Kinetics of Swelling of Polyether-Modified Poly(acrylic acid) Microgels

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Kinetics of Swelling of Polyether-Modified Poly(acrylic acid) Microgels with Permanent and Degradable Cross-Links Lev Bromberg,† Marina Temchenko,† Valery Alakhov,‡ and T. Alan Hatton*,† Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Supratek Pharma, Inc., 531 Boulevard des Prairies, Building 18, Laval, Quebec, Canada H7V 1B7 Received August 23, 2004. In Final Form: November 11, 2004 Spherical particles of 50-100 µm size composed of poly(acrylic acid) networks covalently bonded to Pluronic polyether copolymers were tested for swelling in aqueous media. The microgels were cross-linked either by permanent ethylene glycol dimethacrylate (EGDMA) cross-links alone or by EDGMA together with reversible disulfide or biodegradable azoaromatic cross-links. Optimum conditions for a rapid, diffusionlimited swelling of the pH- and temperature-sensitive microgels with nondegradable cross-links were found. The microgels cross-linked by disulfide groups and equilibrium-swollen in the buffer solution exhibited degradation-limited kinetics of swelling under physiological conditions, with a first-order reaction constant, k1, linearly proportional to the concentration of reducing agents such as dithiotreitol and tris(2-carboxyethyl)phosphine (TCEP). A severalfold faster swelling in the presence of more powerful reducing agent, TCEP, was observed, indicating the chemical specificity of the microgel swelling. The reoxidation of the thiol groups into disulfide cross-links by sodium hypochlorite led to the restoration of the microgels’ diameter measured prior to the reduction-reoxidation cycle, which confirms the shape memory of the microgels. Enzymatically degradable azoaromatic cross-links enabled slow microgel swelling due to degradation of the cross-links by azoreductases from the rat intestinal cecum. The low rate of swelling of the Pluroniccontaining microgels can enable sustained drug release in colon-specific drug delivery.

Introduction Hydrogel particles of submillimeter size (microgels) that are capable of changing their volume many times in response to changes in their aqueous environment are gaining prominence as stimuli-responsive materials in diverse areas such as drug delivery and microelectromechanical devices, biorecognition-based separation media, and sensors.1-6 The stimulus-induced volume transitions can be triggered by changes in the three major components of the osmotic pressure that dictate gel swelling: the elastic energy of the cross-links (Πel), the polymer-solvent mixing free energy (Πm), and, in cases when the gel is ionized, the translational energy of the counterions (Πion).7 By altering the hydrophobicity or charge density of the gel components, affecting Πm or Πion, respectively, one can design temperature-sensitive and pH- and ion-sensitive gels. We have recently introduced such a family of hybrid microgels that comprise a poly(acrylic acid) (PAA) network onto which neutral, amphiphilic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic) chains are attached.8-10 Because ionization of such Pluronic-PAA * To whom correspondence should be directed: phone, 617 2534588; fax, 617 253-8723; e-mail, [email protected]. † Massachusetts Institute of Technology. ‡ Supratek Pharma, Inc. (1) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1-33. (2) Cao, R.; Gu, Z.; Patterson, G. D.; Armitage, B. A. J. Am. Chem. Soc. 2004 126, 726-727. (3) Cao, R.; Gu, Z.; Hsu, L.; Patterson, G. D.; Armitage, B. A. J. Am. Chem. Soc. 2003, 125, 10250-10256. (4) Murthy, N.; Xu, M.; Schuck, S.; Kunisawa, J.; Shastri, N.; Frechet, J. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4995-5000. (5) Plunkett, K. N.; Kraft, M. L.; Yu, Q.; Moore, J. S. Macromolecules 2003, 36, 3960-3966. (6) Plunkett, K. N.; Moore, J. S. Langmuir 2004, 20, 6535-6537. (7) Shibayama, M.; Tanaka, T. Adv. Polym. Sci. 1993, 109, 1-62. (8) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2002, 18, 4944-4952.

microgels is pH-dependent, they swell and collapse dramatically in response to changes in pH, and the presence of poly(propylene oxide) (PPO) segments that have temperature-dependent water solubility renders them temperature-responsive. The benign nature of such microgels and their ability to adhere to mucosal surfaces11 and solubilize and stabilize drugs8,9 make them a convenient vehicle for pharmaceutical dosage forms such as tablets or capsules, in oral drug delivery.12 At the low pHs found in the stomach, the microgels are in a collapsed state and, thus, can protect the loaded drug from degradation, but as the dosage form passes down the gastrointestinal tract (GIT), where pHs are significantly higher, swelling increases due to water diffusion into the gel. This swelling serves to lower Πion, which arises because of the ionization of the carboxylic groups of PAA at these pHs. The diffusion and release rates of the drug increase in the swollen gel. In contrast to our previous works where we relied on changing Πm and Πion to effect these changes in swelling behavior, herein we explore the volume transitions that arise from changes in Πel through controlled, in situ changes in the microgel cross-link density. Specifically, we show that the introduction of disulfide cross-links that can be degraded by reduction to thiols, and can be restored by subsequent reoxidation, provides control over the swelling properties of these Pluronic-PAA gels, and imparts certain shape memory properties to them. We also exploit the observation that azo-cross-links in hy(9) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2003, 19, 8675-8684. (10) Bromberg, L.; Temchenko, M.; Moeser, G. D.; Hatton, T. A. Langmuir 2004, 20, 5683-5692. (11) Bromberg, L.; Temchenko, M.; Alakhov, V.; Hatton, T. A. Int. J. Pharm. 2004, 282, 45-60. (12) Alakhov, V.; Pietrzynski, G.; Patel, K.; Kabanov, A. V.; Bromberg, L.; Hatton, T. A. J. Pharm. Pharmacol. 2004, 56, 1233-1241.

10.1021/la047893j CCC: $30.25 © 2005 American Chemical Society Published on Web 01/20/2005

Kinetics of Swelling of Microgels

drogels can be cleaved by azoreductases present in the colon13-20 to develop azobenzene-cross-linked biodegradable Pluronic-PAA hydrogels with slow swelling kinetics, which is desirable for site-specific drug delivery applications. These gels contain a small fraction of permanent cross-links such that they do not dissolve completely, which is important to ensure that they are not absorbed by the intestinal walls and are effectively cleared by passage through the GIT itself.

Langmuir, Vol. 21, No. 4, 2005 1591 Scheme 1. Chemical Structures of Pluronic-PAA Microgels and Cross-Linkers Copolymerized with the Vinyl Monomers

Experimental Section Materials. Nonionic copolymers Pluronic F127 NF and L92 were obtained from BASF Corp. and used without further treatment. Acrylic acid (99%), ethylene glycol dimethacrylate (EGDMA, 98%, CAS 97-90-5), N,N′-bis(acryloyl)cystamine (BAC, g97%, CAS 60984-57-8), 4-aminoacetanilide (99%), 4-allyl-2methoxyphenol (eugenol, 99%), methacryloyl chloride (98+%), benzyl viologen (97%), dithiothreitol (DTT, 99%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), sodium hypochlorite solution (available chlorine, 13%), Ninhydrin Reagent solution, glycine hydrochloride (99%), and all other fine chemicals were purchased from Sigma-Aldrich Chemical Co. and used as received. All other solvents, buffers, and gases were obtained from commercial sources and were of the highest purity available. Microgel Synthesis. Microgels composed of PAA and Pluronics were synthesized by emulsion/dispersion free-radical polymerization as described in detail previously.8-10 The weight ratio of Pluronic to poly(acrylic acid) in all of the microgel particles was 45:55. In the present work, we employed microgel particles with a diameter of 50-100 µm in the dry state (d0). The molar ratio of the cross-linkers to acrylic acid set in the reaction mixture designates the degree of cross-linking of the microgels (XL, mol % ) 100 × (number of moles of cross-linker/number of moles of acrylic acid)) in what follows. In the microgels cross-linked by EGDMA only, XL varied from 0.01 to 1.0 mol %, while, for microgels cross-linked by both EGDMA and BAC, XL was fixed at 0.1 and 1.0% for EGDMA and BAC, respectively. To confirm the incorporation of BAC into the microgel structure, a control fraction of the microgels was reduced by excess sodium borohydride following argentometric titration as described elsewhere.21 There was no significant discrepancy between the calculated and measured contents of the thiol groups. The presence of a small permanent cross-linking by EGDMA in the microgels containing BAC ensured that the microgel did not dissolve completely upon reduction of the disulfide bonds, as described below. The chemical structures of the Pluronic-PAA microgels and the cross-linkers used in this work are presented in Scheme 1. Synthesis of Microgels with Azoaromatic Cross-Links. The azoaromatic cross-linker, 4,4′-di(methacryloylamino)azobenzene, was synthesized by reaction of 4,4′-diaminoazobenzene with methacryloyl chloride as described by Brøndsted.14,22 The precursor, 4,4′-diaminoazobenzene, was obtained from 4-aminoacetanilide as described in detail elsewhere.23 Then the 4,4′diaminoazobenzene (4.25 g, 20 mmol) was dissolved in 0.1 wt % eugenol solution in pyridine resulting in a 10 wt % solution. (13) Pr´adny, M.; Kopecˇek, J. Makromol. Chem. 1990, 191, 18871897. (14) Brøndsted, H.; Kopecˇek, J. Biomaterials 1991, 12, 584-592. (15) Yeh, P. Y.; Kopecˇkova´, P.; Kopecˇek, J. J. Polym. Sci., Polym. Chem. 1994, 32, 1627-1637. (16) Yeh, P. Y.; Kopecˇkova´, P.; Kopecˇek, J. Macromol. Chem. Phys. 1995, 196, 2183-2202. (17) Yeh, P.-Y.; Berenson, M. M.; Samowitz, W. S.; Kopecˇkova´, P.; Kopecˇek, J. J. Controlled Release 1995, 36, 109-124. (18) Ghandehari, H.; Kopecˇkova´, P.; Kopecˇek, J. Biomaterials 1997, 18, 861-872. (19) Shantha, K. L.; Ravichandran, P.; Rao, K. P. Biomaterials 1995, 16, 1313-1318. (20) Van den Mooter, G.; Maris, B.; Samyn, C.; Augustijns, P.; Kinget, R. J. Pharm. Sci. 1997, 86, 1321-1327. (21) Wulff, G.; Schulze, I. Isr. J. Chem. 1978, 17, 291-297. (22) Brøndsted, H. Hydrogels for colon-specific peptide drug delivery, Ph.D. Thesis, University of Utah, 1991. (23) Alva, K. S.; Lee, T.-S.; Kumar, J.; Tripathy, S. K. Chem. Mater. 1998, 10, 1270-1275.

Methacryloyl chloride (5.9 mL, 60 mmol) was added dropwise into the solution under stirring, and the solution was kept at 60 °C under stirring for 1 h. Eugenol addition prevented a possible premature free-radical polymerization of methacryloyl chloride. The solution was then poured into 100 mL of chilled (4 °C) deionized water, and the resulting mixture was acidified to pH 4 by addition of concentrated HCl. The precipitate was filtered under vacuum, washed with excess aqueous 5% NaHCO3 and deionized water, dried, and repeatedly recrystallized from ethanol. The chemical structure of 4,4′-di(methacryloylamino)azobenzene was confirmed by NMR (1H NMR (400 MHz, pyridined5): δ, 5.55 (d, dCH2), 5.87 (d, dCH2), 1.96 (s, H3C-Cd), 7.85 (q, H4C6) ppm) and elemental analysis (Anal. found (calculated): C, 68.83 (68.95); H, 5.81 (5.79); N, 15.89 (16.08)). Microgels with azobenzene cross-links cleavable by azoreductases from the rat intestine microflora were synthesized by free-radical copolymerization of acrylic acid and 4,4′-di(methacryloylamino)azobenzene with simultaneous grafting of Pluronic chains on the forming network of poly(acrylic acid), in a freeradical procedure analogous to that described previously.8,9 In the Pluronic-PAA microgels, the cross-linking ratio, XL, was set at 0.1 and 1.0 mol % for the EGDMA and azoaromatic (Azo) cross-links, respectively. Using analogous procedures, but without Pluronic, a batch of poly(acrylic acid) microgels cross-linked by the azoaromatic cross-linker was also synthesized (designated PAA-Azo). The cross-linking ratio, XL, was set at 0.1 and 0.56 mol % for the EGDMA and Azo, respectively, in the PAA-Azo microgels. The amount of the azobenzene cross-linking agent incorporated in the microgel particles was determined spectroscopically.15 The dry microgel particles (5-10 mg) were hydrolyzed in 2 M NaOH solution (5 mL) at room temperature for 4 days. The solution was then diluted with 5 mL of dimethyl sulfoxide, and electronic absorbance at 443 nm was assayed. The concentration of the cross-linker in the microgel was expressed in mmol/g dry polymer using the absorbanceconcentration calibration curve developed with 4,4′-diaminoazobenzene in dimethyl sulfoxide/2 M NaOH (1:1) solution. The concentrations of the cross-links in the microgels based on Pluronics L92 and PAA without Pluronics were found to be 72 ( 8.3 and 71 ( 5.8 µmol/g, respectively, which is in excellent agreement with the calculated concentration of 76 µmol/g.

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Bromberg et al. 300 g) were sacrificed, and midline incisions were made. The cecal segment of the intestine contents was cut open, and its content was collected and dispersed in isotonic phosphate buffer (pH 7.4) that had been deoxygenated by nitrogen bubbling, resulting in a 10% w/w suspension, which was lyophilized and kept dry on ice. In the microgel swelling experiments, a 6 mg/mL cecum suspension in isotonic phosphate buffer (pH 7.4) was prepared that contained 1.2 mM benzyl viologen and 2.5 mg/mL of R-D-glucose.17 The suspension was incubated at 37 °C under anaerobic conditions for 14 h and at time t ) 0 was added to microgel particles loaded into a capillary, which was subsequently sealed to prevent penetration of oxygen. The swelling kinetics were measured at 37 °C as described above. The concentration of pendent amino groups originating from the degrading cross-linker was measured spectroscopically using the ninhydrin method as described by Yeh et al.17 In brief, a 1 wt % microgel suspension was incubated under gentle shaking in a sealed vial containing 6 mg/mL cecum suspension in isotonic phosphate buffer (pH 7.4) at 37 °C. The suspension samples (1 mL each) were withdrawn intermittently; the microgels were removed from the samples using syringe filters (5-µm pore diameter, Pall Corp., East Hills, NY) and lyophilized. The dry samples (4-7 mg) were placed into a vial containing 1 M acetate buffer at pH 5.0 (1 mL) following addition of 1 mL of ninhydrin solution, mixing, equilibration at 100 °C for 15 min, and addition of 50% aqueous ethanolic solution (15 mL). The samples were then allowed to equilibrate at room temperature for 1-1.5 h, and the amino group concentration was measured by assaying electronic absorbance at 570 nm. Glycine aqueous solution was used to develop calibration curves, generated simultaneously with the assay samples. In the microgel degradation experiments, microgels preswollen in isotonic phosphate buffer were added to a 6 mg/mL cecum suspension in isotonic phosphate buffer (pH 7.4) containing 1.2 mM benzyl viologen and 2.5 mg/mL of R-D-glucose, to result in 1 wt % microgel suspension.

Results and Discussion

Figure 1. Video stills obtained from replay of the recorded process of typical microgel swelling upon placing a microgel particle in a 10 mM phosphate buffer (L92-PAA-EGDMA, XL ) 1 mol %, pH 7.4, T ) 15 °C). Diameter in dry state, d0 ) 41 µm; diameter in equilibrium-swollen state, d∞ ) 149 µm. Microgel Swelling Experiments. Microgel swelling was studied using a volumetric method as described previously.8 In brief, single microgel particles were placed into glass capillary tubes (0.5-2.3 mm i.d.) using suction pressure applied by a filler/ dispenser. The tubes were placed into a glass thermostated cuvette and observed under a Nikon TMS inverted microscope equipped with a model IV-550 video microscaler and a Hitachi color video monitor. The boundaries of the spherical particles were fitted with the microscaler, and the particle diameters were measured with an accuracy of (0.5 µm or better. Initially, the diameter (d0) of a dry particle or a particle swollen under the initial pH and temperature was measured. Either the capillary tube was then filled gently with water of appropriate pH or the particle was removed from the initial tube and placed into another tube, which itself was then filled with a different solution or cell suspension. In the case of microgels with degradable cross-links, their swelling was studied only in buffer solutions deaerated by nitrogen bubbling. The diameter of the swollen particle (dt) was videotaped, and the diameter changes were measured at lowspeed replays. A typical replay is shown in Figure 1, which illustrates the appearance of the dry microgel particle (t ) 0) followed by rapid swelling in an aqueous medium until an equilibrium diameter is reached. All measurements under given conditions were conducted in triplicate. Testing of Microgels with Azoaromatic Cross-Links. Adult, pathogen-free male Sprague-Dawley rats (Harlan, 250-

Swelling of Microgels with Nondegradable CrossLinks. We have shown previously8 that the kinetics of swelling of the spherical Pluronic-PAA microgel particles are described satisfactorily by the model proposed by Tanaka and coauthors.24,25 In that model, at relatively large swelling times the gel diameter dt approaches exponentially its equilibrium value d∞ according to

(

ln

)

dt - d∞ ) B - t/τ d0 - d∞

(1)

where d0 is the initial gel particle diameter, B is a constant depending only on the gel geometry and the ratio of the shear modulus over the osmotic longitudinal modulus,26 and τ is the relaxation time of the slowest mode in the swelling process. Plots of the kinetic swelling data in terms of eq 1 yield values of τ which, in turn, enable calculation of the effective, cooperative diffusion coefficient of the gel (De)24,25

De ) d∞2/4π2τ

(2)

We have demonstrated previously8 that De for the permanently cross-linked, nonporous Pluronic-PAA hydrogels increases with the degree of cross-linking at constant pH. In this work we describe the effect of pH on the swelling kinetics of microgel particles prepared using both the hydrophobic Pluronic L92, which gives particles (24) Tanaka, T.; Fillmore, D. J. Chem. Phys. 1979, 70, 1214-1218. (25) Li, Y.; Tanaka, T. in Dynamics and Patterns in Complex Fluids; Onuki, A.; Kawasaki, K., Eds.; Springer Proceedings in Physics; Springer-Verlag: Berlin, 1990; Vol. 52, pp 44-54. (26) Skouri, R.; Schosseler, F.; Munch, J. P.; Candau, S. J. Macromolecules 1995, 28, 197-210.


Figure 2. Kinetics of swelling of L92-PAA-EGDMA microgels at various pH values in a 10 mM phosphate buffer (L92-PAAEGDMA, XL ) 0.05 mol %, T ) 37 °C).

Figure 3. Effect of pH on the effective diffusion coefficient (De) of L92-PAA-EGDMA and F127-PAA-EGDMA microgels in a 10 mM phosphate buffer (XL ) 0.05 mol %, T ) 37 °C). Arrows show onsets of ionization in respective microgels. The ionization degree vs pH has been reported elsewhere.9

with a significant porous structure, and the relatively hydrophilic Pluronic F127, which yields smooth, nonporous microgel particles.9,10 Figure 2 shows the pH-dependent swelling kinetics of the macroporous L92-PAA-EGDMA microgels, which are described well by eq 1 (R2 > 0.97 in all cases) and are extremely rapid, reaching equilibrium swelling diameters within 10 s at elevated pH. The effective diffusion coefficients, De, extracted from such fits are shown for both microgels as a function of pH in Figure 3. Characteristic of ionization-driven processes, the De values changed little when the ionization degree was zero, while with the introduction of charges into the gels with increasing pH, De increased steadily. The pH value at which this increase first occurred is about one pH unit below the pKa values for the respective microgels, which have been reported to be 6.27 and 4.95 for L92-PAAEGDMA and F127-PAA-EGDMA, respectively.9 Similar effects of ionization in polyelectrolyte hydrogels have been reported in both acidic27-29 and basic30,31 gels. The effect of the increasing basicity of the solution on the swelling (27) Schosseler, F.; Ilmain, F.; Candau, S. J. Macromolecules 1991, 24, 225-234. (28) Ilmain, F.; Candau, S. J. Prog. Colloid. Polym. Sci. 1989, 79, 172-186. (29) De, S. K.; Aluru, N. R.; Johnson, B.; Crone, W. C.; Beebe, D. J.; Moore, J. J. Microelectromech. Syst. 2002, 11, 544-555. (30) Siegel, R. A.; Johannes, I.; Hunt, C. A.; Firestone, B. A. Pharm. Res. 1992, 9, 76-81.

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Figure 4. Effect of cross-linking density on the effective diffusion coefficient (De) of porous (L92-PAA-EGDMA) and nonporous (F127-PAA-EGDMA) microgels in a 10 mM phosphate buffer (T ) 15 °C, pH 7.4).

kinetics of a gel with fixed -COOH groups can be explained mechanistically in terms of the dissociation of the carboxyl groups in the gel, which leads to the availability of protons that form water on combination with the hydroxyl ions. The electroneutrality of the hydrogel is maintained by cations such as Na+ entering the gel along with the OHions. The increased Na+ and OH- concentrations within the gel give rise to an osmotic pressure that causes the gel to swell until the osmotic pressure is just balanced by the elasticity of the cross-links. The higher the initial difference in the concentration of the base between the solutions inside and outside the gel, the higher is the osmotic pressure and thus the faster are the kinetics of the gel swelling. It is interesting to observe that the De values of the microgels based on Pluronic L92 were higher than those of their F127-based counterparts throughout the pH range (Figure 3). This result is in accord with the observation that the structure of the L92-PAA-EGDMA microgels contains permanent pores, while the F127-PAA-EGDMA microgel structure is devoid of discernible pore structure.8,9 The L92-PAA-EGDMA microparticles consist of a fractal structure10 composed of interconnected micro- or nanospheres with gaps between the dense gel components that widen upon swelling, yielding a well-developed porous structure with pores on the order of 100 nm across. Similar porous gels with interconnected pores have been reported to swell via both diffusion in the polymer struts and convection through the pores.32 Since the rate of the solvent convection into the gel is much faster than the rate of diffusion, porous gels with interconnected pores swell much more rapidly than do nonporous gels. To elucidate the effects of the differences in microgel structure on the kinetics of swelling, the swelling of both porous and nonporous microgels was studied as a function of the cross-linking density (Figure 4).The temperature was maintained at 15 °C, which is below the critical aggregation temperature of the PPO segments,10 to avoid the formation of physical cross-links through intraparticle association.8 As is seen in Figure 4, dramatic differences of up to 30-fold were observed in the De values for porous and nonporous gels at XL < 0.1 mol %, but these differences diminished as the cross-linking density increased, both (31) Siegel, R. A. In Pulsed and Self-regulated Drug Delivery; Kost, J., Ed.; CRC Press: Boca Raton, FL, 1990; pp 129-155. (32) Kabra, B. G.; Gehrke, S. H.; Spontak, R. J. Macromolecules 1998, 31, 2166-2173.

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due to the decline of the De values for L92-PAA-EGDMA and the steady increase of the De values for the F127PAA-EGDMA microgels. The increase in the De values with increasing cross-linking density is typically observed in nonporous gels,8,29,34 since the elastic chains between cross-links become shorter with increasing XL, yielding shorter relaxation times (see eq 2). In the porous microgels, at lower XL values the effective diffusion coefficient De was on the order of 10-5 cm2/s; i.e., the collective motion of the polymer chains proceeded at about the same rate as the restoration of the Donnan equilibrium between the inside of the microgel particles and the outside bulk solution.29 As the cross-linking density increased, the volume fraction of the pores (which themselves possess fast relaxation times34) evidently decreased, thus leading to De values equal to the collective diffusion coefficients of the nonporous gels, i.e., De ≈ 106 cm2/s (Figure 4). These results are insightful for the design of gels with degradable cross-links. For instance, if the total cross-linking of the microgel allows for polymer diffusion-limited swelling before the cross-links are degraded (XL g 1 mol %), and if degradation of the cross-links to give XL , 1 mol % leads to conditions wherein the polymer diffusion is extremely fast, and does not limit the kinetics of swelling, as with L92-PAA microgels, then the swelling can be controlled kinetically by the rate at which these crosslinks are degraded. We show below that swelling upon cross-link degradation can be limited kinetically by the rate of that degradation. Swelling of Microgels with Reversible Disulfide Cross-Links. Up to this point, we have reported on microgels that can respond to changes in temperature or the pH or ion composition of their aqueous environment due to the presence of both ionizable groups (carboxyls) and segments with temperature-dependent water solubility (propylene oxide).8-10 In this paper, we modify our microgels to impart sensitivity toward certain biological analytes. The most convenient way of modifying our Pluronic-PAA microgels without substantially changing the unique synthetic route that we have developed8-10 is to introduce reversible cross-links that can be cleaved by the target analytes. In particular, we have explored N,N′bis(acryloyl)cystamine (BAC) as a reversible cross-linker, which provides disulfide bonds acting as cross-links that can be cleaved via reduction to thiols.5,38 The process of swelling that is coupled with the crosslink reduction can be described by the following scheme, assuming that the cross-link (XL) cleavage and swelling can be presented as two separate steps5 k1

k2

} 2XH 98 {\ XL 2P k shrunken gel -1 swollen gel Under steady-state approximation, the kinetics of the disappearance of the shrunken gel are described simply by (33) Buchholz, F. L. In Modern Superabsorbent Polymer Technology; Buchholz, F. L., Graham, A. T., Eds.; Wiley-VCH: New York, 1998; Chapter 5, pp 167-227. (34) Ikkai, F.; Shibayama, M. J. Polym. Sci., Part B: Polym. Phys. 1996, 34, 1637-1645. (35) Hiratani, H.; Alvarez-Lorenzo, C.; Chuang, J.; Guney, O.; Grosberg, A. Y.; Tanaka, T. Langmuir 2001, 17, 4431-4436. (36) Cleland, W. W. Biochemistry 1964, 3, 480-482. (37) Jocelyn, P. C. Methods Enzymol. 1987, 143, 246-256. (38) Chatterjee, A., N., Yu, Q.; Moore, J. S.; Aluru, N. R. J. Aerospace Eng. 2003, 16, 55-64.

Bromberg et al.

-

k2 d[XL] 1 d[P] ) ) k1[XL] - k-1[XH]2 ) [XH] (3) dt 2 dt 2

Since our L92-PAA microgels enable rapid polymer diffusion when the cross-linking density diminishes (XL , 1 mol %, see Figure 4), we can assume that when a microgel that is initially equilibrium-swollen under nonreducing conditions is placed in a reducing medium and the microgel diameter changes are monitored, the crosslink cleavage will be rate-limiting (i.e., -d[XL]/dt ) k1[XL]) and that the changes in XL will be proportional to the diameter changes ([XL]t ∼ dt - d∞). As is shown below, most of the kinetic experiments with reversible crosslinks were specifically designed to monitor the ratelimiting cross-link degradation. Hence, eq 3 can be presented in its integral form as

ln

( ) [XL]t

[XL]0

) -k1t

(4)

Equation 4, closely resembling eq 1, allows for evaluation of the cross-link cleavage rate constant, k1, from the variation of the microgel diameter with time, when this degradation reaction is rate-limiting. Typical kinetics of microgels with degradable and nondegradable cross-links and identical initial degrees of cross-linking are shown in Figure 5. The microgels were first allowed to equilibrium-swell in a nondegrading medium and were then placed in a solution of dithiothreitol (DTT), a well-known agent capable of reacting with disulfides via thiol-disulfide interchange, which can be described as an exchange of the thiolate anion (XS-)39,40

XS- + RSSR f RSSX + RSXS- + RSSX f XSSX + RSIn the case of DTT the second reaction is intramolecular, with DTT being converted to a stable cyclic disulfide. Formation of the DTT anion, which facilitates the thioldisulfide interchange reaction, is pH-dependent, the pKa of DTT being 9.2.5 Therefore, the reduction of disulfide by DTT proceeds with a high equilibrium constant39 The swelling of microgels that were placed initially as dry beads in a nonreducing buffer solution until they were fully swollen to their equilibrium sizes and were then transferred to DTT solutions is shown in Figure 5. The transfer into the reducing environment at about 1000 s after the initial exposure of the dry beads to the buffer solution had no effect on the microgels cross-linked by EGDMA only but resulted in significant additional swelling of the microgels cross-linked by BAC. The timedependent microgel diameter changes were analyzed in terms of either eq 1 or 4 and resulted in linear curves (R2 > 0.97 in all cases) (Figure 6). Notably, the swelling kinetics in nonreducing buffer was much faster than in the DTT-containing buffer for the same gels at all DTT concentrations studied. This observation supported the notion that the swelling in the presence of DTT was kinetically limited by the disulfide reduction, as the diffusion of a small, water-soluble molecule such as DTT (molecular weight, 154 Da), into the microgels proceeds much more rapidly (DDTT ) 7.5 × 10-5 cm2/s38) than the apparent collective diffusion of the degrading gel (De ) 6 (39) Burns, J. A.; Butler, J. C.; Moran, J.; Whitesides, G. M. J. Org. Chem. 1991, 56, 2648-2650. (40) Kizek, R.; Vacek, J.; Trnkova´, L.; Jelen, F. Bioelectrochemistry 2004, 63, 19-24.


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P(CH2CH2COOH)3 + R-S-S-R + H2O f OdP(CH2CH2COOH)3 + 2R-SH

Figure 5. Swelling kinetics of nondegradable and degradable L92-PAA microgels at pH 7.4 and 37 °C. The nondegradable microgels were cross-linked by EGDMA (XL ) 1.1 mol %), while degradable microgels were cross-linked by both BAC (XL ) 1.0 mol %) and EGDMA (XL ) 0.1 mol %). The microgels were initially placed in a 10 mM phosphate buffer and allowed to equilibrium swell, then at the time indicated by an arrow the microgels were placed in the same buffer containing 10 mM DTT.

Figure 6. Swelling kinetics of a degradable L92-PAA microgel at pH 7.4 and 37 °C expressed in terms of eqs 1 and 4. The microgel was cross-linked by both BAC (XL ) 1.0 mol %) and EGDMA (XL ) 0.1 mol %). The microgel was initially placed in a 10 mM phosphate buffer and allowed to equilibrium swell, and then at time t2 was placed in a 100 mM solution of DTT in the same buffer (pH adjusted to 7.4). The swelling curve for the DTT solution is shifted by the time t2 to the beginning of the axis for clarity of presentation.

× 10-6 cm2/s at 100 mM DTT concentration, corresponding to a disulfide cleavage rate constant of k1 ) 0.034 s-1). Similarly, the degradation of the disulfide cross-links has been reported as a limiting step in the swelling of poly(2-hydroxyethyl methacrylate-co-acrylic acid) hydrogels with a large content of acrylic acid, which provides for the driving force toward swelling.5 To explore the potential discrimination of our microgels between different analytes, we studied the effect of a second reducing agent, tris(2-carboxyethyl)phosphine (TCEP), on the rate of microgel swelling. TCEP has emerged as a much more powerful reductant for disulfide bonds in proteins than DTT.42,43 In aqueous solutions, the reduction reaction is42 (41) Burmeister Getz, E.; Xiao, M.; Chakrabarty, T.; Cooke, R.; Selvin, P. R. Anal. Biochem. 1999, 273, 73-80. (42) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. J. Org. Chem. 1977, 42, 332-338. (43) Han, J. C.; Han, G. Y. Anal. Biochem. 1994, 220, 5-10.

This reaction is irreversible due to the stability of the phosphorus-oxygen bond in the tris(2-carboxyethyl)phosphine oxide reaction product.43 The swelling rates for the equilibrium-swollen BACcross-linked microgels after they were transferred to solutions containing either DTT (see Figure 6) or TCEP as the reducing agent were analyzed using eq 4 to yield effective disulfide cleavage rate constants, k1. The effect of the reductant concentration on the swelling rate constants at pH 7.4 is shown in Figure 7. At low concentrations, below about 10 mM, the rate constant is directly proportional to the reducing agent concentration, but the dependence on concentration becomes less pronounced as the concentration increases beyond this value. At concentrations above 10 mM, the reducing agents are clearly in excess, and at these higher concentrations there is a shift in their dissociation equilibria and the appearance of significant fractions of either DTT anions or phosphine carboxylate anions, whose diffusion into the anionic microgels is impeded. Our choice of the physiological pH was dictated by the fact that even though DTT is more effective in disulfide reduction at higher pH, it is much less stable toward oxidation at these elevated pHs. TCEP (pKa 7.6636), which is a much faster and stronger reductant than DTT at physiological pH,39,40 has been reported to oxidize quite significantly at pH 7.4 in the presence of phosphate ions.40 Oxidation can diminish the fraction of the reducing agent available for the disulfide cleavage in the reducing-agent-limiting regime, thus changing the dependence of k1 on the reducing agent concentration at concentrations exceeding 10 mM (Figure 7). Owing to its overall higher reducing power, TCEP has a much more dramatic effect on the disulfide cleavage rates than does DTT (Figure 7). Indeed, the slopes of the k1 vs reductant concentration yielded effective swelling constants of 9.6 and 1.6 M-1 s-1 for concentrations less than 10 mM for TCEP and DTT, respectively. The 5-6-fold faster swelling rates clearly point to the higher reducing power of TCEP and also indicate certain chemical selectivity toward the reducing solutes. In that regard, our microgels possess a sensitivity toward certain solutes, swelling at different rates depending on the analyte by which they are being challenged. We further observed that the microgels swollen by cleavage of the disulfide cross-links could be collapsed to their original, equilibrium-swollen state by subsequent oxidation of the resulting thiol groups by sodium hypochlorite (Figure 8). If one thiol group reacts with an -SH partner other than in the original disulfide bond, the resulting reoxidized gel should have a conformation different from that of the initial gel, and therefore, it should show a different degree of swelling.35 Such shape memory effects have been observed previously with disulfide-crosslinked gels.21,35 To confirm the reoxidation of the microgels by NaOCl, we conducted an iodometric titration as described by Wulff and Schulze.21 The L92-PAA microgels that had undergone reduction by DTT following oxidation by 100 mM NaOCl were washed with excess phosphate buffer on a filter and compared with microgels of the same batch that were equilibrium-swollen in buffer without the reduction-oxidation cycle. No significant differences in disulfide content between original and reoxidized microgels was observed. Judging by these titration results, it appears that despite the high swelling of the reduced gels, the site separation of the thiol groups was insufficient to

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Figure 7. Effect of concentration of reducing agents, DTT and TCEP, on the swelling rate constant, k1. The degradable L92PAA microgels cross-linked by both BAC (XL ) 1.0 mol %) and EGDMA (XL ) 0.1 mol %) were initially placed in a 10 mM phosphate buffer and allowed to equilibrium swell at pH 7.4 and 37 °C, and then were placed in a solution of the reducing agent in the same buffer (pH adjusted to 7.4). The rate constant is found using eq 4.

Figure 8. Effect of hypochlorite (NaOCl) on swelling kinetics of the degradable L92-PAA microgels cross-linked by both BAC (XL ) 1.0 mol %) and EGDMA (XL ) 0.1 mol %) at 37 °C and pH 7.4. The microgels were initially placed in a 10 mM phosphate buffer and allowed to equilibrium swell, at which point they were placed into a 50 mM DTT solution (pH 7.4) and allowed to swell to a new equilibrium state corresponding to the microgel degradation. Then the microgels were removed from DTT solution and placed into NaOCl solution in the same phosphate buffer. The concentration of sodium hypochlorite in millimolar is indicated. Arrows show the moment of microgel placement into either DTT or NaOCl solutions.

prevent complete restoration of the original disulfide crosslinks and thus the size of the microgels. Analogously, Wulff and Schultze21 observed quantitative reoxidation of the thiol polymer prepared from disulfide. As expected, in our experiments the kinetics of the microgel collapse via disulfide group restoration were dependent on the concentration of the oxidation agent, becoming faster with increasing concentration of the latter. Although the timedependent, intraparticle restoration of disulfide cross-links has been observed previously,21 we are unaware of a theory describing the kinetics of the “shape memory effect” in sufficient detail. We will propose such a theory in a future communication. Thus far, we have tested swelling under degrading conditions specifically designed to reveal the cross-link degradation-controlled kinetics. However, what would happen if dry microgel particles were placed into a

Bromberg et al.

Figure 9. Effect of initial conditions on swelling kinetics of the degradable L92-PAA microgels cross-linked by both BAC (XL ) 1.0 mol %) and EGDMA (XL ) 0.1 mol %) at 37 °C and pH 7.4. The microgels were initially placed in a 10 mM phosphate buffer or in 100 mM DTT or 100 mM TCEP solutions in the same buffer. The d∞ was measured at equilibrium swelling under degrading conditions in all cases. The equilibrium swelling level in buffer only is shown by a dotted line.

degrading medium? To address this question, a series of experiments was conducted, wherein different dry L92PAA microgels cross-linked by BAC (1 mol %) and EGDMA (0.1 mol %) were placed either in 10 mM phosphate buffer (pH 7.4) alone or in the same buffer containing either 100 mM DTT or 100 mM TCEP (Figure 9). After equilibrium swelling was reached, the microgels that swelled in buffer only were placed in 100 mM DTT, to measure their equilibrium diameter (d∞) in the degrading medium (data not shown). As is seen in Figure 9, in the beginning of the swelling process all microgels started to swell rapidly with approximately the same rate that characterized the diffusion-limited kinetics (compare with Figure 6). The microgel that was placed in the buffer only proceeded to swell at the same rate until its equilibrium swelling was reached, which is 40% of the d∞ attained under degrading conditions. The microgel placed in TCEP solution also swelled at the same rate (De ) 1.3 × 10-5 cm2/s) until equilibrium swelling, which, notably, corresponded to the maximum swelling with the BAC cross-links degraded. Hence, with TCEP the cross-link degradation rate was not limiting and the swelling rate was confirmed to be controlled by polymer diffusion. However, the swelling rate in the DTT medium changed to a slower pace after the brief rapid swelling period, with an effective rate constant, k1, of about 0.035 s-1, until about 90 s. This latter rate is typical for the kinetics of microgels under degradation-limiting conditions at the same DTT concentration (compare with Figure 7). The last 1 or 2% of the swelling to equilibrium occurred at a significantly lower rate, which may reflect some disulfide linkages being inaccessible to the reductant. It is thus evident that in the case of DTT both the polymer diffusion and the cross-link degradation can be limiting, but at different stages of the swelling process. Swelling of Microgels with Biodegradable Azoaromatic Cross-Links. As was described in the Introduction, in the present work we aimed at pH-sensitive microgels that could swell sufficiently in the neutral pH range characteristic of the lower part of the human GIT, where the gels can be susceptible to the influence of enzymes such as azoreductases. The high degree of swelling enables sufficiently rapid diffusion of enzymes into the hydrogel, where the enzyme can facilitate the


Figure 10. Swelling kinetics of nondegradable and degradable L92-PAA microgels at pH 7.4 and 37 °C. The nondegradable microgels were cross-linked by EGDMA (XL ) 1.1 mol %), while degradable microgels were cross-linked by both 4,4′-di(methacryloylamino)azobenzene (XL ) 1.0 mol %) and EGDMA (XL ) 0.1 mol %). The microgels were initially placed in an isotonic phosphate buffer and allowed to equilibrium swell, then at the time indicated by an arrow the microgels were placed in the cecum suspension.

cleavage of biodegradable cross-links. Herein, we employed 4,4′-di(methacryloylamino)azobenzene as a cross-linker (Scheme 1) yielding aromatic azo bonds degradable by azoreductase activity. Figure 10 depicts the effect of placing L92-PAA microgels, equilibrium-swollen in phosphate buffer, into a suspension of azoreductase-containing cecal contents of the rat intestine; similar experiments were performed using PAA microgels with no L92 content. Note that the initial swelling rates for the azo-cross-linked microgels from the dry to the equilibrium-swollen state were faster than those for the EGDMA microgels for the same total cross-linking density but that the equilibrium swelling of the EGDMA microgels was higher than that of their azocross-linked counterparts. This observation is consistent with the supposition that the azo cross-linker provides more of a cross-linking effect than does EGDMA (and indeed also BAC, which showed essentially the same behavior as EGDMA), possibly through some intramolecular association between the aromatic rings of these cross-linkers yielding a higher effective cross-linking density within the gel particle. When these microgels were placed in the cecum suspension, the swelling degree of the nondegradable microgels cross-linked by EGDMA did not change appreciably, while the microgels cross-linked by azobenzene swelled 1.5-fold in the cecum due to the azo-group degradation. The swelling kinetics in cecum followed eq 4 in all cases, yielding linear fits (R2 > 0.97) (Figure 11). Notably, the degradation-limited swelling in cecum was in all instances much slower than the diffusionlimited swelling in buffer, with the kinetic constants of (1.5 ( 0.4) × 10-2 (n ) 4) and (3.7 ( 0.5) × 10-2 (n ) 3) found for L92-PAA microgels and PAA microgels without Pluronic, respectively. An over 2-fold, statistically significant difference of identically cross-linked microgels with and without Pluronic can be attributed to the impeding effect of the Pluronic dangling chains on the microgel/water interface. The Pluronic polymers are protein-resistant, because hydrated hydrophilic PEO chains extend out into the solution inhibiting the protein adsorption by steric repulsion.45,46 Therefore, the diffusion (44) Lee, J.; Martic, P. A.; Tan, J. S. J. Colloid Interface Sci. 1989, 131, 252-266.

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Figure 11. Typical swelling kinetics of degradable L92-PAA and PAA microgels at pH 7.4 and 37 °C expressed in terms of eqs 1 and 4. The microgels were cross-linked by both 4,4′-di(methacryloylamino)azobenzene and EGDMA. The microgels were initially placed in an isotonic phosphate buffer and allowed to equilibrium swell, and then at time t2 were placed in a cecum suspension. The kinetics in cecum suspension are shifted by the time t2 to the beginning of the axis for clarity of presentation.

Figure 12. Kinetics of the appearance of the amino groups via degradation of the azobenzene in rat cecum suspension at 37 °C and pH 7.4. The amino groups were measured by ninhydrin assay (see Experimental Section). The L92-PAA and PAA gels had identical cross-linking density and were cross-linked by both 4,4′-di(methacryloylamino)azobenzene and EGDMA.

of the azoreductase into the Pluronic-containing microgels might be inhibited, leading to the lower rate of the degradation-limited swelling. To further elucidate differences between degradation rates of the Pluronic-modified and unmodified PAA microgels, an independent method of titration of the amino groups resulting from the azo group degradation was used (Figure 12). The kinetics of the appearance of the amino groups measured by the ninhydrin assay (see Experimental Section) afforded estimates of the effective rate of azo cross-link degradation, found to be 1.8 × 10-10 and 3.8 × 10-10 mol/h for the L92-PAA and PAA-based microgels, respectively. These results thus confirm the 2-fold faster degradation of the PAA-based microgels. Yeh at al.17 observed a rate of the azo groups degradation in rat cecum of about 2 × 10-9 mol/h in pH-sensitive hydrogels crosslinked by N,N′-(ω-aminocaproyl)-4,4′-diaminoazobenzene. A much slower degradation of the Pluronic-modified (45) Norman, M. E.; Williams, P.; Illum, L. Biomaterials 1993, 14, 193-202. (46) Li, J.-T.; Caldwell, K. D.; Rapoport, N. Langmuir 1994, 10, 44754482.

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microgels in the colon, coupled with their mucoadhesive properties,11 could provide means for a sustained, sitespecific release. Conclusions Dynamic changes in the cross-link density of stimuliresponsive microgels enable novel opportunities for the control of gel swelling, of importance for drug delivery and microgel sensoric applications. We have found optimum conditions for the rapid, diffusion-limited swelling of pH- and temperature-sensitive Pluronic-PAA microgels. The introduction of disulfide cross-links into such microgels enables degradation-limited swelling under physiological conditions, with a first-order reaction constant, k1, with microgels equilibrium-swollen in the buffer solution. The constant k1 depends nonlinearly on the concentration of reducing agents such as dithiothreitol and tris(2-carboxyethyl)phosphine. We demonstrated a “kinetic selectivity” of the disulfide-cross-linked microgels, that is, a severalfold faster swelling in the presence of the more powerful reducing agent, TCEP, than in the presence

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of DTT. This observation may prove to be a route toward sensing of other reducing agents of biological importance such as thiol-containing peptides and enzymes. The reoxidation of the thiol groups into disulfide cross-links by sodium hypochlorite leads to the restoration of the microgels’ diameter measured prior to the reductionreoxidation cycle, which confirms the shape memory of the microgels. Such an effect is useful in MEMS devices utilizing mechanical properties of the gels associated with triggered volume transitions. Introduction of enzymatically degradable azo cross-links into our microgels allowed for control of slow swelling due to degradation of the crosslinks by azoreductases from the rat intestinal cecum. The presence of Pluronics in the microgels structure can impede the enzyme diffusion into the microgel and further reduce the rate of swelling, which is an effect potentially conductive to sustained release during colon-specific drug delivery using degradable microgels. LA047893J

Kinetics of Swelling of Polyether-Modified Poly(acrylic acid) Microgels

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