2.2. Synthesis and Characterization of Folate-PEG

For the synthesis of folate-PEG conjugated nanoparticles, firstly, the carboxylate group of FA was activated by NHS and N’,N’-dicyclohexyl carbodiimide (DCC) for conjugation with the amine group of bifunctional PEG (Figure S1A) [22]. Briefly, a mixture of FA (1.5 g) and DMSO (20 mL) was reacted with DCC (0.65 g) and NHS (1 g) for 12 h at 25 °C in a nitrogenous atmosphere. A solution of NH₂–PEG–COOH (0.65 g) and DMSO (5 mL) was mixed with activated FA and kept for 5 h at 25 °C in a nitrogenous atmosphere. The resultant solution was diluted with acetone (45 mL) (non-solvent for FA) and centrifuged; subsequently, the supernatant was dialyzed using a dialysis bag (Spectra Por 6, MW cutoff, 1000) in deionized water; finally, it was freeze-dried and stored until use in the preparation of PTX/Zein-FA. Before it had been used in the experiments, the yellowish freeze-dried powder of FA–PEG–COOH was characterized for chemical purity by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), ¹H NMR, and proton nuclear magnetic resonance (Varian Inc., Palo Alto, CA, USA) analysis, using d₆-DMSO as a solvent (Figure S1B). The amount of FA conjugated in FA–PEG–COOH was calculated by using the standard curve of free FA, which was measured at 365 nm using an ultraviolet (UV) spectrophotometer (PerkinElmer U-2800, Hitachi, Tokyo, Japan) [23]. The absorbance of the polymer-conjugated FA was also measured under the same UV-visible spectroscopy conditions for free FA (Figure S1C).