WO2009148405A1 - Formation of hydrogel in the presence of peroxidase and low concentration of hydrogen peroxide - Google Patents

Abstract

A hydrogel can be formed by mixing a polymer comprising a crosslinkable phenol group, horseradish peroxidase, and hydrogen peroxide (H₂O₂) in a solution. The H₂O₂ has a molarity of about 1 mM or less. The polymer may be a conjugate of hyaluronic acid and tyramine (HA-Tyr). The solution may also comprise a drug or a protein. The H₂O₂ molarity may be selected so that the hydrogel formed from the solution has a pre-determined storage modulus. It has been found that varying H₂O₂ molarity below about 1 mM does not substantially affect the gelation rate.

Description

FORMATION OF HYDROGEL IN THE PRESENCE OF PEROXIDASE AND

LOW CONCENTRATION OF HYDROGEN PEROXIDE

FIELD OF THE INVENTION

[0001] The present invention relates to hydrogel formation, particularly to formation of hydrogels in the presence of peroxidase and hydrogen peroxide.

BACKGROUND OF THE INVENTION

[0002] Phenol-containing hydrogels, such as hyaluronic acid-tyramine (HA-Tyr) hydrogels, are useful in many applications, including drug or protein delivery and tissue regeneration. Such hydrogels can be formed from a phenol-containing polymer, such as a HA-Tyr conjugate, in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂) as catalysts. In conventional techniques, the gelation rate and the crosslinking density in the hydrogel can be adjusted by changing the concentration of HRP or H₂O₂ in the precursor solution. Such a change typically affects both the gelation rate and crosslinking density.

SUMMARY OF THE INVENTION

[0003] According to an aspect of the present invention, there is provided a solution for forming a hydrogel, comprising a polymer comprising a crosslinkable phenol group; horseradish peroxidase (HRP), of an effective amount for crosslinking the polymer to form the hydrogel; and hydrogen peroxide (H₂O₂), having a molarity of about 1 mM or less. The H₂O₂ molarity may be selected so that the hydrogel formed from the solution has a pre-determined storage modulus. The hydrogel formed from the solution may have a storage modulus from about 10 to about 4000 Pa. The H₂O₂ molarity may be from about 0.146 to about 1.092 mM, such as from about 0.16 to about 0.728 mM. The solution may comprise from about 0.025 to about 1.24 unit/ml of the HRP, such as from about 0.032 to about 0.124 unit/ml of the HRP, or about 0.062 unit/ml of the HRP. The polymer may be a conjugate of hyaluronic acid and tyramine (HA-Tyr). A molar ratio of the H₂O₂ to the tyramine may be about 0.4 or less. The solution may comprise from about 0.1 to about 20 w/v% of the HA-Tyr conjugate, such as about 1.75 w/v% of the HA-Tyr conjugate. The hyaluronic acid has a molecular weight of about 90,000 Da. The tyramine in the solution may have a molarity of from about 0.42 to about 21 mM, such as about 2.57 mM. The solution may have a pH of about 4 to about 8. The solution may be at a temperature of about 293 to about 313 K. The solution may further comprise a drug. The solution may further comprise a protein. The solution may comprise water. The solution may comprise phosphate buffer saline.

[0004] In accordance with another aspect of the present invention, there is provided a method of forming a hydrogel, comprising mixing a polymer comprising a crosslinkable phenol group, HRP, and H2O2 in a solution, to form the hydrogel, the H2O2 having a molarity of about 1 mM or less. The solution may be as described in the preceding paragraph.

[0005] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] In the figures, which illustrate, by way of example only, embodiments of the present invention,

[0007] FIG. 1 is a schematic diagram representing a scheme for forming an HA- Tyr hydrogel, exemplary of an embodiment of the present invention;

[0008] FIGS. 2, 3, 4 and 5 are line graphs showing representative measurement results obtained from samples prepared according an exemplary embodiment of the present invention; and

[0009] FIG. 6 is a bar graph showing swelling ratio measured from different samples. DETAILED DESCRIPTION

[0010] In brief overview, it has been surprisingly discovered that in the formation of a phenol-containing hydrogel in the presence of horseradish peroxidase (HRP) and a low concentration of hydrogen peroxide (H₂O₂), the crosslinking density in the formed hydrogel can be conveniently varied by varying the concentration of H₂O₂, without substantially affecting the gelation rate.

[0011] Some exemplary embodiments of the present invention are related to solutions for forming a hyaluronic acid (HA)-tyramine (Tyr) hydrogel. The solution may be referred to as the precursor solution. The precursor solution contains a conjugate of HA and Tyr (HA-Tyr conjugate), HRP, and a low concentration of H₂O₂. The H₂O₂ may have a molarity of about 1 mM or less in the solution. The ratio of H₂O₂ to HRP in the solution may be about 1.8 mol/g or less. The molar ratio of H₂O₂ to tyramine moiety in the solution may be about 0.4 or less.

[0012] The HA-Tyr conjugate is crosslinkable to form a HA-Tyr hydrogel. The solution may have any suitable concentration of the HA-Tyr conjugate ([HA-Tyr]). In one embodiment, the concentration of HA-Tyr may be selected to obtain desired properties in either the precursor solution or the final hydrogel, or both. For example, the concentration of HA-Tyr may be selected to achieve a desired or suitable viscosity of the precursor solution, such as for injection. The suitable concentration of HA-Tyr may be dependent on the molecular weight of the HA used. For example, in one embodiment, the HA may have a molecular weight of about 9OK Da and [HA-Tyr] may be about 1.75 w/v% (weight-volume percent). When the HA has a higher molecular weight, the concentration may need to be lowered to achieve a similar viscosity. In some embodiments, the HA-Tyr concentration may vary in the range of about 0.1 to about 20 w/v%.

[0013] The degree of conjugation may also vary, such as from about 1 to about 50. In one embodiment, the degree of conjugation may be 6. The degree of conjugation or substitution (the number of tyramine molecules per 100 repeating units of HA) may be calculated from ¹H NMR measurement by comparing the ratio of the relative peak integrations of phenyl protons of tyramine (peaks at 7.2 and 6.9 ppm) and the methyl protons of HA (1.9 ppm). [0014] The concentration or molarity of the tyramine moiety ([Tyr]) in the solution may vary depending on the application. In one embodiment, the molarity of tyramine moiety in the solution may vary from about 0.42 to about 21 mM. For example, it may be about 2.57 mM.

[0015] The solution contains an effective amount of HRP for crosslinking the conjugate to form the hydrogel. The amount of HRP is typically specified or measured in units (U). One unit of HRP is the amount of HRP that catalyses the reaction of 1 μmol of the substrate in 1 minute under the standard conditions. For example, the solution may contain from about 0.025 to about 1.24 unit/ml, or from about 0.032 to about 0.124 unit/ml, of HRP. The concentration of HRP may also be expressed alternatively in g/ml. For example, HRP may be available in 100U/mg, in which case the solution may contain from about 0.25 to about 12.4 μg/ml of HRP, such as from about 0.32 to about 1.24 μg/ml.

[0016] The concentration of HRP may be selected in order to reach the gel point at a pre-determined time, as will be explained further below. In some embodiments, it may be advantageous to select an optimal HRP concentration to achieve the desired gelation time. For example, to obtain a gelation time of about 40 seconds, the solution may contain about 0.062 unit/ml or 0.62 μg/ml of HRP. When the concentration of HRP in the solution is high, for example at above about 0.032 unit/ml in one embodiment, varying the HRP concentration can change the gelation rate/speed without substantially changing the crosslinking density in the formed hydrogel. When the HRP concentration is low, its variation may affect the gelation rate and the crosslinking density. However, the crosslinking density may be further adjusted by varying the concentration of H₂O₂.

[0017] In one embodiment, the initial molarity of H₂O₂ in the solution prior to gelation ([H₂O₂]) may be about 1 mM or less, such as in the range of from about 0.146 to about 1.092 mM, or from about 0.16 to about 0.728 mM. In this case, when the molarity of tyramine is about 2.57 mM, the molar ratio of H₂O₂ to tyramine in the solution is about 0.4 or less, such as from about 0.057 to about 0.425, or from about 0.006 to about 0.283. [0018] The solution may further contain other desired additive such as a drug or a protein, depending on the application. The drug may include a therapeutic protein. For example, interferon, herceptin, or the like may be included in the solution. Non-therapeutic proteins, such as α-amylase, lysozyme, or the like, may also be included in the solution. The amount of other additive(s) may be selected depending on the particular application. It should be noted, however, that the addition of other additive(s) may impact on the mechanical strength or other properties of the formed hydrogel or on the formation process, such as the gelation rate. Thus, depending on which and how much other additive(s) are included, the concentration of H₂O₂ or HRP, or both, may need to be adjusted to off-set such impact.

[0019] The solution may be at a temperature of about 293 to about 313 K, such as at about 310 K (37 ⁰C).

[0020] The pH of the solution may be from about 4 to about 8, such as about 7.4.

[0021] The solvent in the solution may be any suitable solvent. In one embodiment, the solvent may include water. The solution may also include a phosphate buffer saline (PBS). Other suitable saline solvents may also be used. The solution may also include suitable cell culture medium, suitable buffer, or other solvents of desired properties.

[0022] Some exemplary embodiments of the present invention relate to methods of forming a hydrogel. A HA-Tyr conjugate, HRP, and H₂O₂ are mixed in a solution to form an HA-Tyr hydrogel. The solution may be any of the precursor solutions described above. In one embodiment, the molarity of H₂O₂ in the solution is about 1 mM or less. In another embodiment, the molar ratio of H₂O₂ to tyramine in the solution is about 0.4 or less.

[0023] The solution may be prepared in any suitable manner. In an exemplary embodiment, an aqueous solution containing a conjugate of HA-Tyr may be first prepared or obtained. The HA-Tyr conjugate and its solution may be prepared in any suitable manner. The concentration of HA-Tyr conjugate in the solution may vary depending on the application. For example, a concentration of HA-Tyr in the range of about 0.1 to about 20 w/v% may be suitable. In some embodiments, HA- Tyr concentration may be in the range of about 1 to about 3 w/v%. Exemplary procedures for forming HA-Tyr conjugate and a suitable solution of HA-Tyr are described in, for example, Kurisawa et al., "Injectable biodegradable hydrogels composed of hyaluronic acid-tyramine conjugates for drug delivery and tissue engineering," Chemical Communications, 2005, pp. 4312-4314 (referred to herein as "Kurisawa¹¹); and F. Lee et al., "An injectable enzymatically crosslinked hyaluronic acid-tyramine hydrogel system with independent tuning of mechanical strength and gelation rate," Soft Matter, vol. 4, pp. 880-887 (referred to herein as "Lee"), the entire contents of each of which are incorporated herein by reference.

[0024] Selected amounts of HRP and H₂O₂ may be then added to the solution. The amount of added HRP is selected to crosslink the conjugate to form a hydrogel at a selected gelation rate. Generally, the higher the HRP concentration, the higher the gelation rate. For example, to form the hydrogel in less than about one minute (i.e. reaching the gel point in about 60 seconds), the concentration of HRP in the solution may be about 0.124 unit/ml. The gel point can be defined as the point where the storage modulus (G') and loss modulus (G") of the gel solution are the same (crossover), i.e. G' = G". The gelation rate may be adjusted from about 1 second to about 20 minutes, such as by varying [HRP] from about 0.025 to about 1.24 unit/ml.

[0025] The amount of H₂O₂ added is selected to adjust or control the crosslinking density in the resulting hydrogel, and thus its mechanical strength which may be measured in terms of its storage modulus (G'). To this end, it has been discovered that when the H₂O₂ concentration is low, variation of [H₂O₂] will have no substantial impact on the gelation rate. The gelation rate may be thus controlled by varying the HRP concentration. However, when the H₂O₂ concentration is too high, its variation can significantly affect both gelation rate and crosslinking density. In some embodiments, reducing H₂O₂ below about 1 mM can increase the crosslinking density without materially changing the gelation rate. For example, the molarity of H₂O₂ may be selected so that the HA-Tyr hydrogel formed from the solution has a pre-determined storage modulus. The pre-determined storage modulus may vary depending on the particular application. In one embodiment, the pre-determined storage modulus may be in the range of about 10 to about 4000 Pa. The storage modulus of the hydrogel may be measured using any suitable technique. For example, it may be measured using a dynamic mechanical analysis technique, such as an oscillatory rheology technique, as can be understood by persons skilled in the art. Exemplary techniques are described below in the Examples and in Kurisawa and Lee.

[0026] In some embodiments, when HRP concentration is varied above a threshold, it may have no substantial impact on the crosslinking density, as discussed above and illustrated in the Examples below.

[0027] After the catalysts (HRP and H₂O₂) are added, the solution will begin gelation and form a hydrogel within a certain period, such as within about one second to about 20 minutes, depending on the [HRP] in the solution. Gelation should automatically begin after both HRP and H₂O₂ are added to the solution and mixed with the HA-Tyr conjugate. However, it should be noted that gelation rate will be dependent on the temperature. At a lower temperature, the gelation process will proceed more slowly.

[0028] The solution may be injected into a living body immediately after the catalysts are added, so that the gelation will mainly occur within the body. The body may be a tissue, organism, human body, or another type of living body.

[0029] A drug or protein may be added to the solution before gelation and before the solution is injected into the body.

[0030] In some embodiments, the precursor solution for the hydrogel may be prepared and the hydrogel may be formed as described in Kurisawa and Lee.

[0031] For example, in one embodiment, an aqueous solution of HA-Tyr conjugate with a suitable HA-Tyr concentration may be formed by dissolving a selected amount of HA-Tyr conjugate in a PBS solvent or another suitable solvent as discussed above. The concentration of HA-Tyr may vary in the range of about 0.1 to about 20 w/v%, such as being about 1.75 w/v%. The pH value of the aqueous solution may be adjusted to from about 4 to about 8, such as about 7.4 when a PBS solvent is used. The solution may also be pre-heated to, for example, about 310 K. Selected amounts of HRP and H₂O₂ may be added to the solution, depending on the desired gelation speed and crosslinking density. To achieve independent control of gelation speed and crosslinking density, the H₂O₂ molarity in the precursor solution may be about 1 mM or less, and the HRP concentration may be 0.032 unit/ml or more. The H₂O₂ molarity refers to the molarity as determined by the amount of H₂O₂ added to the solution. As would be understood, the molarity of H₂O₂ in the solution will change over time due to reaction with HRP. HRP and H₂O₂ may be added sequentially or at the same time. Either one of HRP and H₂O₂ may be added first. The solution may be mixed by stirring or vortexing during addition of the various ingredients, and optionally thereafter.

[0032] Without being limited to any particular theory, it is expected that the hydrogel is formed from the HA-Tyr conjugate in a gelation/crosslinking process in which the tyramine moieties are oxidatively coupled/crosslinked. This crosslinking process is catalyzed by HRP and H₂O₂. The crosslinking process is expected to involve two successive steps: first, HRP is oxidized by H₂O₂ to form an intermediate; this intermediate then oxidizes the phenol in the conjugate to trigger the crosslinking or coupling of the phenol groups.

[0033] Without being limited to any particular theory, it is postulated that when the H₂O₂ concentration is too high, it may negatively affect the catalytic activities of the HRP in the solution. When the H₂O₂ has a sufficiently low concentration, it likely has negligible effect on HRP activity, and thus will not affect the gelation rate significantly.

[0034] When the concentration of HRP is increased, more HRP enzymes become available to catalyze the crosslinking of tyramines, thus increasing the gelation speed. As the HRP concentration can be used to adjust the gelation rate, fast gelation time can be achieved. As can be appreciated, a faster gelling time may be desirable in some applications. For example, when the solution is administered into a living body to form the hydrogel, such as by subcutaneous injection, faster gelation can provide more localized gel formation than slower gelation. Faster gelation can also reduce uncontrolled diffusion of the gel precursors and the drug molecules to the surrounding tissues, thus reducing the risk of loss of drug material, delivery to unintended site, or overdose.

[0035] As can be appreciated, the mechanical strength of the HA-Tyr hydrogel is dependent on its crosslinking density. A stronger hydrogel may be desired as it will degrade slower. Further, the rate of diffusion release of any drug, protein or other molecules encapsulated in the hydrogel is also dependent on the crosslinking density. Thus, by adjusting the crosslinking density, a desired delivery or release rate may be obtained. As the crosslinking density may be tuned by adjusting [H₂O₂] without materially affecting the gelation rate, desired mechanical strength or delivery rate may be achieved without compromising, such as slowing down, the gelation rate.

[0036] It has been demonstrated by tests that the concentration of H₂O₂ could be used to control the release rate of proteins, such as α-amylase and lysozyme. The release of α-amylase was diffusion-controlled and the release rate decreased with increasing crosslinking density. Lysozymes were shown to interact with hyaluronic acid electrostatically; hence, degradation of the hydrogel network by hyaluronidase was required to achieve sustained release.

[0037] Hydrogels formed according to embodiments of the present invention may be used to provide sustained-release systems, such drug release systems that are designed to prolong the effects of therapeutic proteins in vivo.

[0038] As can be appreciated, the hydrogels may be formed in-situ, and the precursors for the hydrogels and other ingredients may be injected or administered to the formation site, such as by using a needle or a syringe.

[0039] The mechanical strength of HA-Tyr hydrogels has been found to strongly affect their degradation rate in the presence of hyaluronidase in vitro. Hydrogels with higher mechanical strength (crosslinking density) tend to degrade slower. Thus, by adjusting the crosslinking density of the hydrogels, the degradation rate can also be conveniently adjusted without affecting the gelation rate.

[0040] It should be understood that embodiments of the present invention is not limited to the formation of HA-Tyr hydrogels. The processes described above can be modified to form other types of hydrogels from a polymer that contains a crosslinkable phenol group. For example, the HA-Tyr conjugate in the above description may be replaced with another polymer that contains a crosslinkable phenol group. The suitable polymers should be water soluable and should have functional groups that can be conjugated with phenol compounds, with a sufficient degree of conjugation, such as about 6 degree of conjugation. For example, the polymer may have functional groups such as hydroxyl, amine, carboxyl groups, or the like. Suitable polymers may include dextran, chitin, chitoson, heparin, gelatin, collagen, or the like.

[0041] Embodiments of the present invention may be advantageous in a wide range of applications, such as applications of injectable hydrogels for drug delivery or tissue regeneration, or the like.

EXAMPLES

[0042] The materials used in the Examples were obtained as follows unless otherwise specified.

[0043] Sodium hyaluronate 90 kDa was provided by Chisso Corporation™ of Tokyo, Japan.

[0044] Tyramine hydrochloride (Tyr HCI), Λ/-Hydroxysuccinimide (NHS), 1-Ethyl- 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC HCI), sodium chloride, 5-aminofluorescein, dimethyl sulfoxide (DMSO), lysozyme from chicken egg white, α-amylase from Bacillus amyloliquefaciens, hyaluronidase from bovine testes, bovine serum albumin (BSA), polyoxyethylene-sorbitan monolaurate (Tween 20) and Micrococcus lysodeikticus were obtained from Sigma-Aldrich™.

[0045] Sodium tetraborate- 10H2O and carbazole and D-glucuronic acid were obtained from Fluka™.

[0046] Alexa Fluor 680 conjugated BSA (SAIVI Alexa Fluor™ 680) and Alexa Fluor 680 carboxylic acid succinimidyl ester were purchased from Invitrogen™.

[0047] Hydrogen peroxide (H₂O₂) was obtained from Lancaster™. [0048] Horseradish peroxidase (HRP, 100 unit/mg) was obtained from Wako Pure Chemical Industries™.

[0049] Polyclonal antibody to Bacillus amyloliquefaciens α-amylase (biotin) was purchased from Acris Antibodies™.

[0050] Polyclonal antibody to chicken lysozyme (biotin) was purchased from United States Biological™.

[0051] Streptavidin alkaline phosphate conjugated and p-Nitrophenyl phosphate (p-NPP) were purchased from Chemicon™.

[0052] PBS (15OmM, pH 7.3) was supplied by the media preparation facility in Biopolis, Singapore.

[0053] EXAMPLE I

[0054] Example I-A (Synthesis of HA-Tyr conjugate)

[0055] Solutions of HA-Tyr conjugate were prepared according to the procedures described in Kurisawa and Lee, with some modifications as described below. The modification was related to the condensation agents and the purification step to more effectively remove un-reacted tyramine.

[0056] HA (1 g, 2.5 mmol) was dissolved in 100 ml of distilled water, forming an initial solution. Tyramine hydrochloride (202 mg, 1.2 mmol) was first added to this solution. EDC HCI (479 mg, 2.5 mmol) and NHS (290 mg, 2.5 mmol) were then added to initiate the conjugation reaction. As the reaction proceeded, the pH of the mixture was maintained at 4.7 with 0.1 M NaOH. The reaction mixture was stirred overnight at room temperature and then the pH was brought to 7.0. The solution was transferred to dialysis tubes with molecular cut-off of 1000 Da. The tubes were dialyzed against 100 mM sodium chloride solution for 2 days; a mixture of distilled water and ethanol (3:1) was used for the first day and distilled water was used for the next day. The purified solution was lyophilized to obtain HA-Tyr conjugates. The degree of substitution was calculated from ¹H NMR measurement by comparing the ratio of the relative peak integrations of phenyl protons of tyramine and the methyl protons of HA. The degree of substitution was found to be 6. [0057] Example I-B (Synthesis of fluorescent-labeled HA-Tyr conjugate)

[0058] HA (1 g, 2.5 mmol) was dissolved in 100 ml of distilled water, forming an initial solution. Tyramine hydrochloride (162 mg, 0.93 mmol) and 5- aminofluorescein (81 mg, 0.23 mmol in 1.62 ml DMSO) were added to this solution. EDC HCI (479 mg, 2.5 mmol) and NHS (290 mg, 2.5 mmol) were then added and the pH of the mixture was maintained at 4.7 with 0.1 M NaOH. The solution was stirred overnight at room temperature and then brought to pH 7.0. The solution was next filtered with grade 1 Whatman™ cellulose filter paper to remove unconjugated aminofluorescein that had precipitated. The filtrate was collected into dialysis tubes of molecular cut-off 3500 Da. Then the dialysis and lyophilization procedures described in Example I-A were carried out. The degree of substitution of tyramine was calculated from 1H NMR and the degree of aminofluorescein conjugated was estimated by comparing the absorbance value at 490 nm of 1 mg/ml fluorescence- conjugated HA-Tyr solution to a set of aminofluorescein standards. The degrees of substitution of tyramine and aminofluorescein were 4 and 0.4, respectively.

[0059] Example I-C (Synthesis of HA-Tyr hydrogels)

[0060] An aqueous solution of HA-Tyr was formed by dissolving 1 ml of a solution of HA-Tyr, as prepared in Examples I-A and I-B, in PBS, where the final concentration of HA-Tyr was 1.75 w/v%. The aqueous solution had a pH of about 7.4 and was pre-heated to about 310 K. Different amounts of HRP and H₂O₂ were added sequentially to the solution. The solution was then vortexed and immediately applied to a bottom plate for a Rheoscope, whereon the HA-Tyr conjugate in the solution was crosslinked to form an HA-Tyr hydrogel.

[0061] The formation of the HA-Tyr hydrogel was schematically represented in FIG. 1. As can be understood, this scheme involves an enzyme-mediated oxidation reaction in which the phenol groups/derivatives of the tyramine were crosslinked.

[0062] Rheological measurements of the hydrogel formation were performed with a HAAKE™ Rheoscope 1 rheometer (Karlsruhe, Germany) using a cone and plate geometry of 6 cm diameter and 0.903° cone angle. The measurements were taken at 310 K in the dynamic oscillatory mode with a constant deformation of 1 % and frequency of 1 Hz. To avoid slippage of samples during the measurement, the bottom plate was made of roughened glass.

[0063] After the solution was applied to the bottom plate, the upper cone was then lowered to a measurement gap of 0.024 mm and a layer of silicon oil was carefully applied around the cone to prevent solvent evaporation during the experiment. The measurement parameters were determined to be within the linear viscoelastic region in preliminary experiments. Measurement was allowed to proceed until G' reached a plateau. Next, a frequency sweep was performed with a constant shear stress predetermined to induce a 10% deformation at 1 Hz. Also, a strain sweep of increasing deformation from 1 to 100 % was performed at 1 Hz.

[0064] Representative measurement results obtained with the oscillatory rheometry, from a solution containing about 1.75 w/v% of HA-Tyr conjugate, about 0.728 mM of H₂O₂, and about 0.025 unit/ml of HRP, are shown in FIG. 2. The results shown in FIG. 2 include measured storage modulus G' (circles), loss modulus G" (squares) and phase angle δ (triangles) as a function of time. As can be seen, at the beginning of the crosslinking process, G" was two orders of magnitude greater than G' and the phase angle was at 90°, indicating a predominantly viscous material. As time progressed, both G' and G" increased and crossover of the two moduli occurred at about 45° phase angle.

[0065] This crossover point can be regarded as the gel point. The gel point is also the transition point from a viscoelastic liquid to a viscoelastic solid. The time period between the beginning of crosslinking and the gel point is used herein as an indicator of the gelation rate or gelation speed.

[0066] After the gel point, G' continued to increase and eventually reached a plateau at which the phase angle was close to zero, indicating a solid-like elastic material.

[0067] Table I lists the gel points and corresponding HRP concentrations for samples tested with the H₂O₂ concentration fixed at about 0.728 mM. Table I. Gel Point and HRP concentration ([H₂O₂]= 0.728 mM)

[0068] Table Il lists the final storage modulus and corresponding H₂O₂ concentrations for samples tested with the HRP concentration fixed at about 0.62 unit/ml.

Table II. Storage Modulus at different H₂O₂ concentrations with [HRP]= 0.062 unit/ml

[0069] Fig. 3 shows the dependency of the gelation rate as indicated by the gel point (squares), the time required for G' to reach the plateau (triangles), and the final G' value (circles), on the H₂O₂ concentration, respectively. These results were obtained from precursor solutions with a constant HRP concentration at about 0.062 unit/ml, and various H₂O₂ concentrations as shown, which increased from about 0.146 mM to about 1.092 mM.

[0070] As can be seen, the gel point remained substantially constant at about 130 seconds, indicating that the gelation rate was independent of H₂O₂ concentration. [0071] The time required for G' to reach the plateau, i.e. the time needed to form all the possible tyramine crosslinks, increased with H2O2 concentration, suggesting that HRP was continuously oxidized by H₂O₂ and reduced by tyramine until all H₂O₂ has been depleted. G' peaked at about 1.092 mM of H₂O₂ and further increase in H₂O₂ concentration resulted in decreased G'. Such decrease may be due to deactivation of HRP by the excessive H₂O₂. At the given concentration of HRP, different H₂O₂ concentrations at about 1 mM or less produced HA-Tyr hydrogels with different crosslinking densities and hence mechanical strengths, without substantially affecting the gelation rate.

[0072] Fig. 4 shows the dependency of the gel point (squares) and the time required for G' to reach the plateau (triangles), and the final G' value (circles) on HRP concentration, at a fixed H₂O₂ concentration of 0.728 mM. Both the gel point and the time required for G' to reach the plateau decreased with an increasing HRP concentration. At 0.124 unit/ml of HRP, the gel point was reached within about 60 seconds. Tests showed that at a concentration of HRP of about 1.24 unit/ml, hydrogel was formed within about one second (data not shown in FIG. 4).

[0073] The values of G¹ remained relatively constant above 0.032 unit/ml of HRP concentration, indicating that the change of HRP concentration above about 0.032 unit/ml did not substantially affect the crosslinking density or mechanical strength of the formed hydrogels.

[0074] The effects of frequency and strain on G' of the sample hydrogels were also investigated. Frequency sweeps were performed on sample HA-Tyr hydrogels formed with various concentrations of H₂O₂ at fixed HRP concentration (about 0.062 unit/ml). The frequency test results indicated that except for the weakest hydrogel sample, G' was independent of the frequencies, indicating rigid and elastic networks. Test results of strain sweeps of HA-Tyr hydrogels indicated that the G' of hydrogels formed with H₂O₂ concentrations between 0.146 and 0.437 mM was independent of strain, demonstrating that these hydrogels were physically stable. Above 0.582 mM of H₂O₂, the hydrogels showed a slight increase in G' at high strain. Furthermore, the hydrogel formed with 0.728 mM of H₂O₂ showed a sudden decrease in G¹ beyond 60 % strain, indicating a yield stress where the hydrogel was deformed irreversibly. The observed yielding is ascribed to the inherent brittle structure of hydrogels possessing high G'.

[0075] Example I-C (Swelling ratio of HA-Tyr hydrogels)

[0076] Swelling ratios were determined for slab-shaped HA-Tyr hydrogels. To form the slab-shaped HA-Tyr hydrogels, lyophilized HA-Tyr was dissolved in PBS at a concentration of 1.75 w/v%.

[0077] 5 μl of a HRP solution with an appropriate concentration was added to 1 ml of HA-Tyr solution to give a final HRP concentration of about 0.124 unit/ml of HRP.

[0078] For different samples, 5 μl of different concentrations of H2O2 solution to give respective final H₂O₂ molarity of about 0.160, 0.291 , 0.437, 0.582 or 0.728 mM.

[0079] Crosslinking of HA-Tyr was initiated by the addition of HRP and H₂O₂.

[0080] The mixture was vortexed vigorously before it was injected between two parallel glass plates clamped together with 1 mm spacing. The crosslinking reaction was allowed to proceed at 310 K for one hour. Hydrogel slabs were formed.

[0081] Round hydrogel disks with diameters of 1.6 cm were cut out from the hydrogel slabs using a circular mold. The hydrogel disks were immersed in PBS at 310 K for 3 days. The swollen disks were then gently blotted dry with Kimwipe and weighed to obtain the swollen weight. The disks were then lyophilized to obtain the dry weight. The swelling ratio is the ratio of the swollen weight to the dry weight.

[0082] Representative results of measured swelling ratio of sample HA-Tyr hydrogels formed with different concentrations of H₂O₂ are shown in Fig. 5. The swelling ratio decreased with increasing concentration of H₂O₂, indicating that the swelling capacity was reduced due to increased crosslinking density.

[0083] Sample Hydrogels formed with 0.728 mM of H₂O₂ were also swollen in PBS for 24 hours to reach a swelling equilibrium and then immersed in different concentrations of hyaluronidase. After incubation for 37 hours in 2.5 unit/ml, 10 hours in 25 unit/ml, or 4 hours in 125 unit/ml of hyaluronidase, the hydrogels were removed from the solution and rinsed extensively with water before swelling in purified water (Milli-Q™ water) for 2 days. Sample hydrogels without exposure to hyaluronidase were used as controls. The swelling ratios were then determined as described above.

[0084] Representative measured results are shown in FIG. 6, which shows the measured swelling ratios of sample hydrogels that had lost 50 % of their initial weight after incubation at different concentrations of hyaluronidase. The swelling ratios of all sample hydrogels incubated with hyaluronidase were greater than the control sample. This result supports the expectation that the decrease in crosslinking density due to bulk degradation facilitates swelling of the hydrogels during degradation. The swelling ratio increased with decreasing hyaluronidase concentration. This result indicates that bulk degradation occurred in concurrence with surface degradation, which was more dominant at high concentrations of hyaluronidase. However, at low concentrations of hyaluronidase, the effect of surface degradation was diminished, which allowed more time for hyaluronidase to diffuse into the hydrogel network and hence bulk degradation became more predominant.

[0085] Example I-D (Morphology study of HA-Tyr hydrogels)

[0086] HA-Tyr hydrogel samples were formed in glass vials with about 0.124 unit/ml of HRP and about 0.437, 0.582 or 0.728 mM of H₂O₂. The hydrogel samples were swelled in MiIIiQ water for 24 hours to reach a swelling equilibrium before being cut into thin slices (2mm x 5mm x 8mm) using a sharp surgical blade. The samples were frozen rapidly by plunging them into liquid nitrogen slush and then freeze-dried for two days.

[0087] Scanning electron microscopy (SEM) images of the lyophilized samples were taken using an FEI Company Quanta™ 200 (Oregon, USA) microscope, which was equipped with a gaseous secondary electron detector, in a low vacuum mode. The SEM images of freeze-dried hydrogel samples revealed that the pore sizes decreased with increasing H₂O₂ concentration, which is consistent with the swelling ratio results. [0088] Example I-E (Effects of gelation rate on the subcutaneous formation of fluorescent HA-Tyr hydrogels)

[0089] Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were used immediately after euthanization. After shaving the dorsal sides, each mouse was injected subcutaneously with 0.4 ml of 1.75 w/v% fluorescence- labeled HA-Tyr with 0.728 mM H₂O₂ and different concentrations of HRP (0, 0.031 and 0.124 unit/ml). Two hours after injection, the locations of fluorescent HA-Tyr hydrogels were detected using GE's eXplore™ Optix fluorescence imaging machine (Waukesha, Wl) equipped with a 470 nm excitation laser. After fluorescence imaging, incisions were made to expose the site of injections and digital photographs were taken.

[0090] Fluorescence images were taken to observe the position of the HA-Tyr hydrogels formed in the mice body. When the HA-Tyr solution was injected without HRP, no gel formation was observed and the injected solution spread out from the administered site, suggesting that the polymer solution diffused readily in the subcutaneous environment. When HRP were added at an increasing concentration from about 0.031 to about 0.124 unit/ml, the surface area showing fluorescence decreased, while the observed fluorescence intensity increased, indicating hydrogel formation was more localized at the injection site, likely due to the faster gelation rate. The hydrogel formed with 0.124 unit/ml of HRP had a better defined 3- dimensional structure, compared to the hydrogel formed with 0.031 unit/ml of HRP.

[0091] Example I-F (Enzymatic degradation of HA-Tyr hydrogels)

[0092] Hydrogel disks were prepared as described above and were swollen in PBS for 24 hours to reach the swelling equilibrium. The disks were sandwiched between plastic nets to facilitate retrieval of the hydrogels during degradation experiment. The hydrogels were immersed in 20 ml of PBS containing hyaluronidase (0, 2.5, 25 or 125 unit/ml) at 310 K in an orbital shaker rotating at 100 rpm. The extent of degradation of the hydrogels was estimated by measuring both the residual hydrogel weight and the amount of uronic acid (a degradation component of HA) in the degradation solution at different times. To measure the residual weight, the hydrogels were removed from the degradation solution with a pair of forceps, gently blotted dry with Kimwipe™, and weighed. To measure the amount of uronic acid released from the hydrogel, 0.350 ml of the degradation solution was removed and stored in microcentrifuge tubes at about 277 K until analysis. 0.350 ml of freshly prepared degradation solution were then added to maintain the total volume of 20 ml. Degradation experiments were continued until no visible signs of gel remained. It was determined that the activity of hyaluronidase remained 90 percent for 2 days. The amount of uronic acid released from the hydrogel into the degradation medium were assayed using a carbazole assay. 0.3 ml of samples were added to 1.5 ml of 0.025 M sodium tetraborate in sulfuric acid and heated at 373 K for 10 minutes. After cooling to room temperature, 0.1 ml of carbazole (0.125 w/w% in ethanol) was added, mixed and heated at 373 K for 15 minutes. After cooling to room temperature, 0.2 ml of the solution was transferred to a 96 well plate and the absorbance of the solution was measured at 530 nm. The amount of uronic acid in each sample was estimated by comparing to the D-glucuronic acid standards.

[0093] Hydrogel samples formed with a fixed HRP concentration (0.124 unit/ml) but different H2O2 concentrations (0. 437, 0.582, 0.728 mM) were used to study the relationship between mechanical strength and degradation. Degradations of sample HA-Tyr hydrogels were carried out in the presence of 2.5, 25 and 125 unit/ml of hyaluronidase, and analyzed by the measurement of uronic acid production and hydrogel weight loss. Bovine testes hyaluronidase was used as a hydrolytic enzyme.

[0094] Test results indicated that degradation did not occur when hyaluronidase was absent. In the presence of hyaluronidase, the degradation rate depended on the mechanical strength of the hydrogel sample: a stronger hydrogel degraded slower than a weaker hydrogel at the same hyaluronidase concentration. Thus, it can be concluded that the degradation rate of HA-Tyr hydrogels may be conveniently adjusted by changing the H₂O₂ concentration in the precursor solution.

[0095] The weight of the sample hydrogels was also measured at selected time points during the degradation period. At 125 unit/ml of hyaluronidase, the hydrogels lost weight linearly with time, in line with the trend of uronic acid production observed in the carbazole assay, suggesting surface degradation. At lower concentrations (2.5 and 25 unit/ml) of hyaluronidase, the hydrogels swelled (negative weight loss) initially before starting to lose weight (all hydrogel samples were swollen in PBS for 24 hours to reach the swelling equilibrium before degradation). The weakest hydrogel sample (formed with 0.437mM H₂O₂) at the lowest concentration (2.5 unit/ml) of hyaluronidase swelled the most. Based on the shape of the hydrogel samples (slab-shaped) and the observed swelling behavior, it appears that crosslinking density decreased with time due to bulk degradation. The fact that more pronounced bulk degradation was observed in weaker hydrogel samples was expected as a lower crosslinking density could allow faster diffusion of hyaluronidase into the matrix of the hydrogel.

[0096] As can be understood, these results show that the mechanical strength of the HA-Tyr hydrogel may be tuned to optimize its degradation profile for different applications.

[0097] The HA-Tyr hydrogels formed in these examples were shown to be injectable and biodegradable. It was also found that independent control of mechanical strength and gelation rate could be achieved by limiting the molarity of H₂O₂ in the precursor solution low and keeping the HRP concentration above a threshold. At a constant HA-Tyr concentration, G' was varied from 10 to 4000 Pa by increasing H₂O₂ concentration while maintaining a constant and rapid gelation rate.

[0098] It was also shown that changing the H₂O₂ concentration can tune the mechanical strength of the hydrogel and thus achieve fine control of hydrogel degradation rate.

[0099] These results also show that rapid gelation could prevent diffusion of injected HA-Tyr polymer solution in the body, thus localizing gelation at the injection site.

[00100] Further measurement results and discussions related to Example I are disclosed in Lee.

[00101] EXAMPLE Il [00102] Example M-A (Synthesis of HA-Tyr hydrogels)

[00103] HA-Tyr conjugates were synthesized as described in Example I. The degree of substitution was 6 as determined by ¹H NMR.

[00104] 5 μl of an HRP solution and 5 μl of an H₂O₂ solution were added sequentially to an HA-Tyr solution containing 0.24 ml of HA-Tyr conjugate (1.82 w/v %) dissolved in a PBS solvent. The concentrations of the HRP and H₂O₂ solutions were selected so that the final concentration of HRP in the resulting HA-Tyr solution was about 0.031 or about 0.124 unit/ml, and the final concentration of H₂O₂ were about 0.437, 0.582, or 0.728 mM.

[00105] The solution was pre-warmed to a temperature of about 310K. The final concentration of HA-Tyr conjugate in the resulting HA-Tyr solution was about 1.75 w/v%.

[00106] The mixture was vortexed immediately after addition of HRP and H₂O₂.

[00107] 0.210 ml of the resulting HA-Tyr solution was applied to the bottom plate of an rheometer. The upper cone was then lowered to a measurement gap of 0.025 mm and a layer of silicon oil was carefully applied around the cone to prevent solvent evaporation. The solution was allowed to form a HA-Tyr hydrogel.

[00108] Rheological measurements of the samples were made in the same manner as in Example I-C. For each sample, the measurement continued until the storage modulus of the sample reached a plateau.

[00109] Example M-B (Synthesis of hydrogels loaded with protein)

[00110] BSA or lysozyme was dissolved in PBS to form protein solutions with different concentration. 0.065 ml of each protein solution was added to 0.175 ml of an HA-Tyr conjugate solution (2.5 w/v %). The mixed solution was mixed gently. 5 μl of HRP and 5 μl of H₂O₂ were next added to the mixed solution. The final concentration of HA-Tyr conjugate in the resulting solution was about 1.75 w/v% and the protein loading concentrations were 0.15, 1.5 or 15 mg/ml respectively. [00111] The resulting solution was used to form hydrogels as described in Example U-A.

[00112] Example M-C (Conjugation of Alexa Fluor 680 fluorescent dye to lysozyme)

[00113] 0.5 mg of amine-reactive Alexa Fluor 680 carboxylic acid succinimidyl ester dissolved in 50 μl DMSO was added to 12.34 mg lysozyme dissolved in 1.5 ml PBS. The mixture was stirred gently at room temperature for 5 hours in darkness. The reaction mixture was passed through PD-10 desalting columns (GE Healthcare) pre-equilibrated with PBS (0.5 ml per column) to remove un-conjugated dyes. The first fluorescent bands containing the labeled proteins were collected and the concentration of proteins in the elution was estimated by bicinchoninic acid protein assay (BCA, Pierce™). The protein conjugate was then diluted to 40 μg/ml and the absorbance of Alexa Fluor 680 at 679 nm was determined using a UV-VIS spectrometer (Hitachi™). The fluorescence to protein (F/P) molar ratio was calculated to be 0.39 according to the manufacturer's instructions.

[00114] Example M-D (Subcutaneous injections of protein-loaded HA-Tyr hydrogels)

[00115] Fluorescence-labeled HA-Tyr was synthesized as described in Lee. The degree of conjugation (substitution) of aminofluorescein or tyramine was 0.5 or 5, respectively. Immediately after euthanization by CO₂, each adult female Balb/c nude mice was injected subcutaneously on its back with 0.3 ml of 1.75 w/v% fluorescence-labeled HA-Tyr solution containing 80 μg of either Alexa 680 conjugated BSA or lysozyme.

[00116] About one hour after injection, fluorescence images were taken using the IVIS imaging system (Caliper Life Science™, Massachusetts, USA) to determine the location of HA-Tyr conjugates (GFP filter set, λ_ex = 445-490 nm, λ_em = 515-575 nm, exposure time = 0.05 second) and the proteins (Cy5.5 filter set, λe_X = 615-665 nm, λ_em = 695-770 nm, exposure time = 0.01 second). For both detections, the binning of the CCD camera was set to 8; field of view (FOV) was 20 cm and the aperture the lens on the camera was set to f/8. [00117] Example H-E (In-vitro protein release from HA-Tyr hydrogels)

[00118] HA-Tyr hydrogels loaded with 0.25 mg/ml of α-amylase or lysozyme were prepared by mixing 0.5 ml of HA-Tyr (3.5 w/v %) with 0.5 ml of protein solution (0.5 mg/ml). 5 μl of an HRP solution and 5 μl of an H2O2 solution was added to form a mixture. The final concentration of HRP in the mixture was about 0.124 unit/ml and the final molarity of H₂O₂ in the mixture was about 0.437, 0.582 or 0.728 mM. The mixture was vortexed gently before being deposited (injected) between two parallel glass plates clamped together with 1mm spacing.

[00119] Gelation was allowed to proceed at 310 K for one hour, forming hydrogel slabs. Round gel disks with a diameter of 1.6 cm were cut out from the hydrogel slab using a circular mold. Each disk was sandwiched between a plastic net and immersed in 20 ml of a release medium containing PBS, with or without hyaluronidase (2.5 unit/ml). At various selected time intervals, 0.2 ml of the release medium was drawn and stored in a microcentrifuge tube containing 0.2 ml of 0.1 mg/ml BSA in PBS to prevent non-specific adsorption of the model proteins to the plastic surface of the microcentrifuge tubes. 0.2 ml of a PBS solution with or without hyaluronidase was added to maintain the total release medium at 20 ml. The collected samples were stored at about 253 K.

[00120] The amount of proteins contained in each sample was determined by enzyme-linked immunosorbant assay (ELISA) which was carried out at room temperature. The washing procedure between each steps was carried out using a plate washer (Amersham Bioscience™) which was programmed to wash the wells three times with 0.3 ml washing buffer (100 mM PBS containing 0.05 % Tween-20). 0.1 ml of each sample solution thawed to room temperature was added to the wells of a 96-well MaxiSorb™ ELISA plate (NUNC™) and the proteins in the samples were bound to the well by incubation for 1.5 hours and then the wells were washed. After washing, the wells were blocked with 0.2 ml of blocking buffer (BSA 2 w/v% in PBS) for 30 minutes to saturate the protein-binding sites and then the wells were washed. Next, 0.1 ml of either biotinylated anti-α-amylase (2 μg/ml) or anti- lysozyme (1.67 μg/ml) antibodies diluted in blocking buffer were added to the wells and incubated for 1 hour. After washing, 0.1 ml of streptavid in-alkaline phosphatase diluted in PBS was added and incubated for 1 hour and then the wells were washed. Finally, 0.1 ml of p-NPP was added to each well and the plate was incubated until sufficient color had developed (approximately 80 min for α-amylase and 35 min for lysozyme). The absorbance at 405 nm was measured using Tecan Infinite™ 200 microplate reader. The amount of proteins contained in each sample was calculated by comparing with a set of protein standards and was converted to percentage of total protein encapsulated in the hydrogel disk. It was observed that the ELISA signal of a solution of α-amylase in PBS (2.66 μg/ml) at 310 K decreased linearly with time and was reduced by 30 % after 24 hours. This might due to adsorption of α-amylase to glass surface or denaturation of the protein. In order to compensate for the loss in signal, the amount of proteins detected by ELISA was manually offset according to the percentage loss observed in the controls.

[00121] Example H-F (Protein Release in different ionic strengths)

[00122] Hydrogel disks (thickness = 1 mm, weight = 57 mg) containing 0.25 mg/ml of lysozyme were immersed in 1 ml of NaCI solution (0, 0.05, 0.15, 0.5 or 1 M) at 310 K on an orbital shaker at 100 rpm. After four hours of incubation, 0.1 ml of the release medium was collected and the amount of proteins contained in the sample was measured using the micro bicinchoninic acid protein assay (microBCA, Pierce) according to the manufacturer's protocol.

[00123] Example N-G (Activities of proteins recovered by degradation of HA- Tyr hydrogels)

[00124] Hydrogel disks (thickness = 1 mm, weight = 57 mg) containing 5 mg/ml of α-amylase or lysozyme were degraded in 5 ml of 200 unit/ml of hyaluronidase in PBS (supplemented with 0.05 % NaN₃) at 310 K on an orbital shaker at 150 rpm. After 24 hours, no visible sign of an hydrogel was observed. The activity of α-amylase was determined by EnzChek™ Ultra Amylase Assay Kit (Invitrogen™).

[00125] The degradation solutions containing the released α-amylase were diluted 200-folds with PBS. 50 μl of the diluted samples were added to the wells of a 96 well fluorescent plate and then 50 μl of the substrates were added. The plate was incubated for 10 minutes on an orbital shaker at room temperature and then the fluorescence intensity (λ_eX = 485 nm, λ_em = 530 nm) was measured using the Tecan Infinite 200 microplate reader. To determine the activities of lysozyme, 20 μl of lysozyme samples was added to the well of a 96-well UV-starplate (Greiner Bio- one™, Germany) followed by 0.1 ml of Micrococcus lysodeikticus (0.15 w/v% in PBS). The plate was incubated for 15 minutes on an orbital shaker at 50 rpm at room temperature. Then the absorbance at 450 nm was measured using the microplate reader.

[00126] It was found from the test results that it is desirable at least in some applications for the hydrogel to form (crosslink) rapidly. It was found that slow gelation could cause diffusions of the gel precursors and the proteins to be encapsulated in tissues surrounding the injection site, which could compromise the therapeutic outcome.

[00127] It was also found that from the tests that about 0.124 unit/ml of HRP was the highest concentration suitable for injection of a 0.3 ml HA-Tyr conjugate solution without clogging of the needle that was used. It should be understood, however, in other applications, the suitable maximum of [HRP] may be higher or lower.

[00128] It was further found from these tests that in the absence of HRP, no crosslinked network was formed and both the HA-Tyr and the proteins diffused away fromjhe injection site, suggesting that both components diffused readily in the subcutaneous environment. Notably, the area detected with proteins were greater than the area detected with HA-Tyr, indicating that the proteins diffuse faster than HA-Tyr conjugate, likely due to the smaller molecular weight of BSA (BSA 66 kDa and HA 9OkDa).

[00129] At about 0.031 unit/ml of HRP, the area detected with HA-Tyr did not reduce, suggesting that the crosslinking reaction was not quick enough to confine the crosslinked network at the injection site. However, the area detected with proteins decreased slightly, indicating that crosslinking of the HA-Tyr conjugates helped trapping the proteins within the network. [00130] By increasing the HRP concentration to 0.124 unit/ml, both the surface areas detected with HA-Tyr and proteins were significantly reduced, indicating that the rapid gelation resulted in localized formation of the hydrogels and effective encapsulation of the proteins within the network.

[00131] As can be appreciated, rapid gelation may be desirable for an injectable hydrogel system to ensure that the delivery would be localized to the site of injection.

[00132] Test results also showed that when the concentrations of HA-Tyr conjugate and HRP were fixed, the storage modulus (G') of the hydrogel, which was related to the crosslinking density, increased with H₂O2 concentration. Some results are listed in Table III (See samples 2-4).

[00133] It was found that when BSA was included in the precursor solution and encapsulated in the hydrogel, G' decreased by about 7 %, indicating that the network integrity was slightly affected by the presence of BSA (Table III, samples 5- 7).

[00134] Encapsulating lysozyme at 15 mg/ml showed a marked decrease in G' (Table III, sample 10). This is likely due to the electrostatic interactions between the negatively-charged HA and the positively-charged lysozymes which interfered with the crosslinking reaction (more discussions about the electrostatic interactions in Section 3.3). However, the gel points of the hydrogels formed with same HRP concentration (0.124 unit/ml), with or without proteins were all less than 90 seconds, indicating that protein encapsulation did not affect the rate of enzymatic crosslinking.

[00135] The release of α-amylase from HA-Tyr hydrogels, regardless of its crosslinking density, exhibited a burst release which was expected primarily due to the protein concentration gradient inside and outside of the hydrogel that caused the proteins near the surface of the gel to diffuse rapidly out of the matrix. The percentage of proteins released during the burst phase decreased as the crosslinking density increased. After the burst release, the release of α-amylase slowed down. [00136] The release profile of lysozymes displayed a similar burst release which decreased with increasing crosslinking density. However, for hydrogels with the same crosslinking density, the percentage of lysozyme released in the burst phase was much lower than that of α-amylase. Furthermore, the release of lysozymes from HA-Tyr hydrogels discontinued after the burst release.

[00137] It is desirable that a hydrogel system for protein delivery not only delivers the protein at a controlled rate but also maintains the activity of the protein from the time of hydrogel preparation to the point of release. In addition, degraded products from the delivery system might cause protein denaturation. Therefore, it may be desirable that the gel-forming process and the degraded products of the hydrogel system maintains the activity of the protein at the therapeutic level.

Table III Representative test results obtained from sample hydrogels

[00138] In summary, the test results described in Example Il show that localized hydrogel formation and efficient encapsulation of proteins could be achieved by rapid gelation of HA-Tyr hydrogels using a high concentration of HRP. Sustained release of negatively-charged α-amylase at different release rates could be achieved by varying the H₂O₂ concentration in the precursor solution. The activity of released α-amylase remained above 95% at different hydrogel crosslinking densities. Sustained release of positively-charged lysozymes was observed only when the hydrogel network was degraded. The activity of the released lysozymes depended on the crosslinking density of the hydrogel, but the minimum activity was 70%. These results showed that the sample hydrogels were suitable for use in an injectable system for sustained release or delivery of proteins or other like materials.

[00139] As illustrated by the Examples, including the rheological data, swelling ratio studies and morphological analysis demonstrated that, when the H₂O₂ concentration in the precursor solution was limited to a low range, variation of H₂O₂ concentration effectively controlled the mechanical strength of the formed hydrogel without substantially affecting the gelation rate. Further, variation of the HRP concentration effectively controlled the gelation rate in the gelation process without substantially affecting the mechanical strength of the formed HA-Tyr hydrogel when the HRP concentration is above a certain threshold. Thus, independent control of gelation rate and mechanical strength (crosslinking density) were conveniently obtained.

[00140] As now can be understood, embodiments of the present invention enables convenient control of the hydrogel crosslinking density and gelation rate/speed. The degradability of the resulting hydrogel and the release rate of protein or drug or another material embedded in the hydrogel may be conveniently controlled by varying the H₂O₂ concentration in the precursor solution.

[00141] Embodiments of the present invention may be advantageously used in many different fields and applications including drug or protein delivery and tissue engineering applications.

[00142] Other features, benefits and advantages of the embodiments described herein not expressly mentioned above can be understood from this description and the drawings by those skilled in the art.

[00143] Of course, the above described embodiments are intended to be illustrative only and in no way limiting. The described embodiments are susceptible to many modifications of form, arrangement of parts, details and order of operation. The invention, rather, is intended to encompass all such modification within its scope, as defined by the claims.

Claims

WHAT IS CLAIMED IS:

1. A solution for forming a hydrogel, comprising: a polymer comprising a crosslinkable phenol group; horseradish peroxidase (HRP), of an effective amount for crosslinking said polymer to form said hydrogel; and hydrogen peroxide (H2O2), having a molarity of about 1 mM or less.

2. The solution of claim 1 , wherein said H₂O₂ molarity is selected so that said hydrogel formed from said solution has a pre-determined storage modulus.

3. The solution of claim 1 or claim 2, wherein said hydrogel formed from said solution has a storage modulus from about 10 to about 4000 Pa.

4. The solution of any one of claims 1 to 3, wherein said H₂O₂ molarity is from about 0.146 to about 1.092 mM.

5. The solution of claim 4, wherein said H₂O₂ molarity is from about 0.16 to about 0.728 mM.

6. The solution of any one of claims 1 to 5, comprising from about 0.025 to about 1.24 unit/ml of said HRP.

7. The solution of claim 6, comprising from about 0.032 to about 0.124 unit/ml of said HRP.

8. The solution of claim 7, comprising about 0.062 unit/ml of said HRP.

9. The solution of any one of claims 1 to 8, wherein said polymer is a conjugate of hyaluronic acid and tyramine (HA-Tyr).

10. The solution of claim 9, wherein a molar ratio of said H₂O₂ to said tyramine is about 0.4 or less.

11.The solution of claim 9 or claim 10, comprising from about 0.1 to about 20 w/v% of said HA-Tyr conjugate.

12. The solution of claim 11 , comprising about 1.75 w/v% of said HA-Tyr conjugate.

13. The solution of any one of claims 9 to 12, wherein said hyaluronic acid has a molecular weight of about 90,000 Da.

14. The solution of any one of claims 9 to 13, wherein said tyramine has a molarity of from about 0.42 to about 21 mM.

15. The solution of claim 14, wherein said tyramine has a molarity of about 2.57 mM.

16. The solution of any one of claims 1 to 15, having a pH of about 4 to about 8.

17. The solution of any one of claims 1 to 16, at a temperature of about 293 to about 313 K.

18. The solution of any one of claims 1 to 17, comprising a drug.

19. The solution of any one of claims 1 to 18, comprising a protein.

20. The solution of any one of claims 1 to 19, comprising water.

21.The solution of any one of claims 1 to 20, comprising phosphate buffer saline.

22.A method of forming a hydrogel, comprising: mixing a polymer comprising a crosslinkable phenol group, horseradish peroxidase (HRP), and hydrogen peroxide (H2O2) in a solution, to form said hydrogel, said H₂O₂ having a molarity of about 1 mM or less.

23. The method of claim 22, wherein said solution is as defined in any one of claims 1 to 21.