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Interaction Between Soft Nanoparticles and Phospholipid Membranes – Effect of the Polymer Grafting Density on Nanoparticle Adsorption Marek Sokolowski, Zehra Parlak, Christoph Bartsch, Stefan Zauscher, and Michael Gradzielski ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01868 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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ACS Applied Nano Materials

Interaction Between Soft Nanoparticles and Phospholipid Membranes – Effect of the Polymer Grafting Density on Nanoparticle Adsorption Marek Sokolowski† Zehra Parlak,‡ Christopher Bartsch,† Stefan Zauscher,‡* Michael Gradzielski†* †Stranski

Laboratorium für Physikalische Chemie, Technische Universität Berlin, Straße des 17 Juni 124, 10623 Berlin, Germany ‡NSF Research Triangle Materials Research Science and Engineering Center, Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708, USA KEYWORDS. Polymer brush, silica nanoparticles, core-shell nanoparticles, charge interaction, quartz crystal microbalance with dissipation, supported lipid bilayers, PDMAEMA

ABSTRACT: Nanoparticles (NPs) have large potential for biological applications as typically they exhibit strongly size-dependent properties. Specifically, the interaction of NPs with phospholipid membranes is of significant relevance to nanomedicine and the related field of nanotoxicology. Therefore, the investigation of NP interactions with model membranes is not only of fundamental importance, but also of practical value to understand NP interactions with more complex cell membranes. Supported lipid bilayers (SLBs) provide a powerful platform to study such interactions. Here, we report on the interaction of SiO2-NPs – covered with cationic polymer (PDMAEMA) of different grafting density but approximately constant polymer layer thickness – with SLBs of differing charge density. We studied binding of the NPs to the SLBs by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). A key result of the study is that at low solution pH and in presence of electrostatic attraction, the amount of adsorbed NPs drastically decreases with increasing polymer brush grafting density beyond a critical value. However, upon increasing the solution pH (thereby lowering the apparent electrostatic attraction) even NPs with high polymer grafting density adsorb. In this transitional range NP adsorption depends strongly on NP concentration becoming reduced at higher concentration. The experimental observations were interpreted by simple models taking into account electrostatic and van der Waals interactions that allow to gain some insights into the prevailing conditions. KEYWORDS: polymer-modified nanoparticles, supported lipid bilayer (SLB), QCM-D, membrane interactions, adsorption

1. INTRODUCTION Phospholipids are amphiphilic molecules composed of a head group with a hydrophilic phosphate unit and typically two hydrophobic alkyl chains.1 In water, phospholipids selfassemble into bilayer membranes already at very low concentrations. Bilayer formation is related to the cylindrical molecular structure of the phospholipids, described by a packing parameter value p between 0.5–1.2-4 In nature phospholipid bilayer membranes often occur as cell membranes and are involved in formation of intracellular compartments. There are several types of ionic and nonionic phospholipids, differing in their head group chemistry and fatty acid chain, that make up lipid membranes. Most eukaryotic cells contain phospholipids with a choline head group5,6 – a lipid with a zwitterionic head group, but some phospholipids have a phosphate group and therefore cells have an anionic surface charge. The thermodynamically stable phase of pure bilayer membranes is lamellar, while lipid vesicles (often also called liposomes) are in most cases only metastable.7-9 Lipid vesicles are frequently used in pharmaceutical and cosmetic applications,10,11 or serve in drug delivery.12,13 Research in the field of nanotechnology increased drastically in the last decades due to many potentially attractive application possibilities.14–23 Nanoparticles (NPs) have large potential for biomedical applications as they typically exhibit strongly sizedependent properties and specifically, the interaction of NPs with phospholipid membranes is of significant relevance to

nanomedicine19 and the related field of nanotoxicology.22,23 Therefore, an understanding of NP interactions with model membranes is not only of fundamental importance but also relevant to understanding NP interactions with compositionally and structurally more complex cell membranes. To this end we investigated the interactions of NPs with model membranes in the form of supported lipid bilayers (SLBs).

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Scheme 1. a) Top left shows the molecular structure of zwitterionic DOPC and anionic DMPA phospholipids used for the formation of anionic vesicles via extrusion. Top right shows a cartoon of SiO2-NPs modified with anionic (–OH), cationic (–NH2) moieties, and brush grafted polyelectrolyte (PDMAEMA). The bottom left cartoon shows the formation of a supported lipid bilayer on a silica surface, and the bottom right cartoon schematically shows the interaction of NPs with the model membranes. Both processes can be studied with QCM-D. b) Cartoon of increasing polymer density.

The strength of NP binding onto a lipid membrane depends on the stiffness of the membrane, Coulombic and van der Waals (vdW) interactions, and the charge and/or polymer modification of the NP surface.24 Here, we employed two different types of silica NPs; i.e., “hard” anionic (SiO2) and cationic (NH2functionalized), and “soft” cationic NPs, decorated with a covalently bound PDMAEMA polyelectrolyte brush. Such polymer brush-modified NPs are interesting because the mechanical properties of the polymeric shell can be controlled by a systematic variation of the grafting density. This in turn allows for the modulation of the extent and strength of the steric repulsions between particles and between particles and a surface. The NP surface charge density is tunable by the grafting density of the amino groups (for “hard” NPs) or the polymer chains (for “soft” NPs) and the solution pH. Investigations involving such systems have not been done previously and are addressed within this work. To study time-resolved NP-membrane interactions we used a quartz crystal microbalance with dissipation monitoring (QCMD). This instrument allows for in-situ monitoring and validation of supported lipid bilayer formation through monitoring a characteristic, time dependent frequency Δf and dissipation

shift ΔD.25,26 SLB charge density was controlled by adjusting the ratio of zwitterionic (DOPC) and anionic phospholipids (DMPA) in the membranes (Scheme 1). In the experiments, where we systematically varied the grafting density of the polymer, pH, the size of the NPs and their concentration, a systematic understanding of the effects of NP binding to SLB membranes could be obtained (Scheme 1).

2. EXPERIMENTAL SECTION Materials. Acetone, acetic acid, KCl, chloroform, disodium hydrogen phosphate, Hellmanex III, methanol, potassium dihydrogen phosphate, sodium chloride, sodium dodecyl sulfate (SDS), and sodium hydroxide were purchased from Sigma Aldrich and used without further purification. 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) and 1,2-dimyristoyl-snglycero-3-phosphate (sodium salt) (DMPA) were obtained from NOF corporation without further purification. Water with a resistivity higher than 18 MΩ-cm was obtained from a commercial Millipore system. Silica Nanoparticles of spherical shape with two different core diameters (50 or 70 nm) were used, containing either an anionic (SiO2) or cationic (SiO2@NH2) surface, or modified covalently


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Table 1. Sample type, amount of grafted polymer pgraft, diameter dNP of the silica core and molecular weight of the MW,NP of the NPs, frequency and dissipations shifts for the 5th overtone number Δf/5 and ΔD/5, mQCM calculated from equation 3, calculated mAFM from AFM height images (equation 2), particle densities from QCM-D and AFM measurements NQCM and NAFM, and the trapped liquid coat of particles H as calculated from these data. Errors for mAFM/AS and NAFM and H are given in parenthesis. Sample

pgraft

dNP

MW,NP

Δf/5

ΔD/5

mQCM/AS

wt%

nm

g/mol

Hz

10

–

50

8.69⸱107

225

8.1

3.0

50

8.99⸱107

325

27.3

SiO2@NH2

–

70

2.06⸱108

250

10

SiO2@NH2

–

140

2.12⸱10

635

50

SiO2@NH2 SiO2@PDMAEMA

9

with the cationic polyelectrolyte poly(2-(dimethylamino) ethyl methacrylate) (SiO2@PDMAEMA).27 The latter are core-shell structures where the shell thickness for different grafted polymer amounts at pH ~ 4 (fully ionized PDMAEMA), were held constant at tshell ~ 16 nm. Under these conditions the shell polymer segment density profile should decay exponentially.27 The grafting density of polymer modified NPs increases with increasing polymer amount (see Table S1, which gives all details about the NPs employed). For a stretched chain the shell thickness of 16 nm corresponds to a degree of polymerization nDMAEMA ~ 64 (length of repeat units: 0.25 nm)28 but this is a lower estimate due to the observed experimental density profile. All cationic NPs (SiO2@NH2 and SiO2@PDMAEMA-NP) were dispersed in water at pH 4 (adjusted with acetic acid). Pure SiO2-NPs were dispersed in water at pH 9 (adjusted with NaOH). If necessary, the pH of the nanoparticle dispersion was preadjusted to the desired value using acetic acid or NaOH. We described the characterization our polymer modified SiO2-NPs previously.27 The charge density of SiO2@NH2-NPs were calculated from titration measurements (Figure S1) and have a charge density of 0.46 nm-2 and 1.33 nm-2 for diameters of 50 and 70 nm. The PBS (1x) buffer was prepared by dissolving 8.00 g NaCl, 0.20 g KCl, 1.42 g Na2HPO4 and 0.24 g KH2PO4 in 1 L Millipore water. The pH was adjusted to a value of 7.4 by adding 1 M NaOH solution.29 Vesicle preparation First, the desired mass of DOPC was added into a glass vial and mixed with 10 mL methanol and chloroform (V/V = 1/1). Next a desired volume of a stock solution of chloroform methanol and DMPA (c = 1 mmol) was added, and the mixture was vigorously stirred and sonicated for two minutes. After complete solvent evaporation, the dried lipid mixture was dispersed in 10 mL PBS (PBS 1X) and adjusted to a concentration of 0.2 wt%. The dispersion was then extruded 30 times through cellulose acetate filters of first 400 nm (15 x) and then 100 nm pore diameter (15 x), using a lipid extruder (Avanti Polar Lipids). Finally, the vesicle solution was diluted with PBS (1x) to a concentration of 0.1 wt% and used as stock solution. Stock solutions were stored at T = 8 °C and used within two weeks. Each solution was equilibrated at RT prior to each measurement. Methods. Atomic force microscopy (AFM) Surfaces were imaged in tapping mode at room temperature in air, using an atomic force microscope (NanoScope V, Bruker, Santa Barbara, CA, E scanner) and triangular Si3N4 cantilevers (Bruker, k = 40 N/m for NP decorated surfaces or k = 0.6 N/m for visualization of a SLB). Freshly prepared SiO2 surfaces were rinsed with Millipore water, treated in a plasma cleaner (Harrick Plasma PDC 32G)

–6

mAFM/AS

NQCM

NAFM

H

ng/cm²

ng/cm²

1/um²

1/um²

–

3685

726 (101)

255

50 (7)

0.803 (114)

4384

853 (64)

281

54 (4)

0.813 (59)

3950

1141 (111)

89

33 (3)

0.711 (63)

8960

4045 (410)

20

9 (1)

0.640 (60)

at medium power for 2 min, rinsed again with Millipore water, and finally dried in a stream of filtered N2. SLBs decorated with NPs were prepared by sequentially placing droplets of different solutions [PBS (1X), vesicles (0.1 wt%), PBS (1X), water at pH 4, NP solution, Milli-Pore water] on the crystal surface while wicking away the supernatant after each addition step, and a final drying step in air. The height and phase differences were determined from cross-sectional analysis at two different locations. Quartz crystal microbalance with dissipation (QCM-D) measurements were performed with a four-chamber Q-Sense E4 instrument (Biolin Scientific) using Q-Sense SiO2-coated (QSX 303) and Au-coated (QSX 300) crystals. Adsorption was monitored by collecting frequency and dissipation shift data, Δf and ΔD, respectively, from the 3rd to the 11th overtone. Before each measurement the resonance frequency of the crystal was determined first in air and then in degassed PBS (1X). Δf and ΔD values were recorded as long as needed to reach an equilibrated state. Each solution was metered into the QCM chamber at a flow rate of 100 μL/min using a peristaltic pump. When changing the solution, the flow was shortly interrupted to prevent the formation of air bubbles in the tubing system. After each measurement the crystals were incubated at 50 °C in a series of cleaning solutions for 13 min plus 2 min of sonication each: first in Hellmanex III (3 wt%), followed by SDS (2 wt%), acetone, and finally Millipore water, keeping the crystal always wet prior to drying in N2. The adsorbed mass from QCM-D measurements was determined by the modified Sauerbrey relation (Eq. 1) using Δf and ΔD values from the 5th overtone after a steady state value was reached:30–38 𝑚ads 𝐴S

𝐶

(

= 𝑛 ∙ 𝛥𝑓 +

𝑓𝐶 ∙ 𝛥𝐷 2

) ∙ (1 ― 𝐻),

(1)

where fC is the resonance frequency of the crystal (fC = 4.95 MHz), C is the crystal constant (C ~ 17.7 ng/cm2), n is the overtone number (n = 5) and H is the fractional trapped liquid coefficient and AS the normalization to the apparent area. The adsorbed mass data were then transferred into particle number densities, from which we calculated surface coverage values β (see section 2 in supporting information). Doing so requires an exact knowledge of NP size, shape and mass, which were known to us from our previous work on exactly the same particles as used here.27 The NPs will always be surrounded by a water layer, the socalled fractional hydration liquid H. Therefore, QCM-D measures the sum of the masses of adsorbed NPs and trapped water (mads = mNP + mH2O) and thus can overestimate the adsorbed mass by a factor of 5–10.32 The fractional trapped liquid is not comparable with the hydration shell and depends



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Figure 1. Typical QCM-D measurement: ΔD (top) and Δf shifts (bottom) for several steps: I–III describe the SLB formation with subsequent buffer change. The new frequency shift at III serves as the new baseline for further mass calculation for NP adsorption. Step IV shows NP injection into the chamber. NPs with high amount of polymer adsorb less compared to NPs with low amount of grafted polymer or SiO2@NH2. At V the measurement chamber is flushed with water (pH 4) and subsequently at step VI with aqueous SDS solution (1 wt%). Finally, the measurement chamber was rinsed with Millipore water (pH 6) at step VII. Description for individual injection step: (I) PBS injection, (II) vesicle injection with spontaneous SLB formation, (III) H2O injection, (IV) NP injection, (V) H2O injection, (VI) SDS injection, (VII) Millipore water injection.

on the surface coverage and shape of the NPs. An empirical model, assuming a constant shape and thickness of the water coat around each adsorbed NP has been remarkably successful in describing measurement results quantitatively.32,36,37 The principal approach to calculate the fractional trapped liquid coefficient H is to compare the adsorbed mass mQCM of the QCM-D experiment with that obtained by another surface sensitive technique, e.g., AFM yielding mAFM:32 𝑚AFM

1 ― 𝐻 = 𝑚QCM.

(2)

For measuring mAFM the particle decorated crystal from a QCMD measurement was rinsed with Millipore water, removed carefully from the QCM-D instrument and gently dried in air without blow drying. QCM-D measurements confirmed that rinsing the measurement chamber with water does not remove adsorbed NPs. Before H is known, values of the adsorbed mass via QCM-D mQCM were calculated using a modified Sauerbrey equation for unknown adsorbed films that considers so called fluid effects to a first approximation as:30,32

𝑚QCM 𝐴S

(

𝐶

= 𝑛 ∙ 𝛥𝑓 +

𝑓c ∙ 𝛥𝐷 2

).

(3)

After the QCM-D measurement the NP decorated crystals were imaged by tapping mode AFM at four different locations on the surface, with measurement areas of 25 μm² each (Figure S2). The adsorbed mass mAFM (Eq. 4) was then calculated by counting the particles at each location using the software image J,39 averaging the numbers from the four locations, and normalizing by the apparent area AS: 𝑚AFM 𝐴S

𝑁 ∙

=

𝑀W 𝑁A

𝐴S

,

(4)

where the molecular weight of individual particles is MW, the total mass of adsorbed NPs is mAFM per unit area AS, number of particles per unit area is N and Avogadro´s constant is NA. H was determined for “strongly adsorbing” NPs (such as SiO2@PDMAEMA with 3.0 wt% grafted polymer d = 50 nm and SiO2@NH2 (d = 50–140 nm) (Figure S2). Once H is known the mass of other absorbed NPs can be calculated with equation 1 for all QCM-D measurements for the specific overtone


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number (n = 5). As the experimental value for H was very similar for the polymer coated NP and SiO2@NH2 we used a constant valued of 0.803 ± 0.115 for H in all calculations (we are aware that H should vary for individual particle types, but assume that the error will not be very large). Corrected values of mQCM and mads from equation 1 result in low surface coverage values (β = 10–30 %) for all NPs. Intrinsically we assume here a homogeneous NP distribution (strong particle-particle repulsion at pH 4) on the sensor surface in water. In general, QCM-D data of polymer grafted NPs exhibit much higher values for the dissipation than bare SiO2@NH2-NPs. We attribute this to the interface between polymer coated NPs and the sensor surface, where the polymer likely acts as a flexible linker. However, H is strongly size dependent and decreases progressively with increasing NP size (see table 1). Our calculation approach is limited by the assumption that the surface coverage is independent of particle size and extent of polymer grafting. Since our data yield relatively low surface coverage values (β = 10–30 %), weak or no overlap of adjacent liquid coats can be expected. Samples with lower surface coverage values may have actually a higher H value than that used in the calculation, and thus mads may be slightly overestimated. Nonetheless, this overestimate should be rather small and can be considered to lie within measurement accuracy. Conversely, calculated values for mads for low charged NPs (high pH values) may be underestimated, as the overlap of liquid coats of highly packed particles should be considered. Supported lipid bilayer (SLB) formation SLBs were formed spontaneously following the protocol of Hardy et al. usually working at high ionic strength of 150 mM with small vesicle sizes of dves ~ 100 nm on SiO2 surfaces.25 Surface charge densities of the SLB were controlled by the ratio of zwitterionic (DOPC) and anionic (DMPA) phospholipid, where the DMPA content was varied from 0–10 mol%. The mean frequency shift for spontaneous SLB formation by vesicle fusion in PBS solution for our membrane system was ΔfSLB = -26 ± 2 Hz, in good agreement with published values. The thickness d of the SLB membrane was calculated from the Sauerbrey relation:40 𝐶

𝑑 = ― 𝑛 ∙ 𝜌SLB ∙ ∆𝑓,

(5)

where we assumed a phospholipid density of 1.01 g/cm³.41 The calculated bilayer thickness of dSLB = 4.50 ± 0.34 nm is in excellent agreement with the theoretically expected value of 4.51 nm.42 To avoid NP precipitation at high ionic strengths (~0.15 M),27 the PBS buffer was exchanged to pH-adjusted Millipore water. This introduced a frequency shift of about +8 ± 2 Hz. This frequency increase is due, in part, to the reduction of solution viscosity upon changing from PBS to low ionic strength water.43,44 After the buffer change, the SLBs remained stable on the surface. The new steady-state frequency shift was then set as the new baseline for the mass calculation of adsorbed NPs (see Eq. 1 and example measurement in Figure 1). Using this new baseline and the knowledge of the mean molecular weight MW of our NPs (reference27 or table S1) we were able to calculate the number density per area N/AS of adsorbed NPs and the surface coverage β as an easily comparable values describing particle packing. With AH being the area occupied by one particle and assuming a hexagonal dense packing24 it can be written as (see also Figure S3):

𝑁

𝛽 = 𝐴S ∙ 𝐴 H =

𝑚ads 𝐴S

𝑁A

∙ 𝑀W ∙

6 ∙ 𝑅2H 3

,

(6)

with the hydrodynamic radius RH, NA Avogadro constant, and mads/AS the adsorbed mass per unit area obtained from AFM or QCM-D measurements. For details see the supporting information in section 3 and Figures S3 and S5.24 ζ-Potential measurements were done with a Litesizer 500 (Anton Paar). The electrophoretic mobility for the vesicles was measured at room temperature in Millipore water and transferred into ζ-potentials with the Smoluchowski equation.45

3. Results and Discussion Our work is focused on investigating the interactions between anionic supported lipid bilayers (SLB) and cationic polymer grafted or surface modified silica NPs. To gain a comprehensive understanding we studied these interactions as a function of the amount of grafted polymer, the pH, SLB charge density, and the concentration of the NPs. Our experiments yielded four interesting and unexpected findings: i) SLB charge density affected NP binding only slightly. ii) NPs did not bind to the SLB above a critical polymer grafting density. iii) NP adsorption increased with increasing pH despite reduced electrostatic attraction. iv) steady state NP adsorption decreased with increasing NP concentration for weakly binding particles and this effect depends on the flow rate. 3.1 Effect of Polymer Graft Density and SLB Charge Density The charge density of the NPs is controlled by the polymer grafting density and pH, i.e., at higher pH, PDMAEMA or amino moieties of SiO2@NH2-NPs become less charged, which reduces their effective surface potential.27 The first set of experiments was carried out in Millipore water at pH 4, i.e., under conditions in which the polymer brush is fully ionized. The surface charge density of the SLB was varied from 0– 10 mol% by admixing DMPA (singly negatively charged) to DOPC (neutral). The surface coverage β, calculated from QCM-D measurements for cationic polymer-coated NPs is plotted in Figure 2a as a function of the amount of polymer grafted onto the NPs. Initially, with increasing polymer amount, more NPs adsorb onto the SLBs. As this increase is small, the number density of adsorbed NPs decreases with increasing polymer grafting density (see Figure S5). An important observation is that for a pure DOPC SLB (i.e., no surface charge) no adsorption takes place for the smaller NPs (d = 50 nm). This suggests that for the smaller NPs the adsorption process is driven by electrostatic interactions. In contrast, for the larger NPs (d = 70 nm) no such effect is seen. We attribute this to the fact that in this case the contact area is larger (directly proportional to the NP size), resulting in a stronger interaction with the flat SLB surface. The bilayer charge density is increasing with increasing DMPA content at solution pH 4 (Figure S4). This weak increase may explain that regardless of NP size, the adsorption behavior is almost independent of SLB charge density, ranging here from 2 to 10 mol% DMPA. This suggests that the surface density of the adsorbed NPs is largely



determined by the charge interactions among the particles rather than the SLB charge density. The most surprising finding is that NP adsorption decreases dramatically above a critical amount of grafted polymer on the NPs (Figure 2a). This observation was corroborated by AFM imaging on the same samples used for the QMC-D measurements (Figure 3a). While for bare NPs (d = 50 nm) and those of low polymer content (regardless of NP size) there is significant adsorption onto SLBs, no adsorption is observed for NPs with high grafted polymer content.


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Figure 2. a) Surface coverage β calculated from QCM-D measurements for cationic SiO2@NH2 (0 nm–2 grafting density) and SiO2@PDMAEMA-NPs at pH 4 for both core sizes (left d = 50 nm and right d = 70 nm) plotted as a function of the PDMAEMA grafting density. b) cartoon of the interaction of SiO2@PDMAEMA NPs of low (i) and high (ii) grafting densities with supported lipid bilayers. Red dots represent the counterions distributed along the backbone of fully ionized polyelectrolyte brush. (i) NP adsorption due to attaching polyelectrolyte chains in molecular contact due to higher flexibility. (ii) non-adsorption of NPs due to less flexibility of polyelectrolyte chains.

Figure 3. a) AFM height images of cationic NPs d = 50 nm on SLB bilayers in the dried state (DOPC/DMPA = 95/5 mol%) show a decreasing surface coverage with increasing polymer grafting density (numbers in the inset). b): Comparison between particle number densities calculated from QCM-D (rectangle) and AFM (sphere) measurements on SLB (DOPC/DMPA 95/5 mol%) for NPs d = 50 nm on different prepared samples. 0 nm–2 represent the SiO2@NH2-NPs.



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Figure 4. Variation of pH for cationically modified and bare NPs with a core diameter of 50 nm. a) hydrodynamic radius RH (indicated is also the point where the samples precipitate (2Φ), b) corresponding ζ-potentials, c)) surface coverage β (finding (iii) which is an increased NP adsorption with decreasing NP charge (pH↑)).

Together, these observations suggest that there is a critical polymer grafting density above which the tendency for binding to the SLB surface is largely suppressed (ii). This threshold is at ~0.07 nm–2 PDMAEMA for the smaller NPs (d = 50 nm) and increases to ~ 0.7 nm–2 for the larger NPs (d = 70 nm) (Figure 2a). The higher critical surface coverage β may result from stronger binding of the larger compared to the smaller NPs. The lack of adsorption for the more densely grafted NPs can be explained by considering that at high grafting density the charges on the polymer chains are no longer able to effectively interact with the opposite charges on the SLB surface (they are effectively buried within the dense brush). In this situation, the dense, brush-like polymer layer allows for only little conformational flexibility of the grafted (see Figure 2b), and hence the steric repulsion induced by the polymer chains dominates the interaction. An important observation is also that once the polymer decorated NPs have adsorbed, they remain strongly adsorbed on the SLB surface, as flushing the measurement chamber with water does not remove them. In contrast, all NPs are rapidly removed from the surface with a SDS solution (1 wt%, see Figure 1). This observation was further confirmed via AFM imaging by comparing the height

and phase images of samples containing SLB with and without adsorbed NPs (see Supporting Information Figure S6 and S7). It might be noted that our SLB shows DOPC rich domains in the fluid state, and DMPA rich domains in the gel state. The DMPA domains have undefined shape and vary widely in size. Even after NP adsorption we can confirm the presence of lipid bilayer domains (Figure S6 and S7). 3.2 Effect of NP Charge Density on Adsorption. Next, we investigated the influence of NP charge density (controlled by pH) on adsorption, while keeping the charge density of the SLB constant at a ratio of DOPC/DMPA 95/5 mol%. Colloidal stability is reduced around the IEP and more strongly so the lower the degree of polymer grafting, as seen in an increasing hydrodynamic radius (Figure 4a).27 While cationic SiO2@NH2 NPs are highly charged in acidic conditions (pH 4), their charge density is reduced with increasing pH until the isoelectric point (IEP) is reached at pH 6.2. Further increasing the pH causes the particles to be increasingly negatively charged (Figure 4b); i.e., adopting the same charge as the SLBs. In contrast, the anionic SiO2-NPs become increasingly more negatively charged with increasing pH (Figure 4b) and thereby enhance their colloidal stability.


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Figure 5. Surface coverage β for cationic NPs on the SLB with DOPC/DMPA 95/5 mol% for different core sizes (at pH 4). Left d = 50 nm, right d = 70 nm. (iv) NP adsorption decreases with increasing NP concentration (all measurements at a flow rate of 100 µL/min).

The QCM-D data (Figures 4c and Figure S8) show increased adsorption of cationic SiO2@PDMAEMA-NPs with increasing pH (i.e., decreasing NP charge) (iii), which suggests that this effect is not purely of electrostatic nature. In contrast, for the bare, anionic SiO2-NPs, adsorption steadily decreases with increasing pH, as expected for electrostatic control. The results for the polymer modified NPs are at first glance surprising as one would expect an increasingly charge-driven adsorption with decreasing pH. They must be due to the polymer shell and one explanation for this behavior may be that the lateral electrostatic repulsion of similarly charged NPs is reduced with increasing pH, i.e., when the particles are less charged. In addition, less charged chains will be more flexible, thereby allowing for more intense interaction with the surface. Similar to the situation discussed in Section 3.1, the adsorption decreases with increasing amount of grafted polymer. Similar observations have been made by Riley et al. for the adsorption of brush grafted SiO2@PDMAEMA-NPs with 15 nm core diameter onto planar anionic surfaces (oxidized silicon wafers), where particles at pH 5 show weaker adsorption affinity compared to their adsorption affinity at pH 9.28,46 Furthermore, Jing et al. observed a reduced NP adsorption potential onto carboxyl functionalized surfaces with decreasing ionic strength.47 3.3 Effect of NP Concentration on the Adsorption Next, we investigated the influence of NP concentration on adsorption, while keeping the charge density of the phospholipid membrane constant at a lipid ratio of DOPC/DMPA 95/5 mol%. Specifically, we studied the surface coverage for cationic SiO2@NH2 and SiO2@PDMAEMA-NP at moderate and high grafting densities for two core sizes (d = 50 and 70 nm, Figure S9). Obtaining the steady state is mainly a function of the particle concentration and it is achieved much more slowly for reduced concentration. Figures S9 and S10 show that the characteristic adsorption time increases from several seconds to hours with a scaling that is inversely proportional to the NP concentration. For NPs with high grafting densities, Δf and ΔD do not change significantly with increasing NP concentration, and essentially

NP adsorption is absent under these conditions (Figure 5). For the 50 nm NPs the pure SiO2@NH2 particles show a relatively high binding already at low particle concentrations, indicating a much higher binding affinity when compared to the polyelectrolyte modified SiO2-NPs. Interesting this behavior is reversed for the 70 nm NPs. A slight increase in the adsorbed amount may be attributed to the rising concentration. In contrast, for intermediate grafting densities we observed a peculiar concentration dependence for NP adsorption. Especially for the 50 nm NPs the adsorbed amount first increases with increasing concentration, followed by a sharp decrease in the adsorbed amount at higher concentrations (iv) (Figure 5). This transition behavior occurs exactly at the grafting densities at which we also observed the transition from adsorption to non-adsorption (see Figure 2a discussed in Section 3.1 (ii)). Apparently, NP adsorption depends subtly on the precise conditions of the interactions between NPs and surface. It is not entirely an effect of SLB interaction as measurements on a SiO2 surface show a similar trend with increasing particle concentration (Figure S12). Apparently, the intermediate range of weak binding, which is easily influenced by external parameters like concentration (iv) or changes of the grafting density (ii). While both core sizes show similar adsorption trends, larger NPs adsorb generally more strongly than smaller ones. In addition, the sharpness of the transition from adsorbed to non-adsorbed state is less pronounced, in agreement with the observations for the dependence on polymer grafting (Figure 2b). To exclude that this behavior is due to changes of the state of the NPs in bulk, we measured their hydrodynamic radius RH as a function of NP concentration. We found that within measurement uncertainty the RH of core-shellNPs remained constant (Figure S13), which means that there are neither substantial changes in the brush structure nor aggregation processes taking place. However, an important aspect here is the fact that the QCMD measurements are steady state results from flowing systems. Accordingly, we investigated the effect of flow rate on adsorption, by reducing the flow velocity from 100 to 15 μL/min during the measurements. In slower flow we found higher surface coverage β for samples with high concentrations



and almost no concentration dependence of adsorption (Figure S14).

3.4 Discussion and Interpretation In the following we discuss and seek to explain the behavior of this rather complex colloidal system (see Figure 6) in the framework of the basic interaction potentials acting between the NPs and phospholipid SLBs. 3.4.1 Electrostatic Conditions and Chain Flexibility For relatively high grafting densities (NPs have grafting densities of 0.04-0.7 chains/nm2, see Table S1) and at low ionic strength of the bulk solution at pH 4 (cion = 1.4*10–4 mol/L, Debye length κ–1 ~ 25 nm) we assume that the polymer shell of our NPs is in the osmotic brush regime (see also Supporting Information). Since the net charge of planar polyelectrolyte brushes is not exactly zero, some compensating counterions (Δn per chain) likely escape from the brush and form a Gouy−Chapman layer of thickness λ.48

Figure 6. Schematic drawing of counter- and coion distribution of SiO2@PDMAEMA-NPs close at the shell thickness. We assume the osmotic brush regime and mobile counterions can escape from the brush.48,49

When NPs approach a charged, planar SLB one would intuitively expect that oppositely charged NPs tend to adsorb on the SLB surface. However, our experiments revealed a decrease of NP adsorption with increasing charge (decreasing pH) and grafting density (Figure 4, (iii)). In order to rationalize this finding we consider the net force a NP experiences, when it is approaching a SLB surface, which may be approximated by the sum of the electrostatic force FП of two overlapping electric double layers, the attractive vdW force (in which we may include also dipole-dipole interactions as they prevail between silica and the phospholipid head groups), and the steric repulsion force, due to the presence of grafted polyelectrolyte chains: 𝐹net = 𝐹П + 𝐹vdW + 𝐹steric.

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We note that in our case the higher steric repulsion at higher grafting density is apparently not exclusively responsible for the lack of adsorption seen in Figure 2 (ii), as the more highly grafted NPs at higher solution pH (where the electrostatic attraction is reduced) become increasingly adsorbed (Figure 4, iii). This behavior is quite surprising, also because at low solution pH only NPs with low grafting densities do adsorb. It can be explained by a subtle interplay between polymerSLB electrostatic attraction/repulsion and vdW attraction, polymer brush ionization, and polymer chain flexibility. We surmise that at low grafting density the polyelectrolyte chains on the NP surface are more flexible and the polyelectrolyte layer as a whole is more deformable and therefore able to interact more strongly with the SLB than at higher grafting density, where the polyelectrolyte chains are conformationally constrained in the dense brush regime (see Figures 2b and 7). Furthermore, with increasing pH the polyelectrolyte brush is less ionized, and the polyelectrolyte chains regain some of their conformational flexibility and are able to interact with the substrate more strongly through non-electrostatic forces. It has been shown previously that SiO2-NPs are strongly attracted to phospholipid bilayers.51 This attraction is mainly caused by the dipole interaction between the phosphocholine head groups and silica surface.52 Interactions with the phosphocholine head groups are expected to be also relevant for adsorption of the NPs with grafted positively charged polymer chains.

Figure 7. Scheme of the NP interactions with SLB. Red dots represent the counterions distributed along the backbone of fully ionized polyelectrolyte brushes. The chain flexibility is schematically shown by the circular blue arrow and it correlates with the degree of ionization. (i) Low grafting density and flexible polyelectrolyte chains: strong particle adsorption at the SLB due to a more effective interaction of the polyelectrolyte chains with the SLBs. (ii) High grafting density and less flexible polyelectrolyte chains: weak interactions with SLB due to reduced effective contact.

3.4.2 Other interactions influencing the surface coverage Once a sufficient number of NPs are adsorbed to the SLB surface, the net charge of the decorated surface is inverted, which makes it more difficult for additional NPs to reach the surface. Furthermore, the electrostatic repulsion of similarly charged NPs explains the relatively low surface coverage observed (Figure 8). Accordingly, we find relatively low surface coverage values β for highly charged NPs compared to that of less charged NPs, in good agreement with results reported by Riley et al.28,46 and Liu et al.47 Finally, we consider the effect of polymer grafting density on adsorption. Because the polyelectrolyte brushes of core-shellNPs are in the osmotic regime, the steric repulsion of polymer chains and the resulting pressure of counterions within the brush, lead to strong repulsion of SiO2@PDMAEMA-NPs when Dcore-surface becomes smaller than the shell thickness


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tshell ~ 16 nm. The repulsive potential increases with increasing grafting density and therefore the gain of free energy during adsorption decreases. For collapsed polyelectrolyte brushes (i.e., at high pH), the osmotic pressure, mediated by counterions within and outside of the brush, essentially vanishes, and SiO2@PDMAEMA-NPs with high polymer densities can adsorb on the SLB surface. However, with increasing grafting density the conformational freedom of “electroneutral” polyelectrolyte chains (i.e., at high pH) decreases which reduces a more intimate contact between local linkage of the polyelectrolyte chains and the SLB surface. Ultimately this leads to a lower surface coverage, in agreement with the data shown in Figure 4d. The higher surface coverage of large (d = 70 nm) NPs likely results from an increased influence of the vdW attraction due to a larger effective contact area.49 Overall, vdW interactions between phospholipid bilayers and NPs are expected to be large and should play a significant role here.

Figure 8. Scheme showing lateral electrostatic NP-NP interactions and the resulting effect of NP charge density on surface coverage (reduced lateral repulsion at higher pH) (iii).

3.4.3 Effect of NP concentration on adsorption The concentration-dependent adsorption behavior of NPs with moderate polymer densities is clearly surprising (iv). As discussed before we postulate that the total free energy of adhesion, Ead, decreases upon increasing polymer density, and particles with a large amount of grafted polymer do not adsorb, because Ead is much smaller than kT due to strong steric and electrostatic repulsion at small separation distances. Interesting is the behavior of particles with intermediate grafting densities where Ead ≈ kT. These conditions result in weakly bound particles at the surface. NP adsorption is a dynamic process and depends on the competition of the affinity of individual particle to adsorb and the particle-particle interactions. The latter is concentration and also shear dependent, as we do not work at equilibrium but all adsorption experiments were done under steady state conditions where flow of the bulk solution takes place parallel to the interface. Weakly bound particles feel the long-range electrostatic repulsion of identically charged particles (Figure 8). Thus, moving particles dispersed in the bulk solution are able to ablate particles from the interface by a “sand paper like” process that depends on the shear rate and NP concentration (Figure 9). Higher shear rates and NP concentrations in solution increase NP ablation from SLB surfaces. However, this effect affects only weakly bound particles (iv), while strongly bound SiO2@NH2-NPs will remain on the surface. This explanation is supported by the observation that the effect clearly depends on the flow rate, being relevant at high (Vsolvent = 100 μL/min) but much less so at low flow rates (Vsolvent = 15 μL/min) (Figure S14).

Figure 9. Schematic drawing of removal of weakly bound NPs by other flowing NPs in solution. (iv) NP adsorption decreases with increasing NP concentration.

4. CONCLUSION We investigated the interaction between nanoparticles (NPs) and supported lipid bilayers (SLBs) of opposite charge, using quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). In our experiments we controlled the charge density of the particles either by the grafting density of the polyelectrolyte brush (PDMAEMA) at constant pH or by pH while keeping the grafting density constant. The charge density of the SLB was controlled by the mixing ratio between nonionic DOPC and anionic DMPA. Our experiments show that the charge density of SLBs only weakly affects NP adsorption onto charged SLBs. Even for highly charged particles the observed surface coverage values remain low, likely due to mutual repulsion of the NPs at the interface. A general and surprising trend is that increasing NP charge density, introduced either by more grafted polymer or by increasing the solution pH, leads to lower surface coverage values. Furthermore, we found that already adsorbed NPs cannot be removed from the interface with acidic water (i.e., good solvent conditions for cationic NPs) but that NPs rapidly desorb in SDS surfactant solution (presumably due to a high affinity of the NPs to SDS). The most interesting observation regarding NP adsorption onto phospholipid bilayers is that the NP adsorption affinity decreases with increasing polymer grafting density, and that above a critical polymer grafting density effectively no adsorption occurs, even though NPs and bilayers are oppositely charged and attract. This critical polymer grafting density increases with increasing size of the NPs. This behavior can be explained by a reduced electrostatic attraction and an increased steric repulsion that occur at high grafting densities. Furthermore, in the intermediate range of weakly bound NPs one also observes a marked dependence of the NP binding on the concentration of the NPs and this effect depends on the flow rate of the NP solution, becoming much less pronounced at low flow rate and apparently resulting from interactions with moving NPs. In summary, polymer modification of silica nanoparticles provides a powerful tool to control their ability to bind to phospholipid bilayers. Future research will address the effects of the thickness of the polymer layer around the NPs, the influence of the elastic properties of the phospholipid bilayers, and the effect of the ionic strength on these NP/bilayer interactions.

ASSOCIATED CONTENT Supporting Information. Details of the particle properties, mass adsorption calculation for NPs systems, calculation of the surface coverage from the mass density, supporting bilayer remaining after NP adoption, ξ-potential of the bilayers, pH and concentration dependent adsorption, and adsorption kinetics are



listed in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] [email protected]

Present Addresses †If an author’s address is different than the one given in the affiliation line, this information may be included here.

Author Contributions The authors declare no competing financial interest.

Funding Sources Any funds used to support the research of the manuscript should be placed here (per journal style).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Marek Sokolowski and Michael Gradzielski thank the German Science Foundation (DFG) for support via the International Research Training Group (IRTG) 1524 (SSNI) at the Technische Universität Berlin. SZ thanks the National Science Foundation (NSF) for support through the Triangle Materials Research Science and Engineering Center (MRSEC) by award DMR-1121107.

ABBREVIATIONS AFM: atomic force microscopy, DOPC: 1,2-dioleoyl-snglycero-3-phosphocholine (zwitterionic phospholipid), DMPA: 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (anionic phospholipid),PDMAEMA: polydimethylaminoethyl methacrylate, SLB: supported lipid bilayer, SiO2-NP: silica nanoparticle, SiO2@NH2-NP: amino functionalized silica nanoparticle, SiO2@PDMAEMA-NP: PDMAEMA brush grafted functionalized silica nanoparticle, TGA: thermo gravimetric analysis, QCM-D: quartz crystal microbalance with dissipation,

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table of content 338x160mm (150 x 150 DPI)


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Scheme 1. a) Top left shows the molecular structure of zwitterionic DOPC and anionic DMPA phospholipids used for the formation of anionic vesicles via extrusion. Top right shows a cartoon of SiO2-NPs modified with anionic (–OH), cationic (–NH2) moieties, and brush grafted polyelectrolyte (PDMAEMA). The bottom left cartoon shows the formation of a supported lipid bilayer on a silica surface, and the bottom right cartoon schematically shows the interaction of NPs with the model membranes. Both processes can be studied with QCM-D. b) Cartoon of increasing polymer density. 661x502mm (96 x 96 DPI)



Figure 1. Typical QCM-D measurement: ΔD (top) and Δf shifts (bottom) for several steps: I–III describe the SLB formation with subsequent buffer change. The new frequency shift at III serves as the new baseline for further mass calculation for NP adsorption. Step IV shows NP injection into the chamber. NPs with high amount of polymer adsorb less compared to NPs with low amount of grafted polymer or SiO2@NH2. At V the measurement chamber is flushed with water (pH 4) and subsequently at step VI with aqueous SDS solution (1 wt%). Finally, the measurement chamber was rinsed with Millipore water (pH 6) at step VII. Description for individual injection step: (I) PBS injection, (II) vesicle injection with spontaneous SLB formation, (III) H2O injection, (IV) NP injection, (V) H2O injection, (VI) SDS injection, (VII) Millipore water injection. 420x345mm (150 x 150 DPI)


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Figure 2. a) Surface coverage β calculated from QCM-D measurements for cationic SiO2@NH2 (0 nm–2 grafting density) and SiO2@PDMAEMA-NPs at pH 4 for both core sizes (left d = 50 nm and right d = 70 nm) plotted as a function of the PDMAEMA grafting density. b) cartoon of the interaction of SiO2@PDMAEMA NPs of low (i) and high (ii) grafting densities with supported lipid bilayers. Red dots represent the counterions distributed along the backbone of fully ionized polyelectrolyte brush. (i) NP adsorption due to attaching polyelectrolyte chains in molecular contact due to higher flexibility. (ii) non-adsorption of NPs due to less flexibility of polyelectrolyte chains. 514x368mm (150 x 150 DPI)



Figure 3. a) AFM height images of cationic NPs d = 50 nm on SLB bilayers in the dried state (DOPC/DMPA = 95/5 mol%) show a decreasing surface coverage with increasing polymer grafting density (numbers in the inset). b): Comparison between particle number densities calculated from QCM-D (rectangle) and AFM (sphere) measurements on SLB (DOPC/DMPA 95/5 mol%) for NPs d = 50 nm on different prepared samples. 0 nm–2 represent the SiO2@NH2-NPs. 485x190mm (150 x 150 DPI)


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Figure 4. Variation of pH for cationically modified and bare NPs with a core diameter of 50 nm. a) hydrodynamic radius RH (indicated is also the point where the samples precipitate (2Φ), b) corresponding ζpotentials, c)) surface coverage β (finding (iii) which is an increased NP adsorption with decreasing NP charge (pH↑)). 251x190mm (150 x 150 DPI)



Figure 5. Surface coverage β for cationic NPs on the SLB with DOPC/DMPA 95/5 mol% for different core sizes (at pH 4). Left d = 50 nm, right d = 70 nm. (iv) NP adsorption decreases with increasing NP concentration (all measurements at a flow rate of 100 µL/min). 499x186mm (150 x 150 DPI)


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Figure 6. Schematic drawing of counter- and coion distribution of SiO2@PDMAEMA-NPs close at the shell thickness. We assume the osmotic brush regime and mobile counterions can escape from the brush.48,49 250x190mm (150 x 150 DPI)



Figure 7. Scheme of the NP interactions with SLB. Red dots represent the counterions distributed along the backbone of fully ionized polyelectrolyte brushes. The chain flexibility is schematically shown by the circular blue arrow and it correlates with the degree of ionization. (i) Low grafting density and flexible polyelectrolyte chains: strong particle adsorption at the SLB due to a more effective interaction of the polyelectrolyte chains with the SLBs. (ii) High grafting density and less flexible polyelectrolyte chains: weak interactions with SLB due to reduced effective contact. 338x131mm (150 x 150 DPI)


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Figure 8. Scheme showing lateral electrostatic NP-NP interactions and the resulting effect of NP charge density on surface coverage (reduced lateral repulsion at higher pH) (iii). 338x100mm (150 x 150 DPI)



Figure 9. Schematic drawing of removal of weakly bound NPs by other flowing NPs in solution. (iv) NP adsorption decreases with increasing NP concentration. 332x135mm (150 x 150 DPI)


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Interaction Between Soft Nanoparticles and ... - ACS Publications

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