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Additional file 1: Table S1. Ingenuity Pathway Analysis-based profiles of transcriptomic network-related genes in MNPs@SiO2(RITC)-treated cells. Table S2. RT-PCR and real-time PCR primer sequences for genes encoding transcriptomic network-related genes. Figure S1. Determination of size and homogeneity for MNPs@SiO2(RITC) and silica NPs using transmission electron microscope (TEM) analysis. TEM images of MNPs@SiO2(RITC) (a) and silica NPs (b). Scale bar = 50 nm. Figure S2. Evaluation of cytotoxicity in silica NPs and MNPs@SiO2(RITC) treated HEK293 cells. HEK293 cells were treated with silica NPs and MNPs@SiO2(RITC) for 12 h. The changes in cell viability were evaluated with MTS. Data represent mean ± SD of three independent experiments. N.S: Not significant. Figure S3. Low magnification laurdan GP images. Merged DIC and TIRFM images (upper panel) of HEK293 cells. Distributions of MNPs@SiO2(RITC) are shown in each lower panel. GP distributions ranged from −1.0 to 1.0. Scale bar= 2.5 µm. Figure S4. ROS generation in silica NPs and MNPs@SiO2(RITC)-treated cells. Evaluation of intracellular ROS generation using DCFH-DA after 12 h silica NPs and MNPs@SiO2(RITC) treatment in HEK293 cells. The intensity of non-oxidised DCFH-DA was used as a blank. Data represent mean ± SD of three independent experiments. *p < 0.05 vs non-treated control, #p < 0.05 compared between 0.1 and 1.0 µg/µl of NPs-treated cells. N.S: Not significant. Figure S5. Transcriptomic analysis of microarray in HEK293 cells treated with MNPs@SiO2(RITC) for 12 h. Network of lipid peroxidation and focal adhesion related genes was constructed algorithmically by IPA. (a) Transcriptome network of 0.1 µg/µl MNPs@SiO2(RITC)-treated cells and (b) prediction analysis for the network. Red and green areas indicate up- and downregulated genes, respectively. Orange and blue colours indicate activation and suppression. The lines indicate indirect (dotted) or direct (solid) relationship. Differentially expressed genes obtained from microarray data (genes with > 3-fold change) are shown. Figure S6. Metabotranscriptomic analysis of microarray and metabolite profiles in cells treated with 0.1 µg/µl MNPs@SiO2(RITC) for 12 h. (a) Lipid peroxidation and focal adhesion related genes and metabolites network were constructed algorithmically by IPA in 0.1 µg/µl MNPs@SiO2(RITC)-treated HEK293 cells and (b) prediction analysis for the network. Red and green areas indicate up- and downregulated genes, respectively. Orange and blue colours indicate activation and suppression, respectively. The lines indicate indirect (dotted) or direct (solid) relationship. Differentially expressed genes obtained from microarray data (> 3-fold change) and disturbances in metabolic profile (> 20% change) are shown. Figure S7. Metabotranscriptomic analysis of microarray and metabolite profile in cells treated with 1.0 µg/µl MNPs@SiO2(RITC) for 12 h. Lipid peroxidation and focal adhesion related genes and metabolites network were constructed algorithmically by IPA in 1.0 µg/µl MNPs@SiO2(RITC)-treated HEK293 cells. Red and green areas indicate up- and downregulated genes, respectively. Orange and blue colours indicate activation and suppression, respectively. The lines indicate indirect (dotted) or direct (solid) relationship. Differentially expressed genes obtained from microarray data (> 3-fold change) and disturbances in metabolic profile (> 20% change) are shown. Figure S8. Z-stack analysis for NPs treated HEK293 cells. The cells were treated with silica NPs and MNPs@SiO2(RITC) for 12 h. The locations of NPs were analysed with z-stack mode of confocal microscopy. Nucleus, blue; NPs, red; ubiquitin, green; lysosome, violet. Scale bar= 10 μm.

Background Nanoparticles are being used increasingly due to their unique physical and chemical properties and small size. It is well-known that nanoparticles cause side effects, however their biophysical assessment remains challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on the biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. Results Overdose (1.0 μg/µl) treatment of MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. During membrane damage, HEK293 cells were morphologically shrunk and aspect ratio of the cells were significantly decreased upon MNPs@SiO2(RITC) treatment. Each of traction force (measured in micropillar) was found to be increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, velocity of the cell movement was significantly decreased in MNPs@SiO2(RITC)-treated HEK293 cells. Moreover, intracellular ATP also decreased in a dose dependent manner upon MNPs@SiO2(RITC) treatment. To understand the biophysical changes in cells, we analysed transcriptome and metabolic profiles and generated metabotranscriptomics network. The network showed relationships among peroxidation of lipid, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipid and suppression of focal adhesion and cell movement.Conclusion Taken together, our results demonstrate that overdosage of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side effects.

BackgroundNanoparticles are being increasingly used in biomedical applications owing to their unique physical and chemical properties and small size. However, their biophysical assessment and evaluation of side-effects remain challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis.ResultsOverdose (1.0 μg/µL) treatment with MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. In addition, HEK293 cells were morphologically shrunk, and their aspect ratio was significantly decreased. We found that each traction force (measured in micropillar) was increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, the velocity of cell movement was also significantly decreased. Moreover, intracellular level of adenosine triphosphate (ATP) was also decreased in a dose-dependent manner upon treatment with MNPs@SiO2(RITC). To understand these biophysical changes in cells, we analysed the transcriptome and metabolic profiles and generated a metabotranscriptomics network, which revealed relationships among peroxidation of lipids, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipids and suppression of focal adhesion and cell movement.ConclusionTaken together, our results demonstrated that overdose of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side-effects.

BackgroundNanoparticles (NPs) can enter cells and cause cellular dysfunction. For example, reactive oxygen species generated by NPs can damage the cytoskeleton and impair cellular adhesion properties. Previously, we reported that cell spreading and protrusion structures such as lamellipodia and filopodia was reduced when cells are treated with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)), even at 0.1 μg/μL. These protruded structures are involved in a cell’s rigidity sensing, but how these NPs affect rigidity sensing is unknown.ResultsHere, we report that the rigidity sensing of human embryonic kidney (HEK293) cells was impaired even at 0.1 μg/μL of MNPs@SiO2(RITC). At this concentration, cells were unable to discern the stiffness difference between soft (5 kPa) and rigid (2 MPa) flat surfaces. The impairment of rigidity sensing was further supported by observing the disappearance of locally contracted elastomeric submicron pillars (900 nm in diameter, 2 μm in height, 24.21 nN/μm in stiffnessk) under MNPs@SiO2(RITC) treated cells. A decrease in the phosphorylation of paxillin, which is involved in focal adhesion dynamics, may cause cells to be insensitive to stiffness differences when they are treated with MNPs@SiO2(RITC).ConclusionsOur results suggest that NPs may impair the rigidity sensing of cells even at low concentrations, thereby affecting cell adhesion and spreading.

Additional file 1: Fig S1. MNPs@SiO2(RITC) intensity per μm2 area of the cell on soft and rigid PDMS surfaces with MNPs@SiO2(RITC) at different concentrations (0-1 µg/μL) for 12 h. (n=5 cells), NS (not significant) P >0.05, *** P

For stem cell-based therapies, the fate and distribution of stem cells should be traced using non-invasive or histological methods and a nanomaterial-based labelling agent. However, evaluation of the biophysical effects and related biological functions of nanomaterials in stem cells remains challenging. Here, we aimed to investigate the biophysical effects of nanomaterials on stem cells, including those on membrane fluidity, using total internal reflection fluorescence microscopy, and traction force, using micropillars of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) labelled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)). Furthermore, to evaluate the biological functions related to these biophysical changes, we assessed the cell viability, reactive oxygen species (ROS) generation, intracellular cytoskeleton, and the migratory activity of MNPs@SiO2(RITC)-treated hBM-MSCs. Compared to that in the control, cell viability decreased by 10% and intracellular ROS increased by 2-fold due to the induction of 20% higher peroxidized lipid in hBM-MSCs treated with 1.0 &micro, g/&micro, L MNPs@SiO2(RITC). Membrane fluidity was reduced by MNPs@SiO2(RITC)-induced lipid oxidation in a concentration-dependent manner. In addition, cell shrinkage with abnormal formation of focal adhesions and ~30% decreased total traction force were observed in cells treated with 1.0 &micro, L MNPs@SiO2(RITC) without specific interaction between MNPs@SiO2(RITC) and cytoskeletal proteins. Furthermore, the migratory activity of hBM-MSCs, which was highly related to membrane fluidity and cytoskeletal abnormality, decreased significantly after MNPs@SiO2(RITC) treatment. These observations indicated that the migratory activity of hBM-MSCs was impaired by MNPs@SiO2(RITC) treatment due to changes in stem-cell biophysical properties and related biological functions, highlighting the important mechanisms via which nanoparticles impair migration of hBM-MSCs. Our findings indicate that nanoparticles used for stem cell trafficking or clinical applications should be labelled using optimal nanoparticle concentrations to preserve hBM-MSC migratory activity and ensure successful outcomes following stem cell localisation.