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Turn-on small molecule fluorescence sandwich immunoassay.

Containing Supplementary Figures S1 - S12

Clinical trials are evaluating the efficacy of anti-TIGIT for use as single-agent therapy or in combination with programmed death 1 (PD-1)/programmed death-ligand 1 blockade. How and whether a TIGIT blockade will synergize with immunotherapies is not clear. Here, we show that CD226loCD8+ T cells accumulate at the tumor site and have an exhausted phenotype with impaired functionality. In contrast, CD226hiCD8+ tumor-infiltrating T cells possess greater self-renewal capacity and responsiveness. Anti-TIGIT treatment selectively affects CD226hiCD8+ T cells by promoting CD226 phosphorylation at tyrosine 322. CD226 agonist antibody-mediated activation of CD226 augments the effect of TIGIT blockade on CD8+ T-cell responses. Finally, mFOLFIRINOX treatment, which increases CD226hiCD8+ T cells in patients with pancreatic ductal adenocarcinoma, potentiates the effects of TIGIT or PD-1 blockade. Our results implicate CD226 as a predictive biomarker for cancer immunotherapy and suggest that increasing numbers of CD226hiCD8+ T cells may improve responses to anti-TIGIT therapy.

Background Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. Methods Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. Results Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. Conclusions Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity. Graphical Abstract

Background Nanoparticles have been used for biomedical applications, including drug delivery, diagnosis, and imaging based on their unique properties derived from small size and large surface-to-volume ratio. However, concerns regarding unexpected toxicity due to the localization of nanoparticles in the cells are growing. Herein, we quantified the number of cell-internalized nanoparticles and monitored their cellular localization, which are critical factors for biomedical applications of nanoparticles. Methods This study investigates the intracellular trafficking of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] in various live single cells, such as HEK293, NIH3T3, and RAW 264.7 cells, using site-specific direct stochastic optical reconstruction microscopy (dSTORM). The time-dependent subdiffraction-limit spatial resolution of the dSTORM method allowed intracellular site-specific quantification and tracking of MNPs@SiO2(RITC). Results The MNPs@SiO2(RITC) were observed to be highly internalized in RAW 264.7 cells, compared to the HEK293 and NIH3T3 cells undergoing single-particle analysis. In addition, MNPs@SiO2(RITC) were internalized within the nuclei of RAW 264.7 and HEK293 cells but were not detected in the nuclei of NIH3T3 cells. Moreover, because of the treatment of the MNPs@SiO2(RITC), more micronuclei were detected in RAW 264.7 cells than in other cells. Conclusion The sensitive and quantitative evaluations of MNPs@SiO2(RITC) at specific sites in three different cells using a combination of dSTORM, transcriptomics, and molecular biology were performed. These findings highlight the quantitative differences in the uptake efficiency of MNPs@SiO2(RITC) and ultra-sensitivity, varying according to the cell types as ascertained by subdiffraction-limit super-resolution microscopy. Graphical Abstract

An integrated multifunctional light-sheet nanoscopy (iMLSN) combined with differential interference contrast, total internal reflection, epifluorescence, a super-resolution radial fluctuation-stream module, and a wavelength-dependent light sheet was developed to simultaneously realize the six-dimensional (6D) vector-valued (three coordinates + rotational dynamics (azimuth and elevation angles) + transport speed) tracking of anisotropic nanoparticles in single living cells. The wavelength-dependent asymmetric scattering of light by gold nanorods was used to trigger signals depending on the polarizer angle, and real-time photo-switching was achieved by turning the polarizer, obtaining a series of super-resolution images, and tracking using different polarization directions and two channels. This technique was employed to directly observe native gold nanorods (AuNRs; 5 nm diameter × 15 nm length) and surface-functionalized AuNRs during their endocytosis and transport at the upper and attaching side membrane regions of single living cells, revealing that the AuNRs bound to the membrane receptors. The nanorods were subsequently internalized and transported away from the original entry spots. Detailed dynamic information regarding the rotation properties and endocytosis speed during the transmembrane process was also acquired for each region. The developed technique can be considered useful for the real-time monitoring of intracellular transport at various regions in single living cells, as well as for 6D vector-valued non-fluorescence super-resolution imaging and tracking. Graphical Abstract

Dysregulation of microRNAs (miRNAs) has been associated with the pathophysiology of myelodysplastic syndrome (MDS). Trisomy 8 is the most frequent chromosomal abnormalities in Korean patients with MDS. We investigated the dysregulation of miR-597-5p, located on chromosome 8, which is reported to induce cell death in numerous cancers.We compared the expression profiles of miR-597-5p among 65 MDS patients and 11 controls, and analyzed the in vitro effects of miR-597 on leukemic cells using an acute myeloid leukemia cell line transfected with miR-597.We found that miR-597-5p levels were upregulated 4.05-fold in MDS patients compared to those in controls. In vitro study results demonstrated that transfection with a miR-597 mimic induced apoptosis through downregulation of FOS like 2 (FOSL2).These findings suggest that upregulation of miR-597 induces apoptosis and that miR-597 has a possible role in the pathophysiology of MDS.

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent fluorescence-free three-dimensional light-sheet super-resolution microscopy (3D LSRM) with dual-wavelength illumination sources was investigated to study the distance of Mito-ER contacts in various live cells. To detect wavelength-dependent scattering, 12 nm gold nanoparticles (AuNPs) and 20 nm silver nanoparticles (AgNPs) as fluorescence-free nanoprobes were conjugated with Mito and ER. The cubic spline algorithm-based method showed improved localization precision in lateral and axial directions compared with that for previously used least squares and least cubic algorithms. The cubic spline-based depth-dependent localization was applied to the spatial localization of nanoprobes in super-resolution images, in which the average distance of Mito and ER was 22.4 nm in HeLa cells, 22.2 nm in RAW264.7 macrophage cells, 21.9 nm in AGS cells, 21.4 nm in HT29 cells, and 21.3 nm in HEK293 cells. The distances were ∼12% larger than those previously determined by electron microscopy, which demonstrated that this method was accessible and reliable for studying the intracellular structures of various live cells at the subdiffraction limit resolution.

BACKGROUND: Pembrolizumab is a highly selective IgG4 kappa isotype monoclonal antibody against the programmed cell death-1 (PD-1) molecule. In the treatment of non-small cell lung cancer (NSCLC), pembrolizumab has demonstrated significant efficacy, significant survival outcomes, long-lasting responses, and a good safety profile compared with cytotoxic chemotherapy. CASE SUMMARY: A 79-year-old Korean male presented with a left side palpable neck mass. An ultrasound-guided core-needle biopsy of the largest neck mass was performed, and squamous cell carcinoma was confirmed based on the histological and immunohistochemical findings. He was diagnosed with squamous cell carcinoma of the lung with multiple lymph nodes and rib metastases (T1N3M1b, Stage IVA) using enhanced chest computed tomography and (18)F-fluorodeoxyglucose positron emission/computed tomography. After 4 cycles of gemcitabine and carboplatin, we clinically judged the disease as progressive. Owing to the high PD-1 expression demonstrated by the patient, pembrolizumab was initiated (200 mg every 3 wk). After 3 cycles of pembrolizumab, a complete response was achieved. At the 4(th) cycle of pembrolizumab, the white blood cell count was markedly elevated. Peripheral blood smear analysis and bone marrow biopsy were performed. The patient was diagnosed with acute myelomonocytic leukemia. CONCLUSION: We present the first report of acute myelomonocytic leukemia during pembrolizumab treatment in an NSCLC patient; the mechanism remains unknown.

Clinical trials are evaluating the efficacy of anti-TIGIT for use as single-agent therapy or in combination with programmed death 1 (PD-1)/programmed death-ligand 1 blockade. How and whether a TIGIT blockade will synergize with immunotherapies is not clear. Here, we show that CD226loCD8+ T cells accumulate at the tumor site and have an exhausted phenotype with impaired functionality. In contrast, CD226hiCD8+ tumor-infiltrating T cells possess greater self-renewal capacity and responsiveness. Anti-TIGIT treatment selectively affects CD226hiCD8+ T cells by promoting CD226 phosphorylation at tyrosine 322. CD226 agonist antibody-mediated activation of CD226 augments the effect of TIGIT blockade on CD8+ T-cell responses. Finally, mFOLFIRINOX treatment, which increases CD226hiCD8+ T cells in patients with pancreatic ductal adenocarcinoma, potentiates the effects of TIGIT or PD-1 blockade. Our results implicate CD226 as a predictive biomarker for cancer immunotherapy and suggest that increasing numbers of CD226hiCD8+ T cells may improve responses to anti-TIGIT therapy.

Activated hypoxia-inducible factor-1alpha (HIF-1α) plays an important role in the adaptive response of tumor cells to oxygen changes through the transcriptional activation of genes that regulate important biological processes required for tumor survival and progression. In this study, we developed an ultrasensitive hypoxia sensor based on read-out with quantum dots on a gold nanodisc (quantum dot-linked immunosandwich assay, QLISA) with excellent selectivity for HIF-1α. The immunoassay platform was established by comparing the immune response results using Qdot525 as a detection nanoprobe instead of a fluorescent dye (Alexa488) (fluorescent-linked immunosandwich assay, FLISA). In addition, using three-dimensional total internal reflection fluorescence microscopy, the platform was optically sectioned along the

The thyroid gland, which regulates the metabolism of the human body, has a sophisticated feedback system that induces the secretion of thyroid-stimulating hormone (TSH) to regulate the levels of triiodothyronine (T3) and thyroxine (T4). In this study, a single-molecule fourplex nanoimmunosensor was developed for the simultaneous quantitative analysis of TSH, T3, and T4. The three thyroid hormones were detected with a high signal-to-noise ratio in an evanescent field using laser-induced total internal reflection fluorescence. Additionally, the use of gold nanoislands for the detection of molecular interactions between thyroid hormones and antibodies labeled with quantum dots minimized the background noise from the substrate compared with the use of microislands or microwells. The nanoimmunosensor exhibited excellent detection limits of 114-193 yM (yoctomolar = 10

The performance of the ASTA MicroIDSys system (ASTA, Suwon, South Korea), a new matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, was evaluated for the identification of viridans group streptococci (VGS) and compared with the results obtained with the Bruker Biotyper system (Bruker Daltonics, Hamburg, Germany). A total of 106 Streptococcus reference strains belonging to 24 species from the bacterial strain bank was analyzed using the two MALDI-TOF MS systems. Of the 106 reference strains tested, ASTA MicroIDSys and Bruker Biotyper correctly identified 84.9% and 81.1% at the species level, 100% and 97.2% at the group level and 100% and 98.1% at the genus level, respectively. The difference between the two systems was not statistically significant (P = 0.289). Out of 24 species, 13 species were accurately identified to the species level with 100% accurate identification rates with both systems. The accurate identification rates at the species level of ASTA MicroIDSys and Bruker Biotyper were 100% and 87.5% for the S. anginosus group; 78.4% and 73.5% for the S. mitis group; 91.7% and 91.7% for the S. mutans group; and 100% and 100% for the S. salivarius group, respectively. The ASTA MicroIDSys showed an identification performance equivalent to that of the Bruker Biotyper for VGS. Therefore, it would be useful for the identification of VGS strains in clinical microbiology laboratories. This article is protected by copyright. All rights reserved.

Studies on the properties of plasmonic materials and new chemical biological sensors are very important to our understanding of biological systems in single cells. Since the advent of nanofabrication techniques, plasmonic nanostructures have been widely used in various research fields including material science, biology, and medicine. The optical, electronic and catalytic properties of plasmonic nanostructures can be tuned very broadly by controlling their size, shape, and composition. Traditional optical biosensors based on the plasmonic properties of nanostructures have led to the development of high sensitivity, temporal-spatially resolved, and high-throughput plasmonic optical imaging techniques through assembly optimization of light sources, detectors, and other optical components. These techniques have been applied to single-cell research. Here, we briefly provide various applications of plasmonic nanostructures used for single-cell analysis and summarize the advanced detection and imaging techniques at the single-cell level.

MicroRNA (miRNA) dysregulation contributes to myelodysplastic syndromes (MDS), and apoptosis is one of the pathogenic features of MDS. We investigated the dysregulation of miRNA expression and its biological significance in MDS. We compared the expression profiles of miRNAs encoded by chromosome 8 in 65 patients with MDS and 11 controls, and analyzed the in vitro effect of the upregulated miRNA expression. We found that compared to the controls, miR-661 was upregulated by 5.28-fold in patients with MDS. Patients with high miR-661 expression showed significantly decreased overall survival. In vitro study results demonstrated that transfection with a miR-661 mimic induced apoptosis through the activation of p53. These findings suggest that high miR-661 expression can be associated with decreased overall survival and recapitulates apoptosis, the characteristic feature of MDS.

A fluorescence-free ultrasensitive norovirus nanoimmunosensor was investigated based on gradient-fitting super-localization by three-dimensional (3D) dual-view light sheet microscopy (DV-LSM). A highly contagious norovirus was detected by immunoreaction with 100-nm gold nanospots (AuNSs) and 40-nm silver nanoparticles (AgNPs) as imaging probes. Dual-view setup was added to the optical path to simultaneously image AgNP-labelled norovirus and AuNSs in real time. A cylindrical lens and dual-view identified axial distance differences between AuNS and AgNP images as a function of an elliptically distorted point spread function (PSF). Differences between PSFs in each plane at 10 nm slicing intervals were distinguished by gradient-fitting algorithm-based super-localization. This approach showed highly enhanced detection sensitivity and super-localization in the axial direction for norovirus. The axial distance between AuNS and AgNP in norovirus immnosensor was 23 ± 3 nm for 7.8 zM, and the distance increased with increased concentration. The method had an excellent limit of detection (LOD) of 7.8 zM with a wide linear dynamic range of 7.8 zM–240 aM. The LOD was 106 to 2,300,000 times lower than previous methods. Norovirus was detected in lettuce leaf extraction at 14.3 zM with 99.87% recovery. The 3D DV-LSM method with gradient-fitting super-localization was an effective method for ultra-trace norovirus detection at the single-molecule level with highly precise axial resolution.

Endogenous fluorescence imaging techniques are key for modern single-molecule quantification without the use of additional labeling probes. However, the drawback of weak fluorescence signal is the primary challenge in meeting the ever-increasing demands of single-molecule detection. Here, we report a simple and reliable method that provides up to ∼100-fold uniform fluorescence enhancement of endogenous fluorescence of the capsaicinoid molecule. The method is based on a single nanoparticle plasmon-amplified endogenous fluorescence nanospectroscopic sensor (PAEFS). This work demonstrated the applicability of PAEFS in refining sensitivity at the single-molecule level by showing ultralow limits of detection (10

Nanoparticles are a useful material in biomedicine given their unique properties and biocompatibility; however, there is increasing concern regarding the potential toxicity of nanoparticles with respect to cell metabolism. Some evidence suggests that nanoparticles can disrupt glucose and energy homeostasis. In this study, we investigated the metabolomic, transcriptomic, and integrated effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] on glucose metabolism in human embryonic kidney 293 (HEK293) cells. Using gas chromatography–tandem mass spectrometry, we analysed the metabolite profiles of 14 organic acids (OAs), 20 amino acids (AAs), and 13 fatty acids (FAs) after treatment with 0.1 or 1.0 µg/µl MNPs@SiO2(RITC) for 12 h. The metabolic changes were highly related to reactive oxygen species (ROS) generation and glucose metabolism. Additionally, effects on the combined metabolome and transcriptome or “metabotranscriptomic network” indicated a relationship between ROS generation and glucose metabolic dysfunction. In the experimental validation, MNPs@SiO2(RITC) treatment significantly decreased the amount of glucose in cells and was associated with a reduction in glucose uptake efficiency. Decreased glucose uptake efficiency was also related to ROS generation and impaired glucose metabolism in the metabotranscriptomic network. Our results suggest that exposure to high concentrations of MNPs@SiO2(RITC) produces maladaptive alterations in glucose metabolism and specifically glucose uptake as well as related metabolomic and transcriptomic disturbances via increased ROS generation. These findings further indicate that an integrated metabotranscriptomics approach provides useful and sensitive toxicological assessment for nanoparticles.

Despite the large body of research on workplace mistreatment, surprisingly few studies have examined the interaction effect of multiple interpersonal stressors on employee outcomes. To fill this gap, our research aimed to test the moderating effects of coworker incivility and customer incivility on the relationship between abusive supervision, emotional exhaustion, and job performance. Analyses conducted on 651 South Korean frontline service employees revealed that abusive supervision exerted a significant indirect effect on job performance through emotional exhaustion. Customer incivility strengthened the positive relationship between abusive supervision and emotional exhaustion, as well as the indirect effect of abusive supervision on job performance through emotional exhaustion. Our post hoc analysis demonstrated a three-way interaction between abusive supervision, coworker incivility, and customer incivility, the relationship between abusive supervision and emotional exhaustion was significantly positive only when coworker incivility was high and customer incivility was low. We discuss the implications of our findings for theory and practice.

Various mechanisms underlying antimicrobial resistance in Acinetobacter baumannii have been reported. There exists controversy regarding the relationships between efflux pump activity, biofilm formation, and antimicrobial resistance in A. baumannii. In this study, we investigated the relative expression of RND efflux pump genes, H33342 efflux activity, and biofilm-forming activity in 120 A. baumannii clinical isolates, examined their potential relationships with one another, and then statistically analyzed their effects on antibiotic resistance. High adeB expression and high H33342 efflux activity were correlated with low biofilm-forming activity. High adeB expression was significantly correlated with resistance to tigecycline and cefotaxime, but not with the multidrug resistance (MDR) phenotype. Importantly, only high adeJ expression was significantly correlated with the MDR phenotype, and was observed to be correlated with resistance to various antibiotics. However, we found no significant correlation between adeJ expression and biofilm-forming activity. Further, adeG expression was found to not be correlated with antibiotic resistance and biofilm-forming activity. The results of multivariate analysis showed that adeB overexpression and high H33342 efflux activity are related to biofilm-forming activity, and only adeJ overexpression is significantly associated with the MDR phenotype, highlighting the importance of adeJ overexpression.

High level of plasma TGF-β is significantly correlated with poor outcomes with approved immune-oncology therapeutics. Poor clinical results for ALK5 inhibitors and bifunctional fusion protein targeting TGF-β and PD-L1 are also being reported in several clinical trials. Therefore, a novel therapeutic with superior efficacy differentiated from previous TGF-β modulators will demonstrate significant clinical outcomes in combination with immuno-oncology therapeutics. In TME, TGF-β and VEGF synergistically induce eradication of immunogenic tumors. Therefore, a dual-inhibitor targeting both ALK5 and VEGFR2 has high potential to demonstrate effective clinical efficacy against TGF-β- and VEGF-enriched tumors as a single or combination treatment. TU2218 is a highly potent, orally available inhibitor against ALK5 and VEGFR2 with an IC50 of 1.2 nM and 4.9 nM respectively. In a human PBMC assay, 0.5-1 μM TU2218 treatment significantly enhanced IFN-γ production or IFN-γ-producing T lymphocytes number by TGF-β-driven immunosuppression, but vactosertib, galunisertib and TGF-β neutralizing antibody did not have significant effects. In a human regulatory T cells assay, CD4+CD25+ Tregs suppressed the proliferation of naïve CD4+CD25- T cells after TCR triggering. In contrast, 0.5 μM TU2218 treated Tregs completely reversed the TCR-mediated proliferation of CD4+CD25- T cells. In addition, the immunomodulatory activity of TU2218 was analyzed on the co-culture system of cancer cells with human PBMC. In this assay, MCF-7 and HT-1080 displayed strong inhibitory effects on IFN-γ secretion of human PBMC, but TU2218 completely restored IFN-γ production compared with the poor effect of either vactosertib or TGF-β neutralizing antibody. In an NK assay, TGF-β reduced the level of surface proteins of NKG2D and NKp30 on CD56dim and CD56bright human primary NK cells and NK92 cell lines. TU2218 treatment completely reversed not only the inhibitory effect of TGF-β on the expression of NKG2D but also TGF-β-driven impairment of NK cytolytic activity for K562 cell lines on the co-culture system. Results show TU2218 possesses potent anti-cancer immune activities through boosting T and NK immunity as well as blocking Treg activity, implicating the mechanism of overcoming immune tolerance and reversing immunosuppression in TME. These effects of TU2218 in human immune cells would be a basis for novel combination options to develop promising therapeutics for patients with advanced cancer. Citation Format: Nam-Hoon Kim, Seong-Ho Kang, Jihyun Lee, Seung-Hyun Kim, Chan-Hee Yu, Jae Won Choi, Soyoun Ahn, Jeongmin Seo, Hun-Taek Kim. Functional characterization of TU2218, ALK5 and VEGFR2 dual inhibitor, on in vitro tumor immunity-mimetic systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5538.

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories.

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.