Research Article Received: 14 July 2014,

Revised: 12 August 2014,

Accepted: 23 August 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.3075

Organ-specific distribution of gold nanoparticles by their surface functionalization Jong Kwon Leea, Tae Sung Kima, Ji Young Baea, A. Young Junga, Sang Min Leea, Ji Hyun Seoka, Hang Sik Roha, Chi Won Songa, Mi Jin Choib, Jinyoung Jeongb, Bong Hyun Chungb, Yun-Geon Leec, Jayoung Jeonga* and Wan-Seob Choc* ABSTRACT: The behavior and fate of intravenously (i.v.) injected nanoparticles (NPs) can be controlled by several physicochemical factors including size, shape and surface charge. To evaluate the role of surface charge on distribution of NPs, we used neutral-charged 15-nm-sized polyethylene glycol-coated gold nanoparticles (AuNPPEG) as a core NP and carboxyl or amine groups were conjugated to AuNPPEG to generate negative (AuNPCOOH) or positive AuNP (AuNPNH2), respectively. Each type of AuNP was i.v. injected into mice (1 mg kg–1) and the concentration of Au was measured in different organs at 30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months post-injection. The organ distribution also showed the higher deposition rate depending on their functional groups: AuNPPEG for mesenteric lymph node, kidney, brain and testis; AuNPCOOH for liver; AuNPNH2 for spleen, lung and heart. The blood circulation time and the major excretion route were different depending on their functional groups. In conclusion, functional groups conjugated on the surface of AuNPs produce differences in blood kinetics, organ distribution and elimination pattern which can be important information for directing NPs to specific organs or improving the kinetic properties. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: gold nanoparticle; functional group; charge; tissue distribution; intravenous injection

Introduction Gold nanoparticles (AuNPs) are promising candidates for biomedical applications such as imaging, diagnostics and therapeutics because of their relative ease of synthesis and surface modification as well as novel physicochemical properties. For example, AuNPs can be used as contrast agents because of their surface plasmon resonance (Ahn et al., 2013; Lee et al., 2013). In addition, cancer treatment by photothermal therapy (Kennedy et al., 2011; Kuo et al., 2012) and delivery of anti-cancer biomolecules (Pissuwan et al., 2011) can be achieved using AuNPs. These novel properties have stimulated the development of multimodal AuNPs for effective diagnostic and therapeutic functions (Kim et al., 2009; Panchapakesan et al., 2011). AuNPs have been studied for in vivo applications and diverse routes of administration are possible such as intravenous (i.v.) injection, inhalation, oral ingestion and topical application. Among these routes, i.v. injection might be a major route for application in vivo than the others. Therefore, understanding the kinetic characteristics of AuNPs, including absorption, distribution, metabolism and clearance, is critical for the success of injectable AuNPs. In addition, the modulation of kinetic properties to target-specific organs may be important to reduce their associated risks and increase their efficacy (Daniel and Astruc, 2004). Representative tailorable physicochemical properties that can modulate AuNP kinetics are shape, size, surface functional groups. Many previous animal studies have addressed the impact of particle size on the tissue distribution of AuNPs by i.v. injection (De Jong et al., 2008; Semmler-Behnke et al., 2008; Sonavane

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et al., 2008; Cho et al., 2010; Hirn et al., 2011). AuNPs were known to accumulate in the reticuloendothelial organs including the liver, spleen and lymph nodes, and their minimal clearance rate via the urinary and hepato-biliary systems (De Jong et al., 2008; Cho et al., 2010). The AuNPs are mainly localized in the phagolysosomes of the reticuloendothelial cells such as macrophages and Kupffer cells (Sadauskas et al., 2009; Cho et al., 2010). Smaller AuNPs show more widespread distribution and higher levels in the liver, spleen and lymph nodes than larger AuNPs (De Jong et al., 2008; Cho et al., 2010). Surface functionalization has recently been highlighted in biological applications aimed at controlling the behavior and *Correspondence to: Professor Wan-Seob Cho, Department of Medicinal Biotechnology, College of Natural Resources and Life Science, Dong-A University, 840 Handan-2dong, Saha-gu, Busan 604-714, Republic of Korea. Email: [email protected] Dr Jayoung Jeong, Toxicological Research Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, 187 Osongsaengmyeong-2ro, Osong-eup, Cheongwon-gun, Chungcheongbuk-do, 363-700, Republic of Korea. Email: [email protected] a Toxicological Research Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong 363-700, Republic of Korea b Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-700, Republic of Korea c Department of Medicinal Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea

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J. K. Lee et al. fate of administered NPs. Recent studies showed that surface functionalization, especially surface charge, is closely related to the toxicity or inflammogenicity of NPs (Donaldson et al., 2013; Kim et al., 2013; Li et al., 2013). In general, positively charged NPs are known to be more toxic or inflammogenic than negatively charged ones (Donaldson et al., 2013). However, these differential toxicities by the role of surface functionalization can also be applied for the targeting or therapeutic effects of NPs. Moreover, relatively little is known about the role of surface functionalization in the kinetics of NPs including blood circulating time, tissue distribution pattern and elimination (Alexis et al., 2008; Nel et al., 2009; du Toit et al., 2011; Zhu et al., 2013). Therefore, to evaluate the role of surface functional groups or surface charge on the behavior and fate of NPs, we synthesized positive-, neutral- and negative-charged AuNPs having the same core and i.v. injected into mice. We then evaluated the role of surface functionalizations on their tissue distribution pattern for up to 6 months.

Materials and Methods Synthesis of Three Types of AuNPs AuNPs were synthesized by the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) as previously described with some modification (Jana et al., 2001). For synthesis of AuNPPEG, 800 mg of HAuCl4 (Sigma-Aldrich, St. Louis, MO, USA) was added to 800 ml of distilled water (DW) with continuous stirring. Then, 140 ml of 1% citric acid (Sigma-Aldrich) was added and stirred for 10 min to obtain AuNPs. Methoxypoly (ethylene glycol)-thiol (M.W. 5 K, mPEG-SH; SunBio, Anyang, Korea) at 400 mg in 80 ml of DW was added and incubated overnight with stirring. AuNPs were then washed by centrifugation (17 900 g; 5 min) to remove unbound polyethylene glycol (PEG). Samples were then resuspended at 1 mg ml–1 in phosphatebuffered saline (PBS, pH 7.4). For synthesis of AuNPCOOH, 800 mg of HAuCl4 (Sigma-Aldrich) was added to 800 ml of DW with continuous stirring. Then, 140 ml of 1% citric acid (Sigma-Aldrich) was added and stirred for 10 min to obtain AuNPs, and 8 ml of 100 mM 11-mercaptoundecanoic acid (MUA; Sigma-Aldrich) was then added with continuous stirring for 4 h. mPEG-SH (M.W. 5 K; SunBio) at 400 mg in 80 ml of DW was added and incubated overnight with continuous stirring. AuNPs were washed by centrifugation (17 900 g; 5 min) to remove unbound PEG and samples were then resuspended in PBS at a concentration of 1 mg ml–1. For synthesis of AuNPNH2, 750 mg of HAuCl4 (Sigma-Aldrich) was added to 1500 ml of DW with continuous stirring. Then 100 mM 11-amino-1-undecanethiol hydrochloride (AUT; SigmaAldrich) was added and incubated for 20 min with stirring, and 1.5 ml of 100 mM NaBH4 (Sigma-Aldrich) was applied and incubated for 4 h. Then, 400 mg of mPEG-SH (M.W. 5 K; SunBio) in 80 ml of DW was added and incubated overnight with continuous stirring. AuNPs were washed by centrifugation (17 900 g; 5 min) to remove unbound PEG. Samples were resuspended in PBS at a concentration of 1 mg ml–1.

infrared spectroscopy (FTIR) analysis was applied to evaluate the presence of organic molecules such as PEG on the surface of the AuNPs. To measure the primary particle size and morphology, transmission electron microscopy (TEM) was applied using a CM20 microscope (Philips, Eindhoven, Netherlands). The mean diameter and size distribution of AuNPs were evaluated in at least 100 AuNPs. The hydrodynamic size of AuNPs in PBS was evaluated using dynamic light scattering (ELS-Z, Otsuka, Japan). The surface charge of the AuNPs was measured as the zeta potential using an ELS-Z (Otsuka). To measure the zeta potential of AuNPs, particles were serially diluted with 0.1 × PBS to evaluate the optimal concentration, and the zeta potential was calculated according to the cumulative method provided in the built-in software. The agglomeration status of AuNPs in 0.1 × PBS was also evaluated using UV-Vis spectroscopy (Beckman Coulter DU 800; Beckman Coulter, Fullerton, CA, USA). The endotoxin concentrations of the AuNPs (1 mg ml–1 in PBS) were measured using an Endpoint Chromogenic Limulus Amebocyte Lysate assay (Cambrex, MD, Walkersville, USA). AuNPs at 1 mg ml–1 were also plated on blood agar and incubated for 24 h at 37 °C to evaluate possible microbial contamination. Animal Handling Male specific-pathogen-free BALB/c mice (6 weeks old) were purchased from Samtako (Osan, Korea) and acclimated for 7 days after arrival at the animal facility at the National Institute of Food and Drug Safety Evaluation of the Ministry of Food and Drug Safety (Osong, Korea). Mice were housed individually in polycarbonate cages at a controlled temperature (21–23 °C), humidity (38–55%) and light cycle (12 h light/dark). Water (autoclaved water) and feed (LabDiet 5002; PMI Nutrition, Richmond, VA, USA) were supplied ad libitum. Animals were maintained in accordance with AAALAC International Animal Care Policies as provided by the Animal Care and Use Committee of the National Institute of Food and Drug Safety Evaluation of the Ministry of Food and Drug Safety. Intravenous Injection of AuNPs Thirteen mice per group were randomly assigned to receive three types of AuNPs dispersed in PBS at 1 mg kg–1 body weight (22.9–23.4 μg per mice) through i.v. injection via the tail vein. The dose of administration was adopted from our previous study (Cho et al., 2010). After injection, the mice were sacrificed at each time point (30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months) by intraperitoneal injection of ketamine. The mice were weighed and blood was collected from the abdominal vein for measurement of Au levels. Several organs including the liver, spleen, mesenteric lymph node, lung, heart, kidneys, brain and testes were then collected and weighted. At least five mice per each group or time point were used for ICP-MS (inductively coupled plasma-mass spectrometry) analysis and others were used for histopathological analysis. Determination of AuNP Levels by ICP-MS Analysis

Physicochemical Characterization The concentration of AuNPs in PBS was confirmed by ICP-AES (inductively coupled plasma-atomic emission spectrometry) (Optima 3300DV; Perkin-Elmer). Fourier infrared transform

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Sample preparation and ICP-MS analysis after necropsy were performed according to a previously described method (Cho et al., 2009, 2010). Briefly, whole blood, liver, spleen, mesenteric lymph node, lung, heart, right kidney, brain and right testis were

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Organ-specific distribution of AuNPs by surface functionalization collected at each time point. Samples were placed in 100-ml Teflon microwave digestion vessels and digested using a microwave digestion system (Milestone, Sorisole, Italy) at a working temperature of up to 190 °C after 8 ml of aqua regia (6 ml of HCl and 2 ml of HNO3) was added. The mixture was then transferred to another vial, and DW was added to a total volume of 50 ml. The levels of gold were measured by ICP-MS (PerkinElmer, Waltham, MA, USA) under the following conditions: 1350 W RF power, 18 l min–1 plasma gas flow rate and 1.20 l min–1 auxiliary gas flow rate. The concentration of gold was calibrated using 197Au.

Hematological and Histopathological Analysis Blood was collected using an EDTA-containing Vacutainer tube (BD Biosciences, San Jose, CA, USA) for hematological analysis. Hematological analysis was performed using a Bayer ADVIA120 hematology analyzer (Leverkusen, Germany) according to the manufacturer’s instructions. Organs were fixed in 10% neutral-buffered formalin and trimmed, and then paraffin-waxed

tissue blocks were prepared using a routine histological process. Paraffin blocks were then sectioned at 3 μm and stained with hematoxylin and eosin (H&E) for histopathological evaluation by toxicologic pathologists (J.K.L. and W.-S.C.). ICP Data Analysis The concentration of gold was presented as gold per organ weight (ng g–1 organ weight), gold per organ (ng per organ), and percentage of gold based relative to the initial dose (%ID). The levels of gold per organ weight were multiplied by the organ weight to calculate the concentration of gold per organ. The concentrations of gold for the paired organs were summed to calculate the percentage of AuNPs relative to the initial dose. The total weight of the blood was calculated as 6% of the body weight. Statistical Analysis Data were analyzed and plotted using GraphPad Prism software (Ver. 5; GraphPad Software Inc., La Jolla, CA, USA). The body

Table 1. The physicochemical properties of three different gold nanoparticles (AuNPs) (mean ± SD) Abbreviation

Functionalization

Primary size (nm)

Hydrodynamic size (nm)

Zeta potential (mV)

Endotoxin or microbial growth

AuNPPEG AuNPCOOH AuNPNH2

PEG-SH MUA/PEG-SH AUT/PEG-SH

15.01 ± 2.70 15.03 ± 2.49 15.38 ± 2.98

29.80 ± 0.39 32.60 ± 2.05 63.70 ± 1.43

0.35 ± 0.26 -28.40 ± 3.03 16.20 ± 0.77

Not detected Not detected Not detected

Figure 1. Synthesis and characterization of gold nanoparticles (AuNPs). Transmission electron microscopy (TEM) of (A) AuNPPEG, (B) AuNPCOOH, and (C) AuNPNH2. Note that all NPs had a diameter of approximately 15 nm and a narrow size distribution. (D) Fourier transform infrared spectroscopy (FTIR) analysis showed that all three types of AuNPs had a peak at 520 nm, which means that all AuNPs had the same polyethylene glycol (PEG) layer. (E) ultraviolet-visible (UV-Vis) spectrometry showed that all AuNPs were well dispersed in phosphate-buffered saline (PBS).

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J. K. Lee et al. weight and hematological data were compared by one-way ANOVA with post-hoc Tukey’s pair-wise analysis. P < 0.05 was considered to be statistically significant.

Results Physicochemical Properties of AuNPs The physicochemical properties of AuNPs including primary particle size, hydrodynamic size, zeta potential and microbial contamination are summarized in Table 1. Three types of AuNPs showed 15 nm as a core. Neutral charged AuNPs (AuNPPEG) were synthesized by conjugation of PEG-SH. The sequential conjugation of MUA and PEG-SH or AUT and PEG-SH on the surface of bare AuNP produced a negative or positive zeta potential, respectively. The primary size of each AuNP was varied by less than 2% (Table 1 and Fig. 1). The hydrodynamic size of AuNPNH2 was larger than AuNPPEG or AuNPCOOH, but all AuNPs showed a size less than 100 nm. Because of the PEG layer and surface functional groups, the hydrodynamic size of the AuNPs was larger than the primary size measured by TEM. Three types of AuNPs showed neither endotoxin contamination measured by LAL analysis nor microbial contamination on the blood agar plate. In Fig. 1D, FTIR analysis showed that all AuNPs were modified with each chemical [1696 cm–1 (C = O stretch) in AuNPCOOH, 1092 cm–1(C-N stretch) in AuNPNH2, and PEG (1092 cm-1(C-O stretch) and 2851 cm-1(CH stretch)]. In UV-Visible spectra in Fig. 1E, three types of AuNPs showed the maximum peak around 520 nm, corresponding to the unique characteristic of welldispersed AuNPs (localized surface plasmon resonance) in PBS.

The Total Amount of Au in the Tested Organs and Blood The total amounts of Au in the tested organs (liver, spleen, mesenteric lymph node, lung, heart, kidney, brain and testis) were calculated by multiplying the amount of Au by the respective organ weight and presented as a percentage of the initial dose (Fig. 2A). The amount of AuNPCOOH and AuNPNH2 showed a continuous increasing trend until 24 h post-injection and a slightly decreasing trend thereafter. In contrast, the peak levels of AuNPPEG were observed at 7 days post-injection, which was consistent with the longer blood circulation time of AuNPPEG (Fig. 2B). AuNPPEG showed about 5700 ng g–1 per blood weight at 30 min post-injection but only 17.8 ng g–1 remained at 24 h post-injection (Fig. 2B). Interestingly, AuNPCOOH and AuNPNH2 showed minimal levels of Au even at 30 min after injection.

Tissue Distribution Pattern of AuNPs After Intravenous Injection As measured by the concentration of Au per organ weight, the i.v. injected AuNPs were mainly distributed in the liver, spleen and mesenteric lymph node whereas the kidney, lung, heart, brain and testis showed much lower amounts (Figs. 3 and 4). AuNPNH2 showed much higher levels of Au in the spleen, lung, and heart than the other two types of AuNPs throughout the study. AuNPPEG showed higher levels of Au in the mesenteric lymph node, kidney, brain and testis than the other two types of AuNPs, whereas AuNPCOOH showed higher levels of Au in the liver than the other NP types.

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Figure 2. Levels of gold nanoparticles (AuNPs) in the tested organs and blood kinetics. (A) The percentage of AuNPs relative to the initial injected dose. Total amounts of AuNPs were calculated by adding the concentration of AuNPs in each tested organ (liver, spleen, mesenteric lymph node, lung, heart, kidney, brain and testis). (B) The kinetics of AuNPs in blood. Each sample was collected at 30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months. n = 5 for each group. Data were expressed as the mean ± SEM.

Elimination Pattern of AuNPs All AuNPs presented very low levels in the urine and bile throughout the study (Fig. 5). AuNPPEG and AuNPNH2 presented relatively higher levels in the urine than AuNPCOOH (Fig. 5A). However, AuNPCOOH presented higher levels in the bile than the other two types of AuNPs (Fig. 5B).

Clinical Observations, Hematology and Histopathology There were no statistically significant body weight changes or organ weight changes in the treatment groups compared with the vehicle control group (data not shown). In addition, there were no significant treatment-related hematological changes compared with the vehicle control (Table S1, see Supporting Information). There were no significant treatment-related histological changes compared with the vehicle control (data not shown).

Discussion Surface functionalization is routine for application of NPs in biomedical applications but little is known about the role of surface functionalization on tissue distribution because of the limitation of optimal model NPs which are non-toxic and chemically stable

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Organ-specific distribution of AuNPs by surface functionalization

Figure 3. Levels of gold nanoparticles (AuNPs) per organ weight in the (A) liver, (B) spleen, (C) mesenteric lymph node and (D) kidney measured by inductively coupled plasma-mass spectrometry (ICP-MS). Each sample was collected at 30 min, 4, 24, 7, 14 days, 1, 3, and 6 months after a single intravenous injection into mice. n = 5 for each group. Data were expressed as the mean ± SEM.

(Almeida et al., 2011). In this study, we selected AuNP as a model NP because it is known to be relatively non-toxic and easy to synthesis and modify (Daniel and Astruc, 2004; Johnston et al., 2010). To evaluate the role of surface functionalization on tissue distribution, three types of AuNPs with the same core but different functional groups (PEG-SH, MUA/PEG-SH and AUT/PEG-SH) were synthesized and injected into the mouse tail vein. The information about the role of surface functionalization on organ distribution in this study might provide important information on directing NPs to specific organs or improving the kinetic properties. AuNPCOOH and AuNPNH2 were rapidly distributed from the bloodstream into the organs, which was consistent with the high levels of Au in the liver and other tested organs at the early time-point. However, AuNPPEG showed a longer blood circulation time. Considering that all NPs had PEG-SH as their functional group, it is interesting that the conjugation of carboxyl and amine groups facilitated distribution into tissues. In previous studies, i.v. injection of pristine AuNPs showed almost complete blood clearance into organs within 24 h post-injection as demonstrated by 15, 20, 50, 100 and 200 nm AuNPs (Sonavane et al., 2008; Balasubramanian et al., 2010). In contrast, i.v. injection of 4 and 13 nm PEG-coated AuNPs showed high levels of Au at 30 min and 4 h post-injection (Cho et al., 2010). The short blood circulation time of AuNPCOOH and AuNPNH2 may indicate that conjugation of carboxyl or amine groups produce higher binding affinity to serum proteins and immunoglobulins, and

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thus facilitate opsonization by immune cells (Pedraza et al., 2008). Likewise, galactose-PEG-conjugated AuNPs showed faster accumulation in the liver than PEG-coated AuNPs by receptormediated endocytosis (Kawasaki et al., 1986; Bergen et al., 2006). Intravenous injection of PEG-oligocholic acid-based micellar NPs having seven different zeta potentials ranging from–-26.9 mV to 37.0 mV showed that NPs with a high surface charge, either positive or negative, were taken up more in the liver and less in the tumor mass which implies a shorter blood circulation time (Xiao et al., 2011). These results were consistent with our study which showed AuNPNH2 and AuNPCOOH had a shorter blood circulation time than AuNPPEG. However, some surface modification can elongate the blood half-life [e.g. i.v. injection of mixed-charge zwitterionic surface modification of AuNPs showed a much longer blood half-life (about 30.6 h) compared with PEGylated AuNPs (about 6.65 h) and a much lower uptake by reticuloendothelial organs] (Liu et al., 2014). The liver, spleen, lymph node and lung were the major organs that showed high levels of Au for all AuNPs, although there was a functional group-specific distribution pattern. Morais et al. found that functionalization of AuNPs with (1) citrate, (2) 11mercaptoundecanoic acid (11-MUA), (3) Cys-Ala-Leu-Asn-Asn (CALNN), (4) Cys-Ala-Leu-Asn-Ser (CALNS) and (5) Cys-Ala-LeuAsn-Asp (CALND) were mainly distributed in the liver, spleen and lung whereas the lymph node was not tested (Morais et al., 2012). Balasubramanian et al. (2010) showed that pristine 20 nm AuNPs were mainly located in the liver, spleen, olfactory

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J. K. Lee et al.

Figure 4. Levels of gold nanoparticles (AuNPs) per organ weight in the (A) lung, (B) heart, (C) brain and (D) testis measured by inductively coupled plasma-mass spectrometry (ICP-MS). Each sample was collected at 30, 4, 24 h, 7, 14 days, 1, 3 and 6 months after a single intravenous injection into mice. n = 5 for each group. Data were expressed as the mean ± SEM.

bulb, kidney and lung. The lower retention rate of AuNPNH2 in the liver compared with AuNPCOOH was consistent with the higher retention rate in the spleen, lung and heart. Similarly, the low retention rate of AuNPPEG in the liver up to 24 h was consistent with the high retention rate in the kidney, brain and testis. Although agglomeration of NPs may increase the lung burden, previous studies using AuNPs of different sizes (4–200 nm) showed that the lung burden decreased as the size of the AuNPs increased (Sonavane et al., 2008; Cho et al., 2010). The AuNPNH2 used in this study had a larger agglomeration size (63.7 nm) than the other two NPs (29.8 nm for AuNPPEG and 32.6 nm for AuNPCOOH). Positively charged NPs showed a higher uptake in the lung cells (Zhang et al., 2011) and longer retention time in the lung tissue (Choi et al., 2010) than neutral or negatively charged NPs because high zeta potential of NPs can bind to the cell membrane more efficiently (Donaldson et al., 2013). Therefore, the higher retention rate of AuNPNH2 in the lung may have resulted from its functional group or surface charge. The surface charge-dependent accumulation of AuNPs in the liver in our study was consistent with a previous study which showed negatively charged nanostructured lipid carriers (NLCs) were taken up more efficiently than positive ones (Beloqui et al., 2013). However, the distribution pattern of NLCs in the kidney, spleen and lung showed a different pattern compared with AuNPs in our study. Therefore, further studies are warranted to evaluate the impact of surface functionality on organ distribution.

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The levels of AuNPs in the liver were continuously decreased with a slow pace which showed still high levels even at 6 months post-injection. In the mesenteric lymph node, both AuNPCOOH and AuNPNH2 showed a significantly lower retention rate than AuNPPEG. The high retention rate of AuNPPEG in the lymph node after 24 h was consistent with our previous study (Cho et al., 2010), but the low retention rate of AuNPCOOH and AuNPNH2 was noteworthy. The higher levels of AuNPPEG in the lymph node may have resulted from a re-distribution rate from the primary retained organs (Borchardt et al., 1994; Yang et al., 2007; Cho et al., 2010). The levels of AuNPs in the liver relative to the initial dose slowly but constantly decreased, and remained at approximately 40% after 24 h. Low elimination of AuNPs from the organs was observed regardless of their surface functional groups, which was consistent with the low levels of AuNPs in the urine and bile also observed in previous studies (Balasubramanian et al., 2010; Cho et al., 2010). However, the surface functional group may affect the clearance route of AuNPs because AuNPNH2 and AuNPPEG presented higher levels in the urine and AuNPCOOH presented higher levels in the bile than the other AuNPs. Therefore, further studies are needed to confirm the effect of surface functional groups on the elimination route. Although AuNPs used in this study showed no toxicity, relatively low concentration of NPs in the brain and testis might have critical toxicity because those organs are highly sensitive to oxidative stress which can be produced by NPs. For example,

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Organ-specific distribution of AuNPs by surface functionalization

Conflict of Interest The Authors did not report any conflict of interest.

References

Figure 5. Elimination pattern of gold nanoparticles (AuNPs) via (A) urine or (B) bile. The concentration of AuNPs was measured at 30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months post-injection. Note that AuNPPEG and AuNPNH2 showed higher levels in the urine but AuNPCOOH showed higher levels in the bile. n = 5 for each group. Data were expressed as the mean ± SEM.

treatment of silica NPs to neuroblastoma cells originated from human and mouse showed neurotoxicity and Alzheimer-like pathology (Yang et al., 2014). Furthermore, i.v. injection of silver NPs increased testicular and serum testosterone levels by affecting Leydig cell function (Garcia et al., 2014) and i.v. injection of silica NPs damaged the maturation process of sperm in the epididymis (Xu et al., 2014). In conclusion, this study found that functional groups conjugated on the surface of AuNPs produced differences in the tissue distribution pattern which can be utilities for directing NPs to specific organs or improving the kinetic properties.

Acknowledgments We are grateful to Dr Byung-Chul Kang at Seoul National University Hospital Biomedical Research Institute and Yong-Hyun Chung at the Occupational Safety and Health Research Institute for assistance with TEM. This work was supported by grants from the Ministry of Food and Drug Safety (10181MFDS601, 11181MFDS555, 12181MFDS608) in 2010-2012. CWS thanks the MEST, Korea (NRF-2013R1A1A1011330).

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Copyright © 2014 John Wiley & Sons, Ltd.

J. Appl. Toxicol. 2014

Organ-specific distribution of gold nanoparticles by their surface functionalization.

The behavior and fate of intravenously (i.v.) injected nanoparticles (NPs) can be controlled by several physicochemical factors including size, shape ...
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