2.4.1. NP Size and Zeta Potential

Photon correlation spectroscopy was used to determine the particle size and zeta potential of FA-CS-5FU-NPs and CS-5FU-NPs at 25 ◦C in quartz cell and zeta potential cell with a detect angle of 90◦, respectively, using a Malvern Zetasizer Nano ZS 90 (Malvern Instruments Ltd., Malvern, UK). In 5.0 mL of deionized water, one mg of NPs was added, and vortex stirring (Velp Scientifica, Usmate Velate, Italy) was used to fortify the mixture [15].

### 2.4.2. Nanoparticle Morphology

The surface morphology of NPs was examined using the scanning electron microscopy (SEM) technique (JSM6360LA, JEOL, Tokyo, Japan). The NPs were fixed with carbon tape onto studs and directly examined under the SEM. Images of the NPs were captured at a 20,000× magnification level [15].

#### 2.4.3. Percentage Yield, Drug Entrapment Efficiency (%EE), and Drug-Loading Efficiency

Precisely weighed 15 mg of FA-CS-5FU-NPs and CS-5FU-NPs was dispersed in 15 mL of distilled water under magnetic stirring at 200 rpm for 2 h in two separate beakers followed by centrifugation (Laboratory Centrifuge, YJ03-0434000, Shanghai, China) at 5000 rpm for 45 min. The supernatant of both formulations was isolated and analyzed for free 5-FU using the UV spectroscopy technique. The percentage (%) yield was calculated using the formula given in Equation (1) [16]:

$$\% \text{ } yield = \text{Mass of NPs obtained} / \text{ total weight of drug and polymer} \times 100\tag{1}$$

Both drug entrapment efficiency (%EE) and drug-loading efficiency were determined indirectly using free drug concentration. After centrifugation, the obtained sediments of the formulations were dissolved in ethanol, aliquot filtered, and analyzed at 265 nm in UV spectroscopy for drug entrapment efficiency using Equation (2).

$$EE\,\left(\%\right) = \frac{5\text{FUtotal} - 5\text{FU Free}}{5\text{FUtotal}} \times 100\tag{2}$$

The total drug load collected from the supernatant and sediment was used to calculate the drug-loading efficiency using Equation (3). Triplicates were conducted and the results were averaged [16].

$$\text{Drug loading efficiency} = \frac{\text{5FUtotal} - \text{5FUfree}}{\text{Weight of 5FU loaded nanopparticles taken}} \times 100 \tag{3}$$

#### *2.5. In Vitro Release of Nanoparticles*

The release rate for the designed formulations was studied for up to 2 h in 900 mL of release media such as simulated gastric fluid (solution of 0.2 MHCL and 0.2 MKCl, pH 1.2) and simulated intestinal fluid (solution of 0.2 M potassium dihydrogen phosphate and 0.1 M sodium hydroxide, pH 6.5) for up to 24 h using a dissolution tester (basket method type 1) at 37.5 ± 0.5 ◦C. The stirring speed was set at 100 rpm. Then, 15 mg of FA-CS-5FU-NPs and CS-5FU-NPs was placed in two separate baskets and run the apparatus. At predetermined time intervals (0.5, 1, 1.5, 2, 4, 8, 12, 16, 20, and 24 h), a 5 mL sample was withdrawn and replaced with a fresh dissolution medium. All the samples were analyzed using a UV–visible spectrophotometer at a wavelength of 265 nm. The cumulative percentage of the drug released was calculated [17].
