BBN conjugated GNPs: a new targeting contrast agent for imaging of breast cancer in radiology

Abstract

Using of targeted contrast agents in X-ray imaging of breast cancer can improve the accuracy of diagnosis, staging, and treatment planning by providing early detection and superior definition of tumour volume. This study demonstrates a new class of X-ray contrast agents based on gold nanoparticles (GNPs) and bombesin (BBN) for imaging of breast cancer in radiology. GNPs were synthesised in spherical shape in the size range of 15 ± 2 nm and conjugated with BBN followed by coating with polyethyleneglycol (PEG). The in vitro and in vivo behaviour of PEG-coated GNPs-BBN conjugate was investigated performing cytotoxicity, binding, and internalisation assays as well as biodistribution and X-ray imaging studies in mouse bearing breast tumour. Cytotoxicity study showed biocompatibility of the prepared bioconjugate. The binding and internalisation studies using T47D cell line approved the targeting ability of new agent. The biodistribution study showed the considerable accumulation of prepared conjugate in breast tumour in mouse model. The breast tumour was clearly visualised in X-ray images taken from the mouse model. The results showed the potential of PEG-coated GNPs-BBN conjugate as a contrast agent in X-ray imaging of breast tumour in humans that need further investigations.

Fig. 1

Schematic representation of bioconjugation of GNPs in spherical shape with BBN and the PEGylation process

Fig. 2

UV–vis spectrum of synthesised GNPs with the λ_max at 515 nm. Inset image: TEM micrograph of produced GNPs in spherical shape with the diameter size of 15 ± 2 nm (scale line = 50 nm)

Fig. 3

UV–vis spectra of free GNPs, GNPs‐BBN, and PEG‐coated GNPs‐BBN conjugates. The red shifting occurred during each step of the bioconjugate synthesis further indicated the successful fabrication of GNPs (a red shift of 5 nm in the absorbance peaks between GNPs (515 nm) and GNPs‐BBN (520 nm) and a red shift of 10 nm in the absorbance peaks between GNPs‐BBN (520 nm) and PEG‐coated GNPs‐BBN conjugates (530 nm)

Fig. 4

FT‐IR spectra of (a) BBN conjugate, (b) GNPs‐BBN conjugate. The NH₂ stretching frequency of the amine group observed at 3423 cm⁻¹ was shifted to the higher wavelength at 3430 cm⁻¹ after the conjugation reaction. The red shifting shows direct binding of nitrogen atoms of free amino group to GNPs

Fig. 5

Optical serum stability of PEG‐coated GNPs‐BBN conjugate. The new bioconjugate showed a high level of optical stability when exposed to the blood serum up to 24 h

Fig. 6

Viability of T47D cells after 24 h incubation with PEG‐coated GNPs‐BBN conjugate (black bars) and PEGylated GNPs (grey bars) at 10–100 µg/ml concentrations (P < 0.05)

Fig. 7

Selective binding of PEG‐coated GNPs‐BBN conjugate to T47D cells (scale bar: 100 µm). Silver staining was performed to enable visualisation during bright field microscopy to study the fate of GNPs in their interaction with the T47D and E8 (AG01522) cells (a) T47D cells incubated with PEG‐coated GNPs‐BBN conjugate, (b) T47D cells incubated with PEGylated GNPs, (c) E8 (AG01522) cells incubated with PEG‐coated GNPs‐BBN conjugate, (d) E8 (AG01522) cells incubated with PEGylated GNPs

Fig. 8

Quantification of GNPs uptake (in conjugation with BBN and in free form) by T47D cells using AAS. The analysis revealed that the cellular uptake of new bioconjugate by T47D cells was more than %50 after 6 h (in comparison with %12 uptake for free GNPs). This study showed that peptide–receptor‐mediated was helpful for cellular uptake of functionalised GNPs

Fig. 9

Biodistribution data of (a) PEG‐coated GNPs‐BBN, (b) Free GNPs (in PEGylated form) in breast‐tumour‐bearing BALB/c mice at 2, 4, 6, 8, 12, and 24 h after IV administration expressed as %ID/g measured by AAS (n  = 5). The uptake of PEG‐coated GNPs‐BBN bioconjugate by the tumour tissue increased gradually as time increased, reaching a maximum at 6 h (P  < 0.05)

Fig. 10

X‐ray images of a representative BALB/c mouse bearing (a) Breast tumour before injection, At different time intervals (b) 2 h, (c) 4 h, (d) 6 h, (e) 8 h, (f) 12 h, and (g) 24 h post injection of PEG‐coated GNPs‐BBN conjugate (exposure parameters: 22 kV and 10 mAs was the same for all images). The marker ‘L’ was used as an unified X‐ray intensity bar