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Temperature effect on thin lipidfilm elasticity and phase separation:insights from Langmuir monolayer andfluorescence microscopy techniquesZ. Khattariab, M. Maghrabia & T. Al-Abdullahaa Department of Physics, Hashemite University, Zarqa, Jordanb Department of Physics, Tabuk University, Tabuk, KSAPublished online: 08 May 2015.

To cite this article: Z. Khattari, M. Maghrabi & T. Al-Abdullah (2015) Temperature effecton thin lipid film elasticity and phase separation: insights from Langmuir monolayer andfluorescence microscopy techniques, Phase Transitions: A Multinational Journal, 88:7, 668-681, DOI:10.1080/01411594.2015.1021348

To link to this article: http://dx.doi.org/10.1080/01411594.2015.1021348

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Temperature effect on thin lipid film elasticity and phase separation:

insights from Langmuir monolayer and fluorescence microscopy

techniques

Z. Khattaria,b*, M. Maghrabia and T. Al-Abdullaha

aDepartment of Physics, Hashemite University, Zarqa, Jordan; bDepartment of Physics,Tabuk University, Tabuk, KSA

(Received 31 October 2014; accepted 16 February 2015)

Langmuir monolayer pressure isotherms and compressibility modulus measurements ofphospholipid mixtures in several Langmuir monolayer systems at the air/water interfacewere investigated in this study. The ultimate aim was to carry out a comparison of theelasticity modulus for monolayers with different mixtures of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) andchicken egg yolk sphingomyelin (eSM), in the presence/absence of cholesterol (Chol).In particular, we were able to propose that the leading force beyond the phaseseparation into liquid expanded (LE-) and liquid condensed (LC-) phases emerges fromthe increasing barrier to incorporate DOPC molecules into a highly ordered LC-phase.In addition, our findings suggest that DOPC lipid molecules have a priority toincorporate in a disordered LE-phase, while DPPC and eSM prefer the ordered one.Also, Chol seems to split almost equally into both phases, indicating that Chol has nopriority for either phase and there are no particular interactions between Chol andsaturated lipid molecules.

Keywords: Langmuir monolayer technique; monolayer ordered phase; monolayercompressibility; phase transition; lipid rafts

1. Introduction

The presence of cholesterol (Chol) or related sterols in different lipid membranes plays a

vital role for normal cell structure and functioning. Chol is often found randomly distrib-

uted in the lipid rafts which uniquely contribute to form liquid/condensed-ordered-like

phase in many plasma membranes.[1�3] The observation of the co-existence of liquidand condensed lamellar domains in the phase diagrams containing Chol and saturated

phosphatidylcholine or sphingolipid has assumed the significance in cell biology such as

membrane sorting or ultimately the entry of pathogens.[4,5] Importantly, Chol was found

to distribute heterogeneously at different concentrations with various intracellular mem-

branes. Its lowest concentration was found in the membrane of endoplasmic reticulum,

while its highest concentration was found in the plasma membrane.[5,6]

McConnell and others [7,8] were very successful in establishing a detailed molecular

explanation for this phenomenon (i.e., co-existence of liquid expanded/liquid condensed

(LE/LC) phases). Successful theoretical models, thermodynamics or microscopic interac-

tion models which describe these phases and domains were proposed by many research

*Corresponding author. Email: [email protected]

� 2015 Taylor & Francis

Phase Transitions, 2015

Vol. 88, No. 7, 668�681, http://dx.doi.org/10.1080/01411594.2015.1021348

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mailto:[email protected]://dx.doi.org/10.1080/01411594.2015.1021348

groups.[8,9] On the other hand, the description of phase equilibrium of the binary or

ternary mixtures of phospholipid and Chol systems was experimentally determined by

Langmuir monolayer and fluorescence techniques.[10,11] In the last two decades, a large

number of publications have been reported with studies on various systems, where the

presence of lateral phase separation into two phases, liquid-expanded (i.e., disordered)

and liquid-condensed (i.e., ordered) phases, has been observed.[12] The experimental

methods described in those studies have used the monolayer’s compressibility modulus

as a useful tool to infer the structure of these phases in ternary lipid mixtures of sphingo-

myelin, unsaturated phospholipids and Chol.[13] Model membrane systems involving

one, two or three phospholipids have been proven to be a powerful approach for investi-

gating the effect of each lipid on phase separation.[14] However, few studies have exam-

ined the phase separation properties of Langmuir monolayer with complex phospholipid

mixtures as found in biological membranes. Moreover, the effect of Chol on the phase

separation of the monolayer systems remains unclear. In simple model system, Chol can

enhance, inhibit or be essential for phase separation.[12�14]In the present work, we investigate the monolayer compressibility modulus in differ-

ent phospholipid systems in order to carry out a comparison of the elasticity modulus for

monolayers with different compositions of l,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk

sphingomyelin (eSM) in the presence/absence of Chol. Such a comparison turned out to

be adequate to gather new information on the elastic behavior of these lipid monolayers

as well as on the lateral phase separation into two phases that were not fully considered

before. Another motivation for the present work relies on the existence of so-called ‘lipid

rafts’. The original idea of Simon and Ikonen [3] suggested that a membrane containing

rafts is enriched in sphingolipids and Chol. Model membranes similar to lipid rafts have

suggested the existence of a liquid-ordered phase that is primarily composed of tightly

packed acyl carbon chains with extended degrees of freedom. These lipid rafts of mam-

malian cells adjust their membranes’ lipid composition when exposed to external temper-

atures that are different from the normal physiological conditions. The two most common

pathways by which cells alter their membrane lipid composition are the modification of

phospholipid structures and/or by adjusting sterol content.[3] Therefore, the present study

of ternary lipid mixture monolayers that mimic the composition of lipid rafts at various

temperatures will shield some light on the elasticity and morphology of these complex

cell compartments.

2. Materials and methods

2.1. Preparation of lipid monolayers

DPPC, DOPC and eSM were used to prepare the phospholipid monolayer systems at the

air/water interface. The molecular structure of these lipids is depicted in Scheme 1. The

mixtures used in this study are the intersection representations of the ellipses in the Venn

diagram. The phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL,

USA). Cholesterol and chloroform were purchased from Sigma (St Louis, MO, USA).

All components are of >99% purity and used without further purification. Stock solutions

(c D 0.1 mg/mL) were prepared by dissolving the lipids in chloroform. Such solutionswere then used to prepare solutions of the desired lipid-mixture ratio. For samples con-

taining two phospholipids, the ratio was held at 1:1 mol% and when Chol was added, its

concentration was kept at 20 mol%.

Phase Transitions 669

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2.2. Langmuir monolayer pressure isotherms technique

2.2.1. Langmuir film balance

A Langmuir trough manufactured by NIMA Technology Ltd (The Science Park,

Coventry CV4 7EZ, England; model 601M) was used in this study. The trough comes

with a 105-cm2 surface area with two mechanically coupled barriers, surface pressure

sensor, computer interface unit, and software for data acquisition and analysis. Surface

purity of the pure subphase (water or buffer) was checked by closing and opening the bar-

riers to ensure that the pressure (p) readings accuracy is within § 0.1 mN/m. The surfacetension was measured by means of a Wilhelmy paper plate hung to the balance. The data

were collected by the operating software and directly converted into interfacial pressure

after subtracting the interfacial tension of the pure aqueous subphase.

2.2.2. Mixed monolayer p�A isothermsMonolayers were formed at the air/water interface by spreading the solution of lipids in

an organic solvent. A Hamilton syringe was cleaned three times with chloroform, and an

Scheme 1. Venn diagram illustration of the lipids used in this study. V represents the total set ofall lipids as shown in the figure. Each subgroup is represented by an ellipse. The intersections of thesubgroups are the phospholipid mixtures investigated experimentally.

670 Z. Khattari et al.

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appropriate volume of solution (usually 60 mL) was drawn. With the syringe just above

the water surface, aliquots of the lipid mixture were deposited in a dropwise manner onto

the aqueous surface with a caution that the pressure value produced by adding the drop

has returned to zero before introducing the next one. After waiting 30 min for the solvent

to evaporate, compression was started at a speed of 0.05 and 0.1 nm2/mol/min and the

pressure�area (p�A) isotherms were recorded. Reproducibility of the results waschecked by repeating each curve at least twice. The trough temperature was maintained

within §1 �C of the desired temperature by a circulating water bath (Lauda, ModelRK20) and a thermoelectric device.

2.2.3. Compressibility modulus of Langmuir monolayer

Mechanical properties of the monomolecular film can be inferred from the compress-

ibility modulus of the monolayer.[15] The compressibility modulus is the ability of the

material to change its physical state when an external force is applied to it. For mono-

layers, it describes the differential change in the interfacial pressure with relative

change in the molecular area at constant temperature. The inverse compressibility mod-

ulus can be evaluated from the interfacial�area isotherms according to the followingequation:

C¡ 1s D ¡A@p

@A;

where A is the area per molecule at specific interfacial pressure and p is the correspond-ing interfacial pressure. It should be noted that the modulus is zero for bare air/water

interface and increases with the amount of surface-active molecules within the mono-

layer. The values of the compressibility for different monolayers provide information

about the elasticity and the compressibility of the corresponding cell membrane.

For example, a higher value of Cs¡1 indicates lower elasticity of the monomolecular

film (i.e., more condensed films) and vice versa.[16]

2.2.4. Fluorescence microscopy imaging of the monolayer

To observe the morphological characteristics of the monolayer at the air/water interface, a

homemade mini trough was constructed. The trough has a working area of 7 £ 5 cm2which can be mounted directly under the microscope. Fluorescence from a monomolecu-

lar film doped with 1% Tex red fluorescent probe at the air/water interface was observed

using a Nikon 20£ long working distance objective on a Leica microscope. Excitation ofthe fluorescent probes was achieved using a 100 W mercury lamp with a Leica blue light

pass filter. The fluorescent images were captured by a Leica video camera (Model DFC

360 FX) attached to the microscope.[12]

2.2.5. Statistical analysis

The data points shown in Figures 1 and 2 are calculated as the mean value § standarderror of three measurements taking into account the systematic and analytical uncertain-

ties. Student’s t-test was employed to compare differences between mean values of the

physical properties. Samples with Chol were compared with samples without Chol at a

significant difference of p � 0.05.


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3. Results and discussion

Typical pressure�area isotherms were obtained for all phospholipid systems reported inthis study, but are not shown here because they contribute little to the purpose of the study.

For detailed measurements of pressure�area isotherms, see [7] and the references therein.The pressure�area isotherms exemplified by one-, two- and three-component(s) lipid sys-tems are depicted in Figure 1(a). Typical isotherms shown for these monolayers at

T D 30 �C are consistent with those previously reported in [17,18]. For example, the mono-layer of pure DOPC, which always exhibits an LE-phase with a lift-off pressure at about

95 A� 2 and a collapse pressure around 40 mN/m, was found in good agreement with the

results of Chang-Chun Hao et al. [17] carried out at a constant temperature. When either

Chol or eSM is mixed with DOPC, the isotherms shift to a lower molecular area per lipid

molecule in comparison with the single-component system. Also, the interfacial collapse

pressure of mixed monolayers has increased to higher values indicating a molecular mixing

between lipids has been achieved. The relationship between Cs¡1 and the interfacial

1 2 3 4 5 6

0

2

4

6

8

Nor

mal

ized

Inve

rse

Com

pres

sibi

lity

DOPC DOPC-Chol DOPC-eSM-Chol

Normalized Area per Molecule60 80 100 120 140 160

0

40

80

120

160

200

240

280

320

Cs-

1 [m

N/m

]

Area per Molecule [A2]

DOPC DOPC-Chol DOPC-DPPC-eSM

60 80 100 120 140 160 1800

5

10

15

20

25

30

35

40

45

50

Area per Molecule [ 2]

Pre

ssur

e [m

N/m

]DOPCDOPC-CholDOPC-DPPC-eSM

Figure 1. (a) Pressure�area isotherms for selected lipid mixture monolayers at T D 30 �C, (b) thecorresponding inverse compressible modulus, and (c) the normalized inverse compressibility as afunction of the lipid molecular area. The normalized values have been calculated with respect tominimum lipid molecular area as obtained from the isotherms. The mean lipid component in theseselected lipid systems is the DOPC.

2025

3035

4045 50

100150200250300350400450500

T [ 0C]

Cs -1 [m

N/m]

=5 mN/m

=10 mN/m

=15 mN/m

=20 mN/m

=25 mN/m

=30 mN/m

=35 mN/m

=40 mN/m

Figure 2. A 3D diagram illustration of the eSM lipid monolayer elasticity as a function of selectedtemperatures and pressures. These values are calculated from pressure�area isotherms analogous tothose shown in Figure 1.


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pressure for these lipid monolayers at T D 30 �C has been calculated based on p�Aisotherms as shown in Figure 1(b). At this temperature, the Cs

¡1 values reported by [19] atp D 30 mN/m are very close to our values Cs¡1 2 {95�125} mN/m owing to a small dif-ference in measured temperatures between our study and their studies (see Table 1). Mixing

eSM or Chol molecules with DOPC molecules increases the Cs¡1 values to a higher col-

lapsing pressure. This is due to the fact that the cis double bond in the hydrocarbon chains

of DOPC prevents close packing of the molecules and therefore a higher Cs¡1 value

(ca. 110�120 mN/m) was observed for these mixed monomolecular lipid films when com-pared with the pure DOPC monolayer. The packing effect of these two lipids on the phos-

pholipid films’ elasticity is evident from the higher value of Cs¡1. This in turn means the

existence of less compressible mixed monolayers with reduced in-plane elasticity despite

the slightly larger cross-sectional molecular area. In effect, the addition of eSM or Chol lip-

ids to the DOPC monolayers makes them more densely packed (more condensed) and more

produces ordered phases. Such an effect can also be attributed to condensation effect of

Chol when mixed with the monomolecular lipid (see Figure 1(c)) film as pointed out by

Smaby et al.[20] On the other hand, normalizing the inverse compressibility with the mini-

mum lipid molecular area obtained from the p�A isotherms revealed that this effect is dueto a geometrical contribution from the phospholipid headgroups.[18] Once these lipid mix-

tures are spread at the air/water interface at certain temperature to form monolayers, a com-

petition between the headgroups in a tiny amount of the available molecular space becomes

essential for a collective effect of all molecules regarding their molecular rigidity, order and

possible chain tilting. Hence, the amount of the available space will be entrapped among

various lipid molecules, leading to an average molecular cross-sectional area for all lipids

participating in the film at a certain pressure.[18,20]

A three-dimensional (3D) illustration of the inverse compressibility as a function of

both pressure and temperature is presented in Figure 2. The 3D ribbon diagram shows the

detailed variation of Cs¡1 observed at several temperature and pressure values (i.e.,

T 2 {20�55} �C, p 2 {5�40} mN/m). Two-dimensional (2D) phase transitions of a LE(chain-disordered) to condensed (chain-ordered) nature were observed at several tempera-

tures. This observation is consistent with the earlier studies conducted by many groups.

[16�18] The sharpness of the 2D phase transition clearly depends on the nature of the

Table 1. Lipid interfacial elastic moduli of area compressibility and minimum area per molecule atselected points (T, p) lipid mixture.

(T, p) Am (A� 2) Cs

¡1 (mN/m) Am (A� 2) Cs

¡1 (mN/m) Ref.

Single-component system

DOPC (20, 30) 59 122 63 118 [17,19]

DPPC (20, 30) 15 255 14 250 [19]

(25, 30) 46 220 45 210 [21]

eSM (20, 30) 47 325 47 301 [18]

(25, 30) 48 290 47 279 [18]

(30, 30) 49 273 48 153 [18]

Two-component system

DOPC/eSM (20, 30) 51 87 49 108 [17]

eSM/Chol (25, 30) 37 77 39 95 [30]

Note: Am is the minimum area per molecule obtained from the pressure�area isotherms.Cs

¡1 is the inverse compressibility moduli at a given (T, p).


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acyl composition of the lipid mixture. For these lipid chains, the acyl chain length and

headgroups shape of the eSM lipid monolayer play a crucial role in the onset behavior of

the phase transition. Obviously, as the temperature of the lipid monolayer is reduced, a

lower interfacial pressure is gained by the monolayer as can easily be seen in the 2D phase

transition. A dramatic change in the Cs¡1 values was observed across the phase transition

appearing at different points (i.e., at an ordered pairs of (T, p)) in the phase space covered

by the experiments. These inflection points in the 2D space of the phase diagram reflect

the contribution of the partial cross-sectional molecular area within the LE/LC co-existing

phases. As the lipid monolayer crosses the 2D phase transition region, much higher Cs¡1

values occur for the condensed phase of the lipid molecules.[20] Further discussion of the

elasticity for various lipid mixture monolayers based on the lipid composition is given

below for a wide range of possible values of temperature and pressure.

3.1. One-lipid systems

The temperature dependence of phospholipid inverse compressibility coefficient for

the various monolayers at the air/water interface is shown in Figure 3 at a pressure of

p D 5 and 30 mN/m for temperature range T 2 {20�55} �C. As can be observed fromFigure 3(a), the single-component lipid monolayers at low pressure produces Cs

¡1 valuesthat decrease for the various phospholipids in the following order: DOPC � DPPC

20 25 30 35 40 45 50 5520

30

40

50

60

70

80

90

100

110

120 DOPC-DPPC-eSM DOPC-DPPC-Chol DPPC-eSM-Chol DOPC-eSM-Chol

T [0C]

Cs-

1[m

N/m

]

20 25 30 35 40 45 50 55

50

100

150

200

250

300

350 DOPC-DPPC-eSM DOPC-DPPC-Chol DPPC-eSM-Chol DOPC-eSM-Chol

T [0C]

30

40

50

60

70

80

DOPC-Chol DPPC-Chol eSM-Chol

Cs-

1[m

N/m

]

50

100

150

200

250

300

DOPC-Chol DPPC-Chol eSM-Chol

20

25

30

35

40

Cs-

1[m

N/m

]

DOPC-eSM DPPC-eSM DPPC-DOPC

0

25

50

75

100

125 DOPC-eSM DPPC-eSM DPPC-DOPC

15

30

45

60

75

90

105

120

Cs-

1[m

N/m

] DOPC DPPC eSM

0

50

100

150

200

250

300

350

400 DOPC DPPC eSM

Figure 3. The phospholipids monolayer inverse compressibility coefficients for the one- (a), (b),two- (c), (d), (e), (f) and three- (g), (h) component system(s). The compressibility data wereobtained at constant interfacial pressure p D 5 (left panels) and p D 30 (right panels) mN/m.


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compared to that of the two PC molecules in the monolayers.[18,23] The saturated fatty

acids in eSM and DPPC also result in denser packing for these molecules than that for the

unsaturated DOPC.[17,21]

Finally, the correlation between the monomolecular film inverse compressibility coef-

ficients and the lateral packing of the hydrocarbon lipid chains should be employed in the

discussion of the more complex monolayer systems.

3.2. Two-lipid systems

Figure 3(c) and 3(d) shows the inverse compressibility coefficient modulus (p D 5 and30 mN/m) for binary monolayers lipid mixture obtained from the p�A isotherms at vari-ous temperatures. The overall increase in Cs

¡1 values is expected when DPPC or eSM ispresent in the lipid monolayer with the largest effect being for eSM at least for low pres-

sure monolayers.[19,20] When a less elastic molecule is introduced into the monolayer, a

large elastic molecule will be stiff down (and vice versa).[23] In view of phospholipid

chain packing, the addition of DOPC to the high temperature melting lipid impairs the

order in monolayers which decreases the Cs¡1 values. Our data show that this effect is

higher for eSM than for DPPC (see Table 1). The saturated lipids exhibit a two-phase co-

existence of fluid and liquid condensed phases at temperatures lower than the melting

temperature (Tm) as confirmed by [22]. This observation is evidenced from the fluores-

cence images in the fluid phase where the large elasticity modulus is due to the highly

elastic relaxation of the lipid molecules (see Figure 4). Therefore, the reported Cs¡1 val-

ues mostly correspond to lipids in the LE-phase. An increase in the Cs¡1 values can be

expected due to lipid mixing as the amount of the liquid-condensed phase increases

within the monolayer persisting at the air/water interface. Therefore, decreasing the

molecular area available per lipid molecule produces much higher Cs¡1 values character-

izing the evolving LC-phase of the monolayer.

This alteration in behavior was observed as a small peak at T D 40 and 35 �C in theinverse compressibility modulus for eSM and DPPC mixtures, respectively. It should be

noted that the melting temperatures for DPPC and eSM are Tm D 35 and 18 �C, respec-tively. A condensed phase formation leads to a gradual depletion of the high melting lipid

Figure 4. Fluorescence microscopy images of DOPC/DPPC/Chol ternary mixture monolayer atthe air/water interface as a function of temperature at two selected pressures of p D 5 andp D 30 mN/m.


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which is miscible in the liquid phase, leaving mainly DOPC molecules in the condensed

phase. This explains the coalescence of the curves seen at intermediate temperatures with

higher Cs¡1 values of the binary systems than that for pure DOPC monolayer due to hin-

dering effects of the hydrophobic chain rigidity.[17,27]

A noticeable increase in the inverse compressibility value was observed when Chol is

mixed with the lipids forming the monolayers (see Figure 3(e) and 3(f)). This hallmark

shows that Chol effectively reduces the free area (i.e., condensation effect of Chol) avail-

able for the phospholipid molecules present in the monolayer. The resulting effect

increases in the following order: DOPC < DPPC �eSM (i.e., the saturated chains aremore sensible to ordering by Chol than the unsaturated ones). It is well known that Chol

intercalates between the lipid chains, thereby stimulating a huge increase in the film

molecular ordering and packing in the monomolecular film.[18,20]

3.3. Three-lipid systems

In the phospholipid ternary systems, we encounter at first glance an overall increase in the

inverse compressibility coefficient values (see Figure 3(g) and 3(h)). This result has been

attributed to a phase separation into domains of LE-phase and LC-phase. In fact, the

observed increase in the inverse compressibility coefficients is independent of the varia-

tions in the monolayer LC or LE domain size (usually > 5 mm) or shape.[10,11] Domains

of such sizes have been visualized in similar systems by fluorescence and Brewster angle

microscopy techniques.[22] A pronounced peak for the systems was observed between

T D 37 and 43 �C at low pressure. At higher pressure values, a reduction in the peakheight was observed which may be attributed to a close packing between the lipid mole-

cules within the monolayers.[7]

The ternary mixtures of DOPC/DPPC/Chol and DOPC/eSM/Chol systems show

monotonic behavior of the compressibility coefficient at high temperature. Meanwhile,

this monotonic behavior is absent in the mixtures of DPPC/eSM/Chol.

At about 37 and 46 �C, large and small Cs¡1 values were, respectively, obtained for all

the mixtures reported here. At the same temperatures, the phospholipid elasticity in the

two-phase co-existence region is very similar to the monolayers containing Chol. Note

that there is an increase in the compressibility coefficient as a function of temperature

indicating changes in the miscibility of the two phases with respect to the overall lipid

composition. Compressibility studies of lipid monolayers at the air/water interface have

shown that the LE-phase is depressed in the DOPC/DPPC/Chol mixture when the concen-

tration of Chol is reduced in this phase.[10,11] Hence, the inverse compressibility values

of these ternary systems reflect those obtained for the binary DOPC/Chol monolayers.

The inverse compressibility values obtained in the DOPC/eSM/Chol system suggest a

similar separation mechanism operates in this system. Our findings are in good agreement

with the elasticity values estimated for DOPC/DPPC/Chol system.[8�11] Moreover, ourresults suggest that Chol has no specific priority for LE-phase or LC-phase. Such results

are supported by several recent reports, where no preferred interactions between Chol

and eSM have been found.[25�27]If one compares the Cs

¡1 values for the LE-phase to those obtained for the binary sys-tems it is obvious that some DOPC molecules must be settled in this phase, since the

phospholipid monolayer is more elastic than that for DPPC/Chol or eSM/Chol systems.

This means that ternary systems tend to phase-separate into a tightly packed LE-phase

including the three components, and an LC-phase containing only DOPC and Chol mole-

cules. Thus, monolayers having two saturated phospholipid molecules and Chol show no


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phase segregation, and the obtained values for the inverse compressibility are very close

to the average values of the binary DPPC/Chol and eSM/Chol systems at temperatures

above 30 �C.[20] The overall conclusions have been confirmed by fluorescence micros-copy on a ternary mixture as shown in Figure 4.

To further discuss the apparent 2D-phase transitions among the various lipid systems

based on the monolayer morphological transition from LE- to LC-phase passing through

the co-existing region LE/LC-phase in connection with the lipid melting chain tempera-

ture (Tm), we will employ the combined results obtained from the elasticity measurements

and the fluorescence microscopy imaging technique. The chain-melting temperatures of

DPPC, eSM and DOPC are 35, 18.8 and ¡5 �C, respectively. Both lipids mix ideallywith DOPC at all ratios, forming a stable fluid phase structure above its chain-melting

point of ¡5 �C. In fact, Tm of these lipid mixtures with or without Chol has been experi-mentally verified to be liquid-ordered phase in a co-existence with the solid-ordered

phase. This observation has been confirmed from fluorescence microscopy on monolayers

and from the two-component quadrupolar 2H-NMR spectra of deuterated chain studies.

Our data show that the Cs¡1 values have a peak at the transition temperature at least for

the one-component systems near the melting temperature with the exception of DOPC

monolayer as expected.[28]

4. Connection between the monolayer results and the lipid

bilayer in the cell membrane

Many interfacial pressure isotherms have been tested for monolayer and bilayer samples

by several techniques such as fluorescence microscopy, Brewster angle microscopy and

atomic force microscopy. An interfacial monolayer is commonly accepted to model the

outer leaflet of the lipid bilayer in mammalian cell membrane.[18] However, the phases and

phase transition presented in lipid monolayer are mostly understood at lower initial pressures

than those found in the cell membrane. A large body of experimental investigations have

revealed that model membranes of binary and ternary mixtures are similar to those found in

natural lipid raft membrane and show a phase transition or separation to produce an LC-

phase of eSM/Chol and LE-phase of PC-rich lipids. The present study has focused on the

distribution of DOPC in these phase-separated mixtures in order to gain a better understand-

ing about the occurring of natural rafts phase and the synthetic cell membranes. Our results

clearly show that including DOPC or Chol monolayers of ternary mixtures leads to LE-rich

elastic phase distributed among LC-phases similar to that found in the natural rafts. The

present results are analogous to earlier investigations that showed similar LE-phase and LC-

phase in PC and PC/Chol monolayers and bilayers.[28] However, these results are slightly

different from other studies of PC/Chol monolayers because of lipid composition ratios and

sample preparation. For example, the fluorescence experiments performed by Radhakrishnan

and McConnell [9] have provided evidence for three separate phases in the presence of

Chol. The current work predicts similar results owing to the fact that different phospholipids

and molar ratios between lipids have been used. However, the present study did not provide

an evidence for the co-existence of three-phase monolayers. Comparing monolayer and

bilayer elastic values for pure and mixed lipid components is not an easy task due to obvi-

ous experimental challenges linking vesicle elasticity to that of the monolayer of the same

lipid mixtures. In general, vesicle membrane bilayers are unstable below Tm. Little is known

about the vesicle bilayer elasticity modules which emerge from bulk compressibility moduli

measurements. These measurements are performed by micropipette aspiration on a limited

number of lipid membrane mixtures such as brain sphingomyelin or eSM with cholesterol.


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Interestingly, the measured systems of both monolayers and bilayers where the comparison

is possible are generally showing similar trends with respect to the lipid mixture composi-

tion or structure at certain temperatures.[20]

There are still many unresolved questions concerning the existence and the role of

unsaturated fatty lipids and cholesterol in forming the rafts in natural membrane.

However, these observations of lipid�cholesterol complexes in model membranes pro-vide important insights in the ability of these phospholipids to organize within LC- or

LE-phase. In addition, factors that trigger lipid monolayers organization in analogy to

cell membrane bilayers are still to be established.

5. Conclusions

The lateral phase separation in the systems studied in this work can be understood in

terms of lipid order and DOPC miscibility in ordered phases. First, we stress that the

interpretation of our experimental outcomes is based on the hypothesis that the monolayer

elasticity is strongly dependent on the lipid packing order and that the specific interactions

between the lipid molecules have to be neglected. Similar results are suggested in the lit-

erature for lipid�Chol complexes or Chol dimmers. It is worth mentioning here that suchinteractions cannot be removed from our system, but we can confidently say that there is

no need to implement them to understand the changes in the monolayer elasticity upon

alterations in its composition. With this assumption it is obvious that saturated lipids

(e.g., DPPC and eSM) form a more ordered phase than unsaturated lipids, and mixing

with Chol greatly enhances the lipid chain ordering, especially for those with saturated

chains. Therefore, we propose that the leading force beyond the phase separation into

LE- and LC-phases is due to the increasing barrier width for DOPC to be integrated into a

highly ordered LC-phase. Our results suggest that DOPC has a choice to be located in a

disordered LE-phase, while DPPC and eSM prefer the ordered LC-phase. Interestingly,

Chol seems to split almost equally into both phases, indicating that Chol has no specific

affinity for any of these phases, and there are no preferred interactions between Chol mole-

cules and the saturated lipids. This behavior probably emerges from the rigid nature of the

sterol structure, making it rather insensitive to the molecular order of the monolayer com-

position. The primary role of Chol molecules in the phase separation process is to increase

the ordering and packing of the Langmuir monolayer to an extent that the system finally

prefers phase separation than miscibility, where every DOPC lipid molecule departs from

the LC-ordered phase. A future work has to be performed on certain lipid system mixtures

that exactly resemble the lipid rafts presented in mammalian cell membranes.

Acknowledgements

Z. Khattari would like to thank Prof. Thomas Fischer (University of Bayreuth, Germany) for hisgenerous hospitality during carrying out of the experiments in his laboratory.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

Financial support from DFG and Hashemite University [grant number Fi 548 11-1] is gratefullyacknowledged.


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Abstract1. Introduction2. Materials and methods2.1. Preparation of lipid monolayers2.2. Langmuir monolayer pressure isotherms technique2.2.1. . Langmuir film balance2.2.2. Mixed monolayer π-A isotherms2.2.3. Compressibility modulus of Langmuir monolayer2.2.4. Fluorescence microscopy imaging of the monolayer2.2.5. Statistical analysis

3. Results and discussion3.1. One-lipid systems3.2. Two-lipid systems3.3. Three-lipid systems

4. Connection between the monolayer results and the lipid bilayer in the cell membrane5. ConclusionsAcknowledgementsDisclosure statementFundingReferences

journal b phase transitions: a multinational · langmuir monolayer pressure isotherms and...

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