Interactions between poly(2-ethylacrylic acid) and lipid bilayer membranes E� ects of cholesterol and grafted poly(ethylene glycol) David Needham,* JeÜ Mills and Gary Eichenbaum Department of Mechanical Engineering and Materials Science Duke University Durham NC 27708-0300 USA Received 9th November 1998 The exchange of the protonatable polymer poly(2-ethylacrylic acid) (PEAA) has been studied with vesicle membranes containing cholesterol from 0 to 60 mol% or PEG2000-lipid (5 mol%). The release of an entrapped dye from 100 nm extruded liposomes was used as an assay for membrane perturbation by the polymer as a function of pH. The inclusion of cholesterol was found to reduce the pH at which the polymer caused release of the dye from the lipid vesicles and the degree of polymer protonation (i.e.degree of hydrophobicity) correlated well with the increase in elastic expansion modulus of the vesicle bilayer. The results are discussed in terms of a balance between polymer solubility and membrane expansion. With respect to the PEG barrier the presence of 5 mol% PEG2000 which represents full surface coverage did not prevent PEAA from inducing contents release demonstrating that highly hydrated polymeric layers are not eÜective barriers for other water soluble polymers and may point to some association between the two polymers. In pure lipid bilayer systems the aqueous solubility for membrane components is so low (of the order of 10~10 M) that bilayer mass can essentially be considered constant at least during the time of an experiment.In contrast in the presence of other membrane compatible materials like lysolecithin and bile salts which can have a signi–cant solubility in the aqueous phase as monomers and micelles the composition and properties of the bilayer can be changed in seconds and at concentrations in excess of the surfactant critical micelle concentration (c.m.c.) can lyse the membranes causing them to fail.1h4 Another important class of molecules that shows partitioning into bilayers are pH-sensitive polymers such as poly(ethylacrylic acid) (PEAA).5 Here protonation of the carboxyl groups of the polymer reduces the solubility of the molecule in aqueous media making it more hydrophobic. Consequently this promotes solution of the polymer in the hydrophobic interior of lipid bilayers.5 This class of molecule shows a well de–ned cooperative conformational transition from an expanded coil to a collapsed coil that is dependent on pH.The existence of this transition (when compared to the continuous condensation of simple polyelectrolytes such as polyacrylic acid) is a direct result of additional attractive interactions between hydrophobic moieties such as the ethyl group in PEAA. For polymers dissolved in aqueous solution the midpoint of this transition is at pH 6.2.6 Interestingly however in the presence of Ladipalmitoylphosphatidylcholine (DPPC) vesicles the midpoint of the transition is shifted up to pH 6.5.6 This shift indicates some adhesive interaction between the polymer and the membrane at pHs above its natural critical pH for the transition and a role of the bilayer in inducing this transition.103 Faraday Discuss. 1998 111 103»110 Up to now most of the work on this polymer has been carried out with DPPC bilayers. The work presented here was motivated by a desire to examine the way in which the properties of the bilayer and its interface may in—uence the polymer»bilayer interaction. We therefore chose to study bilayers containing cholesterol and bilayers containing a grafted layer of a diÜerent water soluble polymer poly(ethylene glycol) (PEG). The inclusion of cholesterol in lipid bilayer membranes causes a condensation of the interface and a dramatic decrease in elastic area compressibility of the bilayer as well as its permeability to water.7h9 The presence of PEG grafted to the bilayer interface via the incorporation of PEG»lipids into the bilayer can inhibit the close approach of globular macromolecules and micelles.10h12 It is of interest then to determine if these two diÜerent kinds of barriers can hinder the approach and interaction of a water-soluble polymer that can be triggered to undergo a conformational transition due to a lowering of solution pH making it less soluble in aqueous media and more soluble in lipid bilayers which it can then permeabilize.In the present paper then we report the measurement of the release of a —uorescent dye entrapped within lipid vesicles that is induced by the exposure of the vesicles to PEAA solutions as a function of solution pH. These measurements are carried out for lipid vesicle bilayers containing either cholesterol (from 0 to 60 mol%) or PEG-lipid (5 mol%).Based on previous mechanical property data,8 the expectation was that cholesterol-rich membranes would possibly inhibit the polymer»bilayer interaction. For the PEG-lipid membranes interest centered on whether the presence of a PEG covered surface (at the mushroom-brush transition) would inhibit approach and intercalation of the PEAA into the membrane and therefore prevent release of internal contents. Experimental Materials Stearoyloleoylphosphatidylcholine (SOPC) and cholesterol (from Avanti polar lipids) in chloroform (10 mg mL~1) were mixed to give cholesterol at 0 20 30 40 50 and 60 mol%. SOPC and distearoylphosphatidylcholine (DSPE)-PEG2000 (10 mg mL~1) were mixed to give DSPEPEG2000 at 5 mol%.20 mM Phosphate buÜered saline (PBS) [0.8% w/w saline (0.137 M)] was prepared using sodium phosphate monobasic sodium phosphate dibasic and sodium chloride from Mallinckrodt. Carboxy—uoroscence (6-CF) was obtained from Aldrich Inc. Liposome preparation The prepared SOPC and cholesterol solutions in chloroform were dried using a Rotavapor onto a 100 mL round-bottomed —ask.13 Lipid vesicles were prepared by hydrating the dried lipid –lm with a solution of 50 mM 6-carboxy—uoroscene (6-CF) (high enough concentration for —uorescence to be self-quenched) in 20 mM PBS.13 The hydrated lipid mixture was extruded through two 100 nm Poretics polycarbonate membrane –lters using a Lipex extruder to give a homogeneous sample of unilamellar lipid vesicles of 100 nm diameter,14 containing the entrapped carboxy —uoroscene dye in a quenched form.Lipid vesicles were then –ltered using a Sephadex G-50 column (Sigma) to remove unencapsulated 6-CF.13 Contents release measurements 20 mM PBS was prepared at pHs ranging from 5.5 to 7.5. PEAA was dissolved in 20 mM PBS at 33.3 mM (1 mg mL~1) over the same pH range. Both sets of solutions were osmotically matched using glucose to the 6-CF solution used to hydrate and form the liposomes to ensure there were no osmotic pressure gradients across the lipid membranes. 10 lL of liposome solutions were suspended in 1 mL of buÜer containing PEAA (time zero). Fluorescence (jex\436 nm jem\520 nm) intensity was measured every 5 s for 15 min at 30 min and 1 h.(Aminco-Bowman Series 2 Luminescence Spectrometer or a Shimadzu RF-1501 Spectro—uorophotometer were used to measure —uorescence.) As 6-CF leaked out of the liposomes and was diluted by the suspending buÜer self-quenching was eliminated resulting in an increased —uorescence signal. After 1 h 20 lL of Triton X-100 was added to completely solubilize the membranes and a –nal —uorescence reading was made. This –nal reading was used as a maximum or 100% release reference and all Faraday Discuss. 1998 111 103»110 104 other readings were divided by this value to obtain a measure of the ììpercent releasedœœ. This procedure was similar to that used by Gon8 i et al. to study the eÜects of surfactants on release from liposomes.15 In separate experiments the —uorescence of 6-CF in Triton X-100 was found to be unaÜected by the presence of surfactant but was dependent on pH.The pH-dependent intensity was controlled by making control measurements at the same pH. Results andiscussion Cholesterol-containing membranes The eÜect of cholesterol on the pH dependence of PEAA-induced release of contents from liposomes was investigated by measuring release of 6-CF (quenched inside the liposome) as indicated above for liposomes of various cholesterol compositions. First consider the release from liposomes composed of pure SOPC. Fig. 1 shows a plot of percent release of 6-CF vs. time in min for 100 nm liposomes composed of pure SOPC in a solution of 1 mg mL~1 PEAA. Each trace is the cumulated release of carboxy- —uoroscene —uorescence as it leaked out of the liposome sample over a period of 1 h.Each curve represents the pH of the bulk media in the range of 5.5»7.5. As shown on the plot the curve at pH 6.7 was the lowest pH at which the liposomes were stable in the polymer suspension. In polymer solutions more basic than or equal to pH 6.7 baseline release was observed rising from 10% to only 30% release over the time course measured. Similar values are noted for control solutions which did not contain PEAA. In contrast polymer solutions more acidic than pH 6.7 exhibited a burst release reaching 100% of entrapped 6-CF within a few minutes. By comparison Fig. 2 shows a plot of percent release of 6-CF as a function of time for 100 nm SOPC/cholesterol liposomes containing 40 mol% cholesterol.Here release was not observed at pH 6.7 but in solutions more acidic than pH 6.4. Thus the pH at which onset of release occurred was shifted from near pH 6.7 (for 0 mol% cholesterol) to near pH 6.4 (for 40 mol% cholesterol). Then by taking the steady state release values (60 min) at each pH for SOPC systems with cholesterol composition 0 20 30 40 50 and 60 mol% as shown in Fig. 3 a plot of steady state release as a function of pH for each composition was constructed. This –gure shows that all compositions achieved 100% release if the pH was acidic enough. Importantly it also shows that the pH at onset of release was monotonically shifted to more acidic pH values with increasing mol% cholesterol from pH 6.7 for a pure lipid system to pH 6.0 for a cholesterol-saturated (i.e.D60 mol%) system. Thus the uptake of protonated polymer by the bilayer membrane is increasingly inhibited as the bilayer is condensed with cholesterol. What these results then suggest is that there is indeed a Fig. 1 Plot of percent release of 6-CF vs. time in min for a system of pure SOPC 100 nm liposomes suspended in solutions of 1 mg ml~1 PEAA at pHs varying from 5.5 to 7.5. Error bars on each data point represent the standard deviation from the mean for 2 measurements. 105 Faraday Discuss. 1998 111 103»110 Fig. 2 Plot of percent release of 6-CF vs. time in min for a system of 100 nm SOPC liposomes with 40 mol% cholesterol incorporated in the membrane suspended in solutions of 1 mg ml~1 PEAA at pHs varying from 5.5 to 7.5.Error bars on each data point represent the standard deviation from the mean for 2 measurements. The intensity drop noted in some of the curves during the –rst 15 min can be attributed to photobleaching of the 6-CF but this artifact did not aÜect the steady state measurements. correlation between the compressibility of the lipid bilayer (determined by the presence of cholesterol) and the interaction of the polymer with the bilayer which is controlled by pH i.e. the degree of protonation of the polymer which in turn determines its degree of hydrophobicity. In order to develop this correlation further what is needed is a measure of the percent protonation of the polymer as a function of pH. Tirrell and colleagues have characterized the pHdependent solution behavior of the pure polymer.5,6 Titration of the polymer indicates that it has a pK of approximately 5.4.16 Graphically then as shown in Fig.4 as the pH is lowered in the a range 7.4»3.4 the percent of polymer that becomes protonated (and therefore hydrophobic) increases from close to zero to 100%. Critical pHs for some important features of polymer behavior and polymer»bilayer interaction from the previous literature and from the experiments reported here are summarized by this plot. At pH 6.2 as observed by Borden et al. using pyrene —uorescence the free polymer in aqueous solution undergoes a sharp conformational transition.6 Thus D15% of the polymer needs to be Fig. 3 Plot of steady state release of 6-CF (60 min after liposome addition to the polymer solution) vs.pH for 100 nm liposomes composed of SOPC and containing 0 20 30 40 50 and 60 mol% cholesterol. Error bars on each data point represent the standard deviation from the mean for 2 measurements. Faraday Discuss. 1998 111 103»110 106 a Fig. 4 Plot of percent protonation of the polymer vs. pH calculated from the Henderson»Hasselbach equation using the data from Tirrell et al.,16 which measured the pK as 5.4. Shown are critical pHs for several features of polymer and polymer»lipid behavior. protonated in order for the polymer to undergo the collapse transition. At pH 7.4 the polymer was found by Seki and Tirrell,17 to cause a broadening of the main acyl chain gel to liquid crystalline transition temperature for DPPC vesicles. Thus at relatively high pH where the polymer is only marginally (1%) protonated it is intimately associated with the bilayer.This association is sufficient to cause perturbation of the gel/liquid crystalline transition but not enough to cause the polymer collapse transition indicating that at this degree of protonation the interaction with the bilayer is insufficient to ììcatalyzeœœ the collapse. Also it seems that this expanded coil polymer cannot traverse to the inner layer of the bilayer in order to cause bilayer disruption. For DPPC vesicles Tirrell and colleagues showed that at pH 6.5 three events were correlated PEAA induced a sharp vesicle to micelle transition signifying massive reorganization of the lipid ;18 pyrene —uorescence increased sharply signifying the premature induction of the expanded coil to collapsed coil transition due to binding of polymer to the bilayer ;6 and the DPPC lipid acyl chain transition showed maximum broadening.6 Taken together these results implicate the collapse transition as a key event that leads to membrane disruption.In our experiments at pH 6.7 the polymer is D5% protonated when it causes release of dye from the SOPC vesicles (a slightly higher pH than that measured for reorganization of DPPC vesicles,5 and by inference and comparison with the DPPC data of Thomas and Tirrell,5 this signi–es the premature induction of the collapse transition by SOPC bilayers. At pH 6.1 where the polymer is 20% protonated it causes release of dye from cholesterol-saturated vesicles. At this pH the polymer is actually collapsed in solution which represents the maximum potential for the polymer to disrupt the bilayer.A Using KA the data from Fig. 4 and previous measurements of the elastic area expansion modulus for SOPC/cholesterol bilayers,8 Fig. 5 shows that the fractional increase in K is in fact found to be directly proportional to the increase in percent protonation required to cause release of dye from the vesicles. In evaluating this data in terms of energetic considerations there is an energy state for the polymer in aqueous solution that is pH dependent and there is an energy state for the polymer in the bilayer which may be changed by the presence of cholesterol and which determines the release 107 Faraday Discuss. 1998 111 103»110 Fig.5 ììFractional change plotœœ showing the fractional increase in percent protonation of the polymer (over and above that which occurs at pH 6.7) at the pH where dye is released vs. the fractional change in the elastic area expansion modulus (over and above that for the pure SOPC bilayer) for bilayers containing increasing amounts of cholesterol (in parentheses next to each data point). of CF. This latter energy determines the ability of the polymer to create defects and perturb the structure of the bilayer. Assuming that cholesterol does not decrease the pH at the bilayer interface relative to the bulk solution its role is therefore limited to its ability to increase the cohesiveness of the membrane. Then a greater amount of work is required to perturb the structure of the membrane and induce leakage of contents which has to be compensated by increasing the protonation of the polymer.The assay used here was simply the release of the marker dye (i.e. the end point of the interaction) and so does not provide any information on the amount of polymer Fig. 6 Plot of steady state release of 6-CF vs. pH for 100 nm liposomes composed of pure SOPC with 5 mol% PEG2000 as well as 100 nm liposomes composed of only SOPC. Faraday Discuss. 1998 111 103»110 108 bound. Thus at this stage in the work it is not possible to distinguish between the eÜect of cholesterol on polymer partitioning and the formation of defects that result in CF release. Critical information that needs to be obtained includes the concentration dependence of the contents release and a lipid to polymer ratio for the failure event.Grafted PEG Data for CF release from the PEG-grafted liposomes was also obtained as described above. A compilation of the results is shown in Fig. 6 as steady state release vs. pH. The curve for the PEG-grafted liposomes was nearly identical to the plot for the pure SOPC non-PEGylated vesicle case with release occurring near pH 6.8 and reaching 100% by pH 6.6. Thus despite the presence of a complete layer of PEG on the bilayer surface [enough to signi–- cantly retard the approach of globular molecules (avidin) and micelles (MOPC)] PEAA can still approach close enough to the bilayer interface to bind and cause membrane disruption. This indicates an ability of the PEAA polymer to reptate through the PEG polymer layer.As mentioned in the Introduction this same PEG layer cannot prevent MOPC monomer from adsorbing into or desorbing from such PEG-grafted bilayers suggesting that monomer can in fact diÜuse through the PEG layer.10 With a cross-sectional area of 40 ”2 the polymer has a similar size to MOPC of 34 ”2. A binding interaction between PEG and PEAA is also not ruled out. Concluding remarks The results of this study then suggest that membrane compressibility dominates the interaction between lipid bilayers and PEAA such that highly expandable membranes are more likely to take up hydrophobic polymer molecules than less expandable membranes with more condensed interfaces. We plan to obtain additional data on the binding and uptake of polymer by lipid bilayers including the use of the micropipet manipulation technique to measure any area change that may accompany polymer insertion into the membrane at pHs slightly higher than those required to initiate membrane disruption to observe CF release directly for a single vesicle and to measure any changes in the membrane compressibility and tensile strength for polymer-adsorbed but stable bilayers.For the PEG-lipid containing membranes no additional barrier eÜect was observed leading to the conclusion that either these aqueous-soluble polymers can in fact entangle such that the PEAA can come into intimate contact with the lipid interface or an attractive interaction occurs between PEG and PEAA especially for the protonated form of PEAA. This work was supported by grant GM 40162 from the NIH and by the National Science Foundation Engineering Research Center Grant CDR-8622201.The authors would also like to acknowledge useful discussions with Drs. Evan Evans Tom McIntosh Sid Simon and Doncho Zhelev and Mr. Patrick Kiser. References 1 E. Evans W. Rawicz and A. F. HoÜman Bile Acids in Gasteroenterology Basic and Clinical Advances ed. A. F. HoÜman G. Paumgartner and A. Stiehl Kluwer Academic Publishers Dordrecht Boston London 1994 pp. 59»68. 2 D. Needham and D. V. Zhelev Ann. Biomed. Egr. 1995 23 287. 3 D. V. Zhelev Biophys. J. 1996 71 257. 4 D. V. Zhelev Biophys. J. 1998 75 321. 5 J. L. Thomas and D. A. Tirrell Acc. Chem. Res. 1992 25 336. 6 K. A. Borden K. M. Eum K. H. Langley and D.A. Tirrell Macromolecules 1987 20 454. 7 M. Bloom E. Evans and O. G. Mouritsen Q. Rev. Biophys. 1991 24 293. 8 D. Needham and R. S. Nunn Biophys. J. 1990 58 997. 9 D. Needham and D. V. Zhelev V esicles ed. 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L. Voycheck Macromolecules 1988 21 2649. Paper 8/08717B Faraday Discuss. 1998 111 103»110 110