Our results show a significant decrease on Young’s modulus, ~ 20%, in cells exposed to low concentrations of graphene flakes (GF), ~ 10% decrease for cells exposed to low concentrations of multiwalled carbon nanotubes (MWCNTs) than the control cells. a. Google ScholarÂ. 3. © 2021 BioMed Central Ltd unless otherwise stated. The cells were then incubated for 24 h at 37 °C under a 5% CO2 atmosphere. https://doi.org/10.1002/jcp.1041190110. This observation suggests that the GF depletes the protein availability for cell growth, faster than MWCNT; leading to metabolic stress and ROS production [32,33,34,35]. Miller G, Wickson F. Risk analysis of nanomaterials: exposing nanotechnology’s naked emperor. 2015;32:485–512. There are solutions at the end. https://doi.org/10.1073/pnas.1306324110. Cells were fixed with 3% formaldehyde for 30 min at room temperature. In addition, the introduced force/volume technique permit to simultaneously quantify a single cells volume variation at different periods that has provided information of the process of cell death. Based on the experimental results of Harris (1998), the ratio of dynamic elastic modulus to the laboratory measured static chord modulus of dam concrete ranged from 0.7 to 1.20, and the average value was 1.0. Article  The Young modulus is often regarded as the quintessential material property, and students can learn to measure it. Conditions. The GFs in the culture media was able to increase the number of cells expressing ROS production up to 21% as quickly as 2 h after exposure, and 32% after 24 h resulting in a greater deleterious effect on cellular metabolism. Plot a graph of mass added against extension. PubMed Central  AppliedStatics (eme1016) Uploaded by. Shvedova AA, Pietroiusti A, Fadeel B, Kagan VE. https://doi.org/10.2217/nnm.11.88. Your response should include a photo of your setup (books on pillow?) Therefore, the cellular uptake will depend more on the nanomaterials properties rather than the cellular activity of engulfing foreign bodies like macrophages [51]. Next, cells were trypsinized, harvested, centrifuged, and the supernatant was removed. The results from live/dead assay confirms a slightly higher cytotoxic effect expected from MWCNT probably secondary to a larger SSA, nickel impurities, and facilitated cell uptake. Tes Global Ltd is Young’s Modulus (Y) =stress/strain Young’s modulus can be used in the following equation: F = 𝒀(∆𝑳 𝑳𝟎)𝑨 In this equation, F is equal to the force applied to the structure, Y is the Young’s modulus for the material, ΔL is the change in length of the material when the force is applied to it, L 0 is the initial length, and A is Significant at p < 0.05, n: 1000 points per group, t-test. Exposure assessment approaches for engineered nanomaterials. Journal of Nanobiotechnology 2009;72:370–7. The cytoskeleton integrity is shaped by three main structures: actin fibers, intermediate fibers, and microtubules. Multimedia University. b Cells exposed for 24 h to MWCNTs. Oops they did it again! By contrast, fibroblasts exposed to MWCNTs exhibited 4% increase in Young’s modulus after 2 h and a dramatic decrease of 10% after 24 h of exposure. Here are some of the subjects to search for on the cover of a lab report: However, other characteristics related to shape and dimensionality can be determinant on NMs behavior into the organisms. Young’s modulus differences between untreated cells and cells exposed to MWCNTs and GFs. Cells were stained for 30 min in the dark and at room temperature before being processed using LEAP. Then the graph can be analysed to determine E. Adv Exp Med Biol. Purpose This experiment is designed to determine Young’s modulus for a wire when it is stretched. We used two concentrations 10 and 50 µg/mL in DMEM enriched with 10% of BSA. These results regarding the relationship of the Young’s modulus of cells to the nanomaterials’ dimensionality are a novel approach to understand the cytotoxic effects. 2010;5:e9445. Illner D, Scherthan H. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton. Academic year. Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos. These considerable changes were directly correlated to the disruption of the cytoskeleton actin fibers. 2014;16:2153. https://doi.org/10.1007/s11051-013-2153-9. Metabolic regulation of stem cell function. https://doi.org/10.1007/978-0-387-98161-1_1. The cantilever spring constant was calculated using the thermal fluctuations noise method [68]. Google ScholarÂ. Our study shows the dependence of the Young’s modulus on exposure time. The relative trigger force used was 2 nN to minimize the cell deformation and the damage to the cells. 2013;10:20130694. https://doi.org/10.1098/rsif.2013.0694. 1984;119:58–64. J Cell Physiol. The dynamic elastic modulus under dynamic loads does not show the phenomenon of low static elastic modulus in the small stress condition. Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms. Antioxid Redox Signal. Pflugers Arch. registered in England (Company No 02017289) with its registered office at 26 Red Lion Then, cells were incubated for an additional 24 h at 37 °C in an atmosphere with 5% CO2 and a relative humidity of 95%. Carbon nanotube uptake changes the biomechanical properties of human lung epithelial cells in a time-dependent manner. Effects of surface curvature and surface characteristics of carbon-based nanomaterials on the adsorption and activity of acetylcholinesterase. De Nicola M, Bellucci S, Traversa E, De Bellis G, Micciulla F, Ghibelli L. Carbon nanotubes on Jurkat cells: effects on cell viability and plasma membrane potential. The cells were exposed to 50 µg/mL of CBNs for 24 h. Then were fixed and prepared for TEM imaging. Correspondence to PubMed  A worksheet to enable students to analyse experimental data from the Young’s Modulus experiment. Our results evidenced an initial decrease of 8.6% after 2 h of exposure contrary to the initial increase observed on cells exposed to MWCNT after 2 h. The decrease of the Young’s modulus, 24.6% lower, is more evident after 24 h. The metabolic impairment of the cells due to the ROS production and cytoskeleton fiber shortening is correlated with the mechanical response of the cells. Furthermore, MWCNT instead of modifying the protein adsorption, it had been shown to interact mechanically with actin cytoskeleton fibers possibly reinforcing its cellular structure resulting in a higher Young’s modulus [23]. A wide range of test methods is available for measuring modulus, but there is currently some uncertainty within parts of the user community about the reliability Nakanishi J, Morimoto Y, Ogura I, Kobayashi N, Naya M, Ema M, et al. The samples were sonicated and incubated at 37 °C, 95% RH, and 5% CO2 overnight. Risk Anal. The medium was then removed, and the wells were washed thoroughly with PBS supplemented with Mg2+. The MWCNTs exhibited a cylindrical shape with diameters 80 ± 7 nm and a length of 3 ± 1.5 μm; see Additional file 1. Sustainable nanotechnology decision support system: bridging risk management, sustainable innovation and risk governance. J Trace Elem Med Biol. Yue H, Wei W, Yue Z, Wang B, Luo N, Gao Y, et al. Our aim is to determine the Young’s modulus of elasticity of the material of a given wire using Searle’s apparatus. Other reports have indicated that the similarity in shape and dimensions of MWCNTs with the actin stress fibers, once in the cell, could emulate the fibers, resulting in reinforcement of the cytoskeleton [31]; this effect could explain the results observed for NIH3T3 fibroblasts exposed to MWCNTs for 2 h. By contrast, a planar shape, such as that of the GFs, results in cellular membrane deformation associated with enhanced protein adsorption, a lower Young’s modulus and higher ROS production compared to those associated with exposure to MWCNTs, despite their lower SSA. We used TR400PB soft microcantilevers (Olympus, Tokyo, Japan) with a nominal spring constant of 0.09 N/m, modified by attachment of a 5 µm borosilicate glass microsphere close to the tip (Novascan, Ames, IA, USA). Schulte PA, Geraci CL, Murashov V, Kuempel ED, Zumwalde RD, Castranova V, et al. Chen J, Rogers SC, Kavdia M. Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay. 2014;276:12–24. https://doi.org/10.1371/journal.pcbi.1003360. The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. PubMed Google Scholar. Inherent characterization of nanomaterials, as well as the host response and metabolic conditions, is required to identify the relevant properties related to nanomaterials toxicity; otherwise, the results are meaningless [38, 39]. The length of the actin fibers can be used as a descriptor for the cytoskeleton disruption that explains the changes on cells Young’s modulus induced by the exposure of CBNs, at a concentration of 50 µg/mL. Risk assessment of the carbon nanotube group. The shape is critical for protein absorption, which may be enhanced on planar nanomaterials such as graphene flakes. The Young's Modulus of a material is a fundamental property of every material that cannot be changed. Common pitfalls in nanotechnology: lessons learned from NCI’s Nanotechnology Characterization Laboratory. 2003;37:1–17. Local viscoelastic properties of live cells investigated using dynamic and quasi-static atomic force microscopy methods. Multiwall carbon nanotubes (US Research Inc, Houston, TX, USA) were suspended in 1× phosphate buffered saline (PBS) (Gibco, Waltham, Massachusetts, USA) at 1 mg/mL. https://doi.org/10.1016/j.neuron.2011.05.011. Zuo G, Zhou X, Huang Q, Fang H, Zhou R. Adsorption of villin headpiece onto graphene, carbon nanotube, and C60: effect of contacting surface curvatures on binding affinity. The calculation method of Young's modulus is particularly suitable for thin specimens. J Funct Biomater. These assays are strongly dependent on the properties of the nanomaterials, for example; specific surface area (SSA), size, surface defects, and surface charge, and the host response. Article  Subsequently, DHE dye was added to the cells. The Young Modulus is also known as Young's Modulus or the elastic modulus or tensile modulus.. PLoS ONE. H. P. acknowledges funding support from the Colciencias Colombia Scholarship Francisco Jose de Caldas 2012. Percent Difference: 15.342% 4. Cells were incubated for an additional 2 h and 24 h. The cells were then washed twice with warm culture medium (37 °C), and 2 mL of fresh medium was added to each dish. Science. All authors read and approved the final manuscript. 2013;52:4986–97. The cells exposed to both CBNs, at a concentration of 50 µg/mL, show an increased ROS production. Each sample, 10 µL at 1 mg/mL in 1× PBS, were placed on agar grids and dried. The cells are exposed to CBN, the presence of these materials in the media induce ROS production, protein adsorption, and F-actin polymerization disruption. Assessment of endothelial dysfunction. The length of actin stress fibers measured using Alexa Fluor 488 conjugated-phalloidin marker evidenced significant shortening on cell exposed to both CBNs. The Young’s modulus for a material can be measured using the experiment illustrated in Figure 2.The reference wire and test wire made of the selected material are hung from the ceiling. Searle’s apparatus consists of two metal frames F1 and F2. Fletcher DA, Mullins RD. NIH3T3 cells were seeded overnight in a specific 96-well plate (Cyntellect, San Diego, CA, USA) for laser-enabled analysis and processing (LEAP™) (Cyntellect, San Diego, CA, USA; located at Bindley, Purdue University) at 5 × 105 cell/mL concentration in culture media. After 2 h of exposure, fibroblasts exposed to GFs exhibited a fast-mechanical regulation, a 10% decrease in modulus and after 24 h, the modulus had decreased by 20%. I’ve made a video of the experiment here youtu.be/xASsYJo3zrM . Figure 1: Young’s Modulus Comparison. After 24 h of exposure to MWCNTs adsorbed 21.5 µg/mL of albumin, in contrast, the exposure to GFs had albumin adsorption of 127 µg/mL, see Fig. 7. It has been previously demonstrated that rather than introduce toxicity by the presence of iron, MWCNTs promotes iron metabolism sequestration inducing cellular inflammation [45, 47]. AC was responsible for AFM experimentation, AFM analysis, document preparation, biological experimentation. Questions 1. When bacteria are exposed to GFs, there is dramatic adenine adsorption on the E. coli membrane by the GF surface destroying the bacteria inducing death [37]. Our experimental protein absorption results demonstrated higher adsorption rates for GFs than for MWCNTs, which led to a significant decrease of the availability of proteins for cell homoeostasis, increasing the ROS production after 24 h of exposure to 32% for cells exposed to GF and 17% for cells exposed to MWCNTs. [59], study the effect SiO2 NP on the cell’s membrane stiffness, cells exposed to NPs evidenced a concentration-dependent decrease of cell membrane modulus. All the AFM imaging and measurements were performed under physiological conditions using culture media at 37 °C. It is more evident when the adsorbed proteins are calculated considering the SSA; in the case of MWCNT, its protein adsorption regarding the SSA is 3.04 µg/m2 while the GF had protein adsorption of 33.5 µg/m2. https://www.concepts-of-physics.com/searle-method/searle-method.php https://doi.org/10.1111/j.1365-2796.2009.02187.x. In light of these problems, international organizations such as the National Institute for Occupational Safety and Health (NIOSH), the Organization for Economic Cooperation and Development (OECD), the European Community (EC), and various non-governmental organizations (NGOs) are urging the development of label-free in vitro methods that avoid interference with NMs and improve assays’ reliability [10, 11]. AR responsible for document preparation, substantial contribution on experimental design, results analysis and technical support during experiment. Each group of measurements includes 13 cells. Single-cell mechanics provides an effective means to probe in vivo interactions between alveolar macrophages and silver nanoparticles. It demonstrates the higher capability for planar CBNs to sequester proteins compared to cylindrical shaped CBNs. 2013;18:2473–507. Current in vitro methods in nanoparticle risk assessment: limitations and challenges. 2010;4:4872–8. Sci Rep. 2015;5:11692. https://doi.org/10.1038/srep11692. HFP was responsible for document preparation, biological experimental set-up, AFM analysis, cytometry measurements, nanomaterials characterization, LEAP systems experiment and protein adsorption. Pulskamp K, Diabaté S, Krug HF. 2010;267:89–105. https://doi.org/10.1021/nn101151x. After 24 h of cell culture, CBNs (MWCNTs or GFs) were added to the medium at a final concentration of 50 g/mL. There are some other numbers exists which provide us a measure of elastic properties of a material. The NMs properties can also interfere with the reagents of the biochemical and optical assays leading to skewed interpretations and ambiguous results related to the NMs toxicity. µl of GF suspension was added to each well to a final concentration media. Dark and at room temperature responsible development of nanotechnology responses of NIH3T3 fibroblast CBNs’. 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