Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 250–256

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

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Interactions of tamoxifen with distearoyl phosphatidylcholinemultilamellar vesicles: FTIR and DSC studies

http://dx.doi.org/10.1016/j.saa.2014.04.0271386-1425/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +90 (232) 311 23 01; fax: +90 (232) 388 10 36.E-mail address: nadide.kazanci@ege.edu.tr (N. Kazanci).

Duygu Bilge a, Ipek Sahin a, Nadide Kazanci a,⇑, Feride Severcan b

a Department of Physics, Faculty of Science, Ege University, 35100 _Izmir, Turkeyb Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey

h i g h l i g h t s

� DSPC MLVs was used to investigatethe effect of tamoxifen on lipidmembrane.� FTIR study shows that low dose of

TAM ordered the system anddecreased the dynamics in bothphases.� It is observed that higher TAM

concentrations disordered the systemand increased the dynamics.� TAM causes dehydration between

C@O and PO�2 groups of lipids.� DSC study indicates that TAM shifts

the main transition of lipids to lowertemperatures.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 December 2013Received in revised form 6 March 2014Accepted 6 April 2014Available online 18 April 2014

Keywords:TamoxifenDSPCMLVsFourier transform infraredDifferential scanning calorimetry

a b s t r a c t

Interactions of a non-steroidal antiestrogen drug, tamoxifen (TAM), with distearoyl-sn-glycero-3-phos-phatidylcholine (DSPC) multilamellar liposomes (MLVs) were investigated as a function of drug concen-tration (1–15 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR)spectroscopy and differential scanning calorimetry (DSC). FTIR spectroscopy results show that increasingTAM concentrations (except 1 mol%) increased the wavenumbers of the CH2 stretching modes, implyingan disordering effect for DSPC MLVs both in the gel and liquid crystalline phases. The bandwidth values ofthe CH2 stretchings except for 1 mol% increased when TAM concentrations increased for DSPC liposomes,indicating an increase in the dynamics of liposomes. The C@O stretching and PO�2 antisymmetric doublebond stretching bands were analyzed to study interactions of TAM with head groups of lipids. As the con-centrations of TAM increased, dehydration occurred around these functional groups in the polar part ofthe lipids. The DSC studies on thermal properties of DSPC lipids indicate that TAM eliminated the pretransition, shifted the main phase transition to lower temperatures and broadened the phase transitioncurve of the liposomes.

� 2014 Elsevier B.V. All rights reserved.

Introduction of other molecules. Phospholipids are quite various depending on

Biological membranes are very complex and composite struc-tures composed of phospholipids. They may contain small amounts

properties such as polar head group, hydrocarbon chain lengthand degree of unsaturation. Hundreds of different lipid speciesare present in the plasma membrane [1–4]. Phospholipids are offundamental importance since they are the main constituents ofbiological membranes and play an important part in their func-tions [5].

D. Bilge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 250–256 251

Due to the complexity of the natural membranes, numerousstudies have used pure or binary lipid mixtures as membrane mod-els. Phospholipids have been the subject of research for over threedecades as the simplified models of biological membranes [6,7].The phosphocholines coexist with other phospholipids in biologi-cal membranes. Among phospholipids, phosphatidylcholines(PCs) or lecithins are the most abundant in eucaryotic organisms[8]. Therefore, PCs are commonly used phospholipids in modelmembrane studies [9].

TAM widely used in management and prevention of cancers,particularly breast cancer, is a non-steroid antiestrogen agent[10,11]. The chemical structure of TAM is shown in Fig. 1. TAM isalso considered an estrogen antagonist. Although TAM is usuallyconsidered as an estrogen antagonist, the anti-proliferative effectsmay involve mechanisms not restricted to the classical estrogenreceptor (ER) binding model [12]. TAM inhibits the growth oftumor cells by expressing estrogen receptors [13,14] and estrogenreceptor-negative cells [15]. Previous studies on neutral and modelmembranes showed that TAM locates in the hydrophobic part ofthe membrane and has various effects on its physical properties[16–19]. In these studies, the effects of TAM on the membranefluidity and phase transition temperature were investigated asphysical properties by using FTIR, turbidity and fluorescence polar-ization techniques. However, little has yet been known of the somepotential mechanisms and behaviors of TAM in the membrane.

Studies regarding the effect of TAM on membrane are limitedand contradictory to each other. Dicko et al. noted conformationaldisorder caused by the addition of TAM to pure saturated lipidbilayers in both their gel and fluid states by using FTIR [20]. Theyfound an increase in the membrane fluidity of striatum and frontalcortex membranes using 1,6-diphenyl-1,3,5-hexatriene (DPH)fluorescence anisotropy measurements whereas Wiseman et al.reported an ordering effect of TAM on both the hydrophobic andhydrophilic regions of the bilayer in artificial membranes preparedfrom ox-brain phospholipids using the same method [21,22]. Cus-todio et al. observed an increase in the fluidity of the gel phase,however, a slight decrease in the liquid-crystalline phase of lipo-somes obtained from different pure phospholipids [17]. In a bacte-rial model system [18], TAM has been demonstrated to induce adisorder in the gel and slight order in the fluid state. Reports areall similar in that DPH fluorescence anisotropy method determinedthe disordering/ordering effects of TAM. The difference betweenthe consequences of these studies may be accounted for by the nat-ure of the lipid and drug concentration used in them.

Dynamical properties are often less well understood in bio-molecular systems, but they have numerous important essentialproperties. Among saturated lecithins, phosphatidylcholine (DPPC)and DSPC are frequently used as model membranes and in the forms

Fig. 1. Chemical structure of TAM.

of potential drug delivery systems as well [23]. Aygun et al. reportedthat liposomes comprised of dimyristoylphosphatidylcholine(DMPC) or DSPC lipids with cholesterol and zinc phthalocyanine(ZnPC) revealed that several fundamental liposome properties areinfluenced by composition and by lipid-specific features. Theysuggested that DSPC lipids are good building blocks for the produc-tion of liposomes [24]. Harvey et al. also indicated that PCs are com-monly used to form liposomes [25]. In their research, DSPC waschosen as a saturated model membrane.

The current study investigated the effects of TAM on both thegel and liquid crystalline phases of DSPC lipid bilayers, for the firsttime, depending on drug concentrations using FTIR and DSC. FTIRcan be used to monitor subtle changes in the structure and func-tion of the lipid assemblies by analyzing the wavenumber or band-width changes of the different vibration modes representing theacyl chains, the interfacial region and head group region of lipo-somes. Previous studies showed that IR spectral parameters arequite sensitive to structural and dynamics properties of mem-brane’s lipid molecules [26]. Using DSC techniques, some thermo-tropic parameters can be determined such as transition enthalpy(DH), transition temperature (Tm), temperature at which the tran-sition is half completed (T1/2). These parameters could be used todesign liposomes [27]. DSC can be also used to study domain for-mation. Many of the applications of DSC in this area have beenthrough the construction of phase diagrams and their interpreta-tion in terms of phase miscibility [28]. In the present work, DSCwas used to determine how TAM influences thermal characteristicsof lipid bilayers [29].

Materials and methods

Materials

TAM and DSPC were obtained from Sigma (St. Louise, Mo) andused without further purification.

Sample preparation

For FTIR measurements, pure lipid samples prepared accordingto the procedure reported in Severcan et al. [30–32]. Briefly, 5 mgDSPC was dissolved in 0.1 ml chloroform and excess of chloroformwas evaporated by a gentle stream of nitrogen. A dried lipid filmwas obtained by exposure of the samples to vacuum drying for2 h with the HETO-spin vac system (HETO, Allerod, Denmark).Lipid films were then hydrated by adding 25 ml of 10 mMphosphate buffer, pH 7.4. Homogeneous vesicles were formed byvortexing the mixture at �75 �C which is above the main phasetransition temperature (Tm) of DSPC (�54.5 �C) MLVs for 20 min.To prepare TAM-containing liposomes, required amount of TAMfor 1, 6, 9 and 15 mol% concentrations from stock solution whereTAM was dissolved in ethanol, was first placed inside a tube. Thesolution in the tube was subjected to a stream of nitrogen toremove excess ethanol and then 5 mg lipid is added. The procedurementioned above for the preparation of DSPC liposomes wasfollowed.

For DSC measurements, pure DSPC or TAM (1, 6, 9 and 15 mol%),containing thin lipid films, were prepared as described above,except that 2 mg of DSPC was hydrated with 50 ll phosphate buf-fer (pH 7.4) [30,32].

FTIR measurements and statistical analyzes

Sample suspensions of 20 ll were placed between CaF2 win-dows with the cell thickness of 12 lm. Infrared spectra wereobtained using a Spectrum 100 (Perkin–Elmer Inc., Norwalk, CT,

252 D. Bilge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 250–256

USA) equipped with a deuterated triglycine sulfate (DTGS)detector. The instrument was under continuous dry air purge toeliminate atmospheric water vapor. Interferograms were averagedfor 50 scans at 2 cm�1 resolution. Temperature was regulated by aGraseby Specac (Kent, UK) digital temperature controller unit. Thesamples were scanned both 40 �C for their gel phases and 65 �C fortheir liquid crystalline phases and incubated at each temperaturefor 5 min before data acquisition. Experiments were repeated fivetimes and spectra of samples were collected at the mentionedtemperatures. Buffer spectra were also collected at the sametemperatures and digitally substracted from the sample spectracollected at identical conditions. Until the bulk water signal at2300 cm�1 was flattened, the substraction procedure was main-tained from which spectra were substracted to use in detailingthem and statistical analysis. Furthermore, normalized averagespectra were compared to visually demonstrate the changes [30].

Spectra were analyzed by Spectrum v5.0.1 software (Perkin–Elmer). Band positions were measured according to the center ofweight. Bandwidth was measured at 3=4 of peak height position.Spectra were normalized in specific regions for visual demonstra-tion of the comparative changes with the same software [33,34].Statistical significance was assessed using Mann Whitney test. Thistest was applied to determine statistical significance of the spectralparameters of the drug containing groups in comparison to thepure lipid membrane. Significant difference was statisticallyconsidered at the level of P 6 0.05. Statistically significant resultswere shown using an asterisk.

DSC measurements

Experiments were carried out in a Universal TA DSC Q 100 (TAInstruments Inc., New Castle, Delaware, USA). Samples wereencapsulated in hermetically sealed standard aluminum DSC pans.An empty pan was used as a reference to exclude calorimetriceffect of pan. Samples were scanned over a temperature range of30–65 �C at a heating rate of 1 �C/min, as reported previously [32].

Results

FTIR studies

FTIR studies were performed to observe the effect of TAM con-centrations on the lipid membranes namely DSPC. FTIR spectra ofDSPC MLVs were analyzed for different TAM concentrations(1 mol%, 6 mol%, 9 mol% and 15 mol%). Information on the order–disorder state (i.e., acyl chain flexibility) of the lipid membraneswas presented by the CH2 antisymmetric and symmetric stretchingvibrations located at 2920 and 2850 cm�1, respectively [30,32,35].The wavenumber of CH2 stretching bands of acyl chains dependson the average trans/gauche isomerization in the system. The shiftsto higher wavenumbers region correspond to an increase in the

Fig. 2. Concentration dependent values for the wavenumber of the CH2 antisymmetric stP < 0.05.

number of gauche conformers, implying a more disordered state[36,37].

Fig. 2 shows a change in the wavenumber of CH2 antisymmetricstretching bands of DSPC MLVs as a function of TAM concentra-tions both in the gel and liquid crystalline phases, respectively. Itillustrates an opposite effect of TAM on the order parameter atlow and high concentrations for DSPC liposomes in both phases.The wavenumber values of CH2 antisymmetric stretching modedecreased for 1 mol% TAM concentration implying an increase inthe order while higher TAM concentrations increased the wave-number to imply a decrease in the order of DSPC MLVs. Becausean increase in the wavenumber is consistent with the increase inthe gauche conformers (or conformal disorder) of the hydrocarbonchains [37], the wavenumber increase observed at high concentra-tions exhibits a disordering effect of TAM on DSPC MLVs. Similarresults were also observed for CH2 symmetric stretching modes.As compared with pure DSPC, the wavenumber variation valuesof CH2 antisymmetric stretching band are 0.39 and 1.52 cm�1 forthe lowest (1 mol%) and the highest (15 mol%) TAM concentrationsat the gel phase whereas these values change between 0.74 and1.79 cm�1 for the lowest (1 mol%) and the highest (15 mol%)TAM concentrations at liquid crystalline phase, respectively. Thesevariations in the wavenumbers enable better understanding of themagnitude of TAM effect on DSPC MLVs.

Information about the dynamics of membrane systems can beobtained by analyzing the variations in the bandwidth of CH2

stretching modes since the bandwidth reflects the changes in themobility of the acyl chains. An increase in the bandwidth is anindication of an increase in the dynamics of the membrane system[30,32,38,39,19]. Fig. 3 presents the variations in the bandwidthvalues of DSPC liposomes as a dependence of different TAMconcentrations for CH2 antisymmetric stretching modes both inthe gel and liquid crystalline phases. It shows that the bandwidthvalues of CH2 antisymmetric stretching mode remarkablydecreased at 1 mol% while they increased significantly at differentconcentration of TAM above 1 mol%. This implies that the dynam-ics of DSPC liposomes decreased at low concentration of TAMwhilst higher TAM concentrations increased dynamics. Similarresults were also seen in the CH2 symmetric stretching bands(not shown).

To obtain information about effect of the drug on the membraneinterfacial region, FT-IR spectroscopy was used and the C@Ostretching band was monitored. The carbonyl absorption bandaround at 1730 cm�1 caused by the stretching vibrations of estercarbonyl groups of phospholipids is structurally sensitive to thelevel of hydration at the membrane interface and influenced byhydrogen bonding [30,32]. Therefore, any effects in the spectra ofthis region can be attributed to an interaction between TAM andthe polar/apolar interfacial region of the membrane. Fig. 4 showsthe changes in the wavenumber values of C@O stretching modesof pure and TAM-containing DSPC MLVs in both phases. As seenfrom Fig. 4, the wavenumber values increased with the addition

retching mode of pure and TAM-containing DSPC liposomes at (a) 40 �C, (b) 65 �C (�)

Fig. 3. Concentration dependent values for the bandwidth of the CH2 antisymmetric stretching mode of pure and TAM-containing DSPC liposomes at (a) 40 �C, (b) 65 �C (�)P < 0.05.

Fig. 4. Concentration dependent values for the wavenumber of the C@O stretching mode of pure and TAM-containing DSPC liposomes at (a) 40 �C, (b) 65 �C (�) P < 0.05.

Fig. 5. Concentration dependent values for the wavenumber of the PO�2 double bond stretching mode of pure and TAM-containing DSPC liposomes at (a) 40 �C, (b) 65 �C (�)P < 0.05.

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of increasing TAM concentrations, implying dehydration aboutthese functional groups in the interfacial region of the lipid mem-branes [34]. This can be accounted for by the presence of free car-bonyl groups in the system.

Information about the hydration state of the polar head-groupsof the phospholipids can be monitored by the analysis of the wave-number of PO�2 antisymmetric double bond stretching band,located at 1220–1240 cm�1 [30,32,39]. The wavenumber of whichshows the strength of hydrogen bonding in the polar region of thebilayer [40]. The wavenumber variations in the PO�2 antisymmetricdouble bond stretching bands of DSPC liposomes as a function ofdifferent TAM concentrations are given in Fig. 5. Upon TAMaddition into the system, the wavenumber shifts to higher valuesfor both gel and liquid crystalline phases, indicating an increasein dehydration of the phosphate group.

DSC studies

Effects of TAM on phase properties of membrane were deter-mined by DSC technique. Influence of drug molecules on liposomesthermodynamic behavior can also be monitored by DSC. The acyl

chains of liposomes can be exposed to phase transitions at specifictemperatures, depending on the structure of the lipid. Very impor-tant information regarding the transitions can be obtained by DSC[41]. The pretransition points out a transformation from a tilted toa rippled chain gel (L0b ? P0b) phase and at the main transition, thereis a transformation from the gel to the liquid crystalline (P0b ? La)phase [42,43]. Fig. 6 shows DSC curves for pure and TAM-contain-ing DSPC liposomes depending on drug concentrations. Thepre-transition and sharp main transition of DSPC liposomes areobserved at �51.2 �C and �54.5 �C, respectively [44]. The pretran-sition considerably broadened and, it was shifted to a lower tem-perature. Meantime, the main transition temperature was shiftedslightly to a lower temperature at 1 mol% TAM addition. However,the pretransition was abolished and main phase transition wasbroadened significantly at the other concentrations of TAM.

Discussion

FTIR spectroscopy is widely accepted to be highly sensitive toolcapable of providing convincing insight into structural and func-tional changes caused by a variety of factors. For example, the

Fig. 6. DSC thermograms for DSPC MLVs in the absence and presence of differentconcentrations of TAM.

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frequency shifts in the corresponding bands of the different spec-tral regions can be used to obtain knowledge of a range of physico-chemical processes in the systems [45,46]. The CH2 stretchingregion of the infrared spectrum (2800–3000 cm�1) shows thatthe hydrocarbon chains containing gauche conformers absorbinfrared radiation at higher frequencies than those with all trans-conformers. Increase of hydrocarbon chain conformational disor-der is thus accompanied by increases in the frequencies of infraredspectrum absorption bands due to the symmetric and antisymmet-ric stretching modes of lipid hydrocarbon chains. This characteris-tic can be utilized as a probe for changes in lipid hydrocarbon chainconformational disorder. It is frequently used to detect and/ormonitor lipid hydrocarbon chain-melting phase transitions [47,48].

Interaction of TAM with DPPC, DMPC and DPPG MLVs werestudied by our group as a function of TAM concentrations [31].The current study investigated the effect of TAM on lipid phasetransition, lipid order and hydration states of the interfacial groupof DSPC as a function of TAM concentrations. Our FTIR results(Fig. 2a and b) indicate that TAM stabilizes DSPC liposomes atlow concentration (1 mol%) in both the gel and the liquid crystal-line phases while TAM in DSPC MLVs leads to a significant increasefor disorder of the phospholipid acyl chains in their phases at highconcentrations (above 1 mol%). This suggests that TAM penetratesthrough the bilayer, perturbing the packing of the lipid molecules.Boyar and Severcan found that TAM is located in the hydrophobiccore of the bilayer, which is confirmed as a hypothesis by the greatdisordering influence of TAM on DSPC acyl chains [49]. At highconcentrations, the presence of the lateral cycle in the structureof TAM (Fig. 1) probably creates a large void between neighboringDSPC molecules, leading to a decrease of the Van der Waals inter-actions, and to more disordered chains. Previous studies showedthat high concentrations of TAM do not stabilize the membranebut stabilizes it at low concentrations [16,31].

Biological membranes, extremely dynamic and complex liquid-crystalline structures, principally form an interface between thecell and environment and co-ordinates the intercellular communi-cation. Some anticancer drugs have a significant influence onmembranes and their strong interactions at cellular surface mayaffect their mechanism of action. Fluidity is of great importancesince the membrane is not solid but rather exists as a fluid mosaicby which dynamic mobility continuously varies in order to meetthe changing cellular requirements. Therefore, motion, particularlythe rotational and translational ones, is essential so that the mem-brane components could seek their optimal interactions andarrangements. Membranes of tumor cells have been found topossess higher fluidity than membranes of nontumor cells. Thereare increasing findings that variations in the plasma membrane

fluidity of cancer cells are greatly related to the capacity of cancercells to form metastases [50–53]. Variations in membrane fluiditymay play a role in the regulation of membrane properties bothunder physiologic circumstances and in the pathogenesis of thedisease [54,55]. It is thus not surprising that the pharmacologicalmodulation of membrane fluidity could have profound conse-quences and, many drugs have been shown to possess the abilityto adjust the fluidity of a membrane [56]. By modulating the mem-brane fluidity through interactions with the phospholipid bilayerdomains, a drug molecule can also have a remarkably effect onthe membrane functions. Addition of TAM at low concentration(1 mol%) into DSPC MLVs decreases membrane fluidity whereas(6, 9, 15 mol%) increase in membrane fluidity both in the gel andliquid crystalline phases are observed at other TAM concentrations(Fig. 3a and b). Wiseman et al. showed TAM-modulated decrease inmembrane fluidity as measured by DPH fluorescence anisotropywhere low concentrations of TAM were used, supporting our studyresults as well [21]. Severcan et al. previously studied effects ofTAM on lipids and showed its opposite effects on DPPC membranesat its low (1 mol%) and high concentrations (30 mol%), which issimilar to our present results [16,19]. Sade et al. examined interac-tions of a celecoxib, a non-steroid anti-inflammatory agent withDSPC liposomes in order to demonstrate its close relationship tothe activities preventing the harmful effects of chemicals in breast[44]. In accordance with our results, they found an opposite effecton the order of membrane for different concentrations of celecoxib,i.e. an increase in the order of the system at low concentrations,but a decrease at high concentrations. Jedrzejczak et al. investi-gated the effect of three different anticancer drugs (the pro-oxida-tive anthracyclines doxorubicin (DOX), aclarubicin (ACL) andantioxidative anthraquinone mitoxantrone (MTX)) on the fluidityof plasma membrane in immortalized fibroblasts. Although thesedrugs are different from TAM in terms of their chemical structures,they penetrate mainly into hydrophobic part of cell membranesdisturbing their structure like TAM. Their results demonstratedthat these drugs influence predominantly core of lipid bilayer,inducing a considerable decrease in fluidity of lipids at low concen-trations. They also noted a decrease in membrane fluidity at thesurface of the lipid bilayer only at a higher concentration of thedrugs [57]. Clarke et al. used TAM to investigate the fluidity ofestrogen receptor (ER) negative in human breast cancer MDA-MB-436 and that of ER positive in human breast cancer MCF7 cells[58]. The authors found TAM to reduce the dynamics of cell mem-branes while increasing it at high doses. Kayyali et al. investigatedthe ability of TAM and cholesterol, to modulate drug efflux fromphosphatidylcholine liposomes. According to their results, bothcompounds were found to decrease the efflux of chloroquine fromphosphatidylcholine liposomes [59]. In another clinic research, it isstated that low doses of TAM decreased side effects when com-pared to its high doses without altering the pharmacologic effectsof chemotherapy on breast and therefore they resulted that its lowdoses could have a function as an antiestrogen and reduce cellularproliferation and further benefit women with breast cancer [60]. Inagreement with these studies, since we also observed TAM to sta-bilize DSPC liposome at low concentration, only its low doses canbe used in decreasing cancer-induced membrane destabilization[55,61,62].

A number of studies have shown that the major differences indrug–membrane interactions may be accounted for by the fact thatthey localize in the membrane and interact with individual phos-pholipids. Therefore, variations in fluidity of cellular membranesinduced by anticancer drugs are controversial and these variationsthese variations depend on such several factors as condition of theculture, state of cells, concentration of a drug and membrane com-position [63,64]. These studies also revealed that the same concen-trations of drugs lead to different degrees of alterations in the

D. Bilge et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 130 (2014) 250–256 255

membrane fluidity, which may be caused by different interactionsof drugs with the lipid components of cellular membranes [57].

Moreover, various clinical studies showed that low dose of TAMare more effective in several cancer diseases. Gonzaga et al. studiedoptimal biological dose of TAM, namely the minimal active dosewhich may maintain benefits while minimizing its toxicity [65].They concluded that since the endometrial effect of TAM is basedon dose [66], lower doses tend to have the way for modulatingbreast cancer and cardiovascular risk biomarkers without increas-ing endometrial proliferation and menopausal symptoms [67].Decensi et al. suggested that using a lower dose of TAM may reduceits side effects while maintaining its therapeutic and preventiveeffectiveness [68]. In order to examine the biologic effects of lowdoses of TAM, they measured biomarkers related to breast cancer,cardiovascular disease and bone fracture risk. Wu et al. reportedlow-dose TAM to block the growth-promoting effects of estrogenin the breast without disturbing estrogen-regulated genes in theliver [69]. All these clinical results are in agreement with ourresults. This study is therefore of great importance at molecularlevel.

Sensitive to variations in the polarity of their local environ-ments, the absorption bands of ester C@O vibration are affectedby hydrogen bonding and other related interactions. Hence, onecan usually assess changes in the contours of C@O absorptionbands of the ester according to structural and/or hydration varia-tions in the bilayer polar/apolar interface [70]. It has been foundthat PO�2 antisymmetric double bond stretching mode is useful tomonitor the hydration state of the polar head group of phospholip-ids. For instance, a ‘‘free’’ PO�2 is characterized by a frequency ofabout 1240 cm�1 whereas a fully hydrated PO�2 group is character-ized by that of nearly 1220 cm�1 [71,72]. We found a significantincrease in the wavenumber of C@O and PO�2 groups for DSPCMLVs in the gel and the liquid crystalline phases. In accordancewith the empirical rules, decreasing frequency values displays anincrease in the strengthening of existing hydrogen bonding or inthe formation of new hydrogen bonding between the components[32]. We showed (Figs. 4 and 5) that the wavenumber values ofC@O and PO�2 groups shift to higher degrees for all samples con-taining TAM, without any evidence of hydrogen bonding. TAMtends to reduce the forming of hydrogen bonding in the interfacialregion of DSPC liposome, implying the existing of the free carbonylgroups in the system. TAM is probable to replace some H2Omolecules from the interfacial region and lead to an increase inthe number of free carbonyl groups.

Any agent interacting with model membranes causes a varia-tion of thermogram of MLVs and usually this variation is depen-dent on the amount of agent interacting with MLVs. As shown inFig. 6, the interaction of TAM with MLVs also produced some var-iation in the DSC curves [73]. In the absence of TAM, two thermaltransitions were observed for the DSPC liposomes. The presence ofsmall amount of TAM can eliminate the pretransition of DSPCMLVs. As the pretransition is highly sensitive to the presence ofother molecules in the phospholipids, the loss of the pretransitioncannot be attributed to any specific molecular changes. The maintransition shows the transition of the bilayer from a highly all transhydrocarbon chain conformation to a state in which some acylchains exist in the gauche conformation, resulting in greaterphospholipid rotational freedom [43]. Broadening of the main tran-sition peak by the presence of TAM especially for 6 mol%, 9 mol%and 15 mol% concentrations is indicative of a destabilization ofthe model membranes, suggesting an intercalation of this drugwithin the lipid bilayer [73]. Engelke et al. reported that thepresence of TAM in DPPC liposomes leads to a broadening ofthermal transition profile and lowering of the main transition tem-perature. They performed DSC measurements on DPPC liposomesin the presence of 0–7 mol% TAM and their results, their results,

in agreement with ours, showed that the main phase transitiontemperature decreased around 0.4–2 �C depending on TAMconcentration [74]. The broadening DSC curves and lowering oftransition temperature shows to be modified both size and packingof bilayers with a consequent disordered system [30]. The confor-mation of an anticancer drug called paclitaxel and its interactionwith DPPC membranes were investigated by Balasubramanianand Straubinger [75]. Consistent with our results, their studiesfound that paclitaxel partitions into the system, disturbs the con-formation of hydrocarbon chains and causes a broadening in DPPCphase transition. Custo dio et al. found this kind of broadeningeffect of the sterol and TAM on DMPC membrane in which thepresence of these two compounds on the membrane order theDMPC acyl chains [61].

The process of cancer is accounted for by the fact that growth ofabnormal cells is uncontrolled and they spread and, this can seri-ously threaten human health and cause of death. The molecularinteraction of anticancer drugs with the cancer cells tends tomodulate the efficacy of them. Therefore, it is of great importancein order to get better understandings of drug–membrane interac-tions for the efficient and correct usage of the drug considering thatanticancer agents can also have an effect on healthy cells. Ourresults indicated the existing strong interactions between TAMand DSPC MLVs and these interactions are TAM-concentrationdependent.

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