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• W1992095148 abstract "A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43–58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43–58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation. A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43–58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43–58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation. A large number of hydrophilic molecules such as peptides, proteins, and oligonucleotides are poorly internalized by cells because they cross the lipid matrix of the plasma membrane rather inefficiently. This is considered to be a major limitation for their use as therapeutic agents in biomedical research and the pharmaceutical industry. However, it has been reported that the use of short (10–30 amino acids) cationic peptides may provide a solution by enhancing the intracellular delivery of such intractable molecules, both in vitro and in vivo (see reviews in Refs. 1Lindgren M. Hällbrink M. Prochiantz A. Langel U. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 2Dunican D.J. Doherty P. Biopolymers. 2001; 60: 45-60Crossref PubMed Scopus (62) Google Scholar, 3Schwarze S.R. Hruska K.A. Dowdy S.F. Trends Cell Biol. 2000; 10: 290-295Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). These cell-penetrating peptides (CPPs) 1The abbreviations used are: CPP, cell-penetrating peptide; calcein-AM, calcein acetoxymethylester; CoxIV, cytochrome oxidase IV mitochondrial import (peptide/presequence); FITC, fluorescein isothiocyanate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; LY, Lucifer Yellow CH; MAP, model amphipathic peptide; NBD, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole; NEM, N-ethylmaleimide; PBS, phosphate-buffered saline; PG-1, protegrin 1; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol; TAMRA, 5-(and-6)-carboxytetramethylrhodamine; Tf, transferrin.1The abbreviations used are: CPP, cell-penetrating peptide; calcein-AM, calcein acetoxymethylester; CoxIV, cytochrome oxidase IV mitochondrial import (peptide/presequence); FITC, fluorescein isothiocyanate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; LY, Lucifer Yellow CH; MAP, model amphipathic peptide; NBD, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole; NEM, N-ethylmaleimide; PBS, phosphate-buffered saline; PG-1, protegrin 1; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol; TAMRA, 5-(and-6)-carboxytetramethylrhodamine; Tf, transferrin. are either basic segments of RNA- or DNA-binding proteins (4Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar, 5Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16077Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar, 6Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1443) Google Scholar) such as pAntp-(43–58) and Tat-(48–60), or artificial peptides such as Transportan (7Pooga M. Hällbrink M. Zorko M. Langel U. FASEB J. 1998; 12: 67-77Crossref PubMed Google Scholar), model amphipathic peptide (MAP) (8Oehlke J. Scheller A. Wiesner B. Krause E. Beyermann M. Klauschenz E. Melzig M. Bienert M. Biochim. Biophys. Acta. 1998; 1414: 127-139Crossref PubMed Scopus (384) Google Scholar), or other sequence-based constructs (9Chaloin L. Vidal P. Heitz A. Van Mau N. Méry J. Divita G. Heitz F. Biochemistry. 1997; 36: 11179-11187Crossref PubMed Scopus (85) Google Scholar, 10Vidal P. Chaloin L. Heitz A. Van Mau N. Méry J. Divita G. Heitz F. J. Membr. Biol. 1998; 162: 259-264Crossref PubMed Scopus (33) Google Scholar). In addition, the SynB peptides constitute a distinct family of vectors that derive from protegrin 1 (PG-1), an antimicrobial peptide (11Kokryakov V.N. Harwig S.S. Panyutich E.A. Shevchenko A.A. Aleshina G.M. Shamova O.V. Korneva H.A. Lehrer R.I. FEBS Lett. 1993; 327: 231-236Crossref PubMed Scopus (438) Google Scholar). As reported previously, PG-1, the amphipathic structure of which is stabilized by two disulfide bridges (12Aumelas A. Mangoni M. Roumestand C. Chiche L. Despaux E. Grassy G. Calas B. Chavanieu A. Eur. J. Biochem. 1996; 237: 575-583Crossref PubMed Scopus (167) Google Scholar), is believed to exert its antibiotic action by forming pores in the lipid matrix of bacterial membranes (13Mangoni M.E. Aumelas A. Charnet P. Roumestand C. Chiche L. Despaux E. Grassy G. Calas B. Chavanieu A. FEBS Lett. 1996; 383: 93-98Crossref PubMed Scopus (118) Google Scholar, 14Sokolov Y. Mirzabekov T. Martin D. Lehrer R.I. Kagan B.L. Biochim. Biophys. Acta. 1999; 1420: 23-29Crossref PubMed Scopus (109) Google Scholar). Because it has been shown that this pore formation depends critically on a cyclic, disulfide-bonded structure (13Mangoni M.E. Aumelas A. Charnet P. Roumestand C. Chiche L. Despaux E. Grassy G. Calas B. Chavanieu A. FEBS Lett. 1996; 383: 93-98Crossref PubMed Scopus (118) Google Scholar), we designed two analogs, SynB1 and SynB3, that lack the cysteine residues. We then investigated whether these linear peptides, which possess the multiple positive charges of PG-1, were able to penetrate into eukaryotic cells without disrupting the cell membrane. This was shown to be the case, with SynB1 and SynB3 efficiently promoting the transport of various drugs such as doxorubicin and benzylpenicillin (15Rousselle C. Clair P. Lefauconnier J.M. Kaczorek M. Scherrmann J.M. Temsamani J. Mol. Pharmacol. 2000; 57: 679-686Crossref PubMed Scopus (297) Google Scholar, 16Rousselle C. Clair P. Temsamani J. Scherrmann J.M. J. Drug Target. 2002; 10: 309-315Crossref PubMed Scopus (72) Google Scholar) across the blood-brain barrier of in vivo models. An unexpected benefit of the “vectorization” of the PG-p substrate doxorubicin was that its accumulation and potency in resistant K562 cells was improved drastically (17Mazel M. Clair P. Rousselle C. Vidal P. Scherrmann J.M. Mathieu D. Temsamani J. Anticancer Drugs. 2001; 12: 107-116Crossref PubMed Scopus (112) Google Scholar). Understanding the mode of entry of the SynB peptides and defining their intracellular localization are of particular interest for the design of conjugates which, once internalized in target cells, may efficiently release an attached drug in its native state. Several previous studies have proposed that the penetration of CPPs (such as pAntp-(43–58), Tat-(48–60), MAP, and Transportan) into eukaryotic cells occurs in a temperature-independent manner (4Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar, 5Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16077Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar, 7Pooga M. Hällbrink M. Zorko M. Langel U. FASEB J. 1998; 12: 67-77Crossref PubMed Google Scholar, 8Oehlke J. Scheller A. Wiesner B. Krause E. Beyermann M. Klauschenz E. Melzig M. Bienert M. Biochim. Biophys. Acta. 1998; 1414: 127-139Crossref PubMed Scopus (384) Google Scholar). Furthermore, by using various metabolic or endocytosis inhibitors, it has been suggested that the cell-penetrating ability of some CPPs is likely to be an energy-independent, non-endocytotic process (5Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16077Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar, 7Pooga M. Hällbrink M. Zorko M. Langel U. FASEB J. 1998; 12: 67-77Crossref PubMed Google Scholar, 18Suzuki T. Futaki S. Niwa M. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2002; 277: 2437-2443Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Additional studies showed that the cell penetration of enantio or inverso forms of peptides such as MAP or pAntp-(43–58) was as efficient as that of the parent peptides (8Oehlke J. Scheller A. Wiesner B. Krause E. Beyermann M. Klauschenz E. Melzig M. Bienert M. Biochim. Biophys. Acta. 1998; 1414: 127-139Crossref PubMed Scopus (384) Google Scholar, 19Scheller A. Oehlke J. Wiesner B. Dathe M. Krause E. Beyermann M. Melzig M. Bienert M. J. Pept. Sci. 1999; 5: 185-194Crossref PubMed Scopus (144) Google Scholar, 20Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar), suggesting that their internalization is not dependent on a chiral receptor. To explain these observations, it has been mainly proposed that the hydrophobicity and amphipathicity of CPPs confers on them the ability to interact with and pass through the lipid matrix of the plasma membrane via an energy-independent process termed translocation (1Lindgren M. Hällbrink M. Prochiantz A. Langel U. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, 7Pooga M. Hällbrink M. Zorko M. Langel U. FASEB J. 1998; 12: 67-77Crossref PubMed Google Scholar, 20Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar). In accord with this model, biophysical studies have indicated that pAntp-(43–58), Transportan, or MAP can interact with lipid vesicles while adopting a helical structure (21Berlose J.P. Convert O. Derossi D. Brunissen A. Chassaing G. Eur. J. Biochem. 1996; 242: 372-386Crossref PubMed Scopus (153) Google Scholar, 22Dathe M. Schümann M. Wieprecht T. Winkler A. Beyermann M. Krause E. Matsuzaki K. Murase O. Bienert M. Biochemistry. 1996; 35: 12612-12622Crossref PubMed Scopus (349) Google Scholar, 23Lindberg M. Jarvet J. Langel U. Gräslund A. Biochemistry. 2001; 40: 3141-3149Crossref PubMed Scopus (101) Google Scholar, 24Magzoub M. Kilk K. Eriksson L.E. Langel U. Gräslund A. Biochim. Biophys. Acta. 2001; 1512: 77-89Crossref PubMed Scopus (151) Google Scholar). Furthermore, based on several reports indicating that amphipathic anti-microbial and venom peptides are able to cross lipid bilayers (see review in Ref. 25Matsuzaki K. Biochim. Biophys. Acta. 1999; 1462: 1-10Crossref PubMed Scopus (854) Google Scholar), we measured the ability of pAntp-(43–58) to pass from the outer to the inner leaflet of a phospholipid membrane. However, our data revealed an absence of spontaneous translocation despite the lipid-binding ability of this CPP (26Drin G. Déméné H. Temsamani J. Brasseur R. Biochemistry. 2001; 40: 1824-1834Crossref PubMed Scopus (114) Google Scholar). In a similar biophysical study, we demonstrate that PG-1 is able to cross the membrane of phospholipidic vesicles but that a linear analogue, SynB5, is not able to do this despite having a similar lipid-binding activity. This suggested, and was subsequently borne out, that similar linear vectors may also lack the translocation property of the protegrins (27Drin G. Temsamani J. Biochim. Biophys. Acta. 2002; 1559: 160-170Crossref PubMed Scopus (38) Google Scholar). Having said that, artificial phospholipid bilayers may not be an ideal, realistic experimental model in which to investigate CPP passage across the plasma membrane. Therefore, it seemed important to directly examine in cells whether the uptake of diverse SynB peptides was related to the translocation mechanism described for others CPPs or depended on a metabolic cellular process. In this study, we examine the cell uptake of the SynB1 and SynB3 peptides (Table I) compared with that of pAntp-(43–58). We also examine the internalization of SynB5, for which we have also measured its translocation activity in lipid vesicles (27Drin G. Temsamani J. Biochim. Biophys. Acta. 2002; 1559: 160-170Crossref PubMed Scopus (38) Google Scholar). Because these four cationic peptides differ in terms of hydrophobicity and/or helical amphipathicity (Table I), this study could also indicate whether their individual physicochemical properties conferred on them specific mechanistic preferences.Table IName, sequence and physicochemical features of cell-penetrating peptidesPeptideSequenceaThe basic residues are indicated in boldfaced type.LengthNet Charge〈H〉bThe mean hydrophobicity H and mean helical hydrophobic moment μH of each peptide was calculated following the Eisenberg formula (31) with the hydrophobic scale of Fauchère and Pliska, based on the octanol/water partition of individual N-acetyl amino acid residues (32).〈μH〉bThe mean hydrophobicity H and mean helical hydrophobic moment μH of each peptide was calculated following the Eisenberg formula (31) with the hydrophobic scale of Fauchère and Pliska, based on the octanol/water partition of individual N-acetyl amino acid residues (32).SynB1RGGRLSYSRRRFSTSTGR186-0.10.158SynB3RRLSYSRRRF105-0.1240.361SynB5RGGRLAYLRRRWAVLGR1860.2380.213pAntp-(43-58)RQIKIWFQNRRMKWKK1670.1930.33a The basic residues are indicated in boldfaced type.b The mean hydrophobicity H and mean helical hydrophobic moment μH of each peptide was calculated following the Eisenberg formula (31Eisenberg D. Weiss R.M. Terwilliger T.C. Nature. 1982; 299: 371-374Crossref PubMed Scopus (830) Google Scholar) with the hydrophobic scale of Fauchère and Pliska, based on the octanol/water partition of individual N-acetyl amino acid residues (32Fauchére J.L. Pliska V.E. Eur. J. Med. Chem. 1983; 18: 369-375Google Scholar). Open table in a new tab We have measured time- and dose-dependence of the CPPs uptake into K562 cells and examined the influence of various metabolic and endocytosis inhibitors on the internalization. We have also carried out complementary experiments to measure the diffusion of pAntp-(43–58) through a lipid bilayer and examined the effect of cell fixation on the intracellular localization of CPPs. Our results indicate that SynB and pAntp-(43–58) peptides penetrate into cells via an adsorptive-mediated endocytosis process rather than a temperature-independent translocation mechanism. Materials—Fmoc-PAL-PEG-PS (High Load) and Fmoc amino acids were purchased from Perseptive Biosystems (Hamburg, Germany). Other reagents used for peptide synthesis included N,N′-diisopropyl-carbodiimide (DIPCDI, Fluka), 1-hydroxybenzotriazole (HOBT, PerkinElmer Life Sciences), N,N-diisopropylethylamine (DIEA, Fluka), dimethylformamide (DMF, Perseptive Biosystems), 4-chloro-7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD-Cl, Fluka), and 5-(and-6)-carboxytetramethylrhodamine (TAMRA; Molecular Probes, Eugene, OR). Sodium dithionite, sodium azide, N-ethylmaleimide, 2-deoxy-d-glucose, vinblastine, nocodazole, cytochalasin D, bafilomycin A1, chloroquine, nystatin, digitonin, Lucifer Yellow (LY), poly-l-lysine, valinomycin, Triton X-100, and trypsin were supplied by Sigma. Calcein-AM, FITC-transferrin (Tf), Texas Red-Tf, and DiSC3 (5Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16077Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar) were purchased from Molecular Probes. Reagents for cell biology including Opti-MEM, RPMI 1640, and fetal bovine serum were obtained from Invitrogen. The lipids 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol (POPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Peptide Synthesis and Fluorescent Labeling—All peptides were synthesized according to the Fmoc-tert-butyl strategy using an AMS 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany). Labeling of the N terminus of peptides with the NBD or TAMRA probe was achieved as follow. Briefly, resin-bound peptide was treated with piperidine (20% (v/v) in dimethylformamide) in order to remove the N-terminal Fmoc protecting group. NBD-Cl or TAMRA was added in dry dimethylformamide (5-fold molar excess) in the presence of diisopropylethylamine (DIEA; 2-fold molar excess) for 6 h under agitation in the dark to selectively label the N-terminal amino group. The resin was washed with dimethylformamide and treated with deprotecting mixture to cleave the peptides from the resin and deprotect the side chains. For labeling the CoxIV peptide, the deprotected peptide was S-alkylated with N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) amide (Molecular Probes) on cysteine residues in dimethylformamide. Peptide purification was carried out by reverse phase high pressure liquid chromatography (Water-prep LC 40, Waters) using a 0.01% trifluoroacetic acid/acetonitrile gradient. Purity, as assessed by reverse phase analytic chromatography (Beckman Gold equipped with a diode array detector), was >95% for all peptides by the criterion of relative UV absorbencies at 220 nm and at either 460 (for NBD-labeled peptides) or 540 nm (for TAMRA-labeled peptides). The molecular masses were validated by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDITOF MS; Voyager Elite, Perseptive Biosystems). The peptide concentration of unlabeled peptide was determined by absorption spectroscopy, considering the extinction coefficient of tryptophan, tyrosine, and phenylalanine equal to 5680, 1270, and 260 m–1 cm–1, respectively, at 280 nm. For NBD- and TAMRA-labeled peptides, the extinction coefficients provided by the probe manufacturer were used. Cell Culture—K562 cells were purchased from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% (v/v) calf serum (Invitrogen) without antibiotics. Cells were grown on 100-mm dishes in an atmosphere of 5% CO2 at 37 °C. Quantification of Internalized NBD-labeled Peptides—K562 cells were diluted to 3 × 105 cells·ml–1 in culture medium 1 day before the experiment. Cell association and internalization of peptides were measured by flow cytometry using a FACSCaliber (BD Biosciences) driven by the CellQuest software. The flow cytometer was periodically calibrated using fluorescent beads (Calibrite3, BD Biosciences). The day of the experiment the cells were counted, centrifuged, and resuspended in pre-warmed Opti-MEM (37 °C) to obtain a suspension at 6 × 105 cells·ml–1. After a 1-h incubation, the NBD-labeled peptides were incubated with K562 cells in the same medium at 37 °C (final volume was 0.5 ml). Thereafter, the cells were washed twice and resuspended in 0.5 ml (final volume) of ice-cold PBS for flow cytometry analysis. Cell-associated fluorophores were excited at 488 nm, and fluorescence was measured at 525 nm. A cytogram (1 × 104 counts) was acquired, and the cells exhibiting a normal morphology (∼95% of the total, unless otherwise specified) were taken into account to obtain a histogram of fluorescence intensity per cell. The calculated mean of this distribution was considered as representative of the amount of cell-associated peptides. A stock solution of dithionite (1 m) was freshly prepared in 1 m Tris solution (pH 10). Following flow cytometry analysis, 5 μl of dithionite stock solution was added to cells maintained at 4 °C, and the fluorescence of internalized peptides was measured after 5 min. Peptide and Endocytosis Marker Visualization—TAMRA-labeled peptides (1–3 μm) were incubated with K562 cells in an Opti-MEM medium at 37 °C for 90 min. Thereafter, the cells were washed twice with ice-cold PBS and resuspended in 20 μl (final volume) in the same buffer. Suspensions were plated on glass slides and observed immediately without fixation. For visualizing the intracellular localization of Tf or LY, the cells were incubated with either Texas Red-Tf (incubated for 30 min at 25 μg·ml–1)orLY(90minat1mg·ml–1), washed with ice-cold PBS, plated on poly-l-lysine-coated slides for 45 min, fixed with 3.7% formaldehyde (Sigma) in PBS for 10 min, washed, and mounted with PermaFluor® (CML). Confocal images were obtained with a DMR-B Leica microscope equipped with a argon-krypton ion laser with the excitation line at 488 nm or 568 nm (the emission filter was at 525 and 600 nm, respectively) and fitted with a PerkinElmer Ultraview module (PerkinElmer Life Sciences). The samples were observed with a 100× oil immersion PL AZO HCX lens. The images were captured with a CoolSnap HQ camera (Photometrics) at 696 × 520 pixel resolution. The whole set was controlled with the Meta-Morph software (Universal Imaging Corporation). Quantification of Receptor-mediated and Fluid Phase Endocytosis— K562 cells were incubated in Opti-MEM at 37 °C with FITC-Tf (25 μg·ml–1) for 30 min or LY (1 mg·ml–1) for 90 min at 37 °C in Opti-MEM. The uptake was stopped by washing the cells twice with ice-cold PBS (the final volume was 0.5 ml). Cell-associated fluorophores were excited at 488 nm, and fluorescence was measured at 525 nm. A histogram was generated, and the calculated mean of the distribution of fluorescence intensity per cell (104 counts) was considered as representative of the amount of markers associated with the cells. Calcein-AM Release Assay—The cells were incubated with 2.5 μm calcein-AM for 60 min in Opti-MEM, washed, resuspended in the same volume of calcein-free Opti-MEM, and incubated with peptide for 90 min at 37 °C. The cells were washed once with ice-cold PBS, and the calcein content of cells was determined by flow cytometry. Digitonin (100 μg·ml–1) induced the complete lysis of K562 cells after an incubation of 90 min. The apparent percent leakage value was calculated according to the equation 100 × (F – F 0)/(F max – F 0). F 0 and F max correspond to the mean fluorescence intensity associated with normal cells and digitonin-treated cells, respectively. F represents the fluorescence of the calcein-loaded cells incubated with the peptides. Metabolic and Endocytosis Inhibitor Studies—For experiments at a low temperature, the cells were maintained for 30 min on ice before peptide incubation and throughout the experiment. To induce ATP depletion, the cells were incubated for 60 min with 0.1% sodium azide and 50 mm 2-deoxy-d-glucose (DOG) in Opti-MEM prior to the addition of peptide. The ATP cellular concentration was determined using the bioluminescent assay kit from Sigma according to the manufacturer's instructions. The luminescent signal was measured in a 96-well plate with a Tecan Spectrafluor reader. The inhibition of microtubule polymerization was induced by treating cells with vinblastine or nocodazole at 10 μm or 20 μm, respectively, 1 h prior to the addition of peptide. Cytochalasin D was added at 10 μm for 1 h prior to the addition of peptide to block actin polymerization. For NEM treatment, cells were exposed to NEM (10 μm) in Opti-MEM for 5 min and then washed in a NEM-free medium containing the peptide. The neutralization of pH in intracellular acidic compartments was achieved by pre-incubating cells with 10 mm NH4CL for 10 min or with 50 μm chloroquine or 200 nm bafilomycin A1 for 30 min before peptide addition. For the nystatin treatment, cells were exposed to the reagent at 25 μg·ml–1 for 30 min before the addition of the peptide. Inhibition assays with poly-l-lysine were performed by incubating the peptide for 15 min with cells in the absence or presence of the compound (300 μm). Prior to these experiments, we made sure that the various inhibitors were used at a concentration that maintained the plasma membrane integrity by performing a calcein-AM assay. Measure of Peptide Translocation in Lipidic Vesicles—Large unilamellar vesicles were prepared by drying a lipid film of POPC and POPG (at a 3:1 molar ratio) for 3 h and then resuspending the lipids in buffer (20 mm Tris and 100 mm KCl, pH 7). The lipid suspension was freeze-thawed for five cycles and then extruded through polycarbonate filters (0.1-μm pore size) 21 times. Valinomycin (22.5 μm in ethanol) was added to the large unilamellar vesicles at a lipid-to-valinomycin molar ratio of ∼10,000. The lipid concentration was determined in duplicate by phosphorus analysis as described previously (28Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Fluorescence measurements were performed in 3-ml quartz cells at 25 ± 0.1 °C under constant magnetic stirring using a SLM AB-2 spectrofluorometer (SLM Instruments, Inc., Urbana, IL). For measuring the NBD-labeled peptide translocation, excitation and emission wavelengths were set at 460–485 nm and 530–540 nm, respectively. Excitation and emission band pass values were set at 4 nm for both cases. In the translocation assays, the membrane potential was generated by diluting 100-fold valinomycin-containing vesicles in KCl-free buffer (20 mm Tris and 100 mm NaCl, pH 7). The presence of the negative potential was assessed in a separate assay by measuring the fluorescence of DiSC3 (5Vivès E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16077Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar) probes (λex = 622 nm; λem = 670 nm) as described previously (29Sims P.J. Waggoner A.S. Wang C. Hoffma J.F. Biochemistry. 1974; 13: 3315-3330Crossref PubMed Scopus (764) Google Scholar). Time Course—In previously published studies, we have described a method based on the chemical quenching of the NBD fluorophore by the membrane-impermeant dithionite, allowing direct measurement of the amount of internalized NBD-labeled peptide (30Drin G. Mazel M. Clair P. Mathieu D. Kaczorek M. Temsamani J. Eur. J. Biochem. 2001; 268: 1304-1314Crossref PubMed Scopus (124) Google Scholar). To obtain information about the time course of the internalization of the SynB peptides and the pAntp-(43–58) peptide (Table I), we have applied the NBD/dithionite assay to K562 cells incubated with each NBD-labeled peptide for different incubation times (from 0 to 120 min). As shown in Fig. 1, A and B, the cell uptake of the SynB and pAntp-(43–58) peptides takes place in the first minutes and reaches a maximum after ∼1 h. Fig. 1A also demonstrates that the d-amino acid analogs of SynB1 and SynB3 penetrate into cells as efficiently as their l-amino acid counterparts for the first hour. Furthermore, it can be seen that, over the 2-h period of exposure, the cumulative amounts of internalized d-SynB1 and d-SynB3 were higher than those of the l-counterparts. We believe this may arise from retention of the d-peptides within the endosome compartment, partly due to their resistance to proteolysis. Concentration Dependence of Peptide Internalization and Cell Lytic Activity—The dependence of the initial rate of cellular uptake on peptide concentration (0–40 μm) was examined in K562 cells over a period of 15 min. To control for cell integrity, we used a calcein-AM retention assay in which we observed that unlabeled SynB1 and SynB3 did not significantly increase plasma membrane permeability (>90% of the cell population retained calcein). In contrast, the SynB5 and pAntp-(43–58) peptides at 40 μm started inducing the cell-lysis (Fig. 2A). The amount of internalized SynB peptides increased linearly with concentration (Fig. 2B), implying the absence of saturation in their cellular accumulation. Both the l- and d-forms of SynB1 and SynB3 showed similar behavior. At low concentrations, we observed a higher level of internalization of pAntp-(43–58) and SynB5 than SynB1 and SynB3. However, the lytic activity of SynB5 and pAntp-(43–58) at concentrations above 12 μm and 30 μm, respectively, prevented an investigation of the full range of concentrations for these peptides. Peptide Localization" @default.
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• W1992095148 date "2003-08-01" @default.
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• W1992095148 title "Studies on the Internalization Mechanism of Cationic Cell-penetrating Peptides" @default.
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• W1992095148 doi "https://doi.org/10.1074/jbc.m303938200" @default.
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