Microcondensate‐Mediated Intracellular Infusion of mRNA Across the Plasma Membrane

Abstract This study presents a strategy for developing micrometer‐sized coacervates comprised of cationic intracellular delivery peptide FcB(L17E) 3 and nucleic acids (termed “microcondensates”) as a lipid nanoparticle (LNP)‐free platform for intracellular messenger RNA (mRNA) delivery. The feasibility of this approach is demonstrated both in vitro and in vivo. This approach is based on our previous finding that FcB(L17E) 3 forms a microcondensate through interactions with IgG labeled with a negatively charged fluorescent dye. Similarly, the negative charge of nucleic acids causes siRNAs to form microcondensates with FcB(L17E) 3 , resulting in the facile infusion of the siRNAs through the plasma membrane with 40% gene knockdown efficiency. mRNAs form aggregates with FcB(L17E) 3 due to their longer chains, which prevents efficient delivery. Remarkably, adding short single‐stranded DNA (ssDNA) to mRNA enables microcondensate formation with FcB(L17E) 3 , facilitating protein expression. Additionally, this microcondensate system successfully facilitated the intracellular delivery of plasmid DNA (pDNA) by optimizing the charge ratio between FcB(L17E) 3 and nucleic acids. Finally, the subcutaneous injection of microcondensates into mice successfully induced protein expression in vivo. The achievement of in vivo mRNA delivery and protein expression using microcondensates may offer a strategy for nucleic acid delivery with potential applicability in cancer immunotherapy and mRNA‐based vaccination.