BACKGROUND: Today, increasing attention is being paid to the application of biocompatible polymers as drug carriers with low cytotoxicity in drug delivery systems to enhance the therapeutic effects of anticancer agents. MATERIALS AND METHODS: In this study, a biocompatible synthetic polymer (grafted on graphene oxide), composed of N-isopropylacrylamide and 1-vinyl-2-pyrrolidone with L-lysine segments (Lys/PNIPAM-PVP/GO), was developed as a nano-vehicle for the drug. This platform was used for the delivery of fluorouracil (FU) to A549 human lung cancer cells. The superior characteristics of the platform included low-cost precursors, easy synthesis, and the presence of many functional groups for loading drugs. To determine and compare the cytotoxic effects of free FU and its formulated form on the A549 cells, MTT assay was performed; the results showed no significant toxicity difference between the two treated groups (free and formulated FU). For further evaluations, cellular uptake assays were performed via fluorescence microscopy and flow cytometry. RESULTS: Both analyses revealed the low internalization of nano-vehicle into the A549 cells, with 4.31% and 8.75% cellular uptakes in the first two and four hours of treatment. Therefore, the low penetration rate reduced the toxicity of drug-loaded nano-vehicle. CONCLUSION: Finally, DAPI staining and Annexin V-FITC staining were performed as complementary techniques to determine cell apoptosis.