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 target 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.