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

• Integration von Proteincontainern in Mikrogelsysteme zur potenziellen Wirkstofffreisetzung und -abgabe

Budiarta, Made; Beck, Tobias (Thesis advisor); Pich, Andrij (Thesis advisor); Simon, Ulrich (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

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 drug delivery purposes, the anti-cancer drug doxorubicin (DOX) was encapsulated into various ferritin variants. The resulting DOX-ferritin variants then were integrated into the microgel system. However, the cell experiments show that the cytotoxicity of DOX-ferritin variants is lower than the free-DOX. One possible reason is the attachment of DOX molecules on the surface of ferritin cages, which obstructs the ferritin uptake into the cells. Further investigations are needed to confirm and overcome this problem. In this work, a protein containers-microgel system is developed for the first time. Interestingly, the same synthesis could be applied to integrate ferritin containers containing different cargos into the microgels. This approach describes an innovative, previously not considered strategy for integrating various materials into the microgels. Thereby, this work expands the areas for the applications of colloidal microgel systems.