BACKGROUND AND OBJECTIVES: The administration of 5-FU as the first chemotherapeutic agent for colorectal cancer, showed difficulties including short half-life and the development of resistance. One prominent approach to overcome these restrictions, is administration of 5-FU in conjunction with nanoparticles, particularly magnetic nanoparticles. In this study, FeO nanoparticles were prepared by the green synthesis and coated with polylactic acid-hyaluronic acid (PLA-HA) copolymer. Then, the HCT116 colorectal cancer cell line was used to assess the cytotoxicity and effectiveness of PLA/FeO and PLA-HA/FeO nanoparticles for the delivery of 5-FU medication. METHODS: The characteristics of these copolymers were investigated by H-NMR, FTIR and Thermogravimetric analysis. The nanoparticles were prepared using solvent diffusion technique and then characterized with different techniques like dynamic light scattering (DLS), TEM images, FTIR, UV-Vis spectroscopy, and VSM (Vibrational Sample Magnetometer). Ultimately, an assessment of drug encapsulation efficacy, the release profile and an in vitro analysis of cytotoxicity were performed to investigate the efficacy of drug delivery to HCT116 cells. RESULTS: The results of NMR, FTIR and TGA analysis confirmed the successful synthesis of copolymers. A zeta potential of -18 mV and a spherical shape with an average size of 235 nm were characteristics of the synthesized PLA-HA/FeO/5-FU nanoparticles. The encapsulation of FeO nanoparticles in PLA-HA copolymer decreased their magnetic saturation, and VSM analysis showed that the nanoparticles possessed superparamagnetic properties. Additionally, the 5-FU encapsulation efficiency was 42%, and it demonstrated a burst and sustained release pattern. It was discovered that the acidic pH was more effective. The MTT assay proved the low toxicity and biocompatibility of drug-free nanocarriers'. Remarkably, compared to PLA/FeO/5-FU micelles, the viability of HCT116 cells was found to be significantly reduced by PLA-HA/FeO/5-FU micelles. This phenomenon can be explained by the unique way that hyaluronic acid interacts with overex CD44 receptors. CONCLUSION: One potential strategy for targeted drug delivery and HCT116 cell line control is to encapsulate 5-FU in magnetite nanoparticles (FeO) made by the green synthesis method and use HA as cell-surface receptors to create PLA-HA/FeO/5-FU nanoparticles. Nanocarriers favorable physicochemical characteristics and potent apoptotic effects make them promising agents for precisely delivering drugs to colon cancer cells. HIGHLIGHTS: • Drug delivery efficiency is significantly improved while utilizing different ligands are functionalized on the surface of nanoparticles. • Encapsulation of FeO nanoparticles into the PLA-HA/FeO/5-FU micelles and PLA-HA/FeO nanoparticles leads to the production of nanoparticles or micelles with appropriate magnetic properties. • Acidic environments cause more drug release than neutral ones, and since cancer cells have a lower pH than healthy cells, this can cause less drug release in healthy cells and ultimately fewer negative effects on healthy cells.