Targeting and Reprogramming Tumor-Associated Macrophages with Polymeric Nanoparticle-Mediated mRNA Delivery for Cancer Treatment

Abstract

As the second leading cause of death globally, cancer has always been a focal point of intense research, particularly B-cell malignancies, which are complex and drug-resistant. These malignancies arise from genetic changes and are influenced by the tumor microenvironment (TME), where tumor-associated macrophages (TAMs) play a key role. TAMs can be divided into pro-inflammatory M1 type macrophages and anti-inflammatory M2 type macrophages. Reprogramming TAMs from an M2-type to an M1-type is considered a promising cancer therapy.Chimeric antigen receptor engineered macrophages (CAR-M) therapy is a significant potential strategy for targeting TAMs. CAR-M cells can maintain a sustainable M1 phenotype and polarize M2 macrophages toward an M1 phenotype. However, this process is costly, time-consuming, and carries risks like insertional mutagenesis, making it inaccessible for many families. One promising solution is in vivo reprogramming using messenger RNA (mRNA). mRNA can directly encode therapeutically proteins without genomic integration, avoiding the risk of insertional mutagenesis. Additionally, direct programming in vivo offers a more cost-effective and quicker alternative. Since mRNA is unstable, a reliable non-viral vector is required. To enhance drug utilization and meet specific delivery requirements, researchers have increasingly turned to versatile polymeric nanoparticles (PNPs). Compared with other nanocarriers like lipid- or inorganic-based nanoparticles, the adaptability and functionality of PNPs make them promising candidates for targeted drug delivery. In this study, we designed a PNP that consists of perfluorinated polyethylenimine (PFHA-PEI), polyethylene glycol and polyethylenimine (PEG-PEI) and heparin (LHP), which can endow nanoparticles with biocompatibility and membrane penetration ability, facilitating the entry of genetic material into cells. The Layer-by-Layer (LBL) strategy offers an effective method of encasing mRNA in nanoparticles. The principle of this strategy is to assemble layers of oppositely charged materials together by electrostatic interaction, enabling scalable, cost-effective, and highly effective cancer immunotherapies. In this study, we developed an optimal formulation for an antibody conjugated polymeric nanoparticle (PNP), named CAR-APP-F4/80, with small size, low toxicity and strong targeting for efficient CAR-mRNA delivery to TAMs. Its excellent mRNA encapsulation efficiency and successful cell uptake and endosome escape in RAW264.7 were proved. CAR-APP-F4/80 showed strong transfection ability and high CAR expression with low toxicity in the murine macrophage cell line RAW264.7 as well. The increased production levels of IFN-γ and TNF-α testing by ELISA and flat-round like morphology were observed, which demonstrated the potential of CAR-APP-F4/80 for M1 phenotype polarization of RAW264.7. Generally, CAR-APP-F4/80 provides a promising strategy for targeting and reprogramming TAMs for cancer treatment.