Smart and sensitive nanocarriers for the delivery of therapeutic proteins are needed as alternatives for covalent modification with the potentially immunogenic PEG. Nanogels as water swollen, highly hydrophilic polymer networks are promising candidates for protein delivery vehicles. However, scalable production, under sensitive and mild conditions, is still an active area of research. Inverse nanoprecipitation, as one of several production methods, offers the potential for the mild and non-destructive encapsulation of sensitive proteins. The gel networks are preferably formed by crosslinking of biocompatible, hydrophilic, and easily obtainable functionalized polymers. A variety of crosslinking chemistries, such as CuAAC, Thiol-Michael addition, and SPAAC have been studied for this purpose. Most of these chemistries, however, suffer from low biorthogonality, toxic catalysts, or the low synthetic accessibility of the precursors. IEDDA has emerged as an alternative for the other click chemistries, with fast reaction kinetics, high biorthogonality and easily accessible precursors. The goal of this study was to design nanogels in a way that most of the mentioned criteria for a successful nanocarrier system are fulfilled. Nanogels, based on the biocompatible, scalable, hydrophilic and easily functionalizable dPG were presented in this work. Inverse nanoprecipitation was used as a mild gelation method, that lacks toxic surfactants or damaging ultrasound. The bioorthogonal and fast iEDDA click chemistry, based on tetrazines and dienophiles, was established for the first time in the use of nanogel production. The first study focused on the search for suitable dienophiles for the iEDDA crosslinking chemistry. Reactivity and scalability were most important. This was achieved by screening of different iEDDA-reactive dienophile macromonomers. For this, the four different dienophile macromonomers dPG-norbonene, dPG-BCN, dPG-cyclopropene, and dPG-DHP were synthesized. As the tetrazine counterpart, the stable but still reactive dPG-metTet was obtained. The macromonomers were compared regarding their ability to form macro-and nanogels. Gelation times were determined and revealed that only dPG-norbonene and dPG-cyclopropene were able to form macrogels, while dPG-BCN showed incomplete, and dPG-DHP no gel formation at all. For nanogel formation, reaction parameters, such as rotation speed, macromonomer concentration, quenching times, and solvent to non-solvent ratios were screened. Solvent to non-solvent ratio and quenching time were the most influential parameters on nanogel size and polydispersity. The nanogels were obtained in the relevant size range of 40 to 200 nm and were stable for at least several months in aqueous solution. Co-precipitation of the small model protein myoglobin was performed with the most promising macromonomer candidates dPG-norbonene and -cyclopropene. Encapsulation efficiencies of above 70% were achieved. Thus, it could be shown that a combination of dPG as the polymer scaffold, together with easily obtainable iEDDA reactive groups, such as norbonene and methyl tetrazine provide the toolbox for the design of a scalable and functional nanocarrier for proteins. The second study aimed at transferring the gained knowledge on nanogel formation parameters, such as quenching time and solvent to non-solvent ratio on a smart, environmentally responsive version of the nanogel system. Environmentally responsiveness was achieved by the introduction of pH-cleavable acetal groups. One which is cleavable at pH values below 5 (benzacetal) and one which cleaves at values below 3 (THP). For this dPG was functionalized with the respective acetal linkers and then further functionalized with the dienophiles norbonene and BCN from the first study. Norbonene was the most promising candidate and BCN was used as a well-established comparison. The macromonomers showed no toxicity up to concentrations of 2.5 mg/mL in three different cell lines. Nanogels in the size range of 47-200 nm were obtained, which were stable in aqueous solution at pH 7.4 for several months, without decomposition or an increase of polydispersity. Upon exposure to acidic conditions, the benzacetal-based nanogels cleaved to small particles at pH 4.5 within 48 h, while the THP acetal-based nanogels cleaved only at pH 3 to small particles after 48 h. This proved the applicability of the nanogels for lysosomal cleavage and intracellular delivery for benzacetal gels and a potential delivery to the small intestine by the THP acetal functionalized gels. Co-precipitation of the therapeutic protein asparaginase led to encapsulation efficiencies of up to 93%. The degradability of the gels, the high encapsulation efficiencies, as well as the synthetic accessibility and biocompatibility of the macromonomer precursors, point out the potential of this nanocarrier platform for biomedical applications. Based on the data that was obtained, the potential of the iEDDA based nanogels is evident. However, scalability must be improved at least for the nanogel production itself. Continuous flow methods, such as microfluidic based nanoprecipitation could potentially be used for the upscaling of the nanogels presented in this work. Furthermore, the addition of active targeting ligands to the nanogels or the macromonomers before inverse nanoprecipitation would even further increase the applicability of these nanogels for biomedical applications. One way of an easily obtainable active targeting moiety would be the sulfation of the dPG-macromonomers, which would introduce L-selectin binding affinity into the nanogels, thus targeting inflamed tissues.
