Novel dual-function near-infrared II fluorescence and PET probe for tumor delineation and image-guided surgery

The first small-molecule based αvβ3-targeted NIR-II/PET dual-modal probes via base-catalyzed thiol-addition chemistry were concisely assembled and evaluated.


Introduction
Maximizing tumor excision and minimizing collateral damage are the primary goals of cancer surgery. Incomplete excision of tumor tissue, however, negatively affects the prognosis of the patient. Evidence from numerous experimental and clinical studies has demonstrated the signicant benets of molecular imaging in targeted surgery with preoperative molecular diagnostic screening, uorescence image-guided surgery and postoperative imaging. [1][2][3][4][5] So far only two small molecules, methylene blue and indocyanine green (ICG), have been approved by the FDA, emitting within the traditional NIR-I region (750-900 nm) and with the desirable features of rapid renal excretion and low toxicity. 6,7 Despite their imaging qualities being superior to those of visible wavelengths in accurate real-time tumor delineation, photostability and the penetration depth of emitted light in biological tissues remain challenging. To address this limitation, uorophores emitting within the second near-infrared region (NIR-II, 1000-1700 nm) show great promise. NIR-II probes possess high spatial resolution and deep-tissue penetration due to reduced photon scattering and diminished auto-uorescence. [8][9][10][11][12][13] At present, several classes of uorescent NIR-II probes including small molecules, 14-17 carbon nanotubes, 18,19 Ag 2 S dots 20,21 and polymers 22 have been actively employed for NIR-II uorescence for superior vascular imaging, cerebral imaging, lymphatic imaging and imaging-guided surgery. 23,24 A surge in dual-modal instrumentation development for image-guided surgery and other clinical applications has sparked the discovery of more dual-modal contrast agents to clearly delineate the localization and expression of biochemical markers, and effectively track the tumor with high-resolution and high-sensitivity. 2,4-6,25-37 Using dual-modality probes for sentinel lymph node mapping and image-guided surgery shows promising results in prostate cancer, 28,35 breast cancer 36 and head and neck cancer. 37 Although radio-guided surgery and intraoperative uorescence imaging are powerful techniques to improve accurate tumor detection and resection, they have their limitations. Specic radiotracers can be used that target tumor tissue and that can be detected using a gamma probe during surgery. Yet this technique cannot provide a precise delineation of the tumor and resection margins. [25][26][27][28][29][30][31][32][33][34][35][36][37] The addition of a NIR-I uorescent label could help to overcome this limitation. Intraoperative NIR-I uorescence imaging could allow accurate realtime tumor delineation, but the penetration depth of emitted light in biological tissue is limited. There is no doubt that developing novel NIR-II dual-modal molecular probes for in vivo multimodal imaging applications thus has high signicance and direct impact on the eld of biomedicine. Although a variety of small-molecule based scaffolds have been successfully explored to construct NIR-I dual-modal imaging probes, widespread applications of these probes in molecular imaging and biomedicine are heavily hampered due to multiple-step synthetic challenges, tedious protection-deprotection strategies and low chemo-selectivity. 28,38,39 Therefore, developing effective molecular platforms for the construction of dualmodal NIR-II probes with high chemo-selectivity is urgently demanded.
In a proof-of-concept study, a NIR-II and PET imaging dualmodality probe 68 Ga-CHS2 ( Fig. 1 and S1 †) for the rst time has been concisely prepared via novel base-catalyzed thiol-yne chemistry for tumor delineation and image-guided surgery. Two thiolated units CH1055 and 68 Ga-1,4,7-triazacyclononanetriacetic acid (NOTA) are controlled and integrated across a simple linear terminal alkyne such as propiolamide (PA) sequentially by base-induction. A variety of light-sensitive molecules (uorescent probes and proteins) can be assembled straightforwardly and by-products such as disuldes can be minimized signicantly. 39 In this paper, a highly innovative method to improve the surgical resection of U87MG tumors in vivo has been demonstrated using an integrin avb 3 targeted dual-label tracer that can be detected with a PET probe, and then with a uorescence NIR-II camera during surgery.
In comparison with previous synthetic strategies, this approach demonstrated two major advantages: (1) the PA scaffold is very simple and available for controllable mono-thioladdition or double-thiol-addition with high regio-selectivity ( Table 2); (2) the reaction can tolerate a wide range of functional groups such as -NH 2 , -CO 2 H and -OH, and the protecting-group-free strategy can be easily realized for the integration of dual-modality probes in a simple and straightforward way.
The rst peptide-based NIR-II/PET dual-modal imaging probe CHS2 was successfully generated via the above methodology. 3351.3, found: m/z 3350.2 (Fig. S10 †)]. The UV-vis-NIR absorption band of the CHS2 probe was at 600-900 nm (Fig. S11 †). Meanwhile, the uorescence emission spectrum of the CHS2 probe indicated a maximum wavelength at 1055 nm in PBS buffer ( Fig. 2a and S12 †). The uorescence quantum yield of the CHS2 probe was $0.20% in water, measured against an IR-26 reference with a nominal quantum yield of 0.5% under 785 nm excitation. CHS2 also exhibited high photostability in PBS, water and mouse serum with negligible decay under continuous excitation for 1 h (Fig. 2b). However, NIR-I probes such as ICG decayed $50% under the same conditions (Fig. 2c). Furthermore, the high viability of U87MG cell lines aer 24 h incubation with different concentrations of CHS2 and nat Ga-CHS2 demonstrated the high biocompatibility of CHS2 in vitro ( Fig. 2d and S13 †). Briey, CHS2 was then incubated with 68 Ga [2 mCi] under mild conditions for 15 min according to a previously reported method, 41 and puried by RP-HPLC resulting in over 95% purity (Fig. S14 †). The specic activity of 68 Ga-CHS2 was determined to be $25 GBq mmol À1 and it demonstrated excellent stability in mouse serum (no release of 68 Ga for 2 h). U87MG cells were chosen to investigate the specicity of 68 Ga-CHS2 toward integrin avb 3 . 42,43 As shown in Fig. S15, † 68 Ga-CHS2 exhibited good uptake in U87MG cells. In the blocking group, the cells were incubated with 68 Ga-CHS2 (1 mCi per well) and unlabeled RGD (2 mg per well) as a blocking agent, and the uptakes were signicantly reduced, indicating that 68 Ga-CHS2 can specically bind to integrin avb 3 receptors on U87MG cells. Thus, the properties of 68 Ga-CHS2 warrant further in vivo applications. The nude mice bearing U87MG xenogras (n ¼ 3) were injected with $85 mCi (1 nmol) of 68 Ga-CHS2 and were imaged with micro-PET/CT and NIR-II separately. As shown in Fig. 3a    and b, at different time points during the scan, the tumor could be clearly visualized on both PET and NIR-II images with a high signal to background ratio (T/N). The in vivo specicity of the dual-modal imaging probe was conrmed by the blocking experiment and the intensity of the tumor signals was signicantly reduced aer co-injection of unlabeled RGD (750 mg per mouse) for both PET and NIR-II imaging. The quantitative analysis of the PET images further revealed that the tumor uptake values of 68 Ga-CHS2 were gradually increased from 0.5 to 1 h, and then reduced at 2 h aer injection, with 0.77 AE 0.13, 2.48 AE 0.32 and 2.14 AE 0.27% ID g À1 at 0.5, 1 and 2 h, respectively (Fig. S16 †). In contrast, signicantly lower tumor uptake values were observed for the blocking group with values of 0.32 AE 0.07, 0.28 AE 0.05 and 0.18 AE 0.04% ID g À1 at 0.5, 1 and 2 h, respectively. From the NIR-II imaging data, the U87MG tumor uorescence signals were successfully reduced at later time points in the blocking group. The T/N ratios obtained by NIR-II imaging for CHS2 are shown in Fig. S17, † and are proven to be much higher than those of the blocking group at all time points. Moreover, the highest tumor contrast was obtained at the 12 h time point with a T/N value of 4.77 AE 0.26, which is 2-fold more than previously reported NIR-I/RGD based probes. 27 Biodistribution studies were evaluated for this dual-modal probe in major organs ( Fig. S18 and S19 †). Pharmacokinetics of 68 Ga-CHS2 demonstrated renal and hepatobiliary excretion, with a trace amount of CHS2 remaining in the kidney and liver for 60 h post-injection (PI) (Fig. S19 and S20 †). These results also matched well with PET and NIR-II imaging data. Thus, a powerful synergy can be achieved by combining radiotracers for the detection of tumor tissue, and NIR-II optical tracers for subsequent accurate delineation of tumor lesions and resection margins. Encouraged by these promising results obtained in vivo PET/NIR-II images, CHS2 was further evaluated for subsequent precise and accurate delineation of tumor lesions, resection margins and image-guided surgery. U87MG tumorbearing mice (n ¼ 3) were injected with 100 mg of CHS2 and the tumor delineation could be notably identied from the surrounding background tissue at 4 h (Fig. S21 †). The T/N ratio reached 4.75 AE 0.22 at 12 h post-injection, and when the tumor was dissected and removed from the so tissue in the leg region, the T/N ratio dropped to 1.16 AE 0.27, indicating that the tumor was thoroughly dissected (Fig. 4a-e). As NIR-II uorescence image-guided surgery was carried out, all uorescent surgical specimens including the whole tumor and para-cancerous tissues (marked as 1-4, right adjacent to the tumor tissue) were stained with hematoxylin and eosin assessed (Fig. 4f-l). Histological analysis of excised paracancerous tissues at different magnications has shown that the histological characteristics of cancer were not detected. The data further veried tumor delineation and resection margins, as well as uorescence signals by NIR-II imaging (Fig. 4f-l). This way, tumor nodules that were missed by conventional surgery but were successfully detected by our uorescent probe CHS2 can be analyzed to assess the sensitivity and specicity of the dual-modality intraoperative imaging approach at the histological level (Fig. 4). Our gamma probe activity measurements and NIR-II uorescence imaging data obtained during surgery along with these hematoxylineosin staining results could lead to a reduced time of examination, which may aid the surgeon in making better decisions during the surgery.

Conclusions
In summary, we have successfully developed a base-catalyzed strategy for the concise construction of an integrin avb 3 -targeted dual-modality imaging probe, 68 Ga-CHS2, for tumor delineation and image-guided surgery. This probe has demonstrated excellent imaging characteristics in vivo and has high clinical and translational potential. Our highly innovative approach to obtain tumor-free resection margins or maximal cytoreduction using specic tumor targeting dual-label tracers that can be detected with a 68 Ga probe and a uorescence NIR-II camera during surgery will affect patient survival. Thus, our rst targeted dual-modality NIR-II/PET image-guided surgery results show the potential to take oncological surgery one step further and may ultimately contribute to the improved survival of cancer patients.
All animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee and approved by the Institutional Animal Care and Use Committee (IACUC), Wuhan University Center for Animal Experiment, Wuhan, China.

Conflicts of interest
There are no conicts to declare.