Blood Biochemical and Hematological Study after Subacute Intravenous Injection of Gold and Silver Nanoparticles and Coadministered Gold and Silver Nanoparticles of Similar Sizes

Background To investigate the effect of subacute intravenous administration AgNP (silver nanoparticles, 10 nm) and AuNP (gold nanoparticles, 12.8 nm) and AgNP/AuNP mixture to blood biochemistry, hematology, and platelet coagulation, subacute toxicity study was conducted. Methods AuNP and AgNP in which their size distribution was not statistically different, mixed or separate, were injected into the caudal vein of male Sprague-Dawley rats for 4 weeks. The rats were allowed to recover for a further 4 weeks in order to examine systemic toxicity expressed in the blood biochemistry and hematology. The dose groups (5 males per group for the administration and 3 males for the recovery) consisted of 7 divisions, i.e., control, AgNP (with a low dose of 10 μg/kg/day and a high dose of 100 μg/kg/day), AuNP (with a low dose of 10 μg/kg/day and a high dose of 100 μg/kg/day), and mixed AgNP/AuNP (with a low dose of 10/10 μg/kg/day and a high dose of 100/100 μg/kg/day). Results There were no significant dose-related changes in the hematology and blood biochemical values for the rats. Coagulation time in terms of the active partial thromboplastin time (APTT) and prothrombin time (PT) did not show any significant changes, when compared to the control group. Conclusion The subacute injection of AuNP and AgNP or their mixture did not induce any noticeable systemic toxicity.


Introduction
The fast-growing number of applications of manufactured nanomaterials in nanomedicine area such as drug delivery systems, medical devices, nanodrug, and nanoenabled medicinal products is expected to increase significantly. In biomedical applications, AuNPs (gold nanoparticles) are applied as potential carriers for drug delivery, imaging molecules, and even genes [1] and for the development of novel cancer therapy products [2]. Nanosilver or silver nanoparticles (AgNP) for their effective antimicrobial properties have been used in many biomedical and consumer products. Safety of these nanoparticles has been tested via many routes of administration such as oral, inhalation, dermal, intravenous, etc., and from short-to long-term durations. For these nanoparticles, intravenous injection of these nanoparticles would be an essential process to use for biomedical applications. The toxicokinetics of AgNPs including tissue distribution studies have been already conducted [3][4][5][6]. However, all these safety and toxicokinetic studies have been always conducted only for exposure to single type of nanoparticles and not for coexposure of different types of nanoparticles.
We have previously studied such coexposures where AuNP (12.8 nm) and AgNP (10 nm) were injected, mixed or separate, into the caudal vein of male Sprague-Dawley rats for 4 weeks. The rats were allowed to recover for a further 4 weeks in order to examine the differences in AuNP 2 BioMed Research International  Group  G1  G2  G3  G4  G5  G6  G7  A d m i n i s t r a t i o n  5  5  5  5  5  5  5  R e c o v e r y  3  3  3  3  3  3  3 AgNP, silver nanoparticles; AuNP, gold nanoparticles; Low, 10 휇g/kg/day; High, 100 휇g/kg/day; Low a , AgNP 10 휇g /kg/day + AuNP 10 휇g/kg/day; High b , AgNP 100 휇g/kg/day + AuNP 100 휇g/kg/day. / AgNP tissue distribution and clearance. As a result, AgNP and AuNP showed different toxicokinetic properties and the mixed administration of AgNP with AuNP resulted in mutual reduction of their tissue distribution which appeared to be due to competitive inhibition. Furthermore, this subacute intravenous injection study has suggested that these nanoparticles were distributed to the organs in particulate instead of ionic forms [7]. These subacute intravenous injection studies also provided data on safe levels of AgNP and AuNP dose levels. The present study provides an indication on systemic toxicity of these treatments, if any, expressed in the blood biochemistry and hematology.

Silver and Gold
Nanoparticles. The colloidal AgNPs (CAS No. 7440-22-4) were purchased from ABC Nanotech (Daejeon, Korea) with at least 99.98% purity. The synthesis of these nanoparticles was based on an inductive coupled plasma (ICP) method, using silver wire as a precursor, where the resulting AgNPs were immediately stabilized using 0.9% citrate. The detailed synthesis process has already been described [8]. The percentage of Ag ions in the silver nanoparticle preparations was determined by centrifugation, through a cellulose filter with a nominal cutoff value of 3 kDa (Ultra-4, Amicon, Millipore, Germany). The total Ag content in the unfiltered AgNP suspensions, as well as their respective filtrates, was measured using ICP-MS. The percentage of soluble Ag + in the AgNP preparations was calculated by dividing the Ag content in the filtrates, by the Ag + content in the unfiltered AgNP suspensions, which was then multiplied by 100 [5]. The percentage of Ag ions in the 10 nm silver nanoparticles was 0.002% [7]. The AuNPs in a 1% citrate solution were provided by MINTEK (Randburg, South Africa) and have already been recognized as a reference sample (NM-330, TMU14G, 100 mg/liter) by the OECD Working Party of the Manufactured Nanomaterials (WPMN) safety testing sponsorship program [9]. The cumulative median diameter (CMD) and geometric standard deviation (GSD) of the AuNP in the 1% citrate solution were analyzed using transmission electron microscopy. The percentage of Au ions in the AuNP preparations was determined by centrifugation through a cellulose filter with a nominal cutoff value of 3 kDa (Ultra-4, Amicon, Millipore, Germany), as described above for AgNP. The percentage of Au ions in the 12.8 nm gold nanoparticles was 0.0008% [7].

Transmission Electron Microscopy.
A transmission electron microscope equipped with an energy-dispersive X-ray analyzer (TEM-EDX) was used to measure the AgNP and AuNP based on National Institute for Occupational Safety and Health [10] analytical method 7402. The AgNP and AuNP and AgNP/AuNP mixture were analyzed in the presence of 10% serum and without serum, both in saline. The AgNP and AuNP were mounted on a TEM grid (copper grid) and visualized under a field emission-transmission electron microscope (FE-TEM, JEM2100F, JEOL, Japan). The particles were measured at a magnification of 100,000 and the AgNP and AuNP were analyzed using an energy-dispersive X-ray spectrometer (EDS, TM200, Oxford, UK) at an accelerating voltage of 75 kV.

Animals.
Six-week-old male specific-pathogen-free (SPF) Sprague-Dawley rats (60 rats) were purchased from OrientBio (Seongnam, Korea) and were acclimated for 1 week before initiating the intravenous injection. During the acclimation and experimental periods, the rats were housed in polycarbonate cages (maximum of 3 rats per cage), which were installed in individually ventilated cage racks. The rats were kept under a controlled temperature (23 ± 2 ∘ C) and humidity (55 ± 7%) and a 12-h light/dark cycle. The rats were fed a rodent diet (Harlan Teklad, Dooyeol Biotech, Seoul, Korea) and provided with filtered water ad libitum. Body weights were 258.12 ± 1.94 g when nanoparticle (NP) administration commenced. Animals were divided into 7 groups (as shown in Table 1), i.e., control, AgNP (with a low dose of 10 g/kg/day and a high dose of 100 g/kg/day), AuNP (with a low dose of 10 g/kg/day and a high dose of 100 g/kg/day), and mixed AgNP/AuNP (with a low dose of 10/10 g/kg/day and a high dose of 100/100 g/kg/day). The doses were based on the blood concentration of silver after 90 days of oral administration, showing 87-419 g/g of silver in the blood [6].
The AgNP, AuNP, and control vehicle (0.9% citrate) in a 1 ml final volume, separately or in a mixture, were injected using a 1 ml syringe into the caudal vein of the rats once a day, five times per week, for 4 weeks. The nanoparticles were prepared freshly every day based on the rat body weights and used immediately. The administration of the AgNP, AuNP, and AgNP/AuNP mixture was ceased after 4 weeks and the rats were allowed to recover for another 4 weeks. A group of animals were sacrificed at the end of the injection period and an additional group also after the 4-week recovery period in order to investigate the clearance of the tissue-accumulated silver and gold. The experiment was approved by the KCL Institutional Animal Care and Use Committee according to the Korean Animal Care Act.

Coagulation Time Measurement.
Following the blood collection, PT and APTT were measured by a coagulation time measurement device (ACL 7000, Instrumentation Laboratory, Bedford, MA).

Organ Weight Measurement.
After blood collection, the rats were sacrificed by cervical dislocation. The testes, heart, thymus, lungs, spleen, liver, kidneys, and brain were all carefully removed. These organs were then weighed and fixed in a 10% formalin solution containing neutral phosphatebuffered saline. The data were expressed as relative weight (organ weight/body weight x 100).

Statistical
Analysis. The statistical analysis was performed using SPSS (Version 19). The statistical evaluation was performed using an analysis of variance (ANOVA) following multiple comparison tests using Dunnett T3 method. The level of statistical significance was set at p < 0.05 and p < 0.01.

Nanoparticle Size Distribution.
The CMD and the GSD for the silver nanoparticles as measured by TEM were 10 nm and 1.28 nm, respectively, with a log-normal distribution between 6.4 and 13.5 nm. In contrast, the CMD and GSD of the gold analyzed TEM were 12.8 nm and 1.14, respectively, with log-normal distribution between 9 and 21. The difference in the average size was not statistically significant. A TEM image of the AgNP/AuNP mixture showed an agglomerated form; however, the particles in the mixture were well dispersed in the presence of the 10% serum in saline [7].

Animal Observation, Food Consumption, and Body Effects.
No distinctive toxic effects from the AgNP or AuNP injections were observed during the administration and recovery period. Also, there were no significant differences in food consumption between the treated and control groups (data not shown). Likewise, there were no significant dose-related body weight changes during and after the AgNP, AuNP, or a combined administration [7]. Generally, organ weight did not show any significant changes during AgNP, AuNP, and AgNP/AuNP mix administration ( Table 2). The exception, however, was a significant increase (P<0.05) in the left lung weight in the silver low-and high-dose group, when compared to the control group. In addition, a significant decrease (P<0.05) in the high-dose AgNP/AuNP mixture in the recovery group, when compared to control, was observed (Table 3).

Effects on Hematology and Blood Biochemistry.
There were no significant dose-related changes in the hematology values for the rats (Tables 4 and 5). After the 4-week administration period of AgNP and AuNP, separately or in a mixture, the percent of eosinophil increased significantly (P<0.05) in the low dose of AgNP, whereas MCV decreased significantly (P<0.05) in the low dose of AuNP, when compared to the control.
The number of lymphocytes decreased significantly in the high-dose AgNP/AuNP mix while the MPV decreased significantly (P<0.05) in the low-dose AgNP/AuNP mixture when compared to the control. In the recovery group, the percent of neutrophils and MPV decreased significantly (P<0.05) in the high-dose AgNP/AuNP mixture when compared to the control. Sodium concentration was decreased significantly (P<0.05) after the 4-week treatment in the lowand high-dose groups of both AgNP and AuNP, as well as in the AgNP/AuNP mixture groups, when compared to the control. After the 4-week recovery period, the level of glucose increased significantly in the low-and high dose of both AgNP and AuNP groups, when compared to the control group (Tables 6 and 7).

Effect of AgNP and AuNP Injection on Blood Coagulation.
Since a large number of nanoparticles were repeatedly injected into the blood stream, blood coagulation may be induced and, therefore, coagulation by nanoparticle injection was evaluated. Coagulation time in terms of the APTT and PT did not show any significant changes, when compared to the control group (Table 8).
Additional studies were also conducted where a single intravenous injection of gold nanoparticles (20 nm) at 10 g/kg b.w. showed that the Au rapidly and consistently accumulated in liver and spleen throughout the entire timeframe of the study (2 months after injection). Significant accumulation of Au in the kidneys and testis could also be seen from 1-month after injection, where a decrease in Au level was observed in urine and feces. Moreover, a significant increase of Au in the blood could be seen after 2 months of injection, coinciding with the delayed accumulation in the kidneys. Although microarray results of liver and spleen showed significant effects on genes related to detoxification, lipid metabolism, cell cycle, defense response, and circadian rhythm, no apparent toxicity was reported [14]. Finally, injection of 13.5 nm AuNP at different concentrations (137.5-2200 g/kg) over 14-28 days showed that AuNP at low concentrations did not cause any appreciable toxicity and no vascular or behavioral reactions were not observed. Necropsy did not show any macroscopic organ changes in the six groups, although the injection of gold nanoparticles caused transient reversible changes in body weight of the mice within 10-14 days after injection. [15].
On the other hand, short-term studies with AgNPs could show some adverse effects. For example, a subacute (28 days) intravenous injection of AgNP (20 and 100 nm) to rats showed growth retardation with 20 nm and 100 nm AgNP treatment [16]. There was also a severe increase in spleen size and weight with increase in T and B cell numbers. Clinical chemistry indicated liver damage (increased alkaline phosphatase (ALP), ALT, and aspartate aminotransferase (AST)) and hematology showed a decrease in several red blood cell parameters. Complete suppression of the natural killer cell activity in the spleen at high doses was noted. In addition, other immune parameters were affected at these high doses such as decreased interferon-and interleukin-(IL-) 10 production by concanavalin-A stimulated spleen cells, increased IL-1ß and decreased IL-6, IL-10, and TNFproduction by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. Suggested critical dose was 0.37 mg/kg b.w. with immune system as being the most sensitive one [16].
After injection of AgNP (average 15 nm) at 7.5, 30, or 120 mg/kg body weight in mice, no obvious acute toxicity was observed except inflammatory reactions in lung and liver at the 120 mg/kg dose level [17]. Intravenous injection of four different doses of AgNP (15-40 nm) at 4, 10, 20, and 40 mg/kg to rats increased liver function enzymes such as alanine ALT, AST, ALP, gamma-glutamyl transpeptidase (GGTP), and bilirubin, significantly at the 40 mg/kg dose. Reactive Oxygen Species (ROS) in blood serum increased in the high-dose group, indicating that AgNP doses less than 10 mg/kg are safe for biomedical application but at high doses (> 20 mg/kg) are toxic [18].
Since major route of biomedical application of these AgNP and AuNP would be intravenous injection, ensuring data on systemic toxicity, toxicokinetics, and safe dose levels of these particles will be very valuable. Subacute toxicity studies will therefore be of relevance to calculate such safe dose levels. However, very few such studies were conducted in the literature. For example, a subchronic silver exposure to oral and inhalation studies to rats indicated that LOAEL (lowest observed adverse effect level) was 125 mg/kg for oral intake [6] and 515 g/m 3 for inhalation [4]. At this LOAEL, liver toxicity was observed at the blood concentrations of silver of 191 ng/g for oral and 4.31 ng/g of tissue for inhalation exposure. In the present coexposure study of AgNP and AuNP, highest blood concentration of Ag was 15.97 ng/g of tissue after 28 days of 100 g/kg of AgNP intravenous administration without apparent liver toxicity. These results suggest that route of exposure may influence the toxicity of AgNP, which are different among intravenous injection or oral gavage or inhalation, thus emphasizing the observation reported earlier that tail vein injection of AuNP showed lower toxicity than oral or intraperitoneal administration [15].
Our previous publication [7] suggested that the injected AgNP and AuNP circulated in the blood mainly in particulate forms; thus the possibility that these nanoparticles may have interacted with blood components and cells to induce coagulative reaction may have existed. Earlier epidemiological studies suggested that air pollutants, for instance, particulate matter (PM) or ultrafine particles, are responsible for decreased as well as increased coagulation [19][20][21]. In contrast, animal exposure studies to particles of PM 2.5 did not show significant adverse effects on blood coagulation [22]. Although nanoparticle exposure has been suspected to cause blood coagulation in animal exposure studies, there were no consistent results for the induction of blood coagulation due to nanoparticle exposure. For example, exposure to amorphous silica nanoparticles (SiNPs) increased the activation of coagulation cascade by adsorbing and stimulating of intrinsic pathway coagulation factors. This effect resulted in shortening of coagulation time in APTT and PT tests, as well as in the increasing of factor X activation by RVV (Russell viper venom) in blood plasma but not in the sample with removed factors XI and XII. SiNPs did not induce platelet aggregation in platelet rich plasma but changed the shape and granularity of resting platelets and inhibited their aggregation [23]. On the other hand, subacute inhalation exposure to synthetic amorphous SiNPs up to 5.386 mg/m 3 showed no significant differences in the blood coagulation time such as PT and APTT between control and SiNPs-exposed groups during all recovery periods [24].
In AgNP exposure studies, high dose (1000 mg/kg) of AgNP induced shortening of APPT after 28 days of oral exposure to AgNP in female rats [3]. High concentration (515 g/m 3 ) of AgNP subchronic inhalation exposure increased the percent of aggregation in female rats when compared with the control [4]. This high dose or concentration had been shown to induce inflammatory reaction with hepatotoxicity in oral administration and hepatotoxicity and lung inflammation after inhalation exposure. Therefore, toxic dose might be implicated in the blood coagulation. Contrary to these observations, in our study with AgNP and AuNP or coadministration of AgNP/AuNP with maximum of 100 g/kg, each did not change PT or APPT with no changes of hepatoxicity markers or lung inflammation. In addition to dose, route of exposure and presence of inflammatory response may determine the effect of administered nanoparticles on blood coagulation parameters.
Taken together, subacute intravenous injection of AgNP or AuNP and their mixture did not result in dose-dependent changes in the hematology and blood biochemical values and blood coagulation time in terms of APTT and PT. The subacute injection of AuNP and AgNP or and their mixture did not induce any noticeable systemic toxicity.