Fig. 3: Preparation, characterization and mitochondrial targeting behavior of 6F-M-LNPs.
A Formulation of 6F-M-LNPs via microfluidic mixing and thiol-maleimide coupling. B Cellular uptake capacity of 6F-M-LNPs with different proportions of MTS (n = 3 biologically independent samples). C Mitochondrial GFP transfection efficiency of 6F-M-LNPs with different proportions of MTS (n = 3 biologically independent samples). D Physicochemical characterization of 6F-LNPs before and after MTS grafting (n = 3 biologically independent samples), EE encapsulation efficiency. E TEM image of 6F-M-LNPs. Scale bar: 200 nm. F Mitochondrial gene delivery of different F-LNPs detected by CLSM. Scale bar: 10 μm. Mitochondria (Mitotracker Green probe): green; gene cargo (Cy5-pDNA): red. White arrows: Cy5-pDNA in the cytosol. G Pearson’s correlation coefficient between Cy5-pDNA and mitochondria (n = 3 biologically independent samples). H The mitochondrial targeting of different F-LNPs detected by MFI of Cy5-pDNA in extracted mitochondria (n = 3 biologically independent samples). I Detection on key mitochondrial membrane proteins (representative images from n = 3 independent experiments). J The mtGFP transfection after gene silencing of Tomm20 and Tomm22 (n = 3 biologically independent samples). K Mitochondrial colocalization of 6F-M-LNP/Cy5-pDNA observed by Multi-SIM (representative images from n = 3 independent experiments). Outer mitochondrial membranes were labeled by Tomm20-mEmerald transfection. Inner mitochondrial membranes were labeled by Mitotracker Red probe. L CLSM images showing 6F-M-LNP/Cy3-pDNA localization inside the mitochondria (representative images from n = 3 independent samples). Outer mitochondrial membranes (mouse mAb to TOMM20): green; mitochondrial matrix proteins (rabbit pAb to SDHA): red; Cy3-pDNA: blue. M Schematic illustration of mitochondrial targeting behavior of 6F-M-LNPs. A and M was created with BioRender.com. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test (two-tailed; B, C, G, H, J) was used to calculate the statistical significance.
