- Email: [email protected]
liquid interface in presence of an antimicrobial peptide
Thin Solid Films 515 (2007) 5687 – 5690 www.elsevier.com/locate/tsf
Studies of phospholipid monolayer at liquid/liquid interface in presence of an antimicrobial peptide E. Saint Martin a,⁎, O. Konovalov a , J. Daillant b a
b
ID 10B European Synchrotron Radiation Facility, 6, rue Jules Horowitz - B.P.220, 38043, Grenoble, Cedex 9, France Laboratoire Interdisciplinaire sur l'Organisation Nanometrique et Supramoleculaire (LIONS),SCM, bat. 125, CEA Saclay, F-91191 Gif-sur-Yvette Cedex, France Available online 16 January 2007
Abstract An application of the antimicrobial peptides (AP) as antibiotics of new generation requires comprehensive studies of the interaction of these peptides with a cell membrane (CM). Despite the big progress in characterization of the AP-CM system, its elastic properties remain unclear. In the present work we present studies of the structure and the bending rigidity of phospholipid monolayers formed at hexadecane/water interface in presence or absence of antimicrobial peptides. It is shown that due to the interactions between the monolayer and the peptide, the bending rigidity decreases after the injection of the peptide. © 2006 Elsevier B.V. All rights reserved. Keywords: Bending rigidity; Langmuir–Blodgett films; X-ray scattering; Liquid–liquid interface
1. Introduction An application of the antimicrobial peptides (AP) as antibiotics of new generation requires comprehensive studies of the interaction of these peptides with a cell membrane (CM). Despite the big progress in characterization of the AP–CM system, its elastic properties remain unclear. Antimicrobial peptides activity is related to their interactions with the lipid bilayer itself, rather than specific protein receptor (s) within the cell membrane [1]. In this work, we study the structure and the bending rigidity of phospholipid monolayers in presence and absence of antimicrobial peptides. DiPalmitoylPhosphatidylGlycerol (DPPG) was chosen to mimic bacterial cell membrane because antimicrobial peptides were shown to discriminate between mammalian and bacterial cell membranes by their lipids composition [2] and DPPG is the main component of bacterial cell membranes. Experiments were performed at liquid/liquid interface because, as it will be explained below, the X-ray measurements in Grazing Incidence geometry are sensitive to surface tension (γ) and bending rigidity (κ) through the height–height cor⁎ Corresponding author. E-mail address: [email protected] (E. Saint Martin). 0040-6090/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2006.12.010
relation function. On the other hand, at liquid/liquid interface, the surface tension can be reduced to the minimum; consequently the diffuse scattering signal will be due mainly to the elastic properties of membranes. So bending rigidity can be studied more accurately [3]. 2. Experimental section 2.1. Materials Phospholipid DiPalmitoyl-PhosphatidylGlycerol (DPPG, MW = 740 g/mol) was purchased from Sigma (France) (purity N99%) and used without further purification. Antimicrobial peptide Peptidyl-Glycyl-Leucine-carboxylamide (PGLa, MW = 1969 g/mol) consisting of 21 amino acids (GMASKAGAIAGKIAKVALKAL-carboxylamide [4]) was purchased from Multiple Peptide System (San Diego, CA). Chloroform was purchased from Sigma (France) and was of HPLC grade. Pure water was obtained with an ELGA LABWATER system. Hexadecane was purchased from Sigma (France) (purity: 99%) and purified before use with two different techniques. Aluminum oxide (Activated, Basic) (Al2O3), for hexadecane purification, was purchased from Sigma (France).
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E. Saint Martin et al. / Thin Solid Films 515 (2007) 5687–5690
2.2. Isotherms measurements Surface pressure–Area (π–A) isotherms were recorded at Air/Water (A/W ) and Hexadecane/Water (H/W ) interfaces, using a home-made Langmuir trough specially designed for X-ray experiments at the liquid/liquid interface [5]. Liquid hexadecane used for the isotherm measurements was purified by five cycles of filtering through a basic Al2O3 column. Stock solution of DPPG lipids was prepared in chloroform at a concentration of 0.25 mg/ml. Water solution of peptide at a concentration of 0.17 mg/ml was prepared in 10 mM NaPhosphate buffer, pH = 7.4. The monolayers were prepared by spreading, with a Hamilton syringe, 120 μL and 150 μL of phospholipids solution on the interfaces A/W and H/W respectively. After deposition, the films were left for at least 20 min before compression in order to ensure complete evaporation of chloroform. The monolayers were compressed at the interface by symmetric move of two barriers with a velocity of 15 cm2/min. The surface pressure (π) was recorded using a Wilhelmy balance (NIMA tensiometer PS4) with 10 mm wide filter paper, connected to a home made electronic that measures with an accuracy of ± 0.2 mN/m. All π−A measurements of lipid monolayers were done at room temperature (20 °C). 2.3. X-ray experiments X-ray Reflectivity (XRR) for structural characterization and Grazing Incidence diffuse X-ray Scattering in the plane of incidence (GIS) for obtaining bending rigidity of monolayers at liquid/liquid interface were performed. For these experiments, the hexadecane purification was done differently than previously. Pure water was added to hexadecane in the bottle and the mixture was shaken to increase the exchange surface between the two immiscible components. After phase separation we took hexadecane above the polluted interface and used it for experiments. As we measure at the H/W interface this technique allows removing impurity surfactants from the bulk of hexadecane. When the purified hexadecane was put in the trough, the bare H/W interface was compressed and sucked a bit to remove last impurities. Monolayer preparation for the X-ray measurements was the same as for the measurements of the π–A isotherm at H/W interface. After spreading of the phospholipids onto the cleaned H/W interface, the vessel containing the trough was closed with a cap to minimize evaporation of hexadecane during the X-ray measurements. The DPPG-peptide system was prepared from the previously formed lipid monolayer by injection in the water subphase of 50 μL of PGLa solution. We waited for 10 min before taking Xray measurements to ensure complete interaction between PGLa and a monolayer. X-ray measurements were carried out at the ID10B (Troika II) beamline of the European Synchrotron Facility (ESRF). The energy of the monochromatic beam was set to 22.5 keV (wavelength λ = 0.055 nm) to allow the X-ray beam to pass
through 70 mm of the oil (Fig. 1). The grazing angle (α) was set to 0.384 mrad for GIS. The X-ray reflectivity measurements were done in the angular interval from 0.384 mrad to 16.93 mrad. At each scattering vector rocking curve measurements were performed in order to subtract background from the specularly reflected beam. The incident beam size was 0.017 mm × 1.0 mm (V × H) defined using conventional slits. All X-ray measurements were performed at room temperature of 23 °C, to be well above of melting point of the hexadecane and to avoid its freezing on the filter paper, and at two surface pressures. One pressure is above (“High”: 20 mN/m corresponding to 30 mN/m at A/W interface) and below (“Low”: 5 mN/m corresponding to 15 mN/m at A/W interface) the collapse pressure (π = 25 mN/m at A/W interface) of PGLa monolayer [6]. Obtained XRR data were fitted using Parratt algorithm (Fig. 3) for calculation of the reflectivity curves [7]. Electron density profile was modeled analytically with a function 8 1 1 2 2 > > > qch2 −Dqm exp − ðz=rm Þ þ Dqh exp − ððz−LzÞ=rh Þ ; > 2 2 > > < 1 qðzÞ ¼ qh2o þ ðDqh þ qch2 −qh2o Þexp − ððz−LzÞ=rh Þ2 ; > 2 > > > 1 > > : qoil −ðDqm −qch2 þ qoil Þexp − ðz=rm Þ2 ; 2
0V z V Lz zNLz : 0bz
This function describes the mean electron densities of the oil (ρoil), film (ρch2) and water (ρh2o) as well as separated with distance Lz Gaussian like positive (at the film/water interface (Δρh, σh)) and negative (at the oil/film interface (Δρm, σm)) contributions to the electron density ρch2 (Fig. 4). Electron density profile obtained through the fit of XRR data was used for fitting the GIS data. The GIS data were analyzed following to the approach described by S. Mora and co-workers [8]. Main contribution to the GIS signal measured on the gas/liquid or liquid/liquid interface comes from the scattering on the interfacial roughness that result from the thermally excited capillary waves. Presence
Fig. 1. Experimental geometry.
E. Saint Martin et al. / Thin Solid Films 515 (2007) 5687–5690
5689
of a film at the interface modifies the capillary wave spectrum due to intermolecular interactions in the film that manifests itself as bending rigidity. Fourier transformation of the interface height calculated on the base of the free energy of the bent surface [9] gives an expression for the height fluctuation spectrum [10]: hzðqjj Þzð−qjj Þi ¼
1 kB T A Dqg þ gq2jj þ jq4jj
ð1Þ
where A is the surface area, γ is the surface tension, κ is the bending rigidity, and Δρ the density difference between adjacent liquids. Height fluctuation spectrum (1) is used further for the computation of the height–height correlation function bz(0)z(r//)N. Finally the differential scattering cross-section for a homogeneous fluctuating Langmuir film is [8]:
dr ˜ zÞ ~jt in j2 jt sc j2 jqðq dX Z 2 q 2 2 þ sub je−qz hz i drjj eqz hzð0Þzðrjj Þi −1 eiqjj rjj iqz
Fig. 3. Experimental curves of XRR made on monolayer of DPPG at “Low” pressure (opened circle) and “High” pressure (line) compared with DPPG/PGLa monolayers at the same surface pressure: “Low” pressure (filled circle) and “High” pressure (dash line). Measurements are performed at T = 23 °C, at hexadecane/water interface.
Fig. 2 presents measured π–A isotherms of the DPPG monolayer formed at the A/W and at the H/W interfaces. The vertical shift of about 10 mN/m of the isotherm measured at the H/ W interface is because we set as zero for all measurement the surface tension at the air/water interface (γ1 = 72 mN/m). However during measurements at the H/W interface the filter paper crosses two interfaces namely the air/hexadecane (γ2 = 27.2 mN/m) and the hexadecane/water (γ3 = 55.2 mN/m). The total surface
pressure value measured in this case is γ1 − (γ2 + γ3) = −10.4 mN/m that brings mentioned above 10 mN/m offset between isotherm recorded at A/W and H/W interfaces. Similar behavior between isotherms of DPPG monolayers without antimicrobial peptide PGLa at the H/W and the A/W interfaces confirms that there is a film at H/W interface similar to the film at the A/W interface and envisage to study the elastic properties of the system with X-rays at the liquid/liquid interface. Advantage of the liquid/liquid interface is that the surface tension here is reduced if compare it with the gas/liquid interface. Reduction of the surface tension, as it is seen from expression (1), makes possible to detect the effect of the bending rigidity at smaller values of scattering vectors where scattering signal is not sensitive to the discrete nature of the liquids. XRR measurements (Fig. 3) together with the reconstructed electron density profiles (Fig. 4) reveal molecular organization upon the pressure increase in the monolayer of DPPG lipids only and of DPPG + PGLa system. With increase of surface pressure the DPPG monolayer becomes thicker due to smaller tilt of molecules and the electron density at the film/water interface increases (phosphatidyl-glycerol group location). The total thickness of the DPPG + PGLa system at the “Low”
Fig. 2. π–A isotherms of DPPG monolayers at air/water interface (dash line) and hexadecane/water interface (filled circle). Measurements were performed a T = 20 °C.
Fig. 4. Density Profile extracted from XRR fitting for DPPG monolayer at “Low” pressure (opened circle) and “High” pressure (line) compared with density profile for DPPG/PGLa monolayer at the same surface pressure (filled circle for “Low” pressure and dash line for “High” pressure).
ð2Þ
where tin and tsc are the Fresnel transmission coefficients for water under hexadecane for the incident and the scattering angle, respectively. The form factor ρ˜ (qz) is the Fourier transform of the electron density profile of the film and ρsub is the electron density of the substrate (water). The X-ray measurements in GIS geometry are therefore sensitive to γ and κ through the height–height correlation function. 3. Results and discussion
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E. Saint Martin et al. / Thin Solid Films 515 (2007) 5687–5690
(with some small adaptations in the values) as a model to fit GIS data (Fig. 5). Obtained values of the bending rigidity show its dependence of the surface pressure and the peptides presence. With increase of the surface pressure of monolayer without peptide, the bending rigidity increases from 55 kbT at “Low” pressure to 145 kbT at “High” pressure. The monolayer becomes more compact that result in the increase of the interaction between hydrocarbon chains and as a result the rigidity value increases too. Addition of the PGLa to the system at “Low” pressure decreases bending rigidity of the monolayer from 55 kbT for DPPG only to 27 kbT. So the lipid membrane becomes softer. The same tendency of softening of the monolayer with peptides was observed at higher pressure. However we do not present this data and a result fit in this publication because of experimental problems we were not able to reach the same “High” pressure value. Fig. 5. GIS experiments made on DPPG at “Low” pressure (circle), DPPG at “High” pressure (triangle), DPPG + PGLa at “Low” pressure (inversed triangle). Lines represent the best fit for DPPG “Low” pressure, DPPG “High” pressure and DPPG + PGLa “Low” pressure with κ = 55 K bT, 145 KbT and 27 KbT respectively.
pressure is larger than for the DPPG monolayer at the same pressure. Additionally the width of the film/water interface is broader which indicates the adsorption and insertion of peptides into the membrane. Upon increase of the surface pressure the total film thickness becomes smaller and the hydrophilic part of the membrane narrows. This could be explained with squeeze out of peptide from the monolayer above the critical pressure for the peptides. The GIS measurements performed on DPPG monolayer without and with antimicrobial peptides at “Low” and “High” surface pressure are shown on the Fig. 5. The electron density profiles reconstructed from the XRR measurements were used
References [1] K. Lohner, in: K. Lohner (Ed.), Development of Novel Antimicrobial Agents: Emerging Strategies, Horizon Scientific Press, Wymomdham, Norfolk, UK, 2001, p. 149. [2] A. Latal, G. Degovics, R.F. Epand, R.M. Epand, K. Lohner, Eur. J. Biochem. 248 (1997) 938. [3] C. Fradin, D. Luzet, A. Braslau, M. Alba, F. Muller, J. Daillant, J.M. Petit, F. Rieutord, Langmuir 14 (1998) 7327. [4] D. Andreu, H. Aschauer, G. Kreil, R.B. Merrifield, Eur. J. Biochem. 149 (1995) 531. [5] J. Daillant's group (CEA Saclay France). [6] O. Konovalov, S.M. O'Flaherty, E. Saint Martin, G. Deutsch, E. Sevcsik, K. Lohner, Physica, B 357 (2005) 185. [7] L.G. Parratt, Phys. Rev. 95 (1954) 359. [8] S. Mora, J. Daillant, D. Luzet, B. Struth, Europhys. Lett. 66 (2004) 694. [9] W. Helfrich, Z. Naturforsch. 28c (1973) 693. [10] C. Gourier, J. Daillant, A. Braslau, M. Alba, K. Quinn, D. Luzet, C. Blot, D. Chatenay, G. Grübel, J.F. Legrand, G. Vignaud, Phys. Rev. Lett. 78 (1997) 3157.
Studies of phospholipid monolayer at liquid/liquid interface in presence of an antimicrobial peptide E. Saint Martin a,⁎, O. Konovalov a , J. Daillant b a
b
ID 10B European Synchrotron Radiation Facility, 6, rue Jules Horowitz - B.P.220, 38043, Grenoble, Cedex 9, France Laboratoire Interdisciplinaire sur l'Organisation Nanometrique et Supramoleculaire (LIONS),SCM, bat. 125, CEA Saclay, F-91191 Gif-sur-Yvette Cedex, France Available online 16 January 2007
Abstract An application of the antimicrobial peptides (AP) as antibiotics of new generation requires comprehensive studies of the interaction of these peptides with a cell membrane (CM). Despite the big progress in characterization of the AP-CM system, its elastic properties remain unclear. In the present work we present studies of the structure and the bending rigidity of phospholipid monolayers formed at hexadecane/water interface in presence or absence of antimicrobial peptides. It is shown that due to the interactions between the monolayer and the peptide, the bending rigidity decreases after the injection of the peptide. © 2006 Elsevier B.V. All rights reserved. Keywords: Bending rigidity; Langmuir–Blodgett films; X-ray scattering; Liquid–liquid interface
1. Introduction An application of the antimicrobial peptides (AP) as antibiotics of new generation requires comprehensive studies of the interaction of these peptides with a cell membrane (CM). Despite the big progress in characterization of the AP–CM system, its elastic properties remain unclear. Antimicrobial peptides activity is related to their interactions with the lipid bilayer itself, rather than specific protein receptor (s) within the cell membrane [1]. In this work, we study the structure and the bending rigidity of phospholipid monolayers in presence and absence of antimicrobial peptides. DiPalmitoylPhosphatidylGlycerol (DPPG) was chosen to mimic bacterial cell membrane because antimicrobial peptides were shown to discriminate between mammalian and bacterial cell membranes by their lipids composition [2] and DPPG is the main component of bacterial cell membranes. Experiments were performed at liquid/liquid interface because, as it will be explained below, the X-ray measurements in Grazing Incidence geometry are sensitive to surface tension (γ) and bending rigidity (κ) through the height–height cor⁎ Corresponding author. E-mail address: [email protected] (E. Saint Martin). 0040-6090/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2006.12.010
relation function. On the other hand, at liquid/liquid interface, the surface tension can be reduced to the minimum; consequently the diffuse scattering signal will be due mainly to the elastic properties of membranes. So bending rigidity can be studied more accurately [3]. 2. Experimental section 2.1. Materials Phospholipid DiPalmitoyl-PhosphatidylGlycerol (DPPG, MW = 740 g/mol) was purchased from Sigma (France) (purity N99%) and used without further purification. Antimicrobial peptide Peptidyl-Glycyl-Leucine-carboxylamide (PGLa, MW = 1969 g/mol) consisting of 21 amino acids (GMASKAGAIAGKIAKVALKAL-carboxylamide [4]) was purchased from Multiple Peptide System (San Diego, CA). Chloroform was purchased from Sigma (France) and was of HPLC grade. Pure water was obtained with an ELGA LABWATER system. Hexadecane was purchased from Sigma (France) (purity: 99%) and purified before use with two different techniques. Aluminum oxide (Activated, Basic) (Al2O3), for hexadecane purification, was purchased from Sigma (France).
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E. Saint Martin et al. / Thin Solid Films 515 (2007) 5687–5690
2.2. Isotherms measurements Surface pressure–Area (π–A) isotherms were recorded at Air/Water (A/W ) and Hexadecane/Water (H/W ) interfaces, using a home-made Langmuir trough specially designed for X-ray experiments at the liquid/liquid interface [5]. Liquid hexadecane used for the isotherm measurements was purified by five cycles of filtering through a basic Al2O3 column. Stock solution of DPPG lipids was prepared in chloroform at a concentration of 0.25 mg/ml. Water solution of peptide at a concentration of 0.17 mg/ml was prepared in 10 mM NaPhosphate buffer, pH = 7.4. The monolayers were prepared by spreading, with a Hamilton syringe, 120 μL and 150 μL of phospholipids solution on the interfaces A/W and H/W respectively. After deposition, the films were left for at least 20 min before compression in order to ensure complete evaporation of chloroform. The monolayers were compressed at the interface by symmetric move of two barriers with a velocity of 15 cm2/min. The surface pressure (π) was recorded using a Wilhelmy balance (NIMA tensiometer PS4) with 10 mm wide filter paper, connected to a home made electronic that measures with an accuracy of ± 0.2 mN/m. All π−A measurements of lipid monolayers were done at room temperature (20 °C). 2.3. X-ray experiments X-ray Reflectivity (XRR) for structural characterization and Grazing Incidence diffuse X-ray Scattering in the plane of incidence (GIS) for obtaining bending rigidity of monolayers at liquid/liquid interface were performed. For these experiments, the hexadecane purification was done differently than previously. Pure water was added to hexadecane in the bottle and the mixture was shaken to increase the exchange surface between the two immiscible components. After phase separation we took hexadecane above the polluted interface and used it for experiments. As we measure at the H/W interface this technique allows removing impurity surfactants from the bulk of hexadecane. When the purified hexadecane was put in the trough, the bare H/W interface was compressed and sucked a bit to remove last impurities. Monolayer preparation for the X-ray measurements was the same as for the measurements of the π–A isotherm at H/W interface. After spreading of the phospholipids onto the cleaned H/W interface, the vessel containing the trough was closed with a cap to minimize evaporation of hexadecane during the X-ray measurements. The DPPG-peptide system was prepared from the previously formed lipid monolayer by injection in the water subphase of 50 μL of PGLa solution. We waited for 10 min before taking Xray measurements to ensure complete interaction between PGLa and a monolayer. X-ray measurements were carried out at the ID10B (Troika II) beamline of the European Synchrotron Facility (ESRF). The energy of the monochromatic beam was set to 22.5 keV (wavelength λ = 0.055 nm) to allow the X-ray beam to pass
through 70 mm of the oil (Fig. 1). The grazing angle (α) was set to 0.384 mrad for GIS. The X-ray reflectivity measurements were done in the angular interval from 0.384 mrad to 16.93 mrad. At each scattering vector rocking curve measurements were performed in order to subtract background from the specularly reflected beam. The incident beam size was 0.017 mm × 1.0 mm (V × H) defined using conventional slits. All X-ray measurements were performed at room temperature of 23 °C, to be well above of melting point of the hexadecane and to avoid its freezing on the filter paper, and at two surface pressures. One pressure is above (“High”: 20 mN/m corresponding to 30 mN/m at A/W interface) and below (“Low”: 5 mN/m corresponding to 15 mN/m at A/W interface) the collapse pressure (π = 25 mN/m at A/W interface) of PGLa monolayer [6]. Obtained XRR data were fitted using Parratt algorithm (Fig. 3) for calculation of the reflectivity curves [7]. Electron density profile was modeled analytically with a function 8 1 1 2 2 > > > qch2 −Dqm exp − ðz=rm Þ þ Dqh exp − ððz−LzÞ=rh Þ ; > 2 2 > > < 1 qðzÞ ¼ qh2o þ ðDqh þ qch2 −qh2o Þexp − ððz−LzÞ=rh Þ2 ; > 2 > > > 1 > > : qoil −ðDqm −qch2 þ qoil Þexp − ðz=rm Þ2 ; 2
0V z V Lz zNLz : 0bz
This function describes the mean electron densities of the oil (ρoil), film (ρch2) and water (ρh2o) as well as separated with distance Lz Gaussian like positive (at the film/water interface (Δρh, σh)) and negative (at the oil/film interface (Δρm, σm)) contributions to the electron density ρch2 (Fig. 4). Electron density profile obtained through the fit of XRR data was used for fitting the GIS data. The GIS data were analyzed following to the approach described by S. Mora and co-workers [8]. Main contribution to the GIS signal measured on the gas/liquid or liquid/liquid interface comes from the scattering on the interfacial roughness that result from the thermally excited capillary waves. Presence
Fig. 1. Experimental geometry.
E. Saint Martin et al. / Thin Solid Films 515 (2007) 5687–5690
5689
of a film at the interface modifies the capillary wave spectrum due to intermolecular interactions in the film that manifests itself as bending rigidity. Fourier transformation of the interface height calculated on the base of the free energy of the bent surface [9] gives an expression for the height fluctuation spectrum [10]: hzðqjj Þzð−qjj Þi ¼
1 kB T A Dqg þ gq2jj þ jq4jj
ð1Þ
where A is the surface area, γ is the surface tension, κ is the bending rigidity, and Δρ the density difference between adjacent liquids. Height fluctuation spectrum (1) is used further for the computation of the height–height correlation function bz(0)z(r//)N. Finally the differential scattering cross-section for a homogeneous fluctuating Langmuir film is [8]:
dr ˜ zÞ ~jt in j2 jt sc j2 jqðq dX Z 2 q 2 2 þ sub je−qz hz i drjj eqz hzð0Þzðrjj Þi −1 eiqjj rjj iqz
Fig. 3. Experimental curves of XRR made on monolayer of DPPG at “Low” pressure (opened circle) and “High” pressure (line) compared with DPPG/PGLa monolayers at the same surface pressure: “Low” pressure (filled circle) and “High” pressure (dash line). Measurements are performed at T = 23 °C, at hexadecane/water interface.
Fig. 2 presents measured π–A isotherms of the DPPG monolayer formed at the A/W and at the H/W interfaces. The vertical shift of about 10 mN/m of the isotherm measured at the H/ W interface is because we set as zero for all measurement the surface tension at the air/water interface (γ1 = 72 mN/m). However during measurements at the H/W interface the filter paper crosses two interfaces namely the air/hexadecane (γ2 = 27.2 mN/m) and the hexadecane/water (γ3 = 55.2 mN/m). The total surface
pressure value measured in this case is γ1 − (γ2 + γ3) = −10.4 mN/m that brings mentioned above 10 mN/m offset between isotherm recorded at A/W and H/W interfaces. Similar behavior between isotherms of DPPG monolayers without antimicrobial peptide PGLa at the H/W and the A/W interfaces confirms that there is a film at H/W interface similar to the film at the A/W interface and envisage to study the elastic properties of the system with X-rays at the liquid/liquid interface. Advantage of the liquid/liquid interface is that the surface tension here is reduced if compare it with the gas/liquid interface. Reduction of the surface tension, as it is seen from expression (1), makes possible to detect the effect of the bending rigidity at smaller values of scattering vectors where scattering signal is not sensitive to the discrete nature of the liquids. XRR measurements (Fig. 3) together with the reconstructed electron density profiles (Fig. 4) reveal molecular organization upon the pressure increase in the monolayer of DPPG lipids only and of DPPG + PGLa system. With increase of surface pressure the DPPG monolayer becomes thicker due to smaller tilt of molecules and the electron density at the film/water interface increases (phosphatidyl-glycerol group location). The total thickness of the DPPG + PGLa system at the “Low”
Fig. 2. π–A isotherms of DPPG monolayers at air/water interface (dash line) and hexadecane/water interface (filled circle). Measurements were performed a T = 20 °C.
Fig. 4. Density Profile extracted from XRR fitting for DPPG monolayer at “Low” pressure (opened circle) and “High” pressure (line) compared with density profile for DPPG/PGLa monolayer at the same surface pressure (filled circle for “Low” pressure and dash line for “High” pressure).
ð2Þ
where tin and tsc are the Fresnel transmission coefficients for water under hexadecane for the incident and the scattering angle, respectively. The form factor ρ˜ (qz) is the Fourier transform of the electron density profile of the film and ρsub is the electron density of the substrate (water). The X-ray measurements in GIS geometry are therefore sensitive to γ and κ through the height–height correlation function. 3. Results and discussion
5690
E. Saint Martin et al. / Thin Solid Films 515 (2007) 5687–5690
(with some small adaptations in the values) as a model to fit GIS data (Fig. 5). Obtained values of the bending rigidity show its dependence of the surface pressure and the peptides presence. With increase of the surface pressure of monolayer without peptide, the bending rigidity increases from 55 kbT at “Low” pressure to 145 kbT at “High” pressure. The monolayer becomes more compact that result in the increase of the interaction between hydrocarbon chains and as a result the rigidity value increases too. Addition of the PGLa to the system at “Low” pressure decreases bending rigidity of the monolayer from 55 kbT for DPPG only to 27 kbT. So the lipid membrane becomes softer. The same tendency of softening of the monolayer with peptides was observed at higher pressure. However we do not present this data and a result fit in this publication because of experimental problems we were not able to reach the same “High” pressure value. Fig. 5. GIS experiments made on DPPG at “Low” pressure (circle), DPPG at “High” pressure (triangle), DPPG + PGLa at “Low” pressure (inversed triangle). Lines represent the best fit for DPPG “Low” pressure, DPPG “High” pressure and DPPG + PGLa “Low” pressure with κ = 55 K bT, 145 KbT and 27 KbT respectively.
pressure is larger than for the DPPG monolayer at the same pressure. Additionally the width of the film/water interface is broader which indicates the adsorption and insertion of peptides into the membrane. Upon increase of the surface pressure the total film thickness becomes smaller and the hydrophilic part of the membrane narrows. This could be explained with squeeze out of peptide from the monolayer above the critical pressure for the peptides. The GIS measurements performed on DPPG monolayer without and with antimicrobial peptides at “Low” and “High” surface pressure are shown on the Fig. 5. The electron density profiles reconstructed from the XRR measurements were used
References [1] K. Lohner, in: K. Lohner (Ed.), Development of Novel Antimicrobial Agents: Emerging Strategies, Horizon Scientific Press, Wymomdham, Norfolk, UK, 2001, p. 149. [2] A. Latal, G. Degovics, R.F. Epand, R.M. Epand, K. Lohner, Eur. J. Biochem. 248 (1997) 938. [3] C. Fradin, D. Luzet, A. Braslau, M. Alba, F. Muller, J. Daillant, J.M. Petit, F. Rieutord, Langmuir 14 (1998) 7327. [4] D. Andreu, H. Aschauer, G. Kreil, R.B. Merrifield, Eur. J. Biochem. 149 (1995) 531. [5] J. Daillant's group (CEA Saclay France). [6] O. Konovalov, S.M. O'Flaherty, E. Saint Martin, G. Deutsch, E. Sevcsik, K. Lohner, Physica, B 357 (2005) 185. [7] L.G. Parratt, Phys. Rev. 95 (1954) 359. [8] S. Mora, J. Daillant, D. Luzet, B. Struth, Europhys. Lett. 66 (2004) 694. [9] W. Helfrich, Z. Naturforsch. 28c (1973) 693. [10] C. Gourier, J. Daillant, A. Braslau, M. Alba, K. Quinn, D. Luzet, C. Blot, D. Chatenay, G. Grübel, J.F. Legrand, G. Vignaud, Phys. Rev. Lett. 78 (1997) 3157.