Preparation and characterization of nsGO/PCP/OVA

Epichlorohydrin was added to the nsGO solution at 40°C for 4 h under N₂ protection. Once the unreacted epichlorohydrin was removed by ultrafiltration, endotoxin-free PCP solution (10 mg/mL) was added to epichlorohydrin-GO solution in water bath at 42°C for 3 h. After the pH of the nsGO/PCP solution was adjusted to weak acidity, a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro/N-hydroxy succinimide (EDC/NHS) mixture was added, followed by incubation for 30 min. Subsequently, the OVA protein solution (1 mg/mL) was added to the prepared solution for 1 h at room temperature. Then, the unreacted protein was removed by ultrafiltration and the lyophilized solid was collected as nsGO/PCP/OVA nanoparticles. The concentration of nsGO/PCP/OVA was quantified by the OVA level using the Bicinchoninic Acid Assay kit (BCA, Biomed Inc, CA, United States). The encapsulation efficiency was measured using the following formula: [(Total OVA–Free OVA)/Total OVA] ⋅ 100%. The nanoparticles were characterized using transmission electron microscopy (TEM, Hitachi, Japan), Fourier Transform infrared spectroscopy (FT-IR spectroscopy, Spectrum 100 FTIR, PerkinElmer, MA, USA), and dynamic light scattering (DLS, Malvern, Germany). TEM images were analyzed for the nanoparticle size distribution using Nano measurer 1.2 software (Fudan University, China).