Interactions of Dihydrochloride Fluphenazine with DPPC Liposomes

J. Phys. Chem. B 2009, 113, 15495–15502

15495

Interactions of Dihydrochloride Fluphenazine with DPPC Liposomes: ATR-IR and 31P NMR Studies Katarzyna Cies´lik-Boczula,*,† Joanna Szwed,† Agata Jaszczyszyn,‡ Kazimierz Gasiorowski,‡ and Aleksander Koll† Faculty of Chemistry, UniVersity of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland, and Department of Basic Medical Science, Wroclaw Medical UniVersity, Kochanowskiego 14, 51-601 Wroclaw, Poland ReceiVed: May 22, 2009; ReVised Manuscript ReceiVed: October 13, 2009

The influence of dihydrochloride fluphenazine (FPh) on the dipalmitoylphosphatidylcholine (DPPC) bilayer structure was investigated using ATR-IR and 31P NMR methods. The ATR-IR results indicate an increase in conformational disorder in the hydrophobic part compared with pure DPPC liposomes and a decrease in temperature of the chain-melting phase transition in FPh/DPPC liposomes. These effects depended on the concentration of the drug in the DPPC bilayer. The dihydrochloride fluphenazine molecules form H-bonds with the proton-acceptor carbonyl groups of DPPC molecules. At a higher concentration of the drug, the lipid bilayer structure is destroyed, and an isotropic phase is observed using 31P NMR spectroscopy. The interactions between FPh and the lipid bilayer have a crucial role in MDR (multidrug-resistant) activity of this drug. These results improve one possible strategy of cancer chemoprevention with FPh accompanied by fluidization and destabilization of the model lipid bilayer structure. 1. Introduction The phenothiazines, to which dihydrochloride fluphenazine (FPh) belongs, are standard antipsychotic (neuroleptic) drugs used in the treatment of schizophrenia and related psychoses (mania, paranoia, and delirium). They exert their neuroleptic activity by the blockade of dopaminergic receptors in the brain, mainly presynaptic D2 receptors. They also inhibit alpha adrenoreceptors and muscarinic, cholinergic, histamine, serotonin, and opioid receptors in the central nervous system.1 It is well documented that schizophrenic patients treated with phenothiazines have a lower incidence of cancer compared with the general population. These observations suggest that phenothiazines could exert a cancer chemopreventive activity.2 Chemoprevention is a treatment strategy aimed at inhibiting carcinogenesis by the intake of natural or synthetic chemical agents. Several strategies of chemoprevention have been designed to protect against the development of cancer (early chemoprevention) and/or to restore the sensitivity of multidrugresistant (MDR) cancer cells to chemotherapeutic drugs (late chemoprevention). We and other authors have reported that chlorpromazine, trifluoperazine, and fluphenazine inhibited proliferation and induced apoptosis in a dose-dependent manner in genotoxically damaged cell cultures and in a variety of cancer cell lines.3-5 Among the proposed mechanism(s) of phenothiazines’ chemopreventive action is inhibition of calmodulin (CaM) and protein kinase C (PKC),6 although the global anticancer activity of the drugs is probably diverse and remains to be elucidated. Phenothiazines were shown to restore the sensitivity of cytostatic drug-resistant cancer cells to chemotherapeutic drugs (reversion of MDR) in experimental carcinogenesis; they are included in the group of agents able to modulate Pgp (Pgp) * To whom correspondence should be addressed. Tel.: +48 71 3757209. Fax: 71 3282348. E-mail: [email protected]. † University of Wroclaw. ‡ Wroclaw Medical University.

transporter function.6-9 Pgp is a transmembrane protein which moves drugs outside of a cell, decreasing the intracellular concentration of the cytostatics. Phenothiazines possess chemical and structural features important for their interaction with this plasma membrane component, such as a protonable nitrogen atom, aromatic rings, a relatively high hydrophobicity, and hydrogen-bond groups.10 Phenothiazine drugs are supposed to block Pgp by several mechanisms, including binding of the drugs to Pgp, influencing the biophysical properties of the lipid matrix of cell membranes around the protein, and inhibiting ATP-ase activity of Pgp.11-15 Modification of the cell membrane structure and permeability could be a major part of the mechanisms involved in the chemopreventive activity of phenothiazines, and it was well established that Pgp is sensitive to changes in the biophysical properties of the lipid bilayer.16 It is generally believed that the resistance of cancer cells to chemotherapeutic drugs depends on the balance between outward (active) and inward (passive) fluxes of cytotoxic substances.12 The efficiency of the Pgp pump is responsible for the outflux and the increase of the membrane permeability for the influx of anticancer drugs into cancer cells. It was reported that fluphenazine, a commonly used neuroleptic drug of the phenothiazine group, affects Pgp activity in human lymphocyte cultures.17,18 It therefore seems important to elucidate the influence of fluphenazine on lipid matrix models of cell membranes. The results could improve the strategy of cancer chemoprevention with phenothiazines. We studied FPh/dipalmitoylphosphatidylcholine (DPPC) liposomes by means of ATR-IR and 31P NMR spectroscopy. Phosphatidylcholines (PCs) are the most prevalent phospholipids among those constituting the basic structure of eukaryotic cell membranes. DPPC lipid is one of the most numerous members of the PC lipid group and is frequently used as a model for mimicking lipid-biomembrane properties.19-23 The structures of the FPh and DPPC molecules are presented in Figure 1. The aim of the present study was to characterize the conformational state in the hydrophobic part of FPh-doped

10.1021/jp904805t CCC: $40.75  2009 American Chemical Society Published on Web 11/02/2009

15496

J. Phys. Chem. B, Vol. 113, No. 47, 2009

Cies´lik-Boczula et al.

Figure 1. Schematic representation of fluphenazine and dipalmitoylphosphatidylcholine (DPPC).

DPPC liposomes, the interactions of the drug’s molecules with the polar groups of the lipids, and changes in the structure of DPPC assemblies in the presence of the drug. 2. Materials and Methods 2.1. Materials. The lipid dipalmitoylphosphatidylcholine (DPPC, of purity > 99.8%) was obtained from Sigma-Aldrich, Germany, and used without further purification. Dihydrochloride fluphenazine (FPh, analytical grade) was supplied by Jelfa (Jelenia Go´ra, Poland). 2.2. Preparation of Liposomes. A chloroform/methanol (0.8 mL/0.2 mL) solution of DPPC and FPh was dried under a stream of nitrogen. The dry FPh/DPPC films were hydrated by addition of 1 mL of 10 mM phosphate buffer, pH 7.2, or water at a temperature 10 °C above the gel-liquid-crystalline phasetransition temperature of DPPC liposomes. The final concentration of DPPC was 10 mg/mL in phosphate buffer or water (for ATR-IR measurement) and 40 mg/mL in D2O (for 31P NMR measurement). The liposomal suspensions were treated 10 times with a cooling/heating process in which the liposomes were incubated for 10 min at 4 °C, then heated to a temperature 10 °C higher than the Tm of the doped liposomes, and incubated at this temperature, also for 10 min. 2.3. ATR-IR (Attenuated Total Reflection Infrared) Measurements. The ATR-IR infrared spectra were recorded on a Nicolet Avatar 360 FTIR spectrometer; 256 scans were collected at a resolution of 2 cm-1. The liposome suspensions were prepared according to the procedure described above and spread on one surface of a ZnSe ATR crystal (face angle: 45°; 6 reflections, Specac). The dry DPPC/PDP films were prepared by spreading 200 µL of chloroform/methanol (0.8 mL/0.2 mL) solution of the FPh/DPPC mixture on one surface of a ZnSe ATR crystal and evaporating the solvent under a stream of nitrogen. The concentration of DPPC in chloroform/methanol solution was 10 mg/mL. The spectra of the liposome suspensions were recorded in a heating cycle from 5 to 60 °C, and those of the dry films were recorded in a heating cycle from 10 to 90 °C. The sample temperature was equilibrated for 5 min before acquisition of each spectrum. The data were analyzed by Grams

software, and the difference spectroscopy and subtraction of water/buffer from the spectrum of liposome suspenions was applied. 2.4. 31P NMR Measurements. The 31P NMR spectra were recorded on a Bruker 300 NMR Fourier transform spectrometer operating at 121.49 MHz. The FPh/DPPC suspensions were prepared according to the procedure described above in a D2O solution and were put into 5 mm thin-walled NMR tubes. The relaxation delay time was 3 s. The spectra were recorded over a large spectral width (36 kHz) using broad-band proton decoupling. The data were analyzed by Grams software. 2.5. DFT Calculations. The quantum chemical calculations were carried out with the GAUSSIAN 98 program package.24 Density functional theory (DFT) method at the B3LYP25 level with the 6-31++G** basis set was used. The global minimum was proved be cause all obtained frequencies to be positive. 3. Results and Discussions 3.1. ATR-IR Studies of FPh-Doped DPPC Membranes. The interactions of FPh with both the hydrophobic and hydrophilic part of the DPPC bilayer were characterized using the ATR-IR method. The main advantage of ATR-IR spectroscopy in hydrated lipid assembly research is that this technique enables the rapid and reproducible recording of high-resolution low-noise spectra.19 The structure and physicochemical properties of lipid assemblies as a function of the temperature of the chain-melting phase transition strongly depend on the level of hydration.19,26,28 We studied FPh-doped DPPC liposomes in phosphate buffer solution or water, which allowed us to mimic the hydration conditions in biological membranes. The surveyable spectra of DPPC liposomes before and after complexation with fluphenazine are shown in Figure 2. 3.1.1. Dihydrochloride Fluphenazine (FPh). The ATR infrared spectrum of the FPh dry film is shown in Figure 3. The most important frequencies are collected in Table 1. The assignment of the fundamental vibrational bands of the FPh drug is based on the quantum chemical calculations at the B3LYP/ 6-31++G**levelandliteraturedataofothersimilarcompounds.29-31 The FPh molecules interact with the DPPC bilayer. In a dry FPh film, the νOH band is broad and centered at around 3300

Interactions of FPh with DPPC Liposomes

J. Phys. Chem. B, Vol. 113, No. 47, 2009 15497

Figure 2. The ATR-IR spectrum of DPPC (solid line) and FPh/DPPC (50/50 mol %) liposomes (dashed line) in phosphate buffer at room temperature.

Figure 3. The ATR-IR spectrum of a dry film of dihydrochloride fluphenazine (FPh) at 25 °C.

cm-1 as a consequence of H-bond formation by the OH groups of the drug molecules; see Figure 4. In the dry FPh/DPPC bilayer, this band is centered at around 3200 cm-1, showing the formation of a new type of hydrogen bond between protondonor OH groups of drug molecules and proton-acceptor groups of DPPC. The red shift of the maximum of the νOH band (about 100 cm-1) observed in FPh/DPPC mixtures indicates the stronger H-bond interactions between drug-DPPC than between drug-drug molecules. The literature data31 and DFT calculations showed the interaction of HCl molecules with nitrogen atoms in an aliphatic ring of FPh. Thus, in a dry FPh film, the broad and complex band of νNH+ vibrations in the 3000-1800 cm-1 frequency region is observed (Figure 4). The shape and position of this band are not changed in FPh/DPPC mixtures, indicating no alterations of HCl-drug interactions in the DPPC matrix; see Figure 4. The spectra of FPh molecules in water and DPPC liposomes are shown in Figure 5. The νsCF3 band shifts from 1120 cm-1 for FPh in water to 1125 cm-1 in FPh/DPPC liposomes. Incorporation of FPh between DPPC molecules changes the surroundings of the drug molecules compared to that of the water solution, which has an influence on the position of the νsCF3 band. Most probably, the nonpolar part of the drug molecules represented by the aromatic rings with the -CF3 group is located in the hydrophobic part of the DPPC bilayer. Additionally, the changes of bending and torsion vibrations of

the CH groups of aromatic and aliphatic rings of FPh molecules are present as a result of drug-DPPC bilayer interactions (Figure. 5). 3.1.1. CH2 Stretching Vibrations in FPh/DPPC Liposomes. Phospholipid molecules contain several IR-active groups that can function as suitable spectroscopic probes of the structure and interactions in the hydrophobic, interfacial, and polar head group regions of lipid assemblies.19 The vibrations of the CH2 groups are commonly used to detect hydrocarbon chain conformational disorder and mobility and, consequently, to observe the lipid hydrocarbon chain-melting phase transitions in phospholipid bilayers.19,21,27-34 The asymmetric and symmetric stretching vibration modes in DPPC bilayers occur near 2920 and 2850 cm-1, respectively. These two absorption bands are diagnostic of the onset of gauche rotomer formation in all-trans polymethylene chains. They are also one of the most intensive bands in the IR spectra of long-chain diacyl PC lipids and therefore still have a high enough spectroscopic quality in fully hydrated samples for analysis. The increase in the gauche conformer population in the lipid hydrocarbon part shifts the absorption maxima of both the νasCH2 and the νsCH2 band to a higher-frequency region and decreases the intensity and increases the bandwidths of these two vibrational modes.19,33,34 The position of the maximum of the νsCH2 band as a function of temperature for DPPC liposomes with different FPh contents is shown in Figure 6. In all of the investigated FPh/DPPC assemblies, the observed chain-melting phase transition was

15498



TABLE 1: Assignment of the Theoretical Wavenumber Values (cm-1) to the Experimental Bands of a Dry FPh Filma experimental 750 819 870 937 967 1039 1080 117 1143 1166 1211 1245 1283 1325 1423 1444 1464 1492 1567 1602 2429; 2549; 2642 2792; 2831; 2889; 2953 3061 3300 a

DFT 747.8 762.3 836.9 890 894.8 944 947 976.4 1025; 1041 1044 1089.6; 1094 1098 118 1153 1160 1231 1264 1272 1304 1335.6 1402 1456.7 1477 1499 1534.6 1552 1647.6 2048 2337 2291; 3034; 3069; 3077; 3083; 3093; 3129; 3133 3197; 3207; 3215 3575

assingment τ ring + τ ring τ ringII + FCH2aliph_prop τ ringI F(CH2)aliph_et + FCH2aliph_prop γ(C1H) ringΙ τ ringII + F(CH2)aliph_prop τ ringII + F(CH2)aliph + ν(C-C) ringI τ(CH2)aliph + νs(N2C3) ω(CH2)aliph + ν(N3C) + δ(COH) ω(CH2)aliph_et + F(CH2)aliph_et δ(CH) ringI + ω(CH2)aliph + F(CH2)aliph ω(CH2)aliph + F(CH2)aliph + δ(CH) ringI + δ(CH) ringII νs(CF3) δ(CH) ringII + δ(CH) ringI + νas(CF3) δ(CH) ringII + νas(CF3) + δ(CH) ringI τ(CH2)aliph δ(C1H) ringΙ + δ(C1H) ringΙΙ + τ(CH2)aliph + νs(N1C3) δ(C1H)ringΙ + δ(C4H)ringΙ + δ(C4H)ringΙΙ + τ(CH2)aliph_prop + νas(N1C3) τ(CH2)aliph + δ(C1H) ringΙ + δ(C4H)ringΙ ν(CC) ringII + ν(CC) ringI + ω(CH2)aliph_prop δ(OH) + ω(CH2)aliph_2 ω(CH2)aliph_prop + ν(CC) ringI + ν(CC) ringII δs(CH2)aliph_et δs(CH2)aliph_prop + δ(CH)ring_II + δ(CH)ring_I δ(Ν3Η+) + ω(CH2)aliph_et δ(Ν2Η+) + ω(CH2)aliph_et ν(CC) ringI + δs(CSC) + ν(CC) ringII ν(Ν3Η+) ν(Ν2Η+) νs,as(CH2)aliph_et,aliph_prop,aliph_ring II

I

ν(CH) ringI + ν(CH) ringII ν(OH)

Abbreviations: ν, stretching; δ, in-plane bending; γ, out-of-plane bending; τ, torsion calculated with B3LYP/6-31++G**.

Figure 4. The ATR-IR spectrum of a dry DPPC film (solid line), a dry FPh film (dotted line), and a dry film of FPh/DPPC (50/50 mol %) mixture (dashed line) at 25 °C in the 4000-1800 cm-1 frequency region.

accompanied by a discontinuous increase in the νsCH2 absorption maxima derived from the increase in the gauche population. The temperature of the chain-melting phase transition in FPhdoped DPPC liposomes decreases with the increase in FPh content (Figure 7). The temperatures of the observed phase transition derived from analysis of the νsCH2 and νasCH2 band positions were not exactly the same (Figure 7). These differences arise mainly from the fact that the νasCH2 vibrational mode can itself be pretreated by Fermi resonance interaction with the first overtones of the methylene scissoring vibration.19 νsCH2 is

considerably freer of such effects and is therefore a main absorption band for detecting lipid hydrocarbon chain-melting phase transitions correctly. Dihydrochloride fluphenazine causes a concentration-dependent increase in conformational disorder in the temperature range both below and above the phase-transition temperature in FPhdoped DPPC liposomes. The largest shift in the maximum of the νsCH2 band toward the higher-frequency region is observed for the equimolar FPh/DPPC mixture, as shown in Figure 6. This blue shift of the maximum of the νsCH2 band indicates an



Figure 5. Difference spectra of the FPh in water and the water spectrum (solid line), the FPh/DPPC (50/50 mol %) liposomes spectrum, and the pure DPPC liposomes spectrum (dashed line) at 25 °C; / marks the position of the νsCF3 band.

Figure 6. Temperature dependence of the νsCH2 band maxima of FPh/ DPPC (3/97 mol %) (9), FPh/DPPC (10/90 mol %) (O), FPh/DPPC (20/80 mol %) (right-pointing triangle), FPh/DPPC (35/65 mol %) (/), and FPh/DPPC (50/50 mol %) (() liposomes in phosphate buffer; pH ) 7.2.

Figure 7. The temperature of the lipid chain-melting phase transition derived from analysis of the νsCH2 and νasCH2 band positions at different temperatures for FPh/DPPC liposomes with increasing amounts (mol %) of dihydrochloride fluphenazine.

increase in the conformation-dependent membrane fluidization observed at higher concentrations of the drug. 3.1.2. CdO Stretching Vibrations in FPh/DPPC Liposomes. The polar-apolar interface in the DPPC bilayer is represented by the ester carbonyl groups. In the 1750-1700 cm-1 region is the CdO stretching band, which is one of the most intensive lipid polar group bands and is useful for probing the infrared

Figure 8. Temperature dependence of the infrared frequency of the CdO stretching band in the spectra of the FPh/DPPC (3/97 mol %) (9), FPh/DPPC (10/90 mol %) (1), FPh/DPPC (20/80 mol %) (O), FPh/DPPC (35/65 mol %) (right-pointing triangle), and FPh/DPPC (50/ 50 mol %) (/) liposomes.

region of lipid assemblies.35-41 The νCdO band is composed of at least two components originating from two ester carbonyl groups at the sn-1 and sn-2 positions present in long-hydrocarbonchain lipids.19,35-41 In the fully hydrated state, when water molecules form hydrogen bonds with ester groups in DPPC molecules, the contour of the νCdO band broadens, and its maximum shifts to a lower-frequency region compared with the dry DPPC film. In the gel state of the hydrated DPPC bilayer, the νCdO band is centered at around 1734.7 cm-1, and in the disordered, more hydrated liquid-crystal phase, this band almost reaches the 1733 cm-1 position.19 At temperatures around the main phase transition in DPPC liposomes, a sharp shift to the lower-frequency region is observed. This is evoked by the increased hydration of the polar-apolar interface in the DPPC bilayer when conformational disorder in the hydrophobic part is introduced. At the main phase transition, the distance between the DPPC molecules increases, and more water molecules are able to go deeper into the DPPC bilayer structure. The relationship between the maximum position of the νCdO band and the temperature for FPh-doped DPPC liposomes is shown in Figure 8. Increasing the mole fraction of FPh in DPPC liposomes causes a decrease in the νCdO frequency compared with that in pure DPPC liposomes. This red shift is present in the temperature range before and after the phase transition. The phase transition observed in all FPh-doped liposomes is accompanied by a sharp decrease in the νCdO frequency, as shown in Figure 8.

15500



Figure 9. (A) The relationship between the position of the maximum of the νCdO band and the mole fraction of FPh in a dry DPPC film at 10 (large 9), 50 (right-pointing triangel), 70 (small 9), and 90 °C (/). (B) The difference spectrum in the νCdO region of the pure DPPC spectrum and the FPh/DPPC (50/50 mol %) spectrum of a dry film at 10 °C.

3.1.3. CdO Stretching Vibrations in the FPh/DPPC Dry Film. The ATR-IR study of dry FPh-doped DPPC films gave us information about the direct interaction between FPh molecules and the ester groups of DPPC lipids. The position of the νCdO band in the function of the mole fraction of FPh in dry DPPC bilayers for different temperatures is shown in Figure 9A. The increase in FPh concentration in the dry DPPC film decreases the position of the νCdO band. This effect is very visible at lower temperatures and vanishes when the temperature is increased (Figure 9A). The red shift of the νCdO group vibrations, shown in Figure 9B by the difference spectrum of the pure DPPC spectrum and the FPh/ DPPC (50/50 mol %) spectrum in dry film, is accompanied by hydrogen-bond formation between dihydrochloride fluphenazine molecules and the ester groups of the lipids. The hydration of the dry DPPC film causes a similar red shift resulting from H-bond formation between water molecules and the lipid ester groups. The interaction of the drug’s molecules with the ester part of DPPC weakens with increasing temperature; this lower-frequency shift vanishes with a rise in temperature. At 90 °C, the νCdO band is around the position characteristic of the free ester group in a dry DPPC film and does not depend on the concentration of the drug (Figure 9A). According to the results obtained from the dry film, the red shift observed in the liposome suspensions is also caused by H-bond interaction between the drug and the CdO groups. Additionally, in liposome suspensions in which the DPPC bilayer is in a fully hydrated state, the water molecules are still able to form a H-bond with “free” ester groups unoccupied by drug molecules. The νCdO band is always more red-shifted in FPh/DPPC liposomes than that in the DPPC dry film with the same mole fraction of FPh and at the same temperature (compare Figures 8 and 9A). It should be mentioned that water molecules can change the surrounding dielectric permittivity of the ester groups, which also can influence the νCdO band position. In the spectrum of DPPC, there are vibrational bands like νasPO2- and νOH, which are informative about the level of hydration of the lipid membrane.19,41 In DPPC liposomes doped with different concentrations of FPh, the position of the νasPO2band was always at around 1220 cm-1, characteristic of the fully hydrate state of the pure DPPC bilayer. Additionally, the intensity of νOH in doped and pure DPPC liposomes also was similar. Thus, the red shift of the νCdO band position in DPPC bilayers in the presence of different concentrations of drug should arise mainly from the direct interaction of FPh molecules with the ester part of the lipid, not from the changes of hydration of the polar head groups of the lipid.

3.1.4. PO2- Stretching Vibrations in FPh/DPPC Liposomes. In DPPC liposomes, there are at least two characteristic stretching vibrations of phosphate moiety, that is, νasPO2- and νsPO2-, which are in fully hydrated DPPC membranes at 1220 and 1085 cm-1, respectively.19,41 Opposite to νCdO band, the increase of hydration of the polar head groups of DPPC molecules, which takes place during the gel-liquid phase transition, does not change the position of νas,sPO2- bands.19 Of course, the maximum of νas,sPO2- vibrations shifts to a lowerfrequency region proportionally to the increase of the level of hydration up to the DPPC-bilayer-rich full hydration state. Further growth (if it occurs) of water molecules in the hydrophilic part of the DPPC bilayer does not change the nearest sphere of hydration of the phosphate groups, to which the position of νas,sPO2- bands is sensitive. In the presence of different concentrations of FPh in DPPC liposomes, the νasPO2- and νsPO2- bands are still at around 1220 and 1085 cm-1. It seems that the dehydration of phosphate groups in the presence of drug molecules is not present. The study of dry FPh/DPPC films presented below shows the H-bond interaction between FPh molecules and proton-acceptor phosphate groups. In liposomes, in the presence of water, this interaction most probably can also exist but does not change the position of νas,sPO2- bands. 3.1.5. PO2- Stretching Vibrations in FPh/DPPC Dry Films. The νasPO2- vibration is commonly used to detect the hydrogenbond interaction between the phosphate moiety of DPPC molecules and proton-donor groups. In a dry film of drug-doped DPPC bilayers where water molecules are absent, the red shift of the νasPO2- band is caused by hydrogen-bond formation with new compounds. The difference spectra obtained by subtraction of the spectrum of the dry FPh film from the spectrum of the dry FPh/DPPC film (with different concentrations of drug) reveal the changes in the region of the antisymmetric stretching vibration of phosphate groups in DPPC molecules upon complexation; see Figure 10. With the increase of concentration of FPh, a new band in the lower-frequency region corresponding to the vibrations of hydrogen-bonded phosphate groups appears. In an equimolar mixture of drug and lipid, two peaks are present at 1255 (arising from non-H-bonded phosphate groups) and at 1237 cm-1 (arising from H-bonded phosphate groups). The rise of FPh concentration in DPPC bilayers causes the red shift of these two bands, more significantly for the lower-frequency one. The frequencies of νasPO2- vibrations in various FPh/DPPC systems are presented in Table 2. The red shift (the smaller one) is also visible on symmetric stretching vibrations of the phosphate group as a result of the hydrogen-bond connection between the drug and phosphate lipid



Figure 10. The region of the νPO2- vibrations in a dry film of FPh/ DPPC mixtures, from the top for 50/50, 40/60, 30/70, 20/80, and 10/ 90 mol % and pure DPPC at 25 °C. The presented regions of the νPO2vibrations was obtained by subtraction of the dry fluphenazine spectrum from the spectrum of the dry FPh/DPPC mixture.

TABLE 2: Position of νasPO2-, νsPO2-, and νasN(CH3)3 Bands in a Dry DPPC Film and in Dry FPh/DPPC Mixtures at 25°C dry FPh/ DPPC film DPPC FPh/DPPC (10/90 mol %) FPh/DPPC (20/90 mol %) FPh/DPPC (30/90 mol %) FPh/DPPC (40/90 mol %) FPh/DPPC (50/90 mol %)

νasPO2-/cm-1

νsPO2-/cm-1

νasN(CH3)3/cm-1

1260 1259

1094 1093

3025 3022

1258; 1245

1092,5

3021

1257; 1244

1091,5

3018

1256; 1242

1091

3017

1255; 1237

1090

3015

part. The positions of νsPO2- bands in different FPh/DPPC mixtures are shown in Table. 2. 3.1.6. Antisymmetric Stretching of the Trimethylammonium Group in the FPh/DPPC Dry Film. In the pure, dry DPPC bilayers, there is the weak interaction between the choline N(CH3)3 and the phosphate group.19 In this state, the νasPO2and νasN(CH3)3 bands lay at 1260 and 3025 cm -1, respectively.19,27,41 After addition of the FPh drug, this interaction is replaced by a drug-phosphate interaction. Concomitant with the PO2- stretching shift, the νasN(CH3)3 shifts to the lower-frequency region, and in an equimolar FPH/DPPC mixture, it reaches the position of around 3015 cm-1; see Table 2. Most probably, the trimethylammonium group goes away from the phosphate part, and this could be the reason for the red shift of the νasN(CH3)3 band in FPh/DPPC bilayers. Additionally, the drug is not making a H-bond with this group, and we do not observe a highfrequency band characteristic of a water OH- trimethylammonium group interaction. 3.2. 31P NMR Studies of Dihydrochloride FluphenazineDoped DPPC Liposomes. X-ray is the classical technique for characterizing the macroscopic structures adopted by hydrated lipid systems, but it has a limitation associated with the problem of detecting the occurrence and amount of the less predominant phase. In such cases, the 31P NMR technique can avoid this problem to some extent. 31P NMR is a useful technique for the study of the polymorphic phase behavior of both model and biological membrane systems.42,43 The line shape of a 31P NMR

Figure 11. The 31P NMR spectra of DPPC dispersions with 10 (A), 30 (B), 40 (C), and 50 (D) mol % of FPh and pure DPPC (E) liposomes. The spectra were measured in D2O solution at 25 °C and obtained by employing broad-band proton decoupling.

spectrum strongly depends on the conformation, orientation, and dynamics of phospholipid molecules.42,43 In nonbilayer phases, additional motional averaging mechanisms appear which result in distinctive 31P NMR spectra for lipids in the bilayer phase, hexagonal (HII) phase, and phases such as the inverted micellar, cubic, and rhombic. DPPC lipids in water spontaneously form a bilayer structure which gives rise to an asymmetric line shape of their 31P NMR spectra, with a low-field shoulder and a highfield peak.21,42,43 Phases such as the micellar, cubic, and rhombic, with isotropic lipid motion through which lateral diffusion results in averaging over all orientations, form a narrow symmetric 31P NMR spectrum centered at around 0 ppm.21,42,43 The proton-decoupled 31P NMR spectra of DPPC dispersions in D2O solution with different mole fractions of FPh drug at room temperature are shown in Figure 11. Up to 30 mol % of FPh, the DPPC molecules aggregate into a lipid bilayer structure. At higher concentrations of the drug (>30 mol %), the coexistence of anisotropic (bilayer) and isotropic phases is observed (Figure 11B). The isotropic phase can be represented by micellar, cubic, or orthorhombic structures of high curvature, and in the 31P NMR spectrum, it appears as a sharp and symmetric peak centered at 0 ppm. Unfortunately, the 31P NMR technique does not allow us to determine which isotropic structure the FPh/DPPC mixture adopts. A further increase in the mole fraction of FPh drastically changes the structure of the DPPC bilayer assemblies. In the equimolar FPh/DPPC mixture, only an isotropic phase is present. 4. Summary Dihydrochloride fluphenazine (FPh) is an antipsychotic drug which additionally demonstrates a strong cancer chemopreven-

15502


tive activity.17,18 Although it has been known in the pharmaceutical industry for many years, there is still no information about the mechanism of its prolonged sensitivity of multidrugresistant (MDR) cancer cells to chemotherapeutic drugs. Studies on structurally related compounds such as phenotiazines showed highly significant changes in the physicochemical properties of the lipid membrane around the transmembrane protein Pgp (Pgp).11-16 In the more disordered liquid-crystalline phase of the lipid bilayer, this protein loses its activity of decreasing the intracellular concentration of cytostatics by transporting drugs outside of cancer cells. ATR-IR and 31P NMR spectroscopy were used to characterize structure and conformational ordering in the hydrophobic part of the DPPC bilayer and drug interaction with the carbonyl group of DPPC in the presence of different concentrations of dihydrochloride fluphenazine. The analysis of the positions of the νs,asCH2 bands in FPh-doped DPPC liposomes as functions of temperature and drug concentration in the lipid bilayer showed a distinct decrease in the temperature of the chainmelting phase transition of the DPPC bilayer, roughly proportional to the rise in FPh content. At lower concentration of drug, the DPPC molecules adopt a bilayer structure (the 31P NMR spectrum has a characteristic low-field shoulder and a highfield peak), but with higher fluidity caused by more conformationally disordered hydrophobic states (gauche-rich membrane structures). In this case, the mechanism of anti-MDR activity (prevention of a decrease in the intracellular concentrations of cytostatics) can be explained as how in a gauche-rich membrane, the transport of chemotherapeutic drugs outside of the cell by Pgp protein is reduced. At higher concentrations of FPh (>30 mol %), new structures (isotropic phase) of lipid assembly take place; in the 31P NMR spectrum, a sharp and symmetric peak centered at 0 ppm appears. Here, the prevention of Pgp protein activity is accompanied by the formation of new and highly conformationally disordered isotropic assemblies of DPPC molecules. Additionally, the destruction of the lipid bilayer structure (increase of membrane permeability) can lead to the increase of concentration of anticancer drugs in the cell by an increase of the inward (passive) fluxes of cytotoxic substances. It is important to mention that the FPh concentration of 30 mol % could not be achieved in vivo, and therefore, the last mechanism cannot take place in the human body but only under specific scientific experimental conditions. References and Notes (1) Lieberman, J. A.; Bymaster, F. P.; Meltzer, H. Y.; Deutch, A. Y.; Duncan, G. E.; Marx, C. E.; Aprille, J. R.; Dwyer, D. S.; Li, X-M. S. P.; Mahadik, S. P.; Duman, R. S.; Porter, J. H.; Modica-Napolitano, J. S.; Newton, S. S.; John, G.; Csernansky, J. G. Pharmacol. ReV. 2008, 60 (3), 358. (2) Gil-Ad, I.; Shtaif, B.; Levkovitz, Y.; Nordenberg, J.; Taler, M.; Korov, I.; Weizman, A. Oncol. Rep. 2006, 15, 107. (3) Gasiorowski, K.; Malinka, W.; S´wiatek, P. Cell. Mol. Biol. Letters 2003, 8, 927. (4) Gil-Ad, I.; Shatif, B.; Levkovitxz, Y.; Dayag, M.; Zeldich, E.; Weizmann, A. J. Mol. Neurosci. 2004, 22, 189. (5) Zhelev, Z.; Ohba, H.; Bakalova, R.; Hadjimitova, V.; Ishikava, Shinohara, Y.; Baba, Y. Cancer Chemother. Pharmacol. 2004, 53, 267. (6) Ford, J. M.; Prozialeck, W. C.; Hait, W. N. Mol. Pharmacol. 1988, 35, 105.

Cies´lik-Boczula et al. (7) Szabo´, D.; Szabo´, G.; Ocsovszki, I.; Aszalos, A.; Molna´r, J. Cancer Lett. 1999, 139, 115. (8) Ko´nya, A.; Andor, A.; Sa´torhelyi, P.; Ne´meth, K.; Kurucz, I. Biochem. Biophys. Res. Commun. 2006, 346, 45. (9) Tsakovska, I.; Pajeva, I. Curr. Drug Targets 2006, 7 (9), 1123. (10) Bisi, A.; Meli, M.; Gobbi, S.; Rampa, A.; Tolomeo, M.; Dusonchet, L. Bioorg. Med. Chem. 2008, 16, 6474. (11) Hidalgo, A. A.; Pimentel, A. S.; Tabak, M.; Oliveira, O. N. J. Phys. Chem. B 2006, 110 (39), 19637. (12) Eytan, G. D.; Regev, R.; Oren, G.; Assaraf, Y. G. J. Biol. Chem. 1996, 271, 12897. (13) Romsicki, Y.; Sharom, F. J. Biochemistry 1999, 38, 6887. (14) Wadkins, R. M.; Houghton, P. J. Biochim. Biophys. Acta 1993, 1153, 225. (15) Hendrich, A. B.; Wesołowska, O.; Motohashi, N.; Molna´r, J.; Michalak, K. Biochem. Biophys. Res. Commun. 2003, 304, 260. (16) Wesołowska, O.; Hendrich, A. B.; Motohashi, N.; Kawase, M.; Dobryszycki, P.; Oz´yhar, A.; Michalak, K. Biophys. Chem. 2004, 109, 399. (17) Dong, X.-W.; Jia, Y.; Lu, S. X.; Zhou, X.; Cohen-Williams, M.; Hodgson, R.; Li, H.; Priestley, T. Psychopharmacology 2008, 195, 559. (18) Jaszczyszyn, A.; Gasiorowski, K. AdV. Clin. Exp. Med. 2008, 17 (5), 525. (19) Mantach, H. H.; Chapman, D. Infrared Spectroscopy of Biomolecules; Wiley-Liss: New York, 1996; p 159. (20) Cannon, B.; Lewis, A.; Metze, J.; Thiagarajan, V.; Vaughn, M. W.; Somerharju, P.; Virtanen, J.; Huang, J.; Cheng, K. H. J. Phys. Chem. B 2006, 110 (12), 6339. (21) Cies´lik-Boczula, K.; Koll, A. Biophys. Chem. 2009, 140, 51. (22) Cies´lik-Boczula, K.; Ku¨pcu¨, S.; Ru¨nzler, D.; Koll, A.; Ko¨hler, G. J. Mol. Struct. 2009, 919, 373. (23) Chia, N.-C.; Vilcheze, C.; Bittman, R.; Mendelsohn, R. J. Am. Chem. Soc. 1993, 115, 12050. (24) Frish, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.; Peterson, G. A.; Montgomery, J. A.; Raghavachari, K.; Al-Laham, M. A.; Zakrzewski, V. G.; Ortiz, J. V.; Foresman, J. B.; Peng, C. Y.; Ayala, P. Y.; Chen, W.; Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart, J. P.; Head-Gordon, M.; Gonzales, C.; Pople, J. A. Gaussian 94, revision B3; Gaussian Inc.: Pittsburgh, PA, 1995. (25) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (b) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1993, 37, 785. (26) Kodoma, M.; Kuwabara, M.; Seki, S. Biochim. Biophys. Acta 1982, 689, 567. (27) Ciesik, K.; Koll, A.; Grdadolnik, J. Vib. Spectrosc. 2006, 41, 14. (28) Cies´lik-Boczula, K.; Czarnik-Matusewicz, B.; Perevozkina, M.; Filarowski, A.; Boens, N.; De Borggraeve, W. M.; Koll, A. J. Mol. Struct. 2008, 878, 162. (29) Bolboaca, M.; Iliescu, T.; Kiefer, W. Chem. Phys. 2004, 298, 87. (30) Pan, D.; Phillips, D. L. J. Phys. Chem. A 1999, 103, 4737. (31) Qin, B.; Lin, J.; Lin, Z.; Ren, H.; Xue, Y. Spectrochim. Acta, Part A 2005, 61, 717. (32) Mantach, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213. (33) Snyder, R. G. J. Chem. Phys. 1979, 47, 1316. (34) Snyder, R. G.; Maroncelli, M.; Strauss, H. L.; Elligier, C. A.; Cameron, D. G.; Casal, H. L.; Mantsch, H. H. J. Am. Chem. Soc. 1983, 105, 133. (35) Kolling, O. W. J. Phys. Chem. 1992, 96, 6217. (36) Bicknell-Brown, E.; Brown, K. G.; Person, W. B. J. Am. Chem. Soc. 1980, 102, 5486. (37) Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 381. (38) Bush, S. F.; Levine, H.; Levine, I. W. Chem. Phys. Lipids 1980, 27, 101. (39) Lewis, L. N. H.; McElhaney, R. N. Biophys. J. 1992, 61, 63. (40) Grdadolnik, J.; Kidric, J.; Hadzi, D. Chem. Phys. Lipids 1991, 59, 57. (41) Grdadolnik, J.; Hadzi, D. Spectrochim. Acta, Part A 1998, 54, 1989. (42) Cullis, P. R.; De Kruijff, B. Biochim. Biophys. Acta 1979, 559, 399. (43) Cullis, P. R.; De Kruijff, B. Biochim. Biophys. Acta 1978, 513, 31.

JP904805T

Interactions of Dihydrochloride Fluphenazine with DPPC Liposomes

Recommend Documents