Toxicity of inorganic nanomaterials in biomedical imaging.

JBA-06779; No of Pages 17 Biotechnology Advances xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

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Research review paper

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Toxicity of inorganic nanomaterials in biomedical imaging

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Jinxia Li a, Xueling Chang a, Zhanjun Gu a, Feng Zhao a, Zhifang Chai a,b, Yuliang Zhao a a

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China b School of Radiological and Interdisciplinary Sciences, Soochow University, Suzhou 215123, China

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Inorganic nanoparticles have shown promising potentials as novel biomedical imaging agents with high sensitivity, high spatial and temporal resolution. To translate the laboratory innovations into clinical applications, their potential toxicities are highly concerned and have to be evaluated comprehensively both in vitro and in vivo before their clinical applications. In this review, we first summarized the in vivo and in vitro toxicities of the representative inorganic nanoparticles used in biomedical imagings. Then we further discuss the origin of nanotoxicity of inorganic nanomaterials, including ROS generation and oxidative stress, chemical instability, chemical composition, the surface modification, dissolution of nanoparticles to release excess free ions of metals, metal redox state, and left-over chemicals from synthesis, etc. We intend to provide the readers a better understanding of the toxicology aspects of inorganic nanomaterials and knowledge for achieving optimized designs of safer inorganic nanomaterials for clinical applications. © 2013 Published by Elsevier Inc.

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Keywords: Medical imaging Inorganic materials Nanomedicine Nanotoxicity Iron nanoparticles QDs Gold nanoparticles Upconversion nanoparticles

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Introduction . . . . . . . . . . . . . . . 1.1. Iron oxide nanoparticles . . . . . . 1.1.1. In vivo and in vitro toxicity 1.1.2. Origins of toxicity . . . . . 1.2. Gold nanoparticles . . . . . . . . 1.2.1. In vivo and in vitro toxicity 1.2.2. Origins of toxicity . . . . . 1.2.3. Quantum dots . . . . . . 1.2.4. In vivo and in vitro toxicity 1.2.5. Origins of toxicity . . . . . 1.3. Upconversion nanoparticles (UCNPs) 1.4. PET imaging nanoprobes . . . . . . 1.4.1. Conclusions and outlook . 2. Uncited references . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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1. Introduction

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In the 21st century, many new concepts have been proposed and introduced in medical sciences, such as theranosis, preventive medicine,

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E-mail address: [email protected] (F. Zhao).

individualized medicine, multimode imagings, etc. All these require supports from the multifunctional platform of excellent performance (e.g., high sensitivity, high specificity, high resolution and high accuracy). Nanomaterials and nanoscale particles, because of their unique nanocharacteristics and novel physicochemical properties, provide promising multifunctional platforms that suffice the requirements of the above medical diagnosis and/or therapy. For example, early diagnosis that can

0734-9750/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.biotechadv.2013.12.009

Please cite this article as: Li J, et al, Toxicity of inorganic nanomaterials in biomedical imaging, Biotechnol Adv (2013), http://dx.doi.org/10.1016/ j.biotechadv.2013.12.009

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interactions of these engineered nanoparticles with biological systems and the consequent toxicity change with not only materials themselves but also nano-characteristics. For example, the particle size and the nanosurface properties can largely govern the bioavailability, transport, biotransformation, cellular uptake and the triggered biological responses (M. Zhu et al., 2012). Herein, the safe implementation of nanoparticles goes beyond conventional hazard, exposure and risk assessment strategies of the classic materials (Nel et al., 2013). In the past decade, many engineered nanoparticles for biomedical applications have undergone the toxicological studies in vitro or in vivo, however, their pharmacokinetics, namely, the processes of absorption, distribution, metabolism and excretion of these inorganic nanoparticles in vivo, has been less systemically understood so far (Ritschel and Kearns, 2004). After the nanoparticles are administrated into the body in biomedical applications, whatever the entry routes (oral, injection, etc.), interactions between nanoparticles and the biological systems (such as proteins, cells, tissues and organs) are inevitable until the nanoparticles are carried to its effective sites via the bloodstream. The pharmacokinetics of the nanoparticles in vivo and their toxicity that might arise have been described in Fig. 2. First, the exogenous nanoparticles can enter into the bloodstream in contact with various serum proteins. The interaction of nanoparticles with serum proteins mostly leads to the formation of protein layers on the nanosurfaces, called as “protein corona”. The physicochemical parameters of nanoparticles have been found to be critical determinants affecting nanoparticle–protein interactions (Tenzer et al., 2011). In turn, protein corona influences the particle biodistribution, biocompatibility and even therapeutic efficacy (Aggarwal et al., 2009). Therefore, the interaction of the exogenous nanoparticles with serum proteins, after they enter into the blood, has to be carefully investigated in the toxicity assessment. Then, with the bloodstream, different nanoparticles distribute into different organs and tissues to different extents depending on both the physio-anatomical features of the vasculature in these tissue and organs and the physicochemical properties of nanoparticles (Almeida et al., 2011; Wang et al., 2013). Due to the small size of the nanoparticles, the blood can transport them via the circulation to many organs and tissues. In some tissues (i.e., liver and spleen), nanoparticles enter the tissues by size-dependent cellular uptake or endocytosis by macrophages or Kupffer cells so that they are selectively and specifically removed from the blood, which is called tissue-specific extravasation (Wang et al., 2013). Afterwards, these nanoparticles are internalized into cells, offering the basis for executing their diagnostic or therapeutic functions in vivo. Therefore, cellular uptake, intracellular trafficking of nanoparticles and cell fate have to be assessed with the physicochemical properties of nanoparticles being taken into account (Zhao et al., 2011). In some organs, extravasation of the nanoparticles may be restricted by the existence of natural barriers formed by the tight junctions between the endothelial cells. However, the penetration of these barriers (blood–brain barrier, the placenta barrier, etc.) is observed in vivo studies where the undesirable effects (neurotoxicity, reproductive and developmental toxicity, etc.) are likely to be induced in some cases. Following biodistribution, nanoparticles then undergo metabolic processes. The activity of cytochrome P450 enzymes, the main redox enzymes involved in metabolic transformations, is likely to be affected (Fröhlich et al., 2010; Sereemaspun et al., 2008). Finally, the nanoparticles can be excreted from the body via the kidneys or/ and in the feces (Chen et al., 2008). But in some cases, parts of the nanoparticles are retained in the body for the long-term period due to the uncompleted excretion and consequently they may disturb the normal functions of the organs or tissues and induce chronic organ toxicity, metabolic toxicity, immunotoxicity or even genotoxicity. Therefore, to minimize and even abolish the possible health risks, the potential toxicity of these nanoparticles has to be scientifically assessed in depth before they are used in the clinic (Fadeel and Garcia-Bennett, 2010).

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detect diseases before health is deteriorated largely depends on the development of smart diagnostic agents, in which the nanostructured and nanoscale materials play important roles (Bharali and Mousa, 2010). With the rapid accumulation of advanced knowledge on the distinctive properties and unique functions of nanomaterials, utilization of nanoparticle characteristics for medical purposes has shown great promising potentials of building smart nanomedicines for the development of theranosis, preventive medicine, individualized medicine, multimode imagings, etc. Nanomedicine, defined as the applications of nanotechnology for diagnosis, monitoring, therapy and the control of the biological systems, is becoming a hotspot in the research fields of chemistry, materials sciences, medical sciences and clinical translation medicines, etc. (Zhang et al., 2008). The current nanomedicine mainly includes the nanoparticle-based nanovehicles for the targeted drug delivery, diagnostic nanoparticle platforms for the biomedical imagings and the therapeutic nanoparticle platforms for treating clinical diseases (Bharali and Mousa, 2010; Lin and Datar, 2006). The changed physicochemical properties of nanoparticles play crucial roles in the excellent performance of nanoparticle-based medicines (Chen et al., 2005; Cho et al., 2010; Kang et al., 2012; Liang et al., 2010). The first is due to the nanoscale size which makes the physicochemical properties of the nanoparticles different from that of the bulk counterparts. For instance, when the size is scaled down to the range of nanometer sizes, iron oxide nanoparticles are endowed with superhigh magnetic susceptibility (Pouliquen et al., 1992). The second is that the small-sized nanoparticles easily penetrate into the tissue and even cells, allowing the clinicians or researchers to track and detect the histopathological, cellular and even molecular changes during the disease treatment or diagnosis with nanomedicines (Chen et al., 2008). The third is the large surface-to-volume ratio and size-related surface activity. They make the nanoparticles apt to various surface chemical modifications to improve their biocompatibility and to enhance the active targeting by conjugating with disease-related biomarkers (Byrne et al., 2008). The fourth is the nanostructure which can be assembled or constructed layer by layer or shell by shell to contain different payloads for realizing multifunctions-at-one-platform in biomedical applications. Nowadays, the noninvasive in vivo imagings include magnetic resonance imaging (MRI), fluorescence imaging (FI), positron emission tomography (PET) and near-infrared fluorescence imaging (NIRFI), etc. However, given that the limitations for each technology in application, multimodal nanoprobes have been advocated for simultaneous imagings to improve efficiency (Lee et al., 2012). Multifunction-at-oneplatform is the key to actualize the human dreams of multi-mode imagings, theranosis, individualized therapy, etc. (Sun et al., 2011; L.M. Wang et al., 2011). For instance, we developed an integrated multimodal assembly strategy and assembled two gold nanoclusters at the ferroxidase active sites of ferritin heavy chain. The obtained gold–ferritin nanostructure not only retained the imaging properties of gold nanoparticles, but also enhanced fluorescent intensity and possessed tunable emissions from green to far-red owning to the coupling interaction between the paired gold clusters within the ferritin shell. The far-red gold–ferritin nanostructure simultaneously achieved ferritin receptor-mediated targeting and biomedical imaging both in vitro and in vivo and showed great potential as a novel biomedical imaging agent (Sun et al., 2011). Additionally, we innovatively designed a bifunctional peptide which could target cell nuclei and meanwhile biomineralize and capture gold clusters, thus acting as a specific probe for nuclei (L.M. Wang et al., 2011). So far, many nanoparticles have shown enormous potentials of building an effective multifunctions-at-one-platform. Among them, gold nanoparticles, quantum dots, iron oxide nanoparticles, the recently-emerging upconversion nanoparticles, PET imaging nanoprobes, etc. are representative ones with promising applications (Fig. 1) (Taylor et al., 2012). Though they possess new medical functions with high efficacy, the safety concern of inorganic nanoparticles is a key determinant factor in their clinical applications. It is imperative to establish structure–activity relationship (SARs) and to develop the risk reduction strategies. The

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Fig. 1. Typical examples of biomedical imagings with inorganic nanoparticles as contrast agents. A) The schemed structure of Doxorubicin-loaded thermally cross-linked superparamagnetic iron oxide nanoparticles (Dox@TCL-SPIONs); B, C) Optical fluorescence images of major organs and allograft tumors from control and Dox@TCL-SPIONs-treated mice: 1 liver; 2 lung; 3 spleen; 4 tumor; 5 heart; 6 kidney. Samples were derived from tumor-bearing mice (n = 3) intravenously administrated with Dox@TCL-SPION (equivalent to 4 mg of Dox) for 1 h and 12 h (B) and the coresponding control mice that were treated with free Dox (C); (a, b, c were reprinted with permission from ref (Yu et al., 2008); Copyright 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim); (D) Schematic illustration of the synthsized gold–ferritin (Au–Ft) complex. Two gold nanoclusters were assembled at the ferroxidase active sites of ferritin heavy chain with the his residues at the ferroxidase centers playing an important role in the in situ assembly of gold clusters; (E) The emission spectra of the farred Au–Ft complex; (F) Fluorescence images of nu/nu female mice injected with far-red Au–Ft or 0.9% NaCl solution as control. The mice were intravenously injected with far-red Au–Ft at a dose of 0.8 nmol/g body weight or 0.9% NaCl solution in the same volume and the images were taken at 4 h. A representive Au–Ft exposed mouse was shown on the right with a saline injected control mouse on the left. (d, e, f were reprinted with permission from ref (Sun et al., 2011); Copyright 2011, American Chemical Society); (G) Comparisons of fluorescence light emissions of the organic dye tetramethylrhodamine isothiocyanate (TRITC; left vial), green QDs (middle vial) and red QDs (right vial) under normal light illumination at the same concentration of 1.0 mM. In contrast to the unobservable light emission from the organic dye, bright fluorescence emission was observed from the QDs due to the increased light emission rate of QDs. (Reprinted with permission from ref (Gao et al., 2005); Copyright 2005, Elsevier); (H) QDs-labeled cancer cells trapped in a mouse lung. (Reprinted with permission from ref (Voura et al., 2004); Copyright 2004, Nature Publishing Group); (I) In vivo simultaneous imaging of multicolor QDs injected into a live mouse. (Reprinted with permission from ref (Gao et al., 2004); Copyright 2004, Nature Publishing Group); (J) A scheme of the excitation/emission profiles of UCNPs. (Reprinted with permission from ref (J. Zhou et al., 2011); Copyright 2009, Royal Society of Chemistry and American Chemical Society); (K) Fluorescence images of HeLa cells after incubated with rabbit anti-CEA8 Ab-conjugated UCNPs. The fluorescence was excited by a 980 nm NIR laser with increasing excitation powers from 100 to 900 mW or in bright field. (Reprinted with permission from ref (M. Wang et al., 2009); Copyright 2009, American Chemical Society). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

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In this review, we first summarize the in vivo and in vitro toxic responses of typical inorganic nanoparticles that are used in biomedical imaging, followed by further discussions on the origins of the toxicities. We intend to draw the toxicological knowledge of these nanoparticles for optimized designs of safer nanoscale platforms in medical imaging of clinical applications.

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1.1. Iron oxide nanoparticles

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Due to good biocompatibility and improved diagnostic performance, magnetic iron oxide nanoparticles (MION) have been demonstrated as a prominent magnetic resonance imaging (MRI) agent. Early in the 1990s,

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based on the sufficient data obtained from the clinical research, Advanced Magnetics (Cambridge, MA, USA) declared “GastroMark®” (ferumoxsil) in Europe, the first MION-based MR imaging agent. In 1996, Feridex® (ferumoxides) was approved as a novel contrast agent for the hepatic MRI with a favorable safety profile by U.S. FDA. Since then, a number of other iron oxide nanoparticle compounds have been developed in succession, such as Resovist® (ferucarbotran), Combidex® (ferumoxtran-10) and Feraheme® (ferumoxytol). To date, about ten iron oxide contrast agents have been commercialized or at the different clinical phases (Qiao et al., 2009). The superparamagnetic iron oxide nanoparticles are generally considered to be low-toxic and hence proposed as an alternative MRI contrast agent for the patients


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Fig. 2. An illustrative scheme for the pharmacokinetics of extraneous nanoparticles in vivo and possible toxicities that may arise. Nanoparticles, after delivered into the body, undergo the process of pharmacokinetics. They are absorbed and brought into the bloodstream where nanoparticle–serum protein interactions frequently occur. Then they spread into tissues and organs via tissue-specific extravasation from the bloodstream. Afterwards, they may be metabolized in the liver by cytochrome P450 enzymes and some may start metabolic process early in the gastrointestinal tract. Finally, the remnant nanoparticles will be removed from the body via the kidney (urine) or bile (feces). At each step of this process, toxicity may arise. Among them, hematological abnormality, organ toxicity and immunotoxicity arise from the long-term retention of nanoparticles in the liver, lung, spleen and kidney et al. Moreover, nanoparticles may cross the biological barrier system (blood–brain barrier, placental barrier, etc.) and neurotoxicity, reproductive toxicity may be induced. Genotoxicity may also appear. In addition, nanoparticles probably disturb the normal metabolic functions and induce metabolic abnormality.

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1.1.1. In vivo and in vitro toxicity Considering that magnetic nanoparticles have hydrophobic surface and easily agglomerate, a proper surface coating, e.g., a sugar or a polymer, has to be used to improve their stability and dispersibility in homogenous ferrofluids (Kim et al., 2003). Notably, the homogenous ferrofluids with proper surface coatings, in normal dosages, have shown little toxicity in the preclinical and even clinical studies. In a preclinical study, the pharmacokinetics of ferumoxtran-10 (a ultrasmall superparamagnetic iron oxide particles for MRI contrast agent) have been studied with five different animal models including mice, rats, rabbits, dogs and monkeys. After injection, superparamagnetic iron oxide nanoparticles were mainly taken up by macrophages in liver, spleen and lymph nodes within 24 h and then underwent progressive metabolism, and toxicity was only observed in the high dose with repeated injections (Bourrinet et al., 2006). In phase III clinical study (152 patients) of ferumoxtran-10 for the diagnosis of lymphatic metastasis, ferumoxtran-10 exhibited satisfactory performances with rare occurrences of the headache, back pain, vasodilatation and urtucaria as side effects (Anzai et al., 2003). However, under some circumstances, surface coatings can be degraded and expose internal iron oxide nanoparticles to biological systems, which probably cause undesired adverse effects. Understanding the in vivo behaviors of ferric oxide nanoparticles is crucial for assessment of their potential health risk. Previously, we focused on the particokinetic profile of the inhaled Fe2O3 nanoparticles (22 nm) in rats at a dose of 4 mg/rat. The intratracheally instilled nanoparticles were detected to penetrate the alveolar–capillary barrier into systemic circulation within 10 min and then selectively and specifically distribute into phagocyte-rich organs (liver, spleen, kidney and testicle)

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at risks of nephrogenic systemic fibrosis caused by the gadoliniumbased contrast agents (Neuwelt et al., 2009). Due to other wide applications of iron oxide nanoparticles in clinical, clinicians and researchers still keep a close eye on the potential side-effects and toxicity in longterm clinical practice to achieve a safer clinical application.

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where they were phagocytosized by monocyte/macrophages. Parts of Fe2O3 nanoparticles were deposited in the interstitial lung. The plasma elimination half-life of the nanoparticles was calculated to be 22.8 days and the lung clearance rate was 3.06 μg/day. Our results pointed out that the inhaled nanoparticles were not only confined to the lungs but also may go through systemic circulation and redistribution (Zhu et al., 2009). Therefore, systemic toxicity has to be cautioned and monitored that may arise from the occupational and other longterm exposure of the inhaled nanoparticles. In particular, a comparative study of toxic responses of nano- and micron-sized ferric oxide nanoparticles could help us to fully understand the nanotoxicity mechanisms and the risk factors for iron oxide nanoparticles. Hence, we explored the impact of the ferric oxide (Fe2O3) nanoparticles on the pulmonary and coagulation system, followed by further investigation on the relationship between the potential toxicity and the important toxicity-related factors (the size, dosage and exposure time). Fe2O3 nanoparticles with the sizes in 22 and 280 nm, respectively, were intratracheally instilled to male Sprague Dawley rats at the low (0.8 mg/kg bw) and high (20 mg/kg bw) doses. Then toxic effects were monitored at 1, 7 and 30 days postinstillation. Resultantly, oxidative stress-related lung injury was induced with nano-sized Fe2O3 particles more potent than submicronsized ones. Alveolar macrophages were overloaded with the phagocytosed nanoparticles and pro-sign of lung fibrosis was triggered. Moreover, Fe2O3 nanoparticles disturbed the coagulation process as shown by the prolongation of prothrombin time (PT) and activated partial thromboplastintime (APTT), the two typical coagulation parameters at the 30th day of post-instillation (Zhu et al., 2008). Consistent with our results, a similar toxicity was reported that a weak pulmonary fibrosis developed at the 30th day of post-exposure in adult male Wistar rats intratracheally instilled with Fe2O3 nanoparticles at a single dose of 5 mg/kg (Szalay et al., 2012). Other organ toxicity of iron oxide nanoparticles has also been widely reported. After a single intravenous injection of γ-Fe2O3 nanoparticles


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intranasally instilled with Fe2O3 nanoparticles (280 ± 80 nm) at a single dose of 40 mg/kg. Detected by synchrotron radiation X-ray fluorescence analysis, Fe contents were significantly increased in olfactory nerve and the trige minus of brain stem at two-week post-exposure (Wang et al., 2007). The ratios of Fe (III)/Fe (II) were increased in the olfactory bulb and brain stem as analyzed by X-ray absorption near-edge structure (XANES). Concomitantly, the neuron fatty degeneration occurred in the CA3 area of hippocampus (Fig. 3A). So, our result demonstrated that the transferred Fe2O3 nanoparticles via olfactory nerve into the brains did pose safe risks to the neurological system (Wang et al., 2007). Later, we explored the potential neurological toxicity in more detail. Fe2O3 nanoparticles were observed to be mainly deposited in the olfactory bulb, hippocampus and striatum where neurological damage was elicited. Microglial, the main participator in the immune system of CNS (central nerve system) when CNS was injured, was found to be recruited to the above specific areas, especially in the olfactory bulb. Meanwhile, substantial ROS and NO were produced. Neuropathological changes in the brain of the exposed mouse were detected including the irregular arrangement of neuron cells in the olfactory bulb, the cellular

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(0.8 mg/kg) into rats, 40% of the administrated γ-Fe2O3 nanoparticles were excreted via urine within 24 h, and in total 75% could be removed from the body at 72 h. However, the remnant γ-Fe2O3 nanoparticles induced inflammation in the liver, kidneys and lungs (Hanini et al., 2011). In a metabolic analysis of the injected Fe2O3 nanoparticles (at a single dose of 25 mmol/L) in rats, at 48 h post-injection, the nanoparticles induced metabolic abnormality in the spleen and kidneys of the rats with the obvious alterations of the metabolism-associated small molecules and the enzymes such as triglycerides, phospholipids, N- and O-acetyl glycoprotein, glucose and glycogen (Feng et al., 2011). Here it is a highly concerned question: during biodistribution, may the iron oxide nanoparticles cross through the biological barriers in the body? The first natural barrier is the blood–brain barrier (BBB) which directly relates to the neurotoxicity and needs to be concerned. Actually, few results indicated that nanoparticles can directly cross the blood–brain barrier (BBB) and induce damages in the brain. Nevertheless, the intranasally instilled Fe2O3 nanoparticles could transfer to animal brains via the olfactory pathway. We have analyzed the iron microdistribution and chemical state in the brain of mice that was

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Fig. 3. The distribution and the potential neurotoxicity of Fe2O3 in mice. (A) Absorption edge, edge shift and Fe (III)/Fe (II) ratio in brain samples determined by X-ray absorption near-edge structure (XANES) analysis. The brain samples derived from 14-day Fe2O3 nanoparticles-exposed mouse. (Reprinted with permission from ref (Wang et al., 2007); Copyright 2007, Humana Press Inc.); (B, C) Immunofluorescence microscopic observation of CD11b (a marker protein of microglia activation) in mouse olfactory bulb (Objectives: 40×). In the inserted picture on the top right corner, arrows showed CD11b-immunreactive microglia both in the control (B) and γ-Fe2O3 nanoparticles-exposed group (C) (B, C were reprinted with permission from ref (Y. Wang et al., 2011); Copyright 2011, Elsevier); TEM images of ultrastructural alterations in the hippocampus section in Fe2O3 nanoparticles-exposed mice at 30 days postinstillation (D, E, F). (D) The Fe2O3 particles existed inside mitochondria, lysosomes and nearby the outer surface of the mitochondria membrane. L: lysosome, M: mitochondria, magnification: 920000; (E) Slight dilation of rough endoplasmic reticulum and the increased lysosomes in hippocampus of mice. L: lysosome, RER: rough endoplasmic reticulum, magnification: 914000; (F) Several vacuoles presented in the nerve cell cytoplasm of the mice, V: vacuoles. (D, E, F were reprinted with permission from ref (B. Wang et al., 2009); Copyright 2008, Springer Science + Business Media B.V.).


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produced highly reactive hydroxyl radicals that mediated the toxicity of nanoparticles (Voinov et al., 2011). L-glutamic acid-coated Fe2O3 nanoparticles induced a disturbance on the cell redox status in CHL (Chinese hamster lung) cells. They caused ROS production, glutathione depletion and inactivation of some antioxidant enzymes including glutathione reductase, superoxide dismutase, but not catalase (J. Zhang et al., 2012). The iron oxide nanoparticles could further induce oxidative stress-related apoptosis with loss of mitochondria membrane potential and the apoptotic chromatin condensation in ECV304 (human umbilical endothelial) cells (Zhu et al., 2010). Furthermore, the nanoparticlegenerated ROS also mediated microtubule remodeling and increased cell permeability in human microvascular endothelial cells, while a pretreatment of a ROS scavenger catalase significantly inhibited the cell permeability (Apopa et al., 2009). The Fe2O3 nanoparticles-induced neurotocixity was also attributed to the significant oxidative stress caused by the nanoparticles, in which the altered activity of oxidative stress-associated enzymes was determined (B. Wang et al., 2009). Hence, it seems that ROS production and the sequent oxidative stressinduced cellular damages are responsible for the potential toxicity of iron oxide nanoparticles in vivo and in vitro. Recently, we have analyzed and summarized the detailed mechanisms of ROS generation by nanomaterials from a chemical viewpoint (Yan et al., 2013). As the size of materials scales down to nanometers, the increasing surface area makes more atoms be exposed on the particle surface. These external atoms have dangling bonds (vacancies and dislocations), which renders the nanoparticles highly reactive with high surface energy. Thus, the outer orbit electrons of the atoms located on the surface readily transfer to the electron-acceptor groups and reduce these groups in biological substance. Meanwhile, the surface vacancies can accept electrons from electron-donor group, thus oxidizing the biomolecules. In this case, the ROS production relies on the direct contact of nanomaterials surface and biomolecules, so we call it the direct pathway of ROS generation. Opposite to the direct pathway, ROS can be indirectly produced when nanomaterials disturb endogenous biochemical/ physiological equilibriums of cells. Due to the high surface energy, nanomaterials have to slow down the surface energy and stabilize themselves. In biological systems, they snatch the biomolecules and absorb them on particle surface where weak interactions such as electrostatic attraction, π–π stacking happen. This process, consequently, affects the functions of enzymes and damages the structure of cellular organelles, particulately mitochondria, and results in the increased production of ROS in cell. The above two pathways served as the chemical basis of the intracellular ROS production by nanomaterials, especially themetal-related inorganic nanoparticles (Yan et al., 2013).

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1.1.2.2. Iron redox state and chemical instability. The iron redox state also directly affects the toxicity. The iron redox state could dramatically modify the cellular uptake of Fe-containing nanoparticles and influence their induction of oxidative DNA damage (Singh et al., 2012). In Escherichia coli., it was observed that chemically stable nanoparticles (γ-Fe2O3) had no apparent cytotoxicity whereas nanoparticles containing ferrous and particularly zero valent iron were highly cytotoxic. And this difference was verified to be attributed to ROS production by the interplay of oxygen with reduced iron species (FeII and/or Fe0) due to their chemical instability (Auffan et al., 2006). Actually, the chemical instability of the reduced iron species has been evidenced by many studies. For example, addition of nanoparticulate zero-valent iron to oxygencontaining water resulted in oxidation of organic compounds (Joo et al., 2005). Fe0- and Fe3O4-based nanoparticles were reported to be easily oxidized in biological media, showing their high sensitivity towards oxidation (Zhang, 2003).

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1.1.2.1. ROS generation and oxidative stress. The production of ROS (reactive oxygen species) is regarded as the main mechanism underlying the toxicity of metallic nanoparticles including iron oxide nanoparticles. For example, the γ-Fe2O3 nanoparticles (20–40 nm in diameter)

1.1.2.3. Chemical composition and the surface modifications. On chemical aspects, surface chemical modifications have a significant impact on the toxicity of iron oxide nanoparticles. For instance, iron oxide nanoparticles coated with various functional groups\OH,\COOH,\NH2

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swelling, nuclear chromatin condensation and fragmentation in the striatum and cerebral cortex, and the damage towards the integrity of neuronal cells in hippocampal CA1 (Y. Wang et al., 2011). Afterwards, we investigated the neurotoxicity and size effect of repeatedly low-dose (130 μg) intranasal exposure of nano- and submicron-sized Fe2O3 particles (21 nm and 280 nm) to mice. Fe2O3 nanoparticles were found to be located in the hippocampus and olfactory bulb, where they induced size-dependent oxidative stress-related nerve cell damages (Fig. 3 B, C). In the olfactory bulb and hippocampus of exposed mice, alterations in the activity of oxidative stress-related biomarkers were detected with a significant elevation in activity of GSH-Px, Cu, Zn-SOD and cNOS and an obvious decrease in total GSH and GSH/GSSG ratio. Coupled with the oxidative stress, ultrastructural alterations in nerve cells occurred as reflected by membranous structure disruption and lysosome increase in the olfactory bulb (B. Wang et al., 2009) (Fig. 3 D, E, F). These studies have suggested that the inhaled iron oxide nanoparticles may cross BBB and enter into the brain where neurotoxicity is induced. Some results obtained from in vivo studies were consistent with the in vitro data. Lots of data reported the cytotoxicity induced by iron oxide nanoparticles in various cells. As we previously reported, in human aortic endothelial cells (HAECs) incubated with Fe2O3 nanoparticles, Fe2O3 nanoparticles penetrated the cell membrane and localized into the cells, leading to cytoplasmic vacuolation, mitochondrial swelling and even cell death. Additionally, they further increased the expression and secretion of the inflammatory factors ICAM-1 and IL-8 (Zhu et al., 2011). In another cell line PC12, Fe2O3nanoparticles were also internalized and resulted in a dose-dependent decrease in cell viability and also in the cell response to nerve growth factor (Pisanic et al., 2007). However, note that the origin of the observed neurotoxicity may be associated with surface modifications of the nanoparticles. Fe2O3 nanoparticles coated either with dextran or various functionalized polyvinyl alcohols (PVAs) were shown almost non-toxic although they were largely taken up into the isolated brain-derived endothelial and microglial cells (Cengelli et al., 2006). Another very important biological barrier is placenta which directly relates to reproductive and developmental toxicity. Fe2O3 nanoparticles were reported to penetrate the placenta barrier and induce the reproductive and developmental toxicity. When the dimercaptosuccinic acid (DMSA)-coated iron oxide nanoparticles were intraperitoneally injected into the pregnant mice, the nanoparticles were then found in the placenta and fetal liver. Particularly, nanoparticles at a dose higher than 50 mg/kg slowed the growth of infants and moreover caused the histological abnormality in these infants' testicles (Noori et al., 2011). Associated with the reproductive and developmental toxicity, the possible genotoxicity is hence highly concerned. Though the experimental data are still limited so far, some evidences have shown that the genotoxicity probably depends on particle size and exposure dosage. For example, in human bronchial epithelial cells BEAS-2B, αFe2O3 nanoparticles were internalized and induced significant genotoxic effects at particle concentrations higher than 50 μg/mL. Also a size-dependent genotoxicity was observed with nanoscale iron oxide particles inducing more potent genotoxicity than their microscale counterparts (Bhattacharya et al., 2012). Moreover, the possible reproductive and developmental toxicity in a new model organism, zebrafish, was reported. When zebrafish of early life stages were exposed to iron oxide nanoparticles with increasing doses, iron oxide nanoparticles at doses higher than 50 mg/L generated significant developmental toxicity including mortality, hatching delay and malformation (X.S. Zhu et al., 2012).


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differed in cytotoxicity and genotoxicity as shown in L-929 fibroblasts. 456 The positively charged nanoparticles showed a dose-dependent 457 genotoxicity on the cells while the bare nanoparticles and\OH modi458 fied nanoparticles caused no obvious genotoxicity at comparable con459 centrations (Hong et al., 2011). Similarly, iron oxide nanoparticles 460 with different coatings showed a different toxic effect on primary corti461 cal neurons. The polydimethylamine (PEA)-coated nanoparticles dam462 aged the integrity of membrane and induced cell death whereas 463 aminosilane-coated nanoparticles had a much lower toxicity and only 464 affected the metabolic activity at higher concentrations. Dextran465 coated nanoparticles partially altered cell viability at higher concentra466 tions (Rivet et al., 2012). However, how surface modifications affect 467 nanotoxicity remains unclear. One possibility exists that different sur468 Q10 face coatings have different stabilities. Factually, despite the fact that 469 iron oxide nanoparticles coated with different polymers are stable in 470 water or PBS buffer, they may differ in stabilities in culture media with 471 or without the presence of serum. Some surface coatings turn to more 472 readily degrade and expose the internal iron oxide nanoparticles to bio473 Q11 logical system (Arbab et al., 2005; Lu et al., 2006). The bare iron oxide 474 nanoparticles are usually toxic due to their surface hyperactivity and 475 thus proper surface coatings may mitigate the toxicity. For instance, 476 by determination of the surface state of the nanoparticles before and 477 after 24 h contact with the cells using the extended X-ray absorption 478 fine structure (EXAFS), it was found that DMSA coating prevented fibro479 blasts from direct contacts with the nanoparticles, and no obvious cyto480 toxicity was observed in human dermal fibroblasts incubated with 481 DMSA-coated maghemite nanoparticles (nano-γFe2O3) with concen482 trations ranging from 10−6 to 10−1 g/L (Auffan et al., 2006).

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Gold nanoparticles possess some prominent advantages as a new biomedical imaging agent. First, with the well-established synthetic methods, gold nanoparticles are readily manipulated with the substantial design considerations related to the physical and chemical properties. Second, the low background concentration of gold in biological system makes it apt to be measured at extremely low level. And the large electron density of gold enables it to be easily located by electron microscopy (Alkilany et al., 2013). Therefore, gold nanoparticles, as a potential biomedical imaging agent, have shown great prospective in medical diagnosis (Boisselier and Astruc, 2009; Pissuwan et al., 2008) and other medical applications such as cancer therapy by selective mitochondria accumulation of the gold nanoparticles in cancer cells (L.M. Wang et al., 2011) and as a versatile platform for vaccine nanoadjuvants or delivery systems (Xu et al., 2012).

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1.2.1. In vivo and in vitro toxicity The results have shown the in vivo toxicity of gold nanoparticles when they were injected into experimental animals. Below we summarize the major aspects of the toxicity responses induced by gold nanoparticles. Organ distribution of gold nanoparticles was found to be related with the particle size. Rats were intravenously injected in the tail vein with gold nanoparticles with a diameter of 10, 50, 100 and 250 nm, respectively, and the gold content in different organs was determined after 24 h. Au-nanoparticles (10 nm) were found to have an extensive tissue distribution, including the blood, liver, spleen, kidney, testis, thymus, heart, lung and brain, while the larger particles were only detectable in the blood, liver and spleen. It shows that the tissue distribution of gold nanoparticles is size-dependent, and the smaller nanoparticles spread to more organs than larger ones (De Jong et al., 2008). The distribution of gold nanoparticles also needs to be investigated in the specific sensitive stages (e.g. the pregnancy). Previously, we investigated the distribution and cytotoxicity of gold nanoparticles in pregnant mice. Our result revealed that fetal exposure to nanoparticles in murine pregnancy was influenced by both the gestational age and nanoparticle surface composition. As for the influence of gestational age, a critical time window existed in murine pregnancy and impacted the extent of fetal exposure to maternally-administrated gold nanoparticles. The time window was found to be between E9.5 and E11.5 when the transfers of gold nanoparticles from the mother to the fetal was dramatically reduced, basically in coincidence with the maturation of the murine placental blood supply and barrier function of the placenta. In addition to gestational age, the cytotoxicity of gold nanoparticles depended on the surface modification (ferritin, PEG and citrate). This novel finding provided a biosafety implication for Au-nanoparticle administration in pregnancy in humans (Yang et al., 2012). From the toxicological viewpoint, the accumulations of gold nanoparticles in organs may be a potential cause of the toxicity. After a single intravenous injection of a 0.2 mL (15.1 μg/mL) gold nanoparticle solution in rats, the gold nanoparticles were found to be rapidly and consistently accumulated in the liver and spleen within 2 weeks. This accumulation significantly altered the in vivo expressions of genes related to detoxification, lipid metabolism, cell cycle, defense response and circadian rhythm (Balasubramanian et al., 2010). Gold nanoparticles caused renal tissue alterations and had potential nephrotoxicity in rats after one-week nanoparticle exposure. They also induced the alterations in renal tubules marked with cloudy swelling, vacuolar degeneration in a particle size-dependent manner. These were possibly related with gold nanoparticle-induced oxidative stress (Abdelhalim and Jarrar, 2011). The similar ROS-induced histological alterations in the cardiac tissue were observed in rats with exposure to gold nanoparticles (Abdelhalim, 2011). When male Wister rats were intraperitoneally injected with gold nanoparticles (20 μg/kg body weight) for 3 days, an oxidative stress-induced DNA damage and cell death were observed in the brain accompanied with the decreased expression in antioxidant

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1.1.2.4. Dissolution of the nanoparticles and release of excess free iron. Iron oxide nanoparticles are biodegradable and hence undergo the physiological iron metabolism by cells. Iron oxide nanoparticles in the lysosomes are easily degraded into iron ions (Ponka, 1999). As reportedly, Fe, once released from the degradation of iron oxide nanoparticles in lysosomes, is stored in ferritin and metallothioneins to reduce the free Fe level in cells (Hohnholt et al., 2012). However, toxicity probably arises once cells fail to handle the excess irons. On one hand, the excess Fe ions may induce the oxidative stress-related damages. In iron overload mouse, hepatic iron, liver-to-body weight ratio and hepatic lipid peroxidation were observed to be dose-dependently increased (Zhao et al., 2005). Iron oxide nanoparticles were reported to dose-dependently suppress the osteogenic differentiation in human mesenchymal stem cells which could be restored by desferrioxamine, an iron chelator, indicating the toxicity of free Fe in osteogenic differentiation (Chen et al., 2010). On the other hand, Fe iron may disturb the homeostasis of trace metals. It was reported that the inhaled Fe2O3 nanoparticles induced an imbalance of Fe, Cu and Zn in sub-brain regions after mice were intranasally instilled with Fe2O3 nanoparticles at a single dose of 400 μg (10 μL) for 30 days (Wang et al., 2008). In the subcellular distribution analysis of metal-containing proteins of Fe, Cu, Zn and Cd in ironoverload mice, an obvious alteration was found in the distributions of Cu, Zn and Cd in the liver samples obtained from in the treated mice. This suggested that the excessive iron accumulation in vivo may affect the metabolism of other elements such as Zn and Cd (Zhang et al., 2009). Hence, it is important to avoid administration of iron oxide nanoparticles in high doses or repeated dosing over a short time interval in order not to exceed the normal capacity for handling of iron by cells. In summary, although no obvious toxicity has been reported in clinic for iron oxide nanoparticles, their potential health risks still exist if the protective surface coatings are degraded. The biodegradable iron oxide nanoparticles may cause toxicity due to the release of excess Fe iron and the subsequent oxidative stress. Especially, in some cases, iron oxide nanoparticles may easily penetrate some biological barriers (blood brain barrier, placental barrier, cell membrane, etc.), posing potential risks for nervous system and reproductive system.

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1.2.2.1. Excess reactants from synthesis. Nanoparticles are multicomponent systems. The simplest gold nanoparticle solution contains the core material (gold), surface-bound stabilizing ligands and the potential excess reactants from the synthesis (Alkilany and Murphy, 2010). Experimental studies revealed that these excess reactants from synthesis were responsible for the potential toxicity of nanoparticles. Among the excess reactants in gold nanoparticles, the most notorious is cetyltrimethylammonium bromide (CTAB), a cationic surfactant used to direct the growth of gold nanorods (Tang et al., 2013). CTAB itself is quite toxic to cells at submicromolar doses (Isomaa and Ekman, 1975). Hence, the toxicity of gold nanorods probably arises due to lack of the adequate purification or desorption of surfactant from the surface of the nanorods. For instance, when HT-29 cells were incubated with CTAB-capped, PAA-coated, PAH-coated gold nanorod solutions at the same concentration of 0.4 nM for five continuous days, the cells incubated with CTAB-capped gold nanoparticles exhibited an obvious decrease in cell viability. Once the CTAB-capped gold nanoparticles were overcoated with PAA or PAH, they became basically non-toxic. In the PAA-coated and PAH-coated gold nanoparticle-treated groups, cell viability was approximate to that of the control medium-incubated cells. In the experiment, the above three nanoparticle solutions were centrifuged to obtain their corresponding supernatants. Surprisingly, toxicities of supernatant solutions were similar to their original nanoparticle solutions. Particularly, the CTAB-capped gold nanorods supernatant was highly toxic with the most obvious decrease in cell viability (Fig. 4). Meanwhile, 0.2–0.3 μM CTAB was detected to be

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endosome/lysosome and facilitated the release of the functional nucleic acids into the cytoplasm while polyelectrolyte itself did little damage to cells (Guo et al., 2010). The human keratinocyte cells were incubated with 1.5 nm gold nanoparticles bearing positive, neutral and negative charges. The results demonstrated that LD50 (the medium lethal concentrations) for the charged gold nanoparticles and the neutral ones were 10 μg/mL, and 25 μg/mL, respectively. Accordingly, compared with the neutral gold nanoparticles, a more pronounced decrease in the mitochondrial membrane potential was observed in cells treated with the charged nanoparticles. Therefore, when considering the toxicological mechanism, the charge interaction between nanoparticles surface and the mitochondrial outer membrane, may account for the difference in cytotoxicity between the charged gold nanoparticles and the neutral ones (Schaeublin et al., 2011). In addition, surface chemistry and aspect ratio also mediate cellular uptake and toxicity of gold nanorods. In our previous study, we determined the contents of the internalized gold nanorods with different surface coatings in MCF-7 cells. It demonstrated that poly (di-allyldimethyl ammonium chloride) (PDDAC)-coated gold nanorods were the most easily uptaken by MCF cells, followed by CTAB-coated ones. The amount of the polystyrene sulfonate (PSS)-coated gold nanorods in cells was the smallest. More significantly, the amount of the proteins absorbed on the nanoparticles surface was found to be correlated with the cellular uptake of these nanoparticles. So, we speculated that nanoparticles with more proteins on surface may possess more ligands which can be recognized by membrane receptors and thus facilitated the receptor-mediated internalization of these nanoparticles, providing a possible explanation for the surface-chemistry-induced difference in cellular uptake. Aspect ratio of the nanoparticles, namely the shape in a more readily understood term, is another key factor in determining the particles' internalization. We observed that the longer gold nanorods were more difficult to be internalized with the substantial formation of larger aggregates in loose and irregular structures, compared with the shorter ones. Despite the lack of the data for explaining the shape-induced difference in internalization extent, it was postulated that more energy was needed for the internalization of the larger aggregates formed by longer nanorods (Qiu et al., 2010).

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enzymes and the appearance of lipid peroxidation (Siddiqi et al., 2012). These toxic responses can be altered by the surface chemistry of gold nanoparticles. Surface coating is considered as a crucial factor in determining the biodistribution, clearance and toxicity of gold nanoparticles. For instance, BSA- and GSH-protected gold nanoparticles differed in the pharmacokinetics. The GSH-protected gold nanoparticles could be quickly removed by renal clearance and thus the toxicity was significantly decreased, however, the BSA-protected ones had low-efficient renal clearance and largely accumulated in the liver and spleen, causing irreparable toxicity response (X.D. Zhang et al., 2012). Due to nano-scale sizes, nanoparticles in most cases are easily uptaken by cells. We previously reported that cell uptake of gold nanoparticles was size-dependent in normal rat kidney cells and a most appropriate particle size for cellular uptake may exist. As determined by ICP-MS, the 50 nm gold nanoparticles were most favorable for cellular uptake, followed by 25 nm and 10 nm gold nanoparticles (Ma et al., 2011). When murine macrophages were incubated with gold nanoparticles (60 nm) for 24 h, a large number of intercellular vacuoles were formed and gold nanoparticles were observed to be clustered in the vacuoles. However, neither obvious cytotoxicity nor increased production of the proinflammatory mediators was observed at the investigated time frames (Zhang et al., 2011). This might lead to a conclusion that cellular uptake of gold nanoparticles does not induce cytotoxicity. However, many other investigations have demonstrated that the cytotoxicity of nanoparticles remains as a complicate issue depending on various factors, including cell type, particle size, surface charge and the surface modifications, etc. Actually, a similar accumulation of gold nanoparticles in large vacuoles was observed in the human dermal fibroblasts, but they induced the toxicity. After exposed to gold nanoparticles, cells grew slowly and declined the expressions of extracellular matrix proteins, an indicator of the damage to the normal function of dermal fibroblasts. The formation of vacuoles disrupted the cytoskeleton and prohibited cell contraction and motility required in cell proliferation and growth (Mironava et al., 2010). When incubated with dendritic cells (a major mediator in both the innate and acquired immune responses), gold nanoparticles did not directly kill the dendritic cells, but perturbed the immune system by modulating the secretion of immune-related cytokines after they were largely internalized into cells by endocytosis process (Villiers et al., 2010). The cytotoxicity of gold nanoparticles depends on nanosize. A study was made with gold nanoparticles ranging from 0.8 to 15 nm in four cell lines representing major functional cell types including connective tissue fibroblasts, epithelial cells, macrophages and melanoma cells. When exposed to 1.4 nm gold nanoparticles, IC50 of all the four cell lines ranged from 30 to 56 μM. In contrast, gold nanoparticles with 15 nm in size were nontoxic at up to 60-fold higher concentrations, clearly suggesting a size-dependent cytotoxicity (Pan et al., 2007). A similar size-dependent cytotoxicity was reported that the internalization of gold nanoparticles size-dependently induced damage to the lysosome degradation capacity by alkalinization of lysosomal pH in normal rat kidney cells (Ma et al., 2011). However, surface modifications of the gold nanoparticles can largely change the correlation between nanosize and cytotoxicity, which can be exploited in lowering the toxicity of gold nanoparticles. For example, the toxicities of 18 nm gold nanoparticles with different surface modifications including citrate, biotin and cetyltrimethylammonium bromide (CTAB) were studied in human K562 cells within 3 days. It showed that citrate and biotin-modified gold nanoparticles exhibited little cytotoxicity up to a concentration of 250 μM whereas the CTAB-coated nanoparticles were toxic even at a concentration of 0.05 μM (Connor et al., 2005). The surface charges also impact the cytotoxicity of gold nanoparticles. We firstly reported the novel application of the charge-reversal polyelectrolyte deposited gold nanoparticles as efficient gene delivery. Charge-reversal polyelectrolyte pH- dependently shifted the charge nature between positive and negative. The charge reversion under acidic environment assisted gold nanoparticle/nucleic acid escaping from the

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Fig. 4. Cytotoxicity induced by the possible impurities contained in gold nanoparticle solution. (A) The simplified process of the experiment to study the impacts of the impurities contained in gold nanoparticle solution on cytotoxicity in HT-29 cells. HT-29 cells were incubated with the supernatant after centrifugation and the original gold nanoparticle solution, respectively, and the cell viability was determined by MTT assay. (B) The comparison on the induced cytotoxicity between the original gold nanoparticle solutions and their corresponding supernatants. This comparison was furthermore performed among gold nanoparticles with different surface modifications (CTAB-coated, PAA-coated and PAH-coated) to highlight the optimization for reduced cytotoxicity by surface modification. CTAB: cetyltrimethylammonium bromide, PAA: poly (acrylic acid, sodium salt), PAH: poly (allylamine hydrochloride). (Reprinted with permission from ref (Alkilany et al., 2013);Copyright 2013, American Chemical Society; Data used with permission from ref (Alkilany et al., 2009); Copyright 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim).

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contained in the supernatant of CTAB-capped gold nanorods while CTAB concentrations in the other two supernatants were below the detection limit. So, it was the CTAB not the nanoparticles that contributed to the toxicity of gold nanoparticles (Alkilany et al., 2009). Furthermore, sodium citrate, another common contaminant present in gold nanoparticles, also exerts toxic effects. With other experimental conditions invariable, nanoparticles bearing more sodium citrate on the surface were reported to decrease cell viability more significantly in A549 cells (Uboldi et al., 2009). So, the purification in preparation processes of gold nanoparticles to reduce the amount of contaminants will improve the biocompatibility and lower the toxicity of gold nanoparticles in vivo and in vitro.

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1.2.2.2. Oxidative stress. Gold is known to be chemically inert and resistant to oxidation at a macroscopic scale, however, when the size of gold particles reaches a few nanometers, a chemically active surface appears, which often induces oxidative damages (Chiang et al., 2006; Fierro-Gonzalez and Gates, 2007). When human lung fibroblasts MRC5 were incubated with gold nanoparticles, the internalized nanoparticles accumulated in endosomes and produced oxidative stress damage such as lipid peroxidation (Li et al., 2010). After lung fibroblasts were treated with gold nanoparticles (0.5and 1 nm) for 72 h, the oxidative DNA damage was produced with the increase in the level of 8hydroxydeoxyguanosine, an established marker of cellular oxidative stress (Li et al., 2008). Oxidative stress induced by gold nanoparticles was also observed in aquatic organisms such as Mytilusedulis (Tedesco et al., 2008). Based on the above discussion, in vivo accumulations of gold nanoparticles in vital organs probably induce oxidative stressrelated organ toxicity. In order to understand the mechanism of toxicity and to minimize or eliminate the toxicity of the gold nanoparticles, it is

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of great significance to quantify and completely remove the excess reactants from the synthesis of Au-nanoparticles. Furthermore, it will be instructive for optimizing the synthesis procedures to obtain the safer Au nanoparticles for biomedical applications.

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1.2.3. Quantum dots Quantum dots (QDs), a kind of semiconductor nanometer materials, are usually composed by II–Vl or III–V family elements with diameters ranging from 1 nm to 10 nm. The common QDs include CdSe, CdS or CdTe. However, a core-shell structure is usually formed by coating QDs with ZnS to improve the stability of QDs in aqueous solution or biological environments. Due to the high biocompatibility, long halflife of fluorescent and good photochemical stability, QDs are widely used in biomedical imaging (Gao and Dave, 2007; Samir et al., 2012). However, the potential health risks of QDs are also highly concerned and cautioned (Rzigalinski and Strobl, 2009).

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1.2.4. In vivo and in vitro toxicity When it comes to toxic effects of QDs on animals, pharmacokinetics of QDs in the body is one of the most important issues that need to be addressed firstly. QDs in biomedical applications are mostly watersoluble. In an early study on the bio-kinetics, we investigated the water-soluble QDs coated by hydroxyl group modified silica networks (21.3 ± 2.0 nm in diameter and have maximal emission at 570 nm). Male ICR mice were intravenously administrated with the watersoluble QDs at a single dose of 5 nmol/mouse. We quantitatively measured the concentrations of QDs in plasma, organs, and excretion samples collected at the indicated time intervals, the distribution and aggregation state of QDs in tissues. The results revealed that QDs had a plasma half-life of 19.8 ± 3.2 h with a clearance speed of

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of reactive oxygen species (ROS). These suggested both the acute and chronic toxicity by CdSe QDs exposure (Liu et al., 2011). The toxicity was observed in pregnant mice exposed to the CdTe/CdS QDs and the fetus due to the transfer of CdTe/CdS QDs from the mother and fetus. QDs were found to be transferred from the female mice to pups, depending on the QDs size, the dosage used as well as the capped materials on QDs. QDs of smaller sizes or at the higher dosages more readily penetrated through the placental barrier and were transferred into the fetuses. Compared with the bare QDs, QDs capped with silica shell or polyethylene glycol reduced QD transfer but did not avoid it (Chu et al., 2010). During the blood transfer process, QDs may also produce toxicity to hematological systems. When carboxyl-QDs (carboxyl surface coating) and amine-QDs (amine surface coating) were intravenously injected in mice at the dosage of 1.44–3600 pmol/mouse, pulmonary vascular thrombosis was developed in QDs-treated mice. The QDs were potential to activate the coagulation cascade via contact activation and induced vascular thrombosis in which the surface charge was found to play a crucial role (Geys et al., 2008). Furthermore, the toxicity is also inevitable even in the local application of CdSe QDs. For instance, when the mice were administrated with CdSe QDs at the concentration of 20 μM by intrastromal injection, QDs were retained with the cornea up to 26 days and induced damage to the cornea damage, indicating the potential toxicity rising from the eye exposure to QDs (Kuo et al., 2011). The in vitro toxicological study is important for understanding the in vivo data as well the nanotoxicity mechanisms. When human umbilical vein endothelial cells (HUVEC) were exposed to CdTe QDs for 24 h, a dose-dependent decrease in cell viability was observed (Yan et al., 2011). The CdTe QDs' (2 nm) exposure for 24 h also caused a dosedependent decrease in cell viability in MCF-7 cells, shrinkage and deformation of nuclei with chromatin aggregation (Lovrić et al., 2005b). In vitro cytotoxicity test of core/shell CdSe/ZnS QDs in bovine corneal stromal cells, cell viability of the QDs-incubated cells was significantly declined at 24 and 48 h. QDs at a concentration as low as 5–20 nM induced a decrease in cell viability by 50% during 48 h (Kuo et al., 2011). In addition to cell damage, the potential genotoxicity was also observed. For instance, when MCF-7 cells (human breast cancer cells) were incubated with 10 μg/mL CdTe QDs for 24 h, the global hypoacetylation of histone 3 accompanied with the alterations of DNA structure were detected, implying an epigenomic response. Meanwhile, p53, the ubiquitous responder to genotoxic stress, was activated by QDs exposure, indicating the potential genotoxicity of the CdTe QDs (Choi et al., 2008). The toxicity of QDs is associated with their own structure and properties, such as the particle size, surface modification, etc. After exposed to N9 murine microglial cell line, the smaller CdTe QDs (2.3 nm) were localized predominantly in the nuclear compartment whereas the

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57.3 ± 9.2 mL/h/kg. The liver and kidney were the main target organs for QDs. It was for the first time determined that QDs in living mice 772 could metabolize through three paths depending on their interactions 773 with native proteins and their aggregation states in vivo. After five 774 days, 8.6% of the injected dose of aggregated QDs still remained in he775 patic tissue and it was difficult for this fraction to clear from the body 776 (Chen et al., 2008) (Fig. 5). This part of deposited QDs in hepatic tissue 777 should become the major entity inducing the toxic effects in vivo. 778 Recently, we studied the in vivo fate and physiological behavior of 779 QDs in Caenorhabditis elegans with various advanced techniques includ780 ing GFP transfection, fluorescent imaging, synchrotron radiation and 781 classic toxicological approaches. The results from elemental imaging 782 and speciation studies for the first time disclosed the in situ metabolism 783 and degradation of QDs in the alimentary system of living model organ784 ism and the long-term toxicity on reproduction are fully assessed (Qu 785 et al., 2011) (Fig. 6). Moreover, the potential toxicity of QDs has also 786 been reported by other research groups. Captopril-conjugated QDs, at 787 6 h after intraperitoneal injection, were distributed into many organs 788 in male ICR mice via systemic blood circulation, including the liver, 789 spleen, kidney and brain. In these organs, QDs were located predomi790 nantly inside the blood vessels where toxicity of QDs may arise (Kato 791 et al., 2010). Studying the pharmacokinetics of ZnS-capped CdSe QDs 792 in BALB/c and nude mice, till 2-year post-injection, the fluorescence of 793 ZnS-capped CdSe QDs was still observed in the liver, spleen and 794 Q14 lymph nodes despite the fact that they had been degraded into the 795 smaller ones as reflected by fluorescence emission shifts from red to 796 blue. This result demonstrated that although the in vivo breakdown of 797 the QDs occurred, the absolute elimination of QDs from the body may 798 be much more difficult than anticipated (Fitzpatrick et al., 2009). There799 fore, the accumulation and retention of QDs in the tissue, whether in the 800 short or the long-term periods, are likely to cause the acute or chronic 801 toxicity. 802 In fact, substantial in vivo studies have documented that QDs can 803 pose toxicity including nephrotoxicity, hepatic damage, reproductive 804 toxicity and hematological abnormality. For example, when mice were 805 intravenously administrated with CdTe QDs, a mild renal damage was 806 detected with an obvious increase in the level of blood urea nitrogen 807 (BUN) and creatinine (CREA) in mice, indicating the risks of quantum 808 dots in inducing nephrotoxicity (Sadaf et al., 2012). In the toxicological 809 investigation of CdSe QDs in mice, the mice were administrated intra810 peritoneally with CdSe QDs at the dosage of 20 nM (30.6 ng/kg body 811 weight). After 48 h, QDs were observed to exhibit a wide distribution 812 profile in many organs with the highest accumulation in the liver. The 813 long-term retention of nanoparticles in the liver finally induced a signif814 icant oxidative stress-related hepatic damage accompanied with the 815 marked morphological alterations to hepatic lobules and the production

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Fig. 5. Plasma kinetics and bio-distribution profile of silica-coated CdSeSQDs in mice. (A) Time course for the plasma concentrations of QDs in mice after intravenous administration within 120 h. Mice were administrated with the QDs solution via tail vein injection at a dose of 5 nmol/mouse (100 μl in total). The plasma samples were collected from mice at the indicated time intervals (1, 6, 12, 24, 48, 72 and 120 h) after QDs exposure; (B) Bio-distribution profile of QDs at the corresponding time points; (C) Accumulations of QDs in the liver (■) and kidney (●) from QDs-exposed mice. The mean values of the control from control mice served as the baseline. All data were represented as mean ± SD, n = 6. (Reprinted with permission from ref (Chen et al., 2008); Copyright 2008, Elsevier).


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Fig. 6. In situ elemental analysis and degradation of QDs in Caenorhabditis elegans. (A) Schemed metabolic fates of MEA-CdSe@ZnS QDs(CdSe@ZnScoreshell QDs stabilized by 2mercaptoethylamine (MEA)); (B) The representative fluorescence versus elemental distributions of an intact Caenorhabditis elegans exposed to MEA-CdSe@ZnS for 12 h. Fluorescent microscope images were subsequently merged with bright field (BF). Microbeam X-ray fluorescence (μ-XRF) mappings of Se and Zn elements in QDs were shown with quantity of each individual element plotted on the respective scale below. (C) and (D) showed that the enlarged head and tail regions indicated in the boxed regions in (B). Arrows pointed to the beam positions of μ-XANES (microbeam X-ray absorbance near edge structure) spectra shown in figure (E). (E) In situ Se K-edgeμ-XANES spectra of QDs within the pharynx or intestine of QDs-exposed Caenorhabditis elegans in (C) and (D), respectively. Exact positions of each spectrum were displayed by a, b. Beam sizes of μ-XRF mappings and μ-XANES spectra are 5 × 5 μm. (Reprinted with permission from ref (Qu et al., 2011); Copyright 2011, American Chemical Society).

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larger ones (5.7 nm) were distributed throughout the cytoplasm of N9 cells without obvious entry to the nucleus within 1 h. Compared with 864 the larger ones, QDs of a smaller size induced much severer cell dam865 ages with chromosome condensation and membrane blebbing (Lovrić 866 et al., 2005a). Similarly, the size-dependent distribution and toxicity of 867 QDs were found in human macrophages where the smaller QDs 868 targeted nuclear histones and nucleoli after active transport across the 869 nuclear membrane whereas the larger ones did not (Nabiev et al., 870 2007). Again, these toxicological properties of QDs can also be altered 871 by surface modifications. With similar particle sizes, QDs coated with 872 mercaptoundecanoic acid caused DNA damages, but the QD-COOH/ 873 OH, QD-NH2, QD-OH did not show any significant DNA damages, 874 which implied that part of the cytotoxicity of QDs came from their sur875 face coatings rather than the simple positive or negative charges 876 (Hoshino et al., 2004). Noteworthy, the chirality of the surface coating 877 molecules also played a dominate role in nanotoxicity assessment as 878 we previously reported. In our study, we investigated the impacts of 879 CdTe QDs coated with either L-GSH or D-GSH on cell autophagy, which 880 was correlated with cell death. Consequently, the induced cell autopha881 gy was found to be chirality-dependent, with the L-GSH-QDs being 882 more potent than the D-GSH-QDs. Herein, our study highlighted the im883 portance of conformation stability of the surface coatings (Li et al., 884 Q15 2011). Taken together, by manipulating their own structure and proper885 ties, we can minimize the potential toxicity of quantum dots and im886 prove their biocompatibility for better biomedical imaging applications. 887


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1.2.5.1. Cadmium released from QDs. QDs contain heavy metal elements such as Cd, Hg, Pb, As and Cd. Especially, cadmium (Cd) is known to be highly toxic (Das et al., 1997). Cadmium breaks up DNA strands and inhibits the synthesis of biomolecules. Moreover, it can induce the dysfunctions of subcellular organelles by conjugating sulphydryl of protein with Cd2+ (Nath et al., 1984). It has been reported that cadmium, in some cases, can be released from the surface of QDs that consequently causes toxicity. Measurement of the cadmium concentration in 0.25 mg/mL QDs solution under UV exposure at various time points, the results showed that free Cd levels in the solution increased as the UV exposure time extended. It well correlated with cellular toxicity of

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the CdSe QDs as observed in hepatocytes. So, the free cadmium released from QDs may account for QDs toxicity, at least partially (Derfus et al., 2004) (Fig. 7). In order to inhibit Cd2+ release, QDs are often coated by other lowtoxic chemicals such as ZnS, polyacrylate or dihydrolipoic acid to form core-shell structures. For example, when the bare QDs are exposed to air, they induced a dose-dependent decrease in cell viability. Once conjugated with mercaptoacetic acid on the QDs surface in inert atmosphere, the coated QDs became non-toxic. This might be attributed to the possibility that surface coating prohibited surface degradation and the sequent release of Cd2+ ions (Derfus et al., 2004). The bare CdScapped CdSe QDs were more toxic than PEG-substituted QDs to the human breast cancer cell line SK-BR-3. Compared with PEG-coated QDs, the bare CdS-capped CdSe QDs induced a higher intracellular Cd2 + concentration, which accounted for the more severe toxicity (Chang et al., 2006).

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1.2.5.2. Production of reactive oxygen species. Another major cause of QDs toxicity lies in the generation of reactive oxygen species (ROS), which could in turn damage cellular proteins, lipids and DNA, etc. For instance, the CdSe QDs could trigger apoptosis of human osteoblast cells, which could be prevented by the inhibition of ROS production (Lu et al., 2006). In MCF-7 cells incubated with the CdTe QDs, a direct observation of ROS production at 4 h exposure in culture medium was reported. At 24 h, the mitochondria turned round and aggregated at the perinuclear region in CdTe QDs-incubated cells whereas the control cells showed ordinary cell viability. However, the addition of N-acetylcysteine (NAC), a commonly used antioxidant, efficiently protected the cells against QDinduced cell death (Lovrić et al., 2005b). The ROS may be responsible for the liver damage of QDs in mice. In hepatocytes, a production of intracellular reactive oxygen species was detected with a significant increase in the malondialdehyde (MDA) level. The pre-treatment of cells with beta-mercaptoethanol (beta-ME), antioxidant agents, apparently alleviated the cytotoxicity induced by QDs (Liu et al., 2011). Together, the uncompleted elimination of QDs from the body may induce oxidative damage-dependent systemic toxicity including nephrotoxicity, hepatic damage, reproductive toxicity and hematological abnormality et al. As shown in Fig. 8, the poisonous Cd2 + released from QDs can induce cellular toxicity. Therefore, the suitable surface

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coatings will be advantageous to prevent the release of the heavy metal ions and thus decline toxicity.

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Fig. 7. The cytotoxicity of free Cd ion released from CdSe QDs by surface oxidation in hepatocytes. (A) The proposed mechanism of CdSe QDs cytotoxicity in hepatocytes. The oxidation of CdSe QDs surfaces facilitated the release of free Cd ion accompanied with an alteration in absorbance spectra as observed from the color changes of QD solutions from red/orange to yellow. Cd released from nanoparticle surfaces was toxic to hepatocytes. (B) Cd concentration in 0.25 mg/mL solutions of QDs determined by inductively coupled plasma optical emission spectroscopy (ICP/OES) under various conditions. With no oxidation, Cd concentration was extremely low. When the QDs solution was exposed to air or UV, Cd levels increased as the exposure time extended. (C) The corresponding cell viability of the hepatocytes under the experimental conditions as shown in (B), in good correlation with the free Cd levels in the solutions. (Reprinted with permission from ref (Derfus et al., 2004); Copyright 2004, American Chemical Society).

1.3. Upconversion nanoparticles (UCNPs)

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Presently, the newly-emerging near-infrared (NIR)-to-visible upconversion nanoparticles have attracted significant interests. They are able to absorb low energy photons (NIR light) and then emit high energy photons (UV/Vis light) for bio-imaging. The upconversion nanoparticles (UCNPs) hold an intriguing advantage and are expected to create the potential for settling the major drawbacks in bioimaging with other nanoparticles. For instance, in QDs-based fluorescence imaging,

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Fig. 8. An illustrative scheme to demonstrate the toxicological mechanisms of QDs. External stimuli such as UV exposure may facilitate the release of the poisonous Cd2+ from QDs which consequently causes oxidative stress-related damages to cells. In order to alleviate toxicity, the proper surface coatings such as ZnS shell are needed to prohibit the release of the heavy metal ions.

it requires direct illumination of the biosamples by using high energy lights as excitation sources such as visible or even UV light, where application limitations arise due to the inevitable photo-damages to biomolecules. Furthermore, the low signal-to-background ratio results from significant auto-fluorescence from bio-tissues, the limited penetration depth from light absorption and light scattering by biological tissues, all these become drawbacks in their applications (Chen and Zhao, 2012; Jayakumar et al., 2012). The near-infrared (NIR) region is known as the transparent window of the biological tissues, where the light absorption and scattering is minimal. NIR irradiation, compared with the UV–visible light, exhibits overwhelming advantages with larger penetration distance in tissue, lower photo-damage effect and higher signal-to-noise ratio. Currently, a representative of UCNPs is the NaYF4-based UCNPs which are most investigated since Y has an innocuous toxicity profile. Moreover, fluoride usually has low photon energy which favors the generation of efficient upconversion luminescence from UCNPs. Therefore, NaYF4-based nanoparticles are regarded as the ideal UCNPs and are widely used in biomedical field (Chen et al., 2011; Jin et al., 2011). In addition to the biomedical imagings, due to their unique upconversion luminescent property, good chemical/physical stability and superior photo-stability, UCNPs are also becoming a new class of powerful tools in other biomedical applications, ranging from cellular labeling, FRET-based biosensors to drug delivery and photodynamic therapy (C. Wang et al., 2011; Xia et al., 2011). Although there are great enthusiasms about biomedical applications of UCNPs, a good understanding of their toxicology profiles in biological systems is urgently needed to weigh the benefits versus the risks before the exploitation in bioscience and medicine in the future. However, as a newly emerging nanomaterial, the potential health risks of UCNPs have been far from adequately investigated and understood. To date, the toxicological data available for UCNPs are few. Up to now, several types of model organisms including mice and C. elegans have been used to evaluate the potential toxicity of UCNPs.


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Positron emission tomography (PET) is a noninvasive medical imaging technique that uses radiolabeled molecules to demonstrate molecular interactions of biological processes. Positron-emitting radionuclides with short half-lives serve as tracers and are incorporated into compounds, usually the biologically active molecules or analogs or targeting molecules, which is the basis of PET imaging science (Phelps, 2000). Nowadays, PET imaging technologies have been widely used in clinic for molecular therapies and molecular diagnostics (Facey et al., 2007). Nanotechnology may further optimize PET imaging technologies. In the past few years, nanotechnology has been introduced into PET imaging and PET imaging nanoprobes emerge. Nanoparticles exhibit overwhelming advantages to combine PET imaging. Due to the surface and interior of the nanoparticles, many functionalities can be realized better. First, radionuclides can be coupled onto the surface of nanoparticles for PET function. Then, due to the easily-manipulated surface modifications of nanoparticles, active targeting can be conducted through conjugations of different tumor-specific cell surface markers to the huge surface of nanoparticles. In addition, nanoprobes can be further optimized for controlled distribution to obtain a satisfactory pharmaceutical profile (Welch et al., 2009). In fact, many PET imaging nanoprobes have demonstrated promising potentials in the further clinical practice. For example, Almutairi et al. used PET for blood vessel imaging in the hide limb ischemia mouse model. For PET, 76Br was combined to the surface of the dendritic positron-emitting nanoprobes. For controllable biodistribution, PEG was conjugated. For targeting, RGD was attached for specific binding with integrins. This nanoprobe was reported to have satisfactory

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was found even when the concentration of UCNPs reached up to 200 μg/mL. The UCNPs could be transferred to water-soluble and biocompatible counterparts via different protocols, which would expand the applications of UCNPs in bioimagings and other biomedical applications (Ren et al., 2012). When rat skeletal myoblasts and bone marrowderived stem cells were treated with PEI/NaYF4:Yb, Er at a range of concentrations from 1, 5, 10, 15, 25, 50 to 100 μg/mL for 24 h, cell viability of both the cell lines declined mildly in a dose-dependent manner. At the highest concentration of 100 μg/mL, cell viability decreased significantly to 65.3%, 71.9%, respectively, in bone marrow-derived stem cells and rat skeletal myoblasts (Rufaihah et al., 2008). As demonstrated above, UCNPs have satisfactory biocompatibility with low toxicity. However, the sparse and fragmentary data are not sufficient to draw the conclusion that the upconversion nanoparticles are safe. More information needs to be obtained, including pharmacokinetics, the acute or chronic toxicity of UCNPs, with factors such as particle size, shape and surface chemistry be taken into considerations.

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Studying the UCNPs toxicity effects to C. elegans from the growth to procreation by assessing life span, egg production, egg viability and the growth rate of UCNPs-treated worms, it was found that UCNPs did not generate obvious toxicity effect in vivo (Chen et al., 2011). Similarly, when N2 hermaphrodites were cultured in B-growth media (50 mL) with the presence or absence of NaYF4:Yb,Tm (50 mL, 2 mg/mL) for 2 h, life span, egg production, egg viability and growth rate showed no statistical significance between the treated group and the control group. These results indicate the subtle toxicity of NaYF4:Yb,Tm to C. elegans (J.C. Zhou et al., 2011) (Fig. 9). The potential health effects of UCNPs in the mice were also tested. For instance, the polyacrylic acid (PAA)-coated UCNPs were intravenously injected into healthy rats for the investigation of their biocompatibility and tissue distribution. Compared with the control groups, the body weight, eating, fur color, activity and neurological status of treated mice showed no significant changes. Biodistribution results indicated that PAA-coated UCNPs primarily accumulated in the liver and the spleen, and the structures of all the exposed organs were normal and hardly different from those of the control groups. In addition, histological, hematological and biochemical analysis results indicated that there was no toxicity to the mice treated with PAA-coated UCNPs over long exposure times (more than 115 days) (Xiong et al., 2010). In a distribution profile study, healthy rats were intravenously administrated with PEI/NaYF4:Yb, Er, a novel polyethylenimine-coated upconversion fluorescent nanocrystal. At 30-minute post-injection, PEI/NaYF4:Yb, Er was found in various organs with the highest accumulation in the lung followed by the heart, spleen, kidney, liver and blood. However, the level of UCNPs in these organs declined significantly at 24 h postinjection. No apparent nanocrystals were detectable at day 7 postinjection, indicating the satisfactory excretion of UCNP nanocrystals from the rats (Rufaihah et al., 2008). In a systematic toxicity study, mice were injected with UCNPs, no animal deaths occurred during the thirty-day observational intervals. Meanwhile, no loss in their body weights was detectable in UCNPstreated mice, as compared with that of the control group. In addition, no obvious Gd3 +/Mn2 + ions release from UCNPs was detectable as shown by the inductively coupled plasma mass spectrometry (ICPMS) analysis. Therefore, it was the rigid host structure that confined Gd3 +/Mn2 + ions and prohibited their release, making UCNPs lowtoxic to mice even if Gd/Mn elements were contained in the nanoparticles (Yin et al., 2012). To date, various cell lines such as A549, HeLa, Panc 1, RAW have been used to evaluate the in vitro cytotoxicity of UCNPs. Most results have indicated that NaYF4-based UCNPs are not toxic for the tested cell lines within a certain range of concentrations and a limited incubation period. When A549 cells were incubated with silica, PEG phospholipid or TWEEN-coated UCNPs, respectively, no obvious decrease in cell viability

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Fig. 9. The impact of NaYF4:Yb,Tm nanocrystals on the behaviors of N2 wild-type hermaphrodites. Life span (A), egg production (B), egg viability and growth rate assay(C) were determined in N2 wild-type hermaphrodites that were incubated with B-growth media in the presence or absence of NaYF4:Yb, Tm nanocrystals. The numbers of total larvas and the proportion of worms that entered into L4 and adult stages were counted at 46 h checkpoint to obtain the egg viability and growth rate. The egg viability was expressed by the ratio of the number of total larvas to that of 30 eggs. The growth rate was calculated by the proportion of L4 and adult stage worms among the total larvas. The inserted in (B) shows the total number of laying eggs from Day 3 to 7. (Reprinted with permission from ref (J.C. Zhou et al., 2011); Copyright 2010, Elsevier B. V.).


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1.4.1. Conclusions and outlook Inorganic nanoparticles have shown great promises as biomedical imaging agents in the future. The bioimaging technologies have been making great progress towards high sensitivity, high spatial and temporal resolution ranging from tissue imaging to cellular imaging and molecular imaging. These are mostly attributed to the unique physicochemical properties of inorganic nanoparticles which contribute to an unprecedented development of biomedical imaging agents in the past decades. However, before considering their medical applications in clinic, it is of great importance to evaluate the biocompatibility of these nanoparticles both in vitro and in vivo. Further, toxicity aspects are a permanent topic that should be highlighted when it comes to translating the laboratory innovations into clinical applications.

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We hence briefly summarize their toxicity with an illustration in Fig. 9. Nanoparticles are multi-component systems, usually containing the core material, surface-bound stabilizing ligands, the released metal ions and the potential excess reactants from the synthesis. The inorganic nanoparticles themselves may trigger toxicity because of their chemical stability in biological microenvironment and the sequent release of free toxic irons of heavy metals. Moreover, the excess reactants and contaminants (surfactant, inorganic salts et al.) from the synthesis process of inorganic nanoparticles, the degradation of surface coatings are also the common origins of direct toxicity by inorganic nanoparticles (Fig. 10A). From the in vitro data, we realize that the inorganic nanoparticles often induce – ROS production – in immediate cell environment or inside the cells following the endocytosis of nanoparticles. Consequently, the inorganic nanoparticles usually induce oxidative stress-related cytotoxicity such as DNA damage, cell death/apoptosis and cell cycle arrest. Hence, it seems that ROS becomes as a major and common mechanism for their nanotoxicity (Fig. 10B). From the in vivo data, we understand that pharmacokinetics of nanoparticles exert decisive influences on nanotoxicity, including organ toxicity, hematological toxicity, genotoxicity, metabolic toxicity and immune system toxicity (Fig. 10C). When we have understood these origins of nanotoxicity, we may minimize or even eliminate the potential toxicity of inorganic nanoparticles by the purification of the nanoparticles and by surface chemical design. Second, the most common index for in vitro toxicity tests is cytotoxicity, which is usually reflected by cell viability, apoptosis or cell death, the membrane integrity and chromosome condensations. Other conventional toxicology indexes, such as genotoxicity, nephrotoxicity, neurotoxicity and metabolic abnormality can be also used, however, it depends on the types of cell lines selected and tested. To select the cell lines for the above investigations, transportation, deposition, clearance and organ toxicity of nanoparticles need to be firstly determined with animal models. Investigations on blood circulation patterns and the tissue-specific extravasation of nanoparticles, after they enter into the body, will shed light on the choice of the cells types to further explore the cytotoxicity towards specific tissue cells (C. Wang et al., 2011). Third, considering that cell models are oversimplified biological systems, the data obtained from cell models can merely serve as supplementary information and they are mostly not reliable enough for the toxicity assessment of the real systems. Nevertheless, the employment

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performances for blood vessel imaging after 24 h injection into the ischemia mouse. This PET imaging nanoprobe was considered as instructive to localize the ischemic tissues, tumor vasculature usually bearing upregulated-expression of integrins (Almutairi et al., 2009). In addition, 18F was coupled to the phospholipid QDs micelles for nanotrackers. After injected into mice, the biodistribution and pharmacokinetics can be distinctly and dynamically monitored using PET imaging. Moreover, owing to QDs properties, the process of cellular uptake can be observed using fluorescence imaging (Duconge et al., 2008). Single-walled carbon nanotubes (SWNTs) were also used as nanocarriers for PET imaging. With DOTA coated on the SWNTs surface for 64 Cu chelation, RGD attached to target integrins, PEG conjugated for controlled distribution, the modified nanoparobes were demonstrated to actively target the integrin-positive tumors in mice. Moreover, the differences in the length of conjugated PEG resulted in a difference in the biodistribution of this PET imaging nanoprobe in vivo with the shorter length of PEG having shorter blood half-life and higher liver and spleen uptake (Singh et al., 2006). Yang et al. used gold and iron oxide heter-nanostructure for the multimodality imaging of tumors. They modified heter-nanostructure with anti-EGFR antibody to realize the active targeting tumors with aberrant EGFR upregulation by simultaneous PET, optical and MR imagings (Yang et al., 2013). As demonstrated above, PET imaging nanoprobes have exhibited great potentials in biomedical application. However, there is little information reporting the potential toxicity of PET imaging nanoprobes, so their potential health risks remain unobserved and should be cautioned.

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Fig. 10. A simplified illustration of inorganic nanoparticles-triggered toxicity both in vitro and in vivo. (A) The components contained in a typical inorganic nanoparticle solution. Nanotoxicity may arise from the released free metal irons, the left-over chemicals and contaminants (surfactant, inorganic salts et al.), the instability and the degradation of surface coatings. The interactions between nanoparticles and the biomolecules (proteins et al.) will pose impacts on the behaviors of nanoparticles. (B) ROS generated by nanoparticles in immediate cell environment or inside the cells induced oxidative stress-related cytotoxicity such as DNA damage, cell death/apoptosis and cell cycle arrest. (C) In vivo study, pharmacokinetics of nanoparticles exerts influences on nanotoxicity including organ toxicity, hematological toxicity, genotoxicity, metabolic toxicity and immune system toxicity. To monitor the potential long-term toxicity of these nano-agents in the clinical practice of biomedical imaging, we need to understand carefully the following aspects in the future. First, the conventional toxicological protocols have to be tested to verify if they are adequate for nanoparticle toxicity assay. We also need to preliminarily screen the potential toxicity of nanoparticles with varied conditions including cell types, the dosage used, the exposure time and the analytic methods used.


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Auffan et al., 2008 Ge et al., 2008 Lu et al., 2007 Wang et al., 2012 Acknowledgments

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This work was financially supported by NSFC (11305182, 21301176, 1237 21277037) and MOST (2012CB932601, 2010CB933904). 1238

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References

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Abdelhalim MA. Exposure to gold nanoparticles produces cardiac tissue damage that depends on the size and duration of exposure. Lipids Health Dis 2011;10:205–14. Abdelhalim MA, Jarrar BM. Renal tissue alterations were size-dependent with smaller ones induced more effects and related with time exposure of gold nanoparticles. Lipids Health Dis 2011;10:163–9. Aggarwal P, Hall JB, McLeland CB, Dobrovolskaia MA, McNeil SE. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv Rev 2009;61:428–37. Alkilany AM, Lohse SE, Murphy CJ. The gold standard: gold nanoparticle libraries to understand the nano-bio interface. Acc Chem Res 2013;46:650–61. Alkilany AM, Murphy CJ. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res 2010;12:2313–33. Alkilany AM, Nagaria PK, Hexel CR, Shaw TJ, Murphy CJ, Wyatt MD. Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects. Small 2009;5:701–8. Almeida JP, Chen AL, Foster A, Drezek R. In vivo biodistribution of nanoparticles. Nanomedicine 2011;6:815–35. Almutairi A, Rossin R, Shokeen M, Hagooly A, Ananth A, Capoccia B, et al. Biodegradable dendritic positron-emitting nanoprobes for the noninvasive imaging of angiogenesis. Proc Natl Acad Sci U S A 2009;106:685–90. Anzai Y, Piccoli CW, Outwater EK, Stanford W, Bluemke DA, Nurenberg P, et al. Evaluation of neck and body metastases to nodes with ferumoxtran 10-enhanced MR imaging: phase III safety and efficacy study. Radiology 2003;228:777–88. Apopa P, Qian Y, Shao R, Guo N, Schwegler-Berry D, Pacurari M, et al. Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling. Part Fibre Toxicol 2009;6:1. http://dx.doi.org/10.1186/1743-8977-6-1. Arbab AS, Wilson LB, Ashari P, Jordan EK, Lewis BK, Frank JA. A model of lysosomal metabolism of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles: implications for cellular magnetic resonance imaging. NMR Biomed 2005;18:383–9. Auffan M, Decome L, Rose J, Orsiere T, De Meo M, Briois V, et al. In vitro interactions between DMSA-coated maghemite nanoparticles and human fibroblasts: a physicochemical and cyto-genotoxical study. Environ Sci Technol 2006;40:4367–73. Auffan M, Achouak W, Rose J, Roncato M-A, Chanéac C, Waite DT, et al. Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli. Environ Sci Technol 2008;42:6730–5. Balasubramanian SK, Jittiwat J, Manikandan J, Ong CN, Yu LE, Ong WY. Biodistribution of gold nanoparticles and gene expression changes in the liver and spleen after intravenous administration in rats. Biomaterials 2010;31:2034–42. Bharali DJ, Mousa SA. Emerging nanomedicines for early cancer detection and improved treatment: current perspective and future promise. Pharmacol Ther 2010;128: 324–35. Bhattacharya K, Hoffmann E, Schins RF, Boertz J, Prantl EM, Alink GM, et al. Comparison of micro- and nanoscale Fe3+-containing (hematite) particles for their toxicological properties in human lung cells in vitro. Toxicol Sci 2012;126:173–82. Boisselier E, Astruc D. Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity. Chem Soc Rev 2009;38:1759–82. Bourrinet P, Bengele HH, Bonnemain B, Dencausse A, Idee JM, Jacobs PM, et al. Preclinical safety and pharmacokinetic profile of ferumoxtran-10, an ultrasmall superparamagnetic iron oxide magnetic resonance contrast agent. Invest Radiol 2006;41:313–24.

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on their respective adsorption capacity (Ge et al., 2011). Hence, although it seems that these interactions between nanoparticles and biological systems damp the enthusiasms towards controlling the nanotoxicity based on the manipulation of the physicochemical properties of nanoparticles, the attempt to minimize or eliminate the toxicity is still intriguing because if we understand well the nanoparticle–biomolecule interactions and modulate nanoparticles by nanosurface design and functionalization. The diversity of functionalization methods offers great opportunities for optimization, thus playing a crucial role in driving nanomaterials towards medical applications (Yan et al., 2011). Therefore, research on the nanosurface design and functionalization is of great significance in medical translation of nanotechnology for practical purposes.

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of cell models exhibits great advantages in exploring the toxicological mechanisms, especially at the molecular level, compared with the realistic biological systems where the involved multifactors are difficult to handle and are always confusing so that the key factors leading to the major nanotoxicity fail to be distilled. If the in vitro toxicity of a nanoparticle was minor or even unobserved, this does not mean that the safety of this nanoparticle is verified. We still need the tests with animal models to obtain in vivo toxicological data which are more reliable for nanotoxicity assessments. Fourth, lots of parameters have to be carefully addressed to obtain comparable data for a nanoparticle, ranging from the dosage used, the exposure time, the administration routes, the animal types, the indexes investigated to the unique characteristics of administrated nanoparticles, etc. For example, with respect to the animal species, mice or rats are the most frequently used animal models because they are easily fed and handled, inexpensive to maintain and easily obtained. However, experimental data from mice and rats are sometimes different, so other animal models are sometimes needed to confirm the data obtained from mice or rats. Fifth, the indexes most frequently used in nanotoxicological study with animal models include behavior observation, body weight measurement, histological and hematology analysis, serum biochemistry assays, and especially the pharmacokinetics. Termed as the process of absorption, distribution, metabolism and excretion (ADME), pharmacokinetics of the nanoparticles has to be fully explored where the undesired toxicities and side effects are likely to arise (Choi and Frangioni, 2010). Certain toxicity, which may not be observed in the in vitro tests, probably happens due to the inorganic nanoparticles' deposition in the body. As a whole, the data obtained from animal models in the classic toxicology show a good predictive toxicological profile in the humans. The in vivo toxicological results from the animal models are of great significance in evaluating the potential human benefits or risks when we are driving the nanoparticles into clinic applications. So, it is needed to understand whether the similar data obtained from nanoparticles toxicity studies could be still valid when we intend to extrapolate them to human toxicity assessment. If they are valid enough, we can use these data to further optimize the inorganic nanoparticles for improving their biocompatibility and lowering their toxicity. Nevertheless, even if the data from animal models are satisfactory, it should be emphasized that limitations will arise in extrapolating from animal models to humans. Compared to the animals, human environment is much more complicated, more variable and more personalized where more physiological parameters have to be considered and assessed dynamically and individually. Sixth, most nanoparticles possess well-defined physicochemical properties, such as nanosize, shape, aspect ratio, and nanosurface (surface charge, surface chemistry, surface area, etc.), all of which have been evidenced to be highly related with the toxicity of nanoparticles. However, to delineate the relationships between these nanocharacteristics and their toxicities is much more difficult than anticipated, because of unavoidable interactions between nanoparticles and biomolecules in vivo, leading to dynamical changes on those nanocharacteristics during ADME processes of nanoparticles in vivo. Once the nanoparticles enter into the biological system, the nanoparticle– biomolecule interactions frequently occur in the biological microenvironment. Due to nanosurface hyperactivity, nanoparticles tend to absorb some biomolecules like proteins on the particle surface to lower the surface energy and thus form a nanoparticle “corona” of biomolecules (Ge et al., 2011; Monopoli et al., 2012). So, what the cells directly see is the corona of the biomolecules rather than the nanoparticle itself. Such a self-coating of the nanoparticle surface by biomolecules in vivo not only alters the properties of the nanoparticles, but also impacts their biological behaviors including biodistribution and toxicity (Aggarwal et al., 2009; Ehrenberg et al., 2009; Ge et al., 2012). Taking SWCTNs as an example, serum proteins could be adsorbed on their surfaces and reduced the toxicity of SWCTNs themselves, which depended

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Hohnholt MC, Geppert M, Luther EM, Petters C, Bulcke F, Dringen R. Handling of iron oxide and silver nanoparticles by astrocytes. Neurochem Res 2012;38:227–39. Hong SC, Lee JH, Lee J, Kim HY, Park JY, Cho J, et al. Subtle cytotoxicity and genotoxicity differences in superparamagnetic iron oxide nanoparticles coated with various functional groups. Int J Nanomedicine 2011;6:3219–31. Hoshino A, Fujioka K, Oku T, Suga M, Sasaki YF, Ohta T, et al. Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification. Nano Lett 2004;4:2163–9. Isomaa B, Ekman K. Embryotoxic and teratogenic effects of CTAB, a cationic surfactant, in the mouse. Food Cosmet Toxicol 1975;13:331–4. Jayakumar MK, Idris NM, Zhang Y. Remote activation of biomolecules in deep tissues using near-infrared-to-UV upconversion nanotransducers. Proc Natl Acad Sci U S A 2012;109:8483–8. Jin J, Gu YJ, Man CW, Cheng J, Xu Z, Zhang Y, et al. Polymer-coated NaYF4:Yb3+, Er3+upconversion nanoparticles for charge-dependent cellular imaging. ACS Nano 2011;5:7838–47. Joo SH, Feitz AJ, Sedlak DL, Waite TD. Quantification of the oxidizing capacity of nanoparticulate zero-valent iron. Environ Sci Technol 2005;39:1263–8. Kang SG, Zhou G, Yang P, Liu Y, Sun B, Huynh T, et al. Molecular mechanism of pancreatic tumor metastasis inhibition by Gd@C82(OH)22 and its implication for de novo design of nanomedicine. Proc Natl Acad Sci U S A 2012;109:15431–6. Kato S, Itoh K, Yaoi T, Tozawa T, Yoshikawa Y, Yasui H, et al. Organ distribution of quantum dots after intraperitoneal administration, with special reference to area-specific distribution in the brain. Nanotechnology 2010;21:335103–10. Kim DK, Mikhaylova M, Zhang Y, Muhammed M. Protective coating of superparamagnetic iron oxide nanoparticles. Chem Mater 2003;15:1617–27. Kuo TR, Lee CF, Lin SJ, Dong CY, Chen CC, Tan HY. Studies of intracorneal distribution and cytotoxicity of quantum dots: risk assessment of eye exposure. Chem Res Toxicol 2011;24:253–61. Lee DE, Koo H, Sun IC, Ryu JH, Kim K, Kwon IC. Multifunctional nanoparticles for multimodal imaging and theragnosis. Chem Soc Rev 2012;41:2656–72. Li JJ, Hartono D, Ong CN, Bay BH, Yung L-YL. Autophagy and oxidative stress associated with gold nanoparticles. Biomaterials 2010;31:5996–6003. Li JJ, Zou L, Hartono D, Ong CN, Bay BH, Lanry Yung LY. Gold nanoparticles induce oxidative damage in lung fibroblasts in vitro. Adv Mater 2008;20:138–42. Li YY, Zhou YL, Wang HY, Perrett S, Zhao YL, Tang ZY, et al. Chirality of glutathione surface coating affects the cytotoxicity of quantum dots. Angew Chem Int Ed Engl 2011;50: 5860–4. Liang XJ, Meng H, Wang YZ, He HY, Meng J, Lu J, et al. Metallofullerene nanoparticles circumvent tumor resistance to cisplatin by reactivating endocytosis. Proc Natl Acad Sci U S A 2010;107:7449–54. Lin H, Datar RH. Medical applications of nanotechnology. Natl Med J India 2006;19:27–32. Liu W, Zhang SP, Wang LX, Qu C, Zhang CW, Hong L, et al. CdSe quantum dot (QD)-induced morphological and functional impairments to liver in mice. PloS One 2011;6:e24406. http://dx.doi.org/10.1371/journal.pone.0024406. Lovrić J, Bazzi HS, Cuie Y, Fortin GR, Winnik FM, Maysinger D. Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots. J Mol Med 2005a;83:377–85. Lovrić J, Cho SJ, Winnik FM, Maysinger D. Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to multiple organelle damage and cell death. Chem Biol 2005b;12:1227–34. Lu AH, Salabas EL, Schüth F. Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem Int Ed Engl 2007;46:1222–44. Lu HY, Shiao NH, Chan WH. CdSe quantum dots induce apoptosis via activation of JNK and PAK2 in a human osteoblast cell line. J Med Biol Eng 2006;26:89–96. Ma XW, Wu YY, Jin SB, Tian Y, Zhang XN, Zhao YL, et al. Gold nanoparticles induce autophagosome accumulation through size-dependent nanoparticle uptake and lysosome impairment. ACS Nano 2011;5:8629–39. Mironava T, Hadjiargyrou M, Simon M, Jurukovski V, Rafailovich MH. Gold nanoparticles cellular toxicity and recovery: effect of size, concentration and exposure time. Nanotoxicology 2010;4:120–37. Monopoli MP, Åberg C, Salvati A, Dawson KA. Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 2012;7:779–86. Nabiev I, Mitchell S, Davies A, Williams Y, Kelleher D, Moore R, et al. Nonfunctionalized nanocrystals can exploit a cell's active transport machinery delivering them to specific nuclear and cytoplasmic compartments. Nano Lett 2007;7:3452–61. Nath R, Prasad R, Palinal V, Chopra R. Molecular basis of cadmium toxicity. Prog Food Nutr Sci 1984;8:109–63. Nel A, Zhao Y, Madler L. Environmental health and safety considerations for nanotechnology. Acc Chem Res 2013;46:605–6. Neuwelt EA, Hamilton BE, Varallyay CG, Rooney WR, Edelman RD, Jacobs PM, et al. Ultrasmall superparamagnetic iron oxides (USPIOs): a future alternative magnetic resonance (MR) contrast agent for patients at risk for nephrogenic systemic fibrosis (NSF)? Kidney Int 2009;75:465–74. Noori A, Parivar K, Modaresi M, Messripour M, Yousefi MH, Amiri GR. Effect of magnetic iron oxide nanoparticles on pregnancy and testicular development of mice. Afr J Biotechnol 2011;10:1221–7. Pan Y, Neuss S, Leifert A, Fischler M, Wen F, Simon U, et al. Size-dependent cytotoxicity of gold nanoparticles. Small 2007;3:1941–9. Phelps ME. PET: the merging of biology and imaging into molecular imaging. J Nucl Med 2000;41:661–81. Pisanic TR, Blackwell JD, Shubayev VI, Finones RR, Jin S. Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 2007;28:2572–81. Pissuwan D, Valenzuela S, Cortie MB. Prospects for gold nanorod particles in diagnostic and therapeutic applications. Biotechnol Genet Eng Rev 2008;25:93–112. Ponka P. Cellular iron metabolism. Kidney Int 1999;55:S2–S11.

N

C

O

R

R

E

C

T

Byrne JD, Betancourt T, Brannon-Peppas L. Active targeting schemes for nanoparticle systems in cancer therapeutics. Adv Drug Deliv Rev 2008;60:1615–26. Cengelli F, Maysinger D, Tschudi-Monnet F, Montet X, Corot C, Petri-Fink A, et al. Interaction of functionalized superparamagnetic iron oxide nanoparticles with brain structures. J Pharmacol Exp Ther 2006;318:108–16. Chang E, Thekkek N, Yu WW, Colvin VL, Drezek R. Evaluation of quantum dot cytotoxicity based on intracellular uptake. Small 2006;2:1412–7. Chen CY, Xing GM, Wang JX, Zhao YL, Li B, Tang J, et al. Multi hydroxylated [Gd@C82(OH)22]n nanoparticles: antineoplastic activity of high efficiency and low toxicity. Nano Lett 2005;5:2050–7. Chen J, Guo CR, Wang M, Huang L, Wang LP, Mi CC, et al. Controllable synthesis of NaYF4: Yb, Er upconversion nanophosphors and their application to in vivo imaging of Caenorhabditis elegans. J Mater Chem 2011;21:2632–49. Chen J, Zhao JX. Upconversion nanomaterials: synthesis, mechanism, and applications in sensing. Sensors 2012;12:2414–35. Chen YC, Hsiao JK, Liu HM, Lai IY, Yao M, Hsu SC, et al. The inhibitory effect of superparamagnetic iron oxide nanoparticle (Ferucarbotran) on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells. Toxicol Appl Pharmacol 2010;245:272–9. Chen Z, Chen H, Meng H, Xing GM, Gao XY, Sun BY, et al. Bio-distribution and metabolic paths of silica coated CdSeS quantum dots. Toxicol Appl Pharmacol 2008;230: 364–71. Chiang CW, Wang A, Mou CY. CO oxidation catalyzed by gold nanoparticles confined in mesoporous aluminosilicate Al-SBA-15: pretreatment methods. Catal Today 2006;117:220–7. Cho EC, Glaus C, Chen J, Welch MJ, Xia Y. Inorganic nanoparticle-based contrast agents for molecular imaging. Trends Mol Med 2010;16:561–73. Choi AO, Brown SE, Szyf M, Maysinger D. Quantum dot-induced epigenetic and genotoxic changes in human breast cancer cells. J Mol Med 2008;86:291–302. Choi HS, Frangioni JV. Nanoparticles for biomedical imaging: fundamentals of clinical translation. Mol Imaging 2010;9:291–310. Chu M, Wu Q, Yang H, Yuan R, Hou S, Yang Y, et al. Transfer of quantum dots from pregnant mice to pups across the placental barrier. Small 2010;6:670–8. Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 2005;1:325–7. Das P, Samantaray S, Rout G. Studies on cadmium toxicity in plants: a review. Environ Pollut 1997;98:29–36. De Jong WH, Hagens WI, Krystek P, Burger MC, Sips AJ, Geertsma RE. Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials 2008;29:1912–9. Derfus AM, Chan WC, Bhatia SN. Probing the cytotoxicity of semiconductor quantum dots. Nano Lett 2004;4:11–8. Duconge F, Pons T, Pestourie C, Herin L, Theze B, Gombert K, et al. Fluorine-18-labeled phospholipid quantum dot micelles for in vivo multimodal imaging from whole body to cellular scales. Bioconjug Chem 2008;19:1921–6. Ehrenberg MS, Friedman AE, Finkelstein JN, Oberdörster G, McGrath JL. The influence of protein adsorption on nanoparticle association with cultured endothelial cells. Biomaterials 2009;30:603–10. Facey K, Bradbury I, Laking G, Payne E. Overview of the clinical effectiveness of positron emission tomography imaging in selected cancers. Health Technol Asses 2007;11. [iii–iv, xi-267]. Fadeel B, Garcia-Bennett AE. Better safe than sorry: understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications. Adv Drug Deliv Rev 2010;62:362–74. Feng JH, Liu HL, Bhakoo KK, Lu LH, Chen Z. A metabonomic analysis of organ specific response to USPIO administration. Biomaterials 2011;32:6558–69. Fierro-Gonzalez JC, Gates BC. Evidence of active species in CO oxidation catalyzed by highly dispersed supported gold. Catal Today 2007;122:201–10. Fitzpatrick JA, Andreko SK, Ernst LA, Waggoner AS, Ballou B, Bruchez MP. Long-term persistence and spectral blue shifting of quantum dots in vivo. Nano Lett 2009;9: 2736–41. Fröhlich E, Kueznik T, Samberger C, Roblegg E, Wrighton C, Pieber TR. Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes. Toxicol Appl Pharmacol 2010;242:326–32. Gao XH, Cui YY, Levenson RM, Chung LW, Nie SM. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol 2004;22:969–76. Gao XH, Dave SR. Quantum dots for cancer molecular imaging. Adv Exp Med Biol 2007;620:57–73. Gao XH, Yang LL, Petros JA, Marshall FF, Simons JW, Nie SM. In vivo molecular and cellular imaging with quantum dots. Curr Opin Biotechnol 2005;16:63–72. Ge CC, Lao F, Li W, Li YF, Chen CY, Mao XY, et al. Quantitative analysis of metal impurities in carbon nanotubes: efficacy of different pretreatment protocols for ICP-MS spectroscopy. Anal Chem 2008;80:9426–34. Ge CC, Li Y, Yin JJ, Liu Y, Wang LM, Zhao YL, et al. The contributions of metal impurities and tube structure to the toxicity of carbon nanotube materials. 4NPG Asia Materials; 2012e32. http://dx.doi.org/10.1038/am.2012.60. Ge CC, Du JF, Zhao LN, Wang LM, Liu Y, Li DH, et al. Binding of blood proteins to carbon nanotubes reduces cytotoxicity. Proc Natl Acad Sci U S A 2011;108:16968–73. Geys J, Nemmar A, Verbeken E, Smolders E, Ratoi M, Hoylaerts MF, et al. Acute toxicity and prothrombotic effects of quantum dots: impact of surface charge. Environ Health Perspect 2008;116:1607–13. Guo ST, Huang YY, Jiang Q, Sun Y, Deng LD, Liang ZC, et al. Enhanced gene delivery and siRNA silencing by gold nanoparticles coated with charge-reversal polyelectrolyte. ACS Nano 2010;4:5505–11. Hanini A, Schmitt A, Kacem K, Chau F, Ammar S, Gavard J. Evaluation of iron oxide nanoparticle biocompatibility. Int J Nanomedicine 2011;6:787–94.

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N C O

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O

F

Wang M, Mi CC, Wang WX, Liu CH, Wu YF, Xu ZR, et al. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4: Yb, Er upconversion nanoparticles. ACS Nano 2009b;3:1580–6. Wang Y, Wang B, Zhu MT, Li M, Wang HJ, Wang M, et al. Microglial activation, recruitment and phagocytosis as linked phenomena in ferric oxide nanoparticle exposure. Toxicol Lett 2011c;205:26–37. Wang YL, Cui YY, Zhao YL, Liu R, Sun ZP, Li W, et al. Bifunctional peptides: precisely biomineralize Au clusters and specifically stain cell nuclei. Chem Commun 2012;48: 871–3. Welch MJ, Hawker CJ, Wooley KL. The Advantages of nanoparticles for PET. J Nucl Med 2009;50:1743–6. Xia A, Gao Y, Zhou J, Li C, Yang T, Wu D, et al. Core-shell NaYF4:Yb3+, Tm3 + @Fe2O3 nanocrystals for dual-modality T2-enhanced magnetic resonance and NIR-to-NIR upconversion luminescent imaging of small-animal lymphatic node. Biomaterials 2011;32:7200–8. Xiong L, Yang TS, Yang Y, Xu CJ, Li FY. Long-term in vivo biodistribution imaging and toxicity of polyacrylic acid-coated upconversion nanophosphors. Biomaterials 2010;31: 7078–85. Xu LG, Liu Y, Chen ZY, Li W, Liu Y, Wang LM, et al. Surface-engineered gold nanorods: promising DNA vaccine adjuvant for HIV-1 treatment. Nano Lett 2012;12: 2003–12. Yan L, Gu ZJ, Zhao YL. Chemical mechanisms of toxicological property of nanomaterials: intracellular ROS generation. Chem Asian J 2013. http://dx.doi.org/10.1002/ asia.201300542. [2013]. Yan L, Zheng YB, Zhao F, Li SJ, Gao XF, Xu BQ, et al. Chemistry and physics of a single atomic layer: strategies and challenges for functionalization of graphene and graphene-based materials. Chem Soc Rev 2012;41:97–114.Yan M, Zhang Y, Xu KD, Fu T, Qin HY, Zheng XX. An in vitro study of vascular endothelial toxicity of CdTe quantum dots. Toxicology 2011;282:94–103. Yang H, Sun CJ, Fan ZL, Tian X, Yan L, Du LB, et al. Effects of gestational age and surface modification on materno-fetal transfer of nanoparticles in murine pregnancy. Sci Rep 2012;2:847–52. Yang M, Cheng K, Qi SB, Liu HG, Jiang YX, Jiang H, et al. Affibody modified and radiolabeled gold–iron oxide hetero-nanostructures for tumor PET, optical and MR imaging. Biomaterials 2013;34:2796–806. Yin WY, Zhao LN, Zhou LJ, Gu ZJ, Liu XX, Tian G, et al. Enhanced red emission from GdF3: Yb3+, Er3+ upconversion nanocrystals by Li+ doping and their application for bioimaging. Chem Eur J 2012;18:9239–45. Yu MK, Jeong YY, Park J, Park S, Kim JW, Min JJ, et al. Drug-loaded superparamagnetic iron oxide nanoparticles for combined cancer imaging and therapy in vivo. Angew Chem Int Ed Engl 2008;47:5362–5. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC. Nanoparticles in medicine: therapeutic applications and developments. Clin Pharmacol Ther 2008;83:761–9. Zhang Q, Hitchins VM, Schrand AM, Hussain SM, Goering PL. Uptake of gold nanoparticles in murine macrophage cells without cytotoxicity or production of proinflammatory mediators. Nanotoxicology 2011;5:284–95. Zhang T, Qian L, Tang M, Xue YY, Kong L, Zhang SS, et al. Evaluation on cytotoxicity and genotoxicity of the L-glutamic acid coated iron oxide nanoparticles. J Nanosci Nanotechnol 2012a;12:2866–73. Zhang WX. Nanoscale iron particles for environmental remediation: an overview. J Nanopart Res 2003;5:323–32. Zhang XD, Wu D, Shen X, Liu PX, Fan FY, Fan SJ. In vivo renal clearance, biodistribution, toxicity of gold nanoclusters. Biomaterials 2012b;33:4628–38. Zhang Y, Li B, Chen CY, Gao ZH. Hepatic distribution of iron, copper, zinc and cadmium-containing proteins in normal and iron overload mice. Biometals 2009;22:251–9. Zhao F, Zhao Y, Liu Y, Chang XL, Chen CY, Zhao YL. Cellular uptake, intracellular trafficking and cytotoxicity of nanomaterials. Small 2011;7:1322–37. Zhao YL, Li HL, Gao ZH, Xu HB. Effects of dietary baicalin supplementation on iron overload-induced mouse liver oxidative injury. Eur J Pharmacol 2005;509: 195–200. Zhou J, Liu Z, Li FY. Upconversion nanophosphors for small-animal imaging. Chem Soc Rev 2011a;41:1323–49. Zhou JC, Yang ZL, Dong W, Tang RJ, Sun LD, Yan CH. Bioimaging and toxicity assessments of near-infrared upconversion luminescent NaYF4:Yb, Tm nanocrystals. Biomaterials 2011b;32:9059–67. Zhu M, Nie G, Meng H, Xia T, Nel A, Zhao Y. Physicochemical properties determine nanomaterial cellular uptake, transport and fate. Acc Chem Res 2012a;46: 622–31. Zhu MT, Feng WY, Wang B, Wang TC, Gu YQ, Wang M, et al. Comparative study of pulmonary responses to nano- and submicron-sized ferric oxide in rats. Toxicology 2008;247:102–11. Zhu MT, Feng WY, Wang Y, Wang B, Wang M, Ouyang H, et al. Particokinetics and extrapulmonary translocation of intratracheally instilled ferric oxide nanoparticles in rats and the potential health risk assessment. Toxicol Sci 2009;107: 342–51. Zhu MT, Wang B, Wang Y, Yuan L, Wang HJ, Wang M, et al. Endothelial dysfunction and inflammation induced by iron oxide nanoparticle exposure: risk factors for early atherosclerosis. Toxicol Lett 2011;203:162–71. Zhu MT, Wang Y, Feng WY, Wang B, Wang M, Ouyang H, et al. Oxidative stress and apoptosis induced by iron oxide nanoparticles in cultured human umbilical endothelial cells. J Nanosci Nanotechnol 2010;10:8584–90. Zhu XS, Tian SY, Cai ZH. Toxicity assessment of iron oxide nanoparticles in zebrafish (Danio rerio) early life stages. PloS One 2012b;7:e46286. http://dx.doi.org/10.1371/ journal.pone.0046286.

E

T

Pouliquen D, Perroud H, Calza F, Jallet P, Le Jeune J. Investigation of the magnetic properties of iron oxide nanoparticles used as contrast agent for MRI. Magn Reson Med 1992;24:75–84. Qiao RR, Yang CH, Gao MY. Superparamagnetic iron oxide nanoparticles: from preparations to in vivo MRI applications. J Mater Chem 2009;19:6274–93. Qiu Y, Liu Y, Wang LM, Xu LG, Bai R, Ji YL, et al. Surface chemistry and aspect ratio mediated cellular uptake of Au nanorods. Biomaterials 2010;31:7606–19. Qu Y, Li W, Zhou YL, Liu XF, Zhang L, Wang LM, et al. Full assessment of fate and physiological behavior of quantum dots utilizing Caenorhabditis elegans as a model organism. Nano Lett 2011;11:3174–83. Ren WL, Tian G, Jian S, Gu ZJ, Zhou LG, Yan L, et al. TWEEN coated NaYF4: Yb, Er/NaYF4 core/shell upconversion nanoparticles for bioimaging and drug delivery. RSC Adv 2012;2:7037–41. Ritschel WA, Kearns GL. Handbook of basic pharmacokinetics-including clinical applications. Washington, DC: American Pharmacists Association; 2004. Rivet CJ, Yuan Y, Borca-Tasciuc DA, Gilbert RJ. Altering iron oxide nanoparticle surface properties induce cortical neuron cytotoxicity. Chem Res Toxicol 2012;25:153–61. Rufaihah A, Sim E, Ye L, Chen N. Biocompatibility Study of PEI-NaYF4: Yb, Er Upconversion Nanoparticles. 4th Kuala Lumpur International Conference on. Biomed Eng 2008;21: 82–5. Rzigalinski BA, Strobl JS. Cadmium-containing nanoparticles: perspectives on pharmacology and toxicology of quantum dots. Toxicol Appl Pharmacol 2009;238:280–8. Sadaf A, Zeshan B, Wang Z, Zhang R, Xu S, Wang C, et al. Toxicity evaluation of hydrophilic CdTe quantum dots and CdTe@SiO2 nanoparticles in mice. J Nanosci Nanotechnol 2012;12:8287–92. Samir TM, Mansour MM, Kazmierczak SC, Azzazy HM. Quantum dots: heralding a brighter future for clinical diagnostics. Nanomedicine 2012;7:1755–69. Schaeublin NM, Braydich-Stolle LK, Schrand AM, Miller JM, Hutchison J, Schlager JJ, et al. Surface charge of gold nanoparticles mediates mechanism of toxicity. Nanoscale 2011;3:410–20. Sereemaspun A, Hongpiticharoen P, Rojanathanes R, Maneewattanapinyo P, Ekgasit S, Warisnoicharoen W. Inhibition of human cytochrome P450 enzymes by metallic nanoparticles: a preliminary to nanogenomics. Int J Pharmacol 2008;4:492–5. Siddiqi NJ, Abdelhalim MA, El-Ansary AK, Alhomida AS, Ong WY. Identification of potential biomarkers of gold nanoparticle toxicity in rat brains. J Neuroinflammation 2012;9:123–30. Singh N, Jenkins GJ, Nelson BC, Marquis BJ, Maffeis TG, Brown AP, et al. The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles. Biomaterials 2012;33:163–70. Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, et al. Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proc Natl Acad Sci U S A 2006;103:3357–62. Sun CJ, Yang H, Yuan Y, Tian X, Wang LM, Guo Y, et al. Controlling assembly of paired gold clusters within apoferritin nanoreactor for in vivo kidney targeting and biomedical imaging. J Am Chem Soc 2011;133:8617–24. Szalay B, Tatrai E, Nyiro G, Vezer T, Dura G. Potential toxic effects of iron oxide nanoparticles in in vivo and in vitro experiments. J Appl Toxicol 2012;32:446–53. Tang J, Huang J, Man S-Q. Preparation of gold nanoparticles by surfactant-promoted reductive reaction without extra reducing agent. Spectrochim Acta A Mol Biomol Spectrosc 2013;103:345–55. Taylor A, Wilson KM, Murray P, Fernig DG, Levy R. Long-term tracking of cells using inorganic nanoparticles as contrast agents: are we there yet? Chem Soc Rev 2012;41:2707–17. Tedesco S, Doyle H, Redmond G, Sheehan D. Gold nanoparticles and oxidative stress in Mytilus edulis. Mar Environ Res 2008;66:131–3. Tenzer S, Docter D, Rosfa S, Wlodarski A, Kuharev J, Rekik A, et al. Nanoparticle size is a critical physicochemical determinant of the human blood plasma corona: a comprehensive quantitative proteomic analysis. ACS Nano 2011;5:7155–67. Uboldi C, Bonacchi D, Lorenzi G, Hermanns MI, Pohl C, Baldi G, et al. Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441. Part Fibre Toxicol 2009;6:18–30. Villiers CL, Freitas H, Couderc R, Villiers MB, Marche PN. Analysis of the toxicity of gold nano particles on the immune system: effect on dendritic cell functions. J Nanopart Res 2010;12:55–60. Voinov MA, Sosa Pagan JO, Morrison E, Smirnova TI, Smirnov AI. Surface-mediated production of hydroxyl radicals as a mechanism of iron oxide nanoparticle biotoxicity. J Am Chem Soc 2011;133:35–41. Voura EB, Jaiswal JK, Mattoussi H, Simon SM. Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nat Med 2004;10:993–8. Wang B, Feng WY, Wang M, Shi JW, Zhang F, Ouyang H, et al. Transportation of intranasal instillation of fine Fe2O3 particles in brain: micro-distribution, chemical states, and histopathological observation. Biol Trace Elem Res 2007;118:233–43. Wang B, Feng WY, Zhu MT, Wang Y, Wang M, Gu YQ, et al. Neurotoxicity of low-dose repeatedly intranasal instillation of nano- and submicron-sized ferric oxide particles in mice. J Nanopart Res 2009a;11:41–53. Wang B, He X, Zhang ZY, Zhao YL, Feng WY. Metabolism of nanomaterials in vivo: blood circulation and organ clearance. Acc Chem Res 2013;46:761–9. Wang B, Wang Y, Feng WY, Zhu MT, Wang M, Ouyang H, et al. Trace metal disturbance in mice brain after intranasal exposure of nano- and submicron-sized Fe2O3 particles. Chem Anal (Warsaw) 2008;53:927–42. Wang C, Tao HQ, Cheng L, Liu Z. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 2011a;32: 6145–54. Wang LM, Liu Y, Li W, Jiang XM, Ji YL, Wu XC, et al. Selective targeting of gold nanorods at the mitochondria of cancer cells: implications for cancer therapy. Nano Lett 2011b;11:772–80.

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