Metabolic Cleavage and Translocation Efficiency of Selected Cell Penetrating Peptides: A Comparative Study with Epithelial Cell Cultures

• Published in: The AAPS journal Vol. 10; no. 2; pp. 349 - 359
• Main Authors: Foerg, Christina, Weller, Kathrin M., Rechsteiner, Helene, Nielsen, Hanne M., Fernández-Carneado, Jimena, Brunisholz, René, Giralt, Ernest, Merkle, Hans P.
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.06.2008
• Subjects: Amino Acid Sequence; Biochemistry; Biological Transport; Biomedical and Life Sciences; Biomedicine; Biotechnology; Cell Line; Cell Membrane Permeability; Culture Media, Serum-Free; Drug Stability; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Flow Cytometry; Humans; Microscopy, Confocal; Molecular Sequence Data; Peptide Fragments - administration & dosage; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Pharmacology/Toxicology; Pharmacy; Research Article; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; cellular translocation; cell penetrating peptides; metabolism kinetics; epithelial cell cultures; metabolic cleavage
• ISSN: 1550-7416; 1550-7416
• DOI: 10.1208/s12248-008-9029-4

We investigated the metabolic stability of four cell penetrating peptides (CPPs), namely SAP, hCT(9-32)-br, [Pα] and [Pβ], when in contact with either subconfluent HeLa, confluent MDCK or Calu-3 epithelial cell cultures. Additionally, through analysis of their cellular translocation efficiency, we evaluated possible relations between metabolic stability and translocation efficiency. Metabolic degradation kinetics and resulting metabolites were assessed using RP-HPLC and MALDI-TOF mass spectrometry. Translocation efficiencies were determined using fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Between HeLa, MDCK and Calu-3 we found the levels of proteolytic activities to be highly variable. However, for each peptide, the individual degradation patterns were quite similar. The metabolic stability of the investigated CPPs was in the order of CF-SAP = CF-hCT(9-32)-br > [Pβ]−IAF > [Pα] and we identified specific cleavage sites for each of the four peptides. Throughout, we observed higher translocation efficiencies into HeLa cells as compared to MDCK and Calu-3, corresponding to the lower state of differentiation of HeLa cell cultures. No direct relation between metabolic stability and translocation efficiency was found, indicating that metabolic stability in general is not a main limiting factor for efficient cellular translocation. Nevertheless, translocation of individual CPPs may be improved by structural modifications aiming at increased metabolic stability.