Nanoparticle Formulation.

All nanoparticle formulation details are listed in SI Appendix, Fig. S2. The 7C1 lipid was synthesized as previously described (14), and C12-200 lipid was purchased from Wuxi AppTec. LNPs were synthesized by mixing a lipid-containing ethanol phase with a nucleic acid-containing aqueous phase at a 1:3 volume ratio in a microfluidic chip as previously described (36). The ethanol phase was prepared by solubilizing a mixture including some of the following components: lipids 7C1 or C12-200, phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and/or lipid-anchored PEG (e.g., C₁₄ PEG 2000, C₁₈ PEG 1000, and others) (Avanti Polar Lipids). The aqueous phase was prepared in 10 mM citrate buffer with DNA barcode or factor VII siRNA. The 7C1:nucleic acid and C12-200:nucleic acid weight ratios were 5:1 and 10:1, respectively. Immediately after mixing the ethanol and aqueous phases, the resultant LNPs were dialyzed against 1× PBS overnight at 4 °C. Formulation parameters for the 30-LNP screen were generated using statistical Design of Experiment software JMP (SAS Institute) using the Custom Design feature. In all cases (except for the in vivo standard curve in Fig. 2D), we administered 0.04 mg/kg DNA barcode per nanoparticle. This dose was selected because the “total dose” ranged between 0.16 mg/kg DNA barcode (e.g., Fig. 2B) and 1.2 mg/kg DNA (e.g., Fig. 3A), which are within commonly used doses for 7C1 and C12-200 (13, 14).