Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

High-Dose Exposure to Polymer-Coated Iron Oxide Nanoparticles Elicits Autophagy-Dependent Ferroptosis in Susceptible Cancer Cells.

Ferroptosis, a form of iron-dependent, lipid peroxidation-driven cell death, has been extensively investigated in recent years, and several studies have suggested that the ferroptosis-inducing properties of iron-containing nanomaterials could be harnessed for cancer treatment. Here we evaluated the potential cytotoxicity of iron oxide nanoparticles, with and without cobalt functionalization (Fe2O3 and Fe2O3@Co-PEG), using an established, ferroptosis-sensitive fibrosarcoma cell line (HT1080) and a normal fibroblast cell line (BJ). In addition, we evaluated poly (ethylene glycol) (PEG)-poly(lactic-co-glycolic acid) (PLGA)-coated iron oxide nanoparticles (Fe3O4-PEG-PLGA). Our results showed that all the nanoparticles tested were essentially non-cytotoxic at concentrations up to 100 µg/mL. However, when the cells were exposed to higher concentrations (200-400 µg/mL), cell death with features of ferroptosis was observed, and this was more pronounced for the Co-functionalized nanoparticles. Furthermore, evidence was provided that the cell death triggered by the nanoparticles was autophagy-dependent. Taken together, the exposure to high concentrations of polymer-coated iron oxide nanoparticles triggers ferroptosis in susceptible human cancer cells.

Native Study of the Behaviour of Magnetite Nanoparticles for Hyperthermia Treatment during the Initial Moments of Intravenous Administration.

Magnetic nanoparticles (MNPs) present outstanding properties making them suitable as therapeutic agents for hyperthermia treatments. Since the main safety concerns of MNPs are represented by their inherent instability in a biological medium, strategies to both achieve long-term stability and monitor hazardous MNP degradation are needed. We combined a dynamic approach relying on flow field flow fractionation (FFF)-multidetection with conventional techniques to explore frame-by-frame changes of MNPs injected in simulated biological medium, hypothesize the interaction mechanism they are subject to when surrounded by a saline, protein-rich environment, and understand their behaviour at the most critical point of intravenous administration. In the first moments of MNPs administration in the patient, MNPs change their surrounding from a favorable to an unfavorable medium, i.e., a complex biological fluid such as blood; the particles evolve from a synthetic identity to a biological identity, a transition that needs to be carefully monitored. The dynamic approach presented herein represents an optimal alternative to conventional batch techniques that can monitor only size, shape, surface charge, and aggregation phenomena as an averaged information, given that they cannot resolve different populations present in the sample and cannot give accurate information about the evolution or temporary instability of MNPs. The designed FFF method equipped with a multidetection system enabled the separation of the particle populations providing selective information on their morphological evolution and on nanoparticle-proteins interaction in the very first steps of infusion. Results showed that in a dynamic biological setting and following interaction with serum albumin, PP-MNPs retain their colloidal properties, supporting their safety profile for intravenous administration.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

Smart Magnetic Nanocarriers for Multi-Stimuli On-Demand Drug Delivery.

In this study, we report the realization of drug-loaded smart magnetic nanocarriers constituted by superparamagnetic iron oxide nanoparticles encapsulated in a dual pH- and temperature-responsive poly (N-vinylcaprolactam-co-acrylic acid) copolymer to achieve highly controlled drug release and localized magnetic hyperthermia. The magnetic core was constituted by flower-like magnetite nanoparticles with a size of 16.4 nm prepared by the polyol approach, with good saturation magnetization and a high specific absorption rate. The core was encapsulated in poly (N-vinylcaprolactam-co-acrylic acid) obtaining magnetic nanocarriers that revealed reversible hydration/dehydration transition at the acidic condition and/or at temperatures above physiological body temperature, which can be triggered by magnetic hyperthermia. The efficacy of the system was proved by loading doxorubicin with very high encapsulation efficiency (>96.0%) at neutral pH. The double pH- and temperature-responsive nature of the magnetic nanocarriers facilitated a burst, almost complete release of the drug at acidic pH under hyperthermia conditions, while a negligible amount of doxorubicin was released at physiological body temperature at neutral pH, confirming that in addition to pH variation, drug release can be improved by hyperthermia treatment. These results suggest this multi-stimuli-sensitive nanoplatform is a promising candidate for remote-controlled drug release in combination with magnetic hyperthermia for cancer treatment.

T Cell Delivery of Nanoparticles-Bound Anti-CD20 Monoclonal Antibody: Successful B Cell Depletion in the Spinal Cord during Experimental Autoimmune Encephalomyelitis.

We developed a nanotechnology based-cell mediated drug delivery system by loading myelin antigen-specific T cells with nanoparticles bound to anti-CD20 monoclonal antibody. Anti-CD20 antibody is a current treatment (ocrelizumab) for multiple sclerosis (MS), a chronic, inflammatory and autoimmune disease of the central nervous system (CNS). CD20-depletion has been associated with efficacy in active relapsing and progressive MS, but may not efficiently target inflammatory cells compartmentalized in the CNS. In our work, the intravenous transfer of T cells containing nanoparticle-anti-CD20 complex in mice causes B cell depletion in the spleen and in the brain, whereas the injection of anti-CD20 alone depletes B cells only in the spleen. Testing this system in Experimental Autoimmune Encephalomyelitis (EAE), animal model of MS, we found that spinal cord B cell depletion ameliorates the disease course and pathology. Graphical Abstract.

Spectroscopic and photoacoustic characterization of encapsulated iron oxide super-paramagnetic nanoparticles as a new multiplatform contrast agent.

Recently, a number of photoacoustic (PA) agents with increased tissue penetration and fine spatial resolution have been developed for molecular imaging and mapping of pathophysiological features at the molecular level. Here, we present bio-conjugated near-infrared light-absorbing magnetic nanoparticles as a new agent for PA imaging. These nanoparticles exhibit suitable absorption in the near-infrared region, with good photoacoustic signal generation efficiency and high photo-stability. Furthermore, these encapsulated iron oxide nanoparticles exhibit strong super-paramagnetic behavior and nuclear relaxivities that make them useful as magnetic resonance imaging (MRI) contrast media as well. Their simple bio-conjugation strategy, optical and chemical stability, and straightforward manipulation could enable the development of a PA probe with magnetic and spectroscopic properties suitable for in vitro and in vivo real-time imaging of relevant biological targets.

EGFR-Targeted Magnetic Nanovectors Recognize, in Vivo, Head and Neck Squamous Cells Carcinoma-Derived Tumors.

Head and neck squamous cell carcinomas (HNSCC) are a diverse group of tumors with high morbidity and mortality that have remained mostly unchanged over the past decades. The epidermal growth factor receptor (EGFR) is often overexpressed and activated in these tumors and strongly contributes to their pathogenesis. Still, EGFR-targeted therapies such as monoclonal antibodies and kinase inhibitors have demonstrated only limited improvements in the clinical outcome of this disease. Here, we take advantage of the extraordinary affinity of EGF for its cognate receptor to specifically target magnetite-containing nanoparticles to HNSCC cells and mediate, in vitro, their cellular upload. On the basis of this, we show efficient accumulation, in vivo, of such nanoparticles in subcutaneous xenograft tumor tissues in sufficient amounts to be able to mediate visualization by magnetic resonance imaging. Overall, our EGF-coated nanosystem may warrant, in the near future, novel and very efficient theranostic approaches to HNSCC.

Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells.

Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe3O4-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 µg/mL of Fe3O4-NPs. Significant dose-response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death.

In vivo anticancer evaluation of the hyperthermic efficacy of anti-human epidermal growth factor receptor-targeted PEG-based nanocarrier containing magnetic nanoparticles.

Polymeric nanoparticles with targeting moieties containing magnetic nanoparticles as theranostic agents have considerable potential for the treatment of cancer. Here we report the chemical synthesis and characterization of a poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-based nanocarrier containing iron oxide nanoparticles and human epithelial growth factor receptor on the outer shell. The nanocarrier was also radiolabeled with (99m)Tc and tested as a theranostic nanomedicine, ie, it was investigated for both its diagnostic ability in vivo and its therapeutic hyperthermic effects in a standard A431 human tumor cell line. Following radiolabeling with (99m)Tc, the biodistribution and therapeutic hyperthermic effects of the nanosystem were studied noninvasively in vivo in tumor-bearing mice. A substantial decrease in tumor size correlated with an increase in both nanoparticle concentration and local temperature was achieved, confirming the possibility of using this multifunctional nanosystem as a therapeutic tool for epidermoid carcinoma.

Magnetism and spin dynamics of novel encapsulated iron oxide superparamagnetic nanoparticles.

Encapsulated Fe3O4 nanoparticles of average diameters d = 12 nm are obtained by coprecipitation, in the presence of 2-methoxyethanol hemiester of poly(maleic anhydride-alt-butyl vinyl ether) 5% grafted with poly(ethylene glycol) (VP-MAG nanoparticles). A complete characterization of nude and encapsulated nanoparticles through structural techniques (namely XRD, TEM, SEM), Raman spectroscopy and magnetic measurements has been performed. These nanoparticles compared with commercial compounds (ENDOREM®) present superparamagnetic behavior and nuclear relaxivities that make them promising as magnetic resonance imaging (MRI) contrast agents (CAs). We found that our nanostructures exhibit r2 relaxivity higher than those of commercial CAs over the whole frequency range. The MRI efficiency of our samples was related to their microstructural and magnetic properties.

Preliminary evaluation of a 99mTc labeled hybrid nanoparticle bearing a cobalt ferrite core: in vivo biodistribution.

Magnetic nanoparticles have become important tools for imaging a wide range of diseases, improving drug delivery and applying hyperthermic treatment. Iron oxide based nanoparticles have been widely examined, unlike cobalt ferrite based ones. Herein, monodisperse and stable CoFe2O4 nanoparticles have been produced, coated and further stabilized using ethyl 12-(hydroxyamino)-12-oxododecanoate, poly(lactic-co-glycolic acid) and bovine serum albumin. The final product, NBRh1, was fully characterized and has been directly radiolabeled with 99mTc using SnCl1 as the reducing agent in high yields. In vitro stability and hyperthermic properties of 99mTC-NBRh1 were encouraging for further application in low frequencies hyperthermia and biomagnetic applications. In vivo evaluation followed after injection in healthy mice. The planar and SPECT imaging data as well as the biodistribution results were in accordance, showing high liver and spleen uptake as expected starting almost immediately after administration. In conclusion the preliminary results for nanoparticles bearing a cobalt ferrite core justify further investigations towards potential hyperthermic applications, drug transportation and liver or spleen imaging.

Synthesis and coating of cobalt ferrite nanoparticles: a first step toward the obtainment of new magnetic nanocarriers.

Monodisperse and stable cobalt ferrite (CoFe2O4) nanoparticles (5.4 nm) have been produced, coated with mono- and difunctional phosphonic and hydroxamic acids, and fully characterized (using thermogravimetric analysis (TGA), dynamic light scattering (DLS), IR spectroscopy, transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) measurements). Cobalt leakage of the coated nanoparticles has been also studied. Magnetic measurements show the possible applications in hyperthermia at low frequencies, and for this reason, water-soluble coated CoFe2O4 can be seen as a first step toward the obtainment of novel systems for biomagnetic applications.