Abstract by Ragna Guldsmed Diedrichsen

Introduction and hypotheses
Oral delivery of therapeutic peptides, such as insulin, is challenged by biological barriers, resulting in poor permeation across the intestinal epithelium. Transepithelial insulin permeation can be enhanced by cell-penetrating peptides, such as penetratin and its analogues shuffle and penetramax, when applied as carriers. Such peptide-mediated insulin delivery is hypothesized to depend on their interactions with both insulin and the epithelium. Further, their amino acid sequence as well as stereochemistry and branching are hypothesized to be important for their delivery efficiency and mechanism of action.

Methods
Three linear carrier peptides, penetratin, shuffle, and penetramax, as wells as their all-D analogues and some dimeric and trimeric branched analogues were studied with insulin used as a relevant therapeutic peptide. Peptide-insulin complexation as well as peptide-liposome interactions were investigated using orthogonal biophysical methods, where liposomes were used as mimics of the cell membrane. Delivery efficiencies were evaluated by studies on the enzymatic stability of both insulin and the peptides as well as on the transepithelial insulin permeation across Caco-2 cell monolayers with evaluation of peptide-induced effects on the epithelium.

Results
Shuffle and penetramax, but not penetratin, interacted with insulin. Their interactions with insulin were, however, not dependent on the peptide stereochemistry. As expected, all three of the all-D peptides were more stable than the all-L counterparts, yet the permeation of insulin was enhanced differently by the different isomers (D-form > L-form of penetratin and penetramax, but not of shuffle). The enhancement was associated with an immediate reduction in epithelial integrity, where the effect induced by the L-forms of shuffle and penetramax, but not that induced by the D-peptides, was reversible during the Caco-2 cell monolayer exposure to the peptides. The carrier peptide-mediated insulin permeation was also dependent on peptide branching (branched trimeric > branched dimeric > linear penetratin and penetramax), which was associated with alterations in the cytoskeleton morphology. All three peptides induced liposome clustering with the effect being reversible at higher concentrations of shuffle and penetramax, but not of penetratin. The peptides appeared to adsorb onto the liposomes and to deform the liposome shape.

Conclusion
Exploring both stereochemistry and branching of peptide excipients appear to be promising strategies to optimize their efficiency in oral therapeutic peptide delivery. Mechanistic explorations support such optimization, as the present work demonstrates the importance of electrostatic interactions with the cell membrane.