The development and in vitro evaulation of topical nanoemulsion formulation

Aim: Topical nanoemulsion (NE) and nanoemulsion-based gel (NEG) formulations containing daidzein (DZ) for the treatment of skin cancer (melanoma) were prepared and in vitro characterization studies (morphological analysis, droplet size, zeta potential, release study, stability study, viscosity and pH measurements) were performed. In addition, cell culture studies were performed to evaluate the cytotoxic effects of the formulations on melanoma (SK-MEL 30/An1) and normal dermal fibroblast (PCS-201-012) cell lines. Materials and Methods: HPLC method for quantification of DZ was developed and validated. The coarse emulsion was prepared using ethyl oleate (10%), DMSO (5%) as the oil phase, Tween 80 (10%) and Lipoid S100 (5%) as surfactant and ultrapure water and then blank nanoemulsion and nanoemulsion containing DZ formulations (B-NE and DZ-NE) were prepared by passing the coarse emulsion through high-pressure homogenizer (2 cycles). Furthermore, nanoemulsion-based gel formulations (B-NEG and DZ-NEG) were developed using 1% Protasan ™ UP G 213 as the gelling agent. And also, blank conventional emulsion and conventional emulsion containing DZ formulations (B-CE, DZ-CE) were also prepared. The droplet size, size distribution, zeta potential, viscosity and pH values of all formulations were determined. Stability (at 4 C and 25C) and in vitro release (in PBS pH 7.4 with %5 SDS) studies have were performed. Using MTT method, the cytotoxic effects of formulations was evaluated against human melanoma and normal fibroblast cell lines. Results: Droplet size and zeta potential values were 151.12±2.70 and 149.80±3.52 nm (p>0.05) and -22.57±0.78 and -19.32±1.06 mV (p<0.05) for B-NE and DZ-NE, 191.48±5.26 and 200.25 ±11.09 nm (p>0.05) and 18.67±0.59 and 19.35±0.66 mV (p>0.05) for B-NEG and DZ-NEG, 2.830±0.011 and 4.420±0.017 μm (p<0.05) and -15.50±0.75 and -14.67±0.43 mV (p<0.05) for B-CE and DZ-CE, respectively. Positive zeta potential values for B-NEG and DZ-NEG were obtained and a significant viscosity increase was achieved in the presence of Protasan ™ UP G 213 compared to the NE formulations. It was determined that the prepared formulations had pH values (pH 4.53-5.54) compatible with the skin. Higher amount of DZ was released from the DZ-NE with the smallest droplet size. During the stability study (90 days), it was determined that the NE and NEG formulations have droplet sizes in the nano-range and their absolute zeta potential values were about 20-40 mV. All formulations maintained the cytotoxic effect of DZ against the melanoma cell line. Moreover, it was also found that pure DZ and all formulations had no cytotoxic effect on normal fibroblast cells. Conclusion: DZ-containing NE and NEG formulations were successfully prepared. The prepared NE and NEG formulations might be useful delivery systems for the topical application of DZ and treatment of skin cancer. Key Words: Daidzein, fibroblast cell line, melanoma cell line, nanoemulsion, nanoemulsion-based gel, skin cancer, stability