The availability of these candidates to liposomes was assessed by examining whether phase separation occurred at the stage of inverted micelle production for 24 hours at room temperature.
When n-hexane and ethyl acetate were used as solvents, respectively, the inverted micelle phases immediately separated (within 1 hour) into micelle-poor and micelle-rich phases that formed densely packed aggregates, and most micelles were destroyed (Figure 2) as n-hexane has lower polarity (polarity index = 0.1) and ethyl acetate higher solubility in water (8.7 g/100mL) than chloroform (polarity index = 4.1 and solubility in water = 0.81 g/100mL).
Then, 100 mL distilled water with BCAAs or curcumin was added to the mixture and sonicated with a 20 kHz probe-type ultrasonicator (ULH-700S; Jeiotech, Korea) for 5 minutes to form inverted micelles; each cycle consisted of 1 second pulse-on and 4 seconds pulse-off with 210 W sonication power at 4[degrees]C.
Ethyl acetate: n-hexane = 4: 1(v/v) was selected as the optimum ratio because it formed stable inverted micelles without phase separation for 24 hours and produced the most transparent liposome solution (Figure 3).
Individual nonhalogenate solvents did not form stable inverted micelles when used alone.
Caption: Figure 2: Inverted micelles prepared using nonhalogenated solvents to dissolve phospholipids.