(229e) Supramolecular Peptide-Protein Granules for Intracellular Protein Delivery

The ability to deliver active proteins across the cell membrane and into the cytosol would open up a new multi-billion dollar market by providing access to drug targets and signaling pathways that are not accessible within the extracellular environment, with faster pharmacokinetics than what are afforded by DNA and mRNA delivery approaches that require transcription and/or translation. Due to their large size, charge, and hydrophilicity, proteins do not efficiently cross the cell membrane to enter the cytosol on their own. Here we will report on the development of supramolecular peptide-protein granules for intracellular protein delivery. This approach is based on pairs of charge-complementary molecules known as, “CATCH(+) peptides” (sequence = XQKFKFKFKQX; X = Q or K), and, “CATCH(-) fusion proteins” (sequence = YQEFEFEQY; Y = Q or E), which associate in water to form β-sheet materials when combined, but remain in the soluble state when alone. Above a critical concentration of ~200 mM, CATCH(+/-) pairs form elongated fibrillar structures, while in the mM range, these fibrils can entangle to form macroscopic hydrogels. Here we will show that introducing a “crowding agent”, such as Tween-80 micelles or poly(ethylene glycol), can induce the formation of 100-200 nm “CATCH(+/-) granules” at peptide concentrations that are ~10-fold lower than the critical assembly limit. CATCH(+/-) granules are rapidly internalized by various adherent and suspended mammalian cell types. Over time, the active protein payload is delivered to the cytosol or other intracellular compartments. For example, CATCH-Cas9 co-delivered with a guide RNA can knockdown GFP signaling with comparable efficiency as “gold standard” lipofectamine vehicles, demonstrating CATCH-mediated protein delivery into the nucleus. Combining the CATCH(+) peptide with two different CATCH(-) fusion proteins provides granules with compositions governed by the molar ratio of the proteins in the mixture. These multi-protein CATCH(+/-) granules allow for co-delivery of the different protein payloads into the same cell, with user-defined control of the relative dose of each protein type. Importantly, this dual-component granule enables co-delivery of both an “effector” (i.e., a protein that confers therapeutic function) along with a “selector”, where the latter can be used to enrich the sub-population of cells that have internalized the CATCH(+/-) granule. CATCH(+/-) granule internalization demonstrates greater delivery efficiency than other state-of-the-art methods, such as cell penetrating peptides or electroporation, without inducing cell death. We envision that this simple supramolecular nanomaterial approach will afford new opportunities for rapid and efficient cell engineering through intracellular protein delivery.