Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Formal lithium fixation improves direct analysis of lipids in tissue by mass spectrometry.

Mass spectrometry imaging is a powerful method for imaging and in situ characterization of lipids in thin tissue sections. Structural elucidation of lipids is often achieved via collision induced dissociation, and lithium-lipid adducts have been widely reported as providing the most structurally informative fragment ions. We present a method for the incorporation of lithium salts into tissue imaging experiments via fixation of samples in formal lithium solutions. The method is suitable for preparation of single tissue sections, or as an immersion fixation method for whole tissue blocks or organs prior to sectioning. We compare lithium adduct detection and MALDI-MSI of murine brain from analysis of tissues prepared in different ways. Tissues prepared in formal solutions containing lithium or sodium salts before coating in matrix via air-spray deposition are compared with fresh samples coated in lithium-doped matrix preparations by either dry-coating or air-spray deposition. Sample preparation via fixation in formal lithium is shown to yield the highest quality images of lithium adducts, resulting in acquisition of more informative product ion spectra in MALDI MS/MS profiling and imaging experiments. Finally, the compatibility of formal lithium solutions with standard histological staining protocols (hemotoxylin and eosin, Van Giessen and Oil Red O) is demonstrated in a study of human liver tissue.

A Datasheet for the INSIGHT University Hospitals Birmingham Retinal Vein Occlusion Data Set.

Purpose: Retinal vein occlusion (RVO) is the second leading cause of visual loss due to retinal disease. Retinal vein occlusion increases the risk of cardiovascular mortality and the risk of stroke. This article describes the data contained within the INSIGHT eye health data set for RVO and cardiovascular disease. Design: Data set descriptor for routinely collected eye and systemic disease data. Participants: All people who had suffered an RVO aged ≥ 18 years old, attending the Ophthalmology Clinic at Queen Elizabeth Hospital, University Hospitals Birmingham (UHB) National Health Service (NHS) Trust were included. Methods: The INSIGHT Health Data Research Hub for Eye Health is an NHS-led ophthalmic bioresource. It provides researchers with safe access to anonymized routinely collected data from contributing NHS hospitals to advance research for patient benefit. This report describes the INSIGHT UHB RVO and major adverse cardiovascular events data set, a data set of ophthalmology and systemic data derived from the United Kingdom's largest acute care trust. Main Outcome Measures: This data set consists of routinely collected data from the hospital's electronic patient records. The data set primarily includes structured data (relating to their hospital eye care and any cardiovascular data held for the individual) and OCT ocular images. Further details regarding the available data points are available in the supplementary information. Results: At the time point of this analysis (September 30, 2022) the data set was composed of clinical data from 1521 patients, from Medisoft records inception. The data set includes 2196 occurrences of RVO affecting 2026 eyes, longitudinal eye follow-up clinical parameters, over 6217 eye-related procedures, and 982 encountered complications. The data set contains information on 2534 major adverse cardiovascular event occurrences, their subtype, number experienced per patient, and chronological relation to RVO event. Longitudinal follow-up data including laboratory results, regular medications, and all-cause mortality are also available within the data set. Conclusions: This data set descriptor article summarizes the data set contents, the process of its curation, and potential uses. The data set is available through the structured application process that ensures research studies are for patient benefit. Further information regarding the data repository and contact details can be found at https://www.insight.hdrhub.org/. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

A Datasheet for the INSIGHT Birmingham, Solihull, and Black Country Diabetic Retinopathy Screening Dataset.

Purpose: Diabetic retinopathy (DR) is the most common microvascular complication associated with diabetes mellitus (DM), affecting approximately 40% of this patient population. Early detection of DR is vital to ensure monitoring of disease progression and prompt sight saving treatments as required. This article describes the data contained within the INSIGHT Birmingham, Solihull, and Black Country Diabetic Retinopathy Dataset. Design: Dataset descriptor for routinely collected eye screening data. Participants: All diabetic patients aged 12 years and older, attending annual digital retinal photography-based screening within the Birmingham, Solihull, and Black Country Eye Screening Programme. Methods: The INSIGHT Health Data Research Hub for Eye Health is a National Health Service (NHS)-led ophthalmic bioresource that provides researchers with safe access to anonymized, routinely collected data from contributing NHS hospitals to advance research for patient benefit. This report describes the INSIGHT Birmingham, Solihull, and Black Country DR Screening Dataset, a dataset of anonymized images and linked screening data derived from the United Kingdom's largest regional DR screening program. Main Outcome Measures: This dataset consists of routinely collected data from the eye screening program. The data primarily include retinal photographs with the associated DR grading data. Additional data such as corresponding demographic details, information regarding patients' diabetic status, and visual acuity data are also available. Further details regarding available data points are available in the supplementary information, in addition to the INSIGHT webpage included below. Results: At the time point of this analysis (December 31, 2019), the dataset comprised 6 202 161 images from 246 180 patients, with a dataset inception date of January 1, 2007. The dataset includes 1 360 547 grading episodes between R0M0 and R3M1. Conclusions: This dataset descriptor article summarizes the content of the dataset, how it has been curated, and what its potential uses are. Data are available through a structured application process for research studies that support discovery, clinical evidence analyses, and innovation in artificial intelligence technologies for patient benefit. Further information regarding the data repository and contact details can be found at https://www.insight.hdrhub.org/. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1-100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5-100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

Mechanisms for cellular uptake of nanosized clinical MRI contrast agents.

Engineered Nanomaterials (NMs), such as Superparamagnetic Iron Oxide Nanoparticles (SPIONs), offer significant benefits in a wide range of applications, including cancer diagnostic and therapeutic strategies. However, the use of NMs in biomedicine raises safety concerns due to lack of knowledge on possible biological interactions and effects. The initial basis for using SPIONs as biomedical MRI contrast enhancement agents was the idea that they are selectively taken up by macrophage cells, and not by the surrounding cancer cells. To investigate this claim, we analyzed the uptake of SPIONs into well-established cancer cell models and benchmarked this against a common macrophage cell model. In combination with fluorescent labeling of compartments and siRNA silencing of various proteins involved in common endocytic pathways, the mechanisms of internalization of SPIONs in these cell types has been ascertained utilizing reflectance confocal microscopy. Caveolar mediated endocytosis and macropinocytosis are both implicated in SPION uptake into cancer cells, whereas in macrophage cells, a clathrin-dependant route appears to predominate. Colocalization studies confirmed the eventual fate of SPIONs as accumulation in the degradative lysosomes. Dissolution of the SPIONs within the lysosomal environment has also been determined, allowing a fuller understanding of the cellular interactions, uptake, trafficking and effects of SPIONs within a variety of cancer cells and macrophages. Overall, the behavior of SPIONS in non-phagocytotic cell lines is broadly similar to that in the specialist macrophage cells, although some differences in the uptake patterns are apparent.

Surface Chemistry-Dependent Evolution of the Nanomaterial Corona on TiO2 Nanomaterials Following Uptake and Sub-Cellular Localization.

Nanomaterial (NM) surface chemistry has an established and significant effect on interactions at the nano-bio interface, with important toxicological consequences for manufactured NMs, as well as potent effects on the pharmacokinetics and efficacy of nano-therapies. In this work, the effects of different surface modifications (PVP, Dispex AA4040, and Pluronic F127) on the uptake, cellular distribution, and degradation of titanium dioxide NMs (TiO2 NMs, ~10 nm core size) are assessed and correlated with the localization of fluorescently-labeled serum proteins forming their coronas. Imaging approaches with an increasing spatial resolution, including automated high throughput live cell imaging, correlative confocal fluorescence and reflectance microscopy, and dSTORM super-resolution microscopy, are used to explore the cellular fate of these NMs and their associated serum proteins. Uncoated TiO2 NMs demonstrate a rapid loss of corona proteins, while surface coating results in the retention of the corona signal after internalization for at least 24 h (varying with coating composition). Imaging with two-color super-resolution dSTORM revealed that the apparent TiO2 NM single agglomerates observed in diffraction-limited confocal microscopy are actually adjacent smaller agglomerates, and provides novel insights into the spatial arrangement of the initial and exchanged coronas adsorbed at the NM surfaces.

Current Application of Capillary Electrophoresis in Nanomaterial Characterisation and Its Potential to Characterise the Protein and Small Molecule Corona.

Due to the increasing use and production of nanomaterials (NMs), the ability to characterise their physical/chemical properties quickly and reliably has never been so important. Proper characterisation allows a thorough understanding of the material and its stability, and is critical to establishing dose-response curves to ascertain risks to human and environmental health. Traditionally, methods such as Transmission Electron Microscopy (TEM), Field Flow Fractionation (FFF) and Dynamic Light Scattering (DLS) have been favoured for size characterisation, due to their wide-availability and well-established protocols. Capillary Electrophoresis (CE) offers a faster and more cost-effective solution for complex dispersions including polydisperse or non-spherical NMs. CE has been used to rapidly separate NMs of varying sizes, shapes, surface modifications and compositions. This review will discuss the literature surrounding the CE separation techniques, detection and NM characteristics used for the analysis of a wide range of NMs. The potential of combining CE with mass spectrometry (CE-MS) will also be explored to further expand the characterisation of NMs, including the layer of biomolecules adsorbed to the surface of NMs in biological or environmental compartments, termed the acquired biomolecule corona. CE offers the opportunity to uncover new/poorly characterised low abundance and polar protein classes due to the high ionisation efficiency of CE-MS. Furthermore, the possibility of using CE-MS to characterise the poorly researched small molecule interactions within the NM corona is discussed.

Imaging In focus: Reflected light imaging: Techniques and applications.

Reflectance imaging is a broad term that describes the formation of images by the detection of illumination light that is back-scattered from reflective features within a sample. Reflectance imaging can be performed in a variety of different configurations, such as confocal, oblique angle illumination, structured illumination, interferometry and total internal reflectance, permitting a plethora of biomedical applications. Reflectance imaging has proven indispensable for critical investigations into the safety and understanding of biomedically and environmentally relevant nano-materials, an area of high priority and investment. The non-destructive in vivo imaging ability of reflectance techniques permits alternative diagnostic strategies that may eventually facilitate the eradication of some invasive biopsy procedures. Reflectance can also provide additional structural information and clarity necessary in fluorescent based in vivo studies. Near-coverslip interrogation techniques, such as reflectance interferometry and total internal reflection, have provided a label free means to investigate cell-surface contacts, cell motility and vesicle trafficking in vivo and in vitro. Other key advances include the ability to acquire superresolution reflectance images providing increased spatial resolution.

Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles.

The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions.

Protein Corona Modulates Uptake and Toxicity of Nanoceria via Clathrin-Mediated Endocytosis.

Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity.