LNP synthesis. An organic phase was prepared by solubilizing with ethanol a mixture of the synthesized cationic lipid, DOPE (Avanti), cholesterol (Avanti) and C14-PEG 2000 (Avanti) at a predetermined molar ratio. The aqueous phase was prepared in 10 mM citrate buffer (pH 3.0, Fisher) with mLuc (firefly mLuc, Translate), antigen mRNA or nontranslating Cy5-labeled FLuc mRNA (TriLink BioTechnologies). All mRNAs were stored at −80 °C, and were allowed to thaw on ice before use. The ethanol and aqueous phases were mixed at a 3:1 ratio and a lipid/mRNA weight ratio of 10:1 in a microfluidic chip device using syringe pumps (Fusion 4000 Chemyx Syringe Pump) as previously described39. Resultant LNPs were dialyzed against 1X PBS in a 20,000 MWCO cassette (Fisher) at 4 °C for 1 h and were stored at 4 °C before injection. For high-throughput screening, the LNPs were prepared in a 96-well plate by directly adding ethanol phase to aqueous phase. For in vitro screening, LNPs were directly incubated with cells without further dialysis. For in vivo batch analysis screening, LNPs in each classification group (for batch analysis) were mixed and dialyzed against 1X PBS before injection into mice.
Dr. Chandrabali Bhattacharya
JDRF Postdoctoral Fellow
Anderson and Langer Lab
Koch Institute for Integrative Cancer Research
Massachusetts Institute of Technology