Effect of Cholesterol and Phospholipid on the Behavior of Dialkyl

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Effect of Cholesterol and Phospholipid on the Behavior of Dialkyl Polyoxyethylene Ether Surfactant (2C18E12) Monolayers and Bilayers Richard D. Harvey,† Richard K. Heenan,‡ David J. Barlow,† and M. Jayne Lawrence*,† Department of Pharmacy, King's College London, Franklin Wilkins Building, 150 Stamford Street, London SE1 9NN, United Kingdom, and ISIS Facility, Rutherford Appleton Laboratory, Chilton, Oxon, OX11 0QX, United Kingdom Received February 20, 2004. In Final Form: July 8, 2004 Surface pressure-area isotherm, neutron specular reflection, and small-angle neutron scattering studies have been carried out to determine the effects of added cholesterol and distearoylphosphatidylcholine (DSPC), on the molecular structures of monolayers and vesicles containing the dialkyl polyoxyethylene ether surfactant, 1,2-di-O-octadecyl-rac-glyceryl-3-(ω-dodecaethylene glycol) (2C18E12). Previous neutron reflectivity studies on 2C18E12 monolayers at the air/water interface have shown them to possess a thickness of ∼24 Å and highly disordered structure with significant intermixing of the polymer headgroups and alkyl chains. SANS studies of 2C18E12 vesicles gave a bilayer thickness of ∼51 Å. Addition of cholesterol to 2C18E12 monolayers (1:1 molar ratio), produced a marked condensing effect coupled with an increased the layer thickness of ∼7 Å, and in vesicles, increased bilayer thickness by ∼16 Å. Monolayers consisting of 2C18E12:DSPC:cholesterol (1:1:2 molar ratio), showed a layer thickness of ∼31 Å, whereas in vesicles, three-component bilayer was found to be only ∼9 Å thicker than those possessed by vesicles composed solely of 2C18E12. Mixing between the molecules in three-component monolayers was shown to be ideal through analysis of the neutron reflectivity data. These findings are discussed in relation to increased ordering and decreased headgroup/hydrophobe intermixing within both monolayers and vesicle bilayers containing 2C18E12. The inferred increase in molecular order within vesicles composed of 2C18E12 with additional cholesterol and phospholipid is used as a model for explaining theoretical differences in bilayer permeability.

* To whom correspondence should be addressed. E-mail: [email protected]. † King’s College London. ‡ Rutherford Appleton Laboratory.

duction of cholesterol may be attributed to an increased ordering of the surfactant molecules, a phenomenon also observed when the sterol is incorporated in phospholipid bilayers at concentrations above 30 mol%.7 In recent years, details of the structure of surfactant monolayers at the air-liquid interface obtained from neutron specular reflection experiments have been used to determine molecular ordering.8 For neutron specular reflection experiments on surfactant monolayers at the air-liquid interface, deuterated surfactants are generally studied at the interface of air and null reflecting water (a mixture of H2O and D2O with a neutron scattering length density equal to that of air, i.e., zero). Such an experimental setup provides the greatest degree of contrast, where the only scattering signal arises from the adsorbed surfactant.9 The thickness of the hydrophobic chain regions normal to the interface in these surfactant monolayers may be directly related to their order parameters, as they are for phospholipid systems.10 In both cases, molecule order is taken to be related to the orientation of molecules with respect to the monolayer normal.11 This simple model needs adaptation in order to be applied in the case of

(1) Lawrence, M. J.; Chauhan, S.; Lawrence, S. M.; Barlow, D. J. S. T. P. Pharma Sci. 1996, 6, 49-60. (2) Lawrence, M. J.; Barlow, D. J.; Harvey, R. D.; Heenan, R. K. In Self-Assembly; Robinson, B. H., Ed.; IOS Press: Amsterdam, 2003; pp 339-347. (3) Hofland, H. E. J.; Boustra, J. A.; Verhoef, J. C.; Buckton, G.; Chowdry, B. Z.; Ponec, M.; Junginger, H. E. J. Pharm. Pharmacol. 1992, 44, 287-294. (4) van Hal, D. A.; Bouwstra, J. A.; van Rensen, A.; Jeremiasse, E.; de Vringer, T.; Junginger, H. E. J. Colloid Interface Sci. 1996, 178, 263-273. (5) Hofland, H. E. J.; Bouwstra, J. E.; Gooris, G. S.; Spies, F.; Talsma, H.; Junginger, H. E. J. Colloid Interface Sci. 1993, 161, 366-376.

(6) Devaraj, G. N.; Parakh, S. R.; Devraj, R.; Apte, S. S.; Roa, B. R.; Rambhau, D. J. Colloid Interface Sci. 2002, 251, 360-365. (7) Yeagle, P. L. Biochim. Biophys. Acta 1985, 822, 267-287. (8) Dynarowicz-Latka, P.; Dhanabalan, A.; Oliveira, O. N. Adv. Colloids Interface Sci. 2001, 91, 221-293. (9) Lu, J. R.; Li, Z. X.; Thomas, R. K.; Binks, B. P.; Crichton, D.; Fletcher, P. D. I.; McNab, J. R.; Penfold, J. J. Phys. Chem. B 1998, 102, 5785-5793. (10) Le´onard, A.; Escrive, C.; Laguerre, M.; Pebay-Peyroula, E.; Ne´ri, W.; Pott, T.; Katsaras, J.; Dufourc, E. J. Langmuir 2001, 17, 20192030.

Introduction Vesicles composed entirely of synthetic nonionic surfactants with short polymeric headgroups, such as 1,2di-O-octadecyl-rac-glyceryl-3-(ω-dodecaethylene glycol) (2C18E12), have been shown to be sterically stable and, as such, have been considered to be ideal for use as longcirculating drug carriers for parenteral delivery.1 However, vesicles composed solely of 2C18E12 have been shown to exhibit low entrapment efficiencies for aqueous solutes.2 Low entrapment of aqueous solutes by nonionic surfactant vesicles has also been observed for vesicles composed of a number of different surfactants with polyoxyethylene headgroups.3,4 This phenomenon has been attributed to a high degree of permeability intrinsic to the surfactant bilayers, which can be greatly reduced by the incorporation of 40-50 mol% cholesterol (Chol).5,6 The reduction in surfactant bilayer permeability observed upon the intro-

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Behavioral Effect of Cholesterol and Phospholipid

monolayers of 2C18E12, because of the effect of the poly(ethylene glycol) (PEG) headgroups upon the behavior of the hydrophobic region of the film. For monoalkyl poly(ethylene glycol) ether surfactants of the series C12Em, neutron reflectivity experiments reported by Lu and Thomas12 show that increasing the length of the headgroup from 2 to 12 ethylene oxide (EO) units has significant effects upon both the interfacial area per molecule and monolayer thickness. For surfactants where m ) 8 or 12, there is a pronounced difference between the thickness of the monolayer and the extended chain length of the molecule.12 This can be explained by examining the behavior of the PEG headgroup and determining its effect upon the alkyl chain. Sarmoria and Blankschtein13 showed that PEG headgroups tethered to alkyl chain moieties need to contain at least 10 EO units in order to obey polymer scaling laws and, thus, exhibit a random coil conformation. Any PEG headgroup in a random coil conformation will not only have a reduced length from its theoretical extended value, but its bulkier nature will also confer a larger area per molecule upon the surfactant. The increased lateral pressure within the monolayer, caused by the steric requirements of the bulky headgroup, allows the alkyl chains to have a greater degree of tilt away from the surface normal, thus decreasing the film thickness.9 Neutron reflectivity studies on monolayers of 2C18E12, have shown that it behaves in a similar way to monoalkyl poly(ethylene glycol) ethers. Increasing the surface pressure on the monolayer from 11 to 34 mN m-1, increases the thickness normal to the surface from ∼20 to ∼30 Å, which is consistent with a decrease in the angle of tilt of the headgroup in relation to the surface normal.14 At a surface pressure of 28 mN m-1, the two C18 chains have been modeled as lying almost flat on the surface, with a tilt of 70° from the normal.15 At surface pressures above 40 mN m-1, which marks the start of a plateau region in the 2C18E12 surface pressure-area isotherm, off-specular neutron reflection has been detected.16 This plateau region of the surface pressure-area isotherm may be attributed to the increasing steric influence of the PEG headgroup, which would dominate the packing state of the whole molecule.17 A detailed investigation using both partially and fully deuterated forms of 2C18E12, with results modeled using kinematic approximation, revealed significant mixing between the alkyl chains and the PEG headgroups, at a surface pressure of 28 mN m-1.18 This phenomenon has also been observed in monolayers of monoalkyl poly(oxyethylene) ethers.9,19 This intermixing of the alkyl (11) Chapman, D.; Kramers, M. T. C.; Restall, C. J. In Sterols and Bile Acids; Danielsson, H., Sjovall, J., Ed.; Elsevier: Amsterdam, Netherlands., 1985; pp 151-174. (12) Lu, J. R.; Thomas, R. K. In Applications of neutron scattering to soft condensed matter; Gabrys, B. J., Ed.; Gordon and Breach: Langhorne, PA, 2000; pp 205-225. (13) Sarmoria, C.; Blankschtein, D. J. Phys. Chem. 1992, 96, 19781983. (14) Barlow, D. J.; Ma, G.; Lawrence, M. J.; Webster, J. P. R.; Penfold, J. Langmuir 1995, 11, 3737-3741. (15) Barlow, D. J.; Muslim, A.-M.; Webster, J. P. R.; Penfold, J.; Hollinshead, C. M.; Lawrence, M. J. Phys. Chem. Chem. Phys. 2000, 2, 5208-5213. (16) Lawrence, M. J.; Chauhan, S.; Lawrence, S. M.; Ma, G.; Penfold, J.; Webster, J. P. R.; Barlow, D. J. In Chemical Aspects of Drug Delivery Systems; Karsa, D. R., Ed.; The Royal Society of Chemistry: London, 1996; pp 65-76. (17) Mathe, G.; Gege, C.; Neumaier, K. R.; Schmidt, R. R.; Sackmann, E. Langmuir 2000, 16, 3835-3845. (18) Barlow, D. J.; Ma, G.; Webster, J. P. R.; Penfold, J.; Lawrence, M. J. Langmuir 1997, 13, 3800-3806. (19) Penfold, J.; Staples, E. J.; Tucker, I.; Thomas, R. K. Colloids Surf. A 1999, 155, 11-26.

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chains and headgroups increases the monolayer roughness, to a level above that which could be accounted for by capillary wave roughness.18,19 Headgroup and hydrophobe mixing may also account for the discrepancy between the intrinsic monolayer thickness and their fully extended chain lengths.12 In this investigation, the structure of 2C18E12 monolayers are compared at different surface pressures, both with and without cholesterol, using a combination of data obtained from surface pressure-area isotherms and neutron specular reflection measurements. The extent to which the PEG polymer may affect film thickness and the degree of headgroup hydration has also been examined. This was carried out in order to yield information about the extension of the headgroup into the subphase and hence provide further indications of the effects of cholesterol. The results are discussed in relation to the effects of added cholesterol on the ordering of the nonionic surfactant and the miscibility of the components, not only in monolayers consisting of 2C18E12:Chol (1:1 molar ratio) but also in three component monolayers, additionally containing distearoylphosphatidylcholine (DSPC). The addition of phospholipid to the monolayers was carried out in order to determine the effect of a reduction in polymer grafting density upon the ordering of the system. Parallel studies on 2C18E12 vesicles using small-angle neutron scattering were also conducted to compare the effects of Chol and DSPC addition on bilayer parameters with those obtained during the monolayer studies. Materials and Methods Materials. Fully protonated L-R-distearoylphosphatidylcholine (h-SPC) was purchased from Sigma (UK), and fully deuterated 1,2-distearoyl-d70-sn-glycero-3-phosphocholine-1,1,2,2d4-N,N,N-trimethyl-d9 (d83-DSPC) was purchased from Avanti Polar Lipids (Alabaster, USA). Fully protonated cholesterol (Chol) was obtained from Fluka (UK). The fully protonated and fully deuterated versions of 1,2-di-O-octadecyl-rac-glyceryl-3-(ω-dodecaethylene glycol), abbreviated, respectively, to h-2C18h-E12 and d-2C18d-E12,18 were synthesized in the Pharmacy Department of King’s College London. Wilhelmy plates were cut from Whatman No.1 filter paper (Merck, UK Ltd.). Throughout all the experiments, spectroscopic grade chloroform (Rathburn, UK) and 18 MΩ cm residual specific resistance ultrapure water (Elgastat Maxima purifier, Elga UK) were used. All D2O used (99.9% purity) was supplied by Fluorochem Ltd. (UK) and was used to prepare null reflecting water (nrw) consisting of 92% H2O and 8% D2O (v/v). All chemicals were used as supplied. Surface Pressure-Area Isotherms. Monolayers of pure amphiphiles and equimolar binary mixtures were formed by spreading 30 µL of 2 mg mL-1 solutions of the various surfactant mixtures in chloroform onto the water surface in a Nima Technologies Langmuir trough (Nima Technologies, Coventry, UK). The temperature of the subphase was thermostatically controlled at 25 °C. The monolayer films were allowed to equilibrate for 10 min prior to compression to allow evaporation of the chloroform. Surface pressure-area isotherms (measured in triplicate) were obtained using a Wilhelmy plate suspended from a microbalance during the computer-controlled compression of the monolayer at a rate of 50 cm2/min. For binary mixtures, mean areas per molecule were calculated from the molar averages of the components. Neutron Specular Reflection at the Air-Liquid Interface. The neutron reflectivity experiments were carried out on the Critical Reflectance Interfacial Spectrometer (CRISP) at the ISIS Facility, Rutherford Appleton Laboratory (Chilton, Oxfordshire, UK). Fifty microliters of various surfactant mixtures

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made up of 2 mg mL-1 in chloroform were added dropwise to the aqueous subphase surface in a Nima Technologies Langmuir trough (Nima Technologies, Coventry, UK), which had previously been cleaned with chloroform and rinsed with ultrapure water. After 10 min, to allow for solvent evaporation and monolayer equilibration, the Teflon barriers of the trough were compressed to give the required surface pressure (measured by Wilhelmy plate and microbalance). A feedback mechanism maintained the surface pressure at a constant pre-set value for the duration of the reflectivity experiment. The CRISP reflectometer uses a time-of-flight (TOF) method to determine the wavelengths (λ) of neutrons in a white beam at a fixed angle of incidence (θ) to the monolayer surface. Measurements were made at 298 K with a single detector at a fixed θ of 1.5° using a range of neutron wavelengths from 0.5 to 6.8Å. The wavelength range covered a momentum transfer (κ ) 4π sin θ/λ) range of 0.048 to 0.5 Å-1. As incoherent background scattering is prevalent at high κ, i.e., above 0.2 Å-1, the background scattering, which was subtracted from the experimental data before analysis, was determined by extrapolation to high κ. The reflectivities of a number of lipid/surfactant mixtures were determined using both D2O and nrw as subphases. Reflectivity measurements were taken from monolayers of h-2C18h-E12:Chol and d-2C18d-E12:Chol (1:1 molar ratios) at a surface pressure of 34 mN m-1. Data for the neutron reflectivity of h-2C18h-E12 and d-2C18d-E12 monolayers on D2O and nrw at 34 and 40 mN m-1 were obtained from previous work carried out by Ma.20 Finally, mixed monolayers containing various combinations of h-2C18hE12, d-2C18d-E12, h-DSPC, d83-DSPC, and Chol were studied at 34 mN m-1 to investigate 2C18E12:DSPC:Chol (1:1:2 molar ratio) monolayers. Small-Angle Neutron Scattering. SANS measurements were performed on the LOQ beam line at the ISIS pulsed neutron source (CCLRC Rutherford-Appleton Laboratory). Wavelengthdependent corrections are made to allow for the incident spectrum, detector efficiencies, and measured sample transmissions to create a composite SANS pattern. The LOQ instrument gives a scattering vector (Q) range of 0.008 to 0.22 Å-1. Comparisons with scattering from a partially deuterated polystyrene standard allow absolute scattering cross sections to be determined, with an error of (2%. Vesicle samples were prepared for SANS by using the thinfilm hydration technique of Kirby and Gregoriadis21 followed by probe sonication (Soniprobe, Lukas Dawe Ultrasonics, UK) in D2O for a maximum of 5 min. After sonication, the suspensions were centrifuged at 12 000 rpm for 2 min (MSE Micro Centaur, Sanyo, UK) to remove any titanium particles which may have been shed by the sonicator probe. The vesicles were then allowed to anneal at room temperature for at least 1 h prior to any experimental treatment. Three vesicle formulations were prepared to make comparisons with the various monolayers studied by neutron specular reflection; specifically: 2C18E12, 2C18E12: Chol (1:1), and 2C18E12:DSPC:Chol (1:1:2) (each with a total lipid concentration of 1 mg mL-1). Vesicle samples were placed in scrupulously cleaned diskshaped fused silica cells of path length 1 mm (for H2O) or 2 mm (for D2O). Using a 12 mm diameter neutron beam, measurements were carried out at 298 K. Backgrounds from pure H2O and pure D2O were subtracted. All fitting procedures included flat background corrections to allow for any mismatch in the incoherent and inelastic scattering between the sample and solvents. Fitted background levels were always checked to ensure that they were of a physically reasonable magnitude.

Results Surface Pressure-Area Isotherms. Surface pressure-area isotherms for pure 2C18E12, DSPC, and cholesterol, together with those of equimolar binary mixtures of 2C18E12:Chol and 2C18E12:DSPC, are shown in Figure (20) Ma, G. F. K. Structural studies of non-ionic surfactant monolayers and vesicles.; University of London: London, 1997. (21) Kirby, C. J.; Gregoriadis, G. In Liposome Technology; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. I, pp 19-27.

Harvey et al.

Figure 1. Surface pressure-area isotherms for pure amphiphiles and mixtures of (A) 2C18E12 and cholesterol and (B) 2C18E12 and DSPC, at the air/water interface at 25 °C.

1. The areas per molecule obtained from the isotherms (Table 1) were used to determine the excess area (Aex) of mixing 22,23 for the binary mixtures at different surface pressures using the equation below.

Aex ) A12 - (X1A1 + X2A2)

(1)

The excess area gives an indication of the miscibility and ideality of mixing of the two components in a binary mixture by determining the deviation of the actual area per molecule of the binary mixture (A12) from the theoretical molecular area of a mixture of pure lipids (A1 and A2) of appropriate molar fractions (X1 and X2). At any given surface pressure, immiscible or ideally mixed components in the monolayer will have an Aex of zero, while positive or negative values for Aex indicate miscibility which is nonideal and therefore suggests molecular interaction between the components.24 For monolayers consisting of equimolar concentrations of DSPC and cholesterol (results not shown), no significant excess area was observed, which is consistent with the (22) Gaines, G. L. Insoluble monolayers at liquid-gas interfaces; Wiley-Interscience: New York, 1966. (23) Costin, I. S.; Barnes, G. T. J. Colloid Interface Sci. 1975, 51, 106-121. (24) Minones-Trillo, J.; Garcia-Fernandez, S.; Sanz-Pedrero, P. J. Colloid Interface Sci. 1968, 26, 518-531.

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Table 1. Areas Per Molecule of Pure Amphiphiles and Their Mixtures for Different Surface Pressures at 25 °C, Obtained from Surface Pressure-Area Isotherms

a

surface pressure (mN m-1)

Chol A0 (Å2)

DSPC A0 (Å2)

2C18E12 A0 (Å2)

2C18E12:Chola A0 (Å2)

2C18E12:DSPCa A0 (Å2)

25 34 40

40.1 ( 0.3 40.1 ( 0.3 40.1 ( 0.3

54.8 ( 0.4 54.5 ( 0.7 53.8 ( 0.2

145.8 ( 3.1 119.3 ( 1.2 107.8 ( 3.4

61.9 ( 4.2 53.9 ( 2.0 53.3 ( 1.4

101.8 ( 2.9 86.3 ( 0.7 79.1 ( 1.2

Area per molecule derived from calculated mean areas per molecule for the mixture. Table 2. Scattering Lengths and Scattering-Length Densities of Materials Used in This Study

material

volumea (Å3)

scattering length, bb (×104Å)

scattering length density, F (×106Å-2)

nrwc D2O Cholesterol h-DSPC d83-DSPC h-2C18hE12 d-2C18dE12

30 30 602 1240 1240 1965 1965

0 1.92 1.32 2.41 88.87 2.60 128.50

0 6.40 0.22 0.19 7.17 0.13 6.54

a Volumes calculated from density data. b Calculated from atomic scattering length data. c nrw, null reflecting water (92% H2O and 8% D2O (v/v)).

results of previous research showing the absence of any condensing effect of the sterol when mixed with DSPC.25 However, the Aex values for 2C18E12:Chol mixtures were -31.1, -25.8, and -20.7 at 25, 34, and 40 mN/m, respectively. In conjunction with the isotherm for the 2C18E12:Chol mixture (Figure 1A), the highly negative excess area values, generally thought to result from attractive interactions between the different molecules,26 demonstrate a marked condensing effect of the sterol upon the surfactant. For the equimolar 2C18E12:DSPC mixture, the excess area values at any given surface pressure were within the limits defined by experimental error on the measured A12 values. The absence of a discernible collapse pressure corresponding to that of DSPC in the isotherm of the mixture (Figure 1B) suggests that the two components mix ideally rather than exhibiting phase separation.22 Neutron Specular Reflection at the Air-Liquid Interface. Analysis of the reflectivity profiles obtained at various surface pressures for each of the monolayers investigated were carried out using the model-independent kinematic approximation.27 The kinematic method describes each system in terms of partial structure factors for the various monolayer components (hAA) and the solvent (hSS), which in turn provide distribution widths of the amphiphiles (σi) and solvent (ξ) along the interface normal (z) (as described in detail by Lu & Thomas).28 The distribution widths (σi) for the nonionic surfactant 2C18E12 and the phospholipid DSPC were modeled as Gaussian distributions according to eq 2. The distribution widths (ξ) for the subphase solvent associated with each system were modeled as a tanh distribution using eq 3. The crossterm (hAS) gives the separation (δAS) between the monolayer component and solvent distributions (4). Table 2 shows the molecular volume, atomic scattering length, (25) Joos, P.; Demel, R. A. Biochim. Biophys. Acta 1969, 183, 447457. (26) Galvez-Ruiz, M. J.; Cabrerizo-Vilchez, M. A. Colloid Polym. Sci. 1991, 269, 77-84. (27) Crowley, T. L.; Lee, E. M.; Simister, E. A.; Thomas, R. K. Physica B 1991, 173, 143-156. (28) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans. 1998, 94, 995-1018.

Figure 2. Semilog plots of ln[hAA(κ)] against κ2 together with calculated regression lines for (A) 2C18E12:CHOL (1:1) (R2 ) 0.997) and (B) 2C18E12:DSPC:CHOL (1:1:2) (R2 ) 0.989) monolayers at a surface pressure of 34 mN m-1.

and scattering length density data for the molecular species involved in the systems analyzed.

hAA(κ) )

( )

-κ2σi2 1 exp 8 Ai2 2

(ξπκ 2 )

hSS(κ) ) nS02

(ξπκ 2 )

cosec2

hAS ) (hAA - hSS)1/2 sin κδAS

(2)

(3) (4)

The determination of the distribution width (σi) (which is analogous to monolayer layer thickness) and area per molecule (A0) at the various surface pressures studied for each monolayer system was carried out using simple Guinier analysis (data not shown) prior to analysis by kinematic approximation. The values of σ and A0 obtained using the two methods were found to be in close agreement. Using the kinematic approximation, the structure of the monolayer system was determined in terms of the partial structure factors for the lipid/surfactant and associated solvent. Semilogarithmic plots (Figure 2) of the partial structure factors ln[hAA(κ)] versus κ2 were used to obtain values for A0 and σi. The data used to derive the semilog plots were obtained from reflectivity of the deuterated lipid/surfactant monolayer on nrw. The molecular area was derived from the ordinate intercept (-2 ln A0), and the distribution width (σi) from the gradient of the semilog plot (-σi2/8). 2C18E12:Chol Monolayers. At 34 mN m-1, the area per molecule for the 2C18E12:Chol monolayer derived from Figure 2 was significantly higher than that previously reported for 2C18E12 at the same surface pressure:20 respectively, 123 versus 117 Å2 (Table 3). Similar differences between areas per molecule of 2C18E12 monolayers

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Table 3. 2C18E12*, 2C18E12:CHOL (1:1), and 2C18E12:DSPC:CHOL (1:1:2) Layer Thickness (σi) and Molecular Areas (Ai) Determined from the Partial Structure Factor Analysis (hAA(K))a

lipid/surfactant monolayer

surface pressure (mN m-1)

surfactant distribution width σA(Å)

area per moleculeb A0 (Å2)

34 40 25 34 40 34

24.0 ( 0.3 27.0 ( 0.2 25.9 ( 0.3 31.3 ( 0.2 32.9 ( 0.3 31.5 ( 0.1

117.0 ( 2.0 100.0 ( 2.2 158.9 ( 4.9 123.4 ( 2.3 112.3 ( 4.4 236.7 ( 4.4

2C18E12a 2C18E12:CHOL 2C18E12:DSPC:CHOL a

surfactant separationc δAB (Å)

solvent distributionc width σA (Å)

solvent separationc δAS (Å)

2.0 ( 0.5

11.0 ( 0.5 15.0 ( 0.5 12.5 ( 0.5 13.5 ( 0.5 15.5 ( 0.5 11.5 ( 0.5

6.5 ( 0.5 8.5 ( 0.5 6.0 ( 0.5 7.0 ( 0.5 7.2 ( 0.5 8.0 ( 0.5

Ma.20 b

Values obtained from Mixture areas per molecule assume the presence of supermolecules. c Error values quoted are only approximate and are derived from visual inspection of the fits between calculated and observed structure factor curves.

with and without cholesterol were also observed at 25 and 40 mN m-1, (Table 3). These results may be expected since the area per molecule of the binary mixture must contain a contribution from the sterol. As in the case of the results obtained from surface pressure-area isotherms for the mixing between 2C18E12 and cholesterol in a monolayer to be considered ideal, areas per molecule for mixed monolayers can be expected to follow a mean additive rule.22 However, in contrast with the surface pressure-area isotherm data, the analysis of the reflectivity data required the scattering unit of the mixed monolayer to be treated as a single supermolecule consisting of the surfactant and sterol in close association. Therefore, ideal mixing of monolayer components may be expected to result in a molecular area which is the sum of those of the constituent molecules. However, if the A0 of cholesterol is considered to be ∼40 Å2, as obtained from its surface pressure-area isotherm, this does not correspond to the difference between the molecular areas of 2C18E12 and 2C18E12:Chol monolayers at 34 mN m-1, as obtained by neutron reflectivity. The negative differences between the theoretical cross-sectional areas of the supermolecules and those determined by analysis of the reflectivity data at the given surface pressures further illustrates the condensing effect of cholesterol upon the surfactant. The plots for the self and cross partial structure factors against momentum transfer, derived from the reflectivity profiles from monolayers consisting of 1:1 molar ratios of 2C18E12 and cholesterol at 34 mN m-1, are shown in Figure 3. The distribution widths and separations derived from these plots are summarized in Table 3. At 34 mN m-1, the distribution width for the 2C18E12: Chol monolayer is ∼31 Å, which is ∼7 Å greater than the width determined at the same surface pressure for the pure 2C18E12 monolayer.20 Since the contributions made to these distribution widths by capillary wave roughness will be identical (given that the two systems were studied at the same surface pressure), the effect of cholesterol incorporation must be to alter the conformation and orientation of the 2C18E12 molecules and/or to increase the structural roughness of the monolayer. The differences between distribution widths of monolayer and solvent and the separations between their centers for 2C18E12 monolayers with and without cholesterol at different surface pressures are summarized in the volume fraction profile shown in Figure 4. The presence of cholesterol in the 2C18E12 monolayer leads to a 1-2 Å increase in the separation between the centers of the surfactant and solvent distributions and to a 1-2 Å decrease in the solvent distribution width at 34 mN m-1. Although these changes are small, the same trend is evident at 40 mN m-1 (Table 3), and therefore they may

Figure 3. Plots for self and cross partial structure factor against momentum transfer for monolayers consisting of 1:1 molar ratios of 2C18E12 and cholesterol at a surface pressure of 34 mN m-1: (A) self partial structure factor hAA(κ) against κ (data fitted assuming a Gaussian profile), (B) cross partial structure factor hAS(κ) against κ, and (C) self partial structure factor hSS(κ) against κ (data fitted assuming a tanh profile). Error bars show the statistical errors calculated from the variance-covariance matrix in the regression analysis.

reflect genuine structural differences between monolayers with and without cholesterol. It is likely that the increase

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Figure 4. Volume fraction profiles for the 2C18E12:CHOL monolayer and the total of all components at the surface pressure of 34 mN m-1 derived by Gaussian (for 2C18E12) and tanh (for solvent) analysis. Data for the volume fraction profile for 2C18E12* was taken from Ma.20

in surfactant distribution width in the presence of cholesterol results from either the extension of the PEG headgroup into the solvent or a reduction in the tilting of the surfactant alkyl chains away from the monolayer normal. 2C18E12:DSPC:Chol Monolayers. Kinematic analysis of the three-component 2C18E12:DSPC:Chol monolayers proved to be quite difficult. Theoretically, six partial structure factors could have been obtained from solving the combined equations measured under the different deuterium-labeling conditions studied. However, there appeared to be a distortion in the true shape of the cross partial structure factor hAB (where A and B refer to the different labeled lipid/surfactants). The self-partial structure factor hAA was therefore used to represent all three components of the monolayer as a supermolecule with a ratio of 1 2C18E12:1 DSPC:2 cholesterol. The cross-partial structure factor hAB and separation between the 2C18E12 and DSPC component of the monolayer (δAB) were obtained by combining the reflectivity measurements from (1) d-2C18d-E12/h-DSPC/nrw, (2) h-2C18h-E12/d83-DSPC/nrw, and (3) d-2C18d-E12/d83-DSPC/nrw.29 This allowed the equation describing the profile of neutron specular reflection for each of the monolayers studied, to be written as

R3 )

R1 )

16π2 2 bAhAA(κ) κ2

R2 )

16π2 2 bBhBB(κ) κ2

16π2 2 [bAhAA(κ) + b2BhBB(κ) + 2bAbBhAB(κ)] κ2

(5)

The cross-partial structure factors derived from eq 5 are shown in Figure 5. The separation between the 2C18E12 and DSPC components of the monolayer obtained from manually fitting the curve to the experimental data is presented in Table 3. Figure 2 shows the semilog ln[hAA(κ)] versus κ2 plot used to derive the molecular area and distribution width for the three-component monolayer, the values for which are given in Table 3. Plots obtained (29) Lu, J. R.; Su, T. J.; Lawrence, M. J.; Barlow, D. J.; Warisnoicharoen, W.; Zuberi, T. J. Phys. Chem. B 1999, 103, 4638-4648.

from kinematic analysis of the 2C18E12:DSPC:Chol monolayer, showing the self and cross partial structure factors, are shown in Figure 5. Table 3 provides a summary of the distribution widths and molecular area data obtained from the kinematic analysis of the three-component system. As in the case of the binary mixture, for the purposes of the semilog ln[hAA(κ)] versus κ2 plot (Figure 2), the monolayer consisting of 2C18E12:DSPC:Chol was also treated as being made up of supermolecules consisting of all three components in close association. In contrast with the results observed for the binary mixture, the derived area per molecule for the ternary mixture at 34 mN m-1, of ∼237 Å2 (Table 3), is equal to its theoretical value. If the total A0 of ∼237 Å2 is considered to contain contributions from one part 2C18E12 (A0 of ∼112 Å2) and two parts cholesterol (A0 of ∼40 Å2), then the value obtained for the molecular area of DSPC is ∼45 Å2, which compares well with the value previously obtained using neutron specular reflection.30 This suggests that the components of the ternary mixture are ideally mixed. The distribution width and solvent separation data presented in Table 3 are summarized in the volume fraction profile shown in Figure 6. Although separate distributions of 2C18E12 and DSPC are not shown, the behavior of these two components may be inferred from the distribution width of the three-component monolayer and its relation to the solvent distribution. The σA of 31.5 ( 0.1 Å for the 2C18E12:DSPC:Chol layer corresponds well with the value of 31.3 ( 0.1 Å obtained for the 2C18E12: Chol monolayer at the same surface pressure. However, the solvent distribution width for the three-component monolayer was 2 Å smaller than that found for the 2C18E12: Chol monolayer at 34 mN m-1. The separation between the centers of the solvent and adsorbed film distributions was 1 Å larger for 2C18E12:DSPC:Chol compared with 2C18E12:Chol. These differences in σS and δAS indicate that solvent penetration into the three-component monolayer is lower than in the 2C18E12:Chol monolayer, suggesting a lower level of headgroup-region hydration for the 2C18E12: DSPC:Chol system. The derived δAB value of 2 Å (Figure 5) indicates that, within the 2C18E12:DSPC:Chol monolayer, the DSPC distribution is centered close to the midpoint of the 2C18E12 distribution. As the solvent separation for the 2C18E12:DSPC:Chol monolayer is closer to that of 2C18E12:Chol, this indicates that the zwitterionic lipid headgroups are located below the level of the 2C18E12 headgroup, which would extend further into the subphase. Small-Angle Neutron Scattering. For each of the three different vesicle samples prepared, the SANS data were modeled assuming mixtures of (isolated) infinite planar sheets and one-dimensional paracrystals of these sheets using the method described in Harvey et al.31 The modeled fits to the SANS data were obtained by leastsquares refinement of eight parameters: lamellar thickness (L), Schultz polydispersity (σ(L)/L), roughness (Rσ), number of lamellae (M), mean lamellar separation (d), distribution width (σ(d)/d), and the absolute scale factors for unilamellar and multilamellar vesicles. The leastsquares refinements were performed using the modelfitting routines provided in the FISH software.32 The scattering curves obtained from the three vesicle samples (in D2O) are shown in Figure 7 together with the (30) Hollinshead, C. M. Biophysical characteristics of vesicle-forming surfactants.; University of London: London, 2001. (31) Harvey, R. D.; Heenan, R. K.; Barlow, D. J.; Lawrence, M. J. Chem. Phys. Lipids 2004, in press. (32) Heenan, R. K. The “Fish” reference manual. Data fitting program for small-angle neutron scattering (Revised 2000); Rutherford Appleton Laboratory: Chilton, UK, 1989.

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Figure 5. Plots for self and cross partial structure factor against momentum transfer for monolayers consisting of 1:1:2 molar ratios of 2C18E12, DSPC, and cholesterol at a surface pressure of 34 mN m-1: (A) self partial structure factor hAA(κ) against κ (data fitted assuming a Gaussian profile), (B) cross partial structure factor hAS(κ) against κ, (C) cross partial structure factor hAB(κ) against κ and (D) self partial structure factor hSS(κ) against κ (data fitted assuming a tanh profile). Error bars show the statistical errors calculated from the variance-covariance matrix in the regression analysis.

Figure 6. Volume fraction profiles for the 2C18E12:DSPC:CHOL monolayer and the total of all components at the surface pressure of 34 mN m-1 derived by Gaussian (for 2C18E12 and DSPC) and tanh (for solvent) analysis.

curves obtained from modeling the data as mixtures of planar bilayer and paracrystalline stacks. Table 4 gives a summary of the parameters modeled for each of the experimental samples. The addition of both cholesterol and DSPC to 2C18E12 vesicles has two notable effects on bilayer parameters. First, there is an increase in the bilayer thickness from ∼51 Å for 2C18E12 alone to ∼68 and ∼60 Å for 2C18E12:Chol and 2C18E12:DSPC:Chol vesicles, respectively. Second there is a decrease in the d spacing from ∼154 Å (2C18E12) to ∼132 Å (2C18E12:Chol) and ∼140 Å (2C18E12:DSPC:Chol). The increase in layer thickness upon incorporation of cholesterol (with and without DSPC) appears to be consistent with the results from neutron specular reflection.

Figure 7. SANS data for vesicles of varying composition dispersed in D2O: (A) 2C18E12, (B) 2C18E12:CHOL (1:1), and (C) 2C18E12:DSPC:CHOL (1:1:2). The open circles show experimental data, and the solid lines show the fitted models. Individual curves are offset by an order of magnitude.

Discussion

and fully deuterated forms of 2C18E12 and DSPC but just the hydrogenous form of cholesterol. As a consequence, the effect of sterol inclusion on the ordering of the alkyl chains could not be elucidated directly. However, inferences about the effect of cholesterol on the behavior of the lipid and surfactant molecules may nevertheless be made. In previous neutron reflectivity studies, 2C18E12 monolayers have been shown to exhibit significant intermixing of the PEG headgroups and alkyl chains.18 This phenomenon has also been observed in monolayers of monoalkyl polyoxyethylene ether surfactants12 and has been attributed to the solubility of PEG in alkyl chains.33 Upon the addition of cholesterol and DSPC to 2C18E12, we see an increase in monolayer thickness, indicating an inter-

The partial structure factor analyses for the binary and ternary systems examined here employed the hydrogenous

(33) Elworthy, P. H.; Patel, M. S. J. Pharm. Pharmacol. 1984, 36, 116-117.

Behavioral Effect of Cholesterol and Phospholipid

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Table 4. Structural Parameters for Vesicle Lamellae in D2O Modeled from SANS Data Using FISH as Mixtures of Single Isolated Lamellae and Paracrystalline Stacksa vesicle

layer L (Å)

σ(L)/L

Rσ

M

d (Å)

σ(d)/d

stack: planar bilayer

2C18E12 2C18E12:CHOL (1:1) 2C18E12:DSPC:CHOL (1:1:2)

51 ( 0.3 68 ( 0.3 60 ( 0.4

0.05 0.25 0.21

300 400 543

2 2 2

154 ( 2.5 132 ( 1.7 140 ( 9.0

0.05 0.53 0.29

1:16 1:1 1:28

a

Symbols used are as defined in the text.

calation of sterol and lipid between the surfactant molecules and a reduction of PEG and alkyl chain intermixing. The miscibility of the components in the binary and ternary mixtures studied was assessed by indirect means, using excess area calculations from surface pressurearea isotherms of binary mixtures of amphiphiles. Applying this technique to examine the behavior of ternary mixtures has previously led to difficulties in interpretation.24,26,34 The apparent ideal miscibility of DSPC with both cholesterol and 2C18E12 in binary mixtures is overshadowed by the marked condensing effect of cholesterol on 2C18E12. This condensing effect of cholesterol on 2C18E12 is also evident from the areas per molecule obtained from analysis of the neutron reflectivity results, wherein the components of the binary mixture were considered as one supermolecule. The observed ideal mixing between 2C18E12 and DSPC differs from the results of a previous study, which found that monolayers of DSPC with up to 10 mol% pegylated distearoylphosphatidylethanolamine (DSPC:DSPE.MPEG2000) mixed nonideally.35 However, this may be attributed to the difference in the length of the PEG headgroups of 2C18E12 and DSPE.MPEG2000. Analysis of the neutron reflectivity results for the ternary 2C18E12: DSPC:Chol mixture treating the three components as a single supermolecule gave an area per molecule which was consistent with the three components being ideally mixed but does not preclude the existence of phaseseparated DSPC:Chol and 2C18E12:Chol domains. Given the apparent interaction between 2C18E12 and Chol, the presence of phase separated domains in the ternary mixture monolayer cannot be discounted and may be formed in a similar way to lipid rafts present in biological membranes.36 Interaction between 2C18E12 and Chol may also provide an explanation for the observed thickness changes between the pure surfactant and binary mixed monolayers. Neutron reflectivity studies have suggested that the tilt of the alkyl chains in 2C18E12 monolayers is ∼75° from the normal.18 A similar situation is found in monolayers of mono-alkyl pegylated surfactants, for which the addition of a perpendicularly oriented oil (dodecane), was found to push the alkyl chains toward the surface normal.9 The ordering effect of >30 mol% cholesterol on DSPC monolayers is well established: the presence of cholesterol eliminates the tilt of the lipid acyl chains.37 This phenomenon may result from a combination of factors, such as the perpendicular orientation of cholesterol at all surface pressures11 and the likelihood of there being strong attractive van der Waals forces between the long acyl chains of the lipid and sterol.38 Cholesterol may exert a similar effect upon the alkyl chains of 2C18E12, especially at high concentration. This hypothesis is also supported (34) Maloney, K. M.; Grainger, D. W. Chem. Phys. Lipids 1993, 65, 31-42. (35) Chou, T.-H.; Chu, I.-M. Colloid Surf. B 2003, 27, 333-344. (36) Silvius, J. R. Biochim. Biophys. Acta 2003, 1610, 174-183. (37) McIntosh, T. J. Biochim. Biophys. Acta 1978, 513, 43-58. (38) Slotte, J. P. Biochim. Biophys. Acta 1995, 1238, 118-126.

by the observation that the interaction between cholesterol and ether-linked alkyl chains is no different from its interaction with ester-linked acyl chains.39 The attractive forces which may, therefore, exist between the alkyl chains of 2C18E12 and cholesterol, the presence of which is indicated by the observed condensing effect, may help to decrease the chain tilt of the surfactant. The additional incorporation of DSPC into monolayers with 2C18E12 and cholesterol effectively decreases the grafting density of the PEG headgroups. The thickness of the monolayer may be expected to decrease,40 therefore, as the polymer chains have less interaction with each other and thus may protrude from the interface to a lesser degree.41 This decrease in monolayer thickness, effected by a more random conformation of polymer, may be compensated for in the three-component monolayer by an increased ordering of the alkyl chains. This provides an explanation for the observation that the layer thickness for the 2C18E12:DSPC:Chol monolayer is equal to that of the 2C18E12:Chol layer at 34 mN m-1 while the hydration of the three component system appears to be lower, as evidenced by the differences in their solvent distribution widths. The suggested ideal mixing of the components of the 2C18E12:DSPC:Chol monolayer may therefore lead to a closer packing and an improved ordering in this system, compared with that found in the 2C18E12:Chol monolayer. The effect of cholesterol and DSPC incorporation on the behavior of 2C18E12 in vesicle bilayers, as studied by SANS, may be deduced from the modeled changes in bilayer parameters. Although changes in bilayer thickness could be modeled from the SANS data, which are in some ways comparable with those observed from the reflectivity experiments, these cannot be discussed in isolation from the modeled bilayer separations. The separation between 2C18E12 bilayers in D2O, determined by SANS, was in the order of 100 Å2. Despite the dense packing and therefore restricted movement of the PEG chains in 2C18E12, which has been considered to weaken repulsive steric forces,42 this is a relatively large separation. The steric forces responsible for maintaining bilayer separation in systems consisting solely of amphiphiles with short PEG chains40 may contain a large contribution from undulation forces known to be prevalent between “rough” surfaces.43 In the case of 2C18E12 bilayers, the roughness is explained by out-of-plane protrusions of the surfactant molecules. This highly disordered surfactant bilayer may also provide an explanation for increased bilayer permeability. Out-ofplane protrusions of lipid molecules in monolayers is known to decrease the thickness and increases the disorder of the hydrophobic layer.40 In lipid bilayers, decreasing (39) Bittman, R.; Clejan, S.; Lund-Katz, S.; Phillips, M. C. Biochim. Biophys. Acta 1984, 772, 117-126. (40) Kuhl, T. L.; Majewski, J.; Howes, P. B.; Kjaer, K.; von Nahmen, A.; Lee, K. Y. C.; Ocko, B.; Israelachvili, J. N.; Smith, G. S. J. Am. Chem. Soc. 1999, 121, 7682-7688. (41) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Isaelachvilli, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101, 3122-3129. (42) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171-199. (43) Israelachvili, J. Intermolecular and surface forces., 2nd ed.; Academic Press: London, 1991.

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hydrophobe layer thickness and packing discontinuities introduced by increased disordering of the hydrophobic chains contribute to a reduced ability to act as a permeability barrier.44,45 For 2C18E12 vesicles, the incorporation of 50 mol% cholesterol increased bilayer thickness by ∼17 Å. This increase in bilayer thickness may be due either to a decrease in chain tilt, an apparent increase in headgroup length, or a combination of the two. The previously reported intermixing between the PEG headgroup and the hydrophobe in 2C18E12 bilayers46 may therefore be reduced in the presence of cholesterol via exclusion of EO units from the hydrophobic core of the bilayer. If, as has been suggested by Kuhl et al.,40 out-of-plane protrusions in pegylated lipids reduce lateral repulsive interactions between PEG chains, it seems reasonable to assume that the ordering of the bilayer imposed by cholesterol may reduce the magnitude of undulation forces between adjacent bilayers. This proposition is supported by the observed changes in the SANS model for 2C18E12 vesicles, wherein bilayer separation decreases from 103 to 64 Å upon addition of 50 mol% cholesterol. However, the condensing effect of cholesterol on 2C18E12 observed from the monolayer experiments reveals nothing about the nature of the binary mixture and does not exclude the presence of surfactant-rich domains, in which packing (44) New, R. R. C. In Liposomes As Tools in Basic Research and Industry; Philippot, J. R., Schuber, F., Eds.; CRC Press: Boca Raton, FL, 1995. (45) Cevc, G. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993; pp 639-661. (46) Ma, G.; Barlow, D. J.; Lawrence, M. J.; Heenan, R. K.; Timmins, P. J. Phys. Chem. B 2000, 104, 9081-9085.

Harvey et al.

flaws may still exist. Packing flaws present in surfactantrich domains of 2C18E12:Chol vesicle bilayers would allow hydrophobic interactions to emerge between adjacent bilayers,43 thus reducing their separation. The SANS studies also showed that 2C18E12:DSPC:Chol vesicles exhibited a bilayer separation of ∼20 Å greater than that seen in 2C18E12:Chol vesicles, which adds weight to the proposition that bilayers of the binary surfactantsterol mixture contain packing flaws. The thickness of 2C18E12:DSPC:Chol vesicle bilayers was ∼8 Å less than that of 2C18E12:Chol. Although a decrease in polymer grafting density should allow for a reduced steric repulsion between the layers,43 this is not consistent with the ∼80 Å bilayer separation obtained for 2C18E12:DSPC:Chol vesicles. It may therefore be concluded that the decrease in bilayer thickness is due to the 50% reduction in PEG grafting density in the 2C18E12:DSPC:Chol vesicles, allowing greater freedom of mobility for the 2C18E12 polymer headgroups. The increased bilayer separation of 2C18E12: DSPC:Chol vesicles may result from further improved bilayer packing, leading to a reduction in interbilayer hydrophobic interactions. With respect to the use of such vesicles in drug delivery, the addition of cholesterol and DSPC would also serve to reduce bilayer permeability to entrapped polar drug molecules. Whether the resultant reduction in polymer grafting density will compromise the theoretical long-circulating properties of such a drug vector is a subject for further investigation. Acknowledgment. R.D.H. was financially supported by the BBSRC (UK). LA0400297