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Supported lipid bilayers lifted from the substrate by layer-by-layer polyion cushions on self-assembled monolayers
Colloids and Surfaces B: Biointerfaces 28 (2003) 319 /329 www.elsevier.com/locate/colsurfb
Supported lipid bilayers lifted from the substrate by layer-bylayer polyion cushions on self-assembled monolayers Chen Ma a, M.P. Srinivasan a,1, Alan J. Waring b, Robert I. Lehrer c, Marjorie L. Longo a, Pieter Stroeve a,* a
Department of Chemical Engineering and Materials Science, University of California Davis, Davis, CA 95616, USA b University of California at Los Angeles School of Medicine and Harbor /UCLA, Los Angeles, CA 90095, USA c Department of Medicine and Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA Accepted 10 October 2002
Abstract The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/ PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2 /2.1) /10 9 cm2/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes. # 2002 Elsevier Science B.V. All rights reserved.
1. Introduction
* Corresponding author. Tel./fax: /1-530-752-8778. E-mail address: [email protected] (P. Stroeve). 1 Present address: Department of Chemical and Environmental Engineering, The National University of Singapore, Singapore.
Since a supported bilayer membrane was first used to investigate cellular immune responses [1], solid-supported lipid bilayers have been a widely studied topic of practical and scientific interest. Being well-defined models of biological membranes, phospholipid bilayers supported on solid
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substrates are important for their roles in fundamental biophysical research as well as in applications such as biosensors [2]. Supported lipid bilayer membranes have been formed onto glass, quartz, and silicon surfaces [1,3 /8], onto nonfunctionalized metal surfaces [9 /14], or onto selfassembled alkanethiol monolayers [15 /22]. Methods for the bilayer formation have included the Langmuir /Blodgett technique [1,2,9,11,12,18,23], vesicle fusion onto the substrate [1,23/25], spontaneous thinning of lipid/decane mixtures [14,25], and adsorption of charged lipids onto oppositely charged surfaces [20,26,27]. A bilayer deposited directly on silica, glass, or gold is a model membrane with a lack of functional integrity [23]. To yield space for accommodating large integral transmembrane proteins in the supported lipid bilayer and to give lateral mobility to membrane components, a flexible polymer layer, preferably a hydrogel, can be inserted between the solid substrate and the bilayer. Hydrated polymer layers [2,24,26 /31] and self-assembled monolayer (SAM)-supported polyelectrolyte films have served as soft cushions for lipid bilayers [32,33]. Although there are extensive studies of polymersupported bilayers, there is scarcity of data on lateral mobility information, and the incorporation of transmembrane protein and peptide protrusion is seldom mentioned. In our previous work, supported, negatively charged lipid (1-stearoyl-2-oleoyl-phosphatidylserine, SOPS) membranes and mixtures with zwitterionic lipid (1-palmitoyl-2-oleoylphosphatidylcholine, POPC) membranes have been successfully constructed on a polyion/alkylthiol cushion layer pair on gold [34 /37]. The substrate was formed by electrostatic adsorption of a hydrated poly(diallyldimethylammonium chloride) (PDDA) polyion layer on an SAM of alkylthiol (11-mercaptoundecanoic acid, MUA). Bilayer formation kinetics and thicknesses of bilayers were measured by using surface plasmon resonance (SPR) [34 /36], and the lateral mobility of the bilayers was measured by fluorescence recovery after photobleaching (FRAP) [34,35]. The insulating properties and the binding and pore-forming activities of regular and mutant protegrin to this model membrane system were investigated by a combined SPR and
cyclic voltammetry (CV) set-up [36], and the multilayer surface morphology was analyzed by atomic force microscopy [37]. The dimensions of most antimicrobial peptides are of the order of the thickness of cell membranes, suggesting that they are capable of spanning the membrane [38]. Although protegrin-1 would not be quite long ˚ bilayer enough to traverse an approximately 40 A as a monomer (a fully extended protegin-1 mole˚ ˚ long /8 A cule measures approximately 25 A ˚ deep [36]), exceptions occurred in wide /8 A case of membrane thinning effect [38] or peptide molecule lengthening effect, as a result of dimer or oligomer formation [39]. For larger membrane proteins, a thicker polymer cushion is needed to lift the lipid membrane away from the solid substrate, which can be achieved by employing the layer-by-layer polyion adsorption technique. Additional layers of positively charged PDDA can be adsorbed by interleaving with a polyanion such as negatively charged polystyrene sulfonate (PSS) [40]. The technique has been shown to be very suitable to prepare polymeric films with welldefined thickness and homogeneity better than 1 nm [41]. The dominant interaction, electrostatic attraction of opposite charges, can be used to deposit a bilayer on top of the multilayer polymeric film [23,34/37]. Cassier et al. [23] studied the electrostatic coupling of charged phospholipid bilayers with previously dried polyelectrolyte multilayers. Membranes in mixtures with 10% charged (DOPA, dioleoylphosphatidic acid, anionic) and 90% uncharged (DMPC, dimyristoylphosphatidylcholine, zwitterionic) lipid were achieved, yielding a fluid DMPC phase when above the main phase transition at 24 8C, with lateral diffusion coefficients in aqueous environment above 1 /108 cm2/s. However, no attempts were made in their work to insert proteins into membranes for further biophysical studies. In this work, we studied lipid membranes lifted up by layer-by-layer polyion cushions on SAMs of alkylthiol on gold using surface analytical techniques such as SPR and FRAP. Using several polyion layers removes the lipid layer further from the substrate and lessens the substrate influence. Since the polyion film contains water, the lipid bilayer is surrounded on both sides by an
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aqueous environment. Negatively charged PDDA and positively charged PSS were used as polyions for the layer-by-layer assembly. The PDDA/PSS/ PDDA layers were consecutively deposited on a self-assembled MUA monolayer on gold by electrostatic interaction. An ion-channel-forming antimicrobial peptide protegrin-1 was used to determine the kinetics of peptide adsorption and insertion. A schematic drawing of this system is shown in Fig. 1. Protegrin-1 is an arginine- and cysteine-rich, b-sheet 18 amino acid cationic peptide, first isolated from porcine leukocytes [42]. Protegrin-1 has potent activity against bacteria, fungi, and certain enveloped viruses [43] and has preference for negatively charged bacterial cell membranes over host membranes [44], which makes it a good candidate to insert into the lipid membranes constructed in our system.
2. Experimental section 2.1. Materials MUA was obtained from Aldrich (Milwaukee, WI). The polymers PDDA and PSS sodium salt were obtained from Polysciences, Inc (Warrington, PA). The lipids SOPS and POPC as well as the fluorescence probe (16:0 /6:0 NBD-PS, 1-palmitoyl - 2 - [6 - [(7 - nitro - 2 - 1,3 - benzoxadiazol - 4 - yl)-
Fig. 1. Schematic drawing of the lifted-up phospholipid bilayer supported on the PDDA/PSS/PDDA/alkylthiol (MUA) layers on gold and subsequent insertion of the antimicrobial peptide protegrin-1.
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amino]-sn-glycero-3-phosphoserine(sodium salt)) were obtained from Avanti Polar Lipids (Alabaster, AL). The peptide protegrin-1 was prepared by solid-phase synthesis as previously described by Harwig et al. [45]. The buffer Tris(hydroxymethyl)aminomethane (Tris) was obtained from Sigma (St. Louis, MO). The refractive index matching fluid 1-iodonaphthalene was obtained from Cargille Laboratories, Inc (Cedar Grove, NJ). All chemicals were used as received without further purification. De-ionized water (resistivity /17.5 MV cm) was obtained from a nanopure reverse osmosis purification system from Barnstead (Dubuque, IA). 2.2. Sample preparation The buffer solution used throughout the experiments was Tris 7.2 (5 mM Tris buffer, pH 7.2). The PDDA and PSS solution, vesicle solutions, and the protegrin solutions were all prepared in the Tris 7.2 buffer. The percentages used in this article are molar fractions unless otherwise stated. The method of making small unilamellar vesicles was described in detail in our previous work [34,35]. Briefly, an appropriate amount from a 10 mg/ml lipid solution in chloroform was added to a clean glass vial and evaporated by a small N2 stream. About 2 ml of Tris buffer solution was added to make the vesicle solutions, with a total lipid concentration of 0.5 mg/ml. After incubation in a 50 8C water bath for 15 min with several vortexing periods of 15 s in between, the milky solution was sonicated with an ultrasonic tip (Branson 250, 10 W output) until clear. For FRAP experiments, 2 mol% of NBD-PS was included in the lipid solution in chloroform before evaporation. All the other steps of vesicle preparation remained the same. The protegrin-1 solution was prepared at a concentration of 20 mM in Tris 7.2 buffer. High-index glass LaSFN9 slides (n /1.85 at l / 633 nm, Schott, Germany) were used as substrates for SPR experiments. About 50 nm of gold was ˚ /s by an deposited on LaSFN9 at a rate of 0.2 A electron beam evaporator (pressure below 5 / 106 mbar). The sample slides used for FRAP experiments were prepared from Fisher Scientific.
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In this case, a 3 nm thick titanium layer was sputtered first on the glass slides to improve the adhesion of gold to glass. The thickness and the deposition rate of the gold layer were the same as that for the LaSFN9 glass slides. An alkylthiol monolayer was formed on the gold layer by immersing the slides in a 5 mM solution of MUA in ethanol for at least 18 h at room temperature. The slides were rinsed with ethanol and dried with N2 before use. 2.3. Methods The adsorption experiments were monitored with a SPR set-up using a He /Ne laser beam with a wavelength of 632.8 nm. The SPR was set up according to the Kretschmann configuration [46]. A gold-coated LaSFN9 slide covered with an MUA monolayer was mounted on a Teflon cell that holds about 0.8 ml of fluid. An LaSFN9 glass prism was mounted on the glass slide, and 1iodonaphthalene was used as the refractive index matching fluid between the prism and the glass slide. Solutions were exchanged by simultaneous injection and withdrawal from the Teflon cell using two syringes. The Tris 7.2 buffer solution was injected into the Teflon cell and incubated for approximately 15 min to equilibrate with the surrounding room temperature before SPR measurements were taken. First, a 0.2 M PDDA solution was exchanged into the cell and sat for at least 20 min before it was rinsed with buffer. The cell was rinsed until there was no further decrease of reflectivity. Second, a 0.05 M PSS solution was subsequently exchanged into the cell and was allowed to sit for at least 1 h and then was rinsed until there was no further decrease of reflectivity. Third, a 0.05 M PDDA solution was alternatively exchanged into the cell for at least 20 min before it was rinsed until there was no further decrease of reflectivity. The freshly prepared vesicle solutions were injected into the cell, and the growth of the bilayer was monitored. The thickness of the organic layers was calculated using the Fresnel equations (SPR software: WASPLAS version 2.1and WINSPALL version 1.0, Max-Plank Institute for Polymer Research, Mainz, Germany), according to the minimum
angle shift of the SPR profile before and after insertion, as described in detail in our previous work [34,35]. The kinetics of adsorption of each layer was obtained by setting the incidence angle 18 below the minimum angle um, determined by reflectivity versus angle measurements on SPR which were conducted before each kinetic measurement. Refractive indices of polymer solutions and vesicle solutions were measured on an Abbe 60 refractometer (Belllingham and Stanley, Ltd, UK) using a sodium spectral lamp with a wavelength of 589.3 nm. The mobility of the supported bilayers was measured at room temperature by FRAP experiments on a Nikon Diaphot 300 fluorescence microscope. The slides were prepared by putting two half-circular Teflon spacers with a thickness of 60 mm on a cleaned microscope cover slide and adding 60 ml of a 0.2 M PDDA solution in the middle of the formed circle. A gold-coated microscope glass slide covered by MUA SAM was then mounted on the spacers. The set-up was incubated in a humid environment for about 30 min, before the PDDA solution captured between the slides was exchanged with Tris 7.2 buffer solution. Subsequently, a 0.05 M PSS solution was introduced to the space by exchanging solutions and left for about 1 h before rinsing with buffer. Then a 0.05 M PDDA solution was exchanged for about 30 min. After rinsing the polymer solution with buffer, a lipid vesicle solution was introduced for at least 3 h. The excess vesicle solution was then exchanged 10 times with Tris 7.2 buffer solution. The fluorescent label was excited by blue light filtered from a mercury lamp and emitted bright green light. Using a 20 / objective lens on the microscope, a spot of 60 mm in diameter was bleached for 3 min. The bleached spot was then viewed digitally using the same objective lens every 15 min during the recovery stage. Image-processing software (SCION IMAGE, Scion Corp, Frederick, MD) was used to analyze the images as described [34,35]. The lateral diffusion coefficient (D ) was calculated by the equation, D (cm2/s) / 0.224v2 (cm)/t1/2 (s), where v is the radius of the bleached spot and t1/2 the half-time of the fluorescence recovery [34,35].
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3. Results and discussion 3.1. Refractive indices of organic layers and solutions With SPR, the reflectivity was measured as a function of the angle. The minimum angle, um, is a function of the thickness and refractive indices of the layers adsorbed on the gold surface and that of the bulk solution [34,35]. SPR measurements (Fig. 2) show a consistent shift of the resonance curves towards higher angles with increasing number of adsorbed layers. After the minimum angle shift, information was obtained from the SPR measurements; refractive index values of each adsorbed layer were needed to calculate the equilibrium thickness. The refractive index of the pure 5 mM Tris 7.2 buffer solution was measured to be 1.332 at room temperature. The refractive indices of the bulk lipid vesicle (0.5 mg/ml) and peptide solutions (20 mM) in Tris 7.2 buffer were measured as 1.332 and 1.333, respectively. The typical literature value of the refractive index for long-chain lipids, 1.49 [20], was used in the simulations. A refractive index of 1.414 was used for the PDDA layers [34,35], which corresponds to a dielectric constant of 2. The refractive index of the 0.2 M bulk PDDA solution was 1.339, and that of the 0.05 M bulk PDDA solution was 1.335.
Fig. 2. Part of the SPR curves near the minimum angles, where the reflectivity is at its lowest value. The curves shift to the right with successive deposition of PDDA, PSS, PDDA and a lipid bilayer. A SPR curve measured after the deposition of a SOPS bilayer is shown as an example.
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A typical refractive index value for the PSS layer, 1.5 [47], was assumed, which corresponds to a dielectric constant of 2.25 and water content of 99%. The corresponding water content was evaluated by measuring the refractive indices of a series of PSS solutions of different concentrations in 5 mM Tris 7.2 buffer. A linear relation was obtained using least-squares regression (n /0.172 w/1.3322, R2 /0.9991; n , refractive index; w , weight fraction of PSS). The refractive index of the 0.05 M bulk PSS solution was 1.334. 3.2. Polymer adsorption on supported monolayers of MUA on gold The first polymer layer, a positively charged PDDA, was adsorbed from a 0.2 M solution (based on its monomer MW /161.7) onto a negatively charged surface formed by a selfassembled monolayer of MUA on gold. The driving force for the adsorption is the coulombic interaction of the positively charged PDDA and the negatively charged MUA surface [34,35]. When a PDDA solution of a lower concentration (20 mM) was used, the adsorbed PDDA amount was not sufficient to change the negative surface charge of MUA layer and therefore could not be used to adsorb the negatively charged PSS (data not shown). As shown in Fig. 3(a), the kinetic profile of PDDA adsorption from a 0.2 M solution was measured by SPR. The reflectivity increased instantaneously upon injection of the 0.2 M PDDA solution, due to the higher refractive index of the bulk PDDA solution (n /1.339) than that of the Tris 7.2 buffer (n/1.332). A relatively slower kinetics occurring in several minutes before a flat equilibrium stage was due to the adsorption of the PDDA layer. After adsorption of PDDA reached equilibrium (about 20 min), the Teflon cell was rinsed with Tris 7.2 buffer, and the reflectivity dropped significantly. The net increase in reflectivity was about 0.024, and simulations gave a ˚ , as corresponding thickness increase of 129/1 A shown in Table 1. This thickness is consistent with measurements reported in the literature [34,40]. The second polymer layer, a negatively charged PSS, was adsorbed from a 0.05 M solution (based on its monomer MW /206.2) onto the positively
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Fig. 3. SPR kinetic profiles of alternative polymer adsorption ((a) PDDA on MUA; (b) PSS on PDDA/MUA; (c) PDDA on PSS/PDDA/MUA) on gold at pH7.2. The first arrow indicates injection of a polymer solution ((a) 0.2M PDDA; (b) 0.05M PSS; (c) 0.05M PDDA). The other arrows correspond to the rinses of the Teflon cell with 5mM Tris 7.2.
charged PDDA surface. When a lower concentration was used, the adsorbed PSS layer was not able to adsorb the second PDDA layer. As shown by the kinetic profile measured by SPR (Fig. 3(b)), the reflectivity increased significantly within seconds upon injection of the 0.05 M PSS solution. The fast and significant increase of reflectivity is due to the higher refractive index of the 0.05 M
PSS solution (1.334) than the Tris 7.2 buffer (1.332). A relatively slower kinetic process in the order of several minutes before a flat equilibrium stage (reached in 50 min) was due to the adsorption of the PSS layer. The reflectivity dropped significantly after several rinses with buffer, and the final increase of reflectivity was about 0.054. The simulations gave a corresponding thickness ˚ , assuming a refractive index of increase of 169/2 A 1.5 for the adsorbed PSS layer. The PSS layer is a little thicker than the first PDDA layer, which can be explained by the water content differences between the buffer solutions. For the first PDDA layer, the water content was estimated to be 63%, assuming a refractive index of 1.41 [34,35], while the water content was 99% for a PSS layer, regardless of the refractive index. Since the swellability (water content) of the polymer film is directly related to its thickness [48], and the water content of the 0.05 M PSS solution is much more than that of the 0.2 M PDDA solution, the PSS layer is thicker than the first PDDA layer. The third polymer layer, PDDA, was adsorbed from a 0.05 M solution onto the negatively charged PSS surface. As shown by the kinetic profile measured by SPR (Fig. 3(c)), adsorption of the second PDDA layer reached equilibrium in 30 min. After several rinses with buffer, the final increase of reflectivity was about 0.035, with a ˚, corresponding thickness increase of 149/2 A
Table 1 Comparison of thickness values Substrate
˚) Equilibrium thickness (A
Au MUA/Au PDDA/MUA/Au PSS/PDDA/MUA/Au PDDA/PSS/PDDA/MUA/Au SOPS/PDDA/PSS/PDDA/MUA/Au (87.5% SOPS/12.5% POPC)/PDDA/PSS/PDDA/MUA/Au (75% SOPS/25% POPC)/PDDA/PSS/PDDA/MUA/Au Protegrin-1/SOPS/PDDA/PSS/PDDA/MUA/Au Protegrin-1/(75%SOPS/25% POPC)/PDDA/PSS/PDDA/MUA/Au
/ 15/ / 12/15/ / 16/12/15/ / 14/16/12/15/ / 40/14/16/12/15/ / 44/14/16/12/15/ / 52/14/16/12/15/ / 58/41/14/16/12/15/ / 71/54/14/16/12/15/ /
Comparison of thickness values of bare gold, MUA monolayer/gold, PDDA/MUA/gold, PSS/PDDA/MUA/gold, PDDA/PSS/ MUA/gold, 100% SOPS bilayer/PDDA/PSS/PDDA/MUA/gold before and after protegrin-1 insertion, (87.5% SOPS/12.5% POPC) bilayer/PDDA/PSS/PDDA/MUA/Au, (75% SOPS/25% POPC) bilayer/PDDA/PSS/PDDA/MUA/gold before and after protegrin-1 insertion is shown. Thicknesses measured by SPR are shown for the individual layers on gold.
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assuming a refractive index of 1.414. Since the second PDDA layer was adsorbed from a less concentrated solution, the water content in the second PDDA layer was more than that of the first PDDA layer, which explains why the second PDDA layer appears to be a little thicker than the first PDDA layer. The thickness values of the polymer layers are reasonable and fall in the range reported in the literature for multilayer thin films, which are about ˚ in thickness [49]. A PDDA/PSS bilayer 5 /20 A ˚ , as measured by optical ellipsothickness of 23 A metry, was reported for 50-bilayer electrostatic self-assembly thin films, assuming a refractive index of 1.539 [50]. In a typical cycle of PDDA/ silicate multilayered films, a PDDA layer of ˚ was adsorbed from a 5 wt% approximately 11 A aqueous solution [51]. An X-ray reflectivity study ˚ four yielded a film thickness of about 200 A double layers (PSS/PAH)4 on PEI-treated glass ˚ or cysteamine-treated gold surface of about 200 A by consecutive adsorption of oppositely charged polyelectrolytes [23].
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Fig. 4. SPR kinetic profiles of lipid adsorption with different compositions of SOPS and POPC (100% SOPS; 87.5% SOPS/ 12.5% POPC; 75% SOPS/25% POPC) on PDDA/PSS/PDDA/ MUA covered gold surfaces at pH 7.2. For clarity purposes, the kinetic profiles shown in the figure connect the bilayer kinetics and the rinsing kinetics. The perpendicular arrows indicate rinses with buffer to eliminate unbound vesicles. The reflectivity changes were negligible before and after rinsing.
3.3. Deposition of lipids on PDDA/PSS/PDDA Lipid layers of pure, negatively charged lipid, SOPS, and lipid mixtures of SOPS and zwitterionic lipid, POPC were deposited on the PDDA/PSS/ PDDA polymer cushions by vesicle fusion. The kinetic profiles of the adsorption processes are shown in Fig. 4. The final membrane thickness values are plotted in Fig. 5 and listed in Table 1. The thickness values were obtained by fitting the SPR curves before and after vesicle fusion (after rinsing) using the WASPLAS 2.1 software. As shown by SPR kinetic profiles, pure SOPS followed a sharp, fast deposition before the reflectivity reached an equilibrium state within an hour’s time. The thickness of the adsorbed SOPS layer ˚ , as simulated by using a refractive was 409/1 A index of 1.49 for the bilayer. The lipid mixtures with 12.5% POPC and 25% POPC followed similar kinetics: a quick steep growth period and a flat equilibrium state, in comparison with pure SOPS. The thickness increased slightly with an increase in POPC content. When the POPC content was ˚, 12.5%, the thickness of the lipid layer was 44 A
Fig. 5. FRAP data curves obtained for bilayers (100% SOPS; 75% SOPS/25% POPC) formed on PDDA/PSS/PDDA/MUA covered gold surfaces at pH 7.2 at room temperature. The fluorescence intensities of the two bilayers were normalized independently.
which falls in the range of a single bilayer thickness. With an increased amount of POPC composition up to 25%, the thickness of the lipid
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˚ , which is in the layers increased to about 52 A upper range for single bilayer thickness, usually ˚ . Different refractive index values about 40 /50 A used for lipid layers in the literature vary from 1.45 to 1.50 [52,53]. Using a refractive index of 1.50 for the bilayers in this work, the simulated SOPS ˚ */a little thinner than a bilayer thickness is 37 A single bilayer. However, using a refractive index of 1.50 as the refractive index for the bilayer made up of 75% SOPS and 25% POPC results in a thickness ˚ . Thus it is reasonable to consider 75% of 48 A SOPS/25% POPC adsorbed on the PDDA/PSS/ PDDA cushions formed bilayers. We observed a trend of increasing bilayer thickness with increasing POPC content (data not shown), which is very similar to that of the thickness values of the lipid layers deposited on only one PDDA polymer layer [34,35]. In vesicle fusion of SOPS, the driving force of adsorption is the electrostatic coupling between the negatively charged SOPS vesicles and the positively charged PDDA surface. The negative charges of the adsorbed lipid layer repelled further adsorption of lipid molecules, resulting in a single bilayer structure. The relatively thinner SOPS ˚ on PDDA/MUA bilayer with a thickness of 32 A layer pair [34,35], lower than the usual bilayer ˚ for long-chain thickness of approximately 40 A phospholipids in the fluid phase, is possibly due to the interdigitation between the two lipid monolayers and small defects in the bilayers. When the SOPS bilayer is lifted up by the PDDA/PSS/ PDDA cushions, the second PDDA layer is deposited from a less concentrated solution (0.05 M) than that of the first PDDA solution (0.2 M), which may result in a reduced surface charge density in the second PDDA layer, therefore reducing the electrostatic interaction between the SOPS molecules and the second PDDA monolayer. The weaker electrostatic attraction may result in less interdigitation between the two lipid monolayers, thus the SOPS bilayer lifted up on the PDDA/PSS/PDDA cushions is a little thicker than that on only one PDDA layer. When mixing the negatively charged lipid with a neutral phospholipid, the bilayer thickness increases due to a reduced charge density of the lipid membrane with an increase of the POPC content [23]. Similarly, the higher the POPC content in the
mixture of SOPS/POPC, the weaker the electrostatic coupling, the less interdigitation between the two lipid monolayers, and the larger the bilayer thickness. Fluorescence images of the lipid membranes formed by both pure SOPS and 75% SOPS/25% POPC on the PDDA/PSS/PDDA cushions indicated homogeneous distribution of the fluorescent probe molecules in the bilayers with no obvious defects (data not shown). Lateral mobility of the two bilayers was quantitatively analyzed as described in the experimental section. For both of the two cases, the fluorescence intensity of the bleached spot recovered more than 85% at 90 min after bleaching. The bilayer formed by pure SOPS recovered slower than that formed with 75% SOPS/25% POPC (Fig. 5). The calculations for the lateral diffusion coefficients (D ) showed that for pure SOPS bilayer D /1.2 /109 cm2/s, whereas for the bilayer with 75% SOPS/25% POPC D /2.1 /109 cm2/s, both of which were very similar to corresponding cases (D /1/109 cm2/s for pure SOPS bilayer, D /2/109 cm2/s for the bilayer with 75% SOPS/25% POPC) on only one PDDA layer [34]. Cassier et al. [23] reported a value of D about 1 / 109 cm2/s for a negatively charged lipid DOPA bilayer coupled to polyelectrolyte multilayers. It is possible that the high surface charge density restricts the mobility of anionic lipid coupled to the cationic polymer surface, and that the addition of neutral lipid helps to dilute the charge density and improve the mobility of the mixed lipid bilayers. The free zwitterionic lipid monolayers or bilayers are known to exhibit values of D near 108 cm2/s for a fluid phase and near 10 10 cm2/s for a gel phase in lipid/polyelectrolyte composites [54,55]. The lateral diffusion coefficients of lipid bilayers for both pure SOPS and 75% SOPS/25% POPC mixture on three polymer monolayers in this system, like that on only one polymer monolayer in previous study [34], are also within the range of diffusion coefficients in fluid bilayers and are well above the characteristic diffusivities for lipid membranes in the rigid Lb phase (partly ordered, untilted, D B/1011 cm2/s) [56].
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3.4. Peptide insertion Protegrin-1 belongs to a family of innate host defense molecules called antimicrobial peptides, which were first discovered in the early 1980s [39]. This peptide is able to alter the cellular membrane permeability by forming ionic pores that present some specific properties in common with human defensin-channel [57]. Since it displays a broadrange antimicrobial activity against bacteria such as Escherichia coli , C. trachomatis , N. gonorrhea, as well as HIV-1, protegrin-1 has excellent potential for pharmaceutical uses [39]. The sequence of protegrin-1 is NH2 /RGGRLCYCRRRFCVCVGR /CONH2. The peptide has a twostranded antiparallel b-sheet with a hairpin structure held by disulfide bonds formed by cysteine [44,58]. Positively charged arginine residues are present at both ends of the hairpin-like molecule, while hydrophobic and uncharged amino acids form the remainder of its structure [36]. Peptide insertion experiments were monitored by SPR on supported bilayers formed by pure SOPS and 75% SOPS/25% POPC (Fig. 6). Two milliliters of 20 mM solution of peptide protegrin-1 in Tris 7.2 buffer was injected into the Teflon cell. The
Fig. 6. SPR kinetic profiles of protegrin-1 insertion into bilayers composed of 100% SOPS and 75% SOPS/25%POPC formed on PDDA/PSS/PDDA/MUA covered gold surfaces. Data of thickness measurements are listed with the curves for bilayer thickness before and after peptide insertion (after rinsing). Thickness data are in angstroms.
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reflectivity increased instantaneously upon injection of the peptide solution and continued to increase more slowly until equilibrium was reached. After 90 minutes (sufficient time to observe the uptake kinetics), the excess protegrin1 solution was rinsed with Tris 7.2 buffer until there was no further decrease in reflectivity. The thickness results after rinsing are listed in Fig. 6 and Table 1. For pure SOPS bilayers, the thickness ˚ , after protegrin-1 increased from 41 to 58 A insertion. In the case of 75% SOPS/25% POPC ˚. bilayers, the thickness increased from 54 to 71 A It should be noted here that the same value of refractive index has been used for lipid bilayers before and after peptide insertion. Therefore, the thickness increases should be regarded as addition of materials to the lipid membranes [34 /36]. The net increases in thickness were the same for both ˚ . This result is in bilayer systems, i.e. 17 A agreement with the report that the pore structure caused by peptide insertion is relatively insensitive to the detailed composition of the membranes [39,59]. However, the net increase in thickness was ˚ for pure SOPS bilayers supported on the 12 A PDDA/MUA cushions and for pure SOPS bilayers and 75% SOPS/25% POPC bilayers on PDDA/ MPA (3-mercaptopropionic acid) cushions [36]. It needs to be pointed out that, using an optical technique such as SPR alone, it is not easy to determine whether protegrin-1 molecules inserted into the lipid bilayers or adsorbed on the bilayer surfaces. We know that the surface charge density of the second PDDA layer is reduced compared to the first PDDA layer. This would correspond to less repulsion between the cationic protegrin-1 inserted into the bilayer and cationic PDDA, which promotes more protegrin-1 inserted in the bilayer as we observed here. The SPR kinetic profiles of peptide insertion showed a two-step interaction of the protegrin-1 molecules and the lipid bilayers. The initial fast increase in reflectivity might be a response to the surface adsorption of protegrin-1 molecules to the lipid membrane. The slow increase of reflectivity may be due to the trans bilayer insertion process of the peptide: adsorbed molecules insert into the membrane and more peptide molecules add to the surface to replace inserted ones [36]. The helical antimicrobial pep-
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tides, alamethicin and magainin, exhibit two distinct states in lipid bilayers, one state is adsorbing in the lipid headgroup region with the helical axis oriented parallel to the plane of the bilayer and the other state exists as trans bilayer insertion into the hydrocarbon region with the helical axis oriented perpendicular to the plane of the bilayer [60,61]. Heller et al. reported that the two different states of protegrin-1 existing in membranes might correspond to the surface state and the insertion state of alamethicin: one state is adsorbed to the headgroup region of a lipid bilayer with the peptide oriented parallel to the horizontal plane of the bilayer, whereas in the other state, the peptide is inserted perpendicular to the bilayer plane, i.e., the transmembrane state [39,44]. This orientation study supports the hypothesis of our results.
step interaction, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/ MUA. The results are comparable to our work on supported lipid membranes without a layer-bylayer cushion [33,35]. The supported lipid bilayer on a cushion is a useful system to be explored for membrane protein insertion.
Acknowledgements The MRSEC program of the National Science Foundation under Award DMR-9808677 supported this work. M. L. Longo acknowledges funding from the NSF through the CAREER Program (BES-9733764). R. I. Lehrer and A. J. Waring acknowledge support through the following NIH grants: AI22839 and AI37945.
4. Conclusions References A supported lipid membrane system lifted up by a cushion of layer-by-layer polyions on an SAM of alkylthiol was investigated by SPR and FRAP. A long-chain alkylthiol (MUA) layer was self-assembled on a gold surface to create the first negatively charged organic layer. A polyelectrolyte film (PDDA/PSS/PDDA) was then deposited on the alkylthiol-covered solid substrate by the technique of layer-by-layer deposition. By the method of vesicle fusion, anionic lipid SOPS and mixtures of SOPS and the zwitterionic lipid POPC (composition up to 25%) formed bilayers on the layer-bylayer polyion/ cushions on an SAM of alkylthiol through electrostatic interaction with the second PDDA layer. Mobility experiments by FRAP showed a slight increase of the lateral diffusion coefficient for the bilayers with 25% POPC compared to those formed by pure SOPS, both fell in the range of those for mobile membranes. The lateral diffusion coefficients of the single bilayers formed on polymer multilayers were very similar to those formed on only one PDDA layer. SPR measurements on the protegrin-1 addition to the bilayers showed no difference in kinetics or thickness changes with pure SOPS and 75% SOPS/25% POPC. In both cases, the pore-forming peptide remained bound to the lipid membranes by a two-
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Supported lipid bilayers lifted from the substrate by layer-bylayer polyion cushions on self-assembled monolayers Chen Ma a, M.P. Srinivasan a,1, Alan J. Waring b, Robert I. Lehrer c, Marjorie L. Longo a, Pieter Stroeve a,* a
Department of Chemical Engineering and Materials Science, University of California Davis, Davis, CA 95616, USA b University of California at Los Angeles School of Medicine and Harbor /UCLA, Los Angeles, CA 90095, USA c Department of Medicine and Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA Accepted 10 October 2002
Abstract The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/ PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2 /2.1) /10 9 cm2/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes. # 2002 Elsevier Science B.V. All rights reserved.
1. Introduction
* Corresponding author. Tel./fax: /1-530-752-8778. E-mail address: [email protected] (P. Stroeve). 1 Present address: Department of Chemical and Environmental Engineering, The National University of Singapore, Singapore.
Since a supported bilayer membrane was first used to investigate cellular immune responses [1], solid-supported lipid bilayers have been a widely studied topic of practical and scientific interest. Being well-defined models of biological membranes, phospholipid bilayers supported on solid
0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 1 7 5 - 3
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substrates are important for their roles in fundamental biophysical research as well as in applications such as biosensors [2]. Supported lipid bilayer membranes have been formed onto glass, quartz, and silicon surfaces [1,3 /8], onto nonfunctionalized metal surfaces [9 /14], or onto selfassembled alkanethiol monolayers [15 /22]. Methods for the bilayer formation have included the Langmuir /Blodgett technique [1,2,9,11,12,18,23], vesicle fusion onto the substrate [1,23/25], spontaneous thinning of lipid/decane mixtures [14,25], and adsorption of charged lipids onto oppositely charged surfaces [20,26,27]. A bilayer deposited directly on silica, glass, or gold is a model membrane with a lack of functional integrity [23]. To yield space for accommodating large integral transmembrane proteins in the supported lipid bilayer and to give lateral mobility to membrane components, a flexible polymer layer, preferably a hydrogel, can be inserted between the solid substrate and the bilayer. Hydrated polymer layers [2,24,26 /31] and self-assembled monolayer (SAM)-supported polyelectrolyte films have served as soft cushions for lipid bilayers [32,33]. Although there are extensive studies of polymersupported bilayers, there is scarcity of data on lateral mobility information, and the incorporation of transmembrane protein and peptide protrusion is seldom mentioned. In our previous work, supported, negatively charged lipid (1-stearoyl-2-oleoyl-phosphatidylserine, SOPS) membranes and mixtures with zwitterionic lipid (1-palmitoyl-2-oleoylphosphatidylcholine, POPC) membranes have been successfully constructed on a polyion/alkylthiol cushion layer pair on gold [34 /37]. The substrate was formed by electrostatic adsorption of a hydrated poly(diallyldimethylammonium chloride) (PDDA) polyion layer on an SAM of alkylthiol (11-mercaptoundecanoic acid, MUA). Bilayer formation kinetics and thicknesses of bilayers were measured by using surface plasmon resonance (SPR) [34 /36], and the lateral mobility of the bilayers was measured by fluorescence recovery after photobleaching (FRAP) [34,35]. The insulating properties and the binding and pore-forming activities of regular and mutant protegrin to this model membrane system were investigated by a combined SPR and
cyclic voltammetry (CV) set-up [36], and the multilayer surface morphology was analyzed by atomic force microscopy [37]. The dimensions of most antimicrobial peptides are of the order of the thickness of cell membranes, suggesting that they are capable of spanning the membrane [38]. Although protegrin-1 would not be quite long ˚ bilayer enough to traverse an approximately 40 A as a monomer (a fully extended protegin-1 mole˚ ˚ long /8 A cule measures approximately 25 A ˚ deep [36]), exceptions occurred in wide /8 A case of membrane thinning effect [38] or peptide molecule lengthening effect, as a result of dimer or oligomer formation [39]. For larger membrane proteins, a thicker polymer cushion is needed to lift the lipid membrane away from the solid substrate, which can be achieved by employing the layer-by-layer polyion adsorption technique. Additional layers of positively charged PDDA can be adsorbed by interleaving with a polyanion such as negatively charged polystyrene sulfonate (PSS) [40]. The technique has been shown to be very suitable to prepare polymeric films with welldefined thickness and homogeneity better than 1 nm [41]. The dominant interaction, electrostatic attraction of opposite charges, can be used to deposit a bilayer on top of the multilayer polymeric film [23,34/37]. Cassier et al. [23] studied the electrostatic coupling of charged phospholipid bilayers with previously dried polyelectrolyte multilayers. Membranes in mixtures with 10% charged (DOPA, dioleoylphosphatidic acid, anionic) and 90% uncharged (DMPC, dimyristoylphosphatidylcholine, zwitterionic) lipid were achieved, yielding a fluid DMPC phase when above the main phase transition at 24 8C, with lateral diffusion coefficients in aqueous environment above 1 /108 cm2/s. However, no attempts were made in their work to insert proteins into membranes for further biophysical studies. In this work, we studied lipid membranes lifted up by layer-by-layer polyion cushions on SAMs of alkylthiol on gold using surface analytical techniques such as SPR and FRAP. Using several polyion layers removes the lipid layer further from the substrate and lessens the substrate influence. Since the polyion film contains water, the lipid bilayer is surrounded on both sides by an
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aqueous environment. Negatively charged PDDA and positively charged PSS were used as polyions for the layer-by-layer assembly. The PDDA/PSS/ PDDA layers were consecutively deposited on a self-assembled MUA monolayer on gold by electrostatic interaction. An ion-channel-forming antimicrobial peptide protegrin-1 was used to determine the kinetics of peptide adsorption and insertion. A schematic drawing of this system is shown in Fig. 1. Protegrin-1 is an arginine- and cysteine-rich, b-sheet 18 amino acid cationic peptide, first isolated from porcine leukocytes [42]. Protegrin-1 has potent activity against bacteria, fungi, and certain enveloped viruses [43] and has preference for negatively charged bacterial cell membranes over host membranes [44], which makes it a good candidate to insert into the lipid membranes constructed in our system.
2. Experimental section 2.1. Materials MUA was obtained from Aldrich (Milwaukee, WI). The polymers PDDA and PSS sodium salt were obtained from Polysciences, Inc (Warrington, PA). The lipids SOPS and POPC as well as the fluorescence probe (16:0 /6:0 NBD-PS, 1-palmitoyl - 2 - [6 - [(7 - nitro - 2 - 1,3 - benzoxadiazol - 4 - yl)-
Fig. 1. Schematic drawing of the lifted-up phospholipid bilayer supported on the PDDA/PSS/PDDA/alkylthiol (MUA) layers on gold and subsequent insertion of the antimicrobial peptide protegrin-1.
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amino]-sn-glycero-3-phosphoserine(sodium salt)) were obtained from Avanti Polar Lipids (Alabaster, AL). The peptide protegrin-1 was prepared by solid-phase synthesis as previously described by Harwig et al. [45]. The buffer Tris(hydroxymethyl)aminomethane (Tris) was obtained from Sigma (St. Louis, MO). The refractive index matching fluid 1-iodonaphthalene was obtained from Cargille Laboratories, Inc (Cedar Grove, NJ). All chemicals were used as received without further purification. De-ionized water (resistivity /17.5 MV cm) was obtained from a nanopure reverse osmosis purification system from Barnstead (Dubuque, IA). 2.2. Sample preparation The buffer solution used throughout the experiments was Tris 7.2 (5 mM Tris buffer, pH 7.2). The PDDA and PSS solution, vesicle solutions, and the protegrin solutions were all prepared in the Tris 7.2 buffer. The percentages used in this article are molar fractions unless otherwise stated. The method of making small unilamellar vesicles was described in detail in our previous work [34,35]. Briefly, an appropriate amount from a 10 mg/ml lipid solution in chloroform was added to a clean glass vial and evaporated by a small N2 stream. About 2 ml of Tris buffer solution was added to make the vesicle solutions, with a total lipid concentration of 0.5 mg/ml. After incubation in a 50 8C water bath for 15 min with several vortexing periods of 15 s in between, the milky solution was sonicated with an ultrasonic tip (Branson 250, 10 W output) until clear. For FRAP experiments, 2 mol% of NBD-PS was included in the lipid solution in chloroform before evaporation. All the other steps of vesicle preparation remained the same. The protegrin-1 solution was prepared at a concentration of 20 mM in Tris 7.2 buffer. High-index glass LaSFN9 slides (n /1.85 at l / 633 nm, Schott, Germany) were used as substrates for SPR experiments. About 50 nm of gold was ˚ /s by an deposited on LaSFN9 at a rate of 0.2 A electron beam evaporator (pressure below 5 / 106 mbar). The sample slides used for FRAP experiments were prepared from Fisher Scientific.
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In this case, a 3 nm thick titanium layer was sputtered first on the glass slides to improve the adhesion of gold to glass. The thickness and the deposition rate of the gold layer were the same as that for the LaSFN9 glass slides. An alkylthiol monolayer was formed on the gold layer by immersing the slides in a 5 mM solution of MUA in ethanol for at least 18 h at room temperature. The slides were rinsed with ethanol and dried with N2 before use. 2.3. Methods The adsorption experiments were monitored with a SPR set-up using a He /Ne laser beam with a wavelength of 632.8 nm. The SPR was set up according to the Kretschmann configuration [46]. A gold-coated LaSFN9 slide covered with an MUA monolayer was mounted on a Teflon cell that holds about 0.8 ml of fluid. An LaSFN9 glass prism was mounted on the glass slide, and 1iodonaphthalene was used as the refractive index matching fluid between the prism and the glass slide. Solutions were exchanged by simultaneous injection and withdrawal from the Teflon cell using two syringes. The Tris 7.2 buffer solution was injected into the Teflon cell and incubated for approximately 15 min to equilibrate with the surrounding room temperature before SPR measurements were taken. First, a 0.2 M PDDA solution was exchanged into the cell and sat for at least 20 min before it was rinsed with buffer. The cell was rinsed until there was no further decrease of reflectivity. Second, a 0.05 M PSS solution was subsequently exchanged into the cell and was allowed to sit for at least 1 h and then was rinsed until there was no further decrease of reflectivity. Third, a 0.05 M PDDA solution was alternatively exchanged into the cell for at least 20 min before it was rinsed until there was no further decrease of reflectivity. The freshly prepared vesicle solutions were injected into the cell, and the growth of the bilayer was monitored. The thickness of the organic layers was calculated using the Fresnel equations (SPR software: WASPLAS version 2.1and WINSPALL version 1.0, Max-Plank Institute for Polymer Research, Mainz, Germany), according to the minimum
angle shift of the SPR profile before and after insertion, as described in detail in our previous work [34,35]. The kinetics of adsorption of each layer was obtained by setting the incidence angle 18 below the minimum angle um, determined by reflectivity versus angle measurements on SPR which were conducted before each kinetic measurement. Refractive indices of polymer solutions and vesicle solutions were measured on an Abbe 60 refractometer (Belllingham and Stanley, Ltd, UK) using a sodium spectral lamp with a wavelength of 589.3 nm. The mobility of the supported bilayers was measured at room temperature by FRAP experiments on a Nikon Diaphot 300 fluorescence microscope. The slides were prepared by putting two half-circular Teflon spacers with a thickness of 60 mm on a cleaned microscope cover slide and adding 60 ml of a 0.2 M PDDA solution in the middle of the formed circle. A gold-coated microscope glass slide covered by MUA SAM was then mounted on the spacers. The set-up was incubated in a humid environment for about 30 min, before the PDDA solution captured between the slides was exchanged with Tris 7.2 buffer solution. Subsequently, a 0.05 M PSS solution was introduced to the space by exchanging solutions and left for about 1 h before rinsing with buffer. Then a 0.05 M PDDA solution was exchanged for about 30 min. After rinsing the polymer solution with buffer, a lipid vesicle solution was introduced for at least 3 h. The excess vesicle solution was then exchanged 10 times with Tris 7.2 buffer solution. The fluorescent label was excited by blue light filtered from a mercury lamp and emitted bright green light. Using a 20 / objective lens on the microscope, a spot of 60 mm in diameter was bleached for 3 min. The bleached spot was then viewed digitally using the same objective lens every 15 min during the recovery stage. Image-processing software (SCION IMAGE, Scion Corp, Frederick, MD) was used to analyze the images as described [34,35]. The lateral diffusion coefficient (D ) was calculated by the equation, D (cm2/s) / 0.224v2 (cm)/t1/2 (s), where v is the radius of the bleached spot and t1/2 the half-time of the fluorescence recovery [34,35].
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3. Results and discussion 3.1. Refractive indices of organic layers and solutions With SPR, the reflectivity was measured as a function of the angle. The minimum angle, um, is a function of the thickness and refractive indices of the layers adsorbed on the gold surface and that of the bulk solution [34,35]. SPR measurements (Fig. 2) show a consistent shift of the resonance curves towards higher angles with increasing number of adsorbed layers. After the minimum angle shift, information was obtained from the SPR measurements; refractive index values of each adsorbed layer were needed to calculate the equilibrium thickness. The refractive index of the pure 5 mM Tris 7.2 buffer solution was measured to be 1.332 at room temperature. The refractive indices of the bulk lipid vesicle (0.5 mg/ml) and peptide solutions (20 mM) in Tris 7.2 buffer were measured as 1.332 and 1.333, respectively. The typical literature value of the refractive index for long-chain lipids, 1.49 [20], was used in the simulations. A refractive index of 1.414 was used for the PDDA layers [34,35], which corresponds to a dielectric constant of 2. The refractive index of the 0.2 M bulk PDDA solution was 1.339, and that of the 0.05 M bulk PDDA solution was 1.335.
Fig. 2. Part of the SPR curves near the minimum angles, where the reflectivity is at its lowest value. The curves shift to the right with successive deposition of PDDA, PSS, PDDA and a lipid bilayer. A SPR curve measured after the deposition of a SOPS bilayer is shown as an example.
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A typical refractive index value for the PSS layer, 1.5 [47], was assumed, which corresponds to a dielectric constant of 2.25 and water content of 99%. The corresponding water content was evaluated by measuring the refractive indices of a series of PSS solutions of different concentrations in 5 mM Tris 7.2 buffer. A linear relation was obtained using least-squares regression (n /0.172 w/1.3322, R2 /0.9991; n , refractive index; w , weight fraction of PSS). The refractive index of the 0.05 M bulk PSS solution was 1.334. 3.2. Polymer adsorption on supported monolayers of MUA on gold The first polymer layer, a positively charged PDDA, was adsorbed from a 0.2 M solution (based on its monomer MW /161.7) onto a negatively charged surface formed by a selfassembled monolayer of MUA on gold. The driving force for the adsorption is the coulombic interaction of the positively charged PDDA and the negatively charged MUA surface [34,35]. When a PDDA solution of a lower concentration (20 mM) was used, the adsorbed PDDA amount was not sufficient to change the negative surface charge of MUA layer and therefore could not be used to adsorb the negatively charged PSS (data not shown). As shown in Fig. 3(a), the kinetic profile of PDDA adsorption from a 0.2 M solution was measured by SPR. The reflectivity increased instantaneously upon injection of the 0.2 M PDDA solution, due to the higher refractive index of the bulk PDDA solution (n /1.339) than that of the Tris 7.2 buffer (n/1.332). A relatively slower kinetics occurring in several minutes before a flat equilibrium stage was due to the adsorption of the PDDA layer. After adsorption of PDDA reached equilibrium (about 20 min), the Teflon cell was rinsed with Tris 7.2 buffer, and the reflectivity dropped significantly. The net increase in reflectivity was about 0.024, and simulations gave a ˚ , as corresponding thickness increase of 129/1 A shown in Table 1. This thickness is consistent with measurements reported in the literature [34,40]. The second polymer layer, a negatively charged PSS, was adsorbed from a 0.05 M solution (based on its monomer MW /206.2) onto the positively
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Fig. 3. SPR kinetic profiles of alternative polymer adsorption ((a) PDDA on MUA; (b) PSS on PDDA/MUA; (c) PDDA on PSS/PDDA/MUA) on gold at pH7.2. The first arrow indicates injection of a polymer solution ((a) 0.2M PDDA; (b) 0.05M PSS; (c) 0.05M PDDA). The other arrows correspond to the rinses of the Teflon cell with 5mM Tris 7.2.
charged PDDA surface. When a lower concentration was used, the adsorbed PSS layer was not able to adsorb the second PDDA layer. As shown by the kinetic profile measured by SPR (Fig. 3(b)), the reflectivity increased significantly within seconds upon injection of the 0.05 M PSS solution. The fast and significant increase of reflectivity is due to the higher refractive index of the 0.05 M
PSS solution (1.334) than the Tris 7.2 buffer (1.332). A relatively slower kinetic process in the order of several minutes before a flat equilibrium stage (reached in 50 min) was due to the adsorption of the PSS layer. The reflectivity dropped significantly after several rinses with buffer, and the final increase of reflectivity was about 0.054. The simulations gave a corresponding thickness ˚ , assuming a refractive index of increase of 169/2 A 1.5 for the adsorbed PSS layer. The PSS layer is a little thicker than the first PDDA layer, which can be explained by the water content differences between the buffer solutions. For the first PDDA layer, the water content was estimated to be 63%, assuming a refractive index of 1.41 [34,35], while the water content was 99% for a PSS layer, regardless of the refractive index. Since the swellability (water content) of the polymer film is directly related to its thickness [48], and the water content of the 0.05 M PSS solution is much more than that of the 0.2 M PDDA solution, the PSS layer is thicker than the first PDDA layer. The third polymer layer, PDDA, was adsorbed from a 0.05 M solution onto the negatively charged PSS surface. As shown by the kinetic profile measured by SPR (Fig. 3(c)), adsorption of the second PDDA layer reached equilibrium in 30 min. After several rinses with buffer, the final increase of reflectivity was about 0.035, with a ˚, corresponding thickness increase of 149/2 A
Table 1 Comparison of thickness values Substrate
˚) Equilibrium thickness (A
Au MUA/Au PDDA/MUA/Au PSS/PDDA/MUA/Au PDDA/PSS/PDDA/MUA/Au SOPS/PDDA/PSS/PDDA/MUA/Au (87.5% SOPS/12.5% POPC)/PDDA/PSS/PDDA/MUA/Au (75% SOPS/25% POPC)/PDDA/PSS/PDDA/MUA/Au Protegrin-1/SOPS/PDDA/PSS/PDDA/MUA/Au Protegrin-1/(75%SOPS/25% POPC)/PDDA/PSS/PDDA/MUA/Au
/ 15/ / 12/15/ / 16/12/15/ / 14/16/12/15/ / 40/14/16/12/15/ / 44/14/16/12/15/ / 52/14/16/12/15/ / 58/41/14/16/12/15/ / 71/54/14/16/12/15/ /
Comparison of thickness values of bare gold, MUA monolayer/gold, PDDA/MUA/gold, PSS/PDDA/MUA/gold, PDDA/PSS/ MUA/gold, 100% SOPS bilayer/PDDA/PSS/PDDA/MUA/gold before and after protegrin-1 insertion, (87.5% SOPS/12.5% POPC) bilayer/PDDA/PSS/PDDA/MUA/Au, (75% SOPS/25% POPC) bilayer/PDDA/PSS/PDDA/MUA/gold before and after protegrin-1 insertion is shown. Thicknesses measured by SPR are shown for the individual layers on gold.
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assuming a refractive index of 1.414. Since the second PDDA layer was adsorbed from a less concentrated solution, the water content in the second PDDA layer was more than that of the first PDDA layer, which explains why the second PDDA layer appears to be a little thicker than the first PDDA layer. The thickness values of the polymer layers are reasonable and fall in the range reported in the literature for multilayer thin films, which are about ˚ in thickness [49]. A PDDA/PSS bilayer 5 /20 A ˚ , as measured by optical ellipsothickness of 23 A metry, was reported for 50-bilayer electrostatic self-assembly thin films, assuming a refractive index of 1.539 [50]. In a typical cycle of PDDA/ silicate multilayered films, a PDDA layer of ˚ was adsorbed from a 5 wt% approximately 11 A aqueous solution [51]. An X-ray reflectivity study ˚ four yielded a film thickness of about 200 A double layers (PSS/PAH)4 on PEI-treated glass ˚ or cysteamine-treated gold surface of about 200 A by consecutive adsorption of oppositely charged polyelectrolytes [23].
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Fig. 4. SPR kinetic profiles of lipid adsorption with different compositions of SOPS and POPC (100% SOPS; 87.5% SOPS/ 12.5% POPC; 75% SOPS/25% POPC) on PDDA/PSS/PDDA/ MUA covered gold surfaces at pH 7.2. For clarity purposes, the kinetic profiles shown in the figure connect the bilayer kinetics and the rinsing kinetics. The perpendicular arrows indicate rinses with buffer to eliminate unbound vesicles. The reflectivity changes were negligible before and after rinsing.
3.3. Deposition of lipids on PDDA/PSS/PDDA Lipid layers of pure, negatively charged lipid, SOPS, and lipid mixtures of SOPS and zwitterionic lipid, POPC were deposited on the PDDA/PSS/ PDDA polymer cushions by vesicle fusion. The kinetic profiles of the adsorption processes are shown in Fig. 4. The final membrane thickness values are plotted in Fig. 5 and listed in Table 1. The thickness values were obtained by fitting the SPR curves before and after vesicle fusion (after rinsing) using the WASPLAS 2.1 software. As shown by SPR kinetic profiles, pure SOPS followed a sharp, fast deposition before the reflectivity reached an equilibrium state within an hour’s time. The thickness of the adsorbed SOPS layer ˚ , as simulated by using a refractive was 409/1 A index of 1.49 for the bilayer. The lipid mixtures with 12.5% POPC and 25% POPC followed similar kinetics: a quick steep growth period and a flat equilibrium state, in comparison with pure SOPS. The thickness increased slightly with an increase in POPC content. When the POPC content was ˚, 12.5%, the thickness of the lipid layer was 44 A
Fig. 5. FRAP data curves obtained for bilayers (100% SOPS; 75% SOPS/25% POPC) formed on PDDA/PSS/PDDA/MUA covered gold surfaces at pH 7.2 at room temperature. The fluorescence intensities of the two bilayers were normalized independently.
which falls in the range of a single bilayer thickness. With an increased amount of POPC composition up to 25%, the thickness of the lipid
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˚ , which is in the layers increased to about 52 A upper range for single bilayer thickness, usually ˚ . Different refractive index values about 40 /50 A used for lipid layers in the literature vary from 1.45 to 1.50 [52,53]. Using a refractive index of 1.50 for the bilayers in this work, the simulated SOPS ˚ */a little thinner than a bilayer thickness is 37 A single bilayer. However, using a refractive index of 1.50 as the refractive index for the bilayer made up of 75% SOPS and 25% POPC results in a thickness ˚ . Thus it is reasonable to consider 75% of 48 A SOPS/25% POPC adsorbed on the PDDA/PSS/ PDDA cushions formed bilayers. We observed a trend of increasing bilayer thickness with increasing POPC content (data not shown), which is very similar to that of the thickness values of the lipid layers deposited on only one PDDA polymer layer [34,35]. In vesicle fusion of SOPS, the driving force of adsorption is the electrostatic coupling between the negatively charged SOPS vesicles and the positively charged PDDA surface. The negative charges of the adsorbed lipid layer repelled further adsorption of lipid molecules, resulting in a single bilayer structure. The relatively thinner SOPS ˚ on PDDA/MUA bilayer with a thickness of 32 A layer pair [34,35], lower than the usual bilayer ˚ for long-chain thickness of approximately 40 A phospholipids in the fluid phase, is possibly due to the interdigitation between the two lipid monolayers and small defects in the bilayers. When the SOPS bilayer is lifted up by the PDDA/PSS/ PDDA cushions, the second PDDA layer is deposited from a less concentrated solution (0.05 M) than that of the first PDDA solution (0.2 M), which may result in a reduced surface charge density in the second PDDA layer, therefore reducing the electrostatic interaction between the SOPS molecules and the second PDDA monolayer. The weaker electrostatic attraction may result in less interdigitation between the two lipid monolayers, thus the SOPS bilayer lifted up on the PDDA/PSS/PDDA cushions is a little thicker than that on only one PDDA layer. When mixing the negatively charged lipid with a neutral phospholipid, the bilayer thickness increases due to a reduced charge density of the lipid membrane with an increase of the POPC content [23]. Similarly, the higher the POPC content in the
mixture of SOPS/POPC, the weaker the electrostatic coupling, the less interdigitation between the two lipid monolayers, and the larger the bilayer thickness. Fluorescence images of the lipid membranes formed by both pure SOPS and 75% SOPS/25% POPC on the PDDA/PSS/PDDA cushions indicated homogeneous distribution of the fluorescent probe molecules in the bilayers with no obvious defects (data not shown). Lateral mobility of the two bilayers was quantitatively analyzed as described in the experimental section. For both of the two cases, the fluorescence intensity of the bleached spot recovered more than 85% at 90 min after bleaching. The bilayer formed by pure SOPS recovered slower than that formed with 75% SOPS/25% POPC (Fig. 5). The calculations for the lateral diffusion coefficients (D ) showed that for pure SOPS bilayer D /1.2 /109 cm2/s, whereas for the bilayer with 75% SOPS/25% POPC D /2.1 /109 cm2/s, both of which were very similar to corresponding cases (D /1/109 cm2/s for pure SOPS bilayer, D /2/109 cm2/s for the bilayer with 75% SOPS/25% POPC) on only one PDDA layer [34]. Cassier et al. [23] reported a value of D about 1 / 109 cm2/s for a negatively charged lipid DOPA bilayer coupled to polyelectrolyte multilayers. It is possible that the high surface charge density restricts the mobility of anionic lipid coupled to the cationic polymer surface, and that the addition of neutral lipid helps to dilute the charge density and improve the mobility of the mixed lipid bilayers. The free zwitterionic lipid monolayers or bilayers are known to exhibit values of D near 108 cm2/s for a fluid phase and near 10 10 cm2/s for a gel phase in lipid/polyelectrolyte composites [54,55]. The lateral diffusion coefficients of lipid bilayers for both pure SOPS and 75% SOPS/25% POPC mixture on three polymer monolayers in this system, like that on only one polymer monolayer in previous study [34], are also within the range of diffusion coefficients in fluid bilayers and are well above the characteristic diffusivities for lipid membranes in the rigid Lb phase (partly ordered, untilted, D B/1011 cm2/s) [56].
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3.4. Peptide insertion Protegrin-1 belongs to a family of innate host defense molecules called antimicrobial peptides, which were first discovered in the early 1980s [39]. This peptide is able to alter the cellular membrane permeability by forming ionic pores that present some specific properties in common with human defensin-channel [57]. Since it displays a broadrange antimicrobial activity against bacteria such as Escherichia coli , C. trachomatis , N. gonorrhea, as well as HIV-1, protegrin-1 has excellent potential for pharmaceutical uses [39]. The sequence of protegrin-1 is NH2 /RGGRLCYCRRRFCVCVGR /CONH2. The peptide has a twostranded antiparallel b-sheet with a hairpin structure held by disulfide bonds formed by cysteine [44,58]. Positively charged arginine residues are present at both ends of the hairpin-like molecule, while hydrophobic and uncharged amino acids form the remainder of its structure [36]. Peptide insertion experiments were monitored by SPR on supported bilayers formed by pure SOPS and 75% SOPS/25% POPC (Fig. 6). Two milliliters of 20 mM solution of peptide protegrin-1 in Tris 7.2 buffer was injected into the Teflon cell. The
Fig. 6. SPR kinetic profiles of protegrin-1 insertion into bilayers composed of 100% SOPS and 75% SOPS/25%POPC formed on PDDA/PSS/PDDA/MUA covered gold surfaces. Data of thickness measurements are listed with the curves for bilayer thickness before and after peptide insertion (after rinsing). Thickness data are in angstroms.
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reflectivity increased instantaneously upon injection of the peptide solution and continued to increase more slowly until equilibrium was reached. After 90 minutes (sufficient time to observe the uptake kinetics), the excess protegrin1 solution was rinsed with Tris 7.2 buffer until there was no further decrease in reflectivity. The thickness results after rinsing are listed in Fig. 6 and Table 1. For pure SOPS bilayers, the thickness ˚ , after protegrin-1 increased from 41 to 58 A insertion. In the case of 75% SOPS/25% POPC ˚. bilayers, the thickness increased from 54 to 71 A It should be noted here that the same value of refractive index has been used for lipid bilayers before and after peptide insertion. Therefore, the thickness increases should be regarded as addition of materials to the lipid membranes [34 /36]. The net increases in thickness were the same for both ˚ . This result is in bilayer systems, i.e. 17 A agreement with the report that the pore structure caused by peptide insertion is relatively insensitive to the detailed composition of the membranes [39,59]. However, the net increase in thickness was ˚ for pure SOPS bilayers supported on the 12 A PDDA/MUA cushions and for pure SOPS bilayers and 75% SOPS/25% POPC bilayers on PDDA/ MPA (3-mercaptopropionic acid) cushions [36]. It needs to be pointed out that, using an optical technique such as SPR alone, it is not easy to determine whether protegrin-1 molecules inserted into the lipid bilayers or adsorbed on the bilayer surfaces. We know that the surface charge density of the second PDDA layer is reduced compared to the first PDDA layer. This would correspond to less repulsion between the cationic protegrin-1 inserted into the bilayer and cationic PDDA, which promotes more protegrin-1 inserted in the bilayer as we observed here. The SPR kinetic profiles of peptide insertion showed a two-step interaction of the protegrin-1 molecules and the lipid bilayers. The initial fast increase in reflectivity might be a response to the surface adsorption of protegrin-1 molecules to the lipid membrane. The slow increase of reflectivity may be due to the trans bilayer insertion process of the peptide: adsorbed molecules insert into the membrane and more peptide molecules add to the surface to replace inserted ones [36]. The helical antimicrobial pep-
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tides, alamethicin and magainin, exhibit two distinct states in lipid bilayers, one state is adsorbing in the lipid headgroup region with the helical axis oriented parallel to the plane of the bilayer and the other state exists as trans bilayer insertion into the hydrocarbon region with the helical axis oriented perpendicular to the plane of the bilayer [60,61]. Heller et al. reported that the two different states of protegrin-1 existing in membranes might correspond to the surface state and the insertion state of alamethicin: one state is adsorbed to the headgroup region of a lipid bilayer with the peptide oriented parallel to the horizontal plane of the bilayer, whereas in the other state, the peptide is inserted perpendicular to the bilayer plane, i.e., the transmembrane state [39,44]. This orientation study supports the hypothesis of our results.
step interaction, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/ MUA. The results are comparable to our work on supported lipid membranes without a layer-bylayer cushion [33,35]. The supported lipid bilayer on a cushion is a useful system to be explored for membrane protein insertion.
Acknowledgements The MRSEC program of the National Science Foundation under Award DMR-9808677 supported this work. M. L. Longo acknowledges funding from the NSF through the CAREER Program (BES-9733764). R. I. Lehrer and A. J. Waring acknowledge support through the following NIH grants: AI22839 and AI37945.
4. Conclusions References A supported lipid membrane system lifted up by a cushion of layer-by-layer polyions on an SAM of alkylthiol was investigated by SPR and FRAP. A long-chain alkylthiol (MUA) layer was self-assembled on a gold surface to create the first negatively charged organic layer. A polyelectrolyte film (PDDA/PSS/PDDA) was then deposited on the alkylthiol-covered solid substrate by the technique of layer-by-layer deposition. By the method of vesicle fusion, anionic lipid SOPS and mixtures of SOPS and the zwitterionic lipid POPC (composition up to 25%) formed bilayers on the layer-bylayer polyion/ cushions on an SAM of alkylthiol through electrostatic interaction with the second PDDA layer. Mobility experiments by FRAP showed a slight increase of the lateral diffusion coefficient for the bilayers with 25% POPC compared to those formed by pure SOPS, both fell in the range of those for mobile membranes. The lateral diffusion coefficients of the single bilayers formed on polymer multilayers were very similar to those formed on only one PDDA layer. SPR measurements on the protegrin-1 addition to the bilayers showed no difference in kinetics or thickness changes with pure SOPS and 75% SOPS/25% POPC. In both cases, the pore-forming peptide remained bound to the lipid membranes by a two-
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