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Conformations of Short-Chain Poly(ethylene oxide) Lipopolymers at the Air-Water Interface: A Combined Film Balance and Surface Tension Study Zhong Xu,† Nolan B. Holland,‡,§ and Roger E. Marchant*,†,‡ Departments of Biomedical Engineering and Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106 Received May 15, 2000. In Final Form: September 22, 2000 The interfacial behavior of a short-chain poly(ethylene oxide) (PEO) lipopolymer, PEO (molecular weight 2000) grafted distearoylphosphatidylethanolamine (PEO2K-DSPE), and its mixtures with distearoylphosphatidylcholine (DSPC) have been studied using Langmuir film balance and surface tension methods. Surface pressure-area isotherms for pure PEO2K-DSPE and PEO2K-DSPE/DSPC mixtures (with 2, 5, 10, 15, or 20 mol % of PEO2K-DSPE) revealed two phase transitions. The simple additivity in the molecular area of lipid mixtures with increasing incorporation of PEO lipopolymer below the low-pressure transition indicates the existence of a two-dimensional thin layer of PEO and lipid. The low-pressure transition is interpreted as desorption of surface-adsorbed PEO molecules into the aqueous subphase. Correlation of film balance experiments with PEO lipopolymer and surface tension experiments with PEO aqueous solutions suggests dehydration of the PEO in the lipopolymer film by expulsion of PEO-associated water molecules as surface pressure increases. Theoretical models, based on scaling theories of polymers at interfaces, fit the observed isotherms well in the low-pressure regime below the first transition but poorly in the high-pressure regime above the first transition. The high-pressure transition is interpreted as ordering of lipid tails in lipopolymers, as driven by the enthalpic gain from lipid alignment and balanced by the entropic loss for lipid ordering and most importantly by dehydration of the PEO chains.

Introduction Poly(ethylene oxide) (PEO, also known as PEG) is a synthetic water-soluble polymer with a wide range of interface engineering applications. Long-chain PEO has been used widely to stabilize aqueous colloids and dispersions and to prepare protein-resistant biomaterial surfaces.1,2 Surface-grafted short-chain PEO, with a molecular weight in the 1000-5000 range, is becoming important in the preparation of sterically stabilized liposomes and vesicles used for drug delivery.3,4 Liposomes composed of short-chain PEO grafted phospholipids have significantly prolonged circulation time in vivo, attributed to steric stabilization of the liposomes. The steric repulsion force created by the grafted PEO prevents aggregation and opsonization of liposomes, which slows down the clearance process of liposomes from the circulation by the immune system. PEO is an unusual molecule in the sense that it is highly water soluble yet exhibits surface-active properties. PEO is water soluble in all proportions at moderate temperatures. This is attributed to hydrogen-bond formation * To whom correspondence should be addressed: Roger E. Marchant, Ph.D., Department of Biomedical Engineering, Wickenden Building, Case Western Reserve University, Cleveland, OH 44106. Phone: (216) 368-3005. Fax: (216) 368-4969. E-mail: [email protected]. † Department of Biomedical Engineering. ‡ Department of Macromolecular Science. § Present address: Lehrstuhl fu ¨ r Angewandte Physik, LudwigMaximilians-Universita¨t, D-80799 Mu¨nchen, Germany. (1) Zalipsky, S.; Harris, J. M. In Poly(ethylene glycol): Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; American Chemical Society: Washington, DC, 1997; p 1. (2) Andrade, J. D.; Hlady, V.; Jeon, S.-I. In Hydrophilic Polymer; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1996; p 51. (3) Lasic, D. D. Nature 1996, 380, 561. (4) Woodle, M.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171.

between the ether oxygens of PEO and water. In aqueous solution, PEO behaves like a highly dynamic random coil, yet it can readily pack and form a crystallized solid under conditions of high concentration, elevated temperature, or high pressure. The ethylene component is sufficiently hydrophobic to significantly reduce the surface tension of water and to form an absorbed PEO surface layer. PEO also can be spread at the air-water interface to form a polymer monolayer. Many studies concerning the surface behavior of PEO and its surfactant derivatives are focused on long-chain PEO, with a molecular weight of at least 20 000.5,6 For short-chain PEO, its surface behavior could be quite different. For example, Cao and Kim6 reported a molecular weight dependence on equilibrium spreading pressures for PEO monolayers. The profiles of surface pressure versus bulk concentration of PEO solutions also are molecular weight dependent. We are most interested in the surface behavior of short-chain PEO covalently attached to the hydrophilic headgroup of a double-chained zwitterionic lipid, such as distearoyl phosphatidylethanolamine (DSPE). This lipopolymer has importance in preparing sterically stabilized liposomes. It is also useful for exploring scaling theories of grafted polymers for shortchain polymers. Studies on monolayers of short-chain PEO lipopolymers are useful in understanding the properties of PEO tethered at solid-aqueous interfaces, including PEO-liposomes systems [e.g., refs 7-12]. This approach offers a structural basis from which to improve important properties such as suppression of protein adsorption. Monolayers containing mixtures of PEO lipopolymers and phospholipids have been used as close approximations of liposome membranes. A key benefit is that monolayers enable the use of sensitive (5) Kuzmenka, D. J.; Granick, S. Macromolecules 1988, 21, 779. (6) Cao, B. H.; Kim, M. W. Faraday Discuss. 1994, 98, 245.

10.1021/la0006770 CCC: $20.00 © 2001 American Chemical Society Published on Web 12/15/2000

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surface analytical methods otherwise not available for analysis of microscopic liposomes. Over the past several years, conformations of short-chain PEO lipopolymers have been studied using film balance,7 surface force apparatus,8 osmotic stress measurement,9 neutron reflectrometry,10 infrared reflection absorption spectroscopy (IRRAS),11 and surface rheological measurement.12 Film balance measurements on monolayers of lipopolymers reveal two phase transitions in their surface pressurearea (π-A) isotherms. The low-pressure transition is generally interpreted as a pancake-to-mushroom conformational change in the PEO chain, but the origin of the high-pressure transition is not fully understood. Baekmark et al.7 originally assigned it to a mushroom-to-brush conformational transition, following theoretical predictions by de Gennes and Alexander13 on the conformational behavior of tethered polymers. However, they reinterpreted the transition as “native” lipid alkyl chain ordering, based on IRRAS studies11 and the fact that isotherms of polystyrene-PEO diblock copolymers do not exhibit a high-pressure transition.14 Recently, a surface rheological study by Naumann et al.12 revealed the possible formation of a physical gel during the high-pressure transition. In this report, we present surface pressure-area (πA) isotherms for monolayers of lipopolymer PEO2K-DSPE (molecular weight of PEO ) 2000) and its mixtures with distearoyl phosphatidylcholine (DSPC) at the air-water interface using a film balance apparatus. The surface activities of aqueous PEO solutions are measured by surface tensiometry. The data are interpreted using the equation of state for polymers in two dimensions and scaling descriptions in two and three dimensions. We interpret the observed phase transitions in the π-A isotherms as the desorption of surface-adsorbed PEO molecules into the aqueous subphase and the ordering of lipids, balanced by dehydration of the PEO molecules in the subphase by expelling PEO-associated water molecules. Experimental Section Materials. Distearoylphosphatidyl ethanolamine (DSPE), PEO (molecular weight 2000) grafted distearoylphosphatidyl ethanolamine (PEO2K-DSPE), and distearoyl phosphatidylcholine (DSPC) (Figure 1) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL) in powder form or in chloroform solution. The purity is greater than 99%. PEO samples of molecular weight 2000 (PEO2K) and 400 000 (PEO400K) were purchased from Aldrich Chemical (Milwaukee, WI). Water was pretreated with an exchange carbon tank and a water softener and purified by a Milli-RO 120 unit. It was then fed through a Milli-Q Plus unit, which treats the water with UV light (185 and 254 nm) followed by a 0.22 µm Millipak 40 filter. Resistance of the purified water is greater than 18.2 MΩ cm. Chloroform (HPLC grade) was purchased from Aldrich Chemical and used without further purification. Film Balance. Pressure-area isotherms for the pure and mixed monolayers were measured on a Langmuir-Blodgett (LB) (7) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann E. Langmuir 1995, 11, 3975. (8) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479. (9) Kenworthy, A. K.; Hristova, K.; Needham, D.; Mcintosh, T. J. Biophys. J. 1995, 68, 1921. (10) Kuhl, T. L.; Majewski, J.; Wong, J. Y.; Steinberg, S.; Leckband, D. E.; Israelachvili, J. N.; Smith, G. S. Biophys. J. 1998, 75, 2352. (11) Wiesenthal, T.; Baekmark, T. R.; Merkel, R. Langmuir 1999, 15, 6837. (12) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Langmuir 1999, 15, 7752. (13) de Gennes, P. G. Adv. Colloid Interface Sci. 1987, 27, 189. (14) Goncalves da Silva, A. M.; Filipe, E. J. M. Langmuir 1996, 12, 6547.

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Figure 1. Structure of the zwitterionic lipid DSPC and the poly(ethylene oxide)-modified lipid PEO2K-DSPE.

Figure 2. Pressure-area isotherms of (A) PEO2K-DSPE, (B) PEO400K, (C) PEO2K, and (D) DSPE at 21 ( 1 °C. The isotherm for PEO400K was scaled from the original isotherm to represent the behavior per PEO2K segment of the PEO400K molecule. Two phase transitions in the isotherm of PEO2K-DSPE (marked by arrows) are due to the conformational changes in the grafted PEO. trough (KSV5000, KSV Instrument Ltd., Finland) with a dimension of 12 × 70 cm2. The trough was placed on an antivibration table in a class 100 clean room, to reduce mechanical distortion and contamination of the films. The trough was carefully cleaned with precision wipe paper dipped in chloroform and rinsed twice with water. The subphase used was pure water, maintained at 21 ( 1 °C. The surface of the liquid subphase was cleaned using a suction system. Solutions of pure or mixed PEO, lipid, and lipopolymers (70-100 µL, 1 mg/mL in chloroform) were spread on the water subphase, and the chloroform was allowed to evaporate for 10 min. The monolayers were compressed symmetrically with two moving barriers at a speed of 48 cm2/ min. Surface pressure was measured by the Wilhelmy-Plate method. All isotherms are averaged from at least duplicated experiments. Surface Tension. The surface-active properties of the PEOs in solutions at the air-water interface were determined from water surface tension measurements. Surface tensions of aqueous PEO solutions were measured by the de Nouy ring method at 25 °C and ambient pressure, using a Sigma703 tensiometer (KSV Instrument Ltd.). The platinum ring was cleaned by flaming before each measurement.

Results Typical pressure-area (π-A) isotherms of PEO2KDSPE, PEO400K, PEO2K, and DSPE monolayers are represented in Figure 2. The isotherm for PEO400K has been scaled from the original isotherm to represent the behavior per PEO2K segment in PEO400K. For PEO2KDSPE, two transitions are observed. The starting pres-

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Figure 3. Pressure-area isotherms for mixtures of PEO2KDSPE and DSPC at 21 ( 1 °C: (A) pure DSPC and (B-E) monolayers containing 2, 5, 10, and 20 mol % of PEO2K-DSPE. The low-pressure transition at π1 exists for all mixtures (π1 occurs at 2.2 nm2 per molecule for E), whereas the high-pressure transition at π2 is visible only when the concentration of PEO2KDSPE exceeds 10 mol %.

sures and molecular areas of these two transitions are measured as 9.4 mN/m at 9.04 nm2 and 22.3 mN/m at 2.33 nm2, respectively. In contrast, only the condensed gel phase is observed for DSPE, which reflects its high phase-transition temperature of 75 °C.15 Because PEO2KDSPE has the same hydrophobic alkyl tail as DSPE, the rise of surface pressure at all areas per molecule is attributed to the grafted PEO molecules at the air-water interface. For PEO2K and PEO400K, their isotherms match very well with that of PEO2K-DSPE at pressures up to 3.5 mN/m (15.32 nm2 per PEO2K). The isotherms for PEO400K and PEO2K-DSPE match at pressures up to 10.0 mN/m (8.56 nm2 per PEO2K). This result is similar to earlier observations6 showing that the lateral pressure of a pure PEO monolayer depends only on the surface mass concentration, not the polymer chain length, up to a surface concentration of 1.4 mg/m2. Unlike that of PEO2K-DSPE, isotherms for both PEO2K and PEO400K reach saturation at high surface concentrations. The equilibrium saturation pressures are measured at 5.1 and 10.7 mN/m at 5 nm2 per PEO2K for PEO2K and PEO400K, respectively. Saturation is due to the collapse of the PEO monolayer film at the air-water interface and dissolution of the PEO into the water subphase. The molecular weight dependence of the equilibrium collapse pressure has been observed before but not understood well.5 A possible explanation is that long-chain PEO should have a higher probability than short-chain PEO to be confined at the air-water interface, which would allow further compression of the monolayer films resulting in the increased surface pressure. A similar situation might also occur for short-chain PEO, when it is confined to the surface by a water-insoluble lipid, like DSPE. As is shown in Figure 2, the isotherms for PEO2K-DSPE and PEO400K match up to higher pressures than that of PEO2K. Figure 3 shows the pressure-area isotherms for DSPC alone and its mixtures with different concentrations of PEO2K-DSPE at 21 ( 1 °C. DSPC shows a similar isotherm to DSPE. Only the condensed gel state was observed. Extrapolation of the isotherm to zero pressure results in a calculated area of 0.50 nm2 per molecule. As (15) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990.

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the molar concentration of PEO2K-DSPE to DSPC increases, the area per molecule at a particular measured pressure increases proportionally. This indicates that the additional surface pressure is attributed to the repulsive lateral interaction of PEO chains at the interface. Again, two transitions are observed. The low-pressure transition at 8.0 mN/m is observed for all mixtures, whereas the high-pressure transition at 18.4 mN/m is visible only when the molar concentration of the PEO2K-DSPE is equal to or greater than 10%. The corresponding area per PEO2K molecule at the transition points matches well with that obtained directly from the isotherm of pure PEO2K-DSPE. At first glance, the observed high-pressure transition might be attributed to a conformational transition (mushroom-to-brush) in the surface-confined PEO. Theoretical predictions for PEO2K suggest that the distance between grafting points needs to be less than 3.5 nm,8 equivalent to ∼5% (molar) of PEO2K-DSPE in the mixture with DSPC, to give a weakly interacting mushroom conformation for the grafted PEO2K molecules. However, as we show in this report, this high-pressure transition is more likely the dehydration of concentrated PEO molecules in the aqueous subphase rather than the mushroom-to-brush transition in the surface-confined PEO molecules. It has been demonstrated that spread PEO monolayers have the same interfacial behavior as PEO films adsorbed from dilute solutions.16 However, it was reported recently that aqueous PEO solution at high polymer concentrations results in additional reductions in surface tension.6,17 This additional reduction in surface tension can be as high as 20 mN/m, depending on the chain length of the PEO. Figure 4a shows the surface pressure versus bulk concentration profile for PEO2K in dilute solutions. We observe an increase in surface pressure between 10-4 and 10-3 g/dl solutions up to a plateau at ∼5 mN/m. A second plateau at ∼10 mN/m begins around 0.1 g/dl. This behavior is intermediate between the data reported for high and low molecular weight PEO6 (dashed lines in Figure 4). Cao and Kim reported that for PEO of molecular weight greater than 20 kDa the plateau at ∼10 mN/m is reached by 10-3 g/dl, whereas no plateau is observed for the 0.4 kDa PEO. The surface pressure versus bulk concentration isotherm at high concentration (>1% PEO) shows a linear dependence with concentration up to 77 g/mL (22 mN/m) (Figure 4b). Above this concentration (which corresponds to a weight fraction of 0.7), the solution is highly viscous, inhibiting surface tension measurements. Discussion Although short-chain PEO lipopolymers have been used to construct sterically stabilized liposomes for prolonged circulation times in vivo, the mechanism of this process is not fully understood. Knowledge about the conformation, surface mobility, and surface dynamics of grafted PEO chains in the steric barrier layer and their respective contributions in reducing protein adsorption is very limited. Recently, the interaction forces between two supported phosphatidyl ethanolamine lipid bilayers with various concentrations of PEO were measured directly by the surface force apparatus.8 Studies on the disjoining pressures between a deposited PEO-DSPE monolayer and its silicon substrate also indicated the existence of PEO chains in different conformations above the lipopolymer monolayer film.7 It has been hypothesized that the transitions correspond to conformational changes, specifically pancake-to-mushroom and mushroom-to-brush. (16) Sauer, B. B.; Yu, H. Macromolecules 1989, 22, 786. (17) Kim, M. W.; Cao, B. H. Europhys. Lett. 1993, 24, 229.

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Figure 4. (a) Surface pressure versus bulk concentration of diluted (A) PEO (MW 2000), (B) PEO (MW 20 000), and (C) PEO (MW 425) solutions. Curves B and C are calculated from Cao’s data (ref 6). (b) Surface pressure versus relative PEO concentration for concentrated PEO2K solutions.

Although this description is helpful, we hope to develop a picture of the layer structure through combined analysis of film balance and surface tension data. Film Balance Analysis. The surface pressure-area isotherms for PEO2K-DSPE (Figure 2) and its mixtures with DSPC (Figure 3) are rich in information which can be used to infer the conformation for PEO2K at the airwater interface. The isotherm of PEO2K-DSPE contains two transitions, which suggests three major conformational states. Understanding each of these structures helps in understanding the transitions. Figure 5 shows the change in the molecular area as a function of the fraction of PEO-lipid at constant surface pressure. Two distinct curves are observed. At low surface pressure (below the first transition), a linear plot is observed, whereas at high surface pressure the plot curves with increasing slope. The linear curve indicates that there is simple additivity in the molecular area with increasing incorporation of PEO-lipid; that is, the PEO and the lipid coexist at the interface and both occupy a region proportional to their respective homogeneous films. This observation could result from uniform mixing or from the formation of islands of PEO-lipid separate and distinct from the pure DSPE, but in either scenario a single monolayer is present. At higher concentrations, simple additivity does not occur. Initially, increasing the fraction of PEO-lipids does not increase the molecular area very much, but once the PEO-lipid fraction reaches 5 mol %, the increase is

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Figure 5. Area occupied per PEO2K molecule (grafted to DSPE) as a function of the mole percentage of PEO2K-DSPE in the lipid mixtures at a surface pressure of (a) 6.1 mN/m and (b) 14.8 mN/m.

significant. One possibility is that there is loss of molecular area (which is the two-dimensional equivalent of the threedimensional excess volume) by the nonideal mixing of the lipid and PEO in a single interfacial layer. However, considering that at this surface pressure pure PEO is not stable at the interface (Figure 2), it is unlikely that the PEO remains in a shared layer with the lipid hydrocarbons. More likely, the PEO blocks are forced to occupy the aqueous region below the lipid film, acting as a tethered polymer layer. When the PEO-lipid fraction is low, the lipid layer dominates the molecular area, whereas at higher fractions, the molecular area occupied under the film by the PEO dictates the total molecular area, and the increase in measured area becomes linear with increasing PEO fraction. This analysis suggests that the film below the first transition consists of a single layer of PEO and lipid whereas above the first transition distinct layers of hydrocarbon and PEO exist. We now compare this structural model to reported thermodynamic measurements of PEO films. Observing the temperature dependence of PEO surface pressure isotherms, Kuzmenka and Granick5 reported that at submonolayer coverage entropy increases with increased surface coverage of PEO. Further, the temperature coefficient becomes positive (i.e., entropy decreases with increased surface coverage) after monolayer formation. The layer reaches an equilibrium pressure at which further compression leads to collapse of the layer into the subphase. At this point, the temperature coefficient reaches a constant positive value. It has been proposed

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Figure 6. Double logarithmic plots of surface pressure (π) and surface PEO concentration (C): (]) PEO2K-DSPE, (4) PEO400K, and (0) PEO2K. The solid line represents the theoretical exponent 2.85 for the dependence of π on C.

that ordering of water around the PEO is responsible for this anomalous behavior. For PEO-lipids at the interface, the temperature coefficient for the first transition7 is the same as at the equilibrium pressure for pure PEO.5 This suggests that the process occurring at the first transition is the collapse of the PEO chain into the subphase, yet the PEO is confined to the interface by the attached lipid, which leads to the further increases in surface pressure. Other evidence supporting the existence of a twodimensional thin layer of PEO and lipid below the first transition comes from the analysis of π-A isotherms using polymer solution theories. In a two-dimensional polymer film, polymer chains interpenetrate each other. On the basis of the scaling concepts for the thermodynamic properties of polymer chains in the relatively concentrated regime, Kawaguchi18 predicted that the osmotic pressure (π) of a polymer solution, or the surface pressure of a polymer monolayer, is related to the polymer concentration (C). The relationship has the general form

π/T ∼ Cdv/(dv-1)

(1)

where T is the temperature, C is the polymer surface concentration, v is the critical exponent of the excluded volume, and d is the space dimension. In the case of a two-dimensional film (d ) 2), the predicted value for v is 0.77 and 0.5 for good solvent and θ solvent conditions, respectively. Also, the π-C profile is molecular weight independent in this regime. Figure 6 shows double logarithmic plots of π and C for PEO2K-DSPE, PEO2K, and PEO400K. For PEO2KDSPE, log π increases linearly with log C between the surface concentration of 0.15 mg/m2 (22.2 nm2 per PEO2K) and 0.29 mg/m2 (11.6 nm2 per PEO2K). Linear regression of log π versus log C in this region results in an exponent of 2.46 for the dependence of surface pressure on surface concentration, which corresponds to a v of 0.84. Because water is known to be a good solvent for PEO at 21 °C with a theoretical v value of 0.77, the experimental exponent is in relatively good agreement with the prediction of Kawaguchi.18 Also, the linear region of PEO2K-DSPE (18) Kawaguchi, M.; Komatsu, S.; Matsuzumi, M.; Takahashi, A. J. J. Colloid Interface Sci. 1984, 102, 356.

matches relatively well with that of PEO2K and PEO400K. This is consistent with the prediction that the π-C profile is molecular weight independent in the semidilute regime. The double logarithmic plot of π and C for pure PEO2K curves from the linear region at a lower surface concentration (0.22 mg/m2 or 15.0 nm2 per PEO2K), most likely caused by the initiation of desorption of the short-chain polymer into the subphase. The extended linear region for PEO2K-DSPE and PEO400K suggests that EO segments in surface-confined short-chain PEO stay within a relatively thin layer at the interface. Conformation of PEO in such a thin layer is also described by the pancake conformation (thickness ∼ 1.5 nm for grafted PEO2K19). The pancake conformation has been predicted by scaling theory for grafted polymers with relatively strong surface adsorption energies at low surface concentration. In comparison, molecular models suggest that the limiting packing density for EO segments in a strict monolayer (thickness ∼ 0.4-0.5 nm) is 0.45 mg/m2 (0.16 nm2 per segment or 7.2 nm2 per PEO2K).20 Surface Tension Analysis. The dependence of surface pressure on the bulk concentration of PEO solutions provides further insight to the interfacial conformation of PEO. It is observed that transitions occur at similar surface pressures, measured from bulk solution as in the spread monolayers, even though the driving force is quite different. In the case of spread monolayers, the molecules are confined to the surface by their amphiphilic nature and the energy for conformational change comes from the lateral pressure applied to the film. From bulk solution, the molecules are driven to the interface to balance the chemical potential until an equilibrium between the bulk and the surface is achieved. Ultimately, the molecular arrangement at the interface determines the surface pressure, so it is not surprising that transitions are observed at similar values. The surface pressure isotherm for dilute solutions of PEO2K contains two plateaus, which correspond to spread monolayer transitions. The first plateau occurs at ∼5 mN/m which is the collapse pressure of a spread layer of PEO2K. The second plateau occurs near the collapse pressure for high molecular weight PEO (which also is the first transition of DSPE-PEO2K). The chemical potential of the solution at 10-4 g/dl must be high enough to drive the adsorption of a complete layer of PEO2K but not to the extent that a high molecular weight PEO monolayer can form. As the solution reaches a concentration of 10-1 g/dl, a complete layer, characteristic of high molecular weight PEO, forms. This surface pressure of 10 mN/m represents the maximum pressure attainable by the compression of a spread PEO layer. PEO confined to the interface by attached hydrophobic tails, such as DSPEPEO2K, can reach surface pressures greater than this value. However, as observed with DSPE-PEO2K, a transition at ∼10 mN/m occurs. This suggests that similar PEO layers are formed by adsorption from bulk solution, compression of high molecular weight PEO, and compression of tethered low molecular weight PEO. This lends credence to the interpretation of the first DSPE-PEO2K transition being dependent on the PEO; that is, the formation of a PEO layer is independent of the lipid tails. The linearity of the surface pressure isotherms at high bulk concentration fits a model for a binary mixture such (19) Lasic, D. D. In Poly(ethylene glycol): Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; American Chemical Society: Washington, DC, 1997; p 31. (20) Shuler, R. L.; Zisman, W. A. J. Phys. Chem. 1970, 74, 1523.

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that the surface tension γ (γ ) γ0 - π) is

γ ) N1γ1 + N2γ2

(2)

where N is the mole fraction of the components at the interface and γ0 is the surface tension of water at 21 °C. The surface tension extrapolated to zero PEO concentration is 64.5 mN/m, which corresponds to a 0.3 surface fraction of PEO. This fraction changes linearly with the bulk concentration. The decrease in surface tension can be related to the number of water molecules available per ethylene oxide monomer. This drops from 20 to 1 H2O molecule per EO unit for the concentrations 11 and 77 g/dl, respectively. This dehydration of the PEO results in a surface pressure of up to ∼22 mN/m. NMR studies of PEO aqueous solutions show transition in the chemical shift for the ethylene protons of PEO, as the volume percentage of water drops to about 50%.21 This suggests that about 3 H2O molecules are required for hydration of each ethylene oxide unit in a PEO chain. Obviously, for an aqueous solution of PEO2K to reach a surface pressure of 22 mN/m, the PEO chain must be dehydrated. Returning to the data from the DSPE-PEO2K spread monolayers, we observe that after the first transition the surface pressure steadily increases to 22 mN/m where the second collapse is observed. This increase in surface pressure, when compared to the surface tension data, can be partially attributed to dehydration of the PEO. Considering the evidence for the lipid alkyl chain ordering at the second transition, we propose that the second transition is the result of the ordering of the lipid tails of the lipopolymers, as driven by the enthalpic gain of the lipid alignment and balanced by (a) the entropic loss for lipid ordering and most importantly (b) the dehydration of the PEO chains, at the critical surface pressure. As the pressure increases and work is put into the system, entropy terms steadily diminish until they are equal to the enthalpic gain for lipid condensation. At this point, the transition will occur by the condensation of lipid molecules, until further compression of the PEO film is too energetically costly. This creates a relatively dehydrated polymer film and a mixed condensed/disordered lipid layer. As compared to the pure lipid, the condensed lipid phase cannot be complete. With this new physical model, several previously observed experimental phenomena can be better understood. For example, Naumann et al.12 have observed the dependence of the pressure and area of this second transition based on the molecular components. The transition of the shorter lipid occurred at a higher pressure. On the basis of our model, this occurs because the enthalpy of the lipid condensation, which drives the transition, is smaller for shorter lipids, and therefore more energy (greater pressure) needs to be applied to the system. When the PEO portion of the lipopolymer is increased, the transition pressure also increases. In this case, the greater thickness of the polymer layer increases the entropic loss in the polymer layer. In another example, both Naumann et al.12 and Baekmark et al.7 observed the increase in transition pressure as temperature increases. On the basis of our model, this is due to the fact that the transition is dependent on the entropy terms being diminished by pressure, whereas increased temperature increases the magnitude of the entropy terms. Theoretical Analysis. Our new model system obviously contends the second transition as the commonly (21) Liu, K.-J.; Parsons, J. L. Poly(ethylene glycols) 1969, 2, 529.

Figure 7. Schematic illustration of different conformations of lipopolymer PEO2K-DSPE at the air-water interface. The filled circle represents the DSPE anchoring ligand. (A) In the low surface pressure regime (π < the first transition pressure, π1), PEO spreads to form a thin two-dimensional layer with a thickness less than 1.5 nm. (B) In the medium surface pressure regime (π1 < π < the second transition pressure, π2), the adsorbed PEO chains desorb from the air-water interface into the aqueous subphase. The thicker lines represent fully hydrated PEO chains. The PEO chains can be in either a mushroom or a brush conformation. (C) In the high surface pressure regime (π > π2), the PEO chains dehydrate by expelling the associated water.

thought mushroom-to-brush transition in surface-confined PEO molecules. A theoretical analysis, based on scaling theory, also is against the second transition as the mushroom-to-brush transition. Scaling theories use grafting density (D, mean distance between grafting points) and the size of the polymer (N, degree of polymerization) to define the conformation of extended polymer chains in solutions.13 In the case of low grafting densities, the polymer may extend up to the Flory radius of the chain to form a mushroom conformation. The radius of the mushroom is comparable to the Flory radius of the chain, given by

RF ) aN3/5

(3)

where a is the size of a monomer. For PEO2K (45 monomers), RF ) 3.5 nm. With an increase in the polymer grafting density and/or length, the polymer chains start to interact at D ≈ RF. This causes the conformational extension of the chains into the brush conformation. On the basis of this model, the transition surface concentration of PEO-DSPE from the mushroom to the pancake conformation can be calculated. Assuming that the density of EO monomers in the pancake layer is constant, when the surface concentration of PEO2K-DSPE is greater than 0.29 mg/m2 (11.6 nm2 per PEO2K) the radius of the mushroom (R) is

R)

( ) Nm 45

3/5

RF

(4)

where Nm is the number of EO units per PEO2K that extend into the subphase to form a polymer mushroom. The mushroom-to-brush transition occurs when the area occupied by the polymer mushroom equals the area occupied by the remaining EO segments in the pancake

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layer; thus

45 - Nm πR2 ) A0 45

(5)

where A0 is the minimum area per PEO2K in the pancake layer, measured as 11.6 nm2. Solving eqs 4 and 5 results in a Nm of 13 and the corresponding surface concentration of 0.4 mg/m2 (8.25 nm2 per PEO2K) at the transition point. This surface concentration is only slightly higher than the first transition (9.04 nm2) and far away from the observed high-pressure transition at 1.4 mg/m2 (2.33 nm2 per PEO2K). The results of our analysis are consistent with another theoretical analysis regarding polymer brushes by end-capped PEO at the air-water interface by Barentin et al.22 They also suggest that the brush conformation occurs well before the second transition. The qualitative description of the structure of the spread monolayer of DSPE-PEO2K as a function of pressure is complete (Figure 7). At low surface densities, the PEO shares a single spread layer with the lipid tails, consistent with the idea of a two-dimensional pancake, a relatively thin layer at the interface (thickness ∼ 1.5 nm for grafted PEO2K19). At molecular areas below 11.6 nm2 (corresponding to the first transition), the PEO layer is forced from the interface, forming a layer below the surface. As the PEO desorbs from the surface during the transition, the interaction of the PEO in the sublayer leads to an increased surface pressure. For 100% DSPE-PEO2K, the PEO sublayer must be continuous with the PEO chains interacting with each other. Above the transition, the existence of what has been called a mushroom structure (22) Barentin, C.; Muller, P.; Joanny, J. F. Macromolecules 1998, 31,2198.

can at best be short-lived with the formation of a polymer brush soon after. The upper transition is the result of ordering of the lipid tails of the lipopolymers combined with dehydration of the PEO molecules. Below the transition, the PEO molecule is extended but hydrated. The lipid fraction is disordered. Above this transition, the PEO has been highly dehydrated. The lipid fraction is a mixture of condensed and disordered lipids. This is consistent with the large entropic loss characteristic to the upper transition.7 Conclusions Conformations of surface-confined short-chain PEO are revealed by combined film balance studies using monolayers of lipopolymer PEO2K-DSPE and its mixtures with DSPC and surface tension studies using PEO2K aqueous solution. The theoretical models based on the scaling theory developed for long-chain PEO fit experimental data well in the pancake conformation regime but poorly in the mushroom and brush regimes. It is proposed that the highpressure transition is caused by the ordering of the lipid tails of the lipopolymers, as driven by the enthalpic gain of the lipid alignment and balanced by (a) the entropic loss of the lipids and most importantly (b) the dehydration of the PEO chains in the subphase by expelling PEOassociated water molecules, but not by the transition from the mushroom to the brush conformation of surfaceconfined PEO molecules. Acknowledgment. We gratefully acknowledge the financial support of this work provided by National Institutes of Health Grant HL-40047 and the use of facilities at the Center for Cardiovascular Biomaterials. LA0006770