Engineered Bacterial Protoplasts for Spatiotemporally Controlled Tumor Immunotherapy

ABSTRACT Cytokine‐expressing bacteria represent a promising candidate for cancer immunotherapy, but their clinical use is limited by systemic toxicity from lipopolysaccharide (LPS) and other endotoxic components. Here, a protoplast‐based delivery platform, designated as FACaC/T@PI, is constructed for tumor immunotherapy. FACaC/T@PI uses protoplast (PI) as the vehicle for tumor cytokine delivery, which is stabilized via glutaraldehyde crosslinking and engineered to overexpress interferon‐γ (IFN‐γ). The PI is coated with a folate‐modified calcium carbonate (CaCO 3 ) layer, which is loaded with the extracellular matrix (ECM) remodeling agent tranilast. In the acidic tumor microenvironment (TME), PI is exposed to serum complement attack after the CaCO 3 protective layer dissolves. The complement can assemble membrane attack complexes on the protoplast membrane, triggering protoplast lysis and the release of IFN‐γ, which induces tumor cell apoptosis and promotes antitumor immune activation. With markedly reduced LPS content and complement‐mediated cargo release, FACaC/T@PI demonstrates efficacy as a low‐toxicity platform for tumor immunotherapy in both breast and pancreatic cancer models. This protoplast‐based delivery vehicle offers valuable perspectives for the development of bacteria‐derived therapeutic platforms.