Physical and chemical functionalization of macro- and nano-gel systems, to improve controlled drug delivery

In this Ph.D. thesis we investigate hydrogels and nanogels (NGs) for controlled drug delivery. In particular, we first focus the attention on macro hydrogels physically functionalized with graphene, and then we consider nanogels chemically functionalized with different aromatic groups. Indeed, new trends in drug delivery strategies have seen lately a considerable interest in the synthesis of graphene-based materials in order to design biomaterials promoting tunable drug release via electric or stretching stimuli. However, the design of a thermosensitive scaffold using pristine graphene has not been investigated yet, and it can be considered a promising approach for several therapeutic treatments, including hyperthermia. In this work, we present the fabrication of thermosensitive hydrogels where pristine nano-layered graphene (few layered graphene, FLG) is used as a nanofiller to investigate the graphene thermal effect in the drug release scenario. Hydrogels are synthesized in two steps: i) esterification between polyacrylic acid and agarose, under microwave irradiation, to activate the sol gel transition, and ii) incorporation of drug loaded FLG during sol-gel transition. Following a first check over hydrogel non-toxicity, according to ASTM 10399-5 standard, the drug release profile of the gels at different temperatures is investigated. The chosen drug is Diclofenac, an anti-inflammatory molecule used to treat musculoskeletal inflammations. The drug release profile is investigated at three different temperatures: 25, 37 and 44 °C. Results show that is possible to tune diclofenac release over time by modifying temperature. This response is not observed in polymeric scaffolds without FLG, suggesting that pristine graphene, due to its thermal conductivity and pi-conjugated chemical structure, is able to generate electronic interactions with both the polymeric matrix and the drug molecules, modulating the release profile of diclofenac. Moreover, the anti-inflammatory performance of the released drug is evaluated in terms of cyclooxygenase (COX) inhibition, resulting in an efficiency comparable to the administration of free diclofenac in aqueous medium, which proves that the graphene interactions do not affect the therapeutic properties. Furthermore, the high biocompatibility of the synthetized graphene-laden hydrogels confirms their potential use as 3D scaffolds for thermally triggered drug release. As regards NGs the goal is to evaluate how NGs with different aromatic coatings may affect biocompatibility, chemical-physical properties, drug loading, drug release and cell uptake in a specific disease scenario. In particular, NGs are tested on representative renal cell carcinoma T-786O. The focus of this work is on NGs cellular internalization with the purpose of developing an innovative approach based on the microfluidic system to mimic the effective condition on a potential in vivo administration. In detail, NGs synthesis is performed in batch by cross-linking reaction via emulsion-evaporation method and subsequently an aromatic surface functionalization is realized by three different aromatic layers. The obtained NGs are compared in terms of size, polydispersity index, biocompatibility and drug encapsulation efficiency. Regarding the latter, NGs are tested as nanocarriers for drug delivery using Sunitinib malate an FDA-approved drug for the treatment of kidney cancer. The NGs-drug interaction is investigated in terms of drug release varying the aromatic coating. In particular, different drug release profiles are obtained for each aromatic coating confirming the possibility of using the nanomaterial for intracellular controlled drug delivery. On the other hand, the NGs internalization, following a preliminary evaluation through the conventional flow cytometry analysis, is evaluated in vitro using the microfluidic system. The microfluidic-assisted NGs cell uptake is evaluated with two different approaches: static and dynamic conditions. In the first instance, NGs internalization is performed in static conditions to compared it with the conventional approach due to cell seeding occurs in a different environment and then under dynamic to estimate cell uptake in injection-like condition. The collected results showed how the selected aromatic coatings on NGs surface ensure a tunable drug delivery of Sunitinib and modulate the cellular internalization in T-786O cell line. Regarding the latter, the evaluation of NGs cell uptake in flow condition through the microfluidic system has revealed a different trend from the traditional approach, more similar to the effective injection-like conditions, suggesting the potential of this approach. Overall, these results demonstrated that through surface decoration with specific aromatic moieties it’s possible to tune NG-cell interactions and the drug delivery performances, designing versatile nanosystems suitable for the definition of future therapeutic approaches. The work is divided into two different parts: • Part I consists of three chapters and it focuses on the current state of the art on hydrogel for drug delivery (Chapter 1), on hydrogels with graphene (Chapter 2) and on nanocarriers for drug delivery (Chapter 3). • Part II is devoted to the research activities, presenting the experimental methodology conducted for hydrogel with physical functionalization (Chapter 4) and Nanogels with chemical functionalization (Chapter 5). Finally, the last Chapter 6 sums up and collects all the conclusions and proposes future developments to the present work.