Synergistic ROS/enzyme dual-responsive oral drug delivery system: A novel multi-mechanistic platform for spatiotemporal control and overcoming drug resistance in colorectal cancer therapy

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, driven by complex interactions between inflammatory pathways, gut microbiota dysbiosis, and tumor microenvironment remodeling. Conventional therapies, particularly single-target oral chemotherapeutics, are hindered by poor bioavailability, systemic toxicity, and drug resistance. To address these limitations, we engineered KGM-PTX/CSM microspheres, a dual-responsive drug delivery system leveraging the elevated reactive oxygen species (ROS) in CRC and β-mannanase overexpression in the colorectum. The system comprises ROS-sensitive prodrug micelles (PSM) encapsulated within konjac glucomannan (KGM). PSM micelles were synthesized by conjugating hydrophilic chitosan oligosaccharides (COS) with the hydrophobic anti-inflammatory agent mesalazine (MSL) via ROS-labile thioether bonds, followed by paclitaxel (PTX) encapsulation. Upon oral administration, KGM undergoes β-mannanase-triggered degradation in the colon, releasing PSM micelles that subsequently disintegrate in the ROS-rich tumor microenvironment, enabling spatiotemporally controlled drug release. <i>In vitro</i> studies demonstrated ROS-responsive drug liberation (91.2 % cumulative release within 48 h) and enhanced cytotoxicity against PTX-resistant SW480/PTX cells (IC₅₀: 9.33 μg/mL vs. 45.68 μg/mL for free PTX). Mechanistic investigations revealed synergistic interactions among the system's components: PTX stabilized microtubules to induce apoptosis, while MSL counteracted COX-2/P-gp-mediated drug resistance and alleviated PTX-associated intestinal inflammation. In the AOM/DSS-induced orthotopic CRC model, KGM-PTX/CSM significantly inhibited colorectal tumor growth, improved survival rates, and suppressed inflammatory cytokine expression (TNF-α, IL-1β, IL-6, and IL-10) in serum and colorectal tissues. Immunomodulatory effects included enhanced CD8⁺ T-cell activity, suppression of Treg-mediated immune evasion, and macrophage polarization toward the tumor-suppressive M1 phenotype. Gut microbiota analysis demonstrated restored operational taxonomic unit (OTU) counts, increased beneficial bacterial populations, elevated alpha and beta diversity, reduced pro-inflammatory bacteria, and increased short-chain fatty acid (acetate, propionate, and butyrate) concentrations, collectively improving intestinal microecology and inhibiting tumor progression. This study synergistically enhanced the anti-CRC effect through multiple mechanisms of action such as chemotherapy, reversal of chemotherapy resistance, regulation of intestinal flora, anti-inflammation, activation of immune cells, etc., which will provide a certain reference for the research of synergistic drug therapy for CRC.