Fluorinated lipid nanoparticles enable in vivo CAR-macrophage therapy in solid tumor and enhance anti PD-L1 immunotherapy.

In vivo CAR-macrophage (CAR-M) therapy exploits macrophages' tumor-tropic homing, phagocytic capacity, and ability to remodel the immunosuppressive tumor microenvironment, with low risk of cytokine release syndrome and potential as an off-the-shelf therapy. However, clinical translation is currently constrained by the lack of efficient non-viral platforms for in situ macrophage engineering. To address this challenge, fluorinated ionizable lipids were rationally designed-capitalizing on fluorine's high electronegativity, low polarizability, and lipophobicity-to enhance lipid nanoparticle (LNP) delivery. Screening a library of 80 fluorinated lipids identified A1F5C5 as the lead candidate, demonstrating superior mRNA delivery to macrophages both in vitro and in vivo. Our investigation into the mechanism revealed that, while fluorination universally enhanced both cellular uptake and subsequent endosomal dissociation, the specific configuration with five fluorine atoms uniquely conferred a superior membrane fusion capability, which proved critical for achieving efficient endosomal escape. F5-LNPs encapsulating mRNA encoding an hPSMA-targeted CAR (F5-CAR) were formulated using this platform. Intravenous F5-CAR administration reprogrammed the tumor microenvironment, reducing M2-like macrophages and boosting granzyme B and perforin expression in CD8 T cells. Notably, when combined with anti-PD-L1 blockade, this strategy achieved 100% complete and durable regression in MC38-hPSMA tumor-bearing mice. This work establishes fluorinated LNPs as a scalable platform for in vivo CAR-M engineering against solid tumors.