Novel bright-emission small-molecule NIR-II fluorophores for in vivo tumor imaging and image-guided surgery

This work presents the establishment of novel bright-emission small-molecule NIR-II fluorophores for in vivo tumor imaging and NIR-II image-guided sentinel lymph node surgery.


Introduction
As uorescence imaging in vivo continues to gain increasing interest and expand within both academic and clinical settings, a transition shied to longer wavelengths in the second nearinfrared window (NIR-II, 1000-1700 nm) region could have clear-cut advantages of deeper tissue imaging, high spatial resolution, and high contrast owing to minimal auto-uorescence and tissue scattering. 1,2Improved imaging quality, superior lymphatic imaging, deeper brain tumor imaging, and higher tumor-to-background ratios have been achieved recently compared to the conventional NIR-I region (750-900 nm). 3,4Developing novel NIR-II emitting agents for biomedical applications thus has high signicance and directly promotes the eld of biomedicine.Thus far, organic and inorganic materials such as small molecules, 5-7 conjugated polymers, 8 carbon nanotubes, 9,10 quantum dots (QDs), [11][12][13] and rare earth nanoparticles, 14,15 have been actively employed for NIR-II uorescence imaging. 16However, reports of NIR-II uorophores are still scarce and the small-molecule uorescent cores are relatively limited compared with their NIR-I counterparts. 6Hence, it prompts us to expand the library of smallmolecule NIR-II uorophores, which will signicantly promote the widespread use of NIR-II imaging modality.
Several types of small-molecule NIR-II dyes with favorable excretion pharmacokinetics have been reported, in which the uorophore units are generally composed of aromatic conjugate units based on a donor-acceptor-donor (D-A-D) structure with a benzobisthiadiazole (BBTD) core. 5,6Among them, a small-molecule probe Q4 was selected as a scaffold for the facile construction of NIR-II agent SCH1100 for targeted prostate cancer imaging. 6,17However, the complexity and multiple synthetic steps with low yields, tedious chromatographic isolation and the weak brightness of Q4 heavily hinder the wide application of such a promising agent in preclinical and clinical studies.Hence, many efforts should be made to simplify the synthetic strategy and optimize the brightness of smallmolecule NIR-II uorophores.
Herein, we report a novel small-molecule NIR-II dye H1 with an improved synthetic protocol and uorescence characteristics.At longer uorescence emission wavelengths in the NIR-II region, the increased bandgap of molecular uorophores generally gives way to reduce interactions between the a State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China.E-mail: xhy78@whu.edu.cnconjugated backbone and other molecules, causing a high uorescence quantum yield (QY). 18,19,20 Therefore, in this work, R 1 substituent groups on the sp 3 carbon of the uorene group are out-of-plane of the p-conjugated system and thus prevent intermolecular stacking that leads to uorescence quenching.Meanwhile, newly introduced 2-amino 9,9-dialkyl-substituted uorene moieties distort the BBTD backbone and thus effectively tune the electrostatic potential distribution and the bandgap to the desired range.Moreover, the uorene moieties act as both the electron donor and protecting groups with the benets of a compact molecular structure and shielding the backbone from aggregation (Fig. 1).Finally, three types of NIR-II probes (SXH, SDH, and H1 NPs) have been facilely prepared according to the H1 scaffold, and demonstrated different biomedical applications such as passive/active tumor targeted imaging, high resolution imaging of blood vessels on tumors and the whole body, and image-guided sentinel lymph node surgery in the NIR-II imaging The novel organic uorescent compound H1 provides unprecedented opportunities for the construction of a variety of NIR-II probes for in vivo molecular imaging.

Results and discussion
As shown in Fig. 1, compound H1 was synthesized by a convergent route in 15% overall yield over 6 steps from commercially available starting materials without tedious chromatographic isolation (see ESI †).All compounds were characterized by NMR and ESI-MS, and also exhibited good solubility in common organic solvents such as CH 2 Cl 2 and THF (see ESI †).By modulating the non-resonant side chains of the carboxylic acid groups, the nal NIR-II uorophores could be systematically altered to tune the hydrophobicity, polarity and efficient conjugation of bio-targets.The UV-vis-NIR absorption band of H1 was at 600-1000 nm (in CH 2 Cl 2 ) due to the formation of a strong charge-transfer structure between the D-A-D units (Fig. 2a).Meanwhile, the uorescence emission spectrum of H1 was obtained and demonstrated a peak emission wavelength at $1100 nm (Fig. 2a).The results indicated that the brightness of the uorescence signals of H1 was superior to that of Q4 (Fig. 2b).Furthermore, the NIR-II signals of H1 were investigated under various LP lters (900-1400 nm) and no signals were observed with the 1300 nm and 1400 nm lters (Fig. 2c).H1 has exhibited high photo-stability compared to IR-26, with negligible decay under continuous excitation for 1 h (Fig. 2d).
The calculated HOMO and LUMO orbital surfaces of H1 have shown a larger band gap compared to that of Q4, leading to a higher performing uorophore (Fig. 2e and Table S1 †).The QY of H1 was $2.0% under 785 nm excitation (in CH 2 Cl 2 , measured against an IR-26 reference with a nominal quantum yield of 0.5%, Fig. S1 †).All these data demonstrated H1 could be a promising NIR-II dye, suitable for further NIR-II imaging applications.
SXH was easily prepared through conjugation of four carboxylic acid groups of H1 with PEG 1000 chains (Fig. 3a and ESI †).SXH was puried using HPLC and characterized using MALDI-TOF-MS (see ESI †).SXH exhibited high aqueous solubility and the uorescence emission spectrum of SXH demonstrated a similar emission wavelength at $1100 nm to that of H1 (Fig. 3b).The results from a cytotoxicity study further indicated the high viability of U87MG and L929 cells aer 24 h of incubation with different doses of SXH (2, 4, 6, and 8 mM), demonstrating the high biocompatibility of SXH (Fig. 3c).Excretion kinetics were investigated by intravenous injection of 100 mg of SXH into U87MG tumor-bearing nude mice (n ¼ 3) for glioblastoma (GBM) imaging and collecting urine during the course of 24 h post-injection (P.I.).Glioblastoma, the most common primary brain tumor in adults, is usually rapidly fatal. 21The care  for patients with a newly diagnosed glioblastoma entails surgical resection and concurrent radiation therapy (RT) and chemotherapy.Pharmacokinetics of SXH demonstrated rapid urine excretion, with $90% of SXH removal through the renal system within the rst few hours of the 24 h post-injection (Fig. 3d and Fig. S2 †).Finally, U87MG tumor-bearing nude mice (n ¼ 3) were injected with 100 mg of SXH and non-invasive NIR-II uorescence imaging of the glioblastoma tumor was conducted at particular time points.Aer 30 min post-injection, the tumor was clearly visible with a T/NT ratio of $4 and showed passive uptake at all time points due to the non-specic diffusion and accumulation of SXH (Fig. 3e).Ex vivo biodistribution studies were further performed at 24 h post-injection of the probe to evaluate the distribution of SXH in major organs (Fig. S3 †).It was found that SXH mainly accumulated in the kidneys, suggesting that the clearance route of SXH was through the renal system.In addition, a high level of accumulation was also observed in the tumor, indicating that SXH can passively target tumors and be used for future cancer theranostic applications (Fig. S3 †).
Although PEGylation of H1 provided a rapidly excreted, versatile contrast agent capable of passive tumor uptake, H1 could provide more tumor-specic targeting by linking to a molecular imaging ligand.We next demonstrated the application of H1 for receptor-targeted glioma imaging.Integrin a V b 3 has high expression levels in several malignant diseases including glioblastoma and are established biomarkers for metastatic diseases. 22The integrin targeting peptide RGD (arginine-glycine-aspartic acid) has shown promising results for non-invasive molecular imaging of integrin a V b 3 expression in the NIR-I region. 23Considering the advantages of NIR-II imaging, a novel integrin a V b 3 -targeted NIR-II uorophore, SDH, was developed and explored to investigate its imaging properties in vivo.SDH was prepared through conjugation of H1 with a mono-c(RGD) targeting peptide (Fig. 4a), and then puried by HPLC and characterized by MALDI-TOF [calcd for C 72 H 79 N 15 O 14 S 4 : 1589.481,found: m/z 1589.669].The uorescence emission spectrum of SDH demonstrates an emission wavelength at $1050 nm (Fig. 4b).The cell toxicity study also indicated the high biocompatibility of SDH in vitro (U87MG and L929 cells aer 24 h incubation with 2, 4, 6, and 8 mM doses of SDH, Fig. S4 †).These results demonstrated that SDH, as a promising and biocompatible NIR-II uorescent probe, is suitable for tumor targeting imaging.
SDH was then intravenously injected (100 mg) into U87MG tumor-bearing mice (n ¼ 3 per group).From NIR-II imaging data, the U87MG tumor could be clearly visualized from the surrounding background tissue during 24-72 h post-injection (P.I.) (Fig. 4d, 1000LP, 200 ms), and the tumor uptake reached a maximum at 48 h.The specicity of SDH for integrin a V b 3 was conrmed by the blocking experiment.The tumors uorescence signals were successfully reduced at all time points aer coinjection of RGD peptide (500 mg) with SDH for NIR-II imaging (Fig. 4d).An ex vivo biodistribution study indicated that high accumulation was observed in the liver and kidneys, which suggested that the clearance routes of SDH were through both hepatobiliary and renal systems (Fig. S5 †).In addition, the uptake of SDH in tumors was far beyond that of other normal organs and no uptake could be observed in the blocking group,  which further conrmed the good integrin a V b 3 -targeted ability and specicity of SDH (Fig. S5 and S6 †).Hence, the excellent translation ability of SDH represents a highly promising uorescent probe for non-invasive monitoring of early stage glioblastoma in the NIR-II region.
An emerging uorescence imaging application of NIR uorophores, such as indocyanine green (ICG), is currently undergoing clinical trials in detecting sentinel lymph nodes (SLNs) for surgical resection. 24Selectively removing sentinel lymph nodes alleviates lymphedema and other ailments that would be caused by total lymph node removal, which is performed to prevent cancer metastasis.Recent advances in NIR-I uorescence molecular imaging (FMI) for intraoperative image-guided cancer resection have introduced new frontiers for FMI-based therapeutic interventions in preclinical research and clinical applications.6][27][28][29][30][31][32][33] To demonstrate the feasibility of H1 for SLN surgery, H1 was encapsulated into a PEGylated surfactant, DSPE-mPEG 5000 , to prepare watersoluble and biocompatible NIR-II nanoprobes, H1 NPs (Fig. 5a).The prepared H1 NPs showed high monodispersity and homogeneity with an average particle size of $70.0 nm by transmission electron microscopy (TEM, Fig. 5b) and a hydrodynamic diameter of $80.0 nm as determined by dynamic light scattering (DLS, Fig. S7 †).The uorescence emission wavelength of H1 NPs was $1100 nm (Fig. S8 †).The result of the cytotoxicity study indicated the high biocompatibility of H1 NPs (Fig. S9 †).The amount of H1 encapsulated in the liposomes was measured by a UV/VIS spectrophotometer at 874 nm (ESI †).The dye encapsulation efficiency of H1 NPs was 79.8 AE 0.6% (n ¼ 3).
The U87MG tumor-bearing nude mice (n ¼ 3) were injected with 150 mL of H1 NPs.Immediately aer injection, the superior imaging of blood vessels of the whole body and tumor could be clearly observed from the surrounding background tissue using NIR-II imaging (Fig. 5c).Based on this promising result, we applied H1 NPs for lymph node imaging and image-guided surgery on the C57BL/6J model (Fig. 5d).C57BL/6J mice were injected with 100 mL of H1 NPs, with the help of NIR-II imaging, a SLN was successfully determined, even covered with so tissue.Aer the SLN was exposed, it was then resected thoroughly.More importantly, the border of the SLN was easily distinguished, avoiding unnecessary damage of surrounding tissue such as nerves, vessels and tendons.

Conclusions
In summary, we have developed a newly designed and facilely prepared NIR-II uorophore H1 with improved uorescence by introducing 2-amino 9,9-dialkyl-substituted uorene as a donor into the backbone.Based on this H1 scaffold, three types of NIR-II imaging probe, SXH, SDH and H1 NPs, have been prepared and allow for integrin a V b 3 -targeted glioma imaging.To the best of our knowledge, this is the rst time that a NIR-II molecular uorophore has been shown to delineate tumours from surrounding normal tissue.Various biomedical applications such as high resolution imaging of blood vessels on tumours and the whole body of living mice using H1 NPs were also performed through a passive targeted probe.With the help of NIR-II imaging, an SLN was successfully determined and resected thoroughly.Future work will focus on performing intraoperative image-guided cancer surgery in orthotopic models and specialized pharmacokinetic studies with the eventual goal of initiating clinical trials with a NIR-II smallmolecule uorophore.

Fig. 3
Fig. 3 (a) A schematic design of SXH showing four carboxylic acid groups of H1 conjugated with PEG 1000 chains.(b) UV absorbance of SXH and NIR-II fluorescence emission of SXH with a peak at $1100 nm under an 808 nm excitation laser (solvents: water, exposure time: 10 ms).(c) Cellular toxicity of SXH with different doses (2, 4, 6, and 8 mM) in U87MG and L920 cells.(d) SXH agglomerated cumulative urine excretion curve during 24 h post-injection.(e) Non-specific targeting imaging of the U87MG tumor based on SXH under an 808 nm excitation (1000 LP and 200 ms).

Fig. 4
Fig. 4 (a) A schematic of SDH showing one of the carboxylic acid groups of H1 conjugated with targeted ligand RGD peptide.(b) UV absorbance and NIR-II fluorescence emission of SDH.(c) NIR-II signals of U87MG cell labelling by SDH and SDH + excess RGD as a blocking agent (block group) under 808 nm excitation (1000LP and 100 ms).(d) NIR-II images of U87MG tumor mice (n ¼ 3) at different time points (1, 6, 24, 48, and 72 h) after tail vein injection of SDH with or without the blocking agent RGD (500 mg) under 808 nm excitation (1000LP and 200 ms).