INTRODUCTION:

Mefenamic acid comes under the non-steroidal anti-inflammatory category of drugs and is used in mild to moderate pain. The aqueous solubility of mefenamic acid is 0.04 mg/ml. Mefenamic acid is belong to class 2 of biopharmaceutical classification. It is highly permeable through biological membrane but have poor water solubility. Nanosponges are a novel drug delivery mechanism that has arisen in response to rapid breakthroughs in nanotechnology and the requirement for precise and targeted drug delivery¹. Nanosponges can be applied topically, taken orally, or injected intravenously².

The medicine can be loaded onto the nanosponge using a variety of techniques. The medication molecules are encapsulated within the core of nanosponges³. As a result, these are enclosing nanoparticles. Nanosponge is a novel and emerging drug delivery technique that offers controlled and sustained drug delivery for topical application^4,5. The nanosponge delivery system is a one-of-a-kind device for the controlled release of topical medicines with long-term drug release and skin retention. Nanosponges were created using the emulsion solvent diffusion process and then put into a gel as a local depot for long-term drug release^6,7.

Material and Method:

Material:

Virupaksha Organics Ltd., Telangana, India provided me with a mefenamic acid API as a gift sample. Ethyl cellulose was procured from SD. Fine Chem ltd., Carbopol 934 and PEG 400 was procured from Quali Chem, New Delhi.

Methods:

Preparation of nanosponge:

The emulsion solvent evaporation process was used to make nanosponges of Mefenamic acid. Ethyl cellulose (EC) polymer was used for the development of nanosponge formulations. The internal phase, which contained Mefenamic acid and ethyl cellulose in 20 ml of dichloromethane (DCM) was slowly added to a 0.3 % w/v of poly vinyl alcohol in 100ml of aqueous external phase with stirring speed fixed at 1000rpm using a magnetic stirrer for 3 hours. Filtration was used to collect the nanosponges, which were then air-dried for 24 hours at 40°C before being packed into vials. The composition of nanosponges was described in table 1.

Table 1: Composition of Nano sponge formulations

Formulation Code*	Drug: Polymer Ratio
N1	1 : 2
N2	1 : 3
N3	1 : 4
N4	1 : 5
N5	1 : 6
N6	1 : 7

*N stand for Nanosponge formulation

Preparation of nanosponge loaded gel:

Mefenamic acid nano sponge loaded gel was prepared by adding 1 gm of carbopol 934 to freshly prepared nano sponge suspension and neutralizing the pH by using the triethanolamine with continuous stirring with glass rod. Composition of Nano sponge loaded topical gel formulations was described in table 2.

Table 2: Composition of Nano sponge loaded topical gel formulations

Formulation Code*	Ingredients
Formulation Code*	Nanosponges Equivalent to	Carbopol 934	Distilled water (ml)	Triethanola -mine (ml)	Propylene glycol (ml)
G1	100 mg drug	100 mg	10	1.0	0.4
G2	100 mg drug	100 mg	10	1.0	0.4
G3	100 mg drug	100 mg	10	1.0	0.4
G4	100 mg drug	100 mg	10	1.0	0.4
G5	100 mg drug	100 mg	10	1.0	0.4
G6	100 mg drug	100 mg	10	1.0	0.4

*G stand for gel formulation contains nanosponge

Characterization and Evaluation of Nano sponges:

Particle size and Polydispersity:

Particle size (z- average diameter) and poly dispersity index of Mefenamic acid-loaded Nanosponge dispersion were estimated by using a Malvern Zeta sizer at 25°C.

Morphology of Nanosponges by Scanning Electron Microscopy:

The surface morphology of Nanosponges can be examined using scanning electron microscopy. Scanning electron microscopy was used to examine nanosponges to evaluate particle size, shape, and surface morphology. Images were obtained at various magnifications using a scanning electron microscope at a 10 kV acceleration voltage after the sample was stuck to the SEM sample holder with double-sided sticking tape⁸.

Drug entrapment efficiency:

Nanosponges' drug entrapment effectiveness was measured spectrophotometrically (max = 284nm). A sample of Mefenamic acid Nanosponge was combined in methanol and diluted to 100ml with phosphate buffer (pH 6.8) before being maintained overnight. The drug content in Nanosponges was assessed and expressed as actual drug content. The following formula is used to compute the percentage entrapment efficiency (percent EE)⁹.:

Percentage Actual drug content in Nanosponges

entrapment = ------------------------------------------- X 100

efficiency Theoretical drug content

Evaluation Parameters Of Nanosponge Loaded Gel:

Determination of pH:

pH of formulated topical gel formulations were evaluated with the help of digital pH meter. 0.5gm of prepared gel was dispersed in 50ml of distilled water. It was kept aside at 25⁰C for 3 hours. After that pH was checked by pH meter by dipping the electrode in to the solution^10-11.

Drug content:

Mefenemic acid content in nanosponge gel was measured by dissolving 100mg of gel in 10ml methanol. Whatmann filter paper no. 42 was used to filter the solution. In a UV - 1800 spectrophotometer, absorbance was measured at 284 nm after dilution.

Homogeneity:

It was checked by visual inspection for the appearance of gel and presence of any aggregates¹².

Extrudability:

Pfizer hardness tester was used to study the extrudability. 20gm of gel was weighed and filled in 20 gm capacity aluminium. tube. The. Plunger. was set up in such a way that the tube was securely held in place. For 30 seconds, a pressure of 1kg/cm2 was applied. The amount of gel coming out of tube was checked and weighed. The operation was repeated three times at equal distances along the tube.^13-14.

Spreadability:

The spreadability of the prepared gel was assessed by measuring the spreading diameter of 0.5g of gel that is placed in between 20 x 20cm glass plates after 1 minute. The upper plate's mass was standardised at 500g¹³.

S = (m) X (l) X (t)

Where, Spreadability is denoted by ‘S’, weight or mass applied to the glass slide¹⁴ is denoted by ‘m’, length of the glass slide is denoted by ‘l’and time in seconds is denoted by ‘t’^15-18.

In-vitro diffusion studies (dialysis membrane):

The Franze diffusion cell was selected for in-vitro diffusion studies by using a dialysis membrane^19-21. The diffusion cell was fabricated by a local vendor. Receptor compartment volume was made to hold 60ml of diffusion media. The dialysis membrane was fixed between the donor and receptor chamber of the Franz diffusion cell. 0.5gm of gel was spread homogeneously over the dialysis membrane. Dialysis membrane was tied over receptor chamber which was filled with phosphate buffer pH 7.4. The temperature was kept at 37⁰C^22-23. The media was stirred with the magnetic bead at 150 RPM. 2ml sample was withdrawn at a periodic time interval and sink condition was maintained by replacing the same with fresh media^24-26. The sample was filtered²⁷ and absorbance was taken by UV double beam spectrophotometer at 284nm.

Result and Discussion:

Particle size and Polydispersity:

The average size of prepared nano sponge formulations was found in the range of 250nm to 894nm. Particle size of different formulations was tabulated in table 3. The polydispersity index (PI) was found in the range of 0.206 to 0.934. The polydispersity index is an estimation of heterogeneity of size of particles in a mixture. As per International Standards Organization (ISO) the polydispersity index value less than 0.05 reveals monodisperse samples, while values greater than 0.7 are common to a broad size range or polydisperse samples.

Table 3: Particle size distribution study of Nano sponge formulations

Formulation. Code	Particle. Size. (nm)	Polydispersity. Index.
N1	893.7	0.934
N2	1170	0.996
N3	119.8	0.265
N4	301.9	0.221
N5	370.9	0.206
N6	250.2	0.221

Figure 1: Particle size distribution study of nanosponges (N3 formulation)

Morphology of Nano sponges by Scanning Electron Microscopy:

The surface morphology of developed nano sponge formulations was studied by using scanning electron microscopy (SEM). The representative scanning electron microscopy photographs of the nano sponge formulation were shown in figure 2. Scanning electron microscopy photographs of developed formulations clearly reveals spongy and porous nature of particles along with spherical shape.

Figure 2: SEM images of prepared nanosponges

Drug entrapment efficiency

Drug entrapment.efficiency was measured for all developed formulations. The entrapment efficiency was found maximum in the N3 formulation having a 1:4 Drug: Polymer ratio. Random % drug entrapment was found when polymer concentration was increased and no correlation can be established between polymer concentration and drug entrapment.

Table 4: Drug entrapment efficiency of different Nano sponge formulations

Formulation. Code*	% Drug. entrapment
N1	40.68±0.89 %
N2	42.20±0.95 %
N3	72.80±0.75 %
N4	64.50±0.65 %
N5	55.40±0.84 %
N6	54.90±0.98 %

*N stand for nanosponge formulations.

Estimated parameter of nanosponge based topical gels:

The nanosponge-loaded gel was evaluated for various parameters like pH, spreadability, homogeneity, % drug content and extrudability. The results were reported in Table 5. Prepared gel formulations revealed pH range between 6.4 to 7.0. All gel formulations showed good homogeneity. % Assay of gel formulations were found in the range of 87.5 to 92.5%. Extrudability was found in an acceptable range. Spreadability was found in the range of 18.32 to 24.12 gm-cm/sec.

Table 5: Characterization of Nano sponge loaded topical gel formulations

Formula-tion code	pH data	Homogeneity	Spreadibility (gm-cm/sec)	% drug content	Extrudability
G1	6.9	Good	24.04±0.52	89.6±1.2	*
G2	6.8	Good	18.32±0.34	88.04±0.85	**
G3	6.4	Good	22.08±0.85	92.25±1.1	*
G4	6.8	Good	19.32±0.64	91.15±0.95	*
G5	6.7	Good	20.42±0.45	92.5±1.5	*
G6	7.0	Good	24.12±0.50	87.5±1.3	*

Symbol: (*) Acceptable, (**) Good, (***) Excellent.

In-vitro diffusion studies:

In-vitro diffusion study was performed by using Franz diffusion cell. In-vitro diffusion data was graphically represented in figure 3. In-vitro diffusion data revealed that % drug release was decreased as concentration of polymer increased in initial hours.

Figure 3: % Drug Release of different gel formulations loaded with Mefenamic acid nanosponge

Conclusion:

The nanosponge of poorly water-soluble drug Mefenamic acid was successfully prepared with ethyl cellulose as polymer. Different ratios of drug and polymer were tried to achieve maximum drug loading and It was found that formulation N3 with a drug-to-polymer ratio of 1:4 has the highest drug loading. Further nanosponge were characterized for different parameters and results revealed acceptable characteristics of N3 formulation. These prepared nanosponges were loaded in topical Carbopol gel and evaluated for in vitro diffusion release. The data were fitted to different kinetic models which reveal typical zero order in vitro drug diffusion with prolonged drug release and therefore it can be effectively used in the treatment of pain. Because the nanocarrier may permeate the drug deeper into the skin layer than other topical semisolid preparations, the nanosponge-based topical gel formulation is suited for effective pain control and anti-inflammatory action.

Conflict of Interest:

The authors have no conflicts of interest regarding this investigation.

Acknoledgement:

The authors would like to thank Prof. (Dr.) Ranjit Singh, Vice Chancellor, Shobhit University for their kind support and all providing other lab facilities.

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Received on 24.01.2022 Modified on 11.03.2022

Research J. Pharm. and Tech 2023; 16(4):1960-1964.

DOI: 10.52711/0974-360X.2023.00321