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Enthalpic Effects of Chain Length and Unsaturation on Water Permeability Across Droplet Bilayers of Homologous Monoglycerides Maria Lopez, Sue Ellen Evangelista, Melissa Morales, and Sunghee Lee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03932 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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Enthalpic Effects of Chain Length and Unsaturation on Water Permeability Across Droplet Bilayers of Homologous Monoglycerides Maria Lopez, Sue Ellen Evangelista, Melissa Morales, and 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 A deeper understanding of unassisted passive transport processes can better delineate basic lipid dynamics in biological membranes. A Droplet Interface Bilayer (DIB) is made by contacting two aqueous droplets covered with a lipid monolayer, and has increasingly been employed as a model artificial biological membrane. In this study, we have investigated the effect of acyl chain structure of amphiphilic monoglycerides on the osmotic permeability of water across DIB membranes composed of these monoglycerides, where the acyl chain length (C14 to C24), number of double bonds (1–4), and the position of double bond are varied systematically along the acyl chains. Both permeability values and activation energies have been extracted for water transport across a lipid bilayer formed of a homologous series of lipids, allowing us to make ready comparisons between the different lipids and potentially better elucidate the contributions that molecular motifs make to the permeation process. Introduction Membranes are intimately involved in a plethora of biological processes, including establishing and maintaining trans-membrane gradients, compartmentalizing of cells, inter- and intra-cellular communication, cell-cell recognition, and energy transduction events. The semipermeable nature of the membrane controls the level of solutes in both aqueous compartments (cytoplasmic and exofacial) bathing the membrane. Movement of solutes across membranes can be divided into two basic types: passive diffusion and active transport. Passive diffusion requires 1

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no additional energy source other than what is found in the solute's electrochemical (concentration) gradient and results in solute reaching equilibrium across the membrane. Passive diffusion can be either simple passive diffusion, where the solute crosses the membrane at an arbitrary point, or facilitated passive diffusion, where diffusion is assisted by solute-specific facilitators or carriers. Active transport requires additional energy and results in a non-equilibrium accumulation of solute.1 Passive transport in particular plays an important role in many biological processes, including transport of small molecules such as oxygen, carbon dioxide, water, and even some drug molecules. Such transport across bilayer membranes will depend on various factors, especially the structure of the membrane and its component lipids. Therefore, a deeper understanding of unassisted passive transport processes can better delineate basic lipid dynamics in biological membranes. In addition, understanding the mechanism of the passive permeation process as a function of membrane composition is necessary for the rational design of drugs and drug delivery systems, since passive transmembrane permeability is known to be a major mechanism for drug absorption.2 Biological systems are replete with complexity, in large part owing to compositional heterogeneity. For example, the dominant type of cellular membranes are asymmetric for a variety of cell types, having different lipid compositions in the inner and outer leaflets of the bilayer.3 In nature, biological membranes consist of lipids having tail groups which comprise not only saturated hydrocarbon chains (C–C bonds), but a significant fraction of unsaturated hydrocarbon chains (C=C bonds).4, 5 It has been established that the number and location of fatty acid double bonds can influence the structure and function of membranes. Living organisms employ a scope of relevant fatty acids in membrane lipids, varying from 14 to 24 carbons and from zero to six sites of unsaturation. This lipid diversity is essential for tailoring the properties of cellular lipid membranes for various tasks and environments, since the structural and dynamical properties of lipid membranes are known to be affected by the lipid acyl chains. The present study builds upon a background of prior experimental and theoretical studies that have investigated the effect of the properties of individual lipids and bilayers upon water permeability. These properties include bilayer thickness, membrane fluidity, area per lipid, and lysis tension.6, 7, 8, 9, 10, 11, 12 These reports demonstrate the sensitivity of membrane mechanical strength and small molecule permeability to acyl chain structure, including length of acyl chain, 2


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degree of unsaturation and the position of a double bond.

7, 13

In particular, studies of water

permeability through bilayers has a long history, embodied in numerous references describing a variety of measurements using systems such as liposomes14 and various planar lipid membranes (e.g., black lipid membranes).15 Both liposomes and planar bilayers have often been employed for osmotic filtration measurements, where a concentration gradient is generated across a membrane (e.g., inner vesicle compartment vs. its outer bathing solution), in order to promote water flux. From osmotic pressure values on the respective sides of the membrane and membrane area, water permeability coefficients can be determined. Alternatively, tracer movement across the bilayer (deuterated or tritiated water) can be followed.16 The determination of transport phenomena (e.g., permeability coefficients) for passive transport of small neutral molecules has now been developed experimentally to a sufficient degree that the broad contours of permeation behavior are known,17 and show that water permeation rates can vary over a wide range. Yet, even in view of the availability of data, the mechanism of passive permeation is still not completely understood. This is likely due in part to one persistent and prominent feature of this history of investigation: water permeability measurements show significant variation in values, depending upon the different techniques which are employed, the conditions employed, and even the different laboratories.10, 11 Thus, systematic tactics need to be elaborated in order to better analyze the effects of membrane lipid structure, e.g., chain length, and degree and position of chain unsaturation, in order to approach a complete understanding of the permeation process. Our approach to overcome the disadvantages in the literature has been through the use of the droplet interface bilayer (DIB) for water permeability determination. A DIB membrane is a lipid bilayer formed between pairs of aqueous compartments upon juxtaposition of micrometersized droplets, immersed in an oil phase, each of which is stabilized by a lipid monolayer. Bilayer formation occurs at the point of contact, forming adherent droplets.18, 19 Such DIB systems have increasingly been employed as model artificial biological membranes. For example, DIB membranes have been employed for a wide variety of uses, such as the study of bioelectric phenomena,20, 21 in microfluidic based systems,22, 23, 24 and for membrane transport studies.25, 26, 27 DIB systems have also been prominently employed in engineering applications, including for the printing of tissue-like materials28 and for microdroplet crystallization systems.29 In our recent publications,30, 31 we have successfully demonstrated that a DIB method provides a convenient 3


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means to attain reliable values for water transport phenomena, such as permeability coefficients and activation energies. A unique feature for permeability studies using DIB systems includes ease of bilayer formation, and sufficient longevity, stability, and reproducibility to provide direct observation of water transport at various temperatures across a single unsupported bilayer. DIBs offer several advantages over other prominent methods for measuring water permeability in model membranes. In particular, droplet interface bilayers offer ease with which bilayers may be formed, and offer great versatility for the controllability of parameters when exploring the effects of the membrane lipid structure upon bilayer transport. DIBs allow for direct visual observation of shrinkage and swelling of droplets, so that the quantity of water transported across the bilayer area into the droplet of higher osmotic pressure can be observed by volume change. Quite frequently, in contrast, the methods employed for determining permeability using vesicles rely on indirect size measurement methods such as light scattering or the self-quenching of an entrapped fluorescent probe, which could leak from certain liposomes. In DIBs, the dimension of the bilayer can be deduced directly through microscopic monitoring of the geometry of the droplets (see the section below for more detailed explanation for droplet dimension analysis); in this way, one can ensure that the bilayer area will remain substantially constant throughout a defined duration of the water filtration process. For vesicles, the membrane area would be constantly changing during shrinkage or swelling. For planar bilayers, bilayer area is limited by the accuracy of formation of an orifice to support the bilayer. In contrast to the prior techniques that employ tracers to determine water permeability across planar supported bilayers, the osmotic water filtration across DIBs depends upon the dynamic mass transport of water between micron-sized droplets, which would mitigate any unstirred layer effects. DIBs can be assembled readily under mild conditions of juxtaposing aqueous droplets, for simplicity of experimental design. Finally, in principle, a droplet interface bilayer system is capable of forming an asymmetric bilayer for enhanced biological relevance, as was employed in our recent paper.31 In general, the water permeability of the lipid bilayer can be used as a probe of the physical state of the membrane, since aggregate transport numbers depend upon the arrangement of the ensemble of component lipids. The study of water transport process of membranes is of considerable importance and may shed light on the underlying lipid bilayer structure responsible for both permeability and overall function of the membrane. Towards this goal, this study presents 4


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an investigation of the effect of acyl chain structure in amphiphilic monoglycerides on the permeability of water across DIB membranes composed of these monoglycerides. The amphiphiles investigated in this study comprise one unsaturated acyl chain, where the acyl chain length, number of double bonds, and the position of double bond are varied systematically along the acyl chains. We believe this is the first time that both permeability values and activation energies have been extracted for water transport across a lipid bilayer formed of such homologous series of lipids, under a controlled experimental platform, allowing us to make ready comparisons between the different lipids and potentially better elucidate the contributions that molecular motifs make to the permeation process. This may satisfy the need in the field for accurate lipid structural properties, heretofore frustrated by the sometimes-conflicting results found in the literature, to delineate the exquisite interplay between membrane properties and structural components and understand the balanced contribution of each.32 Experimental Section System of Study For the formation of droplet bilayers, we have chosen to employ, as lipids, a series of 1monoglycerides (MG) having different chain length (1424) in the acyl chain, and with varying degrees and types of double-bond unsaturation (Table 1). These lipids are characterized by their high solubility in hydrocarbon oils, surface active behavior, and, especially, their ease in rapid formation of droplet bilayers. 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 depicted in this Table 1 as an exemplary MG; the stereochemistry is arbitrary as we employ racemic mixtures in all of our MG studies herein).

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Table 1. Monoglycerides (MG) Used in This Study

C18:1 Δ9 cis Chain Length: Degree of Unsaturation C14:1

Position of Unsaturation Δ9 cis

Nu-Chek Product No. M209

Bulk Melting Point (℃) 21.0

C16:1

Δ9 cis

M219

21.9

C18:1

Δ6 cis

M229

39.8

C18:1

Δ9 cis

M239

35.9

C18:1

Δ11 cis

M249

36.4

C18:2

Δ9, 12 cis

M254

13.9

C18:3

Δ9, 12, 15 cis

M264

11.4

C18:3

Δ6, 9, 12 cis

M269

-39.2

C20:1

Δ11 cis

M274

42.5

C20:2

Δ11, 14 cis

M284

26.5

C20:3

Δ8, 11, 14 all cis

M289

8.9

C20:3

Δ11, 14, 17 all cis

M294

22.8

C20:4

Δ5, 8, 11, 14 all cis

M299

-35.7

C22:1

Δ13 cis

M304

49.5

C24:1

Δ15 cis

M319

54.3

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; "SqA"), which was used as the immiscible organic (oil) phase, and all other chemicals (of the highest purity available) were purchased from Sigma-Aldrich and used without additional 6


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purification. All samples were stored at -20 ℃ until use and freshly prepared immediately before the experiment. The monoglyceridecontaining organic phase (squalane) was prepared by dissolving an appropriate amount of monoglyceride directly into squalane, followed by brief vortexing and bath sonication for ∼1030 min above the melting point of each monoglycerides until a clear solution is achieved to ensure a homogeneous mixture. For all of the experiments conducted herein, the total monoglyceride concentration in the solution was approximately 12 mM. Aqueous solutions using osmotic agents (NaCl at nominally 0.1 M) were prepared from purified, deionized water (18.2 MΩ·cm) 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 solutions were freshly prepared each time prior to use. 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. The bulk melting temperatures of monoglycerides were determined using a Texas Instruments Q2000 DSC with a rate of 5 °C min−1. All monoglycerides (MG) used in this study were prepared in amber glass vials immediately prior to use. 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. However, if any vortexing or bath sonication was conducted on monounsaturated or 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 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).33 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.34

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System and Procedure for Permeability Measurement using DIB Method A detailed description of our system has been reported.30 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 (Figure 1). The recorded videos and images were analyzed subsequently to measure the dimensions of droplets and corresponding volume changes using custom-built image analysis software. Images were collected with a pixel size of 0.16 μm using the entire field of 1920 × 1080 pixels. Thus, the uncertainty in any measurement of droplet diameter is 0.32 μm (2 pixels × 0.16 μm/pixel). We generally have maintained droplets in the diameter size range of ∼100 μm for all experiments. Hence the error associated with the diameter measurement is 0.32%, which propagates to 0.55% relative uncertainty in the calculation of volume of a droplet. This error is far smaller than the reproducibility of permeability measured for each experiment. The reported value in this study represents the average result of at least 30 individual permeability experiments for each monoglyceride system. In order to derive bilayer (contact) area from the imaged droplet sizes, the geometric methodology developed by Dixit et al. was employed.26 In accordance with this method, values for droplet volume and radius of the bilayer were determined by fitting circles to the droplet outlines (while taking care to correct for tilt in the bilayer interface as well as truncations at the bilayer and substrate surfaces). From the geometry of the droplet outlines can be derived the true center-to-center distance and the bilayer contact angle, and finally the bilayer radius. Each experiment took place over a time course (∼5−10 min) for osmotic water movement across the droplet bilayer, during which time the droplet contact area remains constant. All droplet pairs had substantially the same initial size relative to each other, in the diameter range of 100 ± 5 μm. While this method is not dependent on the initial droplet size regime, our choice of ca. 50 μm radius droplet provides a convenient time scale for each experiment (up to ~10 min for each permeability measurement), since the time required to observe a given volume change is proportional to r3, where r is initial radius.

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Figure 1. 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; and (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 lipids. Each micropipet used for the formation and manipulation of aqueous microdroplets was prepared using a commercially available micropipet puller and subsequently hydrophobized using hexamethyldisilazane [(CH3)3SiNHSi(CH3)3); HMDS] to inhibit wetting of the micropipet glass surface by an aqueous solution. To achieve a hydrophobic coating of the micropipet, about 2 to 3 drops of HMDS was added to the center of an enclosed container having freshly pulled micropipets inside, and held for at least 30 min. The HMDS must be handled using gloves in a fume hood and stored in a refrigerator. It is recommended to read the MSDS information on handling HMDS. When two osmotically imbalanced droplets were made to adhere at a bilayer, water transport immediately commenced (Figure 2). Figure 2 shows typical sequential images of DIB pairs formed by a monoglyceride membrane undergoing an osmotic water transport process, for two systems exhibiting two dramatically different permeability rates: one (a) which is relatively slower than the other (b). This is a manifestation of the direct visual observation of water transport across a single unsupported bilayer formed at the contact zone between two droplets. The rightmost droplet in all images in Figure 2 contains NaCl (0.1 M) with an accurately known osmolality of 9


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approximately 200 mOsm/kg. The osmotic gradient drives water transport through the droplet bilayer, and the direction of water transport is shown with the arrow on the image. Any electrolyte flux is expected to be negligible compared to that of water, as typical ion permeation is almost 10 orders of magnitude slower than that of water. Changes in droplet size due to this water transport were thus measured from the commencement of the process. Control experiment using osmotically balanced droplets was performed to confirm that no volume change occurs without an osmotic imbalance. With respect to the stability of DIBs formed from unsaturated monoglycerides, we have found them to be exceeding stable: DIBs formed from unsaturated monoglycerides in squalane oil have a far greater lifetime (up to one day) than each experimental time (~ 5 minutes). The failure rate of bilayer collapse has been minimal. (a) pure water salt water

t=0 s

t=300 s

t=0 s

t=240 s

(b)

Figure 2. Image sequences for typical DIB permeability measurement are shown. The osmotic gradient between two droplets is set to 200 mOsm/kg. The arrow on the image indicates the direction of water transport. Image (a) shows water transport through DIB made from MG of C20:1 Δ11 in Squalane at 30ºC at t=0s and t=300s. The permeability coefficient for this system is calculated to be ~55 m/s. Image (b) shows water transport through DIB made from MG of C20:4 Δ5, 8, 11, 14 in Squalane at 30ºC at t=0s and t=240s. The permeability coefficient for this system is calculated to be ~260 m/s. The scale bar on the image represents 100 μm.

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Figure 3 (a) depicts the relative volume change (V/Vo) for both the swelling and shrinking droplets. Also shown is the sum of the volumes of the two droplets, which demonstrates that total volume remains constant during the duration of each experiment (~5 min). This is evidence of water transport mainly occurring through the droplet bilayer, with negligible water dissolution into the surrounding squalane due to very low solubility of water in this hydrocarbon. The reported solubility of water in squalane is 0.0055% at 25°C.35 Hence, measuring the changes in droplet diameter as a function of time (dV/dt) allowed us to determine water transport through the bilayer contact area, which is then used to determine water permeability coefficient based on the following relationship (1): (1) where A is geometric bilayer area, νw is the molar volume of water, ∆C(t) is the time-dependent osmolarity gradient between two droplets, and Pf is the bilayer permeability coefficient of water. When the bilayer contact area is constant, as we have ensured in our studies here, the time evolution of the swelling droplet can be obtained from the following equation derived from the integration of eqn. 1, with the simplifying assumption that: since one of the droplets (the shrinking droplet) contains no osmotic agent, its concentration does not change:36

V 2 2Pf AvwCo      t + 1 Vo   Vo 

(2)

Using the known (measured) values for: initial size of the osmotic (swelling) droplet; bilayer contact area (A); and initial osmolarity of the osmotic droplet (Co), then the coefficient Pf for bilayer water permeability may be derived from eqn. 2 from the slope of the curve obtained by plotting (V/Vo)2 as a function of time. All data presented is an averaged (n ≥ 30) time course (∼5  10 min) of osmotic water movement in the droplet bilayer, during which time the droplet contact area remains constant. This is demonstrated in the data shown in the solid triangles in Figure 3a. Figure 3b shows the square of the relative volume change versus time for ~5 min for C20:1 DIB vs C20:4 DIB in squalane at 30°C; dramatically different rates of relative volume change over time indicates different permeability coefficient for these two systems. 11


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(a)

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(b)

Figure 3. (a) Relative volume change for a pair of droplets over time (Vo is initial volume) for swelling droplet (filled circles), shrinking droplet (open circles), and total volume (hatched circles) over the time course of 5 min. The DIB radius during the same time period remains constant (solid triangles). (b) The relative volume change squared vs. time for DIB formed by C20:1 vs C20:4 in squalane at 30°C. The corresponding images are shown in Figure 2. Activation Energy Determination for Water Permeabilities In order to determine the activation energies (Ea) for water permeation process, permeability coefficients were measured at four to six different temperatures between 25 °C and 40 °C. For those monoglyceride having an acyl chain length of 22–24 (C22 and C24), the upper limit for the temperature range was 60 °C. The results are presented as an Arrhenius plot from which the activation energy was determined. 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.

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Results and Discussion 1. Water Permeability and Acyl Chain Length Figure 4 shows water permeability coefficients at 40 C for monoglyceride (MG) membranes having one cis double bond with varying acyl chain length. Our results show that overall, water permeability values change as a function of acyl chain length of monoglycerides. This trend holds true at all measured temperatures but here we report the values for 40 C to increase physiological relevance. The permeability coefficient gradually decreases with increasing acyl chain length, about 10 times lower value moving from MG of C14 to MG of C24; from 305 ± 20 µm/s for the shortest acyl chain length of C14 MG, to 32 ±12 m/s for the longest acyl chain length of C24 MG investigated in this study at the same temperature. Temperature dependence of water permeability coefficient of the same series of MG has been measured and activation energy (Ea) for the water permeation process was determined (Figure 5a and b). As acyl chain length in MG increases, the activation energies (Ea) were found to be markedly increasing: from 7.7 ± 0.5 kcal/mol for C14 MG to 17.0 ± 1.2 kcal/mol for C24 MG. Our results shown in Figures 4 and 5 indicate that the MG membrane represents a higher resistance to transport of water molecules as acyl chain length increases.

Figure 4. The water permeability coefficient (m/s) of mono-unsaturated monoglycerides with varying acyl chain length measured by DIB method in squalane at 40 C.

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(b)

(a)

Figure 5. (a) Temperature dependence of water permeability: a plot of the natural log of the permeability coefficient (Pf, expressed as 10-5 m/s for scaling) versus the reciprocal of absolute temperature, T (scaled to give abscissa values greater than unity) and (b) Activation energy (kcal/mol) for a series of mono-unsaturated monoglycerides with varying acyl chain length measured by DIB method. It is generally accepted that as acyl chain length increases, the thickness of the bilayer concomitantly increases linearly for both monoglycerides and phospholipids.32 Figure 6 shows a plot illustrating the relationship in the present study between the hydrocarbon thickness of the bilayer and the observed water permeability coefficient (Fig. 6a) and activation energy (Fig. 6b). The thickness of the bilayer is based on the reported literature thickness values of the hydrophobic (or hydrocarbon tail group) region of the bilayer for monoglycerides with monounsaturated hydrocarbon chains from carbon atom 14 to 22 with cis double bonds

37

These values were

recovered from high-precision capacitance measurements on planar lipid membranes composed of the involved monoglyceride and dispersed in squalene, a 30-carbon hydrocarbon solvent reported to promote the formation of "solvent-free" bilayers.38 Note that although capacitance measurements of bilayer thickness can only provide the thickness of the hydrophobic region of a bilayer, to the partial or complete neglect of the thickness contribution of the headgroups, this hydrocarbon tail group thickness may be representative of the portion of the bilayer which most 14


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acts as a "barrier" to water transport.39 The literature has in fact performed a critical comparison of capacitance measurements for bilayer thickness, versus optical reflectance methods for bilayer thickness determination.40 It was found that the two methods are consistent with each other, except that the optical reflectance method gives a thickness that is about 0.5 nm greater on average, which is an expected thickness contribution from the polar regions of the monoglyceride lipids studied. For purposes of this study, references to "bilayer thickness" generally will refer to the literature thicknesses measured for the same MG lipids by capacitance measurements, meaning, thickness of hydrocarbon region only. The present permeability values, when expressed as ln Pf, display a nearly linear negative relationship to hydrophobic-region thickness, becoming noticeably smaller with increasing bilayer thickness (See Fig. 6a). Akin to this, the activation energy Ea for the water permeation process, plotted as a function of the hydrocarbon-chain region thickness, exhibits a monotonic increase with increasing thickness, which is also approximately linear (Fig. 6b). (a)

(b)

Figure 6. Dependence of (a) log [water permeability] and (b) activation energy on thickness of hydrophobic region of membrane. Blue circle data points are from this work, as a function of thickness of monoglyceride bilayers (from C14 to C22, thickness data are taken from Ref 37). These data suggest a striking demonstration of the effect of the hydrocarbon-tail length of a lipid upon the net passive permeability of a water molecule through a lipid bilayer. In order to provide some context for these data, it would be useful to compare to the body of permeability values of prior investigators. However, the range of literature experimental values for water permeability measurements has been wide, owing to the various methods of determination (e.g., 15


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osmotic, NMR, radiotracer), and the difficulty to estimate membrane area, and volume. Also, numerous studies have been performed on biological membranes which have inserted proteins, which may inhibit the determination of passive permeability.41 Thus, while the quantitative evaluation of permeability values as a function of acyl chain length are difficult to compare, the overall trends observed in our studies seem to be consistent, qualitatively, with what has been observed in previous experimental and theoretical studies by various research groups. Fettiplace investigated water permeability for black lipid membranes of MG of C18:1 and C22:1, and reported that membranes formed from monoglycerides of longer chain length were thicker and had lower permeabilities.13 It was postulated that changes in thickness of -- and fluidity within -- the bilayer will both influence the permeability, insofar that fluidity may be altered in like manner to the increase in viscosity for longer-chain hydrocarbons. However, these studies are incomplete in that they were based on bilayer membranes which contain decane, a solvent known to be retained in appreciable quantities within the bilayer. Thus the increased thickness and reduced permeability may not be solely attributable to the longer hydrocarbon tail length,13 since the retention of decane solvent will contribute to thickness as well. In our "solvent-free" situation, we may be more confident that the MG tails of higher carbon number should have increased thickness owing solely to the tailgroup itself. Paula et al.10 measured the permeability coefficients for bilayer transport of small molecules as a function of membrane thickness, by study of the kinetics of liposomal volume change under osmotic gradient observed by scattered light intensity. In a manner similar to Fettiplace, Paula et al. concluded that the permeability of water and neutral small molecules showed modest dependence on the membrane thickness. In particular, Paula's study focused on water permeability at 30 oC through bilayers composed of phosphocholine lipids with chain lengths of 14–24 having one site of cis-unsaturation. The permeability for lipids of carbon length 24 was found to be five times lower than for carbon length 14. The paper includes estimates of the dependence of water permeability upon bilayer thickness, for both a solubility-diffusion model, and for a pore-dependent model. The experimental result, which indicated that although thicker bilayers indeed had lower permeability, it was still within the same order of magnitude. This was taken to be inconsistent with a rival pore model, the latter which would require a nearly exponential decrease in permeability upon thickening of a bilayer. These rival models are discussed in somewhat more detail below. 16


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A recent theoretical study using MD calculations by Sugii et al. studied the effects of the lipid hydrocarbon chain length of saturated phosphatidylcholines (DLPC, DPPC, DSPC) on the permeability of H2O, O2, CO and NO.9 The calculations demonstrated that lipid membranes with longer chains display wider free energy barriers. Yet, the local diffusion coefficients in the bilayers were not altered significantly by the chain length. They also estimated the water permeability coefficient and found that it decreases slightly with increasing chain length. The overall thrust of Sugii's study seems to lay in there being a wider (although not higher) free energetic barrier in water transport with increasing chain length, even though the local diffusivity (i.e., diffusion constant in any one location of the bilayer) for the test water molecule was unchanged. Similarly, Pan has discussed42 that lipids having longer chains have greater order than shorter chains, given the same degree of unsaturation, since longer chains will have larger van der Waals cohesive interactions. It appears, then, that our activation energy Ea values which progressively increases with increasing chain length of the hydrophobic tail may be a reflection of the greater van der Waals attraction between tail groups which are in proximity to one another within the bilayer.32 Such cohesiveness should provide barrier function against water transport. For completeness' sake, we note that a contrasting work exists, that of Jensen and Blume,8 who investigated the permeation characteristics of DMPC, DPPC, and DSPC bilayers, and they found that variations in the thickness of the hydrophobic region had only a minor influence on water permeability, the major observed effects on water permeability being a result of the size and charge of the lipid head group, the type of chain linkage, and the chemical structure of chains. However, the studies were for saturated PC, some of which were in gel phase under the conditions studied.8 Models for the transport of water through bilayer membranes include solubility-diffusion models and pore models. Descriptions of the solubility-diffusion mechanism for water permeability across a lipid bilayer have envisioned the bilayer membrane to be thin slab of material with characteristics of a liquid hydrocarbon (hydrophobic matter), separating two aqueous regions. A permeating molecule (from the first region) will cross the headgroup region to partition into the hydrophobic region, and cross by a diffusion process to depart the bilayer by re-partitioning into the second aqueous region.43 A form of the integrated expression for the permeability coefficient of bilayer transport is: P = KD/d

(3) 17


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where K is the water/liquid hydrocarbon partition coefficient of the permeating molecule, D is diffusion coefficient for the permeant, and d is bilayer thickness. The diffusion coefficient of water has been measured in hydrocarbons,35 which may provide a reasonable approximation for D, but actual coefficients of diffusion within the bilayer per se are not easily accessible by experiment. The bilayer thickness is given as d, which equals the thickness of the hydrophobic region, the assumption being that the hydrophobic region of the bilayer constitutes the bulk of the "barrier region" of the bilayer.39 If the membrane thickness as well as the diffusion and partition coefficients of the permeating species are known, the permeability coefficient can be calculated. The systematic studies of Walter et al. in 198644 sought to answer pertinent questions regarding the mechanism for permeation of solutes across a bilayer, including determining the rate-limiting step for translocation (whether into the bilayer or diffusion across the bilayer), and determining the hydrophobicity of the rate-limiting barrier. Their results indicate that very small solutes permeate 2–15 times faster than predicted by their solubility alone (Overton's rule). All of their results were said to be consistent with the solubility-diffusion model. An alternative mechanism to the solubility-diffusion description has been proposed, which suggests that permeation across a bilayer membrane occurs through hydrated transient defects produced by thermal fluctuations.8, 45, 46, 47 By passing through pores, the permeating particle can avoid the Born energy barrier associated with the solubility-diffusion mechanism. Furthermore, protons can be translocated by a mechanism along water "wires" spanning the membrane in such defects. Because this process is intrinsically much faster than diffusion, the transient pore model can account for the high permeability observed for protons. According to the pore model, transient defects should become significantly less common as bilayer thickness increases, leading to a pronounced drop in permeability. The solubility-diffusion model, however, predicts a much smaller effect of bilayer thickness on permeability.43 One prominent further school of thought has set forth evidence that water permeability through bilayers of different lipids is most strongly correlated with the area per lipid, rather than with other structural quantities such as the thickness, saturation, or composition of the headgroup.11, 48 These papers present a three-layer theory that incorporates the area dependence but also includes the thickness as a secondary modulating parameter. Thus, we consider here whether area per lipid may have any relevance to our observed results.

18


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For the case of saturated PC lipids, Kučerka has discussed that a general consequence of increased acyl chain length was a decrease in lipid area, probably due to the attractive van der Waals forces between hydrocarbon tails.32

Similarly, it has been detailed how increased

hydrocarbon chain length results in increased van der Waals attraction, which in turn leads to an ordering of the hydrocarbon chains that effectively reduces the area/lipid. 49 The data regarding monounsaturated phospholipids appears to point to a non-monotonic behavior. Pioneering work by Lewis and Engelman50 estimated values for lipid area in PC bilayers for monounsaturated PC of n = 18, 22, and 24 (both chains of equal length and monounsaturated). All values fell in the range of 0.68–0.70 nm2, with molecular area decreasing slightly for increasing n. This range for change in area was considered as "almost constant" by these investigators. Kučerka & Gallova et al.49 recently studied the structural properties of bilayers formed of monounsaturated diCn:1PC, where n = 14, 16, 18, 20, 22, and 24, by simultaneous analysis of high-resolution x-ray and contrast-varied neutron scattering data. Surprisingly, lipid area changes as a function of n by first increasing up to n < 18 and then decreasing with n > 18, with a maximum near n = 18. This behavior was explained in terms of the position of double bond: for n = 14, 16, 18, the double bond is at 9–cis, while for n = 20, 22, 24 bilayers, the double bond is Δ11, Δ 13, and Δ 15 (all cis). Since lipid chain disorder is postulated to be a function of double bond position,51 the lipid having double bond in the middle of the hydrocarbon chain (C18) would be most disordered. Thus, for phosphocholine double-chain lipids with monounsaturated chains, chain length does not have a simple influence on area per lipid. In the case of bilayers prepared from monoglyceride lipids, there exists some area per lipid data pertaining to the effect of MG chain length upon the packing of the lipid molecules in the bilayer. Waldbillig has prepared "solventless" planar lipid bilayer membranes using monounsaturated monoglyceride amphiphilic lipids dispersed in triglyceride oils.52 The triglyceride molecules are of sufficient molecular bulk as to be excluded from the bilayers, to form a nearly solvent-free planar bilayer. This reference postulates that the planar bilayers formed using monoglyceride lipids in triglyceride oils, likely contains even less solvent than one prepared from a lipid dispersion in squalene (C30H50), heretofore considered the standard type of oil for forming "solvent free" planar bilayers.38 The resulting thin membranes (ascertained by high-precision capacitance measurements) were used to estimate the lipid area per molecule for monoglycerides

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in the bilayer itself. A slight negative dependence (area decrease with increasing chain length) of area per lipid upon monoglyceride acyl chain length may be discerned as shown in Table 2. Table 2. Area per molecule for various monoglycerides 52 Monoglycerides (MG)

Area per Molecule (nm2)

C16:1 MG

0.413

C18:1 MG

0.418

C20:1 MG

0.381

C22:1 MG

0.373

These values are similar to that reported (0.42 nm2) for planar bilayers of C18:1 MG in hexadecane, in which an aliquot of a planar bilayer itself was sampled by passage of an Hg microdroplet therethrough.53 However, the Waldbillig study has cautioned that the calculation of area per lipid depends on an assumption regarding value of lipid chain molecular volume (which itself may be a function of packing). Regardless, the area per lipid seems to have a maximum at C18:1, in which the site of cis-unsaturation is at the middle of the chain (Δ9). In sum, then, the prior data surrounding dependence of monounsaturated lipid packing in bilayers upon chain length seems to converge on there being non-monotonic change of area per lipid as a function of chain length. Therefore, despite the fact that areas per lipid are not available for the monoglycerides used here employed in the squalane solvent of the present study, it appears to be a presumption that a similar trend is entailed in the present case. Permeabilities are not correlated with the available area data. In principle it is possible that the bilayers may thin with increasing T, as is seen with phospholipid bilayers.54 However, this may not necessarily carry over to monoglycerides, owing to a type of headgroup which is much simpler (the headgroup is a 1,2-diol for monoglycerides and a phosphocholine for PC lipids) and smaller (38.4 Å2 at 30 °C vs. DOPC which is 72.4 Å2 at the same temperature). But, the monoglyceride literature reveals that the thickness of the glyceryl monooleate bilayer is not very sensitive to increase of temperature (thickness decrease over the range of 20 – 40 °C was a mere 0.007 Å/K).38 As for area per lipid, the solvent-free planar bilayers formed of glyceryl monooleate had a molecular area expansivity of 0.05 Å2/K, while Pan et al.54 found a molecular area expansivity of 0.21 Å2/K for DOPC. Thus, we feel confident that our Ea values (which required Pf measurements at increasing T) are not spurious artifacts of a putative temperature-induced thinning of the bilayer. 20


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The significance of the present studies lay in them being the first systematic investigation of the energetics of water permeability through lipid bilayers as a function of chain length (and hence, thickness) of the lipids from which the bilayer is composed. We have derived the classical Arrhenius activation energy Ea for the process, which has a direct relationship to the enthalpy of activation ΔH for the water permeation process,55 via the relation of Equation 4: Ea = ΔH + RT

(4)

The enthalpic change for water permeation is believed to be representative of the losses which are concomitant to the desolvation of water when it is removed from its bulk water phase and enters the hydrophobic interior. It would be valuable if one could make direct comparisons between the present Ea values and the growing body of computed values for free energy of activation for water permeability across bilayers (ΔG). Indeed, computational studies pertaining to water permeability across lipid bilayer membranes have recently been reviewed. These studies usually rely largely on an inhomogeneous version of the solubility-diffusion model, and compute resistance to water transport as an integrated summation of free energy profile of the water molecule within the bilayer, coupled with its positional-dependent diffusion coefficients.56 However, it is not a straightforward task to experimentally evaluate free energy of activation for the water permeability process across membranes. Therefore, our discussion will be limited to a qualitative assessment of the significance of the available Arrhenius activation energy data. Recalling the MD study of Sugii et al.,9 in which a free energy profile was calculated for the transport of a water molecule through saturated PC lipid bilayers of varying chain length, we find that there is but a very slight difference in the free energy barrier for the molecule to traverse the bilayer: at most, the difference in barrier height for the respective lipids is ~2 kJ/mol (< 0.5 kcal/mol). If this value is considered to be negligible, then there should be essentially no differences in free energy of activation (ΔG) for water permeability for bilayers composed of lipids of different chain length. The free energy of activation is related57 to the enthalpy of activation by the following relation in Equation 5: ΔG = ΔH – TΔS

(5)

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If the free energy differences for different chain length is essentially nil (as per Sugii), then our observed increases in Ea (and thus ΔH) for increasing chain length should be accompanied by a concomitant relative increase in entropy of activation for the same process. We are endeavoring to perform similar MD computational studies for bilayers formed from monounsaturated monoglycerides, in order to clarify whether the results of Sugii are valid for our system. Since in the present study, the observed values for Ea is more than doubled (progressively increased from 7.7 kcal/mol to 17 kcal/mol) upon going from MG 14:1 to MG 24:1, then it could be presumed that the activation entropy would also have to increase in order to keep the ΔG at a constant value, given Sugii's result that the free energy difference for different chain length is essentially nil. Such a progressively increasing activation entropy would be an appealing result, based on the common presumption that the ordering in the tailgroup structure should increase for bilayers made of lipids of increasing tail length. Since the traverse of a single water molecule through a bilayer, while permeating, should result in the disruption of the ordering of a bilayer (if porosity is assumed to not be the mechanism),55 then activation entropy would perforce increase for increasingly ordered bilayers. An increase in x-ray chain order parameter (Sxray) for bilayers monounsaturated PC lipids has been seen42 for the comparison of DOPC (two monounsaturated C18 chains) to diC22:1 PC, where Sxray increases from 0.25 to 0.4. Therefore, it seems reasonable that an increased activation entropy would be seen for a disruptive permeant species upon traversing an increasingly ordered assemblage of lipids. However, a postulated relative increase in activation entropy does not imply that the activation entropy of the entirety of the permeation process, is a positive value. The total activation entropy should be a sum of (a) the entropy for the partition of water from an initial position in the bulk water to a position inside the hydrocarbon core of the bilayer (ΔSº); and (b) the entropy for the process of diffusion of water through the bilayer interior (ΔS): ΔSº + ΔS. Although the value of ΔSº will be negative, since it corresponds to a water molecule being partitioned away from a bulk location where it enjoys a greater number of degrees of freedom, it remains a difficult task to acertain the absolute magnitude for each of ΔSº and ΔS, respectively. Nevertheless, it may be possible to estimate differences in total activation entropy, Δ(ΔSº + ΔS), for diffusion of water through fluid bilayers of different-acyl chain-length lipids, through the use of Eyring-Zwolinski absolute reaction rate theory for diffusion of a solute through a bilayer.58, 59 According to this theory, the diffusion process is modeled as a series of successive jumps of solute from a first 22


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equilibrium position to a second position. If a boundary condition is imposed upon the theory, such that the main barrier for water permeation is passage through the hydrocarbon core rather than the interfacial region, then the Eyring-Zwolinski equation becomes: P=(λ2kT/dh) * exp(ΔS/R) * exp(-ΔH/RT) where λ is the distance between the successive equilibrium positions (usually assumed to be about 0.25 nm), d is membrane thickness, k is Boltzmann constant, h is Planck constant, R is gas constant, T is absolute temperature, ΔS is entropy of activation and ΔH is enthalpy of activation, respectively, for permeation. Then, by plotting ln(P/T) vs. 1/T, the slope should be ΔH/R and the intercept will be ln(λ2kT/dh) +ΔS/R. For our data, we plotted ln(P/T) vs. 1/T for bilayers in the monounsaturated MG series (chain length 14-22) and ascertained the differences in the respective intercepts. We found that the apparent ΔS would progressively increase for increasing thickness of the bilayers. The difference in apparent ΔS increased by about 50 J/mol-K per nm of greater thickness in the hydrocarbon region in the bilayer. However, the applicability of the Eyring-Zwolinski theory to lipid bilayers likely suffers from severe inadequacies, including the assumption of an arbitrary value for jumping distance  that would be the same for different bilayers; the neglect of interfacial resistance to water entry in the bilayer; and assumption that area per lipid is unchanged. Still, the derived result for relative difference in entropy may be suggestive of reversible loosening of acyl chain segments arising when water molecules are permeating in a bilayer. If a lipid with longer acyl chain has a larger number of attractive interactions between tailgroups, these interactions would be reversibly disrupted, leading to greater entropy of diffusion manifested with longer chain lipids. The above-described method has been used by Petersen60 to estimate the entropy of activation for water transport across a planar bilayer composed of glycerol monooleate/nhexadecane bilayer, in comparison with activation entropy for water transport through hydrocarbon liquid: their difference in ΔS for the bilayer process was about 63 J/mol-K more positive than for permeation through hydrocarbon liquid. Their relatively more positive value for the apparent ΔS for the black lipid bilayer was attributed to be the result of water transport through the bilayer necessiating the disruption of the alkyl chain order.

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2. Water Permeability and Unsaturation of Acyl Chain 2.1 Effects of number and position of double bonds Figure 7 a–f shows the values which were obtained for permeability coefficients of osmotic water transport (7a, 7b); temperature dependence of water permeability coefficient (7c, 7d); and activation energy (Ea) for the water permeation process (7e, 7f), each employing MG lipid having acyl chain length of C18 and C20, with increasing degree of acyl chain unsaturation. Regardless of whether the MG had an acyl chain length of C18 (as in Figure 7a) or C20 (as in Figure 7b), we observed that an increasing degree of chain unsaturation would dramatically enhance water permeability across these membrane bilayers. Specifically, a roughly three-fold increase (from ~90 m/s to ~260 m/s) was seen for the case of C18 MG going from one double bond to three double bonds. Similarly, an about four-fold increase was observed (from ~70 m/s to ~300 m/s) for C20 MG when comparing a monounsaturated MG to a tetraunsaturated MG. These increases in value of permeability coefficient for increasing unsaturation were accompanied by a concomitant smooth, but modest, decrease in activation energy, as shown in Figure 7c–7f. What was observed was an overall decrease in activation energy for increasing degree of double bond unsaturation. We also investigated the effect of the position of double bonds on the permeability values and activation energy. For C18 acyl chain 1-monoglyceride, three positional isomers were investigated, all of which had a sole double bond: MG of C18 with a double bond in position 6, 9, and 11. The permeability values at 35 C did not exhibit significant differences for these monoglycerides, being 87, 95 and 91 m/s, respectively. However, the values for activation energy exhibit a slight enhancement for the positional isomer having a double bond in 6 position: C18:16 MG had activation for water permeability of 10.7 kcal/mol, whereas the respective values were 9.9 and 10 kcal/mol for C18:19 and C18:111. Acyl chains of 18 carbons and having three cis double bonds (i.e., C18:3) are commonly found as a component of phospholipids in many biological membranes.5 Ordinarily, the double bond positions are either at 6, 9, 12 (ω–6) or at 9, 12, 15 (ω–3). Both of these positional isomers have 18 carbons and three unsaturations and differ only in the arrangement (location) of their double bonds. Thus, in this study we compared water transport behavior for droplet interface bilayers composed of 1-monoglyceride lipids with the following acyl chains: MG C18:3 6, 9, 12 versus MG C18:3 9, 12, 15. Conceptually, one may consider these respective lipids to differ 24


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merely in the location of one of their double bonds, since each has double bonds in common 9 and 12 positions. From this vantage point, MG C18:3 6, 9, 12 has its "third" site of unsaturation to be proximal to the carboxyl head of the acyl chain, whereas MG C18:3 9, 12, 15 has its "third" site (15) as distal from the carboxyl head. Bilayers composed of the tri-unsaturated isomer with unsaturation in the 15 position appeared to have a relatively more facilitated water transport (Pf of 262 µm/s and Ea of 7.8 kcal/mol) as compared to MG C18:3 6, 9, 12 (Pf of 248 µm/s and Ea of 8.5 kcal/mol). These values are at 35 C.

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(b)

(a)

(d)

(c)

(d) (e)

(f)

26


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Figure 7. Osmotic Water Permeability (35 ℃) of monoglycerides of acyl chain length of (a) C18 and (b) C20 with varying degree of unsaturation (all cis). A plot of the natural log of the permeability coefficient (Pf expressed as 10-5 m/s for scaling) versus the reciprocal of absolute temperature, T, illustrating temperature dependence of water permeability of (c) C18 and (d) C20 with varying degree of unsaturation. Activation energy for water permeation through monoglycerides of acyl chain length of (e) C18 and (f) C20 with varying degree of unsaturation. A similar trend has been observed for tri-unsaturated 1-monoglycerides having a 20 carbon chain: the isomer MG C20:3 8, 11, 14 was compared with MG C20:3 11, 14, 17. Again, if the 11, 14 sites of unsaturation are considered as common to the two positional isomers, then the isomer with a more distal "third" site for unsaturation (MG C20:3 with 11, 14, 17) exhibited higher water permeability (Pf = 238 m/s) and lower activation energy (Ea = 9.1 kcal/mol) than MG C20:3 with 8, 11, 14 (Pf of 200 m/s, Ea of 9.9 kcal/mol). (b)

(a)

Figure 8. (a) Permeability coefficient (Pf) at 35 C and (b) activation energy (Ea) for a series of MGs having C18 and C20 with either 1, 2, 3 or 4 double bonds as a function of number of double bonds. Figure 8 shows permeability coefficient (Pf) (Figure 8a) and activation energy (Ea) (Figure 8b) for a series of MGs having C18 and C20 with any of 1, 2, 3 or 4 double bonds, set in relation to the number of double bonds. The positional isomers are also shown, where available. This plot 27


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indicates that Pf for water transport (at 35 ºC) increases roughly linearly with increasing number of double bonds, on the order of ~80 m/s per each additional double bond, for both C18-MG and C20-MG (Figure 8a). A common trend is also seen in which the additional double bond lowers Ea: on the order of 1 kcal/mol per additional double bond (Figure 8b). It is also interesting to note that in each positional isomers of MG chain length of C18 and C20, ω–3 tri-unsaturated shows greater permeability with lower activation energy compared to ω–6 of the same chain length.

Figure 9. Activation energy as a function of acyl chain length of monoglycerides of various degree of unsaturation. Mono-unsaturation is represented with solid circle, poly-unsaturation is shown with open circle (two double bonds), hatched circle (three double bonds), and solid triangle (four double bonds). Figure 9 is a recapitulation of all the present results for activation energy of the water permeability process, in a graphical format which is designed to emphasize the enthalpic differences as a function of chain length, and as a function of unsaturation for a given chain length. As can be seen, filled circles depict the progressive increase in Ea for increasing acyl chain length for monounsaturated monoglycerides (MG) (note: we studied two positional isomers of mono-cisunsaturated C18 MG); whilst open circles represent Ea for di-unsaturated, hatched circles for triply unsaturated, and triangle for four double bonds. Similarly, Figure 10 summarizes the osmotic permeability coefficient Pf at 35 °C for MG with acyl chain C18 and C20, but with varying degree of unsaturation. An apparent increase in Pf is observed for increased number of double bonds. Generally, prior studies have indicated that increased rates of water permeation are related to differences in lipid packing.7,

8, 11, 16

For example, perturbation of lipid packing by chain 28


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polyunsaturation can influence water permeability. The overall activation energy of water permeation is found to be in the range of 7–12 kcal/mol for the system we studied, which is consistent with the studies using phospholipid reported by Huster et al.12 by

17

O NMR. The

combination of degree of unsaturation and the chain length of the unsaturated chain determine water permeation rate.

Figure 10. Permeability coefficient at 35 °C for acyl chain C18 and C20 with varying degree of unsaturation. Solid columns represent C18 and hatched columns represent C20. A reliable set of data exists for the effect of increasing polyunsaturation upon water filtration in PC vesicles. Olbrich et al.7 used micropipette aspiration of giant unilamellar vesicles to measure water filtration. They found that two or more cis-double bonds along a chain led to significant increases in (apparent) water permeability: PC C18:0/2 exhibited ∼49 μm/s, while PC diC18:2 (DOPC) gave values of ∼90 μm/s, increasing to ∼146 μm/s for diC18:3, all at 21°C. They concluded that the observed results were consistent with a concept relating water partitioning into the hydrophobic region of the bilayer on the one hand, and chain-ordering on the other. In the work of Huster et al.,12 diffusional water permeation rates across bilayers for polyunsaturated phospholipids was measured by

17

O NMR. The coefficients Pd were very

significantly increased upon introduction of greater unsaturation, although activation energies only increased slightly. They argued that the increase in Pd for polyunsaturated hydrocarbon chains correlates with less tight packing for these lipids, as well as a possibly deeper penetration of water 29


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into these bilayers, suggesting that polyunsaturation lowers resistance to water transport in the upper third of lipid hydrocarbon chains (i.e., distal from headgroup). Calculational studies of electron density within bilayers formed of highly polyunsaturated phosphocholine lipids (using MD) have uncovered a remarkably suggestive rationale for increased water permeability across such bilayers. Saiz et al. performed electron density calculations upon fully hydrated SDPC lipid bilayer (S=stearoyl, D=docosahexaenoyl), having six double bonds on one chain, and found significant overlap between the electron density attributable to water molecules (the water distribution), and the double bond region of the lipid chain. That is, water molecules were relatively localized near sites of unsaturation. The water molecules interact mainly with the unsaturated chain as compared to the saturated chain under identical conditions, which is put forth as a possible cause of enhanced permeability. The greater interaction may manifest itself in a more favorable thermodynamic component (i.e., solubility of water in the hydrocarbon region of the bilayer) of the solubility-diffusion model.61 Direct hydrocarbon-region thickness data are not available for bilayers composed of monoglycerides of increasing unsaturation in our solvent of choice (squalane). However, analogous studies have been performed,13 on monoglyceride (MG) planar lipid membranes formed from n-hexadecane, a solvent molecule which is substantially (although not completely) excluded from the bilayer. Glyceryl monooleate (MO), which is a C18:1–cis-Δ-9 MG, has a hydrocarbon region thickness (d) of 3.28 nm; for di-unsaturated ML (monolinolein) (C18:2), the value was 2.97 nm and for triply unsaturated MLL (monolinolenin) (C18:3), d=2.59 nm. Thus, these capacitance studies indicate that the hydrocarbon region of the bilayer undergoes a modest thinning upon increased levels of unsaturation. This thinning could also contribute to the greater facility of water transport for increasingly unsaturated MG.

Conclusion The study of water transport process of biological membranes is of considerable importance in view of understanding the underlying lipid bilayer structure responsible for both permeability and for the overall function of the membrane. Hence it is desirable to establish a consistent and reliable method for quantifying water transport through bilayers. Herein we have 30


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demonstrated that the relevant data are readily extracted by simple DIB methods. We have determined the effect of acyl chain structure of monoglycerides on the osmotic water permeability; the monoglycerides investigated include fifteen different kinds of lipids comprising acyl chain length of 14 to 24, with varying degree of unsaturation and different position of double bond. By systematically modulating the acyl chain of lipids with identical headgroup in each membrane, the thermodynamic parameters associated with the observed water permeabilities can correlated with chain structure. The results are consistent with the prevailing solubility-diffusion mechanism for water transport. The significance of the present studies lay in them being the first systematic investigation of the energetics of water permeability through lipid bilayers as a function of chain length (and hence, thickness) of the lipids from which the bilayer is composed. Acknowledgments The authors would like to acknowledge the financial support from the National Science Foundation (NSF-CHE-1609135). References 1. 2. 3. 4. 5. 6. 7. 8. 9.

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Table of Contents Graphic

Water Trasport through Droplet Interface Bilayer formed from Monoglycerides (MG)

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Enthalpic Effects of Chain Length and Unsaturation ... - ACS Publications

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