Preparation and characterization of drug-loaded nanoparticles.

Drug-loaded nanoparticles (FR-np) were prepared by an emulsification solvent evaporation technique⁶¹. Briefly, PLGA/PLGA-b-PEG (50/50 wt/wt) was dissolved in dichloromethane to make 10% w/v solutions, then 5% (wt/wt) of chemicals (F and R with the same molar ratio) to the polymer weight were co-dissolved in the polymer solution. The resulting solution was added to a stirred 1% (w/v) PVA solution using a vortex mixer at 2000 rpm for 2 minutes and then sonicated with a 20% amplitude (Sonic Dismembrator 500, Thermo Fisher Scientific) for 40 seconds. After sonication, the emulsion was added dropwise into 1% PVA and stirred for 3 hours at room temperature to remove the residual organic solvent. The nanoparticles were collected and washed three times with distilled water by centrifugation at 10,000xg for 5 minutes at 4°C. Particle diameter was measured by dynamic light scattering (DLS), and the surface morphology was observed by SEM with gold electrospray. The drug release rate from drug-loaded nanoparticles in PBS (pH 7.4) at 37 °C was measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS, Agilent 6460), as previously described⁶¹.