Team:NJU China/Project/Liver

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<h1>Liver: <span>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.
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<h1>Liver: </h1>
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Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.</span>
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<p>For liver targeting, we need to first find a protein specifically recognize hepatic cells. Since Hepatitis 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.</br>
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<img style="float:left" src="https://static.igem.org/mediawiki/2013/d/d9/Pcdna3.1_pres1_small.png">
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Therefore we cloned the pre-S1 into lamp 2b, and we choose pcDNA 3.1(+) as our backbone.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 plasmids encoding the fusion protein of lamp 2b and pre-S1 peptide.</p>
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</br><img width="460px"; height="300px" src="https://static.igem.org/mediawiki/2013/3/3e/%E8%AF%86%E5%88%ABpre_s1_small.png">
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<p>Results for liver targeting:</BR>
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<h1>Experimental procedures and Results :</h1>
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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.</p>
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<p>1. Exosome morphology under TEM</p>
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The exosomes produced by the transfected HEK 293T cells was collected 24h post transfection. The morphology and diameter of the exosomes were examined by TEM.</p>
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<p>1. Exosome morphology under SEM and TEM</br>
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<p>As shown in Fig.1, the diameter of the exosomes is around 50nm and it was round.</p>
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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.</br>
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As shown in Fig.1 and Fig.2, the diameter of the exosomes is around 50nm and it was round.</p>
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<img src="https://static.igem.org/mediawiki/2013/e/e8/M3-project-liver_targeting.png"></br></br>
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<img width="400px"; height="400px" src="https://static.igem.org/mediawiki/2013/3/35/Njuliver1.jpg"></br></br>
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<p>Figure.1 SEM micrographs of the exosomes isolated from 293T cell culture medium after transfection of pre-S1 plasmid.<p>
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<p>Figure.1 TEM micrographs of the exosomes isolated from 293T cell culture medium after transfection of pre-S1 plasmid.<p>
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<img src="https://static.igem.org/mediawiki/2013/0/05/M3-Project-liver_targeting_results-figure1b.png"></br></br>
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<p>2. In vitro evidence for the entry of pre-S1 exosomes into the HepG2 cell</p>
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<p>Figure.2 TEM micrographs of the exosomes isolated from 293T cell culture medium after transfection of pre-S1 plasmid.<p>
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As shown in Fig.2, by labeling the exosomes with DiI-C16 (red) and HepG2 nucleus with DAPI(blue), it can be seen that the exosomes successfully get into the HepG2 cells.</p>
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<p>2. In vitro evidence for the entry of pre-S1 exosomes into the hep G2 cell</br>
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<img width="400px"; height="400px" src="https://static.igem.org/mediawiki/2013/1/17/%E5%9B%BE%E7%89%876.jpg"></br>
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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.</p>
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<p>Fig.2 Confocal microscopy image of the internalization of fluorescently labeled MVs into HepG2 cells. 293T cells were labeled with DiI-C16 (red) and then cultured in RPMI 1640 medium supplemented with 10% FBS. After 4 hours, the supernatants were collected and centrifuged to harvest exosomes. 293T exosomes were resuspended in MCDB-131 medium and incubated with HepG2 cells at 37°C. After incubation for 2 hours, HepG2 cells were washed, fixed, and observed under confocal microscopy.<p>
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<img width="500px"; height="500px" src="https://static.igem.org/mediawiki/2013/c/c3/NjuLiver.png">
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<p>3. In vivo evidence for the liver-specific delivery of siRNA by pre-S1 exosome </p>
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<p>In this part of experiments, we investigated the potential for exosome-mediated systemic siRNA delivery to liver. </p><p>
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<a name="com">&nbsp;</a>
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To characterize the tissue distribution, we chose GFP(Green Fluorescent Protein) mice as our animal model and anti-GFP siRNA(which can silence the GFP gene ) as our ‘killing device’.
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<p>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.</p>
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We first collected three kinds of exosomes:1) empty exosomes (no targeting peptide outside and no anti-GFP siRNA inside)2) exsosomes with anti-GFP siRNA inside only(no targeting ability) 3) exsosomes with both anti-GFP siRNA inside and targeting peptides pre s1 on the surface.
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<p>3. Simulation of pre-S1 exosome distribution within human body by pharmacokinetic model</p>
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Three groups of mice were injected intravenously with three kinds of exosomes respectively. Finally, by using histological section technique and fluorescence microscope, we directly observed the expression level of GFP gene in mice liver from different experimental groups.
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<img src="https://static.igem.org/mediawiki/2013/8/85/M3-Project-liver_targeting_results-figure3.png"></br>
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<p>Figure.3 Exosome concentration change along the time in different compartments.<p>
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Given the same cell number and different expression level of GFP gene, we could concluded that injection of empty exosome did not lead to any significant change of GFP expression in mice liver (Fig.3A). Similarly, the siRNA-contained-only exosome induced a slight, but non-significant GFP silencing in liver (Fig.3B). By contrast, injection of siRNA-pre-S1 exosomes resulted in significant knockdown of GFP mRNA in liver, where the target of preS1 expressed (Fig.3C). These results successfully demonstrated that our modified exosomes could specifically deliver siRNA into liver and thus silence target gene.</p>
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<p>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.<p>
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<img width="742px"; height="400px" src="https://static.igem.org/mediawiki/2013/6/6e/55555.jpg"></br>
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<p>Fig.3 In vivo evidence for the liver-specific delivery of siRNA by pre-S1 exosome. Mice were intravenously injected with anti-GFP siRNA encapsulated in pre-S1 exosomes. Figures above show the section of liver tissue. Upper ones were captured under dark field and lower ones were captured under light field in the same visual field. From the comparison between dark field picture and light field picture, we can easily see the different expression level of GFP between different groups. A was injected wih empty exosomes (no targeting peptide outside and no anti-GFP siRNA inside). B was injected with exsosomes with anti-GFP siRNA inside only(no targeting ability) C was injected wih exsosomes with both anti-GFP siRNA inside and targeting peptides pre-s1 on the surface.</p>
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Latest revision as of 16:50, 28 October 2013

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Liver:

For liver targeting, we need to first find a protein specifically recognize hepatic cells. Since Hepatitis 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.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 plasmids encoding the fusion protein of lamp 2b and pre-S1 peptide.



Experimental procedures and Results :

1. Exosome morphology under 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 TEM.

As shown in Fig.1, the diameter of the exosomes is around 50nm and it was round.



Figure.1 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 HepG2 cell

As shown in Fig.2, by labeling the exosomes with DiI-C16 (red) and HepG2 nucleus with DAPI(blue), it can be seen that the exosomes successfully get into the HepG2 cells.


Fig.2 Confocal microscopy image of the internalization of fluorescently labeled MVs into HepG2 cells. 293T cells were labeled with DiI-C16 (red) and then cultured in RPMI 1640 medium supplemented with 10% FBS. After 4 hours, the supernatants were collected and centrifuged to harvest exosomes. 293T exosomes were resuspended in MCDB-131 medium and incubated with HepG2 cells at 37°C. After incubation for 2 hours, HepG2 cells were washed, fixed, and observed under confocal microscopy.

3. In vivo evidence for the liver-specific delivery of siRNA by pre-S1 exosome

In this part of experiments, we investigated the potential for exosome-mediated systemic siRNA delivery to liver.

To characterize the tissue distribution, we chose GFP(Green Fluorescent Protein) mice as our animal model and anti-GFP siRNA(which can silence the GFP gene ) as our ‘killing device’.

We first collected three kinds of exosomes:1) empty exosomes (no targeting peptide outside and no anti-GFP siRNA inside)2) exsosomes with anti-GFP siRNA inside only(no targeting ability) 3) exsosomes with both anti-GFP siRNA inside and targeting peptides pre s1 on the surface.

Three groups of mice were injected intravenously with three kinds of exosomes respectively. Finally, by using histological section technique and fluorescence microscope, we directly observed the expression level of GFP gene in mice liver from different experimental groups.

Given the same cell number and different expression level of GFP gene, we could concluded that injection of empty exosome did not lead to any significant change of GFP expression in mice liver (Fig.3A). Similarly, the siRNA-contained-only exosome induced a slight, but non-significant GFP silencing in liver (Fig.3B). By contrast, injection of siRNA-pre-S1 exosomes resulted in significant knockdown of GFP mRNA in liver, where the target of preS1 expressed (Fig.3C). These results successfully demonstrated that our modified exosomes could specifically deliver siRNA into liver and thus silence target gene.


Fig.3 In vivo evidence for the liver-specific delivery of siRNA by pre-S1 exosome. Mice were intravenously injected with anti-GFP siRNA encapsulated in pre-S1 exosomes. Figures above show the section of liver tissue. Upper ones were captured under dark field and lower ones were captured under light field in the same visual field. From the comparison between dark field picture and light field picture, we can easily see the different expression level of GFP between different groups. A was injected wih empty exosomes (no targeting peptide outside and no anti-GFP siRNA inside). B was injected with exsosomes with anti-GFP siRNA inside only(no targeting ability) C was injected wih exsosomes with both anti-GFP siRNA inside and targeting peptides pre-s1 on the surface.