*Article* **mRNA-Loaded Lipid Nanoparticles Targeting Dendritic Cells for Cancer Immunotherapy**

**Kosuke Sasaki † , Yusuke Sato \*,†, Kento Okuda, Kazuki Iwakawa and Hideyoshi Harashima \***

> Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-Ku, Sapporo 060-0812, Japan; kskssk.sw.77@gmail.com (K.S.); okuken@eis.hokudai.ac.jp (K.O.); kazukiiwak78@eis.hokudai.ac.jp (K.I.)

**\*** Correspondence: y\_sato@pharm.hokudai.ac.jp (Y.S.); harasima@pharm.hokudai.ac.jp (H.H.);

Tel.: +81-11-706-3734 (Y.S.); +81-11-706-3919 (H.H.)

† These authors contributed equally to this work.

**Abstract:** Dendritic cells (DCs) are attractive antigen-presenting cells to be targeted for vaccinations. However, the systemic delivery of mRNA to DCs is hampered by technical challenges. We recently reported that it is possible to regulate the size of RNA-loaded lipid nanoparticles (LNPs) to over 200 nm with the addition of salt during their formation when a microfluidic device is used and that larger LNPs delivered RNA more efficiently and in greater numbers to splenic DCs compared to the smaller counterparts. In this study, we report on the in vivo optimization of mRNA-loaded LNPs for use in vaccines. The screening included a wide range of methods for controlling particle size in addition to the selection of an appropriate lipid type and its composition. The results showed a clear correlation between particle size, uptake and gene expression activity in splenic DCs and indicated that a size range from 200 to 500 nm is appropriate for use in targeting splenic DCs. It was also found that it was difficult to predict the transgene expression activity and the potency of mRNA vaccines in splenic DCs using the whole spleen. A-11-LNP, which was found to be the optimal formulation, induced better transgene expression activity and maturation in DCs and induced clear therapeutic antitumor effects in an E.G7-OVA tumor model compared to two clinically relevant LNP formulations.

**Keywords:** lipid nanoparticles; particle size; dendritic cells; mRNA; delivery; cancer immunotherapy
