Abstract
Objectives: Triple-negative breast cancer (TNBC) is a form of BC with a poor prognosis and which has a high risk for metastatic progression. There is a critical need to develop novel targeted therapies for TNBC that could prevent formation of metastases or treat small metastases before they impact patient outcome. Our objective was to develop a novel radiation nanomedicine (Auger-gold) composed of gold nanoparticles modified with polyethylene glycol (PEG) polymers that provide stealth and minimize plasma protein opsonization. These particles also enable the attachment of DOTA for complexing the Auger electron-emitter, 111In, and the addition of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, panitumumab (PmAb) to the AuNP. EGFR is overexpressed on 50-90% of TNBC. Auger electrons are ideal for eradicating small metastases due to their subcellular range, high linear energy transfer (LET) and powerful lethal DNA-damaging effects. Methods: AuNP (15, 30, 50 nm) were linked via a lipoic acid (LA) end group to PEG chains (3 and 5 kDa) that either harboured DOTA complexed to 111In or were conjugated directly to PmAb. Stealth properties were assessed by changes in AuNP size measured by dynamic light scattering (DLS) before and after incubation for 1 h with mouse plasma at 37 oC. The effect of AuNP size, the amount of LA-PEG-DOTA and LA-PEG-PmAb reacted with AuNP on stealth properties were studied. The uptake of Auger-gold by BC cells with different EGFR density was measured by darkfield and fluorescence microscopy and compared to AuNP without PmAb modification. The cytotoxicity of Auger-gold in vitro on luciferase-transfected MDA-MB-231/Luc BC cells overexpressing EGFR was determined by clonogenic assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence for γ-H2AX. Targeting of Auger-gold to MDA-MB-231/Luc lung metastases in nude mice was studied by microSPECT/CT and biodistribution studies. Metastases were visualized by bioluminescence imaging (BLI). Results: The smallest change in size (1 nm) after incubation with mouse plasma was demonstrated for 30 nm unconjugated AuNP using the reaction ratio of 0.05 μg LA-PEG-DOTA and 28.6 μg LA-PEG-PmAb per cm2 AuNP surface area. A higher amount of LA-PEG-DOTA, but not LA-PEG-PmAb, linked to Auger-gold resulted in more absorption of plasma protein to AuNP. Darkfield microscopy showed binding and internalization of Auger-gold into EGFR-positive MDA-MB-468, MDA-MB-231, and MDA-MB-231/Luc, but not EGFR negative BT-474 BC cells. There was no internalization of non-targeted AuNP in EGFR-positive BC cells. Overnight exposure to Auger-gold (2.5MBq/1.8 x 1011 AuNP, 7.1 x 1011 AuNP/mL) was significantly more effective in decreasing the survival fraction (SF) of MDA-MB-231/Luc cells than non-targeted AuNP-111In or unlabeled PmAb-AuNP (SF=0.34±0.04, 0.61±0.05, 1.15±0.07, respectively; P<0.001 for PmAb-AuNP-111In vs PmAb-AuNP and P<0.05 for PmAb-AuNP-111In vs AuNP-111In). Auger-gold caused 2-fold more DNA DSBs in MDA-MB-231/Luc cells than AuNP-111In or unlabeled PmAb-AuNP. MicroSPECT/CT images showed more numerous foci of uptake in the lungs of mice at 3 h p.i. of Auger-gold than non-targeted AuNP-111In (4.3MBq, 3.3 x 1012 of 30 nm AuNP). Metastases established by i.v. injection MDA-MB-231/Luc cells were visualized by BLI. Biodistribution studies revealed 3-fold significantly greater lung uptake of Auger-gold than AuNP-111In (2.1±0.1 vs 6.3±0.8 %ID/g, P=0.03) at 24 h p.i. Conclusion: Auger-gold is a promising radiation nanomedicine for treatment of small lung metastases from EGFR-positive TNBC.