Development of a high quantum yield dye for tumour imaging
A fluorescent dye, FEB, with high fluorescence quantum yield for tumour imaging is reported. FEB dyes can be efficiently synthesized in three steps and then easily modified with either PEG or PEG-iRGD to yield FEB-2000 or FEB-2000-iRGD, respectively. Both modified dyes showed negligible toxicity and were thus able to be adopted for in vivo tumour imaging. PEG modification endowed the dye FEB-2000 with both long circulating times and good tumour targeting properties in a MDA-MB-231 xenograft model. Further conjugation with iRGD to generate FEB-2000-iRGD showed minimal targeting enhancement. These results provide a template for the efficient preparation of FEB dyes for use in tumour imaging, thus providing a foundation for future modifications.
Introduction<!>Results and discussion<!>Conclusions
<p>It has remained a high priority to develop sensitive and noninvasive diagnostic tools for cancer-a collection of the most life-threatening diseases. 1 Conventional approaches such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and optical imaging have already shown great promise for non-invasive, real-time, and high-resolution tumour imaging. [2][3][4] Among these, uorescence imaging in the far red and NIR window has several advantages including minimal photo-damage to biological samples, satisfying penetration depth, and negligible interference from background auto-uorescence. [5][6][7] Continuous work is being invested in exploring better uorescent dyes for use in imaging. To date, numerous dyes have been developed and marketed for various biomedical applications, including tumour diagnosis and molecular image-guided surgeries. 8 However, lots of them suffer from low biocompatibility, high toxicity, and low quantum yields. 9,10 Some dyessuch as Rhodamine B-have small Stokes shis (typically less than 35 nm) that cause systematic errors due to excitation backscattering effects. 11 In addition, most tumour-targeting uorescent probes require a combination of targeting partners (such as metabolic substrates), 12 cell-surface receptor targeting peptides, 13 growth factors, 7,14 antibodies, 15 and cancer-specic cell-surface biomarkers. 16,17 Recently, uorene and 3,4-ethoxylene dioxythiophene (EDOT) were employed as the shielding and donor units, respectively, to construct a donor-acceptor-donor-based uorophore that absorbs in the NIR-II window. It was found that using both uorene and EDOT could reduce the intermolecular or intramolecular interactions in the uorophore molecules, enhancing the uorescence quantum yield. Recent work from our lab developed a molecular uorophore (IR-E1) that contained this donor-acceptor-donor structure. 18 Specically, we used benzo[1,2-c:4,5-c 0 ]bis([1,2,5] thiadiazole) (BBTD) as the acceptor and thiophene-based units as the donor. This uorophore was used to characterize dynamic vascular changes in a mouse model of traumatic brain injury. Herein, we report a new uorescent dye, FEB, that is easily synthesized and was found to be suitable for in vivo tumour imaging (Scheme 1). We noticed that benzo-2,1,3-thiadiazole (BTD) has been utilized as a core unit with good uorescence in both solution and solid state. 10,19 By replacing the acceptor unit BBTD with BTD, new uorophores with an emission peak at 670 nm were synthesized. The non-planar, EDOT-BTD-EDOT conguration decreased inter-and intramolecular interactions. 20 The introduction of long alkyl chains reduced intermolecular interactions and enhanced the dye's quantum yield. 21 These uorophores demonstrated a high uorescence quantum yield of 35% in aqueous solution and a large Stokes shi of 120 nm, which are promising for bioimaging applications. We intentionally retained a bromine atom in the alkyl chain, allowing for further modications. 22 We further explored the tumour imaging application with the PEGylated uorophore and the one labelled with iRGD. Interestingly, PEGylated FEB itself was shown to have a tumour targeting effect.</p><!><p>A non-planar EDOT-BBTD-EDOT conguration could avoid inter-and intramolecular interactions. 18 The 3,4-ethylenedioxy groups on EDOT improve dye dispersity in water. Here, a nonplanar EDOT-BTD-EDOT conguration was introduced. Fluorescent BTDs are usually efficient and stable, and show a good signal-to-noise ratio. The uorene and long alkyl chains also served as donors, thereby reducing intermolecular interactions and avoiding uorescence quenching. The FEB dye was synthesized in three steps using a palladium-catalyzed Stille coupling reaction as its key step (Scheme 2). Compound 4 was obtained by di-acylation of compound 3, which was derived from cross-coupling between compounds 1 and 2 (see ESI †). Compound 4 was then coupled with the core part of FEB, namely compound 5, to derive FEB. This coupling step is facile and has a high yield. FEB retained four alkyl bromide moieties for further modications. Azidation was conducted to obtain FEB-N 3 , which can be modied via 'click' chemistry.</p><p>FEB-N 3 showed an absorbance peak at $550 nm and an emission peak at $675 nm in toluene (Fig. 1a). The absolute uorescence quantum yield of FEB-N 3 was 86% in toluene. To further protect the dye core and increase its aqueous solubility, FEB-N 3 was allowed to react with 2 kDa alkyne-PEG-COOH via 'click' chemistry. 23 Successful PEGylation was conrmed using GPC (Gel Permeation Chromatography, see GPC data in ESI †). Fluorescence spectroscopy data showed that FEB-2000 had an absorbance peak at $550 nm and an emission peak at $670 nm (Fig. 1b), which is signicantly better than Rhodamine dyes with small Stokes shis.</p><p>Previous reports have shown that the iRGD peptide c(CRGDK/RGPDC) can interact with both integrin and neuropilin-1 receptors, thereby enabling better cellular and deep tissue penetration. 24,25 To increase the cancer cell targeting ability, we prepared the cyclic iRGD peptide and conjugated it with alkyne-PEG-COOH by amide bond formation. The PEGylated peptide was then conjugated to FEB via 'click' chemistry, producing FEB-2000-iRGD. FEB-2000-iRGD showed an absorbance peak at $550 nm and an emission peak at $670 nm in water (Fig. 1c).</p><p>As shown in Fig. 1, both the PEG and iRGD modications did not interfere with the absorption behaviour. The absorption coefficients of FEB-2000 and FEB-2000-iRGD in water were 28 530 M cm À1 and 27 300 M cm À1 , respectively (Fig. S1 †). Both FEB-2000 and FEB-2000-iRGD showed good stability (Fig. S2 †). As the BTD acceptor was weaker in comparison to the NIR-II uorophore IR-E1, the emission spectrum of the FEB derivatives exhibited a blue shi. However, the reduction in conjugation resulted in a high uorescence quantum yield in water. Using Rhodamine B (QY ¼ 31%) as a reference, 26 the quantum yields of FEB-2000 and FEB-2000-iRGD in water were determined to be 35% and 36%, respectively (Fig. S3 †). The quantum yields of the FEB derivatives were therefore on par with most commercial deep red uorescence dyes, making them suitable for in vivo imaging. Similar to typical benzothiadiazole derivatives, the designed compounds displayed large Stokes shis (120-125 nm). In addition, they showed longer emission wavelengths.</p><p>Fluorescent molecule uptake was rst examined using in vitro systems. 27 MDA-MB-231 cells were incubated with 22 mM FEB-2000 and FEB-2000-iRGD in serum-free media for 6 h, washed, and then imaged using confocal microscopy (Fig. 2a). Both FEB-2000 and FEB-2000-iRGD showed high cellular uptake. We compared the FEB derivatives with the commercially available near-infrared dye IR-783, 28,29 which preferentially targets tumour cells (Fig. S4 †). Although the FEB derivatives showed a poorer ability to label living cells than IR-783, they could be easily prepared in three steps and conveniently modied via 'click' chemistry. Notably, both FEB-2000 and FEB-2000-iRGD showed negligible cytotoxicity in the MDA-MB-231 and HeLa cell lines at a concentration of 100 mM (Fig. 2b and S5 †).</p><p>To evaluate FEB-2000 distribution in vivo, we next performed uorescence imaging using a MDA-MB-231 xenogra model. With intravenous injection of 100 mL FEB-2000 (270 mM), specic tumour uptake of the dye was observed within 6 h. The tumour was clearly visible with a tumour/muscle (T/M) ratio of 2.0 (see Fig. 3a). Aer 24 h, the T/M ratio increased to 2.5. Tumour accumulation was still observed aer 96 h. At 48 h postinjection, we quantied the dye accumulation in different critical organs, including the tumour, heart, liver, spleen, lung, and kidney. As shown in Fig. 3b, there was little dye accumulation in organs other than the liver. It has been reported that the PEG chain itself showed noticeable tumour accumulation through EPR effects. 30,31 FEB-2000 accumulation in the tumour could be attributed to EPR effects caused by PEGylation.</p><p>As previously reported, some dyes require targeting partner conjugation for efficient tumour imaging. 27 We explored the inuence of the iRGD peptide on tumour-targeted optical imaging of the FEB derivatives. The in vivo performance of FEB-2000-iRGD was evaluated to determine any enhanced targeting abilities. However, no statistically signicant targeting enhancement was observed when comparing FEB-2000 with FEB-2000-iRGD (Fig. 3c). As a positive control, 100 mL of Cy5 (50 mM) was also injected. According to the optical imaging results, Cy5 showed a stronger background than that of FEB-2000. We also prepared Cy5-iRGD by conjugating Cy5-NHS and the iRGD peptide (conrmed by LC-MS, presented in ESI †). Except for the liver, direct imaging of dissected tissues and organs revealed that the FEB derivatives showed lower absorption in major organs when compared with either Cy5-iRGD or Cy5 (Fig. 3d and S6 †). In contrast to some earlier reports, recent work has shown that co-administration of iRGD has little effect on the permeability of the chemotherapeutic agent doxorubicin (DOX). 32,33 Our results also suggest that iRGD peptides have little effect on FEB tumour accumulation.</p><p>Taken together, our results indicate that PEGylated FEB alone has high tumour accumulation and long-lasting properties, while further attachment of iRGD provides no increase in FEB tumour accumulation. This indicates that FEB-2000 is  a powerful dye for use in tumour imaging. Pathological data using H&E staining from heart, liver, spleen, lung, and kidney also showed its good biocompatibility properties (Fig. 4).</p><p>NIR uorescence agents generally have low background autouorescence, which helps give better imaging contrast. 34,35 Near-infrared dyes including MH-148 and IR-783 showed a higher cell uptake and tumour/muscle ratio than the FEB derivatives. 29 Therefore, future work will focus on developing high-quantum-yield NIR-II uorophores based on the core structure of FEB. 36</p><!><p>Fluorescence imaging is a useful tool for cancer diagnosis and tumour image-guided surgery. Our study introduces an easily accessible uorescent dye, FEB, which is suitable for in vivo tumour imaging. The PEG modied FEB-2000 is water soluble and stable, showing high tumour accumulation and longcirculating properties. In vitro studies demonstrated that FEB dye derivatives penetrated MDA-MB-231 cells without conjugation of targeting peptides in addition to showing minimal cellular toxicity above the requisite imaging doses. Notably, targeting modications showed negligible enhancement of FEB-2000, suggesting that further development of FEB could potentially lead to an efficient, small molecule tumour imaging agent.</p>