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.
