WO2009148405A1 - Formation d'hydrogel en présence de peroxydase et d'une faible concentration de peroxyde d'hydrogène - Google Patents

Formation d'hydrogel en présence de peroxydase et d'une faible concentration de peroxyde d'hydrogène Download PDF

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WO2009148405A1
WO2009148405A1 PCT/SG2008/000204 SG2008000204W WO2009148405A1 WO 2009148405 A1 WO2009148405 A1 WO 2009148405A1 SG 2008000204 W SG2008000204 W SG 2008000204W WO 2009148405 A1 WO2009148405 A1 WO 2009148405A1
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solution
hydrogel
tyr
hrp
concentration
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PCT/SG2008/000204
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English (en)
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Motoichi Kurisawa
Fan Lee
Joo Eun Chung
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Agency For Science, Technology And Research
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Priority to PCT/SG2008/000204 priority Critical patent/WO2009148405A1/fr
Priority to JP2011512418A priority patent/JP5563563B2/ja
Priority to US12/545,004 priority patent/US8287906B2/en
Publication of WO2009148405A1 publication Critical patent/WO2009148405A1/fr
Priority to US13/336,783 priority patent/US8691206B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/738—Cross-linked polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof

Definitions

  • the present invention relates to hydrogel formation, particularly to formation of hydrogels in the presence of peroxidase and hydrogen peroxide.
  • Phenol-containing hydrogels such as hyaluronic acid-tyramine (HA-Tyr) hydrogels
  • HRP horseradish peroxidase
  • H 2 O 2 hydrogen peroxide
  • the gelation rate and the crosslinking density in the hydrogel can be adjusted by changing the concentration of HRP or H 2 O 2 in the precursor solution. Such a change typically affects both the gelation rate and crosslinking density.
  • 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 (H 2 O 2 ), having a molarity of about 1 mM or less.
  • HRP horseradish peroxidase
  • H 2 O 2 hydrogen peroxide
  • the H 2 O 2 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 H 2 O 2 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 H 2 O 2 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.
  • 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.
  • FIG. 1 is a schematic diagram representing a scheme for forming an HA- Tyr hydrogel, exemplary of an embodiment of the present invention
  • 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.
  • FIG. 6 is a bar graph showing swelling ratio measured from different samples.
  • 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 H 2 O 2 .
  • the H 2 O 2 may have a molarity of about 1 mM or less in the solution.
  • the ratio of H 2 O 2 to HRP in the solution may be about 1.8 mol/g or less.
  • the molar ratio of H 2 O 2 to tyramine moiety in the solution may be about 0.4 or less.
  • 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]).
  • the concentration of HA-Tyr may be selected to obtain desired properties in either the precursor solution or the final hydrogel, or both.
  • 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.
  • the HA may have a molecular weight of about 9OK Da and [HA-Tyr] may be about 1.75 w/v% (weight-volume percent).
  • concentration may need to be lowered to achieve a similar viscosity.
  • the HA-Tyr concentration may vary in the range of about 0.1 to about 20 w/v%.
  • 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 1 H 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).
  • 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.
  • 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 ⁇ mol of the substrate in 1 minute under the standard conditions.
  • 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.
  • HRP may be available in 100U/mg, in which case the solution may contain from about 0.25 to about 12.4 ⁇ g/ml of HRP, such as from about 0.32 to about 1.24 ⁇ g/ml.
  • 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 ⁇ g/ml of HRP.
  • 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 H 2 O 2 .
  • the initial molarity of H 2 O 2 in the solution prior to gelation 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.
  • the molar ratio of H 2 O 2 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.
  • 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.
  • interferon interferon, herceptin, or the like may be included in the solution.
  • Non-therapeutic proteins such as ⁇ -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 H 2 O 2 or HRP, or both, may need to be adjusted to off-set such impact.
  • the solution may be at a temperature of about 293 to about 313 K, such as at about 310 K (37 0 C).
  • the pH of the solution may be from about 4 to about 8, such as about 7.4.
  • the solvent in the solution may be any suitable solvent.
  • the solvent may include water.
  • the solution may also include a phosphate buffer saline (PBS).
  • PBS phosphate buffer saline
  • 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.
  • Some exemplary embodiments of the present invention relate to methods of forming a hydrogel.
  • a HA-Tyr conjugate, HRP, and H 2 O 2 are mixed in a solution to form an HA-Tyr hydrogel.
  • the solution may be any of the precursor solutions described above.
  • the molarity of H 2 O 2 in the solution is about 1 mM or less.
  • the molar ratio of H 2 O 2 to tyramine in the solution is about 0.4 or less.
  • the solution may be prepared in any suitable manner.
  • 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%.
  • 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 “Kurisawa 11 ); 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.
  • Selected amounts of HRP and H 2 O 2 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.
  • the concentration of HRP in the solution may be about 0.124 unit/ml.
  • 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.
  • the amount of H 2 O 2 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').
  • G' storage modulus
  • the molarity of H 2 O 2 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.
  • HRP concentration when varied above a threshold, it may have no substantial impact on the crosslinking density, as discussed above and illustrated in the Examples below.
  • 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 H 2 O 2 are added to the solution and mixed with the HA-Tyr conjugate.
  • gelation rate will be dependent on the temperature. At a lower temperature, the gelation process will proceed more slowly.
  • 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.
  • a drug or protein may be added to the solution before gelation and before the solution is injected into the body.
  • the precursor solution for the hydrogel may be prepared and the hydrogel may be formed as described in Kurisawa and Lee.
  • 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.
  • 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.
  • H 2 O 2 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 H 2 O 2 molarity refers to the molarity as determined by the amount of H 2 O 2 added to the solution. As would be understood, the molarity of H 2 O 2 in the solution will change over time due to reaction with HRP.
  • HRP and H 2 O 2 may be added sequentially or at the same time. Either one of HRP and H 2 O 2 may be added first.
  • the solution may be mixed by stirring or vortexing during addition of the various ingredients, and optionally thereafter.
  • 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 H 2 O 2 .
  • the crosslinking process is expected to involve two successive steps: first, HRP is oxidized by H 2 O 2 to form an intermediate; this intermediate then oxidizes the phenol in the conjugate to trigger the crosslinking or coupling of the phenol groups.
  • 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.
  • the rate of diffusion release of any drug, protein or other molecules encapsulated in the hydrogel is also dependent on the crosslinking density.
  • the crosslinking density may be tuned by adjusting [H 2 O 2 ] without materially affecting the gelation rate, desired mechanical strength or delivery rate may be achieved without compromising, such as slowing down, the gelation rate.
  • 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.
  • 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.
  • HA-Tyr hydrogels have 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Sodium hyaluronate 90 kDa was provided by Chisso CorporationTM of Tokyo, Japan.
  • Tyramine hydrochloride (Tyr HCI), ⁇ /-Hydroxysuccinimide (NHS), 1-Ethyl- 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC HCI), sodium chloride, 5-aminofluorescein, dimethyl sulfoxide (DMSO), lysozyme from chicken egg white, ⁇ -amylase from Bacillus amyloliquefaciens, hyaluronidase from bovine testes, bovine serum albumin (BSA), polyoxyethylene-sorbitan monolaurate (Tween 20) and Micrococcus lysodeikticus were obtained from Sigma-AldrichTM.
  • Alexa Fluor 680 conjugated BSA SAIVI Alexa FluorTM 680
  • Alexa Fluor 680 carboxylic acid succinimidyl ester were purchased from InvitrogenTM.
  • Hydrogen peroxide H 2 O 2
  • LancasterTM Hydrogen peroxide
  • Horseradish peroxidase HRP, 100 unit/mg was obtained from Wako Pure Chemical IndustriesTM.
  • Streptavidin alkaline phosphate conjugated and p-Nitrophenyl phosphate were purchased from ChemiconTM.
  • PBS (15OmM, pH 7.3) was supplied by the media preparation facility in Biopolis, Singapore.
  • 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.
  • 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.
  • 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 WhatmanTM 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.
  • 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 H 2 O 2 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.
  • FIG. 1 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.
  • 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.
  • 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.
  • FIG. 2 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 H 2 O 2 , 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 ⁇ (triangles) as a function of time.
  • G' was two orders of magnitude greater than G' and the phase angle was at 90°, indicating a predominantly viscous material.
  • both G' and G" increased and crossover of the two moduli occurred at about 45° phase angle.
  • 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.
  • Table I lists the gel points and corresponding HRP concentrations for samples tested with the H 2 O 2 concentration fixed at about 0.728 mM.
  • Table Il lists the final storage modulus and corresponding H 2 O 2 concentrations for samples tested with the HRP concentration fixed at about 0.62 unit/ml.
  • 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 H 2 O 2 concentration, respectively.
  • the gel point remained substantially constant at about 130 seconds, indicating that the gelation rate was independent of H 2 O 2 concentration.
  • G' peaked at about 1.092 mM of H 2 O 2 and further increase in H 2 O 2 concentration resulted in decreased G'. Such decrease may be due to deactivation of HRP by the excessive H 2 O 2 .
  • different H 2 O 2 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.
  • 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 H 2 O 2 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).
  • hydrogel formed with 0.728 mM of H 2 O 2 showed a sudden decrease in G 1 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'.
  • Example I-C (Swelling ratio of HA-Tyr hydrogels)
  • Swelling ratios were determined for slab-shaped HA-Tyr hydrogels.
  • lyophilized HA-Tyr was dissolved in PBS at a concentration of 1.75 w/v%.
  • FIG. 5 Representative results of measured swelling ratio of sample HA-Tyr hydrogels formed with different concentrations of H 2 O 2 are shown in Fig. 5. The swelling ratio decreased with increasing concentration of H 2 O 2 , indicating that the swelling capacity was reduced due to increased crosslinking density.
  • Sample Hydrogels formed with 0.728 mM of H 2 O 2 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-QTM water) for 2 days. Sample hydrogels without exposure to hyaluronidase were used as controls. The swelling ratios were then determined as described above.
  • FIG. 6 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.
  • Example I-D Morphology study of HA-Tyr hydrogels
  • 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 H 2 O 2 .
  • 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.
  • Nonobese diabetic/severe combined immunodeficiency 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 H 2 O 2 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 eXploreTM 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.
  • Example I-F Enzymatic degradation of HA-Tyr hydrogels
  • 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.
  • the hydrogels were removed from the degradation solution with a pair of forceps, gently blotted dry with KimwipeTM, and weighed.
  • 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.
  • 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 H 2 O 2 ) at the lowest concentration (2.5 unit/ml) of hyaluronidase swelled the most.
  • 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 H 2 O 2 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 H 2 O 2 concentration while maintaining a constant and rapid gelation rate.
  • HA-Tyr conjugates were synthesized as described in Example I. The degree of substitution was 6 as determined by 1 H NMR.
  • 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%.
  • 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 ⁇ l of HRP and 5 ⁇ l of H 2 O 2 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.
  • the protein conjugate was then diluted to 40 ⁇ g/ml and the absorbance of Alexa Fluor 680 at 679 nm was determined using a UV-VIS spectrometer (HitachiTM). The fluorescence to protein (F/P) molar ratio was calculated to be 0.39 according to the manufacturer's instructions.
  • Example M-D Subcutaneous injections of protein-loaded HA-Tyr hydrogels
  • 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 CO 2 , 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 ⁇ g of either Alexa 680 conjugated BSA or lysozyme.
  • HA-Tyr hydrogels loaded with 0.25 mg/ml of ⁇ -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 ⁇ l of an HRP solution and 5 ⁇ l 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 H 2 O 2 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.
  • the amount of proteins contained in each sample was determined by enzyme-linked immunosorbant assay (ELISA) which was carried out at room temperature.
  • ELISA enzyme-linked immunosorbant assay
  • the washing procedure between each steps was carried out using a plate washer (Amersham BioscienceTM) 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 MaxiSorbTM ELISA plate (NUNCTM) and the proteins in the samples were bound to the well by incubation for 1.5 hours and then the wells were washed.
  • 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.
  • BSA 2 w/v% in PBS blocking buffer
  • 0.1 ml of either biotinylated anti- ⁇ -amylase (2 ⁇ g/ml) or anti- lysozyme (1.67 ⁇ g/ml) antibodies diluted in blocking buffer were added to the wells and incubated for 1 hour.
  • 0.1 ml of streptavid in-alkaline phosphatase diluted in PBS was added and incubated for 1 hour and then the wells were washed.
  • 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 ⁇ -amylase and 35 min for lysozyme).
  • the absorbance at 405 nm was measured using Tecan InfiniteTM 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 ⁇ -amylase in PBS (2.66 ⁇ g/ml) at 310 K decreased linearly with time and was reduced by 30 % after 24 hours. This might due to adsorption of ⁇ -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.
  • microBCA micro bicinchoninic acid protein assay
  • Example N-G Activity of proteins recovered by degradation of HA- Tyr hydrogels
  • lysozyme 20 ⁇ l of lysozyme samples was added to the well of a 96-well UV-starplate (Greiner Bio- oneTM, 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.
  • rapid gelation may be desirable for an injectable hydrogel system to ensure that the delivery would be localized to the site of injection.
  • 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 H 2 O2 concentration.
  • 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.
  • 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.
  • 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.
  • Example Il shows 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 ⁇ -amylase at different release rates could be achieved by varying the H 2 O 2 concentration in the precursor solution. The activity of released ⁇ -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.
  • 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 H 2 O 2 concentration in the precursor solution.
  • Embodiments of the present invention may be advantageously used in many different fields and applications including drug or protein delivery and tissue engineering applications.

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Abstract

On peut former un hydrogel en mélangeant un polymère comprenant un groupe phénol réticulable, de la peroxydase de raifort et du peroxyde d'hydrogène (H2O2) dans une solution. Le H2O2 présente une molarité maximale d'environ 1 mM. Le polymère peut être un conjugué d'acide hyaluronique et de tyramine (HA-Tyr). La solution peut également comprendre un médicament ou une protéine. La molarité de H2O2 peut être choisie de sorte que l'hydrogel formé à partir de la solution ait un module de conservation prédéterminé. On a remarqué que le fait de faire varier la molarité de H2O2 en-deçà d'environ 1 mM n'affecte pas sensiblement la vitesse de gélification.
PCT/SG2008/000204 2008-05-06 2008-06-05 Formation d'hydrogel en présence de peroxydase et d'une faible concentration de peroxyde d'hydrogène WO2009148405A1 (fr)

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JP2011512418A JP5563563B2 (ja) 2008-06-05 2008-06-05 ペルオキシダーゼおよび低濃度の過酸化水素の存在下でのヒドロゲルの形成方法
US12/545,004 US8287906B2 (en) 2008-05-06 2009-08-20 Formation of hydrogel in the presence of peroxidase and low concentration of hydrogen peroxide
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