Fig. 3. LNP^HNSCC efficiently delivers mRNA to cancer cells in vitro.

(A) A lead LNP from screen 2, LNP^HNSCC, was identified and characterized. (B) Diameter (nm), polydispersity index (PDI), pKa, and zeta potential of LNP^HNSCC. (C) Composition, diameter (nm), and PDI of LNP^First, the winner of the first screen with highest tumor and hepatocyte delivery. (D) LNP^HNSCC was formulated with a GFP mRNA and an anchored VHH (aVHH) mRNA and used to transfect FaDu cancer cells in vitro at 0.50, 1, and 2 μg/80K cells. Sixteen hours later, (E) GFP and (F) aVHH expression (% transfected and MFI) were quantified via flow cytometry. (G) aVHH transfection of FaDu cells was also validated via immunohistochemical staining and microscopy. LNP^HNSCC and LNP^First were formulated with aVHH mRNA and used to transfect (H) FaDu human cancer cells, (I) murine AML-12 hepatocytes, and (J) human embryonic kidney (HEK-293) cells in vitro at 0.10, 0.25, and 0.50 μg/80K cells. LNP^HNSCC was better at transfecting human cell lines, while LNP^First showed superior transfection of murine hepatocytes. 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.