Synthesis and in Vitro Degradation of New ... - ACS Publications

Bioconjugate Chem. 1997, 8, 686−694

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Synthesis and in Vitro Degradation of New Polyvalent Hydrazide Cross-Linked Hydrogels of Hyaluronic Acid Koen P. Vercruysse,†,‡ Dale M. Marecak,†,‡ James F. Marecek,‡ and Glenn D. Prestwich*,†,‡ Department of Medicinal Chemistry, Room 201, The University of Utah, 30 South, 2000 East, Salt Lake City, Utah 84112-5820, and Department of Chemistry, The University at Stony Brook, Stony Brook, New York 11794-3400. Received June 5, 1997X

New polyvalent hydrazide cross-linkers were synthesized, characterized, and used to prepare hydrazide cross-linked hydrogels derived from hyaluronic acid (HA). First, the chemical synthesis and characterization of the di-, tri-, tetra-, penta-, and hexahydrazides are presented. Second, HA concentration, buffer type and concentration, and ratio of HA to carbodiimide to cross-linker were varied to obtain HA-hydrogels with different chemical and physical properties. Third, two new assays are described to monitor the stability of HA-hydrogels toward hyaluronidase (HAse) and other media. These assays were used to evaluate the stability of cross-linked HA-hydrogels to HAse solutions and different pH values. Hydrophobic cross-linkers gave the most stable gels, and the susceptibility of the gels to HAse was independent of cross-linker concentration. HAse does not significantly penetrate the HA-hydrogels and acts primarily at the gel-solution interface. The HA-hydrogels are stable in acid environments and dissolve gradually above pH 7.0.

INTRODUCTION

Hyaluronic acid (Figure 1) is a naturally occurring linear polysaccharide that is abundant in the vitreous and in synovial fluid and plays pivotal roles in wound healing, cell differentiation, and cell motility. In addition, aberrant HA1 receptors are involved in cancer metastasis (1, 2). A component of the extracellular matrix, it is a fully biocompatible candidate for modification with drugs and other effector molecules. The use of biocompatible polymers in the treatment of various ailments has expanded rapidly in the past two decades (3). Moreover, derivatization of such polymers with reporter groups (4) and drugs (5, 6) has emerged as a powerful method for controlling delivery and release of these various compounds. Small drug molecules can be linked to the polymer by a method that allows controlled release of the free bioactive group. Highly water-soluble polymers are of added benefit in helping increase the amount of a hydrophobic drug that can be effectively delivered into a living system. The general features of these systems must therefore include complete biocompatibility, water solubility, ability to be chemically modified to allow high substrate loading with a designed release profile, and an economical, scalable process chemistry. Hydrogels have many unique properties and advantages for the development of novel controlled drug delivery systems (7). HA and its derivatives have been used as drug delivery systems with a wide variety of drugs (8, 9). To extend the utility of HA hydrazide derivatives (10-12) to include hydrogels with selected physical properties, we required a selection of polyfunc* Author to whom correspondence should be addressed [telephone (801) 585-9051; fax (801) 585-9053; e-mail gprestwich@ deans.pharm.utah.edu]. † The University at Stony Brook ‡ The University of Utah. X Abstract published in Advance ACS Abstracts, August 15, 1997. 1 Abbreviations: EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; CAS-RN, Chemistry Abstracts Service Registry Number; HA, hyaluronic acid; HAse, hyaluronidase; PEG, poly(ethylene glycol); XL, cross-linker.

S1043-1802(97)00109-2 CCC: $14.00

Figure 1. Structure of native hyaluronic acid.

tional cross-linking agents (13, 14). Herein we describe the synthesis and applications of hydrazide-containing cross-linkers in which the length, hydrophilicity, and number of functional groups have been varied. We also describe the effects of varying pH, buffer type, HA concentration, and cross-linker/carbodiimide/HA ratios to obtain hydrogels with desired physicochemical properties. In vivo, enzymes play a major role in the biodegradation process of biomaterials. Even the most inert polymers can undergo some degradation under physiological conditions (7). In vivo, HA is degraded by HAse, which is ubiquitous in cells and in serum (15, 16). In vitro, the biodegradation of hydrogels has been studied by monitoring the release of microspheres from a matrix (17), by monitoring the release of radiolabeled compounds (18, 19), by monitoring the loss of weight or swelling properties (20, 21), or by visual inspection (22-25). In this paper, we also describe the biodegradation of new crosslinked HA-hydrogels. Two new methods for monitoring the degradation of the HA-hydrogels by HAse were developed and used to examine the effect of the chemical composition of the gels upon their degradation. MATERIALS AND METHODS

General. Fermentation-derived hyaluronan (HA, sodium salt) was provided by Clear Solutions Biotechnology, Inc. (Stony Brook, NY). 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), succinic dihydrazide, fastgreen FCF, and carboxylic acids and esters for the hydrazide syntheses (used without purification) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Bovine testicular hyaluronidase (HAse; 880-1000 units/mg) and bis-tris hydrochloride were obtained from Sigma Chemi© 1997 American Chemical Society

Polyvalent Hydrazide Cross-Linked Hyaluronate Hydrogels

cal Co. (St. Louis, MO). Coomassie Brilliant blue R250 was obtained from Fisher Scientific Co. (Santa Clara, CA). Adipic dihydrazide was obtained from Eastman Kodak Co. (Rochester, NY). Suberic and terephthalic dihydrazide were obtained from Lancaster Synthesis, Inc. (Windham, NH). Methyl acrylate, ethylenediamine, and 1,4-butanediamine were purified by distillation under nitrogen. Other amines were used as received. The H2N-(CH2CH2-O)n-CH2CH2NH2 was purchased from Shearwater Polymers, Inc. (Huntsville, AL) and had a nominal molecular weight of 3400. All other chemicals used were of analytical grade. Analytical Instrumentation. 1H-NMR spectra were obtained on either a Bruker AC-250 or a GE QE-300 spectrometer at 250 or 300 MHz, respectively. 13C-NMR spectra were measured using a Bruker AM-300 spectrometer at 75 MHz. IR spectra were recorded using a Mattson Galaxy Model 3000 FTIR spectrometer. General Procedure for the Preparation of Hydrazides. Free carboxylic acid (5 g) was dissolved in dry alcohol (methanol or ethanol) (50 mL) containing 1-3 drops of concentrated H2SO4, refluxed under nitrogen for 1 h, concentrated under reduced pressure to 265 °C; EI-MS (M+) 252; CAS-RN 36997-31-6. Tri(methyl propanoate)amine [β-Alanine, N,N-Bis(3methoxy-3-oxopropyl)-methyl ester] (11): CAS-RN 333009-4. This ester was prepared in the same manner as 2

All CAS-RN have been provided by the author.

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the first stage of the starburst dendrimer synthesis as described by Tomalia (27, 28). General Procedure for the Preparation of the Tetramethyl r,ω-Alkyldiaminotetrapropanoates. Methyl acrylate (12.9 g, 150 mmol, 13.5 mL) was dissolved in 30 mL of methanol and cooled (4 °C) under nitrogen. The diamine (30 mmol), dissolved in 5-20 mL of methanol depending on its solubility, was added dropwise over 5 min to the stirred acrylate solution. The ice was allowed to melt, and the solution was stirred at room temperature overnight. The solvent and excess methyl acrylate were evaporated under reduced pressure, and the oil remaining was stirred in vacuo for a few hours to remove any residual volatiles. The crude colorless oily products were isolated in essentially quantitative yield and used without further purification for conversion to the hydrazide (26, 29). Tetramethyl 1,2-Ethanediyldiamino-N,N,N′,N′-tetrapropanoate (12): 1H NMR (CDCl3) δ 3.58 (s, 12H, CH3), 2.68 (t, 8H, J ) 7.9 Hz, CH2), 2.40 (s, 4H, CH2), 2.35 (t, 8H, J ) 7.9 Hz, CH2); 13C NMR (CDCl3) δ 172.7, 52.1, 51.3, 49.6, 32.5. Tetramethyl 1,4-Butanediyldiamino-N,N,N′,N′-tetrapropanoate (13): 1H NMR (CDCl3) δ 3.58 (s, 12H, CH3), 2.67 (t, 8H, J ) 11.4 Hz, CH2), 2.36 (t, 8H, J ) 11.4 Hz, CH2), 2.32 (m, 4H, CH2), 1.30 (m, 4H, CH2); 13C NMR (CDCl2) δ 172.9, 53.5, 51.3, 49.1, 32.4, 24.9. Tetramethyl 1,6-Hexanediyldiamino-N,N,N′,N′-tetrapropanoate (14): 1H NMR (CDCl3) δ 3.49 (s, 12H, CH3), 2.58 (t, 8H, J ) 7.2 Hz, CH2), 2.26 (t, 8H, J ) 7.2 Hz, CH2), 2.19 (t, 4H, J ) 7.2 Hz, CH2), 1.23 (m, 4H, CH2), 1.07 (m, 4H, CH2); 13C NMR (CDCl3) δ 172.7, 53.5, 51.2, 49.0, 32.3, 27.0, 26.9. Tetramethyl 1,12-Dodecanediyldiamino-N,N,N′,N′-tetrapropanoate (15): 1H NMR (CDCl3) δ 3.63 (s, 12H, CH2), 2.73 (t, 8H, J ) 7.2 Hz, CH2), 2.40 (t, 8H, J ) 7.2 Hz, CH2), 2.36 (t, 4H, J ) 7.3 Hz, NCH2), 1.37 (m, 4H, CH2), 1.22 (m, 16H, CH2); 13C NMR (CDCl3) δ 172.9, 53.7, 51.3, 49.1, 32.4, 29.5, 29.45, 29.4, 27.2, 27.0. Tetramethyl 1,2-Dihydroxyethanediyl(diethylaminoN,N,N′,N′-tetrapropanoate) (16): 1H NMR (CDCl3) δ 3.51 (s, 12H, CH3), 3.42 (s, 4H, OCH2CH2O), 3.37 (t, 4H, J ) 6.8 Hz, OCH2), 2.67 (t, 8H, J ) 8.0 Hz, CH2), 2.52 (t, 4H, J ) 6.8 Hz, OCH2), 2.30 (t, 8H, J ) 8.0 Hz, CH2); 13C NMR (CDCl3) δ 172.7, 70.2, 69.4, 53.0, 51.3, 49.7, 32.4. Pentamethyl Spermidine-N,N,N′,N′′,N′′-pentapropanoate [Pentamethyl (N′-(3-Aminopropyl)-1,4butanediamine)-N,N,N′,N′′,N′′-pentapropanoate] (17). Methyl acrylate (4.30 g, 50 mmol) was dissolved in 10 mL of methanol and cooled in an ice bath under nitrogen. Spermidine (1.09 g, 7.5 mmol) in 3 mL of methanol was added dropwise over 2 min to the stirred solution. Stirring was continued at ice bath temperature for 1 h and then at room temperature for 16 h. The solvent and excess methyl acrylate were removed in vacuo to give 4.06 g (96%) of a colorless oil: 1H NMR (CDCl3) δ 3.49 (s, 15H, CH3), 2.58 (t, 10H, J ) 7.8 Hz, CH2), 2.26 (t, 10H, J ) 7.8 Hz, CH2), 2.21 (m, 8H, N-CH2), 1.37 (m, 2H, CH2), 1.20 (m, 4H, CH2); 13C NMR (CDCl3) δ 172.96, 172.74, 53.51, 53.48, 51.54, 51.22, 51.19, 49.10, 49.04 (probably two overlapping peaks), 32.34, 32.28, 32.07, 24.80, 24.68, 24.64. Hexamethyl Cascade 6 Aza(3):1-azapropane:propanoate (18). Methyl acrylate (18.1g, 210 mmol, 19 mL) was dissolved in 40 mL of methanol and cooled at 4 °C under nitrogen. Tris(2-aminoethyl)methane (4.39 g, 30 mmol) dissolved in 3 mL of methanol was added with stirring for 3 min. Stirring was continued at 4 °C for 2 h and at room temperature overnight, and then solvent and excess methyl acrylate were removed in vacuo at

688 Bioconjugate Chem., Vol. 8, No. 5, 1997

room temperature to give 19.6 g (98%) of a yellow oil: 1H NMR (CDCl ) δ 3.58 (s, 18H, CH ), 2.70 (t, 12H, J ) 3 3 7.1 Hz, CH2), 2.42 (bs, 12H, -NCH2CH2N-), 2.36 (t, 12H, J ) 7.1 Hz, CH2); 13C NMR (CDCl3) δ 172.6, 53.2, 52.0, 51.2, 49.5, 32.3. Tetramethyl PEG-Diamine-N,N,N′,N′-tetrapropanoate (19). PEG-diamine (1.02 g, 0.3 mmol) was dissolved in 5 mL of dry methanol with gentle warming. The solution was cooled to room temperature under nitrogen and methyl acrylate (0.16 g, 1.8 mmol, 0.17 mL) added. The solution was stirred under nitrogen at room temperature for 18 h, and then the solvent and excess methyl acrylate were evaporated in vacuo at room temperature to give 1.1 g of a white solid. 1H-NMR (CDCl3) showed two triplets at 2.82 and 2.45 ppm, indicating that the propanoate was present. Tris(propanoic hydrazide)amine (20; CAS-RN 91933-31-2 (β-Alanine, N,N-Bis(3-hydrazino-3-oxopropyl)-, Hydrazide). To a 40 °C solution of hydrazine hydrate (40 mL, 0.80 mol) in methanol (30 mL) was slowly added a solution of tris(methyl propanoate) amine (40 mmol, 12.4 g) in methanol (30 mL). The solution was stirred at 40 °C for 1 h, then cooled to room temperature for 1 h, concentrated in vacuo to 20 mL, and recrystallized from isoamyl alcohol at 0 °C to give 12.0 g (97%) of a crystalline white solid, mp 56-57 °C. General Procedure for the Preparation of Acyl Hydrazides [1,2-Ethanediyldiamino-N,N,N′,N′-tetrakis(propanoic hydrazide) (21)]. Tetramethyl ethanediaminetetrapropanoate (12) (2.02 g, 5.0 mmol) was dissolved in 3 mL of absolute ethanol, and hydrazine hydrate (2.00 g, 40 mmol, 2.00 mL) was added. The solution was stirred under nitrogen at 45 °C for 2 h and then cooled to room temperature; ethanol and excess hydrazine were removed in vacuo to give a viscous syrup. The crude material was dissolved in 15 mL of boiling 2-propanol and cooled slowly to room temperature (fast cooling caused the product to oil out) to give a white crystalline solid, which was isolated by filtration and then vacuum dried to give 1.95 g (95%) of 21: mp 140-141 °C (dec); 1H NMR (D2O) δ 2.74 (t, 8H, J ) 11.2 Hz, CH2), 2.52 (s, 4H, NCH2CH2N), 2.33 (t, 8H, J ) 11.2 Hz, CH2); 13C NMR (D O) δ 174.5, 50.7, 49.7, 31.5; HRMS calcd for 2 C14H32N10O4 405.2686, found 405.2700. 1,4-Butanediyldiamino-N,N,N′,N′-tetrakis(propanoic hydrazide) (22) was prepared by the general procedure using 2.16 g (5.0 mmol) of tetraester 13 in 3 mL of ethanol. The product separated as a solid during the reaction. Evaporation of the solvent and excess hydrazine left a white powder that was crystallized from 100 mL of 2-propanol to give 2.13 g (98%) of 22: mp 126-127 °C; 1H NMR (D2O) δ 2.64 (t, 8H, CH2), 2.34 (bs, 4H, CH2), 2.23 (t, 8H, CH2), 1.29 (bs, 4H, CH2); 13C NMR (D2O) δ 174.7, 53.3, 49.3, 31.4, 24.5; HRMS calcd for C16H36N10O4 433.2999, found 433.2976. 1,6-Hexanediyldiamino-N,N,N′,N′-tetrakis(propanoic hydrazide) (23) was prepared by the general procedure using 2.30 g (5.0 mmol) of tetraester 14 in 4 mL of ethanol. Evaporation of the solvent and excess hydrazine left a viscous syrup that was dissolved in 5 mL of 2-propanol, concentrated in vacuo, redissolved in 30 mL of boiling 2-propanol, and cooled (4 °C) overnight. The mixture was then cooled to -10 °C for 2 h, and the solid was isolated by filtration and washed with cold 2-propanol to give 2.34 g of a sticky white solid. Recrystallization from 2-propanol gave 2.0 g (87%) of 23: mp 105108 °C (dec); 1H NMR (D2O) δ 2.65 (t, 8H, J ) 7.2 Hz), 2.32 (m, 4H), 2.24 (t, 8H, J ) 7.2 Hz), 1.32 (m, 4H), 1.15

Vercruysse et al.

(m, 4H); 13C NMR (D2O) δ 174.7, 53.3, 49.3, 31.3, 27.4, 26.2; HRMS calcd for C18H40N10O4 461.3312, found 461.3297. 1,12-Dodecanediyldiamino-N,N,N′,N′-tetrakis(propanoic hydrazide) (24) was prepared from 1.36 g (2.5 mmol) of tetraester 15 and 1.00 g (20 mmol) of hydrazine hydrate in 2 mL of ethanol. After heating for 1 h, the solution set to a waxy solid; the solvent and excess hydrazine were removed in vacuo using 5 mL of 2-propanol. The crude product was dissolved in 50 mL of boiling 2-propanol, cooled slowly to room temperature (crystallization occurred), stored at 5 °C overnight, and filtered, and the solid was washed with 2 × 5 mL of propanol and vacuum dried to give 1.10 g of 24 (81%): mp 124-128 °C (dec); 1H NMR (D2O) δ 2.66 (t, 8H, J ) 7.7 Hz, CH2), 2.33 (m, 4H, NCH2), 2.26 (t, 8H, J ) 7.7 Hz, CH2), 1.33 (m, 4H, CH2), 1.16 (m, 16H, CH2); 13C NMR (D2O) δ 174.5, 53.6, 49.4, 31.4, 29.7, 29.6, 27.8, 26.3; HRMS calcd for C24H52N10O4 545.4251, found 545.4255. 1,2-Dihydroxyethanediyl[diethylamino-N,N,N′,N′-tetrakis(propanoic hydrazide)] (25) was prepared by the general procedure from 2.46 g (5.0 mmol) of tetraester 16 in 6 mL of ethanol. The crude product was crystallized twice from 25 mL of boiling 2-propanol by cooling to -20 °C to give 2.0 g (80%) of 25, as a syrupy liquid at room temperature: 1H NMR (D2O) δ 3.53 (s, 4H, OCH2CH2O), 3.45 (t, 4H, CH2O), 2.69 (t, 8H, CH2), 2.55 (t, 4H, NCH2), 2.23 (t, 8H, CH2); 13C NMR (D2O) δ 174.5, 70.3, 68.8, 52.5, 49.8, 31.4; HRMS calcd for C18H40N10O6 493.3211, found 493.3217. Spermidine Pentapropanoic Pentahydrazide (26). A solution of pentamethyl spermidine pentapropanoate (17, 1.15g, 2.0 mmol) in 3 mL of ethanol containing hydrazine monohydrate (1.00 g, 20 mmol) was stirred at 45 °C for 2 h, ethanol and excess hydrazine were removed in vacuo at room temperature, and the syrup remaining was concentrated using 5 mL of 2-propanol. The crude product was dissolved in 12 mL of boiling 2-propanol and cooled to -10 °C; the solvent was decanted from the solid and the crystallization repeated. Solid 26 (1.0 g, 86%) was dried in vacuo at -10 °C, but was a syrup above 0 °C: 1H NMR (D2O) δ 2.60 (t, 10H, CH2), 2.31 (m, 8H, NCH2), 2.23 (t, 10H, CH2), 1.48 (m, 2H, CH2), 1.29 (m, 4H, CH2); 13C NMR (D2O) δ 174.58, 53.51, 53.25, 51.69, 51.46, 49.41, 49.32, 31.41, 31.33, 30.97, 24.50, 24.27, 23.09; HRMS calcd for C22H49N13O5 576.4058, found 576.4023. Cascade 6 Aza(3):1-azapropane:propanoic Hexahydrazide (27). Hexamethyl cascade 6 aza(3):1-azapropane: propanoate (18, 3.31 g, 5.0 mmol) was dissolved in 5 mL of absolute ethanol, and hydrazine hydrate (3.00 g, 60 mmol, 3.0 mL) was added. The solution was stirred at 45 °C for 2 h, and the ethanol and excess hydrazine were removed in vacuo. The crude oil was dissolved in 30 mL of boiling 2-propanol, cooled to 40 °C, chilled quickly to -78 °C to give a filterable solid that was isolated by filtration through a prechilled funnel at -10 °C, and washed with cold 2-propanol. It was dried under vacuum at -10 °C to give 2.85 g (85%) of 27 (the material melted at temperatures above 0 °C): 1H NMR (D2O) δ 2.65 (t, 12H, J ) 7.1 Hz, CH2), 2.46 (bs, 12H, NCH2CH2N), 2.23 (t, 12H, J ) 7.1 Hz, CH2); 13C NMR (D2O) δ 173.6, 51.0, 49.7, 48.9, 30.6; HRMS calcd for C24H54N16O6 663.4491, found 663.4469. PEG-Diaminetetrapropanoic Tetrahydrazide (28). Tetramethyl PEG-diaminetetrapropanoate (19, 1.1 g, 0.3 mmol) was dissolved in 3 mL of absolute ethanol, and hydrazine hydrate (0.12 g, 2.4 mmol) was added. The



solution was stirred at 45 °C for 3.5 h and at room temperature overnight, and solvent and excess hydrazine were removed in vacuo to give 1.1 g as a white powder. The 1H-NMR (CDCl3) had two triplets at 2.39 and 2.75 ppm and two smaller triplets at 2.55 and 2.84 ppm (the large PEG peak was evident at 3.55 ppm as a broad absorption). Cross-Linking of HA. The cross-linker (XL) was added to an aqueous solution of sodium hyaluronate. The pH was adjusted to the desired value by dissolving the appropriate amount of bis-tris HCl or by addition of 0.1 N HCl. EDC was dissolved in water and added to the mixture. After stirring vigorously for 1 min, the mixture was left at room temperature to gel. All XL equivalents (mol equiv) mentioned in this paper are expressed as the molar ratio of XL to the carboxylic functions present in the glucuronic acid subunits of HA. Degradation of HA-Hydrogel by HAse: Plate Assay. After the reagents were mixed, the gelling solution was poured into a Petri dish and left at room temperature for 24 h to complete gelation. The gel was then washed with water, and six 0.5-cm-diameter wells were excised with a cork borer. One well was loaded with 25 µL of buffer (30 mM citric acid/150 mM Na2HPO4/150 mM NaCl; pH 6.3), and the other wells were loaded with 25 µL of HAse samples dissolved in the same buffer. The gel was incubated at 37 °C for 24 h, at which point the remaining gel was stained with a solution of fastgreen FCF. Clear rings of digested gel could be observed against a green background. Degradation of HA-Hydrogel by HAse: Spectrophotometric Assay. The cross-linking of HA was performed in the presence of 40 µM Coomassie Brilliant blue R250. After all of the reagents were mixed, 1 mL of the gelling mixture was placed in a plastic cuvette such that the volume of gel did not block the light path. When gelation was complete (24 h of standing at room temperature), the gel was washed and 1 mL of enzyme solution was overlayered on top of the gel, in the light path of the spectrophotometer. The absorbance of the hyperphase solution was monitored at 590 nm as a function of the reaction time. pH Stability Experiments. HA was cross-linked in the presence of 50 µM Coomassie Brilliant blue R250. Upon gelation, the gels were cut in pieces of approximately 2 g and washed several times with water (pH 5.6). After the last washing, the water was replaced by a 50 mM phosphate buffer solution (mono- or dibasic phosphate or a mixture of both) with a pH of 4.5, 6.5, 6.9, 7.25, 7.65, or 9.1. One piece of gel was stored in 0.1 N HCl solution (pH 1.3). All samples were incubated at 37 °C. At several time intervals, 2-mL samples were taken, the absorbance was measured at 590 nm, and the samples were returned to the solutions again. RESULTS

Synthesis of Polyvalent Hydrazides. Mono-, di-, and trihydrazides (Figure 2) were prepared by esterification followed by reaction with excess hydrazine hydrate in alcohol by modification of standard procedures (29). The polyvalent hydrazides (Figure 3) were prepared by first reacting a polyamine with a 25% excess of methyl acrylate (26). The resulting polymethyl alkylpolyaminopolypropanoates were treated with an excess of hydrazine hydrate in absolute ethanol to yield the acyl hydrazides. The crude products could usually be crystallized from 2-propanol, albeit often at low temperature. Hydrazinolysis reactions proceeded in near quantitative yields, and recrystallization gave high recovery of purified

Figure 2. Selected commercially available hydrazides and synthetic mono-, di-, and trihydrazides. Table 1. Limiting Reaction Parameters and Physicochemical Properties of Resulting HA-Hydrogelsa reagents HA 0.1 N HCl (fast reaction) bis-tris HCl, pH 4.75 (slow reaction) cross-linker (XL) EDC buffer

pourable gels

solid gels

2.5 mg/mL pH 4.7

8.0 mg/mL pH 3.5

100 mM

500 mM

0.1 mol equiv 0.5 mol equiv tris (pKa ) 8.1)

1.5 mol equiv 1.0 mol equiv bis-tris (pKa ) 6.5)

a A variety of gels were prepared with intermediate properties by varying the parameters indicated.

polyhydrazides. With the exception of trimesic trihydrazide, each of the polyhydrazides was water soluble and was readily employed for HA modification and gelation reactions. Cross-Linking of HA. Depending on the reaction conditions, gels with physicochemical properties ranging from soft and pourable to solid and readily shattered were obtained. The pH of the reaction was maintained between 3.5 and 4.7 by addition of either 0.1 N HCl or 100500 mM bis-tris HCl. Gels would form very rapidly (2040 s) with HCl as the reaction initiator, but would solidify in 5-30 min when bis-tris HCl was used. Table 1 presents an overview of the limiting reaction parameters involved and their effect on the properties of the gels obtained. Hydrogels with properties ranging between both extremes given in Table 1 were obtained by varying the parameters between the values indicated. Plate Assay for Degradation of HA-Hydrogels. Figure 4A illustrates diagrammatically the protocol for this assay. Following hydrogel formation, circular wells were excised and loaded with either buffer () blank) or an HAse solution. After incubation at 37 °C for 24 h and staining with fastgreen FCF solution, clear rings of digested gel appear against the colored background (Figure 4B). Importantly, the assay is highly reproducible as seen in Figure 4B, which compares five wells containing identical HAse concentrations with a single blank. When using different amounts of HAse, the diameter of the digested rings can be plotted against the amount of enzyme added. A linear relationship was obtained, and the slope reflected the sensitivity of the


Vercruysse et al.

Figure 3. Structures of amine-based polyhydrazides.

Figure 5. Degradation of HA-gels by HAse using the plate assay. Comparison of the degradation of gels made with XL-6 and XL-23 (see Figures 2 and 3) is shown by the effect of the amount of XL-6 and the amount of HA in the gel upon their degradation by HAse. The slope of the plot of the average (n ) 6) diameter (in cm) of the digested wells as a function of the amount of HAse (in units) is plotted against the type of gel investigated.

Figure 4. (A, top) Schematic representation of the plate assay for the investigation of the degradation of HA-hydrogels by HAse as described under Materials and Methods. (B, bottom) Photograph (side angle view) showing endpoint of representative plate assay with one blank (at right) and five identical HAse solutions.

gel toward HAse, with a larger slope indicating greater susceptibility to HAse digestion. Figure 5 illustrates a comparison of the degradation of two hydrogels (4.8 mg/ mL HA) prepared using 0.3 mol equiv of cross-linker (XL) 6 or XL-23. The diamine-derived tetrahydrazide XL-23 afforded a more readily degraded gel than the disulfidecontaining dihydrazide XL-6. Next, using XL-6, the effects of the amount of crosslinker and the amount of HA in the gel on the degradation by HAse were examined. For each gel recipe, six Petri plates were prepared and the wells were loaded with different amounts of HAse, varying from 0 to 5.5 units. The average diameters of the digested rings

(standard deviations varied from 2.4 to 4.2%) were plotted as a function of the amount of HAse added; linear regressions (r2 ranged from 0.93 to 0.99) were performed. Figure 5 summarizes the slope values obtained for the gel recipes investigated. Decreasing the HA concentration made the gels more susceptible to HAse degradation, but doubling the XL concentration at a given HA concentration had no effect on the gel degradation. On the basis of these results, the degradation of HAgels made with 10 different types of cross-linkers (XL-1, -2, -3, -4, -6, -9, -21, -22, -23, -24; see Figures 2 and 3) was investigated. Gels containing 4.8 mg/mL HA and 0.5 mol equiv cross-linker were prepared in Petri dishes. Six wells were excised, and 25 µL of buffer () blank) was added to one well, while 25 µL of HAse solution (104 units/mL) was added () addition of 2.6 units) to the other five wells. After incubation at 37 °C for 24 h, the gels were stained and the diameters of the wells were measured (see Figure 4B). The percentage increase in diameter vs the blank was averaged (n ) 5) for each type



Figure 6. Degradation of HA-gels by HAse using the plate assay. A comparison of the effect of 10 different types of crosslinker on the degradation of the HA-gel by HAse is shown. Gels were made containing 4.8 mg/mL HA and 0.5 mol equiv crosslinker. The degradation was investigated at a single enzyme concentration (104 units/mL), and the average (n ) 5) diameter of the digested rings was compared to the diameter of the blank (see Figure 4B). The percent increase in diameter is plotted as a function of the type of cross-linker. For chemical structures and numbering of the cross-linkers, see Figures 2 and 3.

of gel, and the results obtained are plotted in Figure 6. Hydrophobic cross-linkers such as XL-2, -3, and -4 and the disulfide XL-6 produced gels that showed the greatest resistance to degradation. Indeed, the terephthalicderived material using XL-4 showed little or no degradation in 24 h. In contrast, the more hydrophilic dihydrazide and tetrahydrazide linkers showed higher susceptibility to HAse. The tartrate-derived linker XL-9 gave the least stable gel. Spectrophotometric Assay for Degradation of HA-Hydrogels. Figure 7 illustrates the scheme for the basic assay as well as two modifications discussed below. The displacement technique described under Materials and Methods allows the transfer of reproducible amounts of gelling mixture on the bottom of the cuvettes. The average (( standard deviation; n ) 30) weight of gel was 1.010 ( 0.003 g. No Coomassie Brilliant blue R250 leached out of the gels when only buffer was added above the gel. When HAse solution was added, the gel was digested at the surface and the dye was released, diffusing into the hyperphase (Figure 7A). Figure 8 presents representative results obtained for the degradation of HA-gel prepared using 4 mg/mL HA and 0.45 mol equiv XL-23 and containing 40 µM Coomassie Brilliant blue 250 by HAse (180 units/mL). The assay was run with one control and three replicates. Only buffer was added above the control gel, and the enzyme solution was added on top of the other three gels. The absorption at 590 nm of the hyperphase was continuously monitored, averaged, and plotted as a function of the reaction time. This assay was not compatible with all cross-linkers. For example, the lower solubility of XL-6 resulted in precipitation during the degradation, thereby removing co-complexed Coomassie Brilliant blue R250. In these cases, no increase in absorbance could be detected as the degradation proceeded, unless the samples were mixed or shaken. Nonetheless, for the gels made with XL-23, reproducible release profiles could be obtained. Using this assay, the effect of the amount of this tetrafunctional cross-linker (0.15-0.60 mol equiv) on the degradation of the gel by HAse was investigated. Each experiment was performed in triplicate. Interestingly, the rate of degradation of the gels made with XL-23 appeared to be independent of cross-linker concentration

Figure 7. Degradation of HA-gels by HAse using the spectrophotometric assay. Panel A shows the basic assay configuration. Panels B and C illustrate two modifications employed to demonstrate the interfacial mode of degradation.

Figure 8. Degradation of HA-gels by HAse using the spectrophotometric assay. One milliliter of gel (containing 4 mg/mL HA, 0.45 mol equiv XL-23, and 40 µM Coomassie Brilliant blue R250) was poured on the bottom of a plastic cuvette. One milliliter of HAse solution (180 units/mL) was added on top of the gel, and the release of Coomassie Brilliant blue R250 was monitored at 590 nm as a function of the reaction time. (9) Data points when only buffer is added on top of the gel; (b) average of data points (n ) 3) when the HAse solution is added on top of the gel.

(results not shown), consistent with HA degradation occurring at the gel-solution interface. pH Stability Experiments. During the water wash of the gels containing 4.8 mg/mL HA, 1 mol equiv of either XL-6 or XL-23, and 50 µM Coomassie Brilliant blue R250, no dye leached out of the gels. The pieces of


Figure 9. Release of Coomassie Brilliant blue R250 from HAhydrogels at different pH values. Samples (2 g) of HA-hydrogel containing 4.8 mg/mL HA, 1 mol equiv XL-6 or XL-23, and 50 µM Coomassie Brilliant blue R250 were stored in 0.1 N HCl (pH 1.3) or in 50 mM phosphate solutions pH 4.5, 6.5, 6.9, 7.25, 7.65, and 9.1. The percentage of dye released from both types of gel after 4 days of storage at 37 °C is plotted as a function of the pH of the medium.

tetrafunctional cross-linked gel kept their shape during the washing process, but the gel pieces from XL-6 exhibited considerable swelling during the wash. Gels were incubated for several days at pH values from 1.3 to 9.1. At pH 9.1, the gels steadily dissolved in the surrounding medium. Much less dissolution could be observed at pH 7.65, and virtually no released dye could be detected when the gels were stored in media with pH values below 7.0. The gels remained stable under acidic conditions. Within 4 days, both types of gels stored at pH 9.1 were completely dissolved in the medium (100% release). Figure 9 illustrates the percent dye released after 4 days of storage of the gel samples at different pH values. DISCUSSION

Modification of carboxylic functions of small molecules and polymeric polycarboxylates as mediated by carbodiimides has been extensively investigated (30, 31). The extent of reaction is determined by the acid consumed; thus, the degree of cross-linking of HA is a function of reaction pH (see Table 1). The rapidity of the reaction is dependent on the amount of acid immediately available for the reaction. When the pH is adjusted with HCl (total dissociation), all of the acid is readily available for reaction, resulting in rapid, but often heterogeneous, gelation. When using buffer salts such as bis-tris HCl or tris HCl as the source of acid, the amount of acid available depends on the pH of the mixture and the pKa of the buffer component. During the reaction, the free acid is consumed and this is counteracted by the release of some additional acid from the buffer components, leading to more controlled gelation and more homogeneous hydrogels in the presence of amine buffer salts. The plate assay described in this paper was developed on the basis of the method of Richman and co-workers for the quantitation of HAse in biological samples (32). This approach allows comparison of different types of gels for their sensitivity toward HAse, or, in principle, in response to any other gel-degrading agent. The results presented in Figure 5 show a relative comparison of the degradation of gels made with two different types of cross-linker, different amounts of cross-linker, and different amounts of HA. The gel made with tetrahydrazide

Vercruysse et al.

XL-23 is more readily degraded by HAse than the gel made with disulfide dihydrazide XL-6. With XL-6, increasing the amount of cross-linker in the gel seemed not to affect the degradation, while decreasing the amount of HA in the gel seems to increase the extent of degradation by HAse. When the type of cross-linker was varied (Figure 6), extensive degradation was observed for the gel crosslinked with the tartaric dihydrazide crosslinker XL-9, while no degradation could be observed with the terephthalic dihydrazide cross-linker XL-4. Again, very little degradation was observed for gels made with the disulfide dihydrazide XL-6. These results suggest that the degradation of the HA-gels is slower for more hydrophobic cross-linkers. Although most types of gels investigated showed clear signs of degradation by HAse, the enzyme concentrations used (30-200 units/mL) were much higher than the concentration of HAse in, e.g., pooled serum (reported to be 2.6 units/mL) (33). The action of HAse on native HA has been extensively investigated using HAse concentrations ranging from 0.3 to 6.6 units/mL (34-36). The large difference in HAse concentration used to monitor the degradation of native HA or cross-linked HA suggests that the cross-linking reaction dramatically reduces the degradability of HA by HAse. It would be anticipated, therefore, that many of the hydrogels from these studies would show in vivo halflives of days or weeks at HAse concentrations 100-fold below those tested in this study. The spectrophotometric assay (Figure 7) has two main advantages over the plate assay. First, it requires smaller amounts of material for testing, and, second, the time kinetics of the degradation process can be monitored. However, as described under Results, it was not applicable to all types of gels. In principle, this assay could be modified to be performed as a “tablet dissolution test”. The release profiles of Coomassie Brilliant blue R250 (see Figure 8) can be approximated by a sigmoidal curve. This is probably due to the fact that the gel surfaces are not perfectly flat during the enzymatic degradation. As with the plate assay, no difference could be observed with different concentrations of the crosslinker XL-23 on the susceptibility of the gel to degradation by HAse. Two explanations can be advanced for the independence of degradation on cross-linker concentration. First, the HAse assays employed might be insensitive toward the subtle changes in the different gels prepared with different amounts of cross-linker. Conceivably, we have not tested low enough or high enough cross-linker concentrations to see the effect. Moreover, the very high concentrations of HAse used to investigate the degradation of the gels yield a very vigorous attack on the gels. Second, the enzyme could act only at the surface of the gel and would therefore be unaffected by the bulk of the gel body. This would be consistent with both the lack of concentration dependence and the importance of the hydrophobicity or hydrophilicity of the cross-linker on the susceptibility of the gel to HAse degradation. To test the second hypothesis, experiments were performed in which the cuvettes were filled with 2 mL of gelling mixture (Figure 7B) instead of the usual 1 mL. This amount of blue-stained gel blocks the light of the spectrophotometer, leading to continuous high absorbance readings. An HAse solution was added on top of the gel. It was reasoned that if the enzyme acted only on the surface of the gel, no change in absorbance readings would be observed until the gel was degraded, surface by surface, to a level that the light of the spectrophotometer was no longer blocked. If HAse dif-


fused into the gel body and acted within the gel, gradual changes in absorbance, due to the solubilization of the gel from below the interface, would be noticed before the level of the gel had regressed below the light of the spectrophotometer. The results observed (data not shown) were in agreement with surface erosion. That is, the change in absorbance was a sharp step function corresponding to solubilization until the level of the beam was reached. A second modification of the spectrophotometric assay was set up to address the same question. A cuvette was filled with 1 mL of a gelling mixture containing Coomassie Brilliant blue as usual. After gelation, 1 mL of the same gelling mixture containing no dye was added on top and allowed to gel. This yielded a cuvette with a piece of blue gel on the bottom and a piece of clear gel on top, the latter in the light path of the spectrophotometer (Figure 7C). Since the top gel is transparent, continuous low absorbances were measured. On top of this “sandwich-type” of gel, the enzyme solution was added. If the enzyme acted only at the surface, nothing would be observed until the top gel was degraded and the enzyme reached the bottom gel. If the enzyme diffused into the gel bodies, dye would be released from the bottom gel and diffuse into the top gel, leading to differences in the absorbance readings. Again, the results (data not shown) were in agreement with surface erosion. The results of the pH stability test (Figure 9) indicated that HA-hydrogels prepared with two different crosslinkers, disulfide dihydrazide XL-6 and diamine-based tetrahydrazide XL-23, were stable in solutions with pH values below 7.0. At pH values above 7.0, a gradual dissolution and consequent release of dye could be observed. Dye release was not due to an increased swelling of the gel but was solely due to a disintegration and dissolution of the gel. Despite the 3-fold difference of these two gels in susceptibility to HAse, the pH stability of the gels appeared to be independent of the nature of the cross-linker. This stability toward acid pH values and instability toward alkaline pH values could be exploited for transporting drugs, peptides, etc., safely through acid environments (e.g., the stomach) and subsequent release in more alkaline environments (e.g., the intestines). In conclusion, the bioconjugate chemistry previously developed for the chemical modification of HA with hydrazide functionalities has been applied to the crosslinking of this biopolymer to form hydrogels. Crosslinking conditions have been varied so that gels with a broad range of physicochemical properties can be obtained. Two new approaches were developed to investigate the biodegradation of these gels in vitro. With these methods, we observed that hydrophobic linkers provided gels with considerable resistance to HAse. Interestingly, hydrogel degradation was independent of cross-linker concentration, suggesting that degradation occurs only at the gel interface. The stability of these HA-hydrogels in acidic media and their slow dissolution above pH 7.0 suggest potential uses for controlled drug delivery in alkaline environments. ACKNOWLEDGMENT

We thank Collaborative Laboratories, Inc. (East Setauket, NY), Clear Solutions Biotechnology, Inc. (Stony Brook, NY), the U.S. Public Health Service, the Center for Biotechnology at The University at Stony Brook, and start-up funds from The University of Utah for financial support of this work. Pioneering contributions by Dr. T. Pouyani are gratefully acknowledged.

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