Multimodality Tumor Imaging Using Self-illuminating Gold Nanoclusters

【Background and aim】Self-illuminating fluorescence imaging is a newly developed imaging technique, overcoming several shortcomings of traditional fluorescence imaging. It has many distinguished imaging advantages, such as: i) no need of excitation from external illumination sources to fluoresce, resulting in no interference of strong background autofluorenscence, and ii) no issue of excitation attenuation caused by photons absorption/scattering in bio-tissue transmission, which serves extremely important for in vivo imaging, as most imaging targets are non-superficial. So far, only a few selfilluminating fluorescence probes are designed and constructed based on three different energy transfer mechanisms. Quantum dots(QDs), due to their high quantum yield, tunable emission peaks, long fluorescence lifetimes, and negligible photobleaching, are only one material that been employed as the energy transfer acceptor. However, self-illuminating QDs are limited for further biomedical applications because most QDs contain heavy metal elements(such as Cd2+, Pb2+, etc.) and tend to self-aggregate inside living cells. The cytotoxicity of the released heavy metal ions in biological systems and potential environmental hazard of these ions always hinder their practical applications. Therefore, it is highly desirable to develop novel self-illuminating systems with non-toxicity and good biocompatibility for further biomedical applications, especially in molecular imaging and therapy.Gold Nanoclusters(Au NCs)are new kind of fluorescent nanomaterials, which have aroused great interest in the development of new types of luminescent probes due to their high fluorescence, good photostability, non-toxicity, excellent biocompatibility and water solubility. However, the self-illuminating property of Au NCs and possibilities of Au NCs served as an energy transfer acceptor have not been reported. This study was designed to use HSA as bio-temple to synthesize Au NCs and developed a novel one-pot isotope labeling method for the first time. The CRET between Au NCs and isotope were discussed, and the multimodality imaging potential of radiolabeled Au NCs were demonstrated by a series of in vitro and in vivo experiments. The aim of this work was to provide critical evidences and show the potentials of Au NCs as a novel multimodality imaging agent.【Methods】1. Au NCs were prepared by a biomimetic synthesis methods using nature HSA as template. Transmission electron microscope(TEM) images were acquired and size was measured. Fluorescence emission spectra were recorded by the fluorescence spectrophotometer, and CD spectra were recorded on a Jasco J-815 spectropolarimeter. 64 Cu was reduced and doped onto the Au NCs surface by a chelator-free methods. The labeling efficiency and stability were calculated by instant thin layer chromatography(ITLC);2. The PET and self-illuminating NIR imaging potentials of 64Cu-doped Au NCs were demonstrated in vitro. The IVIS imaging system was applied to evaluate CRET effects in 64Cu-doped Au NCs by several chosen filter sets: no filters, > 590 nm, < 510 nm, 515- 575 nm, 575- 650 nm, 695- 770 nm, and 810- 875 nm;3. The subcutaneous tumor xenograft model was established by injection of U87 MG cells into nude mice. 64Cu-doped Au NCs were administrated by tail vein injection, then PET and self-illuminating NIR(CRET-NIR) imaging were conducted at different time points to evaluate the multimodal imaging ability of 64Cu-doped Au NCs in vivo. Regions of interests(ROIs) were drawn over the tumors and the signal intensities from PET and IVIS were measured and compared;4. Biodistribution study and histological examination were applied to evaluate the pharmacokinetics, targeting efficacy and toxicity of 64Cu-doped Au NCs.【Results】1. Au NCs were successfully synthesized using HSA as bio-template. The TEM image showed the size of Au NCs was about 0.93 ± 0.25 nm. The maximum emission peak was at ~680 nm. The CD spectropolarimeter showed that the structure conformation of HSA has not changed much even after Au NCs synthesis. The ITLC indicated that almost all the 64 Cu was reduced and stably doped onto the Au NCs;2. In vitro PET and self-illuminating images showed satisfied dual-modality imaging capability of 64Cu-doped Au NCs. IVIS imaging analysis demonstrated that the CRETNIR would happen within 64Cu-doped Au NCs, and the intensity was much higher than the Cerenkov radiation of 64 Cu Cl2 of same radioactivity. Specifically, in the no filter group, the 64Cu-doped Au NCs showed an intensity of 1.6-that of 64 Cu Cl2, and in the filter of >590 nm, the intensity of 64Cu-doped Au NCs was 9.40 × 106 photon/s, which was 2.7 times than that of 64 Cu Cl2(3.42 × 106 photon/s). More importantly, in 695- 770 nm region, which covered parts of the emission spectrum of Au NCs, the intensities of 64Cu-doped Au NCs were 4.3-fold that of 64 Cu Cl2;3. 64Cu-doped Au NCs exhibited good synergistic dual-modality PET and self-illuminating NIR tumor imaging. PET imaging and ROIs quantification identified evident tumoruptake of 64Cu-doped Au NCs at 1 h post injection, which increased to 14.9 %ID/g and 15.2 %ID/g at 18 h and 24 h, respectively. IVIS images showed an enhanced tumor uptake of 64Cu-doped Au NCs after injection, reaching maximum at 8 h post injection. Importantly, the CRET-NIR imaging(measured by>590 nm filter) showed satisfactory tumor uptake compared to Cerenkov imaging(measured by filter <510 nm), in which tumor signal was only 1/7 of the total and tumor uptake was barely detected. Because of CRET, the ROI measurement displayed good linear correlation between PET and total signal(no filter, r2 = 0.9340) as well as PET and CRET-NIR signal(>590 nm; r2 = 0.9687). In contrast, a weaker linear correlation was found between PET signals and Cerenkov radiation, due to parts of Cerenkov energy were transferred(<510 nm; r2 = 0.7616);4. Organ distribution results indicated that the liver and kidneys may be responsible for the route of clearance of 64Cu-doped Au NCs. Histological examination showed no obvious organ damage at 1 day and 7 days after injection of 64Cu-doped Au NCs.【Conclusions】In summary, we developed a novel self-illuminating gold nanocluster, 64Cu-doped Au NCs, for dual-modality PET and CRET-NIR fluorescence imaging. To the best of our knowledge, it is the first time that noble metal nanoclusters have been reported as energy transfer acceptors for multimodality imaging. The 64 Cudoped Au NCs we developed have the following features: 1) small size, high solubility and stability in aqueous media; 2) both PET and self-illuminating NIR imaging capabilities; 3) in vivo self-illumination, converting short wavelengths of Cerenkov radiation into longer wavelengths, and 4) non-toxicity and good biocompatibility. These self-illuminating nanoclusters provide novel biomedical research tools, especially for molecular imaging and cancer therapy.