Patient-personalized magnetic nanovectors for the treatment of glioblastoma multiforme

Glioblastoma multiforme (GBM), the most aggressive tumor in the central nervous system, poses significant challenges for its treatment due to its invasiveness, genetic heterogeneity, and limited drug delivery across the blood-brain barrier (BBB). Nanotechnology has emerged as an innovative therapeutic approach to fight this devastating disease by enhancing drug bioavailability, facilitating drug delivery across the BBB, and targeting tumor cells, allowing for precise delivery while minimizing damage to healthy tissue. A previous research introduced cell-derived magnetic nanovectors (CDMNVs), which consist of a lipid core encapsulating iron oxide nanoparticles and the drug regorafenib, coated with cell membranes extracted from patient-derived GBM cell cultures to provide an effective targeting of GBM cells in vitro exploiting homotypic affinity, a phenomenon where cancer cells recognize each other via specific membrane proteins. Upon the application of an alternated magnetic field (AMF), the nanovectors were demonstrated to mediate magnetic hyperthermia. In this thesis, CDMNVs were characterized to understand if the coating process could damage the secondary structure of membrane proteins crucial in the homotypic targeting affinity. Fluorescence, Fourier transform Infrared spectroscopy, and Raman spectroscopy were used to determine changes in protein secondary structures before and after the coating. Then, nanovector stability was assessed in different conditions (water, culture medium, and cytoplasm buffer), and the protein corona formation upon contact with serum proteins was examined. Patient-derived GBM cell culture was characterized via immunocytochemistry. The internalization of CDMNVs into patient-derived GBM cells was quantitatively evaluated through an iron colorimetric assay, while their intracellular distribution was qualitatively described using iron staining techniques. Moreover, the effective homotypic targeting of the nanovectors towards various GBM cell sources was investigated in static and dynamic conditions utilizing, for that purpose, a home-made bioreactor previously developed by the research group. Eventually, the impact of CDMNVs on cellular metabolism was monitored using fluorescence lifetime imaging (FLIM), with and without the application of AMF to patient-derived GBM cells. The study revealed that the nanovectors are stable, especially in the presence of serum proteins. When exposed to the intracellular content, instead, enzymes fostered nanovectors degradation after three days of incubation. The functional structure of the cell membrane coating is preserved during the functionalization process and the presence of these proteins seems to prevent the formation of the protein corona on the nanovectors with respect to the bare nanoparticles. Regarding cellular uptake, most of the nanoparticles are internalized within the first 6 h, tending then to accumulate in the perinuclear area. The FLIM results suggest that the nanovectors may play a role in mitochondrial impairment that could be attributed to increased levels of intracellular oxidative stress, probably associated to Fenton reactions mediated by iron oxide nanoparticles and magnetic hyperthermia. The affinity between the nanovectors and their source cells, the preservation of the protein conformation in the coating, and the effect on the cells metabolism hold promise for improving this system as a tool for GBM personalized treatment and diagnosis.