CN115626928B - 一种靶向α型叶酸受体的培美近红外荧光分子作为荧光示踪剂的用途 - Google Patents
一种靶向α型叶酸受体的培美近红外荧光分子作为荧光示踪剂的用途 Download PDFInfo
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Abstract
本发明公开了一种靶向α型叶酸受体的培美近红外荧光分子作为荧光示踪剂的用途，所述培美近红外荧光分子的结构式为：这种主动靶向近红外荧光分子在保留对α型叶酸受体高亲和性的同时，又对β型叶酸受体具有较低亲和性，有效区分肿瘤和炎症，同时具有水溶性好和荧光量子产率高等优点，在近红外荧光影像肿瘤手术导航成像及医学细胞标记领域具有巨大的发展潜力。
Description
技术领域
本发明涉及一种新型靶向α型叶酸受体的培美近红外荧光示踪剂用于区分α型叶酸受体高表达非小细胞肺癌和炎症的应用，涉及近红外手术导航荧光分子及细胞标记成像等领域。
背景技术
近年来，近红外(NIR)成像已广泛应用于临床肿瘤成像。吲哚菁绿(ICG)是目前临床唯一一款获得中国国家食品药品监督管理总局(CFDA)和美国食品和药物管理局(FDA)批准的用于人体医学成像和临床诊断的荧光剂。ICG是一种亲水性三碳菁染料，分子量为776，它在水溶液中的最大吸收峰位于785nm，最大发射峰位于810-820nm。据报道，ICG可以提供肿瘤和转移***的微创成像，包括手术期间的实时成像。人们普遍认为ICG可以通过与血浆中的白蛋白结合，形成4-6nm的纳米颗粒，通过其增强的通透性和滞留(EPR)效应在肿瘤富集。然而，ICG并不优先在肿瘤组织中积聚，因为它不是受体特异性的示踪剂，已经发现ICG不仅能在肿瘤中积累，而且能在其他高通透性组织中积累，如炎症组织。有研究表明，ICG同样会在肿瘤附近的炎症组织表现出荧光，已成为ICG应用的主要限制。为了改善这一现状，需要设计具有主动靶向性的近红外荧光示踪剂。
利用叶酸和叶酸受体的特异性结合特性，以实现荧光探针向肿瘤癌细胞的靶向投递受到国内外学者广泛关注。然而，叶酸受体有四种亚型：FRα，β，γ和δ，而癌组织主要以FRα，β两种亚型为主，其中FRα主要在卵巢癌、肺癌、子宫癌等上皮组织的恶性肿瘤中过度表达；FRβ在胎盘、单核细胞和巨噬细胞上可上调，且这两种亚型在同一有机体上可同时表达。因为恶性肿瘤患者可能会同时受到炎症性疾病折磨，而在炎症区域积累活化巨噬细胞而高度表达FRβ。叶酸和叶酸偶联物对这两种亚型的叶酸受体具有相近的亲和力，使得它不能选择性地识别肿瘤细胞或炎症区域。因此对叶酸及叶酸偶联物改性以改善其对肿瘤靶向作用的特异性具有重要的实际应用意义。
研究发现，培美曲塞表现出对FRα和FRβ的亲和力存在较大的差异。利用这一特性，针对高度表达叶酸受体a亚型的肿瘤癌细胞，选定培美曲塞衍生物为靶向基团，可得到一种FRα高效介导的靶向标记***。与传统的靶向标记***相比，这一靶向标记***可更加高效地输运至表达FRα的肿瘤癌细胞，同时也可以减少表达FRβ亚型的炎症细胞对标记***靶向性的干扰，这对快速高效检测肿瘤细胞具有重要的意义。
发明内容
本发明的目的是提供一种靶向α型叶酸受体的培美近红外荧光分子作为荧光示踪剂的用途，以实现快速高效检测肿瘤细胞。
为实现上述目的，本发明采用如下技术方案：
一种靶向α型叶酸受体的培美近红外荧光分子作为荧光示踪剂的用途，所述培美近红外荧光分子的结构式为：
所述荧光示踪剂利用对不同叶酸受体亚型的特异性不同，有效将肿瘤和炎症组织区分。
所述荧光示踪剂能够区分α型叶酸受体高表达的非小细胞肺癌和炎症。
所述荧光示踪剂在保留对α型叶酸受体高亲和性的同时，又对β型叶酸受体具有较低亲和性。
有益效果：本发明利用有机全合成的方法制备了培美曲塞二钠及其衍生物为主动靶向基团的主动靶向近红外荧光小分子，并将其创新性的应用在肿瘤和炎症组织成像的区分上，这种主动靶向近红外荧光分子在保留对α型叶酸受体高亲和性的同时，又对β型叶酸受体具有较低亲和性，有效区分肿瘤和炎症，同时具有水溶性好和荧光量子产率高等优点，在近红外荧光影像肿瘤手术导航成像及医学细胞标记领域具有巨大的发展潜力。
附图说明
图1是培美近红外荧光分子合成流程图；
图2是培美近红外荧光分子靶向α型叶酸受体高表达细胞图；
图3是培美近红外荧光分子靶向β型叶酸受体高表达细胞图；
图4是培美近红外荧光分子α型叶酸受体高表达和低表达肿瘤活体成像图；
图5是培美近红外荧光分子区分α型叶酸受体高表达非小细胞肺癌和炎症荧光图；
图6是肿瘤切片HE，FRα，FRβ免疫组化和荧光图；
图7是炎症组织切片HE，FRα，FRβ免疫组化和荧光图。
具体实施方式
下面结合附图对本发明做更进一步的解释。
如图1所示，首先制备培美近红外荧光分子，包括以下步骤：
1、氯代ICG衍生物母体的合成：
(1)4-肼苯磺酸(1.6g,31.9mmol)，3-甲基-2-丁烷(2.10ml,90mmol)冰醋酸(50ml)混合加热到120℃在氮气氛下18h。在乙酸乙酯中沉淀后，以粉红色固体的形式过滤和收集粗产物,所得产物(6.5g,25.4mmol)溶解在甲醇(50mL)中。在温和的条件下，溶解液滴加如以氢氧化钾(1.7g,30mmol)和异丙醇(20ml)的溶液中，粗混合物过滤洗涤，得到棕色固体。(97％)
(2)将化合物2(2.3g,8.3mmol)和1,4-丁磺酸内酯在氮气氛加入甲苯溶液中加热110℃，48h。将混合料冷却至室温，溶剂析出。在粗混合物中加入甲醇(10ml)搅拌30分钟:粗混合物过滤，收集，在2:1(v/v)水混合物中溶解(10ml)和甲醇(50ml)。用滴液漏斗将混合溶液慢慢加入乙腈(160ml)中。沉淀物被过滤并收集为粉红色固体。
(40％)
(3)将化合物3(1.5g,2.79mmol)、Vilsmeier-Haack试剂(0.5g,1.39mmol)和无水乙酸钠(0.342g,4.17mmol)在20mL的无水乙醇中，在氮气气氛下回流加热6h。反应混合物冷却至室温，然后过滤，用乙醇和甲醇洗涤，收集为棕绿色固体。(90％)
2、培美靶向药物合成：将培美水解酸(1.05g,3.52mmol)溶于DMF中，搅拌至溶解，将HATU(2.007g,5.28mmol)O-叔丁基-L-酪氨酸叔丁基酯盐酸盐(1.161g,3.52mmol)DIEA(1.364g,10.56mmol)依次加入烧瓶中，搅拌至完全溶解，氮气保护，室温反应30min将反应后的溶液滴加在0.1N aq.HCl(1.0L,0.14M)，产生淡黄色沉淀，抽滤真空干燥，得化合物52.04g固体，产率95％。
将化合物5(2.04g,3.34mmol)放入圆底烧瓶中，加入TFA：H2O(95：5 10mL),搅拌两小时，加入甲基叔丁基醚中，沉淀过滤，真空干燥。得1.507g,产率98％。
3、合成培美近红外荧光分子7：在23℃下向S0456(2.909g，3.276mmol，)的水
(18mL)溶液中，滴加pH 11的Pemetrexed-Tyr(1.507g，3.276mmol，)三阴离子溶液。将反应混合物的温度升至90℃，在90℃下搅拌45分钟，并通过TLC监测化合物7的形成。产物形成完成后，将反应混合物冷却至室温，并通过套管作为稳定流转移至搅拌的丙酮(0.5L)中，得到绿色沉淀。沉淀物在抽气机真空下在烧结漏斗上过滤，用丙酮(3×500mL)洗涤。将绿色粉末状固体在高真空下干燥12h，定量获得化合物7(4.34g)。
4、为了验证本发明的效果，进行了如下验证实验：
(1)培美近红外荧光分子在区分α型叶酸受体高表达肿瘤和炎症方面的应用培美近红外荧光分子特异性识别α型叶酸受体高表达细胞系：选取α型叶酸受体高表达细胞系H1299和低表达细胞系A549,以β型叶酸受体高表达细胞系RAW264.7。细胞在37℃下，在5％CO2和95％空气的潮湿空气中，以及含有10％FBS以及1％双抗的RPMI培养基中培养。利用浓度为100nM培美近红外荧光示踪剂在共聚焦皿孵育一小时，用PBS清洗，DAPI染色，多聚甲醛固定，拍照。
培美近红外荧光示踪剂可以在H1299细胞的整个细胞质中看到而在α型叶酸受体阴性A549细胞中未观察到这一事实，这证实了培美近红外荧光示踪剂被高表达α型叶酸受体的细胞内化，并支持了培美近红外荧光示踪剂对α型叶酸受体高表达肿瘤细胞特异性靶向的论点。在RAW264.7未检测到荧光，表明培美近红外荧光示踪剂对β型叶酸受体无靶向性。
(2)培美近红外荧光分子用于特异性识别α型叶酸受体高表达非小细胞肺癌组织：尾静脉注射10nmol剂量培美近红外荧光示踪剂到H1299与A549荷瘤鼠体内，两小时后通过小动物成像仪观测成像效果以及分析其组织生物分布。培美近红外荧光分子能够特异性识别α型叶酸受体高表达H1299荷瘤鼠并观察到良好的肿瘤背景信号比，而α型叶酸受体低表达A549荷瘤鼠未检测到靶向效果。
(3)培美近红外荧光分子用于区分α型叶酸受体高表达非小细胞肺癌组织与炎症：建立H1299荷瘤鼠模型，并每日在腹膜三角区注射过硫酸盐刺激产生腹膜炎，如图5所示，炎症所在部位的三角区在肿瘤组织附近。利用免疫组化切片验证炎症模型的建立。通过肿瘤和炎症切片的免疫组化切片可以看到，褐色部分表示受体表达阳性，肿瘤α叶酸受体高表达，炎症组织β型叶酸受体高表达，如图6所示，α型叶酸受体高表达非小细胞肺癌切片荧光强度较高，图7显示了β型叶酸受体高表达的炎症组织切片基本无荧光。利用对不同叶酸受体亚型的特异性不同，有效将肿瘤和炎症组织区分。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种靶向α型叶酸受体的培美近红外荧光分子作为荧光示踪剂的用途，所述培美近红外荧光分子的结构式为：
所述荧光示踪剂利用对不同叶酸受体亚型的特异性不同，有效将肿瘤和炎症组织区分。
2.根据权利要求1所述的用途，其特征在于：所述荧光示踪剂能够区分α型叶酸受体高表达的非小细胞肺癌和炎症。
3.根据权利要求2所述的用途，其特征在于：所述荧光示踪剂在保留对α型叶酸受体高亲和性的同时，又对β型叶酸受体具有较低亲和性。
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