Formulation And Characterization of Aceclofenac Loaded Mucoadhesive Microspheres for Controlled Gastric Delivery

Drug delivery systems have evolved dramatically over recent decades, transforming from conventional formulations that rely on simple oral or parenteral administration into sophisticated, engineered platforms designed to improve therapeutic outcomes, patient compliance, and safety. Among the most promising approaches in this evolution is microencapsulation technology, which enables precise control over the release profile, targeting, stability, and bioavailability of active pharmaceutical ingredients (APIs) [1]. Microencapsulation involves enclosing active agents within a polymeric or lipid-based matrix to create tiny, discrete units—microparticles or microspheres—that can protect the drug from degradation, mask unpleasant tastes, control release rates, or deliver the drug to specific sites in the body.

1. MATERIALS AND METHODS
   1. Material: Drugs, Chemicals and Reagents

Aceclofenac was obtained as a gift sample from M/s M Sea Pharmaceuticals Pvt. Ltd., while other essential reagents such as calcium chloride, methanol, ethanol, dichloromethane, polyvinyl alcohol, propylene glycol, PVP, PAA, and various acids and salts including potassium dihydrogen phosphate, sodium hydroxide, hydrochloric acid, and picric acid were sourced from reputed suppliers like Merck, Sigma-Aldrich, Loba Chem, and S.D Fine Chemicals. Carrageenan and double distilled water were also used in the study.

1. 2. Drug-excipients compatibility study

To confirm the chemical compatibility of Aceclofenac with excipients intended for sustained-release mucoadhesive microspheres, FTIR and DSC studies were conducted. FTIR spectra of the pure drug, Okra mucilage, Eudragit RS100, and their physical mixtures were recorded using a Shimadzu IR Affinity-1 spectrometer.

2.3 Formulation of mucoadhesive microspheres: Preparation of mucoadhesive microspheres

The Aceclofenac-loaded mucoadhesive microspheres were prepared using a solvent diffusion and interpolymer complexation technique designed to ensure uniform drug distribution and sustained-release characteristics. Initially, accurately weighed quantities of Aceclofenac, Okra mucilage, and Eudragit RS100 were dissolved in a mixed solvent system of ethanol and water under continuous stirring to create a homogeneous polymer-drug solution. Span 80, serving as a non-ionic surfactant at 1% w/v concentration, was added to reduce interfacial tension and prevent droplet coalescence during emulsification. This prepared drug–polymer solution was then introduced slowly in a thin stream into 100?mL of light liquid paraffin, which acted as the continuous oil phase, maintained at room temperature under constant mechanical stirring at 500?rpm. The gradual addition and steady stirring promoted the formation of fine, discrete droplets, which underwent solvent diffusion, leading to solidification of the microsphere matrix. After 2?hours of continuous stirring to ensure complete solvent removal and droplet stabilization, 2.5% v/v glutaraldehyde solution was added carefully as a crosslinking agent to strengthen the polymeric network and improve mechanical stability of the microspheres. Stirring was maintained for an additional 30?minutes to ensure adequate crosslinking. The resulting hardened microspheres were then collected by filtration, thoroughly washed with n-hexane to remove residual liquid paraffin and any unreacted materials, and finally dried at 60?°C in a hot-air oven for 12?hours. The dried microspheres were stored in airtight containers at room temperature until further characterization and evaluation [2].

The practical yield of the prepared Aceclofenac-loaded mucoadhesive microspheres was determined to evaluate the efficiency and reproducibility of the production process. After preparation and drying, the microspheres were accurately weighed using an analytical balance. The percentage yield was calculated relative to the total expected weight of non- volatile components used in the formulation, which included the weights of the drug and polymers. The yield (%) was computed using the formula:

% Yield = actual weight of product/total weight of excipients and drug × 100

1. 1. 2. Particle Size Determination

The mean particle size of the prepared mucoadhesive microspheres was determined using optical microscopy. A small quantity of dried microspheres was dispersed on a clean glass slide and examined under a calibrated optical microscope at appropriate magnification. Approximately 100 microspheres were randomly selected and measured for their diameter using an ocular micrometer. The recorded measurements were averaged and expressed as mean particle size ± standard deviation. This assessment ensured uniformity in size distribution, which is critical for consistent drug release behavior, mucoadhesive performance, and patient acceptability[3].

2.4.3 Morphological Study using SEM

The surface morphology and structural characteristics of the prepared microspheres were examined using Scanning Electron Microscopy (SEM). A small quantity of dried microspheres was mounted on metal stubs using double-sided adhesive carbon tape and then coated with a thin layer of gold using a sputter coater to enhance electrical conductivity. The samples were observed under a scanning electron microscope at various magnifications to capture detailed images of the microspheres’ surface topography. This analysis provided insights into particle shape, surface smoothness, integrity, and the absence of cracks or aggregation, factors essential for predicting drug release kinetics and mucoadhesive behavior[4].

2.4.4 Drug Loading and Drug Entrapment

Drug loading and entrapment efficiency of the Aceclofenac-loaded mucoadhesive microspheres were determined to evaluate the formulation’s capacity to incorporate and retain the active pharmaceutical ingredient. Accurately weighed microsphere samples were dissolved in an appropriate volume of 0.1?N HCl (pH?1.2) with continuous stirring or mild sonication to ensure complete drug extraction from the polymeric matrix. The resulting solution was filtered and analyzed spectrophotometrically at 275?nm against a pre- established calibration curve of Aceclofenac. Drug loading was expressed as the percentage of drug content relative to the total weight of microspheres, while entrapment efficiency (%) was calculated as the ratio of the actual drug content to the theoretical drug load multiplied by 100. These evaluations were critical for assessing the formulation’s effectiveness in achieving the desired therapeutic dose and ensuring batch-to-batch consistency[5][6][11].

2.4.5 In-vitro Wash-off Test for Mucoadhesion

The in-vitro mucoadhesive properties of the prepared Aceclofenac-loaded microspheres were evaluated using a wash-off test employing excised rat stomach mucosa as a model tissue. Freshly isolated stomach tissue was carefully cleaned with physiological saline to remove adhering debris and then mounted on a glass slide with the mucosal side exposed. Approximately 100 microspheres were evenly spread onto the mucosal surface. The prepared slide was then fixed to the base of a USP disintegration apparatus basket, which was subsequently immersed in a beaker containing 0.1?N HCl (pH?1.2) maintained at 37?±?0.5?°C to simulate gastric conditions. The apparatus was operated with gentle, controlled up-and-down movements to mimic the peristaltic motion of the gastrointestinal tract. At predetermined time intervals up to 12?hours, the number of microspheres still adhering to the mucosal surface was counted. The percentage of retained microspheres at each interval was calculated to assess mucoadhesive strength. Stronger retention indicated improved mucoadhesive properties, suggesting prolonged gastric residence time for the formulation [5].

2.4.6 In-vitro Release Study

The in-vitro drug release profile of Aceclofenac from the mucoadhesive microspheres was assessed using a USP Type I dissolution apparatus. Accurately weighed samples of microspheres were placed in the dissolution baskets and immersed in 900?mL of 0.1?N HCl (pH?1.2), maintained at 37?±?0.5?°C to simulate gastric conditions. The baskets were rotated at 100?rpm to ensure uniform mixing and sink conditions. At predetermined time intervals over a 24-hour period, 5?mL aliquots of the dissolution medium were withdrawn and immediately replaced with an equal volume of fresh pre-warmed medium to maintain constant volume and sink conditions. The withdrawn samples were filtered through 0.45?μm membrane  filters  and  analyzed  spectrophotometrically  at  275?nm  to  determine  the concentration of Aceclofenac released. The cumulative percentage of drug released was calculated and plotted against time to characterize the release behaviour of the microspheres [6][7][8][11].

2.4.7 Release Kinetics: Pharmacokinetic modelling

To understand the mechanism of drug release from the prepared mucoadhesive microspheres, the in-vitro release data were subjected to mathematical modeling using various kinetic models. The release profiles were fitted to Zero-order, First-order, Higuchi, Korsmeyer–Peppas, Hixson–Crowell, and Weibull models. The equations for each model were applied to the cumulative release data, and regression analysis was performed to determine the best-fit model based on the highest coefficient of determination (R²). For the Korsmeyer–Peppas model, the release exponent (n) was also calculated to interpret the mechanism of drug release, where n values indicated Fickian diffusion, anomalous (non- Fickian) transport, or case II transport. This kinetic analysis provided insights into whether the release was controlled primarily by diffusion, erosion, swelling, or a combination of mechanisms, informing formulation design and potential in-vivo performance[9][10][11].

2.5 Statistical analysis

All characterization data of Aceclofenac-loaded mucoadhesive microspheres were statistically analyzed to ensure accuracy and reliability. Experiments were conducted in triplicate, and results were expressed as mean ± SD. One-way ANOVA was used to determine significant differences among formulations (p < 0.05), followed by Dunnett’s post-hoc test and Student’s t-test for specific comparisons. Regression analysis assessed the fit of drug release data to kinetic models using R² values. Design-Expert® softwaresupported experimental design and optimization, while GraphPad Prism and Excel were used for plotting and analysis. This ensured a robust and systematic evaluation of formulation performance.

3. RESULT AND DISCUSSION

3.1 Drug-excipients compatibility study

Fourier-transform infrared  spectroscopy  (FT-IR)  was used to evaluate possible interactions between Aceclofenac and the selected excipients (Okra mucilage and Eudragit RS100).

The FT-IR spectra of pure Aceclofenac showed characteristic peaks at ~3310?cm?¹ (N–H stretching), ~1770?cm?¹ (C=O stretching of carboxylic acid), and ~1515?cm?¹ (aromatic C=C stretching). The physical mixture of Aceclofenac with Okra mucilage and Eudragit RS100 retained all key functional peaks without significant shifts or disappearance, indicating no chemical interaction between the drug and polymers. Minor broadening observed around~3310?cm?¹ was attributed to hydrogen bonding with Okra mucilage but did not indicate incompatibility.

Figure 1. FT-IR spectra of pure Aceclofenac, Okra mucilage, Eudragit RS100, and their physical mixture. Peaks confirm retention of functional groups without significant interaction.

3.2 Characterizations of the mucoadhesive microspheres

1. 1. 1. Percentage Yield

The percentage yield for all formulations ranged from 76.60?±?1.4% (AMF3) to 82.27?±?1.4% (AMF5). Most formulations demonstrated high yields exceeding 80%, confirming efficient recovery of microspheres during the preparation process. Formulations AMF2, AMF4,  AMF5,  and  AMF6  consistently  showed yields above  81%, reflecting optimized process conditions such as appropriate polymer concentrations, effective crosslinking, and controlled stirring during solvent diffusion. AMF3 exhibited the lowest yield (76.60?±?1.4%), suggesting minor process loss potentially due to increased viscosity at higher polymer ratios, leading to droplet coalescence or incomplete crosslinking. High percentage yields across most formulations indicate excellent efficiency and reproducibility of the solvent diffusion technique combined with interpolymer complexation. The use of Okra mucilage and Eudragit RS100 provided a stable matrix, while the controlled addition of glutaraldehyde ensured effective crosslinking without excessive hardening or clumping.

All formulations demonstrated high practical yields, generally exceeding 80%, with values ranging from 76.60?±?1.4% to 82.27?±?1.4%. AMF5 exhibited the highest yield (82.27?±?1.4%), reflecting effective matrix formation, optimal polymer concentration, and stable emulsion during solvent diffusion. Formulations AMF1, AMF2, AMF4, and AMF6 consistently achieved yields above 80%, indicating robust and reproducible process conditions. AMF3 showed a slightly lower yield (76.60?±?1.4%), which may be attributed to increased polymer content leading to higher viscosity, droplet coalescence, or minor losses during filtration and washing steps. These findings confirm the efficiency and reliability of the selected preparation method. The combination of Okra mucilage (providing natural mucoadhesive properties) and Eudragit RS100 (offering pH-independent matrix stability) formed a consistent crosslinked structure when treated with glutaraldehyde. High practical yields across most batches demonstrate the suitability of the solvent diffusion technique for scalable manufacturing. The low standard deviations highlight good batch-to-batch reproducibility, an essential quality attribute for pharmaceutical production.

Figure 2. Practical yield of the mucoadhesive microspheres

Table 5.3 presents the average particle size of the Aceclofenac-loaded mucoadhesive microspheres, demonstrating controlled and consistent microparticulate dimensions suitable for gastroretentive delivery. The mean particle sizes ranged from 112.5?±?2.3?μm (AMF1) to 124.3?±?2.7?μm (AMF5), with low standard deviations indicating uniform size distribution within each batch. Formulations with higher polymer content, such as AMF3 and AMF5, tended to exhibit slightly larger particle sizes, likely due to increased solution viscosity during emulsification, which stabilizes larger droplets before solidification. Despite these variations, all formulations maintained particle sizes well within the desirable 100–130?μm range for oral mucoadhesive systems, ensuring adequate surface area for interaction with the gastric mucosa while avoiding premature transit from the stomach. The controlled particle size also contributes to predictable drug release kinetics and reproducible manufacturing performance. Overall, these results confirm the effectiveness of the solvent diffusion method and formulation design in producing consistently sized, well-defined microspheres that support prolonged gastric residence and sustained Aceclofenac release. Table 3. Average particle size of the mucoadhesive

 Microspheres.

*Data presented as Mean ± SD

1. 1. 1. Morphological Study using SEM

The morphological study of the optimised Aceclofenac-loaded mucoadhesive microspheres was conducted using scanning electron microscopy (SEM) to assess surface characteristics and structural integrity. SEM images revealed that the microspheres were predominantly spherical with smooth, well-defined surfaces, confirming successful formation through the solvent diffusion technique. Minimal surface irregularities or cracks were observed, indicating effective crosslinking with glutaraldehyde and stable matrix formation without structural collapse.

The uniform shape and compact surface morphology suggest homogenous polymer distribution of Okra mucilage and Eudragit RS100 within the microsphere matrix, essential for controlled drug release and reproducible mucoadhesion. Additionally, the absence of visible drug crystals on the surface implies successful encapsulation of Aceclofenac within the polymer network, reducing the risk of burst release.

Figure 3. SEM photographs of the mucoadhesive microspheres (Magnification 200x for left image and 400x for right image)

Table 5.4 summarizes the percentage drug loading and  entrapment efficiency of the Aceclofenac-loaded mucoadhesive microsphere formulations, demonstrating effective incorporation of the drug into the polymeric matrix. Drug loading values ranged from 18.4?±?0.6% (AMF1) to 21.8?±?0.8% (AMF5), while entrapment efficiencies spanned from 76.8?±?2.1% to 87.5?±?2.3%. Higher polymer concentrations in formulations such as AMF3, AMF5, and AMF6 were associated with improved drug entrapment, likely due to enhanced matrix density and reduced diffusion of drug into the external phase during preparation. AMF5 exhibited the highest drug loading and entrapment efficiency, reflecting optimal ratios of Okra mucilage and Eudragit RS100, as well as effective crosslinking with glutaraldehyde. The consistently high values across all formulations indicate robust and reproducible encapsulation performance of the solvent diffusion method. This effective entrapment is critical for achieving sustained drug release, minimizing dose dumping, and ensuring therapeutic consistency in gastroretentive delivery systems.

Table 4. Drug loading and drug entrapment of the mucoadhesive microspheres

Figure 4. Drug loading and drug entrapment of the mucoadhesive microspheres formulations (AMF1-AMF6)

Table 5.6 presents the in-vitro cumulative percentage drug release profiles of Aceclofenac- loaded mucoadhesive microsphere formulations over 12?hours in 0.1N HCl (pH?1.2), simulating gastric conditions. All formulations exhibited a sustained, controlled-release pattern, with initial release of ~10–12% in the first hour, indicating a minimal burst effect and good polymer encapsulation. Gradual and consistent release continued over time, with approximately 46–49% cumulative release achieved by 4?hours and 66–70% by 6?hours across all batches. By 12?hours, cumulative release ranged from 93.9?±?3.0% (AMF5) to 97.2?±?2.8% (AMF2), demonstrating near-complete drug delivery while maintaining prolonged release. Formulations with higher polymer content (AMF5 and AMF3) showed slightly slower initial release rates, attributed to denser matrix networks formed by Okra mucilage and Eudragit RS100, which effectively delayed diffusion. This controlled release profile is essential for gastroretentive systems, ensuring prolonged residence in the stomach and sustained therapeutic levels of Aceclofenac. The low standard deviations across replicates confirm excellent batch-to-batch reproducibility, validating the robustness of the preparation method. Overall, these in-vitro release profiles indicate that the optimised microspheres successfully meet the design objective of providing extended, controlled Aceclofenac release in acidic gastric environments.

Figure 5. In–vitro drug release for Mucoadhesive Microspheres formulations (AMF1- AMF6) in 0.1 N HCL (pH 1.2)

Formulations with higher polymer content, such as AMF5 (n?=?0.61), demonstrated stronger matrix integrity and slower, more controlled diffusion, confirming the role of Okra mucilage and Eudragit RS100 in modulating release rates. The high R² values for the Higuchi model also support significant diffusion-driven release from the swellable matrix system. Overall, these kinetic results validate the design strategy of achieving sustained, controlled drug release via a combination of diffusion and matrix erosion, confirming that the Korsmeyer– Peppas model best describes the release behavior of these gastroprotective mucoadhesive microspheres.