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

Sigward E, Mignet N, Rat P, Dutot M, Muhamed S, Guigner JM, Scherman D, Brossard D, Crauste-Manciet S - Int J Nanomedicine (2013)

Bottom Line: The multiple emulsion stability was found to increase from 24 hours to 2 and 6 months with Labrasol, glycerol, and Cremophor, respectively.The formulation including glycerol, investigated between 1 and 100 mg/mL concentration of nanoemulsion, did not affect cell viability.This last formulation would therefore be of major interest for further developments.

View Article: PubMed Central - PubMed

Affiliation: Chemical, Genetic and Imaging Pharmacology Laboratory; INSERM U1022, CNRS UMR8151, Chimie ParisTech, Faculty of Pharmacy, Paris Descartes University, Sorbone Paris Cité, Paris, France.

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.

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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.
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f8-ijn-8-611: 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.

Mentions: From these results, we can conclude that P2X7 receptor activation is one of the first steps of apoptosis induced by formulation A (which leads to chromatin condensation and cell death). An example of fluorescence results obtained for formulation A after 1 hour of incubation is given in Figure 8. For formulation B, after 1 hour of incubation, results are in accordance with Hoechst 33342 results, whereas after 24 hours, the fluorescence increase corresponded to significant apoptosis induction. We can conclude that for formulations A and B, P2X7 receptor activation is associated with apoptosis induction, observed by chromatin condensation. For formulation C, there was neither chromatin condensation nor P2X7 receptor activation.


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

Sigward E, Mignet N, Rat P, Dutot M, Muhamed S, Guigner JM, Scherman D, Brossard D, Crauste-Manciet S - Int J Nanomedicine (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569110&req=5

f8-ijn-8-611: 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.
Mentions: From these results, we can conclude that P2X7 receptor activation is one of the first steps of apoptosis induced by formulation A (which leads to chromatin condensation and cell death). An example of fluorescence results obtained for formulation A after 1 hour of incubation is given in Figure 8. For formulation B, after 1 hour of incubation, results are in accordance with Hoechst 33342 results, whereas after 24 hours, the fluorescence increase corresponded to significant apoptosis induction. We can conclude that for formulations A and B, P2X7 receptor activation is associated with apoptosis induction, observed by chromatin condensation. For formulation C, there was neither chromatin condensation nor P2X7 receptor activation.

Bottom Line: The multiple emulsion stability was found to increase from 24 hours to 2 and 6 months with Labrasol, glycerol, and Cremophor, respectively.The formulation including glycerol, investigated between 1 and 100 mg/mL concentration of nanoemulsion, did not affect cell viability.This last formulation would therefore be of major interest for further developments.

View Article: PubMed Central - PubMed

Affiliation: Chemical, Genetic and Imaging Pharmacology Laboratory; INSERM U1022, CNRS UMR8151, Chimie ParisTech, Faculty of Pharmacy, Paris Descartes University, Sorbone Paris Cité, Paris, France.

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.

Show MeSH
Related in: MedlinePlus