ENHANCING EFFECTIVENESS OF OXIDATIVE DRUGS COMBINATION (ATO/D-VC) IN KRAS MUTANT COLORECTAL CANCER TREATMENT WITH PROTEASE-FUNCTIONALIZED NANOCARRIERS: A 2D AND 3D CELL CULTURE STUDY

Abstract

KRAS mutations are associated with treatment resistance and poor prognosis in colorectal cancer (CRC), highlighting the need for more effective therapeutic approaches. The combination of arsenic trioxide (ATO) and D-vitamin C (D-VC) has shown promise as an oxidative therapy that targets KRAS-mutant CRC cells by disrupting mitochondria and antioxidant defenses to trigger apoptosis. Although this combination has shown strong efficacy in 2D cell cultures, animal models, and early clinical trials, insight into its behavior in systems that closely replicate the structural features of the tumor microenvironment remains limited. Therefore, 3D in vitro models offer a valuable platform for bridging this gap. This study evaluated the cytotoxicity of ATO/D-VC in 3D cell models known as clusteroids, derived from two KRAS-mutant CRC cell lines, HCT116 and SW620, and directly compared their response to corresponding 2D cultures. Clusteroids closely mimic key aspects of the tumor environment, including cell–cell interactions, extracellular matrix (ECM) deposition, and limited drug diffusion, offering a more predictive platform for assessing treatment efficacy. While ATO/D-VC showed a synergistic cytotoxic effect in 2D, the response in 3D clusteroids was significantly reduced, suggesting that drug access to inner cell layers was limited by ECM barriers. To improve delivery, ATO and D-VC were individually encapsulated in shellac–poloxamer nanoparticles functionalized with Savinase, a protease known to degrade ECM components. These nanoparticles exhibited colloidal stability, a positive surface charge to enhance interaction with negatively charged cancer cells, and enzymatic activity to access cells in the core of 3D models. In clusteroids, co-delivery of the two nanoparticle formulations restored and enhanced the cytotoxic effect of ATO/D-VC, outperforming both free drug and uncoated formulations. This dual-nanoparticle approach preserved the synergistic interaction and led to a substantial reduction in viability and proliferation, particularly in CRC cells. These results highlight the importance of 3D models in preclinical evaluation and demonstrate the potential of functionalized nanoparticles to enhance oxidative drug delivery in solid tumors.