Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Anal Chem. 2010 Sep 15;82(18):7533-41. doi: 10.1021/ac1003085.
In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis.
在本文中，我们描述了一种筛选针对癌细胞具有特定识别能力的肽的新方法。将 70 个肽以阵列形式合成在纤维素膜上，并开发了一种直接研究肽-全细胞相互作用的方法。使用结合细胞的 CyQUANT 荧光，评估了细胞对不同肽与通过噬菌体展示鉴定的先导 12 肽 p160 的相对结合亲和力。筛选确定了至少五个新肽，与 p160 肽相比，对 MDA-MB-435 和 MCF-7 人癌细胞的亲和力更高（高达 3 倍）。这些肽与对照（非癌性）人脐静脉内皮细胞（HUVEC）的结合非常少。这三个肽分别合成并标记有荧光素异硫氰酸酯（FITC），以使用共焦激光扫描显微镜和流式细胞术研究它们与癌细胞和对照细胞的摄取和相互作用。结果证实了 11 肽 11（RGDPAYQGRFL）和 10 肽 18（WXEAAYQRFL）对癌细胞与 HUVEC 的高特异性亲和力。肽 11 通过其序列包含多个识别基序，与靶癌细胞上的不同受体结合，而肽 18 主要与假定的 p160 受体结合。本文报道的肽阵列-全细胞结合测定法是噬菌体展示的补充方法，用于进一步筛选和优化用于癌症治疗和诊断的癌症靶向肽。
