WO2001075089A1 - Methodes et compositions correspondantes relatives a l'utilisation de catalase dans des hydrogels et des biocapteurs - Google Patents

Methodes et compositions correspondantes relatives a l'utilisation de catalase dans des hydrogels et des biocapteurs

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Publication number
WO2001075089A1
WO2001075089A1 PCT/US2001/010612 US0110612W WO0175089A1 WO 2001075089 A1 WO2001075089 A1 WO 2001075089A1 US 0110612 W US0110612 W US 0110612W WO 0175089 A1 WO0175089 A1 WO 0175089A1
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WIPO (PCT)
Prior art keywords
catalase
enzyme
biosensor
analytic
polymer matrix
Prior art date
Application number
PCT/US2001/010612
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English (en)
Inventor
Han In Suk
Jung Dal-Young
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M-Biotech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by M-Biotech, Inc. filed Critical M-Biotech, Inc.
Publication of WO2001075089A1 publication Critical patent/WO2001075089A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems

Definitions

  • This invention is generally related to the use of hydrogels containing oxidoreductase enzymes in biosensors and controlled drug delivery systems, and more particularly to the inclusion of catalase in such biosensors and drug delivery systems.
  • hydrogels are sometimes called "stimulus responsive polymers".
  • a pH-sensitive hydrogel undergoes very large and reversible volume changes in response to pH changes within the hydrogel.
  • Two main types of pH- sensitive hydrogels are acidic hydrogels and basic hydrogels. Acidic hydrogels by definition will be ionized and hence swollen at high pH, and uncharged and unswollen at low pH (Ghandehari H et al 1996 J. Macromol. Chem. Phys. 197:965; Brondsted H et al Polyelectrolyte gels: Properties, Preparation, and Application, Harland RS., Prud Homme P K, eds ACS: 285, 1992).
  • Swelling behavior of a basic hydrogel has the opposite dependence on pH.
  • the pH sensitivity is caused by pendant acidic and basic groups such as carboxylic acids, sulfonic acids, primary amines, and quaternary ammonium salts.
  • pendant acidic and basic groups such as carboxylic acids, sulfonic acids, primary amines, and quaternary ammonium salts.
  • Carboxylic acid groups for example are charged at high pH and uncharged at low pH, whereas the reverse is true for primary amine groups and quaternary ammonium salts.
  • the transition pH for a given pendant group is primarily determined by the pKa value for that pendant group, and by the hydrophobicity of nearby monomers in the polymer chain.
  • oxidoreductase currently being investigated for use in biosensors are glucose oxidase (for sensing blood sugar levels), cholesterase (for sensing cholesterol levels), alcohol dehydrogenase (for sensing alcohol levels), and penicillinase (for sensing penicillin levels).
  • glucose oxidase for sensing blood sugar levels
  • cholesterase for sensing cholesterol levels
  • alcohol dehydrogenase for sensing alcohol levels
  • penicillinase for sensing penicillin levels
  • a pH-sensitive hydrogel containing glucose oxidase (GOx) enzyme is called a glucose-sensitive hydrogel (GSH) due to its responsiveness to environmental glucose concentrations.
  • GSH glucose-sensitive hydrogel
  • Thermally stable GOx is a flavin- containing glycoprotein which catalyzes a reaction which is very specific for glucose, and which produces gluconic acid and hydrogen peroxide in the presence of glucose and oxygen as shown below. Therefore, increases in the environmental glucose concentration lower the pH value within the GSH.
  • Glucose biosensors based on amperometric methods are the most highly developed.
  • an electrode is used which produces a current proportional to the diffusional flux of hydrogen peroxide to the electrode surface, or, alternatively, proportional to the diffusional flux of oxygen to the electrode surface.
  • the diffusional flux of hydrogen peroxide to the electrode surface equals the rate at which hydrogen peroxide is produced by the GOx reaction in the hydrogel adjacent to the electrode.
  • the hydrogels in amperometric glucose biosensors do not swell in response to pH changes.
  • An important physical property of pH-sensitive GSHs is the ability to change volume in response to changes in environmental glucose concentrations, due to changes in pH within the hydrogel caused by the reaction of the GOx enzyme.
  • the catalase reaction produces oxygen, which helps meet the oxygen requirement of the GOx enzymatic reaction. Furthermore, the removal of hydrogen peroxide has been shown to reduce peroxide-induced degradation of the GOx enzyme.
  • a primary objective of the present invention is to provide improved pH-sensitive hydrogels and polymers containing oxidoreductase enzymes, incorporating appropriate amounts of catalase to remove hydrogen peroxide and to produce oxygen and water in situ, thereby enhancing the swelling kinetics of the hydrogels.
  • a further objective of the invention is to provide methods for making such improved hydrogels.
  • a still further objective is to provide a hydrogel- based glucose biosensor having improved swelling kinetics and longer useful life.
  • the invention comprises a hydrogels containing an analyte-sensitive enzyme which generates hydrogen peroxide, co-immobilized with catalase, with the catalase being present in amounts ranging from about 100 units/ml to about 1000 units/ml.
  • the term "hydrogel" is intended to encompass any polymer matrix suitable for use in hydrated conditions.
  • the analyte is glucose and the analyte-sensitive enzyme is glucose oxidase.
  • the invention is applicable any analyte-sensitive enzyme which generates hydrogen peroxide as part of the reaction. These include monoamine oxidase as well as many oxidoreductases.
  • the invention further encompasses biosensors incorporating these hydrogels.
  • the hydrogels may preferably be formulated such that swelling of the gel permits flow of a drug such as insulin out of the gel.
  • the invention encompasses analyte-responsive drug delivery devices containing hydrogels which meet the above description.
  • the hydrogels may be used with biosensors or drug-delivery devices which use pressure transducers or amperometric means to register analyte concentration.
  • Hydrogels according the invention may also be used with devices employing gas reservoirs or semi-permeable membranes.
  • the invention further includes methods for using catalase in hydrogels, biosensors and analyte-responsive drug delivery devices.
  • FIGURE 1 depicts the equilibrium degree of swelling as a function of pH at fixed ionic strength, for a hydroxypropyl methacrylate/ N,N- dimethylaminoethyl methacrylate/ tetraethyleneglycol dimethacrylate (HPMA/DMA/TEGDMA) (70:30:2 mol ratio) hydrogel of dimensions 0.9 mm x 0.9 mm x 0.4 mm, containing a fixed concentration of GOx of 1000 unit/ml of pre-gel solution.
  • Curve A is the profile with 600 unit/ml incorporated catalase
  • curve B is for the hydrogel with no catalase.
  • FIGURE 2 displays kinetic swelling profiles of a 0.4 mm-thick hydroxypropyl methacrylate/ N,N-dimethylaminoethyl methacrylate/tetraethyleneglycol dimethacrylate (HPMA/DMA/TEGDMA) (70:30:2 mol ratio) hydrogel containing a fixed concentration of GOx of 1000 unit/ml (pre-gel solution), with (A) and without (B) catalase. The catalase concentration was 600 unit/ml (pre-gel solution). Measurements were made with the hydrogel immersed in 50 mM Tris buffer at fixed ionic strength of 0.15 M and at room temperature, pH 7.4 and 6.2.
  • HPMA/DMA/TEGDMA N,N-dimethylaminoethyl methacrylate/tetraethyleneglycol dimethacrylate
  • FIGURE 3 displays kinetic swelling profiles for the hydroxypropyl methacrylate/ N,N-dimethylaminoethyl methacrylate/tetraethyleneglycol dimethacrylate (HPMA DMA/TEGDMA) (70:30:2 mol ratio) hydrogel, with 2 mol % of TEGDMA cross-linker.
  • GOx at a concentration of 1000 unit/ml (pre-gel solution) was co-immobilized with various concentrations of catalase (0, 300, and 600 unit/ml, pre-gel solution).
  • the hydrogels were incubated with PBS (pH 7.2) containing 150 mg/dl glucose at 25 C.
  • Curves A,B,C are under air exposure: A is 0 unit catalase/ml; B, 300 unit catalase/ml; C, 600 unit catalase/ml, all for air-exposed conditions.
  • FIGURE 4 represents free GOx activity in a hydrogel containing glucose at 500 mg/dl in PBS buffer (pH7.2), measured as a function of time at 25 °C.
  • A 300 unit of GOx/ml
  • B 300 unit of GOx/ml and 150 unit of catalase/ml.
  • GOx activities were calculated from the absorbance of GOx enzyme assay, and each point was taken individually at fixed time interval.
  • glucose is the analyte and glucose oxidase is the analyte-sensitive enzyme.
  • the hydrogel for this application is relatively thin, between about 0.1 mm and 0.4 mm, and lightly crosslinked.
  • glucose oxidase (GOx) will generally be used at a concentration ranging from about 500 units/ml to 1500 units/ml, as this concentration range is suitable for measuring blood glucose levels in human beings.
  • the useful range of catalase concentration is from about 100 units/ml to about 1000 units/ml. The best results were achieved by about 600 units/ml; 900 units/ml did not produce improvement in swelling kinetics and in fact appeared to produce less of an improvement than 600 units/ml. It is thought that at 900 units/ml, the amount of catalase is high enough to possibly cause protein aggregation which denatures the enzyme, or by limiting substrate diffusion.
  • the hydrogel is selected to have properties suitable for an analyte-responsive drag delivery device.
  • swelling of the hydrogel in response to increased glucose concentration would permit diffusion of insulin out of the gel into the bloodstream.
  • HPMA/DMA hydrogels are known to be useful for insulin delivery in response to the concentration of glucose (Ishihara K et al., Polym. J. 16:625, 1984), and are used for the experiments presented in the Figures.
  • other hydrogels and polymer matrices having suitable swelling and diffusional properties could be used.
  • glucose-sensitive hydrogels responsive to both pH value and glucose concentration are prepared by polymerizing solutions containing hydroxypropyl methacrylate, N,N- dimethylaminoethyl methacrylate, and tetraethyleneglycol dimethacrylate in the mole ratio 70:30:2.
  • Glucose oxidase at concentrations of between 500 units/ml and 1500 units/ml, and catalase at concentrations of from 100 units/ml to 900 units/ml are included in the pre-gel solution and become physically immobilized within the hydrogel matrix.
  • Hydroxypropyl methacrylate HPMA, Polysciences, Inc.
  • DMA N,N-dimethylaminoethyl methacrylate
  • TEGDMA tetra- ethyleneglycol dimethacrylate
  • EG ethyleneglycol
  • TEMED N,N,N',N'-tetramethylethylenediamine
  • APS ammonium persulfate
  • glucose oxidase GOD, Sigma
  • catalase Sigma
  • Sodium dihydrogen phosphate, potassium hydrogen phosphate, TRIS HC1 (ICN), TRIS Base (Sigma), KC1, and NaCl were used as received. Buffers were prepared from citric acid, Tris HC1, Tris Base, and PBS with pH adjusted with NaOH or HC1 to the desired range. Measurement of pH was performed using a Corning pH meter with G-P Combo w/ RJ pH probe. Calculated amounts of NaCl were added to the buffer solutions in order to adjust the ionic strength to 0.15 M, mimicing physiological conditions. Glucose-Sensitive Hydrogel (GSH) Preparation.
  • GSH Glucose-Sensitive Hydrogel
  • Each glucose-sensitve hydrogel was prepared by redox polymerization between two glass plates (10 cmxlO cm) with the gap set using a teflon spacer (0.40 mm). These slide- molds were held together by metal clamps to provide a uniform internal cavity for the pregel solutions.
  • Each pregel solution contained HPMA, DMA, TEGDMA, EG, TEMED, and APS in the mole ratio 70:30:2:10:0.03:0.001. The concentration of these species in the solution was adjusted to insure complete monomer conversion in the final product. Calculated amounts of glucose oxidase and catalase solutions were prepared separately and added into the pregel solution.
  • Pregel solutions were degassed by bubbling nitrogen for 10 min and/or by stirring under a rough vacuum for 5-10 min and then injected into the slide-mold.
  • the molds were kept at 4 °C for 12-16 h to facilitate complete polymerization.
  • the hydrogel slab was separated from the two glass plates with a razor blade and cut into a 9.0 mm by 9.0 mm square disk using a long-blade cutter. All gel disks were washed in 0.5 X and 1.0 X PBS at least 3 days (2-3 times a day) and then stored in 1.0 X PBS buffer at 4 °C overnight or until use. pH Swelling Studies.
  • Hydrogel disks were immersed in 50 mM Tris buffer (pH 10) overnight or until they reached a constant weight value at room temperature (22-23 °C). The total ionic strength of each buffer at each pH was adjusted to the same value (0.15 M) with a calculated amount of NaCl. Periodically, disks were withdrawn from the buffer solution and weighed after removal of excess surface solution by light blotting with a laboratory tissue. The disk weights were individually monitored in this way until they reached a constant value. This typically required from 6 h to 24 h depending on the value of the pH and the composition of the sample, and the buffer was replaced several times to maintain a constant pH during the experiment.
  • Hydrogel disks were immersed in PBS (pH 7.2) overnight or until they reached a constant weight value.
  • a series of glucose solutions (75, 150, and 300 mg/dL) were prepared in a 100 mL bottle prior to use.
  • the hydrogel disks were placed in the glucose solution and the temperature was controlled using a Precision Scientific Inc. dual chamber water bath and a Thermolyne 42000 Incubator. Oxygen saturation was accomplished by vigorous bubbling of oxygen through the glucose solution with constant stirring during the experimental period. In certain other experiments, no oxygen bubbling was performed, and the solution was exposed to oxygen only at the air/solution interface at the top of the solution container.
  • Cross-linking density is also important for pH-sensitive swelling, with an increase in cross-linking density reducing the equilibrium degree of swelling.
  • Glucose-sensitive hydrogels (GSHs) containing a fixed amount of GOx (1000 unit/mL) and five different catalase concentrations (0, 100, 300, 600, 900 unit/mL) show identical equilibrium swelling behavior in response to pH changes.
  • the equilibrium data demonstrate that different concentrations of catalase do not influence equilibrium swelling in the
  • HPMA/DMA-based hydrogels with approximately 2 mol % TEGDMA cross- linker HPMA/DMA-based hydrogels with approximately 2 mol % TEGDMA cross- linker.
  • a pH-sensitive hydrogel cannot react to changes in glucose concentration until the pH value inside the hydrogel changes. Once the pH value inside the gel changes, mass transfer of water into or out of the gel will occur. The mass transfer of water is driven by osmotic pressure forces, which in turn are generated by ion exchange processes and polymer conformational changes within the gel.
  • pH- sensitive hydrogels have ionizable groups such as carboxylic acids, tertiary amine groups, and sulfanilamide groups. lonization of these groups generates strong osmotic pressure forces and swells the gel; deionization of these groups de-swells the gel.
  • preferred embodiments are thin and lightly cross-linked hydrogels without macropores, such as those used for the experiment of Figure 2.
  • the HPMA/DMA-based hydrogels containing GOx and catalase reversibly contract and dilate when the external pH value is cycled.
  • the hydrogel was equilibrated with a pH value of 7.4.
  • the hydrogel was suddenly subjected to a pH value of 6.2, and the swelling was followed as a function of time.
  • the external pH was switched back to 7.4, and the shrinking (de-swelling) kinetics was observed.
  • Figure 2 Several important points are apparent from Figure 2. Firstly, the degree of swelling returns to its initial value after the de-swelling is complete; i.e., the swelling process is reversible.
  • the swelling and shrinking process is reproducible and identical for various concentrations of catalase (0, 100, 300, and 600 unit/mL) in the GSH.
  • catalase and GOx there is no influence of catalase and GOx on the swelling kinetics of the basic hydrogel with 2 mol % cross-linking ratio, at least in the enzyme concentration range studied.
  • the effect of catalase is significant.
  • the relative swelling ratio after 24 h of glucose incubation increases from 4.96 ⁇ 0.042, 5.21 ⁇ 0.072, to 5.44 ⁇ 0.043, at 25 °C.
  • the latter value is increased by about 11 % when the experiment is performed at 37 °C.
  • the swelling ratio decreases when the catalase concentration in the GSH is increased from 600 unit/mL to 900 unit/mL, possibly by protein aggregation which denatures the enzyme, or by limiting substrate diffusion.
  • a catalase loading of 600 unit/mL appears to be optimum.
  • Catalase may also increase the rate of swelling by reducing peroxide-induced degradation of the GOx enzyme.
  • Our experiments indicate that the activity of free GOx decreases by almost 60 % after only four hours of incubation in glucose solutions, and that this decay in activity can be retarded by adding free catalase to the solution (Figure 4). Since the GOx reaction is irreversible, the final equilibrium degree of swelling should be the same for all of the curves in Figure 3, provided that oxygen is replenished by dissolution at the air interface. Curves A, B, and C never reach this plateau value. However, if the gel samples corresponding to curves A, B, and C are withdrawn from the glucose solution and placed in pH buffer at 7.2, all three samples take about ten hours to return to the slightly swollen equilibrium state at pH 7.2.
  • biosensors with a reduced oxygen depletion problem can also be constructed by using catalase together with a semipermeable membrane which permits sufficient diffusion of oxygen and limited diffusion of glucose to the GSHs from exterior fluids such as blood.
  • catalase substantially enhances the swelling kinetics even under conditions of oxygen saturation.
  • catalase concentrations in the range studied are also very useful for that type of device.

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Abstract

Cette invention a trait à des hydrogels contenant de la catalase co-immobilisés à une enzyme sensible à un analysat tel que la glucose oxydase. Ces hydrogels, qui peuvent être sensibles au pH, sont, de préférence, fins et faiblement réticulés. La concentration en catalase est comprise généralement entre 100 et environ 1000 unités par millilitre. Ces hydrogels, qui ont un temps de réponse au gonflement bien plus court que celui des hydrogels dépourvus de catalase, se révèlent des plus utiles dans des biocapteurs et dans des systèmes de délivrance de médicaments sensibles à un analysat. Ces hydrogels ont également une durée de vie utile accrue, du fait de la protection assurée par l'enzyme immobilisée sensible à un analysat contre la dégradation occasionnée par le peroxyde d'hydrogène.
PCT/US2001/010612 2000-04-03 2001-04-02 Methodes et compositions correspondantes relatives a l'utilisation de catalase dans des hydrogels et des biocapteurs WO2001075089A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011097586A1 (fr) 2010-02-08 2011-08-11 Glumetrics, Inc. Protection antioxydante d'un capteur chimique
CN114886871A (zh) * 2022-04-15 2022-08-12 北京林业大学 一种自驱动药物载体的构建及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4655880A (en) * 1983-08-01 1987-04-07 Case Western Reserve University Apparatus and method for sensing species, substances and substrates using oxidase
US5431160A (en) * 1989-07-19 1995-07-11 University Of New Mexico Miniature implantable refillable glucose sensor and material therefor
WO1999017095A1 (fr) * 1997-09-30 1999-04-08 M-Biotech, Inc. Biocapteur destine a mesurer la concentration d'une molecule organique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4655880A (en) * 1983-08-01 1987-04-07 Case Western Reserve University Apparatus and method for sensing species, substances and substrates using oxidase
US5431160A (en) * 1989-07-19 1995-07-11 University Of New Mexico Miniature implantable refillable glucose sensor and material therefor
WO1999017095A1 (fr) * 1997-09-30 1999-04-08 M-Biotech, Inc. Biocapteur destine a mesurer la concentration d'une molecule organique

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011097586A1 (fr) 2010-02-08 2011-08-11 Glumetrics, Inc. Protection antioxydante d'un capteur chimique
CN114886871A (zh) * 2022-04-15 2022-08-12 北京林业大学 一种自驱动药物载体的构建及其制备方法

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