3.3. Characterization of the PPHE Polymeric Nanoparticles

The γ-PGA-Pheo a and HA-EAE7 structures were analyzed by ¹H NMR spectroscopy (ADVANCE III 400, Bruker BioSpin, Billerica, MA, USA). The morphology of the resultant PPHE polymeric nanoparticles was observed by transmission electron microscopy (TEM, H-7600, Hitachi, Tokyo, Japan) after sputter-coating the samples with platinum. The average diameter of the PPHE polymeric nanoparticles was determined by analyzing the TEM images with Image-Pro Plus (Media Cybernetics Inc., Rockville, MD, USA). In addition, the particle size distribution of the PPHE polymeric nanoparticles was determined by the dynamic light scattering (DLS) technique using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The UV–visible spectra were recorded on a Hitachi U-2900 spectrometer (Tokyo, Japan), while the fluorescence emission spectra were measured using a Perkin-Elmer LS55 spectrofluorophotometer (Waltham, MA, USA) at room temperature.

The in vitro release studies of Pheo a from the PPHE polymeric nanoparticles were performed using a dialysis method in a thermostatic shaking incubator (NB-205, N-BIOTEK, Bucheon, Korea). A weighed amount (10 mg) of the PPHE polymeric nanoparticles was dispersed in 10 mL DPBS and then transferred into a dialysis membrane (molecular weight cut-off 2 kDa). The dialysis membrane was immersed into 100 mL DPBS (pH 4.5 or 7.4) and placed in a shaking incubator (200 rpm, 37 °C). The supernatant was collected from the DPBS solution at preset time points. The cumulative release amount of Pheo a from the PPHE polymeric nanoparticles was determined by measuring the absorption of the samples at 403 nm using a UV–visible spectrometer. The percentage of released Pheo a was then calculated based on the initial weight of Pheo a conjugated in the PPHE polymeric nanoparticles.