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Team:NJU China/Project/Liver
From 2013.igem.org
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Liver: For liver targeting, we need to first find a protein specifically recognize hepatic cells. Since Heptitis B virus can infect hepatic cells distinctively, and from recent study[1], we knew that HBV recognizes the hepatic cells via the interaction between the pre-S1 of the HBV envelop protein and NTCP receptor of the hepatic cells. We tried to engineer the pre-S1 from HBV envelope protein to the lamp 2b.
Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.
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 37C. After incubation for 2 hr, hep G2 cells were washed, fixed, and observed under confocal microscopy.
3. Simulation of pre-S1 exosome distribution within human body by pharmacokinetic model
Figure.3 Exosome concentration change along the time in different compartments.
As shown in figure.3, the concentration of exosomes in the blood falls down along the time while the concentration of exosomes in the peripheral compartment goes up first then falls down. Comparing the concentration change of exosomes over time in the liver(our target organ) with other tissues, we can easily see that the peak concentration of exosomes in liver is much higher than that of non-target tissues. Thus we can assure that the after add the liver targeting protein to the exosome (the percentage of exosomes get into the liver increases), the concentration of exosomes in the liver greatly outnumber that in other tissues.
