Tumor mRNA-lipid nanoparticles via chimeric nanobody-lipid co-assembly

<b>Background:</b> Targeted mRNA-lipid nanoparticles (LNP) show great potential for cancer immunotherapy by delivering neoantigen-encoding messages to tumor cells, prompting immune responses against tumors. However, the challenges of inefficient production of targeting ligand-grafted LNPs and the immunogenic effects of polyethylene glycol (PEG) hinder their therapeutic effectiveness. <b>Methods:</b> We introduced a simplified, one-step technique for creating PEG-free, human epidermal growth factor receptor 2 (HER2)-targeted mRNA-LNPs. This method incorporates self-assembled palmitoylated nanobodies (pNB), lipids, and mRNA that encode spike proteins (SP). We engineered cells to produce pNB, which were then mixed with lipids and mRNA at various ratios. Through hydrophobic interactions between the lipid tails and the palmitoyl groups, we assembled tumor-targeting mRNA-LNPs. We optimized both the lipid components and the quantity of pNB, and determined the optimal formulation based on a series of physicochemical characterizations of the LNP as well as in vitro cel assays. Building on this, we further conducted in vitro cytotoxicity assays targeting SP-expressing cells, followed by in vivo immune killing experiments. <b>Results:</b> In vitro, SP-expressing tumor cells triggered strong immune responses and effective tumor cell destruction. Additionally, these pNB-LNPs demonstrated improved tumor-specific delivery, extended tumor retention, and considerable tumor volume reduction in vivo. <b>Conclusion:</b> This streamlined, PEG-free LNPs platform that utilizes pre-existing immunity presents a flexible strategy for targeted cancer immunotherapy and applications in infectious diseases.