Engineering a library of inulin based polyplexes and core-shell nanoparticles: Inside the targeted siRNA delivery to colorectal cancer cells

In this study, we present the systematic design and evaluation of a library of inulin-based graft copolymers for siRNA delivery. Starting from native inulin, we progressively modified the polymer through a modular strategy: first, by grafting branched polyethylenimine (bPEI) at two different densities; then, by introducing polylactic acid (PLA) segments to create amphiphilic copolymers; and finally, by conjugating folic acid (FA) as a targeting moiety for colorectal cancer (CRC) cells. This stepwise approach led to the development of a library of eight structurally related copolymers, enabling a comprehensive investigation of how specific copolymers composition features affect nanoparticles formation, stability, hemo- and cytocompatibility and transfection efficiency.Notably, we observed counterintuitive structure–function relationships, such as a negative impact of high bPEI density on biological stability, and previously unreported roles of FA in improving both colloidal and biological stability, beyond its classical targeting function. The amphiphilic nature imparted by PLA significantly influenced nanoparticle self-assembly, colloidal behaviour, siRNA encapsulation and protection, and release compared to PLA free nanosystems of the polyplex type. Nanoparticles, compared to polyplexes, demonstrated improved siRNA protection and improved transfection despite a reduced intracellular uptake, suggesting a difference in intracellular trafficking and endosomal escape. A multi-omics analysis revealed significant alterations corroborating the active internalization and confirming safety of the treatment at the cellular level. Overall, our work provides new insight into the fine structural tuning of polysaccharide-based vectors, offering valuable guidelines for next-generation nanocarriers.