QbD aided optimization and molecular docking of pH-sensitive hydrogels for targeted colon delivery in ulcerative colitis

Abstract

Oral formulations of aminosalicylates are widely used in the treatment of ulcerative colitis (UC), but their efficacy is limited by low absorption and bioavailability due to first-pass metabolism and gastric degradation. pH-sensitive hydrogels were formulated for colon-targeted, delayed drug release to address this concern. Moreover, molecular docking was conducted to evaluate the binding affinity between β-cyclodextrin (oligomer) and balsalazide disodium. 15 formulations were developed using a Box–Behnken design in Design-Expert® 11, varying the concentrations of cross-linker, oligomer, and monomer while keeping the initiator constant. Hydrogels were developed via free radical polymerization using N,N-methylene bisacrylamide (MBA), β-cyclodextrin, potassium persulfate (KPS), and acrylic acid. Formulations were evaluated for swelling behavior, drug release, entrapment efficiency, porosity, and sol–gel fraction. The hydrogels exhibited maximum swelling at pH 7.2 (3783.4%) and a low sol fraction (10%), achieving 96% drug entrapment. FTIR suggested no interaction between the drug and excipients used. DSC and SEM analyses revealed stable thermal behavior and porous morphology. Molecular docking confirmed promising binding between balsalazide disodium and β-cyclodextrin. In vitro release studies showed sustained drug release for up to 8 hours, with kinetic modeling best fitting the Hixson-Crowell model, indicating drug release by change in geometry, surface area, and swelling of the hydrogel matrix. In the current research work, the optimized pH-sensitive hydrogel providing an effective sustained-release system for balsalazide disodium has been successfully developed, exhibiting promising pH-responsive drug-release characteristics.

Oral formulations of aminosalicylates are widely used in the treatment of ulcerative colitis (UC), but their efficacy is limited by low absorption and bioavailability due to first-pass metabolism and gastric degradation.

1. Introduction

Oral dosage forms are among the most convenient and cost-effective drug delivery systems, and they enhance patient compliance. However, several challenges are associated with the oral route, including first-pass hepatic metabolism, poor bioavailability of the active metabolite, and drug degradation in the acidic gastric environment. To overcome these limitations, innovative drug delivery systems have been developed, such as hydrogels, which provide protection and enable localized delivery of the drug.

Hydrogels are defined as three-dimensional crosslinked structures having polymer chains that can absorb relatively large amounts of fluid. Being porous, soft, and hydrophilic, they are very similar to living body tissues.¹ The hydrophilic functional groups included in the primary polymer chain of hydrogels include sulfate (SO₃H⁻), amine (NH₂), carboxyl (COOH), and hydroxyl groups (OH⁻).² Hydrogels can be synthesized through either physical or chemical cross-linking of monomers and polymers. The interaction is achieved in various ways, such as interpenetrating network formation, radiation method, free radical polymerization mechanism, bulk polymerization, and solution mixing.³ The main component of a hydrogel is a polymeric network, which can be made from natural biopolymers (polysaccharides, proteins) or synthetic polymers. Cross-linkers (chemical or physical) provide structural integrity, while water (70–90%) ensures swelling and biocompatibility. Functional additives like drugs, growth factors, nanoparticles, and porogens enhance drug delivery, tissue engineering, and mechanical strength. Additionally, stimuli-responsive agents (temperature/pH-sensitive polymers) and biodegradable linkages allow hydrogels to adapt to environments and degrade after use.⁴

Balsalazide disodium is chemically identified as the disodium salt of (E)-5-[[-4-[[(2-carboxyethyl) amino] carbonyl] phenyl] azo]-2-hydroxybenzoic acid dihydrate. It is a prodrug formed by linking mesalazine to an inactive carrier molecule, 4-aminobenzoyl-β-alanine, through an azo linkage, as shown in Fig. 1. The azo linkage is metabolized by coliform bacteria in the colon, resulting in the formation of 5-ASA from the inert carrier, 4-aminobenzoyl-β-alanine, along with N-acetylated metabolites.⁵ The active metabolite imparts immunosuppressive and anti-inflammatory activities, which could contribute to its therapeutic effects.

Fig. 1. Chemical structure of balsalazide disodium, indicating the presence of nitrogen atoms capable of forming hydrogen bonding with the hydrogel components, resulting in a stable host–guest interaction.

UC is a chronic condition that causes repeated inflammation of the colon.⁶ It commonly affects the colon and rectum, triggered by an abnormal immune response to various chemical substances within the intestinal lumen in genetically predisposed individuals.⁷ Anti-inflammatory medications, such as corticosteroids (systemic and topical) and 5-aminosalicylate (5-ASA) compounds (e.g., sulfasalazine, olsalazine, mesalamine, and balsalazide), are commonly used to treat ulcerative colitis. Immune response modulation is achieved with immunomodulators (azathioprine, 6-mercaptopurine), immunosuppressants (cyclosporine), and other anti-antigen therapies. Probiotics are also beneficial, particularly for mild to moderate UC.

Design-Expert® software, developed by State Ease, is a statistical software package widely used for Design of Experiments (DOE), response surface methodology, and formulation optimization. It was first created to carry out experimental designs, such as the determination of the optimum formula of preparation. Apart from optimization, it can also interpret the factors in the experiment. Design Expert is divided into three options based on the experimental design to be carried out. These are characterization, screening, and optimization. Optimization is carried out by selecting the concentration of components as an independent variable factor, while the responses, e.g., swelling, porosity, gel fraction, are added as the dependent variables; hence, an optimized formulation is obtained, thus reducing the cost and effort of repeated experimental trials to reach an optimized formulation having the required characteristics. The software analyzes the experimental data using statistical models and ANOVA, identifies significant factors and interactions, and generates response surface plots to support interpretation.⁸

Molecular docking is an important computational tool in structural molecular biology and computer-assisted drug design. The goal of docking is to predict the predominant binding mode and affinity of a ligand with a polymer of known three-dimensional structure. Successful docking methods find high-dimensional spaces effectively and use a scoring function that indicates the binding affinity of the molecules by physical interactions such as hydrogen bonding, electrostatic attractions, or chemical interactions, including covalent bonding. In the present study, molecular docking was used to evaluate the possible interaction between balsalazide disodium and β-cyclodextrin (β-CD).⁹

The current work includes loading balsalazide disodium into a sustained-release hydrogel formulation that was optimized with Design Expert software and formulated using the free radical polymerization process.

2. Materials and methods

Balsalazide disodium was generously provided by, CCL Pharmaceuticals (Lahore, Pakistan). β-CD (oligomer), acrylic acid (monomer), potassium persulfate (initiator), KH₂PO₄, K₂HPO₄, triethanolamine, and HCl were all procured from Sigma-Aldrich (Steinheim, Germany). The cross-linker N,N-methylenebisacrylamide was sourced from Merck KGaA, Germany. Distilled water utilized during the study was freshly prepared in the research laboratory of Riphah International University, Lahore. All the chemicals used were of high-grade purity.

2.1. Formulation of hydrogel

The formulation of hydrogels was carried out using Design-Expert software (Version 13, Stat-Ease Inc., Minneapolis, MN, USA). The trials were systematically planned and optimized using a Box–Behnken design. During these experimental trials, β-CD, acrylic acid, and MBA were considered variable factors, while the concentration of potassium persulfate remained constant, as mentioned in Table 1. Firstly, using a magnetic stirrer with a hot plate, the weighed amount of β-CD was dissolved in 2–3 milliliters of distilled water and constantly swirled at 40 °C. The following stage involved dissolving 0.02 g of the initiator, potassium persulfate (KPS), in 1–2 mL of distilled water while stirring constantly. While ensuring consistent stirring, the initiator solution was introduced gradually to the monomer in a different beaker. The prepared monomer-initiator mixture was gradually poured into the β-CD dispersion while maintaining continuous agitation. Next, the cross-linker (MBA) was added while stirring, and distilled water was used to make up the total volume to 10 mL. For the hydrogel formulation, the prepared solution was transferred into glass test tubes. Afterwards, the solutions were allowed to congeal at 70 °C. The gelation time varied depending on the amount of the cross-linker. After solidifying, the mixture was cooled down to room temperature. Sections of the solidified hydrogels were made into cylindrical discs of 2 mm thickness. After that, an ethanol–water (30 : 70) mixture was used to wash the hydrogel discs to get rid of any monomer and cross-linker molecules that had not reacted. Following purification, the formulations were dried for two to three days at 50 °C in an oven. After the samples were fully dried, they were taken out of the hot air oven.¹⁰

Table 1. Composition of ingredients used in the hydrogel formulation, as designed using Design Expert software.

Sr. no.	(A) 1st factor β-CD (g)	(B) 2nd factor acrylic acid (g)	(C) 3rd factor MBA (g)
1	0.225	4	0.04
2	0.15	4	0.03
3	0.3	3.75	0.02
4	0.3	3.5	0.03
5	0.3	4	0.03
6	0.3	3.75	0.04
7	0.225	4	0.02
8	0.15	3.75	0.02
9	0.15	3.5	0.03
10	0.225	3.75	0.03
11	0.225	3.75	0.03
12	0.225	3.75	0.03
13	0.225	3.5	0.04
14	0.225	3.5	0.02
15	0.15	3.75	0.04
O.F	0.300	3.974	0.02

2.2. Post-loading of the drug

The drug was loaded in the hydrogels by the post-loading method. The post-loading method was used instead of in situ drug loading to minimize the exposure of balsalazide disodium to heat, free radicals, initiator, and polymerization conditions, which may lead to degradation and decreased therapeutic efficacy of the drug. For loading the drug, 80 mg of balsalazide disodium was dissolved in 7.2 pH phosphate buffer at 25 °C to make the total volume up to 10 mL, thus forming a 0.8% drug solution. At 30 °C, a dried hydrogel disc (2 mm in size, 0.11 ± 0.03 g) was immersed in the balsalazide solution until its weight remained constant. The hydrogel disc was taken out of the solution when it was completely loaded with the medication, rinsed with purified water to wash out any leftover drug, and then dried in a drying oven set at 35 °C for one-two days.¹¹

2.3. Evaluation of hydrogels

2.3.1. Organoleptic assessment

The hydrogel formulations were visualized for their physical features, including texture, color, homogeneity, and shape. The samples were visually assessed for surface consistency and checked for any irregularities such as aggregation, discoloration, spotting, cracks, or air bubbles.^12,13

2.3.2. Swelling studies

Swelling studies were performed to assess the hydration capacity and expansion behavior of hydrogels, which directly impact drug release and mechanical properties. The degree of swelling for each hydrogel was examined with buffer media set at various pH levels (1.2, 6.8, and 7.2) to simulate physiological conditions. For this purpose, the dried weight of the hydrogel disc was recorded before immersion in buffer solutions. The samples were allowed to swell until they reached a constant weight. The swollen discs were taken out of the buffer medium at predetermined intervals. The sample was weighed after the extra liquid was carefully blotted away with filter paper. The degree of swelling was determined using the following eqn (1):¹⁴

1

In the above equation, W_d is the weight of the dried hydrogel, whereas W_s is the weight of the swollen hydrogel.

2.3.3. Sol–gel fraction

The percentage of cross-linked and uncross-linked polymers in the hydrogels was determined to assess the extent of cross-linking. The amount of uncrosslinked polymer is indicated by the sol fraction, while the gel fraction represents the cross-linked polymer. The weight of the dried hydrogel was calculated, and then the hydrogels were immersed in purified water for 48 hours at 35 °C. After that, the formulations were dried in a drying oven at a temperature of 45 °C until a uniform weight was achieved. The weight of the formulations after drying was noted and used to determine the sol fraction using eqn (2) and (3): ¹⁵

2

Gel fraction (%) = 100 − Sol fraction (%) 3

In the above eqn (2), W_d is the weight of the sample following extraction, and W_o is the dried weight.

2.3.4. Porosity

Porosity measurement was performed to assess the internal structure, void spaces within the hydrogels, and volume of liquid absorbed by the hydrogels. The solvent replacement approach was utilized to determine the apparent porosity. To achieve full penetration, the weight of the dry hydrogel disc was determined first and then submerged in ethanol for a whole day. Following the immersion period, excess ethanol was blotted off, and the samples were reweighed. The apparent porosity of the formulations was computed using the following formula (eqn (4)):¹⁶

4

The mass of the sample before and after immersion in alcohol is indicated by M₁ and M₂, respectively, in the equation. The density of ethanol is represented by the symbol ρ, and V is used to express the hydrogel's volume.

2.3.5. Calibration curve of balsalazide disodium

A calibration curve for balsalazide disodium was constructed using UV-Visible spectrophotometry. A 1000 µg mL⁻¹ solution of balsalazide disodium was prepared by dissolving 5 mg of the drug in 5 mL of distilled water. From this standard solution, 1 mL was taken and diluted to 10 mL with distilled water, resulting in a concentration of 100 µg mL⁻¹. Then, a 10 µg mL⁻¹ solution was prepared by taking 1 mL from the previously prepared 100 µg mL⁻¹ solution and diluting it to 10 mL with distilled water. This solution was analyzed using a UV spectrophotometer between 200 and 400 nm wavelength ranges to identify the peak absorbance wavelength of balsalazide disodium. To construct the standard calibration curve, further dilutions were prepared from the stock solution. Dilutions with concentrations of 10, 20, 30, 40, and 50 µg mL⁻¹ were made by taking 1, 2, 3, 4, and 5 mL, respectively, from the 100 µg mL⁻¹ solution and bringing the total volume to 10 mL with solvent. Using the UV spectrophotometer, absorbance readings were taken at λ_max for all dilutions, and a standard graph was plotted of concentration versus absorbance for the drug sample. The linearity of the calibration curve was evaluated using the value of the correlation coefficient (R²), while LOD and LOQ were calculated to assess the sensitivity of the analytical method.

2.3.6. Drug entrapment efficiency

The concentration of balsalazide disodium released in the buffer solution of pH 6.8 was compared to the original concentration of the drug used in the hydrogel disc to calculate the entrapment efficiency of the drug. The hydrogel's entrapment effectiveness was evaluated by immersing discs (2 mm in diameter) loaded with balsalazide disodium in 50 mL of phosphate buffer at pH 6.8 and allowing them to swell for 1 day. After swelling, the discs were ground and placed into the same buffer solution. Following five minutes of homogenization at a speed of 8000 rpm, the mixture was centrifuged for ten minutes at a speed of 6000 rpm. The supernatant was filtered, and its absorbance was measured using a UV-visible spectrophotometer. The concentration of the drug was calculated using a pre-constructed calibration curve for balsalazide disodium. The percentage drug entrapped was calculated using the given equation (eqn (5)):⁴

5

2.3.7. In vitro drug release study

One crucial evaluation technique for determining the effectiveness, safety, and quality of a medication delivery system is the percentage of drug released in in vitro testing.¹⁷ The amount of drug released was determined using the USP Type 2 dissolution apparatus (six-paddle system). 900 milliliters of phosphate buffer having 6.8 pH were used as the dissolution medium for the drug-loaded disk. The system ran at 100 rpm, at a temperature of 37 °C. Aliquots of 10 mL were taken out of the dissolution medium after an interval of every one hour till 24 hours i.e. after 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, and 24 hours. To keep sink conditions stable, 10 mL of fresh medium was promptly added after each withdrawal. The absorbance of collected samples was determined using a UV-Visible spectrophotometer, and the amount of medication released at each time point was calculated using the equation of straight line from the calibration curve.¹⁸

2.3.8. Drug-release kinetics

The following kinetic equations were used to investigate the release of balsalazide disodium from the hydrogels:

2.3.8.1. Zero-order kinetics

Q _t = k₀t 6

In the above eqn (6), k₀ is the rate constant, and Q_t represents the proportion of medication released at time t.

2.3.8.2. 1st order kinetics

ln(100 − Q_t) = ln 100 − k₁t 7

where k₁ represents the release rate constant for the 1st order kinetics (eqn (7)).

2.3.8.3. Higuchi's model

Q _t = k_Ht^1/2 8

In the above eqn (8), k_H represents the rate constant of Higuchi's kinetics.

2.3.8.4. Hixson–Crowell model

(100 − Q_t)^1/2 = 100^1/3 − k_HCt 9

In the above eqn (9), k_HC denotes the Hixson–Crowell release rate constant.

2.3.8.5. Korsmeyer–Peppas model

However, the Korsmeyer–Peppas model was preferably used for ideal characteristics of drug release (eqn (10)).

10

Q_t/Q_e is the proportion of the concentration of drug released at time t, where n is the release exponent, and kKP is a constant associated with the device's geometric and structural characteristics.¹⁹

2.3.9. Molecular docking studies

Molecular docking was conducted to determine the binding affinity and pattern of interaction between β-CD and balsalazide disodium. The docking simulations were performed using AutoDock software. A β-CD oligomer was used as the host structure, while balsalazide disodium was selected as the ligand. Grid parameters were optimized to encompass the entire binding cavity of β-CD.

2.4. Numerical optimization

The swelling behavior, porosity, drug entrapment effectiveness, in vitro drug release, and sol–gel fraction of the hydrogels were among the various evaluated responses that were considered during numerical optimization. ANOVA was used in Design Expert Software (version 11) to assess all of these responses and find correlations between the experimental outcomes of the trial formulations.²⁰ Based on these findings, the optimized hydrogel formulation with the desired properties was identified. The final optimized formulation had 0.020 g of MBA, 3.974 g of AA, and 0.300 g of β-CD.

2.5. Evaluation of the optimized formulation

The optimized formulation obtained through Design-Expert® software was prepared and subjected to further evaluation, including its degree of swelling, porosity, gel fraction, in vitro drug release, and drug entrapment efficiency. Subsequently, the optimized formulation was further characterized using Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) to investigate its physicochemical properties, thermal behavior, and surface morphology.

2.5.1. FTIR Analysis

FTIR analysis was performed to investigate possible chemical reactions among the drug and excipients of the formulation, as well as to ensure the efficient incorporation of the drug into the optimized formulations. The characteristic functional group peaks of the pure drug and individual components were compared with those of the formulated hydrogels. An Agilent Cary 630 FTIR Spectrometer (Agilent Technologies, USA) operating over a 4000–650 cm⁻¹ spectrum region was used for the analysis.²¹

2.5.2. SEM

ZEISS Scanning Electron Microscope (Germany), operated at an accelerating voltage of 20 kilovolts, was used to perform the SEM analysis of the optimized formulation. The core purpose of SEM analysis was to observe the texture of the surface, pores, and cross-linking of the samples, which are important aspects affecting drug entrapment, release behavior, and mechanical strength.²²

2.5.3. DSC

DSC is used to show how heat moves through a substance with respect to time or temperature. Simultaneous Thermal Analyzer (SDT Q600, TA Instruments Inc., USA) was used to thermally analyze balsalazide disodium, as well as the optimized hydrogel formulation. For this analysis, accurately weighed samples (ranging from 0.3–5 mg) were finely powdered and placed in platinum crucibles attached to a high-sensitivity microbalance. A temperature interval ranging from 25–500 °C was employed for the thermal analysis. During the procedure, a constant flow of nitrogen gas at 10 mL min⁻¹ was maintained to provide a non-reactive atmosphere and prevent degradation of samples due to oxidation.²³

2.5.4. Stability studies

One-month stability studies were conducted on the optimized formulation, and the degree of swelling at pH 6.8 and 7.2, apparent porosity, and sol–gel fraction were analyzed after that.²⁴

2.5.5. In vitro drug release study

A sequential pH-change dissolution study of the optimized formulation was performed using the USP Type 2 dissolution apparatus to determine the amount of drug released in acidic and alkaline environments. For this purpose, the drug-loaded optimized formulation was placed in 900 mL HCl solution having pH 1.2 for 1 hour; then that disc was transferred to phosphate buffer having pH 6.8 for 3 hours and then to buffer of pH 7.2 for 4 hours, simulating gastric, small intestine, and colonic environments, respectively. The system ran at 100 rpm, at a temperature of 37 °C. Aliquots of 10 mL were taken out of the dissolution medium after an interval of every one hour. To keep sink conditions stable, 10 mL of fresh medium was promptly added after each withdrawal. The absorbance of collected samples was determined using a UV-Visible spectrophotometer, and the amount of medication released at each time point was calculated using the equation of the straight line from the calibration curve.²⁵

3. Results

3.1. Organoleptic evaluation

All hydrogel formulations were flat and disc-shaped. Upon drying, hydrogels appeared translucent to opaque. The formulations were uniform in color, shape, and surface texture, with no visible signs of discoloration or surface imperfections.

3.2. Swelling studies

The formulations showed the greatest swelling behavior at pH 7.2. The extent of swelling in different buffer solutions is illustrated in Fig. 2, represents that the formulations have the least swelling in pH 1.2 and the highest at pH 7.2.

Fig. 2. Graph showing the degree of swelling at pH (1.2, 6.8, and 7.2) of all 15 formulations (F1–F15), indicating greater swelling of the hydrogel network at higher pH, i.e., 7.2, confirming the pH-sensitive behavior of the prepared formulations.

3.3. Sol–gel analysis

Determination of sol–gel fraction expressed sol fractions ranging from (10 ± 0.2) % to (30 ± 0.8) %, while gel fractions were found to be between (70 ± 0.9) % and (90 ± 0.9) %. Formulation F3 showed the highest sol fraction and the lowest gel fraction. The sol fraction results are depicted in Fig. 3(A), and gel fraction results are depicted in Fig. 3(B)

Fig. 3. (A) Sol fraction indicating the proportion of the non-crosslinked portion of formulations (F1–F15), (B) gel fraction of formulations (F1–F15), showing the proportion of the firmly crosslinked portion in each hydrogel formulation.

3.4. Porosity measurement

The formulated hydrogels exhibited apparent porosity values ranging from (1296 ± 14) % to (2421 ± 11) %. The formulation F3 showed the highest porosity value. The values of apparent porosity are graphically represented in Fig. 4. Formulation F3 possesses the highest porosity, while F10 and F11 have the lowest porosity values.

Fig. 4. Percentage apparent porosity of formulations (F1–F15), indicating not only the already existing voids but also the total volume of solvent adhered to the hydrogel network, i.e., apparent porosity.

3.5. Calibration curve of balsalazide disodium

3.5.1. Determination of max. absorbance wavelength of balsalazide disodium

The peak absorbance wavelength λ_max of balsalazide disodium, identified with a UV-Visible spectrophotometer, was 357 nm.

3.5.2. Determination of the linearity curve of balsalazide disodium

The absorbance values measured at 357 nm ranged from 0.48 to 2.28, corresponding to the concentration ranging from 10 to 50 µg mL⁻¹. These values were plotted against their respective concentrations, with concentration values along the abscissa and corresponding absorbance along the ordinate to construct a calibration curve, as shown in Fig. 5. Beer–Lambert's law was obeyed over the concentration range of 10–50 µg mL⁻¹ with a correlation coefficient (R²) value of 0.9943. The limit of detection (LOD) and limit of quantification (LOQ) were 0.782 and 2.369 µg mL⁻¹, respectively, indicating the sensitivity of the method.

Fig. 5. Calibration curve of balsalazide disodium.

3.6. Entrapment efficiency

The observed entrapment efficiency ranged from 92.01 ± 1.01% to 96 ± 2%. These findings are presented in Fig. 6, indicating that formulations F14 and F15 have the greatest values of entrapped drug.

Fig. 6. Drug entrapment efficiency of formulations (F1–F15).

3.7. In vitro drug release studies

The in vitro drug release study showed that within eight hours, the maximum amount of medication was released from the hydrogels. Percentage drug release from F1–F15 was examined at various intervals ranging from one to twenty-four hours. Percentage drug release of formulations in the initial 8 hours ranged from 34.03 ± 1% to 96.87 ± 0.87%, as shown in Fig. 7(A) for F1–F8, and Fig. 7(B) for F9–F15.

Fig. 7. Drug release profiles of initial 8 hours of formulations (A) (F1–F8) (B) (F9–F15).

3.8. Drug release kinetics

The values of R² of all the developed formulations were computed using several kinetic models, which were based on earlier research in this field (Table 2).²⁶

Table 2. R ² and coefficient values for pH-sensitive hydrogels in the first-order, Higuchi, Hixson–Crowell, Korsmeyer–Peppas, and zero-order models.

	First order	Higuchi	Hixson–Crowell	Korsmeyer–Peppas		Zero order	Best fit model
	R 2	R 2	R 2	n	R 2	R 2
F1	0.9769	0.9163	0.9709	0.187	0.9268	0.8262	First order
F2	0.963	0.965	0.963	0.251	0.941	0.925	Higuchi
F3	0.9781	0.9779	0.9790	0.268	0.9613	0.9384	Hixson–Crowell
F4	0.7572	0.7867	0.8954	0.107	0.7339	0.6731	Hixson–Crowell
F5	0.8748	0.8704	0.9027	0.125	0.7728	0.8148	Hixson–Crowell
F6	0.8699	0.8788	0.8810	0.247	0.7601	0.8496	Hixson–Crowell
F7	0.1607	0.7417	0.8785	0.073	0.7627	0.5933	Hixson–Crowell
F8	0.1993	0.1132	0.9719	0.260	0.9659	0.9251	Hixson–Crowell
F9	0.8639	0.6086	0.8570	0.363	0.7143	0.6953	First order
F10	0.4572	0.8943	0.2633	0.250	0.8619	0.8030	Higuchi
F11	0.9530	0.9020	0.9745	0.182	0.8823	0.8151	Hixson–Crowell
F12	0.7515	0.1460	0.5624	0.264	0.8297	0.7623	Korsmeyer–Peppas
F13	0.3654	0.9195	0.0168	0.231	0.8935	0.8423	Higuchi
F14	0.5560	0.8029	0.9456	0.175	0.7944	0.6667	Hixson–Crowell
F15	0.9719	0.9039	0.9805	0.202	0.8862	0.8148	Hixson–Crowell

3.9. Molecular docking studies

The results of molecular docking showed a strong binding affinity, with the best binding pose exhibiting a binding energy of −25.0 kcal mol⁻¹. This negative binding energy indicates the favorable host–guest interaction between balsalazide disodium and β-CD, as illustrated in Fig. 8. In the molecular docking image, conventional hydrogen bonds were observed with bond distances of approximately 2.27, 2.42, 2.54, and 2.58 Å.²⁷ These hydrogen bonds were mainly formed between the oxygen- and nitrogen-containing functional groups of balsalazide disodium and the hydroxyl groups of β-CD. A lone pair interaction was also present, affirming the host–guest interaction.²⁷ The observed high affinity suggests that β-CD can act as a suitable host molecule for increasing drug encapsulation and sustained release of the drug from the formulated hydrogel system.

Fig. 8. Molecular docking simulation of balsalazide disodium with β-CD showing the presence of a lone pair, and the formation of hydrogen bonds between balsalazide disodium and β-CD, responsible for a stable host–guest interaction between them.

3.10. Use of mathematical modeling

The responses were mathematically modeled using a quadratic model in Design Expert software. The polynomial equations (eqn (11)) that resulted for each response are shown below:

Y = A + B + C + AB + AC + BC + A² + B² + C² 11

Various key responses, including swelling degree at pH 6.8 and 7.2, gel fraction, and porosity, were analyzed, and their respective polynomial equations were developed to assess the influence of various formulation variables.

3.10.1. Degree of swelling at 6.8

3.10.1.1. Polynomial equation

Y = 218 100 + 434 100 + 1 890 000 + 48 598.20 + 68 985.02 + 218 600 + 335 200 + 572 900 + 1 090 000 12

The polynomial eqn (12), (3D) surface (Fig. 10A), and contour plots (Fig. 9A) of the pH-sensitive hydrogel revealed a positive and significant influence on the degree of swelling at pH 6.8. A rise in the levels of factors A (β-cyclodextrin) and B (acrylic acid) led to an improvement in the swelling behavior of the hydrogel, while factor C (MBA) exhibited a negative effect on this response.

Fig. 10. 3D graphs showing the impact of changing the concentration of oligomer (β-CD) and monomer (acrylic acid) on (A) swelling behavior at pH 6.8, (B) at pH 7.2, (C) porosity, (D) sol fraction, and (E) gel fraction of hydrogel formulations.

Fig. 9. Contour plot showing the impact of changing the concentration of oligomer (β-CD) and monomer (acrylic acid) on (A) swelling behavior at pH 6.8, (B) at pH 7.2, (C) porosity, (D) sol fraction, and (E) gel fraction of hydrogel formulations.

3.10.2. Degree of swelling at 7.2

3.10.2.1. Polynomial equation

Y = 225 100 + 755 200 + 3 361 000 + 409 200 + 83 405.44 + 355 400 + 876 000 + 1 501 000 + 707 000 13

A notable effect on the swelling degree at pH 7.2 was observed based on the polynomial eqn (13), (3D) surface plots (Fig. 10B), and contour diagrams (Fig. 9B) of the pH-sensitive hydrogels. Increasing concentrations of factor A (β-cyclodextrin) and factor B (acrylic acid) enhanced the swelling capacity, whereas factor C (MBA) showed a diminishing impact on this parameter.

3.10.3. Porosity

3.10.3.1. Polynomial equation

Y = 374 500 + 12 561.13 + 77 224.50 + 342.25 + 110 900 + 81.00 + 530 300 + 7.85 + 158 500 14

Based on polynomial eqn (14), along with the contour (Fig. 9C) and 3D surface plots (Fig. 10C), it was observed that the overall effect on porosity was positive. Variables A and B showed a direct relationship with porosity, indicating that an increase in their values led to enhanced porosity. In contrast, variable C exhibited a negative effect, suggesting that higher levels of C result in a reduction in porosity.

3.10.4. Sol fraction

3.10.4.1. Polynomial equation

Y = 177.66 + 0.2450 + 10.81 + 0.8100 + 5.06 + 13.69 + 31.86 + 109.17 + 0.4852 15

A significant impact on the sol fraction was evident from the polynomial eqn (15), (3D) surface plots (Fig. 10D), and contour diagrams (Fig. 9D) of the pH-sensitive hydrogels. An increase in the concentrations of factor A (β-CD), factor B (acrylic acid), and factor C (MBA) resulted in a reduction in the sol fraction.

3.10.5. Gel fraction

3.10.5.1. Polynomial equation

Y = 177.66 + 0.8450 + 3.51 + 0.8100 + 5.06 + 2.89 + 43.63 + 130.17 + 2.75 16

A considerable influence on the gel fraction was observed based on the polynomial eqn (16), (3D) surface plots (Fig. 10E), and contour diagrams (Fig. 9E) of the pH-sensitive hydrogels. An increase in the concentrations of factor A (β-CD), factor B (acrylic acid), and factor C (MBA) led to an enhancement in the gel fraction.

3.11. ANOVA

3.11.1. Degree of swelling at 6.8

The model is indicated as significant by its F-value of 9.85. The possibility that an F-value this great may be caused by noise is only 1.07%. The significant model values are B, B², C², and C.

3.11.2. Degree of swelling at 7.2

The model is significant, according to its model F-value of 11.22. The chance of an F-value this large due to noise is only 0.80%. Significant model variables in this test are C, A², B², B, and C².

3.11.3. Porosity

The model appears to be significant based on its F-value of 15.95. The probability that an F-value greater than this is merely 0.36%. In this study of porosity, significant model terms are A, C, AC, A², and C².

3.11.4. Sol fraction

The model appears to be significant based on its F-value of 6.68. The chance that an F-value this significant is due to noise is only 2.50%. The significant model values are A and B².

3.11.5. Gel fraction

The model appears to be significant based on its F-value of 4.94. The chance that an F-value this great may be because of noise is only 4.66%. The significant model values are B² and A. The F and p values of analysis of variance for degree of swelling at pH 6.8, 7.2, sol–gel fraction, drug entrapment, and porosity of pH-sensitive hydrogels are illustrated in Table 3.

Table 3. Represents F, P, and R² values of ANOVA for degree of swelling at pH 6.8, 7.2, sol–gel fraction, drug entrapment, and porosity of pH-sensitive hydrogels.

	F-value	P-value	Significance	R 2
Swelling index 1.2	20.85	0.0019	Significant	0.9740
Swelling index 6.8	9.85	0.0107	Significant	0.9466
Swelling index 7.2	11.22	0.0080	Significant	0.9528
Sol-fraction	6.68	0.0250	Significant	0.9232
Gel-fraction	4.94	0.0466	Significant	0.8989
Porosity	15.95	0.0036	Significant	0.9663
Drug entrapment	12.13	0.0067	Significant	0.9562

3.12. Results of optimized formulation

3.12.1. FTIR Analysis

The FTIR spectra indicated the presence of characteristic functional groups in the individual components, balsalazide disodium, and the drug-loaded hydrogel. Acrylic acid showed broad O–H stretching (3190 cm⁻¹) and C C stretching (1623–1631 cm⁻¹),²⁸ while β-CD exhibited strong O–H stretching (3382–3457 cm⁻¹) and C–H stretching of methylene groups (2927–2886 cm⁻¹).²⁹ MBA displayed N–H stretching (3436–3293 cm⁻¹), aliphatic C–H stretching (2927 cm⁻¹), C C stretching (1617–1638 cm⁻¹), and an amide C O band at 1653 cm⁻¹.³⁰ Potassium persulfate showed S O stretching (1200–1000 cm⁻¹) and peroxo linkage vibrations (890–950 cm⁻¹).³¹ These absorption bands are illustrated in Fig. 11.

Fig. 11. FTIR spectrum of β-CD, acrylic acid, MBA, and KPS.

For balsalazide disodium, broad O–H and N–H stretching (3300–3500 cm⁻¹), C–H stretching (2927 cm⁻¹), and distinct bands for C O (1653 cm⁻¹) and N N (1583 cm⁻¹) were consistent with the presence of hydroxyl, amide, carboxylate, and azo groups,³² as expressed in Fig. 12.

Fig. 12. FTIR spectrum of balsalazide disodium and drug-loaded optimized hydrogel disc.

The O–H/N–H stretching band, observed at approximately around 3457 cm⁻¹ in the spectra of the individual components, shifted to a broader range of 3322–3436 cm⁻¹ in the drug-loaded hydrogel, showing a downfield shift of approximately 21–135 cm⁻¹. This shift towards lower wavenumber is consistent with a weakening of the O–H/N–H bond, which typically occurs when these groups participate in hydrogen bonding.³³ Such a shift suggests hydrogen-bonding between hydrogen bonding functional groups of β-CD, acrylic acid, MBA, and polar groups of balsalazide disodium. In addition, the broadening of this band from a single defined peak in the pure components to a wider absorption band is indicative of multiple hydrogen-bonding environments, suggesting the formation of hydrogen bonds of varying strength at various sites between the drug and polymer network. The reduced intensity of the C C stretching band, present in the acrylic acid and MBA spectra, in the drug-loaded hydrogel is consistent with the utilization of the vinyl group in free-radical polymerization.³⁴ The peak at 1653 cm⁻¹ is assigned to the carbonyl (C O) stretching vibrations of carboxylate or amide groups, while the peak at 1583 cm⁻¹ corresponds to azo (N N) stretching vibrations, which are typical for azo compounds. Additionally, peaks in the 900–700 cm⁻¹ range, such as those at 854, 754, and 706 cm⁻¹, are linked to out-of-plane bending of the benzene ring, consistent with the presence of aromatic structures, likely from the drug molecule, as shown in Fig. 8. The retention of these characteristic balsalazide peaks suggested the drug had retained its chemical identity during incorporation. The absence of any new major absorption bands in the drug-loaded hydrogel suggested that no new covalent bonds were formed, and the forces involved in the whole drug loading and crosslinking process are physical interactions, including hydrogen bonding and electrostatic interactions between components.

3.12.2. SEM Analysis

The image of the blank formulation showed a smooth and compact surface, indicating a highly cross-linked polymer matrix. A distinct crystalline or flake-like structure can be found embedded within the matrix, as shown in Fig. 13(A). The absence of visible interlinked pores or a highly porous network indicates a non-porous, dense network. These structural characteristics aid in the diffusion-controlled release of the medicine that is entrapped within the formulation. The SEM image of the optimized formulation demonstrates noticeable alterations in its surface structure when compared to the blank hydrogel disc. The image reveals a highly porous and coarse morphology, with irregular cavities and interconnected pores dispersed throughout the matrix. This structural pattern resembles a swollen, sponge-like configuration, as illustrated in Fig. 13(B). The observed rise in surface irregularity and porosity indicates improved water absorption and swelling capacity, which can contribute to more effective drug diffusion.³⁵

Fig. 13. (A) SEM image of blank formulation at 40×; (B) SEM image of drug-loaded formulation at 26×.

3.12.3. DSC

The thermogravimetric analysis curve (blue), as shown in Fig. 14(A), demonstrates an initial, gradual reduction in weight beginning around 50 °C and continuing up to 160 °C, with an overall weight loss of approximately 5–15%. Most likely, the evaporation of moisture or leftover solvents is the cause of this initial loss. A significant decomposition phase initiates at about 200 °C and progresses until 350 °C, during which a substantial weight reduction of roughly 30–35% occurs, indicating the onset of thermal degradation of the drug substance. Beyond 400 °C, a more pronounced weight decline is observed, leading to a total weight loss of nearly 70–75%, suggesting the complete breakdown of the drug's molecular framework.

Fig. 14. (A) TGA of balsalazide disodium, blank hydrogel, and balsalazide disodium-loaded hydrogel (B) DSC of balsalazide disodium, blank hydrogel, and balsalazide disodium-loaded hydrogel.

The differential scanning calorimetry (DSC) curve (red) displays an endothermic transition between 80–100 °C, which likely corresponds to moisture evaporation. The M.P of the pure substance is shown by a sharp, unmistakable endothermic peak at 410–430 °C, as given in Fig. 14(B), followed by thermal decomposition at higher temperatures. These findings indicate that the pure drug maintains thermal stability up to around 200–250 °C, beyond which significant degradation begins.³⁶ The thermogravimetric analysis (TGA) profile (red curve) of the unloaded hydrogel exhibits an initial weight reduction starting around 50 °C, which gradually extends to nearly 120 °C. This early mass loss, accounting for approximately 15–18%, is likely due to the evaporation of residual moisture or loosely bound water within the hydrogel structure. A more significant degradation phase is evident between 200 °C and 350 °C, during which the hydrogel undergoes considerable thermal decomposition, resulting in a total weight loss of about 45–50%. Beyond 400 °C, the curve levels off with a slight decline, indicating minimal remaining material, likely carbonaceous residue, and the end of the decomposition process.

The differential scanning calorimetry (DSC) trace (purple curve) shows a mild endothermic peak between 80 °C and 110 °C, consistent with the moisture loss observed in the TGA curve. Another broader and less intense thermal event occurs between 180 °C and 220 °C, as shown in Fig. 14(B), possibly corresponding to the glass-transition temperature (T_g) of the hydrogel.³⁷

The TGA thermogram (purple curve) as represented in Fig. 14(A) shows an initial reduction in weight starting near 50 °C, continuing up to approximately 120 °C, with a total loss of around 10–12%. This early mass reduction is due to the evaporation of surplus or weakly bound water molecules present in the hydrogel structure. A second, more prominent weight loss is observed between 200 °C and 350 °C, indicating substantial thermal degradation of the hydrogel. This phase results in a total mass loss of nearly 50–55%, which can be ascribed to the decomposition of the crosslinked matrix and potential thermal degradation of the encapsulated drug. Beyond 400 °C, the curve stabilizes, suggesting the presence of a minor amount of thermally stable residue, likely consisting of carbonaceous material.

The DSC thermogram (blue curve) exhibits notable thermal events. An endothermic peak from 80 °C to 110 °C, as illustrated in Fig. 14(B), corresponds to the loss of moisture observed in the TGA data, confirming the elimination of absorbed water. A more defined endothermic signal around 200 °C indicates the glass transition temperature (T_g) of the polymer. The lack of significant transitions above 300 °C further supports the thermal stability of the system up to the point of degradation.³⁸ The melting peak of the drug at 410–430 °C was absent in the drug-loaded hydrogel formulation.

3.12.4. Stability studies

Stability studies were performed on the optimized hydrogel formulation. The optimized formulation was stored at 40 °C and 75% RH for a period of one month.²⁴ Results (Table 4) depicts there is notably less variation after a 1 month study, which suggests that the formulations are stable over a specified period of time.

Table 4. Results of characterization of optimized formulation after 1 month stability study.

Parameter	After 1 month at 40 °C/75% RH
Degree of swelling at pH 6.8	2638.9
Degree of swelling at pH 7.2	3295.7
Apparent porosity	2169.8
Sol fraction	29.1
Gel fraction	70.9

3.12.5. In vitro drug release study

The in vitro drug release study of the optimized formulation indicated only a minimal amount of drug released, i.e., 13%, in the gastric condition due to the non-swollen state of the hydrogel. Upon transfer to the buffer of pH 6.8 (1–4 h), the drug release reached 53% within the duration of 3 hours due to ionization of acrylic acid, resulting in swelling of the hydrogel in alkaline pH. Following transfer to the pH 7.2 medium (4–8 h), drug release continued to increase steadily, reaching 95% within the duration of 4 hours, as shown in Fig. 15. This sustained, phase-dependent increase in drug release confirms the pH-responsive behaviour of the hydrogel, with minimal release under gastric conditions and progressively enhanced release under intestinal and colonic pH environments, supporting its suitability for colon-targeted drug delivery.³⁹

Fig. 15. In vitro drug release of the optimized formulation at pH 1.2, 6.8, and 7.2.

4. Discussion

To develop a pH-sensitive hydrogel, various monomers having the ability to ionize and swell selectively in the alkaline environment of the intestine were investigated. Since balsalazide disodium, the selected drug for encapsulation, is primarily absorbed in the colon, a monomer responsive to higher pH levels was required. Acrylic acid was selected due to its suitable pKa range (approximately 4.5–5), which enables it to ionize and swell through electrostatic repulsion in alkaline conditions. At low pH, the carboxylic groups present in acrylic acid remain unionized, while at higher pH, the –COOH groups are ionized, resulting in the formation of negatively charged carboxylate ions. These carboxylate ions repel each other, resulting in the expansion of the hydrogel matrix and release of the drug at higher pH, i.e., 6.8 and 7.2. This selection aligns with findings reported by Muhammad Rizwan et al., who extensively discussed appropriate polymers, monomers, and their formulations for pH-sensitive hydrogel systems.⁴⁰ Based on studies from previous research, β-CD was selected as the polymer because it contains a large number of –OH groups, providing it with hydrophilicity, resulting in the formation of stable host–guest interactions with balsalazide disodium, MBA as the cross-linker, and KPS as the initiator to be used in combination with acrylic acid for optimal hydrogel formation. Following a successful initial formulation, 15 experimental trials were conducted using different concentrations of monomer, polymer, and cross-linker, while maintaining a fixed concentration of initiator.⁴¹ Formulations prepared with higher concentrations of polymer and monomer exhibited favorable characteristics—they were non-sticky, dried efficiently, and could be cut cleanly because of the formation of a firm hydrogel crosslinked network between monomer and polymer. In contrast, formulations with lower amounts of these components, including the cross-linker, resulted in sticky hydrogels that were difficult to dry and challenging to section because of their sticky nature, indicating the formation of a less firm crosslinked hydrogel network due to a lack of monomer, polymer, or cross-linker. The purpose of the research was to increase the absorption and bioavailability of balsalazide disodium, a drug known for its limited oral bioavailability. To address this issue, the drug was incorporated into hydrogels designed to release the active compound specifically in the intestinal environment. Accordingly, pH-sensitive hydrogels loaded with balsalazide disodium were successfully developed to meet the desired therapeutic outcomes.

4.1. Organoleptic evaluation

The hydrogel formulations were transparent and glossy in appearance, consistent with earlier studies on β-CD-based hydrogels by Jiayue Liu et al. (2021).⁴² The color of the formulations after loading the drug changed from transparent to orange due to the orange-yellow color of balsalazide disodium. Similar findings regarding a change in color of formulation after loading colored drug were reported by Ying Liu, Xin Tang, et al., 2022.⁴³

4.2. Degree of swelling

The swelling behavior of the formulations was assessed at various pH values (1.2, 6.8, and 7.2) for all 15 formulation trials. The impact of different amounts of excipients on the swelling index was measured. All formulations showed pH-responsive swelling, more prominently at alkaline pH values than at lower pH (acidic conditions). This pH-sensitive behavior is attributed to the existence of acrylic acid in the formulation, which ionizes at higher pH values, i.e., when the pH of the environment is higher than the pK_a value of acrylic acid, resulting in electrostatic repulsion and enhanced water uptake. Thus, the carboxylate ions present in acrylic acid are responsible for the swelling behavior.⁴² According to studies reported by Mohammad, Atassi, & Tally et al. 2017, the hydrogel's tendency to absorb water is pH-dependent.⁴⁴

4.3. Effect of concentration of cross-linker on swelling of hydrogels

The amount of the cross-linker is crucial in hydrogel formulation, as it directly affects the swelling capacity. This study observed an inverse relationship: increasing the amount of cross-linker decreased swelling because it formed a more tightly cross-linked hydrogel network with fewer voids. Ninciuleanu et al. also reported similar results, noting that increasing the amount of MBA caused decreased water absorption and swelling in poly (methacrylic acid) hydrogels, across a range of pH conditions (1.2, 5.4, 7.4).⁴⁵ The extent of decrease was, however, less pronounced in their system than in ours, because of the polymer-to-monomer ratio and network architecture, with our system being based on a β-CD-grafted cyclic network rather than a linear poly (methacrylic acid) backbone.

4.4. Effect of monomer concentration on swelling of hydrogels

The degree of swelling of hydrogels was significantly affected by the concentration of acrylic acid (monomer). An increase in its content led to a noticeable enhancement in the swelling index. This trend can be due to the greater ionization of carboxylic acid groups at elevated pH levels, which promotes increased water uptake. Similar details were presented by Sindhu et al., who observed that a higher monomer concentration contributed to improved swelling because of enhanced ionization of the acidic functional group under alkaline conditions.⁴⁶

4.5. Effect of concentration of polymer, monomer, and cross-linker on gel fraction of hydrogels

In this study, it was noted that increasing the amount of the polymer, monomer, and crosslinking agent led to a corresponding rise in the gel fraction percentage due to enhanced crosslinking between components, resulting in a more gel-like consistency. According to research conducted by Barkat et al., an increase in gel fraction was reported with increased concentration of monomer, cross-linker, polymer, and vice versa.⁴⁷

4.6. Effect of the amount of monomer and cross-linker on the porosity of hydrogels

A higher concentration of monomer or polymer was associated with an increase in porosity, whereas opposite behavior was noted when the quantity of MBA was raised. This is because acrylic acid contains abundant carboxylic groups, which increase the hydrophilicity and the hydrogen-bonding ability, resulting in enhanced solvent penetration into the polymer network. β-CD also contains abundant hydroxyl groups contributing to its hydrophilicity and increasing the solvent penetration into the network. The high value of apparent porosity indicates that the solvent has not only entered the pre-existing pores, but it has also penetrated the polymeric network of hydrogel because of the presence of hydrophilic groups, i.e., acrylic acid and β-CD. Comparable findings were presented by Shabir et al. that a rise in monomer content elevated the solution's viscosity, which hindered bubble escape, thus resulting in the formation of interconnected channels that enhanced porosity. However, raised MBA concentration led to reduced porosity.⁴⁸

4.7. Drug entrapment efficiency

The findings related to the entrapment of the drug showed that entrapment efficiency increases with an increase in both the polymer and monomer concentrations, as the penetration of liquid increases into the hydrogel matrix, also evident by the value of porosity. The greater the penetration of the drug solution into the hydrogel network, the greater will be the drug entrapment. The increase in entrapment efficiency with an increase in concentration of monomer and polymer is not only related to the increase in solvent penetration and porosity; the drug loading is also due to increased β-CD binding sites capable of anchoring the drug through hydrogen bonding, as evident from molecular docking studies. The change in mesh size in alkaline conditions due to ionization of acrylic acid and crosslinking density further restricts drug diffusion out of the network, resulting in an overall increased drug entrapment. These findings are comparable to those of Nautiyal et al., who found that increased monomer and polymer concentrations enhance the entrapment efficiency of drug delivery systems.⁴⁹ Similar findings were also reported by Suhail et al., who reported that an increase in concentration of monomer and polymer leads to increased swelling and drug loading in hydrogels, while swelling was significantly enhanced due to deprotonation of ionizable functional groups in an alkaline environment.³⁹

4.8. Effect of concentration of monomer, cross-linker, and polymer on drug entrapment efficiency

The impact of varying monomer and polymer concentrations on entrapment efficiency was assessed, revealing that higher levels of both components led to improved drug entrapment. This observation is the same as the findings reported by Salawi et al., where enhanced entrapment was attributed to a more expanded and interconnected hydrogel matrix. The broader network formed due to increased polymer and monomer concentrations likely provided more binding sites and space, thereby facilitating more effective encapsulation of the drug within the hydrogel structure.⁴¹ An increase in cross-linker concentration led to enhanced drug entrapment efficiency within the hydrogels. N. S. et al. also reported similar findings in a study involving chitosan/xanthan gum (CS/XG)–based hydrogels, where crosslinking played a critical role in modulating drug entrapment performance.⁵⁰

4.9. Effect of the concentration of components on in vitro drug release

The results of the in vitro study are consistent with those of Ramadan, Elbakry et al., who noted that the concentrations of the monomer, crosslinking agent, and polymer utilized in the formulation have a major impact on drug release profiles.⁵¹ The in vitro drug release was improved by a raised concentration of β-CD. Greater drug diffusion inside the gel matrix and into the surrounding medium is made possible by a larger hydrogel network, which is facilitated by higher β-CD levels. Dong et al. found that dual-crosslinked hydrogels showed quicker drug release as polymer concentration increased, which is consistent with this conclusion.⁵² An increase in the monomer concentration led to an enhanced drug release from the hydrogel, whereas the cross-linker exhibited an opposing effect by reducing the release rate. Similar outcomes were observed in a study by Bueno et al. on GG/PVP-co-poly (acrylic acid) hydrogels, where a higher concentration of acrylic acid improved drug release and an increased amount of cross-linker reduced the drug release.⁵³

4.10. FTIR spectroscopy

The FTIR spectrum of the blank formulation exhibited the appearance of new peaks, showing efficient crosslinking and formation of the polymer matrix. These spectra also assured the compatibility of the excipients with one another. The emergence of new peaks in the FTIR of the loaded formulation confirms the reaction and bonding between the components and the drug, resulting in the incorporation of the drug within the polymer network.

4.11. DSC

The endothermic peaks in the drug, blank formulation, and optimized formulation DSCs were different. Furthermore, a small thermal event at about 200 °C signifies the glass transition temperature or the drug-polymer network interaction. Drug dispersion within the polymeric network is shown by the absence of an endothermic peak at the drug's melting point. The indications of this entire thermal profile demonstrate the formulation's thermal stability while also indicating the drug's successful integration into the polymer matrix. Akram Ashames et al. (2022) conducted a study where similar thermal output was obtained, supporting the incorporation of the drug and its thermal stability within the polymeric matrix.⁵⁴ The melting point of the drug was observed at 410–430 °C, while in the optimized hydrogel, the melting peak was observed at 225 °C. The disappearance of the melting peak of the drug may indicate the incorporation of the drug into the polymer matrix, reduced crystallinity, and molecular dispersion of the drug. The reduced melting temperature also indicates drug-polymer interaction through physical interactions, i.e., hydrogen bonding. A similar interpretation was reported by Yu et al., where the DSC thermogram of acyclovir-loaded nanofibers did not show the characteristic melting peak of acyclovir, indicating that the incorporated drug may be converted into an amorphous state within the polymeric system.⁵⁵

4.12. SEM

A detailed visualization of the surface morphology and internal structure of blank and optimized formulations was given by SEM micrographs. The drug-unloaded formulation displayed a relatively compact and smooth surface, indicating a dense and uniform polymer network formed by effective crosslinking. Conversely, the SEM image of the drug-loaded formulation revealed significant morphological changes, with the surface becoming more porous and irregular, attributed to an interlinked network of channels and cavities. This porous, irregular appearance is likely due to the incorporation of the drug into the polymer matrix. As the drug is loaded by the post-loading method, when the drug solution gets absorbed into the blank hydrogel, it results in chain relaxation and an increase in volume compared to the blank volume to accommodate the drug solution, resulting in swelling of the hydrogel. Upon drying, water evaporates from the swollen hydrogel, but the drug remains in the network, preventing the network from condensing back to the exact same volume, resulting in a porous and irregular appearance of the hydrogel network.⁵⁶ The same SEM results were published for VCL-MAA-based hydrogels by Ozkahraman et al. 2023, showing increased swelling and porosity upon drug loading, which enhanced diffusion and swelling.⁵⁷

4.13. ANOVA

The Design Expert software's projected values and the parameters of the optimized pH-sensitive hydrogels were compared. The expected results and the experimental values did not differ much, which indicates that the optimized formulation met the desired criteria. The optimized formulations showed favorable properties of swelling, porosity, entrapment efficiency, and gel fraction. Table 5 presents the optimized results alongside the predicted outcomes.

Table 5. Comparison of optimized and experimental results^a.

Parameter	Predicted outcomes %	Experimental results %
Degree of swelling at 6.8	2839.2	2794.6
Degree of swelling at 7.2	3501.01	3488.9
Porosity	2336.04	2314.6
Sol fraction	27.4	27
Gel fraction	72.6	73

^aA paired t-test is applied to determine any significant difference between the experimental and predicted outcomes. The value of the t-statistic is 1.89, and the p-value is 0.13. Since p is greater than 0.05, this highlights no prominent difference between predicted and experimental outcomes.

4.14. Drug release kinetics

Using several kinetic models and DD solver software, the pattern of drug release from the pH-sensitive hydrogels was studied in a 6.8 pH phosphate buffer. The best-suited kinetic model was assessed by comparing the R² values of all kinetic models. Out of the 15 formulations (F1–F15), 9 formulations adhered to the Hixson–Crowell kinetic model as the best-fit model.⁵⁸ The best fit to the Hixson–Crowell model indicates that the drug is released from the hydrogel matrix by changes in effective surface area, matrix geometry, and swelling of the hydrogel network rather than only by simple diffusion.⁵⁹ The value of n below 0.5 indicates drug release by Fickian diffusion from the disc-shaped hydrogels.⁶⁰ Similar results were reported by Khan et al., who developed chemically crosslinked pH-sensitive PEG/acrylic acid hydrogels and showed that the Hixson–Crowell model exhibited the highest R² value for hydrogels.⁵⁸

Zero-order and first-order kinetics were also observed for each sample. The first-order kinetics dominated the zero-order kinetics for all 15 formulations (F1–F15). This implies that the sustained release of the drug was observed; a study reported by Ge, Li et al. explained that the sustained release behavior of the drug followed first-order kinetics. According to the study, as it shows first-order kinetics, it is evident that the drug release will be sustained.⁶¹

5. Conclusion

The goal of the study was successfully achieved as oral hydrogels delivered the drug for a duration of 8 hours. The application of a statistical approach appeared useful in optimizing the formulations. Because the results of the optimized formulation are similar to those of the formulation optimized by Design Expert Software, the experimental procedure was determined to be reproducible. Hence, it can be concluded that hydrogel can be used for the sustained release of balsalazide disodium. Moreover, Design Expert will help save time and money by reducing pointless trials to produce an optimal formulation.

Author contributions

Conceptualization, M. Z. and W. S.; methodology, Z. S, and W. S.; software, W. S.; validation, H. A and A. A.; formal analysis, Z. R.; investigation, M. I.; resources, M. I.; writing – original draft preparation, Z. S.; writing – review and editing, W. S. and B. A.; supervision, M. Z.; funding acquisition, M. I. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

All of the data analyzed during the current research project are already incorporated within the manuscript.