*2.7. In Vivo Screening of RNA-Loaded LNPs*

C57BL/6N mice were intravenously injected with the RNA-loaded LNPs at a dose of 1 mg RNA/kg (0.05 mg Nluc mRNA/kg). At twenty-four hours after the injection, the liver and half of the spleen tissues were harvested, frozen in liquid nitrogen, and stored at −80 ◦C for use in Nluc assays. Splenocytes were dissociated from the remaining spleen tissues and were then passed through a cell strainer (40 µm pore, BD Falcon, CA, USA). The recovered cells were spun down (400× *g*, 4 ◦C, 5 min) to remove the supernatant, resuspended in red blood cell (RBC) Lysis buffer (1 mL, BioLegend) and incubated for 5 min at room temperature. The resulting treated cells were washed with Hanks' balanced salt solution (HBSS(-)) by spinning (400× *g*, 4 ◦C, 5 min). The concentration of cells was adjusted to <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>7</sup> cells/mL with fluorescence-activated cell sorting (FACS) buffer, and the resulting cells were treated with a 10 µg/mL solution of an anti-mouse CD16/32 antibody followed by incubation at 4 ◦C for 10 min. The resulting sample was then treated with fluorophoreconjugated anti-mouse antibodies at 4 ◦C for 30 min. Cells were washed with FACS buffer twice, filtered via a nylon filter, stained with propidium iodide (PI) (BioLegend), and analyzed for cellular uptake and cell sorting (SONY SH800 cell sorter, SONY, Tokyo, Japan). Three thousand living B cells (defined as PI−CD19<sup>+</sup> cells), macrophages (Mac, defined as PI−F4/80 <sup>+</sup>CD11c<sup>+</sup> cells), and DCs (defined as PI−I-A/I-E+CD19<sup>−</sup> cells) were collected in a sample tube containing 20 µL of 2 × passive lysis buffer.

Nluc activity was measured using the Nano-Glo Luciferase Assay Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Luminescence was measured using a luminometer (Luminescencer-PSN, ATTO, Tokyo, Japan). For tissues, Nluc activity was corrected for protein quantity using a Pierce BCA Protein Assay Kit (ThermoFisher, Waltham, MA, USA) and expressed as relative light units (RLU) per mg protein. Cellular Nluc activity was expressed as RLU per 3000 cells.
