EXPERIMENTAL 1. Chemicals

The monomers used in this study namely, acrylamide(AAm), maleic acid(MA), and N-vinyl 2-pyrrolidone(VP), Itaconic acid(IA) were obtained from BDH and Aldrich, respectively. KH2PO4, K2HPO4 and H3PO4 used to prepare phosphate buffers and tri-sodium

citrate, sodium dihydrogen citrate and citric acid used to prepare citrate buffers were obtained from BDH. The model drug Mehtylene Blue was obtained from Ficher and BSA (Fraction V) and D-amylase and invertase from Aldrich. Pure terbinafine hydrocloride was used after purification of the coomertial drug Lamisil of Novarsis Company. The chemical formulea of monomers and drugs are given in Scheme 1.

Scheme 1.

n-vinyl-2 pyrrolidone Itaconic acid Terbinafine hydrocloride

For the preparation of Acrylamide/maleic acid hydrogels, acrylamide (AAm) weighted 1g was dissolved in 1ml aqueous solution of 0, 20, 30, 40, 50, 60mg maleic acid respectively.

For the preparation of poly (n-vinyl-2 pyrrolidone/itaconic acid) hydrogels, 2ml n-vinyl-2 pyrrolidone was dissolved 1ml aqueous solution of 0, 60, 120, 180, 240 mg itaconic acid. In order to increase the amount of IA in the VP monomer and to be prepare different network properties having hydrogels, 2ml n-vinyl-2 pyrrolidone was dissolved 1ml aqueous solution of 60, 120, 180 and 240 mg itaconic acid and 0.25% (v/v, EGDMA/VP) cross-linking agent EGDMA added in the mixture. These solutions were placed in PVC straws of 3mm diameter and irradiated at different doses in air at ambient temperature in a ⁶⁰Co Gammacell 220 type J irradiator.

Hydrogels obtained in long cylindrical shapes were cut into pieces of 2–3 mm long.

Hydrogels were dried in vacuum oven at 315K to constant weight and subjected to soxhlet extraction with water as solvent Uncross-linked polymer and/or residual monomer were removed with this extraction from the gel structure. Extracted gels were dried again in vacuum oven at 315K to constant weight. Percentage gelation i.e. percentage conversion of monomers into insoluble network was based on the total weight of the VP or AAm and diprotic acid in the initial mixture. The amount of uncross-linked acid was determined by titration of extract against NaOH(0.05N) to phenolphthalein end point.

CH₂ CH

2.3. Swelling studies

Dried hydrogels were left to swell in a solution of desired pH (2–9), ionic strength I(0.01–0.20M), and temperature (4.0–65.0^oC). Swollen gels removed from the swelling media at regular intervals were dried superficially with filter paper, weighed and placed in the same bath. The measurements were continued until a constant weight was reached for each sample.

The weight fraction of polymer in swollen gel w, was used to calculate the volume fraction Q_2m and equilibrium degree of swelling (EDS), Q, of the gel sample equilibrated in the buffer solution.

> @

Q_2m = 1+ /U U_w(w -1)^{-1 -1} (1)

where U and U_w are the densities of swollen gel and water. The equilibrium degree of swelling (EDS) was defined as Q = 1/Q_2m.

The percentage mass swelling of hydrogels was calculated from the following relation;

S%(m) =

>

@

where mo is the mass of dry gel at the time 0 and mt is the mass of swollen gel at time t.

2.4. Adsorption studies

2.4.1. Adsorption of proteins onto AAm/MA hydrogels

For the adsorption of bovine serum albumin (BSA) Fibrinogene and J-globulin onto AAm/MA hydrogels, 0.1g of dry gel samples were first swollen to equilibrium and then placed in 5 mL solution of 1.0–8.0 mg/mL protein in phosphate buffer (pH2.0–8.0) and allowed to equilibrate for 12 h at different temperatures The amount of adsorbed protein was determined by Commassie Brilliant Blue binding method (Scopes, 1984).

2.4.2. Adsorption of enzymes onto P(AAm/MA) hydrogels

For the adsorption of enzymes onto P(AAm/MA) hydrogels, 0.1g of the dry gel was placed in 20 mL solution of 100 mgdL^-1 D-amylase or invertase in phosphate buffer (10 mM, pH7.0) and allowed to equilibrate for 48 h at 25qC. The enzymatic activities of free and adsorbed D-amylase and invertase were determined by an enzyme reaction using starch as substrate and starch-iodine and Folin — Wu methods respectively (Yoo, et al., 1987).

2.4.3. Adsorption of drugs onto P(AAm/MA) and P(VP/IA) hydrogels

For the investigation of drug release behaviour of P(AAm/MA) and P(VP/IA) hydrogels prepared in this study, Methylene Blue(MB) and Terbinafine hydrocloride(TER-HCl) were used as model drugs. Dry polymer discs (2 mm thickness, 4 mm diameter) were loaded with MB by immersion into aqueous solution of MB (0.5 g/dL) at ambient temperature at pH7 for 2 days. The drug loaded hydrogels were stored for later evaluations in MB solution at ambient temperature.

In order to obtain pure TER-HCl firstly, 250 mg Lamisil was dissolved in 20 ml distilled water. After removing undissolved part the solution was dried in a vacuum oven at 315 K to constant weight. For the investigation TER-HCl adsorption capacities of hydrogels, dry polymer discs(2mm thickness, 4 mm diameter) were loaded with TER-HCl by immersion into aqueous solution of TER-HCl (0.16–3.75 mg TER-HCl/mL) at 4 qC for 2 days.

2.4.4. Controlled release of methylene blue and terbinafin hydrocloride from P(AAm/MA) and P(VP/IA) hydrogels

The controlled release of MB and TER-HCl from hydrogel matrices was measured after a MB or TER-HCl loaded, swollen gel was placed in a vessel containing 50 mL of phosphate buffer solution (0.1 mole/L) at 25qC and at a constant shaking rate. At various times aliquots of 3 mL were drawn from the medium to follow the MB and TER-HCl release and placed again into the same vessel so that the liquid volume was kept constant. MB release was determined spectrophotometrically using a Unicam 8715 spectrophotometer at 641 nm. and TER-HCl at 222 nm. The controlled release of non-specifically adsorbed MB and TER-HCl were followed at pH7 and pH6.0–2.0 were used for the controlled release of specifically bonded MB and TER-HCl from hydrogels. After release at pH2, the hydrogels were immersed in 0.1 mole/L HCl for 2 days to remove any remaining MB and TER-HCl in the gel system.

3. RESULTS AND DISCUSSION