Integration of protein containers into microgel systems for potential drug release and delivery

The application of ferritin containers as a promising drug delivery vehicle is hampered by proteolytic degradation in systemic circulation. Here, a novel strategy to improve the stability of ferritin containers against protease is presented: Ferritin containers were integrated into the polymeric network of polyelectrolyte microgels. First, ferritin was labeled with fluorophores and nanoparticles (NPs) to enable tracking their integration and release from microgels by fluorescence microscopy and transmission electron microscopy (TEM), respectively. To obtain a high fluorophore loading and yield, a new encapsulation strategy based on the cysteine-maleimide coupling reactions was developed. The integration of ferritin into microgel systems was performed by the electrostatic interactions between the charged co-monomers of microgels and the surface charges of ferritin. Initially, ferritin was integrated into microgels with randomly distributed ionizable groups. However, the integration was characterized by precipitation, and the resulting microgels could not be purified from the excess ferritin. Therefore, next, ferritin was integrated into a charged core-neutral shell microgel. Although the neutral shell prevented precipitation, ferritin could leak out from the microgels and ferritin was inhomogeneously distributed among the microgels. All these problems were solved when ferritin was integrated during the microgel synthesis. The integration was very effective because about 80% of the applied ferritin containers were integrated into the microgels. To enable ferritin release from microgels, an acid-degradable crosslinker was applied in the synthesis. It is shown that about 85% and 50% of the integrated ferritin can be released rapidly in buffer with pH 2.5 and 4.0, respectively. However, total degradation of the microgels was not observed due to the self-crosslinking of N-isopropylacrylamide (NIPAM). Finally, proteolytic degradation by chymotrypsin proved that the microgels could protect ferritin against protease.For ...