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Structural Changes to Lipid Bilayers and Their Surrounding Water Upon Interaction With Functionalized Gold Nanoparticles Xavier Toledo-Fuentes, Dan Lis, and Francesca Cecchet J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b05460 • Publication Date (Web): 23 Aug 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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The Journal of Physical Chemistry

Structural Changes to Lipid Bilayers and their Surrounding Water Upon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Interaction with Functionalized Gold Nanoparticles

Xavier Toledo-Fuentes, Dan Lis, and Francesca Cecchet* Research Centre in Physics of Matter and Radiation (PMR) and NAmur Research Institute for LIfe Sciences (NARILIS), University of Namur (UNamur), 61 rue de Bruxelles, B-5000 Namur, Belgium *corresponding author: [email protected], Phone number 0032 (0)81 725487

Abstract Structural changes of solid-supported lipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and of their interfacial water surrounding have been probed with vibrational sum frequency generation (SFG) upon interaction with functionalized gold NPs, carrying negative or positive surface charges. Switching the substrate/DPPC zeta potential from strongly negative to weakly negative or positive values by using SiO2 or CaF2 supporting surfaces has enabled elucidating interfacial charge effects, intermolecular interactions and interaction mechanisms regulating those nano-bio-interfaces. At SiO2/DPPC surfaces (ζ = -30 mV), negative NPs have reinforced the average interfacial water alignment, while no interaction has occurred with DPPC bilayer. Oppositely, positive NPs interact with DPPC, with a probable two-steps mechanism involving the formation of pores within the bilayer followed by a reorganization of a quasi-ordered DPPC bilayer around NPs. At CaF2 /DPPC surfaces (ζ = ±15 mV), both negative and positive NPs have clearly damaged the lipid bilayer structure, together with a drastic disruption of the water organization at the interface. Importantly, all major interfacial structural changes have occurred within the first 15-90 minutes after exposure to NPs, whatever the lipid is supported on SiO2 or CaF2.

Introduction 1

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New technologies based on nanoscale materials have drastically increased the exposure of humans to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

nanoparticles (NPs). Indeed, due to their composition, size and shape, NPs possess novel physicochemical properties making them useful in many goods, such as paints, cleaning products, cosmetics, clothes or fuel additives.1 Moreover, engineered NPs are extensively used as imaging contrast agents and drug-delivery carriers in several biomedical applications and diseases treatments.2 The intense use of NPs makes the study of their cytotoxicity a critical issue. To assess NPs toxicity, in vitro assay protocols have been extensively used, such as viability tests, oxidative stress and inflammatory assays, which measure the survey or uptake of the cellular response upon exposure to NPs.3,4 Today, with the aim to follow the pathways of NPs cytotoxicity, numerous bioanalytical techniques owing nanoscale sensitivity are used, such as imaging and flow cytometry, or (X-ray) fluorescence microscopy.5,6 However, these bioanalytical approaches suffer the lack of detailed knowledge of the molecular scale parameters driving the physicochemical interactions between nanoparticles and cells. Though the mechanisms of cytotoxicity are diverse and complex, and depend on the chemical environment and on the nature of NPs,7,8,9,10 numerous studies converge to prove that the first contacts of NPs with cell membranes may cause cell damages, triggering toxicity pathways.11,12 Due to the complexity of cell membranes, which are composed of a plethora of biological species (lipids, carbohydrates, peripheral and transmembrane proteins), and regulated by multiple dynamical processes, the mechanisms and the physicochemical properties which drive NPs to attach, penetrate and possibly disrupt cell membranes are still not well understood. Obtaining an in-depth molecular view of the NPs/cell membranes system is a key challenge, because one shall probe interactions occurring in very thin (nanometer) regions, comprising the nanoparticle and the membrane, surrounded by a macroscopic physiological environment. Elucidating these first steps, in which NPs meet membranes, is consensually recognized as necessary to understand physiological responses13,14 and to predict biological effects.15 To overcome the problem of the complex composition and dynamics of membranes, it may be advantageous to use biological models of known and controlled composition. Most of the time, the study, at the molecular level, of the NPs/membrane models interactions is carried out with using mono- or bilayers of lipid molecules, which represent one main component of cell membranes.16,17,18,19,20,21,22,23 Both theoretical approaches, based on molecular mechanics simulations for instance,16,17,24,25 and experimental works have addressed the study of the fundamental interactions of 2


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NPs/membrane model interfaces. Imagery tools, such as atomic force microscopy (AFM), transmission electron 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

microscopy (TEM), confocal microscopy,7,11,12,26,27,28,29 have enabled to highlight the NPs-induced damages to the membranes. Spectroscopic methods, such as fluorescence microscopy, have elucidated the NPs-induced surface reconstruction of phospholipids membranes,30 while infrared spectroscopy has shown the structural changes following NPs penetration within the lipid layer.29 Meanwhile, in the last decade, nonlinear optical techniques, such as second harmonic generation (SHG),31,32 sum-frequency generation (SFG),33,34 coherent Anti-Stokes Raman scattering (CARS),18,19,20 or third-harmonic generation35 have also remarkably contributed to the study of model biointerfaces. Among them, SFG spectroscopy has been intensively used to probe the structure, the interactions and the dynamics of membrane models through the measure of their vibrational second order optical response. Being based on a second order nonlinear phenomenon, SFG spectroscopy provides information only about interfacial regions where the centrosymmetry, typical of bulk media, is broken. SFG spectroscopy has elucidated, for example, the symmetry of the two leaflets of a phospholipid bilayer,36 the orientation of lipids and proteins at liquid and solid interfaces,37,38,39 the kinetics of the flip-flop process within bilayers40, the condensation effect of cholesterol to the lipid structure,41 the interaction of DNA with lipid layers,42 the structure and orientation of water molecules close to the polar head of lipid layers,43,44,45 the chirality of proteins and peptides,46,47 and also the interactions of proteins and peptides with membrane models.48,49,50 More recently, the SFG response has been measured from nano-objects dispersed in a condensed phase, demonstrating the potentiality of this spectroscopy to be applied also to 3D nano-interfaces.51 For instance, the SFG response of asymmetric phospholipids vesicles has been probed in a liquid environment.52 The study of NPs/cell membranes interactions with nonlinear optical spectroscopies is a rather new and still unexplored research area, in which second order nonlinear optical spectroscopies may contribute to shed new light on the first contacts of lipid bilayers with NPs. Recently, it has been shown that SFG and SHG spectroscopies can unravel new understandings of the nano-bio-interface composed of a membrane model in interaction with nanoparticles.53,54,55 In this work, SFG spectroscopy is used to characterize the interactions between lipid membrane models and gold NPs, with a specific focus on the role of the relative surface charges on their interactions. In detail, the 3



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membrane model is a bilayer of phosphatidylcholine (a zwitterion globally neutral lipid) adsorbed either on a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

strongly negative SiO2 surface, or on a rather positive CaF2 surface (as schematically represented in Figure 1). Nanoparticles are hydrophilic 5 nm gold NPs, either positively or negatively charged depending on their ligand. In this work, for the first time, the conformational order of the lipid structure and of its hydration layer have been analyzed upon interaction with NPs. Then, combining the structural damages induced to DPPC membrane models with changes in the organization of interfacial water have enabled highlighting, with an outstanding sensitivity, the key role of relative charges in driving the interactions, and unravelling a molecular-scale picture of the interaction mechanisms regulating the above nano-bio-interfaces.

Materials and Methods Materials. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Figure 1) was supplied from Avanti®Polar Lipids, Inc. (Alabaster, AL). Acetone, ethanol, dichloromethane, sulfuric acid and hydrogen peroxide were purchased from Sigma-Aldrich. Gold nanoparticles were purchased from Nanopartz Inc. (Loveland, CO). Charges to the NPs surface are induced by polymer ligands, owing a carboxyl head giving a negative (zeta potential ζ = -40 mV) or an amine head giving a positive (ζ = +20 mV) to NPs. Here, spherical gold nanoparticles with an averaged core diameter of 5 nm and approximately 1 nm of ligand thickness have been used. The ligand density is 5 molecules/nm2 for negative NPs, and 8 molecules/nm2 for positive NPs, as provided by the customer. NPs solutions have a concentration of 0.050 mg/ml. Substrates were calcium fluoride (CaF2) – (111) cleavage structure – and IR-grade fused silica (SiO2) prisms, and were obtained from Crystran Ltd (Poole, UK) and UAB Altechna (Vilnius, Lithuania), respectively. Sample Preparation. SiO2 prisms were cleaned by immersion in warm “piranha” solution (H2SO4:H2O2, 2:1) for at least a half an hour, and then rinsed with large amount of Milli-Q water (18.2 MΩ.cm, pH = 5.5). Due to the CaF2 chemical reactivity with H2SO4 and the risk of damages when immersed in warm “piranha” solution, CaF2 prisms were cleaned by short (5-10 s) immersion in diluted “piranha” solution (H2SO4:H2O2:H2O, 2:1:1) cooled down to room temperature. Milli-Q water has been used for all measurements at the neat prism (SiO2 or CaF2)/water interface. Lipid bilayers were formed with the spontaneous vesicle adsorption technique, following 4


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a protocol adapted from Kasemo et al. and Ogier et al.56,57 A solution of lipid vesicles was prepared by i) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

solubilizing 2.5 mg of DPPC powder in 300 µl of dichloromethane (CH2Cl2), ii) evaporating CH2Cl2 under N2 flow, and iii) adding 1 ml of Milli-Q water. The solution was then sonicated for 10 min and centrifuged at 6000 rpm for 10 min. The so obtained vesicles solution was kept at 4°C for use for 2 days at maximum. To form the bilayer, 1.5 ml of vesicles solution was put in contact with the prisms in a Teflon cell for 3 hours. The duration of vesicles fusion has been set as the time after which the SFG intensity of the OH maximum intensity was stable. Then, the vesicles solution was diluted several times in Milli-Q water in order to exchange the remaining vesicles with pure water, and leave only the fused bilayer at the surface of the prism. This procedure was realized by taking care to do not expose the bilayer to air, in order to avoid damaging of the bilayer itself. The DPPC bilayers supported on the CaF2 and SiO2 surfaces were then characterized by SFG spectroscopy. Interaction of the bilayers with gold nanoparticles was obtained by injecting the NPs solution in the water surrounding the bilayer so that a concentration of 0.025 mg/ml NPs was reached. The lipid bilayer was then left in contact with the NPs during up to 20 hours. SFG spectra were recorded at several interaction times. Finally, after the investigation of the solid/liquid interface, the NPs solution was removed; the solid/air interface was rinsed by Milli-Q water, dried with moderate N2 flow and again characterized by SFG. SFG Measurements. The 1064 nm pump beam of the SFG spectrometer is a 15 ps pulsed laser, obtained with the combination of active and passive mode-locking of a Nd:YAG laser source. The repetition rate of each train of pulse is 25 Hz. This beam synchronously pumps two optical parametric oscillators (OPOs), generating the tunable IR and visible beam. The power of the final IR beam is around 25 mW (corresponding to a peak power of 0,66 MW), while that of the visible beam is about 10 mW. In this work, the frequency range of the tunable IR OPO was between 3700 cm-1 and 2800 cm-1, while the visible OPO was fixed at 532 nm. More details about the SFG spectrometer can be found in literature.58,59 SFG measurements of the substrates/DPPC and substrates/DPPC/NPs interfaces were recorded in total internal reflection geometry through a prism, at both the solid/water and solid/air interfaces (Figure 1), with the SFG, Vis and IR beams in p polarization (ppp). This polarization has been chosen since it probes all the polarization combinations together (i.e. ppp, ssp, sps, pss), which are active for azimuthal isotropic surfaces.60 To perform comparison between SFG spectra recorded in water, SFG responses at the solid/liquid interface have been first normalized by the IR and Vis beams 5



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intensities. Then, the SFG responses of the neat SiO2/water and CaF2/water interfaces have been recorded 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

before any further surface functionalization, and normalized by the OH maximum intensity of the CaF2/water interface, which has been set equal to 1. The intensity of all subsequent SFG spectra describing surface functionalization by DPPC and interaction with NPs are referred to the normalized intensity of this neat interface. Since SFG spectra recorded in air were not normalized by a reference signal - because no comparison between spectra has been performed -, those spectra are plotted without intensity scales on the y-axis. All measurements have been recorded at room temperature (23.5°C), so that DPPC films (whom melting point is around 41.0°C) are in the gel phase. This phase corresponds to a lipid bilayer structure more packed than in the fluid phase. All interactions mechanisms and/or kinetics discussed later have therefore to be reported to the gel phase, since the biological responses of membranes are known to be dependent on the bilayer fluidity.61 The substrates/NPs interfaces were also probed (see Supporting Information) in order to characterize their own SFG response, and overcome the lack of information given by the owner about the chemical composition of the ligands, which could give rise to a vibrational signature interfering with that of the DPPC bilayers. The SFG response of each interface has been investigated at least twice, in order to confirm the reproducibility of the observed behaviors. Analysis of SFG Spectra. SFG spectroscopy is based on a second order nonlinear optical process62 of a radiation-matter interaction. In a SFG experiment, when two laser beams, one in the visible and one in the infrared frequency ranges, are focused on a sample, there is generation, via the nonlinear part of the polarization, of a third beam, whose frequency is the sum of the two incident beams frequencies (ωSFG = ωIR + ωVis) (fig. 1). The SFG intensity is proportional to the intensities of each incident beam and to the square of the modulus of the effective second order nonlinear susceptibility, χ(2), as follows: (1)

2

I SFG ∝ χ (2) IVis I IR

(2)

(2) χ (2) = χ NR + χ R(2)

where χ(2) is a rank 3 tensor related to the second order nonlinear polarization and is equal to the sum of a nonresonant part (χ(2)NR) and a resonant part (χ(2)R).The χ(2)NR element is related to the electronic transitions of the substrate and therefore depends on the visible beam frequency, while χ(2)R depends on the vibrational transitions 6


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of the probed interface. Within the harmonic approximation, the expression of the components χ(2)R,ijk of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

resonant part is:

χ R(2),ijk ∝ ∑ q

(3)

Aq ,ijk

ωq − ωIR − iΓ q

where

Aq ,ijk = Aq ,ijk e

(4)

iϕq

It results that the SFG intensity may be modeled as follows:

I SFG ∝ χ

(2) NR

+∑ q

Aq ,ijk e

iϕq

ωq − ωIR − iΓ q

(5)

2

IVis I IR

All SFG spectra have so been fitted assuming a lorentzian behavior of the measured vibrations, following equations 2-5, thus obtaining the resonant part of the nonlinear susceptibility in function of the infrared frequency (ωIR). For each vibration q, four free parameters are used to fit the experimental profiles: the modulus of the amplitude Aq, the relative phase ϕq, the vibrational peak width Γq and the vibration frequency ωq. All fitting parameters of the spectra shown in the paper can be found in the Supplementary Information (SI).

Results and Discussion Substrates/DPPC Interfaces. Figure 2a shows the SFG spectra obtained at the SiO2 (black curves) and SiO2/DPPC (grey curves) interfaces in water. At the SiO2/water interface two large bands, centered at 3350 cm1

and 3150 cm-1, are due to OH vibrations coming from water molecules, which organize with a preferential

conformation at the interface, compared to randomly oriented molecules in the bulk.63,64,65,66 The fusion of a DPPC bilayer on the SiO2 surface, is accompanied by a decrease of the SFG intensity of OH modes, indicating that the thickness and/or the alignment of interfacial water is less after the lipid adsorption. Water alignment at interfaces is known to be related to the strength of the interfacial zeta potential and of the corresponding electric field.67 The SFG response of OH modes corresponds to an average over all organized water molecules. At the 7



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substrate/DPPC interfaces distinct hydration layers - i.e. between the substrate and the inner leaflet of DPPC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

bilayer, within the polar heads of DPPC, and between the outer leaflet of DPPC bilayer and the bulk water - can contribute to the whole signal, with positive or negative interferences depending on the relative signal phases associated to water dipoles orientations. The zeta potential of SiO2 and SiO2/DPPC interfaces in water has been estimated equal to -90 mV and -30 mV, respectively (Table 1) (see Supplementary Information SI for more details).68,69,70 Therefore, passing from -90 mV to -30 mV makes the water alignment poorer. Figure 2b shows the SFG responses of CaF2 (black curves) and CaF2/DPPC (grey curves) interfaces in water. The OH intensity at the lipid bilayer interface fluctuates around that of the neat substrate. This behavior is due to close absolute zeta potentials values of CaF2 and CaF2/DPPC interfaces in water. Indeed, while CaF2 zeta potential has been set between -15 mV and +15 mV- the range being justified by the strong reactivity of the CaF2 surface upon environment conditions as well as surface carbonation -,71,72,73 the zeta potential of DPPC bilayers on CaF2 can be estimated approximately equal to +15/+20 mV (see SI). Asides OH peaks, in the SFG spectra recorded at the SiO2/DPPC and CaF2/DPPC interfaces in water (Figure 2a and b, grey curves) no CH2 or CH3 vibration of the aliphatic groups of DPPC have been detected, thus testifying that methyl and methylene groups are globally in a centrosymmetric environment, typical of a bilayer structure with a high degree of conformational order of the aliphatic chains. However, irrespective of the substrate, when the same interfaces have been exposed to air (Figure 2c), intense CH3 peaks appear at 2959 cm1

, 2933 cm-1 and 2870 cm-1, which come from the degenerate asymmetric stretching modes, the Fermi

resonance and the symmetric stretching mode of CH3 groups in DPPC, respectively.40 The SFG activity of CH3 vibrations indicates that the symmetry of the bilayer has been broken upon exposure to air. This behavior is well known as the unfolding of the bilayer on itself, forming domains of monolayers and tri-layers of lipid molecules, which are no longer centrosymmetric74,75. However, the absence of CH2 peaks attests that the hydrophobic tails of DPPC have kept an all-trans conformation upon the unfolding process. Indeed, in the alltrans conformation CH2 groups are locally in a centrosymmetric conformation (i.e. an inversion point is in the center of each C-C bond), and therefore they do not lead to a SFG signal. Alongside, residual OH vibrations are still detected, suggesting that aligned water molecules have been trapped between the substrate and the inner leaflet of the lipid bilayer.76,77 The same spectral behavior of the SiO2/DPPC/water interface when exposed to 8


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air (Figure 2c) has also been observed for the CaF2/DPPC/water interface (see SI). This ability of the lipid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chains to remain in the all-trans conformation after exposure to air when starting from a highly ordered bilayer - or on the contrary to show gauche defects when starting from a disordered bilayer -, will be used as an indirect probe of the possible structural damages induced by NPs to the lipid bilayer. Substrates/NPs Interfaces. The SFG response of NPs has been firstly characterized at the neat substrates. Figures 3a-b and Figures 3c-d shows the SFG responses of SiO2 and CaF2 prism interfaces, respectively, in contact with water before and after injection of negative (Figure 3a,c) and positive NPs (Figure 3b,d). The absence of vibrational features after NPs injection (for any time) testifies that NPs do not give rise to their own SFG signature in the probed frequency range, when in contact with both substrates. The fact that no vibrational features from the NPs ligands have been measured, neither from the ligand core in the CH region or from the ligand heads in the COOH/COO- or NH2/NH3+ stretching regions for instance, can be mainly due the polymer structure of the ligands themselves, which is highly disordered and randomly oriented, while the intrinsically weak SFG response of carboxyl and amine groups would be responsible for the silent behavior of the ligands heads. 78 Instead, NPs at the neat substrates have modified the organization of interfacial water. Indeed, at the SiO2 interface, the OH signal intensity has slightly decreased after injection of negative NPs (Figure 3a), while it has almost vanished after injection of positive NPs (Figure 3b). At the CaF2 interface, the OH signal sensibly decreases after injection of both charged NPs. Whatever the relative substrates/NPs charges, the OH intensity has initially driven towards a loss of signal intensity, either due to a decrease of the interfacial potential gradient or to a screening of the surface zeta potential. The time evolution of the OH intensities at those interfaces is shown in SI. The overall OH decrease confirms that, though the NPs did not give an own CH signature, they have reached the interface and modified the organization of the interfacial water layer as a result of their proximity to the substrates, which triggers the electrical properties of the interface due to the charges carried by NPs themselves. Substrates/DPPC/NPs Interfaces. The characterization of the substrates/DPPC interfaces and substrates/NPs interfaces has set the basis for detecting and analyzing their interactions. SiO2/DPPC/NPs. Figure 4 shows the evolution of the intensity of OH bands after injection of negative (blue curves) and positive (red curves) NPs at the SiO2/DPPC/water interface. Immediately after injection of negative NPs, the OH signal increases by 1.5 9



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times in 15 minutes, and then stabilizes around a constant value for the next 20 hours of interaction. No CH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

feature appears for any interaction time (Figure 5a), and the dried interface is characterized by CH3 vibrations, which dominate over very weak CH2 features (Figure 5b). Instead, injecting positive NPs goes together with a drastic increase of OH intensity in the first 15 minutes, which goes back to approximately the same value as before interaction in the next 20 hours. Moreover, after 30 minutes of positive NPs interaction (corresponding to point a in Figure 4, red curve, and to Figure 6a) new vibrations show up at 2960 cm-1 and 2888 cm-1, whose frequencies correspond to a methyl stretching and to the methylene Fermi resonance, respectively. However, they are no longer detected after 20 hours of interaction, and the OH intensity has also decreased (point b in Figure 4, red curve, and Figure 6b). The spectrum recorded after exposure of the interface to air (Figure 6c) shows strong CH3 vibrations, weak CH2 peaks and residual OH bands. Though the overall OH intensity has increased by the NPs approaching both substrates/DPPC interfaces, the mechanisms and the effects of interaction are quite different Negative NPs. When negative NPs (ζ = -40 mV, Table 1) face the SiO2/DPPC interface (ζ = -30 mV), repulsive electrostatic forces set the limiting approach to DPPC, so that NPs do not go close enough to impact the core conformation of the lipid bilayer. However, the gradient of potential at the interface has varied from Δζ = 30 mV for the SiO2/DPPC interface in pure water, to Δζ = 10 mV in presence of NPs (Table 1). The lower potential gradient should have given rise to a less ordered and/or thinner water layer, and therefore to a decrease of the OH signal, which instead has not been observed in the spectra. It can be reasonably excluded that NPs in the bulk may have contributed to increase the water signal, since they have been proved to make the signal mostly decreasing at the neat substrates (Figure 3a and SI). Instead, it is known that the zwitterion polar head of DPPC may reorient upon interaction with charged NPs.30,79,80 In DPPC bilayers, the phosphate-choline axis is likely to be almost perpendicularly tilted with respect to the DPPC hydrophobic body,38 so that in SSLBs it results almost parallel to the interface. NPs may have gone as close to DPPC as to interact with its polar heads. Negative NPs would have preferentially interacted with the positive charge of the choline group of DPPC, with pulling the phosphate-choline axis more perpendicular to the surface (Figure 7, left part). The reorientation of the polar head upon charged NPs has been experimentally30 and theoretically,79,80 and it has been proved that head reorientation goes together with a denser packing of the lipid tails. The vertical orientation of the polar head – and its associated dipole – may 10


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drive the alignment of water molecules over several layers, compared to a parallel orientation of the polar head 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dipole with respect to the surface. The overall balance between the potential gradient decrease and the polar head reorientation appears to give rise to moderate increase of the OH intensity at the interface after injection of negative NPs. Positive NPs. When positive NPs (ζ = +20 mV) are injected at the neat SiO2 interface (ζ = -90 mV), the OH signal has been shown to suddenly disappear (Figure 3b and SI). Instead, when injected at the SiO2/DPPC/water interface (ζ = -30 mV), a strong increase of the OH signal is observed along with the appearance of methyl and methylene vibrations in the SFG spectrum (Figure 4, red curve, and Figure 6a). At the neat SiO2 surface, positive NPs have very fast accumulated, due to strong attractive forces, and this has screened the interfacial potential, and consequently lessened the water alignment and the resulting OH signal intensity. At the SiO2/DPPC interface, while attractive forces still operate, the presence of DPPC bilayer drives a different interaction mechanism. As for negative NPs, also positive NPs make DPPC polar head reorienting. While negative NPs pull out the phosphate-choline dipole, conversely, positive NPs are known to force the same dipole towards the lipid tails (Figure 7, right part). This reorientation goes together with the reduction of the lipid film density.30 The so-increased membrane fluidity may favor NPs penetration. It is indeed known that positive NPs can penetrate the membrane through the transient local disruption of the bilayer and the formation of channel-like regions. The formation of pores around small (

Structural Changes to Lipid Bilayers and Their ... - ACS Publications

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