Course. Kroll A, Dierker C, Rommel C, Hahn D, Wohlleben W, Schulze-Isfort C, et al. The cells exposed to MWCNT had their fibers six times shorter (2.1 ± 1 µm) (Fig. 4c). The medium was then removed, and the wells were washed twice with PBS 1× containing Mg2+. Nevertheless, after 24 h of exposure to MWCNT the Young’s modulus dramatically decrease by 13.6% compared with 2 h results. CAS  Simultaneous measurement of lateral and normal forces with an optical-beam-deflection atomic force microscope. The more detailed results are provided below. Part Fibre Toxicol. 2014;11:18. https://doi.org/10.1186/1743-8977-11-18. 2. New label-free approaches to identify NMs’ effects on biological structures include characterization of mechanical cell response due to deleterious changes in the cytoskeleton, impedance variations due to the loss of cells’ membrane integrity and proteomics to identify gene expressions under metabolic stress has been developed [12,13,14,15,16]. Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape. Sokolov I, Dokukin ME, Guz NV. Cite this article. Mrs Wilkins shows you how to determine the Young Modulus of a metal wire.00:00 Experiment set up04:30 Reading Vernier scale05:12 Plotting graph & analysis CAS  L9774, Sigma-Aldrich, USA). Zimmer CC, Liu YX, Morgan JT, Yang G, Wang K-H, Kennedy IM, et al. Perfect for A-level physics students on any specification. 2010;4:4872–8. 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]. Johnson KL. Square Exposure assessment approaches for engineered nanomaterials. Hilal N, Bowen WR, Alkhatib L, Ogunbiyi O. The length of actin stress fibers measured using Alexa Fluor 488 conjugated-phalloidin marker evidenced significant shortening on cell exposed to both CBNs. The cells exposed to MWCNT shows almost two times the number of mitochondria and some vesicles with MWCNT inside, Fig. 1b. J Cell Physiol. Therefore, it is a shape dependence of the ability of CBNs to adsorb media proteins. J Phys Chem B. https://doi.org/10.1016/j.carbon.2007.03.033. We confirmed the production of radical oxygen species (ROS) on cells exposed to CBNs, which is related to the disruption of the cytoskeleton. We comparatively evaluated the albumin adsorption between GF and MWCNT to determine the potential extracellular effect of both nanomaterials in cells metabolism due to protein sequestration. 15 µL of MWCNTs or GFs at 1 mg/mL was added to each well to a final concentration of 50 µg/mL. 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It is a measure of the stiffness of a material; however, in practice, other properties of materials, scientists and engineers are often interested in, such as yield stress, have more influence on the selection of materials for a particular purpose. Thus, we observed that the shape of CBN strongly affects the cellular cytotoxicity than their SSA. Young's modulus (E or Y) is a measure of a solid's stiffness or resistance to … This worksheet is ideal for home learning since it has an accompanying video explanation of the experiment, the analysis of results and solutions to all questions are included in the document. Nakanishi J, Morimoto Y, Ogura I, Kobayashi N, Naya M, Ema M, et al. The medium was then removed, and the wells were washed thoroughly with PBS supplemented with Mg2+. Your response should include a photo of your setup (books on pillow?) Variation of mechanical property of single-walled carbon nanotubes-treated cells explored by atomic force microscopy. b The cell exposed to GF for 24 h; the F-actin integrity is compromised and is only observed somewhat organized close to the nucleus. An important assumption in the analysis using this model is that the microsphere indentation is much smaller than the finite cell height (10–20% of the cell thickness) to minimize artifacts in extracted elasticity maps originating from contributions of the hard substrate due to the finite cell thickness [71]. Subsequently, DHE dye was added to the cells. 2010;463:485–92. 2005;310:1139–43. Evaluation of cytotoxicity, biophysics and biomechanics of cells treated with functionalized hybrid nanomaterials. 'Young's Modulus' or modulus of elasticity is a measurement of the rate of change of strain as a function of stress. https://doi.org/10.1016/j.jtemb.2017.01.005. 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. Pastrana, H.F., Cartagena-Rivera, A.X., Raman, A. et al. In particular, shorter stress fibers are associated with reduced cell adhesion and motility [54], which are critical cellular processes for cell division and migration. 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. The fiber length was measured by image processing on ImageJ skeleton algorithm. On the other hand, the live/dead assay showed non-significant variations on the viability of cells exposed to MWCNTs and GFs; despite the lower concentration of CBNs tested. 2016;110:2528–39. Adjei IM, Sharma B, Labhasetwar V. Nanoparticles: cellular uptake and cytotoxicity. The percentage of cells expressing ROS production was two times higher on cells exposed to GF than in those exposed to MWCNT, Fig. 3. Biological interactions of graphene-family nanomaterials: an interdisciplinary review. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. It is a measure of the stiffness of a material that is independent of the particular sample of a substance. https://doi.org/10.1021/jp208967t. Our results of physicochemical characterization (size, SSA, and impurities) of the CBNs differ from the ones provided by manufacturers. In other words, it is how easily it is bended or stretched. Secondary to the glutathionylation, the F-actin cytoskeleton no longer possesses straight filaments across the cytoplasm, but instead an aggregation of actin monomers. PubMed Google Scholar. 2014;16:2153. https://doi.org/10.1007/s11051-013-2153-9. The lower availability of media protein can be considered as a major cause of cells toxicity induced by GF on NIH3T3 fibroblasts since the presence of these nanomaterials into the cells were not proved by TEM images. The excess of ROS results in additional glutathione residues to the actin monomers during the polymerization process of F-actin [19]. Flow cytometer results of the live/dead assay. Gavara N, Chadwick RS. Our study shows the dependence of the Young’s modulus on exposure time. https://doi.org/10.1016/j.biomaterials.2012.02.021. 2010;7:22. https://doi.org/10.1186/1743-8977-7-22. Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays. Applying both conventional biochemical approach and cells mechanics, we were able to detect differences in the actin networks induced by MWCNT inside the cells and GF outside the cell’s membrane. Questions 1. 2003;37:1–17. Abbott LC, Maynard AD. volume 17, Article number: 32 (2019) 2010;30:1634–44. Detecting viability transitions of umbilical cord mesenchymal stem cells by Raman micro-spectroscopy. 2016;18:89. https://doi.org/10.1007/s11051-016-3375-4. Cells were then seeded on a culture dish (Fluorodish, 50 mm, WPI, Sarasota, FL, USA) pre-coated with 0.1% gelatine in water (STEMCELL Technologies, Vancouver, British Columbia, Canada) at a concentration of 1.2 × 105 cells/mL. A concentration-dependent decrease of cell viability is observed. Figure 2 presents the dispersion diagrams of the populations expressing calcein-AM and propidium iodine in the live/dead assays. J Nanoparticle Res. Lawen A, Lane DJR. 2017;43:153–60. Young’s Modulus or Elastic Modulus or Tensile Modulus, is the measurement of mechanical properties of linear elastic solids like rods, wires, etc. Karlsson M, Kurz T, Brunk UT, Nilsson SE, Frennesson CI. New approach to investigate the cytotoxicity of nanomaterials using single cell mechanics. Freestone B, Krishnamoorthy S, Lip GY. 2013;110:16027–32. Crist RM, Grossman JH, Patri AK, Stern ST, Dobrovolskaia MA, Adiseshaiah PP, et al. as well as a plot. The concentrations of these structures change on different cells depending on their biological function. However, our cytotoxicity study, which combines biochemical assays with evaluations of the response of mechanical cell properties to exposure to CBNs, demonstrates that, in the case of GFs, a smaller SSA results in a substantial change in elasticity and ROS production. To satisfy this assumption, data were analyzed up to indentations of 20%-pixel cell height. After 24 h, 100 µL of MWCNTs or GFs at 1 mg/mL were added to each well to a final concentration of 50 µg/mL. statement and We measured the actin stress fiber length using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) by segmenting the continuous line in the actin image representing stress fibers. The F-actin measurement indicated a time depending disruption as well as dimensionality relation. There is more protein adsorption, ten times, in GF regarding their SSA. Pulskamp K, Diabaté S, Krug HF. The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. 2013;18:2473–507. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lévy R, Maaloum M. Measuring the spring constant of atomic force microscope cantilevers: thermal fluctuations and other methods. In: Puleo D, Bizios R, editors. J Phys Chem C. 2011;115:23323–8. Elastic Young’ modulus maps and three-dimensional topographic reconstruction of live NIH3T3 in culture media before and after exposure to CBNs: a unexposed cells, b cells exposed 2 h to MWCNTs, c cells exposed 24 h to MWCNTs, d cells exposed 2 h to GFs and e cells exposed 24 h to GFs. Mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress. The slide with its slits is most commonly purchased from a manufacturer who provides a measured value for the slit separation distance - the d value in Young's equation. Subsequently, cells were treated in osmium tetroxide (Sigma-Aldrich, Cat 75632, CA, USA) and dehydrated in a graded series of ethanol. The Young’s modulus of the cells exposed to MWCNT after 2 h had a slight increase, this effect has been reported by different authors after the administration of SWCNT, mentioning that it can be related to the reinforcement by nanomaterials to the cytoskeleton fibers (actin stress fibers) [63, 64]. 2010;8:557–71. 2015;10:2423–50. https://doi.org/10.1007/978-0-387-98161-1_1. Manage cookies/Do not sell my data we use in the preference centre. Therefore, it is possible to have stressed cell also labeled as viable by calcein marker. Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos. Eur J Pharm Biopharm. https://doi.org/10.1016/j.ejpb.2008.08.009. Both the wires should be supported from same rigid support. Samples were not sonicated to simulate a direct biological contact under normal NMs exposure in the environment. We propose that cell toxicity is affected by CBNs’ shape, and not only by their SSA. 2015;32:485–512. Cytoskeleton damage is a sign of cellular homeostasis dysfunction, due to cytotoxic products such as high concentration of radical oxygen species (ROS) [18]. 2015;3:3983–92. We used the standard force–volume AFM mode to generate topographic, and elasticity map sought to determine the cell’s stiffness (Young’s modulus) of untreated and CBN-treated (MWCNT and GF) fibroblasts after 2 and 24 h of exposure, to 50 µg/mL concentration, Fig. 5. Single-cell mechanics provides an effective means to probe in vivo interactions between alveolar macrophages and silver nanoparticles. Therefore, the cellular uptake will depend more on the nanomaterials properties rather than the cellular activity of engulfing foreign bodies like macrophages [51]. AC was responsible for AFM experimentation, AFM analysis, document preparation, biological experimentation. To the best of our knowledge, this report is the first work to assess ROS production, cell’s mechanics and viability with CBNs dimensionality as a direct result of the disruption of actin stress fibers. The Young's Modulus of a material is a fundamental property of every material that cannot be changed. Young's Modulus Experiment Lab report for Youngs Modulus Experiment. Strikingly, after cells exposed to carbon-based nanomaterials for only 2 h a considerable reduction in cellular mechanical properties is observed, whereas no significant production in ROS is measured. The Young Modulus is also known as Young's Modulus or the elastic modulus or tensile modulus.. Later, cells were harvested using trypsin and centrifuged at 200×g. TEM characterization of Multiwall Carbon Nanotubes and Graphene Oxide Flakes are showed. Subramanian V, Semenzin E, Hristozov D, Zabeo A, Malsch I, McAlea E, et al. Kay AR. The increased production of ROS by CBNs can be linked to the biomechanical changes on fibroblast, specifically to lower Youngs modulus. Here are some of the subjects to search for on the cover of a lab report: We used two concentrations 10 and 50 µg/mL in DMEM enriched with 10% of BSA. Previous works have concentrated on studying the toxicity effects based on the SSA [41]. Schematic of experimental setup using AFM. Illner D, Scherthan H. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton. PLoS Comput Biol. The samples were sonicated and incubated at 37 °C, 95% RH, and 5% CO2 overnight. 2012;261:121–33. Gourlay CW, Ayscough KR. Carbon nanotube uptake changes the biomechanical properties of human lung epithelial cells in a time-dependent manner. This AFM mode is based on the acquisition of low-speed, quasi-static F–Z curves predefined by the user grid of points, resulting in the simultaneous acquisition of topography and material properties maps [69]. J Trace Elem Med Biol. Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments. Casey A, Herzog E, Davoren M, Lyng FM, Byrne HJ, Chambers G. Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity. Risk Anal. Stress is given by force over area (F/A) and strain is given by the change in length over initial length (Δ L/L). When compared to the accepted value this represents a percent difference of 94.5%. J Phys Chem B. Cartagena-Rivera AX, Logue JS, Waterman CM, Chadwick RS. Article  Occupational safety and health criteria for responsible development of nanotechnology. 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]. 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. The F–Z curve rate was set to 1 Hz (1 s) to minimize viscoelastic contributions [60]. https://doi.org/10.1016/j.neuron.2011.05.011. 2007;38:S26–32. London WC1R 4HQ. Nakada D, Levi BP, Morrison SJ. https://doi.org/10.1111/j.1365-2796.2009.02187.x. Journal of Nanobiotechnology Altogether, the changes in mechanical properties and the length of F-actin fibers confirmed that disruption of the F-actin cytoskeleton is a major consequence of cellular toxicity. Nanotechnology. Cells were maintained at 37 °C during the AFM experiments using a Petri dish holder and a heater on the MFP-3D scanner X–Y stage (Asylum Research, Santa Barbara, CA, USA). The SSA is a critical parameter in in vitro assays that evaluate the response of cells to nanomaterial exposure, as detected on the basis of catalytic activity, acidity/alkalinity, absorption, fluorescence signaling, adsorption capacity, and metabolic dynamics [3]. We established a correlation between the CBN toxicity and cells’ physical parameter (shape and elasticity) that has not been previously identified by the conventional live/dead viability assay. Proc Natl Acad Sci USA. 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. Nat Rev Mol Cell Biol. Cells were fixed with 3% formaldehyde for 30 min at room temperature. 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].