Cellular Effects and Delivery Propensity of Penetratin Is Influenced by Conjugation to Parathyroid Hormone Fragment 1‑34 in Synergy with pH Mie Kristensen Line Hagner Nielsen Kinga Zor Anja Boisen Malene Vinther Christensen Jens Berthelsen Hanne Mørck Nielsen 10.1021/acs.bioconjchem.7b00687.s001 https://acs.figshare.com/articles/journal_contribution/Cellular_Effects_and_Delivery_Propensity_of_Penetratin_Is_Influenced_by_Conjugation_to_Parathyroid_Hormone_Fragment_1_34_in_Synergy_with_pH/5769414 The cell-penetrating peptide (CPP) penetratin has demonstrated potential as a carrier for transepithelial delivery of cargo peptides, such as the therapeutically relevant part of parathyroid hormone, i.e., PTH­(1-34). The purpose of the present study was to elucidate the relevance of pH for PTH­(1-34)–penetratin conjugates and coadministered penetratin with PTH­(1-34) regarding transepithelial permeation of PTH­(1-34) and cellular effects. Transepithelial permeation was assessed using monolayers of the Caco-2 cell culture model, and effects on Caco-2 cellular viability kinetics were evaluated by using the Real-Time-GLO assay as well as by microscopy following Tryphan blue staining. Morphological Caco-2 cell changes were studied exploiting the impedance-based xCELLigence system as well as optically using the oCelloscope setup. Finally, the effect of pH on the folding propensity of the PTH­(1-34)–penetratin conjugate and its ability to disrupt lipid membranes were assessed by circular dichroism (CD) spectroscopy and the calcein release assay, respectively. The transepithelial PTH­(1-34) permeation was not pH-dependent when applying the coadministration approach. However, by applying the conjugation approach, the PTH­(1-34) permeation was significantly enhanced by lowering the pH from 7.4 to 5 but also associated with a compromised barrier and a lowering of the cellular viability. The negative effects on the cellular viability following cellular incubation with the PTH­(1-34)–penetratin conjugate were moreover confirmed during real-time monitoring of the Caco-2 cell viability as well as by enhanced Tryphan blue uptake. In addition, morphological changes were primarily observed for cells incubated with the PTH­(1-34)–penetratin conjugate at pH 5, which was moreover demonstrated to have an enhanced membrane permeating effect following lowering of the pH from 7.4 to 5. The latter observation was, however, not a result of better secondary folding propensity at pH 5 when compared to pH 7.4. 2017-11-20 00:00:00 pH 5 CPP Cellular Effects Morphological Caco -2 cell changes Delivery Propensity cell-penetrating peptide coadministration approach oCelloscope setup Transepithelial permeation Caco -2 cell culture model transepithelial delivery coadministered penetratin PTH Caco -2 cell viability lipid membranes viability kinetics impedance-based xCELLigence system transepithelial permeation calcein release assay cargo peptides parathyroid hormone conjugate latter observation Real-Time-GLO assay conjugation approach Caco -2