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Effects of Acyl Chain Unsaturation on Activation Energy of Water Permeability Across Droplet Bilayers of Homologous Monoglycerides: Role of Cholesterol Maria Lopez, Jacqueline Denver, Sue Ellen Evangelista, Alessandra Armetta, Gabriella Di Domizio, and Sunghee Lee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03590 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Effects of Acyl Chain Unsaturation on Activation Energy of Water Permeability Across Droplet Bilayers of Homologous Monoglycerides: Role of Cholesterol Maria Lopez, Jacqueline Denver, Sue Ellen Evangelista, Alessandra Armetta, Gabriella Di Domizio, Sunghee Lee* Department of Chemistry, Iona College, 715 North Avenue, New Rochelle, New York 10801, USA *To whom correspondence should be addressed. Tel: 914-633-2638. Fax: 914-633-2240. E-mail: [email protected] Abstract Cholesterol is an important component of total lipid in mammalian cellular membranes, hence the knowledge of its association with lipid bilayer membranes will be essential to understanding membrane structure and function. A Droplet Interface Bilayer (DIB) provides a convenient and reliable platform through which values for permeability coefficient and activation energy of water transport across the membrane can be extracted. In this study, we investigated the effect of acyl chain structure in amphiphilic monoglycerides on the permeability of water across DIB membranes composed of cholesterol and these monoglycerides, where the acyl chain length, number of double bonds, and the position of double bond are varied systematically along the acyl chains. To elucidate the role of cholesterol in these membranes, we investigated its influence on water permeability and associated activation energies, at two different cholesterol concentrations. Our systematic studies show dramatic sensitivity and selectivity of specific interaction of cholesterol with the monoglyceride bilayer having structural variations in acyl chain compositions. Our findings allow us to delineate the exquisite interplay between membrane properties and structural components and understand the balanced contribution of each.

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Introduction The lipid bilayer is the essential structural component of the cellular membrane and serves as a barrier between the intracellular and extracellular environments. The transport of water between a cell and its surroundings can have profound effects, especially under conditions of osmotic imbalance in which cell can change its shape,1 for example. Transport of small molecules across the lipid bilayer is of importance for the principal functions of natural cellular systems. In particular, the transport of water molecules across cellular membranes is critical for balanced maintenance of organism homeostasis. Although the passive diffusion of water across a cell membrane can be considered energetically unfavorable,2 the predominant route for water permeation through plasma membranes is considered to be through the lipid bilayer, even those containing channel proteins.3 For non-facilitated water transport, among many factors that influence the transport of water molecules, the close relationship between the composition of lipid bilayer membranes and its permeability can provide greater understanding of the molecular mechanism of water transport across the lipid bilayer membranes.4 The rate for water permeability has been established to be controlled by a free energy barrier within the membrane, providing a rate-determining step being permeation into the densest region of the lipid chains.3 Hence, studies of water permeability can deliver insights pertaining to factors that mediate lipid packing, such as presence and absence of cholesterol. Biological membranes are not homogeneous mixtures of lipid and protein but composed of various lipidic constituents, including cholesterol. Cholesterol is widely distributed in mammalian cellular membranes as a high fraction of total lipid (∼20−50 mol %), and plays a crucial role as a major and essential component of the plasma membrane. The major role of cholesterol in a membrane has been considered as principally in its organizing effect upon other lipidic components of the membrane thereby modulating the structural, dynamical and physico-chemical properties of the plasma membrane lipid bilayer.5-7 Additionally, the nature and degree of effects of cholesterol and its location on bilayer membranes have been recognized to be dependent upon the structure and composition of the lipid bilayer.8-10 While many studies reveal that cholesterol modifies lipid membranes in complex ways, it is generally characterized that sterols have an influence upon the level of both acyl chain packing and rigidity of the bilayer, thus resulting relatively lower membrane permeability for small solutes, including permeability to water.11-12 Typically, the 2


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presence of cholesterol will reduce water permeability by a factor of 2−4, and often much higher, in proportion to the cholesterol molar ratio. However, despite extensive studies by methods both computational12-17 and experimental,9, 18 on the role of cholesterol with phospholipids and other lipidic substances in natural and artificial membranes, the mechanism by which cholesterol imposes its ordering effect and concomitant effect upon permeability has not always been entirely elucidated. Biological membranes consist of complex lipid species, with unsaturated lipids being probably the most common lipids in nature.19 The degree and position of unsaturation in an acyl chain have been known to strongly influence the structure and function of membranes by modification of membrane physical properties including phase behavior, fluidity, chain order in bilayers.20 In general, it has been observed that typically a lesser degree of membrane condensation and a lower degree of ordering was induced by cholesterol in membranes where the lipids of the membrane are unsaturated rather than saturated.8, 21-22 While good generalizations have been made on the stronger impact of cholesterol on membrane containing saturated lipids compared to that on unsaturated lipids, overall relatively less systematic work has been reported on the interaction of cholesterol with polyunsaturated lipids. Model membrane systems have been widely used to explore the molecular consequences of the presence and absence of cholesterol in biomembranes. Our approach has been to investigate the effect of membrane structure on water permeability using a model biological membrane made by two aqueous droplets in oil, termed the droplet interface bilayer (DIB).23-24 A DIB is formed by contacting two aqueous droplets, each possessing a lipid monolayer, in an immiscible solvent containing lipids (Figure 1).

Figure 1. Schematic description of the droplet interface bilayer (DIB) 3


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In our recent papers, we have demonstrated a DIB as a reliable, convenient, and versatile model membrane with which important biophysical properties, such as water permeability coefficients and activation energies therefore can be determined.25-27 A unique feature for permeability studies using DIB systems includes ease of bilayer formation, sufficient longevity, stability, and reproducibility to provide direct observation of water transport at various temperatures across a single unsupported bilayer. In this article, we report the differential influence of cholesterol depending on varying degrees of polyunsaturation in the lipid bilayers, on the water permeation process in terms of both permeability coefficient and activation energy. The system investigated includes monoglyceride (MG) acyl chains of hydrocarbon 18 and 20, where the degree of unsaturation and the position of the double bond are systematically translated along the chain. Our goal is to contribute systematic experimental evidence for a differential affinity of cholesterol for polyunsaturated chains having diverse degree and position of unsaturation, under a controlled experimental platform, in terms of activation energy for the water permeation process. The systematic nature of these studies can allow us to make ready comparisons of such affinity. The resulting permeability coefficients and activation energy value can serve as a probe for the underlying bilayer structure, which may foster better understanding of the specific interaction between cholesterol and monoglycerides with various acyl chain characteristics, and delineate the exquisite interplay between membrane properties and structural components. As models for lipid bilayer membranes, various constituents, e.g., phospholipids, glycerides, and cholesterol, are often used independently and in combination to simulate basal membrane composition. Particularly monoglycerides, having a single acyl chain, have been a common choice for various planar lipid membranes (e.g., black lipid membranes) as well as for DIBs, since this type of lipid provides a relatively simple and effective alternative to phospholipids in studies of membrane architecture, due to higher stability and shorter time needed to form a stable membrane.28-29 Unlike DIBs prepared using phospholipids, the DIBs formed by unsaturated monoglycerides in this paper have several unique advantages: (1) the formation of the DIB is rapid, and no curing time needed to form a DIB; (2) the DIBs are extremely stable with rarely any failure, and DIBs formed from unsaturated monoglycerides in squalane oil have a sufficiently long lifetime relative to the permeation experimental time (~510 min); and (3) the droplet bilayer contact area 4


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remains constant with time, a critical parameter for the determination of water permeability in this study. Experimental Section System of Study For the formation of droplet bilayers, we employed a mixture of lipids and cholesterol in varying molar ratio. As lipids, a series of 1-monoglycerides (MG) having acyl chain length of 18 and 20 with varying degrees and position of double-bond unsaturation was chosen. The list of lipids used along with the product number (Nu-Chek Prep) is shown in Table 1. A general molecular structure of monoglycerides consists of a glycerol headgroup and an acyl chain, connected at the sn-1 position of glycerol group (C18:1 Δ9 cis, monoolein, is shown in Table 1 as an example; the stereochemistry is arbitrary as we employ racemic mixtures in all of our MG studies herein). Also shown in Table 1 is the structure of cholesterol which consists of a rigid planar tetracyclic ring structure with a hydroxyl group at one end and a short hydrocarbon tail at the other.

Table 1. Monoglycerides (MG) and Cholesterol Used in This Study

C18:1 Δ9 cis (Nu-Chek M239): an exemplary monoglyceride

Cholesterol (Sigma-Aldrich C8667) Chain Length:Degree of Position of Unsaturation Nu-Chek Product No. Unsaturation for MG (all cis) C18:1 Δ6 M229 C18:1 Δ9 M239 C18:1 Δ11 M249 C18:2 Δ9, 12 M254 C18:3 Δ9, 12, 15 M264 C18:3 Δ6, 9, 12 M269 C20:1 Δ11 M274 C20:2 Δ11, 14 M284 5


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C20:3 C20:3 C20:4

Δ8, 11, 14 Δ11, 14, 17 Δ5, 8, 11, 14

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M289 M294 M299

Materials and Sample Preparations All series of monoglycerides (MG) used in this study were purchased from Nu- Chek Prep, Inc. and used as received (purity ≥99%). Squalane (99%, 2,6,10,15,19,23-hexamethyl tetracosane; C30H62; "SqA"), which was used as the immiscible organic phase, and all other chemicals, including cholesterol (CHOL), of the highest purity available, were purchased from Sigma-Aldrich and used without additional purification. All samples were stored at -20 ℃ until use and mixtures were freshly prepared immediately before each experiment. Cholesterol was delivered to the bilayer via a homogeneous bulk oil solution containing cholesterol and monoglyceride at a molar ratio, relative to monoglyceride, from 1:2 to 2:1. Monoglyceride solutions containing cholesterol have been prepared by codispersion of the sterol with monoglycerides in the squalane solvent, followed by brief vortex and bath sonication for up to 30 min held above melting point of each monoglycerides until clear solution is achieved to ensure a homogeneous mixture. For all experiments, the total concentration of lipids (monoglyceride + CHOL) was held constant in the regime of ~12 mM. Aqueous solutions using osmotic agents (NaCl at nominally 0.1 M) were prepared from purified, deionized water (18.2 MΩꞏcm) using a Millipore water purification system (Direct Q-3). The osmolality (in mOsm/kg) of all solutions used was measured by a vapor pressure osmometer (VAPRO model 5600). All experiments were carried out using a custom built temperature-controlled microchamber which was thermostatted via an external circulating water bath. This allows variation and control of temperature from 16 to 65 °C within ±1 °C accuracy. Special care was given for handling of polyunsaturated monoglycerides. In general, the polyunsaturated MG are liquid at or slightly above room-temperature, and are also readily soluble in the squalane solvent, and so therefore minimal energetic input was required to disperse, even in the presence of cholesterol. However, if any vortexing or bath sonication was conducted on polyunsaturated MG, it was limited to several minutes in duration. In order to assay whether polyunsaturated MG undergoes autoxidation under our conditions of sample preparation and experimental time, we employed a UV spectrophotometric test for decomposition damage. Specifically, diunsaturated monoglyceride MG C18:2 converts upon autoxidation to result in a 6


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conjugated diene having absorption at 232-236 nm (peak 233 nm, =2700/M-cm), while MG 18:3 and MG 20:4 decompose to form conjugated trienes with absorptions at 268 nm (=43400/M-cm) and 278 nm (=3350/M-cm).30 Our polyunsaturated MG samples were tested by this assay at each stage of preparation, including before and after vortexing/sonication for 30 min, and no detectable oxidative damage was found. This result is consistent with literature showing negligible autoxidation for polyunsaturated monoglycerides even when concentrated solutions (25% (w/w) in 1-undecanol) were held in air at 65 ºC for 4 hrs.31 Experimental Set-Up We employ the droplet interface bilayer (DIB) as a model system for biological membranes for the study of the influence of cholesterol on a lipid bilayer. Our experimental set-up and procedure for permeability measurement using the DIB method has been described in detail in previous papers and an essentially identical set-up has been used for this experiment.27 In brief, as shown in Figure 2, the system consists of an inverted microscope (Olympus IX 51) combined with two hydraulic micropipet manipulators (Narishige), supported on a vibration-isolated workstation (Newport), with a CCD camera directly attached to the microscope for real time recording of the droplets and their size changes. Recorded videos and images were analyzed subsequently to measure the dimensions of droplets and corresponding volume changes using custom-built image analysis software.

(a)

(b)

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Figure 2. Schematic of the droplet interface bilayer generation system: (a) For the creation and manipulation of the droplets, an inverted microscope combined with two hydraulic micropipet manipulators are used in a temperature-controlled microchamber; (b) Two droplets adhere to form a bilayer when a pair of aqueous droplets, each of which are stabilized by a lipid monolayer, are brought into contact in a squalane solution containing lipid and cholesterol. Osmotic water transport across the lipid bilayer, resulting in swelling of NaCl droplet and shrinking of pure water droplet (original droplets are depicted in dotted line). The arrow indicates the direction of water transport. Image Analysis In accordance with the method of Dixit,32 values for droplet volume and radius of the bilayer were determined by initially fitting circles to the droplet outlines. We derive the true center-to-center distance and the bilayer contact angle from the geometry of the droplet outlines, and subsequently the bilayer radius of the circle that defines the bilayer. Based on image analysis of the geometry of the droplets, the bilayer area is determined to be constant over time for all our experiments reported in this paper. All images were collected having a pixel size of 0.16 m using a field of 1920 × 1080 pixels. The uncertainty in the diameter measurement is 0.32 m (2 pixels x 0.16 m/pixel). Since we generally have maintained droplets in the diameter size range of ~100 m, the error associated with the diameter measurement is 0.32 %, which propagates to ~0.6 % relative uncertainty in volume calculation. The typical diameter of DIB region is ~50 m, and so error associated with measurement contributes ~0.9% relative uncertainty in the calculation of the area of the bilayer. When the uncertainties in optical resolution are taken together, these correspond to an uncertainty in permeability of ~1.3 %. The reported permeability value in this study represents an average result of at least 30 individual permeability experiments for each mixture of monoglyceride and cholesterol system. We have found that the experimental deviation for values of water permeability coefficient (Pf) is larger than any error which can be ascribed to the optical resolution. Determination of Water Permeabilities and Activation Energies for Water Transport using DIB as Model Biological Membranes

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We have shown that DIB can be employed as a reliable and configurable platform for a model membrane to explore structural effects on the permeation of water molecules through lipid bilayer.25-27 A DIB is formed by contacting two aqueous droplets, each possessing a lipid monolayer, in an immiscible solvent containing lipids (Figure 1). When two osmotically unbalanced droplets, each covered with lipid monolayer, were made to adhere at a bilayer, water transport immediately commenced and corresponding changes in droplet volume over time (dV/dt) is measured optically by microscopic observation (Figure 2); and the behavior of the system follows the expression of equation (1): (1) where A is the geometric bilayer area, νw is the molar volume of water (taken as 18 mL/mol), ΔC(t) is the osmolality gradient between two droplets, and Pf is the bilayer permeability coefficient of water. The volume change with time (dV/dt) is related to the bilayer permeability coefficient of water, Pf, as expressed in the Equation (1). In addition, permeability coefficients were measured at four different temperatures between 25 and 50 °C and the activation energies for water permeation process were determined from an Arrhenius plot. In a plot of the natural log of the permeability coefficient (ln Pf) versus the reciprocal of absolute temperature (1/T), the slope of the curve is equal to Ea/R, where Ea is the activation energy and R is the gas constant. Results 1. Degree of double bonds and effect of cholesterol on activation energy of water permeation: C18 and C20 MG We studied the effect of cholesterol on osmotic water permeability coefficients Pf (35 °C) across DIBs formed from monoglycerides (MGs) in squalane solvent, with increasing degree of MG acyl chain unsaturation. Squalane is chosen as the oil solvent owing to its large molecular size and thus scant residual presence within the bilayer. Figure 3 shows these effects for acyl chain length C18 (Figure 3a) and C20 (Figure 3b). The molar ratio of cholesterol to MG in the bilayer-forming oil solution was 1:2 and 2:1. For comparison, water permeability coefficients for single component (pure) MGs systems27 are also shown. We have also tested 4:1 molar ratio of cholesterol to lipid and obtained modest but progressive decreases in Pf relative to that with 2:1 molar ratio (data shown in Supplemental Materials). Therefore, the monolayer and bilayer may not yet be saturated 9


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even when operating under such high cholesterol concentrations, consistent with the relevant literature for monoglycerides.33 (a)

(b)

Figure 3. Osmotic water permeability coefficients (35 °C) across DIBs of monoglycerides with acyl chain length of (a) C18 and (b) C20, with varying degree of unsaturation in the absence and presence of Cholesterol. As shown in Figure 3, when cholesterol is added to the MG bilayer, an overall decrease in water permeability coefficients compared to that of pure MG27 has been observed for both C18 and C20 MG, across all levels of unsaturation. In general, cholesterol is found to have an inhibitory effect on the permeation of water at all cholesterol concentrations investigated (1:2 to 2:1 molar ratio of cholesterol:lipid), and the level of reduction in water permeability coefficient is relative to the amount of cholesterol in the system, showing greater reduction in the presence of 2:1 cholesterol to lipid molar ratio compared to 1:2 molar ratio. The reason for the observed reduction in water permeability upon addition of cholesterol is likely associated with cholesterol's ability to increase the orientational order, thereby engendering a laterally more condensed membrane.5-7 In turn, the bilayer of increased ordering will pose a higher energetic barrier to the transport of water molecules, thus lowering permeability.34 Additionally, an increased degree of acyl chain unsaturation is shown to enhance water permeability of membrane bilayers formed from both pure and from cholesterol-containing MG of acyl chain length of C18 (Figure 3a) and C20 (Figure 3b). Figure 4a displays the osmotic water permeability coefficients at 35 °C as a function of number of double bonds in C18 and C20 MG. This plot indicates that Pf for water transport (at 35 °C) increases roughly monotonically with increasing degree of acyl chain unsaturation for both C18 10


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MG and C20 MG in the absence and presence of cholesterol. For DIB formed from pure C18 MG, there was observed an approximately three-fold increase in water permeability (from 95 m/s to 262 m/s)

upon changing from one double bond to three double bonds. Similarly, an

approximately four-fold increase in permeability (from 70 m/s to 306 m/s) for C20 MG having one double bond to four double bonds, is observed.27 But, in the presence of 2:1 molar ratio of cholesterol:MG for the same two systems, the difference in water permeability among MG having varying degree of unsaturation is shown to be even more intensified. For example, a roughly fourfold increase in Pf was observed (from 60 m/s to 240 m/s) for cholesterol-containing C18 MG, upon a change from one double bond to three double bonds. For C20 MG containing cholesterol (CHOL:MG = 2:1), an approximately six-fold increase (from 36 m/s to 230 m/s) of Pf was observed, when comparing a monounsaturated MG to a tetraunsaturated MG. Thus, in broad outline, an increased level of unsaturation is correlated with increased water permeability, for both cholesterol-containing and pure MG lipid systems. When taken from the vantage point of increasing level of cholesterol, Figure 4b summarizes the level to which the presence of cholesterol, delivered from the oil, decreases the permeability of a given lipid DIB system: this Figure displays the percentage decrease for water permeability coefficients at 35 °C as a function of number of double bonds for both C18 and C20 MG. As is well established, the addition of cholesterol to a lipid bilayer system will generally reduce its passive water permeability,11 but it is notably apparent from Figure 4b that the percentage reduction in Pf induced by the presence of 2:1 cholesterol, is greater for monounsaturated MG compared to MG with a higher degree of unsaturation. In general, lipids of increasing levels of unsaturation will confer a correspondingly increased fluidity to bilayers composed of these lipids.20 Fluidity in bilayers is correlated with high water permeability since for molecules of higher fluidity there is a looser packing of lipids. Bilayer permeability is expected to depend strongly on the creation of porous voids in the hydrocarbon region of the tailgroups, brought about by fluctuations in phospholipid acyl chain packing at ambient temperature.35-36 But, while addition of cholesterol will lower water permeability across bilayers owing to its ability to increase lateral condensation of the membrane,8, 11

the effect of cholesterol is known to be reduced depending on level of unsaturation. Cholesterol

interacts with mono- and diunsaturated PC lipids to an extent, but multiple unsaturation decreases the interaction. For example, it has been observed that the ordering effect of cholesterol is lesser for unsaturated PC as compared to saturated PC, since unsaturation in an acyl chain engenders its 11


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bending, inhibiting the close packing of molecules and weakening interactions with cholesterol.18, 22

(a)

(b)

Figure 4. (a) The osmotic water permeability coefficients at 35 °C, and (b) the percentage decrease in water permeability coefficient due to the presence of cholesterol (2:1 CHOL to MG molar ratio) as a function of number of double bonds in MG acyl chain. We have also determined the permeability coefficient as a function of temperature for C18 and C20 MG systems in the presence of different concentrations of cholesterol. In order to extract the activation energies (Ea) for the water permeation process, permeability coefficients were measured at four different temperatures between 25 °C and 50 °C. The results are presented as an Arrhenius plot (logarithm of the Pf values vs. inverse absolute temperature), from which the slope is proportional to activation energy (Figure 5a and 5b). A summary of activation energy (Ea) values for the water permeation process across DIB is shown in Figure 5c and 5d. For a monoglyceride of given chain length (C18 in Figure 5c, and C20 in Figure 5d) and defined level of unsaturation, Ea values are given for different molar ratio of cholesterol to MG, namely, 0:1; 1:2; and 2:1; with increasing degree of acyl chain unsaturation. The reduction in permeability coefficients upon the addition of cholesterol, previously described in relation to Figure 3, corresponds to a concomitant increase in activation energy for the same system, as presented in Figures 5c and 5d.

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Overall, there is a decrease in activation energy for water transport (shown in units of kcal/mol) with an increasing number of double bonds in an acyl chain, as depicted in a plot of Ea against number of double bonds in C18 and C20 MG, for both pure and cholesterol-containing MG system (Figure 6). The reduction in activation energy with increased degree of unsaturation is relatively modest for pure MG of both tail lengths studied (C18 and C20). For example, the Ea value of 9.9 kcal/mol for pure singly-unsaturated MG C18:1Δ9 is reduced to 7.8 kcal/mol for triply-unsaturated MG C18:1Δ9,12,15. Similarly, the Ea of 11.4 kcal/mol for pure C20:1Δ11 is reduced to 8.6 kcal/mol for quadruply-unsaturated C20:4Δ5,8,11,14. However, in the presence of 2:1 molar ratio of cholesterol:MG in the bulk oil solution, for increasing number of double bonds (in both C18 and C20 acyl chains) the reductions in activation energies with increased unsaturation, are more dramatic. For example, the Ea value is 20.0 kcal/mol for C18 MG having one double bond, in admixture with cholesterol. This value is reduced to 14.0 kcal/mol for cholesterol-containing C18 MG having three double bonds. Similarly, the value for Ea of 21.0 kcal/mol for cholesterol-containing C20 MG having one double bond is lowered to a value of almost half --- 10.5 kcal/mol --- for C20 MG having four double bonds, in admixture with cholesterol 2:1. Thus, as discussed above and represented in Figure 6(b), the extent to which cholesterol can bring about changes in activation energy for water transport depends on the properties of the unsaturated lipid. The most intense change is seen when comparing bilayers composed of mono-unsaturated C20 MG having cholesterol versus tetraunsaturated C20 MG. But this change is undoubtedly due to the compounded effects of two factors. Firstly, the tetraunsaturated C20 MG will have greater fluidity and thus lessened interaction with cholesterol; and, the tetraunsaturated 20 MG also contains a position for its first double bond (as measured from acyl carbon) at the 5-position in the chain. As we shall explain in more detail below, the interaction of cholesterol with unsaturated lipid also depends on position of first double bond.

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(a)

(b) 2:1 CHOL:MG

2:1 CHOL:MG

(c)

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(d)

Figure 5. (a and b) Temperature dependence of permeability for 2:1 molar ratio of CHOL:MG is shown as a plot of the natural log of the permeability coefficient (Pf) versus the reciprocal of absolute temperature, 1/T (scaled to give abscissa values greater than unity). (c and d) Activation energy for water permeation through monoglyceride bilayers with (and without) cholesterol for acyl chain length of C18 and C20 with varying degree of unsaturation.

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(b)

(a)

Figure 6. Change in activation energy with increasing number of double bonds in (a) C18 MG and (b) C20 MG is shown. Lines are used for guidance only. 2. Effect of position of double bond on the activation energy of water permeability across cholesterol-containing bilayers 2.1 C18 MG with Monounsaturation We studied three positional isomers for the monounsaturated C18 acyl chain 1-monoglyceride (C18:1 MG): double bond being located at one of the 6, 9, or 11 positions. The water permeability coefficients at 40 °C for each of these positional isomers are shown in Figure 7a, for both pure MG and in admixture with 2:1 molar ratio of cholesterol to MG. There are little significant differences in water permeability coefficients among C18:1 MG positional isomers in the pure system; all span values in the range of 114–126 µm/s. But, the cholesterol containing MG membranes show dramatic differences in permeability coefficients compared to their pure counterparts. A prominent example is C18:1 11 MG ("monovaccenin"), which shows the largest reduction in permeability coefficient attendant to the addition of cholesterol, from 118 m/s for pure MG to 36 m/s for cholesterol mixed MG. The other positional isomers studied, C18:1 6 MG and C18:1 9 MG, show more modest reductions in permeability in comparing pure MG to cholesterol-mixed MG, from 114 m/s to 97 m/s for C18:1 6; and from 126 m/s to 90 m/s for C18:1 9, respectively. This will be discussed in greater detail in the Discussion section. These positional isomers show specific increases in activation energy upon addition of cholesterol

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(Figure 7b and 7c), but for pure MG, the three positional isomers of C18:1 all exhibit activation energy values in the range of 9.9–10.7 kcal/mol. More particularly, in the presence of 2:1 molar ratio of cholesterol to MG, there are substantial and specific activation energy differences among these positional isomers; 15.0 kcal/mol for C18:1 6 MG, 20.0 kcal for C18:1 9 MG, and 25.0 kcal/mol for C18:1 11 MG. To summarize, for monounsaturated C18:1 with double bond in either 6, 9, or 11, the activation energy for water permeability in the presence of cholesterol is shown to increase upon "moving" the double bond from position 6 to 11. There is a more dramatic influence seen with the 2:1 molar ratio of cholesterol to MG compared to cholesterol to MG molar ratio of 1:2. To illustrate this difference more clearly, Figure 8 shows the plot of activation energy (Ea) as a function of position of double bond. When viewed in terms of the activation energy increase in comparing pure MG to cholesterol-containing MG (at 2:1 molar ratio of cholesterol to MG), there is radically significant increase in activation energy for C18:1 11 (from 10.0 kcal/mol to 25.0 kcal/mol), while a more modest increase is observed for C18:1 6 (from 10.7 kcal/mol to 15.0 kcal/mol) and for C18:1 9 (from 9.9 kcal/mol to 20.0 kcal/mol). This could be indicative of a more favorable interaction between cholesterol and the MG in the 11 position, as compared to MG having double bond closer to the headgroup.

(a)

(c)

(b) 2:1 CHOL:MG

Figure 7. (a) The osmotic water permeability coefficients at 40 °C, (b) temperature dependence of water permeability for permeability 2:1 molar ratio of CHOL:MG (as a plot of the natural log of the permeability coefficient (Pf) versus the reciprocal of absolute temperature, 1/T), and (c) 16


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activation energy for water permeation process for three positional isomers of C18 monounsaturation for MG in the absence and presence of cholesterol.

Figure 8. Activation energy for positional isomers of MG with monounsaturation of C18 acyl chain length as a function of double bond position. Lines are used for guidance only. Any lack of apparent error bars indicate that error bars are the same size or smaller than the symbols. 2.2 C18 and C20 MG with Polyunsaturation Studies were performed to determine the effect upon the activation energies for water permeability, for the addition of cholesterol to droplet bilayers composed of polyunsaturated monoglyceride lipids. Figure 9 shows activation energies for tri-unsaturated C18 MG and C20 MG. There are two commonly-occurring positional isomers for each tri-unsaturated monoglyceride of a given chain length: C18:3 6, 9, 12 and C18:3 9, 12, 15; and C20:3 8, 11, 14 and C20:3 11, 14, 17. Overall, in the presence of cholesterol, the activation energy is higher than that of pure polyunsaturated MG, for all positional isomers. Typically, a higher concentration of cholesterol will increase the activation energy for water permeability.

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(b)

(a)

Figure 9. Activation energy (kcal/mol) for MG with three double bonds of positional isomers with acyl chain length of (a) C18 and (b) C20. Figure 10 shows the plot of activation energy (Ea) as a function of position of the first double bond in acyl chain, to illustrate the significance of the position of first double bond in determining interaction of polyunsaturated lipids with cholesterol.

Figure 10. Activation energy for positional isomers of MG with three double bonds with acyl chain length of C18 and C20 as a function of first double bond position. Any absence of apparent error bars indicate that error bars are the same size or smaller than the symbols. There is a specific isomer effect for the activation energy of water transport across cholesterolcontaining membranes. For example, the activation energy for bilayers composed of C18:3 9, 12, 15 is 7.8 kcal/mol for 0% CHOL, but is enhanced to 14.0 kcal/mol in the presence of 2:1 molar 18


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ratio of CHOL to MG, which is an ~80% increase in Ea. On the other hand, lipid C18:3 6, 9, 12, which also has three double bonds in its acyl chain but they are in a different position, shows only a small increase for the activation energy: from 8.5 kcal/mol (without cholesterol) to 10.9 kcal/mol (with 2:1 CHOL), merely ~30% increase (Figure 9a and 10). This result evidences a striking difference for the influence of cholesterol on activation energy of water permeation in polyunsaturated lipids, depending on positional isomer. It is convenient to posit that this difference is due to the position of the first double bond in these polyunsaturated isomers (as measured from the headgroup). The evidence is strengthened upon study of the C20 triunsaturated system. As shown in Figure 9b and 10, the addition of cholesterol (2:1) to C20:3 8, 11, 14 increased the activation energy from 9.9 to 11.7 kcal/mol (~20% increase), whilst for C20:3 11, 14, 17, the inclusion of CHOL served to increase Ea from 9.1 to 18.5 kcal/mol, which is an increase of almost ~100%. It appears that "moving" a first double bond position from 8 to 11 in a triunsaturated MG having C20 acyl chain length, engendered a pronounced difference in activation energy when cholesterol is present. Discussion Research on the effects of cholesterol with membranes has been under continuous focus in the literature7, 25 with a primary role of cholesterol being recognized for its ability to modulate the molecular organization of membranes, such as the ordering of lipid hydrocarbon chains (the ordering effect) and membrane packing (the so-called condensing effect). As a consequence, cholesterol is generally known to decrease membrane permeability to water, gases, and small organic molecules.8 Cholesterol's effect within bilayers has often been characterized in terms of its "interaction" with neighboring lipids to result in an overall change in bilayer structure relative to its absence. In general, it is believed that the interaction of cholesterol with unsaturated lipids is relatively weaker than that with a fully saturated counterpart, as inferred by a lesser degree of membrane condensation and a lower degree of ordering induced by cholesterol in membranes consisting of unsaturated lipids. Several idealized models have been proposed to explain the interaction between cholesterol and lipids, especially phospholipids. In the so-called umbrella model, the head group of the phospholipid provides a region under which the phospholipid acyl chains and an adjoining 19


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cholesterol must share a limited amount of space. Since most of the cholesterol molecule is hydrophobic, it is compelled to be together with the acyl chains. Another model posits a template effect, in which phospholipid acyl chains are sufficiently flexible to form a molecular complement to the shape of the planar portion of a cholesterol neighbor, which gives rise to tight packing and minimization of the contact of hydrophobic regions of the lipid with the aqueous phase. But the molecular flatness of the major portion of cholesterol (its rigid sterol ring) would disfavor interaction with the bulkiness of unsaturated lipids.37-40 As shown in the present study, cholesterol generally interacts with both mono- and polyunsaturated MG, to result in an overall lowered water permeability coefficient and increasing activation energy for water permeation. But our systematic variation of the acyl chain structure also indicates that an appreciably different level of cholesterol interaction is apparent, depending on the structure of acyl chains for both C18 and C20 MG, as summarized below: (1) Inclusion of cholesterol reduces permeability coefficients and increases water transport activation energy to a varying extent. If there is a higher overall degree of unsaturation in MG, this leads to lesser extent to which water permeability coefficients are reduced by cholesterol, and a lesser increase in activation energy induced by cholesterol, compared to MG having monounsaturation. In the presence of 2:1 molar ratio of cholesterol:MG, for example, the difference in activation energy for water permeation among MG having varying degree of unsaturation is shown to be much more intensified for both C18 and C20 MG, compared to pure MG. (2) Even in the presence of the same degree of unsaturation, there are observed wide ranges for reductions in water permeability coefficients by cholesterol, and increases in activation energies induced by this sterol. This indicates that the degree of unsaturation alone is not the only determining factor for the water permeation process for the lipid bilayer containing cholesterol. (3) The position of the first double bond appears to be correlated well with the observed reduction in permeability coefficients and increased activation energies for the systems we have studied. For monounsaturated monoglyceride (Figure 8) and polyunsaturated monoglyceride (Figure 9), activation energies in the presence of cholesterol are shown to increase upon the conceptual "moving" of a first double bond from position 6 to position 11. If the first double bond is too close to the headgroup, then the effect of cholesterol is lessened. 20


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By introducing cholesterol into the MG lipid bilayer, our present studies show that the reduction in water permeability can range widely from ~15% to ~70%. However, some clear patterns emerge from the data. An increasing level of unsaturation leads to less pronounced reduction in water permeability coefficient (owing to the presence of cholesterol) compared to that of lesser degree of unsaturation. It has already been shown that an increased degree of unsaturation correlates with a larger water permeability coefficient, which can be explained in terms of the presence of double bonds in the acyl chain leading to reduction in average orientational order in the bilayer and suppression in chain ordering.41 Water permeability across membranes appears to generally correlate to the chain ordering of the lipid membrane, which in turn is largely dependent upon the structural factor of the acyl chain: there is increased permeability observed for lipids of lesser chain ordering and a loose packing of lipids.27 Fluorescence measurements on polyunsaturated lipids also confirm that water content in the bilayer hydrocarbon region increases with increasing chain unsaturation,42 which could also contribute to greater solubility of water and thus favorable thermodynamic impetus for the process. Hydrophobicity profiles of lipid membranes (i.e., ability of water to penetrate) have been correlated with the permeability of small polar molecules, including water.43 In the case of bilayers containing cholesterol and monoglyceride lipids having the same degree of unsaturation but different positions for the sites of unsaturation, ranges for the reduction in water permeability are observed. In order to recapitulate this phenomenon more clearly, Figure 11 shows the activation energy (Ea) as a function of the position of first double bond(s) in the acyl chain of C18 and C20 MG, each containing cholesterol (2:1 molar ratio of CHOL:MG). For the case of monounsaturated C18:1 MG, the Ea ranges from 15, 20, and 25 kcal/mol for Δ6, Δ9, and Δ11 (respectively), thus markedly depending on the position of double bond. Similarly, the Ea for water transport has a value of 10.9 kcal/mol for C18:3 MG, Δ6, 9, 12, and 14.0 kcal/mol for C18:3 Δ9, 12, 15. Similarly, the respective Ea values are 11.7 kcal/mol and 18.5 kcal/mol for C20:3 MG, Δ8, 11, 14 and C20:3 MG Δ11, 14, 17. It is unmistakable that the interaction of cholesterol with the MG membrane is highly specific in terms of structural variations in a given acyl chain, and cannot be generalized with the number of double bonds present in the membrane. An illuminating example is found in the literature44 which regards the interaction of cholesterol with PC 21


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monolayers and bilayers containing 18 carbons and three unsaturations, with different arrangement of their double bonds. It was reported that a larger cholesterol-induced condensation is found for 18:0--18:3 (9, 12, 15) compared to that of 18:0--18:3 (6, 9, 12), as determined by measurement of area per molecule and fluorescence experiments. It was also reported that relative cholesterol-induced condensation of phospholipid/cholesterol (1:1) monolayers at 30 C under lateral pressure of 20 mN/m is 31.7 Å2/molecule for 18:0--18:3 (9, 12, 15) versus a condensation value of 7.8 Å2/molecule for 18:0--18:3 (6, 9, 12). Each such phospholipid had a tail with three double bonds, but the PC 18:0--18:3 (9, 12, 15) exhibited a more effective condensation.

Figure 11. Activation energy (Ea) due to cholesterol inclusion (CHOL:MG = 2:1) as a function of the position of first double bond(s) in an acyl chain of C18 and C20 MG. At the microscopic level, cholesterol in general orients in a bilayer with its hydroxyl group facing the aqueous phase, and its sterol ring is positioned approximately parallel and adjacent to the lipid hydrocarbon chains. Cholesterol's short side-chain extends towards the center of the membrane.45 Viewed in terms of its time-averaged position in the bilayer, the cholesterol ring will associate with the first 8−10 carbons of acyl chains of a neighboring lipid. As such, cholesterol increases the orientational order and decreases the rate of motion of the hydrocarbon chains. Higher ordering engenders a laterally more condensed membrane, with increased mechanical strength and decreased permeability.8, 11 22


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In this respect, the location of the cis double bond(s) appears to be critical, in view of the observed variations in the permeability coefficients and activation energies shown herein. This likely indicates that, in order for the rigid sterol ring to exert significant effects on ordering of the acyl chains, cis double bond(s) of neighboring lipids have to be located at positions that do not interfere with van der Waals contacts. The simplified illustration shown in Figure 12 emphasizes the interaction of cholesterol ring and the location of double bond in an adjoining acyl chain.46 The kinks introduced by the cis double bonds diminish a capacity of the rigid steroid ring to interact with unsaturated chains and limit the favorable van der Waals interactions. Hence the nature and extent of the cholesterol interaction with the lipid bilayer, expressed as activation energy for the water permeation process, are very responsive to the acyl chain structure in MG. Consistent with our findings, Stillwell et al.47 showed that cholesterol binds weakly to the PCs containing a fatty acid with a cis double bond closer to headgroup that the carbon 9 position, and binds more efficiently to PCs lack a cis double bond at carbon 9. Atomistic molecular dynamics simulation studies48 dealing with monounsaturated C18 PC lipids reveal the significance of position of the double bond on membrane properties, such as area per molecule, chain ordering, and thickness of membrane. All of these changes are much more pronounced for cholesterol-containing PC lipids compared to that of pure PC lipids.46 Studies employing a coarse grained simulation49 of the interaction of cholesterol with polyunsaturated fatty acid (PUFA)-containing phospholipids have disclosed that the hydroxyl of the sterol actually resides at the center of the bilayer instead of its usual orientation anchored in the bilayer. This is indicative of the ability for the high disorder in polyunsaturated lipids to deter close proximity to the rigid steroid moiety of cholesterol.50-51 It is notable to recognize various structural differences between phospholipid and monoglycerides. Monoglycerides have a 1,2-diol headgroup as compared to the phosphocholine zwitterionic assembly for PC lipids. Monoglycerides thus have a smaller polar head group than that of phosphatidylcholines, leading to smaller area per lipid. Monoglycerides are uncharged whereas phospholipids contain zwitterionic or charged head groups. And, the glycerol-water head group region of the monoglycerides has a relatively high static dielectric constant. Therefore, given these differences, as compared with phospholipids, bilayers formed from monoglycerides can exhibit different characteristics with respect to their interaction with cholesterol. The main relevant difference between monoglycerides and phosphocholines is the hydrogen bonding environment of 23


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cholesterol. In MG, the presence of primary and secondary alcohol groups can act as H-donor to cholesterol. In cholesterol-containing monoglyceride bilayers, the cholesterol headgroup is known to form hydrogen bonds to the glycerol portion, and a greater extent of cholesterol-lipid hydrogenbond formation in monoolein membranes have been reported compared with the DMPC membranes.52 In DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers cholesterol predominately interacts with monoolein. Despite such differences, we believe that overall trends we have seen in the series of monoglycerides with increasing polyunsaturation are qualitatively consistent with what has been reported for PC.

Figure 12. Illustration shows an example of interaction of cholesterol with C18:1 MG containing double bond in different position, 9 vs 11. The presence of kink caused by double bond before C11 position interferes with close-packing of cholesterol and weakens beneficial van der Waals interactions. The resulting lipid bilayer is more permeable to water with lower activation energy. Conclusion In this study, the differential nature of cholesterol's interaction with the acyl chain component of monoglyceride bilayers, in terms of degree and position of unsaturation, has been probed by using water permeability coefficient and associated activation energy, in a quantitative and highly reproducible manner, via a model membrane assembled by the droplet interface bilayer (DIB) as a reliable lipid bilayer model system. We investigated how cholesterol interacts with monoglycerides of C18 and C20 hydrocarbon chain length with varying degrees of unsaturation 24


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where the position of the double bond is varied systematically along the acyl chains. By using water permeability and activation energy as a probe for the packing arrangements in the lipid bilayer, our results demonstrate that monoglyceride acyl chain structure will have a pronounced effect on activation energy of water permeability across the lipid membrane. An overall increased degree of unsaturation resulted in increased osmotic water permeability coefficient and decreased activation energy for the water permeation process. However, in contrast to pure MG, the presence of 2:1 molar ratio of cholesterol:MG will intensify the difference in activation energy for water permeation among MG having varying degree of unsaturation, indicative of the sensitivity of the cholesterol interaction with lipid composition. In addition to degree of polyunsaturation, the position of first double bond is correlated well with the observed activation energy in the cholesterol-containing systems, with higher activation energy values corresponding to systems where the first double bond of the lipid is moved away from the headgroup. Having the first double bond position at C11 for monounsaturated MG provides a maximum in activation energy when in the presence of cholesterol, indicating a specific positional effect of acyl chain structure upon interaction with cholesterol. While the effect of positional isomerization on water permeability were subtle in pure MG, having cholesterol present in the lipid bilayer intensified the difference in activation energy, which dramatically varied depending on the position of the first double bond(s) in an acyl chain. Our systematic studies provide an important basis for better understanding the fundamental molecular interactions that determine the effects of cholesterol on critical membrane properties, such passive permeability across the bilayer, significant for the discernment of cellular life processes as well as for understanding drug transport into cells. We believe that the knowledge gleaned from our studies on the effect of unsaturation on the interaction between cholesterol and lipid bilayer will enhance understanding of membrane compositional complexity and the differential affinity cholesterol has for lipids, since cholesterol and unsaturated fatty acid content can vary widely in cellular membranes. Supporting Information

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The relative permeability coefficient (Pf/Pfo) of C18:1 ∆9 MG and C20:4 ∆5,8,11,14 MG as a function of the molar ratio of cholesterol to monoglycerides in the bilayer forming oil solution, at 25°C. Acknowledgments The authors would like to acknowledge the financial support from the National Science Foundation (NSF-CHE-1609135). JD thanks the Patrick J. Martin Foundation for a scholarship. References 1.

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Effect of cholesterol on membrane water permeability depends strongly on the position of first double bond in an acyl chain in a lipid membrane.

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