Authors:

Madisen R. Domayer

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About Paper:

Intracellular delivery of protein therapeutics has many challenges and limited availability until now. The current methods utilize electroporation, delivery vehicles that facilitate protein escape from intracellular vesicles during endocytosis (i.e., endosomal escape), or highly charged peptides that mediate direct protein crossing of the cell membrane [1]. Key challenges with these approaches include low delivery efficiency, cytotoxicity, or both. In addition to high delivery efficiency and cytocompatibility, an ideal intracellular delivery vehicle would require low amounts of protein, maintain protein activity during internalization, and work universally with various cell types (e.g., adherent and suspended). Here we will present our research developing co-assembled peptide-protein granules for intracellular delivery. These granules are based on a series of charge-complementary co- assembling peptide pairs, known as "CATCH (+/-)". When combined in solution, CATCH peptide pairs co-assemble into supramolecular biomaterials, including nanofibers and fibrillar hydrogels [2-4]. Here we will show that the CATCH(+) peptide and protein with a CATCH(-) fusion tag can co-assemble into nanoscale granules in the presence of macromolecular crowders. A mixture of CATCH(6+) peptide, CATCH(-)-GFP, and PEG produces nano-scale supramolecular granules. Granule shape, size, and assembly kinetics are affected by the molecular weight and concentration of PEG used, demonstrating that the system can be controlled. As the molecular weight of PEG increases, the granules form faster and are larger in size. Higher concentrations of PEG produce the same effect. Granules formed in mixtures of CATCH(6+) peptide, CATCH(6e-)-GFP, and PEG did not induce death of NIH3T3 fibroblasts (i.e., were cytocompatible). After 24 hours of treatment with granules, more than 90% of NIH3T3 cells were GFP positive. CATCH peptide-protein granules yielded greater GFP delivery efficiency and induced less cell death than other methods, such as cell penetrating peptides and electrophoresis.

Source:

University of Florida / Madisen R. Domayer, Renjie Liu, Gregory A. Hudalla / 2023

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Co-authors:

Madisen R. Domayer