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 24 hours to 2 and 6 months with Labrasol, glycerol, and Cremophor, respectively. Cytotoxicity was found for formulations including Labrasol and Cremophor EL. The concentration of emulsion inhibiting 50% cell viability (IC(50)) was determined using the alamarBlue(®) test, giving after 24 hours of incubation, IC(50) = 10.2 mg/mL for the Labrasol formulation and IC(50) = 11.8 mg/mL for the Cremophor EL formulation. Corresponding calculated IC(50) values for surfactants were 0.51 mg/mL for Labrasol and 0.59 mg/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 100 mg/mL concentration of nanoemulsion, did not affect cell viability. Moreover, neither chromatin condensation nor P2X7 activation was found between the 10 and 30 mg/mL final concentration of the emulsion. This last formulation would therefore be of major interest for further developments.

Keywords: Cremophor EL; Labrasol; P2X7 receptor; apoptosis; glycerol; polysorbate.

Figure 1

Schematic representation of W/O/W multiple emulsion. Abbreviation: W/O/W, water-in-oil-in-water.

Figure 2

Schematic representation of W/O/W nanoemulsion preparation. Abbreviations: W/O/W, water-in-oil-in-water; W/O, water-in-oil.

Figure 3

Pseudoternary phase diagrams of Polysorbate-85/Labrasol^®, oil (MCT) and water, with different ratios of Polysorbate-85:Labrasol^®. (A) 6:4; (B) 7:3; (C) 8:2; (D) 9:1. Note: ■ = microemulsion phase. Abbreviation: MCT, medium chain triglycerides.

Figure 4

Transmission electron microscopy micrographs of W/O/W nanoemulsions. (A) Polysorbate-85/Labrasol^®; (B) Polysorbate-85/Cremophor^® EL; (C) glycerol/Polysorbate-85. Abbreviation: W/O/W, water-in-oil-in-water.

Figure 5

Cell viability of W/O/W nanoemulsion formulations using alamarBlue^® test at 1-hour and 24-hour incubation times. (A) Polysorbate-85/Labrasol^®; (B) Polysorbate-85/Cremophor^® EL; (C) glycerol/Polysorbate-85. Notes: Each value represents the mean and standard deviation (n = 3). *Cell viability was significantly (P < 0.001) below cell viability of the control without nanoemulsion. Abbreviation: W/O/W, water-in-oil-in-water.

Figure 6

Apoptosis chromatin condensation assessment (Hoechst 33342 test) of W/O/W nanoemulsion formulations after 1- and 24-hour incubation. (A) Polysorbate-85/Labrasol^®; (B) Polysorbate-85/Cremophor^® EL; (C) glycerol/Polysorbate-85. Notes: Hoechst/AB ratio significantly (**P < 0.001; *P < 0.05) compared with the control without nanoemulsion. Each value represents the mean and standard deviation (n = 3). Abbreviations: AB, alamarBlue^®; W/O/W, water-in-oil-in-water.

Figure 7

Apoptosis P2X7 cell death receptor activation (YO-PRO^®-1 test) of W/O/W nanoemulsion formulations after 1- and 24-hour incubation. (A) Polysorbate-85/Labrasol^®; (B) Polysorbate-85/Cremophor^® EL; (C) glycerol/Polysorbate-85. Notes: Each value represents the mean and standard deviation (n = 3). YO-PRO^®-1/AB ratio significantly (**P < 0.001) compared with the control without nanoemulsion. Abbreviations: AB, alamarBlue^®; W/O/W, water-in-oil-in-water.

Figure 8

Apoptosis P2X7 cell death receptor activation (YO-PRO-1 test). Examples of fluorescence records observed after 1 hour of incubation, of cells incubated with Labrasol^® nanoemulsion at three final concentrations: (A) 10 mg/mL; (B) 20 mg/mL; and (C) 28.5 mg/mL.