Intracellular Biomacromolecule Delivery by Stimuli-Responsive Protein Vesicles Loaded by Hydrophobic Ion Pairing

Gray, Mikaela A.; de Janon, Alejandro; Seeler, Michelle; Heller, William T.; Panoskaltsis, Nicki; Mantalaris, Athanasios; Champion, Julie A.

doi:10.1021/acsomega.4c07666

Intracellular Biomacromolecule Delivery by Stimuli-Responsive Protein Vesicles Loaded by Hydrophobic Ion Pairing

Journal Article · Tue Jan 14 00:00:00 EST 2025 · ACS Omega

DOI:https://doi.org/10.1021/acsomega.4c07666· OSTI ID:2538134

^[1]; de Janon, Alejandro ^[1]; Seeler, Michelle ^[1]; ^[2]; ^[3]; ^[4]; ^[1]

Georgia Institute of Technology, Atlanta, GA (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Georgia Institute of Technology, Atlanta, GA (United States); Trinity College Dublin (Ireland); St. James’s Hospital, Dublin (Ireland)
Georgia Institute of Technology, Atlanta, GA (United States); Trinity College Dublin (Ireland); National Institute for Bioprocessing Research and Training, Dublin (Ireland)

Proteins can perform ideal therapeutic functions. However, their large size and significant surface hydrophilicity and charge prohibit them from reaching intracellular targets. These chemical features also render them poorly encapsulated by nanoparticles used for intracellular delivery. In this work, a novel combination of protein vesicles and hydrophobic ion pairing (HIP) was used to load protein cargo and achieve cytosolic delivery to overcome the limitations of previous protein vesicle properties. Protein vesicles are thermally self-assembling nanoparticles made from elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly, physiological stability, and small size, the ELP sequence was modified to include histidine and tyrosine residues. HIP was used to load and release protein cargo requiring endosomal escape for cytosolic function. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and a significant reduction in viability in both a traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. By examining the uptake of positively and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cytosolic cargo delivery and how HIP loading influences protein vesicle self-assembly and disassembly using microscopy, small-angle X-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins as therapeutics for various diseases and extend protein vesicles to deliver other biomacromolecules, as the strategy developed here resulted in the first cytosolic protein cargo delivery using protein vesicles.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 2538134

Journal Information:: ACS Omega, Journal Name: ACS Omega Journal Issue: 3 Vol. 10; ISSN 2470-1343

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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