Prediction of protein corona on nanomaterials by machine learning using novel descriptors

Engineered nanomaterials (ENMs) have been widely used in various fields due to their novel physicochemical properties. However, the use of ENMs has led to an increased exposure in humans, and the safety of ENMs has attracted much attention. It is universally acknowledged that ENMs could enter the human body via different routes, eg, inhalation, skin contact, and intravenous injection. Studies have proven that ENMs can cross or bypass the blood–brain barrier and then access the central nervous system and cause neurotoxicity. Until now, diverse in vivo and in vitro models have been developed to evaluate the neurotoxicity of ENMs, and oxidative stress, inflammation, DNA damage, and cell death have been identified as being involved. However, due to various physicochemical properties of ENMs and diverse study models in existing studies, it remains challenging to establish the structure-activity relationship of nanomaterials in neurotoxicity. In this paper, we aimed to review current studies on ENM-induced neurotoxicity, with an emphasis on the molecular and cellular mechanisms involved. We hope to provide a rational material design strategy for ENMs when they are applied in biomedical or other engineering applications.

In October 2010 the National Organic Standards Board recommended that engineered nanomaterials (ENMs) be prohibited from food products bearing the U.S. Department of Agriculture’s coveted Organic label.1 If the department adopts the recommendation, ENMs will find themselves in the same officially taboo category as genetically modified organisms when it comes to organic foods—nanotechnology-enabled innovations like flavor- and texture- enhancing ingredients and shelf life– extending packaging will be off the menu. Prior to issuing its recommendation, the board received thousands of public comments and petition signatures supporting the ban and virtually none opposing it. Although an official decision could take years, supporters are confident the recommendation will be adopted, and it will go down as one of the first lines drawn in the sand when it comes to the reach of this relatively new and potentially transformative technology in the American marketplace. Nanotechnology-enabled products are quietly proliferating on U.S. store shelves, despite nagging questions about the safety of synthetic nanoparticles and the products that contain them. “[I]n our regulation of food and most consumer products, we don’t implement the precautionary principle. Things go to market before we know whether or not they’re really safe for human beings over the long term,” says Alexis Baden-Mayer, a lawyer with the Organic Consumers Association, an advocacy group, who attended the meeting and campaigned for the ban. Baden-Mayer and other observers perceive a distinct lack of public awareness about how common ENMs are becoming in the market-place, and she hopes discussion among consumers of organic products will help change that. “Consumers don’t know much about nanotechnology, and the first time they may hear about it is now when they learn that the organic regulations are going to prohibit [it],” she says. The International Organization for Standardization defines a nanomaterial as a material with any external dimension between 1 and 100 nm.2 (By comparison, a double strand of DNA is about 2 nm thick.) Nanoparticles, which have been the focus of most nanotoxicology studies to date,3 are one subset of nanomaterials. Nanoparticles include structures of various shapes, such as nanotubes, nanowires, quantum dots, and fullerenes. They also occur naturally in substances like air, smoke, and sea spray, and “incidental” nanoparticles are created during processes such as combustion and food milling, churning, freezing, and homogenization. (Naturally occurring and incidental nanoparticles were not included in the National Organic Standards Board’s recommendation to ban ENMs.) Nanotechnology—the deliberate synthesis and manipulation of nanomaterials—began in the 1980s. Today thousands of ENMs are manufactured in a kaleidoscope of substances, shapes, and sizes for use in a wide range of products and industrial processes that take advantage of their novel physical, thermal, optical, and biological properties. These properties may be determined by the ENM’s chemical composition, size or shape, crystal structure, solubility, adhesion (the force that holds the nanoparticle components together), or surface chemistry, charge, or area.3 Industry analysts have been forecasting “game-changing” advances as a result of nanotechnology in renewable energy, computers, communications, pollution cleanup, agriculture, medicine, and more.4 Clothing, sunscreens, cosmetics, sporting equipment, batteries, food packaging, dietary supplements, and electronics are just a few of the types of nanotechnology-enabled goods in use by U.S. consumers. But safety questions arise around the nanoparticles in some of these products. The novel biological and physical properties of some ENMs pose unique challenges to comprehensive safety research, and investigators are working to figure out just how hazardous they might be to people, wildlife, and the environment. Compared with larger particles, nanoparticles’ tiny size means tissues may take them up more readily. It also can give them an unusual ability to travel throughout the body, including into cells and cell nuclei, and across the placenta and the blood–brain barrier, as demonstrated in rodent studies.5,6 No cases of human illness or death have been definitively attributed to ENMs. However, a number of researchers and consumer and environmental advocates have warned that the abundant unknowns make it necessary to proceed with caution lest we repeat the history of asbestos, polychlorinated biphenyls, the insecticide DDT, and other innovations that seemed valuable when they were introduced, proceeded with little oversight, and ultimately caused major health or environmental problems.

Interest in the use of engineered nanomaterials (ENMs) as either nanomedicines or dental materials/devices in clinical dentistry is growing. This review aims to detail the ultrafine structure, chemical composition, and reactivity of dental tissues in the context of interactions with ENMs, including the saliva, pellicle layer, and oral biofilm; then describes the applications of ENMs in dentistry in context with beneficial clinical outcomes versus potential risks. The flow rate and quality of saliva are likely to influence the behavior of ENMs in the oral cavity, but how the protein corona formed on the ENMs will alter bioavailability, or interact with the structure and proteins of the pellicle layer, as well as microbes in the biofilm, remains unclear. The tooth enamel is a dense crystalline structure that is likely to act as a barrier to ENM penetration, but underlying dentinal tubules are not. Consequently, ENMs may be used to strengthen dentine or regenerate pulp tissue. ENMs have dental applications as antibacterials for infection control, as nanofillers to improve the mechanical and bioactive properties of restoration materials, and as novel coatings on dental implants. Dentifrices and some related personal care products are already available for oral health applications. Overall, the clinical benefits generally outweigh the hazards of using ENMs in the oral cavity, and the latter should not prevent the responsible innovation of nanotechnology in dentistry. However, the clinical safety regulations for dental materials have not been specifically updated for ENMs, and some guidance on occupational health for practitioners is also needed. Knowledge gaps for future research include the formation of protein corona in the oral cavity, ENM diffusion through clinically relevant biofilms, and mechanistic investigations on how ENMs strengthen the tooth structure.

Among beneficial applications of nanotechnology, nanomedicine offers perhaps the greatest potential for improving human conditions and quality of life. Engineered nanomaterials (ENMs), with their unique properties, have potential to improve therapy of many human disorders. The properties that make ENMs so useful could also lead to unintentional adverse health effects. Challenges arising from physicochemical properties of ENMs, their characterization, exposure, and hazard assessment and other key issues of ENM safety are discussed. There is still scant knowledge about ENM cellular uptake, transport across biological barriers, distribution within the body, and possible mechanisms of toxicity. The safety of ENMs should be tested to minimize possible risk before the application. However, existing toxicity tests need to be adapted to fit to the unique features related to the nanosized material and appropriate controls and reference material should be considered.

Commentary: Revisiting nanoparticle-assay interference: There's plenty of room at the bottom for misinterpretation.

Approaches to the safety assessment of engineered nanomaterials (ENM) in food

Characterizing risk assessments for the development of occupational exposure limits for engineered nanomaterials

Chapter 1 - Interactions with the Human Body

The production of engineered nanomaterials (ENMs) is a scientific breakthrough in material design and the development of new consumer products. While the successful implementation of nanotechnology is important for the growth of the global economy, we also need to consider the possible environmental health and safety (EHS) impact as a result of the novel physicochemical properties that could generate hazardous biological outcomes. In order to assess ENM hazard, reliable and reproducible screening approaches are needed to test the basic materials as well as nanoenabled products. A platform is required to investigate the potentially endless number of biophysicochemical interactions at the nano/bio interface, in response to which we have developed a predictive toxicological approach. We define a predictive toxicological approach as the use of mechanisms-based high-throughput screening in vitro to make predictions about the physicochemical properties of ENMs that may lead to the generation of pathology or disease outcomes in vivo. The in vivo results are used to validate and improve the in vitro high-throughput screening (HTS) and to establish structure-activity relationships (SARs) that allow hazard ranking and modeling by an appropriate combination of in vitro and in vivo testing. This notion is in agreement with the landmark 2007 report from the US National Academy of Sciences, "Toxicity Testing in the 21st Century: A Vision and a Strategy" (http://www.nap.edu/catalog.php?record_id=11970), which advocates increased efficiency of toxicity testing by transitioning from qualitative, descriptive animal testing to quantitative, mechanistic, and pathway-based toxicity testing in human cells or cell lines using high-throughput approaches. Accordingly, we have implemented HTS approaches to screen compositional and combinatorial ENM libraries to develop hazard ranking and structure-activity relationships that can be used for predicting in vivo injury outcomes. This predictive approach allows the bulk of the screening analysis and high-volume data generation to be carried out in vitro, following which limited, but critical, validation studies are carried out in animals or whole organisms. Risk reduction in the exposed human or environmental populations can then focus on limiting or avoiding exposures that trigger these toxicological responses as well as implementing safer design of potentially hazardous ENMs. In this Account, we review the tools required for establishing predictive toxicology paradigms to assess inhalation and environmental toxicological scenarios through the use of compositional and combinatorial ENM libraries, mechanism-based HTS assays, hazard ranking, and development of nano-SARs. We will discuss the major injury paradigms that have emerged based on specific ENM properties, as well as describing the safer design of ZnO nanoparticles based on characterization of dissolution chemistry as a major predictor of toxicity.

With the development of nanotechnology, a growing number of people are expected to be exposed to its products, the engineered nanomaterials (ENMs). Some physico-chemical properties of ENMs, linked to their size in the nanoscale (1-100 nm), make them potentially more reactive, and therefore raise concern about possible adverse effects in humans. In this article, I discuss human diseases which may be predicted after exposure to ENMs, and how their pathogenetic mechanisms may be linked to exposure; in this regard, special emphasis has been given to the triad of oxidative stress/inflammation/genotoxicity and to the interaction of ENMs/proteins in different biological compartments. The analysis of possible adverse effects has been made on an organ-by-organ basis, starting from the skin, respiratory system and gastrointestinal tract. These sites are in fact not only those exposed to the highest amounts of ENMs, but are also the portals of entry to internal organs for possible systemic effects. Although the list and the relevance of possible human disorders linked to ENM exposure are at least as impressive as that of their direct or indirect beneficial effects for human health, we must be clear that ENM-linked diseases belong to the realm of possible risk (i.e. cannot be excluded, but are unlikely), whereas ENMs with proven beneficial effects are on the market. Therefore, the mandatory awareness about possible adverse effects of ENMs should in no way be interpreted as a motivation to disregard the great opportunity represented by nanotechnology.

Nanomaterials have attracted great attention for numerous applications in chemical, biological, and industrial world because of their fascinating physicochemical properties. In particular, understanding the mechanism of engineered nanomaterials (ENs) uptake by cells is important for various biomedical applications including for biosensors, imaging, intracellular drug and gene delivery, and toxicity studies (Han et al. 2007). Spontaneous penetration of functionalized cationic Au NPs has shown cell membrane disruption and cytotoxicity, thus limiting their utility (Chen et al. 2009). The objective of this work is to study and quantify the cellular uptake of three types of ENs (CNTs, Au nanoparticles (NPs), Silica and ZnO NPs) and their corresponding mechanism of action using immunofluorescence staining, ultrastructural characterization (HRTEM), Electrochemical Cell-substrate Impedance Sensing (ECIS) and bioanalytical analysis (ICP-OES). Another recent literature have indicated that Au NPs (5 nm diameter with “special” surface chemistries or arrangements) protected by an amphiphilic monolayer can non-disruptively penetrate cell membrane to deliver drugs, nutrients or biosensors (Verma et al. 2008). Although it seems that ENs can be taken up by cells, the evidence is disparate and the mechanism of uptake is either unclear or in their infancy. This is because of lack of accurate data on the physicochemical properties such as size including surface area, size distribution, chemical composition (purity crystallinity, electronic properties, etc.), solubility, shape and aggregation; and surface structure including surface reactivity, groups and protein corona and their relationship to cellular uptake. Of particular importance is the protein corona, which affects how nanoparticles are internalized by cells and cleared from the body. When NPs are exposed to biological fluid such as media in a cell culture, proteins can bind to the surface of the nanoparticle to form a protein corona. In this study, we first comprehensively characterized the starting EN dispersed in cell media (using DLS, BET, FTIR, Raman, XRD and HRSEM). Next, NPs were exposed to different cell types (A549, NIH 3T3 and PC12 cells) for either 1 or 2 days. The goal here is to address three questions – (a) Do ENs penetrate into cells and how does intracellular trafficking of ENs occur? (b) Does uptake of ENs vary with cell type? (c) Is uptake driven by physiochemical factors such as size, aggregation, shape, functionalization, corona, composition, dose and time? Figure 1 shows typical studies of cellular uptake of EN analyzed using VP-SEM, ICP-OES, immunofluorescence, and ECIS. Our initial evidence shows nanoparticle transport and uptake (see ICP-OES) and cell morphological disruptions. Furthermore, ECIS studies indicate the ability to longitudinally monitor impedance data of exposed cells over 5 days. In the paper, we will present detailed investigation on the influence of other EN physicochemical properties on cellular transport and uptake.

Nanotechnologies, engineered nanomaterials and occupational health and safety – A review

BackgroundRT-qPCR is routinely used in expression profiling of toxicity pathway genes. However, genetic and molecular level studies used to determine, understand and clarify potential risks of engineered nanomaterials (ENMs) are still incomplete. Concerns regarding possible interference caused by intracellular ENMs during analyses have been raised. The aim of this study was to verify a qPCR procedure for gene expression assays, which can be used in toxicity and exposure assessments.ResultsAmplification of ten reference genes was performed to test the expression stability. A preliminary study was performed on RNA from BEAS-2B cells that had been treated with AuNPs. Also, a reference total RNA standard from ten cell lines was spiked with various amounts of the same AuNP. This treatment mimics exposure assessment studies, where assay-interference may be caused by intracellular residual ENMs still being present in the biological samples (during and after isolation/purification procedures). Both types of RNA samples were reverse transcribed and then amplified by qPCR. The qPCR-related software and statistical programs used included BestKeeper, NormFinder, REST and qBase+. These results proved that using standard qPCR analysis and statistical programs should not be the only procedure applied to verify the assay for gene expression assessment related to ENMs. A comparison of SYBR Green to EVA Green was discussed, in addition to a comparison to the latest reports regarding the influence of ENM thermal conductivity, surface interactions with ENMs, effects of ENM size and charge, as well as, the limit of detection in a qPCR assay.ConclusionsAuNPs have the potential to interfere with the assay mechanism of RT-qPCR, thus, assay verification is required for AuNP-related gene expression studies used to evaluate toxicity. It is recommended to use HSP90 and YWHAZ as reference genes, i.e. these were the most stable in our study, irrespective of the source of the RNA, or, the point at which the AuNPs interacted with the assay. This report describes steps that can be utilised to generate a suitable method for gene expression studies associated with toxicity testing of various ENMs. For example, RNA standards that have been spiked with known amounts of ENMs should be run in conjunction with the unknown samples, in order to verify any RT-qPCR assay and determine the degree of error.

Engineered nanomaterials (ENMs) are materials with at least one dimension between 1–100 nm. The small size of ENMs results in a large surface area to volume ratio, giving ENMs novel characteristics that are not traditionally exhibited by larger bulk materials. Coupled with large surface area is an enormous capacity for surface functionalization of ENMs, e.g., with different ligands or surface changes, leading to an almost infinite array of variability of ENMs. Here we explore the effects of various shaped (spheres, rods) and charged (negative, positive) gold ENMs on Daphnia magna (D. magna) in terms of survival, ENM uptake and production of reactive oxygen species (ROS), a key factor in oxidative stress responses. We also investigate the effects of gold ENMs binding to the carapace of D. magna and how this may induce moulting inhibition in addition to toxicity and stress. The findings suggest that ENM shape and surface charge play an important role in determining ENM uptake and toxicity.

Over the last decade or so, one question about engineered nanomaterials (ENMs) has been constantly asked: Are nanomaterials inherently toxic? It is because characteristics such as “nano” scale size, surface charge, surface plasmon resonance, greater surface area, and propensity to ligand with (in)organic and/or polymeric molecules set ENMs physicochemically apart from their bulk/parent analogs. Related to unique properties, which enable greater functionality in a wide range of consumer applications, is the uncertainty about whether unique risk is posed to the environment, health, and safety (EHS) as ENMs are anthropogenically released into the environment. Recognized as the major sinks, soil, water, and air contamination of ENMs, including their leachable or modified by-products, is inevitable. Understanding of potential impacts on terrestrial plant species has remained unclear as anomalies in morphological, anatomical, and physiological endpoints, which have potential for impairing later development in life, are not routinely screened for, however. In this chapter, we report valuable information synthesized via thorough literature review of the current understanding of potential implications of ENM release and exposure to plants via soil, water, and atmospheric deposition. In particular, we report potential fate, biouptake, site of translocation/associated mechanisms, in vivo transformation, and toxicity (germination rate, growth and development, anatomical and physiological anomalies, and yield) of metal-based ENMs. Additionally, potential mechanisms and factors influencing ENMs’ toxicity are explained. Such information is critical to direct future research aimed at uncovering better understanding of nanotoxicology in plants, and to determine whether risk to public health exists from exposure to ENMs through the dietary route.