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Colloids and Surfaces B: Biointerfaces 75 (2010) 34–41

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

hermodynamic and elastic fluctuation analysis of langmuir mixed monolayersomposed by dehydrocholic acid (HDHC) and didodecyldimethylammoniumromide (DDAB)

aula V. Messinaa,∗, Gerardo Prietob, Juan Manuel Rusoc, Marcos D. Fernández-Leyesa,ablo C. Schulza, Félix Sarmientob

Departamento de Química, Universidad Nacional del Sur, CONICET-INQUISUR, Av Alem 1253, (8000) Bahía Blanca, ArgentinaBiophysics & Interfaces Group, Departamento de Física Aplicada, Facultade de Física, Universidad de Santiago de Compostela, (15782) Santiago de Compostela, SpainSoft Matter and Molecular Biophysics Group, Departamento de Física Aplicada, Facultade de Física, Universidad de Santiago de Compostela,15782) Santiago de Compostela, Spain

r t i c l e i n f o

rticle history:eceived 22 October 2008eceived in revised form 5 August 2009ccepted 5 August 2009vailable online 13 August 2009

eywords:ehydrocholic acidDAB

a b s t r a c t

The physicochemical and elastic properties of Langmuir mixed monolayers composed by dehydrocholicacid (HDHC) and didodecyldimethylammonium bromide (DDAB) were evaluated. The experiments wereperformed with a constant surface pressure penetration Langmuir balance based on Axisymmetric DropShape Analysis (ADSA).

The behavior of such amphiphiles in monolayer was clearly non-ideal and would be seriously influencedby the amount of HDHC molecules present. The presence of bile acid type molecules caused the monolayerbe more condensed (Ac diminution) and the intermolecular attractive interactions be stronger (high ε0

values). This fact would be related to H-bond formation between water and carboxilate and carbonile

angmuir-monolayersDSAhermodynamic parameterstatic elasticity modulus

groups in the cholesteric ring and agreed with the existence of laterally structured microdomains atthe monolayer (determined by the analysis of the first virial coefficient, b0 < 1, of the state equation).The miscibility of both surfactants in the monolayer, their high bulk hydrophobicity (�c > 35 mJ m−2)just with the obtained negative values of the free energy of mixing �Gmix, and the excess second virialcoefficient (b1)E obtained allows us to infer that net attractive interaction existed between HDHC andDDAB molecules at the monolayer and that mixed systems would be able to be used in the formulation

blies.

of supramolecular assem

. Introduction

Bile salts with their rigid ring structures are in many respectsnusual surfactants, and are usually described as having both aydrophilic and a hydrophobic faces. The physical chemistry ofile salts micellization has been, and still is, an active area ofesearch. The high specificity and capacity for bile salts trans-ort systems during their enterohepatic circulation might form theasis of a current research on drug–bile acids delivery for specificrug targeting to the liver and for improving the intestinal adsorp-ion of poorly adsorbed or non-adsorbed drugs, such as peptides

1,2]. A variety of techniques have been employed to gain morensight into the structure/size/shape of bile salts aggregates. Sev-ral studies which involved the interactions of such surfactantsn mixtures with cationic amphiphiles have been performed and

∗ Corresponding author. Tel.: +54 291 4551447; fax: +54 291 4551447.E-mail address: pau423ve@yahoo.com.ar (P.V. Messina).

927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2009.08.008

© 2009 Elsevier B.V. All rights reserved.

different types of supramolecular assemblies have been obtained[3]. Swanson-Vethamuthu et al. [4] have studied the behavior ofsodium cholate (NaC, with three hydroxyl groups on the hydropho-bic face) and sodium desoxycholate (NaDOC, with two hydroxylgroups) in combination with C16TAB and C16TAC. In such mixed sys-tems NaC gave mixed micelles that decrease monotonously in size,whereas NaDOC induced a transition to rod-like micelles, at closeto equimolar concentration a separation into two phases occurred[4]. According to that, the aim of this article is the evaluation of thephysicochemical and elastic properties of Langmuir mixed mono-layers composed by a bile salt type (dehydrocholic acid, HDHC) anda double tailed cationic surfactant (didodecyldimethylammoniumbromide, DDAB) as a previous step in their use as structural unitsfor the construction of new delivery systems. The selection of both

amphiphiles was because of their biocompatibility [5,6] and of theirpacking parameters [7].

Although a bilayer cannot simply be considered to consist oftwo independent monolayers, the head group–water interface inthe two systems may be sufficiently similar to make monolayers

P.V. Messina et al. / Colloids and Surfaces

Fd

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2

2

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2

sScp(wcobbaTmbdyailamstfaavemoe

ig. 1. Surfactant molecular structures: (A) dehydrocholic acid (HDHC) and (B) dido-ecyldimethylammonium bromide (DDAB).

ffective bilayers surrogates [8,9]. Spread monolayers could beonsidered the simplest model to get molecular information andence the first step to understand the properties and the behav-

or of selected molecules in future arrangements which can lead toromising materials with biological and medical applications.

. Experimental

.1. Materials

Dehydrocholic acid (HDHC) was from Dr. Theodor SchuchardtMunich) and didodecyldimethylammonium bromide (DDAB) wasbtained from Sigma. Both compounds were of analytical grade99% pure) and were used as purchased, as a reference their struc-ures are shown in Fig. 1.

.2. Apparatus and operation conditions

The experiments were performed with a constant surface pres-ure penetration Langmuir balance based on Axisymmetric Drophape Analysis (ADSA) [10]. The whole setup, including the imageapturing, the micro-injector, the ADSA algorithm, and the fuzzyressure control, is managed by a Windows integrated programDINATEN). A solution droplet is formed at the tip of a capillary,hich is outer one of an arrangement of two coaxial capillaries

onnected to the different branches of a micro-injector. These canperate independently, permitting one to vary the interfacial areay changing the drop volume, and to exchange the drop contenty through flow. The software first detects the drop and with anppropriate calibration, transforms it into physical coordinates.hen the experimental drop profiles, extracted from digital dropsicrographs, are fitted to the Young–Laplace equation of capillarity

y using ADSA. This process is performed automatically, the liquidensity difference and the local gravity being the only inputs andielding as outputs the drop volume V, the interfacial tension �nd the surface area A in about 0.3–5 s for each picture, depend-ng on the required precision. Area control uses a modulated fuzzyogic PID (proportional, integral and derivative control) algorithmnd is controlled by changing the drop volume. During the experi-ent, the drop is immersed in a thermostated and vapor-saturated

tandard spectrophotometer cuvette (Hellma®) minimizing con-amination and drop evaporation. The surface pressure is obtainedrom the relationship � = �0 − � , where � is the surface pressure; �nd �0 are the surface tension of the subphase liquid covered withnd without the monolayer. The setup is placed on a pneumatic

ibration-damped optical bench table in a clean laboratory. Allxperiments were performed at (25.0 ± 0.1) ◦C. Temperature wasaintained by a thermostat bath with recycling water through-

ut all the experiment. The curves were highly reproducible: eachxperiment was done three times, the standard deviation [10c] on �

B: Biointerfaces 75 (2010) 34–41 35

and A was estimated to be ± 0.01 mJ m−2 and ±0.005 nm2 mole c−1,respectively. Equation fitting was done from non-linear proceduresusing ORIGIN® computer package (release 7.0).

2.3. Monolayers

Spreading solutions of HDHC and DDAB and their mixtureswere prepared dissolved in a methanol:chloroform mixture (1:4)to obtain solutions of (7 × 10−5 M) total concentration. Then analiquot of 1.2 �L was spread on the surface using a microsyringe.Once the spreading solvent has evaporated (4 min were allowed)and consequently the amphoteric molecules were confined to amonomolecular film at the subphase–air interface, the programstarted the expansion until a volume of 25 �L at a rate of 0.2 �L s−1.When expansion was finished, the drop area was maintained con-stant for 118 s (the necessary time for the tested monolayers toreach the thermodynamic equilibrium) and then the compressionstarted at the same rate of expansion process.

3. Theoretical methods

With the aim to analyze the response of the adsorbed moleculesto interface compression [11] the static elasticity modulus (ε0) hasbeen estimated from the equilibrium relationship between inter-facial pressure (�) and interfacial area (A):

ε0 =∣∣∣∣(

d�

d ln A

)Teq

∣∣∣∣ (1)

On basis of the �–A dependencies, the two-dimensional com-pressibility Cs of a monolayer at a given molecular area, orequivalently at a given surface pressure, is defined as the partialchange of the area strain with respect to the surface pressure, thatis Cs = −(1/A)(dA/d�). Its reciprocal is the area modulus C−1

s andboth are valuable tools which can be used to classify the monolayerphysical state [12]. For an ideal monolayer, the compressibility isassumed to be additive with respect to the product (Cs,i, Ai, i = 1, 2,3, . . ., etc.). Thus, Cs,ideal is obtained as follows [13]:

Cs,ideal =(

1A�,ideal

)[(Cs1A�,1)X1 + (Cs2A�,2)X2] (2)

where Cs1 and Cs2 are the compressibility of the pure DDAB and thepure HDHC monolayer at a specific surface pressure (�), respec-tively. However, the experimental data Cs obtained from �–Aisotherm of the mixed monolayer is usually different from the idealvalue. The change in compressibility due to the presence of HDHC(�Cs,HDHC) is as follows:

�Cs,HDHC = Cs − Cs1 (3)

The change in compressibility due to interaction non-ideality(�Cs,real) is as follows:

�Cs,real = Cs − Cs,ideal (4)

The interaction between amphiphilic molecules at the air-aqueous solution interface is composed by contributions ofelectrostatic, hydrophobic and hydration forces [14–17]. For mixedsystems, we examined the influence of such forces, analyzing theireffect on the collapse area values. An ideal mixed monolayer and acompletely immiscible monolayer are absolute opposite. However,

both follow Eq. (5). In an ideal mixed monolayer of components1 and 2, the intermolecular force F11 = F12 = F22 whereas in a com-pletely immiscible monolayer F11 � F12 � F22, where F12 representsthe attractive forces between molecules of the two dissimilar com-ponents.

3 rfaces B: Biointerfaces 75 (2010) 34–41

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6 P.V. Messina et al. / Colloids and Su

The collapse area per molecule for an ideal two-componentixed monolayer, Aid

c , can be calculated from:

idc = X1A1,c + X2A2,c (5)

here X1 and X2 are the mole fractions of the components 1 (DDAB)nd 2 (HDHC) at the mixed spread monolayer, A1,c and A2,c are theollapse area per molecule of the pure components.

The excess area, AEc , for a binary monolayer can be expressed as

ollows:

Ec = A1,2,c − (X1A1,c + X2A2,c) (6)

here A1,2,c is the collapse area per molecule of the mixed mono-ayer. A1,2,c, A1,c and A2,c can be obtained from the corresponding–A isotherms and were taken as the point where the upper partf the curve starts to deviate from the straight line [18].

The net interaction between two components in a mixed mono-ayer, at a constant surface pressure � and absolute temperature, can be evaluated from the calculation of excess Gibbs energy�GE

mix) and the interaction parameter (˛) [12,13,16,19], which areiven by:

GEmix =

�∫0

[A1.2 − (X1A1 + X2A2)]d� (7)

nd

= �GEmix

RT(X1X22 + X2X2

1 )(8)

The Gibbs energy of mixing is given by:

Gmix = �Gidmix + �GE

mix (9)

here the first term, the ideal Gibbs energy of mixing (�Gidmix), can

e calculated from the equation:

Gidmix = RT(X1 ln X1 + X2 ln X2) (10)

here R is the universal gas constant.In order to carry out a quantitative analysis of the molecular

nteractions in the mixed monolayers, the equation of state for two-imensional systems was employed:

A = �T (11)

here � is the Boltzmann constant. If it is assumed that theolecules at the monolayers cannot be compressed to a zero area,

he equation of state can be expressed [20] as follows:

(A − A0) = a�T (12)

r in its linear form:

�A

�T= a + �A0

�T(13)

here “a” is a factor related to the aggregation between moleculesn the monolayer and A0 is the minimum area occupied by a

olecule in the monolayer. A value of a = 1 is attributed to the idealas monolayer behavior.

The surface pressure, area and temperature behavior of non-deal monolayers have been accurately represented using the two-imensional virial equation of state [21–23]:

�A

�T= b0 + b1� + b2�2 (14)

here b0, b1 and b2 are the virial coefficients. The value of b0 isttributed to the aggregation state of the film-forming molecules.hen � tends towards zero, the �A product would tend towards �T,

r a fraction of �T for an aggregated system [15]. On the other hand,he value of b1 provides information about the exclusion volumes

Fig. 2. �–A isotherms for HDHC:DDAB systems at water subphase, 25 ◦C: (1)XHDHC = 0; (2) XHDHC = 0.2; (3) XHDHC = 0.4; (4) XHDHC = 0.6; (5) XHDHC = 0.8; (6) XHDHC = 1.

and the interaction between the molecules in the film. Negative orpositive values indicate attractive or repulsive interaction betweenthe molecules, respectively. The rest of the virial coefficients are notsignificant.

4. Results and discussion

4.1. �–A isotherms

A Langmuir film is a two-dimensional system. The compressionisotherm of a spreading film refers to a plot of surface pressure (�)as a function of area per molecule (A). Monolayer characterizationat air/water interface can be directly obtained though the analysisof such representation.

Fig. 2 shows the � vs. A isotherms of mixed and pureHDHC:DDAB monolayers on water subphase. The pure HDHCmonolayer behaves as an expanded film, exhibited low pres-sures (2–3 mJ m−2) for surface areas greater than 1.2 nm2 mole c−1.With monolayer compression, the isotherm lifts off at a molec-ular area of 1.25 nm2 mole c−1 after which further compressionresulted in progressive increase in � values. These findings areconsistent with the molecules lying flat at the air–water interface,with carbonile groups of the steroid nucleus and the carboxylicacid group anchored in the subphase [9]. With continued com-pression, collapse points occurred at a � value of 40 mJ m−2 andat A = 0.48 nm2 mole c−1. In such conditions, it was assumed thatsteroidal nucleus acquired a vertical position remaining almostperpendicular to the aqueous interface. Such fact, suggested thatinterfacial pressure induced a tilting of the molecules during com-pression. There is a general agreement that bile acid moleculesand the fully ionized bile salts associated to form dimmers or largeaggregates within the hydrophobic portion of the membranes andwere thus oriented with the steroid nuclei perpendicular to theinterface [24].

Mixed and pure DDAB monolayers showed a typical �–A curvefor a liquid expanded (LE) film followed by a LE–LC (liquid con-densed) transition. All isotherms display low interfacial � valuesat molecular areas greater than 1.50 nm2 mole c−1. Upon compres-sion, the characteristics of isotherms (even for XHDHC = 0.8) becamedistinctly different from pure bile acid behavior. This fact suggested

that the presence of DDAB acyl chains highly influenced the mono-layer phase transitions.

The mean molecular areas of the HDHC:DDAB mixtures werestrongly dependent on composition as well as surface pressure,which is an indication of a miscible monolayer. So, for XHDHC = 0.2

P.V. Messina et al. / Colloids and Surfaces B: Biointerfaces 75 (2010) 34–41 37

F HC:D(

maaastmtettXt

itltacd

4

ltittb

tDs

ig. 3. Static elasticity modulus (ε0) as a function of mean molecular area (A) for HD4) XHDHC = 0.6; (5) XHDHC = 0.8; (6) XHDHC = 1.

ixed monolayer LE–LC transition began at A ≈ 0.84 nm2 mole c−1

nd at � ≈ 34 mJ m−2; for XHDHC = 0.4 the same transition occurredt A ≈ 0.76 nm2 mole c−1 and at � ≈ 38 mJ m−2 and for XHDHC = 0.6ppeared at A ≈ 0.74 nm2 mole c−1 and at � ≈ 26 mJ m−2. The mixedystem composed by XHDHC = 0.8 with a high content of the bile acidype compound showed a dissimilar behavior. It presented a LE

onolayer until A ≈ 0.84 nm2 mole c−1 followed by a LE–LC transi-ion and finally a LC phase from A ≈ 0.65 nm2 mole c−1. Such phasexisted until an area of A ≈ 0.61 nm2 mole c−1 were a new phaseransition appeared. Monolayers did not show a visible transitiono the solid state (S), the second transition that was appreciated forHDHC = 0.8 could be associated to a rearrangement of molecules inhe LC phase (LC–LC’) [25].

Notice that a stable film of pure DDAB spread on water/airnterface was obtained by the employ of ADSA based pene-ration surface balance, at difference of that informed in theiterature [26,27], where it was inferred that DDAB (contraryo other longer double-alkylammonium bromide salts, suchs ditetradecyldimethylammonium bromide [28], or dioctade-yldymethylammonium bromide [26,27]) showed a poor qualityesorbing monolayer [26].

.2. Monolayer elasticity

The static elasticity modulus provides information about theayer structures; high elasticity values are associated with a filmhat has strong cohesive interfacial structure. A maximum in ε0s related to a configurational transition in the monolayer struc-ure [29]. Actually the ε0 vs. A curve is another form to evaluatehe change of surface pressure during compression and would be

etter to expose the monolayer state.

Fig. 3 shows the variation of static elasticity modulus as a func-ion of molecular area for different HDHC:DDAB mixtures. PureDAB and HDHC monolayer static elasticity modulus curves pre-

ented very different profiles, which are in agreement with the

DAB systems at water subphase, 25 ◦C: (1) XHDHC = 0; (2) XHDHC = 0.2; (3) XHDHC = 0.4;

structures of both molecules. The variation of ε0 for pure DDABmonolayer showed a maximum of 80 mN m−1 which appeared atA ≈ 1.0 nm2 mole c−1. This peak corresponded to the LE–LC mono-layer transition. Similar behavior could be appreciated for mixedfilms with XHDHC = 0.2, 0.4 and 0.6. For such mixtures, the corre-sponding curves presented a peak with a maximum elasticity of70–100 mN m−1 that appeared at the area of 0.8–1.0 nm2 mole c−1.The pure HDHC ε0 vs. A curve did not show maximum or minimumpeaks. In the area range of 1.28–0.85 nm2 mole c−1, it was almost ahorizontal line with an elasticity value of about 25 mN m−1. Startingfrom the area of 0.85 nm2 mole c−1, the elasticity increased until avalue of 50 mN m−1 (0.62 nm2 mole c−1). At this point there was anew and a steeper increase of ε0 until a final value of 300 mN m−1

which corresponded to a molecular area of 0.50 nm2.For the special case of XHDHC = 0.8 two maximum peaks appeared

at 0.85 and 0.62 nm2 mole c−1 which corresponded to the LE–LCand LC–LC’ transitions. In such conditions with a further monolayercompression, the film-forming molecules become closer one otherand the intermolecular attraction gets stronger. As a result the staticelasticity modulus increased distinctively till nearly 350 mN m−1.

The elasticity of the monolayers with high content of HDHC(XHDHC = 0.8 and XHDHC = 1) was larger and transitions occurredat less molecular areas than that of the monolayer rich in DDAB(XHDHC = 0.6, 0.4, 0.2, 1). These results showed that the inclusionof bile acid type molecules has a notable effect on the monolayerintermolecular interactions.

In Fig. 4, plots of C−1s vs. mole fraction of HDHC at three different

surface pressure values are presented. Values of C−1s ranging from

0 to 12 mJ m−2 indicate the gaseous state of a monolayer, varyingfrom 12 to 100 mJ m−2 prove the liquid-expanded state, from 100 to

250 mJ m−2 are characteristics of liquid-condensed state, whereasgreater than 250 mJ m−2 indicate the solid state of a film. At low sur-face pressures (� = 5–10 mJ m−2) for all HDHC:DDAB mixtures, theC−1

s values oscillated between 13 and 50 mJ m−2, which was char-acteristic of the LE state. At high pressures (� = 30 mJ m−2) for the

38 P.V. Messina et al. / Colloids and Surfaces B: Biointerfaces 75 (2010) 34–41

Fb

XiwTm

mcaaeeTbr

4

ecl

Table 2Monolayer pressures and areas at collapse for pure and mixed HDHC:DDAB systemson water subphase, 25 ◦C.

XHDHC Ac/nm2 mole c−1 Ac,ideal/nm2 mole c−1 a �c/mJ m−2

0 0.77 0.77 38.130.2 0.84 0.71 34.580.4 0.76 0.65 38.950.6 0.72 0.59 36.380.8 0.62 0.53 38.811 0.48 0.48 40.85

ig. 4. Area modulus (Cs−1) vs. bile acid mole fraction (XHDHC) for the investigated

inary Langmuir-monolayers.

HDHC = 0, 0.2, 0.4 and 0.6 mixtures, Cs varied from 75 to 100 mJ m−2

ndicating that the monolayers were most expanded. Nevertheless,hen XHDHC = 0.8 and 1 a high condensing effect can be observed.

he values of C−1s were superior that 300 mJ m−2, so a solid like

onolayer would exist.The compressibility data computed by the HDHC:DDAB mixed

onolayers are summarized in Table 1. The measured change inompressibility due to HDHC (�Cs,HDHC) or due to non-ideal inter-ctions (�Cs,real) presented positive values at almost all mixturesnd surface pressures. Such facts implied that mixed monolay-rs were less elastic than pure ones and that such condensationffect was highly influenced by the presence of bile acid molecules.his fact would be related to the possibility of H-bond formationetween water and carboxilate and carbonile groups in cholestericing.

.3. Surface thermodynamic analysis

Table 2 shows the obtained areas and pressures for the differ-nt HDHC/DDAB mixtures spread on water subphase. The idealomposition dependence of the collapse molecular area calcu-ated by Eq. (5) is also included. The effective molecular collapse

Table 1Parameters of compressibility in HDHC:DDAB monolayers, 25 ◦C.

XHDHC Cs,ideal/mJ−1 m2

� = 5 mJ m−2

0 0.0219 ± 0.00260.2 0.0229 ± 0.00320.4 0.0239 ± 0.00340.6 0.0251 ± 0.00120.8 0.0264 ± 0.00151 0.0280 ± 0.0026

� = 10 mJ m−2

0 0.0144 ± 0.00590.2 0.0158 ± 0.00580.4 0.0174 ± 0.00610.6 0.0192 ± 0.00720.8 0.0213 ± 0.00731 0.0239 ± 0.0086

� = 30 mJ m−2

0 0.0159 ± 0.00780.2 0.0150 ± 0.00770.4 0.0140 ± 0.00820.6 0.0128 ± 0.00390.8 0.0112 ± 0.00181 0.0092 ± 0.0035

The error on � and A was estimated to be ± 0.01 mJ m−2 and ±0.005 nm2 mole c−1,respectively.

a Computed by Eq. (5).

area of pure HDHC (Ac ≈ 0.48 nm2 mole c−1) was similar to that ofcholanoic acid [30] and a slightly superior to the cross-sectionalarea of the steroid nucleus (about 0.40 nm2 mole c−1 [31]). Weinfer that at low surface pressure the steroid nucleus of bileacid lied parallel to the aqueous interface but assumed a verti-cal position on compression, remaining almost perpendicular tothe aqueous interface. The obtained value of Ac for pure DDABmonolayer (ADDAB

c ) was about 0.77 nm2 mole c−1, which is the usualcollapse area value obtained for dialkyldimethylammonium bro-mide monolayers [25–27]. Comparing the obtained ADDAB

c valuewith the molecular cross-sectional areas of the close-packed doublechain quaternary ammonium salts (c.a. 0.40 nm2 mole c−1 [32]), weassumed that the DDAB molecules acquired a conformation withboth alkyl chains lying with a certain angle at the surface.

For all HDHC:DDAB mixtures, the mixed Ac values were sig-nificantly higher from the ideally computed ones. Such positivedeviations were related to the existence of less attractive inter-actions at the mixed monolayers.

Collapse pressures (see Table 2) indicate the strength of thehydrophilic attachment to the subphase, as well as the interactionamong hydrophilic head groups within the monolayer. Pure andmixed monolayers presented a collapse pressure value (�c) of about34 mJ m−2 or higher. Such values were related to a high hydrophilic-ity of the molecules. Thus the strength of hydrophilic anchor at

interface was very important, showing that both molecules hadrelatively small areas of hydrocarbon/water contact. This resultedin higher collapse pressures but lower bulk hydrophilicities.

The values of excess area (AEc ) obtained by using Eq. (6) vs. sur-

face pressures at various compositions are shown in Fig. 5. There

�Cs,HDHC/mJ−1 m2 �Cs,real/mJ−1 m2

0 0−0.0044 ± 0.0006 −0.0054 ± 0.0006−0.0049 ± 0.0006 −0.0069 ± 0.0006

0.0313 ± 0.0007 0.0278 ± 0.00080.0211 ± 0.0006 0.0165 ± 0.0006

0 0

0 00.0024 ± 0.0005 0.00102 ± 0.00340.0020 ± 0.0004 −0.0009 ± 0.00020.0144 ± 0.0026 0.0096 ± 0.00120.0041 ± 0.0006 −0.0028 ± 0.0009

0 0

0 00.0050 ± 0.0006 0.0058 ± 0.0003

−0.0040 ± 0.0002 −0.0025 ± 0.00030.0220 ± 0.0007 0.0253 ± 0.0039

−0.0050 ± 0.0006 0.0002 ± 0.000010 0

P.V. Messina et al. / Colloids and Surfaces B: Biointerfaces 75 (2010) 34–41 39

FTa

amAeiAlm(wbmvicuatcam2eomabcs(

c

Fig. 6. Interaction parameter (˛) vs. bile acid mole fraction for the investigatedbinary Langmuir-monolayers.

TE

ig. 5. Excess areas values (AEc ) obtained for the different HDHC/HDC mixtures.

he standard deviation [10c] on � and A was estimated to be ± 0.01 mJ m−2

nd ± 0.005 nm2 mole c−1, respectively.

re always net intermolecular forces among the molecules in theonolayer. It is a matter of magnitude of forces that determine

Ec . For a mixed monolayer, if net attractive intermolecular forcesxist, AE

c will be negative. If AEc is positive, it suggests that the net

nteractions between the two components are repulsive. PositiveEc values can be appreciated at all surface pressures when mono-

ayer is rich in DDAB (XHDHC = 0.2 and 0.4). As the content of HDHColecules augment the AE

c values decreased and become negativeXHDHC = 0.6 and 0.8). The nonzero value of AE

c and their dependenceith the monolayer composition indicated the existence of misci-

le monolayer at all surface pressures. Another way to estimate theonolayer behavior during compression is by the evaluation of the

alues of �GEmix and ˛ at different pressures (calculated accord-

ng to Eqs. (7) and (8)). The interaction parameter (see Fig. 6) islose to �GE

mix (see Table 3) and has a similar meaning. The val-es of ˛ provide some additional information to that from �GE

mixnd to affirm at which proportion of both component the interac-ions are more attractive. The minimum value of ˛ indicates theomposition of a mixed monolayer at which the strongest inter-ction between both components occurs. The negative sing of ˛eans that the interactions among unlike molecules in a binary

D-mixture are more attractive than among like molecules. Thevaluation of such parameters showed that over a surface pressuref 30 mJ m−2, �GE

mix and ˛ were negative for all tested HDHC:DDABixtures. This fact let us to infer that the amphiphiles behavior atbilayer would be thermodynamically favored. Several studies onilayers [33–37] suggested that the areas for molecules in bilayers

orrespond more closely to those in monolayers at collapse pres-ures (35–45 mJ m2) [38]. The preferential mixture compositionminimum ˛ value) was XHDHC = 0.6.

Finally, the thermodynamic stability of the mixed monolayersomposed of two miscible materials at the air/liquid interface was

able 3xcess of mixing Gibbs energy (�GE) of HDHC:DDAB mixtures on water subphase, 25 ◦C.

� (mJ m−2) �GE (kJ mol−1)

XHDHC = 0.2 XHDHC = 0.4

5 0.098 ± 0.002 0.058 ± 0.0010 0.287 ± 0.004 0.191 ± 0.0015 0.204 ± 0.004 0.039 ± 0.0020 0.371 ± 0.006 0.115 ± 0.0025 0.121 ± 0.003 0.043 ± 0.0030 −0.483 ± 0.003 −0.043 ± 0.0035 −8.960 ± 0.008 −2.623 ± 0.0040

Fig. 7. Gibbs energy of mixing (�Gmix) obtained for the different HDHC/HDC mix-tures.

determinate by the free energy of mixing, �Gmix. This parame-ter indicated the energetic tendency to form a mixed monolayerfrom an unmixed. The values of �Gmix are shown in Fig. 7. Appar-ently �Gmix were all negative, suggesting that no phase separationoccurs in the mixed monolayers and confirming that a misciblemonolayer exists. Minimum values of such parameter were found

from � = 25 mJ m−2. The existence of a minimum will indicate amixture composition of the greatest thermodynamic stability incomparison with pure component monolayer. It was also observedthat the values of �Gmix become progressively more negative as

XHDHC = 0.6 XHDHC = 0.8

1 −0.241 ± 0.001 −0.234 ± 0.0043 −0.457 ± 0.004 −0.228 ± 0.0044 −0.540 ± 0.003 0.123 ± 0.0082 −0.243 ± 0.004 0.966 ± 0.0063 −0.519 ± 0.005 1.111 ± 0.0076 −5.288 ± 0.009 −1.969 ± 0.0059 −1.728 ± 0.007

−2.336 ± 0.006

40 P.V. Messina et al. / Colloids and Surfaces B: Biointerfaces 75 (2010) 34–41

Table 4Virial and correlation coefficient from the fitting values of equation.

XHDHC

0 0.2 0.4 0.6 0.8 1

bo 0.054 ± 0.004 0.032 ± 0.003 0.141 ± 0.007 0.164 ± 0.007 0.174 ± 0.014 0.168 ± 0.003−1 −3 −3 10−3 −3 −3 −3

.9) × 1

slm

4

cvtTTeitoftDticpt

twtu

(

wpo

Fp

b1 (m mN ) (312 ± 0.6) × 10 (336 ± 0.4) × 10 (295 ± 1) ×b2 (m2 mN−2) (−350 ± 1.8) × 10−5 (−380 ± 1.4) × 10−5 (−290 ± 21/b0 18 31 7r 0.995 0.993 0.983

urface pressure increases. This may be due to shorter intermolecu-ar distances at higher surface pressures as the monolayer becomes

ore condensed [39].

.4. Equation of state analysis

The virial coefficients (b0, b1, b2) and the correlation coeffi-ients obtained are listed in Table 4. Since the values of the firstirial coefficient (b0) were lower than 1 for all studied systems,wo-dimensional molecular aggregates existed at low pressures.hus their aggregation number (1/b0) could be estimated [19], seeable 4. This could be indirectly related to the existence of lat-rally structured microdomains. Small et al. [31] proposed thatonized bile acids are oriented with their long axis perpendicularo membrane surface and that could then form reverse dimmersr tetramers in biological membranes, with their hydrophilic sur-aces in opposition, and their hydrophobic surface interacting withhe acyl moieties of long-chain phospholipids. For XHDHC = 0, pureDAB monolayer, aggregation number could be estimated within

he order of 18, the presences of a few number of HDHC moleculesncreased the aggregation number to about 31 molecules but as theontent of HDHC augmented the following HDHC:DDAB mixturesresented an aggregation number of about 6 that corresponded tohe aggregation number of HDHC pure monolayers.

The values of the second virial coefficient (b1) were all posi-ive indicating that the predominant interactions in the monolayerere repulsive. To evaluate the nature of the interactions between

wo molecules in a mixed monolayer the following equation can besed [40]:

b1)12 = (b1)1 X2 + (b1)2 X2 + 2(b1)12 X1X2 (15)

here (b1)1, (b1)2 and (b1)12 are the second virial coefficients of theure components 1 (DDAB), 2 (HDHC) and that due to interactionf both components in the mixture, respectively. In ideal condition

ig. 8. Excess second virial coefficients, (b1)E, for different HDHC/HDC mixture com-osition.

(264 ± 1.5) × 10 (254 ± 2) × 10 (285 ± 1.5) × 100−5 (−370 ± 6.2) × 10−5 (−288 ± 5.1) × 10−5 (−167 ± 2.7) × 10−5

6 6 60.998 0.991 0.996

(b1)12 = [(b1)1 + (b1)2]/2 and Eq. (15) becomes:

(b1)id12 = (b1)1 X1 + (b1)2 X2 (16)

where (b1)id12 is the (b1)12 value in an ideal mixed monolayer.

Also, by similarity to Eq. (6) we can define the excess secondvirial coefficient of the mixed monolayer as follows:

(b1)E = (b1)12 − (b1)1 X1 − (b1)2 X2 (17)

The values of (b1)E, for all HDHC/DDAB mixture compositions,are shown in Fig. 8.

Except for XHDHC = 0.2, the values of (b1)E were negative indicat-ing net attractive interactions.

5. Concluding remarks

By joining the results of the preceding sections, a detailed pic-ture of the characteristics of HDHC–DDAB mixed monolayers maybe obtained. The behavior of such amphiphiles in monolayer wasclearly non-ideal and would be seriously influenced by the amountof HDHC molecules.

The HDHC �–A curve agrees well with those found in the litera-ture and in a previous works. Pure HDHC monolayer behaved as anexpanded film with a collapse area (Ac) of about 0.48 nm2 mole c−1,similar to that of cholanoic acid and a slightly higher than the cross-sectional area of the steroid nucleus (0.40 nm2 mole c−1). It can beinferred that at high surface pressure the steroid nucleus of HDHCassumed a vertical position remaining almost perpendicular to theaqueous interface.

On the other hand, pure DDAB showed a stable monolayer witha typical �–A curve for a liquid expanded (LE) film followed by aLE–LC (liquid condensed) transition. The Ac value was the usualobtained for double-alkyl ammonium salts (≈0.77 nm2 mole c−1).

All mixed HDHC:DDAB monolayers had a similar behaviorthat DDAB ones with a higher dependence of molecular areas oncomposition and surface pressure. This indicated the formationof a miscible monolayer. The analysis of collapse pressure val-ues (�c about 34 mJ m−2 or higher) implied that at the interfacethese molecules had relatively small hydrocarbon/water contactareas. This resulted in higher collapse pressures but lower bulkhydrophilicities.

The mixed collapse areas (Ac) were significantly different fromthose ideally computed. For the tested conditions, Ac was highlysuperior to the cross-sectional area of a steroid group and to thearea occupied by a double-alkyl chain implying less condensedmonolayers. Furthermore, high elasticity values (associated witha film that has strong cohesive interfacial structure) have beenobtained as the content of HDHC augment at the monolayer. Theseresults showed that the inclusion of bile acid type molecules hascaused the monolayer disruption and those intermolecular attrac-tive interactions stronger which agreed with the obtained values

of excess area (AE

c ). This fact would be related to the possibility ofH-bond formation between water and carboxilate and carbonilegroups in cholesteric ring and was in accord with the existenceof structured microdomains at the monolayer (determined by theanalysis of the first virial coefficient of state equation).

rfaces

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[37] A. Skibinsky, R.M. Venable, R.W. Pastor, Biophys. J. 89 (2005) 4111.[38] M. Rosoff (Ed.), Vesicles, Marcel Dekker, Inc., New York, 1996, p. 43.

P.V. Messina et al. / Colloids and Su

The miscibility of both surfactant on monolayer, their high bulkydrophobicity just with the negative values of (b1)E obtained gives to infer that net attractive interaction existed among bile acidnd double-alkyl ammonium salts molecules, being the preferen-ial mixture XHDHC = 0.6. A large diminution of �Gmix occurred at≈ 25 mJ m−2 and such parameter became more negative as sur-

ace pressure augmented. Taking into account that monolayersimic bilayers at surface pressures of �c = 35–45 mJ m−2, we are

ble to infer that net attractive interaction would favor the formu-ation of supramolecular assemblies based on mixed HDHC:DDABystems.

cknowledgements

The authors acknowledge the financial support from the Univer-idad Nacional del Sur, Agencia Nacional de Promoción Científica yecnológica (ANPCyT), Concejo Nacional de Investigaciones Cientí-cas y Técnicas de la República Argentina (CONICET), the SpanishMinisterio de Educación y Ciencia” (Project MAT 2008-04722). J.. R. Thanks “Consellería de Educación e Ordenación Universitaria

e Xunta de Galicia” and “Dirección Xeral de Promoción Científica eecnolóxica do Sistema Universitario de Galicia”. PM is an adjunctesearcher of (CONICET). MFL has a fellowship of the CONICET.

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