Abstract 479: Engineering LNP formulations for mRNA delivery to macrophages

Macrophages play a pivotal role in the microenvironment of solid tumors, contributing to cancer progression and metastases through various functions. mRNA technologies may afford opportunities to reprogram these cells towards an anti-tumorigenic phenotype for therapeutic interventions in cancer. This study focuses on optimizing lipid nanoparticle (LNP) formulations for mRNA delivery to macrophages. We first investigated the impact of six ionizable lipids, three helper lipids, and two sterols on the transfection efficiency of mRNA-LNPs in RAW264.7 macrophages in vitro. Among the ionizable lipids, SM-102 demonstrated the highest reporter protein expression, significantly outperforming other lipids except ALC-0315, with the ranking as follows: SM-102 > ALC-0315 = OF-C4-Deg-Lin > CL1 > cKK-E12 > MC3. For helper lipids, DOPE yielded higher protein expression compared to DSPC and 9A1P9, though the difference was not statistically significant relative to DSPC. Among the sterols tested, increasing the proportion of β-sitosterol in β-sitosterol:cholesterol mixtures resulted in a corresponding increase in protein expression. The formulation comprising SM-102, DOPE, and β-sitosterol emerged as the top candidate, surpassing formulations prepared with the same composition as commercially available LNPs, with a 700-fold and 250-fold increase in reporter luciferase (Fluc) and eGFP expression, respectively. We also evaluated the extent of particle internalization, which did not correlate with protein production. Our analysis revealed that nearly 100% of macrophages took up the LNPs with the optimum formulation within 4 hours, with the peak in protein expression observed at 24 hours post-transfection. To extend these findings, the optimum formulation was tested on murine bone marrow-derived monocytes differentiated into M0 [F4/80+], M1-like [F4/80+CD80+CD206-], and M2-like [F4/80+CD206+CD80-] macrophages, as well as RAW264.7 cells polarized into these same subtypes. The results confirmed the superior transfection efficiency of the optimum formulation across all tested phenotypes. To evaluate in vivo performance, we quantified the bioluminescence expression of Fluc mRNA in BALB/c mice following an intraperitoneal injection of 0.5 µg of the mRNA-loaded LNPs. Bioluminescence was measured 6 hours post-injection for whole-body imaging and organs ex vivo. The optimum formulation demonstrated efficient mRNA delivery without any observed toxicity. Organ distribution studies indicated accumulation of the LNPs in the liver and spleen. Overall, our findings demonstrate the significant effect of LNP formulation on its ability to transfect macrophages, achieving efficient transfection in M0, M1-like, and M2-like macrophage subtypes in vitro and effective mRNA expression in vivo. These results highlight the potential of optimized LNPs to advance macrophage-targeted mRNA therapies for the treatment of solid tumors. Citation Format: Yasir A. Alshehry, Raneem R. Aldaqqa, Matthew E. Fernandez, Asma M. Al-Terawi, Douglas H. Sweet, Sandro R.P. da Rocha. Engineering LNP formulations for mRNA delivery to macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 479.