Mechanism of Lysozyme Uptake in Poly(acrylic acid) Microgels - The

Apr 14, 2009 - Thus, the shell which is initially formed as a result of fast lysozyme transport to the gel network and fast protein−microgel interac...
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J. Phys. Chem. B 2009, 113, 6183–6193

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ARTICLES Mechanism of Lysozyme Uptake in Poly(acrylic acid) Microgels Christian Johansson,* Per Hansson, and Martin Malmsten Department of Pharmacy, Uppsala UniVersity, P.O. Box 580, SE-751 23 Uppsala, Sweden ReceiVed: January 23, 2009; ReVised Manuscript ReceiVed: March 10, 2009

The uptake of lysozyme by oppositely charged poly(acrylic acid) microgels was investigated by micromanipulator-assisted light microscopy and confocal microscopy. Lysozyme was observed to distribute nonuniformly within the microgels, forming a core-shell structure with considerably higher lysozyme concentration in the shell than in the core. The core-shell formation can be divided into two periods. During the first of these, the shell is formed during rapid microgel deswelling, and with no lysozyme diffusing into the microgel core. This is followed by a second period, during which microgel deswelling is negligible and lysozyme diffuses into the microgel core. Thus, the shell which is initially formed as a result of fast lysozyme transport to the gel network and fast protein-microgel interactions is able to carry a mechanical load and prevents deswelling during the latter core diffusion period. These two distinct regimes of lysozyme loading were also successfully described theoretically, demonstrating the importance of lysozyme cluster formation for the observed phenomena. 1. Introduction Microgels are slightly cross-linked polymer network particles in the micrometer size range, with an ability to swell and deswell in response to changes in their environment. The uptake of oppositely charged molecules into a charged microgel causes osmotic deswelling, an effect which has been studied for microgels interacting with surfactants,1,2 low Mw drug molecules,3 peptides,4-6 and proteins.7 The capability of microgels to store large amounts of oppositely charged molecules in a small volume makes them potential drug delivery systems,8-14 particularly for macromolecular drugs. To provide information on factors influencing protein binding to, and distribution within, such systems, we previously investigated uptake of the model protein lysozyme into poly(acrylic acid) microgels. For a range of conditions, lysozyme distributed nonuniformly within the microgels, forming a shell in the outer parts of the microgel. The shell had considerably higher lysozyme concentration than the inner part of the microgel, and the core-shell structure appeared to constitute an end state of the lysozyme uptake.7 Core-shell formations have been observed also in systems where microgels interact with peptides,4 surfactants,1 and low Mw drugs,3 and have been found to depend on factors such as pH, ionic strength, degree of cross-linking,1,4 and charged group distribution.3 Given the above complexities in solute incorporation into microgels, additional investigations are needed to clarify transport processes of macromolecules within microgels. In the present study, we therefore further investigate core-shell formation for lysozyme in poly(acrylic acid) microgels, addressing in more detail the interplay between lysozyme diffusion through the microgel network and the resulting network contraction. In doing so, detailed experimental studies are * Corresponding author. E-mail: [email protected].

combined with theoretical analysis of the transport processes involved to provide a comprehensive description of these processes. 2. Experimental Section 2.1. Materials. Lysozyme from chicken egg white (95% purity, Lot 051K7028) was from Sigma-Aldrich (Germany), and used without further purification. Dimethylformamide (DMFA) and fluorescein isothiocyanate (FITC) were both from Acros Organics (Belgium), while Oregon Green 488 (Lot 26793W) and Alexa Fluor 647 (Lot 25563W) were from Invitrogen (USA). A solution of poly(acrylic acid) microgel particles (3.7 wt % cross-linker in the reaction mixture), synthesized by inverse suspension polymerization using cyclohexane as the continuous phase and Span 60 as stabilizer,7 was used. The concentration of the microgel solution was 2.3 mg of gel/g solution, and microgels selected for the micropipet-assisted experiments all had diameters between 60-80 µm at pH 7.0 and ionic strength 220 mM (and in the absence of lysozyme). All other chemicals used were of analytical grade. Purified Milli-Q water was used throughout. For pH control, buffer solutions of sodium phosphate monobasic/sodium phosphate dibasic were used, and the ionic strengths reported are from the contribution of the buffer only. 2.2. Fluorescent Labeling of Lysozyme. FITC labeling: Lysozyme was labeled with FITC, as previously described.7 Oregon Green 488 labeling: Oregon Green 488 was dissolved in DMFA to a concentration of 2 mg/mL, while lysozyme was dissolved in 0.1 M NaHCO3 to a concentration of 5 mg/mL. 80 µL of the Oregon Green 488 solution was added to 2 mL lysozyme solution, and the mixture was stirred at room temperature for 2 h. Unreacted Oregon Green 488 was then removed by repeated size exclusion chromatography using PD10 columns (GE Health Care, Sweden). Lysozyme and Oregon Green 488 concentrations were determined by absorbance

10.1021/jp900706k CCC: $40.75  2009 American Chemical Society Published on Web 04/14/2009

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measurements at 280 and 496 nm, respectively, using a Helios v 4.60 spectrophotometer (ThermoSpectronic, UK), and the molar ratio Oregon Green 488:lysozyme was found to be about 1:3. This degree of labeling was found not to affect the length of the shell formation period (results not shown). Alexa Fluor 647 labeling: Alexa Fluor 647 was dissolved in 0.1 M NaHCO3 to a concentration of 1 mg/mL, while lysozyme was dissolved in 0.1 M NaHCO3 to a concentration of 5 mg/mL. 75 µL of Alexa solution was added to 2 mL of lysozyme solution, and the mixture was stirred at room temperature for 1 h. Unreacted Alexa Fluor 647 was then removed by repeated size exclusion chromatography using PD-10 columns (GE Health Care, Sweden). Lysozyme and Alexa Fluor 647 concentrations were determined by absorbance measurements at 280 and 650 nm, respectively, using a Helios v 4.60 spectrophotometer (ThermoSpectronic, UK), and the molar ratio Alexa Fluor 647: lysozyme was found to be about 1:30. This degree of labeling was found not to affect the length of the shell formation period (results not shown). 2.3. Micromanipulator-AssistedLightMicroscopy.Lysozymedependent microgel deswelling, as well as lysozyme diffusion fronts within individual microgel particles, was studied by micromanipulator-assisted light microscopy. A Bx-51 light microscope (Olympus, Japan) was used, equipped with an ONM-1 manipulator (Narishige, Japan), a DP 50 digital camera (Olympus, Japan), and the software DP-soft (Olympus, Japan). Micropipets (15-30 µm in diameter) were pulled with a Narishige PC-10 puller and smoothened with an MF-9 microforge. Microgels (60-80 µm in diameter at pH 7.0 and ionic strength 220 mM) were captured by suction onto micropipets using an IM-5A injector (Narishige, Japan) and placed inside a flow pipet (2 mm in diameter). Each experiment consisted of capturing the microgel in a solution of pH 7.0 and ionic strength 220 mM, followed by flushing with buffer solution for 3 min and subsequent flushing with lysozyme solution until the lysozyme diffusion front was no longer visible. The flow rate used was 1.8 mL/min, obtained by a P-1 peristaltic pump (Pharmacia, Sweden). Experiments were performed at ionic strengths of 40 and 100 mM, and at lysozyme concentrations 0.063, 0.125, and 0.25 mg/mL. The starting time, t0, was defined as the start of the flushing with lysozyme solution. Pictures were taken every 10 s during the first 2 min, and every 30 s from 2 min onward. The starting volume of the microgel, V0, was defined as the volume at t0, and volumes are given as V/V0. 2.4. Confocal Microscopy. Confocal microscopy experiments were performed to investigate the distribution of lysozyme in microgels, using a Confocal Leica DM IRE2 laser scanning microscope (Leica Microsystems, Germany), equipped with an Ar/HeNe laser, and software Leica TCS SL. Given the low cross-linking density and high water content, intensity distributions obtained for the presently investigated microgels are not distorted by light scattering and require no contrast matching.15 2.4.1. Measurement of Shell Formation Period and Diffusion Front Velocity. Lysozyme solutions of concentrations 0.5, 1.0, and 2.0 mg/mL were prepared by dissolving lysozyme in phosphate buffer (ionic strength 40 or 100 mM), while buffered microgel solutions were prepared by mixing the concentrated microgel solution (mentioned above) with phosphate buffers (ionic strength 40 or 100 mM) to the volume ratio 1:200 of microgel solution:buffer. 0.5 mL of lysozyme solution and 0.5 mL of buffered microgel solution was then added sequentially to a cylindrical container with cover glass bottom, after which the mixture was stirred for 10 s and the container placed in the confocal microscope. The start of each experiment, t0, was

Johansson et al. defined as the time when the microgels were added to the container. After the microgels had sunk to the cover glass bottom, individual microgels were chosen and photographed every 30 s. The pictures used were images of a “cut” through the center of the microgel, perpendicular to the glass bottom. Lysozyme uptake resulted in core-shell formation in the microgels, and the uptake process could be divided into two distinct time periods. The first of these, denoted shell formation, was defined as the time from t0 to the first observation of a diffusion front moving from the shell toward the microgel center, while the second period, denoted core diffusion (during which microgel deswelling was negligible), was defined as the period when the diffusion front was observed to move through the microgel core. Results on the length of the shell formation period represent average data from at least four measurements (at each studied lysozyme concentration and ionic strength). Also the diffusion front velocity was determined by confocal microscopy, through measurements of the volume of the gel core inside the sharp diffusion front at different times during core diffusion. The latter was performed by measuring the diameter of the “sphere” inside the diffusion front in two directions (parallel and perpendicular to the glass bottom), followed by calculations of the average sphere volume. Only pictures taken before the diffusion front reached the center of the microgel were used. The slope of the volume decrease with time was defined as the diffusion front velocity. The diffusion front velocity, at each studied lysozyme concentration and ionic strength, is given as average and standard deviation of at least four measurements. 2.4.2. Addition of Two Differently Labeled Lysozyme Fractions to Microgel Solution. In order to differentiate between lysozyme binding to the microgel early and later in the protein uptake process, experiments were performed with two differently labeled lysozyme fractions added to the same microgel. “Alexalysozyme” (labeled with Alexa Fluor 647) was detected at wavelengths 650-750 nm after excitation with 633 nm, while “Oregon-lysozyme” (labeled with Oregon Green 488) was detected at wavelengths 500-530 nm after excitation with 488 nm, and high selectivity was observed in the detection of the two lysozyme fractions (Figures S1.1 and S1.2, Supporting Information). 250 µL of microgel solution (diluted 100 times with phosphate buffer of pH 7.0 and ionic strength 40 mM) was mixed with 250 µL Oregon-lysozyme solution (1 mg/mL lysozyme, pH 7.0, and ionic strength 40 mM) and 500 µL Alexalysozyme solution (0.5 mg/mL lysozyme, pH 7.0, and ionic strength 40 mM). The two lysozyme solutions were either added simultaneously, or Oregon-lysozyme was added 1 min before Alexa-lysozyme. In both types of experiment, the conditions through the whole experiment were pH 7.0, ionic strength 40 mM, and 0.5 mg/mL lysozyme. The mixture was then positioned under the confocal microscope, where a microgel was located and photographed every 30 s. The delay of 1 min ensured that, for every single microgel examined, the second lysozyme fraction was added before the end of the shell formation period. Intensity profiles in the shell: The Oregon and Alexa intensity profiles through the shell, in the last picture before the core diffusion layer was observed (∼3 min after the initial lysozyme addition), were measured. Intensity ratio in shell and in core diffusion layer: From each experiment, a picture where the diffusion front had reached 1/3 of the way through the microgel core (∼4 min after the initial lysozyme addition) was used to measure Alexa and Oregon fluorescence intensities in the shell and in the core diffusion layer. From each picture, the intensity ratio in the shell was calculated from the maximum intensities along a line through the shell, and an average ratio calculated from 10

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Figure 1. Figure showing how observations from confocal microscopy experiments are translated to a theoretical model. (a) Microscopy picture of a microgel at one moment during lysozyme uptake. The picture shows the shell, the core diffusion layer (cdl) and the protein-free core. (b) Gel model showing the position of the outer boundary of the protein-free core (r0), the cdl (r1), and the shell (r2).

such lines (in every picture). The intensity ratio in the core diffusion layer was calculated using the average Oregon and Alexa intensities along a 15 µm line in the core diffusion layer, and the average ratio from 10 such lines was calculated from each picture. Every reported intensity ratio in shell and core diffusion layer, in turn, represents the average from five different microgels. 3. Theoretical Analysis The model employed assumes spherical geometry, where a protein-free core of radius r0 is surrounded by a core diffusion layer (cdl) between r0 and r1, and a protein-rich shell between r1 and r2, as shown in Figure 1. The conditions considered are the following: the shell and the cdl have uniform and constant concentration of polymer and “stationary” protein. Stationary proteins do not contribute to the diffusiVe mass transport due to strong interactions with the polymer network and/or other stationary protein molecules. Instead, mass transport is governed by “mobile” protein molecules diffusing through the layers. The shell and the cdl grow as mobile protein molecules become incorporated into the structures. Incorporation occurs only at r1 and r0, respectively, where it is immediate (consistent with a fixed composition of the stationary structures and sharp diffusion fronts). Deswelling is associated exclusively with shell growth. Furthermore, the evolution of the gel structure is strictly consecutive, so that core diffusion starts when shell growth ends. The shell growth is assumed to end abruptly at some point (e.g., when the lysozyme aggregation in the shell is sufficient to form a stress-bearing network). Under these conditions, the kinetic problem amounts to describing the movement of the boundaries r1 and r0. Note that the displacement of r2 from its initial position is directly related to r1 and the composition of the shell structure. The movement of r1 depends not only on the rate of mass transport to the boundary but also on the relaxation rate of the gel network and the shell structure, the latter effect difficult to model. However, with support from previous work it will be assumed that the protein transport is rate determining and obeys steady-state kinetics.2,5,16 This means that all other processes, including the transport of simple ions, take place on shorter time scales, so that gel and liquid solution are at osmotic balance during binding.2,16 With fixed composition of the stationary structures, but allowing for concentration gradients of mobile protein (and simple ions), there is strong resemblance to the case of “diffusion with a moving boundary” of class A described by Crank,17 for

which there is an exact solution to the one-dimensional diffusion problem. Importantly, when the mobile fraction of the molecules is small, a quasi-steady state characterized by nearly linear concentration gradients is maintained during the movement of the boundary, giving credit to the steady-state approach. One should keep in mind, however, that in the present case also the boundary r2 moves during shell growth. This means that the diffusion is affected by a “counter flow” of water. Previous applications of the model have involved only simple core-shell structures.2,5,16,18,19 An extension to include the cdl is straightforward. A full derivation is given in Supporting Information, section S2. The result can be written as follows:

R30 t) 3V0Z(Cbulk - C0)

-1 -1 ∫0β {(r-1 0 - r1 )Pcdl + -1 -1 -1 -1 (r-1 1 - r2 )Pshell + r2 D′ } dβ

(1)

Equation 1 gives the time t for a gel to reach a certain state as a function of β, the overall protein-to-polymer charge ratio in the gel. R0 is the radius of the microgel in swelling equilibrium with the liquid prior to protein binding, and V0 the volume per charge on the network in that state. Z is the net charge of the protein, Cbulk the protein concentration in the bulk of the liquid, and C0 the concentration (activity) of the protein at r0. The two first terms inside the integral are associated with the transport through the cdl and the shell, where Pcdl and Pshell are the permeabilities in the respective layers. These are defined in the following way:

Pcdl ) Dcdlkcdl/liq

(2)

Pshell ) Dshellkshell/liq

(3)

where Dcdl and Dshell are the diffusion constants of the protein in the respective regions, and kcdl/liq and kshell/liq are partition coefficients describing the distribution of protein between cdl and liquid, and between shell and liquid, respectively. The third term inside the integral is associated with mass transfer of protein from the bulk liquid to the gel surface. D′ is defined by

D ′ ) (1 + Sh ⁄ 2)D

(4)

where D is the protein diffusion constant in the liquid and Sh the Sherwood number related to an effective stagnant layer thickness in the liquid just outside the gel.16,20 The gel dimensions r0, r1, and r2 are functions of β and the compositions of the different parts of the gel. To be consistent

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Figure 2. Images of a microgel, sucked onto the tip of a micropipet, at different times during flushing with a solution of unlabeled lysozyme (pH 7.0, ionic strength 40 mM, and 0.125 mg/mL lysozyme). After 230 s, a diffusion front of lysozyme is observed to move from the outer part toward the inner of the microgel (indicated by the black arrows). The time from t0 to the time when the diffusion front is first observed (t230s) is defined as the shell formation period, which is followed by the core diffusion period during which the diffusion front moves through the inner of the microgel.

with notations used earlier,2,16 the composition of the stationary shell structure, assumed to be constant during binding, is expressed by the volume of the shell per polyion charge Vshell and the polymer-to-protein charge ratio in the shell fshell. The corresponding quantities for the core are Vcore and fcdl. To evaluate the integral, we define βshell as the ratio between the protein (net) charges in the shell and the total polyion charge in the gel, and let βshell,∞ be the ratio when the protein structure in the shell becomes arrested, which in the model represents the final degree of binding to the shell and the end of the shell formation period. Note that β and βshell include only the stationary protein molecules. The integral is evaluated differently during the two distinct binding periods:

shell formation period :

core diffusion period :

β g βshell,∞

{

β ) βshell r0 ) r1

(5a)

βshell ) βshell,∞ r1 ) r1(βshell,∞) r2 ) r2(βshell,∞)

(5b)

0 e β e βshell,∞

{

During binding the protein-free part of the core will be assumed to act as a sink (i.e., C0 ) 0). The magnitudes of Vshell and fshell influence kshell/liq and the concentrations of simple ions in the shell. These are calculated from the Donnan equilibrium between shell and liquid at r2 (Supporting Information, section S2). It should be pointed out that kshell/liq is not a true equilibrium constant. Instead, at low concentrations of mobile protein, it can be regarded as a conditional distribution constant determined only by Vshell and fshell. This holds for most of the situations encountered here. In the general case, however, it depends also on the mobile protein concentration through eqs S:19-S:21 in Supporting Information. It has been demonstrated earlier21 that the rubber elasticity of the shell network gives rise to an increased pressure in the core, so that Vcore e V0 at swelling equilibrium. If necessary, the effect can be taken into account in a straightforward, but cumbersome, way.2,16 Here we neglect this effect since (1) theoretical calculations show21 that the effect is small for the Vshell and βshell values encountered, and (2) the dynamic pressure on the core should be smaller than the equilibrium pressure because of the internal friction in the shell, particularly when proteins are aggregated. Neglecting the effect and putting Vcore ) V0, allows us to write

Vshell r23 - r13 r23 - (r2 - δ)3 ) ) 3 V0 R - r 3 R 3 - (r - δ)3 0

1

0

(6)

2

where δ ) r2 - r1 is the shell thickness. Equation 6 allows Vshell/V0 to be determined from the initial gel volume R0, the

observed final gel radius r2,∞, and the final shell thickness δ∞, all of which can be considered as time independent. After that, fshell can be determined by fitting the model to the deswelling (time dependent) part of the curves. This must be done by taking into account eqs S:19-S:21 in Supporting Information, regulating the local equilibrium of the nonaggregated protein and simple ions at the gel boundary. Finally, fcdl can be determined by fitting eq 1 to data of r0 as a function of time. 4. Results 4.1. Micromanipulator-Assisted Light Microscopy. Poly(acrylic acid) microgels, sucked onto a micropipet, were flushed with lysozyme solutions and photographed using a light microscope. At 0.063-0.25 mg/mL lysozyme, the microgel shows an initial deswelling followed by the movement of a diffusion front from the outer part of the microgel toward the center (Figure 2). Since the deswelling is essentially halted before the diffusion front is observed to move through the microgel (Figure 3), it is convenient to define two separate periods of lysozyme uptake. During the first period, ranging from t0 to the onset of a diffusion front moving toward the microgel center, the microgel deswells and the shell is formed. This shell formation period is followed by a core diffusion period, during which microgel deswelling is negligible and a lysozyme diffusion front moves through the microgel. In the micromanipulator-assisted experiments, shell formation lasted for 5 min at ionic strength 40 mM and 0.063 mg/mL lysozyme, decreasing to about 1.5 min as the lysozyme concentration was increased to 0.25 mg/mL (Figure 4). Deswelling is initially faster at higher lysozyme concentration, an effect less apparent at lower ionic strength (Figure 3a,b). Interestingly, deswelling after 600 s is larger at lower lysozyme concentration, an effect most distinct at 40 mM and apparently a result of the deswelling stopping at an earlier stage at the higher lysozyme concentration. When flushing the microgel with higher lysozyme concentrations, 0.5 and 1 mg/mL (the two highest concentrations examined in the confocal microscopy experiments), the microgel wrinkles and collapses, an effect starting in the end of the microgel opposite to the micropipet, changing into an unmeasurable nonspherical shape with V/V0 value somewhere below 0.1 (not shown). 4.2. Confocal Microscopy. Confocal microscopy experiments confirmed the existence of separate shell formation and core diffusion periods seen in the micromanipulator-assisted experiments (Figure 5). Due to limitations in the experimental setup, the shell formation period (where the major volume change takes place; Figure 2) could not be followed in detail with this methodology. Core diffusion, on the other hand, could

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Figure 3. Microgel volume versus time and as a function of lysozyme concentration, from micropipet experiments, at an ionic strength of (a) 40 mM and (b) 100 mM.

Figure 4. Length of the shell formation period in the micromanipulatorassisted experiments as a function of lysozyme concentration and ionic strength.

be monitored in considerable detail. As an illustration of this, Figure 6 shows the FITC-lysozyme fluorescence intensity profile through 1/2 of a microgel at one moment during lysozyme uptake. The peak on the left side of the diagram is the intensity from the shell. The intensity profile is “plateau-like” in between the shell and the diffusion front, and the diffusion front constitutes a rather sharp change in fluorescence intensity, enabling measurements of the diffusion front velocity. 4.2.1. Length of Shell Formation Period, and Diffusion Front Velocity. The length of the shell formation period and the core diffusion front velocity were measured at lysozyme concentrations 0.25, 0.5, and 1.0 mg/mL (Figures 7 and 8). Shell formation lasted around 11 min at an ionic strength of 40 mM and 0.25 mg/mL lysozyme, and decreased to roughly 2 min as the lysozyme concentration was increased to 1 mg/mL. An increase in ionic strength, from 40 to 100 mM, extended the shell formation period. The volume inside the diffusion front decreased approximately linearly with time, allowing determination of the diffusion front velocity, which was observed to be higher at higher outer lysozyme concentrations and lower ionic strength (Figure 8).

At lysozyme concentration e0.125 mg/mL, a diffuse concentration profile rather than a sharp diffusion front was observed, and hence no quantitative results are reported for lysozyme concentrations below 0.25 mg/mL. It should also be reiterated that the transport of lysozyme to the microgel is faster in the micromanipulator-assisted experiments, where the microgel is being flushed with lysozyme solution, than it is in the confocal microscopy experiments where the lysozyme solution is unstirred. This difference between techniques explains why microgel deswelling in the confocal microscopy studies occurred at preserved spherical shape also at 0.5 and 1 mg/mL lysozyme, whereas the same concentrations in the micromanipulatorassisted experiments caused the microgel to wrinkle. The difference is also the reason for the shorter shell formation period in the micromanipulator-assisted experiments. 4.2.2. Addition of Two Differently Labeled Lysozyme Fractions. In order to gain further knowledge of the diffusion mechanisms through the shell, an experiment using two differently labeled lysozyme fractions was performed. In the case of simultaneous Oregon- and Alexa-lysozyme addition, the two labeled fractions were similarly distributed in the shell at the end of the shell formation period, both with slightly higher intensity in the outer half of the shell (Figure 9). In the case of Alexa-lysozyme addition delayed by 1 min, however, the concentration of Alexa-lysozyme was higher in the inner part of the shell than in the outer. Pictures where the diffusion front had reached 1/3 of the way through the microgel core (∼4 min after the initial lysozyme addition) were also used to measure the Alexa and Oregon intensities in the shell and in the core diffusion layer (between shell and front) (Figure 10a). In the core diffusion layer, the intensity ratio was the same whether the lysozyme fractions were added simultaneously or if the Alexa-lysozyme addition was 1 min delayed, whereas the intensity ratio in the shell was significantly lower in the case of delayed Alexa-lysozyme addition (Figure 10b). Taken together, these results show that protein molecules entering the gel at a later stage are transported through a layer formed by molecules that have entered at an earlier stage. 5. Discussion 5.1. Two Binding Periods. The present investigation shows that the binding of lysozyme occurs in two consecutive steps. During shell formation, the microgel deswells rapidly (Figure

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Figure 5. Confocal microscopy images showing time-dependent uptake of FITC-labeled lysozyme into a microgel at pH 7.0, ionic strength 40 mM, and 1 mg/mL lysozyme. Each picture represents a “cut” through the center of the microgel, perpendicular to the glass bottom. The pictures show that, initially, a shell with a high concentration of lysozyme is formed, followed (after 180 s) by a diffusion front of lysozyme moving from the lysozyme-microgel shell toward the inner parts of the microgel. The microgel is somewhat deformed by sticking to the cover glass bottom.

Figure 6. FITC-lysozyme fluorescence intensity through half a microgel during lysozyme uptake. The intensity along the line through the microgel is shown in the diagram, which (from left to right) represents the intensity (i) outside the microgel, (ii) in the shell, (iii) in the “plateau” between the shell and the diffusion front, and (iv) inside the diffusion front. The figure shows that the diffusion front constitutes a rather distinct borderline.

3), while no lysozyme is observed to diffuse into the microgel core (Figure 5). This is followed by core diffusion, when lysozyme diffuses through the microgel core with a sharp diffusion front (Figures 2 and 5), accompanied by little or no additional microgel deswelling. The arrest of the gel in a semiswollen core-shell state can be related to the mechanical properties of the shell, determined in turn by its composition and microstructure. It is interesting therefore that, while no significant variation of the final shell thickness can be observed, the length of the shell formation period and the final degree of deswelling decreases with increasing lysozyme concentration in the liquid (Figures 3, 4, and 7). Since also the ionic strength has a large influence, it appears that both the rate of protein binding (expected to increase with increasing protein concentration in solution) and the strength of the electrostatic interactions are of importance. To explore this further we analyze the mechanism of protein transport, the properties of the shells in the arrested gel state, and the information obtained from theoretical analysis of the kinetic data. 5.2. Transport Mechanism. The experiments where two lysozyme solutions labeled with different fluorescent markers

Figure 7. Duration of the shell formation in the confocal microscopy experiments, as a function of lysozyme concentration and ionic strength.

were added to the microgel solution showed that, in the case of delayed Alexa-lysozyme addition, the Alexa-lysozyme intensity was primarily decreased in the outer half of the shell at the end of the shell formation period. It was also observed that IAlexa/IOregon decreased in the shell when going from simultaneous addition to delayed Alexa-lysozyme addition, whereas the ratio in the core diffusion layer was independent of the order of addition. Had the protein molecules first entering the outer part of the microgel also been the first to reach the center, the Alexa-lysozyme intensity profile would be highest in the outer part of the shell in the case of delayed Alexa-lysozyme addition. Also, there would have been relative excess of Oregon-lysozyme in the core diffusion layer in the case of delayed Alexa-lysozyme addition. Instead, the finding of IAlexa/IOregon core diffusion layer being independent of type of addition suggests that this ratio is largely dictated by the concentrations outside the microgel, and that the lysozyme molecules in the core diffusion layer have predominantly diffused through the shell, without adsorbing to the microgel network on their way through the shell. This is contrary to what would be expected from the “relay-race” mechanism proposed earlier22 based on the observations of sequential binding of lyzosyme and cytochrome c to macroscopic poly(acrylic acid) hydrogels.23 The latter experiments

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Figure 8. Measurement of diffusion front velocity. (a) Example images showing the volume inside the diffusion front (indicated by white arrows) for a microgel at times 210 and 240 s. The volumes were plotted as a function of time (b), as shown for four microgels at pH 7.0, ionic strength 40 mM, and 0.5 mg/mL lysozyme. (c) Diffusion front velocity is presented as a function of lysozyme concentration at two different ionic strengths.

Figure 9. Representative Oregon and Alexa fluorescence intensity profiles through the microgel shell, 3 min after initial lysozyme addition (the time when microgel deswelling is halted and lysozyme diffusion through the core follows). The intensity profiles represent lines through the microgel shell, as exemplified in (a), and the diagrams show the intensity profiles in the case of (b) simultaneous Oregon-lysozyme and Alexa-lysozyme addition and (c) Oregon-lysozyme added 1 min before Alexa-lysozyme. The left end of each diagram represents a point outside the microgel and the right end is a point in the microgel core. It is observed that the concentration of Alexa-lysozyme is higher in the inner part of the shell than in the outer, in the case of delayed Alexa-lysozyme addition. The experiment was performed at pH 7.0, ionic strength 40 mM, and 0.5 mg/mL lysozyme.

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Figure 10. Solutions of Alexa-lysozyme and Oregon-lysozyme were added to microgel solution either (i) simultaneously, or (ii) Oregonlysozyme added 1 min before Alexa-lysozyme. (a) The Alexa and Oregon intensities were measured in the shell and in the diffusion front (between shell and front), ∼4 min after the initial lysozyme addition (when the diffusion front had reached 1/3 of the way through the microgel). The experiment was performed at pH 7.0, ionic strength 40 mM, and 0.5 mg/mL lysozyme. (b) The Alexa/Oregon intensity ratio in the shell was significantly lower in the case of delayed Alexalysozyme addition, as compared to simultaneous addition, while the intensity ratio in the diffusion front was the same whether the lysozyme fractions were added simultaneously or if the Alexa-lysozyme addition was 1 min delayed.

were, however, performed with gels substantially larger (cubic, 0.1-1.5 cm3) than the ones in the present study, and at different conditions, which may lead to a different mechanism. Also, since the separate layers in the larger gels may be the result of segregative phase separation of cytochrome c-rich and lysozyme-rich domains due to the chemical differences between the proteins (cf. “polymer incompatibility”), it is not possible to draw conclusions about the mechanism in the respective one-protein systems. (The perturbation of our system by the labeling of a small fraction of the lysozyme molecules with Alexa Fluor and Oregon Green is expected to be minor.) Our observation that the majority of the molecules transported from the liquid solution to the core-shell boundary does not bind to, or exchange with, the molecules forming the structure of the shell indicates that the interior shell structure is “saturated” with protein, and protein molecules in the shell “stationary”. The other fraction,

Johansson et al. responsible for diffusive mass transport, is mobile. The situation can be referred to the case of “diffusion with a moving boundary. The observation of sharp diffusion fronts during core diffusion suggests a similar mechanism in this region. Since no gel deswelling is involved, the process should be similar to protein diffusion through mechanically rigid ion exchange chromatography particles.24-28 Indeed, it has been observed that the uptake of lysozyme by SP Sepharose FF, an agarosebased cation exchanger, at low ionic strength occurs with a self-sharpening lysozyme diffusion front moving toward the particle center, whereas the diffusion front is more diffuse at higher ionic strength.26,27 This is in agreement with the present observations at lower lysozyme concentrations, where the diffusion fronts were sharp at ionic strength 40 mM (Figures 5 and 6), but diffuse at 100 mM. Diffusion in ion exchange particles has been described using a two-state model of “free” molecules in local equilibrium with an “adsorbed” phase allowing for diffusion in both subphases. The time evolution of theoretical concentration profiles have been shown to depend on the nature of the local equilibrium and the relative magnitudes of the diffusion constants. Steplike profiles, like those observed at low ionic strength, are typically obtained for negligible diffusion in the adsorbed phase and a local equilibrium characterized by a large binding constant and rapidly saturating binding isotherms.26,27,29,30 In these systems, higher outer protein concentration and higher ionic strength give faster moving diffusion fronts.26,27 Faster core diffusion fronts at increased protein concentration is observed also in the present system (Figures 8c and 11), but with the front moving slower with increasing ionic strength (Figure 8c). A faster moving diffusion front at higher ionic strength is expected as a result of a weaker binding of the proteins to the polyion chains due to the screening effect of salt. The reverse behavior in our system can be attributed to a dependence also on the transport through the shell and “stagnant” layers surrounding the gels, as further discussed in section 5.4. 5.3. Arrested Gel State. The most intriguing behavior of the present system is that shell formation stops before the entire core has been consumed. Binding to the core taking place after that point is expected to be associated with a reduction of the swelling pressure in the network since each protein molecule replaces several simple counterions. The absence of deswelling during this process suggests that the shell is not deformed by the resulting stress. A previous study on the same system showed shell formation to be initiated by the formation of small shell fragments with high lysozyme content,7 suggesting that lysozyme aggregation takes place during shell formation. Considering lysozyme’s ability to aggregate,31-38 it is possible that clustering of lysozyme can lead to the formation of a stress-bearing protein network (“gelation”) in the shell, thereby arresting the gel in a semiswollen state. A ramified protein network, i.e., a porous structure, would allow fast diffusion of protein through it, consistent with the observation of rapidly moving core diffusion fronts. The latter observation, together with the results from the experiments with sequential binding of the fluorescently labeled proteins, preclude explanations of the gel arrest based on high viscosity (or hydrodynamic jamming) of the shell material due to dense packing of proteins. Also supporting such a mechanism is the absence of shell formation in the case of cytochrome c,39 a small spherical

Lysozyme Uptake in Poly(acrylic acid) Microgels

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Figure 11. Gel radius r2 (solid line, filled symbols), the core-shell boundary r1 (dashed line), and the core diffusion front r0 (dotted line, open symbols) plotted as a function of time. Symbols are experimental data. Lines are eq 1 calculated with the parameters given in Table 1 obtained from fits of the equation to the data points. Ionic strength (mM): 40 (a-c) and 100 (d-f). Protein concentration (g/L): 0.063 (a, d), 0.125 (b, e), 0.25 (c, f).

TABLE 1: Results from Model Fittinga Csalt (mM)

C (g/L)

Vshell (L/mol)

fshell

fcdl

φshell

CL2+shell (µM)

βshell,∞

φcdl

V0 (L/mol)

r2,∞/R0

40 40 40 100 100 100

0.063 0.125 0.25 0.063 0.125 0.25

2.1 2.9 3.7 1.3 1.6 1.7

1.018 1.015 1.025 1.037 1.036 1.042

1.3 1.4 1.3 1.3 1.3

0.093 0.068 0.053 0.14 0.12 0.12

12 16 36 16 26 54

0.75 0.67 0.58 0.80 0.76 0.74

0.014 0.013 0.014 0.021 0.020

10 10 10 7.5 7.5 7.5

0.73 0.80 0.85 0.68 0.72 0.74

a

Fits of eq 1 are presented in Figure 11; δ∞ ) 5 µm, Dshell ) Dcore ) 1.2 × 10-10 m2/s; D′-1 ) 0.

protein very much comparable to lysozyme, except for its lack of self-assembly.

5.4. Model Predictions. In order to shed further light on the processes involved, theoretical modeling was performed

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as described above. As can be seen in Figure 11, the model can be fitted quite well to the experimental data, using an experimentally determined final shell thickness of 5 µm. As a first result, we note that the binding process is not influenced by stagnant layer diffusion. This is concluded from the observation that the theory cannot describe the initial part of the deswelling curves unless there is negligible external mass transfer resistance (Sh f ∞). One explanation to this could be that the liquid flow creates a streaming potential at the gel surface, speeding up the transport by migration in the electric field.40 The values of parameters obtained from the fits are presented in Table 1 together with other quantities calculated. Here, the volume fraction of stationary protein in the shell φshell was calculated from

φshell )

VlysNA ZfshellVshell

(7)

where Vlys is the volume of a lysozyme molecule, taken as the volume of a sphere of radius 20 Å, and NA is Avogadro’s number. The protein volume fraction in the core diffusion layer φcdl was calculated in an analogous way, and βshell,∞ calculated using eq S:26 (Supporting Information). Z was put equal to +10. Dshell and Dcdl were both put equal to 1.2 × 10-10 m2 s-1, calculated from the protein radius using the Stokes-Einstein equation, in good agreement with literature data.41 Since φshell is low, no corrections for obstruction effects were made. Looking first at the composition of the shell at 40 mM salt, we find that both the protein concentration (φshell) and the polymer molar concentration (1/Vshell) decrease significantly with increasing protein concentration outside the microgel. At the same time, the polymer-to-protein charge ratio (fshell) is fairly invariant and close to unity. At 100 mM salt, fshell is only slightly larger but the protein volume fraction is markedly higher than at 40 mM salt, and display little dependence on the protein concentration. In this case the gels are more contracted in the final state than at 40 mM salt as evident from the r2,∞/R0 ratios. Independent of salt concentration, there is a correlation between the amount of protein in the shell (βshell,∞) and the degree of contraction in the final state. This correlation is directly related to the invariance of fshell and expected since every protein molecule added to the shell contributes to the consumption of the swollen core network. However, rather than increasing δ∞, a larger βshell,∞ is accompanied by a denser shell structure. This conclusion holds for any reasonable variation of δ∞ within the studied samples (see Supporting Information, section S5). Obviously, when the protein molecules aggregate to form denser clusters, higher degrees of binding can be reached before the shell structure is sufficiently rigid to prevent further contraction of the gel. Since the protein volume fraction in the shell is far from close packing, the rigidity is most likely due to aggregation of the protein into a stress-bearing network, and related to the inward motion of all structures in the shell accompanying the deswelling process, resulting in a lateral densification of the protein structure and possibly also percolation. (Evidence for the existence of individual clusters at the initial stages of shell formation was reported in our previous paper.7) The latter would rationalize the observed relationship between the density of the shell structure and the final gel volume. Thus, the size of individual clusters increases faster the more porous the internal protein structure (lower φshell) and therefore start to overlap at a lower degree of gel contraction (i.e., the arrested state is reached at a higher r2,∞/R0).

In dilute homogeneous systems the tendency of colloidal gel formation increases with increasing concentration and magnitude of the attraction between particles.42 In analogy, it appears that the higher the attractive forces between the proteins in the microgel, the lower the concentration required for arresting the shell structure, and the more porous the structure of the aggregates. This can explain our observations that φshell values are consistently lower at 40 mM salt, where the attractive forces are expected to be largest, than at 100 mM salt. At 40 mM, there is also a relationship between φshell and CL2+shell, the mobile protein concentration in the outermost shell layers. In the present gels, where fshell > 1 and is nearly constant, CL2+shell increases with increasing protein concentration in the liquid. A large CL2+shell means a large concentration gradient of mobile protein in the shell and thus a high transport rate of protein to r1. Fast transport means that the protein molecules have less time to rearrange at the site of binding, which is expected to lead to a more open structure. The effect should be less important when the attractive forces are weaker, which may explain the absence of the effect at 100 mM salt. Turning to the core diffusion layer, its composition is little affected by the protein concentration outside the gel. The polymer-to-protein charge ratio (fcdl) is clearly higher than in the shells but about the same for both salt concentrations. The reason why fcdl is higher than in the shells is not clear, but may be related to the network being prohibited from contracting. Under these conditions, when the polymer and protein cannot neutralize each others charge to the same extent as in the shell, the binding is expected to be driven mainly by the gain in entropy of releasing counterions bound to the polyion chains. Then fcdl should be approximately equal to 1 - ξ-1, where ξ is the Manning linear charge density parameter.43 For polyacrylate at 298 K in water, ξ ) 2.85, giving fcdl ) 1.5, in reasonable agreement with the values in Table 1. Although the experimental cdl data are rather scattered (Figure 11) due to the low resolution of the micrographs, the slope of the cdl curves is very sensitive to the value of fcdl, explaining why fcdl is given with three significant numbers in Table 1. 6. Conclusions Poly(acrylic acid) microgels display core-shell formation upon lysozyme uptake, with considerably higher lysozyme concentration in the shell than in the core. Lysozyme incorporation into the microgels takes place in two stages. During the first of these, the lysozyme-microgel shell is formed at rapid deswelling of the microgel, with no lysozyme diffusing into the microgel core. This is followed by a second stage, during which microgel deswelling is negligible and lysozyme diffuses into the microgel core. The latter process is comparable to protein diffusion in nondeswelling ion exchange chromatography particles, both systems displaying faster diffusion through the particles at higher protein concentration, as well as diffusion fronts which are sharp at low ionic strength but diffuse at higher ionic strength. At low ionic strength and high lysozyme concentration, the lysozyme-microgel shell “arrests” the microgel in a state of larger V/V0 value due to lysozyme aggregation in the shell, forming a stress-bearing protein network. These findings are not compatible with a “relay-race” mechanism, in which the protein molecules that are the first to enter the outer part of the microgel also are the first to enter the center. Instead, lysozyme molecules entering into the microgel core diffuse predominantly through the preformed shell.

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