*2.2. Conjugation of Folic Acid (FA) with Chitosan (CS)*

The conjugation process of folic acid (FA) to chitosan (CS) is described as follows. FA and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) solution in anhydrous dimethylsulfoxide (DMSO) (20 mL), with 1:1 molar ratio, was made and stirred at room temperature until the EDC and FA were mixed well. The solution was then slowly added to 0.5% (*w*/*v*) CS in an aqueous solution of 0.1 M of acetic acid with a pH of 4.7, and then stirred at 25 ◦C in the dark area for 16 h to let the FA conjugate onto the CS molecules. Then, 1 M of NaOH was added to adjust the pH of the solution to 9.0. The solution was centrifuged at 2500 rpm to settle down the FA–CS conjugate. The sediment was first dialyzed against a phosphate buffer with a pH of 7.4 for 3 days, and then against water for 4 days. Finally, the FA–CS conjugate was collected as a sponge by freeze-drying and kept for further study [13].

#### 2.2.1. Fourier Transform Infrared Spectroscopy

The Fourier transform infrared spectroscopy (FTIR) was performed using an ATR FTIR spectrometer (L1600300, PerkinElmer, Beaconsfield, UK). The FTIR spectra of chitosan, folic acid, and its conjugate (FA–CS) were obtained. The recording range of the spectrum was 600–4000 cm−<sup>1</sup> at 32 scans per minute with a resolution of 4 cm−<sup>1</sup> in absorbance mode. After recording, the spectra were baseline, corrected, and normalized using Spectra software to identify the characteristic peaks and differences [14].

#### 2.2.2. H-NMR

For NMR spectroscopic analyses, solutions of CS, FA, and their conjugates were prepared in 1.97 mL of CDCl3 and kept at room temperature until their complete dissolution. Acetic acid was used as a co-solvent for the solubility of chitosan in CDCl3. 1H-NMR spectra were obtained using a Bruker AV-500 MHz NMR spectrometer. Bruker–Topspin software (version 4.1.1) was used for the analysis of NMR spectra.

#### 2.2.3. Determination of Folate (FA) Content

The FA–CS conjugates were accurately weighed and then dissolved in 50 mL of 0.2 molar sodium bicarbonate buffer solution with a pH of 10 at 25 ◦C with magnetic stirring. The solution was centrifuged at 3500 rpm for 10 min (Laboratory Centrifuge, YJ03-0434000, Shanghai, China). The supernatant was tested for determining folate (FA) using a UV– visible spectrophotometry technique with a wavelength of 365 nm. The folate content was calculated as the percentage of FA in a unit weight of conjugate. For each experiment, at least three duplicates were carried out and the results were averaged.
