ARTICLE pubs.acs.org/Langmuir
Insertion Mechanism of a Poly(ethylene oxide)-poly(butylene oxide) Block Copolymer into a DPPC Monolayer Danielle L. Leiske,† Brian Meckes,† Chad E. Miller,‡ Cynthia Wu,† Travis W. Walker,† Binhua Lin,§ Mati Meron,§ Howard A. Ketelson,|| Michael F. Toney,‡ and Gerald G. Fuller*,† †
Chemical Engineering Department, Stanford University, Stanford, California 94305, United States Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025, United States § CARS, University of Chicago, Chicago, Illinois 60637, United States Alcon Research, Ltd., Fort Worth, Texas 76134, United States
)
‡
ABSTRACT: Interactions between amphiphilic block copolymers and lipids are of medical interest for applications such as drug delivery and the restoration of damaged cell membranes. A series of monodisperse poly(ethylene oxide)-poly(butylene oxide) (EOBO) block copolymers were obtained with two ratios of hydrophilic/hydrophobic block lengths. We have explored the surface activity of EOBO at a clean interface and under 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers as a simple cell membrane model. At the same subphase concentration, EOBO achieved higher equilibrium surface pressures under DPPC compared to a bare interface, and the surface activity was improved with longer poly(butylene oxide) blocks. Further investigation of the DPPC/EOBO monolayers showed that combined films exhibited similar surface rheology compared to pure DPPC at the same surface pressures. DPPC/EOBO phase separation was observed in fluorescently doped monolayers, and within the liquid-expanded liquid-condensed coexistence region for DPPC, EOBO did not drastically alter the liquid-condensed domain shapes. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XRR) quantitatively confirmed that the lattice spacings and tilt of DPPC in lipid-rich regions of the monolayer were nearly equivalent to those of a pure DPPC monolayer at the same surface pressures.
’ INTRODUCTION A number of groups have studied the behavior of amphiphilic block copolymers at interfaces because of their potential applications in drug delivery, cell repair, and other medical fields. The commercially available family of molecules known as poloxamers (triblock copolymers of two poly(ethylene oxide) blocks sandwiching one poly(propylene oxide) block) has been of particular interest.1,2 When considering block copolymers for pharmaceutical use, biocompatibility and surface activity are important factors. The diblock copolymer of poly(ethylene oxide)-poly(butylene oxide) (EOBO) could be a valuable tool in the medical field because it is nontoxic and can be synthesized with good control over the molecular weight of the blocks.3 Although the insertion mechanism of poloxamers into lipid monolayers has been extensively studied by Lee and others,46 interactions between lipids and other block copolymers, such as EOBO, have received less attention. An advantage of EOBO over poloxamer is the ability to customize the total molecular weight and the ratio of the two blocks, thus tuning the hydrophiliclipophillic balance for a particular application. To begin to understand how EOBO might interact with a cell membrane, the effects of EOBO insertion into a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer were explored. DPPC was chosen as a representative biological membrane because phospholipids are r 2011 American Chemical Society
the major component of cell membranes. In addition, DPPC has been well studied, making these results comparable to others in the literature. Here, EOBO was synthesized with two different poly(butylene oxide) block lengths to explore the effects on the surface activity and insertion mechanism. Surface pressure versus area isotherms, interfacial viscoelasticity, fluorescence microscopy, grazing incidence X-ray diffraction, and X-ray reflectivity were utilized to measure the interfacial mechanical properties and molecular structure of EOBO, mixed DPPC/EOBO, and pure DPPC monolayers. Emphasis was placed on comparisons between pure DPPC and mixed DPPC/EOBO monolayers. Our results show that EOBO is surface-active and compressible at the interface. When EOBO adsorbed to an airwater interface in the presence of DPPC, phase separation was observed and the resulting DPPC-rich phase in the mixed monolayer exhibited similar molecular and mechanical properties to pure DPPC at equivalent surface pressures. These results imply that EOBO interacts with DPPC through a mechanism similar to that of poloxamer in spite of several differences between the polymers.5 Received: May 6, 2011 Revised: August 4, 2011 Published: August 11, 2011 11444
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Langmuir First, EOBO is more surface active than poloxamer, which reduces the concentration necessary to achieve similar surface pressures. Second, it was surprising that the polymer architecture (triblock versus diblock) did not significantly alter the configurations at the interface.
’ MATERIALS AND METHODS Materials. Poly(ethylene oxide)-poly(butylene oxide) (EOBO) samples were synthesized and characterized by Alcon Research, Ltd. The length of the poly(ethylene oxide) (EO) block was held constant (45 repeat units), and the poly(butylene oxide) (BO) block length was either 10.3 or 16.2 repeat units, denoted as EOBO-10.3 and EOBO-16.2 in the text. DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was purchased from Avanti Polar Lipids (Alabaster, AL). Texas red 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium (TR-DHPE) was purchased from Molecular Probes (now Invitrogen, Carlsbad, CA). Monolayers were spread from 1 mg/mL stock solutions in ethanol-stabilized chloroform (Sigma-Aldrich, St. Louis, MO). All experiments were performed at room temperature. Isotherms. DPPC was spread at a clean airwater interface in a Langmuir trough by touching microdrops of chloroform solutions to the surface. The spreading pressure was always less than 0.5 mN/m. A Langmuir minitrough (KSV, Espoo, Finland), equipped with symmetric barriers and a Wilhelmy plate, was used to measure the surface pressure. The barriers were placed 10 mm from the outside edges of the trough so that EOBO could be injected into the subphase from behind the barriers without disturbing the lipids. Chloroform was allowed to evaporate for 15 min; then EOBO was injected to a final subphase concentration of 0.070.5 ppm. Monolayers containing DPPC and adsorbed EOBO will be described as DPPC/EOBO or mixed monolayers in the text. The same experiments were completed with no lipids present for comparison. The subphase concentrations presented here are below the critical micelle concentration, meaning that the equilibrium surface pressure will vary with the subphase concentration.7 In addition, utilizing such low subphase concentrations results in a metastable state. As EOBO adsorbs to the airwater interface, the concentration of EOBO in the subphase, or reservoir, is depleted, thus the thermodynamic state is undesirable. However, the purpose of this study was to explore the surface activity of EOBO from an application perspective. Specifically, we chose to understand in detail how EOBO interacts with a model biological membrane over a variety of surface pressures. Because low dosages are typically desirable in most biological systems, low EOBO subphase concentrations were utilized. In addition, the high surface activity of EOBO necessitated low subphase concentrations in order to sample a wide range of surface pressures. All experiments were performed after a sufficient time had been given for EOBO to adsorb to the interface such that the surface pressure did not change over the time scale of the experiments. The results were found to be reproducible, and good agreement between methods was observed. To produce EOBO or mixed isotherms, the surface pressure was monitored until it reached equilibrium (Πequil). Once equilibrium was achieved, the barriers were compressed at a speed of 0.75 cm2/min. The trough area was expanded at a rate of 7.5 cm2/min to minimize the time needed for EOBO to return to the interface in the event that it was excluded. For surface pressure versus area isotherms, a subphase concentration of 0.07 ppm was utilized to ensure low driving forces of EOBO to the interface so that exclusion at high surface pressures would be evident in the expansion isotherms. The subphase concentration was increased to 0.5 ppm for other experiments (unless otherwise specified) to decrease the time required to reach equilibrium. Interfacial Shear Rheology. An interfacial stress rheometer (ISR)8 was used to measure the interfacial rheological properties of pure DPPC and mixed DPPC/EOBO films (prepared in the same
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manner as the isotherms). The Boussinesque numbers (the ratio of surface forces to bulk forces) for these materials were quite high, greater than 100, ensuring that the measured stress was explicitly due to the viscosity of the material at the interface. For the DPPC monolayers, barriers were compressed at a rate of 1.5 cm2/min while the interfacial rheology was monitored. For DPPC/EOBO films, measurements were taken while the polymers adsorbed to the interface. Once the equilibrium surface pressure was achieved, the barriers were compressed at the same speed up to 42 mN/m. Pressures greater than 42 mN/m were not achievable because of space constraints in the trough. Experiments were carried out at a frequency of 1 Hz with a strain amplitude of 0.029, which was found to be in the linear viscoelastic regime for these materials. Fluorescence Microscopy. An inverted epifluorescence microscope (Zeiss, Germany) with a 10 objective and a handmade Teflon Langmuir trough was used to image structures at the interface. Material was added to the interface from a 1 mg/mL solution of 99.8 mol % DPPC and 0.2 mol % TR-DHPE in chloroform. With this particular fluorophore, TR-DHPE is excluded from more ordered phases so that ordered domains appear dark.9,10 This trough was equipped with only one barrier; compression was not symmetric. A subphase concentration of 0.15 ppm EOBO was used for these experiments in order to observe the effects of EOBO on the shape and size of the liquid-condensed (LC) domains in the liquid-expandedliquid-condensed coexistence region of DPPC. After the injection of EOBO, 1 h was required for the surface pressure to reach 4 to 5 mN/m. (The surface pressure did not change if an additional half hour was given for adsorption.) Films were compressed slowly by hand while images of the interface were recorded with a CCD camera. Interfacial X-ray Scattering. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XRR) experiments were performed at the Advanced Photon Source of Argonne National Laboratory in Argonne, IL using ChemMatCARS Sector 15-ID-C. The wavelength was 1.24 Å, corresponding to 10 keV. This particular beamline is dedicated to liquid surface measurements and is equipped with a single-barrier Langmuir trough with a minimum compression speed of 10 cm2/min. GIXD and XRR have been reviewed elsewhere.1113 Experiments were done on a bare water interface, equilibrium EOBO16.2 (a surface pressure of 24 mN/m), an EOBO monolayer compressed to 35 mN/m, pure DPPC, and a DPPC/EOBO-16.2 interface at 30 mN/m. The DPPC/EOBO-16.2 interface was prepared as described above, and the subphase concentration of EOBO-16.2 was 0.5 ppm for all experiments. The equilibrium surface pressure under DPPC was 30 mN/m, and the pure DPPC monolayer was also measured at 30 mN/m so that the effect of EOBO on the structure of DPPC compared to a pure monolayer at equivalent surface pressures could be determined. The trough was kept in a helium environment and moved transverse to the X-ray beam between scans to avoid radiation damage to the samples. The surface pressure was monitored during all scans and was found to remain constant. DPPC forms a distorted hexagonal cell (Figure 1), resulting in two Bragg peaks.14 The peak at lower qxy corresponds to the {01}/{10} reflections, and the higher-q peak is the {11} reflection, where qxy = 2π/d. The lengths of the sides, a and b, and the corresponding angle, γ, can be calculated using the following equations: 2 2d11 jaj ¼ jbj ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ð2d11 Þ2 d10, 01
0qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1 2 2 B ð2d11 Þ d10, 01 C γ ¼ 2 arctan@ A d10, 01
ð1Þ
ð2Þ
The reflectivity was measured by a series of scans with overlapping wave vectors. The reported errors are a combination of statistical errors 11445
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Figure 1. Representation of a distorted hexagonal unit cell, where a = b and γ is not equal to 120°.
Figure 2. (a) Compressionexpansion profiles of EOBO-16.2 under a clean interface. The mean molecular area (MMA) of EOBO is a minimum possible area; it assumes that all EOBO in the system is present at the interface. The actual MMA is likely to be higher than the values shown here because some EOBO is likely to remain in the subphase. (b) A pure DPPC isotherm (black) is shown for reference compared to a mixed monolayer of DPPC and EOBO-16.2 (gray). Notice that the expansion profile of the mixed monolayer resembles a pure DPPC isotherm, indicating that EOBO was excluded from the interface at high surface pressures. The subphase concentration was 0.07 ppm. and the error obtained from patching the overlapping scans. The statistical errors include both counting statistics for the signal and the subtracted backgrounds. Patching error depends on how well the individual scans matched in the overlap regions. Note that a conservative approach was taken with the patching errors, which were added in quadrature. Simulations of XRR profiles utilized Parratt3215 and Motofit16 software packages. These packages allow the user to select a number of boxes with homogeneous electron density and thickness throughout the X-ray beam footprint. Because DPPC contains vertically aligned tail and headgroup regions with laterally homogeneous electron densities, two-layer box models were used to describe DPPC and DPPC/EOBO monolayers. The model also quantifies the root-meansquare roughness, σ, at the interfaces between the boxes.
’ RESULTS Isotherms. Characterization began with experiments to measure the surface activity of EOBO at bare interfaces and under DPPC. Initially, very low subphase concentrations (0.07 ppm) of EOBO were used to minimize the forces needed to drive EOBO to the interface in the event of exclusion from the surface into the subphase. The mean molecular area (MMA) reported in Figure 2a assumes that all EOBO in the system is present at the interface. This is the minimum possible MMA; the actual MMA is likely to be higher because of some EOBO remaining in the subphase. Even at these low subphase concentrations, EOBO was surface-active and compressible (Figure 2a). In fact, the gradual steepening of the isotherm near 500 Å2/molecule indicates a gradual transition from an expanded phase to a condensed one. The compression/expansion hysteresis shows that this transition was either irreversible on the timescale of the
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Figure 3. Equilibrium adsorption surface pressures of EOBO-10.3 and EOBO-16.2 under air (black) and DPPC (gray). The subphase concentration was 0.5 ppm.
Figure 4. Interfacial shear rheology of pure DPPC (squares), DPPC with EOBO 10.3 (triangles), and EOBO-16.2 (circles) in the subphase. Solid symbols represent the elastic modulus (G0 ); open symbols represent the viscous modulus (G00 ). The subphase concentration of EOBO was 0.5 ppm.
expansion (10 min) or that some EOBO was ejected from the interface into the subphase at pressures greater than 23 mN/m. At the same subphase concentration under high-moleculararea DPPC (Figure 2b), EOBO-16.2 adsorbed to an equilibrium surface pressure of 10 mN/m. Upon compression, the isotherm began to resemble that of pure DPPC near 25 mN/m or 42 Å2 per DPPC molecule. The expansion curve of the mixed isotherm regained the liquid-expandedliquid-condensed phase transition that is characteristic of DPPC. Similarities between the isotherms for pure DPPC and DPPC/EOBO imply that EOBO was excluded from the interface at high surface pressures in favor of DPPC, which is insoluble in the subphase. To explore the effects of changing the molecular weight of the BO block, equilibrium adsorption pressures were measured for the two EOBO lengths under air and DPPC with a subphase concentration of 0.5 ppm (Figure 3). The equilibrium pressures associated with EOBO-10.3 were roughly 10 mN/m lower than those for EOBO-16.2. In addition, the surface pressures in the presence of DPPC were 35 mN/m greater than the surface pressures at a clean interface. This difference was greater for EOBO-16.2 than for EOBO-10.3. Interfacial Shear Rheology. The interfacial mechanical properties of DPPC are shown in Figure 4. Below 10 mN/m, the mechanical properties were below the sensitivity limit of the instrument. Starting at 10 mN/m, DPPC was dominated by viscous or fluid behavior with no measurable elasticity. As the monolayer was compressed, the interfacial viscous modulus increased with surface pressure. Near 30 mN/m, the interfacial elastic modulus became measurable and increased rapidly with compression; however, the monolayer remained predominantly fluid with the interfacial viscous modulus greater than the elastic modulus for up to 42 mN/m, the limit of the experiment. The 11446
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Figure 5. Fluorescence micrographs of (ad) pure DPPC and (eh) DPPC/EOBO-16.2 monolayers at (a, e) 6, (b, f) 7, (c, g) 8, and (d, h) 9 mN/m. The scale bar is 50 μm. The subphase concentration was 0.15 ppm.
surface pressure at which the interfacial elastic modulus became measurable varied with different trials. When EOBO was added to the subphase, the viscosity was slightly reduced but the surface pressure dependence of the interfacial viscous modulus closely mimicked pure DPPC for both BO block lengths. The reduced interfacial elastic modulus of mixed monolayers was consistent with the overall reduction in viscosity; however, the interfacial elastic modulus values were somewhat inconsistent because they were near the sensitivity limit of the instrument. The surface rheology of EOBO alone was not measurable. Fluorescence Microscopy. Fluorescence micrographs of DPPC and DPPC/EOBO monolayers (Figure 5) were used to explore the insertion mechanism of EOBO. At 6 mN/m, the liquid-condensed (LC) domains of DPPC were just visible as small circular shapes (a); however, the DPPC/EOBO-16.2 monolayer (e) contained large circular domains roughly 50 μm in diameter in addition to smaller (10 μm) domains with distinct shapes. Because large circular domains have not been reported in pure DPPC monolayers, we suspect that these circular domains were primarily EOBO-16.2. The irregularly shaped domains were likely regions of ordered DPPC. Note that the irregular domains appeared to be both free on the surface and tethered to the circular domains. As the pure DPPC monolayer was compressed to 7 and 8 mN/ m, the dark domains (b, c) in the pure monolayer grew to roughly 8 μm in diameter and acquired shapes distinct to DPPC. The domain size was homogeneous throughout the interface, and the shapes were within the range reported by others.10,17 DPPC/ EOBO monolayers at equivalent surface pressures still contained large dark areas with rounded edges that were elongated, which we attribute to the asymmetric compression of the interface. The other domains resembled pure DPPC domains in shape; however, these domains were larger than pure DPPC, closer to 15 μm. Similar to the observations at 6 mN/m, these irregular domains were observed to be distributed throughout the surface and were in contact with the larger domains. By 9 mN/m, the pure DPPC domains (d) began to show sharp edges as the lipids became more condensed. The edges between the condensed and expanded phases began to blur in the mixed monolayer as well (h). The same experiments were performed with EOBO-10.3 and poloxamer 188 in the subphase, and similar structures were observed (not shown).
Figure 6. GIXD of DPPC (black) and DPPC/EOBO-16.2 (gray) at 30 mN/m. The lines are present to guide the eye. The subphase concentration was 0.5 ppm.
GIXD. EOBO-16.2 did not produce any Bragg peaks at the equilibrium pressure of 24 mN/m or when the surface film was compressed to 35 mN/m, indicating that the block copolymer formed an amorphous surface layer. DPPC at 30 mN/m produced two Bragg peaks, the {01}/{10} peak at qxy= 1.36 Å1 and the {11} peak at 1.47 Å1 (Figure 6). These values correspond to a distorted-hexagonal cell with side lengths of a = b = 5.08 Å with an angle, γ, of 114.8° (Figure 1). The resulting area per chain was 23.4 Å2, which is equivalent to 46.8 Å2/ molecule. The lipid molecules were tilted toward their nearest neighbor (NN) at an angle of 29.5° from the surface normal.18 These results are consistent with the literature.5 For the mixed interface, DPPC was spread over an area per DPPC molecule of 90.5 Å2. With the addition of EOBO-16.2, the equilibrium surface pressure was 30 mN/m. In the DPPC-rich phase, the {01}/{10} peak shifted to 1.38 Å1, resulting in a decrease in the size of the unit cell to a = b = 5.05 Å and in γ to 115.8°. Compared to pure DPPC at the same surface pressure, the area was reduced by 1.9% to 45.9 Å2/DPPC molecule. The increased breadth of the {01}/{10} peak indicates that EOBO decreased the correlation length of DPPC compared to that of a pure monolayer. XRR. The XRR profiles of DPPC-rich phases with and without EOBO-16.2 were subtly different (Figure 7a). The headgroup of pure DPPC at 30 mN/m was found to be 10.5 Å thick; this value was fixed for fitting the reflectivity profile of the mixed monolayer. The tail thickness, 14.5 Å, did not change significantly with the addition of EOBO (Table 1). However, the laterally averaged 11447
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Figure 7. (a) Normalized X-ray reflectivity of DPPC alone (black) and with EOBO-16.2 (gray) at 30 mN/m. The points represent data, and the curves were calculated using two-layer box model fits to the data, with the values given in Table 1. (b) Normalized electron density profiles using the above fits, where DPPC is black and DPPC/EOBO is gray. The EOBO subphase concentration was 0.5 ppm.
Table 1. Parameters Obtained from XRR Fits for DPPC and DPPC/EOBO-16.2 at 30 mN/m dhead (Å) F/FH2O σ (Å) dtail (Å) F/FH2O σ (Å) DPPC
10.5
1.31
4.2
14.5
0.90
3.1
DPPC/EOBO-16.2
10.5
1.30
2.9
14.7
0.82
2.9
electron density of both the headgroup and the tail decreased, which is apparent from Figure 7b. Although there was insufficient time to take measurements with EOBO-10.3 in the subphase, we expect that the results would be similar. The DPPC-rich phases would have a similar structure to that of pure DPPC at the same surface pressure, but this equilibrium surface pressure would be around 16 mN/m for the same subphase concentration. XRR of EOBO alone at the interface was also performed, but reflectivity profiles were only weakly oscillatory and good fits could not be obtained with model or model-independent techniques (not shown).
’ DISCUSSION Amphiphilic block copolymers show interesting behavior at interfaces. This behavior is characterized by the surface concentration, which is dependent on the solubility, molecular weight, amphiphilicity, and additional factors such as the block chemistry.19 At an airwater interface, amphiphilic block copolymers have been shown to adopt a brush conformation where the hydrophobic block is anchored to the interface and the hydrophilic block dangles in the water.20 Small-angle neutron scattering experiments confirmed that poly(ethylene oxide)poly(butylene oxide) block copolymers adopt the expected brush configuration when adsorbed onto hydrophobic solid surfaces;21 therefore, we expect EOBO to be present in brush form at an airwater interface. The poly(butylene oxide) block
should reside at the surface, and the poly(ethylene oxide) block dangles into the subphase. The behavior of EOBO block copolymers at interfaces has been studied by others with some differences in materials and methods.22,23 In studies by Hodges and co-workers, the molecular weight of each block was roughly half the length of the EOBO presented here (EO-23 BO-8 vs EO-45 BO-16.2). Their methods also differed; their material was spread directly on the interface from a chloromethane solution rather than introducing EOBO from the water subphase. It has been shown that soluble block copolymers are more effective at reducing surface tension when spread directly on the interface compared to equivalent volume concentrations adsorbed from the subphase, in part because soluble block copolymers tend to remain at the interface and do not desorb.24 It should be noted that the minimum area per molecule reported here (assuming that all EOBO was present at the interface) is similar to that of Hodges. Because it is unlikely that EOBO-16.2 would occupy the same molecular area with the same reduction in surface tension as a block copolymer with half the molecular weight, we expect that EOBO remained in the subphase during our experiments. In spite of these differences, we observed similar behavior in the surface pressure versus area isotherms at clean interfaces compared to Hodges et al. With both molecular weights, EOBO was found to be compressible with isotherms that exhibited an increase in slope with compression, an indication of the transition from an expanded phase to a more condensed phase.22 However, the lack of GIXD peaks showed that the polymer did not form an ordered layer. In the case of mixed monolayers, the adsorption of EOBO to the interface compressed DPPC, causing it to form a more condensed phase. Although some interactions between EOBO and DPPC were observed, the two chemical species appeared to remain largely phase separated, as evidenced by the fluorescence micrographs. Wu et al. were able to confirm the phase separation of P188 and DPPC by XRR and GIXD.5 They observed heterogeneous mixed monolayers that contained regions similar to ordered DPPC and amorphous P188. Although we did not observe two distinct phases in the X-ray experiments, in our case EOBO was injected from behind the barrier. We expect that the high surface activity of EOBO caused it to insert at the interface near the barrier before it had time to diffuse throughout the trough. Meanwhile, measurements were taken at the opposite end of the trough in a DPPC-rich region. Phase separation was visible during the fluorescence experiments because the trough volume was much smaller (25 mL compared to 260 mL) and EOBO was able to insert into all portions of the interface. The fact that EOBO did not enhance the rheological properties of DPPC indicates that interactions between EOBO and DPPC were limited to the interface and that EOBO was not able to couple with DPPC in a way that enhanced its mechanical strength. In addition, isotherms, fluorescence micrographs, GIXD, and XRR confirmed that interactions between EOBO and DPPC headgroups were minimal: only subtle differences between DPPC and DPPC/EOBO at 30 mN/m were quantifiable by GIXD and XRR. The addition of EOBO caused a shift in the {01}/{10} peak to higher qxy and a slight decrease in the DPPC tilt compared to that of the pure monolayer. However, these changes in DPPC packing were not significant enough to alter the interfacial rheological properties. The slight reduction in the viscosity of mixed monolayers compared to that of pure DPPC monolayers may be attributed to low-viscosity EOBO occupying 11448
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Langmuir a significant surface area of the monolayer, particularly at lower surface pressures. To our knowledge, this is the first study to report how block copolymers influence the interfacial rheological properties of lipid monolayers. The restoration of native mechanical properties after the insertion of EOBO would be desirable in cell membrane repair because dramatic changes in membrane fluidity could severely alter cell functionality.25 In spite of several fundamental differences between EOBO and poloxamers, these results unexpectedly demonstrated that the two EOBO samples in this study interact with DPPC through a mechanism similar to that for P188.5,26 EOBO adsorbs to the interface but remains phase-separated from DPPC at low surface pressures. At higher surface pressures, it is ejected from the interface into the subphase as indicated by expansion isotherms. One major difference between EOBO and poloxamer is the polymer architecture. Poloxamer is a triblock copolymer, whereas EOBO is a diblock copolymer. The additional EO block in the subphase could limit the packing abilities of poloxamer at the interface, but this did not contribute to major changes in the physical properties at equilibrium pressures. The effects of the increased bulkiness of poloxamer compared to EOBO may become more evident at higher surface pressures; however, these conditions were not studied in detail here. Second, EOBO can be synthesized to have a low polydispersity. This means that the observed surface behavior is applicable to all molecules, rather than being a result of some ensemble average, as is the case with the highly polydisperse poloxamer. Ultimately, one can elicit specific control over the surfactant properties of EOBO compared to those of poloxamer. Finally, and most importantly, the enhanced hydrophobicity of poly(butylene oxide) compared to that of poly(propylene oxide) makes EOBO far more effective at reducing surface tension compared to poloxamer. To obtain an equilibrium surface pressure of 30 mN/m in the presence of DPPC, a subphase concentration of 420 ppm was necessary for P188.26 In comparison, only 0.5 ppm of EOBO was required to reach the same surface pressure. We expect that enhanced surface activity would make EOBO advantageous over poloxamer for most medical applications because the dosages could be lowered by orders of magnitude. In addition, altering the length of the poly(butylene oxide) block allows one to tune the surface activity depending on the application. In the system studied here, changing the BO block length by six poly(butylene oxide) units reduced the equilibrium surface pressure by 10 mN/m.
’ SUMMARY We have shown that poly(ethylene oxide)-poly(butylene oxide) blocks are surface active and capable of being compressed. When introduced into the subphase of a DPPC monolayer with a high molecular area, EOBO adsorbs to the interface but remains phase separated from DPPC. The compression of the DPPC phase results in a mixed monolayer with regions of amorphous polymer and ordered DPPC, resulting in a surface with mechanical properties similar to those of pure DPPC. The similar lipid insertion mechanism between EOBO and poloxamer implies that EOBO, and possibly other block copolymer systems, could be substituted for a multitude of applications that have been studied for poloxamer. If such results hold true for materials with unique chemistry and architecture, then this would enable researchers to choose a diblock copolymer system on the basis of the desired chemistry and surface activity.
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’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT ChemMatCARS is principally supported by the National Science Foundation/Department of Energy under grant no. NSF/CHE-0822838. The Advanced Photon Source is supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-Eng-38. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program (P41RR001209). The project was also funded by Alcon Research, Ltd., and D.L.L. was funded by a Stanford graduate fellowship. ’ REFERENCES (1) Lee, R. C.; River, L. P.; Pan, F.; Ji, L.; Wollmann, R. L. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4524–4528. (2) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189–212. (3) Yang, Z.; Pickard, S.; Deng, N.; Barlow, R. J.; Attwood, D.; Booth, C. Macromolecules 1994, 27, 2371–2379. (4) Lee, R. C.; River, P. R.; Pan, F.; Ji, L.; Wollmann, R. L. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4524–4528. (5) Wu, G.; Majewski, J.; Ege, C.; Kjaer, K; Weygand, M. J.; Lee, K. Y. C. Biophys. J. 2005, 89, 3159–3173. (6) Chang, L.; Chang, Y.; Gau, C. J. Colloid Interface Sci. 2008, 322, 263–273. (7) Linse, P.; Hatton, T. A. Langmuir 1997, 13, 4066–4078. (8) Brooks, C. F.; Fuller, G. G.; Frank, C. W.; Robertson, C. R. Langmuir 1999, 15, 2450–2459. (9) Peters, R.; Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7183–7187. (10) Weis, R. M.; McConnell, H. M. Nature 1984, 310, 47–49. (11) Als-Nielsen, J.; Jacquemain, D.; Kjaer, K.; Leveiller, F.; Lahav, M.; Leiserowitz, L. Phys. Rep. 1994, 246, 251–313. (12) Schlossman, M. L.; Synal, D.; Guan, Y.; Meron, M.; SheaMcCarthy, G.; Huang, Z.; Acero, A.; Williams, S. M.; Rice, S. A.; Viccaro, P. J. Rev. Sci. Instrum. 1997, 68, 4372. (13) Meron, M.; Gebhardt, J.; Brewer, H.; Vicarro, J. P.; Lin, B. Eur. Phys. J. 2009, 167, 137–142. (14) Brezesinski, G.; Dietrich, A.; Struth, B.; B€ohm, C.; Bouwman, W. G.; Kjaer, K.; M€ ohwald, H. Chem. Phys. Lipids 1995, 76, 145–157. (15) Braun, C. Parratt32 program; Berlin Neutron Scattering Center (BENSC), Hahn-Meitner Institute: Berlin, 1999. (16) Nelson, A. J. Appl. Crystallogr. 2006, 39, 273–276. (17) McConlogue, C. W.; Vanderlick, T. K. Langmuir 1997, 13, 7158–7164. (18) Kaganer, V. M.; Peterson, I. R.; Kenn, R. M.; Shih, M. C.; Durbin, M.; Dutta, P. J. Chem. Phys. 1995, 102, 9412–9422. (19) F€orster, S.; Antonietti, M. Adv. Mater. 1998, 10, 195–217. (20) Bowers, J.; Zarbakhsh, A.; Webster, J. R. P.; Hutchings, L. R.; Richards, R. W. Langmuir 2001, 17, 131–139. (21) Griffiths, P. C.; Cosgrove, T.; Shar, J.; King, S. M.; Yu, G.; Booth, C.; Malmsten, M. Langmuir 1998, 14, 1779–1785. 11449
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