Transfection in PAS_70:30 and plasma is best with mRNA-LNP formulated with PC helper lipids and low molar percentage of PEGylated lipid. (A) Schematic of an mRNA-LNP and the various optimized components. (B-C) Relative NanoLuc expression in platelets transfected with mRNA-LNP formulated with a library of structural phospholipids at 4 hours (shaded bars) and 24 hours (open bars) in PAS_70:30 (B) or plasma (C). (D and E) Heat map showing the relative NanoLuc expression fold change between structural phospholipids used in the mRNA-LNP for platelets transfected in PAS_70:30 (D) and plasma (E). (F) mRNA-LNP size (nm) (bars, left y-axis) and polydispersity index (PDI) (yellow circles, right y-axis) after formulation with various PEGylated lipids and at various molar percentages. (G and H) Relative NanoLuc expression in platelets transfected with NTX-001-POPC mRNA-LNP formulated with various molar percentages of PEGylated lipids after 4 hours (shaded bars) and 24 hours (open bars) in PAS_70:30 (G) or plasma (H). (I) NanoLuc expression in platelets transfected with preoptimized and optimized mRNA-LNP. Platelets were transfected at a concentration of 250 × 10⁶ mL⁻¹. P values were determined by the 2-way analysis of variance and applied to 24-hour data. Values are reported as mean ± standard error of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. n = 3. Ns, not significant.