Ultrascan 895

The structure and morphology of the RM-PL were visualized using both transmission electron microscope (TEM) and cryogenic electron microscopy (Cryo-EM). For TEM imaging, RM-PL at a concentration of 0.1 mg/mL was deposited onto a glow-discharged carbon-coated grid and was then stained with 1% uranyl acetate. The grid was dried at room temperature and visualized using a TECNAI G2 S-TWIN microscope (FEI, USA). For Cryo-EM imaging, RM-PL was plunge-frozen on holey film grids. A 626 cryo-specimen holder was used for imaging. Data were collected on a JEOL 2100 electron microscope (Japan). Images were recorded under low electron dose conditions (5–20 electron/Å2) using a 4096 × 4096 pixel CCD camera (UltraScan 895, GATAN, Inc., Pleasanton, CA, USA) at a nominal magnification of 20,000 × .
A dynamic light scattering (DLS) detector (Zetasizer, Nano-ZS, Malvern, UK) was employed to measure the size, polydispersity index, and ζ potential of RM-PL. Samples were diluted 20 folds with distilled water at room temperature. The measurements were performed in triplicate. To examine the stability of the formulations in solution, RM-PL was suspended in 1 × PBS or PBS containing 5% fetal bovine serum (FBS) at 4 °C at a concentration of 2 mg/mL. The particle size and polydispersity index were monitored by DLS daily for 6 or 5 days.

Cryo-electron microscopy (cryo-EM) was utilized to examine oxcarbazepine-loaded nanoparticles and to ensure the absence of any free oxcarbazepine crystals that might crystallize out during the fabrication process. Oxcarbazepine-loaded nanoparticles were vitrified on holey carbon film grids (C-flat,™ Protochips, Raleigh, NC, USA) to minimize any potential morphological changes during specimen preparation and imaging.31 (link),32 (link) In brief, nanoparticle dispersions were applied to the holey films in a volume of ~2 μL, blotted with filter paper, and plunged into liquid ethane cooled in a liquid nitrogen bath. Frozen grids were stored under liquid nitrogen and transferred to a cryo-specimen 626 holder (Gatan, Inc., Pleasanton, CA, USA) under liquid nitrogen before loading them into a JEOL 2200 electron microscope, with a field emission gun operating at 200 keV. Grids were maintained at near-liquid nitrogen temperature (−172°C to −180°C) during imaging. Nanoparticles were imaged at 25,000× indicated microscope magnification with a 4 k ×4 k slow-scan CCD camera (UltraScan 895, Gatan, Inc.) using a low-dose imaging procedure.