Comparison of DLPC and DPPC in Controlling the Dynamic

Results of pressure drop across bubble interfaces and bubble deformation by gravity leave no doubt that DST's can be quite low. Detailed analysis of I...
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Comparison of DLPC and DPPC in Controlling the Dynamic Adsorption and Surface Tension of Their Aqueous Dispersions Aurora Pinazo,† Xinyun Wen,‡ Ying-Chih Liao, Alissa J. Prosser, and Elias I. Franses* School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907-1283 Received May 20, 2002. In Final Form: August 6, 2002 The adsorption of dilauroylphosphatidylcholine (DLPC) at the air/ water interface was investigated with tensiometry, infrared reflection-absorption spectroscopy (IRRAS), and ellipsometry. The tension dynamics at 25 °C at constant and pulsating area depends strongly on the concentration (10-1000 ppm) and sizes of dispersed DLPC particles, which are liposomes or vesicles. Dynamic surface tensions as low as 1-5 mN/m are observed for DLPC. For dipalmitoylphosphatidylcholine (DPPC), such tensions are also observed, but only for certain special preparation procedures. Direct probing of the surface by IRRAS and ellipsometry indicates that DLPC adsorbs by a molecular adsorption mechanism, controlled largely by the rate of dissolution of the dispersed particles. The surface layer is a monolayer with no particles attached to it, unlike DPPC in which the surface monolayer forms by particles which reach the interface and remained attached to it (Wen, X.; Franses, E. I. Langmuir 2001, 17, 3194). Spectroturbidimetry and dialysis experiments, used to determine the DLPC solubility in water as 4 ( 1 ppm and the DPPC solubility as essentially zero, support the above mechanisms. For DLPC, a simple diffusion/adsorption model with molecular diffusion and variable effective diffusion length can account for the dynamic surface tension data.

1. Introduction Aqueous dispersions of lipids, alone or in combination with proteins, are important in many biological systems and in food processing. Examples include food stabilizers and emulsifiers in food industries,1,2 and lung surfactants in medicine.3-5 DPPC (or dipalmitoylphosphatidylcholine) is an important lipid component of many biological cell membranes. It is also a key ingredient of lung surfactant, which controls the dynamic surface tension (DST) and helps maintain the lung alveoli healthy.3-5 Lung surfactant is a complex mixture of various lipids and four lungspecific proteins. DPPC, by itself, is slow to adsorb at 25 or 37 °C for certain preparation protocols, because it forms solid hydrated particles below 41 °C, the chain melting transition temperature.5 This property is thought to be linked to its ability to form aqueous surface layers of surface tension as low as 1 mN/m, under surface compression. DPPC forms liposomes (multilamellar liquid crystalline droplets) and vesicles (particles with one bilayer surrounding an aqueous core), above 41 °C.6 As these * To whom correspondence should be addressed. Telephone: (765) 494-4078. Fax: (765) 494-0805. † Current address: Institut d’Investigacions Quimiques i Ambientals de Barcelona, CSIC; Jordi Girona 18-26, 08034 Barcelona, Spain. ‡ Current address: Syngenta, P.O. Box 18300, Greensboro, NC 27419. (1) Darling; D. F.; Birkett, R. J. In Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry, Burlington House: London, 1986; Special Edition No. 58, p 1. (2) Dickinson, E.; Woskett, C. M. In Food Colloids; Bee, R. D., Richmond, P., Mingins, J., Eds.; Royal Society of Chemistry, Thomas Graham House: Cambridge, 1989; Special Edition No. 75, p 74. (3) Clements, J. A.; Hustead, R. F.; Johnson, R. P.; Gribetz, I. J. Appl. Physiol. 1961, 16, 444. (4) Notter, R. H.; Finkelstein, J. N. J. Appl. Physiol. 1984, 57, 1613. (5) Notter, R. H. Lung Surfactants; Marcel Dekker: New York, 2000. (6) Park, S. Y.; Peck, S. C.; Chang, C.-H.; Franses, E. I. In Dynamic Properties of Interfaces and Association Structures; Pillai, V., Shah, D. O. Eds.; AOCS Press: Champaign, IL, 1996; p 1.

dispersions are cooled, they form “frozen” liposomes or “frozen” vesicles of lower stability. For DPPC, we have studied the effect of the protocol of preparation and have found cases where DPPC can produce very low DST’s after adsorption from aqueous systems.6,7 Some new results are shown here. We have also used IRRAS and ellipsometry to directly probe the DPPC surface layer and study the mechanism of adsorption. We have concluded that DPPC particles reach the surface directly, form a monolayer, and remain attached to the surface because of their essentially zero solubility (see Figure 10 later).7 To address solubility and slow adsorption problems, we have studied dilauroylphosphatidylcholine (DLPC), a homologous lipid with shorter fatty alkyl chain length by four CH2 groups. DLPC, having a chain melting transition temperature of around 5 °C, forms conventional liposomes and vesicles at 25 and 37 °C.8 It does have a finite solubility. More importantly, it maintains an ability of producing low DST’s.8 Here we develop a hypothesis that this molecule is mostly in dispersion but has a finite solubility and ultimately adsorbs with a molecular mechanism and faster than DPPC at comparable conditions. In section 3, we present results on tensiometry, IRRAS, and ellipsometry on spread monolayers of DLPC and compare their behavior to that of DPPC. Results of pressure drop across bubble interfaces and bubble deformation by gravity leave no doubt that DST’s can be quite low. Detailed analysis of IRRAS data shows that DLPC forms monolayers which are quite close-packed and can generate low surface tensions. Dialysis results confirm that the DLPC solubility is finite, albeit small, ca. 4 ppm, and that DPPC solubility is smaller than the sensitivity of our measurements. The above hypothesis is supported (7) Wen, X.; Franses, E. I. Langmuir 2001, 17, 3194. (8) Pinazo, A.; Infante, M R.; Park, S Y. ; Franses, E I. Colloids Surf. B 1996, 8, 1.

10.1021/la020476r CCC: $22.00 © 2002 American Chemical Society Published on Web 10/05/2002

Comparison of DLPC and DPPC

strongly by the above data, and further buttressed by a simple mass-transfer model. The results could lead to lung surfactant substitutes, especially if inhibition (prevention of lipid adsorption) by serum proteins (such as albumin and fibrinogen) can also be addressed.9,10 2. Experimental Section 2.1. Materials. Synthetic L-R-dilauroylphosphatidylcholine (DLPC, 99% pure) and l-R-dipalmitoylphosphatidylcholine (DPPC, 99% pure) were purchased from Sigma Chemical Co. (St. Louis, MO). The lipid dispersions were prepared on a weight basis. The 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. DLPC spread monolayers were produced from 0.5 mg/mL in n-hexane (99+%, from Sigma) and ethyl alcohol (200 proof, from Pharmcoproducts, Brookfield, CT) in 9/1 volume ratio. 2.2. Protocols for Preparing Dispersions. Two preparation protocols were used, to vary primarily the sizes of the dispersed particles and possibly their microstructure or morphologies.6-8 In protocol 1, a DLPC dispersion was shaken vigorously by hand at room temperature for about 5-10 min; a DPPC dispersion was shaken similarly but at about 50 °C, above the chain melting transition temperature of 41 °C. In protocol 2, after such shaking, the dispersions were sonicated for about 40 min, for further decreasing the particle size and for producing vesicles. Sonication was done in a sonicator bath (Branson 1200 Ultrasonic cleaner, Branson Cleaning Equipment Co., Shelton, CT) at 25 °C for DLPC and at 50 °C for DPPC. Before testing by spectroturbidimetry, dispersions were stirred for at least 3 h to minimize settling effects. 2.3. Surface Tensiometry. The pulsating bubble surfactometer (PBS), purchased from Electronetics Co. (Amherst, NY), was used for measuring the dynamic surface tension of DLPC dispersions. The instrument uses a 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 for spherical interfaces.11 Constant area tension measurements with R ) 0.40 mm are recorded every 50 ms starting 1 s after 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 ) 0.40-0.55 mm. When the surface area was pulsating, there were instances where the calculated surface tensions were below 10 mN/m, at times close to the point of minimum surface area. Then, the bubble shape would be deformed to nearly oblate spheroidal,12,13 and even become nonaxisymmetric by moving to one side. By adjusting the leveling of the instrument, the latter behavior could be avoided. The bubble shape was monitored continuously by using a CCD camera (Model MN401X, from ELMO Corp., Plainview, NY), and the images were digitized with a frame grabber (Model ALL-IN-WONDER 128 PRO, from ATI Technologies Inc., Marlborough, MA). When the bubble shape was deformed nearly or completely axisymmetrically, the Laplace-Young equation for axisymmetric nonspherical surfaces was used for calculating the surface tensions.14,15 A KSV Langmuir mini-trough, from KSV Instruments, Finland, with a roughened platinum Wilhelmy plate, was used to obtain Π-t (surface pressure vs time) data of spread DLPC monolayers at 25 °C. 2.4. Infrared 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 (9) Wen, X.; Franses, E. I. Colloids Surf. A 2001, 190, 319. (10) Herna´ndez, E. M.; Phang, T. Z.; Wen, X.; Franses, E. I. J. Colloid Interface Sci. 2002, 250, 271. (11) Enhorning, G. J. Appl. Physiol. 1977, 43, 198. (12) Hall, S. B.; Bermel, M. S.; Ko, Y. T.; Palmer, H. J.; Enhorning, G. A.; Notter, R. H. J. Appl. Physiol. 1993, 75, 468. (13) Chang, C.-H.; Franses, E. I. J. Colloid Interface Sci. 1994, 164, 107. (14) Hartland, S.; Hartley, R. W. Axisymmetric Fluid-Liquid Interfaces; Elsevier: New York, 1976. (15) Rotenberg, Y.; Boruvka, L.; Neumann, A. W. J. Colloid Interface Sci. 1983, 93, 169.

Langmuir, Vol. 18, No. 23, 2002 8889 external reflection attachment (Graseby Specac Inc.), which has a removable Teflon Langmuir trough. An incident angle of 40°, relative to the surface normal, 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 (taking about 480 s), or 64 scans (taking about 30 s) for the first spectrum, at 8 cm-1 resolution. The Happ-Genzel apodization and one level of zero filling were employed, yielding the same data spacing as when the spectra were taken at 4 cm-1 resolution. The peak resolution was usually (4 cm-1, and often (2 cm-1. Possible local heating effects were examined and were found to be not significant in our experiments.7 IRRAS data are reported as plots of reflectance-absorbance (RA) vs wavenumber. Reflectance-absorbance is defined as -log(R/R0), where R0 and R are the reflectivities of the pure and the film-covered water surfaces, respectively. The ATR spectra of dry lipid were obtained by using a custom built ATR accessory (Purdue University, W. Lafayette, IN) with the same Nicolet Prote´ge´ instrument. Germanium trapezoidal ATR plates, which have dimensions of 2 × 20 × 50 mm with 45° ends, were purchased from Wilmad Glass Co. (Buena, NJ). Sample solutions or dispersions were placed on the plates and dried in a vacuum. Spectra were collected for 1024 scans at 2 cm-1 resolution. 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 DLPC layers at the air/water interface. Measurements were taken at wavelengths of 633, 546, and 405 nm, at an angle of incidence of 70° measured from the surface normal. A Petri dish, filled with aqueous samples, was placed on the standard sample stage. Ten or more measurements of ∆0 and Ψ0 pairs for the solvent, and of ∆ and Ψ for the solutions, were made at a given wavelength and then averaged. Then, ∆-∆0, which depends primarily on the monolayer properties,16,17 was calculated. The values of δΨ were normally smaller than the average error and are not reported. 2.6. UV-Vis Spectroturbidimetry. A Perkin-Elmer (Norwalk, CT) Lambda 900 UV-vis spectrometer was used. The DLPC dispersions were placed in quartz cells of 1 cm path length, and the spectra were collected from 600 to 250 nm. Since generally smooth spectra were obtained in this range, only the absorbances at 500 and 350 nm are reported. 2.7. Ultrafiltration and Dialysis. Ultrafiltration of some dispersions was done using 12 cm3 sterile syringes (PGC Scientifics, Frederick, MD) with disposable filter units of 0.22 µm Nuclepore PTFE (poly(tetrafluoroethylene)) membranes (Nuclepore Corp., Pleasanton, CA). For dialysis, disposable sterile float-dialyzer cellulose tubes of either 3500 or 5000 molecular weight cutoff, from Spectrum Laboratory Inc. (Rancho Dominguez, CA), were presoaked in water for 1 h. About 7 -10 mL of a DLPC or DPPC dispersion was introduced in the tube, which was placed in contact with pure water at 25 °C for 5-14 days. Prolonged exposure may ensure more complete equilibration in the permeate solutions but may also allow more time for molecular hydrolysis, which is known to occur for DPPC and DLPC after prolonged exposure to water.18 The permeate solutions were analyzed by two methods. In the first method, the solution was freeze-dried, and the resulting white powder was dissolved in CDCl3. 1H NMR spectra (at 300 MHZ, with a Varian Unity 300 spectrometer) at 25 °C were used to determine DLPC concentrations, using 1,4-dioxane as an internal standard. Calibration with two different molar ratios of DLPC to dioxane showed that the error was less than 10%. In the second method, the permeate was partially dried in a vacuum. The resulting concentrated dispersion was then spread on a Germanium ATR plate and totally dried in a vacuum. The remaining white solid on the plate was probed by ATR spectroscopy. (16) De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (17) Walsh, C. B.; Wen, X.; Franses, E. I. J. Colloid Interface Sci. 2001, 233, 295. (18) Chung, J. B.; Shanks, P. C.; Hannemann, R. E.; Franses, E. I. Colloids Surf. 1990, 43, 223.

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Figure 1. Dynamic surface tensions of aqueous DLPC dispersions, at constant area with the bubble method at 25 °C: 2, 10 ppm, protocol 1; 9, 50 ppm, protocol 1; [, 100 ppm, protocol 1; 4, 1000 ppm, protocol 1; ], 1000 ppm, protocol 2; O, filtrate from 1000 ppm (protocol 2) dispersion, actual concentration of order of 20 ppm.

Figure 2. Dynamic surface tension of 1000 ppm aqueous DPPC and DLPC at 25 °C, with bubble area pulsating at 20 cycles/ min (see section 2.3): dashed line, DPPC; solid line, DLPC. Points a and c, at local maxima, and points b and d, at local minima, represent bubbles, the photographs of which are shown in Figure 3.

Results and Discussion

measurements less reliable without the knowledge of the equilibrium bulk concentration. We infer that the dispersed particles affect the mechanism of dynamic tension lowering, but not the equilibrium surface tension. At pulsating area conditions at 20 rpm, shown in Figure 2 (or 80 rpm, not shown), the dynamic surface tension of 1000 ppm DLPC dispersion is as low as 1 mN/m. This suggests that aqueous DLPC may have applications in lung surfactant replacement (if it passes other biological and physiology tests), even though at 25 °C it is above the chain melting temperature. The γmin tension depends on the pulsating frequency and on the DLPC concentration. By contrast, the lowest tension obtained upon surface compression of a spread DLPC monolayer is 7 mN/m in ref 7, apparently because the compressed monolayer is given more time to collapse. The behavior of aqueous DPPC dispersions is much more sensitive, however, to the protocol of preparation. Aqueous DPPC can also have quite low dynamic surface tensions under certain conditions of concentration and sample preparation. We have reported values of tension minima, γmin, of 67 mN/m (protocol 1) or about 40 mN/m (protocol 2) in the first few cycles at 20 rpm. In this paper, the low dynamic tensions of DPPC were obtained by pulsating for the first 30 min at 80 rpm at the same concentration, followed by decreasing the pulsation frequency to 20 rpm. The DST’s obtained this way are much lower than those we report in Figure 2 of ref 7. The differences of the states of the dispersed particles, in the bulk and close to the surface layer, are the subjects of current and future work. Nonetheless, the trends in the tension data for the various different protocols and their variations are reproducible and indicate that DPPC can indeed produce quite low dynamic surface tensions, similarly to DLPC. This points to the importance of the choline polar group and of the two saturated hydrocarbon chains in producing such low tensions. The plateau, or “shoulder”, near the tension maxima of the DPPC data is not due to any surface phase transition (see ref 7 for the surface pressure-area isotherm), but it correlates to the presence of the dispersed particles. It is noteworthy that in most of the literature it is assumed that DPPC alone cannot reach the surface fast enough to produce low ( 10; NL ) 10 effectively represents an infinite stagnant diffusion layer.26 With a typical diffusivity D ) 3.0 × 10-10 m2/s,25 we have calculated the dynamic surface tension γ(t) for different values of NL (Figure 11). The solution was obtained numerically using a finite element method with a stretched grid and adaptive time step for efficient calculations with controlled accuracy. Calculation details are reported elsewhere.26 The effect of decreasing NL, or l, is analogous to the effect of increasing the concentration of dispersed particles, or of decreasing the dispersed particle size (inferred from Figure 1). The results show (26) Liao, Y.-C.; Franses, E. I.; Basaran, O. A. J. Colloid Interface Sci., submitted.

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Figure 11. Calculated dynamic surface tensions for DLPC (see text for details) with parameters c0 ) 4 ppm, Γm,L ) 3.5 × 10-6 mol/m2, D ) 1 × 10-10 m2/s, and KL ) 3.5 · 104 m3/mol: line 1, effective diffusive length l ) 5.5 mm; line 2, l ) 0.55 mm; line 3, l ) 55 µm; line 4, l ) 5.5 µm.

that the model can roughly fit the data of 100 ppm DLPC if one uses NL ) 1 or l ) 0.55 mm (close to our maximum l). The data for 1000 ppm DLPC dispersions in Figure 1 lie between the predictions for NL ) 0.1 and 0.01 or for l between 55 and 5.5 µm. Clearly, the above model can account qualitatively for the observed decrease in the equilibration time with increasing particle concentration with plausible values of l. More detailed models, in which the particle sizes and transport and dissolution rates are accounted for explicitly, are under development and may elucidate further and describe more quantitatively the roles of the particles on the dynamic adsorption at constant or pulsating area. 4. Conclusions Since the aqueous solubility of DLPC at 25 °C is quite small, about 4 ppm, only above the solubility does the dynamic surface tension drop substantially over times of 1 h or less. The dynamic adsorption and surface tension of aqueous DLPC dispersions for 4 ppm < c e 1000 ppm are controlled by the dispersed particle concentration and sizes, which depend on the preparation protocols. The dispersed particles dissolve and replenish dissolved DLPC, which is adsorbed at the air/water interface. The overall dissolution rate and the rate of tension equilibration increase with increasing particle concentration and with

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decreasing particle size. Evidence from ellipsometry and IRRAS supports the hypothesis that the surface DLPC layer is primarily a monolayer without attached particles, by contrast to the surface DPPC layer, which is a monolayer with attached particles. Aqueous DLPC is capable of producing at 25 °C very low dynamic surface tensions, γmin, when the bubble surface area pulsates at 20 or 80 rpm at the area amplitude used. Even under area pulsation and flow, these low surface tensions have a sound static or quasi-static mechanical significance; i.e., viscous effects are not major. This is evidenced by their gravity-induced deformation and the low Laplace-Young pressure jump at the interface. DPPC can also achieve low γmin tensions at 25 °C, but under a more restrictive method of sample preparation and history of surface area pulsation. The results show that for both DPPC and DLPC there is no molecular restriction in achieving low γmin, but there can be transportrate restrictions. The differences in the adsorption mechanisms may be important in practice if these molecules are considered, alone or in combination with other lipids or proteins, as potential lung surfactant replacement drugs.5 The differences may be also important in interactions with serum proteins such as albumin. We have shown recently that albumin can inhibit adsorption and tension lowering by DPPC.9 The effectively zero solubility of DPPC leads to an exclusively particulate adsorption mechanism, which hampers the adsorption of DPPC in the presence of a BSA monolayer. Preliminary observations indicate that DLPC, unlike DPPC, can overcome the BSA inhibition because of the molecular adsorption mechanism. Thus, the small but finite DLPC solubility, of the order of 4 ppm in water at 25 °C, is important and can certainly play a key role on the dynamic tension behavior. Acknowledgment. This research was supported in part by the National Institute of Health (Grant HL-5464102) and the National Science Foundation (Grants CTS 96-15649 and 01-35317). We thank Professor O. A. Basaran for collaboration on the modeling of mass transfer and Dr. L. Pe´rez for assistance with the DLPC NMR analysis. Supporting Information Available: Figures showing the surface pressure of a spread DLPC monolayer being constant in time after an initial rise and the RA intensity for 1000 or 100 ppm DLPC dispersions, prepared with protocol 2, remaining constant after an initial transient. This material is available free of charge via the Internet at http://pubs.acs.org. LA020476R