Freeze‐Thaw Microfluidic System Produces “Themis” Nanocomplex for Cleaning Persisters‐Infected Macrophages and Enhancing Uninfected Macrophages

Abstract Macrophages are the primary effectors against potential pathogen infections. They can be “parasitized” by intracellular bacteria, serving as “accomplices”, protecting intracellular bacteria and even switching them to persisters. Here, using a freeze‐thaw strategy‐based microfluidic chip, a “Themis” nanocomplex (TNC) is created. The TNC consists of Lactobacillus reuteri ‐derived membrane vesicles, heme, and vancomycin, which cleaned infected macrophages and enhanced uninfected macrophages. In infected macrophages, TNC releases heme that led to the reconstruction of the respiratory chain complexes of intracellular persisters, forcing them to regrow. The revived bacteria produces virulence factors that destroyed host macrophages (accomplices), thereby being externalized and becoming vulnerable to immune responses. In uninfected macrophages, TNC upregulates the TCA cycle and oxidative phosphorylation (OXPHOS), contributing to immunoenhancement. The combined effect of TNC of cleaning the accomplice (infected macrophages) and reinforcing uninfected macrophages provides a promising strategy for intracellular bacterial therapy.