Gold Nanoparticles with Organic Linkers for …...Synthetic strategies to produce different shapes of Au NPs include use of surfactants (cetyltrimethylammonium bromide [CTAB] and cet-yltrimethylammonium

47

3Gold Nanoparticles with Organic Linkers for Applications in Biomedicine

Olga Shimoni and Stella M. Valenzuela

3.1 Introduction

Gold nanoparticles (Au NPs) have a rich history of drawing the attention, at first, of alchemists followed by scientists from the fields of chemistry, physics, photonics, and, in more recent times, biology and medicine. Such wide scientific attention has arisen due to the unique physical and chemical properties of these nanoparticles, including their optical and electronic virtues dependent on size, shape, and high surface-to-volume area with facile surface chemistry. Added to these favorable physicochemical properties is their highly desirable and now increasingly accepted biocompatible pro-file. These factors converge to provide the rationale for the current growing interest in their pursuit as new breakthrough therapeutic and prophylactic agents. The range of applications are being explored, which include their use as drug (Cesbron et al. 2015, Danesh et al. 2015, Khandelia et al. 2015) and gene delivery agents (Li et al. 2015); photothermal therapeutic agents (Pissuwan et al. 2006, 2008); contrast dyes for in vivo imaging (Chen et al. 2015); adjuvants in vaccine development (Safari et al. 2012); and antibacterial (Huo et al. 2014), antiparasitic (Pissuwan et al. 2007, 2009), and antiviral agents (Paul et al. 2014).

Our focus in this chapter is the use of organic linkers to modify Au NPs and the subsequent interactions and effects these modified particles have, when used within biological systems.

CONTENTS

3.1 Introduction .......................................................................................................................... 473.2 Synthesis ................................................................................................................................483.3 Gold-Linker Chemistry ....................................................................................................... 493.4 Gold Nanoparticle Cellular Interactions ..........................................................................503.5 In Vitro Cytotoxicity Studies ............................................................................................... 51

3.5.1 In Vitro Studies Reveal PEG-Coating Density Regulates Cellular Uptake of Au NPs .................................................................................................... 51

3.6 In Vivo Biokinetic and Biodistribution Studies ................................................................53References .......................................................................................................................................55

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48 Nanotechnology in Biology and Medicine

3.2 Synthesis

The earliest scientific mentioning of the preparation of spherical Au NPs appeared in the mid-nineteenth century. Nonetheless, their use has been known for over two mil-lenniums. Most of the modern synthesis of spherical Au NPs is based on the modified Turkevich (one-phase method) (Turkevich et al. 1951, Frens 1973) or Brust–Schiffrin (two-phase method) (Brust et al. 1994) methods (Figure 3.1). In both methods, chloroauric acid is reduced by means of a reducing agent in the presence of capping agents. In the case of the Turkevich method, citrate molecules play a double role as a reducing and as a stabilizing agent. The size of the resultant spherical Au NPs can be controlled by varying the con-centration or ratio of gold ions, reducing agent, or stabilizing agent. In the Brust–Schiffrin method, surfactant acts as a capping or protecting layer and sodium borohydride is usu-ally used as a reducing agent. Production of Au NPs via the Turkevich method results in their formation in an aqueous environment, which is convenient for subsequent bioappli-cations. When synthesis occurs via the Brust–Schiffrin method, Au NPs are found in the organic phase (e.g., toluene, hexane, etc.), which produces smaller-sized NPs with narrow size distribution.

Preparation of molecular-sized gold nanoclusters [Au55(PPh3)12Cl6] was reported by Schmid et al., where phosphine-stabilized Au NPs were prepared by reduction of PPh3AuCl with diborane gas in organic solvents (Schmid et al. 1981). Later, the same group described formation of water-soluble nanoclusters by exchange of PPh3 with monosulfo-nated triphenylphosphane (Ph2PC6H4SO3Na) (Schmid et al. 1988). This discovery has led to the investigation of these nanoclusters in bioapplications, discussed later in this chapter.

Citrate

Turkevich method(a)

(b) Brust–Schiffrin method

TolueneTOAB

TolueneAu NPs

NaBH4

Au NPsAuCl–

AuCl–aq. aq.

FIGURE 3.1Two most common methods of spherical gold nanoparticle synthesis. (a) Turkevich method, where chloro-auric ions are reduced and stabilized by citrate molecules. (b) Brust–Schiffrin method involves reduction of chloroauric ions with sodium borohydride (NaBH4) and produced gold nanoparticles are stabilized by the surfactant, tetraoctylammonium bromide (TOAB). Formed and stabilized gold nanoparticles are then eventually dispersed in the organic phase.



Over the last decade, there have been multiple reports on “green” synthesis of Au NPs, a process that avoids the use of harsh or undesirable molecules, such as surfactants, organic solvents, and others (Mukherjee et al. 2001, Raveendran et al. 2006, Narayanan and Sakthivel 2008, Xie et al. 2009, Park et al. 2011). The “green” method involves a use of naturally occurring molecules, including starch, proteins, biopolymers, plant extracts, and more, to play a role as capping and reducing agents simultaneously. Although “green” synthesis is somewhat desirable for application of Au NPs in biomedicine, there are still some concerns about reproducibility of high-quality nanoparticles.

From the 1990s, Brumlik and Martin began a new era of shape diversity of Au NPs (Brumlik and Martin 1991). They were the first to show the possibility of obtaining porous Au NPs with high aspect ratio by templating from porous alumina. To date, numerous articles have been published on the synthesis of Au NPs of various shapes, including rods, shells, cubes, triangular bipyramids, octahedra, hollow cubes, or spheres, and many more (Oldenburg et al. 1998, Jana et al. 2001, Sun and Xia 2002, Liang et al. 2003, Kim et al. 2004, Shankar et al. 2004, Personick et al. 2011). Synthetic strategies to produce different shapes of Au NPs include use of surfactants (cetyltrimethylammonium bromide [CTAB] and cet-yltrimethylammonium chloride [CTAC]), template-assisted electrochemical deposition, galvanic displacement, or vapor phase deposition (Dreaden et al. 2012).

3.3 Gold-Linker Chemistry

As discussed previously, one of the most popular methods to synthesize Au NPs is based on citrate-assisted chloroauric ionic reduction method. Despite the fact that this method has been in use for almost 60 years, it was not until recently that the exact conforma-tional structure of citrate molecules on the surface of Au NPs was elucidated (Park and Shumaker-Parry 2014). Specifically, it was established that partially protonated citrate mol-ecules (dihydrogen citrate anions) are adsorbed onto the gold surface through a coordi-nation complex with a central carboxylate group. Citrate molecules create a layer with a thickness of ∼10 Å through hydrogen bonding and van der Waals forces with adjacent and adsorbed molecules.

One of the most abundant techniques to obtain organic linkers on the surface of Au NPs is through thiol–gold reaction. This reaction originates from bonding between gold atoms sitting on the surface of Au NPs (adatoms) and sulfur atoms in thiol (Jadzinsky et al. 2007). Previously, it was assumed that the bond between adatoms and sulfur is purely ionic; however, more recently it has been established by Riemers et al. via density function theory (DFT) calculations that the bonding is essentially covalent and relatively strong (Reimers et al. 2010).

A plethora of publications now report the use of a thiol–gold chemistry to functional-ize surface of Au NPs with different species (Figure 3.2). Regardless of the initial capping agent on the surface of the Au NPs (citrate, surfactant, or other thiols), tethered molecules can be attached via a ligand exchange reaction (Yeh et al. 2012). Specifically, introduced molecules with thiol end groups displace the existing species of Au NPs in an equilibrium process. Using this method, researchers have successfully functionalized the surface of Au NPs with organic molecules (PEG, fluorophores, drugs, or analytes) (Gu et al. 2003, Lytton-Jean and Mirkin 2005, Hwu et al. 2008, Perni et al. 2011), biomolecules (proteins, peptides, DNA, iRNA, antibodies, or antigens) (Thanh and Rosenzweig 2002, Giljohann et al. 2010,



Rana et al. 2012, Zhang et al. 2012), and various nanoparticles (Kinoshita et al. 2007, Gandra et al. 2012) (Figure 3.2).

Although gold–thiol bonding is considered to be relatively stable, thiol is able to dissoci-ate from the gold surface under physiological conditions with changing redox conditions. For some time, ligands with dithiolates have been considered superior in their chemi-cal stability compared with mono-thiol ligands, due to their multivalent binding ability. However, it was found that dithiolates are in fact more susceptible to oxidative desorption as a result of inefficient packing and ease of disulfide formation (Hou et al. 2009).

An additional approach to the attachment of organic molecules onto the surface of Au NPs involves using the favorable interaction between gold and amine groups. Amine groups create a complex with gold atoms, but the stability of this interaction is rather weak. For instance, bond strength of gold–amine is ∼6 kcal/mol, while thiol–gold is 47 kcal/mol (Hoft et al. 2007). Although the bonding affinity is significantly lower, it has the advan-tage of easy “release,” which can be exploited as a mechanism for the delivery of drug in therapeutic applications (Vigderman and Zubarev 2013).

3.4 Gold Nanoparticle Cellular Interactions

Despite numerous potential uses of Au NPs and their great promise as a wonder drug, there still remains the obligation by researchers to thoroughly examine their biokinetics and biocompatibility. This includes characterization of the interactions and effects of Au NPs on different cell and tissue types, ranging from changes to protein and gene expres-sion profiles, for understanding how they impact and are distributed within and across the various body systems.

There is growing evidence in the literature that when used within defined concentrations (typically ∼10–100 µM) (Shukla et al. 2005) Au NPs appear to be inert and demonstrate low levels of cell and tissue toxicity; however, this is also highly dependent upon particle size,

PEG

Drugs

ProteinsNa

nopa

rticle

s

Organicmolecules

DNA/RNA

Pept

ides

S

S

SS

S

S SFIGURE 3.2Schematic representation of attachment of different molecules using gold–thiol bond.



particle shape, and surface-coating chemistries (Shukla et al. 2005, Pan et al. 2007). Moreover, attention is now turning to unraveling the specific effects of various surface coatings on the Au NPs, which greatly influence uptake, tissue retention, and circulation half-life (Wang et al. 2015). Great promise is being given to the organic polymer linkers, such as polyethylene glycol (PEG) and PEG variants, with growing support for their apparent ability to confer increased biocompatibility properties to Au NPs (Bogdanov et al. 2014). The following sec-tions will focus on the role of different coatings and other critical parameters, such as particle size and charge, in nanoparticle biodistribution and retention within biological systems.

3.5 In Vitro Cytotoxicity Studies

The common approaches used to assess cell toxicity in in vitro systems are via cell viability assays, such as MTT assay, which assesses cell metabolic activity. The MTT assay uses the drug 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is readily taken up by cells grown in culture (Mosmann 1983). Within viable cells, the MTT is enzy-matically reduced to formazan, resulting in its conversion from a yellow to purple color that is readily detected at 570 nm using a UV–Vis microplate reader. It should, however, be noted that assay systems such as MTT and others employ similar wavelengths to detect color changes by the converted drug of interest (e.g., 570 nm for formazan), which can overlap with the peak emission spectra for the Au NPs, dependent on their size, shape, and concentration (Pan et al. 2007, Kroll et al. 2012). Therefore, the imperative for inclusion of relevant controls in these assays is to exclude interference and background attributable to the Au NPs themselves.

Cellular uptake of particulate material can occur via a variety of processes ranging from phagocytosis to receptor-mediated endocytic uptake. A number of excellent review articles discuss Au NPs cellular interactions in detail (Vonarbourg et al. 2006, Alkilany and Murphy 2010, Zhao et al. 2011). As expected, Au NP cellular cytotoxicity is greatly influenced by nanoparticle size and surface charge (Zhao et al. 2011). A study of varying sized Au NPs ranging between 0.8 and 15 nm in diameter, coated with monosulfonated triphenylphos-phane, demonstrated that particles of 1.4 nm diameter were the most toxic with an IC50 of 46 µM. Similarly sized particles were found to be less toxic with 0.8, 1.2, 1.8, and larger 15 nm Au NPs having IC50 values of 250, 140, 230, and 6300 µM, respectively (Pan et al. 2007). This same study also showed that the cellular effects of the toxicity differed between the parti-cles, with the 1.4 nm particles conferring rapid cell death via necrosis within 12 hours, while the similar-sized 1.2 nm Au NPs induced death via an apoptotic pathway (Pan et al. 2007).

A more recent report showed that 4.5 nm PEG-coated Au NPs added to cultured mouse myoblastoma cells were also found to be noncytotoxic up to a concentration of 5 × 1013 particles/mL (Leite et al. 2015). However, when the same Au NPs were added to cells in addition to the drug staurospaurin, they induced a greater cell susceptibility to the effects of the drug. The authors concluded that the PEG-coated Au NPs potentiate the effects of the drug via apoptotic means (Leite et al. 2015).

3.5.1 In Vitro Studies Reveal PEG-Coating Density Regulates Cellular Uptake of Au NPs

As mentioned previously, coating Au NPs with PEG has been shown to reduce in vitro cellular uptake and improve biocompatibility of the particles (Vonarbourg et al. 2006).



Attention has now turned to studying the underlying mechanisms responsible for this biocompatibility, in order to better understand how these effects arise, which will ulti-mately allow for the fine-tuning and rational design of tailored nanoparticles destined for biological applications. Some recent developments are outlined below.

The cellular uptake of PEG-coated Au NPs and the effect of PEG-coating density were studied in detail using murine macrophage cell line J774A.1 (Walkey et al. 2012). The study found that the degree of serum protein adsorption onto the particles directly correlated with particle uptake by the cells (Walkey et al. 2012). The researchers showed that as they increased PEG-coating density (0–1.25 PEG/nm2), the amount of adsorbed serum proteins onto the particles decreased. They then demonstrated that not only was the amount of bound protein different, but also there was a concomitant change in the types of serum proteins bound to the particles. Detailed analysis revealed that they could group the pro-teins in clusters that correlated with the various PEG-coating densities and these clusters provided insight into the cellular uptake mechanisms and efficiencies of uptake. Overall, they demonstrated that not only Au NPs size but also PEG-grafting density ultimately controls the cellular uptake mechanism and uptake efficiency (Walkey et al. 2012). Even at high PEG-coating density that greatly reduced nonspecific serum protein binding, it did not totally eliminate Au NPs uptake by the cells.

Comparison of the cellular uptake of PEG-coated nanospheres (50 nm, negatively charged) compared to nanorods (10 × 45 nm, near neutral charge) by macrophage cell line RAW264.7 demonstrated that shape and surface charge remain critical factors in cellular interactions (Janát-Amsbury et al. 2011). Specifically, the researchers found that there were around four times as many PEG-coated nanospheres compared to nanorods in compa-rable cell samples (Janát-Amsbury et al. 2011). Their in vivo studies also demonstrated a significant reduction of nanorod accumulation in the liver and a longer blood circulation half-life compared to the spherical particles. They explained these difference largely in terms of the varied surface charge of the particles and indicated that particle geometry is also likely to influence these processes; however, they did not expound a mechanism for this, instead concluding that more work needs to be done (Vonarbourg et al. 2006).

Stability of the PEG surface coatings on the Au NPs is another critical aspect of the bio-compatibility characterization process. Research has now emerged to indicate that physi-ological concentrations of cysteine can displace methoxy-PEG-thiol molecules from the Au NPs surface (Larson et al. 2012). Researchers showed that once the PEG particles were placed in cell culture media containing serum, the PEG coating was displaced by cysteine molecules, allowing for the adsorption of proteins onto the particle surface. To overcome this problem, the group included a small alkyl chain, which they refer to as a “hydropho-bic shield,” between the Au NPs surface and the outer hydrophilic PEG layer. In doing so, they found that mPEG–alkyl-thiol-coated Au NPs had greatly reduced protein adsorption and this also led to reduced uptake in vitro, by macrophage cells presumably mediated via the protein corona that normally forms on the mPEG–thiol particles (Larson et al. 2012).

Similarly, a combined in vivo and in vitro study of 5 nm polymer-coated Au NPs revealed that the polymer coating was partially removed following introduction of the particles to biological system (Kreyling et al. 2015). In vivo, the injected Au NPs were largely retained within the liver of the rats, while fragments of the coatings were excreted through the kidney (Kreyling et al. 2015). Complementary in vitro studies suggested that the poly-mer coating was degraded via enzymatic proteolytic cleavage within cells of the liver (Kreyling et al. 2015). This was supported by demonstration that the internalized particles were localized to lysosomal and endosomal compartments within HUVEC and Kuppfer macrophage cells (Kreyling et al. 2015).



3.6 In Vivo Biokinetic and Biodistribution Studies

Controlling in vivo Au NP localization has obvious benefits, with active and passive target-ing approaches becoming better understood and exploited. An increasing number of studies demonstrate that coating of Au NPs with molecules, such as PEG, results in reduced surface fouling of the particles and steric stabilization (Shao et al. 2011). This has been suggested to be due to the inhibition or blocking of nonspecific attachment by serum proteins onto the Au NPs surface preventing the formation of a protein corona and reduced opsonization. In turn, it is believed that PEG coating results in a reduced incidence of uptake and clearance of these PEG-coated Au NPs by cells of the reticulo-endothelial system (Shao et al. 2011, Walkey et al. 2012). The coating appears to act as a “stealth” or “invisibility cloak” around the Au NPs, increasing their circulation half-life within the body and bloodstream. Given the advantages provided by these coatings in making Au NPs more biocompatible and better targeted, a clearer understanding of their mechanism of action is obviously needed.

In vivo studies using animal models to determine Au NPs biocompatibility, clearance from the body, and accumulation within discrete cell and tissue types have usually been assessed by isolation of the tissues of interest, followed by approaches to quantify the pres-ence of Au NPs. Detection methods include scanning and transmission electron micros-copy (Pissuwan et al. 2009, Chen et al. 2013), along with quantification methods, such as inductively coupled plasma mass spectrometry (ICP-MS) (Chen et al. 2013) and radioactive labeling of the particles themselves (Hirn et al. 2011, Kreyling et al. 2014). The follow-ing section summarizes a collection of studies carried out principally by a team based at the Helmholtz Zentrum Munich, Germany, that demonstrate the power of well-designed systematic research that can begin to form the basis for developing models to accurately predict the behavior of Au NPs when introduced into the body.

The three, 24-hour biokinetic studies were carried out in female rats, using monodis-persed, spherical Au NPs across a range of core diameter sizes (monosulfonated triphe-nylphosphane [TPPMS]-coated 1.4, 5, 18, 80, 100 nm Au NPs as negatively charged surface), as well as 2.8 nm size (either carboxyl-coated negative surface or amino-coated positive surface), administered by following methods:

1. Intravenous (IV) injection into the tail vein, and following the movement of the particles from the bloodstream into body organs, tissues, and excrement (Hirn et al. 2011)

2. Oral ingestion by delivery of the particles intra-oesophageally, and following the absorption and uptake of the particles through the gastrointestinal tract (GIT) into the body systems and excrement (Schleh et al. 2012)

3. Inhaled Au NPs uptake, through the lung air–blood barrier, delivered by intratra-cheal instillation to determine accumulation of the particles in the body organs, tissues, and excrement of the animals (Kreyling et al. 2014)

The three studies confirmed the importance of size and surface charge on the in vivo distribution and accumulation of the particles, as well as demonstrating that the admin-istration route was also a critically important determining factor. Specifically, the par-ticles administered orally demonstrated the lowest uptake of all three procedures with <0.4% present in the internal organs and tissues, while between 99.63% and 99.99% of the different-sized and charged particles were found to be located in the GIT and feces at the 24-hour time point post-ingestion (Schleh et al. 2012). Overall, the study showed



that the smaller-sized and more negatively charged particles tended to have higher lev-els of absorption and accumulation in tissues and organs. However, the researchers take care to point out that the 18-nm particles were more readily absorbed compared to the 5-nm particles and were the most highly accumulated particle in the brain of all the Au NPs tested. Moreover, they discussed the role that the types of adsorbed proteins forming the “corona” also likely influence particle accumulation. They speculated that the profile of adsorbed proteins onto the particle surface would be different based on the route of administration and, hence, also led to different tissue localization and accumulation sites. They concluded that individual tailoring and bespoke design of Au NPs for specific organ targeting is necessary (Schleh et al. 2012).

The study of body distribution 24 hours post-IV injection of the various Au NPs showed a rapid movement of the particles from the blood circulation, predominantly into the liver (Hirn et al. 2011). The researchers found that the vast majority of 5–200 nm particles ended up in the liver (91.9%–96.9%), while 81.6% of the negatively charged 2.8 nm particles, 72% for the positively charged 2.8 nm particles, and only 51.3% of the 1.4 nm particles localized to the liver. They demonstrated the existence of a linear relationship between the par-ticles sized 1.4–5 nm in diameter, when plotted against liver retention at 24 hours post-IV injection. They, however, found little size dependency of accumulation in other tissues for particles sized between 18 and 200 nm.

Moreover, some differences have been identified in the accumulation of positively ver-sus negatively charged particles. The results obtained for the spleen, for example, had a 2% accumulation of all particles ranging in size from 1.4 to 200 nm that had a negative TPPMS-coated surface, while the 2.8-nm particles surface coated either carboxyl-negative or amino-positive showed accumulation rates of 8.6% and 11.4%, respectively. This was considered as an unexpected result, given that the spleen, which is part of the reticulo-endothelial system, has previously been shown to accumulate other particles in a size-dependent fashion (Moghimi et al. 1991, Hirn et al. 2011). Again, surface charge was raised as a potential difference resulting in distinct proteins binding onto the particle to form the “corona.” Others have also shown variation in organ distribution of 15-nm PEG-coated Au NPs with differing surface charges (Lee et al. 2015). This “dynamic protein binding and exchange” in turn is suggested to influence the ultimate site of particle accumulation and retention (Hirn et al. 2011).

The final study of this trilogy was aimed at examining the Au NPs distribution and accumulation within the body post-inhalation (Kreyling et al. 2014). The researchers mea-sured biodistribution at 1, 3, and 24 hours post-intratracheal administration of the various-sized particles. They found a strong size dependency, with smaller-sized particles much more likely to cross the air–blood barrier compared to the larger-sized particles up to 80 nm in diameter (Kreyling et al. 2014). The percentage of Au NPs translocated across the air–blood barrier at 24 hours was ∼6%–7% of 1.4 nm Au NPs, <0.1% of 80 nm Au NPs, and ∼0.2%–0.3% of the 200 nm Au NPs. Therefore, of the total 100% administered, only a relatively small percentage of particles had in fact entered the body past the air–blood barrier within a 24-hour time frame. Interestingly, the 200 nm Au NPs did not follow this size-dependency relationship, as they translocated much more readily compared with the 80-nm particles. However, relative to the amount of Au NPs that crossed the air–blood barrier, retention within secondary organs and tissues was found to be independent of the Au NPs size. The researchers also importantly point out that the processes of Au NP translocation from the air into the bloodstream and subsequent accumulation and reten-tion within organs and tissues should be viewed as two distinct processes, with each influenced independently by Au NP size and surface charge (Kreyling et al. 2014).



An additional important finding of the researchers was that Au NP retention in the rat carcass (comprising skeleton, soft tissue, and fat) was greater than that of all the secondary organ retention across all different-sized Au NPs (Kreyling et al. 2014). Of these total carcass retained Au NPs, 10%–20% were within the skeleton and presumed to be located within the bone marrow, as they most likely arrived there via the bloodstream. It is highlighted that these Au NPs particles are, thus, in direct contact with the pluripotent stem cells located within the bone marrow, which are known to be highly sensitive to exogenous stimuli (Kreyling et al. 2014). These findings serve to remind us of the complexities such studies face in attempting to unravel the myriad of processes involved in particle accumulation within tissues and organs.

Independent studies by researchers at the University of Technology, Sydney, Australia, also support the dependency between in vivo accumulation and size distribution of Au NPs, upon their initial route of administration. A study of 20–30 nm citrate-coated Au NPs administered via intraperitoneal injection in mice resulted in significant accumula-tion of the particles in the abdominal fat pad as well as in the liver, with no evidence of accumulation in brain, kidney, and heart tissues, at 72 hours post-injection (Chen et al. 2013). Another group of researchers using 25-nm-sized Au NPs (polyvinyl alcohol stabi-lized) compared particle distribution following oral or intravenous administration in rats over a relatively long period (Bednarski et al. 2015). After 10 days post-administration, researchers showed the injected Au NPs accumulated principally in the liver (>50%), with relatively smaller amounts in the lungs (5.7%) and spleen (2%) and only minor amounts recovered from the urine and feces during the 10-day period. The orally administered Au NPs showed a completely different distribution profile, with the majority of the particles excreted in the feces (55.8%) within the first 4 days of administration, and ∼50-fold fewer particles detected within internal organs (Bednarski et al. 2015). The particles were princi-pally located within the heart (0.6%), serum (0.3%), and brain (0.23%) (Bednarski et al. 2015). Recovery from the organs, of the initial dose of Au NPs, post 10 days, was 60% from the injected rats and only 1.4% from the orally administered rats (Bednarski et al. 2015).

Results of an in vivo study examining Au NPs toxicity undertaken in mice further extend this correlation in support of the importance of administration route on Au NPs activity and fate (Zhang et al. 2010). The study used unmodified 13.5 nm Au NPs that were admin-istered via three different routes: oral, peritoneal injection, and tail vein injection, over a range of concentrations. The study found that of the three administration routes, the least toxic was the intravenous injection route (Zhang et al. 2010).

It is clear that a much deeper understanding of how Au NPs interact within biological systems is needed before they can be accepted as medical and mainstream drug and thera-peutic agents. However, this journey of discovery is also proving to be highly valuable for equally important reasons of basic biological and medical research. In vivo and cell in vitro studies of these particles have already led to a number of novel and serendipitous findings (Vonarbourg et al. 2006, Chen et al. 2013), which in the long run can only serve to enrich our fundamental understanding of the natural and physical world, as well as providing new possibilities in the treatment of disease.

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