NIR AIE luminogens for primary and metastasis tumor imaging and tracking applications

Abstract

Modern lifestyle changes, including irregular diets and late-night activities, have contributed to a significant rise in cancer rates, particularly among younger demographics, highlighting the pressing need for early detection and treatment. Fluorescence imaging techniques play a crucial role in tumor diagnosis, yet traditional organic fluorescent materials suffer from limitations such as poor photostability and fluorescence quenching in aggregates. This paper introduces the design and synthesis of four aggregation-induced emission (AIE) molecules with near-infrared I emission, which are aimed at overcoming fluorescence quenching in the molecular aggregation state. The photophysical properties of these molecules (BTA-TT, BTA-TTM, BTA-FT, and BTA-FTM) were investigated and they exhibited TICT and AIE behaviors in varying water fractions, along with notably large Stokes shifts. In vitro imaging of the four molecules successfully imaged lysosomes within 4T1 cells and they displayed minimal dark toxicity. Moreover, these AIEgens exhibited excellent anti-photobleaching properties, which were superior to those of commercial dyes. In addition, BTA-FTM nanoparticles coated with PEG-2000 showed biosafety and enabled tumor imaging in mice for 59 hours, revealing the tumor metastases in the heart and lungs of mice. This research contributes to the development of novel near-infrared molecules for advanced diagnostic applications.

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Introduction

In light of societal advancements and evolving lifestyles, cancer has emerged as a significant health concern affecting humans.^1–4 Currently, various treatment modalities are available for tumors, including immunotherapy,^5,6 targeted radiotherapy,⁷ and surgical tumor resection.⁸ Among these methods, surgical resection is a common treatment for patients with tumors. During surgical procedures, imaging technology serves as a valuable diagnostic and therapeutic tool.⁹ Presently, ultrasonography (US),¹⁰ positron emission tomography (PET),¹¹ magnetic resonance imaging (MRI),¹² and computed tomography (CT)¹³ are frequently utilized in the imaging field. However, these imaging modalities have several disadvantages, including low resolution, high costs, and potential adverse effects on the human body. Fluorescence imaging offers several advantages, such as high sensitivity, multicolor labelling capability, strong signal intensity, and low experimental costs.^14–17 Compared to visible light imaging, near-infrared fluorescence imaging can effectively resolve biological background fluorescence, minimize light scattering, reduce light-induced damage to cells, and facilitate biological imaging.^18–22 Currently, near-infrared fluorescence probes are primarily categorized into organic and inorganic compounds. Inorganic materials, such as semiconductor nanocrystals and quantum dots, are associated with potential toxicity due to heavy metals.^23–26 In contrast, organic near-infrared probes exhibit diverse structures, low biological toxicity, and biodegradability.^27,28 Conventional fluorophores, however, are often quenched in the aggregation state due to their rigid planar structures, which promotes strong π–π stacking interactions.^29–31 Examples include fluorescein, rhodamine, coumarin, and BODIPY.^32–35 Fluorescence quenching in the aggregation state presents a significant challenge for the application of organic probes in bioimaging. Surprisingly, since Professor Tang's group coined the concept of aggregation-induced emission (AIE) in 2001, it has garnered considerable attention over the past two decades in biological fields.³⁶ Therefore, designing near-infrared organic probes with AIE characteristics represents an effective strategy to address the issue of fluorescence quenching in the aggregation state.³⁷

In this study, we synthesized near-infrared (NIR) probes with aggregation-induced emission (AIE) characteristics, specifically BTA-TT, BTA-TTM, BTA-FT and BTA-FTM. These D–π–A type molecular structures were constructed via the Knoevenagel reaction, utilizing furan or thiophene as the π bridge, benzo[d]thiazole-2-acetonitrile as the acceptor, and triphenylamine or 4-methoxy-N-(4-methoxyphenyl)-N-phenylaniline as the donor. We investigated the photophysical properties of these compounds, which exhibited both twisted intramolecular charge transfer (TICT) and AIE characteristics in varying water fractions. Additionally, we demonstrated the biological imaging capabilities of these new molecules, which localize in lysosomes and exhibit negligible dark toxicity, favorable biocompatibility, and high photostability. Furthermore, BTA-FTM nanoparticles (NPs) coated with PEG-2000 showed excellent fluorescence emission in mice bearing 4T1 tumors for a duration of 59 hours. Meanwhile, BTA-FTM NPs revealed the tumor metastases in the heart and lungs of mice.

Results and discussion

Synthesis, characterization and photophysical properties

The molecular structures of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM are illustrated in Scheme 1. The detailed synthesis procedures and the characterization results of their structures obtained utilizing NMR and high-resolution mass spectrometry are provided in the ESI,† specifically in Fig. S1–S8 (ESI†). To validate the feasibility of the molecular design, the photophysical properties of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM were assessed using UV-Vis and photoluminescence spectroscopy.

Scheme 1 (A) The molecular structures of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM. (B) Schematic of the preparation of BTA-FTM nanoparticles. (C) Schematic diagram of tumor imaging and metastases in mouse.

The UV-Vis spectra of the four molecules in tetrahydrofuran (THF) solution are shown in Fig. 1A. The maximum absorption peaks of BTA-TT and BTA-FT were observed at 484 and 489 nm, respectively. In contrast, the maximum absorption peaks of BTA-TTM and BTA-FTM, which contain the 4-methoxy-N-(4-methoxyphenyl)-N-phenylaniline group, were located at 508 and 512 nm, resulting in red shifts of 24 and 23 nm, respectively. This observation indicates that the stronger electron-donating ability of the 4-methoxy-N-(4-methoxyphenyl)-N-phenylaniline group increases the electron cloud density of the molecules, effectively reducing the absorption energy band and leading to a red shift in the absorption wavelength. As illustrated in Fig. 1B, the emission peaks of BTA-TT, BTA-TTM, BTA-FT, and BTA-FTM in THF were observed at 640, 709, 625, and 702 nm, respectively. Correspondingly, their Stokes shifts were observed at 156, 201, 136, and 190 nm, respectively, which are advantageous for bioimaging and diagnosis.

Fig. 1 (A) The UV-vis spectra of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM in THF solution, respectively (concentration: 1 × 10⁻⁵ mol L⁻¹). (B) Fluorescence emission spectra of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM in THF solution, respectively (concentration: 1 × 10⁻⁵ mol L⁻¹). (C) Plots of the wavelength and water fraction in H₂O/THF mixtures of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM respectively. (D) Plots between the ratio of I/I₀ and water fraction in H₂O/THF mixtures; I₀ and I were the maximum PL intensity of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM in pure THF and in H₂O/THF mixtures (concentration: 1 × 10⁻⁵ mol L⁻¹), respectively; inset: fluorescence photograph of BTA-TT in pure THF and in 80% H₂O/THF mixtures taken under 365 nm UV irradiation.

We explored the photophysical properties of these new molecules with D–π–A structures in mixed THF/H₂O solutions with varying proportions. The relationship of emission wavelength and the water content is shown in Fig. 1C. Clearly, the emission wavelengths of BTA-TTM and BTA-FTM fall within the near-infrared I (NIR-I) window, while those of BTA-TT and BTA-FT do not. Such results should be attributed to the 4-methoxy-N-(4-methoxyphenyl)-N-phenylaniline group with more electron-rich characteristics. For example, as the water content of BTA-TT increased to 60%, the emission wavelength was red-shifted by 37 nm, due to the increase in solvent polarity inducing TICT. However, when the water fraction further increased (≥70%), the emission peak slightly blue-shifted. This may be attributed to the decrease in molecular conjugation in the crowded aggregation state. Regarding the emission intensity, it slightly decreased before 80% water content. While the water content reached 80%, the fluorescence emission intensity increased sharply, which was 6.5-fold higher than that in THF solution. This phenomenon due to the intermolecular interactions in BTA-TT aggregates in water restricted the molecular motions and enhanced the proportion of radiative decay in the excited state. Thus, the AIE of BAT-TT was active in a high water fraction. For other molecules, there were similar photophysical properties to BAT-TT, with both TICT and AIE being active with increasing water fraction. These results demonstrated that the design of D–π–A molecules was effective, not only achieving emission in the near-infrared I, but also suppressing fluorescence quenching in the aggregation state.

Single-crystal structural analysis

To gain further insight into the effect of the molecular structures on the fluorescence emission of BTA-TTM and BTA-FT, their single crystals were grown in DCM/EtOH and analyzed by X-ray diffraction. The crystal data and the collection of parameters are summarized in Table S1 (ESI†). From the single-crystal structural analysis results of BTA-TTM and BTA-FT shown in Fig. 2, it can be seen from the side view that benzo[d]thiazole-2-acetonitrile and the thiophene or furan ring were nearly coplanar in their connection. The twist angle between the furan ring and its adjacent groups was found to be only 1.44° (Fig. 2B), a value significantly smaller than that observed for the corresponding thiophene ring (18.68°). This observation suggested that compounds incorporating furan rings exhibit good conjugation compared to those with thiophene rings. Specifically, the absorption wavelength of the compound featuring a furan ring showed a slight red-shift of 4–5 nm relative to its thiophene counterpart. Additionally, the torsion angle of two phenyl groups in BTA-TTM was 70.5° and 81.75°, which effectively increased steric hindrance and impeded strong intermolecular interactions. Furthermore, numerous weak interactions such as CN⋯H (2.582 Å), N⋯H (2.649 Å), S⋯O (3.157 Å) and CH⋯π (2.847, 2.853, 2.863, and 2.894 Å) effectively suppressed molecular motions in the aggregation state and enabled the dissipation of excited-state energy through radiation transition. Similar to BTA-TTM, BTA-FT also has twist angles of two phenyl groups (30.08° and 62.89°), meanwhile multiple H⋯H (2.372 Å), CN⋯H (2.546 Å), N⋯H (2.629 Å), and CH⋯π (2.803, 2.890, and 3.351 Å) interactions exist in the crystal lattice.

Fig. 2 Single-crystal X-ray diffraction analysis of (A) BTA-TTM and (B) BTA-FT, respectively.

Theoretical calculations

Density functional theory (DFT) calculations were conducted to gain a deeper understanding of the molecular structures and electronic properties using the B3LYP method. As shown in Fig. 3, the electron cloud density of the highest occupied molecular orbital (HOMO) was primarily concentrated in the thiophene (or furan) units and the triphenylamine units (or 4-methoxy-N-(4-methoxyphenyl)-N-phenylaniline units). In contrast, the electron cloud density of the lowest unoccupied molecular orbital (LUMO) was predominantly located on the 2-benzothiazoleacetonitrile components. The energy gaps of BTA-TTM and BAT-FTM were 2.44 and 2.47 eV, respectively, which were smaller than those of BAT-TT and BAT-FT (2.59 and 2.6 eV). The enhancement of electron donation significantly elevated the HOMO energy level of molecules. Consequently, this alteration leads to a decrease in the energy band gap and a redshift of the emission wavelength, aligning with the above photophysical properties.

Fig. 3 The distributions of HOMO–LUMO levels, in addition to the energy levels of BTA-TT, BTA-TTM, BTA-FT and BTA-FTM were presented herein.

In vitro bioimaging

Given the desirable photophysical properties, we first investigated the cellular imaging applications of the developed AIEgens, and 4T1 cells were selected as the representatives. Initially, we investigated the cytotoxicity by co-incubating four AIEgens with 4T1 cells. The results demonstrated the treatment of these AIEgens presented a faint effect on the growth of 4T1 cells with the concentrations ranging from 1 to 128 μM, indicating their good biocompatibility. Furthermore, good biosafety could also be achieved under light irradiation (Fig. S12, ESI†). Then, we studied their bioimaging behavior. As shown in Fig. 4A, these molecules could cross the cell membrane efficiently, and further colocalization analysis revealed that the developed AIEgens showed good colocalization with Lysotracker. Taking BTA-FTM as an example, after coincubation with Lysotracker blue, a co-localization coefficient of 0.76 was obtained for BTA-FTM-treated 4T1 cells, indicating the enrichment in lysosome. Moreover, linear analysis was also performed to verify the localization of BTA-FTM (Fig. 4B), and the perfect merging of BTA-FTM and Lysotracker blue could be observed, indicating the perfect targeting ability of BTA-FTM towards lysosome. In addition, a similar phenomenon was also observed in 4T1 cells incubated with BTA-TT, BTA-TTM, BTA-FT, respectively, revealing their good lysosome targeting behavior (Fig. S15, ESI†). Additionally, the photostability property of the dye is crucial for imaging applications. As illustrated in Fig. 4C, upon continuous irradiation with a 405 nm laser at 20% power for 5 minutes, the fluorescence intensity of Lysotracker blue diminished significantly to 63% of its initial value, whereas the emission intensity of all AIEgens persisted above 80%. Notably, BTA-FT exhibited the highest photostability, maintaining an emission intensity of 93% after laser irradiation. These results suggested that the developed AIEgens possess superior photostability compared to commercial dyes, which may be attributed to the incorporation of the CN group that enabled enhanced photostability.³⁸ Thus, the developed AIEgens not only exhibit good biocompatibility but also perfect lysosome targeting ability, showing high potential for bioimaging applications.

Fig. 4 (A) CLSM images of 4T1 cells were incubated with BTA-TT, BTA-TTM, BTA-FT, and BTA-FTM (AIEgens), respectively, and then cells were treated with Lyso-tracker blue for 30 min [AIEgens] = 12.5 μM, [Lyso-tracker blue] = 200 nM. (B) The line-plot graphs exhibit the fluorescence intensity profiles of the BTA-FTM (red) and Lyso-tracker (blue). (C) Photostability of four AIEgens and Lyso-tracker blue in 4T1 cells under a 405 nm laser at 20% power continuous irradiation.

In vivo imaging ability

Based on the outstanding in vitro bioimaging ability, we further evaluated the in vivo imaging ability. Herein, BTA-FTM was selected as the representative luminogen. A mouse model was constructed by subcutaneously injecting 4 T1 cells bilaterally into the buttocks of female BALB/c mice. As shown in Fig. 5A, nanoparticles were administered to mice bearing 4T1 tumors via intravenous injection. Due to the enhanced permeability and retention (EPR) effect, a bright emission of BTA-FTM could be detected, demonstrating a significant accumulation of particles at the solid tumor. Moreover, the fluorescence signal at the tumor site could be observed after 12 hours, which remained about 71% even 59 hours after the intravenous injection (Fig. 5B). These findings suggested that the nanoparticles show a long term retention effect, which was beneficial for in vivo imaging and monitoring applications. Additionally, the biosafety of the nanoparticles is a critical consideration. After 48 hours post-injection, we investigated the function of kidneys and the liver by evaluating the expression level of biomarkers in mice with different treatments. The expression values for ALB, ALP, and UREA in mice without any treatment were evaluated to be 35.13 g L⁻¹, 62.79 U L⁻¹, and 7.13 mmol L⁻¹, respectively, meanwhile, the similar phenomena were observed for BTA-FTM NP treated-mice with the values of 35.01 g L⁻¹, 64.29 U L⁻¹, and 7.76 mmol L⁻¹ for ALB, ALP, and UREA, respectively (Fig. S15, ESI†). These findings indicated that the liver and kidney functions of the experimental mice were normal, further demonstrating good biocompatibility of the nanoparticles. We also investigated the distribution of nanoparticles in the main organs of heart, liver, spleen, lungs, and kidneys, and tumors of the mice after seven days post-injection. Fig. 5C shows bright fluorescence observed in both the tumor and liver. It should be noted that a considerable signal could also be detected in the heart and lungs. To elucidate the reasons for the accumulation of nanoparticles in different organs, we performed H&E staining on the tumor, heart, liver, spleen, lungs, and kidneys. The results revealed metastatic tumors in the heart and lungs, while no lesions were observed in other organs. This confirmed that the fluorescence emission in the heart and lungs was attributable to the presence of metastatic tumors, whereas there were no metastatic tumors detected in the spleen and kidneys. These results indicated that BTA-FTM nanoparticles can accumulate at the tumor site through the EPR effect, facilitate long-term imaging, exhibit good biocompatibility, and trace tumor metastasis.

Fig. 5 (A) and (B) Fluorescence imaging and intense light intensity of mice injected with BTA-FTM nanoparticles at different times. (C) Distribution of BTA-FTM NPs in varied organs of 4T1 tumor bearing mice 7 days after intravenous injection. (D) H&E staining of tumors and the main organs including the heart, liver, spleen, lung and kidneys. Scale bar: 100 μm.

Experimental

Synthetic procedures

(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-(4-(diphenylamino) phenyl)thiophen-2-yl)acrylonitrile (BTA-TT). Benzothiazole-2-acetonitrile (0.3 g,1.72 mmol), 4-(diphenylamino)benzaldehyde (0.734 g, 2.07 mmol) and basic aluminum oxide (2.63 g, 25.83 mmol) were dissolved in 20 mL toluene into a 100 mL single-neck flask. And the mixture was heated for 16 hours at 110 °C. After the reaction had cooled to room temperature, the reaction was quenched by water and extracted with dichloromethane. The organic layer was removed under vacuum, and the crude product was purified by column chromatography with petrol ether/dichloromethane as the eluent. The pure red powder was obtained with a yield of 32%. ¹H NMR (500 MHz, CD₂Cl₂), δ (ppm): 8.40 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 4.1 Hz, 1H), 7.56 (dd, J = 27.4, 8.1 Hz, 3H), 7.44 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 4.1 Hz, 1H), 7.32 (t, J = 7.7 Hz, 4H), 7.12 (dd, J = 18.9, 7.7 Hz, 6H), 7.05 (d, J = 8.3 Hz, 2H),¹³C NMR (125 MHz, CD₂Cl₂), δ (ppm): 164.50, 158.08, 155.08, 140.25, 139.47, 136.09, 135.61, 128.47, 128.06, 126.86, 125.85, 124.83, 124.31, 123.92, 122.91, 120.13, 119.99, 118.23, 116.61, 116.05, 101.20, 56.75. HR-MS(C₃₂H₂₁N₃S₂): m/z 511.1132 (M⁺: calcd 511.1177).

(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-(4-(bis(4-methoxyphenyl)amino)phenyl)thiophen-2-yl)acrylonitrile (BTA-TTM). The procedure was analogous to that described for BTA-TT. A deep red solid of BTA-TTM was obtained in 36% yield. ¹H NMR (500 MHz, CD₂Cl₂), δ (ppm): 8.29 (s, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 4.0 Hz, 1H), 7.52 (t, J = 7.8 Hz, 3H), 7.46–6.83 (m, 12H), 3.80 (s, 6H), ¹³C NMR (125 MHz, CD₂Cl₂), δ (ppm): 164.56, 154.81, 154.45, 148.28, 140.54, 139.52, 135.99, 131.91, 130.79, 128.51, 128.21, 127.03, 126.57, 125.99, 125.26, 124.54, 124.26, 123.48, 123.00, 120.23, 118.16, 101.47. HR-MS(C₃₄H₂₅N₃O₂S₂): m/z 571.1331 (M +: calad 571.1388).

(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-(4-(diphenylamino)phenyl)furan-2-yl)acrylonitrile (BTA-FT). The reaction followed the same procedure as that used for the synthesis of BTA-TT. The synthesis of a rose red solid of BTA-TTM was achieved in 39% yield. ¹H NMR (500 MHz, CD₂Cl₂), δ (ppm): 8.06–7.96 (m, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.52 (t, J = 7.7 Hz, 1H), 7.45–7.39 (m, 1H), 7.32 (t, J = 7.9 Hz, 4H), 7.26 (d, J = 3.8 Hz, 1H), 7.18–7.05 (m, 8H), 6.84 (s, 1H), ¹³C NMR (125 MHz, CD₂Cl₂), δ (ppm): 164.65, 160.62, 155.16, 150.51, 149.23, 148.22, 136.21, 131.73, 130.80, 128.08, 127.50, 126.83, 126.72, 125.33, 125.03, 124.28, 123.22, 123.11, 122.94, 118.33, 109.11, 100.31. HR-MS (C₃₂H₂₁N₃OS): m/z 496.1450 (M⁺: calcd 496.1405).

(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-(4-(bis(4-methoxyphenyl)amino)phenyl)furan-2-yl)acrylonitrile (BTA-FTM). An identical procedure to that employed during BTA-TT synthesis was followed. A red solid of BTA-FTM was produced in a 48% yield. ¹H NMR (500 MHz, CD₂Cl₂), δ (ppm): 8.01 (d, J = 8.2 Hz, 1H), 7.97 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.67 (s, 2H), 7.54–7.50 (m, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.29–6.72 (m, 12H), 3.81 (s, 6H), ¹³C NMR (125 MHz, CD₂Cl₂), δ (ppm) 164.83, 161.14, 158.14, 155.15, 151.50, 148.95, 140.96, 136.15, 131.64, 129.33, 128.05, 127.51, 126.75, 125.33, 124.19, 122.92, 121.27, 119.95, 118.45, 116.15, 108.57, 99.65, 56.77. HR-MS (C₃₄H₂₅N₃O₂S): m/z 555.1561 (M⁺: calcd 555.1617).

Preparation of BTA-FTM NPs

BTA-FTM (1.0 mg) and DSPE-PEG₂₀₀₀ (2.0 mg) were fully dissolved in THF (1.0 mL). The mixture was quickly injected into 9.0 mL of deionized water using an ultrasonic crusher with 40% power for 2 min, which was stirred in fume hood for two days. The crude NPs were further filtered through a membrane filter (diameter = 200 nm) for further usage.

Cell cultures

4T1 cells were cultured in DMEM medium containing 10% FBS and antibiotics (100 units per mL penicillin and 100 μg mL⁻¹ streptomycin) in a humidified incubator with 5% CO₂ at 37 °C.

Cell imaging

4T1 cells were grown overnight in a cell culture dish for 24 h. The cells were further imaged using a CLSM using different combinations of excitation wavelengths for each dye.

MTT assays

4T1 cells were seeded in a 96-well flat-bottomed microplate. Cells treated with BTA-TTM were incubated with concentrations ranging from 0 to 128 μM at 37 °C under a 5% CO₂ atmosphere for 12 h. The microplate was irradiated at 450 nm (light dosage = 40 mW cm⁻²) for 20 min, while another microplate was kept in the dark for 20 min. After the treatment, the cell was incubated for 20 h. MTT in PBS (10 μL, 5 mg mL⁻¹) was added to each well. The microplate was incubated at 37 °C under a 5% CO₂ atmosphere for another 4 h. The growth medium was then removed, and DMSO (100 μL) was added to each well. The absorbance of the solutions at 520 nm was measured with a Powerwave XS MQX200R microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT). For the MTT assays involving BTA-TT, BTA-FT and BTA-FTM, the procedure was similar.

Live cell confocal imaging

4T1 cells were grown in a confocal imaging dish at 37 °C. After incubation with medium containing 12.5 μM BTA-TTM for 2 h, the medium was removed and washed with PBS (1 mL × 3). The cells were then incubated with Lyso-tracker blue (200 nM) in a growth medium at 37 °C under a 5% CO₂ atmosphere for 30 min. The medium was then removed, and the cell layer was gently washed with PBS (1 mL × 3). The excitation wavelength of Lyso-tracker blue was 405 nm. Pearson's correlation coefficients (PCC) were determined using the program ImageJ. For the confocal imaging involving BTA-TT, BTA-FT and BTA-FTM, the procedure was similar.

Efficacy of BTA-FTM-NPs in vivo

Female BALB/c mice (5 weeks old) were purchased from China Boryxin Biotechnology Co. (Hunan, China). All animal procedures have been approved by the Animal Ethics Committee of The University of South China. The mice were subcutaneously injected with 4T1 cells (1 × 10⁷ cells) in PBS buffer. When the tumor size was about ∼5 to 7 mm, the 4T1 cell-bearing mice were injected with BTA-FTM-NPs.

Conclusions

In summary, four NIR-I fluorescence imaging AIE agents (BTA-TT, BTA-TTM, BTA-FT, and BTA-FTM) with a donor-π-acceptor (D–π–A) structure were designed and synthesized. The absorption peaks of BTA-TTM and BTA-FTM in THF were found at 508 and 512 nm, respectively, while their emission peaks were observed at 709 and 702 nm, respectively. Due to the D–π–A structure and highly twisted donor groups, these four NIR-I molecules exhibited both TICT and AIE behaviors as the water fraction increased. Moreover, single crystal analysis demonstrated that the highly twisted donor groups, TPA and 4-methoxy-N-(4-methoxyphenyl)-N-phenylaniline, effectively inhibit intermolecular π–π interactions. Concurrently, multiple weak interactions suppress intermolecular motions, resulting in bright emission in the aggregated state. Additionally, theoretical calculations revealed the electron cloud distribution of the molecules' LUMO and HOMO, along with their energy gaps. In vitro experiments demonstrated that all AIEgens exhibited minimal dark toxicity and the ability to target lysosomal localization. Furthermore, these AIEgens possessed robust photobleaching resistance compared to commercial Lyso-tracker blue. Among them, BTA-FTM nanoparticles can image tumors for up to 59 hours and successfully trace metastatic tumors of mice in the heart and lungs. Additionally, the nanoparticles exhibited excellent biocompatibility due to negligible damage to the liver and kidneys of mice. The development of these new NIR-I window molecules provides a promising platform for surgical tumor removal and the tracking of tumor metastasis.

Author contributions

Y. Zhu synthesized materials, performed all measurements, and wrote the manuscript. H. Liu analysed the photophysical properties and single crystal data. Y. Zeng and Y. Yin performed cell imaging and tumor imaging in vivo. B. Chen and R. Hu provided intellectual input and revised the manuscript. All the authors discussed the project and analysed the results.

Data availability

The data supporting this article are available within the article and the ESI.† The crystallographic data for BTA-TTM and BTA-FT have been deposited at CCDC with deposition numbers 2378963 and 2378964, respectively.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (22205120), YongJiang Talent Introduction Programme and Hunan Provincial Natural Science Foundation (2024RC3206).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. CCDC 2378963 and 2378964. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d4qm00943f

‡ These authors contributed equally to this work.

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