Gatanultrascan 4000 ccd camera

Nanoparticles were characterized for hydrodynamic diameter and morphology using photon correlation spectroscopy and transmission electron microscopy (TEM), respectively. In brief, nanoparticle dispersion in deionized water (0.1 ​mg/mL) was used to measure particle size using a Nanobrook Omni (Brookhaven Instruments, Holtsville, NY) operating at a 165° constant scattering angle. For TEM, a dispersion of nanoparticles was added onto a copper grid covered by a continuous thin carbon film (400 mesh, Ted Pella Inc., Redding, CA, USA) and air-dried overnight. Images were acquired on a FEI Tecnai G2 F30 ​S-TWIN instrument at 300 ​kV with a Gatan UltraScan 4000 CCD camera (Gatan, Pleasanton, CA, USA) and using Gatan Digital Micrograph 3.9.5 software (Gatan, Pleasanton, CA, USA). A 3–6 ​μm of under-focus was used to improve the phase contrast. Drug loading in nanoparticles was determined following extraction in methanol. Nanoparticle dispersion in methanol (1 ​mg/mL) was agitated overnight using a tube rotator (VWR International, Radnor, PA). The following day, dispersion was centrifuged at 13,000 ​rpm for 30 ​min to remove nanoparticles. The supernatant was quantified for PTX amount using reversed-phase high-performance liquid chromatography (HPLC) as described previously (Agilent Technologies, Santa Clara, CA) [22 ].