Metabolic cleavage and translocation efficiency of selected cell penetrating peptides: a comparative study with epithelial cell cultures.

We investigated the metabolic stability of four cell penetrating peptides (CPPs), namely SAP, hCT(9-32)-br, [Palpha] and [Pbeta], when in contact with either subconfluent HeLa, confluent MDCK or Calu-3 epithelial cell cultures. Additionally, through analysis of their cellular translocation efficiency, we evaluated possible relations between metabolic stability and translocation efficiency. Metabolic degradation kinetics and resulting metabolites were assessed using RP-HPLC and MALDI-TOF mass spectrometry. Translocation efficiencies were determined using fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Between HeLa, MDCK and Calu-3 we found the levels of proteolytic activities to be highly variable. However, for each peptide, the individual degradation patterns were quite similar. The metabolic stability of the investigated CPPs was in the order of CF-SAP = CF-hCT(9-32)-br > [Pbeta]-IAF > [Palpha] and we identified specific cleavage sites for each of the four peptides. Throughout, we observed higher translocation efficiencies into HeLa cells as compared to MDCK and Calu-3, corresponding to the lower state of differentiation of HeLa cell cultures. No direct relation between metabolic stability and translocation efficiency was found, indicating that metabolic stability in general is not a main limiting factor for efficient cellular translocation. Nevertheless, translocation of individual CPPs may be improved by structural modifications aiming at increased metabolic stability.

On the biomedical promise of cell penetrating peptides: limits versus prospects.

The cell membrane poses a substantial hurdle to the use of pharmacologically active biomacromolecules that are not per se actively translocated into cells. An appealing approach to deliver such molecules involves tethering or complexing them with so-called cell penetrating peptides (CPPs) that are able to cross the plasma membrane of mammalian cells. The CPP approach is currently a major avenue in engineering delivery systems that are hoped to mediate the non-invasive import of problematic cargos into cells. The large number of different cargo molecules that have been efficiently delivered by CPPs ranges from small molecules to proteins and even liposomes and particles. With respect to the involved mechanism(s) there is increasing evidence for endocytosis as a major route of entry. Moreover, in terms of intracellular trafficking, current data argues for the transport to acidic early endosomal compartments with cytosolic release mediated via retrograde delivery through the Golgi apparatus and the endoplasmic reticulum. The focus of this review is to revisit the performance of cell penetrating peptides for drug delivery. To this aim we cover both accomplishments and failures and report on new prospects of the CPP approach. Besides a selection of successful case histories of CPPs we also review the limitations of CPP mediated translocation. In particular, we comment on the impact of (i) metabolic degradation, (ii) the cell line and cellular differentiation state dependent uptake of CPPs, as well as (iii) the regulation of their endocytic traffic by Rho-family GTPases. Further on, we aim at the identification of promising niches for CPP application in drug delivery. In this context, as inspired by current literature, we focus on three principal areas: (i) the delivery of antineoplastic agents, (ii) the delivery of CPPs as antimicrobials, and (iii) the potential of CPPs to target inflammatory tissues.

Differentiation restricted endocytosis of cell penetrating peptides in MDCK cells corresponds with activities of Rho-GTPases.

PURPOSE: Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a gamma-zein related sequence. METHODS: Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-gamma/TNF-alphaa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. RESULTS: We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-gamma/TNF-alphaa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. CONCLUSION: Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery.

One-step preparation of polyelectrolyte-coated PLGA microparticles and their functionalization with model ligands.

This work aimed at the development of a novel surfactant-free, one-step process for the concomitant formation of poly(lactide-co-glycolide) (PLGA) microparticles (MP) and surface coating with the polyelectrolyte chitosan, which is suitable for subsequent covalent conjugation of bioactive ligands. The technology is based on solvent extraction from an O/W-dispersion using a static micromixer. Surface coating occurred through interaction of the negatively charged, nascent PLGA MP with the polycationic chitosan, which was dissolved in the aqueous extraction fluid. Particles of 1-10 mum in diameter were produced with excellent reproducibility. The chitosan-coated PLGA MP were spherical and showed a smooth surface without pores, as demonstrated by scanning electron microscopy (SEM). The chitosan coatings were characterized by zeta potential measurements and X-ray photoelectron spectroscopy (XPS). The functional amino groups of chitosan were used to conjugate two model ligands to the coating, i.e. fluorescamine and NHS-PEG-biotin. The presence of the conjugated ligands was revealed by confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). Evidence for biotinylation was demonstrated through binding of fluorescently labelled streptavidin. The developed platform technology is straightforward and flexible. Future studies will focus on the design of microparticulate carriers with bioactive surfaces, e.g. as antigen delivery systems.

Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape.

Cellular entry of peptide, protein, and nucleic acid biopharmaceuticals is severely impeded by the cell membrane. Linkage or assembly of such agents and cell-penetrating peptides (CPP) with the ability to cross cellular membranes has opened a new horizon in biomedical research. Nevertheless, the uptake mechanisms of most CPP have been controversially discussed and are poorly understood. We present data on two recently developed oligocationic CPP, the sweet arrow peptide SAP, a gamma-zein-related sequence, and a branched human calcitonin derived peptide, hCT(9-32)-br, carrying a simian virus derived nuclear localization sequence in the side chain. Uptake in HeLa cells and intracellular trafficking of N-terminally carboxyfluorescein labeled peptides was studied by confocal laser scanning microscopy and flow cytometry using biochemical markers in combination with quenching and colocalization approaches. Both peptides were readily internalized by HeLa cells through interaction with the extracellular matrix followed by lipid raft-mediated endocytosis as confirmed by reduced uptake at lower temperature, in the presence of endocytosis inhibitors and through cholesterol depletion by methyl-beta-cyclodextrin, supported by colocalization with markers for clathrin-independent pathways. In contrast to the oligocationic SAP and hCT(9-32)-br, interaction with the extracellular matrix, however, was no prerequisite for the observed lipid raft-mediated uptake of the weakly cationic, unbranched hCT(9-32). Transient involvement of endosomes in intracellular trafficking of SAP and hCT(9-32)-br prior to endosomal escape of both peptides was revealed by colocalization and pulse-chase studies of the peptides with the early endosome antigen 1. The results bear potential for CPP as tools for intracellular drug delivery.