Hydrogel‐Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane‐Impermeable Cargo

Intracellular delivery of membrane‐impermeable cargo offers unique opportunities for biological research and the development of cell‐based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane‐impermeable cargo. Using this platform, which is termed Hydrogel‐enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)–dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC‐dextran delivery in primary human T cells, outperforming state‐of‐the‐art electroporation‐mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone‐binding nanobody as well as the enzymes granzyme A and Cre‐recombinase. Finally, HyPore‐mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1‐weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost‐effective technique for high‐throughput, ex vivo manipulation of primary cells and cell lines. Crosslinked cationic dextran nanogels are identified as unique polycationic nanomaterials capable of mediating direct cytosolic delivery of membrane‐impermeable cargo. The efficient delivery of various macromolecules in HeLa cells and primary cells is demonstrated. Moreover, this approach outperforms state‐of‐the‐art electroporation for the delivery of neutral macromolecules and the MRI contrast agent gadobutrol in primary human T cells.