Fig. 5: Liposome-facilitated delivery of Cre- and ABE-RNPs to reporter cells. | Nature Communications

Fig. 5: Liposome-facilitated delivery of Cre- and ABE-RNPs to reporter cells.

From: A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina

Fig. 5: Liposome-facilitated delivery of Cre- and ABE-RNPs to reporter cells.

a Pictorial depiction of the liposome formulation. b The size distribution of the CBB11-alone (black) and CBB11-doped liposomes (Lipo1, dark green), in TAS buffer. Average of 3 replicates. c Fluorescence microscopic quantification of the recombination activity triggered by Cre-recombinase delivery, mediated by Lipo1 (dark green) or Lipo2 (dark yellow). CBB14 (red) and Lipofectamine 3000 (LF, purple) served as positive controls. C, Cre recombinase only; NE, no enzyme. Data are presented as the mean of 2 replicates. d The zeta potentials of Lipo1 (blue) and Lipo2 (yellow) in the absence (-, light color) or the presence (+, dark color) of ABE-RNP. The ratio of ABE-RNP to CBB11 is 1:8. Means of two biological replicates. e Representative images of cryo-TEM of Lipo1 (upper right), ABE-RNP only (upper left) and Lipo1 with ABE-RNP (lower). The blue arrows point to ABE-RNP aggregates, and the dark points labeled with the yellow arrow are assumed to be the protein on the liposome surface. Scale bar, 100 nm. N, 8 replicates. f Titration of in vitro delivery of ABE8e-RNPs mediated by CBB11 (blue), CBB14 (red), and Lipo1 (dark green) ~ 5 h after the RNP was complexed with the CBB compounds and liposomes. The ratio [CBB]/[RNP] = 8:1. The total lipid amounts of Lipo 2 (dark yellow) and Lipofectamine 3000 (LF, purple, as control) were comparable to those for Lipo1. Data are presented as mean ± SD; n = 2 replicates. g, h Titration of in vitro delivery of ABE8e-RNPs mediated by Lipo3 (dark blue)/Lipo4 (orange) (g); and Lipo5 (light purple)/ Lipo6 (yellow) (h), respectively, after allowing 15 min for complex formation in each case. The ionizable lipid concentration was always 80-fold greater than the concentration of ABE-RNP. ABE-RNP in TAS buffer (TAS, green) served as a negative control. Data are presented as mean ± SD; n = 4 replicates. i Fluorescence-microscopic images of rd12-reporter cells treated with liposome-RNP complex. The RNP concentration was 100 nM. Representative of 2 biological replicates. Scale bar, 200 μm. Source data are provided as a Source Data file.

Back to article page