Micellar Aggregates Formed Following the Addition of

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Micellar Aggregates Formed Following the Addition of Hexafluoroisopropanol to Phospholipid Membranes Sue M. Ennaceur and John M. Sanderson* University Science Laboratories, Department of Chemistry, South Road, Durham DH1 3LE, United Kingdom Received July 27, 2004. In Final Form: October 22, 2004 The addition of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) to aqueous phospholipid membranes leads to perturbation of the bilayer. In the case of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), calorimetric and small-angle X-ray scattering analyses indicate that effects are already apparent at bound molar HFIP/lipid ratios of less than 1:150, with a pronounced decrease in the temperature of the main (gel to liquid crystalline) phase transition and a decrease in the intensity of the first- and second-order scattering reflections. As the HFIP concentration is raised further, at bound molar HFIP/lipid ratios >2:1, uniform isotropic particulate structures are formed with higher intrinsic curvature than the parent liposomes. These observations are supported by the results of thin-film experiments and are consistent with the formation of DMPC/HFIP adducts that are detergent-like in nature. In the case of 1,2-dioleoyl-sn-glycero3-phosphocholine (DOPC) the effects are much less marked, with no blebbing observed over a comparable range of HFIP concentrations. Although HFIP interacts strongly with DOPC membranes, it appears that membrane rupture is not promoted as readily with this lipid. Data from electron microscopy, laser correlation spectroscopy, and marker release experiments suggest that some of the immediate (nonequilibrium) effects of HFIP on membranes are the consequence of microinhomogeneity in water/HFIP mixtures. On the basis of our observations, we propose a model for the interaction of HFIP with phospholipid membranes.

Introduction The modifications to membrane properties produced by a range of nonpeptidic organic compounds, including steroids, detergents, drugs, and small organic solutes, have been well described over many years. Interaction of these species with membranes may lead to a number of macroscopic outcomes (such as blebbing, leakage, fusion) that are usually accompanied by changes in the thermotropic and fluidic properties of the bilayer and perturbation of the ordering of the phospholipid molecules. It is convenient to consider these molecules according to their relative hydrophobicity, frequently assessed by means of their octanol/water partition coefficient (KOW). Hydrophobic compounds, of high KOW, include steroids such as cholesterol1 and volatile anesthetics such as halothane. With respect to the latter, there is an ongoing debate concerning their mechanism of action, with opinions shifting between a mode of action that involves binding to receptors and one that involves modification of membrane properties such as fluidity.2,3 Amphiphilic compounds such as detergents, which possess both hydrophobic and hydrophilic groups, form micellar aggregates in aqueous solution and frequently exhibit a high affinity for phospholipid membranes. Nonionic detergents, such as alkylglycosides and Triton X-100,4 partition into membranes to form mixed lipidic (lamellar) phases at low concentrations and clear micellar or bicellar solutions at high concentrations following * Author to whom correspondence should be addressed: tel. +44 (0)191 3342107, fax +44 (0)191 3844737, e-mail j.m.sanderson@ dur.ac.uk. (1) New, R. R. C. In Liposomes: a Practical Approach; New, R. R. C., Ed.; IRL Press: Oxford, 1990; p 19. (2) Schreier, S.; Malheiros, S. V. P.; de Paula, E. Biochim. Biophys. Acta 2000, 1508, 210-234. (3) Rubin, E., Miller, K. W., Roth, S. H., Eds. Molecular and Cellular Mechanisms of Alcohol and Anesthetics. Ann. N.Y. Acad. Sci. 1991, 625. (4) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146-163.

saturation of the bilayer with detergent. This has been put to good effect both in the preparation of liposomes by detergent dialysis and in the solubilization of membrane components, most notably proteins. Short-chain ionic surfactants, such as dihexylphosphatidylcholine, are particularly effective at promoting bicelle formation5 and are beginning to find uses in membrane protein crystallography.6 The nature of the van der Waals interactions between amphiphiles and phospholipids is a crucial factor in determining their membrane activity. Perfluorinated amphiphiles, for example, have unique properties that arise largely from the particular nature of fluorocarbons, namely, weak intermolecular interactions and lower conformational flexibility. This is reflected in their nonideal mixing behavior with hydrocarbons.7 In mixed surfactant preparations, such as liposomes composed of normal and perfluorinated lipids, the two will often segregate.8 The interactions of small hydrophilic species of low KOW with membranes tend to produce less dramatic effects than their larger hydrophobic counterparts. The effects of alcohols upon phospholipid membranes have been studied in some detail. At relatively high concentrations (30% v/v), ethanol is able to induce temporary lesions in phospholipid membranes that can be used to load watersoluble solutes into the liposomal interior.9 NMR10,11 and fluorescence12 studies have demonstrated that ethanol molecules locate preferentially in a narrow region around (5) Sanders, C. R.; Prosser, R. S. Structure 1998, 6, 1227-1234. (6) Faham, S.; Bowie, J. U. J. Mol. Biol. 2002, 316, 1-6. (7) Kraft, M. P.; Riess, J. G. Biochimie 1998, 80, 489-514. (8) Elbert, R.; Folda, T.; Rinsdorf, H. J. Am. Chem. Soc. 1984, 106, 7687-7692. (9) Dos Santos, N.; Cox, K. A.; McKenzie, C. A.; van Baarda, F.; Gallagher, R. C.; Karlsson, G.; Edwards, K.; Mayer, L. D.; Allen, C.; Bally, M. B. Biochim. Biophys. Acta 2004, 1661, 47-60. (10) Feller, S. E.; Brown, C. A.; Nizza, D. T.; Gawrisch, K. Biophys. J. 2002, 82, 1396-1404. (11) Holte, L. L.; Gawrisch, K. Biochemistry 1997, 36, 4669-4674. (12) Rottenberg, H. Biochemistry 1992, 31, 9473-9481.

10.1021/la048109y CCC: $30.25 © 2005 American Chemical Society Published on Web 12/14/2004

Hexafluoroisopropanol/Phospholipid Aggregates

the glycerol part of the phospholipid headgroup, interacting via hydrogen bonding to the phosphate group, with only weak hydrophobic interactions with the lipid chains. It has been proposed that this is also the preferential binding site for other alcohols, including trifluoroethanol.13 Consistent with this, calorimetric analysis14 demonstrates that the interaction of alcohols with membranes is dependent on the local structure around the phospholipid headgroups, which in turn is influenced by the composition of the membrane and its thermotropic properties. Interestingly, it appears that, at least for straight-chain aliphatic alcohols, longer chain lengths decrease the binding constants with lipids while at the same time increasing the partition coefficient into the membrane,15 suggesting a distinction between simple binding to the membrane and partitioning into it. Systematic studies of the effects of a series of alcohols on the stability of the membrane protein KcsA have demonstrated that some, especially trifluoroethanol, are able to influence the lateral pressure of the membrane through interactions in the headgroup region of the bilayer, leading to dissociation of the tetrameric protein. In related work,16 it was shown that the same alcohols increased the level of disorder in model membranes and were able to induce leakage of liposomal contents. Of the alcohols studied, 1,1,1,3,3,3hexafluoroisopropanol (HFIP) was the most effective at promoting marker release and had particularly pronounced effects on the stability of the bilayer, producing complete disruption at a concentration of 0.2 M. HFIP is commonly used for the solvation of peptides, particularly those that are hydrophobic or amyloidogenic.17 In common with other fluorinated alcohols, such as trifluoroethanol, is the solubilizing ability of HFIP, which is attributed to the formation of a hydrophobic microenvironment in polar solvents such as water. The combination of a hydrophilic alcoholic functionality alongside bulky hydrophobic trifluoromethyl groups endows the molecule with particularly unique properties, including a high KOW (40,18 compared with 200 for halothane and 1.8 for isopropanol, for example)19 combined with a high aqueous solubility. During the course of work directed toward the insertion of hydrophobic peptides into phospholipid membranes,20 we observed that the addition of even small amounts of HFIP to liposomal preparations containing an internal marker led to a partial release of the marker, consistent with the reports described above. In separate work, we observed that HFIP has a high affinity for liposomal membranes.21 We decided to investigate the interactions of HFIP with phospholipid membranes in more detail, to characterize the morphological and physical changes produced in response to addition of this solute, and in this paper we report our findings. Experimental Section Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Bachem (U.K.), Ltd., and 1,2(13) van den Brink-van der Laan, E.; Chupin, V.; Killian, J. A.; de Kruijff, B. Biochemistry 2004, 43, 4240-4250. (14) Trandum, C.; Westh, P.; Jørgensen, K.; Mouritsen, O. G. Biochim. Biophys. Acta 1999, 1420, 179-188. (15) Westh, P.; Trandum, C. Biochim. Biophys. Acta 1999, 1421, 261272. (16) van den Brink-van der Laan, E.; Chupin, V.; Killian, J. A.; de Kruijff, B. Biochemistry 2004, 43, 5937-5942. (17) Tennant, G. A. Methods Enzymol. 1999, 309, 26-47. (18) Verschueren, K. Handbook of Environmental Data on Organic Chemicals, 3rd ed.; John Wiley & Sons: New York, 1996. (19) Kalisan, R.; Markuszewski, M. Int. J. Pharm. 1996, 145, 9-16. (20) Sanderson, J. M.; Yazdani, S. Chem. Commun. 2002, 11541155. (21) Sanderson, J. M.; Ward, A. D. Chem. Commun. 2004, 11201121.

Langmuir, Vol. 21, No. 2, 2005 553 dioleoyl-sn-glycero-3-phosphocholine (DOPC) was from Sigma; both were used without further purification. Egg phosphatidylcholine (EPC, Sigma) was obtained as a solution (10 mg/mL) in CHCl3. HPLC-grade water was obtained from Fisher, and ultrapure water was purified using a Milli-Q purification system (Waters). 5(6)-Carboxyfluorescein (CF), HFIP, and tris(hydroxymethyl)aminomethane (Tris) were obtained from Lancaster Synthesis (Morecombe, U.K.). Determination of Lipid/Water Partition Coefficients. Multilamellar vesicles (MLVs) were prepared by evaporating a solution of lipid (30 mg) in CHCl3 (1 mL) to dryness in vacuo to form a thin film. The film was then hydrated with D2O (300 µL) to form a lipid suspension (containing ∼9% lipid by weight). Lipid/ water partition coefficients were obtained by addition of varying concentrations of liposomes to a solution of HFIP in D2O (0.09 M) containing NaF (0.3 M) as the reference and monitoring the intensity of the 19F signals by NMR at 376 MHz using a Varian Mercury-400 spectrometer. Experiments were performed at 20 °C. The intensity of the 19F signal arising from free HFIP was measured as a function of lipid concentration, with the intensity of the F- signal used as a reference, and the partition coefficient (KLW) calculated according to eq 1:

KLW ) [W]IL/[L]TIB

(1)

where [W] is the concentration of D2O (55.3 M), [L]T is the lipid concentration, IL is the signal intensity of the HFIP in the lipid phase, and IB is the signal intensity from HFIP in the bulk solution. Values were measured for HFIP/lipid ratios of 1:2 and 1:1. Small-Angle X-ray Scattering (SAXS). MLVs were prepared by evaporating a solution of lipid (30 mg) in CHCl3 (1 mL) to dryness in vacuo to form a thin film. The film was then hydrated with HPLC-grade H2O (300 µL) to form the lipid suspension (containing ∼9% lipid by weight). A series of MLV samples were prepared containing HFIP over a range of concentrations up to 0.05 M by adding an appropriate quantity of HFIP to a sample of the MLV suspension and allowing the mixture to equilibrate for at least 15 min, with periodic agitation to ensure sample homogeneity. The sample was then injected into a quartz capillary and placed in the sample holder of a small-angle diffractometer [a Bruker NanoStar, equipped with a Kristalloflex 760 X-ray source (Cu KR, 0.3 × 0.3 mm2 source point, 6 kW power) and a two-dimensional position-sensitive gas detector], with a path length of 64 cm from the X-ray source and sample to the detector. The beam path was evacuated and the position of the sample optimized by measuring the intensity of the scattered transmission through the sample using glassy carbon. Scattering data for each of the samples were collected for 1 h at 20 °C, and the raw data were integrated to the one-dimensional scattering function I(Q) using the instrument software (Bruker SAXS, System V4.1.09) according to eq 2:

Q ) (4π/λ) sin θ/2

(2)

where λ is the wavelength and θ is the scattering angle. The program Origin (Microcal, version 6) was used to analyze peak areas and prepare the data for publication. Differential Scanning Calorimetry (DSC). Samples of MLVs were prepared by the same method described above for SAXS experiments at a concentration of 250 mg lipid/mL H2O. The lipid solution was divided into 10-µL aliquots and thoroughly mixed with HFIP at concentrations ranging up to 0.05 M. The lipid samples were sealed in aluminum pans and subjected to repeated heating/cooling cycles using a Perkin-Elmer Pyris 1 differential scanning calorimeter. Each sample was scanned a minimum of three times between 4 and 30 °C at a rate of 2 °C/ min. Pressure-Area Isotherms. Experiments were carried out using a Nima Langmuir film balance Type 601 with a total area of 540 cm2. Data were collected and analyzed using Nima 516 software. Prior to monolayer formation, a Π-A isotherm was obtained from each subphase to ensure that no artifacts resulting from impurities were produced. Films were prepared by dropwise addition of a solution of lipid (DOPC or DMPC) in CHCl3 at a concentration of 1 mg/mL to the surface of the aqueous phase in

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the trough. The quantity of material spread on the surface was the same for all experiments, and the temperature of the subphase was maintained at 20 °C throughout. After a period of 15 min following spreading, Π-A isotherms were obtained by compression of the monolayer at a barrier speed of 50 cm2/min. Each lipid monolayer was compressed to a point just below its collapse pressure and expanded several times (three to four cycles) before finally being compressed beyond its collapse area. Isotherms were obtained for subphase compositions ranging from ultrapure H2O to 0.38 M HFIP/H2O. The compressional modulus, κA, was calculated according to eq 3:

κA ) -Am(∆π/∆Am)T

added at the end of each experiment to a final concentration of 1% (v/v), to measure the signal corresponding to 100% CF release and to permit the observed emission intensities to be converted into percentage release according to eq 4:

% release ) 100(Fs - F0)/(Fm - F0)

(4)

where Fs is the measured steady-state emission signal, F0 is the residual emission at the start of the experiment, and Fm is the emission following addition of Triton X-100.

Results (3)

where Am is the molecular area and π is the lateral surface pressure. Electron Microscopy (EM). Unilamellar liposomes were prepared by evaporating a solution of DMPC (0.37 mg) dissolved in CHCl3 (50 µL) to dryness in vacuo to form a thin film. The film was hydrated with HPLC-grade H2O (1 mL) to form a MLV dispersion. The MLV suspension was subjected to five freezethaw cycles between -196 and 30 °C and extruded 10 times through laser-etched polycarbonate membranes (Whatman, 100nm pore size) at 30 °C using a thermobarrel extruder (Lipex Biomembranes). The large unilamellar vesicle (LUV) preparation was mixed with HFIP to form dispersions containing 0.07, 0.5, 1, and 4% HFIP by volume (corresponding to a concentration range of 7 mM to 0.38 M) and spotted, either immediately or after a mixing time of 30 min, onto a carbon-coated copper grid covered with a Formvar layer. Excess LUV/HFIP dispersion was removed, and a droplet of 1% uranyl acetate was deposited onto the grid. After a period of 30 s the excess solution was drawn off from the edge of the grid using blotting paper, and the sample was observed by transmission electron microscopy (TEM) using a Philips 400+ transmission electron microscope at an accelerating voltage of 80 kV. Images were recorded photographically using Kodak 4883 film. Scanning electron microscopy (SEM) analysis was performed on samples prepared in a similar fashion using an Hitachi S-5200 microscope equipped with a field emission gun. Laser Correlation Spectroscopy. Samples of LUVs were prepared by the same method described above for TEM experiments. Size measurements were performed on liposomes at a typical lipid concentration of 2 mM and HFIP/water solutions at concentrations up to 0.3 M. Size experiments on HFIP dispersions in water were performed at concentrations up to 0.57 M by sequential addition of HFIP aliquots to the dispersion. All experiments were performed in replicate (×6) at 25 °C using a ZetaPlus zeta potential analyzer (Brookhaven Instrument Corp.), with a laser wavelength of 658 nm and a scattering angle of 90°. Marker Release Experiments. LUVs containing entrapped CF were prepared by diluting a solution of EPC in CHCl3 (10 mg/mL, 100 µL) with CHCl3 (50 µL) and evaporating the solution to dryness in vacuo to form a thin film. The lipid film was hydrated with 1 mL of a solution of CF (35 mM) and NaCl (∼75 mM) in Tris buffer (10 mM, pH 7.4) with prolonged vortex mixing to ensure complete lipid dispersal. The liposome suspension was subjected to five freeze-thaw cycles between -196 and 30 °C and extruded 10 times through laser-etched polycarbonate membranes (Whatman, 100-nm pore size) at 30 °C using a thermobarrel extruder (Lipex Biomembranes). The LUV suspension was passed through a G10 column (Pharmacia PD-10) equilibrated with a solution of NaCl (∼150 mM) in Tris buffer (10 mM, pH 7.4) and used for fluorescence experiments immediately. Prior to desalting, the ionic strengths of the Tris solutions were measured using a cryoscopic osmometer (Osmomat 030) and adjusted by addition of NaCl to ensure that they were isotonic. Fluorescence experiments were performed with the CFcontaining liposomes in isotonic NaCl/Tris buffer at a lipid concentration of 0.05 mg/mL. Fluorescence emission at 518 nm was measured at an excitation wavelength of 491 nm using a Jobin Yvon-Spex Fluorolog SpectrACQ instrument, with excitation and emission slit widths of 1 mm, respectively, following either addition of HFIP to the liposome preparation (the “forward” experiment) or the addition of liposomes to pre-prepared HFIP/ buffer mixtures (the “reverse” experiment). Triton X-100 was

Lipid/Water Partition Coefficients. For DMPC and DOPC, the lipid/water partition coefficient was estimated according to the change in signal intensity of the HFIP 19 F signal with reference to inorganic fluoride, which was assumed not to partition into membranes. Both DMPC and DOPC produced similar spectra, with the intensity of the sharp doublet arising from HFIP at -76.23 ppm decreasing in relative intensity with increasing lipid concentration. The lipid-bound HFIP exhibited a broad signal that was superimposed on the free HFIP signal, indicative of a slow equilibration process between free and bound HFIP. Nevertheless, as a result of the differing forms of the signals, it was possible to estimate the intensity of the peak from free HFIP to determine KLW. The values we obtained were 463 ( 46 and 669 ( 66 for DMPC and DOPC, respectively. These values were used to estimate bound HFIP/lipid ratios in subsequent experiments. Lipid Properties in HFIP/Water Phases. To determine the effects of HFIP on the bilayer thickness, MLVs composed of DMPC and DOPC were prepared and SAXS data were obtained from them in the presence of a range of HFIP concentrations. The data for DMPC in pure water were characterized by first- and second-order reflections at Q ) 0.098 and 0.195 Å-1 (Figure 1A), corresponding to a lamellar repeat distance (calculated as 2π/Q) of 6.43 ( 0.02 nm, consistent with earlier reports.22 The addition of HFIP to MLV preparations produced progressive changes in the nature of the scattering data, with both first- and second-order reflections becoming broader and decreasing in intensity with increasing HFIP concentration. While these reflections remained in the same position, some splitting of the signals was observed, suggesting either incomplete mixing following HFIP addition or the presence of separate HFIP/lipid phases within the MLV population. Similar experiments with DOPC (Figure 1B) produced a slight decrease in reflection intensity without any significant broadening, with firstand second-order reflections at 0.100 and 0.198 Å-1 consistent with a lamellar repeat distance of 6.33 ( 0.02 nm in pure water. At the highest concentration studied (with a bound HFIP/lipid ratio of 1:4), there was a slight increase in the lamellar repeat distance to 6.50 ( 0.02 nm. These observations were consistent with the formation of a disordered HFIP/DMPC phase, although it was unclear from these data alone whether the increasing disorder resulted from modification of the hydration state of the lipid, changes in lipid packing, or the formation of more complex mixed lipid/HFIP systems, particularly if the interaction of HFIP with membranes was curvaturedependent. Calorimetric analysis of DMPC/water mixtures enabled us to observe the main transition (Tm), corresponding to the change from the rippled gel phase (Pβ′) to the liquid-crystalline phase (LR) centered at 23 °C, and (22) Tristram-Nagle, S.; Nagle, J. F. Chem. Phys. Lipids 2004, 127, 3-14.


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Figure 2. DSC isotherms for DMPC and DMPC/HFIP mixtures. The lipid concentration in these experiments was 0.25 M. The HFIP concentration in each sample is indicated above the corresponding isotherm, with the bound HFIP/lipid ratio at 20 °C indicated in parentheses. The positions of the main (Tm) and pre-transitions (Tp) for DMPC are indicated in italics.

Figure 1. SAXS data for lipid/HFIP mixtures at 20 °C. Each sample contained 9% lipid by weight. Added HFIP concentrations are indicated next to each plot, with the calculated bound HFIP/lipid ratio in parentheses. The scale bar corresponds to the units of the abscissa. A, DMPC/HFIP; B, DOPC/HFIP.

the smaller pre-transition (Tp), corresponding to the change from the gel phase (Lc) to the Pβ′ phase at 12 °C (Figure 2). In the presence of HFIP, the pre-transition disappeared and the main transition moved progressively to lower temperatures as the HFIP content of the sample was raised. The effects of HFIP upon Tm and ∆H were most marked for the first addition of HFIP, during which the enthalpy decreased to ∼50% of its original value for DMPC. The SAXS and DSC data together suggested that the interaction between HFIP and DMPC membranes was disruptive, leading to the formation of disordered aggregate phases. To characterize these phases further, pressure-area (Π-A) isotherms for both DMPC and

DOPC were obtained over a number of HFIP/H2O subphases. Because it was impractical to perform these experiments using molar [HFIP]/[DMPC] ratios in accordance with the SAXS and DSC data, which used high lipid concentrations, experiments were restricted to subphases containing HFIP at concentrations in the range of 0.5-4 vol % (5 mM to 0.38 M), corresponding to bound molar ratios of 1:2 to 3:1. DMPC monolayers over pure H2O produced typical Π-A isotherms (Figure 3A), with a compressional modulus “κA” for the condensed phase (112.0 mN/m, Table 1) consistent with the literature.23 The presence of HFIP in the subphase, however, produced a marked reduction in the collapse pressure (Πc) of the monolayer and marked changes in the collapse area (Ac). The systematic changes in Πc and Ac were found to follow different trends (Figure 3C), with Πc decreasing steadily with increasing HFIP concentration and Ac reaching a maximum at a bound HFIP/lipid ratio of around 2, before decreasing at higher concentrations. Both the compressional modulus of the liquid crystalline phase (Table 1) and the lift-off area (A0) per molecule (Figure 3E) exhibited downward trends with increasing HFIP concentration, consistent with the monolayer becoming increasingly fluidic. The lift-off area (A0) initially decreased to a steady value for subphase HFIP concentrations in the range 0.050.19 M (corresponding to bound HFIP/lipid ratios of 1:2 to 2:1), before decreasing markedly at higher concentrations. Similar experiments were performed using DOPC. The Π-A isotherm for the pure lipid with a pure water subphase had a collapse pressure and a compressional modulus in agreement with the literature values (Figure 3B and Table 1). The DOPC monolayer adopted a smooth transition to condensed states on compression and exhibited similar trends in collapse pressure to DMPC over both pure H2O and HFIP/H2O subphases (Figure 3D), although at slightly lower pressures. The collapse area for DOPC over HFIP/H2O (extrapolated from the collapse (23) Cevc, G.; Marsh, D. Phospholipid Bilayers. Physical Principles and Models. In Cell Biology: A Series of Monographs; Bittar, E. E., Ed.; John Wiley & Sons: New York, 1987; Vol. 5.

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Figure 3. A, B: Π-A isotherms for DMPC and DOPC, respectively, over HFIP/H2O subphases. The figures by each isotherm correspond to the molar HFIP concentration in the subphase, with the bound HFIP/lipid ratio indicated in parentheses. C, D: Variation of the collapse pressures (Πc, solid line) and area (Ac, dashed line) of DMPC and DOPC, respectively, with HFIP concentration in the subphase. E, F: Variation of the lift-off areas (A0) of DMPC and DOPC, respectively, with HFIP concentration in the subphase. Table 1. Data for the Compressional Modulus (KA) of DMPC and DOPC Monolayers over HFIP/H2O Subphases [HFIP] (M) DMPC κA (mN/m) DOPC κA (mN/m)

0

0.05

0.09

0.19

0.38

112.0 134.7

90.2 111.9

72.4 92.9

64.9 76.5

44.9 36.2

pressure) initially decreased at low HFIP concentrations in the subphase and then increased up to a bound HFIP/ lipid ratio of 2, before remaining constant at higher concentrations. The lift-off area (A0) also decreased steadily over the range of HFIP concentrations used (Figure 3F). Liposome Size and Morphology. Samples of DMPC LUVs were examined by TEM in the absence and presence of HFIP. In the former case, the LUVs displayed normal morphologies, with a mean diameter of 123 ( 35 nm and no obvious deformations (Figure 4A). At an HFIP con-

centration of 0.05 M (Figure 4B), three distinctive particulate structures could be seen: unmodified liposomes (indicated by a dashed arrow) with a mean diameter of 105 ( 26 nm, smooth spheroidal particles (indicated by solid arrows) with a mean diameter of 60 ( 17 nm, and structures in which the two were attached (indicated by hollow arrows). Examination of the liposome/HFIP mixture corresponding to Figure 4B after a period of mixing of 30 min indicated that the smooth spheroidal particles were generally absent. When the HFIP concentration was increased to 0.38 M, many of the liposomes separated into loosely adhered aggregates of particle diameter 87 ( 30 nm (Figure 4C), with many of the particles in the aggregates displaying unusually smooth morphologies. At higher magnification (Figure 5), each particle in the mixture appeared to consist of an aggregate of smaller structures.


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Figure 4. TEM images of DMPC liposomes in (A) H2O (×80 000); (B) 0.05 M HFIP/H2O (×46 000); for a description of the markers, see the main text. The calculated bound HFIP/lipid ratio in this sample is 1:2. (C) 0.38 M HFIP/H2O (×22 000). The calculated bound HFIP/lipid ratio in this sample is 3:1. All of the samples were observed immediately following HFIP/liposome mixing. Scale bars represent 100 nm in all cases.

Figure 5. TEM image of DMPC liposomes in 0.38 M HFIP/ H2O, ×60 000. The samples were observed immediately following HFIP/liposome mixing. Examples of liposomes with a blebby appearance are indicated by arrows. The scale bar represents 100 nm.

To examine the effects observed in Figure 4B more closely and to try to ascertain whether some of the structures observed were artifacts, we examined the TEM images produced by both the stain solution on its own and the stain solution containing HFIP. The stain solution produced an essentially featureless image, with occasional patches of high stain density on a uniform background. Interestingly, examination of HFIP/water solutions (Figure 6) indicated that the origin of the smooth spheroidal structures observed in Figure 4B was HFIP. Images obtained by both TEM and SEM, under conditions identical to those used to prepare Figure 4B, displayed the presence of these structures. Some were isolated, with a mean diameter of 29 ( 16 nm, while many appeared as

aggregates. We were also able to observe similar particulate structures in aqueous HFIP samples at concentrations of 0.09 and 0.19 M. In light of these results, we decided to investigate aqueous HFIP solutions by laser correlation spectroscopy. Using a 0.38 M solution of HFIP, we were able to observe particles with a mean effective diameter of 40 nm and a polydispersity of 1.15, although we experienced difficulties in obtaining reproducible results at this concentration because of the low intensity of the scattered signal. At the higher concentration of 0.57 M, however, reproducibility was better, and we obtained a value of 298 nm as the mean effective diameter, with a polydispersity of 0.34. Laser correlation spectroscopy was also used to confirm the effects of HFIP on the liposome morphology observed in the TEM experiments. DMPC LUVs prepared by extrusion were found to have a mean hydrodynamic diameter of 140 nm, with a polydispersity (0.19) typical of liposomes prepared by extrusion. As the HFIP concentration was increased, a significant increase in LUV size was observed, with a concurrent slight increase in polydispersity (from 0.19 to 0.24 for the range of concentrations in Figure 7). It was also noted that the intensity of the light scattering at 658 nm decreased progressively as the HFIP concentration increased (data not shown). Similar experiments with DOPC LUVs indicated a slight decrease in liposome diameter at very low HFIP concentrations and only a very small increase in diameter at higher concentrations. Throughout these experiments, the optical density of the

Figure 6. Images of (A) 0.38 M HFIP in H2O, ×46 000 observed by TEM, and (B) 0.38 M HFIP in H2O, ×110 000 observed by SEM. The scale bar represents 100 nm in both cases.

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examine whether the release of CF was a consequence of inefficient mixing producing regions of high HFIP concentration, we performed the reverse experiment by addition of liposomes to the premixed HFIP/buffer solution in the cuvette. In this case, a similar trend in marker release was observed, albeit at slightly reduced levels when compared to the forward addition. The steady release of CF following HFIP addition was consistent with our earlier results, although the observation of marker release following the addition of LUVs to HFIP solutions was unexpected. Discussion

Figure 7. Size of DMPC LUVs as a function of the HFIP content of the sample determined by laser correlation spectroscopy. The lipid concentration was 2 mM in both cases. A, DMPC/ HFIP; B, DOPC/HFIP.

Figure 8. Release of CF from EPC LUVs, either by addition of HFIP to LUV preparations (dashed line) or by addition of LUVs to HFIP solutions (solid line). For all data points, the lipid concentration following mixing was 0.05 mg/mL.

solution did not change significantly, and there was a similar change in the polydispersity of the sample (from 0.08 to 0.18 across the range of concentrations used). Marker Release Experiments. The initial observation that prompted the work reported in this paper was the apparent promotion of the escape of liposomal contents by HFIP. To characterize this further in light of our other results, we prepared EPC liposomes containing entrapped CF. Addition of HFIP to these liposomes, in a fashion similar to earlier reports,16 produced an immediate loss of entrapped material, indicated by an increase in fluorescence emission intensity. The release of CF following HFIP addition occurred over a very short time period, after which the fluorescence intensity remained stable. The percentage release was determined from this stable value (Figure 8). Addition of increasing quantities of HFIP produced concomitant increases in the extent of marker release. The plot of marker release as a function of HFIP concentration was nonlinear, with a shoulder at an HFIP concentration of ∼0.012 M, indicating that the mechanism of marker release was more complex than simple partitioning of HFIP into the membrane. To

Nature of HFIP in Solution. TEM images of LUVs in the presence of 0.05 M HFIP (Figure 4B) appear to indicate the presence of uniform spherical particles distributed non-randomly throughout the suspension. While some of these particles are isolated, many appear to be adhered to the surface of otherwise normal LUVs. We initially thought that they represented objects produced by budding from LUVs, although we could not rule out the possibility that they were normal LUVs that were out of focus in the image. However, observation of these particles was reproducible in this concentration range and they were not observed in other samples. Furthermore, when the same sample was imaged after a mixing period of 30 min, the particles were no longer present, suggesting that the image in Figure 4B was obtained before equilibrium conditions had been attained. Somewhat surprisingly, upon investigation of HFIP/water mixtures by EM in the absence of liposomes, similar structures were apparent (Figure 6A). From these images alone it was not possible to determine their content or their nature; in particular we were concerned that, because HFIP is volatile, it seemed unlikely that they represented aggregates of HFIP molecules and they may have formed as a result of the presence of high-molecular-weight contaminants in the HFIP solution, arising from impurities either in the stock HFIP used or acquired during sample preparation. We, therefore, repeated these experiments with freshly distilled HFIP using glass syringes that had been extensively washed with this fresh HFIP for all manipulations. However, despite these precautions, we were still able to observe these objects at concentrations as low as 0.09 M. Laser correlation spectroscopic experiments on aqueous HFIP at a concentration of 0.38 M produced results consistent with the formation of particulate structures of size similar to those observed by EM, although we were not able to observe any evidence for particles at lower concentrations by these techniques. Whether this reflected an absence of particles or a lack of sensitivity in the equipment used is open to question, although similar patterns of light scattering have been observed for trifluoroethanol.24 Nevertheless, on the basis of these observations, we speculate that HFIP forms phaseseparated aggregates or clusters in the concentration range 0.38-0.57 M in aqueous solution and possibly also at lower concentrations. The EM images would then reflect disruption of the Formvar surface by localized high concentrations of HFIP, which leave behind visible surface deformations as evidence of their presence. Further work will be required to determine the precise nature of these aggregates, especially because HFIP does not have the properties considered typical of a micelle-forming surfactant. We do note, however, that other researchers have observed inhomogeneities in aqueous HFIP and trifluo(24) Gast, K.; Zirwer, D.; Muller-Frohne, M.; Damaschun, G. Protein Sci. 1999, 8, 625-634.


roethanol solutions over a wide range of concentrations by X-ray scattering methods,25,26 which molecular modeling approaches indicate may form through a combination of hydrogen bonding and segregation of the -CF3 groups.27 It has also been concluded elsewhere that some alcohols, most notably tert-butyl alcohol, are able to form aggregates in aqueous mixtures through self-association.28 In our experiments, the effects of inhomogeneities in aqueous HFIP solutions are only of consequence in those experiments where nonequilibrium conditions are important, namely, the TEM and marker release experiments. In the case of the former, we were able to observe samples both before and after equilibration, and in the latter, marker release was most likely promoted by nonequilibrium effects, reflected in the attainment of a steady-state fluorescence emission after a period of a few minutes following mixing. Interaction of HFIP with DMPC. From our results it is clear that HFIP has a relatively favorable interaction with liposomes composed either of pure DMPC or of pure DOPC. This is reflected in the high lipid/water partition coefficients observed for both of these lipids, which are an order of magnitude greater than the documented octanol/ water partition coefficient. In the case of DMPC it appears that even very small quantities of HFIP introduced to the aqueous medium induce measurable changes in the physical properties of the bilayer that are consistent with a disruptive interaction between the two. As a result of the nature of the equilibrium process, direct comparisons between each of the experiments are not trivial, because the extent of HFIP bound for each datum is obviously dependent on the absolute concentrations of both the alcohol and the lipid, which are by necessity different for each of the experimental techniques used. Nevertheless, when the KLW value for HFIP is considered, it is possible to reach meaningful conclusions when the data are considered as a whole. Bound HFIP/Lipid Ratios < 2:1. The SAXS data are consistent with the formation of a mixed lipid/HFIP phase that either becomes progressively nonlamellar with increasing HFIP concentration or retains a bilayer structure with a polydisperse lamellar repeat distance. It is important to note that, during the course of these experiments, the optical density of the mixture did not change significantly, ruling out the possibility that the decrease in signal intensity was the result of the formation of clear lipid/HFIP phases. Calorimetric analysis supported these data, with the enthalpy and midpoint of the main phase transition of the bilayer decreasing markedly with increasing HFIP concentration. Additionally, the half-height width of this transition increased significantly, and the pre-transition disappeared completely. The presence of the pre-transition and the position of the main transition (Tm) are very sensitive to the presence of solutes in the bilayer and may be used to assess the binding efficacy of drugs to membranes.29 The disappearance of the pre-transition in our case is consistent with at least partial integration into the bilayer, but the changes in the main transition are much more striking. Following the interactions of hydrophobic drugs with bilayers, (25) Hong, D.; Hoshino, M.; Kuboi, R.; Goto, Y. J. Am. Chem. Soc. 1999, 121, 8427-8433. (26) Kuprin, S.; Graslund, A.; Ehrenberg, A.; Koch, M. H. J. Biochem. Biophys. Res. Commun. 1995, 217, 1151-1156. (27) Fiorini, M.; Burger, K.; Mark, A. E.; Roccatano, D. J. Phys. Chem. B 2001, 105, 10967-10975. (28) Price, W. S.; Ide, H.; Arata, Y. J. Phys. Chem. A 2003, 107, 4784-4789. (29) Fildes, F. J. T.; Oliver, J. E. J. Pharm. Pharmacol. 1978, 30, 337-342.

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perturbations seen in the temperature of the midpoint of the main transition tend to be of the order of 1-2 °C at bound drug/lipid ratios of 10 mol %,30 considerably less than observed in our case, and are ascribed to the interaction of the drugs with the fatty acyl chains in the region of the low dielectric interior of the bilayer.31 Similar changes are reported following the binding of peptides to membranes.32,33 Our data are more consistent with those observed for solutes with specific binding sites in the region of the lipid headgroups, in which large changes in enthalpy and temperature of the main transition may result from binding, especially for saturated lipids such as DMPC, where these interactions are far more disruptive to the tight packing of lipid headgroups. This is supported by our thin-film experiments, which produced data consistent with a disruptive incorporation of the HFIP into the monolayer at low concentrations (e bound HFIP/lipid ratios of 2:1), having the effect of increasing Ac and decreasing Πc (Figure 3C). In this concentration regime, there is also a slight reduction in A0 (Figure 3E), the reason for which is unclear. Bound HFIP/Lipid Ratios > 2:1. At higher HFIP concentrations, monolayer destabilization in the thin film experiments occurred to a higher degree. Both the lift-off and collapse areas of the film decreased significantly, as did the collapse pressure. Ac was maximal when the composition of the subphase was approximately 0.19 M, corresponding to a bound HFIP/lipid ratio of 2:1. The simplest explanation for these observations is an equilibrium process between a mixed HFIP/lipid monolayer and HFIP/lipid particles in the subphase. While this renders difficult a comparison of thin-film results in this concentration range with the data at lower bound HFIP/ lipid ratios, we have nevertheless included these data to illustrate the dramatic effects that occur in this concentration range. In the TEM images obtained in 0.38 M HFIP, the liposomes appeared to form aggregates of featureless particles. Laser correlation spectroscopic analysis indicated that the average liposome size doubled as the HFIP concentration was increased to 0.3 M. In addition, the optical density of the solution was considerably lower in this concentration range when compared with the initial value in the absence of HFIP. The observation of considerably weaker scattering from HFIP solutions at higher concentrations (g0.38 M) was not considered to detract from these results. Taken together, these observations suggest that aggregation of mixed HFIP/DMPC particles is a real phenomenon. The morphology of the particles observed by TEM was interesting, because a number appeared to be clusters of smaller particles (Figure 5). The size of each cluster of particles was the same (within experimental error) as the LUVs in the sample preparation, which is consistent with a mechanism for their formation that involves liposomal breakdown by blebbing or division. In light of the EM experiments on aqueous HFIP solutions, some caution needs to be maintained in this analysis, because the observed clusters may also reflect properties induced by HFIP prior to liposome deposition or be features induced by sample preparation. Nevertheless, the structures visible in Figure 5 are nonuniform, and the regular spherical deformations observed to arise from the action of HFIP were not present; we are, therefore, confident that they reflect the mor(30) Arrowsmith, M.; Hadgraft, J.; Kellaway, I. W. Int. J. Pharm. 1983, 16, 305-318. (31) Jain, M. K.; Wu, N. M. J. Membr. Biol. 1977, 34, 157-201. (32) Prenner, E. J.; Lewis, R.; Kondejewski, L. H.; Hodges, R. S.; McElhaney, R. N. Biochim. Biophys. Acta 1999, 1417, 211-223. (33) van Kan, E. J. M.; Ganchev, D. N.; Snel, M. M. E.; Chupin, V.; van der Bent, A.; de Kruijff, B. Biochemistry 2003, 42, 11366-11372.

560


Figure 9. Schematic model for the interaction of HFIP with DMPC thin films.

phologies of lipid-containing structures. As a consequence, the trends in LUV size obtained by laser correlation spectroscopy reflect the formation of large aggregates of small particles. On the basis of the above results, we propose a mechanism for the interaction of HFIP with DMPC. This is shown schematically in Figure 9 for the case in which DMPC is in the form of a thin film, although all of the arguments apply equally well to liposome samples. At low HFIP concentrations (bound HFIP/lipid ratios e 2:1), partitioning of the alcohol into the membrane produces localized regions of disruption (structure B) prior to the establishment of equilibrium conditions. Binding to the membrane is promoted by the formation of hydrogen bonds between the hydroxyl group of HFIP and the phosphate oxygens of the headgroup. This is consistent with the mode of binding of other alcohols to phospholipids10-14 and is justified on the grounds that HFIP is a better hydrogenbond donor than these alcohols (and by turn, phosphate is an extremely good hydrogen bond acceptor). Although we have not investigated the stoichiometry of the binding of HFIP to phospholipids, it is reasonable to assume that at least two HFIP molecules are capable of binding to each phosphate. Binding is reversible, as analysis by 19F NMR spectroscopy demonstrated that HFIP could be completely removed from a liposome sample by dialysis (data not shown). Binding would disrupt the normal headgroup interactions in the lipid and consequently


destroy the cooperativity of the thermotropic phase changes observable by DSC. HFIP has a sufficiently large molecular volume so that the lipid/HFIP adducts would have an inverted-conical shape and, thus, display detergent-like behavior, preferring to locate in regions of positive membrane curvature.34 Because fluoroalkyl groups do not mix ideally with either water or alkanes,7,8 there will exist a driving force for the lipid-bound HFIP molecules to associate. Such association would produce membrane patches of high intrinsic curvature and, consequently, lead to curvature strain35 within the membrane. This would account for the increase in the collapse area and decrease in the collapse pressure at low HFIP subphase concentrations in thin-film experiments. The slight decrease in lift-off area that we observed may also reflect the ability of parts of the monolayer, particularly those rich in HFIP, to adopt configurations with some curvature. The presence of some ordered (HFIP-poor) and disordered (HFIP-rich) domains is consistent with the decrease in scattering intensity observed in SAXS experiments as well as broadening and splitting of the scattering signal. The boundaries between areas of membrane rich in HFIP and poor in HFIP are likely to be most marked following mixing and before the equilibration of HFIP throughout the sample has occurred. These sites would contain many defects and, therefore, be of higher porosity than normal, and they are consequently most likely to be the sites of leakage of CF in the marker release experiments. It should be noted that the range of HFIP concentrations that we have investigated in this paper includes values significantly smaller than those described elsewhere, where complete disruption of the membrane was reported.16 In our experiments, interaction with the membrane prior to the establishment of equilibrium conditions becomes increasingly disruptive, particularly at concentrations higher than ∼0.12 M. The EM results are particularly revealing here, because the apparent observation of particles bound to the surface of liposomes indicates a mechanism by which localized high concentrations of HFIP can occur in the membrane, assuming that the particles reflect the formation of HFIP clusters in the aqueous solution. The shoulder at 0.12 M would than reflect the concentration at which significant proportions of HFIP begin to exist in a heterogeneous phase. Furthermore, if marker release was simply the result of inefficient mixing during sample preparation, we would not have expected to observe release in the reverse experiment, in which liposomes were added to HFIP solutions. The observation of marker release in these experiments is consistent with heterogeneous clusters of HFIP molecules associating with the membrane surface and is difficult to explain by other means. As the concentration of HFIP in the aqueous medium is raised above 0.09 M, HFIP aggregates begin to associate with the monolayer surface and the phospholipid binding sites for HFIP become increasingly saturated, enhancing the ability of the monolayer to adopt curved geometries (structure C). In the case of thin films, it is likely that before saturation is reached, removal of material from the monolayer under equilibrium conditions in the form of micellar structures (structure D) of higher curvature than the parent membrane will occur to leave behind a more stable low-curvature phospholipid monolayer depleted in HFIP (structure E). In the thin-film experiments, saturation occurs at a bound HFIP/lipid ratio of 2:1, (34) Heerklotz, H. J. Phys.: Condens. Matter 2004, 16, R441-R467. (35) Epand, R. M.; Epand, R. F. Biophys. J. 1994, 66, 1450-1456.


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for incorporation of HFIP molecules into the headgroup region without disruption of membrane packing. In fact, the thin film experiments indicate that at subphase HFIP concentrations e 0.02 M (corresponding to a bound HFIP/ lipid ratio of 4:1), incorporation of HFIP into the bilayer decreases the collapse area of the film (structure B), consistent with the formation of favorable intermolecular contacts. At concentrations g 0.02 M HFIP, the collapse area of the film increases steadily to a bound HFIP/lipid ratio of around 2:1 at a subphase concentration of 0.19 M and then increases more slowly as the HFIP concentration is raised further. The lift-off area of the film decreases steadily to a bound HFIP/lipid ratio of 2:1 and then remains stable, suggesting that HFIP binding to the headgroup region is approaching saturation at this concentration (structure C). This is again consistent with the proposed binding of two HFIP molecules per lipid headgroup. The SAXS data are consistent with a nondisruptive incorporation of HFIP into the DOPC bilayer, because only small changes in scattering intensity were apparent over the range of concentrations studied. Conclusions Figure 10. Schematic model for the interaction of HFIP with DOPC thin films.

because at higher HFIP concentrations the collapse of the film occurs at significantly smaller surface areas. This ratio is consistent with our suggestion that each lipid molecule is capable of binding two HFIP molecules. In the case of liposomes, those rich in HFIP divide into the smaller structures of higher curvature observed by TEM. These are likely to be micellar (L1) in nature, because this phase is favored by molecules with an inverted conical shape. More complex mixed phases cannot be ruled out completely, however; for instance, it has been demonstrated that the membrane activity of some peptides leads to the formation of small unilamellar vesicles of diameter 2040 nm.36-38 In these examples, in common with ours, lamellar structures are postulated to form under the influence of positive curvature strain, with a concomitant increase in isotropy of some membrane components. Interaction of HFIP with DOPC. The interaction of HFIP with DOPC in the form of a thin film is shown in Figure 10. HFIP is incorporated much more easily into the DOPC monolayer than into the DMPC monolayer. This is consistent with the proposed interaction of HFIP with phospholipid headgroups. Because DOPC is in the fluid liquid crystalline (LR) phase at room temperature, with the major intermolecular interactions determined by the packing of the acyl chains, there is sufficient space (36) Bonev, B. B.; Lam, Y.-H.; Anderluh, G.; Watts, A.; Norton, R. S.; Separovic, F. Biophys. J. 2003, 84, 2382-2392. (37) Agirre, A.; Flach, C.; Goni, F. M.; Mendelsohn, R.; Valpuesta, J. M.; Wu, F. J.; Nieva, J. L. Biochim. Biophys. Acta 2000, 1467, 153164. (38) Batista, U.; Jezernik, K. Cell. Biol. Int. Rep. 1992, 16, 115-123.

In these experiments we have demonstrated that HFIP interacts favorably with phosphatidylcholines, although the effects of this interaction depend strongly upon the degree of saturation of the lipid. Unsaturated lipids are able to bind HFIP without deleterious effects on membrane integrity, whereas those that are saturated become unstable and denature to form mixed HFIP/lipid particles. We have not speculated on the precise nature of these particles, although they are clearly isotropic and display a tendency to aggregate. It seems likely that they display some micellar properties, although lamellar structures cannot be ruled out. Further experimentation, particularly characterization by techniques such as neutron scattering, should help to clarify this issue. The effects produced by HFIP arise from a combination of the unique properties of this molecule, particularly its relatively high octanol/ water partition coefficient combined with the ability to form strong hydrogen bonds to phosphocholine headgroups. The results are consistent with the binding of two HFIP molecules per lipid headgroup. The ability of HFIP to form distinct particulate structures in aqueous mixtures has been suggested before, but it will be of some interest to pursue these properties further, particularly because HFIP is commonly used to solvate hydrophobic molecules for applications in biological assays. Acknowledgment. We thank the Royal Society (U.K.) for an equipment grant that supported part of this work (RSRG 24281), D. Carswell (DSC measurements), and A. C. Richardson (EM). Supporting Information Available: Sample 19F NMR spectra from partition coefficient determination. This material is available free of charge via the Internet at http://pubs.acs.org.

LA048109Y

Micellar Aggregates Formed Following the Addition of

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