In vivo tumor vasculature targeting and PET imaging with reduced graphene oxide

Objectives Graphene-based materials exhibit unique properties for biomedical applications including cancer therapy. Our goal was to employ reduced graphene oxide (RGO) for in vivo tumor targeting, and quantitatively evaluate pharmacokinetics and tumor targeting with positron emission tomography (PET), using ⁶⁴Cu as the radiolabel.

Methods RGO sheets, with amino group-terminated PEG chains on the surface, were conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid, for ⁶⁴Cu labeling) and TRC105 (an antibody that binds to CD105, a receptor overexpressed on tumor vasculature). FACS analyses, size measurements, and serum stability studies were performed to characterize the RGO conjugates before in vivo investigation (PET, biodistribution, and blocking studies) in 4T1 murine breast tumor-bearing mice. Imaging results were validated by histological assessment.

Results The RGO conjugate, ⁶⁴Cu-NOTA-RGO-TRC105, had a size range of 20-80 nm. It exhibited CD105 specificity and superb stability in vitro and in vivo. Serial PET imaging and biodistribution studies revealed that 4T1 tumor uptake of ⁶⁴Cu-NOTA-RGO-TRC105 was clearly visible at 0.5 h post-injection (p.i.) and remained stable over time (5.0±0.6, 5.6±0.2, 5.7±0.2, 4.5±0.4, and 4.0±0.5 %ID/g at 0.5, 3, 6, 24, and 48 h p.i.; n = 4), which gave excellent tumor contrast and was several fold higher than the non-targeted RGO conjugate. Various in vivo (e.g. blocking with TRC105), in vitro (e.g. flow cytometry), and ex vivo (e.g. histology) studies further confirmed the specificity of ⁶⁴Cu-NOTA-RGO-TRC105 for targeting CD105 on the tumor vasculature. Little extravasation was observed, confirming that tumor vasculature targeting is an ideal approach for RGO.

Conclusions This is the first report of in vivo tumor targeting and imaging with RGO. This proof-of-principle study opened up new perspectives for future research and cancer theranostics using graphene-based materials, which are desirable nanoplatforms for biomedical applications because of the versatile chemistry and low toxicity.