Development and in vitro cytotoxic profiles of multistage systems containing irinotecan hydrochloride and bevacizumab for the treatment of human colorectal carcinoma.

Multistage drug delivery systems are promising formulations able to enhance the therapeutic efficacy and tolerability of entrapped active compounds by integrating multiple functional components able to efficiently interact with biological barriers. In this study, we developed a multistage (MS) system made up of a poloxamine 908 (PL908) thermo-sensitive hydrogel containing drug-loaded zein nanoparticles (NPs) for the co-delivery of bevacizumab (BVZ) and irinotecan (Iri). The NPs were stabilized with DSPE-PEG2000, while ion pairing with dextran sulfate (DXS) enhanced Iri retention. NPs prepared with Iri or BVZ (0.2 mg/ml) showed distinct properties: Iri-NPs were characterized by a mean diameter of 93 nm (PDI: 0.099), a Zeta potential of -37 mV and 51% drug retention, whereas BVZ-NPs demonstrated mean sizes of 191 nm (PDI: 0.179), -8 mV of surface charge and 45% retention of active compound. Rheological characterization confirmed the thermo-sensitive behavior and mechanical stability of the MS systems. In vitro release studies on the NPs and MS system demonstrated a sustained drug leakage, with 40% Iri and 20% BVZ released after the first 8 h. Permeability assays using a 3D intestinal model revealed an enhanced drug permeation across the barrier. Furthermore, the cytotoxicity of the systems on 2D monolayers and 3D multicellular colorectal cancer (CRC) spheroids demonstrated the pharmacological efficacy of the active compounds retained by the MS system, showing a decrease of cell viability up to 50%, and confirmed the high biocompatibility of the formulations. These results highlight the potential of the proposed MS system as an advanced platform for localized and prolonged co-delivery of chemotherapeutic and antiangiogenic agents in CRC therapy.