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.

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Future healthcare requires development of novel theranostic agents that are capable of not only enhancing diagnosis and monitoring therapeutic responses but also augmenting therapeutic outcomes. Here, a versatile and stable nanoagent is reported based on poly(ethylene glycol)‐b‐poly(l‐thyroxine) (PEG‐PThy) block copolypeptide for enhanced single photon emission computed tomography/computed tomography (SPECT/CT) dual‐modality imaging and targeted tumor radiotherapy in vivo. PEG‐PThy acquired by polymerization of l‐thyroxine‐N‐carboxyanhydride (Thy‐NCA) displays a controlled Mn, high iodine content of ≈49.2 wt%, and can spontaneously form 65 nm‐sized nanoparticles (PThyN). In contrast to clinically used contrast agents like iohexol and iodixanol, PThyN reveals iso‐osmolality, low viscosity, and long circulation time. While PThyN exhibits comparable in vitro CT attenuation efficacy to iohexol, it greatly enhances in vivo CT imaging of vascular systems and soft tissues. PThyN allows for surface decoration with the cRGD peptide achieving enhanced CT imaging of subcutaneous B16F10 melanoma and orthotopic A549 lung tumor. Taking advantages of a facile iodine exchange reaction, 125I‐labeled PThyN enables SPECT/CT imaging of tumors and monitoring of PThyN biodistribution in vivo. Besides, 131I‐labeled and cRGD‐functionalized PThyN displays remarkable growth inhibition of the B16F10 tumor in mice (tumor inhibition rate > 89%). These poly(l‐thyroxine) nanoparticles provide a unique and versatile theranostic platform for varying diseases.