CN114478304A - 一种具有肿瘤乏氧检测的指示剂及其制备方法用途 - Google Patents
一种具有肿瘤乏氧检测的指示剂及其制备方法用途 Download PDFInfo
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Abstract
本发明公开了一种具有肿瘤乏氧检测的指示剂及其制备方法用途。指示剂由联硼酸频哪醇酯、1‑(4‑溴苯基)‑1，2，2‑三苯乙烯、4，4‑二溴偶氮苯、1‑(4‑苯硼酸频哪醇酯)‑1，2，2‑三苯乙烯制备而成，具有肿瘤乏氧检测的性质，能够实现在乏氧条件下荧光点亮的效果。此外本指示剂还可以检测光动力治疗所产生的乏氧环境，对其进行精准检测具有重要意义。
Description
技术领域
本发明涉及分子材料及制备方法和用途，特别涉及一种具有肿瘤乏氧检测的指示剂及其制备方法用途。
背景技术
恶性肿瘤可以破坏组织，器官结构和功能，引起坏死出血合并感染，患者最终可能由于器官功能衰竭而死亡，恶性肿瘤已经严重威胁人类健康，令人谈癌色变，因此肿瘤细胞的早期准确诊断具有重要意义。由于肿瘤过度生长导致内部血液氧气供应不能满足肿瘤细胞快速增殖的代谢需求，使肿瘤内部形成显著的乏氧微环境。肿瘤乏氧意味着肿瘤内的氧气含量低于正常的生理水平。乏氧现象普遍存在于实体肿瘤中，是所有肿瘤的普遍特征。在某些实体肿瘤中氧气的含量甚至能低至0，而在正常组织中的含量通常为30mmHg。乏氧细胞远离血管并在治疗过程中不能有效地暴露在抗癌药物中，导致实体瘤中的乏氧细胞不能被药物有效地杀灭。因此，准确的乏氧成像不仅可以使临床医生诊断出乏氧肿瘤患者并确定乏氧肿瘤的位置，且可以根据结果制定合理的治疗策略，有助于提高治疗效果。荧光成像因其操作简单、非侵入性、高灵敏度和高时空分辨率而被认为是最有前途的成像手段之一。荧光成像可以在分子水平上对分析物进行特异的检测，观察动态的生物过程。疏水性的聚集诱导发光分子(AIEgen)在水溶液中可以聚集成发光明亮的聚集体，在生物体系中表现出优异的荧光成像性能。
与还原剂类似，还原酶可以催化还原反应，维持细胞内氧化还原动态平衡。当细胞内氧化还原平衡因过量产生的活性氧(ROS)破坏时，还原酶可以作为有效的抗氧化物质对抗氧化应激。此外在生物医学领域，还原酶通常被认为是潜在的诊断生物标志物，用来评估疾病(如恶性肿瘤)的存在或进展。当血管功能障碍时，还原酶表达水平有所提高。因此，建立具有还原酶敏感检测能力的分子体系有利于肿瘤的早期检测。偶氮化合物是研究最多的光开关之一，其在偶氮还原酶的作用下，偶氮键会断裂并恢复分子的荧光发射性能。不同于传统的含硝基基团的分子，降解过程会产生有毒的硝基苯，偶氮基团的降解只产生毒性较小的氨基苯，细胞毒性大大降低。
目前的乏氧检测指示剂检测效率不高。利用偶氮基分子作为乏氧检测指示剂能实现肿瘤细胞的快速检测。
发明内容
发明目的：本发明的目的是提供具有肿瘤乏氧检测的指示剂。
本发明的另一目的是提供所述具有肿瘤乏氧检测的指示剂的制备方法和用途。
该指示剂包括偶氮基团的乏氧检测分子，具有肿瘤乏氧检测的性质，能够实现在乏氧环境下荧光增强的效果，对于肿瘤乏氧的精确检测，癌症的早期诊断具有重要意义。
技术方案：本发明的具有肿瘤乏氧检测的指示剂，化学结构式为：
其中，R选自：
从化学结构式来看，乏氧检测指示剂分子具有偶氮基团。由于偶氮键基团的存在，分子在溶液和固体中都具有不发射荧光的特性。该指示剂可以被活细胞胞吞，并在乏氧条件下通过偶氮还原酶还原恢复发光性能。当细胞内氧浓度降低时，其荧光增强，成为检测乏氧环境的探针，此外本指示剂还可以检测光动力治疗所产生的乏氧环境。
所述的具有肿瘤乏氧检测的指示剂的制备方法，包括以下步骤：
(1)将联硼酸频哪醇酯和1-(4-溴苯基)-1，2，2-三苯乙烯原料混合，加热，反应后冷却，分离，纯化，得到白色固体，即1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯；其中1-(4-溴苯基)-1，2，2-三苯乙烯原料的化学结构式为：
(2)将4，4-二溴偶氮苯和1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯原料反应，得到指示剂。
指示剂由联硼酸频哪醇酯、1-(4-溴苯基)-1，2，2-三苯乙烯、4，4-二溴偶氮苯、1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯制备而成，具有肿瘤乏氧检测的性质。
进一步地，在步骤(2)反应中将4，4-二溴偶氮苯和1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯溶解在四氢呋喃中，加入催化剂和饱和碳酸钾溶液，混合，在氮气保护下搅拌加热，在保持恒定温度的情况下连续反应，得到乏氧检测指示剂。
进一步地，所述的具有肿瘤乏氧检测的指示剂在肿瘤乏氧环境检测中的用途。
进一步地，所述检测步骤为：将乏氧指示剂纳米颗粒与人类宫颈癌细胞株共孵育，分别于不同氧浓度的环境下培养，后在激光共聚焦显微镜上进行成像观察。
进一步地，所述不同氧浓度为21％O2或0％O2。
本发明上述的指示剂的用途中，肿瘤乏氧检测的用途通过偶氮基团的快速还原并引起显著的荧光增强。指示剂分子具有良好的细胞相容性，且没有细胞暗毒性，用于进入活细胞。指示剂分子在体外与连二亚硫酸钠反应，随着反应时间增加荧光强度不断增强，用于体外偶氮键断裂，荧光增强。指示剂分子通过肿瘤细胞内的偶氮还原酶断裂偶氮键，实现在乏氧状态下荧光增强的效果。
有益效果：本发明与现有技术相比，具有如下优势：
1、偶氮基团与荧光基团通过化学键相连生成分子后，可以猝灭分子的荧光。
2、由于乏氧肿瘤细胞中存在偶氮还原酶，能将偶氮键断裂，从而实现在乏氧状态下荧光增强，检测肿瘤细胞乏氧。
附图说明
图1为本发明第一实施例所制备的TPE-doe的氢谱图；
图2为本发明第一实施例所制备的TPE-bi-Aze的氢谱图；
图3为TPE-bi-Aze乏氧检测指示剂与连二亚硫酸钠反应，偶氮键断裂，荧光强度随着反应时间增强的变化趋势结果图；
图4为TPE-bi-Aze和聚氧乙烯聚氧丙烯反应后，通过动态光散射测得的粒径分布图；
图5为TPE-bi-Aze与A375细胞共同孵育12h，再在0％O2、21％O2下共同孵育4h，用MTT法检测细胞存活率的示意图；
图6为在405nm的激发波长下经过200μM TPE-bi-Aze共孵育12h，再分别在0％O2、21％O2下，共同孵育4h的宫颈癌细胞系HeLa细胞的激光共聚焦成像图。
具体实施方式
本发明优选的肿瘤乏氧检测指示剂的制备方法，包括以下步骤：
步骤1，将联硼酸频哪醇酯和1-(4-溴苯基)-1，2，2-三苯乙烯原料混合；并一起加热至60-100℃，然后在保持恒定温度的情况下连续反应12-24h，再经冷却，分离，纯化，得到白色固体，即1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯；
其中，1-(4-溴苯基)-1，2，2-三苯乙烯原料的化学结构式为：
步骤2，将4，4-二溴偶氮苯和1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯原料反应；从而得到指示剂；
进一步，在上述技术方案中，联硼酸频哪醇酯和1-(4-溴苯基)-1，2，2-三苯乙烯通常在有机溶剂中混合，在一定条件下，也可以直接混合。当两者反应时，有机溶剂采用四氢呋喃。这样，在步骤1中可以将联硼酸频哪醇酯和1-(4-溴苯基)-1，2，2-三苯乙烯溶解在四氢呋喃中，加入催化剂和饱和碳酸钾溶液，并在三颈圆底烧瓶中混合，然后在氮气保护下搅拌加热至85℃，然后在保持恒定温度的情况下连续反应24h。反应结束后，冷却至室温，减压蒸馏除去溶剂，再通过硅胶柱色谱法进一步纯化，从而得到白色固体，即1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯。
在步骤2中，将4，4-二溴偶氮苯和1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯溶解在四氢呋喃中，加入催化剂和饱和碳酸钾溶液，并在三颈圆底烧瓶中混合，然后在氮气保护下搅拌加热至80℃，然后在保持恒定温度的情况下连续反应24h。反应结束后，冷却至室温，减压蒸馏除去溶剂，再通过硅胶柱色谱法进一步纯化，从而得到淡黄色固体，即乏氧检测指示剂。
在这里，催化剂采用四(三苯基膦)钯。指示剂分子可以被活细胞胞吞，并在乏氧条件下通过还原酶还原。当细胞内氧浓度降低时，其荧光增强，成为检测缺氧环境的探针。
为了使得发明的技术方案、技术目的以及技术效果更为清楚，以使得本领域技术人员能够理解和实施本发明，下面将结合附图及具体实施例对本发明做进一步详细的描述。
第一实施例
在本实施例中，乏氧检测指示剂分子的化学结构式为：
本实施例还提出了上述乏氧检测指示剂分子的制备方法，包括以下步骤：
1)TPE-doe的合成
在三颈圆底烧瓶中将联硼酸频那醇酯(0.38g，1.5mmol)、1-(4-溴苯基)-1，2，2-三苯乙烯(0.41g，1.0mmol)、四(三苯基膦)钯(0.058g，0.05mmol)溶解于脱气的四氢呋喃(50mL)溶液中。然后，在搅拌下将饱和碳酸钾溶液(3.6mL)加入到四氢呋喃溶液中，得到混合物。混合物在氮气氛围下回流反应过夜，随后冷却至室温过滤，减压除去溶剂，得到粗产物。粗产物用二氯甲烷和盐水萃取三次，分离出有机相并用无水硫酸钠干燥，再通过硅胶柱色谱纯化，得到白色固体产物(0.32g，产率为70.0％)，即TPE-doe。1H NMR(600MHz，CD2Cl2)δ7.49，7.48，7.12，7.11，7.11，7.10，7.10，7.10，7.04，7.03，7.03，7.02，7.02，7.02，7.01，7.00，7.00，1.29.。如图1所示。
2)TPE-bi-Aze的合成
在三颈圆底烧瓶中将4，4-二溴偶氮苯(0.34g，1.0mmol)、1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯(1.15g，2.5mmol)、四(三苯基膦)钯(0.058g，0.05mmol)溶解于脱气的四氢呋喃(50mL)溶液中。然后，在搅拌下将饱和碳酸钾溶液(3.6mL)加入到四氢呋喃溶液中，得到混合物。混合物在氮气氛围下回流反应过夜，随后冷却至室温过滤，减压除去溶剂，得到粗产物。粗产物用二氯甲烷和盐水萃取三次，分离出有机相并用无水硫酸钠干燥，再通过硅胶柱色谱纯化，得到棕黄色固体粉末(65mg，77.1％)，即TPE-bi-Aze。1H NMR(600MHz，CDCl3)：δ7.96(d，J＝8.5Hz，2H)，7.71(d，J＝8.5Hz，2H)，7.43(d，J＝8.3Hz，2H)，7.09(dddd，J＝19.1，9.5，7.9，4.5Hz，17H)ppm.。如图2所示。
第二实施例
在本实施例中，乏氧检测指示剂与连二亚硫酸钠反应，偶氮键断裂，荧光强度随着反应时间增强。
在实验中，将TPE-bi-Aze溶于二甲基亚砜(DMSO)/4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)溶液中，在室温下与连二亚硫酸钠反应，在350nm的光激发下，记录不同反应时间荧光强度的变化，以此证明偶氮键断裂恢复分子的荧光发射，结果如图3所示。
指示剂纳米粒子粒径大小检测
将TPE-bi-Aze和聚氧乙烯聚氧丙烯溶于四氢呋喃中，在快速搅拌下，并将其快速打入H2O中，挥发除去四氢呋喃，最终形成均一澄清的溶液，使用滤膜过滤除去粒径较大的颗粒。通过动态光散射测得其水合半径为103nm，结果如图4所示。
指示剂对A375细胞的毒副作用检测
在96孔板中铺好HeLa细胞，培养24h。将TPE-bi-Aze与细胞分别在0％O2、21％O2，共同孵育4h(放置培养箱中，37℃)后，加入MTT(3-(4，5-dimethyl-2-thiazolyl)-2，5-diphenyl-2-H-tetrazolium bromide，噻唑蓝)继续培养4小时。最后将上清液吸出并加入DMSO将产生的甲瓉溶解。将96孔板置于振动台上摇振30秒后使用酶联免疫检测仪测量96孔板中各孔在570nm处的吸收值，检测细胞的存活率，结果如图5所示。
指示剂的肿瘤乏氧检测
将宫颈癌细胞系HeLa细胞与TPE-bi-Aze共孵育12h，再分别在0％O2、21％O2，共同孵育4h(放置培养箱中，37℃)，除去染料培养基并用PBS轻柔洗涤3次；激光共聚焦显微镜成像并分析，激发波长：405nm；发射滤光片：450-550nm结果如图6所示。
Claims (6)
1.一种具有肿瘤乏氧检测的指示剂，化学结构式为：
其中，R选自：
2.权利要求1所述的具有肿瘤乏氧检测的指示剂的制备方法，其特征在于：包括以下步骤：
(1)将联硼酸频哪醇酯和1-(4-溴苯基)-1，2，2-三苯乙烯原料混合，加热，反应后冷却，分离，纯化，得到白色固体，即1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯；其中1-(4-溴苯基)-1，2，2-三苯乙烯原料的化学结构式为：
(2)将4，4-二溴偶氮苯和1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯原料反应，得到指示剂。
3.根据权利要求2所述的具有肿瘤乏氧检测的指示剂的制备方法，其特征在于：在步骤(2)反应中将4，4-二溴偶氮苯和1-(4-苯硼酸频哪醇酯)-1，2，2-三苯乙烯溶解在四氢呋喃中，加入催化剂和饱和碳酸钾溶液，混合，在氮气保护下搅拌加热，在保持恒定温度的情况下连续反应，得到乏氧检测指示剂。
4.权利要求1所述的具有肿瘤乏氧检测的指示剂在肿瘤乏氧环境检测中的用途。
5.根据权利要求4所述的应用，其特征在于：所述检测步骤为：将乏氧指示剂纳米颗粒与人类宫颈癌细胞株共孵育，分别于不同氧浓度的环境下培养，后在激光共聚焦显微镜上进行成像观察。
6.根据权利要求5所述的应用，其特征在于：所述不同氧浓度为21％O2或0％O2。
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