Biomedical Research

Abstract

The rapid emergence of gold nanoparticles (AuNPs) technology holds great promise for future applications due to their large volume specific surface areas with high diverse surface activities than bulk gold. These properties have made AuNPs of great importance in the development of excellent nanoelectronic chips, promising vehicle for a wide range of biomedical and environmental applications. However, the huge impact arising from the physiochemical properties has given rise to new concerns for future health status. Currently, there is dearth information on AuNPs health effects and no regulatory safety and guidelines relating their properties to toxicities. This review, therefore, focuses on the potential toxicological aspect of AuNPs experienced so far and their interactions with biological systems. These can be applied as measures to improve their biomedical applications and risk assessment. However, assessing the safety issues of nanoparticles is quite challenging, because of the vast physiochemical properties that confound their biomedical and toxicological profiles. Therefore more research with standardized NPs physicochemical properties is needed based on the different types of AuNPs to establish both in vitro and in vivo nanotoxicities. The establishment of each size with specific ligand properties will update the complex conflicting ideas emanating from the different AuNPs safety studies thereof.

Keywords

Gold Nanoparticles, Physiochemical Properties, Cells interaction, Toxicity

Introduction

Nanotechnology is the study of matter at the atomic molecular level with attention focused from 1 to 100 nm diameter nanoscale size [1-3], Other scientist envisage it in terms of its volume specific surface area (VSSA) greater than 60 m²/cm³, reflecting the critical importance of surface reactivity of nanomaterials rather than size [4]. Nanomaterials especially engineered gold nanomaterials, hold great promises for future applications due to it large VSSA thereby amplifying their electrical, chemical, mechanical, thermal and optical properties [5-6] that differ from bulk gold. Bulk gold is considered bio-inert, a property found only at the macroscopic level, but at nanoscale size, gold exhibit different properties due to its surface plasmon resonance excitation characteristics [7- 9]. Gold nanomaterials are currently used to enhance solar cells [10] and as liquid crystal that acts as flash memory devices [11]. They also have extensive potential biomedical applications in drug delivery, gene therapy, photothermal and radio-therapy, biosensing as well as contrast agents for cancer, diagnostic tracers, immobilization of enzymes and cell imaging [1,6,12-15]. Other uses include water and hydrogen purification, pollution control and as catalysts in carbon monoxide oxidation [16-19]. However, despite their huge potential benefits in the realm of environmental, biomedical and industrial applications, very little is known about the short and long term health effects in organisms and the environment. Reports show that synthesized NPs can circulate in the body for extended periods of time without being rejected by the body's immune system. All these behaviours are guided by the small size, shape and surface charges. This is of concern because during syntheses and applications, gold nanoparticles (AuNPs) of various sizes, shapes and surface charges are generated that may be of health risk. Currently, there are very limited data and no safety and regulatory guidelines concerning the manufacture and application of nanomaterials. This review, therefore, focuses on the properties of gold nanomaterials and their interactions with biological systems. This will provide information on how AuNPs physicochemical properties, including those of attached functional groups influence cellular responses both in vitro and in vivo.

Literature Search Information

Data for the current study were obtained from various indexing journal sites such as PubMed, Medline, Embase, Global Health, SCOPUS/Elsevier, Web of Science, Springer, Langmuir, Google Scholar, Scientific and peerreviewed reports, conference proceedings published in English. With the search terms as “gold nanoparticles, production, synthesis, biomimetic synthesis of gold nanoparticles, toxicity, uses of gold nanoparticles, functionalization of gold nanoparticles, types and shapes of gold nanoparticles, toxicity of spherical and rod nanoparticles, toxicity of biomaterials of gold nanoparticles, gold nanoparticles ligands, effect of gold nanoparticle aspect ratio on its toxicity’ toxicity of chemically synthesized gold nanoparticle, biologically synthesized gold nanoparticles, effect of gold nanoparticles on cells. Others include toxicity of gold nanoparticles biconjugates, bioaccumulation of aggregation and agglomeration of gold nanoparticles, cellular toxicity of gold nanoparticles’

Synthesis of Gold nanoparticles

In general, AuNPs are synthesized by the chemical reduction of chloroauric acid (HAuCl4) using various reducing agents [20-25]. The reduction process causes Au3+ to be reduced to neutral gold atoms which further become supersaturated and precipitated as more gold atoms aggregate to form sub-nanogold particles [19]. There are several methods involved in the syntheses of AuNPs, with HAuCl4 as the main source of the gold atoms [22]. These methods include the following modifications (1) The Turkevich method which produces monodispersed spherical AuNPs suspended in water with citrate ions acting as both reducing and capping agents [26]. (2) The Brust method that produces AuNPs in organic liquids which are normally not miscible with water [27]. (3) The Perrault method which uses hydroquinone to reduce HAuCl4 in an aqueous solution to produce AuNP seeds [28]. (4) The Martin method which generates “naked” monodisperse AuNPs in water due to the reduction of HAuCl4 by sodium boron tetrathydride (NaBH4) [29]. (5) The Sonolysis process that produces AuNPs based on ultrasounds, reacting in an aqueous solution of HAuCl4 in glucose using hydroxyl and sugar pyrolysis radicals as reducing agents [30]. (6) Other friendly and cheaper methods of AuNPs synthesis include the use of biological agents such as microbial enzymes, plant phytochemicals or microorganisms such as bacteria and yeast cells [31-32]. For example chickpea leaf reduces 0.1mM HAuCl4 solution to AuNPs at room temperature as well as capping the AuNPs from aggregating [33]. Escherichia coli K12 have a fantastical behaviour to biosynthesis AuNPs at room temperature without the addition of growth media, pH adjustments or the inclusion of electron donors and stabilizing agents [34]. This biomimetic processes have revolutionized the nanotechnology field entirely.

The role played by microbial systems in AuNPs synthesis is vital because of their natural ability and mechanism to detoxified metallic gold ions through the reduction process either extracellularly or intracellularly as opposed to chemical synthesis of AuNPs[34-35]. Therefore are more environmentally friendly to exposed gold nanomaterial than chemical synthesis [34,36]. However, microbial and biological synthesis suffers from poor mono-dispersity, random aggregation, non-uniform shapes scale up as compared to chemical synthesis. [36]. Therefore production efficiency and specificity of AuNPs using biological processes is poor and improvement in the design and production of AuNPs by biomimetic technique is needed.

Currently, these methods are being modified to produce AuNPs of various sizes using various reducing agents [22,31,34,37]. The synthesized AuNPs differ in several forms with some existing as branched nanocrystals of varying shapes i.e. monopod, bipod, tripod or tetrapod structures [38]. After production, these different forms are stabilized from aggregation and agglomeration with organic ligands such as peptides, proteins, fungus [39] and polymers such as polyethylene glycol (PEG) [23,40,41]. Furthermore, these different forms of ligands stabilized AuNPs can be modified by attaching other functional groups based on the application of choice [20,40].

The size of AuNPs in terms of the proportion of width to height that is the aspect ratio (AR) in relation to toxicity is still debatable. Different AR of AuNPs gives different plasmon bands and wavelengths that equally exhibit different colours [42-43]. For example the study by Zhang et al [44] showed that various encapsulated AuNPs surface charges, sizes and shapes to human HEp-2 and canine MDCK cells exhibit different cytotoxic effects. However, the differences were exhibited by the shapes, where CTAB encapsulated gold nanorods (AuNRs) were relatively higher in cytotoxicity than citrate stabilized gold nano-spheres. Within the AuNRs there was no significant difference between the AuNRs different ARs [44] but increasing AR of AuNPs are difficult in cells uptake than those with lower AR [44-46]. Currently the clearance findings of AuNPs AR by phagocytic cells are still under investigation because of the encompassed complexity exhibited the physiochemical properties of both the AuNPs and their bio-conjugates cellularly. Studies by Qiu et al [47] have shown that cellular uptake is highly dependent on the AR and functional groups attached because ligands such as CTAB can equally enter cells with or without AuNPs, destroy mitochondria, and induce apoptosis [47]. The cationic PDDAC-coated AuNPs with an AR of 4 have been shown to possess both an insignificant toxicity with high cellular uptake, showing excellent photothermal therapeutic properties [47]. The method of AuNPs synthesis also affects toxicity [42-43], however AR in terms of cellular toxicity should be explained with caution because of the dearth information available in terms of AuNPs toxicological considerations. Therefore more systematic AuNPS toxicity studies are essential to decide the role of AR properties relative to cellular responses.

Routes of exposures to Gold Nanoparticles

Exposure to AuNPs can occur during development, synthesis, and applications by direct injection or ingestion into the system, and waste disposal [48-49]. Exposure can also arise from AuNP-composite bound to consumer products in markets, homes and outdoor activities [50]. Such exposes can account for their accumulation in the soil, water bodies and environment. Other main potential routes of exposure include inhalation, absorption through skin contact and release from implants [49,51-52]. Furthermore, the approval of AuNPs for various biomedical applications by the Food and Drug Administration (FDA) has led to increased applications as drug carriers, cancer therapy and biological applications [51,53-55].

Other mode of AuNPs exposures include airborne and surface materials adherence which sometimes are difficult to detect. They can therefore, persist and bioaccumulate in such environment making them readily to translocate into the food chain thereby influencing both biotic and abiotic processes [56]. This enhances the uptake of AuNPs by other environmental organisms such as algae and fish which can further be consumed by animals and humans.

The effect of size and shape of Gold nanoparticles toxicity

Synthesized AuNPs come in a variety of sizes and shapes ranging from 1 nm to 500 nm: some as rods, spheres, tubes, wires, ribbons, plate, cubic, hexagonal, triangular, tetrapods, etc [38,57]. The small size and their ‘needlelike’ penetrating ability into cells have also made AuNPs excellent carriers in biomedical and molecular biology techniques [58]. This needle like feature as reported by De Jong et al [53] have ease the absorption, penetration, circulation and distribution of AuNPs in bio-systems as a size dependent factor. These findings were similar to those earlier reported by Connor et al [59] who found that AuNPs of approximately 18 nm in diameter could penetrate the cells without cell injury and toxicity. A study by Tsoli et al [60] also demonstrated that AuNPs of approximately 1 nm in diameter could penetrate the cell and nuclear membranes and attach to DNA without cell injury and cell death. The mechanism of entry into cells without cell injury has not been elucidated, but it seems the small nanosize plays a major role. The small size of the AuNPs therefore, facilitates their incorporation into biological systems for subsequent probing and modification [61]. These unique features of AuNPs have led them to various chemical properties transducing into dissimilar cellular studies where some are reported either as toxic or non toxic. Some display size dependent toxicity due to the presence of coated surface ligands [5,62-63], while others because of their large surface area to volume ratio provide platforms for increase surface particle activity [54]. This therefore, contributes an easy flexible pathway of penetration and reactivity in biological system than bulk gold material.

In terms of size, De Jong et al [53] found that 10 nm AuNP when administered to experimental animals can circulate more within 24h than other sizes. The mechanism of this 10 nm AuNP widespread has not been elucidated. Apart from the fact that AuNPs circulations in the system are highly size dependent, earlier findings by Hauck et al [64] also showed that other sizes such 50 nm AuNPs when exposed within 30 min can be the most abundant cellular AuNPs in the a system.

The interactions of AuNPs with biological systems are often related to their physiochemical characteristics which enable them to be internalized within cells, a situation which is not possible for larger particles. This is one of the reasons why AuNPs may be toxic than larger particles when compared on a mass dosage. This emphasizes lies in the importance of their dimension, the large surface area to volume ratio which enables them applicable in biomedical systems [65,66].

Other studies of size-dependent cytotoxicity have been demonstrated in triphenylphosphine stabilised AuNPs using four cell lines such as tissue fibroblasts (L929), epithelial cells (HeLa), macrophages (J774A1) and melanoma cells (SK-Mel-28) [67]. Data obtained from these studies shown that cellular response is size dependent. For example 1.4 nm AuNP was observed as the most toxic responsible for rapid cell death by necrosis [67] as compare to 15 nm which was shown to be non-toxic [68]. This suggests that “larger” NPs are non-toxic in vitro. Furthermore, other in vitro studies on AuNPs of 20 and 100 nm in diameters have been shown to have no apparent effect on viability of human retina microvascular endothelial cells [69]. Although some studies have shown AuNPs not having an effect on cell viability, it is important to note that genotoxicity can occur without cytotoxicity and may result in genetic damage and transcription alterations which are not phenotypically expressed. A study on the effect of 5nm to 20 nm AuNPs on MRC-5 human fetal lung fibroblast cells have showed no influence on the viability of MRC-5 treated cells [70- 71]. However, cell proliferation was inhibited which was linked to downregulation of cell cycle genes. More so, oxidative DNA damage has been observed in conjunction with a downregulation of DNA repairs [71]. Furthermore, other reports have revealed that AuNPs of 2-4 nm, 5-7 nm and 20-40 nm are non-toxic to MRC-5 cells however when they were ≥ 10 ppm induced apoptosis and up-regulated the expressions of pro-inflammatory genes interlukin-1 (IL-1), interlukin-6 (IL-6) and tumor necrosis factor (TNF-alpha) [72]. Table 1 summaries some AuNPs in vitro toxicity studies that examined size, type of cell culture and their biological effects.

Table 1: Summary of some selected in vitro gold nanoparticles cytotoxicity studies.

The effect of AuNPs shows that the smaller the AuNP the higher the probability of it to cause toxicity as well as bind easily on cellular surfaces. For example 1.4 nm AuNP in diameter was found to bind with DNA and affect genes (mutation) as comparable to their larger ccounterparts.

Some studies have shown that AuNPs between 30 and 110 nm when exposed to rats for up to 15 days, can accumulate in the lungs, olfactory bulb, spleen, oesophagus, tongue, kidney, aorta, heart, septum and blood. This was quantified by means of inductively coupled plasma mass spectrometry (ICP-MS) [85]. The results obtained so far were similar to those earlier reported by Takenaka et al [86] where engineered gold nanomaterials of 5-8 nm were found retained in rat’s lungs before translocating into other tissues. These were also in agreement with those reported by Lasagna-Reeves et al [87] who used ICP-MS and GF-AAS to determine a significant amount of AuNPs in the liver, blood, brain, kidney, spleen, and lungs. Sadauskas et al [88] also demonstrated the amount of 2, 40 and 100 nm AuNPs in mice with similar tools with the liver as site of huge bioaccumulation, with only a small fraction translocating into the blood circulation and macrophage endosomes. The 2 nm AuNPs were further found to be the most translocated particles within the liver cells. The in vivo behavioural activities of these particles were due to their large surface area per unit mass. This shows that AuNPs bio-distribution vary with different sizes due to the availability of atoms ready to take part in various chemical reactions.

The mechanisms of biodistribution of AuNPs so far described are via endocytotic-exocytotic activity and to a lesser extend by paracellular transport (transport of molecules around cells and via tight junctions of epithelial cells) [88-89]. Such mechanisms are due to differences in the surface properties of the AuNPs, the type of animals used and the route of exposures. Table 2 summaries some of the in vivo AuNPs toxicity studies which examine the size, route of exposure and biological effect.

Table 2: Summary of some selected in vivo gold nanoparticles toxicity studies.

Gosens et al [93] believes that single AuNPs can pose greater health effects than their agglomerates and aggregates counter parts. Because of the agglomerates and aggregates relative larger sizes, they are restricted from translocating easily across membranes as compared to single nanogold particles. Although, when Gosens et al [93] intratracheally instilled AuNPs agglomerates and spherical single dose of 1.6 mg/kg AuNPs (50 nm or 250 nm) into rat lungs, both particles gave mild pulmonary inflammation at the same dosage. Meanwhile, earlier reports by Mühlfeld et al [89] and Sadauskas et al [88] showed that when AuNPs are inhaled and deposited in the lungs, only a small fraction (both single and agglomerates) can be phagocytozed with a small part translocated across the alveolar epithelium. Nevetheless, the nanosize factor is a major significant feature in determining the deposition, translocation, distribution and fate of AuNPs. These facilitate the crossing of the blood brain barrier by AuNPs, which accumulate in neural tissues as well as in the placenta and fetus [87,94-95]. Earlier reports by Takahashi and Matsuoka [94] reported the uptake of colloidal AuNPs of 5 and 30 nm after maternal intravenous injection in rats. Other studies by Lee et al [96] and Myllynen et al [95] have also showed the internalization of 10-30 nm PEGlyated AuNPs in the placental cells which are comparable to immunoglobulins that cross the placenta (IgG). The findings from these studies also showed AuNPs with sizes up to 240 nm crossing the human placental barrier without affecting the viability of the placental explants. Other findings by Sadauskas et al [88], however, showed that AuNPs of 2 and 4 nm when injected intravenously or intraperitoneally respectively did not seem to penetrate either the placenta barrier or the blood - brain barrier but were found in the macrophages and Kupffer liver cells. Information from literature envisages size as the most significant physical property responsible for inducing AuNP toxicities [97].

Furthermore, the influence of AuNP toxicity has also been shown to vary due to the different particle shapes. Among the shapes, rods shaped AuNPs have been reported to demonstrate more toxicity than their spherical counterparts. Research on gold nanorods has shown that they are more toxic to human keratinocyte cells (HaCaT) as compared to spherical gold nanomaterials [62]. The mechanisms of less toxicity of spherical AuNPs compared to nanorods are yet to be demonstrated; however, they are all engulfed on their surface properties. Studies investigating the cytotoxicity and cellular uptake of gold nanorods on human breast adenocarcinoma cell line (MCF- 7) also reported loss of mitochondrial integrity in cells treated with nanorods [47] as compared to spherical shapes [77]. Li et al [70] also showed that naked AuNPs (20 nm in diameter) when taken up by MRC-5 human lung fibroblast in vitro can induce autophagy (degradation of a cell's own components via lysosomal machinery) concomitant with oxidative stress, stimulating upregulation of antioxidants, stress response genes and protein expression. Other studies have also shown nanorods toxicity to be highly associated with surface layer used for the synthesis of nanorods such as CTAB [97]. Therefore the association of surface stabilizers and functional ligands chemistry or composition should not be overlooked. Also as the application of AuNPs are increasing in medicine to diagnose and treat diseases detailed data on the possible toxic effect of various sizes, shapes and ligands of the AuNPs are needed because the current available information are limited and inconsistent.

The effect of ligands and bio-conjugates on the toxicity AuNPs

In nanotechnology, ligands are functional groups attached onto the surfaces of NPs thereby modifying their surface activities. The functional groups are usually attached either covalently or non-covalently onto the NPs by chemical processes (98-99). Place-exchange reaction is the most versatile and widely used method for introducing functional groups to AuNPs (57,92). The widely attached functional groups onto AuNPs are highly available for further conjugation (57). Polyethylene glycol (PEG), poly-L- lysine (PLL), poly- D- L- lactic-co- glycolic acid (PLGA) and their co- polymers have been successfully applied to develop novel biocompatible AuNPs [14]. Of these, PEG has gained popularity as a modifying agent due to its amphiphilic and solubility characteristics [12].

Hetero-functionalized PEGylated mono protected clusters (MPCs) with a thiol group on one terminal and a reactive functional group on the other have become popular for AuNP applications [100]. Preferred end groups for hetero- functional PEG AuNPs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids, hydroxyl, methoxy and esters [101]

Functionalization of AuNPs increases the circulation period of the NPs in the blood stream [12]. A study investigating the bio- distribution of PEG modified and non- modified gold nanorods in mice reported a larger percentage of modified AuNPs in the blood in contrast to unmodified particles over the same time period [102]. Other data have shown that surface modified AuNPs have the ability to reduce cellular toxicity associated with chemical surfactants used during the synthesis of the NPs [42,92]. Takahashi et al [94] reported the modifying of gold nanorods with phosphatidylcholine to reduce cytotoxicity associated with the CTAB molecule on the nanorods surfaces. Another study by Goodman et al [103] investigated the hazardous effect of AuNPs modified with an amine and carboxyl groups on Cos-1 cells, red blood cells, and E. coli cells. Their results showed that anionic AuNPs species are non-toxic to cells, whereas cationic species can cause moderate toxicity in all cells lines. The authors suggested that toxicity was related to the interaction of a positive charge on the ammonium species with a negative charge on the lipid bilayer of cell membranes.

Other findings reported by et al [67] showed that 1.4 nm AuNPs in diameter capped with triphenylphosphine monosulfonate can cause necrosis via the oxidative stress and mitochondrial damage, while Gu et al [104] found that 3.7 nm AuNPs in diameter modified poly(ethylene glycol) (PEG) were non toxic when internalized in the cell nucleus of human cervical cancer (HeLa). This shows that functionalized molecules play a significant role in AuNPs toxicity.

Some findings showed that functionalized AuNPs are not cytotoxic but can cause a slight reduction in the reactive oxygen and nitrite species [59,63,105]. However, according to the findings by Bar-Ilan et al and Pan et al [61,67], functionalized AuNPs with a concentration of 62.5 mg mL^_1 of triphenylphosphine monosulfonate (TPPMS) utilized as ligand is non-toxic, whereas concentration higher than 625 mg mL^_1 can result in morphological malformations of zebrafish embroyos. Earlier studies by Tsoli et al [60] and Pan et al [67] revealed that 1.4 nm AuNPs functionalized with TPPMS are able to bind on dsDNA major groove and disrupt cellular function. However, their findings failed to indicate whether the effect was specific to gold 1.4 nm or to all AuNPs coated with TPPMS. In other related study it was found that the toxicity and biodistribution of PEG-coated AuNPs 20 nm with TA-terminated PEG5000 has more stability with lower toxicity than 40 or 80 nm AuNPs functionalized with TA-terminated PEG5000 [90]. It is therefore, evident that functional groups on AuNPs affect toxicity. An investigation into the effect of size and the presence or absence of sodium citrate residues on the cytoxicity and uptake of AuNPs in alveolar type-II cells has showed the reduction in cell viability due to sodium citrate residues on AuNPs [52].

However, when Boca et al [106] examined the cytotoxic effect of chitosan capped-AuNPs on chinese hamster ovary cells in vitro, the conjugated particles were found to tranverse the cell membrane by endocytosis; and using dark field microscopy imaging, it revealed ≥ 85% of the cells were viable even after long period of exposure. This, therefore, shows that chitosan-conjugated AuNPs can be deemed to have great potential in cellular imaging or photothermal therapy as they are non-toxic compared to other coated AuNPs.

Oberdorster et al [107] earlier showed that, partial surface composition coupled with size of the NP is accountable for the observed toxic effects. However, other studies by Bar-Ilan et al [61] demostrated that zebra-fish embryo toxicity depends more on its surface chemical composition rather than on particle size. This implies that surface functionalization or coatings of AuNPs has a very huge impact on the toxicity of nanomaterials.

Furthermore, Cho et al [108] showed that PEG-coated 13 nm AuNPs when injected intravenously in to BALB/C mice can elicit an immune response and apoptosis with further accumulation in the liver and spleen after a week of administration. The findings of Wang et al [97] involving the intravenously injected CTAB- capped gold nanorods into rats circulated in blood as the main route of bio-distribution. Similarly, Hirn et al [109] also found the accumulation of AuNPs mainly in the liver, spleen and to a lesser extend in the kidney, brain, muscle and bone. Those found in the spleen and Kupffer cells lymphocytes form aggregates within the lysosomes [97]. The formation of aggregation scenario can result into complex biological system of an unknown response and toxicity in vivo. This indicates the need to study the biological effect of NPs in biological systems.

The effects of surface charge on the toxicity of gold nanoparticles

Surface charge which is measured by zeta potential is one of the major physical characteristic influencing AuNPs toxicity [108]. The application of zeta potential provides useful information on the stability of colloid nanomaterials. It is thus, essential to always state whether the zeta potential of colloid NPs is positively or negatively charged. Surface charges determine the properties and functions of NPs. AuNPs have charged (negatively or positively) surfaces which make them highly reactive and receptive to surface modifications due to either cations or anions interaction, thus, creating a net surface charge [40]. Based on surface charges, AuNPs can promote protein refolding through electrostatic interactions between the exposed charged residues on the un folded protein and the oppositely charged ligands on the AuNPs [110]. The overall high negative charge of the NP-protein complex prevents the proteins from aggregating; the NP thereby promotes refolding which can be used to refold proteins in a chemical denatured state [110].

It is important to note that modifications of NP surfaces may cause undesirable ionic interactions with biological systems [73], due to changes in surface charges. Many AuNPs are stabilized with surface charges to prevent aggregation via electrostatic repulsion [42], playing a significant role in toxicity of the NP. Aggregated AuNPs have modified surface charges which intend influence changes of cellular environment and thus altering the cellular behaviour and cellular toxicity [79].

Apart from earlier reports that cationic are moderately toxic than anionic AuNPs [103], other reports by Schaeublin et al [111] have shown that both cationic and anionic AuNPs are toxic to cells. Schaeublin et al [111] further showed that both positively and negatively charged AuNPs can alter the mitochondrial membrane potential resulting into oxidative stress. The oxidative stress according to current findings by Oikawa et al [112] enhances the production of reactive oxygen species, various immunologic stimuli, inflammation, some human diseases such as neurodegenerative disorders, and cancers. Apart from these the anionic and cationic surface charges of AuNPs can stimulate lymphoid cells phagocytosis to an extend greater than neutral AuNPs [79]. Therefore, the wider the charge differences on the AuNP surfaces, the greater the opportunity for it phagocytic activities and the inflammatory responses.

Other studies have shown that surfaces charges of NPs enhance their uptake into cells. For example findings by Chithrani et al [62] using incubated citric acid coated AuNPs have shown that NPs surface charges can potential influence their uptake by mammalian cell line HeLa. Furthermore, He et al [113] also found that although the surface charges play a significant role in phygocytoses they also aid phagocytic clearance due to the NPs small size and high diffusible nature. The interaction of AuNPs with serum proteins therefore alter the physiochemical properties of the NP, which can intend affect uptake and target drug delivery processes [114]. Other factors such as ionic strength (charges) of AuNPs can also affect their biocompatibility, thereby interfering with the biokinetics of the cells, resulting in a reduction in cell viability [75,115].

However, citrate stabilized AuNPs toxicity test using MTT assay have shown that 20 nm AuNPs at a concentration of 300 μM have no significant effect on cell human dermal fibroblast-fetal [74] which was similar to earlier findings by Connor et al [59]. In other study purified and citrate sterilized AuNPs have shown rather milder cytotoxicity in A549 and NCIH441 cells as compared to the particles with excess citrate. This indicates that functionalized side chain can interfere with the activities of the AuNPs depending on the shape and surface charge [62,103]. This, therefore, indicates that further more in vitro studies on cell viability concerning charge AuNPs properties are required to ascertain their toxicity.

As discussed only a few studies of AuNPs toxicities have been conducted using transformed cells lines with only a limited number using primary cell cultures which are prototype systems closer to in vivo studies. AuNPs surface modifications (surface charges) have been found to influence particle uptake in vitro. Some of the reports have shown that in vitro studies are the easiest toxicology studies which can be used to better understand the molecular events underlying cellular effects [66] as shown in Table 1. However, Donaldson et al [66] have stressed that in vitro studies on cell culture alone are highly limited due to narrow range of biological effects which do not reflect the range of pathological effects observed in vivo. Apart from that, the issue of NPs translocation into host tissues and their toxicokinetics in vivo is an important underlying principle in understanding nanotoxicology because in vitro testing has shown less convincing results. Apart from that there are few in vivo studies on the biodistribution and biological effects of AuNPs that can serve as a basis for assessing its health impact due to surface charges. Furthermore, in vivomethods are very important because they determine the whole body health effects in animals although this will depend on the route (nasal, oral or dermal) of exposure (49). The biodistribution and biological processes after exposures are all tied down to the surface physiochemical properties that make them chemically reactive upon interaction with biological systems [93]. Therefore, the attributes of AuNPs and its coated surface charges must be examined with care to ascertain toxicities.

Conclusion and Challenges

In AuNPs and gold nanomaterials applications, the most important features stimulating their compositions array are sizes, shapes, surface area/porosity, surface charges, aggregation, surface modifications and host cell interactions. We can say these unique properties of AuNPs profiles are size-dependent and provide the challenge of determining their biological toxicities. However, for a better understanding of the acute, subchronic and chronic health effects of AuNPs toxicity, recommendations of methods for testing of reproductive toxicity, genotoxicity or carcinogenicity effects have been made available by the Organization for Economic Cooperation and Development (OECD). Furthermore, studies gathered so far show that AuNPs nanotoxicity studies and their health effects/implication are currently limited and insufficient to determine their health status to both human and the environment. Therefore, more in vitro and in vivo AuNPs and gold nanomaterials toxicity studies are highly recommended to augment the current limited controversial data (whether toxic or no-toxic). This is due to the fact that gold nanotechnology is a relative new field and its findings are budding. Also their easy aggregations arising from the fragile capped stabilized surfaces make physical handling difficult thus limiting applications. Different sizes, shapes and surface ligands exhibit different properties, therefore, when considering toxicity testing for AuNPs these factors should be taken into account. Furthermore, in depth research should be done to understand the chemical processes of size, shape and or surface ligands because they exhibit different properties both in vitro and in vivo. In addition, before, AuNPs and other NPs application are safely and widely applied in biomedical systems or at best in clinical trials, information on their biocompatibility, bio-distribution and biodegradability nature of Nanomaterials when applied into biological systems. Also it will be of paramount importance to understand the long term persistent behavior of AuNPs in vivo before their applications in biological settings.

Acknowledgement

The author acknowledges the financial support from the Department of Science and Technology (DST) South Africa.

References

1. Chandra P, Das D, Wahab AAA, Gold nanoparticles in molecular diagnostics and therapeutics. Digest Journal of nanomedicine and biostructures. 2010. 5(2): 363-367
2. Simate GS, Iyuke SE, Ndlovu S, Yah CS, Walubita LF. The Production of Carbon Nanotubes from Car- bon Dioxide – Challenges and Opportunities: Journal of Natural Gas Chemistry. 2010. 19 (5): 453-460
3. Yah CS, Iyuke SE, Simate GS, Unuabonah EI, Bath- gate G, Matthews G, Cluett JD. Continuous synthesis of multiwalled carbon nanotubes from xylene using the swirled floating catalyst chemical vapor deposition technique. Journal of Materials Research. 2011. 26 (5): 623 -632.
4. Napierska D, Thomassen LCJ, Lison D, Martens JA, Hoet PH. The nanosilica hazard: another variable en- ity. Particle and Fibre Toxicologhy. 2010. 7:39
5. Jennings T, Strouse G. Past, present, and future of gold nanoparticles. Adv. Exp. Med. Biol. 2007; 620: 34.
6. Bracamonte MV, Bollo S, Labbe P, Rivas GA, fer- reyra NF. Quaternized chitosan as support for the as- sembly of gold nanoparticles and glucose oxidase. Physiochemical characterization of the platform and evaluation of its biocatalytic activity. Electrochmica Acta. 2011. 56: 1316-1322.
7. Aillon KL, Xie Y, El-Gendy N, Berkland CJ, Forrest ML. nanomaterials physiochemical properties on in vivo toxicity. Advanced drug Delivery Reviews. 2009. 61(8): 457-466.
   
8. Marsich E, Travan A, Donati I, Luca AD, Benincasa M, Crosera M, Paoletti. Biological response of hydrogels embedding gold nanoparticles. Collods and SurfaceB: Biointerfaces. 2011. 83: 331-339.
9. Jenkins JT, Halaney DL, Sokolov KV, Ma LL, Ship- ley HJ, Mahajan S, Louden CL, Asmis R, Milner TE, Johnsons KP, Feldman MD. 2013. Excretion and tox- icity of gold–iron nanoparticles . Materials Science and Engineering: C. 33(1): 550–556
10. Prime D, Paul S, Joseph-Franks PW. Gold nanoparti- cle charge trapping and relation to organic polymer memory devices. Philos transact A Math Phys Eng Sci. 2009; 367(1905):4215-25.
11. Tsoukalas D. From silicon to organic nanoparticle memory devices. Philos transact A Math Phys Eng Sci. 2009; 367(1905): 4169-79.
12. Pissuwan D, Niidome T, Cortie MB. The forthcoming applications of gold nanoparticles in drug and gene de- livery systems, Journal of Controlled Release. 2011; 149(1): 65-71.
13. Surendra N, Nidhi G, Ramesh C. Cationic Polymer Based Nanocarriers for Delivery of Therapeutic Nucleic Acids. Journal of Biomedical Nanotechnol- ogy. 2011. 7 (4): 504-520.
14. Zhang XD, Wu HY, Wu D, Wang YY, Chang JH, Zhai ZB, Meng AM, Liu PX, Zhang LA, Fan FY. Toxicologic effects of gold nanoparticles in vivo by different administration routes. International Journal of Nanomedicine. 2010; 5: 771–781
15. Li X, Zhou H, Yang L, Du G, Pai-Panandiker AS,Huang X, Yan B. 2011. Enhancement of cell recogni- tion in vitro by dual-ligand cancer targeting gold na- noparticles. Biomaterials. doi:10.1016/j.biomaterials. 2010.12.031
16. Hashmi, A. S. K.; Hutchings, G. J. Gold catalysis.Angew. Chem., Int. Ed. 2006; 45: 7896–7936.
18. McPherson JS; Thompson DT. Selectivity of gold catalysis for application of commercial interest. Trop- ics in Catalysis. 2009; 52: 743-750.
19. Sardar R, Funston AM, Mulvaney P, Murray RW.Gold Nanoparticles: Past, Present, and Future. Lang- muir. 2009; 25(24): 13840–13851
20. Tshikhudo RT, Demuru D, Wang Z, Brust M, Secchi A, Pochini A. 2005. Molecular recognition by Ca- lix[4]arene-modified gold nanopaerticles in aqueous solution. Agew. Chem. Int. Ed. 44:2913-2916.
21. Krause RWM, Mamba BB, Malefetse TJ, Bambo FM,Malefetse TJ. Cyclodectrins: Chemistry and Physics. ISBN: Chapter 9 cyclodextri polymers: Synthesis and application in water treatment. Editor. JIe Hu: Trans- world Research Network, Kerala. India. 2010;Pp 185- 209.
22. Low A, Bansal V. A visual tutorial on the synthesis of gold nanoparticles. Biomed Imaging Interv J. 2010; 6(1):e9
23. Lu X, Tuan HY, Korgelc BA, and Xia Y. Facile Syn-thesis of Gold Nanoparticles with Narrow Size Distri- bution by Using AuCl or AuBr as the Precursor .Chemistry. 2008; 14(5): 1584–1591.
24. Mallick K, Witcomb M, Erasmus RM, Strydom AM. Low temperature magnetic property of polymer encap- sulated gold nanoparticles. Journal of Applied Physics. 2009; 106:074303-074209.
25. Sharma V, Kyoungweon P, Mohan S. "Colloidal dispersion of gold nanorods: Historical background, optical properties, seed-mediated synthesis, shape separation and self-assembly". Material Science and Engineering Reports. 2009; 65 (1-3): 1–38.
26. Kimling J, Maier M, Okenve B, Kotaidis V, Ballot H, and Plech A. 2006.; Turkevich Method for Gold Na- noparticle Synthesis Revisited. J. Phys. Chem. B. 110 (32): 15700–15707.
27. Brust M, Walker M, Bethell D, Schiffrin DJ, Whyman R. "Synthesis of Thiol-derivatised Gold Nanoparticles in a Two-phase Liquid-Liquid System". Chem. Commun.: 1994; 801.
28. Perrault SD, Chan WCW. "Synthesis and Surface Modification of Highly Monodispersed, Spherical Gold Nanoparticles of 50-200 nm". J. Am. Chem. Soc. 2009; 131 (47): 17042.
29. Martin MN, Basham JI, Chando P, Eah SK. "Charged gold nanoparticles in non-polar solvents: 10-min synthesis and 2D self-assembly". Langmuir. 2010; 26 (10): 7410.
30. Vinodgopal K, Neppolian B, Lightcap IV, Grieser F, Ashokkumar M, Kamat PV. 2010. Sonolytic Design of Graphene−Au Nanocomposites. Simultaneous and Se- quential Reduction of Graphene Oxide and Au(III) J. Phys. Chem. Lett., 2010, 1 (13), pp 1987–1993.
31. Prathna TC, Mathew L, Chandrasekaran N, Raichur AM, Mukherjee A. Biomimetic Synthesis of Nanopar- ticles: Science, Technology & Applicability.School of Bio Sciences & Technology, VIT University, Depart- ment of Materials Engg., Indian Institute of Science. India. www.intechopen.com
32. Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC, Azam A, Owais M. 2011. Fungus- mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer. 2011(6): 2305 – 2319
33. Singh A, Sharma MM, Batra A. 2013. Synthesis of gold nanoparticles using chick pea leaf Extract using green chemistry. Journal of Optoelectronics and Bio- medical Materials. 5(2): 27 – 32.
34. Srivastava SK, Yamada R, i Ogino C, Kondo A.2013.Biogenic synthesis and characterization of gold nanoparticles by Escherichia coli K12 and its hetero- geneous catalysis in degradation of 4-nitrophenol. Nanoscale Research Letters. 8:70
35. Ogi T, Saitoh N, Nomura T, Konishi Y. 2010. Room- temperature synthesis of gold nanoparticles and nano- plates using Shewanella algae cell extract. J Nanopart Res, 12:2531–2539.
36. Narayanan KB, Sakthivel N. 2010. Biological synthe- sis of metal nanoparticles by microbes. Adv Colloid Interface Sci. 156:1–13.
37. Balasubramanian SK, Jittiwat J, Manikandan J, Ong CN, Yu LE, Ong WY. Biodistribution of gold nano- particles and gene expression changes in the liver and spleen after intravenous administration in rats. Bioma- terials. 2010; 31:2034–2042.
38. Wu HY, Liu M, and Huang MH. Direct Synthesis of Branched Gold Nanocrystals and Their Transforma- tion into Spherical Nanoparticles. J. Phys. Chem. B. 2006;110: 19291-19294.
39. Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar SR, Khan MI, Ramani R, Parischa R, Ajaya- kumar PV, Alam M, Sastry M, Kumar R. Bioreduction of AuCl4- ions by the fungus Verticillum species and surface trapping of gold nqanoparticles formed. Angew. Chem. Int. Ed. 2001; 40:3585–3588.
40. Pillay J, Ozoemena KI, Tshikhudo RT, Moutloali RM. Monolayer-Protected Clusters of Gold Nanoparticles: Impacts of Stabilizing Ligands on the Heterogeneous Electron Transfer Dynamics and Voltammetric Detec- tion. Langmuir. 2010; 26(11): 9061–9068.
41. Simpson CA, Salleng KJ, Cliffel DE, Feldheim DL. 2013. In vivo toxicity, biodistribution, and clearance of glutathione-coated gold nanoparticles. Nanomedicine: Nanotechnology, Biology and Medi- cine. 9(2): 257–263.
42. Alkilany AM, Murphy . 2010. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res .12:2313–2333.
43. Thakor AS, Jokerst J, Zavaleta C, Massoud TF, Gambhir SS. 2011. Gold Nanoparticles: A Revival in Precious Metal Administration to Patients. Nano Lett. 11(10):4029-36.
44. Zhang Y, Xu D, Li W, Yu J, Chen Y. 2012.. Effect of Size, Shape, and SurfaceModification on Cytotoxicity of Gold Nanoparticles to Human HEp-2 and CanineMDCK Cells. Journal of Nanomaterials. ID 375496, 7doi:10.1155/2012/375496
45. Murphy CJ, Gole AM, Stone JW, Sisco PN, Alkilany AM, Goldsmith EC, Baxter SC. 2008. Gold nanopar- ticles in Biology: Beyond Toxicity to Cellular Imag- ing. Acc Chem Res. 41(12):1721-30.
46. Mironava T, Hadjiargyrou M, Simon M, Jurukovski V, Rafailovich MH. 2010.Gold nanoparticles cellular toxicity and recovery: Effect of size, concentration and exposure time. Nanotoxicology, 4(1): 120–137.
47. Qiu Y, Liu Y, Wang L, Xu L, Ba R, Ji Y, Wu X, Zhao Y, i Y, Chen C Surface chemistry and aspect ra- tio mediated cellular uptake of Au nanorods. Biomate- rials. 31(30): 7606–7619.
48. Lewinski N, Colvin V, Drezek R. Cytotoxicity of na-noparticles. Small. 2008; 4(1): 26-49.
49. Yah CS, Iyuke SE, Simate GS. 2012. Nanoparticles toxicity and their routes of exposures. PJPS: 25(2): 477-491.
50. Weinberg H, Galyean A, Leopold M. Evaluating engi-neered nanoparticles in natural waters. TrAC Trends in Analytical Chemistry. 2011; 30(1): 72-83.
51. Byrne HJ, Lynch I, de Jong WH, Kreyling WG, Loft S, Park MVDZ, Riediker M, Warheit D. Protocols for assessment of biological hazards of engineered nano- materials. The European Network on the Health and Environmental Impact of Nanomaterials. 2010;Pp.1- 30.
52. Uboldi C, Bonacchi D, Lorenzi G, Hermanns MI, Pohl C, Baldi G, Unger RE and Kirkpatrick CJ. Gold nano- particles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441. Particle and Fibre Toxicol- ogy. 2009;6:18
53. De Jong WH, Hagens WI, Krystek P, Burger MC, Sips AJAM, Geertsma RE. Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials. 2008 29: 1912-1919.
54. Van Doren EAF, Temmerman PRHD, Francisco AD,Mast J. Determination of the volume specific surface area by using transmission electron tomography for characterization and definition of nanomaterials. Jour- nal of Nanobiiotechnology. 2011. 9:17
55. Patra CR, Bhattacharya R, Mukhopadhyay D, Muk- herjee P. Fabrication of gold nanoparticles for targeted therapy in pancreatic cancer. Advanced Drug Delivery Reviews. 2010; 62: 346–361.
56. Renault S, Baudrimont M, Mesmer-DudonsN, Gon- zalez P, Mornet S, Brisson A. Impacts of gold Thakor AS, Jokerst J, Zavaleta C, Massoud TF, Gambhir SS. 2011. Gold Nanoparticles: A Revival in Precious Metal Administration to Patients. Nano Lett. 11(10):4029-36.
57. Ghosh P, Han G, De M, Kim CK, Rotello VM. Gold nanoparticles in delivery applications. Advanced Drug Delivery Reviews. 2008; 60: 1307–1315.
58. Yum K, Wang N, Yu MF. Nanoneedle: A multifunctional tool for biological studies in living cells. Nanoscale. 2010; 2: 363-372.
59. Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanoparticlesv are taken up by human cells but do not cause acute cytotoxicity. Small. 2005;1: 325–327.
60. Tsoli M, Kuhn H, Brandau W, Esche H, Schmid G.Cellular Uptake and Toxicity of Au55 Clusters. Small. 2005; 1(8-9): 841–844.
61. Bar-Ilan O, Albrecht RM, Fako VE, and Furgeson DY. Toxicity Assessments of Multisized Gold and Silver Nanoparticles in Zebrafish Embryos. Small. 2009;5(16): 1897–1910.
62. Chithrani BD, Ghazani AA, Chan WCW. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 2006; 6: 662.
63. Khan JA, Pillai B, Das TK, Singh Y, Maiti S. Molecu- lar Effects of Uptake of Gold Nanoparticles in HeLa Cells . ChemBioChem. 2007; 8: 1237.
64. Hauck T.S, Ghazani A.A, Chan W.C. Assessing the Effect of Surface Chemistry on Gold Nanorod Uptake, Toxicity, and Gene Expression in Mammalian Cells. Small, 2007; 4: 153.
65. Fanord F, Fairbairn K, Kim H, Garces A, Bhethanabotla V, Gupta VK. 2011. Bisphosphonate- modified gold nanoparticles: a useful vehicle to study the treatmentNanotechnology. 22. 035102 doi: 10.1088/0957-4484/22/3/035102
66. Donaldson K, Borm PJA, Castranova V, Gulumian M. The limits of testing particle-mediated oxidative stress in vitro in predicting diverse pathologies; relevance for testing of nanoparticles. Particle and Fibre Toxicology. 2009; 6:13
67. Pan Y, Neuss S, Leifert A, Fischler M, Wen F, Simon U, Schmid G, Brandau W, Jahnen-Dechent W. Size- dependent cytotoxicity of gold nanoparticles. Small. 2007; 3: 1941.
68. Chen YS, Hung YC, Liau I, Huang GS. Assessment of the In Vivo Toxicity of Gold Nanoparticles. Nanoscale Res Lett. 2009; 4:858–864.
69. Kim GY, Shim J, Kang MS, Moon SH. Optimized coverage of gold nanoparticles at tyrosinase electrode for measurement of a pesticide in various water sam- ples. Journal of Hazardous Materials. 2008; 156: 141– 147.
70. Li JJ, Hartono D, Ong CN, Bay BH, Yung LYL. Au- tophagy and oxidative stress associated with gold na- noparticles. Biomaterials. 2010; 31: 5996-6003.
71. Coradeghini R, Gioria S, García CP, Nativo P, Fran- chini F, Gilliland D, Ponti J, Rossi F. 2013. Size- dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts. Toxicology Letters. 217(3) : 205–216
72. Yen HJ, Hsu SH, Tsai CL. Cytotoxicity and immu- nological response of gold and silver nanoparticles of different sizes. Small. 2009; 5(13):1553-61.
73. Tedesco S, Doyle H, Redmond G, Sheehan D. Gold nanoparticles and oxidative stress in Mytilus edulis. Marine Environmental Research. 2008; 66: 131–133.
74. Qu Y, Lu X. Aqueous synthesis of gold nanoparticles and their cytotoxicity in human dermal fibroblast – fe- tal. Biomedical Materials. 2009; 4(2):025007.
75. Dobrovolskaia MA, Patri AK, Zheng J, Clogston JD, Ayub N, Aggarwal P, Neun BW, Hall JB, McNeil SE. Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles. Nanomedicine: Nanotechnol- ogy, Biology, and Medicine. 2009; 5: 106–117.
76. Mao Z, Wang B, Ma L, Gao C, Shen J. The influence of polycaprolactone coating on the internalization and cytotoxicity of gold nanoparticles. Nanomedicine: Nanotechnology, Biology, and Medicine. 2007; 3: 215–223.
77. Wei XL, Mo ZH, Li B, Wei JM. Disruption of HepG2 cell adhesion by gold nanoparticle and Paclitaxel dis- closed by in situ QCM measurement. Colloids and Surfaces B: Biointerfaces. 2007; 59: 100–104.
78. Wang C, Wang J., Liu D, Wang Z. Gold nanoparticle-based colorimetric sensor for studying the interactions of β-amyloid peptide with metallic ions. Talanta. 2010; 80: 1626–1631.
79. Arnid, Malugina. Ghandeharia H. Cellular uptake and toxicity of gold nanoparticles in prostate cancer cells: a comparative study of rods and spheres. J. Appl. Toxicol. 2010; 30: 212–217.
80. Glazer ES, Massey KL, Zhu C, Curleybn SA. Pancre- atic carcinoma cells are susceptible to noninvasive ra- dio frequency fields after treatment with targeted gold nanoparticles. Surgery. 2010; 148: 319-324.
81. Hartono D, Yang HKL, Yung LYL. The effect of cho-lesterol on protein-coated gold nanoparticle binding to liquid crystal-supported models of cell membranes. Biomaterials. 2010; 31: 3008–3015.
82. Rayavarapu RG, Petersen W, Hartsuiker L, Chin P,Janssen H, van Leeuwen F, Otto C, Manohar S, Van Leeuwen TG..In vitro toxicity studies of polymer- coated gold nanorods. Nanotechnology. 2010; 21 (14):145101.
83. Brandenberger C, Rothen-Rutishauser B, Mühlfeld C, Schmid O, Ferron GA, Maier KL, Gehr P, Lenz AG.Effects and uptake of gold nanoparticles deposited at the air–liquid interface of a human epithelial airway model. Toxicology and Applied Pharmacology. 2010; 242: 56–65.
84. Wiwanitkit V, Sereemaspun A, Rojanathanes R. Effect of gold nanoparticles on spermatozoa: the first world report. Fertility and Sterility. 2009; 91(1): e7-e8.
85. Yu LE, Yung LYL, Ong CN, Tan YL, Balasubrama- niam KS, Hartono D, Shu G, Wenk MR, Ong WY. Translocation and Effects of Gold Nanoparticles after Inhalation Exposure in Rats. Nanotoxicology. 2007; 1(3): 235 – 242.
86. Takenaka S, Karge E, Kreyling WG, et al. Distribution pattern of inhaled ultrafine gold nanoparticles in the rat lung. Inhalation Toxicol. 2006; 18:733–40.
87. Lasagna-Reeves C, Gonzalez-Romero D, Barria MA, Olmedo I, Clos A, Ramanujam VMS, Urayama A, Vergara L, Kogan MJ, Soto C. Bioaccumulation and toxicity of gold nanoparticles after repeated admini- stration in mice. Biochem Biophys Res Commun. 2010; 393:649–655.
88. Sadauskas E, Jacobsen NR, Danscher G, Stoltenberg M, Vogel U, Larsen A, Kreyling W, Wallin H. Bio- distribution of gold nanoparticles in mouse lung fol- lowing intratracheal instillation. Chemistry Central Journal. 2009; 3:16
89. Mühlfeld C, Rothen-Rutishauser B, Blank F, Van-hecke D, Ochs M, Gehr P. Interactions of nanoparti- cles with pulmonary structures and cellular responses. Am J Physiol Lung Cell Mol Physiol. 2008; 294:817- 29.
90. Lipka J, Semmler-BehnkeM, Sperling RA, Wenk A, Takenaka S, Schleh C, Kissel T, Parak WJ, Kreyling WG. Biodistribution of PEG-modified gold nanoparti- cles following intratracheal instillation and intrave- nous injection. Biomaterials. 2010;31:6574–6581.
91. Sousa F, Mandal S, Garrovo C, Astolfo A, Bonifacio A, Latawiec D, Menk RH, Arfelli F, Huewel S, Legname G, Galla HJ, Krol S. Functionalized gold nanoparticles: a detailed in vivo multimodal micro- scopic brain distribution study. Nanoscale. 2010; 2: 2826-2834
92. Simpson CA, Huffman BJ, Gerdon AE, Cliffe DE. Unexpected Toxicity of Monolayer Protected Gold Clusters Eliminated by PEG-Thiol Place Exchange Reactions. Chem. Res. Toxicol. 2010; 23: 1608–1616.
93. Gosens I, Post JA, de la Fonteyne LJJ, Jansen EHJM, Geus JW, Cassee FR, de Jong WH. Impact of agglom- eration state of nano- and submicron sized gold parti- cles on pulmonary inflammation. Particle and Fibre Toxicology. 2010; 7:37.
94. Takahashi S, Matsuoka O. Cross Placental Transfer of 198Au-colloid in Near Term Rats. J Radiat Res. 1981; 22:242-249.
95. Myllynen PK, Loughran MJ, Howard CV, Sormunen R, Walsh RA, Vähäkangas KH. Kinetics of gold na- noparticles in the human placenta. Reproductive Toxi- cology. 2008; 26 (2): 130-137.
96. Lee, K-B, Park S-J, Mirkin CA, Smith JC, Mrksich M. 2002. Protein Nanoarrays Generated By Dip-Pen Nanolithography. Science. 295 (5560): 1702-1705
97. Wang S, Lu W, Tovmachenko O, Rai US, Yu H, Ray PC. Challenge in understanding size and shape de- pendent toxicity of gold nanomaterials in human skin keratinocytes. Chemical Physics Letters. 2008; 463: 145–149.
98. Ngoy JM, Iyuke SE, Neuse WE, Yah CS. 2011. Cova- lent Functionalization for Multi-Walled Carbon Nano- tube (f-MWCNT) -Folic Acid bound bioconjugate. Journal of Applied Sciences. 11(15) : 2700-2711
99. Pan Y, Leifert A, Ruau D, Neuss S, Bornemann J, Schmid G, Brandau W, Simon U, Jahnen-Dechent W. Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small. 2009; 5(18):2067-76.
100. Shenoy D, Fu W, Li J, Crasto C, Jones G, DiMarzio C,Sridhar S, Amiji M. Surface Functionalization of Gold nanoparticles using hetero- bifunctional poly (ethylene glycol) spacer for intracellular tracking and delivery. International Journal of Nanomedicine. 2006; 1(1): 51-
     57
101. Grubbs et al., 2007 Grubbs RB. 2007. Roles of polmer ligands in naoparticle stabilization. Polymer Review. 47: 197-215.
102. Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Takahashi H, Kawano T, Katayama Y, Niidome Y. 2006. PEG-modified gold nanorods with a stealth character for in vivo applications.
103. Journal of Controlled Release. 343-347.
104. Goodman CM, Mccusker CD, Yilmaz T, Rotello VM. Toxicity of Gold Nanoparticles Functionalized with Cationic and Anionic Side Chains. Bioconjugate Chem. 2004;15: 897-900.
105. Gu YJ, Cheng J, Lin CC, Lam YW, Cheng SH, Wong WT. Nuclear penetration of surface functionalized gold nanoparticles. Toxicology and applied Pharma- cology. 2009; 231(1): 6-04
     106 Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compart- ment: a microscopic overview. Langmuir. 2005; 21: 10644.
106. 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; 21: 10644.
107. Boca SC, Potara M, Toderas F, Stephan O, Baldeck PL, Astilean S. Uptake and biological effects of chito- san-capped gold nanoparticles on Chinese hamster Overy cells. Materials Science and Engineering. 2011; 31 (2): 184-189.
108. Oberdorster G, Oberdorster E, Oberdorster J. Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles. Environ. Health Perspect. 2005; 113: 823.
109. Cho WS, Cho M, Jeong J, Choi M, Cho HY, Han BS, Kim SH, Kim HO, Lim YT, Chung BH, Jeon J. Acute toxicity and pharmacokinetics of 13 nm-sized PEG- coated gold nanoparticles. Toxi Appl Pharmacol. 2009; 236(1):16-24.
110. Hirn S, Behnke MS, Schleh C, Wenk A, Lipka J,Schäffler M, Takenaka S, Möller W, Schmidn G, Si- mon U, Kreyling WG. Particle size-dependent and sur- face charge-dependent biodistribution of gold nanoparticles after intravenous administration. Eur J of Pharm Biopharm. 2011; 77(3):407-16.
111. De M, Rotello VM. Synthetic “chaperones”: nanoparticle-mediated refolding of thermally denatured proteins. Chem. Commun. 2008, 30: 3504-3506
112. Schaeublin NM, Braydich-Stolle LK, Schrand AM, Miller JM, Hutchison J, Schlagera JJ, Hussain SM. Surface charge of gold nanoparticles mediates mecha- nism of toxicity. Nanoscale. 2011; 3: 410 - 420.
113. Oikawa D, Akai R, Tokuda M, Takao I. A transgenic mouse model for monitoring oxidative stress. Scientific report. 2012, 2: 229
114. He C, Hua Y, Lichen Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomate- rials. 2010 31: 3657-3666.
115. Ehrenberg MS, Friedman AE, Finkelstein JN, Ober-dorster G, Mcgrath JL. The influence of protein ad- sorption on nanoparticle association with cultured en- dothelial cells. Biomaterials. 2009;30: 603 - 610.
116. Kunzmann A, Andersson B, Thurnherr T, Krug H, Scheynius A, Fadeel B. Toxicology of engineered nanomaterials: Focus on biocompatibility, biodistribu- tion and biodegradation, Biochim. Biophys. Acta. 2011; 1810(3): 361-373.