CN111777615B - 一种检测环氧合酶2的光动力治疗探针及其制备 - Google Patents
一种检测环氧合酶2的光动力治疗探针及其制备 Download PDFInfo
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Abstract
本发明公开了一种检测环氧合酶2的光动力治疗探针及其制备方法，所述的探针化合物结构如式Ⅰ所示。该前药分子将癌症标志物环氧合酶2的有效抑制剂消炎痛，利用柔性丁烷与双光子萘酰胺荧光团连接，并且引入Ce6光敏剂用于光动力治疗。探针在缓冲溶液和正常细胞中无明显荧光，而在过表达环氧合酶2的溶液和癌细胞中荧光和单线态氧量子产率剧烈增加，实现荧光成像协同光动力治疗。优势在于，探针兼具分子靶向作用和光动力疗效，是一种鉴定肿瘤并在光激发下生成单线态氧杀死肿瘤的有效工具。
Description
技术领域
本发明涉及一种肿瘤标志物抑制剂与光敏剂结合的双光子荧光探针化合物，更具体地说是一种基于环氧合酶2抑制剂消炎痛与光敏剂Ce6相结合的荧光探针化合物的制备方法，及其在荧光成像协同光动力治疗肿瘤中的应用，属于生物医药领域。
背景技术
癌症是世界范围内的主要疾病，影响癌症死亡率的一个重要因素是难以获得准确的早期诊断。此外，由于手术过程中切除不彻底导致的肿瘤复发是癌症治疗的另一挑战。因此，迫切需要对癌症进行早期和准确的诊断，以达到对肿瘤和扩散的癌变组织进行可视化的定位。环氧合酶2是一种在癌细胞中过量表达而在正常细胞中很少表达的酶，临床数据表明，环氧合酶2的过度表达存在于癌症早期到转移和扩散的所有阶段，且其含量随着癌症的进展而增加。因此，环氧合酶2有希望成为癌症成像的特异性标志物，特别是在胃癌，胰腺癌，结肠癌等肿瘤中。
值得注意的是，腹腔镜内窥镜手术已在许多胃肠道疾病的治疗方面取得了重大进展，它具有减少手术创伤和并发症，术后恢复快等优点。最近，已开发出肿瘤特异性成像试剂，以图像指导更有效地治疗胃肠道癌。特别是，腹腔镜可以使光很容易地传递到疾病部位，用光敏剂(PSs)通过一定波长范围的光产生活性氧(ROS)利用光动力疗法(PDT)治疗胃癌。理想的光敏剂应易溶于水，并且能够准确地靶向疾病部位。同时，活性探针的荧光成像能够对恶性肿瘤进行可视化，并且具有高选择性，高分辨率，无创性，有效的避免了当前成像技术(如正电子发射断层扫描，单光子发射计算机断层扫描方法和X射线成像)所造成的放射危害。
本发明公开了一种检测环氧合酶2的光动力治疗探针及其制备方法，所述的探针化合物结构如式Ⅰ所示。该前药分子将癌症标志物环氧合酶2的有效抑制剂消炎痛，利用柔性丁烷与双光子萘酰胺荧光团连接，并且引入Ce6光敏剂用于光动力治疗。探针在缓冲溶液和正常细胞中无明显荧光，而在过表达环氧合酶2的溶液和癌细胞中荧光和单线态氧量子产率剧烈增加，实现荧光成像协同光动力治疗。优势在于，探针兼具分子靶向作用和光动力疗效，是一种鉴定肿瘤并在光激发下生成单线态氧杀死肿瘤的有效工具。
发明内容
本发明要解决的技术问题：一是提供一种检测环氧合酶2的光动力治疗探针及其制备方法，所述前药化合物具有对癌症进行双光子荧光成像的优点。二是提供一种荧光诊断试剂对环氧合酶2进行有效，准确和原位的响应。三是提供一种光敏剂化合物，具有对癌症进行光动力治疗的优点。四是提供一种在胃癌小鼠模型中进行荧光成像协同光动力治疗指导腹腔内窥镜手术的应用。
为了解决上述技术问题，采取的技术方案如下：
本发明提供一种检测环氧合酶2的光动力治疗探针，具有如下分子结构式：
化合物Npco-Ce6
本发明还提供一种检测环氧合酶2的光动力治疗探针的制备方法，步骤包括：
(1)(a)将4-溴-1,8-萘酐(1eq)和1,4-丁二胺(3-3.5eq)溶于无水二恶烷溶液中，将混合物用氩气吹扫并加入三乙胺(7-10mL)，在氩气中回流8-12小时，冷却后在室温下搅拌10-14小时，用冷水沉淀产物并真空过滤，得到固体化合物1；(b)将化合物1(1eq)溶解于DMF溶液中，向其中加入消炎痛(1-1.5eq)，EDCl(1eq)，HOBt(1.5-2eq)和DMAP(1-1.5eq)，将混合溶液在室温氮气中搅拌20-24小时，柱纯化(二氯甲烷/乙醇)得到固体化合物2；
(2)(c)将化合物2(1eq)溶解于2-甲氧基乙醇溶液中，并向其中添加1,4-丁二醇(1-1.5eq)，将混合物在黑暗下回流12-16小时，冷却至室温后，柱纯化(二氯甲烷/乙醇)得到固体化合物3；(d)将化合物3(1eq)溶于DCM溶液中，向其中加入Ce6(1.1-1.5eq)，DCC(1.3-1.8eq)和DMAP(1.3-1.8eq)，待混合物完全溶解后，在室温下搅拌20-24小时，通过在乙醚溶液中沉淀三次将反应混合物纯化，然后真空干燥后得到化合物Npco-Ce6。
本发明的优点:
本发明的前药分子，具有通过环氧合酶2抑制剂靶向胃癌细胞，将光敏剂探针准确输送到肿瘤部位的优势。
本发明的前药分子，通过消炎痛部分与环氧合酶2结合后产生折叠的构象，使光敏剂在肿瘤部位有效积聚。
本发明的前药分子，解决了目前光敏剂激发光为可见光，无法对深层肿瘤进行光动力治疗的缺陷。
本发明的前药分子，作为一种新型的环氧合酶2抑制剂结合光敏剂荧光探针，能够增强单线态氧量子产率，产生协同的抗肿瘤效果。
因此，本发明是一种非侵入性，靶向肿瘤细胞后实现单线态氧持续释放，并伴随荧光成像协同光动力治疗的有效工具。在生物医药分析检测领域具有广阔的应用前景。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。
实施例1
一种检测环氧合酶2的光动力治疗探针的制备方法，步骤包括：
1)化合物1的合成：
将4-溴-1,8-萘酐(0.97g，3.5mmol)和1,4-丁二胺(1.02g，11.6mmol)溶于无水二恶烷溶液(100mL)中，将混合物用氩气吹扫并加入三乙胺(10mL)，在氩气中回流11小时，冷却后在室温下搅拌10小时，用冷水沉淀产物并真空过滤，得到固体化合物1，产率80％。
化合物2的合成：
将化合物1(0.16g，0.45mmol)溶解于DMF(15mL)溶液中，向其中加入消炎痛(0.19g，0.54mmol)，EDCl(0.08g，0.4mmol)，HOBt(0.12g，0.8mmol)和DMAP(0.07g，0.6mmol)，将混合溶液在室温氮气中搅拌22小时，减压蒸发除溶剂，柱纯化(二氯甲烷/乙醇＝30/1)得到固体化合物2，产率60％。
2)化合物3的合成：
将化合物2(4.316g，6.3mmol)溶解于2-甲氧基乙醇溶液(30mL)中，并向其中添加1,4-丁二醇(0.567g，6.3mmol)，将混合物在黑暗下回流16小时，冷却至室温后，减压蒸发除溶剂，柱纯化(乙醇/二氯甲烷＝10/1)得到固体化合物3，产率35％。
化合物Npco-Ce6的合成：
将化合物3(0.28g，0.4mmol)溶于DCM溶液(10mL)中，向其中加入Ce6(0.3g，0.52mmol)，DCC(0.12g，0.56mmol)和DMAP(0.07g，0.6mmol)，待混合物完全溶解后，在室温下搅拌20小时，通过在乙醚溶液中沉淀三次将反应混合物纯化，然后真空干燥后得到化合物Npco-Ce6。
实施例2
Npco-Ce6在不同浓度的环氧合酶2中的荧光响应
取实施例1合成的Npco-Ce6(50μM)溶解在Tris-HCl缓冲溶液(100mM)中制备储备溶液。在光谱扫描模式下使用共聚焦多光子显微镜进行双光子荧光光谱变化的测定。可调滤光器自动从400到700nm以1nm的增量步进。使用锁模钛：蓝宝石激光源(CoherentChameleon，90MHz，200fs)的激发波长为800nm，输出功率为1230mW，在每个波长间隔处依次捕获双光子荧光光谱，通过荧光强度(I575nm/I450nm)之比获得比率型荧光光谱。在低浓度的环氧合酶2(<0.5μg/mL)中，荧光发射在450nm处，并且在0.0-0.3μg/mL范围内，荧光强度和环氧合酶2浓度之间存在线性关系。然而，当环氧合酶2的浓度超过0.5μg/mL时，随着酶浓度的增加，在450nm处的荧光发射逐渐降低，并在575nm处出现新的发射峰并逐渐增加，经测定荧光比值(I575nm/I450nm)在环氧合酶2的浓度在0.5-3.5μg/mL范围内有良好的线性关系，结果表明Npco-Ce6对环氧合酶2的浓度变化表现出高度敏感的响应。
实施例3
评估Npco-Ce6产生单线态氧的能力
为了获得有效的光动力治疗，取实施例2中Npco-Ce6储备溶液(10μM)，通过激光有效地产生单重态氧。未修饰Ce6的双光子荧光化合物不能产生单线态氧，游离Ce6表现出低的单线态氧生成速率，而不受激光照射的影响，这是由于Ce6具有疏水性导致聚集效应的产生。当激光照射时，Npco-Ce6产生的单线态氧比游离Ce6高2.67倍，该结果表明，充分分散在水中的Npco-Ce6可以在激光照射下主动产生ROS。
实施例4
活细胞中环氧合酶2的比率荧光成像
取实施例2中Npco-Ce6储备溶液(10μM)，对三种癌细胞系HT-29，HeLa和MCF-7进行染色，通过单光子和双光子诱导的荧光显微镜显示出快速而强烈的荧光响应。相反，两种正常细胞系COS-7和HEK 293没有表现出明显的荧光。结果表明荧光强度与这些细胞系中存在的环氧合酶2浓度相关。当将0、1.0、2.5、5.0μg/mL的Npco-Ce6递送至HeLa细胞时，红色通道(575nm)的荧光强度增加，而绿色通道(450nm)处荧光强度降低。
实施例5
Npco-Ce6对肿瘤生长和体内抗癌作用效果的测定
用PBS和680nm辐照治疗的小鼠中的肿瘤迅速增大，由于可以忽略的暗毒性，游离的Npco-Ce6没有显示出抑制肿瘤的作用，680nm照射后，Npco-Ce6导致肿瘤异常消退，且无肿瘤复发。然而，用游离Ce6治疗的小鼠仅观察到轻微的肿瘤抑制作用。同时，在观察期结束时，Ce6治疗小鼠的肿瘤大小约为Npco-Ce6治疗小鼠的16倍。
上述仅为本发明的优选具体实施方式，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但本发明的设计构思并不局限于此，凡利用此构思对本发明进行非实质性的改动，均应属于侵犯本发明保护范围的行为。
Claims (3)
1.一种检测环氧合酶2的光动力治疗探针的制备方法，其特征在于，所述化合物结构如式Ⅰ所示：
该方法的制备步骤如下：
(1)(a)将4-溴-1,8-萘酐和1,4-丁二胺溶于无水二恶烷溶液中，将混合物用氩气吹扫并加入三乙胺，在氩气中回流8-12小时，冷却后在室温下搅拌10-14小时，用冷水沉淀产物并真空过滤，得到固体化合物1；(b)将化合物1溶解于DMF溶液中，向其中加入消炎痛，EDCl，HOBt和DMAP，将混合溶液在室温氮气中搅拌20-24小时，柱纯化得到固体化合物2；
(2)(c)将化合物2溶解于2-甲氧基乙醇溶液中，并向其中添加1,4-丁二醇，将混合物在黑暗下回流12-16小时，冷却至室温后，柱纯化得到固体化合物3；(d)将化合物3溶于DCM溶液中，向其中加入Ce6，DCC和DMAP，待混合物完全溶解后，在室温下搅拌20-24小时，通过在乙醚溶液中沉淀三次将反应混合物纯化，然后真空干燥后得到化合物Npco-Ce6
2.根据权利要求1所述的一种检测环氧合酶2的光动力治疗探针的制备方法，其特征在于：所述的步骤(1)(a)中4-溴-1,8-萘酐和1,4-丁二胺摩尔比范围为1：(3～3.5)；4-溴-1,8-萘酐溶于二恶烷溶液的摩尔浓度范围为0.03～0.04mol·L-1；(b)中化合物1和消炎痛摩尔比范围为1：(1～1.5)；EDCl，HOBt和DMAP摩尔比范围为1：(1.5～2)：(1～1.5)；化合物1溶于DMF的摩尔浓度范围为0.03～0.035mol·L-1；柱纯化中二氯甲烷和乙醇体积比为(50～25)：1。
3.根据权利要求1所述的一种检测环氧合酶2的光动力治疗探针的制备方法，其特征在于：所述的步骤(2)(c)中化合物2与1,4-丁二醇摩尔比范围为1：(1～1.5)；化合物2溶于2-甲氧基乙醇的摩尔浓度范围为0.2～0.25mol·L-1；柱纯化中乙醇和二氯甲烷体积比为(10～5)：1；(d)中化合物3，Ce6，DCC和DMAP摩尔比范围为1：(1.1～1.5)：(1.3～1.8)：(1.3～1.8)；化合物3溶于DCM的摩尔浓度范围为4～5mmol·L-1。
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