Brain

For brain targeting, we chose to use RVG, which is a short peptide from Rabies Virus, as our targeting protein. RVG can specifically recognize acetylcholine receptor in the central nervous system [2], thus we engineer the RVG peptide into the lamp 2b and we use pcDNA 3.1(+) as our vector.
To produce the brain-specific RVG exosomes, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmids encoding the fusion protein of lamp 2b and RVG peptide.
Also, we use a siRNA as an indicator to assess the targeting effect of RVG-exosomes and loaded the siRNA into the RVG-modified exosomes by direct transfection of the HEK 293T cells with siRNA probe.

Brain targeting Results：
To produce the exosomes that have RVG on their surface for brain targeting, we first transfected the exosome-producing cells, HEK 293T cells, with the plasmid encoding the fusion protein of lamp 2b and RVG peptide.
To check if the RVG-containing exosomes can be delivered to the brain, we use a siRNA as probe. We first encapsulated the siRNA into the RVG-modified exosomes by direct transfection of the HEK 293T cells with siRNA probe.

1.Quantification of siRNA contained in the exosomes
We first quantify the amount of siRNA encapsulated into the exosomes. We transfected the HEK 293T cells (transfected with RVG plasmids before) with siRNA before collecting the exosome. We used the exosomes collected from the HEK 293T cells (transfected with RVG plasmids before) without transfection of siRNA as negative control.
By quantitative PCR analysis of a series of siRNA with known concentration, we drew a standard curve. By referring to this curve, we calculate the absolute amount of siRNA in the exosomes. As shown in Fig.1, the amount of siRNA in the negative control is quite low (background) while the siRNA contained in the RVG exosomes transfected with siRNA probe reaches as high as 0.8 fmol (RNA)/μg (exosome).

Figure.1 Empty exosome is collected from HEK 293 T cells without transfection of siRNA probe while the RVG exosome +siRNA is collected from the HEK 293T cells after transfection of siRNA probe. The amount of siRNA contained in the exosome is measured by qPCR.

In vitro evidence for the entry of RVG exosomes into the primary cortical neuron
From the quantification of siRNA contained in the exosome，we can conclude that siRNAs can be encapsulated into RVG exosomes. Then we co-culture the siRNA-containing RVG exosomes with primary cortical neurons, and then measure the amounts of siRNA probe in the neurons to see if our RVG exosome can successfully get into the neurons.
From Fig.2, we can see that siRNA probe labeled with Alexaflour 555（red ）successfully get into the neurons.

Figure.2 Confocal microscopy image of the internalization of Alexaflour 555（red ）labeled siRNA into HepG2 cells.

Then we use qPCR to measure the relative siRNA probe level in the neurons co-cultured with both empty RVG-exosomes and siRNA containing RVG-exosomes. We can easily see from Fig.3 that almost no siRNA was detected from the neurons co-cultured with 40 μg empty RVG-exosomes, while the siRNA detected in neurons co-cultured with siRNA probe containing RVG-exosomes show both significant and dose dependent increase.

Figure.3 The RNA was extracted from the primary cortical neurons co-cultured 24h with 40 μg empty exosome, 20μg siRNA containing RVG-exosomes,40μg siRNA containing RVG-exosomes, respectively. And the RNA extracted was measured by qPCR using probe for the siRNA encapsulated in the exosomes.

In vivo evidence for the entry of RVG-exosomes entry into the brain
To further investigate whether the RVG-exosome can get into brain, we intravenously injected the empty RVG-exosomes, siRNA containing RVG-exosomes, respectively, into the mouse. Then we took the brain out and measure the siRNA level in the cortex and medulla.
As shown in Fig.4, no siRNA was detected in both cortex and medulla after the injection of empty RVG-exosomes, while for the siRNA containing RVG-exosomes, the siRNA detected in the cortex and medulla are significant higher than that of the empty exosomes.

Figure.4 The mice were intravenously injected with 200 μg empty and siRNA containing RVG-exosomes, respectively once a day, and continued for four days. On the fifth day, the mice were killed and their brains were taken out. The RNA from their cortex and medulla were measured using siRNA probe for the siRNA encapsulated in the exosome.