2.4. Nanoparticle Characterization

The size, polydispersity (PDI), and zeta potential of the nanoparticles were characterized immediately after FNP and after filtration using dynamic light scattering (Malvern Zetasizer ZS, Malvern Instruments Ltd., Malvern, United Kingdom). The nanoparticle size and polydispersity index (PDI), a measure of uniformity, were measured by averaging 4 measurements at a scattering angle of 173°. Nanoparticles populations with a PDI of less than 0.400 were considered uniform [37]. The nanoparticle size stability at 4 °C was observed by measuring size and PDI for up to 3 weeks after formulation. The concentration of the nanoparticle dispersion following filtration was determined by thermogravimetric analysis (TGA) (Pyris 1 TGA, Perkin Elmer, Waltham, MA, USA).

Transmission electron microscopy (TEM) samples were prepared by diluting the filtered nanoparticle dispersions with DI water 1:20 by volume ratio and pipetting 5 μL three times onto a TEM grid with Formvar/Carbon support films (200 mesh, Cu, Ted Pella, Inc., Redding, CA, USA) and dried under ambient conditions. Dilution was necessary to prevent aggregation during drying. The samples were imaged with a JEOL JEM-1230 (JEOL USA, Inc., Peabody, MA, USA) at 120 kV.

To determine the drug content of the nanoparticles, acetonitrile (1.8 mL) was added to nanoparticles (50 μL) filtered with Amicon filter, as previously described, and the sample was vortexed so that the nanoparticles would disassemble. The sample was centrifuged at 10,000× g rpm for 6 min, and then the supernatant was collected for reverse-phase high-performance liquid chromatography (RP–HPLC) (1260 HPLC with Quaternary Pump and UV–Vis Diode Array Detector, Agilent, Santa Clara, CA, USA) fitted with a Luna^® 5 µm C18 100 Å, LC Column 250 × 4.6 mm (Phenomenex, Torrance, CA, USA). The sample was eluted with degassed acetonitrile and water gradient at a flow rate of 1 mL/min (0–1 min at 80:20, 1–6 of ramp up to 0:100, 6–8 min at 0:100, and ramp down to 80:20 between 8 and 9 min). PTX was measured at a wavelength of 228 nm with a retention time of ~8 min and LAP was measured at 332 nm with a retention time of ~9 min. The concentration of each drug was determined by comparing the peak areas with the standard calibration curve. Encapsulation efficiency (EE%) and drug loading (DL%) were calculated based on Equations (1) and (2), respectively, and the values reported are the average and standard deviation of three trials.