Characterization of Nanoemulsion and Nanoemulgel Formulations

Malvern Zetasizer Nano ZS90, UK was used to measure particle size and polydispersity. The measurements were performed in triplicate at 25°C and fixed angle (90°). The test samples were poured in sample cells after suitable dilution. Polydispersity index values were used to ensure particle diameter uniformity. Malvern Zetasizer Nano ZS (Malvern Instruments, Ltd, UK) Series ZEN3600 with electrophoretic light scattering along with a particle size analyzer was used to measure the zeta potential of NE formulae by applying electric field of one volt. For the measurement of zeta potential, the samples were diluted appropriately with prefiltered double distilled water and readings were taken in triplicate. Digital pH meter (pH 7110 Xylem Analytics Germany GmbH) was used to determine pH of both blank and drug-loaded NE formulations and taken in triplicate. The type of NE (either o/w or w/o) was verified by performing a conductivity test. For this purpose, the electrode of digital conductometer (HANNA^® Instruments, HI9811-5, Romania) was dipped in the NE formulations and the readings noted in triplicate. Viscosity of NE formulae was determined by Brookfield viscometer (DV-II+ Pro) using spindle 63 with spindle speed of 100 rpm. The formed emulgel formulations were subjected to tests similar to those performed for NE (pH, viscosity and conductivity).

All animal handling procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and followed the guidelines of the Animal Welfare Act. All protocols were also reviewed by the Institutional Animal Care and Use Committee Government College University Faisalabad (Faisalabad, Pakistan) prior to granting approval for the study (GCUF/ERC/2043). Male albino rat skin (shaved dorsal skin) was used to perform an in vitro skin permeation study. The rats were sacrificed and dorsal hair was removed followed by surgical removal of skin. The excised skin was then stored at −20°C after washing with phosphate buffer solution. For the skin permeation study, Franz diffusion cell (9 mL) with effective diffusion area of 1.76 cm² was used. Fat and other associated subcutaneous tissues were removed by scalpel from the dermal edge and the processed patch was placed between receptor and donor compartments of a cell with dermal layer towards receptor chamber and the stratum corneum (horny layer) was facing the donor compartment.

One mL of each test formulation was placed on the epidermal skin side. The PBS (pH 7.4) was filled in the receptor chamber. After placing Franz diffusion cell on magnetic stirrer, the magnetic bead was rotated at fixed speed of 300 rpm at temperature 37 ± 0.5°C.¹⁷ Samples were taken at predetermined time intervals for the next 24 h and analyzed spectrophotometrically at 251.5 nm wavelength after suitable dilution. Equal volume of fresh dissolution medium was replenished after each sampling. The mean cumulative quantity of permeated drug from the formulations through skin (per cm²) was plotted against time to obtain DIF permeation profile. For each formulation, permeability coefficient (Kp) and flux (J) were calculated separately.

By using linear regression analysis, the cumulative quantity of DIF that permeated through rat epidermal membrane per cm² at steady state was plotted against time and the slope of resultant plot was used to calculate flux (μg/cm².h). The permeability coefficient (Kp) was calculated using the following equation:

Where J indicates the flux and C denotes the DIF concentration in the donor chamber.²¹

The in vitro skin permeation study for nanoemulgel formulations was performed under the same conditions as used for nanoemulsion formulations.

The rats were divided into five groups (n=5) for evaluating anti-inflammatory effect as:

Group 1: (Control): untreated inflammation induced group.

Group 2: (Standard): received diclofenac diethylamine emulgel.

Group 3: (treated with DIF-loaded nanoemulgel).

Group 4: (treated with DIF-IC-loaded nanoemulgel).

Group 5: (Healthy rats).

To minimize the number of animals used in the experiments, the same animals were reused after executing one acute inflammatory model. Before use in the next model, a washout period of 2 weeks was given to experimental rats and only those animals having no signs of inflammation/necrosis or any physical disability were reused.

1% carrageenan (0.1 mL) and histamine were administered to each rat of every group through subcutaneous administration of right hind paw in the sub-plantar surface. The test formulations and standard were applied half an hour before the administration of carrageenan/histamine. The paw volume was monitored at regular time intervals i.e. 0, 1, 2, 3 and 4 husing a digital Vernier caliper. Percent inhibition of paw volume was determined through equation 2 and represented as mean ± SD.²²

The rats were pretreated with test formulations and standard 30 min before the formalin administration. 0.1 mL of formaldehyde (2%) was injected in the sub-plantar region of left hind paw of all rats on days 1 and 3 of the study for edema induction. Digital Vernier caliper was used for paw volume measurement for 13 consecutive days. Percentage inhibition was calculated using equation 2. Results are presented as mean ± SD of percentage inhibition of paw edema.²³