Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial activity towards both Gram-positive and Gram-negative bacteria as well as eukaryotic cell viability. All examined analogs retained the ability to cross eukaryotic membranes giving rise to a distribution within the vacuolar apparatus. Interestingly, a series of shuffled analogs of penetratin with the cationic residues in conserved positions, attain the same α-helical conformation as native penetratin in the presence of cholesterol-containing liposomes, while conformational differences were observed in the presence of highly anionic liposomes. While the antibacterial effect of the two groups of peptides was similar, the eukaryotic cellular uptake of the shuffled analogs was noticeably lower than for native penetratin. Moreover, a point substitution of Met to Leu in native penetratin had no influence on eukaryotic cellular uptake and antimicrobial effect, and only a minor effect on cytotoxicity, in contrast to the fact that the same substitution in the shuffled analog gave rise to reduced eukaryotic cellular uptake while increasing the antibacterial effect and cytotoxicity.

ABSTRACT. Penetratin is a widely used carrier peptide showing promising potential for mucosal delivery of therapeutic proteins. In the present study, the importance of specific penetratin residues and pH was investigated with respect to complexation with insulin and subsequent transepithelial insulin permeation. Besides penetratin, three analogues were studied. The carrier peptide-insulin complexes were characterized in terms of size and morphology at pH 5, 6.5, and 7.4 by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. At pH 7.4 mainly very large complexes were present, while much smaller complexes dominated at pH 5. Presence of arginine residues in the carrier peptide proved to be a prerequisite for complexation with insulin as well as for enhanced transepithelial insulin permeation in vitro. Rearrangement of tryptophan residues resulted in significantly increased insulin permeation as compared to that of the parent penetratin. In general, precomplexation with penetratin and its analogues at pH 5 gave rise to increased insulin permeation as compared to that observed at pH 7.4; this finding was further supported by a preliminary in vivo study using the parent penetratin.

PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development.

The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating peptide, penetratin, were synthesized, one with selenomethionine (SeMet) added at the N-terminal of the peptide (N-PenM(Se)) and the other with the internal methionine (Met) replaced with SeMet (i-PenM(Se)). The purity of the synthesized peptides was 92% for N-PenM(Se) and 89% for i-PenM(Se) as determined by liquid chromatography (LC)-ICP-MS. The selenium-labelled peptides were investigated by cell uptake studies in HeLa WT cells. The stability of the peptides was monitored in water, cell medium and during cell uptake studies. Total uptake of selenium was quantified by flow injection (FI)-ICP-MS. Speciation analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation was observed during cell uptake studies. The major degradation products were determined by LC-electrospray ionization mass spectrometry (ESI-MS). The labelling method in combination with FI-ICP-MS, LC-ICP-MS and LC-ESI-MS techniques provided detailed information on the fate of penetratin in cellular uptake studies. Most pharmaceutical peptides, including penetratin, are synthetic analogues of endogenous peptides, and incorporation of selenium may improve the critical assessment of the native drug or drug delivery candidate early in the drug development process.

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