3,4-Ethylenedithio thiophene donor for NIR-II fluorophores with improved quantum yields

Abstract

Bright fluorophores are vital for bioimaging in the second near-infrared (NIR-II, 1500–1700 nm) window. Donor engineering has been demonstrated to be effective at significantly improving the brightness of donor–acceptor–donor (D–A–D) NIR-II fluorophores. Developing new donor units to construct NIR-II fluorophores with improved quantum yields (QYs) is still challenging. Herein, 3,4-ethylenedithio thiophene (EDST) is employed as the donor unit for the first time to construct a new NIR-II fluorophore, IR-nFES. Compared with the fluorophore IR-nFE with the 3,4-ethylenedioxy thiophene (EDOT) donor, sulphur atom replacement can significantly increase the conjugation backbone distortion, affording an enhanced fluorescence QY of 5.5% in toluene for IR-nFES. Water soluble nanofluorophores (NFs) of IR-nFES are fabricated by encapsulating with an amphiphilic copolymer, and IR-nFE NFs show a QY of 1.3%, 4.8 times higher than that of the IR-nFE counterpart. It is further demonstrated that IR-nFES NFs are significantly brighter than IR-nFE NFs and indocyanine green (ICG), and also exhibit good optical stability. Consequently, IR-nFES NFs realize superior in vivo imaging of mouse cerebral vessels with a high signal-to-background ratio (SBR) of 4.99. Our studies reveal that sulphur substitution can afford new NIR-II molecular fluorophores with enhanced brightness.

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Introduction

Due to enhanced tissue penetration and decreased background signals, fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) has shown great promise for biological research and clinical applications.^1–11 However, the development of NIR-II fluorophores lags behind that of the visible and first near-infrared counterparts, showing significantly lower quantum yields (QYs). Organic fluorophores are favourable for biological applications due to their improved biocompatibility and structural tunability. To date, numerous organic molecular fluorophores, including polymethine fluorophores,^12–14 donor–acceptor–donor (D–A–D) fluorophores,^15–23 and A–D–A fused-ring acceptor fluorophores,^24–27 have been developed for NIR-II bioimaging. Among them, D–A–D fluorophores are an interesting type as their fluorescence emission wavelengths can be effectively tuned by varying D/A units.²⁸ They also exhibit appreciable fluorescence QYs, large Stokes shifts, and good photostability.^29–33

It has been demonstrated that molecular engineering of the donor unit plays a key role in improving QYs for D–A–D type NIR-II fluorophores, especially in aqueous conditions.^16–19 Thiophene- and benzene-based donor units are generally used to construct NIR-II fluorophores, and thiophene is more favourable than benzene as it could afford larger conjugation for longer emission wavelengths. Unfortunately, NIR-II fluorophores with pristine thiophene donors exhibit poor QYs in aqueous conditions.¹⁷ To overcome this problem, 3,4-ethylenedioxy thiophene (EDOT) was utilized to replace thiophene as the donor unit to construct S–D–A–D–S (S: shielding unit) type molecular fluorophores, and the QY was exponentially increased from 0.002% of IR-FTP with the thiophene donor to 0.2% of IR-FEP with the EDOT donor in aqueous solutions (the QY values were recalculated with IR-26's QY of 0.050% in 1,2-dichloroethane as the reference).¹⁷ 3-Alkoxy or 2-alkyl thiophene donors were also found to be effective to improve the QY, for example, IR-FTAP with 3-octyl thiophene as the donor exhibited a QY of 0.53% in water.¹⁸ Recently, a dioctyl chain-substituted 3,4-propylenedioxy thiophene (PDOT) unit was utilized as the donor, and the constructed fluorophore IR-FP8P not only showed an improved QY of 0.60%, but also exhibited a higher molar extinction coefficient than IR-FTAP.¹⁶ The interactions between fluorophores and water molecules were found to be a key issue for the low QYs in aqueous solutions. Moreover, such donor modifications afford increased distortion between the donor and acceptor units when compared with the pristine thiophene donor. This could reduce the interactions between the molecular cores and water molecules, leading to higher QYs.¹⁸ In the aforementioned fluorophores, aqueous solubility was enabled by conjugation with water soluble side chains. Besides, encapsulating the fluorophores with amphiphilic polymers is another way to prepare water soluble nanofluorophores (NFs).²⁷ NIR-II NFs generally exhibit higher QYs due to the weaker interactions between the molecular fluorophores and water molecules. However, tight intermolecular interactions in NFs often result in the aggregation caused quenching (ACQ) phenomenon.²⁹ Until now, only a few NIR-II molecular NFs could exhibit QYs of over 1% in aqueous conditions. Therefore, it is also of great significance to explore new donor units with optimized structures to further improve the QYs of NIR-II NFs.

Herein, a 3,4-ethylenedithio thiophene (EDST) unit is utilized as the donor for the first time to construct a new S–D–A–D–S type NIR-II molecular fluorophore, IR-nFES (Scheme 1). Replacing the oxygen atoms of the EDOT unit with sulphur atoms can significantly increase conjugated backbone distortion, resulting in an increased QY of 5.5% in toluene, much higher than those of reported NIR-II fluorophores in organic solvents.^{25–27,29–33} Encapsulating IR-nFES in an amphiphilic copolymer yields water soluble IR-nFES NFs, exhibiting the maximum wavelength of the fluorescent band of ∼1040 nm and a Stokes shift of ∼280 nm, which is more red-shifted compared to most organic NIR-II NFs. Benefiting from the weaker intermolecular interactions of IR-nFES molecules, the QY of IR-nFES NFs reaches 1.3%, which is 4.8 times higher than 0.27% of IR-nFE NFs and is among the highest QYs for NFs. When excited at 808 nm, the IR-nFES NF aqueous solution demonstrates higher brightness than IR-nFE NFs and good photostability. Consequently, IR-nFES NFs demonstrate significantly better results for in vivo imaging of mouse cerebral vessels with a signal-to-background ratio (SBR) of 4.99, much higher than 1.70 of indocyanine green (ICG).

Scheme 1 Molecular structures of IR-nFE and IR-nFES.

Results and discussion

Design and synthesis of fluorophores

As shown in Scheme 1, EDST is an analogue of EDOT when changing two oxygen atoms to sulphur atoms. Due to the larger size of sulphur than oxygen atoms, EDST is expected to afford larger conjugated backbone distortion in IR-nFES than in IR-nFE. It may be able to weaken the intermolecular interactions, thus improving the QY. The synthesis of IR-nFES is similar to that of IR-nFE (Scheme S1, ESI†). After converting EDST to the corresponding key intermediate EDST-SnBu₃, it is coupled with a fluorene unit. Finally, the Stille coupling reaction with the acceptor unit of dibromo-benzobisthiadiazole yields the final S–D–A–D–S type NIR-II fluorophore IR-nFES. The structures of IR-nFE and IR-nFES are validated by ¹H/¹³C NMR and high-resolution mass spectroscopy (see details in the ESI†).

Geometry and electronic properties

To investigate the influence of donor units on electronic properties and geometries, density functional theory (DFT) calculations were performed at the optimally tuned ωB97XD*/6-31G(d) level with Gaussian 16 software (computational details are shown in the ESI†).^34–37 As illustrated in Fig. 1, after replacing the EDOT unit with an EDST unit, the highest occupied molecular orbital (HOMO)/lowest unoccupied molecular orbital (LUMO) energy levels are lower-shifted from −5.85/−4.29 eV of IR-nFE to −6.25/−4.49 eV of IR-nFES. In this regard, the electronic energy gap is calculated to be 1.76 eV for IR-nFES, relatively larger than 1.56 eV for IR-nFE. The optimized ground-state (S₀) and excited-state (S₁) geometries reveal that the dihedral angles between the donor and acceptor units of IR-nFES are calculated to be 63°/43° at the S₀/S₁ state, significantly larger than 44°/31° of IR-nFE. The dihedral angles between the donor and shielding units of IR-nFES are also larger than those of IR-nFE (Fig. 1). The larger dihedral angles between the donor and acceptor/shielding units can afford larger conjugated backbone distortion in IR-nFES. On the other side, the side chains of the fluorene unit in IR-nFES orient more vertically to the conjugated backbone than those in IR-nFE. These could provide better protection of the conjugated backbone core in IR-nFES from intermolecular interactions, leading to higher QYs.

Fig. 1 (a) HOMOs/LUMOs and (b) optimized ground-state (S₀)/first singlet excited state (S₁) geometries of IR-nFE and IR-nFES. The alkyl chains on fluorene shielding units are simplified with methyl groups to reduce computational requirements.

Photophysical properties

The optical properties of IR-nFE and IR-nFES were investigated in toluene solution (Fig. 2). Compared to IR-nFE with an absorption peak at 760 nm and a molar extinction coefficient of 23.7 × 10³ M⁻¹ cm⁻¹, the IR-nFES exhibits a blue-shifted absorption and a lower molar extinction coefficient of 16.3 × 10³ M⁻¹ cm⁻¹ (Table 1), which can be ascribed to the larger calculated energy gap and larger conjugated backbone distortion, respectively (Fig. 1). It should be noted that IR-nFES exhibits more red-shifted absorption and a higher molar extinction coefficient than the previously reported IR-FA with an alkyl thiophene donor.¹⁷ The maximum wavelength of the fluorescent band of IR-nFES is located at 990 nm, a 28 nm blue-shift compared to 1018 nm of IR-nFE (Fig. 2b and Table 1). Both IR-nFE and IR-nFES display Stokes shifts over 250 nm. Notably, a QY of 5.5% is calculated for IR-nFES, relatively higher than 3.10% of IR-nFE.

Fig. 2 (a) Absorption spectra and (b) fluorescence emission spectra of the molecular fluorophores of IR-nFE and IR-nFES in toluene; the absorption spectra were measured at 30 μM. (c) Schematic illustration of the preparation of water soluble nanofluorophores (NFs) of IR-nFE and IR-nFES. (d) Hydrodynamic size of IR-nFE NFs and (e) IR-nFES NFs measured by the dynamic light scattering method. (f) Absorption spectra and (g) fluorescence emission spectra of IR-nFE NFs and IR-nFES NFs in water; the absorption spectra were measured at 0.05 mg mL⁻¹. The emission spectra were measured with an OD of 0.08 at 808 nm.

Table 1 Optical properties of molecular fluorophores in toluene and nanofluorophores (NFs) in water. ε_max: molar extinction coefficient, λ_abs: wavelength of the absorption peak, λ_em: maximum wavelength of the fluorescent band, QE = QY × ε_max. The QY is calculated in the range of 900–1400 nm, and the QY of IR-26 is 0.050% in dichloroethane as the reference

Fluorophores	ε max (10^3 M^−1 cm^−1)	λ abs (nm)	λ em (nm)	Stokes shift (nm)	QY (%)	QE
IR-nFE	23.7	760	1018	258	3.1	73
IR-nFES	16.3	715	990	275	5.5	89
IR-nFE NFs	17.5	768	1053	285	0.27	4.7
IR-nFES NFs	12.5	724	1043	319	1.3	16

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Nanofluorophores

The IR-nFE NFs and IR-nFES NFs are prepared by encapsulating IR-nFE and IR-nFES molecules with a biocompatible amphiphilic copolymer, 1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjugate-2000 (DSPE-mPEG2000), as illustrated in Fig. 2c. After encapsulation, a uniform and transparent NF solution can be obtained, suggesting the good dispersion of fluorophores in water. The hydrodynamic particle sizes of the two NFs are characterized by the dynamic light scattering (DLS) method and it is shown that IR-nFE NFs and IR-nFES NFs display a similar particle size of ∼45 nm (Fig. 2d and e). Similar to the absorption in toluene, IR-nFES NFs exhibit an absorption peak at 724 nm, a 44 nm blue-shift compared to 768 nm of IR-nFE NFs (Fig. 2f). A molar extinction coefficient of 12.5 × 10³ M⁻¹ cm⁻¹ is calculated for IR-nFES NFs, slightly lower than 17.5 × 10³ M⁻¹ cm⁻¹ of IR-nFE NFs (Table 1). The maximum wavelength of the fluorescent band of IR-nFES NFs is located at 1043 nm, similar to 1053 nm of IR-nFE NFs (Fig. 2g). The Stokes shifts of IR-nFE NFs and IR-nFES NFs are both over 280 nm. Impressively, the QY of IR-nFES NFs is calculated to be 1.3%, 4.8 times higher than 0.27% of IR-nFE NFs, which can be attributed to the weaker intermolecular aggregation of IR-nFES than that of IR-nFE in NFs. In addition, the significantly higher QY of IR-nFES NFs in water may also be ascribed to the better protection of fluorophores from interaction with water molecules due to the larger size of sulphur atoms in EDST units.^17,18,27 The brightness (QE) of fluorophores correlates with the product of QY and molar extinction coefficient (QE = QY × ε_max). Benefiting from the significant improvement of the QY of IR-nFES NFs, the QE of IR-nFES NFs is calculated to be 16, which is 3.4 times higher than 4.7 of IR-nFE NFs.

In vitro and in vivo imaging

Aqueous solutions of IR-nFES NFs, IR-nFE NFs and ICG with the same fluorophore mass concentration of 20 μg mL⁻¹ were prepared and imaged using an NIR-II fluorescence imaging system with excitation of an 808 nm laser (Fig. 3). The IR-nFES NF aqueous solution displays the highest brightness under both 900 and 1000 nm LP filters (Fig. 3a–c). It is worth noting that the brightness of IR-nFES NF aqueous solution can be further enhanced if the excitation wavelength of the laser is changed to shorter ones that can match the peak absorption wavelength of IR-nFES NFs. Long-term optical stability is also of vital significance for their application in NIR-II in vivo bioimaging. The fluorescence intensities of aqueous solutions of ICG, IR-nFE NFs and IR-nFES NFs were monitored under continuous 808 nm laser irradiation. As shown in Fig. 3d, after irradiation for 60 min, the fluorescence intensities of IR-nFE NF and IR-nFE NF aqueous solutions remain unchanged, while the fluorescence intensity of the ICG solution decreases rapidly, indicating the superior stability of the D–A–D NFs of IR-nFE and IR-nFES.

Fig. 3 Imaging performance of ICG, IR-nFE NFs and IR-nFES NFs with the same concentration (20 μg mL⁻¹) in water under 900 (a) and 1000 nm (b) long pass (LP) filters. (c) The fluorescence intensity of fluorophores under different LP filters. An 808 nm laser with a power density of 180 mW cm⁻² was used for excitation. Exposure times of 15 and 20 ms were used for imaging with 900 and 1000 LP filters, respectively. (d) Optical stability of ICG, IR-nFE NFs and IR-nFES NFs in water under continuous 808 nm laser irradiation with a power density of 50 mW cm⁻².

The cytotoxicity of IR-nFES NFs was assessed by the MTT assay (Fig. S13, ESI†). The cellular viability of 4T1 and bEnd.3 cells shows a survival range above 90% even at a concentration of 100 μg mL⁻¹, confirming their good biocompatibility for in vivo biological imaging. As shown in Fig. 4a, after an intravenous injection of IR-nFES NFs at a dose of 150 μL (1 mg mL⁻¹), sophisticated cerebral blood vessel networks could be clearly discriminated at 1300 nm LP at 2 min post-injection. In contrast, the imaged vessels are very blurry after injection of ICG with the same dose. Moreover, the in vivo time-course imaging using IR-nFES NFs still exhibits clear vessels after 20 min, while the vessels could not be observed for imaging using ICG (Fig. 4a and Fig. S14, ESI†). These results indicate the superior imaging performance of IR-nFES NFs to ICG in brightness and optical stability. A cross-sectional intensity profile displays a feature size of 413 μm and a maximum SBR of 4.99 for the cerebral vessel of IR-nFES NF treated mice (Fig. 4b), better than those of the ICG treated mice (over 570 μm and 1.70). In addition, the organs of BALB/c mice were stained with hematoxylin and eosin (H&E) (Fig. S15, ESI†), and the results demonstrate the good in vivo biosafety of n-FES NFs. IR-nFES NFs also exhibit relatively long blood circulation time (Fig. S16, ESI†).

Fig. 4 (a) Non-invasive in vivo NIR-II imaging of cerebral vessels of IR-nFES NFs and ICG with the same concentration (150 μL, 1 mg mL⁻¹) in PBS under a 1300 nm long-pass (LP) filter. The red line represents the cross-section of the vessels. (b) Cross-section profile of the cerebral vessel signal-to-background ratio across the solid red line. An 808 nm laser was used for excitation, providing a power density of 150 mW cm⁻² and an exposure time of 100 ms.

Conclusions

In summary, an EDST unit is formed by replacing two oxygen atoms of the EDOT unit with two larger sulphur atoms, and it is utilized as the donor moiety to construct a new S–D–A–D–S type NIR-II fluorophore, IR-nFES. The conjugated backbone distortion in IR-nFES is increased when compared with IR-nFE, which can effectively reduce the intermolecular interactions and thus significantly increase the QY of IR-nFES. Impressively, the encapsulated IR-nFES NFs exhibit a high QY of 1.3% in aqueous solutions, which is 4.8 times higher than 0.27% of IR-nFE NFs. IR-nFES NFs are brighter than IR-nFE NFs and also show good optical stability and biocompatibility. Cerebral vessels with a SBR of 4.99 are successfully observed clearly for imaging with IR-nFES NFs. The appreciable brightness and absorption of IR-nFES NFs in a shorter wavelength region can benefit the combination with other bright NIR-II fluorophores with absorption in longer wavelength regions for multi-colour imaging.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 21772084), Fundamental Research Layout of Shenzhen (No. JCYJ20180504165657443), Guangdong Provincial Natural Science Foundation-Yueshen Joint Funding (Youth Project) (No. 2019A1515110464), Shenzhen San-Ming Project (SZSM201809085) and Shenzhen Science and Technology Commission-free exploration/general project (No. JCYJ20190812151209348).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2qm01278b

‡ These authors contributed equally to this work.

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