FIGURE SUMMARY
Title

Extracellular Nanovesicle Enhanced Gene Transfection Using Polyethyleneimine in HEK293T Cells and Zebrafish Embryos

Authors
Zhang, Z., Wen, K., Zhang, C., Laroche, F., Wang, Z., Zhou, Q., Liu, Z., Abrahams, J.P., Zhou, X.
Source
Full text @ Front Bioeng Biotechnol
Figure 1

(A) Schematic representation for EV generation from cells. (B) EV isolation process by multiple centrifugation. (C) SDS PAGE gel of EVs, showing a thick band with molecular weight of ~200 kD and a few thin bands between 50–200 kD using Coomassie Blue staining. (D) Band-shift assay of DNA, PEI/DNA complexes, and PEI/DNA/EVs complexes, at different N/P ratios (the molar ratio of nitrogen in PEI to phosphor in DNA). In (D) PEI60kD was used in lanes 3–9 and PEI25kD was used in lanes 10–15.
Figure 2

AFM images of (A) EVs, (B) PEI25kD/DNA, (C) PEI25kD/DNA/EVs, (E) PEI60kD/DNA, (F) PEI60kD/DNA/EVs, and (D) TEM images of EVs. In (A), the EVs were adsorbed on positively charged mica that was functionalized with (3-amino- propyl)triethoxysilane. In (B,C,E,F), the complexes were adsorbed on freshly cleaved mica. In (D), the left image shows EVs that were fixed with PFA, stained with PTA, and adsorbed on positively charged carbon grid; the middle image showed the EVs that were fixed with PFA, stained with PbAc, and adsorbed on positively charged carbon grid; the right image shows the EVs that were fixed with PFA, stained with PTA, and adsorbed on formvar grid.
Figure 3

(A–C) TEM images of PEI25kD/DNA/EVs; (E–G) TEM images of PEI25kD/DNA; (D,H) Schematic representation of the complex formation of (D) EVs/DNA/PEI and (H) PEI/DNA.
Figure 4

Optical (left) and fluorescent (right) images of HEK293T cells transfected with plasmid EGFP using (a–f) PEI60kD at N/P = 80 and (a'–f') PEI25kD at N/P = 160, at a post transfection time of 48 h. EV solution of different volume (0, 0.5, 1, 2, 5, and 10 μL) were added into each well of the 96-well plate for images (a–f) and for (a'–f'), respectively. The protein concentration in EV solution was 3 ng/μL−1 All the images were taken at the same exposure time (500 ms).
Figure 5

Transfection efficiency of HEK293T cells by plasmid EGFP using (A) PEI60kD and (B) PEI25kD; Luciferase activity after transfection using (C) PEI60kD and (D) PEI25kD; PTEN expression after transfection using (E) PEI60kD and (F) PEI25kD; determined by MTT assay, at a post transfection time of 48 h for different N/P ratios. EV solution of different volume (0, 0.5, 1, 2, 5, and 10 μL) with protein concentration of 3 ng/μL−1 were added into each well of the 96-well plate.
Figure 6

Cell viability of HEK293T cells transfected with plasmid EGFP using (A) PEI60kD and (B) PEI25kD; Cell viability of HEK293T cells transfected with plasmid ELuc using (C) PEI60kD and (D) PEI25kD; Cell viability of HEK293T cells transfected with plasmid EpTEN using (E) PEI60kD and (F) PEI25kD, determined by MTT assay, at a post transfection time of 48 h for different N/P ratios. EV solution of different volume (0, 0.5, 1, 2, 5, and 10 μL) was added into each well of the 96-well plate. The protein concentration in EV solution was 3 ng/μL−1.
Figure 7

Fluorescence images of (A–C) PEI25kD/EGFP, (D–F) PEI25kD/EGFP/EVs, (G–I) PEI60kD/EGFP, and (J–L) PEI60kD/EGFP/EVs in zebrafish embryos 8 h after injection.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Front Bioeng Biotechnol
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