2.

4 Characterization of thhe gel formulation:

2.4.1 Quality evaluation
The optimal in situ gel formula containing nanosystems is evaluated for quality including
the following criteria:
- Appearance: The optimal in situ gel formula should be transparent, homogeneous,
and free of clumps. It should be easy to apply and remove from the skin.
- pH: The gel should have a pH between 4 and 9.
- Stability: The gel's stability is evaluated using thermal cycling. After six cycles
between 4°C and 40°C, the gel remains transparent, colorless, and maintains a
solid form without precipitation or separation.
- Spreadability test: thickness of the preparation must be equivalent to the
comparison placebo, the average thickness of the three measurements is used for
comparison.
According to thermal cycling research, the in situ gel exhibits a thermoresponsive nature.
At 4°C, the gel becomes a transparent, colorless liquid. As the temperature increases, the
gel gradually forms. At room temperature, the gel is thick, soft, and easily adheres to the
skin. At 40°C, the gel has a thicker consistency compared to room temperature.
2.4.2 Stability study
Nano gel formulas are considered to meet the requirements if they simultaneously satisfy
the quality standards for organoleptic properties, particle size, pH, phase stability after
undergoing heat cycles, and sedimentation under centrifugation.
For phase stability, the gels must remain stable after six heat cycles, with no separation or
precipitation of active ingredients occurring. The gels are kept in a humidity chamber at
40°C and 75% humidity. They are tested at time points of 0, 1, 2, 4, and 8 weeks.
2.4.3 In vitro study of antimicrobial activity
Six test solutions were prepared containing different concentrations of ibuprofen,
fluconazole, tween 80, ethanol, and distilled water. A placebo solution was also included
for comparison. All solutions were diluted 1:10 with a buffer solution at pH 6.8.
The antimicrobial activity of these solutions was tested using the agar diffusion method.
Candida fungi were inoculated onto a Sabouraud Dextrose Agar (SDA) plate and
incubated at 37°C for 36-48 hours. After incubation, a small amount of the fungi was
then transferred from the SDA plate to a fresh plate of Sabouraud Dextrose Broth (SDB).
This SDB culture was incubated overnight to obtain a denser cell suspension, then
measuring its density using a spectrometer.
The Candida cell suspension from the SDB culture was then used to inoculate petri dishes
containing 15 mL of fresh SDA. Wells with a 6 mm diameter were created in the agar of
these plates. Each well was then filled with 50 µL of a different test solution. The plates
were incubated for 2 days at refrigeration temperature (around 4°C). After incubation, the
zones of inhibition around each well were measured and analyzed with Fiji software.