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Cite this: DOI: 10.1039/c4sm02502d
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Hybrid copolymer–phospholipid vesicles: phase separation resembling mixed phospholipid lamellae, but with mechanical stability and control Dong Chena and Maria M. Santore*b Vesicles whose bilayer membranes contain phospholipids mixed with co-polymers or surfactants comprise new hybrid materials having potential applications in drug delivery, sensors, and biomaterials. Here we describe a model polymer–phospholipid hybrid membrane system exhibiting strong similarities to binary phospholipid mixtures, but with more robust membrane mechanics. A lamella-forming graft copolymer, PDMS-co-PEO (polydimethylsiloxane-co-polyethylene oxide) was blended with a high melting temperature
phospholipid,
DPPC
(1,2-dipalmitoyl-sn-glycero-3-phosphocholine),
over
a
broad
compositional range. The resulting giant hybrid unilamellar vesicles were compared qualitatively and quantitatively to analogous mixed phospholipid membranes in which a low melting temperature phospholipid, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), was blended with DPPC. The mechanical properties of the hybrid vesicles, even when phase separated, were robust with high lysis stresses and strains approaching those of the pure copolymer vesicles. The temperature-composition phase diagram of the hybrid vesicles closely resembled that of the mixed phospholipids; with only slightly greater nonidealities in the hybrid compared with DOPC/DPPC mixed membranes. In both systems, it was demonstrated that tension could be used to manipulate DPPC solidification into domains of patchy or striped morphologies that exhibited different tracer incorporation. The patch and stripeReceived 11th November 2014 Accepted 4th February 2015
shaped domains are thought to be different solid DPPC polymorphys: ripple and tilt (or gel). This work demonstrates that in mixed-phospholipid bilayers where a high-melting phospholipid solidifies on
DOI: 10.1039/c4sm02502d
cooling, the lower-melting phospholipid may be substituted by an appropriate copolymer to improve
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mechanical properties while retaining the underlying membrane physics.
Introduction Multicomponent phospholipid vesicles have long held scientic interest because of the insight they provide into biological membranes and their utility as drug delivery agents. Giant vesicles made of copolymers, called “polymersomes” by some,1,2 have attracted recent attention because of their exceptional mechanical properties in comparison with phospholipids,1,2 including tunable viscoelasticity, membrane diffusion,3 and bending mechanics;4 large lysis strains and tough behavior;1 and resistance to surfactants.5 While parallels have been drawn between the polymer and phospholipid vesicles, less work has focused on hybrid membranes containing phospholipids and copolymers (here we distinguish hybrid vesicles as those whose primary components form lamellae individually). Factors working against the formation of hybrid vesicles include
a
Department of Physics, University of Massachusetts at Amherst, 120 Governors Drive, Amherst, MA 01003, USA
b
Department of Polymer Science and Engineering, University of Massachusetts at Amherst, 120 Governors Drive, Amherst, MA 01003, USA. E-mail: [email protected]. umass.edu; Tel: +1-413-577-1417
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incompatibilities between the two types of lamella such as differing thicknesses, and chemical incompatibility between the components themselves (producing high line tensions and cohesive failure in phase separated membranes). Indeed, some descriptions of hybrid vesicle systems mention broad compositional ranges where vesicles (or at least hybrid vesicles) do not form.6–8 Recent formation of hybrid vesicles has been facilitated by careful choice of copolymer chemistry and architecture. Beyond studies of submicron liposomes,9 giant unilamellar vesicles are appealing because membrane mechanics can be probed and membrane physics visualized. Hybrid membrane systems include a copolymer of polyethylene oxide (PEO)-co-polybutadiene mixed with DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), or DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) with and without cholesterol;10 a PEO-co-polyisobutylene copolymer mixed with DPPC;11,12 a polyoxozaline–PDMS–polyoxozaline ABA triblock mixed with phosphatidylethanolamine or DPPC,9 and a gra PDMS–PEO mixed with DPPC and POPC.7 These works report a variety of phase behaviors: complete miscibility over the compositional range investigated,11 phase separation producing
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DPPC-rich phases over a narrow composition window at room temperature,11,12 and potential suppression of either nucleation or domain growth following nucleation.11,12 Open questions include how each species contributes to the overall membrane features and how the hybrid vesicles compare with mixed phospholipid bilayers in terms of mechanical stability and biomimicry. The prospects look good, as hybrid vesicles have been shown to have improved delivery properties13 and exhibit key biomimetic functionality, such as adhesion-triggered phase separation.14 On the other hand, phase behavior in these systems is far from understood: compared with entirely phospholipid systems where phase diagrams have been well-studied, we are not aware of attempts to map the temperature composition phase space of hybrid systems containing lamella forming polymers and phospholipids. Current reports focus mostly on evidence for phase separation based on appearance at room temperature. The current work develops a qualitative and quantitative account of the physical features that underlie the biomimicry and robust mechanics in a potentially powerful hybrid system: poly(dimethyl siloxane)-co-poly(ethylene oxide) [PDMS-co-PEO] gra copolymer, mixed with DPPC, over the full compositional range. The silicone gra copolymer, Xiameter OFX-5329, previously sold under the name Dow Corning 5329, is established to form lamellae and giant unilamellar vesicles.15,16 It has been well studied by us17,18 and others.15,16 Important to the current work, the gra copolymer, while polydisperse, has an overall molecular weight of 3250 g mol1 with 2–3 PEO side arms per each PDMS backbone, and each PEO arm containing 12 EO units. When hydrated, it spontaneously forms lamellae.15 The hybrid copolymer/DPPC membrane is benchmarked here against a well-studied two-component membrane system, DOPC/DPPC. This pair has long held scientic interest because its two components are prominent in cell membranes. Pure DPPC is a solid at room temperature while DOPC is a uid. When mixed, the pair produce a single membrane phase at temperatures above the melting point of DPPC, and exhibit uid–solid coexistence in the membrane at cooler temperatures.19,20 Such uid–solid coexistence is typical of many membrane systems,21–24 and is distinguished from model cholesterol-containing membranes that exhibit uid–uid coexistence as a result of immiscibility.25–27 Fluid–solid phase separation in systems such as DOPC/DPPC is driven by the tendency of one of the components, here DPPC, to order on its own. While membranes exhibiting uid–uid immiscibility have spurred research on how the overall membrane shape is dictated by the competition between elasticity and line tension,28,29 a different physics governs the behavior of solid domains in uid membranes.30–32 Elaborating on the mechanisms that dictate solid domain shape on a curved membrane is beyond the current scope; however, we point out here that the current paper documents the domain shapes that form for the hybrid and DOPC/DPPC systems at low and high membrane tensions, laying groundwork for future studies. Fluid–solid coexistence in giant vesicles is one example in the general class of so material physics problems concerning crystal formation on curved surfaces.
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DPPC's relatively large head group, compared with its hydrophobic tails, facilitates a molecular tilt within the solidlike bilayer, a feature common to other saturated phosphatidylcholines including DLPC and DMPC.33,34 Cholesterol-containing membranes comprised of DPPC and other phospholipids display a variety of phases including a mixed uid (La), liquid condensed, and “gel” or solid-like phases, depending on composition.25,27,35,36 Without cholesterol, membranes containing DPPC and low melting phospholipids exhibit a stunning gallery of patterned solid domains including owers, blobs, stripes, and hexagons.21,22,24,37 Important in determining domain morphology in mixed vesicles are the properties of DPPC itself. Pure (but hydrated) DPPC melts at 41.5 C and mixed membranes containing DPPC are usually single La phase above this temperature.38 Below 41.5 C but above the pretransition temperature of 35.5 C, DPPC lamellae exist in a “ripple” or “corrugated” structure38 where the molecules are tilted at 20 degrees from the local membrane surface normal but their overall projection is roughly perpendicular to the macroscopic membrane. At temperatures below the pretransition, DPPC forms a “gel” phase in which the membrane is relatively smooth, and the molecules exhibit a 30 degrees tilt. The gel and ripple solids appear similarly in NMR and FRET but are distinct in X-ray and DSC studies. Ultimately, the control of a membrane's molecular organization and the domain shape is important because the solid type and the domain shape determine the ability of different membrane phases to connectedly span large distances, affecting transport along the membrane. Additionally, curvature at the domain edges may inuence biochemical reactions at these locations.39 In developing a comparison between the hybrid copolymer/ DPPC membranes and the DOPC/DPPC membrane system, this work attends to the mechanical and thermodynamic properties, and in particular membrane tension, which has been shown to profoundly inuence domain morphology in DOPC/DPPC.40–42 This study addresses membrane mechanics, the phase transition temperatures, and the utility of membrane tension in selecting the solid DPPC molecular organization and domain shape.
Experimental description Lipids 1,2-Dioleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-snglycero-3-phosphocholine (DOPC and DPPC respectively) from Avanti Polar Lipids (catalog numbers 850375C and 850355C) were used as received. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamineN-(Lissamine rhodamine B sulfonyl) ammonium salt, [Rh-DOPE, catalog number 810150C] and 1-2-dipalmitoy-sn-glycero-3-phosphoethanolamine-N-(Lissamine rhodamine B sulfonyl) ammonium salt, [Rh-DPPE, catalog number 810158C] from Avanti were employed as tracer lipids. Copolymer A gra copolymer having a PDMS (polydimethylsiloxane) backbone with PEO (polyethylene oxide) gra side chains was a
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gi from Xiameter. This compound is the same as previously sold under the name Dow Corning 5329. This study was initiated with DC5329 and completed with a fresh sample from Xiameter. Data (mechanical properties, images in Fig. 1 and 3, and tension data) from both samples were compared and found quantitatively indistinguishable within experimental error. Thus the ndings here are not conned to a particular batch of sample. Vesicles Giant unilamellar vesicles were prepared by electroformation.43 Copolymer and DPPC, or DOPC and DPPC, in the desired molar proportions, were dissolved in chloroform near a concentration of 1 mg ml1. Rh-DPPE or Rh-DOPE tracers were employed at a concentration of 0.1 mol%. 10 mL of solution was placed on the platinum wire electrodes and, aer drying, the chamber was lled with DI (de-ionized) water or sucrose solution. An alternating current was applied to the electrodes at 3 V and 10 Hz for 1 hour, while the chamber was maintained at 52 C, ensuring the compositional uniformity.
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diameter, contributed to the overall uncertainty on tension of 0.02 mN m1. Phase separation Vesicle phase separation was studied by uorescence microscopy in a sealed chamber that contained vesicle suspensions in a 125 mm gap between two coverslips. Temperature-control was achieved using a custom thermal manifold. Images were obtained using a Nikon Diaphot 300 inverted uorescence microscope with a CoolSnap HQ CCD camera. In the sealed chamber, osmotic and other conditions were chosen to produce targeted membrane tensions, as described below. Osmotic pressure control of near-zero Some studies focused on vesicles whose membrane tensions were manipulated osmotically to 0 0.02 mN m1. These vesicles were electroformed in a 200 mOsm sucrose solution, and equilibrated in 250 mOsm sucrose solution. Over a few tens of minutes, water diffusion out of the vesicles made them accid, as described below.
Micropipettes Micropipettes, with inner diameters of 3–6 mm, were employed to quantitatively manipulate vesicle tension in studies of membrane mechanics. Straight tips were employed and their shapes rened to produce nearly constant inner diameters in the region where vesicles projected into the pipettes. Bovine serum albumin (Sigma catalog number A7511) was adsorbed to prevent vesicle adhesion. Vesicles were chosen in the 15–50 mm diameter range to avoid error in calculating tension and area. The vesicle tension was controlled by a siphon manometer. The uncertainty on the suction pressure was less than 1 mm water. Small errors (less than 10%) in measuring pipette and vesicle
Vesicles with spontaneous elevated tensions In contrast to osmotically-conditioned vesicles, most of the vesicles in the current program were formed and equilibrated in DI water at elevated temperatures prior to cooling. Phospholipid and hybrid vesicles handled in this way were observed not to exhibit excess area and were, instead, found to be tensed at short times aer controlled cooling. It is not obvious that this treatment should produce elevated membrane tensions; however, characterization studies described previously40,41 and applied below to hybrid vesicles demonstrated that this was indeed the case, as detailed in the Results section.
Fig. 1 Appearance of copolymer/DPPC (“hybrid”) vesicles compared with DOPC/DPPC (“lipid”) vesicles for different compositions and two cooling rates from the one-phase region. The scale bars are 10 mm. Rh-DOPE is employed as the tracer.
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The hypothesized mechanism for development of membrane stress, key to the design of studies to measure tension, involves a starting point at elevated temperatures, for instance near 45 C in our studies, where vesicles exhibit little, if any, excess area.41 Cooling causes both the vesicle membrane and its water contents to contract thermally.44 The membrane contracts more extensively than the aqueous vesicle center, causing development of membrane stress. The membrane stress relaxes as water diffuses out of the vesicles across the membrane, on a timescale of minutes.45–48 Thus the actual values of the membrane stress and its variation with time depend on the cooling rate relative to that of water diffusion. If cooling is extremely fast, the membrane ruptures and reseals,49–51 making it difficult to anticipate whether fast or slow cooling will produce higher tensions. Extremely slow cooling, however, always maintains low tensions.40,41 Because of this complexity, membrane tension cannot be anticipated by calculations based only on osmotic pressure differences. Instead, membrane tension is measured, as described below.
Characterizing vesicle stress and strain Micropipette aspiration was employed to probe two different vesicle features: the traditional stress–strain curve, including the area expansion modulus, Ka, and the membrane stress relevant to phase separation. Both studies employed open micropipette-accessible chambers made of coverslips supported in a 1 mm gap conguration. The chamber's ends were coupled to a constant temperature reservoir. Stress–strain curves were measured following established procedures.18,44 A test vesicle was aspirated at low suction and, aer stretching to ensure good lubrication with the micropipette, the tension was set close to zero. Subsequent 10 second step-wise increases in suction were recorded on video. If the vesicle did not break, the tensions could be stepped back down and then back up to conrm reversibility. Vesicle images were analyzed to determine the membrane area and the strain at each step. The membrane tension, s, was calculated from the Laplace equation: P s Rp s¼ 2Rp 2 Rv
(1)
Rp and Rv are the micropipette and vesicle radius (outside the micropipette). Ps is the suction pressure on the micropipette. Separate studies quantied the combined impact of cooling history and osmotic conditioning on the hybrid membrane stress, at conditions similar to the initial instants of phase separation. Vesicles were cooled at the rate of interest to 35 C, a temperature within range of the phase transition, and then immediately transferred to the micropipette chamber that was heated to the same temperature. Measurements were made within minutes of transfer to the micropipette chamber, allowing some tension loss, but minimizing the loss as much as possible. The vesicle was characterized by rst attempting to aspirate at extremely low suctions, corresponding to stress in the range 0.02–0.04 mN m1, and areal strains of less than 0.1%. For accid vesicles, a projection Soft Matter
would appear in the micropipette. Tensed vesicles, however, would be pulled to the mouth of the pipette but not produce a projection. Subsequent increase in suction would produce a projection only once the suction pressure balanced the opposing force from the membrane tension. The suction pressure corresponding to the rst appearance of a projection allowed determination of the membrane tension of the stressed vesicles according to the Laplace equation, above.
Results Vesicle morphology Fig. 1 contains images of hybrid copolymer/DPPC vesicles photographed at room temperature aer cooling at different rates from the single-phase region at 43 C. These data demonstrate how the resulting vesicle morphology depends on composition and the thermal history during the approach to room temperature. Fig. 1 also presents remarkable parallels between copolymer/DPPC and DOPC/DPPC vesicle suggesting that copolymer and DOPC play similar roles as the lower melting uid membrane component (the images of the DOPC/ DPPC vesicles are reproduced from a paper focusing on DOPC/ DPPC phase separation41 for comparison with hybrid vesicles). In Fig. 1, single-phase uid membranes are consistently found, for both hybrid and DOPC/DPPC vesicles at low overall DPPC content. Single-phase hybrid membranes are also observed at temperatures above 41.5 C (the melting point of DPPC), as expected (images of single-phase uid vesicles at elevated temperatures are not included because they appear identical to those having low DPPC content). Dark domains, which exclude the tracer dye Rh-DOPE, are visible at room temperature in Fig. 1, and occupy an increasing membrane area with greater DPPC content in either vesicle type. The observed greater solid (dark) domain area with increased DPPC content indicates that the solid domains predominantly DPPC. This is in fact established for the DOPC/ DPPC system19,35,52 and suggests that the solid domains in the hybrid system have similarly high DPPC content. Vesicles electroformed and cooled in DI water to room temperature at a rate of 5 C min1 from about 43 C display patchy solid domains which, upon close examination, oen have hexagonal facets. Vesicles cooled more slowly, for instance at 1 C min1, can exhibit stripes or patches, depending on composition. Interesting in both hybrid and phospholipid systems, there are distinct ranges of compositions where cooling history, not the composition itself, determines whether striped or patchy solid domains persist. Quantitative differences distinguish the phase separation in the hybrid and DOPC/DPPC bilayers. Most notably, at the cooling rate of 1 C min1, in the phospholipid system there is a broader composition window, above about 45 mol% DPPC, where stripes are the solid morphology. In the case of the copolymer/DPPC vesicles, the compositional window for the striped solid morphology is narrower, starting around 70 mol% DPPC (at 70 mol% DPPC, a solid patch is seen among the solid stripes. The composition of 70 mol% DPPC in the hybrid vesicles apparently lies close to the boundary between the two solid
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morphologies). We will argue below that whether vesicles display patched or striped domain morphology depends on the membrane tension and, further, that the differences between the appearance of stripes or patches in lipid vs. hybrid vesicles cooled at 1 C min1 (the lower part of Fig. 1) resulted from modest differences in the tension of the two vesicle types. Molecular organization in striped and patchy solid domains Differences in the interaction of tracer lipid with the striped and patchy solid domains provide convincing evidence for differences in the molecular organization within the two domain types in either vesicle system. Fig. 2 compares the appearance of copolymer/DPPC and DOPC/DPPC vesicles containing either Rh-DPPE or Rh-DOPE tracer (the latter was employed in all the vesicles of Fig. 1). The patchy domains exclude both tracers, but the striped domains take up Rh-DPPE selectively, in either hybrid or phospholipid vesicles. Thus, depending on the tracer but not on the vesicle system (hybrid or strictly phospholipid), the stripes can appear light or dark. This argues for different molecular organization in the two types of solid domains and suggests that the two domain types are similar in the two vesicle systems. Membrane mechanics Fig. 3 compares room temperature stress–strain curves for copolymer, hybrid, and phospholipid vesicles aspirated in micropipettes. The mixed membranes contain 70 mol% DPPC and were manipulated to be patchy. Vesicle deformations were generally reversible. Because entry of solid domains (especially stripes) into the micropipette can sometimes interrupt the progress of aspiration and membrane stretching, we chose data
Appearance of vesicles containing 70 mol% DPPC, containing two different tracer dyes. Comparison is made between hybrid and phospholipid vesicles with cooling history related to tension. Images E and F only are reproduced from ref. 40. The scale bar is 10 mm.
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sets for the phase-separated vesicles where domains did not happen to hang up upon entry into the micropipette. Without over-emphasizing the quantitative aspects of stress–strain curves of the phase-separated vesicles, we included these data because they appear reasonable. We also included the single component vesicles as a reference. Fig. 3 makes two important distinctions between phospholipid and copolymer-containing membranes. First, the lysis stress and strain for the copolymer and hybrid vesicles far exceeds that of the pure phospholipid and especially the phaseseparated phospholipid. Lysis strains for the copolymer-containing vesicles exceed 8%, compared with 2% for the mixed phospholipid vesicles. A second take home point from the mechanical experiments is the difference in moduli between the hybrid and phospholipid membranes. Area expansion moduli of 200 mN m1, observed for our phospholipid vesicles, are typical of these liposomes.53,54 The copolymer-containing vesicles have area expansion moduli a factor of two lower. Additionally, the copolymer dominates the mechanics of mixed vesicles: hybrid vesicles containing 70% DPPC have an area expansion modulus only slightly greater than that of the pure copolymer. The stress–strain curves and the resulting area expansion moduli, Ka, in Fig. 3 are representative of at least 10 vesicles of each type within 10% error, and are typical of uid phospholipid53–55 and copolymer vesicles.55 Indeed, for copolymers there is little dependence of the Ka on membrane thickness or the choice of copolymer.5,55 The consistency of our ndings with the literature and the relatively tight reproducibility argue that these vesicles are unilamellar. Multilamellar or onion-skin vesicles have integer multiples of the Ka values of unilamellar vesicles. Such anomalous specimens were rare (less than 5%) of the vesicles we encountered and were easily distinguished by their mechanical properties and appearance (which allowed us not to choose them in micrographs or other studies).
Fig. 2
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Fig. 3 Comparison of stress–strain curves for pure DOPC, 30/70 DOPC/DPPC, 30/70 copolymer DPPC, and copolymer vesicles. The asterisk indicates rupture. Ka values are shown.
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Taken together these results suggest that for a given strain, the copolymer and hybrid vesicles will experience lower stress than the pure phospholipid system. Additionally, hybrid membranes, even when phase separated, exhibit a smaller tendency to rupture compared with entirely phospholipid membranes.
Tension and mechanism of morphological fate Fig. 1 illustrates how, in both membrane types, the cooling rate appears to dictate the morphological vesicle fate over a range of membrane compositions. Indeed the cooling rate turns out to be among the most experimentally-accessible means of tuning morphology. For DOPC/DPPC vesicles; however, it was demonstrated that membrane tension, imposed osmotically or using micropipettes, was the underlying factor governing the morphology.40,41 If the tension of DOPC/DPPC membranes was controlled independently, the inuence of cooling rate on the initial appearance of stripes or patchy solid domains was removed. The exception was that large domains either type could be kinetically trapped at low temperatures. Fig. 4A summarizes membrane tensions, s, at the intermediate temperature of 35 C. This temperature chosen to be in the middle of cooling runs near the onset of phase separation, as will be demonstrated below in Fig. 5. Fig. 4 reports that tension similarly inuences hybrid and DOPC/DPPC vesicles, though there are quantitative differences. Then Fig. 4B demonstrates that tension is the dominant factor in establishing the type of solid formed. Towards an understanding of Fig. 4A, we rst review the features of the cooling/tension measurement study: prior to cooling, vesicles are annealed in the single phase region at 43 C and this was found to produce s 0.41 Also in general, at long times aer cooling, membrane tension has relaxed to zero. At intermediate times, cooling causes the membrane to contract. Thermal contraction can be substantial for phospholipids, a 5%
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area decrease is expected for each 10 C of cooling.44 So, if vesicles are not accid initially (for instance as a result of osmotic conditioning), thermal membrane contractions produce stress. Membrane stresses relax by water diffusion from the vesicle center, a process that equilibrates on the order of tens of minutes for phospholipids.45–48 So during periods of cooling and also later at constant temperature, whenever there is membrane stress, there is stress relaxation via water diffusion. As a result of the two competing processes (thermal contraction to produce stress and diffusion to relax it), membrane stress goes through maximum with time, but starts and ends near zero. If thermal quenching is too rapid relative to diffusive timescales, stresses may exceed the lysis conditions reported in Fig. 3 and water release may occur by membrane rupture. This behavior is difficult to detect directly, but the breaking and resealing of stressed membranes has been previously reported.49–51 We are interested in these intermediate elevated tensions because they determine the domains formed during phase separation. An approximation of the membrane tension in the early moments of phase separation comes from measurements of membrane tension at an intermediate temperature. We chose 35 C. At this temperature, cooling runs were interrupted and vesicles transferred to the micropipette chamber, at a constant temperature of 35 C. Measurements of membrane tension were made immediately upon transfer because even at constant temperature, water diffusion across the membrane causes a continued reduction in membrane stress. Further, because tension is decreasing from the instant vesicles were transferred to the stress measurement chamber, the stresses measured are lower than they were just prior to transfer at 35 C. For this reason, we say that the measured stresses represent a lower bound on the stresses occurring at 35 C in phase separation runs. Fig. 4A rst shows that vesicles, conditioned with a higher sucrose osmolarity on the outside of the vesicle compared with
Fig. 4 (A) Membrane tension after cooling vesicles containing 70% DPPC, osmotically conditioned or handled in DI water, to 35 C, a temperature near that of the initial phase separation. 10–15 vesicles were studied at each condition and error bars reflect the full range of tension values observed. For the vesicles conditioned in sucrose, the tension was zero for all vesicles and so no error bars appear. (B) Images of hybrid vesicles containing 70% DPPC subject to different cooling and osmotic control, indicated. Scale bars are 10 mm.
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(A) observed temperatures for the first appearance of patches on cooling in phospholipid and hybrid vesicles. Lower black line summarizes melting point depression calculation in which the fluid phase is an ideal solution (B) same experiments but with additional data on hybrid vesicles to facilitate a mass-fraction representation on the x-axis.
Fig. 5
the interior, do not experience stress at the intermediate temperature of 35 C, during a cooling run. This is the case for both vesicle types because such osmotic conditioning produces excess membrane area (the vesicles are made oppy). With thermal contraction of the membrane during cooling, this area is consumed; however, there must be sufficient excess area that a tension increase is not observed. By contrast, for vesicles cooled in DI water, Fig. 4A reveals that substantial membrane stresses at 35 C, aer transfer to the stress measurement chamber. Because stress is decreasing continually during the transfer, the values in Fig. 4A are lower than those actually occurring at 35 C during the cooling run in which phase separation is occurring. Membrane stress develops for vesicles in DI water but not for sucrose because only the latter have excess area (as a result of osmotic conditioning). Fig. 4A reports that higher membrane tensions are sustained at the cooling rate of 1 C min1 compared with lower near-zero tensions at 5 C min1. These differences in tensions are statistically signicant within 99% certainty. Fig. 4A also reports differences in the tension in phospholipid compared with hybrid vesicles. The impact of cooling rate of membrane tension may result from the greater tendency for vesicle lysis upon rapid thermal quenching. Additionally, differences between the two vesicle types could be explain by the observed lower area expansion moduli, in Fig. 3. For a given membrane contraction on the x-axis, the hybrid vesicles sustain a lower membrane stress. By isolating the impacts of tension and cooling rate, Fig. 4B illustrates, for hybrid copolymer vesicles, that tension is the important variable in dictating domain morphology. In hybrid vesicles osmotically-manipulated to maintain zero membrane tension, solid patches form, independent of cooling rate. However, for the same cooling rate but two different tensions, stripes appear at high tensions while patchy solid domains are seen at low tensions. A preference for patch-shaped solid domain formation was previously reported in mixed DOPC/ DPPC vesicles maintained near zero tension.40,41 The similar
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roles of tension in both vesicle types suggest more broadly parallel underlying thermodynamics for hybrid and purely phospholipid vesicles. Cooling rate is a convenient processing variable through which tension, and therefore the solid morphology can be tuned. Once solid domains form, however, their morphology is retained for hours, on the order of vesicle lifetime. A large energy barrier opposes the transformation of one domain type to another, even when stress is applied (at room temperature). In obtaining the mechanical data in Fig. 3, we observed no qualitative alteration of the dark solid domains. Composition-temperature space Fig. 5 illustrates the thermodynamic parallel between the two classes of vesicles in terms of the low-tension temperaturecomposition space for uid–solid phase separation. Fig. 5 plots the temperatures for the initial appearance of solid domains in vesicles of varying composition that were cooled sufficiently quickly from the one-phase region to produce patches. The domains start as small nuclei that become clearly visible when they reach a size of about a micron.42 The temperature of rst appearance is not signicantly dependent on the cooling rate as long as the rate produces tensions sufficiently low for patchy domain formation. Results for the DOPC/DPPC system are in quantitative agreement with literature studies employing NMR,19 supporting the sensitivity of the uorescence method. Fig. 5A demonstrates that for hybrid and phospholipid vesicles the temperature-composition state diagram appears similar, except that phase separation occurs at slightly elevated temperatures in the hybrid system. Both sets of experimental data in Fig. 5A extrapolate to the melting temperature of pure DPPC (41.5 C). Additionally, the observed dark domain areas in phase separated DOPC/DPPC vesicles having patch shaped domains were previously found to conform to the inverse lever arm rule with a solid phase containing 95% or more DPPC, arguing that the amounts of domains represented a local equilibrium.42 Together these
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points motivate the calculation of the standard colligative property behavior for melting point depression in an ideal solution uid mixture with a pure solid, shown as the lower black curve in Fig. 5A. It is apparent that the DOPC/DPPC system exhibits modest deviations from ideal solution. While the DPPC/copolymer interaction favors mixing less than the DOPC/DPPC interaction, neither system is sufficiently non-ideal to produce uid–uid de-mixing.
Discussion The behavior of these hybrid copolymer–phospholipid vesicles is remarkable in several regards. First, our results provide a counter-example to the idea that bilayer-forming polymers are fundamentally incompatible with phospholipid lamellae.6–8 Phospholipid–copolymer compatibility requires chemical compatibility along with some matching of lamellar thicknesses. Our hybrid lamellae are almost as compatible as mixed phospholipid membranes. Phase separation in membranes such as ours, where DPPC is mixed with a low melting lamella former, results from the fundamental tendency for DPPC to crystallize below 41.5 C, rather than from a fundamental incompatibility between the two components. The region of thermodynamic space in which DPPC crystallizes in hybrid vesicles is only slightly larger than the two-phase region for DOPC/DPPC mixtures, arguing for substantial miscibility of both systems. Indeed, the solubility parameter of PDMS is 14.9 J cm3,56 close to that of alkanes (14– 16 J cm3) and likely resembling interactions in the hybrid membrane core. Miscibility is further favored by similar DPPC and copolymer bilayer thicknesses, 5.5 (ref. 57) and 7.7 nm (ref. 15) respectively (the copolymer includes a PEG corona while the DPPC includes only hydrated polar headgroups). Additionally in the copolymer membrane, the PDMS chains assume a random conformation, facilitating nanometer-scale changes in lamellar thickness to accommodate “objects” such as solid DPPC domains. Worth considering, copolymer polydispersity introduces uncertainty in the molecular weight and therefore uncertainty in the mole fractions of the mixed hybrid membranes. Polymer blend compositions are oen prescribed as mass or volume fractions. If mass fraction is employed as the independent variable, the state diagram appears as Fig. 5B. In Fig. 5B, the hybrid membrane phase diagram shows further apparent deviations from ideal solution behavior, but the underlying parallel behavior is preserved. Domains in DPPC-containing membranes likely assume at least one of the distinct solid polymorphs exhibited by DPPC. These include a family of corrugated ripple (Pb0 ) morphologies that persist, for pure (but hydrated DPPC) between 38.5 and 41.5 C, and a planar tilt gel (Lb0 ) phase below 38.5 C down to room temperature. The assignment of these different bilayer solids to patches and stripes within mixed DPPC-containing lamellae has been debated.22,58 Striped regions with corrugated morphology found by AFM within DPPC-containing membranes immobilized on mica suggest a similar relationship in vesicles.59 Our more recent data, with mixed DOPC/DPPC vesicles, argue
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differently:40,41 we document the formation of stripe solid DPPC domains at elevated tensions above a triple point and patchy solid domains at lower tensions. The observed tension-dependence of the phase transition temperatures of DOPC/DPPC vesicles was found to be in quantitative agreement with fundamental thermodynamic arguments (somewhat similar to Clausius–Clapeyron) when the patchy solid domains were assigned the properties of the DPPC ripple phase. This strongly suggests the ripple Pb0 solid phase within the patchy DPPC domains and a gel Lb0 phase in the stripes. The most straight-forward argument, however, for the ripple Pb0 phase (with its greater areal density of DPPC) within the patchy solid, is the tendency of the membrane to favor patches at low tension and stripes at high tension, for example in Fig. 1, 2 and 4. Simply put, the less dense membrane phase Lb0 persists at higher tensions. Fig. 1 therefore suggests that the patches and stripes in the hybrid vesicles are ripple and gel DPPC solid domains, and that the patchy DPPC solid domains have similar DPPC content in both cases. In this sense, manipulation of the tension of a polymer membrane, in our hands, directs the crystallization and morphology of a phospholipid. Other observed parallels between the vesicle morphology in the hybrid and phospholipid systems include similar domains sizes and numbers, and a similar inuence of cooling history. This suggests similar membrane thermodynamics and similar mechanisms for domain formation. Patchy DPPC domains in phospholipid membranes are found to result from nucleation and growth,42 and the same mechanism is likely to produce DPPC domains during cooling of hybrid membranes. To the extent that the domain sizes and numbers are similar for a given compositions and cooling rate, the nucleation rate, with its dependence on the enthalpy of domain formation and the line tension around nucleated domains, suggests that line tensions in the hybrid system are not unusual. Indeed the mechanical strength of phase-separated hybrid vesicles in Fig. 3B suggests that line tension is not high. An important difference between hybrid and phospholipid membranes, evident in Fig. 1, is the greater tendency for patch formation and smaller compositional window for stripe formation in hybrid vesicles. With the correlation between low tension and stripe formation in both systems, this suggests lower tensions in the formative instants of phase separation of hybrid vesicles. This hypothesis is affirmed, at least in part by the mechanical data of Fig. 4A. For a given membrane strain (such as a thermal contraction), the soer copolymer-containing vesicles experience a lower stress and a greater tendency to produce patches rather than stripes. We expect that separate manipulation of the hybrid vesicles to sustain large membrane tensions will produce stripes over a larger compositional range.
Conclusions This study examined multicomponent membranes that contained copolymer and phospholipid mixtures to probe how well a mechanically-robust hybrid system could approximate the behavior of mixed phospholipid membranes. Giant unilamellar hybrid vesicles and analogous phospholipid vesicles containing
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DPPC were in many ways similar over the full compositional range. Similarities included the general appearance of vesicles having analogous compositions and thermal histories. Phase separation in both systems resulted from the tendency for DPPC to solidify in the membrane to form nearly pure DPPC domains. The DPPC domains appeared patchy or stripe-shaped, depending on composition, tension, and thermal history, with striking similarity between the two types of membranes. The temperatures observed for the onset of phase separation was, to rst order the same in the two systems. Small differences included slightly higher solidication temperatures in copolymer-containing membranes, indicative of slightly less ideal mixing in the hybrid compared with the phospholipid system. The mixing non-idealities were insufficient to produce uid– uid de-mixing. Tension was shown to play a dominant role, (tuned through cooling history), in determining the types of membrane domains to form. Parallel dye-incorporation behavior between hybrid and DOPC/DPPC membranes suggested that the striped and patchy domains in either system were a result of differences in their molecular organization. A ripple DPPC polymorph in the patchy domains along with a tilt gel structure in the stripes was the best explanation for the differences in the domains. The current study demonstrated how a copolymer can replace a low-melting phospholipid to produce mechanicallyrobust vesicles with similar thermodynamic and tension response to phospholipid membranes, including domain morphology and connectivity. These new materials represent tools for further study of biological mechanisms and may form the basis for future materials that communicate adhesively with cells.
Acknowledgements This work was supported by NSF-0820506 and NSF-1264855.
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Cite this: DOI: 10.1039/c4sm02502d
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Hybrid copolymer–phospholipid vesicles: phase separation resembling mixed phospholipid lamellae, but with mechanical stability and control Dong Chena and Maria M. Santore*b Vesicles whose bilayer membranes contain phospholipids mixed with co-polymers or surfactants comprise new hybrid materials having potential applications in drug delivery, sensors, and biomaterials. Here we describe a model polymer–phospholipid hybrid membrane system exhibiting strong similarities to binary phospholipid mixtures, but with more robust membrane mechanics. A lamella-forming graft copolymer, PDMS-co-PEO (polydimethylsiloxane-co-polyethylene oxide) was blended with a high melting temperature
phospholipid,
DPPC
(1,2-dipalmitoyl-sn-glycero-3-phosphocholine),
over
a
broad
compositional range. The resulting giant hybrid unilamellar vesicles were compared qualitatively and quantitatively to analogous mixed phospholipid membranes in which a low melting temperature phospholipid, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), was blended with DPPC. The mechanical properties of the hybrid vesicles, even when phase separated, were robust with high lysis stresses and strains approaching those of the pure copolymer vesicles. The temperature-composition phase diagram of the hybrid vesicles closely resembled that of the mixed phospholipids; with only slightly greater nonidealities in the hybrid compared with DOPC/DPPC mixed membranes. In both systems, it was demonstrated that tension could be used to manipulate DPPC solidification into domains of patchy or striped morphologies that exhibited different tracer incorporation. The patch and stripeReceived 11th November 2014 Accepted 4th February 2015
shaped domains are thought to be different solid DPPC polymorphys: ripple and tilt (or gel). This work demonstrates that in mixed-phospholipid bilayers where a high-melting phospholipid solidifies on
DOI: 10.1039/c4sm02502d
cooling, the lower-melting phospholipid may be substituted by an appropriate copolymer to improve
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mechanical properties while retaining the underlying membrane physics.
Introduction Multicomponent phospholipid vesicles have long held scientic interest because of the insight they provide into biological membranes and their utility as drug delivery agents. Giant vesicles made of copolymers, called “polymersomes” by some,1,2 have attracted recent attention because of their exceptional mechanical properties in comparison with phospholipids,1,2 including tunable viscoelasticity, membrane diffusion,3 and bending mechanics;4 large lysis strains and tough behavior;1 and resistance to surfactants.5 While parallels have been drawn between the polymer and phospholipid vesicles, less work has focused on hybrid membranes containing phospholipids and copolymers (here we distinguish hybrid vesicles as those whose primary components form lamellae individually). Factors working against the formation of hybrid vesicles include
a
Department of Physics, University of Massachusetts at Amherst, 120 Governors Drive, Amherst, MA 01003, USA
b
Department of Polymer Science and Engineering, University of Massachusetts at Amherst, 120 Governors Drive, Amherst, MA 01003, USA. E-mail: [email protected]. umass.edu; Tel: +1-413-577-1417
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incompatibilities between the two types of lamella such as differing thicknesses, and chemical incompatibility between the components themselves (producing high line tensions and cohesive failure in phase separated membranes). Indeed, some descriptions of hybrid vesicle systems mention broad compositional ranges where vesicles (or at least hybrid vesicles) do not form.6–8 Recent formation of hybrid vesicles has been facilitated by careful choice of copolymer chemistry and architecture. Beyond studies of submicron liposomes,9 giant unilamellar vesicles are appealing because membrane mechanics can be probed and membrane physics visualized. Hybrid membrane systems include a copolymer of polyethylene oxide (PEO)-co-polybutadiene mixed with DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), or DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) with and without cholesterol;10 a PEO-co-polyisobutylene copolymer mixed with DPPC;11,12 a polyoxozaline–PDMS–polyoxozaline ABA triblock mixed with phosphatidylethanolamine or DPPC,9 and a gra PDMS–PEO mixed with DPPC and POPC.7 These works report a variety of phase behaviors: complete miscibility over the compositional range investigated,11 phase separation producing
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DPPC-rich phases over a narrow composition window at room temperature,11,12 and potential suppression of either nucleation or domain growth following nucleation.11,12 Open questions include how each species contributes to the overall membrane features and how the hybrid vesicles compare with mixed phospholipid bilayers in terms of mechanical stability and biomimicry. The prospects look good, as hybrid vesicles have been shown to have improved delivery properties13 and exhibit key biomimetic functionality, such as adhesion-triggered phase separation.14 On the other hand, phase behavior in these systems is far from understood: compared with entirely phospholipid systems where phase diagrams have been well-studied, we are not aware of attempts to map the temperature composition phase space of hybrid systems containing lamella forming polymers and phospholipids. Current reports focus mostly on evidence for phase separation based on appearance at room temperature. The current work develops a qualitative and quantitative account of the physical features that underlie the biomimicry and robust mechanics in a potentially powerful hybrid system: poly(dimethyl siloxane)-co-poly(ethylene oxide) [PDMS-co-PEO] gra copolymer, mixed with DPPC, over the full compositional range. The silicone gra copolymer, Xiameter OFX-5329, previously sold under the name Dow Corning 5329, is established to form lamellae and giant unilamellar vesicles.15,16 It has been well studied by us17,18 and others.15,16 Important to the current work, the gra copolymer, while polydisperse, has an overall molecular weight of 3250 g mol1 with 2–3 PEO side arms per each PDMS backbone, and each PEO arm containing 12 EO units. When hydrated, it spontaneously forms lamellae.15 The hybrid copolymer/DPPC membrane is benchmarked here against a well-studied two-component membrane system, DOPC/DPPC. This pair has long held scientic interest because its two components are prominent in cell membranes. Pure DPPC is a solid at room temperature while DOPC is a uid. When mixed, the pair produce a single membrane phase at temperatures above the melting point of DPPC, and exhibit uid–solid coexistence in the membrane at cooler temperatures.19,20 Such uid–solid coexistence is typical of many membrane systems,21–24 and is distinguished from model cholesterol-containing membranes that exhibit uid–uid coexistence as a result of immiscibility.25–27 Fluid–solid phase separation in systems such as DOPC/DPPC is driven by the tendency of one of the components, here DPPC, to order on its own. While membranes exhibiting uid–uid immiscibility have spurred research on how the overall membrane shape is dictated by the competition between elasticity and line tension,28,29 a different physics governs the behavior of solid domains in uid membranes.30–32 Elaborating on the mechanisms that dictate solid domain shape on a curved membrane is beyond the current scope; however, we point out here that the current paper documents the domain shapes that form for the hybrid and DOPC/DPPC systems at low and high membrane tensions, laying groundwork for future studies. Fluid–solid coexistence in giant vesicles is one example in the general class of so material physics problems concerning crystal formation on curved surfaces.
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DPPC's relatively large head group, compared with its hydrophobic tails, facilitates a molecular tilt within the solidlike bilayer, a feature common to other saturated phosphatidylcholines including DLPC and DMPC.33,34 Cholesterol-containing membranes comprised of DPPC and other phospholipids display a variety of phases including a mixed uid (La), liquid condensed, and “gel” or solid-like phases, depending on composition.25,27,35,36 Without cholesterol, membranes containing DPPC and low melting phospholipids exhibit a stunning gallery of patterned solid domains including owers, blobs, stripes, and hexagons.21,22,24,37 Important in determining domain morphology in mixed vesicles are the properties of DPPC itself. Pure (but hydrated) DPPC melts at 41.5 C and mixed membranes containing DPPC are usually single La phase above this temperature.38 Below 41.5 C but above the pretransition temperature of 35.5 C, DPPC lamellae exist in a “ripple” or “corrugated” structure38 where the molecules are tilted at 20 degrees from the local membrane surface normal but their overall projection is roughly perpendicular to the macroscopic membrane. At temperatures below the pretransition, DPPC forms a “gel” phase in which the membrane is relatively smooth, and the molecules exhibit a 30 degrees tilt. The gel and ripple solids appear similarly in NMR and FRET but are distinct in X-ray and DSC studies. Ultimately, the control of a membrane's molecular organization and the domain shape is important because the solid type and the domain shape determine the ability of different membrane phases to connectedly span large distances, affecting transport along the membrane. Additionally, curvature at the domain edges may inuence biochemical reactions at these locations.39 In developing a comparison between the hybrid copolymer/ DPPC membranes and the DOPC/DPPC membrane system, this work attends to the mechanical and thermodynamic properties, and in particular membrane tension, which has been shown to profoundly inuence domain morphology in DOPC/DPPC.40–42 This study addresses membrane mechanics, the phase transition temperatures, and the utility of membrane tension in selecting the solid DPPC molecular organization and domain shape.
Experimental description Lipids 1,2-Dioleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-snglycero-3-phosphocholine (DOPC and DPPC respectively) from Avanti Polar Lipids (catalog numbers 850375C and 850355C) were used as received. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamineN-(Lissamine rhodamine B sulfonyl) ammonium salt, [Rh-DOPE, catalog number 810150C] and 1-2-dipalmitoy-sn-glycero-3-phosphoethanolamine-N-(Lissamine rhodamine B sulfonyl) ammonium salt, [Rh-DPPE, catalog number 810158C] from Avanti were employed as tracer lipids. Copolymer A gra copolymer having a PDMS (polydimethylsiloxane) backbone with PEO (polyethylene oxide) gra side chains was a
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gi from Xiameter. This compound is the same as previously sold under the name Dow Corning 5329. This study was initiated with DC5329 and completed with a fresh sample from Xiameter. Data (mechanical properties, images in Fig. 1 and 3, and tension data) from both samples were compared and found quantitatively indistinguishable within experimental error. Thus the ndings here are not conned to a particular batch of sample. Vesicles Giant unilamellar vesicles were prepared by electroformation.43 Copolymer and DPPC, or DOPC and DPPC, in the desired molar proportions, were dissolved in chloroform near a concentration of 1 mg ml1. Rh-DPPE or Rh-DOPE tracers were employed at a concentration of 0.1 mol%. 10 mL of solution was placed on the platinum wire electrodes and, aer drying, the chamber was lled with DI (de-ionized) water or sucrose solution. An alternating current was applied to the electrodes at 3 V and 10 Hz for 1 hour, while the chamber was maintained at 52 C, ensuring the compositional uniformity.
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diameter, contributed to the overall uncertainty on tension of 0.02 mN m1. Phase separation Vesicle phase separation was studied by uorescence microscopy in a sealed chamber that contained vesicle suspensions in a 125 mm gap between two coverslips. Temperature-control was achieved using a custom thermal manifold. Images were obtained using a Nikon Diaphot 300 inverted uorescence microscope with a CoolSnap HQ CCD camera. In the sealed chamber, osmotic and other conditions were chosen to produce targeted membrane tensions, as described below. Osmotic pressure control of near-zero Some studies focused on vesicles whose membrane tensions were manipulated osmotically to 0 0.02 mN m1. These vesicles were electroformed in a 200 mOsm sucrose solution, and equilibrated in 250 mOsm sucrose solution. Over a few tens of minutes, water diffusion out of the vesicles made them accid, as described below.
Micropipettes Micropipettes, with inner diameters of 3–6 mm, were employed to quantitatively manipulate vesicle tension in studies of membrane mechanics. Straight tips were employed and their shapes rened to produce nearly constant inner diameters in the region where vesicles projected into the pipettes. Bovine serum albumin (Sigma catalog number A7511) was adsorbed to prevent vesicle adhesion. Vesicles were chosen in the 15–50 mm diameter range to avoid error in calculating tension and area. The vesicle tension was controlled by a siphon manometer. The uncertainty on the suction pressure was less than 1 mm water. Small errors (less than 10%) in measuring pipette and vesicle
Vesicles with spontaneous elevated tensions In contrast to osmotically-conditioned vesicles, most of the vesicles in the current program were formed and equilibrated in DI water at elevated temperatures prior to cooling. Phospholipid and hybrid vesicles handled in this way were observed not to exhibit excess area and were, instead, found to be tensed at short times aer controlled cooling. It is not obvious that this treatment should produce elevated membrane tensions; however, characterization studies described previously40,41 and applied below to hybrid vesicles demonstrated that this was indeed the case, as detailed in the Results section.
Fig. 1 Appearance of copolymer/DPPC (“hybrid”) vesicles compared with DOPC/DPPC (“lipid”) vesicles for different compositions and two cooling rates from the one-phase region. The scale bars are 10 mm. Rh-DOPE is employed as the tracer.
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The hypothesized mechanism for development of membrane stress, key to the design of studies to measure tension, involves a starting point at elevated temperatures, for instance near 45 C in our studies, where vesicles exhibit little, if any, excess area.41 Cooling causes both the vesicle membrane and its water contents to contract thermally.44 The membrane contracts more extensively than the aqueous vesicle center, causing development of membrane stress. The membrane stress relaxes as water diffuses out of the vesicles across the membrane, on a timescale of minutes.45–48 Thus the actual values of the membrane stress and its variation with time depend on the cooling rate relative to that of water diffusion. If cooling is extremely fast, the membrane ruptures and reseals,49–51 making it difficult to anticipate whether fast or slow cooling will produce higher tensions. Extremely slow cooling, however, always maintains low tensions.40,41 Because of this complexity, membrane tension cannot be anticipated by calculations based only on osmotic pressure differences. Instead, membrane tension is measured, as described below.
Characterizing vesicle stress and strain Micropipette aspiration was employed to probe two different vesicle features: the traditional stress–strain curve, including the area expansion modulus, Ka, and the membrane stress relevant to phase separation. Both studies employed open micropipette-accessible chambers made of coverslips supported in a 1 mm gap conguration. The chamber's ends were coupled to a constant temperature reservoir. Stress–strain curves were measured following established procedures.18,44 A test vesicle was aspirated at low suction and, aer stretching to ensure good lubrication with the micropipette, the tension was set close to zero. Subsequent 10 second step-wise increases in suction were recorded on video. If the vesicle did not break, the tensions could be stepped back down and then back up to conrm reversibility. Vesicle images were analyzed to determine the membrane area and the strain at each step. The membrane tension, s, was calculated from the Laplace equation: P s Rp s¼ 2Rp 2 Rv
(1)
Rp and Rv are the micropipette and vesicle radius (outside the micropipette). Ps is the suction pressure on the micropipette. Separate studies quantied the combined impact of cooling history and osmotic conditioning on the hybrid membrane stress, at conditions similar to the initial instants of phase separation. Vesicles were cooled at the rate of interest to 35 C, a temperature within range of the phase transition, and then immediately transferred to the micropipette chamber that was heated to the same temperature. Measurements were made within minutes of transfer to the micropipette chamber, allowing some tension loss, but minimizing the loss as much as possible. The vesicle was characterized by rst attempting to aspirate at extremely low suctions, corresponding to stress in the range 0.02–0.04 mN m1, and areal strains of less than 0.1%. For accid vesicles, a projection Soft Matter
would appear in the micropipette. Tensed vesicles, however, would be pulled to the mouth of the pipette but not produce a projection. Subsequent increase in suction would produce a projection only once the suction pressure balanced the opposing force from the membrane tension. The suction pressure corresponding to the rst appearance of a projection allowed determination of the membrane tension of the stressed vesicles according to the Laplace equation, above.
Results Vesicle morphology Fig. 1 contains images of hybrid copolymer/DPPC vesicles photographed at room temperature aer cooling at different rates from the single-phase region at 43 C. These data demonstrate how the resulting vesicle morphology depends on composition and the thermal history during the approach to room temperature. Fig. 1 also presents remarkable parallels between copolymer/DPPC and DOPC/DPPC vesicle suggesting that copolymer and DOPC play similar roles as the lower melting uid membrane component (the images of the DOPC/ DPPC vesicles are reproduced from a paper focusing on DOPC/ DPPC phase separation41 for comparison with hybrid vesicles). In Fig. 1, single-phase uid membranes are consistently found, for both hybrid and DOPC/DPPC vesicles at low overall DPPC content. Single-phase hybrid membranes are also observed at temperatures above 41.5 C (the melting point of DPPC), as expected (images of single-phase uid vesicles at elevated temperatures are not included because they appear identical to those having low DPPC content). Dark domains, which exclude the tracer dye Rh-DOPE, are visible at room temperature in Fig. 1, and occupy an increasing membrane area with greater DPPC content in either vesicle type. The observed greater solid (dark) domain area with increased DPPC content indicates that the solid domains predominantly DPPC. This is in fact established for the DOPC/ DPPC system19,35,52 and suggests that the solid domains in the hybrid system have similarly high DPPC content. Vesicles electroformed and cooled in DI water to room temperature at a rate of 5 C min1 from about 43 C display patchy solid domains which, upon close examination, oen have hexagonal facets. Vesicles cooled more slowly, for instance at 1 C min1, can exhibit stripes or patches, depending on composition. Interesting in both hybrid and phospholipid systems, there are distinct ranges of compositions where cooling history, not the composition itself, determines whether striped or patchy solid domains persist. Quantitative differences distinguish the phase separation in the hybrid and DOPC/DPPC bilayers. Most notably, at the cooling rate of 1 C min1, in the phospholipid system there is a broader composition window, above about 45 mol% DPPC, where stripes are the solid morphology. In the case of the copolymer/DPPC vesicles, the compositional window for the striped solid morphology is narrower, starting around 70 mol% DPPC (at 70 mol% DPPC, a solid patch is seen among the solid stripes. The composition of 70 mol% DPPC in the hybrid vesicles apparently lies close to the boundary between the two solid
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morphologies). We will argue below that whether vesicles display patched or striped domain morphology depends on the membrane tension and, further, that the differences between the appearance of stripes or patches in lipid vs. hybrid vesicles cooled at 1 C min1 (the lower part of Fig. 1) resulted from modest differences in the tension of the two vesicle types. Molecular organization in striped and patchy solid domains Differences in the interaction of tracer lipid with the striped and patchy solid domains provide convincing evidence for differences in the molecular organization within the two domain types in either vesicle system. Fig. 2 compares the appearance of copolymer/DPPC and DOPC/DPPC vesicles containing either Rh-DPPE or Rh-DOPE tracer (the latter was employed in all the vesicles of Fig. 1). The patchy domains exclude both tracers, but the striped domains take up Rh-DPPE selectively, in either hybrid or phospholipid vesicles. Thus, depending on the tracer but not on the vesicle system (hybrid or strictly phospholipid), the stripes can appear light or dark. This argues for different molecular organization in the two types of solid domains and suggests that the two domain types are similar in the two vesicle systems. Membrane mechanics Fig. 3 compares room temperature stress–strain curves for copolymer, hybrid, and phospholipid vesicles aspirated in micropipettes. The mixed membranes contain 70 mol% DPPC and were manipulated to be patchy. Vesicle deformations were generally reversible. Because entry of solid domains (especially stripes) into the micropipette can sometimes interrupt the progress of aspiration and membrane stretching, we chose data
Appearance of vesicles containing 70 mol% DPPC, containing two different tracer dyes. Comparison is made between hybrid and phospholipid vesicles with cooling history related to tension. Images E and F only are reproduced from ref. 40. The scale bar is 10 mm.
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sets for the phase-separated vesicles where domains did not happen to hang up upon entry into the micropipette. Without over-emphasizing the quantitative aspects of stress–strain curves of the phase-separated vesicles, we included these data because they appear reasonable. We also included the single component vesicles as a reference. Fig. 3 makes two important distinctions between phospholipid and copolymer-containing membranes. First, the lysis stress and strain for the copolymer and hybrid vesicles far exceeds that of the pure phospholipid and especially the phaseseparated phospholipid. Lysis strains for the copolymer-containing vesicles exceed 8%, compared with 2% for the mixed phospholipid vesicles. A second take home point from the mechanical experiments is the difference in moduli between the hybrid and phospholipid membranes. Area expansion moduli of 200 mN m1, observed for our phospholipid vesicles, are typical of these liposomes.53,54 The copolymer-containing vesicles have area expansion moduli a factor of two lower. Additionally, the copolymer dominates the mechanics of mixed vesicles: hybrid vesicles containing 70% DPPC have an area expansion modulus only slightly greater than that of the pure copolymer. The stress–strain curves and the resulting area expansion moduli, Ka, in Fig. 3 are representative of at least 10 vesicles of each type within 10% error, and are typical of uid phospholipid53–55 and copolymer vesicles.55 Indeed, for copolymers there is little dependence of the Ka on membrane thickness or the choice of copolymer.5,55 The consistency of our ndings with the literature and the relatively tight reproducibility argue that these vesicles are unilamellar. Multilamellar or onion-skin vesicles have integer multiples of the Ka values of unilamellar vesicles. Such anomalous specimens were rare (less than 5%) of the vesicles we encountered and were easily distinguished by their mechanical properties and appearance (which allowed us not to choose them in micrographs or other studies).
Fig. 2
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Fig. 3 Comparison of stress–strain curves for pure DOPC, 30/70 DOPC/DPPC, 30/70 copolymer DPPC, and copolymer vesicles. The asterisk indicates rupture. Ka values are shown.
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Taken together these results suggest that for a given strain, the copolymer and hybrid vesicles will experience lower stress than the pure phospholipid system. Additionally, hybrid membranes, even when phase separated, exhibit a smaller tendency to rupture compared with entirely phospholipid membranes.
Tension and mechanism of morphological fate Fig. 1 illustrates how, in both membrane types, the cooling rate appears to dictate the morphological vesicle fate over a range of membrane compositions. Indeed the cooling rate turns out to be among the most experimentally-accessible means of tuning morphology. For DOPC/DPPC vesicles; however, it was demonstrated that membrane tension, imposed osmotically or using micropipettes, was the underlying factor governing the morphology.40,41 If the tension of DOPC/DPPC membranes was controlled independently, the inuence of cooling rate on the initial appearance of stripes or patchy solid domains was removed. The exception was that large domains either type could be kinetically trapped at low temperatures. Fig. 4A summarizes membrane tensions, s, at the intermediate temperature of 35 C. This temperature chosen to be in the middle of cooling runs near the onset of phase separation, as will be demonstrated below in Fig. 5. Fig. 4 reports that tension similarly inuences hybrid and DOPC/DPPC vesicles, though there are quantitative differences. Then Fig. 4B demonstrates that tension is the dominant factor in establishing the type of solid formed. Towards an understanding of Fig. 4A, we rst review the features of the cooling/tension measurement study: prior to cooling, vesicles are annealed in the single phase region at 43 C and this was found to produce s 0.41 Also in general, at long times aer cooling, membrane tension has relaxed to zero. At intermediate times, cooling causes the membrane to contract. Thermal contraction can be substantial for phospholipids, a 5%
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area decrease is expected for each 10 C of cooling.44 So, if vesicles are not accid initially (for instance as a result of osmotic conditioning), thermal membrane contractions produce stress. Membrane stresses relax by water diffusion from the vesicle center, a process that equilibrates on the order of tens of minutes for phospholipids.45–48 So during periods of cooling and also later at constant temperature, whenever there is membrane stress, there is stress relaxation via water diffusion. As a result of the two competing processes (thermal contraction to produce stress and diffusion to relax it), membrane stress goes through maximum with time, but starts and ends near zero. If thermal quenching is too rapid relative to diffusive timescales, stresses may exceed the lysis conditions reported in Fig. 3 and water release may occur by membrane rupture. This behavior is difficult to detect directly, but the breaking and resealing of stressed membranes has been previously reported.49–51 We are interested in these intermediate elevated tensions because they determine the domains formed during phase separation. An approximation of the membrane tension in the early moments of phase separation comes from measurements of membrane tension at an intermediate temperature. We chose 35 C. At this temperature, cooling runs were interrupted and vesicles transferred to the micropipette chamber, at a constant temperature of 35 C. Measurements of membrane tension were made immediately upon transfer because even at constant temperature, water diffusion across the membrane causes a continued reduction in membrane stress. Further, because tension is decreasing from the instant vesicles were transferred to the stress measurement chamber, the stresses measured are lower than they were just prior to transfer at 35 C. For this reason, we say that the measured stresses represent a lower bound on the stresses occurring at 35 C in phase separation runs. Fig. 4A rst shows that vesicles, conditioned with a higher sucrose osmolarity on the outside of the vesicle compared with
Fig. 4 (A) Membrane tension after cooling vesicles containing 70% DPPC, osmotically conditioned or handled in DI water, to 35 C, a temperature near that of the initial phase separation. 10–15 vesicles were studied at each condition and error bars reflect the full range of tension values observed. For the vesicles conditioned in sucrose, the tension was zero for all vesicles and so no error bars appear. (B) Images of hybrid vesicles containing 70% DPPC subject to different cooling and osmotic control, indicated. Scale bars are 10 mm.
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(A) observed temperatures for the first appearance of patches on cooling in phospholipid and hybrid vesicles. Lower black line summarizes melting point depression calculation in which the fluid phase is an ideal solution (B) same experiments but with additional data on hybrid vesicles to facilitate a mass-fraction representation on the x-axis.
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the interior, do not experience stress at the intermediate temperature of 35 C, during a cooling run. This is the case for both vesicle types because such osmotic conditioning produces excess membrane area (the vesicles are made oppy). With thermal contraction of the membrane during cooling, this area is consumed; however, there must be sufficient excess area that a tension increase is not observed. By contrast, for vesicles cooled in DI water, Fig. 4A reveals that substantial membrane stresses at 35 C, aer transfer to the stress measurement chamber. Because stress is decreasing continually during the transfer, the values in Fig. 4A are lower than those actually occurring at 35 C during the cooling run in which phase separation is occurring. Membrane stress develops for vesicles in DI water but not for sucrose because only the latter have excess area (as a result of osmotic conditioning). Fig. 4A reports that higher membrane tensions are sustained at the cooling rate of 1 C min1 compared with lower near-zero tensions at 5 C min1. These differences in tensions are statistically signicant within 99% certainty. Fig. 4A also reports differences in the tension in phospholipid compared with hybrid vesicles. The impact of cooling rate of membrane tension may result from the greater tendency for vesicle lysis upon rapid thermal quenching. Additionally, differences between the two vesicle types could be explain by the observed lower area expansion moduli, in Fig. 3. For a given membrane contraction on the x-axis, the hybrid vesicles sustain a lower membrane stress. By isolating the impacts of tension and cooling rate, Fig. 4B illustrates, for hybrid copolymer vesicles, that tension is the important variable in dictating domain morphology. In hybrid vesicles osmotically-manipulated to maintain zero membrane tension, solid patches form, independent of cooling rate. However, for the same cooling rate but two different tensions, stripes appear at high tensions while patchy solid domains are seen at low tensions. A preference for patch-shaped solid domain formation was previously reported in mixed DOPC/ DPPC vesicles maintained near zero tension.40,41 The similar
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roles of tension in both vesicle types suggest more broadly parallel underlying thermodynamics for hybrid and purely phospholipid vesicles. Cooling rate is a convenient processing variable through which tension, and therefore the solid morphology can be tuned. Once solid domains form, however, their morphology is retained for hours, on the order of vesicle lifetime. A large energy barrier opposes the transformation of one domain type to another, even when stress is applied (at room temperature). In obtaining the mechanical data in Fig. 3, we observed no qualitative alteration of the dark solid domains. Composition-temperature space Fig. 5 illustrates the thermodynamic parallel between the two classes of vesicles in terms of the low-tension temperaturecomposition space for uid–solid phase separation. Fig. 5 plots the temperatures for the initial appearance of solid domains in vesicles of varying composition that were cooled sufficiently quickly from the one-phase region to produce patches. The domains start as small nuclei that become clearly visible when they reach a size of about a micron.42 The temperature of rst appearance is not signicantly dependent on the cooling rate as long as the rate produces tensions sufficiently low for patchy domain formation. Results for the DOPC/DPPC system are in quantitative agreement with literature studies employing NMR,19 supporting the sensitivity of the uorescence method. Fig. 5A demonstrates that for hybrid and phospholipid vesicles the temperature-composition state diagram appears similar, except that phase separation occurs at slightly elevated temperatures in the hybrid system. Both sets of experimental data in Fig. 5A extrapolate to the melting temperature of pure DPPC (41.5 C). Additionally, the observed dark domain areas in phase separated DOPC/DPPC vesicles having patch shaped domains were previously found to conform to the inverse lever arm rule with a solid phase containing 95% or more DPPC, arguing that the amounts of domains represented a local equilibrium.42 Together these
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points motivate the calculation of the standard colligative property behavior for melting point depression in an ideal solution uid mixture with a pure solid, shown as the lower black curve in Fig. 5A. It is apparent that the DOPC/DPPC system exhibits modest deviations from ideal solution. While the DPPC/copolymer interaction favors mixing less than the DOPC/DPPC interaction, neither system is sufficiently non-ideal to produce uid–uid de-mixing.
Discussion The behavior of these hybrid copolymer–phospholipid vesicles is remarkable in several regards. First, our results provide a counter-example to the idea that bilayer-forming polymers are fundamentally incompatible with phospholipid lamellae.6–8 Phospholipid–copolymer compatibility requires chemical compatibility along with some matching of lamellar thicknesses. Our hybrid lamellae are almost as compatible as mixed phospholipid membranes. Phase separation in membranes such as ours, where DPPC is mixed with a low melting lamella former, results from the fundamental tendency for DPPC to crystallize below 41.5 C, rather than from a fundamental incompatibility between the two components. The region of thermodynamic space in which DPPC crystallizes in hybrid vesicles is only slightly larger than the two-phase region for DOPC/DPPC mixtures, arguing for substantial miscibility of both systems. Indeed, the solubility parameter of PDMS is 14.9 J cm3,56 close to that of alkanes (14– 16 J cm3) and likely resembling interactions in the hybrid membrane core. Miscibility is further favored by similar DPPC and copolymer bilayer thicknesses, 5.5 (ref. 57) and 7.7 nm (ref. 15) respectively (the copolymer includes a PEG corona while the DPPC includes only hydrated polar headgroups). Additionally in the copolymer membrane, the PDMS chains assume a random conformation, facilitating nanometer-scale changes in lamellar thickness to accommodate “objects” such as solid DPPC domains. Worth considering, copolymer polydispersity introduces uncertainty in the molecular weight and therefore uncertainty in the mole fractions of the mixed hybrid membranes. Polymer blend compositions are oen prescribed as mass or volume fractions. If mass fraction is employed as the independent variable, the state diagram appears as Fig. 5B. In Fig. 5B, the hybrid membrane phase diagram shows further apparent deviations from ideal solution behavior, but the underlying parallel behavior is preserved. Domains in DPPC-containing membranes likely assume at least one of the distinct solid polymorphs exhibited by DPPC. These include a family of corrugated ripple (Pb0 ) morphologies that persist, for pure (but hydrated DPPC) between 38.5 and 41.5 C, and a planar tilt gel (Lb0 ) phase below 38.5 C down to room temperature. The assignment of these different bilayer solids to patches and stripes within mixed DPPC-containing lamellae has been debated.22,58 Striped regions with corrugated morphology found by AFM within DPPC-containing membranes immobilized on mica suggest a similar relationship in vesicles.59 Our more recent data, with mixed DOPC/DPPC vesicles, argue
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differently:40,41 we document the formation of stripe solid DPPC domains at elevated tensions above a triple point and patchy solid domains at lower tensions. The observed tension-dependence of the phase transition temperatures of DOPC/DPPC vesicles was found to be in quantitative agreement with fundamental thermodynamic arguments (somewhat similar to Clausius–Clapeyron) when the patchy solid domains were assigned the properties of the DPPC ripple phase. This strongly suggests the ripple Pb0 solid phase within the patchy DPPC domains and a gel Lb0 phase in the stripes. The most straight-forward argument, however, for the ripple Pb0 phase (with its greater areal density of DPPC) within the patchy solid, is the tendency of the membrane to favor patches at low tension and stripes at high tension, for example in Fig. 1, 2 and 4. Simply put, the less dense membrane phase Lb0 persists at higher tensions. Fig. 1 therefore suggests that the patches and stripes in the hybrid vesicles are ripple and gel DPPC solid domains, and that the patchy DPPC solid domains have similar DPPC content in both cases. In this sense, manipulation of the tension of a polymer membrane, in our hands, directs the crystallization and morphology of a phospholipid. Other observed parallels between the vesicle morphology in the hybrid and phospholipid systems include similar domains sizes and numbers, and a similar inuence of cooling history. This suggests similar membrane thermodynamics and similar mechanisms for domain formation. Patchy DPPC domains in phospholipid membranes are found to result from nucleation and growth,42 and the same mechanism is likely to produce DPPC domains during cooling of hybrid membranes. To the extent that the domain sizes and numbers are similar for a given compositions and cooling rate, the nucleation rate, with its dependence on the enthalpy of domain formation and the line tension around nucleated domains, suggests that line tensions in the hybrid system are not unusual. Indeed the mechanical strength of phase-separated hybrid vesicles in Fig. 3B suggests that line tension is not high. An important difference between hybrid and phospholipid membranes, evident in Fig. 1, is the greater tendency for patch formation and smaller compositional window for stripe formation in hybrid vesicles. With the correlation between low tension and stripe formation in both systems, this suggests lower tensions in the formative instants of phase separation of hybrid vesicles. This hypothesis is affirmed, at least in part by the mechanical data of Fig. 4A. For a given membrane strain (such as a thermal contraction), the soer copolymer-containing vesicles experience a lower stress and a greater tendency to produce patches rather than stripes. We expect that separate manipulation of the hybrid vesicles to sustain large membrane tensions will produce stripes over a larger compositional range.
Conclusions This study examined multicomponent membranes that contained copolymer and phospholipid mixtures to probe how well a mechanically-robust hybrid system could approximate the behavior of mixed phospholipid membranes. Giant unilamellar hybrid vesicles and analogous phospholipid vesicles containing
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DPPC were in many ways similar over the full compositional range. Similarities included the general appearance of vesicles having analogous compositions and thermal histories. Phase separation in both systems resulted from the tendency for DPPC to solidify in the membrane to form nearly pure DPPC domains. The DPPC domains appeared patchy or stripe-shaped, depending on composition, tension, and thermal history, with striking similarity between the two types of membranes. The temperatures observed for the onset of phase separation was, to rst order the same in the two systems. Small differences included slightly higher solidication temperatures in copolymer-containing membranes, indicative of slightly less ideal mixing in the hybrid compared with the phospholipid system. The mixing non-idealities were insufficient to produce uid– uid de-mixing. Tension was shown to play a dominant role, (tuned through cooling history), in determining the types of membrane domains to form. Parallel dye-incorporation behavior between hybrid and DOPC/DPPC membranes suggested that the striped and patchy domains in either system were a result of differences in their molecular organization. A ripple DPPC polymorph in the patchy domains along with a tilt gel structure in the stripes was the best explanation for the differences in the domains. The current study demonstrated how a copolymer can replace a low-melting phospholipid to produce mechanicallyrobust vesicles with similar thermodynamic and tension response to phospholipid membranes, including domain morphology and connectivity. These new materials represent tools for further study of biological mechanisms and may form the basis for future materials that communicate adhesively with cells.
Acknowledgements This work was supported by NSF-0820506 and NSF-1264855.
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