Metabolic cleavage and translocation efficiency of selected cell penetrating peptides: a comparative study with epithelial cell cultures

Abstract

We investigated the metabolic stability of four cell penetrating peptides (CPPs), namely SAP, hCT(9-32)-br, [Palpha] and [Pbeta], 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 > [Pbeta]-IAF > [Palpha] 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.

Fig. 1

Quenching effect through the addition of Trypan blue. MDCK cells were incubated with [Pβ]-IAF in the absence or in the presence of Trypan blue and analyzed by FACS (a) and CLSM in the absence (b) or in the presence (c) of Trypan blue. Cell nuclei (in blue) were stained with Hoechst 33342. CPPs are displayed in green. Bar = 50 μm

Fig. 2

Translocation of CPPs in epithelial cell models. Each of the cell models, HeLa (a, d, g, j), MDCK (b, e, h, k) and Calu-3 (c, f, i, l), was incubated for 2 h with the investigated CPPs: CF-SAP (a, b, c), CF-hCT(9-32)-br (d, e, f), [Pα]-IAF (g, h, i) or [Pβ]-IAF (j, k, l) and cells were observed by CLSM. Cell nuclei (displayed in blue) were stained with Hoechst 33342. Bar = 50 μm. Translocation of CF-SAP (a) and CF-hCT(9-32)-br (d) in HeLa was previously reported (23) but added for completeness

Fig. 3

Quantification of translocated CPPs in cell models. Each cell model, HeLa, MDCK and Calu-3, was incubated for 2 h with the investigated CPPs: CF-SAP, CF-hCT(9-32)-br, [Pα]-IAF or [Pβ]-IAF and analyzed by FACS analysis. Mean cell fluorescence ± SD (n = 3)

Fig. 4

Typical MALDI-TOF MS and RP-HPLC profiles of metabolites upon metabolic degradation of [Pβ]-IAF in contact with Calu-3 layers. After 1 min, [Pβ]-IAF was practically fully intact and represented by the main peak corresponding to the original CPP in the RP-HPLC diagram as well as in the MALDI-TOF MS spectrum. After 2 h of incubation with Calu-3 cells, the increase in number of metabolite peaks as obtained by RP-HPLC was consistent with that in the MALDI-TOF MS spectra

Fig. 5

Identical metabolite patterns of [Pβ]-IAF in all three cell models. MALDI-TOF MS spectra of [Pβ]-IAF after incubation with HeLa, MDCK or Calu-3 cell models, respectively, demonstrate identical metabolite patterns as shown by selected MALDI spectra within the first hour after incubation

Fig. 6

Scheme of suggested metabolic cleavage sites of CPPs upon incubation with the three cell models. Individually shaded arrowheads correspond to the corresponding cleavage sites between the respective amino acids. The resulting metabolites are indicated through equally shaded bars as identified by MALDI-TOF MS