Fig. 2.
Generation of AP-EXOR&L&S and evaluation of their ability to promote axon elongation in vitro. (a) Flow cytometry for measuring the loading efficiency of RVG-CP05, ILP-CP05 and ISP-CP05 to AP-EXO. Cy5.5 labeled RVG-CP05 (10 μg), FAM labeled ILP-CP05 (10 μg) and MCA labeled ISP-CP05 (10 μg) were co-incubated with AP-EXO (10 μg) together at 4 °C for 6 h, the non-specific binding peptides were removed by ultracentrifugation. AP-EXORVG indicated AP-EXO loaded with RVG-CP05, AP-EXOISP indicated AP-EXO decorated by ISP-CP05, AP-EXOILP indicated AP-EXO decorated by ILP-CP05. AP-EXOR&L&S represents AP-EXO simultaneously painted by RVG-CP05, ILP-CP05 and ISP-CP05. Left 3D images show the simultaneously binding efficiency of three peptides on AP-EXO, right 2D images show the analysis process. Cy5.5 as the indicator of horizontal axis and MCA as the indicator of vertical axis for those four exosomes showed in the middle panel of Fig. 2a, and also FAM as the indicator of the horizontal axis in the right panel of Fig. 2a. (b) Representative TEM images of AP-EXO and AP-EXOR&L&S. Scale bar: 200 nm. TEM, Transmission electron microscope. (c) Size distribution of AP-EXO and AP-EXOR&L&S. (d) Representative confocal images showing binding and colocalization of rhodamine-labeled ISP-CP05, FAM-labeled ILP-CP05 and Alexa Fluor 405-labeled RVG-CP05 with DiR-labeled AP-EXO on polystyrene beads. Scale bar: 1 μm. (e–g) Axonal guidance spot assays to evaluate the ability of AP-EXOR&L&S in promoting axonal elongation toward the CSPG-spot barrier. e, representative images of primary cortical neurons cross the CSPG-spot barrier after AP-EXOR&L&S incubation. Scale bar:10 μm. f, quantitative analysis of axons crossing the CSPG barrier per primary cortical neuron (n = 34, *p < 0.05, one way ANOVA on ranks post Dunn's Method). g, the comparison of cortical neurons crossing the CSPG barrier across 3 groups (n = 4, **p < 0.001, one-way ANOVA post Student-Newman-Keuls test). (h–j) Axon elongation of primary cortical neurons cultured in CSPG medium. h, representative images of axon length and branches (asterisk) number after AP-EXOR&L&S treatment. Scale bar: 20 μm. i, quantification of axon length (NC: n = 51, AP-EXO: n = 95, AP-EXOR&L&S: n = 135. *p < 0.05, one way ANOVA on ranks post Dunn's Method). j, quantification of branches number shown in (h) (NC: n = 48, AP-EXO: n = 57, AP-EXOR&L&S: n = 69. *p < 0.05, one way ANOVA on ranks post Dunn's Method).