A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

Non-Animal Strategies for Toxicity Assessment of Nanoscale Materials: Role of Adverse Outcome Pathways in the Selection of Endpoints.

Faster, cheaper, sensitive, and mechanisms-based animal alternatives are needed to address the safety assessment needs of the growing number of nanomaterials (NM) and their sophisticated property variants. Specifically, strategies that help identify and prioritize alternative schemes involving individual test models, toxicity endpoints, and assays for the assessment of adverse outcomes, as well as strategies that enable validation and refinement of these schemes for the regulatory acceptance are needed. In this review, two strategies 1) the current nanotoxicology literature review and 2) the adverse outcome pathways (AOPs) framework, a systematic process that allows the assembly of available mechanistic information concerning a toxicological response in a simple modular format, are presented. The review highlights 1) the most frequently assessed and reported ad hoc in vivo and in vitro toxicity measurements in the literature, 2) various AOPs of relevance to inhalation toxicity of NM that are presently under development, and 3) their applicability in identifying key events of toxicity for targeted in vitro assay development. Finally, using an existing AOP for lung fibrosis, the specific combinations of cell types, exposure and test systems, and assays that are experimentally supported and thus, can be used for assessing NM-induced lung fibrosis, are proposed.

Cause-and-Effect Analysis as a Tool To Improve the Reproducibility of Nanobioassays: Four Case Studies.

One of the challenges in using in vitro data to understand the potential risks of engineered nanomaterials (ENMs) is that results often differ or are even contradictory among studies. While it is recognized that numerous factors can influence results produced by nanobioassays, there has not yet been a consistently used conceptual framework to identify key sources of variability in these assays. In this paper, we use cause-and-effect analysis to systematically describe sources of variability in four key in vitro nanobioassays: the 2',7'-dichlorofluorescein assay, an enzyme-linked immunosorbent assay for measuring interleukin-8, a flow cytometry assay (Annexin V/propidium iodide), and the Comet assay. These assays measure end points that can occur in cells impacted by ENMs through oxidative stress, a principle mechanism for ENM toxicity. The results from this analysis identify control measurements to test for potential artifacts or biases that could occur during conduct of these assays with ENMs. Cause-and-effect analysis also reveals additional measurements that could be performed either in preliminary experiments or each time the assay is run to increase confidence in the assay results and their reproducibility within and among laboratories. The approach applied here with these four assays can be used to support the development of a broad range of nanobioassays.

Uptake of label-free graphene oxide by Caco-2 cells is dependent on the cell differentiation status.

BACKGROUND: Understanding the interaction of graphene-related materials (GRM) with human cells is a key to the assessment of their potential risks for human health. There is a knowledge gap regarding the potential uptake of GRM by human intestinal cells after unintended ingestion. Therefore the aim of our study was to investigate the interaction of label-free graphene oxide (GO) with the intestinal cell line Caco-2 in vitro and to shed light on the influence of the cell phenotype given by the differentiation status on cellular uptake behaviour. RESULTS: Internalisation of two label-free GOs with different lateral size and thickness by undifferentiated and differentiated Caco-2 cells was analysed by scanning electron microscopy and transmission electron microscopy. Semi-quantification of cells associated with GRM was performed by flow cytometry. Undifferentiated Caco-2 cells showed significant amounts of cell-associated GRM, whereas differentiated Caco-2 cells exhibited low adhesion of GO sheets. Transmission electron microscopy analysis revealed internalisation of both applied GO (small and large) by undifferentiated Caco-2 cells. Even large GO sheets with lateral dimensions up to 10 µm, were found internalised by undifferentiated cells, presumably by macropinocytosis. In contrast, no GO uptake could be found for differentiated Caco-2 cells exhibiting an enterocyte-like morphology with apical brush border. CONCLUSIONS: Our results show that the internalisation of GO is highly dependent on the cell differentiation status of human intestinal cells. During differentiation Caco-2 cells undergo intense phenotypic changes which lead to a dramatic decrease in GRM internalisation. The results support the hypothesis that the cell surface topography of differentiated Caco-2 cells given by the brush border leads to low adhesion of GO sheets and sterical hindrance for material uptake. In addition, the mechanical properties of GRM, especially flexibility of the sheets, seem to be an important factor for internalisation of large GO sheets by epithelial cells. Our results highlight the importance of the choice of the in vitro model to enable better in vitro-in vivo translation.

Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay.

An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications.

Transfer studies of polystyrene nanoparticles in the ex vivo human placenta perfusion model: key sources of artifacts.

Nanotechnology is a rapidly expanding and highly promising new technology with many different fields of application. Consequently, the investigation of engineered nanoparticles in biological systems is steadily increasing. Questions about the safety of such engineered nanoparticles are very important and the most critical subject with regard to the penetration of biological barriers allowing particle distribution throughout the human body. Such translocation studies are technically challenging and many issues have to be considered to obtain meaningful and comparable results. Here we report on the transfer of polystyrene nanoparticles across the human placenta using an ex vivo human placenta perfusion model. We provide an overview of several challenges that can potentially occur in any translocation study in relation to particle size distribution, functionalization and stability of labels. In conclusion, a careful assessment of nanoparticle properties in a physiologically relevant milieu is as challenging and important as the actual study of nanoparticle-cell interactions itself.

Toward the development of decision supporting tools that can be used for safe production and use of nanomaterials.

Although researchers have intentionally produced and used nanomaterials for more than a century, nanotechnology has made its mark in most areas of daily life in the past 20 years. Now thousands of products contain nanoparticles, nanofibers, or nanostructured parts. Because some chemical products have caused severe problems to human health and to the environment, we should consider the overall biological and toxicological effects of nanomaterials as we decide whether to use them in various products. We should also reflect on the mechanisms for making these decisions, which may greatly influence the development, production, and use of such products. The preselection of appropriate materials during the early product design state should allow industry and applied researchers to mitigate the risks of these new materials. However, currently the human and ecological risks of the applied nanomaterials during their life cycle are unknown. A large set of physicochemical characteristics can determine the potential human and environmental exposure to and hazards from nanomaterials. Thus, researchers will need many years to gather and analyze all the data to perform a comprehensive risk assessment for engineered nanomaterials and to develop a sound decision making process. The ideal risk assessment approach would include cost-effective screening processes to target resources toward the risks of greatest concern. The outcome of the risk assessment is only as good as the quality of the data used. Unfortunately, the actual review process of most journals that publish on nanotoxicology focuses on "mechanistic studies and results" rather than a toxicologically relevant outcome. For example, journals often do not include studies that show no effect as worthy of publication ("no-effect-studies" dilemma), which can lead to misleading interpretations of toxicological data for hazard identification. However, even with insufficient data sets, researchers can produce a preliminary comparable risk assessment ("approximate" risk assessment). Researchers have already performed risk-based evaluations of nanomaterials grounded on the comparison of exposure concentrations with no-effect levels (as required for chemical risk assessment), examining generic nanomaterials such as "nano-TiO2" but not specific forms or modifications. Even though these data sets on hazard and exposure are incomplete, they already provide the basis to illustrate the current state of knowledge and uncertainties. Therefore industry and applied researchers can calculate the probability that an adverse effect might occur and begin to balance the benefits and potential risks of an innovation. Based on the increasing numbers of nanotoxicology publications and funding programs, this Account reviews the decision support approaches that already exist to safely implement engineered nanomaterials during an early phase of innovation.

Nanosafety research--are we on the right track?

The number of studies that have been published on the topic of nanosafety speaks for itself. We have seen an almost exponential rise over the past 15 years or so in the number of articles on nanotoxicology. Although only a couple of hundred papers had appeared on the topic of "Nanomaterials: environmental and health effects" before 2000, this number has exploded to over 10 000 since 2001. Most of these studies, however, do not offer any kind of clear statement on the safety of nanomaterials. On the contrary, most of them are either self-contradictory or arrive at completely erroneous conclusions. Three years ago in this Journal we underscored the deficiencies in the way these studies were designed and pointed out the sources of error in the methods used. Now, on the basis of a comprehensive review of the literature and with the help of selected toxicological end points, we attempt to indicate where the significant weaknesses of these studies lie and what we must improve in the future.

Classification framework for graphene-based materials.

Graphing graphene: Because the naming of graphene-based materials (GBMs) has led to confusion and inconsistency, a classification approach is necessary. Three physical-chemical properties of GBMs have been defined by the GRAPHENE Flagship Project of the European Union for the unequivocal classification of these materials (see grid).

The DaNa projects: public communication of (nano)material safety data-from conspiracy theories to study quality.

In this perspective, the authors give their view on the developments and experiences on communicating on (nano)materials safety. We would like to share our experiences with the scientific community in order to make them useful for future communication activities. We present the long-term work of the science communication projects DaNa, DaNa2.0 and DaNa4.0, running from 2009 to 2023. Starting in the early 2000s with the beginnings of nanotechnology research, communication on the safety of nanomaterials with the public was still very new and faced the projects with many challenges. Today, science communication is indispensable for the dissemination of scientific findings and a fact-based approach like the DaNa "Knowledge Base Materials" creates a trustworthy dialogue with the public. This long-term project series has made a significant contribution to communication on the safety of nanomaterials, perhaps even the largest among publicly funded project series worldwide.

Comparability of in vitro tests for bioactive nanoparticles: a common assay to detect reactive oxygen species as an example.

The release of reactive oxygen species (ROS) during the electron transport of mitochondrial aerobic respiration is the major source of ROS. However, contact between cells and nanoparticles (NPs) can also induce release of ROS, leading to an imbalance towards the pro-oxidative state. At low levels of ROS production, cells initiate a protective response to guarantee their survival, but an excess of ROS can damage cellular compounds such as membranes and various organelles, or directly cause genotoxicity. Thus an elevated level of ROS is an important indicator of cellular stress and an accurate recording of this parameter would be very informative. ROS can be measured by various assays, but all known assays measuring and quantifying ROS possess certain weaknesses. The problems and challenges of quantitatively detecting ROS in vitro using the 2',7'-dichlorodihydrofluorescein (DCF) assay is discussed as an example. In addition, we debate the difficulties in finding a suitable and stable chemical reaction control for the DCF assay (or other ROS-detecting assays). As a conclusion, we believe that using 3-morpholinosydnonimine hydrochloride (Sin-1) as a ROS inducer in the DCF assay is feasible only qualitatively. However, a quantitative measurement of the absolute amount of ROS produced and a quantitative comparison between experiments is (at the moment) impossible.

Effects of subtoxic concentrations of TiO2 and ZnO nanoparticles on human lymphocytes, dendritic cells and exosome production.

Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investigate if commercially available TiO(2) and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO(2) or ZnO nanoparticles at concentrations from 1 to 100µg/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO(2) nanoparticles. Non-toxic exposure, 10µg/mL, to TiO(2) and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO(2) nanoparticles induced a down regulation of FcγRIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on FcγR-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cultures. TiO(2) or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO(2) and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions.

Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro.

BACKGROUND: Increasing concern has been expressed regarding the potential adverse health effects that may be associated with human exposure to inhaled multi-walled carbon nanotubes (MWCNTs). Thus it is imperative that an understanding as to the underlying mechanisms and the identification of the key factors involved in adverse effects are gained. In the alveoli, MWCNTs first interact with the pulmonary surfactant. At this interface, proteins and lipids of the pulmonary surfactant bind to MWCNTs, affecting their surface characteristics. Aim of the present study was to investigate if the pre-coating of MWCNTs with pulmonary surfactant has an influence on potential adverse effects, upon both (i) human monocyte derived macrophages (MDM) monocultures, and (ii) a sophisticated in vitro model of the human epithelial airway barrier. Both in vitro systems were exposed to MWCNTs either pre-coated with a porcine pulmonary surfactant (Curosurf) or not. The effect of MWCNTs surface charge was also investigated in terms of amino (-NH2) and carboxyl (-COOH) surface modifications. RESULTS: Pre-coating of MWCNTs with Curosurf affects their oxidative potential by increasing the reactive oxygen species levels and decreasing intracellular glutathione depletion in MDM as well as decreases the release of Tumour necrosis factor alpha (TNF-α). In addition, an induction of apoptosis was observed after exposure to Curosurf pre-coated MWCNTs. In triple cell-co cultures the release of Interleukin-8 (IL-8) was increased after exposure to Curosurf pre-coated MWCNTs. Effects of the MWCNTs functionalizations were minor in both MDM and triple cell co-cultures. CONCLUSIONS: The present study clearly indicates that the pre-coating of MWCNTs with pulmonary surfactant more than the functionalization of the tubes is a key factor in determining their ability to cause oxidative stress, cytokine/chemokine release and apoptosis. Thus the coating of nano-objects with pulmonary surfactant should be considered for future lung in vitro risk assessment studies.

A Systematic Review on the Hazard Assessment of Amorphous Silica Based on the Literature From 2013 to 2018.

Background: Nanomaterials are suspected of causing health problems, as published studies on nanotoxicology indicate. On the other hand, some of these materials, such as nanostructured pyrogenic and precipitated synthetic amorphous silica (SAS) and silica gel, have been used for decades without safety concerns in industrial, commercial, and consumer applications. However, in addition to many in vivo and in vitro studies that have failed to demonstrate the intrinsic toxicity of SAS, articles periodically emerge, in which biological effects of concern have been described. Even though most of these studies do not meet high-quality standards and do not always use equivalent test materials or standardized test systems, the results often trigger substance re-evaluation. To put the results into perspective, an extensive literature study was carried out and an example of amorphous silica will be used to try to unravel the reliability from the unreliable results. Methods: A systematic search of studies on nanotoxicological effects has been performed covering the years 2013 to 2018. The identified studies have been evaluated for their quality regarding material and method details, and the data have been curated and put into a data collection. This review deals only with investigations on amorphous silica. Results: Of 18,162 publications 1,217 have been selected with direct reference to experiments with synthetically produced amorphous silica materials. The assessment of these studies based on defined criteria leads to a further reduction to 316 studies, which have been included in this systematic review. Screening for quality with well-defined quantitative criteria following the GUIDE nano concept reveals only 27.3% has acceptable quality. Overall, the in vitro and in vivo data showed low or no toxicity of amorphous silica. The data shown do not support the hypothesis of dependency of biological effects on the primary particle size of the tested materials. Conclusion: This review demonstrates the relatively low quality of most studies published on nanotoxicological issues in the case of amorphous silica. Moreover, mechanistic studies are often passed off or considered toxicological studies. In general, standardized methods or the Organization for Economic Cooperation and Development (OECD) guidelines are rarely used for toxicological experiments. As a result, the significance of the published data is usually weak and must be reevaluated carefully before using them for regulatory purposes.

Collection of Controlled Nanosafety Data-The CoCoN-Database, a Tool to Assess Nanomaterial Hazard.

Hazard assessment is the first step in nanomaterial risk assessment. The overall number of studies on the biological effects of nanomaterials or innovative materials is steadily increasing and is above 40,000. Several databases have been established to make the amount of data manageable, but these are often highly specialized or can be used only by experts. This paper describes a new database which uses an already existing data collection of about 35,000 publications. The collection from the first phase between the years 2000 and 2013 contains about 11,000 articles and this number has been reduced by specific selection criteria. The resulting publications have been evaluated for their quality regarding the toxicological content and the experimental data have been extracted. In addition to material properties, the most important value to be extracted is the no-observed-adverse-effect-level (NOAEL) for in vivo and the no-observed-effect-concentration (NOEC) for in vitro studies. The correlation of the NOAEL/NOEC values with the nanomaterial properties and the investigated endpoints has been tested in projects such as the OECD-AOP project, where the available data for inflammatory responses have been analysed. In addition, special attention was paid to titanium dioxide particles and this example is used to show with searches for in vitro and in vivo experiments on possible lung toxicity what a typical result of a database query can look like. In this review, an emerging database is described that contains valuable information for nanomaterial hazard estimation and should aid in the progress of nanosafety research.

120 years of nanosilver history: implications for policy makers.

Nanosilver is one nanomaterial that is currently under a lot of scrutiny. Much of the discussion is based on the assumption that nanosilver is something new that has not been seen until recently and that the advances in nanotechnology opened completely new application areas for silver. However, we show in this analysis that nanosilver in the form of colloidal silver has been used for more than 100 years and has been registered as a biocidal material in the United States since 1954. Fifty-three percent of the EPA-registered biocidal silver products likely contain nanosilver. Most of these nanosilver applications are silver-impregnated water filters, algicides, and antimicrobial additives that do not claim to contain nanoparticles. Many human health standards for silver are based on an analysis of argyria occurrence (discoloration of the skin, a cosmetic condition) from the 1930s and include studies that considered nanosilver materials. The environmental standards on the other hand are based on ionic silver and may need to be re-evaluated based on recent findings that most silver in the environment, regardless of the original silver form, is present in the form of small clusters or nanoparticles. The implications of this analysis for policy of nanosilver is that it would be a mistake for regulators to ignore the accumulated knowledge of our scientific and regulatory heritage in a bid to declare nanosilver materials as new chemicals, with unknown properties and automatically harmful simply on the basis of a change in nomenclature to the term "nano".

Evidence maps: communicating risk assessments in societal controversies: the case of engineered nanoparticles.

The transparent and fair characterization of scientific evidence for reporting the results of a hazard assessment is a demanding task. In this article, we present an approach for characterizing evidence--the evidence map approach. The theoretical starting point is to view evidence characterization as a form of argumentation. Thus, evidence maps are designed to depict the evidence base, the pro and con arguments, and the remaining uncertainties, which together lead experts to their conclusions when summarizing and evaluating the scientific evidence about a potential hazard. To illustrate its use, the evidence maps approach is applied to characterizing the health-relevant effects of engineered nanoparticles. Empirical data from an online survey suggests that the use of evidence maps improves the reporting of hazard assessments. Nonexperts prefer to receive the information included in an evidence map in order to come to an informed judgment. Furthermore, the benefits and limitations of evidence maps are discussed in the light of recent literature on risk communication. Finally, the article underlines the need for further research in order to increase quality of evidence reporting.

Nanotoxicology: an interdisciplinary challenge.

The increasing consumption of products containing nanomaterials that can be currently observed and forecasts of new developments and applications fan the fear of individuals and organizations regarding new risks to health. Considering experiences gained from previous technology developments, such fears are not completely unfounded. But are they really justified? And is it justified, moreover, to speak of "nanotoxicology" as a new discipline? This Review seeks to cast light on the phenomena that may occur as nanoobjects interact with cells, tissues, and organisms. Furthermore, we will demonstrate that the many data made available on the biological effects of nanomaterials do not always come from studies that can be considered reliable. We will point out the aspect of reliability with specific examples from the literature and will not address specific (nano)materials. In particular, inadequate methods will be described together with recommendations how to avoid this in the future, thereby contributing to a sustainable improvement of the available data.

A methodology for developing key events to advance nanomaterial-relevant adverse outcome pathways to inform risk assessment.

Significant advances have been made in the development of Adverse Outcome Pathways (AOPs) over the last decade, mainly focused on the toxicity mechanisms of chemicals. These AOPs, although relevant to manufactured nanomaterials (MNs), do not currently capture the reported roles of size-associated properties of MNs on toxicity. Moreover, some AOs of relevance to airborne exposures to MNs such as lung inflammation and fibrosis shown in animal studies may not be targeted in routine regulatory decision making. The primary objective of the present study was to establish an approach to advance the development of AOPs of relevance to MNs using existing, publicly available, nanotoxicology literature. A systematic methodology was created for curating, organizing and applying the available literature for identifying key events (KEs). Using a case study approach, the study applied the available literature to build the biological plausibility for 'tissue injury', a KE of regulatory relevance to MNs. The results of the analysis reveal the various endpoints, assays and specific biological markers used for assessing and reporting tissue injury. The study elaborates on the limitations and opportunities of the current nanotoxicology literature and provides recommendations for the future reporting of nanotoxicology results that will expedite not only the development of AOPs for MNs but also aid in application of existing data for decision making.

The adsorption of biomolecules to multi-walled carbon nanotubes is influenced by both pulmonary surfactant lipids and surface chemistry.

BACKGROUND: During production and processing of multi-walled carbon nanotubes (MWCNTs), they may be inhaled and may enter the pulmonary circulation. It is essential that interactions with involved body fluids like the pulmonary surfactant, the blood and others are investigated, particularly as these interactions could lead to coating of the tubes and may affect their chemical and physical characteristics. The aim of this study was to characterize the possible coatings of different functionalized MWCNTs in a cell free environment. RESULTS: To simulate the first contact in the lung, the tubes were coated with pulmonary surfactant and subsequently bound lipids were characterized. The further coating in the blood circulation was simulated by incubating the tubes in blood plasma. MWCNTs were amino (NH2)- and carboxyl (-COOH)-modified, in order to investigate the influence on the bound lipid and protein patterns. It was shown that surfactant lipids bind unspecifically to different functionalized MWCNTs, in contrast to the blood plasma proteins which showed characteristic binding patterns. Patterns of bound surfactant lipids were altered after a subsequent incubation in blood plasma. In addition, it was found that bound plasma protein patterns were altered when MWCNTs were previously coated with pulmonary surfactant. CONCLUSIONS: A pulmonary surfactant coating and the functionalization of MWCNTs have both the potential to alter the MWCNTs blood plasma protein coating and to determine their properties and behaviour in biological systems.