In vitro uptake and stability study of pVEC and its all-D analog

A key step in the development of new hydrophilic pharmaceuticals is to get them through biological barriers. Cellpenetrating peptides, CPPs, have been shown previously to enter cells both in vitro and in vivo by a nonendocytotic mechanism and to be able to carry large cargo molecules with them. Recently, we showed that a small peptide, pVEC, from murine vascular endothelial cadherin, has the characteristics to be classified as a protein derived CPP. Here we have further investigated pVEC together with its allD analog for cellular uptake, intra and extracellular stability, and their enzymatic degradation. The two peptides, pVEC and allD pVEC, translocate into aortic endothelial cells and murine fibroblasts by a nonendocytotic mechanism. In phosphate buffer, pVEC remains intact while the Cterminal lysine is quickly removed in human serum and serumcontaining media. Both pVEC and pVEC without the Cterminal Lys were detected by mass spectrometry inside the two cell types tested. The pVEC halflife is 10.5 min in phosphate buffer containing 10 units of trypsin and 44.6 min in phosphate buffer containing 4.2 units of carboxypeptidase A and 18 units of carboxypeptidase B. In contrast to pVEC, the allD analog remains intact in serum and resists enzymatic degradation.