Abstract
Traditional organic luminogens, such as aggregation-caused quenching or aggregation-induced emission luminogens, only suitable to exhibit bright luminescence in the single state (i.e., solution or aggregated state), restricting their applications in heterogeneous environments. Herein, we propose a class of luminogens, aggregation-caused quenching / aggregation-induced emission dual property multimodal luminogens, which can simultaneously balance radiative and non-radiative decay processes in both the solution and aggregation states, bridging the gap between aggregation-caused quenching and aggregation-induced emission luminogens. By manipulating the rigidity planes and twisted groups of the molecules, we successfully develop a series of dual-property multimodal dyes DPM-HD1-3 with excellent second near-infrared window (NIR-II) fluorescent, photoacoustic, and photothermal properties signals. Based on the dual-property multimodal characteristics of DPM-HD3, we construct a CO-activated multimodal luminogen, DPM-HD3-CO, for the step-imaging guided therapy in the tumor-bearing mice. DPM-HD3-CO can overcome the interference of tumor heterogeneity, and reveal the relationship between CO levels and treatment response in the different treatment steps via multimodal imaging. We expect that the introduction of the concept of dual-property multimodal luminogens would open up a innovative avenue for dye chemistry, offering greater possibilities for future widespread applications in the areas such as chemistry, biomedical imaging, and energy.
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Introduction
Organic luminescent materials have extensive applications in biomedical imaging, chemical sensors, organic light-emitting diodes (OLEDs), and optoelectronic devices1,2,3,4 Nevertheless, practical implementation is still hindered by various limitations. Conventional organic luminogens usually emit light solely in diluted solutions but often experience aggregation-induced quenching (ACQ) in the aggregated/solid state5,6,7. In contrast, aggregation-induced emission luminogens (AIEgens) typically exhibit strong emission in the aggregated/solid states but weak fluorescence in diluted solutions8,9. These mean that the ACQgens and AIEgens are only suitable for efficient luminescence in a single state (solution or solid)10,11,12,13, thus limiting their applications in complex or heterogeneous environments.
Heterogeneity occurs at the subcellular, cellular, and organismal levels, due to genetic and phenotypic differences14,15. The biological systems heavily relies on the internal microenvironment (pH, viscosity, polarity, etc.) and bioactive species (reactive oxygen species, reactive sulfur species, metal ions, etc.) to maintain normal physiological processes16,17. Abnormal changes in the microenvironment and bioactive molecules can affect the homeostasis of biological systems, leading to the occurrence and progression of diseases18,19,20,21. Therefore, unraveling biological heterogeneity is of great significance for studying fundamental biology and human diseases. In this respect, biomedical imaging technology plays a crucial role in deciphering biological phenomena, structures, and mechanisms across various spatial scales and has found widespread application in disease theranostics and biological research22,23,24,25,26,27,28. Cancer is typically heterogeneous, and its environment and molecular fingerprints change over time29,30, making precise detection and management particularly challenging. Ideal imaging-guided of cancer treatment requires for multiple imaging approaches at different stages of treatment to obtain accurate cancer heterogeneous detection and treatment response, and immediate customization of individualized treatment strategies. Nevertheless, most existing multi-modal luminescent materials ACQgens or AIEgens are prone to uneven aggregation and non-uniform concentration distribution in complex environments, resulting in compromised precision and reliability of imaging outcomes. So far, the dyes with efficient multimode luminescence in solution and aggregated state are still lacking.
In this work, we introduce the innovative concept of ACQ/AIE dual-properties multimodal luminescent agents (DPMgens), aiming to bridge the gap between ACQgens and AIEgens. We construct a series of multimode luminescent agents, DPM-HD1-3, with NIR-II fluorescent (FL), photoacoustic (PA) and photothermal properties in aggregation-liquid state by regulating the rigid plane and twisted groups of molecular engineering strategy. Leveraging the unique ACQ/AIE dual-properties multimodal luminescent properties of DPM-HD3, we customized a CO-activated multimodal luminescence (DPM-HD3-CO) for the step-imaging guided therapy of cancer. DPM-HD3-CO can overcome the interference of cancer heterogeneous environment, providing high-fidelity multimodal diagnostic information for different treatment stages of cancer, thereby improving cancer treatment options and rendering the personalized therapy. This study demonstrates that DPMgens can effectively integrate the advantages of ACQgens and AIEgens into a single molecule, overcoming the heterogeneous effects and facilitating the applications of chemical dyes in heterogeneous and complex environments.
Results
Design and synthesis of multimodal luminogens with ACQ/AIE dual properties
Traditional fluorophores commonly exhibit ACQ effects, where they have strong fluorescence in dilute solutions but weak or quenched fluorescence in aggregation states, thus favoring non-radiative relaxation processes (PA and heat signals)9 In contrast, AIEgens exhibit weak emission or non-emissive in dilute solutions, yet they emit intense light in the aggregated state (Fig. 1a)8,31. Consequently, the single-state high emission of conventional organic materials may cause uncertainty of imaging in practical applications, as the luminogens usually aggregates in varying degrees in complex environments. We envisioned that we could tailor a class of multi-modal luminescent agents (DPMgens) with ACQ/AIE dual-properties that can simultaneously balance the radiative and non-radiative decay processes in solution/aggregation state. This aims to bridge the gap between ACQgens and AIEgens and thus to promote high-fidelity dual-state multi-modal applications of chemical dyes in heterogeneous environments. At the molecular level, NIR fluorophores with large planar conjugated structures usually exhibit strong luminescence in solution, while fluorescence quenching occurs in the aggregated state due to strong intermolecular π-π stacking effect5,32. At the morphological level, the AIEgens with twisted rotor groups exhibit weak/non-fluorescent state (non-radiative decay) in solution, whereas in the aggregated state (morphological level), restricted molecular vibrations and rotations lead to intense fluorescence emission (Fig. 1b)33. Therefore, we decided to develop DPMgens through manipulating rigid planes and twisted groups at the molecular and morphological levels. To construct DMPgens with NIR-II FL, PA, and photothermal properties, the molecular design should consider the following requirements: 1) Incorporation of the large steric hindrance groups to create more space for the rigid frameworks and twisted rotors, facilitating FL performance in both solution and aggregated states. 2) Introduction of the twisted rotor groups (such as diphenylamine, triphenylamine, tetraphene, etc.) to provide appropriate intramolecular motion and promote the photothermal and photoacoustic properties in both aggregated and liquid states. 3) Enhancement of the intramolecular donor-acceptor (D-A) interactions to achieve NIR-II absorption and emission. Based on this strategy, we used the hemicyanine dye (HD) as the skeleton to construct the NIR-II DPMgens (Fig. 1c). On the one hand, expanding the conjugated skeleton of HD would yield a dye with a long wavelength (ACQ-HD). The ACQ-HD dye possesses a large D-A conjugated rigid structure, potentially exhibiting good fluorescence properties in solution, while being susceptible to ACQ phenomenon in the aggregated state. On the other hand, the introduction of twist groups may convert the HD dyes into a dye with AIE characteristics (AIE-HD). The AIE-HD would have a high fluorescence signal in the aggregated state, while in the solution state the non-radiative transition may dominate due to high intramolecular vibration or rotation (strong photothermal and PA efficiency). By manipulating molecular strategies for twisting groups and rigid planes at the molecular and morphological levels, we could design a series of dual-state multimodal dye (DPM-HD1-3) with NIR-II FL, PA and photothermal properties by combining the advantages of ACQ-HD and AIE-HD dyes into a single molecule. Based on DPMgens dual-property multimodal properties, we planned to design an activatable theranostic agent DPM-HD3 for the step-imaging guided cancer heterogeneity precision therapy (Fig. 1d). The dual-state multi-modal probes can overcome non-uniform aggregation effects in complex tumor microenvironments, exhibiting high-fidelity dual-state multi-modal luminescent characteristics, thus providing the promising tools for accurate detection and personalized treatment of cancer.
a The optical characteristics of ACQgens and AIEgens in aggregation and solution states. b Molecular design strategy of multimodal luminogens with AIE/ACQ dual properties. c Rational design of NIR-II multimodal luminogens DPMgens with AIE/ACQ dual properties. d The activatable NIR-II multimodal theranostic agents for step-imaging guided personalized photothermal therapy. DPMgens AIE/ACQ dual properties of multimodal luminogens, PA photoacoustic signal, FL fluorescence, PTT photothermal therapy.
To gain insight into the electronic and geometry structure of the molecules, density functional theory (DFT) calculations were performed with the Gaussian 09, Revision C.01 program at the B3LYP/3-31 G(d) level. The optimized ground and excited state molecular geometry of HD, AIE-HD, ACQ-HD and DPM-HD1-3 were shown in Supplementary Fig. 1a and Supplementary Data 1–6. Compared to HD, the introduction of the rotor group enhances the molecular geometry distortion of AIE-HD in the S0 and S1 states (−22.09° and −17.14°, respectively), which may facilitate dissipating the excited-state energy from free high-frequency rotation in solution, thus may reducing emission in solution. When introducing a large conjugated structure into ACQ-HD molecules, the dihedral angle of the ground S0 and S1 state rhodamine-anthracene core framework are 14.19° and 12.36°, respectively. This relatively planar conjugated structure may promote the fluorescence emission of ACQ-HD in solution, while it was prone to aggregation-induced quenching in the aggregated or solid states. When introducing the twisted groups, the dihedral angle of the S0 state rhodamine-anthraquinone core skeleton in DPM-HD1-3 molecules gradually decreases with the addition of the twisted structures, measuring 22.34°, 16.87°, and −12.45° respectively. Furthermore, the dihedral angles of DPM-HD1-3 in the S1 state gradually approach planarity, measuring 12.89°, 0.47°, and −1.16°, which may aid the emission in solution. Notably, compared to DPM-HD1, DPM-HD2 and DPM-HD3 exhibit the smaller dihedral angles in the S1 state, which may be due to the greater steric hindrance of triphenylamine (TPE) and tetraphenylethylene (TPA) compared to diphenylamine (DPA), thus promoting the formation of the molecules to adopt a relatively planar conformation.
In addition, the free rotational and vibration properties of the end rotor groups offer additional opportunities for intra-molecular motion in both the aggregated and liquid states, which can potentially coordinate the photo-thermal and photo-acoustic conversion efficiencies of the solution and aggregated states. Enhancing the electron-donating ability of the molecules can strengthen the efficient intra intramolecular charge transfer (ICT) effect of AIE-HD and ACQ-HD, reduce the energy gaps (∆E), and favor wavelength extension (Supplementary Fig. 1b). The highest occupied molecular orbitals (HOMOs) of DPM-HD1-3 exhibit delocalized wave functions along the diphenylamine, tetraphenylethylene, and triphenylamine units, while the lowest unoccupied molecular orbitals (LUMOs) primarily resided in the rhodamine-oxygen heterotricycles core structures. This indicated that the DPM-HD1-3 molecules possess strong D-A characteristics and could undergo efficient intra ICT. Correspondingly, with the electron donor groups become more pronounced, the ∆E decrease gradually in the order of DPM-HD1 (1.87 eV) > DPM-HD2 (1.63 eV) > DPM-HD3 (1.59 eV). This trend may facilitate their long-wavelength absorption/emission within the NIR-II biological window. Therefore, through the manipulating rigid plane and twisted structure strategy at the molecular and morphological levels, it is possible to obtain dual-state multimode luminogens with NIR-II fluorescence, photoacoustic, and photothermal properties. To achieve the DPMgens design objective, ACQ-HD, AIE-HD, and DPM-HDS (DPM-HD1, DPM-HD2 and DPM-HD3) were synthesized following the synthetic routes described in Supplementary Figs. 2–6. The final compounds were characterized via high-resolution mass spectrum (HRMS), 1H NMR, and 13C NMR (Supplementary Figs. 7–33).
Photophysical property studies of DPMgens
Subsequently, we studied the photophysical properties of AIE-HD, ACQ-HD, and DPM-HD1-3 in the various solvents using UV-Vis-NIR absorption spectroscopy and fluorescence spectra (Supplementary Table 1). As expected, all the dyes exhibited redshift emissions. In the solvent dichloromethane (DCM), the AIE-HD, ACQ-HD, and DPM-HD1-3 exhibited maximal absorption/emission wavelengths located at 701/808, 754/915, 881/943, 813/930, and 820/932 nm, respectively (Supplementary Figs. 34–36). Notably, the absorption/emission wavelength of DPM-HD3 in EtOH could be extended to 900/950 nm. Compared to the previously constructed NIR hemicyanine dyes HD, the maximal emission wavelength of DPM-HD3 is red-shifted more than 230 nm. These results suggest that increasing the ICT effect and rigid conjugated structure can extend both absorption and emission wavelengths. To investigate the optical properties of AIE-HD, ACQ-HD, and DPM-HD1-3 dyes, we examined their emission in both the solution and aggregated states. As shown in Fig. 2a, ACQ-HD displayed robust NIR-II FL efficiency in the dilute solution with a fluorescence quantum yields (ΦF) of 0.404%. Conversely, the FL signal of ACQ-HD was obviously quenched in the solid state, possibly due to the aggregation-induced quenching effect caused by π-π stacking between molecules. In contrast, AIE-HD exhibited weaker fluorescence emission efficiency (ΦF of 0.048%) in the EtOH dilute solution, whereas it shows higher fluorescence emission (915 nm) in the solid state (ΦF = 0.105%), potentially due to the aggregation-induced emission effect. To compare the photophysical properties of AIE-HD and ACQ-HD molecules in their aggregated states, we investigated their fluorescence spectra in the various THF/H2O (v/v) compositions. As shown in Supplementary Fig. 37a, b, when the water content (fw) increased from 0% to 40%, the FL923 signal of ACQ-HD gradually strengthens, which may be due to the enhanced solvent polarity. However, when the fw exceeds 40%, the FL923 signal decreases significantly until it reaches the baseline, possibly due to the ACQ effect and/or a reduction in solubility during aggregate formation. The FL815 signal of AIE-HD gradually intensified with increasing fw (0–60%), which may be attributed to the AIE effect (Supplementary Fig. 37c, d). However, when fw exceeds 70%, the FL815 signal gradually weakens, possibly due to molecular deposition at higher water fractions. Additionally, we further tested the absorption spectra of ACQ-HD and AIE-HD at the different water contents. The absorption of ACQ-HD begins to decrease when fw exceeds 40%, while AIE-HD only starts to decrease when fw exceeds 60% (Supplementary Fig. 38), indicating that their absorption reduction is also related to fluorescence quenching at high water content. At high water content (fw = 70%), the fluorescence lifetimes of AIE-HD and ACQ-HD are 1.195 ns and 6.986 ns, respectively (Supplementary Fig. 39). The calculated acoustic loudness factor (ALF)34 were 3.35 × 1013 and 3.04 × 1012, respectively, (Supplementary Table 2), indicating that they have potential for photoacoustic imaging. These results indicate that ACQ-HD and AIE-HD only exhibit effective emission in a single state (dilute solution or aggregated/solid state), limiting their imaging applications in heterogeneous environments. In practice, this single-state emission characteristic increases the uncertainty of imaging, as luminescent materials typically aggregate to varying degrees in complex biological environments. Next, we examine the optical performance of DPM-HD1-3 in the liquid and solid systems. In EtOH solution, DPM-HD1-3 exhibited good NIR-II fluorescence emission at 908, 920, 950 nm, respectively, indicating that expanding conjugation and enhancing ICT effect contributed to lengthening the emission wavelength of the dye (Fig. 2b). In addition, compared with AIE-HD (ΦF = 0.048%), DPM-HD1-3 show higher fluorescence luminescence efficiency, with ΦF of 0.38%, 0.25% to 0.28%. These findings suggest that the rigid conjugated backbones of DPM-HD1-3 molecules facilitates luminescence in liquid state. The optical stability experiments (Supplementary Fig. 40) indicate that AIE-HD, ACQ-HD, and DPM-HD1-3 exhibit excellent photostability, which may be favorable for long-term imaging. Notably, the DPM-HD1-3 display strong solid-state fluorescence emissions at 1115, 1020, and 1045 nm (Fig. 2c). The DPM-HD2 (ΦF = 0.067%) and DPM-HD3 (ΦF = 0.072%) both exhibited higher solid-state FL efficiency compared to DPM-HD1 (ΦF = 0.026%). This may be attributed to the presence of the larger sterically hindering groups (TPE and TPA) in DPM-HD2 and DPM-HD3, which may reduce face-to-face π-π stacking and thus enhance the solid-state luminescence. Furthermore, the solid-state emission of DPM-HD1-3 showed a redshift of approximately 100 nm compared to their liquid state, likely due to the existence of the excimer. The fluorescence lifetime of DPM-HD1-3 in the solid state were longer (2-8 times) than in the solution state (Supplementary Fig. 41 and Supplementary Table 2), further supporting the occurrence of the excimer emission in the solid state13. The solid-state fluorescence spectra at the different temperatures showed that DPM-HD1-3 were not very sensitive to temperature change in the solid state. (Supplementary Fig. 42). We employed X-ray diffraction (XRD) to investigate the solid-state stacking of DPM-HD1-3. The DPM-HD1-3 exhibit the strong scattering peaks at around 2θ = 25° (Supplementary Fig. 43). The interlayer distance calculated using the Bragg’s equation was around 3.5 Å, suggesting that DPM-HD1-3 have a strong π-π stacking that promotes the excimer emission in the solid state13. These findings illustrate that DPM-HD1-3 demonstrate excellent NIR-II FL efficiency in both solution and solid states, which is advantageous for their imaging application in heterogeneous environments.
a FL spectra of AIE-HD and ACQ-HD in EtOH solution and solid state. The inset depicts the corresponding fluorescence images. AIE-HD solution λex = 760 nm, solid state λex = 808 nm; ACQ-HD solution and solid state λex = 808 nm. b Normalized NIR-II FL spectra of DPM-HD1-3 in EtOH solution. The inset shows the corresponding solution NIR-II fluorescence images. λex = 808 nm. c FL spectra of DPM-HD1-3 in solid state. The illustration is the corresponding solid state NIR-II fluorescence images. λex = 808 nm. d PA spectra and e corresponding PA intensity of ACQ-HD, AIE-HD, and DPM-HD1-3 in PBS (10 μM, pH 7.4, containing 50% EtOH). (n = 4 independent samples). f Linear relationship between PA intensity and DPM-HD1-3 of different concentrations in EtOH solution. (n = 4 independent samples). g Photothermal heating-cooling process of DPM-HD1-3 solutions (20 μM, pH 7.4, containing 50% EtOH) under continuous irradiation of 808 nm light (1 W cm−2). h Relative PA intensity of DPM-HD1-3 in DMF/glycerol mixture with different glycerol fractions. Ex: 880 nm. (n = 4 independent samples). i The PA strength of DPM-HD1-3 in solid state. Ex: 900 nm Data are presented as the means ± s.d. (n = 3 independent samples). Source data are provided as a Source Data file.
In addition, we further investigated the dual emission characteristics of DPM-HD1-3 in the presence of both the solution and aggregated states. We doped an appropriate concentration (0.1 wt%) of DPM-HD1-3 into lauric acid (LA) to construct a temperature-responsive fluorescence system (DPM-HD1-3@LA). LA is a natural fatty acid with a unique melting point (approximately 44 °C)35. As shown in Supplementary Fig. 44, when the temperature of DPM-HD1@LA was lowered from 60 to 30 °C, its maximum emission peak shifts from 910 to 1050 nm, resembling the fluorescence transition from the solution to solid state. DPM-HD2@LA and DPM-HD3@LA also exhibit the redshift emission from the solution to solid state. However, HD@LA, ACQ-HD@LA and HD-AIE@LA emit maximally only in the solution or solid state. This phenomenon confirms that DPM-HD1-3 possesses the advantages of both ACQgens and AIEgens, enabling dual emission in both the solution and aggregated/ solid states. To understand the aggregation-state luminescence mechanism of the molecules in the aggregated state, molecular dynamics (MD) simulations of ACQ-HD, AIE-HD, and DPM-HD1-3 were performed using the GROMACS program36. The snapshots were taken from the production simulations to illustrate the stacking modes of the molecular aggregates. As shown in Supplementary Fig. 45, the aggregates of ACQ-HD, AIE-HD, and DPM-HD1-3 all exhibit disordered amorphous forms. The dimers of ACQ-HD display face-to-face π-π stacking, which is prone to the aggregation-induced quenching. However, due to the presence of the twisted steric groups, the dimers of AIE-HD and DPM-HD1-3 exhibit the staggered π-π stacking, which is favorable for the formation of the excimer emission in the aggregated/solid state.
Next, we further investigated the PA and photothermal properties of AIE-HD, ACQ-HD, and DPM-HD1-3. First, we recorded the PA spectra of the AIE-HD, ACQ-HD, and DPM-HD1-3 by measuring the PA intensity at different wavelengths. The data show good correlation between the PA spectra and absorption curves, indicating that the PA signals originate from the absorption of NIR chromophores (Fig. 2d, e). In the 50% PBS/EtOH solution, AIE-HD and DPM-HD1-3 exhibit higher PA intensities than ACQ-HD, with the PA intensity order being ACQ-HD < AIE-HD < DPM-HD1 < DPM-HD2 < DPM-HD3. This could be due to the twisted rotor groups (such as DPA, TPE, and TPA groups) present in AIE-HD and DPM-HD1-3 molecules, which may induce appropriate intramolecular motion and thereby enhancing the PA signal in solution. In addition, the PA intensity of DPM-HD1-3 exhibits a good linear relationship with molar concentration (Fig. 2f), indicating its potential for quantitative analysis. These results indicate that DPM-HD1-3 show excellent bimodal performance of NIR-II FL and PA in a monodispersed state. To investigate the origin of the PA signal of ACQ-HD, AIE-HD, and DPM-HD1-3, we examined their photothermal properties. Under near-infrared light irradiation, the maximum temperatures of AIE-HD, ACQ-HD, and DPM-HD1-3 solution were 41.0, 49.3, 66.8, 57.6, and 62.7 °C, respectively (Fig. 2g). The photothermal conversion efficiencies (η) were 35.6, 42.2, 69.0, 58.5, and 65.3% respectively (Supplementary Figs. 46–50). It is worth noting that the photothermal conversion efficiency (PCE) of DPM-HD3 is 23.1% higher than that of ACQ-HD, indicating that enhanced intramolecular motion contributes to coordinating the non-radiative transition process of the dye in the liquid state. To assess the photothermal stability of DPM-HD1-3, we recorded the temperature changes in the DPM-HD1-3 solutions under continuous laser irradiation. The experimental results showed that the maximum temperature of DPM-HD1-3 does not decrease significantly after four heating-cooling cycles under irradiation of 808 nm (1 W/ cm2) laser (Supplementary Figs. 51–53), while the maximum of ICG (a commercial indocyanine green dye) decreased significantly (Supplementary Fig. 54). These results suggest that DPM-HD1-3 exhibit excellent photothermal stability, which is beneficial for achieving prolonged imaging and photothermal therapy. Moreover, DPM-HD1-3 exhibit the favorable acoustic loudness factor (ALF) in both the solution and aggregated states (Supplementary Table 2), which is advantageous for PA imaging applications in heterogeneous environments. Next, we studied the PA properties of DPM-HD1-3 at various viscosities. As shown in Fig. 2h, the PA of the DPM-HD1-3 molecules increases with the enhancement in glycerol content in the N, N-dimethylformamide (DMF)-glycerol mixture. In addition, their absorbance and fluorescence emission were also gradually increased with the increasing viscosity (Supplementary Figs. 55 and 56). This phenomenon may be attributed to the enhanced conjugated rigid planes of DPM-HD1-3 under the high viscosity conditions, which increases the molar absorptivity and promotes the output of the PA and FL signal.
To verify the multimodal properties of DPM-HD1-3 in the solid-state, we further examined the photothermal and PA performance of the solid-state DPM-HD1-3. The experimental findings demonstrated that the highest temperatures reached by the solid-state DPM-HD1-3 were 95.6, 98.9, and 92.6 °C respectively, which facilitate the generation of photoacoustic signals (Supplementary Fig. 57). Next, we also investigated the PA performance of DPM-HD1-3 in the solid state. The result demonstrate that solid state DPM-HD1-3 still exhibits strong PA signals (Fig. 2i and Supplementary Fig. 58). These research findings demonstrate that the innovative DPM-HD1-3 dyes can effectively regulate the radiative and non-radiative transition processes in both solid and solution states, exhibiting efficient NIR-II FL, PA, and photothermal multimode properties in solution/solid state environments. This ACQ/AIE dual-properties multimodal luminescent feature of the DPM-HDs chemical dyes holds great promise for applications in heterogeneous environments. Next, the size and morphology of DPM-HD1-3 in the aggregated state were examined using dynamic light scattering (DLS) and scanning electron microscopy (SEM). As depicted in Supplementary Figs. 59–61, the hydrodynamic diameters of the DPM-HD1-3 dyes were 108, 100, and 120 nm, respectively, with polydispersity indices (PDI) of 0.246, 0.318 and 0.226, respectively. The SEM images showed that DPM-HD1-3 had a near-spherical morphology with an average size similar with the measurements obtained by DLS. Moreover, we assessed the pKa values of DPM-HD1-3 and found them to be 4.1, 7.1, and 6.7, respectively (Supplementary Figs. 62–64). This indicates that these dyes are suitable for application over a wide pH range. Overall, the DPM-HDS can effectively balance the radiative and non-radiative transition processes in both the solid and liquid states, with unique dual-state NIR-II FL, PA, and photothermal multimodal properties, thereby potentially bridging the gaps between ACQgens and AIEgens.
Design and spectral studies of activated multimodal theranostic agents based on DPMgens
Cancer is typically heterogeneous, with its environment and molecular fingerprints changing over time, making precise detection and management particularly challenging29,30. Toward this end, activatable multimodal molecular theranostic agents may enable multidimensional, real-time, non-invasive assessment of oncogene-driven alterations and their therapeutic response, strengthening the effectiveness of cancer management37,38,39. However, most of the existing multimodal molecular theranostic agents rely on the use of ACQgens or AIEgens dyes, which are susceptible to heterogeneous aggregation effects in complex and variable environments, leading to false positive or negative imaging results. Therefore, the development of activated multimodal theranostic agents is of great significance for the accurate detection and personalized treatment of complex diseases. Endogenous CO, produced by the family of heme oxygenase (HOs), serves as a vital gas-signaling molecule that plays a regulatory role in various physiological processes and pathologies40. CO is involved in the regulation of key signaling pathways in physiological systems, exerting anti-inflammatory, anti-apoptotic and anti-proliferation effects41,42. It is worth noting that abnormal levels of the endogenous CO are closely associated to the development process of cancer. Thus, spatiotemporal dynamic monitoring of the relationship between CO levels and treatment response during different treatment stage is crucial for diagnosing, managing tumor heterogeneity. Leveraging the unique dual-property multimodal luminescence characteristics of DPM-HD3, we have constructed a CO-activated multimodal theranostic agent, DPM-HD3-CO, for the step-imaging guided therapy of tumor heterogeneity (Fig. 3a). The theranostic agent DPM-HD3-CO uses an allyl chloroformate functionalized DPM-HD3 dye as the CO sensing moiety and PdCl2 as the additive to capture CO. The sensing mechanism of DPM-HD3-CO relies on CO converting Pd2+ to Pd0, which then triggers the Tsuji–Trost reaction to remove the allyl group43,44. The agent DPM- HD3-CO initially would not exhibit NIR-II FL/PA/PTT signals as the dye DPM-HD3 was in the caged state. When the probe system (DPM-HD3-CO + PdCl2) specifically responds to tumor-associated CO, DPM-HD3 would be released, thus switching on NIR-II FL, ratio-PA, and photothermal signals for the step-imaging guide therapy of tumor heterogeneity. The NIR-II FLI enables rapid tumor site localization, while PA imaging can provide the detailed information, such as tumor 3D geometry and depth, providing clear guidance for personalized treatment. PTI can provide sensitive and rapid feedback to the phototherapy response. More importantly, DPM-HD3-CO can overcome the heterogeneous accumulation effect in a heterogeneous disease environment to provide high-fidelity multimodal diagnostic information. Therefore, the developed activatable multimodal theranostic agent DPM-HD3-CO was expected to decipher tumor information in pathological settings at the different treatment stages, timely adjust the treatment plan of tumors, and improve the precision of tumor treatment. The synthetic pathway of DPM-HD3-CO was depicted in Supplementary Fig. 65, and its structure was characterized using 1H NMR, 13C NMR and HRMS (Supplementary Figs. 66–68).
a Schematic illustrations of CO-activated multifunctional theranostic agent (DPM-HD3-CO) for multimodal diagnose guided personalized therapy. b Absorption spectra of probe system (DPM-HD3-CO + PdCl2, 10 μM each) after adding CORM-3 at different concentrations in PBS (10 mM, pH 7.4, containing 50% EtOH). c The relationship between PA900/PA690 and CORM-3 concentration. (n = 3 independent samples). d Fluorescence spectra of probe system (DPM-HD3-CO + PdCl2, 10 μM each) responding to CORM-3 (0–100 μM). The inset is the fluorescence imaging of the DPM-HD3-CO solution before and after the reaction. λex = 808 nm. e The relationship between FL950 and CORM-3 concentration. (n = 3 independent samples). f The heating and cooling photothermal curves before and after the reaction of DPM-HD3-CO with CORM-3. g Photothermal stability of DPM-HD3-CO responding to CO in aqueous solution during four on/off irradiation cycles with an 808 nm laser (1 W cm−2). Data are presented as the means ± s.d. Source data are provided as a Source Data file.
To investigate the feasibility of DPM-HD3-CO for NIR-II FL/ratio PA sensing of CO, we evaluated the optical properties and targeted recognition capabilities of the probe DPM-HD3-CO in vitro. First, the probe DPM-HD3-CO response to CO was tested in the presence of PdCl2. Commercially available [Ru(CO)3Cl (glycinate)] (CORM-3, a classic CO-releasing molecule) was used as the CO donor43,45,46. As shown in Fig. 3b, the free DPM-HD3-CO exhibits a maximum absorption wavelength of 690 nm. Upon the addition of CORM-3 (0–100 μM), the absorption at 690 nm gradually decreased, while a characteristic peak corresponding to the DPM-HD3 dye appeared at 900 nm. When the CORM-3 concentration was 100 μM, the absorbance ratio Abs900/Abs690 increased from 0.02 to 2.39, suggesting that DPM-HD3-CO can be utilized for the ratiometric detection of CO, thereby enhancing the accuracy and reliability of the measurement. The time-dependent study found that the intensity of Abs900 gradually increased with prolonged time, reaching a plateau at 25 min, indicating the probe DPM-HD3-CO possesses the ability to detect CO rapidly (Supplementary Fig. 69). Furthermore, we also investigated the PA properties of the DPM-HD3-CO. The PA spectra were measured in the wavelength range of 680 to 1064 nm before and after the reaction of DPM-HD3-CO with CORM-3. The free DPM-HD3-CO displayed a strong PA signal at 690 nm (PA690), while the 900 nm PA signal was very weak (PA900/PA690 ~ 0.11). Upon reaction with CORM-3, the PA900 signal significantly increased, while the PA690 signal decreased, resulting in an approximately 24-fold enhancement of the PA900/PA690 signal (Supplementary Fig. 70). A dose-dependent enhancement of the PA900/PA690 signal was observed when DPM-HD3-CO was treated with different concentrations of CORM-3 (Fig. 3c and Supplementary Fig. 71). The PA900/PA690 exhibited a good linear relationship with CORM-3 concentrations from 0–30 μM, with a detection limit of 8.2 × 10−7 M, indicating that DPM-HD3-CO exhibits excellent PA ratio. Additionally, the NIR-II fluorescence intensity at 950 (FL950) increased by approximately 26.7-fold after incubation with various concentrations of CORM-3 (Fig. 3d) and demonstrated a positive linear relationship with CORM-3 concentration (0–40 μM), with a detection limit of 4.7 × 10−7 M (Fig. 3e). These results suggest that the probe DPM-HD3-CO exhibits sensitive response to CO in both NIR-II FL and PA dual-modal modes. The response mechanism of the probe to CORM-3 was verified by high-resolution mass spectrometry (HRMS) and liquid chromatography-mass spectrometry (LC-MS). The HRMS analysis revealed the presence of a distinct mass spectral peak at m/z 829.3846 after the reaction between the probe and CORM-3, corresponding to the molecular weight of the [DPM-HD3] dye (Supplementary Fig. 72). In addition, the LC-MS results showed that there was a major ion peak of m/z 829.3606 at the retention time of 19.17 min (Supplementary Fig. 73), which matches the expected compound DPM-HD3 ([M]+, calculated m/z 829.3636). The ion peak of m/z 913.3819 is attributed to the probe DPM-HD3-CO ([M]+, calculated m/z 913.3847) at the retention time of 19.60 min. These results demonstrate that the probe DPM-HD3-CO can react with CO to release the dye DPM-HD3 under physiological conditions, thereby activating NIR-II FL950 and PA900/PA690 signals. Next, we investigated the selectivity of DPM-HD3-CO. The results showed that only CO exhibited the significant Abs900 signal enhancement, while the absorption changes of other analytes were almost negligible (Supplementary Fig. 74a). We also further examined the chemical stability of the probe in the presence of the different concentrations of ONOO-. The results showed that the DPM-HD3-CO structure may be partially damaged at a concentration of 100 μM ONOO- (Supplementary Fig. 74b). However, as shown in Supplementary Fig. 74c, the Abs900 values of the probe treated with 15 μM CORM-3 are similar in the presence or absence of 15 μM ONOO-, suggesting that the probe DPM-HD3-CO has a selective response to CORM-3. Moreover, the pH sensitivity of DPM-HD3-CO was also examined. As depicted in Supplementary Fig. 75, the Abs900 signals of the probe exhibited negligible changes within the pH range of 3.0–9.0. However, upon the addition of CORM-3, the DPM-HD3-CO solution exhibited excellent responses within the physiological pH range of 6.5–8.5, indicating that the DPM-HD3-CO is capable of detecting CO within a wide range of physiological pH values. Furthermore, the photothermal efficacy of DPM-HD3-CO was assessed before and after its response to CO by monitoring the temperature variation in the probe solution upon exposure to an 808 nm laser with a power of 1 W/cm². As shown in Fig. 3f and Supplementary Fig. 76, the temperature of the CO-activated probe increased significantly (58.3 °C) within 8 min, whereas the free probe exhibited negligible temperature changes under the same conditions. Additionally, the results of the reversible heating-cooling experiments showed that the maximum temperature of the CO-activated probe remained nearly constant for at least four cycles, indicating the superior photothermal stability of DPM-HD3-CO (Fig. 3g). Taken together, these results confirm that the probe DPM-HD3-CO could serve not only as an activatable NIR-II FL/ratio-PA probe for CO detection but also as an efficient photothermal agent.
Assessment of imaging and photothermal therapy of activated theranostic agent in cancer cells
To investigate the response of the DPM-HD3-CO toward CO in cells, FL and PA imaging was performed on the hepatocellular carcinoma cells (Hepa1-6) and normal hepatocytes (HL-7702). Prior to the cell imaging, the cytotoxicity of the DPM-HD3-CO was evaluated using the Cell Counting Kit-8 (CCK-8) method in both Hepa1-6 and HL-7702 cell lines. After incubation with the DPM-HD3-CO (50 μM) for 24 h, the survival rate of both cell lines was still higher than 82% (Supplementary Fig. 77), indicating that the probe DPM-HD3-CO had low toxicity for both the normal and cancer cells. Subsequently, the ability of the probe DPM-HD3-CO to image CO in cells was investigated. The previous studies have indicated that cancer cells release more endogenous CO than normal cells47. As shown in Supplementary Fig. 78, the signal intensity of FL950 and ratio PA900/PA690 in Hepa1-6 cells was significantly higher than that in HL-7702 cells, indicating that the levels of CO in the cancer cells may be higher than the normal cells. Additionally, after pre-treating Hepa1-6 cells with zinc protoporphyrin (ZnPP, a typical endogenous CO scavenger) for 1 h, the FL950 and ratio PA900/PA690 signals decreased significantly. These results further indicate that DPM-HD3-CO can selectively recognize the increased CO level in the cancer cells, thereby activating FL and PA dual-modal signals. To further validate that DPM-HD3-CO can effectively identify CO levels in cancer cells, we used a commercial CO probe system, 3’, 6’-Bis (allyloxy)-Fluoran, for confocal imaging of the cells (Supplementary Fig. 79). The results showed that the green fluorescence signal of Hepa1-6 cancer cells was significantly higher than that of HL-7702 normal cells. The imaging results from the commercial CO probe were consistent with those from DPM-HD3-CO, further confirming that DPM-HD3-CO can detect the increased endogenous CO level in the cancer cells. To investigate the PTT effect of DPM-HD3-CO on HL-7702 and Hepa1-6 cells, we used a dual-staining method with Calcein-AM (staining live cells, green fluorescence) and propidium iodide (PI, staining dead cells, red fluorescence) to evaluate the phototoxicity of DPM-HD3-CO (Supplementary Fig. 79). As expected, significant green fluorescence was observed in the cells treated with PBS or PBS + laser, indicating low phototoxicity of the utilized laser. Meanwhile, the cells cultured with DPM-HD3-CO without laser irradiation also showed significant green fluorescence, further suggesting low toxicity of DPM-HD3-CO to both the cancer cells and normal cells. In contrast, when the cells were treated with DPM-HD3-CO + laser, significant red fluorescence was observed in Hepa1-6 cells, while the HL-7702 cells exhibited green fluorescence, indicating that the probe can selectively kill the cancer cells. The viability of the cells was further assessed by the MTT assay. The data in Supplementary Fig. 80 showed that the viability of HL-7702 and Hepa1-6 cells was still 82% after PBS + Laser or the probe treatment, indicating that only the laser irradiation or probe treatment is less toxic to the normal or cancer cells. The results of the results of Probe + Laser treatment demonstrated that the viability of HL-7702 cells was still higher than 80%, while the viability of Hepa1-6 cells was about 10%, indicating that the probe had targeted photothermal therapeutic effect on the cancer cells. These results suggest that DPM-HD3-CO can serve as a CO-activated therapeutic diagnostic agent for in-situ photothermal therapy of cancer cells, with minimal side effects.
In vivo multimodal imaging using activated theranostic agent
Next, to evaluate the in vivo NIR-II FL imaging performance, DPM-HD3-CO was injected separately into the normal muscle tissue and tumor of the thighs of the mice, and then recorded and analyzed their NIR-II FL950 signal. Prior to the administration of DPM-HD3-CO, no significant FL950 signal was observed in the normal muscle tissue and tumor in the thighs of the mice (Fig. 4a). After 0.5 h of injecting the DPM-HD3-CO, the FL950 intensity in the tumor site was significantly higher (4.2 times) than that in the normal muscle tissue (Fig. 4b). This indicates that the level of CO in the tumor is higher than that in the normal muscle tissue, and that the abundance of CO in the tumor microenvironment can activate the fluorescence signal of the DPM-HD3-CO. Over the course of time, there was a gradual decrease in the NIR-II FL signal at the tumor, likely due to the metabolism of the activated probe within the mouse. Remarkably, even after 48 h, the significant NIR-II FL signal continued to be observed at the tumor site, suggesting the DPM-HD3-CO tremendous potential for monitoring and tracking tumor development over an extended period. Additionally, we conducted the 3D ratio PA imaging of the tumors. As shown in Fig. 4c, d, a strong PA690 signal and a weak PA900 signal were observed at the tumor site following probe injection (0 h), with a PA900/PA690 ratio of 0.34. As time progressed, the PA690 signal gradually decreased while the PA900 signal gradually increased, and the PA900/PA690 ratio increased about 4.2-fold at 0.5 h. Over time, the signal intensity of PA900/PA690 decreased to about 0.89, which may be due to the metabolism, clearance or different properties upon cellular uptake of the probe in the heterogeneous tissues. Notably, due to its good retention ability in the tumor tissue, the signal was still clearly identifiable even after 48 h. In addition, the 3D PA imaging depth analysis showed that the imaging depth of the DPM-HD3-CO was about 10 mm (Supplementary Fig. 81), indicating that the probe had a good tissue penetration depth. These results suggest that that the probe could depict the spatial profile of deep tumor tissues through 3D PA imaging, providing deep non-invasive anatomical information for the detection of cancer. By taking full advantage of the high sensitivity of FL technology, and the good penetration ability and spatial resolution of PA imaging, DPM-HD3-CO could provide comprehensive diagnostic information for cancer, which is beneficial to guide doctors to customize personalized treatment plans. Next, we investigated the photothermal imaging performance of the probe DPM-HD3-CO in the tumor-bearing mice. As shown in Fig. 4e, the photothermal signal of the probe + laser group exhibited gradual increased with the illumination time, reaching 55.9 °C within five minutes (Fig. 4f). Moreover, the temperature at the tumor site only showed a slight increase in the PBS + laser group after 6 min of irradiation. The above results show that the increased CO level within the tumors can activate the photothermal properties of DPM-HD3-CO, thus reducing the photothermal damage to the normal tissues and realizing the effect of activating the photothermal therapy in situ. Furthermore, fluorescence imaging of metabolism and major organs in vivo show that the probe could respond at the tumor site and can be metabolically cleared through the liver in vivo (Supplementary Figs. 82 and 83). These results suggest that the activated probe DPM-HD3-CO can perform NIR-II FL, PA, and photothermal tri-modal imaging of increased endogenous CO level within the tumors, with the potential for long-term companion diagnostics in vivo.
a The representative NIR-II FL imaging of the normal muscle tissue (ROI A) and tumor (ROI B) in the thighs of the mice at different times points (0, 0.5, 6, 12, 48 and 60 h) after intratumoral injection of 20 μM DPM-HD3-CO (100 μL, including 10 μM PdCl2). Imaging parameters: 808 nm laser, 880 nm long-pass filters. b Corresponding mean intensity of NIR-II FL imaging. (n = 5 mice). c PA imaging of tumor regions at different times points (0, 0.5, 6, 12, 48 and 60 h) after intratumoral injection of 20 μM DPM-HD3-CO (100 μL, including 10 μM PdCl2). The meaning of the colors were indicated in the color bars: The red and green represent PA900 channel. The orange and purple represent PA690 channel. d Corresponding mean intensity of the PA imaging. (n = 5 mice). e PTI of the tumor exposed to continuous laser irradiation (808 nm, 1 W cm−2, 0–7 min) after post-injection of PBS and 20 μM DPM-HD3-CO (100 μL, including 10 μM PdCl2). (n = 5 mice). The yellow represent PTI channels. Imaging effects of NIR-II FLI, PAI, and PTI before and after photothermal treatment (Laser: 808 nm, 1 W cm−2). f The corresponding temperature intensity in (e). The excitation source is an 808 nm laser and FLI was collected by 880 nm long-pass filter with an exposure time of 100 ms. Data are presented as the means ± s.d. (n = 5 mice). Source data are provided as a Source Data file.
Tracking the heterogeneous distribution of theranostic agent in cancer cells and tumor tissue
In order to investigate the distribution of theranostic agent DPM-HD3-CO in complex cancer cells and tumors tissue microenvironment, we conducted confocal image on cancer cells and tumors tissue with different treatments (Fig. 5a). First, to investigate the distribution of the probe DPM-HD3-CO in Hepa1-6 cells under the different treatments, DPM-HD3-CO (20 μM) was incubated with Hepa1-6 cells for 30 min, followed by the laser treatment. Then, the laser confocal microscopy was used to image the Hepa1-6 cells, and the aggregate size was measured using the Nano Measurer software. As shown in Fig. 5b and Supplementary Fig. 84, compared to the PBS group, DPM-HD3-CO was distributed in the aggregated state within the cancer cells with the aggregate sizes mainly at 600 nm. However, upon the 808 nm laser irradiation, the aggregate size of DPM-HD3-CO reduced to approximately 300 nm. These findings indicate that the alterations in the cancer cell microenvironment are likely to affect the heterogeneous distribution of DPM-HD3-CO. Next, we further examined the possible impact of the probe DPM-HD3-CO heterogeneous distribution on imaging performance (Fig. 5c). The Hepa1-6 cells treated with PBS, probe, or probe + laser were digested with trypsin and centrifuged to obtain the cell pellets for FL and PA imaging. The results show that probe DPM-HD3-CO treatment group exhibited prominent FL and PA signals, indicating that the probe has good dual-mode emission in the aggregation state. Furthermore, after probe + laser treatment, the cells still exhibited bright NIR-II FL/PA dual-modal signals (Supplementary Fig. 85). These indicate that the FL and PA signals of the probe were not easily affected by changes in the aggregation state. Next, we further compared the imaging performance of DPM-HD3-CO with AIE-HD and ACQ-HD in the Hepa1-6 cells (Supplementary Fig. 86). AIE-HD showed aggregated distribution in the cells and exhibited stronger FL signals in the cell pellets, while AIE-HD + Laser exhibited smaller aggregates in the cells and showed stronger PA signals in the cell pellets. ACQ-HD showed aggregated distribution in the cells and exhibited stronger PA signals in the cell pellets, while ACQ-HD + Laser exhibited smaller aggregates in the cells and showed stronger FL signals in the cell pellets. These observations suggest that the FL/PA signal outputs of AIE-HD and ACQ-HD are sensitive to the changes in the aggregate sizes, which may affect the accuracy of the imaging. In comparison, the DPM-HD3-CO treatment group exhibited aggregated distribution in Hepa1-6 cells and showed strong FL/PA dual-mode signals in the cell pellets, while the DPM-HD3-CO + Laser group showed smaller aggregates in the cells and still displayed the stable FL and PA dual-mode signals in the cell pellets, indicating that DPM-HD3-CO can overcome the impact of heterogeneous distribution on imaging performance. Next, we also further compared the imaging performance of DPM-HD3-CO with AIE-HD and ACQ-HD in the tumor tissue sections (Supplementary Fig. 87). AIE-HD, ACQ-HD, or DPM-HD3-CO (each 20 μM, 100 μL) were intratumorally injected into the tumors, respectively, followed by laser exposure. AIE-HD showed aggregated distribution in the tumor tissue section and exhibit stronger FL signals in the tumor, while AIE-HD + Laser exhibited smaller aggregates in the tumor tissue section and showed stronger PA signals in the tumor. ACQ-HD showed aggregated distribution in the tumor tissue section and exhibited stronger PA signals in the tumor, while ACQ-HD + Laser exhibited smaller aggregates in the tumor tissue section and showed stronger FL signals in the tumor. The FL and PA signal outputs of AIE-HD and ACQ-HD were susceptible to the changes in the aggregation state, which was consistent with the observations in the Hepa1-6 cells. In contrast, the DPM-HD3-CO treatment group exhibited aggregated distribution in the tumor tissue section and showed strong FL/PA dual-mode signals in the tumor, while the DPM-HD3-CO + Laser group showed smaller aggregates in the tumor tissue section and still exhibited the stable FL and PA dual-mode signals in the tumor. These results further suggest that DPM-HD3-CO can overcome the impact of heterogeneity distribution on imaging performance in the tumor tissues, improving the accuracy of cancer detection and treatment evaluation. To assess the impact of the heterogeneous distribution on therapeutic efficacy, we conducted the tests on the photothermal performance of DPM-HD3-CO under various tumor tissue microenvironments (Fig. 5d). Compared to the PBS control group, the probe-treated group showed a predominant aggregated distribution within the intercellular spaces of the tumor tissue (size approximately 600 nm), accompanied by a noticeable green fluorescent signal. In contrast, the probe + laser-treated group exhibited the smaller aggregates (size around 300 nm) but had intense red fluorescence signals. These results indicate that the probe’s heterogeneous distribution does not affect its photothermal therapeutic efficacy in the different pathological environments. Therefore, based on its unique dual-property multimodal properties, the agent DPM-HD3-CO can overcome the influence of heterogeneous distribution on FL, PA, and PTT performance, helping improve the accuracy of disease detection and treatment in complex physiological/pathological environments.
a Illustration of the heterogeneous distribution of the DPM-HD3-CO in tumor tissue at different treatment stages following intratumoral injection of DPM-HD3-CO. b Representative confocal bright field images of Hepa1-6 cells treated with PBS, only probe system (20 μM DPM-HD3-CO + 10 μM PdCl2), and probe system (20 μM DPM-HD3-CO + 10 μM PdCl2) with laser irradiation (808 nm). Each experiment was repeated independently 5 times (n = 5), and similar results were obtained. Green circles show the aggregates of the probe DPM-HD3-CO, and the blue circles indicate the smaller aggregates of the DPM-HD3-CO. c Representative naked-eye, NIR-II FL and PA images of the Hepa1-6 cell pellets treated with PBS, probe system (20 μM DPM-HD3-CO + 10 μM PdCl2), probe system (20 μM DPM-HD3-CO + 10 μM PdCl2) + laser. The meaning of the colors were indicated in the color bars: The gray and white represent FL channels. The red and green represent PA900 channel. The orange and purple represent PA690 channel. FL imaging parameters: λex: 808 nm, 880 nm long-pass filters. PA900 images excitation wavelength: 900 nm. PA690 images excitation wavelength: 690 nm. Each experiment was repeated independently 5 times (n = 5), and similar results were obtained. d Representative confocal bright field images and live/dead cell staining fluorescence images of the tumor tissue sections from the PBS, probe system (20 μM DPM-HD3-CO + 10 μM PdCl2), probe system (20 μM DPM-HD3-CO + 10 μM PdCl2) + laser groups. Each experiment was repeated independently 5 times (n = 5 mice), and similar results were obtained. Green fluorescence indicates the signal from Calcein-AM staining (λex: 490 nm); representing living cells, while red fluorescence indicates propidium iodide staining (λex: 535 nm); represents dead cells. Green circles show the bigger aggregates of the probe DPM-HD3-CO, and the blue circles indicate the smaller aggregates of the probe DPM-HD3-CO + laser. Data were expressed as mean ± SD. Source data are provided as a Source Data file.
Activatable multimodal theranostic agents for step-imaging guide personalized photothermal therapy
Cancer is a complex and heterogeneous disease characterized by intricate interactions among different tumor cells and their changing microenvironment48. This heterogeneity results in each type of cancer, and even each group of cancer cells, being unique in its characteristics. Hence, it is imperative to consider this aspect during cancer treatment and devise “personalized” treatment plans tailored to individual patients. To address this challenge, we have proposed a step-imaging guided therapy approach that can monitor the treatment response of patients at different stages by detecting specific biomarkers, thus enabling the development of personalized treatment plans for different patients. Based on its unique dual-property multimodal characteristics, the agent DPM-HD3-CO can overcome the influence of heterogeneous aggregation, and provide highly accurate diagnostic information for different stages of cancer treatment. We investigated the phototherapy effect of the theranostic agent DPM-HD3-CO for the cancer step-imaging guided therapy (Fig. 6a). The Hepa1-6 tumor-bearing nude mice were randomly divided into four groups: PBS, PBS + laser, DPM-HD3-CO, and DPM-HD3-CO + laser groups. Given the heterogeneity within the tumor, multimodal imaging of the treatment group mice is necessary at different stage of treatment to guide precise photothermal therapy. In clinical practice, the most challenging problem in the process of tumor treatment is to judge whether there are residual tiny tumors after treatment of large tumors and accurately decipher the spatial distribution of tiny tumors. The integration of NIR-II FL and PA imaging has comprehensive advantages such as excellent sensitivity, high spatial resolution, and deep imaging depth, making it an ideal diagnostic method in cancer treatment. During the pre-treatment stage, the probe DPM-HD3-CO (20 μM, 100 μL) was intratumorally injected into the tumor, followed by NIR-II FL, PA, and PT imaging. The experimental results showed the enhanced NIR-II FL (5.89 times), PA900/PA690 (5.31 times), and PT signals at the tumor site of the Hepa1-6 tumor-bearing mice compared to the muscle tissue in the thighs of the normal mice (control group) (Fig. 6b, Supplementary Fig. 88). Diagnostic information such as tumor location, depth, and spatial distribution at the pre-treatment stage could be obtained based on activated 3D PA signals (Supplementary Movie 1). At the same time, the response of the tumor mice to photothermal therapy was evaluated through thermographic imaging. In the first-step treatment, the mice were treated with intratumoral injection of 100 µL DPM-HD3-CO (20 μM) and then subjected to segmented irradiation at the tumor site (808 nm, 1 W/cm2) for 6 min each time, repeated 4 times with a 20-min interval between each treatment. On the 9th day after the completion of the first-step treatment, NIR-II FL, PA900/PA690, and PT imaging were performed to assess the therapeutic effect and prognosis of the tumor. After the first-step of the treatment, there should be no significant enhancement of FL and PA900/PA690 signals at the treatment site if the tumor has been completely eradicated. However, NIR-II FL, PA900/PA690, and PT signals were still observed in the mice, indicating the residual tiny tumors in their bodies. At this point, 3D PA imaging could be used to observe the spatial position and depth of residual tumors at the microscopic spatial scale (Supplementary Movie 1), guiding further treatment. In addition, we conducted the ex vivo and hematoxylin and eosin (H&E) analysis of the tumors in the pre-treatment and the first-step treatment groups. The experimental results showed that, the first-step treatment group still had the residual tumors, compared to the pre-treatment group (Supplementary Fig. 89). After confirming the presence of the residual tumor, the second-step PTT treatment was conducted to eradicate the residual tumor. The DPM-HD3-CO (20 μM, 100 μL, including 10 μM PdCl2) were intratumorally injected directly into the area of the residual tumor, followed by fractional irradiation of the tumor site with an 808 nm laser, three times per session, repeated twice with a 10-min interval between each session. After the completion of the second-step PTT treatment, the NIR-II FL, PA900/PA690, and PT signal intensities in the Hepa1-6 tumor-bearing mice were nearly identical to those in the normal control group (Supplementary Fig. 90), indicating essentially complete eradication of the tumor. These results suggest that DPM-HD3-CO can overcome the interference of cancer heterogeneous environments, providing high-fidelity multimodal diagnostic information for the different stages of cancer treatment, thereby enhancing the accuracy of cancer treatment and achieving personalized therapy.
a Schematic illustration of DPM-HD3-CO for step-imaging guided personalized photothermal therapy in the tumor mice. b Representative NIR-II FLI/PAI/PTI of the Hepa1-6 tumor-bearing mice at pre-treatment, first-step PTT and second-step PTT. (n = 8 mice) FL imaging parameters: λex: 808 nm, 880 nm long-pass filters. PA900 images excitation wavelength: 900 nm. PA690 images excitation wavelength: 690 nm. PTI images excitation wavelength: 808 nm. The meaning of the colors were indicated in the color bars: The gray and white represent FL channels. The red and green represent PA900 channel. The orange and purple represent PA690 channel. The yellow represent PTI channels. c Survival curves of the tumor-bearing mice after treatments with PBS, PBS + laser, DPM-HD3-CO (20 μM, 100 μL), and DPM-HD3-CO (20 μM, 100 μL) + laser. (n = 8 mice per group). d Representative photographs of the mice from the PBS, PBS + laser, DPM-HD3-CO, and DPM-HD3-CO + laser groups at 0 and 18 days. (n = 8 mice). e Representative photographs of the excised tumors at 18 days after treatments with PBS, PBS + laser, DPM-HD3-CO (20 μM, 100 μL), and DPM-HD3-CO (20 μM, 100 μL) + laser. (n = 8 mice). f The relationship between the relative tumor volume and treatment duration in the PBS, DPM-HD3-CO (20 μM, 100 μL), and DPM-HD3-CO (20 μM, 100 μL) + laser groups. (n = 8 mice). g The relationship between the mouse body weight and treatment duration in the PBS, DPM-HD3-CO, and DPM-HD3-CO + laser groups. Data are presented as the means ± s.d. (n = 8 mice, similar results were obtained.). Source data are provided as a Source Data file.
To examine the efficacy of the step-imaging guided therapy strategy, the survival rate study was performed for the different treated mice. As depicted in Fig. 6c, following 36 days of treatment, the survival rates of the mice in the PBS, DPM-HD3-CO and PBS + laser treated groups all decreased to 0%. In sharp contrast, the DPM-HD3-CO + laser group exhibited a high survival rate of 87.5% after 25 days of treatment. Furthermore, throughout the 50-day study period, the survival rate of the mice remained consistently at 87.5%, with no observed recurrence. The significant improvement in the survival rate for the DPM-HD3-CO + laser treatment group reflects the substantial advantage of the step-imaging guided therapy strategy. To further evaluate the efficacy of the step-imaging guided therapy strategy, the tumor volume changes were monitored following the photothermal therapy. The results of the treatment revealed that PBS and DPM-HD3-CO groups did not exhibit a significant therapeutic effect on the tumor (Fig. 6d, e). Conversely, the DPM-HD3-CO + laser group demonstrated significant inhibition effect of tumor growth, demonstrating that the photothermal properties of the probe could be specifically activated at the tumor site and possess excellent anti-tumor capability. The tumor size and weight of the mice were measured every 2 days during the treatment period to assess the treatment effect and side effects. As shown in Fig. 6f, the tumor growth remained stable in the absence of laser irradiation. However, when the probe was intratumorally injected and subjected to light therapy, the tumor growth was effectively suppressed, showing a trend towards complete elimination. In addition, the body weight of the mice in the probe + laser group slightly decreased at the beginning of treatment, but gradually increased with prolonged treatment duration, indicating the probe DPM-HD3-CO promising anti-tumor activity (Fig. 6g). The three control groups steadily gained the body weight during the treatment period, indicating the good biocompatibility of the probe. In addition, H&E histological analysis showed that only the tumor area showed significant damage after the photothermal treatment, while no lesions or injuries in the normal muscle tissues and major organs of heart, spleen, liver, lung, and kidney (Supplementary Figs. 91 and 92). This also further confirms that DPM-HD3-CO has low phototoxicity to the normal muscle tissues and does not produce significant side effects during the treatment. Consequently, the dual-property multimodal probe with CO activation developed in this study can be used as a companion diagnostic tool for the different treatment stages of cancer to achieve precise treatment of the individual tumors.
Discussion
In summary, we have proposed the concept of ACQ/AIE dual-properties multimodal luminescent materials, DPMgens. Significantly, DPMgens can simultaneously balance radiation and non-radiative transitions in both solution and solid states, exhibiting stable ACQ/AIE dual-properties multimodal luminescence. To demonstrate the advantages of DPMgens, we constructed a series of luminescent agents DPM-HD1-3 with NIR-II FL, PA, and PT multimodal signals by manipulating the planar skeleton and twisted groups at the molecular and morphological levels. DPM-HDs can overcome the influence of heterogeneity accumulation effects, thus achieving high-fidelity multimodal luminescence in complex environments. Leveraging the unique ACQ/AIE dual-properties multimodal luminescent properties of DPM-HDs, we successfully customized an activatable dual-property multimodal therapeutic agent DPM-HD3-CO for step-imaging guided cancer heterogeneous therapy. The agent DPM-HD3-CO could overcome the interference caused by tumor heterogeneity and be utilized to reveal the relationship between CO levels and treatment response during the different stages of therapy, achieving accurate tumor detection and personalized therapy. The development of solid-liquid states multimodal probes not only provides an approach for the high-fidelity detection and accurate treatment of heterogeneous tumors, but also may open up the opportunity for monitoring other heterogeneous diseases and complex environments. More importantly, the development of DPMgens has addressed the impact of aggregation and dispersion on the optical performance of traditional single state dyes, thus heralding an era for dual-state multimodal chemical dyes. This breakthrough offers promising possibilities and forward-looking directions for research and applications in the areas such as biomedical imaging, environmental monitoring, chemical sensors, optoelectronic devices, and beyond.
Methods
Experimental animals
All animal experiments were approved by the Animal Experiment Ethics Committee of Guangxi University (Protocol Number: Gxu-2022-027). The 4-weeks-old female BALB/c-nu mice were purchased from the Laboratory Animal Center of the Guangxi Medical University (Nanning, China). Animals were kept in a controlled environment with a temperature of around 25 °C, humidity of 50 ± 10% and a light/dark period of 12 h. The maximum allowable tumor load was 2,000 mm3, and the maximum tumor size was not exceeded in all trials.
Tracking the heterogeneous distribution of DPM-HD3-CO in cancer cells and tumor tissues
The Hepa1-6 cancer cells were seeded in confocal cell culture dishes at a density of 1 × 105 cells/dish for 12 h. To investigate the distribution of the probe DPM-HD3-CO in the cells, the Hepa1-6 cells were incubated with DPM-HD3-CO (20 μM, containing 10 μM PdCl2) for 30 min. Then, the Hepa1-6 cells of the light exposure group were irradiated with 808 nm laser for 10 min. PBS group: the Hepa1-6 were incubated with PBS for 30 min. Probe group: the Hepa1-6 cells were incubated with 20 μM DPM-HD3-CO (containing 10 μM PdCl2) for 30 min. Then, the laser confocal microscopy was used to image the Hepa1-6 cells treated with PBS, DPM-HD3-CO, or DPM-HD3-CO + Laser. The diameter of aggregates was analyzed using the Nano Measurer software. To investigate the potential impact of the probe DPM-HD3-CO heterogeneous distribution on the imaging performance, the Hepa1-6 cells from the PBS, probe DPM-HD3-CO, or probe DPM-HD3-CO + Laser treatment groups were digested with trypsin, then collected and centrifuged to obtain the cell pellets for FL and PA imaging. To study the distribution and therapeutic effect of the probe in the tumor/ tissue sections, 100 μL of DPM-HD3-CO (20 μM, containing 10 μM PdCl2) was intratumorally injected into the tumor. For the specified light treatment group, the tumor region was irradiated with an 808 nm laser for 15 min. PBS (0.01 M, 100 μL) was intratumorally injected into the tumor as a control group. The tumor tissues from the PBS, probe DPM-HD3-CO, and probe DPM-HD3-CO + laser treatment groups (5 mice per group) were then excised, frozen sectioned, and subject to the confocal imaging. To evaluate the photothermal therapy effect of DPM-HD3-CO, the tumor tissue sections from the different treatment groups were stained with Calcein-AM or propidium iodide (live/dead cell staining reagents) for fluorescence imaging. To investigate the potential impact of the DPM-HD3-CO heterogeneous distribution in the tumor on the imaging performance, the tumors from the DPM-HD3-CO and DPM-HD3-CO treatment groups were excised for FL and PA imaging.
In vivo NIR-II FL, PA, and photothermal imaging
The Hepa1-6 tumor-bearing mice model was established by subcutaneously injecting a suspension of 1 × 106 Hepa1-6 cells into the back of the right hind leg. The Hepa1-6 tumor-bearing mice were intratumorally injected with 100 μL DPM-HD3-CO (20 μM, containing 10 μM PdCl2). Then, at 0, 0.5, 6, 12, 24, 48 and 60 h post-injection, the mice were anesthetized using 2% isoflurane in oxygen and imaged through a commercial Series II 900/1700 imaging system with the long pass filter of 880 nm and quantitative analyses by Image J. Additionally, in vivo photoacoustic imaging was conducted using LOIS-3D photoacoustic imaging system (Tomo Wave Laboratories, USA) at designated time intervals following the intratumoral injection of 20 μM DPM-HD3-CO (100 μL, containing 10 μM PdCl2). The infrared thermal images of mice were acquired using an IR camera during the irradiation of 808 nm laser (1 W/cm2) for 10 min at 30 min after intratumorally injected with 20 μM DPM-HD3-CO (100 μL, containing 10 μM PdCl2). The mouse intratumorally injected with PBS under the same irradiation condition were used as the control. To evaluate the metabolism of DPM-HD3-CO in vivo, we administered 100 μL of the probe DPM-HD3-CO (20 μM, 100 μL, containing 10 μM PdCl2) via intratumoral injection to monitor its metabolism within the body. After 24 h post-injection, major organs (including the heart, liver, spleen, lungs, and kidneys) and tumor tissues were excised. Their surfaces were rinsed multiple times with saline for NIR-II fluorescence imaging.
In vivo photothermal therapy
The Hepa1-6 tumor-bearing mice were randomly divided into four groups (eight mice per group)): PBS group, PBS + laser group, DPM-HD3-CO group, and DPM-HD3-CO + laser group. For the first-step treatment, the mice were intratumorally injected with 100 µL of DPM-HD3-CO (20 μM, containing 10 μM PdCl2). Subsequently, the tumor site was segmented irradiated (808 nm, 1 W/cm2) for 5 min per session, repeated four times with a 20-min interval between each irradiation. On the 9th day following the completion of the first-step treatment, tumor therapeutic effects were assessed using NIR-II FL, PA, and photothermal imaging. For the second-step treatment, targeted therapy was administered based on the multimodal imaging results to completely eradicate residual microscopic tumors. The 20 μM DPM-HD3-CO (100 μL, containing 10 μM PdCl2) were injected directly into the areas of residual tumor, followed by fractional irradiation of the residual tumor site with an 808 nm laser, three times per session, repeated twice with a 10-min interval between each session. On the 18th day following the completion of the treatment, tumor eradication and absence of recurrence were confirmed through NIR-II FL, PA, and photothermal imaging. To visualize the antitumor effect, the tumor-bearing mice were photographed on day 0 and 18. After a variety of treatments, the mouse body weight and tumor volume were recorded every 3 days during 28-day study duration. The tumor volume was measured by a vernier caliper and calculated as V = a × b2/2. (a: tumor length; b: tumor width). Relative tumor volume was calculated as RTV = (V − V0)/V0 (V0 was the initial tumor volume). Relative body weight was calculated as RBW = (W − W0)/W0 (W0 was the initial mouse body weight).
Statistics and reproducibility
Unless otherwise specified in figure legends, each experiment was repeated at least three times independently, and the results were similar. All images shown are representative results from biological replicates. Microsoft Excel 2016 and Origin 2018 was used to analyze the data in this study. The results are expressed as mean ± standard deviation (SD).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The data discussed in this study are presented in the manuscript, Supplementary information. All data are available from the corresponding author upon request. The Cartesian coordinates of optimized molecular geometry are provided in Supplementary Data 1-6. Source data are provided with this paper.
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Acknowledgements
This work was financially supported by National Natural Science Foundation of China (22277014, W. Lin, and 22077048, W. Lin), Guangxi Natural Science Foundation (2021GXNSFDA075003, W. Lin, AD21220061, W. Lin), and the startup fund of Guangxi University (A3040051003, W. Lin).
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Weiying Lin (W. Lin) and Wenxiu Li (W. Li) conceived project and designed experiments; W. Li, Sixin Ai (S. A.) and Huayong Zhu (H. Z.) performed research; W. Li, S. A. and H. Z. analyzed the data; W. Li and W. Lin wrote the original draft and revised the manuscript. W. Lin contributed to project supervision and funding acquisition. All authors have read and approved the final version of the manuscript.
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Li, W., Ai, S., Zhu, H. et al. Activatable second-near-infrared-window multimodal luminogens with aggregation-induced-emission and aggregation-caused-quenching properties for step-imaging guided tumor therapy. Nat Commun 16, 2471 (2025). https://doi.org/10.1038/s41467-025-57673-1
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DOI: https://doi.org/10.1038/s41467-025-57673-1
