*Article* **5-Fluorouracil-Loaded Folic-Acid-Fabricated Chitosan Nanoparticles for Site-Targeted Drug Delivery Cargo**

**Shafi Ullah 1,†, Abul Kalam Azad 2,\*,†, Asif Nawaz <sup>1</sup> , Kifayat Ullah Shah 1, Muhammad Iqbal <sup>1</sup> , Ghadeer M. Albadrani <sup>3</sup> , Fakhria A. Al-Joufi <sup>4</sup> , Amany A. Sayed <sup>5</sup> and Mohamed M. Abdel-Daim 6,7,\***


**Abstract:** Nanoparticles play a vital role in cancer treatment to deliver or direct the drug to the malignant cell, avoiding the attacking of normal cells. The aim of the study is to formulate folicacid-modified chitosan nanoparticles for colon cancer. Chitosan was successfully conjugated with folic acid to produce a folic acid–chitosan conjugate. The folate-modified chitosan was loaded with 5-FU using the ionic gelation method. The prepared nanoparticles were characterized for size, zeta potential, surface morphology, drug contents, entrapment efficiency, loading efficiency, and in vitro release study. The cytotoxicity study of the formulated nanoparticles was also investigated. The conjugation of folic acid with chitosan was confirmed by FTIR and NMR spectroscopy. The obtained nanoparticles were monodispersed nanoparticles with a suitable average size and a positive surface charge. The size and zeta potential and PDI of the CS-5FU-NPs were 208 ± 15, 26 ± 2, and +20 ± 2, respectively, and those of the FA-CS-5FU-NPs were 235 ± 12 and +20 ± 2, respectively, which are in the acceptable ranges. The drug contents' % yield and the %EE of folate-decorated NPs were 53 ± 1.8% and 59 ± 2%, respectively. The in vitro release of the 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 cytotoxicity of the nanoparticles was enhanced in the presence of folic acid. The presence of folic acid in nanoparticles shows much higher cytotoxicity as compared to simple chitosan nanoparticles. The folate-modified nanoparticles provide a potential way to enhance the targeting of tumor cells.

**Keywords:** colon cancer; targeted delivery; folate-conjugated nanoparticles; cytotoxicity
