Phase Separation in Phosphatidylcholine ... - ACS Publications

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Phase Separation in Phosphatidylcholine Membrane Caused by the Presence of a Pyrimidine Analogue of Fluphenazine with High AntiMultidrug-Resistance Activity ‡ ́ Katarzyna Cieślik-Boczula,*,† Piotr Swiątek, Agata Jaszczyszyn,§ Patrycja Zawilska,∥ Kazimierz Gąsiorowski,§ Wiesław Malinka,‡ and Gottfried Köhler⊥ †

Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland Department of Drug Chemistry, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland § Department of Basic Medical Science, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland ∥ Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland ⊥ Max F. Perutz Laboratories, Department of Computational and Structural Biology, University of Vienna, Campus Vienna Biocenter 5/1, Vienna 1030, Austria ‡

ABSTRACT: Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, 31P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazinerelated compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of antiMDR drugs by providing very effective inhibitors of the pump activity of Pgp. cells.10 The really high nonspecificness to the chemical structure of substances transported by Pgp practically excludes finding effective anticancer drugs which will not be transported by Pgp.11,12 Previous reports from our and other laboratories showed that fluphenazine (FPh), an antipsychotic (neuroleptic) drug from the phenothiazine family, was both a chemosensitizer (MDR modulator, Pgp inhibitor) and a selective inducer of apoptosis in cancer cells or genotoxically damaged cells.13−18 Recently it was shown that FPh is able to reverse the multidrug resistance in cancer cells and could exert significant chemopreventive activity at least in experimental carcinogenesis.19,20 A number of statistical and epidemiological studies documented lower cancer rates among psychiatric patients treated with neuroleptic medications, fenothiazines, and among them also treated with FPh.21,22 It should be mentioned that two phenothiazines, FPh

1. INTRODUCTION Multidrug resistance (MDR), i.e., the resistance of tumor cells to most chemotherapeutic agents, cytostatic drugs, is a major limiting factor in cancer chemotherapy.1−3 Among various mechanisms involved, the main reason of MDR is thought to be overexpression of membrane protein P-glycoprotein (Pgp), which mediates efflux of divergent xenobiotics, also cytostatic drugs, outside cancer cells.1−6 Also, inhibition of apoptosis of transformed cells in vitro seems to be closely related to MDR.6,7 It was observed that in several human cancer cell lines many chemotherapeutic drugs decreased the level of Pgp expression and the effect correlated significantly with observed apoptosis arrest.8 Restoring sensitivity to a chemotherapy of multidrugresistant cancer cells by an inhibition of Ppg transporter function and by a reopening of the normal apoptosis signaling/ pathways are important aims of a chemoprevention.6,9 The current strategies of chemoprevention are focused on a development of new agents, which can inhibit Pgp activity and, simultaneously, overcome apoptosis deficiency of cancer © 2014 American Chemical Society

Received: November 5, 2013 Revised: March 2, 2014 Published: March 6, 2014 3605

dx.doi.org/10.1021/jp410882r | J. Phys. Chem. B 2014, 118, 3605−3615

The Journal of Physical Chemistry B

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structure of eukaryotic cell membranes.29 Together with other lipids such as DMPC or POPC, DPPC is commonly used in many scientific laboratories for studying processes associated with phase transitions and/or states of a membrane below and above the transition temperature.25,27

and chlorpromazine (CPZ), together with haloperidol are on the World Health Organization (WHO) list of Essential Medicines of 2009, as three essential drugs in antipsychotic treatments.23 Chemical modifications of the structure of the FPh molecule performed at the Medical University of Wroclaw led to production of a new group of FPh analogues, among which a few of them possessed 10−20% higher activity of the transport inhibition function of Pgp than one found for the parent compound, FPh.16,17 Presented in this work the pyrimidine analog of fluphenazineFPh-prm (the structure shown in Figure 1) inhibited at the highest level the transport activity

2. EXPERIMENTAL METHODS 2.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine was purchased from Avanti Polar Lipids, Hamburg, Germany, with a purity of >99.8%. The fluorescence probe Laurdan (2-(dimethylamino)-6-dodecanoylnaphthalene)was purchased from Molecular Probes (Eugene, OR, USA). All compounds were used without further purification procedure. Synthesis of the 10-{3-[4-(2-Pyrimidyl)piperazyn-1-yl]-2hydroxypropyl}-2-(trifluoromethyl)phenothiazine Dihydrochloride (FPh-prm). The main steps of the synthesis of FPhprm was performed according to refs 15 and 30. The key intermediate 10-(2,3-epoxypropyl)-2-(trifluoromethyl)phenothiazine was prepared via the treatment of commercially available 2-(trifluoromethyl)phenothiazine with 1-bromo-2,3epoxypropane at room temperature in the presence of sodium hydride. The product, FPh-prm, was obtained in the oxirane ring-opening reaction. From the postreaction mixture, the FPhprm compound was separated by column chromatography. For pharmacological purposes the obtained free base of the FPhprm product was converted to the corresponding water-soluble hydrochloride. The steps of reaction were monitored by thinlayer chromatography on 0.25 mm Merck silica gel (60 F254) and visualized by UV light. Melting point was uncorrected. 1H NMR was recorded in CDCl3 at 300 MHz using a Bruker spectrometer. 1H chemical shifts were reported in δ (ppm). Elemental analyses were within ±0.4% of the theoretical values and were performed on a Carlo Erba NA-1500 analyzer. The reaction was monitored by thinlayer chromatography on 0.25 mm Merck silica gel (60 F254) and visualized by UV light (λ = 264 or 365 nm). Flash chromatography was performed on silica gel Kieselgel 60 (70− 230 mesh) from Merck. A solution of 0.4 g (0.00124 mol) of 10-(2,3-epoxypropyl)-2(trifluoromethyl)phenothiazine and 0.00124 mol of 4-(2pyrimidyl)piperazine in 10 mL of ethanol was refluxed under stirring for 6 h. Then the solvent was evaporated under reduced pressure, and the residue was purified through flash chromatography. The obtained product was in the next step transformed to the corresponding salt by means of ethanol saturated with hydrogen chloride gas. Formula: C24H26Cl2F3N5OS. MW: 560.46. Transition temperature (Tt): 81−83 °C; 43% yield. CC [ethyl acetate/n-hexane (1:1)]: Rf = 0.45. 1H NMR (base): δ 2.45−2.68 (m, 12H, CH2N(CH2CH2)2NCH2), 3.97−4.22 (m, 3H, N10-CH2CH), 6.46−6.52 (m, 1H, H5-pyrimidine), 6.97−7.04 (m, 2H, PhtH), 7.16−7.24 (m, 5H, PhtH), 8.28−8.32 (m, 2H, H4,6pyrimidine). 2.2. Preparation of Liposomes. Small unilamellar vesicles (SUV) with different molar ratios of FPh-prm and DPPC have been prepared. Mixed compounds were dissolved in methanol/ chloroform (1:1 (w/w)) and then were dried under a stream of nitrogen. The residual solvent was removed under vacuum for 2 h. Dry lipid films were hydrated by the addition of 1 mL of water obtained from a Millipore filtration device (Millipore, Milli Q) during 10 of the cooling/heating processes. Finally liposomal suspensions were extruded through the filter with

Figure 1. Schematic representation of the structure of FPh and its pyrimidine analogFPh-prm.

of Pgp in cultures of lymphocytes genotoxically damaged by pretreatment with benzo[α]pyrene (B[α]P). It was documented that FPh-prm caused an increase of rhodamine 123 accumulation (an indicator of the range of Pgp inhibition) of about 36% compared to the control cells in which this compound was not present.17 In resistant cells the outward drug transport is higher than the drug influx. To obtain the accumulation of anticancer drugs in cancer cells, the efficiency of efflux pumps must be reduced and/or the permeability of the cell membrane should be increased. It was documented in the literature that various Pgp inhibitors (MDR modulators) perturbed a lipid bilayer of the plasma membrane and, because the activity of Pgp strongly depends on the state of its lipid environment, chemotherapeutic drugs would circumvent the Pgp gate.24−26 Previously, we established an interaction of FPh with model biological membranes and supposed it to be an important mechanism of a chemopreventive activity of that drug.27,28 Description of the character of structural changes of lipid membranes caused by the presence of highly active phenothiazine analogues will allow one to determine the role of membrane alterations in the Pgp-related mechanism of a multidrug resistance of cancer cells. It is interesting to check if a newly synthesized strong anti-MDR agent can change the structure of lipid membranes and what type of membrane alterations are responsible for their increasing the Pgp-related anti-MDR activity. The main goal of presented studies was to examine the effect of FPh-prm on the membrane structure of dipalmitoylphosphatidylcholine (DPPC) by means of fluorescence, 31P nuclear magnetic resonance spectroscopy (NMR), attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) and microcalorimetry (differential scanning calorimetry (DSC)) studies. The DPPC lipid is one of the most numerous members of the phosphatidylcholine (PC) group which is the most prevalent lipid group among those constituting the basic 3606



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for water solution of FFh-prm. Cuvettes of 1 cm light path length were used with a slit width of 2 nm and interval of 0.1 nm. To reduce the effect of background signals and improve resolutions of overlapped signals, second derivatives of absorption spectra were calculated in GRAMS software based on the Savitzky−Golay method, in which the cubic polynomial convolution of 29 points was employed.33,34 2.5. 31P NMR Measurements. 31P NMR spectra were recorded at room temperature on a Bruker 300 NMR Fourier transform spectrometer operating at 121.49 MHz with 3 s of relaxation delay time. The FPh-prm/DPPC liposomes were prepared in D2O solution without extruding procedure with the final lipid concentration of 20 mg/mL and were put into 5 mm thin-walled NMR tubes. The spectra were recorded over a large spectral width (36 kHz) using broad-band proton decoupling. The raw 31P NMR spectra were fitted with Gaussian/Lorentz functions in GRAMS program.

100 nm in the diameter of pores (LiposoFast with polycarbonate filter, Avestin, Ottawa, ON, Canada). Inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to determine the total amount of phosphate (originated from DPPC molecules) and sulfur (from FPh-prm) in liposomal suspensions. The ARL 3410 spectrometer was operated at the wavelengths of 213 and 182 nm for phosphorus and sulfur detection, respectively. The final amounts of both molecules recovered after liposome preparation were equal to 90−88% for DPPC and 91−87% for FPh-prm. The mean diameter (volume weighting) of liposomes was measured using particle size analyzer Zetasizer, Nano-ZS model (Malvern Instruments Ltd., Southborough, U.K.). Liposome size was obtained by dynamic light scattering using a He−Ne laser at 633 nm and ranged between 78 ± 2 and 74 ± 3 nm for pure DPPC and FPh-prm-rich DPPC liposomes, respectively. DSC Measurements. The FPh-prm/DPPC liposomes with the final DPPC concentration of 5 mg/mL were prepared as previously described. The calorimetric scans were recorded using the VP-DSC microcalorimeter (MicroCal). Individual samples were scanned six times for heating up scans and six times for cooling down scans with a scan rate of 90 and 60 °C/ h, respectively. The prescan and postscan thermostats were for 15 and 10 min, respectively. Samples were measured immediately after preparation. The area under the transition profiles was used to calculate the molar enthalpy change accompanying phase transition (called further the transition enthalpy) by using the Grams software, and after consideration of the retained lipid amount during preparation. 2.3. Fluorescence Measurements. The DPPC liposomes in the presence of 18.2 and 171 μM of fluphenazine analogue were prepared as described in Preparation of Liposomes. Liposomes were incubated with the Laurdan fluorescence probe in darkness for 30 min at room temperature with the final concentrations of 1.5 μM Laurdan and 180 μM phospholipid. The steady-state emission and excitation spectra of Laurdan were performed with a FLSP920 fluorescence spectrometer (Edinburgh Instruments Ltd., U.K.) equipped with a thermostatted cell holder. The emission spectra were acquired over the range of 400−560 nm. The excitation wavelength was 320−400 nm for the fluorescence excitation wavelength dependence studies and 390 nm when the temperature dependent fluorescence intensity measurements were performed. The Laurdan generalized polarization (GP) was calculated using the following equation proposed by Parasassi et al.:31,32 GP = (I445 − I490)/(I445 + I490)

3. RESULTS 3.1. Microcalorimetric Studies of FPh-prm/DPPC Liposomes. The thermal behavior of the DPPC liposomes mixed with different content of FPh-prm is summarized in Figures 2− 4. On heating, pure DPPC liposomes exhibit two

(1)

where I440 and I490 are the fluorescence emission intensities at the blue and red edges of the emission spectrum, respectively. In Laurdan anisotropy measurements performed for different temperatures the fluorescence intensity and excitation were set at 445 and 350 nm, respectively. 2.4. UV−Vis Absorption Measurements. The absorption spectra of FPh-prm molecules in water solution and liposome dispersions were carried out on the UV−vis spectrophotometer, Cary100 Bio. The samples were prepared with concentrations such as those for fluorescence and DSC experiments. The FPh-prm concentration in water solution was 20 μM. The absorptions spectrum of the sample solution was measured against the reference solution: pure DPPC liposomes for samples with doped DPPC liposomes and water

Figure 2. DSC thermograms of water dispersion of DPPC liposomes in the presence of different concentration of FPh-prm. Numbers represent FPh-prm:DPPC mole ratios. Insert: example of thermogram (FPh-prm:DPPC, 0.112) deconvolution.

endothermic transitions: a pretransition at around 35 °C broad, with low-enthalpy changes, and a main transition represented by a sharp transition peak with the maximum at around 41.5 °C accompanied by high-enthalpy changes. The pretransition, arisen from the conversion of a lamellar gel phase (Lβ) to a rippled gel phase (Pβ), appeared to be very sensitive to the presence of a pyrimidine analog of fluphenazine. Even in very small amounts of FPh-prm (≥0.02 mole ratio) the pretransition 3607



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to or higher than 0.05, when a one-endotherm peak splits into a few subcomponents, the decrease of Tm was more distinct and has a similar slope of line for each subcomponent; see Figure 3. Thus, all three phases have a similar trend of thermal behavior with increasing concentration of the doped molecules. Furthermore, the increase of the relative enthalpy changes (ΔH/ΔH(DPPC)), calculated as the division of the value of the transition enthalpy (ΔH) for the FPh-prm/DPPC liposomes under scrutiny by the value of the DPPC transition enthalpy (ΔH(DPPC)), sharply increased in a low range of FPh-prm concentration, as it is shown in Figure 4. The character of changes of ΔH/ΔH(DPPC) also depends on the phase separation process. Similarly to the correlation of Tm versus mole ratio of FPh-prm to DPPC, the values of ΔH/ ΔH(DPPC) have a biphasic behavior in relation to the content of the doped compound. The relative enthalpy becomes almost constant with only a slightly increasing deviation from the point when the phase separation appeared in the studied systems. 3.1. Laurdan Fluorescence Studies of FPh-prm/DPPC Liposomes. To further study the effect of FPh-prm on the phase behavior of phospholipid membranes, we employed Laurdan as a fluorescence probe sensitive to the polarity and mobility of its lipid environment. Formation of a charge transfer excited state of the fluorescence naphthalene residue of Laurdan, stabilized by the water dipole reorientation process, takes place in the liquid-crystalline conditions of lipid bilayer and results in the red-shifted emission.31,32,35,36 The dipolar relaxation of probe molecules is not present in apolar solvents and also in the gel phase of the lipid environment, which produces the emission with the maximum in the blue region.31,32,35,36 Additionally, Laurdan can be used for monitoring the phase separation if new-forming lipid domains differ in their water content and/or molecular dynamics of water molecules accompanied by changes of polarity and/or packing of a lipid bilayer.31,32,35,36 In experiments presented here, the blue emission band with the maximum at approximately 445 nm arrived from Laurdan molecules placed in the DPPC bilayer in the gel phase, while the increase of the fluidity in the liquid-crystalline state of lipids results in the highwavelength shift of the maximum of fluorescence spectra to the position at around 490 nm. The plot of the GP values, calculated according to eq 1, as a function of the excitation wavelength provides useful information about the homogeneity of Laurdan lipid environment and tells us when phase coexistence takes place in studied systems. At the temperature of the main phase transition of the pure DPPC membrane, where both gel and liquid-crystalline phases coexist, the generalized polarization is a characteristic ascending function of excitation wavelength; see Figure 5A, the plot for 41 °C. Simultaneously, Laurdan emission spectra recorded at temperatures of the homogeneous gel phase show the highest GP values which are not dependent on λext. The generalized polarization values are negative and decrease with the increase of excitation wavelength in pure liquid-crystalline state, as was shown in Figure 5A for pure DPPC membrane. Differently from pure DPPC, the GP(λext) lines obtained for FPh-prm/ DPPC mixtures in the temperature range of the gel phase are no longer flat but become an ascending function of λext, characteristic for the heterogeneous system. This positive slop of GP(λext) lines is more distinct when the membrane concentration of the pyrimidine analog of fluphenazine is higher and values of GP, especially for lower wavelengths of excitation, are clearly lower (around 0.3 for DPPC liposomes

Figure 3. Effect of the increase of concentration of FPh-prm on the temperature of phase transitions of DPPC liposomes. ΔTm: the difference of the temperature of the main phase transition in pure DPPC liposomes and the temperature of the phase transition defined for each DSC subcomponent as a function of FPh-prm concentration inside of the DPPC membrane of the liposomes.

Figure 4. Influence of FPh-prm on the relative molar enthalpy, ΔH/ ΔH(DPPC), in DPPC liposomes as were derived from the microclimatic studies. Errors are given a standard deviation value of six heating measurements.

peak was not detectable, leading us to assume that structural differences between a rippled and not-rippled lamellar gel state were no longer present. The sharp peak of the lipid main transition associated with the trans/gauche isomerization broadens and its intensity goes down as a result of the decrease of cooperativity of the observed transition in FPh-prm-mixed DPPC systems. In FPh-prm/DPPC liposomes, when the mole ratio of both compounds was ≥0.05, the shape of the main transition peak was modified and at least three main subpeaks appeared. A similar complex-peak structure had also existed for liposomes measured in the presence of a high amount of fluphenazine analog, when the ratio content ranged from 0.112 to 0.429. The changes of the temperature of each of the three subphases present in the DPPC membrane doped with increasing FPh-prm content are shown in Figure 3. In the whole studied concentration range the temperatures of transitions (Tm) decreased with the rise of the FPh-prm concentration. This reduction of Tm was less expressed in the low ratio range, when phase separation was not yet present. On the other hand, in samples with the content of FPh-prm equal 3608



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negative slope starting from slightly higher values of GP than for not-mixed membranes; see Figure 5C. The changes of fluorescence emission spectra of Laurdan in pure and FPh-prm-doped DPPC membranes at different temperatures allowed us to monitor the phase transition at which the sharp drop of GP values were observed. Figure 6A presents GP plotted as a function of temperature in studied liposomes, where the generalized polarization was calculated for λext = 390 nm. The temperature of the phase transition is dependent on the concentration of FPh-prm inside of the DPPC bilayer and, similarly to DSC results, also decreases with the increase of the FPh-prm content. To extend our knowledge on the effect of incorporation of FPh-prm into the structure of the DPPC bilayer, the steadystate Laurdan anisotropy measurements were performed. The Laurdan anisotropy as a function of temperature is presented in Figure 6B for pure and FPh-prm-mixed DPPC liposomes. Transition of the phospholipid from gel to liquid-crystalline phase was accompanied by a decrease in anisotropy values from about 0.25 (in gel state) to 0.1 (in fluid state), typical for DPPC bilayer-forming liposomes37 . Also, in this case the temperature of the phase transition, designated from the sigmoidal shape of the temperature relation of Laurdan anisotropy, decreases in mixed liposomes. Similarly to GP results, the incorporation of fluphenazine analog into the lipid bilayer affects Laurdan anisotropy more visibly in the gel phase than in the hightemperature liquid-crystalline state. The increase of a contribution of FPh-prm causes the decrease of anisotropy in both phases but lower concentration of FPh-prm (18.2 μM) has no effect on anisotropy in the liquid-crystalline state, which is accompanied by almost no changes in GP values in such conditions. For fluorescence studies, there was more water phase present in the samples; i.e., the whole system was more diluted than in other experiments used in this work. To obtain the concentration of FPh-prm inside a lipid membrane comparable to that one in FPh-prm-concentrated experiments we had to use higher FPh-prm:DPPC mole ratios. The changes of the partition of FPh-prm between lipid bilayer vesicles and water, demonstrated by the shift of the maximum of the second derivative of absorption spectra of FPh-prm, as a function of sample concentration are shown in Figure 7. It is commonly known that the maxima of absorption bands of many different compounds with phenothiazine ring shift toward lower wavelength with the increase of the polarity of the environment.33,34 A dilution of FPh-prm-mixed liposomes moved the maximum of the second derivative to the lower wavelength region as a result of migration of FPh-prm molecules from a lipid to water phase. Irrespective of a sample concentration and FPh-prm:DPPC mole ratios, in all cases, the spectra did not achieve a position characteristic for a pure water condition, which means that in the presence of DPPC liposomes a part of the FPh-prm molecules are present inside of a lipid bilayer. Thus, fluorescence results show us the trend of changes caused by the increase of membrane concentration of FPh-prm, without the exact assignment to the real concentration of FPhprm inside the lipid bilayer. 3.3. 31P NMR Studies of FPh-prm/DPPC Liposomes. 31P NMR technique is frequently used to determine a type of lipid aggregation in hydrated states.27,38,39 When lipid molecules form a bilayer structure, in which they have rapid rotational motion along their long axis, the 31P NMR signal has a characteristic broad shape with a high-field peak and low-field

Figure 5. Generalized polarization (GP) values for Laurdan in pure DPPC (A) and DPPC liposomes mixed with 18.2 μM (B) or with 171 μM FPh-prm (C) as a function of excitation wavelength measured at different temperatures. DPPC and Laurdan concentrations were 180 μM and 1,5 μM, respectively.

with 18.2 μM of FPh-prm and around 0.1 for DPPC liposomes with 171 μM of FPh-prm) than the one for the gel phase of pure DPPC liposomes (around 0.45); compare the pictures from A to C in Figure 5. Additionally, in the range of higher values of λext, irrespective of the presence of different concentrations of FPh-prm, the GP values become unaffected compared to values obtained for pure DPPC bilayer in similar conditions. The liquid-crystalline phase of lipid membrane is less influenced by the presence of admixture, as both the shape of GP(λext) and GP values observed for DPPC liposomes and mixed with 18.2 μM FPh-prm resemble each other. Farther increase of the content of FPh-prm results in a more distinct 3609



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Figure 6. Laurdan generalized polarization changes as a function of the increase of temperature for pure and FPh-prm-mixed DPPC vesicles. The excitation wavelength was 390 nm (A); function of Laurdan anisotropy changes with the increase of temperature for water dispersions of pure DPPC liposomes and in the presence of 18.2 and 171 μM FPh-prm (B).

Figure 7. Second derivative spectra of FPh-prm calculated from the absorption spectra of FPh-prm in water (dotted line), in diluted (dashed line), and in concentrated DPPC liposomes (solid line): (A) liposomes with FPh-prm:DPPC mole ratio equal to 0.10; (B) liposomes with FPh-prm:DPPC mole ratio equal to 0.95.

shoulder.27,38,39 On the other hand, a hexagonal (HII) phase is manifested by the reversible shape of the 31P NMR line with a high-field shoulder and a low-field peak.38 Narrow, symmetric, and centered at around the 0 ppm micellar signal, cubic and rhombic phases arrive from isotropic lipid motion through which lateral diffusion results in averaging over all orientations.27,38,39 As different lipid structures are represented by completely different 31P NMR signals one of the biggest advantages of this method is the ability to easily show the coexistence of different phases in one sample. The proton-decoupled 31 P NMR spectra of DPPC dispersions in D2O solution with different mole fractions of FPh-prm at room temperature are shown in Figure 8. Generally, the lipid bilayer structure is in all systems containing admixture. Only for the highest FPh-prm concentration does the isotropic phase begins to appear in the membrane. In the DPPC liposomes mixed with high concentration of FPh-prm (0.428 mole ratio) for the first time still the bilayer signal

Figure 8. Gray lines represent the row 31P NMR spectra of pure DPPC dispersion in D2O (A) and the FPh-prm:DPPC (0.112) mixture (B) or the FPh-prm:DPPC (0.429) mixture (C). Dark lines represent subpeaks obtained by fitting the spectra with Gaussian/ Lorentz function. The measurements were done at 25 °C by employing broad-band proton decoupling. (∗) Peak for isotropic phase.

coexists with the isotropic one centered at around 0 ppm, but the bilayer is the dominant phase. Unfortunately, as the isotropic 31P NMR signal can be represented by different lipid aggregates such as micelle, liposomes with small diameters, and so on, this technique does not allow us to determine which isotropic structure the FPh-prm-rich DPPC mixture is able to adopt. In each case two main subpeaks characteristic for bilayer structure are present in the 31P NMR spectra: see Figure 8 and Table 1 for parameters of deconvolution of spectra with 3610



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an example in Figure 9B. Conformational fluidization of a hydrophobic region of pure and lipid-doped membranes are also accompanied by broadening and decreasing intensity of the νas,s(CH2) bands; see Figure 9. The difference spectra in the ν(CH) region of pure or mixed DPPC in the gel and in the liquid-crystalline phase, collected in Figure 10, compare the

Table 1. Parameters of the Deconvolution of 31P NMR Spectra for Pure DPPC and FPH-prm-Mixed DPPC Liposomes FPh-prm:DPPC mole ratio pure DPPC 0.11 0.428

center/ppm

height/au

width/ppm

area/au

30.1 −18.9 25.9 −18.4 37.3 −0.7 −21.6

6953.1 30798.1 4075.2 31358.5 964.0 4182.5 33223.2

39.9 35.1 39.7 21.6 28.7 1.2 16.9

38171176 1.77 × 108 21391649 1.23 × 108 4375503.5 658666.7 1.03 × 108

Gaussian/Lorentz functions. With the increase of FPh-prm content, the high-field peak centered at around −20 ppm, decreases in width and slightly increases in height. On the other hand, the height of the low-field peak goes down with the smaller alterations of a bandwidth. Thus, changes of lipid motion and orientation within the lipid bilayer are caused by the FPh-prm molecules. 3.4. CH2 Stretching Vibrations in FPh-prm/DPPC Liposomes. In the 2800−3000 cm−1 frequency region of the infrared spectrum of water dispersion of DPPC liposomes there are very well defined asymmetric and symmetric stretching vibrations of CH2 groups of lipid alkyl chains.27,39,41 In DPPC membranes, during the chain-melting main phase transition, the decrease in the ratio of the population of trans to gauche conformers shifts the asymmetric and symmetric CH 2 stretching bands (νas,s(CH2)) from 2920 and 2950 cm−1 (in gel phase) to the position of 2922 and 2953 cm−1 (in liquidcrystalline state), respectively.27,39−41 This characteristic blue shift is also present in all FPh-prm-mixed DPPC liposomes, as it is shown for FPh-prm−lipid mixture with 0.429 mole ratio as

Figure 10. Difference spectrum of pure DPPC liposomes spectrum at the gel phase temperature and the spectrum for one at the liquidcrystalline state (red line), the difference spectrum of the FPh-prm/ DPPC (0.05) liposomes spectrum at the gel phase temperature and the spectrum for one at the liquid-crystalline state (green line), the difference spectrum of the FPh-prm/DPPC (0.112) liposomes spectrum at the gel phase temperature and the spectrum for one at the liquid-crystalline state (dark yellow line), and the difference spectrum of the FPh-prm/DPPC (0.429) liposomes spectrum at the gel phase temperature and the spectrum for one at the liquidcrystalline state (violet line), in the range of lipid ν(CH) vibrations.

character of conformational changes during phase transition in pure and FPh-prm-doped DPPC bilayers of liposomes. The difference absorption profile (Δabs) obtained by subtraction from the spectrum of FPh-prm-mixed DPPC (0.05) in the gel

Figure 9. Changes of the FTIR-ATR spectra of pure (A) and FPh-prm-mixed DPPC vesicles in water solution as a function of an increase of temperature. 3611



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phase from the spectrum of this sample but in liquid-crystalline state has similar shape and intensity to one calculated in the same way for the pure DPPC system. The changes of inter- and intramolecular interactions in the hydrophobic part during phase transition are similar only in the case of a small amount of FPh-prm present in the DPPC bilayer. The increase in content of fluphenazine analogue results in the decrease in the intensity of difference profiles with no changes of the positions of their maxima; see Figure 10. Thus, the chain-melting phase transition is accompanied by a smaller contribution of the alteration of the trans/gauche ratio in FPh-prm-rich lipid systems.

Parent compoundFPhis one of the members of the phenothiazine-related group of chemoprevention modulators, and as shown in FTIR-ATR studies performed by us in a publication in 2009,23 it also introduces conformational fluidization in the hydrophobic region of the DPPC bilayer. Although for many years the anti-MDR activity of FPh has been known, there are only residual mentions about its influence on lipid membrane structure and connection of this with its ability to inhibit Pgp in cancer cells. In its influence on membrane fluidization presented in this work a newly synthetized pyrimidine analog of FPh is found to be similar to FPh and other phenothiazine derivatives. Furthermore, appearing at ≥0.05 molar ratio of FPh-prm to DPPC, the splitting of the endothermic peak of the main phase transition into a few subcomponents (see Figure 2) arises from phase separation in mixed lipid bilayers. In all cases the thermograms for FPh-prm/ DPPC mixtures, irrespective of the FPh-prm concentration, were fitted with three Gaussian/Lorentz functions. The presence of three separated transition peaks can mean either that the whole homogeneous system undergoes three separate transitions one after another or that there are at least three distinct populations of species undergoing the same transitions or the system is more complicated and that different regions of the lipid membrane are a subject of a different type of phase transition with the participation of various lipid structures such as bilayer, micelle, and hexagonal, etc. The second hypothesis seems to be confirmed in our systems by Laurdan fluorescence studies. The presence of pure gel phase represented by membrane with homogeneously dissolved FPh-prm is excluded by the positive GP(λext) slope at temperatures below Tm and GP values in the range too low for pure lipid gel phase, around 0.1−0.3. Because the ascending function of generalized polarization versus wavelength excitation is a consequence of the coexistence of different states, the FPh-prm/DPPC membrane contains a mixture of different quasi-gel phases present in separate domains. Since the nature of the fluorescence of Laurdan in the lipid bilayer depends on the polarity (water content) and arrangement of lipid and water molecules (in tightly packed lipid membranes water molecules have less content and they are more fixed and have less freedom of motion), the new-forming phases in mixed membranes should be different in their water-related polarity and/or packing influenced the mobility. The heterogeneity derived from phase separation is not present in the DPPC bilayer mixed with lower FPh-prm concentration in a higher temperature range; over Tm the pure liquid-crystalline phase is represented by descending function of GP(λext), and even the GP values are typical for pure DPPC in this phase; see Figure 5. However differences become more evident when more doped compounds are present in the membrane; the descending function becomes clearer and starts from a little bit higher values of GP for 171 μM FPh-prm than for pure DPPC. On the basis of these observations we concluded that FPh-prm strongly affects the gel phase and has a minor influence on the liquid-crystalline state. The hypothesis that phenothiazine derivatives can induce phase separation in phospholipid model bilayers has already been suggested by Frenzel et al.42 for CPZ-PC systems or by Hendrich et al.43 for TFP in DPPC and DOPC:SM:Cho liposomes. Nevertheless, it was always difficult to clearly define the character of coexisting phases. Hendrich et al. in a study published in 2011 for the first time visualized directly that the phase separation in DOPC:SM:Cho giant unilamellar lip-

4. DISCUSSION Results presented above show strong interaction of the newly synthetized amine analog of fluphenazine with phospholipid membrane. As the microcalorimetric studies proved, even the lowest FPh-prm concentration used abolished the pretransition (Tp) by perturbation of a Tp-related packing of lipid acyl chain and head groups. Additionally, with further increase of the membrane concentration of the doped compound we observed the downward shift of the temperature of the chain-melting phase transition accompanied by the broadening of the DSC peak of the main phase transition of mixed bilayer with lower cooperativity. These microcalorimetric variations are similar to the effect of many other phenothiazine-related compounds like CPZ,42 TFP (trifluoperizine)43 on model lipid membranes, for which the anti-MDR activities were also proved. Decrease of Tm with broadening of the transition peak observed in lipid bilayers mixed with phenothiazine-related compounds is commonly recognized to be the sign of increased membrane fluidization caused mainly by the growth of the population of gauche conformers and mobility of lipid chains.29,45 Because the outward transport activity of Pgp is strongly connected with the packing of lipids in membranes, that is, the active efflux Pgp pump is in the rigid and closely packed gel phase of its lipid environment while, in more-disordered liquid-crystalline form, this protein loses its pumping function,45,46 it is postulated that the increase of membrane fluidization, which almost always accompanies phenothiazine compounds, is responsible for their membrane-related inhibition of the outer transport of many different molecules by Pgp. Pgp has two hydrophobic domains, each one containing six putative transmembrane α helices; see Figure 1A in ref 45. They cross through the membrane and are involved in the drug-binding process, which induces conformational changes within Pgp.47 Because Pgp can recognize substrates from the membrane phase, the physicochemical properties of the lipid environment can modulate the membrane concentration of Pgp substrates around this protein. The partition coefficient of substrates between membrane and water (environment of living cells) changes with the phase state of lipid bilayer. Additionally, the ATPase activity of Pgp, which also participates in efflux pumping of many anticancer drugs out of the tumor cells, is preserved in fluid lipid mixtures.45,48,49 This is a specific feature for many other membrane ATPases. The main place of sitting of Pgp, in which this protein has a high pumping activity, is rigid domains of living membranes sphingomyelin rafts50although a reconstruction of Pgp in pure phosphatidylcholine bilayer of vesicles showed that Pgp still maintains their pumping function in such model membranes.46 It was stated that the lipid rigid gel phase decreases and the fluid liquid-crystalline state inhibits a drug transport by Pgp through PC membranes.46 3612



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pure, and middle content of FPh-prm in the DPPC membrane. All of them are present in the range of 0.05−0.429 mole ratio of FPh-prm to DPPC, have a bilayer structure, and demonstrate a similar character of reduction of the temperature of the phase transition with the increase of the membrane concentration of the pyrimidine analog. (2) The significant increase of anti-MDR activity of a pyrimidine analog of FPh can come from the ability of FPh-prm to regional accumulation by which it can obtain high local concentration in FPh-prm-rich domains in lipid membrane. It was documented that the ability of FPh and its different analogues to restore the sensitivity to chemotherapy of multidrug-resistance cancer cells by inhibition of the Pgp transporter function increases with the rise of the concentration of those compounds in tumor cell cultures.28,58 Thus, local accumulation of FPh-prm can allow use of a smaller drug concentration in order to obtain satisfactory results and reduces side effects which accompany chemotherapy. (3) For the pyrimidine analog of FPh the membrane fluidization of regions of the lipid bilayer with high FPh-prm concentration for which a decrease of the chain-melting phase temperature was observed is an important step in the membrane-related mechanism of the inhibition of the transport activity of the membrane protein Pgp.

osomes (GUV) caused by the presence of TFP is represented by domains.51 They were able to demonstrate the shape of them and concluded that TFP forms smaller in size domains than that in pure DOPC:SM:Cho membranes. The comparison of our results from DSC and Laurdan fluorescence with 31P NMR experiments allowed us to prove the phase separation in FPh-prm/DPPC membranes represented by domains with the structure of lipid bilayer but with different FPh-prm contribution. The characteristic 31P NMR signal, coming from the phosphorus nucleus of the phosphate group of DPPC, with a high-field shoulder and low-field peak obtained at the temperature of the gel phase indicated the presence of lipid aggregation in mixtures with FPh-prm only in the form of the bilayer but with different FPh-prm/DPPC contribution. Of course when we distinctly increased the FPh-prm content, up to 0.428 mole ratio an additional isotropic phase starts to appear in the still dominated bilayer structure. This nonbilayer phase is a result of the beginning of membrane solubilization caused by a high admixture concentration at which the membrane is saturated with a doped compound. Different temperatures of phase transitions for domains which the retained bilayer structure in FPh-prm/DPPC systems should originate from alterations in FPh-prm/DPPC composition. During the heating process all FPh-prm/DPPC mixtures undergo the lipid chain-melting phenomenon accompanied by the increase of population of the gauche conformers of CH2 groups in lipid tails as the FTIR-ATR studies showed; see Figure 9. Additionally, the increase of the FPh-prm concentration lowers the contribution of the trans/gauche isomerization, which takes place during a phase transition; see Figure 10. Structurally different inhibitors and substrates of Pgp strongly interact with various lipid membranes. Many researchers proved the changes of bilayer organization in the presence of Pgp modulators. Studies performed in Tulkens’ laboratory on DPPC and others modelling membranes in the presence of ciprofloxin and azithromycin showed a decrease in temperature of the lipid phase transition and changes in the fluidity and permeability of doped membranes.52−55 Due to a high affinity of Pgp modulators to lipid membrane, these compounds are present in the membrane, which is the main place from where Pgp takes its substrates. The vacuum cleaner model of a mechanism of the drug transport by Pgp requires the presence of substrates inside the membrane, especially in the inner leaflet of the bilayer. It is interesting that this hypothesis can be applied to multidrug transporters in animal and bacterial cells, as it was stated by numerous investigations in Konnings’ laboratory.56,57 Our studies proved the presence of FPh-prm inside the DPPC membrane, which allowed us to classify the FPh-prm as a potential Pgp substrate transported by this protein form the membrane medium to out of the cell through the vacuum cleaner mechanism. Additionally, this compound can work as an Pgp inhibitor by modulation of membrane properties.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +48 71 375 7209. Fax: +48 71 328 2348. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Foundation for Polish Sciencethe programme POMOST, cofinanced by the European Union within the European Regional Development Fund (V edition, 2012), and by the grant nr DEC-2012/05/B/ ST4/02029 (OPUS) from the National Science Centre. We thank the Ö AD (Project No. PL 07/2013) and Polish Ministry of Science and Higher Education (grant-in-aid for scientific research and development for young scientists, 2012) for additional financial support.

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REFERENCES

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