Initial entrapment of nucleic acids in lipid nanoparticles (LNPs) is dependent on the use of ionizable cationic lipids, which draw nucleic acids into lipid particles at low pH in the presence of ethanol. Manufacturing of fully formed LNPs is completed by buffer exchange and removal of ethanol. We studied particle morphology at intermediate pH values during buffer exchange using an fluorescence resonance energy transfer (FRET) assay to indicate particle interactions, particle sizing, cryo-electron microscopy (cryo-EM), and small-angle X-ray scattering (SAXS). We compared LNPs formed by different ionizable lipids, including DLin-MC3-DMA, ALC-0315, and SM-102, used, respectively, in the Food and Drug Administration (FDA)-approved products, Onpattro, Comirnaty, and Spikevax. FRET and cryo-EM studies confirmed that particle interaction and fusion occurred during buffer exchange. By dialyzing LNPs in various buffers, we found that stable particles with bleb-like protrusions were formed at intermediate pH (i.e., pH 5.5). Fusion and particle growth occurred at higher pH values for SM-102 LNPs, reflecting the higher pKa of SM-102. SAXS profiles showed that, when fully formed at the final pH of 7.4, MC3 and ALC-0315 LNPs had lost bilayer-like structures, which were present after particle formation at pH 4. In contrast, LNPs produced with 1,2-dioleyloxy-3-dimethylaminopropane (DODMA) and SM-102 retained some bilayer-like structures at pH 7.4.
