Effects of physicochemical properties of nanomaterials on their toxicity.

Effects of physicochemical properties of nanomaterials on their toxicity Xiaoming Li a, *, Wei Liu a, Lianwen Sun a, Katerina E. Aifantis b, Bo Yu c, **, Yubo Fan a, ***, Qingling Feng d

, Fuzhai Cui , Fumio Watari

a

Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological

d

e

Science and Medical Engineering, Beihang University, Beijing 100191, China b

Department of Civil Engineering-Engineering Mechanics, University of Arizona, Tucson, AZ 85721,

USA c

Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282,

China d

State Key Laboratory of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084, China

e

Department of Biomedical Materials and Engineering, Graduate School of Dental Medicine, Hokkaido

University, Sapporo 060-8586, Japan

* Corresponding author. E-mail address: [email protected] ** Corresponding author. E-mail address: [email protected] *** Corresponding author. E-mail address: [email protected]

Abstract: Due to their unique size and properties, nanomaterials have numerous applications, which range from electronics, cosmetics, household appliances, energy storage and semiconductor devices, to medical products such as biological sensors, drug carriers, bioprobes, and implants. Many of the promising properties of nanomaterials arise from their large surface to volume ratio and, therefore, nano-biomaterials that are implantable have a large contact area with the human body. Before, therefore, we can fully exploit nanomaterials, in medicine and bioengineering, it is necessary to understand how they can affect the human body. As a step in this direction this review paper provides a comprehensive summary of the effects that the physicochemical properties of commonly used nanobiomaterials have on their toxicity. Furthermore, the possible mechanisms of toxicity are described with the aim to provide guidance concerning the design of the nanobiomaterials with desirable properties.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbma.35384 This article is protected by copyright. All rights reserved.

Journal of Biomedical Materials Research: Part A

Keywords: Physicochemical properties; Nanomaterials; Nanoparticles; Toxicity

John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

Page 2 of 27

Page 3 of 27


1. Introduction With the astonishing development of nanotechnology, nanomaterials have become more and more popular and important in our daily life. It has been estimated that the production of nanomaterials will increase in 2020 by more than 25 times what it is today [1]. This is due to the wide range of applications that they have in numerous fields, ranging from commercial products such as electronic components, cosmetics, household appliances, semiconductor devices, energy sources, food color additives, surface coatings and medical products such as biological sensor, drug carriers, biological probes, implants, and medical imaging [2-4]. Despite this future dependence of our society on nanomaterials, studies regarding their safe incorporation in our lives are very limited [5]. Wiesner et al [6] made an elaborate assessment of the risks of manufactured nanomaterials. They discussed several sources of nanoparticles and various ways by which nanomaterials could enter into the body, and they pointed out that large concentrations of nanoparticles might cause damage to our body, especially in the occupational environments. Nanoparticles can get into the human body through various ways, such as skin penetration, inhalation or injection, and due to their small size and diffusion abilities they have the potential to interact with cells and organs. In addition to involuntary exposure to nanoparticles by means of contacting nanomaterials-based products, there are the cases where nanoparticles will interact with the human body for biomedical purposes. For example, using nanoparticles for targeted drug delivery requires nanoparticles to traverse the cell membranes and interact with specific cells; hence the success rate of drug delivery is based to a large extent on the biocompatibility of nanoparticles. Moreover, some implants containing specific nanoparticles may undergo biodegradation in the cellular environment or release active bioparticles, leading to potential damage of the physiological activity of cells, such as cell membrane integrity and the activity of mitochondria. Research has shown that different physicochemical properties of nanoparticles result in different cellular uptake [7]. Particularly, the size and surface area greatly



influence the course of cellular uptake in the case of liposomes [8], silicon microparticles [9], quantum dots [10], hydroxyapatite nanoparticles [11], carbon nanotubes [12], polymeric nanoparticles [13, 14], and gold nanoparticles [15]. Furthermore, the different crystallinity [16] and concentration [17] of nanoparticles significantly affect their toxicity. The main mechanism by which nanoparticles are toxic for the human body is through the generation of reactive oxygen stress, which causes DNA damage, unregulated cell signaling, and changes in cell motility, cytotoxicity, apoptosis, and cancer initiation and promotion [18-20]. Reactive oxygen is necessary for cell function, but excessive amounts cause the aforementioned effects. In the present article an overview is presented regarding the toxicity of nanoparticles comprised of different materials such as porous silicon particles, gold, hydroxyapatite, PLGA, Fe3O4, or ZnO. A summary is given of how the physicochemical properties of these nanomaterials such as size, structure, shape, surface chemistry and composition affect their toxicity, while at the same time an introduction is given about the mechanisms of action of nanomaterial toxicity. This work can provide guidelines for the design of nanomaterials with desirable biocompatible properties.

2. In vitro and in vivo toxicity tests on nanomaterials In order to assess the toxicity of nanoparticles and examine the effects of their physicochemical properties, cell culture tests are performed on designated nanomaterials. Toxicity tests in vitro are very useful and necessary to characterize the toxicity of nanomaterials and greatly contribute to the understanding of the toxic mechanisms. For example, Lactate dehydrogenase (LDH) release is an extremely common assay employed in present toxicity studies [21-23]. By means of measuring the content of released LDH, it is possible to obtain a qualitative measure for the extent of cell damage [24]. Moreover, an oxidation-reduction reaction can occur only in active mitochondria having dehydrogenase enzymes, so it is possible to take advantage of this reaction in order to determine the activity of mitochondria, and John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

Page 4 of 27

Page 5 of 27


MTT is the most accepted assay method at present [25-32]. However, care must be taken concerning the dyes used, since the degree of accuracy of toxicity assays greatly depends on the interaction between nanomaterials and dyes. For instance，some researchers have found that carbon nanotubes can interact with individual dyes, such as Alamar Blue and Neutral Red [24,33-36]. Compared to animal studies, cellular testing is ethically less ambiguous, easier to control and reproduce, and less expensive [37]. In the case of cytotoxicity measurements, the cell cultures are influenced by fluctuations of external environment (temperature, waste concentrations, pH and so on), so repeated experiments are necessary for obtaining accurate results. Compared to in vitro systems, toxic kinetics, bio-distribution and toxic kinetics are more likely to be revealed for in vitro systems (Figure 1), hence although animal experiments are generally more costly and time-consuming, they are particularly useful. A complete animal experiment could offer a significant amount of important data with regard to dermal and gastrointestinal toxicities, pulmonary accumulation connected with the original deposition of nanomaterials by means of different routes. In addition, the toxicity of cardiovascular, immunological and neurological function could be determined by in vivo studies, as mentioned above.

3. Effect of physicochemical properties of nanomaterials on their toxicity It is of considerable importance to perform physicochemical characterization of nanoparticles, such as surface properties, surface charge, size, shape, structure, composition, crystalline and other parameters, since these properties have a great effect on the interaction with specific cells and their toxicity. In the sequel hence a detailed introduction regarding the toxicity of nanomaterials in terms of their various physicochemical properties is given.

3.1. Effect of particle size on toxicity The size of nanomaterials has a direct and significant impact on the physiological John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


activity. If a particle has a size below 1µm, it will be transmitted into cells causing unknown effects. However, if the particle is greater than 1µm, the particles will not enter the cells easily, but instead large amounts of certain proteins will absorb on their surface, and then the nanoparticles will react with cells in a specific manner. Hence, the nanoparticles’ size plays a critical role in cellular uptake, efficiency of particle processing in the endocytic pathway and physiological response of cells to nanoparticles [38-44]. For example, Kim et al [45] investigated the size-dependent cellular toxicity of Ag nanoparticles using three different characteristic sizes against several cell lines including MC3T3-E1 and PC12. They demonstrated that the toxicity of Ag nanoparticles was precisely size-and dose-dependent in terms of cell viability, intracellular reactive oxygen species generation, lactate dehydrogenase release, and ultra structural changes in cell morphology. In the end, it was concluded that the smallest sized Ag nanoparticles (10 nm) had a greater ability to induce apoptosis in the MC3T3-E1 cells than the other sized Ag nanoparticles (50 and 100nm). Furthermore, it was found that the Ag nanoparticles induced cytotoxic effects against tissue cells, which were particle size-dependent, and therefore, the particle size needs careful consideration for biomedical applications. In a similar case, Park et al [46] studied the toxicity effects of Ag nanoparticles of different sizes and made a comparison in terms of cytotoxicity, inflammation, genotoxicity and developmental toxicity. They concluded that Ag nanoparticles of 20 nm were more toxic than the larger nanoparticles in all toxicity endpoints studied, moreover they ingeniously compared the toxicity of Ag nanoparticles and that of Ag-ions and came to the conclusion that L929 fibroblasts were more sensitive to the effects of 20 nm nanoparticles, than the Ag-ions, while the Ag particles that were over 20nm were less toxic than Ag-ions. In addition, the effects of Ag nanoparticles compared to Ag-ions on four human cell lines were also dependent on the size of the nanoparticles in accord with previous reports [47]. Besides the cytotoxic effects of Ag nanoparticles, Dey et al [16] synthesized hydroxyapatite particles (HAp) via three different routes to achieve micro and nanosized powders, different morphologies and crystallinity (Figure 2) were obtained and they explored the inhibitory effect of John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

Page 6 of 27

Page 7 of 27


nano-HAps on the in vitro growth of human cells HCT116. The crystallinity of the HAps played a significant role. They proved that decreasing the hydroxyapatite powder crystallite size between 11 nm and 22 nm significantly increased the HCT116 cells' inhibition, moreover the HAp crystallinity and morphology also made a great difference in the cellular inhibition of human cancer cells. He et al [48] studied the toxicity of polymeric nanoparticles (rhodamine B). They modified rhodamine B with carboxymethyl chitosan and chitosan hydrochloride, forming decorated rhodamine B with negative charge and positive charge, respectively. Eventually, they concluded that nanoparticles with slight negative charges and a particle size of 150 nm tended to accumulate in tumors more efficiently. Generally speaking, biodegradable nanoparticles are likely to be safer than non-biodegradable nanomaterials. Semete et al [49] investigated the in vivo toxicity and bio-distribution of PLGA (poly (D,L-lactic-coglycolic acid)) NPs with a size of 200-300 nm. About 40% percent of PLGA particles were localized in the liver after a seven-day oral administration in mice, while remains were found in the brain and kidney without clear toxicity. Apart from size dependent toxicity due to ROS-generating capability, particle size can affect the degradation of the polymer matrix. With the decrease of particle size, the surface area to volume ratio increases greatly, leading to an easier penetration and release of the polymer degradation products [50]; it is not that the nanoparticles entering the brain did not increase in this case. Even though it can be assumed that the smaller the nanoparticle size, the more likely it can enter into cells and cause potential damages, the mechanisms of toxicity are very complicated, so the size factor cannot be viewed as the only influence parameter. For instance, Yin et al [51] examined the effects of particle size and surface coating on the cytotoxicity of nickel ferrite in vitro using the Neuro-2A cell line as a model. They concluded that for nickel ferrite particles prepared by ball milling without use of oleic acid, the cytotoxicity was independent of particle size within the given mass concentrations and surface areas. For nickel ferrite nanoparticles coated with oleic acid, the cytotoxicity significantly increased when one John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


or two layers of oleic acid were deposited. Large nanoparticles (with coatings of oleic acid) showed a higher cytotoxicity than smaller particles. It was also noted that if oleic acid molecules were present in the form of a monomer, they were not cytotoxic. However, if they formed micelles or coated the ferrite particles, when their functional groups were aligned in space, cytotoxicity would be observed. Moreover, the cytotoxic effect was greater for large particles than for smaller particles, which indicated that the size effect could be connected with the surface energy. The effect that the size of hydroxyapatite nanoparticles has on the anti-tumor activity and apoptotic signaling proteins was investigated by Yuan et al [52]. For the purpose of studying the effect of particle size on cell apoptosis, the HepG2 cells were stained with Hochest for 48h, and their morphology changes were examined using confocal microscopy. It was demonstrated that notable changes occurred in the nuclear chromatin by means of HAPN treatment. Untreated cells (in the control group) contained round nuclei with homogeneous chromatin and exhibited less bright blue color (Fig. 3a), whereas for cells treated with HAPN, in particular with the hydroxyapatite nanoparticles of 45nm (Fig. 3c) and 78nm (Fig. 3d), the intensity of the blue light observed in the apoptotic cells was higher compared to untreated cells. Classical morphology characteristics of apoptosis were noted, such as a reduction in nuclear size, chromatin condensation and DNA fragmentation (Fig.3b, 3c, 3d, 3e). In the end, it was concluded that the anti-tumor activity and HAPN-induced apoptosis highly depended on the size of HAPNs in HepG2 cells.

3.2 Effect of nanomaterial structure and shape on toxicity In addition to size, the structure and shape of nanomaterials are two additional vital factors that influence their toxicity. Nanomaterials may have different shapes and structure, such as tubes, fibers, spheres, planes, and poly-hydra. These distinctions may lead to differences in their toxicity effects. A most distinct example of the effect of shape on toxicity is for carbon-based [53]. Grabinski et al [54] investigated the cellular effects of different carbon-based materials using mouse keratinocytes. The carbon materials tested included carbon nanofibers, multi-walled carbon nanotubes


Page 8 of 27

Page 9 of 27


(MWCNT), and single-walled carbon nanotubes (SWCNT). They concluded that carbon nanofibers did not significantly affect cell viability; however, MWCNT and SWCNT reduced cell viability in a time-dependent manner. After 24 h, cells exposed to MWCNT produced three times the reactive oxygen than those exposed to SWCNT. Zhang et al [55] conducted a study comparing toxicity of graphene and carbon nanotubes. They concluded that both graphene and SWCNTs induce cytotoxic effects, and these effects are concentration-dependent and shape-dependent, moreover, graphene induced a stronger metabolic activity than that of SWCNTs at low concentrations, indicating the effect of shape on cellular toxicity. Li et al made a further study on the effect of carbon nanotubes on bone formation in vivo [56]. They evaluated the attachment, proliferation, osteogenic gene expression, ALP/DNA, protein/DNA and mineralization of human adipose-derived stem cells cultured in vitro on multi-walled carbon nanotubes (MWNTs) and graphite (GP) compacts with the same dimension. Moreover, they assessed the effect of these two kinds of compacts on ectopic bone formation in vivo and finally considered that MWNTs might stimulate inducible cells in soft tissues to form inductive bone by concentrating more proteins, including bone-inducing proteins. In addition to examining the shape of carbon based materials, different toxicity behavior has also been observed for TiO2 NPs with different crystal structures. For instance, Gurr et al [57] reported that TiO2 NPs can induce oxidative DNA damage, lipid peroxidation, and micronuclei formation in the absence of light, where anatase NPs of the same size and chemical composition are introduced. Furthermore, shape dependent toxicity of nickel NPs has been reported by Ispas et al [58].

3.3 Effect of nanomaterial surface on toxicity Surface also plays a role in toxicity, as it influences the adsorption of ions and biomolecules that may change the organism or cellular responses towards particles. In addition, surface charge is a major determinant of colloidal behavior, which influences the organism response by changing the shape and size of NPs through aggregate or agglomerate formation. For example, Shahbazi et al [59] evaluated the



impact of mesoporous silicon nanoparticles surface chemistry on immune cells and human RBCs both in vitro and in vivo. Aiming to explore the effect of surface chemistry and surface charge of several mesoporous silicon nanoparticles on different cells, including Raji (B-cell), Jurkat (T-cell), U937 (monocyte) and RAW264.7 (macrophage) cells, further studies where performed for thermally oxidized porous silicon nanoparticles (TOPSi), thermally carbonized porous silicon nanoparticles (TCPSi), and triethoxysilane functionalized thermally carbonized porous silicon nanoparticles (APSTCPSi) (Figure 4). It was concluded that less ATP depletion and genotoxicity will be caused for negatively charged hydrophilic and hydrophobic mesoporous silicon nanoparticles than the positively charged amine modified hydrophilic mesoporous silicon nanoparticles, proving the significance of the silicon nanoparticles surface charge on the immunocompatibility. Moreover, Calatayud et al [60] studied the effect of surface charge of functionalized Fe3O4 nanoparticles on protein adsorption and cell uptake. It was demonstrated that the functional groups on the magnetic nanoparticle surface determined the formation of protein-magnetic nanoparticle clusters and suggested the ability to modify the surface of magnetic nanoparticles in order to control the non-specific protein adsorption. Grabowskia et al conducted a study on effects of surface chemistry of mesoporous silicon nanoparticles on

their immunotoxicity and

biocompatibility[61]. They assayed the toxicity of PLGA nanoparticles using human lung epithelial cells. Three different PLGA NPs were acquired using different stabilizers, namely, neutral, positively or negatively charged NPs, respectively. Finally, Nadege Grabowskia et al demonstrated that in terms of the inflammatory response, negative PLGA NPs led to a higher inflammatory response of cells. This may be correlated to a higher uptake of these NPs, implying the significance of performing a detailed characterization of the PLGA NPs together with their potential involvement in the inflammatory response. Finally, it should be noted that the concept of "nanomaterial surface" includes more aspects, such as surface area, pore, surface chemical bond and potential. For instance, El Badawy et al [62] studied the various factors that influence toxicity of John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

Page 10 of 27

Page 11 of 27


silver nanoparticles, and they concluded that the surface charge was one of the most importantones. Furthermore, it was shown that silver nanoparticles exhibited an obvious surface charge dependent toxicity on the different bacillus species.

3.4 Effect of nanomaterial concentration on toxicity A final vital parameter to consider regarding the toxicity of nanomaterials is their concentration. A study by Santos et al [63] concluded that when the concentration of thermally hydrocarbonized and carbonized porous silicon particles was greater than 2 mg/ml, they were toxic for cells; while for thermally oxidized porous silicon particles, the non-toxic threshold concentration was 4 mg/ml. Hussain et al [64] conducted research on the toxicity of different kinds of nanoparticles (Fe3O4, Al, MoO3 and TiO2) in BRL 3A rat liver cells in vitro. They concluded that when exposed to Ag nanoparticles of 5-50 µg/ml concentration, the function of mitochondria will suffer a dramatic decline. For Ag nanoparticles, when its concentration was up to 100-250 µg/ml, the LDH leakage increased in cells. Similarly, Usenko et al [65] evaluated the toxicity of carbon fullerene using embryonic zebrafish, and they concluded that exposure to 200 µg/ml C60 and C70 induced a significant increase in malformations.

4. Potential mechanism of toxicity At present, distinct mechanisms and hypothesis are used to reveal the toxicity of nanomaterials, however, the mechanisms by which reactive oxygen is generated is one of the most popular theories in the academic circle. Reactive oxygen generally includes superoxide anion radicals, hydroxyl radicals, singlet oxygen, and hydrogen peroxide [66]. In case of appropriate content, reactive oxygen contributes to the regulation of signaling systems, proliferative response, and protein redox and gene expression in cells [67-69]. However, various negative effects, such as peroxidizing lipids, damage of specific protein and DNA, will appear with superlative content of reactive oxygen [70]. In most cases, reactive oxygen species are produced in the course of synthesis of ATP, accompanied by the transfer of protons



and electrons in the mitochondria. Moreover, the mitochondria are the targeted endpoint of nanomaterials once they smoothly enter the cells, either through diffusion or endocytosis [71]. Particularly when nanoparticles enter the cells they gather around the mitochondria, causing the dysfunction of corresponding mitochondria, which will break the balance between the production and consumption of reactive oxygen. This results in the overproduction of reactive oxygen species, which can induce oxidative stress that will further disturb the normal physiological functions [72] of cells, giving rise to inactivation of specific proteins [73], peroxidation of lipids [74-76], and DNA damage [77] that result in cell death and genotoxic effects (Figure 5). It should be noted here that the existence of the high levels of unsaturated fatty acids, makes the central nervous system vulnerable to peroxidation [78]. Many diseases are closely related with oxidative stress including arthritis, cardiovascular disease, Parkinson’s disease, cancer and other diseases [79-85]. Also another mechanism by which nanoparticles aid in the production of oxidative stress is that they may catalyze the production of reactive oxygen, which in turn results again in oxidative stress (Figure 5). Furthermore, it is suggested that tiny nanoparticles could interact with membrane proteins and activate signaling pathways, thus affecting the function of cells [86, 87]. Only once we have a good understanding of the toxic mechanism of nanomaterials, can we take targeted measures to reduce the toxicity effects to the greatest degree. For example, to alleviate ROS effects, some new steps have been taken in NP design. Recently, cerium oxide nanoparticles have been developed that incorporate oxygen defects that could scavenge free radicals. It was found that the cerium oxide NPs prevented oxidative stress, as well as N-acetyl cystine in mice with tetrachloride-induced liver toxicity. Owing to the accumulation of ROS, the DNA of normal cells tends to suffer mutation, which results in cells becoming cancerous. However, the existence of antioxidants can decrease the accumulation of reactive oxygen species greatly, thus reducing the probability of cancer formation. Finley [88] suggested that cruciferous vegetables such as broccoli and cauliflower seem to be especially protective against cancer precisely because of the antioxidant effect of cruciferous vegetables. John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

Page 12 of 27

Page 13 of 27


5 Conclusions and Perspectives With the development of nanomaterial-based products and their exponenential production, it is extremely necessary to have a better understanding regarding the effects they have to the human body. Similar to bulk materials, the composition of nanomaterials imposes a tremendous effect on their toxicity. In the present review, however, it was shown that in addition to composition, the physicochemical properties of nanomaterials, such as size, structure and shape, surface properties and concentration, also play an important role in influencing their toxicity. Thus, the toxicity of nanomaterials can be altered significantly by the manipulation of several physicochemical properties. A fundamental understanding of the biological interactions of NPs with cells, proteins, and tissues, is vital to the future design of safe nanotechnologies. Only once we have a good understanding of the mechanisms by which NPs interact with cells, can we design safer and more effective nanomaterials. However, there are not sufficient data on the interpretation of toxicity of nanomaterials in vivo, which is extremely important for understanding the route of nanomaterials in our body. Moreover, the long-term toxic effects of exposure on the condition of nanomaterials are still unknown. Hence, until the aforementioned are examined in detail, the manufacturing of nanomaterial-based products should be more prudent and careful in case of releasing toxic nanoparticles during their use or disposal.

Conflict of interests We declare that we have no financial or personal relationship with any people or organization that may inappropriately influence our work, there is no professional or commercial interest of any kind in all the commercial identities mentioned in our paper.



Acknowledgements The authors acknowledge the financial supports from the National Basic Research Program of China (973 Program, No. 2011CB710901), the National Natural Science Foundation of China (No. 31370959, 81371931 and 61227902), Beijing Natural Science Foundation (No. 7142094), Fok Ying Tong Education Foundation (no. 141039), Program for New Century Excellent Talents (NCET) in University from Ministry of Education of China, State Key Laboratory of New Ceramic and Fine Processing (Tsinghua University), International Joint Research Center of Aerospace Biotechnology and Medical Engineering, Ministry of Science and Technology of China, and the 111 Project (No. B13003).

Reference [1] Wright J. Nanotechnology: Deliver on a promise. Nature 2014; 509: 58-59. [2] Mahmoudi M, Azadmanesh K, Shokrgozar MA, Journeay WS, Laurent S. Effect of nanoparticles on the cell life cycle. Chemical reviews 2011; 111: 3407-3432. [3] Li XM, Liu X, Fan YB, Cui FZ. Biomedical Investigation of CNT based Coatings. Surface & Coatings Technology 2011; 206: 759-766. [4] Li XM, Wang L, Fan YB, Feng QL, Cui FZ, Watari F. Nanostructured scaffolds for bone tissue engineering. Journal of Biomedical Materials Research Part A 2013; 101A: 2424-2435. [5] Thilo P, Dietmar S, Dieter W, Vince C, Val V, Qamar R. Human health implications of nanomaterial exposure. Nanotoxicology 2008; 2: 9-27. [6] Wiesner MR, Lowry GV, Alvarez P, Dionysiou D, Biswas P. Assessing the risks of manufactured nanomaterials. Environmental science & technology 2006; 40: 4336-4345. [7] Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 2008; 5: 505-515. [8] Chono S, Tanino T, Seki T, Morimoto K. Uptake characteristics of liposomes by rat alveolar macrophages: influence of particle size and surface mannose modification. J Pharm Pharmacol 2007; 59: 75-80.


Page 14 of 27

Page 15 of 27


[9] Santos HA, Riikonen J, Salonen J, Mäkilä E, Heikkilä T, Laaksonen T, Peltonen L, Lehto VP, Hirvonen J. In vitro cytotoxicity of porous silicon microparticles: Effect of the particle concentration, surface chemistry and size. Acta Biomaterialia 2010; 6: 2721-2731. [10] Osaki F, Kanamori T, Sando S, Sera T, Aoyama T. A quantum dot conjugated sugar ball and its cellular uptake on the size effects of endocytosis in the subviral region. J Am Chem Soc 2004; 126: 6520-6521. [11] Sun JS, Tsuang YH, Chang WH, Li J, Liu HC, Lin FH. Effect of hydroxyapatite particle size on myoblasts and fibroblasts. Biomaterials 1997; 18: 683-690. [12] Li XM, Fan YB, Watari F. Current investigations into carbon nanotubes for biomedical application. Biomedical Materials 2010 ; 5: 022001. [13] Wina KY, Feng SS. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005; 26: 2713-2722. [14] Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm 2005; 298: 315-322. [15] Chithrani BD, Ghazani AA, Chan WCW. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 2006; 6: 662-668. [16] Dey S, Das M, Balla VK. Effect of hydroxyapatite particle size, morphology and crystallinity on proliferation of colon cancer HCT116 cells. Materials Science and Engineering C 2014; 39: 336-339. [17] Krajewski S, Prucek R, Panacek A, Avci-Adali M, Nolte A, Straub A, Zboril R, Wendel HP, Kvitek L. Hemocompatibility evaluation of different silver nanoparticle concentrations employing a modified Chandler-loop in vitro assay on human blood. Acta Biomaterialia 2013; 9: 7460-7468. [18] Nel A, Xia T, Mädler L, Li N. Toxic potential of materials at the nanolevel. Science 2006; 311: 622-627. [19] Li XM, Wang L, Fan YB, Feng QL, Cui FZ. Biocompatibility and Toxicity of Nanoparticles and Nanotubes. Journal of Nanomaterials 2012; no. 548389. [20] Zhu X, Hondroulis E, Liu W, Li CZ. Biosensing approaches for rapid genotoxicity and cytotoxicity assays upon nanomaterial exposure. Small 2013; 9: 1821-1830. [21] Sayes CM, Fortner JD, Guo W, Lyon D, Boyd AM, Ausman KD , Tao YJ, Sitharaman B, Wilson LJ, Hughes JB, West JL, Colvin VL. The differential cytotoxicity of water-soluble



fullerenes. Nano letters 2004; 4: 1881-1887. [22] Muller J, Huaux F, Moreau N, Misson P, Heilier JF, Delos M, Arras M, Fonseca A, Nagy JB, Lison D. Respiratory toxicity of multi-wall carbon nanotubes. Toxicology and applied pharmacology 2005; 207: 221-231. [23] Uo M, Tamura K, Sato Y, Yokoyama A, Watari F, Totsuka Y, Tohji K. The cytotoxicity of metal-encapsulating carbon nanocapsules. Small 2005; 1: 816-819. [24] Haslam G, Wyatt D, Kitos P A. Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities. Cytotechnology 2000; 32: 63-75. [25] Flahaut E, Durrieu MC, Remy-Zolghadri M, Bareille R, Baquey C. Investigation of the cytotoxicity of CCVD carbon nanotubes towards human umbilical vein endothelial cells. Carbon 2006; 44: 1093-1099. [26] Monteiro-Riviere NA, Inman AO. Challenges for assessing carbon nanomaterial toxicity to the skin. Carbon 2006; 44: 1070-1078. [27] Li XM, Feng QL, Wang WJ, Cui FZ. Chemical characteristics and cytocompatibility of collagen-based scaffold reinforced by chitin fibers for bone tissue engineering. Journal of Biomedical Materials Research Part B 2006; 77B: 219-226. [28] Magrez A, Kasas S, Salicio V, Pasquier N, Seo JW, Celio M, Catsicas S, Schwaller B, Forro, L. Cellular toxicity of carbon-based nanomaterials. Nano letters 2006; 6: 1121-1125. [29] Tian F, Cui D, Schwarz H, Estrada GG, Kobayashi H.Cytotoxicity of single-wall carbon nanotubes on human fibroblasts. Toxicology in vitro 2006; 20: 1202-1212. [30] 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: 325-327. [31] Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview. Langmuir 2005; 2: 10644-10654. [32] Takahashi H, Niidome Y, Niidome T, Kaneko K, Kawasaki H, Yamada S. Modification of gold nanorods using phosphatidylcholine to reduce cytotoxicity. Langmuir 2006; 22: 2-5. [33] Casey A, Herzog E, Davoren M, Lyng FM, Byrne HJ, Chambers G. Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity. Carbon 2007; 45: 1425-1432.


Page 16 of 27

Page 17 of 27


[34] Wörle-Knirsch JM, Pulskamp K, Krug HF. Oops they did it again! Carbon nanotubes hoax scientists in viability assays. Nano letters 2006; 6: 1261-1268. [35] Herzog E, Casey A, Lyng FM, Chambers G, Byrne HJ, Davoren M. A new approach to the toxicity testing of carbon-based nanomaterials-the clonogenic assay. Toxicology letters 2007; 174: 49-60. [36] Li XM, Gao H, Uo M, Sato Y, Akasaka T, Feng QL, Cui FZ, Liu XH, Watari F. Effect of carbon nanotubes on cellular functions in vitro. Journal of Biomedical Materials Research Part A 2009; 91A: 132–139. [37] Li XM, Huang Y, Zheng LS, Liu HF, Niu XF, Huang J, Zhao F, Fan YB. Effect of substrate stiffness on the functions of rat bone marrow and adipose tissue derived mesenchymal stem cells in vitro. Journal of Biomedical Materials Research Part A 2014; 102A: 1092-1101. [38] Chithrani BD, Chan WCW. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett 2007; 7: 1542-1550. [39] Li XM, Liu HF, Niu XF, Fan YB, Feng QL, Cui FZ, Watari F. Osteogenic differentiation of human adipose-derived stem cells induced by osteoinductive calcium phosphate ceramics. Journal of Biomedical Materials Research Part B 2011; 97B: 10-19. [40] Chen M, Mikecz A. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles. Exp Cell Res 2005; 305: 51-62. [41] Rejman J, Oberle V, Zuhorn IS, Hoekstra D. Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis. Biochem. J 2004; 377: 159-169. [42] Lanone S, Boczkowski J. Biomedical applications and potential health risks of nanomaterials: molecular mechanisms. Current molecular medicine 2006; 6: 651-663. [43] Powers KW, Brown SC, Krishna VB, Wasdo SC, Moudgil BM, Roberts SM. Research strategies for safety evaluation of nanomaterials. Part VI. Characterization of nanoscale particles for toxicological evaluation. Toxicological Sciences 2006; 90: 296-303. [44] Li XM, Yang Y, Fan YB, Feng QL, Cui FZ, Watari F. Biocomposites reinforced by fibers or tubes, as scaffolds for tissue engineering or regenerative medicine. Journal of Biomedical Materials Research Part A 2014; 102A: 1580-1594. [45] Kim TH, Kim M, Park HS, Shin US, Gong MS, Kim HW. Size-dependent cellular toxicity of silver nanoparticles. Journal of Biomedical Materials Research Part A 2012; 100A: 1033-1043.



[46] Park MV, Neigh AM, Vermeulen JP, de la Fonteyne LJ, Verharen HW, Briedé JJ, van Loveren H, de Jong WH.The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles. Biomaterials 2011; 32:9810-9817. [47] Liu W, Wu Y, Wang C, Li HC, Wang T, Liao CY, Cui L, Zhou QF, Yan B, Jiang GB. Impact of silver nanoparticles on human cells: effect of particle size. Nanotoxicology 2010; 4: 319-330. [48] He CB, Hu YP, Yin LC, Tang C, Yin CH. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010; 31:3657-3666. [49] Semete B, Booysen L, Lemmer Y, Kalombo L, Katata L, Verschoor J, Swai HS. In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems. Nanomedicine: Nanotechnology, Biology and Medicine 2010; 6: 662-671. [50] Panyam J, Dali MM, Sahoo SK, Ma W, Chakravarthi SS, Amidon GL, Levy RJ, Labhasetwar V. Polymer degradation and in vitro release of a model protein from poly(d,l-lactide-co-glycolide) nano-and microparticles. Journal of Controlled Release 2003; 92: 173-187. [51] Yin H, Too HP, Chow GM. The effects of particle size and surface coating on the cytotoxicity of nickel ferrite. Biomaterials 2005; 26: 5818-5826. [52] Yuan Y, Liu CS, Qjan JC, Wang J, Zhang Y. Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials 2010; 31: 730-740. [53] Li XM, Gao H, Uo M, Sato Y, Akasaka T, Abe S, Feng QL, Cui FZ, Watari F. Maturation of osteoblast-like SaoS2 induced by carbon nanotubes. Biomedical Materials 2009; 4: 015005. [54] Grabinski C, Hussain S , Lafdi K, Braydich-Stolle L , SchlagerJ. Effect of particle dimension on biocompatibility of carbon nanomaterials. Carbon 2007; 45: 2828-2835. [55] Zhang Y, Ali SF, Dervishi E, Xu Y, Li Z, Casciano D, Biris AS. Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. Acs Nano 2010; 4: 3181-3186. [56] Li XM, Liu HF, Niu XF, Yu B, Fan YB, Feng QL, Cui FZ, Watari F. The use of carbon nanotubes to induce osteogenic differentiation of human adipose-derived MSCs in vitro and ectopic bone formation in vivo. Biomaterials 2012; 33: 4818-4827. [57] Gurr JR, Wang AS, Chen CH, Jan KY. Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 2005; 213: 66-73.


Page 18 of 27

Page 19 of 27


[58] Ispas C, Andreescu D, Patel A, Goia DV, Andreescu S, Wallace KN. Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish. Environmental science & technology 2009; 43: 6349-6356. [59] Shahbazi MA, Hamidi M, Mäkilä EM, Zhang H, Almeida PV, Kaasalainen M, Salonen JJ, Hirvonen JT, Santos HA. The mechanisms of surface chemistry effects of mesoporous silicon nanoparticles on immunotoxicity and biocompatibility. Biomaterials 2013; 34: 7776-7789. [60] Calatayud MP, Sanz B, Raffa V, Riggio C, Ibarra MR, Goya GF. The effect of surface charge of functionalized Fe3O4 nanoparticles on protein adsorption and cell uptake. Biomaterials 2014; 35: 6389-6399. [61] Grabowski N, Hillaireau H, Vergnaud J, Santiago LA, Kerdine-Romer S, Pallardy M, Tsapis N, Fattal E.Toxicity of surface- modified PLGA nanoparticles toward lung alveolar epithelial cells. International Journal of Pharmaceutics 2013; 454: 686-694. [62] El Badawy AM, Silva RG, Morris B, Scheckel KG, Suidan MT, Tolaymat TM. Surface charge-dependent toxicity of silver nanoparticles. Environmental science & technology 2010; 45: 283-287. [63] Santos HA, Riikonen J, Salonen J, Mäkilä E, Heikkilä T, Laaksonen T, Peltonen L, Lehto VP, Hirvonen J. In vitro cytotoxicity of porous silicon microparticles: Effect of the particle concentration, surface chemistry and size. Acta Biomaterialia 2010; 6: 2721-273. [64] Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicology in Vitro 2005; 19: 975-983. [65]Usenko CY, Harper SL, Tanguay RL. In vivo evaluation of carbon fullerene toxicity using embryonic zebrafish. Carbon 2007; 45: 1891-1898. [66] Yin JJ, Liu J, Ehrenshaft M, Roberts JE, Fu PP, Mason RP, Zhao B. Phototoxicity of nanotitanium dioxides in HaCaT keratinocytes-generation of reactive oxygen species and cell damage. Toxicol Appl Pharmacol 2012; 263: 81-88. [67] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39: 44-84. [68] Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006; 160: 1-40.



[69] Dalle-Donne I, Rossi R, Giustarini D, Colombo R, Milzani A. S-glutathionylation in protein redox regulation. Free Radical Biology and Medicine 2007; 43: 883-898. [70] Oberdörster G, Oberdörster E, Oberdörster J.Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environmental health perspectives 2005; 113: 823-39. [71] Xia T, Kovochich M, Brant J, Hotze M, Sempf J, Oberley T, Sioutas C, Yeh JI, Wiesner MR, Nel AE. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano letters 2006; 6: 1794-1807. [72] Meng H, Xia T, George S, Nel AE. A predictive toxicological paradigm for the safety assessment of nanomaterials. ACS Nano 2009; 3: 1620-1627. [73] Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chem Res Toxicol 1997; 10: 485-494. [74] Butterfield DA, Kanski J. Brain protein oxidation in agerelated neurodegenerative disorders that are associated with aggregated proteins. Mech Ageing Dev 2001; 122: 945-962. [75] Poli G, Leonarduzzi G, Biasi F. Oxidative stress and cell signalling. Curr Med Chem 2004; 11: 1163-1182. [76] Poon HF, Calabrese V, Scapagnini G, Butterfield DA. Free radicals and brain aging. Clin Geriatr Med 2004; 20: 329-359. [77] Evans MD, Dizdaroglu M, Cooke MS. Oxidative DNA damage and disease: induction, repair and significance. Mutat Res 2004; 567: 1-61. [78] Adibhatla RM, Hatcher JF. Lipid oxidation and peroxidation in CNS health and disease: from molecular mechanisms to therapeutic opportunities. Antioxidants & redox signaling 2010; 12: 125-169. [79] Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002; 82: 47-95. [80] Sohal RS, Mockett RJ, Orr WC. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic Biol Med 2002; 33: 575-586. [81] Liu XH, Li XM, Fan YB, Zhang GP, Li DM, Dong W, Sha ZY, Yu XG, Feng QL, Cui FZ, Watari F. Repairing goat tibia segmental bone defect using scaffold cultured with mesenchymal stem cells. Journal of Biomedical Materials Research Part B 2010; 94B: 44-52. [82] Li XM, Liu XH, Dong W, Feng QL, Cui FZ, Uo M, Akasaka T, Watari F. In vitro evaluation


Page 20 of 27

Page 21 of 27


of porous poly(L-lactic acid) scaffold reinforced by chitin fibers. Journal of Biomedical Materials Research Part B 2009; 90B: 503-509. [83] Kawanishi S, Hiraku Y, Murata M, Oikawa S. The role of metals in site-specific DNA damage with reference to carcinogenesis. Free Radic Biol Med 2002; 32: 822-832. [84] Niedowicz DM, Daleke DL. The role of oxidative stress in diabetic complications. Cell Biochem Biophys 2005; 43: 289-330. [85] Sachidanandam K, Fagan SC, Ergul A. Oxidative stress and cardiovascular disease: antioxidants and unresolved issues. Cardiovasc Drug Rev 2005; 23: 115-132 [86] Li XM, Van Blitterswijk CA, Feng QL, Cui FZ, Watari F. The effect of calcium phosphate microstructure on bone-related cells in vitro. Biomaterials 2008; 29: 3306-3316. [87] Dobrovolskaia MA, Clogston JD, Neun BW, Hall JB, Patri AK, McNeil SE. Method for analysis of nanoparticle hemolytic properties in vitro. Nano letters 2008; 8: 2180-2187. [88] Finley JW. The antioxidant responsive element (ARE) may explain the protective effects of cruciferous vegetables on cancer. Nutrition reviews 2003; 61: 250-254.



Figure Legends

FIGURE 1. Influential factors and evaluation methods of nanomaterials' toxicity

FIGURE 2. FESEM images of starting nano-hydroxyapatite powders with different morphologies by different synthesis methodologies. (a) nano-hydroxyapatite powders with fine and irregular shape (b) nano-hydroxyapatite with rod-like structure. (Adapted with permission from Ref. 16. Copyright 2014 Elsevier Ltd).

FIGURE 3. Fluorescent micrographs of HepG2 cells after 48 h exposure at 100mg/mL with (a) vehicle (b) HAPN-26nm (c) HAPN-45nm (d) HAPN-78nm (e) HAPN-175nm (400 × magnification). Via cell staining to visualize nuclear morphology, the vehicle-treated HepG2 cells contained round nuclei with homogeneous chromatin, while, cells treated with HAPN showed chromatin condensation, reduction of nuclear size as well as nuclear fragmentation. (Adapted with permission from Ref. 52. Copyright 2009 Elsevier Ltd).

FIGURE 4. SEM images of interactions between the porous silicon nanoparticles and immune cell in vitro. TOPSi and TCPSi NPs showed the lowest effect on the cell membrane integrity and morphology, while APSTCPSi caused the highest rate of toxicity in terms of changes occurred in the shape and membrane integrity of the cells. Scale bars are 4µm. (Adapted with permission from Ref. 59. Copyright 2013 Elsevier Ltd).

FIGURE 5.The potential mechanism of nanomaterials' toxicity


Page 22 of 27

Page 23 of 27


FIGURE 1 284x168mm (300 x 300 DPI)

John Wiley & Sons, Inc.

This article is protected by copyright. All rights reserved.


FIGURE 2 193x80mm (300 x 300 DPI)



Page 24 of 27

Page 25 of 27


FIGURE 3 197x114mm (300 x 300 DPI)




FIGURE 4 197x158mm (300 x 300 DPI)



Page 26 of 27

Page 27 of 27


FIGURE 5 282x131mm (300 x 300 DPI)



Effects of water and cell culture media on the physicochemical properties of ZnMgO nanoparticles and their toxicity toward mammalian cells.

Contribution of engineered nanomaterials physicochemical properties to mast cell degranulation.

Physicochemical properties of nanomaterials: implication in associated toxic manifestations.

Role of Physicochemical Properties in Nanoparticle Toxicity.

Antibacterial properties and toxicity from metallic nanomaterials.

The Developmental Toxicity of Complex Silica-Embedded Nickel Nanoparticles Is Determined by Their Physicochemical Properties.

Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs).

Effects of antibiotic physicochemical properties on their release kinetics from biodegradable polymer microparticles.

Physicochemical properties of some organophosphates in relation to their chronic toxicity.

Applications and toxicity of graphene family nanomaterials and their composites.

Techniques for physicochemical characterization of nanomaterials.

Toxicity of Nanomaterials.

The influence of lysosomal stability of silver nanomaterials on their toxicity to human cells.

Transcriptional profiling identifies physicochemical properties of nanomaterials that are determinants of the in vivo pulmonary response.

Rational engineering of physicochemical properties of nanomaterials for biomedical applications with nanotoxicological perspectives.

Effects of salting treatment on the physicochemical properties, textural properties, and microstructures of duck eggs.

On the physicochemical properties of gellan gum.

On the physicochemical properties of pyridohelicenes.

Assessment of the exhalation kinetics of volatile cancer biomarkers based on their physicochemical properties.

N-[(2,6-Dimethylphenoxy)alkyl]aminoalkanols-their physicochemical and anticonvulsant properties.

Physicochemical characteristics of nanomaterials that affect pulmonary inflammation.

Toxicity of inorganic nanomaterials in biomedical imaging.

Toxicity of nanomaterials; an undermined issue.

Effects of Kaolinite (Macsumsuk) and Herb Mixtures on the Quality and Physicochemical Properties of Pork.