Self-assembled NIR-II dye conjugated small peptides for bone imaging

Objectives Research on the second near-infrared window (NIR-II, 1000 - 1700 nm) fluorescence imaging is a new rising field. It shows advantages of superior temporal resolution, reduced scattering, deep tissue penetration, and negligible tissue autofluorescence comparing to conventional NIR-I window (650 - 950 nm) imaging. Recently we developed a novel NIR-II dye CH1055 that is composed of D-π-A-π-D type of chromophores and demonstrated its high potential for tumor imaging. In this study, we report the use of self-assembled CH1055 dye conjugated small peptides (CH1055-P1 and CH1055-P3) for optical imaging of bone (Fig. 1a). This novel NIR-II imaging technique may find important applications for imaging of bone diseases, such as cancer bone metastases, rheumatoid arthritis, related rheumatic diseases, etc.

Methods The four carboxyl NIR-II dye CH1055 was activated with NHS/EDC, and reacted with different ratio of the peptide to obtain one or three peptide(s) modified CH1055 (CH1055-P1, CH1055-P3). For comparison, a similar conjugate CH1055-4PEG2000-P was synthesized. These three probes were intravenously (iv) injected into nude mice (n=3 per group) for NIR-II fluorescent imaging. Images were acquired at different time points using an in-house NIR-II imaging system with a 808nm laser and 1100nm long pass filter.

Results CH1055-P1 and CH1055-P3 were successfully synthesized and characterized by MALDI-TOF-MS and HPLC. When dissolving in pH7.4 PBS, CH1055-P1 and CH1055-P3 self-assembled to ~10nm and ~20nm nanoparticles, respectively, whereas CH1055-4PEG2000-P remained as single molecule form (Fig. 1b). After injection of the CH1055-P3 to the nude mice, sternum, thoracic vertebrae, lumbar vertebrae, femur, tibia, and knee joint of the mice were clearly visualized from the surrounding tissue with NIR-II imaging even at 0.5h. The bone-to-normal (B/N) tissue ratio reached a peak at 3h p.i. (Fig. 1c, d). Image quantification analysis indicated that the B/N ratio reached a maximum ~3.5 and maintained ~2 after 48h p.i.. CH1055-P1 showed similar bone imaging property but lower B/N value, with maximum of ~2.5. As a comparison, the small molecular probe CH1055-4PEG2000-RGD showed no targeting to the bone.

Conclusions CH1055-P1 and CH1055-P3 with self-assembling ability are successfully prepared. They show excellent bone targeting ability and may find important applications for imaging of bone abnormality.

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