Glucose-sensitivity of glucose oxidase-containing cationic copolymer hydrogels having poly(ethylene glycol) grafts

Abstract

Glucose oxidase and catalase were immobilized on poly(diethylaminoethyl methacrylate-g-ethylene glycol) gels by copolymerization of the constituent monomers and the functionalized enzyme solutions. The hydrogels were prepared in the form of discs and microparticles. The amount and the activity of enzymes immobilized in the matrix were determined. The hydrogels were tested for their response to glucose by exposing microparticles to varying concentrations of glucose. The generation of gluconic acid as a result of the reaction of glucose with oxygen was investigated as a function of polymer parameters, such as crosslinking ratio and enzyme loading. Pulsatile variation of the glucose concentration was used to confirm the glucose-dependent swelling properties of these hydrogels.

Introduction

Glucose-sensitive membranes and hydrogels can be used effectively to achieve feedback-controlled release of insulin. These polymeric gels demonstrate a reversible swelling behavior in response to glucose concentrations in the surrounding medium. The resulting changes in the mesh size modulate release of insulin physically imbibed in the gels. Since insulin diffusion through these gels is dependent on the changes in mesh size caused by the varying concentration of glucose in the medium, these gels are effective in bringing about feedback-responsive release.

Glucose oxidase (GOD) immobilization on polymers has been investigated in detail in attempts to produce materials for the construction of new types of glucose sensors [1]. Work has also been published on the use of GOD for stimulation of pH-responsive release from polymer matrices and membranes [2]. GOD has been successfully immobilized on a wide variety of polymers, such as polyacrylates [3], polymethacrylates [4], polyethylene [5], polypyrroles [6], silica [7], and poly(vinyl alcohol) [8], [9]. The activity of immobilized enzymes is an important parameter that determines the performance of the hydrogel membrane. In most cases, there is a significant loss of enzyme activity due to harsh methods employed during immobilization and polymerization. Stability of these enzymes is also a major concern as irreversible degradation might occur with time [9].

The next important criterion determining the feasibility of these gels in insulin delivery is their optimal performance under physiological conditions. Heller and co-workers [10], [11] observed that the glucose-sensitivity of poly(ortho esters) could be improved by incorporating tertiary amines into the structure. Albin et al. [12] studied GOD-immobilized polyacrylamide gels and poly(dimethylaminoethyl methacrylate) (PDMAEM) gels in the form of macroporous and microporous matrices. The kinetics of solute transport in these membranes was found to be limited by the solubility of oxygen in the surrounding medium.

The studies conducted by Goldraich and Kost [13] on GOD-immobilized copolymers of 2-hydroxyethyl methacrylate (HEMA) and dimethylaminoethyl methacrylate (DEAEM) crosslinked with tetra(ethylene glycol) dimethacrylate (TEGDMA) indicated very slow deswelling rates. These gave rise to significant gel irreversibilities as the gels were unable to recover their original conformation when transferred between two solutions containing varying concentrations of glucose. This was further confirmed by Ishihara and co-workers using GOD-immobilized polyamides [14], [15] and polymethacrylates [16]. The slow collapse was attributed to the small range of pH variation (between 6.2 and 6.6) attained in the matrix.

In our previous work [17], [18], we studied the pH sensitivity of poly(diethylaminoethyl-g-ethylene glycol) (P(DEAEM-g-EG)) hydrogels. It was shown that these hydrogels had a distinct transition behavior, characterized by a steep change in swollen volume at pH 7.0. Below this pH, the hydrogels exhibited high swelling ratios. Above the transition pH, the hydrogels were relatively collapsed with much smaller swelling ratios. Both of these states were related to their corresponding mesh sizes The ionization of the tertiary amine side groups under these conditions resulted in the swelling of the polymer through two mechanisms: (i) increase in the hydrophilicity of the polymer and (ii) electrostatic repulsion between the positively charged groups. In the absence of glucose, the pH in the microenvironment of the gels was found to remain constant at 7.4 and the gel remained collapsed. For applications, two different geometries were studied, namely, discs and microparticles. Of course, the dynamics of the pH-sensitive swelling/deswelling behavior was considerably faster in the case of microparticles.

In our research, GOD was immobilized in the hydrogel to impart glucose-sensitivity to the matrix. To improve the rate of reaction and, therefore, the swelling rates within the gel matrix, one option was to immobilize catalase in the hydrogel along with GOD. Low conversions and reduced rates of reactions might be due to the inhibitory effect of hydrogen peroxide that is formed as a byproduct of the glucose oxidation reaction. Catalase reduces hydrogen peroxide and eventually removes it from the system. By this reaction, some oxygen is recovered and made available for the formation of acid. Thus, an efficient redox system is set up within the gel which operates to minimize unwanted products and provide maximum availability of reactants for the formation of hydrogen ions [12].

Incorporation of poly(ethylene glycol) grafts on the main chains of the hydrogel was an innovative approach based on previous work done in our laboratory [19]. These grafts were expected to retard the degradation of enzymes and proteins within the gel. For an internally implantable device, the presence of certain ‘stealth’ groups helps to minimize the immunoreaction and subsequent rejection by the body. PEG is known to have such stealth properties, which can help maximize the lifetime of the hydrogel in the body [20].

In this work, the glucose sensitivity of P(DEAEM-g-EG) hydrogels was investigated. The effect of a glucose stimulus and the eventual decrease in the pH of the hydrogel on swelling were studied. The change in the swelling ratio and the mesh size under a glucose stimulus were determined.