**3. Results and Discussion**

#### *3.1. Synthesis of FA–CS Conjugate*

The synthesis of the FA–CS conjugates was carried out by means of carbodiimide chemistry using the water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Figure 1). The EDC is a "zero-length" crosslinked chemical. It is used in the formation of conjugate via amide linkage without leaving a spacer molecule [18]. The EDC reacted with the COO− of the FA and 5-FU to form an intermediate of active ester. The intermediate reacted further with the primary amine (NH2) group of the CS, giving rise to an amide (N– H) bond, with an isourea by-product that was removed easily by filtration or dialysis [18]. The FTIR and 1H-NMR spectra (Figures 2 and 3) successfully confirmed the conjugation of folic acid onto chitosan molecules.

**Figure 1.** A scheme illustrating the reaction of chitosan with folic acid.

**Figure 2.** FTIR spectra of (**a**) pure chitosan, (**b**) pure FA, and (**c**) FA–CS conjugate.

**Figure 3.** *Cont*.

**Figure 3.** 1H-NMR spectra of (**A**) pure chitosan, (**B**) pure FA, and (**C**) FA–CS conjugate.

#### 3.1.1. FTIR Studies

In an FTIR of chitosan (Figure 2), a strong band at the region of 3400 cm−<sup>1</sup> represents NH functional groups (primary amine). The absorption band at around 2977 cm−<sup>1</sup> can be attributed to CH symmetric stretching. Bands at 1401 cm−<sup>1</sup> indicate a methyl group (CH3). Symmetrical bending in the range of 1260–800 cm−<sup>1</sup> belong to the glycosidic ring; in particular, the band at 1156 cm−<sup>1</sup> corresponds to the glycosidic linkage. Similarly, an FTIR of pure folic acid showed the IR spectrum at 3100–3500 cm−<sup>1</sup> which can be attributed to the OH carboxylic of glutamic acid moiety and the NH group of the pterin ring stretching. Absorption at 1760 cm−<sup>1</sup> represents C=O carboxylic acid in pure FA. Similarly, the absorption band at 1432 cm−<sup>1</sup> represents the phenyl and the pterin ring. The band at 3321 cm−<sup>1</sup> pure folic acid is absent/overlapped in the conjugate formulation, indicating the coupling of folate with a chitosan polymer [19]. The primary amine of the chitosan reacted with the carboxylic acid group of folic acid, forming an amide bond. The amide bond formation between the chitosan and folic acid was evidenced by a shift of the FTIR wavenumber of folic acid from 1760 to 1680 cm<sup>−</sup>1. The assignment of FTIR peaks was

correlated with earlier studies [20–22]. The peaks at 2.07 ppm attributed to the acetamino group CH3, and the CH peak appeared at 3.50–3.95 ppm, corresponding to carbons 3, 4, 5, and 6 of the glucosamine rings of CS.

#### 3.1.2. H-NMR Study

FA has two active –COOH groups at its end point. Among these, γ-COOH is more sensitive to the reaction, owing to its high reactivity [23]. The final product of FA-CS was synthesized by the reaction between the activated FA ester and the primary amine NH2 groups of CS through the formation of an amide bond under homogeneous conditions. The peaks at 2.08 ppm attributed to the hydrogen atom of the methyl group (CH3) of the acetamino groups of chitosan, as well as CH peaks at 3.77–3.8 ppm, can be explained by hydrogen bonded to carbons 3, 4, 5, and 6 of the glucosamine rings of CS [24]. The CS conjugation was confirmed by the peculiar signals at 2.5 ppm, which attributed to the aromatic protons of the FA, and characteristic peaks at 2.84 ppm corresponded to the FA proton from the H22 [25]. This was ascribed to the development of amide linkage after the folic acid–chitosan conjugation. Ji et al. previously reported similar results at 2.45 ppm in relation to the FA proton from the H10 and H22 [24], respectively, which is in line with the current study.
