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Magnetic Solid Lipid Nanoparticles as a Multifunctional Platform against Glioblastoma Multiforme

Periodic Reporting for period 3 - SLaMM (Magnetic Solid Lipid Nanoparticles as a Multifunctional Platform against Glioblastoma Multiforme)

Reporting period: 2020-03-01 to 2021-08-31

Central nervous system (CNS) tumors are an important cause of morbidity and mortality worldwide. Among them, glioblastoma multiforme (GBM) is the most aggressive and lethal, characterized by extensive infiltration into the brain parenchyma. Under the standard treatment protocols, GBM patients can expect a median survival of 14.6 months, while less than 5% of patients live longer than 5 years. This poor prognosis is due to several factors, including the highly aggressive and infiltrative nature of GBM, resulting into incomplete resection, and the limited delivery of therapeutics across the blood-brain-barrier (BBB).
The present project aims at addressing these therapeutic challenges by proposing a nanotechnology-based approach for the treatment of GBM, focused on the selective uptake of drug-loaded multifunctional magnetic lipid nanovectors able to cross the BBB and to be selectively internalized by cancer cells. A synergic attack against GBM will be performed, consisting of a pharmaceutical treatment owing to the chemotherapy drug delivered by the nanovectors, and of a physical treatment originating from the hyperthermia, achieved thanks to the stimulation of the magnetic nanoparticles with an appropriate alternating magnetic field.
By demonstrating the effectiveness of the proposed platform to cross the BBB and to support tumor regression, a huge impact on human healthcare is envisioned. Moreover, further outcomes of this project are expected by considering the development of nanotechnology-based, multi-functional solutions that can easily be adapted to many other high-impact diseases at CNS level.
In the first period of the project (M1-M30) attention has been focused on the development and characterization of the lipid nanovectors, on the set-up of the in vitro BBB model, and on in vitro experiments.
Three different in vitro BBB models have been set-up and characterized: a classic static model for preliminary testing of the developed nanomaterials, a dynamic multi-compartmental and multi-cellular fluidic system that allows testing the drivability of magnetic vectors upon remote guidance, and a real-scale biohybrid model. The latter represents the first example in the literature of a biomimetic BBB system, since its design was inspired from the brain microcapillaries at real scale, yet also biohybrid, since the developed structures are scaffolding the endothelial cells, and the combination of the artificial component with the biological one is essential for a correct in vitro modeling of the blood-brain barrier functionalities.
Concerning the nanovectors, different prototypes have been developed, characterized, and in vitro tested.
The first example concerns encapsulation and delivery of nutlin-3a, a potent activator of the p53 tumor suppressor. Nutlin-3a and superparamagnetic nanoparticles were encapsulated in solid lipid nanoparticles, and the obtained hybrid nanovectors were widely characterized by analyzing both their physicochemical properties and their biological effects on U-87 MG glioblastoma cells. In particular, they showed good colloidal stability, the ability to cross an in vitro BBB system upon magnetic targeting, and a superior pro-apoptotic activity with respect to the free drug.
The second approach proposes the fabrication of biomimetic lipid-based magnetic nanovectors with a good loading capacity and a sustained release profile of the chemotherapeutic drug temozolomide. The fabricated nanovectors demonstrated an enhanced release after exposure to an alternating magnetic field, and a complete release of the encapsulated drug after the synergic effect of low pH, increased concentration of hydrogen peroxide, and increased temperature. The nanovectors have been moreover fully tested on a multi-cellular complex model resembling the BBB and the tumor microenvironment. Investigations of cancer therapy based on hyperthermia and on chemotherapy have been extensively carried out, highlighting anti-proliferative and pro-apoptotic effects on GBM 3D models. A systematic study of transcytosis and endocytosis mechanisms has been moreover performed with multiple complimentary investigations, besides a detailed description of local temperature increments following hyperthermia application.
In order to develop real multi-functional nanovectors, we explored the possibility to include magnetic and antioxidant features into a single nanoplatform. Nanocubes composed of magnetite and manganese dioxide coated with U-251 MG glioblastoma cell-derived membranes (CM-NCubes) have been synthesized and fully characterized. CM-NCubes demonstrated a concentration-dependent oxygen generation; moreover, for the first time in the literature, an intracellular increase of temperature due to the exothermic scavenging reaction of hydrogen peroxide was showed. Internalization studies demonstrated that CM-NCubes are internalized at a higher extent by homotypic U-251 MG cells with respect to other cerebral cell lines. The ability of the CM-NCubes to cross an in vitro BBB model and to be magnetically-accumulated in GBM cells under flow condition was demonstrated. Finally, drug-loaded (in this case sorafenib) CM-NCubes resulted able to induce apoptosis in U-251 MG spheroids upon exposure to an alternating magnetic field, with a higher activity of apoptosis-related enzymes caspases.
Major obstacles to the successful treatment of GBM are also related to the acquired resistance to chemotherapy drugs. As an innovative anticancer approach, low-intensity electric stimulation represents a physical treatment able to reduce multidrug resistance and to induce remarkable anti-proliferative effects by interfering with Ca2+ and K+ homeostasis and by affecting the organization of the mitotic spindles. In the framework of this project, we proposed the exploitation of ultrasound-sensitive piezoelectric barium titanate nanoparticles to remotely deliver electric stimulations to GBM cells. Nanoparticles have been functionalized with an antibody against the transferrin receptor in order to obtain a dual targeting of BBB and of GBM cells. The remote ultrasound-mediated piezostimulation allowed to significantly reduce the in vitro proliferation of glioblastoma cells and, when combined with a sub-toxic concentration of temozolomide, to induce an increased sensitivity to the chemotherapy treatment and remarkable anti-proliferative and pro-apoptotic effects.
During the project first period (M1-M30) the following points provided an actual breakthrough in the literature:
- preparation and characterization of dynamic multi-cellular BBB models, including the tumor microenvironment;
- preparation, characterization, and in vitro validation of multi-functional hybrid magnetic lipid nanovectors, able to cross in vitro models of the BBB and to induce anti-proliferative and pro-apoptotic effects in GBM cells owing to a combination of chemotherapy and hyperthermia;
- first example of “piezoelectric” stimulation in GBM cells, aiming at reducing multidrug resistance and at enhancing anti-proliferative effects of traditional chemotherapy drug.
Following results are envisioned to be achieved during the project second period (M31-M60):
- transcriptomic and proteomic investigations about the mechanisms of action of the optimized nanovectors;
- nanovector in vivo biocompatibility and biodistribution;
- in vivo validation of the proposed multi-functional nanoplatform.
Lipid-based magnetic nanovectors enhance apoptosis and reduce proliferation of brain cancer cells.