Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes

Abstract

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3 μg luciferase-encoding mRNA in mice.

mCherry on Top: A Positive Read-Out Cellular Platform for Screening DMD Exon Skipping Xenopeptide–PMO Conjugates

“…BafA1 is an inhibitor of vacuolar-type H + -ATPase (V-ATPase) and reduces endosomal acidification. Previous works could show that transfection with delivery systems based on ionizable structural motifs can be inhibited by BafA1. − By using the mCherry-DMDEx23 reporter cells, we evaluated the dependency of successful PMO delivery on the endosomal pH on a functional level.…”

Dynamic carriers for therapeutic RNA delivery

“…5 ). These carriers that combine the benefits of both polymer- and lipid-based delivery modules were found as up to several hundred-fold more potent than previous carrier generations ( 125 ). They were designed to contain at least two novel lipo amino fatty acids (LAFs) as cationizable motifs with tunable polarity in combination with polar cationizable Stp aminoethylene units.…”

Loading of extracellular vesicles with nucleic acids via hybridization with sponge-like lipid nanoparticles

“…In addition, we investigated the endosomal remodeling in HeLa cells stably expressing the Galectin-8 mRuby3 fusion protein (Gal8-mRuby3). NP-mediated disruption of the endosomal membrane causes the recruitment and binding of Gal8 to β-galactoside sugars located on the inner leaflet of endosomes (60,61). The binding of Gal8-mRuby3 can then be followed by a transition from a diffuse cytosolic signal to bright punctate patterns.…”