**1. Introduction**

Colon cancer (CC) is one of the leading causes of mortality and morbidity in the world. Nine percent of all cancer cases are colon cancer. Throughout the world, CC is the third most common cancer type and the fourth most common cause of death [1]. CC is the most common type of cancer among all cancer types in western countries. Cases of colorectal cancer have increased remarkably in the past fifty years, and such cases have become the second highest in females and the third highest in males. The primary means of treatment for CC is surgery, with chemotherapy and/or radiotherapy

**Citation:** Ullah, S.; Azad, A.K.; Nawaz, A.; Shah, K.U.; Iqbal, M.; Albadrani, G.M.; Al-Joufi, F.A.; Sayed, A.A.; Abdel-Daim, M.M. 5-Fluorouracil-Loaded Folic-Acid-Fabricated Chitosan Nanoparticles for Site-Targeted Drug Delivery Cargo. *Polymers* **2022**, *14*, 2010. https://doi.org/10.3390/ polym14102010

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 18 March 2022 Accepted: 9 May 2022 Published: 13 May 2022

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being indicated depending on the nature and severity of the disease. Chemotherapy using different anticancer agents can be used as an adjuvant treatment on the second number after surgery, as a neo-adjuvant treatment before surgery, or as a main treatment to abate tumor size and growth as well as metastasis risk [2]. The 5-fluorouracil (5-FU) drug is an analog of the pyrimidines, and therefore uses the same metabolic routes as uracil and thymine. It is classified as an antimetabolite drug, interfering with nucleoside metabolism in RNA and DNA, and is used for the treatment of various tumors, such as those found in breast adenocarcinoma, the gastrointestinal tract, the ovary, the head, and the neck [3]. Despite its effectiveness, 5-FU presents some drawbacks. After oral administration, 5-FU bioavailability is highly variable due to its inconsistent absorption in the gastrointestinal tract and first-pass metabolism through the liver. Thus, the 5-FU half-life is extremely short (6–20 min), and frequent and high doses are required to maintain adequate plasma concentrations [4]. An alternative to overcoming these drawbacks and improving drug bioavailability, promoting controlled drug release, and choosing for more cell selectivity is the application of polymeric nanoparticles as 5-FU carriers. The oral delivery of drugs is of tremendous interest for patients seeking safe and controlled drug delivery. Compared to injections, the oral administration of anticancer drugs via oral route is cost-effective, reducing the hospitalization duration of the patient, as well as improving the patient's quality of life. Examples of some drugs that are used for cancer treatment are as follows: 5-flourouracil (5-FU), hexacarbonyl-(5-FU), and N4 pentoxylcarbonyl-5-deoxy-5flourocytidine (capecitabine) [5].

Chitosan is a semi-crystalline, linear polysaccharide that is composed of (1-4)-2 acetamido-2-deoxy-β-D-glucan (*N*-acetyl D-glucosamine) and (1-4)-2-amino-2-deoxy-β-D-glucan (D-glucosamine) units. Chitosan is not extensively present in the environment in its original form but can be derived easily from chitin (a natural polymer) by removing its acetyl group. The ratio of D-glucosamine to the sum of D-glucosamine and N-acetyl D-glucosamine gives the degree of deacetylation (DD) of chitosan. DD indicates the number of amino (NH2) groups along the chains [6]. Chitosan provides a valuable tool for the current system of novel drug delivery owing to its intrinsic biological and physicochemical properties. The characteristics of chitosan nanoparticles (NPs), such as their small size, better stability, inexpensiveness, easy manufacturing process, lower toxicity, and versatile method of administration, made them favorable drug and gene delivery carriers. Chitosan can be easily chemically modified due to the presence of its active functional groups such as amine (NH2) and hydroxyl (OH) groups. Due to pH changes and electrostatic interactions throughout the gastrointestinal tract (GIT) that are vital for maintaining the NP's stability, the permanent positive charge of chitosan favors their mucoadhesion property in the intestinal mucosa layer. This characteristic has been used to develop enhanced drug delivery systems that could help in CC treatment [7].

The surface morphology of nanoparticles (NPs) can be modified with the conjugation of targeting ligands, such as folic acid (FA), antibodies, integrins, transferrin, and polysaccharides, to improve receptor affinity and internalization by target tissues. Many tumor cell surfaces overexpress folate receptors (FRs), which are less often expressed in normal and healthy cells. This feature makes tumor cells an excellent target for tumor-targeting drug delivery [8]. Folic acid (FA) has emerged as an optimal targeting ligand for the selective delivery of attached imaging and therapeutic agents to cancer cells and inflammation sites. The use of FA as a target ligand has arisen primarily from its following features: (1) its easy conjugation to both therapeutic and diagnostic agents; (2) its great affinity for the folate receptors (FRs); and (3) the distribution of folate receptors (FRs) in limited numbers in normal tissues. Folic acid as a targeting ligand has been investigated by many scientists [9]. In one study, it was demonstrated that the folic-acid-modified chitosan NPs were excellent vectors for the colon-specific delivery of 5-aminolevulinic acid (5-ALA) for fluorescent endoscopic detection [10]. The FA decoration upheld the establishment of a genuine affinity for FRs+ cancer cells even when co-cultured closely with higher numbers of healthy cells [11]. In one study, FA was evaluated in vitro, in which it was conjugated with carboxymethyl chitosan, and its nanoparticles were loaded with doxorubicin for targeted drug delivery. These FA-modified NPs manifested FA feasibility as an excellent targeted delivery carrier [12]. In the present study, folic-acid–chitosan-conjugated nanoparticles for oral delivery were prepared and evaluated for in vitro release and cytotoxicity studies.

#### **2. Materials and Methods**

#### *2.1. Materials*

Chitosan (deacetylation degree—83% and mol wt—310,000–375,000), 5-flourouracil and folic acid were obtained from Sigma-Aldrich (lot# A263299) (Sigma-Aldrich, Inc. St. Louis, MO, USA). TPP (85%), potassium dihydrogen phosphate, calcium chloride, 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC), and sodium hydroxide were obtained from Sigma Chemicals (Merck Pte. Ltd. 2 Science Park Drive, Singapore). Acetic acid, hydrochloric acid, ethanol, and DMSO were obtained from Merck (Merck KGaA, Darmstadt, Germany).
