PHARMACEUTICALLY OPTIMIZED TOPICAL NANOGEL FORMULATION 53 CS, it was found appropriate to continue working with these formulations because tur- bidity was observed even if they were clear at the time they were prepared. Gel formulations are preferred for topical applications b ecause of their ability to release drug active ingredients from systems and because they are absorbed faster than creams and oint- ments. In addition to this, nanogels have the characteristics of high stability, high drug loading capacities, targeting of active substances, and reducing the side effects with the potential for controlled release (19). In this direction the aim of our study was to develop nanogel formulations containing vitamin C for topical use to carry out in vitro characteriza- tion studies and to investigate the antioxidant activity of selected formulations in vitro (20). VITAMIN C LOADING TO THE OPTIMUM FORMULATIONS One of the most important effects of oxidative stress is that reactive oxygen species, that is, free radicals, can cause DNA damage and infl ammation because of lipid peroxidation, re- sulting in aging. Antioxidants can prevent these events by reducing free radicals and their effects (21). Vitamin C is a well-known antioxidant and anti-infl ammatory, anti-aging, and anticarcinogenic agent. Vitamin C has been used in various formulations because of its an- tioxidant properties, and its effectiveness has been proven. The reason why we use vitamin C as an active substance in our study is to prepare a stable and high–penetration rate formu- lation with this substance, which is widely used in commercially available products (22). The dose information regarding the antioxidant use of vit amin C in topical application is 10 mg/mL in the literature (23). The active substance is in the form of a light white crystal. There was no change in color and appearance of the formulations after the ac- tive ingredient was added. CHARACTERIZATION STUDIES Characterization studies were pe rformed to determine the physicochemical properties of the formulations as presented next. DETERMINATION OF PARTICLE/DROPLET SIZE AND PI OF FORMULAT IONS The vitamin C active substance was charged, and the parti cle size of the loaded formula- tions was made as described in the Method section. The particle size results of the nano- gels are shown in Table II. Table II Particle Size PDI and Zeta Potentials of the Nanogel Formulations Formulation code Particle size (nm) ± SD PI Zeta potential (mV) ± SD F1 211.3 ± 68.45 0.732 ± 0.274 -16.1 ± 3.05 F9 551.0 ± 10.74 0.179 ± 0.013 23 ± 1.84 F19 119.9 ± 13.71 0.191 ± 0.006 15.6 ± 0.541 F27 148.9 ± 1.019 0.208 ± 0.006 22 ± 1.36 F31 1.688 ± 223.5 0.762 ± 0.172 20.82 ± 0.965

JOURNAL OF COSMETIC SCIENCE 54 Table III Content Quantifi cation Results of Formulations Formulation code Drug content (%) ± SD F1 86.077 ± 1.294 F9 113.911 ± 2.520 F19 103.488 ± 1.352 F27 107.928 ± 1.043 F31 89.457 ± 0.373 The particle sizes of the formulations were in the range of 148.9 ± 1,019 and 1.688 ± 223.5 nm. In literature review, this value was generally lower than 400 nm for topical nanogel formulations, and in our study, formulations with similar particle size (F1, F19, and F27) were selected optimally (24,25). The PI describes the unequal degree of size distribution of the particles. The PI value ranging from 0.1 to 0.25 indicates that the particle size distribution is narrow, but if it is greater than 0.5, it shows a very wide dis- tribution. As the PI approaches 1, the particle size distribution increases. When long- term stability is desired, it is important to ensure that the PI parameter is as low as possible. Formulations with a PI of less than 0.5 were selected optimally. DETERMINATION OF ZETA POTENTIAL OF FORMULATIONS The zeta po tential of the formulations without vitamin C ac tive substance loading was performed, as described in the Method section. The zeta potentials of the nanogels are shown in Table II. Zeta potential is a scientifi c term for electrokinetic pote ntial in colloidal systems and a measure of surface charge. It can affect particle stability as well as cellular uptake and intracellular processes. The stability of the colloidal system depends on the balance be- tween the two opposing forces. These are van der Waals forces and electrical double lay- ers. The photon correlation spectroscopy technique was chosen to measure zeta potential. Positive zeta potentials or negative zeta potentials greater than 30 mV can lead to mono- dispersity. On the other hand, values less than 5 mV can lead to agglomeration. The zeta potential is infl uenced not only by the properties of the nanoparticles but also by the nature of the solution, such as pH and ionic strength. The formulations prepared have zeta potential between -16.01 ± 3.05 and 23 ± 1.84. The zeta potential of the formulations is expected to be positive because of the cationic charge of CS. However, only zeta potential of F1 formulation was negative. It suggests that there is a problem during preparation. The zeta potentials of nanogel formulations prepared by Islam et al. (26) were found to be positive because of the cationic charge of CS. They observed a reduction in zeta potential by the addition of alginate. According to the obtained characterization results, F19 formulatio n which is selected optimally based on particle size and zeta potential contains low–molecular weight CS. When medium– and high–molecular weight CS species are used, turbidity and pre- cipitate are formed, and particle size and PDI are higher than those of low–molecular weight CS formations.