Effect of three monoglyceride based cubosomes systems on the viability of human keratinocytes

Three lipid-based cubic liquid crystalline nanoparticles (or cubosomes) systems were formulated and prepared by emulsifying surfactants Brij® 30 and Brij®35 with aqueous solution of poly(ethylene) glycol derivatives and glyceryl monooleate: one formulation containing Monomuls® 90-O18 and Pluronic® PE 10500, another one containing Monomuls® 90-O18 and Brij®35, and the third one based on Pluronic® PE 6800, Brij®30 and Brij® 35. Toxicity of formulations was evaluated by measuring the effect of those cubosomes on human keratinocytes (NCTC2544) viability. Cells were seeded at densities of 15,000 and 30,000 cells per well, respectively, into a 24-well tissue culture dish. The cell viability test was performed using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay by measuring oxidative activity of mitochondrial succinate dehydrogenase enzyme. Effect on cell viability for each formulation was determined as function v(t, c) of time (24, 48 and 72 hours) and cubosomes concentrations (0, 5, 10, 20, 25 and 50 μg/mL). Viability of cells decreased with increasing the concentration of cubosomes at all three measuring times but the results were highly variable and the dependence on concentration was far from being linear. Replacement of analysis of dependence of viability at the three independent times, with analysis of areas under time viability curves (AtV) led to a much more statistically significant dependence on concentration. As a general rule, AtV dependence on concentration was linear. Decreasing of the number of cells or increasing of toxicity led to linear biphasic dependence: a rapid initial decrease followed by a slower final decline. Classification of compounds as toxic and non-toxic was undertaken using the method of classification of plasma levels curves in bioequivalence studies. Based on comparison of time integral of viability (AtV)-concentration of cubosomes curves, it was found that first formulation is not toxic; the second is at the frontier between toxic and nontoxic, the third is toxic. From comparison of formulations composition, appeared that toxicity is determined by the content of Brijs.