Development of vitamin e-enriched nanoemulsion for wound healing

Wound problem is a global health care issue that can burden the individuals and countries. Vitamin E is a potent natural antioxidant and tocopherols are the common form to be used in skin-care products. However, studies found that the antioxidant potency of tocotrienols is 60 times than tocopherols which believe can benefit for the wound problems. Red palm oil (RPO) rich in vitamin E (70-80% tocotrienols; 20-10% tocopherols) and carotenoids, is a favourable choice to be used as a natural source of tocotrienols. In our work, a tocotrienol-rich oil-in-water-nanoemulsion (NE) formulation was optimized using response surface methodology (RSM) and formulated using high- pressure homogenizer. Effect of the concentration of three independent variables [surfactant (5-15 wt%), co-solvent (10-30 wt%) and homogenization pressure (500-700 bar)] toward two response variables (droplet size, polydispersity index) was studied using central composite design (CCD) coupled to RSM. RSM analysis showed that the experimental data could be fitted into a second-order polynomial model and the coefficients of multiple determination (R²) is 0.9115. The optimized formulation of NE consisted of 6.09 wt% mixed surfactant [Tween 80/Span 80 (63:37, wt)], 20 wt% glycerol as a co-solvent via homogenization pressure (500 bar). The optimized NE response values for droplet size and polydispersity index were 119.49nm and 0.286, respectively. The actual values of the formulated nanoemulsion were in good agreement with the predicted values obtained from RSM, thus the optimized compositions have the potential to be used as a nanoemulsion for cosmetic formulations. Characteristics and stability studies were evaluated for 6 months at three different temperatures (4°C, 25°C and 40°C), which included drop let size, polydispersity index, viscosity, antioxidant activity, active contents (carotenoids and vitamin E) and colour. It was found that optimized nanoemulsion has fewer changes over the time. No significant difference (P>0.05) was found in terms of droplet size, polydispersity index, viscosity, antioxidant activity and active contents at 4°C storage condition over the time. These findings indicate that optimized nanoemulsion was more stable than the nanoemulsions without co-solvent (glycerol) and emulsions without co-solvent and surfactant (Tween 80 and Span 80). Keratinocytes migration and zebrafish tail regeneration experiments were used to evaluate the wound healing effect of NE. MTT assay provided a concentration range of 0.35-8.75 μg/ml of nanoemulsion that produced cell viability more than 100%. The wound closure of keratinocytes treated with 3.50 μg/ml and 1.75 μg/ml of NE was significantly faster than the blank (35.56%); they increased to 73.76% and 67.37%, respectively, after 24 hours of treatment. The lethal concentration at 50% (LC50 value) obtained from acute and prolonged toxicity was almost similar, which was 4.6 mg/ml and 5.0 mg/ml, respectively. Growth of zebrafish tail regeneration treated with NE at a concentration of 2.5 mg/ml was significantly faster than the untreated zebrafish, which regenerated to 40% on the fifth day, more than 60% on the tenth day of treatment and fully recovered at the 20ᵗʰ day. In conclusion, these results indicate that tocotrienols-rich nanoemulsion formulated was stable and showed potential in enhancing wound healing through accelerated wound closure.

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