Consistent with the finding that the binding between AuNP and 211At is tight and stable, the labeling rate of 211At-AuNP-S-mPEG and the size of the particles did not appear to change in the present study even after a time period equivalent to 6 half-lives of 211At. An in vitro experiment evaluating cytotoxicity against tumor cells showed that unlabeled AuNP-S-mPEG at nanoparticle sizes of 5 to 120 nm did not affect cell viability even after the cellular internalization of the nanoparticles had been confirmed. Thus, the toxicity of AuNP-S-mPEG itself was suggested to be negligible.
In C6 glioma cells, significant cytotoxicity was observed after in vitro treatment with 211At-AuNP-S-mPEG with a diameter of 120 nm labeled with 1 MBq/mL of radioactivity. In the PANC-1 cells, the cytotoxicity of 211At-AuNP-S-mPEG was similar to that seen in the C6 glioma cells. Thus, the cell toxicity of 211At-AuNP-S-mPEG as a non-targeted agent may not depend on the cell type.
The degree of cellular internalization of the 120 nm AuNP-S-mPEG was shown to be very high (Fig. 2G). For smaller AuNP-S-mPEG, however, higher concentrations were required for cellular internalization (Fig. 2B, D, F). Although the mechanism responsible for cellular internalization remains unknown, the 120 nm 211At-AuNP-S-mPEG might have precipitated to the bottom of the well used in the presently reported in vitro system, creating an extremely elevated concentration around cells that had adhered to the bottom of the culture. As a result, frequent cellular contact caused by close proximity with a high concentration of AuNP-S-mPEG might be involved in cellular internalization. Cytotoxicity was only confirmed (Fig. 1H) under conditions where internalization was observed (Fig. 2G). These facts suggest that the cytotoxicity of 211At-AuNP-S-mPEG is caused by α irradiation, which is enhanced by the cellular internalization of the nanoparticles. When injected into cancer tissue, the 211At-AuNP-S-mPEG are thought to be first distributed in the intercellular fluid. Intercellular fluid accounts for about 15% of the whole tissue, and the diffusion capability of nanoparticles is lower than that of a membrane-permeable solvent. Therefore, at the time of injection, the AuNP is probably spatially concentrated around the tumor cells. The prolonged contact of cells in tissues with a high concentration of 211At-AuNP-S-mPEG may cause the nanoparticles to be internalized, thereby suppressing tumor cell growth.
Observations of the intratumoral distribution using autoradiography revealed that the 30 nm 211At-AuNP-S-mPEG had a high diffusivity in the tumor tissue, while the 120 nm 211At-AuNP-S-mPEG had a relatively low diffusivity. These observations suggest that the smaller the nanoparticle size, the greater the diffusivity in the tumor tissue. The tumor growth suppression effect of the α-rays from 211At-AuNP-S-mPEG was proportional to the size of the particles under the conditions of this study. Thus, for particles that are a minimum of 5 nm in diameter or larger, smaller 211At-AuNP-S-mPEG are considered to have greater diffusivity in tumor tissues and hence a greater tumor growth suppression effect. Unlabeled AuNP-S-mPEG did not show obvious cytotoxicity in either in vivo or in vitro studies. Thus, the antitumor property of the nanoparticles is clearly due to the cytotoxic effect of α-rays. 211At-AuNP-S-mPEG with diameters ranging between 5 and 120 nm did not accumulate systemically in any organs for at least 42 hours, i.e. 6 half-lives of 211At after intratumoral administration. These results show that 211At-AuNP-S-mPEG does not undergo back-diffusion from the tumor tissue into blood vessels. On the other hand, AuNP with a diameter of 10 nm or more reportedly exhibit vascular permeability and can be dispersed to various organs in a size-dependent manner after intravenous or gastrointestinal administration.[22–24] These inconsistencies with previous studies indicate that the vascular permeability of AuNP may depend on particle modification.
The effectiveness of targeting a specific protein for the purpose of nanoseed brachytherapy is controversial.[15, 25] In this study, even non-targeted nanoseeds were shown to have a tumor growth inhibitory effect and to exert cytotoxicity in two different types of tumor cells both in vitro and in vivo. In principle, this method is likely to be independent of the histological type of the malignant tumor and could therefore be easily used as a brachytherapy for many tumor types. The labeling of 211At-AuNP-S-mPEG is completed by a simple operation involving the stirring of AuNP-S-mPEG and 211At together. Therefore, the nanoparticles can be generated immediately before administration, allowing them to be labeled with a high specific radioactivity. In the present study, 211At-AuNP-S-mPEG showed a high and exclusive retention at the injected site, where 211At radioactivity was detected. These findings and the properties of α-rays suggest that very high-dose treatments with simple nanoparticles 211At-AuNP-S-mPEG might be feasible on an outpatient basis. In addition, Au and PEG are already clinically used as medicines, and their low toxicity has already been recognized.[26, 27]
The present study had the following limitations. First, the mass concentration, particle concentration, and osmotic pressure of the administered solutions were difficult to control because the administration of a constant radioactivity concentration was prioritized. Second, since the radioactivity could not be further increased due to restrictions on the supply of 211At, the radioactivity level administered to the tumor was set at around 1.4 MBq. In a study examining the intratumoral administration of Lu-177-labeled AuNP-S-mPEG, however, a much stronger tumor growth suppression was observed at an administration level of 4.5 MBq, compared with 3.0 MBq administration. Further study is thus necessary to optimize the dose in experiments using higher doses.