Toxicity of Plasmonic Nanomaterials and Their Hybrid Nanocomposites

Toxicity of Plasmonic Nanomaterials and Their Hybrid Nanocomposites

CHAPTER FIVE Toxicity of Plasmonic Nanomaterials and Their Hybrid Nanocomposites Ahmed Nabile Emam*,1, Emad Girgis†,1, Wagdy K.B. Khalil{, Mona Bakr ...

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CHAPTER FIVE

Toxicity of Plasmonic Nanomaterials and Their Hybrid Nanocomposites Ahmed Nabile Emam*,1, Emad Girgis†,1, Wagdy K.B. Khalil{, Mona Bakr Mohamed} *Biomaterials Department, National Research Centre, Dokki, Giza, Egypt † Solid State Physics Department, National Research Centre, Dokki, Giza, Egypt { Cell Biology Department, National Research Centre, Dokki, Giza, Egypt } National Institute of Laser Enhanced Sciences, Cairo University, Giza, Egypt 1 Corresponding authors: e-mail address: [email protected]; [email protected]

Contents 1. 2. 3. 4. 5.

Introduction Toxicity Testing and Risk Assessment of Nanomaterials Toxicity Evaluation Animal Testing for Toxicity Factors Affecting Nanotoxicology 5.1 Effect of size, concentration, and surface area 5.2 Effect of animal model 5.3 Purity 5.4 Surface charge and chemistry 6. Genotoxicology 7. The Most Common Genotoxicological Tests 7.1 8-Hydroxydeoxyguanosine DNA adducts 7.2 Micronucleus test 8. Genotoxicity of Plasmonic Nanomaterials 8.1 Gold nanoparticles 9. Evaluation of Genotoxicity of Magneto-Plasmonic Hybrid Nanocomposites 9.1 Genotoxicity of the Au–Co NPs 10. Conclusion References

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Abstract Nanomaterials are defined as particles, fibers and tubes, composite materials, and nanostructured surfaces with at least one dimension smaller than 100 nm (i.e., 109 m). Over the last decades, interest for engineered nanomaterials with specific physicochemical properties has grown dramatically. This opens great opportunities for use and is attractive in large number of applications that are being developed as well as by the

Advances in Molecular Toxicology, Volume 8 ISSN 1872-0854 http://dx.doi.org/10.1016/B978-0-444-63406-1.00005-2

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2014 Elsevier B.V. All rights reserved.

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increasing number of already marketed products. Therefore, due to their novel properties, a progressive concern about the potential effects of nanomaterials on human health has increased among toxicologists. Consequently, a great effort is underway to investigate the possible adverse effects of engineered nanomaterials, including genotoxicity. Plasmonic nanomaterials such as gold and silver and their composites have many biomedical applications, especially as drug delivery, cancer therapy, and diagnosis agents. This chapter summarizes the toxicological studies of gold nanoparticles and their dependence on size, shape, and capping materials. More light will be focused on our recent study on the genotoxicity investigations on nanomaterials especially gold and their alloys such as magneto-plasmonic nanoalloys (i.e., Au–Co alloy). In general, the toxicity of nanocomposite materials depends not only on the properties of their individual components but also on their morphology, dispersion, and interfacial characteristics.

1. INTRODUCTION In the last few decades, the field concerning nanotechnology applications is rapidly growing and promises substantial benefits that will have significant economic and industrial impacts. It will be applicable to a whole host of areas, ranging from engineering, photoelectronic devices, environmental remediation, and medical healthcare [1]. The most validated definition for the concept of nanotechnology, as subsequently presented by National Nanotechnology Initiative (NNI) [2], is the ability to work at the molecular level, in order to engineer and create new materials, and devices with a functional structure possessing fundamentally new molecular aspects [3]. Compared to the behavior of materials in the bulk phase, the behavior, and the structural features of the materials in the range of about 109 to 107 m (i.e., 1000 times smaller than the diameter of a human hair) is not necessarily predictable from that observed at large size scales, but they exhibit and possess a newly observed phenomena and unique intrinsic properties, that differ remarkably from bulk materials of the same composition [2,4]. These unique properties have been recognized and provide new opportunities in numerous scientific and technological areas [5]. Nanomedicine is known as the medical application of nanoscience [6]. Another definition of this term is summarized in the statement that “the science and technology of diagnosing, treating and preventing disease, and traumatic injury, of relieving pain, and of preserving and improving human health, using molecular tools and molecular knowledge of the human body.” [7] Nanomedicine seeks to deliver a valuable set of research tools

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and clinically useful devices in the near future [6,8]. Due to the superior and favorable physical and chemical properties of different nanomaterials (i.e., plasmonic [9], magnetic [10–12], quantum dots [13,14], and carbon nanomaterials [15]) that provide new efforts for a variety of biomedical applications involving both the industrial and commercial sectors [16–23] including advanced drug delivery systems [24–26], new therapies [27–30], and in vivo imaging [31–36]. In case of medical applications using nanomaterials, nanoparticles can be introduced into the living organism either accidentally or intentionally through different routes (i.e., inhalation [37], swallowing [38], absorption through skin [39], releasing from different implants in living tissue [40]) as shown in Fig. 5.1. In the meantime, nanoparticles can be moved through different sites such as blood, or brain via the circulatory system [41]. In addition, it can pass through cell membranes in organisms through different

Figure 5.1 Pathways of exposure to nanoparticles and associated diseases as suggested by epidemiological, in vivo, and in vitro studies. The figure has been used with a copyright permission from Bazco [45].

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diffusion mechanisms and interact with cellular machinery [42–44]. Hence, it is very important to consider the nanomaterial as a new material which must be tested with complete toxicological evaluation even though its bulk form has a known studied toxicological data, which will be an important issue especially for those with carcinogenic potential.

2. TOXICITY TESTING AND RISK ASSESSMENT OF NANOMATERIALS The schematic for analysis and action with new materials is depicted in Fig. 5.2, following Handy and Shaw [46]. These procedures are indeed a complex process, and the exact details of assessment depend on the targeted application of the new material [46]. In the assessment process, the as-prepared nanomaterial undergoes several physical and chemical characterization tests in order to investigate their physicochemical properties (i.e., size, shape, optical properties, etc.), and a range of toxicity tests before carrying out the hazardous doses calculation [46]. Then, the next two stages in safety evaluation procedures are

Figure 5.2 A safety evaluation and assessment for the as-prepared nanomaterials.

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related to risk assessment and risk management of nanomaterials in order to generate preferred plans to deal with these new materials (i.e., safe handling, storage, etc.) depending on hazards, effects, and exposure information [46]. Hence, these steps have been translated into several safety documents such as certificate of analysis and material safety data sheet. In order to test the toxicity of the nanomaterials and their nanocomposites, there are several types of toxicological tests that must be used in the safety assessment of nanomaterials, include mammalian toxicity (i.e., acute and chronic, oral toxicity, dermal toxicity, skin irritation), tests for mutagenicity (mostly, in vitro tests using cancerous cells or bacteria as the “microbial test”), and ecotoxicity tests to enable some assessment of the risk to the environment (e.g., acute toxicity to fish, invertebrates, and algae) [46–48]. In medical applications, additional series of tests in the form of clinical trials and more in depth investigations of mammalian toxicity (i.e., metabolism, cellular uptake, and side effects investigation) will be carried out [49,50]. From the toxicological point of view, the dose versus response relationship is essential for any toxicological test. In this sense, there are many fundamental assumptions related to this role. One of these assumptions is focused on the concentration of the toxicant at the target is related to dose [46]. In highly ionic solutions, nanoparticles do not form colloidal solutions (i.e., aggregation and adsorption on the surface) [51]. Thus, the nanoparticles will initially be trapped into the mucous layer on epithelial surfaces rather than being absorbed into the cells [52]. This adds an extra uncertainty factor to risk calculations and requires invention of new tests for nanomaterials in order to satisfy the fundamentals of the dose–response relationship [53]. In order to achieve the risk characterization for any new substance, more information on the exposure concentration for these nanomaterials should be available. Also, it is not ethical to expose humans to nanomaterials to obtain toxicological data, but animal studies can be used instead. Even with simplified approaches to risk calculations for single substances such as [54]: Risk ¼ C  T

(5.1)

where C is concentration of the substance, T is from the slope of a dose– response curve, the problem of lack of measurement of environmental concentrations remains. Other approaches to risk calculation are also problematic. The hazard quotient (HQ) also relies on quantifying the exposure concentration [54]:

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HQ ¼ E=Rf ðdoseÞ

(5.2)

where E is exposure or intake, Rf(dose) is the reference dose. Moreover, the Rf(dose) is defined as an estimate of the daily exposure for humans that will result in no significant adverse health effect in the most sensitive part of the population [54].

3. TOXICITY EVALUATION Most chemicals are now subject to stringent criteria for safety testing before being marketed. This is especially true for pharmaceuticals, food additives, pesticides, and industrial chemicals. Knowledge of toxicity is primarily obtained in three approaches [55]: 1. By observation of people during normal use of a substance or from accidental exposures. 2. By experimental studies using animals. 3. By studies using cells (human, animal, plant). This chapter will describes work accomplished, using the second approach in which experimental animal studies are employed to obtain the genotoxicological evaluation of plasmonic (i.e., Au NPs) and hybrid nanocomposites such as magneto-plasmonic nanoalloys (i.e., Au–Co NCs).

4. ANIMAL TESTING FOR TOXICITY In order to use nanomaterials in biomedical applications, some toxicological tests including animal tests are performed prior to human clinical trials as part of the nonclinical laboratory tests. Animal test results are often the only means by which toxicity in humans can be effectively predicted. These tests provide many benefits such as indicating ease with which to control chemical exposure and environmental conditions, and virtual evaluation for any type of toxic effect. Finally, it is of course necessary to study the mode of action of different nanomaterials (i.e., toxicity mechanism) [55]. Some procedures for routine safety testing have been standardized. Standardized animal toxicity tests are highly effective in detecting toxicity that may occur in humans. Principal types of animal-based toxicity tests include [56]: 1. Acute toxicity 2. Subacute toxicity 3. Subchronic toxicity 4. Chronic toxicity

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5. Carcinogenicity 6. Reproductive toxicity 7. Developmental toxicity 8. Dermal toxicity 9. Ocular toxicity 10. Neurotoxicity 11. Geneotoxicity The selection of animal species may vary with the toxicity test. Rodents and rabbits are the most commonly used laboratory species due to their availability, low costs in breeding and housing, and past history in producing reliable results [57].

Acute toxicity tests Acute toxicity tests are generally the first tests conducted. They provide data on the relative toxicity likely to arise from a single or brief exposure. Standardized tests are available for oral, dermal, and inhalation exposures [55]. Subacute toxicity tests Subacute toxicity tests are employed to determine toxicity likely to arise from repeated exposures of several weeks. Standardized tests are available for oral, dermal, and inhalation exposures [55]. Subchronic toxicity tests Subchronic toxicity tests are used to determine toxicity likely to arise from repeated exposures of several weeks to several months. Standardized tests are available for oral, dermal, and inhalation exposures [55]. Chronic toxicity tests Chronic toxicity tests determine toxicity from exposure for a substantial portion of a subject’s life. They are similar to the subchronic tests except that they extend over a longer period of time and involve larger groups of animals [55].

5. FACTORS AFFECTING NANOTOXICOLOGY Nanotoxicology is the study of the toxicity of nanomaterials. Because of quantum size effects and large surface area to volume ratio, nanomaterials have unique properties compared with their larger counterparts [45,58]. Therefore, the assessment procedures for nanomaterials are different from the traditional toxicological evaluation usually followed with bulk materials. For nanotoxicology, new factors can induce adverse effects on the biological system such as nanoparticle size, shape, capping materials, target organ, animal model, and mode of administration. The size of nanomaterials and the animal model were found to be the most effective factors affecting the toxicological study and must be taken into consideration.

5.1. Effect of size, concentration, and surface area Once the particle size is reduced to nanoscale, the surface-to-volume ratio which determines the prospective number of reactive groups increases

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dramatically compared to the larger bulk counterparts. Enhanced activities that may occur could be either useful (e.g., carrier capacity for drugs, increased uptake, and interaction with biological tissues) or harmful (e.g., toxicity, instability, induction of oxidative stress) depending on the intended application [42,57]. Marilyn et al. [59] studied the risk assessments of nanomaterials. He reported that any toxic effects of nanoparticles will be very specific to the type of base material, size, substituent, and coatings. Hallock et al. [59] and Oberdorster et al. [60] reported that nanoparticles with size <100 nm, showed greater toxicity than fine particulates with size <2.5 μm of the same material on a mass basis. This has been observed with different types of nanoparticles, including TiO2, Al2O3, carbon black, cobalt, and nickel [59,60]. In addition, Warheit et al. [61] found that the toxicity for cytotoxic crystalline quartz not only depended on the particle size, but related to surface reactivity as measured by hemoglobin release from cells in vitro [42].

5.2. Effect of animal model Although the particle size and concentration can influence the toxicological assessment of the nanomaterials, the type of animal model plays an important role in toxicological study. Some models are more sensitive than others resulting in variation in the resultant adverse effects. For example, the rat model is known to be an extremely sensitive species for developing adverse lung responses to nanoparticles, particularly at overload concentrations. The tumor-related effects are unique to rats and have not been reported in other particle-exposed rodent species (i.e., mice or hamsters) under similar chronic conditions. For the mechanistic connection, it has been postulated that the particle-overload effects in rats result in the development of “exaggerated” lung responses, characterized by increased and persistent levels of pulmonary inflammation, cellular proliferation, and inflammatory-derived mutagenesis in the rat, and this ultimately results in the development of lung tumors following high-dose, long-term exposures to a variety of particulate types [62–64]. In contrast to the response in rats, evidence from numerous studies demonstrate that particle-exposed mice and hamsters do not develop sustained inflammation, mesenchymal cell alterations, and consequent lung tumors following high-dose, long-term exposures to low-toxicity dusts. Therefore, species differences in lung responses to inhaled particles are important

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considerations for assessing the health risks to nanoparticles. To complicate further our perceptions of nanoparticle toxicity, some recent evidence suggests that, on a mass basis, not all nanoparticle types are more toxic than microsized particles of similar chemical composition [62–64].

5.3. Purity Nanomaterial purity is also an important consideration as residual contaminating metals may actually be responsible for genotoxicological responses rather than the actual nanomaterial itself, the quantity of which are dependent upon the synthesis procedure employed. Although postproduction processing removes most of these metal catalysts, even purified nanomaterials may still contain up to 15% residual metal by mass. While researchers have made encouraging attempts to purify the nanomaterials under investigation in order to rule out the effects of impurities on the observed toxicity, often the damaging effects of the purification process have been overlooked [1].

5.4. Surface charge and chemistry The surface chemistry of nanomaterials is one of the most critical factors for providing more information regarding their behavior under different experimental conditions. Firstly, surface charge and chemistry will point out the formation of agglomerates depending on several factors such as the pH of the aqueous environment [65]. Hence, this information can be used to gain insight into these aggregation/disaggregation kinetics that may occur during the in vitro experiment, or according to the specific biological compartment in which the nanomaterials may become concentrated. Another factor, which plays a substantial role in cellular uptake of nanomaterials, is known as surface charge. According the cellular diffusion mechanism through the plasma cell membrane, which is negatively charged (i.e., due to the phospholipids on the outer surface), as is the intracellular environment, so that anionic nanomaterials may be endocytosed at a lower rate than those that are cationic. Although this has been observed in practice using PEGylated polylactide and hydrogel nanoparticles of similar sizes but Gratton et al. [66] and Harush-Frenkel et al. [67] show that at different charges [66,67], this is not always the rule and it does not preclude the uptake of negatively charged nanoparticles [68]. However, nanoparticles with cationic surface charges appear to be associated with greater cytotoxic responses as compared to those with anionic charges, although it is unclear as to whether

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the cell death is the direct result of the surface charge or if it is because of the increased uptake often associated with cationic nanoparticles [69]. Additionally, DNA is negatively charged; thus, cationic nanomaterials may be more likely to interact with the genetic material.

6. GENOTOXICOLOGY Genotoxicity is one toxicological manifestation; this branch is related to study the chemical and physical disorders or damage in nucleic acids (i.e., DNA and RNA) following exposure to endogenous or exogenous agents. This damage is characterized by formation of small lesions arising at very specific sites within the DNA strands (i.e., DNA adducts, DNA cross-links, and resultant mutations). Alternatively, gross abnormalities can arise at the chromosomal level including the alteration in chromosome copy number, fragmentation, and structural chromosomal rearrangements [70]. The type of genotoxic effect is entirely dependent upon its mechanism of action [71,72] as shown in Fig. 5.3. Primary mechanisms for genotoxicity. Primary mechanisms for genotoxicity are imparted by nanomaterials themselves at the level of the single cell. It requires cellular internalization of the nanomaterials and often subsequent interaction with biomolecules. This type of mechanism is divided into two subcategories (i.e., direct and indirect acting agent). Direct acting agents. They come into a direct contact with the genetic material causing physical or chemical damage. This might include the formation of DNA lesions at the specific sites within the DNA molecule, which lead to mutagenesis as they may destabilize the nucleotide they damage resulting in its loss and therefore an a basic site which is often subject to error prone repair leading, thus leading to strand breakages [71,72]. Direct DNA disorder can only be induced if the nanomaterials are able to penetrate the cell nucleus, or free in cytoplasm may have a chance to bound in a direct contact with DNA during mitosis (i.e., cell division) when the nuclear membrane breaks down [73,74]. Indirect acting agents. They induce genetic damage via intermediate biomolecules that are often components of the cell division cycle (Fig. 5.4) [75]. DNA replication and cell division are complex multifactorial processes that involve large number of proteins, which are good targets for indirect acting genotoxic substances [70]. Consequently, damage to such components often results in structural or numerical chromosomal aberrations. Aside from

Figure 5.3 Mechanism for nanomaterial-induced DNA damage. The figure has been used with a copyright permission from Elsevier Publishing. Ref. [70].

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Figure 5.4 Indirect mechanisms that can lead to genotoxicity. The figure has been used with a copyright permission from Elsevier Publishing. Ref. [1].

interfering with the DNA replication process, indirect DNA damage may arise as a result of the induction of oxidative stress [76,77]. Secondary mechanisms for genotoxicity. This refers to the ability of the genotoxins to induce an inflammatory response in vivo that results in excessive reactive oxygen species (ROS) generation by macrophages, and neutrophil cells recruited to the exposure site. This is a defense mechanism within the body to protect against invading pathogens—acute inflammation often enables the removal of foreign bodies by the immune cells, but if this fails due to bio-persistence then the result is chronic inflammation, promoting oxidative stress, which is responsible for surrounding cells damage within the tissue [70]. In this model, the nanomaterial itself may exhibits no genotoxic action within in vitro tests, but their physicochemical features may be make it to promote a chronic immune response in in vivo testing that, in turn generates ROS responsible for inducing genotoxicity within the tissue cells at the nanomaterial deposition site [76].

7. THE MOST COMMON GENOTOXICOLOGICAL TESTS 7.1. 8-Hydroxydeoxyguanosine DNA adducts 8-Hydroxydeoxyguanosine (8-OHdG) adducts are considered as one of the most common genotoxicological effects, which form due to the indirect

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mechanism induced by cellular uptake of nanoparticles that cause oxidative stress. Due to this oxidative stress, a new class of the reactive radicals known as ROS has been generated which has an ability to attack DNA and modify, most frequently, the guanine (G) base resulting in the formation of an 8-OHdG modification (known as a DNA adduct) as depicted in Fig. 5.5 [1]. This DNA adduct can lead to mutations in DNA when replication occurs as DNA polymerases do not recognize it as guanine. Therefore, 8-OHdG is an excellent biomarker to determine the extent of oxidative damage in our body. It will not show us which part of the body is damaged by free radical activity but it does enable us to evaluate our overall health status in relation to oxidative stress [1,77,78]. Various methods are available for the detection of the 8-OHdG DNA adduct both in vivo and in vitro. The most sensitive methods used for the detection of this DNA adduct are HPLC and mass spectrometry (GC– MS) based techniques, which are frequently utilized to screen body fluids for oxidative DNA damage. Alternative methods are antibody based techniques such as immunofluorescence, immunohistochemistry, or DNA dot blots which involve the detection of an antibody attached to the 8-OHdG adduct [1,79,80].

7.2. Micronucleus test A micronucleus (MN) test is a test used in toxicological screening for potential genotoxic compounds. The assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens. This test is based on the formation of number of MN in treated cells [80,81]. In this test, MN are formed during anaphase from chromosomal fragments or whole chromosomes that are left behind when the nucleus divides (Fig. 5.6). Over time, the assay has evolved to include a pretreatment with

Figure 5.5 Formation process of 8-OHdG adduct.

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Figure 5.6 Formation process of micronuclei (MN).

cytochalasin-B (Cyt-B), a cytokinesis blocking agent that inhibits cell division, thereby giving the cells a binucleated appearance. This enables more accurate scoring and the ability to sieve out the dividing cells from the nondividing ones, thereby reducing the incidence of false positives [80].

8. GENOTOXICITY OF PLASMONIC NANOMATERIALS In this section, we review the literature to date that has alluded to the ability of plasmonic nanomaterials to induce DNA damage. All appropriate studies identified are as in following section.

8.1. Gold nanoparticles Gold nanoparticles (Au NPs) (i.e., 20 nm) display unique optical and catalytic activity properties compared to the bulk phase [82]. These nanoparticles often become internalized by endocytosis and by 24 h are enclosed in lysosomal bodies arranged around the perinuclear region [83]. However, when the particle size decrease to less than 20 nm (i.e., 3–8 nm), the particles did not show any cytotoxic or immunogenic effects

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and reduce ROS and reactive nitrogen species (RNS) in a time-dependent (4 h) and dose-dependent (100 mM) manner in macrophage cells as reported by Shukla et al. [83] Another evidence has been reported by Kataoka et al. in which the antioxidant activity of gold nanoparticles may be due to the ability of gold ions (i.e., Au1+) to inhibit the DNA-binding activity of AP-1 and NF-κB transcription factors thus in turn downregulating the expression of proinflammatory cytokines, which are involved in the generation of ROS and RNS [84]. However, in contrast, a recent study has been carried out by Li et al. using gold nanoparticles (i.e., Au NP) with average size of 20 nm on embryonic lung fibroblasts indicated a significant oxidative DNA damage due to 8-OHdG adducts, at concentrations as low as 25 mg/mL Au NP [84]. This was accompanied by decreased expression of DNA repair genes and the cell cycle checkpoint genes MAD2, cyclin B1, and cyclin B2, which is of concern as lowering the cellular DNA damage response pathways could promote genetic instability, particularly if the cells are subject to further insults. It therefore appears that despite the inert nature of gold, Au NPs are capable of inducing DNA damage indirectly through an oxidative stress response, albeit in a cell type, or size-dependent manner. Of importance, was the fact that regardless of the underlying genetic damage and transcription alterations observed by Li et al. in the Au NP treated cells, no cytotoxicity was observed in this study [85]. In vivo genotoxicity, however, has been investigated only once. Schulz et al. reported that a single dose (i.e., 18 μg) of Au NPs was not genotoxic in rats—as assessed by the comet assay and the MN test. Genotoxicity could not be identified after the treatment with three different particle sizes: 200, 20, and 2 nm [86].

9. EVALUATION OF GENOTOXICITY OF MAGNETO-PLASMONIC HYBRID NANOCOMPOSITES In this section, an overview of the achieved work which was done using experimental animal studies to obtain the genotoxicological evaluation of hybrid nanomaterials such as magneto-plasmonic nanoalloys (i.e., Au–Co NCs) [87].

9.1. Genotoxicity of the Au–Co NPs In this section, we will give an overview of genotoxic investigation for pure Au and Au–Co NPs, if they could: (a) alter the expression of

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tumor-initiating genes, (b) induce MNs formation, (c) induce genomic damage caused by oxidative stress (8-OHdG), and (d) inhibit the activity of antioxidant enzymes in male mice. To investigate the influence of these nanoparticles characteristics on the toxicity, the morphology (size and shape) of the particles used within different media was determined prior to performing the toxicity studies [87]. 9.1.1 Gene expression The tumor-initiating (CYP3A, p53, and p27) and antioxidant (GST) genes in liver tissues of male mice after treatment with gold (Au) and gold–cobalt (Au–Co) nanoparticles have been investigated as shown in Figs. 5.7–5.10. In case of tumor initiating (CYP3A, p53, and p27), a significant increase in the hepatic mRNA level, and the expression of CYP3A and p27 gene in mice has been observed due to the exposure to the medium and highest doses of gold–cobalt (Au–Co) NPs (i.e., 160 and 320 mg/kg bw) for 14 days as depicted in Fig. 5.7 and 5.9, respectively. Furthermore, at the highest dose of Au–Co NPs (i.e., 320 mg/kg bw) for 7 and 14 days, there is a significant increase the expression level of p53 gene as shown in Fig. 5.8. While at the lowest dose of Au–Co NPs, there is no any significant alteration or increase in the expression levels of CYP3A, p53, and p27 genes at both time intervals as shown in Figs. 5.7–5.9 [87]. On the other hand, in case of exposure to

Relative expression of CYP3A gene

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 7 days

Control 0.65

CP 1.52

Au/Co80 0.81

14 days

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1.51

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Au/Co160 Au/Co320 0.98 1.09 1.24

1.33

Au80 0.71

Au160 0.85

Au320 0.98

0.87

0.97

1.09

Nanoparticles treatment

Figure 5.7 Expression of CYP3A gene in the liver of mice exposed to Au–Co and Au NPs. Adapted with permission from Ref. [87], Copyright (2012) American Chemical Society.

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Control

CP

7 days

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1.42

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0.99

1.27

Au80

Au160

Au320

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0.82

0.93

0.77

0.93

1.09

Nanoparticles treatment

Relative expression of P27 gene

Figure 5.8 Expression of p53 gene in the liver of mice exposed to Au–Co and Au NPs. Adapted with permission from Ref. [87], Copyright (2012) American Chemical Society.

1.4 1.2 1 0.8 0.6 0.4 0.2 0

Control

CP

7 days

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14 days

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Au/Co80 Au/Co16 Au/Co32 0 0 0.68 0.78 0.99 0.72

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Au80

Au160

Au320

0.65

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0.82

0.73

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Nanoparticles treatment

Figure 5.9 Expression of p27 gene in the liver of mice exposed to Au–Co and Au NPs. Adapted with permission from Ref. [87], Copyright (2012) American Chemical Society.

gold (Au) NPs there is not any significant alteration in the expression levels of CYP3A, p53, and p27 genes at low and higher doses at both time intervals. While at the highest dose, it is clear that a significant increase in the expression levels of CYP3A, p53, and p27 genes occurs especially at the 14th day of treatment [87].

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Relative expression of GSTP1 gene

1.4 1.2 1 0.8 0.6 0.4 0.2 0

Control

CP

7 days

1.2

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Au/Co80 Au/Co16 Au/Co32 0 0 1.1 0.82 0.73 0.84

0.65

0.57

Au80

Au160

Au320

1.15

0.99

0.81

1.1

0.81

0.67

Nanoparticles treatment

Figure 5.10 Expression of GST gene in the liver of mice exposed to Au–Co and Au NPs. Adapted with permission from Ref. [87], Copyright (2012) American Chemical Society.

In the case of the antioxidant gene (GST), the expression level upon the exposure to Au and Au–Co NPs is illustrated in Fig. 5.10. It is clear that a significant decrease in the GST expression has been obtained upon the treatment for 14 days with Au–Co NPs with medium and highest doses (i.e., 160 and 320 mg/kg bw). In contrast, there is not any significant alteration in the expression level when the mice were exposed to low or medium doses after 7 days of treatment as shown in Fig. 5.10 [87]. On the other hand, in the same trend at the highest dose (320 mg/kg bw) with Au NPs, there is a significant decrease in the expression level of GST gene that occurred after 14 days of treatment as shown in Fig. 5.10 [87]. While exposure to low and medium doses after 7 and 14 days of treatment results in no significant decrease of the expression levels (Fig. 5.10) [87]. 9.1.2 Micronucleus assay Figure 5.11 demonstrates the influence of exposure with Au–Co and Au NPs on micronuleated polychromatic erythrocytes (MnPCEs) formation in the bone marrow cells of male mice. As mentioned earlier, the MN test is one of the powerful tests that has been used in toxicological screening for potential genotoxic compounds and carcinogens. It is clear that, as shown in

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25 20 15 10 5 0

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CP

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Au/Co3 20 12.6

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Au160

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Au320

7.1

7.8

9.8

20.4

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10.7

13.9

7.9

8.1

11.1

Nanoparticles treatment

Figure 5.11 Micronucleated polychromatic erythrocytes (MnPCEs) of male mice exposed to Au–Co and Au NPs at 7 and 14 days. Adapted with permission from Ref. [87], Copyright (2012) American Chemical Society.

the illustrated results, a significant increase in incidence of MnPCEs has been obtained upon the exposure to medium and highest doses compared with control group at both 7 and 14 days of treatment [87]. In contrast, there is not any significant alteration in the incidence of MnPCEs for both treatment time intervals at the low dose of exposure [87]. On the other hand, a significant increase in the incidence of MnPCEs formation has been obtained only at the highest dose of Au NPs at both time intervals of treatment (i.e., 7 and 14 days) in comparison with control group. While with low and medium dose of exposure using Au NPs, it is clear that there is not any change in the MnPCEs formation at both time intervals without significant differences (Fig. 5.11) [87]. 9.1.3 Determination of glutathione peroxidase activity Determination of glutathione peroxidase (GPx) activity is considered as another genotoxic test has been investigated in our study. GPx is an enzyme family with peroxidase activity, which have an important role in protection of the organism from oxidative damage. The biochemical function of GPx is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water [88]. As demonstrated in Fig. 5.12, it is clear that at medium and highest doses upon exposure to Au–Co NPs after 7 and 14 days of treatment, a significant low levels of the GPx activity exists compared to control group (Table 5.1). One the other hand, upon exposure to Au NPs with the highest dose at both time intervals indicates the same trend as in Au–Co NPs a significant low GPx activity level compared to control group (Fig. 5.12 and Table 5.1).

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6

GPx activity

5 4 3

7 days 14 days

2 1

Au -3 20

Au -1 60

Au -8 0

Au -C o16 0 Au -C o32 0

o80 -C Au

C on C yc tro lo l ph os ph am id e

0

Nanoparticles treatment

Figure 5.12 Glutathione peroxidase (GPx) activity of male mice exposed to Au–Co and Au NPs at 7 and 14 days. Table 5.1 The glutathione peroxidase (GPx) activity in mice upon exposure to Au–Co and Au NPs (mg/kg) at 7 and 14 days of treatment GPx activity (U/mg tissues/min) Treatment*

7 days

14 days

Control

5.7  0.03

5.5  0.03

Cyclophosphamide

1.6  0.10

1.4  0.10

Au–Co-80

5.2  0.14

4.1  0.14

Au–Co-160

3.7  0.13

3.0  0.13

Au–Co-320

3.2  0.11

2.8  0.11

Au-80

5.4  0.12

4.5  0.12

Au-160

4.6  0.11

4.3  0.11

Au-320

4.0  0.12

3.2  0.12

However, at low and medium doses at both time intervals, there was no significant alteration in levels of enzyme activity with respect to the control group (Fig. 5.12 and Table 5.1) [87]. 9.1.4 Generation of 8-hydroxy-2-deoxyguanosine Also in our pervious published studies, the 8-OHdG generation in hepatic mice genome after Au-, Au–Co NPs treatment as an alternative for

193

Generation of 8-OHdG adduct

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16 14 12 10 8 6 4 2 0

Control

CP

Au/Co8 0

Au/Co1 60

Au/Co3 20

Au80

Au160

Au320

7 Days

3.6

14.1

3.7

5.4

7.8

3.5

3.8

4.9

14 Days

3.5

14.2

5.4

10.7

12.3

3.9

5.2

9.1

Nanoparticles treatment

Figure 5.13 Generation of 8-OHdG adduct in hepatic mice genome following Au and Au–Co NPs at 7 and 14 days. Adapted with permission from Ref. [87], Copyright (2012) American Chemical Society.

oxidative stress induced damage has been depicted in Fig. 5.13. It is clear that 8-OHdG levels in control liver tissues were ranged between 3.6 8-OHdG per 105 dG and 3.5 8-OHdG per 105 dG. In the case of treatment with Au NPs, it is clear that, at low dose of treatment at 7 and 14 days was relatively similar to that of the control group. While at the medium dose of Au NPs, the 8-OHdG/2-dG generation ratio has been increased by 1.5-fold after 14 days of treatment compared to that of the control group. In addition, this ratio increased from 1.4-fold at 7 days to 2.6-fold at 14 days of treatment (Fig. 5.13) [87]. In the same trend, the ratio of 8-OHdG/2-dG generation increased slightly after treatment at a low dose of Au–Co NPs treatment for 7 and 14 days compared with that in the control group. However, this has been increased to 1.5- and 2.2-fold at 7 days to 3.1- and 3.6-fold at 14 days following medium and high doses of Au–Co NPs treatment, respectively (Fig. 5.13) [87]. These results were in agreement with the results which were obtained by Balasubramanian et al. [88]. They reported that that several genes such as those of the cytochrome P450 family (i.e., CYP4a and CYP3) were significantly upregulated upon treatment with Au NPs treatment leading to increase in their expressions [88]. According to all of these findings, it is clear that upon the exposure of mice to Au nanoparticles, the cytochrome 450s are localized in the inner mitochondrial membrane or endoplasmic

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reticulum of cells, which will catalyze multiple reactions, which accounts for their central importance in metabolizing and detoxification of extremely large number of endogenous and exogenous molecules as which found in liver (i.e., metabolic products, drugs, and toxic compounds) which play direct or indirect role in alteration of the metabolic genes/pathways existing in those tissues [88]. In order to monitor the gene expression changes of the genes involved in cell cycle regulation in relation to Au NPs treatment, Cho et al. [89] investigated the gene expression profiles in mouse liver after a single intravenous injection of PEG-coated Au NPs. Their results indicate that the commonly expressed genes were categorized as cell cycle, apoptosis, inflammation, and metabolic process. These findings are consistent with our results which found upregulation of cell cycle regulation genes (p53 and p27) in liver tissues exposed to Au and Au–Co NPs [89]. These changes in the gene expression could be explained due to the fact that Au NPs cause alterations in the gene expression in liver tissues, which is cited in numerous studies [88–91]. It has been reported that injection of different sizes of Au NPs in rats resulted in accumulation mainly in the liver [88–90]. In addition, Garnett and Kallinteri [91] reported that organs of the reticuloendothelial system (RES) including the liver and spleen could efficiently accumulate NPs via opsonization, that is, NPs could bind to antibodies in the plasma and are subsequently recognized by the phagocyte-rich RES. This may explain the persistent accumulation of Au NPs in hepatic cells that cause gene expression changes. In the case of Au–Co nanoalloys, these particles were able to induce significant alterations in the tumor-initiating genes associated with an increase of MNs formation and a reduction in the GPx activity more than pure gold nanoparticles themselves. Despite that Au NPs are recognized by Cho et al. [92] as being nontoxic, some reports on their toxicity indicates that the toxicity depends on the particle size, shape, coating material, bioactivity, surface chemistry of the nanoparticles. The fact that Au–Co nanoalloy induces more toxicity than pure Au NPs is attribute to the presence of cobalt in the alloy. A previous study reported that in oral administration of nanoparticles, they tend to agglomerate when suspended in a low pH liquid producing an alteration of their surface ionic composition [93]. This might cause the nanoparticles to separate from their agglomerates and produce an increased surface area, potentially increasing the generation of a cellular ROS. In the current study, Au–Co NPs were exposed to low pH because they were

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administered orally to the male mice and then passed to the gastric tract. As a result, Au–Co NPs may be degraded, and their coating molecules are damaged between 7 and 14 days of treatment. Accordingly, it may be assumed that induction of MnPCEs formation, DNA damage, and generation of high amounts of 8-OHdG were most likely caused by the alteration of Au–Co NPs surface ionic composition and/or high intracellular ROS generation. These results are in agreement with those of Colognato et al. [94], who demonstrated that Co NPs were capable of inducing clastogenicity in human peripheral blood leukocytes. This was also accompanied with a reduction in cell viability. Additionally, cobalt ions were able to produce single strand breaks, chromosomal aberrations, sister-chromatid exchanges in human MCF-7 cells [95,96]. Also, Zhu et al. [97] reported that cobalt chloride was able to induce apoptosis of RGC-5 cells and increase the expression of tumor-initiating P53 gene. A key mechanism that is proposed to be responsible for the genetic alterations exerted by Co NPs involves oxidative stress. This refers to a redox imbalance within cells usually as a result of increased intracellular ROS and decreased antioxidants. Another explanation for the effect of Co NPs toxicity is due to the release of Co2+ ions from the Co NPs [98]. These ions increase ROS release with the potential enhanced generation of 8-OHdG induced DNA damage and alterations in the gene expression. In addition, Singh et al. [1] demonstrated that Co2+ ions released from certain nanoparticles can cause the conversion of cellular oxygen metabolic products such as H2O2 and superoxide anions to OH radical. Hydroxyl radical represents one of the primary DNA damaging species, which can cause Thymine–Tyrosine (DNA–histone protein) cross-links in chromatin [99]. Accordingly, free metals ions can result in OH-induced purine and pyrimidine modifications [100]. Similarly, Au–Co NPs inhibit the antioxidant activity of the GPx enzyme and downregulate the expression of GST gene. Although these findings show that Au NPs are less toxic than Au–Co NPs, the highest dose of gold nanoparticles was able to induce toxicity and increase the expression changes of CYP3A, p53, p27 genes, MNs formation, and generation of DNA adducts. These results are consistent with several previous studies in which it is reported that neutral Au NPs caused DNA damage and increase in both nuclear and cytoplasmic p53 expression in a human keratinocyte cell line (HaCaT) [101,102]. Numerous studies attempted to explain the regulation of gene/enzyme changes and genetic toxicity including micronuclei formation in relation to Au NPs treatment. Johnston et al. [103] reported that Au NPs to bind to DNA, which may be exploited within the treatment of diseases, but may

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also contribute to genotoxicity, or affect gene expression (block transcription). Molecular simulations and TEM analyses were used to determine the ability of Au NPs to interact with DNA molecules [104]. Tsoli et al. [105] found that Au NPs were able to traverse the plasma membrane with subsequent subcellular distribution. It was revealed that Au NPs were distributed within the cytoplasm and nucleus, where they were capable of binding to DNA. This finding therefore not only confirmed that NPs were strongly bound to DNA but also demonstrated their cytotoxic potential. In addition, Goodman et al. [106] illustrated the ability of Au NPs to bind to DNA, which caused a conformational change within the structure of DNA. This was suggested to impact on DNA transcription, and this was exemplified by the finding that RNA polymerase activity was inhibited with subsequent enzyme level changes. The interaction of Au NPs with serum proteins is likely to enable their transportation within the body. In addition, interactions with proteins may have detrimental consequences for particle behavior or normal protein structure and function, and therefore impact on normal cell function, especially antioxidant defense. Furthermore, the ability of Au NPs to interrupt transcription and translation is of concern. On the other hand, hepatic cells are able to detoxify a variety of substances that engage in ROS formation [107] and also have potential defense mechanisms, including intracellular antioxidants and antioxidant enzymes such as GPx, superoxide dismutase, and catalase [108]. These observations are in agreement with the current results, where mouse hepatic cells exposed to Au NPs under the present study could not decrease the level of the antioxidant GPx enzyme except at the highest dose of Au NPs (320 mg/kg bw). Consequently, the ROS levels caused by Au NPs exposure concentrations used in this study were not high enough to overcome antioxidant defense mechanisms and so cause immediate gene expression alterations and MNs formation. Further experiments are needed to determine the long-term effects, such as DNA damage, that are known to be caused by elevated ROS production [108].

10. CONCLUSION In conclusion, the genotoxicological evaluation of gold nanoparticles and their hybrid nanocomposite such as magneto-plasmonic nanoalloys (i.e., Au–Co NPs) has been explored. In case of the genotoxic effects of Au and Au–Co NPs, we report that the Au–Co alloy NPs caused more alterations in the gene expression, DNA damage (MNs), and DNA adduct than

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Au NPs. These influences were accompanied by a reduction in the antioxidant defense and may consequently cause increases in ROS formation which induce gene expression alterations and genetic toxicity. However, Au NPs could not induce this toxicity on the DNA and administration was accompanied by a level of the antioxidant GPx enzyme similar to the control group except at the highest dose. Therefore, it would appear that ROS levels caused by Au NPs exposure were not high enough to overcome antioxidant defense mechanisms. These results might be useful for designing NPs for biomedical applications such as cancer therapy and imaging, drug delivery, and smart targeting.

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