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Abstract Background During inhalation, airborne particles such as particulate matter ≤ 2.5 μm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 μm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 μm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL− 1 ( 0 to 44 µg.cm− 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. Results We could detect the translocated fraction of DEPs across the PET membranes with 3 μm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL− 1) and 75% by LIT (LOD: 0.20 µg.cm− 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL− 1 (11 and 22 µg.cm− 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. Conclusion We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.

Nanomaterials hold immense potential for numerous applications in energy, health care, and environmental sectors, playing an important role in our daily lives. Their utilization spans from improving energy efficiency to enhancing medical diagnostics, and mitigating environmental pollution, thus presenting a multifaceted approach towards achieving sustainability goals. To ensure the sustainable and safe utilization of nanomaterials, a thorough evaluation of potential hazards and risks is essential throughout their lifecycle—from resource extraction and production to use and disposal. In this review, we focus on understanding and addressing potential environmental and health risks associated with nanomaterial utilization. We advocate for a balanced approach with early hazard identification, safe-by-design principles, and life cycle assessments, while emphasizing safe handling and disposal practices, collaboration, and continuous improvement. Our goal is to ensure responsible nanotechnology development, fostering innovation alongside environmental and community well-being, through a holistic approach integrating science, ethics, and proactive risk assessment.

Antimony-doped tin oxide nanoparticles (ATO NPs) have emerged as a promising tool in biomedical applications, namely robust photothermal effects upon near-infrared (NIR) light exposure, enabling controlled thermal dynamics to induce spatial cell death. This study investigated the interplay between ATO NPs and macrophages, understanding cellular uptake and cytokine release. ATO NPs demonstrated biocompatibility with no impact on macrophage viability and cytokine secretion. These findings highlight the potential of ATO NPs for inducing targeted cell death in cancer treatments, leveraging their feasibility, unique NIR properties, and safe interactions with immune cells. ATO NPs offer a transformative platform with significant potential for future biomedical applications by combining photothermal capabilities and biocompatibility.

Introducing particles as additives, specifically engineered nanoparticles, in the food industry has improved food properties. Since 2014, alongside the presence of these added particles, there has been a mandatory requirement to disclose if those additives are nanomaterials in the ingredient list of food products. However, detecting and characterizing nanomaterials is time-consuming due to their small sizes, low concentrations, and diverse food matrices.We present a streamlined analytical process to detect the presence of silica and titania particles in food, applicable for food regulation and control. Using X-ray Fluorescence Spectrometry for screening enables quick categorization of inorganic particles labeling accuracy, distinguishing products with and without them. For the former, we develop matrix-independent digestion and introduce time-effective statistics to evaluate the median particle size using a reduced number of particles counted, ensuring accurate “nano” labeling.Through the implementation of this work, our objective is to simplify and facilitate verifying the proper labeling of food products.

Abstract Background Toxicity assessment for regulatory purposes is starting to move away from traditional in vivo methods and towards new approach methodologies (NAM) such as high-throughput in vitro models and computational tools. For materials with limited hazard information, utilising quantitative Adverse Outcome Pathways (AOPs) in a testing strategy involving NAM can produce information relevant for risk assessment. The aim of this work was to determine the feasibility of linking in vitro endpoints to in vivo events, and moreover to key events associated with the onset of a chosen adverse outcome to aid in the development of NAM testing strategies. To do this, we focussed on the adverse outcome pathway (AOP) relating to the onset of pulmonary fibrosis. Results We extracted in vivo and in vitro dose–response information for particles known to induce this pulmonary fibrosis (crystalline silica, specifically α-quartz). To test the in vivo–in vitro extrapolation (IVIVE) determined for crystalline silica, cerium dioxide nanoparticles (nano-CeO2) were used as a case study allowing us to evaluate our findings with a less studied substance. The IVIVE methodology outlined in this paper is formed of five steps, which can be more generally summarised into two categories (i) aligning the in vivo and in vitro dosimetry, (ii) comparing the dose–response curves and derivation of conversion factors. Conclusion Our analysis shows promising results with regards to correlation of in vitro cytokine secretion to in vivo acute pulmonary inflammation assessed by polymorphonuclear leukocyte influx, most notable is the potential of using IL-6 and IL-1β cytokine secretion from simple in vitro submerged models as a screening tool to assess the likelihood of lung inflammation at an early stage in product development, hence allowing a more targeted investigation using either a smaller, more targeted in vivo study or in the future a more complex in vitro protocol. This paper also highlights the strengths and limitations as well as the current difficulties in performing IVIVE assessment and suggestions for overcoming these issues.

Abstract Slime expelled by velvet worms entraps prey insects within seconds in a hardened biopolymer network that matches the mechanical strength of industrial polymers. While the mechanic stimuli-responsive nature and building blocks of the polymerization are known, it is still unclear how the velvet worms’ slime hardens so fast. Here, we investigated the slime for the first time, not only after, but also before expulsion. Further, we investigated the slime’s micro- and nanostructures in-depth. Besides the previously reported protein nanoglobules, carbohydrates, and lipids, we discovered abundant encapsulated phosphate and carbonate salts. We also detected CO2 bubbles during the hardening of the slime. These findings, along with further observations, suggest that the encapsulated salts in expelled slime rapidly dissolve and neutralize in a baking-powder-like reaction, which seems to accelerate the drying of the slime. The proteins’ conformation and aggregation are thus influenced by shear stress and the salts’ neutralization reaction, increasing the slime’s pH and ionic strength. These insights into the drying process of the velvet worm’s slime demonstrate how naturally evolved polymerizations can unwind in seconds, and could inspire new polymers that are stimuli-responsive or fast-drying under ambient conditions.

Abstract Background In the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type. Methods Here we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes. Results We evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson’s and Manders’), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson’s, but also Manders’ correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up. Conclusions The results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes. Graphical Abstract

Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs.Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC.Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC.Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.

The complex interaction between tumor-associated macrophages (TAMs) and tumor cells through soluble factors provides essential cues for breast cancer progression. TAMs-targeted therapies have shown promising clinical therapeutical potential against cancer progression. The molecular mechanisms underlying the response to TAMs-targeted therapies depends on complex dynamics of immune cross-talk and its understanding is still incomplete. In vitro models are helpful to decipher complex responses to combined immunotherapies. In this study, we established and characterized a 3D human macrophage-ER+ PR+ HER2+ breast cancer model, referred to as macrophage-tumor spheroid (MTS). Macrophages integrated within the MTS had a mixed M2/M1 phenotype, abrogated the anti-proliferative effect of trastuzumab on tumor cells, and responded to IFNγ with increased M1-like polarization. The targeted treatment of MTS with a combined CSF1R kinase inhibitor and an activating anti-CD40 antibody increased M2 over M1 phenotype (CD163+/CD86+ and CD206+/CD86+ ratio) in time, abrogated G2/M cell cycle phase transition of cancer cells, promoted the secretion of TNF-α and reduced cancer cell viability. In comparison, combined treatment in a 2D macrophage-cancer cell co-culture model reduced M2 over M1 phenotype and decreased cancer cell viability. Our work shows that this MTS model is responsive to TAMs-targeted therapies, and may be used to study the response of ER+ PR+ HER2+ breast cancer lines to novel TAM-targeting therapies.

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell–nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell–substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell–matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment.

The study of plastic particles, particularly those in the micro-, sub-micro-, and nano-size ranges, within food and beverages has gained increasing interest within recent years. However, many analytical techniques have limits of detection which hinder their use for the study of these particles in these sample matrices. In addition, remaining contaminants from the matrices can interfere with the signals from plastic particles. Thus, great care must be given to sample preparation and data interpretation to ensure accurate results. This study proposes the use of sample purification through chemical digestion protocols to facilitate the study of plastic particles present in tea samples, and serves to highlight technical limitations which must be overcome in future studies.

The understanding of the engineered nanomaterials (NMs) potential interaction with tissue barriers is important to predict their accumulation in cells. Herein, the fate, e.g., cellular uptake/adsorption at the cell membrane and translocation, of NMs with different physico-chemical properties across an A549 lung epithelial tissue barrier, cultured on permeable transwell inserts, were evaluated. We assessed the fate of five different NMs, known to be partially soluble, bio-persistent passive and bio-persistent active. Single exposure measurements using 100 µg/ml were performed for barium sulfate (BaSO4), cerium dioxide (CeO2), titanium dioxide (TiO2), and zinc oxide (ZnO) NMs and non-nanosized crystalline silica (DQ12). Elemental distribution of the materials in different compartments was measured after 24 and 80 h, e.g., apical, apical wash, intracellular and basal, using inductively coupled plasma optical emission spectrometry. BaSO4, CeO2, and TiO2 were mainly detected in the apical and apical wash fraction, whereas for ZnO a significant fraction was detected in the basal compartment. For DQ12 the major fraction was found intracellularly. The content in the cellular fraction decreased from 24 to 80 h incubation for all materials. Repeated exposure measurements were performed exposing the cells on four subsequent days to 25 µg/ml. After 80 h BaSO4, CeO2, and TiO2 NMs were again mainly detected in the apical fraction, ZnO NMs in the apical and basal fraction, while for DQ12 a significant concentration was measured in the cell fraction. Interestingly the cellular fraction was in a similar range for both exposure scenarios with one exception, i.e., ZnO NMs, suggesting a potential different behavior for this material under single exposure and repeated exposure conditions. However, we observed for all the NMs, a decrease of the amount detected in the cellular fraction within time, indicating NMs loss by cell division, exocytosis and/or possible dissolution in lysosomes. Overall, the distribution of NMs in the compartments investigated depends on their composition, as for inert and stable NMs the major fraction was detected in the apical and apical wash fraction, whereas for partially soluble NMs apical and basal fractions were almost similar and DQ12 could mainly be found in the cellular fraction.

The gastrointestinal tract is the main target of orally ingested nanoparticles (NPs) and at the same time is exposed to noxious substances, such as bacterial components. We investigated the interaction of 59 nm silica (SiO2) NPs with differentiated Caco-2 intestinal epithelial cells in the presence of cholera toxin subunit B (CTxB) and compared the effects to J774A.1 macrophages. CTxB can affect cellular functions and modulate endocytosis via binding to the monosialoganglioside (GM1) receptor, expressed on both cell lines. After stimulating macrophages with CTxB, we observed notable changes in the membrane structure but not in Caco-2 cells, and no secretion of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) was detected. Cells were then exposed to 59 nm SiO2 NPs and CtxB sequentially and simultaneously, resulting in a high NP uptake in J774A.1 cells, but no uptake in Caco-2 cells was detected. Flow cytometry analysis revealed that the exposure of J774A.1 cells to CTxB resulted in a significant reduction in the uptake of SiO2 NPs. In contrast, the uptake of NPs by highly selective Caco-2 cells remained unaffected following CTxB exposure. Based on colocalization studies, CTxB and NPs might enter cells via shared endocytic pathways, followed by their sorting into different intracellular compartments. Our findings provide new insights into CTxB’s function of modulating SiO2 NP uptake in phagocytic but not in differentiated intestine cells.

Abstract Background Air pollution is killing close to 5 million people a year, and harming billions more. Air pollution levels remain extremely high in many parts of the world, and air pollution-associated premature deaths have been reported for urbanized areas, particularly linked to the presence of airborne nano-sized and ultrafine particles. Main text To date, most of the research studies did focus on the adverse effects of air pollution on the human cardiovascular and respiratory systems. Although the skin is in direct contact with air pollutants, their damaging effects on the skin are still under investigation. Epidemiological data suggested a correlation between exposure to air pollutants and aggravation of symptoms of chronic immunological skin diseases. In this study, a systematic literature review was conducted to understand the current knowledge on the effects of airborne particulate matter on human skin. It aims at providing a deeper understanding of the interactions between air pollutants and skin to further assess their potential risks for human health. Conclusion Particulate matter was shown to induce a skin barrier dysfunction and provoke the formation of reactive oxygen species through direct and indirect mechanisms, leading to oxidative stress and induced activation of the inflammatory cascade in human skin. Moreover, a positive correlation was reported between extrinsic aging and atopic eczema relative risk with increasing particulate matter exposure.

Commercial static cell culture substrates can usually not change their physical properties over time, resulting in a limited representation of the variation in biomechanical cues in vivo. To overcome this limitation, approaches incorporating gold nanoparticles to act as transducers to external stimuli have been employed. In this work, gold nanorods were embedded in an elastomeric matrix and used as photothermal transducers to fabricate biocompatible light-responsive substrates. The nanocomposite films analysed by lock-in thermography and nanoindentation show a homogeneous heat distribution and a greater stiffness when irradiated with NIR light. After irradiation, the initial stiffness values were recovered. In vitro experiments performed during NIR irradiation with NIH-3T3 fibroblasts demonstrated that these films were biocompatible and cells remained viable. Cells cultured on the light stiffened nanocomposite exhibited a greater proliferation rate and stronger focal adhesion clustering, indicating increased cell-surface binding strength.

Oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) are short synthetic nucleic acid molecules with a promising potential to treat a wide range of diseases. Despite considerable progress in the field, the development of safe and effective delivery systems that target organs and tissues other than the liver is challenging. While keeping possible off-target oligonucleotide interactions and toxicity related to chemical modifications in mind, innovative solutions for targeted intracellular delivery are highly needed. Herein, we report on the design, synthesis and testing of a novel multi-modified and multi-functionalized heteroduplex oligonucleotide (HDO) with respect to its intracellular delivery and its ability to silence genes in HeLa cells. Simultaneously, folic acid- and peptide- labeled HDO show proficient silencing of the green fluorescent protein (GFP) gene with an 84% reduction in the GFP fluorescence. In addition, the Bcl2 HDO achieved effective Bcl2 gene knockdown in the cells. The data show the proficiency of the multi-functionalization strategy and provide an example for advancing the design of safe and efficient forthcoming oligonucleotide therapeutics, such as HDO.

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.

A large number of prevalent lung diseases is associated with tissue inflammation. Clinically, corticosteroid therapies are applied systemically or via inhalation for the treatment of lung inflammation, and a number of novel therapies are being developed that require preclinical testing. In alveoli, macrophages and dendritic cells play a key role in initiating and diminishing pro-inflammatory reactions and, in particular, macrophage plasticity (M1 and M2 phenotypes shifts) has been reported to play a significant role in these reactions. Thus far, no studies with in vitro lung epithelial models have tested the comparison between systemic and direct pulmonary drug delivery. Therefore, the aim of this study was to develop an inflamed human alveolar epithelium model and to test the resolution of LPS-induced inflammation in vitro with a corticosteroid, methylprednisolone (MP). A specific focus of the study was the macrophage phenotype shifts in response to these stimuli. First, human monocyte-derived macrophages were examined for phenotype shifts upon exposure to lipopolysaccharide (LPS), followed by treatment with MP. A multicellular human alveolar model, composed of macrophages, dendritic cells, and epithelial cells, was then employed for the development of inflamed models. The models were used to test the anti-inflammatory potency of MP by monitoring the secretion of pro-inflammatory mediators (interleukin [IL]-8, tumor necrosis factor-α [TNF-α], and IL-1β) through four different approaches, mimicking clinical scenarios of inflammation and treatment. In macrophage monocultures, LPS stimulation shifted the phenotype towards M1, as demonstrated by increased release of IL-8 and TNF-α and altered expression of phenotype-associated surface markers (CD86, CD206). MP treatment of inflamed macrophages reversed the phenotype towards M2. In multicellular models, increased pro-inflammatory reactions after LPS exposure were observed, as demonstrated by protein secretion and gene expression measurements. In all scenarios, among the tested mediators the most pronounced anti-inflammatory effect of MP was observed for IL-8. Our findings demonstrate that our inflamed multicellular human lung model is a promising tool for the evaluation of anti-inflammatory potency of drug candidates in vitro. With the presented setup, our model allows a meaningful comparison of the systemic vs. inhalation administration routes for the evaluation of the efficacy of a drug in vitro.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studied in vitro and adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells.

The approval of new nanomedicines requires a deeper understanding of the interaction between cells and nanoparticles (NPs). Silica (SiO2) and gold (Au) NPs have shown great potential in biomedical applications, such as the delivery of therapeutic agents, diagnostics, and biosensors. NP-cell interaction and internalization can trigger several cellular responses, including gene expression regulation. The identification of differentially expressed genes in response to NP uptake contributes to a better understanding of the cellular processes involved, including potential side effects. We investigated gene regulation in human macrophages and lung epithelial cells after acute exposure to spherical 60 nm SiO2 NPs. SiO2 NPs uptake did not considerably affect gene expression in epithelial cells, whereas five genes were up-regulated in macrophages. These genes are principally related to inflammation, chemotaxis, and cell adhesion. Nuclear receptor NR4A1, an important modulator of inflammation in macrophages, was found to be up-regulated. The expression of this gene was also increased upon 1 h of macrophage exposure to spherical 50 nm AuNPs and 200 nm spherical SiO2 NPs. NR4A1 can thus be an important immediate regulator of inflammation provoked by NP uptake in macrophages.

Research in nanoscience continues to bring forward a steady stream of new nanomaterials and processes that are being developed and marketed. While scientific committees and expert groups deal with the harmonization of terminology and legal challenges, risk assessors in research labs continue to have to deal with the gap between regulations and rapidly developing information. The risk assessment of nanomaterial processes is currently slow and tedious because it is performed on a material-by-material basis. Safety data sheets are rarely available for (new) nanomaterials, and even when they are, they often lack nano-specific information. Exposure estimations or measurements are difficult to perform and require sophisticated and expensive equipment and personal expertise. The use of banding-based risk assessment tools for laboratory environments is an efficient way to evaluate the occupational risks associated with nanomaterials. Herein, we present an updated version of our risk assessment tool for working with nanomaterials based on a three-step control banding approach and the precautionary principle. The first step is to determine the hazard band of the nanomaterial. A decision tree allows the assignment of the material to one of three bands based on known or expected effects on human health. In the second step, the work exposure is evaluated and the processes are classified into three “nano” levels for each specific hazard band. The work exposure is estimated using a laboratory exposure model. The result of this calculation in combination with recommended occupational exposure limits (rOEL) for nanomaterials and an additional safety factor gives the final “nano” level. Finally, we update the technical, organizational, and personal protective measures to allow nanomaterial processes to be established in research environments.

Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold (AuNPs) and iron oxide nanoparticles (FeOxNPs) either alone or combined. Environmental scanning electron microscopy revealed that single NPs of both types bound within minutes on the cell surface but with a distinctive difference between FeOxNPs and AuNPs. Uptake analysis studies based on laser scanning microscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry revealed intracellular appearance of both NP types in all exposure scenarios and a time-dependent increase. This increase was higher for both AuNPs and FeOxNPs during co-exposure. Cells treated with endocytotic inhibitors recovered after co-exposure, which additionally hinted that two uptake mechanisms are involved. Cross-talk between uptake pathways is relevant for toxicological studies: Co-exposure acts as an uptake accelerant. If the goal is to maximize the cellular uptake, e.g., for the delivery of pharmaceutical agents, this can be beneficial. However, co-exposure should also be taken into account in the case of risk assessment of occupational settings. The demonstration of co-exposure-invoked pathway interactions reveals that synergetic nanoparticle effects, either positive or negative, must be considered for nanotechnology and nanomedicine in particular to develop to its full potential.

Abstract Background The lung represents the primary entry route for airborne particles into the human body. Most studies addressed possible adverse effects using single (nano)particles, but aerosolic nanoparticles (NPs) tend to aggregate and form structures of several hundreds nm in diameter, changing the physico-chemical properties and interaction with cells. Our aim was to investigate how aggregation might affect the biodistribution; cellular uptake and translocation over time of aerosolized NPs at the air-blood barrier interface using a multicellular lung system. Results Model gold nanoparticles (AuNPs) were engineered and well characterized to compare single NPs with aggregated NPs with hydrodynamic diameter of 32 and 106 nm, respectively. Exposures were performed by aerosolization of the particles onto the air-liquid interface of a three dimensional (3D) lung model. Particle deposition, cellular uptake and translocation kinetics of single and aggregated AuNPs were determined for various concentrations, (30, 60, 150 and 300 ng/cm2) and time points (4, 24 and 48 h) using transmission electron microscopy and inductively coupled plasma optical emission spectroscopy. No apparent harmful effect for single and aggregated AuNPs was observed by lactate dehydrogenase assay, nor pro-inflammation response by tumor necrosis factor α assessment. The cell layer integrity was also not impaired. The bio-distribution revealed that majority of the AuNPs, single or aggregated, were inside the cells, and only a minor fraction, less than 5%, was found on the basolateral side. No significant difference was observed in the translocation rate. However, aggregated AuNPs showed a significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h. Conclusions Our studies revealed that aggregated AuNPs showed significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h, but the uptake rate was similar at later time points. In addition, aggregation did not affect translocation rate across the lung barrier model since similar translocation rates were observed for single as well as aggregated AuNPs.

Abstract Determination of the cell type specific response is essential towards understanding the cellular mechanisms associated with disease states as well as assessing cell-based targeting of effective therapeutic agents. Recently, there have been increased calls for advanced in vitro multi-cellular models that provide reliable and valuable tools correlative to in vivo. In this pursuit the ability to assess the cell type specific response is imperative. Herein, we report a novel approach towards resolving each specific cell type of a multi-cellular model representing the human lung epithelial tissue barrier via multi-colour flow cytometry (FACS). We proved via ≤ five-colour FACS that the manipulation of this in vitro model allowed each cell type to be resolved with no impact upon cell viability. Subsequently, four-colour FACS verified the ability to determine the biochemical effect (e.g. oxidative stress) of each specific cell type. This technique will be vital in gaining information upon cellular mechanics when using next-level, multi-cellular in vitro strategies.

Abstract: Nanoparticles (NPs) possess unique properties useful for designing specific functionalities for biomedi- cal applications. A prerequisite of a safe-by-design and effective use in any biomedical application is to study NP–cell interactions to gain a better understanding of cellular consequences upon exposure. Cellular uptake of NPs results mainly in the localization of NPs in the complex environment of lysosomes, a compartment which can be mimicked by artificial lysosomal fluid. In this work we showed the applicability of lysosomal fluid as a platform for a fast assessment of gold, iron oxide and silica NP stability over 24 h in a relevant biological fluid, by using multiple analytical methods.

Abstract: Besides conducting excellent fundamental research in domains of strategic importance, the National Centers of Competence in Research (NCCRs) also aim to become centers of reference for education, equal opportunities, and knowledge and technology transfer. These activities are supported by a communication strategy focused on specific target groups. This article describes some of the main strategic goals and achievements of the NCCR Bio-Inspired Materials, presents the main activities launched by the Center throughout its first funding phase, and provides a glimpse of new plans and directions for the second phase.

Abstract: The main cause of cancer-related death is due to cancer cell spreading and formation of secondary tumors in distant organs, the so-called metastases. Metastatic cancer cells are detectable in the blood of cancer patients as circulating tumor cells (CTC) and may be exploited for prognostic and monitoring purposes, including in breast cancer. Due to their very low frequency, however, their quantitative detection remains a challenge in clinical practice. Nature has developed mechanisms to amplify rare biological events or weak signals, such as intracellular signaling pathways, cytokine networks or the coagulation cascades. At the National Center for Competence in Research (NCCR) in Bio-Inspired Materials we are coupling gold nanoparticle-based strategies with fibrinogen and DNA bio-inspired amplification cascades to develop an in vitro test to specifically and sensitively detect CTCs in patients' blood. In this article, we describe the biological context, the concept of bio-inspired amplification, and the approaches chosen. We also discuss limitations, open questions and further potential biomedical applications of such an approach.

Abstract: Nanoparticles (NPs) have sizes that approach those of pathogens and they can interact with the membranes of eukaryotic cells in an analogous fashion. Typically, NPs are taken up by the cell via the plasma membrane by receptor-mediated processes and subsequently interact with various endomembranes. Unlike pathogens, however, NPs lack the remarkable specificity gained during the evolutionary process and their design and optimization remains an expensive and time-consuming undertaking, especially considering the limited information available on their molecular interactions with cells. In this context, molecular dynamics (MD) simulations have emered as a promising strategy to investigate the mechanistic details of the interaction of NPs with mammalian or viral membranes. In particular, MD simulations have been extensively used to study the uptake process of NPs into the cell, focusing on membrane vesiculation, endocytic routes, or passive permeation processes. While such work is certainly relevant for understanding NP–cell interactions, it remains very difficult to determine the correspondence between generic models and the actual NP. Here, we review how chemically-specific MD simulations can provide rational guidelines towards further bio-inspired NP optimization.

Abstract: Pore-forming peptides are of interest due to their antimicrobial activity and ability to form gateways through lipid membranes. Chemical modification of these peptides makes it possible to arrange several peptide monomers into well-defined pore-forming structures using various templating strategies. These templated super-structures can exert antimicrobial activity at significantly lower total peptide concentration than their untemplated equivalents. In addition, the chemical moieties used for templating may be functionalized to interact specifically with targeted membranes such as those of pathogens or cancer cells. A range of molecular templates has been explored, including dimerization of pore-forming monomers, their covalent attachment to cyclodextrin, porphyrin or fullerene scaffolds as well as attachment of amino acid linkers or nucleic acid constructs to generate assemblies of 4 to 26 peptides or proteins. Compared to free peptide monomers, templated pore assemblies showed increased membrane affinity, prolonged open-state lifetimes of the pores and more frequent pore formation due to higher local concentration. These constructs are useful model systems for biophysical studies to understand porin and ion channel proteins and their mechanisms of insertion into lipid membranes. Recently designed DNA-templates are expanding the usefulness of templated pore assemblies beyond applications of cell killing and may include targeted drug delivery and accelerate the emerging field of single-molecule detection and characterization of biomolecules by nanopore-based resistive pulse sensing.

Abstract: We investigate the structure and the dynamics of dense suspensions of NIH 3T3 fibroblast cells. Using two-photon microscopy we obtain three dimensional (3D) images from which the size and the packing structure of the dense cell suspensions can be extracted. In addition, we analyse the global time-dependent behaviour of the suspensions by time-lapse measurements of cell sedimentation. Since cell adhesion is a non-equilibrium living process the interplay can be influenced by cell viability interfering with cell–cell interactions.

Abstract: Understanding the interaction between cells and their environment is fundamental for mechanobiology. To mimic the behavior of cells in physiological and pathological conditions, synthetic substrates must have topographical and/or mechanical properties that evolve in time. Dynamic substrates mainly rely on stimuli-responsive materials where an external stimulus induces controlled variations in topography or mechanics. Herein, we describe the development of a dynamic cell culture substrate where mechanical properties are reversibly tuned in situ using magnetically-responsive superparamagnetic iron oxide nanoparticles (SPIONs).

Small plastic particles such as micro- (

Silica nanoparticles (NPs) are widely used in various industrial and biomedical applications. Little is known about the cellular uptake of co-exposed silica particles, as can be expected in our daily life. In addition, an inflamed microenvironment might affect a NP’s uptake and a cell’s physiological response. Herein, prestimulated mouse J774A.1 macrophages with bacterial lipopolysaccharide were post-exposed to micron- and nanosized silica particles, either alone or together, i.e., simultaneously or sequentially, for different time points. The results indicated a morphological change and increased expression of tumor necrosis factor alpha in lipopolysaccharide prestimulated cells, suggesting a M1-polarization phenotype. Confocal laser scanning microscopy revealed the intracellular accumulation and uptake of both particle types for all exposure conditions. A flow cytometry analysis showed an increased particle uptake in lipopolysaccharide prestimulated macrophages. However, no differences were observed in particle uptakes between single- and co-exposure conditions. We did not observe any colocalization between the two silica (SiO2) particles. However, there was a positive colocalization between lysosomes and nanosized silica but only a few colocalized events with micro-sized silica particles. This suggests differential intracellular localizations of silica particles in macrophages and a possible activation of distinct endocytic pathways. The results demonstrate that the cellular uptake of NPs is modulated in inflamed macrophages but not in the presence of micron-sized particles.

Synthetic colloidal nanoparticles are nowadays omnipresent. Nonetheless, adequately characterizing them and interpreting the data is challenging, as their surrounding environment, e.g. the medium they are dispersed in, is often an active contributor to their size, morphology and structural integrity. In this regard, cryo-transmission electron microscopy (cryo-TEM) is an ideal methodology. This article provides a general guidance for beginners and experts encountering this technique on the common benefits and pitfalls when characterizing synthetic nanoparticles. Illustrative experimental examples are presented which cover the importance of water as a supportive and structural component, along with contrast generation and electron beam damage.

Precision-cut lung slices (PCLS) are an established ex vivo alternative to in vivo experiments in pharmacotoxicology. The aim of this study was to evaluate the potential of PCLS as a tool in nanotoxicology studies. Silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles as well as quartz particles were used because these materials have been previously shown in several in vitro and in vivo studies to induce a dose-dependent cytotoxic and inflammatory response. PCLS were exposed to three concentrations of 70 nm monodisperse polyvinylpyrrolidone (PVP)-coated Ag-NPs under submerged culture conditions in vitro. ZnO-NPs (NM110) served as ‘soluble’ and quartz particles (Min-U-Sil) as ‘non-soluble’ control particles. After 4 and 24 h, the cell viability and the release of proinflammatory cytokines was measured. In addition, multiphoton microscopy was employed to assess the localization of Ag-NPs in PCLS after 24 h of incubation. Exposure of PCLS to ZnO-NPs for 4 and 24 h resulted in a strong decrease in cell viability, while quartz particles had no cytotoxic effect. Moreover, only a slight cytotoxic response was detected by LDH release after incubation of PCLS with 20 or 30 µg/mL of Ag-NPs. Interestingly, none of the particles tested induced a proinflammatory response in PCLS. Finally, multiphoton microscopy revealed that the Ag-NP were predominantly localized at the cut surface and only to a much lower extent in the deeper layers of the PCLS. In summary, only ‘soluble’ ZnO-NPs elicited a strong cytotoxic response. Therefore, we suggest that the cytotoxic response in PCLS was caused by released Zn2+ ions rather than by the ZnO-NPs themselves. Moreover, Ag-NPs were predominantly localized at the cut surface of PCLS but not in deeper regions, indicating that the majority of the particles did not have the chance to interact with all cells present in the tissue slice. In conclusion, our findings suggest that PCLS may have some limitations when used for nanotoxicology studies. To strengthen this conclusion, however, other NP types and concentrations need to be tested in further studies.

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of −20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles.

The interfacing of colloidal nanoparticles with mammalian cells is now well into its second decade. In this review our goal is to highlight the more generally accepted concepts that we have gleaned from nearly twenty years of research. While details of these complex interactions strongly depend, amongst others, upon the specific properties of the nanoparticles used, the cell type, and their environmental conditions, a number of fundamental principles exist, which are outlined in this review.

Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

In the emerging market of nano-sized products, silver nanoparticles (Ag NPs) are widely used due to their antimicrobial properties. Human interaction with Ag NPs can occur through the lung, skin, gastrointestinal tract, and bloodstream. However, the inhalation of Ag NP aerosols is a primary concern. To study the possible effects of inhaled Ag NPs, an in vitro triple cell co-culture model of the human alveolar/airway barrier (A549 epithelial cells, human peripheral blood monocyte derived dendritic and macrophage cells) together with an air–liquid interface cell exposure (ALICE) system was used in order to reflect a real-life exposure scenario. Cells were exposed at the air–liquid interface (ALI) to 0.03, 0.3, and 3 µg Ag/cm2 of Ag NPs (diameter 100 nm; coated with polyvinylpyrrolidone: PVP). Ag NPs were found to be highly aggregated within ALI exposed cells with no impairment of cell morphology. Furthermore, a significant increase in release of cytotoxic (LDH), oxidative stress (SOD-1, HMOX-1) or pro-inflammatory markers (TNF-α, IL-8) was absent. As a comparison, cells were exposed to Ag NPs in submerged conditions to 10, 20, and 30 µg Ag/mL. The deposited dose per surface area was estimated by using a dosimetry model (ISDD) to directly compare submerged vs ALI exposure concentrations after 4 and 24 h. Unlike ALI exposures, the two highest concentrations under submerged conditions promoted a cytotoxic and pro-inflammatory response after 24 h. Interestingly, when cell cultures were co-incubated with lipopolysaccharide (LPS), no synergistic inflammatory effects were observed. By using two different exposure scenarios it has been shown that the ALI as well as the suspension conditions for the lower concentrations after 4 h, reflecting real-life concentrations of an acute 24 h exposure, did not induce any adverse effects in a complex 3D model mimicking the human alveolar/airway barrier. However, the highest concentrations used in the ALI setup, as well as all concentrations under submerged conditions after 24 h, reflecting more of a chronic lifetime exposure concentration, showed cytotoxic as well as pro-inflammatory effects. In conclusion, more studies need to address long-term and chronic Ag NP exposure effects.

When nanomaterials enter biological fluids, they are immediately covered by biomolecules, particularly proteins, forming the so-called protein corona. The dynamic nature and complexity of the protein corona can impact upon the biological effects and distribution of nanomaterials with an organism. Therefore, the protein corona is an important factor in determining the biological impact of any nanomaterials. The protein adsorption pattern is determined by various factors, including the bio-fluids’ protein composition, the nanomaterials’ physicochemical properties, as well as the time and type of exposure. Predominantly, research has focused upon spherical nano-objects, however, due to their ever-increasing potential use within human based applications, and, therefore, heightening and inevitable exposure to the human body, little is known regarding how proteins interact with nanofibers. Therefore, the present review focuses on the current knowledge as to how the geometry of man-made (nano)fibers, carbon nanotubes (in comparison with asbestos fibers), affects their interaction with proteins within biological fluids. Summarizing state-of the art methodologies applied to dissect protein-binding signatures, it is further discussed whether the protein corona composition of fibrous and non-fibrous materials differ, as well as what impact the protein corona has on (nano)fiber uptake, intracellular distribution and their subsequent toxicity.

Polydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction.

Polyelectrolyte multilayer microcapsules around 3.4 micrometers in diameter were added to epithelial cells, monocyte-derived macrophages, and dendritic cells in vitro and their uptake kinetics were quantified. All three cell types were combined in a triple co-culture model, mimicking the human epithelial alveolar barrier. Hereby, macrophages were separated in a three-dimensional model from dendritic cells by a monolayer of epithelial cells. While passing of small nanoparticles has been demonstrated from macrophages to dendritic cells across the epithelial barrier in previous studies, for the micrometer-sized capsules, this process could not be observed in a significant amount. Thus, this barrier is a limiting factor for cell-to-cell transfer of micrometer-sized particles.

The key findings of a workshop jointly organized by the Swiss Centre of Applied Ecotoxicity, the Swiss Centre for Applied Human Toxicology (SCAHT), and the Federal Office of Public Health (FOPH) are summarized and provide a critical analysis of the current regulatory framework for nanomaterials and a snapshot of some hot topics in nanoscience.

Due to the constant expansion within the nanotechnology industry in the last decade, nanomaterials are omnipresent in society today. Nanotechnology-based products have numerous different applications ranging from electronic (e.g., advanced memory chips) to industrial (e.g., coatings or composites) to biomedical (e.g., drug delivery systems, diagnostics). Although these new nanomaterials can be found in many “everyday” products, their effects on the human body have still to be investigated in order to identify not only their risk, but also their potential benefits towards human health. Since the lung is commonly thought to be the main portal of entry into the human body for nanomaterials released within the environment, this review will attempt to summarise the current knowledge and understanding of how nanomaterials interact with the respiratory tract. Furthermore, the advantages and disadvantages of different experimental model systems that are commonly used to study this exposure route to the human body will be discussed.

Since the early 1990s nanofibers, particularly those of a carbonaceous content [1] have received heightened interest due to their advantageous physico-chemical characteristics (e.g., high strength, stiffness, semi-conductor, increased thermal conductivity and one of the highest Young’s modulus [2]).[...]

Cellulose nanocrystals exhibit an interesting combination of mechanical properties and physical characteristics, which make them potentially useful for a wide range of consumer applications. However, as the usage of these bio-based nanofibers increases, a greater understanding of human exposure addressing their potential health issues should be gained. The aim of this perspective is to highlight how knowledge obtained from studying the biological impact of other nanomaterials can provide a basis for future research strategies to deduce the possible human health risks posed by cellulose nanocrystals.

In this article we present an overview of some of our research in the field of nanoscience. By combining two different scientific backgrounds (chemistry and biology), we investigate nanoparticle–cell interactions from different angles. This requires an interdisciplinary approach involving material synthesis and characterization, cell biology (biochemistry) and microscopy. In particular, we describe the synthesis and magnetic properties of superparamagnetic iron oxide nanoparticles as well as their behavior in cell culture, evaluate different visualization and detection methods, and investigate the interaction of such magnetic particles with immune cells.