New frontiers of engineered nanovectors to improve treatment efficacy and safety in glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary human brain tumor in the adult, associated with very poor prognosis and a median overall survival of 14 months. The presence inside the tumor of Glioblastoma Stem-Like Cells (GSC) and the presence of the Blood-Brain Barrier (BBB) represent major obstacle for the effective treatment of GBM. Advances in nanotechnology have led to the development of new nanovectors with multiple functionalities that opens new possibilities in the definition of novel therapeutic strategies. Major object of the present PhD study was the development of innovative engineered liposomes (LPs) able to cross BBB and specifically target the GBM tumor environment, limiting systemic side effects on peripheral organs and brain healthy parenchyma. The designed strategy involves the double functionalization of drug-loaded LPs with (i) a modified peptide of the Apolipoprotein-E (mApoE) to favor the BBB crossing and GSC uptake, and (ii) an MMP-2 and -9 sensible lipopeptide (M2-9SLP) to increase tumor specificity. Given that aberration in MEK/ERK and PI3K/akt/mTOR pathway predominate and confer GSCs resistance to therapy, the small MEK kinase inhibitors trametinib (TRAM) and pimasertib (PIMA), and the mTOR inhibitor Rapalink-1 (RL1) were considered as key therapeutic molecules to target the GSCs. Anti-tumor activity was explored in patient-derived GSC primary lines established in the lab and characterized for relevant genomic mutations and transcriptomic profile. TRAM, PIMA and RL1 were evaluated as free-drugs and embedded into both single mApoE and double M2-9SLP/mApoE functionalized LPs. A survey of the three drugs in eight GSC lines indicated a significant induction of GSC apoptosis upon 72 hours of treatment in approximately 50% of the tested lines with an overall higher sensitivity to TRAM compared to the other two drugs. After TRAM and PIMA treatment, a reduction of p-ERK and increase of phosphorylation of mTOR related target molecules was observed pointing out a probable ERK/mTOR redundant activity. An impaired ability of the MEK inhibitors to efficiently cross an in vitro BBB model was observed. Whereas RL1 cytotoxicity on endothelial cells suggests a possibly harmful effect on the healthy BBB. These data support the encapsulation of these molecules into delivering LPs. To strengthen therapeutic efficacy and lower off-target effects, double functionalized M2-9SLP/mApoE drug loaded LPs were developed. M2-9SLP efficacy was evaluated on GSCs displaying different MMP2 enzymatic activities by mean of Calcein-loaded M2-9SLP/mApoE-LPs. Data shown that calcein uptake correlates with MMP2 activity levels and increases upon M2-9SLP/mApoE-LPs treatment, thus demonstrating the M2-9SLP strategy to be selective against MMP2 enriched microenvironment. Given the higher GSC sensitivity to TRAM treatment compared to PIMA and RL1, TRAM was encapsulated into M2-9SLP/mApoE-LPs with consequent increase of apoptosis 72 hours after the treatment indicating that the encapsulation process did not alter TRAM efficacy. Overall, these results demonstrate the ability of TRAM, PIMA and RL1 to inhibit GSCs proliferation and survival, also once encapsulated into double functionalized LP. The presented drug delivery strategy has been proved to be particularly specific for GBM therapy, were an enrichment of MMP2 is observed and the presence of the BBB limit drug delivery efficacy. Indeed, the encapsulation of MEK and mTOR inhibitors into M2-9SLP/mApoE-LP could represent a promising therapeutic strategy to circumvent brain limited access of these drugs and to limit toxicity on the healthy parenchyma.