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Moreover, before FB is displaced by DLPC at the interface, there is a substantial initial enhancement in the FB adsorption, consistent with some inter...
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Langmuir 2005, 21, 10140-10147

Displacement of Fibrinogen from the Air/Aqueous Interface by Dilauroylphosphatidylcholine Lipid† Tze-Lee Phang, Scott J. McClellan, and Elias I. Franses* School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette, Indiana 47907-2100 Received February 18, 2005. In Final Form: May 11, 2005 Fibrinogen (FB) and other serum proteins leak into the aqueous alveolar lining layer due to lung injuries. The adsorption of these serum proteins at the air/aqueous interface can produce higher surface tensions than the pulmonary lipids, and acute respiratory distress syndrome (ARDS) can ensue. By having a molecular adsorption mechanism, as compared to a particulate adsorption mechanism of other longer chain lipids, dilauroylphosphatidylcholine (DLPC) lipid can expel FB from the air/aqueous interface at 25 °C, in water or in phosphate-buffered saline, as proven by tensiometry (also at 37 °C), ellipsometry, and infrared reflection-absorption spectroscopy. Moreover, before FB is displaced by DLPC at the interface, there is a substantial initial enhancement in the FB adsorption, consistent with some interaction or binding of DLPC with FB to produce a more hydrophobic protein surface. After the FB molecules have been displaced by DLPC, or when DLPC has already adsorbed at the interface, FB molecules are less favored to adsorb near the DLPC monolayer with the lecithin headgroups facing toward them. The results have implications for possible uses of DLPC lipid in potential lung surfactant formulations in treating patients with ARDS.

1. Introduction Displacing an already adsorbed fibrinogen (FB) layer and reducing the adsorption of FB at various surfaces are important in many applications. These include lung surfactant formulations1 and biomaterial applications.2,3 FB is a serum protein with a molecular weight of 340 kDa and dimensions of 5 × 5 × 46 nm.4 Leakage of serum proteins into the alveolar aqueous lining layer due to lung injuries can degrade and inhibit the function of lung surfactant by preventing the adsorption of dipalmitoylphosphatidylcholine (DPPC), the major lung surfactant lipid, and hence preventing low surface tensions.1,5-8 High surface tensions caused by serum proteins, or by removal of some lung surfactants by excess fluid accumulation, cause the lung alveoli to collapse and subsequently leads to difficulty in breathing, as observed in the patients with acute respiratory distress syndrome (ARDS), a life threatening disease with high mortality.1 Many patients with severe acute respiratory syndrome (SARS) also develop ARDS.9-12 Mechanical ventilation, which has been used for treating ARDS, is not effective.13 Moreover, the surfactant formulations used for treating †

Part of the “Bob Rowell Festschrift” special issue. * To whom correspondence should be addressed. Tel: (765)4944078. Fax: (765)494-0805. (1) Notter, R. H. Lung Surfactants; Marcel Dekker: New York, 2000. (2) Okazaki, Y.; Tateishi, T.; Ito, Y. Mater. Trans. 1997, 38, 78. (3) Ratner, B. D. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: Toronto, 1996; p 1. (4) Sober, H. A. CRC Handbook of Biochemistry Selected Data for Molecular Biology; The Chemical Rubber Co.: Ohio, 1968; p 53. (5) Holm, B. A.; Notter, R. H. In Lung Surfactant Treatment of Lung Diseases; Ross Laboratories: Ohio, 1988. (6) Cheng, C.-C.; Chang, C.-H. Langmuir 2000, 16, 437. (7) Wen, X.; Franses, E. I. Colloids Surf. A 2001, 190, 319. (8) Liu, Y.-L.; Chang, C.-H. Colloid Polym. Sci. 2002, 280, 683. (9) Rubenfeld, G. D. J. Am. Med. Assoc. 2003, 290, 397. (10) Joynt, G. M.; Antonio, G. E.; Lam, P.; Wong, K. T.; Li, T.; Gomersall, C. D.; Ahuja, A. T. Radiology 2004, 230, 339. (11) Lew, T. W. K.; Kwek, T. K.; Tai, D.; Earnest, A.; Loo, S.; Singh, K.; Kwan, K. M.; Chan, Y.; Yim, C. F.; Bek, S. L.; Kor, A. C.; Yap, W. S.; Chelliah, Y. R.; Lai, Y. C.; Goh, S. K. J. Am. Med. Assoc. 2003, 290, 374.

infants with respiratory distress syndrome (RDS) have not been shown to work with ARDS patients. Hence, new lung surfactant formulations are needed to treat ARDS. The newly developed Surfaxin drug by Discovery Laboratories, which is in clinical trials for possible treatments of ARDS, contains various lipids and an SP-B protein analogue and is targeted for ARDS.14 From previous studies, we discovered that the inhibition of DPPC by serum proteins is due to the essentially zero aqueous DPPC solubility.7 This implies that the DPPC particles, liposomes or vesicles, adsorb at the air/water interface via a particulate mechanism, in which liposomal or vesicular particles reach the interface, unfold or “unzip”, and release their molecules while remaining attached to the surface.15 This mechanism is easily disrupted by fast adsorbing serum proteins, which, if present, compete with lung surfactants for the surface and adsorb during area expansion. When the surface is covered by serum proteins, the DPPC particles cannot reach the surface, and hence they cannot release their molecules to generate low surface tensions.7 This discovery leads to the idea of suggesting the use of dilauroylphosphatidylcholine (DLPC), a homologue of DPPC with a shorter chain length and a finite aqueous solubility of ∼4 ppm in water, as a possible lung surfactant replacement lipid. DLPC adsorbs to interface via a molecular adsorption mechanism.16,17 It can generate low surface tensions even in the presence of serum albumin.18 DLPC has a low toxicity, as shown in animal tests.19 Moreover, DLPC liposomes were used as carriers (12) Peiris, J. S. M.; Chu, C. M.; Cheng, V. C. C.; Chan, K. S.; Hung, I. F. N.; Poon, L. L. M.; Law, K. I.; Tang, B. S. F.; Hon, T. Y. W.; Chan, C. S.; Chan, K. H.; Ng, J. S. C.; Zheng, B. J.; Ng, W. L.; Lai, R. W. M.; Guan, Y.; Yuen, K. Y. Lancet 2003, 361, 1767. (13) Marshall, E. Science 2003, 300, 1225. (14) Wiswell, T. E.; Smith, R. M.; Katz, L. B.; Mastroianni, L.; Wong, D. Y.; Willms, D.; Heard, S.; Wilson, M.; Hite, R. D.; Anzueto, A.; Revak, S. D.; Cochrane, C. G. Am. J. Resp. Crit. Care 1999, 160, 1188. (15) Wen, X.; Franses, E. I. Langmuir 2001, 17, 3194. (16) Phang, T.-L.; Liao, Y.-C.; Franses, E. I. Langmuir 2004, 20, 4004. (17) Pinazo, A.; Wen, X.; Liao, Y.-C.; Prosser, A. J.; Franses, E. I. Langmuir 2002, 18, 8888. (18) Phang, T.-L.; Franses, E. I. J. Colloid Interface Sci. 2004, 275, 477.

10.1021/la0504412 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/16/2005

Displacement of Fibrinogen

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of asthma drugs, inhaled by healthy adult volunteers.20-22 In this article, we show that DLPC can displace already adsorbed FB layers at the air/aqueous interface, and has the potential to be used therapeutically to remove FB and produce low dynamic surface tensions appropriate for treating RDS and ARDS. These findings have, therefore, implications for future lung surfactant formulations. 2. Experimental Section 2.1. Materials. Synthetic L-R-dilauroylphosphatidylcholine (DLPC, 99% pure) was purchased from Sigma Chemical Co. (St. Louis, MO). Fibrinogen (FB, type I-S: from bovine plasma) was purchased in powder form from Sigma Chemical Co. (St. Louis, MO). The powder contains 75 wt % FB, 15 wt % sodium chloride, and 10 wt % sodium citrate, used in the isolation and stabilization of the protein. Experiments were done with water or with aqueous protein or lipid in phosphate-buffered saline (“buffer”). Sodium chloride (NaCl) and sodium dihydrogen phosphate (NaH2PO4‚ H2O) were analytical reagent grade from Mallinckrodt Specialty Chemicals Co. (Paris, KY). Disodium hydrogen phosphate dodecahydrate (Na2HPO4‚12H2O) was purchased from Fluka Chemical Corp. (Milwaukee, WI). All materials were used as received. The lipid dispersions and protein solutions were prepared on a weight basis. The buffer solution with pH of 7.2 contained 150 mM of NaCl, 32 mM of NaH2PO4‚H2O, and 93 mM of Na2HPO4‚12H2O. All FB sample preparation and handling procedures were done in a hood, with a respirator and gloves. All experiments were completed within 8 h after FB solution preparations, except for a few transmission IR experiments in which FB solutions were “aged” for 1 week. The pure water used for all samples was first distilled and then passed through a Millipore four-stage cartridge system, resulting in a water resistivity of 18 MΩ cm at the exit port. 2.2. DLPC Dispersion Preparation and Injection Protocols. DLPC dispersions were shaken vigorously at room temperature, ∼25 °C, for about 2 min, decreasing the size of the liposome droplets. Then, dispersions were magnetically stirred for 30 min to provide a narrower range of liposome sizes, and possibly some vesicles. Finally, dispersions were sonicated using a sonicator bath (Branson 3510 Ultrasonic cleaner, Branson Ultrasonics Corporation, Danbury, CT) until they appeared translucent, further decreasing the size of the liposomes and producing mostly vesicles. The temperature of the samples prepared using this protocol was maintained in the range of 25-40 °C. In several experiments, protein was allowed to be adsorbed first, from aqueous solutions, for about 1 h. Then, a long-needle syringe was inserted into the solution from the air/water surface, and a concentrated lipid dispersion was injected slowly, to avoid perturbing the surface layer. Although no additional stirring was used, the turbulent flow during injection produced some mixing and a somewhat homogeneous dispersion. 2.3. Surface Tension Measurements. Dynamic surface tension (DST) of samples at constant area was measured with the Wilhelmy plate method. A platinum plate is connected to a KSV electrobalance (KSV Instruments, Finland). A sample solution is poured into a glass dish. The temperature of the solution can be controlled via an external circulator. The plate is lowered until the bottom edge of the plate touches the solution. The vertical force (F) resulting as the plate is pulled upward is due to the surface tension (γ)

F ) Pγ cos θ

(1)

where P is the wetted perimeter of the plate and θ is the contact angle. If θ is assumed to be zero (as done here), or if it is known independently, then γ can be determined. This method has a (19) Gilbert, B. E.; Seryshev, A.; Knight, V.; Brayton, C. Inhal. Toxicol. 2002, 14, 185. (20) Vidgren, M.; Waldrep, J. C.; Arppe, J.; Black, M.; Rodarte, J. A.; Cole, W.; Knight, V. Int. J. Pharm. 1995, 115, 209. (21) Waldrep, J. C.; Gilbert, B. E.; Knight, C. M.; Black, M. B.; Scherer, P. W.; Knight, V.; Eschenbacher, W. Chest 1997, 111, 316. (22) Saari, M.; Vidgren, M. T.; Koskinen, M. O.; Turjanmaa, M. H.; Nieminen, M. M. Int. J. Pharm. 1999, 181, 1.

“deadtime” of ∼1 min needed for the sample loading and for the sample flow to stop. The standard error for the measured DST values is (2 mN/m. 2.4. Ellipsometry. A Rudolph Research (now Rudolph Technologies, Flanders, NJ) Auto ELII automatic null ellipsometer was used for measuring the ellipsometric angles, ∆ and Ψ, which are defined in standard monographs,23,24 of adsorbed lipid and protein layers at the air/water interface. Measurements were taken at a wavelength (λ) of 633 nm with an incident angle (φo) of 70° measured from the surface normal. A Petri dish, filled with aqueous sample, was placed on the standard sample stage. The dynamic ellipsometry angles, ∆(t) and Ψ(t), were made at a given wavelength. Then ∆o and Ψo of the solvent (water or buffer) were subtracted from the measured ∆ and Ψ values of the samples to yield δ∆ ≡ ∆ - ∆o and δΨ ≡ Ψ - Ψo, which increase as the surface density (mg/m2) or the thickness of the adsorbed surface layer increases.25 To calculate the thickness (d1) and the refractive index (n1) of adsorbed layers at the air/water interface, δ∆ and δΨ (or ∆ and Ψ) are used along with standard ellipsometry theory,23,24 and a three-layer model consisting of air (0), isotropic adsorbed layer (1), and aqueous solution (2). The ellipsometric angles are related to the total reflection coefficients for p- and s-components of the amplitude beam wave, Rp and Rs

F)

Rp ) tan Ψei∆ ) tan Ψ(cos ∆ + i sin ∆) Rs

(2)

where

tan ∆ )

imag(Rp/Rs) real(Rp/Rs)

(3)

and

tan Ψ )

[ ( )]

Rp 1 real cos ∆ Rs

(4)

In general, the coefficients Rp and Rs are a function of the Fresnel reflection coefficients at the interface between air and the adsorbed layer, r01,p and r01,s, and those at the interface between the adsorbed layer and aqueous solution, r12,p and r12,s.23,24 These coefficients depend on λ, φο, n1, d1, no (the ambient air refractive index), and n2 (the aqueous solution refractive index). Given λ, φο, n1, d1, no, and n2, one can obtain Rp and Rs. From eqs 2-4, one gets the ∆ and Ψ values, or the “trajectory”.23,24 Examples of such trajectories for various d1 and n1 are shown later (see Figure 4 later). If the measured values of δ∆ and δΨ are large, then n1 and d1 can be calculated directly. The accuracy of calculating n1 is roughly (0.01 if d1 ranges from 20 to 60 nm. If the δΨ values are too small to be measured precisely, then n1 cannot be obtained with sufficient reliability. Thus n1 has to be assumed and d1 is estimated. In either case (of large or small δΨ values), the surface density Γ is calculated from the following expression:26

Γ)

(n1 - n2)d1 (dn/dc)

(5)

where dn/dc is the refractive index increment, normally measured by using differential refractometry. Literature values of dn/dc for FB protein range from 0.18 to 0.192 cm3/g.27-31 In this paper, dn/dc of 0.19 cm3/g is used. For DLPC, we determine the dn/dc (23) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1979. (24) Tompkins, H. G. A User’s Guide to Ellipsometry; Academic Press: New York, 1993. (25) Walsh, C. B.; Wen, X.; Franses, E. I. J. Colloid Interface Sci. 2001, 233, 295. (26) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (27) Malmsten, M. Interface Sci. 1997, 5, 159. (28) Bernocco, S.; Ferri, F.; Profumo, A.; Cuniberti, C.; Rocco, M. Biophys. J. 2000, 79, 561.

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Figure 1. Surface tension γ for 750 ppm FB solution for 1 h; then 1 mL of 30 000 ppm of DLPC dispersion was injected into the 30 mL of FB solution to yield a mixture of ∼730 ppm FB and 970 ppm DLPC: (0) in water at 25 °C; (4) in buffer at 25 °C; ([) in buffer at 37 °C.

Figure 2. Ellipsometric parameter |δ∆| at φo ) 70° and λ ) 633 nm: (0, ]) in water (AfBfCfDfEfFfG); (2, b) in buffer; (0, 2) 750 ppm FB in solution at the first 1 h; (], b) 1 mL of 30 000 ppm of DLPC dispersion was then injected into the 30 mL of FB solution to yield a mixture of ∼730 ppm FB and 970 ppm DLPC. The standard error is (0.05°. values by spreading a DLPC monolayer, on water or on buffer, with a surface density Γ of that of a close-packed monolayer. By measuring the ellipsometry angles (∆ and Ψ), we determine d1 and n1. Then, from eq 5 with n1 ) 1.46 for DLPC, we obtain dn/dc ) 0.12 cm3/g. This value is close to that of 0.119 cm3/g reported for DPPC.32 The value n1 of 1.46 is also close to that of 1.47 used in the literature for DPPC.33 The value of Γ is determined with lower uncertainty than that of n1 or d1, because as n1 increases d1 decreases.25 2.5. Fourier Transform Infrared Spectroscopy. All infrared spectra were taken using a Nicolet Prote´ge´ 460 Fourier transform infrared spectrometer equipped with a liquid nitrogen cooled mercury-cadmium-telluride (MCT) detector. The transmission spectra of aqueous FB solutions (4 wt %) were collected using a Specac Omni-Cell (Specac Inc., Woodstock, GA) with a (29) Ho¨o¨k, F.; Vo¨ro¨s, J.; Rodahl, M.; Kurrat, R.; Bo¨ni, P.; Ramsden. J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf. B 2002, 24, 155. (30) Armstrong, J.; Salacinski, H. J.; Mu, Q. S.; Seifalian, A. M.; Peel, L.; Freeman, N.; Holt, C. M.; Lu, J. R. J. Phys. Condensed Matter 2004, 16, S2483. (31) Vo¨ro¨s, J. Biophys. J. 2004, 87, 553. (32) Charron, J. R.; Tilton, R. D. J. Phys. Chem. 1996, 100, 3179. (33) Polverini, E.; Arisi, S.; Cavatorta, P.; Berzina, T.; Cristofolini, L.; Fasano, A.; Riccio, P.; Fontana, M. P. Langmuir 2003, 19, 872.

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Figure 3. Ellipsometric angle |δΨ| at φo ) 70° and λ ) 633 nm: (0, ]) in water; (2, b) in buffer; (0, 2) 750 ppm FB in solution at the first 1 h; (], b) 1 mL of 30 000 ppm of DLPC dispersion was then injected into the 30 mL of FB solution to yield a mixture of ∼730 ppm FB and 970 ppm DLPC. The vertical lines indicate error bars.

Figure 4. Calculated trajectories of ellipsometric parameters |δ∆| and |δΨ| for a three-layer model at λ ) 633 nm and φο ) 70°, for various values of n1 and d1; x-symbols indicate increments of 5 nm in d1. The dynamic ellipsometric parameters data for 750 ppm fibrinogen in water (AfB; b) and after injection of a concentrated DLPC into the fibrinogen solution (BfC fDfEfFfG; 2) are shown. See Figures 2 and 3. The calculated values of d1 and n1 are shown in Figures 5 and S1 in the Supporting Information. 6 µm Mylar spacer. The water or buffer spectra were subtracted from the transmission spectra, as outlined by Chittur.34 Infrared reflection-absorption spectroscopy (IRRAS) experiments were performed by using an external reflection attachment (Graseby Specac Inc., Smyrna, GA), which has a removable Teflon Langmuir trough. An incidence angle of 40°, measured relative to the surface normal, was used. IRRAS data are reported as plots of reflectance-absorbance (RA) vs wavenumber. RA is equal to -log 10(R/Ro), where Ro and R are the reflectivities of the pure and the film-covered water (or buffer) surfaces, respectively. The instrument was continuously purged with dry air from a Balston purge gas generator, for reducing the water vapor and carbon dioxide in the sample chamber. Spectra were collected either at 4 or 8 cm-1 resolution with the Happ-Genzel apodization and one level of zero filling to yield the same data spacing as when the spectra were taken at either 2 or 4 cm-1 resolution. The numbers of scans varied depending on the sampling time. The spectrum of the water vapor was subtracted, to eliminate (34) Chittur, K. K. Biomaterials 1998, 19, 357.

Displacement of Fibrinogen the water vapor bands interference in the polar group region. The spectra were taken using unpolarized light. Fourier self-deconvolution (FSD) and second derivative routines were used to resolve the underlying bands, or components, in the experimental spectra.35 For transmission IR (TIR), the full bandwidth at half-height (fwhh) of 15-19 cm-1 and a resolution enhancement factor (K) of 2.1-2.4 are used. For IRRAS, values of fwhh of 11-13 cm-1 and K-values of 1.3-1.4 are used. Once the component amide bands were identified on the basis of the band-narrowing and second derivative spectra, an iterative curve fitting with Gaussian band shapes (by adjusting the positions, intensities, and shapes of the component bands) to the experimental spectrum was employed.

3. Results and Discussion 3.1. Tensiometry Results. In water, at constant area and at 25 °C, the DST of 750 ppm FB alone dropped from 72 to 52 mN/m within 1 min, and remained nearly steady for 60 min (Figure 1; 0). The DST behavior for FB in buffer at 25 and at 37 °C is similar to that in water (Figure 1; ∆ and (). These results indicate that FB adsorbs fast to the interface. The measured steady-state surface tension of FB, 52 mN/m, is in good agreement with those reported previously.36-39 After FB was allowed to adsorb for 1 h, a DLPC dispersion was injected into the FB solution (see section 2.2) to yield a mixture of ∼730 ppm FB and 970 ppm DLPC. For the DLPC injection process, there was a “deadtime” of ∼1 min during which no measurements were taken. After the DLPC injection, the surface tension decreased to an equilibrium surface tension of 24 mN/m as with DLPC alone.16-18,40 The surface tension behavior approaches that of DLPC alone at longer times, suggesting that DLPC has adsorbed and probably expelled most or all of the FB protein layer from the surface. The expulsion is apparently driven by the lowering of the system surface free energy, which is reduced more by a complete DLPC monolayer than by a fibrinogen layer. It took a shorter time to form an adsorbed layer consisting mostly of DLPC at the physiological temperature of 37 °C (∼10 min after the DLPC injection) than at 25 °C (∼30-50 min). This is because DLPC dissolves from the vesicles, diffuses, and adsorbs faster at 37 than at 25 °C.16,40 The overall equilibration time may include a few minutes that is takes for the dispersion to be fully homogenized after injection, since no additional stirring was used, to avoid perturbing the surface. We infer that, even though FB molecules adsorbed first, DLPC molecules were still able to adsorb at the interface. At equilibrium, the adsorbed layers from FB/DLPC mixtures consist mostly of DLPC, in water and in buffer. These inferences are tested with other methods. Several studies have reported partial or full displacement of proteins, β-lactoglobulin or casein, from the air/water or oil/water interface, respectively,41,42 by using soluble nonionic ethoxylated surfactants. In these studies, the surface tensiometry or the solution depletion method were used to infer protein desorption or expulsion. Most (35) Jackson, M.; Mantsch, H. H. Crit. Rev. Biochem. Mol. 1995, 30, 95. (36) Igoshin, V. A.; Kuzmin, S. M.; Bening, G. P.; Yampol’skaya, G. P. Colloid J. 1983, 45, 57. (37) Baszkin, A.; Boissonnade, M. M. ACS Symp. Ser. 1995, 602, 209. (38) Hernandez, E. M.; Phang, T.-L.; Wen, X.; Franses, E. I. J. Colloid Interface Sci. 2002, 250, 271. (39) Hernandez, E. M.; Franses, E. I. Colloids Surf. A 2003, 214, 249. (40) Pinazo, A.; Infante, M. R.; Park, S. Y.; Franses, E. I. Colloids Surf. B 1996, 8, 1. (41) Coke, M.; Wilde, P. J.; Russell, E. J.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489. (42) Courthaudon, J.-L.; Dickinson, E.; Dalgleish, D. G. J. Colloid Interface Sci. 1991, 145, 390.

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proteins tend to adsorb irreversibly to surfaces,43,44 especially if they are “soft” and unfold or deform substantially after adsorption. The irreversibility of adsorption means that, if the solution is depleted from a soluble protein, protein does not desorb to any appreciable extent. However, as shown here and in the above references, and as known from liquid chromatography, proteins can still be displaced by small molecules. Such displacement is thermodynamically favorable if the surface energy decreases upon adsorption of the small molecules. 3.2. Ellipsometry. Experimental Results. For 750 ppm of FB in water, the value of the ellipsometric parameter |δ∆| increased within 60 min from 2.54° to 4.70° (Figure 2, 0, AfB), and |δΨ| increased from 0.17° to 1.25° (Figure 3, AfB). The continuous increases in these parameters indicate that FB continues adsorbing at the interface and that no steady-state condition has been established in the adsorption density, even though the surface tension showed no variation. This is apparently because in the adsorbed FB layer, only a portion of the large FB molecule closest to the surface contributes to the surface tension. Moreover, as the protein adsorbs, additional time-dependent denaturation may occur43,44 and may cause changes in the surface tension. After a DLPC dispersion was injected into the FB solution, to yield a mixture with 730 ppm FB and 970 ppm DLPC in water, the |δ∆| and |δΨ| values increased temporarily to 5.54° and 2.05° for about 20 min (BfCfD), and then dropped precipitously within about 5 min (DfEfF) to 0.42° and 0.01°. The initial enhancement is a novel observation for FB and has also been observed with albumin.18 The enhancement implies either (i) that the DLPC molecules adsorb at the air/water interface and fill the empty spaces between the FB molecules, thereby increasing the refractive index and the total surface density of the adsorbed layer, or (ii) that DLPC molecules bind to the FB surface and form a more hydrophobic lipoprotein complex than FB alone. In the latter case, the adsorption of FB to the surface is enhanced, either to a more concentrated monolayer or to a loosely packed second layer. We have used quantitative ellipsometry below (section 3.3 and IRRAS in section 3.5) to establish the second possibility. At longer times, the ellipsometric parameters were similar to those of DLPC alone (FfG),16,18 indicating that DLPC expelled FB fully from the interface and formed an equilibrium DLPC monolayer, consistently with the tensiometry results. The |δ∆| and |δΨ| values were smaller for 750 ppm FB in buffer than in water, and nearly steady at 1.86° and 0.15° over 1 h (Figures 2 and 3). After a DLPC dispersion was injected into the FB solution, the values first increased again but only slightly to 2.06° and 0.19°. Then they decreased a lot to 0.3° and 0.03°. At equilibrium, the values were the same as those of DLPC alone.16,18 Hence, DLPC can expel FB in buffer as well. 3.3. Ellipsometry. Quantitative Analysis. A threelayer ellipsometry model is used to calculate the effective thickness d1, refractive index n1, and surface density Γ1 of the adsorbed surface layer, as detailed in section 2.4. For water systems, the calculated δ∆/δΨ trajectories for films with n1 ranging from 1.38 to 1.42 and for d1 ranging from 0 up to 80 nm are shown in Figure 4, along with the experimental data points (AfBfCfDfEfFfG). For FB alone, inspection of Figure 4 plainly shows that d1 increased and n1 decreased with time (AfB). After the (43) Tripp, B. C.; Magda, J. J.; Andrade, J. D. J. Colloid Interface Sci. 1995, 173, 16. (44) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988, 125, 246.

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Figure 5. Calculated values for the thickness d1 of the adsorbed layers: (0, ]) in water; (2, b) in buffer; (0, 2) before DLPC injection; (], b) after DLPC injection. The insert is for times from 80 to 120 min.

Figure 6. Calculated values for surface density Γ of the adsorbed layers: (0, ]) in water; (2, b) in buffer; (0, 2) before DLPC injection; (], b) after DLPC injection.

DLPC injection, there was a transition period, where d1 increased temporarily while n1 decreased (BfC), and then d1 started decreasing while n1 increased (CfD). Finally, d1 dropped to a value (data points F and G) that was lower than the initial value of FB alone (data point A). For 750 ppm FB in water, the increase in d1 from 12 to 54 nm (Figures 4 and 5, AfB) and the decrease in n1 from 1.451 to 1.405 (Figure 4, AfB; see also Figure S1 in the Supporting Information) indicate that as more FB molecules adsorb to the interface, the adsorbed FB layer undergoes a transformation to a thicker but less dense layer. After the DLPC injection, d1 increased rapidly to ∼80 nm (BfC), whereas n1 remained at ∼1.40, suggesting that the adsorption of FB to the interface was accelerated after introducing DLPC into FB solution. A second adsorbed layer may have formed since d1 ≈ 80 nm is greater than all dimensions of the FB molecule. The rapid enhancement in the FB adsorption may be attributed to the binding of DLPC to the FB molecules to form a more hydrophobic FB-DLPC complex. This binding may be electrostatic in nature. At these conditions, FB has a small net negative charge, but its surface contains multiple charged sites, positive or negative. The zwitterionic DLPC headgroup, which contains positive and negative groups, may have access to several binding sites, which remain to be identified. Then, n1 started to increase to ∼1.41 (CfD), implying that the DLPC molecules started ad-

Figure 7. IR spectra (amides region) of FB in buffer (solid lines) and the spectra reconstructed (dotted lines); the Gaussian components are also shown. (A) fresh solution, TIR; (B) aged solution, TIR; (C) adsorbed layer, IRRAS. See also Table 1.

sorbing at the air/water interface and filled the empty spaces between the FB molecules, hence increasing the film refractive index. At the same time, d1 started to decrease to 68 nm, indicating that the DLPC molecules started displacing the FB molecules from the adsorbed layer. Finally, the dramatic drop in the value of the ellipsometric parameters (FfG) to the values that were similar to those for DLPC alone16,17 indicates that the adsorbed layer consisted mostly of DLPC. Moreover, since in this region the δΨ value was too small to accurately determine n1 and d1 simultaneously as above, we had to fix n1 to the value of 1.46 of DLPC in order to determine d1 (see section 2.4). The calculated d1 value was ∼2.4 nm (Figure 5), which is consistent with a fully extended hydrocarbon chains of 1.5 nm and lecithin headgroups thickness of about 1.0 nm.45-50 The abrupt change in the film thickness suggests that the first surface layer of FB is expelled from the surface. (45) Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid and Surface Chemistry; Marcel Dekker: New York, 1997; p 369. (46) Bu¨ldt, G.; Gallym H. U.; Seelig, J.; Zaccai, G. J. Mol. Biol. 1979, 134, 673. (47) Zaccai, G.; Bu¨ldt, G.; Seelig, A.; Seelig, J. J. Mol. Biol. 1979, 134, 693. (48) Nagle, J. F.; Zhang, R. T.; Tristram-Nagle, S.; Sun, W. J.; Petrache, H. I.; Suter, R. M. Biol. J. 1996, 70, 1419. (49) Petrache, H. I.; Tristram-Nagle, S.; Nagle, J. F. Chem. Phys. Lipids 1998, 95, 83. (50) McIntosh, T. J.; Simon, S. A. Biochemistry 1986, 25, 4948.

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Langmuir, Vol. 21, No. 22, 2005 10145

Table 1. Results of Spectral Decomposition of the Amide I Region Indicated by Fourier Self-Deconvolution and Second Derivative of the FTIR Spectra of FB in buffer

in water

wavenumber, cm-1

area fraction

wavenumber, cm-1

area fraction

conformations

TIR 4 wt % fresh

1689 1683 1670 1652 1634 1616

0.06 0.08 0.12 0.37 0.27 0.10

1691 1681 1667 1650 1633 1615

0.04 0.09 0.20 0.33 0.20 0.15

turns β-sheet aggregates turns R-helical β-sheets β-sheet aggregates

TIR 4 wt % aged for 1 week

1697 1684 1670 1653 1635 1617

0.02 0.14 0.16 0.29 0.22 0.17

1690 1679 1668 1654 1637 1614

0.08 0.10 0.12 0.20 0.28 0.22

turns β-sheet aggregates turns R-helical β-sheet β-sheet aggregates

IRRAS 750 ppm adsorbed for 50 min

1695 1680 1667 1649 1629 1615

0.09 0.15 0.24 0.31 0.14 0.07

1695 1681 1665 1653 1635 1622

0.07 0.12 0.23 0.30 0.23 0.05

turns β-sheet aggregates turns R-helical β-sheets β-sheet aggregates

method/conc/states

Any second or third layer, which is loosely associated with the first layer, can no longer remain adsorbed. The surface tension drop is more gradual, possibly because DLPC continues to adsorb as FB is expelled. For 750 ppm of FB in buffer, n1 and d1 were nearly steady at 1.42 and 16 nm, respectively, implying that the adsorbed FB layer is denser and thinner in buffer than in water (Figure 5 and Figure S1). After the DLPC injection, d1 temporarily increased to ∼30 nm, whereas n1 decreased, indicating that more FB molecules adsorbed to the interface to form a loosely bound layer. These values dropped to that of DLPC alone (the region where δΨ is small), suggesting that DLPC molecules have expelled FB molecules from the interface and a DLPC monolayer has formed at the interface. The calculated d1 (with n1 ) 1.46) was ∼1.5 nm which is less than that in water. The smaller value is due either to a different chain orientation, or to overestimating the value of n1 ) 1.46, if the adsorbed surface density of DLPC is less in buffer than in water.16 The surface density for FB in water increased with time, from 8 to 21 mg/m2 within 1 h (during which no steadystate value was reached), whereas that in buffer remained constant at about 6.5 mg/m2 (Figure 6). After the DLPC injection, the surface density (we presume to be mostly FB; see section 3.5) increased to ∼30 mg/m2 (40% increase) in water and ∼7.4 mg/m2 (only 14% increase) in buffer. At longer times, the surface density of the adsorbed layer, presumed to be mostly DLPC, was ∼2.7 mg/m2 in water and 1.5 mg/m2 in buffer. These values are indeed similar to those of close-packed monolayer of DLPC (2.5 mg/m2 or 4.0 µmol/m2 in water and 1.9 mg/m2 or 3.0 µmol/m2 in buffer).16 3.4. Spectral Analysis for FB. Figure 7 (and Figure S2) shows the FTIR spectra in the amide I region for FB (solid lines), compared with the spectra reconstructed (dotted lines) on the basis of the Gaussian components computed using the FSD and second derivative procedure. The assigned wavenumbers of the amide I components in Table 1 are based on refs 35 and 51-57. We did not use the amide II band for the protein conformation analysis because the FB amino acid side chains absorb in this region. Moreover, the amide II band is less sensitive in the details of the protein secondary structure.58 The values for the percentages of the different secondary structures are estimated from the relative areas of each peak. We found that “freshly” prepared FB in buffer contains ∼37% R-helical (at 1651 cm-1), ∼27% intramolecular β-sheet (at

1634 cm-1), ∼18% intermolecular β-sheet aggregates (at 1616 and 1683 cm-1), and ∼18% turns (at 1670 and 1689 cm-1) conformations (Table 1). In water, the freshly prepared FB solution contains a lower percentage of R-helical structures (∼33% vs ∼37%) and a higher percentage of β-sheet aggregates (∼18% vs ∼24%) than that in buffer, indicating that the protein denatures faster and more in water.57 The amide I region of the FB spectra, in water and in buffer, does not show the random coil component around 1644 cm-1, possibly because its area fraction is small and is masked by the two strong peaks at 1651 and 1633 cm-1. When the FB solutions were aged for one week, the R-helical structure decreased from ∼37% to ∼29% in buffer and from ∼33% to ∼20% in water; the β-sheet aggregates increased from ∼18% to ∼31% in buffer and from ∼24% to ∼32% in water, suggesting that FB denatures with time as it forms intermolecular aggregates. Even though the IRRAS spectra were noisy (due to the interference from the water vapor that cannot be totally eliminated), the adsorbed FB from water shows a higher percentage of β-sheet conformations (intramolecular and possibly intermolecular β-sheets) than the adsorbed FB from buffer, indicating that the adsorbed FB from water unfolds more at the interface. The conformational states of the adsorbed FB proteins are different from those of the bulk proteins.44 Whether in the adsorbed state or in the bulk, FB tends to denature faster in water. 3.5. IRRAS Results. The IRRAS spectrum for 750 ppm of FB in buffer, after 50 min of adsorption time shows the amide I and amide II bands of the protein (Figure 8, spectrum a).38,39,59 The RA intensities for these bands were 0.0069 ( 0.0002 and 0.0052 ( 0.0002, respectively, which are slightly lower than those reported previously (probably because of some small difference in the method of (51) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469. (52) Susi, H.; Byler, D. M. Methods Enzymol. 1986, 130, 290. (53) Azpiazu, I.; Chapman, D. Biochim. Biophys. Acta 1992, 1119, 268. (54) Istokkum, I. H. M.; Linsdell, H.; Hadden, J. M.; Haris, P. I.; Chapman, D.; Bloemendal, M. Biochemistry 1995, 34, 10508. (55) Yang, P. W.; Mantsch, H. H.; Arrondo, J. L. R.; Saint-Girons, I.; Guillou, Y.; Cohen, G. N.; Baˆrzu, O. Biochemistry 1987, 26, 2706. (56) Pelton, J. T.; McLean, L. R. Anal. Biochem. 2000, 277, 167. (57) Green, R. J.; Hopkinson, I.; Jones, R. A. L. Langmuir 1999, 15, 5102. (58) McClellan, S. J., Ph.D. Thesis, Purdue University, West Lafayette, IN, May 2005. (59) Arrondo, J. L. R.; Gon˜i, F. M. In Lipid-Protein Interactions; Watts, A., Ed.; Elsevier Science Pub. Co.: New York, 1993; p 321.

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Phang et al.

Figure 9. RA intensities of amide I (0, 9) and νa-CH2 (4, 2) for DLPC injection experiment in water (0, 4) and in buffer (9, 2).

Figure 8. RA intensities of IRRAS spectra for FB and DLPC/ FB mixture in buffer: (a) 750 ppm FB after adsorption for 50 min; (b-l) after DLPC was injected into the FB solution to yield a mixture of ∼750 ppm FB and 1000 ppm DLPC. The intensity of the CO2 band in the vapor phase is strong for 1 and 4 min after in DLPC injection. Then, the intensity of CO2 band decreased with time, and it disappeared after 10 min. Vibration bands, 1-5, are νa-CH2, νs-CH2, carbonyl CdO, amide I, and amide II, respectively.

subtracting the reference background).39 Then, a DLPC dispersion was injected into the solution. During the first 10 min, the RA intensities of the amide bands remained similar to those of FB alone (Figure 8, b-e). After that, the RA’s increased temporarily to 0.0131 ( 0.0002 and 0.0087 ( 0.0002 (Figure 8, f-h), indicating that the FB surface densities at the interface increased. At t ) 25 min, the RA intensities of the amide bands decreased (Figure 8, i), and there was simultaneous evidence of the DLPC bands 1-3, which come from the antisymmetric methylene stretching vibration νa-CH2, the symmetric methylene stretching vibration νs-CH2, and the lipid carbonyl stretching CdO vibration, respectively.16,17,59 The RA intensities of these bands were ∼0.0021, 0.0011, and 0.0024, suggesting that DLPC adsorbs at the interface as FB desorbs. After 30 min, the amide bands were no longer observed, indicating that no FB remained in the surface layer (Figure 8, j-l), or that its amount, if any, was too small to be detected. Thereafter, the RA intensities of the lipid bands remained constant and similar to that of DLPC,16,18 showing that, as FB was expelled from the surface, DLPC adsorbed. In water, a similar result was obtained (Figure 9; Figure S3 shows the actual spectra). The RA intensity of amide I of FB alone was higher in water than in buffer (Figure 9; 0 vs 9), consistent with the ellipsometry results. After the DLPC injection, the RA intensity of the amide I band increased temporarily, then decreased, and finally dropped to zero (∆). The intensity of the νa-CH2 band of the DLPC lipid increased from 0 to 0.0019 and remained nearly steady. Again, consistently with tensiometry and ellipsometry, these results show that DLPC can expel the adsorbed FB layer from the aqueous interface. 3.6. Discussion. There was a substantial initial enhancement in the FB adsorption before FB was displaced by DLPC from the air/aqueous interface, in either water or in buffer, as probed by the ellipsometry and

Figure 10. Schematic (not to scale for liposomes and vesicles) of expulsion of 750 ppm FB by 1000 ppm DLPC in aqueous dispersion: (a) FB is allowed to adsorb for an hour, and then DLPC is injected into the FB solution; (b) DLPC starts adsorbing and it also possibly binds to FB making it more hydrophobic. Hence, more FB adsorbs to the surface; (c) DLPC continues adsorbing, and finally displaces or expels FB.

IRRAS methods. Our hypothesis for explaining the overall mechanism of the expulsion process is as follows (Figure 10). When a DLPC dispersion is injected into the FB solution, dissolved DLPC molecules not only start adsorbing at the surface, but they also bind with FB molecules to form FB-DLPC lipoprotein complexes, which are more hydrophobic than FB, resulting in an initial enhancement in the total FB adsorption. As more DLPC molecules adsorb to the interface and are replenished from dispersed vesicles, the surface tension decreases or the surface pressure increases, causing a thermodynamic (and a mechanical) driving force for expelling FB from the surface. The ability of DLPC to expel or displace FB from the air/water interface is important for obtaining effective lung surfactant formulations in the presence of such serum proteins. In our experiments, DLPC can easily displace FB. If the FB layer has been “aged” for 1 day or longer, it is possible that it may aggregate further and become more

Displacement of Fibrinogen

difficult to dislodge. We speculate that DLPC will still displace aged FB layers with the same mechanism, since DLPC can displace “aged” albumin layers,18 and because FB in water is displaced even when it is substantially aggregated. In future experiments, one can test this hypothesis further. Once DLPC expels FB and forms a complete monolayer at the interface, no FB is detected by IRRAS. These results imply that the FB molecules are less favored to adsorb to a surface with the lecithin headgroups facing toward them. This is analogous to the results in which the adsorption of serum proteins onto a biomaterial surface is significantly reduced when the surface is coated with phosphatidylcholine groups.27,60 A reduction in the adsorption of these serum proteins onto biomaterial surfaces is important for improving the life cycle of implantable materials. Hence, the results suggest possible uses of DLPC coatings of biomaterial surfaces. 4. Conclusions FB in water adsorbed continuously at the air/aqueous interface, whereas FB in buffer adsorbed and reached a plateau within 1 h of adsorption. The continuous adsorption of FB in water may arise because FB unfolds or (60) Vermette, P.; Gauvreau, V.; Pe´zolet, M.; Laroche, G. Colloids Surf. B 2003, 29, 285.

Langmuir, Vol. 21, No. 22, 2005 10147

denatures continuously in water, as supported by the FTIR spectral analysis results. The already adsorbed FB layers, in water or in phosphate-buffered saline at 25 or 37 °C, can be displaced by DLPC. This can be important in lung surfactant formulations for treating ARDS. There is an initial enhancement in the FB adsorption after DLPC injection, probably due to the formation of an FB-DLPC lipoprotein complex, which is more hydrophobic than FB. After the DLPC expels the FB from the interface and forms a complete monolayer, it prevents adsorption of FB to the interface, and helps maintain lower surface tensions than solutions of FB. Acknowledgment. This research is supported in part by the National Institutes of Health (Grant HL-5464102), the National Science Foundation (Grants CTS 0135317 and 0457289), and the Indiana 21st Century Research and Technology Fund. Supporting Information Available: The refractive index n1 of the adsorbed layers, in water and in buffer, as calculated from |δ∆| and |δΨ| are show in Figure S1. Figure S2 shows the IR spectra (amides region) of FB in water and the spectra reconstructed; the Gaussian components are also shown. Figure S3 shows the RA intensities of IRRAS spectra before and after the DLPC injected into the FB solution in water. This information is available via the Internet at http://pubs.acs.org. LA0504412