Membrane Permeabilization and Efficient Gene Transfer by a Peptide

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Bioconjugate Chem. 1998, 9, 260−267

Membrane Permeabilization and Efficient Gene Transfer by a Peptide Containing Several Histidines Patrick Midoux,* Antoine Kichler, Vale´rie Boutin, Jean-Claude Maurizot, and Michel Monsigny Centre de Biophysique Mole´culaire, CNRS et Universite´ d’Orle´ans, rue Charles-Sadron, F-45071 Orle´ans Cedex 02, France. Received September 4, 1997; Revised Manuscript Received December 10, 1997

We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH; at pH 6.8, the peptide permeabilized 50% of the cells at 20 °C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum. Therefore, this peptide containing several histidines that become positively charged when the pH decreased to less than 7.0 is a suitable helper for delivering molecules into the cytosol upon either permeabilization of the plasma membrane induced by lowering the extracellular medium to pH 6.4 or permeabilization of the endosomal membrane induced by acidification of endosomes.

INTRODUCTION

The introduction of nucleic acids into cells is of great interest for the development of gene therapy. Various nonviral vectors are available. Cationic lipids such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (1) or dioctadecylamidoglycylspermine (DOGS) (2) and cationic polymers such as polylysine conjugates (3-6) or polyethylenimine (7) are synthetic vectors that form lipoplexes and polyplexes with DNA (8), suitable for gene delivery into eukaryotic cells. Recognition signals, including asialoglycoprotein, transferrin, insulin, immunoglobulins, growth factors, carbohydrates, and metabolites, linked to polylysine (for reviews, see refs 3-6) and to polyethylenimine (9) increased the cell specificity of polyplexes. Cationic lipids and cationic polymers are also suitable for oligonucleotide delivery; DOTMA (10, 11) and polyethylenimine (7) enhanced oligonucleotide cellular uptake. An antisense oligonucleotide complexed with polylysine substituted with either asialoglycoprotein (12), transferrin (13), folate (14), or fucoside (15) inhibited hepatitis B viral, myc, myb, and ICAM-1 gene expression, respectively. Nucleic acids must be delivered into the cytosol and/ or the nucleus. To increase the transmembrane passage of the DNA into the cytosol, several helper molecules, including lipids, chloroquine, glycerol, or fusogenic peptides, were used. Dioleoylphosphatidylethanolamine (DOPE), a neutral fusogenic lipid, was mixed with DOTMA to give lipofectin, which is commonly used for gene and oligonucleotide delivery (1). Chloroquine, a weak base which prevents the delivery of the endosomal content to lysosomes, induces the formation of large endosome-derived vacuoles, and destabilizes their membrane, enhancing the polyfection with transferrin-polyl* To whom correspondence should be addressed. Fax: 33 2 38 69 00 94. Phone: 33 2 38 25 55 95. E-mail: [email protected].

ysine (16) and glycosylated polylysine (17, 18); in addition, chloroquine allows the dissociation of glycosylated polylysine-plasmid complexes (19). The inhibition of ICAM-1 gene expression by antisense oligonucleotidefucosylated polylysine complexes increased drastically in the presence of chloroquine (15). Enhancement of the polyfection with transferrin-polylysine was also reported in the presence of glycerol (20). Amphiphilic peptides exhibiting fusogenic properties in acidic media were used in the development of synthetic virus-like gene-transfer vehicles. Amphiphilic anionic peptides such as GLFEAIAGFIENGWEGMIDGGGC (21, 22), GLFEAIEGFIENGWEGMIDGWYG (23) derived from the sequence of the N-terminal segment of the HA-2 subunit of the influenza virus hemagglutinin involved in the fusion of the viral envelope with the endosomal membrane, increased the polyfection with transferrin-polylysine complexes. The amphiphilic anionic peptide GLFEAIAEFIEGGWEGLIEG (E5) corresponding to the N-terminal segment of the HA-2 subunit of the A/PR/8/34 influenza virus hemagglutinin in which G-4, G-8, T-15, and D-19 were replaced by glutamyl residues and M-17 was replaced by a leucyl residue, which induces membrane fusion and permeabilization at acidic pH (24-26), increased the polyfection with lactosylated polylysine (17). Other amphiphilic anionic peptides such as GLFEALLELLESLWELLLEA (27), NPVENYIDEVLNEVLVVPNININSSNC [the sequence of the N-terminal segment of VP1 of HRV2 rhinovirus (28)], or WEAALAEALAEALAEHLAEALAEALEALAA (GALA) (23, 29) were also found to enhance the gene-transfer efficiency of various polylysine conjugate-DNA complexes. These amphiphilic anionic peptides which contain acid residues (mainly glutamate) are supposed to increase the transmembrane passage of the plasmid into the cytosol upon the membrane disruption of the acidic endocytotic vesicles; this permeabilization is attributed to the protonation of the acid residues of the peptides, and the protonation allows the peptide to

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Permeabilizing Activity of a Histidylated Peptide

change its conformation from random to R-helix as shown by circular dichroism. Similarly, amphiphilic basic peptides such as melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2), a peptide isolated from the venom of the european honey bee Apis mellifera (30), and GLFKAIAKFIKGGWKGLIKG (K5), an amphiphilic cationic peptide in which the glutamyl residues of the anionic peptide E5 in positions 4, 8, 11, 15, and 19 were replaced by lysyl residues (25), and WKAALAKALAKALAKHLAKALAKALKALAA (KALA) (31) exhibit membrane fusion and permeabilization activities at neutral pH. Melittin coupled to dioleoylphosphatidylethanolamine and complexed with DNA was reported to be efficient in transfecting cells (32). Nevertheless, it is noteworthy to emphasize that those cationic peptides as well as the above-mentioned anionic peptides are inefficient when the transfection step is conducted in the presence of serum. Knowing that the imidazole group of histidine has a pK of around pH 6.0 and thus becomes cationic in a slightly acidic medium, we hypothesized that changing the lysyl residues of the peptide K5 by histidine residues uncharged at neutral pH but cationic in slightly acidic medium (at a pH close to that found in the lumen of the endocytotic vesicles) will not permeabilize the plasma membrane of the cells under classical conditions, medium at pH 7.4, but will selectively disrupt the membrane of acid vesicles and will lead to the transmembrane passage of plasmids into the cytosol. In this work, we describe the synthesis and characterization of the peptide GLFHAIAHFIHGGWHGLIHGWYG (H5WYG1 ), which is analogous to the N-terminal segment (GLFGAIAGFIEGGWTGMIDGWYG) of the HA-2 subunit of the influenza virus hemagglutinin in which G-4, G-8, E-11, T-15, and D-19 were replaced by histidyl residues and M-17 was replaced by a leucyl residue. We show that this peptide exhibits a very efficient membrane permeabilization activity at pH 6.8; at such a very slightly acidic pH, the amphiphilic anionic E5WYG peptide was completely ineffective. We also demonstrate that this peptide increases the gene-transfer efficiency of cells by using glycosylated polylysine-DNA complexes in a pH-dependent manner, and that the efficient gene transfer is not impaired by the presence of serum. MATERIALS AND METHODS

Chemicals. Luciferin, chloroquine, bafilomycin A1, and Triton X-100 were from Sigma (St. Louis, MO). L-Glutamine, dimethyl sulfoxide, ATP, glycerol, and MgCl2 were from Merck (Darmstadt, Germany). Dithiothreitol was from Serva (Heidelberg, Germany). Poly(L-lysine) hydrobromide 30000-50000 (average degree of polymerization of 190) was from Bachem Feinchemikalien (Bubendorf, Switzerland). Poly(L-lysine) hydrobromide (1 g in 200 mL of H2O) was transformed to its p-toluenesulfonic acid salt as described previously (33). Polylysine was partially substituted with an average number of 60 lactosyl residues as previously described (17). Peptide H5WYG. H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) and E5WYG (GLFEAIAEFIEGGWEGLIEGWYG) were purchased from Severn Biotech Ltd. (Kidderminster, Worcester, U.K.); alternatively, peptides were synthesized with the Fmoc strategy. E5WYG was 1 Abbreviations: BET, ethidium bromide; BVC, p-[β-[7-(dimethylamino)-1,4-benzoxazin-2-on-3-yl]vinyl]phenylpropenoic acid; E5WYG, GLFEAIAEFIEGGWEGLIEGWYG; FBS, fetal bovine serum; H5WYG, GLFHAIAHFIHGGWHGLIHGWYG; Lact, lactosyl residue; pLK, poly(L-lysine); RLU, relative light unit.

Bioconjugate Chem., Vol. 9, No. 2, 1998 261

purified as previously described (17). H5WYG was purified by HPLC on a C18 Spherisorb ODS, 5 mm column by using a linear gradient in H2O containing 0.5% trifluoroacetic acid over the course of 40 min from 0 to 80% acetonitrile. The peptide was eluted with a retention time of 23.4 min. The peptide was characterized by mass spectrometry using a matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometer (calcd 2584.6 Da, found 2584.7 Da). The peptide concentration was determined by using an 280 nm of 12 200 M L-1 cm-1. Spectrofluorimetry. Fluorescence measurements were performed at 25 °C with a Shimadzu RF-5000 spectrofluorimeter (Roucaire, Courtaboeuf, France). Solutions were contained in a 5 × 5 mm quartz cuvette; their absorbance (A1cm) was lower than 0.1 at the excitation wavelength. Circular Dichroism. Circular dichroism measurements were carried out on a Jobin-Yvon Mark IV dichrograph. Data acquisition and analysis were performed on a microcomputer interfaced to the spectrometer. The instrument had been calibrated with camphorsulfonic acid-d10. Optical quartz cells with a path length of 0.1 cm were used under nitrogen. The temperature of the cell was maintained at 20 ( 0.5 °C with a circulating water bath. Curves were signal-averaged with a minimum of four repeats and presented with a similarly signal-averaged buffer baseline subtracted. Cells and Cell Culture. U937 promonocytic cells (a human histiocytic lymphoma cell line, ATCC CRL 1593, ATCC, Rockville, MD) were cultured in RPMI 1640 medium (Gibco, Renfrewshire, U.K.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco); HepG2 cells (a human hepatoma cell line, ATCC 8065 HB) in MEM (Gibco) supplemented with 10% FBS and 1% non-essential amino acids; and Rb-1 cells [rabbit smooth muscle cells, kindly provided by M. Nachtigal (University of South Carolina, Columbia, SC)] (34) and B16 cells (a murine melanoma cell line, ATCC CRL 6322) in DMEM medium (Gibco) supplemented with 10% FBS and new-born calf serum (Gibco), respectively. All the culture media were supplemented with 2 mM L-glutamine (Merck), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Eurobio, Paris, France). Cells were cultured at 37 °C in a humidified atmosphere (95% air and 5% CO2). Cells were mycoplasma-free as shown by using the bisbenzimide Hoechst 33258 (35) and the BVC-kanamycin A staining methods (36). Membrane Permeabilization Studies. The membrane permeabilization activity of H5WYG was assessed by using flow cytofluorometry as described previously (26). U937 cells growing in RPMI complete medium were collected in the exponential-growth phase and washed in sheath fluid [134 mM NaCl, 3.7 mM KCl, 15.2 mM NaF, 1.9 mM KH2PO4, 16.5 mM Na2HPO4, and 0.2% 2-phenoxyethanol (pH 7.4)]. The sheath fluid (1 mL) containing 5 × 105 U937 cells was made 12.5 µM in ethidium bromide (BET, Molecular Probes, La Jolla, CA) (5 µL of a 1 mg/mL stock solution in H2O) at 20 °C and then 36 µM in H5WYG (50 µL of a 0.84 mM stock solution in H2O). The pH of the cell suspension was then lowered by adding aliquots (10-70 µL) of a 0.3 N HCl solution. The pH values were controlled by using a pH meter. After a 5-15 min incubation, the cell fluorescence intensity was measured by using a FACSort cytometer (Becton Dickinson, Sunnyvale, CA). A 488 nm excitation wavelength was produced by a 15 mW cold air argon laser. A 585 ( 44 nm long-pass filter was used to collect the emitted red fluorescence. The side scatter, the forward

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scatter, and the red fluorescence were simultaneously recorded in the list mode on a minimum of 10 000 cells at 300 events per second. The data were analyzed by using the Cell Quest software (Becton Dickinson). Plasmid. pSV2LUC plasmid (5 kb), kindly provided by A. B. Brasier (Massachusetts General Hospital, Boston, MA) (37), is an expression vector encoding the firefly luciferase gene under the control of the SV40 T large antigen promoter. pUT650 plasmid (5149 bp) from CAYLA (Toulouse, France) is an expression vector encoding the firefly luciferase gene under the control of the human cytomegalovirus promoter. Supercoiled DNA plasmids were isolated by a standard alkaline lysis method followed by CsCl gradient centrifugation in the presence of BET, extensive extraction with n-butanol, and precipitation with ethanol. Gene Transfer. HepG2 cells (3 × 105 cells per 4 cm2 well) and Rb-1 cells (1 × 105 cells per 4 cm2 well) were plated (day 0) into 12-well tissue culture plates; B16 cells (8 × 104 cells per 2 cm2 well) were plated (day 0) into 24-well tissue culture plates. Polyplexes between a plasmid and lactosylated polylysine (LactpLK) were made with the lowest polymer to DNA molar ratio giving a complete retardation of all the DNA in electrophoresis and used to transfect cells as previously described (18). pSV2LUC or pUT650 (10 µg, 3 pmol) in 0.7 mL of serumfree DMEM was mixed with LactpLK (30 µg, 340 pmol) in 0.3 mL of serum-free DMEM, and the solution was kept for 30 min at 20 °C. Then the solution was made either 100 µM in chloroquine, 10 µM in E5WYG, or 10 µM in H5WYG and then supplemented with 1, 20, or 50% FBS, unless otherwise stated. On day 1, the medium was removed and the polyfection solution was added into each well: 1 mL in the case of 12-well tissue culture plates or 0.5 mL in that of 24-well tissue culture plates. After incubation for 4 h at 37 °C in a humidified atmosphere (95% air and 5% CO2), the medium was removed and cells were further incubated for 48 h at 37 °C in 2 mL of fresh complete culture medium containing 10% serum, in a humidified atmosphere (95% air and 5% CO2). Experiments were carried out in triplicate. Luciferase Assay. Luciferase gene expression was measured by monitoring its luminescence activity according to De Wet et al. (38) as previously described (18). The luminescence was recorded for 4 s in a Lumat LB 9501 luminometer (Berthold, Wildbach, Germany). Measurements were carried out in duplicate. The data shown correspond to the number of relative light units (RLU) from 106 cells. The number of RLU from a 1 pg/mL luciferase solution was 2000. RESULTS

Peptide Characterization. The peptide used, GLFHAIAHFIHGGWHGLIHGWYG (H5WYG), is an analogue of the N-terminal segment (GLFGAIAGFIEGGWTGMIDGWYG) of the HA-2 subunit of the influenza virus hemagglutinin in which G-4, G-8, E-11, T-15, and D-19 were replaced by histidyl residues and M-17 was replaced by a leucyl residue. The absorbance and fluorescence spectra of a 16 µM peptide solution in various buffers in the 9.0-4.5 pH range were studied. The absorbance spectrum of H5WYG depended on the pH of the medium (Figure 1). The absorbance at the maximal wavelength (279 nm) decreased when the pH decreased with a midpoint at pH 5.7, and the ratio of the maximal absorbance at 279 nm to the minimal absorbance between 252 and 260 nm (Amax/Amin) increased when the pH decreased with a midpoint at pH 5.7. These data indicate that (i) H5WYG was slightly associated or

Midoux et al.

Figure 1. pH dependence of the absorbance of H5WYG. A stock solution (1 mg/mL in H2O) was diluted to 16 µM with the following buffers: from pH 4.5 to 7.0, 0.15 M NaCl, 50 mM citrate phosphate buffers and 0.15 M NaCl, 50 mM sodium phosphate buffer (pH 7.4); and from pH 8.0 to 9.0, 0.15 M NaCl, 50 mM Tris-HCl buffers. (b) Absorbance at 280 nm and (9) the 280 nm absorbance/255 nm absorbance ratio.

Figure 2. pH dependence of the fluorescence of H5WYG. A stock solution (1 mg/mL in H2O) was diluted to 16 µM with the following buffers: from pH 4.5 to 7.0, 0.15 M NaCl, 50 mM citrate phosphate buffers and 0.15 M NaCl, 50 mM sodium phosphate buffer (pH 7.4); and from pH 8.0 to 9.0, 0.15 M NaCl, 50 mM Tris-HCl buffers. The fluorescence spectra were recorded under a 290 nm excitation wavelength. (b) Maximal fluorescence intensity and (9) the wavelength at the maximal emission intensity.

aggregated at neutral pH, (ii) peptide aggregates dissociated between pH 6.5 and 5.0, and (iii) H5WYG was not associated or aggregated at pH