Kärt Padari

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)

Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides

Cell‐penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class‐A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14‐oligonucleotide nanocomplexes in the HeLa pLuc705 splice‐correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to < 50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14‐oligonucleotide nanocomplexes and suggest for the first time that some CPP‐based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.—Ezzat, K., Helmfors, H., Tudoran, O., Juks, C., Lindberg, S., Padari, K., El‐Andaloussi, S., Pooga, M., Langel, Ü. Scavenger receptor‐mediated uptake of cell‐penetrating peptide nanocomplexes with oligonucleotides. FASEB J. 26, 1172‐1180 (2012). www.fasebj.org

Direct Translocation as Major Cellular Uptake for CADY Self-Assembling Peptide-Based Nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.

New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids

Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160nm. Such nanoparticles have a negative surface charge (-11 to -18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine™ 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

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.

PepFect14 Peptide Vector for Efficient Gene Delivery in Cell Cultures

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.

Differential Endosomal Pathways for Radically Modified Peptide Vectors

In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect HeLa cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.

Liquid-phase synthesis of a pegylated adenosine–oligoarginine conjugate, cell-permeable inhibitor of cAMP-dependent protein kinase

An adenosine-oligoarginine conjugate (ARC) was assembled in a stepwise manner on a poly(ethylene glycol) carrier. The pegylated conjugate inhibited cAMP-dependent protein kinase with IC(50)=460 nM and the cellular uptake of its BODIPY FL derivative was demonstrated and compared to that of free ARC with fluorescence microscopy.

Identification of the ability of highly charged nanomolar inhibitors of protein kinases to cross plasma membranes and carry a protein into cells

A fluorescently labeled adenosine-oligoarginine conjugate (ARC), nanomolar bisubstrate analogue-type inhibitor of basophilic protein kinases PKA and PKC, readily enters cells of different origin and localizes into cytoplasm and nucleus. Moreover, the biotinylated derivative of ARC is able to deliver avidin, a non-covalently attached protein cargo, into cells.

Insights into cell entry and intracellular trafficking of peptide and protein drugs provided by electron microscopy.

For widening the arsenal of protein and peptide therapeutics that act within cells, their cell-entry mechanisms, intracellular trafficking and distribution need to be characterized in detail. Immunofluorescence microscopy has been a prevalent tool for these studies. However, due to the limited resolution, it is often complemented with other methods. This article focuses on the perspectives of electron microscopy in tracking the intracellular delivery and trafficking of proteins, peptides and their carriers. This review introduces the electron microscopy techniques and labeling methods currently used for studying the cellular whereabouts of peptides and proteins with a focus on their intracellular trafficking. Since cell-penetrating peptides have widely been harnessed as carriers for proteins and peptides, and their usage is rapidly expanding, a particular emphasis has been placed on their applications and cell-entry mechanisms.