50:10:38.5:1.5, respectively, and total concentration of 5.5 mM and 20-fold excess by mass compared to mRNA) was combined with an aqueous mix containing mRNA, water, and 100 mM pH 4 citrate bu↵er (3-fold excess by volume compared to lipid solution) using a microfluidic mixer. For DSC, the formulation did not contain any mRNA and its volume was replaced with equivalent volume of DI water. Nanoparticles were then dialyzed twice against 3 L of pH 7 PBS bu↵er and concentrated using appropriate centrifuge filters. Nanoparticle size was determined using su ciently diluted solutions in PBS via dynamic light scattering (DLS), and mRNA encapsulation e ciency was determined using a modified Quant-iT RiboGreen RNA reagent (Invitrogen).
In Vitro transfection. Cells were plated in white, clear-bottom 96-well plates. HeLa cells were plated at 4,000 cells/well and transfected 24 hours after initial plating with a known quantity of mRNA. The transfection results were then read 24h later using CellTiter-Fluor Cell Viability and ONE-Glo Luciferase Assays (Promega).
Cryo-electron microscopy. CryoEM acquisition was performed at 300kV using Falcon III and K3 cameras with DED. 3-5 ul of the sample was dispensed on a plasma cleaned grid in the Vitrobot chamber at 95% relative humidity and allowed to rest for 30 seconds. Then, the grid was blotted for 3 seconds with filter paper and plunged into liquid ethane cooled by liquid nitrogen. The frozen grids were then checked for visible defects and assembled into cassettes. The collected images were then processed and analyzed manually using Fiji. 3 LNP populations contained at least 85 particles. Only unobstructed LNPs with clearly defined edges were included in the statistical analysis to ensure accurate classification of LNP populations and to avoid counting structural deformities due to sample prep or ice thickness issues. Polydispersity index (PDI) was calculated as P dI = ( /µ) 2 , wherestandard deviation, µ -mean of particle size.
Thermal analysis. Di↵erential scanning calorimetry (DSC) was performed using formulations containing no mRNA and either DSC Q2000 or 2920 instrument (TA Instruments) in 0-100 C range at 10 C/min. The collected data was processed by subtracting PBS background signal and analyzed using TA Universal Analysis software.
Membrane fluidity assay. TMA-DPH assay was performed as described elsewhere. 4 Briefly, LNP solution was diluted to a total lipid concentration of 0.5 mg/ml and mixed in a microcuvette with equal volume of TMA-DPH, diluted from 1mM stock solution in EtOH to 0.25-2 ug/ml final concentration (ca. 100-700 lipid:fluorophore ratio) with pH 7 PBS bu↵er. The fluorescence anisotropy was recorded using temperature-controlled automatic polarization setup at wavelengths 355 nm (excitation) and 430 nm (emission), ramping the temperature at 5 C/min.
Prof. G. Sahay conceived the idea, directed the research, and reviewed the paper. Dr. Y.
Eygeris wrote the paper, formulated the particles, performed transfection study, conducted the cryoEM imaging and the TMA-DPH assay, and analyzed the DSC data. S. Patel conceived the idea of TMA-DPH experiments, formulated the particles, and revised the paper.
A. Jozic conducted DSC experiments. 