Biomacromolecules 2002, 3, 1304-1311
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Disulfide Cross-Linked Hyaluronan Hydrogels Xiao Zheng Shu,‡ Yanchun Liu,‡ Yi Luo,‡,† Meredith C. Roberts,§ and Glenn D. Prestwich*,‡ Department of Medicinal Chemistry, The University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah 84108-1257, and Department of Bioengineering, The University of Utah, 20 South 2030 East, Room 144, Salt Lake City, Utah 84112 Received June 25, 2002; Revised Manuscript Received August 22, 2002
A new disulfide cross-linking strategy was developed to prepare hyaluronic acid (HA) hydrogel from thiolmodified HA. First, dithiobis(propanoic dihydrazide) (DTP) and dithiobis(butyric dihydrazide) (DTB) were synthesized and then coupled to HA with carbodiimide chemistry. Next, disulfide bonds of the initially formed gel were reduced using dithiothreitol (DTT) to give, after exhaustive dialysis, the corresponding thiol-modified macromolecular derivatives HA-DTPH and HA-DTBH. The degree of substitution of HADTPH and HA-DTBH could be controlled from 20% to 70% of available glucuronate carboxylic acid groups. The pKa values of the HA-thiol derivatives were determined spectrophotometrically to be pKa ) 8.87 (HADTPH) and pKa ) 9.01 (HA-DTBH). The thiol groups could be oxidized in air to reform disulfide linkages, which resulted in HA-DTPH and HA-DTBH hydrogel films. Further oxidation of these hydrogels with dilute H2O2 created additional cross-links and afforded poorly swellable films. The disulfide cross-linking was reversible, and films could be again reduced to sols with DTT. Release of blue dextran from crosslinked films was used as a model for drug release. The rapid gelation of the HA-DTPH solution under physiological conditions was also achieved, which demonstrated the capacity for in situ cell encapsulation. Thus, L-929 murine fibroblasts were encapsulated in HA-DTPH hydrogel; these cells remained viable and proliferated during 3 days of culture in vitro. Introduction Hyaluronic acid (HA) is a nonsulfated glycosaminoglycan (GAG) consisting of repeating disaccharide units (R-1,4-Dglucuronic acid and β-1,3-N-acetyl-D-glucosamine) with molecular weights (Mw) up to 10 000 kDa. Present in all connective tissues as a major constituent of extracellular matrix (ECM),1 this polyanionic polymer has unique physicochemical properties and distinctive biological functions.2 HA has been implicated in water homeostasis of tissues, in the regulation of the permeability of other substances by steric exclusion phenomena, and in the lubrication of joints.3 HA also binds specifically to proteins in the ECM, on the cell surface, and within the cell cytosol, thereby stabilizing the cartilage ECM3,4 and mediating cell adhesion, cell motility,5,6 growth factor action,7 morphogenesis, embryonic development,8 and inflammation.9 Unmodified HA has been used in drug delivery and surgery, such as an adjuvant in ophthalmic drug delivery,10 enhancement of the absorption drugs and protein via mucosal tissues,11-14 and also in the field of viscosurgery, viscosupplementation, and wound healing.15-17 However, the poor biomechanical properties of this soluble natural polymer and its rapid degradation in vivo preclude many direct clinical applications. Therefore, to obtain materials that are more * To whom correspondence should be addressed. Phone: 801 585-9051. Fax: 801 585-9053. E-mail:
[email protected]. ‡ Department of Medicinal Chemistry. † Present address: Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139. § Department of Bioengineering.
mechanically and chemically robust, a variety of hydrophobic modifications and chemical cross-linking strategies have been explored to produce insoluble or gel-like HA materials. Generally, the target groups in modification chemistries involve the carboxyl and hydroxyl groups of the sugar moieties.18 For example, the HA-esterified materials, collectively called HYAFF, are prepared by alkylation of the tetrabutylammonium salt of HA with an alkyl or benzyl halide in dimethyl formamide solution.19 Cross-linked HA has been prepared using divinyl sulfone,20 1,4-butanediol diglycidyl ether,21 glutaraldehyde,22 watersoluble carbodiimides,23 and a variety of other bifunctional cross-linkers. Extensive efforts have also been made to produce HA derivatives having unique properties for specific biomedical applications.24-35 For instance, HA can be modified with hydrazides, and in some cases with amines, through carbodiimide-mediated coupling to form novel HA derivatives, which may then be cross-linked by various reagents to form hydrogels.26-29,31 However, the cross-linking agents are often relatively cytotoxic small molecules, and the hydrogels have to be extracted or washed extensively to remove traces of unreacted reagents and byproducts,36 thus precluding use in many medical applications. We now describe a novel and physiologically compatible strategy to prepare HA hydrogels for potential in situ encapsulation of cells and in situ wound repair. In this strategy, a latent crosslinking agent is incorporated into the soluble polymer. Crosslinking can be induced simply by exposure to air. Moreover, this process is fully reversible and offers both enzymatic and nonenzymatic routes for biodegradation. Thus, we first
10.1021/bm025603c CCC: $22.00 © 2002 American Chemical Society Published on Web 09/27/2002
Hyaluronan Hydrogels
Figure 1. Structure of disulfide-containing dihydrazides.
prepared thiol-modified HA using carbodiimide-mediated hydrazide chemistry,37 and then HA hydrogels were formed under physiological conditions by air oxidation of thiols to disulfides. This research represents the first disulfide crosslinked hydrogels prepared from HA or any GAG, although there is precedent for the general approach for fabrication of biocompatible materials using thermosensitive polyacrylamides 38 and thiolated collagen.39-41 Materials and Methods Materials. Fermentation-derived hyaluronan (HA, sodium salt, Mw ) 1.5 MDa) was obtained from Clear Solutions Biotech, Inc. (Stony Brook, NY). 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI), 3,3′-dithiobis(propanoic acid), 4,4-dithiobis(butanoic acid), and hydrazine hydrate were from Aldrich Chemical Co. (Milwaukee, WI). Dulbecco’s phosphate-buffered saline (DPBS), bovine testicular hyaluronidase (HAse, 330 U/mg), and blue dextran (Mw ) 200 000) were from Sigma Chemical Co. (St. Louis, MO). Dithiothreitol (DTT) was from Diagnostic Chemicals Limited (Oxford, CT). 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was from Acros (Houston, TX). Analytical Instrumentation. Proton NMR spectral data were obtained using a Varian INOVA 400 at 400 MHz. UVvis spectral data were obtained using a Hewlett-Packard 8453 UV-visible spectrophotometer (Palo Alto, CA). Gel permeation chromatography (GPC) analysis was performed using the following system: Waters 515 HPLC pump, Waters 410 differential refractometer, Waters 486 tunable absorbance detector, and Ultrahydrogel 250 or 1000 columns (7.8 mm i.d. × 130 cm) (Milford, MA). The eluent was 200 mM phosphate buffer (pH 6.5)/MeOH ) 80:20 (v/v), and the flow rate was 0.3 or 0.5 mL/min. The system was calibrated with standard HA samples provided by Dr. U. Wik (Pharmacia, Uppsala, Sweden). Fluorescence images of viable cells were recorded using a Nikon Eclipse TE300 with epi-fluorescence capabilities. Cell proliferation was determined using a biochemical assay (Cell-Titer 96 Proliferation Kit, Promega, Madison, WI) at 550 nm, which was recorded on an OPTI Max microplate reader (Molecular Devices, Sunnyvale, CA). Synthesis of Thioacid Dihydrazides. The oxidized forms of the required thiol cross-linkers 3,3′-dithiobis(propanoic hydrazide) (DTP) and 4,4′-dithiobis(butanoic hydrazide) (DTB) (Figure 1) were synthesized from their diacids as described previously for DTP.37 Thus, free dicarboxylic acids were converted into diesters by refluxing in ethanol with acid catalysis. The diesters were hydrazinolyzed with hydrazine hydrate to form the corresponding dihydrazides. DTP:37 yield, 92%. 1H NMR (400 MHz, DMSO-d6): δ 9.05 (s, 2H,
Biomacromolecules, Vol. 3, No. 6, 2002 1305
N-NH-C(O)), δ 4.21 (s, 4H, NH2-N-C(O)), δ 2.88 (t, 4H, C(O)-C-CH2-S), δ 2.40 (t, 4H, N-C(O)-CH2-C). DTB: yield, 52%. 1H NMR (400 MHz, DMSO-d6): δ 8.95 (s, 2H, N-NH-C(O)), δ 4.15 (s, 4H, NH2-N-C(O)), δ 2.66 (t, 4H, C-C-CH2-S), δ 2.10 (t, 4H, C(O)-CH2-CC), δ 1.82 (p, 4H, C(O)-C-CH2-C-S). MS-EI, m/z 266.0 (M+, 1.44); 133.0 (SC3H6CON2H3+, 46.78); 101.0 (C3H6CON2H3+, 100.0). HRMS for C8H18O2S2N4: found 266.0864; calcd. 266.0871. Preparation of Low Molecular Weight (LMW) HA by Acid Degradation. High molecular weight HA (1.5 MDa) (20 g) was dissolved in 2.0 L of distilled water, and the solution pH was adjusted to ca. 0.5 by the addition of concentrated HCl. The degradation was carried out at 37 °C, 130 rpm stirring for 24 h. After that, the pH of the solution was adjusted to 7.0 by the addition of 1.0 N NaOH before transfer of the solution to dialysis tubing (Mw cutoff ) 3500), and the solution was dialyzed against water for 4 days. The solution was then centrifuged, and the supernatant was lyophilized to give 15 g of LMW HA (Mw ) 246 kDa, Mn ) 120 kDa, polydispersity index ) 1.97). Preparation of Thiolated HA. Thiolated HA derivatives with different loadings were prepared following a general protocol (Figure 2). In a representative example, LMW HA (20 g, 50 mmol) was dissolved in 2.0 L of water; 23.8 g of DTP or 26.6 g of DTB (100 mmol) was added while the solution was stirred. The pH of the reaction mixture was adjusted to 4.75 by the addition of 1.0 N HCl. Next, 19.2 g of EDCI (100 mmol) in solid form was added. The pH of the reaction mixture was maintained at 4.75 with aliquots of 1.0 N HCl. The reaction was stopped by addition of 1.0 N NaOH, raising the pH of the reaction mixture to 7.0. Then, 100 g of DTT (ca. 650 mmol) in solid form was added, and the pH of the solution was raised to 8.5 by addition of 1.0 N NaOH. After the mixture was stirred for 24 h, the pH of reaction mixture was adjusted to pH 3.5 by the addition of 1.0 N HCl. The acidified solution was transferred to dialysis tubing (Mw cutoff ) 3500) and dialyzed exhaustively against dilute HCl (pH 3.5, approximately 0.3 mM) containing 100 mM NaCl, followed by dialysis against dilute HCl, pH 3.5. The solution was then centrifuged, and the supernatant was lyophilized. The purity of thiolated HA (HA-DTPH and HADTBH) was measured by GPC and 1H NMR, and the degree of substitution (SD) was determined by 1H NMR. The free thiols on the side chain of HA-DTPH and HA-DTBH were determined by a modified Ellman method.42 SD (%) and thiol content (%) were defined as the number of DTP (or DTB) residues and thiols per 100 disaccharide units, respectively. Representative results: HA-DTBH (Mw ) 165 kDa, Mn ) 63 kDa, polydispersity index ) 2.62, SD ) 72%) and HADTPH (Mw ) 136 kDa, Mn ) 61 kDa, polydispersity index ) 2.23, SD ) 58%). pKa Determination. The pKa of thiols in HA-DTPH and HA-DTBH was determined spectrophotometrically on the basis of the UV absorption of thiolates as proposed by Benesch and Benesch.43 Solutions of HA-DTPH and HADTBH (ca. 5 mg) were dissolved in 100 mL of 0.001 N HCl containing 0.1 N NaCl (stable ionic strength). Freshly
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Figure 2. Synthetic scheme and structure of thiolated HA derivative.
prepared solutions were immediately measured in the UV region with a scan from 190 to 300 nm. Gelation of Thiolated HA Solutions. The solution (flow)-gel (no flow) transition was determined by a flow test utilizing a test tube inverting method reported by Jeong et al.44 HA-DTBH and HA-DTPH were dissolved in DPBS to give 3.0% (w/v) solutions under N2 protection. The solution pH was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 by addition of 1.0 N NaOH. Freshly prepared solutions (1.0 mL) with different pH were immediately injected into glass tubes (0.8 cm in diameter, 7.5 cm in length). After exposure to air at room temperature for 15 or 30 min, the test tube was inverted. If no fluidity was visually observed in 1 min, we concluded that a gel had formed. Preparation of Disulfide-Cross-Linked HA films. HADTBH and HA-DTPH were dissolved in DPBS to give 3.0% (w/v) solutions, and the solution pH was adjusted to 7.4 by the addition of 1.0 N NaOH. For drug-loaded gels, 0.15% (w/v) blue dextran (Mw ) 200 000) was included as a model drug. Next, 25 mL of the solution was poured into a 9-cm Petri dish and allowed to dry at room temperature. After ca. 3 days, a film was ready. As required, the film was further oxidized by immersion in 0.3% H2O2 for 1 h. The film was then rinsed with distilled water, cut into 6-mm diameter disks, and dried at room temperature for 1 day and then at 1 mmHg for one week to give films with 0.1 mm thickness. Swelling Determination. Disks of HA-DTBH and HADTPH film (6 mm in diameter) were weighed (W0), immersed in glass vials containing 10 mL of DPBS (pH 7.4), and placed in a shaking incubator at 37 °C at 300 rpm. At predetermined time intervals, the wet films were weighed (Wt) immediately after the removal of the surface water by
blotting between two pieces of filter paper. Swelling ratio (R) was defined as Wt/W0. Disulfide Content Determination. Disks of HA-DTBH and HA-DTPH film were degraded by acid hydrolysis (0.1 N HCl). The total sulfur content (disulfide plus thiol) was measured using 2-nitro-5-thiosulfobenzoate (NTSB) according to the method of Thannhauser et al.45 In addition, the free thiol content was measured by the Ellman method.46 Disulfide content was calculated from the difference between total sulfur content and free thiol content. Blue Dextran Release Studies. Drug-loaded 6-mm disks of HA-DTBH and HA-DTPH film were immersed in glass bottles containing 2 mL of release media and placed in an incubator at 37 °C at 300 rpm. At predetermined time intervals, 1 mL of supernatant solution was removed, and the blue dextran content was determined by UV-vis absorption at 625 nm. Then, 1 mL of blank release media was added back to maintain constant volume. Release media is DPBS containing 0, 10, and 50 mM DTT (the media pH was adjusted to pH 7.4 by adding 1.0 N NaOH) or DPBS containing hyaluronidase (HAse, 100 U/mL). In Situ Cell Encapsulation. HA-DTPH (Mw ) 136 kDa, Mn ) 61 kDa, polydispersity index ) 2.23, SD ) 58%) was dissolved in DMEM/F-12 medium (GIBCO, Rockville, Maryland) to give a 3.0% (w/v) solution under N2 protection, and the solution pH was adjusted to 7.4 by adding 1.0 N NaOH. Then the solution was sterilized under UV light for 25 min under N2 protection. Murine fibroblasts (L-929, ATCC, Manassas, VA) were cultured in a triple flask (Fisher, Springfield, NJ) until 90% confluence and then trypsinized and mixed with HA-DTPH solution to a final concentration of 2 × 106/mL. Next, 0.5 mL of the HA-DTPH solution
Hyaluronan Hydrogels
was added into each well of a 12-well plate. The cell-loaded plates were incubated (37 °C, 5% CO2, 4 h) until a solid hydrogel formed, and then 2 mL of DMEM/F-12 medium with 10% of newborn calf serum (GIBCO, Rockville, MD) was added into each well. The plates were transferred to an incubator (37 °C, 5% CO2, 3 days) without a change of medium. Cell Viability and Proliferation. The viability of murine L-929 fibroblasts in the hydrogel was determined by a double-staining procedure using fluorescein diactate (F-DA) and propidium iodide (PI).47 F-DA (Molecular Probes, Eugene, OR), a nonfluoresent fluorescein derivative, diffuses through the membrane of living cells and is hydrolyzed by intracellular esterase to produce a green fluorescence. PI (Sigma Chemical Co., St. Louis, MO), which is excluded by intact cell membranes but was able to diffuse across a damaged cell membrane, binds to nucleic acids to produce a bright red fluorescence. Briefly, a 5 mg/mL solution of F-DA in acetone was diluted to 20 µg/mL in DPBS that contained 0.2 µg/mL PI. After 1 and 3 days culture with encapsulated cells in vitro, the hydrogels were rinsed twice with DPBS, immersed in the diluted F-DA/PI solution for 10 min at room temperature and then washed with DPBS for 5 min. Then, live and dead cells were observed on a Nikon TS 100 microscope (Nikon, Melville, NY) with triple (DAPI/FITC/CY3) filter. After different culture times, the number of viable cells in each hydrogel was determined using a biochemical assay (Cell-Titer 96 Proliferation Kit, Promega, Madison, WI) as previously described.48 In this method, a tetrazolium salt (MTS) is reduced by the mitochondria of living cells into a colored formazan product of which the presence can be detected spectrophotometrically. The hydrogels in 12-well plates were rinsed twice with DPBS buffer, and then 900 µL of DMEM/F-12 medium with 5% of newborn calf serum and 180 µL of Cell Titer 96 Proliferation Kit solution were added into each well. After 2 h of incubation with gentle shaking (37 °C, 5% CO2), a 125-µL aliquot of each of the solutions was transferred individually into a 96-well plate and read at 550 nm with an OPTI Max microplate reader (Molecular Devices). The absorbance reading was converted into a cell number based on standard curves generated from the assay of known numbers of cells. Data sets were compared using two-tailed, unpaired t-tests. P-values less than 0.05 were considered to be significant. Results and Discussion Synthesis and Characterization of Thiolated HA. Carbodiimides have been widely used for activation of carboxyl groups of GAGs under the acidic conditions that are necessary for protonation of the carbodiimide nitrogens, leading to nucleophilic attack of the carboxylate anion at the central carbon to form an initial O-acylisourea.49 The modification of HA by carbodiimides is usually performed at pH 4.75, at which carbodiimide nitrogens appear to be sufficiently protonated while HA mainly exists as the carboxylate.50 However, a number of studies have demon-
Biomacromolecules, Vol. 3, No. 6, 2002 1307 Table 1. Optimization of DTP Modification of HA molar ratio of HA/DTP/EDCI
reaction time (min)
degree of substitution (%)
1:2:2 1:2:2 1:2:2 1:2:2 1:2:2 1:1:2 1:4:2 1:2:1 1:2:0.5
5 10 30 60 120 120 120 120 120
28.8 41.7 49.2 54.4 66.8 38.9 42.5 31.1 26.8
strated that for viscous macromolecules the unstable intermediate O-acylisourea can rearrange to a stable N-acylurea in competition with capture by exogenously added amines, largely because of the very low fraction of free amine in the acidic environment.31,49,50 In contrast, hydrazides retained nucleophilicity at acidic pH and effectively couple to the O-acylisourea to give hydrazide functionalized HA derivatives useful for a variety of biomedical applications.26-29 We describe herein the use of the EDCI-hydrazide chemistry to introduce thiol groups into HA. This was accomplished by EDCI-mediated reaction of HA with disulfide containing dihydrazides followed by reduction of the first-formed gels with DTT to give soluble thiolated HA (Figure 2). Three disulfide-containing diacids were used to prepare the corresponding dihydrazides, in which the distance between the disulfide and amide groups varied from one to three methylenes (Figure 1). Some examples in the literature reveal that the shift of pKa of thiols can be induced by electrostatic interactions with neighboring ionizable groups or polar residues or both.51-53 We expected that the proximity of the amide group would affect the ease of oxidation of the free thiol to the corresponding disulfide. DTP (n ) 2) and DTB (n ) 3) were readily synthesized, and their structures were confirmed spectroscopically by 1H NMR and MS. The lower homologue with 2,2′-dithiobis(ethanoic acid) dihydrazide (n ) 1, DTE) was prepared but was unstable at room temperature and not studied further. Using EDCI chemistry, we coupled DTP and DTB to the HA carboxylates at pH 4.75 (Figure 2). Thus, LMW HA (10 mg/mL) was mixed with DTP or DTB at various molar ratios (Table 1), the pH was adjusted to 4.75, and then different quantities of EDCI in solid form were added to initiate the reaction. The pH of the reaction mixture was maintained at 4.75 by addition of 1.0 N HCl. The viscosity of the solution increased significantly, and usually a gel formed. The reaction was stopped by the addition of 1.0 N NaOH. Then, DTT in solid form was added to reduce the disulfide bonds. The gel dissolved gradually, and the pH of the reaction mixture was maintained at 8.5 at room temperature while the mixture was stirred overnight. At least a 3-fold excess of DTT (relative to DTP or DTB) was required to dissolve the gel, and complete reduction of all disulfides required at least a 5-fold excess of DTT. The mixture was then purified by dialysis against a dilute HCl solution at pH 3.5, which was selected to avoid the oxidization of thiols at higher pH and the degradation of thiolated HA at lower pH. The dialyzed solution was then centrifuged, and the super-
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Figure 4. Absorption at 242 nm as a function of pH (a) for HA-DTPH and HA-DTBH solution and (b) logarithmic plot of log[(Amax - Ai)/Ai] vs pH. The pKa values correspond to the intercept with the abscissa.
Figure 3. 1H NMR spectra in D2O of (a) HA-DTPH (SD ) 40%) and (b) HA-DTBH (SD ) 72%).
natant was lyophilized to give HA-DTPH and HA-DTBH in yields of 50%-70%. The purity and molecular weight distribution of the thiolated HA were measured by GPC and 1H NMR. A monodisperse GPC profile was detected by both UV and refractive index, indicating that both large and small impurities had been completely removed. After lyophilization, the thiolated HA was completely soluble in water. The reduction of disulfide also influenced the GPC profile. When the disulfides were only partially reduced, a broad-peak GPC profile with side-peak was observed, and after lyophilization, the thiolated HA was not soluble in water. The structure of HA-DTPH and HA-DTBH shown in Figure 2 was confirmed by 1H NMR spectra in D2O (Figure 3). Compared to the spectrum of HA (the N-acetyl methyl protons of HA were at δ 1.95), new resonances for HADTPH appeared at δ 2.72 and δ 2.58, corresponding to the two side chain methylenes (CH2CH2SH) (Figure 3a). For HA-DTBH, the new protons seen at δ 2.55 and δ 2.44 were from the CH2SH and that R to the amide group (NHCOCH2),
respectively, while the β-methylene of CH2CH2CH2SH overlapped with the N-acetyl methyl protons of HA (Figure 3b). The SD was determined by integration of the methylene signals of DTP or DTB residues using the N-acetyl methyl resonances as an internal standard. The SD determined by 1 H NMR was quite close to values of the free thiols obtained by the modified Ellman method.42 For example, the SD of HA-DTP measured by 1H NMR was 58%, while the free thiol content measured by modified Ellman method was 60%. In these experiments, SD was mainly controlled by the molar ratios of HA/DTP/EDC and reaction time (Table 1). By selecting suitable reaction parameters, we can control the degree of substitution over a wide range (28%-67%) (Table 1). Similar results were also observed in the case of DTBmodified HA. Because the nucleophilic thiolates rather than thiols are reported to be the reactive species in many kinds of reactions, it was important to obtain the pKa values for the free thiols in HA-DTPH and HA-DTBH. These were determined spectrophotometrically on the basis of the UV absorption of thiolates.43 With increasing pH, the absorption of solutions increased abruptly, especially at the pH near the pKa of thiols (Figure 4a). According to the procedure reported by Lutolf and co-workers,53 the intercept with the abscissa in a graphical representation of log[(Amax - Ai)/Ai] vs pH yielded the pKa value. There was good linear approximation of the five central points both for HA-DTPH and HA-DTBH, giving a value of 8.87 for HA-DTPH and 9.01 for HADTBH. The pKa of HA-DTPH was slightly lower than that
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Hyaluronan Hydrogels Table 2. The Air-Induced Gelation of HA-DTPH and HA-DTBH Solution (3.0% w/v) Determined by a Test Tube Inversion Methoda HA-DTPH
HA-DTBH
pH
15 min
30 min
15 min
30 min
5.0 6.0 7.0 8.0 9.0
S S G G G
S S G G G
S S VS VS VS
S S VS G G
a
S ) solution; G ) gel; VS ) highly viscous solution.
Figure 6. Swelling of HA-DTPH and HA-DTBH films in DPBS at pH 7.4. Films were prepared with 3.0% (w/v) solutions of HA-DTPH (Mw ) 136 kDa, Mn ) 61 kDa, polydispersity index ) 2.23, SD ) 58%) and HA-DTBH (Mw ) 165 kDa, Mn ) 63 kDa, polydispersity index ) 2.62, SD ) 72%), and the films (n ) 3) were oxidized in air only (solid symbols) or further oxidized with 0.3% (v/v) H2O2 for 1 h (open symbols).
Figure 5. Disulfide content in HA-DTPH and HA-DTBH films. Films were prepared with 3.0% (w/v) solutions of HA-DTPH (Mw ) 136 kDa, Mn ) 61 kDa, polydispersity index ) 2.23, SD ) 58%) and HA-DTBH (Mw ) 165 kDa, Mn ) 63 kDa, polydispersity index ) 2.62, SD ) 72%), and the films (n ) 3) were either air oxidized only or further oxidized with 0.3% (v/v) H2O2 for 1 h.
of HA-DTBH because the thiol in HA-DTPH was more easily activated by the proximity of the amide group (Figure 4b). Compared to HA-DTBH, the lower pKa of thiols in HADTPH suggested increased reactivity and increased capability to regenerate the disulfide structure under the same conditions. A qualitative procedure was used to evaluate the reformation of disulfide. When HA-DTPH and HA-DTBH solutions were in contact with air, the viscosity increased and a gel formed because of the oxidation of thiols to disulfide by O2. At higher pH, both HA-DTPH and HADTBH solutions more easily formed gels because thiols were converted to more reactive thiolates at higher pH (Table 2). For instance, with 3.0% HA-DTPH solution (SD ) 58%), the solution at pH 7.0, 8.0, and 9.0 gelled within 15 min, while at pH 5.0 and 6.0 no obvious increase in viscosity of solution was observed after 30 min (Table 2). The thiols of HA-DTBH were less reactive, and thus the gelation of HADTBH solution (3.0% w/v, SD ) 72%) was sluggish (Table 2). Disulfide Cross-Linked HA Films. Thiolated HA films were prepared by pouring HA-DTPH or HA-DTBH solution (pH 7.4) into a Petri dish followed by drying in air. To strengthen the films, some were further incubated in 0.3% H2O2 for 1 h to increase the number of disulfide bonds formed. The surface of all films was very smooth, and the inside structure was very dense (data not shown). The disulfide content in the film was determined by NTSB45 after complete acid hydrolysis. As seen in Figure 5, oxidation with dilute H2O2 indeed increased the number of disulfide linkages. For example, the disulfide content in
HA-DTPH film increased from 0.175 to 0.212 mmol/g after the oxidation with H2O2. In the case of HA-DTBH film, fewer disulfide linkages were formed because of air oxidation because the thiol was less reactive (the value was 0.125 mmol/g); however, this could be increased significantly to 0.25 mmol/g by oxidation with H2O2. However, following the H2O2 oxidizing procedure, no additional thiol groups are detected within both the HA-DTPH and the HA-DTBH films, and only ca. 30%-40% of the available thiols formed disulfide bonds. This suggests that H2O2 oxidation of thiol groups not only created new disulfide bridges but also led to the production of S-oxidized sulfenic or sulfonic acids that would not be detected using NTSB and DTNB.54 The swelling of HA-DTPH and HA-DTBH films in DPBS was in accordance with the disulfide content in the films and was shown in Figure 6. The air-oxidized films swelled significantly because of the low degree of cross-linking with a swelling ratio at 5.5 h of 16.2 for HA-DTBH film and 9.5 for HA-DTPH. These ratios are similar to PEG-dialdehyde cross-linked HA adipic dihydrazide hydrogels used for drug release29 and wound repair.55 After H2O2 oxidation, the swelling ratio decreased to 3.5 for the HA-DTBH film and 2.9 for HA-DTPH film. To further confirm that the HA-DTPH and HA-DTBH films were cross-linked by reversible disulfide linkages, the hydrogel films were incubated in DPBS that contained different concentrations of DTT at pH 7.4 (data not shown). Even with DTT concentration as low as 10 mM, films generated from both air and H2O2 oxidation swelled significantly and dissolved gradually because of reduction of disulfide by DTT. As the gels dissolved, a model drug (blue dextran, Mw ) 200 000) that had been noncovalently entrapped in the hydrogel films was released. Thus, within 100 min, both HA-DTPH films that had been further oxidized with H2O2 were dissolved, and consequently the blue dextran model drug was released completely in the presence of DTT concentration of 10 and 50 mM. The release of blue dextran occurring in the absence of DTT (Figure 7) was negligible. Furthermore, the enzyme HAse also accelerated the release
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assay (Cell-Titer 96 Proliferation Kit, Promega, Madison, WI). The results indicated that cells proliferated in hydrogel after culture of 2 and 3 days and the cell number increased ca. 15% at day 3, which was significant with p < 0.05 (Figure 8). Conclusions
Figure 7. Release of blue dextran from HA-DTPH in DPBS containing different concentrations of DTT at pH 7.4. Films were prepared with 3.0% (w/v) solutions of HA-DTPH (Mw ) 136 kDa, Mn ) 61 kDa, polydispersity index ) 2.23, SD ) 58%), and the films (n ) 3) were oxidized in air only and then further oxidized with 0.3% (v/v) H2O2 for 1 h.
Figure 8. Fibroblast proliferation in HA-DTPH hydrogel after in vitro culture of 0, 1, 2, and 3 days. The cells were encapsulated in 3.0% (w/v) HA-DTPH solution according to the methods described in the text, and the healthy cell number in the hydrogel (n ) 5 or 6) was determined by MTS assay. After in vitro culture of 2 and 3 days, the number of cells in the hydrogel increased significantly (p < 0.05).
of model drug (blue dextran) from films. For example, in 48 h, the release percentage of blue dextran from air-oxidized HA-DTPH film in DPBS at 37 °C at 300 rpm was less than 7%, while under the same conditions in DPBS with 100 U/mL HAse, 30% of the blue dextran was released with concomitant partial degradation of the film. After 48 h, approximately 36% of the film had been lost because of enzymatic digestion, as determined gravimetrically. In Situ Cell Encapsulation. The rapid gelation of HADTPH solution under physiological conditions exhibits potential utility for many biomedical applications, for example, wound healing, defect filling, prevention of postsurgical adhesions, and cell encapsulation for tissue repair. Murine fibroblasts were entrapped within a cross-linking HADTPH hydrogel, and the encapsulated cells were examined after 24 and 96 h of culture. Viable cells, indicated by green fluorescence upon F-DA staining, were evident after 96 h of culture. Fewer than 5% dead cells were observed as red fluorescence from PI staining (data not shown). Unlike twodimensional culture in flasks, the fibroblasts in the hydrogel maintained a round shape. In addition, clumps of cells, as well as individual cells, were observed in hydrogel. After in vitro culture for 1, 2, and 3 days, the number of viable cells residing in the hydrogel were determined by MTS
A novel disulfide cross-linking method was developed for the preparation of HA hydrogel films. The HA hydrogel was cross-linked through thiolated HA (HA-DTPH and HADTBH) by air oxidation with subsequent additional oxidation by dilute H2O2 solution increasing the number of cross-links. The thiols of HA-DTPH had lower pKa than that of HADTBH and thus were more readily reoxidized to give hydrogels. The disulfide linkage is cleavable in the presence of DTT. This disulfide cross-linked HA hydrogel can be prepared under physiological conditions, and no cross-linking agents were needed, and no byproducts were produced. Murine fibroblasts were encapsulated in situ during gelation of HA-DTPH, and the cells remained viable and proliferated in hydrogel during 3 days of cell culture in the threedimensional matrix. These unique properties suggest that thiolated HA has many potential clinical applications in wound healing and tissue repair. Acknowledgment. We thank The University of Utah and the NIH (Grant DC04663 to S. D. Gray and G. D. Prestwich) for financial support. Valuable discussions with Dr. J. Shelby, Ms. K. R. Kirker, and Dr. Gray contributed to the design and evaluation strategies for these new materials. References and Notes (1) Knudson, C. B.; Knudson, W. Semin. Cell DeV. Biol. 2001, 12, 6978. (2) Laurent, T. C.; Laurent, U. B.; Fraser, J. R. Ann. Rheum. Dis. 1995, 54, 429-432. (3) Fraser, J. R.; Laurent, T. C.; Laurent, U. B. J. Intern. Med. 1997, 242, 27-33. (4) Dowthwaite, G. P.; Edwards, J. C.; Pitsillides, A. A. J. Histochem. Cytochem. 1998, 46, 641-651. (5) Hardwick, C.; Hoare, K.; Owens, R.; Hohn, H. P.; Hook, M.; Moore, D.; Cripps, V.; Austen, L.; Nance, D. M.; Turley, E. A. J. Cell Biol. 1992, 117, 1343-1350. (6) Collis, L.; Hall, C.; Lange, L.; Ziebell, M.; Prestwich, R.; Turley, E. A. FEBS Lett. 1998, 440, 444-449. (7) Cheung, W. F.; Crue, T. F.; Turley, E. A. Biochem. Soc. Trans. 1999, 27, 135-142. (8) Toole, B. P. J. Intern. Med. 1997, 242, 35-40. (9) Gerdin, B.; Hallgren, R. J. Intern. Med. 1997, 242, 49-55. (10) Saettone, M. F.; Monti, D.; Torracca, M. T.; Chetoni, P. J. Ocul. Pharmacol. 1994, 10, 83-92. (11) Moore, A. R.; Willoughby, D. A. Int. J. Tissue React. 1995, 17, 153156. (12) Morimoto, K.; Yamaguchi, H.; Iwakura, Y.; Morisaka, K.; Ohashi, Y.; Nakai, Y. Pharm. Res. 1991, 8, 471-474. (13) Miller, J. A.; Ferguson, R. L.; Powers, D. L.; Burns, J. W.; Shalaby, S. W. J. Biomed. Mater. Res. (Appl. Biomater.) 1997, 38, 25-33. (14) Gowland, G.; Moore, A. R.; Willis, D.; Willoughby, D. A. Clin. Drug InVest. 1996, 11, 245-250. (15) Juhlin, L. J. Intern. Med. 1997, 242, 61-66. (16) Davidson, J. M.; Nanney, L. B.; Broadley, K. N.; Whitsett, J. S.; Aquino, A. M.; Beccaro, M.; Rastrelli, A. Clin. Mater. 1991, 8, 171177. (17) Prestwich, G. D.; Vercruysse, K. P. Pharm. Sci. Technol. Today 1998, 1, 42-43. (18) Prestwich, G. D. Glycoforum. http://glycoforum.gr.jp/science/hyaluronan/HA18/HA18E.html (2001).
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