Poster X15 - Supramolecular Peptide-Protein Granules for Intracellular Protein Delivery

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Stephanie Herrera, MS

Graduate Student Researcher
University of Florida
Gainesville, Florida, United States

Co-Author(s)

Madisen Domayer

Graduate Student
Vanderbilt University
Ormond Beach , Florida, United States

Primary Investigator(s)

Gregory A. Hudalla, PhD (he/him/his)

Associate Professor
University of Florida
Gainesville, Florida, United States

Co-Author(s)

AA

Alex Adolphson

Graduate Student Researcher
University of Florida Biomedical Engineering, United States

Introduction: The ability to deliver active proteins across the cell membrane and into the cytosol would provide access to druggable targets that are not available within the extracellular environment. However, 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 present the development of supramolecular peptide-protein granules for intracellular protein delivery.

Materials and

Methods: This approach is based on pairs of charge-complementary molecules known as, “CATCH(+) peptides” and “CATCH(-) fusion proteins”, which associate in water to form beta-sheet fibrils when combined but remain in the soluble state when alone. Introducing a “crowding agent”, such as Tween-80 micelles, poly(ethylene glycol), or excess CATCH(+) peptide, can induce the formation of 100-200 nm “CATCH(+/-) granules” at peptide concentrations that are ~10-fold lower than the critical fibrillization limit in dilute conditions (~200 M). We use fluorescence, confocal, and electron microscopy to capture the formation of these granules and the trafficking of representative GFP granules into various cell types include NIH 3T3 Fibroblasts and Jurkats. Flow cytometry is used to measure granule toxicity and protein uptake efficiency.

Results, Conclusions, and Discussions: CATCH(+/-) granules are rapidly internalized by various adherent and suspended mammalian cell types. Different CATCH-fusion proteins can be co-assembled into multicomponent granules with tunable composition. Multicomponent granules allow for 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 and lower cell death than other state-of-the-art methods, such as cell penetrating peptides or electroporation. Collectively, these data establish CATCH(+/-) granules as a simple and flexible nanomaterial platform cell engineering via intracellular protein delivery.

Acknowledgements (Optional):