MATERIALS AND METHODS

Several solutions containing different concentrations of diethyleneglycoldimethacrylate (DEGDMA-Aldrich) and glycidylmethacrylate (GMA-Sigma) in ethylpropionate (EP-Aldrich) were prepared, degassed with nitrogen and irradiated on a ^6oCo gamma source at room temperature without stirring, with a dose rate of 15 kGy/h and with total doses from 1 to 50 kGy.

The obtained microspheres were washed several times with the solvent, centrifuged and freeze dried. The characterization was performed by spectroscopy (ATI Mattson RS-1 FTIR spectrometer, equipped with an MTEC 300 photoacoustic detector) and electron microscopy (JEOL JSM 5600LV).

The amount of epoxy group incorporated in microspheres was visualized by copper staining, and their bioactive material binding capacity was tested with histidine and lysosyme in the following way:

Microsphere samples were immersed in 1 M IDA dissolved in dimethyl sulfoxide/water (1:1). The reaction was performed at 80oC for 8 h. After washing with water, the microspheres were dried until constant weight. Measured quantities were immersed in 0.2 M CuSO4. After washing with deionized water, copper was released from the microspheres by shaking them with 0.1 M EDTA, pH7.0, at room temperature for 8 h. Copper content was determined spectroscopically comparing the absorbance of the supernatant at 715 nm with that of 0.1 M EDTA with Cu(II) at various concentrations. For the reaction with histidine, a total volume of 1–10 mg of microspheres was immersed for 24 h in an excess of histidine in 20 mM sodium phosphate buffer, pH7.0, 250 mM NaCl, in a f nal volume of 1 ml. The concentration was caiculated from the measured absorption at 220 nm.

The microspheres were also tested for affnity purification of peroxidase from A.rusticana roots and Gycine max seed coats in aqueous two-phase system. In this method, the microspheres were mixed with several different PEG/phosphatase aqueous two-phase system and the crude extract, equilibrated, then recovered and washed. The peroxidase was eluted with a-D mannopiranose and the amount of high-puriti peroxidase was determined by SDS-PAGE analysis.

4. RESULTS AND DISCUSSION

When a solution of DEGDMA is irradiated in an adequate solution, due to the homogeneous initiation, monodisperse co-polymer spheres are formed. An adequate solution is not only a good solvent for the monomer, but it also has to allow a streched-out configuration for the monomer in order to allow independent solvation of the two methyl groups, thus enhancing the intermolecular over intramolecular crosslinking⁷. The monomer concentration is another important factor for microsphere formation, and Fig.1 presents the products obtained from irradiation of several DEGDMA solutions of different concentration in ethyl propionate.

FIG. 1. SEM photographs of microspheres prepared by irradiation of a 10% (upper left); 35% (upper right); 45% (lower left); and 60% (lower right) DEGDMA solution in ethyl propionate.

The pictures illustrate the fact, that for microsphere formation only monomer concentrations below 30% are suitable. When the monomer concentration lies between 30 and 50%, so called "monolith" is formed. (Such monoliths are useful for affinity separation and microfiltration, and we are now investigating the effect of various parameters on the pore size and flux trough such monoliths⁸. This work is beong carried out in collaboration with Argentina.) When the monomer concentration was increased further, glassy homopolymer formed.

To enable covalent binding of variety of bioactive molecules to the microspheres, we introduced epoxy groups by compolymerization of DEGDMA with glycidylmethacrylate (GMA) in their common solvent, ethyl propionate. The investigated factors of influence on the microsphere yield and size were: irradiation temperature, absorbed dose and dose rate, and co-monomer ratio in the feed solution.

Table I and Fig. 2 illustrate the effect of GMA content on the yield and size of microspheres for a 10% monomer solution, irradiated at room temperature, with dose of 20 kGy.

The increase in the size of the microspheres with increasing GMA content was expected. Since the growth of a particle is influenced by competition of polymerization and crosslinking reactions, with the increasing content of a monofunctional monomex, the the probability of propagation over crosslinking will also increase, leading to bigger particles.

Further increase of the GMA content caused the particles to aggregate. When only GMA is irradiated, no precipitate formed.

The size of the microspheres does not depend on the absorbed dose, as shown on Fig. 3.

The microspheres irradiated with 3 and with 25 kGy have the same size (10% DEGDMA solution in EP), but the size distribution is slightly broader for lower dose.

TABLE I. PROPERTIES OF GMA/DEGDMA MICROSPHERES

GMA content (%) Yield (%) Diameter (µm)

0 98.7 0.94 20 68.8 1.04 40 56.1 1.32 60 51.2 2.91

FIG. 2. SEM photographs of microspheres prepared by irradiation of a 10% co-monomer solution with different GMA contents of 0% (upper left); 20% (upper right); 40% (lower left); and 60% (lower right), with 20 kGy.

FIG. 3. SEM photographs of microspheres irradiated with 3 kGy (left) and 25 kGy (right).

Due to their similar chemical structure, the infrared spectra of DEGDMA and GMA are almost identical. The incorporation of GMA in the microspheres is seen only from the appearance of a new peak at around 910 cm-l, assígned to epoxy groups (Figs 4. and 5).

It is also possible to confirm this incorporation by the reaction with copper, both visually and spectroscopically (Fig. 6). The usefulness of the microspheres for immobilization of various bioactive molecules was tested with histidine and lysosyme⁹, also shown on Fig. 6.

FIG. 4. FTIR spectra of pure monomers, DEGDMA (lower spectrum) and GMA (upper spectrum).

FIG. 5. FTIR/PAS spectra of microspheres with different GMA content.

FIG. 6. Binding capacity of GMA microspheres.

Aqueous two-phase systems are formed when PEG salts and inorganic salts are dissolved above certain concentrations. The characteristic feature of such a system is that it allows the partitioning of biomolecules and cell particles of diverse origin under nondenaturing conditions, therefore it is specially suited for enzyme extraction and purification from biological media. When the microspheres were added to this system, they distribution depended on the molecular weights of the PEG, as shown on Table II.

TABLE II. PARTITION OF A.RUSTICANA CRUDE EXTRACT PROTEINS AND MICROSPHERES IN PEG-PHOSPHATE SYSTEMS AT PH7

PEG M.W.

Material 20.000 6.000 1.540 600

Microspheres Whole system Top phase Interphase Interphase

K HRP 0.02 0.03 0.60 37.70

K Total Proteins 0.79 0.63 0.63 5.20

For the affinity separation, PEG 600 system was chosen, as there the peroxidase was completely in the top phase¹⁰. After addition of microspheres, only 10–15% of peroxidase remained soluble. By elution, 75–80% of high purity enzyme was obtained. These results showed that by using the functional microspheres, it was possible to achieve a selective recovery of enzymes with good yield and high purification level.

The microspheres, beside the narrow size distribution, have another advantage: they are stable in dry state and can be used in both aqueous and organic solvents. Presently, immobilization of several proteins for purification purposes and monoclonal antibodies of schistosoma for immunodiagnosis are under way.