Part:BBa_K1633004:Experience

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Applications of BBa_K1633004

USAGE AND BIOLOGY

We package MOR siRNA into exosomes by transfecting HEK293 cells with a a Lamp2b-RVG plasmid and the MOR siRNA-2 plasmid and then collect siRNA-encapsulated exosomes. When inject the modified exosomes into the bloodstream, exosome will specifically recognize acetylcholine receptors and fuse with neurons under the direction of the RVG peptide. Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed function will result in the inhabitation of the secretion of GABA and the suppression of the dopaminergic reward pathway, which ultimately have some therapeutic effects on opioid dependence.

CHARACTERIZATION

Interference efficiency of MOR siRNA-2 plasmid

To ensure the interference efficiency,MOR siRNA-2 plasmid was transfected into the mouse neuroblastoma cell line Neuro2A. Efficient knockdown of MOR by MOR siRNA-2 in Neuro2A cells is observed.

Package of MOR siRNA into exosomes

The levels of MOR siRNA in isolated exosomes were assayed by a quantitative RT-PCR assay. The MOR siRNA concentration in exosomes was calculated to be approximately 0.14 pmol/μg. The results showed that MOR siRNA can be successfully packaged into exosomes, no matter the exosomes were modified on the outside membrane with or without RVG peptide.

Figure 5. The concentration of MOR siRNA in unmodified or RVG-modified exosomes.

TEM photographs of exosomes carrying MOR siRNA inside and RVG on membranes

We next characterized the RVG exosomes loaded with MOR siRNA using transmission electron microscopy (TEM). The TEM photographs showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 90 nm and a double-layer membrane surrounded. These characteristics indicate that the exosome properties were not affected by the modifications.

Figure 6. TEM photographs of the exosomes with outside RVG modification and inside siRNA loading.

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