Biodistribution of gold nanoparticles synthesized by γ-irradiation after intravenous administration in mice

Hydrogen tetrachloroauratetrihydrate (HAuCl₄ · 3 H₂O), pure water from Merck, Germany and sodium alginate (M_n = 4.35 × 10⁵ g mol⁻¹) from Hayashi Pure Chemical Industry Ltd, Japan were used as received. Glassware was treated with regia solution (1 V HNO₃ : 3 V HCl), washed with pure water and dried.

Two stock solutions, particularly 10 mM Au³⁺ and 2% alginate were prepared by dissolving HAuCl₄ · 3H₂O and alginate into water. Au³⁺ 1 mM/alginate 0.5% solution was prepared by pouring Au³⁺ solution into alginate solution with desired concentration while stirring for about 10 min. Then the prepared Au³⁺/alginate solution of 200 ml was put into a glass bottle with plastic cap. Irradiation was carried out on the gamma Co-60 irradiator STSVCo-60/B at a dose of 8 kGy with dose rate of 1.3 kGy h⁻¹ at VINAGAMMA Center in Ho Chi Minh City.

The absorption spectrum of AuNPs solution, which was diluted by water to 0.1 mM calculated as Au³⁺ concentration, was taken on a UV-Vis spectrophotometer model UV-2401PC, Shimadzu. The size and size distribution of the AuNPs were characterized by TEM images on transmission electron microscope (TEM) model JEM1010 (JEOL, Japan) and statistically calculated from about 300 particles.

Mice of the Swiss line were supplied by Pasteur Institute of Ho Chi Minh City and fed at Institute of Malarialogy-Parasitology-Entomology in Ho Chi Minh City for 8 weeks and their average body weight was about 30 g. The mice were then injected with 0.5 ml AuNPs solution containing 1 mg gold via a tail vein. The control mice were also intravenously injected by 0.5 ml of 0.5% alginate solution without AuNPs.

The blood of mice after intravenous AuNPs injection at 0, 1, 3, 6 and 12 h was collected and put into tubes containing heparin for analysis. Haematology indexes including haematocrit (Htc), haemoglobin (Hb), total white (WBC) and red (RBC) blood cell counts, lymphocytes (Lympho), monocytes (Mono), and granulocytes (GR) counts, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) were analyzed by the 18 parameters celltac α (Nihon Kohden, Japan) automatic haematology analyzer.

The blood of injected mice was collected in the same way as haematology analysis. The blood was then centrifuged at 5000 rpm for 10 min to separate the serum for analysis. Serum clinical chemistry indexes including glucose (Glu), creatine (CR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), globin (GLB) and albumin (ALB) were analyzed by BioSystem A17 analyzer (Belgium).

The mice after intravenous AuNPs injection were killed and the liver, kidney, lung and blood at 0, 1, 3, 6 and 12 h were collected. The tissues were then dried at 180 °C for 5 h and the content of gold in each tissue was quantitatively analyzed at Da Lat Nuclear Research Institute by k₀-neutron activation analysis (k₀-NAA) method as described by Abd et al [17].

The UV-Vis spectrum of AuNPs solution in figure 1(a) showed the maximum absorption wavelength (λ_max) at 530 nm for Au³⁺ concentration of 1 mM. The TEM image and particles size distribution are shown in figures 1(b) and (c). The average diameter of AuNPs was determined to be about 20 nm with a fairly narrow Gaussian distribution.

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Different AuNPs sizes can be pre-selected by varying Au³⁺ concentration and/or seed enlarge-ment approach. [12–14, 16] According to Anh et al [12] gamma Co-60 irradiation method is suitable for production of AuNPs with controllable size and high purity.

Since the intravenous administration was proposed as the best way for application of AuNPs in medical fields such as cancer diagnostics and therapy [4–8], carriers for transmucosal delivery of insulin [9], x-ray contrast agent [10] etc. The toxicity test of AuNPs after intravenous administration is very important. In this study, 10 blood haematology indexes (Htc, Hb, WBC, RBC, Lympho, Mono, GR, MCV, MCH and MCHC) of intravenous AuNPs injected mice and control mice were analyzed at 1, 3, 6 and 12 h after injection. The results in table 1 show that there was no significant difference in blood haematology indexes between control and AuNPs injected mice.

Table 2 records the serum clinical chemistry indexes of AuNPs injected and control mice. The results also showed that the contents of glucose, creatine, aspartate aminotransferase, alanine aminotransferase, total protein, total bilirubin and albumin of AuNPs injected mice were not significantly different from those of control mice. The analyzed results on haematology and serum clinical chemistry of tested mice proved that AuNPs were not toxic by the intravenous administration at a dose of 1 mg/mouse (30 g). The results are in good agreement with those reported by Hainfeld et al [10].

The distribution of AuNPs in different tissues of mice is displayed in figure 2. The results indicated that the concentration of AuNPs in blood decreased with time, the gold content almost disappeared after 6 h injection (0.01 mg per 1 gram fresh tissue) and the blood became normal at 12 h after injection. The results in figure 2 also indicated that at 1 h after injection, AuNPs penetrated into liver, kidney and lung. At 6 h after post injection, AuNPs were mainly distributed in liver and lung tissue, in particular, the gold content in liver tissue was found to be about 0.56 mg per 1 gram fresh tissue and attained 76.33% of injected dose, while at the same time the gold content in lung tissue was found to be about 0.45 mg per 1 gram fresh tissue and attained 11.86% of injected dose. The gold content in kidney was lower than that in liver and lung (0.06 mg per 1 gram fresh tissue and only 2.23% of injected dose). At 12 h after injection, the gold content decreased in all tissues. The results in figure 2 also indicated that after injection, AuNPs transferred from blood into other tissues and mostly accumulated in liver and lung with the maximal concentration at 6 h after injection.

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Thus, the AuNPs/alginate product with particle diameter of about 20 nm synthesized by gamma Co-60 irradiation using alginate (0.5%) as stabilizer is not toxic at a dose of about 33.3 mg kg⁻¹ body weight of mice. Thus, it can be potentially applied as x-ray contrast agent, especially for blood and liver. In addition, Nghiem et al also studied in vivo toxicity of 15 nm AuNPs capped by bovine serum albumin (BSA) by tail vein injection for mice with a dose from 1.16 to 5.84 mg kg⁻¹ body weight [18]. They concluded that the AuNPs did not produce any mortality or gross behavioral changes in mice at the doses studied; all animals experimented on were healthy and had normal weight development. Porkharkar et al reported that LD₅₀ (dose required to kill half of the population) value of AuNPs/chitosan was found to be greater than 2000 mg kg⁻¹ body weight of rats by oral administration [19]. They concluded that AuNPs/chitosan produced no toxicity in rats that can be exploited for potential therapeutic applications. Furthermore, spherical AuNPs of different sizes (5–70 nm) were reported not to be toxic to human skin cells [20]. Thus, AuNPs/alginate product is promising to apply in biomedicine, cosmetics and in other fields as well.