Institute for Biostructures and Bioimages, CNR, Molecular Biotechnology Center, University of Turin, Via Nizza 52, I-10126 Torino, Italy.
Mol Pharm. 2011 Oct 3;8(5):1750-6. doi: 10.1021/mp2001044. Epub 2011 Aug 5.
Murine melanoma B16 cells display on the extracellular side of the plasma membrane a large number of reactive protein thiols (exofacial protein thiols, EPTs). These EPTs can be chemically labeled with Gd-DO3A-PDP, a Gd(III)-based MRI contrast agent bearing a 2-pyridinedithio chemical function for the recognition of EPTs. Uptake of gadolinium up to 10(9) Gd atoms per cell can be achieved. The treatment of B16 cells ex vivo with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) results in an increase by 850% of available EPTs and an increase by 45% of Gd uptake. Blocking EPTs with N-ethylmaleimide (NEM) caused a decrease by 84% of available EPTs and a decrease by 55% of Gd uptake. The amount of Gd taken up by B16 cells is therefore dependent upon the availability of EPTs, whose actual level in turn changes according to the extracellular redox microenvironment. Then Gd-DO3A-PDP has been assessed for the labeling of tumor cells in vivo on B16.F10 melanoma tumor-bearing mice. Gd-DO3A-PDP (or Gd-DO3A as the control) has been injected directly into the tumor region at a dose level of 0.1 μmol and the signal enhancement in MR images followed over time. The washout kinetics of Gd-DO3A-PDP from tumor is very slow if compared to that of control Gd-DO3A, and 48 h post injection, the gadolinium-enhancement is still clearly visible. Therefore, B16 cells can be labeled ex vivo as well as in vivo according to a common EPTs-dependent route, provided that high levels of the thiol reactive probe can be delivered to the tumor.
鼠黑色素瘤 B16 细胞在质膜的细胞外表面显示大量反应性蛋白巯基（细胞外蛋白巯基，EPTs）。这些 EPT 可以用 Gd-DO3A-PDP 进行化学标记，Gd-DO3A-PDP 是一种基于 Gd(III)的 MRI 对比剂，带有 2-吡啶二硫化学官能团，用于识别 EPTs。可以达到每个细胞高达 10(9)个 Gd 原子的摄取量。用还原剂（如三（2-羧乙基）膦（TCEP））处理 B16 细胞，可使 EPTs 的可用性增加 850%，Gd 摄取量增加 45%。用 N-乙基马来酰亚胺（NEM）阻断 EPTs 可使可用 EPTs 减少 84%，Gd 摄取量减少 55%。因此，B16 细胞摄取的 Gd 量取决于 EPTs 的可用性，而 EPTs 的实际水平又根据细胞外氧化还原微环境而变化。然后，在 B16.F10 黑色素瘤荷瘤小鼠体内评估了 Gd-DO3A-PDP 对肿瘤细胞的标记。以 0.1 μmol 的剂量水平将 Gd-DO3A-PDP（或作为对照的 Gd-DO3A）直接注入肿瘤区域，并随时间跟踪磁共振图像中的信号增强。与对照 Gd-DO3A 相比，Gd-DO3A-PDP 从肿瘤中的洗脱动力学非常缓慢，并且在注射后 48 小时，仍然可以明显看到钆增强。因此，B16 细胞可以根据共同的 EPTs 依赖性途径进行体外和体内标记，只要可以将高浓度的硫醇反应性探针递送到肿瘤。
