Small, Traceable, Endosome-Disrupting, and Bioresponsive Click Nanogels Fabricated via Microfluidics for CD44-Targeted Cytoplasmic Delivery of Therapeutic Proteins.

Nanogels (NG) are among the most ideal cytoplasmic protein delivery vehicles; however, their performance is suboptimal, partly owing to relatively big size, poor cell uptake, and endosomal entrapment. Here, we developed small, traceable, endosome-disrupting, and bioresponsive hyaluronic acid NG (HA-NG) for CD44-targeted intracellular delivery of therapeutic proteins. With microfluidics and catalyst-free photo-click cross-linking, HA-NG with hydrodynamic diameters of ca. 80 and 150 nm, strong green fluorescence and efficient loading of various proteins including saporin (Sap), cytochrome C, herceptin, immunoglobulin G (IgG), and bovine serum albumin could be fabricated. Interestingly, 80 nm-sized HA-NG revealed clearly better cellular uptake than its 150 nm counterparts in both CD44-negative U87 cancer cells and CD44-positive 4T1 and MDA-MB-231 cells. Moreover, small NG exhibited accelerated endosomal escape, which was further boosted by introducing GALA, a pH-sensitive fusogenic peptide. Accordingly, Sap-loaded small and GALA-functionalized HA-NG showed the highest cytotoxicity in CD44-positive MDA-MB-231, 4T1, A549, and SMMC-7721 cancer cells. The biodistribution studies demonstrated that 80 nm-sized HA-NG displayed significantly greater tumor uptake as well as penetration in MDA-MB-231 human breast tumor xenografts than its 150 nm counterparts, whereas the introduction of GALA had no detrimental effect on tumor accumulation. Small, endosome-disrupting, and bioresponsive HA-NG with easy and controlled fabrication hold a great potential for targeted protein therapy.