Fig. 4. LNP^HNSCC shows preferential systemic delivery of mRNA to human solid tumors while de-targeting the liver in vivo.

(A) LNP^HNSCC and LNP^First, the winner LNP of the first screen, were formulated with an anchored NanoLuc mRNA and administered intravenously to Nu/J mice carrying FaDu xenograft tumors at 2 mg of RNA/kg. Thirty-six hours later, luciferase expression was quantified via whole-organ imaging in (B) FaDu tumors and (C) livers. Fold change total flux and radiance over control organs’ background (p/s/cm²/sr) in tumors were comparable while LNP^HNSCC had lower NanoLuc expression in the livers, demonstrating de-targeted delivery to the liver. (D) Tumor tropism was quantified by computing the tumor-to-liver ratio using fold change and radiance over control for both LNPs, demonstrating LNP^HNSCC had superior tumor preferential delivery. (E) LNP^HNSCC and LNP^First were formulated with an aVHH mRNA and administered intravenously to Nu/J mice carrying FaDu xenograft tumors at 2 mg of RNA/kg. Sixteen hours later, aVHH protein expression (% transfected and MFI) was quantified via flow cytometry in (F) FaDu tumors (human FaDu cells, ECs, and immune cells) and (G) livers (immune cells, ECs, and hepatocytes). LNP^HNSCC and LNP^Llver both functionally delivered aVHH mRNA to tumor cell types comparably while LNP^HNSCC had lower aVHH expression in liver ECs and hepatocytes, demonstrating de-targeted delivery to the liver. (H) Tumor tropism was quantified by computing the tumor-to-liver ECs and FaDu-to-hepatocyte ratios using % transfected and MFI for both LNPs, demonstrating LNP^HNSCC had superior tumor preferential delivery ECs: Endothelial cells, MFI: Mean fluorescence intensity. Two-way ANOVA, Tukey’s multiple comparisons test, *P < 0,05, **P < 0.01, ***P < 0.001, ****P < 0.001, average ± S.D.