8
Acyl chain length and charge effect on Tamoxifen–lipid model membrane interactions Duygu Bilge a , Nadide Kazanci a,, Feride Severcan b a Department of Physics, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey b Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey highlights " Different lipids were used to examine the effect of TAM on acyl chain length and charges of lipids. " FTIR technique was used for spectral analysis and DSC technique was used for calorimetric analysis. " This study indicated there are strong interactions between TAM and the lipid membranes. graphical abstract article info Article history: Received 18 January 2013 Received in revised form 20 February 2013 Accepted 26 February 2013 Available online 6 March 2013 Keywords: Tamoxifen FTIR DSC Chain length Charge effect abstract Tamoxifen (TAM), which is an antiestrogenic agent, is widely used during chemotherapy of breast, pan- creas, brain and liver cancers. In this study, TAM and model membrane interactions in the form of mul- tilamellar vesicles (MLVs) were studied for lipids containing different acyl chain length and different charge status as a function of different TAM (1, 6, 9 and 15 mol%) concentrations. Zwitterionic lipids namely dipalmitoyl phosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC) lipids were used to see the acyl chain length effect and anionic dipalmitoyl phosphtidylglycerol (DPPG) lipid was used to see the charge effect. For this purpose Fourier transform-infrared (FTIR) spectroscopic and differential scanning calorimetric (DSC) techniques have been conducted. For zwitterionic lipid, concen- tration dependent different action of TAM was observed both in the gel and liquid crystalline phases by significantly increasing the lipid order and decreasing the dynamics for 1 mol% TAM, while decreasing the lipid order and increasing the dynamics of the lipids for higher concentrations (6, 9 and 15 mol%). However, different than neutral lipids, the dynamics and disorder of DPPG liposome increased for all TAM concentrations. The interactions between TAM and head group of multilamellar liposomes was monitored by analyzing the C@O stretching and PO 2 antisymmetric double bond stretching bands. Increasing Tamoxifen concentrations led to a dehydration around these functional groups in the polar part of the lipids. DSC studies showed that for all types of lipids, TAM eliminates the pre-transition, shifts the main phase transition to lower temperatures and broadened the phase transition curve. The results indicate that not the acyl chain length but the charge status of the polar head group induces different effects on lipid membranes order and dynamics. Ó 2013 Elsevier B.V. All rights reserved. 0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molstruc.2013.02.031 Corresponding author. Tel.: +90 2323112301. E-mail address: [email protected] (N. Kazanci). Journal of Molecular Structure 1040 (2013) 75–82 Contents lists available at SciVerse ScienceDirect Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc

Acyl chain length and charge effect on Tamoxifen–lipid model membrane interactions

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Journal of Molecular Structure 1040 (2013) 75–82

Contents lists available at SciVerse ScienceDi rect

Journal of Molec ular Stru cture

journal homepage: www.elsevier .com/ locate /molst ruc

Acyl chain length and charge effect on Tamoxifen–lipid model membrane interactions

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

a Department of Physics, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey b Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey

h i g h l i g h t s

" Different lipids were used to examine the ef fect of TAM on acyl chain length and charges of lipids.

" FTIR technique was used for spectral analysis and DSC technique was used for calorimetric analysis.

" This study indicated there are strong interactions between TAM and the lipid membra nes.

0022-2860/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.molstruc.2013.02.031

⇑ Corresponding author. Tel.: +90 2323112301.E-mail address: [email protected] (N. Kaz

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 18 January 2013 Received in revised form 20 February 2013 Accepted 26 February 2013 Available online 6 March 2013

Keywords:TamoxifenFTIRDSCChain length Charge effect

a b s t r a c t

Tamoxifen (TAM), which is an antiestrogenic agent, is widely used during chemotherapy of breast, pan- creas, brain and liver cancers. In this study, TAM and model membrane interactions in the form of mul- tilamellar vesicles (MLVs) were studied for lipids containing different acyl chain length and different charge status as a function of different TAM (1, 6, 9 and 15 mol%) concentrations. Zwitterionic lipids namely dipalm itoyl phosphatidylcholine (DPPC), and dimyristoylphospha tidylcholine (DMPC) lipids were used to see the acyl chain length effect and anionic dipalmitoyl phosphtidylglycer ol (DPPG) lipid was used to see the charge effect. For this purpose Fourier transform-infrared (FTIR) spectroscopic and differential scanning calorimetric (DSC) techniques have been conducted. For zwitterionic lipid, concen- tration dependent different action of TAM was observed both in the gel and liquid crystalline phases by significantly increasing the lipid order and decreasing the dynamics for 1 mol% TAM, while decreasing the lipid order and increasing the dynamics of the lipids for higher concentrations (6, 9 and 15 mol%).However, different than neutral lipids, the dynamics and disorder of DPPG liposome increased for all TAM concentrations. The inter actions between TAM and head group of multilamellar liposomes was monitored by analyzing the C@O stretching and PO �2 antisymmetric double bond stretching bands.Increasing Tamoxifen concentrations led to a dehydration around these functional groups in the polar part of the lipids. DSC studies showed that for all types of lipids, TAM eliminates the pre-transition, shifts the main phase transition to lower temperatures and broadened the phase transition curve. The results indicate that not the acyl chain length but the charge status of the polar head group induces different effects on lipid membranes order and dynamics.

� 2013 Elsevier B.V. All rights reserved.

ll rights reserved.

anci).

76 D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82

1. Introduction

Tam oxif en (TAM) is a non -ster oid al est rog eni c com poun d pre- scr ibed for the man agem ent of bre ast can cer [1]. Expr ess ing est ro- gen rec ept ors and estro gen rec ept or- negat ive cell s as wel l, TAM inh ibit s the grow th of tum or cell s [2–5] . Pre vio us studi es rev eal ed tha t TAM stro ngly inte rac ts wit h lipi ds and pro tein s in bio me m- bra nes [6]. Ind eed , TAM ten ds to trig ger ear ly modi fication s in the mo rphol ogy and str uctu re of bre ast tum or cell memb ran es and this can be rela ted to its ant i prol ifer ativ e pro ces s [7]. The pert urb atio n of the lipi d bila yer str uctur e by TAM may cont ribut e to its mu ltip le me chani sm of ant ican cer acti on [8]. Alt houg h TAM is often reg ard ed as an est rog en anta goni st, sev era l stu dies sugg est som e alte rna tive me chani sm of act ion [9]. Cus tod io et al., usin g sar cop lasm ic reti cu- lum me mbran es and rat liv er mito chond ria , repo rted tha t mult iple cell ula r eff ects of TAM and cyt otox ic effe cts are cons ist entl y rel ated wit h mult iple inte rac tion s at the lev el of the cell memb ran e [10 ,11] .Lux o et al. have use d a micr oor gani sm gro wn und er lab ora tor y-co n- tro lled cond iti ons as a mode l sys tem in ord er to clar ify the mole cul ar me chani sms of TAM and its ant i prol ifer ativ e effe cts [12 ,13] .Pre vio us studi es repo rted that TAM at hig h conce ntr atio ns can acc u- mu late in bio memb ran es [14 ,15] . Exp erim ent al stu dies perfo rmed on arti ficial and bio log ical me mbra nes show that TAM is enr ich ed in lipi d bil aye rs and aff ects me mbra ne phys ical prop erti es [14 ,16,17–20] and the chem ical compo siti on [4,1 2,1 4,18 ,20,21–26]. For exa mpl e Mon teri o et al. repo rted the mole cul ar mec hani sms of acti on of TAM and hyd roxy tam oxi fen on bac teri al pola r lipi ds by usi ng fluoresc ence pol ariz atio n [27 ]. In othe r study [28 ] DM PC lipi ds wer e use d to obs erv e how par titi on of TAM and OH TAM depe nds on me mbran e com pos ition and on drug con cent rati on and Wi sema net al. [17 ] rep ort ed the abi lit y of TAM and the ir com pou nds to dec rea se memb ran e fluidity . They use d mo del me mbra ne sys tem cons ist ing of lipo somes form ed from ox-br ain pho spho lipi ds dis -per sed in aque ous medi um. Cus todio et al. repo rted a dif fere nt eff ect of TAM on neutr al lipi ds (DMPC, DPPC and DSP C) in the gel and the liq uid cry sta llin e pha se. Acc ordi ng to this wor k, TAM sta bil ize s the me mbran e (for low con cent rati on) in the liq uid crys tall ine pha se whi lst a str ong fluidizin g eff ect is obs erv ed in the gel pha se as me asure d by fluores cence pola riza tion of DPH [16 ]. Mor eove r, in our pre viou s stu dies , conc entr atio n dep ende nt diff ere nt acti on of TAM on memb ran e fluidity was repo rted both in the gel and in the liq uid cry sta llin e pha se usin g DPPC mo del memb ran es by Fou rier tran sfo rm- infr are d (FTIR) spec tro scopy [14 ,20 ]. The se stu die s re- port ed tha t TAM des tabi liz es the memb ran e at high conce ntr atio ns,howe ver , it has an oppos ite effec t on memb ran e at low conce ntr a- tion [14 ,20 ]. Diff ere nt than the prev iou s stu dies , in the cur ren t stu dy the effe cts of acy l chai n len gth on stru ctur al para met ers such as lipi dorde r and the stre ngth of hydr ogen bond ing and the eff ect of char ge sta tes of lipid s on Tam oxif en–memb ran e inte rac tion s in term s of both stru ctur al and func tion al par ame ters wer e inv est iga ted . For this purp ose mode l me mbra nes con stit uted by MLVs pre pare d from zwi tter ion ic DPP C, DMPC , and ani oni c DPP G wer e inv est igat ed usin gtwo non inv asive techn iqu es, name ly FTIR and diff ere ntia l sca nni ng calo rim etry (DSC). We sho wed for the first time the com para tive eff ects of TAM on memb ran e dyna mics , memb rane ord er, natu re of hydr oge n bond ing and pha se-t ran siti on beha vio r amo ng dif fere nt pho spho lipi ds whi ch have diff ere nt acy l chai n and char ge grou ps,cont ain ing dif fere nt TAM con cent rati ons.

2. Materials and methods

2.1. TAM, DPPC, DMPC and DPPG were obtained from sigma (St.Louise, Mo) and used without further purification

For infrared measurements , multilamellar MLVs were prepared according to the procedure reported by Severcan et al. [21]. In

order to prepare the liposomes, 5 mg lipid was dissolved in 0.1 ml chloroform, inside an round bottom tube, then the excess chlorofor m was evaporated by nitrogen stream and the remaining solvent in the round bottom tube was spin vacuumed (HETO-spinvac) dried for 2 h. The dry thin films were then hydrated by adding 25 ml of 10 mM phosphat e buffer, pH 7.4. Homogeneous vesicles were formed by vortexing the mixture for 20 min at 20 �C above the main phase transition temperature (Tm) of the lipids. To prepare TAM-containing liposomes, required amount of TAM from stock solution was initially put into the sample tube, excess of organic solvent was evaporated by nitrogen flux and then 5 mg of lipid was added. The similar procedure was then applied for pre- paring TAM containing liposomes.

Twenty microliter of liposomes samples were placed in be- tween CaF 2 windows using a spacer with 12 lm sample thickness.Infrared spectra were obtained using a Perkin–Elmer Spectrum 100 equipped with a DTGS detector. Interferogram s were averaged for 50 scans at 2 cm �1 resolution. The temperature was controlled by aGraseby Specac digital temperature controlle r unit and the sam- ples were incubated for 5 min at each temperature before the scan of the spectrum. The selected temperature s were 25 �C, 41 �C, 55 �Cfor DPPC liposomes, 10 �C, 23 �C, 35 �C for DMPC liposome s and 25 �C, 40 �C, 55 �C for DPPG liposome s. These temperat ures repre- sent the gel phase, ripple phase and the liquid crystalline phase of the lipid membranes , respectively. Membrane samples for each li- pid were prepared five times and their spectra were collected at the mentioned temperature s and buffer substract ion procedure was performed. Buffer spectra were also collected at the same tem- perature s and digitally substracted from the sample spectra col- lected at identical condition s. The substraction procedure is performed till the bulk water signal at 2300 cm �1 is flattened.These substracted spectra were used for detailed and statistical analysis. However , for visual demonstration of the changes, nor- malized average spectra were compared [22,29].

The FTIR spectra were analyzed by using Spectrum v5.0.1 soft- ware (Perkin–Elmer). The vibrational band of CH 2 antisymmetr ic stretchin g mode was sufficiently seperated after water substrac- tion procedure and therefore no band deconvolution or fit routines was used to evaluate their bandwid ths for relative measureme nts in this model membrane study [23,30–33]. The band positions were evaluated accordin g to the center of weight, and bandwidth was measured at 0.75 � peak height position. Normalizati on was applied to the spectra only for visual demonstrat ion of the struc- tural changes by using Perkin–Elmer software. Mann–Whitney Utest was applied to determine statistical significance of the spectral paramete rs of the drug containing groups in comparison to the pure lipid membran es. Significant difference was statistically taken into account at the level of P 6 0.05. Final results which were sta- tistically significant were shown by an asterisk on the figures.

The lipid mixtures for the DSC measure ments were prepared accordin g to the same procedure as for the infrared study; how- ever, this time, thin films were obtained by hydrating 2 mg of phospho lipids with 50 ll phosphate buffer. TA Q 100 DSC instru- ment was used with heating rate of 1 �C/min.

3. Results

3.1. DSC studies

The effect of TAM on membrane phase behavior was determined by DSC technique. Fig. 1a–c demonstrat e DSC curves for DMPC,DPPC and DPPG liposomes respectively, in the absence and pres- ence of different TAM concentrations . The characteri stic pre-transi- tion and sharp main transition are seen respectively for DPPC liposome s at �35.20 �C and �41.20 �C [21,24], for DMPC liposome at �13,40 �C and �22.80 �C [34,35], and for DPPG liposomes at

D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82 77

�33 �C and �40 �C, which are in agreement with previous studies [25,35]. Our DSC study revealed that for all lipid membranes with the addition of TAM, the main phase transition temperature de- creased and the pre-transition temperature disappeared. Further- more, the main DSC curves decreased in intensity and broadened.

3.2. FTIR studies

FTIR studies carried out to monitor the effect of acyl chain length on the Tamoxifen–membrane interactions using two different lipid membranes constituted from namely DPPC and DMPC. These are neutral lipids with different acyl chain length. The charge effect on the TAM-membran e interaction was studied by comparing the results of DPPC and DPPG MLVs. DPPG has the same acyl chain length with DPPC, but has negative charge. FTIR spectra of lipo- somes were analyzed in the absence and presence of four different TAM concentrations (1 mol%, 6 mol%, 9 mol% and 15 mol%). The CH2 antisymm etric and symmetric stretching vibrations which are located at 2920 and 2850 cm �1, respectively and the C@Ostretching mode located around 1730 cm �1 which reflects the ester group vibrations and PO �2 antisymm etric double bond stretching mode in the 1200–1260 cm �1 region, were taken into consider- ation. Order–disorder state of the system was investigated by wavenumber analysis of the CH 2 stretchin g modes [21,26,36,37].The dynamic state of system was studied by monitoring the band- width of the CH 2 antisymmetric stretching band [21,24,38]. The wavenumber variation of the C@O and PO �2 antisymmetr ic double bond stretchin g bands gives informat ion about the strength of hydrogen bonding around these functiona l group [21,22,25].

Fig. 2 shows the average FTIR spectra of DMPC, DPPC and DPPG MLVs in the absence and presence of TAM in their liquid crystalline phases. Each spectrum was normalized with respect to the CH 2antisymmetr ic stretching band at 2920 cm �1 to give a visual dem- onstration of the TAM-induced variations. It is clearly seen from the figures that TAM induces changes in the wavenumber and the bandwidth values.

Fig. 3a and b show TAM dependence of the wavenumber of the CH2 antisymm etric stretching modes of DMPC, DPPC and DPPG MLVs, respectively in their gel phase and liquid crystalline phase.The results revealed that TAM shows opposite effect on lipid order at low and high concentratio ns for DPPC and DMPC MLVs. The wavenumber values of CH 2 antisymmetr ic stretching mode de- creased with 1 mol% TAM addition implying an increase in the or- der, whilst, the wavenumber increased higher TAM concentrations ,implying a decrease in the order of DPPC and DMPC MLVs. How- ever the wavenumber values in DPPG membranes increased for all TAM concentr ations in the both phases, indicating a decrease in the lipid membran es order. The same results were also observed for the CH 2 symmetr ic stretching modes (not shown).

These results indicate that acyl chain length difference between DMPC and DPPC MLVs has no effect on the order–disorder state of the model membranes . In contrast, for DPPG, TAM induces a differ- ent effect at low concentratio n (1 mol%) with a decrease of the acyl chain order.

The changes in the bandwidth values or membran e fluidity as afunction of concentratio n of the DMPC, DPPC and DPPG liposomes for the CH 2 antisymmetric stretching modes in their gel and liquid crystalline phase are given in Fig. 4a and b. The bandwidth values of CH 2 antisymmetr ic stretching mode significantly decreased at 1 mol% but significantly increased as the TAM concentratio n in- creased for DPPC and DMPC liposome s, which shows that the dynamics of DPPC and DMPC liposome s decreased at low concen- tration while an increase in dynamics observed at higher TAM con- centrations. However , the dynamics of DPPG liposome increased for all TAM concentratio ns. Similar results were also observed for the CH 2 symmetric stretching bands (not shown).

The interaction between TAM with glycerol backbone near the head group of DMPC, DPPC and DPPG multilam ellar liposome swas monitored by analyzing C@O stretching modes. Fig. 5a and bshow the concentration- dependent changes in the wavenumber of C@O stretchin g modes in their gel and liquid crystalline phases respectively . It can be seen from the figures that the wavenumber values of samples increased as the TAM concentration increased,which implies that increasing TAM concentration caused dehydra- tion around these functional groups in the polar part of the lipid membran es [23] and this behavior is not affected from acyl chain length and the charge status of the membrane.

The polar head groups of phospho lipids were monitored using PO�2 antisymm etric double bond stretchin g band [38]. The wave- number of this band indicates the strength of hydrogen bonding between phosphate group and hydrogen atoms of water or biolog- ical macromolec ules [39]. Fig. 6a and b show TAM-dependen ce of the wavenumber of the PO �2 antisymmetr ic double bond stretching bands of DMPC, DPPC and DPPG liposome s respectively in their gel and liquid crystalline phases. In the current study, the wavenum -ber values shifted to higher values as TAM concentration increased.This indicates an increase in dehydrati on.

4. Discussion

TAM is the endocrin e treatment of choice for advanced breast cancer [40]. TAM can be used in the treatment of each phases of breast cancer due to its antiestrogeni c effects on breast tissue. In spite of beneficial estrogen-agon istic influences of TAM on bone density and serum lipids, there are several deleterious effects of it on endometr ium [41–43]. Besides these clinical data, under- standing of the acting mechanism of TAM is very important for the therapy of the diseases and determination of the suitable dose of it. Therefore several research have been conducted so far on TAM-bio logical and model membrane interactio ns [10–26].

Biologica l membran es are complex structures containing a vari- ety of phospholipids molecules that differ with respect to the structure of the polar head group and hydrocarbo n chain length,to accomplis h optimum lipid and protein packing into the bilayer and to guarantee membrane stability. Because of the complexi ty of biomembranes , investiga tions are concentrated on model mem- branes. Lipid order and fluidity are significant parameters for avail- able functions of biologica l membran es in terms of impacts of cellular processes and the disease. Liposomes are very important formulat ions in drug delivery. Physical consistence of liposome shas been found to be a function of acyl chain length of lipids [44]. Phospholipids with high transition temperature and long chain length are more stable than those with low transition tem- perature and short chain length [45,46]. It is known that acyl chains and polar head groups of phospho lipids have a significantinfluence on lipid bilayers. Therefore, we studied molecular inter- actions between TAM and neutral and charged lipid membran es based on acyl chain length and charge of phospho lipids.

In the present study, we, for the first time, tested the effect of TAM on different lipid membran es which have different chain length and charge status. Lipids that are used in our study namely DPPC (with 16 carbons) and DMPC (with 14 carbons) exhibit the same characterist ics in many respects having two saturated acyl chains, having a choline head group, and being neutral [47]. Fur- thermore , their order parameter profiles are essentially similar to each other [48]. DPPG has the same acyl chain length with DPPC,but has negative charge. Our results reveal that the interaction of TAM with lipid MLVs depends markedly on the charge status of their phosphat e head groups. At neutral pH, zwitterionic DPPC and DMPC head groups are polar but neutral [49]. DPPG differs from DPPC only in the substitution of a glycerol moiety for choline

Fig. 1. (a) DSC curves of DMPC liposomes in the absence and presence of different concentration of TAM. (b) DSC curves of DPPC liposomes in the absence and presence of different concentration of TAM. (c) DSC curves of DPPG liposomes in the absence and presence of different concentration of TAM.

78 D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82

Fig. 2. FTIR spectra of: (a) DMPC (at 35 �C), (b) DPPC (at 55 �C) and (c) DPPG (at 55 �C) MLVs in the absence and presence of 15 mol% TAM in the CAH stretching region.

D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82 79

in the head group. At neutral pH, the DPPG head group has nega- tive charge. Although there are various differences on the physical properties of the interactions of lipid membranes having different acyl chain length with drugs [35,50–52], in the current study no significant difference was observed due to lipid acyl chain length.However, it is observed that charged status of polar head groups of liposomes have significant effects on order and dynamics of lipid systems compared to the neutral counterpart. Interaction of an- other anticancer agent derived from 1-aryl-3-(2-chloroethyl) ureas (CEUs) with neutral and charged counterparts (DMPC and DMPG)were previously studied by Saint-Laurent et al. [53]. The tempera- ture profiles of the CH 2 symmetric stretching mode indicated that the presence of CEU in DMPG membranes causes a smaller increase of the wavenumber compare d to the same DMPC/CEU systems.

They also explained this case by the difference in the lipid polar head group which can modify the degree of incorporation of CEU molecule s in the hydrophobic lipid region. In another study Bensikadd our et al. [49] compared the binding of ciprofloxacin,synthetic chemothera py antibiotics on DPPC and DPPG liposome s(or mixtures of phospholipid s [DOPC:DPPC ], and [DOPC:DPPG])using quasi-elasti c light scattering and steady-state fluorescenceanisotropy. The results of them indicated that the interactions of ciprofloxacin with lipids depend significantly on the nature of their phosphat e head groups and that ciprofloxacin interacts preferen- tially with anionic lipid compounds, like phosphat idylglycerol,present at a high content in these membran es.

The concentratio n dependent variations in bandwidths reflectchanges in the mobility of the acly chains. The increase in

Fig. 3. Tamoxifen concentration dependence of the wavenumber of the CH 2 anti symmetric stretching mode for: (a) DMPC liposomes at 10 �C, DPPC liposomes at 25 �C, DPPG liposomes at 25 �C (in their gel phases), (b) DMPC liposomes at 35 �C,DPPC liposomes at 55 �C, DPPG liposomes at 55 �C (in their liquid crystalline phase)(�) P < 0.05.

Fig. 4. Tamoxifen concentration dependence of the bandwidth of the CH 2 anti symmetric stretching mode for: (a) DMPC liposomes at 10 �C, DPPC liposomes at 25 �C, DPPG liposomes at 25 �C (in their gel phases). (b) DMPC liposomes at 35 �C,DPPC liposomes at 55 �C, DPPG liposomes at 55 �C (in their liquid crystalline phase)(�) P < 0.05.

80 D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82

bandwidth is associate d to an increase of lateral mobility of the li- pid acyl chains while the wavenum ber shift is related to the intro- duction of gauche conformers in the hydrocarbon chains [21,22,26,54]. In other words, membran e fluidity increases by an increase in the rates of lateral diffusion of lipid molecules. Similar to our studies, several studies have displayed a correlation be- tween the different antitumor agents and membran e fluidity[14,20,55,56 ]. Since amphiphilic drugs, such as TAM can easily interact with membrane lipids directly, it is of the interest to inves- tigate the mechanism of their interaction with lipid membranes in order to design more potent and useful drugs. Similar to our find-ings on neutral lipids, Severcan et al. previously reported the oppo- site effect of TAM on membrane fluidity of DPPC liposomes at low (1 mol%) and high (30 mol%) TAM concentr ations [14,20]. Mem- brane fluidity is a typical characteri stic of cellular membranes and based on their lipid and protein components. Plasma mem- brane fluidity of cancerous cells is greatly associate d with their likelihood of malignancy [57]. There is considerable evidence that any variations in plasma membrane fluidity of cancer cells are clo- sely related to their capacity to metastases. Fluidity is an important characterist ic of biologic membran es at molecular level. Changes in membrane fluidity may play a role in reregulation of membrane properties [57]. Clarke et al. [58] investigated the fluidity of estro- gen receptor (ER) negative human breast cancer MDA-MB -436 cells and ER positive human breast cancer MCF7 cells by using TAM. They reported that TAM reduced fluidity of cellular mem- branes but an increase was occurred in membrane fluidity with high dose of TAM. Another clinic study on TAM reported that its low doses decreased side effects as compared to high doses

without changing pharmacolo gic activity of chemotherapy in breast, adding that TAM in low doses could act as an antiestrogen and reduced cellular proliferation and therefore further benefittedwomen with breast cancer [5]. Since we have also found stabilizing effect of TAM at low concentration, this drug can be successfu lly used at only low doses to decrease cancer-indu ced membrane destabili zation [17,57,59].

Although zwitterionic lipids are the major lipid components of eukaryot ic cell membranes , anionic phospholipid s are also present in considerable quantities and are known to be essential structural and functional components of such membranes [60,61]. The non- zero net charge of PGs makes them different from generally stud- ied zwitterio nic lipids. Because of this, PGs have been suggested as one of the possible components in immune liposome s, generic drug delivery vehicles [62]. Anionic phospholipids like phosphati- dylglycerol possess a larger head group area and are less densely packed than zwitterionic phospholipids like phosphatidylch olines [63]. DPPG membranes have charged head groups that would be expected to repel each other, leading to decreased chain–chaininteractio ns. Because of this, net van der Waals forces between the hydrocarbo n chains reduce. This makes the mono layers less ri- gid and more compressib le. Thus, we can say that DPPC mem- branes are less fluid or more stable than DPPG membran es although both contain two hydrocarbo n chains with 16 carbon atoms. Such a stability differenc e may explain why TAM has more profound influence on the DPPG membranes than DPPC and DMPC membran es. Previous studies showed that TAM localized in hydro- phobic part of the membrane in lipid bilayers, by affecting both its physical [14,16,17–20] and chemical propertie s [4,12]. Therefore,TAM may be located in the hydrophobic part of less packed DPPG

Fig. 5. Tamoxifen concentration dependence of the wavenumber of the C@Ostretching mode for: (a) DMPC liposomes at 10 �C, DPPC liposomes at 25 �C, DPPG liposomes at 25 �C (in their gel phases), (b) DMPC liposomes at 35 �C, DPPC liposomes at 55 �C, DPPG liposomes at 55 �C (in their liquid crystalline phase) (�)P < 0.05.

Fig. 6. Tamoxifen concentration dependence of the wavenumber of the PO �2 double bond stretching mode for: (a) DMPC liposomes at 10 �C, DPPC liposomes at 25 �C,DPPG liposomes at 25 �C (in their gel phases). (b) DMPC liposomes at 35 �C, DPPC liposomes at 55 �C, DPPG liposomes at 55 �C (in their liquid crystalline phase) (�)P < 0.05.

D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82 81

membranes and thus it may have a more significant effect on the order of chains which could explain the stability difference be- tween DPPC, DMPC and DPPG membran es. Their comparison of FTIR spectra and DSC curves shows similar effects on membran eproperties. In this study, our results show that TAM-neutral lipid MLVs have not been affected from the lipid acyl chain length.

Sade et al. [29] recently investigated interactions of neutral 1,2- distearoyl-sn-glycero-3-pho sphatidylch oline DSPC lipids with celecoxib, a non-steroid anti-inflammatory agent reported that the usage of celecoxib is related with the activities which prevent the harmful effects of chemicals in breast and also its inhibitory activity influences a variety of cancer-relat ed pathways. Their study is consistent with ours in that when celecoxib concentratio nincreased, pre-transition temperature disappea red and main tran- sition temperature shifted to lower values. Similar to our study,their results also showed the opposite effect on the molecular or- der of membrane for different celecoxib concentratio ns. Similar to our studies they reported an increase on the order of the system at low concentrations while a decrease on the order of drug at high concentratio ns. The broadening of peak profile and lowering of transition temperat ure demonst rate that both size and packing of bilayers are modified, and the system is disordered [22]. Balasubr- amanian and Straubinger [64] investigated the conformation of paclitaxel which is an anticance r drug and its interaction with DPPC liposomes. Supporting our studies, they reported that paclit- axel, was found to partition into the bilayer, disturbing the hydro- carbon chain conformation and inducing a broadeni ng of the DPPC phase transition. They also found that the position of the drug in the bilayer depends on drug concentration and incorporation of

paclitaxel into the DPPC bilayer affects other physical propertie sof the bilayer such as the lipid order parameter.

We found a significant increase in the wavenumber of C@O and PO�2 groups for all MLVs in the gel and the liquid crystalline phases.According to the empirical rules, a decrease in the frequency values shows either the strengthening of existing hydrogen bonding or formatio n of new hydrogen bonding between the components [21,65]. In the current study, the frequenc y shifts to higher values for all of the samples containing TAM, indicating that there is no evidence of hydrogen bonding. TAM decrease s the strength of hydrogen bonding in the interfacial region. This implies that there are free carbonyl groups in the system. It is possible that TAM dis- places some H2O molecules from the interfacial region resulting in an increase in the number of free carbonyl groups.

5. Conclusion s

Cancer is caused by uncontrolled growth and spreading of abnormal cells. It can seriously threaten human health and is aleading cause of death. The efficacy of an anticancer drug is mod- ulated by its molecular interactio n with the cancer cells. Therefore it is very important to better understand drug-mem brane interac- tion for the effective and correct use of the drug since anticancer drugs can also affect healthy cells.

The present study has for the first time investiga ted whether the effect of acyl chain length and charge state of the lipid head group would have any impacts on the TAM-model membrane interactio ns using DPPC, DMPC and DPPG liposome s. Our results indicated that there is strong interactions between TAM and the

82 D. Bilge et al. / Journal of Molecular Structure 1040 (2013) 75–82

lipid membranes and these interactions are TAM-concentr ation dependent.

Acknowled gement

This work was supported by Ege Universit y Research Fund 2007 Fen 039.

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