Article pubs.acs.org/Langmuir
Interactions between Phospholipids and Organic Phases: Insights into Lipoproteins and Nanoemulsions Ellen Hildebrandt,†,‡ Alberto Dessy,‡ Jan-Hendrik Sommerling,†,‡ Gisela Guthausen,† Hermann Nirschl,† and Gero Leneweit*,‡,§ †
Karlsruhe Institute of Technology (KIT), Institute of Mechanical Process Engineering and Mechanics, 76131 Karlsruhe, Germany Abnoba GmbH, 75177 Pforzheim, Germany § Carl Gustav Carus-Institute, Association for the Promotion of Cancer Therapy, 75223 Niefern-Oeschelbronn, Germany ‡
S Supporting Information *
ABSTRACT: The adsorption of phosphatidylcholines (PCs), dissolved in squalene or squalane as an organic phase, was studied at the interface with water. Using profile analysis tensiometry, the equilibrium adsorption isotherms, minimum molecular interfacial areas, and solubility limits were derived. For squalene, differences in PC solubility and interfacial adsorption were found, depending on PC saturation. Compared to saturated PCs, unsaturated PCs showed a 3fold-lower interfacial density but up to a 28-fold-higher critical aggregation concentration (CAC). In addition, the solubility limit of unsaturated PC in squalene and in its saturated form squalane diverged by a factor of 739. These findings provided evidence for steric repulsion or π−π interactions of π bonds in both solvent and solute or both effects acting complementarily. In squalane, low solubilities but high interfacial densities were found for all investigated PCs. Changes in fatty acid chain lengths showed that the influence of the increases in entropy and enthalpy on solubility is much smaller than solvent/solute interactions. Oxidation products of squalene lowered the interfacial tension, but increasing concentrations of PC expelled them from the interface. The CAC of saturated PC was increased by oxidation products of squalene whereas that of unsaturated PCs was not. Our findings indicate that the oxidation of triglycerides in lipoprotein cores can lead to increased solubility of saturated phospholipids covering the lipoproteins, contributing to destabilization, coalescence, and terminally the formation of atherosclerotic plaques. The consideration of solvent/solute interactions in molecular modeling may contribute to the interfacial tension and the corresponding kinetic or thermodynamic stability of lipoproteins. Measured areas per molecule prove that PCs form monolayers of different interfacial densities at the squalene/water interface but multilayers at the squalane/water interface. These findings showed that combinations of solvent or solute saturation affect the outcome for nanoemulsions forming either expanded or condensed monolayers or multilayers.
1. INTRODUCTION
established and well-accepted method for the characterization of liquid interfaces.7−11 However, the adsorption of PCs at oil/ water interfaces is still not fully understood, considering its fundamental importance for medical nanoemulsions, lipoproteins, and biotechnologies. Fundamental studies of adsorption phenomena with phospholipids such as DPPC, DMPE, and EPC exist7−11 but were performed with organic phases not relevant for pharmaceutical emulsions. In contrast, Miglyol, squalene, and plant oils such as soybean oil, sesame oil, cottonseed oil, and castor oil have been successfully applied for injectable drugs and dietary supplementation formulations12 with emulsifiers EPC, POPC, DOPC, DSPC, DPPC, DMPC, and DLPC,13 although interfacial tension studies do not exist.
Phospholipids are the key emulsifying components for milk fat globules and fat digestion including transport and metabolism, especially by chylomicrons and lipoproteins.1−3 Moreover, phospholipids are among the most commonly used emulsifiers in dietary, cosmetic, and pharmaceutical products where the nanometric structure essentially contributes to the product’s characteristics and functionalities. The engineering of asymmetric vesicles4,5 and applications in digital microfluidics6 requires oil and water emulsified by phospholipids. Classically, the adsorption of phospholipids is studied at the air/water surface by a Langmuir film balance. More recently, the development of drop profile analysis tensiometry (PAT) and liquid/liquid Langmuir troughs stimulated phospholipid studies at the chloroform/water,7,8 1,2-dichloroethane/water,9 ndodecane/water,10 and triolein/water11 interfaces. Today, the measurement of dynamic interfacial tension with PAT is an © XXXX American Chemical Society
Received: March 11, 2016 Revised: May 4, 2016
A
DOI: 10.1021/acs.langmuir.6b00978 Langmuir XXXX, XXX, XXX−XXX
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discussed. Finally, consequences for nanoemulsions using phospholipids as emulsifiers are considered regarding the density of adsorbed interfacial layers.
Studies of interfacial tension with pharmaceutically relevant oils are therefore needed to elucidate the adsorption mechanisms involved. For the preparation of nanoemulsions, especially for pharmaceuticals, the choice of a suitable oil phase is, besides the hydrophilic−lipophilic balance (HLB) and the concentration of emulsifying components, one critical parameter in optimizing their stability. Squalene is a triterpene hydrocarbon that is extracted from shark liver or natural sources such as olive oil. It is often used for parental vaccine adjuvant emulsions and drug delivery.13−15 However, it is known that squalene has different natural impurities depending on plant or animal sources.16 Squalene is a main component of human sebum17 and a precursor of cholesterol biosynthesis.18 Because of its six double bonds, squalene is highly sensitive to chemical degradation caused by oxygen, ultraviolet A radiation, and ozone.19,20 Squalene is commonly used for emulsion-based preparations because of its very low viscosity and high surface tension compared to those of other vegetable, animal, and mineral oils.14 Its hydrogenated form, squalane, is frequently used for topical pharmaceutical formulations of ointments and creams. However, the interactions of both squalene or squalane with emulsifying components, especially phospholipids, have so far not been discussed intensively. For oily compounds used in medical applications, comprehensive descriptions of the interfacial properties and emulsifying mechanisms are still lacking. In general, the interfacial tension and surfaces pressure for monolayers formed at oil/water interfaces with physiologically relevant lipid compositions are an important issue, e.g., for fat metabolism. In lipoproteins and chylomicrons, phospholipids and cholesterol emulsify mostly triglycerides and cholesterol esters, but this natural mixture would be too complex to elucidate molecular interactions. Therefore, squalene and squalane are chosen instead of triglycerides because of their chemical simplicity as pure saturated and unsaturated hydrocarbons. This allows us to model specific interactions between the phospholipid monolayer and the hydrophobic core of lipoproteins, especially the saturated and unsaturated fatty acids of triglycerides. The choice of this model allows us to contribute to the understanding of some fundamental properties of phospholipid emulsions, especially lipoproteins. This study shows how the oxidation of unsaturated hydrocarbons influences their emulsification, which is investigated by the oxidation of squalene in the present work. Therefore, a comparison between squalene with and without oxidation products is performed in order to identify their respective adsorption behavior and monolayer formation at an oil−water interface. The physical presence of oxidation products was investigated using PAT, gas chromatography (GC), and nuclear magnetic resonance (NMR) spectroscopy. The effect of oil oxidation on the stability of the emulsifying phospholipid monolayer of lipoproteins is also considered. Furthermore, the influence of saturation or unsaturation of the dissolving organic bulk phase and the phospholipid’s fatty acids are analyzed with respect to the interfacial layer density, i.e., the area per phospholipid molecule. Adsorption isotherms are used to quantify the solubility limits by an identification of the critical aggregation concentrations (CACs), which reveal the interactions between the solvent and the solute. On the basis of the biophysical interactions of phospholipids with organic phases, the properties of lipoproteins such as their size, surface coverage with phospholipids, stability, and destabilization are
2. EXPERIMENTAL SECTION 2.1. Materials. Phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) were provided by Lipoid (Ludwigshafen, Germany). The lipids were received in powder form and dissolved in either squalene or squalane to prepare stock solutions. Squalene with a purity of ≥98% and squalane with a purity of >99% were obtained from Sigma-Aldrich (Taufkirchen, Germany), as well as hydrogen peroxide (30% H2O2). Activated magnesium silicate (MgSiO3, 60−100 mesh), used as adsorbent for the purification of squalene, was acquired from Merck (Darmstadt, Germany). Highly purified water (for HPLC application) was purchased from VWR International (Darmstadt, Germany). 2.2. Oil-Phase Preparation. The dissolution of phospholipids in the oil phase was prepared with energetic input using a Sonorex RK 514 Transistor ultrasonic water bath from Bandelin Electronics (Berlin, Germany). By photon correlation spectroscopy (PCS), using the Zetasizer Nano ZS90 from Malvern Instruments (Worcestershire, U.K.), the dissolution of the initial phospholipid aggregates, most likely inverted micelles, was monitored. In the final organic bulk phase with dissolved phospholipids, all phospholipid particles had to be eliminated. Therefore, the size and number of measured particles had to be identical to those of the pure oil phase as a control, i.e., allowing particle sizes ≤30 nm (z average) and a derived count rate of ≤5 kcounts per second, as already shown for POPC in squalene.21 When squalene is used as purchased, it is referred to as nonpurified squalene (Sqen.p.). Squalene was purified (Sqep.) by incubation with 2 g of MgSiO3 in five repetitive cycles. For more details on purification and induced oxidation, see Supporting Information. 2.3. Tensiometry. The dynamic interfacial tension was measured with the PAT-1 drop shape tensiometer from Sinterface Technologies (Berlin, Germany). The lipid concentration was varied in the organic phase to examine the adsorbed monolayers at liquid−liquid interfaces. To regulate the bulk concentration of the phospholipid exactly, 3 mL of an oil phase was used as the surrounding phase with the water phase, forming a pendent drop of a volume of typically 20−30 μL. This configuration was proven to be more accurate compared to a buoyant oil drop in water in order to avoid a depletion of the phospholipid bulk concentration in oil by diffusion to the aqueous phase.22 The applicability of our tensiometric data of millimeter-sized water in oil drops for the interpretation of nanometer-sized oil in water drops can be justified by the weak dependence of curvature for droplet radii >5 nm (Ollila et al.2) because interfacial properties of nanoscopic liquid drops are affected only by curvature for radii of nanoemulsions and lipoproteins ≤60 σ, where σ is the characteristic length of the Lennard-Jones potential.23 Adsorption isotherms of POPC, DPPC, and DMPC were measured at 20.0 ± 0.1 °C with a thermostatically controlled measuring chamber in an air-conditioned laboratory. The validity of the optical calibration was confirmed before each measurement series by testing the surface tension of pure liquids in air. With values of 72.7 mN/m for water/air, 31.7 mN/m for squalene/air, and 28.8 mN/m for squalane/air, surface tensions were obtained in concordance with the literature after ≥2000 s and 20 °C.24 Equilibrium interfacial tensions γe were extrapolated from measured data in a γ(1/√t) plot for t → ∞ using the long-time approximation for diffusion-controlled adsorption25 given by the Hansen−Joos equation
⎡ dγ ⎤ RT Γ 2 = ⎢ ⎥ ⎣ d(1/ t ) ⎦t →∞ c0
π 4D
(1)
where γ(t) is the dynamic interfacial tension, R is the gas constant, T is the absolute temperature, Γ is the interfacial concentration, c0 is the B
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Langmuir Table 1. Overview of Interfacial Tension at Oil/Water Interfacesa γ (0) ± SD (mN/m)
sample
oil
A B C D E F G H
Sqen.p. Sqep. Sqep. + arg (7 d) + RT Sqep. + arg (16 m) + 4 °C Sqep. + 0.2% H2O2 Sqep.+ 2% H2O2 Sqep. + air (21 m) + RT Sqan.p.
23.9 47.4 46.2 47.2 45.3 43.8 7.4 46.1
± ± ± ± ± ± ± ±
γ (2000) ± SD (mN/m)
0.7 1.3 0.3 1.6 0.3 1.8 0.01 1.7
17.0 46.7 42.1 39.5 38.3 36.8 5.9 44.6
± ± ± ± ± ± ± ±
0.8 0.8 0.8 1.2 0.4 2.5 0.03 1.8
γe ± SD (mN/m)
n
Δγ/Δt μN/(m s)
± ± ± ± ± ± ± ±
3 3 2 3 2 2 3 3
3.45 0.35 2.65 3.93 4.55 5.30 20.74 0.75
15.3 46.6 39.5 36.7 36.1 35.1 5.5 44.2
1.1 1.1 1.1 0.9 0.5 1.9 0.05 1.5
The mean values of the interfacial tension at time point zero γ(0) and after 2000 s, γ(2000), as well as γe were determined from n independent PAT measurements. The respective standard deviations (SD) are also shown.
a
Figure 1. Profile of a drop for tensiometry analysis to model adsorption of surface-active compounds. (a) Real image of a pendent water drop in a squalene environment for profile analysis tensiometry (PAT). (b) Schematic drawing of a monolayer of phospholipids at the water/squalene interface. (c) Hypothetical multilayer formation at the interface of water and squalane. bulk concentration of the emulsifier (phospholipid), and D is the diffusion coefficient. By use of the Gibbs adsorption isotherm, Γ is given by the following equation:
Γ=−
c0 ⎛ ∂γ ⎞ ⎜ ⎟ RT ⎝ ∂c0 ⎠
=− p,T
1 ⎛ ∂γ ⎞ ⎜ ⎟ RT ⎝ ∂ ln c0 ⎠
p,T
at the interface and lower the surface tension. For that reason, we purified squalene with magnesium silicate in order to remove the surface-active compounds in five cycles using a method described as a single-step procedure.27,28 Our results, however, show that a single-step purification is insufficient for squalene whereas five cleaning cycles lead to γe = 46.6 ± 1.1 mN/m and only a marginal averaged reduction of γ(t) in time of Δγ/Δt = 0.35 μN/(m s). This proves the stepwise cleaning procedure of our protocol to be adequate in order to remove practically all surface-active impurities from squalene, as illustrated in Figure S1 and summarized in Table S1 in the Supporting Information. GC measurements were performed in order to quantify the amount of contamination before and after the purification process (shown in Figure S2 in the Supporting Information). The highly purified squalene (Sqep.) allows the formation of an interfacial monolayer of added phospholipids to be characterized in appropriate accuracy. 3.2. Oxidation of Squalene. The storage stability of Sqep. was proven to be limited by oxidation as shown in Table 1. Squalene was tested as sample A as purchased, sample B after purification, and under diverse conditions applied to purified squalene, which are listed as follows: (C) 1 week of storage in an argon atmosphere at room temperature (RT); (D) 16 months of storage under an argon atmosphere and cool conditions (4 °C); (E) after incubation for 18 h with 0.2% H2O2; (F) after incubation for 18 h with 2% H2O2; and (G) a 21-month-old squalene sample, stored under nonoxygenavoiding conditions (air, at RT). Nonpurified squalane was measured additionally (sample H). Storage under oxygenavoiding conditions (argon atmosphere) but at room temperature leads to an averaged decrease rate of interfacial tension Δγ/Δt = 2.65 μN/(m s) in a testing period of 2000 s (sample C), as presented in Table 1 (third row). Contamination from the storage container was excluded because different materials (heat-cleaned glassware and polypropylene) lead to equal results. Measurements of long-time storage (16 months) of
(2)
By the use of eq 2, Γ was derived from experimental data in a γ vs log c0 diagram that allows us to determine the gradients of interfacial tension with respect to bulk concentration c0, i.e., ∂γ/γ ln c0. The minimum surface area per lipid molecule A was calculated by the relation
A=
1 NΓ
(3)
where N is Avogadro’s number. 2.4. Other Techniques. For a detailed description of squalene purification and GC and NMR spectroscopy, see the Supporting Information.
3. RESULTS AND DISCUSSION 3.1. Interfacial Tension between Water and Squalene. The dynamic interfacial tension γ = γ(t) of Sqen.p. is detected initially at 23.9 mN/m, decreases to 17 mN/m after 2000 s, and approaches an equilibrium of γe = 15.3 mN/m, as shown in Table 1 (sample A). The measurement setup of a pendent drop used for profile analysis tensiometry is visualized in Figure 1a, and an idealized monolayer is illustrated in Figure 1b. In contrast to the experimentally obtained results, the theoretically expected interfacial tension γc for pure squalene was calculated with a Gibbsian model by Marmur and Valal,26 and an empirical correlation (according to eq 25) is based on two different sets of experimental data: γc = 47.6 mN/m derived from all determined data or 53.1 mN/m derived by using parameters from only three liquid groups. This comparison shows that nonpurified squalene contains a significant quantity of surface-active contaminants that adsorb C
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Langmuir squalene under an argon atmosphere at 4 °C proved to be adequate to decrease oxidation. After 16 month of storage, the decrease rate is 3.93 μN/(m s). Chemical oxidation by exposure to 0.2 and 2% H2O2 over 18 h leads to average decrease rates of Δγ/Δt = 4.55 and 5.3 μN/(m s), respectively, over 2000 s of interfacial tension measurement. Measurements of long-time-stored squalene under nonappropriate conditions (air, at RT) lead after 21 months to a decrease rate of 20.74 μN/(m s) with the 2000 s of measurement (sample G). These results prove that an induced oxidation of squalene leads to an increased reduction of interfacial tension that consequently displays an increased number of surface-active compounds. Petrick and Dubowski identified the oxidation products of squalene after exposition to indoor air at room temperature.20 Oxidation leads to the formation of surface-active products (6methyl-5-hepten-2-one, geranyl acetone, and long-chain ketones and aldehydes) as well as gas-phase products (formaldehyde, acetone, 4-oxopentanal, glyoxal, and pyruvic acid). Owing to the polarity of the CO double bonds in all ketones and aldehydes, the oxidation products are surfaceactive, comparable to the effects at the triolein/water interface studied by Mitsche et al.11 They showed that triolein is surfaceactive at low film pressures at the air/water interface but forms a bulk phase (oil lens) at film pressures higher than the collapse film pressure (>12.1 mN/m). Analogously, we conclude that the surface-active oxidation products of squalene cause a decrease in the interfacial tension of a pure squalene/water interface from 47 down to about 5 mN/m (Table 1). The concentration of phospholipids in the oil phase correlates with the displacement of the oxidation products from the interfacial monolayer. To elucidate the respective effects of the oxidation products and phospholipids on the interfacial tension and monolayer formation, we performed all measurements with both Sqep.and Sqen.p. for comparison. Squalene and its derivatives were studied by Pogliani et al.29 and Naziri et al.30 To characterize the oxidation of squalene and gain insight into the process of oxidation (or aging31), NMR was applied. NMR experiments allowed us to monitor the formation of squalene oxidation products and a chemical shift in the double-bond region. NMR provides clear evidence that oxidation processes occur when oxidation is not avoided, e.g., sample G in Table 1 and Figure S4 in Supporting Information. Because no other chemical groups or degradations were identified by NMR in samples A−G, we conclude that the increased surface activity of samples A and C to G is exclusively caused by oxidation. 3.3. Isotherms of POPC and DPPC at the Squalene/ Water Interface. The adsorption of POPC and DPPC is studied at the squalene/water interface with Sqep. and Sqen.p.. For the determination of adsorption isotherms, equilibrium interfacial tensions are used and are presented in Figure 2. From the slopes of the Gibbs adsorption isotherms (thin lines), the minimum area per molecule at the CAC was derived according to eqs 2 and 3. The CAC of POPC is almost identical in both squalene qualities, i.e., 1.256 mM in Sqep. and 1.259 mM in Sqen.p.. In the case of DPPC, the CAC is 0.045 mM in Sqep. (Hildebrandt et al.22) and 0.103 mM in Sqen.p.. The areas per molecule for POPC and DPPC are 132 and 42 Å2 in Sqep.(22) and 119 and 44 Å2 in Sqen.p., respectively. Irrespective of squalene’s purity, the isotherm of each phospholipid has almost the same slope, and as a result, the respective minimum areas per molecule are almost identical. The large differences between the areas per molecule and the CAC of POPC and
Figure 2. Equilibrium interfacial tension γe as a function of initial bulk concentration c0 of (a) POPC and (b) DPPC in squalene. The extrapolated interfacial tension data with error bars of the standard deviation (SD) for ≥2 independent measurements are shown for water droplets in (■) nonpurified squalene (Sqen.p.) and (○) purified squalene (Sqep.). Solid black lines: Gibbs adsorption isotherm according to eq 2. Broken gray vertical lines: CAC, i.e., the intersection of the adsorption isotherm with the lowest interfacial tensions.
DPPC are regarded as a consequence of the difference in fatty acid saturation. Comparing the adsorption isotherms of POPC for both degrees of purity of squalene, it can be stated that they match almost exactly. As a consequence, the interfacial density of all surface-active agents at the Sqen.p./water interface must be practically identical to that formed by POPC at an Sqep./water interface. This means that POPC completely expels the impurities for all concentrations as long as its own film pressure equals or exceeds the film pressure of the maximum adsorption density of the impurities. In contrast to POPC, the adsorption isotherm of DPPC in Sqep. and Sqen.p. are identical only with respect to the slope of the curves but differ by approximately a factor of 2.3 regarding the CAC. As a consequence, for Sqen.p. a 2.3 times higher bulk concentration of DPPC is necessary to achieve the same surface activity as is obtained by DPPC in Sqep.. There is evidence of attractive interactions between DPPC and the impurities that enhance the solubility limit. The CAC of POPC differs from that of DPPC by factors of 27.9 and 12.2 for the Sqep. and Sqen.p. oil phases, respectively. Given the fact that POPC and DPPC are identical in their hydrophilic headgroup and differ only in one of their two fatty acids, differences in the minimum interfacial tension can be attributed only to the CC double bond in the oleoyl chain. This difference can lead to higher attractive interactions between POPC and squalene or to higher repulsive interactions between DPPC and squalene or even both effects acting complementarily. The hypothetical attractive interactions are illustrated in a model oil drop in Figure 3, which demonstrates the consequence of an D
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3.5. Interfacial Density at Squalene/Water Interfaces. The minimum interfacial tension in the equilibrium of POPC is γe < 1 mN/m in both Sqep. and Sqen.p., whereas for DPPC, it is γe ≥ 2 mN/m in purified and γe ≥ 5 mN/m in nonpurified squalene (Figure 2). This result for an adsorbed monolayer is in contrast to the findings for spread monolayers at the air/water interface.33 There, POPC collapses at a surface tension of γ ≈ 28 mN/m, whereas DPPC collapses at only γ ≈ 22 mN/m. The minimum area per molecule of 42 Å2 for DPPC at the interface of purified squalene with water is in reasonable agreement with that of DPPC found at the air/water interface. For pure DPPC at the air/water interface, Jurak and Conde measured33 an area per molecule of 46 Å2 at the collapse film pressure of Πmax ≈ 50 mN/m. Distearoylphosphatidylcholine (DSPC) with its fatty acids having 18 C atoms and no double bonds shows a minimum area per molecule of 40 Å2 at the 1,2-dichloroethane/ water interface.9 A comparison of the cited results with the adsorption layer of DPPC at the squalene/water interface and its molecular area of 42 Å2 per molecule at a film pressure of Π ≈ 40−42 mN/m proves a reasonable similarity of both molecular area and film pressure. But why is the POPC adsorption layer at the Sqep./water interface so different from a compressed monolayer at the air/ water interface? The minimum area per molecule of POPC adsorption layers at the purified squalene/water interface is 119 Å2 at a maximum film pressure of Π ≈ 45 mN/m. Our results are in contrast to an area of 60 Å2 at the air/water interface determined at the collapse film pressure of Πmax ≈ 43 mN/m on a Langmuir trough.33 This comparison shows that adsorbed monolayers of phospholipids of (partially) unsaturated fatty acids differ much more from compressed monolayers at the air/ water interface than those of saturated fatty acids such as DPPC. Obviously, POPC reaches higher film pressures at the squalene/water interface by occupying a 2-fold-higher area per molecule compared to a compressed monolayer. This is interpreted as a consequence of attractive interactions. 3.6. Interfacial Behavior of PCs at the Squalane/Water Interface. The dynamic interfacial tension of water droplets in squalane is initially 46.1 mN/m, decreases to 44.6 mN/m after 2000 s, and approaches an equilibrium of 44.2 mN/m (Table 1). Thus, squalane contains many fewer impurities than does squalene without any purification step and is not subject to auto-oxidation. Applying the approach by Marmur and Valal,26 we determined the theoretical expectations of the interfacial tension for a squalane/water interface to be 48.9 mN/m (derived by all data) and 52.5 mN/m (derived by using parameters from only three liquid groups), in good agreement with our measured data for nonpurified squalane (Sqan.p.). The adsorption isotherms obtained for water droplets in a squalane environment with phospholipids POPC, DPPC, and DMPC are presented in Figure 4. The CAC is found to be 0.0017, 0.0322,22 and 0.0907 mM for POPC, DPPC, and DMPC, respectively. Unlike the results in squalene, the slopes of the adsorption isotherms of all three phospholipids are almost equal. Using the Gibbs adsorption isotherm (eq 2), the minimal area per lipid molecule is found to be 27, 25,22 and 23 Å2 per molecule for POPC, DPPC, and DMPC. Apparently, the area per molecule given by the Gibbs adsorption isotherm is much lower than the cross-sectional area of the adsorbed phospholipids, which is estimated to be around 36 Å2/molecule as confirmed by crystallography and molecular modeling (Saupe et al.34). Shchipunov and Kolpakov found results35 similar to ours and concluded that phospholipids form not only
Figure 3. Model of a lipoprotein or oil droplet with adsorbed and dissolved phospholipids whose bulk concentration is either below the CAC (left hemisphere) or above the CAC (right hemisphere). The applicability of our tensiometric data of millimeter-sized water in oil drops for the interpretation of nanometer-sized oil in water drops is described in section 2.3. The different concentration ranges lead to either expanded or condensed monolayer coverage. (a) POPC with hypothetical π−π interactions between unsaturated CC double bonds, indicated by the frame in the drop and its enlargement above. (b) DPPC with weaker π−π interactions by PO and CO π bonds.
interaction-mediated solubility on the interfacial density. The oil droplets with POPC (Figure 3a) and DPPC (Figure 3b) are presented in combination with the adsorption isotherms, indicating the different concentration ranges for a full monolayer coverage. Section 3.4 will discuss the hypothesis of attractive interactions, and the hypothesis of repulsive interactions is discussed in section 3.7. 3.4. π−π Interactions. The sole qualitative difference between phospholipids DPPC and POPC is the double bond or π bond (CC) in the oleoyl chain of POPC. Because pure squalene possesses six double bonds, this provides evidence that the π bonds of POPC and squalene interact by π−π interactions as sketched in Figure 3 and in the table of contents graphic (TOC). Sherrill provided an overview of numerical techniques to quantify π−π interactions.32 However, theoretical research has not yet achieved specific results applicable to the π−π interactions of squalene with different phospholipids to account for differences in solubility and CAC. Therefore, the empirical results on the CAC of phospholipids with or without CC π bonds dissolved in organic oils with or without π bonds (squalene vs squalane) will be used for a quantitative estimate on the effect of π−π interactions on solubility in section 3.7. E
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POPC is 18.9 times lower than that of DPPC, which does not consist of any CC bonds but like POPC contains two CO double bonds and one PO double bond. In squalene, POPC is 27.9 times more soluble than DPPC. This indicates that the CO and PO bonds also contribute to the π−π interactions with the squalene solvent but do not impede their solubility in squalane. The CAC of DPPC was reported by Li et al. to be about 0.03 mM in chloroform, a common solvent for phospholipids.7 Compared to the CAC of DPPC in squalane, which is 0.0322 mM, this indicates that squalane has similar dissolution properties for phospholipids with saturated fatty acids. Comparing the CAC of DPPC in squalane with that of DMPC, it becomes obvious that the shorter chain lengths of the fatty acids of DMPC (14:0, 14:0) lead to an increase in the solubility limit by a factor of 2.8. This demonstrates the influence of both entropy and enthalpy when comparing phospholipids of saturated fatty acids but different chain lengths. The increase in entropy can be attributed to the higher diffusivity, and the gain in enthalpy, to the stronger van der Waals interactions between solute and solvent. 3.7. Relative Effects of Interactions for Solubility Limits of Phospholipids. A comparison of the different solubility limits of saturated and unsaturated phospholipids in saturated or unsaturated hydrocarbons offers the possibility to quantify the effects using a relative scale. Regarding the reference of the scaling, it is most suitable to choose the solubility of a saturated phospholipid in a saturated hydrocarbon because this excludes all repulsive or attractive effects. Figure 5 shows a comparison of the relative CAC values expressed as ratios of the CAC of DPPC in squalane. Figure 5 allows us to assess the relative intensity of the different potential effects that are indicated by numbers in circles and briefly listed in the captions of the figure. As can be seen in Figure 5, ① indicates a repulsive effect between the solvent and solute with different degrees of order, which could explain the lower solubility of POPC in squalane (first column) compared to that of DPPC and DMPC (second and third columns). In water, steric repulsion stemming from entropic and enthalpic effects is well-known.37 In organic solvents, however, the existence of such effects would need further investigation. Another effect that could increase the solubility of unsaturated phospholipids in their solvent originates from the dynamic properties of the molecular structure: the C−C bonds that link the cis-locked double bonds with the methylene carbons have
Figure 4. Equilibrium interfacial tension γe as a function of bulk concentration c0 of POPC, DPPC and DMPC in squalane. The extrapolated interfacial tension data with the standard deviation for ≥2 independent measurements are shown for (■) POPC, (●) DPPC, and (▲) DMPC. Solid lines according to eq 2. Broken gray vertical lines indicate the CAC, as shown in Figure 2.
monomolecular layers at oil/water interfaces but in some cases also form multilayers at oil/water interfaces consisting of several monolayers, at least with a thickness of three monolayers.35 This hypothesis of complex structures being formed at oil/water interfaces is supported by tensiometry and X-ray scattering summarized by Schlossman and Tikhonov.36 They showed that alkanols can form multilayers at alkane/ water interfaces36 of two, three, or four monomolecular layers. Given the very low solubility of the tested phospholipids in squalane, we conclude that the multilayer has an odd number of monomolecular layers. In a piled interfacial structure of three layers with a combined interfacial area of three phospholipid molecules of 23−27 Å2/molecule, the area occupied in each of the three monolayers is around 69−81 Å2/molecule. This derived area in each single monolayer is in good correlation to typical densities of phospholipid leaflets of a phospholipid bilayer membrane. The CACs of the three phospholipids in squalane strongly differ from those in squalene and in addition depend on the saturation or nonsaturation of the phospholipid. The CAC of POPC in purified squalene is 739-fold higher than its CAC in squalane, but for DPPC it is only 1.4-fold higher in purified squalene compared to its CAC in squalane. These ratios indicate that for POPC the lack of π bonds in squalane leads to a dramatic decrease in solubility. In squalane, the solubility of
Figure 5. Comparison of the different solubility limits of saturated and unsaturated phospholipids in relation to the CAC of DPPC in squalane. Different potential effects are indicated by numbers in circles: ①, steric (entropic and enthalpic) repulsion; ②, dynamic properties of unsaturation;38,39 ③, self-interaction of π bonds between unsaturated fatty acids in PL; ④, increase in entropic and enthalpic energy due to reduced chain length; ⑤, solubilization by oxidized, surface-active squalene; and ⑥, interaction of π bonds in phospholipids with π bonds in oil. F
DOI: 10.1021/acs.langmuir.6b00978 Langmuir XXXX, XXX, XXX−XXX
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Langmuir
regarding flat surfaces compared to experimental results,11 the interfacial tensions then correspond to 11 mN/m for HDL and 16 mN/m for LDL.2 However, typical experimental results produced by spinning drop tensiometry show interfacial tensions of
