Understanding the Collapse Mechanism in Langmuir Monolayers

Jun 27, 2013 - Chondroitin sulfate interacts mainly with headgroups in phospholipid monolayers. Lucinéia F. Ceridório , Luciano Caseli , Osvaldo N. ...
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Understanding the Collapse Mechanism in Langmuir Monolayers through Polarization Modulation-Infrared Reflection Absorption Spectroscopy Thiago Eichi Goto and Luciano Caseli* Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil S Supporting Information *

ABSTRACT: The collapse of films at the air−water interface is related to a type of 2D-to-3D transition that occurs when a Langmuir monolayer is compressed beyond its stability limit. Studies on this issue are extremely important because defects in ultrathin solid films can be better understood if the molecular mechanisms related to collapse processes are elucidated. This paper explores how the changes of vibration of specific groups of lipid molecules, as revealed by polarization modulationinfrared reflection absorption spectroscopy (PM-IRRAS), are affected by the monolayer collapse. Different mechanisms of collapse were studied, for those lipids that undergo constant-area collapse (such as stearic acid) and for those that undergo constant-pressure collapse (such as DPPC, DPPG, and DODAB). Lipid charges also affect the mechanism of collapse, as demonstrated for two oppositely charged lipids.

1. INTRODUCTION Monomolecular films that organize at air−water interfaces have received special attention in the last several decades because of the possibility of investigating nanoscale molecular interactions. Langmuir monolayers are suitable as systems for investigating the processes related to molecular recognition1−3 and as models for biomembranes.4−6 Furthermore, these films can be transferred to solid supports through the so-called Langmuir− Blodgett (LB) and Langmuir−Schaefer (LS) techniques. Monolayers that are transferred to solid matrices not only enlarge the variety of techniques to characterize these systems, but also make viable the production of sensors6 and optoelectronic devices7 based on ultrathin solid films. One of the main approaches to characterizing Langmuir monolayers is obtaining surface pressure−area (π−A) isotherms, which are obtained by compressing films laterally with movable barriers and following the increase in π for as long as the molecular area decreases. By analyzing the profile of the resultant curves, two-dimensional (2D) states can be determined, which then enable the identification of gaseous, liquid-expanded (LE), liquid-condensed (LC), and solid states for films located at air−water interfaces. Monolayer compression decreases the available area per surfactant molecule, which in general will cause an increase in the surface pressure until the monolayer reaches a limiting value above which it cannot be further compressed. Then, with continued compression, the material is forced out of the interface, a situation that is known as collapse. The surface pressure at this limiting value is called the collapse pressure (πc), and with further compression the monolayer transitions from a 2-D to a 3-D structure. During the collapse, surfactants are forced out of the interface and form a 3-D entity. The © 2013 American Chemical Society

collapse occurs above the equilibrium spreading pressure (the pressure at which the monolayer is in equilibrium with the bulk phase), which means that this transition occurs when the monolayer is in a supersaturated (metastable) state. Investigating the collapse mechanism is important not only for better understanding of the fundamentals in surface science, but also because collapse plays an important role in the regulation of surface tension in lung alveolar air−liquid interfaces,8 which are crucial for maintaining low surface tension values during breathing. Additionally, collapse is related to defects in ultrathin films in some manner. Collapse in monolayers has been characterized experimentally9−11 and studied with theoretical models12 in the last several decades. Despite the growing interest in characterizing monolayer collapses, the mechanisms involved in this process are not fully understood. It is not yet completely clear which properties of the constituent material of the monolayer determine the mechanism that leads a 2-D structure to form a 3-D one. Furthermore, the experimental characterization of a monolayer at the collapse state is not trivial. In contrast, theoretical models normally consider the monolayer as an elastic continuum, disregarding the molecular structure of the film at the air−water interface.12 In general, collapse mechanisms appear to be complex processes, with a dependence on molecular structure,9 degree of ionization,10 rate of compression,11 and subphase conditions, including ions and subphase pH.10,13 These variables affect the rheological properties of the monolayer, such as the monolayer Received: March 14, 2013 Revised: June 26, 2013 Published: June 27, 2013 9063

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bilayers suspended below the film can be reincorporated into the interface during expansion. However, when the monolayer is compressed at a high rate of compression, multiple folds extend across the trough, in a direction perpendicular to the compression. For both cases, the process of buckling must occur, which begins at boundaries between 2D phases because of the instability caused by height differences between phases. However, because multiple folds also occur in monolayers that present one single phase during compression, this explanation is controversial.23 Defects that arise below the collapse pressure may occur independently of coexisting phases; thus, the existence of precollapse instabilities, which may appear because of compression beyond the equilibrium surface pressure, must be considered. This instability may lead the monolayers to bend instead of break, and such folding enables collapse to occur at high surface pressures. Additionally, such folding does not occur without in-plane resistance to shear.14 Therefore, fracture and nucleated collapse can be distinguished with the existing mechanism that is proposed for a collapse transition. Usually liquid-condensed or solid phase monolayers collapse via fracture followed by loss of material,24 and liquid-expanded phase monolayers collapse with solubilization of materials into the subphase. The monolayer can then be considered a “plate”. After reaching limiting values, this plate must first fracture and break, followed by buckling or loss of material. Furthermore, the mechanisms of monolayer collapse also appear to be dependent on the amount of surfactant spread25 and on the capacity for forming nematic clusters.26 However, none of these mechanisms have been proven conclusively. For instance, it has been reported that for some phospholipids showing phase coexistence, large and isolated folds rather than vesicles are observed at the boundaries between the two phases;14 these folds remain connected to the monolayers, which would permit the film to reverse when expanded. In addition, it has been suggested11 that a behenic acid monolayer formed ridges prior to the formation of the 3-D phase. Schief et al.20 also observed that dipalmitoylphosphatidylcholine (DPPC) monolayers collapse, forming buds above the interface, away from the water, thus allowing the lost material to be reincorporated into the monolayer during expansion. Therefore, the mechanism by which a monolayer collapse occurs is not straightforward, and it is of interest to find other strategies to investigate the steps that leads to this transition at the molecular level. Experimental techniques such as other than the classical surface pressure−area isotherms, imaging techniques, such as Brewster angle microscopy (BAM),27 light scattering,20 electron microscopy,16 fluorescence microscopy (FM),28 and atomic force microscopy (AFM) on monolayers transferred to solid supports29 have been extensively employed to study collapse in Langmuir monolayers. In this sense, one of the most recent techniques for investigating monolayers at the air−water interface is polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS). This powerful tool for obtaining detailed orientations of chemical groups at an interface decisively identifies the vibration transition dipoles that are parallel or perpendicular to the interface, being specific for surfaces. More detail on this technique can be found elsewhere.30,31 In addition, it has previously shown32 the possibility of investigating the collapse process of Langmuir monolayers with this technique, in which it has been shown that the increase in the intensity of PM-IRRAS signal bands for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dio-

elasticity. For elasticity, it has been reported that fluid monolayers that do not support static shear stress can develop viscous shear stress caused by in-plane flows.14 In other words, for monolayers with relatively high elasticity, collapse occurs with out-of-plane folding because of anisotropic shear stress, and for monolayers with relatively low elasticity collapse occurs with in-plane rearrangements. It is also important to mention that as the value of pH in the subphase alters the net charge of the hydrophilic group of the monolayer, the surface elasticity should be affected, and consequently the collapse mechanism. For the origins of collapse studies, Ries and Swift were one of the pioneers in investigating the phenomenon,15−17 postulating a simple model in which the monolayer must pass through three steps: weakening, folding, and bending, which leads to the formation of trilayers, as shown in Figure 1. Afterward, a

Figure 1. Mechanism for the monolayer collapse suggested by Ries et al.16 In the limit of the packed monolayer (A), the surface is further compressed leading to the weakening (B), buckling (C), bending (D), and collapse of the monolayer (E).

sequence of articles from Kundu and co-workers10,13,18,19 showed the formation of structures thicker than three layers with subsequent compression, showing the existence of islands of multilayers along the trilayer. These structures were related to collapses that presented a sharp decrease in the surface pressure in π−A isotherms after reaching πc. The authors called this case “constant area collapse”, in which the number of layers increased as the collapse progressed.13 When the collapsed structure was maintained in coherent trilayers, the π−A isotherms presented a constant value of surface pressure (or a constant slow increase relative to the area decrease), which the authors called “constant surface pressure”. In this case, the monolayers obeyed a mechanism called “folding-sliding”. The authors also suggested that these mechanisms can be influenced by the dissociation degree of the polar group of the investigated fatty acids, with the collapse characteristics changing if the pH or the ions in the subphase changed. The formation of multilayers near the air phase successfully explained the collapse for some surfactants, such as fatty acids. However, for other surfactants, such as some phospholipids, a monolayer that is compressed to its limit buckles near the aqueous subphase instead of at the air phase. The bilayer that is formed then folds, leading to the formation of 3-D buds and vesicles.9,20−22 During this process, the loss of material to the subphase can be reversible or irreversible, depending on whether the interface can reincorporate the lost material by expansion of the monolayer. Usually, slow compression rates lead to the formation of giant folds in the aqueous subphase, which arise at defects at the air−water interface.23 For this case, 9064

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least three independent experiments were carried out. A fixed angle of incidence (80°) was used. In this angle, the reflectivity is maximized, the noise is minimum, and the signal is maximum. The frequency of wavelength modulation was 1500 cm−1, and the resolution of the instrument is 8 cm−1. Atomic Force Microscopy (AFM) was also employed for further characterization, and the images were obtained in the tapping mode, employing a resonance frequency of approximately 300 kHz, a scan rate of 1.0 Hz, and scanned areas of 5.0 × 5.0 μm. For these measurements, a Digital AFM-Nanoscope IIIA instrument was employed. The tip was made from silicon. The images were carried out on Langmuir−Schaefer films, with the monolayers transferred horizontally to silicon substrates after collapse attainment. All experiments were carried out keeping the area constant at a temperature of 25.0 ± 0.5 °C.

leoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoylsn-glycero-3-phospho-L-serine (DOPS) during collapse supports the conclusion that these lipids form multilayer structures. Furthermore, an increase in an optical effect in the spectra that is related to the dispersion of the refractive index of water could be attributed to an increase in the layer thickness. Therefore, the discussion of this study32 focused on the formation of trilayer or multilayer structures rather than on the loss of material to the aqueous subphase that resulted from the formation of buds or vesicles. Considering the necessity of a better understanding on the process that leads from 2-D to 3-D structures at the air−water interface, in this paper, the collapse of monolayers of selected lipids was investigated through PM-IRRAS and correlated to the collapse mechanism. To do so, stearic acid (HSt) was chosen as a pattern fatty acid, representing the constant area mechanism. For lipids presenting mechanisms in which the surface pressure is maintained upon collapse, three phospholipids with different charges in the polar head were chosen: a zwitterionic lipid, dipalmitoylphosphatidylcholine (DPPC); a negatively charged lipid, dipalmitoylphosphatidylglycerol sodium salt (DPPG); and a positively charged lipid, dimethyldioctadecylammonium bromide (DODAB).

3. RESULTS AND DISCUSSION The profile of the isotherms for the pure stearic acid is wellknown in the literature34 and is reported in the Supporting Information, and we will describe the profile focusing on the area of the collapse. The monolayer compression can lead to values of surface pressure that extrapolated the equilibrium value, and with overcompression, the surface pressures reaches values as high as 60 mN/m. With further compression, the monolayer collapses, which results in a downward drift of the surface pressure until a constant surface pressure of 20−25 mN/m. The surface pressure starts decreasing because multilayers form. The currently existing mechanism proposes a model known as “constant area collapse”,10 by which the HSt molecules must form trilayers with an island of multilayers to reach a stable state. The proposed mechanism subsequently follows three main steps: buckling that forms bilayers, folding of the buckled structures, and formation of bilayers above the existing monolayer. Instability leads to an island of multilayers that characterizes the constant-area mechanism. To obtain additional information about the orientation of the chemical groups that is supposed in this model, PM-IRRAS spectroscopy was then employed for an HSt monolayer before (molecular area of 26 Å2) and after collapse (molecular area of 17 Å2), as shown in Figure 2. The bands at 2916 and 2836 cm−1 (panel A) are attributed to antisymmetric and symmetric C−H stretching in CH2 groups, respectively. The intensities of both bands increase with compression, and the highest value is attained after collapse. In addition, when the monolayer is permitted to relax 30 min after the collapse, the intensities of these bands decrease because of the relaxation of the monolayer. In contrast, the intensity of the band at 2882 cm−1, attributed C−H stretches of CH3, increases after relaxation, and the band is shifted to higher energies. This finding indicates a definite increase in the disorder of the multilayer formation after relaxation, which can lead to voids and cracks, as supposed in the literature.14 Panel B of Figure 2 shows a band centered at 1682 cm−1, attributed to the CO stretch of carboxylic acid, which indicates that HSt may already be in the form of a dimer.35 Because the HSt monolayer is found in the condensed phase, this finding is indicative of a precollapse process, as suggested in the literature.20 In this case, a 3D nucleus can be formed before reaching the maximum surface pressure during compression. The band at 1652 cm−1, attributed to bending of the surface water,31 can be considered an optical effect related to the change in the refractive index that occurs with the formation of the monolayer. Consequently, the geometrical arrangement upon monolayer spreading and the alteration in the hydration

2. MATERIALS AND METHODS HSt, DPPC, DPPG, and DODAB were purchased from Sigma-Aldrich, with purity degree higher than 99%, and used without further purification, and dissolved in chloroform (HSt and DPPC) or chloroform/methanol (9:1 v/v) for a concentration of 0.5 mg/mL. Aliquots of these solutions were spread on the interfaces of aqueous solutions contained in a Langmuir trough, model Mini from KSV Instruments (Finland), and allowed to evaporate for 20 min. Afterward, the monolayer was compressed at a rate of 0.5 Å2·molecule−1·s−1. The compression of the monolayers continued beyond the collapse point until the barriers attained the minimum possible area. To obtain the desired pH values, the subphase was adjusted with diluted NaOH (Merck) or HCl (Merck). When not mentioned, the monolayers were formed on water purified with MilliQ system (pH ∼ 5.5). For several subphases, chitosan was obtained from Galena (Brazil), with a 25% degree of acetylation (determined by 1 H NMR spectroscopy). The chitosan was purified through dissolution in HCl medium at pH 3.0, followed by filtering in an NaOH medium at pH 10. The chitosan had a molecular weight, Mn, of 125 kDa, with a polydispersity index of 3.5, as determined by size exclusion chromatography under previously described conditions.33 Dextran sulfate (DS) from Sigma-Aldrich was also used as a subphase for DODAB and dissolved in pure water for a concentration of 0.2 mg/ mL. For all experiments, water was purified with a Milli-Q system (resistivity of 18.2 Ω cm−1 and surface tension of 72.0 mN/m at 25 °C). Polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were conducted with a KSV PMI 550 instrument (KSV instrument Ltd., Finland). The Langmuir trough was set up so that the light beam reached the monolayer at a fixed incidence angle of 80°. The incoming light was continuously modulated between s- and p-polarization, and spectra were measured for both polarizations. The difference between the spectra provided surface-specific information, and the sum provided the reference spectrum. More information about this technique can be seen in refs 30 and 31. Absorption from the parallel polarized light beam is sensitive mostly to vertically oriented dipoles, while absorption of the perpendicularly polarized beam is sensitive to horizontally oriented dipoles with respect to the interface. At least 6000 spectra were accumulated to provide each spectrum that was presented in this paper. The spectra were selected in the desired regions which presented the lowest signal/noise ratios. Vertical offsets of the curves were carried out in order to provide more clarity in the figures and to better compare the spectra intensities. For each group of spectra, at 9065

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Figure 2. PM-IRRAS spectra for HSt monolayers, showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000−1800 cm−1.

Figure 3. PM-IRRAS spectra for DPPC monolayers, showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000− 1800 cm−1.

of the carbonyl groups when the monolayer is highly packed can alter the band at 1652 cm−1. The band at 1270 cm−1 is assigned to a coupled mode of C− O stretching and the OH in-plane bending vibrations of the trans configuration, which suggests that the trans configuration is preferable. Kimura et al.36 reported that this configuration is preferred by trilayer LB films, which might also indicate tri- (or multi-) layer formation. The isotherms for DPPC, DPPG, and DODAB on pure water are reported in the Supporting Information, with the isotherms showing a constant surface pressure mechanism. For DPPC, the monolayer presents a collapse at high surface pressures (about 68 mN/m); for DPPG, the surface pressure of collapse is 65 mN/m; and DODAB presents the collapse with the lowest surface pressure: 44 mN/m, presenting a decrease to 43 mN/m just after the collapse attainment with values remaining approximately constant with further compression. This mechanism means that the surface pressure tends to remain constant after collapse. Figure 3 shows the DPPC PM-IRRAS spectra. The bands at 2917 and 2847 cm−1, similar to those for HSt, are attributed to CH stretches in CH2 groups for antisymmetric and symmetric vibrations, respectively. Estrela-Lopes et al.37 found these peaks for DPPC at 40 mN/m at 2915 and 2850 cm−1. The spectra show that the intensity of these bands increase with compression before collapse, and tends to decrease slightly when collapse occurs, which may be indicative of the loss of order in the film at the interface. Panel B in Figure 3 reveals the main bands for the polar groups and the CH bending of DPPC. The band at 1471 cm−1, caused by CH2 bending with collapse. Although it is believed that there is loss of materials from the interface because of the formation of buds and vesicles in the aqueous subphase,20,21 this result shows that there is an increase in the surface density of DPPC with collapse. In fact successive cycles of compression−decompression leads to hysteresis pointing to

shift to lower areas, indicating loss of material as already known in the literature. In contrast, the band at 1737 cm−1, attributed to CO stretching, increases with compression before collapse, but its intensity does not change after the collapse occurs. This finding indicates that this group is not significantly affected by the 2D-3D phase transition. The band at 1671 cm−1, caused by water bending or CO hydration, is less evident before collapse but becomes more evident afterward. At 1266 cm−1, the band is positive relative to the baseline before collapse but becomes negative afterward, which is a consequence of the relative orientation of the groups with respect to the interface. Together, all these results indicate a possible bending of the bilayers toward the aqueous subphase, which may affect the intensity of the main groups and also invert the position of the phosphate groups. However, because the CH and phosphate vibrations do not decrease, a complete loss of material to the subphase would be possible only if the compression was able to restore the lost materials to the subphase. An indicator of whether this restoration occurs is the band for water vibrations at 1569 cm−1. This band is said to be proportional to the thickness of the layer at the air−water interface.38 Because this band increases with collapse, it is probable that 3D structures form and remain attached to the interface to some extent, forming a type of subsurface below the DPPC monolayer. Galla et al.39 have reported the formation of stacks of lung surfactant molecules for a mixed protein−lipid monolayer before the collapse attainment. Although we are studying the formation of 3D structures during the collapse process of pure lipid monolayers, it is probable that that work corroborates in some way to fact that certain structures can be attached to monolayer instead to be merely protruded toward the aqueous phase. Figure 4A shows the PM-IRRAS spectra for DPPG monolayers before (50 Å2/molecule) and after collapse (30 and 20 Å2/molecule). The band in 2917 cm−1 is attributed to the asymmetric C−H stretch for CH2, and the band in 2847 9066

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1266 cm−1 was not affected to a significant extent by the collapse. At 1738 cm−1, the carbonyl stretch band increases with collapse but decreases with further compression to 20 Å2. This oscillation may be a consequence of the formation of vesicles and 3D buds that occurs in the subphase; the surface density is not sufficiently compensated because of the progressive monolayer compression. In addition, changes in the refractive index must be considered, which may also affect the band intensities. A large band at 1523 cm−1, more evident for the lowest molecular areas, is attributed to water bending and should result from the accumulation of water at the interface because of lipid buckling. This band overlaps the one at approximately 1450 cm−1, which is attributed to CH2 deformation. The band at 1397 cm−1 is also more evident with collapse and is attributed to the C−OH in-plane bending vibration from the lipid polar head. This band usually does not appear (or is not clear) for a noncollapsed Langmuir monolayer and should be enhanced by the accumulation of water at the interface. A water molecule is possibly attracted by the increase in negative charges that occurs during monolayer compression. It is probable that the negative charge of DPPG influences the process of collapse. Consequently, to infer the role of the charges on the collapse, DPPG monolayers at low pH values were investigated because these pH values may reduce the amount of free negative charges from phosphate. Figure 5 shows that the bands in the 2800−3000 cm−1 region are not significantly affected by the decreased pH in relation to

Figure 4. PM-IRRAS spectra for DPPG monolayers (pH 5.5), showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000−1800 cm−1.

cm−1 is attributed to the symmetric C−H stretch for CH2. From 50 to 30 Å2/molecule, the intensity of the band increases as a consequence of the surface density. However, the antisymmetric/symmetric ratio varies from 1.05 for the area of 50 Å2 to 1.50 for the area of 30 Å2. This change should be a consequence of the disorder provided by the collapse. This ratio is not as clear for DPPC as it is for DPPG. Because DPPG should also form 3D-buds near the aqueous subphase, one would expect the level of organization of the resting lipids to remain constant, with the lipids forming a coherent monolayer at the water-interface. However, these results indicate that at least a certain degree of disorder is imposed on the resting lipid at the interface upon collapse. With further compression to reach the molecular area of 20 Å2, the intensity of the antisymmetric peak remains practically identical to that obtained for the area of 30 Å2. In contrast, the intensity of the symmetric band is relatively lower, which is an unexpected indicator of more order. Because a collapsed monolayer is being compressed, it is possible that the formation of 3D buds and vesicles at the aqueous subphase allows the lipids that remain at the monolayer to have a low degree of packing. It is probable that this low degree of packing is a combination of effects that involve favorable loss of material to the aqueous interface; the loss of density at the molecular surface that occurs during compression is not stoichiometrically compensated. Because the surface pressure does not decay just after collapse, effects that are related to the viscoelasticity of the monolayer should most likely be considered. Reports in the literature14 have already considered this fact when explaining certain collapse mechanisms. It is also important to emphasize that, for our case, as each spectrum should take at least 10 min to be obtained, the data shown is reported only for conditions in the equilibrium, and dynamic aspects related to kinetically limited structural states could not be assessed. The effect of collapse on the polar groups is more clearly shown in panel B of Figure 4. The phosphate stretch band at

Figure 5. PM-IRRAS spectra for DPPG monolayers (pH 2.0), showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000−1800 cm−1.

that obtained for Figure 4. However, we have to mention that the bands at 20 and 30 Å2, which are superimposed, are more intense than at 50 Å2, and after collapse their relative intensity remains constant. This fact indicates that the neutralization of the charges of DPPG does not lead to a significant loss of order with collapse as observed for DPPG at pH 5.5. In the 1100−1800 cm−1 region, the strong negative band to the baseline for water bending appears in 1680 cm−1. This band 9067

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occurs because of the difference in the reflectivity of covered and uncovered monolayers. Although this band is very sensitive to artifacts, such as evaporation or water leaking, this result is consistently repeated. Therefore, it is likely that this band is associated with changes in the geometry of the surface water upon spreading of the lipid monolayer. For the case of DPPG in higher pH values (Figure 4), it is probable that the negative charge maintains the orientation of the surface water molecules. This orientation is maintained for DPPG before collapse. However, upon collapse, there is a clear tendency for this band to become positive relative to the baseline. Furthermore, all the other bands tend to decrease upon compression, which indicates the monotonic behavior ascribed to the decrease in the reflectivity of the monolayer. These bands are in 2917 and 2848 cm−1, attributed to C−H stretches in CH2; 1744 cm−1, attributed to CO stretches; 1561 cm−1 attributed to surface waters bedings; 1458 cm−1, attribured to C−H bendings in CH2, and 1269 cm−1, attributed to PO stretches. At this point, it is difficult to infer a conclusive model that is specifically related to the polar groups. For this reason, a type of “marker” with particular affinity for polar groups was employed. Chitosan, with its positive charge at low pH values, has specific affinity for negatively charged phospholipids at the air−water interface.40 Thus, this polysaccharide was employed to interact with the polar group of DPPG, and the effect of collapse was further investigated. Chitosan (or any other polyelectrolyte) must affect the collapse of phopholipid monolayers by neutralizing the charges of the polar heads and consequently altering the surface elasticity prior to collapse. The compression isotherms on the chitosan-containing subphase are already reported in the literature,40,41 and our isotherms (not shown) present similar results. The presence of carbohydrate and hydroxylated polymers in the subphase produces large changes of the packing areas and collapse pressure. The assignment of the bands in Figure 6 is similar to that in Figure 5. The spectra show that, in contrast to the results obtained for DPPG at pH ∼ 5.5, the CH2 stretching bands are progressively decreased with compression, even after the collapse has occurred, which could be related to the irreversible disorder imposed by chitosan. Also, the bands are not as welldefined for the noncollapsed monolayer, and their intensities increase after collapse. The antisymmetric/symmetric ratio increases from 1.7 at a molecular area of 30 Å2 to 2.1 Å2 for 20 Å2, which indicates that the monolayer becomes less ordered with sequential compression of the collapsed monolayer. Chitosan, despite its high polarity and low surface activity, contains some hydrophobic groups in its chain that may penetrate the alkyl chains of the phospholipid to some extent. Usually chitosan imposes order on phospholipid monolayers, as demonstrated with sum frequency generation spectroscopy and dynamic surface elasticity.41 Such order occurs because the incursion of the chains geometrically restricts the lipid monolayer. The results indicate that the collapse causes a decrease in the relative amount of chitosan at the interface, which is an effect of the preference of the polysaccharide for vesicles that are formed in the aqueous subphase over lipid monolayers. This finding is reasonable because, as a macromolecule (the sample employed had a molecular weight above 100 kDa), chitosan should prefer an interface with less conformational restrictions. The buds of the 3-D DPPG system appear to provide this type of interface. More detail on the effect of chitosan on DPPG collapse should be obtained if the 1100−1800 cm−1 region is analyzed.

Figure 6. PM-IRRAS spectra for DPPG monolayers (pH 2.0) in the subphase containing chitosan (0.1 mg/mL), showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000−1800 cm−1. Reference spectrum was taken with the subphase without chitosan.

The negative band for water bending at approximately 1670 cm−1 appears more pronounced, which clearly indicates that chitosan may influence the geometry of water molecules at the air−water interface. This finding is predictable if the hydrophilicity of this polysaccharide is taken into account. Chitosan contains several OH groups in its structure and may form hydrogen bonds with and even replace the water molecules at the interface. In addition, carbonyl bands are no longer clearly distinguishable in the spectrum, which may result from sheltering of the carbonyl bands by chitosan. Phosphate bands appear to show more relevant changes. First, the band is shifted to lower energies than those in the spectra for DPPG in pure water (Figures 4 and 5). However, the band at 1236 cm−1 is shifted to higher energies with collapse (1256 cm−1), and its relative intensity increases. This finding could be explained as follows: chitosan, which is expelled to the aqueous subphase because of the formation of 3D buds, must avoid the screening of phosphate groups, as reported in recent literature.41 This explanation confirms the proposed model in which chitosan prefers to interact with 3D buds in the aqueous subphase upon collapse than with 2D layers at the air−water interface. Positively charged lipids were also assayed. The DODAB spectra (Figure 7A) show that the CH2 stretching bands increase with collapse, with a simultaneous increase in the monolayer, a phenomenon similar to that observed for DPPG. The assignment of the bands in Figure 7A is similar to that in Figure 5A.The 1100−1800 cm−1 region includes less relevant bands because only bands that do not change significantly with collapse appear in this region. These bands are for water bending (about 1550 and 1670 cm−1) and CH2 bending (1458 cm−1). Because the vibrational bands of quaternary ammonium are not within the measurable range for PM-IRRAS, dextran sulfate (DS), a negatively charged polysaccharide, was also employed 9068

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revealing that, in contrast to the observations for chitosan/ DPPG, DS has a great affinity for the DODAB monolayer at the air−water interface, even after collapse. Therefore, this finding provides evidence that the collapse mechanism depends on several factors, including the nature of the polar head, the number of alkyl chains, and even the presence of other substances in the subphase. To finish, although many questions on molecular mechanisms involved in monolayer collapse remains open, this work does provide support for reconsidering and refining the existing models. To better correlate the data obtained with vibrational spectroscopy to the organization of the film after collapse, the structures at the air−water interface were transferred to solid supports through the Langmuir−Schaefer technique and analyzed with atomic force microscopy (Figure 9). The formation of large domains for HSt (panel A) is related to the formation of noncoherent multilayers, as described in the constant-area collapse mechanism. For DPPC (panel B), the image is more homogeneous, pointing to the formation of bilayer stacks below the previous monolayer at the air−water interface. The relative roughness of the images, however, points also to the fact that the small domains of the monolayer must be irregular in height, with some molecules being protruded toward one of the phases more than other molecules. With DPPG (panel C), the film is even rougher, confirming the model in which buds can expelled irreversible to the aqueous subphase. The expelling of molecules from the interface may disturb the monolayer originating such defects. Panel D shows that the formation of these defects is influenced by the charges of the lipid since for DPPG on a pH 2.0 suphase the film is smother considering that the root mean squared (RMS) roughness value is higher for the lowest pH value. This fact occurs without and with chitosan (panel E) in the subphase, pointing here to the preferential formation of buds and vesicles during the collapse. With chitosan, however, the image is more homogeneous, with less plateaus, pointing to a less disturbing 2D-3D transition. With DODAB (panels F and G), the film becomes rougher again (although not so much as for DPPC), suggesting the stacking of bilayers suspended below the monolayer in the event of collapse. This present work then proposes that for some cases (e.g., phospholipids) the model for collapse in multilayers must be revised with the PM-IRRAS spectra presented here. Especially for two-alkyl lipids, the formation of bilayers suspended below the monolayer or buds or vesicles protruding from the monolayer is highly dependent on nature of the polar head, including charge and adsorbed substance. Also, the orientation of molecular groups has been identified during collapse, and this fact was fundamental to confirm the models proposed in this work.

Figure 7. PM-IRRAS spectra for DODAB monolayers (pH 5.5), showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000−1800 cm−1.

as a subphase for DODAB monolayers to further investigate the effect of this lipid on polar groups. Figure 8 shows that the CH2 stretches are not significantly affected by the presence of DS. The assignment of the bands in Figure 8 is similar to that in Figure 7. Panel B shows the enhanced intensity of the polysaccharide band (C−O−C vibration at 1266 cm−1),

4. CONCLUSIONS The results presented in this paper detail the effects that occur on specific chemical groups when lipid monolayers are subjected to 3D structures because of a collapse event. For monolayers that form multilayers near the air phase, such as those formed from stearic acid, hydrophilic groups are strongly affected. This effect occurs because these groups are protruded in a new geometric configuration to allow a trilayer (or multilayer) structure with an evident degree of dehydration. For lipids with two alkyl chains, which are believed to form 3D buds near the aqueous phase, significant changes are observed for the alkyl groups. These groups tend to be less ordered as a

Figure 8. PM-IRRAS spectra for DODAB monolayers (pH 5.5) on a dextran sulfate subphase (0.1 mg/mL), showing the effect of the collapse. Panel A: 2800−300 cm−1 region. Panel B: 1000−1800 cm−1. 9069

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Figure 9. AFM images for the monolayers collapse deposited as Langmuir−Schaeffer films. (A) HSt; (B) DPPC; (C) DPPG; (D) DPPG on pH 2.0 suphase; (E) DPPG on chitosan solution subphase; (F) DODAB; (G) DODAB on DS subphase. RMS roughness values are indicated in the insets for each image.

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consequence of the loss of molecules to the subphase; the extent of the loss is greater than the surface molecular density that is recovered with compression. The presence of oppositely charged macromolecules on the polar groups of these lipids causes different effects, depending on the charge and chemical nature of the lipid and the macromolecules. For DPPG/ chitosan, the polysaccharide tends to stabilize the 3D structures in the aqueous subphase. In contrast, the studies of the DODAB/DS pair show a clear tendency for DS to continue interacting with the lipid at the air−water interface.

ASSOCIATED CONTENT

S Supporting Information *

Additional figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone +55 (11) 3319-3568. Fax: +55 (11) 4043-6428. Notes

The authors declare no competing financial interest. 9070

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ACKNOWLEDGMENTS The authors thank FAPESP (2008/10851-0) and INEO-CNPq for financial support. T.E.G. received a fellowship from nBioNet: Films and Sensors (CAPES).

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