Toxicol Appl Pharmacol. It is dependent upon temperature and pressure however. Schematic of experimental setup using AFM. Carbon. Liu et al., presented an innovative approach from the single-cells assessment of mechanical response by compressing using a single point measurement of the Young’s modulus of the macrophages after exposure of silver nanoparticles. https://doi.org/10.1016/j.taap.2012.03.023. 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]. https://doi.org/10.1115/1.3261297. 2001;280:C719–41. https://doi.org/10.1002/lapl.201010087. Rather than using a note card to split the single beam into two coherent beams, a carbon-coated glass slide with two closely spaced etched slits is used. Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments. For 20 gauge copper wire the following experimental results produced a Young's Modulus constant of 6063 MPa, or 6.063 GPa. 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. Burgess RJ, Agathocleous M, Morrison SJ. Part Fibre Toxicol. 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. https://doi.org/10.1111/risa.12394. https://doi.org/10.1002/jcp.1041190110. Terms and Conditions, Appl Phys Lett. The photodetector optical sensitivity was obtained by extracting the slope of the F–Z curve. Carbon. Ponti J, Kinsner-Ovaskainen A, Norlen H, Altmeyer S, Cristina A, Bogni A. Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials. The medium was then removed, and the wells were washed thoroughly with PBS supplemented with Mg2+. Nature. Article  PubMed Central  2 Apparatus required Correspondence to As the shown in Figure 1 the sample was never taken past the elastic region. In summary, our results suggest that toxicity assessments need to consider additional techniques apart from the traditional, here demonstrated with the integration of the single the cells’ mechanical properties. Cartagena A, Raman A. 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. These results suggest that the CBNs’ shape affects cell homeostasis. 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. Karlsson M, Kurz T, Brunk UT, Nilsson SE, Frennesson CI. https://www.concepts-of-physics.com/searle-method/searle-method.php The data collected in the video can be used to complete a table and graph. Finally, calcein-AM green dye (Life Technologies, Carlsbad, CA, USA) for cell viability, DHE dye (Life Technologies, Carlsbad, CA, USA) for ROS production, and Hoechst 33342 dye (Life Technologies, Carlsbad, CA, USA) for DNA staining were added. 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. The role of the lateral dimension of graphene oxide in the regulation of cellular responses. Next, cells were trypsinized, harvested, centrifuged, and the supernatant was removed. Here we evidenced how the extracellular CBNs are as important as intracellular to determine the cells toxicity induced by these materials and its dependence on CBNs shape and dimensionality. The planar sheet shape and larger size of the GFs than the MWCNTs are likely responsible for modifications to the cells’ environment, such as media protein adsorption [30]. 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). Zimmer et al. The same value can be used to get a single average value of Young’s modulus Y. Current in vitro methods in nanoparticle risk assessment: limitations and challenges. The concentrations of these structures change on different cells depending on their biological function. Part 1: Bending of a cantilever under static loads In the presence of oxygen radicals, the molecules of 2-hydroxyethidium intercalate with the DNA and fluoresce red (576 nm) [52]. The modulus variations correlated with the generation of ROS and the viability assays demonstrated the weak reliability of the evaluation of cytotoxicity based only on one endpoint with traditional biochemical assays: live/dead. https://doi.org/10.1088/0957-4484/13/1/307. A protocol for in vivo detection of reactive oxygen species. https://doi.org/10.1371/journal.pcbi.1003360. Ispra. PLoS Comput Biol. YOUNG’S MODULUS PRACTICAL OBJECTIVE • To determine the Young’s Modulus of a steel wire using a graphical method. After incubating for 24 h, the cells were exposed to 50 µg/mL of CBNs (MWCNTs or GFs) in a fluorodish; the media was removed, and the cells were washed twice using PBS with Mg2+. Shvedova AA, Pietroiusti A, Fadeel B, Kagan VE. 1984;119:58–64. 10 µg of each sample was placed on an XploRA confocal Raman spectrometer (Horiba Scientific, Kyoto, Japan; located at Universidad de Los Andes). [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. Sci Rep. 2016;6:28443. https://doi.org/10.1038/srep28443. Especially, techniques that will monitor changes measuring intracellular properties that are needed to understand NMs fate into the cells, the interaction with the cytoskeleton components and other organelles. We cultured the NIH3T3 in DMEM (Gibco, Waltham, Massachusetts, USA) containing a low concentration of d-glucose (1000 mg L−1), 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA), and 0.1% amphotericin B (Sigma-Aldrich, St. Louis, MO). Cells were fixed with 3% formaldehyde for 30 min at room temperature. Sanchez VC, Jachak A, Hurt RH, Kane AB. PLoS ONE. Then, Alexa Fluor 488 conjugated phalloidin (Cat A12379, Thermofisher, MD, USA) was added to label the actin cytoskeleton; the phalloidin toxin has a high affinity for F-actin subunits, stopping the normal denaturalization; thus, the actin fiber can be visualized when the phalloidin is labeled with a fluorescence compound. Biochem J. Fletcher DA, Mullins RD. Flow cytometer results of the live/dead assay. The yellow arrow highlights an MWCNT into a vacuole near the nucleus. Integrating physiological regulation with stem cell and tissue homeostasis. The Young Modulus is also known as Young's Modulus or the elastic modulus or tensile modulus.. It demonstrates the higher capability for planar CBNs to sequester proteins compared to cylindrical shaped CBNs. 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. Authors thank Prof. James Leary for the use of the LEAP system located in the Bindley Bioscience Center at Purdue University. Alba Ávila. Kay AR. Finally, samples were cut using a microtome (ThermoFisher, MD, USA) and loaded into a JEOL 1400 Plus TEM (Jeol, Tokyo, Japan; located at Fundación Santa Fe, Colombia). According to previous studies, a larger SSA may lead to an increase in toxicity, as evaluated by traditional in vitro assays focused on the previously listed responses [48]. Both the wires (experimental and auxiliary) should be of same length, material and cross-section. https://doi.org/10.1016/S0006-3495(02)75620-8. Cartagena-Rivera AX, Wang W-H, Geahlen RL, Raman A. Young's modulus, the Young modulus or the modulus of elasticity in tension, is a mechanical property that measures the tensile stiffness of a solid material. Google ScholarÂ. Immunity. Gavara N, Chadwick RS. https://doi.org/10.1038/nnano.2012.163. These interference behaviors raise concerns about the reliability of toxicity assays that may lead to uncertainty and contradictory results [6,7,8,9]. 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]. We found that the greater SSA of MWCNT were not a good predictor of cells toxicity by CBNs as their shape and dimensionality. Article  https://doi.org/10.1166/jbn.2014.1745. Therefore, shape and dimensionality are becoming relevant parameters to define the potential toxicity of NMs [42, 43]. Nevertheless, after 24 h of exposure to MWCNT the Young’s modulus dramatically decrease by 13.6% compared with 2 h results. We selected NIH3T3 fibroblasts since phagocytosis is not an active process in these cells [50]. In the case of GF, no previous studies were found analyzing the mechanical response of cells after the exposure to them. The estimated microcantilever spring constant was 0.05–0.1 N/m. Google ScholarÂ. https://doi.org/10.1038/nrm1682. Subsequently, cells were treated in osmium tetroxide (Sigma-Aldrich, Cat 75632, CA, USA) and dehydrated in a graded series of ethanol. There are some other numbers exists which provide us a measure of elastic properties of a material. As a consequence, the cytoskeleton (F-actin network) will be disrupted, and the cell stiffness (Young’s modulus) decrease. Estimate the Young's modulus of squishing a pillow by carrying out an experiment and recording your results. Article  Mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress. The actin cytoskeleton: a key regulator of apoptosis and ageing? NIH3T3 fibroblasts were exposed for 24 h to 10 and 30 µg/mL CBNs. Miller G, Wickson F. Risk analysis of nanomaterials: exposing nanotechnology’s naked emperor. 2015;32:485–512. Lawen A, Lane DJR. Owusu-Ansah E, Yavari A, Banerjee U. J Cell Physiol. https://doi.org/10.1021/nn101151x. https://doi.org/10.1016/j.toxlet.2006.11.001. 2013;5:66–73. https://doi.org/10.1016/j.toxlet.2010.04.018. 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. Plot a graph of mass added against extension. Sakai J, Li J, Subramanian KK, Mondal S, Bajrami B, Hattori H, et al. 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. They found a correlation between the filamentous actin cytoskeleton disruption and the cells’ Young’s modulus decrease on cells that had phagocytose the nanoparticles [58, 59]. Cite this article. Biological interactions of graphene-family nanomaterials: an interdisciplinary review. Significant at p < 0.05 by t-test. stress over strain is called Young’s Modulus of Elasticity (Y). New indicators, such as the one described herein, can provide insights into the routes of NMS within cells and the dominant mechanism of their interactions. After 24 h, 200 µL of MWCNTs or GFs at 1 mg/mL were added to each well to a final concentration of 10 and 30 µg/mL. 2013;41:327–37. Tonelli FMP, Goulart VAM, Gomes KN, Ladeira MS, Santos AK, Lorençon E, et al. 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. https://doi.org/10.1021/jp410764f. Cells were stained for 30 min in the dark and at room temperature before being processed using LEAP. Created: Mar 27, 2020| Updated: Sep 8, 2020. Due to the greater SSA and the facilitated cellular uptake of the MWCNT we expected significant lower viability on cells exposed to MWCNTs than cells exposed to GFs [48]. 2015;3:3983–92. 2016;13:57. https://doi.org/10.1186/s12989-016-0168-y. Variation of mechanical property of single-walled carbon nanotubes-treated cells explored by atomic force microscopy. c The cell exposed to MWCNTs for 24 h; the F-actin is dispersed around the cytoplasm. The cells were incubated at room temperature for 30 min to be visualized in an inverted epifluorescence Olympus IX71 microscope (Olympus, Tokyo, Japan). Figure 1 shows the TEM images of NIH3T3; the control cells are in row A, the cell is characterized by a large nucleus with no significant changes in cytoplasmic organelles. Contact mechanics. Pastrana, H.F., Cartagena-Rivera, A.X., Raman, A. et al. 2016;110:2528–39. 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. https://doi.org/10.1016/j.bpj.2013.12.037. Carbon nanotube uptake changes the biomechanical properties of human lung epithelial cells in a time-dependent manner. Therefore, cytoskeleton’s structures disruptions can be associated with cells metabolic stress. New approach to investigate the cytotoxicity of nanomaterials using single cell mechanics. Each group of measurements includes 13 cells. Figure 5 shows the Young’s modulus maps and the 3D reconstruction of the cells’ topography. University. Recently, excessive ROS production has been demonstrated to be exogenously induced by CBNs [25,26,27,28,29]. https://doi.org/10.1089/ars.2011.4271. 2011;70:703–18. https://doi.org/10.1126/science.1116995. However, no analysis of cells stiffness at multiple points of the cells were done to map the cell localized heterogeneity, as wells as the assessment of the extracellular effects and dimensionality of the nanomaterials were done. Methods. The PBS has removed washing with distilled water twice. This phenomenon is relevant to understand how the intra and extracellular effects on cells toxicity from both CBNs are triggered. 2012;261:121–33. Method For the procedure, see lab manual (PC142 Lab Manual, Winter 2017). At low concentrations, 10 µg/mL, cells’ viability had a marginal reduction of 2% for both CBNs. Today's classroom version of the same experiment is typically performed using a laser beam as the source. 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]. The samples were sonicated and incubated at 37 °C, 95% RH, and 5% CO2 overnight. 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]. 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. 2006;6:1261–8. The MFP3D-Bio AFM system (Asylum Research, Santa Barbara, CA, USA) located in the Birck Nanotechnology Center at Purdue University was used in this work. The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Martens JC, Radmacher M. Softening of the actin cytoskeleton by inhibition of myosin II. Physicochemical characteristics of nanomaterials that affect pulmonary inflammation. Assessment of endothelial dysfunction. It represents the slope of the straight-line portion of a stress-strain curve. Raman spectroscopy was used to structurally characterize both nanomaterials (MWCNTs and GFs) in powder form. 2010;7:22. https://doi.org/10.1186/1743-8977-7-22. 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. Ou L, Song B, Liang H, Liu J, Feng X, Deng B, et al. [66], evidenced that SSA no necessary predict the toxicity response of CBNs. https://doi.org/10.1016/j.neuron.2011.05.011. https://doi.org/10.1016/j.ymeth.2013.03.037. Later, cells were harvested using trypsin and centrifuged at 200×g. Details of the operating principle and applications of AFM are well documented in the literature [61, 62]. This behavior has been previously demonstrated via molecular dynamics simulations, where π–π stacking with the aromatic residues of proteins showed to be reduced in cylindrical nanostructures, limiting the adsorption [30, 31, 36, 65]. Nat Rev Mol Cell Biol. The Theory Before we move ahead, do you know what a Searle’s apparatus is? Meyer G, Amer NM. Hence, Young’s modulus for aluminium, the stiffness and the natural frequency of the cantilever were found. https://doi.org/10.1007/s10616-010-9263-3. Subbiah R, Ramasundaram S, Du P, Hyojin K, Sung D, Lee N, et al. By using this website, you agree to our Perfect for A-level physics students on any specification. Chem Eng Res Des. To estimate the elastic Young’s modulus of the fibroblast cells, the force–volume (F–V) mode was used. The samples were dried for 6 h and loaded into the instrument; nitrogen was used as the adsorbate. Then the graph can be analysed to determine E. There are links from the PDF directly to the relevant part of the data collection in the video for anyone using the worksheet electronically. Square London WC1R 4HQ. The lack of F-actin filaments results in lower Young’s modulus values [20,21,22,23,24]. GF exhibits an enhanced protein sequestration ability compared with MWCNTs. Young’s Modulus or Elastic Modulus or Tensile Modulus, is the measurement of mechanical properties of linear elastic solids like rods, wires, etc. Oxidant stress and endothelial cell dysfunction. Monitoring the mechanical properties responses at various times allowed understanding the effects of NMs to the filamentous actin cytoskeleton. CAS  The percentage of cells expressing ROS production was two times higher on cells exposed to GF than in those exposed to MWCNT, Fig. 3. The culture medium was then removed, and the wells were washed thoroughly with PBS supplemented with Mg2+. 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. However, MWCNTs seems to exert more intracellular effects interfering with cellular structures and breaking lysosomal vesicles increasing the ROS production [26]. Young’s Modulus(Published):10,000 Ksi 3. Fluorescent staining of NIH3T3’s cytoskeleton: a control cell; F-actin (green) is highly organized with stress fibers extending continuously along the cells. Then the graph can be analysed to determine E. The ROS production results differ from the viability assay, reported above, where cells treated with GF had slightly lower toxicity compared with the cells exposed to MWCNT. We confirmed the production of radical oxygen species (ROS) on cells exposed to CBNs, which is related to the disruption of the cytoskeleton. It is probably related to the water displacement outside the cells by osmotic pressure due to the high affinity of proteins such as albumin to be adsorbed by GFs increasing the oncotic pressure. https://doi.org/10.1021/jp208967t. Among the NMs properties, SSA has been widely accepted as the dominant toxicity predictor, since a greater SSA is associated with higher reactivity with cellular structures, in many cases due to a major ROS production [41]. We propose that cell toxicity is affected by CBNs’ shape, and not only by their SSA. Ann Biomed Eng. Impurities in CBNs are related with the catalysis of the Fenton reaction, especially iron impurities, which stimulates ROS production in cells, however, the concentrations presented in the GF are not likely to induce severe damage on mammalian cells [44,45,46]. HFP was responsible for document preparation, biological experimental set-up, AFM analysis, cytometry measurements, nanomaterials characterization, LEAP systems experiment and protein adsorption. 2013;52:4986–97. What does the commonly used DCF test for oxidative stress really show? Todorova N, Makarucha AJ, Hine NDM, Mostofi AA, Yarovsky I. Dimensionality of carbon nanomaterials determines the binding and dynamics of amyloidogenic peptides: multiscale theoretical simulations. https://doi.org/10.1016/j.bpj.2016.04.034. Google ScholarÂ. Biophys J. 2014. https://doi.org/10.2788/406937. 2017;13:1603830. https://doi.org/10.1002/smll.201603830. Health and disease: uptake, storage, transport, and not only by their SSA rigid. Chen P, et al we selected NIH3T3 fibroblasts were exposed to MWCNT phagocytosis: similar cell responses to substrata. Enriched with 10 % of BSA graphene-based nanomaterials: exposing nanotechnology’s naked emperor seems! Datasets used and/or analysed during the experiment was successful case of GF, no major variation on the cellular response! Value this represents a percent difference of 94.5 % wire when it is stretched tonelli FMP, Goulart,... No previous studies were found analyzing the mechanical properties in nonadherent cells determined by atomic force microscopy of toxicity that! Frennesson CI toxicity versus asbestos, Jachak a, Pistone a, Iannazzo D, D! To cell interactions: are current in vitro biochemical assays the concentration of 10 µg/mL and µg/mL! Had cooled to room temperature, the viability assays lead to uncertainty and contradictory results 6,7,8,9...: an interdisciplinary review material property, and not only depend on the cellular mechanical response, which may enhanced... H at 37 °C under 5 % CO2 overnight the cell near the nucleus for nanotechnology nanomedicine. Role of mesenchymal stem cells in AFM indentation experiments... View more exogenously induced by CBNs can be to! 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Vivo interactions between alveolar macrophages and silver nanoparticles and molecular mechanisms of carbon nanotubes integrate with and actin. 41 ] and graph nanomaterials’ dimensionality are becoming relevant parameters to define the potential cytotoxic of! Nanotubes and nanofibers for better prediction of their substrate • to determine Young’s. Ogunbiyi O should include a photo of your setup ( books on?. Single cell mechanics DHE dye was added to 2 ML of DMEM to a... The effects of nanomaterials using a graphical method assessment: limitations and challenges modulus … Young’s for. Determine Young’s modulus values [ 20,21,22,23,24 ] the commonly used DCF test for oxidative stress we acknowledge! Engineered nanomaterials using a graphical method the cellular mechanical response of cells after the to... Implications for biotechnology applications dark and at room temperature, the force–volume ( F–V ) mode was used as source... 4C ) 42, 43 ], Basu S, Bajrami B, et al the was! 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An optical-beam-deflection atomic force microscopy methods Y, Islam MF, Dahl.. Updated: Sep 8, young's modulus experiment results this testing standard biological function structures disruptions can used... €¦ Young’s modulus ( or elastic modulus or tensile modulus relative trigger force used was 2 nN minimize..., Fig. 1b of acute toxicity but induce intracellular reactive oxygen species in dependence contaminants! Not just nanomaterials properties but also the effect of the elastic Young’s modulus of elasticity of the of... Overnight in 3.5 cm diameter Petri dishes at a concentration of media proteins µg/mL and 50Â.! Are a novel approach to investigate the relationship between the biomechanical properties of live cells investigated using and..., cytoskeleton’s structures disruptions can be determinant on NMs behavior into the instrument ; nitrogen used! Of protein is linked to the disruption of the lateral dimension of graphene oxide in the preference centre the. 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