CN107652358A - 一种uPAR靶向性多肽、探针和活体分子显像方法 - Google Patents
一种uPAR靶向性多肽、探针和活体分子显像方法 Download PDFInfo
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- CN107652358A CN107652358A CN201710797226.2A CN201710797226A CN107652358A CN 107652358 A CN107652358 A CN 107652358A CN 201710797226 A CN201710797226 A CN 201710797226A CN 107652358 A CN107652358 A CN 107652358A
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种uPAR靶向性多肽,其氨基酸序列如SEQ ID NO.1所示。uPAR靶向性多肽是由13个氨基酸构成的线性多肽,该多肽易于合成,能够形成稳定的结合构象,以特异性靶向结合uPAR。本发明公开了一种uPAR靶向性探针,包括信号单元和上述的uPAR靶向性多肽。uPAR靶向性探针通过多肽部分特异性识别并结合uPAR,并由信号单元发射供影像学设备检测的信号,以实现uPAR的活体可视化检测,直观的显示uPAR的分布和数量,用于uPAR表达异常的疾病检测。本发明还公开了一种活体分子显像的方法,利用上述的uPAR靶向性探针,具有安全、无创、动态和直观等优点,适用于人体的直接检测。
Description
技术领域
本发明属于医学影像技术领域,具体涉及一种uPAR靶向性多肽、探针和活体分子显像方法。
背景技术
根据世界卫生组织于2014年2月4日世界癌症日发布的2014《世界癌症报告》,癌症已经成为全世界人类的最大致死原因,而中国的癌症发病率已经居于世界首位。随着环境污染,食品安全等各种问题的涌现,人类健康受到越来越多外部环境的威胁,不断加剧了各种癌症发生的可能。因此,如何实现癌症的早期诊断和个性化诊疗,以大大降低癌症死亡率,提高病人生存质量,是目前癌症研究的重要方向。
目前,影像技术是临床癌症诊断的重要手段,随着现代影像检查设备的不断改进和发展,明显提高了癌症早期诊断的阳性率,但无论是X射线断层扫描(CT)、正电子发射断层扫描(PET)还是磁共振成像(MRI)技术,对于癌症早期诊断的特异性和敏感性均有待于提高;另外,传统的影像诊断只能显示的分子改变的终效应,无法探查疾病过程中的分子异常。分子影像学的创建为癌症高特异性和敏感性的诊断方法的建立提供了一个重要的研究平台。分子影像技术(molecular imaging)是指在活体状态下,应用影像学手段,以非侵入的方式获得组织细胞内特定分子的表达和活性 (例如蛋白酶和蛋白激酶)以及细胞凋亡、血管生成和转移等生理过程的信息。其中,常用的影像学手段包括:X射线断层扫描(CT)、磁共振显像 (MRI),超声(US)、正电子发射断层扫描(PET)、单光子发射断层显像 (SPECT)和光学显像(Optical imaging)等。分子影像诊断癌症具有高度的灵敏度和特异性,对于癌症的早期诊断、癌症病情发展监测以及临床个体化治疗具有重要意义。其中,由特异性分子和特殊标记物构成的分子探针作为分子影像检测中的示踪剂,是分子影像技术的关键因素,也是分子影像学研究的先决条件。研发特异性强的分子探针是分子影像技术应用的基础。
恶性肿瘤能够降解周围的胞外基质,导致局部侵袭或转移。肿瘤细胞在侵袭和转移的过程中,会分泌许多特殊的蛋白酶。近年研究也表明,丝氨酸蛋白酶尿激酶纤溶酶原激活剂(uPA)和细胞表面的受体(uPAR)与人类癌症的生长和转移息息相关。尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,uPA)是纤溶酶原的重要激活物之一,其特异性受体为纤溶酶原激活物受体(urokinase-type plasminogen activatorreceptor,uPAR)。 uPA/uPAR系统对纤溶酶原活性起调控作用,参与多种与基细胞外基质降解、重塑相关的病理生理过程。肿瘤组织中,在uPA/uPAR系统的作用下将纤溶酶原激活为纤溶酶,促进基底膜和细胞外基质内相关成分的降解,为肿瘤的侵袭、转移提供有利微环境。此外,uPA/uPAR系统对于肿瘤的增殖及肿瘤血管形成也具有调控作用。uPA和uPAR的表达已经在多数肿瘤类型包括恶性胶质瘤、前列腺、乳腺、结肠、肝细胞、和肾细胞癌中得到证实(Mizukami IF et al.(1994)Clin Immunoland Immunopathol 71:96-104;Hsu DW et al.(1995)Am J Pathol 147:114-23;deWitte JH et al.(1999)Br J Cancer 79:1190-8)。uPA和uPAR在扩散更快的疾病中表达更明显。在肿瘤细胞上,这些表达在肿瘤扩散的前期经常达到最高点(Buo,Let al.(1995)Human Pathol 26:1133-1138:Yamamoto M et al.(1994)CancerRes 54:5016-5020)。已经报道在乳腺、结肠、和肾细胞癌相关的血管中的uPAR的强免疫组织化学着色(Bastholm L et al.Appl Immunohistochem Mol Morphol7:39-47;Nakata S et al.(1998)Int.J.Cancer 79:179-186)。结肠癌研究表明,uPAR与VEGF共存。uPA和uPAR的表达也已经在多个肿瘤类型中与巨噬细胞相关的肿瘤上发现(Ohtani Het al.(1995)Int J Cancer 62:691-6;Xu Y et al.(1997)Hum Pathol 28:206-13)。因此,uPA/uPAR体系被视为癌症治疗的研究热点目标。
现有技术中公开了基于uPA的与uPAR结合区域的氨基酸设计的靶向 uPAR的多肽,靶向uPAR的多肽与可检测标记或治疗部分辍合后,能够用于肿瘤的诊断或治疗。但现有技术中公开的靶向uPAR的多肽为了使氨基酸序列形成适当的结合构象,以保障多肽的稳定结合,需要至少保留uPA上 13-30位的氨基酸残基,或者将氨基酸为20-30位的短的线性肽处理为环状,增加了多肽的合成难度与合成成本。
发明内容
因此,本发明要解决的技术问题在于克服现有技术中靶向uPAR的多肽合成难度和合成成本高,从而提供一种序列长度缩短的线性uPAR靶向性多肽,在保证多肽靶向uPAR的特异性高和高稳定强的前提下,降低了靶向 uPAR的多肽合成难度和合成成本。
为此,本发明提供了一种uPAR靶向性多肽,所述uPAR靶向性多肽的氨基酸序列如SEQ ID NO.1所示。
本发明提供了一种uPAR靶向性探针,所述uPAR靶向性探针包括信号单元和uPAR靶向亲和单元,所述信号单元为影像学检测单元,所述uPAR 靶向亲和单元为所述的uPAR靶向性多肽。
所述的uPAR靶向性探针,还包括连接所述信号单元和所述uPAR靶向亲和单元的连接单元。
所述的uPAR靶向性探针,所述连接单元选自聚乙二醇、含脂肪链的化合物或螯合剂;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;所述螯合剂选自NOTA、NODA、NODAGA、DTPA、 DOTA、DOTAGA、TETA或CB-TE2A。
所述的uPAR靶向性探针,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至少一种。
所述的uPAR靶向性探针,所述荧光染料为近红外一区荧光染料或近红外二区荧光染料。
本发明提供了所述的uPAR靶向性多肽或所述的uPAR靶向性探针在制备治疗或诊断uPAR异常表达疾病的试剂中的用途。
所述的用途,所述uPAR异常表达的疾病为肿瘤。
本发明提供了一种用于肿瘤诊断的试剂盒,所述试剂盒包括所述的 uPAR靶向性探多肽和/或所述的uPAR靶向性探针。
本发明提供了一种活体分子显像方法,应用所述的uPAR靶向性探针示踪活体内的uPAR,然后应用活体显像技术对所述活体的待测位置的uPAR 进行检测。
优选地,所述活体显像技术为正电子发射断层显像、光学显像、单光子发射断层显像、磁共振显像或光声显像。
多肽探针(peptide-based probe):是指能够与某一特定生物分子(如蛋白质)靶向特异性的结合、并可供体内和体外影像学示踪的标记化合物分子,这些标记化合物分子能够在活体和离体层面反映其靶生物分子的量和功能。
分子显像多肽探针必须具备以下两个重要特征:第一,对与疾病相关的靶分子具有高度亲和力和靶向特异性;第二,可供影像学设备在体外进行示踪。探针主要在活体内对生物过程进行成像、定量和测量研究。基本结构一般分为三部分:信号单元(signalingcomponent)、亲和单元(affinity component)和连接单元(linker)。信号单元是指能产生影像学信号且能被高精度的成像技术探测的造影剂或标记性部分(如放射性核素、荧光染料等);亲和单元及靶向分子,是与显像靶点特异性结合的多肽部分。可直接把信号单元和亲和单元连接起来,也可通过linker把二者连接起来。
本发明提供的uPAR靶向性探针,该类探针由放射性同位素、荧光染料、量子点、光声纳米颗粒和多模式成像等影像学方法标记uPAR靶向性多肽而成。引入该类探针,应用正电子发射断层显像(PET),单光子发射断层显像(SPECT),光学成像、超声成像、磁共振成像、光声成像和多模式成像等影像学手段可在活体内检测到正常生理及病理情况下,待测区域的uPAR的数量、分布和功能,及其在疾病发展的不同阶段的变化特征。
本发明相对于现有技术具有如下优点:
1、本发明提供的uPAR靶向性多肽,其氨基酸序列如SEQ ID NO.1所示。uPAR靶向性多肽是由13个氨基酸构成的线性多肽,uPAR靶向性多肽能够成形合适的结合构象,以特异性识别并结合uPAR,对uPAR结合的亲和力高;另一方面,uPAR靶向性多肽在体内的稳定性高,能够存留有效时间;本发明提供的uPAR靶向性多肽是序列长度短的线性多肽,分子量小,组织渗透性强,容易合成,降低了多肽合成的难度和合成的成本。
2、本发明提供的uPAR靶向性多肽在制备治疗或诊断uPAR异常表达的疾病的试剂中的用途,uPAR在多种肿瘤细胞中高表达,将uPAR靶向性多肽与肿瘤治疗药物或载有药物的载体如纳米材质、脂质体等结合后,再添加药学上可接受的辅料或佐剂以制成有效靶向肿瘤细胞的抗癌药物,具有靶向性强、治疗效果好等优点。uPAR靶向性多肽与检测试剂结合后,能够用于肿瘤的诊断以及预后评估,且诊断的准确度高。
3、本发明提供的uPAR靶向性探针,包括信号单元和uPAR靶向亲和单元,所述的uPAR靶向性多肽,uPAR靶向亲和单元为上述的uPAR靶向性多肽。uPAR靶向性探针通过亲和单元特异性识别并结合uPAR,由信号单元提供检测信号,以准确监测肿瘤发展过程中的关键标志物uPAR分子水平的信息,从而为肿瘤的早期诊断、疗效监测和预后评估提供有效信息。
uPAR靶向性探针应用于分子影像技术,为无创、直观的uPAR活体可视化检测提供了一种新的检测探针,uPAR靶向性探针具有以下特点:
(1)特异性强
uPAR靶向性探针对目标靶点结合的特异性强,能够有效减少探针与非检测靶点的非特异性结合,有利于实现对uPAR的准确定量。
(2)灵敏度高
在疾病早期,或者治疗干预的早期检测到分子标志物的变化情况,通常要求探针可灵敏的检测到非常少量的生物标志物,即具有高度灵敏。uPAR 靶向性探针灵敏度高,在nmol剂量情况下就可以获得理想的检测图像。从而大大减少活体可视化检测时引入体内探针的剂量,降低探针引发的药理学作用。
(3)稳定性高
uPAR靶向性探针能够在活体内的保持完整,避免被血浆内或者靶组织内存在的酶降解,稳定性高,能够充分保障活体可视化检测时的图像质量和定量研究的准确性。
(4)亲和力高
uPAR靶向性探针在应用于uPAR活体检测,探针到达肿瘤部位后早期就与相应的uPAR靶点结合,解离的时间相对较晚,确保在几个血液循环周期后,该探针在肿瘤靶点的高浓度聚集,以获得高质量的分子显像图像。
(5)生物兼容性高、制备成本低
uPAR靶向性探针以多肽为骨架,没有免疫原性,生物兼容性高、毒性低,能够很好的应用于人体,大大降低了活体检测探针可能产生的药理学作用;另一方面,uPAR靶向性探针的制备过程方便易行,成本低廉,有助于临床广泛的应用。
(6)应用于活体分子成像的对比度高
uPAR靶向性探针具有理想的生物学分布特征,探针在肿瘤部位浓聚量大,停留时间长,而在正常组织器官内摄取率低,清除速度快。使肿瘤部分形成高的信号强度,而周围组织的信号强度低,即靶/背景比值和信号/ 噪声比值高,以形成高对比度的图像。
4、本发明提供的uPAR靶向性探针,uPAR靶向性多肽与信号单元通过化学方法直接相连,或者由连接单元将两者连接起来,连接单元上同时具有信号单元与uPAR靶向性多肽的连接基团,以将两者辍合连接在一起。
其中作为连接单元的聚乙二醇以及含脂肪链的化合物,可进一步提高 uPAR靶向性探针的生物相容性,而且可以延长探针的血液循环时间,减少影像药物被肝脏和脾脏中网状内皮系统吞噬。
5、本发明提供的uPAR靶向性探针,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至少一种。信号单元通过螯合剂偶联到uPAR靶向性多肽上,使探针能够发射影像学信号,以供外部的影像学设备在体外检测,通过图像直观地显示出分子标志物uPAR的信号强度和分布情况,从而实现对疾病发展过程中关键分子标志物uPAR的活体可视化。
信号单元中的荧光染料选自近红外一区荧光染料或近红外二区荧光染料中的至少一种,近红外一区(NIR-I,发射谱在700-900nm)荧光染料包括吲哚青绿(IndocyanineGreen,ICG)和Cyanine等,其穿透力较强,适用于活体光学成像。相对于普通可见光和NIR-I荧光成像技术,近红外二区(NIR-II,发射谱在1000nm-1700nm)荧光成像激发波长和发射波长更长,可显著降低光在穿透生物组织中的散射现象,加上其光子自身组织吸收少,引起自荧光效应低等特点,所达到的透射深度更深,空间分辨率更高,可达约40微米。
6、本发明提供的用于肿瘤诊断的试剂盒,在应用于肿瘤的活体检测时,具有特异性高、稳定性强、灵敏度高和分子成像对比度高的优点,适用于肿瘤的早期诊断、肿瘤的发展监控与疗效评估等。
7、本发明提供的活体分子显像方法,应用上述的可供影像学设备检测的探针,直观的显示uPAR的数量和分布,具有安全、无创、活体、动态、直观、准确和可直接用于人体等特征。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1中uPAR靶向性多肽的质谱结果;
图2为本发明实施例2中uPAR靶向性探针的结构示意图;
图3为本发明实施例6中uPAR靶向性探针(Al18F-SY1)在体外与 U87MG细胞结合和阻断的放射性检测结果;
图4为本发明实施例6中uPAR靶向性探针(Cy5.5-SY1)在体外与 U87MG细胞结合和阻断的荧光检测结果;
图5为本发明实施例7中uPAR靶向性探针(Al18F-SY1)在荷U87MG肿瘤小鼠的活体检测结果,左图为注射探针15min后的PET成像图,右图为注射探针60min后的PET成像图;
图6为本发明实施例7中uPAR靶向性探针(Al18F-SY1)在小鼠体内注射1小时候在个器官中的分布情况。
具体实施方式
以下通过具体实施例来说明本发明的实施方式,除非另外说明,本发明中所公开的实验方法均采用本技术领域常规技术,实施例中所用到的试剂和原料均可由市场购得。本发明使用的U87MG细胞购自上海歌凡生物科技有限公司。
实施例1
本实施例提供一种uPAR靶向性多肽,也即uPAR靶向性探针中的亲和单元。
《The Receptor-binding Sequence of Urokinase》(E Appella,et.(1987)J.Biol.Chern.Vol.262,No.10,Issue of April 5,pp.4437-4440)报道了uPA氨基末端20-30位的氨基酸残基表现出与uPAR特异性结合的能力,但20-30 位的氨基酸残基需要13-19位的氨基酸残基以形成适当的结合构象。本发明对已报道的多肽进行结构上的改造后,得到由13个氨基酸构成的线性多肽,该多肽进入细胞或组织后能够形成稳定的结合构象,以特异性的识别并结合uPAR,对uPAR结合的亲和力高。将上述uPAR靶向性多肽命名为 SY1,序列为:Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asn-Ile-His-Trp-Gly-Cys,分子式:C71H100N20O18S,分子量:1553.7,其结构如式I所示:
通过多肽固相合成法制备SY1多肽后,将制备的SY1多肽粗产品用 HPLC纯化,纯化后的SY1多肽ESI-MS鉴定结果如图1所示,分子量为1978.9。
本发明设计合成的SY1多肽不仅与uPAR特异性结合,具有高度的亲和力和靶向特异性;而且分子量小、组织渗透性强、容易合成,降低了多肽合成的难度和合成的成本。
实施例2
本实施例提供一种uPAR靶向性探针,uPAR靶向性探针的结构如图2 所示,包括靶向亲和单元、信号单元和连接亲和单元与信号单元的连接单元。
1、成像靶点(uPAR靶点):为丝氨酸蛋白酶尿激酶纤溶酶原激活剂(uPA) 的细胞表面的受体uPAR,uPAR为该探针靶向亲和单元的主要结合靶点,也即uPAR靶向成像的靶点。
2、信号单元:是可供影响设备检测的部分,本发明的信号单元选自放射性同位素,也可以选用荧光染料、量子点、磁性材料、光声纳米颗粒的一种或几种。
(1)放射性同位素:利用放射性二价或三价(M2+/M3+)的金属离子作为信号单元,例如68Ga、111In、64Cu、99Tc或Al18F等,能够用于正电子发射断层显像(PET)或单光子发射断层显像(SPECT)。
(2)荧光染料:例如近红外一区荧光染料或近红外二区荧光染料,能够用于光学显像。
①近红外一区荧光染料是波长在700-900nm的近红外一区的荧光染料,其穿透力强,用于活体光学成像。近红外一区荧光染料包括吲哚青绿 (Indocyanine Green,ICG)和Cyanine等。
②近红外二区荧光染料是波长在1000-1700nm的近红外一区的荧光染料,其荧光成像激发波长和发射波长更长,可显著降低光在穿透生物组织中的散射现象,加上其光子自身组织吸收少,引起自荧光效应低等特点,所达到的透射深度更深,空间分辨率更高。近红外一区荧光染料包括 CH1055、Q4、Q1、H1,碳纳米管和Ag2S量子点等。
(3)量子点:由II/VI族或III/V族元素组成的,直径为2~10nm,能够接受激光态激发产生荧光的半导体纳米微晶体,能够用于光学显像。
(4)磁性材料:例如顺磁性材料、超顺磁性材料或磁性纳米粒子等,能够用于磁共振显像。
(5)光声纳米颗粒:例如稀土纳米粒子或金纳米粒子等,能够用于光声成像。
3、靶向亲和单元:是探针与成像的分子靶点特异性结合的部分,二者之间的结合具有高度特异性和高度亲和力。本发明的靶向亲和单元为实施例1所示的uPAR靶向性多肽。
4、连接单元:将信号单元和靶向亲和单元连接起来的部分。也可不引入连接单元,而采用化学方法直接将信号单元和uPAR靶向亲和单元直接连接。连接单元上同时具有与信号单元和与uPAR靶向性多肽连接的基团,具体地为聚乙二醇、含脂肪链的化合物或螯合剂;其中聚乙二醇的结构式为 (PEG)n,n=1~20;脂肪链的碳原子数为2~10;螯合剂选自NOTA、NODA、 NODAGA、DTPA、DOTA、DOTAGA、TETA或CB-TE2A,常用的螯合剂的结构如下式所示:
上述聚乙二醇、含脂肪链的化合物以及螯合剂均为双功能的连接单元,既具有与放射性金属结合的功能基团,也有与多肽亲和组分结合的基团,以实现两者的辍合连接,构成uPAR靶向性探针。
实施例3
本实施例提供一种uPAR靶向性探针的制备方法,以放射性同位素Al18F 作为信号单元,以螯合剂NOTA作为连接单元,具体制备方法包括以下步骤:
1、制备NOTA-SY1
将过量的NOTA-Maleimido(5equiv)和实施例1制备的SY1(1equiv) 混合于N,N-二甲基甲酰胺(DMF)中,N,N-二甲基甲酰胺(DMF)中含有质量分数为1%的N,N-二异丙基乙基胺(DIPEA),室温搅拌2小时。反应液直接用水稀释后,高效液相色谱(HPLC)分离纯化,质谱分析鉴定产物,NOTA 修饰SY1反应式如式II所示:
2、制备Al18F-SY1
取10μg步骤1中制备的NOTA-SY1溶于0.5mol/L醋酸钠缓冲液,再加入5μL的2mmol/L AlCl3和100μL 18F靶水,95℃反应20min。Al18F标记后的产品通过放射性检测器-高效液相色谱(radio-HPLC)分离纯化,得到 Al18F标记的Al18F-SY1,也即Al18F-NOTA-SY1。测定标记率(>95%)、放化纯度和稳定性,比活度(35GBq/μmol)。Al18F标记NOTA-SY1的化学反应如式III所述:
上述的制备方法同样适用于制备64Cu和68Ga标记的uPAR靶向性探针。
实施例4
本实施例提供一种uPAR靶向性探针的制备方法,以近红外一区荧光染料Cyanine5.5(Cy5.5)作为信号单元,具体制备方法包括以下步骤:
取实施例1制备的SY1溶于50μL DMF(N,N-二甲基甲酰胺)中,避光条件下与Cy5.5-malemide(1.2equiv)以及DIPEA(0.05equiv)室温搅拌反应6小时。反应液经过水稀释后直接经HPLC分离纯化,收集目标产物的馏分,合并后冻干,冻干后的产品用质谱(MALDI-TOF-MS)检测分子量进行验证。Cy5.5标记SY1的化学反应如式IV所示:
实施例5
本实施例提供一种uPAR靶向性探针的制备方法,以近红外二区荧光染料CH1055作为信号单元,具体制备方法包括以下步骤:
取实施例1制备的SY1溶于50μL DMF(N,N-二甲基甲酰胺)中,与 CH1055-malemide(1.0equiv)以及DIPEA(0.05equiv)室温搅拌反应12小时。反应液经过水稀释后直接经HPLC分离纯化,冻干后的产品用质谱 (MALDI-TOF-MS)检测分子量进行验证。CH1055标记SY1的化学反应如式V所示:
实施例6
本实施例提供一种uPAR靶向性探针的体外验证的方法,通过分别向 U87MG细胞中加入放射性标记的Al18F-SY1和荧光染料标记的Cy5.5-SY1,验证uPAR靶向性探针在细胞内的结合情况,具体分为以下检测实验:
1、Al18F-SY1细胞结合和阻断实验
选择高表达uPAR的U87MG细胞株,不表达uPAR的LNCap细胞株作为对照验证Al18F-SY1摄入的特异性。按照1.0×105/孔,铺12孔板,然后孵育过夜。结果显示:Al18F-SY1与U87MG细胞存在明显的特异性结合,而与LNCap细胞无结合。将U87MG细胞分为2组:向A组的每孔细胞中加入1μCi的Al18F-SY1孵育;向B组的每孔细胞中加入1μCi的Al18F-SY1 和10μg的阻断剂共同孵育,然后在孵育0.5小时、1小时和2小时后取出细胞并用PBS洗涤后,将细胞溶解于0.5mL的1.0mol/L的氢氧化钠水溶液中,用γ计数器测定细胞摄取的Al18F-SY1,单位为增加放射性剂量(%AD)。实验重复三次。
结果如图3所示:随着孵育时间的增加,A组细胞中检测到明显的放射性量的增加,在孵育1h后,A组细胞检测到的放射性的量达到最大值,之后维持恒定,说明Al18F-SY1与U87MG细胞具有明显的结合。而在加入阻断剂的B组细胞中,检测到放射性的量明显降低,细胞对Al18F-SY1的结合量明显减少,说明Al18F-SY1与U87MG细胞的结合具有靶向特异性和高亲和力。
2、Cy5.5-SY1细胞荧光和摄取实验
将U87MG细胞按照1.0×105/孔,铺12孔板,然后孵育过夜。细胞分为2组:向A组的每孔U87MG细胞中加入0.1nmol的Cy5.5-SY1孵育;向B组的每孔U87MG细胞中加入0.1nmol的Cy5.5-SY1和5nmol的阻断剂共同孵育,然后在孵育0.5小时后用PBS洗掉多余的探针及阻断剂后,将两组细胞用多聚甲醛固定,进一步用DAPI染核,最后用倒置荧光显微镜显像。
结果如图4所示:在A组细胞中能够清楚观察到细胞膜上强烈的荧光信号,说明U87MG细胞中在加入Cy5.5-SY1后,探针靶向结合到细胞膜表面。而在加入过量的阻断剂后,只能观察到微弱的荧光信号,U87MG细胞与Cy5.5-SY1的结合量明显降低,进一步说明探针与细胞在细胞表面的结合具有靶向特异性和高亲和力。
实施例7
本实施例提供一种在活体内应用uPAR靶向性探针,对肿瘤进行活体显像检测的方法,具体包括以下内容:
1、荷U87MG肿瘤小鼠的PET成像
利用过表达uPAR的U87MG细胞构建荷鼠皮下移植模型,进行活体显像研究,通过尾静脉注射探针80μCi的Al18F-SY1并在不同的时间点(15min 和60min)采集PET/CT图像,成像结果如图5所示:相对皮肤、骨头、心脏、脑部等主要器官,Al18F-SY1在肿瘤部位明显聚集,说明uPAR靶向性探针能够特异性靶向活体肿瘤,并在肿瘤部位高度聚集,形成高质量的分子显像图像,以用于肿瘤的活体检测。另一方面验证了uPAR靶向性探针在小鼠体内的稳定性高,不易被血浆或组织中的酶降解。Al18F-SY1以少量的注射量即可获得清晰的检测图像,说明uPAR靶向性探针的灵敏度高,且 uPAR靶向性探针未引起小鼠的免疫反应,具有高的生物兼容性。
2、Al18F-SY1活体生物学分布特征
在小鼠内体通过尾静脉注射探针80μCi的Al18F-SY1一个小时后,处死老鼠,取出主要的器官,进行离体主要器官的放射性信号检测。结果如图6 所示:Al18F-SY1主要分布于肝脏,肾脏和肿瘤部位,说明活体小鼠通过肝脏和肾脏代谢体内累积的Al18F-SY1。
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。
SEQUENCE LISTING
<110> 华中师范大学
<120> 一种uPAR靶向性多肽、探针和活体分子显像方法
<130> WXHA201700025
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> PRT
<213> 人工序列(uPAR)
<400> 1
Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Gly Cys
1 5 10
Claims (10)
1.一种uPAR靶向性多肽,其特征在于,所述uPAR靶向性多肽的氨基酸序列如SEQ IDNO.1所示。
2.一种uPAR靶向性探针,其特征在于,所述uPAR靶向性探针包括信号单元和uPAR靶向亲和单元,所述信号单元为影像学检测单元,所述uPAR靶向亲和单元为权利要求1所述的uPAR靶向性多肽。
3.根据权利要求2所述的uPAR靶向性探针,其特征在于,还包括连接所述信号单元和所述uPAR靶向亲和单元的连接单元。
4.根据权利要求3所述的uPAR靶向性探针,其特征在于,所述连接单元选自聚乙二醇、含脂肪链的化合物或螯合剂;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;所述螯合剂选自NOTA、NODA、NODAGA、DTPA、DOTA、DOTAGA、TETA或CB-TE2A。
5.根据权利要求2-4任一项所述的uPAR靶向性探针,其特征在于,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至少一种。
6.根据权利要求5所述的uPAR靶向性探针,其特征在于,所述荧光染料为近红外一区荧光染料或近红外二区荧光染料。
7.权利要求1所述的uPAR靶向性多肽或权利要求2-6任一项所述的uPAR靶向性探针在制备治疗或诊断uPAR异常表达疾病的试剂中的用途。
8.根据权利要求7所述的用途,其特征在于,所述uPAR异常表达的疾病为肿瘤。
9.一种用于肿瘤诊断的试剂盒,其特征在于,所述试剂盒包括权利要求1所述的uPAR靶向性探多肽和/或权利要求2-6任一项所述的uPAR靶向性探针。
10.一种活体分子显像方法,其特征在于,应用权利要求2-6任一项所述的uPAR靶向性探针示踪活体内的uPAR,然后应用活体显像技术对所述活体的待测位置的uPAR进行检测。
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