Accepted Manuscript

Concentration-dependent effect of melatonin on DSPC membrane

Ipek Sahin, Duygu Bilge, Nadide Kazanci, Feride Severcan

PII: S0022-2860(13)00749-7

DOI: http://dx.doi.org/10.1016/j.molstruc.2013.08.060

Reference: MOLSTR 19995

To appear in: Journal of Molecular Structure

Received Date: 17 July 2013

Revised Date: 29 August 2013

Accepted Date: 30 August 2013

Please cite this article as: I. Sahin, D. Bilge, N. Kazanci, F. Severcan, Concentration-dependent effect of melatonin

on DSPC membrane, Journal of Molecular Structure (2013), doi: http://dx.doi.org/10.1016/j.molstruc.2013.08.060

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Concentration-dependent effect of melatonin on DSPC membrane

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

aDepartment of Physics, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey bDepartment of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey Corresponding Author: nadide.kazanci@ege.edu.tr Abstract

The concentration-induced effects of melatonin on distearoyl phosphatidylcholine

(DSPC) model membranes were investigated by using two different non-invasive techniques,

namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry

(DSC). An investigation of the C-H, C=O and PO2- double bond stretching mode in FTIR

spectra and DSC studies reveals that the inclusion of melatonin changes the physical

properties of the DSPC multilamellar liposomes (MLVs) by shifting the main phase transition

to lower temperatures, abolishing the pretransition, ordering the system in the gel phase and

slightly disordering the system in the liquid crystalline phase, increasing the dynamics both in

the gel phase and liquid crystalline phases. Melatonin also causes strong hydrogen bonding

between C=O and PO2- groups of lipids and the water molecules around.

Keywords: Melatonin; Distearoyl phosphatidylcholine; MLVs; Fourier transform infrared;

Differential scanning calorimetry.

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1. Introduction

Melatonin (N-acetyl-5-methoxytryptamine), a hormone principally produced and

released by the pineal gland under influence of the environmental light/dark cycle is a

ubiquitously acting direct free radical scavenger [1]. It participates in several important

physiological functions, including detoxifying the devastatingly reactive hydroxyl radical [2],

biologic regulation of circadian rhythms, sleep, mood, reproduction and

neuroimmunomodulation [3]. Moreover, melatonin has also been shown to have diverse other

functions, e.g. influence of immune function as well as antiproliferative and antioxidative

actions [4-7]. It also lowers age-related oxidative damage in the Alzheimer’s disease [8]

Parkinson’s disease [9] and central nervous system [10].

Several mechanisms are combined to explain the protective efficiency of melatonin:

(a) a direct antioxidant action inhibiting the lipid peroxidation, (b) inducing changes on lipidic

bilayers fluidity, (c) protecting intracellular components against peroxidative damage [11].

However, the exact action mechanisms underlying its effect is not well understood and thus

several studies are in progress. One of the mechanisms responsible for the valuable effects of

melatonin could be its membrane action. Despite its potential importance, limited number of

studies are available in the literature about the interaction at molecular level of melatonin with

membranes [11–13]. These electron spin resonance (ESR), fluorescence, differential scanning

calorimetry (DSC), and ultraviolet (UV) studies used rat microsomal membranes and

phosphatidylcholine model membranes in the form of multilamellar, unilamellar, and reversed

micelles. They mainly report the effect of melatonin on membrane dynamics, which are not

always consistent with each other.

Understanding the mechanism of interactions between drugs and cell membranes at

the molecular level has critical importance in a wide variety of scientific disciplines. Many

drugs, displaying a great variety of applications and with various structures, can cross or bind

to lipid membranes and possibly alter the physical properties of that membrane. Biological

membranes are extremely complex supramolecular liquid-crystalline structures principally

composed of phospholipids [14]; thus, in order to better understand the function of melatonin

at molecular level, it is important to study its interaction with membrane components and

specifically with phospholipids. Owing to their structural similarity with biological

membranes, phospholipid liposomes have been used as model membranes for a long time.

Especially, they have been made advantage of investigations of drug/membrane interaction

[15–19].

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In our previous studies, we investigated the effect of melatonin on lipid phase

transition, lipid order and hydration states of the head group of zwitterionic dipalmitoyl

phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG), in the form of

multilamellar vesicles (MLVs) as a function of temperature and different melatonin

concentrations [20,21]. In the current study, the effect of different concentrations of melatonin

on model membranes composed of another zwitterionic lipid, 1,2-distearoyl-sn-glycero-3-

phosphocholine (DSPC) MLVs, has been investigated by using two different noninvasive

techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning

calorimetry (DSC). These techniques have been widely used in model membrane studies [20,

22,23] and allowed us to obtain detailed information about the concentration dependent

effects of melatonin in two different membrane phases not only on membrane dynamics, but

also on structural parameters, such as lipid phase-transition profile, membrane acyl chain

order, and hydration status of the head group and glycerol backbone regions, which, to the

best of our knowledge, has not been reported previously.

Phospholipids are differentiated from each other by headgroup, chain length, and

unsaturation. It is obvious that the structural differences of the lipids can have important

effects on the drug/membrane interactions. DPPC used in our previous study [20], and DSPC

are both zwitterionic phospholipids with the same polar headgroup and they differ from each

other only in the length of their hydrophobic chain moiety. These phospholipids also are fully

saturated lipids, and they all exhibit many different physical and chemical properties. In this

study, we conducted an intensive investigation on the molecular interactions between

melatonin and lipid by applying liposome as model cell membranes. For this study the

phospholipid distearoyl phosphatidylcholine (DSPC) was considered for liposome

preparations.

2. Materials and methods

Melatonin and DSPC were purchased from Sigma (St.Louis, MO, USA) and used

without further purification. For the infrared measurements, pure phospholipid MLVs were

prepared according to the procedure, reported by Severcan et al. [20]. To prepare DSPC

MLVs, 5 mg of phospholipid was dissolved in chloroform in a round-bottom flask. A dried

lipid film was obtained by evaporating it with a nitrogen flux and then pumping it for at least

2 h under vacuum by using Heto spin vac. The film was hydrated by adding 25 μl of 10 mM

phosphate buffer, pH 7.4. Liposomes were formed by vortexing the mixture at a temperature

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above the gel-to-fluid phase transition for 20 min. In order to prepare melatonin containing

liposomes, appropriate amount of melatonin was taken from the stock solution, in which

melatonin was dissolved in ethanol, and put in a round-bottom flask. The excess ethanol was

evaporated by nitrogen stream and then 5 mg of phospholipid was added and dissolved in the

same round-bottom flask by chloroform. The same procedure for the preparation of pure

DSPC liposomes was then followed. Sample suspensions of 20 μl were placed between CaF2

windows with the cell thickness of 12 μm. Infrared spectra were obtained using a Spectrum

100 (Perkin-Elmer Inc., Norwalk, CT, USA) equipped with a deuterated triglycine sulfate

(DTGS) detector. The instrument was under continuous dry air purge to eliminate

atmospheric water vapor. Interferograms were averaged for 50 scans at 2 cm-1 resolution.

Temperature was regulated by a Graseby Specac (Kent, UK) digital temperature controller

unit. The samples were incubated for 5 min at each temperature before data acquisition. The

selected temperatures were 40 ºC and 65 ºC for DSPC liposomes. These temperatures

represent the gel phase and the liquid crystalline phase of the lipid membranes, respectively.

Membrane samples were prepared five times and their spectra were collected at the mentioned

temperatures and buffer substraction procedure was performed. Buffer spectra were also

collected at the same temperatures and digitally substracted from the sample spectra collected

at identical conditions. 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, normalized average spectra were

compared [24]. The FTIR spectra were analyzed by using Spectrum v5.0.1 software (Perkin-

Elmer). The vibrational band of CH2 antisymmetric stretching mode was sufficiently

separated after water substraction procedure and therefore no band deconvolution or fit

routines was used to evaluate their bandwidths for relative measurements in this model

membrane study [20,21]. The band positions were evaluated according to the center of

weight, and bandwidth was measured at 0,75x peak height position. Normalization was

applied to the spectra only for visual demonstration of the structural changes by using Perkin

Elmer software. Mann-Whitney U test was applied to determine statistical significance of the

spectral parameters of the drug containing groups in comparison to the pure lipid membrane.

Significant difference was statistically taken into account at the level of P≤0.05. Final results

which were statistically significant were shown by an asterisk on the figures.

The lipid mixture for the DSC measurements were prepared according to the same

procedure as for the infrared study; however, this time, thin films were obtained by hydrating

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2 mg of phospholipid with 50 μl phosphate buffer. The samples were scanned over a

temperature range of 30–65°C. TA Q 100 DSC (TA Instruments Inc., New Castle, Delaware,

USA) instrument was used with a heating rate of 1 ºC/min [20,24].

3. Results

In the current study DSPC MLVs, both pure and containing different concentrations of

melatonin varied from 1 to 15 mol%, were investigated by FTIR spectroscopy, and DSC. In

the analysis of the FTIR spectra, CH2 antisymmetric, C=O stretching, and PO2- double

stretching bands were studied. The C-H stretching modes at 2800–3000 cm-1, the C=O

stretching mode at 1735 cm-1, and the PO2- double bond stretching mode at 1220–1240 cm-1

were considered.

3.1. DSC Studies Calorimetric investigation of DSPC liposomes with or without melatonin was carried

out in a temperature range of 30–65 ºC. The corresponding heating flow as a function of

temperature curve is shown in Figure 1. The thermogram for pure DSPC liposomes shows

two endothermic peaks corresponding to the pre- and main phase transitions (Tm) occurring at

51.2 and 54.5°C, respectively, which is in agreement with previous studies [25,26]. As seen

from Figure 1, increasing melatonin concentrations eliminates the pretransition peak and

lowers the main phase-transition temperature.

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Figure 1. DSC curves of DSPC liposomes in the absence and presence of different concentrations of melatonin. 3.2. FTIR Studies FTIR spectroscopy was used to determine the changes in membrane structure and

dynamics by analyzing the wavenumber and bandwidth of different vibrational modes, which

represent the acyl chains, head group, and interfacial region of lipid molecules [20,22,27].

Figure 2a displays the wavenumber variation of the CH2 antisymmetric stretching bands of

DSPC liposomes in the absence and presence of different melatonin concentrations in the gel

phase. Figure 2a gives information also about the order-disorder state (i.e., acyl chain

flexibility) of the membrane [20,24]. The wavenumber of the CH2 stretching bands of acyl

chains depend on the average trans/gauche isomerization in the system, and shifts to higher

wave numbers correspond to an increase in the number of gauche conformers, which implies

a more disordered state [28].

Figure 2. Melatonin concentration dependence of the wavenumber of the CH2 anti symmetric stretching

mode for DSPC liposomes at a) 40 ºC (in the gel phase) b) 65 ºC (in the liquid crystalline phase) (*) P<0.05.

As seen from the Figure 2a, at temperature ranges corresponding to the gel phase (<

Tm), the addition of melatonin results in a decrease in the wavenumber, which indicates an

increase in the number of trans conformers. The increase in the number of trans conformers

implies an increase in the order of the bilayer [23,27-29,]. In contrast, as seen from Figure 2b,

in the liquid crystalline phase (>Tm), the incorporation of different concentrations of

melatonin into the phospholipid system slightly shifts the wavenumber to higher values,

which indicates an increase in the number of gauche conformers. The increase in the number

of gauche conformers implies a decrease in the order of bilayer in liquid crystalline phases

[23,30].

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Information about the dynamics of membrane systems can be obtained by analyzing

the variations in the bandwidth of the CH2 stretching modes, since bandwidth reflects the

changes in the mobility of the acyl chains. An increase in the bandwidth is an indication of an

increase in the dynamics of the membrane system [24,31-33 ].

Figure 3. Melatonin concentration dependence of the bandwidth of the CH2 anti symmetric stretching mode

for DSPC liposomes at a) 40 ºC (in the gel phase) b) 65 ºC (in the liquid crystalline phase) (*) P<0.05.

Figure 3 shows the bandwidth variation of the CH2 antisymmetric stretching bands of

DSPC MLVs with varying melatonin concentrations in the gel and liquid crystalline phases.

Bandwidth was measured at 0.75x peak height position. Qualitatively similar results were also

obtained at 0.50x peak height position (not shown). It is evident from Figure 3 that the

bandwidth increases with the addition of melatonin both in the gel and liquid crystalline

phases, and thus, melatonin increases the dynamics of the membrane.

In order to examine the interaction of melatonin with glycerol backbone near the head

group of phospholipids in interfacial region, the C=O stretching band was analyzed [20,29].

The wavenumber variation of this band is shown in Figure 4. According to the empirical

rules, a decrease in the wavenumber implies either the strengthening of existing hydrogen

bonding or formation of new hydrogen bonding between the components [20,34]. As seen

from the figures, both in the gel and liquid crystalline phase, the addition of melatonin

produces a decrease in wavenumber, which indicates that melatonin increases hydrogen

bonding around this functional group.

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Figure 4. Melatonin concentration dependence of the wavenumber of the C=O stretching mode for DSPC

liposomes at a) 40 ºC (in the gel phase) b) 65 ºC (in the liquid crystalline phase) (*) P<0.05.

The interaction between melatonin and head group of DSPC liposomes was monitored

by PO2- double bond stretching mode which is located at 1230 cm-1. Figure 5a and 5b show

variation of the wavenumber of the PO2- double bond stretching mode for DSPC liposomes in

the absence and presence of different concentrations of melatonin in the gel and liquid

crystalline phases, respectively. As seen from the figure, the wavenumber of this band also

shifts to lower values with the addition of different concentrations of melatonin into the DSPC

MLVs, which indicates hydrogen bonding in between phosphate group of DSPC and

melatonin or water molecules [29].

Figure 5. Melatonin concentration dependence of the wavenumber of the PO2- double bond stretching mode for

DSPC liposomes at a) 40 ºC (in the gel phase) b) 65 ºC (in the liquid crystalline phase) (*) P<0.05.

4. Discussion

The interactions between biological cell membranes and drugs at molecular level have

a constitutive effect on the pharmacokinetics of the drugs. They specify partitioning,

9

orientation, and conformation of the drugs in the membranes and hence play an important role

in transport, distribution, accumulation, and finally efficacy of the drugs [35-37]. These

interactions can also alter the physicochemical properties and thus the functioning of the

membranes. Melatonin is one of the most important molecule among the endogenous

molecules that could change membrane properties, because it penetrates all physiologic

barriers [11,38,39]. Moreover, melatonin and its metabolites have antioxidant properties that

prevent damage induced by reactive species through several mechanisms, principally as a

scavenger of these oxidative agents [40–46].

Interaction of melatonin with DPPC and DPPG MLVs has been studied in detail,

previously by our group, as a function of temperature and melatonin concentrations [20,21].

In the current study, we investigated the effect of melatonin on lipid phase transition, lipid

order, and hydration states of the head group of DSPC as a function of melatonin

concentrations varying from 1 to 15 mol%. In this study two non-perturbing techniques,

namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry

(DSC) were used. DSC is a thermodynamic technique convenient for studying phase

transitions of membrane bilayers with and without inserted drug molecules. It has been widely

used to investigate the thermal changes caused by the incorporation of the drugs into the

membrane bilayers. FTIR spectroscopy is based on monitoring the energy absorbed between

vibrational energy levels of different functional groups belonging to macromolecules in

biological systems. For this reason, it gives a spectrum unique to the system where the bands

assigned to different functional groups can be monitored simultaneously. The change in the

frequencies of the infrared bands in the membrane, owing to drug administration, bring out

relative structural information, such as lipid acyl chain flexibility and nature of the chemical

bonds. In the present study, in the interpretation of the results of FTIR spectra, we were

particularly careful in distinguishing between structural parameters describing molecular

order and motion parameters such as bandwidth describing molecular dynamics as suggested

by others [47]. We investigated effects of melatonin on hydrophilic portion of the membrane

by analyzing C = O stretching band and PO2- double bond stretching mode on hydrophobic

portion of the membrane by analyzing C-H stretching region.

The most largely studied phospholipids are the phosphatidylcholines (PCs) [48,49]

due to their widespread inclusion in cell membranes as a structural component. They are

major components of biological membranes and other biological lipid-containing structures

and fluids. The PCs are generally the major zwitterionic phospholipid classes, and found in

eucaryotic plasma membranes. For example, a saturated diacyl phosphatidylcholine, DPPC

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used in our previous study [20], is the major component of pulmonary surfactant, where it

exists as amonolayer and undergoes rapid changes from liquid-expanded to liquid-condensed

states with each respiratory cycle. The PCs with different hydrocarbon chain length (e.g., C16

and C18) are of different surface properties and exhibit an increase in the cooperativity of their

phase transition as a function of chain length. Each has its effect on the properties of the

membrane. Saturated PCs, especially DPPC and DSPC, are frequently incorporated into

liposomes to be used as model membranes and as potential drug delivery systems. DSPC used

in this study belongs to a family of saturated, symmetric, dialkylphosphocholines (PCs)

having alkyl chain lengths of 18 carbon atoms (distearoyl-PC or DSPC) . In this study we

aimed to contribute to a more thorough understanding of the effect of melatonin on the major

components of biological lipid assemblies by systematically investigating the interaction of

melatonin with model lipid assemblies composed of phosphatidylcholine. We have extended

our previous investigations [20,21] to the interactions of melatonin with DSPC which has

different chain length as compared with DPPC.

Our DSC and FTIR data reveal that melatonin eliminates the pretransition temperature

and shifts the main phase-transition temperature (Tm) of DSPC membranes gradually to lower

temperatures as drug concentration is increased. Previously Saija et al. [11], Severcan et al.

[20] and Sahin et al. [21] also reported a decrease in Tm in the presence of melatonin for

DMPC, DPPC and DPPG MLVs respectively, which have shorter acyl chains than that of

DSPC.

The analysis of the wavenumber of the CH2 antisymmetric stretching mode in FTIR

spectra showed that melatonin decreases acyl chain flexibility, i.e., increases order of DSPC

membranes in the gel phase and slightly decreases in the liquid crystalline phase (Fig. 2a and

2b). The same result in the gel phase was observed in our previous study where we

investigated interactions of melatonin with DPPC MLVs [20]. The increase in the order

means an increase in the number of trans conformers on the acyl chains of membrane which

indicates an increase in the thickness of the membrane.

The bandwidth of the CH2 symmetric and antisymmetric stretching bands gives

information about lipid fluidity in membranes [20,22,24,31,32]. The results of this analysis

revealed that melatonin increases the fluidity of pure DSPC membranes at all concentrations.

The fluidity is expected to influence bilayer permeability property, which is required for

normal and optimal activity of membrane associated structures. There are limited number of

studies in the literature about the effect of melatonin on membrane dynamics whose results

were not always consistent with each other [11,13,20,50,51]. For example, Drolle et al. used

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small-angle neutron diffraction (SAND) from oriented lipid multi-layers, small-angle neutron

scattering (SANS) from unilamellar vesicles experiments and Molecular Dynamics (MD)

simulations to elucidate non-specific interactions of melatonin and cholesterol with 1,2-

dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-snglycero-3-

phosphocholine (DPPC) model membranes and they concluded that melatonin decreases the

thickness of both model membranes by disordering the lipid hydrocarbon chains, thus

increasing membrane fluidity [51]. Another study using DSC technique, also reported a

significant fluidizing effect of melatonin on dimyristoylphosphotidylcholine (DMPC) MLVs

and LUVs, at concentrations of melatonin varied from 1.5 to 18 mol% [11]. These results are

in agreement with our previous DPPC MLVs study [20] and with our present study.

Conflicting these studies including ours, Costa et al. reported a decrease in membrane

dynamics with the addition of melatonin in DMPC [13]. It is wellknown that melatonin is a

powerful endogenous antioxidative molecule protecting cells from oxidative damage and it

has strong anti lipid peroxidation properties [52-55]. It is also known that there is a strong

relevance between lipid peroxidation and fluidity of cell membranes [56,57]. As reported

previously, lipid peroxidation stress decreases membrane fluidity in cellular membranes [58-

60]. In addition to its antioxidative protection, the biophysical effects of melatonin on the

membrane may also contribute to its cell protective properties. The influence of melatonin on

membrane fluidity could be connected with possible mechanism for the observed reduction in

membrane damage. Melatonin’s fluidifying effect may thus reduce the effect of lipid

peroxidation on the membrane dynamics [61-63].

In the present study, melatonin showed an increase in the hydrogen bonding of C=O

and PO2- groups in both the gel and liquid crystalline phases. The electronegative atom is the

nitrogen of the furanose ring of melatonin, which has a partial negative charge. The hydrogen

in the N–H group in this ring has a partial positive charge. This hydrogen can make hydrogen

bonding with the oxygen atoms in the C=O and PO2- functional groups of the lipids. The

possibility of melatonin-induced hydrogen bonding between the oxygen molecules of both

carbonyl and phosphate groups of DSPC and nearby water molecules should also be

considered. In another study wherein interaction of melatonin with DMPC model membrane

was analyzed by UV spectroscopy, Ceraulo et al. reported that melatonin strongly interacts

with polar head region of the membrane and forms strong hydrogen bonds [12]. These results

are consistent with our previous studies where we investigated the interactions of melatonin

with DPPC and DPPG MLVs [20,21].

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The strong hydrogen bonding induced by melatonin at the carbonyl and phosphate

groups in DSPC membranes (Figs. 4 and 5) and the lack of broadening in the phase transition

curve (Fig. 1) suggests that melatonin does not perturb entirely the cooperativity region (C2–

C8) of the acyl chains and melatonin molecule positions itself in the bilayer with a preferential

location in the interfacial region [20,64]. Since melatonin is a small amphiphilic molecule, it

is soluble both in fats and in water [65,66], preferably located at hydrophilic/hydrophobic

interfaces and can readily cross all the anatomic barriers. These specifications make melatonin

able to protect all the cellular structures from oxidant agents.

In conclusion, we found melatonin, when incorporated in the lipid bilayers, to interact

actively with the lipids and to induce changes in their physico-chemical properties. In

addition, a possible location of melatonin in the interfacial region of the membrane has been

proposed. The data presented here clarify, to a certain extent, the molecular interactions of

melatonin with membrane systems and may additionally contribute to a better understanding

of melatonin’s physiologic properties and the development of therapeutic advanced systems.

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Highlights:

►The interaction of melatonin on DSPC MLVs was investigated.

►Spectral and calorimetric analysis techniques were used in this study.

►This study shows there are strong interactions between melatonin and lipid membranes.

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