Span 20

Ketorolac tromethamine (KT) was obtained from TCI chemicals (Tokyo, Japan). Fertilized white horn eggs were purchased from Heath Farm (Fakenham, UK). Transcutol P, Labrafac PG, Labrafac lipophile WL 1349, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrafil M 2130 CS, and Labrasol ALF were received as a gift from Gattefosse SAS (Leon, France). Tween 20, Tween 60, Tween 80, Span 20, triacetin, ethyl oleate, dimethyl isosorbide, ethylene glycol, propylene glycol and isopropyl myristate were obtained from ACROS organics (Fair Lawn, NJ, USA). Cremophor RH 40, Cremophor EL, butylene glycol, pentylene glycol, hexylene glycol, Span 80, 2-butyl-2-ethyl-1,3-propanediol, (2,3-butanediol), NaOH were purchased From Sigma-Aldrich (Taufkirchen, Germany). Sodium chloride, sodium bicarbonate, and calcium chloride dehydrate were from Fisher Scientific UK Ltd. (Loughborough, UK). Other chemicals used were of analytical grade.

Bipolymeric NCs were prepared in two steps according to a previously reported method with some modifications [34 ]. The first step, emulsification, was used to prepare oil in water NE. For the oily phase, 30 mL ASH extract was mixed with 19 mL castor oil (Acros Organics, USA) and 1 g Span 20 (Acros Organics, USA) using a homogenizer (GLH 850, Omni Inc., USA) at 5000 rpm for 15 min. The oily phase was added gradually to a 100 mL solution of Tween 80 (1% w/v, Acros Organics, USA) using a homogenizer at 10,000 rpm for 45 min and then sonicated using a probe sonicator (Model LC 60/H, Elma, Germany) at 40% amplitude for 15 min with 5 s on/off and temperature adjusted to 15 °C.
In the second step, ionic gelation, a 100-mL alginate solution (0.03 or 0.06% w/v, Sigma-Aldrich, USA) was added gradually to ASH extract NE while stirring for 30 min. A 20-mL calcium chloride solution (0.06% w/v, Acros Organics, USA) was gradually added and stirred for 30 min. Finally, a 20-mL chitosan solution (0.03% or 0.06% w/v, Sigma-Aldrich, USA) in 1% acetic acid (pH 4–4.5, Acros Organics, USA) was gradually added with stirring for 30 min. The obtained NC suspension was sonicated to unify the particle size using a probe sonicator at 40% amplitude for 5 min with 5 s on/off and temperature adjusted to 15 °C. The preparation was stored at 4 °C. All samples were prepared using deionized distilled water.