Gold nanorods for target selective SPECT/CT imaging and photothermal therapy in vivo

Abstract

The development of theranostic agents with high detection sensitivity and antitumor efficacy at low concentration is a challenging task for target selective imaging and therapy of cancers. In this study, folate-conjugated and radioactive-iodine-labeled gold nanorods (GNRs) were designed and synthesized for target selective SPECT/CT imaging and subsequent thermal ablation of folate-receptor-overexpressing cancers. Both (ortho-pyridyl) disulfide-poly(ethylene glycol)-folate and a short peptide, H(2)N-Tyr-Asn-Asn-Leu-Ala-Cys-OH, were conjugated on the surface of the GNRs through thiol chemistry. The tyrosine in the peptide sequence was introduced for radioactive-iodine labeling through an iodine-tyrosine interaction. The labeling efficiency of radioactive iodine was more than 99%. Radiochemical stability tests on iodine-125-labeled GNRs in human serum showed that 91% of the iodine-125 remained intact on the GNRs after incubation for 24 h. In vitro and in vivo results in this study confirmed the potential utility of folate-conjugated and iodine-125-labeled GNRs as smart theranostic agents. This type of platform may also be useful for the targeted SPECT/CT imaging and photothermal therapy of inflammatory diseases such as atherosclerosis and arthritis, in which folate-receptor-overexpressing macrophages play pivotal roles.

Keywords: SPECT/CT; Target selective imaging; gold nanorods; photothermal therapy.

Scheme 1.. Synthetic procedure for folate-conjugated and radioactive-iodine-labeled GNRs, FA-¹²⁵I-GNR.

Figure 1.. A. Absorption spectrum and transmission electron microscopy (TEM) image of FA-GNR; and B. Dispersion stability of FA-GNR in deionized water (DW), phosphate-buffered saline (PBS) solution, and RPMI 1640 (without phenol red) containing 10% fetal bovine serum (FBS).

The solutions were maintained at 25 °C and observed for 168 h. Photographs and absorption spectra shown above were taken after 168 h of incubation.

Figure 2.. A. Intracellular uptake of FA-¹³¹I-GNR against SKOV3 cell line (n=3) and A549 cell line (n=3).

FR-positive SKOV3 and FR-negative A549 cells were both incubated with FA-¹³¹I-GNR for 4 h in the absence (▪) or presence (□) of free FA (1 mM) as a competitor. Dotted line: radioactivity measured from the cells treated with free I-131; B. Cell viability of SKOV3 cells treated with FA-GNR at various concentrations (0, 0.25, 0.5, 1, and 2 nM, based on GNR, n=3).

Figure 3.. In vitro photothermal therapy using FA-GNR at various concentrations (0, 0.25, 0.5 and 2 nM, based on GNR) against SKOV3 cells.

SKOV3 cells were incubated with FA-GNR for 4 h in the absence or presence of free FA (1 mM) as a competitor, washed, and received light illumination with an 810-nm laser (2 W/cm², 4 min). Light was applied to the area inside the dotted white line. The cells were then stained with calcein AM. Live cells and the nuclei of dead cells are shown in green and red, respectively.

Figure 4.. Radio TLC chromatogram of FA-¹²⁵I-GNR.

Labeling efficiency was measured by instant thin layer chromatography (ITLC) with saline as the developing eluant, and was shown to be more than 99%.

Figure 5.. Radiochemical stabilities of FA-¹²⁵I-GNR in human serum.

ITLC of FA-¹²⁵I-GNR showed high radiochemical stability in human serum at 37 °C after incubation for 24 h (n=3). The radiostability was maintained at 90.9±0.3% for up to 24 h.

Figure 6.. A. Coronal and sagittal plane SPECT images of the mice at different time points.

The mice received intravenous injections of FA-¹²⁵I-GNR at a dose of 40.7 MBq/mouse (equal to 0.841 pmol FA-GNR/mouse). White arrows indicate tumor sites. B. Representative SPECT/CT fusion image of SKOV-3 xenograft-bearing mouse at 24 h after injection of FA-¹²⁵I-GNR (Th: thyroid, B: bladder, T: tumor).

Figure 7.. A. Thermographic monitoring in the tumors of PBS-treated mice (n=4, 90 µL/mouse) and FA-¹²⁵I-GNR-treated mice (n=3, dose: 40.7 MBq/mouse) during 810-nm light illumination (+L) for 5 min (2 W/cm²); B. Thermographic images captured after 5 min of 810-nm light illumination; C. Tumor size after the therapy session.

The mice received intravenous injection of either PBS or FA-¹²⁵I-GNR on day 0, and the tumors were then illuminated with light on day 1. Points, mean; bars, standard deviation; PBS+L (n=4) and FA-¹²⁵I-GNR +L (n=3); n = the number of mice.