*2.1.7 Magnetization studies using (SQUID) analysis*

The saturation magnetization of Fe3O4 NPs, CS-Fe3O4-OCS ferrogel was measured by vibrating sample magnetometer VSM (SQUID model MPMS XL 7) from Quantum at room temperature between magnetic fields of −14,000 (Oe) to 14,000 (Oe).

#### *2.1.8 Scanning electron microscopy (SEM)*

The chitosan-based hydrogels cross-linked with oxidized chitosan was frozen at −75°C for 24 h and then lyophilized (by Alpha 1–2 LDplus) for 48 h. The lyophilized sample was obtained and then examined by a scanning electron microscopy (SEM) (Mini SEM Hirox SH-4000).

#### *2.1.9 Hydrogel swelling*

Freeze-dried CS/OCS hydrogel were immersed in phosphate-buffered saline (PBS) at 37°C (pH = 1.2, pH = 5.8 and pH = 7.4). The samples were weighed before being put into PBS (W0). After the vial was sealed and held at 37°C for 24 h, and the excess solution on the surface of the hydrogels was quickly absorbed with filter paper [55]. The equilibrium-swelling ratio (SR) was calculated using the following equation:

$$\mathbf{S} \mathbf{R} \mathbf{\%} = \frac{\mathbf{W}\_{\text{s}} - \mathbf{W}\_{\text{d}}}{\mathbf{W}\_{\text{d}}},\tag{1}$$

where, Wd is the weight of dry hydrogel after lyophilization and Ws is the weight of swollen hydrogel.

#### *2.1.10 In vitro drug release from the hydrogels and ferrogels*

(5-FU, caffeine or ascorbic acid) was selected and used as a model drug in the release experiments. In vitro drug release test was performed in a phosphate buffer solution PBS (pH 7.4 at 37°C) under shaking. The hydrogels and ferrogels (m = 1.14 g) (3 cm x 4 cm) were placed in a cartouche before immersing in 1000 mL of phosphate-buffered saline (PBS). At predetermined time intervals, 5 mL of release medium was withdrawn. Then 5 mL of fresh buffer was added to the original to maintain the total volume. The drug release was determined by UV–Vis spectrophotometry at λmax (5-Fluorouracil (266 nm), caffeine (273 nm) and ascorbic acid (265 nm)). The concentration of the active ingredient in the (PBS, pH = 7.4 at 37°C) has been achieved from the calibration curve, and the amount of drug released at time t (Mt) was calculated by accumulating the total active ingredient release up to that time. In vitro drug release tests were performed in triplicate (n = 3). There are a few steps, which mainly control drug release phenomena from the polymer matrix, dissolution of the drug, liquid penetration into the matrix and diffusion of the drug from the drug encapsulated in the matrix. In order to understand the release kinetics and the mechanism of the active ingredient release, release kinetics data obtained in vitro using ferrogels and hydrogels are fitted with kinetics model. The release data are best fitted with the Korsmeyer−Peppas (KP) model. The (KP) model deal with Eq [57]:

$$\% \text{Cumulative release} = \frac{\mathbf{M}\_t}{\mathbf{M}\_o} \times \mathbf{100} \tag{2}$$

where "Mt" is the amount of drug released at time (t), "M0" is the maximal amount of the drug released at maximum interval. It is interesting to note that three drugs (5-FU, caffeine and ascorbic acid) in hydrogels exhibit a Fickian nature of drug diffusion. However, the interaction of the drug molecules with the matrix play an important role in the drug release kinetics occurring through a diffusion mechanism.

#### **2.2 Statistical analysis**

The experimental data are expressed as the mean values of at least three replicates ± standard deviation (SD). The results were analyzed and showed usage Kaleida graph.
