Formulation And In-Vitro Evaluation of Celecoxib Nanosponges Loaded Topical Gels

Table No 1: Formulation table of Celecoxib loaded nanosponges

Nanosponges using different proportions of β-cyclodextrin, Ethyl Cellulose (mg), Eudragit RS100 (mg) as rate retarding polymer and co-polymers like polyvinyl alcohol were prepared by solvent evaporation method. Disperse phase consisting of Celecoxib (100mg) was dissolved in 20ml solvent  (Dimethylformamide) and was  slowly  added  to  a  definite  amount  of PVA in 100ml of aqueous continuous phase, prepared by using magnetic stirrer. The reaction mixture was stirred at 1000 rpm for three hours on a magnetic stirrer for 2hours. The nanosponges formed were collected by filtration through whatman filter paper and dried in oven at 50^oC for 2 hours. The dried nanosponges were stored in vaccum desicator to ensure the removal of residual solvent.

Evaluation parameters of Nanosponges¹⁷

Drug Content

An accurately weighed quantity of each nanosponge formulation equivalent to 50 mg of celecoxib was dissolved in isotonic solution, filtered, suitably diluted, and analyzed at 252 nm using a UV–Visible spectrophotometer. Drug content was calculated from the calibration curve.

Entrapment Efficiency

Entrapment efficiency was determined by dispersing an accurately weighed quantity of celecoxib-loaded nanosponges equivalent to 100 mg in distilled water, followed by centrifugation at 500 rpm for 5 min. The drug content in the clear supernatant was measured at 252 nm using a UV–Visible spectrophotometer. The amount of entrapped drug was calculated by subtracting the free drug from the total drug content, and the entrapment efficiency (%) was calculated using the following equation:

Entrapment Efficiency (%) = [(Total Drug − Free Drug) / Total Drug] × 100

Scanning electron microscopy

The morphological features of prepared nanospongess are observed by scanning electron microscopy at different magnifications.

Particle size and shape

Average particle size and shape of the formulated nanospongess was determined by using Malvern Zetasizer ZS using water as dispersions medium. The sample was scanned for determination of particle size.

Preparation of Celecoxib Nanosponge Topical Gel

Celecoxib-loaded nanosponges were incorporated into a topical gel using Poloxamer 407, chitosan, and sodium alginate as gelling agents. The polymers were dispersed in water and allowed to hydrate overnight. Propylene glycol, methyl paraben, and propyl paraben were then added, followed by the dispersion of celecoxib-loaded nanosponges. The pH of the formulation was adjusted with triethanolamine to obtain a homogeneous gel. The composition of the nanosponge gel is presented in the corresponding table.

Table No 2: Composition of Celecoxib Nanosponge Gel

Evaluation parameters of nanosponges incorporated topical insitu gels

pH:

The pH of the formulation was measured using a digital pH meter (Lab India, 6E404). The pH of the topical gel formulation should be between 3 to 9.

Drug content:

1 gm of the gel was dissolved in 100 ml of phosphate buffer pH 7.4, a sample (5ml) was taken from this solution and diluted to 25 ml. Fluconazole concentration was determined by measuring the absorbance at 264 nm using UV-visible Spectrophotometer (Shimadzu, UV2600).

Viscosity:

The viscosity of prepared gel was measured using Brookfield viscometer (Brookfield Engineering, spindle LV6) at different RPM viscosity. The measurement was made over a whole range of speed settings from 5-100 rpm with 10 seconds between two successive speeds.

Spreadability test:

The spreadability of the gel formulation was determined by using a sliding plate apparatus and by measuring the diameter of 1 gm of gel between horizontal plates after 1 minute. The standardized weight tied on the upper plate was 125 gm. An excess of gel is placed between two glass slides and a 1000 gm weight is placed on them for 5 minutes, to compress the sample to a uniform thickness. The bottom slide is anchored to the apparatus and weights are placed in the pan. The time in seconds needed to separate the two slides is taken as a measure of spreadability. A shorter time interval indicates better spreadability. Spreadability was determined by using a formula.

S = M*L/ T

S = Spreadbility, M = weight tied to the upper slide, L = length of a glass slide, T = Time taken to separate two slides (sec).

Diffusion Studies Procedure:^18,19

In-vitro diffusion study of nanosponge formulation was performed through the cellulose membrane by using Franz diffusion cell. The receptor compartment was filled with 7.4 pH phosphate buffer and kept at 37 ± 0.5 °C with continuous stirring with help of a magnetic stirrer.100 mg of the gel was placed over the cellulose membrane. An interval of 1, 2, 3, 4, 5, 6, 7, and 8, 9 hour 1 ml sample was withdrawn and suitably diluted. The withdrawn sample was replaced with the same amount of 7.4 pH phosphate buffer to maintain the sink condition. Diluted samples were analyzed for fluconazole content with help of UV at 252 nm.

Kinetic Studies: Mathematical models:

Different release kinetic equations (zero-order, first-order, Higuchi's equation and Korsmeyer-peppas equation) were applied to interpret the release rate of the drug from matrix systems for the optimized formulation. The best fit with higher correlation (r2) was calculated.

Zero-order model:

Drug dissolution from dosage forms that do not disaggregate and release the drug slowly can be represented by the equation

Qt = Q0 + K0t

First Order Model:

The first order equation describes the release from systems where the dissolution rate is dependent upon the concentration of the dissolving species.

Log C= Log Co-kt/2.303

Higuchi model: The first example of a mathematical model aimed to describe drug release from a system was proposed by Higuchi in 1961. Initially conceived for planar systems, it was then sustained to different geometrics and porous systems. This model is based on the hypothesis that

Q = KH - t^1/2

Korsmeyer-Peppas model: Korsmeyer et al.(1983) derived a simple relationship which described drug release from a polymeric system equation. To find out the mechanism of drug release, first60% drug release data were fitted in Korsmeyer-Peppas model,

Mt / M∞ = Ktⁿ

Table No 3: Drug transport mechanisms suggested based on ‘n’ value.

RESULTS & DISCUSSIONS

Celecoxib Characterization:

Solubility: Solubility of Celecoxib was carried out in different solvents like, methanol, Ethanol, 7.4 pH phosphate buffer, 0.1N HCl and 6.8 pH phosphate buffer. Solubility of the drug is more in 7.4pH Phosphate Buffer.

Fig2: Solubility studies of Celecoxib

Determination of absorption maximum (λmax): Determination of Celecoxib  λ-max was done in 7.4pH Phosphate buffered Saline for accurate quantitative assessment of drug dissolution rate.  The maximum absorbance of the Celecoxib in 7.4pH Phosphate Buffer was found to be 252 nm. The wavelength of 252 nm was selected for analysis of drug in dissolution media.

Fig.3: λ-max in 7.4pH Phosphate Buffer

Calibration curve:                 Table No 4: Calibration curve data of Celecoxib

Fig.4:Calibration Curve of Celecoxib  in 7.4pH Phosphate Buffer

The linearity was found to be in the range of 2-12μg/ml in 7.4 pH Phosphate Buffer. The regression value was closer to 1 indicating the method obeyed Beer-lambert’s law.

Drug excipient compatibility:

Drug and excipient compatibility was confirmed by comparing spectra of FT-IR analysis of Pure drug with that of various excipients used in the formulation. The functional groups that were presented in the pure drug were present in the optimized formulation with very minute changes.

Fig.5.: FTIR Spectra of Pure Drug

Fig.6: FTIR Spectra of drug and excipient

Drug Content: The Nanosuspension Formulation from F1-F12 was found to be in between 93.18±1.78%-99.26±1.46% and the drug content of Nano sponge formulation F8 which was having more drug content when compared to the remaining formulations.

Table.No.5: Drug content of formulations

Entrapment efficiency: The entrapment efficiency of formulation F1-F12 was found in the range of 51.24±1.58% to 79.79±1.15%

Table No 6: % Entrapment Efficiency of Nano sponges

Morphology determination by scanning electron microscopy (SEM):

Scanning electron microscopy (SEM) was used to determine the Morphology of the prepared nano sponges. The surface morphology of the Celecoxib nanosponge F8 was studied by SEM. Surface morphology of the Celecoxib nanosponge was found to be porous, irregularly shaped structures.

Fig.7: Nanosponges structure optimized formulation (F8)

Particle size analysis of Nanosponges:

The particle size of the nanosponges was determined by optical microscopy. As the ratio of polymer was increased, the mean particle size of Celecoxib nanosponges had also decreased. The significant decrease may be due to the increase in the viscosity of the droplets. Celecoxib nanosponges having a size range of 510 to 600 nm (nanometer) with normal frequency distribution was obtained.

Table No.7: Mean Particle size of Nano sponges

pH of the formulated gel

The pH of gel formulation was determined by using the pH meter. The results were reported in Table. The pH of all formulations was in the range compatible with the normal pH range of the skin. Hence the preparation was non-irritant.

Table No.8: pH of the formulated gel

Drug Content: The prepared formulation was analyzed for drug content. It was observed that the drug content in the prepared nanosponges gel was satisfactory and the drug was uniformly distributed in all the formulation. The percentage of drug content was found to be between 84.15% to 98.27%.

Table No.9:  Drug Content

Viscosity

The viscosity of gel was measured by Brookfield viscometer with spindle LV6. The result shows a decrease in viscosity as a shear rate (RPM) is increased which indicates Gel has pseudoplastic flow. The result indicates the viscosity of gel formulation was consistent.

Table No.10: Viscosity values

Spreadability: Spreadability is an important characteristic of topical formulation and it’s responsible for correct dosage transfer to the target site. Spreadability is an important factor to consider in the formulation of gel. The viscosity and spreadability are inversely proportional to each other.

Table No.11: Spreadability

In vitro diffusion studies of prepared Nanosponges:

In vitro release studies were performed in triplicate using USP basket method at 50 rpm and 37±0.2^oC in 900ml of phosphate buffer (pH 7.4), 100 mg of the formulated Nanosponge gels is used for each experiment. Samples were taken at appropriate time intervals for 1,2,3,4,5,6,7,8,9, hour. The samples were measured spectrophotometrically at 252 nm. Fresh dissolution medium was replenished each time when sample is withdrawn to compensate the volume. By comparing the above diffusion studies of formulations F8G1-F8G9 Maximum drug release was found in F8G6 formulation containing Drug: Chitosan in 1:2 ratio. So F8G6 formulation was taken as the optimized formulation, and drug release kinetics were performed for F8G6 formulation.

Table No.12: Percentage of drug release of Nanosponge gels (F8G6)

Fig.8 :  In vitro drug release of formulations F8G1- F8G9

Release Kinetics Analysis for Optimized Formulation F8G6:

The optimized formulation F8G6 has coefficient of determination (R²) values of 0.967, 0.805, 0.932 and 0.713 for Zero order, First order, Higuchi and Korsmeyer Peppas respectively. A good linearity was observed with the First order, indicates the rate of drug release through the mode of diffusion and to further confirm the diffusion mechanism, data was fitted into the Korsmeyer Peppas equation which showed linearity with n value of 1.223 for optimized formulation. Thus n value indicates the super case II transport mechanism, the release kinetics of the optimized formulation was best fitted into Zero order with the super case II transport mechanism.

Zero Order

Fig.9: Zero Order Plot for F8G6

First Order

Fig.10: First Order Plot for F8G6

Higuchi Plot

Fig.11: Higuchi Plot for F8G6

Peppas Plot

Fig.12: Peppas Plot for F8G6

Regression values of F8G6:

Table No.13: Regression values