Mattias Hällbrink

Cell penetrating peptides

The present invention relates to a method for predicting or designing, detecting, and/or verifying a novel cell-penetrating peptide (CPP) and to a method for using said new CPP and/or a novel usage of a known CPP for an improved cellular uptake of a cellular effector, coupled to said CPP. Furthermore, the present invention also relates to a method for predicting or designing, detecting and/or verifying a novel cell-penetrating peptide (CPP) that mimics cellular effector activity and/or inhibits cellular effector activity. The present invention additionally relates to the use of said CPP for treating and/or preventing a medical condition and to the use of said CPP for the manufacture of a pharmaceutical composition for treating a medical condition.

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Gαs and Gαi/Gαo activation

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Gαs proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Gαs but also to the Gαi/Gαo type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.

Deletion analogues of transportan

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Cellular translocation of proteins by transportan

Proteins with molecular masses ranging from 30 kDa (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein–peptide constructs to penetrate into Bowes melanoma, BRL, and COS‐7 cells. After 0.5–3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS‐7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.

Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties

UNLABELLED Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

Cell entry and antimicrobial properties of eukaryotic cell-penetrating peptides

Antimicrobial drug action is limited by both microbial and host cell membranes. Microbes stringently exclude the entry of most drugs, and mammalian membranes limit drug distribution and access to intracellular pathogens. Recently, cell‐penetrating peptides (CPPs) have been developed as carriers to improve mammalian cell uptake. Given that CPPs are cationic and often amphipathic, similar to membrane active antimicrobial peptides, it may be possible to use CPP activity to improve drug delivery to microbes. Here, two CPPs, TP10 and pVEC, were found to enter a range of bacteria and fungi. The uptake route involves rapid surface accumulation within minutes followed by cell entry. TP10 inhibited Candida albicans and Staphylococcus aureus growth, and pVEC inhibited Mycobacterium smegmatis growth at low micromolar doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damage microbes, and this effect was sufficient to clear HeLa cell cultures from noninvasive S. aureus infection. Also, conversion of the cytotoxicity indicator dye SYTOX Green showed that TP10 causes rapid and lethal permeabilization of S. aureus and pVEC permeabilizes M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC can enter both mammalian and microbial cells and preferentially permeabilize and kill microbes.

Molecular Parameters of siRNA–Cell Penetrating Peptide Nanocomplexes for Efficient Cellular Delivery

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery efficiency. However, the molecular basis of this increased activity is poorly understood. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA-CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luciferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP-siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP-siRNA complexes acquired a negative ζ-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle-cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based oligonucleotide transfection agents.

The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake

Although cell‐penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have sug‐gested that there might be a direct penetration of peptides in parallel with different forms of endocyto‐sis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in tar‐get cell apoptosis—perforin, granulysin, and gran‐zymes—share many features common in uptake of cell‐penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma mem‐brane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high‐performance liquid chroma‐tography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell‐penetrating peptide up‐take, thus preventing leakage of endogenous molecules out of the cell.—Palm‐Apergi, C., Lorents, A., Padari, K., Pooga, M., and Hallbrink, M. The membrane repair response masks membrane disturbances caused by cell‐penetrating peptide uptake. FASEB J. 23, 214‐223 (2009)

Passage of cell-penetrating peptides across a human epithelial cell layer in vitro

Cell barriers are essential for the maintenance and regulation of the microenvironments of the human body. Cell-penetrating peptides have simplified the delivery of bioactive cargoes across the plasma membrane. Here, the passage of three cell-penetrating peptides (transportan, the transportan analogue transportan 10, and penetratin) across a Caco-2 human colon cancer cell layer in vitro was investigated. The peptides were internalized into epithelial Caco-2 cells as visualized by indirect fluorescence microscopy and quantified by fluorimetry. Studies of peptide outflow from cells showed that the peptides were in equilibrium across the plasma membrane. The ability of the peptides to cross a Caco-2 cell layer was tested in a two-chambered model system. After 120 min, 7.0%, 2.8% and 0.6% of added transportan, transportan 10 and penetratin respectively was detected in the lower chamber. Both transportan and transportan 10 reversibly decreased the trans-epithelial electrical resistance of the barrier model, with minimum values after 60 min of 46% and 60% of control respectively. Penetratin did not affect the resistance of the cell layer to the same extent. Although transportan markedly increased the passage of ions, the paracellular flux of 4.4 kDa fluorescein-labelled dextran was limited. In conclusion, the results indicate that the transportan peptides pass the epithelial cell layer mainly by a mechanism involving a transcellular pathway.

Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects.

Cell-penetrating peptides (CPPs) are carriers developed to improve mammalian cell uptake of important research tools such as antisense oligonucleotides and short interfering RNAs. However, the data on CPP uptake into non-mammalian cells are limited. We have studied the uptake and antimicrobial effects of the three representative peptides penetratin (derived from a non-mammalian protein), MAP (artificial peptide) and pVEC (derived from a mammalian protein) using fluorescence HPLC in four common model systems: insect cells (Sf9), gram-positive bacteria (Bacillus megaterium), gram-negative bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). We demonstrate that non-mammalian cells internalize CPPs and a comparison of the uptake of the peptides show that the intracellular concentration and degradation of the peptides varies widely among organisms. In addition, these CPPs showed antimicrobial activity.