Characterization of drug-loaded micelles

The zeta potential, hydrodynamic diameter and particle size distribution of the Rh₂-M were measured by Malvern system (ZEN-3600, Malvern Instruments, Worcestershire, UK). Micellar solutions were filtered with a 0.45 µm filter prior to the measurement. All the values were the average of at least three parallel measurements.

Transmission electron microscopy (TEM) pictures of the copolymer micelles were captured on a transmission electron microscopy (TEM; JEM-200CX, JEOL, Tokyo, Japan). Briefly, A drop of Rh₂ micelles solution (1.0 mg/mL) was dipped into the copper grid, mixed with a drop of 2% (w/v) phosphotungstic acid. After the deposition, samples were observed under TEM.

The drug-loading efficiency (LE%) and encapsulation efficiency (EE%) were determined using a 4.6 mm × 250 mm Hypersil ODS C18 5.0 μm column (Thermo, Waltham, MA) by HPLC (Agilent 1260, Palo Alto, CA). Briefly, the Rh₂-M solution was diluted with methanol to dissociate the micellar nanoparticles and centrifugation at 15,000 rpm for 15 min. Then the supernatant was filtrated by 0.45 μm filter membrane and the concentration of Rh₂ was determined by HPLC. The composition of the mobile phase was acetonitrile: water: phosphoric acid (65:35:0.2, v/v/v) with a flow rate of 1.0 mL/min at 35 °C. Quantitation was achieved using UV detection at 203 nm. A typical injection was 20 μL. The method was linear over a wide concentration range of 0.02–1.0 mg/mL. Assay method had been verified by methodology.

Then, use the following formula to calculate the LE% and EE%: