CN113209315B - 靶向肿瘤的多肽探针及应用 - Google Patents
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Abstract
本发明属于医学影像技术领域，具体涉及一种靶向肿瘤的多肽探针及其在肿瘤成像方面的应用。一种靶向肿瘤的多肽探针，所述多肽探针通式为：R‑X‑YW7，其中YW7为在体内特异性靶向ANXA2多肽，序列为YWRGVYN；X为linker，R为信号单元，X与R共价连接。所述多肽探针在制备肿瘤诊断试剂、肿瘤成像试剂、肿瘤的手术引导剂和/或肿瘤主动靶向治疗相关药物示踪剂中的应用。实验证明，本发明提供的靶向肿瘤多肽探针特异性强，灵敏度高，先后通过培养的裸鼠皮下胰腺癌模型、裸鼠皮下胶质瘤模型以及裸鼠原位胶质瘤模型明确该多肽探针能被胶质瘤特异性吸收，并在2小时内快速聚集于肿瘤细胞或肿瘤组织部位，与对照组相比有显著肿瘤靶向优势，在临床上用于肿瘤诊断和治疗。
Description
技术领域
本发明属于医学影像技术领域，具体涉及靶向肿瘤的多肽探针及其在肿瘤成像方面的应用。
背景技术
癌症是世界上主要的死亡原因之一。其发病率和死亡率高、诊疗难度大、易复发和转移，仅在2018年，美国约有609640人死于癌症，相当于每天有近1700人死亡。在过去的几十年里，在治疗癌症方面取得了重大进展。包括手术、化疗和放疗在内的传统方法极大地提高了癌症患者的存活率。然而，复发和多药耐药仍然是限制癌症成功根除和长期治疗的不可逾越的障碍。膜联蛋白A2为脊椎动物膜联蛋白家族中的一员。在肿瘤细胞常表现异常高表达，提示与肿瘤细胞的粘附、增殖、侵袭和迁移等生理活动发生、转移、病例分级和预后息息相关。癌症发病机理中膜联蛋白A2在多种肿瘤种的过表达和细胞表面易位提示膜联蛋白A2是肿瘤相关特异性靶标。因此，与膜联蛋白A2特异性结合的配体可用作成像探针的靶向基团。以多肽作为靶向基团是一种常用的靶向策略，在肿瘤靶向治疗领域已有较多应用，在肿瘤靶向成像的研究也逐渐增多。与单抗相比，小分子多肽具有免疫原性弱、体内快速分布、穿透性强 、易于合成和修饰等优点。部分基于多肽的肿瘤靶向成像探针，如BLZ-100、CRGDP-ZW800-1等已进入临床试验阶段。
分子影像技术的快速发展使得对肿瘤的诊断由传统的解剖形态向前推进到功能、代谢，甚至是受体和基因的蛋白分子水平。其中成像设备可通过探针中有效信号的传递，实现可视化肿瘤快速检测，已成为肿瘤诊断和治疗研究领域的热点。研究发现，荧光信号波长越接近900nm穿透能力越强，因此，吸收及发射光谱均处于700-1000nm的近红外荧光（NIRF)应是观测生理指标的最优选择。许多近红外染料如Cy7和IR800-CW与肽或特异性抗原标记后成功用于肿瘤模型的可视化诊断。
膜联蛋白A2表达量较高的胰腺癌是一种发病隐匿、进展快、疗效不佳、中位生存期短、预后极差的一种恶性肿瘤，素有“癌中之王”"称号。目前，手术切除是胰腺癌病人获得治愈机会和长期生存的唯一有效方法。然而，多数胰腺癌病人因病期较晚而失去手术机会。近年来，随着外科手术新技术和新理念的发展，局部治疗手段以及抗肿瘤药物的应用等，以胰腺癌综合治疗为核心的多种治疗手段联合应用得到广泛关注并取得了显著成果，进一步改善了胰腺癌病人的整体预后。而影响癌症患者治疗效果和预后最为关键的是早发现、早诊断和早干预。
膜联蛋白A2在胶质瘤（Glioblastoma，GBM）中的的表达量与正常脑组织相比差异较大。而GBM是中枢神经系统最常见、最具侵袭性、预后较差的原发性恶性肿瘤，占颅内肿瘤的50%~60%。GBM是成人最常见的颅内原发性恶性肿瘤，具有病程短、预后差、病死率和复发率较高等特点。临床上迫切需要可用于检测预后和预测的非侵入性生物标志物的有效手段，尤其是胶质瘤的检测试剂不但要具备其他肿瘤显影剂高灵敏度的特点，同时还要具备可穿越血脑屏障（BBB）的能力和较高的安全性。
发明内容
为了克服现有技术存在的问题，本发明提供了一种靶向肿瘤的R-X-YW7多肽探针及其应用，本发明将多肽YW7（YW7为之前专利中所提供的一种肿瘤靶向多肽，其序列为YWRGVYN）与信号分子通过linker连接合成多肽探针，该多肽探针有望成为肿瘤靶向性多肽探针，以用于各种ANXA2高表达的肿瘤，尤其是胰腺癌的早期精准诊断和靶向治疗。实验证明该类多肽探针可以在活体上靶向ANXA2高表达的胰腺癌细胞和组织，因此可以作为潜在的肿瘤显像剂、手术术中引导剂和追踪剂，在临床上用于肿瘤诊断和治疗。
为实现上述发明的目的，本发明采用以下技术方案。
一种靶向肿瘤的多肽探针，所述多肽探针通式为：R-X-YW7，其中YW7为在体内特异性靶向ANXA2多肽，序列为YWRGVYN；X为linker，R为信号单元，X与R共价连接。
进一步地，所述信号单元选自放射性同位素、荧光染料、量子点、磁性材料和光声纳米颗粒中的一种或多种。
优选的，所述信号单元为荧光染料。
所述荧光染料选自可见光区(400-700nm)荧光染料、近红外一区(NIR-I，700-900nm)荧光染料以及近红外二区(NIR-II，1000nm-1700nm)荧光染料中的一种。其中可见光区包括ALEXA FLUOR 488、ALEXA FLUOR 500、异硫氰酸荧光素（FITC）、香豆素类染料、DyLight荧光染料等，近红外一区包括菁染料类（ICG、Cyanine等）、BODIPY类、ALEXA FLUOR790、ALEXA FLUOR 750、IRDye 800CW等，近红外二区包括方酸类、IR1048及其衍生物、IR1050及其衍生物、IR1061及其衍生物等。
进一步地，所述linker选自聚乙二醇、6-氨基己酸、天冬氨酸、甘氨酸、聚乙烯、鸟嘌呤肽核酸单体GB、含脂肪链的化合物或螯合剂中的。其中，所述聚乙二醇的结构式为(PEG)n，n＝1-20；所述脂肪链的碳原子数为2-10；所述螯合剂为NOTA、NODA、DTPA或DOTA。其中以聚乙二醇作为linker，可进一步提高上述肿瘤靶向荧光显像剂的生物相容性，增强水溶性，延长其血液循环时间。GB通过其氢键相互作用增强荧光猝灭效果，降低起始荧光强度。
所述多肽探针在靶向肿瘤细胞中的应用，其靶点为ANXA2。实验证明，该多肽探针在体内外靶向肿瘤细胞的应用。
上述多肽探针在制备肿瘤诊断试剂、肿瘤成像试剂、肿瘤的手术引导剂和/或肿瘤主动靶向治疗相关药物示踪剂中的应用。
进一步地，所述肿瘤为ANXA2基因高表达的肿瘤，包括胰腺癌、肝癌、肺癌、乳腺癌、脑胶质瘤或卵巢癌等。
优选的，所述肿瘤为胰腺癌和脑胶质瘤。
本发明还提供了多肽探针制备成荧光显像剂后，在荧光系统引导下胰腺癌、脑胶质瘤的术中定位，实现术中肿瘤的检测和切缘的确定，提高手术精准性，有望引导肿瘤的根治性切除。
与现有技术比，本发明的有益效果如下。
1）本发明提供的肿瘤靶向多肽探针特异性强，灵敏度高，先后通过培养的裸鼠皮下胰腺癌模型、裸鼠皮下胶质瘤模型以及裸鼠原位胶质瘤模型明确该多肽探针能被胶质瘤特异性吸收，并在2小时内快速聚集于肿瘤细胞或肿瘤组织部位，与对照组相比有显著肿瘤靶向优势。
2）本发明提供的肿瘤靶向多肽探针，抗原性小，制备难度不高，成本低，高效低毒，有助于临床广泛的应用。
3）本发明提供的肿瘤靶向多肽探针结构稳定，在体内甚至跨BBB和BBTB后，仍保持多肽的靶向性，便于体外探测或体内肿瘤成像。
4）本发明提供的肿瘤靶向多肽探针可用于ANXA2高表达的大多数肿瘤细胞，用于影像学体外检测时，可以实现肿瘤相关标志物的可视化，有希望广泛应用于多种肿瘤的显像剂的制备。
附图说明
图1为R-X-YW7的结构通式。
图2为ANXA2的mRNA在各种肿瘤中的表达情况。
图3为 Cy7-YW7的结构式。
图4为 Cy7-YW7的质谱表征图。
图5-6为胰腺癌异位荷瘤鼠Cy7-YW7和Cy7组成像图及T/N统计图。
图7-8为胰腺癌异位荷瘤鼠Cy7-YW7各脏器成像图。
图9-11为胶质瘤异位荷瘤鼠Cy7-YW7和 Cy7组成像图及T/N统计图。
图12为胶质瘤异位荷瘤鼠Cy7-YW7组各脏器成像图。
图13-15为胶质瘤原位荷瘤鼠Cy7-YW7和 Cy7组成像图及T/N统计图。
图16为胶质瘤原位荷瘤鼠Cy7-YW7组各脏器成像图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
一种靶向肿瘤的多肽探针，所述多肽探针通式为：R-X-YW7，其中YW7为在体内特异性靶向ANXA2多肽，序列为YWRGVYN；X为linker，R为信号单元，X与R共价连接。R-X-YW7的结构通式如图1所示，R为信号分子，为检测提供探测信号，优选为NIRF家族，X为连接R和多肽YW7的linker。多肽靶点ANXA2 在各种肿瘤中的表达情况如图2所示。
实施例1 靶向胰腺癌的多肽探针Cy7-YW7。
本发明提供的靶向胰腺癌的多肽探针，以Cy7-YW7为例，由之前专利201910766853.9中报道的多肽YW7（YWRGVYN）和近红外荧光染料Cy7，通过PEG3偶联，委托生工生物工程（上海）有限公司合成纯度为95%的荧光多肽探针。Cy7-YW7的结构式和质谱表征如图3-4所示。
实施例2体内靶向小鼠胰腺癌异位荷瘤模型实验。
首先构建胰腺癌皮下瘤小鼠模型，将处于对数生长期的PANC-1细胞胰酶消化，调整细胞浓度为1×108/mL，接种至裸鼠右背侧近腋部皮下，接种后饲养于SPF级，定期观察肿瘤大小，待肿瘤大小约为100mm3时，筛选出无坏死、肿瘤形状规则的荷瘤裸鼠，分组进行试验。 以8nmol的剂量将Cy7、Cy7-YW7溶于生理盐水，通过尾静脉注入荷瘤裸鼠动物模型体内，用小动物活体成像仪分别于0、1、1.5、2、2.5、4小时时间点检测肿瘤的荧光分布，其中对照组成像图和实验组成像图，如图5所示。取2小时后的实验组小鼠的器官进行成像，统计数据肿瘤相对于腿部肌肉荧光值得到T/N柱状图如图6所示。
实验结果显示：尾静脉注射1.5小时后，与对照组相比，Cy7-YW7荧光肽在肿瘤明显富集；尾静脉注射2小时后，与对照组相比Cy7-YW7荧光肽在肿瘤中的单位重量肽含量明显大于其他脏器，如图5所示，实验组小鼠器官成像也进一步证明该结果证明Cy7-YW荧光肽在肿瘤中的单位重量肽含量明显大于其他脏器如图7-8所示。
实施例3 构建U-87 MG细胞皮下瘤动物模型。
构建U-87 MG细胞皮下瘤动物模型：体内动物实验中，将胶质瘤U-87 MG细胞转染荧光素酶基因。扩增转染后的胶质瘤U87细胞，以2×108/mL的密度皮下接种至5-6周的裸鼠右背侧近腋部，定期观察裸鼠状态及肿瘤大小，当肿瘤体积约为100mm3时，筛选出无坏死、肿瘤形状规则的荷瘤裸鼠进行实验。
8nmol的Cy7和Cy7标记多肽（Cy7-YW7）分别通过尾静脉注入到胶质瘤U-87 MG细胞腋下异位荷瘤裸鼠，分别于0、1、2、2.5小时后通过小动物成像仪成像，发现肿瘤位置Cy7-YW7有特异性聚集，而对照Cy7肿瘤位置显著聚集不明显，如图9所示，且对照Cy7组与实验Cy7-YW7组T/N有显著性差异（*P<0.05、**P＜0.01、***P＜0.001）如图10所示。之后，Cy7-YW7尾静脉注射2h后，将荧光素酶底物工作液腹腔注射到每只裸鼠体内。通过小动物成像仪双通道拍摄，merge结果如图11所示。拍照后，将裸鼠解剖，取各个脏器和肿瘤组织，显像如图12所示，说明该多肽对GBM有较好的靶向性。
实施例4 构建U-87 MG原位瘤动物模型。
体内动物实验中，将转染荧光素酶基因胶质瘤U-87 MG细胞扩增转染后，以1.25×108/ml的密度接种至5-6周的裸鼠脑内，一周后腹腔注射荧光素酶底物并采用小动物活体成像系统确定脑内成瘤大小及位置。
8nmol的Cy7和Cy7标记多肽（Cy7-yw7）通过尾静脉注入到胶质瘤U-87 MG细胞原位荷瘤裸鼠体内，0, 1, 1.5, 2, 2.5小时后小动物成像仪成像，Cy7-yw7有明显聚集，而对照Cy7组肿瘤信号弱如图13所示。之后将裸鼠解剖，取各个脏器及其脑组织并成像计算T/N如图14、图16所示。通过小动物成像仪双通道拍摄，merge结果如图15所示。结果表明该多肽探针可以进入脑部并有效聚集于脑组织中小鼠脑原位瘤模型的瘤组织部分。
从实施例中以多肽探针Cy7-YW7为例可以得出，本发明中基于肿瘤靶向多肽YW7设计的靶向多肽探针在小动物肿瘤模型中，具有良好的靶向胰腺癌细胞脑胶质瘤细胞和组织的特性，其在小鼠异位和原位瘤中均显示出良好特异性聚集，可成为胰腺癌、脑胶质瘤的显像剂的潜在候选化合物，并有可能广泛应用于ANXA2高表达的各种肿瘤的早期诊疗和术中引导中，为提高胶质瘤的临床诊疗效率带来了希望。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (1)
1.一种靶向肿瘤的多肽探针在制备肿瘤诊断试剂、肿瘤成像试剂、肿瘤的手术引导剂和/或肿瘤主动靶向治疗相关药物示踪剂中的应用，其特征在于，所述肿瘤为脑胶质瘤；所述多肽探针具有穿越血脑屏障的能力；所述探针为多肽YW7和近红外荧光染料Cy7通过PEG3偶联；所述多肽YW7序列为YWRGVYN。
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