S413-PV Cell-Penetrating Peptide Forms Nanoparticle-Like

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Bioconjugate Chem. 2010, 21, 774–783

S413-PV Cell-Penetrating Peptide Forms Nanoparticle-Like Structures to Gain Entry Into Cells Ka¨rt Padari,‡,† Kaida Koppel,‡,† Annely Lorents,† Mattias Ha¨llbrink,§ Miguel Mano,| Maria C. Pedroso de Lima,| and Margus Pooga*,†,⊥ Department of Developmental Biology, Institute of Molecular and Cell Biology, University of Tartu, EE51010 Tartu, Estonia, Department of Neurochemistry, Stockholm University, SE10691 Stockholm, Sweden, Center for Neuroscience and Cell Biology and Department of Biochemistry, University of Coimbra, 3001-401 Coimbra, Portugal, and Estonian Biocentre, EE51010 Tartu, Estonia. Received December 30, 2009; Revised Manuscript Received February 17, 2010

Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S413-PV. We demonstrate that S413-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S413-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S413-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins’ activity reduce the internalization of S413-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.

INTRODUCTION 1

Cell-penetrating peptides (CPPs) constitute an expanding peptide family with a common denominatorsthe ability to cross biological membranes and gain access into living cells. These peptides have been applied to drag various molecules into cells, like siRNA (1, 2), PNA oligomers (3, 4), peptides (5), proteins (6, 7), plasmids (8), and bigger particles (9–11). Although the mechanism of CPP cellular uptake is far from being fully understood yet, their potential to target cargo molecules into cells has been widely appreciated and used. Nevertheless, unraveling of the uptake mechanism of CPPs is of high importance for both achieving the specific targeting and increasing the biological response, i.e., the activity of delivered cargo in the cellular interior. Initially, the internalization of CPPs was suggested to occur by direct translocation across the plasma membrane and not by receptor-mediated endocytosis. After the discovery of artifactual redistribution of membrane-associated CPPs into cells and nucleus upon fixation, the “endocytosis only” concept was * Correspondence should be addressed to Prof. Margus Pooga, Institute of Molecular and Cell Biology, Riia 23, 51010 Tartu, Estonia, Tel: (372)7 375 049, Fax: (372)7 420 286, E-mail: [email protected]. ‡ These authors contributed equally to this work † University of Tartu. § Stockholm University. | University of Coimbra. ⊥ Estonian Biocentre. 1 Abbreviations: CPP, cell-penetrating peptide; GAG, glycosaminoglycan; HSPG, heparan sulfate proteoglycan; LAMP-2, lysosome associated membrane protein 2; MRR, membrane repair response; RR, ruthenium red; TEM, transmission electron microscopy; MVB, multivesicular body.

introduced, and ideas about passage of peptides directly across the plasma membrane were neglected (12, 13). Experiments in living nonfixed cells implicated the involvement of different endocytic pathways in the internalization of CPPs and their conjugates: clathrin-mediated endocytosis (14), lipid raft/caveolae-mediated endocytosis (15, 16), and macropinocytosis (17, 18). However, a number of recent studies demonstrated the switching between different cellular distribution patterns of CPPs (from the punctuate/vesicular to the diffuse/uniform characteristic to penetration mechanism) in response to the shift of experimental conditions, like the concentration of CPP, the temperature, and the size of cargo (5, 19–21). Currently, CPPs are considered to use a combination of multiple mechanisms for cell entrysdirect translocation and endocytosis. Due to the high positive charge, CPPs interact electrostatically with the negatively charged extracellular matrix, probably glycosaminogycans (GAG) at the cell surface. Membraneassociated heparan sulfate containing proteoglycans has been shown to be a primary interaction site of CPPs with the plasma membrane mediating their uptake by cells (14, 18, 22, 23). Moreover, recent investigations indicate that cationic peptides reorganize the extracellular matrix and the binding of CPPs to the negatively charged GAGs induces their clustering at the surface of cells thereby stimulating the endocytotic uptake of formed aggregates (24, 25). Although it is generally appreciated that GAGs are essential for cellular translocation of CPPs, information about other anionic molecules, which are also suggested as possible uptake mediators of CPPs, is still lacking. In our study, we focused on the S413-PV peptide, its interaction with the cell surface, and translocation into cells. S413PV is a chimeric CPP that contains a 13 amino acid sequence derived from dermaseptin S4 and the nuclear localization signal

10.1021/bc900577e  2010 American Chemical Society Published on Web 03/05/2010

Internalization Mechanism of Cell-Penetrating Peptides

of the simian virus 40 (SV40) large T antigen (26). We have previously shown that the S413-PV peptide is efficiently taken up by cells in a rapid dose-dependent manner, which is distinct from endocytosis and most likely involves direct penetration across cell membranes (19, 27). Circular dichroism analysis and the change in fluorescence emission spectra of the S413-PV peptide upon interaction with target membranes implicated that the peptide acquires a helical conformation in the presence of target membranes and is partially inserted in the hydrophobic environment of lipid bilayer (28). The high translocation ability and the cellular delivery potential of S413-PV peptide was recently corroborated by successful transfection of cells with plasmid DNA (8). The vast majority of studies examining the CPPs’ uptake and cellular localization have used fluorophore-labeled peptides. Fluorescence microscopy is a very powerful technique for visualizing the uptake and location of peptides in cells. However, it does not provide sufficiently detailed information regarding whether CPPs associate first with the extracellular matrix or directly with the plasma membrane, what is the size and shape of loci of initial contacts of CPPs with cells, which type of vesicles mediate the uptake, and so forth. In addition, the fluorescence signal might be quenched in the cellular environment, especially in vesicles with low pH or when associated with polyanions, as well as on the cell surface upon interaction with heparan sulfate proteoglycans (HSPGs) (29) complicating the interpretation of fluorescence microscopy results. Transmission electron microscopy, on the other hand, provides resolution necessary to unambiguously assign the localization of CPPs in relation to subcellular compartments and, moreover, to examine how peptides interact with the plasma membrane and extracellular matrix components. Still, quite a few studies have used electron microscopy to assess the mechanism of CPP uptake. In this study, we used transmission electron microscopy to characterize the cellular uptake of a cell-penetrating peptide S413PV and its analogues with reversed NLS and scrambled sequence. We demonstrate that S413-PV peptides form nanoparticle-like regular spheric structures on the cell surface to enter HeLa cells. Our data indicate that these CPPs associate first with the cell surface anionic glycosaminoglycans and assemble in spherical clusters followed by the interaction with the plasma membrane. Accumulation of S413-PV particles on/in the plasma membrane leads to their cell entry and membrane repair response, which is only induced at high peptide concentrations. The cellular uptake of S413-PV peptide is markedly reduced at low temperature or by the inhibition of dynamins’ activity, but not completely ceased. We provide ultrastructural insight into the membrane interaction and cellular uptake of S413-PV peptides, which will contribute to our current understanding of how CPPs cross biological membranes and enter cells.

EXPERIMENTAL PROCEDURES Cell Culture. Human cervical carcinoma cell line HeLa was cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, Invitrogen, UK) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, Austria), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Gibco, Invitrogen, UK). Wildtype CHO-K1 and glycosaminoglycan mutant CHO cell line pgsA-745 was cultured in Ham’s F12 culture medium (Gibco, Invitrogen, UK) containing 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Cells were grown in a humidified atmosphere containing 5% CO2 at 37 °C. Peptides. High-purity (>95%) S413-PV peptide (ALWKTLLKKVLKAPKKKRKVC), reverse NLS peptide (ALWKTLLKKVLKAVKRKKKPC), and scrambled sequence (KTLKVAKWLKKAKPLRKLVKC) were obtained from Thermo Electron (Thermo Electron GmbH, Karlsruhe, Germany). The

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peptides were modified with an acetyl group at the N-terminus and had an amide group at the C-terminus. Peptides were tagged with Nanogold (Monomaleimido Nanogold, Nanoprobes, NY, d 1.4 nm) by forming a covalent bond between the thiol group of peptide and the maleimide group of label by incubation at a molar ratio 2.5:1 in 50% methanol at 30 °C for 90 min. Methanol was removed and the conjugate concentrated by speed-vac to reach 50-100 µM concentration of labeled peptide. The conjugate with 1:1 peptide/Nanogold ratio was purified to homogeneity by size-exclusion chromatography on Superdex Peptide HR10/30 in buffer containing 35 mM Tris-HCl pH 7.5, 105 mM NaCl, and 30% (v/v) acetonitrile or by reversed phase chromatography on Pro RPC HR5/2 (Pharmacia, Sweden) in water-acetonitrile gradient in the presence of 0.1% TFA and concentrated again. The analysis of conjugate by both abovementioned chromatography methods yielded a single peak. Treatment of Cells with Peptides. Cells were seeded onto glass coverslips in 24-well plates, grown to 80-90% confluence, and incubated with culture medium containing 0.1-3 µM goldlabeled peptides at 37 °C for 10 min to 24 h depending on the experimental setup. After incubation, the cells were washed twice with cell culture medium and processed for electron microscopy (see below). For low-temperature experiments, cells were preincubated in culture medium at 10 °C for 30 min, followed by treatment with peptides at the same temperature for 1 h. Staining of Cell Surface Acidic Mucopolysaccharides with Ruthenium Red. After incubation with Nanogold-labeled peptides, cells were washed twice with cell culture medium and fixed with 2.5% glutaraldehyde containing 1 mg/mL ruthenium red (Sigma-Aldrich, Germany) in the cacodylate buffer (0.1 M sodium cacodylate, pH 7.4) for 1 h. The ruthenium red-stained cells were then washed twice for 10 min with the cacodylate buffer and processed for TEM as described below. Treatment of Cells with Hyaluronidase. Hyaluronan chains were removed from the surface of pgsA-745 CHO cells by treating with 300 µg/mL hyaluronidase (Sigma-Aldrich, Germany) in serum-free (SF) medium at 37 °C for 30 min. Thereafter, the cells were incubated with 1 µM gold-labeled peptides for 1 h in fresh SF medium containing 300 µg/mL hyaluronidase. Plasma Membrane Repair Assay. Cells were washed with SF culture medium and incubated with 0.5-10 µM peptides in SF medium for 30 min at 37 °C. Treated cells were washed with PBS, fixed with 3% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) for 30 min, and washed with PBS. The control cells were permeabilized with methanol at -20 °C for 15 min to expose all intracellular LAMP-2 antigens. The sites of unspecific binding were blocked with 10% nonfat dry milk in PBS for 1 h. LAMP-2 was stained with monoclonal antibody (H4B4, DSHB, 1:100) for 1 h and Alexa Fluor 488-conjugated goat antimouse antibody (Invitrogen, 1:400) for 30 min at room temperature in dark. The solution of secondary antibody was supplemented with 1 µg/mL propidium iodide to assess the intactness of plasma membrane. The coverslips were washed, mounted on glass slides with Fluoromount G (Electron Microscopy Sciences, Hatfield, PA, USA), and analyzed with fluorescence microscopy. Images were recorded using an Olympus BX61 microscope equipped with CCD camera DP70 or Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). Obtained images were processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA). Transmission Electron Microscopy. After treatment with Nanogold-labeled or unlabeled peptides, the cells were fixed with 2.5% glutaraldehyde in cacodylate buffer at room temperature for 1 h and washed two times for 10 min with cacodylate buffer. The Nanogold label of peptides was magnified

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Figure 1. Interaction of S413-PVwt and its analogues with the plasma membrane of HeLa cells. Cells were incubated with 1 µM Nanogold-labeled S413-PVwt (A), S413-PVrev (B), and S413-PVscr (C) for 30 min. Insets in A and B illustrate the enlarged nanoparticles of S413-PVwt (100 nm) and S413-PVrev (150 nm) peptides, respectively. For clarity, note that the black circles in B and C indicate some glycogen granules, which may misleadingly be referred to as the peptide nanoparticles. (D) Distribution of S413-PVwt peptide in cell-free system. Two micromolar Nanogold-labeled S413-PVwt in cell culture medium was placed onto blank sections on TEM grid and allowed to air-dry. The Nanogold (1.4 nm) particles were magnified by silver deposition to ∼10 nm. (E) Quantitative analysis of the size of the nanoparticles formed by S413-PVwt and S413-PVrev peptide. Scale bars 0.5 µm.

by silver enhancement for 2-5 min with HQ Silver kit from Nanoprobes (Yaphank, NY, USA) following the protocol suggested by manufacturers. The silver deposition was stabilized by toning with 0.05% gold chloride (30). Cells were postfixed with 1% osmium tetroxide in the cacodylate buffer containing 15 mg/mL potassium ferrocyanate at room temperature for 1 h. After osmification, the cells were washed three times for 5 min with cacodylate buffer and dehydrated with 70%, 96%, and twice with 100% ethanol. Finally, the coverslips were dipped into acetone and embedded in epoxy resin (TAAB Laboratories Equipment Ltd., UK). Ultrathin sections were cut in parallel with the coverslip and contrasted in 2% uranyl acetate in 50% ethanol for 2 min and in lead citrate for 2 min. The sections were examined with a JEM-100S (JEOL, Tokyo, Japan) transmission electron microscope at 80 kV. The scanned electron microphotos were analyzed and processed with Adobe Photoshop 7.0 software. Statistical analysis was performed using program R 2.6.0. Treatment of Cells with Dynasore and Transferrin. For electron microscopy, the activity of dynamins was inhibited by pretreating cells with 80 µM dynasore (AKos GmbH, Steinen, Germany) in SF culture medium at 37 °C for 30 min (31). The peptides were diluted to the concentration of 1 µM in the same solution and incubated for 1 h. For transferrin uptake, 5 × 104 HeLa cells were seeded onto glass coverslips in 24-well plate one day before experiment. Cells were washed twice with SF medium and pretreated for 30 min at 37 °C with 80 µM dynasore in SF culture medium or SF medium only. For visualizing endosomes, cells were incubated with 10 µg/mL transferrin labeled with Alexa Fluor 594 (Molecular Probes, Invitrogen, OR, USA) in the corresponding medium for 2 min at room temperature, washed 3 times with the corresponding medium without transferrin, and incubated for an additional 15 min at 37 °C. Treated cells were washed twice with cold 0.2 M glycine buffer containing 0.15 M NaCl (pH 3.0) for 30 s, followed by washing with cold PBS, and fixed with 4% paraformaldehyde in PBS on ice for 30 min. Fixed cells were washed with PBS and mounted to microscope

slides with 30% glycerol in PBS. The images were recorded and processed as described above.

RESULTS S413-PV Peptides Assemble into Small Nanoparticle-Like Spherical Structures Upon Interaction with the Plasma Membrane of HeLa Cells. In order to characterize the cellular uptake of the S413-PV peptides at the ultrastructural level, we tagged the original “wild-type” peptide (S413-PVwt) and its analogues with reversed NLS (S413-PVrev) and scrambled sequence (S413PVscr) covalently with Nanogold clusters of 1.4 nm in diameter. Incubation of HeLa cells with 1 µM S413-PVwt-nanogold conjugates led to the association of peptides into spherical structures, which accumulated on the plasma membrane and entered cells. These structures had regular shape and relatively uniform size of about 80-100 nm reminiscent of nanoparticles (Figure 1A). The assembly of wild-type peptide into nanoparticles was rather prevailing, but still a fraction of peptides bound to the extracellular matrix without formation of spherical structures. S413-PVrev peptide formed similar nanoparticle-like structures, albeit somewhat bigger and more variable in size and shape (Figure 1B). Quantitative analysis revealed that the average size of particles formed by S413-PVrev peptide was about 1.8 times bigger (142.6 ( 22.7 nm) than those formed by wildtype peptide (79.8 ( 16.4 nm) (Figure 1E). In contrast to S413PVwt and S413-PVrev, the peptide with scrambled sequence (S413PVscr) could not assemble into spherical structures and typically formed bigger irregular aggregates on the plasma membrane of cells (Figure 1C). This suggests that the peptide with scrambled sequence cannot acquire a regular structure similar to the wild-type and reverse NLS peptides. The spherical structures of wild-type and reversed NLS peptides seem to form only in the presence of cell membranes, since the incubation of peptides in the cell culture medium (Figure 1D) with or without calf serum did not lead to assembly of nanoparticles. Next, we assessed whether the characteristic spherical assemblies are induced by the Nanogold cluster used

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Figure 2. Association of the nanoparticles of S413-PVwt with cell surface mucopolysaccharides. Nanoparticles on the surface of HeLa cells after incubation with 4 µM unlabeled (A,B) or 1 µM Nanogold-labeled (C,D) S413-PVwt peptides for 1 h and stained with ruthenium red (RR). Arrows in A and B indicate the S413-PVwt nanoparticles formed by unlabeled peptide. Arrow in D points to the surface region lacking both the binding of S413-PVwt peptides and RR-staining. Note that the glycogen granules in the cytoplasm (some of them surrounded by black circles in A, B, and C) may look similar to the peptide nanoparticles. Scale bars 0.5 µm.

for peptide labeling and not by the peptide. As expected, the cationized Nanogold was rarely detected in association with cells after incubation in identical conditions, and no spherical assemblies were ever detected (data not shown), showing that the specific clustering was indeed induced by the peptides. To further confirm this, we incubated cells with 1 µM of goldlabeled peptide and added unlabeled peptide at 2, 4, or 9 µM concentration. At all used concentrations, similar regular structures formed on/in the cell surface in analogy with the goldlabeled peptides only. However, in the presence of unlabeled peptide the number of gold labels per nanosphere was lower than in nanostructures obtained by incubating cells with Nanogold-labeled peptide only (data not shown). This demonstrates that both the gold-labeled and unlabeled S413-PVwt peptides are capable of forming nanoparticles and intermix with one another. In line with this, when we incubated cells with unlabeled peptides only, either at 4 (Figure 2A,B) or 10 µM concentration, we also detected similar distinct electron-dense spherical structures on the surface of HeLa cells, which probably represent the same particles. Analogous results were also obtained with S413-PV peptide with reversed NLS sequence (data not shown). Cell Surface Glycosaminoglycans Are Involved in the Formation of Peptide Nanostructures. The electron microphotos revealed that the peptide nanoparticles always had an electron-dense background. Binding of polycationic molecules, e.g., cell-penetrating peptides, to the HSPGs is known to induce the clustering and concentration of peptides to the cell surface as dense aggregates followed by their internalization (24, 25). Therefore, we visualized the extracellular matrix with ruthenium red (RR), a polycationic cytochemical stain for cell surface mucopolysaccharides. Ruthenium red markedly increased the staining of the plasma membrane and especially the extracellular matrix surrounding cells. In line with the staining of extracellular matrix, the electron density of S413-PV clusters’ background was markedly increased by RR treatment (Figure 2C,D). In addition, we noticed that S413-PV peptides associated preferentially with the membrane areas marked with ruthenium red, but not with regions lacking the intense stain of RR (arrow in Figure 2D). To further assess the involvement of glycosaminoglycans, we analyzed the association of S413-PVwt with the cell surface and internalization in wild-type and glycosaminoglycan-deficient Chinese hamster ovary (CHO) cells. In wild-type CHO cells,

Figure 3. Effect of hyaluronidase treatment on the formation of S413PV nanostructures. Wild-type CHO-K1 (A) and glycosaminoglycan (GAG) mutant CHO cells (pgsA-745) (B,C) were incubated with 1 µM Nanogold-labeled S413-PVwt peptide for 1 h. (C) The GAG-deficient CHO cells were pretreated with 300 µg/mL hyaluronidase in serumfree medium for 30 min at 37 °C to remove hyaluronan chains from the cell surface and incubated with 1 µM gold-labeled S413-PVwt in the same solution for 1 h. Arrows in C indicate the few peptides that bound to the plasma membrane and internalize into cells after hyaluronidase treatment. Scale bars 0.5 µm.

the peptide accumulated extensively to the cell surface and formed nanospheres with similar size and shape as in HeLa cells (Figure 3A). The nanoparticles of S413-PV were also present at the surface of mutant CHO-pgsA-745 cells, which are defective in glycosaminoglycan biosynthesis (32) and lack specific GAG assemblies (Figure 3B). However, the amount of peptide particles on the surface of GAG deficient cells was markedly reduced in comparison to the wild-type CHO. Treatment of mutant CHO cells with hyaluronidase, which specifically cleaves glycosidic bonds in hyaluronic acid and thereby removes the hyaluronan strands and the hyaluronan filament-associated proteoglycans from the cell surface abolished the formation of S413-PV nanoparticles. However, some peptide particles were able to associate with the cell surface and internalize into cells even after treatment with hyaluronidase (arrows in Figure 3C). In order to determine more specifically the electron-dense membrane constituents associating with S413-PV nanostructures on the cell surface, we incubated peptides in a cell-free system with different isolated acidic glycosaminoglycans (i.e., chondroitin sulfate, heparin, hyaluronan) in the cell culture medium. Unexpectedly, under these conditions none of the examined

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Figure 5. Translocation and intracellular localization of S413-PVwt peptide. HeLa cells were incubated with 0.5 µM (A,B) or 1 µM S413PVwt (D,E) for 1 h, and with 1 µM S413-PVwt for 24 h (C,F). Accumulation of S413-PVwt nanoparticles to the cell surface (A), internalization in small vesicles (arrows in B), and localization in caveosome-like structures (C) or multivesicular bodies (D,E). Arrow in F and enlarged section (G) indicate the peptide-containing vesicles with a discontinuous membrane after 24 h of S413-PVwt incubation with cells. Scale bars 0.5 µm.

Figure 4. Concentration- and time-dependent formation of S413-PV nanostructures. HeLa cells were incubated with 0.1 µM S413-PVwt for 10 min (A), 0.5 µM (B) or 3 µM S413-PVwt (C) for 1 h. Single clusters of S413-PVwt (arrows in A) at the cell surface formed at a lower concentration; peptide particles of typical size (B) and large irregular aggregates (C) formed at higher concentrations, 0.5 µM and 3 µM respectively. Scale bars 0.5 µm.

GAGs alone were sufficient to induce the formation of nanoparticle-like structures of this peptide (data not shown). Formation of S413-PV Nanostructures Is Dependent on the Peptide Concentration and Incubation Time. The clusters of S413-PV were detectable by electron microscopy as early as 10 min after the addition of peptides to HeLa cells at the concentration of 0.1 µM. As expected, at low concentration only the “germs” of the nanostructures were detected attached to the cell membrane (arrows in Figure 4A). The peptide-containing structures were small and contained only a few gold particles per cluster. Peptides were taken up by cells also at this concentration and they localized in small (e50 nm) vesicles under the plasma membrane. Longer incubation (1 h) at this peptide concentration resulted in a slight increase in the surfacebound as well as internalized S413-PV particles. Upon longer incubation, the size of peptide-containing structures did not

increase and remained similar to that observed after 10 min incubation. However, at 0.5 and 1 µM concentrations the assemblies of S413-PV peptides exhibited a characteristic spherical shape and size, had higher background density, and comprised more gold particles (Figure 4B). These peptide nanoparticles accumulated extensively at the plasma membrane during 1 to 24 h of continuous incubation with cells. A negligible fraction of peptide particles at 1 µM concentration formed bigger conglomerates during 4 to 24 h, but most of these still retained regularity and a uniform size in bigger structures. However, when we increased the concentration of labeled peptides to 2-3 µM, the large aggregates (up to 0.5-1 µm diameter) started to form already in 1 h (Figure 4C). S413-PV Peptides Interfere with the Organization of the Plasma Membrane and Efficiently Enter the Cells. The S413PVwt and S413-PVrev peptides associated preferentially with specific loci on the plasma membrane. Upon longer incubation, the peptide nanostructures continued to accumulate in these particular regions on the cell surface. Accumulation of peptides interfered with the distinct ultrastructure of the lipid bilayer turning it hardly detectable by electron microscopy. In parallel with the destabilization of the lipid bilayer, the peptide particles inserted into the cortical cytoplasm (Figure 5A) and entered deeper into cells.

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Figure 6. Induction of the membrane repair response by S413-PV peptide. HeLa cells were incubated with 1 µM (A), 3 µM (B), 5 µM (C) S413-PVwt or 1 µM (E), 3 µM (F), 5 µM (G) S413-PVrev at 37 °C for 30 min. LAMP-2 was visualized with monoclonal LAMP-2 antibody and Alexa Flour 488-conjugated antimouse antibody. The membrane repair response is not induced at lower concentration of S413-PVwt (A) and S413-PVrev (E) peptides. LAMP-2 signal appeared at the plasma membrane higher concentrations of S413-PVwt (B,C) and S413-PVrev (F,G) peptides. (D) DIC image of cells in panel A. Localization of LAMP-2 protein in control cells permeabilized with methanol (H). Scale bar 10 µm.

In parallel with the interaction of S413-PV nanostructures with the plasma membrane, the formation and pinching off of small vesicles with diameter of 50-100 nm into cells was induced and some of these also contained peptides (Figure 5B). In addition, peptides were found in bigger rosette-like structures, morphologically reminiscent of caveosomes (Figure 5C). The peptide nanoparticles were present also in multivesicular bodies (MVB), especially after longer incubation. Interestingly, even inside the MVB the distinct spherical organization of peptides was retained for at least 1-4 h (Figure 5D,E). The peptide structures usually located on the inner surface of the vesicle and were mostly in direct contact with the membrane of vesicles. Continuous uptake of S413-PV peptides by cells during 24 h led to their high accumulation in large vacuoles (Figure 5F). This was accompanied by impaired membrane integrity of some peptide-containing vesicles (arrow in Figure 5F,G) indicating that the high concentration of S413-PV can destabilize the membrane and induce the escape of peptides into the cytosol. S413-PV Peptides Enter Cells without Triggering the Membrane Repair Response, which Requires Higher Peptide Concentrations. Next, we assessed whether S413-PV peptides caused plasma membrane disturbances at the extent of enabling calcium influx into cells and thereby triggering the plasma membrane repair response (MRR). Under these circumstances, the intracellular vesicles donate membranes to reseal the injured plasma membrane, and this can be detected by the exposure of lysosomal membrane protein LAMP-2 to the plasma membrane of intact cells (33). Incubation of HeLa cells with 0.5-1 µM wild-type S413-PV did not trigger the plasma membrane repair response (Figure 6A). It is remarkable that efficient cellular uptake of S413-PVwt peptide takes place without disturbing the ordered packing of the plasma membrane or evoking the membrane repair response. Higher concentrations of peptide starting from 3 µM led to marked fusion of lysosomal membranes with the plasma membrane, and LAMP-2 was detected all over the plasma membrane as distinct punctae (Figure 6B,C). At 10 µM concentration, the plasma membrane resealing was triggered in more than 80% of cells. The peptide with reversed NLS sequence induced MRR at somewhat lower concentration than the wild-type peptide. LAMP-2 was targeted to the plasma membrane in about 10% of cells already after incubation with 2 µM peptide (data not

shown). At 3 µM peptide concentration, LAMP-2 was present at the plasma membrane of 50% of cells (Figure 6F). Increasing the S413-PVrev concentration to 5 µM induced appearance of LAMP-2 on the plasma membrane of almost all cells in specimen (Figure 6G). However, even at 5 µM S413-PVrev concentration the integrity of the plasma membrane was not impaired since propidium iodide could not enter cells (data not shown). S413-PV analogue with scrambled sequence was the least potent to induce targeting of LAMP-2 to the outer leaflet of the plasma membrane. Very few cells became positive for the lysosomal protein at the cell surface starting from 5 µM concentration and reaching a maximum at 10 µM concentration where almost all of the population was stained. However, not all LAMP-2 antigens became accessible to antibodies at the peptide concentration with the highest induction ability (in case of all used peptides), because the removal of membrane lipids by methanol fixation led to a drastically elevated staining of this lysosomal protein all over the cell (Figure 6H). Cellular Uptake of S413-PV Peptides Is Reduced at Low Temperature. At 10 °C, the S413-PVwt and both of its analogues associated with the plasma membrane to a lesser extent than at the physiological temperature, and their translocation into cells was diminished. The S413-PVwt and S413-PVrev peptides were still able to assemble into nanostructures, although the size of peptide clusters was more variable and their shape less uniform (Figure 7A,B). At low-temperature condition, the peptides associated with the plasma membrane/extracellular matrix as either irregular aggregates or single clusters. Surprisingly, despite the markedly decreased amount of surface-bound peptides the vesicular uptake of S413-PV peptides was not blocked at 10 °C. However, only a few or single gold particles were in small vesicles or bigger vesicular structures (arrows in Figure 7B,C), which located in the cortical cytoplasm or in the close proximity of the plasma membrane but not deeper in cells. Dynamin Inhibition Does Not Abolish the Vesicular Uptake of S413-PV by HeLa Cells. Dynasore, an inhibitor of dynamin GTPase activity, blocks the formation and fission of endocytic vesicles in most pathways (31). Therefore, we examined the effect of dynasore on the translocation efficiency of S413-PV. Inhibition of dynamins decreased but did not abolish the vesicular uptake of S413-PV nanoparticles. Even in the presence of dynasore, the S413-PV peptides induced the formation and

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Figure 7. Effect of low temperature and dynamin inhibition on cellular translocation of S413-PV peptides. HeLa cells were incubated with 1 µM S413-PVwt (A), S413-PVrev (B), or S413-PVscr (C) at 10 °C for 1 h. HeLa cells incubated with 1 µM S413-PVwt for 1 h in the presence of dynasore (80 µM) at 37 °C (D,E). Arrows indicate internalized peptides in small vesicles formed at low temperature (B,C) and in dynasoretreated cells (D). Scale bars 0.5 µm.

fission of small vesicles. The formed small vesicles localized mainly in the cortical cytoplasm near the plasma membrane and contained typically a few labeled peptides (Figure 7D). Remarkably, S413-PVwt peptides were also taken up into large endosomal structures (g0.5 µm) despite the presence of dynasore (Figure 7E). The control cells incubated with Alexa Fluor 594 conjugated transferrin, as a marker for dynamin-dependent endocytic pathway, corroborated the inhibition of endocytosis by dynasore, since only a negligible part of protein was taken up by cells (Supporting Information Figure S1). These findings suggest that S413-PV peptides can also gain entry into cells by endocytic processes that do not necessarily require dynamin.

DISCUSSION Cell-penetrating peptides have been under intense investigation from the moment of their discovery 15 years ago, but the discussion about their mechanism of association with cells, uptake, and cargo delivery is still continuing, and consensus has not yet been reached in some key questions. Currently, a growing number of studies support the view that CPPs enter cells by different mechanisms, which act in parallel, and for most CPPs, more than one cellular pathway exists. Previously, we have shown that the internalization of S413-PV cellpenetrating peptide proceeds by at least two different mechanisms: an endocytic pathway, which prevails at lower peptide concentrations, and an alternative endocytosis-independent mechanism that is induced preferentially at higher peptide concentrations (19). In addition, we demonstrated that the localization of this peptide is not markedly changed upon fixation with aldehydes (19), which enables studies at an ultrastructural level by transmission electron microscopy in fixed specimen. Electron microphotos revealed that the Nanogold-labeled (1.4 nm gold cluster) S413-PVwt peptide assembles into small spherical structures at the plasma membrane of HeLa cells. Considering the relatively uniform size of 80-100 nm and regular shape of the particles formed by S413-PV peptides, we called these structures “nanoparticles” for brevity. The ability to form nanoparticles was also observed for analog of S413-PV peptide with reversed NLS sequence, which clustered in regular

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spherical structures although with bigger and less uniform size (∼140 nm). In contrast, the peptide with scrambled sequence, S413-PVscr, failed to form such spherical structures despite having an identical amino acid composition. The scrambled analogue of S413-PV formed bigger irregular aggregates and entered cells less efficiently than S413-PVwt and S413-PVrev peptides, indicating that not only the amino acid composition, but also their positions influence the mode of interaction with cell surface and subsequent internalization. In general, we observed that the formation of small and regular peptide-containing structures is in good correlation with their ability to enter cells, i.e., the peptides forming regular particles are more efficiently taken into cells. This observation could explain our earlier reports (27, 28) demonstrating that the cellular uptake of the scrambled peptide was significantly less efficient than that of S413-PVwt or with reversed NLS peptides. On the basis of the study of peptide conformational changes, we have previously attributed the better internalization properties of the S413-PV and reverse NLS peptide, compared to the scrambled peptide, to the ability of these peptides to adopt an amphipathic R-helical conformation upon interaction with the negatively charged membranes. The changes in the secondary structure of CPPs could in turn induce rearrangement of the plasma membrane lipids leading to peptide internalization (27). The electron microscopy results described here are quite well in line with this conceptsthe plasma membrane of HeLa cells becomes less distinct upon interaction with S413-PVwt or S413-PVrev but not with S413-PVscr. Both the modulation of plasma membrane properties and the different size of peptide assemblies on the cell surface might be responsible for differences in cell-translocation ability of S413PV peptides. The first hint that CPPs may assemble in clusters upon association with cells can be found in a publication demonstrating the receptor-independent internalization of the third helix of the Antennapedia homeodomain in 1996 (34). Although it was not discussed by the authors at that time, the visualization of Antp(43-58) by colloidal gold particles in electron microscopy reveals that this peptide tends to localize in cells as small clusters in parallel to its even/diffuse distribution. The clustering of peptides to produce particles or aggregates has also been described for other CPPs. Some amphipathic CPPs, like SAP and Pep-1, self-assemble in aqueous solution (35–37). On the other hand, the aggregation and clustering can be induced by the binding of CPPs to HSPGs on the cell surface (24). Remarkably, not only CPPs, but also some amyloidogenic polypeptides form aggregates to gain access into cells. For example, the large fibrillar polyglutamine peptide aggregates (0.2-0.5 µm in diameter) are able to penetrate the cytosolic compartment of mammalian cells where they can nucleate the aggregation of other soluble proteins containing polyglutamine sequence (38). The cell-penetrating peptide Pep-1, which analogously to S413-PV peptide is amphipathic and comprises a NLS sequence, forms discrete nanometer-sized particles when complexed with cargo protein in solution (37). The Pep-1/cargo nanoparticles were of about 80-120 nm diameter as characterized by scanning electron microscopy and light scattering measurements. Analogously to Pep-1, some other short amphipathic peptides, e.g., MPG and CADY, have been demonstrated to form stable nanoparticles with proteins and/or nucleic acids in solution (39). Considering these results and taking into account that different cargo molecules can influence the properties of its carrier peptide (40), we assessed whether the Nanogold tag, which we conjugated to the peptide could induce/facilitate the assembly of S413-PV nanoparticles or determine their size and structure. The Nanogold cluster is very stable, has no overall charge, and is considered of no affinity to proteins. Still, its diameter is 1.4

Internalization Mechanism of Cell-Penetrating Peptides

nm, and it could alter the bulk properties of peptide more than typically used fluorophores. Although we cannot completely exclude some impact of the gold label on the formation of S413PV nanoparticles, it cannot be the main assembling force, since after incubation of cells with unlabeled peptides, we were still able to clearly detect the distinct spherical structures. The particles of “naked” S413-PV were reminiscent of structures formed with S413-PV-Nanogold conjugates. The similar size of formed particles suggests that the Nanogold tag does not contribute markedly to the assembly of S413-PV nanoparticlelike structures. It remains nonetheless unclear whether the formation of the nanoparticle-like structures by the S413-PVwt or S413-PVrev peptides is somehow related to the ability of these peptides to adopt a helical conformation. CPPs may also bunch on the surface of cultured mammalian cells. Ziegler et al. demonstrated that peptides and other cationic compounds can cluster GAGs and induce formation of particles on the surface of cells (24, 25). The initial study revealed the presence of strongly absorbing aggregates (