Novel Organoid Culture System for Improved Safety Assessment of Nanomaterials.

Over the past few decades, the increased application of nanomaterials has raised questions regarding their safety and possible toxic effects. Organoids have been suggested as promising tools, offering efficient assays for nanomaterial-induced toxicity evaluation. However, organoid systems have some limitations, such as size heterogeneity and poor penetration of nanoparticles because of the extracellular matrix, which is necessary for organoid culture. Here, we developed a novel system for the improved safety assessment of nanomaterials by establishing a 3D floating organoid paradigm. In addition to overcoming the limitations of two-dimensional systems including the lack of in vitro-in vivo cross-talk, our method provides multiple benefits as compared with conventional organoid systems that rely on an extracellular matrix for culture. Organoids cultured using our method exhibited relatively uniform sizing and structural integrity and were more conducive to the internalization of nanoparticles. Our floating culture system will accelerate the research and development of safe nanomaterials.

Monitoring lipid alterations in Drosophila heads in an amyotrophic lateral sclerosis model with time-of-flight secondary ion mass spectrometry.

Lipid alterations in the brain are well-documented in disease and aging, but our understanding of their pathogenic implications remains incomplete. Recent technological advances in assessing lipid profiles have enabled us to intricately examine the spatiotemporal variations in lipid compositions within the complex brain characterized by diverse cell types and intricate neural networks. In this study, we coupled time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an amyotrophic lateral sclerosis (ALS) Drosophila model, for the first time, to elucidate changes in the lipid landscape and investigate their potential role in the disease process, serving as a methodological and analytical complement to our prior approach that utilized matrix-assisted laser desorption/ionization mass spectrometry. The expansion of G4C2 repeats in the C9orf72 gene is the most prevalent genetic factor in ALS. Our findings indicate that expressing these repeats in fly brains elevates the levels of fatty acids, diacylglycerols, and ceramides during the early stages (day 5) of disease progression, preceding motor dysfunction. Using RNAi-based genetic screening targeting lipid regulators, we found that reducing fatty acid transport protein 1 (FATP1) and Acyl-CoA-binding protein (ACBP) alleviates the retinal degeneration caused by G4C2 repeat expression and also markedly restores the G4C2-dependent alterations in lipid profiles. Significantly, the expression of FATP1 and ACBP is upregulated in G4C2-expressing flies, suggesting their contribution to lipid dysregulation. Collectively, our novel use of ToF-SIMS with the ALS Drosophila model, alongside methodological and analytical improvements, successfully identifies crucial lipids and related genetic factors in ALS pathogenesis.

Background free in vivo29Si MR imaging with hyperpolarized PEGylated silicon nanoparticles.

This study demonstrated the potential of 50 nm PEGylated Si NPs for high-resolution in vivo29Si MR imaging, emphasizing their biocompatibility and water dispersibility. The acquisition of in vivo Si MR images using the lowest reported dose after subcutaneous and intraperitoneal administration opens new avenues for future 29Si MR studies.

Uncertainty evaluation of photoluminescence quantum yield measurement in an integrating hemisphere-based instrument.

We present an integrating hemisphere-based (i.e., a variant of integrating spheres) implementation of the indirect illumination method for absolute photoluminescence quantum yield measurements, which is a recommended method in the international standard IEC 62607-3-1:2014. We rigorously formulated a mathematical model and a measurement procedure for the absolute photoluminescence quantum yield measurement in the integrating hemisphere-based system. The measurement system was calibrated using an Hg-Ar discharge lamp and spectral irradiance standard lamps for wavelength and relative spectral radiant flux scales, respectively. Furthermore, we identified and evaluated uncertainty components involved in the photoluminescence quantum yield (PLQY) measurement. To validate our measurement system, we applied it to the two de facto standard dyes: quinine bisulfate (QBS) and fluorescein (FLS). Consequently, their PLQY values were determined to be 0.563±0.024 (k=2) and 0.876±0.032 (k=2) for, respectively, QBS and FLS, which are consistent with previous reports.

Simultaneous Multiplexed Imaging of Biomolecules in Transgenic Mouse Brain Tissues Using Mass Spectrometry Imaging: A Multi-omic Approach.

The importance of multi-omic-based approaches to better understand diverse pathological mechanisms including neurodegenerative diseases has emerged. Spatial information can be of great help in understanding how biomolecules interact pathologically and in elucidating target biomarkers for developing therapeutics. While various analytical methods have been attempted for imaging-based biomolecule analysis, a multi-omic approach to imaging remains challenging due to the different characteristics of biomolecules. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool due to its sensitivity, chemical specificity, and high spatial resolution in visualizing chemical information in cells and tissues. In this paper, we suggest a new strategy to simultaneously obtain the spatial information of various kinds of biomolecules that includes both labeled and label-free approaches using ToF-SIMS. The enzyme-assisted labeling strategy for the targets of interest enables the sensitive and specific imaging of large molecules such as peptides, proteins, and mRNA, a task that has been, to date, difficult for any MS analysis. Together with the strength of the analytical performance of ToF-SIMS in the label-free tissue imaging of small biomolecules, the proposed strategy allows one to simultaneously obtain integrated information of spatial distribution of metabolites, lipids, peptides, proteins, and mRNA at a high resolution in a single measurement. As part of the suggested strategy, we present a sample preparation method suitable for MS imaging. Because a comprehensive method to examine the spatial distribution of multiple biomolecules in tissues has remained elusive, our strategy can be a useful tool to support the understanding of the interactions of biomolecules in tissues as well as pathological mechanisms.

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

Utilization of chromogenic enzyme substrates for signal amplification in multiplexed detection of biomolecules using surface mass spectrometry.

MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

Length difference of multi-walled carbon nanotubes generates differential cytotoxic responses.

Carbon nanotubes have recently been rated as an effective biomaterial owing to their functionalization ability. However, the safety of multi-walled carbon nanotubes (MWCNTs) has yet to be clearly understood. To investigate how cells differentially react to minor geometric differences, we prepared well-dispersed and stable long and short MWCNTs showing an approximately 100-nm length difference in an in vitro system. Through an optimal combination of bovine serum albumin (BSA) and fetal bovine serum (FBS) biosurfactants and ultrasonication, we first confirmed that the MWCNTs were maintained without aggregation throughout the experiments. Internalized MWCNTs in human coronary artery smooth muscle cells were then quantified in a label-free manner using coherent anti-Stokes Raman scattering, followed by an analysis of their localization via two-photon excitation fluorescence. Intracellular MWCNTs were found to primarily localize in mitochondria with abnormal morphologies. Mitochondrial dysfunction, which was found to result from early stages of oxidative stress that consequently lead to cell death, was then proved via decreasing mitochondrial membrane potentials, with short MWCNTs showing significantly greater cytotoxicity than long MWCNTs. Our results suggest that even small length differences of MWCNTs may lead to differential responses in cells.

Toxicity of orally administered food-grade titanium dioxide nanoparticles.

This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose-related changes in the Organisation for Economic Cooperation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte-macrophage colony-stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24-h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress-related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.

Molecule-Resolved Visualization of Particulate Matter on Human Skin Using Multimodal Nonlinear Optical Imaging.

Precise measurement of particulate matter (PM) on skin is important for managing and preventing PM-related skin diseases. This study aims to directly visualize the deposition and penetration of PM into human skin using a multimodal nonlinear optical (MNLO) imaging system. We successfully obtained PM particle signals by merging two different sources, C-C vibrational frequency and autofluorescence, while simultaneously visualizing the anatomical features of the skin via keratin, collagen, and elastin. As a result, we found morphologically dependent PM deposition, as well as increased deposition following disruption of the skin barrier via tape-stripping. Furthermore, PM penetrated more and deeper into the skin with an increase in the number of tape-strippings, causing a significant increase in the secretion of pro-inflammatory cytokines. Our results suggest that MNLO imaging could be a useful technique for visualizing and quantifying the spatial distribution of PM in ex vivo human skin tissues.

Amorphous silica nanoparticle-induced pulmonary inflammatory response depends on particle size and is sex-specific in rats.

Due to mass production and extensive use, the potential adverse health effects of amorphous silica nanoparticles (ASiNPs) have received a significant attention from the public and researchers. However, the relationship between physicochemical properties of ASiNPs and their health effects is still unclear. In this study, we manufactured two types of ASiNPs of different diameters (20 and 50 nm) and compared the toxic response induced in rats after intratracheal instillation (75, 150 or 300 µg/rat). There were no dose-related differences in mortality, body weight gain or organ weight between the groups. However both types of ASiNPs significantly decreased the proportion of neutrophils in male rats, whereas the levels of hemoglobin and hematocrit were markedly reduced only in female rats instilled with 20 nm-ASiNPs. ASiNPs-induced lung tissue damage seemed to be more evident in the 20 nm ASiNP-treated group and in female rats than male rats. Similarly, expression of caveolin-1 and matrix metalloproteinase-9 seemed to be most notably enhanced in female rats treated with 20 nm-ASiNPs. The total number of bronchial alveolar lavage cells significantly increased in rats instilled with 20 nm-ASiNPs, accompanying a decrease in the proportion of macrophages and an increase in polymorphonuclear leukocytes. Moreover, secretion of inflammatory mediators clearly increased in human bronchial epithelial cells treated with 20 nm-ASiNPs, but not in those treated with 50 nm-ASiNPs. These results suggest that pulmonary effects of ASiNPs depend on particle size. Sex-dependent differences should also be carefully considered in understanding nanomaterial-induced adverse health effects.

Surface modification of solid-state nanopore by plasma-polymerized chemical vapor deposition of poly(ethylene glycol) for stable device operation.

Biopolymer adsorption onto a membrane is a significant issue in the reliability of solid-state nanopore devices, since it degrades the device performance or promotes device failure. In this work, a poly(ethylene glycol) (PEG) layer was coated on a silicon nitride (SiNx) membrane by plasma-polymerized vapor deposition to inhibit biopolymer adsorption. From optical observations, the deposited PEG layer demonstrated increased hydrophilicity and anti-adsorption property compared to the SiNx surface. Electrical properties of the PEG/SiNx nanopore were characterized, showing Ohmic behavior and a 6.3 times higher flicker noise power due to the flexible conformation of PEG in water. Antifouling performance of each surface was analyzed by measuring the average time from voltage bias to the first adsorption during DNA translocation experiments, where the modified surface enabled two times prolonged device operation. The time to adsorption was dependent on the applied voltage, implying adsorption probability was dominated by the electrophoretic DNA approach to the nanopore. DNA translocation behaviors on each surface were identified from translocation signals, as the PEG layer promoted unfolded and fast movement of DNA through the nanopore. This work successfully analyzed the effect of the PEG layer on DNA adsorption and translocation in solid-state nanopore experiments.

Oral toxicity of titanium dioxide P25 at repeated dose 28-day and 90-day in rats.

BACKGROUND: Nanotechnology is indispensable to many different applications. Although nanoparticles have been widely used in, for example, cosmetics, sunscreen, food packaging, and medications, they may pose human safety risks associated with nanotoxicity. Thus, toxicity testing of nanoparticles is essential to assess the relative health risks associated with consumer exposure. METHODS: In this study, we identified the NOAEL (no observed adverse effect level) of the agglomerated/aggregated TiO2 P25 (approximately 180 nm) administered at repeated doses to Sprague-Dawley (SD) rats for 28 and 90 days. Ten of the 15 animals were necropsied for toxicity evaluation after the repeated-dose 90-day study, and the remaining five animals were allowed to recover for 28 days. The agglomerated/aggregated TiO2 P25 dose levels used included 250 mg kg- 1 d- 1 (low), 500 mg kg- 1 d- 1 (medium), and 1000 mg kg- 1 d- 1 (high), and their effects were compared with those of the vehicle control. During the treatment period, the animals were observed for mortality, clinical signs (detailed daily and weekly clinical observations), functional observation battery, weekly body weight, and food and water consumption and were also subjected to ophthalmological examination and urinalysis. After termination of the repeated-dose 28-day, 90-day, and recovery studies, clinical pathology (hematology, blood coagulation time, and serum biochemistry), necropsy (organ weights and gross findings), and histopathological examinations were performed. RESULTS: No systemic toxicological effects were associated with the agglomerated/aggregated TiO2 P25 during the repeated-dose 28-day, 90-day, and recovery studies in SD rats. Therefore, the NOAEL of the agglomerated/aggregated TiO2 P25 was identified as 1000 mg kg- 1 d- 1, and the substance was not detected in the target organs. CONCLUSION: Subacute and subchronic oral administration of the agglomerated/aggregated TiO2 P25 was unlikely to cause side effects or toxic reactions in rats.

Hepatocyte CREBH deficiency aggravates inflammatory liver injury following chemokine-dependent neutrophil infiltration through upregulation of NF-κB p65 in mice.

Fulminant hepatitis is a serious inflammatory condition of the liver characterized by massive necrosis of liver parenchyma following excessive immune cell infiltration into the liver, and possibly causing sudden hepatic failure and medical emergency. However, the underlying mechanisms are not fully understood. Here, we investigated the role of cyclic AMP-responsive element-binding protein, hepatocyte specific (CREBH) in concanavalin A (ConA)-driven hepatitis-evoked liver injury. C57BL/6J (WT) and Crebh knockout (KO) mice injected with ConA (7.5 or 25 mg/kg) and bone marrow (BM) chimeric mice, generated by injection of BM cells into sub-lethally irradiated recipients followed by ConA injection (22.5 or 27.5 mg/kg) 8 weeks later, were used for in vivo study. Primary mouse hepatocytes and HEK293T cells were used for a comparative in vitro study. Crebh KO mice are highly susceptible to ConA-induced liver injury and prone to death due to increased neutrophil infiltration driven by enhanced hepatic expression of neutrophil-attracting chemokines. Notably, BM chimera experiment demonstrated that Crebh-deficient hepatocytes have an enhanced ability of recruiting neutrophils to the liver, thereby promoting hepatotoxicity by ConA. Intriguingly, in vitro assays showed that p65, a subunit of NF-κB and common transcription factor for various chemokines, dependent transactivation was inhibited by CREBH. Furthermore, p65 expression was inversely correlated with CREBH level in ConA-treated mice liver and TNFα-stimulated primary mouse hepatocytes. This is the first demonstration that CREBH deficiency aggravates inflammatory liver injury following chemokine-dependent neutrophil infiltration via NF-κB p65 upregulation. CREBH is suggested to be a novel therapeutic target for treatment of fulminant hepatitis.

Hepatocyte SHP deficiency protects mice from acetaminophen-evoked liver injury in a JNK-signaling regulation and GADD45ß-dependent manner.

Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure. Prolonged c-Jun N-terminal kinase (JNK) activation plays a central role in APAP-induced liver injury; however, growth arrest and DNA damage-inducible 45 beta (GADD45ß) is known to inhibit JNK phosphorylation. The orphan nuclear receptor small heterodimer partner (SHP, NR0B2) acts as a transcriptional co-repressor of various genes. The aim of the present study was to investigate the role of SHP in APAP-evoked hepatotoxicity. We used lethal (750 mg/kg) or sublethal (300 mg/kg) doses of APAP-treated wild-type (WT), Shp knockout (Shp-/-), hepatocyte-specific Shp knockout (Shphep-/-), and Shp and Gadd45ß double knockout (Shp-/-Gadd45ß-/-) mice for in vivo studies. Primary mouse hepatocytes were used for a comparative in vitro study. SHP deficiency protected against APAP toxicity with an increased survival rate, decreased liver damage, and inhibition of prolonged hepatic JNK phosphorylation in mice, which was independent of APAP metabolism regulation. Furthermore, Shphep-/- mice showed diminished APAP hepatotoxicity compared with WT mice. SHP-deficient primary mouse hepatocytes also showed decreased cell death and inhibition of sustained JNK phosphorylation following toxic APAP treatment. While SHP expression declined, GADD45ß expression increased after APAP treatment in WT mice. In Shp-/- mice, APAP-evoked GADD45ß induction was significantly enhanced. Notably, the ameliorative effects of SHP deficiency on APAP-induced liver injury were abolished in Shp-/-Gadd45ß-/- mice. The current study is the first to demonstrate that hepatocyte-specific SHP deficiency protects against APAP overdose-evoked hepatotoxicity in a JNK signaling regulation and GADD45ß dependent manner. SHP is suggested to be a novel therapeutic target for APAP overdose treatment.

Protein Food Matrix⁻ZnO Nanoparticle Interactions Affect Protein Conformation, but May not Be Biological Responses.

Because of their nutritional value, zinc oxide (ZnO) nanoparticles (NPs) are applied as a dietary source of zinc, by direct addition to complex, multiple-component food matrices. The thereby occurring interactions of NPs with food matrices may have biological or toxic effects. In particular, NP interactions with food protein can lead to structural deformation of the latter, potentially changing its digestive efficiency and gastrointestinal absorption. In this study, interactions between ZnO NPs and a representative complex protein food matrix, skim milk, were compared with those between NPs and individual components of this food matrix (i.e., protein, saccharide, and mineral). The effects of the interactions on biological responses were investigated in terms of cytotoxicity, cellular uptake, intestinal transport, structural deformation for proteins, and digestive efficiency. The results demonstrated that the physicochemical properties of ZnO NPs were strongly influenced by the protein matrix type, leading to an increased dispersion stability in the complex protein matrix. However, these interactions did not affect cell proliferation, membrane damage, cellular uptake, intestinal transportation, or protein digestive efficiency, although a slight conformational change of proteins was observed in the presence of ZnO NPs. In conclusion, no toxic effects were observed, suggesting the safety of NPs when added to complex food matrices.

On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening.

Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteine-tethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)T315I kinase inhibitor, henceforth referred to as KR135861. KR135861 showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315I tyrosine kinase, with an IC50 value as low as 1.3 µM. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABLT315I, in contrast to imatinib's inhibitory effects only on Ba/F3 cells expressing wild-type BCR-ABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy, sensitivity, and specificity.

Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity.

Bare gold nanospheres have been shown to have anti-angiogenic effects but are optically unfavorable because their resonant wavelength lies in the visible spectrum. Here, we design gold nanodisks with a higher scattering capability than gold nanorods and with a resonant wavelength at near-infrared region - the area where the source of light utilized by optical coherence tomography (OCT) lies. With a physical synthesis system, we then fabricate 160-nm-sized gold nanodisks exhibiting resonant wavelength at 830 nm. The synthesized nanoparticles were successfully visualized in in vivo OCT at concentrations as low as 1 pM. After demonstrating their binding ability to vascular endothelial growth factor (VEGF), we show that they suppress VEGF-induced migration of endothelial cells. Finally, we demonstrate that intravitreally injected gold nanodisks attenuate neovascularization of oxygen-induced retinopathy in mice, in a dose dependent manner, such that they are cleared from the vitreous within 2 weeks without histologic or electrophysiologic toxicity.

Probing nanoparticles and nanoparticle-conjugated biomolecules using time-of-flight secondary ion mass spectrometry.

Bio-conjugated nanoparticles have emerged as novel molecular probes in nano-biotechnology and nanomedicine and chemical analyses of their surfaces have become challenges. The time-of-flight (TOF) secondary ion mass spectrometry (SIMS) has been one of the most powerful surface characterization techniques for both nanoparticles and biomolecules. When combined with various nanoparticle-based signal enhancing strategies, TOF-SIMS can probe the functionalization of nanoparticles as well as their locations and interactions in biological systems. Especially, nanoparticle-based SIMS is an attractive approach for label-free drug screening because signal-enhancing nanoparticles can be designed to directly measure the enzyme activity. The chemical-specific imaging analysis using SIMS is also well suited to screen nanoparticles and nanoparticle-biomolecule conjugates in complex environments. This review presents some recent applications of nanoparticle-based TOF-SIMS to the chemical analysis of complex biological systems.

Study on the deformation of endothelial cells using a bio-inspired in vitro disease model.

A bio-inspired in vitro disease model was developed to investigate the basic biophysics of atherosclerotic diseases. In vivo study was conducted in advance using zebrafish fed with a normal diet and a cholesterol-enriched diet. The endothelial cells (ECs) of the zebrafishes fed with a normal diet are tightly attached and aligned. Their collagen has a fiber-like structure. By contrast, the endothelial cells of the zebrafish on high cholesterol diet are disorganized and their collagen has broken structures. In vitro models of human umbilical vein endothelial cells (HUVECs) were established on collagen films to mimic such in vivo experimental results. The normal collagen film simulates the extracellular matrix (ECM) in the blood vessels of a normal zebrafish, and the collagenase-treated collagen film mimics the ECM in blood vessels of an abnormal zebrafish. The HUVECs cultured on the normal collagen film are tightly attached, similar to those of a normal zebrafish. However, the cells cultured on the collagenase-treated collagen film are aggregated and biomarkers of endothelial dysfunction are expressed on the surface of the abnormal endothelial cells established on the denatured collagen film. The present in vitro model using a bio-inspired collagen film has a great potential for the design of novel therapies for clinical treatments of atherosclerosis through better understanding on the outbreak mechanism of atherosclerosis.