CN105688230A - 七甲川吲哚花菁染料-聚乙二醇-叶酸复合物及制备方法和应用 - Google Patents
七甲川吲哚花菁染料-聚乙二醇-叶酸复合物及制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种七甲川吲哚花菁染料-聚乙二醇-叶酸复合物及制备方法和应用，所述七甲川吲哚花菁染料-聚乙二醇-叶酸可以改进小分子近红外荧光染料普遍存在的脂溶性问题，同时增强对有关组织的靶向特异性。其方法反应条件温和，步骤简单，易于操作，原料成本低；用本方法制备的七甲川吲哚花菁染料-聚乙二醇-叶酸复合物，水溶性好，应用安全性提高，有利于减小对正常细胞的毒性；七甲川吲哚花菁染料-聚乙二醇-叶酸对具有高表达叶酸受体的肺癌的靶向性。
Description
技术领域
本发明属于生物技术领域，特别涉及一种七甲川吲哚花菁染料-聚乙二醇-叶酸的制备方法以及用于高表达叶酸受体的肺癌细胞的检测。
背景技术
荧光成像在医学和生物学研究中具有独特的优势，其中近红外区域(600nm-1000nm)由于生物分子的光吸收最低，自发荧光最弱，大量的红外光可以穿透组织和皮肤而被检测到。由于近红外区域灵敏度高，可以提供实时动态肿瘤活体成像，因此近年来，近红外活体成像技术引起了越来越多学者和外科医生的关注。
在近红外荧光成像技术中，荧光探针的质量至关重要。目前所研究的近红外小分子荧光染料中，普遍存在一个水溶性差、在生物体内代谢时间短的问题，应用时需要事先溶于二甲基亚砜(DMSO)等有机溶剂中。申请人在前期研究中合成了一系列七甲川吲哚花菁染料(史春梦等.一组碳花青染料的类的近红外荧光化合物的用途.CN101518528A；史春梦等.七甲川吲哚花菁染料及其合成方法和应用.CN102268191A)，虽然这种染料具有良好的近红外荧光特性、一定的靶向肿瘤杀伤作用和光敏/热敏效果(CaixiaYue,PengLiu,MingbinZheng，etc.IR-780dyeloadedtumortargetingtheranosticnanoparticlesforNIRimagingandphotothermaltherapy.Biomaterials,2013,34:6853-6861；XuTan,ShenglinLuo,DechunWang，etc.ANIRheptamethinedyewithintrinsiccancertargeting,imagingandphotosensitizingproperties.Biomaterials,2012,33(7):2230-2239)，然而由于该类花菁染料是脂溶性的，在应用时需要事先溶于DMSO中。而DMSO是一种氢键破坏剂，存在严重的毒性作用，与蛋白质疏水基团发生作用，导致蛋白质变性，具有血管毒性和肝肾毒性；同时对人体皮肤具有很好的渗透性，可以将一些溶于其中的有毒物质带入肌肤，所以在应用时存在潜在的安全性风险。
发明内容
本发明的目的是提供一种七甲川吲哚花菁染料-聚乙二醇-叶酸复合物和制备方法及应用，所述七甲川吲哚花菁染料-聚乙二醇-叶酸复合物改进了小分子近红外荧光染料普遍存在的脂溶性问题，同时增强对叶酸受体肺癌的靶向特异性。该方法反应条件温和，步骤简单，易于操作，原料成本低；用本方法改性所得的近红外荧光染料，水溶性好，应用安全性提高，有利于减小对正常细胞的毒性；七甲川吲哚花菁染料-聚乙二醇-叶酸对具有高表达叶酸受体的肺癌的靶向性将有所增强。
为实现上述目的，本发明的技术方案为：
七甲川吲哚花菁染料-聚乙二醇-叶酸复合物(IR-808-PEG-FA)，该复合物的结构式为：
七甲川吲哚花菁染料-聚乙二醇-叶酸复合物(IR-808-PEG-FA)的制备方法，有以下步骤：
a.取七甲川吲哚花菁染料(IR-808)、双氨基聚乙二醇(PEG)、N,N'-二环己基碳酰亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)，加入二氯甲烷(CH2Cl2)、四氢呋喃(THF)和N,N二甲基甲酰胺(DMF)混合溶剂，惰性气氛，室温下避光搅拌反应，得到IR-808-PEG；
b.取叶酸(FA)、N,N'-二环己基碳酰亚胺和N-羟基琥珀酰亚胺，加入二氯甲烷、四氢呋喃和N,N-二甲基甲酰胺混合溶剂，惰性气氛，室温下避光搅拌反应，活化叶酸，得到叶酸活性酯(FA-NHS)；
c.步骤a得到的IR-808-PEG溶液与步骤b中得到的叶酸活性酯FA-NHS混合，惰性气氛，室温下避光搅拌反应，得到IR-808-PEG-FA；
d.步骤c得到的IR-808-PEG-FA纯化。
所述七甲川吲哚花菁染料为其N-烷基侧链不同链长(n＝1-12)，且末端为羧基的七甲川吲哚花菁染料类似物。
步骤a所述N,N'-二环己基碳酰亚胺：N-羟基琥珀酰亚胺：七甲川吲哚花菁染料的摩尔比为3:3:0.5-3，所述七甲川吲哚花菁染料：双氨基聚乙二醇的摩尔比为1:2-4。
所述的聚乙二醇两端为氨基封端的直链聚合物且分子量为600-10000。
步骤b所述N,N'-二环己基碳酰亚胺：N-羟基琥珀酰亚胺：叶酸的摩尔比为在3:3:0.5-3。
步骤b所述活化反应时间为10-100h。
IR-808-PEG，和FA-NHS的摩尔比为1:2-4。
步骤a所述混合溶剂二氯甲烷：四氢呋喃：N,N-二甲基甲酰胺的体积比为3:1-1.5:1-1.5。
步骤b所述混合溶剂二氯甲烷：四氢呋喃：二甲基亚砜的体积比为3:1-1.5:1-1.5。
步骤a和步骤c所述的惰性气氛，其惰性为氮气或氩气。
步骤d所述的纯化在避光条件下进行，其方法是：所得IR-808-PEG-FA溶液高速离心，除去沉淀，旋蒸溶液后，柱层析，收集洗脱部分。
所述柱层析的洗脱剂为二氯甲烷和甲醇混合溶剂，其体积比为V二氯甲烷：V甲醇＝20:0.1-2。
七甲川吲哚花菁染料-聚乙二醇-叶酸复合物在制备用于高表达叶酸受体的肿瘤细胞检测中的试剂盒的应用，如：肺癌等肿瘤细胞的检测等。
本发明对现有的七甲川吲哚花菁染料进行了改性，提高其水溶性。另外，该制备工艺条件温和，操作方便，原料经济，易于实施。
本发明的有益效果：
通过聚乙二醇(PEG)修饰解决了染料的水溶性问题，降低了染料潜在的毒副作用，提高了其临床应用的潜能；
通过叶酸(FA)修饰增强了荧光探针对是对高表达叶酸受体的肺癌、卵巢癌、乳腺癌等肿瘤细胞的主动靶向性，可作为肿瘤细胞检测的试剂盒。
附图说明
图1为IR808-聚乙二醇-叶酸(IR-808-PEG-FA)的结构表征核磁氢谱；
图2为IR808-聚乙二醇-叶酸(IR-808-PEG-FA)荧光光谱图；
图3为IR808-聚乙二醇-叶酸(IR-808-PEG-FA)的细胞毒性评价；
图4为IR808-聚乙二醇-叶酸(IR-808-PEG-FA)被叶酸受体高表达的肺癌细胞高效摄取。
具体实施方式
实施例中所采用的试剂与仪器：
溶剂二氯甲烷、THF、DMF、甲醇均需经过蒸馏纯化与分子筛干燥处理。其他所涉及的化学试剂为市售的分析纯产品；
所有反应均处于氩气及避光保护条件下进行，且硅胶薄层层析监测至反应结束。硅胶薄层层析使用烟台市芝罘黄务硅胶开发试验厂所生产的高效薄层层析硅胶板(型号CF-254)，采用254nm荧光下直接检测或采用磷钼酸、茚三酮、溴甲酚绿、碘显色等官能团显色剂进行检测。
柱层析用于染料分子的最终纯化，采用烟台市芝罘黄务硅胶开发试验厂生产的层析硅胶(10－40μ)。层析用有机溶剂均为分析纯，且经过重蒸干燥处理。目标化合物1HNMR由美国Varian公司生产的MercuryPlus-400核磁共振谱仪测定，TMS作内标，CDCl3作溶剂。
所用A549和H460为两种肺癌肿瘤细胞，均从美国ATCC公司购买，且按照其要求相应培养基进行细胞培养。
所举实施例是为了更好的描述本发明的内容，并不是本发明的内容仅限于所举实例。该领域的相关人员根据上述发明内容对实施方案进行非实质的改进和调整，仍属于本发明的保护范围。
实施例1.IR808-PEG的制备(步骤a)
按照摩尔比1：1：1.3：1依次称取七甲川吲哚花菁染料IR-808(其结构见上述反应式)，氨基封端PEG(分子量3400)，N,N'-二环己基碳酰亚胺和N-羟基琥珀酰亚胺置于干燥锥形瓶中后，依次量取二氯甲烷，四氢呋喃，N,N-二甲基甲酰胺的体积为15mL，3mL，3mL作为反应的混合溶剂，充入氩气1-2min后，密封，避光，室温，持续搅拌12-48h；
实施例2.叶酸活性酯(FA-NHS)的制备(步骤b)
按照摩尔比1：1.2：1.1依次称取叶酸(FA)，N,N'-二环己基碳酰亚胺和N-羟基琥珀酰亚胺置于干燥锥形瓶中后，依次量取二氯甲烷，四氢呋喃，N,N-二甲基甲酰胺15mL，3mL，3mL作为反应的混合溶剂，密封避光，室温，持续搅拌12-24h；所得产物即为叶酸活性酯FA-NHS.
实施例3.IR-808-PEG-FA的制备(步骤c)
将实施例1中反应所得IR-808-PEG与叶酸活性酯(FA-NHS)充分混合，通入氩气1-2min，密封，避光，持续搅拌24-72h，高速离心除去沉淀，旋蒸除去有机溶剂后层析法进行纯化，洗脱剂组成比例V二氯甲烷：V甲醇＝20：1-0：1，收集最后洗脱部分即为制备目标产物IR-808-PEG-FA。以氘代氯仿为溶剂核磁共振波谱测定产物结构，结果如图1所示；以PBS(pH＝7.4)为溶剂检测产物的荧光性能，结果如图2所示。
实施例4.细胞毒性评价
A549和H460细胞接种于96孔板，37℃，5％CO2孵育过夜，分别用2.6μM与5.2μM的IR-808-PEG-FA和IR-808处理48h后，CCK-8法检查各自肿瘤细胞活力，细胞活力(％)＝(实验组OD值-空白组OD值)/(对照组OD值-空白组OD值)×100％。
结果如图3所示。
实施例5.A549和H460肺癌细胞对IR-808-PEG-FA的摄取检测
A549和H460细胞接种于6孔板，37℃，5％CO2孵育过夜，分别加入2.6μM与5.2μM的IR-808-PEG-FA和IR-808(其结构上述见反应式)，20h后胰酶消化收集细胞，PBS漂洗两遍后重悬细胞，流式细胞仪分别检查10000个各自肿瘤细胞的平均荧光强度，633nm激发，780nm收集信号，结果如图4所示。
Claims (10)
1.一种七甲川吲哚花菁染料-聚乙二醇-叶酸复合物，其特征在于，该复合物的结构式为：
2.七甲川吲哚花菁染料-聚乙二醇-叶酸复合物(IR-808-PEG-FA)的制备方法，其特征在于，有以下步骤：
a.取七甲川吲哚花菁染料、双氨基聚乙二醇、N,N'-二环己基碳酰亚胺N-羟基琥珀酰亚胺，加入二氯甲烷、四氢呋喃和N,N二甲基甲酰胺混合溶剂，惰性气氛，室温下避光搅拌反应，得到IR-808-PEG；
b.取叶酸、N,N'-二环己基碳酰亚胺和N-羟基琥珀酰亚胺，加入二氯甲烷、四氢呋喃和N,N-二甲基甲酰胺混合溶剂，惰性气氛，室温下避光搅拌反应，活化叶酸，得到叶酸活性酯(FA-NHS)；
c.步骤a得到的IR-808-PEG溶液与步骤b中得到的叶酸活性酯FA-NHS混合，惰性气氛，室温下避光搅拌反应，得到IR-808-PEG-FA；
d.步骤c得到的IR-808-PEG-FA纯化。
3.根据权利要求2所述的制备方法，其特征在于：所述七甲川吲哚花菁染料为其N-烷基侧链不同链长(n＝1-12)，且末端为羧基的七甲川吲哚花菁染料类似物。
4.根据权利要求2所述的制备方法，其特征在于：步骤a所述N,N'-二环己基碳酰亚胺：N-羟基琥珀酰亚胺：七甲川吲哚花菁染料的摩尔比为3:3:0.5-3，所述七甲川吲哚花菁染料：双氨基聚乙二醇的摩尔比为1:2-4；所述混合溶剂二氯甲烷：四氢呋喃：N,N-二甲基甲酰胺的体积比为3:1-1.5:1-1.5。
5.根据权利要求2所述的制备方法，其特征在于：所述双氨基聚乙二醇两端为氨基封端的直链聚合物且分子量为600-10000。
6.根据权利要求2所述的制备方法，其特征在于：步骤b所述N,N'-二环己基碳酰亚胺：N-羟基琥珀酰亚胺：叶酸的摩尔比为在3:3:0.5-3；混合溶剂二氯甲烷：四氢呋喃：二甲基亚砜的体积比为3:1-1.5:1-1.5。
7.根据权利要求2所述的制备方法，其特征在于：步骤b所述活化反应时间为10-100h。
8.根据权利要求2所述的制备方法，其特征在于：步骤c所述的IR-808-PEG：叶酸活性酯的摩尔比为1:2-4。
9.根据权利要求2所述的制备方法，其特征在于：步骤d所述的纯化在避光条件下进行，其方法是：所得IR-808-PEG-FA溶液高速离心，除去沉淀，旋蒸溶液后，柱层析，收集洗脱部分，柱层析的洗脱剂为二氯甲烷和甲醇混合溶剂，其体积比为V二氯甲烷：V甲醇＝20:0.1-2。
10.七甲川吲哚花菁染料-聚乙二醇-叶酸复合物在制备用于高表达叶酸受体的肿瘤细胞检测中的试剂盒的应用。
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