Figure 1.

In Vitro Confirmation of Exosome Display Targeting of Model Ag to Exosomes

(A) Schematic diagram showing expression plasmid design. Enhanced green fluorescent protein (EGFP) was engineered into pcDNA3.1SSmut_C1C2 to generate pcDNA3.1SSmut-EGFP_C1C2 (EGFP_C1C2) or pcDNA3.1SSmut-EGFPΔ (EGFPΔ), respectively. (B and C) Following transfection of Expi293F cells with EGFPΔ (B) or EGFP_C1C2 (C), exosomes were purified by ultracentrifugation (100,000 × g) and size exclusion chromatography (SEC). ELISAs using 20 μL of each SEC fraction confirmed enrichment of exosome markers, tetraspanins CD9, CD63, and CD81 on expected fractions (exosomes are 8–13) for EGFPΔ and EGFP_C1C2, and EGFP on the surface of EGFP_C1C2-transfected cells only. (D) SEC fractions enriched for CD9, CD63, and CD81 were validated using nanoparticle tracking analysis (NTA: Malvern Panalytical) to confirm ∼150-nm size and concentration. The mode value for size is shown, which is the size in nanometers of the majority of particles. (E) ELISA on 1 μg/well of pooled, purified exosomes to confirm the presence of tetraspanins CD9, CD63, and CD81 and the presence of EGFP on the surface of exosomes purified from EGFP_C1C2-transfected cells only. Data shown represent background subtraction of matched isotype control Abs. Positive controls for tetraspanins included exosomes purified from Du145 cells (provided by Prof. A Clayton and Dr. J. Webber, Cardiff University, Cardiff, UK). Wells coated with 1 μg/mL recombinant EGFP protein (Abcam, USA) were used as a positive control for EGFP Ab binding. Dashed line separates exosome samples from control samples. (F) Western blot on purified exosomes (Exo) and cleared cell lysate (CL) from the corresponding transfected cells to detect exosome-enriched endosomal protein ALIX and to show minimal detection of exosome-excluded endoplasmic reticulum protein GRP94.