pH-Degradable Polyglycerol-Based Nanogels for Intracellular Protein Delivery

The growing interest in therapeutic proteins for biomedical applications is still limited by their tendency to degrade and undergo opsonization when exposed to physiological conditions. To protect them from external stimuli and mask their immunogenic sites, poly(ethylene glycol) can be covalently attached to the biomolecules. While this process requires a chemical modification of the original structure and can cause immunogenic reactions, therapeutic proteins can be masked by noncovalent encapsulation into nanocarriers such as nanogels (NGs). Here, we present the synthesis of four new pH-degradable NGs and evaluate their potential to serve as a cytochrome C (CC) delivery platform. Our surfactant-free encapsulation protocol for CC relies on forming NGs through inverse nanoprecipitation using inverse electron-demand Diels–Alder cyclizations (iEDDA) between methyl tetrazines and norbornenes. We compare an aliphatic acetal with different benzacetals and evaluate the substituents’ influence on the NGs’ cleavability and the subsequent intracellular release of CC. Introducing an aromatic π-system beside the acetal framework increased the NG stability against buffer ions and shifted the acetal hydrolysis to the desired lyso- and endosomal pH values. Increasing the degree of substitution in the meta-position with methoxy groups, smaller NGs and more acid-stable acetals were obtained. The BA(OMe)-NGs emerged as the most promising candidate, exhibiting great stability at pH 7.4 and acetal hydrolysis at pH ≤ 6.5 without aggregation. Remarkably, this system released up to 60% of the loaded CC after 96 hours of incubation at endosomal pH values, causing apoptosis of McF7 cells with IC50 values of 8.25 μM.