Acclaim 120 c18

The obtained formulations were centrifuged (4500 rpm for 15 min) (Model 5804, Eppendorf, Hauppauge, NY, USA). The supernatants were diluted in methanol (1:2) to promote the release of MB encapsulated in PMs. The obtained samples were analysed with HPLC. Chromeleon 7.2 software was used for data acquisition. The chromatographic conditions included a commercially available Acclaim^TM 120 C18 (100 × 4.6 mm) column with a particle size of 5 µm from Thermo Fisher Scientific (Bremen, Germany). The optimized mobile phase was water: methanol (25:75, v:v) following an isocratic flow of 1.0 mL/min for 10 min, and the temperature of the column was set at room temperature. The injection volume was 10 μL, and the detection was performed at 238 nm. A calibration curve for MB was prepared in methanol from five standard solutions: 28 μg/mL, 32 μg/mL, 40 μg/mL, 56 μg/mL and 61 μg/mL. Through interpolation of the calibration curve, the MB concentration in the supernatant was obtained. The theoretical concentration of MB was calculated considering the initial amount of MB added and the dilutions performed throughout the procedure. Thus, the encapsulation efficiency (EE) was calculated as follows: $$\mathrm{E}\mathrm{E}\left(\%\right)=\frac{\mathrm{N}\mathrm{P}\mathrm{C}}{\mathrm{T}\mathrm{C}}\times 100$$
where TC is the theoretical concentration of the MB if the entrapment efficiency is 100% (μg/mL) and NPC is the final concentration of the MB in the nanoparticle (μg/mL).

Chromatographic analysis and separation were carried out by using Nexera UC SFC system (Shimadzu, Japan) with an SPD-20A UV detector. Furthermore, the system was equipped with a column manager, a convergence manager controlling the pressure of supercritical carbon dioxide, an auto-sampler (5 μL loop for injection), an online degasser and a backpressure regulator (BPR). With an LC-20ADXR pump, the modifier could be delivered and then mixed with supercritical carbon dioxide. Lab Solutions Ver5.8 was used for instrument control, data collection and processing.
The final SFC conditions were as follows: A Thermo Scientific^TM Acclaim^TM 120 C18 (5 μm, 4.6 mm × 250 mm) column was used to separate twelve target analytes. The mobile phase comprised of supercritical CO₂ (A) and methanol (B). The gradient elution program was set as follows: 0–5 min, 5% B; 5–10 min, 3% B. The flow rate was maintained at 1.4 mL/min. The column temperature was set at 40 °C and the backpressure was 10 MPa. The injection volume was 5 μL. The UV detection wavelength was 220 nm.