Langmuir 2002, 18, 7299-7308
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Effect of Surfactant on Unilamellar Polymeric Vesicles: Altered Membrane Properties and Stability in the Limit of Weak Surfactant Partitioning Maria M. Santore* Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 01003
Dennis E. Discher Departments of Chemical Engineering and Mechanical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104
You-Yeon Won and Frank S. Bates Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
Daniel A. Hammer Departments of Chemical Engineering and BioEngineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received February 7, 2002. In Final Form: July 8, 2002 Surfactant incorporation into bilayer vesicle membranes made of diblock copolymers was examined for the case of weak affinity between the membrane [poly(ethyl ethylene)-co-poly(ethylene oxide)] and the surfactant [Pluronic L31]. For copolymer vesicles formed in the absence of surfactant and subsequently exposed to surfactant solutions, a micropipet aspiration technique was employed to monitor surfactant incorporation kinetics and to characterize surfactant-induced changes in the membranes. Though the surfactant incorporation was weak and reversible, it reduced the area expansion modulus by almost a factor of 2, while dramatically increasing the vesicles’ susceptibility to lysis. Additionally, water permeability was increased by a factor of 2. The surfactant incorporation rates were proportional to the free surfactant concentration, and other features of the membrane response to surfactant exposure were consistent with transport-limited surfactant attack of the membranes. The most plausible mechanism for surfactant interaction with the membrane is that the surfactant resides at the interface between the hydrophobic membrane core and the poly(ethylene oxide) corona, reducing the interfacial tension of the lamella and allowing the membrane to thin slightly, also increasing its area per unit mass. Observations suggest slow surfactant penetration of the hydrophobic core such that this particular surfactant acts on both inner and outer leaflets.
Introduction The incorporation of surfactants and peptides into bilayer membranes is of interest for several reasons: First, it provides perspective on the mechanisms by which viruses infect living cells using surface-active fusion proteins and the mechanisms by which lysis proteins (also surfactant-like, such as those associated with beetle and bee toxins) alter cell membranes. Second, since liposomes and other vesicles have found use in drug delivery and potential gene therapy applications, it is important to understand how amphiphilic molecules, especially those occurring naturally in the body, interact with these carriers. Third, with the delivery concept in mind, the exposure of surfactants to cargo-containing vesicles may alter their permeability and lysis behavior, affording precise control over the release of vesicle contents. The attack of fusion peptides on phospholipid bilayers has been a focus of recent study, especially with the growing need to understand the mechanisms by which viruses operate.1-7 From such works, it is now understood that fusion peptides incorporate into SOPC (1-stearoyl(1) Longo, M. L.; Waring, A. J.; Gordon, L. J.; Hammer, D. A. Langmuir 1998, 14, 2385-2395.
2-oleoyl phosphatidylcholine) membranes and ultimately form pores at appropriate pHs.1,2 There have also been reports of biphasic uptake kinetics suggestive of a mechanism where the surfactants first permeate the outer leaflet of a bilayer layer and then, with a slower rate, access the inner leaflet by traversing the hydrophobic core or by taking advantage of fluctuating membrane density.3 Other work, focusing on the morphologies in suspensions of surfactants and egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), or polyoxyethylene-8lauryl ether (C12E8), revealed unusual membrane phases including planar membrane fragments.8 Also relevant is that certain charged synthetic polymers and surfactants are known to destabilize cell membranes and liposomes.9-11 Investigators therefore are exploiting (2) Longo, M. L.; Waring, A. J.; Hammer, D. A. Biophys. J. 1997, 73, 1430-1439. (3) Needham, D.; Zhelev, D. V. Ann. Biomed. Eng. 1995, 23, 287298. (4) White, J. M. Science 1992, 258, 917-924. (5) Hoekstra, D. J. Bioenerg. Biomembr. 1990, 22, 121-155. (6) Stegmann, T.; Delfino, J. M.; Richards, F. M.; Helenius, A. J. Biol. Chem. 1991, 266, 18404-18410. (7) White, Annu. Rev. Physiol. 1990, 52, 675-697. (8) Johnsson, M.; Silvander, M.; Karlsson, G.; Edwards, K. Langmuir 1999, 15, 6314-6325.
10.1021/la0201319 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/29/2002
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the electrostatic attractions between cationic polymers or surfactants and negatively charged phospholipid membranes to produce bacteriocides12 and antibiotic surfaces13 that cause leakage in cellular membranes. More recently, the leakage induced by electrostatic-driven adsorption (or incorporation) of proteins, peptides, and surfactants has formed the basis for a highly sensitive chromatographic method.14 While these works demonstrate the importance of electrostatic interactions to drive incorporation of foreign molecules into membranes, hydrophobic interactions also are known to play a key role in the attack of cell membranes by the sided helices within viral fusion peptides.10 The current paper explores the static and dynamic features of hydrophobically driven nonionic surfactant interactions with uncharged bilayer membranes, for the case of polymeric rather than phospholipid or low molecular weight surfactant vesicles. Motivation stems from the discovery that certain nonionic diblock copolymers spontaneously form lamellar phases and, with appropriate mechanical agitation, can produce giant unilamellar vesicles, in the size range of living cells.15-19 The polymeric vesicles, which have been the focus of several recent articles,15,20 consist of poly(ethyl ethylene) (PEE) hydrophobic cores and poly(ethylene oxide) (PEO) coronas and are extremely robust. In particular, they can sustain on the order of 20% strain to lysis, despite area expansion and bending moduli that are similar to those of phospholipid vesicles such as SOPC. The long-time stability of the polymeric vesicles greatly exceeds that of phospholipid membranes, and the permeability to water of the polymeric vesicles is several orders of magnitude lower. To exploit these new materials whose promise for delivery applications is so great, it is a high priority to develop means by which the substances they carry can be released on demand. Attack by surfactants is one strategy. There are an enormous number of options for the choice of surfactant to alter the vesicle membranes. As a starting point, we considered Pluronics, a family of nonionic triblocks with established biomedical applications. The particular surfactant, L31, was chosen because of its modest hydrophobicity: L31 is a triblock surfactant with a nominal molecular weight of 900 and a nominal poly(propylene oxide) (PPO) composition of 90 mass %, which makes up the middle block. The end blocks are PEO, comprising the remaining 10 mass % of the molecule. The nominal PEO content is only 100 molecular weight units, or roughly one EO monomer on each PPO chain end. Because the L31 surfactant possesses only modest hydrophobicity, it was expected to exhibit limited micelle (9) Rafalski, M.; Ortiz, A.; Rockwell, A.; van Ginkel, L. C.; Lear, J. D.; DeGrado, W. F.; Wilshchut, J. Biochemistry 1991, 30, 10211-10220. (10) Oren, Z.; Shai, Y. Biopolymers 1998, 47, 451-463. (11) Zauner, W.; Orgis, M.; Wagner, E. Adv. Drug Delivery Rev. 1998, 30, 97-113. (12) Tew, G. N.; Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll, P. J.; Klein, M. L.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5110-5114. (13) Tiller, J. C.; Leao, C.-J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981-5985. (14) Boukobza, E.; Sonnenfeld, A.; Haran, G. J. Phys. Chem. B 2001, 105, 12165-12170. (15) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143-1146. (16) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Curr. Opin. Colloid Sci. 2000, 5, 125-131. (17) Lee, J. C.-M.; Burmudez, H.; Discher, B. M.; Sheehan, M. A.; Won, Y.-Y.; Bates, F. S.; Discher, D. E. Biotechnol. Bioeng. 2001, 63, 135-145. (18) Lee, J. C.-M.; Law, R.; Discher, D. E. Langmuir 2001, 17, 35923597. (19) Lee, J. C.-M.; Santore, M.; Bates, F.; Discher, D. E. Macromolecules 2002, 35, 323-326. (20) Aranda-Espinoza, H.; Bermudez, H.; Bates, F. S.; Discher, D. E. Phys. Rev. Lett. 2001, 87, 208301.
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formation, an attractive feature for the current investigation. A relatively high critical micelle concentration (cmc) (or negligible micellization) is significant because it facilitates examination of single-molecule interactions with the membrane over a broad range of surfactant concentrations. Other surfactants, which are more hydrophobic, possess lower cmc’s, limiting the concentrations of individual molecules interacting with vesicle membranes. Greater surfactant hydrophobicity restricts one to the dilute limit of single-molecule concentration. Modest hydrophobicity also stipulates weak partitioning between the L31 surfactant and the nonionic vesicle membrane, providing the focus of the current work. The regime of weak partitioning, with limited surfactant concentration in the membrane phase and substantial surfactant in free solution, is distinctly different from the opposite case of strong surfactant partitioning into the membrane. With strong partitioning, a two-component membrane phase is present while the free solution surfactant concentration is negligible.21,22 Of particular technological relevance, in the limit of weak partitioning, the surfactant incorporation should be reversible. This article presents micropipet aspiration experiments23-25 demonstrating that even with weak surfactant-membrane partitioning, vesicle membrane properties such as permeability, area expansion modulus, and susceptibility to lysis are substantially altered. We show that the surfactant incorporation is reversible, as are changes in the vesicle properties themselves, suggesting even greater versatility in using weakly associating surfactants to tune vesicle membrane properties. Also addressed are the dynamics and mechanism of surfactant attack on the membrane, here found to be consistent with transport-limited behavior. Experimental Section The PEO-PEE diblock copolymer constituting the polymer vesicles was OE-7, whose use was described previously.15-20,26,27 On average, it contains 40 EO and 37 EE segments, with an overall polydispersity of 1.1. Chloroform was purchased from Fisher. Solutes such as sucrose and glucose were purchased from Sigma (St. Louis, MO). The Pluronic L31 surfactant, a gift from BASF, was used as received. The cmc for L31 was not revealed by our literature search, and BASF staff scientists informed us that L31 does not possess a true cmc. Nonetheless, a surface tension study, employing the bubble tensiometer method, revealed apparent cmc’s near 650 ppm for L31 in deionized water and 250 mOsm glucose solutions. The cmc value was associated with the break in the plot of surface tension as a function of concentration. Vesicles of the OE-7 diblock copolymer were prepared by electroforming on platinum electrodes. A film of the copolymer was deposited on platinum wires by evaporation of 20 µL of a carbon tetrachloride solution of the copolymer. The film was dried under vacuum for several hours, and then the electrodes were submerged in a 260 mOsm sucrose solution. A 10 V, 10 Hz current was applied to the electrodes for up to 8 h, after which it was reduced to 3 V and 3 Hz for 10-30 min. The vesicles, most of which were unilamellar as evidenced by area expansion moduli of 120 mN/m,15 were harvested in a syringe and stored for later use. These stock solutions contained sucrose solution both inside and outside the vesicles. When the vesicles were studied in the (21) Bagatolli, L. A.; Gratton, E. Biophys. J. 2000, 78, 290-305. (22) Jorgenson, K.; Mouritsen, O. G. Biophys. J. 1995, 69, 942-954. (23) Evans, E.; Needham, D. J. Phys. Chem. 1987, 91, 4219-4228. (24) Olbrich, K. C.; Rawicz, W.; Needham, D.; Evans, E. A. Biophys. J. 2000, 79, 321-327. (25) Needham, D.; Zhelev, D. In Vesicles; Rosoff, M., Ed.; Marcel Dekker: New York, 1996; pp 372-443. (26) Hillmyer, M. A.; Bates, F. S. Macromolecules 1996, 29, 69947002. (27) Hajduk, D. A.; Kossuth, M. B.; Hillmyer, M. A.; Bates, F. S. J. Phys. Chem. B 1996, 102, 4269-4276.
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Figure 1. Two-chambered transfer cell with aspiration (coming in from the right) and transfer (coming in from the left) pipets shown. The vesicle-containing fluid resides in the center region of each chamber. The stacked regions are layers of microscope slide pieces, while the bottom of each chamber has been cut from a cover slip. The transfer pipet comes all the way through the left chamber into the right chamber to mate with the aspiration pipet. Then the entire cell is shifted so the mated tips of the pipets finish in the left chamber. various experiments, they were typically transferred to glucose solutions, such that the sugar external to the membrane capsules was replaced by glucose. This generated a refractive index difference that allowed the vesicles to be imaged in a light microscope. Two-chambered observation cells (see Figure 1) were employed in kinetic studies of surfactant incorporation and water permeability. These were fashioned from glass pieces cut from cover slips and microscope slides. The two parallel chambers were spaced roughly 2-3 mm apart, each with approximate dimensions of 10 × 5 × 3 mm. Micropipets were pulled from capillary glass (Friedrich and Dimmock, Millville, NJ) on a Kopf Instruments (Tujnga, CA) puller. The pipets were forged on a Technical Products International (St. Louis, MO) microforge. The inner diameters, which typically ranged from 6 to 8 microns, were calibrated in an optical microscope using a micrometer grid. This method was shown to give results consistent with previous pipet calibration methods.1 For simple measurements of the area expansion modulus, Ka,23 single-chambered observation cells were employed. These consisted of a single observation chamber, identical to one-half of the pair shown in Figure 1. For studies focusing on the area expansion modulus, Ka, surfactant uptake kinetics, or water permeability, the chamber preparation was important. The chamber surfaces were passivated with albumin solution for up to 1 h prior to sample testing, as were the micropipet surfaces. Prior to vesicle studies, each chamber was rinsed several times with the appropriate glucose/ surfactant solution to ensure that the solute concentrations within the chamber were those intended. The current micropipet aspiration studies largely employed equipment and followed procedures used in the Hammer lab.1,2 Experiments were conducted on an inverted Nikon Diaphot-TMD microscope with Hoffman optics (Modulation Optics, Greenvale, NY) and a Videoscope International CCD-200 camera (Washington, DC). Of note, the Hoffman optics are sensitive to edge features of objects and rely on the difference in refractive index between the inside (sucrose solution) and outside (glucose/ surfactant solution) of the vesicles. All video frame images contained a video overlay of time and suction pressure and were recorded on a Sony SVO-9500VTR machine. Labview software (National Instruments, Austin, TX) was employed to obtain vesicle dimensions and projection lengths. During vesicle aspiration, pressure was controlled using a twochambered manometer, with Validyne pressure transducers (Northridge, CA) to monitor the suction, which was overlaid on the video. The micropipets themselves were manipulated within the test chamber using Narishige micromanipulators. Area Expansion Moduli. To determine the influence of L31 surfactant on the area expansion modulus, Ka, of the copolymer vesicles, the latter were incubated in surfactant solutions for 2-4 days prior to modulus measurements. Typically, 50 µL aliquots of the stock vesicle solution were added to 1 mL samples of glucose solutions containing L31 surfactant. The greater density of the sucrose inside the vesicles caused them to slowly settle to the bottom of the glucose-surfactant solutions, where they could be collected for further study. Vesicles kept in
Langmuir, Vol. 18, No. 20, 2002 7301 surfactant solutions for 7-10 days showed similar properties to those of vesicles incubated for 4 days. To measure the area expansion moduli, samples of vesicles in L31 surfactant/glucose solutions were transferred to singlechambered observation cells. From the vesicles present, the larger ones, typically greater than 25 µm, were chosen and each aspirated into a micropipet, as shown in frame A of Figure 2. Vesicles were prestressed up to tensions of 5-10 mN/m to smooth any wrinkles and incorporate any tethers back into the membrane. The suction was then reduced to a low level, near 2 cm of water, just sufficient to hold the vesicle in the pipet. The suction pressure was then increased stepwise (∼5-8 cm water per step, as convenient and reproducible), nearly to the point of lysis. The suction was then reduced, and a second series of measurements were made to check reproducibility. The second series of steps was conducted up to the point where the vesicles broke. Videotape of each experiment was analyzed to determine the vesicle diameter and the projection length, L, inside the pipet at each incremental stress/strain point. The isotropic membrane tension, τ, for each point was then calculated according to25
τ ) PsRp/(2 - 2Rp/Rv)
(1)
where Rp is the inner pipet radius and Rv is the vesicle radius external to the pipet. Ps is the suction pressure. The area change, ∆A, was calculated according to
∆A ) 2πRp(1 - Rp/Rv) ∆L
(2)
Tension was plotted as a function of fractional area change, and the slope of the curve is, by definition, Ka. Equation 2 assumes that each vesicle is substantially larger than the pipet diameter. 23 In our studies, Rv/Rp was typically 3 or greater. We ensured the applicability of eq 2 by aspirating vesicles of different sizes. The fact that they all gave the same Ka values and lysis strains, within a small margin of experimental error, confirmed that our procedures were appropriate. Typically 10 vesicles were sampled for each surfactant solution studied. Usually there was no notable effect of the rate of stretching on the resulting stress-strain behavior, and data were typically taken by waiting 4-5 s between steps. In some cases in the presence of surfactant, the time spent at each incremental stress/strain point did affect the results. Such details are noted specifically in the discussion below. Measurements of Surfactant Uptake Kinetics. The kinetics of surfactant uptake into the OE-7 membranes was studied, one vesicle at a time, for over 60 vesicles using a transfer pipet technique adapted from those previously described.1,2 In the current version of the experiment, a test vesicle was chosen from many vesicles kept in glucose solution in the right-hand side of a two-chambered microscope observation cell, as in Figure 1. The suction pressure was increased stepwise to measure Ka prior to the transfer. Any vesicles not having Ka’s in agreement with previously reported values for OE-7 were discarded (because they were likely multilamellar). The suction was decreased to a low level just necessary to hold the vesicle in the pipet, 2 cm of water, and a transfer pipet, which had been prefilled with the solution from the right chamber, was brought in close proximity to the test vesicle. The test vesicle and the tip of its micropipet were then moved inside the transfer pipet, and the pair of pipets in this configuration was shifted into the left observation chamber. Next, at time zero, the transfer pipet was pulled back and away from the test vesicle, exposing it to the surfactant solution in the left chamber. The test vesicle was held at fixed tension, and its subsequent size and shape changes were recorded. Photos of this process are found in Figure 2. In studies of surfactant uptake, the glucose solution in the right observation chamber was chosen to have an osmotic pressure identical to that of the solution of surfactant and glucose in the left observation chamber, with both solutions near 275 mOsm. The equality of osmotic pressures in these experiments, along with the pre-equilibration of the vesicles in the right chamber prior to the transfer, ensured negligible water permeation of the vesicles during the experiment. Therefore, while the test vesicle was held in the surfactant solution, its volume was constant. Incorporation of surfactant into the membrane, how-
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Figure 2. Vesicle transfer to 2680 ppm L31 Pluronic: (A) before the transfer; (B,C) moving into the transfer pipet (which gives some optical distortion); (D) transferring into Pluronic solution (focus lost while in air); (E,F) removing from the transfer pipet; (G-I) holding in Pluronic solution while the projection grows. Note that in (G) the lines highlight the projection evolution, which has actually begun in (F), immediately upon removal from the transfer pipet. above for surfactant uptake kinetics, but in permeability studies, both chambers contained the same surfactant concentrations but different amounts of glucose to give different osmotic pressures. Vesicles were pre-equilibrated in the first chamber, and their Ka values were checked prior to their transfer and permeability measurement. Since the surfactant concentration was the same in both observation cells, the membrane area should have been constant during and after transfer. The water diffusion across the membrane, driven by the osmotic difference, however, caused volume changes that could be quantified by measuring the change in the projection, L, in the pipet:24
V(t) ) π[2Rp3/3 + Rp2(L - Rp) + Rv3(2 + 3u - u3)/3]
Figure 3. Stress as a function of areal strain for OE-7 vesicles incubated in L31 Pluronic solutions at different concentrations: 0, 15, 150, 900, and 3000 ppm. ever, increased the membrane area, causing the projection to protrude further into the aspiration micropipet. From measurements of the projection length and the diameter of the vesicle outside the micropipet, the area evolution was determined using eq 2. It was also possible to establish that during the time frame of interest, the vesicle volume was indeed roughly constant. To the extent possible, long experiments were avoided because evaporation from the test chambers tended to increase the glucose and surfactant concentrations of the test solutions, reducing the vesicle volume and artificially lengthening the projection in the holding micropipet. Measuring Water Permeability. We followed the procedure described by Olbrich et al.,24 in which vesicles were transferred between glucose solutions of differing osmotic pressures, and changes in the projection in the pipet were measured. The manipulation of test vesicles between two observation chambers using a transfer pipet was similar to the procedure described
(3)
Here u is a correction factor, defined to be (1 - (Rp/Rv)2)0.5. The assumption of constant area in our studies was shown to be valid by calculating the vesicle surface area. From eq 3, the permeability was ultimately determined. Olbrich et al.24 describe a simple model leading to the following equation for the determination of the permeability, P:
(V* - 1) exp(V*) ) (V0 - 1) exp(V0 - tPACiνw/Vi) (4a) or, defining the quantity Y for ease of graphical analysis,
Y ) (V* - V0) ln[(V* - 1)/(V0 - 1)] ) -tPACiνw/Vi (4b) Here V* is the dimensionless vesicle volume, V(t)/V(t ) ∞), νw is the specific volume of water, A is the vesicle area, Ci is the concentration external to the vesicle, and permeability is defined by the rate of volume change or water flow across the membrane:
dV/dt ) PAνw ∆c
(5)
The only assumptions leading to eq 4 are that osmotic gradients within and outside the vesicle are negligible, such that the
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Figure 4. The effect of Pluronic L31 bulk solution concentration on the OE-7 membrane area expansion modulus. The main figure is a pseudoisotherm representation, while the inset shows the logarithmic scale. primary resistance to water diffusion is across the membrane. Olbrich has shown that this is a reasonable approximation for phospholipid vesicles24 whose permeabilities are up to 2 orders of magnitude greater than those of the polymeric vesicles in this study. Therefore, this approximation is even more robust for our study. Also, eq 4 can be applied for vesicles transferred to solutions of higher or lower osmotic pressure than the original. We examined both in this study, with changes in osmolarity on transfer ranging from 5 to 20 mOsm and the original osmolarity near 275 mOsm.
Results Mechanics and Isotherms. Figure 3 illustrates the membrane tension as a function of the fractional area strain for several different vesicles after incubation in solutions of L31 Pluronic at different concentrations. The slope of the τ versus ∆A/A0 plot yields the area expansion modulus, Ka. For the native OE-7 vesicle, we find a Ka of 120 mN/m, reproducing previously measured values for OE-7.15 With increasing amounts of L31 surfactant, Ka appears to decrease and τ is no longer linear in ∆A. Rather, τ curves turn over gently with increased areal strain. In situations with curved stress-strain plots, we determined Ka from the apparent zero strain limit, with the vesicle held gently but fully aspirated into the pipet. (While an even smaller strain regime,
