SiO2 Fibers of Two Lengths and Their Effect on Cellular Responses of Macrophage-like Cells.

The immunoreactivity or/and stress response can be induced by nanomaterials' different properties, such as size, shape, etc. These effects are, however, not yet fully understood. This study aimed to clarify the effects of SiO2 nanofibers (SiO2NFs) on the cellular responses of THP-1-derived macrophage-like cells. The effects of SiO2NFs with different lengths on reactive oxygen species (ROS) and pro-inflammatory cytokines TNF-α and IL-1ß in THP-1 cells were evaluated. From the two tested lengths, it was only the L-SiO2NFs with a length ≈ 44 ± 22 µm that could induce ROS. Compared to this, only S-SiO2NFs with a length ≈ 14 ± 17 µm could enhance TNF-α and IL-1ß expression. Our results suggested that L-SiO2NFs disassembled by THP-1 cells produced ROS and that the inflammatory reaction was induced by the uptake of S-SiO2NFs by THP-1 cells. The F-actin staining results indicated that SiO2NFs induced cell motility and phagocytosis. There was no difference in cytotoxicity between L- and S-SiO2NFs. However, our results suggested that the lengths of SiO2NFs induced different cellular responses.

The Effects of TiO2 Nanoparticles on Cisplatin Cytotoxicity in Cancer Cell Lines.

There have been many studies on improving the efficacy of cisplatin and on identifying safe compounds that can overcome multi-drug resistance (MDR) acquired by cancer cells. Our previous research showed that polyethylene glycol-modified titanium dioxide nanoparticles (TiO2 PEG NPs) affect cell membrane receptors, resulting in their aggregation, altered localization and downregulation. TiO2 PEG NPs may affect P-glycoprotein (P-gp), a membrane efflux channel involved in MDR. In this study, we investigated the effect of TiO2 PEG NPs on cisplatin cytotoxicity. We used HepG2 cells, which highly express P-gp and A431 cells, which show low expression of P-gp. The results showed that 10 µg/mL 100 nm TiO2 PEG NPs increased intracellular cisplatin levels and cytotoxicity in HepG2 cells but not in A431 cells. TiO2 PEG NPs treatment decreased the expression level of P-gp in HepG2 cells. Our findings indicate that TiO2 PEG NPs enhance cisplatin cytotoxicity by down regulating P-gp and that TiO2 PEG NPs are promising candidates for inhibiting P-gp and reversing drug resistance acquired by cancer cells.

EGF Conjugation Improves Safety and Uptake Efficacy of Titanium Dioxide Nanoparticles.

Titanium dioxide nanoparticles (TiO2 NPs) have a strong potential for cancer therapeutic and bioimaging applications such as photodynamic therapy (PDT) and photodynamic diagnosis (PDD). Our previous results have shown that TiO2 NPs have a low cellular uptake and can induce cell proliferation. This suggests that TiO2 NPs could increase the risk of tumor overgrowth while being used for PDD and PDT. To solve this problem, we constructed epidermal growth factor-ligated polyethylene glycol-coated TiO2 NPs (EGF-TiO2 PEG NPs). In this work, we studied the effect of EGF conjugation on the cellular uptake of TiO2 PEG NPs. Then, we investigated the effect of both non-conjugated and EGF-TiO2 PEG NPs on the A431 epidermal cancer cell line, proliferation and growth via the investigation of EGFR localization and expression. Our results indicated that TiO2 PEG NPs induced EGFRs aggregation on the A431 cells surface and induced cell proliferation. In addition, EGF-TiO2 PEG NPs induced the internalization of EGFRs inside of cells with increased cellular NPs uptake and decreased cellular proliferation compared to TiO2 PEG NPs-treated cells. These findings suggest that EGF conjugation can increase the efficacy of TiO2 PEG NPs for biomedical applications such as PDD and PDT with decreased risk of tumor overgrowth.

Polystyrene Nanoparticles Induce Apoptosis or Necrosis With or Without Epidermal Growth Factor.

Polystyrene nanoparticles (PS NPs) are biocompatible and low toxic material to biological systems. In this mind, PS NPs are widely used as a model for studying the interaction between nanoparticles and cells. Even PS NPs showed low toxicity, they could affect to some cellular responses. In this study, we investigated the influence of PS NPs on the epidermal growth factor (EGF)-response in the A431 human epithelial carcinoma cell line. The results showed that PS NPs interfered with the normal EGF-response of A431 cells in a dose-dependent manner. In addition, EGF significantly increased the uptake of PS NPs in A431 cells. Localization studies of PS NPs and EGF receptor (EGFR) indicated that changes in the EGF-response of A431 cells are related to the interaction between PS NPs and the EGF-EGFR complexes. The viability of cells exposed to PS NPs or combination of PS NPs and EGF decreased due to PS NPs induced cell death. The results also suggested that without EGF, PS NPs internalized in the cells cause cell death by necrosis, whereas EGF enhances the uptake ratio of PS NPs, and PS NPs in the cytoplasm together with EGF-EGFR complexes may inhibit receptors recycling, leading to apoptosis. This finding could be useful for the safe and effective use of nanoparticles in clinical applications.

Size-Dependent Effect of Silver Nanoparticles on the Tumor Necrosis Factor α-Induced DNA Damage Response.

Silver nanoparticles (AgNPs) are widely used in many consumer products due to their anti-inflammatory properties. Therefore, the effect of exposure to AgNPs should be investigated in diseased states in addition to healthy ones. Tumor necrosis factor-α (TNFα) is a major cytokine that is highly expressed in many diseased conditions, such as inflammatory diseases, sepsis, and cancer. We investigated the effects of two different sizes of AgNPs on the TNFα-induced DNA damage response. Cells were exposed to 10 and 200 nm AgNPs separately and the results showed that the 200 nm AgNPs had a lower cytotoxic effect with a higher percent of cellular uptake compared to the 10 nm AgNPs. Moreover, analysis of reactive oxygen species (ROS) generation and DNA damage indicated that TNFα-induced ROS-mediated DNA damage was reduced by 200 nm AgNPs, but not by 10 nm AgNPs. Tumor necrosis factor receptor 1 (TNFR1) was localized on the cell surface after TNFα exposure with or without 10 nm AgNPs. In contrast, the expression of TNFR1 on the cell surface was reduced by the 200 nm AgNPs. These results suggested that exposure of cells to 200 nm AgNPs reduces the TNFα-induced DNA damage response via reducing the surface expression of TNFR1, thus reducing the signal transduction of TNFα.

Change in Renal Glomerular Collagens and Glomerular Filtration Barrier-Related Proteins in a Dextran Sulfate Sodium-Induced Colitis Mouse Model.

Renal disease is not rare among patients with inflammatory bowel disease (IBD) and is gaining interest as a target of research. However, related changes in glomerular structural have rarely been investigated. This study was aimed at clarifying the changes in collagens and glomerular filtration barrier (GFB)-related proteins of glomeruli in a dextran sulfate sodium (DSS)-induced colitis mouse model. Acute colitis was induced by administering 3.5% DSS in Slc:ICR strain mice for eight days. Histological changes to glomeruli were examined by periodic acid-Schiff (PAS) and Masson's trichrome staining. Expressions of glomerular collagens and GFB-related proteins were analyzed by immunofluorescent staining and Western blot analysis. DSS-colitis mice showed an elevated disease activity index (DAI), colon shortening, massive cellular infiltration and colon damage, confirming that DSS-colitis mice can be used as an IBD animal model. DSS-colitis mice showed increased glycoprotein and collagen deposition in glomeruli. Interestingly, we observed significant changes in glomerular collagens, including a decrease in type IV collagen, and an increment in type I and type V collagens. Moreover, declined GFB-related proteins expressions were detected, including synaptopodin, podocalyxin, nephrin and VE-cadherin. These results suggest that renal disease in DSS-colitis mice might be associated with changes in glomerular collagens and GFB-related proteins. These findings are important for further elucidation of the clinical pathological mechanisms underlying IBD-associated renal disease.

The Influence of a Nanopatterned Scaffold that Mimics Abnormal Renal Mesangial Matrix on Mesangial Cell Behavior.

The alteration of mesangial matrix (MM) components in mesangium, such as type IV collagen (COL4) and type I collagen (COL1), is commonly found in progressive glomerular disease. Mesangial cells (MCs) responding to altered MM, show critical changes in cell function. This suggests that the diseased MM structure could play an important role in MC behavior. To investigate how MC behavior is influenced by the diseased MM 3D nanostructure, we fabricated the titanium dioxide (TiO2)-based nanopatterns that mimic diseased MM nanostructures. Immortalized mouse MCs were used to assess the influence of disease-mimic nanopatterns on cell functions, and were compared with a normal-mimic nanopattern. The results showed that the disease-mimic nanopattern induced disease-like behavior, including increased proliferation, excessive production of abnormal MM components (COL1 and fibronectin) and decreased normal MM components (COL4 and laminin α1). In contrast, the normal-mimic nanopattern actually resulted in cells displaying normal proliferation and the production of normal MM components. In addition, increased expressions of α-smooth muscle actin (α-SMA), transforming growth factor ß1 (TGF-ß1) and integrin α5ß1 were detected in cells grown on the disease-mimic nanopattern. These results indicated that the disease-mimic nanopattern induced disease-like cell behavior. These findings will help further establish a disease model that mimics abnormal MM nanostructures and also to elucidate the molecular mechanisms underlying glomerular disease.

Silver nanoparticles reduce the apoptosis induced by tumor necrosis factor-α.

Silver nanoparticles (AgNPs) are widely known to have anti-inflammatory properties, but the exact mechanism underlying this anti-inflammatory effect is not clearly understood. Tumor necrosis factor-α (TNFα) is a major pro-inflammatory cytokine that is expressed in the early stage of cell inflammation and induces apoptosis by several known pathways. Our study aimed to investigate the effect of AgNPs on the response of lung epithelial cells to TNFα and the molecular mechanism of this response. Lung epithelial cell line NCI-H292 cells were exposed to AgNPs (5 µg/mL) and/or TNFα (20 ng/mL) for 24 h, then cellular uptake was analyzed using flow cytometry. Our results showed that AgNPs were taken up by cells in a dose-dependent manner and that the cellular uptake ratio of AgNPs was significantly increased in the presence of TNFα. Apoptosis assays indicated that exposure to AgNPs significantly decreased the apoptotic effect of TNFα. Confocal microscopy was used to localize the tumor necrosis factor receptor 1 (TNFR1) and revealed that TNFR1 localized on the surface of cells exposed to TNFα. In contrast, TNFR1 localized inside cells exposed to both AgNPs and TNFα, with very few receptors scattered on the cell membrane. The results indicated that AgNPs reduced the cell surface TNFR1 expression level. The results suggested that the reduction of surface TNFR1 reduced cellular response to TNFα, resulting in an anti-apoptotic effect.

PEGylated TiO2 nanoparticles mediated inhibition of cell migration via integrin beta 1.

Nanoparticles (NPs) elicit various physiological responses in cellular environment, and the effect of NPs on cell migration is of high interest. In this work, the effects of NPs on cell migration and their possible mechanisms were studied. Here, we showed that after exposure to pegylated titanium dioxide nanoparticles (TiO2-PEG NPs, where PEG stands for the polyethylene glycol), NCI-H292 cells exhibited slower migration than control cells. Furthermore, larger NPs inhibited cell migration much stronger than smaller NPs. Following NP exposure, the cells showed decreased expression of integrin beta 1 and phosphorylated focal adhesion kinase (pFAK), and disrupted F-actin structures. We demonstrated that a possible mechanism involved NP-mediated promotion of the lysosomal degradation of integrin beta 1, thus leading to reduced expression of pFAK and cytoskeletal disruption and inhibited cell migration. Therefore, our results showed that inhibition of NCI-H292 cell migration by NPs is mediated through integrin beta 1, which provides useful information for the application of NPs in cancer therapy and related fields.

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells.

This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO2 nanoparticles (TiO2-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 µg ml-1) of TiO2-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO2-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO2-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO2-PEG NPs. In conclusion, our results indicate that TiO2-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses.

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche.

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.

Molecular mechanism of DNA damage induced by titanium dioxide nanoparticles in toll-like receptor 3 or 4 expressing human hepatocarcinoma cell lines.

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) are widely used in the biological sciences. The increasing use of TiO2 NPs increases the risk of humans and the environment being exposed to NPs. We previously showed that toll-like receptors (TLRs) play an important role in the interactions between NPs and cells. Our previous results indicated that TLR4 increased the DNA damage response induced by TiO2 NPs, due to enhanced NP uptake into the cytoplasm, whereas TLR3 expression decreased the DNA damage response induced by TiO2 NPs because of NP retention in the endosome. In this study, we explored the molecular mechanism of the DNA damage response induced by TiO2 NPs using TLR3 or TLR4 transfected cells. We examined the effect of TLR3 or TLR4 over-expression on oxidative stress and the effect of DNA damage induced by TiO2 NPs on gene expression levels. RESULTS: Our results showed evidence for elevated oxidative stress, including the generation of reactive oxygen species (ROS), with increased hydrogen peroxide levels, decreased glutathione peroxidase, and reduced glutathione and activated caspase-3 levels in cells exposed for 48 h to 10 µg/ml TiO2 NPs. These effects were enhanced by TLR4 and reduced by TLR3 over-expression. Seventeen genes related to DNA double-strand breaks and apoptosis were induced, particularly IP6K3 and ATM. CONCLUSION: Our results indicated that TiO2 NPs induced ROS, and the above molecules are implicated in the genotoxicity induced by TiO2 NPs.

The effect of physical and chemical cues on hepatocellular function and morphology.

Physical topographical features and/or chemical stimuli to the extracellular matrix (ECM) provide essential cues that manipulate cell functions. From the physical point of view, contoured nanostructures are very important for cell behavior in general, and for cellular functions. From the chemical point of view, ECM proteins containing an RGD sequence are known to alter cell functions. In this study, the influence of integrated physical and chemical cues on a liver cell line (HepG2) was investigated. To mimic the physical cues provided by the ECM, amorphous TiO2 nanogratings with specific dimensional and geometrical characteristics (nanogratings 90 nm wide and 150 nm apart) were fabricated. To mimic the chemical cues provided by the ECM, the TiO2 inorganic film was modified by immobilization of the RGD motif. The hepatic cell line morphological and functional changes induced by simultaneously combining these diversified cues were investigated, including cellular alignment and the expression of different functional proteins. The combination of nanopatterns and surface modification with RGD induced cellular alignment and expression of functional proteins, indicating that physical and chemical cues are important factors for optimizing hepatocyte function.

Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles.

Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs) and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO2 NP) agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO2 NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO2 NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO2 NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.

Comparison of cellular uptake and inflammatory response via toll-like receptor 4 to lipopolysaccharide and titanium dioxide nanoparticles.

The innate immune response is the earliest cellular response to infectious agents and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) play an important role in these interactions. We have already shown that TLRs are involved with the uptake of titanium dioxide nanoparticles (TiO2 NPs) and promote inflammatory responses. In this paper, we compared role of cellular uptake and inflammatory response via TLR 4 to lipopolysaccharide (LPS) and TiO2 NPs. In the case of LPS, LPS binds to LPS binding protein (LBP) and CD 14, and then this complex binds to TLR 4. In the case of TiO2 NPs, the necessity of LBP and CD 14 to induce the inflammatory response and for uptake by cells was investigated using over-expression, antibody blocking, and siRNA knockdown experiments. Our results suggested that for cellular uptake of TiO2 NPs, TLR 4 did not form a complex with LBP and CD 14. In the TiO2 NP-mediated inflammatory response, TLR 4 acted as the signaling receptor without protein complex of LPS, LBP and CD 14. The results suggested that character of TiO2 NPs might be similar to the complex of LPS, LBP and CD 14. These results are important for development of safer nanomaterials.

Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter.

Nanotechnology is becoming increasingly important for products used in our daily lives, such as the masses of titanium dioxide nanoparticle agglomerates (TiO(2) NPs) used in the pharmaceutical industry, for cosmetic products, or for pigments. Meanwhile, a serious lack of detailed information concerning the interaction between the nanomaterials and cells limits their biological and medical applications. Sensing technology is very important for understanding these interactions. We have shown that TiO(2) NPs induce heat shock protein 70B' (HSP70B') mRNA [Okuda-Shimazaki et al., 2010. Int J Mol Sci 11:2383-2392]. In the current work, sensor cells for detection of cellular responses to NPs were prepared by transfecting an HSP70B' promoter-reporter plasmid. First, to find suitable cells for detection, five different mammalian cell lines were chosen as potential sensor cells. The results showed TiO(2) NP response in some cell lines, although different sensor cells had different TiO(2) NP response levels, as heat shock response ability is important for the detection. Then, we studied the TiO(2) NP time-course response and dose response. The results indicated that our sensor cells can detect TiO(2) NP cellular responses. Our work should aid in understanding the interactions between bio-nanomaterials and cells.

Substrate stiffness affects skeletal myoblast differentiation in vitro.

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

Preparation and biological evaluation of hydroxyapatite-coated nickel-free high-nitrogen stainless steel.

Calcium phosphate was formed on nickel-free high-nitrogen stainless steel (HNS) by chemical solution deposition. The calcium phosphate deposition was enhanced by glutamic acid covalently immobilized on the surface of HNS with trisuccinimidyl citrate as a linker. X-ray diffraction patterns and Fourier transform infrared spectra showed that the material deposited on glutamic acid-immobilized HNS within 24 h was low-crystallinity calcium-deficient carbonate-containing hydroxyapatite (HAp). The biological activity of the resulting HAp-coated HNS was investigated by using a human osteoblast-like MG-63 cell culture. The HAp-coated HNS stimulated the alkaline-phosphate activity of the MG-63 culture after 7 days. Therefore, HAp-coated HNS is suitable for orthopedic devices and soft tissue adhesion materials.

Effect of polyethylene glycol modification of TiO2nanoparticles on cytotoxicity and gene expressions in human cell lines.

Nanoparticles (NPs) are tiny materials used in a wide range of industrial and medical applications. Titanium dioxide (TiO(2)) is a type of nanoparticle that is widely used in paints, pigments, and cosmetics; however, little is known about the impact of TiO(2) on human health and the environment. Therefore, considerable research has focused on characterizing the potential toxicity of nanoparticles such as TiO(2) and on understanding the mechanism of TiO(2) NP-induced nanotoxicity through the evaluation of biomarkers. Uncoated TiO(2) NPs tend to aggregate in aqueous media, and these aggregates decrease cell viability and induce expression of stress-related genes, such as those encoding interleukin-6 (IL-6) and heat shock protein 70B' (HSP70B'), indicating that TiO(2) NPs induce inflammatory and heat shock responses. In order to reduce their toxicity, we conjugated TiO(2) NPs with polyethylene glycol (PEG) to eliminate aggregation. Our findings indicate that modifying TiO(2) NPs with PEG reduces their cytotoxicity and reduces the induction of stress-related genes. Our results also suggest that TiO(2) NP-induced effects on cytotoxicity and gene expression vary depending upon the cell type and surface modification.