Microfluidic‐Assisted Metal‐Polyphenol Cloaked Bacteria Enable CRISPR/dCas9‐Mediated Pyroptosis Cascade Amplification for Effective Tumor Immunotherapy

ABSTRACT Pyroptosis triggered by pore‐forming Gasdermin proteins in cancer cells facilitates anti‐tumor immune activation by releasing pro‐inflammatory cytokines and immunogenic contents following cellular rupture. However, selectively triggering pyroptosis in tumors still remain limited in clinical applications. Here, it is reported a microfluidic‐assisted bacterial delivery system using attenuated Salmonella typhimurium VNP20009 encapsulated with metal‐phenolic networks composed of ferric ions (Fe 3 + ) and tannic acid (TA) to enhance intracellular gasdermin D (GSDMD) expression through targeted CRISPR/dCas9 delivery, thereby inducing robust tumor pyroptosis. Mechanistically, this system achieves cascade amplification of pyroptotic cell death through coordinated multi‐modal mechanisms. Following systemic administration, VNP20009 specifically accumulates in hypoxic tumor regions while the coated Fe 3 + –TA nanofilm undergoes pH‐responsive dissolution in the acidic tumor microenvironment (TME), simultaneously generating ROS through Fenton reaction and releasing CRISPR/dCas9 system to upregulate GSDMD expression. Concurrently, the abundant flagella of VNP activate caspase‐1, which in turn cleaves the overexpressed GSDMD proteins into its active form, thereby triggering robust pyroptosis in tumor cells. Taken together, by coupling bacterial adjuvanticity with ROS‐mediated stress and CRISPR‐driven GSDMD upregulation, this strategy achieves efficient amplification of pyroptosis and promotes antitumor immune activation.