Promoting near‐infrared II fluorescence efficiency by blocking long‐range energy migration

Generally, long wavelength absorbed near‐infrared II (NIR‐II) dyes have a low fluorescence efficiency in aggregate states for aggregate‐caused quenching effect, simultaneously enhancing efficiency and extending absorption is a challenging issue for NIR‐II dyes. Here, three benzo[1,2‐c:4,5‐c']bis[1,2,5]thiadiazole (BBT) derivatives (TPA‐BBT, FT‐BBT, and BTBT‐BBT) are used to clarify fluorescence quenching mechanisms. When the BBT derivatives are doped into a small molecule matrix, they show quite different fluorescence behaviors. Structure‐distorted TPA‐BBT displays fluorescence quenching originating from short‐range exchange interaction, while FT‐BBT and BTBT‐BBT with a co‐planar‐conjugated backbone exhibit concentration‐dependent quenching processes, namely changing from long‐range dipole‐dipole interaction to exchange interaction, which could be majorly ascribed to large spectral overlap between absorption and emission. By precisely tuning doping concentration, both FT‐BBT and BTBT‐BBT nanoparticles (NPs) present the optimal NIR‐II fluorescence brightness at ∼2.5 wt% doping concentration. The doped NPs have good biocompatibility and could be served as fluorescence contrast agents for vascular imaging with a high resolution under 980‐nm laser excitation. Those paradigms evidence that molecular doping can promote fluorescence efficiency of long wavelength‐absorbed NIR‐II fluorophores via suppressing long‐range energy migration.

However, except for some polymethine dyes and semiconducting polymers, the maximum absorption of most organic NIR-II dyes is located in the range of 650-900 nm, the short excitation wavelength would strongly restrict imaging resolution and depth. [1,2,[14][15][16][17][18][19][20] Although organic fluorophores with donor-acceptor-donor (D-A-D) structure could easily tune photophysical properties, the long-wavelength-absorbed NIR-II fluorophores display severely aggregate-caused quenching (ACQ) effect in aggregate states. Many efforts have been devoted to molecular structure modification to improve NIR-II fluorescent efficiency. [1,2] For example, in the most typical benzo[1,2-c:4,5-c']bis( [1,2,5]thiadiazole) (BBT)-based organic NIR-II dyes, 3-substituted or 3,4di-substituted thiophene could cause largely conformation distortion between BBT and thiophene moieties for steric hindrance repulsion effect, which effectively hampered intermolecular π-π stacking and reduce ACQ effect. [21][22][23][24][25][26][27][28][29][30] As a consequence, the NIR-II fluorescence quantum yield (QY) was over 11% in an aqueous solution after carefully molecular structure optimization. [25,31] While the distorted conjugated backbones would not only lead to a blue shift of the maximum absorption peak but also decrease the molar extinction coefficient, both are detrimental to construct long-wavelength excited NIR-II dyes. [26,27] In addition, there is a trade-off between NIR-II fluorescence efficiency and absorption wavelength in this molecular design strategy. So, it is hard to simultaneously design NIR-II fluorophores with high fluorescence efficiency and long-wavelength absorption, an alternative strategy should be developed to solve this contradictory issue.
BBT-based NIR-II fluorophores with quasi-coplanar or coplanar conformation could facilely prolong absorption profiles >800 nm, even to NIR-II region, and showed moderate to high fluorescent QY (>2.0%) in organic solvents, while their NPs suffered from ACQ issues in aqueous solution, which was commonly ascribed to intermolecular π-π stacking. [22,24,32,33] Some bulky substitutions, such spirobifluorene, tetraphenylethene, and triaryl amine were tried to functionalize BBT-based fluorophores to overcome ACQ. [34,35] However, it was unexpected that the fluorescence quenching could not be prevented effectively, the QY of spirobifluorene-modified BBT derivative dramatically dropped from 5.9% in tetrahydrofuran to 0.04% in NPs, indicating that the quenching effect should come from other nonradiative pathways, not merely from intermolecular π-π stacking. [34] Herein, three BBT derivatives with distorted or co-planar conjugated backbones were synthesized to clarify fluorescence quenching mechanisms in the aggregate state (Scheme 1). The results reveal that fluorescence quenching mainly originates from long-range dipole-dipole interaction for FT-BBT and BTBT-BBT with co-planar structures, while from short-range exchange interaction for TPA-BBT with distorted structure. So, doping BTBT-BBT into a matrix with a low content could block long-range energy migration, and the BTBT-BBT doped NPs have high fluorescence brightness under 980-nm laser excitation, which could be further used as NIR-II fluorescence contrast agent to visualize the vascular system.

RESULTS AND DISCUSSION
To extend the absorption profile of organic dyes, donoracceptor type molecular structure with co-coplanar configuration is an effective design principle; meanwhile, bulky groups should be fixed to molecular skeleton to reduce unexpected intermolecular π-π stacking. With this philosophy, a BBT derivative (BTBT-BBT) was designed and synthesized ( Figure 1). BTBT-BBT was successfully synthesized via successive four-step reactions, and its molecular structure was solidly confirmed by nuclear magnetic resonance and matrix-assisted laser desorption ionization time-of-flight mass spectrum (Figures S1-S14). Both FT-BBT and BTBT-BBT have high thermal stability with a thermal decomposition temperature of 388 and 375 • C, respectively ( Figure S15). In addition, the compoumd DBT was also synthesized as the molecular matrix, crystal data indicate that DBT has a fully coplanar and rigid structure, butylphenyl moieties locate on both sides of the conjugated plane, so it is reasonable to infer that BTBT-BBT also has coplanar structure, and the bulk side chains can impede π-π stacking ( Figure 2D and Table S1). As expected, theoretic calculation by density function theory indicates that TPA-BBT has a highly distorted structure with torsion angles of ∼26 o and 50 o , while the molecular structures gradually planarize in FT-BBT and BTBT-BBT, and BTBT-BBT adopts fully coplanar structure with torsion angles of 0 o (Figure 2A-C). The frontier molecular orbitals were evaluated by cyclic voltammetry ( Figure 2E,F), the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are −5.16 and −3.84 eV for FT-BBT, and −5.05 and −3.85 eV for BTBT-BBT, so the bandgap is 1.32 and 1.20 eV for FT-BBT and BTBT-BBT, respectively. Those data are consistent with theoretic results ( Figure S16), indicating that LUMO mainly locates on BBT group, and electron-donating groups mainly influence HOMO in BBT derivatives. As a result, planar conformation and strong electron-donating ability can decrease bandgap in BBT derivatives and extend their absorption profiles to NIR region. In addition, the molecular length of the conjugated backbone is about 28.4, 27.3, and 32.9 Å for TPA-BBT, FT-BBT, and BTBT-BBT, respectively.
The solvent effect of absorption and fluorescence were further investigated. As shown in Figure 3, BBT derivatives display a absorption band at near infreared region due to intramolecular charge-transfer effect. With an increase in solvent polarity, the absorption profiles are slightly blueshifted, the maximum absorption peaks in toluene and dichloromethane are 730 and 712 nm for TPA-BBT, 846 and 836 nm for FT-BBT, and 947 and 932 nm for BTBT-BBT, respectively. The optical gaps of FT-BBT and BTBT-BBT are ∼1.29 and ∼1.18 eV, respectively, calculated from their optical absorption edges, which are consistent with the results of cyclic voltammetry. In toluene solution, the molar extinction coefficient of TPA-BBT, FT-BBT, and BTBT-BBT is 2.0 × 10 4 , 3.8 × 10 4 , and 4.5 × 10 4 L mol −1 cm −1 , respectively, suggesting that molecular conformation planarization can obviously enhance absorption ability of the dyes. In comparison with TPA-BBT, there is ∼110 and 220-nm red-shift in the maximum absorption peak of FT-BBT and BTBT-BBT due to coplanar molecular structures promoting electron delocalization over conjugated backbones. For the strong electron-accepting ability of the BBT group, TPA-BBT, FT-BBT, and BTBT-BBT display solvent polarity-dependent NIR-II fluorescence emission, the maximum emission peak in toluene and dichloromethane centers on 998 and 1045 nm for FT-BBT, and 1092 and 1178 nm for BTBT-BBT, respectively. Importantly, BTBT-BBT has a fluorescence QY of 7.4% in toluene calculated by using FT-BBT as the reference fluorophore (QY FT-BBT = 19% in toluene, Figure S17). [33,36] While TPA-BBT has strong fluorescence peak centered at 992 nm in toluene, and weak fluorescence peak at 1022 nm in dichloromethane, showing that fluorescent properties of TPA-BBT are more sensitive to solvent polarity for its strong intramolecular charge transfer effect.
The NPs with pure dye molecules are fabricated by encapsulating TPA-BBT, FT-BBT, and BTBT-BBT into DSPE-mPEG 2000 micelles, respectively. As shown in Figure 3C,F, similar to the literatures report, TPA-BBT NPs shows strong NIR-II fluorescence intensity for high QY of ∼9%, and FT-BBT NPs possess very weak NIR-II fluorescence for low QY of 0.02%. [22,25] Unexpectedly, BTBT-BBT with eight bulk and steric n-butylphenyl groups only emits weak fluorescence in NPs under 980-nm laser excitation ( Figure 3I). The results support the idea that fluorescence quenching is hard to be prevented in BBT-based NIR-II fluorophores with planar molecular structure, that is why the researchers developed D-A-D-structured NIR-II dyes with distorted molecular conformation. [1,2] So, we want to explore the reasons that cause severe fluorescence quenching in those NIR-II dyes except for π-π stacking-related quenching species.
Previously, Dai and Li groups reported an intriguing and robust way to improve NIR-II efficiency by encapsulating NIR-II dyes into amphiphilic polymers with poly(styrene) structures, and the brightness could be enhanced about one to two magnitudes. [14,[36][37][38] This gave us a hint that isolating NIR-II dyes by organic matrix may reduce fluorescence quenching in an aggregate state, so we tried to use DBT as a matrix, and TPA-BBT, FT-BBT, or BTBT-BBT as a dopant to fabricate doped NPs. As indicated in Figure 3B,E,H and S18, the absorbance of TPA-BBT, FT-BBT, and BTBT-BBT shows a linear relationship with the doping content, while the maximum absorption peaks keep unchanged at 730, 840, and 957 nm for TPA-BBT, FT-BBT, and BTBT-BBT NPs, respectively, suggesting that the molecular conformation in the ground state is the same in different doping content systems. Interestingly, when the total mass (dye + DBT) concentration keeps unchanged, the fluorescence intensity of FT-BBT-and BTBT-BBT-doped NPs gradually enhances as the decrease of doping content, and the optimal doping content is ∼2.5 wt%, which displays the highest fluorescence intensity ( Figure 3F,I). Unlike FT-BBT and BTBT-BBT, the fluorescence intensity always goes down as doing content decreases in TPA-BBT-doped NPs ( Figure 3C (Table S2). The results clearly demonstrate that the doping strategy is a robust way to improve NIR-II fluorescence efficiency. In addition, as shown in Figure 3C,F,I, the maximum emission peak gradually blue-shifts from 1081 to 975 nm for FT-BBT, and from 1120 to 1062 nm for BTBT-BBT as the doping concentration decrease from 100 wt% to 1 wt%, while TPA-BBT-doped NPs show unchanged and well-resolved emission bands at 937, 1011, and 1100 nm, corresponding to 0-0, 0-1 and 0-2 vibronic transitions, respectively, the intensity ratio of 0-0 to 0-1 emission bands gradually increases as the decrease of doping content. The spectral change of those BBT derivative NPs can be ascribed to a weak self-absorption effect in low doping content, and the low-energy excimer/exciplex emission does not be observed in the long wavelength region.
To further uncover the origin of differences in fluorescence behaviors, NIR-II fluorescence imaging was conducted under identical conditions except for doping concentration. As shown in Figure 4A-C, when the doping content drops from 100 wt% to 25 wt%, the fluorescence intensity of the NPs increases or decreases slowly, while the trend changes dramatically below 25 wt%, indicating that ∼25 wt% is a critical doping content. As illustrated in Figure 4G, since DBT matrix and NIR-II dyes have similar and large aromatic structures, the dyes should be uniformly dispersed into the matrix, the critical intermolecular distance (D c ) was calculated by the following equation [39] : where V is the unit cell volume of DBT (∼5794.96 Å 3 ), X c is the critical doping molar content (X c = 0.202, 0.208, and 0.137 for TPA-BBT, FT-BBT, and BTBT-BBT, respectively), N is number of DBT in the unit cell (N = 4), 2 V/N is used to evaluate the volume of dyes. According to the above data, D c was calculated to be 25.5, 25.2, and 28.4 Å for TPA-BBT, FT-BBT, and BTBT-BBT, respectively. The real intermolecular distance may be larger than D c as DBT is amorphous and loosely packed in NPs. Since D c is well comparable to the molecular length of conjugated backbones (Figure 2A), indicating the ACQ can be ascribed to exchange interaction (Dexter energy transfer). Interestingly, as indicated in Figure S21 as the doping content decreases from 25 wt% to 1 wt%, the fluorescence intensity of FT-BBT and BTBT-BBT sharply increases and peaks at ∼2.5 wt%, then decreases at lower doping content, which means 2.5 wt% is another critical doping content. According to the above equation, D c1 was calculated to be 51.9 and 61.2 Å for FT-BBT and BTBT-BBT, respectively, which was about two times molecular length. Since D c1 is quite large, the nonradiative energy transfer process is not the result of exchange interaction. Therefore, the ACQ of FT-BBT and BTBT-BBT in low doping content should be assigned to multipolar interaction. Moreover, at the equivalent mass of NIR-II emitters, as doping content decreases from 100 wt% to 1 wt%, the brightness of TPA-BBT exhibits only 2.6 times enhancement, while the brightness of FT-BBT and BTBT-BBT-doped NPs enhances more than two orders of magnitude ( Figure S22 Since FT-BBT or BTBT-BBT-doped NPs have similar concentration quenching phenomena to rare-earth ion doping systems, [40,41] the interaction types between NIR-II dyes can be estimated by the following equation: where I is fluorescence intensity, x is molar doping content, k and β are constants for the same excitation condition, θ is the interaction type, θ = 3, 6, 8, and 10 refers to exchange interaction with the nearest neighbor molecules, dipole-dipole interaction, dipole-quadrupole interaction, and quadrupole-quadrupole interaction, respectively. As presented in Figure 4D-F, the relationship of I/x and x is plotted at logarithmic plot, −θ/3 is the slope of linear fitting, so the θ values are 2.6, 5.1, and 4.1 for TPA-BBT, FT-BBT, and BTBT-BBT, respectively. The θ value of TPA-BBT is close to 3, demonstrating that the concentration quenching originates from short-range exchange interaction. While the θ values of FT-BBT and BTBT-BBT are larger than 3 and close to 6, indicating that the concentration quenching mainly results from dipole-dipole interaction, namely Föster-like energy migration. While the distance D c1 is quietly different from the Föster distance R 0 , the R 0 is the distance at which energy transfer efficiency is 50% in the donor and acceptor system, and the D c1 is the distance of between dyes at which the doping NPs have the highest brightness. The above experimental results suggest the NIR-II fluorescence quenching processes are quite different between structure-distorted TPA-BBT and structure-planarized FT-BBT and BTBT-BBT. As depicted in Figure 2, TPA-BBT has a large Stokes shift (∼290 nm), there is little overlap between absorption and emission spectra, and the exciton energy migration in NPs occurs only through short-range exchange interaction ( Figure 4H). Unlikely, due to structural planarization and rigidity, both FT-BBT and BTBT-BBT have a small Stokes shift (135 nm for FT-BBT, 102 nm for BTBT-BBT), there is a large spectral overlap between absorption and emission in aggregate state, the exciton energy migration majorly originates from long-range dipole-dipole interaction, which leads to severely ACQ in aggregate states ( Figure 4I). So, doping long-wavelength absorbed NIR-II dyes into a matrix is an effective approach to overcoming the long-range quenching effect and enhancing fluorescence efficiency.
Since BTBT-BBT NPs with 2.5 wt% doping concentration have a maximum absorption peak of 957 nm and the highest fluorescence brightness under 980-nm laser excitation ( Figure S23), which are used to perform further experiments. First, the size distribution and morphology of NPs were investigated by dynamic light scattering and transmission electron microscope (TEM), BTBT-BBT-doped NPs show irregular aggregated NPs, and the hydrodynamic size and zeta potential are 72.2 ± 19.1 nm, and −32.61 mV ( Figure 5A), respectively. In addition, as shown in Figures  S24 and S25, both the fluorescence intensity and particle size keep unchanged for 7 days at room temperature, indicating the as-prepared NPs have a good stability and can be served as NIR-II fluorescence imaging contrast agent for blood vessels and tumors. Then, the biocompatibility of NPs was assessed by 2-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay, the viability of triple-negative breast cancer cells is still about 90% when the incubation concentration is up to 100 μg/mL ( Figure 5B). In addition, the living/dead co-staining assay also shows that the cells keep a high survival rate ( Figure 5C). The above experimental data indicate 2.5 wt% doped BTBT-BBT NPs have good biocompatibility and should be safely used as imaging contrast in vivo. Third, fluorescence intensity is a linear relationship with concentration under 980-nm laser excitation, suggesting that 2.5 wt% doped BTBT-BBT NPs are suitable for fluorescence signal quantification ( Figure S26). Fourth, the imaging depth experiments of NPs were also performed under 980-nm laser excitation ( Figure S27), the fluorescence  [42] intensity rapidly drops from ∼4.1 × 10 4 to ∼1800 as the depth increase from 0 to 8 mm, and the penetration depth reaches ∼6 mm. Finally, using NIR-II fluorescence imaging to visualize blood vessel and lymphatic systems is an important tool for evaluating vascular heterogeneity and tumor metastasis. Whole-body blood vessel imaging using BTBT-BBT-doped NPs as contrast agents was conducted under 980-nm laser excitation. As shown in Figure 5D, distinct vascular anatomy is observed after 15 min postinjection of tail-intravenous administration, and the apparent width of the major blood vessel of the mouse hindlimb is ∼0.42 mm with a signal-to-background ratio of ∼1.8 ( Figure 5E). Furthermore, the pharmacokinetic processes of BTBT-BBT doped NPs can be directly visualized via blood vessel imaging. The fluorescence intensity of the major blood vessel of the hindlimb decreases rapidly within 1 h post-injection, then gradually fades off ( Figure 5D). Fluorescence imaging of ex vivo organs was performed at 8 h postinjection to evaluate the biodistribution of 2.5 wt% doped BTBT-BBT NPs ( Figure S28). The results indicate that the NPs mainly accumulate in the liver and spleen. The hematoxylin-eosin (H&E) staining of the major organs reveals no noticeable pathological change at 48 h after intravenous administration ( Figure  S29), suggesting that the NPs have a good bio-safety. In vivo imaging experiments indicated that BTBT-BBT-doped NPs can be served as an excellent NIR-II fluorescence probe for visualizing vascular systems.

CONCLUSION
In summary, the novel NIR-II fluorophore BTBT-BBT has a strong absorption peak centered at ∼947 nm for fully coplanar conformation and a QY of 7.4% in solution, while it shows severe fluorescence quenching in aggregate. To clarify the quenching mechanism, the photophysical properties of three BBT derivatives were investigated in detail. The experimental results reveal that fluorescence quenching mainly originates from long-range dipole-dipole interaction for FT-BBT and BTBT-BBT with co-planar conformation, while from short-range exchange interaction for TPA-BBT with distorted structure. The reasons for those phenomena may lie in the spectral overlap degree between absorption and fluorescence, and the large spectral overlap will promote Föster-like energy migration and energy dissipation via nonradiative pathways. It can be concluded that doping BTBT-BBT into a molecular matrix in a low content (∼2.5 wt%) can effectively block the long-range energy migration pathway, and the BTBT-BBT-doped NPs have high fluorescence brightness under 980-nm laser excitation, which can be further used as NIR-II fluorescence contrast agent to investigate vascular system.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.