Results for liver targeting:
To produce the exosomes that have pre-S1 on their surface for liver targeting, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmid encoding the fusion protein of lamp 2b and pre-S1 peptide.

1. Exosome morphology under SEM and TEM
The exosomes produced by the transfected HEK 293T cells was collected 24h post transfection. The morphology and diameter of the exosomes were examined by both SEM and TEM.
As shown in Fig.1 and Fig.2, the diameter of the exosomes is around 50nm and it was round.

Figure.1 SEM micrographs of the exosomes isolated from 293T cell culture medium after transfection of pre-S1 plasmid.

Figure.2 TEM micrographs of the exosomes isolated from 293T cell culture medium after transfection of pre-S1 plasmid.

2. In vitro evidence for the entry of pre-S1 exosomes into the hep G2 cell
As shown in Fig.3, by labeling the exosomes with DiI-C16 (red) and hep G2 nucleus with DAPI(blue), it can be seen that the exosomes successfully get into the hep G2 cells.

Fig.3 Confocal microscopy image of the internalization of fluorescently labeled MVs into hep cells. 293T cells were labeled with DiI-C16 (red) and then cultured in RPMI 1640 medium supplemented with 10% FBS. After 4 hr, the supernatants were collected and centrifuged to harvest exosomes. 293T exosomes were resuspended in MCDB-131 medium and incubated with hep G2 cells at 37􏰀C. After incubation for 2 hr, hep G2 cells were washed, fixed, and observed under confocal microscopy.

3. In vivo evidence for the entry of pre-S1 exosomes into the liver