Journal of Controlled Release 67 (2000) 203209 www.elsevier.

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Polymer-induced leakage of cations from dioleoyl phosphatidylcholine and phosphatidylglycerol liposomes

James L. Thomas*, David A. Tirrell
Received 24 September 1999; accepted 19 January 2000
1

Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, USA

Abstract The amphipathic polyacid, poly(2-ethylacrylic acid) (PEAA) has recently been shown to form uctuating channels in patch-clamp measurements of phospholipid bilayers [J.C. Chung, D.J. Gross, J.L. Thomas, D.A. Tirrell, L.R. Opsahl-Ong, Macromolecules 29 (1996) 46364641.]. To explore this phenomenon further, we have quantied the PEAA-mediated pH-dependent release of sodium and calcium ions from phospholipid vesicles. Permeability to calcium increases linearly with polymer concentration and exponentially with decreasing pH. Permeabilization of negatively charged phosphatidylglycerol (PG) liposomes occurs to a similar extent and with a similar pH dependence to that of zwitterionic phosphatidylcholine (PC) liposomes, implying that a charge neutral species is responsible for the leakage. The pH dependence of leakage shows that the cooperative protonation of from three to ve carboxylate anions is required for permeabilization. Such neutralization could result in a neutral segment of polymer chain of sufcient length to span the bilayer. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Liposome; Permeabilization; Polyelectrolyte; Micellization

1. Introduction Interactions of synthetic polymers with lipid membranes are currently of substantial interest. Lipid vesicles are increasingly being used for DNA transfection and drug delivery, and the ability to modulate the properties of vesicles by means of covalently attached, adherent, or entrapped polymers is im-

*Corresponding author. Present address: Department of Chemical Engineering and Applied Chemistry, Columbia University, 500 W 120th St, New York, NY 10027, USA. 1 Present address: Departments of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.

portant to the development of novel responsive delivery systems. Synthetic polypeptides and polymers have many different effects on lipid bilayers, including induction of membrane fusion [15], selective membrane permeabilization [610], catalysis of lipid ip-op [11], formation of laterally phase-separated membrane domains [12,13], membrane strengthening and stabilization [14], transformations in vesicle shape [15], and phase transformations to micelles [16] or lamellar gels [17]. In many cases, these effects may be dependent on variables such as pH or temperature [18,19]. We have studied the pH-dependent behavior of poly(2-ethylacrylic acid) (PEAA (1), see Scheme 1), which binds to lipid bilayer membranes, solubilizing

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N 2 , hydration with buffer by vortexing, followed by extrusion through polycarbonate lters with 100-nm pores (Nuclepore, Pleasanton, CA) using compressed N 2 at 400 p.s.i., at least 10 times. This procedure produces predominantly single-bilayer vesicles [23].
Scheme 1.

2.1. Calcium leakage measurements

DOPC vesicles were hydrated at 10 g / l in 140 mM CaCl 2 , extruded, and then run over a size exclusion column (50 mm39 mm; Sepharose CL4B, Pharmacia, Uppsala, Sweden) into a buffer (Buffer A) of 20 mM N -[2-hydroxyethyl]piperazineN 9-[2-ethanesulfonic acid]) (Hepes), 20 mM NaCl, and 360 mM glucose (for osmotic support). In experiments designed to test the linearity of leakage rates as a function of PEAA concentration, a small percentage (5%) of the anionic lipid dioleoylphosphatidylglycerol was included in the liposomes, to mitigate the effects of changing liposome surface charge at changing polymer concentrations [24]. Cuvettes were prepared using 60 ml of this liposome stock, 60 ml of a 10 g / l PEAA stock, 1100 ml buffer A, and HCl to adjust pH. A calcium-sensitive uorescent indicator, Mag-fura-2 (Molecular Probes, Eugene, OR) was added to a nal concentration of 0.4 mM. Mag-fura-2 responds to the binding of calcium by a shift in excitation spectrum; by measuring the uorescence emission (500 nm) under 332 and 378 nm excitation, the calcium concentration can be determined. A calibration was performed to yield R 2 R min 21 [Ca ] 5 300 mM 3 ]]] R max 2 R where R is the ratio of the emission intensity under 332 nm illumination to the intensity under 378 nm illumination, and R min and R max are determined from zero and saturating calcium concentrations [25]. Cuvettes were covered with paralm to prevent evaporation between measurements. Calcium, as a divalent cation, can interact strongly with negatively charged liposomes and induce fusion and membrane disruption [26]. For this reason, we chose to examine the leakage of sodium ions from both phosphatidylglycerol and phosphatidylcholine membranes. Sodium concentrations were measured with an ion-selective electrode (Accumet sodium combination electrode, Fisher Scientic, Pittsburgh,

dipalmitoyl and dimyristoyl phosphatidylcholine liposomes to form polymerphospholipid mixed micelles at pH 6.5 [16,20]. This pH-switchable membrane activity may be useful in pharmaceutical formulations requiring targeted or controlled release of liposomally encapsulated agents. Recently, ion channel activity has been observed in intact membranes treated with low concentrations of this polymer [21]. To further explore the mechanism of ion conduction, we have used a uorescent calcium indicator and a sodium-sensitive electrode to characterize the rates of leakage of ions from large, unilamellar vesicles treated with PEAA. Calcium and sodium are biologically important ions: calcium is a ubiquitous intracellular second messenger, while sodium ux through axonal membranes is crucial in the propagation of nerve action potentials. Consequently, methods of controlling the uxes of these ions in biological membranes may be useful, and inferences drawn from studies of ion transport may serve to guide the design and development of new liposomal controlled delivery systems.

2. Materials and methods Poly(2-ethylacrylic acid) was prepared by freeradical polymerization as described elsewhere [22]. The polymer had a weight-average molecular weight of 30 kDa (300 monomer units), determined by gel permeation chromatography (TSK6500 and TSK6300 columns) with poly(ethylene oxide) standards, 50 mM aqueous Tris(hydroxymethyl)aminomethaneHCl solvent. Dioleoyl phosphatidylcholine (DOPC) and dioleoyl phosphatidylglycerol (DOPG) were purchased from Avanti Polar Lipids (Birmingham, AL), and used as received. Liposomes were prepared by drying a chloroform solution of phospholipid under

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PA.) At the sodium concentrations and pH values used, the electrode response was Nernstian to within 5% (i.e., interference from the sub-micromolar proton concentrations was negligible.) Liposomes were loaded with 200 mM NaCl, and run over the Sepharose column into a buffer of 20 mM Hepes and 400 mM glucose, pH adjusted with ammonium hydroxide. Samples were prepared with 1 g / l vesicles and 1 g / l PEAA. DOPG liposomes were found to contain approximately three times as much sodium as did DOPC liposomes. The difference was approximately equal to the concentration of lipids on the inner leaets of the vesicles, and likely reects the binding of sodium to the negatively charged lipid membrane. To determine the initial rates of leakage in a model independent way, leakage was calculated from linear ts to the rst data points of the leakage curves, specically, points at less than one-third of the total observed leakage. In all cases, the rate of leakage from untreated vesicles was subtracted from the raw rates.

3. Results Poly(2-ethylacrylic acid) binds to dioleoyl phosphatidylcholine liposomes in a pH-dependent manner and causes conversion to mixed polymerlipid micelles. Evidence for this interaction can be seen in a plot of the optical density of extruded liposomes treated with polymer as a function of pH (Fig. 1). Liposomes (2 g / l) were treated with PEAA (2 g / l) at various pH values and left overnight. An abrupt decrease in turbidity is observed at pH 6.4. Negative stain electron microscopy shows that, at pH 6.1, DOPC membranes have been converted by PEAA into small, disc-shaped micelles (Fig. 2). The increase in turbidity on further reduction of the pH below 6.4 is due, we believe, to aggregation of the resulting micellar products. If the pH of the DOPC / PEAA samples at pH 6.0 and 6.2 is raised by the addition of a small amount of sodium hydroxide, these samples clarify rapidly (within 5 min, open circles in Fig. 1). Similar micellization has been observed for both dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine [16]. PEAA-treated dioleoyl phosphatidylcholine liposomes exhibit signicant leakage of calcium at a pH

Fig. 1. Optical density (500 nm) of 2-g / l suspensions of extruded, unilamellar DOPC vesicles treated for 16 h with PEAA (2 g / l) in a buffer of 20 mM Hepes and 400 mM glucose. (Glucose was included since it is used for osmotic support in all ion leakage measurements.) Vesicles that are untreated show no variation in optical density with pH ( d ). In the presence of PEAA, optical density is unaffected above pH 6.4, but at pH 6.4 there is an abrupt decrease in turbidity, and at lower pH the optical density increases ( h ). The optical density increase at low pH is likely due to aggregation of polymerphospholipid micelles. By adding NaOH to these samples to raise the pH to 6.9, they are readily claried to the same extent as the pH 6.4 sample, as indicated by the dashed arrows (------ s ).

as high as 7.3 in 20 mM NaCl / 20 mM Hepes / 360 mM glucose buffer (Fig. 3). Note that signicant leakage occurs at a pH almost one unit higher than that which causes micellization, which suggests that calcium leakage occurs via a pore or channel, rather than as a by-product of the vesicle-to-micelle transition. Such pores could well be simply local uctuations into non-lamellar phases; direct measurement of current through PEAA-induced pores has shown the existence of unitary conductance states, however [21]. The initial rates of calcium leakage are plotted in Fig. 4. The initial rate of calcium leakage varies exponentially with pH; i.e., as a power law in the hydrogen ion concentration. In higher ionic strength medium, there is a shift in the leakage curve to lower pH, also shown in Fig. 4. This shift is qualitatively

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[P] Keq 5 ]]] [P0 ][H 1 ] n log[P] 5 log Keq [P0 ] 2 n 3 pH where P0 is a polymer which cannot cause leakage, P is a polymer which can, and n is the number of hydrogen ions that must bind to the anionic polymer to convert it to a leakage competent form. The observed power-law dependence on hydrogen ion concentration implies that several hydrogen ions must bind to the polymer (or a segment of the polymer) to render it leakage-competent. An estimate of this number, n, can be made from the slope of the line in Fig. 4, which is 2 4.560.3 for the sample in 20 mM NaCl, and 2 3.360.5 for the sample in 200 mM NaCl. To examine the role of lipid surface charge in modulating PEAA behavior, we determined the rates of leakage of sodium from both DOPC and DOPG extruded vesicles. (As discussed in Section 2, the divalent calcium ion is known to perturb the structure of anionic liposomes [26].) Initial rates of leakage were estimated as described above; the results are shown in Fig. 6. The increase in leakage rate with decreasing pH is consistent with the leakage behavior of calcium, indicating that cooperative binding of several protons are needed to allow the polymer to cause leakage from DOPC vesicles. (The slopes of the best t lines are 2 4.660.8 for the DOPC vesicles, and 2 4.761.0 for the DOPG vesicles.) Interestingly, there is only a small reduction in the rate of leakage in the acidic DOPG membranes, compared with the zwitterionic DOPC. In this context, it is notable that the pH values observed for micellization pH of DPPC and DPPG membranes are also very similar (pH 6.55 and pH 6.45, respectively [27]). These results strongly suggest that it is a largely neutralized segment of the PEAA that produces the pore, since charged sites on the polymer would experience strong Coulombic repulsion from the DOPG membrane. The power-law dependence of leakage activity of hydrogen ion concentration is intriguing in light of previous measurements on the polymer ionization in aqueous solution. The fraction of dissociated carboxylic acids on the polymer is about 40% at pH 7.0 at this ionic strength, and varies by only a few

Fig. 2. Negative stain (2% phosphotungstic acid) electron micrograph of DOPC:PEAA (1:1 mixture) at pH 6.1. Bar 5 100 nm. Arrow indicates micellar structures, which sometimes aggregate in stacks.

consistent with increased ionization of the polymer at higher ionic strengths, and a similar shift in pH with ionic strength has been observed in the ability of PEAA to effect disruption of multi -lamellar vesicles: Eum et al. [24] found that PEAA disrupted multilamellar aggregates of DOPC at a pH that decreased from 7.1 to 6.5 as ionic strength was increased. To determine whether cooperativity among several polymer molecules is required to cause leakage, the leakage rate was measured as a function of the concentration of added polymer. The leakage rate was found to be proportional to the polymer concentration (Fig. 5. The variation in leakage rate with changing pH and polymer concentration can be described with a simple model based on chemical equilibrium: P0 1 n H 1 P

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Fig. 3. PEAA-induced leakage of Ca 2 1 from extruded DOPC vesicles, in 20 mM Hepes, 20 mM NaCl, and 360 mM glucose. Vesicles (0.5 g / l) contained 140 mM CaCl 2 and were treated with PEAA (0.5 g / l). Calcium in the surrounding medium was measured using the uorescent dye, mag-fura-2.

Fig. 4. Initial rates of leakage of Ca 2 1 from PEAA-treated DOPC vesicles. In moderate ionic strength buffer (same as that used in Fig. 3), the leakage is detectable above background at ca. pH 7.4, while in high ionic strength buffer (20 mM Hepes, 200 mM NaCl) the onset is at ca. pH 6.9. In both buffers, leakage increases exponentially with decreasing pH, indicating a power-law dependence on [H 1 ]. The slope of the best-t line to the 20 mM [NaCl] data is 2 4.560.3; for the 200 mM [NaCl] data, the best slope is 2 3.260.3.

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Fig. 5. Initial rate of leakage of Ca 2 1 from PEAA-treated DOPC vesicles as a function of added polymer. DOPC:DOPG (20:1) vesicles were extruded in 140 mM Ca 2 1 , and leakage measurements were performed with 0.5 g / l lipid in a buffer of 20 mM Hepes, 20 mM NaCl, and 360 mM glucose at pH 6.88. A linear dependence on polymer concentration indicates that cooperativity among several polymer molecules is not required to cause leakage.

Fig. 6. Initial rates of leakage of Na 1 ions from PEAA-treated DOPC ( s ) and DOPG ( h ) vesicles, in a buffer of 20 mM Hepes and 400 mM glucose. Vesicles were extruded in 200 mM NaCl. The leakage of Na 1 in DOPC is similar to that of Ca 2 1 . The leakage from DOPG vesicles is shifted only slightly in pH.

percent over the range pH 6.97.3 (U.K.O. Schroder, D.A. Tirrell, K.H. Langley, unpublished results). (The presence of membranes shifts the titration curve only slightly toward greater neutralization [28].) The protonation of ve additional carboxylate anions could produce a segment of ca. 14 uncharged monomer units, in the oversimplied case in which the charged carboxylate sites are initially distributed uniformly along the chain. Such a neutralized segment would have an extended length of 14 3 2.5 / monomer 5 35 A, long enough to traverse the A [29]. DOPC bilayer, which has a thickness of 36.1 A In fact, however, little is known about the charge distribution in partially ionized electrolytes, and PEAA undergoes a hydrophobically driven conformational collapse at pH values only slightly lower than those examined in this work (ca. pH 6.3) [24]. Large uctuations in charge density would be expected close to this transition, since the free energy of the energy of a cooperatively collapsed polymer segment will necessarily be close to the free energy

of the expanded coil. As a consequence, it is possible that quite large segments of neutralized polymer could be formed by the addition of only four or ve hydrogen ions at pH 7.0. Finally, it must be acknowledged that the leakage of sodium and calcium ions observed in this work could in principle be due to either a carrier-based mechanism, or the formation of transient membrane channels, or both. In a previously published study using the patch-clamp technique, clear evidence for ion-transport through uctuating channels was found [21]. However, in that work a transmembrane holding potential was applied, and the possible role of the transmembrane potential in establishing channel activity was not examined. Moreover, a very slow transport process, such as a carrier-mediated transport of ions, would not have been detected by the patch-clamp methodology used. The patch-clamp measurements did demonstrate the existence of channels with open states of less than 200 ms in duration. Clearly, slow leakage of ions from vesicles would require that such channels are either very slow to form or that they reseal very rapidly upon

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forming, since complete leakage from a vesicle with a single open channel could occur in less than 1 s [30]. Because the dynamics of adsorbed polymer chains (with many degrees of freedom) can be extremely slow [31,32], conformational reorganization of a membrane-adsorbed molecule to form a channel-competent state is likely to be rate limiting, and may account for the fact that leakage is observed on a timescale of hours.

4. Conclusions We have examined the PEAA-mediated transport of cations through dioleoyl phosphatidylcholine and phosphatidylglycerol bilayer membranes. We nd that, although PEAA micellizes phosphatidylcholine membranes at ca. pH 6.5, at substantially higher pH the polymer can permeabilize intact membranes to sodium and calcium ions. A linear dependence of leakage rate on polymer concentration indicates that aggregation of PEAA is not required to form carriers or pores. The similar behavior of PEAA in zwitterionic and negatively charged membranes implies that a largely neutralized segment of the polymer chain is responsible for the permeabilization. Lastly, the number of carboxylate anions that must be neutralized to enable the chain to form a pore is about three to ve; this may be sufcient to neutralize a stretch of the polymer long enough to traverse the bilayer.

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