CN112500386B - 基于吡罗红肟的近红外HClO荧光探针、制备及其应用 - Google Patents
基于吡罗红肟的近红外HClO荧光探针、制备及其应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种基于吡罗红肟的近红外HClO荧光探针、制备及其应用。
背景技术
癌症与正常细胞/组织的区别对癌症的早期诊断和治疗至关重要。在目前已经开发的肿瘤影像工具中,基于荧光探针的肿瘤成像技术因其具有可视化、无侵袭性、高灵敏度、无电离辐射、活体实时成像等优点而受到广泛关注,此类荧光探针最常见的设计策略是将荧光团与配体,包括化学分子、多肽、蛋白质和抗体等,进行化学偶联,这些配体可以特异性地结合到癌细胞过表达的表面标记物上。利用这类探针来影像癌症和正常细胞/组织的差异有助于患者个体化治疗,但由于癌细胞和肿瘤的遗传或表型异质性,利用这种差异来诊断广谱性癌症依然很困难。
因此,科研工作者把注意力集中在肿瘤的异常代谢上,即有氧糖酵解(也称为Warburg效应),癌细胞这种特殊的代谢方式会创造一个与癌症类型无关的癌细胞特有的微环境,如细胞外pH值降低、细胞内谷胱甘肽(GSH)和活性氧(ROS)水平升高等。然而,由于癌细胞和正常细胞之间的细胞外pH和细胞内GSH水平并没有太大的差异(pH分别为6.5和7.4;谷胱甘肽均为mM级别),迄今为止,通过该策略达到区分目的的例子非常少。
相反,癌细胞内的ROS浓度,包括O2 -、H2O2、OH·、ONOO-和ClO-等,大约是正常细胞的10倍,因此,ROS敏感型探针应该对癌细胞比对正常细胞/组织具有更高的敏感性。事实上,癌细胞的这一特性已被广泛应用在癌症治疗相关的ROS响应药物递送系统。尽管在过去的十年中有大量的ROS荧光探针被报道,但是到目前为止,只有少数的ROS荧光探针可以达到区分癌和正常细胞/组织的效果。其中一个可能的原因是由于探针敏感性不足,这使得大多数探针难以对癌细胞中本底的ROS作出响应;另一个原因可能是由于大部分ROS探针的吸收和发射波长位于可见区,与近红外光(650-900nm)相比,可见光的组织穿透性差,严重限制了该类探针在组织或活体中的成像应用。因此,开发能够同时克服以上缺点的ROS荧光探针将对于诊断广谱性肿瘤具有重要意义。
发明内容
本发明提出了一种基于吡罗红肟的近红外HClO荧光探针、制备及其应用,该探针在近红外区显示出对HClO的快速、高选择性和高灵敏的荧光响应。这些优异的特性使其在区分癌细胞和正常细胞/组织的实际应用中获得成功。
实现本发明的技术方案是:
一种基于吡罗红肟的近红外HClO荧光探针(PyOX),荧光探针的结构式如下:
所述的荧光探针的制备方法,步骤如下:
(1)在0℃、氮气环境中,将氧杂蒽酮溶于无水四氢呋喃中,向上述反应液中缓慢加入甲基溴化镁的THF溶液,室温搅拌过夜,反应结束后用水猝灭、DCM萃取、减压去除溶剂,得到的产物重新溶解在乙腈和高氯酸的水溶液中,搅拌10分钟后再次用DCM萃取,有机相用无水Na2SO4干燥,过滤蒸发得到粗产品,柱色谱纯化得到中间体2;
(2)将步骤(1)得到的中间体2和I2溶解于CHCl3,回流反应0.5h,向上述溶液中加入二甲亚砜继续回流48h,反应结束后冷却至室温,混合物用饱和Na2S2O3淬灭、二氯甲烷萃取,有机相经无水Na2SO4干燥、过滤蒸发,柱色谱纯化得到中间体3;
所述步骤(1)中氧杂蒽酮的结构式如下:
所述中间体2的结构式如下:
所述中间体3的结构式如下:
所述步骤(1)中氧杂蒽酮和甲基溴化镁的摩尔比为1:1.25;步骤(2)中间体2和I2的摩尔比为1:1,步骤(3)中间体3和盐酸羟氨的摩尔比为1:10。
所述的荧光探针在近红外区域检测HClO的应用,最低检测限为2.4nM。
本发明的探针的合成及对HClO的传感机理所示:
所述的荧光探针在区分癌细胞和正常细胞中的应用,通过检测细胞内ROS水平区分癌细胞和正常细胞。
使用PyOX探针区分癌细胞和正常细胞/组织的原理如图所示:
本发明的有益效果是:本发明将肟反应基团与吡罗红荧光团的9位相结合构建的探针,在近红外区域对HClO显示出快速、显著的荧光off-on响应,灵敏度极高,已成功应用于癌细胞/组织与正常细胞/组织的区分。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例中间产物2的1H NMR图;
图2为实施例中间产物2的13C NMR图;
图3为实施例中间产物2的HRMS图;
图4为实施例中间产物3的1H NMR图;
图5为实施例中间产物3的13C NMR图;
图6为实施例中间产物3的HRMS图;
图7为PyOX的1H NMR图;
图8为PyOX的13C NMR;
图9为PyOX的HRMS图;
图10(A,B)为PyOX(2μM)经ClOˉ处理前后的吸收和发射光谱图,B中插图为PyOX(2μM)经2当量ClOˉ处理后在680nm处荧光强度的变化;C为随着ClOˉ用量的增加,PyOX(2μM)荧光光谱的变化;D为PyOX(2μM)经不同物质(包括2μM的ClOˉ和ONOOˉ;20μM的O2 ·ˉ,·OH,1O2,H2O2和NO;200μM的Cys,Hcy,GSH,SHˉ和HSO3 ˉ;200μM的HCO3 ˉ,AcOˉ,NO3 ˉ,NO2 ˉ,Clˉ,Cu2+,Ca2+,Fe2+,Fe3+和Zn2+)处理后的荧光光谱变化;
图11(A)预先用PyOX(2μM)处理20分钟后正常细胞和癌细胞的荧光成像图;(B)为(A)的定量数据;(C)混合培养的A549和RAW264.7细胞经PyOX(2μM)处理20分钟后的荧光成像图;收集波长为650-750nm(λex=633nm);
图12为不同条件下RAW264.7细胞荧光成像图。(i)为仅被PyOX(2μM)处理的RAW264.7细胞,(ii-iv)为预先被PyOX(2μM)处理、随后被2μM ClO-、SIN-1和H2O2处理的RAW264.7细胞;(v)为预先用LPS(1mg/mL)/IFN-γ(50ng/mL)培养12h、随后经PyOX(2μM)处理的RAW264.7细胞;(vi)为预先用抑制剂ABAH(300μM)、LPS(1μg/mL)/IFN-γ(50ng/mL)培养12h、随后经PyOX(2μM)处理的RAW264.7细胞,收集波长为650-750nm(λex=633nm);
图13中(A)荷瘤小鼠经尾静脉或瘤内注射PyOX(20μM,50μL)的活体成像图;(B)为HepG2荷瘤小鼠经尾静脉注射PyOX(20μM,50μL)后荧光随时间变化图;采用630nm的激发滤光片和700nm的发射滤光片;
图14为MCF-7荷瘤小鼠暴露的肿瘤和周围正常的组织喷洒PyOX(20μM)5分钟后的活体成像图。(a)明亮图像;(b)荧光图像;(c)喷洒PyOX后的荧光图像;(d)切除肿瘤后的荧光图像。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例
基于吡罗红肟的近红外HClO荧光探针的制备,步骤如下:
(1)在0℃、氮气环境中,将氧杂蒽酮(1.01g,3mmol)溶于无水四氢呋喃(30mL)中,向上述反应液中缓慢加入甲基溴化镁(3.75mmol)的THF溶液,室温搅拌过夜,反应结束后用水猝灭(50mL)、DCM萃取(3×50mL)、减压去除溶剂,得到的产物重新溶解在乙腈和高氯酸的水溶液中,搅拌10分钟后再次用DCM萃取(3×50mL),有机相用无水Na2SO4干燥,过滤蒸发得到粗产品,柱色谱纯化得到中间体2(1.05g,80%)。
1H NMR(600MHz,CDCl3)δ7.96(d,J=9.6Hz,2H),7.03(d,J=9.6Hz,2H),6.70(s,2H),3.63(q,J1=7.2Hz,J2=6.6Hz,8H),2.88(s,3H),1.34(t,J=6.6Hz,12H);13C NMR(150MHz,CDCl3)δ152.39,140.597,123.53,123.06,115.71,115.02,66.78,49.03,21.55;ESI-MS[M+H]+:calcd for 337.2274,Found 337.2276.
(2)将步骤(1)得到的中间体2(1.5g,3.43mmol)和I2(0.87g,3.44mmol)溶解于CHCl3,回流反应0.5h,向上述溶液中加入二甲亚砜(8mL)继续回流48h,反应结束后冷却至室温,混合物用饱和Na2S2O3(50mL)淬灭、二氯甲烷萃取(3×50mL),有机相经无水Na2SO4干燥、过滤蒸发,柱色谱纯化得到中间体3(0.81g,52%);
1H NMR(600MHz,CDCl3)δ10.98(s,1H),8.20(d,J=9.6Hz,2H),7.12(d,J=9.6Hz,2H),6.90(s,2H),3.70(q,J1=7.2Hz,J2=6.6Hz,8H),1.38(t,J=6.6Hz,12H);13C NMR(150MHz,CDCl3)δ191.35,158.42,155.45,140.42,129.47,115.69,111.87,97.55,46.43,12.69;ESI-MS[M+H]+:calcd for 351.2067,Found 351.2068.
(3)将步骤(2)得到的中间体3(45mg,0.1mmol)、NH2OH·HCl(69mg,1mmol)和分子筛(5粒)加到乙腈(10ml)中,室温搅拌6h,反应液经过滤、水洗、DCM萃取(3×50mL),有机相经干燥、过滤蒸发,柱色谱纯化得到探针PyOX(25mg,55%)。
1H NMR(600MHz,DMSO-d6)δ13.01(s,1H),9.10(s,1H),8.26(d,J=9.6Hz,2H),7.23(d,J=9.6Hz,2H),6.91(s,2H),3.68(d,J=6.6,8H),1.23(t,J=6.6Hz,12H);13C NMR(150MHz,DMSO-d6)δ157.76,155.35,144.60,144.17,131.49,114.98,111.72,96.60,45.76,12.98;ESI-MS[M+H]+:calcd for 366.2176,Found 366.2180.
性能测试
1.溶液配制
将探针PyOX用乙腈配成2mM的储液,随后用20mM的PBS(pH 7.4)稀释至相应浓度。
次氯酸盐溶液(ClO-)用商业化NaClO溶液在去离子水中稀释制备,其浓度通过测量溶液在292nm处的吸收来确定(ClO-在去离子水中的摩尔消光系数为350M-1cm-1)。
过氧亚硝酸盐溶液(ONOO-)根据文献报道制备(R.M.Uppu,W.A.Pryor,Synthesisof peroxynitrite in a two-phase system using isoamyl nitrite and hydrogenperoxide,Anal.Biochem.1996,236,242-249.),其浓度通过测量溶液在302nm处的吸收来确定(ONOO-溶液在0.1M NaOH中的摩尔消光系数为1670M-1cm-1)。
过氧化氢溶液(H2O2)用商业化H2O2溶液在去离子水中稀释制备。
2.细胞培养和荧光成像
所有细胞系均购自GeneFull生物技术有限公司(中国)。
所有细胞均培养在37℃、含5%的二氧化碳的培养箱中,其中Raw 264.7细胞和Cos-7细胞培养在含有10%的胎牛血清、100U/mL青霉素G钠和100μg/mL链霉素的RPMI 1640培养基中;A549细胞、HepG2细胞、T98G细胞、BEAS-2B细胞和HUCEC细胞培养在含有10%的胎牛血清、100U/mL青霉素G钠和100μg/mL链霉素的DMEM(高糖)培养基中;进行细胞影像实验前,预先将细胞置于30mm的玻底细胞培养皿上,静置12小时待细胞贴壁,用磷酸缓冲液(PBS)洗涤细胞3次,然后使用Ceiss LMS 710共聚焦显微镜进行荧光成像,收集波长为650-750nm(λex=633nm)。
3.细胞毒性
采用CCK-8细胞增殖实验研究探针PyOX的细胞毒性。简单地说,首先将贴壁生长的A549细胞消化为细胞悬液,细胞以每孔5.0×103个细胞的密度接种于96孔板中,用100μL的DMEM培养基孵育过夜,细胞贴壁。然后将PyOX储液(2mM)加入到上述培养基中,终浓度保持在0~10μM,对照及测试组均重复6次,继续孵育细胞;细胞培养24h后,弃去旧培养基,PBS洗涤2次,更换含10%CCK-8的新鲜培养基,孵育0.5h,用iMarkTMMicroplateAbsorbanceReader测定450nm处的吸光度。
4.亚细胞定位
实验前,将细胞置于30mm的玻底细胞培养皿上,静置12小时至细胞贴壁,PBS洗涤3次;在37℃、PBS缓冲溶液中,分别用PyOX(2μM)和LysoTracker Green DND-26(50nM,10min)/MitoTracker Green FM(0.2μM,10min)染色细胞,PBS洗涤3次后,进行荧光成像。PyOX的收集波长为650-750nm(λex=633nm),LysoTracker Green DND-26/MitoTrackerGreen FM的收集波长为500-600nm(λex=488nm)。
5.活细胞中HClO成像
在影像细胞中外加HClO的实验中,Raw 264.7细胞首先用PyOX(2μM)预处理20分钟,然后分别用2μM的ClO-、SIN-1(商品化的ONOO-供体)和H2O2处理20分钟;在影像细胞中内生HClO的实验中,Raw 264.7细胞用LPS(1μg/mL)/IFN-γ(50ng/mL)预处理12h,然后用PyOX(2μM)处理20分钟,PBS洗涤三次后,进行荧光影像;在抑制实验中,细胞用MPO特异性抑制剂ABAH(300μM)、LPS(1μg/mL)/IFN-γ(50ng/mL)预处理12h,然后用PyOX(2μM)处理20分钟,PBS洗涤三次后,进行荧光影像。
6.用PyOX对荷瘤鼠模型进行肿瘤成像
所有的动物实验都是按照山西大学伦理委员会颁布的相关法律和指南进行的。BALB/c雄性裸鼠(6-8周龄)购自北京维通利华实验动物技术有限公司。
将A549细胞、HepG2细胞或MCF-7细胞(1×106个细胞)经皮下注射于裸鼠左腋(或左腿)处,接种15天后,荷瘤小鼠经尾静脉或瘤内注射PyOX。活体动物成像是在Bruker多模式活体成像系统中进行的,选择630nm的激发滤光片和700nm的发射滤光片。
7.利用PyOX的影像引导切除术
MCF-7荷瘤鼠经解剖暴露出肿瘤及正常组织,暴露肿瘤和周围正常组织喷洒PyOX(2μM)5分钟进行影像;将肿瘤摘除,再次影像。活体动物成像是在Bruker多模式活体成像系统中进行的,选择630nm的激发滤光片和700nm的发射滤光片。
测试结果
1.水溶性和光稳定性
由于探针在水中具有良好的溶解性(高达60μM),故体外测试体系均选取纯PBS缓冲体系。首先在PBS(20mM,pH 7.4)中研究了PyOX(2μM)与HClO反应前后吸收光谱的变化。如图10A所示,在PBS(20mM,pH 7.4)中,PyOX的最大吸收峰值为581nm,明显比经典吡罗红染料的吸收峰长,这是由于肟的吸电子作用增加了染料激发态的分子内电荷转移(ICT)过程;如图9所示,向PyOX溶液中加入ClO-后,探针的最大吸收峰红移至649nm处,HRMS数据证实,反应生成产物PyCNO(m/z=365.173)。
在相同条件下,进一步研究了PyOX(2μM)与HClO反应前后荧光光谱的变化。如图10B所示,由于C=N异构化导致的非辐射跃迁过程,PyOX在PBS中几乎没有荧光;然而,向PyOX溶液中加入ClO-后,在近红外区引起了显著的荧光增强,最大发射峰位于680nm处,反应可以在10s内完成。荧光滴定实验表明,ClO-的浓度与680nm处的荧光强度呈良好的线性关系,检测限低至2.4nM(图10C)。
此外,PyOX对ClO-的选择性高于其他ROS/RNS以及生物相关阳离子、阴离子和生物硫醇(图10D)。
2.细胞培养和细胞成像
如图11A所示,用PyOX处理癌细胞,包括A549细胞、HepG2细胞和T98G细胞20分钟后影像,所有细胞均有明亮的红色荧光;而用PyOX处理正常细胞,包括BEAS-2B细胞、HUVEC细胞和COS-7细胞20分钟后影像,所有细胞均显示微弱的荧光。荧光定量分析显示,癌细胞的荧光增强约为正常细胞的3倍(图11B),这表明PyOX有可能根据细胞内ROS水平的差异区分癌细胞和正常细胞。
在癌细胞和正常细胞共培养实验中,A549细胞和RAW264.7细胞在同一共聚焦皿中共同培养24小时,细胞贴壁后用PyOX处理20分钟,然后在共聚焦激光扫描显微镜(CLSM)下影像。如图11C所示,A549细胞(绿色箭头)可见明显的红色荧光信号,而正常的Raw264.7细胞仅可见微弱的荧光(白色箭头),该实验说明PyOX在活体肿瘤诊断中具有一定的潜力。
3.细胞中HClO的选择性成像和细胞毒性
在RAW264.7细胞中检测了PyOX对HClO的影像能力。如图12所示,预先孵化PyOX的RAW264.7细胞用633nm的激光激发时,红色荧光几乎可以忽略;当预先孵化PyOX的RAW264.7细胞继续孵化几种具有代表性的ROS时,只有HClO能诱导细胞发出明亮的红色荧光;当RAW264.7细胞预先被LPS诱导12h,继续孵化PyOX后,可以观察到明亮的红色荧光。这些结果表明,PyOX具有良好的细胞膜通透性,并且可以选择性影像细胞内源性及外源性的HClO。
此外,CCK-8细胞增殖实验研究结果显示,PyOX在0-10μm的浓度范围内具有较低的细胞毒性,细胞存活率大于85%。
4.肿瘤成像
分别将PyOX(20μM,50μL)经皮下注射到A549荷瘤鼠、HepG2荷瘤鼠和MCF7荷瘤鼠的肿瘤区域及正常组织区域,随后在小动物活体成像系统下成像。如图13A所示,肿瘤区域显示出明显的荧光信号,而正常组织区域显示出微弱的荧光信号;另外,将PyOX(20μM,50μL)经尾静脉注射到HepG2荷瘤鼠体内,只有肿瘤区域出现了显著的荧光信号,该信号在10min时达到最大,30min后由于代谢逐渐消失(图11B)。这些结果表明,由于癌细胞本底的ROS水平高于正常细胞,PyOX可以选择性影像荷瘤鼠的肿瘤部位。
5.利用PyOX的影像引导切除术
如图14所示,将PyOX喷洒于暴露的肿瘤和周围正常组织,只有肿瘤区域出现明亮的荧光信号,而周围正常组织的荧光可以忽略;将小鼠的肿瘤完全切除后,在剩余的组织中几乎看不到荧光。结果表明,PyOX在外科肿瘤切除术中有很好的应用前景。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
2.权利要求1所述的荧光探针的制备方法,其特征在于步骤如下:
(1)在0℃、氮气环境中,将氧杂蒽酮溶于无水四氢呋喃中,向上述反应液中缓慢加入甲基溴化镁的THF溶液,室温下搅拌过夜,反应结束后用水猝灭、DCM萃取、减压去除溶剂,得到的产物重新溶解在乙腈和高氯酸的水溶液中,搅拌10分钟后再次用DCM萃取,有机相用无水Na2SO4干燥,过滤蒸发得到粗产品,柱色谱纯化得到中间体2;
(2)将步骤(1)得到的中间体2和I2溶解于CHCl3,回流反应0.5h,向上述溶液中加入二甲亚砜继续回流48h,反应结束后冷却至室温,混合物用饱和Na2S2O3淬灭、二氯甲烷萃取,有机相经无水Na2SO4干燥、过滤蒸发,柱色谱纯化得到中间体3;
(3)将步骤(2)得到的中间体3、NH2OH·HCl和分子筛加到乙腈中,室温搅拌6h,反应液经过滤、水洗、DCM萃取,有机相经干燥、过滤蒸发、柱色谱纯化得到探针PyOX;所述步骤(1)中氧杂蒽酮的结构式如下:
所述中间体2的结构式如下:
所述中间体3的结构式如下:
3.根据权利要求2所述的荧光探针的制备方法,其特征在于:所述步骤(1)中氧杂蒽酮化合物和甲基溴化镁的摩尔比为1:1.25;步骤(2)中间体2和I2的摩尔比为1:1,步骤(3)中间体3和NH2OH·HCl的摩尔比为1:10。
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