Difference between revisions of "Part:BBa K1180002:Experience"

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[[File:NJU-China-parts-RVG-confocal.jpg|350px]]
 
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Figure 6. Confocal microscopy images of fluorescent-labeled oligonucleotide (Alexa Fluor 555, red) in untreated control cells or in cells incubated with RVG exosomes (RVG exosome), unmodified exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-exosome) or RVG exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-RVG exosome). Images of four cell lines (Neuro2A, C2C12, A549 and MCF-7) were acquired.
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Figure 3. Confocal microscopy images of fluorescent-labeled oligonucleotide (Alexa Fluor 555, red) in untreated control cells or in cells incubated with RVG exosomes (RVG exosome), unmodified exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-exosome) or RVG exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-RVG exosome). Images of four cell lines (Neuro2A, C2C12, A549 and MCF-7) were acquired.
  
 
=== RVG exosomes specifically deliver MOR siRNA into neuronal cells ===
 
=== RVG exosomes specifically deliver MOR siRNA into neuronal cells ===
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[[File:NJU-China-parts-fig 7.png|350px]]
 
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Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentrations in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).
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Figure 4. Quantitative RT-PCR analysis of MOR siRNA concentrations in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).
  
 
=== GFP levels in the brains of GFP transgenic mice decrease after tail vein injection of a plasmid that expresses GFP siRNA ===
 
=== GFP levels in the brains of GFP transgenic mice decrease after tail vein injection of a plasmid that expresses GFP siRNA ===
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[[File:NJU-China-PARTS-Figure15.jpg|450px]]
 
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Figure 8. Fluorescence confocal microscopy images showing sections from different tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with saline (control) or with GFP siRNA loaded into normal exosomes (siRNA-exosome) or RVG exosomes (siRNA-RVG exosome).
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Figure 5. Fluorescence confocal microscopy images showing sections from different tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with saline (control) or with GFP siRNA loaded into normal exosomes (siRNA-exosome) or RVG exosomes (siRNA-RVG exosome).
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== Silencing capability validation (in vitro and in vivo) ==
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=== RVG exosomes loaded with MOR siRNA specifically reduce MOR expression in neuronal cells ===
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We next evaluated the effect of RVG exosome-delivered siRNA on MOR expression in vitro. MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were dramatically reduced in cells treated with RVG exosome-delivered siRNA, while no reduction in the MOR protein and mRNA levels were observed in cells treated with exosomes without the RVG peptide on their surface. These results suggest that the RVG peptide modification on the exosome membrane can specifically guide exosomes to target neuronal cells, allowing for the delivery of MOR siRNA into neuronal cells to reduce MOR expression levels.
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===User Reviews===
 
===User Reviews===

Revision as of 19:38, 18 September 2015


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Applications of BBa_K1180002

Targeting capability validation (in vitro and in vivo)

RVG exosomes specifically deliver fluorescent-labeled oligonucleotide into neuronal cells

To determine whether RVG exosomes can deliver siRNAs into neuronal cells, Neuro2A cells were selected as the recipient cells to incubate with RVG exosomes loaded with Alexa Fluor 555-tagged oligonucleotide (red fluorescence). First, untreated Neuro2A cells or cells treated with RVG exosomes but without loading the fluorescent-labeled oligonucleotide, which served as the controls, were not fluorescently labeled under fluorescence confocal microscopy. In contrast, significant fluorescence signals were observed in Neuro2A cells treated with RVG exosomes loaded with Alexa Fluor 555-tagged oligonucleotide, whereas the fluorescence signals were dramatically lower in cells treated with unmodified exosomes loaded with Alexa Fluor 555-tagged oligonucleotide. The results suggest that RVG exosomes can specifically deliver siRNA to cells of neuronal origin, while unmodified exosomes are generally rejected by neuronal cells. Interestingly, a greater number of Alexa Fluor 555-tagged oligonucleotides accumulated in non-neuronal cells including C2C12 (skeletal muscle origin), A549 (lung origin) and MCF-7 (breast origin) when these cells were incubated with unmodified exosomes compared with those with RVG exosomes, suggesting that RVG exosomes are, in contrast, rejected by non-neuronal cells. In summary, the results indicate that RVG peptide on the exosomal membrane efficiently guides exosomes to enter neuronal cells bearing the acetylcholine receptor on their membranes but prevents exosomes from entering non-neuronal cells lacking the surface acetylcholine receptor.

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Figure 3. Confocal microscopy images of fluorescent-labeled oligonucleotide (Alexa Fluor 555, red) in untreated control cells or in cells incubated with RVG exosomes (RVG exosome), unmodified exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-exosome) or RVG exosomes loaded with fluorescent-labeled oligonucleotide (oligonucleotide-RVG exosome). Images of four cell lines (Neuro2A, C2C12, A549 and MCF-7) were acquired.

RVG exosomes specifically deliver MOR siRNA into neuronal cells

Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells incubated with RVG exosomes loaded with MOR siRNA. The siRNA concentrations were barely detected in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, C2C12 cells were treated with RVG exosomes loaded with MOR siRNA, and MOR siRNA was barely detected. Taken together, these results clearly demonstrate that the RVG peptide modification on the exosome membrane specifically guides exosomes to target neuronal cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of MOR siRNA into the recipient cells.

NJU-China-parts-fig 7.png

Figure 4. Quantitative RT-PCR analysis of MOR siRNA concentrations in Neuro2A and C2C12 cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA (siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).

GFP levels in the brains of GFP transgenic mice decrease after tail vein injection of a plasmid that expresses GFP siRNA

To determine whether siRNA delivered via RVG exosomes can pass through the BBB and regulate endogenous gene expression, we packaged siRNA against green fluorescent protein (GFP) into RVG exosomes and injected them into GFP-transgenic mice through the tail vein. Then, the GFP levels in various tissues were determined by measuring fluorescence emission using a fluorescence microscope. Compared with control mice, injection of the RVG exosomes loaded with GFP siRNA dramatically reduced GFP levels in different parts of the brain of GFP-transgenic mice. In contrast, unmodified exosomes loaded with GFP siRNA did not induce obvious GFP silencing in mouse brain. However, while unmodified exosomes loaded with GFP siRNA had a significant effect on GFP levels in the lungs, livers and spleens of GFP-transgenic mice, RVG exosomes loaded with GFP siRNA only induced slight but non-significant GFP silencing in these tissues. The results successfully demonstrate that exosome-packaged siRNA can be delivered to various tissues, thus silencing endogenous gene expression. The results also indicate that RVG peptide on the surface of exosomes has some selectivity for neuronal tissues, which may simultaneously prevent siRNA from spreading to non-neuronal tissues.

NJU-China-PARTS-Figure15.jpg

Figure 5. Fluorescence confocal microscopy images showing sections from different tissues of GFP-transgenic mice. GFP-transgenic mice were intravenously injected with saline (control) or with GFP siRNA loaded into normal exosomes (siRNA-exosome) or RVG exosomes (siRNA-RVG exosome).

Silencing capability validation (in vitro and in vivo)

RVG exosomes loaded with MOR siRNA specifically reduce MOR expression in neuronal cells

We next evaluated the effect of RVG exosome-delivered siRNA on MOR expression in vitro. MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were dramatically reduced in cells treated with RVG exosome-delivered siRNA, while no reduction in the MOR protein and mRNA levels were observed in cells treated with exosomes without the RVG peptide on their surface. These results suggest that the RVG peptide modification on the exosome membrane can specifically guide exosomes to target neuronal cells, allowing for the delivery of MOR siRNA into neuronal cells to reduce MOR expression levels.



User Reviews

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