Periodic Reporting for period 1 - MagGraphZyme (Design of novel Magnetic Graphene Oxide Nanozyme platform for Theranostic applications)
Periodo di rendicontazione: 2023-06-01 al 2025-05-31
Cancer is among the leading causes of death worldwide. According to data compiled by the International Agency for research on cancer (https://gco.iarc.fr/en(si apre in una nuova finestra)) In 2022, there were almost 20 million new cases and 9.7 million cancer-related deaths worldwide. By 2040, the number of new cancer cases per year is expected to rise to 29.9 million and the number of cancer-related deaths to 15.3 million. Due to this rapid growth, it is imperative to combine all necessary efforts to mitigate the evolution of this disease and to eliminate cancer cell more efficiently. Thanks to advancements on immunotherapy, targeted therapies, nanotechnology among others, the new strategies to cancer research has gradually shifted its focus to a combination of them for enhanced treatment effectiveness. Specifically, the rise of catalytic medicine through nanozymes (nanomaterials that mimic enzymatic activity) shows promise in combination with therapies such as photothermal therapy (PTT), photodynamic and sonodynamic therapy (PDT/SDT), and magnetic fluid hyperthermia (MFH), where the common point is to provoke the cancer cells death (through the oxidative stress ) enhancing the reactive oxygen/nitrogen species (ROS/RNS) production, by the stimulation of multifunctional hybrid nanomaterials (MFHNM) previously loaded on cancer cells, with an external source: Near infrared laser (NIR), ultrasounds or alternate magnetic field (AMF), by the decomposition of the cellular endogenous hydrogen peroxide H2O2 through Fenton and Heber-Weiss reactions. Modelling the cell death mechanisms by any of these approaches requires identifying in first place the best configuration of MFHNM to use (that with the best features such as heating efficiency as catalytic performace) by which it necessary to carried out an in-depth characterization of them. In second place the intracellular distribution of the agents (MFNMs) that provokes metabolic or physical cell damage and in third place the evaluation of damage that they generate on the specific place on the cellular environment.
With this synergistic approach in mind, we objectives are focused on production, characterization in depth of MFHNM based on magnetic nanoparticles (MNPs) as heating agents for ROS production and its evaluation on cancer cells.This project looking to advance on the understanding of the molecular mechanisms of cancer cell death pathways which is essential for understanding their role in cancer progression, which represent a highly ambitious objective, with a global impact and perfectly aligned with Pillar 2, "Global Challenges and European Industrial Competitiveness" (health), of the Horizon Europe (HE) program. Additionally, the scientific challenges involved represent research at the frontier of knowledge, aligning with Pillar 1, "Excellent Science," within HE
With this synergistic approach in mind, we objectives are focused on production, characterization in depth of MFHNM based on magnetic nanoparticles (MNPs) as heating agents for ROS production and its evaluation on cancer cells.This project looking to advance on the understanding of the molecular mechanisms of cancer cell death pathways which is essential for understanding their role in cancer progression, which represent a highly ambitious objective, with a global impact and perfectly aligned with Pillar 2, "Global Challenges and European Industrial Competitiveness" (health), of the Horizon Europe (HE) program. Additionally, the scientific challenges involved represent research at the frontier of knowledge, aligning with Pillar 1, "Excellent Science," within HE
We successfully synthesized and thoroughly characterized two series of magnetic nanoparticles vanadium and aluminum ferrites with average sizes ranging from 8 to 36 nm. These nanoparticles were obtained via the thermal decomposition of vanadium, aluminum, and iron acetylacetonates in the presence of oleic acid and oleylamine. Electron microscopy techniques, including SEM-EDS, TEM, and HRTEM, confirmed the expected stoichiometry and morphology: smaller particles were predominantly spherical, while larger ones exhibited faceted structures. Selected-area electron diffraction (SAED) patterns obtained from HRTEM images confirmed a face-centered cubic (FCC) spinel crystal structure across all samples. Compositional analysis by STEM-EELS revealed a core shell structure with a vanadium-rich shell in vanadium ferrite nanoparticles, whereas aluminum ferrite nanoparticles displayed a more homogeneous elemental distribution. These observations were further supported by X-ray photoelectron spectroscopy (XPS) performed on powder samples. Magnetic properties were assessed using SQUID magnetometry. Smaller particles exhibited superparamagnetic behavior below room temperature, while the largest particles showed a blocked regime extending up to room temperature. All samples demonstrated magnetic properties comparable to bulk magnetite, with effective anisotropy constants similar to or lower than bulk values, as determined through ferromagnetic resonance (FMR) experiments. These properties correlated well with heating efficiency, assessed via both calorimetry and AC magnetic hysteresis loop measurements, yielding specific loss power (SLP) values of up to 1000 W/g. Electron spin resonance (ESR) spectroscopy detected hydroxyl radical (•OH) concentrations as high as 2300 nM, indicating strong peroxidase-like activity in vanadium ferrite samples. In contrast, aluminum ferrites showed reduced activity, likely due to partial substitution of divalent iron ions by trivalent aluminum ions in the crystal lattice. The best-performing samples (15 and 30 nm) were successfully transferred into aqueous media using a glucose coating and incubated with pancreatic cancer cells for biological testing. TEM imaging confirmed efficient nanoparticle uptake, with localization in lysosomal and endosomal compartments. Cell viability remained above 100% at concentrations up to 100 µg/mL of magnetic nanoparticles. The effects of intracellular nanoparticle excitation by external stimuli such as alternating (AC) magnetic fields and near-infrared (NIR) laser irradiation are still under investigation. However, in some cases, loss of cell membrane integrity was observed via TEM in samples prepared by high-pressure freezing and freeze substitution, suggesting increased production of reactive oxygen species (ROS) potentially induced by nanoparticle excitation.
The evaluation of the effects induced in cancer cells loaded with magnetic nanoparticles (MNPs) following irradiation with near-infrared (NIR) light and/or alternating current (AC) magnetic fields is still ongoing. The chosen sample preparation strategy, based on high-pressure freezing (HPF) followed by freeze substitution, aims to preserve the native hydration state of the cellular models. This methodology minimizes structural artifacts commonly introduced by conventional chemical fixation methods. The ultimate objective is to enable high-resolution analysis of cellular ultrastructure at the molecular level, representing an approach that goes beyond the current state of the art and therefore requires further research for full validation. We are confident that this line of work is appropriate and expect to achieve significant progress in this direction during the final phase of the project.