Evaluation of Histidine–Octamer-Modified Hyaluronic Acid as a Cytosolic Drug Delivery Material via CD44-Mediated Cellular Uptake and Endosomal Escape

Background: Although intracellular delivery via endocytosis is a promising strategy for drugs acting in the cytosol or nucleus, many macromolecular therapeutics remain trapped within endosomal/lysosomal compartments, limiting efficient cytosolic delivery. In this study, we designed a novel functional hyaluronic acid (HA)-based polymer, HA–octahistidine (His8), by conjugating His8 to CD44-targeting HA, and evaluated its physicochemical properties, cellular uptake, and endosomal escape capability. Methods: HA–His8 was synthesized via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS)-mediated coupling and characterized using 1H NMR spectroscopy. Particle size and zeta potential were measured via dynamic light scattering, and buffering capacity was evaluated using acid–base titration. Cellular uptake and intracellular localization were investigated in CD44-high MDA-MB-231 and CD44-low MCF-7 cells via confocal laser scanning microscopy. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Successful conjugation of His8 was confirmed with a degree of substitution of 4.66 mol% relative to HA carboxyl groups. HA–His8 exhibited a nanoscale hydrodynamic diameter under physiological conditions, which increased under acidic conditions together with changes in zeta potential. HA–His8 exhibited higher buffering capacity than free His8 and was preferentially internalized by CD44-high MDA-MB-231 cells compared with MCF-7 cells. Compared with unmodified HA, HA–His8 exhibited lower colocalization with LysoTracker that decreased over time, indicating reduced retention within acidic vesicles. HA–His8 also exhibited low cytotoxicity over the tested concentration range. Conclusions: His8 modification alters HA intracellular localization while preserving CD44 targeting, thereby facilitating endosomal escape. These findings highlight HA–His8 as a potential platform for the cytosolic delivery of macromolecular therapeutics, including proteins, peptides, and nucleic acids.