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Fig. 4 Characterization of AMBP-modified LNPs interaction with C2C12 myoblasts. Confocal images of C2C12 cells (250 000 cells/dish) after exposure to (a) Texas red (TR)-labeled 4-LNPs, (b) mCherry-loaded 4-LNPs, (c) 1-LNPs, (d) 1:2-LNPs, and (e) 1:3-LNPs (approximately 1.5 × 1011 LNPs). GFP is shown in green, and mCherry or Texas red is shown in red. Scale bars: a–b, 25 μm; c–e, 50 μm. (f) Flow cytometry quantification of GFP (shown in green) and mCherry (shown in red) in C2C12 cells after 2 h of incubation with mCherry-loaded LNPs (n = 15) or (g) LNPs modified with protein fusions 1, 1:2, and 1:3 (n = 12). Box and whiskers plots display min to max error bars and data points. Each data point is the mean of fluorescence of 10 000 events. Multiple unpair t tests are displayed as n.s. for nonsignificance, **p < 0.01 and ****p < 0.0001. (h) Cell viability of C2C12 myoblasts (50 000 cells/well, 96-well plate) over 7 days after exposing them to different conditions (20 μL of 1 μM proteins or approximately 1.5 x 1010 vesicles) for 2 h. Cell viability (n = 3) was calculated via extrapolation of the fluorescence data obtained from the Alamar Blue (AB) metabolic activity assay from a previously calculated standard curve. The mean and standard deviation are reported. LNP diagrams were created with Biorender.com.