CN116284147B - 选择性检测肿瘤细胞内高表达的hNQO1的荧光探针及其应用 - Google Patents
选择性检测肿瘤细胞内高表达的hNQO1的荧光探针及其应用 Download PDFInfo
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Abstract
本发明公开了一种选择性检测肿瘤细胞内高表达的hNQO1的荧光探针及其应用,该探针与hNQO1酶发生反应后能开启配合物640nm处的红色荧光发射,公开了所述探针具有在磷酸盐缓冲溶液中有效识别hNQO1酶的能力,检出限为0.4239ng/ml,并且不受其他生物相关的金属离子、阴离子、生物分子的干扰。本发明的探针具有高的选择性和灵敏性以及优良的水溶性、膜通透性,且细胞毒性低,可作为直观监测活细胞中过表达的hNQO1酶的工具,并且能有效的将NQO1阳性癌细胞与NQO1阴性细胞区分开来,可应用于hNQO1过表达肿瘤组织切片的术中病理诊断,实现微米级高分辨率实时成像肿瘤中hNQO1,有利于hNQO1过表达肿瘤的早期诊断和及时治疗。
Description
技术领域
本发明涉及一种荧光探针,尤其涉及一种用于选择性检测肿瘤细胞内高表达的NAD(P)H:醌氧化还原酶(hNQO1)的荧光探针及其应用。
背景技术
癌症一直是最严重的公共卫生问题之一,癌症患者的总体生存率非常低,且与肿瘤初期诊断的分期密切相关。因此,准确识别癌细胞对于恶性肿瘤的早期诊断和及时治疗至关重要。基于探针的光致发光成像技术不仅具有较高的选择性、敏感性和时空分辨率,而且能够对病变组织细胞微环境进行实时无创监测,是识别癌细胞的极佳选择。然而,拥有以肿瘤标志物(酶、基因、蛋白和小分子)为靶点且快速启动能够准确识别癌细胞的探针是实现这一目标的关键。人类NAD(P)H:醌氧化还原酶1(NQO1)与癌症密切相关,与正常细胞相比高度表达(5-200倍)在肺癌等各种肿瘤细胞,结肠癌,肝癌、乳腺癌、黑素瘤、胰腺癌、胃癌等,在癌症进展中发挥着重要作用,可作为生物传感器设计的理想靶点。近年来,一系列基于三甲基苯醌锁定效应,含多种荧光团的可激活h NQO1的荧光探针被开发出来用于肿瘤成像。然而,大多数已报道的荧光团存在一些局限性,例如,紫外线激发容易对生物样本造成伤害;小的斯托克斯位移会导致灵敏度和精度降低;光漂白导致探针的荧光猝灭;低的水溶性也阻碍了其在生物领域的实际应用。因此,构建有效识别肿瘤细胞内hNQO1酶的新型荧光探针仍然是至关重要的。
发明内容
针对现有技术的不足,本发明要解决的问题是提供一种用于肿瘤细胞内高表达的NAD(P)H:醌氧化还原酶(hNQO1)检测的荧光探针。
本发明所述用于肿瘤细胞内高表达的NAD(P)H:醌氧化还原酶(hNQO1)检测的荧光探针,所述钌(II)配合物的磷光探针[Ru(TLQ-PIPY)(bipy)2]2+的结构如(I)所示:
上述用于肿瘤细胞内高表达的NAD(P)H:醌氧化还原酶(hNQO1)检测的荧光探针化合物制备方法概述如下:
为增加探针的水溶性以及在生理环境下的实用性同时获得相对较好的光物理性质,发明人将2,5-二溴吡嗪和N-Boc-哌嗪溶解在N-甲基吡咯烷酮中(NMP),回流反应24h,得到化合物1(BRPY),然后将其加入到含有2-三丁基锡基吡啶的甲苯溶液中室温搅拌反应10h得到化合物2(PIPY)。随后,将得到的化合物2(PIPY)溶解在二氯甲烷中与三甲基醌酸反应,加入2-(7-偶氮苯并***-1-基)-N,N,N',N'-四甲基六氟磷酸铵(HATU)和N,N-二异丙基乙胺(DIPEA)反应24h,合成新型配体TLQ-PIPY。最终的钌(II)配合物荧光探针的合成需要在惰性气体保护下将顺-二(2,2”-联吡啶)二氯化钌(II)二水合物与三氟甲烷磺酸银和配体TLQ-PIPY(II)在甲醇中回流反应4-8h,冷却至室温过滤沉淀,加入***得到红色粉末[Ru(TLQ-PIPY)(bipy)2]2+,具体的制备反应式如下:
本发明所述探针在磷酸盐缓冲溶液中检测hNQO1酶的光学性质。
该探针与hNQO1酶发生反应后能开启配合物640nm处的红色荧光发射;为了评价探针[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1的传感能力,在PBS缓冲液(0.01mol/L,pH 7.4)中,测定其在25℃下的吸收光谱和发射光谱的变化。在[Ru(TLQ-PIPY)(bipy)2]2+的吸收光谱(图1a)中,450nm左右的吸收峰属于金属到配体的电荷转移跃迁(MLCT)。在286nm附近的强吸收带是由配体基团π-π*跃迁引起的,同时在300-350nm处的肩峰,源自于配体间的跃迁(LPIPYLTLQCT和LbipyLTLQCT)。图中可见[Ru(TLQ-PIPY)(bipy)2]2+和NADH(100μmol/L)混合溶液在260nm处有强吸光度,在337nm处有中等吸光度。探针[Ru(TLQ-PIPY)(bipy)2]2+在450nm的激发下,640nm处显示弱荧光(图1b)。在NADH的存在下,探针[Ru(TLQ-PIPY)(bipy)2]2+与hNQO1的反应在640nm处引起了强的荧光发射,并伴有较大的斯托克斯位移(190nm)。如图2a和2d所示,探针[Ru(TLQ-PIPY)(bipy)2]2+在NADH和hNQO1存在的情况下,在640nm处,荧光强度在20min内快速增强,这表明[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1具有较强的反应性和快速的反应能力,随后荧光强度的上升速度逐渐放缓趋于不变。在图3a和3b中,随着hNQO1的加入,荧光强度逐渐增强,最终大约增加了8倍,在0.4μg/mL的hNQO1浓度下达到饱和状态。探针对hNQO1检出限(LOD)是0.4239ng/ml,说明探针[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1高度敏感,具有监测细胞中微量hNQO1的潜力。
本发明所述探针在磷酸盐缓冲溶液检测hNQO1酶的酶反应动力学参数。
通过绘制不同浓度[Ru(TLQ-PIPY)(bipy)2]2+溶液的荧光强度随时间的变化曲线(4a),得到反应速率V(μmol min-1mghNQO1-1)。通过Michaelis-Menten分析,确定了表观动力学参数为:Michaelis常数(Km)=1.66±0.24μmol,最大速度(Vmax)=2.83±0.15μmolmin-1mghNQO1-1,催化常数(kcat)=56.63S-1,特异性常数(kcat/Km)=34.07μmol-1S-1。
本发明所述探针对hNQO1酶的选择性。
探针[Ru(TLQ-PIPY)(bipy)2]2+在加入到分别含有等摩尔的Na+、K+、Mg2+、Zn2+、Cu2+、Co2+、Ni2+、Ca2+、Cd2+、Mn2+、Pb2+、Fe3+、Cr3+、F-、Cl-、Br-、I-、AC-、SO3 2-、S2-、SO4 2-、HCO3 -、CO3 2-、NO3 -、NO2 -、PO4 3-、H2PO2 -、S2O3 2-、半胱氨酸、谷胱甘肽、赖氨酸、丙氨酸、苏氨酸、天冬酰胺酸、丝氨酸、谷氨酸、色氨酸、组氨酸时,荧光强度不变。如图4b所示,探针[Ru(TLQ-PIPY)(bipy)2]2+溶液中只有NADH和hNQO1共存时,在640nm处荧光增强明显,而其他干扰物种的荧光响应可以忽略,结果表明探针[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1具有好的选择性。
本发明所述探针的细胞毒性测定
其中:所述细胞为A549和MDA-MB-231细胞。
[Ru(TLQ-PIPY)(bipy)2]2+对A549细胞的细胞毒性实验(图5)表明,[Ru(TLQ-PIPY)(bipy)2]2+(10、20、50、100、500μM)对A549细胞的毒性很小,细胞活力>0.95。随着[Ru(TLQ-PIPY)(bipy)2]2+浓度升高至1000μΜ,细胞活力略低,但仍有90%以上的细胞存活。MDA-MB-231细胞也几乎不受[Ru(TLQ-PIPY)(bipy)2]2+的影响,细胞活力>0.90。这些数据表明[Ru(TLQ-PIPY)(bipy)2]2+不具有任何明显的细胞毒性。
本发明所述探针用于活细胞中hNQO1检测成像应用
其中:所述细胞为A549和MDA-MB-231细胞。
实验结果证实,利用本发明所述钌(II)配合物的荧光探针标记后的共聚焦图像显示,探针[Ru(TLQ-PIPY)(bipy)2]2+处理后的NQO1阳性A549细胞由于细胞内的hNQO1活性而显示出明亮的红色荧光,而处理后的NQO1阴性MDA-MB-231细胞仅显示微弱的荧光(图6)。此外,NQO1阳性的A549细胞经双香豆素(hNQO1抑制剂)预处理后再用探针[Ru(TLQ-PIPY)(bipy)2]2+培养后的荧光成像显示的荧光极弱,进一步证实了[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1的特异性识别。这些结果表明[Ru(TLQ-PIPY)(bipy)2]2+具有良好的膜通透性,且细胞毒性低,可作为直观监测活细胞内过表达的hNQO1酶的工具,并且能有效的将NQO1阳性癌细胞与NQO1阴性细胞区分开来,有利于恶性肿瘤的早期诊断和及时治疗。
本发明所述探针用于hNQO1高表达的活体肿瘤检测成像应用
如图7a所示,瘤内注射[Ru(TLQ-PIPY)(bipy)2]2+1min后,肿瘤部位在670nm处可见明显荧光信号。在hNQO1作用下下,肿瘤区域的荧光强度在1~50分钟内逐渐增强。瘤内注射40~50分钟后荧光达到最大值,然后在50~90分钟荧光缓慢衰减。结果表明,[Ru(TLQ-PIPY)(bipy)2]2+具有在活体内被NQO1酶快速激活的能力,可进一步用于检测活体瘤内NQO1水平,有利于恶性肿瘤的早期诊断和及时治疗。
本发明所述探针用于hNQO1高表达的活体肿瘤切片检测成像应用
在共聚焦显微镜下,A549肿瘤切片显示出明显的红色荧光,分辨率达到7-9μm(图7b)。在透射电镜(TEM)下,[Ru(TLQ-PIPY)(bipy)2]2+在A549肿瘤切片中呈现不规则的黑色球形分布(图7c),证明[Ru(TLQ-PIPY)(bipy)2]2+可应用于hNQO1过表达肿瘤组织切片的术中病理诊断,而且在微米级高分辨率实时成像肿瘤中hNQO1活性方面具有相当大的潜力,有利于hNQO1过表达肿瘤的早期诊断和及时治疗。
附图说明
图1:探针[Ru(TLQ-PIPY)(bipy)2]2+的吸收(a)和荧光(b)光谱。
图2:加入hNQO1/NADPH后探针[Ru(TLQ-PIPY)(bipy)2]2+随时间的荧光强度变化(a,b)。
图3:在探针[Ru(TLQ-PIPY)(bipy)2]2+和NADH存在下,加入不同浓度hNQO1后探针荧光光谱(a);荧光强度与hNQO1浓度的线性关系(b)。
图4:(a)探针[Ru(TLQ-PIPY)(bipy)2]2+在pH7.4,0.01mol/L含NADH的PBS缓冲液中对hNQO1的酶动力学曲线;(b)探针[Ru(TLQ-PIPY)(bipy)2]2+在hNQO1,NADH和不同的干扰因素(金属离子(Na+、K+、Mg2+、Zn2+、Cu2+、Co2+、Ni2+、Ca2+、Cd2+、Mn2+、Pb2+、Fe3+、Cr3+),阴离子(F-、Cl-、Br-、I-、AC-、SO3 2-、S2-、SO4 2-、HCO3 -、CO3 2-、NO3 -、NO2 -、PO4 3-、H2PO2 -、S2O3 2-)、生物分子(半胱氨酸、谷胱甘肽、赖氨酸、丙氨酸、苏氨酸、天门冬氨酸、丝氨酸、谷氨酸、色氨酸、组氨酸)、NADH、NADH+hNQO1)存在下的荧光强度变化。
图5:探针的细胞毒性测试。
图6:(a)A549细胞,双香豆素预处理后的A549细胞和MDA-MB-231细胞,在37℃与[Ru(TLQ-PIPY)(bipy)2]2+孵育后的荧光图像。(b)a组细胞成像的相对荧光强度。
图7:(a)在瘤内给药[Ru(TLQ-PIPY)(bipy)2]2+(1000μM In,50μL,λex=460nm,λem=670nm)后不同时间(0~90min)A549荷瘤小鼠内源性hNQO1的体内荧光成像。(b)肿瘤内给药[Ru(TLQ-PIPY)(bipy)2]2+(1000μM in,50μL,λex=488nm,λem=550-690nm)后40-50minA549荷瘤小鼠肿瘤组织冰冻切片荧光成像。(c)肿瘤内给药[Ru(TLQ-PIPY)(bipy)2]2+(1000μM,50μL)后40-50min A549荷瘤小鼠肿瘤组织透射电镜显微照片。
具体实施方式
以下结合具体实施例,对本发明进行详细说明。
实施例1:2-溴-5-(哌嗪-1-基)吡嗪(1)(BRPY)合成
2,5-二溴吡嗪(1.0g,4.20mmol)和N-Boc-哌嗪(0.93g,5.0mmol)溶解在50mL NMP中搅拌在110℃,然后滴加DIPEA回流2h,反应混合物冷却至室温后过滤,产生淡黄色固体,溶解在三氟乙酸/二氯甲烷的混合液中(1:1,v/v)。反应混合物在室温下搅拌6小时,去除溶剂,得到淡黄色固体,产量:0.8g(78.7%)。1H NMR(600MHz,Chloroform-d)δ8.12(d,J=1.4Hz,1H),7.86(d,J=1.5Hz,1H),3.56–3.47(m,4H),3.04-2.94(m,4H),1.88(s,1H).13CNMR(151MHz,Chloroform-d)δ154.16,143.84,130.17,125.80,45.70,45.66.
实施例2:2-(哌嗪-1-基)-5-(吡啶-2-基)吡嗪(2)(PIPY)的合成
将2-溴-5-(哌嗪-1-基)吡嗪(2.1g,8.67mmol)添加到2-三丁基锡基吡啶(3.8g,10.4mmol)的甲苯(100毫升)溶液中搅拌在110℃,回流24小时在氮气保护下。将反应混合物冷却至室温后,浓缩后硅胶柱色谱纯化,得到淡黄色固体产物。产量:1.25g(60%)。1H NMR(600MHz,DMSO-d6)δ9.00(s,1H),8.62-8.59(m,1H),8.38(s,1H),8.12(d,J=8.3Hz,1H),7.88(dd,J=7.6,2.0Hz,1H),7.34(d,J=5.0Hz,1H),3.65-3.62(m,4H),2.91-2.84(m,4H),1.24(t,J=7.2Hz,1H).13C NMR(151MHz,DMSO-d6)δ155.04,154.84,149.66,139.89,139.00,137.67,130.30,123.28,119.36,52.55,45.37,45.11,7.72.
实施例3:配体TLQ-PIPY(3)的合成
2-(哌嗪-1-基)-5-(吡啶-2-基)吡嗪(0.48g,2.0mmol)在氮气保护下,混合2-(7-偶氮苯并***-1-基)-N,N,N',N'-四甲基六氟磷酸铵(HATU)(0.91g)和N,N-二异丙基乙胺(DIPEA)(0.52g)在二氯甲烷(20mL)中搅拌反应30min。随后三甲基醌酸被添加到混合中,并在室温下搅拌24h。反应结束后经硅胶柱色谱纯化得到淡黄色固体产品。产量:0.57g(60%)1HNMR(600MHz,Chloroform-d)δ9.10(d,J=1.6Hz,1H),8.64-8.60(m,1H),8.15-8.11(m,2H),7.78-7.73(m,1H),7.24-7.20(m,1H),3.75(dd,J=4.5,2.4Hz,2H),3.70-3.59(m,6H),3.04(s,2H),2.13(s,3H),1.93(t,J=1.2Hz,3H),1.89(q,J=1.1Hz,3H),1.45(s,6H).13C NMR(151MHz,Chloroform-d)δ191.47,187.72,170.88,155.01,154.32,154.23,149.30,143.18,140.76,140.58,138.24,136.91,136.72,129.08,122.81,119.87,47.10,45.11,44.70,44.51,40.87,37.84,28.95,14.29,12.75,12.21.
实施例4:探针[Ru(TLQ-PIPY)(bipy)2]2+(4)的合成
将顺-二(2,2”-联吡啶)二氯化钌(II)二水合物(0.06g,0.12mmol)、三氟甲烷磺酸银(0.144mmol)和配体TLQ-PIPY置于5ml甲醇中在氮气保护下回流4小时将反应液浓缩,加入***得到红色粉末状的[Ru(TLQ-PIPY)(bipy)2]2+,产量:0.064g(36%)。1H NMR(600MHz,DMSO-d6)δ9.39(s,1H),8.85(t,J=8.1Hz,3H),8.81(d,J=8.1Hz,1H),8.61(d,J=8.3Hz,1H),8.26-8.14(m,4H),8.12-8.02(m,2H),7.77(d,J=5.6Hz,1H),7.70(d,J=5.7Hz,1H),7.66(d,J=5.6Hz,1H),7.58(q,J=7.0Hz,2H),7.53(p,J=6.2,5.0Hz,3H),7.35(t,J=6.7Hz,1H),6.96(s,1H),3.57(s,2H),3.49-3.37(m,6H),2.95(s,2H),2.02(s,3H),1.86(s,3H),1.78(s,3H),1.34(s,6H).13C NMR(151MHz,DMSO-d6)δ190.96,187.42,170.94,157.24,157.17,156.84,156.80,156.55,155.68,154.84,152.25,152.08,151.55,151.47,150.85,143.80,143.76,139.45,138.69,138.55,138.14,137.18,135.46,131.45,128.47,128.42,128.30,128.18,126.02,125.26,125.01,124.93,124.85,121.75,46.01,44.15,43.99,43.54,37.92,28.55,14.29,13.05,12.23.
实施例5:探针[Ru(TLQ-PIPY)(bipy)2]2+的紫外吸收和荧光发射光谱
将[Ru(TLQ-PIPY)(bipy)2]2+溶于PBS缓冲液(pH7.4,0.01mol/L)中。测试浓度为:[Ru(TLQ-PIPY)(bipy)2]2+(5μmol/L),hNQO1/NADPH(0.4μg/mL,100μmol/L)。如图1a所示,金属到配体的电荷转移跃迁(MLCT)的特征吸收峰出现在450nm左右。如图1b所示,在NADH的存在下,探针[Ru(TLQ-PIPY)(bipy)2]2+与hNQO1的反应在640nm处引起了强的荧光发射,并伴有较大的斯托克斯位移(190nm)。
实施例6:探针[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1酶响应时间测定
将[Ru(TLQ-PIPY)(bipy)2]2+溶于PBS缓冲液(pH7.4,0.01mol/L)中。测试浓度为:[Ru(TLQ-PIPY)(bipy)2]2+(5μmol/L),hNQO1/NADPH(0.2μg/mL,100μmol/L)。在室温下采用450nm作为激发波长,连续测定不同时间点(0-120min)的荧光强度。如图2所示,[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1具有较强的反应性和快速的反应能力。
实施例7:探针[Ru(TLQ-PIPY)(bipy)2]2+的荧光滴定实验
将不同浓度hNQO1(0-0.5μg/mL)加入含NADH(100μmol/L)和[Ru(TLQ-PIPY)(bipy)2]2+的(5μmol/L)PBS缓冲液(0.01mol/L,pH 7.4)中,在室温下采用450nm作为激发波长测定溶液荧光强度的改变。如图3所示,随着hNQO1的加入,荧光强度逐渐增强,最终在0.4μg/mL NQO1时达到饱和状态,荧光强度大约增加了8倍。而且,在0-0.4μg/mL范围内,溶液中hNQO1浓度与[Ru(TLQ-PIPY)(bipy)2]2+荧光强度变化呈现良好的线性关系,其对hNQO1检测线LOD=0.4239ng/mL,说明[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1拥有较高的信噪比和灵敏度。
实施例8:探针[Ru(TLQ-PIPY)(bipy)2]2+的酶动力学分析
在25℃下使用荧光分光光度计完成[Ru(TLQ-PIPY)(bipy)2]2+的酶动力学实验。将不同浓度[Ru(TLQ-PIPY)(bipy)2]2+(0.5-6μmol/L)加入含NADH(100μmol/L)和hNQO1(0.25μg/mL)的PBS缓冲液(0.01mol/L,pH 7.4)。测定640nm处的荧光强度,间隔时间为1min持续10min。如图4a所示,通过Michaelis-Menten分析,得到Michaelis常数(Km)、最大反应速度(Vmax)、催化常数(kcat)和特异性常数(kcat/Km)。
实施例9:探针[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1酶的选择性
将金属离子(100equiv.hNQO1)(Na+、K+、Mg2+、Zn2+、Cu2+、Fe2+、Co2+、Ni2+、Ca2+、Cd2+、Mn2+、Pb2+、Fe3+、Cr3+、Al3+)、阴离子(F-、Cl-、Br-、I-、AC-、SO3 2-、S2-、SO4 2-、HCO3 -、CO3 2-、NO3 -、NO2 -、PO4 3-、H2PO2 -、S2O3 2-)和生物分子(半胱氨酸、谷胱甘肽、赖氨酸、丙氨酸、苏氨酸、天冬氨酸、丝氨酸、谷氨酸、色氨酸、组氨酸)加入到含有[Ru(TLQ-PIPY)(bipy)2]2+(5μmol/L),NADH(100μmol/L)和hNQO1(0.5μg/mL)的PBS缓冲液(0.01mol/L,pH 7.4)测试其荧光强度,荧光光谱记录在500-900nm,激发在450nm。如图4b所示,探针[Ru(TLQ-PIPY)(bipy)2]2+对hNQO1具有好的选择性。
实施例10:A549和MDA-MB-231细胞的培养
首先将A549和MDA-MB-231细胞分别注入到含有双抗(青霉素和链霉素)和10%的小牛血清的F12K和L-15养基中,然后置于含有5% CO2的恒温(37℃)细胞培养箱中培养。当细胞在培养瓶中生长到对数期,再接片培养:用铬酸洗液浸泡盖玻片半小时,再用清水、超纯水冲洗三遍后,烘干,灭菌后放到无菌一次性培养皿中。将培养瓶中的培养液倒出后,细胞用PBS缓冲液冲洗三遍,再用1毫升浓度为0.25%的胰酶消化3分钟,倒掉胰酶溶液,再加入少量培养基溶液,然后用移液枪鼓气吹打均匀,细胞计数,留取适当密度的细胞后,加入培养基,再次吹打均匀,然后将细胞加入到提前准备好的含有载玻片的培养皿中,把接种好的培养皿放到细胞培养箱中,待其贴壁生长。在CO2恒温细胞培养箱中培养12-36小时,待细胞密度达到50%-70%,即可用于细胞染色实验。
实施例11:探针对A549和MDA-MB-231细胞的细胞MTT实验
首先采用细胞计数板来检测细胞浓度,滴一滴细胞培养液到含有四块16格1mm×1mm的大方格的计数板上,数一下每个方格上的细胞个数,取平均值n(细胞浓度为n/mm3)。将细胞的浓度控制在5000~50000个/mm3,然后对细胞培养液进行稀释,再放入96孔板培养。待96孔板中的细胞生长一天后,分别在孔板中加入10、20、50、100,500,1000μM的探针,然后在孔板中加入20μL浓度为5mg/ml的3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,再继续培养24小时。依次把孔板内的培养液吸去,再向每个空格中分别加入150μL DMSO,将孔板放到摇床上低速振荡使结晶物得到充分的溶解。最后在酶标仪上读取各孔的吸光值(OD490 nm)进行计算。对照孔(培养液、细胞、浓度相同的药物溶解介质、二甲基亚砜和MTT)调零孔(二甲基亚砜、培养基和MTT)。如图5所示,细胞毒性实验结果可以看出:A549和MDA-MB-231细胞在含有1000μM染料的培养基中孵育24h后细胞的存活率在90%以上,可以认为染料对细胞没有明显毒性。
实施例12:探针对A549和MDA-MB-231细胞中hNQO1成像检测
A549细胞用探针[Ru(TLQ-PIPY)(bipy)2]2+(1000μM)的PBS缓冲溶液(0.01mol/L,pH 7.4)在37℃下染色5h,MDA-MB-231细胞在同样条件下孵育。同时进行了hNQO1酶抑制试验。首先在37℃下用双香豆素(100μM)孵育A549细胞12h,然后在37℃下用探针[Ru(TLQ-PIPY)(bipy)2]2+(1000μM)的PBS缓冲溶液(0.01mol/L,pH 7.4)处理A549细胞4h。共聚焦显微镜图像由458nm氩激光和1000×油浸物镜在ZEISS LSM880共聚焦显微镜上拍摄.如图6所示,本发明所述钌(II)配合物的荧光探针标记后的共聚焦图像显示,探针能很好的穿透活A549和MDA-MB-231细胞的胞膜进入细胞,直观监测细胞内的hNQO1酶,能有效的区分NQO1阳性癌细胞与NQO1阴性细胞。
实施例13:探针用于hNQO1高表达的活体肿瘤检测成像
取对数生长期的A549细胞,将5×106细胞皮下注射到小鼠体内。为检测内源性hNQO1活性,将[Ru(TLQ-PIPY)(bipy)2]2+溶解于PBS缓冲溶液(0.01mol/L,pH 7.4)中注入肿瘤(1000μmol/L,50μL)。利用PerkinElmer IVIS成像***在不同时间间隔对动物进行成像。如图7a所示,探针在小鼠体内的成像,瘤内注射[Ru(TLQ-PIPY)(bipy)2]2+1min后,肿瘤部位在670nm处可见明显荧光信号。在hNQO1作用下下,肿瘤区域的荧光强度在1~50分钟内逐渐增强。瘤内注射40~50分钟后荧光达到最大值,然后在50~90分钟荧光缓慢衰减。肿瘤内给药[Ru(TLQ-PIPY)(bipy)2]2+(1000μM in,50μL)后40-50min A549荷瘤小鼠肿瘤组织冰冻切片,在-20℃的低温恒温器(Leical 1950冷冻切片机,德国)中,切割成15μm厚的切片。荧光成像采用尼康A1R HD25共聚焦显微镜(λex=488nm,λem=550-690nm)(图7b)。并且取A549荷瘤小鼠组织切片(1mm3),在2.5%戊二醛4℃下快速固定2h,然后在含1%锇酸的0.1mol/LPBS中室温固定2h。样品经脱水、浸润、包埋、切片、染色后,在80kV的透射电子显微镜(HITACHI,HT7800/HT7700)下观察拍照(图7c)。结果表明,[Ru(TLQ-PIPY)(bipy)2]2+可应用于hNQO1过表达肿瘤组织切片的术中病理诊断,而且在微米级高分辨率实时成像肿瘤中hNQO1活性方面具有相当大的潜力,有利于hNQO1过表达肿瘤的早期诊断和及时治疗。
应当理解的是,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (5)
1.一种选择性检测肿瘤细胞内高表达的hNQO1的荧光探针,其特征在于:所述荧光探针的结构通式如(Ι)所示:
2.权利要求1所述的荧光探针的制备方法,其特征在于:包括以下步骤:将2,5-二溴吡嗪和N-Boc-哌嗪溶解在N-甲基吡咯烷酮中(NMP),回流反应24h,得到化合物1(BRPY),然后将其加入到含有2-三丁基锡基吡啶的甲苯溶液中室温搅拌反应10h得到化合物2(PIPY);随后,将得到的化合物2(PIPY)溶解在二氯甲烷中与三甲基醌酸反应,加入2-(7-偶氮苯并***-1-基)-N,N,N',N'-四甲基六氟磷酸铵(HATU)和N,N-二异丙基乙胺(DIPEA)反应24h,合成新型配体TLQ-PIPY;最终的钌(II)配合物荧光探针的合成需要在惰性气体保护下将顺-二(2,2”-联吡啶)二氯化钌(II)二水合物与三氟甲烷磺酸银和配体TLQ-PIPY(II)在甲醇中回流反应4-8h,冷却至室温过滤沉淀,加入***得到红色粉末Ru(TLQ-PIPY)(bipy)2,具体的制备反应式如下:
3.一种如权利要求1所述的荧光探针的应用,其特征在于,所述荧光探针应用于制备NQO1酶的检测试剂及检测试剂盒。
4.权利要求3所述的应用,其特征在于,通过选择性检测hNQO1酶成像,有效的将NQO1阳性癌细胞与NQO1阴性细胞区分开来。
5.权利要求4所述的应用,其特征在于,所述NQO1阳性细胞为A549细胞,所述NQO1阴性细胞为MDA-MB-231细胞。
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