Background/Aim: Cancer is a leading cause of death worldwide. Conventional treatments as surgery, chemotherapy, radiotherapy, and combined therapies are commonly used. However, these therapies have several limitations and side effects. To address these issues, innovative research is being conducted on nanocarriers (NCs) functionalized with antineoplastic agents. These NCs aim to overcome limitations and improve patients' lives. However, before they can be used clinically, these NCs are primarily assessed on a lab scale to determine their efficacy. Materials and Methods: A primary cell culture was established from a lymphoblastic neoplasm in the maxilla. After characterization, the cells were cultured in 2D to evaluate the dose-effect of nanoparticles (NPs), such as Zinc oxide (ZnO) and Magnesium oxide (MgO), as well as those of free drugs of 5-fluorouracil (5-FU) and cisplatin (Cis). Based on the results, a 3D spheroid culture was used for further study. Finally, the spheroids were histologically processed for immuno-morphological observation. Results: To evaluate spheroid cell viability, we conducted an MTT assay. Treatment of cell spheroids with ZnONPs, 5-FU, and NPs conjugated with antitumor agents such as 5-FU-ZnO and Cis-ZnO decreased cell viability by >25%, >60% and >10% and <20% at a concentration of 0.06, 0.015 and 0.015 & 0.03 mg/ml, respectively. Conclusion: Nanoparticles conjugated with antitumor agents showed promising antineoplastic effects on both 2D and 3D cell cultures. However, the efficacy of the nanoparticles varied between the different models. This highlights the importance of selecting appropriate in vitro culture models for the evaluation of biomedical agents.Cancer remains a significant public health problem and is one of the leading causes of mortality and morbidity worldwide. In the United States, it is estimated that there will be an increase of 1,958,310 new cases and 609,820 cancer deaths by 2023 (1). Hematological neoplasms are the third most common type of cancer after lung and breast neoplasms. In Mexico, cancer is the ninth leading cause of death, with lymphomas, leukemias, and myelomas being the most frequent types, accounting for 10% of all cancer diagnoses (2-5).In recent years, cancer nanotechnology has become increasingly important for the diagnosis and treatment of cancer, with the goal of improving human well-being. The field has seen substantial development, with the approval of over 50 nano-based drugs by the US Food and Drug Administration (FDA). These drugs have shown promise in improving the effectiveness of cancer treatment while minimizing side effects (6-8). Malignant neoplasms of the head and neck are traditionally treated with a combination of surgery, radiation therapy, or both, depending on the size, type, and location (9-11). In recent years, nanoparticles (NPs) have received increasing attention due to their unique and promising characteristics. Among the different types of nanoparticles, nanocarriers (NCs) have shown potential in 1658