Mats Hansen

Distinct uptake routes of cell-penetrating peptide conjugates.

Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.

Penetration without cells: Membrane translocation of cell-penetrating peptides in the model giant plasma membrane vesicles

The cellular internalization of cell-penetrating peptides (CPPs) is proposed to take place by both endocytic processes and by a direct translocation across the plasma membrane. So far only scarce data is available about what determines the choice between the two uptake routes, or the proportion of used pathways when both are active simultaneously. Furthermore, the mechanism(s) of membrane penetration by peptides is itself still a matter of debate. We have introduced the giant plasma membrane vesicles (GPMVs) to study the interaction of six well-described CPPs (fluorescently labeled nona-arginine, Tat peptide, Penetratin, MAP, Transportan and TP10) in a model system of native plasma membrane without the interference of endocytic processes. The membranes of GPMVs are shown to segregate into liquid-ordered and liquid-disordered phases at low temperatures and we demonstrate here by confocal microscopy that amphipathic CPPs preferentially associate with liquid-disordered membrane areas. Moreover, all tested CPPs accumulate into the lumen of GPMVs both at ambient and low temperature. The uncharged control peptide and dextran, in contrary, do not translocate from the medium into the lumen of vesicles. The absence of energy-dependent cellular processes and the impermeability to hydrophilic macromolecules makes the GPMVs a useful model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis.

CPP-protein constructs induce a population of non-acidic vesicles during trafficking through endo-lysosomal pathway

The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters - cell-penetrating peptides (CPPs) - are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP-cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs - transportan, oligoarginine and Tat - complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.

Protein delivery with transportans is mediated by caveolae rather than flotillin-dependent pathways

Delivery of large bioactive cargoes into cells with the help of cell-penetrating peptides (CPPs) is mostly based on endocytic processes. Here we map the cellular pathways used by transportan and transportan 10 (TP10) for protein transduction in HeLa cells. CPP-mediated cellular delivery is often suggested to be lipid-raft-dependent; therefore, we used flotillin-1, caveolin, Rab5, and PI3P as markers to elucidate the involvement of these particular endosomal pathways in the protein uptake process. Confocal laser scanning and electron microscopy reveal only a negligible overlap of avidin/neutravidin conveyed into cells by transportans with the raft marker flotillin-1 or early endosomal markers Rab5 and PI3P. However, about 20% of protein-CPP complexes colocalize with the caveolar/caveosomal marker caveolin, and down-regulation of caveolin-1 by siRNA treatment leads to the inhibition of the CPP-mediated protein uptake by 30-50%. On the contrary, the lack of flotillin-1 increases rather than decreases the CPP-mediated protein transport. The participation of the caveolin-1-dependent pathway in CPP-mediated protein delivery was also corroborated by using caveolin-1 knockout mouse embryonic fibroblasts.

Uptake of cell-penetrating peptides in yeasts

The uptake of different cell‐penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC‐analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)3K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.

Cellular Uptake of Cell-Penetrating Peptides

Cellular machinery is protected from the surrounding by two-layer lipid membrane that is impermeable for most substances unnecessary for cellular metabolism. Unfortunately, from a cellular point of view, most new generation drugs, designed to act on gene regulation and transcription, are also considered to be unnecessary for metabolism and therefore showing poor, if any, intracellular localization. To overcome this obstacle, several chemical and physical methods have been developed, improving the uptake, but, on the other hand, also showing some unwanted side effects or limitations for in vivo applications. This dictates the continuing need for improved drug delivery and one way seems to be the relatively new class of compounds – cell-penetrating peptides (CPPs). Discovered approximately a decade ago, the content of this class is growing rapidly, containing now more than 100 compounds, which shows the intensity of work in this field. CPPs have already been shown to translocate cellular membranes in an unknown, seemingly receptor-independent and non-endocytotic manner. Moreover, they are able to deliver cargoes exceeding their own size up to 100-fold into a cellular milieu both in vitro and in vivo. The variety of different cargoes includes, but is not limited to: DNA, antisense PNA, oligonucleotides and small proteins. Recent data argues though that endocytosis is involved and contributes in some cases to the main part of the translocation. This review summarizes data on mechanisms of cell-penetrating peptides.

Distribution of CPP-protein complexes in freshly resected human tissue material

Interest in cell-penetrating peptides (CPPs) as delivery agents has fuelled a large number of studies conducted on cultured cells and in mice. However, only a few studies have been devoted to the behaviour of CPPs in human tissues. Therefore, we performed ex vivo tissue-dipping experiments where we studied the distribution of CPP-protein complexes in samples of freshly harvested human tissue material. We used the carcinoma or hyperplasia-containing specimens of the uterus and the cervix, obtained as surgical waste from nine hysterectomies. Our aim was to evaluate the tissue of preference (epithelial versus muscular/connective tissue, carcinoma versus adjacent histologically normal tissue) for two well-studied CPPs, the transportan and the TAT-peptide. We complexed biotinylated CPPs with avidin-β-galactosidase (ABG), which enabled us to apply whole-mount X-gal staining as a robust detection method. Our results demonstrate that both peptides enhanced the tissue distribution of ABG. The enhancing effect of the tested CPPs was more obvious in the normal tissue and in some specimens we detected a striking selectivity of CPP-ABG complexes for the normal tissue. This unexpected finding encourages the evaluation of CPPs as local delivery agents in non-malignant situations, for example in the intrauterine gene therapy of benign gynaecological diseases.