Conversion of rod-shaped gold nanoparticles to spherical forms and their effect on biodistribution in tumor-bearing mice
© Akiyama et al.; licensee Springer. 2012
Received: 28 August 2012
Accepted: 4 October 2012
Published: 11 October 2012
Gold nanorods that have an absorption band in the near-infrared region and a photothermal effect have been used as nanodevices for near-infrared imaging and thermal therapy. Choice of the optimal shape of gold nanorods which relates optical properties and in vivo biodistribution is important for their applications. In the present study, to investigate the relationship between the shape of gold nanorods and their biodistribution after intravenous injection, we first prepared two types of gold nanorods that had distinct aspect ratios but had the same volume, zeta potential, and PEG density on the gold surface. Biodistributions of the two types of gold nanorods after intravenous injection into tumor-bearing mice were then compared. Although a slight difference in accumulation in the spleen was observed, no significant difference was observed in the liver, lung, kidney, and tumors. These results suggest that biodistribution of the gold nanorods in the aspect ratio range of 1.7 to 5.0, diameter of 10 to 50 nm, and volume of approximately 4 × 103 nm3 was dependent mainly on surface characteristics, PEG density, and zeta potential.
KeywordsAspect ratio Gold nanoparticles Gold nanorods Laser Biodistribution
Gold nanorods are tiny, rod-shaped gold particles. They have two distinctive different absorption bands derived from the transverse and longitudinal surface plasmon resonance of free electrons in the visible and near-infrared (NIR) regions, respectively[1, 2]. Gold nanorods absorb NIR light, which is suitable for in vivo applications such as imaging and for photoradiation therapy because of maximal penetration of light into tissues. Gold nanorods have a photothermal effect, i.e., the adsorbed light energy is converted to heat. Therefore, gold nanorods are expected to be contrast agents for NIR imaging and exothermic nanodevices for photothermal therapy.
Gold nanorods are prepared in the presence of the cationic detergent 1-hexadecyltrimethylammonium chloride (cetyltrimethylammonium chloride; CTAB), which acts as a stabilizer of gold nanorods. To apply gold nanorods as medical nanodevices, biocompatible gold nanorods have been prepared by coating with phosphatidylcholine or by modifying gold nanorods with polyethylene glycol (PEG)[6, 7]. PEG-modified gold nanorods show high dispersion stability, high circulation stability in the blood after intravenous injection into mice, and accumulation in tumors mediated by the enhanced permeability and retention (EPR) effect[8, 9]. The PEG-modified gold nanorods have been applied to NIR imaging and photodynamic/photothermal therapy of tumors[8, 10–13].
The size and aspect ratio of gold nanorods are important factors of their uptake into cells[14–16] and biodistribution[17–20]. Arnida et al. reported that rod-shaped gold nanoparticles accumulated more efficiently in tumors and less efficiently in the liver compared with sphere-shaped gold nanoparticles. However, the compared gold nanorods and gold nanospheres had different volumes and zeta potentials that affected cellular uptake (e.g., phagocytosis). Therefore, if we examine the effect of the geometry of gold nanoparticles on biodistribution, other parameters should be equivalent.
In the present study, we prepared two types of gold nanorods that had distinct aspect ratios but had an identical volume, zeta potential, and surface structure. Biodistributions of these nanorods after intravenous injection into tumor-bearing mice were then compared.
Animal experiments were undertaken according to the Guidelines for Animal Care and Use Committee, Kyushu University (Fukuoka, Japan). Male ddY mice (Kyudo Co., Ltd., Saga, Japan) aged 5 to 6 weeks were used. Mice were maintained in a temperature-controlled environment at 24°C with a 12-h light-dark cycle. They were provided with drinking water and food ad libitum.
Preparation of gold nanorods of lower aspect ratio from higher ones
Gold nanorods prepared by our research team were obtained from Mitsubishi Materials Corp. (Tokyo, Japan) and Dai Nippon Toryo Co. Ltd. (Tokyo, Japan). The mean length and width of the as-obtained gold nanorods were 49.6 ± 9.7 nm and 10.6 ± 3.2 nm, respectively (aspect ratio, 5.0 ± 1.2). A suspension of gold nanorods containing CTAB was stored at a constant temperature (37°C). Gold nanorods were modified with PEG chains. Briefly, the gold nanorods in the suspension were centrifuged at 14,000 × g for 10 min at room temperature, decanted, and re-suspended in water to remove excess CTAB. Thiol-terminated mPEG solution (molecular weight approximately 5,000 Da; NOF Corporation, Tokyo, Japan) was added to the gold nanorod suspension at a PEG/gold molar ratio of 1.0. The mixture of PEG solution and gold nanorod suspension at a final gold concentration of 1 mM was stirred for 24 h at room temperature. NIR pulsed laser light (Nd:YAG 1064 nm, 750 mW, 20 Hz, approximately 6 mm in beam diameter) irradiated the mixture for 5 min. The mean length and width of gold nanorods irradiated with the NIR pulsed laser light were 27.0 ± 8.2 nm and 17.2 ± 3.7 nm, respectively (aspect ratio, 1.7 ± 0.9). A control mixture was not irradiated with the NIR pulsed laser light. These mixtures were stirred for another 24 h at room temperature and centrifuged twice at 14,000 × g for 10 min at room temperature, decanted, and re-suspended in water to remove remaining CTAB and excess PEG reagent.
Characterization of PEG-modified gold nanorods
The absorption spectra of gold nanorods in the visible-to-NIR light regions were measured with a V-670 spectrophotometer (Jasco, Tokyo, Japan). Gold nanorods and the PEG layer on the gold nanorods were imaged with a transmission electron microscope (JEM-2010, JEOL, Tokyo, Japan) after staining the PEG layer with 1% phosphotungstic acid. Particle size distribution, surface area, and volume were calculated from transmission electron microscopy (TEM) images of 100 particles. Amounts of PEG modified on the gold nanorods were evaluated by elemental analyses according to our previous publication. Zeta potentials of gold nanorod suspensions in 0.2 M sodium phosphate buffer (pH 7.4) were evaluated using a Malvern Zetasizer Nano ZS (Malvern, Worcestershire, UK).
Biodistribution of gold nanorods
Tumor-bearing mice were used in biodistribution experiments and were prepared as follows: Colon-26 cells: mouse colon carcinoma cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Cells were incubated at 37°C under a 5% CO2-humidified atmosphere. Incubated colon-26 cells were injected subcutaneously into the abdomen at 2 × 106 cells in 100 μL of Hank's balanced salt solution per mouse (ddY, male, 5 weeks, 29 to 35 g). They were allowed to grow for 5 days, wherein the tumors reached approximately 5 to 7 mm in diameter. PEG-modified gold nanorods were injected into tumor-bearing mice via the tail vein. Doses of PEG-modified gold nanorods with an aspect ratio of 5.0 and PEG-modified gold nanorods with an aspect ratio of 1.7 were at 1.01 and 1.05 mM of gold concentration in 10 μL/g body weight of 5% glucose solution, respectively. Mice were sacrificed 72 h after injection. Several hundred microliters of blood were collected from the heart and immediately mixed with 1 mg ethylenediamine tetra-acetic acid. The liver, lung, spleen, and kidney, as well as tumors, were collected. These samples were added to approximately 4 mL aqua regia and then heated overnight at 90°C to 100°C. Samples were diluted in 0.5 M HCl to the appropriate volume. Gold concentrations were quantified by inductively coupled plasma mass spectrometry (ICP-MS) using an Agilent 7500c system (Agilent. Tokyo, Japan). ICP-MS measurement was done with thallium as the internal standard.
Results and discussion
Preparation of PEG-modified gold nanorods with distinct aspect ratios
Characteristics of PEG-modified gold nanorods treated without and with near-infrared pulsed laser light
Particle size of short axis (nm)
10.6 ± 3.2
17.2 ± 3.7
Particle size of long axis (nm)
49.6 ± 9.7
27.0 ± 8.2
5.0 ± 1.2
1.7 ± 0.9
Surface area (×10−3 nm2)
1.61 ± 0.43
1.42 ± 0.40
Volume (×10−3 nm3)
4.02 ± 1.81
4.32 ± 1.70a
Number of modified PEG on one particle (×10−2)
9.50 ± 4.28
8.25 ± 3.25
Area occupied by one PEG molecule (nm2)
1.82 ± 0.29
1.82 ± 0.33a
Zeta potential (mV)
0.48 ± 1.66
1.83 ± 3.34a
The amount of modified PEG on gold nanorods was measured by elemental analyses (Table1). The number of modified PEG molecules on one particle decreased from 9.50 × 102 to 8.25 × 102 by laser irradiation. The actual release of PEG from the gold surface to supernatant has been already confirmed at a similar condition in our previous report. However, the area occupied by one PEG molecule as PEG density on the gold nanorods and the zeta potentials of the gold nanorods did not change after laser irradiation. These data suggested that the laser irradiation of gold nanorods resulted in transformation from a rod shape to a spherical shape without changing the volume, PEG density, or zeta potential.
Biodistribution in tumor-bearing mice
Figure4B shows the concentration of accumulated gold nanorods in the organs as percentage of injected dose per gram of tissue or milliliter of blood. The concentration is an important parameter for photothermal therapy and bioimaging mediated by gold nanorods. Accumulation of gold nanorods with an aspect ratio of 1.7 in the spleen was slightly lower than that of gold nanorods with an aspect ratio of 5.0, although statistical significance was low (0.1 > p > 0.05; Figure4B). In the case of the liver, lung, and kidney, no difference between the gold nanorods with aspect ratios of 1.7 and 5.0 was observed. No difference was observed in the tumors, suggesting that a change in the shape of the gold nanorods was not sufficient to influence the EPR effect in the tumors. The slight difference observed in the spleen may have been due to physical filtration in this organ as observed for larger particles (>200 nm)[29, 30]. It may not have been due to changes in the contribution of the RES in the spleen because a significant difference in the accumulation in the liver (where many Kupffer cells (macrophage-like cells) constitute the RES at sinusoids) was not observed. These results suggested that the shape of the gold nanorods barely affected the biodistribution of gold nanorods after intravenous injection into tumor-bearing mice except for that in the spleen (though the change in biodistribution was slight). Previously, we compared biodistributions of several types of gold nanorods, which have different zeta-potentials and PEG densities on the gold surface but have the same aspect ratio[8, 9]. In those studies, the surface structure of the gold nanorods strongly affected their biodistributions after intravenous injection. Therefore, it can be concluded that the biodistribution would be more affected primarily by the surface characteristics of the gold nanorods than the aspect ratio.
In the present study, the shape of the original gold nanorods was changed by irradiation with NIR light to a spherical form. Then, we obtained two types of PEG-modified gold nanorods with distinct aspect ratios but with the same volume, PEG density, and zeta potential. The change in the aspect ratio of the gold nanorods did not affect the biodistribution appreciably. These results suggest that the biodistribution of gold nanorods in the aspect ratio range of 1.7 to 5.0 and size of 10 to 50 nm at a volume of approximately 4 × 103 nm3 is dependent mainly on the surface characteristics, PEG density, and zeta potential. If the application of gold nanorods to therapeutic use is considered, the gold nanorods circulating in the blood or other tissues such as tumors are irradiated with NIR light. Irradiation with the pulsed laser light will induce the shape change of the gold nanorods and release of molecules bound on the gold nanorods without changing their biodistribution. On the other hand, irradiation with continuous wave laser light can heat the gold nanorods continuously without changing the shape of the gold nanorods. Thus, combinations of gold nanorods and different modes of light irradiation provide us various drug delivery/release and heating systems, and it will be an advantage of gold nanorods over other types of nanoparticles. The results obtained in this study will be used as basic information on the in vivo application not only of gold nanorods with various aspect ratios, but also of other types of anisotropic nanoparticles.
cetyltrimethylammonium chloride 1-hexadecyltrimethylammonium chloride
enhanced permeability and retention
inductively coupled plasma mass spectrometry
This research was supported by a Grant-in-Aid for Scientific Research (B, grant number no. 22300158) from the Japan Society for the Promotion of Science (JSPS) and by a grant from the Precursory Research for Embryonic Science and Technology (PRESTO) of the Japan Science and Technology Agency (JST).
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