Laurdan emission study of the cholesterol-like effect of long-chain alkylresorcinols on the structure of dipalmitoylphosphocholine and sphingomyelin membranes

Biophysical Chemistry 221 (2017) 1–9

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Laurdan emission study of the cholesterol-like effect of long-chain alkylresorcinols on the structure of dipalmitoylphosphocholine and sphingomyelin membranes Patrycja Zawilska a, Katarzyna Cieślik-Boczula b,⁎ a b

Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, F. Joliot-Curie 14a, 50-383 Wroclaw, Poland Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The cholesterol-like effect of ARs on DPPC and SM membranes • High cooperativity of the phase lipid transition in ARs-mixed membranes • The increase in Tm of ARs-mixed DPPC and SM membranes

a r t i c l e

i n f o

Article history: Received 5 August 2016 Received in revised form 6 November 2016 Accepted 10 November 2016 Available online 12 November 2016 Keywords: Laurdan fluorescence DPPC membranes Sphingomyelin (SM) Cholesterol Long chain alkylresorcinols Lipid main phase transition Hydration of lipid membrane Lipid chain order

a b s t r a c t Long-chain alkylresorcinols (ARs) are commonly found in plant and bacteria cells, and they exhibit a wide variety of biological effects, including antifungal, antitumor, and antiphrastic activities. The cholesterol (Chol)-like effect of ARs with hydrocarbon side-chain lengths ranging from C15 to C25 on the structure of pure and Chol-doped dipalmitoylphosphocholine (DPPC) and sphingomyelin (SM) membranes was investigated by Laurdan fluorescence spectroscopy. The Laurdan emission generalized polarization parameter was analyzed as a function of the temperature and excitation wavelength in DPPC (or SM)/Chol, DPPC (or SM)/AR, and DPPC/Chol/AR systems. It was found that AR incorporation into both DPPC and SM bilayers induces an increase in the temperature of the main lipid phase transition, similar to the effect of Chol molecule incorporation. The phase separation, lipid-chain ordering, and membrane hydration are discussed for the AR-mixed membranes and compared with DPPC (or SM)/Chol membranes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Abbreviations: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; SM, sphingomyelin; AR, alkylresorcinols; Chol, cholesterol; Tm, main phase transition; GP, generalized polarization parameter; FT-IR, Fourier-transform infrared spectroscopy. ⁎ Corresponding author. E-mail address: [email protected] (K. Cieślik-Boczula).

http://dx.doi.org/10.1016/j.bpc.2016.11.004 0301-4622/© 2016 Elsevier B.V. All rights reserved.

Cholesterol (Chol) is an abundant and essential lipid component of biological membranes [1]. Although Chol has several different functions in living cells, one of its main roles is as a modulator of the

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physicochemical properties of the phospholipid bilayer structure of plasma membranes. One of the main effects of Chol insertion into a lipid membrane is connected with Chol-mediated changes in the temperature, which widens or even eliminates the main lipid phase transition [2,3]. Additionally, Chol is one of the main molecules responsible for the heterogeneous structure of biological membranes. There are indicators that prove that lipid rafts are Chol-rich domains with properties of the liquid-ordered (Lo) phase [4]. The structural properties of the Lo phase triggered by Chol molecules have been estimated in many studies based on a liposomal model of biological lipid membranes [2,5–7]. The Chol-rich Lo state is characterized by an increase in the lipid chain order, a restricted rate of lateral diffusion, a reduced area per molecule, and a decrease in membrane hydration. Cholesterol not only plays an important role in biological membranes, it is also one of the crucial components of the lipid membrane of liposomes, which are used as a very effective drug delivery system. The presence of Chol molecules in the lipid membrane of liposomes increases the long-time stability of vesicles and prolongs their blood circulation time by decreasing the membrane permeability [8–10]. Searching for new chemical compounds with a Chol-like effect on the structure of lipid membranes is an interesting and important task for designing liposomal drug carriers. Chol molecules in the membrane wall of liposomes can be replaced by these new compounds, which can also have important biological functions. The resulting lipid membrane of the liposomes will have the same or similar structural properties to Chol-mixed membranes, but it will possess additional biological features related to the inserted compound. In a presented work we discuss the Chol-like influence of long chain alkylresorcinolic (ARs) lipids on a structure of dipalmitoylphosphocholine (DPPC) membranes, see their structures in Fig. 1. DPPC lipids are one of H3C N

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the most numerous members of the most prevalent lipid group among those lipids constituting the basic structure of the lipid bilayer wall of liposomes [8,9]. Additionally, DPPC liposomes are frequently used as a model for lipid biomembranes [11–17]. Given that Chol molecules are located in sphingomyelin (SM) rafts, which are present in the biomembranes of living cells and rich in SM lipids, investigating the effect of AR molecules on SM membranes could provide information about whether AR molecules have a Chollike effect on lipid biomembranes. AR lipids with a wide range of biological activities naturally occur in plants, bacteria, fungi, and animals, but bran cereals are their main source. AR lipids possess antibiotic [18], antifungal [19], and antitumor [20] activities, which makes them attractive for the agriculture, pharmaceutical, and nutrition industries [21–24]. As an amphiphilic compound, they can easily interact with lipid membranes and change the properties and activities of various membrane-associated enzymes [25]. ARs are mainly found in the aleurone layers of the kernel [26]. Interestingly, they can replace phospholipids during encystment of Azotobacter vinelandii membranes [27]. All of the facts suggest that ARs can interact with biological lipid membranes, but their role in plant and bacterial cells is still not clear and needs further investigation. ARs are a common component of the diet of human and animals, and they can interact with their biomembranes. It has been suggested that AR compounds can be incorporated into erythrocyte membranes [28] and stored in adipocyte tissues as other lipids [29]. Nevertheless, the Chol-like effect of ARs on plant and animal physiology has not been reported. Studies presented in this paper show a Chol-like effect of ARs on a liposomal model of sphingomyelin rafts of biomembranes. ARs can be used as part of novel liposomal formulations for drug delivery [30]. It has been shown that the presence of ARs in SM/Chol, PC,

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Fig. 1. Structure of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), SM (sphingomyelin), AR (15:0) (5-n-pentadecylresorcinol) and Chol (cholesterol). The AR homologs differ in the hydrocarbon side-chain length (C15–C25).

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and PC/PE liposomes increases the entrapment volume and their size stability. Additionally, retention of the captured solute in AR-mixed liposomes is generally lower than in control phospholipid vesicles [31]. All of these positive effects of ARs on liposomal drug delivery systems, together with proposed by us in this paper their Chol-like effect, make them very promising components for effective liposomal drug carriers. Many methods have been successfully used to investigate the structure of lipid membranes. One of the most frequently used methods is fluorescence spectroscopy. This method can not only be used to investigate small molecules with internal fluorescence [32], but it can also be used to investigate non-fluorescent materials, such as lipid membranes, by taking advantage of fluorescence probes [33–36]. The aim of the current study was to determine whether long-chain ARs have the same effect on the structures of DPPC and SM membranes as Chol using the Laurdan fluorescence method. Based on the temperature and excitation wavelength dependence of the Laurdan emission generalized polarization (GP) parameter, the temperature and cooperativity of the main lipid phase transition, and the lack of evidence of domain coexistence in DPPC (or SM)/Chol, DPPC (or SM)/AR, and DPPC/Chol/AR membranes were characterized. Based on a comparison of the effect of ARs on DPPC and SM membranes it was stated that a Chol-like effect of ARs on a structure of lipid membranes was not significantly dependent on a type of used lipids. 2. Materials and methods 2.1. Materials DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and egg SM were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The fluorescence probe Laurdan (2-(dimethylamino)-6-dodecanoylnaphthalene) was obtained from Molecular Probes (Eugene, OR, USA). Chol, high-performance liquid chromatography (HPLC) grade methanol, and water were purchased from Sigma-Aldrich (Poznan, Poland). All of the other chemicals were of analytical grade and used without further purification. Rye bran (Secale cereale) was purchased from Vitacorn (Lagiewniki Koscielne, Poland). 2.2. Isolation of ARs from rye bran A mixture of saturated and unsaturated homologs of ARs with sidechain lengths ranging from C15 to C25 was extracted from rye bran according to a previously described procedure [37]. The resulting acetone extract was purified by liquid chromatography using chloroform:methanol (95:5, v/v) as the mobile phase on a glass column (60 cm × 6 cm) packed with Si60 Geduran 0.063–0.2 mm silica gel (Merck, Darmstadt, Germany). The purity of the isolated materials was verified by the colorimetric method according to a previously described procedure [38]. The alk(en)ylresorcinols with a purity of about 90% were hydrogenated [39]. Separation of the individual homologs was achieved by gradient reversed-phase HPLC (Waters, Milford, MA, USA). To obtain the individual homologs of the 5-n-ARs, 2 ml of methanol solution containing the hydrogenated ARs (30 mg/ml) was added on a column (C18 Luna 10 μ (2) AXIA 100 Å, 250 mm × 21.2 mm, 10 μm, Phenomenex, Torrance, CA, USA). The separation was performed at room temperature at a flow of 12 ml/min and monitored at 280 nm. The gradient used for the analysis was as follows: 0– 3 min, 93–95% methanol; 3–68 min, 95–100% methanol; 68–70 min, 100% methanol; 70–75 min, 100–93% methanol; and 75–80 min, 93% methanol. The individual homologs were separated at 15, 20, 28, 39, 56, and 62 min for compounds from AR (15:0) to AR (25:0). The purities of the obtained homologs were confirmed by analytical HPLC (methanol:water 95:5 v/v and flow rate 1.5 ml/min) on a Luna 5 μ C18 (2) 100 Å column (Phenomenex) at 220 nm. The structures of the isolated homologs were confirmed by gas chromatography–mass spectroscopy according to a previously published method [40,41]. The pKa values of the AR homologs were determined by ACD/Percepta 14.0.0 software (ACD/Labs, Toronto,

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Ontario, Canada). The pKa values were 9.5 and 11.1 for all of the AR homologs. 2.3. Preparation of liposomes Chloroform solutions of DPPC or egg SM mixed with ARs and/or Chol in an appropriate molar ratio were prepared. The chloroform was then evaporated under nitrogen gas, and the resulting thin lipid film was redissolved in tert-butanol. These mixtures were frozen in liquid nitrogen and freeze-dried overnight at a low pressure using a Savant Modulyo apparatus (Savant, USA). The dry samples of DPPC or SM mixed with ARs and/or Chol were then hydrated with MilliQ water. The hydration process was performed in a water bath at a temperature of 10 °C above the respective gel–liquid-crystalline phase transition temperatures. The obtained multilamellar vesicles were subjected to 10 cycles of freezing in liquid nitrogen and thawing in a sonic bath at 60 °C without further calibration. The mean diameter of the vesicles was determined by a Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK). Their average size ranged from 350 to 500 nm. 2.4. Fluorescence measurements Liposome suspensions with a DPPC concentration of 68 μM were incubated with Laurdan (1.25 μM) in the dark for 50 min at room temperature. The steady state emission of Laurdan was measured by a Cary Eclipse fluorescence spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a thermostated cell holder in the range 10– 65 °C. For the temperature-dependent fluorescence intensity measurements, the excitation wavelength was 390 nm. The excitation wavelength was 320–400 nm for the fluorescence excitation wavelength dependence studies. The Laurdan GP parameter was calculated using the following equation: GP ¼ ðI440 −I490 Þ=ðI440 þ I490 Þ

ð1Þ

where I440 and I490 are the fluorescence emission intensities at the blue and red edges of the emission spectrum, respectively. Because of the high pKa values of the AR homologs, these compounds were in the uncharged state in the neutral pH water suspensions for all of the liposomes. The presence of ARs in the DPPC membranes should therefore not change the membrane potential, surface charge, or local pH, which can affect the Laurdan fluorescence. 3. Results and discussion 3.1. Laurdan emission study of DPPC/Chol liposomes To determine whether AR compounds have a similar effect on DPPC membranes to Chol, a Laurdan fluorescence study of DPPC liposomes mixed with different molar percentages of Chol was performed. The emission and excitation spectra of Laurdan shift according to changes in the environment close to the fluorescence probe [42]. Laurdan is very sensitive to the polarity and mobility of its lipid surrounding, especially to the presence of water molecules, which can reorient around the Laurdan fluorescent moiety [33,42]. In polar (highly hydrated) lipid membranes, formation of the Laurdan charge transfer excited state is determined by dipolar reorientation of water molecules around the dipole of the probe. Because the gel and liquid-crystalline lipid phases differ in the level of hydration and water mobility, Laurdan can be used as a fluorescence probe to monitor the transition between these two phases with good precision [5,33–35]. Additionally, this probe allows domain formation in lipid membranes to be observed [6,34,36,43]. In the liquid-crystalline phase, an increase in hydration of the glycerol backbone of the phospholipids, which is the region of the lipid bilayer where the fluorophore of Laurdan is located, together with a decrease in the membrane order both cause formation of the charge transfer excited state of

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Fig. 2. (A) Laurdan emission GP as a function of temperature in pure and Chol-mixed DPPC liposomes: pure DPPC (blue squares), and 10 mol% (orange circles), 25 mol% (grey stars), 30 mol% (yellow triangles), 35 mol% (navy blue rhombuses), and 40 mol% (green crosses) Chol in the DPPC/Chol liposomes. (B) Temperature of the chain melting phase transition (Tm) for the DPPC/Chol liposomes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the naphthalene residue, which is stabilized by the water dipole reorientation process. This effect can be seen in the red-shifted emission of Laurdan in the liquid-crystalline lipid phase. For the pure DPPC liposomes under study, the maximum of the Laurdan fluorescence spectrum is at 490 nm (data not shown). At this condition, the excitation spectrum has a peak maximum at the blue edge of the spectrum (340 nm). In the gel lipid phase, the blue shift of the emission band (to 445 nm for the pure DPPC liposomes) and the red shift of the maximum of the excitation spectrum (to 390 nm) are a consequence of a strong decrease of the dipolar relaxation process caused by both a decrease in the water concentration inside the DPPC membranes and a decrease in the molecular motion of water molecules in the tightly packed membrane. The spectroscopic properties of Laurdan are described by the GP parameter (Eq. (1)). In the pure DPPC bilayer, the homogeneous gel phase has a GP value of 0.5, and the pure liquid-crystalline phase has GP values ranging from −0.2 to −0.4 with increasing temperature (see Fig. 2A). In all of the Chol-mixed DPPC liposomes, the low-temperature phase is represented by the same GP value (around 0.55), which is slightly higher than that of the gel phase of pure DPPC liposomes (see Fig. 2A). Thus, in this temperature range, the GP value is not dependent on the concentration of Chol. This is in agreement with other Laurdan fluorescence studies of PC/Chol systems [5,6]. Based mainly on IR studies presented in a literature for PC/Chol membranes, it has been suggested that the presence of Chol molecules decrease membrane hydration at the level of the lipid glycerol backbone [44]. Conversely, some researchers have suggested that Chol molecules have no significant effect on hydration of the polar/apolar interface of PC membranes in the temperature range of the gel phase [2,7]. Additionally, a Laurdan fluorescence anisotropy study of DPPC/Chol liposomes by Bell and co-workers [5] clearly showed that Chol molecules increase the Laurdan anisotropy as a consequence of an increase in the order of the DPPC membrane in the gel phase. The ordering effect of Chol molecules has been confirmed by FT-IR studies [2,45], which showed that Chol induces an increase in the conformational order of the hydrocarbon lipid chains of gel phospholipid bilayers. Therefore, it is concluded that this slight increase in the GP value for DPPC/Chol liposomes in the gel phase (see Fig. 2A) is mainly because of an increase in the membrane order, and it is most probably accompanied by a slight decrease in the water content at the level of the glycerol lipid backbone. Chol molecules have a much more significant effect on the liquidcrystalline phase than the gel phase of the DPPC bilayer. The GP values clearly increase with increasing Chol concentration at higher temperatures (Fig. 2A). This is a consequence of formation of the liquid-ordered

phase (Lo) [46,47] in the Chol-rich DPPC membranes rather than the disordered liquid-crystalline phase (Lo), which is the characteristic phase for the pure DPPC system. In this Chol-induced high-temperature phase, the amount of water-mediated dipolar relaxation of Laurdan clearly decreased compared with the relaxation phenomenon of Laurdan in the liquid-crystalline phase of the pure DPPC bilayer. The Chol-induced Lo phase is characterized by high lipid-chain ordering [2,45], which is similar to that observed in the gel phase of pure phospholipids. Additionally, the Lo phase is characterized by a restricted rate of lateral diffusion and a reduced area per lipid molecule [2]. In a Laurdan study of DMPC/Chol membranes by Parasassi et al. [6], it was concluded that the main effect of Chol addition to lipid bilayers in the liquid-crystalline state is a decrease in the molecular motion of water molecules rather than a decrease in the water concentration. At 41 °C, there is a sharp decrease in the GP value of the main phase transition (Tm) for the pure DPPC membranes (see Fig. 2A). An increase in the Chol content in the DPPC liposomes progressively widens the range of temperatures at which the lipid phase transition occurs with a simultaneous decrease in the difference between the GP values at high and low temperatures, which agrees with the results reported in Refs. [5,6]. The Chol-induced increase in the Tm value of the mixed DPPC membranes is shown in Fig. 2B. Plots of the Laurdan emission GP as a function of the excitation wavelength at the temperatures of the gel state, liquid-crystalline state, and Tm of the Chol-mixed DPPC liposomes are shown in Fig. 3. The independence between the GP value and the wavelength, which is characteristic of the homogenous gel phase of pure DPPC membranes [34–36,42], is not affected by addition of Chol. This means that at low temperatures, Chol molecules can mix with lipid molecules, or Cholrich and Chol-poor domains form in Chol/DPPC mixtures but with no significant difference in their order and/or hydration. Based on the phase diagrams of PC/Chol membranes [7,48], it can be concluded that in the low-temperature region, the gel phase, which is the characteristic phase for pure PC bilayers, can coexist with the Chol-induced Lo state only for the medium range of Chol content. This phase separation in the low-temperature state of the DPPC/Chol mixtures was also shown by Redondo-Marata et al. [49] using atomic force microscopy. Thus, the results in Fig. 3 show that if this phase separation is also present in the Chol/DPPC membranes in the gel phase, these domains cannot be distinguished by the Laurdan fluorescence. At the highest Chol concentration, it is suggested that only one phase is present [7,48]. Accordingly, the Chol-rich systems have a constant linear function of GP (λex) in the low-temperature range.

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Fig. 3. Laurdan emission GP as a function of the excitation wavelength at the temperatures of the gel (stars), Tm (triangles), and liquid-crystalline (circles) states of Chol-mixed DPPC liposomes: pure DPPC (blue), and 10 mol% (orange), 25 mol% (grey), 30 mol% (yellow), 35 mol% (navy blue), and 40 mol% (green) Chol in the DPPC/Chol liposomes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

From the temperature dependence of the Laurdan GP (Fig. 2A), an increase in the Chol content decreases the difference between the low- and high-temperature GP values. This means that the low- and high-temperature Chol-rich lipid membranes become more similar with increasing membrane content of Chol. Therefore, for the Chol/DPPC system at the temperature of the phase transition, where the low-temperature phase coexists with the high-temperature state, the wavelength dependence of the GP value for domain coexistence is only slightly visible (Fig. 3). A slightly decreasing function of GP(λex) for the liquid-crystalline phase of the pure DPPC membranes is only observed for Chol-poor systems. For Chol-rich DPPC membranes, the high-temperature phase is represented by an almost constant function of GP(λex), which is characteristic of a homogenous lipid mixture. Indeed, Chol-rich membranes are mainly in the Lo phase at higher temperatures [47,49]. 3.2. Laurdan emission study of DPPC/AR liposomes To monitor the effect of long-chain homologs of AR (see Fig. 1 for their structures) on the structure of DPPC bilayers, the Laurdan emission GP values were analyzed as a function of temperature (see Fig. 4 for the C15 homolog of AR as an example) and excitation wavelength (data not shown). It is interesting that all of the homologs of AR considered in this study (hydrocarbon chain lengths of C15, C17, C19, C21, C23, and C25) incorporated in the DPPC bilayers in the AR concentration range 5– 50 mol% have the same effect on the GP value at low temperatures (data not shown). Similar to the DPPC/Chol mixtures, for the ARmixed DPPC membranes in a gel phase, the GP values only slightly increase (by ~ 0.1) compared with those for the pure DPPC membranes (see Fig. 4). The liquid-crystalline phase is much more modified by the presence of ARs than the gel phase. In this case, the changes in GP values are strongly dependent on the AR concentration, and there are only slight differences in the GP values between AR molecules with different hydrocarbon chain lengths (data not shown). Modification of the structures of the gel and liquid-crystalline phases of the DPPC membranes by ARs is similar to that of the DPPC/Chol system. The Chol-concentrationdependent increase in the GP value at high temperature is mainly because of an increase in the membrane order [2,6,45]. A molecular dynamics study of DMPC/AR mixtures by Siwko et al. [50] showed that the presence of AR molecules can also markedly increase the ordering of the DMPC tails. Conversely, it has been suggested that the length of

Fig. 4. Laurdan GP as a function of temperature in DPPC liposomes doped with 0 mol% AR (15:0) (blue squares), 5 mol% AR (orange circles), 10 mol% AR (grey stars), 15 mol% AR (yellow triangles), 30 mol% AR (navy blue rhombuses), and 50 mol% AR (green crosses). The emission GP parameters were calculated from the emission spectra with an excitation wavelength of 390 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the AR chain does not affect the ordering [50]. Additionally, the degree of hydration of this more ordered AR-mixed lipid membrane decreases [50], especially at the level of the glycerol backbone of the phospholipids where the fluorophore of Laurdan is located. This dehydration process in the AR-mixed DPPC membranes can be easily detected by Laurdan fluorescence spectroscopy. An AR-concentration-dependent increase in the GP value for the DPPC/AR system in a high-temperature range is thus accompanied by both an increase in the membrane order and a decrease in membrane hydration. The Chol-like rigidifying effect of AR molecules on the bilayer fluidity has also been found in ESR and differential scanning calorimetry (DSC) studies [50–54]. The relationships between the temperature of the main phase transition of DPPC/AR (derived from a sigmoidal curve of the temperature dependence of the GP value) and the concentration of the AR homologs are shown in Fig. 5. All of the AR homologs clearly increase the Tm value of the DPPC membrane. Short-chain AR molecules (C15 and C17) have a similar effect on the Tm to Chol. In this case, the phase transition temperature is around 49–50 °C and only slightly increases with increasing AR concentration. With increasing AR chain length, the effect of the ARs on the Tm is less similar to that of the DPPC/Chol mixtures. For longchain AR homologs, the Tm value is more dependent on the AR concentration (see Fig. 5). Interestingly, the phase transition of the DPPC/AR system retains its high cooperativity even for a very high AR content, independently of the AR chain length (see the results for the C15 homolog in Fig. 4). This is different from the DPPC/Chol mixtures, where a high Chol concentration almost prevents the phase transition.

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The slopes of the GP(λex) functions for all of the DPPC/AR membranes (data not show) correspond to the slopes of the DPPC/Chol membranes at the corresponding temperatures. Similar to the DPPC/ Chol system, there is no clear evidence of domain formation in the DPPC/AR system. 3.3. Laurdan emission study of DPPC/Chol/AR liposomes To investigate the change of the structure of the DPPC bilayer in the presence of both Chol and AR molecules, the ternary lipid complex was investigated. In the DPPC/Chol/AR system, Chol molecules were

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progressively replaced by AR molecules. The temperature-dependent evolution of the Laurdan emission GP values for DPPC/Chol/AR membranes with different Chol and AR contents is shown in Fig. 6. Substituting Chol molecules with AR molecules leads to a slight increase in the GP value for the gel phase and a decrease in the GP value for the high-temperature phase. This effect is similar for each AR homolog and more visible for AR-rich membranes. Although Chol and AR molecules can separately affect the lipid bilayer structure in a similar way, replacement of Chol by AR changes the properties of the lipid membrane. The gel phase in the AR-rich membranes is more ordered than in the pure DPPC membranes. In contrast, the high-temperature state of the

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Fig. 6. Laurdan GP as a function of temperature in DPPC liposomes doped with Chol and AR homologs: (A) AR (15:0), (B) AR (17:0), (C) AR (19:0), (D) AR (21:0), (E) AR (23:0), and (F) AR (25:0). The symbols represent the AR concentration: 0 mol% (squares), 5 mol% (circles), 10 mol% (stars), 15 mol% (triangles), and 30 mol% (rhombuses). The emission GP parameters were calculated from the emission spectra with an excitation wavelength of 390 nm.

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the surrounding lipids, and therefore mixtures with the same molar ratio of all components but different AR tail lengths give different Tm temperatures. In the Chol-poor systems, AR molecules mainly affect the Tm value. Almost constant GP(λex) linear functions, similar to the functions shown in Fig. 2, were obtained for all of the DPPC/Chol/AR mixtures at all of the temperatures studied (data not shown). Irrespective of the chemical composition of the Chol- and AR-mixed DPPC bilayer and the chain length of the AR homolog, the DPPC/Chol/AR mixtures are thus quite homogenous in terms of the membrane order and/or hydration in the whole temperature range. 3.4. Laurdan emission study of SM/Chol and SM/AR liposomes

DPPC:Chol:AR [mol:mol:mol] Fig. 7. Tm values of the DPPC/Chol/AR liposomes.

mixed membranes gradually becomes disordered and/or rehydrates with increasing AR content. This is surprising because AR molecules induce an increase in the lipid chain order and cause dehydration of the membrane (see Fig. 4), similar to Chol molecules. The ability of AR lipids to maintain the relatively high cooperativity of the phase transition, which is the opposite effect to Chol, is retained in the ternary complexes. An increase in the AR concentration accompanied by a decrease in the Chol content in the DPPC/Chol/AR membranes resulted in a decrease in the temperature range of the observed phase transition. The effect of changes in the molecular composition of the DPPC/ Chol/AR system on the Tm temperature is shown in Fig. 7. The Tm temperature changes in two ways in the Chol-rich ternary complexes. In this case, the Tm temperature is dependent on the chain length of the AR molecule. Short-chain AR homologs increase the Tm of the DPPC/ Chol/AR membranes more than long-chain homologs (see Fig. 7). The effect of the lipid chain length on the Tm value of the Chol-mixed PC membranes was investigated by McMullen et al. [3] using DSC. They found that Chol progressively decreases the Tm temperature of PC membranes with saturated hydrocarbon chains containing 18 or more carbon atoms. In contrast, Chol molecules increase the Tm value in the presence of PC lipid with a chain length of 16 carbon atoms or less [3, 55]. A similar chain-length-dependent effect is observed in the DPPC/ Chol/AR mixtures in this study. The final Tm value is affected by both Chol and AR molecules. In the Chol-rich membranes, the Chol molecules mainly determine the Tm value, which is sensitive to the chain length of

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The relationships between the GP value and the temperature for pure SM, and Chol- and AR-mixed SM liposomes are shown in Fig. 8A. Additionally, to compare the effect of Chol and AR molecules on SM membranes and the DPPC systems, the temperature-related changes in the GP values for pure DPPC, DPPC/Chol, and DPPC/AR with 30 mol% Chol and AR molecules in the lipid membranes are shown in Fig. 8B. The gel phase of the pure SM membrane has a GP value of around 0.4 and the liquid-crystalline state has a GP value of − 0.55. These values are slightly lower than the corresponding values in the gel and liquid-crystalline phases of the pure DPPC membrane. This is because the egg SM used in this study was a mixture of lipids with different hydrocarbon chain lengths, whereas DPPC molecules have only C16 hydrocarbon chains. The egg SM membrane is thus less homogenous than the DPPC membrane with less tightly packed lipids and a more hydrated membrane. These features lead to lower GP values in both the gel and liquid-crystalline phases of the SM membranes than the corresponding values in the more ordered and more tightly packed DPPC membranes. The main phase transition is less cooperative in the pure SM membrane than in the pure DPPC membrane. Incorporation of 30 mol% Chol in the SM and DPPC membranes has a similar effect (see Fig. 8A and B). The gel phase of these both systems has similar GP values, and in the liquid-crystalline phase GP values are also almost the same. On the other hand, the main phase transition is less cooperative in the case of SM/Chol than in DPPC/Chol. AR homologs with C15, C17, C19 and C21 hydrocarbon chain lengths modify the gel phase of the pure SM membrane in the same way to Chol molecules (see Fig. 8A). Conversely, for the long-chain homologs of AR molecules (C23 and C25), the GP values are considerably lower as a consequence of AR-triggered fluidization and/or hydration of the SM membrane in

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Fig. 8. Laurdan emission GP as a function of temperature in (A) DPPC and (B) SM liposomes. Pure DPPC and SM liposomes (red squares), and DPPC and SM liposomes doped with 30 mol% of Chol (black crosses), AR(15:0) (blue squares), AR(17:0) (orange circles), AR(19:0) (grey stars), AR(21:0) (yellow triangles), AR(23:0) (navy blue rhombuses), and AR(25:0) (green crosses). The emission GP parameters were calculated from the emission spectra with an excitation wavelength of 390 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

P. Zawilska, K. Cieślik-Boczula / Biophysical Chemistry 221 (2017) 1–9

Temperature [°C]

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55

Acknowledgments

50

This work was supported by the National Science Centre, Poland (decision numbers 2015/17/B/ST4/03717 (OPUS) and DEC-2012/05/B/ ST4/02029 (OPUS)). We thank the OAD (project number PL 07/2013) and the Foundation for Polish Science (Pomost/2012-5/2), the POMOST program, co-financed by the European Union within the European Regional Development Fund (V edition, 2012) for additional financial support. Assistance from Dr. Rikard Landberg (Swedish University of Agricultural Sciences) is gratefully acknowledged.

45 40 35 30

Fig. 9. Tm values of the mixed DPPC (blue squares) and SM (green circles) liposomes. L means the DPPC or SM lipid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the gel phase. Similarly to the DPPC systems, the presence of all of the AR molecules in the liquid-crystalline phase of the SM membrane results in higher GP values than those for the pure SM membranes. Thus, in both types of membrane (DPPC and SM), ARs increase the membrane order and/or dehydration, which is characteristic for Cholmixed membranes. The exception of this is only for the SM membrane in the gel phase mixed with the shot-chain AR homologs. Characteristic for DPPC/AR systems higher cooperativity of the phase transition compared with DPPC/Chol membranes, is still observed for the SM/AR systems (compare images A and B in Fig. 8). Similar to the Chol-mixed SM membrane, an increase in the Tm value is also observed for the ARs-mixed SM membranes (see Fig. 9). The Tm values for the SM/AR samples are less than for DPPC/AR systems (see Fig. 9). Almost constant GP(λex) linear functions are obtained for the SM/AR membranes, similar to the Chol- and AR-mixed DPPC liposomes (data not shown). The SM/AR (or Chol) mixtures are thus relatively homogenous in terms of the membrane order and/or hydration in the whole temperature range without any evidence of domain formation. 4. Conclusions The results can be summarized as follows: 1. AR homologs have a Chol-like effect on both DPPC and SM bilayers in terms of the lipid chain ordering, membrane dehydration, and the increase in the Tm value. From these results, we conclude that longchain homologs of AR are promising candidates for replacing Chol molecules in DPPC-based liposomal drug carriers. A Chol-like effect on SM bilayers suggests the same effect on the biomembranes of living cells, which should increase research interest in AR molecules as a promising modulator of lipid biomembranes. 2. The main difference between lipid (DPPC or SM) membranes mixed with Chol and ARs is the ability of the AR homologs to maintain the relatively high cooperativity of the main lipid phase transition, even at high AR concentrations. 3. A characteristic of the Chol-mixed membrane is that the increase in Tm is more distinct for DPPC/AR mixtures and is dependent on the AR concentration and the AR hydrocarbon chain length. 4. The ability of AR lipids to maintain the relatively high cooperativity of the phase lipid transition, which is the opposite effect to Chol, is maintained in DPPC/Chol/AR membranes. The Tm value of the ternary lipid complexes changes depending on the relative contributions of each membrane component and the chain length of the AR homolog.

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