Chain and Pore-Blocking Effects on Matrix Degradation in Protein

Aug 21, 2014 - Lina Nyström , Randi Nordström , Jane Bramhill , Brian R. Saunders , Rubén Álvarez-Asencio , Mark W. Rutland , and Martin Malmsten...
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Chain and Pore-Blocking Effects on Matrix Degradation in ProteinLoaded Microgels Ronja Widenbring,* Göran Frenning, and Martin Malmsten Department of Pharmacy, Uppsala University, P.O. Box 580, SE-751 23 Uppsala, Sweden S Supporting Information *

ABSTRACT: Factors affecting matrix degradation in protein-loaded microgels were investigated for dextran-based microgels, the sugar-binding protein Concanavalin A (ConA), and the dextran-degrading enzyme Dextranase. For this system, effects of enzyme, protein, and glucose concentrations, as well as pH, were considered. Microgel network degradation was monitored by micromanipulator-assisted light microscopy, whereas enzyme and protein distributions were monitored by confocal microscopy. Results show that Dextranase-mediated microgel degradation increased with increasing enzyme concentration, whereas an increased ConA loading in the dextran microgels caused a concentration-dependent decrease in microgel degradation. In the presence of glucose, competitive release of microgel-bound ConA restored the microgel degradation observed in the absence of ConA. To clarify effects of mass transport limitations, microgel degradation was compared to that of non-cross-linked dextran, demonstrating that ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. The experimentally observed effects were qualitatively captured by a modified Michaelis−Menten approach for spherical symmetry, in which network blocking by ConA was included. Taken together, the results demonstrate that matrix degradation of protein-loaded microgels depends sensitively on a number of factors, which need to be considered in the use of microgels in biomedical applications.

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INTRODUCTION Current progress in biotechnology has resulted in a dramatic increase in protein and peptide drugs and drug candidates. However, such biomacromolecular drugs are sensitive to deactivation through aggregation and conformational changes, as well as to chemical and enzymatic degradation. For these drugs to reach a wider therapeutic applicability, appropriate drug delivery vehicles are needed. The role of such delivery systems is to encapsulate the drug and protect it from deactivation or degradation and subsequently to release the drug in a controlled manner. One approach to encapsulate and protect protein and peptide drugs is by using microgels,1−5 lightly cross-linked hydrogel particles. Microgels are able to bind and store proteins and peptides and to release them upon stimuli, in turn related to a capacity for swelling transitions in response to changes in the external environment.3 They therefore offer great potential as drug delivery vehicles. Analogously, microgels offer opportunities as surface coatings when adhered to biomaterials or surgical implants to reduce infection and inflammation and to improve biocompatibility, particularly when containing antimicrobial and anti-inflammatory drugs.2,6−9 For most therapeutic applications, microgels need to be readily biodegradable in order to avoid accumulation-related adverse effects. Through choice of chemistry, biodegradable materials can be designed to degrade according to the requirements of the particular application at hand, from minutes to weeks or longer.10−14 Although drug release in some cases is determined © 2014 American Chemical Society

by carrier degradation rate only, biodegradable systems generally display increased complexity compared to nondegradable ones, as drug release is given by both physicochemical interactions and degradation kinetics and since the presence of the drug may potentially affect the latter. Because of their good biocompatibility, dextran-based microgels have been studied as potential protein and peptide drug carriers.15−17 Dextran consist predominantly of α-1,6 glucosidic linkages that are hydrolyzed by Dextranase enzymes,18,19 thereby generating linear oligosaccharides.15,20−23 Dextranases are found in several organs in the body, such as in the liver, spleen, kidney, and colon. In man, semiquantitative results have demonstrated that liver displays high Dextranase activity, with intermediate activity found in spleen, and low activity in kidney. In the gastrointestinal tract, the highest activty is found in the distant part of the jejunum.24,25 Dextranase is not, however, present in blood to any large extent. Consequently, Dextranase-mediated hydrolysis of dextran has not only attracted interest for controlled and targeted release of drugs from dextran hydrogels17,21,26−30 but also for dextran conjugates with therapeutic agents,24,31,32 for the treatment of dental plaque, in vaccines, and in the cosmetic, detergent, and food industries.15 Despite being widely investigated from an application perspective, systematic Received: July 1, 2014 Revised: August 8, 2014 Published: August 21, 2014 3671

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DP 50 digital camera (Olympus, Tokyo, Japan). Micropipettes (10−20 μm in diameter) were prepared with a PC-10 puller and a MF-9 forger (both Narishige, Tokyo, Japan). Gel particles were captured by micropipette suction using an IM-5A injector (Narishige, Tokyo, Japan), placed inside a 2 mm diameter flow pipet, and flushed with enzyme solution using a Peristaltic pump P-1 (Pharmacia, Uppsala, Sweden) at a flow rate of 1.9 mL/min. This experimental setup ensures that the solute composition surrounding the microgel remains constant and unaffected by solution uptake in gels and enables the solution composition (e.g., enzyme and glucose concentration) to be switched during the run of the experiment. Furthermore, the setup provides laminar flow with a controllable (flow-dependent) unstirred layer thickness, enabling theoretical analysis of diffusion.39 Captured gel particles were photographed and analyzed using Olympus cellSens Dimension (Olympus, Tokyo, Japan). The volume ratio is expressed as V/V0, were V is the volume of a gel particle after buffer or enzyme exposure for a certain time, and V0 is the volume of a gel particle in 10 mM phosphate buffer, pH 7.4, prior to enzyme addition. The volume response of single dextran microgels (90−100 μm in diameter when fully swollen) upon enzymatic degradation was investigated by flushing the microgels with Dextranase solution of 0.1, 0.2, 0.5, or 1 units/mL in phosphate (pH 7.4), acetate (5.4), or carbonate (pH 9.4) buffer until microgels were completely disintegrated. Before exposure to Dextranase, if stated, microgels were equilibrated with 1, 10, or 100 μM ConA solution in phosphate buffer, pH 7.4, for 24 h. The results are presented as the average and standard deviation of deswelling ratios obtained from at least triplicate experiments. Confocal Laser Scanning Microscopy (CLSM). Enzyme Labeling. Dextranase was labeled with Alexa Fluor-488 dye according to a standard protocol recommended by the supplier. In brief, ∼5−10 μg of dye per milligram of enzyme was reacted for 1 h in phosphate buffer, pH 7.4. Unreacted dye was removed by size-exclusion chromatography using PD-10 columns (GE Healthcare, Uppsala, Sweden). The concentration of Alexa 488 was determined by absorbance measurements at 495 nm with a Helios γ 4.60 spectrophotometer (Thermospectronic, Cambridge, UK), and the enzyme concentration was measured spectrophotometrically after complexation with bisinchoninic acid.40 Absorbance measurements were performed on a Saphire plate reader (Tecan, Männedorf, Switzerland) at 562 nm. The labeling density (Alexa/Dextranase molar ratio) thus measured was 0.003 (μM Alexa488/U/mL Dextranase). Protein and Enzyme Distribution. The distribution and intensity of the labeled ConA and Dextranase within the microgel particles were monitored with a confocal Leica DM IRE2 laser scanning microscope (CLSM; Leica Microsystems, Wetzlar, Germany) equipped with an Ar laser, using a 63 × 1.2 water objective and Leica TCS SL software (Leica Microsystems, Wetzlar, Germany). (Note that in contrast to more heavily cross-linked polymer beads, which complicate confocal microscopy due to optical effects,41 these relatively weakly cross-linked microgels display only minor complications of this type.) Ten microliters of microgel solution (0.1 w/w %) was equilibrated for 24 h with 200 μL of either 1, 10, or 100 μM ConA/Alexa Fluor 633 conjugate (ConA-633). To investigate enzymatic degradation of single gel particles, 20 μL of ConA-microgel solution was vortexed with 750 μL of Dextranase solution of 100 U/mL for 10 s at 1400 rpm. The solution was then transferred into a confocal microscopy cuvette, and a gel particle was chosen for analysis. For glucose experiments, 20 μL of ConA−microgel solution was equilibrated with 750 μL of 0 or 40 mM glucose in phosphate buffer for 30 min on a shaking board, followed by analysis. To evaluate the average fluorescence intensity in the microgels, region of interest (ROI) analysis was performed. Enzyme Assay. Dextranase activity on non-cross-linked dextran was monitored by measuring the amount of released dye complex (Cibacron Blue20) from Blue Dextran 2000, using the method of Koh and Khouw19 with slight modifications. The reaction was started by the addition of one volume part of Dextranase solution to one volume part of freshly prepared blue dextran (1% w/v) to a final enzyme concentration of either 0.1, 0.2, 0.5, or 1 units/mL and was left on a shaking board at room temperature for the specified time. The reaction was terminated by the addition of four volume parts of ice-cold ethanol (99.7%), followed by

studies of factors affecting matrix degradation in dextran (and other) microgels and related systems, particularly in the presence of a protein load, are surprisingly sparse. In a previous investigation, effects of network properties on enzymatic degradation of encapsulated peptides in nondegradable microgels were investigated.33 Shifting perspective, the present study instead outlines how protein loading affects the degradation of the polymeric matrix in microgels. Because of its wide application interest in protein drug delivery, dextran− Dextranase was chosen as a suitable matrix and enzyme, respectively. Additional advantages offered by this combination is that Dextranase degrades only the matrix and not incorporated proteins. Furthermore, it can be combined with Concanavalin A (ConA) as a model protein, which provides high-affinity binding with the dextran matrix to obtain more pronounced effects of matrix blocking, as well possibilities for competitive release by addition of glucose, which in itself does not affect the matrix network. Using this system, the effects of enzyme, protein, and glucose concentration was investigated, together with those of pH-dependent enzyme inactivation. Microgel network degradation/deswelling were monitored by micromanipulator-assisted light microscopy, whereas Dextranase and ConA distribution were monitored through confocal microscopy. Experimental results were compared to those from a modified Michaelis− Menten approach for spherical symmetry, in which network blocking by ConA was included. To demonstrate effects of confinement and mass transport limitations, degradation of dextran microgels was furthermore compared to that of noncross-linked dextran. Taken together, the results demonstrate that matrix degradation of protein-loaded microgels depends sensitively on a number of factors, which need to be considered in the use of microgels in biomedical applications.

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EXPERIMENTAL SECTION

Materials. Dextran microgels/microparticles, Sephadex G-200 (Particle size: 40−120 μm), were obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden). Although being more highly crosslinked and less responsive than many traditional microgels, these are nevertheless highly swollen and water-rich and are therefore referred to as microgels throughout for convenience. The pore size distribution of Sephadex G-200 is quite wide, separating dextran in the molecular weight range of 1000−200 000 Da,34 corresponding to a radius of gyration of ≈6−15 nm.35−37 Dextran microgels were suspended in phosphate buffered saline (PBS) buffer (10 mM, 140 mM NaCl, 3 mM KCl, pH 7.4), prepared from PBS tablets from EC Diagnostics AB (Uppsala, Sweden), to the final microgel concentration of 0.1 w/w % and left to swell for at least 72 h on a shaking board prior to use. Dextranase from Penicillium sp. (product no. D8144) and Concanavalin A (ConA) from Canavalia ensiformis (Jack bean) Type VI (product no. L7647) were from Sigma-Aldrich (Schnelldorf, Germany). Alexa Fluor488 carboxylic acid succinimidyl ester, mixed isomer, and Concanavalin A/Alexa Fluor 633 conjugate (product no. C21402) were from Invitrogen (Eugene, OR, USA), and the bisinchoninic acid (BCA) assay kit was from Pierce (Rockford, USA). D-Glucose was obtained from Amresco (Solon, USA), and Blue Dextran 2000 (Mw ≈ 2000 kDa) was from GE Healthcare (product no. 17-0360-01). All other chemicals were of analytical grade. Purified Milli-Q water was used throughout. For pH control, acetate (10 mM acetate buffer, 140 mM NaCl, pH 5.4), phosphate (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, pH 7.4), or carbonate (10 mM carbonate buffer, 140 mM NaCl, pH 9.4) buffer was used. Enzyme-Induced Microgel Degradation/Deswelling. Changes in microgel volume upon enzyme degradation were monitored by micromanipulator-assisted light microscopy, as described previously,38 using an Olympus Bx-51 light microscope (Olympus, Tokyo, Japan) equipped with an ONM-1 manipulator (Narishige, Tokyo, Japan) and a 3672

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immersing the solution in an ice bath for 15 min. After centrifugation at 8000 g for 25 min, the absorbance was read at 631 nm with a Helios γ 4.60 spectrophotometer (Thermospectronic, Cambridge, UK) against a blank from which Dextranase was omitted. The rate of conversion of dextran-bound Cibacron Blue dye (ethanol-insoluble) to an ethanolsoluble form20 on dextran digestion by Dextranase19 could thus be measured, and the amount of released dye was set equivalent to the degree of activity. Analysis of Gel Degradation. Background. Various models have been used to describe enzymatic degradation of polymer gels, gel particles, and related systems. For example, Nyvall Collén et al. investigated degradation of κ-carrageenan variants by κ-carrageenase as a function of iodide binding.42 The latter induces an order transition of κcarrageenan but also causes direct blocking of the polysaccharide chains for enzymatic degradation. Analysis of these effects were performed at the Michaelis−Menten (MM) level and discussed in terms of an iodidedependent decrease of enzyme−substrate complexes.42 Expanding from the single-component MM analysis, Franssen et al. studied the enzymatic degradation of intramolecularly polymerized methacrylated dextran and of methacrylated dextran hydrogels.22 Experimental degradation rates were interpreted with a multiple substrate model, in which unsubstituted, monosubstituted, and multiply substituted chains have different degradation rates and MM constants.22 Somewhat related, Mahammad et al. developed a MM-based model for enzymatic degradation of guar galactomannan.43 Effect of branching was accounted for by describing guar as a substrate consisting of three types of bonds with different MM kinetic parameters. Degradation was assumed to occur by random scission of the three types of bonds, although allowing also nonrandom scission through rate constants depending on both chain length and the position of the hydrolyzable bond in the chain. Using this model, it was demonstrated that the enzymatic reaction of this system follows zero-order MM kinetics and that polydispersity increases with time. Subsequently, polydispersity decreases and the degradation obeys first-order kinetics.43 Also addressing the issue of reaction order, Cheng et al. investigated enzymatic degradation of guar galactomannan.44 In dilute polymer solutions, reaction rate was found to follow MM kinetics, initially displaying first-order kinetics with substrate concentration but saturating enzyme/polymer binding at intermediate concentrations, resulting in zero-order degradation. At yet higher polymer concentrations, precluded enzyme diffusion reduces degradation. A scaling-based reaction−diffusion model was proposed to incorporate both enzyme reaction and diffusion effects, allowing polymer concentration regimes to be identified for substrate-limited, enzyme-limited, and diffusion-limited reactions.44 Mass transport effects on degradation were described also by Vernerey et al., although for cellmediated enzymatic degradation of hydrogels.45 On the basis of MM kinetics and models for mixtures and rubber elasticity, the approach taken allows for coupling among gel mechanical properties, enzyme diffusion, and degradation kinetics. The occurrence of two degradation regimes was demonstrated, in which degradation is dominated by either diffusion or reaction kinetics.45 Moving to enzymatic degradation of more complex substrates, Tzafriri et al. reported on a reaction−diffusion model for enzymatic degradation of fibrillar matrices.46 Steric hindrance was incorporated by considering the fibrillar gel as a solid porous network. The fibrils, in turn, were assumed to consist of cylinders of tightly packed monomeric rods. When in contact with the enzyme solution, enzyme molecules diffuse into the gel and bind to monomers located at the fibril surface, but they are unable to enter into the tightly packed individual rods due to their size. Once a monomer at the surface of a fibril is cleaved, it detaches and goes into solution. These authors were furthermore able to demonstrate the instantaneous diffusion limit to be roughly valid for previously reported experiments on collagenasemediated erosion of fibrillar collagen, allowing an estimate of the MM constant of that system. Using this estimate to simulate additional erosion experiments subsequently allowed elucidation of the role of diffusion in the enzymatic degradation of this system.46 Model. An analysis of simultaneous enzyme diffusion and gel degradation results in a nonlinear reaction−diffusion problem with a moving boundary. For this reason, a simplified analysis is presented, in which degradation is assumed to be confined to the gel surface and the

erosion rate is assumed to be proportional to the concentration of enzyme−substrate complexes at the surface. The starting point is the standard Michaelis−Menten scheme (1)

E+S⇌C→E+P

in which the enzyme E combines with its substrate S to form a complex C that irreversibly degrades the substrate, thereby forming a degradation product P and releasing the enzyme. The equations corresponding to the MM reaction Scheme 1 take the form dS = − k1ES + k −1C dt

(2)

dC = + k1ES − (k −1 + k 2)C dt

(3)

where S, C, and E represent the substrate, complex, and free enzyme concentrations, respectively, and k1 is the rate of complex formation, k−1 is the rate of the reverse process, and k2 is the rate of product formation. Defining the total substrate concentration T = S + C and adding eqs 2 and 3, one obtains dT = − k 2C dt

(4)

Hence, the rate of degradation is proportional to the complex concentration. The enzyme concentration is kept fixed at a certain value E0 at the surface of the gel. When gel degradation occurs at the surface only, the total substrate concentration will also remain fixed at a certain value T0. Hence, eq 3 implies that dC = + k1E0(T0 − C) − (k −1 + k 2)C dt = + k1[E0T0 − (E0 + K m)C ]

(5)

at the gel surface, where Km = (k−1 + k2)/k1 is the MM constant. Equating the right-hand side of eq 5 to zero, the maximal complex concentration is obtained as Cmax = E0T0/(E0 + Km). Since the complex concentration is zero initially, integration of eq 5 yields

C = Cmax[1 − e−κ1t ]

(6)

where κ1 = k1(E0 + Km). The erosion rate dR/dt is assumed to be proportional to the complex concentration at the surface dR = − k 3C dt

(7)

where k3 is a proportionality constant. Combining eqs 6 and 7, one obtains

R − R 0 = −k3

∫0

t

C dt = −

k 3Cmax (κ1t + e−κ1t − 1) κ1

(8)

Since the gel has a spherical shape, its relative volume change can be expressed as

⎤3 ⎛ R ⎞3 ⎡ kC V = ⎜ ⎟ = ⎢1 − 3 max (κ1t + e−κ1t − 1)⎥ κ1R 0 V0 ⎣ ⎦ ⎝ R0 ⎠

(9)

Inserting the expressions for κ1 and Cmax, the final equation takes the form ⎫3 ⎧ k 3E0T0 V −k1(E0 + K m)t ⎬ [ k ( E K ) t e 1] = ⎨1 − + + − 1 0 m V0 k1R 0(E0 + K m)2 ⎭ ⎩

(10) For the calculations of the gel degradation in the absence of ConA, the rate constants k1 and k3 were estimated from the experimental result for an enzyme concentration of 1 U/mL. The MM constant Km was set equal to 0.2 U/mL, as suggested by the results in Figure 7a. However, 3673

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Dextranase (Mw = 41 kDa47), which is supported also by CLSM results in Figure 4. The importance of surface-localized degradation was reported also by Ghugare at al. for dextran methacrylate microgels.16 Quantitatively, increasing the enzyme concentration 10-fold from 0.1 to 1 U/mL results in an approximately 3-fold faster time until complete microgel disintegration. In comparison, Dextranase degradation of noncross-linked dextran in solution displayed an essentially 10-fold increase in activity with a 10-fold increase in enzyme concentration (Figure S1). Clearly, therefore, there are access limitations for Dextranase to degrade dextran microgels, suggesting the importance of surface confinement. Apart from previous results by Ghugare et al. showing that degradation of dextran-based microgels depends upon the relative enzyme amount,16 increased degradation with increasing Dextranase concentration has previously been reported for dextran macrogels by Franssen et al., Hennink et al., and Moriyama and Yui.21,26,27 Effects of ConA Concentration. Next, the effects of ConA concentration was investigated by CLSM. As shown in Figure 2, an increased protein concentration results in an increased loading in the dextran microgels, in analogy to previous findings by Chern et al. for dextran-modified latex particles.48 Intensity profiles in Figure 2a show that ConA binds throughout the dextran microgels, possibly with a slightly higher binding of ConA at the outer regions of the microgel compared to binding in the core, although optical effects cannot be fully excluded as contributing to the minor intensity minimum observed at the microgel core.41 As shown in Figure 3, microgel degradation decreases with an increasing ConA load in the microgels. These results suggest that ConA binding restricts Dextranase to enter the dextran network, followed by suppressed matrix degradation restrictions from dextran-bound ConA, in which bound ConA effectively protects the dextran chains from degradation through substrate access limitations. Further supporting this, Figure 4 shows that increased ConA loading results in decreased Dextranase microgel binding, and that bound Dextranase in ConA-loaded microgels is largely confined to their outer region. In analogy, incorporation of ConA in dextran macrogels has previously been observed to result in a tighter network structure due to induced carbohydrate linkages by the tetravalent ConA.49 Thus, while Dextranase degradation of non-cross-linked dextran shows no substrate access restrictions from the enzyme

Figure 1. (a) Enzymatic degradation of dextran microgels at different Dextranase concentrations in 10 mM phosphate buffer, pH 7.4. Also shown are results for microgels exposed to buffer solution without enzyme. (b) Exemplifying light microscopy images taken at different stages of the degradation of dextran microgels upon addition of 0.2 U/ mL Dextranase in 10 mM phosphate buffer, pH 7.4. the model results were relatively insensitive to the value of Km as long as sufficiently small values were used. Finally, the effect of chain blocking was incorporated in the model in a simplified manner by reduction of T0. This results in a corresponding reduction of the maximal complex concentration Cmax without affecting the form of eq 7 since any explicit dependence of the erosion rate on T0 has been subsumed in the constant k3.

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RESULTS AND DISCUSSION Effects of Dextranase Concentration. Effects of enzymatic degradation of dextran-based microgels were first determined through micromanipulator-assisted light microscopy. As shown in Figure 1a, dextran microgels are degraded by Dextranase in a dose-dependent manner. Exemplifying light microscopy images in Figure 1b furthermore show that the microgel becomes gradually more diffuse with time, indicating that the degradation is not exclusively affecting the surface. Having said that, matrix degradation is expected to be particularly important at the microgel surface or outer region due to the relatively large size of

Figure 2. (a) CLSM images and corresponding intensity profiles displaying the distribution of 1, 10, and 100 μM ConA-633 in microgels after equilibration with labeled protein solution for 24 h in 10 mM phosphate buffer, pH 7.4, i.e., in the absence of Dextranase. (b) Effect of protein concentration on the mean fluorescence intensity from at least 10 separate gels. 3674

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Effects of Competitive ConA−Glucose Binding. Since ConA has a higher binding affinity for glucose than for dextran, Dglucose is an interesting candidate for competitive displacement of dextran-bound ConA.50−53 As shown by CLSM experiments in Figure 5, preadsorbed ConA can indeed be released from dextran microgels by addition of glucose. As a result of the decreased ConA loading in the presence of glucose, degradation of ConA-loaded dextran microgels increased (Figure 6). The higher the glucose concentration, the faster the Dextranase induced degradation. Interestingly, a small initial swelling of the microgel can be seen when glucose is added, which is not seen for experiments without glucose (Figure 3). As observed previously in related systems, this is likely to be a result of gel swelling due to specific replacement of ConA cross-links.49,51,54 Once degradation causes the microgel volume to decrease after the small initial swelling, the degradation pattern is more similar to that of unloaded microgels (Figure 1a) than to that of ConA-loaded ones (Figure 3), supporting the conclusion that the initial swelling is caused by competitive release of microgel-bound ConA. As seen in Figure 6, increased glucose concentration also results in a decreased time until complete microgel disintegration. Partly, this is expected to be due to an increase in mesh size of the gel network with increasing glucose concentration due to displacement of ConA cross-links, in turn leading to increased enzyme accessibility.54 In addition, glucose-induced ConA release results in bare dextran chains, hence increasing substrate accessibility, in turn causing an increased degradation rate. As previously demonstrated, dextran microgels combined with ConA can consequently be used for glucose-responsive drug delivery, e.g., in relation to self-regulated insulin delivery.49,55,56 Comparison between Dextran Microgels and NonCross-Linked Dextran. In order to obtain further information on mass transport limitations in dextran microgel degradation, as well as on the relative importance of mesh shrinkage and single chain blocking caused by ConA, a series of experiments was performed in which Dextranase-induced degradation of dextran microgels was compared to that of non-cross-linked dextran (Mw ≈ 2000 kDa). As shown in Figure 7a, degradation of non-crosslinked dextran scales monotonically with Dextranase concentration, as expected if all binding sites at the substrate remain accessible throughout the degradation process, and products are efficiently diffusing away to avoid reaction poisoning. In contrast, dextran microgels displays a quantitatively much weaker dependence on Dextranase concentration, demonstrating mass transport limitations for the degradation, either through restrictions in Dextranase penetration through the entire microgel network and access to dextran chains, through restrictions in product diffusion away from the microgel, or both. In the presence of ConA, CLSM demonstrates accentuated restrictions for Dextranase penetration throughout the dextran microgels (Figure 4), suggesting pore blocking. At the same time, however, ConA may preclude degradation also by its simple binding to the dextran chains, effectively blocking these for Dextranase scission. In order to clarify the relative importance of these two blocking effects of ConA, degradation experiments were performed with non-cross-linked dextran. As shown in Figure 7b, the degradation of both cross-linked and non-crosslinked dextran decreases with increasing ConA concentration; however, a significantly smaller obstruction by ConA was found for non-cross-linked dextran. Since non-cross-linked ConA only allows direct blocking of the dextran chain, the weaker dependence on the ConA concentration for the non-crosslinked dextran shows such direct chain blocking to be relatively

concentration (Figure S1), void dextran microgels show clear signs of such limitations (Figure 1a), an effect further accentuated in the presence of ConA (Figure 3).

Figure 3. (a) Enzymatic degradation of ConA-loaded and void dextran microgels at 0.2 U/mL Dextranase in 10 mM phosphate buffer, pH 7.4. Before exposure to Dextranase, microgels were equilibrated with either (□) buffer or (▲) 1 μM, (◊) 10 μM, or (●) 100 μM ConA solution for 24 h. (b) Enzymatic degradation of ConA-loaded dextran microgels at different Dextranase concentrations in 10 mM phosphate buffer, pH 7.4. Before exposure to Dextranase, microgels were equilibrated with 10 μM ConA solution for 24 h. Also shown are results for ConA-loaded gels exposed to pure buffer solution without enzyme. For clarity, error bars at each time point were omitted in both figures a and b, and standard deviation is instead shown at the last point.

Figure 4. CLSM images and corresponding intensity profiles for 1, 10, and 100 μM ConA-633 in microgels, displaying the distribution of ConA-633 (red, top) and Dextranase-488 (green, bottom) at 10 min after mixing with enzyme solution of 100 U/mL in 10 mM phosphate buffer, pH 7.4. Before exposure to labeled Dextranase, microgels were equilibrated with labeled protein solution (ConA-633) for 24 h. 3675

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Figure 5. (a) CLSM images and corresponding intensity profiles displaying the distribution of ConA-633 in dextran microgels after 30 min of equilibration with either 0 or 40 mM glucose solution. Before exposure to glucose, microgels were equilibrated with labeled ConA solution (10 μM ConA-633) for 24 h in 10 mM phosphate buffer, pH 7.4. (b) Mean fluorescence intensity from at least 10 separate gels after 30 min of equilibration with either 0 or 40 mM glucose solution. For 40 mM glucose solution, no intensity was detected.

Figure 6. Enzymatic degradation of protein-loaded dextran microgels at 0.2 U/mL Dextranase in 10 mM phosphate buffer, pH 7.4, at different glucose concentrations. Before exposure to Dextranase, microgels were equilibrated with 10 μM ConA solution for 24 h.

minor. Instead, ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. Although the cross-linked nature of the dextran microgels thus precludes enzymatic degradation, it may also result in degradation-promoting effects under special circumstances. As seen in Figures 7c and S2, Dextranase activity is completely lost at pH 9.4 for both cross-linked and non-cross-linked dextran, an effect due to Dextranase undergoing a transition from its native conformation to an unfolded state at pH ≈ 8.57 At pH 7.4, however, there is a striking difference between the dextran microgels and non-cross-linked dextran, the degradation of the latter, but not of the former, being much suppressed compared to that at pH 5.4. This may be taken as an indication of the conformational transition under weakly alkaline conditions having been initiated already at pH 7.4. In such a scenario, the higher remaining activity observed for the dextran microgel is due to conformational stabilization of the enzyme, as previously reported for other proteins incorporated in both microgels and

Figure 7. Comparison of Dextranase degradation of dextran microgels with that of non-cross-linked dextran. (a−c) Relative degradation rate as a function of Dextranase concentration, ConA concentration, and pH, respectively. The relative degradation rate was obtained by normalizing the degradation rate (the degradation rate is set equivalent to the degree of activity and to the inverted time to reach V/V0 = 0.6 for non-crosslinked and cross-linked, respectively) with that at 0.1 U/mL, in the absence of ConA, and at pH 5.4, respectively.

other porous materials.2,58 It should also be remembered, however, that the net charge of Dextranase increases with pH above its IEP (IEP = 4.647). Therefore, conformational and translational entropy penalty on enzyme binding, in combination with a weakly repulsive net electrostatic interaction, prevents Dextranase from binding to non-cross-linked dextran at pH 7.4. For dextran microgels, however, the entropic penalty on enzyme 3676

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CONCLUSIONS Qualitatively similar to the non-cross-linked system, but partially restricted by mass transport limitations, degradation of dextran microgels increases with Dextranase concentration. With time, particularly the outer region of the microgel is continuously degraded/dissolved, resulting in a microgel volume decrease. ConA binds throughout the dextran microgels, resulting in decreased network degradation in a loading-dependent manner. Comparison of microgel degradation to that of non-cross-linked dextran demonstrated that ConA limits enzyme substrate access in dextran microgels primarily through pore blocking and induction of pore shrinkage. Presence of glucose in the buffer competes with dextran for ConA binding, causes a concentration-dependent decrease of microgel-bound ConA, and restores matrix susceptibility to Dextranase degradation. A modified Michaelis−Menten approach for spherical symmetry, in which network blocking by incorporated protein was included, was able to qualitatively capture these effects. Although ConA is used only as a model protein to clarify basic effects of chain and pore blocking on matrix degradation of protein-loaded microgels, analogous effects could be anticipated for a wide range of therapeutically interesting lectins, including mannose-binding lectins involved in the immune system.54 In addition, as the reduced matrix degradation is mainly an effect of loadingdependent substrate inaccessibility and pore blocking, analogous effects could be expected for any microgel loaded with a protein/ peptide with high binding affinity to the polymer matrix. Having said that, design of such therapeutically more interesting delivery systems also requires consideration of various applicationspecific factors, including microgel size and cross-linking density, as well as loading degree, depending also on requirements on administration route (intravenous, oral, pulmonary, etc.), dose, release kinetics, and other factors.

Figure 8. Calculations from the modified Michaelis−Menten approach for spherical symmetry, allowing blocking by incorporated protein. (a, b) Effects of enzyme concentration and chain blocking, respectively. In figure b, the enzyme concentration was set to 0.2 U/mL. (c, d) Calculations from model computation were compared to those observed experimentally, normalizing degradation rate at the lowest Dextranase and ConA concentration investigated, respectively.

binding is much lower due to the presence of cross-links in the microgels, and van der Waals interactions (although weak in this system) may provide an additional driving force for complexation between dextran and Dextranase, a prerequisite for dextran hydrolysis. Apparently, these different factors are very close to balance at pH 7.4, resulting in the observed higher degradation for the cross-linked dextran microgels under these special circumstances. Model Calculations. Most of these experimental observations are qualitatively captured by a simple Michaelis−Menten model for spherical symmetry, in which chain blocking by loaded protein is incorporated. Thus, as shown in Figure 8a, increasing Dextranase concentration results in an increased degradation rate. In the presence of blocking, degradation is suppressed, an effect increasing with increasing ConA concentration (Figure 8b). Given simplifying assumptions in the model employed, notably regarding the degradation process being confined to the surface region only and dextran chain degradation products not suffering from exceedingly slow diffusion away from the degradation zone, care should be taken not to push the quantitative analysis too far. Having said that, it should still be noted that the Dextranase concentration dependence is comparable to that observed experimentally (Figure 8c). With a blocking of about 60% at the highest ConA concentration investigated, the ConA concentration dependence from the model calculations similarly tracks that observed experimentally (Figure 8d). Together, these findings give further support for the importance of Dextranase diffusion limitations within the microgels, as well as pore blocking effects of ConA.

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ASSOCIATED CONTENT

* Supporting Information S

Dextranase activity as a function of enzyme concentration, time, and pH. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Lise-Britt Wahlberg is gratefully acknowledged for technical support with activity measurements and Pia Krö ger for performing early trial experiments on these systems. This work was financed by the Swedish Research Council.

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