Haruhiko Kamada

Silica and titanium dioxide nanoparticles cause pregnancy complications in mice

The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.

Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes.

BackgroundClarifying the physicochemical properties of nanomaterials is crucial for hazard assessment and the safe application of these substances. With this in mind, we analyzed the relationship between particle size and the in vitro effect of amorphous nanosilica (nSP). Specifically, we evaluated the relationship between particle size of nSP and the in vitro biological effects using human keratinocyte cells (HaCaT).ResultsOur results indicate that exposure to nSP of 70 nm diameter (nSP70) induced an elevated level of reactive oxygen species (ROS), leading to DNA damage. A markedly reduced response was observed using submicron-sized silica particles of 300 and 1000 nm diameter. In addition, cytochalasin D-treatment reduced nSP70-mediated ROS generation and DNA damage, suggesting that endocytosis is involved in nSP70-mediated cellular effects.ConclusionsThus, particle size affects amorphous silica-induced ROS generation and DNA damage of HaCaT cells. We believe clarification of the endocytosis pathway of nSP will provide useful information for hazard assessment as well as the design of safer forms of nSPs.

Carbon Nanotubes Elicit DNA Damage and Inflammatory Response Relative to Their Size and Shape

Carbon nanotubes (CNTs) have been one of the most extensively researched and developed nanomaterials. However, little concern has been placed on their safety. The biological effects of CNTs are believed to differ relative to size and shape. Thus, the relationship between the characteristics of CNTs and their safety needs to be evaluated. In this study, we examined the biological effects of different-sized multi-walled CNTs (MWCNTs) and single-walled CNTs (SWCNTs). Long and thick MWCNTs induced the strongest DNA damage while similar SWCNTs caused little effect. Comparison of inflammatory responses of various types of CNTs found that peritoneal CNT administration of long and thick MWCNTs increased the total cell number in abdominal lavage fluid in mice. These results indicate that long and thick MWCNT, but not short and thin MWCNT, cause DNA damage and severe inflammatory effects. These findings might provide useful information for constructing novel CNTs with safety.

Systemic distribution, nuclear entry and cytotoxicity of amorphous nanosilica following topical application.

Currently, nanomaterials (NMs) with particle sizes below 100 nm have been successfully employed in various industrial applications in medicine, cosmetics and foods. On the other hand, NMs can also be problematic in terms of eliciting a toxicological effect by their small size. However, biological and/or cellular responses to NMs are often inconsistent and even contradictory. In addition, relationships among NMs physicochemical properties, absorbency, localization and biological responses are not yet well understood. In order to open new frontiers in medical, cosmetics and foods fields by the safer NMs, it is necessary to collect the information of the detailed properties of NMs and then, build the prediction system of NMs safety. The present study was designed to examine the skin penetration, cellular localization, and cytotoxic effects of the well-dispersed amorphous silica particles of diameters ranging from 70 nm to 1000 nm. Our results suggested that the well-dispersed amorphous nanosilica of particle size 70 nm (nSP70) penetrated the skin barrier and caused systemic exposure in mouse, and induced mutagenic activity in vitro. Our information indicated that further studies of relation between physicochemical properties and biological responses are needed for the development and the safer form of NMs.

Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-α antagonist

Tumor necrosis factor-α (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.

Functionalization of Tumor Necrosis Factor-α Using Phage Display Technique and PEGylation Improves Its Antitumor Therapeutic Window

Purpose: In this study, the optimization of antitumor therapy with tumor necrosis factor-α (TNF-α) was attempted. Experimental Design: Using the phage display technique, we created a lysine-deficient mutant TNF-α (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding. Results: The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF-α (wTNF-α). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF-α and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH2 terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-α, whereas the randomly mono-PEGylated wTNF-α had 6% of the bioactivity of the wTNF-α. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-α. Conclusions: These results indicated that this functionalized TNF-α may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.

Comparative study on transduction and toxicity of protein transduction domains

Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs.

In vivo biotinylation of the vasculature in B-cell lymphoma identifies BST-2 as a target for antibody-based therapy

The discovery of accessible markers of lymphoma may facilitate the development of antibody-based therapeutic strategies. Here, we describe the results of a chemical proteomic study, based on the in vivo biotinylation of vascular proteins in lymphoma-bearing mice followed by mass spectrometric and bioinformatic analysis, to discover proteins expressed at the tissue-blood border of disseminated B-cell lymphoma. From a list of 58 proteins, which were more than 10-fold up-regulated in nodal and extranodal lymphoma lesions compared with their levels in the corresponding normal host organs, we validated BST-2 as a novel vascular marker of B-cell lymphoma, using immunochemical techniques and in vivo biodistribution studies. Furthermore, targeting BST-2 with 2 independent monoclonal antibodies delayed lymphoma growth in a syngeneic mouse model of the disease. The results of this study delineate a strategy for the treatment of systemic B-cell lymphoma in humans and suggest that anti-BST-2 antibodies may facilitate pharmacodelivery approaches that target the tumor-stroma interface.

Interleukin-1 Family Cytokines as Mucosal Vaccine Adjuvants for Induction of Protective Immunity against Influenza Virus

ABSTRACT A safe and potent adjuvant is needed for development of mucosal vaccines against etiological agents, such as influenza virus, that enter the host at mucosal surfaces. Cytokines are potential adjuvants for mucosal vaccines because they can enhance primary and memory immune responses enough to protect against some infectious agents. For this study, we tested 26 interleukin (IL) cytokines as mucosal vaccine adjuvants and compared their abilities to induce antigen (Ag)-specific immune responses against influenza virus. In mice intranasally immunized with recombinant influenza virus hemagglutinin (rHA) plus one of the IL cytokines, IL-1 family cytokines (i.e., IL-1α, IL-1β, IL-18, and IL-33) were found to increase Ag-specific immunoglobulin G (IgG) in plasma and IgA in mucosal secretions compared to those after immunization with rHA alone. In addition, high levels of both Th1- and Th2-type cytokines were observed in mice immunized with rHA plus an IL-1 family cytokine. Furthermore, mice intranasally immunized with rHA plus an IL-1 family cytokine had significant protection against a lethal influenza virus infection. Interestingly, the adjuvant effects of IL-18 and IL-33 were significantly decreased in mast cell-deficient W/Wv mice, indicating that mast cells have an important role in induction of Ag-specific mucosal immune responses induced by IL-1 family cytokines. In summary, our results demonstrate that IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce Ag-specific immune responses for protection against pathogens like influenza virus.

Effect of surface properties of silica nanoparticles on their cytotoxicity and cellular distribution in murine macrophages.

Surface properties are often hypothesized to be important factors in the development of safer forms of nanomaterials (NMs). However, the results obtained from studying the cellular responses to NMs are often contradictory. Hence, the aim of this study was to investigate the relationship between the surface properties of silica nanoparticles and their cytotoxicity against a murine macrophage cell line (RAW264.7). The surface of the silica nanoparticles was either unmodified (nSP70) or modified with amine (nSP70-N) or carboxyl groups (nSP70-C). First, the properties of the silica nanoparticles were characterized. RAW264.7 cells were then exposed to nSP70, nSP70-N, or nSP70-C, and any cytotoxic effects were monitored by analyzing DNA synthesis. The results of this study show that nSP70-N and nSP70-C have a smaller effect on DNA synthesis activity by comparison to unmodified nSP70. Analysis of the intracellular localization of the silica nanoparticles revealed that nSP70 had penetrated into the nucleus, whereas nSP70-N and nSP70-C showed no nuclear localization. These results suggest that intracellular localization is a critical factor underlying the cytotoxicity of these silica nanoparticles. Thus, the surface properties of silica nanoparticles play an important role in determining their safety. Our results suggest that optimization of the surface characteristics of silica nanoparticles will contribute to the development of safer forms of NMs.