Hydrogel Layers As Revealed by Fluoresce - ACS Publications

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Dynamics in Stimuli-Responsive Poly(N‑isopropylacrylamide) Hydrogel Layers As Revealed by Fluorescence Correlation Spectroscopy Apostolos Vagias,† Peter Košovan,‡,§ Kaloian Koynov,† Christian Holm,‡ Hans-Jürgen Butt,† and George Fytas*,†,/ †

Max-Planck-Institute for Polymer Research, 55128 Mainz, Germany Institute für Computerphysik, Universität Stuttgart, 70569 Stuttgart, Germany § Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 120 00 Praha 2, Czech Republic / Department of /Materials Science University of Crete and IESL-FORTH, 71110 Heraklion, Greece ‡

S Supporting Information *

ABSTRACT: We employ fluorescence correlation spectroscopy (FCS) to study the translational mobility of molecular tracers in stimuli-responsive grafted poly(N-isopropylacrylamide) (PNiPAAm) hydrogels, under variable solvency conditions. Tracer−matrix interactions were tuned by selecting three different molecular tracers. In contrast to a noninteracting tracer (Alexa 647), the mobility of a weakly (Alexa 488) and a strongly interacting (Rhodamine 6G) tracer deviates from a simple single Fickian diffusion. In addition to pure crowding effects, the mobility of both Alexa488 and Rhodamine 6G is influenced by tracer−polymer interactions. We interpret the observed trends in tracer mobility in terms of the interplay between Coulombic repulsions and short-range attractions. Although tracer dynamics and hydrogel swelling ratio are interdependent properties, their relation turns out to be nontrivial and does not allow predictions of tracer dynamics on the basis of polymer structural information. Hence, a universal scaling behavior is not possible, due to tracer−polymer interactions. environments has attracted strong interest,17 as its understanding is pivotal for several biosensor-related applications13,18,19 and drug delivery.20 The mobility of a tracer in dense macromolecular environments can be significantly influenced by the tracer size, shape and rigidity (particle vs polymer),21−25 matrix concentration and molecular weight,21,23 presence of cross-links,26−31 pore− tracer size ratio,32 solvency conditions (temperature),4,33 pH,5,34 ionic strength,35,36 as well as tracer−polymer interactions.4,35,36 Crowded environments, due to both pronounced matrix concentration and to possible tracer−matrix interactions,18,37 render a thorough investigation of tracer diffusivity not an easy task.38 Under dense matrix conditions, the tracer dynamics may deviate from a single Fickian diffusive mode.39−41 The number of theoretical works concerning tracer diffusion in hydrogels and dense polymeric networks in general, still remains rather limited.30,42−44 In addition, most experimental papers that deal with the mobility of molecular

I. INTRODUCTION A scientifically active area in soft matter are the stimuliresponsive polymer materials,1,2 spanning from micelles and brushes to cross-linked grafted films. Such materials exhibit specific response to external stimuli, such as temperature,3,4 pH,5 magnetic or electric fields and ionic strength.6−8 Polymer hydrogels possess numerous advantages, such as biocompatibility,3,9 inherent ability to swell by absorbing significant amounts of water and finally pronounced mechanical properties10 (tunable porosity and elasticity11). Among different stimuli-responsive polymer materials, thermoresponsive polymer networks12 that can potentially exhibit phase separation from the solvent they are embedded into have drawn special attention. A quite popular polymer of this class is PNiPAAm, exhibiting a lower critical solution temperature (LCST) in water close to human body temperature; as such, PNiPAAm has been frequently employed in biosensors13 and drug delivery applications.14,15 Although mechanical properties of thermoresponsive PNiPAAm networks have been systematically studied,16 the complexity of the network and the underlying tracer−network interactions render the exact elucidation of the tracer dynamics through such networks rather nontrivial. On the other hand, tracer diffusion in hydrogels and other crowded © 2014 American Chemical Society

Received: May 6, 2014 Revised: July 4, 2014 Published: July 17, 2014 5303

dx.doi.org/10.1021/ma500928p | Macromolecules 2014, 47, 5303−5312

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Scheme 1. Chemical Structures: (A) Weakly Interacting Tracer (A488 Acid Derivative), (B) Strongly Interacting Tracer (Rh6G), and (C) Repeat Unit, for the Poly(N-isopropylacrylamide)-Based Terpolymer (PNiPAAm)

tracers4,31 or nanoparticles27,28,45 in cross-linked matrices focus on noninteracting systems. To get the best benefit from the application of thermoresponsive polymers in biosensor-related applications13 and in drug delivery, a stringent control of how different physicochemical parameters may be influencing complex solute dynamics in such polymer networks is required. Experimental and theoretical studies investigating solute transport under realtime human body conditions are highly desired. An investigation of the influence of temperature and salt on the tracer diffusion in thermoresponsive grafted polyelectrolyte layers has been missing so far. A fundamental prerequisite for rationalizing tracer mobility in grafted systems is to scrutinize the response of tracer dynamics in the bulk of the grafted network (micrometer-sized gel thicknesses), before studying any effects related with tracer mobilities in polymer networks in close proximity to the substrate.46 In this work, fluorescence correlation spectroscopy47 (FCS) has been employed to probe tracer mobility with varying attractive strength in grafted cross-linked PNiPAAm hydrogels. To address the issue of attractive strength, two different molecular tracersa strongly (Rhodamine 6G) and a weakly (Alexa 488) interacting tracer have been employed and their diffusion in grafted PNiPAAm hydrogel layers has been compared with each other and with that of a noninteracting tracer (A647); the important role of A647 consists in being the reference tracer, exhibiting “normal simple diffusion” behavior,4 without all peculiarities present in the other two cases. Tracer diffusion has also been investigated under good and poor solvency conditions, using temperature or ionic strength as the external stimuli that change the solubility of PNiPAAm in water. The aim of this paper is to enrich current information available for thermoresponsive cross-linked networks4,13,19,32,48 and it is 3-fold: (i) To interpret the nature of tracer−polymer interactions, assessed by the perturbation of different external stimuli on tracer dynamics and network swelling ratio, (ii) to illustrate the effects of the network collapse and/or cross-link density on molecular tracer mobility in hydrogels, and (iii) to

discuss about possible scaling relations by comparing the findings with analogous results reported in the literature. The paper is outlined as follows. The fabrication of the hydrogels, the FCS technique and the protocol for acquiring the stimulidependent hydrogel thicknesses are described in section II. The results concerning translational dynamics for the interacting tracers at both varying and constant swelling ratios of salt-free hydrogels are outlined in sections IIIB and III C respectively. Next, the effect of monovalent salt is addressed in section IIID. Rationalization of the complex diffusivity of the molecular tracers is discussed in Ssection IV, and the paper concludes with section V.

II. EXPERIMENTAL SECTION The studied terpolymer has been synthesized as a custom precursor for the preparation of multiresponsive hydrogels. The NiPAAm groups provide the thermoresponsive features. The role of the methacrylic acid is to provide the ionizable group which makes the gel responsive to pH. The presence of charges on the gel increases its swelling ratio, which is a much desired feature. In addition, the incorporation of methacrylic acid along the copolymer backbone prevents from the socalled “skin-barrier” effect.48 This is a process by which a thin, dense layer is formed at the gel’s outer surface acting as a barrier against the escape of water molecules as the LCST is approached, thus hindering the phase transition. The examined PNiPAAm terpolymer consisted of 94% mol poly(N-isopropylacrylamide) as well as of hydrophilic (5% mol of methacrylic acid) and hydrophobic (1% mol of benzophenone methacrylate) groups. It was synthesized by free radical polymerization as described elsewhere.48 Its polydispersity index, PI = 2.7 and the weight-averaged molecular weight, Mw = 280 kg/mol, were obtained from gel permeation chromatography. In good solvents, the hydrodynamic radius is 15 nm and the overlap concentration, c* = 3 × 10−3 g/mL. The benzophenone groups served as the cross-linking agent between the polymer chains, upon illumination with UV light at wavelength λ = 365 nm. The chemical structure of the PNiPAAm terpolymer unit and publicly available structures for the fluorescent tracers used in this work, namely A488 and Rh6G, are shown in Scheme 1. The chemical structure of A647 (Alexa Fluor 647 cadaverine, disodium salt, A30679) is not publicly available. As A647 lacked any attractive interactions with the examined PNiPAAm,4 it was simultaneously present in the studied polymer samples together with 5304


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Figure 1. Normalized fluorescence intensity profiles, IF(z)/IF,max, in HG-4 for Alexa488 (left) and Rh6G (right) vs distance, z, normal to the substrate, at 25 °C. In both panels, experimentally measured profiles (red dots), convolution fits (dashed green lines), and tracer profiles assuming a step distribution (dotted blue lines) are denoted. The maximum intensity obtained from the fit was used for normalization in each shown panel. the other dyes, acting as a “control” tracer. Regarding the tracer surface charges, Rh6G is a positively charged tracer,49 while ζ-potential measurements in Milli-Q water revealed that A488 and PNiPAAm are strongly negatively charged and slightly negatively charged, respectively. The different strength of interactions with the polymer matrix become evident from the shape of the fluorescence intensity correlation function vs time at low polymer concentrations, as shown in our earlier paper for PNiPAAm solutions.50 Preparation of Grafted PNiPAAm Layers. Round microscope cover glass slides (2.5 cm diameter, 160 μm thickness) were treated with a 1 mM ethanol solution of 4-(3-triethoxysilyl) propoxybenzophenone ethanolic solution, in order to functionalize the glass slide with benzophenone groups. The benzophenone groups would then serve as anchoring agents, thus enabling the spin-coated PNiPAAm terpolymer to covalently anchor onto the glass substrate at a later step. A 10 wt % PNiPAAm solution in ethanol was spin coated at room temperature onto the prefunctionalized round microscope cover glass slides at certain spinning speed (250 rpm) and spinning time (60 s). After spin coating, the slides were annealed in vacuum for 1 h at a temperature (T = 170 °C) higher than the polymer’s glass transition temperature, in order to relieve the polymer system from possible stresses. The slides were subsequently dried overnight at T = 50 °C and directly cross-linked by UV irradiation (Stratalinker 2400, Stratagene) at λ = 365 nm (1 h of cross-linking corresponds to an irradiation energy dose of 6.28 J/cm 2 . Consecutively, the slides were rinsed 15 times in situ with absolute ethanol, to remove any un-cross-linked chains; between all steps before cross-linking, the slides were kept in argon atmosphere. The FCS measurements in the grafted hydrogels were performed 30 min after addition of the fluorescent tracer aqueous solution, to ensure that the gel has reached equilibrium in its fully swollen state. For all studies described in this paper, only ultrapure deionized water was used (filtered through a Milli-Q water purification system, resistivity at 18.2 MΩ·cm), i.e., no buffers. Determination of the Swelling Ratio. The swelling ratio, Rs, was determined as the ratio of the thickness of a fully swollen gel under given conditions (temperature, salt) to that of the dry gel. Because the gel was grafted to a planar substrate, the swelling effectively proceeds only in the z direction (normal to the substrate). With the assumption that the polymer is space-filling in the dry gel, we determined the PNiPAAm volume fraction ϕ, in the samples as ϕ = Rs−1. The dry thickness was measured by a profilometer (KLA-Tencor Stylus P-16+) in five to six different locations of the dried sample, after the crosslinked polymer had been rinsed in ethanol. The thickness of the fully swollen hydrogels was determined in situ on the FCS setup by measuring the average fluorescence intensity as a function of the position of the microscope objective along the z-direction (z-scan) with a step of 1 μm. The concentration profile of the tracer and the gel profile were determined by deconvoluting the fluorescence intensity

profile and the FCS observation volume as described in more detail in the Results. Sample Holders. A reusable Attofluor steel chamber was used as a sample holder for the round microscope cover glass slides with grafted PNiPAAm hydrogel layers. In order to prevent solvent evaporation during the experiments, the Attofluor sample chamber was covered with an additional round microscope cover glass slide. Fluorescence Correlation Spectroscopy (FCS). The measurements were performed on a commercial FCS setup (Carl Zeiss, Jena, Germany) consisting of the module ConfoCor2, and an inverted microscope, Axiovert 200. A 40× Plan Neofluar objective was used, bearing the following features: high numerical aperture-NA (NA = 1.2), working distance 0.28 mm, and water as immersion liquid. An argon ion (Ar+) laser at λ = 488 nm, a HeNe laser at λ = 543 nm, and a HeNe laser at λ = 633 nm were used to excite A488, Rh6G, and A647, respectively. The fluorescent emission was collected by the same objective and after passing through an emission filter and a confocal pinhole, was delivered to an avalanche photodiode detector capable of single-photon counting. The temporal fluctuations of the detected fluorescence intensity, δIF(t), caused by the diffusion of the fluorescent tracers through the confocal limited observation volume were recorded and evaluated in terms of an autocorrelation function,

G(t) =

⟨δ IF(t)· δ IF(t + τ )⟩ ⟨IF(t)⟩2

(1)

The experimentally measured autocorrelation curves for all fluorophores employed in this paper were represented by the multicomponent diffusion function,41 ⎞−1/2 −1 n ⎛ T ′ −t / τT⎞⎟ t⎞ ⎛ t ⎟ 1 ⎛⎜ G′(t ) = 1 + ⎜1 + e ⎟∑ Fi ⎜1 + τ ⎟ ⎜1 + 2 ⎟ N⎝ 1 − T′ S τi ⎟ ⎠ i=1 ⎝ i⎠ ⎝ ⎠

(2) T′ and τT are the fraction and the decay time of the triplet state, N represents the average number of diffusing fluorescent species through the FCS observation volume, Fi and τi are the amplitude and the lateral diffusion time through the diffraction-limited FCS observation volume of the ith species with Di = w02/4τi being the respective diffusion coefficient. Next, S = z0/w0 is the so-called structural parameter given as the ratio between the axial (2z0) and the lateral (2w0) dimensions of the Gaussian confocal observation volume. For each excitation wavelength, the values of (2z0) and (2w0) have been calibrated by reference measurements in dilute (10 nM) aqueous solutions of the molecular tracers Alexa488 (A488), Rhodamine 6G (Rh6G), and Alexa647 (A647), using published values of their diffusion coefficients in pure water.51 Furthermore, in all fits the triplet time and fraction 5305


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were used as free fit parameters. The obtained triplet times have been in the range 1−3 μs, as expected for the studied dyes.52 The multicomponent Fickian diffusion model (eq 2) has been used to fit the correlation curves, using either two (n=2) or three (n=3) components. The latter case (n=3) has been employed for some of the Rh6G autocorrelation curves, to account for the minor contribution (F3 ≤ 0.1) of an extremely slow process with characteristic times at about 1s, related to aggregates or some rare-strong adsorption process. As such, the physical significance of that process is not further discussed. On the basis of the presence of a second slower process and its relative amplitude (Figure 4b below) in weakly cross-linked PNiPAAm hydrogels, we will refer to the three tracers as noninteracting (A647), weakly interacting (A488) and strongly interacting (Rh6G). In addition, the z-scans in Figure 1 are an additional evidence for favorable interactions concerning Rh6G.

Figure 2. Swelling ratios, Rs(T), of four different examined HGs (Table 1) as a function of temperature.

III. RESULTS A. Swelling Ratios and Permeation of the Tracers. To determine the swelling ratio, we used the z-scans as described in the Experimental Section and exploited the fact that the tracers partition differently between the gel and the supernatant solutions. In particular, Rh6G accumulates in the gel, while A488 is depleted in the gel. The interface between the gel and solution can be identified by a drop (Rh6G) or by an increase (A488) in fluorescence intensity profile of the z-scan (Figure 1). We represent the normalized intensity profile, which is a function of the distance normal to the glass substrate, z, as IF(z)/IF,max; IF,max is the maximum intensity, corresponding to the intensity either in the supernatant solution (A488, Figure 1a) or in the gel (Rh6G, Figure 1b). The range of z starts at the glass substrate (z = 0) and propagates away from it, into the bulk of the grafted hydrogel and consecutively toward the corresponding supernatant solution. It should be noted that the analysis of z-scans was limited by a number of factors. Not in all cases could the interface be uniquely identified e.g. due to the low signal-to-noise ratio, as well as the relatively large dimension (2z0 ∼ 1.5 μm) of the FCS probing volume in the normal direction. In order to overcome some of these problems and to obtain a more reliable value for the swollen gel thickness, we have deconvoluted the experimentally measured profiles as shown in Figure 1 and described in the Supporting Information. We finally characterized our gel samples in terms of the swelling ratio or polymer volume fraction (Table 1, Figure 2). In spite of some inaccuracy in the determination of the swelling ratio, we distinguish two types of gels according to Figure 2 and Table 1: (i) weakly cross-linked gels (HG-1, HG-2) which do not collapse upon an increase in temperature and (ii) strongly

cross-linked gels (HG-3, HG-4) which shrink upon a temperature increase. The remaining gels were quite compact already at room temperature, which is an anticipated trend from the phase diagram of PNiPAAm in water.53 HG-5 and HG-6 samples were measured at a single temperature but at different ionic strengths. In this case we observed that the gel swelling is not affected up to 0.1 M KNO3, while 1 M KNO3 causes a gel collapse. Swelling behavior is determined by the interplay of the shortrange hydrophobic interactions (solvent quality), long-range electrostatics and by the cross-link density of the gel.54 With respect to an ideal gel under Θ conditions, the presence of charged acrylic groups provides electrostatic repulsion and enhances the swelling; the electrostatic interaction can be suppressed by the addition of salt. The hydrophobicity of PNiPAAm is controlled by temperature, which eventually leads to collapse around the LCST. The temperature at which the collapse occurs depends on cross-linking and increase of crosslinking suppresses the gel swelling; the cross-linking was controlled by the UV irradiation dose4 (Table 1). From the phase diagram of PNiPAAm in water,53 it follows that at low polymer volume fractions ( 100 only, but no conclusive information could be obtained about its variation with gel swelling. On the contrary, partitioning of A488 in different gels

Table 1. Polymer Volume Fractions ϕ(T) (±10%) Values for the Different PNiPAAm HGs Examined (Swollen in Milli-Q Water) at the Respective UV Irradiation Doses (E in J/cm2) ϕ(T) HG-1 HG-2 HG-3 HG-4 HG-5 HG-6 HG-7

E [J/cm2]

25 °C

29 °C

32 °C

35 °C

37 °C

0.52 0.52 1.05 1.57 6.28 6.28 6.28

0.023 0.030 0.09 0.15 0.18 0.21 0.21

0.023 0.03 0.10 0.17 − − 0.26

0.027 0.03 0.20 0.23 − − 0.28

− 0.031 0.21 0.48 − − −

0.042 − − − − − − 5306


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Figure 3. Fluorescence intensity autocorrelation functions, G(t), for (a) the strongly (Rh6G) and (b) weakly (A488) interacting dyes in HG-4 at different temperatures. The dashed and solid vertical lines indicate the fast and slow diffusion times, respectively. Insets: The diffusion time, τslow(T), of the slow process for (a) Rh6G and (b) A488 in HG-4 at various temperatures.

Figure 4. Variation of the swelling ratio, Rs(T), diffusion slowdown D(T)/D0(T) and amplitude of the fast diffusion Ffast(T) for (a) Rh6G (red squares) and A488 (blue triangles) in HG-4 and (b) the same quantities for the same tracers in HG-1 (for Rh6G, red squares) and in HG-2 (for A488, blue triangles). The fast and the slow diffusion process are denoted by the empty and solid symbols in the middle panel, respectively. Dashed and dashed-dotted lines in the middle panel of (a) indicate the temperature dependent slowdown for the slow process in HG-1 and HG-2, i.e., from panel b. Dashed and dashed-dotted lines in part b indicate the diffusion slowdown for the slow Rh6G and A488 process at 25 °C in HG-1 and HG-2, respectively.

the concentration of cations inside the gel is greater than in the bulk, while the opposite is true for the anions. With the gel collapse occurring upon an increase of temperature, the density of chargeable acrylic groups increases, which should enhance the electrostatic partitioning. On the other hand, this is counteracted by a decrease in dissociation of the acrylic groups. Because of their weak acid character, the resulting change of the charge density is smaller than that of the volume fraction. The observation that P of A488 increases with increasing polymer ϕ in HG-4 (Figure S2) contradicts the trend which was predicted by the simple Donnan theory and confirmed by simulations.57 This indicates the presence of an additional specific tracer− polymer attraction of nonelectrostatic origin, consistent with our recent study50 of solutions of the same PNiPAAm terpolymer. We assume that this specific attraction is of hydrophobic origin. We emphasize, however, that the hydrophobic tracer−polymer and polymer−polymer interactions are not the same and can have a completely different dependence on temperature. The collapse of PNiPAAm at higher temperatures has been explained in terms of intramolecular hydrogen bonding,55 while the precise origin of the tracer−

(cf. Figure 1 and Figure S1, Supporting Information) provides insights into the nature of tracer−polymer interactions. In the weakly cross-linked HG-2, we obtained P ∼ 0.02, and both the partition coefficient and the swelling ratio were practically independent of temperature (Figure S2). In the more crosslinked HG-3 and HG-4, an increase in temperature resulted in increasing polymer volume fraction (Figure 2). Concurrently, for A488, P increased from P ∼ 0.1 at 25 °C to P ∼ 1 at 32 °C (Figure S2). Addition of salt KNO3 to HG-6 at 25 °C caused P of A488 to increase from 0.02 (no salt) to about 0.3 (100 mM KNO3), while the gel swelling ratio was unaffected. Further salt addition (1 M) caused gel collapse and a further increase of P to ∼1 (Figure S2). A qualitatively similar trend was observed also for A647 (Figure S3). The qualitative differences in partitioning of Rh6G and A488, i.e., accumulation vs depletion, as well as the effect of salt could be explained within the framework of Donnan theory,57 invoking electrostatic interactions between the negatively charged PNiPAAm, the positively charged Rh6G, the negatively charged tracer A488 and the almost noninteracting A647. When an anionic hydrogel swells in a solution containing ions, 5307


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system can be found either in the free volume, which results in fast diffusion similar to the pure solvent or be captured within a collapsed polymer dense domain. The domain size increases with increasing PNiPAAm hydrophobicity (and hence temperature), however, it should not exceed the focal volume size (∼300 nm), unless the gel is completely collapsed; in the opposite case, the fast process and a measurable Dslow would be hardly conceivable. Therefore, the slow process in HG-4 probably corresponds to the residence time in the collapsed domain, rather than tracer diffusion inside the domain. Consistently, a raise of temperature results in an increased domain size and prolonged residence time (hence Dslow). It is worth mentioning that single molecule tracking of Rh6G in surface bound PNiPAAm films61 revealed both large degree of probe confinement and diffusion slowdown comparable to the one shown in Figure 4. Moreover, statistical analysis of the trajectories infers both fast and slow diffusion in agreement with the analysis of the FCS functions with two-component diffusion (eq 2). The inherent dynamics of the hydrogels is manifested in the fast cooperative diffusion (∼10−7cm2/s) driven by the osmotic pressure and a second diffusive mode62 which is about 2 orders of magnitude slower. The origin of the slow diffusion in chemical networks was in dispute until recently.63 It is now assigned to the relaxation of slow density fluctuations arising either from the aforementioned collapsed dense domains or long correlation lengths between them.63−65 In similar PNiPAAm hydrogel layers, both the fast cooperative diffusion (∼4 × 10−7cm2/s) and the slow gel diffusion (∼5 × 10−9cm2/ s) were resolved by a microphoton correlation spectroscopy technique.62 In this gel picture (see Figure 5 below), the slow

polymer interactions is not clear. The molecular structure of the tracers might allow for hydrogen bonding as well as for van der Waals interactions between the aromatic units in the tracers and the benzophenone cross-linker. The observed trend in P of A488 upon the variation of temperature in HG-4 is a combination of all the above-mentioned counteracting effects. The interactions heavily impact the tracer mobility. B. Tracer Dynamics in the Strongly Cross-Linked Hydrogels. In this subsection, we describe the effect of the gel collapse on the tracer mobility as revealed by the analysis of the fluorescence intensity autocorrelation functions. We have recently shown50 that Rh6G binds reversibly to the PNiPAAm terpolymer in dilute polymer solutions. The reversible binding results in a two-component diffusion of the tracer, where the fast component is interpreted as free tracer diffusion while the slow component relates to the dynamics of tracer bound to a polymer chain. The latter is typically slightly faster than the polymer self-diffusion in solution, because the tracer is not tightly bound to the polymer and short-term unbinding events cannot be resolved. The two-component diffusion was also observed for A488 but only above the overlap concentration of the polymer, in solution. The PNiPAAm terpolymer in the aforementioned work, though, was used at much higher dilution and without cross-linking. The discussion of tracer partitioning in the preceding section further corroborates the model used for the interpretation of the solution results.50 Similar ideas about specific tracer−polymer interactions will be used for the rationalization of the results presented below. In Figure 3 we present the FCS functions, normalized as G(t) = (G′(t) − 1)N, for Rh6G and A488 measured in the collapsing gel HG-4 at several temperatures. The FCS curves display very broad decays that cannot be represented by a single component fit (Figure S4). Hence the tracer dynamics for A488 and Rh6G exhibits deviations from a single Fickian diffusion; the case of single decay was realized for A647, hence the term noninteracting. In line with the solution study,50 we used a twocomponent Fickian diffusion model (eq 2) to represent G(t) for each tracer. Under this assumption and in spite of the complex origin of τslow, both τfast and τslow were converted to diffusion coefficients (section II) for the purpose of simplicity and quantitative comparison. The temperature dependence of the obtained diffusion coefficients D(T) normalized to the corresponding values D0(T) of the tracers in pure water51 is shown in Figure 4a; for A647, D0 = 3.3 × 10−6cm2/s, and for both A488 and Rh6G, D0 = 4.1 × 10−6cm2/s at 25 °C, while D0(T) at different temperatures was scaled to the water viscosity. The fast process for either tracer is only weakly affected by the gel collapse as can be seen from Figure 3 and from the middle panel of Figure 4a, but the slowdown of the fast tracer diffusion relatively to the free solution does depend on the cross-linking density (cf. Figure 4, parts a and b). On the basis of the same figures, the slow process exhibits a 100-fold retardation as the gel collapses. If τslow indeed corresponds to the dynamics of polymer-bound tracer, then at first glance the observed trend is the expected behavior. Simulation studies58,59 have shown that the hydrophobic collapse of branched polyelectrolytes should proceed through formation of collapsed domains as also suggested experimentally.60 In particular, at initial stages of the collapse, these domains are formed near branch points, where the local polymer density is greater than the bulk average, while most of the volume occupied by the polymer is still filled by solvent. A tracer diffusing in such a

Figure 5. Schematic presentation of dense domains around network junctions whose size increases as the temperature increases and trajectories exemplifying fast (blue) and slow (in red) tracer mobility.

tracer diffusion of Figure 4 can be associated with the slow gel mode assuming comparable diffusion coefficients. While the amplitude of the slow process (1 − Ffast) in A488 is virtually independent of temperature, it increases with cross-linking density (cf. Figure 4, parts a and b). We should note, however, that heterogeneities are not a prerequisite for the observation of the slow process, since two-component diffusion of Rh6G was observed even in dilute solutions of the same terpolymer,50 where long-range heterogeneities cannot be present. Hence, the inhomogeneities should be envisaged as locally collapsing (dense) domains. C. Tracer Dynamics in Weakly Cross-Linked Hydrogels. At low ϕ (and hence low cross-link densities) exemplified by HG-1 and HG-2, the swelling ratio is virtually temperature independent (top panel of Figure 4b). In these noncollapsing gels, we studied the effect of temperature on the diffusion of A488 and Rh6G. 5308


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Figure 6. (a) Experimental G(t) for Rh6G in water (dashed curve) and in HG-5 along with z-scan for Rh6G in HG-5 (inset: normalized Rh6G fluorescence intensity profile, IF(z)/IF,max, in HG-5 (white) and in the supernatant solution (gray) vs distance, z, normal to the substrate (gray)), for different ionic strength (I) values, at 25 °C. (b) Diffusion slowdown, D(I)/D0(I) for the fast (white triangles) and slow components (black triangles), and amplitude, Ffast(T), for the system in part a. The values of the listed composition, ϕ, correspond to the swollen and collapsed (at I = 1 M salt) HG-5, at 25 °C.

presence of the very small volume fraction of the collapsed domains, again being stronger for Rh6G in HG-1 than for A488 in HG-2 (middle panel of Figure 4b). Despite quantitative differences between the tracer dynamics in highly cross-linked (collapsing) and weakly cross-linked (noncollapsing) gels, and between different tracers, the observed trends in tracer dynamics can be rationalized within the same framework of locally inhomogeneous hydrogels. However, we have observed the two-component diffusion of the Rh6G even in dilute solutions of the same terpolymer,50 where long-range heterogeneities cannot be present. Therefore, we believe that the presence of inhomogeneities does not significantly affect the presented observations. It is the formation of collapsed nanodomains, in which the tracers are captured to the polymer, by specific attraction of hydrophobic origin. Both Dslow and Fslow seem to reflect the strength of this attraction, while small difference in the swelling ratios of the two weakly cross-linked gels (HG-1 and HG-2) is manifested in the temperature dependence of Dslow. At low cross-link densities, differences in the tracer−polymer interactions are clearly discernible in the slow process (Dslow and Fslow in Figure 4b), whereas in highly cross-linked hydrogels (HG-4 in Figure 4a) this disparity is diminished resembling the behavior of the slow process for the same tracers in PNiPAAm solutions.50 D. Monovalent Salt (KNO3) Effects. To further support our interpretation and get more insight into the tracer− polymer interactions, we examined the influence of monovalent salt on the tracer dynamics also in relation with the swelling properties of the hydrogels. The addition of salt can elucidate possible influence of electrostatics on the tracer−PNiPAAm interactions. Hence, the dynamics of the strongly interacting molecular tracer (Rh6G) and of the noninteracting A647 were investigated in the grafted hydrogels at good solvency conditions (T = 25 °C), using a monovalent salt (KNO3) as an additional external stimulus. As discussed above, the addition of salt screens the electrostatic interactions, resulting in the gel collapse. However, this collapse is expected to proceed differently than by the variation of temperature. A hydrophobic interaction varied by temperature, is short-ranged on molecular length scales, and hence the collapse due to hydrophobicity can be assumed to be local. On the other hand, electrostatics, are long-ranged, and their screening is a nonlocal effect. Therefore, the collapse can be assumed to be homogeneous. This notion is corroborated by the suppression

Similar to the situation in the collapsing gels, we observed two diffusion processes characterized in terms of the corresponding diffusion coefficients shown in the middle panel of Figure 4b. Like the diffusion of noninteracting molecular tracers,4 we observed that the normalized Dfast(T)/ D0(T) is essentially independent of temperature and is only slightly retarded (Dfast(T) < D0(T)) compared to the collapsing (higher cross-linking density) hydrogels (Figure 4a). The slow diffusion process of A488 in HG-2 (middle panel of Figure 4b) is virtually temperature independent, in accordance with the negligible temperature dependence of the swelling ratio for this gel (top panel of Figure 4b). The slow process of Rh6G in HG1 shown in Figure 4b is, in absolute terms, about 5 times slower and almost twice stronger than that of A488. In addition, it is weakly slowed down with temperature, which well correlates with the weak decrease of the swelling ratio of HG-1 (top panel of Figure 4b). These observations can be rationalized in the same framework of a locally collapsed hydrogel as discussed in section IIIB. Network junctions serve as nuclei for nanodomain formation, which macroscopically results in very little collapse of the weakly cross-linked HG-1 as seen in Figure 4b. We illustrate this situation in Figure 5. A tracer in the free volume undergoes fast diffusion (blue trajectory) similar to the pure solvent while, when a tracer with specific attraction to the polymer is captured in a dense (collapsed) domain, its diffusion (red trajectory) is significantly retarded. For collapsing gels with increasing temperature, the dense domain size increases enhancing the slowdown effect (Figure 4a).The noninteracting A647 exhibits a single diffusion only, irrespectively of the swelling ratio, but it slows down when the hydrogel collapses due to crowding effects (Figure S5). Since HG-2 exhibits no collapse at all, but the A488 dynamics still contain the slow component, we may anticipate that there is a restricted formation of collapsed domains within the studied temperature range. This is in accordance with the low amplitude Fslow (Figure 4b) compared with its value in highly cross-linked HG-4 (Figure 4a). Conversely, the mild collapse of HG-1 in the same temperature range hints toward formation of the domain nuclei which is again less extensive than in HG-4 based on the Fslow of Rh6G in these two hydrogels (Figure 4a,b). Correspondingly, there is no change in Dslow of A488 in HG-2, while there is a mild decrease of Dslow of Rh6G in HG-1, which shrinks considerably more than HG-2. Moreover, the fast process is expectedly unaffected by the 5309


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reinforces the initial speculation that the two interacting tracers display different overall interactions with PNiPAAm. Moreover, the observed differences in Rh6G dynamics and permeations using salt or temperature (T) as stimulus hint to the dual character of tracer−PNiPAAm interactions. Regarding a possible “structure−interaction” relationship, we conjecture that hydrophobic attractions lead to tracer binding on PNiPAAm. The different partitioning of A488 and Rh6G in the hydrogels is a nontrivial result of the competition between the electrostatic interactions (repulsive for A488 and attractive for Rh6G) and the hydrophobic attraction. Finally, the temperature dependence of Dslow(T) is dominated by the formation of collapsed domains,60 which grow with increasing temperature and temporarily capture the tracer. In such case, the slow process probably reflects the residence time of the tracer in these domains. As an alternative, taking into account the presence of slow density fluctuations in gels, the tracer Dslow may also be related to the gel slow diffusion mode.62 In this context, FCS tracer diffusion in and dynamic light scattering from hydrogels, yield structural information on this complex system. In absence of specific tracer−polymer attractive interactions, a master plot of molecular tracer diffusion slowdown could be obtained by geometric “crowding” arguments in polymer solutions21 and gels,4 under good solvency conditions. The single component diffusion of such noninteracting tracers is a local process and hence scales with polymer concentration in physical networks leading to a “master” plot (dashed line in Figure 7). Diffusion of the same noninteracting tracers is slower

of the slow gel mode with salt concentration observed by dynamic light scattering.66 At the same time, the addition of salt suppresses the influence of the electrostatics on the tracer partitioning between the gel and the solution. Figure 6a shows G(t) for Rh6G in HG-5 at varying ionic strength (I) and the inset shows the tracer intensity profile IF(z)/IF,max in the same hydrogel under the same conditions. At the ionic strength of 0.1 M and below, there was no significant gel collapse revealed by the IF(z)/IF,max plot. Higher resolution surface plasmon resonance and optical waveguide spectroscopy for similar hydrogel layers have shown a 30% increase of water uptake compared to the pure water with NaCl concentration around 0.01 M.7,66 At the highest examined ionic strength (I = 1 M), the thickness of the grafted PNiPAAm HG-5 is about 8.5 μm, compared to its thickness of about 12 μm in salt-free solution at 25 °C. Similar salt-dependent shrinkage is also reported in recent works on identical grafted PNiPAAm layers using surface sensitive techniques.7,67,66 The dependence of the fast and slow diffusion on the ionic strength in Figure 6b reveals that upon addition of salt, the Rh6G dynamics is not affected, as long as the gel is fully swollen. The dynamics still contains the fast and slow component, but the slow one strongly dominates. In the collapsed gel (at I = 1 M of salt), we observe a speedup of the fast process and a simultaneous increase of its contribution to G(t). This observation appears commensurable to analogous findings4 concerning an additional speed-up exemplified by a noninteracting molecular tracer (A647) in identical hydrogel layers, however, upon thermal collapse; for the latter case, the observed speed-up of A647 diffusion reflects diffusion through solvent-rich regions. As in the case of thermally collapsed HG4(Figure 4a), the slow process becomes significantly slowed down in the collapsed gel also in the salt-induced collapse of HG-5 (Figure 6b). The overall trend in Rh6G dynamics upon salt-induced collapse is in contrast with its behavior in HG-4 (Figure 4) where the collapse was stimulated by temperature changes: Dslow assumed a lower value in the thermally collapsed HG-4 than in HG-5; Ffast was insensitive to the variation of temperature (or ϕ), while it changes dramatically when HG-5 is collapsed by the addition of salt; HG-4 undergoes stronger shrinkage at 35 °C (ϕ from 0.15 to 0.33) than HG-5 at 1 M salt (ϕ from 0.18 to 0.22). These differences indicate that the microscopic mechanisms of gel collapse induced by temperature change and/or by addition of salt may be different in the present polymer. As already mentioned, variation of temperature and addition of monovalent salt can lead to different hydrogel structures in the collapsed state. On the other hand, the addition of salt screens the long-ranged electrostatic interactions between all charged species, which homogeneously deswells the gel. On top of these generic interactions, one has to add effects which have been shown56 to alter the LCST of both pure PNiPAAm and its copolymer with acrylic acid. This picture is supported by the reduced amplitude of the slow gel process66 in similar PNiPAAm hydrogel layers upon addition of salt as observed by microphoton correlation spectroscopy.62

Figure 7. Mobility slowdown presented as D(ϕ)/D0 vs ϕ, in the case of strong attractions exemplified by Rh6G (black symbols) in HGs. The Dslow (ϕ)/D0 data sets correspond to the same HG shown in Table 1. HG-1 (squares), HG-3 (rhombi), HG-4 (triangles), and HG7 (circles). Fast and slow processes are denoted by empty and solid symbols, respectively. Dashed and solid curves denote stretched exponential dependences vs ϕ for the recently reported noninteracting molecular tracer diffusion slowdown of D(ϕ)/D0, in solutions21 and in HGs,4 accordingly.

due to the presence of permanent cross-links, but still follows a universal scaling function (solid line in Figure 7) in cross-linked hydrogel networks. This situation changes qualitatively in the presence of specific interactions. In such case, the slow component of the tracer dynamics reflects various local processes, such as the formation of collapsed network domains, or internal dynamics of the chains to which the tracer binds. Since the size of the collapsed domains and their dynamics do not exhibit a simple relation to the polymer volume fraction, the latter turns out not to represent a universal scaling variable for the Dslow(ϕ)/Do of the interacting tracers. This becomes

IV. DISCUSSION Using temperature as the external stimulus, at low HG crosslink densities (ϕ < 0.1), we evidenced that Rh6G exhibits stronger attractions to PNiPAAm than A488 (Figure 4b).The observation that at low cross-link densities Dslow(T) for A488 manifests qualitative differences compared to that of Rh6G, 5310


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clear from the plot of Dslow(ϕ)/Do as a function of ϕ for the interacting tracers (Rh6G in Figure 7 and/or A488 in Figure S6). In contrast to that, superposition on the universal scaling plot holds for the Dfast(ϕ)/Do data of A488 and Rh6G in both PNiPAAm HGs68 and in PNiPAAm aqueous solutions.50

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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V. CONCLUDING REMARKS By means of FCS, we studied the effects of external stimuli on the mobility of molecular tracers in grafted PNiPAAm hydrogel layers as a function of cross-link density, polymer volume fraction and tracer−polymer interactions. Different stimuli for the gel collapse lead to substantial differences in the tracer− polymer interactions and associated tracer mobilities. Moreover, the power of FCS is also demonstrated, by using different molecular tracers as “spy” for the hydrogel’s microscopic behavior and also by taking advantage of the variability in tracer−polymer interactions. The interactions of A488 and Rh6G with PNiPAAm vary differently with changes in external conditions, as revealed by their respective differences in permeation and dynamics, using temperature as external stimulus. At low concentrations in the “as-prepared” state, e.g., low ϕ at 25 °C, the temperature dependent slow diffusion in HGs reveals that the dynamics of A488 and Rh6G seem to be related with evolving collapsed microdomains in the gel, whereas the tracer−polymer interaction potential seems to involve a more substantial contribution from crowding effects, as shown by increasing ϕ (25 °C). An additional key finding of our study is that the tracer mobility in the collapsed state may depend on the applied stimulus, as demonstrated for Rh6G. The conclusive message from this paper is 2-fold: (i) an attempt for superposition of slow diffusion data vs polymer concentration fails even for molecular, yet interacting, tracers, calling for alternative scaling attempts; (ii) the slow diffusion most likely represents a combined mobility between weak adsorption/desorption events and free diffusion beyond the diffraction-limited FCS resolution, whereas the overall slow diffusivity resolved by FCS captures information about tracer entrapment into evolving hydrophobic nanodomains, polymer strand dynamics and binding energy of the tracer on the strands. To gain further information we suggest to use smaller observation volumes (STED-FCS, with higher axial resolution), and combine FCS with simulations. This combination can provide further quantitative information about associated thermodynamic parameters and binding constants, and also verify the appropriateness of the employed two-component Fickian model, when deviations from single Fickian diffusion are observed.

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ACKNOWLEDGMENTS The authors would like to acknowledge the Deutsche Forschungsgemeinschaft for financial support in the framework of SPP1259 “Intelligence Hydrogele”, SPP 1066 and MSMT of the Czech Republic, Grant LK21302 (P.K.). We would also like to thank Uli Jonas for helpful discussions, Katja Nilles for synthesis of the PNiPAAm polymer, Andreas Best and Gabi Hermann for technical support, and Karmena Jaskiewicz for ζpotential measurements.

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ABBREVIATIONS A488, Alexa488; A647, Alexa647; ε, tracer−PNiPAAm interaction potential; E, UV-irradiation energy dose; HG, hydrogel; P, tracer permeation coefficient in hydrogel; Rh6G, Rhodamine 6G; STED, stimulated emission depletion

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ASSOCIATED CONTENT

* Supporting Information S

A488 density profile in HG-5 for different ionic strength (I) values at 25 °C, A488 partitioning in different gels, A647 density profile and the associated G(t) in HG-6 for different ionic strength (I) values at 25 °C, interpretation of the z-scans, and diffusion slowdown vs ϕ for A488. This material is available free of charge via the Internet at http://pubs.acs.org.

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*E-mail: (G.F.): [email protected]. 5311


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