Synthesis and Characterization of Acrylamide/Acrylic Acid Co-Polymers and Glutaraldehyde Crosslinked pH-Sensitive Hydrogels

This project aims to synthesize and characterize the pH-sensitive controlled release of 5-fluorouracil (5-FU) loaded hydrogels (5-FULH) by polymerization of acrylamide (AM) and acrylic acid (AA) in the presence of glutaraldehyde (GA) as a crosslinker with ammonium persulphate as an initiator. The formulation’s code is named according to acrylamide (A1, A2, A3), acrylic acid (B1, B2, B3) and glutaraldehyde (C1, C2, C3). The optimized formulations were exposed to various physicochemical tests, namely swelling, diffusion, porosity, sol gel analysis, and attenuated total reflection-Fourier transform infrared (ATR-FTIR). These 5-FULH were subjected to kinetic models for drug release data. The 5-FU were shown to be soluble in distilled water and phosphate buffer media at pH 7.4, and sparingly soluble in an acidic media at pH 1.2. The ATR-FTIR data confirmed that the 5-FU have no interaction with other ingredients. The lowest dynamic (0.98 ± 0.04% to 1.90 ± 0.03%; 1.65 ± 0.01% to 6.88 ± 0.03%) and equilibrium swelling (1.85 ± 0.01% to 6.68 ± 0.03%; 10.12 ± 0.02% to 27.89 ± 0.03%) of formulations was observed at pH 1.2, whereas the higher dynamic (4.33 ± 0.04% to 10.21 ± 0.01%) and equilibrium swelling (22.25 ± 0.03% to 55.48 ± 0.04%) was recorded at pH 7.4. These findings clearly indicated that the synthesized 5-FULH have potential swelling characteristics in pH 6.8 that will enhance the drug’s release in the same pH medium. The porosity values of formulated 5-FULH range from 34% to 62% with different weight ratios of AM, AA, and GA. The gel fractions data showed variations ranging from 74 ± 0.4% (A1) to 94 ± 0.2% (B3). However, formulation A1 reported the highest 24 ± 0.1% and B3 the lowest 09 ± 0.3% sol fractions rate among the formulations. Around 20% drug release from the 5-FULH was found at 1 h in an acidic media (pH1.2), whereas >65% of drug release (pH7.4) was observed at around 25 h. These findings concluded that GA crosslinked 5-FU loaded AM and AA based hydrogels would be a potential pH-sensitive oral controlled colon drug delivery carrier.


Synthesis Hydrogel
The mechanism of polymerization of acrylic acid and acrylamide in the presence of glutaraldehyde (GA) cross-linker was adapted from Wang et al. and partially modified the synthesis reaction mechanism shown in Scheme 1. Acrylic acid (1), acrylic acid sodium (2) and acrylamide (3) can react to Poly(acrylic acid-co-acrylamide) (4) using KPS as a radical initiator. The persulfate initiator was decomposed under heating to generate sulfate anion radical. The cross-linker glutaraldehyde (5) was added after polymerization finished in order to avoid the crosslinking reaction of Poly(acrylic acid-co-acrylamide) (4) during polymerization. Through heat-treatment at 110-160 • C the amide groups and hydroxyl groups can react with aldehyde groups from glutaraldehyde (5) to form poly(AA-co-AM) (6) network structure. Grafted hydrogels exhibit pH-sensitive swelling properties and pH-controlled drug release behavior.

Solubility Study
The 5-FU is a nucleobase analogue having a pKa value of (8 ± 0.1). It has a water solubility of 12.2 g/L at 20 °C. (repeated) It is freely soluble in a phosphate buffer of pH Scheme 1. General mechanism for polymerization of AA and AM in the presence of glutaraldehyde cross-linker [31].

Solubility Study
The 5-FU is a nucleobase analogue having a pKa value of (8 ± 0.1). It has a water solubility of 12.2 g/L at 20 • C. (repeated) It is freely soluble in a phosphate buffer of pH 7.4 as well as in distilled water compared to pH 1.2 ( Table 1). The solubility study is an important parameter which conforms to the dissolution experiments.

FTIR
FTIR spectral analysis of 5-FU and 5-FULH is shown in Figure 1. Pure 5-FU, drug loaded, and unloaded hydrogel samples were assessed using FTIR in the array of 400-4000 cm −1 . FTIR represents the distinct peak at 3177 cm −1 , 2927 cm −1 , 1658 cm −1 in pure 5-FU spectra and 3196 cm −1 , 2924 cm −1 and 1654 cm −1 in 5-FULH. In the case of pure 5-FU, the bands at 3412 cm −1 , 1165 cm −1 show evidence of N-H stretching (free) and -C-F band; after drug loading some of the bands disappear and N-H stretching (free) appears at the same wave number, while -C-F band appears at 1165 cm −1 . This indicates that the 5-FU is molecularly dispersed into the prepared hydrogels [32]. In the case of unloaded hydrogel, see Figure 1c, the AM peak appears at 1647 cm −1 for primary amide (C=O) stretching. The peak for the alkene group (HC=CH) appears at 3065 cm −1 . In the band of GA, the peak assigned to the C-OH stretching at 1243 cm −1 is very clear, suggesting that the desired material has been successfully prepared [33]. Owing to the electrostatic interaction among the various functional groups of AM, AA and GA, the characteristic peaks of AM, AA and GA are shifted to developed 5-FULH. Figure 1b shows that the loading of 5-FU by the developed hydrogel was successful and no interaction between the 5-FU and hydrogel ingredients was detected [34].

Swelling Studies
The release of the drug from the 5-FULH occurs once the polymer network is dissolved, followed by drug diffusion from the surface of the structure which is associated with the swelling behavior of the 5-FULH [35]. The advantage of hydrogel is that it can swell in the surrounding medium due to its chemical structure, which allows affinity with the water molecules [36]. Swelling studies of 5-FULH were conducted using various pH mediums of 1.2, 6.8, and 7.4. Equilibrium and dynamic swelling proportions of different 5-FULH are depicted in Table 2. developed hydrogel was successful and no interaction between the 5-FU and hydrogel ingredients was detected [34].

Swelling Studies
The release of the drug from the 5-FULH occurs once the polymer network is dissolved, followed by drug diffusion from the surface of the structure which is associated with the swelling behavior of the 5-FULH [35]. The advantage of hydrogel is that it can swell in the surrounding medium due to its chemical structure, which allows affinity with the water molecules [36]. Swelling studies of 5-FULH were conducted using various pH mediums of 1.2, 6.8, and 7.4. Equilibrium and dynamic swelling proportions of different 5-FULH are depicted in Table 2.   The swelling values of these gels were measured at 37 • C and medium pH of 1.2, 6.8, and 7.4. The swelling increased with an increase in AM concentration and a raise in medium pH. This swelling behavior of the polymer was due to its hydrophilic nature. The physical nature and hydrogen bond formation restricts the release of the drug. The swelling values of these gels were measured at 37 °C and medium pH of 1.2, 6.8, and 7.4. The swelling increased with an increase in AM concentration and a raise in medium pH. This swelling behavior of the polymer was due to its hydrophilic nature. The physical nature and hydrogen bond formation restricts the release of the drug.   The swelling values of these gels were measured at 37 °C and medium pH of 1.2, 6.8, and 7.4. The swelling increased with an increase in AA concentration and a raise in medium pH. This swelling behavior of the polymer was again due to its hydrophilic nature. The physical nature and hydrogen bond formation again restricts the release of the drug. The swelling values of these gels were measured at 37 • C and medium pH of 1.2, 6.8, and 7.4. The swelling increased with an increase in AA concentration and a raise in medium pH. This swelling behavior of the polymer was again due to its hydrophilic nature. The physical nature and hydrogen bond formation again restricts the release of the drug.

Effect of GA
Introduction of a crosslinking agent such as GA affects the swelling behavior of 5-FULH. Figures 6 and 7 show GA at different concentrations and the effect over the swelling of hydrogel at 37 • C in medium pH of 1.2, 6.8, and 7.4. Results show that when the concentration of GA increases, the swelling ratio decreases. This might be due to increased cross linking of GA. Generally, mobility of the polymer chain is affected by the crosslinking, ensuring the low solubility of the polysaccharide [37]. GA promotes the degree of crosslinking which in turn results in folding of the polymeric chains and the subsequent attainment of reticulation point, thus affecting the aqueous absorption capacity [38].

Effect of GA
Introduction of a crosslinking agent such as GA affects the swelling behavior of 5-FULH. Figures 6 and 7 show GA at different concentrations and the effect over the swelling of hydrogel at 37 °C in medium pH of 1.2, 6.8, and 7.4. Results show that when the concentration of GA increases, the swelling ratio decreases. This might be due to increased cross linking of GA. Generally, mobility of the polymer chain is affected by the crosslinking, ensuring the low solubility of the polysaccharide [37]. GA promotes the degree of crosslinking which in turn results in folding of the polymeric chains and the subsequent attainment of reticulation point, thus affecting the aqueous absorption capacity [38].

Porosity Measurement
The porosity values of 5-FULH range from 34% to 62% with different weight ratios of

Porosity Measurement
The porosity values of 5-FULH range from 34% to 62% with different weight ratios o AM, AA, and GA. It was found that the higher the concentration of polymer and monome

Porosity Measurement
The porosity values of 5-FULH range from 34% to 62% with different weight ratios of AM, AA, and GA. It was found that the higher the concentration of polymer and monomer, the higher the porosity values. While enhancing the crosslinker concentration, the porosity values decrease. Table 3 (B3) shows a higher % of gel fraction (94 ± 0.2%) with a higher % of porosity (62 ± 0.06%); a similar type of relationship was found in previous studies. The controlled release of the drug, around 65% at 25 h, could be attributed to the reduction of water entry and subsequent diffusion of the drug from the hydrogel network [39].  Table 3 shows the results of gel and sol fraction. In relation to the value of gel fraction, it was observed that the gel fraction depended on the AM and AA. The gelling strength of the prepared 5-FULH may increase with the higher content of AM, AA, and GA. As the concentration of the AA is increased, the polymerization reaction is also enhanced due to the accessibility of more binding sites. The higher gel fraction is attributed to the increased bulk density of the hydrogel structure. A firm and robust hydrogel network is established due to the higher concentration of polymer, resulting in a greater degree of crosslinking which in turn leads to a lower porous structure of hydrogels [40]. However, sol fraction is decreased since the concentration of AA and GA is increased due to the inverse relationship with gel fraction [41].

Drug Loading
Drug loading was determined by using swelling, extraction, and weight values. The amount of 5-FU was presented as g/g of dry gel and the data were presented in Table 4.

Cumulative Drug Release Measurement
From the polymeric 5-FULH, it can be seen that the release pattern of a formulation depends on the swelling behavior of these hydrogels. This section discusses some of the factors which influence the release of 5-FU from the prepared hydrogels.

Effect of AM
These 5-FULH were designed by varying AM concentrations at 37 • C and pH of 1.2 and 7.4. The release of 5-FU is very low in an acidic medium, increasing to 67% in a basic medium. This might be due to greater swelling in a basic medium and less swelling in an acidic medium. Mundargi et al. reported that the release of 5-FU increases when AM content increases in the matrix [42]. Figure 8 shows that drug release was varied based on the pH of the medium i.e., around 20% drug release was found at 1 h in an acidic media (pH 1.2), whereas >65% of drug release (pH 7.4) was observed at around 25 h from or with? the 5-FULH containing a higher amount of AM due to increased polymer chain flexibility, as shown in Figure 9. This release behavior of the drug was in agreement with the previous study where single polymeric chains in pure AM allowed higher drug transportation compared to the co-polymeric chain [43].

Effect of GA
Figures 12 and 13 describe the release of 5-FU under different concentrations of GA. The release of 5-FU in an acidic medium was almost the same irrespective of different concentrations of GA. The release of 5-FU was lower with a higher concentration of GA when it comes to basic medium pH 7.4. This could be attributed to the higher H-bonding and lower swelling of 5-FULH with a high concentration of crosslinking agent. It has been reported that the gelation ratio is faster in GA crosslinked hydrogel, therefore higher GA concentration increases the drug loading capacity [45]. As seen in Figure 13, the 5-FU release percentages from the 5-FULH at pH 7.4 were more than 50% of the pH 1.2 at 18% in

Effect of GA
Figures 12 and 13 describe the release of 5-FU under different concentrations of GA. The release of 5-FU in an acidic medium was almost the same irrespective of different concentrations of GA. The release of 5-FU was lower with a higher concentration of GA when it comes to basic medium pH 7.4. This could be attributed to the higher H-bonding and lower swelling of 5-FULH with a high concentration of crosslinking agent. It has been reported that the gelation ratio is faster in GA crosslinked hydrogel, therefore higher GA concentration increases the drug loading capacity [45]. As seen in Figure 13, the 5-FU release percentages from the 5-FULH at pH 7.4 were more than 50% of the pH 1.2 at 18% in concentration increases the drug loading capacity [45]. As seen in Figure 13, the 5-FU release percentages from the 5-FULH at pH 7.4 were more than 50% of the pH 1.2 at 18% in every formulation. The present findings are in line with the previous data which investigated 5-FU loaded GA blended pH-responsive hydrogel and found 5-FU release of around 64.0% to 85.99% at pH 7.4 but only 13.33% to 19.64% at pH 1.2 [46].  The protonation of sulfonate ions occurs at acidic pH 1.2, which results in the generation of strong hydrogen bonds and physical interaction between the functional groups of the hydrogel, causing a subsequent reduction in swelling [47]. However, at pH 7.4, deprotonation of sulfonate ions occurs, owing to the enhanced electronic density on the polymeric structure and less physical interaction among the -SO −3 moieties, thereby generating a greater degree of swelling of AA 5-FULH [48]. All of these factors cause reduction in intermolecular hydrogen bonding, resulting in higher swelling [49]. Likewise, the COOH groups of AA are protonated at pH 1.2 which leads to a fall in swelling. However,  The protonation of sulfonate ions occurs at acidic pH 1.2, which results in the generation of strong hydrogen bonds and physical interaction between the functional groups of the hydrogel, causing a subsequent reduction in swelling [47]. However, at pH 7.4, deprotonation of sulfonate ions occurs, owing to the enhanced electronic density on the polymeric structure and less physical interaction among the -SO −3 moieties, thereby generating a greater degree of swelling of AA 5-FULH [48]. All of these factors cause reduction in intermolecular hydrogen bonding, resulting in higher swelling [49]. Likewise, the COOH groups of AA are protonated at pH 1.2 which leads to a fall in swelling. However, at higher pH 7.4, carboxylic acid (CA) functionalities are deprotonated, resulting in higher swelling of the 5-FULH [50]. Also, the osmotic pressure of the ions is increased due to the increase in protonation of the COOH groups [51]. The carboxylic acid (CA) moieties were The protonation of sulfonate ions occurs at acidic pH 1.2, which results in the generation of strong hydrogen bonds and physical interaction between the functional groups of the hydrogel, causing a subsequent reduction in swelling [47]. However, at pH 7.4, deprotonation of sulfonate ions occurs, owing to the enhanced electronic density on the polymeric structure and less physical interaction among the -SO −3 moieties, thereby generating a greater degree of swelling of AA 5-FULH [48]. All of these factors cause reduction in intermolecular hydrogen bonding, resulting in higher swelling [49]. Likewise, the COOH groups of AA are protonated at pH 1.2 which leads to a fall in swelling. However, at higher  [50]. Also, the osmotic pressure of the ions is increased due to the increase in protonation of the COOH groups [51]. The carboxylic acid (CA) moieties were converted to the salt form which led to maximum swelling, as the pH of the medium was elevated from a lower to a higher number [52].

Release Kinetics
Several kinetic models were employed to help understand the release mechanism of the 5-FU from the hydrogels, and the regression coefficients (r) were selected for the evaluation of the most suitable drug release [53]. The (r) values of most samples were greater for zero-order kinetics compared to the first-order kinetics, which could be ascribed to the variation in the amounts of AA and GA. From the results of the Higuchi model, it was obvious that the release of the drug follows the diffusion-controlled release mechanism. Among the models, the (r) values of 5-FULH for zero order (0.9632-0.9954) was comprehensively higher than the first order (0.9378-0.9932). The results indicated that the release pattern of the drug corresponded to the zero-order kinetics.

Conclusions
The 5-FULH were processed for oral delivery using AM, AA and GA crosslinked. These prepared 5-FULH were physio-chemically characterized. ATR-FTIR shows no such interaction between the excipients and the model drug (i.e., 5-FU). Swelling studies show that minimum swelling was achieved at acidic pH and maximum at alkaline Ph, which depends on the monomer, polymer, and crosslinking agent. By increasing AM and AA concentrations, swelling increased, whereas by increasing GA, it decreased. In vitro release studies show that the most drug was released on alkaline pH, whereas the least drug was released on acidic pH. The release of the drug also increases with increasing AM and AA content but decreases with an increase in cross linker. The drug release kinetics models followed a non-Fickian order of release. These findings concluded that AM and AA based 5-FULH would be appropriate for controlled drug delivery with pH reactive characteristics.

Pre-Formulation Studies and Standard Curve
The stock solution was prepared using 10 mg of 5-FU dissolved in 100 mL of phosphate buffer pH 7.4. A series of dilutions were made in descending order using phosphate buffer pH 7.4. Samples from such mixtures were taken and spectrophotometrically analyzed at 256 nm (Shimadzu 601, Japan). The results were taken in triplicate and plotted.
where: Y = the absorbency of the solution, M = the angle of standard curve of identified concentration, C = concentration that must be calculated and B = the curve's cut off.

Solubility Study of Drug
The solubility study of 5-FU was conducted using different solvents of varying pH at 37 • C.

Preparation of 5-FULH
The 5-FULH were produced by a simple crosslinking method, with slight modifications as presented in Table 5. In the first step a specific amount of distilled water was put into a beaker and placed on a magnetic stirrer and heated. Thereafter the AM was poured into the beaker and heated up till a clear solution was achieved. Similarly, by applying slight modifications in temperature as well as in revolution time, an AA solution was obtained. These solutions were then thoroughly mixed with continuous stirring on a magnetic stirrer until a clear homogeneous liquid was produced. Finally, by adding distilled water, the volume of the solution was made up to a hundred milliliters. Consequently, in a drop wise method and with regular stirring, cross linker was added to the mixture, to produce a uniform clear solution. This solution was then poured into the deoxygenized test tubes and left to congeal in them for four days. The 5-FULH, once they were formed, were then removed from the tubes. With the help of sharpened blades, these gels were cleaved as well as properly scrubbed with Methanol. The discs were then put into petri dishes and desiccated in an oven at 40 • C for seventy-two hours [54,55].

Dynamic Swelling Study
Dried slices of these preparations were properly weighed and immersed into buffers of various pH (i.e., pH 7.4, pH 6.8, and pH 1.2). By removing discs from the medium, readings were noted over regular time periods [57,58]. The surface water was removed from the 5-FULH using tissue paper. The discs were dipped again in the pH 6.8 and 7.4 buffer, after having been weighed at definite intervals [59]. The following formula was used to obtain the dynamic S: where: Ws = weight of swollen gel at specific time, Wd = weight of dry hydrogel, and S = swelling ratio.

Equilibrium Swelling Study
ES studies were also performed on the 5-FULH, which were prepared for frequent swelling. The % ES was determined using the following equation [60,61]. where:

Diffusion Coefficient (DC)
DC is the quantity of solvent that was absorbed/diffused across the unit area in unit time through a concentration gradient [62,63]. The diffusion coefficient was calculated by Formula (4): where: D = DC of the 5-FULH, Qeq = the hydrogel's swelling at equilibrium, θ = the angle of straight portion of swelling curves and h = early sample thickness.

Sol Gel Fraction Analysis
Sol-gel analysis technique was performed for predisposing reactant quantity used during the preparation of the 5-FULH. The prepared 5-FULH were dried without washing and placed in deionized water at ambient temperature until constant weight was attained. After that, extracted hydrogel was taken out and dried in an oven at 60 • C [64,65].
where: M 2 = Final/extracted gel wt, M 1 = Initial wt of dry gel and Gel f raction = 100 − SF

Porosity Measurement
Porosity is an important consideration mainly affecting the swelling attributes of the 5-FULH. The % porosity was calculated by solvent replacement technique. Hydrogel discs were dried and soaked in ethanol (100%) overnight. Extra ethanol was removed using blotting paper and then the 5-FULH were weighed [66]. The porosity was calculated and attained by using the following equation: where: M t = weight before immersion, M o = weight after immersion, ρ = density of absolute ethanol and v = volume of hydrogel.

Drug Loading
Samples which showed maximum swelling were used for drug loading and release studies. The drug loading into the discs of hydrogel was achieved by soaking them for one week in a solution of the drug. A 1% w/v 5-FU in pH 7.4 solution was used for drug loading. After achieving the equilibrium value, the swelled 5-FULH were removed from the drug solution, blotted with filter paper, first dried at room temperature, and then placed in an oven at 40-45 • C for one week to remove the absorbed solvent. To determine the percentage of drug-loading, weighed drug loaded samples were extracted repeatedly using a phosphate buffer solution of pH 7.4 up to exhaustion, and the concentration of the drugs in pooled extract was determined spectrophotometrically at λ max 256 nm. The quantity of drug loaded into the 5-FULH was also determined by the swelling method [67].

In Vitro Release Study
To assess the amount of released drug from formulated 5-FULH, in vitro release studies were performed, using dissolution apparatus (pharma test; Pt-Dt 7). These hydrogel discs were positioned in a dissolution medium of 500 mL at 37 • C temp in a pH of 1.2 and 7.4 and shaken at a hundred revolutions per minute. Samples were selected at specific time periods and replaced. These samples were then evaluated at 256 nm using a UV-spectrophotometer (Shimadzu) [68].

Release Kinetics
To investigate the discharge of medicament from the gels and its mechanism, various kinetic models were applied [58], which are given below.

Statistical Analysis
Findings from all of the experiments were made in triplicate and presented as the means ± S.D. These samples were analyzed statistically with the help of one-way ANOVA and t-test. At p < 0.05, differences were considered as significant.