Role of Subsurface Particulates on the Dynamic Adsorption of

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Role of Subsurface Particulates on the Dynamic Adsorption of Dipalmitoylphosphatidylcholine at the Air/Water Interface Xinyun Wen and Elias I. Franses* School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907-1283 Received October 25, 2000. In Final Form: March 9, 2001 Dipalmitoylphosphatidylcholine (DPPC) is the major component of lung surfactant, which stabilizes the lung by reducing greatly the surface tension at the air/liquid interface of the alveoli. In this study, the adsorption behavior of DPPC at the air/water interface was investigated with tensiometry, infrared reflection-absorption spectroscopy (IRRAS), and ellipsometry. Two different preparation protocols were used to vary the size and microstructure of dispersed particles, and their effects were assessed. The tension results indicate that sonication of DPPC above the main gel-to-liquid-crystal transition temperature, to break the large liposomes into smaller vesicles, greatly improved the adsorption rate and tension-reduction ability of DPPC dispersions. In IRRAS, the intensity of the νa-CH2 band was found to be much higher for DPPC dispersions than the value for spread DPPC monolayers at the maximum monolayer surface density, suggesting that after surface film formation there may be substantially more material than a monolayer associated with the air/water interface. The extra material detected by IRRAS is probably due to some DPPC vesicles or liposomes close to the interface or attached to the monolayer. Although the extra material does not contribute to the ellipsometry or the surface tension at constant area, it influences the dynamic tension under area oscillation. These results are consistent with the idea that formation of a surface film from DPPC dispersions involves two steps: diffusion of particles (vesicles or liposomes) from the bulk phase, followed by their partial disintegration to form an insoluble surface film and a surfaceassociated reservoir.

1. Introduction Dipalmitoylphosphatidylcholine (DPPC) is the most abundant component of lung surfactant, which is a complex lipid-protein mixture in the lining layer of the alveolar fluid.1,2 Lung surfactant stabilizes the lung by reducing the surface tension at the air/liquid interface of the alveoli. Understanding of phospholipid transport to the air/water interface is basic to the comprehension of dynamic surface tension properties in the alveolar lining layer and the overall dynamics of respiration. At temperatures below Tc ) 42 °C, DPPC molecules are in the gel phase and are essentially insoluble in water, forming a particulate dispersion. Because there is only a negligible amount of dissolved DPPC molecules in solution, spontaneous adsorption of dissolved molecules from the bulk phase is expected to be quite slow or insignificant. Vilallonga and Tajima and Gershfeld observed that the surface tension of a DPPC dispersion measured below 42 °C was nearly equal to that of water,3,4 indicating no detectable adsorption by tension measurements. However, surface tension lowering was observed at temperatures below 42 °C by other authors and by our group.5-10 For 1350 ppm DPPC dispersions in saline at 37 °C, dynamic * To whom correspondence should be addressed. Tel: (765) 4944078. Fax: (765) 494-0805. (1) Notter, R. H.; Shapiro, D. L. Pediatrics 1981, 68, 781. (2) Jobe, A.; Ikegami, M. Am. Rev. Respir. Dis. 1987, 136, 1256. (3) Vilallonga, F. Biochim. Biophys. Acta 1968, 163, 290. (4) Tajima, K.; Gershfeld, N. L. Biophys. J. 1985, 47, 203. (5) King, R. J.; MacBeth, M. C. Biochim. Biophys. Acta 1979, 557, 86. (6) Notter, R. H.; Smith, S.; Taubold, R. D.; Finkelstein, J. N. Pediatr. Res. 1982, 16, 515. (7) Bois, A. G.; Albon, N. J. Colloid Interface Sci. 1985, 104, 579. (8) Chung, J. B.; Shanks, P. C.; Hanneman, R. E.; Franses, E. I. Colloids Surf. 1990, 43, 223. (9) Launois-Surpas, M. A.; Ivanova, Tz.; Panaiotov, I.; Proust, J. E.; Puisieux, F.; Georgiev, G. Colloid Polym. Sci. 1992, 270, 901.

tensions as low as 34 mN/m were observed.10 The discrepancies in the surface tension measurements are due primarily to the differences in dispersion preparation and probably to effects of impurities. The mechanism of the lipid entering the air/water interface is mainly related to short-range hydrophobic molecular interactions and consequently should depend on the bulk physical state of the dispersion, which is a function of temperature, time since mixing, and the protocol of dispersion preparation.1,10 Two thermal transitions have been observed for aqueous DPPC dispersions: a “pretransition” at 36 °C and the main gel-to-liquid-crystal transition near 42 °C. These transitions reflect changes in the fluidity of the hydrocarbon chains. The surface tensions of DPPC dispersions were found to decrease significantly above the DPPC transition temperature.4,7 In addition to temperature, the state of dispersed DPPC particulates depends on the dispersion preparation protocol used. Different methods of mixing and heating the dispersions may affect the DPPC bulk phase structures. Shaking or stirring DPPC dispersions above Tc produces large multilamellar liposomes, which freeze when cooled to 25 or 37 °C. By contrast, sonication of these liposomes above Tc produces smaller unilamellar vesicles with average diameters as low as 250 Å.1,11-14 When left standing below 42 °C, vesicles may “freeze” (become less fluid) and fuse or form nonspherical particulate aggregate structures with dimensions ranging from 0.07 to 0.1 µm and also larger particles visible to the eye.15,16 (10) Park, S. Y.; Peck, S. C.; Chang, C.-H.; Franses, E. I. In Dynamic Properties of Interfaces and Association Structures; Pilai, V., Shah D. O., Eds.; AOCS Press: Champaign, IL, 1996; p 1. (11) Huang, C. Biochemistry 1969, 8, 344. (12) Sheetz, M. P.; Chan, S. I. Biochemistry 1972, 11, 4573. (13) Suurkuusk, J.; Lentz, B. R.; Barenholz, Y.; Bittonen, R. L.; Thompson, T. E. Biochemistry 1976, 15, 1393. (14) Finkelstein, M.; Weissman, G. J. Lipid Res. 1978, 19, 289.

10.1021/la001502t CCC: $20.00 © 2001 American Chemical Society Published on Web 04/24/2001

Adsorption of DPPC at the Air/Water Interface

The formation of a phospholipid monolayer from vesicles or liposomes in bulk dispersions is a process more complex than that of a soluble monolayer. Previously proposed models have generally included initial diffusion of the vesicles to the interface, followed by the subsequent disintegration of the vesicles to form an interfacial film.9,17-19 Regarding the nature of the surface films, some researchers report evidence of monomolecular films,17,20 and others infer inhomogeneous superficial mesophases including bilayers or more complex structures.9,21 Schu¨rch used the term surface-associated reservoir to indicate that there is more material at or connected with the air/liquid interface than just a monolayer.22 A surface-associated reservoir and particulate adsorption have been postulated either indirectly, based on surface tension evidence,22 or more directly based on evidence from surface potential and radiotracer methods.9 In this paper, tensiometry, infrared reflection-absorption spectroscopy (IRRAS), and ellipsometry were used for probing the role of subsurface particulates on the adsorption behavior of aqueous DPPC at the air/water interface. For comparing the results with different experimental techniques, all experiments were done at 25 °C, instead of 37 °C which is the physiological temperature of the alveolar layer fluid in the lungs. The results will demonstrate that for an aqueous lipid dispersion although only the monolayer is responsible for reducing the surface tension, there is more than a monolayer associated with the air/liquid interface. 2. Materials and Experimental Methods 2.1. Materials. Synthetic L-R-dipalmitoylphosphatidylcholine (DPPC) (99+%) was purchased from Sigma Chemical Co. (St. Louis, MO). The lipid dispersions were prepared on a weight basis. The pure water used for all samples was first distilled and then passed through a Millipore four-stage cartridge system, which has an organic adsorption column, two mixed ion-exchange columns, and an ultrafiltration unit, resulting in water resistivity of 18 MΩ cm at the exit port. n-Hexane (99+%) from Sigma Chemical Co. and ethyl alcohol (200 proof) from Pharmco Products, Inc. (Brookfield, CT) were the solvents used to prepare DPPC solutions for making spread monolayers. 2.2. Protocols for Preparing Dispersions. To study the effect of dispersion state on the dynamic adsorption behavior of DPPC, two preparation protocols were used, to vary primarily the size of the dispersed particles and possibly their microstructure or morphology. In protocol 1, a DPPC dispersion was heated to 55-60 °C to form lamellar liquid crystal liposomes. The dispersion was then shaken vigorously, decreasing the liposome droplet size, and then cooled to the room temperature to yield frozen liposomes. In protocol 2, the dispersion was heated as above to form liposomes, and then it was sonicated, for further decreasing the droplet size and for producing vesicles, and finally cooled to the room temperature. Sonication was done in a sonicator bath (Branson 1200 Ultrasonic cleaner, Branson Cleaning Equipment Co., Shelton, CT) at 55 °C for 45 min. 2.3. Surface Tension Measurements. The pulsating bubble surfactometer (PBS), purchased from Electronetics Company (15) Schullery, S. E.; Schmidt, C. F.; Felgner, P.; Tillack, T. W.; Thompson, T. E. Biochemistry 1980, 19, 3919. (16) Wong, M.; Anthony, F. H.; Tillack, T. W.; Thompson, T. E. Biochemistry 1982, 21, 4126. (17) Schindler, H. Biochim. Biophys. Acta 1979, 555, 316. (18) Vassilieff, C. S.; Panaiotov, I.; Manev, E. D.; Proust, J. E.; Ivanova, Tz. Biophys. Chem. 1996, 58, 97. (19) Walters, R. W.; Jenq, R. R.; Hall, S. B. Biophys. J. 2000, 78, 257. (20) Salesse, C.; Durcharme, D.; Leblanc, R. M. Biophys. J. 1987, 52, 351. (21) Ivanova, T.; Panaiotov, I.; Georgiev, G.; Surpas, M. A.; Proust, J. E.; Puisieux, F. Colloids Surf. 1991, 60, 263. (22) Schu¨rch, S.; Bachofen, H. In Surfactant Therapy for Lung Disease; Robertson, B., Taeusch, H. W., Eds.; Marcel Dekker: New York, 1995; p 3.

Langmuir, Vol. 17, No. 11, 2001 3195 (Amherst, NY), was used for measuring the dynamic surface tension of DPPC dispersions. The instrument uses a sensitive pressure transducer for measuring the pressure drop ∆P across the air/water interface of a bubble. The surface tension γ is then calculated from the Laplace-Young equation, ∆P ) 2γ/R.23-25 Constant area tension measurements with R ) 0.40 mm are recorded every 50 ms after an initial 1 s delay upon forming a new bubble. At pulsating area conditions, the area of the bubble can be changed (nearly) sinusoidally at frequencies from 1 to 100 cycles/min with the radius of the bubble varying from R ) 0.400.55 mm. 2.4. Infrared Reflection-Absorption Spectroscopy. Infrared reflection-absorption spectra were obtained with a Nicolet Prote´ge´ 460 Fourier transform infrared spectrometer equipped with a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) detector and an external reflection attachment (Graseby Specac Inc.), which has a removable Teflon Langmuir trough. The angle of incidence was measured relative to the surface normal, and an incident angle of 40° was used. For water vapor and carbon dioxide to be reduced in the sample chamber, the instrument was continuously purged with dry air from a Balston purge gas generator. All spectra were collected using 1024 scans at 8 cm-1 resolution with Happ-Genzel apodization and one level of zero filling, resulting in the same data spacing as when the spectra were taken at 4 cm-1 resolution. IRRAS data are reported as plots of reflectance-absorbance (RA) versus wavenumber. Reflectance-absorbance is defined as -log10(R/R0), where R0 and R are the reflectivities of the pure and the film-covered water surfaces, respectively.27-30 Sakai and Umemura studied the effect of infrared radiation on the IRRAS spectra of Langmuir monolayers by using a band path filter to reduce some heating due to IR radiation.42 They claimed (as one reviewer pointed out) that using a filter is necessary to prevent local heating (about 1 °C was measured by them) which can lead to the depletion of the monolayer density. They observed that for stearic acid monolayers at low surface densities, the RA intensities measured without the filter were smaller than those measured with the filter. Without the filter, no peaks were observed in the spectral region between 3000 and h ) 25.2 Å2/ 2800 cm-1 until the monolayer was compressed to A molecule (or Γ ) 6.6 × 10-6 mol/m2). In our experiments, we had no problem observing DPPC monolayer spectra at any surface density above 1.0 × 10-6 mol/m2. We also examined stearic acid monolayers, at the same surface densities at which Sakai and Umemura noticed a problem, both without and with an aperture that reduces the sample area by about 20-fold. No difference was observed. Without using a filter, we observed both the antisymmetric and the symmetric methylene stretching vibration bands h ) 42 Å2/molecule). at surface densities as low as 4 × 10-6 mol/m2 (A Moreover, the RA intensities for the antisymmetric methylene stretching vibration band, measured without a filter, were similar to those measured by Sakai and Umemura who used a band path filter. Hence, any possible local heating effects were not significant in our experiments. 2.5. Ellipsometry. A Rudolph Research (now Rudolph Technologies, Flanders, NJ) Auto ELII automatic null ellipsometer was used for measuring ∆ and Ψ of adsorbed or spread surfactant layers at the air/water interface. Measurements were taken at wavelengths of 633 nm (and also at 546 and 405 nm) and an angle of incidence of 70° measured from the surface normal. The diameter of the beam is 2 mm. A Petri dish, filled with aqueous samples, was placed on the standard sample stage. Ten or more measurements of ∆ and Ψ pairs were made at a given wavelength and then averaged. Then, ∆ for water was (23) Enhorning, G. J. Appl. Physiol. 1977, 43, 198. (24) Chang, C.-H.; Franses, E. I. J. Colloid Interface Sci. 1994, 164, 107. (25) Chang, C.-H.; Franses, E. I. Chem. Eng. Sci. 1994, 49, 313. (26) Park, S. Y.; Chang, C.-H.; Ahn, D. J.; Franses, E. I. Langmuir 1993, 9, 3640. (27) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (28) Flach, C. R.; Gericke, A.; Mendelsohn, R. J. Phys. Chem. 1997, 101, 58. (29) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (30) Wen, X.; Lauterbach, J.; Franses, E. I. Langmuir 2000, 16, 6987.

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Figure 1. Dynamic surface tensions of aqueous DPPC dispersions measured with the bubble method at 25 °C for protocol 1 (O) and protocol 2 (0); estimated uncertainty is (2 mN/m.

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Figure 2. Dynamic tensions at pulsating area of aqueous DPPC dispersions with the bubble method at 25 °C at a pulsating rate of 20 rpm (cycles per minute): 1, protocol 1; 2, protocol 2.

3.1. Dynamic Tension and Adsorption Behavior of DPPC Dispersions. The bubble method was used for measuring the dynamic tension of 1000 ppm DPPC dispersions for two protocols at 25 °C (Figure 1). For protocol 1 samples, the surface tension remained around 72 mN/m for 103 s and decreased slightly to 68 mN/m after 104 s (∼3 h). The tension did not seem to reach an equilibrium or steady value after 104 s, indicating that the adsorption dynamics of DPPC dispersions is quite slow. This might be attributed to the slow release of DPPC molecules from frozen liposomes. Freshly prepared protocol 2 samples, which consisted mostly of vesicles or particles produced from cooling the vesicular dispersion, exhibited stronger and faster dynamic adsorption than protocol 1 samples. The surface tension reached 55 mN/m after 104 s. The dynamic tension behavior of DPPC dispersions was also studied at pulsating area conditions. Usually, during the expansion cycle the surfactant molecules in the bulk phase adsorb onto the interface. When the bubble area is compressed back to its original size, the excess molecules move from the interface to the bulk phase via a desorption mechanism or other mechanism involving monolayer deformation. If the desorption is not fast enough, the molecules accumulate at the interface and the surface density becomes transiently higher than the equilibrium surface density. This, in turn, results in the dynamic surface tension being lower than the equilibrium surface tension. This mechanism is termed “dynamic adsorption hysteresis” and can be accounted for by diffusion, adsorption, and desorption steps.24-26 For protocol 1 samples, when the bubble was pulsated at 20 cycles/min the maximum tension, γmax, was 72 mN/m, which is close to the surface tension of water, and the minimum tension,

γmin, was 67 mN/m, which is close to the initial surface tension (Figure 2). The high γmax values suggest that there was negligible adsorption of additional molecules during the area expansion. This is expected because the adsorption time scale of DPPC is much larger than the pulsation period (3 s here). Moreover, adsorption hysteresis was negligible, as shown by the minimum tension being close to the initial tension. For protocol 2 DPPC dispersions, after the bubble was pulsated for 30 s the maximum tension, γmax, was 66 mN/m and the minimum tension, γmin, was 40 mN/m. The low γmin values indicate that the surface density was increased during the compression stage because of dynamic adsorption hysteresis. The maximum and minimum tensions were found to decrease with successive pulsation cycles. After the bubble was pulsated for the second time at 20 cycles/min, the maximum and minimum tensions were lower, 60 and 28 mN/m, respectively, than in the first stage (curves not shown, more details will be given elsewhere34). Because the adsorption time scale of DPPC vesicles is large compared to the pulsation period, it seems unlikely that the additional material transferred to the air/water interface during the expansion cycle was adsorbed from the bulk. Our hypothesis is that the “excess” surfactant material was probably incorporated into the surface-active film from a surface-associated reservoir, consistent with the hypothesis of Schu¨rch.22 Overall, the surface tension results under constant and pulsating area conditions show that sonication of DPPC above 42 °C greatly improved the adsorption rate, most probably by breaking the large liposomes into smaller vesicles. It has been postulated that upon sonication the conversion of the liposomes to small vesicles may involve a two-stage process: fragmentation into open sheets of bilayers and subsequent spontaneous closure of the sheets into closed shells.35,36 Because the stability of the various possible intermediate forms is expected to depend on the balance between edge energy and bending energy of open or closed bilayers, various structures may be present ranging from small disks to almost-closed vesicles.36-38

(31) Manning-Benson, S.; Bain, C. D.; Darton, R. C. J. Colloid Interface Sci. 1997, 189, 109. (32) Goates, S. R.; Schodield, D. A.; Bain, C. D. Langmuir 1999, 15, 1400. (33) Walsh, C. B.; Wen, X.; Franses, E. I. J. Colloid Interface Sci. 2001, 233, 295.

(34) Wen, X. Ph.D. Thesis, Purdue University, West Lafayette, IN, expected in 2001. (35) Lasic, D. D. Biochem. J. 1988, 256, 1. (36) Cornell, B. A.; Middlehurst, J.; Separovic, F. Faraday Discuss. Chem. Soc. 1986, 81, 163. (37) Fromherz, P. Chem. Phys. Lett. 1983, 94, 259.

subtracted from the measured ∆ values of the DPPC samples to yield δ∆, which depends primarily on the monolayer properties.31-33

3. Results


Figure 3. Data of surface tension vs surface density (actually applied or produced by area compression) of spread DPPC monolayers at 25 °C. Sections A, B, and C correspond to the smoothly changing regions between certain monolayer breakpoints and were fitted to polynomial equations (fitted curves not shown) for purposes of interpolation.

Several of the postulated intermediate structures would be expected to be surface active, if they have exposed edges from which lipid could initiate spreading to the air/water interface. To determine the dynamic surface densities of the adsorbed DPPC layers from the dynamic tensions, the plot of surface tension versus surface density of spread DPPC monolayers was obtained (Figure 3), and it was fitted to a fourth-order polynomial in three sections (A, B, and C), corresponding to the smoothly changing regions between certain monolayer break-points. The fitting provided empirical Γ(γ) relationships, which were then used to calculate Γ(t) plots from the experimentally determined γ(t) of dispersion experiments with the bubble method. For protocol 1 DPPC dispersions, the thuscalculated surface density increased from 1.7 × 10-6 to 1.9 × 10-6 mol/m2 after about 2500 s (Figure 4). For protocol 2 samples, the surface density reached a higher value of 3.3 × 10-6 mol/m2 after 3000 s. The calculated surface densities indicate that the adsorbed DPPC layer was in a liquid-expanded (LE) phase (of high compressibility) for protocol 1 samples and a liquid-condensed (LC) phase (of low compressibility) for protocol 2 samples. Figure 5 shows the calculated surface densities at 20 cycles/min. The maximum surface densities at minimum bubble area were 2.1 × 10-6 and 3.6 × 10-6 mol/m2 for samples from protocols 1 and 2, respectively. 3.2. IRRAS and Ellipsometry of Spread DPPC Monolayers. IRRAS was used to study the properties of spread DPPC monolayers first. The results were then compared to those of adsorbed layers from DPPC dispersions. The spectra were taken using both polarized and unpolarized light. Typical IRRAS spectra in the hydrocarbon stretching region (3000-2800 cm-1), with unpolarized light, are shown in Figure 6 for DPPC monolayers on H2O. As is well-known, the two major bands at ∼2920 and ∼2850 cm-1 are due to the antisymmetric methylene stretching vibration (νa-CH2) and the symmetric methylene stretching vibration (νs-CH2), respectively.27,30 The frequencies of the CH2 stretching vibration bands are (38) Qiu, R.; MacDonald, R. C. Biochim. Biophys. Acta 1994, 1191, 343.

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Figure 4. Calculated dynamic surface densities ((0.2 × 10-6 mol/m2) for the data of Figure 1: O, protocol 1; 0, protocol 2.

Figure 5. Calculated surface densities for the data of Figure 2: 1, protocol 1; 2, protocol 2.

sensitive to the conformational order of the hydrocarbon chains. Whereas lower wavenumbers are characteristic of highly ordered almost all-trans conformations, higher wavenumbers indicate an increasing number of gauche conformers per chain (a measure of the “disorder” of the chains). For DPPC monolayers on a regular water (H2O) subphase, the frequency of the νa-CH2 band decreased continuously from 2922 cm-1 at Γ ) 1.9 × 10-6 mol/m2 (87 Å2/molecule) to 2918 cm-1 at Γ ) 3.1 × 10-6 mol/m2 (54 Å2/molecule) (Figure 7). These results are in good agreement with those of Mitchell and Dluhy.39 The frequency of 2922 cm-1 is very similar to those observed for bulk DPPC multilayer dispersions above the melting point, indicating that the hydrocarbon chains are disordered and fluidlike.39 This result is consistent with the surface pressure-molecular area isotherm that the DPPC monolayer is in the liquid-expanded (LE) phase at Γ ) 1.9 × 10-6 mol/m2. As the DPPC monolayer goes from the LE phase to the LC phase, the hydrocarbon chains become more trans-like, ordered, and close-packed, as indicated by the decrease in the νa-CH2 band frequency. For a (39) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc. 1988, 110, 712.

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Figure 6. IRRAS spectra of the C-H stretching vibration region for spread DPPC monolayers. An unpolarized IR beam was used, and the incidence angle was 40°. The surface densities of the monolayers are 1.9, 2.3, 2.7, 3.1, 3.5, 3.9, 4.2, 4.6, 5.8, 7.7, and 9.7 × 10-6 mol/m2, from top to bottom.

Figure 7. Dependence of the peak frequency of the νa-CH2 band on the surface density of spread DPPC monolayers.

surface density Γapplied (density of actual material spread) ranging from 3.5 × 10-6 mol/m2 (47 Å2/molecule) to 4.6 × 10-6 mol/m2 (36 Å2/molecule), where the DPPC monolayer is in the LC phase, the νa-CH2 band frequency remains nearly constant at 2917.6 ( 0.4 cm-1, indicating an almost all-trans conformation. At Γapplied > 4.6 × 10-6 mol/m2, where part of the DPPC surface layer is in a collapsedmonolayer state, the νa-CH2 band frequency of 2916 cm-1 is consistent with that of the DPPC gel bulk phase. The RA intensities (|RA|) of the νa-CH2 band increase with increasing surface density Γapplied (Figure 8), and the values measured with p-polarized light are higher than those with s-polarized or unpolarized light. The slope of the RA versus Γ curve is smaller at surface densities lower than 2 × 10-6 mol/m2. Above 2 × 10-6 mol/m2, the slope is large and remains nearly constant up to about 3.9 × 10-6 mol/m2. The increase in slope can be attributed to several factors: either (i) the conformational order of the hydrocarbon chains increases with increasing surface density or (ii) the average tilt angle of the hydrocarbon

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Figure 8. Dependence of the RA intensity of the νa-CH2 band on the surface density of spread DPPC monolayers: [, measured with p-polarized light; 9, measured with s-polarized light; b, measured with unpolarized light; 2, expected intensities for unpolarized light, calculated by using the RA intensities for p- and s-polarized light.

chains decreases with increasing surface density. For Γapplied ranging from 3.9 × 10-6 to 4.6 × 10-6 mol/m2, which is the surface density range corresponding to a closepacked monolayer, the νa-CH2 band intensities remain nearly constant. At Γapplied > 4.6 × 10-6 mol/m2, where a partial monolayer collapse (or potentially a multilayer) could be expected, the νa-CH2 band intensities increased further but by less than 20% for an increase of 120% in Γapplied. This indicates that a portion of the applied material does not appear in the monolayer, implying a monolayer collapse, in which some portion of the monolayer may be expelled from the surface to form three-dimensional collapse structures on the surface or just “hanging” beneath the water surface. The small further increase in RA intensities suggests that some of the expelled DPPC monolayer might still remain close to the surface, contributing slightly to the reflectance-absorbance signal but much less than if it were on the surface. With polarized light, the average tilt angle θ of the hydrocarbon chain can be estimated by using an anisotropic-layer model27,28,41 and a method introduced by Tung et al.41 Calculations from the anisotropic-layer model show that at any given tilt angle θ, an approximately linear relationship exists between the monolayer extinction coefficient kmax and the RA intensities for both s- and p-polarization.41 For determination of the average tilt angle θ, the experimental RA intensities were first used to calculate s- and p-kmax values at all angles θ. The true value of θ was obtained by minimizing the difference between s- and p-kmax. Using this method, we found that the average chain tilt angle is 60 ( 4° at Γ ) 1.06 × 10-6 mol/m2, and then it decreases with increasing surface density to 20 ( 4° for Γ ) 3.87 × 10-6 mol/m2. At Γ ) 3.52 × 10-6 mol/m2, the tilt angle and the monolayer extinction coefficient were found to be 25° and 0.55, respectively, which compare well to the results obtained by Gericke et al.40 Using the RA intensities for s- and p-polarized light, (40) Gericke, A.; Flach, C. R.; Mendelsohn, R. Biophys. J. 1997, 73, 492. (41) Tung, Y.-S.; Gao, T.; Rosen, M. J.; Valentini, J. E.; Fina, L. J. Appl. Spectrosc. 1993, 47, 1643. (42) Sakai, H.; Umemura, J. Langmuir 1997, 13, 502.


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Figure 10. IRRAS spectra of the C-H stretching vibration region for aqueous DPPC dispersions: 1, protocol 1; 2, protocol 2.

Figure 9. Ellipsometry data for spread DPPC monolayers at the air/water interface: O, at λ ) 633 nm; 0, at λ ) 546 nm; ], at λ ) 405 nm. The angle of incidence was 70°.

we estimated the expected intensities for unpolarized light and compared them to the measured intensities (Figure 8). The difference was (0.0004, which was within the experimental error. We infer that the presence of the beam splitter and other optical elements in the optical path did not introduce any significant polarization in the unpolarized beam. To further probe the surface layer, ellipsometry measurements were done. First, DPPC layers were prepared with different surface densities for constructing a calibration curve showing how δ∆, the difference in the ∆ values between the film-covered surface and the clean water surface, changes with surface density Γapplied (Figure 9). Measurements were taken at wavelengths of 633, 546, and 405 nm, at an angle of incidence of 70° measured from the surface normal. The |δ∆| values increased with decreasing wavelength. At all three wavelengths, the average |δ∆| values increased almost linearly with increasing Γapplied until they remained fairly constant at about Γapplied ) 4.0 × 10-6 mol/m2, where the monolayer reached its maximum packing density. The values for |δ∆| at Γapplied ) 4.0 × 10-6 mol/m2 are 0.57°, 0.67°, and 0.92° for λ ) 633, 546, and 405 nm, respectively. When the surface density was increased further to 7.7 × 10-6 mol/m2, the |δ∆| values showed no further increase. These results imply that at Γapplied > 4.0 × 10-6 mol/m2 the collapsed portion of the monolayer does not contribute to ellipsometry measurements and hence is present not as a multilayer parallel to the surface but as some other structure. 3.3. IRRAS and Ellipsometry of Adsorbed Layers from DPPC Dispersions. IRRAS was then used for studying the adsorption of DPPC from bulk dispersions. Representative IRRAS spectra of adsorbed DPPC layers are shown in Figure 10. The frequencies of the νa-CH2 band are 2915 ( 1 and 2916 ( 1 cm-1 for protocols 1 and 2, respectively. For both protocols, the frequency is much lower than the wavenumber 2918 cm-1 of a complete monolayer (Figure 7) and very similar to that of the gelphase DPPC, indicating that some bulklike material is involved. The intensities of the CH2 stretching vibration bands, measured with unpolarized light, are plotted as a function of time in Figure 11. For the samples prepared by protocol 1, the νa-CH2 band intensity increased slowly with time

Figure 11. Comparison of measured RA intensities of the νaCH2 band for aqueous DPPC dispersions for protocol 1 (O) and protocol 2 (0, measured with regular experimental setup; ], measured with an aperture with a diameter of 2 mm) to the RA intensities calculated from the measured surface tensions, the surface equation of state (Figure 3), and the measured RA intensities of spread monolayers (Figure 5) for (b) protocol 1 and (9) protocol 2 dispersions.

and did not reach a steady value even after 6000 s, indicating that the adsorption was quite slow. Because DPPC is denser than water and the frozen liposomes were large, gravity could cause particles to migrate away from the surface and probably would be expected to contribute to a decrease in the adsorption rate. Freshly prepared protocol 2 samples exhibited much faster dynamic adsorption behavior than protocol 1 samples, indicating that the size and the microstructure of the DPPC particles affect greatly the adsorption process, consistent with the tension results. By using the surface density data inferred from tension results (Figure 4) and the calibration curve of RA versus Γ for the spread DPPC monolayers (Figure 8), we calculated the expected RA intensities if the surface contained only a monolayer contributing to the tension (Figure 11). The measured RA intensities are much higher than the calculated values. For the 1000 ppm sonicated DPPC sample, the intensity of the νa-CH2 band reached a nearly constant value of 0.022 (with unpolarized light) after 10 min. With p- and s-polarization, the steady-state RA intensities were 0.028 and 0.016, respectively. These RA intensities are much higher than the values observed

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Figure 12. Ellipsometry data for aqueous DPPC dispersions: O, protocol 1; 0, protocol 2.

for spread DPPC monolayers at the maximum monolayer surface density, suggesting that there might be more than a monolayer detected at the air/water interface along with the monolayer. Schindler studied the adsorption of DOPC vesicles at the air/water interface and proposed a possible mechanism for adsorption from lipid vesicles.17 He proposed that vesicles first diffuse to and then disintegrate at an open air/water interface. This disintegration leads to monolayer formation at the interface. Once the monolayer is formed, no further vesicular disintegration may occur because of the absence of an available interface, and a layer of vesicles which may come in contact with the monolayer may form under the monolayer. The vesicle layer is presumed to be in diffusional exchange with the bulk and in lipid (molecule) exchange with the monolayer. Our IRRAS results seem to be consistent with Schindler’s model. The high RA intensities cannot be due to a monolayer alone and can be attributed to some DPPC vesicles or other particles close to the air/water interface. The effect of IR beam size was also studied to investigate whether the large RA intensities were due to the large beam area used in IRRAS. An aperture with a diameter of 2 mm was used. For a 1000 ppm sonicated DPPC dispersion, the RA intensities for the νa-CH2 band, measured with the aperture, were about the same as those measured with the regular instrumental setup (beam diameter of about 1 cm) (Figure 11), indicating that the large RA intensities were not due to the large sample area. The surface layer of 1000 ppm DPPC dispersions was also probed with ellipsometry. The values of |δ∆| at λ ) 633 nm as a function of time are shown in Figure 12. For protocol 1 samples, which consisted mostly of large liposomes, the average value of |δ∆| was about 0.3° after 1 h. On the basis of the calibration curve for spread DPPC monolayers (Figure 9), the corresponding surface density is 2 × 10-6 mol/m2. For protocol 2 samples, the average |δ∆| was 0.5°, which corresponds to a surface density of 3.2 × 10-6 mol/m2. These surface densities are in good agreement with the values estimated from tension data by using the Γ(γ) relationship, 1.9 × 10-6 and 3.3 × 10-6 mol/m2 for protocols 1 and 2, respectively. Unlike the IRRAS results, ellipsometry results showed no higher |δ∆| values for DPPC dispersion experiments, and the presence of only a monolayer could be inferred from the data. One of the possible reasons why IRRAS

Wen and Franses

detects the surface-associated structures but ellipsometry does not is that DPPC does not absorb light at the wavelength used in ellipsometry, as is the case for IRRAS, and changes little the refractive index of the subphase. In IRRAS, the intensity of RA peaks depends strongly on the extinction coefficient of the absorbing species. If the absorbing species is very close to the air/water interface, it can contribute substantially to the measured IR reflectance-absorbance but not to the ellipticity (or δ∆). To examine how a possible presence of a DPPC sublayer can affect the IRRAS intensity but not the ellipsometry measurements, we did a simple sample calculation, as follows. For aqueous DPPC dispersions, if the sublayer has a thickness d (possibly related to the DPPC particulate size), a DPPC volume fraction φ, and a refractive index n(φ) + ik(φ) and if one assumes that d ) 50 nm and φ ) 0.02, then the calculated values at ν ) 2916 cm-1 are n ) 1.402, k ) 0.026, and |RA| ) 0.014. Similarly, for d ) 100 nm and φ ) 0.03, n ) 1.402, k ) 0.031, and |RA| ) 0.022. Thus, with plausible values for d and φ of a sublayer, |RA| can be substantial and comparable to the observed values. In ellipsometry, at λ ) 633 nm and with φ ) 0.03, n changes from 1.330 (for pure water) to 1.335, and the corresponding |δ∆| value for the adsorbed layer changes from 0.50° to 0.48°; this change is within the experimental error. Hence, the proposed model of the presence of a DPPC sublayer is supported by such calculations. Of course, more accurate calculations would have to be based on more direct evidence of sublayer thicknesses and volume fractions. Because δ∆ is expected to be proportional to the thickness of the adsorbed layer,33 the ellipsometry results suggest that the larger RA intensities detected by IRRAS are due to DPPC vesicles or metastable aggregates which are close to the air/water interface but are not due to DPPC multilayers. 4. Discussion Adsorption of phospholipid liposomes at the air/water interface is a complex process, in which many factors (molecular, colloidal, and thermodynamic) may be involved. Surface tension results show that sonication of DPPC above the main gel-to-liquid-crystal transition temperature improved the adsorption rate substantially. At both constant and pulsating area conditions, the tensions were lower for protocol 2 samples than for protocol 1 samples. Moreover, more pronounced dynamic adsorption hysteresis was observed for protocol 2 samples. The excess DPPC molecules adsorbed during the surface expansion cycle were probably from a surface-associated reservoir. A simplified model sketch for vesicles is depicted in Figure 13 (a similar sketch can be postulated for liposomes or frozen vesicles or liposomes). Completely intact vesicles or partially closed bilayers diffuse to the air/water interface, where they open or disintegrate to form patches of an insoluble monomolecular layer, which may be stretched or may diffuse covering the surface. After some disintegration of vesicles, surface aggregates of various shapes and dimensions may remain attached to the monolayer. The surface layer contains lipid monomers in a stable monomolecular film and associated nonequilibrium aggregates attached to the monolayer. If this surface layer is perturbed by surface expansion and subsequent compression, these aggregates can act as a surface source or reservoir of lipid monomers. For aqueous DPPC dispersions, IRRAS results cannot be accounted by just a monolayer and require the hypothesis that there is a lot more material than a DPPC monolayer at the air/ water interfacial region. This material does not contribute to the tension directly but can influence the dynamic


Figure 13. Schematic of a two-step hypothesis for monolayer formation from vesicles of an insoluble lipid. Vesicles diffuse to the interface and then disintegrate or open up to form an interfacial film in the second step. 1, closed vesicle structure; 2, open or disintegrated vesicle structure. Evidence indicates that a subsurface reservoir of vesicles remains attached to the monolayer; see text.

tension under area oscillation. The frequency of the νaCH2 band also indicates that the extra material is bulklike. By contrast, ellipsometry results are affected not by this extra material but only by the monolayer which affects the surface tension.

Langmuir, Vol. 17, No. 11, 2001 3201

5. Conclusions The tension results of aqueous DPPC dispersions indicate that for nearly insoluble lipids, the dispersed particles play quite an important role in adsorption dynamics. Sonication of DPPC above the main gel-toliquid-crystal transition temperature to break the large liposomes into smaller vesicles greatly improves the tension-reduction ability of DPPC dispersions. Thus, dispersion preparation protocols need to be carefully controlled. The tension-derived hypothesis of “surfaceassociated reservoir” has been better established by using infrared reflection-absorption spectroscopy and ellipsometry. The extra material associated with the air/liquid interface does not seem to contribute to the tension directly but can act as a dynamic reservoir for lipid molecules under area expansion/compression. The ellipsometry results suggest that the extra material can be due not to a lipid bilayer or multilayer but to attached lipid vesicles or metastable aggregates. Acknowledgment. This research was supported in part by the National Institutes of Health (Grant HL 5464102) and the National Science Foundation (Grant CTS 96-15649). LA001502T

Role of Subsurface Particulates on the Dynamic Adsorption of

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