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Effect of cis-(Z)-flupentixol on DPPC membranes in the presence and absence of cholesterol
Chemistry and Physics of Lipids 198 (2016) 61–71
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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Effect of cis-(Z)-flupentixol on DPPC membranes in the presence and absence of cholesterol Dilek Yonara,* , M.Maral Sunnetcioglub a b
Middle East Technical University, Department of Biological Sciences, 06800, Ankara, Turkey Hacettepe University, Department of Physics Engineering, 06800 Beytepe, Ankara, Turkey
A R T I C L E I N F O
Article history: Received 10 February 2016 Received in revised form 21 May 2016 Accepted 2 June 2016 Available online 6 June 2016 Keywords: Phospholipid membrane Flupentixol EPR spin labeling FTIR and DSC
A B S T R A C T
Cis-(Z)-flupentixol dihydrochloride (FLU), a thioxanthene drug, is used in therapy of schizophrenia as well as in anxiolytic and depressive disorders. Since the action mechanism of FLU is not completely understood, the main objective of present study is to provide a detailed evaluation of flupentixolphospholipid membrane interactions at molecular level. FLU-dipalmitoylphosphatidylcholine (DPPC) interactions in presence and absence of cholesterol (CHO) were investigated as a function of temperature. The changes in upper part of membrane were more pronounced than those in central part of membrane, as indicated by EPR and FTIR. FLU was proposed to incorporate into phospholipid membranes with its triple ring parallel to head group and its chain toward alkyl chain of phospholipids. According to DSC results, the incorporation of 10 mol% FLU into DPPC caused a shoulder in transition peak, suggesting the occurence of a phase separation, and formation of this new phase is still observable in presence of CHO. It is well known that, structure and dynamics of lipids have significant influence on the function of membrane bound proteins, and consecutively their actions. Based upon these, it was proposed that FLU may modify membrane associated receptors and transport proteins, which would form the basis of its clinical efficiency. ã 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The investigation of membrane-drug interactions is of critical importance for pharmacological science because it is directly related to the mechanisms of drug action (Bourgaux and Couvreur, 2014). Many pharmacologically active compounds, with a number of different applications (antibiotics, antifungal, antidepressants, antihistamines, local anesthetics, anticancer drugs, etc.), can cross or bind to cell membranes and possibly alter the physical properties of the membrane (Seddon et al., 2009). Since biological membrane is a quite complex structure, model membranes composed of cellular membrane lipids are used to evaluate membrane-drug interactions. Membrane lipids participate in cellular signaling, hence regulate the important cell functions. Therefore, molecules which interact with membrane lipids may cause alterations in membrane composition, protein function or gene expression etc (Vigh et al., 2005). Flupentixol has two geometric isomers, namely cis-(Z) and trans-(E), however only cis form is pharmacologically active
* Corresponding author. E-mail address: [email protected] (D. Yonar). http://dx.doi.org/10.1016/j.chemphyslip.2016.06.002 0009-3084/ã 2016 Elsevier Ireland Ltd. All rights reserved.
(Woodruff and Freedman, 1981). cis-(Z)-flupentixol dihydrochloride (FLU), (Z)-4-[3-[2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]-1-piperazineethanol dihydrochloride, a catamphiphilic thioxanthene drug, has structural characteristics related to those of the phenothiazines (Fig. 1). FLU, like other thioxanthene compounds, is used in the acute and long term therapy of schizophrenia as well as in the lower dose range for the treatment of anxiolytic and depressive disorders. Although the action mechanism of flupentixol is not completely understood, it is found that flupentixol binds with high affinity to various dopamine receptors, to 5-HT2A, and to an alpha1-adrenergic receptors (Leysen et al., 1993). The studies regarding the interaction of flupentixol with the molecular components of cells and biological membranes were limited. Ford et al. (1990) studied the effects of the neuroleptic drugs cis and trans isomers of flupentixol on primary drug resistance and drug accumulation in several cell lines. Similar binding affinity to P-glycoprotein (Pgp), but different multidrug resistance (MDR) reversing activity has been proposed for trans and cis-flupentixol. In another study, Dey et al. (1997) provided direct evidence in favor of two non-identical drug-interaction sites within P-glycoprotein and proposed a model for the role of these two sites in drug translocation using a photoaffinity substrate
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2.2. Methods 2.2.1. Electron paramagnetic resonance (EPR) spectroscopy
Fig. 1. Chemical structure of cis-(Z)-flupentixol dihydrochloride (FLU).
analog and cis-flupentixol, a known modulator of Pgp function. When cis and trans-flupentixol-phosholipid interaction studies were performed, it was found that trans-flupentixol interacts about twice as strongly with lecithin as do cis-flupentixol. This indicates a stereospecific interaction of these catamphiphiles with phospholipids (Seydel et al., 1991). The same group also proposed in another study that both stereoisomers interacted more strongly with the negatively charged PS than with the neutral PC and a different orientation of trans and cis-flupentixol may be expected when they entered the lipid bilayer by the ring system (Pajeva and Wiese, 1997). The alterations in vitamin A metabolism in vivo as a result of flupentixol and cefotiam administration were investigated. It was concluded that the flupentixol treatment resulted in vitamin A depletion (Schindler et al., 2004). Since the action mechanism of FLU is not clear yet, the main objective of the present study is to provide a detailed evaluation of the interactions between FLU and phospholipid membranes at the molecular level. The interaction of FLU with DPPC membranes in presence and absence of cholesterol is investigated by FTIR, EPR spin labeling and DSC techniques. EPR spin labeling spectroscopy provides site specific information on ordering, mobility and polarity of membranes. Moreover, the insertion depth and orientation of the incorporated molecule within the membrane can be obtained with this technique (Gordon-Grossman et al., 2012). In addition to direct evaluation of EPR spectra, computer simulations of the spectra are performed to obtain more precise description of the membrane characteristics (Štrancar, 2007; lu, 2014). The information regarding the Yonar and Sünnetçiog effect of drug molecule on lipid order, lipid dynamics, phase transition behaviour and hydration of the head group and interfacial region can also be obtained from FTIR spectroscopy (Lewis et al., 2005; Cong et al., 2009). DSC technique has been extensively used to study the precise phase behaviour of hydrated phospholipid membranes (McElhaney, 1982) and to investigate the thermal changes caused by drug incorporation into phospholipid bilayers (Momo et al., 2005; Zhao et al., 2007; How et al., 2014). 2. Materials and methods 2.1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids, cholesterol (CHO), spin labels 5- and 16-doxyl stearic acid (5-DS, 16-DS) and antidepressant drug cis-(Z)-flupentixol dihydrochloride (FLU) were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Phosphate buffered saline (PBS) (without Ca, Mg) was obtained from Dr. Zeydanlı (Dr. Zeydanlı Life Sciences, Ltd. Şti., Ankara, Turkey).
2.2.1.1. Sample preparation. DPPC membranes were prepared in the absence and presence of 30 mol% CHO. 1 mol% concentration of 5- and 16-DS spin labels was used. The drug, FLU, concentration incorporated into the DPPC membranes was 1, 5, 10 mol%. Phospholipids, CHO and FLU were dissolved in chloroform stock solution containing spin label. Chloroform was evaporated first with nitrogen gas stream and then samples were kept under vacuum for overnight to remove residual chloroform. The obtained dry films were hydrated by 0.15 ml of PBS at pH 7.4, mixed with vortex at a temperature above phase transition temperature of the lipids to get multilamellar vesicles (MLVs) and then centrifuged (Eppendorf 5804-R; Eppendorf-Netheler-Hinz GmbH, Hamburg, lu, 2014). Pellets were Germany) at 20 C (Yonar and Sünnetçiog used in the studies. EPR measurements were performed on a Bruker EMX-131 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) with ER4103TM cylindirical cavity in a temperature range 10–55 C using the following spectral conditions: modulation frequency 100 kHz, modulation amplitude 0.2 mT and microwave power 10 mW. Sample temperature was controlled to 1 C by Bruker VT4111 temperature controller. Each sample was prepared and measurements were repeated at least two times. 2.2.1.2. Computer simulation of EPR spectra. EPR spectra are composed of several superimposed spectral components, since membrane is a heterogeneous structure composed of the regions with different motional characteristics and polarities. Therefore, except from direct spectral evaluation, EPR spectra of the studied samples were simulated by a computer program called EPRSIMC using a multi-component fast restricted wobbling motion approximation to get further information about the physical parameters of the membrane domains. The model used for fitting procedure allows up to four spectral components with different spectral parameters (Štrancar, 2007; Štrancar et al., 2003). The model parameters provided for each spectral component are the open cone angle q of the wobbling motion, asymmetry angle of the cone f, one effective rotational correlation time tc, which describes the rate of motion, additional broadening constant W, polarity correction factor on hyperfine splitting (A) tensor, pa, which is used as a scalar to correct all the A tensor components, describing the differences in polarity of the spin label surroundings, and the weight of each spectral component which describes the relative amount of the spin probes with particular motional mode. The order parameter S, free rotational space of nitroxide V, and the normalized rotational diffusion rate of nitroxide Dr constructed from the model parameters and definitions used in the cone model of wobbling motion are given below. S ¼ 0:5 cos2 u þ cosu
V ¼ uf=ðp=2Þ2 Dr ¼ uf=4tc Simulations were performed for each sample in the temperature range studied.
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2.2.2. Fourier transform infrared (FTIR) spectroscopy The samples were prepared in the same way with those for the EPR studies but without incorporation of the spin label. FTIR measurements were done by using water-insoluble CaF2 windows. Sample was placed between the CaF2 windows and 10 mm sample thickness was obtained by mylar spacers. Spectra were recorded within a temperature range of 10–55 C using a Perkin Elmer Spectrum One FTIR spectrometer with temperature-stabilized fast recovery deuterated triglycine sulfate (FR-DTGS) detector. Temperature was controlled by Unicam Specac Digital Temperature controller mounting the sample holder. Interferograms were averaged for 100 scans at 4 cm1 resolution. All samples were prepared at least two times and their spectra were acquired. The water bands stemming from the presence of buffer were subtracted in order to provide a better resolution of the bands. During the subtraction process, the water band located around 2125 cm1 was flattened. The normalization of spectra in the studied regions was carried out for the representation of relative changes using peak normalization. 2.2.3. Differential scanning calorimetry (DSC) DSC was used to monitor the influence of FLU on thermotropic properties of DPPC MLVs. The samples were prepared in the same way with those for the FTIR studies. Samples were placed in a sealed aluminum pans. An empty pan was also used as reference during the measurements, so that its calorimetric effect was extracted by the computer program. Calorimetric measurements were carried out using a Perkin Elmer Diamond DSC at a scan rate lu, 5 C/min as described in previous studies (Yonar and Sünnetçiog 2014; Rubio et al., 2011). Each sample was prepared and analyzed at least three times to check the repeatibility of the results. Two heating and cooling scans were performed for each analysis to ensure the reproducibility. Only heating curves are evaluated and presented here. The temperature at the peak maximum is defined as the transition temperature (Tm) and the area under the peak after baseline adjustment and normalization to the sample amount represents the enthalpy change (DHm) during the transition. 2.2.4. Statistical analysis The results were represented as the mean values of parameters and the errors were calculated as standard deviation. Statistical significance was evaluated by using two-way analysis of variance (2way ANOVA) and Dunnett’s multiple comparison test. The degree of significance (p values) with respect to the pure DPPC was p < 0.05.
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3. Results 3.1. EPR results The effect of FLU on lipid order and dynamic was investigated by using 5- and 16-DS spin labels with nitroxide group localized at different depths in DPPC membranes. Experimental and calculated spectra of the samples were given at 45 C for 5-DS labeled and at physiological temperature (37 C) for 16-DS labeled DPPC membranes in Fig. 2a and b. The given temperatures were selected from the region where the greater changes exist. The changes in the maximum hyperfine splitting constant (2Amax) for 5-DS and in the mid-field line width (DBpp) for 16-DS labeled membranes were analyzed directly from EPR spectra. 2Amax values reflect the average ordering of the alkyl chains and DBpp is related to the dynamics of the spin label’s nitroxide group. Temperature dependence of 2Amax of 5-DS in DPPC MLVs at different concentration of FLU was given in Fig. 3a and b. The addition of FLU to DPPC membranes gave rise to a shift towards the lower temperatures in the main phase transition temperature of DPPC. This shift was more pronounced for 10 mol% FLU. For 5-DS labeled DPPC MLVs, incorporation of FLU did not cause any significant changes in 2Amax values in the gel phase, but in the liquid crystalline phase all concentrations of FLU induced gradual decrease in 2Amax values. The changes in 2Amax with temperature were distributed in a broader range for 5 and 10 mol% FLU (Fig. 3a). There was a slight decrease in 2Amax values at lower temperatures in the CHO incorporated DPPC MLVs with 5 and 10 mol% of FLU, whereas a more pronounced decrease was observed at higher temperatures (Fig. 3b). Besides direct evaluation of EPR spectra, the spectra of the studied samples were simulated to get more information on domain (spectral components with different motional pattern) properties of membrane and to obtain the physical parameters of these components. Some of the physical parameters were given in Fig. 3c and d for 5-DS labeled DPPC MLVs. Generally two motional patterns of 5-DS were observed in DPPC MLVs. The number of domains was more than two at some temperatures. A shift in the phase transition temperature towards lower temperatures was observed with the addition of FLU. FLU caused a decrease in the order parameter (S), a slight decrease in the rotational diffusion rate (Dr) and a slight increase in the free rotational space of nitroxide (V) especially at temperatures above phase transition temperature, but it did not make any significant effect on polarity (pa) (Fig. 3c). The observed changes in the physical parameters of
Fig. 2. Examples of experimental (black line), calculated (red line) total and domain's spectra (blue, green, magenta lines) of a) 5-DS and b) 16-DS spin labeled DPPC MLVs at 45 and 37 C, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Temperature dependence of 2Amax of 5-DS in (a) pure (b) 30 mol% of CHO incorporated DPPC MLVs at various concentrations of FLU. Temperature dependence of 10 mol % FLU induced changes in the order parameters (S) and rotational diffusion rate (Dr) of 5-DS in (c) pure, (d) 30 mol% CHO incorporated DPPC MLVs. Diameter of circles denotes the proportion of spin probe in different domain types. Bars indicate second moment in the distribution of values obtained from the GHOST condensation technique.
CHO incorporated DPPC membranes were less pronounced than the pure ones. However, a significant decrease in the rotational diffusion rate of nitroxide (Dr) for less ordered domain was observed at 37 C and above by incorporation of 10 mol% FLU (Fig. 3d). 16-DS spin label was employed to monitor the changes in the alkyl chain region of DPPC membranes. Temperature dependence of DBpp of 16-DS in DPPC MLVs at various concentrations of FLU is given in Fig. 4a and b. The addition of 5 and 10 mol% FLU to DPPC
MLVs caused a significant decrease in DBpp values at temperatures below phase transition, which implies an increase in the dynamics of the nitroxide, but it did not cause any significant change at temperatures above phase transition (Fig. 4a). In CHO incorporated DPPC MLVs, FLU incorporation caused a decrease in DBpp values at all temperatures. This decrease was more pronounced for 5 and 10 mol% FLU incorporated samples (Fig. 4b). Concerning the simulation results of alkyl chain region of membranes, some of the physical parameters (S, pa and Dr) for
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Fig. 4. Temperature dependence of DBpp of 16-DS in (a) pure, (b) 30 mol% of CHO incorporated DPPC MLVs at various concentrations of FLU. Temperature dependence of 10 mol% FLU induced changes in the order parameters (S), polarity correction factor (pa) and rotational diffusion rate (Dr) of 16-DS in (c) pure, (d) 30 mol% CHO incorporated DPPC MLVs. Diameter of circles denotes the proportion of spin probe in different domain types. Bars indicate second moment in the distribution of values obtained from the GHOST condensation technique.
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pure and CHO incorporated DPPC membranes are shown in Fig. 4c and d. Generally two motionally different patterns and a shift in the phase transition temperature towards lower temperatures with the addition of FLU were observed. The effect of 10 mol% FLU on DPPC MLVs was seen as an increase in the polarity of the most ordered domain in the gel phase. There was no change in the liquid crystalline phase with the addition of FLU as in the direct evaluation of spectra (Fig. 4c). In CHO incorporated DPPC MLVs, the order parameter of the less ordered domain decreased and polarity correction factor of the same domain increased slightly with the addition of FLU below 28 C. There was a significant abnormal increase in the rotational diffusion rate of nitroxide around the phase transition temperature of DPPC in the presence of CHO. This increase in diffusion was repressed when 10 mol% FLU was added (Fig. 4d). This repression was also seen in 1 and 5 mol% FLU incorporated samples (data not shown). 3.2. FTIR results Both pure and CHO incorporated DPPC MLVs containing 1– 10 mol% of FLU were investigated as a function of temperature by FTIR spectroscopy. Fig. 5 shows the normalized FTIR spectra of DPPC MLVs in the absence and presence of 1, 5, and 10 mol% FLU at 37 C in 3000–1000 cm1 region with enlarged panel zoomed into C¼O stretching band. The normalized spectra is demonstrated for the representation of comparative changes in the frequency, intensity and bandwidth of the mentioned bands in the absence and presence of FLU. The changes in the lipid hydrocarbon chain conformational order-disorder and hydrocarbon chain-melting phase transitions were obtained by using the symmetric and asymmetric stretching vibrations of the methylene (CH2) group (2850 and 2920 cm1, respectively) on lipid hydrocarbon chains (Mantsch and McElhaney, 1991). Fig. 6a and b shows temperature dependence of the frequency of CH2 asymmetric stretching band of DPPC MLVs in the absence and presence of different concentrations of FLU. As seen from the figure, the broadening of phase transition profile was observed by the addition of higher concentrations of FLU. The frequency values of concerned band were measured at the center of the peaks. The gradual shifts towards lower temperatures in the main phase transition temperature (Tm) of DPPC were observed with increasing FLU concentration. The same results were obtained for the CH2 symmetric stretching band (data not shown).
An increase in the frequency of CH2 stretching band reflects the lipid hydrocarbon chain’s conformational disorder. As FLU concentration was gradually increased, the frequency of the CH2 stretching band decreased below Tm which ascribes to the order of the system in the gel phase. But at 35 C and above up to phase transition, the frequency of the CH2 stretching band increased significantly with the addition of 10 mol% of FLU, which implies a decrease in the order of the system during the phase changes, and there was no significant change in the liquid crystalline phase (Fig. 6a). This is due to the further reducing of the phase transition temperature by the addition of 10 mol% of FLU. The results for CHO incorporated DPPC MLVs are shown in Fig. 6b. Significant increase in CH2 stretching frequency were seen by the addition of 10 mol% FLU. This result indicates that higher concentrations of FLU caused a decrease in the order of the CHO incorporated DPPC MLVs (Fig. 6b). The temperature dependence of the bandwidth of CH2 asymmetric stretching band for pure and CHO incorporated DPPC MLVs in absence and presence of FLU is shown in Fig. 6c and d. The bandwidths of CH2 asymmetric stretching band were measured at 75% of height of the peaks. The bandwidth is related to the motional rates of the molecule, so it is the evidence of membrane dynamics (Mantsch and McElhaney, 1991). At 35 C and above up to 41 C, an increase in the bandwidth values were seen especially with 10 mol% incorporation of FLU. However, there were no significant changes in the liquid crystalline phase by FLU incorporation (Fig. 6c). As seen in Fig. 6d, the bandwith of CH2 asymmetric stretching band at all temperature range increased by incorporation of 5 and 10 mol% FLU, which shows the increased dynamic of the membrane. The degree of significance with respect to pure DPPC MLVs was p < 0.05. Temperature dependence of the frequency of C¼O stretching band of DPPC MLVs in the absence and presence of different concentrations of FLU is shown in Fig. 7. In the drug-membrane interaction studies, the changes in the C¼O stretching absorption band located between 1740 and 1730 cm1 might provide important clues to the structural and/or hydration changes of bilayer polar-apolar interfacial region (Cong et al., 2009; Lewis and McElhaney, 1998). The frequency of C¼O stretching band shifts towards higher values as compared to pure DPPC MLVs in the gel phase for the samples containing 5 and 10 mol% FLU, which may indicate an increase in dehydration of ester carbonyl groups. At 35 C and above up to phase transition, a dramatic decrease in
Fig. 5. FTIR spectra of DPPC MLVs in the absence and presence of 1, 5 and 10 mol% FLU in 3000–1000 cm1 spectral region with enlarged panel zoomed into C¼O stretching band at 37 C.
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Fig. 6. Temperature dependence of the frequency and bandwidths of CH2 asymmetric stretching bands of (a–c) pure, (b–d) CHO incorporated DPPC MLVs in the absence and presence of 1, 5, 10 mol% FLU.
frequency was observed for 10 mol% FLU incorporated DPPC MLVs, which implies a probable increase in the hydrogen bonded C¼O groups (Fig. 7a). After phase transtition of pure DPPC, a decrease was observed with incorporation of 5 mol% FLU. In the presence of CHO, the addition of 5 and 10 mol% FLU caused a decrease at 35 C and above (Fig. 7b). 3.3. DSC results The changes in the phase transition temperature and the shape of the DSC thermograms were investigated to study the termotropic aspect of drug-membrane interactions. DSC thermograms obtained for DPPC and 1, 5, 10 mol% FLU incorporated DPPC in absence and presence of 30 mol% CHO are presented in Fig. 8a and b and the thermodynamic parameters associated with lipid phase transitions are summarized in Table 1. The incorporation of increased amounts of FLU caused progressive decrease in the main phase transition temperature of DPPC and the addition of 10 mol% of FLU produced the largest decrease around 3.3 C (Fig. 8a and Table 1). While pre-transition peak was still observed with 1 mol% FLU incorporation, it disappeared at the concentrations above 1 mol%. Concentrationdependent and asymmetric broadening of transition peaks were seen by increasing FLU concentration. As consistent with the literature, the transition enthalpies of the pre-transition (DHp) and main transition (DHm) of pure DPPC were 0.97 and 6.23 kcal/mol,
respectively (Yeagle, 2011; Fa et al., 2006). DHp of DPPC significantly decreased at 1 mol% FLU and DHm significantly decreased at all FLU concentrations. The incorporation of 10 mol % FLU into DPPC also caused a shoulder in the transition peak on the left side. In addition, the presence of 30 mol% CHO caused a significantly broad heat flow peak and maximum heat flow observed at higher temperature (2.3 C) than pure DPPC MLVs (Fig. 8b and Table 1). CHO incorporation caused a decrase in DHm of pure DPPC (1.47 kcal/mol). Any significant changes in the transition enthalpy of CHO incorporated DPPC were observed either at low or at high concentrations of FLU as indicated by Table 1. Furthermore, incorporation of FLU into the CHO containing DPPC MLVs caused a decrease in the temperature, at which maximum heat flow was observed. The shoulder in the transition peak on the left side in presence of 10 mol% FLU was still seen with the addition of CHO. 4. Discussion Spectroscopic and calorimetric measurements pointed out that FLU incorporation into phospholipid membranes gives rise to some changes in the physical properties of the membrane such as phase transition, order, and dynamic. The first pronounced effect of FLU was on the phase transition behaviour of DPPC. The lamellar gel (Lb) to lamellar liquid crystalline (La) chain melting phase transition (Tm), a cooperative phase transition, covers the conversion of a highly ordered gel state to a relatively disordered
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Fig. 7. Temperature dependence of the frequency changes at the C¼O stretching band for (a) pure and (b) CHO incorporated DPPC MLVs in the absence and presence of 1, 5, 10 mol% FLU.
liquid crystalline bilayer. While the hydrocarbon chains of the phospholipids are packed in a highly ordered manner below Tm, the chains are disordered and possess more motional freedom above Tm. The incorporation of the drug into phospholipid membrane may alter the phase transition temperature and broaden the thermotropic peak. A shift towards lower temperatures in Tm of DPPC membranes was observed with the increasing concentration of FLU which indicates the occurence of more motional freedom at lower temperatures in presence of FLU (Figs. 3, 4,6–8). A shift approximately 3.3 C towards lower temperatures in Tm was observed with incorporation of 10 mol% of FLU into DPPC MLVs (Fig. 8a). The intercalation of amphiphilic molecules between the polar head groups of a phospholipid membrane is able to depress the main transition temperature. Shifts in both directions in Tm were reported in previous studies with different drugs (Paiva et al., 2012; Pentak, 2014; Sinha et al., 2014; Alsop et al., 2015). DPPC has a pre-transition at around 35 C. While pre-transition peak was still observed with 1 mol% FLU incorporation, it disappeared by the addition of 5 mol% FLU and above. The disappearance of the pre-transition is thought to be caused by the flupentixol interference with the tilting of DPPC alkyl chains (de Lima et al., 1990). The line width at half maximum of the peaks of the heat flow (DT1/2) is also very sensitive to the presence of any additives and it may indicate the changes in the cooperativity. The cooperativity is inversely proportional to DT1/ 2. The addition of FLU broadened the calorimetric peaks and provided more asymmetric peaks (Fig. 8a), indicating a decrease in the transition cooperativity. The polar interactions in the interfacial region of phospholipids and hydrophobic interactions between alkyl chains might be blocked by the addition of the drug. This may cause the formation of different regions in terms of interactions. As a result of these different regions, lipids do not melt at the same temperature and the transition zone broadens (Zhao et al., 2007). The changes in 2Amax and frequency of CH2 asymmetric stretching band which distributed in a broader range for 5 and 10 mol% FLU supports the observed broadening of phase transition profile by DSC. A dramatic decrease in the main transition enthalpy (DHm) values were observed with incorporation of 1, 5 and 10 mol% FLU into DPPC membranes. The enthalpy change associated with the main lipid chain melting is related to molecular packing of the alkyl chain. So, a decrease both in Tm and DHm of the gel to liquid crystalline phase transition in presence of drug indicates the
Fig. 8. DSC thermograms of (a) pure and (b) 30 mol% CHO incorporated DPPC MLVs in the absence and presence of 1, 5, 10 mol% FLU.
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Table 1 Main phase transition temperatures (Tm) and enthalpies of the main phase transition (DHm) for pure and FLU incorporated DPPC membranes in the absence and presence of 30 mol% CHO. FLU amount (mol%)
0 1 5 10
DPPC
CHO incorporated DPPC
Tp ( C)
DHp (kcal/mol)
Tm ( C)
DHm (kcal/mol)
Tm ( C)
DHm (kcal/mol)
37.6 0.1 35.0 0.2 – –
0.97 0.03 0.09 0.01 – –
42.2 0.3 42.0 0.5 40.6 0.4 38.8 0.1
6.23 0.42 4.81 0.59 4.43 0.53 4.18 0.71
44.5 0.6 44.3 0.5 40.0 0.5 31.3 0.7
1.47 0.12 2.14 0.49 1.66 0.09 –
instability of the phospholipid bilayer (Sarpietro et al., 2014; Lucio et al., 2009). The incorporation of 10 mol% FLU into DPPC caused a shoulder in the transition peak on the left side. This type of appearence on either side indicates the domain formation and may indicate the phase separation and immiscibility (Straubinger and Balasubramanian, 2016). According to EPR results, there were some changes in domain percentages by incorporation of FLU (Fig. 3c) which support this phase separation behaviour. Complex effects of cholesterol incorporation on phospholipid thermotropic phase behaviour has been revealed in literature (McMullen and McElhaney, 1995; Vist and Davis, 1990; Epand et al., 2006). The chain length dependent shift direction in the heat flow peak maxima was observed previously. For PCs having hydrocarbon chains of 16 or fewer carbon atoms, a shift towards higher temperature side was expected (McMullen et al., 1993). Broadening in the heat flow peaks, a shift toward higher temperatures in its maximum and a decrease in transition enthalpy were obtained for 30 mol% of CHO containing DPPC MLVs relative to pure ones consistent with literature (Fig. 8b, Table 1). The incorporation of FLU into the CHO containing DPPC MLVs caused a decrease in the temperature, at which maximum heat flow was observed. Up to 5 mol% FLU there is an increase in the linewidth of heat flow peak and a narrowing was observed for 10 mol% FLU. However, the addition of FLU either at low or at high concentrations did not cause significant changes in the transition enthalpy of CHO incorporated DPPC. EPR results obtained from direct evaluation of EPR spectra indicated that FLU was an effective drug both in the interfacial and the alkyl chain region of DPPC membranes. In the interfacial region of DPPC membranes, the gradual decrease in 2Amax values in the liquid crystalline phase (Fig. 3a), decrease in the order parameter (S), slight increase in the free rotational space (V) and the rotational diffusion rate of nitroxide (Dr) especially at temperatures above Tm (Fig. 3c) indicated the alterations in the order and mobility in the liquid crystalline phase caused by FLU. Although the observed changes in the physical parameters of CHO incorporated DPPC membranes were less pronounced than the pure ones, a decrease in the order for both domains and a significant increase in the free rotational space of nitroxide for less ordered domain was observed at 37 C and above by 10 mol% FLU addition (Fig. 3d). Such an increase in the free rotational space of nitroxide for both pure and CHO incorporated DPPC might be the result of the interactions between FLU and lipids via hydrogen bonds, or water molecules. At the membrane/water interface, the hydroxyl group of CHO interacts with oxygen atoms of PC via direct H bonds and water bridges (Pasenkiewicz-Gierula et al., 2000). In the presence of FLU, the drug-PC and CHO interactions in similar ways results in providing a free rotational space for 5-DS spin label, which is reflected as a decrease in the order parameter. FTIR results also imply an increase in the hydration of ester carbonyl groups (Fig. 7b), which is the confirmation of the EPR results mentioned above. The degree of hydration and hydrogen bonding of water to the segments located at or near lipid polar/apolar interfaces can strongly influence the properties of lipid bilayers (Lewis et al., 1996).
In the alkyl chain region of DPPC membranes, the observed decrease in DBpp values at temperatures below phase transition induced by the addition of 5 and 10 mol% FLU implies an increase in the dynamics of the nitroxide, but FLU did not cause any significant change at temperatures above phase transition due to the totally free motion of alkyl chains (Fig. 4a). According to FTIR results, a decrease in the frequency of the CH2 stretching bands which implies the decreased number of gauche conformers that is increased hydrocarbon chain order, was seen below Tm especially at higher concentrations of FLU. However, an increase was observed in the vicinity of phase transition, indicating the increased hydrocarbon chain conformational disorder and mobility (Fig. 6a). Concerning EPR simulation results of the alkyl chain region of membranes, the order of the higher populated domain increases below Tm as confirmed by FTIR and the polarity of the most ordered domain increases below Tm by the addition of FLU (Fig. 4c)., Higher concentration of drug may increase the water penetration into the membrane. As proposed previously in the other studies, the water penetration into the hydrophobic region of bilayer leads to changes in the polarity and hydration profiles across lipid membranes (Kurad et al., 2003; Bartucci et al., 2003). The observed increase in polarity was also proposed by a molecular dynamic study, which demonstrated that the anesthetic drug caused an increase of the area per lipid, compared to pure DMPC, allowing the penetration of the water molecules deeper into the membrane (Höghberg et al., 2007). Additionally, in a study on the behaviour of small solutes and large drugs in a lipid bilayer, hydration even in the middle part of the bilayer was observed (Bemporad et al., 2005). In fact, our FTIR results clearly indicated a gradual increase in the water amount with increasing FLU concentration. The presence of CHO suppressed this penetration. However, we could only observe this increase with EPR in the alkyl chain region, possibly due to the conformation of the nitroxide. Octanol/water partition coefficient (logP) of FLU is 4.51 (Scott and Clymer, 2002). Lipophilicity of an organic compound is very often evaluated by its partition coefficient (logP) in the octanol/water system which is used to model biological membrane (Esteves et al., 2013). The logP value of the drug indicates high lipophilicity, accordingly high partition of it into lipid phase. Therefore, FLU is expected to be effective on investigated membranes which was corroborated by our studies. In CHO incorporated DPPC MLVs, the decrease in DBpp values (Fig. 4b) and the increase in the bandwith of CH2 asymmetric stretching band at all temperatures caused by FLU (Fig. 6d) point out the increased dynamic of the membrane. Increase in the frequency of the CH2 stretching bands at all temperatures by the addition of FLU implies the increased disorder in the lipid chains that occurs with the onset of gauche rotamer formation and the accompanying decline in the number of all-trans rotamers (Fig. 6b) (Lewis and McElhaney, 2013). Incorporation of cholesterol into liposome bilayers increases the orientational and conformational order of the phospholipid chains in PC-CHO bilayers. This might be responsible for the reduced partitioning of small molecules (Xiang and Anderson, 1995). This downfall partitioning was shown in some previous studies (Yonar and lu, 2014; Wisniewska and Wolnicka-Glubisz, 2004; Sünnetçiog
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Zhang et al., 2007). EPR simulation results showed that the addition of FLU into CHO incorporated DPPC still has a contribution to the physical parameters (Fig. 4d). As previously noticed, in the presence of CHO, there was a significant abnormal increase in the rotational diffusion rate of nitroxide in the temperature region, where the phase transition of pure DPPC was observed. The increase in diffusion means that small clusters could appear in the vicinity of phase transition when the lipid packing was not so homogeneous, making the phase not so compact (Yonar et al., 2011). Therefore, the diffusion in such a phase might be expected to be higher. This increase in diffusion was repressed when the FLU concentration was gradually increased, confirming the homogenizing effect of FLU (Fig. 4d). 5. Conclusions It was clearly pointed in this work that FLU was an effective drug both in the interfacial and the alkyl chain region of DPPC membranes. Moreover, FLU was observed to be highly effective in the presence of cholesterol even less pronounced than pure samples. The ordering of lipid membrane decreased and dynamics increased by the FLU addition. The incorporation of 10 mol% FLU into DPPC also caused a shoulder in the transition peak, suggesting the occurence of a phase seperation, and the formation of this new phase is still observable in presence of CHO. As the changes in the upper part of the membrane were even more pronounced than those in the central part of the membrane, FLU was proposed to be incorporated into the phospholipid membranes with its triple ring parallel to the head group of phospholipids and its chain toward the alkyl chain of phospholipids. It was concluded in accordance with our results that FLU caused notable alterations in the order, packing and dynamics of membrane lipids. It is well known that the structure and dynamics of lipids have significant influence on the function of membrane bound proteins, and consecutively on their actions. Based upon these, it was proposed that in the studied dose range which covers some therapeutic concentrations of flupentixol, FLU may modify the membrane associated receptors and transport proteins, which would form the basis of its clinical efficiency. Acknowledgments We would like to thank Hacettepe University, Scientific Research Projects Coordination Unit for providing financial support to the project numbered 012T06604001. References Alsop, R.J., Toppozini, L., Marquardt, D., Ku9cerka, N., Harroun, T.A., Rheinstädter, M. C., 2015. Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes. BBA 1848, 805–812. Bartucci, R., Guzzi, R., Marsh, D., Sportelli, L., 2003. Intramembrane polarity by electron spin echo spectroscopy of labeled lipids. Biophys. J. 84, 1025–1030. Bemporad, D., Luttmann, C., Essex, J.W., 2005. Behaviour of small solutes and large drugs in a lipid bilayer from computer simulations. BBA 1718, 1–21. Bourgaux, C., Couvreur, P., 2014. Interactions of anticancer drugs with biomembranes: what can we learn from model membranes? J. Controlled Release 190, 127–138. Cong, W., Liu, Q., Liang, Q., Wang, Y., Luo, G., 2009. Investigation on the interactions between pirarubicin and phospholipids. Biophys. Chem. 143, 154–160. Dey, S., Ramachandra, M., Pastan, I., Gottesman, M.M., Ambudkar, S.V., 1997. Evidence for two nonidentical drug-interaction sites in the human Pglycoprotein. Proc. Natl. Acad. Sci. U.S.A. 94, 10594–10599. de Lima, M.C.P., Chiche, B.H., Debs, R.J., Duzgunes, N., 1990. Interaction of antimycobacterial and anti-pneumocystis drugs with phospholipid membranes. Chem. Phys. Lipids 53, 361–371. Epand, R.F., Ramamoorthy, A., Epand, R.M., 2006. Membrane lipid composition and the interaction of pardaxin: the role of cholesterol. Protein Pept. Lett. 13, 1–5. Esteves, F., Moutinho, C., Matos, C., 2013. Correlation between octanol/water and liposome/water distribution coefficients and drug absorption of a set of pharmacologically active compounds. J. Liposome Res. 23 (2), 83–93.
Fa, N., Ronkart, S., Schanck, A., Deleu, M., Gaigneaux, A., Goormaghtigh, E., MingeotLeclercq, M.P., 2006. Effect of the antibiotic azithromycin on thermotropic behavior of DOPC or DPPC bilayers. Chem. Phys. Lipids 144, 108–116. Ford, J.M., Bruggemann, E.P., Pastan, I., Gottesman, M.M., Hait, W.N., 1990. Cellular and biochemical characterization of thioxanthenes for reversal of multidrug resistance in human and murine cell lines. Cancer Res. 50, 1748–1756. Gordon-Grossman, M., Zimmermann, H., Wolf, S.G., Shai, Y., Goldfarb, D., 2012. Investigation of model membrane disruption mechanism by melittin using pulse electron paramagnetic resonance spectroscopy and cryogenic transmission electron microscopy. J. Phys. Chem. B 116, 179–188. Höghberg, C.J., Maliniak, A., Lyubartsev, A.P., 2007. Dynamical and structural properties of charged and uncharged lidocaine in a lipid bilayer. Biophys. Chem. 125 (2–3), 416–424. 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Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy. Chem. Phys. Lipids 57, 213–226. McElhaney, R.N., 1982. The use of differential scanning calorimetry and differential thermal analysis in studies of model and biological membranes. Chem. Phys. Lipids 30, 229–259. McMullen, T.P.W., McElhaney, R.N., 1995. New aspects of the interaction of cholesterol with dipalmitoylphosphatidylcholine bilayers as revealed by highsensitivity differential scanning calorimetry. BBA 1234 (1), 90–98. McMullen, T.P.W., Lewis, R.N.A.H., McElhaney, R.N., 1993. Differential scanning calorimetric study of the effect of cholesterol on the thermotropic phase behavior of a homologous series of linear saturated phosphatidylcholines. Biochemistry 32, 516–522. Momo, F., Fabris, S., Stevanato, R., 2005. Interaction of fluoxetine with phosphatidylcholine liposomes. Biophys. Chem. 118, 15–21. Paiva, J.G., Paradiso, P., Serro, A.P., Fernandes, A., Saramago, B., 2012. Interaction of local and general anaesthetics with liposomal membrane models: a QCM-D and DSC study. Colloids Surf. B 95, 65–74. Pajeva, I.K., Wiese, M., 1997. QSAR and molecular modelling of catamphiphilic drugs able to modulate multidrug resistance in tumors. Quant. Struct. -Act. Relat. 16, 1–10. Pasenkiewicz-Gierula, M., Rog, T., Kitamura, K., Kusumi, A., 2000. Cholesterol effects on the phosphatidylcholine bilayer polar region: a molecular simulation study. Biophys. J. 78, 1376–1389. Pentak, D., 2014. Physicochemical properties of liposomes as potential anticancer drugs carriers. Interaction of etoposide and cytarabine with the membrane: spectroscopic studies. Spectrochim. Acta A 122, 451–460. Rubio, L., Rodríguez, G., Alonso, C., López-Iglesias, C., Cócera, M., Coderch, L., de la Maza, A., Parra, J.L., López, O., 2011. Structural effects of flufenamic acid in DPPC/ DHPC bicellar systems. Soft Matter 7, 8488–8497. Sarpietro, M.G., Accolla, M.L., Cova, A., Prezzavento, O., Castelli, F., Ronsisvalle, S., 2014. DSC investigation of the effect of the new sigma ligand PPCC on DMPC lipid membrane. Int. J. Pharm. 469, 88–93. Schindler, R., Fielenbach, T., Rave, G., 2004. Flupenthixol and cefotiam: effects on vitamin A metabolism in rats. Br. J. Nutr. 92, 597–605. Scott, D.C., Clymer, J.W., 2002. Estimation of distribution coefficients. Pharm. Technol. 30–40. Seddon, A.M., Casey, D., Law, R.V., Gee, A., Templer, R.H., Ces, O., 2009. Drug interactions with lipid membranes. Chem. Soc. Rev. 38 (9), 2509–2519. Seydel, J.K., Cordes, H.P., Wiese, M., Chi, H., Schaper, K.J., Coats, E.A., Kunz, B., Engel, J., Kutscher, B., Emig, H., 1991. QSAR: Rational Approaches to the Design of Bioactive Compounds. In: Silipo, C., Vittoria, A. (Eds.), Elsevier, Amsterdam, pp. 367–376.
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Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Effect of cis-(Z)-flupentixol on DPPC membranes in the presence and absence of cholesterol Dilek Yonara,* , M.Maral Sunnetcioglub a b
Middle East Technical University, Department of Biological Sciences, 06800, Ankara, Turkey Hacettepe University, Department of Physics Engineering, 06800 Beytepe, Ankara, Turkey
A R T I C L E I N F O
Article history: Received 10 February 2016 Received in revised form 21 May 2016 Accepted 2 June 2016 Available online 6 June 2016 Keywords: Phospholipid membrane Flupentixol EPR spin labeling FTIR and DSC
A B S T R A C T
Cis-(Z)-flupentixol dihydrochloride (FLU), a thioxanthene drug, is used in therapy of schizophrenia as well as in anxiolytic and depressive disorders. Since the action mechanism of FLU is not completely understood, the main objective of present study is to provide a detailed evaluation of flupentixolphospholipid membrane interactions at molecular level. FLU-dipalmitoylphosphatidylcholine (DPPC) interactions in presence and absence of cholesterol (CHO) were investigated as a function of temperature. The changes in upper part of membrane were more pronounced than those in central part of membrane, as indicated by EPR and FTIR. FLU was proposed to incorporate into phospholipid membranes with its triple ring parallel to head group and its chain toward alkyl chain of phospholipids. According to DSC results, the incorporation of 10 mol% FLU into DPPC caused a shoulder in transition peak, suggesting the occurence of a phase separation, and formation of this new phase is still observable in presence of CHO. It is well known that, structure and dynamics of lipids have significant influence on the function of membrane bound proteins, and consecutively their actions. Based upon these, it was proposed that FLU may modify membrane associated receptors and transport proteins, which would form the basis of its clinical efficiency. ã 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The investigation of membrane-drug interactions is of critical importance for pharmacological science because it is directly related to the mechanisms of drug action (Bourgaux and Couvreur, 2014). Many pharmacologically active compounds, with a number of different applications (antibiotics, antifungal, antidepressants, antihistamines, local anesthetics, anticancer drugs, etc.), can cross or bind to cell membranes and possibly alter the physical properties of the membrane (Seddon et al., 2009). Since biological membrane is a quite complex structure, model membranes composed of cellular membrane lipids are used to evaluate membrane-drug interactions. Membrane lipids participate in cellular signaling, hence regulate the important cell functions. Therefore, molecules which interact with membrane lipids may cause alterations in membrane composition, protein function or gene expression etc (Vigh et al., 2005). Flupentixol has two geometric isomers, namely cis-(Z) and trans-(E), however only cis form is pharmacologically active
* Corresponding author. E-mail address: [email protected] (D. Yonar). http://dx.doi.org/10.1016/j.chemphyslip.2016.06.002 0009-3084/ã 2016 Elsevier Ireland Ltd. All rights reserved.
(Woodruff and Freedman, 1981). cis-(Z)-flupentixol dihydrochloride (FLU), (Z)-4-[3-[2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]-1-piperazineethanol dihydrochloride, a catamphiphilic thioxanthene drug, has structural characteristics related to those of the phenothiazines (Fig. 1). FLU, like other thioxanthene compounds, is used in the acute and long term therapy of schizophrenia as well as in the lower dose range for the treatment of anxiolytic and depressive disorders. Although the action mechanism of flupentixol is not completely understood, it is found that flupentixol binds with high affinity to various dopamine receptors, to 5-HT2A, and to an alpha1-adrenergic receptors (Leysen et al., 1993). The studies regarding the interaction of flupentixol with the molecular components of cells and biological membranes were limited. Ford et al. (1990) studied the effects of the neuroleptic drugs cis and trans isomers of flupentixol on primary drug resistance and drug accumulation in several cell lines. Similar binding affinity to P-glycoprotein (Pgp), but different multidrug resistance (MDR) reversing activity has been proposed for trans and cis-flupentixol. In another study, Dey et al. (1997) provided direct evidence in favor of two non-identical drug-interaction sites within P-glycoprotein and proposed a model for the role of these two sites in drug translocation using a photoaffinity substrate
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2.2. Methods 2.2.1. Electron paramagnetic resonance (EPR) spectroscopy
Fig. 1. Chemical structure of cis-(Z)-flupentixol dihydrochloride (FLU).
analog and cis-flupentixol, a known modulator of Pgp function. When cis and trans-flupentixol-phosholipid interaction studies were performed, it was found that trans-flupentixol interacts about twice as strongly with lecithin as do cis-flupentixol. This indicates a stereospecific interaction of these catamphiphiles with phospholipids (Seydel et al., 1991). The same group also proposed in another study that both stereoisomers interacted more strongly with the negatively charged PS than with the neutral PC and a different orientation of trans and cis-flupentixol may be expected when they entered the lipid bilayer by the ring system (Pajeva and Wiese, 1997). The alterations in vitamin A metabolism in vivo as a result of flupentixol and cefotiam administration were investigated. It was concluded that the flupentixol treatment resulted in vitamin A depletion (Schindler et al., 2004). Since the action mechanism of FLU is not clear yet, the main objective of the present study is to provide a detailed evaluation of the interactions between FLU and phospholipid membranes at the molecular level. The interaction of FLU with DPPC membranes in presence and absence of cholesterol is investigated by FTIR, EPR spin labeling and DSC techniques. EPR spin labeling spectroscopy provides site specific information on ordering, mobility and polarity of membranes. Moreover, the insertion depth and orientation of the incorporated molecule within the membrane can be obtained with this technique (Gordon-Grossman et al., 2012). In addition to direct evaluation of EPR spectra, computer simulations of the spectra are performed to obtain more precise description of the membrane characteristics (Štrancar, 2007; lu, 2014). The information regarding the Yonar and Sünnetçiog effect of drug molecule on lipid order, lipid dynamics, phase transition behaviour and hydration of the head group and interfacial region can also be obtained from FTIR spectroscopy (Lewis et al., 2005; Cong et al., 2009). DSC technique has been extensively used to study the precise phase behaviour of hydrated phospholipid membranes (McElhaney, 1982) and to investigate the thermal changes caused by drug incorporation into phospholipid bilayers (Momo et al., 2005; Zhao et al., 2007; How et al., 2014). 2. Materials and methods 2.1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids, cholesterol (CHO), spin labels 5- and 16-doxyl stearic acid (5-DS, 16-DS) and antidepressant drug cis-(Z)-flupentixol dihydrochloride (FLU) were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Phosphate buffered saline (PBS) (without Ca, Mg) was obtained from Dr. Zeydanlı (Dr. Zeydanlı Life Sciences, Ltd. Şti., Ankara, Turkey).
2.2.1.1. Sample preparation. DPPC membranes were prepared in the absence and presence of 30 mol% CHO. 1 mol% concentration of 5- and 16-DS spin labels was used. The drug, FLU, concentration incorporated into the DPPC membranes was 1, 5, 10 mol%. Phospholipids, CHO and FLU were dissolved in chloroform stock solution containing spin label. Chloroform was evaporated first with nitrogen gas stream and then samples were kept under vacuum for overnight to remove residual chloroform. The obtained dry films were hydrated by 0.15 ml of PBS at pH 7.4, mixed with vortex at a temperature above phase transition temperature of the lipids to get multilamellar vesicles (MLVs) and then centrifuged (Eppendorf 5804-R; Eppendorf-Netheler-Hinz GmbH, Hamburg, lu, 2014). Pellets were Germany) at 20 C (Yonar and Sünnetçiog used in the studies. EPR measurements were performed on a Bruker EMX-131 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) with ER4103TM cylindirical cavity in a temperature range 10–55 C using the following spectral conditions: modulation frequency 100 kHz, modulation amplitude 0.2 mT and microwave power 10 mW. Sample temperature was controlled to 1 C by Bruker VT4111 temperature controller. Each sample was prepared and measurements were repeated at least two times. 2.2.1.2. Computer simulation of EPR spectra. EPR spectra are composed of several superimposed spectral components, since membrane is a heterogeneous structure composed of the regions with different motional characteristics and polarities. Therefore, except from direct spectral evaluation, EPR spectra of the studied samples were simulated by a computer program called EPRSIMC using a multi-component fast restricted wobbling motion approximation to get further information about the physical parameters of the membrane domains. The model used for fitting procedure allows up to four spectral components with different spectral parameters (Štrancar, 2007; Štrancar et al., 2003). The model parameters provided for each spectral component are the open cone angle q of the wobbling motion, asymmetry angle of the cone f, one effective rotational correlation time tc, which describes the rate of motion, additional broadening constant W, polarity correction factor on hyperfine splitting (A) tensor, pa, which is used as a scalar to correct all the A tensor components, describing the differences in polarity of the spin label surroundings, and the weight of each spectral component which describes the relative amount of the spin probes with particular motional mode. The order parameter S, free rotational space of nitroxide V, and the normalized rotational diffusion rate of nitroxide Dr constructed from the model parameters and definitions used in the cone model of wobbling motion are given below. S ¼ 0:5 cos2 u þ cosu
V ¼ uf=ðp=2Þ2 Dr ¼ uf=4tc Simulations were performed for each sample in the temperature range studied.
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2.2.2. Fourier transform infrared (FTIR) spectroscopy The samples were prepared in the same way with those for the EPR studies but without incorporation of the spin label. FTIR measurements were done by using water-insoluble CaF2 windows. Sample was placed between the CaF2 windows and 10 mm sample thickness was obtained by mylar spacers. Spectra were recorded within a temperature range of 10–55 C using a Perkin Elmer Spectrum One FTIR spectrometer with temperature-stabilized fast recovery deuterated triglycine sulfate (FR-DTGS) detector. Temperature was controlled by Unicam Specac Digital Temperature controller mounting the sample holder. Interferograms were averaged for 100 scans at 4 cm1 resolution. All samples were prepared at least two times and their spectra were acquired. The water bands stemming from the presence of buffer were subtracted in order to provide a better resolution of the bands. During the subtraction process, the water band located around 2125 cm1 was flattened. The normalization of spectra in the studied regions was carried out for the representation of relative changes using peak normalization. 2.2.3. Differential scanning calorimetry (DSC) DSC was used to monitor the influence of FLU on thermotropic properties of DPPC MLVs. The samples were prepared in the same way with those for the FTIR studies. Samples were placed in a sealed aluminum pans. An empty pan was also used as reference during the measurements, so that its calorimetric effect was extracted by the computer program. Calorimetric measurements were carried out using a Perkin Elmer Diamond DSC at a scan rate lu, 5 C/min as described in previous studies (Yonar and Sünnetçiog 2014; Rubio et al., 2011). Each sample was prepared and analyzed at least three times to check the repeatibility of the results. Two heating and cooling scans were performed for each analysis to ensure the reproducibility. Only heating curves are evaluated and presented here. The temperature at the peak maximum is defined as the transition temperature (Tm) and the area under the peak after baseline adjustment and normalization to the sample amount represents the enthalpy change (DHm) during the transition. 2.2.4. Statistical analysis The results were represented as the mean values of parameters and the errors were calculated as standard deviation. Statistical significance was evaluated by using two-way analysis of variance (2way ANOVA) and Dunnett’s multiple comparison test. The degree of significance (p values) with respect to the pure DPPC was p < 0.05.
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3. Results 3.1. EPR results The effect of FLU on lipid order and dynamic was investigated by using 5- and 16-DS spin labels with nitroxide group localized at different depths in DPPC membranes. Experimental and calculated spectra of the samples were given at 45 C for 5-DS labeled and at physiological temperature (37 C) for 16-DS labeled DPPC membranes in Fig. 2a and b. The given temperatures were selected from the region where the greater changes exist. The changes in the maximum hyperfine splitting constant (2Amax) for 5-DS and in the mid-field line width (DBpp) for 16-DS labeled membranes were analyzed directly from EPR spectra. 2Amax values reflect the average ordering of the alkyl chains and DBpp is related to the dynamics of the spin label’s nitroxide group. Temperature dependence of 2Amax of 5-DS in DPPC MLVs at different concentration of FLU was given in Fig. 3a and b. The addition of FLU to DPPC membranes gave rise to a shift towards the lower temperatures in the main phase transition temperature of DPPC. This shift was more pronounced for 10 mol% FLU. For 5-DS labeled DPPC MLVs, incorporation of FLU did not cause any significant changes in 2Amax values in the gel phase, but in the liquid crystalline phase all concentrations of FLU induced gradual decrease in 2Amax values. The changes in 2Amax with temperature were distributed in a broader range for 5 and 10 mol% FLU (Fig. 3a). There was a slight decrease in 2Amax values at lower temperatures in the CHO incorporated DPPC MLVs with 5 and 10 mol% of FLU, whereas a more pronounced decrease was observed at higher temperatures (Fig. 3b). Besides direct evaluation of EPR spectra, the spectra of the studied samples were simulated to get more information on domain (spectral components with different motional pattern) properties of membrane and to obtain the physical parameters of these components. Some of the physical parameters were given in Fig. 3c and d for 5-DS labeled DPPC MLVs. Generally two motional patterns of 5-DS were observed in DPPC MLVs. The number of domains was more than two at some temperatures. A shift in the phase transition temperature towards lower temperatures was observed with the addition of FLU. FLU caused a decrease in the order parameter (S), a slight decrease in the rotational diffusion rate (Dr) and a slight increase in the free rotational space of nitroxide (V) especially at temperatures above phase transition temperature, but it did not make any significant effect on polarity (pa) (Fig. 3c). The observed changes in the physical parameters of
Fig. 2. Examples of experimental (black line), calculated (red line) total and domain's spectra (blue, green, magenta lines) of a) 5-DS and b) 16-DS spin labeled DPPC MLVs at 45 and 37 C, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Temperature dependence of 2Amax of 5-DS in (a) pure (b) 30 mol% of CHO incorporated DPPC MLVs at various concentrations of FLU. Temperature dependence of 10 mol % FLU induced changes in the order parameters (S) and rotational diffusion rate (Dr) of 5-DS in (c) pure, (d) 30 mol% CHO incorporated DPPC MLVs. Diameter of circles denotes the proportion of spin probe in different domain types. Bars indicate second moment in the distribution of values obtained from the GHOST condensation technique.
CHO incorporated DPPC membranes were less pronounced than the pure ones. However, a significant decrease in the rotational diffusion rate of nitroxide (Dr) for less ordered domain was observed at 37 C and above by incorporation of 10 mol% FLU (Fig. 3d). 16-DS spin label was employed to monitor the changes in the alkyl chain region of DPPC membranes. Temperature dependence of DBpp of 16-DS in DPPC MLVs at various concentrations of FLU is given in Fig. 4a and b. The addition of 5 and 10 mol% FLU to DPPC
MLVs caused a significant decrease in DBpp values at temperatures below phase transition, which implies an increase in the dynamics of the nitroxide, but it did not cause any significant change at temperatures above phase transition (Fig. 4a). In CHO incorporated DPPC MLVs, FLU incorporation caused a decrease in DBpp values at all temperatures. This decrease was more pronounced for 5 and 10 mol% FLU incorporated samples (Fig. 4b). Concerning the simulation results of alkyl chain region of membranes, some of the physical parameters (S, pa and Dr) for
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Fig. 4. Temperature dependence of DBpp of 16-DS in (a) pure, (b) 30 mol% of CHO incorporated DPPC MLVs at various concentrations of FLU. Temperature dependence of 10 mol% FLU induced changes in the order parameters (S), polarity correction factor (pa) and rotational diffusion rate (Dr) of 16-DS in (c) pure, (d) 30 mol% CHO incorporated DPPC MLVs. Diameter of circles denotes the proportion of spin probe in different domain types. Bars indicate second moment in the distribution of values obtained from the GHOST condensation technique.
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pure and CHO incorporated DPPC membranes are shown in Fig. 4c and d. Generally two motionally different patterns and a shift in the phase transition temperature towards lower temperatures with the addition of FLU were observed. The effect of 10 mol% FLU on DPPC MLVs was seen as an increase in the polarity of the most ordered domain in the gel phase. There was no change in the liquid crystalline phase with the addition of FLU as in the direct evaluation of spectra (Fig. 4c). In CHO incorporated DPPC MLVs, the order parameter of the less ordered domain decreased and polarity correction factor of the same domain increased slightly with the addition of FLU below 28 C. There was a significant abnormal increase in the rotational diffusion rate of nitroxide around the phase transition temperature of DPPC in the presence of CHO. This increase in diffusion was repressed when 10 mol% FLU was added (Fig. 4d). This repression was also seen in 1 and 5 mol% FLU incorporated samples (data not shown). 3.2. FTIR results Both pure and CHO incorporated DPPC MLVs containing 1– 10 mol% of FLU were investigated as a function of temperature by FTIR spectroscopy. Fig. 5 shows the normalized FTIR spectra of DPPC MLVs in the absence and presence of 1, 5, and 10 mol% FLU at 37 C in 3000–1000 cm1 region with enlarged panel zoomed into C¼O stretching band. The normalized spectra is demonstrated for the representation of comparative changes in the frequency, intensity and bandwidth of the mentioned bands in the absence and presence of FLU. The changes in the lipid hydrocarbon chain conformational order-disorder and hydrocarbon chain-melting phase transitions were obtained by using the symmetric and asymmetric stretching vibrations of the methylene (CH2) group (2850 and 2920 cm1, respectively) on lipid hydrocarbon chains (Mantsch and McElhaney, 1991). Fig. 6a and b shows temperature dependence of the frequency of CH2 asymmetric stretching band of DPPC MLVs in the absence and presence of different concentrations of FLU. As seen from the figure, the broadening of phase transition profile was observed by the addition of higher concentrations of FLU. The frequency values of concerned band were measured at the center of the peaks. The gradual shifts towards lower temperatures in the main phase transition temperature (Tm) of DPPC were observed with increasing FLU concentration. The same results were obtained for the CH2 symmetric stretching band (data not shown).
An increase in the frequency of CH2 stretching band reflects the lipid hydrocarbon chain’s conformational disorder. As FLU concentration was gradually increased, the frequency of the CH2 stretching band decreased below Tm which ascribes to the order of the system in the gel phase. But at 35 C and above up to phase transition, the frequency of the CH2 stretching band increased significantly with the addition of 10 mol% of FLU, which implies a decrease in the order of the system during the phase changes, and there was no significant change in the liquid crystalline phase (Fig. 6a). This is due to the further reducing of the phase transition temperature by the addition of 10 mol% of FLU. The results for CHO incorporated DPPC MLVs are shown in Fig. 6b. Significant increase in CH2 stretching frequency were seen by the addition of 10 mol% FLU. This result indicates that higher concentrations of FLU caused a decrease in the order of the CHO incorporated DPPC MLVs (Fig. 6b). The temperature dependence of the bandwidth of CH2 asymmetric stretching band for pure and CHO incorporated DPPC MLVs in absence and presence of FLU is shown in Fig. 6c and d. The bandwidths of CH2 asymmetric stretching band were measured at 75% of height of the peaks. The bandwidth is related to the motional rates of the molecule, so it is the evidence of membrane dynamics (Mantsch and McElhaney, 1991). At 35 C and above up to 41 C, an increase in the bandwidth values were seen especially with 10 mol% incorporation of FLU. However, there were no significant changes in the liquid crystalline phase by FLU incorporation (Fig. 6c). As seen in Fig. 6d, the bandwith of CH2 asymmetric stretching band at all temperature range increased by incorporation of 5 and 10 mol% FLU, which shows the increased dynamic of the membrane. The degree of significance with respect to pure DPPC MLVs was p < 0.05. Temperature dependence of the frequency of C¼O stretching band of DPPC MLVs in the absence and presence of different concentrations of FLU is shown in Fig. 7. In the drug-membrane interaction studies, the changes in the C¼O stretching absorption band located between 1740 and 1730 cm1 might provide important clues to the structural and/or hydration changes of bilayer polar-apolar interfacial region (Cong et al., 2009; Lewis and McElhaney, 1998). The frequency of C¼O stretching band shifts towards higher values as compared to pure DPPC MLVs in the gel phase for the samples containing 5 and 10 mol% FLU, which may indicate an increase in dehydration of ester carbonyl groups. At 35 C and above up to phase transition, a dramatic decrease in
Fig. 5. FTIR spectra of DPPC MLVs in the absence and presence of 1, 5 and 10 mol% FLU in 3000–1000 cm1 spectral region with enlarged panel zoomed into C¼O stretching band at 37 C.
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Fig. 6. Temperature dependence of the frequency and bandwidths of CH2 asymmetric stretching bands of (a–c) pure, (b–d) CHO incorporated DPPC MLVs in the absence and presence of 1, 5, 10 mol% FLU.
frequency was observed for 10 mol% FLU incorporated DPPC MLVs, which implies a probable increase in the hydrogen bonded C¼O groups (Fig. 7a). After phase transtition of pure DPPC, a decrease was observed with incorporation of 5 mol% FLU. In the presence of CHO, the addition of 5 and 10 mol% FLU caused a decrease at 35 C and above (Fig. 7b). 3.3. DSC results The changes in the phase transition temperature and the shape of the DSC thermograms were investigated to study the termotropic aspect of drug-membrane interactions. DSC thermograms obtained for DPPC and 1, 5, 10 mol% FLU incorporated DPPC in absence and presence of 30 mol% CHO are presented in Fig. 8a and b and the thermodynamic parameters associated with lipid phase transitions are summarized in Table 1. The incorporation of increased amounts of FLU caused progressive decrease in the main phase transition temperature of DPPC and the addition of 10 mol% of FLU produced the largest decrease around 3.3 C (Fig. 8a and Table 1). While pre-transition peak was still observed with 1 mol% FLU incorporation, it disappeared at the concentrations above 1 mol%. Concentrationdependent and asymmetric broadening of transition peaks were seen by increasing FLU concentration. As consistent with the literature, the transition enthalpies of the pre-transition (DHp) and main transition (DHm) of pure DPPC were 0.97 and 6.23 kcal/mol,
respectively (Yeagle, 2011; Fa et al., 2006). DHp of DPPC significantly decreased at 1 mol% FLU and DHm significantly decreased at all FLU concentrations. The incorporation of 10 mol % FLU into DPPC also caused a shoulder in the transition peak on the left side. In addition, the presence of 30 mol% CHO caused a significantly broad heat flow peak and maximum heat flow observed at higher temperature (2.3 C) than pure DPPC MLVs (Fig. 8b and Table 1). CHO incorporation caused a decrase in DHm of pure DPPC (1.47 kcal/mol). Any significant changes in the transition enthalpy of CHO incorporated DPPC were observed either at low or at high concentrations of FLU as indicated by Table 1. Furthermore, incorporation of FLU into the CHO containing DPPC MLVs caused a decrease in the temperature, at which maximum heat flow was observed. The shoulder in the transition peak on the left side in presence of 10 mol% FLU was still seen with the addition of CHO. 4. Discussion Spectroscopic and calorimetric measurements pointed out that FLU incorporation into phospholipid membranes gives rise to some changes in the physical properties of the membrane such as phase transition, order, and dynamic. The first pronounced effect of FLU was on the phase transition behaviour of DPPC. The lamellar gel (Lb) to lamellar liquid crystalline (La) chain melting phase transition (Tm), a cooperative phase transition, covers the conversion of a highly ordered gel state to a relatively disordered
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Fig. 7. Temperature dependence of the frequency changes at the C¼O stretching band for (a) pure and (b) CHO incorporated DPPC MLVs in the absence and presence of 1, 5, 10 mol% FLU.
liquid crystalline bilayer. While the hydrocarbon chains of the phospholipids are packed in a highly ordered manner below Tm, the chains are disordered and possess more motional freedom above Tm. The incorporation of the drug into phospholipid membrane may alter the phase transition temperature and broaden the thermotropic peak. A shift towards lower temperatures in Tm of DPPC membranes was observed with the increasing concentration of FLU which indicates the occurence of more motional freedom at lower temperatures in presence of FLU (Figs. 3, 4,6–8). A shift approximately 3.3 C towards lower temperatures in Tm was observed with incorporation of 10 mol% of FLU into DPPC MLVs (Fig. 8a). The intercalation of amphiphilic molecules between the polar head groups of a phospholipid membrane is able to depress the main transition temperature. Shifts in both directions in Tm were reported in previous studies with different drugs (Paiva et al., 2012; Pentak, 2014; Sinha et al., 2014; Alsop et al., 2015). DPPC has a pre-transition at around 35 C. While pre-transition peak was still observed with 1 mol% FLU incorporation, it disappeared by the addition of 5 mol% FLU and above. The disappearance of the pre-transition is thought to be caused by the flupentixol interference with the tilting of DPPC alkyl chains (de Lima et al., 1990). The line width at half maximum of the peaks of the heat flow (DT1/2) is also very sensitive to the presence of any additives and it may indicate the changes in the cooperativity. The cooperativity is inversely proportional to DT1/ 2. The addition of FLU broadened the calorimetric peaks and provided more asymmetric peaks (Fig. 8a), indicating a decrease in the transition cooperativity. The polar interactions in the interfacial region of phospholipids and hydrophobic interactions between alkyl chains might be blocked by the addition of the drug. This may cause the formation of different regions in terms of interactions. As a result of these different regions, lipids do not melt at the same temperature and the transition zone broadens (Zhao et al., 2007). The changes in 2Amax and frequency of CH2 asymmetric stretching band which distributed in a broader range for 5 and 10 mol% FLU supports the observed broadening of phase transition profile by DSC. A dramatic decrease in the main transition enthalpy (DHm) values were observed with incorporation of 1, 5 and 10 mol% FLU into DPPC membranes. The enthalpy change associated with the main lipid chain melting is related to molecular packing of the alkyl chain. So, a decrease both in Tm and DHm of the gel to liquid crystalline phase transition in presence of drug indicates the
Fig. 8. DSC thermograms of (a) pure and (b) 30 mol% CHO incorporated DPPC MLVs in the absence and presence of 1, 5, 10 mol% FLU.
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Table 1 Main phase transition temperatures (Tm) and enthalpies of the main phase transition (DHm) for pure and FLU incorporated DPPC membranes in the absence and presence of 30 mol% CHO. FLU amount (mol%)
0 1 5 10
DPPC
CHO incorporated DPPC
Tp ( C)
DHp (kcal/mol)
Tm ( C)
DHm (kcal/mol)
Tm ( C)
DHm (kcal/mol)
37.6 0.1 35.0 0.2 – –
0.97 0.03 0.09 0.01 – –
42.2 0.3 42.0 0.5 40.6 0.4 38.8 0.1
6.23 0.42 4.81 0.59 4.43 0.53 4.18 0.71
44.5 0.6 44.3 0.5 40.0 0.5 31.3 0.7
1.47 0.12 2.14 0.49 1.66 0.09 –
instability of the phospholipid bilayer (Sarpietro et al., 2014; Lucio et al., 2009). The incorporation of 10 mol% FLU into DPPC caused a shoulder in the transition peak on the left side. This type of appearence on either side indicates the domain formation and may indicate the phase separation and immiscibility (Straubinger and Balasubramanian, 2016). According to EPR results, there were some changes in domain percentages by incorporation of FLU (Fig. 3c) which support this phase separation behaviour. Complex effects of cholesterol incorporation on phospholipid thermotropic phase behaviour has been revealed in literature (McMullen and McElhaney, 1995; Vist and Davis, 1990; Epand et al., 2006). The chain length dependent shift direction in the heat flow peak maxima was observed previously. For PCs having hydrocarbon chains of 16 or fewer carbon atoms, a shift towards higher temperature side was expected (McMullen et al., 1993). Broadening in the heat flow peaks, a shift toward higher temperatures in its maximum and a decrease in transition enthalpy were obtained for 30 mol% of CHO containing DPPC MLVs relative to pure ones consistent with literature (Fig. 8b, Table 1). The incorporation of FLU into the CHO containing DPPC MLVs caused a decrease in the temperature, at which maximum heat flow was observed. Up to 5 mol% FLU there is an increase in the linewidth of heat flow peak and a narrowing was observed for 10 mol% FLU. However, the addition of FLU either at low or at high concentrations did not cause significant changes in the transition enthalpy of CHO incorporated DPPC. EPR results obtained from direct evaluation of EPR spectra indicated that FLU was an effective drug both in the interfacial and the alkyl chain region of DPPC membranes. In the interfacial region of DPPC membranes, the gradual decrease in 2Amax values in the liquid crystalline phase (Fig. 3a), decrease in the order parameter (S), slight increase in the free rotational space (V) and the rotational diffusion rate of nitroxide (Dr) especially at temperatures above Tm (Fig. 3c) indicated the alterations in the order and mobility in the liquid crystalline phase caused by FLU. Although the observed changes in the physical parameters of CHO incorporated DPPC membranes were less pronounced than the pure ones, a decrease in the order for both domains and a significant increase in the free rotational space of nitroxide for less ordered domain was observed at 37 C and above by 10 mol% FLU addition (Fig. 3d). Such an increase in the free rotational space of nitroxide for both pure and CHO incorporated DPPC might be the result of the interactions between FLU and lipids via hydrogen bonds, or water molecules. At the membrane/water interface, the hydroxyl group of CHO interacts with oxygen atoms of PC via direct H bonds and water bridges (Pasenkiewicz-Gierula et al., 2000). In the presence of FLU, the drug-PC and CHO interactions in similar ways results in providing a free rotational space for 5-DS spin label, which is reflected as a decrease in the order parameter. FTIR results also imply an increase in the hydration of ester carbonyl groups (Fig. 7b), which is the confirmation of the EPR results mentioned above. The degree of hydration and hydrogen bonding of water to the segments located at or near lipid polar/apolar interfaces can strongly influence the properties of lipid bilayers (Lewis et al., 1996).
In the alkyl chain region of DPPC membranes, the observed decrease in DBpp values at temperatures below phase transition induced by the addition of 5 and 10 mol% FLU implies an increase in the dynamics of the nitroxide, but FLU did not cause any significant change at temperatures above phase transition due to the totally free motion of alkyl chains (Fig. 4a). According to FTIR results, a decrease in the frequency of the CH2 stretching bands which implies the decreased number of gauche conformers that is increased hydrocarbon chain order, was seen below Tm especially at higher concentrations of FLU. However, an increase was observed in the vicinity of phase transition, indicating the increased hydrocarbon chain conformational disorder and mobility (Fig. 6a). Concerning EPR simulation results of the alkyl chain region of membranes, the order of the higher populated domain increases below Tm as confirmed by FTIR and the polarity of the most ordered domain increases below Tm by the addition of FLU (Fig. 4c)., Higher concentration of drug may increase the water penetration into the membrane. As proposed previously in the other studies, the water penetration into the hydrophobic region of bilayer leads to changes in the polarity and hydration profiles across lipid membranes (Kurad et al., 2003; Bartucci et al., 2003). The observed increase in polarity was also proposed by a molecular dynamic study, which demonstrated that the anesthetic drug caused an increase of the area per lipid, compared to pure DMPC, allowing the penetration of the water molecules deeper into the membrane (Höghberg et al., 2007). Additionally, in a study on the behaviour of small solutes and large drugs in a lipid bilayer, hydration even in the middle part of the bilayer was observed (Bemporad et al., 2005). In fact, our FTIR results clearly indicated a gradual increase in the water amount with increasing FLU concentration. The presence of CHO suppressed this penetration. However, we could only observe this increase with EPR in the alkyl chain region, possibly due to the conformation of the nitroxide. Octanol/water partition coefficient (logP) of FLU is 4.51 (Scott and Clymer, 2002). Lipophilicity of an organic compound is very often evaluated by its partition coefficient (logP) in the octanol/water system which is used to model biological membrane (Esteves et al., 2013). The logP value of the drug indicates high lipophilicity, accordingly high partition of it into lipid phase. Therefore, FLU is expected to be effective on investigated membranes which was corroborated by our studies. In CHO incorporated DPPC MLVs, the decrease in DBpp values (Fig. 4b) and the increase in the bandwith of CH2 asymmetric stretching band at all temperatures caused by FLU (Fig. 6d) point out the increased dynamic of the membrane. Increase in the frequency of the CH2 stretching bands at all temperatures by the addition of FLU implies the increased disorder in the lipid chains that occurs with the onset of gauche rotamer formation and the accompanying decline in the number of all-trans rotamers (Fig. 6b) (Lewis and McElhaney, 2013). Incorporation of cholesterol into liposome bilayers increases the orientational and conformational order of the phospholipid chains in PC-CHO bilayers. This might be responsible for the reduced partitioning of small molecules (Xiang and Anderson, 1995). This downfall partitioning was shown in some previous studies (Yonar and lu, 2014; Wisniewska and Wolnicka-Glubisz, 2004; Sünnetçiog
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Zhang et al., 2007). EPR simulation results showed that the addition of FLU into CHO incorporated DPPC still has a contribution to the physical parameters (Fig. 4d). As previously noticed, in the presence of CHO, there was a significant abnormal increase in the rotational diffusion rate of nitroxide in the temperature region, where the phase transition of pure DPPC was observed. The increase in diffusion means that small clusters could appear in the vicinity of phase transition when the lipid packing was not so homogeneous, making the phase not so compact (Yonar et al., 2011). Therefore, the diffusion in such a phase might be expected to be higher. This increase in diffusion was repressed when the FLU concentration was gradually increased, confirming the homogenizing effect of FLU (Fig. 4d). 5. Conclusions It was clearly pointed in this work that FLU was an effective drug both in the interfacial and the alkyl chain region of DPPC membranes. Moreover, FLU was observed to be highly effective in the presence of cholesterol even less pronounced than pure samples. The ordering of lipid membrane decreased and dynamics increased by the FLU addition. The incorporation of 10 mol% FLU into DPPC also caused a shoulder in the transition peak, suggesting the occurence of a phase seperation, and the formation of this new phase is still observable in presence of CHO. As the changes in the upper part of the membrane were even more pronounced than those in the central part of the membrane, FLU was proposed to be incorporated into the phospholipid membranes with its triple ring parallel to the head group of phospholipids and its chain toward the alkyl chain of phospholipids. It was concluded in accordance with our results that FLU caused notable alterations in the order, packing and dynamics of membrane lipids. It is well known that the structure and dynamics of lipids have significant influence on the function of membrane bound proteins, and consecutively on their actions. Based upon these, it was proposed that in the studied dose range which covers some therapeutic concentrations of flupentixol, FLU may modify the membrane associated receptors and transport proteins, which would form the basis of its clinical efficiency. Acknowledgments We would like to thank Hacettepe University, Scientific Research Projects Coordination Unit for providing financial support to the project numbered 012T06604001. References Alsop, R.J., Toppozini, L., Marquardt, D., Ku9cerka, N., Harroun, T.A., Rheinstädter, M. C., 2015. Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes. BBA 1848, 805–812. Bartucci, R., Guzzi, R., Marsh, D., Sportelli, L., 2003. Intramembrane polarity by electron spin echo spectroscopy of labeled lipids. Biophys. J. 84, 1025–1030. Bemporad, D., Luttmann, C., Essex, J.W., 2005. Behaviour of small solutes and large drugs in a lipid bilayer from computer simulations. BBA 1718, 1–21. Bourgaux, C., Couvreur, P., 2014. Interactions of anticancer drugs with biomembranes: what can we learn from model membranes? J. Controlled Release 190, 127–138. Cong, W., Liu, Q., Liang, Q., Wang, Y., Luo, G., 2009. Investigation on the interactions between pirarubicin and phospholipids. Biophys. Chem. 143, 154–160. Dey, S., Ramachandra, M., Pastan, I., Gottesman, M.M., Ambudkar, S.V., 1997. Evidence for two nonidentical drug-interaction sites in the human Pglycoprotein. Proc. Natl. Acad. Sci. U.S.A. 94, 10594–10599. de Lima, M.C.P., Chiche, B.H., Debs, R.J., Duzgunes, N., 1990. Interaction of antimycobacterial and anti-pneumocystis drugs with phospholipid membranes. Chem. Phys. Lipids 53, 361–371. Epand, R.F., Ramamoorthy, A., Epand, R.M., 2006. Membrane lipid composition and the interaction of pardaxin: the role of cholesterol. Protein Pept. Lett. 13, 1–5. Esteves, F., Moutinho, C., Matos, C., 2013. Correlation between octanol/water and liposome/water distribution coefficients and drug absorption of a set of pharmacologically active compounds. J. Liposome Res. 23 (2), 83–93.
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