Allegra 64r centrifuge

To promote drug entrapment within the oily droplets of the nanoemulsions, the selected drugs (doxorubicin, irinotecan, cisplatin) underwent HIP formation with AOT, following literature methods. Briefly, doxorubicin hydrochloride and irinotecan hydrochloride were separately dissolved in distilled water, at 10 and 4 mg/mL, respectively. Their corresponding HIPs were precipitated via the addition of an AOT solution (4.5 mg/mL) in a 1:1 molar ratio, followed by centrifugation at 34,000× g for 5 min (Allegra^® 64R centrifuge, Beckman Coulter, Palo Alto, CA, USA) [21 (link),22 (link)]. In the case of cisplatin, a 2 mg/mL solution in water was added to 10 mg/mL AgPF₆ (1:2 molar ratio), and left under stirring overnight, in order to precipitate AgCl, which was then removed via centrifugation at 53,000× g for 30 min (Allegra^® 64R centrifuge, Beckman Coulter, Palo Alto, CA, USA). The supernatant, containing the platinum, was added to a 4.5 mg/mL AOT solution (platinum–AOT molar ratio 1:2), and kept under magnetic stirring for 30 min, which allowed cisplatin–AOT precipitation to occur, prior to centrifugation at 34,000× g for 10 min (Allegra^® 64R centrifuge, Beckman Coulter, Palo Alto, CA, USA) [23 (link)]. The three precipitated HIPs were further washed with distilled water, and then dried under a nitrogen flow.

Fresh chicken eggs (medium-sized) were bought from a local supermarket in Xuanwu County, Nanjing, China. The eggs were checked for cracks after they were washed with tap water and cleaned with tissue paper. Cleaned and unbroken eggs were selected for the experiment. The eggs were cracked open, and then the egg white was carefully separated from the egg yolk and chalazae using an egg separator. The egg white was then homogenised in a beaker (sealed with aluminium foil) by a magnetic stirrer (CrystalMS2-P1H, Suzhou Jiemei Electronics Co., Ltd., Suzhou, China) at 4 °C for 2 h. Subsequently, 300 mL of the homogenised egg white was diluted with three times deionised water (DIW) then was gently stirred manually using a glass stirring rod. The egg white protein was quantified as 24.36 mg/mL using the Biuret reagent method at a pH (S20 SevenEasy pH meter, Mettler Toledo, OH, USA) of 8.79. Whereas an electric conductivity meter (INESA DDBJ-350, INESA Scientific Instrument Co., Ltd., Shanghai, China) was used to determine electric conductivity of 2.37 mS/cm, total dissolved solids (TDS) of 1182 mg/L and salinity of 0.14%. The diluted egg white solution was then centrifuged (Allegra 64R Centrifuge, Beckman Coulter, IN, USA) at 11,000× g for 30 min at 4 °C to remove impurities and insoluble proteins.

The lignin dissolved by the eutectic systems during the process was recovered via precipitation. In detail, the framework of ChLA, ChGly, ChLAGly, ChEug, ChCat and Ch4Hba was suppressed by water addition. 75% (w/w) of distilled water was added to approximately 1 g of DES liquid fraction. Conversely, ChZn/GVL fractions were separated from lignin by antisolvent procedure, keeping a 9:1 ratio of water addition, according to a previous work [12 (link)]. Thirdly, lignin was precipitated from alkaline solutions by acidification. In detail concentrated H₂SO₄ was added to reach pH 2, according a previously presented protocol [17 (link)].
The precipitation process was accelerated by means of ultracentrifugation (26,000 rpm, 5 min, Allegra 64R Centrifuge, Beckman Coulter, CA, USA), performed at RT. The resulting solid underwent a washing/centrifuging cycle for three times with distilled water and, in the case of ChEug and Ch4Hba, with ethanol. Finally, the recovered precipitate was dried under vacuum and weighted.
The liquid fraction resulting from the separation protocol (see Figure S15) was freeze-dried to remove excess water and tested for the residual antioxidant power. Water and pH adjustments were performed where required.