Lateral Distribution of a Poly(ethylene glycol)-Grafted Phospholipid in

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Lateral Distribution of a Poly(ethylene glycol)-Grafted Phospholipid in Phosphocholine Monolayers Studied by Epifluorescence Microscopy Kanwal Tanwir and Valeria Tsoukanova* Department of Chemistry, York UniVersity, Toronto, Ontario, Canada M3J 1P3 ReceiVed July 10, 2008. ReVised Manuscript ReceiVed September 10, 2008 Mixed monolayers of distearoylphosphatidylcholine (DSPC) and a poly(ethylene glycol)-(PEG)-grafted distearoylphosphatidylethanolamine with a PEG molecular weight of 2000, DSPE-PEG2000, spread on phosphatebuffered saline (PBS) were used as models of bio-non-fouling membrane-mimetic surfaces in order to visualize the lateral distribution of PEG2000-phospholipid in the host phospholipid matrix. Epifluorescence microscopy (EFM) was used to locate DSPE-PEG2000 molecules in the DSPC matrix by detecting the fluorescence from a fluorescein fluorophore attached to the distal end of the PEG2000 chain. Comparative analysis of surface pressure-area isotherms and EFM images revealed that DSPE-PEG2000 mixes nonideally with DSPC in monolayers on a PBS subphase. A transition from a phase-separated monolayer to a homogeneous mixture was observed with increasing surface pressure and PEG content. The effect of nonideal mixing behavior of DSPE-PEG2000 on its lateral distribution in the DSPC matrix was interpreted in terms of excluded volume interactions between the PEG2000 chains and a mismatch in the tilt of aliphatic chains on DSPC and DSPE-PEG2000 molecules.

1. Introduction Phospholipids bearing chemically grafted poly(ethylene glycol) (PEG) chains have been the focus of much interest because of their potential use in surface modifications for enhanced biocompatibility of various materials.1,2 Incorporating PEGgrafted phospholipids into self-assembled lipid matrixes (i.e., monolayers, bilayers, and vesicles/liposomes) is a versatile approach to engineering an array of bio-non-fouling membranemimetic surfaces for use as tissue-contacting layers on the surfaces of implantable materials, ligand-supporting membranes for biosensors, and vesicles for drug delivery. Bulky, highly hydrated PEG chains are believed to provide a steric repulsive barrier that prevents the nonspecific binding of dissolved biomolecules onto the membrane-mimetic surface,1,3,4 yet a number of studies have reported that membrane-mimetic surfaces bearing PEG chains with molecular weights of 1000-5000 Da are not always efficient at repelling serum proteins.1,5 This has stimulated a lot of research aimed at elucidating the effect of grafting density, conformation, packing modes, and interactions of grafted PEG chains on their bio-non-fouling properties.3-12 However, it is intuitively clear that for PEG chains to be successful at repelling dissolved biomolecules they should form a continuous, uniform graft completely shielding the membrane-mimetic surface. In the vast * To whom correspondence should be addressed. E-mail: [email protected]. (1) Vermette, P.; Meagher, L. Colloids Surf., B 2003, 28, 153. (2) Kim, K.; Shin, K.; Kim, H.; Kim, C.; Byun, Y. Langmuir 2004, 20, 5396. (3) Rex, S.; Zuckermann, M. J.; Lafleur, M.; Silvius, J. R. Biophys. J. 1998, 75, 2900. (4) Bianco-Peled, H.; Dori, Y.; Schneider, J.; Sung, L. P.; Satija, S.; Tirrell, M. Langmuir 2001, 17, 6931. (5) Efremova, N. V.; Sheth, S. R.; Leckband, D. E. Langmuir 2001, 17, 7628. (6) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. 1997, 101, 3122. (7) Kuhl, T. L.; Majewski, J.; Howes, P. B.; Kjaer, K.; von Nahmen, A.; Lee, K. Y. C.; Ocko, B.; Israelachvili, J. N.; Smith, G. S. J. Am. Chem. Soc. 1999, 121, 7682. (8) Baekmark, T. R.; Wiesenthal, T.; Kuhn, P.; Albersdorfer, A.; Nuyken, O.; Merkel, R. Langmuir 1999, 15, 3616. (9) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Langmuir 1999, 15, 7752. (10) Xu, Z.; Holland, N. B.; Marchant, R. E. Langmuir 2001, 17, 377. (11) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975. (12) Bickel, T.; Marques, C.; Jeppesen, C. Phys. ReV. E 2000, 62, 1124.

majority of studies, the lateral structure of PEG grafts on membrane-mimetic surfaces was assumed rather known; it was generally presumed that PEG-phospholipids were evenly distributed throughout the host phospholipid matrix.1,4-6 Within the past decade, however, it has become increasingly clear that PEG-grafted phospholipids do not distribute uniformly over the host phospholipid matrix but rather cluster at some locations into domains that are tens to hundreds of nanometers in diameter.2,3,13 This may lead to a graft structure where some locations on the membrane-mimetic surface will have an insufficiently dense PEG coating or will be directly exposed to dissolved biomolecules. Such gaps, even small, in PEG grafts on membrane-mimetic surfaces may have a tremendous effect on their interactions with dissolved biomolecules (e.g., serum proteins). The fact that the interactions of PEG-grafted membranemimetic surfaces with proteins were also found to depend on the size of binding proteins1,14 seems to support this idea. However, to elucidate the effect that the lateral structure may have on the interactions of PEG-grafted membrane-mimetic surfaces with dissolved biomolecules, one first has to address (i) whether the partitioning of PEG-phospholipid conjugates in phospholipid matrices is the rule, (ii) on what scale it occurs, and (iii) what factors control it. So far, the direct microscopic visualization of lateral structure was performed for only a few PEG-grafted monolayers in a very limited range of PEG grafting densities,2,13 which did not allow for full characterization and quantification of the lateral distribution of constituents in PEG-grafted membrane-mimetic surfaces. In this study, mixed monolayers of distearoylphosphocholine (DSPC) and poly(ethylene glycol)-(PEG)-grafted distearoylphosphoethanolamine with a PEG molecular weight of 2000, DSPE-PEG2000, were used as models of bio-non-fouling membrane-mimetic surfaces to visualize the lateral distribution of the PEG2000-phospholipid in the host phospholipid matrix. DSPC and DSPE-PEG2000 are among the components most commonly used in liposomal formulations.1,2 Binary mixtures (13) Tsukanova, V.; Salesse, C. 18th Canadian Conference on Surfaces, Ottawa, Canada, 2002. (14) Rahmati, K.; Koifman, J.; Tsoukanova, V. Colloids Surf., A 2008, 321, 181.

10.1021/la802205y CCC: $40.75  2008 American Chemical Society Published on Web 11/21/2008

Lateral Distribution of a PEG2000-Phospholipid

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Figure 1. Chemical structures of DSPE-PEG2000 and DSPC molecules.

of DSPC and DSPE-PEG2000 have also been extensively studied in monolayers, bilayers, and vesicles/liposomes as ideal models of biocompatible coatings, biosensor platforms, and drug-delivery systems.1,5,10,15,16 For none of these models, however, has the lateral distribution of DSPE-PEG2000 in the DSPC matrix been visualized to date. Mixtures of DSPC and DSPE-PEG2000 are especially suitable for monolayer studies because stearoyl chains prevent the dissolution and loss of these phospholipids from the interface.11 The advantage of monolayers as model systems is that they allow the precise control of parameters such as lateral (surface) pressure and area per phospholipid molecule, which is not possible with bilayer and vesicle models. Then, a wide range of variations in the PEG grafting density can be easily simulated in a well-controlled manner by monolayer compression. Moreover, the continuous observation of monolayer morphology upon compression with imaging techniques offers a unique opportunity to visualize the partitioning of PEG-phospholipid conjugates in phospholipid matrixes and how it changes with increasing polymer grafting density. However, most of the monolayer studies with DSPC/DSPE-PEG2000 mixtures have been performed so far at the air/water interface.1,10,15,16 Thus, we attempted this study to gain insight into the mixing behavior of DSPE-PEG2000 in DSPC monolayers spread on a subphase of physiological relevance (e.g., on phosphate-buffered saline (PBS)). The major emphasis of the present report will be on the lateral distribution of DSPEPEG2000 in the host DSPC matrix. We used epifluorescence microscopy (EFM) to locate DSPE-PEG2000 molecules in the DSPC matrix by detecting the fluorescence from a fluorescein fluorophore attached to the distal end of the PEG2000 chain. On the basis of EFM image analysis, we quantitatively assessed the partitioning of DSPE-PEG2000 molecules between coexisting phases in mixed monolayers at various PEG2000-phospholipid contents. We also identified factors controlling the lateral distribution of DSPE-PEG2000 in the DSPC matrix.

2. Materials and Methods 2.1. Materials. Poly(ethylene glycol)-grafted phospholipid with a PEG average molecular weight of 2000 {1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[poly(ethylene glycol)2000], i.e., DSPEPEG2000}, its fluorescent analog {1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[poly(ethylene glycol)2000-N′-carboxyfluorescein], i.e., DSPE-PEG2000Fluo}, and 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) were obtained from Avanti Polar Lipids and used as received. The chemical structures of DSPC and DSPE-PEG2000Fluo are presented in Figure 1. Stock solutions of DSPC and PEG2000-grafted phospholipids were prepared at concentrations of 0.1-0.6 mg/mL by dissolution in chloroform. Stock solutions were mixed in various molar ratios to obtain spreading solutions containing 1, 3, 6, and 9 mol % PEGphospholipid. All solutions were stored in the dark at 4 °C. Chloroform (15) Chou, T. H.; Chu, I. M. Colloids Surf., A 2002, 211, 267. (16) Chou, T. H.; Chu, I. M. Colloids Surf., B 2003, 27, 333.

was HPLC grade (Fisher Scientific Co., Whitby, Ontario, Canada). Phosphate-buffered saline (PBS) from Sigma containing 0.01 M phosphate salt, 0.12 M NaCl, and 0.0027 M KCl at pH 7.4 was used as the subphase. Deionized water produced by a Milli-Q Synthesis water purification system was used for buffer preparation. The specific resistivity of water was 18 × 106 Ω · cm (pH 5.6 in equilibrium with atmospheric carbon dioxide). 2.2. Methods. A KSV2000SP Langmuir trough (KSV Instruments Ltd., Finland) with an effective surface area of 75 × 760 mm2 was used to study the monolayer behavior of DSPC, PEG2000-grafted phospholipids, and their mixtures. The trough was thermostatted to maintain the subphase temperature at 20 ( 1 °C. A filter paper Wilhelmy plate was used to measure the surface pressure, π, to an accuracy of 0.1 mN/m. Mixed monolayers were spread onto the PBS subphase from a DSPC/DSPE-PEG2000 or DSPC/DSPE-PEG2000Fluo solution with various mol % PEGphospholipid. After 15 min to allow for evaporation of the solvent, monolayers were compressed at a rate of 10 mm/min. The surface pressure-area (π-A) isotherms were obtained via symmetric compression of mixed monolayers by two barriers. Area, A, in isotherm plots represents the mean area per phospholipid molecule. The lateral distribution of the PEG2000-phospholipid in the host DSPC matrix was visualized by epifluorescence microscopy (EFM). In our EFM experiments, the contrast was derived from differences in composition between regions in which the fluorescent analog of the PEG2000-phospholipid, DSPEPEG2000Fluo, preferentially partitions and DSPE-PEG2000Fluoexcluded domains. EFM images were acquired using a custom NIMA trough (NIMA Technology Ltd., U.K.) with an effective surface area of 70 × 460 mm2 interfaced with an upright Nikon Eclipse FN1 epifluorescence microscope (Nikon, Japan). The π-A isotherm measurement parameters were adjusted to precisely match those discussed above for the KSV2000SP trough. To observe the fluorescence from the fluorescein fluorophore attached to the distal end of the PEG2000 chain on the DSPE-PEG2000Fluo molecule, Nikon CFI infinity optics, a blue excitation filter set (B-1E filter combination, 480CWL excitation filter, 505LP dichroic mirror, and 540CWL barrier filter) and a 10× objective (Nikon CFIPlan 10) were used. The images were captured by a Hamamatsu CCD camera, ORCA ER(AG) (Hamamatsu, Japan) directly onto a computer screen using Simple PCI 6 software (Compix Inc., PA). Monolayer compression and EFM imaging were performed simultaneously. In each series of measurements, images most representative of the monolayer morphology were selected for analysis. Image analysis in terms of the percentage of dark domains, % dark domains, and the domain number density, Nd, was performed with the Quantify package of Simple PCI 6. In the Quantify package, the value of % dark domains is found by relating the area occupied by domains to the area of the image. The value of Nd represents the percentage of domains of a certain diameter and is calculated by relating their total area to the area occupied by all domains in the image. Plotted as a function of domain diameter, Nd provides the distribution of domain diameters in monolayer images. To obtain the values of % dark domains and Nd, at least three images were analyzed from different parts of the monolayer.

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Tanwir and TsoukanoVa

3. Results 3.1. Surface Pressure (π-A) Isotherms of Mixed DSPC/ DSPE-PEG2000 Monolayers on the PBS Subphase. The major goal of this study was the visualization of the morphology of mixed DSPC/DSPE-PEG2000 monolayers with an emphasis on the lateral distribution of the PEG2000-grafted phospholipid in the host DSPC matrix. This required the use of a fluorescent analog of the PEG2000-phospholipid, DSPE-PEG2000Fluo, to enable us to locate PEG2000-phospholipid molecules in mixed monolayers through the direct EFM detection of fluorescence from the fluorescein fluorophore attached to the distal end of the PEG2000 chain. However, when introduced into monolayers to enable EFM imaging, fluorescent analogs of phospholipids may alter the isotherms of phospholipid monolayers and may sometimes behave as impurities that also affect the monolayer morphology.17,18 That is why we performed a series of control measurements to evaluate the effect of DSPE-PEG2000Fluo on the isotherms as well as on the morphology of mixed DSPC/DSPE-PEG2000 monolayers. In these measurements, three types of mixed monolayers were used: (i) monolayers containing 1, 3, 6, and 9 mol % DSPE-PEG2000 in the host DSPC matrix, (ii) monolayers containing the same mol % of DSPE-PEG2000Fluo instead of DSPE-PEG2000, and (iii) monolayers in which only 0.5 mol % of their total PEG2000phospholipid content was replaced with DSPE-PEG2000Fluo to enable EFM imaging. At any given PEG2000-phospholipid content, the isotherms measured for the three types of mixed monolayers were virtually identical and superimposed on each other within the experimental error (data not shown). Mixed monolayers of types ii and iii containing DSPE-PEG2000Fluo also exhibited the same types of morphology patterns in their EFM images at comparable surface pressures and PEG2000phospholipid contents (data not shown). At 3-9 mol % of the fluorescent DSPE-PEG2000Fluo phospholipid in mixed monolayers (type ii), which would normally be considered to be an excessive amount of fluorescent probe,17,18 no artifacts were observed, and the morphology of these monolayers was absolutely identical to that of mixed monolayers containing only 0.5 mol % DSPE-PEG2000Fluo (type iii). Plausibly, the fluorescein fluorophore attached to the distal end of the flexible PEG2000 chain is somehow accommodated underneath the monolayer and does not interfere with the intermolecular interactions and molecular organization in the mixed monolayers to any significant extent. Thus, for clarity of discussion in this report, only the results obtained with the type ii mixed monolayers containing DSPC and DSPE-PEG2000Fluo as the PEG2000-phospholipid will be presented. We believe that conclusions about the lateral distribution of the PEG2000-grafted phospholipid in the host DSPC matrix based on these results are valid for mixed DSPC/ DSPE-PEG2000 monolayers as well. Figure 2 shows the π-A isotherms of mixed DSPC/DSPEPEG2000Fluo monolayers containing 1-9 mol % of the PEG2000-phospholipid spread on the PBS subphase. As seen in the Figure, all of the isotherms exhibit three major regions: (i) an expanded region below ∼8 mN/m, (ii) a pseudoplateau with a midpoint at ∼11 mN/m, and (iii) a low-compressibility region above ∼20 mN/m. Increasing PEG2000-phospholipid content shifted the mixed monolayer isotherms to the right by increasing the mean molecular area. It also caused a significant broadening of the pseudoplateau with a slight decrease in the π value at the onset of the plateau from 9 mN/m at 1 mol % PEG to 8 mN/m (17) McConnell, H. M. Annu. ReV. Phys. Chem. 1991, 42, 171. (18) Worthman, L. A.; Nag, K.; Davis, P. J.; Keough, K. M. W. Biophys. J. 1997, 72, 2569.

Figure 2. π-A isotherms of mixed DSPC/DSPE-PEG2000Fluo monolayers on the PBS subphase at 20 ( 1 °C with different mol % PEG: (a) 1, (b) 3, (c) 6, and (d) 9. Horizontal bars indicate the points of miscibility transition in mixed monolayers corresponding to the change in their morphology from heterogeneous “domain appearance” to a single homogeneous phase as observed with EFM.

at 9 mol %. In the low-compressibility region, the effect of PEG2000-phospholipid content on the isotherms of mixed monolayers was negligible. Above 40 mN/m, the isotherms of mixed monolayers containing 1-6 mol % PEG were virtually superimposed on each other whereas the isotherm for the monolayer containing 9 mol % PEG2000-phospholipid was shifted to larger areas by ∼0.03 nm2. The collapse pressure of the mixed monolayers increased from 59 mN/m at 1 mol % PEG2000-phospholipid in the host DSPC monolayer to 63 mN/m at 9 mol %. 3.2. Morphology of Mixed DSPC/DSPE-PEG2000 Monolayers on the PBS Subphase. As discussed above, the morphology of mixed monolayers was visualized with EFM by detecting the fluorescence from DSPE-PEG2000Fluo bearing a fluorescein fluorophore at the distal end of the PEG2000 chain. Hence, fluorescent areas in the images presented in Figure 3 directly report on the lateral distribution of the PEG2000-grafted phospholipid in the host DSPC matrix. Figure 3 shows typical morphologies seen in mixed monolayers containing 1 and 9 mol % DSPE-PEG2000Fluo (images A-C and D-F, respectively). As seen in the Figure, the morphology of mixed monolayers exhibits three types of lateral DSPE-PEG2000Fluo distributions. Dark circular DSPE-PEG2000Fluo-excluded domains in the fluorescent background seen in images A and D are typical of images captured from mixed monolayers at surface pressures below 10 mN/m. Upon further compression, “melting” of the domain boundaries occurred continually, and a morphology change to a single homogeneous fluorescent phase was observed for all mixed monolayers. However, the surface pressure at which this morphology change took place varied significantly depending on the PEG2000-phospholipid content in the monolayers. For the mixed monolayer containing 1 mol % PEG, the dark circular domains persisted throughout the isotherm plateau and the lowcompressibility region as seen in images B and C captured at π ≈ 13 and 40 mN/m, respectively. The monolayer eventually converted to a single homogeneous fluorescent phase at a surface pressure as high as ∼48 mN/m (i.e., immediately before the monolayer collapse, image not shown). By contrast, dark DSPEPEG2000Fluo-excluded domains disappeared rapidly upon


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Figure 3. Typical epifluorescence microscopy images of mixed DSPC/DSPE-PEG2000Fluo monolayers containing 1 mol % (A-C) and 9 mol % (D-F) PEG2000-phospholipid: (A, D) at the midpoint of the isotherm plateau at π ) 10 mN/m; (B, E) at the end of the isotherm plateau at π ) 13 mN/m, and (C, F) in the low-compressibility region at π ) 40 mN/m. Bright areas correspond to the phase containing fluorescent DSPEPEG2000Fluo. The image size is 250 × 250 µm2.

compression of the mixed monolayer containing 9 mol % PEG, and a single fluorescent phase was formed by the end of its isotherm plateau (i.e., at π ≈ 13 mN/m, Figure 3E). Surface pressures at which the single fluorescent phase appeared in mixed monolayers containing 3 and 6 mol % PEG fall in between those for 1 and 9 mol %. The points corresponding to the morphology change from a heterogeneous domain appearance to a single homogeneous fluorescent phase are marked with horizontal bars on the isotherms in Figure 2. For monolayers containing 3 and 6 mol % PEG, this morphology change was observed at ∼20 and ∼17 mN/m, respectively (images not shown). In the lowcompressibility region, yet another type of morphology was visualized for mixed monolayers containing 6 and 9 mol % DSPEPEG2000Fluo. Slightly above 30 mN/m, irregularly shaped, highly fluorescent DSPE-PEG2000Fluo-rich domains similar to the one seen in Figure 3F were observed sporadically in these monolayers. The change in monolayer morphology upon compression was examined in more detail by estimating the percentage of dark domains, % dark domains, in mixed monolayers at various surface pressures. The percentage of dark domains was calculated from EFM images taken at several points along the isotherms of mixed DSPC/DSPE-PEG2000Fluo monolayers and is plotted in Figure 4 as a function of surface pressure. As noted above, all mixed monolayers show the same trend of gradually decreasing percentage of dark DSPE-PEG2000Fluo-excluded domains as the surface pressure increases. Yet, as seen in the Figure, much higher surface pressure is required to convert the mixed monolayer containing 1 mol % PEG into a single fluorescent phase (0% dark domain) than in the case of monolayers containing 3-9 mol % PEG. In fact, at 1 mol % PEG the percentage of dark domains was rather high at all surface pressures whereas increasing PEG2000-phospholipid content in mixed monolayers resulted in a significantly decreased number of DSPEPEG2000Fluo-excluded domains at low surface pressures and

Figure 4. Percentage of dark DSPE-PEG2000Fluo-excluded domains per image as a function of surface pressure for mixed DSPC/DSPEPEG2000Fluo monolayers containing 1, 3, 6, and 9 mol % DSPEPEG2000Fluo as indicated. All data are for monolayers spread on the PBS subphase at T ) 20 ( 1 °C. Dashed lines are guides to the eye. Error bars indicate ( standard deviation.

their complete disappearance at high π. Indeed, at 1 mol % PEG, dark DSPE-PEG2000Fluo-excluded domains constitute approximately 52% of the monolayer at low surface pressure (π ≈ 5 mN/m). Upon increasing the PEG2000-phospholipid content to 3 mol %, the percentage of dark domains drops to 37% and then monotonously decreases to 28 and 21% with PEG2000phospholipid content increasing to 6 and 9 mol %, respectively. Overall, at any given π, the percentage of dark DSPEPEG2000Fluo-excluded domains was noticeably lower for mixed

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content increasing from 1 to 9 mol % obtained at the same π ≈ 5 mN/m. In image A in Figure 5 captured from a mixed monolayer containing 1 mol % PEG, large DSPE-PEG2000Fluo-excluded domains ranging from 15 to 27 µm in diameter coexist with many intermediate ones (5-10 µm in diameter) and fewer small domains of ∼2 µm in diameter. With increasing PEG2000phospholipid content, large domains disappear, and a morphology change to noticeably smaller DSPE-PEG2000Fluo-excluded domains is observed (see images A-D in Figure 5). At 9 mol % PEG, the mixed monolayer features predominantly small DSPE-PEG2000Fluo-excluded domains of rather uniform size (∼2-5 µm in diameter) that are evenly distributed in the fluorescent background (image D in Figure 5). This change in monolayer morphology with increasing PEG2000-phospholipid content is more clearly demonstrated by examining the distribution of DSPE-PEG2000Fluo-excluded domains of different diameters shown in Figure 6. Clustered columns in Figure 6 represent the domain number density, Nd, plotted as a function of diameter for DSPE-PEG2000Fluo-excluded domains observed in EFM images in Figure 5. As seen in Figure 6A, at 1 mol % PEG2000phospholipid in the mixed monolayer the distribution of DSPEPEG2000Fluo-excluded domain diameters is very asymmetric. It exhibits a maximum at ∼8 µm with a high domain number density of DSPE-PEG2000Fluo-excluded domains of 5-10 µm in diameter and a significant weight of large domains of various diameters (with a total domain number density of ∼18% for domain diameters ranging from 15 to 27 µm). With increasing PEG2000-phospholipid content, the domain diameter distribution function becomes less asymmetric by gradually losing weight on the side of large-diameter domains. The diameter distribution covers a range of ∼2-19 µm at 3 mol % PEG (Figure 6B) and becomes even narrower at 6 mol % (∼2-13 µm, Figure 6C). Eventually, at 9 mol % PEG the Nd function no longer shows a wide distribution of domain diameters but rather a maximum centered at ∼4 µm.

4. Discussion

Figure 5. Typical epifluorescence microscopy images of mixed DSPC/ DSPE-PEG2000Fluo monolayers in the expanded region of their isotherms. Images are captured at π ) 5 mN/m for monolayers containing 1 (A), 3 (B), 6 (C), and 9 mol % (D) PEG2000-phospholipid. Bright areas correspond to the phase containing fluorescent DSPE-PEG2000Fluo. The image size is 250 × 250 µm2.

DSPC/DSPE-PEG2000Fluo monolayers with higher PEGphospholipid contents. Observations discussed above suggest that the lateral distribution of DSPE-PEG2000Fluo in the host DSPC matrix varies depending not only on the surface pressure but also, to a large extent, on the PEG2000-phospholipid content. Figure 5 shows EFM images of mixed monolayers with PEG2000-phospholipid

The mixing of DSPC with PEG2000-grafted DSPE has been studied extensively in monolayers at the air/water interface, and a detailed discussion of isotherms of DSPC monolayers containing various PEG mol % can be found elsewhere.10,15,16 Compared to monolayers on water, spreading mixed DSPC/DSPE-PEG2000 and DSPC/DSPE-PEG2000Fluo monolayers on the PBS subphase results in isotherms with similar features that are slightly more expanded and shifted to larger molecular areas at low and intermediate surface pressures. Above 40 mN/m, the difference between the isotherms on water reported previously10,15,16 and the isotherms measured on the PBS subphase in the present study diminishes. Thus, the isotherms of mixed DSPC/DSPE-PEG2000 monolayers on the PBS subphase are comparable to the previously studied isotherms of similar DSPC/DSPE-PEG2000 contents on water, so only the most essential details will be discussed here. In brief, DSPC used in our study as the matrix for the PEG2000phospholipid forms a condensed-type monolayer on the PBS subphase at 20 °C. Similar to the previously reported data,15,16 DSPC isotherm lift-off was observed at 0.65 nm2/molecule and was immediately followed by a steep rise in surface pressure to the point of monolayer collapse at A ≈ 0.4 nm2/molecule and π ≈ 57 mN/m (data not shown). Introducing the PEG2000phospholipid into the DSPC matrix significantly increases the area per phospholipid molecule in mixed monolayers. For instance, 1 mol % DSPE-PEG2000Fluo added to the DSPC matrix induces a shift in the lift-off area of the π-A isotherm from 0.65 nm2/molecule (for a pure DSPC monolayer) to 0.9 nm2/molecule,


Figure 6. Domain number density, Nd, as a function of domain diameter for DSPE-PEG2000Fluo-excluded domains observed in mixed DSPC/ DSPE-PEG2000 monolayers containing 1, 3, 6, and 9 mol % PEG as indicated. Values of Nd are the percentage of domains of typical diameters seen in images in Figure 5. Each value of Nd was calculated by relating the area occupied by domains of a certain diameter to the total area covered with DSPE-PEG2000Fluo-excluded domains in the image. Calculations of domain number density for domain diameters in the range of 2-11 µm were performed with a domain diameter increment of 1.5 µm whereas for larger-diameter domains an increment of 2.5-3 µm was used. All data are for mixed monolayers at π ≈ 5 mN/m.

which is likely due to the need for extra area to accommodate grafted PEG2000 chains extended at the interface in the pancake conformation.6-10,19 Moreover, all mixed monolayer isotherms develop a pseudoplateau in a surface pressure range of ∼8-13 mN/m (Figure 2). A similar plateau was observed in the isotherms of PEG2000-grafted phospholipid monolayers on various subphases and is attributed to a conformational change in grafted PEG2000 chains.6-11,19 Also, given the fact that the plateau becomes more pronounced with increasing PEG2000-phospholipid content in mixed monolayers (Figure 2), it may be interpreted as the characteristic pseudoplateau associated with the transition (19) Jebrail, M.; Schmidt, R.; DeWolf, C. E.; Tsoukanova, V. Colloids Surf., A 2008, 321, 168.

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from pancake to pseudobrush conformation19,20 in the PEG2000 chain of the DSPE-PEG2000. Above the plateau, when the conformational transition in PEG2000 chains has occurred, the isotherms of DSPC and all of the mixed monolayers virtually converge, thus implying that grafted PEG2000 chains no longer require extra area in the monolayer plane (i.e., they are submerged in the subphase and buried underneath the phospholipid monolayer). For the PEG2000 chain to be completely buried underneath the monolayer, the area mismatch between the PEG2000 moiety and the phospholipid part of the DSPE-PEG20000 molecule has to be compensated for by DSPC molecules filling “gaps” between aliphatic chains of neighboring DSPE-PEG2000Fluo molecules. To estimate the area mismatch, one has to relate the effective cross-sectional area of the PEG2000 pseudobrush to the crosssectional area of two closely packed aliphatic chains. For the latter, a value of 0.38 nm2 was reported in the literature.21 By contrast, the effective cross-sectional area of the PEG2000 pseudobrush can be somewhere in between 2.6 and 12.3 nm2.3,14,19 A comparison of these values suggests that the area of the PEG2000 pseudobrush projected at the interface exceeds the area of the two aliphatic chains on the DSPE-PEG20000 molecule by at least a factor of 7. Hence, to bridge gaps in the aliphatic chain region due to the area mismatch and to enable the formation of entire condensed monolayers, at least six DSPC molecules should be accommodated above each PEG2000 chain and around each DSPE-PEG2000 molecule. This implies that, for the mixed monolayer isotherms to converge to that of DSPC in the lowcompressibility region, the two components, DSPC and the PEG2000-phospholipid, must be miscible to some extent. 4.1. Miscibility in Mixed DSPC/DSPE-PEG2000 Monolayers on the PBS Subphase. Miscibility in phospholipid monolayers containing PEG2000-grafted phospholipids has been investigated previously at the air/water interface.15,16 In particular, for mixed DSPC/DSPE-PEG2000 monolayers on water, nonideal mixing behavior was reported on the basis of the analysis of their isotherms in terms of the excess area of the mixture, Aexc. In general, assuming that the two components, DSPC and PEG2000phospholipid (either DSPE-PEG2000 or DSPE-PEG2000Fluo) form an ideal binary mixture or behave as completely immiscible, the mean molecular area, Aid/im, in their mixed monolayers should be the sum of the areas of separated individual components. At any given surface pressure, the relation between Aid / im and the mole fractions of the components, χDSPC and χDSPE-PEG2000, should then obey the equation

Aid ⁄im ) χDSPCADSPC + χDSPE-PEG2000 ADSPE-PEG2000

(1)

where ADSPC and ADSPE-PEG2000 are the molecular areas of DSPC and PEG2000-phospholipid in their pure monolayers, respectively, at the same surface pressure.15,16,22 Consequently, if the mixing behavior does not obey eq. 1, then the deviation from this additivity rule can be quantitatively expressed in terms of the excess area of the mixture, Aexc, as

Aexc ) A - Aid ⁄im ) A - (xDSPCADSPC + xDSPE-PEG2000 ADSPE-PEG2000) (2) where A is the actual area per molecule in the mixed monolayer as inferred from its isotherm.15,16 By definition, Aexc is zero if the two components form an ideal mixture or behave as completely (20) Tsukanova, V.; Salesse, C. J. Phys. Chem. B 2004, 108, 10754. (21) Helm, C. A.; Mo¨hwald, H.; Kjaer, K.; Als-Nielsen, J. Europhys. Lett. 1987, 4, 697. (22) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley: New-York, 1966.

14084 Langmuir, Vol. 24, No. 24, 2008


Table 1. Excess Area, Aexc, Calculated Using Equation 1 for Mixed DSPC/DSPE-PEG2000Fluo Monolayers at Various Surface Pressures Aexc, nm2 π, mN/m

1 mol % PEG

3 mol % PEG

6 mol % PEG

9 mol % PEG

5 10 15 25 35 50

0.04 0.03 0.03 0.02 0.02 0.02

-0.02 -0.03 -0.03 -0.02 -0.01 0

-0.08 -0.06 -0.05 -0.04 -0.03 -0.01

-0.12 -0.10 -0.08 -0.06 -0.04 -0.01

Each value of Aexc is an average over a set of five isotherm measurements. For all mixed monolayers, standard deviations for Aexc were within (0.01 nm2.

immiscible whereas any deviation from zero, either positive or negative, is evidence of nonideal miscibility in mixed monolayers.22 By analyzing isotherms in terms of Aexc, Chou et al.15,16 obtained positive deviations for mixed DSPC/DSPE-PEG2000 monolayers in the expanded region and a pseudoplateau of their isotherms. In the low-compressibility region, Aexc values became negative, thus indicating nonideal mixing behavior.15,16 Miscibility analysis for mixed DSPC/DSPE-PEG2000 and DSPC/ DSPE-PEG2000Fluo monolayers on the PBS subphase in the present study has yielded somewhat similar results that are summarized in Table 1. The mixed monolayer containing 1 mol % PEG2000-phospholipid showed positive Aexc values at all surface pressures, which might be an indication of repulsive interactions and/or poor miscibility between the two components. For monolayers containing 3-9 mol % PEG, negative Aexc values were obtained with negative deviations becoming more pronounced at higher PEG2000-phospholipid contents in monolayers. Presumably, this suggests more favorable interactions and/or better miscibility between the two components with increasing PEG2000-phospholipid content. Another argument in support of miscibility in mixed DSPC/ DSPE-PEG2000 monolayers comes from the observed trend of varying collapse pressure with increasing PEG2000-phospholipid content. According to the surface phase rule, if the two components are miscible, then the collapse pressure of their monolayers will change depending on the composition.15,16,22 By contrast, in the case of two completely immiscible components, their monolayers will exhibit two collapse points, each occurring at the same surface pressure regardless of monolayer composition.15,16,22 As discussed above and seen in Figure 2, the collapse pressures of mixed DSPC/DSPE-PEG2000 monolayers were higher than that of pure DSPC (∼57 mN/m) and increased gradually with increasing PEG2000-phospholipid content from 59 mN/m at 1 mol % PEG to 63 mN/m at 9 mol %. Such continuous growth of collapse pressure with increasing PEG2000phospholipid content is an indication of miscibility between the two components. Although increasing collapse pressures and nonzero Aexc values are clearly an indication of miscibility in the mixed DSPC/DSPEPEG2000Fluo monolayers, the two components might be nonideally miscible as noted above. In particular, immiscible “mixed phases”11,13,18 of different compositions may coexist in these monolayers, which is in fact suggested by the EFM images in Figures 4 and 5. As discussed above, dark DSPEPEG2000Fluo-excluded domains coexisted with the fluorescent DSPE-PEG2000Fluo-rich background in all of the mixed monolayers at π e 13 mN/m. In EFM images of binary mixtures containing nonfluorescent DSPC and fluorescent DSPEPEG2000Fluo, the dark domains obviously identify the DSPC phase. Then, the fluorescence background might be a mixed

Table 2. Values of % Dark Domains for the Case of Two Completely Immiscible Components Calculated Using Equation 4 for Mixed DSPC/DSPEPEG2000Fluo Monolayers at π ≈ 5 mN/m mol % PEG

XDSPCADSPC, nm2

A, nm2

Aexc, nm2

% dark domains (immiscible)

1 3 6 9

0.55 0.54 0.52 0.50

0.70 0.86 1.13 1.42

0.04 -0.02 -0.08 -0.12

81 ( 3 61 ( 1 42 ( 3.5 31 ( 4

a Standard deviations for the percentage of dark domains, (SD, were calculated as (SD ) [(Aexc/2A)2]1/2 in order to take into account a possible effect of Aexc on the mean molecular area of DSPC in mixed monolayers. (A more detailed explanation is provided in the text.)

phase rich in DSPE-PEG2000Fluo that also contains DSPC molecules. To determine whether the fluorescent background is indeed a mixed phase, we compared the percentage of dark domains observed in the images in Figure 5 to theoretical values calculated for the case of two completely immiscible components that separate between two single-component phases (i.e., dark DSPC domains and the fluorescent DSPE-PEG2000Fluo background). When the two components are completely immiscible, it is logical to assume that the percentage of dark domains, % dark domains, at a given surface pressure should simply depend on the mean molecular area of DSPC in mixed monolayers, χDSPCADSPC, and A as

% dark domains (immiscible) )

(

)

χDSPCADSPC 100% (3) A

In this equation, A is the mean molecular area of mixed monolayer inferred from its isotherm. Yet, as discussed above, for all of the mixed monolayers A deviates from the mean molecular area of completely immiscible mixture, Aid/im, by Aexc. Among other factors, this deviation might be due to a lateral organization in mixed monolayers that alters the area occupied by each DSPC molecule compared to ADSPC in the pure DSPC monolayer at the same surface pressure. To take this into account, the percentage of dark domains for the case of two completely immiscible components was calculated from

% dark domains (immiscible) )

(

)

χDSPCADSPC Aexc 100% + A 2A (4)

which is an average between the two extreme values, χDSPCADSPC/A and (χDSPCADSPC + Aexc)/A. The former corresponds to a lateral organization when each DSPC molecule in mixed monolayers occupies the same area as in the pure DSPC monolayer at a given surface pressure. The mean molecular area for DSPC in mixed monolayers then equals χDSPCADSPC. If, however, the area occupied by each DSPC molecule in mixed monolayers differs from that in a pure DSPC monolayer, then Aexc can account for this. In the latter case, the mean molecular area of DSPC in mixed monolayers will be χDSPCADSPC + Aexc. Eventually, relating both χDSPCADSPC and χDSPCADSPC + Aexc to A and taking the average as given by eq. 4 yields the percent of dark domains in mixed monolayers for the case of two completely immiscible components. The values for % dark domains (immiscible) together with χDSPCADSPC (where ADSPC ) 0.55 nm2),15,16 Aexc, and A used in our calculations are summarized in Table 2 for mixed monolayers at π ≈ 5 mN/ m. To demonstrate the difference between % dark domains calculated for the case of completely immiscible components and the actual percentage of dark DSPE-PEG2000Fluo excluded domains observed in the images in Figure 5, the two sets of data are plotted together in Figure 7. Curve a in Figure 7 shows the


Figure 7. Percentage of dark DSPE-PEG2000Fluo-excluded domains as a function of mol % PEG in mixed monolayers at π ) 5 mN/m. Points were plotted on the basis of the data for % dark domains (a) calculated for the case of two completely immiscible components in Table 2 and (b) obtained from image analysis in Figure 4. For the latter, only the starting points of curves in Figure 4 (at π ) 5 mN/m) were used.

calculated values for % dark domains (immiscible) plotted as a function of PEG content whereas curve b contains the values for % dark domains obtained from the image analysis in Figure 4 for all mixed monolayers at π ≈ 5 mN/m. As seen in Figure 7, the percentage of dark DSPE-PEG2000Fluo-excluded domains in the images of mixed monolayers (curve b) is noticeably smaller than what would be expected from theoretical calculations (curve a) if the two components were completely immiscible and DSPC molecules resided exclusively in DSPE-PEG2000Fluo-excluded domains. Interestingly, the comparison of values for data points on curve a to those on curve b in Figure 7 reveals that in all of the mixed monolayers at π ≈ 5 mN/m approximately 65% of DSPC molecules partition into domains. This implies that the rest of the DSPC molecules then must mix with DSPEPEG2000Fluo to stay outside the domains. Thus, the fluorescent background indeed contains both DSPC and DSPE-PEG2000Fluo, which means that at π e 13 mN/m the two components phaseseparate between the DSPC domains and mixed DSPC/DSPEPEG2000Fluo phase. The above analysis of miscibility in terms of Aexc, collapse pressures, and % dark domains has therefore demonstrated that the PEG2000-grafted phospholipid mixes nonideally with DSPC in monolayers on the PBS subphase, which is in good agreement with the previously reported data obtained at the air/water interface.15,16 In the expanded region and plateau of mixed monolayer isotherms, two immiscible phases have been identified as a single-component DSPC phase and a mixed DSPC/DSPEPEG2000Fluo phase, yet upon further compression, the mixing behavior changes remarkably. An almost ideal mixture of the two components is formed in monolayers containing 3-9 mol % PEG just above their isotherms plateau. By contrast, the monolayer containing 1 mol % PEG remains phase-separated at all surface pressures. A somewhat homogeneous mixture of the two components appears in this monolayer only immediately prior to monolayer collapse. 4.2. Evolution of the DSPE-PEG2000Fluo-Excluded Domains upon Compression. The findings above suggest that although DSPC does mix with the PEG2000-phospholipid the mixing behavior of the two components is significantly surfacepressure-dependent. Upon spreading, a phase-separated mono-

Langmuir, Vol. 24, No. 24, 2008 14085

layer was formed at all PEG2000-phospholipid contents studied. Hence, most of the DSPC molecules (∼65%) initially tend to assemble into single-component domains whereas only ∼35% of them reside in the mixed DSPC/DSPE-PEG2000Fluo phase. For a transition from the phase-separated monolayer to a homogeneous mixture to begin, the monolayer had to be compressed beyond the isotherm pseudoplateau. Thus, the following question arises: what is the driving force for the phase separation and miscibility transition in mixed DSPC/DSPEPEG2000 monolayers? A plausible answer can be found by comparing the properties of the two phospholipids as discussed below. Previous studies of the monolayer behavior of DSPC on aqueous subphases have shown that because of the strong cohesive interactions between C18 aliphatic chains, DSPC molecules form a predominantly liquid-condensed (LC) phase immediately upon spreading.10,15,16,23 Such an LC phase is characterized by a tight packing of aliphatic chains in the all-trans conformation.17 By contrast, DSPE-PEG2000 molecules, although bearing the same C18 aliphatic chains, tend to form a liquid-expanded (LE) phase.19 This is mainly because of the grafted PEG2000 chain that extends at the interface in the pancake conformation, thus creating a spatial barrier to the cohesive interactions between the aliphatic chains on DSPE-PEG2000 molecules. Indeed, each PEG2000 chain containing 45 -(CH2-CH2-O)- monomers with a crosssectional area of 0.35 × 0.9 nm2 will require approximately 14.2 nm2 in the headgroup region of the monolayer to accommodate all of its monomers at the interface in the pancake conformation.11,20 This will keep C18 chains at a large separation distance in a disordered/tilted state, thus making the cohesive interactions between them extremely weak, if possible at all.19,20 It is therefore not surprising that, when mixed with the LC phase forming phospholipids such as DSPC, DSPE-PEG2000 molecules are excluded from the LC domains because otherwise they would introduce disorder into the aliphatic chain packing17,18,24-26 in the LC phase of DSPC. As a result, a separation between two phases will first take place upon spreading. Then, the mismatch in the tilt of aliphatic chains of DSPC and DSPE-PEG2000 molecules at the phase boundaries will introduce line tension between the two phases and stabilize the domains.17,24-26 That is why the domains, although noticeably decreasing in diameter, persisted throughout the entire expanded region and pseudoplateau of mixed monolayer isotherms. For domain boundaries to disappear, the line tension had to decrease.24-26 From the discussion above, it follows that the line tension between the LC domains of DSPC and the DSPE-PEG2000-rich LE phase originates mainly from an interaction barrier associated with the mismatch in the ordering, in particular, tilt, of aliphatic chains in the two phases. To overcome this barrier preventing the two phases from mixing with each other, the tilt in the LE phase should adjust to match that in the LC phase.24-26 For this to occur, the C18 chains on DSPE-PEG2000 molecules should be brought close to each other, which is possible only after the PEG2000 chains undergo a conformational transition to pseudobrushes and submerge in the subphase, thus eliminating the spatial barrier holding the C18 chains at large separation distances. Indeed, as soon as the conformation transition in PEG2000 chains began (at the onset of the plateau region of mixed monolayer isotherms, 80.

(23) Kubo, I.; Adachi, S.; Maeda, H.; Seki, A. Thin Solid Films 2001, 393,

(24) Kuzmin, P. I.; Akimov, S. A.; Chizmadzhev, Y. A.; Zimmerberg, J.; Cohen, F. S. Biophys. J. 2005, 88, 1120. (25) Frolov, V. A. J.; Chizmadzhev, Y. A.; Cohen, F. S.; Zimmerberg, J. Biophys. J. 2006, 91, 189. (26) Coban, O.; Popov, J.; Burger, M.; Vobornik, D.; Johnston, L. J. Biophys. J. 2007, 92, 2842.

14086 Langmuir, Vol. 24, No. 24, 2008

π ≈ 8 mN/m), the DSPC domains decreased in diameter (cf. image A in Figure 5 and images A and B in Figure 3 captured below the isotherm plateau and in the plateau region for the mixed monolayer containing 1 mol % PEG), indicating that the line tension decreased and the two phases slowly started to interpenetrate each other. Upon further compression, increasing cohesive interactions between C18 chains of DSPC and DSPEPEG2000 should eventually eliminate the disorder and tilt mismatch in the aliphatic chain region of mixed monolayers. Consequently, this must render the line tension zero and trigger the miscibility transition from domains of micrometer diameters to a somewhat homogeneous mixture, which was indeed observed (images D and F in Figure 3 for the mixed monolayer containing 9 mol % PEG and Figure 4). At this point, however, our experimental data do not allow us to conclude whether the mixtures are truly homogeneous at the molecular level or contain nanodomains26 that are not distinguishable by EFM imaging (limited to 2 µm resolution) used in this study. Nevertheless, the interpretive scheme above seems to explain reasonably well the evolution of DSPE-PEG2000Fluo-excluded domains and the miscibility transition in mixed DSPC/DSPE-PEG2000 monolayers containing 3-9 mol % PEG. Yet the concept of the mismatch in the tilt of aliphatic chains as the driving force for the phase separation in mixed DSPC/DSPE-PEG2000 monolayers cannot satisfactorily describe the mixing behavior of the two components at 1 mol % PEG. In this monolayer, for the miscibility transition to occur, the surface pressure had to rise to as high as ∼48 mN/m, which is approximately 30 mN/m higher than the miscibility transition pressure in monolayers containing 3-9 mol % PEG. Apparently, some other mechanism(s) might control the lateral distribution of the PEG2000-phospholipid in the host DSPC monolayer, which is discussed below. 4.3. Effect of PEG Content on the Lateral Distribution of DSPE-PEG2000 in the DSPE Matrix. A comparative analysis of the EFM images in Figure 5 clearly indicates that the lateral distribution of DSPE-PEG2000 in the host DSPC matrix varies depending not only on surface pressure but also, to a large extent, on the PEG2000-phospholipid content. As summarized in Figure 7 (curve b), the percentage of dark DSPE-PEG2000Fluo-excluded domains in mixed monolayers at π ≈ 5 mN/m decreases from 52 to 21% with increasing PEG2000-phospholipid content from 1 to 9 mol %. The domains become noticeably smaller with a narrower distribution of their diameters (Figures 5 and 6). This demonstrates that, with increasing mol % PEG, the PEG2000phospholipid molecules do not merely take over a larger monolayer area but also become more evenly distributed in the host DSPC matrix. A similar trend was predicted by Rex et al.3 from Monte Carlo (MC) simulations of the properties of PEG2000-lipid-containing membrane surfaces at grafting densities comparable to PEG2000-phospholipid contents in mixed monolayers in the present study. Snapshot views of the MCsimulated surfaces3 revealed that, at all grafting densities, PEG2000-lipids tend to cluster together at random grafting points rather than distribute evenly throughout the membrane. For membranes containing 2 mol % PEG2000-lipids, large areas free from PEG2000 chains are present on the surface, whereas at 10 mol % the surface is densely populated with PEG2000 chains. At higher PEG contents, the “clustering pattern” in the lateral distribution of PEG2000 chains is less apparent yet still seen in the MC snapshots. Nevertheless, both theoretical results by Rex et al.3 and our experimental data indicate that higher PEG contents induce more homogeneous mixing in PEG2000containing phospholipid matrixes, which may have something to do with the conformational freedom and configurational entropy


of individual PEG2000 chains. In particular, excluded from LC domains of DSPC, DSPE-PEG2000 molecules are forced to cluster together in the LE phase. This will lead to local PEG “crowding” and steric repulsion between grafted PEG2000 chains as a result of excluded volume interactions.1,19 With increasing PEG2000-phospholipid content, steric repulsion will become stronger as a result of increasing excluded volume interactions between spatially confined PEG2000 chains that strive to preserve their conformational freedom and configurational entropy. Consequently, with increasing PEG content, PEG2000 chains will seek to stay as far apart as possible, thus forcing DSPEPEG2000 molecules to distribute more evenly in the host DSPC matrix. Therefore, the phase separation in mixed DSPC/DSPEPEG2000 monolayers will be controlled by two opposing processes. On one hand, mismatch in the tilt of aliphatic chains will cause the exclusion of DSPE-PEG2000 molecules from the LC phase of DSPC. On the other hand, steric repulsion due to excluded volume interactions between PEG2000 chains crowding the LE phase will promote the “infiltration” of DSPE-PEG2000 molecules into the LC phase. The latter is especially true for mixed monolayers at surface pressures above the isotherm plateau in which the PEG2000 chains underwent a conformational transition into pseudobrushes. However, for excluded volume interactions to become the dominating factor in the lateral distribution of the PEG2000-phospholipid in the host DSPC matrix, PEG2000 chains on neighboring DSPE-PEG2000 molecules must in fact exert a noticeable lateral pressure on each other. This can only be the case if the area available per PEG2000 chain in the mixed monolayer, APEG2000, is comparable to the effective cross-sectional area of the PEG2000 pseudobrush that was reported in the range of 2.6-12.3 nm2.3 The area APEG2000 can be assessed from the mean molecular area, A, in mixed monolayers assuming that, above the pseudoplateau, PEG2000 chains are completely submerged into the subphase and consequently occupy all of the area available underneath the monolayer while phospholipid molecules do not contribute to A “underneath the monolayer plane”.14 The latter implies that

APEG2000 )

A χDSPE-PEG2000

(5)

From this equation and the data in Figure 2, a value of 52 nm2 was obtained for APEG2000 in the mixed monolayer containing 1 mol % PEG at 20 mN/m (i.e., slightly above the isotherm plateau corresponding to the pancake-to-pseudobrush conformational transition). This value exceeds the effective cross-sectional area of the PEG2000 pseudobrush by at least a factor of 4. Most likely, at 1 mol % PEG, the excluded volume interactions will not be effective at preventing DSPE-PEG2000 molecules from being excluded from the DSPC LC phase because there is an area of 2.6-12.3 nm2 available per individual PEG2000 chain in the LE phase. This offers a plausible explanation as to why the mixed monolayer containing 1 mol % PEG2000-phopholipid in the host DSPC matrix remained phase-separated up to the point of monolayer collapse. By contrast, with increasing PEG2000-phospholipid content, values of APEG2000 calculated at π ≈ 20 mN/m abruptly decrease down to 18 nm2 at 3 mol % PEG and then to 9.5 and 7 nm2 at 6 and 9 mol % PEG, respectively. A comparison of these values to the effective cross-sectional area of the PEG2000 pseudobrush (2.6-12.3 nm2) implies that at 3-9 mol % PEG there is barely enough room underneath the mixed DSPC/DSPE-PEG2000 monolayers to accommodate PEG2000 chains. Under such circumstances, driven by excluded volume interactions, PEG2000 chains will force DSPE-PEG2000 molecules to distribute evenly over the entire host DSPC matrix.


That is why the transition from phase-separated to homogeneous mixtures takes place in mixed DSPC/DSPE-PEG2000 monolayers containing 3-9 mol % PEG at 13 g π g 20 mN/m (i.e., almost immediately after the conformational transition in PEG chains). Although the results above may seem to fall in a general line of thought that increasing PEG2000-phospholipid content favors a more homogeneous distribution of DSPE-PEG2000 molecules in the host DSPC matrix, one has to be cautious about high PEG contents because they may compromise the structural integrity and high-pressure stability of mixed monolayers.19,20 Indeed, the isotherm of the mixed monolayer containing 9 mol % PEG in Figure 2 exhibits a second slope discontinuity at ∼26 mN/m. This slope discontinuity is reminiscent of the high-pressure transition in the DSPE-PEG2000 monolayer, which was attributed to the monolayer collapse.19 Hence, at 9 mol % PEG in DSPC matrix, the excluded volume interactions between PEG2000 pseudobrushes can be so significant that they do not merely promote the homogeneous mixing of the two components but in fact might squeeze some of the DSPE-PEG2000 molecules out of the mixed monolayer. The appearance of bright, highly fluorescent DSPE-PEG2000Fluo-rich domains similar to the one seen in Figure 3F in a homogeneous monolayer seems to support this idea. However, this needs to be investigated further using imaging techniques such as Brewster angle microscopy or atomic force microscopy that report on the difference in height between the coexisting phases.19 However, this observation implies that there might exist a rather narrow range of mixed monolayer compositions with PEG content between 3 and 6 mol % where both a somewhat homogeneous distribution of DSPE-PEG2000 molecules and high pressure stability can be achieved. These findings may be useful for selecting mixture compositions while developing membrane-mimetic surfaces with controlled bio-nonfouling properties.

Langmuir, Vol. 24, No. 24, 2008 14087

5. Conclusions The lateral distribution of distearoylphosphoethanolamine grafted with PEG chains with a molecular weight of 2000, DSPEPEG2000, in the DSPC matrix was visualized with EFM in mixed monolayers spread on the PBS subphase at various surface pressures and PEG2000-phospholipid contents. A comparative analysis of surface pressure-area isotherms and EFM images revealed that DSPE-PEG2000 mixes nonideally with DSPC. At low surface pressures, two immiscible phases have been identified in mixed monolayers as a single-component DSPC phase and a mixed DSPC/DSPE-PEG2000 phase. Above 20 mN/m, an almost ideal mixture of the two components is formed in monolayers containing 3-9 mol % PEG. By contrast, the monolayer containing 1 mol % PEG2000-phospholipid remains phase-separated at all surface pressures. These observations led us to a novel suggestion that the lateral distribution of DSPEPEG2000 in the DSPC matrix is controlled by two opposing processes. On one hand, the mismatch in the tilt and the conformational order of aliphatic chains on the two molecules promotes the exclusion of DSPE-PEG2000 from the liquidcondensed DSPC domains and phase separation. On the other hand, steric repulsion due to excluded volume interactions between grafted PEG2000 chains tends to distribute DSPEPEG2000 molecules evenly in the DSPC matrix. At low PEG contents, the exclusion of DSPE-PEG2000 from the DSPC phase dominates, thus resulting in phase-separated monolayers whereas with increasing PEG2000-phospholipid content enhancing excluded volume interactions between grafted PEG2000 chains lead to a homogeneous distribution of DSPE-PEG2000 throughout the entire DSPC matrix. Acknowledgment. We thank the Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation for the financial support of this study. LA802205Y

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