Sviluppo in vitro di una nanopiattaforma innovativa per la veicolazione di un inibitore della tirosina chinasi e di un siRNA per il trattamento del glioblastoma

Glioblastoma multiforme (GBM) is a severe and aggressive cancer that affects the brain and central nervous system. Multimodal therapy, which includes surgery, chemotherapy, and radiotherapy, is traditionally used to extend patient survival. However, the prognosis remains very poor. Several factors contribute to the low survival rate, including the aggressiveness of the treatments on the already frail patient, the localisation of the tumour which often prevents complete removal (leading to frequent recurrence), and the high infiltration capability of cancer cells. In addition, tumour resistance is a major limitation due to the heterogeneity of the tumour microenvironment and also by the presence of the blood-brain barrier (BBB), a selective barrier that limits the penetration of drug treatments. Therefore, it is extremely important to develop an efficacious delivery system to allow the drugs penetrate across the BBB and to easily reach the target they were designed for. For this purpose, nanomedicine is deeply investigated because different nanoparticles can efficiently load the drug of interest and release it in the target region in a time-controlled manner. Nanosponges made from beta-cyclodextrin polymer can be useful because of their above average loading capacity and modifiability. In this thesis, we have investigated the efficacy of these Nanosponges as a delivery system to load Ibrutinib (Ib), an irreversible inhibitor of a particular family of kinases able to overstimulate the autophagy in glioblastoma cells. Furthermore, a panel of several beta-cyclodextrin-based polyplexes (BCDI) were tested in order to carry a siRNA against the transcript of the Serum Glucocorticoid – regulated Kinase-1 (SGK-1) protein, a kinase with an important role in tumour growth. All the experiments have been carried out on murine glioblastoma (GL-261) cells. The cytotoxicity of free Ib, Nanosponges alone or Nanosponges loaded with IB were tested on Gl-261 cells grown in two-dimensional (2D) culture. Furthermore, on the same cell model, the cytotoxicity of several BCDI polyplexes that carry the siRNA delivered against SGK-1 were investigated. Results show a significant cytotoxicity of Ib alone (p < 0.01) at the highest concentration used (100 M) at 24 and 72 h after the incubation, while a significant reduction of cell growth was already detected when Ib (10 M) was loaded in Nanosponges. No significant cytotoxicity was detected in Nanosponges conditions alone. In parallel, an investigation of cytotoxicity of different BCDI polyplexes was performed and BCDI-Q was then chosen as an ideal candidate thanks to its physical and chemical characteristics. In addition, because BCDI polyplexes carry siRNA against SGK-1, a preliminary gene expression analysis of SGK-1 mRNA was performed on GL-261 cells in 2D culture. However, no statistically significant gene expression of SGK-1 was observed in 2D cultures of GL-261. Therefore, the same mRNA gene expression was investigated on GL-261 three-dimensional (3D) culture, since it has been postulated in literature that the stress condition, observed in the core of the spheroids, can affect the SGK-1 gene expression. Indeed, a statistically significant mRNA SGK-1 gene expression was detected on GL-261 spheroids at 7 days post seeding. In conclusion, from our data, the -cyclodextrin-based Nanocould be considered as a potential candidate to deliver Ib and BCDI for a targeted therapy in future experiments.