Abstract
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Objectives Graphene, an emerging nanomaterial with single-layered two-dimensional carbon atom network, has attracted tremendous interest due to its unique electronic, optical, mechanical and chemical properties, and has been applied as a versatile platform for cancer imaging and therapy. Here, we propose a novel chelator-free radiolabeling mechanism, by which 64Cu can directly label onto the graphene surface with great in vivo stability via transition metal-pi interactions. By eliminating the influences of chelator, chelator-free radiolabeled graphene can maintain the native properties of nanoparticles, which enables a more precise control over their in vivo fate.
Methods To study the influence of metal-pi interactions on the chelator-free radiolabeling, reduced graphene oxide (RGO) and graphene oxide (GO) nanosheets were labeled by directly mixing the nanosheets with 64Cu at different concentrations and temperatures. Serving as a comparison, the conventional chelator, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), was conjugated onto RGO and labeled with 64Cu at the same conditions. Anti-cancer drug doxorubicin (DOX)-loaded RGO-PEG was also investigated to understand the influence of drug loading on the labeling efficiency. Thin layer chromatography (TLC) was employed to study and compare their labeling yields. Serum stability studies and in vivo positron emission tomography (PET) imaging were conducted to examine their labeling stability and tumor imaging capacity. However, since PET only displays the distribution of radioisotopes rather than the nanoparticles, photoacoustic imaging was also applied as a complementary modality to confirm the PET results. The mechanism of chelator-free labeling was proven by Fourier transform infrared spectroscopy.
Results Systematic studies indicated that 40-80 % of 64Cu was intrinsically labeled onto RGO-PEG at different concentrations and temperatures. In contrast, the labeling yields of GO-PEG remained low (5-20 %), due to incomplete graphene structure with less pi bonds for coordination with 64Cu, thereby confirming the role of transition metal-pi interactions. This mechanism was further validated by labeling of DOX-loaded RGO-PEG. The labeling yield of (DOX)RGO-PEG (0.2 mg/ml at 37 ºC) slightly decreased to 32.7 ± 4.2 %, due to the competition between DOX and 64Cu for pi-bonds on the surface of RGO. Traditional radiolabeling with NOTA-RGO-PEG was found to be more efficient (93.1 ± 1.1 % at 0.2 mg/ml at 37 ºC). However, serums stability studies indicated comparable radiostability for both 64Cu-RGO-PEG and 64Cu-NOTA-RGO-PEG (>75% 64Cu remaining stably after 24 h incubation). More importantly, higher tumor uptake was observed with 64Cu-RGO-PEG (6.6 ± 0.7 %ID/g at 6 h post injection) in comparison with 64Cu-NOTA-RGO-PEG (4.9 ± 0.8 %ID/g at 6 h post injection) via PET imaging, suggesting that chelator-free labeled RGO-PEG possesses higher in vivo radiostability and is better suited for tumor imaging with graphene nanomaterials. Photoacoustic imaging also confirmed the successful tumor retention of RGO-PEG after 6 h post-injection, which showed stronger signal than that in blank control mice.
Conclusions Herein, we reported the first example of chelator-free radiolabeling of RGO nanosheets with 64Cu, which exhibited excellent radiostability and enhanced imaging capacity. Importantly, chelator-free radiolabeling can maintain the native properties of the nanoparticles, making it more suitable for nanoparticle-based radiolabeling. By investigating the mechanism of chelator-free radiolabeling of graphene, our study provided important guidance for the future research on radiochemistry and in vivo applications of graphene-based nanomaterials.