PHARMACEUTICALLY OPTIMIZED TOPICAL NANOGEL FORMULATION 51 DETERMINATION OF PARTICLE/DROPLET SIZE AND PI OF FORMULATIONS The particle size/droplet sizes a nd PI of nanogel formulations containing vitamin C were measured at room temperature (25 ± 2°C) with a 173° pain using the Malvern Zetasizer Nano ZS instrument (Malvern, United Kingdom). Five attempts were made from each sample, and each measurement was repeated fi ve times. DETERMINATION OF ZETA POTENTIAL OF F ORMULATIONS The zeta potentials of nanogel formu lations containing vitamin C were measured at 78.5 dielectric constant and 25 ± 2°C with Malvern Zetasizer Nano ZS. Five attempts were made from each sample, and each measurement was repeated fi ve times. PH MEASUREMENTS OF FORMULATIONS The pH of nanogel formulations containi ng vitamin C was measured at room temperature with a pH meter (Thermo Fisher Scientifi c, United Kingdom). Then, 0.1 M NaOH solution was used for formulations requiring pH adjustment. MORPHOLOGICAL ANALYSIS WITH ATOMIC FORCE MICROSCOPY (AFM) The morphological properties of nanogel formulations containing vitamin C were made by AFM using a commercial instrument of Pico Plus (Molecular Imaging, Tempe, AZ). Topography images were obtained using intermittent mode, a spring constant of 48 N/m, and silicon symbol probes having a frequency of 190 kHz. All measurements were per- formed at room temperature. DETERMINATION OF RHEOLOGICAL PROPERTIES OF FORMULATIONS The nanogel formulations were examined r heologically using a rheometer device (TATX Discovery HR1). Studies were performed using a 40-mm-diameter steel probe, with 1-mm fi xed spacing (16,17). The fl ow properties of the formulations were determined at 25°C and 32°C. VISCOSITY MEASUREMENT OF FORMULATIONS The viscosities of nanogel formulations we re measured using a single point viscometer (Vibro Viscometer SV-10 A&D Company, Tokyo, Japan). IN VITRO DRUG RELEASE STUDIES OF NANOGELS The dialysis membrane was used for in vitr o release studies of nanogel formulations contain- ing vitamin C. For the study, 2 mL of nanogel formulations containing 10 mg/mL vitamin

JOURNAL OF COSMETIC SCIENCE 52 C were placed into the dialysis membrane, and the end of the dialysis membrane was closed with a sealer. Then, 200 mL of buffer solution with a pH of 7.4 was prepared. Dialysis membranes were placed in 10 mL of solution placed on a multi-heater mixer set at 37 ± 1°C and set at 150 rpm. In the in vitro release study, 1 mL sample was taken at 30 min, 1, 2, 3, 4, 5, and 6 h. After the sample was taken, 1 mL of buffer solution was added to the medium. The samples were evaluated by HPLC, and the concentration of vitamin C in the medium was determined (n = 3). IN VITRO 2,2-DIPHENYL-1-PICRYLHYDRAZYL (DPPH) ASSAY FOR ANTIOXIDANT ACTIVITY OF VITAMIN C–LOADED NANOGELS The antioxidant activity of vitamin C–loaded n anogels was assessed by evaluating its ability to scavenge DPPH (18). DPPH was dissolved in methanol to yield a 200 μM concentration. Then, 100 μL of DPPH solution was mixed with 100 μL of vitamin C solution (10 μg/mL and 1 mg/mL in methanol) or vitamin C–loaded nanogels (1 mg/mL in methanol) or empty nanogels (1 mg/mL in methanol). A control sample was prepared by mixing 100 μL of DPPH solution with 100 μL of methanol. All the samples were stored at room temperature for 30 min. Then, the absorbance was measured at 517 nm against blank (methanol). All the experiments were performed in triplicate. The percent inhibition (or antioxidant activ- ity) was calculated according to the following formula: ¯ ¡ ° q100 ¡ °, ¡ ° DPPH Sample A A % Inhibition ADPPH where ADPPH is the absorbance of DPPH control and ASample is the absorbance of the sample. Data were analyzed using analysis of variance (ANOVA) . In the case of a signifi cant ANOVA, post hoc analysis was performed using Tukey’s test. Values were expressed as mean ± standard error (SE). The level of p 0.05 was considered to be statistically sig- nifi cant. Statistical analyses were performed with Statistical Package for the Social Sci- ences for Windows (SPSS, version 15.0). STABILITY TESTS The selected nanogel formulations were evaluated in a s tability cabinet containing 60% relative humidity at room temperature at 25°C and 75% relative humidity at 40°C. The parameters used in characterization studies (particle size, PI, zeta potential, and pH) were evaluated at certain time intervals. RESULTS AND DISCUSSION After the nanogel formulations mad e as described in the M ethod section were examined visually, studies were continued with the selected formulations. Because of turbidity and precipitation in the formulations prepared by Method B, this method was not continued. In the formulations made with Method A and prepared using medium–molecular weight