Ligand-Dependent Nanoparticle Clustering within Lipid Membranes

Article pubs.acs.org/JPCB

Ligand-Dependent Nanoparticle Clustering within Lipid Membranes Induced by Surrounding Medium Suzana Šegota,*,† Danijela Vojta,‡ Dania Kendziora,§ Ishtiaq Ahmed,§ Ljiljana Fruk,§ and Goran Baranović‡ †

Division for Marine and Environmental Research and ‡Division of Organic Chemistry and Biochemistry, Rudjer Bošković Institute, POB 180, Zagreb 10000, Croatia § DFGCenter for Functional Nanostructures, Karlsruhe Institute for Technology (KIT), Karlsruhe, 76131 Germany S Supporting Information *

ABSTRACT: The interactions between hydrophobic or semihydrophobic gold and silver nanoparticles (NPs) and a dimyristoylphosphatidylcholine (DMPC) bilayer as a model cell membrane in two ionic solutions result in the structural reorganization within the bilayer manifested as locally increased nanomechanical compaction in the vicinity of NP clusters as well as changed overall thermotropic properties. The effects of NP surface charge and hydrophobicity were examined using AFM imaging, force spectroscopy and IR spectroscopy. The NP clustering occurred during hydration process of dry films containing both the DMPC molecules and the NPs by the mechanism in which the number of bilayer deformations was reduced by NP clustering. The force spectroscopy showed increased bilayer density around (semi)hydrophobic NP clusters and thus locally increased lateral compaction of the bilayer. The strengthening effect was observed for both the silver and the gold NPs in a high ionic strength solution such as seawater, while it was absent under physiological conditions. The local lipid rearrangement induces the longrange lipid reorganization resulting in the bilayer phase transition shifting toward lower or higher temperatures depending on the solution ionic strength (at the most by −1.0 °C in phosphate buffered saline and at the most by +0.5 °C in seawater). temperature. Bothun et al.15 also demonstrated that the incorporation of hydrophobic, decanethiol stabilized 6 nm AuNPs into a liposomal membrane decreases the melting temperature by 2 °C at 1:1 (w/w) lipid to NP (L/N) loading ratios, that is, increases the bilayer fluidity of the gel phase by reduction of lipid ordering. The opposite effect was also observed, but for bilayer-decorated magnetoliposomes.16 Another interesting fact is the occurrence of reversible hydrophobic NP clustering within the bilayer. The models developed for hydrophobic protein interaction within a membrane were shown to be applicable also for hydrophobic NPs.17 Computational simulations suggested that although the size of the NPs plays an important role in determining the effects on the biomembrane, the surface properties of NPs (roughness, charge, hydrophobicity) are a determining factor in disruption and defect formation within a lipid bilayer.18−20 In these studies no molecules were attached to the NP and semihydrophobicity is simulated by putting spherical charges onto the NP surface. In another approach, there are neutral and charged surfacemodifying molecules on the NP. AuNPs differing in signs and densities of surface charges either spontaneously adhere to the

1. INTRODUCTION The biological effects of nanoparticles (NPs) are dependent on their unique properties such as size, hydrophobicity, chemical composition, shape, surface charge density and a level of clustering. These parameters affect cellular uptake and translocation from point of entry to the target site as well as the protein binding, which might hinder NP efficacy.1,2 Therefore, the basic understanding of NPs’ interactions with biological systems, especially with cell membranes, is crucial for the determination of the cell uptake efficiency and consequently NP cytotoxicity.3−6 In a number of recent studies, different response mechanisms of the liposomal bilayer to the surface charge of NPs and various biomacromolecules such as proteins and sugars have been reported.7−10 In short, it is the problem of gaining deeper insight into the effects of size, shape, surface charge, and extent of NP loadings on bilayer thickness, fluidity, and phase transition. When positioned within a lipid bilayer, a hydrophobic NP induces the “unzipping” in the bilayer due to the disruption of the packing of the lipid alkyl tails.11 The enrichment of the lipid bilayer interior by the hydrophobic silicon NPs has been explained by the interactions of NPs with the hydrophobic tails of the lipid molecules.12 Park et al.13,14 found by measuring fluorescence anisotropy that hydrophobic Ag and Au NPs have an effect in increasing of the DPPC (dipalmitoyl-phosphatidylcholine) membrane fluidity and in decreasing the phase transition © XXXX American Chemical Society

Received: January 28, 2015 Revised: April 1, 2015

A

DOI: 10.1021/acs.jpcb.5b00898 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B bilayer surface or penetrate into the bilayer.20 The phase transition temperature can also be regulated by encapsulating hydrophobic NPs differing in surface properties.21 Increased density of surface-modifying molecules on the NPs decreases the main phase transition temperature of a lipid membrane. Besides the size of NPs which plays a key role in determining the effects on the biomembrane; that is, larger NPs may result in different morphological changes than smaller ones,8 surface charge density of NPs seems to be equally important,22 as well as the inclusion of long-range electrostatic interactions.23 Along with the reported strong correlation between the membrane permeability and the solubility of various types of small NPs,17,19,24,25 the restructuring of membranes associated with NPs functionalized by hydrophobic ligands whose diameters are smaller than the membrane thickness has also been given due attention. In this study, however, the restructuring of membranes will be approached by two not yet employed techniques, namely, force and IR spectroscopy. Within this study, the influence of the hydrophobic and semihydrophobic gold and silver NPs on the supported lipid bilayer (SLB) lateral compaction at low (phosphate buffered saline (PBS)) and high (seawater (SW)) ionic strength conditions was investigated by atomic force microscopy (AFM) imaging, force spectroscopy (FS), and IR spectroscopy. SW was used as a naturally occurring realization of the high ionic strength conditions. Taking into account that many NPs, in particular those from sunscreen creams or various cosmetic products, end up in SW,26 it should obviously be included in studies of the nanotoxicological aspect of the membrane uptake. Gold and silver NPs containing different stearyl-based ligands were explored. The average diameter of the NPs prepared for this study was somewhat less than 3 nm, that is, smaller than the normal thickness of the bilayer which is about 5 nm, and it is expected that the tendency of NPs toward clustering will be observed. IR spectroscopy27 was used to quantify the effect of an aqueous ion mixture as well as the effect of the acquired surface charge density of hydrophobic NPs on the thermotropic phase behavior of zwitterionic (neutral) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipids. The main phase transition involves variations in the mobility of acyl chains and an increase in the lipid lateral mobility. The headgroups have an effect through the electrostatic (dipolar) interactions. The changed character of the main transition is thus caused by the interactions between lipid molecules and the ligands bound to NPs. Alternately, AFM imaging and force spectroscopy were employed to quantify the nanomechanical compaction of SLBs without28−31 and with embedded NPs. These methods were used to explore local (in the vicinity of an encapsulated NP cluster) nanomechanical lateral compaction of the membrane that is supposed to be significantly affected by the interactions between NP ligands and lipid molecules. Furthermore, the presence of divalent ions (Ca2+ and Mg2+) in SW is expected to increase the mechanical lateral compaction of a membrane with embedded charged NPs in both the gel and the liquid phase. For example, electrostatic interactions between the negative charged phosphatidylserine (PS) and divalent ions have a much more pronounced effect on the membrane compaction than for neutral DMPC (the area per lipid for DPPS is 0.402 nm2 which is 0.04 nm2 less than for the DPPS-Ca2+ system32). Although a substantially lower effect was expected for DMPC in comparison to PS, the electrostatic

contribution to the DMPC membrane compaction was measurable31 as it will also be demonstrated below.

2. EXPERIMENTAL METHODS 2.1. Materials. 1,2-Dimyristoyl-phosphatidylcholine (DMPC, Lipoid GmbH, Germany, > 98% purity, gift sample) with saturated C14 length chains and chloroform (Merk, Darmstadt, Germany) was used as received for liposome preparation for AFM and force spectroscopy measurements. DMPC (Sigma-Aldrich, >99% purity) was used as received for film and dispersion preparation for IR-spectroscopy measurements. Both chemicals were of the highest purity commercially available. Phosphate buffered saline (PBS) was prepared at pH 7.4 and ionic strength I = 150 mM. SW samples were collected from the offshore station in the Northern Adriatic (salinity S = 37.42 %, pH 8.2, I = 550 mM with respect to NaCl only, dissolved organic compounds (DOC) = 1 mg dm−3, C(Ca2+) = 10.4 mM, C(Mg2+) = 53.3 mM) and filtered (0.22 μm Whatman). The SW ionic strength was thus nearly four times greater than that of PBS. A part of the DOC is surface active substances (SAS) of very diverse composition, from hydrophilic polysaccharides and conditionally hydrophobic humic substances to very hydrophobic fatty acids.33 The SAS covers a range of concentrations, on average 10% of DOC concentration34 which is in the present case around 0.0001 g dm−3. Since the overall ionic content of SW amounts to 30 g dm−3, electrostatic interactions between a loaded lipid membrane and cations could not be appreciably perturbed by the presence of SAS. All chemicals required for synthesis of Au NPs, (HAuCl4· H2O, lipoic acid, and NaBH4) and their functionalization by coupling via NHS-EDC cross-linking reaction (stearylamine, EDC, NHS), further referred as functionalization A (Supporting Information, Figure S1) or AuNP_A, were acquired from Sigma-Aldrich. Both coated silver NPs (AgNP_A and AgNP_B) were synthesized according to the same protocol described in the Supporting Information (SI). Semihydrophobic linker B (Figure S2) for functionalization of gold and silver NPs was synthesized as described in the SI and attached to the NPs by ligand exchange. The term semihydrophobic is here used for charged hydrophobic, and thus a semihydrophobic NP is mostly hydrophobic with charges randomly distributed on its surface.24 Functionalized NPs were further characterized by UV−vis spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Zeta (ζ) potentials of NPs, pure and NP-loaded liposomes were measured as described in the SI. Nanoparticle homoligand coverage, for example, the degree of surface functionalization of gold and silver NPs is calculated by assuming the following: (a) an arrangement of alkanedithiolates on the flat faces is in agreement with the coverage γ = 0.33 observed for alkanethiolate monolayers on hexagonally close-packed planar gold;35,36 (b) in self-assembled monolayers of dithiolate on surface silver (111) atoms there are 26% more chains per unit area than on gold (111) on-top sites;37 (c) NPs are spherical and have narrow size distribution. The obtained ligand densities of NPs are shown in the SI, Table S2. 2.2. Formation of Liposomes and SLBs. DMPC was dissolved in chloroform solutions of gold or silver NPs. The (weight of lipids)/(weight of NPs) loading ratios, L/N, are shown in SI, Table S2. The ratios were calculated from the gold and silver NP molecular weights taking into account the difference in arrangement of alkanethiolates on the flat faces of B


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The Journal of Physical Chemistry B

Figure 1. (a) Scheme of the preparation of two types of NPs used in this study by employing amide coupling of commercial stearyl amine A and ligand exchange methodology with synthesized linker B. (b) TEM images of AuNPs and AgNPs (upper right), AFM images of AuNP_A (middle left), and AuNP_B (middle right), and their corresponding vertical profiles (bottom).

NP_A was prepared by amide coupling of commercially available stearyl amine A to lipoic acid-coated NPs using EDC/ NHS coupling strategy and extraction of NP_A using chloroform. Semihydrophobic NP_B containing an additional side chain amine group capable of carrying positive charge was prepared using the ligand exchange method, that is, by replacing lipoic acid with synthesized ligand B (see SI for synthetic details). The size and shape of the monolayer-protected gold and silver NPs were investigated both by TEM and AFM. AFM particle size analyses was performed on a 2 × 2 μm2 imaged surface area for each NP sample confirming that NPs are spherical and similar in size (Figure 1b, middle, and Table 1). The lateral dimension of the bright spots in the AFM images (Figure 1b) is around 30 nm. This is an order of magnitude less

gold and silver as explained. After rotary evaporation of the solvent, the remaining lipid film with NPs was dispersed in (a) phosphate buffer saline (PBS) or (b) in filtered SW (for details see SI). All SLB samples were prepared under the same experimental conditions by the drop deposition method on freshly cleaved mica attached to the metal disc (vesicle fusion method). The preparation of gold or silver loaded liposomes followed closely the procedure described in refs 12−14. Each SLB sample was washed with solvent in order to remove unreacted stearyl amine or lipoic acid and nonencapsulated NPs from solution above the SLB. 2.3. Nanomechanical Properties of SLBs. Force curves were obtained in liquid contact mode only and under equilibrium conditions. All the curves presented here were obtained with the very sharp tip (SNL, Bruker, nom. freq 65 kHz, nom. spring constant 0.35 N/m, nom. tip radius 2 nm and the tip speed of 1 μm s−1). The spring constant of the cantilever was calibrated using the reference cantilever method38 (Bruker, CLFC-NOBO). The time to record an approach and retraction force curve cycle was about 1 s. During a typical force spectroscopy experiment, the tip successively approaches and leaves the surface in a cyclic manner. It was imperative to have the continuous bilayer during such measurements since otherwise the tip could easily become contaminated at the mica−lipid edges.

Table 1. Mean Height of a Single Functionalized NP within Each Sample As Observed by AFM and the Mean Diameter As Determined by TEM sample AgNP_A AgNP_B AuNP_A AuNP_B

3. RESULTS AND DISCUSSION Preparation and Properties of NPs. Two different stearyl-based ligands (ligands A and B, Figure 1) were used to prepare hydrophobic and semihydrophobic nanoparticles, respectively, to aide the lipid bilayer embedment. Hydrophobic

AFM mean height (nm) 2.8 2.8 3.1 3.3

± ± ± ±

0.8 0.7 0.8 1.0

TEM mean diametera (nm) 2.2 2.2 1.9 1.9

± ± ± ±

1.4 1.4 1.1 1.1

Δd (nm)

ligand densityb (nm2)

± ± ± ±

5.3 5.2 4.2 4.2

0.6 0.6 1.2 1.4

2.2 2.1 1.9 2.1

a

Metal core diameter of the NPs before their functionalization with A or B ligands. bNanoparticle ligand density is calculated using the ratio γ = 0.33 of alkanedithiolates to surface gold atoms of a NP.36 It was also further assumed that there are 26% more chains per unit area on silver than on gold.37 C


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Figure 2. Zeta potential of liposomes with efficiently loaded hydrophobic AgNP_A and semihydrophobic AgNP_B in PBS (pH 7.4) buffer and in SW (pH 8.2). The error bars show the standard deviation between three independent samples.

follows that an average number of ligands per NP is 80 and 50 for AgNPs and AuNPs, respectively. The 3 nm diameter NPs are suitable for entrapping within the lipid (DMPC) bilayer, even with the surface coatings made of flexible ligands. According to the previously published results,43 alkyl chains of both ligands are typically in all-trans configuration. Since the alkyl chain of DMPC contains 14 and the chains of ligands A or B 17 CH2 groups, the expected overall diameter of the NP_A or NP_B would be around 9 nm, that is, larger than the bilayer thickness. The conformations of the chains should consequently be twisted toward the closer contacts with the metal core if the NPs are to be entrapped within the bilayer. This, however, seems to be energetically very costly. Instead, interdigitation of lipid and NP chains accompanied by bilayer deformations can take place ensuring in such a way stable position of NP within the bilayer. Such structures have been described as bilayer unzipping.11 A picture in which a cluster of NPs is enveloped by the upper monolayer of a SLB44 is consistent with such an interpretation. Such an arrangement requires practically no bending of the NP ligand chains and also is likely the only possible one for an entrapped NP cluster. In any case, interdigitation of the lipid chains and the NP chains should take place ensuring the stable position of a NP within the bilayer. Zeta (ζ)-Potential of NPs and of NP Loaded Liposomes. The zeta-potential measurements for NPs in chloroform at room temperature showed that after replacing the hydrophobic ligand A by the semihydrophobic charged ligand B, the zeta potential significantly changes from the negative, ζ = −12.3 ± 1.2 mV (AuNP_A) and ζ = −13.3 ± 1.5 mV (AgNP_A) to positive ζ = +15.9 ± 0.8 mV (AuNP_B) and ζ = +45.8 ± 2.1 mV (AgNP_B). The studied functionalized NPs are genuinely neutral and yet they exhibit nonzero zeta potential when dispersed in chloroform which is a nonprotonic solvent having a low donor number and dielectric constant. If traces of water in chloroform or other impurities, most likely metal cations that easily form complexes with amines,45 are assumed, then the negative charge on NP_A should be attributed to the traces of unreacted −COOH linkers during the NP preparation. The surface charge of the NP_B is then primarily determined by the amine NH2 end group of the side chain causing different charge potential relative to NP_A.

than what has been measured in SLBs (see later). Presumably, individual NPs are seen here, while in the bilayer they tend to be clustered. According to TEM, the diameter of the metal core was approximately 2 nm (Figure 1b, Table 1) and there were observable size changes of 1 nm in the AFM determined NP heights due to the ligand addition. The TEM diameters are the same irrespective of the metal and that can be the consequence of the synthesis pathway. The measured AFM increase of 1 nm is ligand-independent within the accuracy of the measurements. We now proceed to estimate the surface coverage of the employed NPs. Our calculated ligand density values (Table 1) are within the reported values. They are practically independent of the metal core for both ligands. They are also in very good agreement with experimental density values obtained by Lanterna et al.39 for small AuNPs functionalized with heterocyclic alkanedithiols of similar chain length. According to Luedtke and Landman,40 self-assembled multilayers on the gold nanocrystallites are characterized by the ratios of 1.55 and 1.87 between the number of exposed surface gold atoms to the numbers of adsorbed sulfur atoms for the smaller and larger nanocrystallites, respectively. They also determined the ligand surface density as a function of the number of ligands per NP and report a value of around 3 ligands nm−2 (ranging from 2 to 5 ligands nm−2) for sulfur heterocyclic compounds and described the density as almost size-independent. However, they postulated a greater ligand density with an increase of the chain length. They explained these results by van der Waals (vdW) interactions between ligand chains. Similarly, Xia et al.41 found for similar to our ligand lengths a decrease of ligand density with an increase of chain length. Packing densities of 4.97 ± 0.01, 4.58 ± 0.01, and 2.20 ± 0.03 ligand nm−2 were determined by use of X-ray photoelectron spectroscopy (XPS) for gold NPs modified with mercaptoundecanoic acid, mercaptohexanoic acid, and thioctic acid, respectively. Hinterwirth et al.42 showed that the size-indpendent but ligand chain length-dependent surface densities on the gold NPs ranged between 6.3 molecules nm−2 and 4.3 molecules nm−2. Using ICP-MS, Hinterwirth et al., 42 were found size independent but ligand chain length-dependent ligand density as well as no significance difference of hydrophilic and hydrophobic ligands with approximately the same ligand length. From the estimated ligand densities (Table 1) it D


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Figure 3. Temperature dependence of the symmetric CH2 stretching frequency in the IR spectra of unloaded31 and AuNP- and AgNP-loaded supported DMPC bilayers in PBS buffer (a and c) and SW (b and d).

Table 2. Phase Transition Temperatures (°C) for NP-Loaded DMPC Films PBS pure DMPCa + Au_A + Ag_A + Au_B + Ag_B average difference a

24.5 23.5 24.6 23.6 23.5

Tm.pure − Tm

SW 24.5 24.6 25.5 24.8 24.5

−1.0 +0.1 −0.9 −1.0 −0.7 ± 0.5

Tm.pure − Tm

Tm.PBS − Tm.SW

+0.1 +1.0 +0.3 0.0 +0.4 ± 0.5

0.0 −1.1 −0.9 −1.2 −1.0 −1.1 ± 0.1

Reference 31.

As for the AgNP loaded liposomes in an aqueous medium, the liposome zeta potential changed from negative in PBS to positive in SW (Figure 2). The absolute value of the zeta potential in the gel phase is always greater than in the fluid phase except for the AgNP_B. It is very well-known that zeta potential of phosphatidylcholine liposomes depends on the ionic strength and ionic composition of the solvent.28,29,46 Divalent cations, present in SW, more strongly bind to the lipid headgroup than monovalent cations (Na+, K+), present in both PBS buffer and SW.31 Thus, the sign change of the zeta potential of the unloaded liposomes from negative (in PBS buffer) to positive (in SW) sign (Figure 2) is primarily due to the underlying processes such as cooperative and competitive ion binding in both the gel and the liquid crystalline phase of the lipid bilayer. It is well documented in the literature13−15,17 that the method used in the present work to prepare NP loaded liposomes ensures the encapsulation of the hydrophobic NPs within the liposome bilayer. Since the side chains of ligands B as possible charge carriers are within the NP monolayer and not so exposed to the solvent, the hydrophobic character of NP_Bs

is more pronounced. Nevertheless, the differences between the two ligand types were observed. The contribution of semihydrophobic ligand B has been reflected in the increasing of liposome zeta potential from more negative −4.78 mV (unloaded liposomes) to −0.45 mV in the gel phase and from −3.62 mV (unloaded liposomes) to −2.98 mV in the liquid phase. The significant difference in zeta potential value (Δζ = −4.33 mV and −0.64 mV in the gel and liquid phase, respectively) and almost 4-fold larger than of hydrophobic ligand A (Δζ = −1.07 mV and −0.26 mV in the gel and liquid phase, respectively) are thus caused by semihydrophobic ligand B. These results can be explained by the pKa value of 10 for primary amine moiety found in ligand B. Since the pH values of PBS and SW are 7.4 and 8.2, respectively, a certain degree of protonation of ligand B is to be expected. As a result, the charged side chains -(CH2)4NH3+ of ligand B might be translocated out of the hydrophobic bilayer region becoming aligned with the phosphate groups of DMPC in the polar region of the bilayer, adapt themselves through electrostatic and hydrophobic interactions to the surrounding lipids, and become more exposed to divalent cations in the surrounding medium. At the same time, the hydrophobic parts E


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Figure 4. 3D and 2D AFM height images of NP_A embedded within DMPC SLB layers and the corresponding section height profiles in denoted circles at 24.3 °C. Vertical scales in height images are 20 nm.

might be the stacked nature of SLBs47 (the number of bilayers forming a film on the surface of an IR window was certainly much greater than 1). The broadening is still present when a SLB is loaded with AgNP_A (Figure 3a,b). In PBS, the transition temperature is downshifted by 0.7 °C on average, while in SW the shift is +0.4 °C. The difference between the two media of −1.0 ± 0.1 °C is not sensitive to the type of NPs. In the case of AgNPs, the effect is more pronounced for semihydrophobic NPs with ligand B. The slight decrease of Tm by 1 °C only in PBS suggests that the incorporation of the hydrophobic NPs at the L/N loading ratio between 2 and 1700 (SI, Table S2) can lead to the increased bilayer fluidity only when the ionic strength is not too high. These loading ratios are comparable with those used by Bothun15 where it was shown by DSC and fluorescence anisotropy measurements that even for the ratio 1:1 for the DPPC and decanethiol modified AgNPs

of the amphiphilic ligands stay within the hydrophobic bilayer region and fuse with surrounding lipid molecules leading to the significant lateral compaction which should thus be reflected in bilayer nanomechanics, which will be demonstrated in what follows. Phase Behavior of SLB with NPs. Shifting of the main phase transition temperature, Tm, by inclusion of hydrophobic and semihydrophobic NPs within the DMPC bilayer has been accurately measured by IR spectroscopy under the equilibrium conditions (Figure 3, Table 2). The overall heating rate from 19 to 30 °C was 0.1 °C min−1 and that included incrementing the temperature, waiting for temperature equilibration, and recording a spectrum. A broadening of the main transition of unloaded DMPC in SW with a transition temperature width of 8−10 °C (Figure 3b) is likely substrate induced because it was not observed in dispersions.31 An additional contributing factor F


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Figure 5. 3D and 2D AFM height images of NP_B embedded within DMPC SLB layers and the corresponding section height profiles in denoted circles at 24.3 °C. Vertical scales in height images are 20 nm.

in PBS, the transition temperature Tm dropped only by 2 °C. Alternately, in the study of magnetic hydrophobic NP concentration influence on the transition temperature of soybean lecitin liposomes,48 the observed decrease for the 1000:1 ratio and 250:1 ratio were 4 and 10 °C, respectively. In our case there was thus a small but observable change in the transition temperature. The main phase transition of an SLB is a first order phase transition and the slope of the ν̃ vs temperature curve shows the extent of cooperativity, that is, the width of the region of phase coexistence. The cooperativity is reduced the most for an unloaded DMPC bilayer in SW and AgNP_A loaded DMPC bilayers. In these cases only a small number of lipid molecules form a cooperative unit which can certainly be broadly interpreted in terms of the overall balance between long and short-range interactions between lipid molecules, cations, and NPs.

There is thus an agreement of our observations with the coarse-grained molecular dynamics results for the 20:1 w/w DPPC/NP system. Although the presence of NPs in a relative low loading ratio induces rearrangement of the lipid bilayer, the hydrophobic NPs immersed in the bilayer core have no significant effect on the main transition temperature.17,21,24 By inclusion of hydrophobic NPs, the orientation of DMPC molecules adjacent to the NPs is perturbed, which has been observed as a small main phase transition shift compared to that of the pure DMPC bilayer (Table 2). This effect has been observed in experimental studies15,17 as well as in MD dynamics simulations.21,24 Lin et al.20 and Lin et al.21 found that by inclusion of hydrophobic NPs, the main phase transition temperature could decrease or increase depending on the surface density of ligands. In our experiments, both NPs, NP_A, and NP_B, have likely the same density (Table S2), and G


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Table 3. Heights of the SLB Protrusions from the Unperturbed SLB Surface and Diameters and Lengths of NP Clusters h/nm

hmin/nm

hmax/nm

Au_A_PBS Au_A_SW Ag_A_PBS Ag_A_SW

3 3 2 5

± ± ± ±

2 1 1 2

1 2 1 2

7 7 4 9

Au_B_PBS Au_B_SW Ag_B_PBS Ag_B_SW

5 2 4 3

± ± ± ±

3 1 2 1

2 1 2 2

12 10 10 6

D/nm

Dmin/nm

Dmax/nm

± ± ± ±

3 7 2 2

141 121 118 144

69 47 55 75

33 33 27 43

87 ± 61 54 ± 29

13 14

L/nm

Lmin/nm

Lmax/nm

202 ± 202 59 ± 51

23 12

1637 382

265 119

Figure 6. The yield threshold of a mica-supported DMPC SLB loaded with gold and silver NPs in PBS (a and c) and SW (b and d) is temperature dependent. All points correspond to the central value of Gaussian fitting to the obtained histograms. The error bars show the standard deviation between three independent samples.

hydrophobic NPs were built into the lipid bilayer and more often as clusters rather than as individual particles. Not only hydrophobic (AgNP_A and AuNP_A), but also semihydrophobic (AgNP_B and AuNP_B) NPs were built into the bilayer. This is in agreement with the experimental results that AuNPs functionalized with cationic headgroups enter the hydrophobic region of the lipid bilayer.50 Altered membrane thickness around the embedded nanoparticle has also been predicted in the simulations with functionalized NPs.51 Since the NP diameter of 10 nm (including ligands) was larger than the normal thickness of the bilayer, the partial encapsulation was only feasible with charged ligand end groups extending into the surrounding water. The effect of the hydrophobic NPs on SLB nanomechanics at a constant loading ratio was further investigated in detail by force spectroscopy. The occurrence of NP clusters in SLBs, clearly seen from the cross section profiles in Figure 4 and 5, must be influenced by the way the NP-loaded bilayers were prepared. All the samples in this study were prepared the same way. The mean cluster heights (∼4 nm and always measured from the unperturbed membrane surface) are comparable to the diameters of a single

consequently the shifts of the main phase transition correlate with the type of ligand, whereas in the same solvent no ligand induced difference was observed for AuNPs. However, for AgNPs the transition with AgNP_A in either PB or SW is wider with a width of ∼5 °C. This is evidence for the changed phase transition kinetics that should be mainly influenced by the metal core. AFM Imaging and Nanomechanics. AFM imaging was applied to locate NPs in a SLB (Figure 4 and 5). Given the strong preference of the NPs for binding into a hydrophobic environment, they are most likely embedded into the bilayer concomitantly modifying surface topography. The homogeneous scabrous bilayer without ruptures is clearly seen irrespective of embedded hydrophobic NPs. The diameter and length values for all examined protrusions from the unperturbed SLB surface are corrected for the convolution effect of the tip49 and presented in Table 3. The height increase which roughly corresponds to the diameter of NPs forming clusters is shown as a vertical section profile. However, lateral dimensions are much larger and amount to a few hundred nanometers. These findings conclusively confirmed that the H


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Figure 7. Force curve thickness analysis of the NP loaded membranes in the vicinity of NP clusters and in the bulk. The ordinate values are calculated as percentage difference, d% = 100 × (1 − dp/dunp) or 100 × (1 − db/dunp) where dunp stands for the thickness of the unperturbed DMPC bilayer,31 dp for the thickness in the vicinity of a NP cluster, and db the thickness far from any cluster (bulk). The thickness of DMPC SLB is 5.3 nm in PBS and 5.4 nm in SW.31

near the lipid phosphate groups modifies the orientation of the dipoles and consequently the mutual interaction between the phospholipids. Moreover, an increased yield threshold value in high ionic strength solutions31 is attributed to a lateral compaction effect of the salts on the lipid bilayer. We also performed a force curve thickness analysis (Figure 7). It is seen that, for example, in PBS and at 21.1 °C, the dp of the Au_A bilayer is 0.5% thinner and the db is 4% thinner than the unperturbed DMPC bilayer. On the other hand, in PBS and at 29.2 °C, the dp of the same bilayer is 5% thicker and the db is only 0.5% thicker. The thinning relative to dunp was observed in PBS at the lowest measured temperature and larger in the regions far from NP clusters. This indicates that bilayer disordering was caused by the NP presence. However, on the other hand, the thickening of both dp and db is thus always present at the highest measured temperature. The difference in the temperature dependence for any of the studied systems is particularly strong when the solvent ionic strength is changed, which is in accord with the IR measured phase transition temperatures (Figure 3). The force spectroscopy results are obtained only around NPs. The fact that no disrupted lipid bilayer far from NPs has been observed is consistent with the simulation study by Lin et al.21 The inclusion of NPs, at moderate concentrations as in our study, can have significant effect on lipid rearrangement, causing the expansion of the nonpolar domains within the bilayer which is seen as an increase of bilayer thickness. The observed rupture force in SW was increased (for gold NPs, Fy = 3.08 nN in gel phase, Fy = 4.4 nN in fluid phase). The main contribution to the increased threshold force could arise from the electrostatic interactions between semihydrophobic B ligands and the polar head groups of lipid molecules. In

NP. On the other hand, since the heights are found in the interval from 1 to 12 nm, the NP clusters appear multilayered. Taking into account lateral dimensions, in particular Dmin or Lmin, the clustering is more pronounced with B ligands. An interesting fact is the obvious tendency of AuNP_Bs to form rod-shaped clusters and especially so in low ionic strength medium. Speaking in terms of relative orientation between two particles it seems that in the case of AuNP_B there is an orientation leading to quasilinear agglomerates. However, this is effective only in PBS, that is, is not the same in all solvents. It is very likely that the most probable way of clustering can also be controlled by the choice of the solvent ionic strength. We further performed force spectroscopic measurements on DMPC SLBs also in two solutions, PBS buffer and SW. The corresponding histograms of yield threshold forces fitted with Gaussian at different temperatures for each solution are summarized in Supporting Information, Figure S1. The average rupture forces were measured only within the regions around the NPs that were chosen among the NPs found in the selected 2 × 2 μm2 areas. The increase in the rupture force for both phases (below and above Tm = 24 °C) in SW relative to PBS (for example, for AuNP_A Fy = 3.7 ± 0.6 nN in PBS, and Fy = 6.8 ± 1.3 nN in SW at 23.4 °C, Figure 6c,d and SI, Figure S1) can be attributed to the increased lipid lateral interactions in SW provoked by the enhanced binding of the divalent cations. In the case of charged semihydrophobic AuNP_B, the significant lateral compaction of the lipids and an increase of the required force (from 10 to 50%) with respect to the pure DMPC bilayer occurs only in SW while in PBS the bilayer becomes softer (50% in both phases) (Figure 6c,d). Although reduced, the same effect is again present in both media for Ag_NPs. The presence of ions I


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ing effect was observed for both the silver and the gold NPs in a high ionic strength solution such as SW, while it was absent under physiological conditions. The effect was more pronounced for the semihydrophobic NPs protected with ligand B. This can be ascribed to the formation of nondisruptive bilayer deformations by the electrostatic and hydrophobic interactions whereby the charged ligand ends are translocated out of the hydrophobic bilayer region. The charge translocation, to a large extent increased by the divalent ions in surrounding medium, has been also reflected in the more positive zeta potential of NP-loaded liposomes relative to that of pure liposomes. At the same time, the hydrophobic parts of the studied ligands stay within the hydrophobic bilayer region and fuse with surrounding fatty acyl chains of lipid molecules. There was presumably a reorganization of the initially homogeneous lipid bilayer involving lipid density increase in the regions surrounding NPs and a decrease in the regions that are halfway between the two first neighbor NP clusters. The observed clustering of semihydrophobic NPs and described characteristics of the lipid−NP interactions parallel to a certain extent the lipid−protein interactions. In either case the stresses are provoked in the lipid bilayer. Our results are yet another confirmation of the similarities in the membranemediated interaction between proteins and NPs that is effective provided two proteins or NPs happen to be close enough. The universal nature of membrane-mediated attraction thus extends to NPs as well.

other words, bulk membrane property is screened by strong ligand−lipid interactions. Water penetration into the bilayer around embedded NPs was thus probably enhanced, and the electrostatic interaction between the primary amine group of ligand B and the ions in SW strengthen. In this context, strong interactions result in a decrease of the membrane mobility. The charge translocation, to a large extent increased by divalent ions in the surrounding medium has been reflected in a more positive zeta potential of NP-loaded than of pure liposomes (Figure 2). On the other hand, the hydrophobic part of the amphiphilic ligands stay within the hydrophobic bilayer region and fuse with surrounding lipid molecules. The local lipid rearrangement induces the long-range reorganization resulting in the bilayer phase transition shifting toward lower temperatures. Such behavior has been observed in the study of polymersomes where their increased functionality and enhanced mechanical strength was recently demonstrated by introducing the NPs into their curved membranes.52 A critical first step in fully understanding the interactions between monolayer-protected AuNPs and cells was recently presented by demonstrating the nondisruptive nature of the inserted state and by showing the importance of AuNP surface properties.51 Therefore, the ion composition, ionic strength of the medium, and the ion binding competition are also directly responsible for the nanomechanics of lipid bilayers particularly in SW, keeping in mind that in this study the low loading ratios of NPs were applied.31 During the phase transition process, hydrophobic NPs embedded among the lipid hydrophobic tails show small impact on the DMPC bilayer that certainly can be ascribed to the zwitterionic (neutral) nature of DMPC lipid molecules.

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ASSOCIATED CONTENT

* Supporting Information S

Synthesis of functionalized NPs; characterization of NPs; formation of liposomes and SLB; histograms of yield threshold forces. This material is available free of charge via the Internet at http://pubs.acs.org.

4. CONCLUSIONS We studied the effects of encapsulated NPs on the morphology of zwitterionic (neutral) supported DMPC bilayer. We focused on the (semi)hydrophobic NP clusters within the lipid bilayer and the factors influencing the nanoparticle−membrane interactions, among them particle size and surface charge. In preparation of liposomes and SLBs encapsulating hydrophobic or semihydrophobic NPs the protocol described in the literature was closely followed. Liposomes that adsorbed onto the mica support were carrying clustered NPs within the bilayers, that is, coated NPs with an effective diameter larger than the bilayer thickness that accommodated themselves into the bilayer hydrocarbon region by the unzipping mechanism.11 Our results, especially the thickness measurements, also confirmed that DMPC SLBs contained embedded NPs. It is found that the NP−lipid interactions were changed by replacing PBS buffer with a high ionic strength medium (SW) toward enhancement of the membrane compaction in the vicinity of encapsulated NPs. Although, in general, hydrophobic NPs were shown to be capable of inducing large local structural deformations, we observed that for small NPs (2 nm core diameter) such effects were not large, were more prominent in SW, and were less dependent on the type of the ligand bound to the NP metal core. On the nonlocal level of phase change, the effect of encapsulated NPs was the lowering of the phase transition temperature in PBS at the most by 1.0 °C and its increase at the most by 0.5 °C in SW, thus emphasizing the more dominant surrounding medium influence. The force spectroscopy results, on the other hand, indicated that hydrophobic NPs can locally increase the lateral compaction of the bilayer. The strengthen-

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AUTHOR INFORMATION

Corresponding Author

*Tel: +385 1 456 1185. Fax: +385 1 468 0242. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by Grant Nos. 0982934-2744, 0982904-2927 from the Ministry of Science, Education and Sports of the Croatian Government and Croatian−German bilateral cooperation project “Interaction of ligand coated NPs with lipid membranes” and DFG-CFN Project A5.7. The authors thank G. Pletikapić for help in nanoparticle synthesis, A. Philippe and M. Sikirić for zeta potential measurements, M. Pocrnić for IR spectroscopy measurements, V. Č adež for graphical abstract preparation, and P. Bockstaller for TEM measurements.

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Ligand-Dependent Nanoparticle Clustering within Lipid Membranes

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