Targeting Efficiency and Specificity of CAP-Exosomes and Lamp2b-Exosomes in the Brain 
To investigate the potential of CAP-exosomes to specifically deliver a transgene,  (GFP gene expression plasmid), we have developed a workflow, as shown in Figure 2A.  First, CAP-exosomes and Lamp2b-exosomes were labeled with 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) dye. To wash away free DiI, exosomes  were resuspended in 1× phosphate- buffered saline (1XPBS) and pelleted by ultracentrifugation. Next, pelleted DiI-labeled exosomes were resuspended in electroporation buffer,  and a total of 50 µg of exosomes corresponding to ~5 × 10^9 particles were mixed with 50  µg of GFP expression plasmid DNA in a final volume of 100 µL electroporation buffer for  a single IV injection to be administered into FVB mice. GFP expression plasmid was inserted into respective exosomes using 4-D-Nucleofector (instruments and reagents from  Lonza, Basel, Switzerland) following the company’s instructions. Mice were sacrificed 10  days after IV injection, and serial coronal sections were made and images were taken with  a confocal microscop. 

4.1.2. Plasmid DNA Transfection in BM-MSC Cells and Loading into Exosomes by Electroporation
BM-MSCs were used for transfection of CAP-Lamp2b, and hygro-Lamp2b expression cassette containing plasmid DNA by electroporation. GFP expression plasmid was inserted into CAP-exosomes using 4-D-Nucleofector (instruments Core Unit: AAF-1001B and reagents from Lonza, Basel, Switzerland, following the company’s instructions). In brief, a P3 primary cell 4D Nucleo factor kit (V4XP3024, catalog no, Lonza) was used first for standardizing the efficiency of plasmid DNA transfection in cells and loading in exosomes. Using 5 × 10^6 cells, 5 µg DNA, and 7 different electroporation program available in the 4D Nucleofactor X unit, highest efficiency was achieved when using program No.1. In the case of both exosomes, 1 × 10^9 particles, 10 µg of GFP-plasmid DNA, 100 µL buffer, and the use of 7 different electroporation program, highest efficiency was achieved by program No. 5, as determined by measuring GFP gene expression in BM-MSCs after transfection with GFP gene-loaded exosomes.