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Investigation of cellular responses upon interaction with silver nanoparticles.

Subbiah R, Jeon SB, Park K, Ahn SJ, Yun K - Int J Nanomedicine (2015)

Bottom Line: When compared with kanamycin, AgNPs exhibited moderate antibacterial activity.The degree to which cellular morphology was altered directly proportional to the level of AgNP-induced cytotoxicity.Together, these data suggest that atomic force microscopy can be used as a potential tool to develop a biomechanics-based biomarker for the evaluation of NP-dependent cytotoxicity and cytopathology.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Republic of Korea ; Department of Biomedical Engineering, Korea University of Science and Technology, Daejon, Republic of Korea.

ABSTRACT
In order for nanoparticles (NPs) to be applied in the biomedical field, a thorough investigation of their interactions with biological systems is required. Although this is a growing area of research, there is a paucity of comprehensive data in cell-based studies. To address this, we analyzed the physicomechanical responses of human alveolar epithelial cells (A549), mouse fibroblasts (NIH3T3), and human bone marrow stromal cells (HS-5), following their interaction with silver nanoparticles (AgNPs). When compared with kanamycin, AgNPs exhibited moderate antibacterial activity. Cell viability ranged from ≤ 80% at a high AgNPs dose (40 µg/mL) to >95% at a low dose (10 µg/mL). We also used atomic force microscopy-coupled force spectroscopy to evaluate the biophysical and biomechanical properties of cells. This revealed that AgNPs treatment increased the surface roughness (P<0.001) and stiffness (P<0.001) of cells. Certain cellular changes are likely due to interaction of the AgNPs with the cell surface. The degree to which cellular morphology was altered directly proportional to the level of AgNP-induced cytotoxicity. Together, these data suggest that atomic force microscopy can be used as a potential tool to develop a biomechanics-based biomarker for the evaluation of NP-dependent cytotoxicity and cytopathology.

No MeSH data available.


Related in: MedlinePlus

Morphology of cells treated with AgNPs.Notes: Optical DIC (A–F), SEM (G–L), and 3D Bio-AFM (M–R) images of control and NP-treated HS-5, NIH3T3, and A549 cells. (M–R) Cells are scanned in an order of 100×100 µm (dark gold), 50×50 µm (light blue), and 10×10 µm (green fluorescence) scale, respectively.Abbreviations: AgNPs, silver nanoparticles; AFM, atomic force microscopy; DIC, differential interference contrast; NPs, nanoparticles; SEM, scanning electron microscopy.
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f3-ijn-10-191: Morphology of cells treated with AgNPs.Notes: Optical DIC (A–F), SEM (G–L), and 3D Bio-AFM (M–R) images of control and NP-treated HS-5, NIH3T3, and A549 cells. (M–R) Cells are scanned in an order of 100×100 µm (dark gold), 50×50 µm (light blue), and 10×10 µm (green fluorescence) scale, respectively.Abbreviations: AgNPs, silver nanoparticles; AFM, atomic force microscopy; DIC, differential interference contrast; NPs, nanoparticles; SEM, scanning electron microscopy.

Mentions: The morphology and topography of untreated HS-5, NIH3T3, A549 cells (control) and those treated with 40 µg/mL of AgNPs were evaluated by optical microscopy, SEM, and AFM, as shown in Figure 3. The cells were widespread, and potential candidates for AFM cell imaging were chosen using optical microscopy (Figure 3A–F). Following optical microscopy, no obvious changes were found in cell structure before and after treatment with AgNPs. In contrast to optical microscopy, SEM images revealed obvious changes in shape and structure following treatment with AgNPs. All cells had obtrusive filopodia, lamellipodia, and invadopodia, as confirmed by SEM imaging (Figure 3G–L). However, measurements of height, roughness, and cell–cell interactions were inconsistent and we concluded that SEM fails to provide any direct quantitative measurement. Hence, we utilized AFM to determine cellular properties such as height and roughness in response to AgNPs (Figures 3M–R and 4A), as this technique has been previously used in a similar context. The height images of control and AgNP-treated cells, obtained by AFM, provide a representative topography of the population. Figure 3M–R displays the 3D height images of control and AgNP-treated cells at 100×100 µm scale (Figure 3, third row; gold), at 50×50 µm (fourth row; blue) and 10×10 µm (fifth row; green), respectively. Morphological observation of the NP-treated cells compared with controls confirmed the changes in cell structure. The average height of cells varied among tested groups, and depended on the stretched and shrunken patterns of individual cell types. The average height of HS-5, NIH3T3, and A549 was 1.7 µm, 1.5 µm, and 2.4 µm, respectively. However, the average height of cells increased to 2.25 µm, 1.7 µm, and 2.7 µm for HS-5, NIH3T3, and A549, respectively, after AgNPs treatment. We inferred that AgNPs interact at the cell surface and induce cell rounding that causes an increase in cell height (Figure 3). These images coincide well with SEM images of cells, in which AgNPs were visualized on the surface of cells (Figure 3H, J, L, N, P, and R). The RMS roughness of the cell surface was a robust indicator of the properties of the cell surface before and after treatment with NPs (Figure 4A). Most importantly, AgNPs treatment increased the RMS roughness of cells, indicating that AgNPs were indeed adhering to the surface of cells (Figure 4A). The roughness of control HS-5, NIH3T3, and A549 cells was ~259 nm, ~347 nm, and ~227 nm, whereas after AgNPs treatment this increased tô360 nm, ~448 nm, and ~365 nm, respectively. We conclude that AgNPs alter the cell surface by adherence or internalization and thereby increase roughness. The size and charge of the AgNPs determine the way that they interact with cells. The interaction of AgNPs with cells was directly proportional to the surface roughness. Several studies have demonstrated that cell morphology is altered upon NPs treatment. For example, Pletikapic et al found that AgNPs interact with marine diatoms and cause local intracellular damage without disintegration of the cell wall.37 Similarly, Ogneva et al demonstrated that silica-based NPs directly interact with mesenchymal stem cell membranes, trigger a structural reorganization of cortical cytoskeleton, and cause morphological cellular changes.16 Our data are consistent with those of these previous studies and indicate that AgNPs remain at the surface of cells or are internalized into the cytoplasmic membrane, but do not disrupt the cell membrane.


Investigation of cellular responses upon interaction with silver nanoparticles.

Subbiah R, Jeon SB, Park K, Ahn SJ, Yun K - Int J Nanomedicine (2015)

Morphology of cells treated with AgNPs.Notes: Optical DIC (A–F), SEM (G–L), and 3D Bio-AFM (M–R) images of control and NP-treated HS-5, NIH3T3, and A549 cells. (M–R) Cells are scanned in an order of 100×100 µm (dark gold), 50×50 µm (light blue), and 10×10 µm (green fluorescence) scale, respectively.Abbreviations: AgNPs, silver nanoparticles; AFM, atomic force microscopy; DIC, differential interference contrast; NPs, nanoparticles; SEM, scanning electron microscopy.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556294&req=5

f3-ijn-10-191: Morphology of cells treated with AgNPs.Notes: Optical DIC (A–F), SEM (G–L), and 3D Bio-AFM (M–R) images of control and NP-treated HS-5, NIH3T3, and A549 cells. (M–R) Cells are scanned in an order of 100×100 µm (dark gold), 50×50 µm (light blue), and 10×10 µm (green fluorescence) scale, respectively.Abbreviations: AgNPs, silver nanoparticles; AFM, atomic force microscopy; DIC, differential interference contrast; NPs, nanoparticles; SEM, scanning electron microscopy.
Mentions: The morphology and topography of untreated HS-5, NIH3T3, A549 cells (control) and those treated with 40 µg/mL of AgNPs were evaluated by optical microscopy, SEM, and AFM, as shown in Figure 3. The cells were widespread, and potential candidates for AFM cell imaging were chosen using optical microscopy (Figure 3A–F). Following optical microscopy, no obvious changes were found in cell structure before and after treatment with AgNPs. In contrast to optical microscopy, SEM images revealed obvious changes in shape and structure following treatment with AgNPs. All cells had obtrusive filopodia, lamellipodia, and invadopodia, as confirmed by SEM imaging (Figure 3G–L). However, measurements of height, roughness, and cell–cell interactions were inconsistent and we concluded that SEM fails to provide any direct quantitative measurement. Hence, we utilized AFM to determine cellular properties such as height and roughness in response to AgNPs (Figures 3M–R and 4A), as this technique has been previously used in a similar context. The height images of control and AgNP-treated cells, obtained by AFM, provide a representative topography of the population. Figure 3M–R displays the 3D height images of control and AgNP-treated cells at 100×100 µm scale (Figure 3, third row; gold), at 50×50 µm (fourth row; blue) and 10×10 µm (fifth row; green), respectively. Morphological observation of the NP-treated cells compared with controls confirmed the changes in cell structure. The average height of cells varied among tested groups, and depended on the stretched and shrunken patterns of individual cell types. The average height of HS-5, NIH3T3, and A549 was 1.7 µm, 1.5 µm, and 2.4 µm, respectively. However, the average height of cells increased to 2.25 µm, 1.7 µm, and 2.7 µm for HS-5, NIH3T3, and A549, respectively, after AgNPs treatment. We inferred that AgNPs interact at the cell surface and induce cell rounding that causes an increase in cell height (Figure 3). These images coincide well with SEM images of cells, in which AgNPs were visualized on the surface of cells (Figure 3H, J, L, N, P, and R). The RMS roughness of the cell surface was a robust indicator of the properties of the cell surface before and after treatment with NPs (Figure 4A). Most importantly, AgNPs treatment increased the RMS roughness of cells, indicating that AgNPs were indeed adhering to the surface of cells (Figure 4A). The roughness of control HS-5, NIH3T3, and A549 cells was ~259 nm, ~347 nm, and ~227 nm, whereas after AgNPs treatment this increased tô360 nm, ~448 nm, and ~365 nm, respectively. We conclude that AgNPs alter the cell surface by adherence or internalization and thereby increase roughness. The size and charge of the AgNPs determine the way that they interact with cells. The interaction of AgNPs with cells was directly proportional to the surface roughness. Several studies have demonstrated that cell morphology is altered upon NPs treatment. For example, Pletikapic et al found that AgNPs interact with marine diatoms and cause local intracellular damage without disintegration of the cell wall.37 Similarly, Ogneva et al demonstrated that silica-based NPs directly interact with mesenchymal stem cell membranes, trigger a structural reorganization of cortical cytoskeleton, and cause morphological cellular changes.16 Our data are consistent with those of these previous studies and indicate that AgNPs remain at the surface of cells or are internalized into the cytoplasmic membrane, but do not disrupt the cell membrane.

Bottom Line: When compared with kanamycin, AgNPs exhibited moderate antibacterial activity.The degree to which cellular morphology was altered directly proportional to the level of AgNP-induced cytotoxicity.Together, these data suggest that atomic force microscopy can be used as a potential tool to develop a biomechanics-based biomarker for the evaluation of NP-dependent cytotoxicity and cytopathology.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Republic of Korea ; Department of Biomedical Engineering, Korea University of Science and Technology, Daejon, Republic of Korea.

ABSTRACT
In order for nanoparticles (NPs) to be applied in the biomedical field, a thorough investigation of their interactions with biological systems is required. Although this is a growing area of research, there is a paucity of comprehensive data in cell-based studies. To address this, we analyzed the physicomechanical responses of human alveolar epithelial cells (A549), mouse fibroblasts (NIH3T3), and human bone marrow stromal cells (HS-5), following their interaction with silver nanoparticles (AgNPs). When compared with kanamycin, AgNPs exhibited moderate antibacterial activity. Cell viability ranged from ≤ 80% at a high AgNPs dose (40 µg/mL) to >95% at a low dose (10 µg/mL). We also used atomic force microscopy-coupled force spectroscopy to evaluate the biophysical and biomechanical properties of cells. This revealed that AgNPs treatment increased the surface roughness (P<0.001) and stiffness (P<0.001) of cells. Certain cellular changes are likely due to interaction of the AgNPs with the cell surface. The degree to which cellular morphology was altered directly proportional to the level of AgNP-induced cytotoxicity. Together, these data suggest that atomic force microscopy can be used as a potential tool to develop a biomechanics-based biomarker for the evaluation of NP-dependent cytotoxicity and cytopathology.

No MeSH data available.


Related in: MedlinePlus