Application of Grazing-Incidence X-ray Methods to Study Terrace-Stepped SiC Surface for Graphene Growth.

The synthesis of graphene by the graphitization of SiC surface has been driven by a need to develop a way to produce graphene in large quantities. With the increased use of thermal treatments of commercial SiC substrates, a comprehension of the surface restructuring due to the formation of a terrace-stepped nanorelief is becoming a pressing challenge. The aim of this paper is to evaluate the utility of X-ray reflectometry and grazing-incidence off-specular scattering for a non-destructive estimate of depth-graded and lateral inhomogeneities on SiC wafers annealed in a vacuum at a temperature of 1400-1500 °C. It is shown that the grazing-incidence X-ray method is a powerful tool for the assessment of statistical parameters, such as effective roughness height, average terrace period and dispersion. Moreover, these methods are advantageous to local probe techniques because a broad range of spatial frequencies allows for faster inspection of the whole surface area. We have found that power spectral density functions and in-depth density profiles manifest themselves differently between the probing directions along and across a terrace edge. Finally, the X-ray scattering data demonstrate quantitative agreement with the results of atomic force microscopy.

3D volume segmentation and reconstruction. Supervised image classification and automated quantification of superparamagnetic iron oxide nanoparticles in histology slides for safety assessment.

This article presents an automated image-processing workflow for quantitative assessment of SPION accumulation in tissue sections stained with Prussian blue for iron detection. We utilized supervised machine learning with manually labeled features used for training the classifier. Performance of the classifier was validated by 10-fold cross-validation of obtained data and by measuring Dice and Jaccard Similarity Coefficients between manually segmented image and automated segmentation. The proposed approach provides time and cost-effective solution for quantitative imaging analysis of SPION in tissue with a precision similar to that obtained via thresholding method for stain quantification. Furthermore, we exploited the classifiers to generate segmented 3D volumes from histological slides. This enabled visualization of particles which were obscured in original 3D histology stacks. Our approach offers a powerful tool for preclinical assessment of the precise tissue-specific SPION biodistribution, which could affect both their toxicity and their efficacy as nanocarriers for medicines.

Nanotechnology in pulmonary medicine.

Nanotechnology in medicine-nanomedicine-is extensively employed to diagnose, treat, and prevent pulmonary diseases. Over the last few years, this brave new world has made remarkable progress, offering opportunities to address historical clinical challenges in pulmonary diseases including multidrug resistance, adverse side effects of conventional therapeutic agents, novel imaging, and earlier disease detection. Nanomedicine is also being applied to tackle the new emerging infectious diseases, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), influenza A virus subtype H1N1 (A/H1N1), and more recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review we provide both a historical overview of the application of nanomedicine to respiratory diseases and more recent cutting-edge approaches such as nanoparticle-mediated combination therapies, novel double-targeted nondrug delivery system for targeting, stimuli-responsive nanoparticles, and theranostic imaging in the diagnosis and treatment of pulmonary diseases.

Aerosolized drug-loaded nanoparticles targeting migration inhibitory factors inhibit Pseudomonas aeruginosa-induced inflammation and biofilm formation.

Aim: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, which has been shown to promote disease severity in cystic fibrosis. Methods: In this study, aerosolized drug-loaded nanoparticles containing SCD-19, an inhibitor of MIF's tautomerase enzymatic activity, were developed and characterized. Results: The aerosolized nanoparticles had an optimal droplet size distribution for deep lung deposition, with a high degree of biocompatibility and significant cellular uptake. Conclusion: For the first time, we have developed an aerosolized nano-formulation against MIF's enzymatic activity that achieved a significant reduction in the inflammatory response of macrophages, and inhibited Pseudomonas aeruginosa biofilm formation on airway epithelial cells. This represents a potential novel adjunctive therapy for the treatment of P. aeruginosa infection in cystic fibrosis.

A complex morphofunctional approach for zinc toxicity evaluation in rats.

Anthropogenic activity causes the introduction of zinc compounds into the biological cycle in mining and processing sites and its accumulation in organs and tissues, causing systemic toxicity. A cumulative effect of zinc is predominantly neurotoxic and it also affects the respiratory, cardiovascular and digestive systems. This study evaluates the effects of single-dose intragastric administration of 100 mg/kg zinc succinate on the structure and function of organs and tissues in male Wistar rats 1 month after treatment. The presented morphofunctional approach for the toxicity evaluation included the study of behavioral responses using the automated Laboras® complex, fluorescent spectral analysis of the NADH and FAD activity and histological evaluation of animal organs and tissues. The results of the behavioral activity assessment showed a significant decrease in animals' motor activity, whereas the fluorescence spectra analysis demonstrated a decrease in coenzyme NADH without the reduction of FAD levels. We detected toxic and dystrophic changes in the cerebral cortex, heart, lungs and liver tissues. Our original multiparametric approach enables a comprehensive assessment of the long-term toxic effects of the metal salts such as zinc succinate, especially in the cerebral cortex at the doses much lower than the acute LD50 reported for the common zinc salts.

Comparing the Effects of Intracellular and Extracellular Magnetic Hyperthermia on the Viability of BxPC-3 Cells.

Magnetic hyperthermia involves the use of iron oxide nanoparticles to generate heat in tumours following stimulation with alternating magnetic fields. In recent times, this treatment has undergone numerous clinical trials in various solid malignancies and subsequently achieved clinical approval to treat glioblastoma and prostate cancer in 2011 and 2018, respectively. However, despite recent clinical advances, many questions remain with regard to the underlying mechanisms involved in this therapy. One such query is whether intracellular or extracellular nanoparticles are necessary for treatment efficacy. Herein, we compare the effects of intracellular and extracellular magnetic hyperthermia in BxPC-3 cells to determine the differences in efficacy between both. Extracellular magnetic hyperthermia at temperatures between 40-42.5 °C could induce significant levels of necrosis in these cells, whereas intracellular magnetic hyperthermia resulted in no change in viability. This led to a discussion on the overall relevance of intracellular nanoparticles to the efficacy of magnetic hyperthermia therapy.

Proinflammatory Effect of Carbon-Based Nanomaterials: In Vitro Study on Stimulation of Inflammasome NLRP3 via Destabilisation of Lysosomes.

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.

Author Correction: Preparation from a revisited wet chemical route of phase-pure, monocrystalline and SHG-efficient BiFeO3 nanoparticles for harmonic bio-imaging.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The phenotype of target pancreatic cancer cells influences cell death by magnetic hyperthermia with nanoparticles carrying gemicitabine and the pseudo-peptide NucAnt.

In this paper we show that conjugation of magnetic nanoparticles (MNPs) with Gemcitabine and/or NucAnt (N6L) fostered their internalization into pancreatic tumor cells and that the coupling procedure did not alter the cytotoxic potential of the drugs. By treating tumor cells (BxPC3 and PANC-1) with the conjugated MNPs and magnetic hyperthermia (43 °C, 60 min), cell death was observed. The two pancreatic tumor cell lines showed different reactions against the combined therapy according to their intrinsic sensitivity against Gemcitabine (cell death, ROS production, ability to activate ERK 1/2 and JNK). Finally, tumors (e.g. 3 mL) could be effectively treated by using almost 4.2 × 105 times lower Gemcitabine doses compared to conventional therapies. Our data show that this combinatorial therapy might well play an important role in certain cell phenotypes with low readiness of ROS production. This would be of great significance in distinctly optimizing local pancreatic tumor treatments.

Multilayered Cultures of NSCLC cells grown at the Air-Liquid Interface allow the efficacy testing of inhaled anti-cancer drugs.

Evidence supports the advantages of inhalation over other drug-administration routes in the treatment of lung diseases, including cancer. Although data obtained from animal models and conventional in vitro cultures are informative, testing the efficacy of inhaled chemotherapeutic agents requires human-relevant preclinical tools. Such tools are currently unavailable. Here, we developed and characterized in vitro models for the efficacy testing of inhaled chemotherapeutic agents against non-small-cell lung cancer (NSCLC). These models recapitulated key elements of both the lung epithelium and the tumour tissue, namely the direct contact with the gas phase and the three-dimensional (3D) architecture. Our in vitro models were formed by growing, for the first time, human adenocarcinoma (A549) cells as multilayered mono-cultures at the Air-Liquid Interface (ALI). The in vitro models were tested for their response to four benchmarking chemotherapeutics, currently in use in clinics, demonstrating an increased resistance to these drugs as compared to sub-confluent monolayered 2D cell cultures. Chemoresistance was comparable to that detected in 3D hypoxic tumour spheroids. Being cultured in ALI conditions, the multilayered monocultures demonstrated to be compatible with testing drugs administered as a liquid aerosol by a clinical nebulizer, offering an advantage over 3D tumour spheroids. In conclusion, we demonstrated that our in vitro models provide new human-relevant tools allowing for the efficacy screening of inhaled anti-cancer drugs.

Preparation from a revisited wet chemical route of phase-pure, monocrystalline and SHG-efficient BiFeO3 nanoparticles for harmonic bio-imaging.

We present two new synthetic routes for bismuth ferrite harmonic nanoparticles (BiFeO3 HNPs). Both phase-pure and mixed phase BiFeO3 materials were produced after improvement of the solvent evaporation and sol-gel combustion routes. Metal nitrates with a series of dicarboxylic acids (tartronic, tartaric and mucic) were used to promote crystallization. We found that the longer the carbon backbone with a hydroxyl group attached to each carbon, the lower the annealing temperature. We also demonstrate that nanocrystals more readily formed at a given temperature by adding glycerol but to the detriment of phase purity, whereas addition of NaCl in excess with mucic acid promotes the formation of phase-pure, monocrystalline nanoparticles. This effect was possibly associated with a better dispersion of the primary amorphous precursors and formation of intermediate complexes. The nanoparticles have been characterized by XRD, TEM, ζ-potential, photon correlation spectroscopy, two-photon microscopy and Hyper-Rayleigh Scattering measurements. The improved crystallization leads to BiFeO3 HNPs without defect-induced luminescence and with a very high averaged second harmonic efficiency (220 pm/V), almost triple the efficiency previously reported. This development of simple, scalable synthesis routes which yield phase-pure and, crucially, monocrystalline BiFeO3 HNPs demonstrates a significant advance in engineering the properties of nanocrystals for bio-imaging and diagnostics applications.

The anticoagulant properties of cadmium telluride quantum dots.

The size-dependent optical properties of quantum dots (QDs) are frequently exploited for use in medical imaging and labelling applications. Similarly, presented here, they also elicit profound size-dependent anticoagulant properties. Cadmium telluride quantum dot (QDs) (3.2 nm) were shown to have a dramatic anticoagulant effect centred on around the intrinsic coagulation pathway, compared to their 3.6 nm counterparts. Several clinically relevant diagnostic tests were carried out over a concentration range of the QDs and demonstrated that the 3.2 nm QDs elicited their response on the intrinsic pathway as a whole, yet the activity of the individual intrinsic coagulation factors was not affected. The mechanism appears also to be strongly influenced by the concentration of calcium ions and not cadmium ions leached from the QDs. Static and shear-based primary haemostasis assays were also carried out, demonstrating a profound anticoagulant effect which was independent of platelets and phospholipids. The data presented here suggest that the physical-chemical properties of the QDs may have a role in the modulation of haemostasis and the coagulation cascade, in a yet not fully understood mechanism. This study has implications for the use of similar QDs as diagnostic or therapeutic tools in vivo, and for the occupational health and safety of those working with such materials.

Towards the Identification of an In Vitro Tool for Assessing the Biological Behavior of Aerosol Supplied Nanomaterials.

Nanoparticles (NP)-based inhalation systems for drug delivery can be administered in liquid form, by nebulization or using pressurized metered dose inhalers, and in solid form by means of dry powder inhalers. However, NP delivery to the lungs has many challenges including the formulation instability due to particle-particle interactions and subsequent aggregation, causing poor deposition in the small distal airways and subsequent alveolar macrophages activity, which could lead to inflammation. This work aims at providing an in vitro experimental design for investigating the correlation between the physico-chemical properties of NP, and their biological behavior, when they are used as NP-based inhalation treatments, comparing two different exposure systems. By means of an aerosol drug delivery nebulizer, human lung cells cultured at air-liquid interface (ALI) were exposed to two titanium dioxide NP (NM-100 and NM-101), obtained from the JRC repository. In parallel, ALI cultures were exposed to NP suspension by direct inoculation, i.e., by adding the NP suspensions on the apical side of the cell cultures with a pipette. The formulation stability of NP, measured as hydrodynamic size distributions, the cell viability, cell monolayer integrity, cell morphology and pro-inflammatory cytokines secretion were investigated. Our results demonstrated that the formulation stability of NM-100 and NM-101 was strongly dependent on the aggregation phenomena that occur in the conditions adopted for the biological experiments. Interestingly, comparable biological data between the two exposure methods used were observed, suggesting that the conventional exposure coupled to ALI culturing conditions offers a relevant in vitro tool for assessing the correlation between the physico-chemical properties of NP and their biological behavior, when NP are used as drug delivery systems.

Culturing substrates influence the morphological, mechanical and biochemical features of lung adenocarcinoma cells cultured in 2D or 3D.

Alternative models such as three-dimensional (3D) cell cultures represent a distinct milestone towards capturing the realities of cancer biology in vitro and reduce animal experimentation in the preclinical stage of drug discovery. Significant work remains to be done to understand how substrates used in in vitro alternatives influence cancer cells phenotype and drug efficacy responses, so that to accurately link such models to specific in vivo disease scenarios. Our study describes how the morphological, mechanical and biochemical properties of adenocarcinoma (A549) cells change in response to a 3D environment and varying substrates. Confocal Laser Scanning (LSCM), He-Ion (HIM) and Atomic Force (AFM) microscopies, supported by ELISA and Western blotting, were used. These techniques enabled us to evaluate the shape, cytoskeletal organization, roughness, stiffness and biochemical signatures of cells grown within soft 3D matrices (PuraMatrix™ and Matrigel™), and to compare them to those of cells cultured on two-dimensional glass substrates. Cell cultures are also characterized for their biological response to docetaxel, a taxane-type drug used in Non-Small-Cell Lung Cancer (NSCLC) treatment. Our results offer an advanced biophysical insight into the properties and potential application of 3D cultures of A549 cells as in vitro alternatives in lung cancer research.

Cadmium nanoparticles citrullinate cytokeratins within lung epithelial cells: cadmium as a potential cause of citrullination in chronic obstructive pulmonary disease.

Objective: The objective of the study was to determine whether the cadmium-derived materials induce intracellular protein citrullination. Methods: Human A549 lung epithelial cells were exposed to cadmium in soluble and nanoparticulate forms represented by cadmium chloride (CdCl2) and cadmium oxide (CdO), respectively, and their combinations with ultrafine carbon black (ufCB) produced by high temperature combustion, imitating cigarette burning. Protein citrullination in cell lysates was analyzed by Western immunoblotting and verified by immunofluorescent confocal microscopy. Target citrullinated proteins were identified by proteomic analysis. Results: CdO, ufCB and its combination with CdCl2 and CdO after high temperature combustion induced protein citrullination in cultured human lung epithelial cells, as detected by immunoblotting with anti-citrullinated protein antibody. Cytokeratins of type II (1, 2, 5, 6A, 6B and 77) and type I (9, 10) were identified as major intracellular citrullination targets. Immunofluorescent staining confirmed the localization of citrullinated proteins both in the cytoplasm and cell nuclei. Conclusion: Cadmium oxide nanoparticle exposure facilitated post-translational citrullination of proteins.

Silver nanoparticles as a medical device in healthcare settings: a five-step approach for candidate screening of coating agents.

Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without detrimental toxic side effects. However, there is not a clear and complete protocol to define and relate the properties of the particles (size, shape, surface charge, ionic content) with their specific activity. In this paper, we propose an effective multi-step approach for the identification of a 'purpose-specific active applicability window' to maximize the antimicrobial activity of medical devices containing silver nanoparticles (Ag NPs) (such as surface coaters), minimizing any consequent risk for human health (safety by design strategy). The antimicrobial activity and the cellular toxicity of four types of Ag NPs, differing in their coating composition and concentration have been quantified. Through the implementation of flow-field flow fractionation, Ag NPs have been characterized in terms of metal release, size and shape. The particles are fractionated in the process while being left unmodified, allowing for the identification of biological particle-specific contribution. Toxicity and inflammatory response in vitro have been assessed on human skin models, while antimicrobial activity has been monitored with both non-pathogenic and pathogenic Escherichia coli. The main benefit associated with such approach is the comprehensive assessment of the maximal effectiveness of candidate nanomaterials, while simultaneously indexing their properties against their safety.

Induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials.

Citrullination, or the post-translational deimination of polypeptide-bound arginine, is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. Recent studies have shown that nanomaterials can trigger protein citrullination, which might constitute a common pathogenic link to disease development. Here we demonstrated auto-antibody production in serum of nanomaterials-treated mice. Citrullination-associated phenomena and PAD levels were found to be elevated in nanomaterials -treated cell lines as well as in the spleen, kidneys and lymph nodes of mice, suggesting a systemic response to nanomaterials injection, and validated in human pleural and pericardial malignant mesothelioma (MM) samples. The observed systemic responses in mice exposed to nanomaterials support the evidence linking exposure to environmental factors with the development of autoimmunity responses and reinforces the need for comprehensive safety screening of nanomaterials. Furthermore, these nanomaterials induce pathological processes that mimic those observed in Pleural MM, and therefore require further investigations into their carcinogenicity.

Targeted polyethylene glycol gold nanoparticles for the treatment of pancreatic cancer: from synthesis to proof-of-concept in vitro studies.

The main objective of this study was to optimize and characterize a drug delivery carrier for doxorubicin, intended to be intravenously administered, capable of improving the therapeutic index of the chemotherapeutic agent itself, and aimed at the treatment of pancreatic cancer. In light of this goal, we report a robust one-step method for the synthesis of dicarboxylic acid-terminated polyethylene glycol (PEG)-gold nanoparticles (AuNPs) and doxorubicin-loaded PEG-AuNPs, and their further antibody targeting (anti-Kv11.1 polyclonal antibody [pAb]). In in vitro proof-of-concept studies, we evaluated the influence of the nanocarrier and of the active targeting functionality on the anti-tumor efficacy of doxorubicin, with respect to its half-maximal effective concentration (EC50) and drug-triggered changes in the cell cycle. Our results demonstrated that the therapeutic efficacy of doxorubicin was positively influenced not only by the active targeting exploited through anti-Kv11.1-pAb but also by the drug coupling with a nanometer-sized delivery system, which indeed resulted in a 30-fold decrease of doxorubicin EC50, cell cycle blockage, and drug localization in the cell nuclei. The cell internalization pathway was strongly influenced by the active targeting of the Kv11.1 subunit of the human Ether-à-go-go related gene 1 (hERG1) channel aberrantly expressed on the membrane of pancreatic cancer cells. Targeted PEG-AuNPs were translocated into the lysosomes and were associated to an increased lysosomal function in PANC-1 cells. Additionally, doxorubicin release into an aqueous environment was almost negligible after 7 days, suggesting that drug release from PEG-AuNPs was triggered by enzymatic activity. Although preliminary, data gathered from this study have considerable potential in the application of safe-by-design nano-enabled drug-delivery systems (ie, nanomedicines) for the treatment of pancreatic cancer, a disease with a poor prognosis and one of the main current burdens of today's health care bill of industrialized countries.

Pharmacokinetics and bio-distribution of novel super paramagnetic iron oxide nanoparticles (SPIONs) in the anaesthetized pig.

Manufactured nanomaterials have a variety of medical applications, including diagnosis and targeted treatment of cancer. A series of experiments were conducted to determine the pharmacokinetic, biodistribution and biocompatibility of two novel magnetic nanoparticles (MNPs) in the anaesthetized pig. Dimercaptosuccinic acid (DMSA) coated superparamagnetic iron oxide nanoparticles (MF66-labelled 12 nm, core nominal diameter and OD15 15 nm); at 0.5, or 2.0 mg/kg) were injected intravenously. Particles induced a dose-dependent decrease in blood pressure following administration which recovered to control levels several minutes after injection. Blood samples were collected for a 5-h period and stored for determination of particle concentration using particle electron paramagnetic resonance (pEPR). Organs were harvested post-mortem for magnetic resonance imaging (MRI at 1.5 T field strength) and histology. OD15 (2.0 mg/kg) MNP had a plasma half-life of approximately 15 min. Both doses of the MF66 (0.5 and 2.0 mg/kg) MNP were below detection limits. MNP accumulation was observed primarily in the liver and spleen with MRI scans which was confirmed by histology. MRI also showed that both MNPs were present in the lungs. The results show that further modifications may be required to improve the biocompatibility of these particles for use as diagnostic and therapeutic agents.

Magnetic Nanoparticles in Cancer Theranostics.

In a report from 2008, The International Agency for Research on Cancer predicted a tripled cancer incidence from 1975, projecting a possible 13-17 million cancer deaths worldwide by 2030. While new treatments are evolving and reaching approval for different cancer types, the main prevention of cancer mortality is through early diagnosis, detection and treatment of malignant cell growth. The last decades have seen a development of new imaging techniques now in widespread clinical use. The development of nano-imaging through fluorescent imaging and magnetic resonance imaging (MRI) has the potential to detect and diagnose cancer at an earlier stage than with current imaging methods. The characteristic properties of nanoparticles result in their theranostic potential allowing for simultaneous detection of and treatment of the disease. This review provides state of the art of the nanotechnological applications for cancer therapy. Furthermore, it advances a novel concept of personalized nanomedical theranostic therapy using iron oxide magnetic nanoparticles in conjunction with MRI imaging. Regulatory and industrial perspectives are also included to outline future perspectives in nanotechnological cancer research.