br The second goal of this study was to

The second goal of this study was to assess the effect of combining an optimized microemulsion with a microneedle roller. Microneedles have been employed for penetration enhancement alone or in combi-nation with deformable vesicles, invasomes and solid lipid nano-particles, among other systems (Badran et al., 2009; Paleco et al., 2014; Qiu et al., 2008). Despite being less explored, its combination with microemulsions was recently reported to increase the skin-to-receptor phase ratio of propranolol in a manner that depended on microemul-sion composition (Kelchen and Brogden, 2018). This result incited us to investigate whether association with microneedles would potentiate the microemulsion-mediated delivery of celecoxib. Finally, the influence of microemulsion on celecoxib cytotoxicity was assessed. With this study, we aim at contributing to the development of a new strategy for che-moprevention that can benefit an increasing number of patients con-sidered high risk for breast cancer development.
2. Materials and methods
Propylene glycol and sodium hydroxide were obtained from Synth (Diadema, SP, Brazil). Monocaprylin and tricaprylin were a kind gift from Abitec Corporation (Janesville, WI, USA), and decylglucoside from BASF (Ludwigshafen, Germany). Phosphatidylcholine was pur-chased from Avanti Polar Liproxstatin-1 (Alabaster, AL, USA). Acetonitrile, ethanol and methanol were purchased from J.T. Baker (Center Valley, PA, USA). Celecoxib was purchased from Cayman (Ann Harbor, MI, USA). Evan’s blue, tablets for PBS and DMSO were purchased from Sigma (St Louis, MO, USA). Other, more specific, reagents (such as those employed in cell culture) are described with the methodology in which they were employed. Ultrapure water (ASTM® type 1) was em-ployed unless stated in the methodology of individual experiments.  International Journal of Pharmaceutics 560 (2019) 365–376
3. Methods
3.1. Development of microemulsions
Ternary phase diagrams were constructed using the water titration method at room temperature to assess the influence of composition on water incorporation and microemulsion area of formation. Phosphatidylcholine (PC) and decylglucoside (DG) were selected as surfactants due to their low irritation potential, and because partial replacement of PC with a hydrophilic surfactant has been reported to aid microemulsion formation, reducing the extent of liquid crystalline phase formation (Cichewicz et al., 2013; Patel et al., 2006; Vozmediano et al., 2000). Ethanol (Et) and propylene glycol (PG) were assessed as co-surfactants (Graf et al., 2008; Savic et al., 2009). The surfactant-co-surfactant mixtures (S-coS) tested were composed of PC:DG:PG or PC:DG:Et at 1:2:1 or 1:2:1.5 (w/w/w).
As oil phase, monocaprylin was selected based on previous reports demonstrating its ability to dissolve other lipophilic compounds (such as lycopene, paclitaxel and vitamin E) and increase their skin pene-tration more pronouncedly than longer monoglycerides and triglycer-ides (Cichewicz et al., 2013; Hosmer et al., 2013; Lopes et al., 2009; Lopes et al., 2010; Pepe et al., 2013). Because these effects were con-centration-dependent, monocaprylin was maintained above 10% in the formulations tested to enable increases in the skin penetration (Hosmer et al., 2011; Lopes et al., 2009). Preliminary studies, conducted to in-vestigate its ability to dissolve celecoxib, demonstrated that when monocaprylin was mixed with the surfactant blend selected, at least 5% (m/m, maximum concentration tested) of the drug could be dissolved, further supporting its choice as oil phase.
The oil phase and S-coS were mixed at 1:9 to 9:1 ratios (w/w), ti-trated at room temperature with deionized water (in 5% increments) under vortexing for 30 s (Genie 2, vortex set at position 10, Scientific Industries, Bohemia, USA), and formulations that were fluid, clear, and did not undergo phase separation were preliminarily classified as mi-croemulsions and assigned to a monophasic region in the phase dia-gram (Carvalho et al., 2017b; Thomas et al., 2014). The isotropy of formulations selected among those classified as microemulsions was confirmed by polarized light microscopy (Phelps et al., 2011; Thomas et al., 2014).