Special Issue "Advancements in Nanotoxicology"

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A special issue of Nanomaterials (ISSN 2079-4991).

Deadline for manuscript submissions: 30 April 2015

Special Issue Editor

Guest Editor
Prof. Dr. Robert Tanguay
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331-4003, USA
Website: http://emt.oregonstate.edu/roberttanguay
E-Mail: robert.tanguay@oregonstate.edu
Phone: +1 541 737 6514
Fax: +1 541 737 0497
Interests: developmental toxicity; high throughput screening; nanotoxicology; zebrafish; gene expression, miRNAs, and systems toxicology

Special Issue Information

Dear Colleagues,

This issue is a continuation of the previous successful special issue "Nanotoxicology".

Nanomaterial science continues to advance with the generation of more complex nanostructures with exciting potential applications. There have been parallel advances in the biological sciences aimed at evaluating the biocompatibility of these novel nanoparticles. Over recent years, we have realized that evaluating nanoparticles and biological interactions is quite complex because local environmental conditions influences particle behavior, and thus biocompatibility. In order to advance the development of safer high performing products, we need to understand the structural basis for these dynamic behaviors.

In this Special Issue, we are especially interested in manuscripts that advance the understanding of the specific nanomaterials attributes that govern or influence nanomaterial behavior and biocompatibility. This Issue invites manuscripts ranging from understanding dynamic behaviors of particles in aqueous environment, cellular toxicity, whole animal toxicity, neurotoxicity, immunotoxicity, genotoxicity, and population scale effects. Manuscripts that define specific biological responses at the organismal, gene expression, proteomic, and genetic levels are also invited.

Prof. Dr. Robert Tanguay
Guest Editor

Submission

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. Papers will be published continuously (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are refereed through a peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Nanomaterials is an international peer-reviewed Open Access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 600 CHF (Swiss Francs). English correction and/or formatting fees of 250 CHF (Swiss Francs) will be charged in certain cases for those articles accepted for publication that require extensive additional formatting and/or English corrections.


Keywords

  • biocompatibility
  • nanotoxicology
  • in vivo
  • in vitro
  • predictive
  • nanotoxicity
  • safety assessment
  • nanoparticle characterization

Related Special Issue

Published Papers (1 paper)

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p. 894-901
by , , , , , ,  and
Nanomaterials 2014, 4(4), 894-901; doi:10.3390/nano4040894
Received: 14 October 2014 / Revised: 3 November 2014 / Accepted: 21 November 2014 / Published: 28 November 2014
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(This article belongs to the Special Issue Advancements in Nanotoxicology)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Type of Paper: Article
Title:
Biological Effect of Single-Walled Carbon Nanotubes on Skeletonema costatum and Prorocentrum donghaiense in seawaters
Authors:
Fangfang Li, Jiangtao Wang *, Liju Tan
Affilication:
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100, China; E-Mail: jtwang@ouc.edu.cn
Abstract:
Carbon nanotubes (CNTs) have been used in a variety of industrial areas as a kinds of new nanometer materials. Algal growth test was developed to determine the biological effect of single-walled carbon nanotubes (SWNTs) on Skeletonema costatum and Prorocentrum donghaiense. The results showed that SWNTs stimulated the growth of S. costatum within 72 h and 24 h as theirs concentrations were smaller than 0.5 mg/L and 10 mg/L respectively, While the growth of S. costatum was inhibited within 48 h and 96 h as theirs concentrations were higher than 10 mg/L and 0.5mg/L respectively (p<0.05). The concentrations of chlorophyll were the same as the density of phytoplankton cells. The growth of P. donghaiense was inhibited as SWNTs concentrations were higher than 100 mg/L within 72 h, while SWNTs promoted the growth of P. donghaiense within 48 h after SWNTs were added as their concentrations were smaller than 5 mg/L(p<0.05). The results of chlorophyll were similar to the density of P. donghaiense cells. The roles of SWNTs on the growth of algae were suggested to be associated with the active of some enzymes and the physical properties, such as agglomeration and shading.

Type of Paper: Article
Title:
Comparative Metal Oxide Nanoparticle Toxicity Using Embryonic Zebrafish
Authors:
Leah C. Wehmas1, Catherine Anders2, Jordan Chess2, Alex Punnoose2, Cliff B. Pereira3, Juliet A. Greenwood4 and Robert L. Tanguay1, *
Affilications:
1 Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, 1007 Agriculture & Life Sciences Building, Corvallis, OR 97331, USA
2
Department of Physics and the Interdisciplinary graduate program in Biomolecular Sciences, Boise State University, 1910 University Drive, Boise, ID 83725, USA
3
Department of Statistics, Oregon State University, Corvallis, OR 97331, USA
4
Department of Biochemistry and Biophysics, Environmental Health Sciences Center, 2011 Agricultural & Life Sciences Building, Corvallis, OR 97331, USA
Abstract:
Engineered metal oxide nanoparticles (MO NPs) are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of the physicochemical properties contributing to whole-animal nanomaterial toxicity is required. We compared the in vivo toxicity of four widely used semiconductor MO NPs: zinc oxide (ZnO), titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would depend on their physicochemical properties. Our experimental results suggest that hydrodynamic size and charge may not be as important as elemental composition and dissolution potential in dictating MO NP toxicity. Only ZnO NP caused significant adverse effects, and only when prepared in pure water (LC50 = 6.39 mg/L). ZnO NP toxicity was life stage dependent. The 24 h toxicity increased greatly (at least 14.7 fold) when zebrafish exposures started at the larval life stage compared to an embryonic exposure. Quantification of ZnO NP dissolution revealed high levels of Zn ion (40-89% of total sample). Exposure to Zn ion equivalents revealed dissolved Zn was the primary cause of toxicity.

Type of Paper: Article
Title:
Removal of Radioactive Cesium Using Prussian Blue Magnetic Nanoparticles
Authors:
Sung-Chan Jang 1,2, Sang-Bum Hong 1, Hee-Man Yang 1, Kune-Woo Lee 1, Jei-Kwon Moon 1, Bum-Kyoung Seo 1,*, Yun Suk Huh 2,* and Changhyun Roh 1,3,*
Affilications:
1 Decontamination and Decommissioning Research Division, Korea Atomic Energy Research Institute (KAERI), 989-111 Daedukdaero Yuseong, Daejeon 305-353, Korea
2
Department of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon 402-751, Korea
3
Biotechnology Research Division, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI), 1266, Sinjeong-dong, Jeongeup, Jeonbuk 580-185, Korea
Abstract:
Radioactive cesium (137Cs) has inevitably become a human concern due to exposure from nuclear power plants and nuclear accident releases. Many efforts have been focused on removing cesium and the remediation of the contaminated environment. In this study, we elucidated the ability of Prussian blue-coated magnetic nanoparticles to eliminate cesium from radioactive contaminated waste. Thus, the obtained Prussian blue-coated magnetic nanoparticles were then characterized and examined for their physical and radioactive cesium adsorption properties. This Prussian blue-coated magnetic nanoparticle-based cesium magnetic sorbent can offer great potential for use in in situ remediation.

Type of Paper: Article
Title:
Gene Expression, Protein Function and Pathways of Arabidopsis thaliana Responding to Silver Nanoparticles in Comparison to Silver Ions, Cold, Salt, Drought, and Heat
Author:
Jane Geisler-Lee
Affilication:
Department of Plant Biology, Southern Illinois University Carbondale, 1125 Lincoln Drive, MC6509, Carbondale, IL 62901, USA
Abstract:
Silver nanoparticles (AgNPs), due to their unique physical and chemical properties, have been widely used in different sectors of industry. Their widespread usage has caused environmental concerns. However, the similarity in the toxicity assays and expression analysis between AgNPs and silver ions (Ag+) may have led to the conclusion that both could pose the similar environmental risks to plants. In order to further understand the differences between AgNPs and Ag+, a comprehensive analysis was performed using six sets of Arabidopsis microarray data (AgNPs, Ag+, cold, drought, heat and salt). Up-regulated and down-regulated as well as shared and unique genes were categorized according to stresses. The encoded proteins of these genes were also subjected to protein protein interaction network analysis, and gene ontology (GO) enrichment analyses in Pfam domain, InterPro protein class and KEGG pathway. The stress of AgNPs was more similar to the stresses of Ag+, cold and salt than to heat and drought in Arabidopsis. However, AgNPs stress encompassed three unique features (from the other five stresses): response to fungus (i.e. enriched beta-1,3-endoglucanase domain), anion transport, and cell wall/plasma membrane related. The similarity of AgNPs (abiotic stress) to the response to fungus (biotic stress) suggests physical/mechanical damage due to AgNPs’ nano size. Anion transport implies the differentiation of AgNPs stress from Ag+ and Na+ (from salt) stresses. All the 60 AgNPs-specific genes could be sorted into two categories, protection from oxidative burst and involvement in cell wall and/or plasma membrane. In sum, despite the shared similarity in gene expression and metabolic pathways to the three abiotic stresses (Ag+, cold, salt), AgNPs not only are a new abiotic stressor but also pose different toxicity risks to Arabidopsis plants.
Keywords:
Silver nanotoxicity, silver nanoparticles vs. silver ions, abiotic stresses, expression, protein functions

Type of Paper: Review
Title:
Antitumoral activities of metal oxide nanoparticles
Authors:
M.Pilar Vinardell, H, Llanas and M. Mitjans
Affilication:
Physiology Departament, Faculty of Pharmacy, Universitat de Barcelona, 08007 Barcelona, Italy
Abstract:
In recent times, nanoparticles have received much attention for their implication in cancer therapy. Studies have shown that different metal oxide nanoparticles induce cytotoxicity in cancer cells but not in normal cells obtained from different sources. In some cases this anticancer activity has been demonstrated for the nanoparticle alone or in combination with different therapies, such as photocatalytic therapy or with some anticancer drugs. Zinc oxide nanoparticles have been demonstrated to have this activity alone or loaded with an anticancer drug such as doxorubicin. Other nanoparticles showing cytotoxic effects on cancer cells are cobalt oxide, iron oxide, cooper oxide and others. The mechanism of this antitumoral activity could be through the generation of ROS or apoptosis and necrosis among other possible. In the present paper we will review the more significant results obtained with different metal oxide nanoparticles in the context of antitumoral activity.

Type of Paper: Article
Title:
Antibacterial Activity Comparison of three Metallic Nano-oxides and their corresponding Dissolved Metal Ion
Author:
Junfeng Niu
Affilication:
School of Environment, Beijing Normal University, Beijing 100875, China
Abstract:
The potential eco-toxicity of nanosized titanium dioxide (TiO2 NPs), copper oxide (CuO NPs), and zinc oxide (ZnO NPs) water suspensions was investigated using Escherichia coli (E. coli) as test organism. As controls for the solubility, the soluble salts (CuCl2 and ZnCl2) were also analyzed. The true particle sizes of nanoparticles (NPs) in medium were significantly larger than the advertised nanoparticle size nominal diameter. Bacterial toxicity tests showed that all the three NPs were harmful to varying degrees, and their antibacterial activities increased with the increase of the concentration. Antibacterial activity generally increased from TiO2 to ZnO to CuO, which is closely related with the nanoparticle sizes. Ion toxicity further demonstrated that dissolved metal ions were also responsible for toxicity of CuO NPs and ZnO NPs. These results highlight the need for caution during the use and disposal of such manufactured nanomaterials to prevent unintended environmental impacts, as well as the importance of further research on the mechanisms and factors that increase toxicity to enhance risk management.

Type of Paper: Review
Title:
Nanoparticle Optical Properties Confound Assays Commonly Employed for High Throughput / Content Toxicity Testing
Authors:
William Polk 1 and Kevin Dreher 2,*
Affilications:
1. Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27514, USA
2.
Cardiopulmonary and Immunotoxicology Branch, Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA
Abstract:
Nanomaterials are a diverse collection of novel materials with one dimension less than 100 nm and display unique chemical and physical properties due to their nanoscale size. Owing to rapid deployment of nanomaterials and their physicochemical novelty, an emphasis has been placed on developing alternative testing methods, often employing high throughput and content screening (HTS/HCS) assays, to characterize and rank the toxicities of these materials in a manner consistent with the vision of the Toxicology in the 21st Century Initiative. Research reported here demonstrates that many of the available HTS/HCS fluorescent based assays were confounded by nanoparticles (NPs) and their cellular interactions; such that, deployment with standard controls resulted in both false positive and false negative errors. We show effects in assays measuring cytotoxicity, oxidative stress, and nuclear content using commercially available metal oxide NPs ranging in size from 7 to 250 nm and varying in crystalline structure. The confounding effects are demonstrated in a variety of primary human endothelial cell types and are shown to be more severe within cells than would be anticipated from analytic analysis. Moreover, the effects are demonstrated to be particle dependent and not predictable by elemental composition, purity, or primary particle size. It is concluded that a majority of available HTS/HCS assays have the potential to be impacted by particle optical interference and quality analysis is necessary for each particle within a toxicity testing model prior to the initiation of screening programs.

Type of Paper: Review
Title:
Metal Nanoparticles as Nanotoxicants for Aquatic Environments
Authors:
M. Larguinho1, A. Cordeiro1, M.S. Diniz1 and P.V. Baptista2,*
Affilications:

1
UCIBIO-REQUIMTE, DQ, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal
2
UCIBIO, Nanomedicine@FCT, DCV, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Quinta da Torre, 2829-516 Caparic, Portugal
Abstract:
The expansion and development of nanotechnology brought forth novel and improved materials with remarkable applicability in industry, research, biomedicine, etc. Metal nanoparticles (NPs), in particular, have been used in a broad range of applications depending on composition and properties, e.g. silver and magnesium oxide as antimicrobial agents, gold for drug and gene delivery, iron oxide for contrast agents, etc.. The growing use of metal NPs has generated a concern regarding elimination, environmental fate and ecotoxicological risk, where dispersion patterns and adverse effects are still unknown. Metal NPs with different characteristics (size, shape, composition and surface coverage) impact numerous biological consequences and show different risks in terms of partition to water systems and persistence in the environment. Moreover, in an aquatic system, each metal NP shows a dynamic equilibrium of dissolution to free metal ions and/or complexation with ligands in water, which influences toxicity. Therefore, in terms of nanotoxicology, it is important to treat each type of NP as a different contaminant. We will focus on metal NPs’ biological consequences to the aquatic biota. Exposure of organisms with different levels of biological organisation (e.g. microalgae, plants, animals) to NPs will be discussed, regarding adverse effects and potential risk of bioaccumulation. Additional insights will be provided in terms of what is being done towards elimination of metal nanoparticles from aquatic systems to prevent extensive persistence of these contaminants in the environment.

Type of Paper: Article

Title: Nanohybrid Toxicity Evaluation: Characterization Challenges for Emergent Properties and Proposed Strategies

Authors: Navid B. Saleh 1*, Nirupam Aich 1, Joseph Bisesi 2, A. R. M. Nabiul Afrooz 1, Jaime Plazas-Tuttle 1, and Tara Sabo-Attwood 2

Affilication: 1 Department of Civil, Architectural and Environmental Engineering, University of Texas at Austin, Austin, TX 78712

2 College of Public Health and Health Professions, Center for Human and Environmental Toxicology, University of Florida, Gainesville, FL 32611

Abstract: Hybridization of nanomaterials has been pursued to achieve multifunctionality or to enhance material performance. Modulation of physicochemical and electronic properties (e.g., bandstructure, dissolution, surface chemistry) or emergence of new properties (e.g., dimensionality, stiffness) via conjugation can result in unique toxicological consequences. While systematic evaluation of nanohybrid (NH) toxicity are desired, deciphering toxicity mechanisms necessitate thorough assessment of physicochemical properties of NHs in complex environmental and biological media as well as need to utilize appropriate and adequate toxicity assays. This article will present the research progress in evaluation of NH toxicity, review state-of-the-art nano-scale characterization and toxicological assays, and identify research needs to characterize the evolving properties of the NHs. Challenges and limitations in the current electron microscopy, spectroscopic, light scattering, and toxicological assays will be determined and strategies will be proposed to modify or develop new techniques.

Type of Paper: Article
Title:
Effect of developmental exposure to nano-sized diesel engine exhaust particle origin secondary organic aerosol on olfactory-based learning performance and related gene expression in brain of preweaning mice
Authors:
Tin-Tin Win-Shwe*, Yuji Fujitani, Hiroshi Nitta, Seishiro Hirano
Affilication:
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Abstract:
The aims of our present study were to establish a novel olfactory-based learning test and to examine the effects of developmental exposure to nano-sized diesel exhaustparticle origin secondary organic aerosol (SOA), a model environmental pollutant, on learning performance in preweaning mice. Pregnant BALB/c mice were exposed to clean air, diesel exhaust (DE) or DE-origin SOA (DE-SOA) from gestational day 13 to postnatal day (PND) 10 in exposure chamber. On PND11, preweaning mice were examined by olfactory-based spatial learning test. Twenty-four hours after completion of spatial learning test, the olfactory bulb was removed from each mouse and examined the neurological and immunological markers using real-time RT-PCR method. First, we have established the preweaning mouse model which could be used for early diagnosis of learning deficit after developmental exposure to environmental pollutants. On the test phase, the mice exposed to DE or DE-SOA took longer time to reach target compared to the control mice. The expression level of neurological markers such as AC3, GOLF and immunological markers such as TNF-, COX2 and Iba1 were increased significantly in olfactory bulb of DE-SOA exposed preweaning mice compared to the control mice. Our results indicate that developmental exposure to DE-SOA may affect olfactory-based spatial earning behavior in preweaning mice by modulating c-AMP signaling pathway genes and the inflammatory markers in the olfactory bulb of preweaning mice. We suggest that although the potential toxic substances contained in DE-SOA may reach the brain via olfactory nerve route or systemic circulation and induce spatial learning deficit.
Keywords:
developmental neurotoxicity, secondary organic aerosol, olfactory-based learning, preweaning mice, olfactory bulb, nanotoxicity

Type of Paper: Review
Title:
Nanotoxicity: An interplay of Oxidative stress, DNA damage and Apoptosis
Authors:
Puja Khanna, Boon-Huat Bay, Gyeong-Hun Baeg
Affilication:
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Abstract:
Nanoparticles are emerging as a useful tool for a variety of biomedical, consumer and instrumental applications such as drug delivery systems, biosensors and environmental sensors. In particular, nanomedicine has been shown to offer greater specificity, enhanced bioavailability as well as less detrimental side effects as compared to the existing conventional therapies. Hence, bionanotechnology has received immense attention in recent years. However, despite the extensive use of nanoparticle today, there is still a limited understanding of nanoparticle-mediated cytotoxicity. Recent in vivo and in vitro studies have shown that nanoparticles often cause cytotoxicity by increasing reactive oxygen species (ROS) levels and/or the expression of pro-inflammatory mediators. In response to nanoparticle-induced oxidative stress, the homeostatic redox state of the host becomes disrupted, leading to cellular genotoxicity. Furthermore, nanoparticles are known to up-regulate the transcription of pro inflammatory genes such as tumor necrosis factor-α (TNF-α) and IL (interleukins)-1, IL-6 and IL-8 by activating nuclear factor-kappa B (NF-κB) signalling. These sequential molecular and cellular events lead to severe DNA damage followed by apoptosis. However, the exact molecular mechanisms of how nanoparticles induce cytotoxicity are not fully understood. This lack of knowledge is a significant impediment in the use of nanoparticles in vivo. In this review, we will provide a comprehensive assessment of signalling pathways that are involved in the nanoparticle-induced oxidative stress and pro-inflammation. Possible strategies to circumvent nanotoxicity and develop safe and effective nanoparticle-based therapy will also be discussed in detail.
Keywords:
Nanoparticles, Nanotoxicity, Oxidative stress, DNA damage, Apoptosis

Type of Paper: Article
Title:
Penetration and toxicity of nanomaterials in higher plants
Authors:
Giuseppe Chichiriccò and Anna Poma
Affilication:
Department of Life, Health and Environmental Sciences, University of L’Aquila, I-67100 L’Aquila, Italy
Abstract:
Nanomaterials (NMs) comprise both inorganic nanoparticles that consist of metals, oxides and salts coming prevalently from the road and air traffic, and engineered particles  consisting of organic compounds (carbon nanotubes),  which are produced in Laboratory only. They have at least one dimension less than 100 nanometers in size. Accordging to shape, size, surface area and charge they have different mechanical, chemical, electrical and optical properties suitable for technological and biomedical applications to be increasingly produced and manipulated in Laboratory. Despite the profitable potentialities, exposure and the use of nanoparticles are hazardous to health of the terrestrial ecosystems owing to their capacity to enter the animal and plant body and interact with their cells. Studies on NMs involve technologists, biologists, physicists, chemists and ecologists, so there are numerous reports dealing with the potentialities of NMs and their action on the micro-and macro-organisms, which significantly raised the level of knowledge especially in the field of nanotechnology, and many aspects concerning the nano-biology remain undiscovered, including the mechanisms for the in vivo interactions with plant biomolecules. In this review we report and discuss with a bit of criticism a number of studies dealing with the uptake, translocation and the adverse action of the NMs in order to update the knowledge on the plant vulnerabilty and stimulate new experiments on this topic.

Type of Paper: Review
Title:
Role of Physiochemical Properties of Nanoparticles Referring to Toxicity
Authors:
Woo Chul Song 1,, Seung Won Shin 1,, In Hyun Song 1, and Soong Ho Um 1,2, *
Affilications:
1 School of Chemical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea; E-Mail: swc1234@skku.edu
2
SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea.
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: sh.um@skku.edu; Tel.: +82-31-290-7348; Fax: +82-31-290-7272.
Abstract:
With recent rapid growth of technological comprehension of nanoscience, researchers have continuously made an effort with aim of adapting this knowledge into research field of engineering and applied science. Especially, the dramatic advance in nanomaterial marked a new epoch in biomedical engineering with expectation of their huge contribution on human healthcare. However, several questions regarding with their safety and toxicity have came to us concurrently due to their relatively short research history. So, with all these reasons, we should be aware of the dangerousness about handling the nanomaterials. In this paper the recent studies of toxicology of nanomaterials will be reviewed from a physiochemical point of view. Variations of those properties such as size distribution, electrostatic status, surface area, general morphology and aggregation status can significantly affect the physiological interactions between nanomaterials and our tissues. Accordingly, it is very important to finely control these properties in order to fit one’s purpose.

Last update: 4 March 2015

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