This work explored different modalities for the optimization of the properties of thermoresponsive nanogels, with regard to their application as drug carriers in anticancer therapy. The ability of nanogels to reversibly bind different chemotherapeutic drugs was investigated, following different strategies. The main focus was the implementation of synthetic procedures that allowed the incorporation of dendritic structures into nanogels, together with the design of multicompartimental nanogels as drug carriers with improved versatility. In order to successfully translate these synthetic efforts into nanogels showing good in vivo compatibility, thermoresponsive nanogels were investigated for their ability to interact with blood proteins in physiological conditions. The assessment of the protein corona around thermoresponsive nanogels, after their incubation in human serum and plasma, was described in Section 3.1 - 3.2 of the present thesis. These studies focused on the understanding of polymer – protein associations in physiological conditions with regard to the role of the single constituents of thermoresponsive nanogels in the interaction with proteins, namely thermoresponsive polymers and dPG. In order to direct the drug carrier to the desired target in vivo and prevent its sequestration from the bloodstream, unwanted interactions with opsonins at a protein corona level should be avoided. The analysis of such interactions help understanding the biocompatibility of thermoresponsive nanogels that are designed for intravenous administration, ideally to bridge a gap between in vitro and in vivo assays. While both nanogel constituents, dPG and pNIPAM, are regarded as polymers with very low protein affinities, their incorporation into nanogels leads to a relevant size increase, which is a common factor associated with increased protein binding. An in-depth analysis of the protein corona of dPG-based thermoresponsive nanogels is thus revealed in this thesis in Section 3.1 - 3.2. Following the need to optimize nanogels to adjust the biological responses they will evoke, the ability to resist unwanted surface interactions with proteins was proven to be enhanced in the presence of dPG, as expected from its neutral, hydrophilic character. dPG provided colloidal stability to thermoresponsive nanogels, without drastically altering the key thermoresponsive properties, as well as inhibiting the unspecific absorption of proteins onto the nanogels’ surface. The inhibition of the protein binding was so successful that only traces of protein were found for these nanogel systems (3 – 7 μg / m2 nanogel). Nevertheless, a protein-dependent aggregation for dPG-pNIPAM nanogels was found above their VPTT, as revealed by DLS measurements in full human serum at 37 °C. The traces of immunoglobulin light chains, found specifically enriched at 37 °C, may signal an immunoglobulin bridging between nanogels as driving force for their aggregation, analogous to what was found for polystyrene nanoparticle systems in the literature. This temperature-dependent protein corona study proved the need to optimize the polymer choice for their use in biology, where unspecific protein binding is ruling the final polymer / nanoparticle distribution and blood availability. In fact, by changing the polymer from pNIPAM to pNIPMAM, the nanogels were found to be safe to use in physiological conditions at 37 °C, as they do not aggregate in serum and reach their hydrophobic transition at a higher VPTT of 46 °C. The generation of innovative nanogel systems for different applications as responsive drug carriers for anticancer therapy is disclosed in Section 3.3 - 3.5 of this thesis. The core of this section is the optimization of the synthesis of pH- and / or thermoresponsive nanogels, by tuning the dendron / pNIPAM comonomer ratio in copolymer nanogels (Section 3.4) or by screening polymer concentrations to obtain an optimal binding strength for the loading and sustained release of therapeutic molecules by SIPN nanogels (Sections 3.3, 3.5). Nanogels with tunable reactivities are designed using dendritic polymers. Dendritic structures allow for a high density of functional groups, that improve nanogel solubility and may mediate multivalent effects. The use of dPG as macromolecular crosslinker helped in previous reports to improve the biocompatibility of thermoresponsive pNIPAM. Linear polymers with tunable responsive functionalities / concentrations were used in combination with dendritic moieties, in a synergistic effort for the optimization of the nanogels’ biological behavior. The use of the nanogels as drug delivery systems was demonstrated for small chemotherapeutic drugs (cisplatin and DOX), as well as for cytochrome c as a therapeutic protein. The changes in nanogel morphology upon chemical modification were thoroughly investigated, in relation with the nanogel’s ability to weakly interact with therapeutic molecules (small drugs or proteins). In this way, an optimal tuning of the binding and release kinetics of the loaded molecule for efficient drug delivery could be achieved. SIPN nanogels were developed as powerful agents for the rational tuning of the interactions between nanogels and the desired therapeutic cargo. Most importantly, the introduction of a secondary network to give SIPN does not directly interfere with the key responsive properties of nanogels, but rather adds new physicochemical responsive modalities. dPG-pNIPAM nanogels were employed as starting materials for the generation of SIPN within the nanogel scaffold. In this way, the advantageous properties of dPG-pNIPAM nanogels were further tuned, with pABC as dendritic secondary polymeric network to achieve SIPN nanogels, which allow the assembly of a therapeutic cytochrome c corona. The loaded nanogels showed therapeutic potency against HeLa cells which could be specifically triggered by a temperature shift. Here, the swelling of thermoresponsive nanogels was used as the mechanical force helping the disruption of the supramolecular protein corona and its intracellular delivery into HeLa cells (Section 3.3). The use of dendritic ABC was further investigated as comonomer for the synthesis of pABC-co-pNIPAM nanogels, which introduced pH-responsiveness in addition to the thermoresponsive behavior of nanogels. This allowed a sustained release of cisplatin only upon reaching acidic compartments (like lysosomes) of cells (Section 3.4). The copolymerization with ABC drastically altered the thermoresponsiveness of the copolymer compared to pNIPAM, to give nanogels with VPTT behavior only in acidic conditions (pH < 5), with VPTT values decreasing in an ABC concentration-dependent manner. The decrease in the VPTT was related to increased intermolecular pABC hydrogen bonding, when pABC was in its protonated state. The loading of the nanogels with cisplatin at pH 7.4 resulted in stable ABC-Pt bonds, which may be disrupted in acidic conditions, as a consequence of H-bond driven nanogel aggregation and subsequent release of the previously bound cisplatin. In an analogous approach to that of Section 3.3, SIPN nanogels showed increased affinity towards the chemotherapeutic drug DOX, with the aid of p(AMPS) as secondary polymeric network within the nanogels, and achieved in vivo anticancer activity in DOX-resistant conditions (Section 3.5). The use of AMPS SIPN nanogels could tune the electrostatic binding of DOX to the carrier and thereby enable the drug to successfully bypass the resistance mechanisms of multidrug resistant tumor cells in vitro and in vivo. All areas of the research discussed in this thesis help to unveil relevant discoveries for the optimization the properties of soft, stimuli-responsive nanogels, for their application as innovative drug carriers. The discoveries published in this work help to push forward the implementation of next generation nanogels and to disclose further information about the modalities of actions of stimuli-responsive nanogels and their translation into clinical therapies. There is no limit for the design of new nanoparticles exhibiting moderate polymer - drug interactions for sustained drug release, as long as the polymer - protein interactions are identified and show compatibility with the envisioned therapeutic purpose.
