DPPC–DOPC Langmuir monolayers modified by hydrophilic silica nanoparticles: Phase behaviour, structure and rheology

Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

DPPC–DOPC Langmuir monolayers modified by hydrophilic silica nanoparticles: Phase behaviour, structure and rheology 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

i n f o

Article history: Received 10 November 2011 Received in revised form 12 December 2011 Accepted 21 December 2011 Available online 27 December 2011 Keywords: Lung surfactant Lipids Silica nanoparticles Langmuir monolayers Compression isotherms Langmuir–Blodgett deposition Atomic force microscopy Dilational rheology Total harmonics distortion

a b s t r a c t Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and mixtures of DOPC with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), at a ratio of 37:63 in weight, spread on pure water and on silica nanoparticle dispersions, have been investigated using a combination of thermodynamic, surface rheology, BAM and AFM diagnostics. The compression surface pressure isotherms were determined in a Langmuir trough as well as the surface pressure response to harmonic area variation of the monolayer. Composite layers were obtained at selected thermodynamic states by transfer from the fluid interface to solid substrates and then analysed by AFM diagnostics. Aim of this study was to evaluate the effect of the incorporation of silica nanoparticles on the phase behaviour and structural properties of these monolayers. In fact, as shown in previous works on similar lipid systems, the hydrophilic silica nanoparticles dispersed in the sub-phase are transferred into the monolayer due to the interaction with lipid molecules which makes them partially hydrophobic. The results here obtained indicate that the appreciable influence of silica nanoparticles, previously observed for DPPC alone, is also important for DOPC and DOPC–DPPC mixture. Moreover, as confirmed by the AFM results on the deposited layers, these effects are mainly due to the disruption of the molecular packing and to the modification of the miscibility between the two lipid components. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The effect of additional components, such as solid nanoparticles, on the properties of Langmuir monolayers of fatty amphiphiles is a topic of increasing interest because of its application as model in several fields involving biological systems, biomembranes response [1] or respiratory physiology [2,3]. Lipid monolayers have been widely investigated by the analysis of the surface pressure–area (˘–A) isotherms [1,4–6] which allows structural features to be identified through essentially thermodynamic information. The phase behaviour of these systems is in fact related to the structural changes induced by the molecular lateral packing in the monolayer [7,8], which is modified by the increasing of the lipid surface concentration. The structure of these monolayers has been investigated in many works using in situ diagnostic techniques, such as X-ray diffraction [9], infrared reflection absorption spectroscopy (IRRAS) [10], fluorescence microscopy [11], laser light scattering [12], and Brewster Angle Microscopy (BAM) [13,14]. The interaction and/or incorporation of external components modifies the phase behaviour and the structure of these monolayers essentially because of the effects on the lateral packing. This is

∗ Corresponding author. Tel.: +39 010 6475731; fax: +39 010 6475700. E-mail address: [email protected] (E. Guzmán). 0927-7757/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2011.12.059

of particular importance for biologically relevant structures which are mostly composed by complex mixtures of surface active components [15], among them, the lung surfactant (LS) [2]. The study of the impact of nanoparticles on the respiratory functionality is a topic of extreme interest in relation to environmental particulate and to the increasing utilisation of nanomaterials [16–19]. Considering that, as show in previous works [20–24], the segregation of nanoparticles at the liquid surface influences the interfacial tension and the dilational rheology of surfactant systems, it is clear the importance to investigate the effects on the surface properties and dilational rheology of lipid Langmuir monolayers, to understand the potential negative effect on the respiratory functionality. LS is in fact a complex mixture of lipids and proteins whose surface tension changes during the respiratory cycle [2,25] ensuring suitable mechanical properties to the lungs. In a previous work [26] the effect of nanoparticles on the properties of lipid monolayers has been investigated spreading 1,2dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and palmitic acid on silica nanoparticle dispersions. These systems were studied from the thermodynamic and structural point of view, using a Langmuir trough technique coupled with Brewster Angle Microscopy diagnostics. The above lipids are relevant in the field of LS. In the present work, the effect of silica nanoparticles has been similarly analysed for Langmuir monolayers of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) and a mixture, 63:37 in weight,

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

of DOPC and DPPC. These lipids are in fact two of the essential components of the LS [2,3] and, in the above proportion, constitute about the 65% in weight of the total composition of the LS in mammals [2]. Besides the thermodynamic study of the lipid monolayers in the Langmuir trough coupled with BAM, we report here a deeper structural investigation based on AFM analysis of Langmuir–Blodgett films obtained by a controlled transfer of the mixed monolayers on solid substrates. The last aspect investigated in this work is the effect of nanoparticles on the surface pressure response to oscillatory variation of the surface area. As already observed in previous works on similar systems [27], nanoparticles may have appreciable effects on the linearity of such surface pressure response when the amplitude of the perturbation increases and becomes comparable with that proper of the respiratory cycles [2,25]. 2. Materials DOPC and DPPC were purchased from Sigma (Germany) with a purity higher than 99% and used without further purification. The molecular weights of these lipids are 786.1 and 734.1 for DOPC and DPPC, respectively. Solutions of lipids for the spreading were prepared using chloroform for HPLC from Sigma (Germany). Dispersions of silica nanoparticles at 1 wt% were obtained by diluting a commercial colloidal dispersion of spherical silica particles, 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 an hydrodynamic radius of 15 ± 2 nm and  = −42 ± 1 mV for the colloidal dispersion [28]. Water was deionised 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 [21], 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. 3. Methods The Langmuir trough used for all the reported experiments was a KSV minitrough (Finland), equipped with two hydrophilic Delrin® barriers allowing symmetric compression/expansion of the free liquid surface. The surface tension, , was measured by a paper Willhelmy 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. 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 [13,14]. 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 nanoparticle–lipid layers.

175

The surface pressure–area (˘–A) isotherms were determined by measuring the surface pressure during a controlled compression of the monolayer free area, at a rate of 2 cm2 /min, equivalent to an area deformation rate, d(A/A0 )/dt, of about 3 × 10−5 s−1 . The Langmuir trough was also used to measure the surface pressure response to harmonic variations of the surface area, A, at controlled amplitude, (A, and frequency, , A(t) = A0 + A sin(2t)

(1)

where A0 is the initial area. This dilational rheology study was performed at increasing deformation amplitude in order to investigate the linearity of the monolayer behaviour. Under viscoelastic linear regime, usually obtained at small amplitude sinusoidal variation of the surface area, the surface pressure response presents a sinusoidal profile as well as the same frequency, ˘(t) = ˘ 0 + ˘ sin(2t + ϕ),

(2)

where ˘ is the amplitude of the response and ϕ a phase shift accounting for a possible viscous character of the layer. Increasing the deformation amplitude, distortions in the response signal may appear which means that the response is no longer linear [29]. The appearance of harmonics of higher order than the fundamental frequency, indicating the non-linearity of the response, are effectively evaluated by analysing the Fourier transform (FFT) of the surface pressure response [30]. A quantitative evaluation of the response non-linearity is provided then by the total harmonic distortion (THD) [31,32], defined as



THD =

k>1

 k 2

1

(3)

 k are the k-Fourier coefficients, that is the amplitude of the korder harmonics in the representation of the signal as a Fourier sum,  1 being, in particular, the amplitude of the fundamental harmonic. Linear systems present responses with vanishing THD, while larger values are associated to an increasingly non-linear behaviour. The Langmuir trough is equipped with an automatic dipper for Langmuir–Blodgett (LB) films deposition onto solid substrates. This device allows the controlled transfer of the monolayer on solid substrates while keeping constant the surface pressure by the movement of the barriers. The dipper was utilised to transfer the LB film on a hydrophilic glass plate of dimensions 20 mm × 20 mm, by a vertical pulling, at a constant velocity of 1 mm/min. AFM images of the LB films were obtained using a Veeco Nanoscope III (Digital Instrument, Santa Barbara, CA). This allows a sample up to about 1 cm2 to be fitted in the operational stage with a maximum scanning area of about 12 ␮m and a Z resolution of about 4 ␮m for imaging. It has been used in air Contact Mode with minimum force and when it was necessary in Tapping Mode. For all the reported experiments the temperature was at a controlled value of 22.0 ± 0.1 ◦ C. 4. Results and discussion 4.1. Equilibrium properties and structure of lipid monolayers Fig. 1 reports the ˘–A isotherms obtained for DOPC monolayer and for the DOPC–DPPC, 37:63 in weight, mixed monolayer. The ˘–A isotherm found in Ref. [27] for pure DPPC is also reported for sake of comparison. The phase behaviour of DPPC monolayer has been widely investigated [33–37]. The typical characteristics of the DPPC isotherm are evident. These are the compression of the liquid expanded (LE) phase at high area per molecules, the surface pressure plateau corresponding to the coexistence of the disordered LE phase with the highly ordered liquid-condensed (LC) phase and the

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

Acollapse per molecule /Å 2

176

Π / mN/m

60

40

48 46 44 42 40 38 36 34 0.0

0.2

20

0

0.4

0.6

0.8

1.0

xDPPC DPPC DOPC DOPC+DPPC

40

60

80

A per molecule/Å2 Fig. 1. ˘–A isotherms for DPPC, DOPC and DPPC + DOPC (ratio 63:37 in weight) Langmuir monolayer. The inset represents the relation between the Acollapse and the molar fraction of DPPC in the monolayer (1 Pure DPPC and 0 pure DOPC). DPPC:  0 = 1.7 ␮mol/m2 ; DOPC:  0 = 1.3 ␮mol/m2 ; DPPC + DOPC:  0 = 1.5 ␮mol/m2 .

compression of the LC phase with increasing surface pressure till the collapse of the monolayer, occurring at ˘ collapse ∼ 70 mN m−1 . The feature of ˘–A isotherm obtained for DOPC monolayer is in agreement with previous literature results [38]. Surface pressure increases continuously following the characteristic feature of an expanded-like monolayer, without plateau of coexistence between phases, till a collapse surface pressure of about 40 mN/m. This apparent difference in the phase behaviour of DOPC, in respect to DPPC, is related to the non-saturated character of the hydrocarbon chains of DOPC molecules which, making weaker the van der Waals cohesive interactions between the hydrophobic chains, reduces the efficiency of the molecular packing in the lipid monolayer, hindering the formation of domains of condensed phase typical of DPPC. Moreover, while the BAM images reported in Ref. [27] evidenced the nucleation and growth of bean-shaped domains of LC phase in the LE matrix, during the compression of DPPC monolayer, it was not possible to reveal any kind of condensed domains, by the same BAM imaging, during the compression of DOPC. The collapse of the DOPC monolayers occurs at a value of ˘ significantly lower than that found for DPPC monolayers and at higher area per molecule. This also indicates the lower degree of packing for the molecules in the collapse conditions [39].

Fig. 2. BAM images (=311 ␮m × 418 ␮m) for DPPC + DOPC monolayer in absence and presence of silica nanoparticles (NP), for different values of surface pressure.

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

The isotherm of mixed DPPC–DOPC (63:37) monolayers falls between those of the two pure lipids and it is in accordance with previously literature results [40,41]. Even at this monolayer composition, with a larger amount of DPPC, the plateau corresponding to the LE–LC phase coexistence was not revealed. However, the BAM images reported in Fig. 2 show that, in the mixed monolayer, the formation of LC domains occurs in similar way than what observed for DPPC alone in Ref. [27]. Considering that DPPC and DOPC at the investigated temperature are immiscible [42], the nucleation of a mixed condensed phase is not favoured. Thus the domains observed are likely DPPC domains in a LE DOPC matrix. This LC phase nucleation starts for higher values of ˘ than those required for pure DPPC monolayers. In fact, as evident from the isotherms reported in Fig. 1, higher surface pressure are necessary for the mixed monolayer to reach the area per molecule necessary for the nucleation of DPPC domains. This is because of the resistance to compression of the coexistent LE phase of DOPC. This accounts for the continuous increasing of the surface pressure during compression. It is worth noticing that there is a linear relation between the area per molecules at the collapse and the DPPC molecular fraction

177

(xDPPC ) in the monolayer (insert in Fig. 1). This relation, already found elsewhere for the same system [40,41], according to the additivity rule [43] confirms the DOPC–DPPC immiscibility and the formation of phase-separated domains in the mixed monolayer. The analysis of the topographical AFM images of LB films deposited on solid substrates provided further information about the interaction between DPPC and DOPC in the mixed monolayers. This study was performed both for pure DPPC and mixed DPPC–DOPC monolayers on LB films obtained at the same surface pressure values. Fig. 3 shows the AFM images of films obtained for ˘ = 7.5 mN/m. In DPPC monolayers (Fig. 3a), small hemispherical shaped domains with an average radius around 50 nm are found which are homogenously distributed along the monolayer structure. Notice that the scale here investigated by AFM techniques is rather different from that of the BAM images (more than one order of magnitude smaller) so that the domains here revealed can be considered to make part of the higher size domains observed in the BAM images. The height of the domains is less than 2.5 nm which is slightly smaller than the theoretical value for the length of the DPPC molecules (2.8 nm) [44], suggesting that the molecules in the LC phase are not yet in a completely vertical orientation. In presence

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

178

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

Fig. 4. AFM images of LB films deposited at ˘ ∼ 20 mN/m. (a) DPPC. (b) DPPC + DOPC. (c) DPPC + NP (Region I). (d) DPPC + NP (Region II). (e) DPPC + DOPC + NP. Note that the scale for the height profile is in nanometers.

of DOPC (Fig. 3b) domains characterised by different shapes and sizes were evidenced and the average height of these domains was found to be around 2 nm, indicating an even less vertical orientation of the lipid molecules. Being the two components immiscible, the presence of DOPC has the effect of hindering the packing of DPPC so that the formation found at this degree of compression of the monolayer are, as a matter of fact, aggregates of DPPC but not yet real LC domains. Continuing with the compression (˘ ∼ 20 mN/m), the DPPC monolayer becomes more homogeneous as result of the coalescence of LC domains (Fig. 4a), whereas in the presence of DOPC (Fig. 4b) the domains appear closer without coalescing. The average height of the structures (around 2 nm) on the monolayer confirms the effect of the immiscibility of DOPC. The close-packing of the molecules is hindered by the presence of DOPC molecules which, due to their non-saturated hydrocarbon chains, make weaker the cohesive interaction between DPPC molecules. Further compression (˘ ∼ 40 mN/m) drives the DPPC monolayers (Fig. 5a) to form structures with a thickness of about 3–4 times the molecular length of the DPPC. This may be due to the transition monolayer–multilayer [45]. For monolayer containing DOPC, the AFM images (Fig. 5b) show the presence of a structure surrounding areas where smaller isolated domains are visible with height of few molecular lengths. As general trend, the compression of multicomponent monolayers containing DPPC to these values of ˘ leads to a refinement of the interfacial layer by selective squeezing out of molecules with lower collapse pressure [45]. The feature presented in Fig. 5b indicates a possible growth of the film in thickness as consequence of the expulsion of DOPC from the bidimensional layer and the

subsequent refinement process of the monolayer (nucleation of DPPC). 4.2. Effect of silica particles on the equilibrium properties and structure In Ref. [27] it was shown that the presence of silica nanoparticles in the sub-phase modifies the typical isotherm of pure DPPC monolayer. The principal changes observed, with a 1 wt% of silica nanoparticle dispersion as sub-phase, were the shifting towards higher areas per molecule and the disappearance of the LE–LC coexistence plateau. These effects were attributed to the transfer of silica nanoparticles into the interfacial layer, due to adsorption of lipid molecules which may make them partially hydrophobic [46]. The formation of these complexes is due to the electrostatic interaction between the dissociated silanol groups with negative charge on the silica surface and the zwitterionic betain headgroup of the DPPC molecules. This is possible because the positive charged ammonium which is at the far end of the lipid headgroup allows the attractive electrostatic DPPC–nanoparticles interaction [46]. The AFM investigation of the LB films obtained by DPPC spreading on the silica nanoparticle dispersions shows that at ˘ = 7.5 mN/m (Fig. 3c) the monolayer structure is less homogeneous than that of pure DPPC with more irregular domains. From the section analysis, the inclusion of nanoparticles in the lipid monolayers is evidenced by the presence of regions with height of the order of the nanoparticles diameter. Further evidences of the penetration of the nanoparticles to the DPPC monolayers are obtained from the AFM images corresponding to ˘ ∼ 20 mN/m and

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

179

Fig. 5. AFM images of LB films deposited at ˘ ∼ 40 mN/m. (a) DPPC. (b) DPPC + DOPC. (c) DPPC + NP. (d) DPPC + DOPC + NP.

50

DOPC+NP DOPC

a

DPPC+DOPC+NP DPPC+DOPC

b

-1

40

Π Π/mN·m

˘ ∼ 40 mN/m (Figs. 4 and 5) where it is clear that the presence of nanoparticles induces a distorted pattern in DPPC monolayers. The state at ˘ ∼ 20 mN/m presents special interest, being observed a structure with a quasi-homogeneous lipid layer (Region I) surrounded for regions where nanoparticles are presented (Region II). All these observations are coherent with the results reported in Ref. [27]. In Fig. 6 the compression isotherms of DOPC and mixed DOPC–DPPC monolayers spread on a 1 wt% of silica nanoparticle dispersion are compared with those obtained without particles. It is noteworthy that for pure DOPC monolayer the presence of nanoparticles induces an important shift towards higher areas without appreciable effects on the feature of the isotherm while for the DPPC–DOPC mixture there is a matching of the two obtained isotherms with and without nanoparticles. The same mechanism of interaction with silica nanoparticles discussed for DPPC is applicable also to DOPC. This means that also in this case there is the formation of hydrophobic nanoparticle–lipid complexes and the incorporation of them into the monolayers. Similarly to the case of DPPC, in presence of particles, already at the beginning of the experiment, the real interfacial

30 20 10 0 0.4

0.6

A/A0

0.8

1.0

0.4

0.6

0.8

1.0

A/A0

Fig. 6. ˘–A/A0 isotherms for different Langmuir monolayers obtained in absence and presence of silica nanoparticles (NP, silica dispersion, concentration ∼1 wt%). (a) DPPC. (b) DOPC. (c) DPPC + DOPC.

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

4.3. Influence of the nanoparticles in the miscibility of DPPC and DOPC Further insights in the interaction between DPPC and DOPC, and how this is affected by the presence of nanoparticles may be obtained recurring to considerations based on the thermodynamic of mixtures [49]. For a mixed monolayer at a given surface pressure, the ideal area per molecule, Aideal , is defined as Aideal = XDPPC ADPPC + XDOPC ADOPC

(4)

100

Aideal (DPPC+DOPC) Aideal (DPPC+DOPC+NP)

90

A per molecule /Å 2

concentration is higher because the liquid surface is partially occupied by the attached particles and the area per molecules consequently reduced. However, while for DPPC the vertical orientation of the dipole plays an essential role in the control of the packing and this is influenced by the introduction of charged particle, for DOPC the reorientation of the hydrophobic tails at the interface is already limited by the presence of double bonds in the hydrocarbon chain, being this the reason of the absence of coexisting LC–LE domains. Thus the presence of charged nanoparticles influences less the packing of DOPC molecules and the only nanoparticle effect on the DOPC monolayer, is that induced by the occupation of the liquid surface. This is confirmed by the BAM images of DOPC monolayers in presence of nanoparticles which, similarly to what observed for pure DOPC on water, showed a homogeneous phase which condenses in the vicinity of the collapse point. While for pure lipids the presence of nanoparticles affects both the phase behaviour and the structure of the monolayers, for mixed DPPC–DOPC monolayers the scenario seems to be different. The matching between the two isotherms obtained with and without nanoparticles presented in Fig. 6b is in fact a noticeable result. In any case it is well-known the impossibility to extrapolate the behaviour of a mixture of lipids from the behaviour of the individual fractions [47]. The BAM images (Fig. 2) evidenced that the presence of nanoparticles modifies the morphology of the mixed monolayers which remains homogeneous until surface pressures around 40 mN/m, which corresponds to the refinement process of the monolayer. A more detailed information about the structure has been obtained from the AFM analysis of the LB films. In fact for mixed monolayers at ˘ ∼ 7.5 mN/m in presence of silica nanoparticles, the film is not completely homogeneous but presents inclusions of nanoparticle aggregates and an ongoing continuous phase in presence of smaller domains. However the formation of these domains is limited in relation to the system in absence of nanoparticles (Fig. 3d). The nanoparticles inclusions are also observed for monolayers obtained at ˘ ∼ 20 mN/m together with larger areas of films with height up to 2 nm which calls for a possible rearrangement of the lipid molecules tilting to the vertical position due to the pressure increase. Arriving to ˘ ∼ 40 mN/m, a network structure is formed which may be related to the refinement process above mentioned. This behaviour may be related to the differences on the packing of DPPC and DOPC in mixed monolayers. As we explained above the interaction of DPPC with the nanoparticles limits the formation of LC domains because hinder the vertical orientation of the dipole of DPPC molecules. Similar effects are induced by nanoparticles on DOPC but, in this case, the orientation of the dipole is not relevant for the molecular packing. Additionally, the presence of DOPC in the monolayers also hinders the packing of DPPC. A synergetic effect is induced by nanoparticles and DOPC which completely hinders the nucleation of DPPC domains characteristic of the pure DPPC and mixed monolayers spread on pure water. This occurs without any evident effects on the feature of the isotherm. Similar effects have observed by Tatur and Badia in their study on the effect of hydrophobic alkylated gold nanoparticles in the phase behaviour and structure of lung surfactant models [48].

A12 (DPPC+DOPC) A12 (DPPC+DOPC+NP)

80

70

60

50

a

40 0

10

20

30

40

Π/mN·m-1 12 10

DPPC+DOPC+NP DPPC+DOPC

8

Aexc per molecule/Å 2

180

6 4 2 0 -2 -4 -6

b

-8 0

10

20

30

40

Π/mN·m-1 Fig. 7. For different states of the mixed monolayer DPPC + DOPC in absence and presence of nanoparticles. (a) Values of Aideal and A12 . (b) Values of Aexc .

where ADPPC and ADOPC are the areas per molecule in pure monolayers of DPPC and DOPC, respectively, at the considered ˘, and XDPPC and XDOPC the molar fractions of DPPC and DOPC in the mixture. The deviations of the behaviour of the real system from that of the ideal mixture may be evaluated using the excess area, Aexc Aexc = A12 − Aideal

(5)

where A12 is the area per molecule in the mixed monolayer at the given value of ˘. For an ideal mixed monolayer, Aexc is zero. Fig. 7 shows the Aexc values as a function of the surface pressure of the mixed monolayer, in absence and presence of nanoparticles. In absence of nanoparticles, Aexc shows positive values in almost the whole surface pressure range. This evidences a repulsive interaction between DPPC and DOPC and confirms the separation of the DPPC and DOPC phases. For high values of ˘, the differences between the ideal and the real behaviour are reduced. This may be related to the refinement process of the monolayer for the selective squeezing-out of DOPC from the layer and the transition from monolayer to multilayer of the DPPC molecules. In presence of nanoparticles, Aexc assumes negative values for the whole range, without any important dependence on the surface pressure. This may mean that nanoparticles induce attractive interactions between DPPC and DOPC. However, as in this case the real system is a ternary one, where the exact concentration

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

DOPC

DOPC+NP

b

60

c

d

-1

Π Π/mN·m

Π Π/mN·m

-1

60

a

40

0

40

20

20

100

120

140

160

100

120

t/s

140

0

160

100

120

140

120

140

160

t/s DPPC+DOPC+NP

f

60

h

g

Π Π/mN·m

-1

-1

60

e

100

160

t/s

t/s DPPC+DOPC

Π Π/mN·m

181

40

20

20

0

40

100

120

140

t/s

160

100

120

140

160

0

100

120

140

100

160

120

140

160

t/s

t/s

t/s

Fig. 8. Effect of the amplitude of deformation on the surface pressure response of DOPC and DPPC–DOPC mixed monolayers to a sinusoidal variation of the area at frequency  = 0.05 Hz for two different deformation amplitude u0 = 0.01 and u0 = 0.4. For DOPC in absence of nanoparticles, u0 = 0.01 (a) and u0 = 0.4 (b), and in presence of nanoparticles, u0 = 0.01 (c) and u0 = 0.4 (d). For DPPC + DOPC in absence of nanoparticles, u0 = 0.01 (e) and u0 = 0.4 (f), and in presence of nanoparticles, u0 = 0.01 (g) and u0 = 0.4 (h).

An important aspect related to the potential implications for lung surfactant physiology, concerns the dilational rheology of the lipid monolayers [50] and the effect that nanoparticles of different nature may have on it [51], under quasi-realistic respiratory conditions. During the respiratory cycle, the LS film in the alveoli is subjected to a periodic area perturbation of about 30–40%, at a frequency within  ∼ 0.04–0.2 Hz, around a reference state characterised by a value of ˘ ∼ 35–40 mN/m [2]. To investigate this aspect we analysed the ˘ response of the different lipid monolayers, with and without nanoparticles, to sinusoidal changes of the interfacial area at a fixed frequency  ∼ 0.05 Hz and different amplitude of the area change, A/A0 , from 1 up 40%. For both the considered monolayers, increasing A/A0 the response becomes non-linear (Fig. 8). This is evident by observing the Fourier spectra of such signals in Fig. 9, where harmonics of higher order appear in addition to the fundamental one. The number of higher order harmonics and their amplitude increase with the increase of the deformation amplitude. Therefore the incorporation of nanoparticles in the lipid monolayers changes the linearity of the oscillatory ˘ response as was observed for other lipid systems [27,52]. Fig. 10 shows the values of THD, calculated from the experimental signal obtained for periodical deformation at different amplitude. The feature of this parameter versus the deformation amplitude allows a more compact representation of the nonlinearity. The results evidence important differences between the

a

DOPC 14 12

FFT(Π)

4.4. Effect of nanoparticles on simulated respiratory cycles

16

10

u0=1%

ν

u0=1%

ν

b

DOPC+NP

u0=40%

u0=40%

8 6 4

2ν

2

2ν

3ν

3ν

4ν 5ν

0

16 14

FFT(Π)

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 a miscible lipid mixture. This may also be at the origin of the homogeneous structural pattern observed in the BAM images.

ν

0.1

0.1

ν/Hz

ν/Hz

c

DPPC+DOPC

DPPC+DOPC+NP ν

12

u0=1%

10

u0=40%

d

u0=1% u0=40%

8 6 4

2ν 3ν

2

4ν

2ν 5ν

3ν

4ν 5ν

0 0.1

0.1

ν/Hz

ν/Hz

Fig. 9. FFT spectra of the surface pressure response to a sinusoidal variation of the area at frequency  = 0.05 Hz for DPPC–DOPC monolayers. In absence of nanoparticles for a deformation amplitude of u0 = 0.01 (a) and u0 = 0.4 (b). In presence of nanoparticles for a deformation amplitude of u0 = 0.01 (a) and u0 = 0.4 (b).

182

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183

0.4 DPPC+DOPC DPPC+DOPC+NP

DOPC DOPC+NP

0.3

THD

0.2

0.1

0.0

b

a

0.0 0.1

0.2

ΔA/A0

0.3

0.4 0.0

0.1

0.2

0.3

0.4

ΔA/A0

Fig. 10. Total harmonic distortion (THD) from surface pressure response to a sinusoidal variation of the area at frequency  = 0.05 Hz as function of the deformation amplitude, obtained for the DPPC, DOPC and DPPC–DOPC, with (a) and without silica nanoparticles (b). The lines are guides for the eye.

effect of the nanoparticles on single lipid monolayer and on mixed monolayer. In fact, as for pure DPPC and DOPC nanoparticles induce an important enhancement of the non-linearity of the rheological response, the contrary is found for the DPPC–DOPC mixed monolayer. Being the linearity of the response to the area perturbation related to the redistribution of material, one can interpret these data considering that the penetration of nanoparticles into the pure lipid monolayers increases the time necessary for the reequilibration. This is clear in particular for the case of pure DPPC, because of the existence of microdomains in the monolayer that may be an important source of non-linearity [53], affected by the presence of nanoparticles. For mixed DPPC–DOPC monolayers, it must be considered that the LC microdomains disappear when the nanoparticles are included to the monolayers. This leads to a more fluid monolayer with a lower re-equilibration time and a consequent increase of linearity (decrease on THD). Thus the THD value can be used to discriminate different regimes of the system behaviour and consequently could be conveniently utilised as one of the parameters quantifying the potential adverse impact on respiratory physiology, based on dilational rheology measurements. 5. Conclusions The effect of silica nanoparticles on the behaviour of DOPC and DOPC–DPPC monolayers has been investigated by a systematic experimental study on the properties of such monolayers spread both on pure water and on silica nanoparticle dispersion. This work was especially focused on the equilibrium and structural surface properties. In order to make this study effective, the Langmuir trough technique was coupled with the AFM analysis of the Langmuir–Blodgett deposited films obtained under controlled thermodynamic conditions. From the results here obtained it is possible to conclude that silica nanoparticles present appreciable effects on the phase behaviour of the investigated lipid systems, essentially related to their incorporation into the monolayer. As already discussed in previous works on DPPC monolayers spread on the same silica dispersion, this nanoparticle transfer to the lipid layer is induced by the modification of nanoparticle hydrophobicity caused by lipid adsorption. The utilisation of this experimental approach, based also on structural characterisation of LB films, allowed the surface segregation of silica nanoparticles to be confirmed and the consequent

structural modification of the lipid monolayer to be evaluated in a more quantitative way. Besides an increase of the structural disorder of the monolayer there is also an effect on the formation of domains due to the electrostatic interactions that influences the molecular orientation at the surface. Another important effect, already observed in previous studies on other lipid–nanoparticle systems concerns the linearity of the surface pressure response to area perturbation. For the present DOPC and DOPC–DPPC systems it was found that when nanoparticles are incorporated into the monolayer, the linearity of the response is strongly modified. The results here obtained can be applied in the field of interaction between particulate materials and lung surfactants. It is, in fact, shown that nanoparticles can have important effects on the physico-chemical properties of lipid monolayers especially concerning their response to periodic area deformations. 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 by European Science Foundation, ESF-COST Action D43 “Colloid and Interface Chemistry for Nanotechnologies”. References [1] H. Möhwald, Phospholipid and phospholipid-protein monolayers at the air/water interface, Annu. Rev. Phys. Chem. 41 (1990) 441–476. [2] R. Wüstneck, J. Perez-Gil, N. Wüstneck, A. Cruz, V.B. Fainerman, U. Pison, Interfacial properties of pulmonary surfactant layers, Adv. Colloid Interfaces Sci. 117 (2005) 33–58. [3] Y.Y. Zuo, R.A.W. Veldhuizen, A.W. Neumann, N.O. Petersen, F. Possmayer, Current perspectives in pulmonary surfactant—inhibition, enhancement and evaluation, Biochim. Biophys. Acta 1778 (2008) 1947–1977. [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] N. Nandi, D. Vollhardt, Effect of molecular chirality on the morphology of biomimetic Langmuir monolayers, Chem. Rev. 103 (2003) 4033–4076. [7] D.G. Dervichian, Changes of phase and transformations of higher order in monolayers, J. Chem. Phys. 7 (1938) 931–948. [8] S. Stallberg-Stenhagen, E. Stenhagen, N. Sheppard, G.B.B.M. Sutherland, A. Walsh, Phase transitions in condensed monolayers of normal chain carboxylic acids, Nature 156 (1945) 239–240. [9] 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. [10] R. Mendelsohn, J.W. Brauner, A. Gericke, External infrared reflection absorption spectrometry of monolayer films at the air-water interface, Annu. Rev. Phys. Chem. 46 (1995) 305–334. [11] C.M. Knobler, Seeing phenomena in flatland: studies of monolayers by fluorescence microscopy, Science 249 (1990) 870–874. [12] W. Lu, C.M. Knobler, R.F. Bruinsma, M. Twardos, M. Dennin, Folding Langmuir monolayers, Phys. Rev. Lett. 89 (2002) 146107. [13] 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. [14] J. Zhao, D. Vollhardt, G. Brezesinski, S. Siegel, J. Wu, J.B. Li, R. Miller, Effect of protein penetration into phospholipid monolayers: morphology and structure, Colloids Surf. A 171 (2000) 175–184. [15] V.M. Kaganer, H. Mohwald, P. Dutta, Structure and phase transitions in Langmuir monolayers, Rev. Mod. Phys. 71 (1999) 779–819. [16] R.K. Harishchandra, M. Saleem, H.-J. Galla, Nanoparticle interaction with model lung surfactant monolayers, J. R. Soc. Interface 7 (2010) S15–S27. [17] L. Mazzola, Commercializing nanotechnology, Nat. Biotechnol. 21 (2003) 1137–1143. [18] P.H.M. Hoet, I. Brüske-Hohlfeld, O.V. Salata, Nanoparticles – known and unknown health risks, J. Nanobiotechnol. 2 (2004) 2–12, doi:10.1186/14773155-2-12. [19] T. Kanishtha, R. Banerjee, C. Venkataraman, Effect of particle emissions from biofuel combustion on surface activity of model and therapeutic pulmonary surfactants, Environ. Toxicol. Pharmacol. 22 (2006) 325–333. [20] B.P. Binks, Particles as surfactant-similarities and differences, Curr. Opin. Colloid Interfaces Sci. 7 (2002) 21–41.

E. Guzmán et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 174–183 [21] F. Ravera, E. Santini, G. Loglio, M. Ferrari, L. Liggieri, Effect of nanoparticles on the interfacial properties of liquid/liquid and liquid/air surface layers, J. Phys. Chem. B 110 (2006) 19543–19551. [22] F. Ravera, M. Ferrari, L. Liggieri, G. Loglio, E. Santini, A. Zanobini, Liquid–liquid interfacial properties of mixed nanoparticle–surfactant systems, Colloids Surf. A 323 (2008) 99–108. [23] L. Liggieri, E. Santini, E. Guzmán, A. Maestro, F. Ravera, Wide-frequency dilational rheology investigation of mixed silica nanoparticle–CTAB interfacial layers, Soft Matter 7 (2011) 7699–7709. [24] E. Santini, E. Guzmán, F. Ravera, M. Ferrari, L. Liggieri, Properties and structure of interfacial layers formed by hydrophilic silica dipersions and palmitic acid, Phys. Chem. Chem. Phys. 14 (2012) 607–615. [25] H. Bachofen, S. Schurch, Alveolar surface forces and lung architecture, Comp. Biochem. Physiol. 129 (2001) 183–193. [26] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Influence of silica nanoparticles on thermodynamic and structural properties of DPPC – palmitic acid Langmuir monolayers, Colloids Surf. A 413 (2012) 280–287. [27] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Effect of hydrophilic and hydrophobic nanoparticles on the surface pressure response of DPPC monolayers, J. Phys. Chem. C 115 (2011) 21715–21722. [28] A. Maestro, E. Guzmán, E. Santini, F. Ravera, L. Liggieri, F. Ortega, R.G. Rubio, Wettability of silica nanoparticle-surfactant nanocomposite interfacial layers, Soft Matter 8 (2012) 837–843. [29] H. Hilles, A. Maestro, F. Monroy, F. Ortega, R.G. Rubio, M.G. Velarde, Polymer monolayers with a small viscoelastic linear regime: equilibrium and rheology of poly(octadecyl acrylate) and poly(vinyl stearate), J. Chem. Phys. 126 (2007) 124904. [30] M. Wilhelm, Fourier-transform rheology, Macromol. Mater. Eng. 287 (2002) 83–105. [31] G. Loglio, P. Pandolfini, R. Miller, A. Makievski, J. Krägel, F. Ravera, Oscillation of interfacial properties in liquid systems: assessment of harmonic distortion, Phys. Chem. Chem. Phys. 6 (2004) 1375–1379. [32] G. Loglio, P. Pandolfini, R. Miller, A. Makievski, J. Krägel, F. Ravera, B.A. Noskov, Perturbation–response relationship in liquid interfacial systems: non-linearity assessment by frequency–domain analysis, Colloids Surf. A 261 (2005) 57–63. [33] R. Wüstneck, N. Wüstneck, D.O. Grigoriev, U. Pison, R. Miller, Stress relaxation behaviour of dipalmitoyl phosphatidylcholine monolayers spread on the surface of a pendant drop, Colloids Surf. B 15 (1999) 275–288. [34] K.J. Klopfer, T.K. Vanderlick, Isotherms of dipalmitoylphosphatidylcholine (DPPC) monolayers: features revealed and features obscured, J. Colloid Interface Sci. 182 (1996) 220–229. [35] M.C. Phillips, D. Chapman, Monolayer characteristics of saturated 1,2-diacyl phosphatidylcholines (lecithins) and phosphatidylethanolamines at the airwater interface, Biochim. Biophys. Acta 163 (1968) 301–313. [36] D.D. Baldyga, R.A. Dluhy, On the use of deuterated phospholipids for infrared spectroscopic studies of monomolecular films: a thermodynamic analysis of single and binary component phospholipid monolayers, Chem. Phys. Lipids 96 (1998) 81–97.

183

[37] G.N. Kretzschmar, J. Li, R. Miller, H. Motschmann, H. Möhwald, Characterisation of phospholipid layers at liquid interfaces. 3. Relaxation of spreading phospholipid monolayers under harmonic area changes, Colloids Surf. A 114 (1996) 277–285. ˜ A.P. Gunning, V.J. Morris, P.J. Wilde, J.M.R. Patino, Effect [38] A. Lucero, M.R.R. Nino, of hydrocarbon chain and pH on structural and topographical characteristics of phospholipid monolayers, J. Phys. Chem. B 112 (2008) 7651–7661. [39] K. Kjaer, J. Als-Nielsen, C.A. Helm, L.A. Laxhuber, H. Möhwald, Ordering in lipid monolayers studied by synchrotron X-ray diffraction and fluorescence microscopy, Phys. Rev. Lett. 58 (1987) 2224–2227. [40] K. Nag, K.M. Keough, Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines, Biophys. J. 65 (1993) 1019–1026. [41] V. Vié, N.V. Mau, E. Lesniewska, J.P. Goudonnet, F. Heitz, C.L. Grimellec, Distribution of ganglioside GM1 between two-component, two-phase phosphatidylcholine monolayers, Langmuir 14 (1998) 4574–4583. [42] M.K. Jain, R.C. Wagner, Introduction to Biological Membranes, John Wiley and Sons, New York, 1980. [43] I. Prigogine, Insoluble Monolayers at Liquid-Gas, Interscience, New York, 1966. [44] X.M. Yang, D. Xiao, S.J. Xiao, Y. Wei, Domain structures of phospholipid monolayer Langmuir-Blodgett films determined by atomic force microscopy, Appl. Phys. A 29 (1994) 139–143. [45] H. Zhang, Q. Fan, Y.E. Wang, C.R. Neal, Y.Y. Zuo, Comparative study of clinical pulmonary surfactants using atomic force microscopy, Biochim. Biophys. Acta 1808 (2011) 1832–1842. [46] C.E. McNamee, M. Kappl, H.-J.r. Butt, K. Higashitani, K. Graf, Interfacial forces between a silica particle and phosphatidylcholine monolayers at the air-water interface, Langmuir 26 (2010) 14574–14581. [47] J. Pérez-Gil, Structure of pulmonary surfactant membranes and films: the role of proteins and lipid–protein interactions, Biochim. Biophys. Acta 1778 (2008) 1676–1695. [48] S. Tatur, A. Badia, Influence of hydrophobic alkylated gold nanoparticles on the phase behavior of monolayers of DPPC and clinical lung surfactant, Langmuir 28 (2012), doi:10.1021/la203439u. [49] K. Gong, S.-S. Feng, M.L. Go, P.H. Soew, Effects of pH on the stability and compressibility of DPPC/cholesterol monolayers at the air–water interface, Colloids Surf. A 207 (2002) 113–125. [50] M.I. Sameh, A. Saad, W. Neumann, E.J. Acosta, A dynamic compressionrelaxation model for lung surfactants, Colloids Surf. A 354 (2010) 34–44. [51] E.J. Acosta, Z. Policova, S. Lee, A. Dang, M.L. Hair, A.W. Neumann, Restoring the activity of serum-inhibited bovine lung extract surfactant (BLES) using cationic additives, Biochim. Biophys. Acta 1798 (2010) 489–497. [52] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Influence of the adsorption of hydrophilic silica nanoparticles on dilational rheology of DPPC – palmitic acid Langmuir monolayers, Soft Matter, submitted for publication. [53] L.R. Arriaga, I. López-Montero, R. Rodríguez-García, F. Monroy, Nonlinear dilational mechanics of Langmuir lipid monolayers: a lateral diffusion mechanism, Phys. Rev. E 77 (2008) 061918.