Increased apoptotic potential and dose-enhancing effect of gold nanoparticles in combination with single-dose clinical electron beams on tumor-bearing mice

Abstract

High atomic number material, such as gold, may be used in conjunction with radiation to provide dose enhancement in tumors. In the current study, we investigated the dose-enhancing effect and apoptotic potential of gold nanoparticles in combination with single-dose clinical electron beams on B16F10 melanoma tumor-bearing mice. We revealed that the accumulation of gold nanoparticles was detected inside B16F10 culture cells after 18 h of incubation, and moreover, the gold nanoparticles were shown to be colocalized with endoplasmic reticulum and Golgi apparatus in cells. Furthermore, gold nanoparticles radiosensitized melanoma cells in the colony formation assay (P = 0.02). Using a B16F10 tumor-bearing mouse model, we further demonstrated that gold nanoparticles in conjunction with ionizing radiation significantly retarded tumor growth and prolonged survival compared to the radiation alone controls (P < 0.05). Importantly, an increase of apoptotic signals was detected inside tumors in the combined treatment group (P < 0.05). Knowing that radiation-induced apoptosis has been considered a determinant of tumor responses to radiation therapy, and the length of tumor regrowth delay correlated with the extent of apoptosis after single-dose radiotherapy, these results may suggest the clinical potential of gold nanoparticles in improving the outcome of melanoma radiotherapy.

Figure 1

Visualization of gold nanoparticles (AuNP) inside B16F10 cells. AuNP localization in cells was visualized by silver enhancement or by fluorescence labeling of AuNP. (a) The localization of AuNP was determined by silver enhancement, and (b) the localization of AuNP‐. Alexa Fluor 594 conjugates were detected directly under a microscope (bar = 50 µm; original magnification 200×). BF, Bright field, DAPI, 4'‐6‐diamidino‐2‐phenylindole.

Figure 2

Gold nanoparticles (AuNP) colocalized with endoplasmic reticulum (ER) and Golgi in cells. B16F10 cells were cultured with AuNP for 20 h. The localization of AuNP was determined by silver enhancement, and the (a) ER‐Tracker Red dye and (b) BODIPY TR Ceramide Golgi Tracker were used for live‐cell ER and Golgi labeling (bar = 50 µm; oiginal magnification 200×; BF, bright field).

Figure 3

Biodistribution of gold nanoparticles (AuNP) in mice. (a) Twenty‐four hours after AuNP injection, tissues of tumor‐bearing mice were excised, processed, and used for AuNP detection using atomic absorption detection. (b) Silver staining of AuNP inside a tumor. Twenty‐four hours after AuNP injection, tumors were excised and paraffin‐embedded. Five‐micrometer thick sections from each representative specimen were obtained and then processed with the silver enhancement kit (bar = 200 µm). PBS, phosphate‐buffered saline.

Figure 4

Gold nanoparticles (AuNP) radiosensitized melanoma cells. B16F10 cells were treated with AuNP (10 nM for 18 h) and assessed for radiosensitization by clonogenic cell survival immediately after irradiation. For the survival curves, each data point represents the average of three independent experiments each plated in triplicate ± SD (solid line, control; dotted line, 10 nM AuNP; *P = 0.02).

Figure 5

Antitumor effects of the combination treatment of gold nanoparticles (AuNP) and radiotherapy in tumor‐bearing mice. C57BL/6 mice were inoculated subcutaneously with B16F10 cells (1 × 10⁶) at day 0. At day 7, tumor‐bearing mice were injected intravenously with 200 µL of 200 nM AuNP, or with 200 µL of phosphate‐buffered saline (PBS) 24 h before irradiation (25 Gy/mouse). Mice were monitored for (a) tumor growth and (b) survival (n = 4–7; *P < 0.05). RT, radiotherapy.

Figure 6

Apoptotic activity was analyzed by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Tumors were excised from mice 6 h postradiotherapy. Five‐micrometer thick representative cryostat sections were obtained and then processed with the TUNEL System. (a) Positive TUNEL straining was observed under a fluorescent microscopy. (b) Quantitative analysis. Apoptotic cells were calculated by averaging the number of positive TUNEL signals from eight fields with highest density of TUNEL signals in each section (n = 8; *P < 0.05; original magnification 40×). RT, radiotherapy.