Cellular uptake but low permeation of human calcitonin-derived cell penetrating peptides and Tat(47-57) through well-differentiated epithelial models

Purpose: To investigate whether cell penetrating Peptides (CPP) derived from human Calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers.

Methods: Cellular uptake of hCT(9-32) and permeation of six hCT-derived Peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 Cell Culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The Peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC.

Results: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested Peptides and their cargo was lower than that of paracellular markers.

Conclusions: The CPP uptake pattern depends on both the type of peptide and the Cell Culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.