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Impact of Reductive Cleavage of an Intramolecular Disulfide Bond Containing Cationic Gemini Surfactant in Monolayers and Bilayers V. Matti J. Sa¨ily,†,‡ Samppa J. Ryha¨nen,†,‡ Hilkka Lankinen,§ Paola Luciani,| Giovanna Mancini,| Mikko J. Parry,† and Paavo K. J. Kinnunen*,† Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, UniVersity of Helsinki, Helsinki, Finland, Peptide and Protein Laboratory, Department of Virology, Haartman Institute, UniVersity of Helsinki, Helsinki, Finland, and CNR, IMC-Sezione Meccanismi di Reazione and Dipartimento di Chimica “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy ReceiVed September 2, 2005. In Final Form: December 7, 2005 The properties of a novel disulfide-bond-containing gemini surfactant bis[N,N-dimethyl-N-hexadecyl-N-(2mercaptoethyl)ammonium bromide] disulfide (DSP) were studied using a Langmuir balance, supported monolayers, differential scanning calorimetry, giant vesicles, and LUVs. In 150 mM NaCl the cmc for DSP was 7.5 µM whereas that of the monomer N,N-dimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide (MSP) was 12.1 µM. Both surfactants exhibited single endotherms upon DSC, with peak temperatures Tm at 21.7 and 20.1 °C for DSP and MSP, respectively. The endotherm for MSP was significantly broader indicating less cooperative melting. Both in monolayers and in vesicles reductive cleavage of the disulfide bond of DSP could be obtained by glutathione (GSH). For Langmuir films of DSP the addition of GSH into the subphase led to a decrease in surface pressure π as well as surface dipole potential Ψ. Although the cleavage by GSH was significantly slower in the presence of a charge saturating concentration of DNA, it did not prevent the reaction. The resulting monomers detached from supported monolayers, leading to loss of affinity of the surface for DNA. Disruption of giant vesicles containing DSP within approximately 30 s following a local injection of GSH was observed, revealing membrane destabilization.

Introduction Promising results with some immunodeficiency syndromes,1 malignant tumors,2 and therapy given in connection to coronary bypass surgery3 have confirmed the therapeutic potential of gene transfer. However, the lack of efficient gene transfer vectors continues to be the major barrier for adapting gene therapy as a routine. Viral vectors were the primary choice; however, they exhibit severe disadvantages such as immunogenicity, limited insert size, difficult production, and biohazards.4,5 Accordingly, lacking most of the problems associated with viral vectors and being relatively easy to make, complexes of DNA with cationic liposomes (lipoplexes) are now considered to represent perhaps the most promising alternative.6,7 So far, however, the transfection efficiency of lipoplexes has been significantly lower than for * To whom the correspondence should be addressed. Tel: 358-9-191 125400. Fax: 358-9-191 25444. E-mail: [email protected]. † Institute of Biomedicine, University of Helsinki. ‡ Contributed equally. § Haartman Institute. | CNR, IMC. (1) Hacein-Bey-Abina, S.; Le Deist, F.; Carlier, F.; Bouneaud, C.; Hue, C.; De Villartay, J. P.; Thrasher, A. J.; Wulffraat, N.; Sorensen, R.; Dupuis-Girod, S.; Fischer, A.; Davies, E. G.; Kuis, W.; Leiva, L.; Cavazzana-Calvo, M. N. Engl. J. Med. 2002, 346, 1185-1193. (2) Weichselbaum, R. R.; Kufe, D. Lancet 1997, 349, 10-12. (3) Mann, M. J.; Whittemore, A. D.; Donaldson, M. C.; Belkin, M.; Conte, M. S.; Polak, J. F.; Orav, E. J.; Ehsan, A.; Dell’Acqua, G.; Dzau, V. J. Lancet 1999, 354, 1493-1498. (4) Hacein-Bey-Abina, S.; Von Kalle, C.; Schmidt, M.; McCormack, M. P.; Wulffraat, N.; Leboulch, P.; Lim, A.; Osborne, C. S.; Pawliuk, R.; Morillon, E.; Sorensen, R.; Forster, A.; Fraser, P.; Cohen, J. I.; de Saint Basile, G.; Alexander, I.; Wintergerst, U.; Frebourg, T.; Aurias, A.; Stoppa-Lyonnet, D.; Romana, S.; Radford-Weiss, I.; Gross, F.; Valensi, F.; Delabesse, E.; Macintyre, E.; Sigaux, F.; Soulier, J.; Leiva, L. E.; Wissler, M.; Prinz, C.; Rabbitts, T. H.; Le Deist, F.; Fischer, A.; Cavazzana-Calvo, M. Science 2003, 302, 415-419. (5) Alesci, S.; Ramsey, W. J.; Bornstein, S. R.; Chrousos, G. P.; Hornsby, P. J.; Benvenga, S.; Trimarchi, F.; Ehrhart-Bornstein, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7484-7489. (6) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413-7417.

viruses. The barriers to cationic lipid-mediated transfection seem to include the difficulty in producing homogeneous complexes, inefficient escape of DNA from the endosomes, poor dissociation of DNA from the complexes, and problems associated with nuclear translocation.8,9 To enhance the efficiency of lipofection an intramolecular disulfide bridge containing cationic lipids has been introduced as one possible solution to overcome the poor release of DNA from lipoplexes and to diminish the cytotoxicity of the cationic vectors.10-18 More specifically, these lipids are expected to release DNA upon reduction by glutathione (GSH), the most abundant intracellular reducing agent present in concentrations up to 10 mM in tissues such as liver.19 Increased transfection efficiencies have indeed been reported for this type of cationic lipid.12 Because of their unique properties, gemini surfactants continue to gain widespread interest both in industry and in academic laboratories.20 The physicochemical properties of geminis are (7) Lasic, D. D., Ed. In Liposomes In Gene DeliVery; CRC Press: Boca Raton, FL, 1997. (8) Zuhorn, I. S.; Hoekstra, D. J. Membr. Biol. 2002, 189, 167-179. (9) Zabner, J.; Fasbender, A. J.; Moninger, T.; Poellinger, K. A.; Welsh, M. J. J. Biol. Chem. 1995, 270, 18997-19007. (10) Hirko, A.; Tang, F.; Hughes, J. A. Curr. Med. Chem. 2003, 10, 11851193. (11) Kumar, V. V.; Chaudhuri, A. FEBS Lett. 2004, 571, 205-211. (12) Tang, F.; Hughes, J. A. Bioconjugate Chem. 1999, 10, 791-796. (13) Tang, F.; Hughes, J. A. Biochem. Biophys. Res. Commun. 1998, 242, 141-145. (14) Wetzer, B.; Byk, G.; Frederic, M.; Airiau, M.; Blanche, F.; Pitard, B.; Scherman, D. Biochem. J. 2001, 356, 747-756. (15) Huang, Z.; Li, W.; MacKay, J. A.; Szoka, F. C., Jr. Mol. Ther. 2005, 11, 409-417. (16) Dauty, E.; Remy, J. S.; Blessing, T.; Behr, J. P. J. Am. Chem. Soc. 2001, 123, 9227-9234. (17) Byk, G.; Wetzer, B.; Frederic, M.; Dubertret, C.; Pitard, B.; Jaslin, G.; Scherman, D. J. Med. Chem. 2000, 43, 4377-4387. (18) Fuchs, S.; Buethe, D.; Khanna, A.; Yadava, P.; Hughes, J. J. Drug Targeting 2004, 12, 347-353. (19) Ostergaard, H.; Tachibana, C.; Winther, J. R. J. Cell Biol. 2004, 166, 337-345.

10.1021/la052398o CCC: $33.50 © 2006 American Chemical Society Published on Web 01/05/2006

ReductiVe CleaVage of a Cationic Gemini Surfactant

Figure 1. Reductive cleavage of the gemini surfactant DSP to yield two MSP monomers. See text for details.

surprisingly complex, and their phase behavior, for example, shows unexpected diversity.21 They are significantly more surface active than conventional surfactants, having very low cmc’s. Interestingly, geminis are potent antimicrobial agents and also hold promise for use in gene therapy.22 Changing the length and hydrophobicity of the spacer has a dramatic impact on the physicochemical properties of geminis and also affects the interaction of cationic geminis with DNA.23 Aligned with the approach described above also reducible gemini surfactants14 have been synthesized; however, their transfection efficiency remains to be verified. In addition to their potential use in transfection reducible gemini surfactants also represent an interesting model for biophysical research. We studied the properties of the cysteamine-based cationic gemini surfactant bis[N,N-dimethyl-N-hexadecyl-N-(2mercaptoethyl)ammonium bromide] disulfide containing an intermolecular disulfide bond (DSP, Figure 1). The cmc values and the thermal phase behavior of DSP and the corresponding monomer were first determined. Subsequently, we investigated the surface properties of DSP using a Langmuir balance and monitored the consequences of its reduction both in the absence and presence of DNA. Whereas in most previous studies dithiothreitol was used as a reducing reagent because of its greater reductive efficiency, we employed the naturally occurring GSH. Complementing the above, we employed surface plasmon resonance (SPR) to study the impact of the disulfide bond cleavage in supported monolayers of DSP. Last, we utilized giant phospholipid vesicles containing DSP to observe the impact of reduction on macroscopic membrane integrity. Materials and Methods Materials. SOPC and GSH were from Sigma, and sodium chloride from J. T. Baker (Deventer, Holland). Herring sperm DNA (average size of e2000 bp) was from Gibco BRL (Carlsbad, CA). The purity of SOPC was checked by thin-layer chromatography on silicic acid coated plates (Merck, Darmstadt, Germany) using chloroform/ methanol/water (65:25:4, by volume) as a solvent system. Examination of the TLC plates after iodine staining revealed no impurities. Lipid concentrations were determined gravimetrically by using a high precision electrobalance (Cahn, Cerritos, CA). Freshly deionized filtered water (Milli RO/Milli Q, Millipore Inc., Jaffrey, NH) was used in all experiments. (20) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39, 19071920. (21) Ryha¨nen, S. J.; Pakkanen, A. L.; Sa¨ily, M. J.; Bello, C.; Mancini, G.; Kinnunen, P. K. J. J. Phys. Chem. B 2002, 106, 11694-11697. (22) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.; Soderman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Garcia Rodriguez, C. L.; Guedat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; van Eijk, M. C. P. Angew. Chem., Int. Ed. 2003, 42, 1448-1457. (23) Chen, X.; Wang, J.; Shen, N.; Luo, Y.; Li, L.; Liu, M.; Thomas, R. K. Langmuir 2002, 18, 6222-6228.

Langmuir, Vol. 22, No. 3, 2006 957 Synthesis of DSP. The reducible cysteamine core based gemini surfactant bis[N,N-dimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide] disulfide (DSP; see Figure 1 for structure) was synthesized as follows. In brief, 0.580 g (2.8 mmol) of N-(2-{[2(dimethylamino)ethyl]dithio}ethyl)-N,N-dimethylamine and 3.41 mL (11.2 mmol) of 1-bromohexadecane were added to 5.6 mL of 2-propanol. The mixture was maintained at room temperature for 2 weeks. The obtained white solid was washed with Et2O. Crystallization from absolute ethanol gave the desired product that was characterized by NMR. 1H NMR (in CDCl ; δ): 4.185 (t, 4H); 3.610-3.484 (m, 8H); 3 3.430 (s, 12H); 1.752 (m, 4H); 1.243 (m, 48H); 0.867 (t, 6H). 13C NMR (in CDCl ; δ): 65.18; 63.93; 51.22; 31.96; 31.70; 29.73; 3 29.70; 29.64; 29.52; 29.43; 29.40; 29.31; 26.31; 22.99; 22.73; 14.16. Anal. Calcd for C39H84N2S2Br2: C, 58.19; H, 10.52; N, 3.48; S, 7.97. Found: C, 58.45; H, 10.53; N, 3.46; S, 8.02. The preparation of N-(2-{[2-(dimethylamino)ethyl]dithio}ethyl)N,N-dimethylamine starting from 2-(dimethylamino)ethanethiol was carried as described.24 The obtained colorless oil was negative in the lead acetate assay25 and was characterized by 1H NMR. 1H NMR (in CDCl ; δ): 2.766 (t, 4H); 2.552 (t, 4H); 2.212 (s, 3 12H). NMR spectra were recorded on a Bruker AC 300 P spectrometer operating at 300.13 and 75.47 MHz for 1H and 13C, respectively, equipped with a thermostated sample tube holder. Signals were referenced with respect to TMS (δ ) 0.000 ppm) used as an internal standard in CDCl3. Determination of Cmc. The cmc for DSP was determined at ambient temperature with a Delta-8 multichannel microtensiometer (Kibron Inc., Helsinki, Finland) and the isotherm analyzed with the Gibbs adsorption model embedded in the software (Delta-8 Manager) provided by the instrument manufacturer. For these measurements serial dilutions in the concentration range from 0.212 µM to 1 mM in deionized water or 130 or 150 mM NaCl were employed, as indicated. To cleave DSP into its monomers 5 mM GSH (final concentration) was added to 1 mM DSP solution and subsequently incubated at +4 °C for 24 h, prior to subjecting to serial dilution and assay for cmc, as described above. Differential Scanning Calorimetry. The surfactant dispersions were hydrated with 150 mM NaCl, vortexed, and maintained on an ice bath for at least 12 h before loading into the calorimeter cuvette (final concentration 1 mM). When indicated, GSH was included to solution prior to incubation on ice. A VP-DSC microcalorimeter (Microcal Inc., Northampton, MA) was operated at a heating rate of 0.5 deg/min, and data were collected during heating scans from 4 to 50 °C. The instrument was interfaced to a PC, and the data were analyzed using the routines of the software provided by the instrument manufacturer. Monolayer Experiments. A computer-controlled Langmuir-type film balance (MicroThrough XS, Kibron Inc.) was used to record compression isotherm (π-A) of DSP. All glassware used was rinsed thoroughly with ethanol and purified water (Millipore). To ensure complete evaporation of the solvents the films were allowed to settle for 4 min before recording of π-A isotherms. DSP was dissolved in chloroform and spread in this solvent onto the surface of 25 mL of 150 mM NaCl at ∼25 °C. The monolayers were compressed at a rate of 4 (Å2/molecule)/min. Surface pressure π is defined as π ) γ0 - γ where γ0 is the surface tension of the air/water interface and γ is the value for surface tension in the presence of a lipid monolayer compressed at varying packing densities. The reciprocal isothermal compressibility, i.e., the elastic modulus of area compressibility (CS-1) was calculated as described previously.26 CS-1 provides an efficient means to determine the onset and completion pressures of possible phase transitions and was thus used to examine the (24) Iranpoor, N.; Zeynizadeh, B. Synthesis 1999, 49-50. (25) Verma, K., K.; Bose, S. Anal. Chim. Acta 1973, 65, 236-239. (26) Smaby, J. M.; Kulkarni, V. S.; Momsen, M.; Brown, R. E. Biophys. J. 1996, 70, 868-877.

958 Langmuir, Vol. 22, No. 3, 2006 compression isotherms in more detail. Accordingly, the higher the CS-1, the lower the interfacial elasticity. Circular wells with Teflon rims and with gold-plated bottom (subphase volume 6 mL, diameter 5 cm) were used for monitoring the changes in surface pressure π and surface dipole potential ψ. When indicated, 330 nmol of glutathione (55 µM final concentration) was added into the magnetically stirred subphase of 150 mM NaCl underneath a DSP monolayer. In some experiments herring sperm DNA (2.5 µM) was included in the subphase prior to inclusion of glutathione. Monolayer dipole potential ψ was measured using the vibrating plate method (µSpot, Kibron Inc.). Surface Plasmon Resonance. SPR measurements were performed with a Biacore 2000 instrument using HPA sensor chips (Biacore AB, Uppsala, Sweden). The surface of the latter is composed of long-chain alkanethiol molecules forming a flat, quasi-crystalline hydrophobic layer. Coating of the HPA sensor surface with DSP was performed at +40 °C following instructions of the manufacturer. In brief, the HPA chip surface was first washed for 5 min with 40 mM n-octyl β-D-glucopyranoside and then coated with 1 mM DSP for 20 min, both at a flow rate of 5 µL/min in water. Reduction of DSP on the HPA chip surface was performed with 3 mM GSH. The binding of herring sperm DNA, 1 µg/mL, was studied for DSP monolayer, after reduction of the surfactant, and for an uncoated HPA surface. The rate of flow in both measurements above was 5 µL/min. Temperature was maintained at 25 °C. Formation of Giant Vesicles. DSP and SOPC were mixed (molar ratio of 1:9) in chloroform after which the solvent was evaporated under a stream of nitrogen. The residue further maintained under reduced pressure for 12 h to remove traces of solvent. The dry lipid residue was subsequently dissolved in diethyl ether: methanol (9:1, by volume) to yield a final total lipid concentration of 1 mM. A 4 µL volume of the above lipid solution was transferred on the surface of the two Pt electrodes in the GUV formation chamber27 and then dried under a gentle stream of nitrogen for at least 10 min.28 Possible solvent residues were removed by evacuation in a vacuum for at least 0.5 h. A glass chamber with the attached electrodes and a quartz window bottom was placed on the stage of an inverted microscope (Olympus IX70, Olympus Optical Co., Tokyo, Japan). An ac field (sinusoidal wave function with a frequency of 4 Hz and an amplitude of 0.2 V) was applied prior to adding 1.6 mL of buffer (0.5 mM Hepes, pH 7.4). During the first 1 min of hydration the voltage was increased to 1.0-1.2 V. The ac field was turned off after 2-4 h, and GUVs were observed with differential interference contrast optics with a 10×/0.30 or 20×/0.40 objective. The sizes of the GUVs were measured using calibration of the images by motions of the micropipet as proper multiples of the step length (50 nm) of the micromanipulator (MX831 with MC2000 controller, SD Instruments, Grants Pass, OR). Images were recorded with a Peltiercooled 12-bit digital CCD camera (C4742-95, Hamamatsu, Japan) interfaced to a computer and operated by the software (HiPic 5.0.1 or Aquacosmos 1.2) provided by the camera manufacturer. When indicated, small aliquots (approximately 50 pL) of glutathione (10 mM) corresponding to 0.5 pmol of GSH were applied from the micropipet onto the outer surface of individual giant vesicles with a pneumatic microinjector (PLI-100, Medical Systems Corp., Greenvale, NY). All experiments were conducted with a Peltiercontrolled thermal microscope stage (TS-4, Physitemp, Clifton, NJ) set to 30 °C. Micropipets29 with inner tip diameters of ∼0.5 µm were drawn from borosilicate capillaries (1.2 mm outer diameter) by a microprocessor-controlled horizontal puller (P-87, Sutter Instrument Co., Novato, CA). 90° Light Scattering. Multilamellar liposomes (MLVs) were prepared by mixing appropriate amounts of the surfactant and lipid stock solutions in dry chloroform to obtain the desired compositions after which the solvent was removed by evaporation under a stream (27) Holopainen, J. M.; Angelova, M.; Kinnunen, P. K. J. Methods Enzymol. 2003, 367, 15-23. (28) Angelova, M.; Dimitrov, D. Faraday Discuss. Chem. Soc. 1986, 81, 303311. (29) Schnorf, M.; Potrykus, I.; Neuhaus, G. Exp. Cell Res. 1994, 210, 260267.

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Figure 2. Surface pressure vs concentration for DSP (solid squares) and MSP (open circles) in 150 mM NaCl. The temperature was ≈24 °C. of nitrogen. For removal of residual amounts of solvent the samples were further maintained under high vacuum for at least 2 h. The resulting dry lipid films were then hydrated with buffer (0.5 mM Hepes, pH 7.4) and thereafter incubated for 30 min above the main transition temperature of the lipid components (approximately 60 °C). To obtain large unilamellar vesicles (LUVs, diameter approximately 100 nm) the hydrated lipid dispersions were vortexed vigorously and then extruded with a LiposoFast small volume homogenizer (Avestin, Ottawa, Canada) by subjecting to 19 passes through polycarbonate filter (100 nm pore size, Nucleopore, Pleasanton, CA). Static light scattering due to the DSP/SOPC 10/90 LUVs was measured with a Perkin-Elmer LS 50B spectrofluorometer with excitation and emission monochromators set at 500 and 501 nm, respectively. A 2 mL volume of the 100 µM LUV solution was placed into a magnetically stirred four-window quartz cuvette thermostated at 25 °C. To observe the effects of GSH addition (630 µL, 32.5 mM) on LUVs containing DSP, scattering values were recorded as a timedrive up to 40 min. Dynamic Light Scattering. Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, U.K.) was used to measure dynamic light scattering, and dedicated software from the instrument manufacturer was used to fit the particle size distribution using standard settings. LUVs were prepared as described above, and 1 mL of the 400 µM LUV solution was placed into a standard plastic cuvette thermostated at 25 °C. To observe the effects of GSH addition (6-100 µL, 32.5 mM) on LUVs containing DSP, scattering values were recorded as a timedrive up to 60 min.

Results Cmc. As the assembly of amphiphiles is critically determined by their cmc values, the current study was initiated by recording Langmuir absorption isotherms for the gemini surfactant DSP and its monomer MSP, produced by cleavage by GSH (Figure 1). The equilibration of DSP between the bulk aqueous phase solution and the air/water interface was rather slow. Accordingly, the absorption of amphiphile to the interface continued up to 180 min, with an equilibrium being reached in approximately 3.5 h. The cmc for the gemini surfactant DSP in 150 mM NaCl was 7.5 ( 0.3 µM, in keeping with the low values reported for geminis20 when compared to conventional surfactants. As the chains of DSP are covalently linked via a disulfide bond, this surfactant can be cleaved by reduction into two monomeric MSPs, representing conventional cationic surfactants. For MSP the cmc was 12.1 ( 0.8 µM (Figure 2). In deionized water the cmc for DSP was ∼10 µM and for MSP ∼220 µM. Similar results were obtained using dithionite as a reductant (data not shown). The difference in the cmc’s readily suggests very different surface

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Figure 3. DSC traces for 1 mM DSP in 150 mM NaCl as such (a) and after incubation for 24 h with 5 mM GSH (b). The calibration bar represents 10 kJ °C-1 mol-1.

properties for these two surfactants which are likely to be reflected in the interactions of these compounds with DNA, for instance. Differential Scanning Calorimetry. Thermotropic phase behavior of DSP and the impact of reductive cleavage of the spacer by GSH to yield two MSPs were determined by DSC. Upon heating, DSP exhibited a main endotherm with a peak at 21.7 °C and two shoulders at approximately 23 and 25 °C, with a total enthalpy of ∼84.39 kJ/mol (Figure 3a). Reduction of DSP with 5 mM GSH caused pronounced changes in the endotherm, decreasing the peak temperature to 20.1 °C and reducing the enthalpy content to 68.54 kJ/mol. The endotherm also became significantly broader thus indicating less cooperative melting (Figure 3b). Monolayer Experiments. Monolayers of DSP on a pure water subphase were unstable, most likely because of lack of screening of the two positive charges in its polar headgroup (data not shown). Accordingly, when 150 mM NaCl was present, stable films were formed. Compression isotherms revealed a smooth π-A curve, lacking indications of phase transitions (Figure 4A). Analysis of the compression isotherms in terms of their compressibility modulus Cs-1 as a function of surface pressure π (Figure 4B) further supported the lack of phase transitions. The maximum for Cs-1, approximately 100 mN/m and representing the lowest interfacial elasticity, was recorded at 32 mN/m, corresponding to 124 Å2/DSP. Surface dipole potential Ψ increased continuously from approximately 100 mV (at π ) 0 mN/m) to 170 mV reached at monolayer collapse observed at a limiting area of ∼118 Å2/ DSP and at a surface pressure of ∼38 mN/m. To monitor the reductive cleavage of DSP in monolayers we injected GSH (55 µM final concentration and yielding saturation with respect to the number of the disulfide bonds in DSP) into the magnetically stirred subphase, while recording changes in π and Ψ. The addition of GSH caused a decrease both in π as well as in Ψ (Figure 5A). The decrement in π started approximately 1 min after the addition of the reductant, with a new equilibrium reached in ∼30 min, with an average decrement of 9.7 mN/m ((3.3 mN/m). Simultaneously, the value for Ψ decreased by approximately 32.9 mV ((15.9 mV). These experiments were reproduced using a range of initial surface pressure values (varying from 10.9 to 18.6 mN/m) yielding surface dipole potentials from 99.9 to 161.1 mV, respectively. The presence of 2.5 µM DNA in the subphase had a pronounced impact on DSP monolayers (Figure 5B). In brief, the values for

Figure 4. (A) Representative π-A (a) and Ψ-A (b) isotherms for a DSP monolayer residing on a 150 mM NaCl subphase. (B) Elastic modulus of area compressibility, CS-1, derived from the π-A isotherm presented in panel A. The temperature was ∼24 °C.

Ψ increased dramatically, at 16 mN/m, for example, from ∼155 to ∼420 mV, yielding a difference of 0.15 fV/molec. The most striking difference was, however, the attenuation in the decrement in ψ and π caused by glutathione. Accordingly, following the addition of GSH it took approximately 3-4 times longer (30 min vs 90-120 min) for the monolayers to reach a new equilibrium in the presence of DNA than in its absence. SPR Experiments. The above studies were substantiated by observing surface plasmon resonance (SPR) for supported DSP monolayers on a HPA sensor surface. Interestingly, when the supported DSP monolayers were subjected to a continuous flow of 3 mM GSH (Figure 6A), decrement from approximately 250 to 80 (RU) was evident, in keeping with reductive cleavage of DSP and subsequent release of MSP from the sensor surface. The kinetics of this process was very similar to that observed for monolayers of DSP residing on air/water interface, with a new steady-state reached in ∼30 min. GSH induced a minor increment in the response level for the uncoated chip, presumably resulting from nonspecific binding of the peptide to the gold surface. As enhanced gene transfection efficiency of cleavable gemini surfactants assumes monomers to have less affinity for DNA, it was of interest to study the attachment of DNA to the supported DSP monolayer and the impact of reduction by GSH (Figure 6B). DNA readily bound to DSP monolayers, as expected, whereas no binding was evident to the GSH-treated DSP membranes or to the uncoated chips. These findings are in keeping with an efficient cleavage of DSP also on supported monolayers as well as the reduced binding of DNA to the monomeric cationic surfactant.

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Figure 5. (A) Changes in surface pressure π (a) and surface dipole potential Ψ (b) as a function of time following the addition (at t ) 0) of a 10 µL aliquot of 33 mM glutathione (corresponding to a final concentration of 55 µM) into the subphase of 150 mM NaCl. T ≈ 24 °C. (B) Similar experiment but recorded in the presence of 2.5 µM DNA (in base pairs) in the subphase. Note the different scale on the x-axis.

Studies with GUVs and LUVs. SOPC was used as a bulk lipid (X ) 0.90), and the mole fraction of DSP was 0.10. With higher contents of DSP formation of GUVs was not observed. The size of a GUV formed varied between approximately 20 and 100 µM. GSH (10 mM) was injected using a micropipet and from a distance of approximately 300 µm with a gentle pressure to avoid the impact of a mechanical pressure pulse on the membrane. Interestingly, in several experiments a bright spot came visible within approximately 5 s after the addition of GSH, moving on the GUV surface. In about 20 s the spot pinched off from the GUV surface and the GUV started shrinking. This process continued at an accelerating rate and led to the disappearance of the GUV in ∼30 s (Figure 7). In control experiments the same amount of the buffer (0.5 mM Hepes, pH 7.4) injected in a similar manner produced no changes in GUV morphology. Prompted by both the monolayer as well as the GUV experiments, we studied if reduction by GSH has an effect DSP/ SOPC (XDSP ) 0.10) LUVs, monitored by static light scattering. Accordingly, if GSH would break the LUVs into micelles, one would expect a decrease in scattering. However, no changes in the scattering were observed. Yet, the addition of anionic detergent SDS (35 mM final concentration) caused sudden and efficient decrease of scattering intensity (data not shown). The effects of GSH on the DSP/SOPC LUVs were further characterized by dynamic light scattering. The addition of GSH did not result in

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Figure 6. (A) SPR response as a function of time following the addition of glutathione (3 mM final concentration, t ) 0) into the aqueous phase rinsing the supported DSP monolayer on the sensor chip surface (a) and with uncoated HPA surface as a control (b). (B) Binding of DNA (1 µg/mL) to a DSP monolayer (a) and after the reduction of the surfactant with glutathione (b) and with uncoated HPA surface (c) used as a control. The above measurements were performed at 25 °C.

decrease of the vesicle size, but instead their diameter slightly increased, from ∼106 to 109 nm, respectively.

Discussion As expected for a gemini surfactant, the cmc for DSP in water was very low, 10 µM, while that for the monomer MSP was ∼0.22 mM. In keeping with increased surface tension of water and augmented screening of the surfactant charges, the cmc’s decreased progressively in the presence of increasing NaCl concentrations. Accordingly, in 150 mM salt the cmc’s for DSP and MSP were 7.5 ( 0.3 and 12.1 ( 0.8 µM, respectively. Adventitious oxidation of MSP could partly explain these rather small differences in cmc’s. GSH caused pronounced alterations also in the thermotropic phase behavior of DSP. More specifically, following cleavage by GSH the endotherms broadened and values for Tm decreased, indicating less cooperative melting and attenuated chain-chain interactions (Figure 3b). The above changes due to reductive cleavage of DSP are readily expected to result from increased charge separation (driven by Coulombic repulsion between the MSP monomers) and diminished interactions between the alkyl chains, thus decreasing both Tm and the

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transition cooperativity. Moreover, both interfacial hydration and the capability for hydrogen bonding change upon dimerization of MSP, which could contribute to the observed behavior as well. Langmuir balance enables to investigate lipids in a systematic manner, with force-area (π-A) isotherms and interfacial elastic moduli of area compressibility (CS-1) providing precise indicators for changes in the film structure.30 Accordingly, it was of interest to use this technique to characterize the surface properties of DSP and monitor the consequences of reductive cleavage by simultaneously assessing π and surface dipole potential Ψ.31 DSP formed stable monolayers only when NaCl was present in the subphase. This is likely to result from screening of the two positive charges of DSP by Cl-, leading to reduced Coulombic repulsion between the surfactant molecules as well as to a change in effective molecular shapes.32 The large lift-off area AL for DSP (225 Å2/molecule) can be explained by assuming the molecules to lie flat with the alkyl chains on the surface as suggested for dihexadecyldimethylammonium bromide,33,34 thus maximizing the distance between the positive charges of DSP. CS-1 exhibited a maximum at 32 mN/m, revealing the minimum in the compressibility of the surfactant films. Geminis have been

concluded to form more coherent interfacial films than their monomeric counterparts.20 The addition of GSH into the subphase underneath a DSP monolayer resulted in a dramatic decrement both in the surface pressure π as well as in surface dipole potential Ψ, as expected from the cleavage of DSP and the resulting escape of MSP into the subphase, in keeping with the observed differences in the cmc’s. SPR measurements complied with the above monolayer results, with DSP coating readily disappearing due to GSH. The loss of DSP from the chip surface had roughly similar kinetics compared to those seen for monolayers (Figure 6A). Coating the SPR surface with DSP caused the signal to increase by 1000 RU whereas flushing the surface with GSH led to reduction only by 120 RU. Accordingly, only a fraction of the surfactant was released by the reductive cleavage of DSP. Previous reports have demonstrated transformation of GUVs subjected to chemical modification, e.g. enzyme action.35-39 Breaking the intermolecular disulfide bond of a gemini by GSH results in the conversion of a two-chain amphiphile into two single-chain amphiphiles. While this at first glance would compare to the action of a lipolytic enzyme such as phospholipase A2, degrading the substrate into lysophospholipid and a fatty acid, this analogy is superficial only. The important difference is that contrasting enzyme action the reductant such as GSH does not act as a catalyst. The microinjection of 10 mM GSH onto the surface of a single giant vesicle of SOPC containing DSP (X ) 0.10) resulted in the shrinkage of GUV within ca. 30 s. In keeping with the monolayer experiments, this should originate from the cleavage of the S-S bond in the spacer of the gemini. The monomers resulting from the reduction appear not to be able to support the bilayer structure required for vesicle stability but instead readily initiate fusion of the GUV with the surrounding lipid matrix as well as an escape of the lipid in the form of clustered monomers, presumably also micelles (Figure 7). Yet, neither static nor dynamic light scattering showed any significant changes after the addition of GSH into DSP containing LUVs. Monomolecular layers residing on the air/water interface and giant vesicles, however, differ in their membrane curvature from LUVs. For an individual gemini surfactant in a giant vesicle the membrane is almost planar and the packing will be closer to that in monolayer than in LUVs. The ability to condense and bind DNA and as well as to release it after reaching the desired compartment in the target cell is crucial when considering the design of lipids for gene delivery. Disulfide-linker strategy aims at an efficient release of DNA by reduction of the linker by intracellular GSH. A disulfide bridge in the spacer of a gemini surfactant may have considerable advantages compared to amphiphiles with a disulfide bridge connecting a hydrophilic headgroup and the hydrophobic chains. Accordingly, for lipoplexes composed of the latter type of surfactants the charged headgroup is likely, at least to some extent, to remain associated with DNA after the reduction of the S-S bridge, thus continuing to screen the negative charges of DNA and keeping it condensed. In keeping with the above, low transfection efficiencies have been reported for cationic lipids of this kind.11 Wetzer et al. showed that lipids bearing disulfide bonds in the hydrophilic part of the molecule were more readily reduced than those with the linker in the alkyl chain. These lipids

(30) Brockman, H. Curr. Opin. Struct. Biol. 1999, 9, 438-443. (31) Brockman, H. Chem. Phys. Lipids 1994, 73, 57-79. (32) Kinnunen, P. K. J. In Handbook of Nonmedical Applications of Liposomes; Lasic, D. D., Barenholz, Y., Eds.; CRC: Boca Raton, FL, 1996; Vol. 1, pp 153-171. (33) Dynarowicz, P.; Vila Romeu, N.; Minones Trillo, J. Colloids Surf., A 1998, 131, 249-256. (34) Sa¨ily, V. M. J.; Ryha¨nen, S. J.; Holopainen, J. M.; Borocci, S.; Mancini, G.; Kinnunen, P. K. J. Biophys. J. 2001, 81, 2135-2143.

(35) Walde, P. In Giant Vesicles; Perspectives in Supramolecular Chemistry Vol. 6; Luisi, P. L., Walde, P., Eds.; Wiley: Zurich, Switzerland, 2000; pp 297311. (36) Holopainen, J. M.; Angelova, M. I.; So¨derlund, T.; Kinnunen, P. K. J. Biophys. J. 2002, 83, 932-943. (37) Jaeger, D. A.; Clark, T., Jr. Langmuir 2002, 18, 3495-3499. (38) Jaeger, D. A.; Zeng, X. Langmuir 2003, 19, 8721-8725. (39) Nurminen, T. A.; Holopainen, J. M.; Zhao, H.; Kinnunen, P. K. J. J. Am. Chem. Soc. 2002, 124, 12129-12134.

Figure 7. Changes in the morphology of a GUV (SOPC:DSP, 9:1 molar ratio) recorded before (A) and 5 (B), 10 (C), 15 (D), 20 (E), and 30 s (F) after the addition of approximately 50 pL aliquot of 10 mM glutathione (corresponding to ∼0.5 pmol of GSH) onto the GUV surface with a micropipet. The temperature was 30 °C. Scale bar ) 20 µm.

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were also amenable to reduction even when complexed to DNA.14 The use of the chemical reduction of the disulfide bond has been claimed to be limited by the required long reaction times (>1 h).40 To this end we observed the reductive cleavage of DSP by GSH both in the absence and in the presence of DNA. Under the conditions used by us the kinetics of the reduction was, however, significantly slower when DNA was present (30 vs 90 min). This can be explained by the DNA bound to the headgroup of the gemini representing a steric hindrance, diminishing the access of GSH to the S-S bond of DSP. Importantly, although attenuating the reduction, the presence of DNA did not inhibit the process. Once the S-S bond in the spacer of DSP becomes reduced (resulting the formation of two single chain amphiphiles), DNA is expected to be released from the complexes. Our SPR experiments provided evidence for diminished affinity of the MSP monomers for DNA (Figure 6B). It is still possible that the MSP monomers would remain bound to DNA in the bulk phase. The avidity of DSP to GSH reduction suggests that this gemini could well be suitable for transfection in vivo. In conclusion, GSH readily cleaved the intramolecular S-S bond of the cationic gemini surfactant DSP in free-standing monolayers on an air-water interface, in supported monolayers immobilized on solid surfaces, and in vesicles. Surface tension measurements confirmed that the gemini surfactant has a lower (40) Jong, L. I.; Abbott, N. L. Langmuir 2000, 16, 5553-5561.

Sa¨ily et al.

cmc than that of the MSP monomer. Thermal phase behavior showed diminished cooperativity and decreased packing for MSP compared to DSP. The kinetics of reduction by GSH were similar both for monolayers residing on an air/water interface and for supported monolayers deposited on a gold surface and monitored by SPR. While the cleavage by GSH was significantly attenuated by charge-saturating concentrations of DNA, the latter did not prevent the reduction. Supported monolayers of DSP readily bound DNA whereas no binding was observed to the DSP monolayers reduced by GSH. In giant vesicles containing DSP, microinjection of GSH caused disruption of the vesicle within approximately 30 s. Interestingly, membrane curvature seems to play a crucial role in the membrane perturbation as for LUVs no changes following the addition of glutathione were observed. Acknowledgment. This study was supported by grants from the Research Foundation of Orion Corp., the Finnish Cultural Foundation, Finnish Medical Society Duodecim, and Finnish Medical Foundation (V.M.J.S.) and Helsinki Biomedical Graduate School (S.J.R.). M. D. Juha-Matti Alakoskela is acknowledged for valuable discussions, and Juha-Pekka Mattila, for help with the chemical nomenclature. The authors thank Kaija Niva and Kristiina So¨derholm for excellent technical assistance. HBBG is supported by the Finnish Academy. LA052398O