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Electrostatic Swelling Transitions in Surface-Bound Microgels Lina Nyström, Rubén Alvarez Asencio, Göran Frenning, Brian Roy Saunders, Mark W. Rutland, and Martin Malmsten ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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ACS Applied Materials & Interfaces

Electrostatic Swelling Transitions in SurfaceBound Microgels Lina Nyström 1,*; Rubén Álvarez-Asencio 2,3, Göran Frenning 1, Brian R. Saunders 4, Mark W. Rutland 2,5 and Martin Malmsten1

1. Department of Pharmacy, Uppsala University, P.O. Box 580, SE-752 32 Uppsala, Sweden 2. Department of Surface and Corrosion Science, School of Chemical Science and Engineering, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden 3. Institute for Advanced Studies, IMDEA Nanoscience, 28049 Madrid, Spain 4. School of Materials, The University of Manchester, MSS Tower, Manchester, M13 9PL, United Kingdom 5. SP Technical Research Institute of Sweden, SP Chemistry, Materials and Surfaces, SE-114 86 Stockholm, Sweden

KEYWORDS: Atomic force microscopy (AFM), Finite element method (FEM), Microgel, pH-responsive, Quartz crystal microbalance (QCM-D), Surface-bound

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ABSTRACT: Herein, electrostatic swelling transitions of poly(ethyl acrylate-co-methacrylic acid) (EA/MAA) microgels, covalently bound to silica surfaces, are investigated. Confined at a solid surface, microgel swelling is anisotropically hindered and the structure flattened, to an extent dictated by pH and microgel composition. Microgel deformation under applied load is also shown to depend on microgel charge density, the highest deformation observed at intermediate charge densities. Two modes of microgel deformation under load were observed, one elastic and one viscoelastic, related to polymer strand deformation and displacement of trapped water, respectively. Results on polymer strand dynamics reveal that the microgels are highly dynamic, as the number of strand-tip interaction points increases 4-fold during a 10 s contact time. Furthermore, finite element modeling captures these effects qualitatively, and shows that stress propagation in microgel network decays locally at the rim of contact with a solid interface or close to tip probe, respectively. Taken together, the results demonstrate a delicate interplay between surface and microgel, which determines the structure and nanomechanical properties of the latter, and which needs to be controlled in applications of such systems, e.g., as pH-responsive surface coatings in biomaterials.


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INTRODUCTION Microgels are cross-linked colloidal gel particles, which frequently display dramatic swelling transitions in response to one or several of a wide range of stimuli. Depending on the characteristics of microgel monomers and other incorporated compounds or nanoparticles, microgels can be designed to be environmentally responsive to a number of key triggers, e.g., changes in temperature, pH, ionic strength, redox conditions, or specific metabolites.1, 2 Due to this, and their fast response time, microgels have interested researchers in a variety of fields. While the majority of microgel research has so far concerned microgel suspensions and their applications, there is increasing interest in surface-bound microgels, which offer a robust and facile alternative for surface modification and functionalization. Microgels and nanogels at interfaces are therefore attracting emerging interest in applications such as biomaterials,3 biosensors,4,

5

and antifouling surfaces.6,

7

In particular, surface-bound microgels offer

opportunities to add biological functionality by loading such structures with bioactive agents, e.g., in the context of drug delivery and functional biomaterials.8-11 In this context, microgels offer a versatile approach for surface modifications with pre-determined size and characteristics compared to more elaborate bottom-up approaches, such as layer-by-layer deposition12 and surface-initiated polymerization.13, 14 Until now, most of the work on surface-bound microgels has been focused on temperatureresponsive microgels, such as those formed by poly(N-isopropylacrylamide) (PNIPAM) and (PNIPAM-co-acrylic acid).5, 15-21 Such studies have demonstrated that pure PNIPAM and comonomer mixture microgels can form well-organized adsorbed layers, either by dip coating or spin coating,15, 16 and that the surface microgel packing density can be controlled by factors such as microgel concentration, pH, and deposition speed.17 The properties of such surfacebound microgels have also been characterized with regards to effects of microgel and surface properties. For example, using atomic force microscopy (AFM), it has been shown that while 3 ACS Paragon Plus Environment


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the phase transitions temperature of individual PNIPAM microgels is not affected by the confinement to a solid interface,18 microgels flatten at surfaces,17, 18 suppressing temperatureinduced volume changes.17 Furthermore, the cross-linking density and co-monomer content of adsorbed PNIPAM-based microgels affect both their swelling transitions and mechanical properties, similar to effects observed in solution.19, 20 For microgels whose swelling is determined by electrostatic interactions, considerably less is known about factors affecting surface-bound microgel swelling/deswelling, as well as surface-induced microgel deformations and nanomechanics. For pH-responsive microgels, such as the poly(ethyl acrylate-co-methacrylic acid) microgels studied in the present work, the amount of incorporated charged monomers, ionic strength, as well as pH, are all likely to affect microgel properties both in solution and when surface-bound.8, 22 However, electrostatic effects for surface-bound microgels are not readily translated from theoretical concepts or findings obtained for nonionic microgels due to long-range Columbic interactions between the charges, as well as complex counterion distribution within the network.23 Nevertheless, there have been a couple of studies addressing properties effecting electrostatic swelling transitions in surface-bound microgels. Thus, FitzGerald et al. investigated pH-dependent swelling of adsorbed poly(2-vinylpyridine) microgels using in situ tapping mode AFM, and found a swelling transition similar to that for the corresponding dispersed microgel system.24 Furthermore, Howard et al. showed, using optical reflectometry and quartz crystal microbalance

(QCM),

that

the

pH-responsive

swelling

of

adsorbed

ethylamino)ethyl methacrylate] microgels was stable over several cycles.

25

poly[2-(di-

However, until

now, little is known about how the underlying surface influences electrostatically triggered structural transitions for surface-bound microgels, their nanomechanical properties, and the interplay between chain and hydrostatic contributions in such systems.


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Given the sparsity of studies addressing these issues, the present study was undertaken to provide a comprehensive investigation of factors affecting electrostatic swelling transitions, including both topography and nanomechanical properties, of surface-bound, charged microgels. For this purpose, electrostatic swelling transitions of methacrylic acid (MAA)based microgels (~ 100 nm diameter), covalently bound to silica surfaces, were investigated as a function of pH and microgel charge density. In doing so, quartz crystal microbalance with dissipation (QCM-D) was used to evaluate mass and viscoelasticity changes related to water and counterion uptake at a fixed surface concentration of (covalently bound) microgels. Furthermore, AFM was used as a powerful label-free technique for monitoring the 3D structure of individual surface-bound microgels under varying conditions.26 In addition, using PeakForce quantitative nanomechanical property mapping (PF-QNM), nanomechanical properties of surface-bound MAA microgels, polymer chain dynamics, as well as induced interactions between polymer chains within the microgels and the cantilever tip, were investigated at the single microgel level. Supporting the experimental studies of electrostatic swelling transition of surface-bound microgels, we also employed finite element modeling (FEM) of microgel swelling transitions, interfacial deformation, microgel nanomechanical properties, as well as internal rearrangements under applied load.



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EXPERIMENTAL SECTION Reagents. Ethyl acrylate (EA), methacrylic acid (MAA), 1,4-butandiol diacrylate (BDDA), sodium dodecyl sulfate, K2HPO4, ammonium persulfate (APS), ammonium hydroxide (NH4OH),

hydrogen

peroxide

(H2O2),

hydrochloric

acid

(HCl),

3-

glycidoxypropyltrimethoxysilane (GOPS), toluene, N,N-Diisopropylethylamine (Hunig´s base) methanol, dichloromethane, and diethyl ether were all of analytical grade and obtained from Sigma-Aldrich (Schnelldorf, Germany). Sodium acetate/acetic acid, Tris HCl, and carbonate/sodium bicarbonate buffers, all at 10 mM ionic strength, were used for pH 4.0, 7.4, and 10, respectively. Purified Milli-Q water was used throughout.

Microgels synthesis. Poly(EA/MAA/BDDA) microgels were synthesized using seed-feed emulsion polymerization, as previously described.8 Briefly, monomer emulsion seed was initially added to nitrogen-purged sodium dodecyl sulfate solution, followed by immediate addition of K2HPO4 (3 g of 7.7 wt% aqueous solution) and ammonium persulfate (APS; 3.6 g of 5wt % aqueous solution) in a four-necked round-bottom flask equipped with a mechanical stirrer and a reflux condenser. The seed solution was left to stir for 30 min at 80° C before the remaining monomer emulsion mixture was added continuously (at 2.6 mL/min) over 90 min. To achieve microgels with varying charge density, monomer emulsion was mixed in either 79/20/1, 66/33/1, or 39/60/1 (EA/MAA/BDDA) w/w. Microgels were abbreviated according to the MAA content in the feed solution, i.e., MAA20, MAA33 and MAA60, respectively. Potentiometric titration yielded the MAA content of these to be 22.1 ± 1.1, 36.9 ± 0.4, and 63.3 ± 1.5 w/w %, respectively. During the polymerization process, the microgel hydrodynamic diameter was continuously measured using photon correlation spectroscopy using a BI-9000 Brookhaven light scattering apparatus (Brookhaven Instrument Cooperation, NY, U.S.A), fitted with a 20 mW HeNe laser, and a detector set at 90° scattering angle. To 6 ACS Paragon Plus Environment

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achieve similar size of the three microgel systems, an additional initiator step (3.2 g of 3 wt % APS) was used in the case of MAA33 microgels, and two more initiator steps (3.2 g of 5 wt % APS) for MAA20 microgels. When the targeted hydrodynamic diameter (~100 nm) was reached, the reaction was cooled and the mixtures extensively dialyzed against water. For further comparisons, MAA20, MAA33, and MAA60 microgels were titrated to determine the degree of dissociation as a function of pH using a Mettler-Toledo D1 15 titrator (Mettler Toledo, Columbus, USA).8 From these results, together with determined MAA content in the microgels, the degree of charge of the microgels at a specific pH could be calculated.

Covalent coupling. To enable covalent coupling of MAA microgels, borosilicate glass coverslips (Fisher Scientific, Västra Frölunda, Sweden) were modified with GOPS to add epoxy functionality to the surface interface, which allowed the carboxyl acid components of the polymer strands to bind covalently to the surface. This was done according to a previously reported protocol.8 In brief, glass substrates were first cleaned in 25% NH4OH, 30% H2O2 and H2O (1:1:5, w/w), and then again in 25% HCl, 30% H2O2 and H2O (1:1:5, w/w), followed by extensive rinsing in water and 99.7% ethanol. Washed glass substrates were placed in a dried glassware, followed by addition of dried toluene (400 ml), GOPS (100 ml) and Hunig´s base (3 ml) under N2 (g). The reaction was refluxed with a condenser at 110° C for 24 h. Samples were thereafter sonicated twice for 15 min in methanol, then rinsed in dichloromethane and diethyl ether, before being submerged in 0.1 w/w microgel solution, pH 5.1, and subsequently incubated overnight at 50° C. Unbound microgels were removed through vigorous rinsing, and samples stored in water until further use.

Quartz crystal microbalance with dissipation (QCM-D). The response of surface-bound microgels to changes in pH and ionic strength was monitored in situ by QCM-D (Q-sense E4 7 ACS Paragon Plus Environment


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microbalance, Q-sense, Gothenburg, Sweden), allowing changes in frequency (f) and dissipation (D) to be simultaneously recorded over time using Q-soft software (Q-sense, Gothenburg, Sweden). The theory underlying the technique has been thoroughly described elsewhere.27, 28 Quartz sensors with a 50 nm silicon dioxide layer, fundamental frequency (f0 ~ 5 MHz), were obtained from Q-Sense (Gothenburg, Sweden). Before use, the sensors were modified in a similar manner as described above for glass coverslips to enable covalent coupling of microgels. First, sensors were cleaned in 25% NH4, 30% H2O2 and H2O (1:1:5, w/w) at 80 °C for 5 min, followed by extensive rinsing in water and ethanol (99.7%). Samples were then GOPS-modified and submerged in microgel dispersion overnight. Unbound microgels were subsequently removed through vigorous rinsing, and samples stored in water until further use. At the start of each measurement, a base line of surface-bound MAA20, MAA33, and MAA60 microgels in water (80 µl min-1) was recorded. After stabilization, shifts in frequency (∆f) and dissipation (∆D) at overtones 3, 5, 7, 9, 11, and 13 were monitored over time (15 min), step-wise cycling buffers of various ionic strength (at a constant pH of 7.4) or pH (at a constant 10 mM ionic strength). Mass changes were modeled using an extended Voight model for viscous layers (QTools v3.1.27, Biolin Scientific AB, Västra Frölunda, Sweden). The modeled mass (∆m) thus obtained for the various ambient conditions refers to the difference in mass compared to base line, i.e., the mass of surface-bound microgels together with associated water (Figure S1). Solvent contributions, in turn, were obtained by measurements for ‘bare’ GOPS-treated sensors, i.e., without surface-bound microgels.

Atomic force microscopy (AFM). Imaging and force measurements were carried out using a Dimension FastScan Atomic Force Microscope (Bruker, Santa Barbara, U.S.A) equipped with a Nanoscope V controller. PeakForce Tapping mode was used to enable quantitative 8 ACS Paragon Plus Environment

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nanomechanical property mapping (QNM) of surface-bound microgels, together with their topography. To investigate swelling and nanomechanical properties, microgels were covalently attached to glass coverslips as described above, and allowed to equilibrate for a minimum of 15 min in the solvent before measurement. Images were obtained using silicon nitride cantilevers (ScanAsyst-Fluid+, Bruker, Santa Barbara, U.S.A), with a nominal radius of ~2 nm and a spring constant ranging from 0.4 to 0.7 Nm-1, according to a calibration procedure described elsewhere.29 Scan rates ranged between 0.5 and 1.5 Hz, and oscillation amplitudes between 50 and 110 nm, using a scanner resonance of 2 kHz. During imaging, which was done in situ throughout, a maximum 800 pN force was used. NanoScope Analysis software V1.50 (Bruker, Santa Barbara, U.S.A) was used for image processing and statistical analysis. Any tilt in the horizontal direction of topography images was corrected by subtraction using a first order polynomial. The force-distance curves presented were measured on individual microgels. Microgels were first viewed and centered in the field of view to ensure that force measurements were performed at the center of each microgel. Individual force measurements (n ≥ 14) at either 2 nN or 5 nN were then performed. A slight change in penetration depth was found at the higher 5 nN force if scanning frequency was changed; therefore all presented data were performed at a constant 0.5 Hz frequency. As shown in Figure S2, no visible effect on microgel topography was detected after force measurements. Further data quantification was performed using MATLAB (version 8.6.0.267246, The MathWorks, Inc., Natick, United States). For calculations of tip-sample interactions during retrace after an added time delay in contact, the number of adhesive bond ruptures (visible as disruptions in the retraction curve, e.g. minima larger than a 0.05 nN cut-off force) were counted and their mean force presented in Table 1. The creep distance reported (Table 1 and Figure S3) was measured as the z displacement of piezo during the added time delay. 9 ACS Paragon Plus Environment


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Modeling procedure. For finite element modeling of the swelling and nanomechanical properties of surface-bound microgels, as well as of effects of tip indentation, the model for pH-sensitive hydrogels put forward by Marcombe et al., and elaborated upon by Li et al., was utilised.30,

31

Elaborative description of the theory behind the model can be found in the

Supplementary Information. Room temperature was assumed and the molecular volumes ‫ݒ‬ and ‫ݒ‬ୱ were both set to 0.03 nm3.32 The microgel was taken to be spherical with a diameter of 100 nm in its protonated, uncharged, state (at low pH). Since all loads were applied in the vertical direction (mirroring the way the AFM experiments were performed), an axisymmetric model, utilising an unstructured mesh consisting of about 2000 quadratic triangular elements, was used. The user subroutine implemented by Marcombe et al. was slightly modified and adapted for the COMSOL (version 5.1; COMSOL AB, Sweden or COMSOL Inc., USA) finite element software that was used for the simulations.30 Two types of simulations were performed: Swelling of surface-bound gels and probe indentation into surface-bound gels. The methods used to attach gels to the surface and to simulate probe indentation are briefly described below. Surface attachment. The shape of the surface-bound microgels was determined in a simplified manner as follows. Firstly, the microgel was pressed against a flat substrate by a body force. Secondly, the part of the microgel that was in contact with the substrate was attached by springs of sufficiently large spring constant to ensure that the microgel essentially remained fixed, mirroring the covalent immobilization of microgels at the substrate in the experimental systems. The body force was thereafter removed and the gel allowed to relax while remaining attached to the substrate. These calculations were performed at a pH of 5.1 and a salt concentration of 10 mM. The later result, however, were found to be insensitive to the salt concentration for this relatively low pH, where the gel was weakly charged. The 10 ACS Paragon Plus Environment

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magnitude of the body force used was determined such that the gel after relaxation had a height/width ratio (h/w) of about 0.4 for the MAA33 gel. Probe indentation. Probe indentation was modelled in a simplified manner by application of a vertical force along the symmetry axis. This force was distributed over a circular area of radius 10 nm in the deformed configuration in such a way that a parabolic stress profile was obtained. At the site of load application, the gel boundary was constrained to move in the vertical direction only, hence mimicking the response to a load applied by an AFM probe.



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RESULTS Microgel Swelling. pH-dependence. The pH dependency of MAA microgels was first investigated by comparing the swelling of surface-bound microgels to that in microgel dispersion (Figure 1). With increasing ambient pH, the acrylic acid residues of the microgels de-protonate (pKa ≈ 6.4-7.0),8 increasing the electrostatic repulsion and causing the microgels to swell (Figure 1a). By assuming a spherical cap shape, and using the mean height and width extracted from AFM topographies, the volume of the flattened surface-bound microgels was calculated. The results show that the volume of the surface-bound microgels increased with both pH and the MAA content (Figure 1b). The volume changes calculated from surfacebound microgels are qualitatively smaller, but otherwise display the same pH and charge density dependence, compared to microgels in the dispersed state (Figure 1c). The mass taken up by surface-bound microgels of different composition and at different pH was also quantified using QCM-D, (Figure 1d). The mass difference shown corresponds to the increase of water and counterions associated with the surface-bound microgel layer in buffer, compared to the dry mass and associated water of the corresponding system in water (schematic of experimental set up shown in Figure S1). Reflecting its relatively less swollen state, the microgel with lowest MAA content (MAA20; 20w/w% MAA monomer added during synthesis) shows very low water uptake, which depends only weakly on pH. In the other end of the spectrum, MAA60 microgels were able to increase its effective mass by almost a factor of 4 when increasing pH from 4.0 to 10.


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a)

b)

c)

d)

Figure 1. a) Representative AFM topography images of surface-bound MAA33 microgels in pH 4.0, 7.4, and 10. b) The microgel volume increase extracted from AFM topography measurements, using data of mean height and width from 5x5 µm areas (≈ 300 individual microgels; n ≥ 5). As can be seen, the higher the charge density (found at higher pH and MAA content) the larger the volume increase (V/V0). c) Volume of MAA20, MAA33, and MAA60 microgels in dispersion, measured by photon correlation spectroscopy. d) QCM-D results on mass (buffer) uptake in surface-bound MAA20, MAA33, and MAA60 at various pH values, compared to microgel water content in water during baseline recording.



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Ionic strength dependence. QCM-D was also used to investigate the microgel response to changes in ionic strength. As shown in Figure 2a the microgel response was fully reversible to changes in ionic strength. When increasing the ionic strength in the surrounding buffer, the frequency shifts to a lower value, corresponding to an effective mass increase, while dissipation increases, i.e., the layer becomes more viscous. Since MQ water is not pH adjusted, the initial swelling, i.e., when going from water to 10 mM Tris, pH 7.4, is due to two effects. The swelling and increased water uptake that is indicated by the mass increase (frequency decrease) is due to both the change in charging (as in Figure 1d) and the increase in ionic strength. Thereafter, pH was kept constant and the changes were solely due to changes in ionic strength. As shown in Figure 2a, the higher the microgel charge density, the larger the response to changes in ionic strength. Somewhat counterintuitively, the mass coupled to the QCM crystal increased with increasing ionic strength. Since the number of surface-bound microgels was constant during the experiment, which was assured through covalent binding and also illustrated by complete reversibility on cycling the ionic strength, the observed changes in frequency and dissipation on varying the ionic strength are exclusively due to uptake/release of water and/or counterions from the media. Thus, the results indicate that increased counterion accumulation within or in close proximity to the surface-bound microgels with increasing ionic strength dominated over any osmotic deswelling effects that occurred simultaneously. The increasing ionic strength, while also expected to provide a screening effect and reducing electrostatic repulsions within the gel, will also tend to increase the charge, which in turn will increase the amount of directly bound water and associated counterions. This is further supported by the finding that dissipation increases with increasing ionic strength for all three microgels. From dissipation-frequency plots, additional information on the processes involved can be observed (Figure 2b). Since a linear correlation between frequency and dissipation is expected for layers with constant 14 ACS Paragon Plus Environment

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mechanical properties, deviations from linearity indicates structural rearrangements.33 As seen in Figure 2b, there is a characteristic gradient representative of each microgel and salt concentration, the change of which is much more pronounced for the gels of higher charge density. This indicates that the latter undergo more pronounced structural rearrangements in response to changes in ionic strength, especially between 1 and 10 mM.



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a)

b)

Figure 2. a) Ionic strength dependency of frequency (upper) and dissipation (lower) from QCM-D measurements of surface-bound MAA20, MAA33, and MAA60 microgels going from MQ water to 1 mM, 10 mM, and 100 mM Tris HCl, pH 7.4. A silane-treated sensor without surface-bound microgels was used as control (red). The figure shows overtones 5 (solid line) and 7 (dashed line). b) Overtone 5 data replotted as ∆D vs. ∆f. The baseline of surface-bound microgels is showed in water at 0 Hz.


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Properties of individual surface-bound microgels. Microgel structure. As a result of the contact between the loosely cross-linked microgels and the silica surface, MAA-microgels deform (‘flatten’) compared to their original spherical shape in solution. The degree of such surface-induced flattening depends on microgel charge density, which in turn is dictated by the MAA content and pH (Figure 3). Thus, the lowest charge density microgel, MAA20, displays the flattest structure, where the height/width ratio for surface-bound microgels is only about 0.25-0.35. On the other hand, the highest charge density microgel, MAA60, is less affected by the surface and forms almost a perfect half sphere, i.e., h/w ≈ 0.5 at all pH values. The surface-induced microgel deformation is further affected by pH, as most clearly seen for MAA20 and MAA33. Quantitatively, however, the effect of pH variation on microgel flattening after covalent immobilization of microgels is relatively modest.



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a)

b)

Figure 3. Flattening of surface-bound microgels. a) Representative microgel cross-sections of individual MAA33 microgel at pH 4, 7.4, and 10, selected from AFM topography measurements as in Figure 1a. b) Surface-induced asymmetry, expressed as the ratio between microgel mean height (h) and width (w) for MAA20, MAA33, and MAA60 at different pH values. As can be seen, the lower the microgel charge density, the ‘flatter’ the microgel structure. Values of h/w were extracted from AFM topography measurements, mean height and width from 5 x 5 µm areas (≈ 300 individual microgels; n ≥ 5).


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Microgel deformation. Next, PF-QNM was used to investigate microgel deformation in the z-direction under applied load and its dependence of microgel charge density. Microgel deformation was plotted against the fraction of de-protonated MAA (Figure 4), assuming that the microgel titration of surface-bound microgels is equivalent to that of microgels in solution. At low charge density, the microgel structure is collapsed and the polymer chains are close together with low amounts of associated water (at pH 4.0 for all microgels, Figure 1d). This results in a rigid structure with low deformability. Increasing the charge density to an intermediate value, around 20-35 % charged MAA monomers, the microgels become softer, able to deform up to around 60% of their original height (relative strain, ε, of ~ 0.6), when an 800 pN force is applied. Upon increasing the charge density further, however, microgel deformability was again observed to decrease. Thus, microgel deformability under applied load displays a distinct maximum at intermediate microgel charge density (Figure 4).



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a)

b)

c)

Figure 4. a) Microgel deformability, expressed as relative strain, as a function of the fraction of charged groups within the microgels. b). Schematic illustration of the swelling progression with increasing charge fraction (caused by a combination of MAA content and pH) and resulting electrostatic repulsion within the microgel. c) Representative (PF-QNM) deformation images of microgels of 0.59, 33.8, and 63.3 % charge density, respectively.


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The response of individual surface-bound MAA33 microgels was further investigated by monitoring force-distance curves at different applied forces at pH 7.4, 10 mM ionic strength (Figure 5). Results show two different regimes in cycling trace/retrace in a force-distance curve. Thus, when a lower force of 2 nN is applied, a fully reversible (elastic) response of the individual microgel is found. When the applied force is increased to 5 nN, however, a hysteresis is observed between the trace (approach; dashed line) and retrace (separation; solid) curves, signifying energy dissipation and work loss during trace-retrace cycling. These results suggest that at low applied force, the AFM tip is only able to push the polymer chains within the microgels elastically, while a higher applied force results in water molecules being squeezed out from, or re-distributed within, the microgel network.



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Figure 5. Force-distance curves for surface-bound MAA33 microgel response at an applied force of 2 and 5 nN (0.5 Hz), using PF-QNM. The graphs show trace (approach; dashed lines) and retraction (separation; solid lines). Contact between sample and tip was defined to occur at a force of 0.2 nN. Data are displayed as median values of 10-15 measurements at the center of one individual MAA33 microgel at pH 7.4, 10 mM ionic strength.


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Polymer chain dynamics. To further investigate polymer chain dynamics within surfacebound microgels, the same experimental set-up was used, but this time with an added contact time delay and a constant compression force of 2 nN. When increasing the contact time between tip and microgel, the individual polymer chains have time to relax and form contact points with the cantilever tip. This results in a considerable increase in the work of adhesion (area between approach and retraction curves), as well as an increased maximum detachment force as a function of time. When retracting the tip away from the microgel, the breaking of individual bonds can be followed and quantified.34 As shown in Figure 6a, an increasing number of interaction points were found with increasing contact time, shown as individual minima in the black retrace lines. The mean numbers of bonds, larger than a defined 0.05 nN cut-off value, as well as the average force of these, was extracted and summarized in Table 1. After the maximum 10 s contact time (which corresponds to the approach of a plateau in the gel relaxation (Table 1 and Figure S3)), the number of detectable interactions between the AFM tip and polymer chains within a single microgel increased about four times on average. The results demonstrate that the polymer chains within the microgel were highly flexible and that equilibration within a single microgel occurs over a time-scale of seconds.



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a)

b)

Figure 6. AFM results on tip-microgel bridging. a) Force vs. distance curves of sample-tip interaction obtained for MAA33 microgel for the indicated time delays in contact (2 nN, 0.5 Hz) at pH 7.4, 10 mM ionic strength. Trace (approach) curves are shown in grey and retrace (separation) curves in black. b) Schematic illustration of tip and microgel components (66 w/w EA, 33 w/w MAA, 1 w/w BDD) used for these experiments.


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Table 1. Quantification of AFM retrace experiments (cf, Figure 6). Time delay

Mean no. of breaks

[s]

Mean force a)

Creep distance b)

[pN]

[nm]

0

5

68.6

0.04 ± 0.03

0.1

10

100.9

1.67 ± 0.19

0.5

11

127.6

3.70 ± 0.23

1

14

147.7

4.09 ± 0.18

5

23

206.4

6.37 ± 0.26

10

20

217.5

± 0.70

a)

Average force of bond breakage, n=7-15; b)Piezo displacement during time delay, ∆(trace-retrace), n=7



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Finite element modeling (FEM; Supplementary Material, Annex 1) of microgel swelling as a function of pH demonstrates that the volume increase is indeed affected by the underlying surface (Figure 7), compared to free swelling microgels (Figure S4). Quantitatively, the volume increase on going from pH 4 to 10 ranges from about 4.4 for the MAA20 microgel to about 7.1 for the MAA60 microgel. Consistent with the experimental data (Figure 1), the surface-bound microgels swell considerably less than unconfined microgels in solution (up to ~10 times less). Furthermore, as shown in Figure 7c, the stress caused by the irreversible attachment of the microgel at the surface occurs primarily in the immediate proximity of the 3-phase line formed at the microgel attachment rim. The latter was most visible at a higher swelling degree (pH 10). With increasing pH and/or microgel charge density, the effect of the underlying surface decreased, and the surface-bound microgels displayed an increasingly spherical shape (Figure 7b,c).


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a)

c)

b)

Figure 7. Finite element modeling results for pH-dependent swelling of surface-immobilized MAA20, MAA33, and MAA60 microgels at a salt concentration of 10 mM. a) Volume changes V/V0, and b) height, h, over width, w. c) Cross-sectional shape of MAA33 microgel covalently immobilized at a solid surface at the indicated pH values, with von Mises stresses color-coded as indicated.



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Modeling was also used to investigate indentation volumes, as well as stress propagation through individual microgels under applied pressures, mirroring AFM tip-microgel interactions. Figure 8a shows an example of a 2D cross-section of the indentation depth under 100 pN applied force of a MAA33 microgel. Further comparisons of the resulting indentation volumes for the three microgel variants at varying pH are shown in Figure 8b, (and also sorted according to fraction of charged groups in Figure S5). The results show that at intermediate charge density at pH 7, all three microgels deform under the 100 pN applied load. Quantitatively, the indentation volume displays a maximum at intermediate pH, analogous to the maximum in strain observed experimentally (Figure 4a). Furthermore, Figure 8c shows that the indentation volume is quite small, at maximum ≈ 0.05, and strain distributions are observed only in the immediate vicinity of the deformation point.


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a

b

c

Figure 8. a) Cross-sectional shape of MAA33 microgel at pH 7 experiencing indentation by an AFM tip mimic under an applied force of 100 pN (10 nm radius). For comparison, the shape of the gel prior to the application of the force is indicated in grey. b) Indentation volume for MAA20, MAA33, and MAA60 microgels at an applied load of 100 pN and c) volume indentation normalized with microgel volume prior applied pressure (Vindentation/V0).



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DISCUSSION Similarly to their nonionic counterparts,35 charged microgels adopt a flattened structure when attached to a solid surface. For the presently investigated microgels, we also note some deviation from perfect circular symmetry in the lateral plane (Figure 1a). However, as this has been observed also with cryoTEM for these microgels in suspension,8 this is an inherent property of the microgels investigated rather than being related to microgel-surface interactions, such as uneven surface tethering or laterally asymmetric swelling. While surfacebound titrating microgels retain the intrinsic pH-dependent swelling displayed in solution, (covalent) attachment to the surface reduces swelling, both in the surface plane and normal to the surface. This renders the volume changes considerably smaller for surface-bound microgels than that of microgels in solution, which can swell freely in all three dimensions. As clearly shown at lower MAA content, swelling occurs primarily normal to the surface. As a result of this, the surface-bound microgels become less flattened with increasing microgel charge density, as demonstrated by both AFM experiments (Figure 3) and FEM modeling (Figure 7). These findings could be compared to those of Höfl et al., who studied temperaturedependent volume phase transitions of adsorbed p(NIPAM-co-vinylacetic acid) microgels and found swelling to occur almost exclusively through an increase in height, with reported h/w changes from 0.2 to 0.05 upon temperature increase and following collapse of the microgel structure.18 Similarly, Burmistrova et al. found that surface-induced flattening of p(NIPAMco-AAc) microgels depends on the cross-linking density of the particle.20 Thus, microgels with a low cross-linking density were found to swell more in the direction normal to the surface, whereas stiffer microgels retain their shape during swelling. By contrast, Wellert et al. reported that ethylene glycol-based microgels adsorbed at a silicon interface without any additional supporting layer swell/deswell more in the lateral direction upon temperature changes.36 30 ACS Paragon Plus Environment

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While increased swelling generally translates into softer structures, nanomechanical properties of surface-bound MAA-based microgels depend in a complex manner on microgel swelling. Thus, deformation of surface-bound microgels only increases with increasing internal electrostatic repulsion (Figure 4a), and resulting water uptake, up to charge densities of around 20-35 %. The electrostatic repulsion at this point causes the microgel mesh size to increase, translating into lower elastic penalty of (small) deformations, such as in the AFM experiments under an 800 pN load. When increasing the charge density further, either by increasing the MAA content of the microgel or by increasing pH, the electrostatic repulsion becomes sufficiently high to overcome the applied load from the cantilever tip and the deformation of the microgels therefore decreases again. To the best of our knowledge, this behavior has not been previously reported for charge dependent surface-bound microgels. However, related findings were obtained by Hashmi and Dufresne in an investigation of changes in microgel modulus as a function of temperature for surface-bound NIPAM microgels. Even though the modulus was found to increase at higher temperatures where the NIPAM particles are collapsed, a minimum of the modulus was reported around the volume transition temperature.37 A decrease in elastic modulus close to volume phase transition was also reported for low cross-linked (2% BIS) p(NIPAM-co-AAc).20 Similarly, Voudouris et al. reported that while the shear modulus (G) increased monotonically with swelling, the compressive elastic modulus (K) and the Poisson ratio of single pNIPAM microgels changed non-monotonically close to volume phase transition temperature (VPTT).38 Furthermore, Sierra-Martín et al. found a lower bulk modulus around VPTT, implying individual microgel particles to exhibit higher compressibility around VPTT.39 Although swelling transitions for electrostatically responsive systems are generally expected to be more pronounced to that of uncharged systems due to the considerably longer decay length of electrostatic interactions, it is nevertheless interesting that the non-monotonic nanomechanical properties of surface31 ACS Paragon Plus Environment


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bound microgels, observed previously for temperature-responding systems, seem to translate also to charge-regulated ones. In analogy with the AFM results, QCM-D results demonstrated that water uptake in surface-bound microgels increased with increasing pH and microgel charge density (Figure 1d). In addition, the data show that on changing the ionic strength at constant pH of 7.4, notable structural changes within the polymer network of the microgel can be detected in the frequency-dissipation plots, especially at higher microgel charge density. Interestingly, the effective mass of the surface-bound microgels increased with increasing ionic strength (Figure 2b), despite expected osmotic deswelling.

23, 40

Here, it should be noted that similar

results have been observed previously by Burmistrova and von Klitzing, using scanning force microscopy, for studies of physisorbed PNIPAM-co-AAc microgels as a function of salt concentration.

17

These findings were proposed to depend on the adhesion between the

polymer and the substrate, where an increase in excess salt decreased the attractive surfacemicrogel interaction responsible for the adsorption. Since the microgel was flattened after physisorption, swelling (caused by partial desorption) was promoted if the attractive interaction between microgel and surface was reduced. Analogously, Nerapusri et al. found the adsorbed (NIPAM-co-AAc) microgels to swell linearly up to a concentration of 1 M NaCl concentration.

15

However, while screening of electrostatically driven microgel adsorption

may be the origin of these previous findings, this is unlikely to provide a mechanism in the presently investigated system. Thus, since the presently investigated microgels were bound covalently to the silica surface, and non-bound microgels removed by vigorous rinsing, the importance of physisorption is dramatically reduced. As shown by finite element modeling, there is certainly stress at the attachment rim (Figure 7c), which may be able to drive partial desorption of the outer part of the adsorbed microgel on reducing non-specific attractive interactions between the microgel and the surface, but this can only occur until the outermost 32 ACS Paragon Plus Environment

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covalent coupling points are extended. Hence, electrolyte-induced partial desorption is unlikely to cause the mass uptake observed with increasing electrolyte concentration for the microgels presently investigated, particularly as both microgels and the silane-coated surface were negatively charged, and physisorption therefore expected to increase with increasing ionic strength (this was also demonstrated experimentally using ellipsometry (Figure S6)). Therefore, other processes are likely to cause the electrolyte-dependence observed in Figure 2, notably enrichment of electrolytes in the vicinity of the surface-bound microgels with increasing ionic strength. Although normalization for background electrolyte was performed, coupling may be different for the microgel-covered surface compared to the control surface. A key issue when probing nanomechanical properties of soft structures, as with the surfacebound microgels presently investigated, is the extent of indentation and stress propagation, as this will affect the volume probed. As shown by FEM (Figure 8), minor indentations only cause relatively local stresses, involving only a minor part of the microgel. With larger indentations/stresses, gradually larger volumes are affected, and this was also probed in nanomechanical experiments. Even at the highest load applied, however, only a small fraction of the microgel was probed (Vindentation/V0 ≈ 0.05). Furthermore, although a larger volume is affected with increasing load, the strain induced by the AFM tip (as well as the surface rim) decays very rapidly (Figure 7 and 8). This shows that strain dissipation can be exceedingly efficient in the highly deformable microgels investigated. As concluded previously by Hong et al., using a theoretical approach based on a field theory in terms of non-equilibrium thermodynamics, a gel will deform in two modes, either a fast process of short-range rearrangement of polymer chains (with constant gel volume), or a slower long-range migration of solvent molecules.41 This mechanism fits well with the experimental results of the present investigation, showing that polymer strands of the individual microgels respond elastically under low applied pressure; whereas, under higher pressures a viscoelastic hysteresis was 33 ACS Paragon Plus Environment


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found (Figure 5), which was interpreted as being due to ‘squeezing out’ water in a sponge-like manner. By varying the time delay in contact at low loads, the polymer chains are provided time to interact with the AFM tip. From the AFM results (Figure 6a) which showed a considerable increase in the work of adhesion and the number of strands interacting with the oppositely charged AMF tip, it is clear that these loosely cross-linked microgels are able to effectively dissipate applied stress. The combination of AFM results and FEM modeling showed that chain re-allocation, which is present within the loosely cross-linked microgels, occurs readily and locally. Finally, although not being of primary focus of the present investigation, the effects discussed above are likely to be of importance for the application of surface-bound microgels as drug delivery matrices. For example, the finding of a force-dependent release of water from the microgels indicates the existence of a threshold for such release, which is likely to apply also to water-soluble drugs, and which may potentially translate into cases where the ‘pressure’ is caused by deswelling rather than by an external probe, in analogy to ‘squashing release’ found previously for other systems.2 Furthermore, swelling- and deformation-related interchannel distances are expected to be of importance for release of encapsulated drugs, together with microgel structure, as well as drug interactions with the microgel network and with the underlying surface.


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CONCLUSIONS AFM PeakForce QNM and QCM-D studies show that both structure and nanomechanical properties of surface-bound poly(ethyl acrylate-co-methacrylic acid) (EA/MAA) microgels depend strongly on electrostatic interactions which are, in turn, determined by microgel charge density as well as ambient pH and ionic strength. When covalently bound to silica these microgels are effectively flattened, in some cases up to a height/width ratio of 0.2. Although the microgels keep their pH-dependent swelling when surface-bound, the swelling is quantitatively smaller than free swelling in microgel dispersion. It is also distinctly asymmetric, with swelling occurring particularly normal to the surface. The latter can be clearly seen particularly at low swelling degrees at low MAA content and pH. Also the microgel deformation under applied load was found to depend on charge density, with microgels of intermediate charge densities displaying the highest deformation. Finally, timedependent force measurements demonstrated an increased number of tip-strand interaction points with time, which increased by a factor of ≈ 4 over a 10 s contact time. This result signifies considerable internal conformational freedom for intra-microgel strands. FEM captured the experimental observation of the effects of the underlying surface on swelling asymmetry and also showed that stress within the microgel network, induced either by the presence of the surface or by tip indentation, decays locally at the rim of contact with a solid interface or close to AFM tip probe, respectively.



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ASSOCIATED CONTENT Supporting Information. Schematic of the QCM-D experimental set up used, AFM topography images, piezo displacements during AFM-tip contact time, complementing FEM calculations of the swelling behavior of dispersed microgels, as well as ellipsometry results of physisorbed amount of MAA-microgels. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding author. * E-mail: [email protected] ACKNOWLEDGEMENT AFM measurements were performed at Albanova Nanofabrication Facility (Stockholm, Sweden). Dr. Deborah Wakeham is gratefully for skillful technical support. This work was financed by the Swedish Research Council grant numbers (2012-1842; 2013-4384) and the Knut and Alice Wallenberg Foundation (KAW 2012.0078).


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TABLE OF CONTENTS GRAPHIC


Electrostatic Swelling Transitions in Surface-Bound ... - ACS Publications

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