Seeler, M. | Search bioRxiv

bioRxiv2025

Hydrophobic Ion Pairing for Simple, Non-Toxic Transfection

Although biomacromolecules require intracellular delivery for therapeutic effect, existing transfection agents are often characterized by high cost, low efficiency, and/or cytotoxicity. Here, we describe a new transfection approach based on hydrophobic ion pairing (HIP), which involves the simple mixing of a hydrophobic counterion with charged biomacromolecules. Among tested cargoes (proteins, siRNA, and pDNA), the HIP siRNA system performed especially well, achieving silencing in fibroblasts (80%), T cells (90%), and neurons (70%). HIP siRNA was also highly potent in mice, with tropism dependent on the route of administration. Most notably, intraperitoneal administration enabled [~]40% LAMP-1 knockdown in the pancreas, and intravenous delivery resulted in a remarkable 80% silencing in the heart. Heart delivery was also highly selectively, with no significant knockdown in the liver. Together, these data demonstrate a new, inexpensive approach to biomacromolecular delivery with the potential to target difficult-to-transfect organs, thus expanding the therapeutic potential of nucleic acids.

bioengineering↗

bioRxiv2024

Intracellular biomacromolecule delivery by stimuli responsive protein vesicles loaded by hydrophobic ion pairing

Therapeutic biomacromolecules are highly specific, which results in controlled therapeutic effect and less toxicity than small molecules. However, proteins and nucleic acids are large and have significant surface hydrophilicity and charge, thus cannot diffuse into cells. These chemical features render them poorly encapsulated by nanoparticles. Protein vesicles are self-assembling nanoparticles made by warming 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 and small size, ELP was modified to include histidine and tyrosine residues. Additionally, hydrophobic ion pairing (HIP) was used to load and release protein and siRNA cargos requiring endosomal escape. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and significant reduction in viability in traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. They also delivered siRNA to knockdown protein expression in a murine fibroblast cell line. By examining uptake of positive and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cargo release and how HIP influences protein vesicle self-assembly using microscopy, small angle x-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins for various diseases and extend protein vesicles to deliver other biomacromolecules.

bioengineering↗