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新型血管内皮生长因子受体Neuropilin-1(NRP-1)和整合素ανβ3在脑胶质瘤中异常表达,且其表达的水平与肿瘤恶性程度呈正相关,表明二者与肿瘤的恶性生物学行为有密切关系[1-2]。目前研究已分别证实含精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)和丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸(Ala-Thr-Trp-Leu-Pro-Pro-Arg,ATWLPPR)的多肽分别能够特异性识别整合素αvβ3和NRP-1[2-3]。然而,分别单独使用RGD和ATWLPPR作为显像剂或治疗探针时存在多种问题:(1)采用单体肽作为显像剂,要求肿瘤表面的受体浓度相对正常组织器官的受体浓度达到一定水平,这种情况不会发生在所有肿瘤的生长、浸润和转移等过程中;(2)单体肽与肿瘤受体靶点结合的亲和力相对较弱,会导致肿瘤摄取不高及药物快速从标靶部位脱离;(3)某些单体肽在体内的药代动力学特性可能不理想。
我们在已有的对RGD多肽研究的基础上,针对脑胶质瘤高表达的整合素αvβ3和NRP-1受体,创造性地将环状多肽c(RGDyK)与ATWLPPR相结合,制备一种新型的双靶点融合肽探针c(RGDyK)-ATWLPPR,并对其进行18F标记,希望该融合肽在肿瘤细胞表面能同时与双靶点结合,提高多肽的肿瘤摄取以及受体-配体亲和力,以构建一种更为高效的脑胶质瘤诊断探针。我们通过一系列的体内外生物学实验,评价探针的细胞结合率、受体亲和力及肿瘤PET显像等方面的性质。同时,比较双靶点融合肽分子探针与对应单体肽分子探针在肿瘤早期诊断方面的区别和特点。
新型αvβ3和Neuropilin-1双靶点正电子成像探针18F-FAl-NOTA-RGD-ATWLPPR用于脑胶质瘤的PET显像研究
Imaging of glioma with an integrin αvβ3 and neuropilin-1 dual-targeted PET probe 18F-FAl-NOTA-RGD-ATWLPPR
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摘要:
目的构建可靶向αvβ3和血管内皮生长因子受体Neuropilin-1(NRP-1)双受体的正电子成像探针,并验证双靶点融合肽探针较之单靶点探针的优越性。 方法采用18F-氟化铝(18F-FAl)配合物的方法实现分子探针的18F标记。在人神经胶质瘤U87MG细胞中,检测αvβ3和NRP-1的表达水平,测定分子探针精氨酸-甘氨酸-天冬氨酸(RGD)-丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸(ATWLPPR)与αvβ3/NRP-1的受体-配体亲和力。在U87MG荷瘤裸鼠模型中,测定18F标记RGD-ATWLPPR的体内肿瘤micro-PET显像特性,并且与其对应单体进行比较分析。采用方差分析和t检验对结果进行统计学分析。 结果αvβ3及NRP-1在U87MG肿瘤细胞、肿瘤组织及肿瘤新生血管中均有较高水平的表达。受体-配体亲和力测定的实验结果显示,18F-FAl-NOTA-RGD-ATWLPPR双靶点融合肽与αvβ3及NRP-1的亲和力并未明显优于其单体,但融合肽在U87MG细胞中的摄取高于相应的单体肽。Micro-PET显像结果显示,融合肽较其单体肽RGD[(4.86±0.48)% ID/g vs.(3.33±0.15)% ID/g,t=10.21,P < 0.05]和ATWLPPR[(4.86±0.48)% ID/g vs.(2.28±0.41)% ID/g,t=32.16,P < 0.05]表现出了更好的显像效果,且融合肽在αvβ3、NRP-1任一受体被未标记“冷”肽阻断的情况下仍能获得肿瘤的阳性显像结果。 结论18F-FAl-NOTA-RGD-ATWLPPR可以灵敏地对整合素αvβ3和NRP-1中任何一个受体高表达的肿瘤进行显像,并且较其单体具有更高的肿瘤摄取,但该融合肽的受体-配体亲和力还有待进一步提高。 -
关键词:
- 整合素αvβ3 /
- 神经纤毛蛋白质1 /
- 正电子发射断层显像术 /
- 分子探针
Abstract:ObjectiveArg-Gly-Asp (RGD) or Ala-Thr-Trp-Leu-Pro-Pro-Arg (ATWLPPR) peptide binds specifically to integrin αvβ3 or neuropilin-1(NRP-1) receptor, respectively.In this study, a novel heterodimer peptide probe containing both RGD and ATWLPPR was designed in one molecule.The in vitro and in vivo biological behavior of the dual-targeted imaging probe RGD-ATWLPPR was compared with its corresponding counterparts. Methods18F labeling was conducted through 18F-FAl approach.In the integrin αvβ3-positive U87MG human glioma cell line, the αvβ3/NRP-1 receptor binding affinity of RGD-ATWLPPR was tested and compared with its counterparts RGD and ATWLPPR.The tumor uptake and distribution pattern of 18F-labeled RGD-ATWLPPR through PET imaging was evaluated and compared with those of RGD and ATWLPPR.The means were compared using one-way ANOVA and t test. ResultsBoth integrin αvβ3 and NRP-1 showed high expression in U87MG glioma cells and tumor tissues.RGD-ATWLPPR exhibited similar in vitro receptor binding affinity to those of RGD and ATWLPPR.Based on the PET imaging study, 18F-labeled RGD-ATWLPPR (denoted as 18F-FAl-NOTA-RGD-ATWLPPR) demonstrated significantly higher tumor uptake than RGD[(4.86±0.48)% ID/g vs.(3.33±0.15)% ID/g, t=10.21, P < 0.05]and ATWLPPR[(4.86±0.48)% ID/g vs.(2.28±0.41)% ID/g, t=32.16, P < 0.05].In the blocking study, 18F-FAl-NOTA-RGD-ATWLPPR showed positive imaging result in the presence of excess unlabeled RGD or ATWLPPR.The tumor uptake decreased to the background level when unlabeled RGD and ATWLPPR were co-injected before administration of 18F-FAl-NOTA-RGD-ATWLPPR. ConclusionsThe dual-targeted PET probe 18F-FAl-NOTA-RGD-ATWLPPR specifically binds to either integrin αvβ3 or NRP-1 receptor and could be a promising PET imaging agent for NRP-1-/αvβ3+and NRP-1+/αvβ3-tumors.The receptor-binding affinity of RGD-ATWLPPR must be further improved. -
Key words:
- Integrin αvβ3 /
- Neuropilin-1 /
- Positron-emission tomography /
- Molecular probes
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图 2 人神经胶质瘤U87MG细胞和组织中整合素αvβ3及NRP-1受体的免疫荧光染色结果图中,A、B分别为整合素αvβ3、NRP-1受体在U87MG细胞中的免疫荧光染色结果(×200),A呈强阳性,B呈阳性;C、D分别为整合素αvβ3、NRP-1受体在U87MG肿瘤组织切片中的免疫组化染色结果(×10);E、F分别为整合素αvβ3、NRP-1受体在肿瘤新生血管的免疫组化染色结果(×20),白色箭头所示处为肿瘤血管。整合素αvβ3及NRP-1受体在人神经胶质瘤U87MG细胞及组织中均呈高表达。NRP-1:neuropilin-1。
Figure 2. Immunofluorescent staining for integrin αvβ3 and neuropilin-1 in U87MG cells, U87MG glioma tissues and tumor vasculature
图 3 NOTA-c(RGDyK)和NOTA-RGD-ATWLPPR竞争结合U87MG细胞表面整合素αvβ3和NRP-1受体结果图中,A:NOTA-c(RGDyK)和NOTA-RGD-ATWLPPR与125I-c(RGDyK)竞争结合U87MG细胞表面整合素αvβ3受体结果,log[M]中M为NOTA-c(RGDyK)或NOTA-RGD-ATWLPPR的浓度;B:NOTA-ATWLPPR和NOTA-RGD-ATWLPPR与125I-ATWLPPR竞争结合U87MG细胞表面NRP-1受体结果,log[M]中M为NOTA-ATWLPPR或NOTA-RGD-ATWLPPR的浓度;NOTA:1,4,7-三氮杂环壬烷-1,4,7-三乙酸;RGD:精氨酸-甘氨酸-天冬氨酸;ATWLPPR:丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸;NRP-1:neuropilin-1。
Figure 3. Inhibition of 125I-c(RGDyK) binding to integrin αvβ3 in U87MG cells by NOTA-c(RGDyK) and NOTA-RGD-ATWLPPR (A). Inhibition of 125I-ATWLPPR binding to NRP-1 in U87MG cells by NOTA-ATWLPPR and NOTA-RGD-ATWLPPR(B).
图 4 U87MG肿瘤细胞中18F-FAl-NOTA-c(RGDyK)、18F-FAl-NOTA-ATWLPPR以及18F-FAl-NOTA-RGD-ATWLPPR在不同时间点的细胞摄取结果图中,FAl:氟化铝;NOTA:1,4,7-三氮杂环壬烷-1,4,7-三乙酸;RGD:精氨酸-甘氨酸-天冬氨酸;ATWLPPR:丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸。
Figure 4. Uptake of 18F-FAl-NOTA-c(RGDyK), 18F-FAl-NOTA-ATWLPPR, and 18F-FAl-NOTA-RGD-ATWLPPR in U87MG cells at different times
图 5 荷人神经胶质瘤U87MG裸鼠行18F-FAl-NOTA-ATWLPPR、18F-FAl-NOTA-c(RGDyK)或18F-FAl-NOTA-RGD-ATWLPPR的micro-PET显像图及肿瘤摄取值比较图中,A:micro-PET显像图,箭头所指处为肿瘤所在;B:18F-FAl-NOTA-RGD-ATWLPPR、18F-FAl-NOTA-c(RGDyK)以及18F-FAl-NOTA-ATWLPPR 3种示踪剂的肿瘤摄取值比较(用%ID/g表示);FAl:氟化铝;NOTA:1,4,7-三氮杂环壬烷-1,4,7-三乙酸;RGD:精氨酸-甘氨酸-天冬氨酸;ATWLPPR:丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸。
Figure 5. Representative micro-PET images and tumor uptakes of nude mice bearing subcutaneous U87MG glioma xenografts injected with 18F-FAl-NOTA-ATWLPPR, 18F-FAl-NOTA-c(RGDyK) and 18F-FAl-NOTA-RGD-ATWLPPR
图 6 用超量非标记的NOTA-ATWLPPR、NOTA-c(RGDyK)及NOTA-RGD-ATWLPPR阻断后,荷人神经胶质瘤U87MG裸鼠的18F-FAl-NOTA-RGD-ATWLPPR micro-PET显像图及肿瘤摄取值比较图中,A:micro-PET显像图,箭头所指处为肿瘤所在;B:18F-FAl-NOTA-RGD-ATWLPPR在U87MG肿瘤模型中对照组、NOTA-ATWLPPR阻断组、NOTA-c(RGDyK)阻断组和NOTA-RGD-ATWLPPR阻断组的肿瘤摄取值比较(以%ID/g的形式表示);FAl:氟化铝;NOTA:1,4,7-三氮杂环壬烷-1,4,7-三乙酸;RGD:精氨酸-甘氨酸-天冬氨酸;ATWLPPR:丙氨酸-苏氨酸-色氨酸-亮氨酸-脯氨酸-脯氨酸-精氨酸。
Figure 6. Representative micro-PET images and tumor uptake of nude mice bearing subcutaneous U87MG glioma xenografts injected with 18F-FAl-NOTA-RGD-ATWLPPR after a blocking dose injection of NOTA-ATWLPPR, NOTA-c(RGDyK), and NOTA-RGD-ATWLPPR
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