2.5. Drug-Loading and Encapsulation Efficiency

The concentration of CK in the CMC-β-CD Ngs was determined using high performance liquid chromatography (HPLC; Agilent 1260 Infinity) [23]. The lyophilized samples were accurately weighed and dissolved in acetonitrile, sonicated for 30 min, filtered with a 0.45 μm membrane filter, and analysed by HPLC. The Eclipse Plus C18 column (4.60 mm × 250 mm, 5 µm) (Agilent, Santa Clara, CA, USA) was used for separation, and gradient elution chromatography was used with mobile phase of water (A) and acetonitrile (B). The flow rate was 1.5 mL/min, the injection volume was 20 µL and the column temperature was set at 35 °C. The ultraviolet wavelength was set to 203 nm. The linear regression equation of the calibration curve for CK in the test range of 5–100 µg/mL was y = 4.4311 x + 0.4386, R2 = 0.9992 (y: the peak area, x: CK concentration, µg/mL). Drug loading (DL) and entrapment efficiency (EE) of CK-Ngs were calculated by Equations (2) and (3).