Article pubs.acs.org/Langmuir
Model Lung Surfactant Films: Why Composition Matters Sahana L. Selladurai, Renaud Miclette Lamarche, Rolf Schmidt, and Christine E. DeWolf* Department of Chemistry and Biochemistry and Centre for NanoScience Research, Concordia University, 7141 Sherbrooke Street West, Montreal, Canada H4B 1R6 S Supporting Information *
ABSTRACT: Lung surfactant replacement therapies, Survanta and Infasurf, and two lipid-only systems both containing saturated and unsaturated phospholipids and one containing additional palmitic acid were used to study the impact of buffered saline on the surface activity, morphology, rheology, and structure of Langmuir monolayer model membranes. Isotherms and Brewster angle microscopy show that buffered saline subphases induce a film expansion, except when the cationic protein, SP-B, is present in sufficient quantities to already screen electrostatic repulsion, thus limiting the effect of changing pH and adding counterions. Grazing incidence X-ray diffraction results indicate an expansion not only of the liquid expanded phase but also an expansion of the lattice of the condensed phase. The film expansion corresponded in all cases with a significant reduction in the viscosity and elasticity of the films. The viscoelastic parameters are dominated by liquid expanded phase properties and do not appear to be dependent on the structure of the condensed phase domains in a phase separated film. The results highlight that the choice of subphase and film composition is important for meaningful interpretations of measurements using model systems.
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INTRODUCTION Lung surfactant (LS) coats the inner surface of the lung and serves to reduce surface tension to prevent alveolar collapse during exhalation.1−3 It also reduces the work necessary for breathing.3,4 LS is composed of 80−90% phospholipids, of which 20−30% is unsaturated and 70−80% is saturated; it also contains 3−10% neutral lipids such as cholesterol and 5−10% surfactant-associated proteins.2,5−7 These proteins include SPA, SP-B, SP-C, and SP-D, where SP-B and SP-C are the surface active proteins that are responsible for membrane−membrane associations and reservoir formation, respectively.8−10 Langmuir monolayers are frequently used as model membranes for lung surfactant systems.7,11 Dipalmitoylphosphatidylcholine (DPPC) is often used to represent the saturated lipid component and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) the unsaturated lipid component,3 although there is a plethora of work using lung surfactant replacement therapies4,7,9,12 as model systems. Multiple factors can influence the biophysical properties of these model membranes. This includes composition and spreading conditions of the system but also composition of the subphase, that is, pH, salt composition, and concentration. Although lung surfactant model systems have been studied extensively throughout the literature, water and buffer have been used interchangeably as the subphase with scarce direct comparisons despite the wealth of data on the impact of pH, salts, and counterions on lipid monolayers.13−19 The focus of this work is a systematic study of the impact of a buffered saline subphase on both lipid only and lipid−protein © XXXX American Chemical Society
lung surfactant models; the choice of subphase is important in order to understand the behavior of these films under physiological conditions. The physiologically relevant buffer used in this study is tris-buffered saline (TBS), where the salt content is exclusively sodium chloride. Sodium and potassium cations are the most abundant in intracellular and extracellular fluids;16,20 sodium cations have been shown to have a more significant impact on lipid membranes than potassium cations.17 Moreover, monovalent cations are more important in the context of plasma membranes,17 while divalent cations are more important in the context of mitochondrial membranes. The impact of the counterion is highly dependent on the composition of the monolayer film, namely, the relative amounts of charged and uncharged lipids and the presence or absence of surfactant proteins, which themselves may act as highly localized counterions. Four model systems were selected and include DPPC:POPG, DPPC:POPG with palmitic acid (PA), Infasurf, and Survanta, where Infasurf and Survanta are lung surfactant replacement therapies and can be used to study the effect of the presence of protein. Both of these therapies contain saturated and unsaturated phospholipids as well as SPB and SP-C, but Infasurf contains approximately 0.9% w/w SPB and 0.7−1.3% w/w SP-C, whereas Survanta contains approximately 0.04% w/w SP-B and 0.9% w/w SP-C.12 Other notable differences in the composition include the lack of Received: August 7, 2016 Revised: September 12, 2016
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Figure 1. Isotherms of DPPC:POPG (top, left), DPPC:POPG:PA (top, right), Infasurf (bottom, left), and Survanta (bottom, right) monolayers on water (−) and TBS (- -) at 23 °C. Note that for the lipid protein mixtures the isotherms are plotted as a function of trough area rather than molecular area, as an average molecular weight for these natural extracts is not known. Technology) with a maximum trough surface area of 80 cm2. Monolayers of all four model lung surfactant membranes were spread on aqueous subphases (ultrapure water or buffered saline) from a 1 mg mL−1 chloroform solution and compressed at a speed of 5 cm2 min−1. For Infasurf and Survanta isotherms, the exact molecular weight is not known. For this reason, the isotherms are presented as changes in surface pressure as a function of trough area (cm2) rather than molecular area. The spreading volume and concentration were kept constant in order to ensure comparisons could be made Surface Rheology. Surface rheological measurements were carried out using a SINTERFACE Technologies profile analysis tensiometer (PAT), where a model lung surfactant monolayer solution in chloroform with a concentration of less than 0.1 mg mL−1 was spread on a 10 μL drop of subphase. The spreading volume ranged from 0.4 to 0.8 μL. After spreading, the surface area of the pendant drop was expanded from 25 to 40 mm2. The drop was then left to equilibrate for an additional 3 min to allow for complete evaporation of the chloroform and possible rearrangement of the membrane components. Rheological measurements were done by using a step through method: from the initial surface area of 40 mm2, the film is compressed to 26.8 mm2 at a compression speed of 0.06 mm2 s−1; the drop was then left to equilibrate for 300 s after which the drop volume was oscillated for 600 s with an amplitude of 0.5 mm2 and a frequency of 0.01 s−1 (100 s period, six oscillations). The drop was then left to equilibrate for 180 s, before compressing to the next surface area, equilibrated (300 s) and oscillated once again using the same parameters. This cycle was continued until a surface tension of around 25 mN m−1 was reached, giving rheological data for surface pressures ranging from 10 to 45 mN m−1; the cycle is depicted in Figure S1 in the Supporting Information. This rheological data yields the dilational surface elasticity and viscosity of the monolayer as described elsewhere.21 Brewster Angle Microscopy. Brewster angle microscopy (BAM) was performed on films spread from a concentration of 1. 0 mg mL−1 in chloroform on a Langmuir film balance (Nima Technology) coupled with an I-Elli2000 imaging ellipsometer (I-Elli2000, Nanofilm Technologies). This instrument is equipped with a 50 mW Nd:YAG laser (λ = 532 nm), and images were obtained using a 20× magnification lens with a lateral resolution of 1 μm and a 53.15° incident angle. For these films, a compression speed of 5 cm2 min−1 was used.
cholesterol and inclusion of the additive palmitic acid in Survanta (a full analysis of lipid and protein composition of these systems is available in ref 12). DPPC:POPG:PA mixtures are often used as a mimetic of this system. DPPC:POPG monolayers have been studied thoroughly on water but less so on buffer.11 On the other hand, DPPC:POPG:PA and Survanta systems have been studied on both water and buffer but with different spreading conditions, that is, DPPC:POPG:PA spread from chloroform and Survanta from a diluted aqueous suspension.3,6,11 Like Survanta, Infasurf is also usually spread from an aqueous suspension, but there is a lack of data on both buffer and water.6 The compositional variations in these four systems will allow us to discern the roles of different membrane phases in lung surfactant functioning and the extent to which they are affected by subphase pH and counterions.
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MATERIALS AND METHODS
Materials. Dipalmitoylphosphatidylcholine (DPPC, >99%) and 1palmitoyl-2-oleoyl-phosphatidylglycerol (POPG, >99%) were purchased from Avanti Polar Lipids. Palmitic acid (PA, >99%), tris(hydroxymethyl)aminomethane (tris, >99. 8%), and NaCl salt (>99%) were purchased from Sigma-Aldrich. Survanta and Infasurf are lung surfactant replacement therapies that were donated by Abbott Laboratories and Ony Inc. respectively. The spreading solvent used in all experiments conducted was HPLC grade chloroform obtained from Fisher Scientific. Preparation of Mixtures, Solutions, and Subphases. The DPPC:POPG and DPPC:POPG:PA mixtures were prepared using stock solutions of DPPC, POPG, and PA to achieve molar ratios of 77:23 and 61:19:20, respectively, all of which were prepared in chloroform. The Infasurf and Survanta solutions were prepared by introducing weighed lyophilized sample into chloroform. Water subphases were comprised of ultrapure water with a resistivity of 18.2 MΩ cm−1 from a Barnstead Easypure II LF purification system. The tris-buffered saline (TBS) was prepared by using 50 mM tris and 150 mM NaCl in ultrapure water, where the pH was adjusted to 7.4 using hydrochloric acid. Surface Pressure−Area Isotherms. Surface pressure−area isotherms were obtained using a Langmuir film balance (Nima B
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Figure 2. BAM images (220 μm width) of surface pressures (left to right): 10, 20, 25, 30, and 40 mN m−1 of DPPC:POPG on water (top) and TBS (bottom).
Figure 3. BAM images (220 μm width) of surface pressures (left to right): 10, 20, 25, 30, and 40 mN m−1 of DPPC:POPG:PA on water (top) and TBS (bottom). 340 cm2 Langmuir−Blodgett trough after spreading solutions similar to the ones used for BAM. Data was then analyzed using Interactive Data Language (IDL) and Origin. The in-plane diffraction peaks (Qxy) and the out-of-plane diffraction peaks (Qz) were fit with Lorentzian and Gaussian functions, respectively. These measurements provide information about the ordering and structure of the film.
Grazing Incident X-ray Diffraction. Grazing incident X-ray diffraction (GIXD) measurements were done at the Advanced Photon Source at Argonne National Laboratories at the ChemMatCARS 15ID-C beamline. The X-ray beam wavelength, incident angle, horizontal size, and vertical size were 1.239 Å, 0.0906°, 20 μm, and 120 μm, respectively. These parameters led to a beam footprint of 20 μm × 7.6 cm. A two-dimensional Swiss Light Source PILATUS 100 K detector was used in single-photon counting mode. In order to minimize intense low-angle scattering, two sets of slits were used. One was placed 292.0 nm from the sample, and the other was placed in front of the detector. Measurements were taken at the air−liquid interface of a
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RESULTS AND DISCUSSION
The compression isotherms of all four lipid and lipid−protein systems on water and on buffered saline are shown in Figure 1. C
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Figure 4. BAM images (220 μm width) of surface pressures (left to right): 10, 20, 25, 30, and 40 mN m−1 of Infasurf on water (top) and TBS (bottom).
Figure 5. BAM images (220 μm width) of surface pressures (left to right): 10, 20, 25, 30, and 40 mN m−1 of Survanta on water (top) and TBS (bottom).
The plateau around 40−50 mN m−1 is generally accepted to be associated with the squeeze-out of material from the interface and the formation of three-dimensional lipid−protein structures (the different regions of the isotherm are well described by H. Zhang et al.,12 for example). A film collapse occurs at much higher surface pressures, although a ribbon-type Langmuir trough is often required to reach these ultrahigh surface pressures and this is not observed in our measurements. Considering first the two simplified lipid-only lung surfactant mimetics, DPPC is zwitterionic and is known to form a
condensed phase at surface pressures above 8 mN m−1,22 while POPG is anionic, and forms a liquid-expanded (LE) phase at all surface pressures above 0 mN m−1.23 The most significant effect of changing the subphase from water to buffered saline for these two systems is a significant expansion of the film. Seifert et al.24 reported fluidization of lung surfactant model membranes comprising DPPC, DPPG and DPPC/DPPG (4:1) with the addition of HEPES buffer to the subphase and with increasing pH. The fluidization resulted in a film expansion and an increase in the phase transition plateau. D
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Figure 6. Surface dilational elasticity (left) and viscosity (right) data of DPPC:POPG on water (■) and TBS (□).
Figure 7. Surface dilational elasticity (left) and viscosity (right) data of DPPC:POPG:PA on water (■) and TBS (□).
shown that PA interacts with DPPC to form a mixed crystalline condensed phase on water.11 Since the carboxylic acid headgroups of PA are deprotonated on both subphases used, the presence of salt may affect domain morphology.19 On the other hand, the BAM images of Infasurf on water and on TBS show very little difference (Figure 4) in terms of area occupied by the LE phase, in good agreement with the isotherms in Figure 1. Comparing the BAM images of Infasurf to those of DPPC:POPG (Figure 2), the differences in domain size and in line tension due to the presence of the cationic SP-B protein are more pronounced when the subphase is buffered; however, the underlying differences seem to occur in the lipid film. Survanta, which contains the additional palmitic acid, is low in SP-B and rich in SP-C protein; it exhibits smaller domains on the water subphase but otherwise similar behavior in the presence or absence of buffer. More striking is the comparison of DPPC:POPG:PA to Survanta which exhibits a clear reduction in domain size when SP-C is present. Moreover, at 40 mN m−1, the LE phase is never completely lost. Contrary to Infasurf, where there appears to be only some residual LE phase present, in Survanta, the LE phase is quite apparent. Thus, SPC, the dominant surfactant protein present in Survanta,12 may act to maintain the LE phase at the interface. Also, because of the similarity at 40 mN m−1 for Survanta on either water or TBS, it appears that SP-C is significantly not affected by the presence of buffered saline in the subphase, as might be expected for a predominantly hydrophobic, transmembrane protein. Thus far, through compression isotherms and BAM images, it has been shown that buffering the subphase in general makes the film more fluid for which the viscosity (resistance to flow) and elasticity (recovery after removal of a stress) will be impacted. Small changes in the LE-C phase ratios can have strong impacts on the surface viscosity of lung surfactant films
These impacts were attributed to both penetration of dipolar HEPES into the film and charge repulsion from increased deprotonation of the DPPG headgroups. In our system, we have used a tris buffered saline, so a contributing factor will also be the intercalation of cations into the headgroup region that has been reported for DPPC18 and the POPG.15 Interpretation of the salt effects on Infasurf and Survanta systems is inherently more complicated due to the complexity of the composition (both charged and uncharged proteins and more complex lipid composition). For Survanta, a clear film expansion is observed, while Infasurf shows almost no change in the isotherm. Infasurf is high in the lung surfactant protein SP-B, while Survanta is depleted in this protein.12 This cationic protein has been proposed to interact specifically with POPG in model lung surfactant membranes,25 and such an interaction could partially screen the charged POPG headgroups, thus minimizing the impact of altering the subphase pH and ionic composition. BAM images of all four systems are shown in Figures 2−5. The images for DPPC:POPG and DPPC:POPG:PA in the liquid expanded-condensed (LE-C) phase coexistence regions show that there is significantly more LE phase on TBS than on water at any given surface pressure, which correlates with the expansion observed in the compression isotherm. There is a loss of contrast at high surface pressures (40 mN m−1) attributed to the loss of the LE-C phase coexistence.11 This occurs at lower surface pressures on water than on buffer which may be because of the counterion stabilization of the charged POPG and/or PA components20 with the buffered saline, resulting in less charge repulsion at higher surface pressures. For DPPC:POPG:PA (Figure 3), there is also a clear change in domain size and shape with the addition of buffer at all surface pressures which is not observed with DPPC:POPG alone. The line tension is significantly lower in the absence of buffer, leading to more flower shaped domains. GIXD has E
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Figure 8. Surface dilational elasticity (left) and viscosity (right) data of Infasurf on water (■) and TBS (□).
Figure 9. Surface dilational elasticity (left) and viscosity (right) data of Survanta on water (■) and TBS (□).
Table 1. Summary of Lattice Type, Tilt Azimuth, and Tilt Angles Measured by GIXD for DPPC:POPG, DPPC:POPG:PA, Infasurf, and Survanta (for Full Data, See Supporting Information Tables S1 and S2) system DPPC:POPG
water
11
buffered saline
DPPC:POPG:PA
water11 buffered saline
Infasurf Survanta
water buffered saline water
π (mN m−1)
lattice
tilt azimuth
tilt angle (deg)
25 35 50 25 35 50 25 40 25 40 45 45 25
oblique oblique centered rectangular oblique oblique oblique oblique centered rectangular oblique oblique oblique oblique centered rectangular hexagonal centered rectangular hexagonal centered rectangular hexagonal centered rectangular hexagonal
intermediate intermediate NN intermediate intermediate intermediate intermediate NN intermediate intermediate intermediate intermediate NN untilted NN untilted NN untilted NN untilted
26 20 10 37 26 9 19 7 26 21 5 7 16 0 10 0 18 0 13 0
35 buffered saline
25 35
due to a near zero slope at the transition and a slope change in the elasticity both of which highlight that the LE-C phase transition occurs at a higher surface pressure on buffer. This phase transition is less obvious in the more complex mixtures where the phase transition is smeared out as described by Bringezu et al.11 The reduction in viscoelastic properties associated with fluidization of the films is more evident in the LE-C coexistence regions, that is, above 15 mN m−1 for DPPC:POPG and 10 mN m−1 for DPPC:POPG:PA (below these surface pressures, the entire film forms a mixed LE
important for both the reversible spreading process during inhalation and exhalation as well as preventing the loss of material from the alveoli due to surface tension gradients.23 Additionally, surface dilational elasticity and viscosity are higher in systems with saturated lipids, since they can pack together to form a condensed phase.26 The fluidization observed for three of the four systems on buffered saline subphases correlates with a reduction in surface elasticity and viscosity (Figures 6−9). For the simplest two-component system (DPPC:POPG), the LE-C phase transition manifests itself as a maximum in the viscosity F
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rectangular unit cell with NN tilt, while, on buffer at the same surface pressure, the peaks remain in an oblique chain lattice which again corresponds with a more fluid film on buffer. Thus, one can conclude that the film expansion is not solely due to the increased amounts of LE phase present (as observed by BAM) due to fluidization but also due to an expansion within the condensed phase domains. Contrary to what is reported in the literature,6 peaks were detected as early as 20 mN m−1 for Infasurf, although the peaks were too broad and diffuse to fit. This is because the area occupied by condensed phase in the footprint region of the Xray beam is much lower in the lipid−protein systems and therefore yields much weaker peaks. Analysis was possible at a higher surface pressure (45 mN m−1, see Table 1) where more of the LE phase is squeezed out. An oblique lattice is observed for both subphases with very little difference in the organization. Also, there were very weak additional peaks at low Qxy values which we believe may be due to a liquid ordered (LO) phase due to the presence of cholesterol in Infasurf.12 Survanta on water has been reported to form two coexisting condensed phases, a tilted phase with approximately 1:1 mol/ mol DPPC:PA and an untilted phase comprising DPPC and tripalmitin.4 The tilted phase was shown to exhibit an oblique lattice at moderate (20 mN m−1) surface pressures and transition to a centered rectangular lattice by 30 mN m−1. Our finding of a centered rectangular lattice for the tilted phase at 25 mN m−1 is in agreement with these previous measurements. A similar phase behavior was observed on the TBS subphase. Stenger et al.27 also reported a main centered rectangular phase which became untilted by 40 mN m−1 for Survanta measured on a bicarbonate buffered saline. The tilt angle on tris buffered saline appears to be slightly larger than that on the bicarbonate buffered saline, although this is likely due to chemical differences in the buffer used and the inclusion of divalent calcium ions in the bicarbonate buffer.27 Of particular note is that for both Infasurf and Survanta the expansion of the condensed phase due to buffered saline is significantly reduced, with only a 2−4° tilt angle increase at each pressure. The more complex lipid composition with charged, uncharged, and neutral lipids appears to limit the impact of pH and salt on the condensed phase. The lack of a significant change to the condensed phase yet a drastic reduction in viscoelastic parameters observed for Survanta is evidence that viscoelastic parameters are dominated by both the ratio of LE to C phases23 as well as the properties of the LE phase (average molecular area and intermolecular interactions) and not by the structure within the condensed phase.
phase). At these higher surface pressures, the LE phase in the lipid only systems is POPG-rich, the consequence of which is that the charge repulsion of the anionic lipids is no longer moderated by the large amount of DPPC in this phase. Thus, the addition of saline provides counterions which can serve this role. The presence of PA in the ternary mixture increases the film’s rheological properties due to closer packing of the film.11 Figures 8 and 9 show the viscoelasticity data for the two protein-containing systems. Survanta, the system with additives such as PA, always reaches higher rheological values than systems without PA whether the subphase is buffered or not. However, systems with surfactant proteins behave differently when salt is present. For example, in terms of elasticity, at low surface pressures, Infasurf and Survanta exhibit similar values only when salt is present. In the case of viscosity, the lipid-only (DPPC:POPG, DPPC:POPG:PA) systems exhibit greater viscosity values than their comparable lipid−protein (Infasurf, Survanta) counterparts on water, but the opposite occurs on buffer. For Infasurf, the subphase does not have a major impact on the viscoelastic properties (Figure 8), as might be expected given the lack of change in the morphology and isotherm, although a small impact on elasticity can be measured at high surface pressures. With regard to viscoelastic parameters, two factors must be considered when assessing the reduced impact of a buffered saline subphase on Infasurf. First, it should be noted that the elasticity and viscosity values are already low on water such that the difference may be minimal when compared to buffer. Second, as noted above, the cationic proteins may already interact electrostatically with the charged phospholipids, thus reducing the impact of the addition of counterions to the subphase. On the other hand, the rheological properties for Survanta shown in Figure 9 are evidently reduced when using a TBS subphase rather than water. Survanta, like Infasurf, is also always in the LE phase. However, there are two noteworthy differences: the presence of the PA additive and the low proportion of SP-B. The PA significantly increases the viscoelastic properties of the system, and thus, any reduction may be amplified. Conversely, for Infasurf, the moderating effect of the protein already lowers the viscoelastic properties significantly and therefore any reduction may be difficult to discern. The PA may also undergo redistribution between the phases with greater charge screening. Finally, the lack of the charged SP-B may leave the film more susceptible to the effects of ionic strength and counterions. GIXD was used to evaluate structural changes in the condensed phase of the monolayers which are summarized in Table 1. Fitted Qz and Qxy peak positions, lattice parameters (a, b, γ), chain tilt (t), area per chain perpendicular to the chain long axis (A0), and projected area per chain (Axy) can be found in Tables S1 and S2 in the Supporting Information. At moderate surface pressures, regardless of subphase, the lipidonly systems exhibit three peaks all at values of Qz > 0, which corresponds to an oblique chain lattice with a tilt azimuth between nearest neighbor (NN) and next nearest neighbor (NNN). Both lipid-only systems show a pronounced expansion of the unit cell on a buffered saline subphase leading to an increased tilt angle of the chain. This effect is greater with the PA present and may indicate the higher impact of counterions on a condensed phase containing a charged species. On water, the DPPC:POPG and DPPC:POPG:PA monolayers by 50 and 40 mN m−1, respectively, exhibit a phase transition to a
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CONCLUSION The significant film expansion induced by buffered saline that is observed for DPPC:POPG, DPPC:POPG:PA, and Survanta is attributed to a combination of increased charge repulsion and intercalation of counterions into the headgroup region. The expansion is observed for both the LE phase (greater area occupied by fluid LE phase in BAM images) and within the condensed phase (greater tilt angle and lattice expansion from GIXD). The consequence of the fluidization and expansion of the LE phase is a significant decrease in viscoelastic parameters. The film expansion due to buffered saline is significantly reduced for Infasurf which is attributed to the strong interaction between POPG and SP-B in the LE phase. This suggests that the electrostatic interaction with SP-B may screen the POPG headgroups from the counterions and that the protein may act G
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(8) Hawgood, S. Surfactant Protein B: Structure and Function. Neonatology 2004, 85 (4), 285−289. (9) Notter, R. H.; Wang, Z.; Egan, E. A.; Holm, B. A. ComponentSpecific Surface and Physiological Activity in Bovine-Derived Lung Surfactants. Chem. Phys. Lipids 2002, 114 (1), 21−34. (10) Sharifahmadian, M.; Sarker, M.; Palleboina, D.; Waring, A. J.; Walther, F. J.; Morrow, M. R.; Booth, V. Role of the N-Terminal Seven Residues of Surfactant Protein B (SP-B). PLoS One 2013, 8 (9), e72821. (11) Bringezu, F.; Ding, J.; Brezesinski, G.; Zasadzinski, J. A. Changes in Model Lung Surfactant Monolayers Induced by Palmitic Acid. Langmuir 2001, 17 (15), 4641−4648. (12) Zhang, H.; Fan, Q.; Wang, Y. E.; Neal, C. R.; Zuo, Y. Y. Comparative Study of Clinical Pulmonary Surfactants Using Atomic Force Microscopy. Biochim. Biophys. Acta, Biomembr. 2011, 1808 (7), 1832−1842. (13) Grigoriev, D.; Krustev, R.; Miller, R.; Pison, U. Effect of Monovalent Ions on the Monolayers Phase Behavior of the Charged Lipid DPPG. J. Phys. Chem. B 1999, 103 (6), 1013−1018. (14) Zhao, W.; Róg, T.; Gurtovenko, A. A.; Vattulainen, I.; Karttunen, M. Atomic-Scale Structure and Electrostatics of Anionic Palmitoyloleoylphosphatidylglycerol Lipid Bilayers with Na+ Counterions. Biophys. J. 2007, 92 (4), 1114−1124. (15) Garidel, P.; Blume, A. 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) Monolayers: Influence of Temperature, pH, Ionic Strength and Binding of Alkaline Earth Cations. Chem. Phys. Lipids 2005, 138 (1−2), 50−59. (16) Huang, Z.; Hua, W.; Verreault, D.; Allen, H. C. Influence of Salt Purity on Na+ and Palmitic Acid Interactions. J. Phys. Chem. A 2013, 117 (50), 13412−13418. (17) Gurtovenko, A. A.; Vattulainen, I. Effect of NaCl and KCl on Phosphatidylcholine and Phosphatidylethanolamine Lipid Membranes: Insight from Atomic-Scale Simulations for Understanding Salt-Induced Effects in the Plasma Membrane. J. Phys. Chem. B 2008, 112 (7), 1953−1962. (18) Aroti, A.; Leontidis, E.; Maltseva, E.; Brezesinski, G. Effects of Hofmeister Anions on DPPC Langmuir Monolayers at the Air−Water Interface. J. Phys. Chem. B 2004, 108 (39), 15238−15245. (19) Tang, C. Y.; Allen, H. C. Ionic Binding of Na+ versus K+ to the Carboxylic Acid Headgroup of Palmitic Acid Monolayers Studied by Vibrational Sum Frequency Generation Spectroscopy. J. Phys. Chem. A 2009, 113 (26), 7383−7393. (20) Mao, Y.; Du, Y.; Cang, X.; Wang, J.; Chen, Z.; Yang, H.; Jiang, H. Binding Competition to the POPG Lipid Bilayer of Ca2+, Mg2+, Na+, and K+ in Different Ion Mixtures and Biological Implication. J. Phys. Chem. B 2013, 117 (3), 850−858. (21) Leser, M. E.; Acquistapace, S.; Cagna, A.; Makievski, A. V.; Miller, R. Limits of Oscillation Frequencies in Drop and Bubble Shape Tensiometry. Colloids Surf., A 2005, 261 (1−3), 25−28. (22) Kim, K.; Choi, S. Q.; Zasadzinski, J. A.; Squires, T. M. Interfacial Microrheology of DPPC Monolayers at the Air-Water Interface. Soft Matter 2011, 7 (17), 7782−7789. (23) Alonso, C.; Waring, A.; Zasadzinski, J. A. Keeping Lung Surfactant Where It Belongs: Protein Regulation of Two-Dimensional Viscosity. Biophys. J. 2005, 89 (1), 266−273. (24) Seifert, M.; Breitenstein, D.; Klenz, U.; Meyer, M. C.; Galla, H. J. Solubility versus Electrostatics: What Determines Lipid/Protein Interaction in Lung Surfactant. Biophys. J. 2007, 93 (4), 1192−1203. (25) Hemming, J. M.; Hughes, B. R.; Rennie, A. R.; Tomas, S.; Campbell, R. A.; Hughes, A. V.; Arnold, T.; Botchway, S. W.; Thompson, K. C. Environmental Pollutant Ozone Causes Damage to Lung Surfactant Protein B (SP-B). Biochemistry 2015, 54 (33), 5185− 5197. (26) Vrânceanu, M.; Winkler, K.; Nirschl, H.; Leneweit, G. Surface Rheology of Monolayers of Phospholipids and Cholesterol Measured with Axisymmetric Drop Shape Analysis. Colloids Surf., A 2007, 311 (1−3), 140−153. (27) Stenger, P. C.; Wu, G.; Miller, C. E.; Chi, E. Y.; Frey, S. L.; Lee, K. Y. C.; Majewski, J.; Kjaer, K.; Zasadzinski, J. A. X-Ray Diffraction
as a buffer for the system, limiting the impact of minor changes in pH, salt, and composition. This work highlights the importance of using physiologically relevant compositions and recognizing that changes in pH and/or ionic composition can have significant impacts on film fluidity and these must be taken into consideration when interpreting changes to model membranes.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02945. Graph of the rheology program, contour plot and fit Qxy and Qz peaks of DPPC:POPG:PA at 25 mN m−1, and tables of all GIXD data obtained and analyzed (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge financial support from the Natural Science and Engineering Research Council (NSERC, grant number 03977-2014) of Canada and the Canada Foundation for Innovation (CFI, grant number 8071). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
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DOI: 10.1021/acs.langmuir.6b02945 Langmuir XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.langmuir.6b02945 Langmuir XXXX, XXX, XXX−XXX
