Copolymerized PEGlyated Acrylate Hydrogels for Delivery of Dicolofenac Sodium

Hydrogels are polymeric networks that can absorb and retain large amounts of water and biological fluids and swell, still maintaining their three-dimensional structure (1, 2). These polymeric networks contain hydrophilic domains that are hydrated in an aqueous environment, thereby creating the hydrogel structure. The term network indicates the presence of cross-links, which help avoid the dissolution of the hydrophilic polymer in an aqueous medium (3–5). Hydrogels have many advantages over other drug delivery systems such as good mechanical and optical properties and biocompatibility (6, 7). The degradation products of hydrogels are usually nontoxic or have lower toxicity. Lower interfacial tension between the surface of the hydrogel and the physiological fluid helps minimize protein adsorption and cell adhesion on the hydrogel's surface. The soft rubbery nature of hydrogels also can minimize mechanical irritation when used as in vivo implants (8).

Drug release from hydrogels can be regulated by controlling water swelling and the cross-linking density of the polymers (9, 10). Because of their matrix form, hydrogels allow drug molecules to be released at a very slow rate, and when given orally, the slow release reduces gastrointestinal side effects. Hydrogels also can be given locally as transdermal drug delivery systems (11–13) and as implants (14) at or near the site of inflammation. Because of local delivery, the dose of the drugs can be further reduced.Therefore, systemic toxicity of drugs such as hepatotoxicity, blood dyscrasias, hypersensitivity, and exacerbation of asthma can be minimized. When given by injection, hydrogels help sustain drug release for a longer duration of time (15, 16).

The integrity of a drug-delivery device during its lifetime is a very important factor for its pharmaceutical use. Changing the degree of cross-linking and copolymerization facilitates achieving the desired rigidity and hardness in the hydrogels. Increasing the degree of cross-linking, however, can create a more brittle structure. Optimizing the concentration of the cross-linker, thus, is a very important factor. Copolymerization also can help achieve the desired properties of hydrogels (8). Many scientists have prepared and studied such hydrogels using copolymerization techniques (17–19). In the present work, copolymerization of poly(ethylene glycol) (PEG) and acrylic acid was carried out in the presence of polyethylene glycol diacrylate (PEGDA). Ethylene glycol diacrylate or dimethacrylate also have been used as an agent for cross-linking of preformed polymers in concentrations <1% (20–22). PEGDA of various molecular weights were used in various concentrations to study the effect the PEG's concentration and molecular weight had on the properties of the hydrogels that were formed.

Materials and methods

PEGs of various molecular weights were procured from Wilson Labs (Mumbai, India). Acrylic acid monomer and dicyclohexylcarbodiimide were procured from Himedia Laboratories (Bombay, India). The solvents and other chemicals were procured from Central Drug House, Ltd.( New Delhi, India). All the solvents and chemicals were of analytical grade. The drug, diclofenac sodium, was a gift sample from Promise Pharmaceuticals (Sagor, India).

Synthesis of polyethylene glycol diacrylate. PEGDA was prepared by using an esterification reaction, involving acrylic acid and PEG of various molecular weights (see Table I). Weighed amounts of PEG were dissolved in dichloromethane and mixed with a twice-molar solution of dicyclohexyl carbodiimide in dichloromethane. Acrylic acid then was added (in five times molar excess) into this solution with stirring. The stirring was continued for 2 h at room temperature followed by 2 h of stirring on an ice bath. The precipitate of dicyclohexyl urea byproduct was filtered out by vacuum filtration, and the volume of the filtrate was reduced using a rotary vacuum evaporator (Superfit, Mumbai, India). The product (PEGDA) was then precipitated by adding this concentrated filtrate to an excess of diethyl ether with continuous stirring. Vacuum filtration again was performed to separate the precipitated product from the solvent. PEGDA then was purified by dissolving in dichloromethane and reprecipitating with diethyl ether. Finally, the purified product was dried under vacuum in a vacuum oven (Jyoti Scientific, Gwalior, India) and then stored in a vacuum desiccator until use. PEGs of various molecular weights were used for the preparation of the copolymers.

Table I: Composition of different hydrogel formulations used in the present study.

IR spectroscopy was performed to confirm the formation of an ester linkage between PEG and acrylic acid. The IR spectra of the reactants (acrylic acid and PEG) and the product (PEGDA) were compared for any change in vital peaks. The samples were compressed with potassium bromide in the form of pellets and analyzed using a Fourier transform IR spectrophotometer (PE 1600, Perkin Elmer, Waltham, MA).

Preparation of hydrogels. Polymerization of an acrylic acid monomer was carried out in the presence of PEGDA by radiation polymerization using a photochemical reactor. Solution polymerization was carried out in double distilled water. In Petri plates, weighed amounts of both PEGDA and acrylic acid up to a concentration range of total polymers of about 50% weight/volume (w/v) were dissolved in water and then exposed to ultraviolet radiations at wavelength (λ) of 365 nm for polymerization using a photochemical reactor (Jain Scientific Glasswares, Ambala, India). The hydrogel wafers then were dried and stored in a well-closed container until further use. To study the effect of concentration of such PEGDA cross-linkers on the properties of the hydrogels, hydrogel formulations were prepared with various ratios of PEGDA to the acrylic acid monomer (Table 1).

The prepared hydrogels were purified by soaking the hydrogels in an excess amount of ethanol or ethanol-and-water mixture 12–24 h to remove the soluble impurities. Spectral analysis of the washing medium confirmed the completion of washing.

For easy handling and storage, the purified hydrogels were dried before drug loading. Drying was performed under a hot-air stream flowing over the hydrogel surface using a hot- air sterilizer oven (YSI-431, Yorco, New Delhi, India) at 60 °C for 2 h followed by drying in a vacuum oven (Jyoti Scientific, Gwalior, India) at 45 °C for 2 h.

Characterization of hydrogels. The physical appearance and texture of the hydrogels were visually evaluated, and their inner morphology was studied using scanning electron microscopy (SEM). The samples were gold-coated and observed under an electron microscope (Leica, Ernst-Leitz-Strasse, Germany) under various magnifications. Glass-transition temperatures of the hydrogels were measured using a differential transition calorimeter (822E, Mettler Toledo, Columbus, Ohio). An aluminum crucible of 40-L capacity held the samples. The samples were heated from 35 °C to 350 °C at a rate of 10 °C/min. Nitrogen gas was used as cooling (at 200 L/min) as well as the purging medium (at 80 L/min).

Absorbing water is one of the vital properties of hydrogels, because it makes them biocompatible (1). The water-absorbing capacity of hydrogels was determined in terms of percent equilibrium water content (% EWC) or swelling ratio (SR). The dry hydrogels were weighed (W_d) and then immersed in distilled water for 24 h at 37 ± 2 °C. The hydrogels then were reweighed (W_s) after removing the excess water by lightly soaking the swollen hydrogels using filter paper. The swelling ratio of the hydrogels was determined using the formula:

Water diffusion study. Release of the drug from a matrix system generally occurs by a diffusion mechanism. The drug diffuses out, along with water, in the form of a solution at pH 7.0 of the release medium. The water-diffusion study, therefore, is indirectly a measure of solute diffusion. It is assumed that diffusion from the matrices follows Fick's law of drug dissolution and diffusion in the medium, both during the sorption and desorption phenomenon (23). In the present study, the diffusion property of the hydrogels was studied using desorption phenomenon. The swollen hydrogels were dried gradually under air at room temperature and weighed after 15-min intervals until a constant weight was reached. The diffusion coefficients of the hydrogels were calculated using the equation:

in which, D is the diffusion coefficient of the hydrogel, δm_t is the weight loss in t time, δm_∞ is the weight loss at infinity, l is the thickness of the dried hydrogel, and t is the time of diffusion of water from the hydrogels during drying.

Drug loading. The drug can be incorporated in the hydrogels by in situ drug-loading during polymerization and by drug-loading by incubation after polymerization (5). In the present experiment, diclofenac sodium was loaded by incubating a known weight of the hydrogel in a 10% w/v aqueous solution of the drug for 24 h at room temperature. The drug-loaded hydrogels then were dried and stored in a well-closed container for further use. The drug remaining in the incubating solution was determined spectrophotometrically at 276 nm after appropriate dilution. The entrapment efficiency of the hydrogel was calculated as follows:

in which, A_o is the initial amount of drug in the incubation medium, A_f is the final amount of drug in the incubation medium, and W_h is the dry weight of the hydrogel incubated for drug loading.

Release kinetics and porosity. Release studies were carried out in distilled water as receptor medium, maintained at 37 ± 2 °C. Sink condition was continuously maintained. The amount of drug released in the dissolution medium was determined spectrophotometrically at 276 nm after every 1-h interval. The study was continued for 72 h. The data of release profiles for each formulation were analyzed further by model fitting with the help of software PCP DissoV2.5 (Pune College of Pharmacy, Pune, India).

All of the properties of the hydrogels, including hydration capacity, diffusion, and drug release, can be correlated to the porosity of the polymer matrix. Porosity of the hydrogels also can be determined by the diffusion studies (24). The diffusion study of the drug from drug-loaded hydrogels and their simple aqueous solutions was carried out using dialysis tubes of pore size 2.4 nm (Himedia, Mumbai, India), and the diffusion coefficients were calculated. The relation between these two diffusion coefficients can be expressed in terms of the Mackie & Meares' equation:

in which d and d_o are the diffusion coefficients of the drug from the hydrogel and aqueous solution, respectively, and ε is the porosity of the hydrogel.

Results and discussion

Acrylate hydrogels are generally prepared using N,N'-methylene-bisacrylamide (BIS) as a cross-linker, and N,N,N',N'-tetramethylene-diamine (TEMED)/ 2, 2-azo-bis-isobutyronitrile (AIBN), and ammonium peroxydisulfate as initiators (25). These reagents may be toxic for living cells (26, 27). In addition, they may lead to the formation of brittle structures if used in high concentrations.

Copolymerization can be used to produce hydrogels of desired mechanical properties (8). In the present work, copolymerization of acrylic acid was carried out in the presence of PEGDA to avoid the chances of toxicity and to prepare hydrogels with good mechanical properties. Being a PEG derivative, PEGDA is biocompatible and nontoxic (28). PEGDA is a bifunctional polymer and as such also can act as a cross-linker. PEGDA mainly functions as a cross-linker using comparatively lesser amounts of PEGDA (1%) (20–22). But in this experiment, it was used as a template for polymerization when the polymerization of acrylic acid monomers was carried out in the presence of PEGDA. Hydrogels of PEGDA also were reported, but those are mainly PEG hydrogels. In the present study, only two acrylate groups were there for each PEG molecule, and the aim was to prepare PEG-acrylic acid copolymer-based hydrogels. Such copolymers can improve the physicochemical properties of polymers comprised of only acrylic acid. In the present study, monomeric acrylic acids were polymerized on the terminals of PEG-acrylates using 25–75% PEGDA as the template for acrylic acid copolymerization. This process then modifies some physical properties of only polyacrylic acid hydrogels. PEGDA of various molecular weights were used in different concentrations to study the effect of concentration and molecular weight on the properties of the hydrogels that were formed.

Synthesis of PEG-diacrylates. The cross-linker, PEGDA, was synthesized in the laboratory by the esterification reaction between PEG and acrylic acid monomer in dichloromethane. The esterification reaction is catalyzed by the addition of dicyclohexyl carbodiimide in the reaction mixture. The schematic representation of this reaction is shown in Figure 1.

Figure 1

Because bifunctional PEGs were used, the reaction could take place at both the ends. Stoichiometrically, all of the other reactants were used in amounts twice as much as the amount of PEG used in the reaction. Addition of some excess of reactants, however, always is preferred in reactions involving macromolecules such as PEG to overcome the steric hindrance provided by their bulky structures. Acrylic acid monomer, therefore, was used in a five times molar excess quantity as compared with the stoichiometrically calculated value. The esterification reaction took place in the reaction mixture of PEG and dichloromethane in dicyclohexyl carbodiimide in 5 min after the addition of acrylic acid. The precipitate of dicyclohexyl urea started appearing, and stirring was continued for 4 h to complete the reaction. An ice-cold temperature was maintained to precipitate out dicyclohexyl urea, completely.

The byproduct was separated from the product-solution by vacuum filtration. The filtrate was concentrated, and the product was precipitated out by adding it to five times excess of diethyl ether. The product was separated by vacuum filtration to separate the ether soluble impurities from the product. The product so formed was purified by recrystallizing it from its solution in dichloromethane. The final purified product was dried under vacuum.

IR spectral analysis was performed to ascertain the completion of the reaction. The IR spectrum of the product was compared with those of pure PEG and pure polyacrylic acid (i.e., acrylic acid polymerized without PEG). The vital peaks present in the IR spectrum of PEG were at 3463 cm^–1 because of the –OH stretch, 2917 cm^–1 for the alkyl–CH stretch, and 1099.3 cm^–1 for the ether (–C–O–C–) group. The main peaks in the IR spectrum of plain polyacrylic acid are: –OH stretch at 3386.5 cm^–1 , –CH stretch at 2926.8 cm^–1 , and –C=O stretch at 1720.6 cm^–1 . Whereas in the spectrum of the synthesized-PEGDA (see Figure 2), no peak was observed in the range of 3200–3600 cm^–1 (i.e., peak because –OH stretch was absent). These interpretations showed that all the –OH groups of PEG have reacted with the –COOH group of acrylic acid. A shift in peak because of the –C=O stretch was observed, from 1720.6 cm^–1 in acrylic acid to 1761.6 cm^–1 in the case of the product. The peak at 1761.6 cm^–1 is because of the carbonyl stretch in the case of the esters. This shift further confirmed the synthesis of PEGDA. Another characteristic peak because of ester linkage, (i.e., a distinct and sharp peak because of the C(=O)–O– group) also was seen at 1199.3 cm^–1 in the spectrum of PEGDA.

Figure 2

Synthesis of hydrogels. Because there are two reactive terminal hydroxyl groups in PEG, these macromers (PEGDA) contain two acrylate groups per molecule. Upon free-radical polymerization, these macromers form a cross-linked three-dimensional gel.

The hydrogels were prepared by the copolymerization of acrylic acid monomers, and the synthesized PEGDA by radiation-induced polymerization. Initially, a heat-initiation technique was adopted for preparing the hydrogels, which was faster compared with the radiation-induced polymerization technique. Because the heating process could not be controlled, preparation of the hydrogels could not be reproduced. For further studies, therefore, the hydrogels were prepared only by radiation polymerization.

Again, both bulk- and solution-polymerization methods were tried for synthesizing the hydrogels. The solution polymerization in aqueous medium was comparatively faster than bulk polymerization. The polymerization of the acrylates was determined by the time presence of –H and –OH radicals in the H₂O molecules in the dispersed phase. This condition may be attributed to the release of –H and –OH free radicals from the water molecules, which helps in the initiation and propagation of polymerization. The PEG molecules further are capped with hydrophobic polymerizable units, thereby forming micelle-like structures in water. The effective concentration of the double bonds within the micelles increases, thereby increasing the rate of the propagation reaction in free-radical polymerization. The radical-termination reactions are diffusion-controlled and are retarded with the increase in viscosity of the medium, which restricts the segmental motion of the involved polymer radical. An increase in the propagation rate and a decrease in the termination rate results in a high polymerization rate and rapid gelation (29).

As the prepared hydrogels were washed and dried before characterization, the type of polymerization technique used (bulk or solution polymerization) imparted no significant change in the physical properties of the hydrogels. The faster technique (the solution-polymerization method) was continued for further preparation of the hydrogels.

Radical polymerization was accomplished by exposing the polymerizing mixture (i.e., the aqueous solution containing the monomer and cross-linker) to UV radiation (365 nm) using a photochemical reactor (Jain Scientific Glasswares, Ambala, India) for 36 h. Completion of polymerization was evident from the change in the physical state of the reaction mixture. The reaction mixture initially was in the form of a liquid, which gradually thickened as the polymerization took place. A flexible solid mass was formed upon the completion of polymerization. In this way, the hydrogels were prepared using PEGDA of various molecular weights and in different weight ratios. PEGDA, being a bifunctional agent, can serve as a cross-linker in the present copolymerization reaction (see Figure 3).

Figure 3

As the molecular weight of the cross-linker is increased, the molar percentage will decrease for the same weight percentage. With the increase in the molecular weight of PEGDA, therefore, the cross-link density would decrease. This change follows the prediction made by the chemical representation of the copolymeric PEG-acrylate hydrogels containing PEGDA of different molecular weights (see Figure 4). It is assumed that for the same molar ratio of PEGDA, the size of the pores might increase with the increase in molecular weight.

Figure 4

Purification and drying of hydrogels. The polymerization was done until a solid mass was formed that may contain some unreacted monomers, either entrapped in the matrix or adsorbed on the surface. Purification of the hydrogels that were formed was essential before their further use. The purification was carried out by soaking the prepared hydrogels in a hydroalcoholic mixture for 24 h. The acrylate monomers, being soluble in water, came out in the aqueous system used for washing. The washing medium was tested intermittently for the presence of any extractive by spectral analysis by scanning in the UV range against a suitable blank. Completion of purification was confirmed by the absence of any absorbance in this range.

Swollen hydrogels have lower mechanical strength and further provide surface for microbial growth. The purified hydrogels were dried to help in their further handling and storage. Drying was performed under hot-air stream flowing over the hydrogel surface using a hot-air sterilizer oven for 2 h at 60 °C, followed by 2 h of drying in a vacuum oven at 45 °C. The two steps for drying were followed because fast drying in a vacuum oven can create a dried, glassy shell around the hydrogel's surface, which can considerably slow down the drying process. Air-drying, therefore, is done because it can uniformly remove the moisture without creating a dried, glassy shell around the hydrogel's surface. A major portion (approximately 70–80%) of the moisture can be removed by air-drying. After this initial drying, the residual moisture (i.e., the remaining 20–30%) can be removed effectively with a vacuum oven (30).

Characterization

Differential scanning calorimetry (DSC). DSC studies of prepared hydrogels were performed to determine their glass-transition temperatures and confirm the formation of hydrogels. The glass-transition temperature of plain PEGDA was 56.42 °C, whereas the DSC of the polyacrylate cross-linked with PEGDA showed an endothermic peak at 252.58 °C. This temperature was quite higher than the glass-transition temperature of polyacrylic acid (113.6 °C) (31). The change in glass-transition temperature indicates that the individuality of PEGDA and polyacrylate had been lost because of the formation of chemical linkage between PEGDA and the acrylic acid polymer. In addition, the increase in glass-transition temperature of polyacrylate from 113.6 °C to 252.58 °C on cross-linking with PEGDA shows that the thermodynamic stability of the acrylic acid polymer increased because of the formation of cross-linkages.

Morphology. The hydrogels were flexible solids, with somewhat transparent or translucent appearance. On swelling, these became totally transparent. After swelling, their flexibility and rigidity were found to be dependent on the concentration of the cross-linker used. As the concentration of the PEGDA was increased, the flexibility of the hydrogels increased. No significant difference in the morphology of hydrogels, however, was found with the increase in the molecular weight of PEGDA.

Scanning electron microscopy (SEM) was performed to study the internal structure of the hydrogels. SEM photographs (see Figure 5) showed that these hydrogels have a porous polymeric network. Drug molecules can be seen in these photographs in the form of crystals embedded in the hydrogel matrix (see Figure 5b).

Figure 5

Water-absorbing capacity. Swelling in the presence of aqueous solutions or body fluids is the most important property of a hydrogel. When immersed in aqueous medium, these hydrogels absorb high amounts of water and swell several times their original size. The water-absorbing (hydration) capacity of the hydrogels was studied in terms of percent equilibrium water content and swelling ratio. The hydrogels formed by copolymerization of PEG and acrylate monomers had a very high water-absorbing capacity, absorbing water roughly 2–10 times of their dry weight (see Table II). The homopolymer of acrylic acid can absorb water up to 1.5 times its dry weight.

Table II: Water uptake and swelling ratio of different acrylic acid hydrogel formulations (n* = 3)

The equilibrium water content and swelling ratio of the hydrogels formed using the same weight percentage of the PEGDA of higher molecular weight were lower than those found for lower molecular weight PEGDA. Among the hydrogels containing 75% w/w PEGDA, formulation B₁₀ absorbed the maximum amount of water (percent equilibrium water content was 61.16%) and B₆₀ showed minimum water-absorbing capacity (percent equilibrium water content was 55.23%). In the case of the higher molecular weight PEGDA, this result may be attributed to a lower molar percentage of PEGDA, corresponding to the same weight percentage of PEGDA.

PEGDA acted as a polymerization template for the polymerization of acrylic acid monomers at both ends, resulting in copolymers where PEG became entangled within the polyacrylate polymeric network. PEGDA is hydrophilic, so with the increase in the ratio of PEGDA in copolymeric structures, the hydrophilicity of the hydrogels increased. This study is different from studies that use the usual other small molecular cross-linkers that are not hydrophilic. These cross-linkers cannot impart any hydrophilicity and at the same time can only increase the compactness of the hydrogel mass by a sewing-like action for end-to-end polymeric compaction with an increase in its amounts within the polymeric networks.The trend in the present study showed that as the hydrophilic nature of PEG decreases with the increase in molecular weight, the hydrophilicity of these copolymeric hydrogels decreased with the increase in molecular weight. For the same weight ratio, the molar percentage of PEGDA decreases with the increase in molecular weight, reducing the hydrophilicity and water-absorbing capacity and swelling tendency of such copolymeric polyacrylates.

Water-diffusion study. The release of a drug from a matrix system usually is governed by diffusion, so determining the diffusion coefficient of the hydrogels is essential. The diffusion coefficient of the swollen hydrogel can be measured fairly easily by either the membrane permeation method or the sorption and desorption method. The membrane permeation time–lag experiment has been widely used; however, it is not always the most practical method (30). In the present work, therefore, desorption phenomenon (32) was used to determine the diffusion coefficient of the prepared hydrogels.

The hydrogels showed good diffusion property, with diffusion coefficients of the order 10^–6 (see Table III). As the molecular weight of the PEGDA was increased, its molar percentage decreased for the same weight percent. The cross-linking density of the system, therefore, decreased with the increase in the molecular weight of PEGDA, thereby increasing the diffusion coefficient. This is evident from the diffusion coefficient of formulation B₁₅, which was 3.32 × 10^–6 cm² /s, whereas that of B₆₀ was 4.57 × 10^–6 cm² /s. With the increase in molecular weight of PEGDA, the porosity might have increased because of the increase in chain length of the PEG cross-linker. Also, the hydrophilicity of PEGs decrease with the increase in molecular weight (33), so the diffusion of water became easier from systems containing PEGs of higher molecular weight. The diffusion coefficient, however, decreased with an increase in the amount of PEGDA(e.g., that for C₁₅ was found to be 2.24 ×10^–6 cm² /s and that for D₁₅ was 1.86 ×10^–6 cm² /s). This decrease may be attributed to the increase in the amount of hydrophilic PEG, which causes an increase in the difficulty of water diffusion out of the hydrogel matrix.

Table III: In vitro characterization of different hydrogel formulations (n* = 3).

These are copolymers of polyacrylates formed at the terminals of PEGs, which attach to polyacrylates at the other terminal of different PEGs, causing interlinkages because of the nonspecificity of interactions every time. Polyacrylate on one PEG unit always is freely attracted and linked with polyacrylates on other PEG units. This condition causes randomly networked structures. In such copolymeric networks, where instead of traditional low-molecular weight cross-linkers, higher molecular weight PEGs were used. This change caused the simple polyacrylate units to entangle with the PEGs within such structures by acrylate initiators at the terminals. Such results, therefore, can be justified for the equilibrium water content and swelling ratios of hydrogels based on the hydrophilic propensity of such hydrophilic PEGDAs of higher molecular weights as PEGs. PEGDA is hydrophilic, so with the increase in the ratio of PEGDA in copolymeric structures, the hydrophilicity of the hydrogels increased. The hydrophilic nature of PEG decreases with the increase in molecular weight; therefore, the hydrophilicity of these copolymeric hydrogels decreased with the increase in molecular weight. Also, for same weight ratio, the molar percentage of PEGDA decreases with the increase in molecular weight, thereby reducing the hydrophilicity of the polyacrylate gel structures. The PEG present in such polyacrylates decreases with the increase in molecular weights at the same weight percentages. The water absorption inside the gel matrix is lower, so the swelling ratio is less. The diffusivity of water or drying of the hydrogels are at the same time controlled by the presence of PEG, so the diffusion coefficient increased with reduced hydrophilicity of the carriers.

Drug loading. Drug loading can be done either during polymerization (in situ drug loading) or after polymerization by incubating the prepared hydrogels in the drug solution. Exposure to radiation, however, may cause a change in the chemical structure of the drug, if the loading is done during polymerization (5). In the present project, the loading of hydrogels with the drug was performed by equilibrium absorption from the concentrated drug solution.

The hydrogel was filled in a dialysis bag and suspended in a drug-solution of known concentration. The volume of loading solution was kept in excess as compared with the volume of the hydrogel to ensure that the external drug concentration remained relatively constant. After equilibration for 24 h or 48 h as described previously, the hydrogels were dried into glassy, dehydrated drug-loaded hydrogels. A part of the loaded drug might get adsorbed on the surface of the hydrogels, and the rest of the druggets entrapped in the hydrogel matrix.

The drug-loading efficiency was estimated by extracting the loaded drug by methanol. The extraction procedure was repeated a few times with small volumes of methanol to extract the drug completely. The extracts then were pooled and dried. The residue was dissolved in a respective solvent system and analyzed spectrophotometrically against a suitable blank. The hydrogels showed good drug-loading capacity, e.g., formulation B₁₀ contained 67.6% w/w% diclofenac (see Table III). The drug may get released from the pores present in the hydrogels, which can accommodate large amounts of drug molecules.

The drug-loading capacity was higher in hydrogels containing higher amounts of PEG. As the extent of PEGylation decreased with the increase in molecular weight of PEGDA, the entrapment efficiency of the hydrogels decreased, as in the case of B₁₀ having 67.6% w/w entrapment efficiency whereas B₆₀ showed entrapment of 32.1% w/w for diclofenac sodium.

Drug-release profile. The release profiles were determined in distilled water by suspending a dialysis membrane containing the drug-loaded system filled in a 20-mL release medium at neutral pH. The sink condition was maintained continuously by replacing the receptor media each time with fresh fluid after sampling. The cumulative amount of the drug released (expressed as a percentage) after several time-intervals was estimated and plotted against time (see Figure 6).

Figure 6

A small burst release initially was observed from all the formulations, approximately 3–4% of the loaded drug was released during the first hour, which may have resulted from the adsorption of some of the drug molecules on the surface of the hydrogels. In the later phase, the release of the drug in the hydrogel matrix was assisted by the diffusion of water through the matrix. The release rate, therefore, decreased after the initial burst release because diffusion through the cross-linked polymer matrices is a time-consuming phenomenon. In each case, however, the release rate was faster for hydrogels made of higher molecular weight PEG, which might be as a result of the decrease in cross-linking density with the increase in molecular weight as the molar composition of PEGDA decreases for the same weight composition. The release rate is slower for higher molar percentages of PEGDA. As with the increase in hydrophilicity, the ease of drug release decreases because the drug diffuses out along with water as solution. For example, after 96 h, 80.38% of the loaded drug was released from B₆₀ whereas 33.23% diclofenac was released from formulation D₆₀ .

Table IV (a): Cumulative percentage of drug released by various hydrogel formulations.

Table IV (b): Cumulative percentage of drug released by various hydrogel formulations.

Drug release from matrix systems generally takes place by diffusion. As the diffusion of water in the present case increased with an increase in molecular weight of PEGDA (as found by the diffusion coefficient studies), the drug-release profile also followed similar trends (see Tables IV[a]) and IV [b]) as the drug was released from the hydrogels in the form of an aqueous solution. In the case of the same weight percentage of PEGDA, an increase in the molecular weight of PEGDA led to an increase in the release rate. For example, after 96 h, 35.24% of the loaded drug was released from B₁₀ , whereas 59.35% diclofenac was released from formulation B₄₀ within the same time period.

The data of release profiles for each formulation further were analyzed by model fitting using the following equations:

in which, % R is the percent of drug release; K is the release rate constant; t is the time of release; and e is the logarithmic exponent.

Table V: Results of data-fitting analysis of release profiles of the hydrogel formulations.

The release data of all the formulations were fitted into these models, and the correlation coefficients, slope, and intercepts were determined (Table V). From the values of the correlation coefficients, the best fitted data can be predicted (34). The curve fitting of the release data was carried out mainly by regression analysis, and for zero-order, the release trend is more significant and has a higher R² value than that at the first order model of analysis. Most of the formulations of these types generally release the drug in a mixed-order basis. In the present experiment, the release profile, however, was found to be best fitting in the zero-order kinetics. It can be assumed, therefore, that the release of the drug from the hydrogel systems follows zero-order kinetics to some extent.

Table VI: Acrylic acid hydrogel formulations prepared according to 32 factorial design and their properties.

Porosity. Like other matrix systems, the porosity (created by the chemical cross-linking using PEGDA in the hydrogels) can be calculated by the diffusion rate coefficients (25). In the present study, the porosity was calculated using the diffusion coefficients of the drug from the drug-loaded hydrogels and its aqueous solution. The results of this study (see Table III) confirmed the prediction that the porosity of the hydrogels increased with the increase in the molecular weight of the PEGDA for the same percentage w/w content. For example, the porosity of formulation B₁₀ (hydrogel containing PEG 1000) was 0.392 and that of B₆₀ (hydrogel containing PEG 6000) was 0.522.

Table VII: Analysis of variance (ANOVA) of regression on the responses of acrylic acid hydrogels.

Statistical analysis. All the data obtained were further analyzed using factorial design and multiple regressions. The effects of weight ratio and molecular weight were studied on all the parameters. Three levels (high, medium, and low) were chosen for these two factors (see Table VI). A 3² factorial design was constructed, and properties of nine formulations (see Table VI) were compared. The analysis of variance (ANOVA) test was applied on the data obtained (see Table VII). The responses were regressed against the factors using the first order with interaction and the following equation was formed.

The F-significance was < 0.05 in all the cases. The ANOVA test, therefore, showed that the models were quite significant. It can be predicted, therefore, that the properties of these hydrogels were determined by both the molar percentage of PEGDA as well as the type of monomer used. From the release pattern observed, it can be suggested that these hydrogel formulations can be used as sustained-release dosage forms.

Conclusion

Generally, in case of cross-linking agents such as divinyl glycol, divinyl benzene, or tripropyleneglycol diacrylate, an increase in cross-linking density is known to reduce the equilibrium swelling (35, 36). In this study, where polyethylene diacrylate (PEDGA) was used as cross-linker, an increase in equilibrium swelling was observed with the increase in the amount of PEGDA. This change may be from PEGDA as it is a water-retaining compound. Because it is serving as a copolymer in the present hydrogel system, the properties of PEGDA affects the properties of these copolymeric hydrogels. The diffusion coefficient and drug-release rate, however, followed the general trend: An increase in the amount of PEGDA reduced the diffusion coefficient and release rate because of an increase in cross-linking density. Apart from the cross-linking density, in this study the larger amounts of cross-linker PEGDA, having somewhat higher affinity for water, made the diffusion a little more difficult. In all the experiments, for the similar weight composition, the increase in molecular weight of PEG led to a decrease in the water-imbibing capacity and drug-loading capacity of the hydrogels because of the reduction in hydrophilicity of the system.

The diffusion coefficient and drug-release rate, however, increased with the increase in molecular weight. These results also were related to the porosity of the hydrogels. The porosity of the hydrogels decreased with an increase of the amount of PEGDA and increased with an increase in molecular weight. This change may be from a significant reduction in molecular availability of poly(ethylene glycol) cross-linkers in the polyacrylate hydrogel matrix. The chemical representation of the hydrogels made up of different molecular weight PEGDA (see Figure 4) also supported this assumption of the porosity of hydrogels.

Acknowledgment

The authors acknowledge the University Grants Commission (New Delhi, India) and the Council of Scientific and Industrial Research (New Delhi, India) for funding the research projects as fellowship grants to some of the authors. The authors extend their deep gratitude to Electron Microscopy Division of All India Institute of Medical Sciences (New Delhi, India), Sun Pharmaceuticals (Baroda, India) and SAIF-RSIC, Central Drug Research Institute (CDRI) (Lucknow, India) for extending their instrumentation facilities for the purpose of the sample analysis.

Sulekha Bhadra,PhD, and Dipankar Bhadra, PhD, are research officers in Sun Pharma's Advanced Research Center, Vadodara, India. Govind Prasad Agrawal* is a professor, Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar, Madhya Pradesh, India, 470003, tel. + 91 7582 265457, bhadrasb28@yahoo.com

*To whom all correspondence should be addressed.

Submitted: Feb. 20, 2006. Accepted: Aug. 28, 2006.

Key words: acrylate, copolymer, diclofenac, hydrogels, PEG

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