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Functional Characterization of an Endosome-disruptive Peptide and Its Application in Cytosolic Delivery of Immunoliposome-entrapped Proteins

2002; Elsevier BV; Volume: 277; Issue: 30 Linguagem: Inglês

10.1074/jbc.m200429200

1083-351X

Enrico Mastrobattista, Gerben A. Koning, Louis van Bloois, Ana C.S. Filipe, Wim Jiskoot, Gert Storm,

Monoclonal and Polyclonal Antibodies Research

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e.peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH2-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes. Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e.peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH2-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes. diphtheria toxin A chain cholesterol 2′,7′-[bis(carboxymethyl)amino]methyl-fluorescein N-succinimidyl-S-acetylthioacetate sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate poly(ethylene glycol) 2000 1,2-distearoyl-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt iso-octylphenoxypolyethoxyethanol monoclonal antibody epidermal growth factor receptor diphtheria toxin HEPES-buffered salt solution phosphatidylcholine phospholipid 2-[N-morpholino]ethanesulfonic acid] egg-derived phosphatidylcholine confocal laser scanning microscopy To exert an optimal therapeutic effect, an administered drug must safely reach not only its target cell but also the appropriate location within that cell. Many biotherapeutic agents (e.g. peptides, proteins, and nucleic acids) act at sites in the cytosol or nucleus that are difficult to reach due to poor membrane permeation characteristics. Therefore, these biotherapeutic agents rely on drug carriers that allow cytosolic delivery of these agents into diseased cells. One attractive strategy to obtain cytosolic delivery is the development of fusion-competent antibody-directed liposomes (immunoliposomes) that, after receptor binding, can fuse with the plasma-membrane of target cells or, after endocytosis of liposomes, with the endosomal membranes. This membrane fusion process should result in cytosolic access of liposome-entrapped drug molecules. Such fusion-competent liposomes can be obtained by disassembling enveloped viruses, isolating their fusion-promoting components, and reassembling these into vesicles to obtain so-called virosomes (1Scheule R.K. Biochemistry. 1986; 25: 4223-4232Crossref PubMed Scopus (22) Google Scholar, 2Stegmann T. Morselt H.W. Booy F.P. van Breemen J.F. Scherphof G. Wilschut J. EMBO J. 1987; 6: 2651-2659Crossref PubMed Scopus (133) Google Scholar, 3Schoen P. Chonn A. Cullis P.R. Wilschut J. Scherrer P. Gene Ther. 1999; 6: 823-832Crossref PubMed Scopus (85) Google Scholar, 4Kaneda Y. Adv. Drug. Del. Rev. 2000; 43: 197-205Crossref PubMed Scopus (161) Google Scholar). Another, more refined approach involves the use of synthetic peptides designed to resemble the putative fusion peptide of viruses (5Plank C. Zauner W. Wagner E. Adv. Drug. Del. Rev. 1998; 34: 21-35Crossref PubMed Scopus (169) Google Scholar, 6Pecheur E.I. Sainte-Marie J. Bienven Hoekstra D. J. Membr. Biol. 1999; 167: 1-17Crossref PubMed Scopus (121) Google Scholar, 7Fujii G. Adv. Drug. Del. Rev. 1999; 38: 257-277Crossref PubMed Scopus (37) Google Scholar, 8Wagner E. Adv. Drug. Del. Rev. 1999; 38: 279-289Crossref PubMed Scopus (188) Google Scholar). It is hypothesized that destabilization of endosomal membranes is less damaging than plasma membrane destabilization. The latter may result in cytotoxicity through eradication of the electrochemical potential generated by the asymmetric distribution of ions across the plasma membrane. Therefore, drug delivery into the cytosol of target cells via endosomal escape is the preferred route. It is expected that fusion and/or membrane-destabilizing mechanisms that allow endosomal escape of liposome-entrapped therapeutic macromolecules will increase the therapeutic availability at the intracellular target site and, consequently, the therapeutic efficacy of liposomal drug formulations. Many viral fusion peptides have been identified. Their mechanistic and structural role in the fusion process occurring between the envelopes of viruses and membranes of host cells has been extensively studied, both with intact viral fusion proteins and with synthetic analogs of the fusion protein domains. One of the best characterized viral fusion peptides is the NH2-terminal domain of influenza virus hemagglutinin subunit HA-2 (9Mechtler K. Wagner E. New J. Chem. 1997; 21: 105-111Google Scholar, 10Oberhauser B. Plank C. Wagner E. Akhtar S. Delivery Strategies for Antisense Oligonucleotide Therapeutics. CRC Press, Inc., Boca Raton, FL1995Google Scholar). Recently, the membrane structure of the NH2-terminal fusion domain of influenza virus hemagglutinin and the low pH-induced conformational change has been resolved by NMR (11Han X. Bushweller J.H. Cafiso D.S. Tamm L.K. Nat. Struct. Biol. 2001; 8: 715-720Crossref PubMed Scopus (402) Google Scholar). It was demonstrated that synthetic analogs of the hemagglutinin fusion domain attached to a hydrophilic sequence inserted into liposomal bilayers in an "inverted V" conformation both at neutral (pH 7.4) and acidic pH (pH 5.0) with residue 12 located at the bilayer surface. At pH 5.0, at which the peptide is rendered fusogenic, the COOH-terminal side of the V-shaped peptide undergoes a conformational change, resulting in the formation of short 310-helix, that allows steeper insertion of the fusion peptide into the bilayer. This steeper insertion is thought to cause membrane destabilization. Studies with synthetic peptides resembling the native sequence of the influenza virus NH2-terminal domain of the HA-2 subunit have clearly demonstrated that such peptides are able to destabilize both model membranes (such as liposomes) and natural membranes in a pH-dependent manner. Fusion peptide-induced lipid mixing and aqueous content mixing between liposomes have been demonstrated, indicating that the peptide analogues have fusogenic capacities (12Matsumoto T. Biophys. Chem. 1999; 79: 153-162Crossref PubMed Scopus (22) Google Scholar,13Düzgünes N. Shavnin S.A. J. Membr. Biol. 1992; 128: 71-80Crossref PubMed Scopus (58) Google Scholar). Influenza virus-derived fusion peptides have been used to enhance the endosomal escape of oligonucleotides (14Pichon C. Freulon I. Midoux P. Mayer R. Monsigny M. Roche A.C. Antisense Nucleic Acid Drug Dev. 1997; 7: 335-343Crossref PubMed Scopus (56) Google Scholar, 15Freulon I. Roche A.C. Monsigny M. Mayer R. Biochem. J. 2001; 354: 671-679Crossref PubMed Google Scholar) and polycation-condensed DNA complexes (5Plank C. Zauner W. Wagner E. Adv. Drug. Del. Rev. 1998; 34: 21-35Crossref PubMed Scopus (169) Google Scholar, 16Kichler A. Mechtler K. Behr J.P. Wagner E. Bioconj. Chem. 1997; 8: 213-221Crossref PubMed Scopus (90) Google Scholar, 17Wagner E. Plank C. Zatloukal K. Cotten M. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7934-7938Crossref PubMed Scopus (660) Google Scholar) after cellular uptake. Surprisingly, despite the wealth of reports on the use of liposomes to functionally characterize fusogenic peptides, only one study reported on the utility of these peptides in enhancing cytosolic delivery of liposome-entrapped drugs (18Baru M. Nahum O. Jaaro H. Sha'anani J. Nur I. J. Drug Target. 1998; 6: 191-199Crossref PubMed Scopus (16) Google Scholar). In this paper, the possibility to utilize co-encapsulation of pH-dependent fusion peptides into liposomal drug formulations as a means to obtain endosomal escape of liposome-entrapped drug molecules and release of these molecules into the cytosol of tumor cells was studied. For this purpose, a dimeric peptide (diINF-7) resembling the NH2-terminal domain of influenza virus hemagglutinin was synthesized. A previous study demonstrated that the use of a dimer is preferred, since dimerization resulted in a strongly increased destabilizing activity toward liposomal bilayers and erythrocytes (10Oberhauser B. Plank C. Wagner E. Akhtar S. Delivery Strategies for Antisense Oligonucleotide Therapeutics. CRC Press, Inc., Boca Raton, FL1995Google Scholar). First, the membrane-destabilizing and fusogenic capacity of diINF-7 either in free form or encapsulated in liposomes was demonstrated by using liposomes as model membranes. Next, the capacity of these peptides to induce endosomal escape of immunoliposome-entrapped bioactive molecules was demonstrated by measuring cytosolic delivery of diphtheria toxin A chain (DTA)1 as a model compound. Co-encapsulation of diINF-7 into DTA-containing immunoliposomes resulted in drastically increased cytotoxicity toward ovarian carcinoma cells, indicating that this peptide can be used to obtain cytosolic delivery of liposome-entrapped drugs with poor membrane permeation capacities. Cholesterol (CHOL), 2′,7′-[bis(carboxymethyl)amino] methyl-fluorescein (calcein),N-succinimidyl-S-acetylthioacetate (SATA), and sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) were obtained from Sigma. Egg phosphatidylcholine and 1,2-distearoyl-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (PEG2000-DSPE) were from Avanti Polar Lipids (Birmingham, AL). Maleimide-PEG2000-DSPE was obtained from Shearwater Polymers (Huntsville, AL). 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PyrPC) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) were purchased from Molecular Probes Europe BV (Leiden, The Netherlands). Iso-octylphenoxypolyethoxyethanol (Triton X-100) was obtained from BDH Laboratory Supplies (Poole, UK). Dithiothreitol was from Pierce. Formaldehyde solution was obtained from Janssen Chimica (Geel, Belgium). Murine mAb 425 of isotype IgG2a (EMD55900) (Merck) directed against the human epidermal growth factor receptor (EGFR) was kindly donated by Dr. G. A. van Dongen (Department of Otolaryngology/Head and Neck Surgery, University Hospital Vrije Universiteit, Amsterdam, The Netherlands). Irrelevant isotype-matched murine mAb directed against the influenza virus HA (clone 12CA5) was kindly donated by E. Boot (Immunology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands). The human ovarian carcinoma cell line NIH:OVCAR-3 originated from the laboratory of Dr. Hamilton (National Cancer Institute, Bethesda, MD) (19Hamilton T.C. Young R.C. McKoy W.M. Grotzinger K.R. Green J.A. Chu E.W. Whang-Peng J. Rogan A.M. Green W.R. Ozols R.F. Cancer Res. 1983; 43: 5379-5389PubMed Google Scholar). OVCAR-3 cells were cultured in Dulbecco's modified Eagle's medium containing 3.7 g/liter sodium bicarbonate, 4.5 g/liter l-glucose and supplemented withl-glutamine (2 mm), HEPES (10 mm), 10% (v/v) fetal calf serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml) at 37 °C with 5% CO2 in humidified air. All cell culture-related material was obtained from Invitrogen. The 24-amino acid peptide INF-7 was synthesized by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase synthesis essentially as described by Plank et al. (20Plank C. Oberhauser B. Mechtler K. Koch C. Wagner E. J. Biol. Chem. 1994; 269: 12918-12924Abstract Full Text PDF PubMed Google Scholar). Crude peptide was precipitated by the dropwise addition of ether and collected by centrifugation. The peptide was washed three times with ether and subsequently dried under a stream of argon followed by high vacuum. Subsequently, the peptide was dissolved in 1 ml of 20 mmammonium bicarbonate, pH 8.5, and freeze-dried. Lyophilized peptide was stored at −20 °C. Purity and identity of the peptide were confirmed by high pressure liquid chromatography (Waters 486; Millipore Corp.), amino acid analysis, and fast atom bombardment mass spectrometry. The presence of a cysteine residue at the carboxyl terminus resulted in dimerization of the INF-7 peptide by disulfide bond formation. DTA was produced essentially as described by Oeltmann and Wiley (21Oeltmann T.N. Wiley R.G. Methods Enzymol. 1988; 165: 204-210Crossref PubMed Scopus (2) Google Scholar) and Carroll et al. (22Carroll S.F. Barbieri J.T. Collier R.J. Methods Enzymol. 1988; 165: 68-76Crossref PubMed Scopus (44) Google Scholar). In short, supernatant of cultures ofCorynebacterium diphtheriae containing high amounts of diphtheria toxin (DT), which was kindly donated by Dr. Kersten (The National Institute of Public Health and The Environment, Bilthoven, The Netherlands) was dialyzed against two 1-liter changes of HEPES-buffered salt solution (HBS; 5 mm HEPES, 150 mm NaCl, pH 7.4) overnight at 4 °C using Slide-A-Lyzer dialysis cassettes (Pierce) with a molecular weight cut-off of 10,000. Four milligrams of dialyzed DT was nicked with trypsin (1 μg/ml in HBS) for 30 min at room temperature, after which the trypsin was inactivated with soybean trypsin inhibitor (5 μg/ml final concentration). DT was reduced and denatured in HBS containing 8 m urea and 0.1 mdithiothreitol and applied to a 1.5 × 50-cm column of Sephacryl S-200 equilibrated in elution buffer (50 mm Tris-HCl, 2m urea, 10 mm dithiothreitol, and 1 mm EDTA, pH 7.5). Fractions of 5 ml were collected and analyzed for the presence of DTA by SDS-PAGE under reducing conditions. Fractions containing DTA were pooled and dialyzed overnight at 4 °C against two 1-liter changes of HBS containing 0.1 m2,2′-dihydroxyethyl disulfide to reversibly block the free sulfhydryl groups. The concentration of DTA was determined spectrometrically,versus appropriate blanks, using the extinction coefficient at 280 nm of 1.5 liters × g − × cm−1. The biological activity of DTA to inhibit protein synthesis was determined with a rabbit reticulocyte lysate in vitrotranslation system (Promega, Madison, WI) (23Carroll S.F. Collier R.J. Methods Enzymol. 1988; 165: 218-225Crossref PubMed Scopus (24) Google Scholar). Lipids used for all liposome preparations were dissolved in CHCl3/MeOH (2:1 (v/v) ratio) and stored at −20 °C under a nitrogen atmosphere. Egg PC and CHOL (30 μmol of lipid) dissolved in the organic solvent were mixed at a molar ratio of 2:1 in a round bottom flask. When indicated, 0.1 mol % (relative to PL) of the lipidic fluorescence probe DiD, 10 mol % PyrPC (relative to PL), or varying amounts of PEG2000-DSPE were added to the lipid mixture prior to solvent evaporation using a rotary evaporator device. The formed lipid films were further dried under a stream of nitrogen for at least 1 h to remove traces of organic solvent. Lipid films were hydrated with 1-ml solutions of HBS, calcein (90 mm), diINF-7 (500 μg/ml in HBS), DTA (250 μg/ml in HBS), or combinations of the latter two substances. Hydration of lipids was facilitated by shaking the flasks in the presence of glass beads. The formed liposomes were subsequently extruded through polycarbonate filters with pore sizes varying from 0.05 to 0.6 μm using a hand extruder from Avanti Polar Lipids. Nonentrapped material was removed from liposomes by centrifugation steps (two 45-min steps at 100,000 × g; 4 °C). Pelleted liposomes were resuspended in 1 ml of HBS. The phospholipid concentration of liposome formulations was determined by the colorimetric method of Fiske and Subbarow (24Fiske C.H. Subbarow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar). The amount of encapsulated protein (DTA and diINF-7) was determined with the BCA protein assay reagent kit (Pierce) after disruption of the liposomes with 0.5% (v/v) Triton X-100. For targeting purposes, conjugates of mAb 425-PEG2000-DSPE were transferred to liposomes essentially as described by Ishida et al. (25Ishida T. Iden D.L. Allen T.M. FEBS Lett. 1999; 460: 129-133Crossref PubMed Scopus (193) Google Scholar). In short, 2.5 mg of mAb 425 was modified with an 8-fold molar excess of SATA to randomly introduce thiol groups. SATA-modified mAb 425 was deacetylated and allowed to react with micelles containing PEG2000-DSPE and maleimide-PEG2000-DSPE at a 4:1 molar ratio. One micromole of liposomes was incubated with 425-PEG-DSPE micelles corresponding to an amount of 30 μg of conjugated protein for 90 min at 40 °C to allow transfer of mAb 425-PEG2000-DSPE to the liposomes. Purification of liposomes was performed on a Sepharose CL-4B column (Amersham Biosciences). With this technique, ∼70% of targeting ligands could be reproducibly transferred into preformed liposomes. OVCAR-3 cells grown to a confluent monolayer were detached from culture flasks by incubating the cells for 5 min at 37 °C with trypsin/EDTA solutions from Invitrogen. Detached OVCAR-3 cells (2 × 105 cells) were incubated with DiD-labeled liposomes in a final volume of 300 μl of culture medium for 1 h at 37 °C. Thereafter, cells were washed with immunofluorescence buffer (1% bovine serum albumin in phosphate-buffered saline, pH 7.4) by two centrifugation steps (5 min at 750 × g) and resuspended into 500 μl of immunofluorescence buffer before being analyzed by flow cytometry with a FACSCalibur flow cytometer (Becton & Dickinson, Mountain View, CA). For confocal laser-scanning microscopy analysis, cells that were incubated with liposomes were washed three times with 1 ml of phosphate-buffered saline before fixation with 2% (v/v) of formaldehyde in phosphate-buffered saline for 20 min at room temperature. After fixation, cells that were adhered to chamber slides were washed twice with phosphate-buffered saline and overlaid with cover slides that were sealed with nail polish. Cells were analyzed on a Leica TCS-SP confocal laser-scanning microscope equipped with a 488-nm argon, 568-nm krypton, and 647-nm HeNe laser. CD measurements were performed with a dual-beam DSM 1000 CD spectrophotometer (On-Line Instrument Systems, Bogart, GA). The subtractive double-grating monochromator was equipped with a fixed disk, holographic gratings (2400 lines/mm, blaze wavelength 230 nm), and 1.24-mm slits. Cuvettes with a path length of 1 mm were used, and the peptide concentration was 100 μg/ml. CD spectra were recorded from 250 to 200 nm at 25 °C. Each measurement was the average of six repeated scans (step resolution 1 nm, 1 s each step) from which the corresponding background spectrum was subtracted. Quantitative prediction of the helical content was accomplished by fitting CD data with the Hennessey-Johnson algorithm (26Hennessey J.P.J. Johnson W.C.J. Biochemistry. 1981; 20: 1085-1094Crossref PubMed Scopus (574) Google Scholar), by the program CDNN in "simple spectra" configuration (27Böhm, G. (1997) CDNN CD Spectra Deconvolution Software, Version 2.1Google Scholar), using a protein data base with known secondary structure as the basis set. Liposomes composed of EPC and CHOL (molar ratio of 2:1) and with increasing amounts of PEG2000-DSPE (0, 2.5, 5, and 10 mol %) were added to a cuvette at a concentration of 300 or 1000 μm in a final volume of 1.5 ml of HBS. Immediately after the addition of diINF-7 (lipid/peptide molar ratio of 50), peptide-induced aggregation of liposomes at the indicated pH values was determined by measuring the increase in optical density of the liposome dispersion with a spectrophotometer at 500 nm over a period of 8–12 min at room temperature. A serial dilution (1:5) of free or liposome-encapsulated diINF-7 peptide was prepared in a 96-well microtiter plate by transferring 20 μl of the initial peptide solution (50 μg/ml for free peptide and 0.3 μg/ml for liposome-entrapped peptide) from one well to the next well containing 80 μl of HBS. Calcein-containing liposomes were added to each well at a final concentration of 40 μm. After a 60-min incubation at room temperature, samples were analyzed for fluorescence with an LS50B fluorimeter (PerkinElmer Life Sciences) set at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Calcein leakage was expressed relative to the difference in the fluorescence of calcein liposomes incubated in the absence of diINF-7 peptide (0% leakage) and the fluorescence of calcein liposomes that had been disrupted with 0.5% (v/v) Triton X-100 (100% leakage). The effect of Triton X-100 on calcein fluorescence intensity was negligible at the tested concentration (28Memoli A. Palermiti L.G. Travagli V. Alhaique F. J. Pharmacol. Biomed. Anal. 1999; 19: 627-632Crossref Scopus (19) Google Scholar, 29Memoli A. Palermiti L.G. Travagli V. Alhaique F. J. Pharmacol. Biomed. Anal. 1994; 12: 307-312Crossref Scopus (4) Google Scholar). diINF-7-induced lipid mixing was monitored with the pyrene excimer assay developed by Galla and Hartmann (30Galla H.J. Hartmann W. Chem. Phys. Lipids. 1980; 27: 199-219Crossref PubMed Scopus (194) Google Scholar). Liposomes (5 μm) labeled with 10 mol % PyrPC (Molecular Probes, Inc., Eugene, OR) were mixed in a total volume of 1.4 ml of HBS with a 20-fold molar excess of nonlabeled donor liposomes in a cuvette kept at 37 °C. diINF-7 peptide was added at the amounts indicated, and after a 2-min equilibration period, the pH of the medium in the cuvette was lowered to 5.2 by adding one-twentieth volume of fusion buffer (0.1 m Mes, 0.1 macetic acid, pH 4.1). Fusion was continuously monitored at 37 °C by measuring the decrease in PyrPC excimer fluorescence with an LS50B fluorimeter set at an excitation wavelength of 345 nm and an emission wavelength of 480 nm while the cuvette contents were continuously stirred. The decrease of fluorescence was expressed relative to the difference in the initial fluorescence and the excimer fluorescence at infinite dilution, which was obtained by disrupting the liposomes with 0.5% (v/v) Triton X-100 (final concentration). The occurrence of diINF-7-induced intermixing of liposome-entrapped solutes was determined with a fluorescent assay based on quenching of calcein with bivalent metal ions (31Kendall D.A. MacDonald R.C. J. Biol. Chem. 1982; 257: 13892-13895Abstract Full Text PDF PubMed Google Scholar). Unilamellar liposomes of 150 nm in size containing calcein (0.8 mm) and CuSO4 (1 mm) in HBS were admixed at a concentration of 50 μm with a 5-fold molar excess of liposomes containing 20 mm EDTA in a stirred cuvette kept at a temperature of 37 °C. diINF-7 was added at a concentration of 5 μg/ml. The pH of the buffer was lowered to 5.2 by adding one-twentieth volume of fusion buffer. Aqueous contents mixing and/or leakage were continuously monitored at 37 °C by measuring the increase in calcein fluorescence with a fluorimeter set at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. To be able to determine the contribution of both leakage and aqueous content mixing to the total increase in calcein fluorescence, one-twentieth volume of 1 mmCuSO4 was added to the external medium during the fusion process. OVCAR-3 cells were plated in 96-well cell culture plates at 5 × 103 cells/well and cultured for 24 h. Culture medium was removed by decanting the plates, and cells were overlaid with 100 μl of fresh culture medium. To the first well of each column 100 μl of liposome formulation was added at a final lipid concentration of 1 mm (corresponding to 0.5 μg/ml of DTA). 2-Fold serial dilutions of liposome formulations were prepared by transferring 100 μl of the first well in a column to the next and so forth. After a 1-h incubation at 37 °C, liposomes were removed from the cells by decanting the plates, and cells were washed with 100 μl/well of fresh culture medium and overlaid with 100 μl of fresh culture medium before incubation was continued. Forty-eight hours after the addition of liposome formulations, cell viability was assessed with the colorimetric XTT assay as previously described (32Arigita C. Zuidam N.J. Crommelin D.J. Hennink W.E. Pharm. Res. (N. Y.). 1999; 16: 1534-1541Crossref PubMed Scopus (128) Google Scholar). The primary amino acid sequence of diINF-7 is presented in TableI. Compared with the native sequence of the NH2-terminal domain of the influenza virus HA-2 subunit, diINF-7 differs in only two amino acids; glycine at position 4 and alanine at position 7 have been substituted by glutamic acid residues. These substitutions favor the pH dependence of the fusogenic activity of the peptide. Since fusogenic activity is thought to be directly correlated with α-helix formation of the peptide (6Pecheur E.I. Sainte-Marie J. Bienven Hoekstra D. J. Membr. Biol. 1999; 167: 1-17Crossref PubMed Scopus (121) Google Scholar,33Düzgünes N. Gambale F. FEBS Lett. 1988; 227: 110-114Crossref PubMed Scopus (34) Google Scholar, 34Wharton S.A. Martin S.R. Ruigrok R.W. Skehel J.J. Wiley D.C. J. Gen. Virol. 1988; 69: 1847-1857Crossref PubMed Scopus (203) Google Scholar, 35Brasseur R. Mol. Membr. Biol. 2000; 17: 31-40Crossref PubMed Scopus (79) Google Scholar), the secondary structure of diINF-7 was determined by circular dichroism spectropolarimetry at both pH 7.4 and pH 5.2 in the presence or absence of detergent-solubilized phospholipids. Since Subbaraoet al. (36Subbarao N.K. Parente R.A. Szoka F.C.J. Nadasdi L. Pongracz K. Biochemistry. 1987; 26: 2964-2972Crossref PubMed Scopus (274) Google Scholar) showed that interpretation of CD spectra below 222 nm in the presence of phospholipid vesicles is ambiguous due to liposome scattering, we used mixed micelles of phospholipids and detergent to determine the effect of a hydrophobic environment on the helical content of the peptide. The CD spectrum (Fig.1) of the free peptide at pH 5.2 shows minima at 208 and 222 nm, typical of a highly α-helical structure; in contrast, at neutral pH, the diINF-7 peptide is primarily randomly coiled (Fig. 1A and Table II). Interestingly, the presence of detergent-solubilized phospholipids can also induce α-helix formation of diINF-7 (Fig. 1B and Table II).Table IAmino acid sequences of fusion peptidesPeptideSequenceInf HA-2GLFGAIAGFIENGWEGMIDGWYG—–diINF-7GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMIDGWYGCINF HA-2, amino-terminal sequence of Influenza virus X-31 (H3N2) hemagglutinin subunit HA-2; diINF-7, peptide analogue resembling Inf HA-2 dimerized at the COOH terminus by disulfide bond formation between COOH-terminal cysteine residues. Differences in amino acid sequence of diINF-7 compared with the native Inf HA-2 sequence are highlighted in boldface type. Open table in a new tab Table IIMolar absorbance difference at 222 nm (Δε222) and helical content of diINF-7 in the absence or presence of mixed micelles and at different pH valuesMixed micellespHΔɛ222Helical contentm−1cm−1%7.4−0.675155.2−2.75131EPC/OG (1 mm/40 mm)7.4−2.53829EPC/OG (1 mm/40 mm)5.2−4.68653 Open table in a new tab INF HA-2, amino-terminal sequence of Influenza virus X-31 (H3N2) hemagglutinin subunit HA-2; diINF-7, peptide analogue resembling Inf HA-2 dimerized at the COOH terminus by disulfide bond formation between COOH-terminal cysteine residues. Differences in amino acid sequence of diINF-7 compared with the native Inf HA-2 sequence are highlighted in boldface type. In this study, neutral liposomes (EPC/CHOL molar ratio 2:1) with and without incorporated PEG2000-DSPE were used as model membranes to test the membrane-destabilizing capacity of diINF-7 and targeted drug carriers to deliver DTA into the cytosol of tumor cells. Liposomes wer