JOURNAL OF COSMETIC SCIENCE 50 CHARACTERIZATION OF NANOGEL FORMU LATIONS All prepared nanogel formulations were fi rst visually examined to see if there is any precipita- tion or turbidity. It was decided that the formulations with turbidity and precipitation were not suitable and would not be used in future studies. Studies were carried out to determine the physicochemical properties of clear formulations which were found to have a constant pH. Particle size, zeta potential, and PI parameters were examined in the pre- pared nanogel formulations. Table I Contents of Nanogel Formulations Formulation code CS solution ratio (%) BSA solution ratio (%) CS molecular weight CS: BSA pH F1 0.75 0.02 Low 1:5 5.4 F2 0.5 0.02 Low 1:5 5.4 F3 1 0.02 Low 1:5 5.4 F4 0.75 0.02 Medium 1:5 5.4 F5 0.5 0.02 Medium 1:5 5.4 F6 1 0.02 Medium 1:5 5.4 F7 0.75 0.02 High 1:5 5.4 F8 0.5 0.02 High 1:5 5.4 F9 1 0.02 High 1:5 5.4 F10 0.75 0.02 Low 1:4 5.4 F11 0.5 0.02 Low 1:4 5.4 F12 0.5 0.5 Low 1:4 5.95 F13 1 0.02 Low 1:4 5.4 F14 0.75 0.02 Medium 1:4 5.4 F15 0.5 0.02 Medium 1:4 5.4 F16 1 0.02 Medium 1:4 5.4 F17 0.75 0.02 High 1:4 5.4 F18 0.5 0.02 High 1:4 5.4 F19 1 0.02 High 1:4 5.4 F20 0.75 0.02 Low 1:6 5.4 F21 0.5 0.02 Low 1:6 5.4 F22 0.5 0.5 Low 1:6 5.95 F23 1 0.02 Low 1:6 5.4 F24 0.75 0.02 Medium 1:6 5.4 F25 0.5 0.02 Medium 1:6 5.4 F26 1 0.02 Medium 1:6 5.4 F27 0.75 0.02 High 1:6 5.4 F28 0.5 0.02 High 1:6 5.4 F29 1 0.02 High 1:6 5.4 F30 0.75 0.02 Low 13:10 5.4 F31 0.5 0.02 Low 13:10 5.4 F32 0.5 0.5 Low 13:10 5.95 F33 1 0.02 Low 13:10 5.4 F34 0.75 0.02 Medium 13:10 5.4 F35 0.5 0.02 Medium 13:10 5.4 F36 1 0.02 Medium 13:10 5.4 F37 0.75 0.02 High 13:10 5.4 F38 0.5 0.02 High 13:10 5.4 F39 1 0.02 High 13:10 5.4 * In all the formulations prepared, the ratio of acetic acid solution was tested both 0.75 and 1%.

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