INTRODUCTION:

Mefenamic acid (MFA) belongs to the class of aminobenzoic acids^1,2. Specifically anthranilic acid with a 2,3-dimethylphenyl group replacing one of the hydrogens is attached to the nitrogen atom^3,4,5. It's categorized as a anti-inflammatory non-steroidal drug (NSAID) and is utilized for alleviating minor to moderate pain, encompassing headaches, osteoarthritis, dental pain, and rheumatoid arthritis^5,6,7. Functioning as an analgesic, xenobiotic, antirheumatic agent, non-steroidal anti-inflammatory drug, antipyretic, and prostaglandin-endoperoxide synthase inhibitor, MFA falls under class II of the Biopharmaceutical Classification System (BCS) due to its Low aqueous solubility and significant permeability^8,9,10.

With a half-life of approximately 2 hours, the challenge lies in prolonging its duration of action, which is addressed through the innovative approach of formulating MFA nanosponges. This novel drug delivery system not only extends the drug's half-life but also enhances its stability and mitigates potential side effects^11,12. Particularly crucial for conditions demanding prolonged pain relief and inflammation reduction, such as osteoarthritis, controlled-release drug delivery systems provide sustained therapeutic effects. Given the need for frequent administration of analgesics, oral administration is preferred due to its convenience and non-invasiveness¹³.

MATERIALS AND METHODS:

Materials used:

Working standard of pharmaceutical grade MFA was gift sample from Flamingo Pharmaceuticals Ltd. Acetone, Methanol and Dichloromethane (DCM) were obtained from S.D. Fine Chemicals, Mumbai, India. PVA (Polyvinyl alcohol), Ethyl Cellulose, Poloxamer 188 and Eudragit RS 100 were procured from Research-Lab fine chem, Mumbai, India.

Preparation of Nanosponges:

The emulsion solvent diffusion method was chosen for its convenience and efficiency in developing nanosponges. In this process, nanosponges were synthesized using varying concentrations of ethyl cellulose (EC) polymer and polyvinyl alcohol (PVA) to achieve customized release profiles and enhance drug loading. The procedure involved dissolving the drug in 10ml of acetone at 50°C and the polymer in 10ml of dichloromethane (DCM)^14,15. The resulting solutions were then slowly added to a specific amount of PVA in a 100ml aqueous external phase while stirring at 1000 rpm on a magnetic stirrer for 2hours. Nanosponges were subsequently collected via filtration, Oven-dried at 40°C for 3-4hours, followed by air drying for 12 hours.Initially, different polymers (ethyl cellulose and Eudragit RS100) and emulsifying agents (PVA and Poloxamer 188) were screened at a 1:1 ratio to determine their suitability for nanosponge development. The investigation concluded that ethyl cellulose and PVA were the most suitable polymer and emulsifying agent, respectively¹⁶.

Table 1: 3² Factorial experimental design levels and variables

Input variables	Level 1	Level 2	Level 3
Input variables	Low (-1)	Medium (0)	High (+1)
Drug:polymer ratio	1:0.75	1:1	1:1.25
Speed of Stirring	800 rpm	1000 rpm	1200 rpm
Response variables
Particle size (μm)	Minimum
PercentIn- vitro drug release	Maximum

Optimization of formulation by factorial design:

To optimize the formulation, a 3-level, 2-factor factorial design was used, involving 9 experimental runs plus 1 center point. The independent variables were the drug:polymer ratio (X1) and speed of stirring (X2), while the response variables were size of particle (Y1) and in-vitro drug release percent (Y2). Responses were analyzed usingExpert software- Statease Design (Trial version 13, Stat-Ease Inc., MN). Table 1 shows the levels of the variables and responses used for optimizing MFA-loaded nano sponges. Additionally, Table 2 provides the recommended batches from the Statease Design Expert software, including their respective responses, based on the specified levels and combinations of the independent variables¹⁷.

Evaluation of Nanosponges:

Physicochemical characteristics:

a) Bulk density and Tapped density¹⁸

b) Flow properties¹⁸The nanosponges were poured into a measuring cylinder to determine their volume for bulk density. For tapped density, the cylinder underwent approximately 100 taps, and both densities were then determined using the formulas.

Flow properties were evaluated by measuring the percentage compressibility, also referred to as Carr's index (C), and by determining the angle of repose. Funnel was used for determining angle of repose, wherein nanosponges were permitted to flow through a funnel onto a level surface, forming a pile on the paper.

In the evaluation of flow property of powder, when the angle of repose has the value 25-30°, the substance shows excellent flowability and performance, while when compressibility index has value ≤10, no aid is needed for enhancing the flowability of powder.

Percent Recovery:

The percent recovery of nanosponges was determined by substituting the weight of raw materials and the weight of the nanosponge in following formula.^19,20,21

Entrapment efficiency:

To calculate the entrapment efficiency, 10mg of nanosponges were mixed infive ml of methanol and sonicated for 10 minutes.The volume was then adjusted to ten ml. The resultant solution underwent filtration, dilution, and the amount of MFA was determinedwith spectrophotometer at 283nm.^22,23

FTIR study:

FTIR is used to find outcompatibilitybetweenMFA andpolymers used in the formulation.IR spectra of MFA loaded nanosponges were seen in between 4000-400 cm^-1.

Table 2: 3² Factorial experimental design with responses

Batch no.	Run	Factor 1 X1: Drug- Polymer ratio	Factor 2 X2: Speed of Stirring	Response 1 Y1: Particle size	Response 2 Y2: % drug release
OP 1	1	1:0.75	800	444	86.91
OP 2	2	1:0.75	1000	396	89
OP 3	3	1:0.75	1200	385	90.96
OP 4	4	1:1	800	592	88.40
OP 5	5	1:1	1000	538	95.92
OP 6	6	1:1	1200	541	93.20
OP 7	7	1:1.25	800	690	76.82
OP 8	8	1:1.25	1000	636	84.32
OP 9	9	1:1.25	1200	614	82.21

Differential Scanning Calorimetry (DSC) study: Samples were placed in standard aluminum pans with lids and exposed to air flow (60ml/min). The temperature was raised by 10°C per minute, and heat flow measurements were taken between 30°C and 300°C under a nitrogen atmosphere to maintain inert conditions. Thermograms were generated for MFA alone, the physical mixture of MFA with polymers, and MFA-loaded nanosponges.

Particle size analysis:

Particle size analysis was performed utilizing a NANOPHOX particle size analyzer, employing cross-correlation laser diffraction as the principle for measurement. The measurements were conducted at a Angle of scattering 90° with respect to the incident light, at a temperature of 25°C, and data were collected continuously for 300 seconds. The Polydispersity Index was utilized to assess the size distribution of the nanosponge population. The mean diameter, correlation function diagram, and distribution diagram of each batch were recorded.²³

Scanning Electron Microscopy (SEM):

It was performed to obtain nanosponges surface morphology.

The overnight dried powder was used for the SEM studies.

Powder X-ray diffraction (PXRD):

Powder X-ray diffraction (PXRD) analysis was carried on optimized nanosponges and pure drug samples using an analytical XRD Bruker D8 instrument equipped with a chromium line as the radiation source. Standard procedures involved operating at a 40 kVvoltage, 25mA a current, and rate of scanning 1° per minute over a range of 10 to 70°. The PXRD patterns of pure MFA exhibited distinct sharp peaks characteristic of a crystalline compound, which were then compared with the PXRD patterns of the nanosponges.

In-vitro drug release:

Percent in-vitro release studies wascarried out on MFA nanosponge capsules employing a USP type-II paddle apparatus (LABINDIA auto sampling) .Temperature was maintained at 37±0.5°C and a stirring speed of 100 rpm. Each capsule contained nanosponges equal to 250 mg of MFA and was submerged in 900ml of tris buffer (pH 9) dissolution medium. Samples were removed at specific intervals of 1, 2, 4, 6, 8, 10, and 12hours throughout a 12hour period, with fresh dissolution fluid added at each interval to sustain sink conditions. Samples were analysed using a Shimadzu 1801 UV-Spectrophotometer equipped with UV-spectrum software, with measurements taken 283 nm. Each experiment was carried out in triplicates, and the outcomes were presented as the mean value.

Drug release kinetics:

Different Kinetic models were utilized to determine the percent in-vitro releaseand explain the release kinetics. Plots were generated to assess the release kinetics, including: cumulative percentage of drug release against time (Zero-order kinetic model); natural logarithm of cumulative percentage of drug remaining against time (First-order kinetic model); cumulative percentage of drug release against the square root of time (Higuchi square root model); the cube root of the initial concentration minus the cube root of the remaining percentage against time (Hixson-Crowell plot); and natural logarithm of drug releasecumulative percentage against the time natural logarithm (Korsmeyer-Peppas model).

Evaluation of capsules:

Disintegration test:

The assembly was placed in900 ml of water in a beaker, which was kept at a temperature of 37±2°C.

Drug content of capsule:

Nanosponges, each containing MFA equal to ten mg of the drug were dissolved in 10ml of methanol.Further dilutions were prepared and UV-spectrophotometer wasused to determine drug concentration at 283nm .

Weight variation test:

The average weight was determined,test criteriasatisfies if not more than two individual capsule weights deviate from the average weight by more than 7.5%, and none exceed a deviation of 15%.

Final formulation:

The calculated amount of MFA-loaded nanosponges from the optimized batch (OP 5) was filled into size 0 hard gelatin capsules. This amount was determined based on the percentage of drug entrapment achieved by the nanosponges.

Stability Studies:

The capsules filled with the optimized batch of formulation (OP 5) underwent accelerated stability testing according to ICH guidelines, at temperatures of 30±2°C/65±5% RH and 40±2°C/75±5% RH. Capsules were retrieved after period of 1, 2, and 3 months from the initiation of placement into the chamber. The appearance, content of drug, and percentage of in-vitro drug release were assessed.^.

RESULTS AND DISCUSSION:

Optimization of formulation by factorial Design:

Factorial design used was 2-factor, 3-level for the optimization of MFA nanosponges. A total of 9 formulations were prepared and the responses of these batches are shown in Table 2.

Table 3: Final formula used in the formulating of MFA nanosponges (OP 5)

Materials and parameters	Quantity
MFA	250 mg
Ethyl Cellulose	250 mg
Polyvinyl alcohol	0.75% w/v
Dichloromethane : Acetone	20 ml
Stirring speed	1000 rpm

The graphical depiction of the in-vitro drug release from 9 optimized batches reveals a controlled release pattern, as depicted in Figure 1.

Fig. 1: Representation illustrating the in-vitro drug release from 9 optimized batches.

The surface response plot generated from Design Expert software for particle size indicated that an increase in particle size correlated with higher drug-to-polymer ratios, while a slight decrease in particle size was noted with increased stirring speed. This relationship stemmed from the thicker polymer coating resulting from higher polymer content, consequently enlarging the particle size. Conversely, higher stirring speeds led to a reduction in particle size, likely due to increased shear forces that fragmented molecules into smaller sizes. The observed particle size ranged from 350 to 690nm.

Regarding the drug releasepercentage, as drug-to-polymer ratio increase drug release decreases, particularly evident in batches with a 1:1.25 drug-to-polymer ratio, attributed to the polymer's retarding effect. However, batches with a 1:0.75 drug:polymer ratio exhibited drug release rates comparable to those with a 1:1 ratio, although these batches couldn't sustain release for the entire 12hour period. Stirring speed showed no significant impact on drug release percentage, which fell within the range of 75% to 96% across all batches, regardless of variations in stirring speeds and drug-to-polymer ratios used in the optimization studies.

Fig. 2: Overlay plot

In figure 2, the overlay plot revealed that the batches formulated from 1:1 drug polymer ratio and with 1000 and 1200rpm stirring speed would produce desired results. Batch OP 5 showed % drug release of 95.92% and particle size of 538nm with less turbulence effect than batch OP 6. Hence, the batch OP 5 with drug: polymer ratio as 1:1 and stirring speed as 1000rpm was finalized.

Final formulation:

The final formula used in formulating MFA nanosponges is shown in Table 3. Further, batches were prepared using this formula and evaluation of nanosponges were carried out. Later, they were manually filled into capsules.

Table 3: Final formula used in the formulating of MFA nanosponges (OP 5)

Materials and parameters	Quantity
MFA	250 mg
Ethyl Cellulose	250 mg
Polyvinyl alcohol	0.75% w/v
Dichloromethane : Acetone	20 ml
Stirring speed	1000 rpm

Evaluation of Nanosponges:

Physicochemical characteristics:

The results of physicochemical characteristics of nanosponges of optimized batch OP 5 were found to be in acceptable range. The bulk density, tapped density, angle of repose and % compressibility of MFA-loaded nanosponges were found to be as 0.7640g/ml, 0.8212 g/ml, 27.34° and 8.68% respectively. Based on the outcomes, the substance showed good flowability and performance. Hence, no aid was needed for enhancing the flowability of powder.

%Recovery yield and %Entrapment efficiency of optimized batch:

%yield recovery and encapsulation efficiency % of optimised batch (OP 5) was found to be 91.25% and 89.92% respectively.

FTIR study:

The drug-polymer interactions were investigated using IR.spectroscopy. It was observed that MFA displayed notable peaks at 1645, 1575, and 1254 cm^-1, representing C=O, C=C, and C-O stretching, respectively. Moreover, a broad peak spanning approximately 2567 to 3308 cm^-1 was assigned to H-bonded O—H and N—H stretching vibrations.

Differential Scanning Calorimetry (DSC) study^23,24:

The pure MFA exhibited a distinct melting endothermic peak at 235.9°C in the DSC curves. In contrast, formulation OP 5 displayed a broad, shallow endothermic peak at 244°C, aligning with the melting point range of ethyl cellulose (240-250°C). Notably, the DSC thermogram of the nanosponge formulation did not reveal any sharp peaks. This observation indicates that MFA was not in a crystalline state but had transitioned into an amorphous state.

Particle size analysis^24,25:

The particle size distribution ranged from 350 to 690nm, with the optimized batch measuring at 538nm. Figure 3 illustrates the distribution diagram of the optimized nanosponge formulation.

Fig. 3: Particle sizedistribution diagram of optimized nanosponge

Scanning Electron Microscopy (SEM):

Figure 4 displays SEM images showing the MFA nanosponges formed in a spherical shape.The porous and spherical characteristics of the particles are advantageous for cellular uptake, as spherical particles are generally more easily absorbed compared to rregularly shaped particles.^26,27

Fig. 4: Surface Electron Microscopy image of MFA nanosponge

Powder X-ray diffraction (PXRD):

Characteristic reflections of MFA were observed at diffraction angles 2θ of 15°, 29°, 40°, and 50°. However, these distinct reflections of MFA were not present in the patterns of the drug-loaded nanosponges. Instead, they were replaced by the typical hump associated with amorphous materials. This suggests that the active ingredient exists in an amorphous state within the nanosponges.^28,29,30

In-vitro drug release of MFA nanosponges^31,32:

The % drug release of MFA-loaded nanosponge formulation (OP 5) was found to be 95.92% and it has successfully controlled the release for about 12 hrs. The in-vitro drug release profile of MFA nanosponge (OP 5) batchb is revealed in Figure 1.

Kinetic models:

The drug releasepercentage data obtained from the optimized batch (OP 5) was analyzed using various pharmacokinetic models, and the coefficient of regression (r2) values are presented in Table 4. Various kinetic models were used to assess the in-vitro drug release profile of OP 5, aiming to explain the drug releasemechanism. Among these models, the zero-order and Korsmeyer-Peppas models demonstrated the best fit, indicated by their highest coefficient of regression values. The superior fit of the zero-order model suggests drug release at a constant rate, ensuring sustained therapeutic drug concentrations over an extended period. Similarly, the Korsmeyer-Peppas model, tailored to describe release of drug from polymeric systems, also displayed a high coefficient of regression. This model accounts for various release mechanisms simultaneously, including water diffusion into the matrix, matrix swelling, and matrix dissolution.^33-36

Table 4: R² values of different kinetic models

Kinetic models	R²
	Zero order	First order	Higuchi	Hixson- Crowell	Koresmeyer- Peppas
	0.991	0.8561	0.9809	0.9669	0.991

Evaluation of capsules:

Disintegration test:

The capsules pass the test if all of them have disintegrated. All the 6 capsules were disintegrated within 10 minutes. Hence, it passes the disintegration test.

Drug Content of capsule:

Optimized capsules drug content was 89.92%^37,38.

Weight variation test:

The average weight of content of 20 capsules of was found to be 445.12mg. As per IP limit, It was found that none of the capsule content deviates outside the range. Therefore, capsules comply weight variation test^39,40.

Stability testing:

The stability testing was performed according to ICH guidelines^41-45 for a 3 months period at the following storage conditions. Table 5 displays the acquired results.

The percentage of drug release of MFA from batch OP 5, which was set aside for stability studies, did not exhibit any notable alteration.It can be concluded that the samples were stable over three months of study.The results were found to be convincing that the nanosponges have the potential to function as an efficient oral delivery system for poorly water-soluble drugs.^46-50

CONCLUSION:

Using the emulsion solvent diffusion method, an oral formulation of MFA-loaded nanosponge capsules based on ethyl cellulose was successfully developed. The primary objective of this formulation was to reduce dosing frequency by achieving controlled drug release and minimizing the adverse effects associated with conventional oral MFA formulations. The nanosponges produced were evaluated for parameters including % recovery yield, % entrapment efficiency, particle size, and in-vitro drug release percentage. Optimization was conducted using a 3² factorial design to streamline the experimental process.

The optimized formulation (OP 5) demonstrated an in-vitro % drug release of 95.92% and an mean particle size of 538.17 nm. Scanning electron microscopy (SEM) analysis indicated that the nanosponges formed were spherical, non-aggregated, fine, free-flowing, and exhibited a porous structure. Powder X-ray diffraction (P-XRD) studies revealed the absence of crystalline peaks from the pure drug, suggesting homogeneous dispersion of MFA within the polymer matrix.

Stability testing of the optimized batch conducted over a three-month period showed no significant alterations in physical characteristics, drug content, or drug release profile. Thus, it can be inferred that the developed formulation remains stable and holds potential as a controlled drug delivery system capable of sustained release for up to 12 hours.

Current and Future developments:

Nanosponge formulations are increasingly prominent in drug delivery systems, particularly in oral and topical applications, due to their ability to provide controlled drug release to targeted areas. Nanosponges offer a versatile medium for drug transport, capable of encapsulating a diverse range of medications including both lipophilic and hydrophilic drugs. Additionally, they find applications in solubility enhancement, gas entrapment (such as oxygen), cosmetics, diagnostics, and adsorbents for poisoning. The future holds promising prospects for the utilization of nanosponges across various fields. As research in this domain progresses, the scope of their applications is expected to expand accordingly.

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Received on 09.05.2024 Revised on 20.09.2024

Accepted on 29.11.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):1968-1974.

DOI: 10.52711/0974-360X.2025.00281

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sr. No.	Evaluation parameters	Storage Conditions	1 month	2 month	3 month
1	% Drug content	30°C/65% RH	87.43 %	88.00 %	85.35 %
1	% Drug content	40°C/75% RH	86.90 %	86.24 %	87.33 %
2	*In- vitro* % drug release	30°C/65% RH	95.59 %	96.63 %	96.05 %
2	*In- vitro* % drug release	40°C/75% RH	96 %	94.08 %	95.92 %