Periodic Reporting for period 4 - SLaMM (Magnetic Solid Lipid Nanoparticles as a Multifunctional Platform against Glioblastoma Multiforme)
Período documentado: 2021-09-01 hasta 2022-12-31
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 ERC SLaMM project aimed at addressing these therapeutic challenges by proposing a nanotechnology-based approach focused on the selective uptake of drug-loaded multifunctional magnetic lipid nanovectors. A synergic attack against GBM has been proposed, 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.
At the end of the project, the demonstrated effectiveness of the proposed platform to support tumor regression both in vitro and in vivo envisions a huge impact on future human healthcare.
The ERC SLaMM project aimed at addressing these therapeutic challenges by proposing a nanotechnology-based approach focused on the selective uptake of drug-loaded multifunctional magnetic lipid nanovectors. A synergic attack against GBM has been proposed, 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.
At the end of the project, the demonstrated effectiveness of the proposed platform to support tumor regression both in vitro and in vivo envisions a huge impact on future human healthcare.
The performed work can be summarized in the development of innovative platforms for in vitro testing, the preparation and characterization of lipid-based magnetic nanovectors, and the assessment of their functionality both in vitro and in vivo.
Different in vitro BBB models have been set-up and characterized: among them, a real-scale biohybrid model representing the first example in the literature of a biomimetic BBB system, with a design inspired from the brain microcapillaries at real scale.
Concerning the nanovectors, different prototypes have been developed, characterized, and in vitro tested.
In the first example, the chemotherapy drug nutlin-3a and superparamagnetic nanoparticles were encapsulated in solid lipid nanoparticles, and the obtained hybrid nanovectors characterized by analyzing both their physicochemical properties and their biological effects on U87-MG glioblastoma cells. In particular, they showed 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 proposed the fabrication of biomimetic nanostructured lipid carriers 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, highlighting anti-proliferative and pro-apoptotic effects on GBM 3D spheroids. 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. Proteomics allowed elucidating the mechanisms of action of chemotherapy and hyperthermia; the latter, in particular, was demonstrated to be particularly effective at lysosomal level, by destabilizing the lysosomal membrane and inducing cytotoxic effects upon release in the cytoplasm of the lysosomal content. Eventually, an in-depth investigation of protein corona conformation during the BBB passage has been carried out through super-resolution microscopy and proteomics.
To evaluate the in vivo therapeutic potential of this multifunctional system against GBM, its efficacy on mice bearing U87-MG Luc glioblastoma has been evaluated. A single temozolomide-loaded nanovectors intra-tumoral administration followed by 3 cycles of magnetic hyperthermia during three consecutive days was investigated. Nanovector administration combined with activation by alternating magnetic fields remarkably inhibited tumor growth (18% with respect to the control groups) and prolonged animal survival (50% of subjects alive at the end of the trial schedule); median survival was eventually significantly longer in the combined treatment (about 66% higher with respect to the control groups). These findings indicate that the developed multifunctional nanoplatform owns important anti-glioma effects stemming from combined hyperthermia and chemotherapy, highlighting the full success of SLaMM.
In parallel to the main research line of this project, we had the opportunity to investigate “homotypic” recognition as an alternative targeting approach, i.e. the ability of cancer cells to recognize each other thanks to specific cell membrane proteins. Different kinds of nanoparticles have been decorated with cell membranes derived from cancer cells, and their targeting towards the same typology of cancer cells assessed. Effective drug delivery and in vitro anticancer activity was demonstrated, giving promising hints also towards personalized and precision nanomedicine.
Eventually, as an innovative anticancer approach, we had the possibility to explore low-intensity electric stimulation mediated by ultrasounds and piezoelectric nanomaterials: the combination of chemotherapy treatment with chronic piezoelectric stimulation resulted in activation of cell apoptosis and anti-proliferation pathways, induction of cell necrosis, inhibition of cancer migration, and reduction of cell invasiveness in drug-resistant GBM cells; an efficient anti-angiogenic activity was demonstrated as well.
On the whole, results of the project have been presented to 25 international conferences, disseminated in many “generic public” events and on social media (LinkedIn, Facebook, Twitter), and gave origin to 22 scientific papers (average Impact Factor 8.5 collected citations 1110).
Concerning exploitation, the results about the real-scale in vitro models gave origin to two ERC Proof-of-Concept Grants (BBBHybrid and MagDock), and to a start-up project under development; a patent has been filed (PCT/IB2020/059365).
Different in vitro BBB models have been set-up and characterized: among them, a real-scale biohybrid model representing the first example in the literature of a biomimetic BBB system, with a design inspired from the brain microcapillaries at real scale.
Concerning the nanovectors, different prototypes have been developed, characterized, and in vitro tested.
In the first example, the chemotherapy drug nutlin-3a and superparamagnetic nanoparticles were encapsulated in solid lipid nanoparticles, and the obtained hybrid nanovectors characterized by analyzing both their physicochemical properties and their biological effects on U87-MG glioblastoma cells. In particular, they showed 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 proposed the fabrication of biomimetic nanostructured lipid carriers 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, highlighting anti-proliferative and pro-apoptotic effects on GBM 3D spheroids. 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. Proteomics allowed elucidating the mechanisms of action of chemotherapy and hyperthermia; the latter, in particular, was demonstrated to be particularly effective at lysosomal level, by destabilizing the lysosomal membrane and inducing cytotoxic effects upon release in the cytoplasm of the lysosomal content. Eventually, an in-depth investigation of protein corona conformation during the BBB passage has been carried out through super-resolution microscopy and proteomics.
To evaluate the in vivo therapeutic potential of this multifunctional system against GBM, its efficacy on mice bearing U87-MG Luc glioblastoma has been evaluated. A single temozolomide-loaded nanovectors intra-tumoral administration followed by 3 cycles of magnetic hyperthermia during three consecutive days was investigated. Nanovector administration combined with activation by alternating magnetic fields remarkably inhibited tumor growth (18% with respect to the control groups) and prolonged animal survival (50% of subjects alive at the end of the trial schedule); median survival was eventually significantly longer in the combined treatment (about 66% higher with respect to the control groups). These findings indicate that the developed multifunctional nanoplatform owns important anti-glioma effects stemming from combined hyperthermia and chemotherapy, highlighting the full success of SLaMM.
In parallel to the main research line of this project, we had the opportunity to investigate “homotypic” recognition as an alternative targeting approach, i.e. the ability of cancer cells to recognize each other thanks to specific cell membrane proteins. Different kinds of nanoparticles have been decorated with cell membranes derived from cancer cells, and their targeting towards the same typology of cancer cells assessed. Effective drug delivery and in vitro anticancer activity was demonstrated, giving promising hints also towards personalized and precision nanomedicine.
Eventually, as an innovative anticancer approach, we had the possibility to explore low-intensity electric stimulation mediated by ultrasounds and piezoelectric nanomaterials: the combination of chemotherapy treatment with chronic piezoelectric stimulation resulted in activation of cell apoptosis and anti-proliferation pathways, induction of cell necrosis, inhibition of cancer migration, and reduction of cell invasiveness in drug-resistant GBM cells; an efficient anti-angiogenic activity was demonstrated as well.
On the whole, results of the project have been presented to 25 international conferences, disseminated in many “generic public” events and on social media (LinkedIn, Facebook, Twitter), and gave origin to 22 scientific papers (average Impact Factor 8.5 collected citations 1110).
Concerning exploitation, the results about the real-scale in vitro models gave origin to two ERC Proof-of-Concept Grants (BBBHybrid and MagDock), and to a start-up project under development; a patent has been filed (PCT/IB2020/059365).
At the end of the project, the following points represented an actual breakthrough in the literature:
• preparation and characterization of dynamic multi-cellular BBB models, including the first 1:1 scale BBB model in the literature (highlighted in the 2018 Annual Report on the ERC Activities and Achievements);
• preparation, characterization, and in vitro validation (even through proteomic analysis) of multi-functional hybrid magnetic lipid nanovectors, able to cross BBB models 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, by exploiting both inorganic and organic piezoelectric nanoparticles, aiming at reducing multi-drug resistance and at enhancing anti-proliferative and anti-angiogenic effects of traditional chemotherapy drugs;
• exploitation of the “homotypic” targeting for nanoparticle delivery, paving the way to personalized and precision nanomedicine;
• in vivo validation of the proposed multi-functional nanoplatform: 66% increment of median survival in treated animals with respect to the controls.
• preparation and characterization of dynamic multi-cellular BBB models, including the first 1:1 scale BBB model in the literature (highlighted in the 2018 Annual Report on the ERC Activities and Achievements);
• preparation, characterization, and in vitro validation (even through proteomic analysis) of multi-functional hybrid magnetic lipid nanovectors, able to cross BBB models 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, by exploiting both inorganic and organic piezoelectric nanoparticles, aiming at reducing multi-drug resistance and at enhancing anti-proliferative and anti-angiogenic effects of traditional chemotherapy drugs;
• exploitation of the “homotypic” targeting for nanoparticle delivery, paving the way to personalized and precision nanomedicine;
• in vivo validation of the proposed multi-functional nanoplatform: 66% increment of median survival in treated animals with respect to the controls.