Lc 20a series instrument

The NPs were centrifuged at 10,000g for 10 min under 4 °C to remove free #2.⁵⁸ The supernatant (containing loaded #2) was transferred to a new tube and diluted 5-fold with methanol. Total #2 was obtained by dissolving an equal volume of the initial suspension in methanol and diluted 5-folds. The entrapment efficiency (EE) and LC of #2 were then calculated according to the following equation
$$\begin{gathered} \text{EE}(\%)=\frac{\text{encapsulated}\#2}{\text{initial}\#2}\times 100 \\ \text{LC}(\%)=\frac{\text{encapsulated}\#2}{\text{weight of unloaded carrier}}\times 100 \end{gathered}$$
The encapsulated and initial #2 were determined by a HPLC system. The HPLC system was an LC-20A series instrument (Shimadzu Corporation, Kyoto, Japan) equipped with a double-solvent manager, an injection needle, a chromatographic column heater, and a DAD detector. Sample separation was performed using a Phenomenex HPLC column (Luna, 5 μm, C18, 250 × 4.6 mm, Phenomenex Ltd., Washington, USA). The flow phase contained methanol (solvent A) and 0.1% trifluoroacetic acid (solvent B). A gradient elution program is as follows: from 42 to 65% A (0–20 min). The flow rate was 1 mL/min, the injection volume was 10 μL, the temperature of column compartment was 40 °C, and the wavelength of the DAD detector was set to 254 nm.