Formulation and cytotoxicity evaluation of new self-emulsifying multiple W/O/W nanoemulsions

Abstract

Three multiple water-in-oil-in-water (W/O/W) nanoemulsions have been designed for potential inclusion of either lipophilic or hydrophilic drugs using a two-step emulsification process exclusively based on low-energy self-emulsification. The W/O primary emulsion was constituted by a blend of oil (medium chain triglyceride), a mixture (7:3) of two surfactants, and a 10% water phase. The surfactants were a mixture of Polysorbate-85/Labrasol®, Polysorbate-85/ Cremophor® EL or glycerol/Polysorbate-85. The final W/O/W nanoemulsions were obtained by the addition of water, with a weight ratio nanoemulsion/water of 1:2. The multiple emulsion stability was found to increase from 24hours to 2 and 6months with Labrasol, glycerol, and Cremophor, respectively. Cytotoxicity was found for formulations including Labrasol and Cre¬mophor EL. The concentration of emulsion inhibiting 50% cell viability (IC50) was determined using the alamarBlue®test, giving after 24hours of incubation, IC50= 10.2mg/mL for the Labrasol formulation and IC50= 11.8mg/mL for the Cremophor EL formulation. Corresponding calculated IC50values for surfactants were 0.51mg/mL for Labrasol and 0.59mg/mL for Cremophor EL. In both cases, cytotoxicity was due to an apoptotic mechanism, evidenced by chromatin condensation and P2X7 cell death receptor activation. The formulation including glycerol, investigated between 1 and 100mg/mL concentration of nanoemulsion, did not affect cell viability. Moreover, neither chromatin condensation nor P2X7 activation was found between the 10 and 30mg/mL final concentration of the emulsion. This last formulation would therefore be of major interest for further developments. © 2013 Sigward et al, publisher and licensee Dove Medical Press Ltd.