绿色)染色,而死细胞被PI(红色)染色;
图22是用5μM PTZSPY染色的MCF-7细胞在连续光照下300秒的实时共聚焦成像;λex=488nm,λem=580-650nm;
图23是MCF-7细胞在连续光照下300秒的实时共聚焦成像,用5μM PTZSPY染色的明亮的领域。
具体实施方式
通过以下实施例并结合附图对本发明的荧光探针作进一步的说明,但这些实施例不对本发明构成任何限制。
一、实施方法
除非另有说明,使用的所有分析级化学品和溶剂均购自商业供应商。有机溶剂为分析纯。NaClO用作ClO-/HClO的来源。9,10-蒽二基-双(亚甲基)二丙二酸(ABDA)购自Sigma-Aldrich并按原样使用。磷酸盐缓冲盐水(PBS)购自Hyclone。胎牛血清(FBS)、青霉素、链霉素和Dulbecco's Modified Eagle培养基(DMEM)均购自Gibco。Mito-TrackerGreen购自Invitrogen。Cell Counting Kit-8(CCK-8)和DAPI购自Dojindo Laboratories。生物成像实验在Leica Microsystems的Leica系统上进行,分析软件为LAS X。1H NMR(400MHz)和13C NMR(100MHz)在JNM-ECZR 400MHz光谱仪上记录,DMSO-d6作为溶剂和TMS作为内标。使用maXisTM 4G UHR-TOF光谱仪(安捷伦)记录质谱。UV-vis光谱是通过UV-6100分光光度计获得的。
水溶液中单线态氧生成检测
ABDA(9,10-蒽二基-双(亚甲基)-二丙二酸)被用作评估在白光照射(100mW/cm2)下溶液中六种AIE-PSs的1O2生成的指标。ABDA(5×10-5M)溶液与AIE-PSs在水(5×10-6M)中混合,然后暴露于白光照射(100mW/cm2)。在不同的照射时间记录ABDA在378nm处的吸光度降低。
细胞培养和成像
MCF-7细胞在补充有10%(v/v)胎牛血清的DMEM(高糖)中在37℃、5%CO2中培养。MCF-7细胞预先铺在35毫米激光共聚焦皿中。然后,将细胞与5μM PTZSPY和DAPI在37℃下孵育30分钟。用1×PBS洗涤大量材料和染料后,通过共聚焦显微镜捕获样品。
细胞毒性
MCF-7细胞和HUVEC细胞分别预先接种于96孔板中。然后,将细胞与含有10%FBS(v/v)、0.1、0.25、0.5、1、2.5、5μM PTZSPY的DMEM在37℃下分别孵育30分钟。随后,将样品用或不用100mW/cm2白光照射30分钟。然后,将丰富的材料置于新鲜培养基中,并将样品在37℃下孵育24小时。根据提案通过CCK-8检测细胞活力,并通过GraphPad Prism 5分析结果。
荧光共定位
为了与MitoTracker Green共染色,首先将细胞与PTZSPY和Mito-Tracker Green在37℃下孵育30分钟。然后除去培养基,用PBS冲洗细胞3次,然后在共聚焦显微镜下成像。对于PTZSPY,发射滤光片为600-650nm;对于MitoTracker Green,激发光为488nm,发射滤光片为490-550nm。
二、实施例
本发明提供的具有次氯酸激活荧光和线粒体靶向功能的光敏剂PTZSPY的合成路线如下:
化合物1:4-甲基吡啶(930mg,10mmol)和甲基碘(1.42g,10mmol)在甲苯中混合,将所得混合溶液在室温下搅拌过夜。反应完成后,过滤,固体用乙醚洗涤并进一步真空干燥,得到浅黄色固体状产物(2.19g,9.3mmol)。产率:93%。1H NMR(400MHz,DMSO-d6)δ8.79(d,J=6.8Hz,2H),7.93(d,J=6.8Hz,2H),4.23(s,3H),2.56(s,3H)。具体如图1所示。
3-溴-10H-吩噻嗪:10H-吩噻嗪(2.1g,0.01mol)溶解在无水THF(20mL)中,将所得混合溶液通过注射器通过恒定的氮气流脱气30分钟,然后冷却至0℃(冰浴/水)。N-溴代琥珀酰亚胺(NBS,1.9g,0.01mol)溶解在干燥脱气的THF(20mL)中,并将所得NBS溶液缓慢滴入上述冷却的10H-吩噻嗪溶液中搅拌1小时。然后,将混合溶液在0℃(冰浴/水)下搅拌12小时并使其达到室温。再向溶液中加入饱和亚硫酸钠水溶液,并用二氯甲烷萃取溶液数次。有机层用无水Na2SO4干燥并过滤。真空除去溶剂,残余物通过硅胶色谱纯化(己烷/乙酸乙酯=4:1),得到产物(1.0g,31%),为无色固体。1H NMR(400MHz,DMSO-d6):8.67(s,1H),7.10(m,2H),6.98(t,J=7.2Hz 1H),6.89(d,J=8.0Hz,2H),6.75(t,J=7.6Hz,1H),6.64(d,J=7.6Hz,1H),6.57(d,J=8.0Hz 1H)。
化合物3:向3-溴-10H-吩噻嗪(830mg,3mmol)、PdCl2(dppf)(146mg,0.2mmol)、K2CO3(1.1g,8mmol)和甲苯(20.0mL)的混合物中,加入(5-甲酰基噻吩-2-基)硼酸(624mg,4mmol)在乙醇(16mL)中的悬浮液,将所得混合物在氩气气氛中加热回流6小时。冷却至室温后,反应液通过硅藻土过滤,有机相用水(3×10mL)洗涤。然后将有机层用Na2SO4干燥并真空浓缩,通过硅胶色谱法使用二氯甲烷/石油醚(v/v,2:1)作为洗脱液纯化粗产物,得到化合物2,为橙色固体:产率68%。1H NMR(400MHz,DMSO-d6):9.81(s,1H),8.89(s,1H),7.94(d,J=3.6Hz 1H),7.55(d,J=4.0Hz,1H),7.39(dd,J=8.4,2.4Hz,1H),7.33(d,J=4.0Hz,1H),6.98(t,J=8.0Hz 1H),6.90(d,J=8.0Hz 1H),6.76(t,J=7.6Hz1H),6.68(m,2H)。13C NMR(100MHz,DMSO-D6):δ184.14,152.98,143.53,141.28,141.06,139.95,128.30,126.81,126.58,126.53,124.19,122.86,117.94,116.25,115.23,115.17。C17H12NOS2[M+H]+的HRMSm/z计算值310.0360,实测值310.0336。具体如图2和3所示。
化合物4:向50mL双颈烧瓶中加入Pd2(dba)3(55mg,0.06mmol)、t-Bu3P(47mg,0.06mmol)、t-BuONa(96mg,1mmol)和无水甲苯(20mL),将所得混合物在室温下搅拌15分钟。再向混合物中加入溴苯(157mg,1mmol)和化合物3(309mg,1mmol)。冷却至室温后,反应液通过硅藻土过滤,有机相用水(3×10mL)洗涤。然后将有机层用Na2SO4干燥并真空浓缩,然后通过硅胶色谱法使用二氯甲烷/石油醚(v/v,2:1)作为洗脱液纯化粗产物,得到化合物3,为橙色固体:产率60%。1H NMR(400MHz,DMSO-d6):9.81(s,1H),7.94(d,J=4.4Hz 1H),7.69(t,J=8.0Hz 2H),7.56(t,J=4.4Hz,2H),7.47(d,J=2.0,1H),7.43(d,J=7.6Hz,2H),7.30(dd,J=8.8,2.4Hz 1H),7.06(d,J=9.2Hz 1H),6.91(m,2H),6.08(m,2H)。13C NMR(100MHz,DMSO-D6):δ184.27,152.17,144.91,143.28,141.62,140.24,139.82,131.82,131.08,129.49,128.01,127.42,126.06,124.84,124.41,123.65,120.39,118.73,116.45,116.42。C23H15NOS2Na[M+Na]+的HRMS m/z计算值408.0493,实测值408.0471。具体如图4和5所示。
PTZSPY:将化合物4(192mg,0.5mmol)、化合物1(118mg,0.5mmol)和0.5mL哌啶在10mL无水乙醇中混合,将所得混合物加热至回流温度过夜反应,然后冷却至室温。滤出沉淀,用冷乙醇洗涤并进一步真空干燥,得到黑色固体状的PTZSPY:产率80%。1H NMR(400MHz,DMSO-d6):8.76(d,J=6.8Hz 2H),8.15(m,3H),7.70(t,J=7.6Hz 2H),7.56(t,J=7.6Hz,1H),7.48(m,5H),7.23(dd,J=8.4,2.0Hz 1H),7.09(t,J=8.4 2H),6.92(m,2H),6.10(t,J=8.8 2H),4.17(s,3H)。13C NMR(100MHz,DMSO-D6):δ152.68,146.41,145.38,144.22,143.38,140.34,139.45,134.23,134.04,131.82,131.07,129.44,127.99,127.21,125.43,125.03,123.84,123.56,123.51,121.94、120.43、118.77、116.55、116.54、47.25。C30H23N2S2 +[M]+的HRMS m/z计算值475.1297,实测值475.1288。具体如图6、7和8所示。
三、效果验证
荧光探针PTZSPY由于分子结构中亲水性吡啶和I-而没有发射,但是分子中的硫醚可以被特定的氧化剂(例如,ClO-)氧化成亚砜,反应后可以观察到红色发射。如图9所示,荧光探针PTZSPY在490nm处显示出最大吸收并且不发光。PTZSPY=O在450nm处显示出最大吸收,在610nm处显示出强烈的红色发射,以及160nm的斯托克斯位移,这有助于避免自吸收和激发光干扰。
在图9中,在没有ClO-的情况下,荧光探针在水溶液中没有荧光。而在与ClO-孵育后,荧光探针PTZSPY在37℃孵育1分钟内表现出非常快速的荧光光谱强度增加(图11)。为了验证荧光探针对HClO的假设检测机制,因为不再观察到PTZSPY的分子离子峰,并且捕获了m/z=491.1216的峰,与[PTZSPY=O]+(m/z=491.1246)。HR-MS证实其机理是二价硫氧化为亚砜。
由于吡啶带正电荷,PTZSPY在乙腈中的溶解性差,在乙腈-水混合物中针对不同体积分数的乙腈记录了PTZSPY=O的荧光发射光谱。如图10所示,随着乙腈含量增加到95%,PTZSPY=O的荧光强度急剧上升,该结果表明PTZSPY=O具有良好的AIE特性。然后,在PBS缓冲液(pH 7.26,10mM,含有0.5%DMSO)中进行探针(10μM)与HClO的荧光滴定实验。随着ClO-的逐渐加入,610nm处的红色发射显着增加(图12B),并在160μM ClO-处达到稳定状态。此外,610nm处的PL强度与ClO-(0-100μM)的浓度呈线性关系(图12C)。PTZSPY对ClO-的检测限估计为16.9nM,通过3σ/B规则的计算方程获得。图13中的结果表明,其他ROS、含氮物质、阳离子和常见氨基酸的存在几乎无法打开PTZSPY的荧光。相比之下,ClO-在610nm处显示出显着的红色发射增强,证明PTZSPY对ClO-显示出可区分的响应。为了测试PTZSPY的应用范围,我们评估了其在一系列不同pH值范围从4到10的缓冲液中与ClO-反应的能力(图14)。在这个pH范围内,发现探针对ClO-的响应在4-10的宽pH范围内相当稳定,表明该测量与大多数生物应用兼容。
耐光漂白性是光敏剂的重要特性之一。当100mW光连续照射7.5分钟时,PTZSPY=O的荧光强度基本不变,证明它们具有较高的抗光漂白性(图15)。光敏剂在光照射下的高效ROS生成能力是一个重要标准。因此,我们通过测量9,10-蒽二基-双(亚甲基)-二丙二酸(ABDA)的光降解率来评估PTZSPY在PBS中的1O2生成能力。如图16所示,当探针PTZSPY和ABDA的溶液暴露在光线(400-800nm)下时,ABDA的吸收逐渐减弱,表明形成了1O2。其中,PTZSPY消耗的ABDA最多,是Rose Bengal的2.68倍。值得注意的是,PTZSPY=O的部分能量是通过荧光损失的,探针仍然具有很大的PDT,这为开发用于FL-PDT的活性荧光探针提供了很好的机会。
通过CCK8测定对MCF-7癌细胞进行定量评估,PTZSPY被进一步用作PDT应用的PS。剂量依赖性细胞毒性研究表明,PTZSPY在黑暗条件下都表现出相对较低的细胞毒性,表明它们具有可接受的生物相容性(图17)。相比之下,在白光照射下,细胞活力逐渐迅速下降,在0.5μM PTZSPY存在下保持80%的细胞活力,2.5μM PTZSPY可导致几乎完全细胞死亡。这些结果表明PTZSPY通过PDT途径对癌细胞消融具有相当强的作用。此外,为了研究PTZSPY相对于正常细胞杀死癌细胞的选择性,使用HUVEC正常细胞作为模型进行了剂量依赖性细胞毒性实验。在白光不存在和存在的情况下,HUVEC细胞的存活率明显高于MCF-7细胞,表明这些PTZSPY在正常细胞中的积累效率很低。这些PTZSPY杀死MCF-7细胞的出色能力使它们在癌症光动力治疗中非常有前途。
PTZSPY的优越性能促使我们进一步评估PTZSPY可以通过ClO-在MCF-7细胞中开启荧光的成像可能性。共聚焦激光扫描显微镜(CLSM)图像显示MCF-7细胞被PTZSPY染色显示出红色发射(图18)。当MCF-7细胞与PTZSPY(5μM)孵育30分钟,然后用NaClO(10μM)处理30分钟时,可观察到明显的红色荧光。然而,加载PTZSPY的细胞用N-乙酰半胱氨酸(NAC,HClO的清除剂,10μM)预处理30分钟,荧光信号几乎不可见。所有这些数据都证实了PTZSPY对MCF-7细胞的渗透性以及PTZSPY对不同浓度ClO-的点亮可视化的适用性。
具有快速增殖和高代谢特征的癌细胞具有比正常细胞更高的ROS水平。与正常细胞相比,ClO-作为活细胞中的活性氧之一,在一些癌细胞中也高表达。因此,我们通过细胞共培养实验测试了PTZSPY区分癌细胞与正常细胞的能力。MCF-7细胞和HUVEC细胞共培养并用PTZSPY(10μM)处理30分钟。如图19所示,MCF-7细胞显示出比HUVEC细胞强得多的荧光强度。
阳离子基团对膜负电位较高的线粒体具有较高的选择性。因此,我们进一步检查了PTZSPY的亚细胞定位,并使用Mito Tracker green进行了共定位研究。如图20所示,线性区域(ROI)(探针PTZSPY和Mito Tracker Green共染色)的强度分布变化是同步的,PTZSPY的荧光信号对ClO-的响应与Mito Tracker green的荧光很好地重叠以及相关图的强度显示高Pearson系数(0.91),证实PTZSPY特异位于活MCF-7细胞的线粒体中。
为了进一步研究PDT处理的效果,进行了PTZSPY用AM和PI杀死MCF-7细胞的活/死染色。在没有光照的情况下,MCF-7细胞检测到明显的绿色荧光,表明MCF-7在黑暗中具有极好的生物相容性(图21)。相反,光照射15分钟后,死细胞的数量显着增加。我们连续照射MCF-7 5分钟,并通过CLSM观察到明显的形态变化(图22-图23)。可以看出,光照15秒后细胞有明显的形态变化,光照30秒后细胞开始吐气泡,光照3分钟后气泡破裂,光照5分钟后荧光强度明显下降。细胞形态的变化证明了机械损伤的原因。同时,细胞活力的降低伴随着荧光强度的降低。这是因为在细胞器受损后,探针在细胞中的靶向能力减弱,不再聚集。
综上所述,本发明中的荧光探针可以通过肿瘤微环境更准确地指导光动力治疗。荧光探针PTZSPY是一种多功能的线粒体靶向探针,包括线粒体靶向能力、高识别特异性、ClO-激活荧光以及伴随的1O2原位产生用于PDT,可用于选择性监测ClO-以区分癌细胞和PDT诱导的癌细胞消融。线粒体靶向和ClO-可激活的AIE活性光敏剂PTZSPY的设计为准确的荧光引导成像提供了新的见解。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可能对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
3.根据权利要求2所述的具有次氯酸激活荧光和线粒体靶向功能的光敏剂的制备方法,其特征在于,它包括以下步骤:
(1)10H-吩噻嗪、N-溴代琥珀酰亚胺与THF混合均匀进行化学反应制备3-溴-10H-吩噻嗪;
(2)在PdCl2(dppf)和K2CO3存在的条件下,3-溴-10H-吩噻嗪与(5-甲酰基噻吩-2-基)硼酸进行化学反应制备化合物3;
(3)在Pd2(dba)3、t-Bu3P和t-BuONa存在的条件下,化合物3与溴苯进行化学反应制备化合物4;
(4)在哌啶存在的条件下,化合物1、化合物4与无水乙醇混合均匀,加热至回流温度进行化学反应制备荧光探针PTZSPY。
4.根据权利要求3所述的具有次氯酸激活荧光和线粒体靶向功能的光敏剂的制备方法,其特征在于,在步骤(1)中,所述10H-吩噻嗪与N-溴代琥珀酰亚胺的摩尔比为1:0.8~1.5,优选为1:1;在步骤(2)中,所述3-溴-10H-吩噻嗪与(5-甲酰基噻吩-2-基)硼酸的摩尔比为1:1~2,优选为1:1.3;所述3-溴-10H-吩噻嗪与PdCl2(dppf)的摩尔比为1:0.03~0.1,优选为1:0.07;所述3-溴-10H-吩噻嗪与K2CO3的摩尔比为1:2~3,优选为1:2.7。
5.根据权利要求3所述的具有次氯酸激活荧光和线粒体靶向功能的光敏剂的制备方法,其特征在于,在步骤(3)中,所述化合物3与溴苯的摩尔比为1:0.8~1.5,优选为1:1;所述化合物3与Pd2(dba)3的摩尔比为1:0.03~0.1,优选为1:0.06;所述化合物3与t-Bu3P的摩尔比为1:0.03~0.1,优选为1:0.06;所述化合物3与t-BuONa的摩尔比为1:0.8~1.5,优选为1:1。
6.根据权利要求3所述的具有次氯酸激活荧光和线粒体靶向功能的光敏剂的制备方法,其特征在于,在步骤(4)中,所述化合物1与化合物4的摩尔比为1:0.8~1.5,优选为1:1。
7.权利要求1所述的具有次氯酸激活荧光和线粒体靶向功能的光敏剂在制备靶向肿瘤细胞线粒体的荧光成像诊断药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述具有次氯酸激活荧光和线粒体靶向功能的光敏剂在次氯酸荧光成像诊断药物中的应用。
9.权利要求1所述的具有次氯酸激活荧光和线粒体靶向功能的光敏剂在制备具有光动力肿瘤治疗药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述光敏剂经过激发光照射后产生单线态氧杀伤肿瘤细胞。
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CN115368415A (zh) * | 2022-08-18 | 2022-11-22 | 徐州医科大学 | 一种用于检测线粒体内ClO-/H2O2的多色荧光探针及其制备方法和应用 |
CN115368415B (zh) * | 2022-08-18 | 2024-03-12 | 徐州医科大学 | 一种用于检测线粒体内ClO-/H2O2的多色荧光探针及其制备方法和应用 |
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