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Cellular Effects and Delivery Propensity of Penetratin Is Influenced by Conjugation to Parathyroid Hormone Fragment 1‑34 in Synergy with pH
journal contribution
posted on 2017-11-20, 00:00 authored by Mie Kristensen, Line Hagner Nielsen, Kinga Zor, Anja Boisen, Malene Vinther Christensen, Jens Berthelsen, Hanne Mørck NielsenThe
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.
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pH 5CPPCellular EffectsMorphological Caco -2 cell changesDelivery Propensitycell-penetrating peptidecoadministration approachoCelloscope setupTransepithelial permeationCaco -2 cell culture modeltransepithelial deliverycoadministered penetratinPTHCaco -2 cell viabilitylipid membranesviability kineticsimpedance-based xCELLigence systemtransepithelial permeationcalcein release assaycargo peptidesparathyroid hormoneconjugatelatter observationReal-Time-GLO assayconjugation approachCaco -2
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