Annely Lorents

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)

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.

Cell-penetrating Peptides Split into Two Groups Based on Modulation of Intracellular Calcium Concentration

Background: Uptake of various cell-penetrating peptides (CPPs) can be toxic to cells. Results: Amphipathic CPPs disorder the plasma membrane inducing the influx of calcium ions that in turn can activate recovery mechanisms. Conclusion: Influx of calcium ions and subsequent toxicity induced by the uptake of CPPs can be averted efficiently. Significance: Membrane-active CPPs can be exploited as efficient transport vectors. Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca2+ levels in Jurkat and HeLa cells. The elevated Ca2+ concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca2+ and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca2+ if its level is not drastically elevated.

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

Insight into Cell-Entry Mechanisms of CPPs by Electron Microscopy

Despite the quickly widening application of cell-penetrating peptides (CPP) for the cellular delivery of various macromolecules, the cell entry mechanisms of these peptides have remained elusive so far. The basic features of the translocation of CPPs into cells have been mapped by fluorescence microscopy and activity-based assays revealing that endocytotic mechanisms are mainly responsible for the uptake at physiological temperature. However, the high concentration of CPP or the lowering of the incubation temperature below 10°C (re)activates a nonvesicular cell entry mode. The fluorescence microscopy can hardly provide detailed information about the interaction of CPP molecules with the extracellular structures, the induced changes in the morphology of the plasma membrane, etc. Therefore, application of electron microscopy could help to shed light on the nature of nonvesicular uptake mechanism. Transmission electron microscopy (TEM) has been a valuable tool for the morphological characterization of biological material at high resolution. It can provide useful information at the ultrastructural level about the interaction and arrangement of CPPs on the cell surface, the entrapment in cellular organelles and the translocation to the cytoplasm. In this chapter, we present a method for the tagging of CPPs covalently with a 1.4 nm gold cluster and provide a flat-embedding protocol for the mapping of Nanogold™-labeled CPPs in cultured cells by TEM. This method enables to retain the cell monolayers in their in situ orientation. The Nanogold™ tag is putatively not interfering with the uptake of CPPs and enables the production of specimens with excellent morphology and good contrast.

Cell-penetrating peptides recruit type A scavenger receptors to the plasma membrane for cellular delivery of nucleic acids.

Scavenger receptors (SRs) are a large family of multifunctional receptors that are involved in a range of physiologic and pathologic processes. The ability of class A scavenger receptors (SR‐As) to bind anionic ligands facilitates the internalization of negatively charged cell‐penetrating peptide (CPP)‐nucleic acid nanocomplexes and thus makes them attractive targets for delivery of various nucleic acids. Recently, we demonstrated that SR‐A3 and SR‐A5 are recruited from intracellular membranes to the plasma membrane after incubation with PepFect 14‐splice‐switching oligonucleotide complexes. Here, we examined the mechanisms responsible for translocation of SR‐As to the cell surface. We demonstrate that, in addition to nanocomplexes, some amphipathic CPPs are able to induce externalization of SR‐A3 and SR‐A5, and this process requires the presence of calcium ions. Furthermore, translocation of SR‐A3 and SR‐A5 requires activity of phosphatidylinositol‐3‐kinase, intact actin cytoskeleton, and the presence of serum proteins in culture medium.—Juks, C., Lorents, A., Arukuusk, P., Langel, Ü., Pooga, M. Cell‐penetrating peptides recruit type A scavenger receptors to the plasma membrane for cellular delivery of nucleic acids. FASEB J. 31, 975–988 (2017). www.fasebj.org

Arginine-Rich Cell-Penetrating Peptides Require Nucleolin and Cholesterol-Poor Subdomains for Translocation across Membranes

Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesicles (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy-dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nucleolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.