Savinase-functionalised oxidative drug-loaded nanocarriers enhance the treatment of solid colorectal tumours in a 3D cell culture model.

Colorectal cancer (CRC) mutations drive resistance and poor prognosis, underscoring the need for more effective therapies. The oxidative drug therapy combining arsenic trioxide (ATO) and D-vitamin C (D-VC) has demonstrated promising efficacy by targeting mitochondrial functions and depleting antioxidant defences to induce apoptosis in CRC cells. ATO and D-VC create a hostile environment for cancer cells by simultaneously targeting mitochondrial metabolism and redox homeostasis, reducing their ability to adapt and survive. This study evaluated the cytotoxic effects of ATO/D-VC in 2D cell cultures and 3D cell models, known as clusteroids, generated from CRC cell lines HCT116 and SW620. In the 2D cultures, the ATO/D-VC combination significantly reduced cell proliferation to 40-60% and viability to below 30% of control levels. In contrast, clusteroids showed a more limited response, with proliferation reduced to 60-80% and viability to 80-90%, highlighting the impact of the extracellular matrix (ECM) and cell-cell interactions in limiting drug diffusion within structured tumour microenvironments. To overcome these drug diffusion barriers, ATO and D-VC were individually encapsulated in poloxamer-stabilized shellac-based nanoparticles (NPs) surface functionalized with Savinase, a protease known to degrade ECM components. The cell viability and cell proliferation assays demonstrated that nanoparticle-mediated delivery significantly enhanced treatment efficacy in clusteroids. Dual treatment of Savinase-coated ATO and D-VC loaded NPs caused pronounced disruption of clusteroid morphology and substantially reduced both viability and proliferation to approximately 30-40% of untreated control levels. Compared to the free drug and uncoated nanoparticle formulations, the Savinase-functionalized nanoparticle formulation achieved nearly twice the reduction in viability and proliferation, indicating a marked improvement in therapeutic effect. Unloaded Savinase-coated nanoparticles showed minimal impact, underscoring their biocompatibility. This approach demonstrates the potential of protease-functionalized nanoparticles to enhance the oxidative drug delivery and efficacy in CRC tumours and could potentially allow targeting the therapeutic resistance in other solid tumours with dense ECM barriers.