Our purpose was to develop a human
epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast
cancers and up to 2-fold higher in locally advanced disease (LABC).
Trastuzumab was derivatized with a
polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce
trastuzumab-PEG-OPSS. These
immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3
breast cancer cells. Reacting
trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per
trastuzumab). Attachment of approximately 7 PEG chains per
trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of
biological molecules on AuNPs we estimate 14.3 ± 2.7
antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 μg of
trastuzumab-PEG-OPSS. Specificity of
trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of
trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of
trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance
DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher
DNA-DSBs with
trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The
trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC
therapy. The design and systematic approaches taken to surface modify and characterize
trastuzumab-PEG-AuNPs described in this study would have application to other molecularly targeted AuNPs for
cancer treatment.