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Roles of Sterol Derivatives in Regulating the Properties of Phospholipid Bilayer Systems Tham Thi Bui, Keishi Suga, and Hiroshi Umakoshi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04343 • Publication Date (Web): 09 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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Roles of Sterol Derivatives in Regulating the Properties of Phospholipid Bilayer Systems Tham Thi Bui, Keishi Suga, Hiroshi Umakoshi* Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka 560-8531, Japan KEYWORDS: Cholestertol; Lanosterol; Ergosterol; Membrane property; Fluorescent probe; Fluid membrane ABSTRACT: Liposomes are considered an ideal biomimetic environment and are potential functional carriers for important molecules such as steroids and sterols. With respect to the regulation of selfassembly via sterol insertion, several pathways such as the sterol biosynthesis pathway are affected by the physicochemical properties of the membranes. However, the behaviors of steroid or sterol molecules (except cholesterol (Chl)) in the self-assembled membranes have not been thoroughly investigated. In this study, to analyze the fundamental behaviors of steroid molecules in fluid membranes, Chl, lanosterol, and ergosterol were used as representative sterols in order to clarify how they regulate the physicochemical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Membrane properties such as surface membrane fluidity, hydrophobicity, surface membrane polarity, inner membrane polarity, and inner membrane fluidity were investigated using fluorescent probes, including 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, 8-anilino-1-naphthalenesulfonic acid, 6-propionyl-2-(dimethylamino) naphthalene, 6-dodecanoyl-2-dimethylaminonaphthalene, and 1,6diphenyl-1,3,5-hexatriene, respectively. The results indicated that each sterol derivative could regulate the membrane properties in different ways. Specifically, Chl successfully increased the packing of DOPC/Chl membrane proportional to its concentration, while lanosterol and ergosterol showed lower efficiencies in ordering of the membrane in hydrophobic regions. Given the different binding positions of the probes in the membranes, the differences in membrane properties reflected the relationship between sterol derivatives and their locations in the membrane.

INTRODUCTION

Biomembranes are composed of various amphiphiles such as phospholipids and many types of sterols with hydrophilic headgroups and hydrophobic steroid bodies. The structural backbone of the natural membrane is constructed of a phospholipid bilayer embedded with molecules such as proteins, glycolipids, glycoproteins, and steroids.1 Membrane composition can differ depending on the organism, cell type, and membrane type.2 As a biomembrane model in the cell, liposomes have been shown to act as a physical boundary separating the inside of the cell from the outside, while also allowing the membrane to selectively interact with the environment.3 By responding to dynamic changes, lipid membrane protect the inner compartments e.g., genes, enzymes, and proteins from the environmental stimulation, including pH, temperature change, and oxidative stress. As reported previously, liposomes may induce a variety of processes under stress conditions: translocation of proteins across membranes,4 membrane fusion,5 and recognition of biomacromolecules.6,7 In addition, liposomes are regarded as effective carriers for nucleic acids such as DNAs and RNAs as well as drugs that are delivered to the target cells through endocytotic or direct membrane fusion pathways.8,9 It was recently reported that 1

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liposome membranes can affect gene expression in the in vitro Escherichia coli cell-free translation system, including transcription, translation, and folding steps,10,11 where cholesterol (Chl) plays an important role in regulating the membrane properties (e.g., fluidity, polarity, surface charge, and domain size). Based on the distribution of sterols in membranes, membrane characteristics can be modulated and applied in bioengineering to investigate various biological processes.12 Sterols are well-known as the third lipid class and contribute to the formation of liquid-ordered membrane states known as lipid rafts,13 as well as play crucial roles in regulating and changing membrane characteristics to create signals for biological processes such as protein translocation, gene expression, or biosynthetic pathway activation.14-16 Considering the roles of sterol molecules in lipid bilayer membranes, the diverse chemical structures of sterols may affect the regulation of membrane properties. In artificial membranes, Chl inserts into and interacts with the plane of the bilayer, with its hydroxyl group near the ester carbonyl of phospholipid molecules and the hydrocarbon tails extending toward the bilayer center;17-21 its tetracyclic ring structure lies near the phospholipid carbons at positions 2–10.22-25 Because of the close proximity of the planar sterol ring system, the hydrocarbon chains are ordered in the inner membrane. Chl interacts with the phospholipid membrane in the headgroup region through hydrogen bonds and with the hydrocarbon chains through van der Waals forces and hydrophobic forces.26-29 However, because of the complexity of the experimental methods and results obtained, few studies have examined these issues compared to studies of bilayer lipid membranes.30 Bilayer lipid membranes formed in aqueous environments (water and oil phases) have advantages over other methods because the composition, molecular packing, and physical state can be controlled. Studies of mixed lipid systems have focused on investigating the nature of biological membranes, which contain high amounts of saturated and unsaturated phosphatidylcholines (PC) such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC), respectively.31 Recently, the effects of sterols (e.g. Chl and lanosterol (Lan)) on the properties of DPPC membranes were compared based on thermal phase behavior,32 NMR,33 and fluorescence anisotropy measurements;34 the interaction of these lipids depends on the structure of the sterol side chain and the tetracyclic nucleus. In particular, they form a liquidordered (lo) phase in membranes and exhibit a condensing effect on different scales. Nevertheless, the effect of these sterols on the characteristics of unsaturated phospholipid (i.e., DOPC) membranes remains unclear because of their complexity, in contrast to that of saturated phospholipid membranes (i.e., DMPC or DPPC).24,32,35,36 In this study, we investigated the influences of Chl, ergosterol (Erg), and Lan on fluid membranes such as DOPC liposomes. To understand the mechanism underlying the interaction between phospholipids and steroids, the membrane properties of steroid-incorporated DOPC liposomes were analyzed using multiple fluorescent probes with different binding depths within the membrane and in each part of the membrane from the exterior to the interior. We estimated the binding depth (ɛ) of each fluorescent probe from the emission wavelength and emission intensity of probes in the phospholipid membranes (see supporting information Figure S1), using including 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5hexatriene (TMA-DPH), 8-anilino-1-naphthalenesulfonic acid (ANS), 6-propionyl-2-(dimethylamino) naphthalene (Prodan), 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), and 1,6-diphenyl-1,3,5hexatriene (DPH). Consequently, the membrane properties evaluated by the probes with different binding location could overview the affinity and location of sterols in the membrane. Thus, the roles of steroids in the membrane were quantitatively analyzed (Figure 1).

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Figure 1. Schematic illustration for the location of sterols or fluorescent probes in DOPC membrane. For the details of the fluorescent probe location, see supporting information Figure S1.

EXPERIMENTAL Materials. DOPC and DPPC were purchased from Avanti Polar Lipids (Alabaster, AL). Chl, Lan, and Erg were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were purchased from Wako Pure Chemical (Osaka, Japan) and were used without further purification. Liposome preparation. A chloroform solution of DPPC and DOPC with Chl, Lan and Erg in 10, 30 and 50 mol% of each sterol was dried in a round-bottom flask by rotary evaporation under a vacuum. The lipid films obtained were dissolved in chloroform twice, and the solvent was evaporated. The lipid thin films were kept under a high vacuum at least 3 hrs and then hydrated at room temperature with 3 ml phosphate-buffered saline (PBS). The vesicle suspension was frozen at -80 °C and thawed at 50 °C. This freeze and thaw cycle was repeated five times The liposome suspensions were extruded11 times through two layers of polycarbonate membranes with mean pore diameters of 100 nm using an extruding device (Liposofast; Avestin Inc,. Ottawa, ON, Canada). Finally, the liposome samples (total lipid: 20 mM) were successfully prepared. Evaluation of the membrane fluidity in the surface and inner membrane. 10 µL of fluorescent probes (100 µM of TMA-DPH or DPH) were added to 12.5 µl liposome solution (total lipid concentration: 20 mM) in 977.5 µL PBS buffer (molar ratio: total lipid/probe=250/1). The fluorescence polarizations of DPH and TMA-DPH (Ex = 360 nm, Em = 430 nm) were measured after 1 hr incubation at 37 °C, by using the fluorescence spectrophotometer (FP-6500; JASCO, Tokyo, Japan). The samples were incubated at least 30 min in the dark. The samples were excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) (0 °, 0 °) and parallel (I∥) (0 °, 90 °) to the excited light were recorded at 430 nm. The polarization (P) of DPH was then calculated by using the following equations: 3


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P = (I∥ - G I⊥)/(I∥ + G I⊥),

(1)

G = i⊥/i∥,

(2)

where i⊥ and i∥ are the emission intensities perpendicular to the horizontally polarized light (90 °, 0 °) and parallel to the horizontally polarized light (90 °, 90 °), respectively, and G is the correction factor. The membrane fluidity was evaluated by the reciprocal of polarization (1/P). The total concentrations of lipid and DPH or TMA-DPH were 100 and 0.4 µM, and the final volume for each sample was 1 mL. Characterization of membrane polarity by using Laurdan and Prodan. 10 µL of fluorescent probes (100 µM of Laurdan or Prodan) were added to 12.5 µl liposome solution (total lipid concentration: 20 mM) in 977.5 µL PBS buffer (molar ratio: total lipid/probe=100/1). The sample solutions were incubated for 2 hrs at 37 °C, then the fluorescence spectrum of Laurdan or Prodan for each liposome was recorded at appropriate emission wavelengths from 380 nm to 600 nm at 37 °C, with the excitation wavelength of 340 nm. For Laurdan, the membrane polarity (GP340,Laurdan) was estimated based on the following equation: GP340,Laurdan = (I440 – I490)/ (I440 + I490),

(3)

where I440 and I490 are the emission intensities of Laurdan at 440 nm and 490 nm, respectively. For Prodan, the membrane polarity (GP340,Prodan) was estimated based on the following equation: GP340,Prodan = (I437 – I510)/ (I437 + I510),

(4)

where I437 and I510 are the emission intensities of Prodan at 437 nm and 510 nm, respectively. The total concentrations of lipid and Laurdan or Prodan were 100 and 1 µM, and the total volume for each sample was 1 mL. Identification of membrane hydrophobicity by using ANS. 10 µL of fluorescent probes (100 µM of ANS) were added to 12.5 µl liposome solution (total lipid concentration: 20 mM) in 977.5 µL PBS buffer (molar ratio: total lipid/probe=100/1). The sample solutions were incubated for overnight at 37 °C, then the emission spectra of ANS for the liposomes were recorded from 375 nm to 600 nm at 37 °C, with the excitation wavelength of 350 nm. The hydrophobicity of membrane was estimated by comparing the fluorescence intensity of ANS at 484 nm (I484). The total concentrations of lipid and ANS were 100 and 1 µM, and the final volume for each sample was 1 mL. The normalization of fluidity, hydrophobicity and polarity membrane. To understand and evaluate the effect of each sterol in phospholipid membranes, the raw data (as shown in Figures 2 and S7) were normalized as the following equation (5). The normalized values z’ for membrane fluidity (measured by TMA-DPH or DPH), hydrophobicity (measured by ANS) and membrane polarity (measured by Prodan or Laurdan) were calculated by using 1/P, I484, and -GP340, respectively: z'=((z-y))/((x-y)),

(5)

where x, y, z are the maximum, minimum and other values, respectively, among the liposomes tested in this study. Herein, the maximum (z=x, thus z’=1) and minimum (z=y, thus z’=0) values were regarded as most-disordered membrane and most-ordered membrane, in order. Finally, these normalized values could be used to construct the membrane properties chart for each liposome to indicate the distinction of each liposome membrane properties from the outer to inner membrane. 4


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RESULTS AND DISCUSSION Phase State of Liposome Membrane in the Hydrophobicity Region Liposome membranes own a hydrophobic gradient in the vertical direction of membrane from the surface into inner membrane,37 where the emission property of the fluorescent probe depends on the binding location in the membrane due to different surrounding environment. We have reported the binding location of DPH,6 Laurdan,6,38 ANS,38 and 2-p-toluidinylnaphthalene-6-sulphonate (TNS)3,6 in liposome systems by analyzing their emission properties in 1,4-dioxane-water systems. Depending on the emission wavelength and intensity of probes such as DPH, Laurdan, ANS, TMA-DPH, and Prodan in DOPC liposome, the distribution of each probe in the membrane could be identified (Figure 1, Supporting information Figures S1 and S2), and these probes are utilized to investigate in situ membrane properties in liposomes.6,37,39-43 At first, Laurdan was used as a molecular probe to monitor liposome heterogeneity.39,44 Fluorescent spectra of Laurdan for liposomes were evaluated (Supporting information Figure S3), and the membrane polarity (GP340,Laurdan) was identified and shown in Figs. 2a, b, and c. The existence of the ordered phase, which was estimated by the Laurdan emission peak at 440 nm, was observed in DOPC/Chl=(7/3) and DOPC/Chl=(5/5), while other DOPC/sterol liposome showed only one emission peak at 490 nm (Supporting information Figure S3). Moreover, the recorded fluorescent spectra of Laurdan in each liposome showed the phase transition of liposomes when Chl concentration was increased, indicating the role of Chl in altering the membrane properties. This indicates that the Chl successfully form the lo phase in proportional to its concentration in DOPC liposome. In addition, DPH, a widely used fluorescent probe used for investigating inner membrane fluidity (1/PDPH), localized almost exclusively in the hydrocarbon cores of the phospholipid membrane. Changes in fluorescence anisotropy of DPH in the intact membranes indicated the fluidity of the inner membranes; the 1/PDPH value for each liposome bilayer membrane was calculated and compared as shown in Figs. 2d, e, and f. On the other hand, effects of Lan or Erg on the membrane properties in the hydrophobic region were not as significant.

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Figure 2. Membrane polarity and fluidity at 37 °C investigated by Laurdan and DPH, respectively. GP340,Laurdan values for (a) DOPC/Chl, (b) DOPC/Lan, (c) DOPC/Erg; 1/PDPH values for (d) DOPC/Chl, (e) DOPC/Lan, and (f) DOPC/Erg. Each data was normalized according to eq. (5). Dotted lines represent the GP340,Laurdan and 1/PDPH values for DOPC and DPPC liposomes as reference.

A Cartesian diagram44 for the liposomes modified with 10, 30, and 50 mol% of sterols was prepared using data for membrane fluidity (1/PDPH, x-axis) and polarity (GP340,Laurdan, y-axis). Based on this Cartesian diagram for sterol-incorporated liposomes, the phase state of each liposome focusing on the hydrophobic region was determined as shown in Figure 3 (also see Supporting information, Figure S4). The cross point of the x- and y-axes is the threshold point of the phase transition in solid-ordered (so) phase and ld phase: using this method, the liposomes plotted in the 4th quadrant of the Cartesian diagram are considered to be in the ld phase.44 The data for phase state measurements of liposomes, obtained using DPH and Laurdan probes in our previous study, have been utilized in this study to estimate the boundary of each phase state (Supporting information Figure S4 and Table S1). In particular, a previous report showed that liposomes in the 1st quadrant such as DOPC/Chl=(7/3) could be in a heterogeneous phase, while liposomes in the 4th quadrant were in a homogeneous ld phase.44 DPPC and DOPC/Chl=(5/5) in the 2nd quadrant were considered in ordered phases. The results also indicate that DOPC/Chl alters both the fluidity and polarity of membranes when increasing the amount of Chl from 0 to 50 mol%, indicating that Chl increases the order of the inner membrane. These results agree with those of previous reports regarding the effect of Chl on the membrane.30 However, Erg slightly changed the fluidity and had little effect on polarity; these trends were opposite those observed for Lan-modified DOPC liposomes. Therefore, changes in both the polarity and fluidity of the interior of the membrane 6


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generally showed specific patterns for the presence of sterols, indicating the vital role of the sterol chemical structure in forming liposome membranes. The Cartesian diagram reflected variations in the membrane properties in the interior (or hydrophobic region) of the lipid bilayer, however, membrane characteristics in the hydrophilic region, the affinity of phospholipid molecules to sterols have not be inferred. Thus, the outer membrane was analyzed using other fluorescent probes with distinctive binding depths; this analysis is discussed in the next section.

Figure 3. Cartesian diagram44 for liposome modified with sterols based on 1/PDPH and GP340,Laurdan values at 37 °C. When the liposome membrane becomes polar (GP340,Laurdan decrease), its fluidity becomes higher (1/PDPH increase).

Characterization of Hydrophilic Regions on the Membrane Surface The surface membrane properties for DOPC/sterol liposomes were estimated by using TMA-DPH, ANS, and Prodan. Because no peak shifts of ANS or TMA-DPH were observed in DOPC/sterol liposomes, it was assumed that the binding location of probes did not change, and that the in situ environment around probes could be altered depending on the type and amount of sterols. The obtained data were shown in supporting information (Figures S5, S6, and S7), and the normalized data are summarized in Figure 4. Herein, the higher (~1) and lower (~0) z’ values indicate the disordered and ordered membrane, respectively.

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Figure 4. Normalized membrane properties (z’) in surface (hydrophilic region) investigated at 37 °C. The surface membrane fluidity, 1/PTMA-DPH values for (a) DOPC/Chl, (b) DOPC/Lan, (c) DOPC/Erg; the membrane hydrophobicity, I484 investigated by ANS for (d) DOPC/Chl, (e) DOPC/Lan, (f) DOPC/Erg; the membrane surface polarity, GP340,Prodan for (g) DOPC/Chl, (h) DOPC/Lan, and (i) DOPC/Erg. Each value was normalized according to eq. (5). Dotted lines represent the z’ values for DOPC and DPPC liposomes as reference.

TMA-DPH-Based Analyses of Membrane Fluidity of Liposome Surfaces TMA-DPH binds to the phospholipid membrane surface, where it is assumed to embed into regions with a dielectric constant ɛ ~ 60.4 (Supporting information Figure S1). This indicates that TMA-DPH reflects membrane surface fluidity. The normalized 1/PTMA-DPH values are shown in Figure 4 (a, DOPC/Chl; b, DOPC/Lan; and c, DOPC/Erg). The results showed that the addition of Chl and Erg reduced membrane fluidity, while Lan had little effect on membrane ordering. As illustrated in Fig. 1, Lan has two more methyl groups at C4 than the other sterols, which protect the β-surface of the planar steroid ring system and make the surface of the molecule rough. Thus, the molecular structure of Lan interferes with the 8


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interactions between phospholipid molecules in the hydrophilic region of the membrane surface, and then Lan reduces membrane order and increases fluidity by more than Chl and Erg. ANS-Based Analysis of Membrane Hydrophobicity ANS shows low fluorescent intensity in polar environments and binds preferentially to hydrophobic cavities (Supporting information Figure S5).45,46 The quantum yield (φ) of ANS in the liposome could be dependent on the physicochemical property of the membrane,47 wherein the φ value of ANS was bigger in DOPC liposome than in DPPC (Figure S5). The location of ANS was estimated by emission peak (Supporting information, Fig. S1):38 the binding location of ANS in DOPC liposome was regarded as the regions with dielectric constants ɛ ~ 26.5. It has also reported that ANS could be located near the phosphate group in the phospholipid membranes.40 The membrane characteristics can be evaluated by comparing the fluorescence intensity of ANS at I484 (Supporting information, Figures S1 and S5). Figures 4d, e, and f show the normalized I484 values for the disparity of ANS fluorescent intensity at 484 nm for DOPC/Chl, DOPC/Lan, and DOPC/Erg. In general, an increase in the percentage of Chl led to the increase of inner membrane packing. On the other hand, Cheng et al have reported that the DOPC/Chl liposome (30 mol% Chl) rather decreased the packing with an increasing of the hydration dynamics near membrane surface.48 Based on our result, the effects of 10 mol% of sterols raised the ANS fluorescent intensity, while 50 mol% of sterols decreased the intensity. This indicates that sterols and phospholipid molecules interacted strongly and the hydrogen bond network was formed at the membrane surface, when the higher amount of sterols are existing in the membrane. Because the interaction between Lan and DOPC in the surface area is weaker than that of other sterols, the surface becomes flexible and contains many hydrogen bonds. Thus, 50 mol% of Lan prevents the attack of ANS deeper into the membrane and leads to lower fluorescent intensity. In contrast, Erg has less affinity with headgroups of phospholipid molecules at 50 mol%; therefore, the surface has more hydrophobic sites than Chl or Lan. This explains why Chl and Erg made the membrane more hydrophobic than Lan, but the degree was different between Chl and Erg. In fact, the addition of one double bond at C7–8 of the B ring in the steroid ring system reduced electron resonance and weakened the interaction between Erg and the membrane in the ANS-binding region. Prodan-Based Analysis of Surface Membrane Polarity By identifying the location of Prodan in the DOPC lipid membrane (Supporting information, Figure S1),39,43 the ɛ value around Prodan was estimated to be 43 in the DOPC membrane, indicating that Prodan binds to deeper (hydrophobic) regions in the membrane than TMA-DPH. Depending on the different fluorescent intensity of Prodan in liposomes and water systems (Supporting information Figure S6), the GP340,Prodan values analyzed by Prodan were calculated (Supporting information Figure S7) and normalized and are shown in Fig. 4g, h, and i. The normalized values for DOPC mixed with Chl ranged from 0 to 50 mol%, demonstrating that Chl increases membrane packing (highly-ordered), while Erg only minimally changed the polarity compared to DOPC. In contrast, DOPC/Lan membranes became hydrophilic around the Prodan-binding area. The structure of Lan contains one methyl group at C14 that protects the α-face, whereas Chl and Erg contain none. Thus, Lan makes the membrane rougher and slightly bent compared to the effects of Chl and Erg.49 Compared with Chl, the surface area in the case of Erg is more flexible, weakening the interaction between Erg and DOPC. Consequently, at the Prodan binding depth, Chl had a greater condensing effect than Erg, while Lan membranes were disordered. In summary, cooperation between sterols in the phospholipid membranes changed the basic characteristics of the membranes from the surface to the interior. We analyzed the effect of sterols on membrane fluidity and polarity using many fluorescent probes such as TMA-DPH, ANS, Prodan, 9


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Laurdan, and DPH. Although the sterols had similar molecule structures, our results revealed that each type of sterol had different and typical effects on each part of the membrane. Figure 5 summarizes the effect of 10 and 50 mol% sterols in DOPC membranes, wherein the normalized values of the surface membrane characteristics, such as surface membrane fluidity (by TMA-DPH), hydrophobicity (ANS), and surface membrane polarity (Prodan), are compared. Given the different phase states of disordered liposomes (e.g., DOPC) and ordered liposomes (e.g., DPPC), the normalized data for DOPC and DPPC were considered as two landmarks, which is useful in comparing the properties of DOPC membranes modified with the sterol variants Chl, Lan, and Erg. Compared to the DOPC membrane itself, liposomes modified with 10 mol% sterols such as Chl, Lan, and Erg show distinct properties. The results for these liposomes suggest that these membranes are more flexible on the surface and condensed in the deeper region. After increasing the amount of each sterol to 50 mol%, the membrane property chart indicated that Chl condensed the membrane to the greatest extent in all areas, indicating that it interacted tightly with phospholipid molecules to decrease fluidity and increase hydrophobicity. Although Lan also condensed the membrane, the degree of its influence was lower than that of Chl, except at the binding location of Prodan which showed that the membrane in this region was more hydrophilic than with DOPC. In contrast, Erg caused ordering of the DOPC membrane, but had the second largest effect on the surface membrane, whereas the membrane characteristics at the ANS and Prodan binding sites were quite similar to those of pure DOPC and the inner regions were more ordered than with Chl; however, the ordering was approximately equal to that observed with Lan.

Figure 5. Variation of membrane property chart for each kind of DOPC/sterol liposomes.

Discussion for Possible roles Sterols in Altering the “Fluid” Membrane Property. In general, animal membranes contain high proportions of Chl, ranging from 10 to 50 mol%, depending on the cell type.50 Chl has a tetracyclic structure with an OH group in the equatorial position on the first ring and a short aliphatic chain, with two branched methyl groups, on the D pentenic ring.14 In contrast to the mammalian membrane, Erg comprises the major percentage of sterols in the fungal membrane,51 the structure of which differs from that of Chl by the presence of additional double bonds at C7–8 on the B-ring and at C22–23 on the acyl chain and the addition of one methyl group at C24.14 In each eukaryotic kingdom, sterol specificity may be related to biological evolution for the precursor Lan, but distinct sterol formation pathways remain unclear, and why Erg is present in the fungal membrane while Chl is present in animal cells requires further investigation.52 In fact, the sterol biosynthesis 10


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pathway includes a dozen metabolic steps and multiple enzymes.53 The precursor of the sterol is squalene, which is converted to Lan in a two-step enzymatic reaction. Lan is therefore regarded as a central molecule in sterol evolution: Erg in fungi (11 steps) and Chl in animals (8 steps);54 furthermore, a recent study found that the Lan pathway was involved in Erg synthesis in plants.55 As the precursor of Chl, the structure of Lan is slightly different as it has two methyl groups in the A ring and another methyl group at C8–9, as well as one double bond at C8–9 in the steroid ring and at C24–25 of the tail. The differences in sterol chemical structures influence the alterations of membrane properties. A wide variety of sterols have been investigated for their phase behaviors in lipid monolayers, such as DPPC and active sterols including Chl, Erg, and lathosterol, which generally produce two regions of immiscible liquid phase, whereas inactive sterols such as Lan, cholestenone, and coprostanol do not.56 The addition of sterols may change the membrane properties in different ways in monolayer and bilayer lipid membranes. The Cartesian diagram in the Fig. 3 shows different sterols generally produce unique phase behaviors in each membrane, depending on the amount added. For example, 10 and 50 mol% Chl results in the formation of homogenous DOPC liposomes in ld and lo phases, respectively, while 30 mol% Chl fixes the membrane in the heterogeneous phase. Using the same percentages of sterol, Erg causes DOPC membranes to take on a disordered phase. In addition, a previous study clarified the role of various sterols in saturated phospholipid membranes such as DPPC and found that Chl, Lan, and Erg contract membranes to different extents. Specifically, Chl has a greater contraction effect than the other sterols.30,49 However, the interaction between these types of sterols in unsaturated phosphatidylcholines such as DOPC, which share the same phospholipid group with DPPC but have different hydrocarbon tails, is unclear. Therefore, we also focused on the role of the hydrocarbon tails of lipid molecules in cooperating with sterols. To take advantage of the dispersion of fluorescent probes in the membrane and determine the membrane characteristics in each binding region, basic membrane characteristics were evaluated. Mixtures of DOPC and Chl consistently showed attractive intermolecular interactions, while lipid films containing Erg and Lan showed expansion. Such different behaviors can be caused by differences in the chemical structures of these sterols. Chl has a higher affinity for both saturated and unsaturated phospholipids than Lan because its additional methyl groups protect the β- and α-surfaces, decreasing the strength of the van der Waal’s interactions. The alterations in the location and the addition of double bonds in the steroid rings and hydrocarbon chains of Lan also indicate a greater expansion effect than that noted with Chl. Therefore, the Lan molecule itself is more mobile and less ordered in the phospholipid bilayer than Chl. Similarly, to the Chl chemical structure, a small change in the molecule leads to a large difference between Chl and Erg. Specifically, the carbon double bond in the steroid ring reduces the interaction between Erg and the membrane at the B ring and increases flexibility in this region. Furthermore, the unsaturated bond distribution and extra methyl group in the steroid tail support that Erg had a less pronounced effect on membrane contraction than Lan. Changes in the membrane properties demonstrate an interaction between sterols and the membrane from the exterior to the interior of the membrane, indicating that sterols reside and disperse along with phospholipid molecules through interactions between the steroid ring and tail with the phosphatidylcholine, glycerol, and fatty acid chain of phospholipid molecules. However, our results refute that sterols reside in the middle of the bilayer membrane, which is composed of polyunsaturated phospholipids because of their extreme disorder.57,58 Our study also evaluated the evolution of sterol biosynthetic pathways in natural membranes, for example, the synthesis of Chl, Lan, and Erg from squalene (Supporting information Figure S8).59 Lan can be transformed into Erg (the most abundant sterol in fungal membranes) or into Chl (a well-known and major sterol in animal membranes). Changes in the membrane properties of the phospholipid bilayer upon mixing with sterols would generate signals for the next transduction into intermediary sterols before transformation into Chl or Erg. Thus, these 11


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signals regulate the process in which several enzymes participate and continuously inhibit or enhance the enzymes to complete the biosynthetic processes. CONCLUSION Using membrane-binding fluorescent probes such as TMA-DPH, ANS, Prodan, Laurdan, and DPH, the physicochemical membrane properties of DOPC/sterol liposomes were investigated. Chl successfully ordered the membrane in a concentration-dependent manner. In contrast, Lan and Erg showed lower efficiencies in ordering the hydrophobic regions of the membrane (monitored by Laurdan and DPH). Focusing on membrane properties near surface (hydrophilic region), the difference in the chemical structure of the sterol molecule was significant. Fluorescent probes that bind to different locations in phospholipid membranes, can reveal the fundamental properties of steroids in the fluid membrane. Considering that the activity of biological molecules can be controlled on lipid membranes,7 alterations in membrane properties depending on the type and amount of sterol could elucidate our understanding of the biological role of sterol derivatives. Based on our results, the effect of sterols on modulating membrane characteristics in each region from the surface into the interior were clarified, and these characterization methods can be applied for a variety of lipid membranes, including those of living cells. Speculatively, it might be possible to characterize the outer leaflet of the lipid bilayer using our method. Although further investigation are needed to characterize living cell membrane or asymmetric lipid bilayer, the analysis of the “hieratical” membrane properties can be applied for a variety of selfassembled membranes.

FIGURE LEGENDS Figure 1. Schematic illustration for the location of sterols or fluorescent probes in DOPC membrane. For the details of the fluorescent probe location, see supporting information Figure S1. Figure 2. Membrane polarity and fluidity at 37 °C investigated by Laurdan and DPH, respectively. GP340,Laurdan values for (a) DOPC/Chl, (b) DOPC/Lan, (c) DOPC/Erg; 1/PDPH values for (d) DOPC/Chl, (e) DOPC/Lan, and (f) DOPC/Erg. Each data was normalized according to eq. (5). Dotted lines represent the GP340,Laurdan and 1/PDPH values for DOPC and DPPC liposomes as reference. Figure 3. Cartesian diagram35 for liposome modified with sterols based on 1/PDPH and GP340,Laurdan values at 37 °C. When the liposome membrane becomes polar (GP340,Laurdan decrease), its fluidity becomes higher (1/PDPH increase). Figure 4. Normalized membrane properties (z’) in surface (hydrophilic region) investigated at 37 °C. The surface membrane fluidity, 1/PTMA-DPH values for (a) DOPC/Chl, (b) DOPC/Lan, (c) DOPC/Erg; the membrane hydrophobicity, I484 investigated by ANS for (d) DOPC/Chl, (e) DOPC/Lan, (f) DOPC/Erg; the membrane surface polarity, GP340,Prodan for (g) DOPC/Chl, (h) DOPC/Lan, and (i) DOPC/Erg. Each value was normalized according to eq. (5). Dotted lines represent the z’ values for DOPC and DPPC liposomes as reference. Figure 5. Variation of membrane property chart for each kind of DOPC/sterol liposomes.

ASSOCIATED CONTENT

Supporting Information 12


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This material is available free of charge via the Internet at http://pubs.acs.org. Characterization of fluorescent probes in 1,4-dioxane/water systems; GP340,Laurdan and GP340,Prodan values for 1,4-dioxane/water systems; Fluorescent spectra of Laurdan for DOPC/sterol liposomes; Cartesian diagram for homogeneous liposome systems and for heterogeneous liposome systems; Fluorescence spectra of ANS for liposomes and the emission intensities I484 at 37 °C; Fluorescent spectra of Prodan for DOPC/sterol liposomes; 1/PTMA-DPH, I484, and GP340,Prodan values for DOPC/sterol liposomes; and Modified biosynthesis pathway of sterols in membrane.

AUTHOR INFORMATION Corresponding Author *Corresponding author: Prof. Dr. Hiroshi Umakoshi Phone: +81-6-6850-6287; E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. Yukihiro Okamoto (Graduate School of Engineering Science, Osaka University) for his constructive comments and technical support. This work was supported by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research A (26249116), JSPS Grant-in-Aid for Research Activity Start-up (25889039), and JSPS Grant-in-Aid for Challenging Exploratory Research (T15K142040). T. T. B. shows her thanks to Japanese Government (Monbukagakusho: MEXT) Scholarship supported for her completing this study. REFERENCES (1) Singer, S.J.; and Nicolson, G.L. The fluid mosaic model of the structure of cell membranes. Science 1972, 175, 720– 731. (2) Spector, A.A.; Yorek, M.A. Membrane lipid composition and cellular function. J. Lipid Res. 1985, 26, 1015-1035. (3) Suga, K.; Tanabe, T.; Tomita, H.; Shimanouchi, T.; Umakoshi, H. Conformational change of singlestranded RNAs induced by liposome binding. Nucleic Acids Res. 2011, 39, 8891-8900. (4) Umakoshi, H.; Yoshimoto, M.; Shimanouchi, T.; Kuboi, R.; Komasawa, I. Model system for heatinduced translocation of cytoplasmic beta-galactosidase across phospholipid bilayer membrane. Biotechnol. Progr. 1998, 14, 218-226.

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Page 14 of 18

(5) Yoshimoto, M.; Walde, P.; Umakoshi, H.; Kuboi, R. Conformationally changed cytochrome cmediated fusion of enzyme- and substrate-containing liposomes. Biotechnol. Progr. 1999, 15, 698-696. (6) Umakoshi, H.; Suga, K. Use liposome as a designable platform for molecular recognition from “Statistical separation” to “Recognitive separation”. Solvent Extr. Res. Dev. Jpn. 2013, 20, 1-13. (7) Walde, P.; Umakoshi, H.; Stano, P.; Mavelli, F. Emergent properties arising from the assembly of amphiphiles: Artificial vesicle membranes as reaction promoters and regulators. Chem. Commun. 2014, 50, 10177-10197. (8) Zohra, F.T.; Chowdhury, E.H.; Tada, S.; Hoshiba, T.; Akaike, T. Effective delivery with enhanced translational activity synergistically accelerates mRNA-based transfection. Biochem. Biophys. Res. Commun. 2007, 358, 373-378. (9) Koynova, R.; Tenchov, B.; Wang, L.; Macdonald, R.C. Hydrophobic moiety of cationic lipids strongly modulates their transfection activity. Mol. Pharm. 2009, 6, 951-958. (10) Bui, H.T.; Umakoshi, H.; Ngo, K.X.; Nishida, M.; Shimanouchi, T.; Kuboi, R. Liposome membrane itself can affect gene expression in the Escherichia coli cell-free translation system. Langmuir 2008, 24, 10537-10542. (11) Umakoshi, H.; Suga, K.; Bui, H.T.; Nishida, M.; Shimanouchi, T.; Kuboi, R. Charged liposome affects the translation and folding steps of in vitro expression of green fluorescent protein. J. Biosci. Bioeng. 2009, 108, 450-454. (12) Aden, H.; Michael, R.; Heinz, A.; Peter, L.; Georg, P. Differential modulation of membrane structure and fluctuations by plant sterols and cholesterol. Biophys. J. 2008, 94, 3935–3944. (13) Ribeiro, N.; Streiff, S.; Heissler, D.; Elhabiri, M.; Albrecht-Gary, A.M.; Atsumi, M.; Gotoh, M.; Desaubry, L.; Nakatani, Y.; Ourisson, G. Reinforcing effect of bi- and tri-cyclopolyprenols on ‘primitive’ membranes made of polyprenyl phosphates. Tetrahedron 2007, 63, 3395–3407. (14) Dufourc, E.J. Sterols and membrane dynamics. J. Chem. Biol. 2008., 1, 63-77. (15) Beck, J.G.; Mathieu, D.; Loudet, C.; Buchoux, S.; Dufourc, E.J. Plant sterols in "rafts": a better way to regulate membrane thermal shocks. FASEB J. 2007, 21, 1714-1723. (16) Dingle, J.T.; Gordon, J.L.; Hazleman, B.L.; Knight, C.G.; Thomas, D.P.; Phillips, N.C.; Shaw, I.H.; Fildes, F.J.; Oliver, J.E.; Jones, G.; Turner, E.H.; Lowe, J.S. Novel treatment for joint inflammation. Nature 1978, 271, 372-373. (17) Bittman, R.; Bittman, R. Ed.; Subcellular Biochemistry Series, vol 28. Plenum Press: New York, pp. 145-172, 1997. (18) Yeagle, P.L., Ed.; The Structure of Biological Membranes, 2nd ed; CRC Press: Boca Raton, FL, pp. 121–146, 2005. (19) Davis, J.H.; Finegold, L., Ed.; Cholesterol in Membrane Models. CRC Press: Boca Raton, FL, pp. 67–136., 1993. (20) Worcester, D.L.; Franks, N.P. Structural analysis of hydrated egg lecithin and cholesterol bilayers. II. Neutrol diffraction. J. Mol. Biol. 1976, 100, 359–378. 14


Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(21) Dufourc, E.J.; Parish, E.J.; Chitrakorn, S.; Smith, I.C.P. Structural and dynamical details for the cholesterol-lipid interaction as revealed by deuterium NRM. Biochemistry 1984, 23, 6062–6071. (22) Morrow, M.R.; Singh, D.; Lu, D.; Grant, C.W. Glycosphingolipid fatty acid arrangement in phospholipid bilayers: cholesterol effects. Biophys. J. 1995, 68, 179-186. (23) Stockton, G.W.; Smith, I.C.; A deuterium nuclear magnetic resonance study of the condensing effect of cholesterol on egg phosphatidylcholine bilayer membranes. I. Perdeuterated fatty acid probes. Chem. Phys. Lipids 1976, 17, 251-263. (24) Davies, M.A.; Schuster, H.F.; Brauner, J. W.; Mendelsohn, R. Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence. Biochemistry 1990, 29, 4368-4373. (25) O'Leary, T.J.; Finegold, L., Ed.; Cholesterol in Membrane Models. CRC Press: Boca Raton, FL, pp. 175-195, 1993. (26) Yeagle, P.L. Cholesterol and the cell membrane. Biochim. Biophys. Acta. 1985, 822, 267–287. (27) Slotte, J.P. Sphingomyelin-cholesterol interactions in biological and model membranes. Chem. Phys. Lipids 1999, 102, 13–27. (28) Yeagle, P.L.; Hutton, W.C.; Huang, C.; Martin, R.B. Phospholipid head-group conformations; intermolecular interactions and cholesterol effects. Biochemistry 1977, 16, 4344–4349. (29) McMullen, T.P.W.; McElhaney, R.N. Physical studies of cholesterol–phospholipid interactions. Curr. Opin. Coll. Interf. Sci. 1996, 1, 83–90. (30) Minones, J.J.; Pais, S.; Minones, J.; Conde, O.; Dynarowicz-Latka, P. Interactions between membrane sterols and phospholipids in model mammalian and fungi cellular membranes — A Langmuir monolayer study. Biophys. Chem. 2009, 140, 69-77. (31) James, A.S.; Sterling, A. Wheaten, Paulo F. Almeida. Phase separation and fluctuations in mixtures of a saturated and an unsaturated phospholipid. Biophys. J. 2012, 102, 2526–2535. (32) Mannock, D.A.; Lewis, R.N.A.; McElhaney, R.N. Comparative colorimetric and spectroscopic studies of the effects of lanosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membrane membranes. Biophys. J. 2006, 91, 3327-3340. (33) Miao, L.; Nielsen, M.; Thewalt, J.; Ipsen, J. H.; Bloom, M.; Zuckermanm, M.J.; Mouritsen, O.G. From Lanosterol to cholesterol: structure evolution and differential effects on lipid bilayers. Biophys. J. 2002, 82, 1429-1444. (34) Bernsdorff, C.; Winter, R. Differential properties of the sterols cholesterol, ergosterol, sistosterol, trans-7-hydrocholesterol and lanosterol on DPPC bilayer order. J. Phys. Chem. 2003, 107, 10658-10664. (35) Endress, E.; Bayerl, S.; Prechtel, K.; Maier, C.; Merkel, R.; Bayerl, T.M. The effect of cholesterol, lanosterol, and ergosterol on lecithin bilayer mechanical properties at molecular and microscopic dimensions: A solid-state NMR and micropipet study. Langmuir 2002, 18, 3293-3299. (36) Czub, J.; Baginski, M. Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol. Biophys. J. 2006, 90, 2368-2382. (37) Cevc, G. Membrane electrostatics. Biochim. Biophys. Acta. 1990, 1031, 311-382. 15


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 18

(38) Ishigami, T.; Suga, K.; Umakoshi, H. Chiral Recognition of l-Amino Acids on Liposomes Prepared with l-Phospholipid, ACS Appl. Mater. Interf. 2015, 7 (38), 21065-21072. (39) Parasassi, T.; Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc. 1995, 5, 59−69. (40) Dhanikula, A.B.; Panchagnula, R. Fluorescence anisotropy, FT-IR spectroscopy and 31-P NMR studies on the interaction of paclitaxel with lipid bilayers. Lipids 2008, 43 (6), 569-579. (41) Krasnowska, E.K.; Gratton, E.; Parasassi, T. Prodan as a membrane surface fluorescence probe: Partitioning between water and phospholipid phases, Biophys. J. 1998, 74 (4), 1984-1993. (42) Suga, K.; Tanabe, T.; Umakoshi, H. Heterogeneous cationic liposomes modified with 3β-N-[(N′, N ′-dimethylamino)ethyl]carbamoylcholesterol can induce partial conformational changes in messenger RNA and regulate translation in an escherichia coli cell-free translation system, Langmuir 2013, 29 (6), 1899-1907. (43) Cwiklik, L.; Aquino, A.J.A.; Vazdar, M.; Jurkiewicz, P.; Pittner, J.; Hof, M.; Lischka, H. Absorption and fluorescence of PRODAN in phospholipid bilayers: A combined quantum mechanics and classical molecular dynamics study, J. Phys. Chem. A 2010, 115(41), 11428-11437. (44) Suga, K.; Umakoshi, H. Detection of Nano-sized Sized Ordered Domains in DOPC/DPPC and DOPC/Ch Binary Lipid Mixture Systems of Large Unilamellar Vesicles Using a TEMPO Quenching Method. Langmuir 2013, 29, 4830-4838. (45) Slavik, J. Anilinonaphthalene sulfonate as a probe of membrane composition and function. Biochim. Biophys. Acta. 1982, 694, 1-25. (46) Stryer, L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J. Mol. Biol. 1965, 13, 482-495. (47) Montich, G.G.; Cosa, J.J.; Maggio, B. Interaction of 1-anilinonaphthalene 8-sulfonic acid with interfaces containing cerebrosides, sulfatides and gangliosides, Chem. Phys. Lipids 1988, 49(1-2), 111117. (48) Cheng, C.-Y.; Olijve, L.L.C.; Kausik, R., Han, S. Cholesterol enhances surface water diffusion of phospholipid bilayers, J. Chem. Phys. 2014, 141 (22), 22D513. (49) David, A.M.; Ruthven, N.A.H.L.; Ronald, N.M. Comparative calorimetric and spectroscopic studies of the effects of Lanosterol and Cholesterol on the thermotrophic phase behavior and organization of dipalmitoylphosphatidinecholine bilayer membranes. Biophys. J. 2006, 91, 3327–3340. (50) Bloch, K.; Neuberger, A. and Van, D.L., Ed.; Biochemistry of Lipids, Lipoproteins and Membranes, vol 20. Elsevier; North Holland, Amsterdam, pp. 363–381, 1991. (51) Barwic, J.; Tancrede, P. The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers. Chem. Phys. Lipids. 1997, 85, 145-155. (52) Parks, L.W.; Casey, W.M. Physiological implications of sterol biosynthesis in yeast. Annu. Rev. Microbiol. 1995, 49, 95–116. 16


Page 17 of 18

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(53) Fakheri, R.J.; Javitt, N.B. Autoregulation of cholesterol synthesis: physiologic and pathophysiologic consequences. Steroids 2011, 76, 211-215. (54) Galea, A.M.; Brown, A.J. Special relationship between sterols and oxygen: were sterols an adaptation to aerobic life?. Free Radic. Biol. Med. 2009, 47, 880–889. (55) Ohyama, K.; Suzuki, M.; Kikuchi, J.; Saito, K.; Muranaka, T. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc. Natl. Acad. Sci. USA. 2009, 106, 725730. (56) Stottrup, B.L.; Keller, S.L. Phase behavior of lipid monolayers containing DPPC and Cholesterol Analogs. Biophys. J. 2006, 90, 3176-3183. (57) Harroun, T.A.; Katsaras, J.; Wassall, S.R. Cholesterol is found to reside in the center of a polyunsaturated lipid membrane. Biochemistry 2008, 47, 7090–7096. (58) Sourav, H.; Ravi, K.K.; Anunay, S.; Amitabha, C. Differential effect of cholesterol and its biosynthetic precursors on membrane dipole potential. Biophys. J. 2012, 102, 1561-1569. (59) David, W.N. Biosynthesis of cholesterol and other sterols. Chem. Rev. 2011, 111, 6423-6451.

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