CN109568593A - 一种叶酸受体介导、光激发光敏药物偶联物及其制备方法 - Google Patents
一种叶酸受体介导、光激发光敏药物偶联物及其制备方法 Download PDFInfo
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Abstract
本发明属于高分子药物技术领域，具体涉及一种叶酸受体介导、光激发光敏药物偶联物及其制备方法。首先将叶酸与氨基聚乙二醇进行酰胺化反应，然后与血卟啉HPD进行酰胺化反应，获得具有叶酸受体靶向光激发血卟啉基团的聚乙二醇柔性链偶联。本发明制备所得的偶联物，能够通过叶酸受体介导靶向肿瘤细胞，经过内吞进入细胞，加快血卟啉肿瘤细胞内的积蓄，经特定波长紫光或红光照射，可以进行精准诊断和治疗；同时本发明为制备靶向血卟啉荧光分子药物偶联物提供了一种简单而有效的途径。
Description
技术领域
本发明属于高分子药物技术领域，具体涉及一种叶酸受体介导，光激发光敏 药物偶联物及其制备方法。
背景技术
光激发的血卟啉光敏药物是癌症诊断和治疗的一类新药，通过紫光照射可激 发荧光，通过荧光检测，可以用癌症早期诊断。它也可以通过光动力学疗法对癌 症进行治疗，且其治疗优于传统的手术、放疗和化疗三大治疗肿瘤手段许多，它 对癌组织都具有非常强的选择性，对正常组织的损伤很小。原因是这种血卟啉药 物注入体内1h后，各组织中放射性含量达高峰，组织分布顺序为肺＞肝＞肾＞血＞ 胃＞肠＞脾＞心＞肌肉＞胸腺＞脑＞骨，肿瘤中的含量为以上12种组织中的第89位； 到72h，肿瘤组织的含量上升到第5位。注射本品5h后，总排泄量占注射量的 73.29％，d 11为91％。由此见可，这药物注入人体后到达肿瘤中不是最高的， 通过72h的代谢排泄后，由于肿瘤组织对血卟啉光敏药物亲合力高，有潴留作用， 才能形成肿瘤的贮存高于周边正常组织，在诊断和治疗中才能起到选择性治疗作 用，而在这光敏药物在形成肿瘤组织浓度比正常组织高，是经过48h＝72h的漫长 的排泄过程，在这过程中正常组织的药物含量是非常高，对人体产生光毒性自然 是大的，为此通过接受治疗患者要在暗室中避光一个月时间，极其不方便。
本发明就针对这类药物在癌症诊断和治疗所存在问题，采用受体靶向策略， 发明出一种能快速在特定肿瘤组织中达到有效光敏药物诊断及治疗浓度的受体 靶向新型偶联物，将把光激发的光敏药物与叶酸受体偶联，一旦注入人体内，就 能与FR特异性结合，选择性浓集于FR表达丰富的组织，通常来说许多恶性肿瘤 都存在FR丰富表达。在这FR在靶组织和非靶组织分布的显著性差异，FR介导 的偶联物可获得靶组织与正常组织高对比度的浓度差异，故能对病变进行诊断、 定位及治疗效。由此看来本发明的受体靶向新型偶联物能为改变以往在癌症诊断 和治疗中所使用血卟啉类光敏药物，利用效率不高，有80％没能利用以上排泄 体外的缺陷，药物能高效快速投放在肿瘤中，正常组织残留小，光毒副作用小，在治疗时中也无需暗室进行避光。因此这种叶酸受体介导靶向、光(紫光、红双 光子)激发的光敏药物偶联物，在临床应用中将会具有广泛发展前景。
目前尚未见到此类叶酸受体介导靶向光敏药物偶联物的文献报道。
发明内容
本发明的一个目的是提供一种叶酸受体介导靶向、光(紫光、红光)激发的 光敏药物偶联物。
本发明的另一个目的是提供所述偶联物的制备方法。
本发明的目的通过一下技术方案来予以实现：
提供一种叶酸受体介导、光激发的光敏药物偶联物，其结构通式：
其中：FA为叶酸，PEG为分子量范围500～5000Da的聚乙二醇。
所述一种叶酸受体介导、光激发光敏药物偶联物的制备步骤：
(1)将叶酸与双端氨基聚乙二醇反应，得到叶酸功能化的单端氨基聚乙二醇；
(2)将叶酸功能化的单端氨基聚乙二醇与反应得到主链末端含有血卟啉的聚乙二醇；
在步骤(1)中，首先在无水二甲亚砜溶液中，采用二环己基碳二亚胺(DCC)、 N-羟基琥珀酰亚胺(NHS)和三乙胺(TEA)组合催化剂，然后将叶酸(FA)、双 端氨基聚乙二醇NH2-PEG-NH2、DCC、NHS和TEA，按摩尔用量为1∶1∶1.5∶1.5∶5 的配比，在室温下搅拌反应10～20小时。
在步骤(2)中，将FA-PEG-NH2溶在N，N-二甲基酰胺(DMF)中，加入血 卟啉(1，3，5，8-四甲基-2，4-二(α-羟乙基)卟酚-6，7-二丙酸)，然后在EDC、NHS组合催化中进行反应，它们的摩尔用量为1∶1∶1.5∶1.5，反应中在室温下进 行搅拌，反应时间为10～20小时。
制备得到抗肿瘤药物偶联物的主要特点：(1)在叶酸的介导下，偶联物有选 择性第富集于肿瘤细胞表面，而在正常细胞表面上较少存在，并通过受体介导内 吞作用进入细胞；(2)偶联物中链接有聚乙二醇柔性链，科技减少与网状内皮细 胞膜的亲和性没，从而避免网状内皮细胞的吞噬，延长药物在血液中的循环时间， 增强被动靶向肿瘤组织效应；(3)此偶联物不仅可以响应紫光，还可以发生红双 光子作用下激发，释放出荧光发现恶性肿瘤，或在红光照射下引发化学反应释放 单态氧毒杀肿瘤细胞起到治疗效果，而叶酸和聚乙二醇是无毒无害的，尤其是在 治疗红光的使用，因为长波长红光对组织细胞损伤较小，且能够增加对组织的穿 透深度，有无比优越。
发明有益效果
本发明提供了简单易行的偶联物制备方法，是基于含有光致断裂基团的聚乙 二醇为链接剂，与细胞特异性配体叶酸等相连，成功合成携带有光致断裂结构和 对肿瘤细胞上的叶酸受体具有高亲和力的靶向光激发光敏药物，自1986年科学 家发现叶酸(FA)是由受体介导的内吞途径进入细胞以来，人们开始对FA作为靶 向载体进行深入研究。现已证实在卵巢癌、子宫内膜癌、间皮组织癌、肾癌、头 颈部肿瘤、肺癌、乳腺癌、结直肠癌等恶性上皮组织来源肿瘤、关节炎部位有过 表达的叶酸受体(FR)。这样本发明的叶酸受体介导、光激发光敏药物偶联物会 主动靶向在特定肿瘤组织细胞表面，并能够在外界环境特定波长光刺激下释放出 荧光用于肿瘤检测。在特定波长激发下发生化学反应产生单态氧毒死癌细胞达到 精准治疗效果，具有良好的医药领域应用推广价值。
附图说明
图1为本发明反应式。
具体实施方式
以下结合实施例来进一步解释本发明，但实施例并不对本发明作任何形式的 限制。
实施例1叶酸-聚乙二醇-血卟啉偶联物的制备，叶酸-聚乙二醇-光敏偶联剂的合成。
1)叶酸功能话的聚乙二醇的合成。
称取双端氨基聚乙二醇(NH2-PEG-NH2，M＝3200Da)640mg溶解在10ml无水 DMSO中，加入2ml溶有叶酸FA和NHS的DMSO溶液，然后依次加入DCC、TEA(各 物质的摩尔用量NH2-PEG-NH2∶FA∶NHS∶DCC∶TEA＝ 1∶1∶1.5∶1.5∶5)，在氮气保护下，室温搅拌、避光反应12h，将反应混合物用20ml 去离子水稀释、在500rpm下离心，除去副产物二环己基脲，再加入10ml丙酮以 除去未反应的叶酸，将上清液放入截留分子量为3000Da的透析管中，用去离子 水稀释48h，冷冻干燥，得到FA-PEG-NH2。
2)连有光敏基团聚乙二醇的合成。
600mg FA-PEG-NH2溶于20ml DMF中，然后加入血卟啉(1，3，5，8-四甲基-2，4- 二(α-羟乙基)卟酚-6，7-二丙酸)、EDC、NHS(摩尔比为 FA-PEG-NH2∶HPD∶EDC∶NHS＝1∶1∶1.5∶5)通入氮气，室温搅拌反应15h，加入20ml 去离子水稀释、在500rpm下离心，将上清液转入透析管中用去离子水透析48h 后，冷冻干燥，得到FA-PEG-HPD。
实施例2光毒性副反应及在肿瘤组织中的靶向性效果观察。
1)光毒性副反应实验
以血卟啉注射液(重庆市华鼎现在生物制药有限责任公司)为对比例。取 ICR小鼠20只，雌雄各半，随机分为2组，每组10只。各组小鼠经尾部静脉给 予实施例和对比例药物(按照血卟啉计算给药剂量为6.26×10-2g·Kg-1·d-1)。 给药前小鼠背部去毛，去毛面积为2cm×2cm。给药后1h，将两组小鼠置于5500K 色温光源(近似室外午后大阳光)3500LX光照条件下照射20min，以小鼠背部皮 肤为主要观察区域，分别于不同的时间观察比较实施例和对比例光毒性副反应。 使用405nm半导体激光，以50mw/cm2的功率照射，观察发射荧光特征的表现。观 察结果见表1。
表1组药物中血卟啉对小鼠光毒性副反应的影响以及荧光特征表现
从表1以看出，将血卟啉用叶酸偶联后，明显降低血卟啉的光敏毒性。而从荧 光特征观察引起光毒反应的确来自于血卟啉药物的光敏反应。
2)药物滞留量实验
以血卟啉注册液(重庆市华鼎现代生物制药有限责任公司)为对比例，比较 实施例和对比例在肿瘤组织中的靶向性及给药24h在肿瘤组织中的滞留量。
将HePG-2细胞接种子ICR小鼠右前肢皮下，建立小鼠肝脏肿瘤模型，分别 将对比例和实施例样品经小鼠尾静脉给药后，不同时间点(0.05h、0.25h、0.5h、 1h、2h、4h、8h、12h、24h)将小鼠处死，取肿瘤块，用HPLC分析肿瘤组织中 血卟啉含量，计算各组药物中血卟啉的相对靶向率及24h在肿瘤组织的滞留量。 结果见表2。
表2组药物中血卟啉在肿瘤组织的靶向性和24h滞留量
从表2可以看出，将血卟啉用叶酸偶联后，明显提高血卟啉在肿瘤组织的靶 向性和滞留量，延长血卟啉在肿瘤组织的滞留时间。
从以上的实验观察可以看出本发明采用受体靶向策略，制成的叶酸受体介 导、光激发光敏药物偶联物与普通血卟啉注射液相比较，正常组织含量少，靶向 特征明显，大大提高血卟啉体内恶性肿瘤中的含量。
以上所述仅是本发明的一种优选实施方式，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前提下，还可以做出若干的变换和改进，这些变换和 改进也应视为本发明的保护范围。
Claims (4)
1.一种叶酸受体介导、光激发光敏药物偶联物，其特征在于其结构式：
其中：FA为叶酸，PEG为分子量范围500～5000Da的聚乙二醇。
2.权利要求1所述一种叶酸受体介导、光激发光敏药物偶联物的制备方法，其特征在于包括以下步骤：
(1)将叶酸与双端氨基聚乙二醇反应，得到叶酸功能化的单端氨基聚乙二醇；
(2)将叶酸功能化的单端氨基聚乙二醇与血卟啉反应得到主链末端含有血卟啉光敏基团的聚乙二醇。
3.根据权利要求2所述的制备方法，其特征在于步骤(1)中，首先在二甲亚砜溶液中，采用二环已基碳二亚胺(DCC)、N-羟基琥珀酰亚胺(NHS)和三乙胺(TEA)组合催化剂，然后将叶酸(FA)、双端氨基聚乙二醇NH2-PEG-NH2、DCC、NHS和TEA，按摩尔用量为1∶1∶1.5∶1.5∶5的配比，在室温下搅拌反应10～20小时。
4.根据权利要求2所述的制备方法，其特征在于步骤(2)中，将FA-PEG-NH2溶在N，N-二甲基酰胺(DMF)中，加入血卟啉，然后在EDC、NHS组合催化中进行反应，它们的摩尔用量为1∶1∶1.5∶1.5，反应中在室温下进行搅拌，反应时间为10～20小时。
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| US20090053247A1 (en) * | 2005-10-07 | 2009-02-26 | Photobiotics Limited | Biological materials and uses thereof |
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| CN103768614A (zh) * | 2013-11-08 | 2014-05-07 | 盐城工学院 | 一种叶酸受体介导、光响应抗肿瘤药物偶联物及其制备方法 |
| CN106924732A (zh) * | 2017-03-09 | 2017-07-07 | 南京理工大学 | 一种超声治疗用肿瘤靶向型血卟啉注射剂及其制备方法 |
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| US20090053247A1 (en) * | 2005-10-07 | 2009-02-26 | Photobiotics Limited | Biological materials and uses thereof |
| US20100297017A1 (en) * | 2008-01-31 | 2010-11-25 | Mark Savellano | Method for Synthesizing and Using Pegylated Peptide-Photoactive Chromophore Conjugates and Micellular Formulations Thereof |
| CN103768614A (zh) * | 2013-11-08 | 2014-05-07 | 盐城工学院 | 一种叶酸受体介导、光响应抗肿瘤药物偶联物及其制备方法 |
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