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.

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.

Multifunctional graphene-based magnetic nanocarriers for combined hyperthermia and dual stimuli-responsive drug delivery.

The synthesis of hydrophilic graphene-based yolk-shell magnetic nanoparticles functionalized with copolymer pluronic F-127 (GYSMNP@PF127) is herein reported to achieve an efficient multifunctional biomedical system for mild hyperthermia and stimuli-responsive drug delivery. In vitro tests revealed the extraordinary ability of GYSMNP@PF127 to act as smart stimuli-responsive multifunctional nanomedicine platform for cancer therapy, exhibiting (i) an outstanding loading capacity of 91% (w/w, representing 910 µg mg-1) of the chemotherapeutic drug doxorubicin, (ii) a high heating efficiency under an alternating (AC) magnetic field (intrinsic power loss ranging from 2.1-2.7 nHm2 kg-1), and (iii) a dual pH and thermal stimuli-responsive drug controlled release (46% at acidic tumour pH vs 7% at physiological pH) under AC magnetic field, in just 30 min. Additionally, GYSMNP@PF127 presents optimal hydrodynamic diameter (DH = 180 nm) with negative surface charge, high haemocompatibility for blood stream applications and tumour cellular uptake of drug nanocarriers. Due to its physicochemical, magnetic and biocompatibility properties, the developed graphene-based magnetic nanocarrier shows high promise as dual exogenous (AC field)/endogenous (pH) stimuli-responsive actuators for targeted thermo-chemotherapy, combining magnetic hyperthermia and controlled drug release triggered by the abnormal tumour environment. The presented strategy and findings can represent a new way to design and develop highly stable added-value graphene-based nanostructures for the combined treatment of cancer.

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.

Salt concentration and solar orientation in two supralittoral sandhoppers: Talitrus saltator (Montagu) and Talorchestia ugolinii Bellan Santini and Ruffo.

The influence of salt concentration in the seawater on solar orientation in Talitrus saltator and Talorchestia ugolinii was studied in a confined environment (transparent plexiglass bowls). Sodium and calcium concentrations strongly affect both sea-land orientation and the sun compass mechanism in T.saltator, whereas the behaviour of T. ugolinii is less influenced. The absence of Na(+) does not influence the sun compass mechanism, but causes an inversion in the mean direction of orientation in T. saltator. In T. ugolinii, there was no influence on the compass mechanism for solar orientation and no inversion in the directional choice. In the absence of Ca(2+), a photonegative tendency was observed for T saltator together with marked reduction in the capacity to go in any direction. However, the effect of Ca(2+) absence on the orientation capacity of T. saltator is reversible and the orientation capacity can be reduced in a few minutes. The different behaviour of the two species of sandhoppers is discussed.

Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

Liquid chromatographic/electrospray ionization tandem mass spectrometric study of polyphenolic composition of four cultivars of Fragaria vesca L. berries and their comparative evaluation.

High-performance liquid chromatography coupled with ion spray mass spectrometry in the tandem mode with both negative and positive ionization was used for investigating a variety of polyphenolic compounds in four genotypes of Fragaria vesca berries. About 60 phenolic compounds belonging to the compound classes of phenolic acids, ellagitannins, ellagic acid derivatives, flavonols, monomeric and oligomeric flavanols, dihydrochalcones and anthocyanins were reported, providing for the first time a quite complete picture of polyphenolic composition of F. vesca berries. Some of the polyphenols herein investigated, such as a tris-galloyl-hexahydroxydiphenoyl-hexose, two castalagin/vescalagin-like isomers and peonidin-malonylglucoside, were described for the first time. Principal component analysis applied on original HPLC-MS/MS data, acquired in multiple reaction monitoring mode, successfully discriminated the four investigated cultivars on the basis of their polyphenolic composition, highlighting the fundamental role of mass spectrometry for food characterization.

Distribution and mass balance of hexavalent and trivalent chromium in a subsurface, horizontal flow (SF-h) constructed wetland operating as post-treatment of textile wastewater for water reuse.

In this study, during a two-year period, we investigated the fate of hexavalent and trivalent chromium in a full-scale subsurface horizontal flow constructed wetland planted with Phragmites australis. The reed bed operated as post-treatment of the effluent wastewater from an activated sludge plant serving the textile industrial district and the city of Prato (Italy). Chromium speciation was performed in influent and effluent wastewater and in water-suspended solids, at different depths and distances from the inlet; plants were also analyzed for total chromium along the same longitudinal profile. Removals of hexavalent and trivalent chromium equal to 72% and 26%, respectively were achieved. The mean hexavalent chromium outlet concentration was 1.6 ± 0.9 µg l(-1) and complied with the Italian legal limits for water reuse. Chromium in water-suspended solids was in the trivalent form, thus indicating that its removal from wastewater was obtained by the reduction of hexavalent chromium to the trivalent form, followed by accumulation of the latter inside the reed bed. Chromium in water-suspended solids was significantly affected by the distance from the inlet. Chromium concentrations in the different plant organs followed the same trend of suspended solids along the longitudinal profile and were much lower than those found in the solid material, evidencing a low metal accumulation in P. australis.