Design & Characterization of Mefenamic Acid Hydrogel Based on Microsphere Drug Delivery System for the Enhancement of Analgesic & Anti-inflammatory Activity

Inflammation is a biological defense response triggered by harmful stimuli such as pathogens, damaged cells, and irritants. NSAIDs act by inhibiting cyclo-oxygenase (COX) enzymes and reducing prostaglandin synthesis. [1]

Mefenamic acid, an anthranilic acid derivative, is effective in treating mild to moderate pain including dysmenorrhea, arthritis, and musculoskeletal disorders. However, oral administration leads to gastrointestinal irritation, bleeding, and frequent dosing due to its short half-life.

To overcome these limitations, controlled drug delivery systems such as microspheres and hydrogels have been explored. Microspheres provide sustained drug release and improved stability, while hydrogels allow transdermal delivery and bypass first-pass metabolism.[2]

Novel drug delivery systems such as microspheres and hydrogels have gained significant attention in recent years for controlled and targeted drug release. Microspheres are small spherical particles composed of natural or synthetic polymers capable of encapsulating therapeutic agents within a polymeric matrix. They offer several advantages including sustained drug release, improved stability, enhanced bioavailability, and reduced dosing frequency. By controlling particle size, polymer concentration, and preparation parameters, microspheres can modulate drug release kinetics effectively.[3,4]

Hydrogels, on the other hand, are three-dimensional cross-linked polymeric networks capable of absorbing substantial amounts of water without dissolving. Due to their high water content, biocompatibility, and similarity to biological tissues, hydrogels are widely used in topical and transdermal drug delivery systems. Transdermal administration provides several advantages over oral delivery, including avoidance of first-pass metabolism, reduction of gastrointestinal side effects, improved patient compliance, and maintenance of steady plasma drug levels.[5,6]

Incorporating drug-loaded microspheres into a hydrogel matrix combines the benefits of both systems. The microspheres act as reservoirs providing sustained drug release, while the hydrogel serves as a suitable carrier for topical application and enhances transdermal penetration. This dual-controlled system can potentially reduce systemic exposure, minimize dosing frequency, and improve therapeutic outcomes.

The formulation is expected to improve solubility, provide prolonged drug release, enhance skin permeation, and reduce gastrointestinal side effects associated with conventional oral therapy. The present study focuses on developing microsphere-loaded hydrogel of mefenamic acid for improved therapeutic efficacy.[7]

Fig.1.1 Mechanism action of NSAIDS

2. MATERIALS AND METHODS

2.1 Materials

2.2 Methods:

2.2.1. Microsphere formulation by solvent evaporation method.

Polymer and Drug Selection.

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Preparation of the Polymer Solution.

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Emulsified in aqueous phase under controlled stirring

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Solvent Evaporation

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Washing and collection

Fig.2.2.1 Formulation of microsphere

Fig.2.2.2 Formulation of hydrogel

2.3. Preformulation Studies: Pre-formulation testing was an investigation of physical and chemical properties of a drug substance alone. It is the first step in rational development of dosage form.

• • • Solubility study: An extra amount of Mefenamic acid was added to 10 mL of different solvents (distilled water, ethanol, methanol, acetone, chloroform, phosphate buffer pH 7.4), agitated at 25°C for 24 hours, then filtered and measured spectrophotometrically at 284 nm.
    • Melting point determination: Melting point of drug was determined by using Melting point apparatus. The melting point of drug was found to be 230.5°C, which validates its purity. It is thermally stable under the normal temperatures used to make microspheres and hydrogels.
    • Organoleptic evaluation: Organoleptic evaluation involves the use of human senses (sight, smell, taste, and touch) to assess the basic physical characteristics of a drug substance. These properties are important in the preliminary identification and quality assessment of pharmaceutical raw materials.
    • Partition coefficient determination: The shaking equal volumes of the aqueous phase and the organic phase (n-octanol) in a separating funnel allowed researchers to calculate the drug's partition coefficient. 50ml of a drug solution containing 1 mg/ml was placed in a separating funnel, shaken with an equal amount of n-octanol for10minutes, and then left to stand for 24 hours while being shaken intermittently. Next, U V Spectra were used to determine the concentration. Using an appropriate blank, the concentration of mefenamic acid in the aqueous phase was measured using UV spectrophotometry at 284 nm.

Fig.2.2.7:Graph of partition coefficient of mefenamic acid.

2.4 Optimization Parameters: Optimizing parameters for microspheres depends on the specific application and materials used, such as drug delivery, diagnostic applications, or industrial use. However, there are Several general optimization parameters that are commonly considered. Three formulations were created by using different organic solvents, including diethyl ether, ethyl acetate, and acetone.

1. Drug: Polymer ratio.

2. Stirring  speed (500, 700, 900 rpm)

3. Organic: Aqueous phase ratio(1:5,1:10,1:15)

1. 2. of Microspheres
      1. Particle size analysis: The diameter of 100 prepared microspheres was measured to establish their particle size. The sizes ranged from 5-8 µm. The average particle size was 6.8 µm, with a standard deviation of 0.5 µm, showing a narrow and homogeneous distribution. Microscopic examination indicated that the microspheres were spherical in shape with a smooth surface and exhibited little aggregation, validating the formulation method's potential for uniform microsphere formation.[8]

Fig.2.5.1:Particle size distribution of microsphere.

Fig.2.5.2: (a) Images of microsphere

Fig.2.5.2:(a)Mean particle size of microsphere by optical microscope and SEM.

(b) ransmission Electron Microscopy (TEM) study: TEM images taken at Korsmeyer-Peppas model: The Korsmeyer-Peppas model showed the strongest correlation (R² = 0.999).[9]

Table: Particle size, shape & surface of drug loaded microsphere by TEM study.

Fig.2.5.2:(b)TEM image of mefenamic acid microspheres.

1. 1. 3. Drug Content and Entrapment Efficiency (EE %):

400 mg of known-quantity microspheres was dissolved in ethanol. Use UV-visible spectrophotometry to filtered and analysed the drug concentration.

Formula: Drug content percentage is equal to (drug amount in sample/microsphere weight)x100.

(Actual Drug in Sample/Weight of Microspheres)×100. Drug Content (%) = (85 mg / 400 mg) × 100 = 21.25%.

Entrapment Efficiency was calculated by using the formula:

Entrapment Efficiency(%)= (Actual Drug Content / Theoretical Drug Content) × 100.

EE%=(85mg/100mg)×100=85%.

Depending on the solvent system and drug-polymer ratio, entrapment efficiency normally falls between 70% and 95%. Sample Microsphere mass analysed (mg) Theoretical drug in sample (mg) Actual drug measured (mg)

Example 400mg 400.00100.00 Drug content (%) Entrapment.

Fig.2.5.3: Drug content and entrapment efficiency value

1. 1. 4. Production yield: The microsphere manufacturing yield was calculated to determine the solvent evaporation method's efficiency. The yield was calculated by weighing the dried microspheres recovered after formulation and comparing them to the total weight of the medication and polymer utilized. The yield in this investigation ranged between 85 and 92%, depending on polymer content and stirring speed.

Formula: Yield % = (Weight of dried microspherere covered/ Total weight of drug and polymer) × 100

Fig.2.5.4: Percentage yield(%) of microsphere

1. 1. 5. Fourier Transform Infrared (FTIR) Spectroscopy: The IR beam going through the ATR cell reflected several times through the sample, yielding IR spectra primarily of surface material. The ATR-FTIR gives information about the surface composition of microspheres based on manufacturing techniques and conditions.

2.6 : Optimization parameter of hydrogel:

1. Polymer Ratio Effect (Drug: Polymer)

2. Effect of Polymer Concentration (Ethyl Cellulose in Microspheres/Carbopol in Hydrogel).

3. Effect of Stirring Speed.

2.7: Characterisation of hydrogel:

2.7.1 Clarity: The clarity of the produced hydrogel compositions was assessed visually. A little amount of each mixture was put in a clear glass container and seen against black and white backgrounds under standard laboratory lighting conditions. The formulations were tested for turbidity, particulate matter, and air bubbles. In addition to clarity, the color, consistency, and smoothness of the hydrogels were evaluated visually and tactilely to guarantee uniformity and aesthetic appeal.

2.7.2 Viscosity: The viscosity of the produced hydrogel compositions was measured with a Brookfield viscometer. Measurements were performed on the spindle LV-4 at a rotating speed of 6 RPM and a temperature of 28 °C. The % torque was recorded to verify that the measurements remained within the instrument's authorized working range. To corroborate the hydrogel's rheological behavior, viscosity measurements were taken with spindle LV-3 at a higher speed of 30 RPM. Apparent viscosity and torque values were recorded for both spindle-speed combinations.

Fig.2.7.2:Graph of torque vs spindle speed of viscosity of hydrogel

2.7.3 PH scale: After carefully weighing 1g of gel, it was combined with 100 mL of pure water. A digital pH meter was used to find the dispersion's pH.

2.7.4 Spreadability: Two rectangular glass plates with the necessary dimensions were taken. A single glass plate held 1g of the material. The second dish was piled on top of the first to sandwich the sample between the two plates. A 100gm weight was placed on top of the upper plate to provide a consistent thin layer of sample between the plates. The weight was removed, and extra gel sample was scraped off the edges. The top plate was then dragged by a thread with a 50-gram weight connected to it. The time taken for the top plate to travel distance move and separate from the bottom plate was measured. Shorter durations suggest more spreadability.

Formulas:                  S= M× L/T

Where, S=spreadability, M=weight(g),L=length moved by the slide(cm),  T =time (s).

Area of Spread: The area of spread measures the gel’s ability to cover a surface under standard conditions. The hydrogel showed a mean spread area of 23.98cm², confirming uniform spreading and suitable viscosity for topical application.

Area=πr2

2.7.5 Extraudability: Extrudability controls how rapidly the hydrogel may be removed from its container (a collapsible aluminum tube) and applied to the skin's surface. It gives information regarding the gel's mechanical strength, viscosity, and spreadability, all of which impact patient compliance and dosage uniformity. A steady weight of 500 g was applied to the crimped end of the tube, which was positioned between two glass slides. The amount of gel that the nozzle extruded in 10 seconds was measured. Extrudability was calculated using the following formulae as extrusion efficiency (%):

Extrusion Efficiency(%)=Wt/We×100

Fig.2.7.5: Mean Values of Spreadability, Area of Spread & Extrudability of Optimized Gel

2.7.6 Drug content uniformity and release: The drug content of uniformity was examined to ensure that medicines were uniformly distributed throughout the gel matrix. A gel contain 100 mg of mefenamic acid was dissolved in phosphate buffer (pH 5.5) and examined using spectroscopy at λmax = 285 nm. The drug concentration in all formulations ranged between 96 to 98%, showed that the microspheres were evenly dispersed in the hydrogel base.

Fig.2.7.6: Drug Content(%) of Hydrogel Formulations

2.7.7 Swelling Index: To understand the diffusion and hydration properties of hydrogels, their swelling capacity was assessed. Weighing the formulations before (W1) and after immersion (W?) in phosphate buffer (pH 5.6) for eight hours allowed us to calculate the swelling index. The swelling in dextrose in direct proportion to the carbopol concentration. For long-term release systems, controlled water absorption and drug diffusion are made possible by the moderate swelling of HG2.[11, 12]

Fig.2.7.7: Comparative Swelling Index of Hydrogel Formulation

2.7.8 In- vitro drug release studies and kinetic modeling: Phosphate buffer saline (PBS, pH5.6) was used as the diffusion medium in the in vitro drug release study to assess the mefenamic acid release properties from the optimized microsphere-loaded hydrogel formulation (codedHG2).

The pH of the skin's surface normally varies between 5.0 and 6.0. PBS (pH 5.6) closely resembles the physiological of topical administration, making release data more relevant for dermal delivery evaluation. The experiment employed a Franz diffusion cell with an effective diffusion area of 3.14 cm² and a 25 mL receptor compartment capacity. A dialysis membrane (MWCO 12,000-14,000 Da, HiMedia, India) was used to divide the donor and receptor compartments. The dialysis membrane was soaked in PBS (pH5.6)for 12 hours prior to use in order to guarantee adequate hydration and remove glycerin and preservatives. The receptor compartment was filled with phosphate buffer saline (pH5.6), kept at 37 ± 0.5 °C, and agitated at 100 rpm with a magnetic bead in order to attain homogeneity. The donor compartment's membrane surface was evenly coated with a hydrogel containing 100 mg of mefenamic acid. After the materials were filtered and appropriately diluted, 2 mL of the sample was removed from the receptor compartment at predetermined intervals (0.5, 1, 2, 4, 6, 8, 10, and 12 hours) and promptly refilled with fresh PBS (pH 5.6) to maintain sink conditions. [12]

Fig.2.7.8: Cumulative% Drug Release of Mefenamic Acid Hydrogel (HG2) in PBS (pH5.6)

2.8.  Stability Studies:

Conducted under accelerated conditions for 3 months: The findings revealed that there were no significant alterations in the analyzed parameters over the research period. These findings validated the microspheres' high stability and indicated adequate polymer-drug compatibility under both storage settings. During the research period, no significant changes were seen in any of the measures tested. The microspheres were consistently distributed inside the hydrogel matrix, with no evidence of aggregation, indicating structural integrity and constant drug release properties. These findings validatedthehydrogelformulation'sacceptablephysicalandchemicalstabilityunderthe investigated storage conditions. [13,14]

Table :Effect of Storage Conditions on Physical Appearance, Drug Content and Drug Release of Microspheres

Table: Effect of Stability Conditions on pH and Viscosity of Hydrogel

2.9. Hemolysis Study:

Performed to evaluate blood compatibility of formulation. The absorbance values obtained were 0.02forthe negative control, 0.98 for the positive control, and 0.06forthe HG2 formulation. Based on these findings, the proportion of hemolysis for HG2 was determined as 4.1%. According to ISO hemocompatibility rules, materials with hemolysis levels of less than 5% are deemed blood compatible. The low hemolysis result for HG2 demonstrated little disruption to red blood cell membranes, confirming the formulation's excellent blood compatibility. This positive behavior was ascribed to the polymer's biocompatibility and the regulated release of mefenamic acid from the hydrogel matrix containing microspheres. Overall, the results revealed that the HG2 formulation was safe and acceptable for topical medication administration, particularly in settings where inadvertent blood contact or damaged skin integrity may occur. [15]

Fig.2.9The graph of hemocompatibility

2.10 Ex-Vivo Skin Permeation Study:

Carried out using animal skin membrane to determine cumulative drug permeation. The total proportion of drug permeated rose gradually over time. Drug permeation rates of 10.6%,19.8%,33.5%,47.2%,59.4%,67.8%,and72.6%were measuredat1,2,4,6,8,10, and 12 hours, respectively. These results suggested that mefenamic acid permeated the skin in a continuous and regulated manner. Overall, the study showed that the medication penetrated the skin effectively via the hydrogel formulation, Indicating that it is suitable for topical distribution with long-term therapeutic effects.[16]

Fig.2.10 Ex-vivo skin penetration

3. RESULT:

Preformulation studies revealed that the drug possesses poor water solubility, which justified the need for developing a controlled drug delivery system. During optimization, a stirring speed of 700 rpm and an organic to aqueous phase ratio of 1:10 were found to provide the highest percentage yield of microspheres. Scanning Electron Microscopy (SEM) analysis demonstrated that the prepared microspheres were spherical in shape with a smooth surface, indicating uniform formulation. The entrapment efficiency was found to be satisfactory, confirming effective incorporation of the drug within the polymer matrix. FTIR studies confirmed the compatibility between the drug and polymer,with no significant interaction observed.

The developed microsphere-loaded hydrogel exhibited acceptable pH within the skin- compatible range, along with suitable viscosity and good spreadability, making it appropriate for topical application. In-vitro drug release studies showed sustained drug release up to 12 hours, indicating prolonged therapeutic action. The release kinetics followed the Higuchi model, suggesting a diffusion-controlled mechanism of drug release from the microspheres. Stability studies demonstrated that the formulation remained stable without significant changes in physical and chemical parameters. Hemolysis studies confirmed good blood compatibility, ensuring formulation safety. Furthermore, ex-vivo permeation studies indicated enhanced transdermal drug penetration. Overall, the microsphere-loaded hydrogel effectively minimized the initial burst release and maintained prolonged drug delivery, thereby improving therapeutic performance.

Table: Summary of Results and Discussion