Therapeutic delivery of proteins and nucleic acids into cells is a difficult goal. Of the many challenges in the delivery process, perhaps the most demanding is providing these biologics with access to the cytosol. Most delivery strategies employ endosomal uptake, requiring endosomal escape for the payload biologics to be effective. In our research, we have developed strategies to deliver proteins and nucleic acids directly to the cytosol through fusion of nanomaterials with the cell membrane. Results and Discussion We have developed two systems for delivering biologics into the cytosol. The first is nanoparticle-stabilized nanocapsules (NPSC), formed via assembly of arginine-functionalized gold nanoparticles (AuNP) around fatty acid nanodroplets. These systems have been used to deliver proteins and siRNA directly to the cytosol in vitro, and have been used for efficient delivery of siRNA targeting TNF-a in vivo, providing effective anti-inflammatory treatment. Our second system uses co-engineering of nanoparticles and proteins to deliver proteins into the cytosol. These studies co-assemble arginine-functionalized AuNPs with Cas9 protein modified with a glutamate (e-tag) chain. We have employed this system to co-deliver complete CRISPR machinery (Cas9 protein and guide RNA) for effective editing both in vitro (Figure 1) and in vivo.

Direct cytosolic delivery of nucleic acids and proteins (including CRISPR/Cas9) through membrane fusion: Who needs endosomes?

256th National Meeting (2018)

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