Mixed DPPC–cholesterol Langmuir monolayers in presence of hydrophilic silica nanoparticles

Colloids and Surfaces B: Biointerfaces 105 (2013) 284–293

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Mixed DPPC–cholesterol Langmuir monolayers in presence of hydrophilic silica nanoparticles Eduardo Guzmán ∗ , Libero Liggieri, Eva Santini, Michele Ferrari, Francesca Ravera CNR – Istituto per l’Energetica e le Interfasi, UOS Genova, Via De Marini 6, 16149 Genoa, Italy

a r t i c l e

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Article history: Received 24 October 2012 Received in revised form 19 December 2012 Accepted 10 January 2013 Available online 18 January 2013 Keywords: Langmuir monolayers Nanoparticles Lipids Lung surfactant Dilational rheology BAM Ellipsometry AFM

a b s t r a c t Langmuir monolayers of Cholesterol (Chol) and a mixture of Chol with 1,2-Dipalmitoyl-sn-glycerol-3phosphocholine (DPPC), at a ratio of 17:83 in weight, spread on pure water and on silica nanoparticle dispersions, have been investigated measuring the compression isotherms as well as the surface pressure response to harmonic area variation of the monolayer. Aim of this study was to evaluate the effects of the interaction of silica nanoparticles with Chol and the conditions for the incorporation in the monolayer. In previous works on different kind of lipid monolayers, it has been shown that hydrophilic silica nanoparticles dispersed in the sub-phase may transfer into the monolayer, driven by the interaction with the lipid molecules that make them partially hydrophobic. The results here obtained indicate that also for Chol and Chol–DPPC mixtures the presence of silica nanoparticles may have important effects on the phase behaviour and structural properties of the monolayer. As confirmed by complementary structural characterisations, BAM, AFM and ellipsometry, the principal effect of the nanoparticle incorporation is the disruption of the monolayer packing, owing to the alteration of the cohesive interactions of lipid components. © 2013 Elsevier B.V. All rights reserved.

1. Introduction In recent years the study of the phase behaviour and structure of Langmuir monolayers composed by fatty amphiphiles have undergone an intense development because these relative simple systems can be assumed as appropriate models to investigate complex structures and processes with biological implications [1,2]. It is noteworthy that the major part of the biologically relevant systems, such as the lung surfactant and the biomembranes, are composed by complex mixtures of surface active components, essentially lipids and proteins. The interaction between these different components determines the packing and orientation of the molecules at the interface [2,3]. This induces different features in the surface pressure–area (П–A) isotherms [3–5] whose study gives information of thermodynamic nature on the phase behaviour of the layer. Such information may be effectively correlated to those, of structural character, accessible by other techniques such as reflectivity techniques (neutron and X-ray) [6], Brewster Angle Microscopy and Ellipsometry [7,8], Infrared Reflection Absorption Spectroscopy (IRRAS) [9], Fluorescence Microscopy [10] or Laser Light Scattering [11].

∗ Corresponding author. Tel.: +39 010 6475731; fax: +39 010 6475700. E-mail address: [email protected] (E. Guzmán). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.01.020

One of the biologically relevant lipid systems of great interest for its implication in the respiratory function is the lung surfactant (LS) that is the complex lipid–protein film overlaying the alveolar walls and ensuring the correct mechanical behaviour during breathing. LS contains principally saturated and unsaturated phospholipids, whose the most important is DPPC (1,2-Dipalmitoyl-sn-glycerol-3phosphocholine), a smaller quantity of specific surfactant proteins and a significant amount, about 10 wt%, of Cholesterol (Chol) [12,13]. This composition ensures the proper functionalities of LS such as the lowering of the surface tension during the respiratory cycles and the mechanical stability of the lung [14,15]. In particular the role of cholesterol is important for the dynamic behaviour of the lipid film as improves the fluidity of the layer avoiding solid segregation [16]. Moreover, the increasing production and utilization of nanomaterials in several industrial fields [17] have arisen the problem of investigating the effect of nano-particulate materials into the environment [18]. This makes interesting the study of the potential impact of nanoparticles on the behaviour and structure of lipids layers which can be considered as models of biological structures. These investigations take special significance since several works have pointed out that the surface segregation of nanoparticles strongly modifies the thermodynamic and kinetic behaviours of surfactant systems, influencing both the interfacial tension and the dilational rheology [19–23]. This makes the study of the

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interactions between nanoparticles and lipid monolayers a point of essential relevance for assessing on a more fundamental basis the potential adverse effects of nanoparticles on the physiological function of different relevant bio-membranes and/or bio-fluids. In previous works, the effect of hydrophilic silica nanoparticles on the properties of lipid monolayers has been investigated for the mixed systems DPPC–Palmitic Acid [24] and DPPC–1,2Dioleoyl-sn-glycero-3-phosphocholine (DOPC) [25]. These systems were studied from the thermodynamic and structural point of view, using a Langmuir trough technique coupled with Brewster Angle Microscopy, ellipsometry and Atomic Force Microscopy. These lipid mixtures present an important relevance in the field of LS [12]. In the present work, the effect of silica nanoparticles in Langmuir monolayers of Cholesterol (Chol) and a mixture, 17:83 in weight, of Chol and DPPC is analysed. These lipids in fact, in this proportion, constitute about the 50% in weight of the total composition of the LS in mammals [12,15]. Moreover Chol is a very important component of different biological systems due to its ability of modulating the properties and the structure of interfacial layers and membranes and for that is one of the most studied lipids in the literature [26–28]. This study is especially aimed at investigating the nanoparticle effects on the phase properties and mechanical behaviour of monolayers containing Cholesterol. For this reason these results complement those obtained in previous works on more simple lipid systems [29,30]. 2. Materials and methods 2.1. Materials 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and Cholesterol (Chol), were purchased from Sigma (Germany) at 99% purity and used without further purification. The molecular weights of these lipids are 734.1 g/mol and 386.7 g/mol for DPPC and Chol respectively. Solutions of lipids for the spreading were prepared using chloroform for HPLC from Sigma (Germany). The absence of surface active impurities in the spreading solvent was tested by the evaluation of the compression isotherm of the bare solvent at the air/water interface. Dispersions of silica nanoparticles at 1 wt% were obtained by diluting a commercial colloidal dispersion of spherical silica particles, Levasil 200/30 (H.C. Starck – Germany), presenting a narrow size distribution around an average diameter of 30 nm and high stability without the addition of any stabilizing components. These characteristics were checked by Dynamic Light Scattering and potential measurements which provided a hydrodynamic radius of 15 ± 2 nm and  = −42 ± 1 mV for the colloidal dispersion [31]. Water was deionized and purified by a multi-cartridge, Elix plus Milli-Q (Millipore) system, providing a resistivity greater than 18 M cm. Its purity was checked by surface tension measurements, which provided a value of 72.5 ± 0.2 mN/m, at 20 ◦ C, without any appreciable kinetics over several hours. The same constant value is found for the silica dispersions [20], proving the absence of surfactant impurity in the bare dispersion and that nanoparticles, being highly hydrophilic, do not segregate at the clean water/air interface. 2.2. Methods All reported experiments have been performed using a Langmuir through (KSV minitrough, Finland) with a total area of 243 cm2 , equipped with two hydrophilic Delrin® barriers allowing symmetric compression/expansion of the free liquid surface. The surface tension, , was measured by a paper Willhelmy

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plate (Whatman CHR1 chromatography paper, effective perimeter 20.6 mm, supplied by KSV), ensuring a zero-angle contact angle. Surface pressure is then obtained as ˘ =  w − , where  w is the surface tension of pure water. The spreading of lipids as Langmuir monolayer was made using a Hamilton syringe to drop a controlled volume of lipid solution on the aqueous sub-phase, pure water or nanoparticle dispersion. From this volume and the solution concentration (typically 1 g/L), it is then possible to control the number of molecules present on the surface after evaporation of the solvent. After the deposition, and before starting any experiments, the monolayer was left to equilibrate for 1 h. This time was checked to be sufficient for the complete evaporation of the solvent and the equilibration of the nanoparticles-lipid layers. The surface pressure–area (˘–A) isotherms was determined by measuring the surface pressure during a controlled compression of the monolayer free area, at a rate of 2 cm2 /min that is equivalent to an area deformation rate, d(A/A0 )/dt, of about 3 10−5 s−1 . A Brewster Angle Microscope, BAM, (Multiskop, Optrel, Germany) is also implemented coupled with the Langmuir trough in order to obtain morphological information on the spread layers. The same device allows performing ellipsometry experiments to estimate the variation of the surface layer thickness during compression [32–34]. Ellipsometry experiments were performed at a fixed angle of 56◦ , well above of the Brewster angle of the pure interface (53.1◦ ), in order to improve the measurement sensitivy. The Langmuir trough is moreover equipped with an automatic dipper for Langmuir–Blodgett (LB) films deposition. This device allows the transfer of monolayers onto solid substrates at controlled constant surface pressure by a vertical pulling, at constant velocity (typically 1 mm/min). This dipper device was here utilized to transfer the investigated films to a hydrophilic glass plate to be analysed by AFM. AFM images were obtained using a Veeco Nanoscope III (Digital Instrument, Santa Barbara, CA) used in air Tapping Mode. Equipped with the suitable scanner, by this technique it is possible to analyse samples in an operational area of more than 100 ␮m2 allowing to provide more details about the features of the systems under investigation. Additionally to these equilibrium characterization of the surface layers, the Langmuir trough is also used for dilational rheology measurement according to the Oscillatory Barrier Method [35,36]. This method allows the modulus of the complex dilational viscoelasticity, which is defined as the variation of the surface tension  due to the dilational deformation u = A/A, i.e. E = ∂/∂u, to be evaluated against the frequency. Details on the specific techniques used in this work are reported in ref. [30]. The measurement is based on the acquisition of the surface pressure response to small amplitude sinusoidal variation of the surface area. The measurements were here performed in a frequency range from 10−3 to 0.15 Hz with a fixed amplitude, u = 0.01, which ensures, as discussed in Ref. [30], the linearity of the layer response.

3. Results and discussion 3.1. Effect of nanoparticles in the equilibrium properties of cholesterol monolayers In this section the changes induced by the presence of nanoparticles on the equilibrium properties such as ˘–A isotherm, quasi static elasticity and surface structure, of Cholesterol Langmuir monolayers are analysed. Fig. 1 reports the compression isotherms obtained for Chol monolayers spread on water and on silica nanoparticles dispersion (concentration ∼1 wt%). The initial area per molecule of the Chol

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50 45

Π/mN·m-1

40

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15

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0

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60

A per molecule/Å2

Chol Chol+NP

10

0 0.5

0.6

0.7

0.8

0.9

1.0

A/Å2 Fig. 1. Surface pressure–area isotherms for Chol Langmuir monolayers spread on water subphase and on 1 wt% silica nanoparticles dispersion. A0 = 243 cm2 is the initial surface area. In the insert the same surface pressure data acquired for pure water are plotted against the area per Chol molecule.

monolayer before the compression were in all cases 75 A˚ 2 obtained by the deposition of 21 ␮g of Chol (21 ␮l of 1 mg/mL lipid solution) on the aqueous surface of initial area A0 = 243 cm2 . The feature of the isotherm of Chol spread on a water subphase is in agreement with those reported in ref. [37]. It is characterized by a large region at area per molecule larger than 50 A˚ 2 (see insert of Fig. 1) where the monolayer behaves as a gaseous phase. With further compression a sudden increase of the surface pressure is observed till the collapse at П ∼ 45 mN m−1 . This latter is the typical behaviour of a solid-like monolayer with a high rigidity. During the compression, the reorientation of the Chol molecules at the interface progresses achieving a close packing conformation where the lipid molecules are oriented perpendicular to the interface [38]. The BAM images (Fig. 2) obtained under different compression degrees of the monolayer evidences that Chol presents a homogeneous phase during the whole phase diagram. This is coherent with the existence of a uniform gaseous phase that continuously changes into a total condensed phase [39]. The formation of this latter is related to the strong cohesive Van der Waals interactions.

When the Chol monolayer is spread on silica nanoparticles dispersion, the compression isotherm presents a different feature, as similarly observed for other lipid monolayers in previous works [24,25], that may be ascribed to the transfer of silica nanoparticles into the interfacial layer. The most noticeable effect of the presence of nanoparticles in Chol monolayers is that the surface pressure starts to increase at lower compression degree or, in other words, the ˘–A isotherm is shifted towards the higher areas. This effect together to the reduction of the collapse pressure may be discussed in analogy with what done for other lipid systems [24,25] as driven by the interaction between the nanoparticles colliding with the surface layer and the lipid molecules. However, here, the interaction between nanoparticles and Chol does not have an electrostatic origin as in the case of DPPC [24] and DOPC [25], but it is driven by the formation of hydrogen bonds between the non-dissociated silanol groups on the surface of the nanoparticles and the hydroxyl group of the Chol molecules. It is in fact well known [16] that the Chol forms insoluble monolayers where the molecules, upon compression, tend to orient themselves with the polycyclic hydrophobic part towards the air phase whereas the hydroxil group remains in the water phase. This mechanism of nanoparticle–lipid monolayer interaction is similar to that previously investigated for palmitic acid–silica nanoparticles system [23]. Also in this case, after interacting with the monolayer, the nanoparticles form partially hydrophobic complexes but, while with palmitic acid part of these complexes are transferred back to the aqueous sub-phase, this does not occur here due to the higher hydrophobicity of Chol. The particle trapped in this way at the interface induce a steric hindrance to compression which leads to the increase of П at areas per molecule higher than those of pure Chol. Under these conditions, the maximum packing of the Chol molecules in the monolayer is achieved for an area per molecule higher than in the case of the pure system. This also explains the decrease of the collapse surface pressure. The BAM images (Fig. 2) show a homogeneous phase texture both in absence and presence of nanoparticles and this is also confirmed by the AFM images of the LB films obtained at the surface pressure of 7.5 mN/m (Fig. 3). The AFM study of Chol monolayers (Fig. 3a) evidences a single phase structure with an average height in the range of the molecular size. In presence of nanoparticles (Fig. 3b), coherently with the BAM images and the compression isotherm, the main feature of the Chol structure is not affected. However, the two characteristic heights observed in the film, the

Fig. 2. BAM images sequences (images size = 311 ␮m × 418 ␮m) of Chol monolayers spread on water subphase and on 1 wt% silica nanoparticles dispersion for different compression degrees.

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δΔ/deg

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a)

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Fig. 4. ı dependence on the surface pressure for monolayers containing Chol in absence and presence of nanoparticles. (a) Chol. (b) DPPC + Chol. Lines are only guides for the eyes.



ε0 = −A

∂˘ ∂A



(1) T

500 500

400

ε0/mN·m

first one in the range of the Chol molecular size and the second one in the range of the nanoparticle size, evidence the penetration of nanoparticles that leads to the formation of nanoparticles + Chol complexes at the interface. Another evidence of the penetration of nanoparticles in the Chol monolayer is provided by ellipsometry experiments. This technique allows evaluating the thickening-thinning of the monolayer as a function of the surface pressure due to the interaction with nanoparticles. A qualitative approach to this problem is the evaluation of the changes in the ellipsometric angle . A decrease in  corresponds to the increase in the monolayer thickness [40–42]. Fig. 4 shows the changes of  as referred to the clean interface (without lipids), ı. The results obtained using ellipsometry evidences, for all the monolayers, a decrease in ı with the increase in the surface pressure which is a strong indication of a monolayer thickening. This can be ascribable to the reorientation of the lipid molecules at the interface. In presence of nanoparticles, ı results always to be higher in absolute value. This can be considered as an additional evidence of the nanoparticles penetration into the lipid film. From the ˘–A isotherms above discussed, it is possible to determine the dilational elasticity, ε0 , under quasi-static compression of the surface area,

250

-1

Fig. 3. AFM images of LB films deposited at ˘ ∼7.5 mN/m. (a) Chol. (b) Chol + NP. (c) DPPC + Chol. (d) DPPC + Chol + NP.

This is an important characteristics related to the elastic energy stored by the monolayer when it is continuously compressed and provides information about its rigidity. For isothermal compression of the monolayer at very low constant rate, this quasi equilibrium quantity can be evaluated by the numerical derivative of the П–A isotherms obtained in the Langmuir trough. Fig. 5 shows the variation of ε0 during the compression and as a function of ˘, obtained for Chol monolayers spread on water and on silica nanoparticles dispersion. When the Chol is spread on pure water, ε0 presents a suddenly increase with the increase of the surface pressure until a maximum value of ε0 around 500 mN m−1 , in according with the high cohesion of closed-packed Chol monolayers that allows classifying the Chol monolayers as solid-like films. When

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Π/mN·m-1 Chol Chol+NP

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0 0.5

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A/A0 Fig. 5. Quasi-static dilational elasticity calculated from ˘–A isotherms for Chol Langmuir monolayers spread on water subphase and on 1 wt% silica nanoparticles dispersion calculated from the ˘–A isotherms of Fig. 1.

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DPPC Cholesterol DPPC+Cholesterol

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Fig. 6. П–A isotherms of DPPC, Chol and DPPC + Chol (ratio 83:17 in weight) Langmuir monolayers.

0

the Chol is spread on silica nanoparticles dispersion, the elasticity curve presents similar feature to that in the case of Chol spread in water but the values of elasticity are lower. This is coherent with assumption of a steric hindrance to the molecular packing and the consequent decrease of the rigidity of the monolayer.

40

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A per molecule/Å2 Fig. 8. Quasi-static dilational elasticity for DPPC (black), Chol (light grey) and DPPC + Chol (ratio 83:17 in weight, dark grey) Langmuir monolayers, calculated from the ˘–A isotherms of Fig. 6.

3.2. Influence of cholesterol on the equilibrium properties of DPPC monolayers The ˘–A isotherm for a mixed Chol–DPPC (17:83) monolayer spread on water is presented in Fig. 6 together with those of the pure components. The phase behaviour of the mixture is significantly different from that of the pure lipids. The introduction of Chol to DPPC monolayers leads to the disappearance of the characteristic plateau corresponding to the LE-LC phase coexistence [43,44], behaving the monolayer as an expanded phase in this part of the phase diagram. The above features result from modifications in the monolayer texture as it is shown in the BAM images (Fig. 7). The introduction of Chol makes less evident the typical LC domains ascribable to DPPC [24] and the interfacial texture can be considered as an almost homogeneous phase during the whole compression. This is due to the molecular re-organization inside the mixed Chol–DPPC monolayer. In fact, due to their high

hydrophobicity, the Chol molecules tend to distribute among the DPPC molecules in order to minimize the contact of the hydrophobic sterol ring with water [16]. This distribution is energetically favoured and leads to a highly ordered structure which is strongly stabilized by the van der Waals cohesive interactions between the sterol rings of Chol molecules and the alkyl chains of DPPC. Under these conditions, the nucleation of DPPC domain is hindered in favour of the formation of a quasi-homogeneous condensed phase [37]. In practice Chol has a condensing effect on DPPC that induces an increased order in the monolayer. This is also confirmed by the AFM images of the LB film (Fig. 3c) obtained at ˘ = 7.5 mN/m, where a rather homogeneous network of aggregate phase can be observed. This is also reflected in the quasi-equilibrium dilational elasticity (Fig. 8) which presents the typical behaviour of a fluid-like system with maximum ε0 around 150 mN m−1 . Such reduction of

Fig. 7. BAM images sequences (images size = 311 ␮m × 418 ␮m) for the monolayers of the mixture DPPC–Chol (87:13) spread on water subphase and on silica nanoparticles dispersion (NP, concentration ∼1 wt%) for different compression degrees.

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100

DPPC+Cholesterol DPPC+Cholesterol+NP

45

30

A per molecule/Å2

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Aideal(DPPC+Chol) Aideal (DPPC+Chol+NP) A12(DPPC+Chol) A12(DPPC+Chol+NP)

90 80 70 60 50

a)

40

15

0 0.4

0.6

0.8

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A/A0 Fig. 9. П–A/A0 isotherms for DPPC + Chol Langmuir monolayers spread on water and on 1 wt% silica nanoparticles dispersion.

Aex per molecule/Å2

8 6

DPPC+Chol DPPC+Chol+NP

4 2 0

b)

-2

elasticity compared to DPPC, points out the fluidizing effect of Chol in DPPC monolayers reducing the elasticity of the films. 3.3. Effect of nanoparticles in the equilibrium properties of DPPC–Chol monolayers Fig. 9 reports, for sake of comparison, the isotherms of the mixture spread on pure water and on nanoparticle dispersion. The presence of nanoparticles shifts the ˘–A isotherm to higher values of the area per molecule. This is explained considering that the nanoparticles occupy the available area of the interface increasing, as a matter of fact, the local lipid surface concentration. Additionally, the change in the slope of the isotherm and the small increase in the collapse pressure observed in presence of nanoparticles suggest that some modifications in the orientation of the molecules at the interface may occur. This is related to the intricate balance of interactions proper of this mixed monolayer where the penetration of the nanoparticles induces complex refinement processes which may influence both the structure and the composition of the monolayers. To explain this it is necessary to consider the different interactions between the monolayer components. The introduction of small quantities of Chol to DPPC monolayers, as discussed above, due to the strong interaction between the sterol chain and the alkyl chains of DPPC molecules, induces a high cohesion between the molecules at the interface. The electrostatic interaction of DPPC with silica nanoparticles, responsible of the formation of lipid-nanoparticles complexes, induces the disruption of the interfacial structure of the monolayer [29,45]. Whereas the interaction between Chol and nanoparticles leads to an important steric hindrance to the packing density of the Chol. This allows proposing that the refinement process induced by the presence of nanoparticles consists in a re-arrangement of the mixed monolayers in a three dimensional structure composed by lipid-nanoparticle complexes that allows higher surface pressures, and collapse pressures, to be achieved during compression. The nanoparticle penetration into the mixed monolayer is also supported by AFM analysis and ellipsometry measurements. In fact, even if the presence of nanoparticles does not change appreciably the morphology of the lipid layer, their penetration is evidenced by the presence of two different characteristic heights in the layer profile (Fig. 3d). It is worth mentioning that in this case, the nanoparticles are more homogenously distributed along the monolayer and less aggregated compared with the pure Chol monolayer.

0

10

20

30

40

Π/mN·m-1 Fig. 10. For different П states of the mixed monolayer DPPC + Chol in absence and presence of nanoparticles. (a) Values of Aideal and A12 . (b) Values of Aex .

Ellipsometry experiments evidences the thickening induced by the nanoparticles on DPPC + Chol monolayers which is bigger than that observed for pure Chol system (Fig. 4). This may be explained considering the stronger electrostatic interaction of SiO2 with DPPC, with respect to the hydrogen bond established between SiO2 and Chol. The changes in the behaviour and cohesion of the monolayer due to the presence of nanoparticles can be analysed based on concepts from the thermodynamics of mixtures [46]. For an ideal mixture, it is possible to define the area per molecule at a given surface pressure, Aideal , as Aideal = XDPPC ADPPC + XChol AChol

(2)

where ADPPC and AChol are the areas per molecule of pure DPPC and Chol in the monolayer at the considered П and XDPPC and XChol are the respective molar fractions. The deviation from the ideal behaviour of the real mixture can be evaluated using the excess area, Aex , Aex = A12 − Aideal

(3)

where A12 is the experimentally evaluated area per molecule in the mixed monolayer at a given value of П. This quantity provides an estimation of the cohesive interaction between the molecules of different species or, in other words of the mutual miscibility. For an ideal mixed monolayer, or vanishing interaction, Aex is zero. Fig. 10 shows the values of Aex for different states of the mixed monolayer, corresponding to different values of the surface pressure, in absence and presence of nanoparticles. In absence of nanoparticles, the dependence of Aex on the surface pressures allows the variation of the cohesive interaction between DPPC and Chol, or their mutual miscibility, to be evaluated during the monolayer compression. For low П, the mixed monolayer shows that the lipids are immiscible while, when П increases the values of Aex become negative which implies an attractive interaction between Chol and DPPC. This can be explained considering that for low degree of compression the high volume of the sterol rings leads to an important steric hindrance that weakens the cohesion between the molecules. However, with the advancing of the

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dilational elasticity discussed above, which can be associated to a frequency of 10−5 Hz corresponding to the surface deformation. The interpretation of these data has been performed by using a theoretical model giving an expression for the complex dilational viscoelasticity which, in terms of surface pressure variation with the surface area, is defined as E = −A0 ∂˘/∂A. This model accounts for dynamic relaxation processes which occur within the interfacial layer [47–51] and provides the following expression for the complex dilational viscoelasticity versus frequency

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ε0/mN·m-1

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E = E0 +

-1

Π/mN·m

DPPC+Chol DPPC+Chol+NP

50

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0.8

1.0

A/A0 Fig. 11. Quasi-static dilational elasticity calculated from ˘–A isotherms for DPPC–Chol Langmuir monolayers spread on water subphase and on silica nanoparticles dispersion, calculated from the ˘–A isotherms of Fig. 6.

compression and the subsequent reorientation of the molecules, the Van der Waals cohesive interactions between the sterol rings of Chol and the alkyl chains of the DPPC molecules are strongly increased. The scenario significantly changes in presence of nanoparticles where Aex assumes positive values for the whole range of the area variation, without any important dependence on the surface pressure. This may mean that nanoparticles induce repulsive interactions between DPPC and Chol resulting in the disruption of the interfacial structure. However, as in this case the real system is a ternary one, where the real concentration of nanoparticles is not exactly known, Aexc has to be considered as an effective parameter. In fact in presence of nanoparticles the system behaves as composed by two lipids mutually immiscible. As shown in Fig. 11, the presence of silica nanoparticles does not modify significantly the feature of ε0 vs. ˘, but it induces a general increase of its values. This is coherent with the assumption of important modifications induced by the nanoparticles on the packing of the lipid molecules at the interface and on the refinement processes. In fact, the increase of the collapse pressure and quasi-equilibrium elasticity of the mixed layer in presence of nanoparticles, which are unusual effects with respect to other lipidnanoparticle systems previously investigated [24,25], recalls for a scenario where the nanoparticles, through a refinement process, induce a re-arrangement of the monolayer in a structure characterized by a higher order degree. 3.4. Effect of nanoparticles in the low frequency dilational rheology of DPPC–Chol monolayer In this section, the effects of nanoparticles on the dynamicmechanical properties of the mixed DPPC–Chol monolayers are analysed by means of dilational viscoelasticity measurements. In our previous works [29,30] it has been evidenced how this kind of rheological investigation are effective to reveal the occurring of surface dynamic processes in the mixed lipid monolayer. Fig. 12 report the modulus of the dilational viscoelasticity, |E|, against frequency obtained for DPPC–Chol monolayers spread on water and on silica dispersions, for different values of the reference П and, then, to different states of the monolayers structure. The latter correspond to different degrees of compression of the surface layer. These data include also the values of the quasi-equilibrium

N  j=1

(Ej − Ej−1 )

1 + i j 1 + j

2

(4)

where j = j / and j is the characteristic frequency of the j-th process occurring in the interfacial layer, and Ej are thermodynamic parameters related to limit elasticities. In particular E0 is the limit of the elasticity when the frequency tends to zero and EN the high frequency limit elasticity. The dilational viscoelasticity obtained for DPPC + Chol monolayers spread on water and onto silica nanoparticles are interpreted by fitting the expression of the dilational viscoelasticity modulus calculated by Eq. (4). This allowed relaxation processes with characteristic frequencies within the investigated frequency range, to be evidenced by the presence of inflection points in the best fit curve. As shown in Fig. 12, the measured values of the modulus of the dilational viscoelasticities are fairly described by this model with the best fit parameters reported in Fig. 13. The model evidences the presence of one or two relaxation processes, depending on the reference monolayer state. As general trend, the introduction of nanoparticles modifies both the limit elasticities and the frequency of the rheological response. Concerning the limit elasticities, both E0 and E1 tend to increase with the surface pressure, as it is expected for systems at increasing packing and, coherently with the above discussion, such increase is enhanced in presence of particles. In absence of nanoparticles a kinetic surface process with characteristic frequency around 0.1 mHz is detected for all the reference states investigated. An additional kinetic process with higher value of the characteristic frequency (∼10 mHz) appear when the monolayer is further compressed, at reference ˘ = 25 mN/m and higher. This behaviour can be explained considering the mutual miscibility of the lipids described above (see Fig. 10). For ˘ < 25 mN/m, the lipid molecules present repulsive interactions (Aex > 0). Thus the monolayer is in an expanded phase state where the kinetic process at low characteristic frequency can be attributed to a surface re-organization with molecular re-orientation occurring in long time scale. The increase of the surface pressure beyond a threshold value, larger than ˘ = 20 mN/m, induces attractive interactions with the consequent enhancing of the lipid miscibility. This makes the monolayer a more aggregate phase as evident also by the slight change of slope in the ˘–A isotherm reported in Fig. 6. Under these conditions the kinetic process with higher characteristic frequency, of the order of 10 mHz, may be associated to a change of state for the lipid molecules in the monolayer that means that some molecules may pass alternately from aggregated to free states during oscillations. In presence of nanoparticles, the features of the dilational viscoelasticity are in general maintained despite some specific effects due to the nanoparticles penetration. In this case, in fact, the first process detected presents values of characteristic frequency in the range 0.1–10 mHz with a significant dependence on the compression degree of the monolayer. This process can be ascribed to the exchange of lipid molecules between the surface of the nanoparticles and the fluid interface but also to re-orientation of lipid molecules at the free liquid surface. A possible explanation of

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400

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Fig. 12. Modulus of the dilational viscoelasticity against frequency by oscillatory barrier experiments for DPPC + Chol monolayers spread on water (a) and on 1 wt% silica nanoparticle aqueous dispersion (b) at different values of ˘, corresponding to different compression states. Lines represent the best fit theoretical curves according to Eq. (4).

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Fig. 13. Best fit parameters obtained using Eq. (4) for DPPC + Chol mixed monolayers spread on water (black symbols) and on 1 wt% silica nanoparticles dispersion (open symbols) (a) low (circles) and high (triangles) frequency limit elasticities vs. surface pressure, (b) characteristic frequencies of the dynamical processes, 1 (circles) and 2 (triangles). The lines are guides for the eyes.

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the trend of the characteristic frequencies, reported in Fig. 13 for ˘ < 20 mN/m, can be that these two processes have comparable characteristic times and consequently appear as single processes. The observed decrease of 1 with the compression degree means that the molecular surface re-orientation process becomes slower with the compression because of the induced increase of the surface concentration. The higher frequency process appearing at ˘ = 20 mN/m can be assumed to be the same, above mentioned, lipid molecule exchange between nanoparticle and free liquid surface that, in this case, has a characteristic time significantly different from the molecular re-orientation process. 4. Conclusions The effect of hydrophilic silica nanoparticles on phase behaviour and structure of lipid monolayer containing Chol is investigated in a Langmuir trough equipped with a Brewster Angle Microscopy, an ellipsometer and a dipper for LB deposition. Different appreciable effects have been evidenced: those of the nanoparticles on the Chol monolayer, of cholesterol in the DPPC monolayer and the change in the properties of the mixed Chol–DPPC monolayers due to the penetration of nanoparticles. The penetration of nanoparticles into the monolayer is essentially driven by the interaction with the lipid molecules. The principal consequence of this incorporation is the modification in the cohesion between the lipid molecules which affects the packing and, consequently, the phase behaviour and rheological response of the mixed systems. Although obtained for lipid model systems, the results here obtained can be useful for a better comprehension of the interaction between particulate materials and lung surfactants or cell membranes whose composition can be, in a first approximation, assimilated to that of the lipid systems here investigated. In particular these results, especially focused on Cholesterol, complement those obtained in previous studies concerning other lipid mixtures. In particular, as already underlined in our previous works on this subject, the study of the rheological and equilibrium properties of such systems may be important to evaluate the potential adverse effect of the nanoparticles on the behaviour of biological relevant layers. Acknowledgements This work was financially supported by IIT – Istituto Italiano di Tecnologia within the Project SEED 2009 “Nanoparticle Impact of Pulmonary Surfactant Interfacial Properties – NIPS” and carried out in the framework of the ERF COST actions CM1101 “Colloidal Aspects of Nanoscience for Innovative Processes and Materials”. References [1] V.M. Kaganer, H. Möhwald, P. Dutta, Structure and phase transitions in Langmuir monolayers, Rev. Mod. Phys. 71 (1999) 779–819. [2] N. Nandi, D. Vollhardt, Effect of molecular chirality on the morphology of biomimetic Langmuir monolayers, Chem. Rev. 103 (2003) 4033–4076. [3] H. Möhwald, Phospholipid and phospholipid–protein monolayers at the air/water interface, Annu. Rev. Phys. Chem. 41 (1990) 441–476. [4] H.M. McConnell, Structures and transitions in lipid monolayers at the air–water interface, Annu. Rev. Phys. Chem. 42 (1991) 171–195. [5] C.M. Knobler, R.C. Desai, Phase transitions in monolayers, Annu. Rev. Phys. Chem. 43 (1992) 207–236. [6] K.Y.C. Lee, A. Gopal, A. von Nahmen, J.A. Zasadzinski, J. Majewski, G.S. Smith, M. Lujan Jr., P.B. Howes, K. Kjaer, Influence of palmitic acid and hexadecanol on the phase transition temperature and molecular packing of dipalmitoylphosphatidyl–choline monolayers at the air–water interface, J. Chem. Phys. 116 (2002) 774–783. [7] S. Sundaram, J.K. Ferri, D. Vollhardt, K.J. Stebe, Surface phase behavior and surface tension evolution for lysozyme adsorption onto clean interfaces and into DPPC monolayers: theory and experiment, Langmuir 14 (1998) 1208–1218.

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