Conjugation of Cell-Penetrating Peptides to Parathyroid Hormone Affects Its Structure, Potency, and Transepithelial Permeation

Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1–34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1–34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1–34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1–34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1–34) as compared to native PTH(1–34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1–34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1–34) as compared to co-administration of CPP and PTH(1–34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1–34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1–34) across an intestinal epithelium.