polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane Tathyana Tumolo a , Marcelo Nakamura b , Koiti Araki b , Mauricio S. Baptista a,∗ a b

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, P.O. Box 26077, São Paulo, Brazil Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, P.O. Box 26077, São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 14 June 2011 Received in revised form 22 September 2011 Accepted 10 October 2011 Available online 17 October 2011 Keywords: Hybrid bilayer membrane Biosensors Inner mitochondrial membrane Cations Surface plasmon resonance

a b s t r a c t We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness ´˚ in contrast to only 2 ± 1.5 A´˚ usually observed in the absence of cations. In the presence of of 19.5 ± 1.8 A, specific concentrations of spermine and Ca2+ the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na+ was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Many events can be characterized and quantified using surfaces that mimic cell membranes including the interactions of solid substrates [1–3], drugs [4], antibodies [5], carbohydrates [6–8] and peptides/proteins [9–13] with membranes. Lipid monolayers and bilayers can be assembled in sensor surfaces by several ways [2,14–16]. The more prevalent methods are Langmuir–Blodgett [2] and vesicle adsorption on solid supports [14–16] that allow the preparation of specific membrane surfaces using synthetic and/or natural phospholipids. However, the process of transferring lipids and proteins to solid supports is not yet well understood. Many studies have evaluated the mode of interaction between liposomes and solid surfaces, such as silica [17], mica [18], gold (self-assembled monolayer) [19], and surfaces chemically modified with polyelectrolytes [12]. The characterization of lipid layers assembled on solid surfaces has been carried out by different techniques, including atomic force microscopy (AFM) [20], quartz

∗ Corresponding author. Tel.: +55 11 3091 8952; fax: +55 11 3091 2186. E-mail address: [email protected] (M.S. Baptista). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.10.013

crystal microbalance with dissipation monitoring (QCM-D) [21] and surface plasmon resonance (SPR) [11]. One of the most employed solid supports to facilitate the adsorption of lipid layers are thiol monolayers with hydrophobic or hydrophilic terminations, which are self-assembled on gold surfaces [22–29]. A system composed of a self-assembled alkanethiol monolayer onto a metal surface (e.g. gold) and an adsorbed phospholipid monolayer on this alkanethiol layer is called hybrid bilayer membrane (HBM) [29–31] (Fig. 1). The main advantages of HBM are its stability and easiness to prepare, allowing the formation of a fluid lipid layer that can mimic cell membranes [30]. If the alkanethiol monolayer has hydrophilic termination the adsorption of vesicles can lead to the formation of a lipid bilayers, but in this case we must consider that the stability of the bilayer is highly dependent on the ionic strength of the solution. The exact mechanism of HBM formation on solid supports is not well understood, although important information are available [24–31]. Lingler et al. [24] proposed a stepwise interaction between the hydrophobic termination of a thiol monolayer and vesicles: (i) adhesion of vesicles to the hydrophobic surface; (ii) partial disruption of vesicle structure; (iii) lateral spreading of lipid monolayer with reorganization of lipid molecules on the surface. Complete surface coverage was achieved within a period of ∼7000 s [25,26].

2

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

giant unilamellar vesicles in different kinds of membrane studies [53,54]. No-one has ever reported phase separation, indicating the miscibility of these phospholipids at these molar ratios. In this work, we used SPR to characterize a mimetic model of the IMM adsorbed on thiol monolayer in the presence of Na+ , Ca2+ and spermine. We characterized its structure and report the best conditions for the effective formation of this HBM at various ionic strengths and periods of adsorption. The effect of the cations and polycations is discussed in face of the available theories of vesicle fusion and creation of HBM. This IMM biomimetic assembly may allow further studies on the interactions that might occur in mitochondria of leaving cells [55,56]. 2. Experimental 2.1. Reagents and samples

Fig. 1. Formation of a hybrid bilayer membrane (HBM) on a gold surface, using a hydrophobic thiol monolayer and vesicles made of different phospholipids.

This long period is explained by a thermodynamically unfavorable adsorption [24], mainly because of the hydration forces. There are many articles showing that divalent cations (Ca2+ and Mg2+ ) and polycations facilitate the fusion of membranes composed of negatively charged phospholipids [32–46]. Hong et al. [34] and Zimmerberg et al. [37], using negatively charged vesicles showed that fusion of these vesicles was facilitated by divalent cations and polyamines. Recently, Schultz et al. [38] employing vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) demonstrated that the interaction between PS head groups and Mg2+ leads to the formation of DPPS microdomains (composed of 10–15 lipids) and realignment planes of the acyl chains depending on the DPPS to DPPC ratio. Experiments in solution were also employed to demonstrate the role of spermine in vesicle fusion [34]. Although the role of cations and polycations on membrane fusion has been elucidated in solution by several solution-based experiments, the literature lacks studies on the effect of these species in membranes supported in solid substrates by using precise techniques such as SPR. Nevertheless, it is not trivial to transfer concepts and conditions described in solution-based experiments to membranes supported in solid substrates. Besides the importance of creating stable lipid layers for sensor technology, the interaction of cations with membranes and the consecutive facilitation of membrane fusion have many important roles in biology [47–49]. However, there is no comprehensive study on the effect of cations or polycations on the kinetics of surface coverage after vesicle adsorption to solid substrates. In addition, no one has used vesicles composed of different phospholipid ratios that mimic IMM. The general interest in understanding processes related with bioenergetics and with the mechanisms of cell death suggests that devising mimetic models of the IMM is highly interesting because they can allow new studies of the biophysics and physical-chemistry of the IMM [50]. A common lipid mimetic of the inner mitochondrial membrane (IMM) consists of a mixture of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL), about 39% PE, 44% PC and 17% CL [51]. This mixture has 44% of a saturated phospholipid (DMPC) and the remaining 56% of unsaturated phospholipids (dimethyl-PE and CL), which is a saturated/unsaturated ratio similar to the ratio found in the majority of the mammalian membranes, showing therefore good fluidity at room temperature [52]. Lipid mixtures, which are identical or similar to the one used here, have been used to prepare small and

The polycation spermine (Acros Organics, Belgium), phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) (Sigma, USA), cardiolipin from bovine heart disodium salt (CL) (Sigma, USA) and 1,2-dioileoyl-sn-glycero-3-phosphatidylethanolamine-N,N-dimethyl (dimethyl-PE) (Avanti Polar Lipids Inc., USA) (their structures are depicted in Scheme 1). All other materials were of the best analytical grade available were used as received. Water was bi-distilled from an all glass apparatus and was further purified through a Millipore Milli-Q system. Vesicles were prepared as follows: lipid solutions with a proper ratio of PC:PE:CL (4.5:3.5:2.0) were prepared in chloroform and dried under an argon flow in a glass tube to form a lipid film. An aliquot (600 ␮L) of Hepes buffer (Sigma, USA) 5 mM, pH 7.4, was added to this film, yielding a vesicle suspension composed of a mixture of phospholipids, which were passed at least 20 times through a 50 nm porous size membrane (Nuclepore-Whatman, USA) using a manual syringe to prepare small unilamellar vesicles (SUVs) by extrusion (extrusion system from Avestin, Canada). Other vesicles solutions were prepared in the same way by addition of NaCl (Synth, Brazil) and CaCl2 (Merck, Germany) solutions, both at concentrations 15 mM and 150 mM, and spermine solutions 0.2 mM and 3 mM – all of them were prepared in Hepes buffer 5 mM pH 7.4. Large unilamellar vesicles (LUVs) were prepared by extrusion through a 1000 nm porous size membrane in the presence of CaCl2 150 mM. The transition temperatures (Tm ) of phospholipids used in this study are CL = 10 ◦ C [57], DMPC = 23.5 ◦ C and dimethyl-PE = 21.2 ◦ C [58]. Our experiments were conducted at room temperature (about 25 ◦ C). 2.2. Dynamic light scattering Determination of the size distribution of the vesicles was performed by dynamic light scattering technique (DLS), using ZetaPALS and Particle Sizing Software 2.29 from Brookhaven Instruments Corp. (NY, USA). This experiment was performed for SUV prepared by extrusion through a 50 nm porous size membrane in Hepes buffer (pH 7.4) in the presence of NaCl 150 mM. Hepes buffer was previously filtered to eliminate dust particles. Two solutions of vesicles containing the same proportions of dimethyl-PE/DMPC/CL, but prepared at different days, were subjected to this analysis. The first solution was prepared one day before the DLS measurement and kept in the refrigerator; the second was prepared and analyzed at the same day. The effective diameter of the first preparation was 108.5 ± 1.2 nm with polydispersity 0.088. The effective diameter of the second one was 107.6 ± 1.2 nm with polydispersity 0.088. Therefore, these results showed that the vesicles are stable for at least 24 h and therefore the vesicles were not fusing or aggregating in solution.

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

O

-

OH P

O

O O

O

P

-

O H3C

H

H

O

O

O

O

O

O

+

N

H3C

P

O

O

H O -O

O

H

O

O

O O

3

O

O

1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N,N-dimethyl (Dimethyl-PE)

O

O

O +

N

P

O

O

H O -O

O O

1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)

H2N

Cardiolipin (CL)

NH

NH

NH2

N,N'-bis(3-aminopropyl)butane-1,4-diamine (Spermine) Scheme 1. Molecular structures of the phospholipids and spermine.

2.3. HBM preparation A miniaturized SPR sensor, the Spreeta Sensor (Texas Instruments, TX, USA), was used to measure the change in refractive index (n) due to adsorption/desorption of molecules. A flow injection system was introduced in the SPR equipment by using a peristaltic pump (model Tris, Teledyne Isco Inc., USA), a manual injector (kindly provided by Prof. Dr. Lucio Angnes, IQ/USP) and Tygon tubing (Masterflex, Cole-Parmer Instrument Co., USA). An octadecanethiol (ODT) (Aldrich, USA) solution (10 mM) was prepared in ethanol (Synth, Brazil), injected in the flow system and kept flowing over the sensor for about 18 h in order to functionalize the gold sensor. After this period, the surface was washed with ethanol (30 mL) followed by water (80 mL) to remove any thiol molecules that were not adsorbed. The thickness value of the ODT layer was calculated to be 2.1 ± 0.3 nm, which is in agreement with previously reported values [59–63]. HBMs were obtained by injecting PC/PE/CL vesicle suspensions at the flow rate 0.35 mL min−1 on the SPR sensor previously modified with ODT. When the vesicle solution reached the sensor, the flow was stopped and the vesicles were kept in contact with the ODT monolayer during 1300–8100 s (∼22 and 35 min, respectively), until the n values reach saturation (change of 10−5 R.I.U. – refractive index units). Hepes solution (5 mM, pH 7.4) was injected (flow rate 1.4 mL min−1 ) to remove non-adsorbed vesicles. All experiments were performed at least three times. HBM composed by single phospholipids, i.e. PC, PE or CL, were also a subject of study: in this case, vesicles suspensions were prepared with each one of these phospholipids in the same Hepes buffer solution, in the absence or in the presence of Ca2+ 150 mM solution. In order to characterize the required time to form an HBM by vesicle adsorption on an ODT monolayer, LUVs (PC/PE/CL) were prepared in the presence of Ca2+ 150 mM, and the same protocol of HBM formation described above was followed. Different periods of

adsorption were used: 500, 600, 850, 1000, 1900, 2200, 3300 and 6500 s (∼8.5, 10, 14, 17, 32, 37, 55 and 108 min). The integrity of the HBM obtained at 1900 s was tested by the adsorption of two proteins: cytochrome c (cyt c) from horse heart (Sigma, USA) and bovine serum albumine (BSA, fraction V) (Acros Organics, USA), both in the concentration of 10 ␮M prepared in Hepes buffer 5 mM. Cyt c has positive charge and BSA has negative charge at the pH at which the experiments were performed (pH 7.4). 2.4. Atomic force microscopy The following equipment was used: MAC ModelTM Scanning Force Microscopy (SFM), Type II MAC Levers (silicon cantilevers and silicon tips, nominal k ∼ 2.8 N/m, resonance frequency ∼75 kHz in air and ∼30 kHz in water), commercial liquid cell for in situ imaging (diameter 2 cm) without flow system, Agilent Series 4500 SPM Microscope (PicoSPM) and PicoScan 2100 Controller (Molecular Imaging Corp., USA). At first, gold substrates were cleaned by immersion and sonication in the following solvents: toluene, acetone and ethanol (all from Synth, Brazil). The modification of the gold surface with ODT followed the protocol described above at HBM Preparation. The gold surface, clean and dry, was placed in the AFM cell and the LUV solution prepared in the presence of CaCl2 150 mM was kept in contact with this surface for 1900s. After this period, the LUV solution was removed and the surface was washed with Hepes buffer pH 7.4. To perform AFM measurements the cell compartment was completely filled with Hepes buffer. 2.5. Theoretical background on SPR measurements At first, it was estimated the decay length of evanescent electromagnetic field (ld ) according to Jung et al. [62]: ld =

/2 [−n4eff /(n2eff

+ εmetal )]

1/2

(1)

4

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

Fig. 2. (a) Changes in refractive index (relative values) as a function of time for the formation of a lipid layer in the presence of Hepes 5 mM, pH 7.4 (absence of added cations). (b) Changes in n as a function of time in the presence of Hepes 5 mM pH 7.4 and spermine 3 mM. (c) Thickness values estimated after all experiments in comparison to those values referred in the literature (hachured area), where 1: Na+ 15 mM; 2: Na+ 150 mM; 3: Ca2+ 15 mM; 4: Ca2+ 150 mM (SUV); 5: Ca2+ 150 mM (LUV); 6: Ca2+ 150 mM (LUV after EDTA flow); 7: spermine 0.2 mM; 8: spermine 3 mM; 9: absence of cation/polycation (R.I.U.: refractive index units). All experiments were conducted in solutions containing Hepes 5 mM pH 7.4.

where  is the wavelength of the light source in the Spreeta Sensor (840 nm), neff is the equivalent refractive index of a system “film + buffer” monitored by the SPR equipment and ε is the gold dielectric constant (23 + 124i). The thickness d (nm) of a saturated layer (thiol or lipid monolayer) is given by [62]: d=−

l  d

2



ln 1 −

neff − nb



na − nb

(2)

where na is the refractive index of the bulk material and nb is the refractive index of solvent or buffer. The difference between the refractive index (n) baseline value for buffer in the absence of salt (nb ) and those n values reached after Hepes buffer flow (neff ) was used to estimate the thickness of lipid layers assembled on thiol monolayers. In these calculations the following values of n were used: na = 1.46 for phospholipids [64], na = 1.45 for thiols [62], nb = 1.3332 for Hepes buffer and nb = 1.36 for ethanol [65]. The value of d was used to estimate the adsorbed amount of phospholipids according to following equation [66]:  =d

n − n  a b dn/dC

(3)

where d is the thickness of the adsorbed layer (in nm) and dn/dC is the refractive index increment of the adsorbed material [67], in this case phospholipids on thiol layer (dn/dC = 0.182 cm3 g for phospholipids [68]). 3. Results and discussion 3.1. Influence of cations in the formation of a PC/PE/CL HBM When vesicles were prepared and injected in Hepes buffer (without any extra salt added), there was an initial increase in the

refractive index, which may be due to several processes, i.e., differences in the refractive index of the vesicle solution, adsorption of integral vesicles, formation of lipids patches weakly adsorbed on the surface, and formation of a monolayer. We cannot accurately quantify anything, unless the solution flowing through the instrument has the same refractive index of the buffer solution used before the injection. Note that there was almost no change in the refractive index after the washing step, showing that the phospholipid adsorption was negligible (Fig. 2a). By using Eq. (2), the estimated average thickness of the remaining lipid monolayer was only 0.2 ± 0.15 nm. Note that this value is much smaller than the thickness expected for a lipid monolayer, which is basically dependent on the lipid length, being 1.6 ± 0.2 nm for a lecithin monolayer (adsorbed on octadecanethiol) and 2.2 nm for a DMPC monolayer (on dodecanethiol) [24,26,31,69]. Although adhesion and rupture of vesicles can occur through a slow process in the absence of cations, this process is inefficient and only a small amount of vesicles coalesce on the thiol layer. A few phospholipid molecules that remained on the hydrophobic surface did not form a homogeneous phospholipid monolayer, thus the lipid layer has a low thickness value. Former studies had described the successful formation of stable HBMs, but in those cases vesicle suspensions were always made in buffers with high salt concentrations, usually NaCl [24,26,30]. Therefore, we decided to investigate the effect of different cations on the efficiency and kinetics of HBM formation by performing experiments in the presence of NaCl, CaCl2 and spermine. Changes in n as a function of adsorption time, which were achieved after the injection of vesicles prepared in the presence of spermine, are shown in Fig. 2b. It is also shown a comparative plot of monolayer thickness obtained at different experimental conditions (Fig. 2c).

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9 Table 1 Thickness values of the phospholipid monolayer and amount of adsorbed phospholipids in the HBM formed on ODT monolayer after injection of vesicles prepared in different conditions. Solution

Thickness (nm)

NaCl 15 mM NaCl 150 mM CaCl2 15 mM CaCl2 150 mM (SUV) CaCl2 150 mM (LUV) CaCl2 150 mM (LUV, after EDTA) Spermine 0.2 mM Spermine 3 mM Cation/polycation absent

0.3 1.3 2.2 1.7 2.2 2.0 2.3 2.2 0.2

± ± ± ± ± ± ± ± ±

0.2 0.1 0.2 0.2 0.3 0.2 0.5 0.6 0.1

Adsorbed amount (ng mm−2 ) 0.2 0.9 1.5 1.2 1.5 1.4 1.6 1.5 0.1

± ± ± ± ± ± ± ± ±

0.2 0.03 0.2 0.2 0.3 0.1 0.3 0.4 0.1

In addition, values of thickness and amount of adsorbed phospholipids are summarized in Table 1. One can observe that injection of vesicles on an ODT monolayer leads to an increase in n, which do not return to its baseline value even after injection of buffer (Fig. 2a and b). By analyzing the plot in Fig. 2c and the results summarized in Table 1 one can observe that the presence of cations affect the layer thickness and the amount of adsorbed phospholipids. In the absence of cations/polycations (Fig. 2c – point 9) we could barely observe any adsorption on the ODT monolayer. At 15 mM of NaCl we quantified formation of a lipid layer with thickness of only 0.3 ± 0.2 nm, which indicates a defective and/or incomplete lipid monolayer. But NaCl 150 mM allowed the formation of a lipid layer with thickness of 1.3 ± 0.1 nm, which is better than that reached with NaCl 15 mM, but still smaller than that predicted for a complete lipid monolayer (1.6–2.2 nm). In the presence of the polycation spermine 3 mM (Fig. 2b), the thickness was estimated in 2.2 ± 0.6 nm. Even at low spermine concentration (0.2 mM) we quantified thickness of 2.3 ± 0.5 nm (see Table 1). One can conclude that spermine induces the formation of lipid monolayer even at low concentrations in a reproducible manner, in contrast to the results obtained with NaCl (Fig. 2c). The thickness of lipid layers formed in the presence of 15 mM or 150 mM of Ca2+ were estimated to be 2.2 ± 0.2 nm and 1.7 ± 0.2 nm, respectively, which are indicative of the formation of lipid monolayers with thickness values in agreement with a complete coverage. When using large unilamellar vesicles (LUV) in the presence of Ca2+ 150 mM, thickness was estimated to be 2.2 ± 0.3 nm, showing that the size of the vesicle does not seem to affect the thickness of the lipid monolayer.

5

Table 2 Estimated values of thickness and amount of adsorbed lipids after different periods of adsorption in the presence of LUV and Ca2+ 150 mM (after EDTA and buffer flow). Adsorption time

Thickness (nm)

500 s (8.5 min) 600 s (10 min) 850 s (14 min) 1000 s (17 min) 1900 s (32 min) 2200 s (37 min) 3300 s (55 min) 6500 s (108 min)

1.8 1.8 1.7 1.9 1.8 1.9 1.9 1.9

± ± ± ± ± ± ± ±

0.2 0.2 0.1 0.1 0.1 0.2 0.2 0.2

Adsorbed amount (ng mm−2 ) 1.3 1.2 1.2 1.4 1.3 1.3 1.3 1.4

± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1

In the earlier 80s, de Kruijff and Cullis [70] showed that calcium ions bind to phospholipids, which might help to explain the mechanism of HBM formation in the presence of calcium (see further discussion below). The Ca2+ /phospholipid binding constants are typically 10–20 M−1 for zwitterionic phospholipids, and in the case of charged phospholipids this value is higher and dependent on the ionic strength. Therefore, calcium ions that remain in the lipid monolayer after HBM formation may, in principle, interfere with the thickness measurements. In the LUV/Ca2+ 150 mM experiments, EDTA (50 mM solution) was injected after the buffer washing step and a new thickness for the lipid monolayer was estimated, which was 2.0 ± 0.2 nm. EDTA is mainly pentaprotic at pH 7.4 and forms stable complexes with Ca2+ ions. Although there is no definitive prove of its effect on membrane mimetics supported on solid substrates, EDTA was used in these systems to remove the excess calcium ions that were bound to the HBM. When comparing the two thicknesses (with and without an EDTA flow) only small differences in the thickness were observed, which were not statistically significant (by performing Student’s t-test) and indicate that the residual calcium ions has a negligible effect in the thickness measurements. It is also important to characterize the time required to allow the formation of a flawless HBM by letting the vesicles in contact with the ODT monolayer. Changes in n as a function of time, using three different periods of adsorption, are shown in Fig. 3a. One can observe that all curves describe the same adsorption pattern. Fig. 3b and Table 2 present the thickness and the amount of adsorbed lipid evaluated using eight different periods of adsorption. The periods of adsorption that allowed the formation of a complete HBM (as low as 8.5 min), at this specific condition (presence of Ca2+ 150 mM), are much smaller than the adsorption periods suggested in the literature, i.e., 7000 s (∼117 min) [24–26].

Fig. 3. (a) Variation of refractive index as a function of time for the adsorption of PC/PE/CL LUV on the thiol surface in the presence of Ca2+ 150 mM, at three different periods of adsorption. Down arrows indicate the specific time in which the injection of buffer was performed. The thickness measurements were performed as indicated in Fig. 2. (b) Calculated thickness of the lipid monolayers formed at eight different periods of adsorption compared with the range of values of lipid monolayers available in the literature (hachured area).

6

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

Fig. 4. Topographic image obtained by AFM (left) for a hybrid bilayer membrane that mimics the inner mitochondrial membrane and its cross-sectional line scan (right). [Ca2+ ] = 150 mM.

A control experiment was performed using vesicle adsorption on a system composed of a positively charged polyelectrolyte, i.e., PDDA (polydiallyldimethylammonium chloride), directly assembled on the gold surface. The estimated thickness of the lipid layers achieved by SUV adsorption (prepared only in Hepes buffer solution) on the PDDA surface was 21.3 nm, which is an indicative of the formation of many lipid layers instead of single lipid monolayer or a bilayer. Therefore, the presence of cations in the vesicle solution has distinct effects than those observed when vesicles are adsorbed directly on charged surfaces covered by cationic polyelectrolytes. Cations and polycations seem to cause subtle changes in the forces that stabilize a lipid bilayer facilitating the HBM formation (see further discussion below).

3.2. Characterization of the HBM formed in the presence of calcium It is important to characterize whether the HBM that mimic IMM are well formed in the presence of calcium, i.e., if the lipids are homogenously distributed and if the charged lipids, which are present in the vesicles, are also present in the HBM. The 2D topographic image obtained by AFM of a HBM formed within 1900 s (32 min) of vesicle adsorption shows good homogeneity, without any apparent domains showing excess of surface modification or exposure of surface areas without modification (Fig. 4, left). A crosssectional line scan obtained from this surface (1.5 ␮m × 1.5 ␮m) is also shown in Fig. 4 (right), with a root mean square (rms) roughness of 3 nm over this area. This value is relatively large considering the thickness of the whole HBM, which is around 4 nm. However, similar roughness values have been reported for supported bilayers in surfaces that have high heterogeneity in the z axis [71]. Although this experiment does not allow an accurate evaluation of the real thickness of the HBM, one can observe the homogeneity and integrity of the lipid layer on the surface [71]. The integrity of this HBM was also confirmed by differential adsorption of two proteins on the HBM: bovine serum albumine (BSA) and cytochrome c (cyt c). Considering that this HBM is 80% zwitterionic (PC and PE) and 20% composed by an anionic phospholipid (CL), one can expect that it will occur a strong interaction between HBM and cyt c, which is positively charged at pH 7.4, but not with BSA, which is negatively charged at this pH [29]. In the plot of Fig. 5 one can verify the interactions with both proteins and observe that after injection of BSA there is a small refractive index change that return to the baseline level after injection of buffer. Because BSA is known to bind strongly to both gold surface and ODT monolayers, the absence of BSA binding is an indicative of

complete phospholipid coverage. The fact that BSA does not bind to this HBM also suggests that the HBM is negatively charged, because there is an electrostatic repulsion between the negatively charged BSA and the headgroup of cardiolipin. On the other hand, the injection of cyt c leads to a considerable change in n, which do not return to the baseline level after buffer injection, an indicative of strong protein adsorption. The cyt c-HBM interaction could only be dissociated by addition of Ca2+ [29]. This profile is expected on the adsorption of cyt c on a surface that mimics IMM. Thus, one can conclude that this HBM exhibit the characteristics of the lipid components of the IMM. 3.3. Kinetics of adsorption The kinetics of phospholipid adsorption were studied by plotting the amount of adsorbed phospholipids as a function of time and as a function of ln (time) (Fig. 6a and b, respectively), considering five different preparations of vesicles: absence of cation, SUVs in NaCl 150 mM, CaCl2 150 mM and spermine 3 mM, and LUVs in CaCl2 150 mM. The plots in Fig. 6a clearly show that there is a relationship between the rate of adsorption and the presence of cations. The amount of adsorbed phospholipids as a function of time in the presence of spermine or calcium is higher than that obtained in the presence of NaCl. It is also clear that the adsorption kinetics do not follow Langmuir type isotherms (Fig. 6b).

Fig. 5. Adsorption of BSA and cyt c on the HBM that mimics IMM. The injection of the solutions (prepared at pH 7.4) of both proteins, buffer and calcium are indicated by arrows.

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

7

Fig. 6. (a) Amount of adsorbed phospholipids as a function of time (min) after injection of vesicles prepared in the absence of cation (curve 1), and after injection of vesicles prepared under different conditions: SUV/Na+ 150 mM (2), SUV/Ca2+ 150 mM (3), SUV/spermine 3 mM (4) and LUV/Ca2+ 150 mM (5). (b) Amount of adsorbed phospholipids as a function of natural logarithm of time, in the same conditions as in (a).

In the initial stages of HBM formation there are several steps of adsorption, which are, the approach and adhesion of vesicles on the thiol monolayer (a fast process) followed by rupture of the vesicular structure and the monolayer formation, which are slow processes because the hydrophobic tail of phospholipids in vesicles must interact with the hydrophobic tail of thiol layer [24]. We decided to arbitrarily separate the process in steps 1 and 2 as the first and the next 30 s of adsorption, respectively. Step 1 is probably related to approach/adhesion of vesicles on the surface, while step 2 must have both components of adhesion and fusion/rupture of vesicular structure. From 1 min to 3 min, the process assumes an almost zero order rate and must be mainly relate to the reorganization of the lipids adsorbed on the surface. The rates obtained for steps 1 and 2 are presented in Table 3. Note that vesicles prepared in the presence of Na+ , even at high concentrations, present extremely small rate values indicating that Na+ is not able to facilitate either adhesion or fusion/rupture steps. Conversely, both calcium and spermine are efficient inductor of both steps. By considering the used concentrations and the observed rates, spermine seems to be the most efficient inductor of both processes. Another effect that seems to be important is the size of the vesicles. LUVs present two times faster steps 1 and 2 compared with SUVs under the same Ca2+ concentrations (compare kinetics 3 and 4). Independently of the vesicle type or the cation used to prepare the vesicles, the faster is step 1 the faster will be step 2 and also the rates measured from 1 to 3 min (0.8, 0.6, 0.4, 0.1 ng mm−2 /min, for kinetics 5, 4, 3, 2, respectively). This indicates that the conditions that stimulate vesicle adhesion will also stimulate the fusion/rupture processes, suggesting again that the effects caused by ions must be related with destabilization of the forces that maintain the membrane integrity [35].

3.4. Mechanism of HBM formation in the presence of calcium An interesting question arises from the experiments showed above: the effects of calcium and spermine are only due to the presence of cardiolipin in the lipid mixture or the other lipids also interact with these cations? It was possible to characterize the effect of Ca2+ on each phospholipid type by using vesicles composed of only one of the phospholipids (Fig. 7). It is clear that in the absence of Ca2+ the phospholipid monolayers are barely formed (very small thickness values are obtained). However, in the presence of Ca2+ , phospholipid monolayers having expected thickness were observed in all cases, indicating that the effect of calcium favoring HBM formation also occurs for vesicles made of single phospholipids. It can also be noted the different thickness among different HBMs, which is due to the fact that each phospholipid has its characteristic packing in the HBM, allowing different thickness and amounts of adsorption. Therefore, all phospholipids interact with calcium causing changes in their respective vesicles that support the HBM formation, demonstrating that the interaction is not pure electrostatic, once the net charge of the lipids is neutral for DMPC and dimethyl-PE and negatively charged for CL. However, all phospholipids have negatively charged phosphate groups that interact specifically with Ca2+ .

Table 3 Estimated rates of phospholipid adsorption in experiments of HBM formation. The numbers in parenthesis designate the experiments that are shown in Fig. 6. Solution

(1) Cation/polycation absent NaCl 15 mM (2) NaCl 150 mM CaCl2 15 mM (3) CaCl2 150 mM (SUV) (4) Spermine 3 mM (5) CaCl2 150 mM (LUV) Spermine 0.2 mM

Rate of adsorption (ng mm−2 /min) 1st kinetic

2nd kinetic

0.1 0.1 0.1 4.5 4.0 7.3 8.9 7.9

– – 0.05 1.1 1.4 2.6 3.5 1.7

Fig. 7. Estimated thickness of monolayers of phospholipids achieved by the adsorption of vesicles prepared with pure phospholipids (PC, PE or CL) in individual adsorption experiments on ODT monolayers, in the presence and absence of Ca2+ 150 mM. Adsorption times, with stopped flow, were 7000 s in the absence of calcium and 700 s in the presence of calcium.

8

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9

Several effects can affect the efficiency of HBM formation, i.e., ionic strength, specific ion interaction, hydration force, lipid reorganization. Although ionic strength seems to play a role, its effect is much smaller than that of ions that specifically interact with the phosphate groups of the membranes. Our results indicate that Ca2+ and spermine are much more efficient on HBM formation, both in terms of final thickness and rate of HBM formation (Fig. 2c and Table 3, respectively), than Na+ ions, indicating the important role of phosphate/cation interactions. Cations can also act as a “bridge” in the binding of phospholipids molecules to the solid support [42], and polycations, such as spermine, are known to efficiently induce vesicle aggregation [35]. The four amino groups in spermine molecule have high pKa values (8.6, 9.3, 10.4 and 11.2) [72]. The pH used in these experiments was 7.4 (buffered with Hepes); therefore, all amino groups in spermine are positively charged, allowing strong interaction with phosphate groups. Although the results with spermine are promising for the purpose of forming a HBM with a percentage of anionic phospholipid, CaCl2 is a more economically affordable reagent and easily found in laboratories. Recently, it was also demonstrated that the formation of continuous supported lipid bilayer (SLB) on borosilicate glass was induced by SLB edges, that is, SLB patches surrounding by integral vesicles, which enhanced vesicle adhesion and induced its rupture [73]. In general terms, results from our work described herein as well as literature articles indicate that molecules and ions that favor vesicle fusion could play important roles in favoring HBM formation. Literature data also indicate that Ca2+ ions can bind to both zwitterionic [41,74] and anionic charged lipids [70,75,76], explaining why the effect of calcium favoring HBM formation is observed both by zwitterionic and charged membranes. These ion/membrane interactions cause an endothermic process observed for a Ca2+ /CL interactions, excluding the occurrence of electrostatic interactions, which should be exothermic [77]. According to Sinn et al. [77], binding of Ca2+ to PC membranes is an entropic driven process. Ion-pair formation, which causes dehydration on the vesicle surface and consequently entropy increase, seems to be the major event favoring membrane reorganization. Ion-pairs can be both solvent-free and solvent-separated, although Oliveira et al. [78] have shown that with a minimum of available water, solvent-separated ion-pairs are already present. The role of hydration forces and lipid reorganization is also evident because both Ca2+ and spermine are known to cause phase separation, destabilization of the hydration forces homogeneity, facilitating the exposure of hydrophobic groups at the vesicle surface. These changes would be of great importance to bring in contact the hydrophobic tail of phospholipids and the alkanethiol layer (therefore, a hydrophobic interaction), making the step of approach lipid-thiol layer more favorable due to hydrophobic attractive forces, and also leading to a decrease in the required time to the formation of a lipid monolayer. According to Leckband et al. [40], the addition of Ca2+ in a system composed of a mixture of phospholipids (PC/PG 3:1) leads to local phase separations into PG-rich and PC-rich domains with expansion of the PC-rich domains. In this latter domain, the hydrophobic region of the membrane bilayer interior can be exposed, resulting in a more hydrophobic surface that facilitates the fusion between bilayers. Therefore, there are reduction of both hydration and electrostatic repulsive forces and enhancement of attractive forces. Fusion is directly attributed to the enhanced hydrophobicity of lipid domains (interaction between exposed hydrophobic regions), which is caused by the binding of Ca2+ to the headgroup of phospholipids. Monovalent ions, such as Na+ , can change the electrostatic forces between bilayers and influence the kinetic of fusion, but without a special role in the process of fusion [79]. At high NaCl concentrations, the hydration of these ions on the surface of the bilayer prevents the approach between the bilayers through a repulsive

interaction (repulsion force by hydration or hydration of shortrange), limiting the fusion between the lipid layers. The fact that Ca2+ facilitate HBM formation not only with vesicles made of mixtures but also with vesicles made of pure phospholipids indicates that the important aspect is to destabilize the homogeneity of the hydration forces in the water/oil interface. The formation of HBM depends on the formation of small defects in the hydration forces at vesicle surface that favor the exposure of the hydrophobic core, allowing the proper surface lipid coverage in HBMs. When these concepts are applied to vesicles containing lipid mixtures, as the one used in this work that mimics IMM, one can quickly obtain homogeneous HBMs. 4. Conclusions In the absence of any type of salt vesicle adhesion and fusion is not favored and HBM formation depends on the interaction with substrate edges. Conversely, vesicles prepared in the presence of Ca2+ 15 and 150 mM and spermine 0.2 and 3 mM efficiently adhere and fuse on thiol monolayer, allowing fast formation of phospholipid monolayers. Ca2+ and spermine interact specifically with phosphate groups of the phospholipids destabilizing the hydration forces of the vesicle interface, favoring both vesicle adhesion and its rupture to allow HBM formation. By using vesicles made of proper lipid mixtures, we have shown and explained the conditions to form a HBM that mimics the inner mitochondrial membrane. Acknowledgements This research was financially supported by the São Paulo Research Foundation (Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo -FAPESP), CNPq and CAPES. References [1] M.I. Fisher, T. Tjärnhage, Biosens. Bioelectron. 15 (2000) 463. [2] S. Yokoyama, Y. Ohta, H. Sakai, M. Abe, Colloids Surf. B Biointerfaces 34 (2004) 65. [3] Y. Takeda, S. Horito, Colloids Surf. B Biointerfaces 41 (2005) 111. [4] Y.N. Abdiche, D.G. Myszka, Anal. Biochem. 328 (2004) 233. [5] I. Vikholm, T. Viitala, W.M. Albers, J. Peltonen, Biochim. Biophys. Acta Biomembr. 1421 (1999) 39. [6] F. Yang, X. Cui, X. Yang, Biophys. Chem. 99 (2002) 99. [7] A.P. Zhu, N. Fang, M.B. Chan-Park, V. Chan, Biomaterials 26 (2005) 6873. [8] P.Y. Tseng, S.M. Rele, X.L. Sun, E.L. Chaikof, Biomaterials 27 (2006) 2627. [9] R. Naumann, E.K. Schmidt, A. Jonczyk, K. Fendler, B. Kadenbach, T. Liebermann, A. Offenhäusser, W. Knoll, Biosens. Bioelectron. 14 (1999) 651. [10] R. Vidu, L. Zhang, A.J. Waring, R.I. Lehrer, M.L. Longo, P. Stroeve, Mater. Sci. Eng. B 96 (2002) 199. [11] K. Glasmästar, C. Larsson, F. Höök, B. Kasemo, J. Colloid Interface Sci. 246 (2002) 40. [12] C. Ma, M.P. Srinivasan, A.J. Waring, R.I. Lehrer, M.L. Longo, P. Stroeve, Colloids Surf. B Biointerfaces 28 (2003) 319. [13] E.J. Choi, E.K. Dimitriadis, Biophys. J. 87 (2004) 3234. [14] M. Tanaka, E. Sackmann, Nature 437 (2005) 656. [15] A. Berquand, P.E. Mazeran, J. Pantigny, V. Proux-Delrouyre, J.M. Laval, C. Bourdillon, Langmuir 19 (2003) 1700. [16] Y. Shao, Y. Jin, J. Wang, L. Wang, F. Zhao, S. Dong, Biosens. Bioelectron. 20 (2005) 1373. [17] R. Rapuano, A.M. Carmona-Ribeiro, J. Colloid Interface Sci. 193 (1997) 104. [18] R.P. Richter, A.R. Brisson, Biophys. J. 88 (2005) 3422. [19] C.A. Keller, B. Kasemo, Biophys. J. 75 (1998) 1397. [20] R.P. Richter, A. Brisson, Langmuir 19 (2003) 1632. [21] R.P. Richter, A. Mukhopadhyay, A. Brisson, Biophys. J. 85 (2003) 3035. [22] Z. Salamon, G. Tollin, Biophys. J. 71 (1996) 848. [23] Z. Salamon, G. Tollin, Biophys. J. 71 (1996) 858. [24] S. Lingler, I. Rubinstein, W. Knoll, A. Offenhäusser, Langmuir 13 (1997) 7085. [25] L.M. Williams, S.D. Evans, T.M. Flynn, A. Marsh, P.F. Knowles, R.J. Bushby, N. Boden, Supramol. Sci. 4 (1997) 513. [26] L.M. Williams, S.D. Evans, T.M. Flynn, A. Marsh, P.F. Knowles, R.J. Bushby, N. Boden, Langmuir 13 (1997) 751. [27] M.A. Cooper, A.C. Try, J. Carroll, D.J. Ellar, D.H. Williams, Biochim. Biophys. Acta Biomembr. 1373 (1998) 101. [28] A.T.A. Jenkins, R.J. Bushby, S.D. Evans, W. Knoll, A. Offenhäusser, S.D. Ogier, Langmuir 18 (2002) 3176.

T. Tumolo et al. / Colloids and Surfaces B: Biointerfaces 91 (2012) 1–9 [29] E. Suraniti, T. Tumolo, M.S. Baptista, T. Livache, R. Calemczuk, Langmuir 23 (2007) 6835. [30] A.L. Plant, M. Brigham-Burke, E.C. Petrella, D.J. O’Shannessy, Anal. Biochem. 223 (1995) 342. [31] J.B. Hubbard, V. Silin, A.L. Plant, Biophys. Chem. 75 (1998) 163. [32] P.W.M. van Dijck, B. de Kruijff, A.J. Verkleij, L.L.M. van Deenen, J. de Gier, Biochim. Biophys. Acta Biomembr. 512 (1978) 84. [33] A. Portis, C. Newton, W. Pangborn, D. Papahadjopoulos, Biochemistry 18 (1979) 780. [34] K. Hong, F. Schuber, D. Papahadjopoulos, Biochim. Biophys. Acta Biomembr. 732 (1983) 469. [35] M.W. Yung, C. Green, Biochem. Pharmacol. 35 (1986) 4037. [36] R. Stevanato, A. Wisniewska, F. Momo, Arch. Biochem. Biophys. 346 (1997) 203. [37] J. Zimmerberg, F.S. Cohen, A. Finkelstein, J. Gen. Physiol. 75 (1980) 241. [38] Z.D. Schultz, I.M. Pazos, F.K. McNeil-Watson, E.N. Lewis, I.W. Levin, J. Phys. Chem. B 113 (2009) 9932. [39] J. Wilschut, D. Hoekstra, Trends Biochem. Sci. 9 (1984) 479. [40] D.E. Leckband, C.A. Helm, J. Israelachvili, Biochemistry 32 (1993) 1127. [41] K. Satoh, Biochim. Biophys. Acta Biomembr. 1239 (1995) 239. [42] G. Puu, I. Gustafson, Biochim. Biophys. Acta Biomembr. 1327 (1997) 149. [43] Y.F. Dufrêne, G.U. Lee, Biochim. Biophys. Acta Biomembr. 1509 (2000) 14. [44] I. Reviakine, A. Simon, A. Brisson, Langmuir 16 (2000) 1473. [45] S. Boudard, B. Seantier, C. Breffa, G. Decher, O. Félix, Thin Solid Films 495 (2006) 246. [46] N. Düzgüne, S. Nir, J. Wilschut, J. Bentz, C. Newton, A. Portis, D. Papahadjopoulos, J. Membr. Biol. 59 (1981) 115. [47] A. Rodríguez, P. Webster, J. Ortego, N.W. Andrews, J. Cell Biol. 137 (1997) 93. [48] A. Rodríguez, M.G. Rioult, A. Ora, N.W. Andrews, J. Cell Biol. 129 (1995) 1263. [49] M.T. Grijalba, A.E. Vercesi, S. Schreier, Biochemistry 38 (1999) 13279. [50] T.G. Frey, C.A. Mannella, TIBS 25 (2000) 319. [51] D.L. Nelson, M.M. Cox, Lehninger – Principles of Biochemistry, W.H. Freeman, New York, 2004. [52] P. John, F.R. Whatley, in: R.D. Preston, H.W. Woolhouse (Eds.), Advances in Botanical Research, vol. 4, Academic Press, London, 1977 (Chapter 2). [53] C. Kawai, F.M. Prado, G.L.C. Nunes, P. Di Mascio, A.M. Carmona-Ribeiro, I.L. Nantes, J. Biol. Chem. 280 (2005) 34709.

9

[54] N. Khalifat, N. Puff, S. Bonneau, J.-B. Fournier, M.I. Angelova, Biophys. J. 95 (2008) 4924. [55] D.R. Green, J.C. Reed, Science 281 (1998) 1309. [56] C. Pereira, R.D. Silva, L. Saraiva, B. Johansson, M.J. Sousa, M. Côrte-Real, Biochim. Biophys. Acta Mol. Cell. Res. 1783 (2008) 1286. [57] D. Hegner, U. Schummer, G.H. Schnepel, Biochim. Biophys. Acta Biomembr. 307 (1973) 452. [58] D. Marsch (Ed.), CRC Handbook of Lipid Bilayers, CRC Press, Boca Raton, 1990. [59] C.D. Bain, J. Evall, G.M. Whitesides, J. Am. Chem. Soc. 111 (1989) 7155. [60] C.D. Bain, E.B. Troughton, Y.T. Tao, J. Evall, G.M. Whitesides, R.G. Nuzzo, J. Am. Chem. Soc. 111 (1989) 321. [61] K.A. Peterlinz, R. Georgiadis, Langmuir 12 (1996) 4731. [62] L.S. Jung, C.T. Campbell, T.M. Chinowsky, M.N. Mar, S.S. Yee, Langmuir 14 (1998) 5636. [63] S. Meucci, G. Gabrielli, G. Caminati, Mater. Sci. Eng. C 8–9 (1999) 135. [64] E.E. Ross, B. Bondurant, T. Spratt, J.C. Conboy, D.F. OˇıBrien, S.S. Saavedra, Langmuir 17 (2001) 2305. [65] R.C. Weast (Ed.), CRC Handbook of Chemistry and Physics, CRC Press, Cleveland, 1979. [66] J.A. de Feijter, J. Benjamins, F.A. Veer, Biopolymers 17 (1978) 1759. [67] T. Tumolo, L. Angnes, M.S. Baptista, Anal. Biochem. 333 (2004) 273. [68] B. Städler, D. Falconnet, I. Pfeiffer, F. Höök, J. Vörös, Langmuir 20 (2004) 11348. [69] A.T.A. Jenkins, T. Neumann, A. Offenhäusser, Langmuir 17 (2001) 265. [70] B. de Kruijff, P.R. Cullis, Biochim. Biophys. Acta Biomembr. 602 (1980) 477. [71] C.W. Meuse, S. Krueger, C.F. Majkrzak, J.A. Dura, J. Fu, J.T. Connor, A.L. Plant, Biophys. J. 74 (1998) 1388. [72] A. Kanavarioti, E.E. Baird, P.J. Smith, J. Org. Chem. 60 (1995) 4873. [73] K.L. Weirich, J.N. Israelachvili, D.K. Fygenson, Biophys. J. 98 (2010) 85. [74] J.G. Stollery, W.J. Vail, Biochim. Biophys. Acta Biomembr. 471 (1977) 372. [75] M. Roux, M. Bloom, Biophys. J. 60 (1991) 38. [76] P. MacDonald, J. Seelig, Biochemistry 26 (1987) 1231. [77] C.G. Sinn, M. Antonietti, R. Dimova, Colloids Surf. A Physicochem. Eng. Aspects 282–283 (2006) 410. [78] C.S. Oliveira, E. Bastos, E. Duarte, R. Itri, M.S. Baptista, Langmuir 22 (2006) 8718. [79] J. Bentz, N. Dusgune, S. Nir, Biochemistry 22 (1983) 3320.