3.1.3. Folate (FA) Content

Folate content was found to be 5% of the total weight of the FA-CS-5FU-NPs formulation. Folic acid is commonly engaged as a ligand for targeting cancer cells, as its receptors are over-expressed on the surface of several human cancer cells. Integrating folic acid into chitosan-based drug delivery inventions directs the systems with a well-organized targeting ability [26].

#### *3.2. Characterization of Nanoparticles*

#### 3.2.1. Size, Zeta Potential, and Surface Morphology

The size of the CS-5-FU NPs was found to be 208 ± 14.65 nm, while the FA-CS-5-FU NPs was 235 ± 11.5 nm, as shown in Table 1 and Figure 4A,B. The poly dispersity index (PDI) was found to be 0.2 and 0.19 for of the FA-CS-5-FU NPs and the CS-5-FU NPs. This small size of NPs is important since such NPs in anticancer drugs can easily escape the leaky tumor vasculature and accumulate within the tumor region to exert cytotoxic effects on proliferating cells [27]. The zeta potential of the FA-CS-5-FU NPs was found to be +20 ± 2. The FA-CS-5-FU NPs show an insignificant decrease in zeta potential as compared to the CS-5-FU NPs (Table 1; *p* > 0.05). Other studies have also found a decrease in the value of zeta potential after folate conjugation. [28–30]. The obtained zeta potential value of +26 ± 2 mV in the instance of folic-acid-modified chitosan NPs indicates that folic acid binds to chitosan quite strongly. The free positive NH2 groups of chitosan molecules may account for the positive value. This positive zeta potential is helpful in crossing the negatively charged membrane of cancer cells. The value of the zeta potential (ZP) indicates the repulsive interactions between suspended particles and can therefore be used to forecast the stability of colloidal aqueous dispersions. The prepared nanoparticles were spherical in shape and smooth in surface, as shown in Figure 4B.

**Table 1.** Physicochemical characterization of folic-acid-modified 5-FU-loaded chitosan NPs. Data were presented as triplicate (*n* = 3) and mean ± SD.


**Figure 4.** (**A**) Size distribution of nanoparticles, (**B**) surface morphology of folic-acid-modified 5-FU-loaded chitosan NPs.

#### 3.2.2. Drug Content, Encapsulation Efficiency, and Drug-Loading Efficiency

TPP was used as a cross linker in folate-modified chitosan nanoparticles loaded with 5-FU. The drug content and %EE of the drug were estimated based on the amount of the drug in the supernatant and the sedimented pellets of dispersed nanoparticles after centrifugation. The FA-CS-5-FU NPs demonstrated 5-FU content of 53 ± 0.14% and EE of 59 ± 0.23%. The drug-loading efficiency was 43 ± 3% and 39 ± 2% for CS-5- FU NPs and FA-CS-5-FU NPs, respectively. A decrease in the loading efficiency of NPs with FA conjugation occurred because the folic acid changed a number of amino groups on the chitosan molecules, lowering their positive charges and thereby attracting drug molecules [13]. Consequently, it emerged that the amount of folic acid conjugations in the mixture had a significant effect on the loading efficiency (LE) (Table 1; *p* < 0.05).

#### *3.3. In Vitro Release*

In vitro drug release was evaluated at a pH of 1.2 and 6.5 to measure the 5-FU release from the FA-CS-5FU-NPs and the CS-5FU-NPs using a USP dissolution apparatus 1. Such conditions were set to simulate the acidic gastric and physiological environment of the intestine. The percentage of drug released from the FA-CS-5FU-NPs and the CS-5FU-NPs was in the range of 10.08 ± 0.45% to 96.57 ± 0.09% and 6 ± 0.31% to 91.44 ± 0.21%, respectively. In artificial gastric liquid, 17.02 ± 0.12% and 14.5 ± 0.41% of 5-FU were released from the FA-CS-5FU-NPs and CS-5FU-NPs, respectively, in the first 2 h. The difference in the release pattern of these two formulations was insignificant, as shown in Figure 5 (*p* > 0.05). The initial release of 5-FU at an acidic pH was followed by a sustained release of up to 24 h. The initial release may be due to weakly bound drugs on the surface of nanoparticles [20].

**Figure 5.** In vitro release study of pure 5-FU, CS-5FU-NPs, and FA-CS-5FU-NPs.

Drug release at a pH of 6.5 within the first 2 h from the FA-CS-5FU-NPs and CS-5FU-NPs was 39.37 ± 3% and 36 ± 2.45%, and the accumulative release in 24 h (1440 min) was 96.57 ± 7% and 91.44 ± 7.45%, respectively. These in vitro values indicate that the FA-decorated nanoparticles can be used as a 5-FU delivery vector with a typical controlled release process. The remarkably high release rate of 5-FU from the folic-acid-conjugated nanoparticles, more interestingly at a pH of 6.5, may be due to the increased acidity of the respective release media caused by the presence of folic acid on the targeted nanoparticles. The improved hydrophilicity of the FA–CS nanoparticles due to the addition of folate was linked to the increased release rate [31]. Taken together, the acidic environments of tumor cells are likely to elicit the release of 5-FU from the developed delivery vehicles, and the sustained drug release profile from the vehicles over time can reduce dosing regimens [32].

#### *3.4. Cytotoxicity Studies*

Cytotoxicity studies of free drug and NPs were performed on caco-2 cell lines. The percentage of cell death was determined and shown in Figure 5. The IC50 value of free 5-FU was found to be 4.21 μg/mL. This value was reduced to 3.43 μg/mL (CS-5-FU-NPs) and 2.67 μg/mL (FA-CS-5-FU-NPs) when 5-FU was incorporated into nanoparticles, showing significantly more cytotoxicity than the free drug. Up to 9% of cell death was induced by the free drug (5-FU solution). The percentage of cell death increased when the CS-5- FU-NPs and FA-CS-5-FU-NPs were applied. This increase might be due to the combined effect of drug and hydrophilicity of the FA–CS nanoparticles due to the addition of folate, which began to increase the release rate of 5-FU from NPs. However, a significant effect (*p* < 0.05) on the percentage of cell death was produced when the FA–CS-conjugated NPs were applied (Figure 6). This was the resultant effect of the combination of the drug and folic acid conjugation with chitosan. The FA receptors are more expressed on cancer cells; therefore, the introduction of folic acid on NPs makes them more targeted and cytotoxic in action.

**Figure 6.** Cytotoxicity study shows the % cell death of 5-FU, CS-5-FU-NPs, and FA-CS-5-FU-NPs.

#### **4. Conclusions**

Nanoparticles were successfully prepared using the ionic gelation method. The size and zeta potential and PDI of the CS-5FU-NPs were 208 ± 15, 26 ± 2, −20 ± 2, respectively, and those of the FA-CS-5FU-NPs were 235 ± 12, +20 ± 2 and 0.25, respectively, which are within acceptable ranges. FTIR and 1H-NMR studies confirmed the conjugation of folic acid with the nanoparticles. The drug contents' % yield and the %EE of folate-decorated NPs were 53 ± 1, 80.8 and 59 ± 2%, respectively. The in vitro release of FA-CS-5FU-NPs and CS-5FU-NPs was in the range of 10.08 ± 0.45 to 96.57 ± 0.09% and 6 ± 0.31 to 91.44 ± 0.21, respectively. The percentage of cell death increased in the presence of folic acid, as compared to the free drug and chitosan nanoparticles due to the overexpression of folate receptors on the cancer cells. The results of all these parameters indicate that folatemodified chitosan 5-FU nanoparticles can be used successfully for the delivery of 5-FU with enhanced cytotoxicity and targeted delivery to the tumors.

**Author Contributions:** Conceptualization, A.K.A. and M.M.A.-D.; Data curation, A.K.A. and A.N.; Formal analysis, M.I.; Funding acquisition, A.K.A. and M.M.A.-D.; Investigation, S.U. and M.I.; Methodology, S.U., A.N. and K.U.S.; Validation, A.A.S.; Visualization, A.K.A.; Writing—original draft, S.U.; Writing—review & editing, A.K.A., A.N., K.U.S., M.I., G.M.A., F.A.A.-J., A.A.S. and M.M.A.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R30), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia; and Faculty of Pharmacy, AIMST University, Kedah, Malaysia.

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Review Board of office of Research, Innovation, and Commercialization (ORIC, 1600/ORIC/2019-ag-394).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R30), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.; and Faculty of Pharmacy, AIMST University, Kedah, Malaysia.

**Conflicts of Interest:** The authors declare no conflict of interest.
