Dimerization extends π‐conjugation of electron donor‐acceptor structures leading to phototheranostic properties beyond the sum of two monomers

Near‐infrared (NIR)‐II fluorescence imaging‐guided photothermal therapy (PTT) has attracted great research interest, and constructing donor‐acceptor (D‐A) electronic configurations has become an established approach to lower bandgap and realize NIR‐II emission. However, very few π‐conjugated phototheranostic agents can realize efficient NIR‐II guided PTT using a clinically safe laser power density, implying that sufficient photothermal performance is still desired. In addition to the continuously refreshed photothermal conversion efficiency levels, the strategies that focus on enhancing light absorptivity have been rarely discussed and endow a new direction for enhancing PTT. Herein, a dimerization π‐extension strategy is raised to synthesize π‐conjugated dimers with A‐D‐A monomers. We observe that the light absorptivity (ε) of the dimers is strengthened three times owing to the enhanced electronic coupling effect as a result of the π‐conjugation extension, thereby surpassing the 2‐fold increase in chromophore numbers from the monomer to dimers. Thanks to the enhancement in light absorption, the dimers could generate much more photothermal heat than the monomer in in vivo PTT treatments. Therefore, an efficient anti‐tumor outcome has been fulfilled by using dimers under a low laser power (0.3 W/cm2). Moreover, the dimers with extended π‐conjugation structures become more favorable to the radiative excited state decay, thus exhibiting a distinguishing improvement in NIR‐II imaging compared with monomer. Collectively, due to the improved light absorptivity, the dimers can gain superior NIR‐II fluorescence brightness and photothermal performance over the recently reported material, which goes beyond the monomer in double doses for in vivo applications. All these results prove that dimerization is an effective strategy for designing high‐performance phototheranostic materials.

[9] Such a current situation implies that the PTT performance of organic phototheranostic agents still needs improvements to meet the clinical requirements.Besides the gradually saturated photothermal conversion efficiency (PCE) levels, light absorptivity is also crucial to be taken into consideration as both PTT and NIR-II imaging depend on the light-harvesting capability of the organic π-conjugated materials, [10] which may provide another direction to substantially improve the materials' NIR-II guided PTT performance. [11,12]onstructing donor-acceptor (D-A) electronic configurations to reinforce intramolecular charge transfer is the most efficient method to develop low-bandgap π-conjugated materials for NIR-II guided PTT applications. [13,14]According to the energy gap law, the low-bandgap NIR organic πconjugated molecules usually favor the non-radiative decay processes, thus showing intrinsically high photothermal conversion efficiency. [15,16]For this reason, the π-conjugated small molecules with A-D-A or D-A-D structures are the most used NIR-II emitting and PTT multifunctional agents for their superior performance and clear structure-property relationship. [17,18]Recently, many groups, including ours, have paid great interest in studying a kind of A-D-A molecule comprised of two terminal electron-deficient indanone (IC) derivatives, and several structural design strategies, such as modulating the electron-donating core or engineering side chains, have been developed. [17,19]The PCE values of these A-D-A molecules could reach 70%-80% due to the extremely strong electron-withdrawing nature of the IC end units.However, very limited space has been left for this kind of molecule to further elevate PCE, especially by enhancing the D-A push-pull effect.On the other hand, extending π-conjugation is an alternate way to narrow the bandgap and enlarge the light-harvesting capability, which has not been investigated in previous studies. [11]The oligomerization strategy is a valid method to extend the π-conjugation skeleton.The resultant oligomers, which possess the advantages of both small molecules and polymers, generally exhibit clear molecular structures, defined molecular weights, and good batch-to-batch reproducibility. [20]Compared with D-A-type conjugated polymers with complicated configurations and conformations, the well-defined molecular structures of oligomers endow us with an opportunity to clarify the structure-property relationship.Moreover, the configuration and conformation of a molecule are critical for molecular packing in the aggregated states, which also play important roles in determining the phototheranostic properties.Accordingly, the dimers of an A-D-A type molecule with well-defined structures were synthesized and investigated in this work.
Herein, a dimerization π-extension strategy (Scheme 1) is developed, and the π-conjugation extending effect on the properties of these NIR-II emitting and PTT multifunctional agents has been investigated. [21,22]Specifically, an A-D-A type π-conjugated molecule BTIC was selected as the monomer structure, which showed a high PCE value of 66.4%. [21,22]Further, we employed different coupling approaches, the single-bond and vinyl-bond links, to synthesize A-D-A-linkage-A-D-A type dimers, dBTIC-S and dBTIC-D.Compared with the monomer, the light-harvesting capability of the dimers was enhanced by three times in the nanoparticle state.Interestingly, although the dimers contain 2-fold chromophores compared to the monomer, the enhancement in their light absorptivity surpasses the simple addition of chromophores and exhibits extra enhancements due to the electronic coupling effect of π-conjugation extension.Therefore, thanks to the enhancement in light absorption, the dimers could generate much more photothermal heat (ε × PCE) than the monomer and the other recently reported NIR-II emitting and PTT multifunctional agents, that is, 95400, 85400, and 31000 for dBTIC-S, dBTIC-D, and BTIC, respectively.Such superior photothermal properties promised a better vivo PTT performance of the dimers.We observed that the dBTIC-D nanoparticles (NPs) could induce 27 • C temperature elevation (ΔT) under 10-min laser irradiation (808 nm, 0.6 W/cm 2 ); on the contrary, the BTIC monomer, even in double doses, only induced a ΔT of 12 • C.These results demonstrated that the in vivo PTT performance of the dimer was significantly enhanced, which goes beyond the sum of two monomers due to the dimerization π-extension strategy.Furthermore, we attempted to conduct a PTT treatment using dBTIC-D NPs by 808 nm laser under a clinically safe power of 0.3 W/cm 2 , and a sufficient tumor-growth suppression outcome was successfully realized.In addition, more than the enhancement in PTT, we also observed that extending the A-D-A repeats would prolong spectral wavelength and promote fluorescence emission.The in vitro NIR-II fluorescence brightness (ε × photoluminescence quantum yield [PLQY]) of dBTIC-D was found to be 8-fold higher than the BTIC monomer, resulting in a distinguishing improvement in NIR-II imaging resolution.Taken together, we demonstrated that the dimerization strategy was very promising for designing NIR-II emitting and PTT multifunctional materials, which can significantly improve the light-harvesting capability, promoting both PTT and NIR-II imaging substantially.

RESULTS AND DISCUSSION
The synthetic routes of BTIC, dBTIC-S, and dBTIC-D are shown in Figure 1A.Monomer BTIC was directly synthesized by utilizing BT2CHO-BO and 2H-IC through the Knoevenagel condensation reaction.For the two dimers dBTIC-S and dBTIC-D, the asymmetrically mono-brominated BTIC-Br was synthesized with both 2H-IC and γ-Br-IC as the terminal groups.
Then, through the Stille-Kelly reaction with Pd (PPh 3 ) 2 Cl 2 as the catalyst, mono-brominated BTIC-Br underwent dimerization with hexamethylditin (Me 6 Sn 2 ) or (E)−1,2-bis (tributylstannyl) ethene (Bu 3 SnCH 2 = CH 2 SnBu 3 ) as the reducing agent to achieve dBTIC-S with a single-bond linkage or dBTIC-D with a vinyl linkage, respectively.More details of the synthesis procedure and structural characterization were listed in the Supplementary Information (Figures S1-S6).Further, density functional theory (DFT) calculations were performed to explore the optimized configurations and electronic structures of the materials using Gaussian 9 software.The undecyl and butyl-octyldo side chains were replaced by methyl and isobutyl groups in order to simplify the calculations.The optimized configurations of BTIC, dBTIC-S, and dBTIC-D are shown in Figure 1 and Figures S7 and  S8.BTIC exhibited good molecular planarity due to the intramolecular S⋅⋅⋅O interactions with a distance of 2.69 Å (Figure S7).Such S⋅⋅⋅O conformational locked effect remained in each monomeric unit of the dimers.Moreover, On the top view of these calculated structures, the twisted angle between A and D groups were similar for the monomer and dimers, that is, 0.17 • in BTIC monomer, 0.58 • and 1.04 • in dBTIC-S, and 0.24 • and 0.77 • in dBTIC-D (Figure S8).However, the different linkages for dimerization resulted in distinct structural conformations in dBTIC-S and dBTIC-D.As shown in the front view, dBTIC-S exhibited nonplanar conformation with a twist angle of ∼35 • between two monomeric units as a result of the rotational flexibility of the single-bond linkage (Figure 1A).In contrast, a complete planar conformation of dBTIC-D was observed due to the rigid nature of the vinyl linkage.In addition, the distributions of electron density in the highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) of dimers were quite different from the monomer (Figure S9).For these dimers, the electron density of HOMO becomes more delocalized on the electron-donating core of each monomeric unit, while the LUMO electron density mainly distributes on the two-linked A groups.The energetic characteristics based on DFT demonstrate that the HOMO energy levels of these two dimers were almost identical (−3.55/−5.51eV for dBTIC-S and −3.56/−5.49eV for dBTIC-D), resulting in energy gaps (1.96 eV for dBTIC-S and 1.87 for dBTIC-D) notably lower than that of the monomer BTIC (2.06 eV).
The ultraviolet-visible absorption spectra of BTIC, dBTIC-S, and dBTIC-D in chloroform solution and their thin films are shown in Figure 1B and Figure S10.In the monodispersed state, the BTIC monomer showed a rigid structure due to the S⋅⋅⋅O conformational locked effect, which resulted in narrowly distributed absorption bands (Figure 1B).These two dimers showed relatively broader absorption bands, which is probably because the dimers possess more conformational flexibility at the linkage positions.The molar absorption coefficients at the maximal absorption wavelengths for BTIC, dBTIC-S, and dBTIC-D were 1.80 × 10 5 , 2.84 × 10 5 , and 2.25 × 10 5 M −1 cm −1 , respectively.However, the light absorptivity of the dimers did not reach two folds of BTIC.As the absorptions of the dimers became broader, we also integrated the area below the absorption curves over the wavelength from 400 to 900 nm (Figure 1B).The integral areas per molar per cm were 2.06 × 10 7 , 4.56 × 10 7 , and 3.80 × 10 7 M −1 cm −1 for BTIC, dBTIC-S, and dBTIC-D, respectively.We found that the absorption areas of the dimers were approximately twice of the monomer.Further, the fluorescent emissions of the three samples in the monodispersed state with normalized absorbance at 710 nm were compared (Figure 1D).The fluorescence spectra revealed that the emissive intensity of the dimers was notably higher than that of the monomer.To obtain an insight into the fluorescence enhancement upon oligomerization, we employed femtosecond transient absorption (fs-TA) to study the excited state decay behaviors of the three molecules (Figure 1F-H).The fitting results of the ground state bleaching decay curves (Figure S11) depicted that their excited state exhibited two decay components.The short-lived (τ 1 ) decay component should correspond to the non-radiative decay lifetime (Figure 1I).The long-lived (τ 2 ) decay component matches the reported fluorescence lifetime of the analogous A-D-A molecules, [23] thus, which can be assigned to the radiative decay component.In comparison with BTIC (A 2 = 69%, τ 2 = 966 ps), the dimers showed enhanced radiative lifetime (τ 2 = 1140 ps for dBTIC-S and τ 2 = 1101 ps for dBTIC-D) and a higher fraction (A 2 = 71% for dBTIC-S and A 2 = 74% for dBTIC-D).These results implied that the π-extension structure of the dimers changed the excited state properties in comparison with the monomer.As both π-π* state (the locally excited [LE] electronic state) and the charge transfer (CT) state contribute to the low-lying excited state of D-A molecules, [24] we assumed that π extension upon oligomerization might increase the LE component in the excited state and accordingly enhance the radiative decay process.
To deliver these hydrophobic fluorophores for biomedical applications, they were encapsulated with the biocompatible amphiphilic copolymer DSPE-PEG 2000 to prepare composite NPs through the nanoprecipitation method.The morphologies and sizes of three NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) (Figure S12).TEM images showed that these NPs displayed uniform spherical morphologies with an average diameter of ≈ 60 nm, which was consistent with the DLS result of BTIC, dBTIC-S, and dBTIC-D as 63, 61, and 59 nm, respectively.[27] Moreover, the long-term storage stability of NPs was further investigated in a phosphate-buffered saline (PBS) solution.As displayed in Figure S13, monomer and dimer NPs could be nicely dispersed in PBS and showed good stability in 30 days.
The absorption and fluorescence spectra (Figure 1C,E) of all NPs were measured in water.We observed that the molar absorptivity of the NPs decreased relative to the monodispersed states, which is a common phenomenon for the nanoprecipitation method. [26,27]The molar absorption coefficients at 808 nm for BTIC, dBTIC-S, and dBTIC-D NPs were determined to be 0.47×10 5 , 1.37×10 5 , and 1.40 ×10 5 M −1 cm −1 , respectively.Interestingly, the absorption capability of the dimers became ∼ 3 folds stronger than the monomer, implying more intermolecular π-π electronic coupling may exist in the dimers' aggregated state.To verify this assumption, different nanoparticles were prepared by varying the weight ratios between the π-conjugated molecules and DSPE-PEG 2000 (w:w = 1:5.0,1:7.5, and 1:10.0), and the molar absorptivity at maximum absorption was tested and summarized in Table S2.As the ratio of π-conjugated molecules increased, their molar absorptivity increased as more intensive π-π interactions would be formed in the nanoparticles.We found that the dimers showed a more significant increase in absorptivity than the monomer, demonstrating that the enlarged π-conjugated planes of the dimers will facilitate intermolecular π-π interactions in the aggregated state in comparison with the monomer.Accordingly, it was revealed that the oligomerization using π-conjugated BTIC as the basic unit through single or double bonds further extended π-conjugation length, resulting in the fully conjugated dimers with extended electronic delocalization structure.Moreover, in the aggregated state, these dimers exhibited an improved intramolecular electronic coupling effect, further increasing the light absorptivity. [28,29]As a result, the light absorptivity of the dimers surpassed the summary of two monomers.Further, the fluorescence spectra of the three NPs were collected by normalizing their absorption intensity at 808 nm (Figure 1E).Consistent with the single-molecular properties, the fluorescence intensity of the dimer NPs was higher than that of the monomer NPs.The PLQYs of BTIC, dBTIC-S, and dBTIC-D NPs were calculated using IR-26 (QY = 0.5%) in dichloroethane as the reference, which was 0.8%, 1.5%, and 2.0%, respectively (Figure S14).It was worth noting that the different linkages for dimer construction also showed visible influences on the fluorescence properties of the aggregated state.The absorption spectra of NPs became notably broader compared with that of the monodispersed state, indicating the coexistence of multiple aggregation states.Depicted by the absorption peaks (Figure 1C), dBTIC-S NPs showed a 0-0/0-1 vibronic peak ratio smaller than unity; in contrast, the 0-0/0-1 peak ratio of dBTIC-D NPs is larger than unity.Such results implied that a relatively higher degree of H-aggregation formed in dBTIC-S NPs, which could well explain the higher PLQY of dBTIC-D NPs in comparison with dBTIC-S NPs. [27,30]We believed such a difference in the aggregated state came from the different conformations of the two dimers.
If the power of 0.6 W/cm 2 was applied, the temperature elevation after 10 min irradiation was 30 and 32 • C for dBTIC-S and dBTIC-D NPs, respectively.Moreover, both dimer NPs showed very robust photothermal stability under repeated laser irradiations.All the above results proved that the dimerization strategy would result in concurrently enhanced light harvesting capability and fluorescence emission.The former effect comes from the facilitated intermolecular interaction as a result of the π-conjugation extension of dimers, and the latter might be attributed to the π-conjugation effect of dimerization that is favorable to the radiative transition processes. [31,32]Therefore, the fluorescence brightness (ε 808 nm × PLQY) and the photothermal performance (ε 808 nm × PCE) were calculated and compared with the recently reported organic phototheranostic agents. [33,34]On one hand, the fluorescence brightness (ε 808 nm × PLQY) of dBTIC-S and dBTIC-D NPs were 2000 and 2900, respectively.
The brightness of dBTIC-D NPs was ∼8 times higher than BTIC monomer, and the brightness of both dimers was superior over most NIR-II emitting organic dyes (Figure 2A,C).On the other hand, although the dimers showed comparable PCE values to the monomer, the enhanced absorptivity determined more heat would be converted by the dimers.The photothermal performance (ε 808 nm ×PCE) for dBTIC-S and dBTIC-D NPs were 94500 and 85400, which was not only higher than BTIC NPs (31000) and surpassed most of the reported organic PTT agents (only 808-nm excited materials were selected to make the comparison reasonable) (Figure 2A,D).Further, the NIR-II FLI-guided PPT performances of the dimer and monomer were compared.Owing to the greater absorptivity and PLQY, dBTIC-D NPs were selected.Considering the dimer structure containing twice more chromophores than the monomer, the BITC monomer of double doses was used for comparison purposes.First, the NIR-II fluorescence images of dBTIC-D NPs (33 μM) and BTIC NPs (66 μM) were compared (Figure 2B and Figure S18), and we observed that the dimer showed notable brighter fluorescence than the monomer under 808 nm laser irradiation.Further, for in vivo NIR-II imaging, Figure 3 showed fluorescence angiography of the mouse vascular at 10 min post-injection of BTIC NPs (66 μM) and dBTIC-D NPs (33 μM) under 808 nm laser irradiation using different filters (1110 and 1319 nm).After tail vein injection of dBTIC-D NPs, the vascular structures were able to be clearly discriminated from the surrounding tissue under both 1100 and 1319 nm long-pass filters (Figure 3A and Figure S19), providing an accurate mapping of the blood circulation system.By contrast, the BTIC NPs with double molar doses still could not provide clear vascular structures under the same imaging conditions (Figure 3B).Furthermore, determined by the femoral artery image, a high signal-to-background ratio (SBR = 4.3) was observed for using dBTIC-D NPs as the imaging agent, while BTIC NPs could only result in an SBR of 2.5.These results implied that the dimer (dBTIC-D NPs) would lead to significantly improved spatial resolution for in vivo imaging compared with the monomer when the dosage ratio of 1:2 was used.
Then, the PTT performance was also compared between the dimer and monomer under a molar ratio of 1:2.Before evaluating the photothermal treatment efficacy in vivo, their intracellular cytotoxicity was evaluated with the standard CCK-8 assays.The results indicated both the dimer and monomer NPs showed no apparent dark cytotoxicity to 4T1 cells (Figure 3C,D).However, under laser irradiation (0.6 W/cm 2 ) for 10 min, both NPs showed concentration-dependent cell-killing capability.Moreover, the dimer showed a higher photo-toxicity than the monomer, that is, a cell-killing rate reached 77.7% when the cells were incubated with dBTIC-D NPs (9.9 μM); the rate decreased to 51.7% when a double molar dosage of BTIC (19.8 μM) was used.For further confirming the PTT effect, live/dead cell staining was also performed by laser scanning confocal microscope.Fluorescence images of propidium iodide (red, the marker for dead cells) and calcein acetoxymethyl ester (green, the marker for live cells) co-stained 4T1 cells with different treatments were obtained (Figures S20 and S21).In comparison, negligible dead cells were observed in the control groups.Subsequently, the in vivo PTT performance was evaluated.After intravenous injection of dBTIC-D NPs (33 μM) and BTIC NPs (66 μM), respectively, the IR thermography result (Figure 3F) demonstrated that the surface temperatures of the tumors treated with dBTIC-D NPs + laser raised rapidly from 35 to ∼60 • C upon 10-min laser irradiation.Under the same condition, the temperature elevation effect of BTIC NPs was relatively weaker (from 35 to ∼45 • C).Therefore, all these results showed that the dimer indeed exhibits better NIR-II fluorescence and photothermal performance in vitro and in vivo compared to the monomer in the double doses, indicating that the phototheranostic performance of the dimer went beyond the sum of two monomers.As the good phototheranostic properties of the dimers were fully disclosed, the in vivo performance of dBTIC-D NPs was  S1. evaluated consequently.First of all, its blood circulation halftime was estimated by the semi-quantified analysis method.A half-time of 3.6 h was revealed, which was sufficient for the PTT treatment (Figure S22).Then, upon intravenous injection of dBTIC-D NPs, the NIR-II fluorescence signal accumulated at tumors was monitored.As time increased, the fluorescence from angiogenic vessels became faint, and the fluorescence signal of the tumor gradually intensified and reached the highest intensity at 24 h (Figure 4A,D).Therefore, the time point of 24 h was identified as the ideal accumulation time for the subsequent PTT treatment.At this time point, the bio-distribution analysis further confirmed the high tumor enrichment ability of the NPs (Figure in contrast to the negligible signal on the heart, lung, and kidney. In addition, NPs were mainly distributed in the tumor, liver, and spleen, demonstrating that the liver and spleen should participate in the metabolism of dBTIC-D NPs.After 48 h of NPs injection, the fluorescence signal of the tumor was still very strong, which indicated that dBTIC-D provided precise and long-term localization for potential NIR-II imaging-guided tumor therapy.To evaluate the PTT treatment in vivo, 4T1 tumor-bearing mice were randomly divided into four groups, that is, PBS-laser, PBS + laser, dBTIC-Dlaser, dBTIC-D + laser, and the tumor sites were irradiated by 808 nm laser (0.6 W/cm 2 ) for 10 min.IR thermography result (Figure 3E) demonstrated that the surface temperatures of the tumors treated with dBTIC-D NPs + laser raised rapidly from 37 to 58 • C in the first two minutes and maintained a steady temperature of 61 • C.During the 14 days, the body weight of mice and tumor volume were monitored every 2 days to assess the in vivo PTT efficacies of the dimer NPs.As presented in Figure 4B and Figure S24, the dBTIC-D + laser group realized complete tumor ablation and no local reoccurrence during the 14-day period.In contrast, the tumors increased nearly 4 folds in volume for the three control groups (PBS-laser, PBS + laser, and dBTIC-D-laser) after 14 days.For further exploring the potential of clinical applications, we also tried to use the FDA-approved maximum permissible exposure dose for 808 nm laser (0.3 W/cm 2 ) to evaluate the PTT performance of dBTIC-D NPs (Figure 4E,F and Figure S25).As shown in Figure 4G, the surface temperatures of the tumors treated with dBTIC-D NPs + laser raised over 48 • C, which resulted in effective tumor-growth suppression. [35]After 14-day treatment, the tumors of the dBTIC-D + laser group were reduced to half of the original sizes (Figure 4E, and Figure S26).Additionally, no significant body weight loss was detected during the treatment period in all the tested groups (Figure 4E,F), suggesting the good biocompatibility of dBTIC-D for in vivo applications.Furthermore, tumors excised from the four groups were analyzed by the histologic section with hematoxylin and eosin (H&E) staining.As shown in Figure 4H, the tumor tissue in the dBTIC-D + laser group showed more massive nucleus absence and vacuolization compared with the other three groups, indicating severe cancer cell apoptosis or necrosis.On the other side, for the other main organs of the mice after the 14-day treatment, the histopathological analysis showed no visible signs of damage (Figure S27).In addition, the mice after the treatment had normal blood parameters with no statistical difference compared with those of the healthy mice (Figure S28).Collectively, these experimental results convinced that dBTIC-D NPs were biologically safe and efficient NIR-II imaging-guided PTT agents.

CONCLUSION
In summary, we proposed the dimerization strategy based on the BTIC monomer to exploit new phototheranostic agents for concurrent high-quality NIR-II FLI and PTT.Two different dimers, dBTIC-S and dBTIC-D, were designed and synthesized, which share the identical A-D-A basic unit but were coupled by different linkage structures.Compared with the monomer, the extended π-conjugation through dimerization can enhance fluorescence emission and facilitate the inter-molecular π electronic coupling, leading to enhanced light-harvesting capability.Therefore, these dimer NPs exhibited leading photophysical performance among recently reported similar materials for photoimaging diagnostics and light-triggered precision therapeutics.Notably, the phototheranostic properties of dBTIC-D NPs could go beyond that of the BTIC monomer of double doses.Accordingly, an 808 nm laser with a low intensity of 0.3 W/cm 2 was sufficient to irradiate dBTIC-D NPs to reach an efficient anti-tumor treatment.This work shows that extending the conjugation of the D-A type conjugated molecules by the dimerization strategy will efficiently promote the performance of NIR-II fluorescence emitting and PTT multifunctional agents, providing a novel idea for developing this kind of material in the future.

Preparation of BTIC, dBTIC-S, and dBTIC-D NPs
In a typical process, photosensitizer (0.5 mg) and DSPE-PEG 2000 (10 times the weight of photosensitizer) were mixed in tetrahydrofuran (THF, 2.0 mL), and then the solution was added into ultrapure water (18.0 mL) under a simultaneous treatment of ultrasonication.Subsequently, THF was removed at 40 • C with mechanical stirring, and water was added into the solution as 20.0 mL.The solution was stored at 4 • C for further use.All NPs were prepared according to the same procedure.

Cellular culture
4T1 cells were maintained as monolayer cultures in DMEM at 37 • C, the media were supplemented with 10% FBS and 1% penicillin/streptomycin.The cells were cultured until confluence was reached before each experiment.

Dark toxicity and phototoxicity
The dark toxicity of dBTIC-D NPs and PBS were evaluated as follows: After cell attachment, the culture media was replaced with 100 μL of fresh one containing different concentrations of dBTIC-D NPs, followed by incubation for another 24 h.The cell viability was assessed by the widely used CCK8 test.All data were based on three parallel experiments.The phototoxicity of dBTIC-D NPs was evaluated according to a similar procedure, but the culture media was replaced with 100 μL of fresh one containing different concentrations of dBTIC-D NPs, followed by exposure to 808 nm laser irradiation at 0.6 W cm −2 for 10 min.Irradiated cells were then incubated at 37 • C for another 24 h and cell viability was also evaluated using the CCK8 test.All data are based on three parallel experiments.

Flow cytometry
200,000 cells were seeded to a well of a 6-well plate and incubated in DMEM media for 24 h (37 • C).After incubation with the dBTIC-D NPs (50 μg mL −1 ) for 12 h, cells were stained with PI and Annexin V-FITC and tested through FCM analysis.Finally, the apoptotic level of cells in each group was analyzed by the FlowJo V10 software.

Animals and tumor mouse model
All animal experiments were in accordance with Institutional Animal Use and Care Regulations, according to protocol No. SUSTC-JY2019068, approved by the Laboratory Animal Ethics Committee of the Southern University of Science and Technology.After being acclimated and tested for infectious diseases for 1 week, 4-week-old BALB/c mice were subcutaneously injected with 4T1 cells (100,0000 cells each mouse) into the right rear hips of the mice.After about one week, mice with tumor volumes of about 150-250 mm 3 were randomized into treatment groups.The tumor size was calculated using the following formula: Volume = (Length×Width 2 )/2.The mice were with 808 nm, 0.6 W/cm 2 laser irradiation 10 min after tail vein injection 12 h.

In vivo NIR-II FLI
The tumor-bearing mouse was anesthetized with isoflurane to remove fur.Next, the aqueous solution (200 μL) of dBTIC-D NPs concentration equivalent to 0.5 mg/mL was intravenously injected into the mouse.After injection, the mouse was imaged with the small animal imaging system at designated time points.

Histological analysis
For H&E staining, the tissues were fixed in 10% neutral buffered formalin, processed routinely into paraffin, sectioned at 4 μm, stained with H&E or TUNEL apoptosis assay kit, and examined by microscope.

Blood routine test
The blood of the tumor-bear mice after 14 days of PTT treatment was collected, and then a blood routine test was conducted on the automatic hematology analyzer (DF52Vet; Dymind Biotech.).

S C H E M E 1
Illustration of the dimerization strategy to extend the π-conjugation of the A-D-A monomer resulting in both enhanced near-infrared (NIR)-II fluorescence brightness and photothermal effect.F I G U R E 1 (A) Synthesis route of BTIC, dBTIC-S, and dBTIC-D; Optimized ground-state (S 0 ) geometries based on density functional theory (DFT) results of dBTIC-S and dBTIC-D.(B, C) Absorption spectra (molar absorptivity (ε) versus wavelength) of BTIC, dBTIC-S, and dBTIC-D in chloroform solutions and their nanoparticles (NPs) dispersed in water.(D, E) The fluorescence spectra of BTIC, dBTIC-S, and dBTIC-D in chloroform solutions (the samples with the same absorbance at 710 nm) and their NPs dispersed in water (the samples with the same absorbance at 808 nm).(F-H) TA spectra signals of BTIC, dBTIC-D, and dBTIC-S on the 1-1000 ps timescales following photoexcitation at 660 nm laser pulse.(I) Fitting results of the ground state bleaching decay curves (A i represents the fraction of the excited population of the associated component; τ i represents the lifetime of each excited component).

F I G U R E 3
Near-infrared (NIR)-II fluorescence images of healthy mice (without tumor implantation) at 10 min post-injection of (A) dBTIC-D nanoparticles (NPs) and (B) BTIC NPs in the different doses (the molar dose of monomer BTIC NPs (66 μM) was twice that of dimeric NPs (33 μM)) under different filters (1100 and 1319 nm); Cross-sectional intensity profiles along the red line in the image with the peaks fitted to Gaussian functions.Cell proliferation of 4T1 cells was incubated with the (C) dBTIC-D and (D) BTIC NPs at various concentrations in the dark and after NIR light irradiation (808 nm, 0.6 W/cm 2 ) for 10 min.(E) Photothermal images and (F) temperature changes of 4T1 tumor-bearing mice in different groups after NIR light irradiation (808 nm, 0.6 W/cm 2 ) for 10 min (BTIC of 66 μM and dBTIC-D of 33 μM).