Interaction of Nonionic PEO−PPO Diblock Copolymers with Lipid

Biomacromolecules 2005, 6, 2678-2687

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Interaction of Nonionic PEO-PPO Diblock Copolymers with Lipid Bilayers Millicent A. Firestone*,† and So¨nke Seifert‡ Materials Science and Advanced Photon Source Divisions, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439 Received February 7, 2005; Revised Manuscript Received May 11, 2005

The relationship between the molecular architecture of a series of poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) diblock copolymers and the nature of their interactions with lipid bilayers has been studied using small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC). The number of molecular repeat units in the hydrophobic PPO block has been found to be a critical determinant of the nature of diblock copolymer-lipid bilayer association. For dimyristoyl-sn-glycero-3phosphocholine (DMPC)-based biomembrane structures, polymers whose PPO chain length approximates that of the acyl chains of the lipid bilayer yield highly ordered, expanded lamellar structures consistent with well-integrated (into the lipid bilayer) PPO blocks. Shorter diblock copolymers produce mixed lamellar and nonlamellar mesophases. The thermotropic phase behavior of the polymer-doped membrane systems is highly influenced by the presence and molecular architecture of the diblock copolymer, as evidenced by shifting of the main phase transition to higher temperatures, broadening of the main transition, and the appearance of other features. Studies of temperature-induced changes in the mesophase structure for compositions prepared with well-integrated PEO-PPO polymers indicate that they undergo reversible changes to a nonlamellar structure as the temperature is lowered. Increasing either the number of repeat units in the PEO block or the polymer concentration promotes a greater degree of structural ordering. Introduction The structural characteristics of aqueous solutions of amphiphilic, nonionic block copolymers have been studied extensively during the last several years. Of particular interest have been poly(ethylene oxide)-b-poly(propylene)oxideb-poly(ethylene oxide) triblock copolymers (abbreviated as PEO-PPO-PEO or EOn-POm-EOn), which have emerged as potentially important agents in biotechnology and molecular medicine.1 Recently, interest has shifted to investigations of their interactions with biological materials, including substrate-supported lipid monolayers,2 lipid bilayers,3 and vesicles.4-6 This interest stems primarily from their potential application as inexpensive, readily available substitutes for more expensive lipid-grafted polymers (lipopolymers) in the preparation of sterically stabilized (“Stealth”) liposomes for drug delivery.7 Another area of potential promise is their use in the sealing of damaged or permeabilized cell membranes following trauma.8-12 PEO-PPO-PEO triblock copolymers have also been shown to serve as an essential component in various biomimetic, self-assembled nanostructured materials.3,13,14 In prior work, we demonstrated that poly(ethylene glycol)grafted, lipid-based complex fluids, which are biomimetic, can be employed as scaffolding for the formation of organized arrays of either soluble or membrane proteins15 * To whom correspondence should be addressed. Phone: (630) 2528298. Fax: (630) 252-9151. E-mail: [email protected]. † Materials Science Division. ‡ Advanced Photon Source Division.

or for inorganic nanoparticles.14 In the latter case, a simple modification of the composition (i.e., increasing the number of repeat units on the lipid-appended PEG moieties) was shown to offer a facile means to modulate the interactions of encapsulated nanoparticles, as reflected in changes in their optical and electronic properties. Given that the physical properties of these materials are partly dependent upon the number of molecular repeat units in the PEG chain, it is clear that far greater tunability of materials properties could be achieved by developing similar formulations employing nonionic block copolymers, which, unlike PEG-lipid conjugates, are available in a wide range of molecular architectures. Essential to increasing the utility of nonionic block copolymers in these applications is an improved understanding of the factors that control their association with lipid bilayers. Recently, we reported a study detailing the parameters (e.g., number of repeat units in the PPO block) that control the mode of interaction of PEO-PPO-PEO triblock copolymers with lipid bilayers.3 In general, triblock copolymers were found to be weakly held within the lipid bilayer. At low temperatures (below ca. 10 °C), in fact, they were found not to be associated with the bilayer. Although the weak interaction of the triblock copolymers with the bilayer may offer advantages in certain applications (e.g., membrane healing), it may be detrimental in others (e.g., the fabrication of robust biomimetic nanostructured materials). This weak association may have its origins in any of several causes, among them poor integration of the PPO unit (due to

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mismatch between block length and acyl chain dimensions of the lipid bilayer), steric constraints imposed by the symmetric, flanking PEO blocks, or increased water solubility of the PPO block at reduced temperatures. The possibility that steric constraints weaken the copolymer-bilayer interactions suggests that replacement of the triblock architecture with the corresponding diblock copolymers, PEO-PPO (I) warrants investigation, particularly because removal of one of the symmetric PEO blocks renders the copolymer structurally similar to well-known alkane-oxyethylene (CnEOm) nonionic surfactants16 or PEG-grafted lipids.

Despite this potential advantage, little work has been directed at characterization of PEO-PPO diblock copolymers in the melt,17 their self-assembly in water, or their interaction with lipid bilayers.18,19 Recently, however, work examining aqueous mesophase formation of other PEO-based diblocks with polybutadiene (PEO-PBD), polyethylethylene (PEOPEE),20 or polybutylene oxide (PEO-PBO)21-23 has been reported. Currently, there is considerable interest in the potential use of PEO-PEE for polymersomes, robust vesicles that exhibit a 10-fold lower permeability to water than do phospholipid-based vesicles.24 In this report, we evaluate the effect of the molecular architecture of PEO-PPO diblock copolymers on their mode of interaction with a model biomembrane, a modification of the quaternary, PEGylated lipid-based complex fluid25 previously developed in this laboratory that (as noted above) has shown considerable potential as the basis of a variety of novel nanostructured materials and that can serve as a robust biomimetic membrane model system. Specifically, we employ small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC) to probe the structural perturbations caused by introducing various PEO-PPO diblock copolymers. Of particular interest are the effect of the molecular weight (length) of both the hydrophobic PPO block and the hydrophilic PEO block, and the impact of their association as a function of concentration and solvent quality (as modulated by variations in temperature). Experimental Section Materials and Methods. Lyophilized dimyristoyl-snglycero-3-phosphocholine (DMPC) and 1,2 dimyristoyl-snglycerol-3-phosphoethanolamine-N-poly(ethylene glycol) (DMPE-EO114 and DMPE-EO45) were purchased from Avanti Polar Lipids (Alabaster, AL) and used as received. Lauryldimethylamine-N-oxide (LDAO) was purchased from Calbiochem-Novabiochem Corp. (LaJolla, CA). All diblock copolymers were purchases from Polymer Sources (Dorval, QC, Canada), and triblock copolymers were obtained from BASF Corp. (Mount Olive, NJ) and used as received. Table 1 summarizes the characteristics of these polymers. Milli-Q (18 MΩ) water was used for sample preparation.

Table 1. Properties of PEO-PPO Diblock Copolymers Used in This Work diblock (EO-PO)

PO MW (g/mol)

no. of PO

EO MW (g/mol)

5000-1100 5200-1700 8000-1700 12 000-1700 6000-3500 F68 F127

1100 1700 1700 1700 3500 1738 3692

19 29 29 29 60 30 63

5000 5200 8000 1200 6000 6600 8624

no. of EO PO/EO 119 124 190 287 143 2 × 75 2 × 98

0.16 0.25 0.15 0.10 0.42 0.20 0.32

Sample Preparation. Block copolymer samples were prepared as quaternary compositions consisting of 0.755 ( 0.018 weight fraction water (Φw), 0.023 ( 0.005 weight fraction surfactant (Φs), 0.132 ( 0.013 weight fraction lipid (ΦL), and 0.0681 ( 0.017 weight fraction polymer (Φp). All samples were prepared such that the polymer-to-phospholipid ratio was held between 1.5 and 13 mol %, as specified in the text. Hydration of the solid components in deionized water was accomplished by repeated cycles of heating (50 °C), vortex mixing, and cooling on an ice bath until sample uniformity was achieved. Physical Methods. Small-angle X-ray scattering (SAXS) measurements were made using the instrument at undulator beamline 12ID-C (11 keV) of the Advanced Photon Source at Argonne National Laboratory. The sample-to-detector distance was such as to provide a detecting range for momentum transfer of 0.0025 < q < 0.6 Å-1. The scattering vector, q, was calibrated using a silver behenate standard at q ) 1.076 Å-1. The 2-D scattering images were first corrected for spatial distortion and sensitivity of the detector, and then radially averaged to produce plots of scattered intensity, I(q), versus scattering vector, q, where q ) 4π/λ(sin θ). The value of q is proportional to the inverse of the length scale, Å-1. Samples were sealed in 1.5 mm quartz capillaries. Temperature control of samples was achieved using a custom-built Peltier cooler. Thermal properties were measured by differential scanning calorimetry (DSC) at heating rates of 1 or 2 °C/min on a TA Instruments Q100 interfaced to a refrigerated cooling system. Instrument calibration was performed using an indium standard. Weighed amounts (5-12 mg) of the various compositions were sealed in aluminum pans and equilibrated at either -10 or 5 °C for 10 min. Phase transitions were recorded over a range from -10 to 70 °C. Successive heating scans were found to yield identical results. The transition temperature (Tm) was taken as the temperature at the peak of the endothermic transition. Transition enthalpies were determined by integration of the endothermic peak using the accompanying software package provided by TA Instruments. Results and Discussion To gain insight into the nature of the association of amphiphilic, nonionic diblock copolymers with lipid bilayers, the interactions of bilayers with PEO-PPO diblock copolymers of varying molecular architectures (Table 1) were examined by SAXS, WAXS, and thermal analysis. The

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Figure 1. Synchrotron small-angle X-ray scattering profiles collected for (A) 30 w/v% DMPC dispersion in water, quaternary mixture employing, (B) 5 mol % PEG5000-DMPE conjugate, (C) 5 mol % PEO5000-PPO1100 diblock copolymer, (D) 5 mol % PEO5200-PPO1700 diblock copolymer (E) 5 mol % PEO6000-PPO3500 diblock copolymer, and (F) 5 mol % F127 triblock copolymer. All measurements were made at 37 °C.

choice of polymers allows for assessment of the effect of the hydrophobic block length (PO molecular weight) and the EO chain length on the nature of association, the influence of the concentration of the incorporated diblock copolymer on the steric barrier produced (and thus, the aggregate structure), and the effect of temperature on the mode of insertion. The SAXS profiles collected at 37 °C on two well-characterized systems, a simple aqueous dispersion of a zwitterionic phospholipid, DMPC, and the PEGylated lipid (PEG5000-DMPE)-based mesophase, are presented in Figure 1A and B, respectively. The scattering profile of the dispersion of DMPC in water (30% (w/v)) is dominated by two Bragg reflections of integral-order spacing (q ) 0.099, 0.199 Å-1), indicative of one-dimensional lamellae with a periodicity, d, of 63 Å. This pattern is consistent with a sheetlike structure composed of lipid bilayers that are separated by a water layer. The addition of other components (e.g., polymer, cosurfactants) to this simple, binary mixture

is known to modify the balance of forces between the lipid molecules, producing new phases, often with interesting physical properties that can be exploited in the development of new materials.14,27 For example, prior work in this laboratory has explored the structure and physical properties of various polymer-grafted, lipid-based mesophases that consist of a quarternary mixture of a phospholipid DMPC, a polymer introduced as poly(ethylene) glycol (PEG) terminally grafted onto a phospholipid headgroup (dimyristoylphosphatidylethanolamine, DMPE-EOn), and a zwitterionic cosurfactant (N,N-dimethyldodecylamine-N-oxide, LDAO) in water.13 This material undergoes a thermoreversible phase transition at 16 °C, converting between a structured, elastic solid (gel) lamellar phase (LRg) and a lowviscosity, 2-D hexagonally ordered array of prolate micelles (H1).13 The SAXS curve (Figure 1B) for a quaternary compositions prepared with PEG5000-DMPE at a grafting density

Nonionic PEO-PPO Diblock Copolymers

Figure 2. Calorimetric heating curves: (A) 30 (w/v)% DMPC dispersion in water, scan rate 1 °C/min, (B) 5 mol % PEG5000 grafted lipid-based complex fluid composition, scan rate 2 °C/min, (C) 5 mol % PEO5000-PPO1100-based complex fluid composition, scan rate 2 °C/min, (D) 5 mol % PEO5200-PPO1700-based complex fluid composition, scan rate 2 °C/min, (E) 5 mol % PEO8000-PPO1700based complex fluids composition, scan rate 2 °C/min, (F) 5 mol % PEO12000-PPO1700-based complex fluid composition, scan rate 2 °C/min, (G) 5 mol % PEO6000-PPO3500-based complex fluid, scan rate 2 °C/min. Identical thermograms were obtained independent of the initial equilibration temperature.

of 5 mol % at 37 °C features five distinct diffraction peaks at integral-order spacing (q ) 0.0287, 0.0581, 0.868, 0.115, 0.145 Å-1), consistent with a highly ordered lamellar structure featuring a significantly increased (vs the DMPC aqueous dispersion) repeat distance of 218 Å. The observed ∼155 Å expansion of the lattice spacing in these materials (vs the simple aqueous dispersion of DMPC) is believed to arise from the steric pressure produced by the grafted PEG chains. (That is, a stable material is formed when the intervening water channel is large enough to accommodate a swollen polymer coil.) The scattering curves for the aqueous dispersion of DMPC and the DMPC/PEG5000DMPE/LDAO water mixture (Figure 1A and B, respectively), along with analogous results for a series of PEOPPO-PEO triblock copolymers,3 can now serve as standards against which to evaluate the effect of introducing various PEO-PPO diblock copolymers on the structure of the formed aggregate. Probing the Influence of PPO Architecture on Structure. The effect of the incorporation of a nonionic, diblock copolymer (PEO-PPO) on the membrane structure was assessed by replacing the PEG5000-DMPE component of the quaternary phases (described above) with 5 mol % of selected diblock (PEO-PPO) copolymers (PEO5000PPO1100, PEO5200-PPO1700, PEO6000-PPO3500), which feature increasing PO molecular weight (chain lengths of 19, 30, and 60, respectively), and PEO5200-PPO1700, PEO8000-PPO1700, and PEO12000-PPO1700, which feature increasing EO molecular weight (chain lengths of 124, 190, and 287). Compositions prepared with the PEO5000-PPO1100 and PEO6000-PPO3500 diblock copolymers were found to yield opaque samples, while the PEO5200-PPO1700 and PEO12000-PPO1700 diblockbased compositions yielded materials that were optically transparent, birefringent gels at room temperature (22 °C).

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PEO8000-PPO1700 diblock-based compositions yielded an optically transparent, birefringent liquid at room temperature. The first of the diblock copolymers, PEO5000-PPO1100 (Table 1), possesses a very short propylene oxide (PO) chain length (only 19 molecular repeat units). The radius of gyration (Rg) of the PPO block can be calculated (assuming a Gaussian chain) as approximately 9 Å, a length substantially less than the predicted dimension of the hydrophobic part of the lipid (here, DMPC) bilayer, which is ca. 20 Å.26,27 A typical SAXS profile collected on the PEO5000-PPO1100 diblock copolymer-containing (5 mol %) sample at 37 °C is presented in Figure 1C. The scattering profile features three sharp diffraction features at q ) 0.0280, 0.111, 0.221 Å-1 and several broad shoulders positioned at ca. q ) 0.060, 0.077, 0.13, 0.195 Å-1. These features reside atop a broad background scattering, which can be attributed to structure arising from the form factor of a micellar component. The complexity of the pattern suggests that the material is likely a mixed phase that comprises both lamellar and nonlamellar structures. The existence of such a phase is likely a reflection of the poor integration of the diblock copolymer arising from a mismatch between the length of the hydrophobic PPO chain and the acyl chain dimensions of the lipid bilayer. Interestingly, unlike prior studies showing that triblock copolymers with short PPO segments (e.g., BASF F38, which has 15 PO repeat units) generate a lamellar system with increased spatial coherence (broad diffraction peaks),3 the structurally analogous diblock copolymer used here produces a mixed phase. Replacement of this diblock copolymer with one that contains 29 molecular repeat units in the PO block (Rg ≈ 12 Å), PEO5200-PPO1700, yields the scattering results shown in Figure 1D. The scattering curve features five, sharp diffraction peaks (at q ) 0.0334, 0.0679, 0.102, 0.136, 0.170 Å-1), corresponding to a well-ordered, one-dimensional lamellar phase with periodicity of 188 Å, a pattern consistent with the formation of a swollen or expanded lamellar structure similar to those produced by incorporating the PEG-lipid conjugate. In contrast, the scattering profile obtained on compositions prepared with Pluronic F68, a triblock copolymer possessing the same number of molecular repeat units in the central PPO unit (30 repeats) but with two symmetrical blocks of PEO (75 repeats), was found to produce an aggregate structure similar to that of a simple aqueous dispersion of DMPC, suggestive of inadequate association of the polymer in the lipid bilayer.3 Thus, although the tri- and diblock copolymers contain the same number of PO units, they give rise to vastly different lamellar phase structures. For the triblock copolymer, the structure suggests poor PPO integration into the hydrophobic acyl chain region of the lipid bilayer. The diblock copolymer, however, appears to be well-anchored in the lipid bilayer. The poor integration of the triblock copolymer is believed to arise from a PPO block length insufficient to fully span the membrane, a configuration allowing for the symmetric ethylene oxide (EO) blocks to protrude into the intervening water channels on either side of the membrane. The “flat” configuration adopted by a poorly inserted triblock copoly-

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mer, it should be noted, is believed to promote lateral spreading of the EO chains along the surface of the membrane, a polymer conformation better suited for sealing defects in a permeablized cell membrane.28 PEO6000-PPO3500, in which the PO block length has been doubled to 60 molecular repeat units, also gives rise to a swollen lamellar structure, as evidenced by a SAXS curve (Figure 1E) that exhibits five sharp Bragg peaks of integralorder spacing (q ) 0.0398, 0.0800, 0.120, 0.161, 0.199 Å-1), corresponding to a periodicity of 158 Å. The scattering curve also contains a small peak at q ) 0.061 Å-1, which may signal the emergence of a second phase, and may thus indicate that the upper limit of the length of the hydrophobic block yielding a stable bilayer has been reached. Interestingly, a remarkably similar scattering profile is observed for the corresponding triblock copolymer, F127 (Figure 1F), which also contains 63 molecular repeat units in the PO central core. Here too, the scattering curve features five Bragg peaks (q ) 0.0431, 0.0869, 0.130, 0.174, 0.200 Å-1) indicative of a lamellar structure of nearly the same lattice dimensions (d ) 146 Å). As in the case of the diblock, a small peak at 0.068 Å-1 is observed again, suggesting the possible emergence of a second phase. Taken together, the results for the diblock copolymers indicate that, although there is a minimum required PPO chain length for optimal integration into the lipid bilayer to produce the swollen lamellar phases, that length is less than that required for the corresponding triblock copolymer. This is not entirely unexpected, given that, structurally, the diblock copolymers are more like the PEG-lipid conjugates in that they lack the geometric constraints imposed by the two symmetric PEO blocks of the triblock copolymers. Therefore, in general, triblock copolymers may be better suited for clinical use in the treatment of permeabilized membranes, while diblock copolymers are more suitable for the synthesis of robust nanoscale biomimetic materials for applications as drug delivery vehicles or scaffolds for the organization of membrane proteins. Influence of Diblock Copolymer Architecture on the Phase Transition. The thermotropic phase behavior of lipid membrane systems is strongly influenced by the presence of guest molecules. In particular, characteristics (e.g., position, enthalpy) of the thermally induced gel-liquid (LβLR) phase transition are determined by the nature of the interactions between the membrane constituents and by the topology of the guest (here, polymer). The DSC heating profile (1 °C/min) obtained for a 30 w/v % aqueous solution of DMPC is presented in Figure 2A. This profile exhibits a pretransition centered at 14.35 °C and a narrow, endothermic peak (fwhm ) 0.64 °C) centered at 23.30 °C corresponding to the main hydrocarbon chain melting transition (i.e., ripple phase, Pβ, to a lamellar fluid phase, LR). As shown in Figure 2B, the differential heating scan for the quaternary phase prepared with 5 mol % PEG5000-DMPE exhibits a more complex phase transition in the temperature range of 1331 °C. This transition features an asymmetric profile comprising two components, a peak centered at Tm ) 23.15 °C and a shoulder at 18.5 °C. The enthalpy associated with the major peak is smaller (3.92 J/g) than that for the DMPC

Firestone and Seifert

alone (11.50 J/g). In addition, the main peak is considerably broader (fwhm ) 6.3 °C) than that of the DMPC dispersion. The width of a transition is an important indicator of the size of the cooperative unit that undergoes the transition and is inversely proportional to the cooperativity of the phase transition.13 Thus, the wide main transition for the PEG5000based quaternary phase signals a highly noncooperative event. Replacement of the PEGylated lipid with the 5 mol % PEO5000-PPO1100 diblock copolymer, a polymer that features only 19 PPO units, yields the DSC profile presented in Figure 2C. The heating scan looks similar to that recorded for compositions prepared with the PEG-lipid conjugate; that is, the thermogram exhibits a broad, asymmetric peak. This endothermic transition, which is centered at 25.2 °C, is broader (fwhm ) 7.07 °C), however, than that observed for the PEG-lipid composition, reflecting greater noncooperativity of the transition. The increase in both the Tm and the enthalpy (8.25 J/g) versus the PEG-lipid composition, while atypical for lipid systems, has been observed previously for certain peptide-lipid systems.29 More importantly, although the scattering data suggest poor integration of this particular diblock copolymer into the lipid bilayer, only subtle changes are observed in the phase transition. The influence of increasing the number of molecular repeats in the PEO block from 124 to 190 or 287 on the characteristics of the phase transition can be seen by comparing the heating scans collected for PEO5200PPO1700, PEO8000-PPO1700, and PEO12000-PPO1700 shown in Figure 2D-F, respectively. Thermotropic scans collected on samples incorporating 5 mol % PEO5200PPO1700 (Figure 2D) exhibit a broad, very complex broad thermotropic phase transition. There is a small pretransition at 10 °C, followed by a sharp peak at 14 °C and the main transition at 24.2 °C. In addition, there is a small transition at 33 °C. In contrast, the thermotropic phase transition for the PEO8000-PPO1700-based composition (Figure 2E) is more consistent with that observed for the PEO5000PPO1100-based composition, exhibiting a broad (fwhm ) 5.45 °C) asymmetric endothermic peak shifted to a higher temperature (26.07 °C). The profile of PEO12000-PPO1700based materials is considerably narrower (fwhm ) 2.85) and centered at 22.02 °C. Clearly, significant changes are observed in the heating curves collected on the diblocks copolymers, but these changes do not readily correlate with any particular change in the molecular architecture of the diblock copolymers. The observed shifts in the position of the phase transition may signal changes in host-guest interactions. Frequently, introduction of a guest species into a lipid bilayer acts as a “defect”, increasing the disorder (increasing local membrane fluidity) of the membrane and thus lowering the Tm.29 If the guest fits well into the membrane, however, the hydrophobic forces may act synergistically to increase the local order and rigidity of the membrane, leading to an increase in Tm and a decrease in the width of the transition.29 Similar thermograms featuring either two peaks or a pronounced shift in a single peak have been observed previously in lipid-DNA and binary lipid mixtures, a result that has been attributed

Nonionic PEO-PPO Diblock Copolymers

to nonideal mixing.30,31 (It should be noted however, that scattering data collected on these samples provide no indication of a distinct structural phase. Moreover, macroscopic-phase separation was not observed.) Prior work has shown that nonideal mixing of phospholipids can occur in systems composed of lipids with a large difference in the acyl chains or by altering the charge of the headgroup region.33,34 DSC studies of the interaction of PEGylated phospholipids with chlolesterol-phospholipid mixtures have also found shifts in the position and broadening of the main transition, as well as the appearance of new shoulders and peaks, all of which were attributed to solubilization of the bilayer to mixed micelles.42 An alternative explanation for the increase in Tm may be the dehydrating effect of poly(ethylene glycol), which has also been shown to increase the Lβ to LR and H11 phase transitions in lipid bilayers.32 Finally, thermograms of compositions prepared with 5 mol % PEO6000-PPO3500 (Figure 2G) were found to show two broad endothermic transitions, one centered at 5.96 °C (5.17 J/g), and a second at 35.26 °C (3.65 J/g). Although the exact origin of the two peaks is unclear at present, DSC studies of the diblock alone dispersed in water (at the same concentration) show a peak positioned at 13.30 °C. Therefore, the lowtemperature peak may reflect a population of PEO6000PPO3500 micelles, while the high-temperature transition may arise from the portion of the diblock associated with the lipid bilayer. Interestingly, the dramatic changes in the thermograms for compositions prepared with the diblock copolymers were not observed for analogous systems incorporating the corresponding triblock copolymers. This obviously indicates that the interaction of the diblock copolymers with the lipid bilayers is significantly different from that with the triblock copolymers. The observed changes in the phase transition may be attributed to a lipid bilayer that fragments (lateral segregation) into domains that are rich in the diblock coplolymer and regions that remain as pure lipid.35 Increased thermodynamic stability of the mesophases was not observed for the triblock copolymer-based systems, and the increase in the transition does not scale with increased number of PEO units, making it is less likely that the observed changes in thermal transitions are due to dehydration of the lipid headgroup region and increased hydration of the grafted PEO.36 Finally, it should be noted that the polyoxyalkylene block copolymers used in this study do have a certain degree of polydispersity, which could influence the observed thermal properties. Further work is clearly required to fully elucidate the origin of the apparent increase in thermodynamic stability of mesophases incorporating certain diblock copolymers. Effect of Temperature on Mesophase Structure. Polymer aggregation processes leading to supramolecular structures depend not only on molecular architecture, but also on polymer-solvent interactions (i.e., solvent quality), which are determined, in part, by temperature. The aqueous solubility of PPO, for example, exhibits dramatic temperature dependence. Below ca. 15 °C (at ambient pressure), water is a good solvent for PPO, while at higher temperatures, PPO becomes more hydrophobic and, hence, less soluble in water.27 Conversely, PEO is hydrophilic and remains water soluble within the temperature range from 0 to 100 °C, but

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Figure 3. Semi-log plot of small-angle X-ray scattering curves collected on quaternary compositions prepared with 5 mol % PEO5000-PPO1100 at 10, 25, and 37 °C. SAXS profiles on quaternary compositions.

the reduced solvent quality that accompanies increasing temperature causes chain contraction.37 Although there have been several reports describing the effect of temperature on PEO-PPO-PEO triblock copolymer association with lipid bilayers,3,6 little information is available concerning the temperature-dependent association of PEO-PPO diblock copolymers with lipid bilayers. A detailed study of the effect of temperature on the structure of the PEO-PPO diblock copolymer-based systems was therefore carried out at three temperatures: 8-10, 25, and (because of the potential in vivo applications of these systems) 37 °C (physiological temperatures). Figure 3 shows the temperature-dependence of the scattered X-ray intensity as a function of scattering vector for compositions prepared with 5 mol % PEO5000-PPO1100. At a sample temperature of 10 °C, the scattering curve exhibits three broad Bragg peaks of integral-order spacing (q ) 0.0989, 0.194, 0.294 Å-1) that index to a lamellar structure with periodicity of 63.5 Å, a dimension typically observed for a simple aqueous dispersion of DMPC (Figure 1A). In addition, two smaller peaks are also observed in the low q region at q ) 0.0377, 0.0642 Å-1, consistent with the presence of a second structural component of nonlamellar symmetry. Increasing the sample temperature to 25 °C (Figure 3) causes significant broadening of the Bragg peaks that arise for the lamellar structural component, as well as a slight upward shift toward higher scattering vector (q ) 0.107 (shoulder at 0.129), 0.216 (0.187), 0.324 Å-1), reflecting a contraction in the lattice dimensions to d ) 58.7 Å. Upon increasing the temperature to 37 °C, continued broadening (and reduction in intensity) of the Bragg peaks associated with the lamellar component and contraction of the lattice (d ) 56.6 Å) is observed. In addition, a peak centered at q ) 0.0280 emerges. The changes observed in the SAXS patterns above and below the endothermic phase transition suggest that this particular composition always comprises a mixture of lamellar and nonlamellar phases, with the relative proportion of the lamellar structure being greatest at low temperatures. These temperature-induced structural changes are the opposite of those found for compositions prepared with the PEG-lipid conjugate, for which a 2D hexagonal structure is formed in the cold phase (