Chitosan as a Removing Agent of β-Lactoglobulin from Membrane

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Chitosan as a Removing Agent of β-Lactoglobulin from Membrane Models Luciano Caseli,*,† Felippe J. Pavinatto,† Thatyane Morimoto Nobre,‡ Maria E. D. Zaniquelli,‡ Tapani Viitala,§ and Osvaldo N. Oliveira, Jr.† Instituto de Fı´sica de Sa˜ o Carlos, UniVersidade de Sa˜ o Paulo, Sa˜ o Carlos, SP, Brazil, Departamento de Quı´mica, Faculdade de Filosofia Cieˆ ncias e Letras de Ribeira˜ o Preto, UniVersidade de Sa˜ o Paulo, Ribeira˜ o Preto, SP, Brazil, and KSV Instruments Ltd, Ho¨tla¨a¨mo¨tie 7, 00380 Helsinki, Finland ReceiVed December 13, 2007. In Final Form: January 16, 2008 Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove β-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

Introduction Chitosan is a natural polysaccharide used in numerous applications, acting as a bactericide,1 in drug2 and gene delivery3 and blood coagulation,4 and as a fat reducer.5 It is biocompatible, biodegradable, and nontoxic, being the only positively charged natural polysaccharide in nature. In most cases, chitosan activity involves interaction with biological interfaces through hydrophobic, dipole, and mainly electrostatic forces. Among the many applications of chitosan, its possible use as anti-allergic agent has motivated the publication of a recent paper, in which chitosan was suggested to remove β-lactoglobulin (BLG) from lipophilic environments.6 This is especially relevant to the dairy industry because in contrast to cow milk that is rich in BLG, human milk contains negligible quantities of this protein, which is a potential allergen for humans.6,7 BLG is the major whey protein of ruminants, whose functions are still not completely understood in spite of extensive physical and biochemical studies.7 It has cell-recognizing properties, including linking to small hydrophobic substances such as fatty acids, phospholipids, and retinol and complexing with water-soluble macromolecules (e.g., polysaccharides). The removal of BLG by chitosan probably

involves complexation between these substances, but the mechanism involved is still unknown. The successful use of chitosan in medical applications implies suitable interactions with cell membranes, in some cases including penetration through the membrane, as in gene-delivery applications.3 The main challenge now is to identify precise molecularlevel mechanisms. In fact, the action of chitosan on bioinspired interfaces has pointed to strong interactions with lipids in liposomes,8 monolayers,9-11 and Langmuir-Blodgett films.10,11 Phospholipid monolayers, in particular, have been extensively used to mimic cell membranes12 and to probe interactions among lipids, proteins, and polysaccharides on the molecular level. Even though chitosan is not surface-active, it adsorbs onto monolayers of cholesterol, in addition to neutral and charged phospholipids.9-11 Being positively charged, chitosan interacts strongly with negatively charged lipids (e.g., dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG)),10,11 and this may have important biological implications because negatively charged lipids comprise up to 20% of cell membranes.13 In this study, we investigated the chitosan ability to remove BLG from interfaces made from negatively charged lipids. For this purpose, we employed Langmuir monolayers at the airwater interface.

* Corresponding author. E-mail: [email protected]. Tel: +55 16 33738061. Fax: +55 16 3371 5365. † Instituto de Fı´sica de Sa ˜ o Carlos, Universidade de Sa˜o Paulo. ‡ Departamento de Quı´mica, Universidade de Sa ˜ o Paulo. § KSV Instruments Ltd.

BLG was purchased from Sigma and dissolved in phosphate buffer (50 mg mL-1, pH ∼7.0). Horseradish peroxidase and urease were also obtained from Sigma. DMPA sodium salt, DPPC, DPPG, and

(1) Liu, H.; Du, Y.; Wang, W.; Sun, L. Int. J. Food. Microbiol. 2004, 95, 147-155. (2) Gupta, K. C.; Kumar, M. N. V. R. ReV. Macromol. Chem. Phys. 2000, C40, 273-308. (3) Kima, T.-H.; Jianga, H.-L.; Jerea, D.; Parka, I.-K.; Chob, M.-H.; Nahc, J.-W.; Choia, Y.-J.; Akaiked, T.; Choa, C.-S. Prog. Polym. Sci. 2007, 32, 726753. (4) Okamoto, Y.; Yano, R.; Miyatake, K.; Tomohiro, I.; Shigemasa, Y.; Minami, S. Carbohydr. Polym. 2003, 53, 337-342. (5) Tsujita, T.; Takaichi, H.; Takaku, T.; Sawai, T.; Yoshida, N.; Hiraki, J. J. Lipid Res. 2007, 48, 358-365. (6) Casal, E.; Montilla, A.; Moreno, F. J.; Olano, A.; Corzo, N. J. Dairy Sci. 2006, 89, 1384-1389. (7) Kontopidis, G.; Holt, C.; Sawyer, L. J. Dairy Sci. 2004, 87, 785-796.

(8) Mertins, O.; Cardoso, M. B.; Pohlmann, A. R.; Silveira, N. P. J. Nanosci. Nanotechnol. 2006, 6, 2425-2431. (9) Pavinatto, F. J.; Dos Santos, D. S., Jr.; Oliveira, O. N., Jr. Polı´m: Cieˆ nc. Tecnol. 2005, 15, 91-94. (10) Pavinatto, F. J.; Pavinatto, A.; Caseli, L.; Dos Santos, D. S., Jr.; Nobre, T. M.; Zaniquelli, M. E. D.; Oliveira, O. N., Jr. Biomacromolecules 2007, 8, 1633-1640. (11) Pavinatto, F. J.; Caseli, L.; Pavinatto, A.; Dos Santos, D. S., Jr.; Nobre, T. M.; Zaniquelli, M. E. D.; Silva, H. S.; Miranda, P. M.; Oliveira, O. N., Jr. Langmuir 2007, 23, 7666-7671. (12) Brezesinski, G.; Mohwald, H. AdV. Colloid Interface Sci. 2003, 1001002, 563-584. (13) Pott, T.; Maillet, J. C.; Dufourc, E. J. Biophys. J. 1995, 69, 1897-1908.

Experimental Details

10.1021/la7038762 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/27/2008

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Figure 2. Change in surface pressure (πf - πi) vs initial surface pressure for BLG (0.25 mg mL-1) adsorbing at a DMPA monolayer.

Figure 1. Kinetics of adsorption for BLG (0.25 mg mL-1) at a clean air-water interface (A) and at a DMPA monolayer with an initial surface pressure, πi, of ca. 12 mN m-1 (B). cholesterol from Sigma were dissolved in HPLC-grade chloroform to render a 0.5-1.0 mg mL-1 solution. Chitosan was obtained from Galena Quı´mica e Farmaceˆutica Ltda (Brazil), with a degree of acetylation of 22% as determined using H1 NMR according to the method in ref 14. Chitosan was purified by dissolution in diluted HCl (0.1%), followed by filtration and precipitation in a NaOH aqueous solution and washing with isopropanol and water until neutrality was achieved. A molecular weight, Mn, of 108 700 Da and a polydispersity index of 6.2 were determined by size exclusion chromatography (SEC, Shimadzu) using Shodex Ohpak SB-G, SB803-HQ, and SB-805-HQ columns, 0.3 M acetic acid/0.2 M sodium acetate as the eluent (flow of 0.8 mL min-1), a temperature of 35 °C, and Pullulan and glucosamine standards. The adsorption kinetics curves were obtained from a Kibron tensiometer (microtrough X), and surface pressure-area isotherms were obtained and the fabrication of LB films was performed in a mini-KSV Langmuir trough. The trough and the tensiometer are equipped with a surface-pressure sensor (Wilhelmy method) and housed in a class 10 000 clean room. Aliquots of lipid solution were spread on an aqueous subphase containing Theorell-Stenhagen buffer (NaOH, citric acid, boric acid, and phosphoric acid, whose pH was adjusted to 3.0 with addition of 2 M HCl). Water for preparing the buffer solution was supplied by a Milli-RO coupled to a Milli-Q purification system from Millipore and had a resistivity of 18.2 MΩ cm at pH ∼6. The chitosan samples were dissolved in the buffer mentioned, using a concentration of 0.20 mg mL-1 and employed as a subphase for lipid monolayers. The ionic strength was kept at 0.03 M, at which chitosan is believed to adopt a random coil conformation.15 For the adsorption kinetics studies, the well of the tensiometer was filled with 500 µL of chitosan solution (0.20 mg mL-1), and aliquots of 10 µL of BLG solution were injected into the bottom of the well. The change in surface pressure was then monitored with time. For adsorption kinetics with a lipid monolayer (14) Signini, R.; Campana, S. P. Polym. Bull. 1999, 42, 159-166. (15) Tsaih, M. L.; Chen, R. H. J. Appl. Polym. Sci. 1999, 73, 2041-2050.

at the air-water interface, aliquots of DMPA or cholesterol were spread on a 500 µL chitosan solution to render the desired initial surface pressure. After a constant surface pressure had been reached (equilibrium), aliquots of 10 µL of BLG solution were injected into the subphase, and the changes in surface pressure were followed with time. Control experiments were also performed with BLG injected under a DMPA monolayer spread on a Theorell-Stenhagen buffer with no chitosan. Surface pressure-area isotherms for the monolayers were obtained with compression using movable barriers with a decrease in area of 5 Å2 molecule-1 min-1. A Langmuir trough with a total capacity of 40 mL was filled with chitosan solution (0.20 mg mL-1), and then DMPA was spread on the air-water interface to render an initial area per lipid molecule of 100 Å2. Polarization-modulation infrared reflection absorption spectroscopy was performed using a KSV PMI 550 instrument (KSV Instruments Ltd, Helsinki, Finland). The Langmuir trough is set up so that the light beam reaches the monolayer at a fixed angle of incidence of 80°. The incoming light is continuously modulated between s and p polarization at a high frequency. This allows the simultaneous measurement of spectra for the two polarizations, with the difference providing surface-specific information and the sum providing the reference spectrum. Because the spectra are measured simultaneously, the effect of water vapor is largely reduced. The dynamic elasticity of DMPA, chitosan-DMPA, and mixed chitosan-BLG DMPA monolayers was studied with the axisymmetric shape drop analysis method (OCA-20 from Dataphysics Instruments GmbH, Germany) with oscillating drop accessory ODG20 as described in the literature.16,17 A solution of ca. 10-4 M DMPA was gently touched to the surface of a reduced-size drop, which was formed with the buffer solution, with or without chitosan (0.20 mg mL-1). The drop was then rapidly expanded up to a predetermined drop area to yield the desired surface pressure. For mixed chitosanBLG subphases, the kinetics of adsorption was taken immediately and 1 h after preparing the solution. After waiting the desired time for diffusion-adsorption-desorption processes, the dynamic surface elasticity data were obtained by using periodic drop oscillation with an amplitude of 0.1 mm (relative area variation ∆A/A of 5.5%) and frequency of 1.0 Hz. The viscous effect on the surface elasticity (i.e., the imaginary part of the elasticity modulus) was estimated from the phase angle. Dynamic light scattering measurements were obtained with a Zetasizer 300 HAS device from Malvern Inc. using a HeNe laser source at 633 nm with 10 mW power. Measurements were made using an angle of 90° with temperature control. The transfer of the monolayers onto different solid supports (see below) was done at a surface pressure of 30 mN m-1 by allowing the solid substrate to emerge perpendicularly to the interface at a dip rate of 3 mm min-1. The formation of LB films was confirmed first by analyzing the transfer ratio and later by fluorescence (16) Caseli, L.; Masui, D. C.; Furriel, R. P. M.; Leone, F. A.; Zaniquelli, M. E. D. J. Braz. Chem. Soc. 2005, 16, 969-977. (17) Nobre, T. M.; Wong, K.; Zaniquelli, M. E. D. J. Colloid Interface Sci. 2007, 305, 142-149.

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Figure 4. Change in surface pressure of a DMPA monolayer caused by the injection of BLG (0.25 mg mL-1) into a chitosan-containing subphase (0.20 mg mL-1). πmax is the maximum surface pressure corresponding to the maximum in Figure 3. πf is the equilibrium surface pressure.

Figure 3. Adsorption kinetics for BLG (0.25 mg mL-1) injected into a chitosan solution (0.20 mg mL-1) with a clear air-water interface (A) and with a DMPA monolayer (B). spectroscopy (Shimadzu FR-5301PC) and nanogravimetry using a quartz crystal microbalance (QCM, Stanford Research Systems Inc.). The solid supports used were optical glass for fluorescence measurements, whereas for QCM nanogravimetry we employed an AT-cut quartz crystal coated with Au (Stanford Research Systems Inc, fundamental frequency of ca. 5 MHz). All of the experiments were performed at 23 ( 1 °C. The final concentration of the protein in all experiments was calculated to be 0.25 mg mL-1. All experiments were carried out at least three times.

Results and Discussion Adsorption at the Air-Water Interface. Figure 1A shows the adsorption kinetics for BLG inserted into a buffer solution without chitosan onto a bare air-water interface. After an induction time of ca. 50 min, which can be ascribed to a diffusive process, the surface pressure increased to 17 to 18 mN m-1, which is an equilibrium value that was reached approximately 4 h after the protein injection. The surface activity of BLG has already been reported in the literature in several concentrations. Trofimova et al.18 reported a surface pressure of 20-25 mN m-1 30 h after spreading for a BLG concentration of 0.035 mg mL-1. Ferna´ndez et al.19 found a surface pressure of 25.9 mN m-1 for a protein concentration of 5.10-5 mg mL-1. Baeza et al.20 reported that BLG adsorbs at a pendant drop surface causing a 15 mN m-1 increase in surface pressure after 17 min for a protein concentration of 1 mg mL-1. For the same concentration, Ganzevles et al.21 measured (18) Trofimova, D.; De Jongh, H. J. Langmuir 2004, 20, 5544-5552. (19) Ferna´ndez, M. C.; Sa´nchez, C. C.; Nino, R. R.; Patino, J. M. R. Langmuir 2007, 23, 7178-7188. (20) Baeza, R.; Pilosof, A. M. R.; Sanchez, C. C.; Patino, J. M. R. AIChE J. 2006, 52, 2627-2638. (21) Ganzevles, R. A.; Zinoviadou, K.; Vliet, T. V.; Stuart, M. A. C.; de Jongh, H. H. J. Langmuir 2006, 22, 10089-10096.

a surface pressure of 25 mN m-1. Bos and Nylander22 showed a saturation effect for a BLG concentration of >6 mg mL-1 with a maximum surface pressure of 20 mN m-1. Miano et al.23 obtained a surface tension versus BLG concentration curve in which a critical concentration was reached at 10-6 M (∼0.0184 mg mL-1). Gauthier et al.24 measured a surface pressure of 19 mN m-1 after 1.5 h for a BLG concentration of 0.1 mg mL-1. The differences among the results mentioned above can be attributed not only to the use of distinct BLG concentrations but also to different methods and conditions under which the measurements were performed. The surface pressures depend on the diffusion of macromolecules through the buffer solution, the area-volume ratio of the “recipients” used (varying from large Langmuir troughs to small pendant drops), and conditions such as pH, ionic strength, and temperature. Nevertheless, in all cases the adsorption of BLG led to surface pressures between 15 and 25 mN m-1, which agree with the values measured here. Injecting BLG under a preformed DMPA monolayer (Figure 1B) leads to a rapid increase in surface pressure, without induction time, with equilibrium reached in ca. 1 h. The surface pressure increased from 11.8 mN m-1, the initial surface pressure due to the DMPA monolayer that had already formed, to approximately 15.4 mN m-1 owing to BLG incorporation from the subphase. These data indicate a clear affinity of the protein for the phospholipids, which explains its rapid adsorption. Also, the isoelectric point of BLG is 5.1,25 from which we infer that the adsorption was performed with the protein carrying a net positive charge. Because DMPA has a negatively charged polar head, electrostatic attractions may drive the protein-lipid interaction. The adsorption of proteins onto lipid monolayers normally depends on the initial surface pressure (πi) (i.e, the surface packing). The increase in surface pressure (πf - πi) due to BLG incorporation in a DMPA monolayer with several πi values is shown in Figure 2. As usually observed for polypeptides,26 the higher the initial surface packing of the phospholipids, the lower the surface pressure increase. The extrapolation of the straight line to a zero surface pressure change yields the so-called exclusion surface pressure.27,28 At this exclusion pressure, the protein can (22) Bos, M. A.; Nylander, T. Langmuir 1996, 12, 2791-2797. (23) Miano, F.; Calcara, M.; Millan, T. J.; Enea, V. Colloids Surf., B 2005, 44, 49-55. (24) Gauthier, F.; Bouhallab, S.; Rinault, A. Colloids Surf., B 2001, 21, 3745. (25) Papiz, M. Z.; Sawyer, L.; Eliopoulos, E. E.; North, A. C. T.; Findlav, J. B.C.; Sivaprasadarao, R.; Jones, T. A.; Newcomer, M. E.; Kraulis, P. J. Nature 1986, 324, 383-385. (26) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109-140.

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Figure 5. Adsorption of BLG (0.25 mg mL-1) at a cholesterol (A), DPPC (B), or DPPG (C) monolayer under a chitosan solution subphase (0.20 mg mL-1).

no longer be incorporated at the air/water interface in a way that contributes to the surface pressure. For BLG, Figure 2 shows an exclusion surface pressure of ca. 30 mN m-1. When BLG is injected into a subphase containing chitosan, the adsorption properties change. Under the conditions used here, chitosan is not surface-active10 (i.e., it does not cause a significant increase in surface pressure). However, in the presence of a DMPA monolayer, chitosan causes the surface pressure to increase,11 which indicates the affinity between the polysaccharide and the phospholipids. For BLG injected into a chitosan solution (without a lipid monolayer at the interface), the surface pressure increases sharply as shown in Figure 3A. After a kink point of 3.5 mN m-1, the surface pressure increases more smoothly until equilibrium is reached in less than 2 h. The absence of an induction time differs from the result obtained for BLG in pure buffer (Figure 1A), thus pointing to an effect from chitosan. The lower surface excess, with an equilibrium surface pressure of ca. 6 mN m-1 when compared to the kinetics for the pure protein, can be explained by the formation of chitosan-protein complexes in solution, which has been already reported by Gu¨zey et al.29 The kink around 3.5 mN m-1 can be ascribed to changes in conformation and/or orientation of protein-polysaccharide complexes adsorbed at the interface. Therefore, the adsorption process may be divided into two steps: an initial one assigned to the rapid adsorption of complexes at the interface, adsorbing faster than BLG alone, and a second, slower process with the adsorption of nonassociated BLG molecules. The faster adsorption of BLG in the presence of chitosan or DMPA when compared with that of BLG alone (Figure 1A) suggests that it may be due to the incorporation of BLG onto the interface. (27) Ronzon, F.; Desbat, B.; Chauvet, J. P.; Roux, B. Colloids Surf., B 2002, 23, 365-373. (28) Caseli, L.; Masui, D. C.; Furriel, R. P. M.; Leone, F. A.; Zaniquelli, M. E. D. Colloids Surf., B 2005, 46, 248-254. (29) Gu¨zey, D.; McClements, D. J. Food Biophys. 2006, 1, 30-40.

The presence of soluble complexes was confirmed in subsidiary experiments of dynamic light scattering measurements with an angle of 90° for BLG-chitosan solutions, with 190 nm aggregates being observed for chitosan-BLG solutions 3 h after preparation. Interestingly, for BLG injected into a chitosan solution with a DMPA monolayer at the air-water interface, unusual behavior is observed in Figure 3B. The initial surface pressure of 21.5 mN m-1 represents a DMPA monolayer on the chitosan solution subphase in equilibrium. After the injection of BLG (time ) 0), the pressure increases rapidly to ca. 22.5 mN m-1, probably owing to the adsorption of protein or protein-chitosan aggregates. Then, the surface pressure begins to decrease and reaches values even lower than the initial one. After 60 min, equilibrium is apparently reached, with a surface pressure of 19.5 mN m-1. This reduction can be attributed to the possible formation of BLG-chitosan complexes at the interface, which can be dissolved into the subphase. As already mentioned, the formation of soluble chitosan-BLG complexes is well established in the literature29,30 and has been confirmed here with light scattering experiments. Hence, the result in Figure 3B is clear evidence that chitosan is able to remove the protein from the monolayer. Because the equilibrium surface pressure is lower than the initial surface pressure, probably some lipid molecules were also dragged with the BLG-chitosan complexes. In subsidiary experiments, we confirmed that this removal occurs for other initial surface pressures (in the range of 6-30 mN m-1). Figure 4 shows that the increase in pressure, πmax πi, of DMPA on a chitosan-containing subphase caused by the injection of BLG, in the early stages, decreases with the initial surface pressure. In contrast, the decrease in pressure, πi - πf, due to BLG and even DMPA desorption increases with the initial surface pressure. (30) Chen, L.; Subirade, M. Biomaterials 2005, 26, 6041-6053.

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Figure 7. Surface pressure-area isotherms for DMPA on different subphases and for different conditions (displayed in the inset for panels A and B). Immed. (immediately), after 1 h, and after 4 h refer to the time elapsed before monolayer compression after BLG injection in a chitosan solution subphase (0.20 mg mL-1). The final concentration of BLG was 0.25 mg mL-1. Figure 6. PM-IRRAS spectra for BLG (0.25 mg mL-1) adsorbing onto DMPA monolayers (initial surface pressure of 20 mN m-1) without (A) or with 0.2 mg mL-1 chitosan (B) in the subphase several times after BLG injection (A) and chitosan injection (B).

To determine if this effect can be extended to other lipids, we performed similar experiments with cholesterol, DPPC, and DPPG instead of DMPA. Figure 5A,B shows that when BLG is inserted into the chitosan solution under a preformed cholesterol or DPPC monolayer, its adsorption leads to an equilibrium plateau, with no reduction in surface pressure. In contrast, with the negatively charged DPPG (Figure 5C), the surface pressure decreased from 19 to ca. 17 mN m-1. Therefore, the removal of chitosan-BLG complexes from the lipid interface occurs only with negatively charged lipids, highlighting the importance of electrostatic interactions. Moreover, the removal effect was observed for BLG but not for the proteins horseradish peroxidase (HRP) and urease (results not shown). All three proteins are amphiphilic and are surface-active, but they differ in molecular weight and isoelectric point. The molecular weights are approximately 480 000 for urease, 44 000 for HRP, and 18 300 for BLG, and the isoelectric points are 5.1 for urease, 6.9 for HRP, and 5.1 for BLG. Figure 6 shows the PM-IRRAS spectra for BLG adsorbing on a DMPA monolayer. Peaks for amide I (1650-1660 cm-1), assigned to CdO stretching, and amide II (1540-1560 cm-1), assigned to NH bending, are typical of polypeptides.31 Figure 6B clearly shows the presence of amide bands due to BLG adsorption at DMPA monolayers. Amide groups with opposite signs to those for the CH2 scissor mode of DMPA (2850-2950 cm-1, inset) indicate that the amide CdO and NH groups are oriented (31) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712-719.

preferentially perpendicular to the membrane surface and parallel to the relatively well ordered acyl chains. Zhang et al.32 found similar results with BLG interacting with liposomes (i.e., CH2 and amide bands opposite to each other) and suggested that the polypeptide R-helix axis is inserted into the lipid monolayer. Upon introducing chitosan into the subphase, the amide peaks started to change, and 20 min after insertion, the amide II band shifted from 1566 to 1555 cm-1 and the amide I band shifted from 1654 to 1664 cm-1. These shifts point to changes in BLG conformation. Also observed were positive peaks at 1526 cm-1 and at 1575-1600 cm-1 due to NH2 from chitosan, indicating the co-adsorption of chitosan at the DMPA monolayer. After 240 min, amide and amine bands disappeared, suggesting the removal of BLG by chitosan. Figure 7 shows the characterization of DMPA monolayers using surface pressure-area isotherms under several conditions. Both chitosan and BLG cause the expansion of DMPA monolayers when they are alone in the subphase (Figure 7A). With BLG, the effect is more pronounced, resulting in a monolayer with a high compressibility or low compressional modulus, Cs-1, defined as -A(dπ/dA), where A is the molecular area.33 For instance, at 30 mN m-1, Cs-1 is 189 mN m-1 for pure DMPA, 85 mN m-1 for mixed chitosan-DMPA, and 39 mN m-1 for mixed BLGDMPA monolayers. Also, there is total suppression of the liquidexpanded to liquid-condensed phase transition for BLG-DMPA monolayers. For pure DMPA, the transition occurs at a surface pressure of ca. 3.5 mN/m. In all cases, the collapse occurs at ca. 37 Å2, which can indicate the expulsion of the macromolecules (32) Zhang, X.; Ge, N.; Keiderling, T. A. Biochemistry 2007, 46, 5252-5260. (33) Davies, J. T.; Rideal, E. K. Interfacial Phenomena, 2nd ed; Academic Press: New York, 1963.

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Table 1. Dynamic Surface Viscoelastic Properties for a Drop Surface Containing BLG (0.25 mg mL-1), Chitosan (0.20 mg mL-1), and/or DMPA (Surface Pressure of 30 mN m-1) material (s)

BLG

BLG + Chi

DMPA

DMPA + BLG

DMPA + Chi

DMPA + BLG + Chi (10 min)

DMPA + BLG + Chi (4 h)

E (mN m-1) Ei (mN m-1)

18.0 0.5

6.3 1.0

79.4 2.5

334.4 9.9

68.3 5.1

269.3 13.5

112.4 2.7

Table 2. Nanogravimetry Data for DMPA LB Films Transferred from Monolayers at the Air-Water Interface monolayer

DMPA

DMPA + Chi

DMPA + Chi + BLG (immed.)

DMPA + Chi + BLG (1 h)

DMPA + Chi + BLG (4 h)

transfer ratio deposited mass (ng)

0.98 109.5

1.05 258.2

0.95 324.0

1.01 405.1

0.98 259.9

from the interface to the subphase or to the subsurface below the phospholipid polar heads. In Figure 7B, chitosan solution (0.20 mg mL-1) was used to fill the trough and then DMPA was spread on the air-water interface, and 2 h elapsed to guarantee chitosan adsorption.11 Then aliquots of BLG were injected into the subphase, and certain periods of time elapsed before compression was performed. When the monolayer was immediately compressed, there was overlap with the isotherm for the DMPA-chitosan monolayer, indicating insufficient time for the protein to incorporate into the lipid monolayer. After 1 h, there is a large expansion of the monolayer due to the adsorption of BLG. After 4 h, the complexation of chitosan with BLG leads to the condensation of the monolayer, indicating the removal of some material from the interface. It is important to note that this kind of experiment differs from those of adsorption kinetics (Figure 3). For the surface pressure-area isotherms, the volume of the trough (∼40 mL) used is larger than that of the wells (∼0.5 mL) used for adsorption kinetics experiments. Long diffusive processes can occur; therefore, the periods of time involved are longer when performing the compression isotherm. Dynamic Measurements. When protein molecules adsorb onto monolayers, the rheological properties of the latter are affected, making it difficult to investigate the direct influence of the protein. A possible alternative is the use of dynamic measurements, such as harmonic oscillation and the axisymmetric drop analysis method, that have already proven suitable in monolayer studies.10,11,34,35 Of particular relevance is how BLG and chitosan can affect the elasticity parameters of DMPA monolayers. Table 1 shows the dilatational elasticity (E) for the interfaces studied in this article. These values are analogous to the compressional modulus (Cs-1) obtained from surface pressure-area isotherms for monolayers in a Langmuir trough. Cs-1 and E (in the drop) can be correlated,16 but the phenomena in the two measurements are different. The Cs-1 measurement is obtained under equilibrium conditions, but E is a nonequilibrium property involving dynamic processes. When the interface is not purely elastic, an imaginary part of the elasticity (Ei) must be considered, which is usually related to viscous effects associated with dissipative and diffusive processes and surface reactions at the interface.36 (See ref 36 for a discussion of controversies about this issue.) For the Gibbs monolayer formed with BLG adsorbing at the interface from the aqueous solution, E is low, probably as a result of the flexibility of the biomacromolecule. Wang et al.37 (34) Cornec, M.; Narsimham, G. Langmuir 2000, 16, 1216-1225. (35) Caseli, L.; Moraes, M. L.; Zucolotto, V.; Ferreira, M.; Nobre, T. M.; Zaniquelli, M. E. D.; Rodrigues Filho, U. P.; Oliveira, O. N., Jr. Langmuir 2006, 22, 8501-8508. (36) Ivanov, I. B.; Danov, K. D.; Ananthapadmanabhan, K. P.; Lips, A. AdV. Colloid Interface Sci. 2005, 114, 61-92. (37) Wang, Z.; Narsimhan, G Langmuir 2005, 21, 4482-4489.

reported an E of 20 mN m-1 for 0.5 wt % BLG for frequencies >1 Hz. The elasticity of protein-containing surfaces can be modified by polymers.20 Upon the introduction of chitosan, the elasticity was reduced as the system became less surface-active with the formation of chitosan-BLG complexes. For DMPA, E is relatively large (ca. 80 mN m-1) owing to the formation of an insoluble compact monolayer at the interface. This elasticity decreases by more than 10 mN m-1 with chitosan in the subphase, which induces some flexibility in the monolayer. However, for the BLG-DMPA system, E is greater than 300 mN m-1. This result is in contrast to the decrease in the compressional modulus caused by BLG under equilibrium conditions (surface pressurearea isotherm: Figure 6A): in the oscillating drop, we observe an elasticity enhancement caused by the incorporation of protein molecules in the phospholipid monolayer. Because surface elasticity in the drop is a nonequilibrium phenomenon, dynamic processes involving BLG penetration in the first steps of adsorption may be undetected in the surface pressure-area curves. Moreover, for a monolayer in a Langmuir trough, the adsorption of BLG occurs at a low state of packing (0 mN m-1), and a surface pressure of 30 mN m-1 is attained by compression. However, in the oscillating drop, DMPA is spread on a BLG-containing interface after the formation of a BLG solution drop. Expanding and compressing the drop will form dynamically new interfaces, and processes of adsorption/ desorption of BLG may occur. The high dynamic surface elasticity value for mixed BLG-DMPA monolayers may be explained by the rapid adsorption of BLG to the interface in a high state of packing, leading to a piston-like effect, as already observed for other systems involving proteins being adsorbed at phospholipid monolayers.28,35 During the expansion/compression process of the drop, with the DMPA monolayer on the air-water interface, the protein penetrates rapidly, thus compressing the monolayer and leading to larger changes in surface pressure. Because elasticity is defined as dγ/d ln A, a sudden compression of the DMPA monolayer because of the fast penetration of the protein yields a higher surface elasticity. Because the compressional modulus is smaller for equilibrium measurements (surface pressure-area isotherms), it is possible that after BLG penetration a rearrangement of protein molecules at the monolayer occurs because of relaxation. This confirms the high affinity of BLG for DMPA interfaces, even at high surfaces pressures. Elasticity values for DMPA monolayers in the presence of chitosan-BLG complexes decrease when compared to those of DMPA-BLG interfaces. The longer the time waited after drop formation, the lower the elasticity: after 1 h, E is less than half the value measured 10 min after DMPA spreading. The formation of chitosan-protein complexes is again the key factor because the interaction between chitosan and BLG hampers the protein’s ability to be incorporated in the phospholipid monolayer.

4156 Langmuir, Vol. 24, No. 8, 2008

Figure 8. Fluorescence spectra of DMPA LB films transferred from monolayers at the air-water interface.

Caseli et al.

can be roughly attributed to the mass of chitosan deposited. The ability of chitosan to be transferred together with DMPA has already been characterized by sum-frequency spectroscopy, atomic force microscopy, and infrared spectroscopy.11 When the DMPA monolayer formed on the chitosan-BLG subphase was transferred to solid supports, a mass of 324.0 ng could be obtained, thus pointing to some BLG being also transferred. After 1 h, a higher amount of BLG could be transferred, which was attributed to a diffusive process. However, after 4 h a smaller mass was measured, which corroborates the surface pressure-area isotherms in Figure 6 in that 4 h is a sufficient time for chitosan to remove BLG adsorbed on DMPA monolayers. Figure 8 shows the fluorescence spectra for LB films, in which we observe a fluorescence emission peak at 330 nm (with excitation at 295 nm) owing to the excitation of tryptophan groups of BLG. Fluorescence spectroscopy is useful in identifying the intrinsic fluorescence of tryptophanyl residues and is particularly sensitive to the polarity of microenvironments along the transition.40 BLG is reported to maintain its native structure and is not fully unfolded at the interface, even in contact with phospholipids.37 The spectra for BLG-containing LB films confirm the presence of BLG if transfer was made immediately and after 1 h of BLG spreading. After 4 h, the peak disappears, again indicating the removal of the protein from the lipid interface.

Conclusions

Figure 9. Model for chitosan action removing BLG from phospholipid monolayers.

The imaginary contribution for dilatational elasticity (Ei) is usually insignificant compared to E. This indicates almost purely elastic behavior. Higher values are found when BLG-chitosan is involved, indicating some viscous or diffusive effect. LB Films. To investigate further the ability of chitosan to remove BLG from DMPA monolayers, we transferred the films from the liquid interface onto solid supports using the LangmuirBlodgett (LB) technique. We performed deposition at a surface pressure of 30 mN m-1 and measured the mass using a quartz crystal microbalance. Table 2 shows a transfer ratio of approximately 1.0 for all cases, which confirms the quality of deposition. The mass deposited of a single layer of DMPA is 109.5 ng, consistent with published values.11,38,39 The mass measured for a mixed chitosan-DMPA LB film was 258.2 ng. The difference (38) Caseli, L.; Zaniquelli, M. E. D.; Furriel, R. P. M.; Leone, F. A. Colloids Surf., B 2002, 25, 119-128. (39) Caseli, L.; Furriel, R. P. M.; De Andrade, J. F.; Leone, F. A.; Zaniquelli, M. E. D. J. Colloid Interface Sci. 2004, 275, 123-130. (40) Viseu, M. I.; Carvalho, T. I.; Costa, S. M. B. Biophys. J. 2004, 86, 23922402.

Upon combining the results from kinetic adsorption curves, surface pressure-area isotherms, infrared spectroscopy, quartz crystal microbalance measurements, and fluorescence spectra, we could draw the following conclusions: (1) BLG and chitosan can diffuse from the subphase onto DMPA monolayers. (2) If chitosan is in the subphase, then BLG may still adsorb onto DMPA but then is removed from the interface. This removal also occurred for DPPG monolayers but not for monolayers of cholesterol and DPPC. Therefore, electrostatic interactions are crucial to the removal mechanism. There is also some specificity in the interaction because no removal occurred for other proteins tested in interaction with DMPA. (3) Surface pressure-area isotherms indicated that chitosanBLG complexes condense DMPA monolayers after a longer diffusion time. The removal of BLG from model membrane systems induced by chitosan may be depicted as in Figure 9. Chitosan complexes with BLG and even drags lipid molecules from a stable monolayer. To the best of our knowledge, this is the first molecular-level evidence of the possible protein-removal action of chitosan, which may be involved in its various biological applications. Acknowledgment. This work was supported by FAPESP, IMMP, FINEP, CNPq, and Rede Biomat (Brazil). We thank KSV Instruments Ltd for the use of the PM-IRRAS instrument. LA7038762