Ay220

Chlorophyll was measured in accordance with Yang et al. [87 ]. The plant leaf samples (0.25 g) were weighed using a digital balance (Shimadzu, model AY220, Kyoto-Japan), dried in liquid nitrogen, and ground with a mortar and pestle. After grinding, the total pigments were extracted in 5 mL of 80% acetone using a 10 mL Pasteur pipette. After centrifugation (Centurion1020 D.E) of each sample at 1500× g for 5 min, a crude extract with a light green to green solution was obtained. The pellet was discarded, and the absorbance of the supernatant was measured using a 1 cm pathlength cuvette at 663.6, 646.6, and 440.5 nm on a UV–VIS spectrophotometer (GENESYS 10 UV–VIS, Thermo Spectronic, Rochester, New York, NY, USA). These wavelengths represent the absorption peaks of chlorophyll-a, chlorophyll-b, and carotenoids, respectively.
The chlorophyll-a, chlorophyll-b, total chlorophyll, and carotenoid contents were calculated using the following equations [88 (link),89 (link)]:

After induction and treatment of the edema, the animals of each model were euthanized with a solution of ketamine (240 mg/kg) and xylazine (45 mg/kg) and 6 mm diameter ear tissue samples were collected from right and left ears using a metallic punch (Richter). Then, the tissue samples were individually weighed on analytical balance (AY220, Shimadzu) and the edema was evaluated by the difference of weight (mg) between right and left (non-inflamed) ears.

For gravimetric analysis of biomass, we transferred 45 mL of the grown cultures to 50 mL conical tubes, and centrifuged at 4,233 g for 10 min to remove the supernatant, followed by two washings using 15 mL of PBS 1X each time. We dried the cell pellets at 60°C until constant weight. We measured the biomass using an analytical balance (Shimadzu AY220), and the dry cells were used for lipid extraction. All experiments were performed in triplicate.

The drug loading (DL%) was defined as the mass of aspirin impregnated per mass of polymer and was obtained using Equation (3):
where m_f is the film mass 5 days after impregnation for the scCO₂ impregnation and the film mass after the drying step for the soaking method, and m₀ is the film mass before the impregnation measured with precision balance (10⁻⁴ g, model AY-220, Marte, Shimadzu, São Paulo, Brazil). For each method, the impregnation was performed at least in triplicate, and the average drug loading is presented.
The difference between the drug loading of each polymer and method was statistically evaluated through ANOVA, using the Tukey test with 5% of significance.

To fabricate the liquid-infused film used for electrowetting, the transparent glass substrate coated with Indium Tin Oxide (ITO) was first cleaned by plasma for ~30 min. After cleaning, the PTFE membrane (thickness ~ 20 μm, Sterlitech Corporation) was gently placed on the top of ITO glass slide. To make the PTFE membrane smooth and be of good contact with the ITO substrate, we wet the membrane using ethanol with the help of capillary wicking effect. Then after the evaporation of ethanol, we added the liquid lubricant with a pre-determined volume into the membrane. In our experiment, the low surface tension 3 M Fluorinert FC-70 (γ ≈ 18 mN/m, η ≈ 12 cSt) and Dupont Krytox GPL 103 (γ ≈ 16 ~ 20 mN/m, η ≈ 80 cSt) were chosen as liquid lubricants to spread on PTFE membrane and form a stable film via capillary wicking. The thickness of the liquid-infused film H is determined by the fluid volume V according to the equation H = (V + m/ρ)/S, where ρ is the PTFE membrane density, m and S are the weight (measured by analytical balance, AY220, Shimadzu) and the surface area of the membrane, respectively. In our experiment, the thickness of the as-prepared liquid-infused film was tailored to ~20 μm and ~50 μm, respectively.

We extracted the lipids from the biomass following the method of Bligh and Dyer (1959 (link)). Dry biomass was suspended in chloroform/methanol (2:1, v/v) and cell lysis occurred using a Turrax homogenizer (ULTRA-TURRAX T18; IKA), with four cycles of homogenizing for 2 min and ice cooling for 1 min to avoid lipid heating. For determination of the total lipid content, solvents were evaporated at 60°C using a rotatory evaporator (Laborota 4,000eco) and dried at 60°C for 24h. Finally, extracted lipid weight was measured using an analytical balance (Shimadzu AY220). We expressed the lipid yield as concentration (g/L medium), the lipid content as a percentage of the lipid weight relative to dry biomass weight (% w/w) and the productivity as g lipid/(g biomass × h).