Part:BBa_K1180002:Experience
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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.
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.
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.
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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