Fig. 2

Optimization of LNP-M formulations to deliver spike. A and B LNP-M with DSPC or DOPE to deliver pGFP. 293T cells were seeded into a 24-well plate and transfected with LNP-M/pGFP, with DSPC replaced by DOPE. FACS of GFP was analyzed after 20 h, showing GFP expression images (A) and percentages of GFP-positive cells (B). C and D LNP-M with DMG-PEG 2000, ALC-0159, or DSPE to deliver pGFP. E and F LNP-M plus NLS or histones to deliver pGFP. pGFP was pre-incubated with NLS (mass ratio at 20:1), histones (mass ratio at 20:1) or NLS + histones (10:1) before microfluidic mixing with four lipids. 293T cells were transfected for 72 h before GFP expression image acquisition. G and H LNP-M plus NLS or histones to deliver pGFP, with DSPC, was pre-incubated with either NLS (mass ratio at 80:1) or histones (mass ratio at 80:1) or NLS + histones (40:1) before microfluidic mixing and transfection. I and J LNP-M delivers the luciferase (Luc) gene into mice. Mice were intramuscularly injected with optimized LNP-M-encapsulated pLuc (40 μg per animal), and bioluminescence was detected after five days. K LNP-M delivers pGFP intramuscularly into mice. L-N LNP-M delivers pSpike into mice. Spike expression in muscle tissues was detected using Western blot, serum levels of IL-6 and TNF-α assessed by ELISA, and body weights measured every three days. O H&E staining of major mouse organs. Mice were immunized three times on days 0, 14, and 28, and sacrificed on day 42 before organ collection and histological analyses. Data were presented as means ± SD. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant