Abstract
Simultaneous acceleration of radiative and restricted non-radiative decay is crucial for constructing fluorescent materials with high luminous efficiency in the near-infrared second window (NIR-II). This paper proposes a dual strategy of π-extension and deuteration to effectively address this challenging issue. By extending the π-conjugation of aromatic groups and introducing isotopic effects in the AIEgen, enhanced oscillator strength is achieved, suppressing excited state deformation and high-frequency oscillation. Under these conditions, faster radiative decay and restricted non-radiative decay can be realized simultaneously. The excellent emission characteristics of AIEgen in molecular states can be well maintained in aggregates. The corresponding NIR-II emitting AIEgen nanoparticles exhibit high brightness, large Stokes shift, and superior photostability, making them suitable for image-guided cancer and sentinel lymph node (SLN) surgeries. This study provides a new approach to enhance the luminescence efficiency of NIR-II fluorophores in the biomedical field.
Introduction
Fluorescence imaging offers a non-radioactive, non-invasive, and high spatiotemporal resolution method for visualizing dynamic changes in biological processes. Among them, imaging in the second near-infrared window (NIR-II, 900 ~ 1700 nm) has attracted significant attention due to its outstanding advantages of deep penetration depth, low background interference, and high spatiotemporal resolution. High luminous efficiency NIR-II fluorophores are key to achieving ideal imaging performance. For an ideal NIR-II fluorophore, fast radiative decay (kr) and suppressed non-radiative decay (knr) are both essential to enhance luminescence efficiency. The former requires a large oscillator strength, while the latter is closely related to vibrational relaxation (VR) and internal conversion (IC) processes. However, achieving both a fast kr and a restricted knr simultaneously is challenging. So far, most design principles for NIR-II fluorophores can be simplified to: (1) expand π-conjugation and (2) enhance intramolecular donor-acceptor (D-A) interactions. Designing D-A or D-π-A structures with one or more donors or acceptors to narrow the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) is another popular strategy. However, due to severe twisted intramolecular charge transfer (TICT), this overlap is often not significant. Photoluminescence and photothermal processes corresponding to radiative and non-radiative decay are the main pathways when the excited state energy returns to the ground state. The advantages of photoluminescence and photothermal imaging determine the practical applications of materials in fluorescence imaging, photothermal therapy, and photoacoustic imaging. Aggregation-induced emission (AIE) luminescent materials provide an effective strategy to solve the above problems, as the active intramolecular motion of AIE luminescent materials in discrete or aggregated states can offer a good platform to modulate the trend of radiative and non-radiative decay. Our previous work has demonstrated that the energy of the excited state can be effectively converted into heat in the aggregated state by inducing long alkyl chains as spacers to promote the motion of active excited state molecules. If aromatic segments are reasonably extended to enhance intermolecular interactions, theoretically, the motion of excited state molecules can be restricted to suppress non-radiative decay. Replacing high-frequency carbon-hydrogen (C−H) with heavier carbon-deuterium (C−D) can reduce non-radiative decay caused by high-frequency oscillations. Additionally, the larger atomic volume of deuterium also increases the steric hindrance of the benzene ring, further suppressing intramolecular motion of the excited state. This paper proposes a simple π-extension and deuteration hybrid strategy to design NIR-II AIEgens with higher luminous efficiency (Scheme 1). By extending π-conjugation, radiative decay can be accelerated while suppressing molecular excited state deformation, thereby limiting non-radiative decay. The reduced high-frequency oscillation due to deuteration further suppresses non-radiative decay. NIR-II emitting AIEgen nanoparticles exhibit high brightness, large Stokes shift, and superior photostability, performing excellently in image-guided cancer and sentinel lymph node (SLN) surgeries.
Experimental Results and Discussion
Design and Synthesis.
Scheme 1 employs π-conjugated large and strong electron-withdrawing naphthalene diimide-2-(1,3-dithiol-2-yl) acetonitrile (NDA) fragments as acceptors. TPE serves as the donor and rotor, directly coupling with NDA to construct NDA-TPE. Due to the strong non-radiative decay of NDA-TPE in the solid state, it has been proven to be a good photothermal material. Although non-radiative decay contributes to producing superior photothermal materials, multifunctional systems balancing photothermal effects and fluorescence performance exhibit more advantages in advanced therapeutic applications or precise treatments, requiring fine-tuning of radiative and non-radiative decay. To enhance radiative decay, a benzene ring is inserted between D and A to expand π-conjugation, establishing compound NDA-PTPE. To further suppress non-radiative decay caused by high-frequency C−H oscillation, some hydrogens in TPE are replaced with deuterium to construct NDA-PDTPE.
Photophysical Properties
All compounds exhibit strong absorption in THF from 450 to 800 nm with similar high molar absorptivity. As shown in Figure 1a, the photoluminescence (PL) intensity of NDA-TPE, NDA-PTPE, and NDA-PDTPE gradually increases. Their quantum yields (QYs) are measured using ICG (QY = 13% in DMSO) as a reference (Figure 1b). Compared to NDA-TPE, NDA-PTPE shows a significant improvement in QY. The luminescence efficiency of NDA-PDTPE is also increased, though not significantly. Furthermore, the transient fluorescence spectra indicate that the average fluorescence lifetimes of NDA-TPE, NDA-PTPE, and NDA-PDTPE are 1.28, 1.62, and 1.70 ns, respectively. From this, the kinetic parameters of kr and knr can be obtained (Figure 1c). NDA-PTPE shows an increased kr value and a decreased knr value compared to NDA-TPE, indicating accelerated radiative decay and suppressed non-radiative decay. On the other hand, NDA-PDTPE’s deuterated kr is similar to NDA-PTPE, but knr decreases, suggesting that deuteration may not significantly affect radiative decay but impacts non-radiative processes. AIE characteristics are verified by collecting PL spectra in a DMF/H2O mixture, as shown in Figure 1d. All compounds exhibit weak emission in DMF due to active molecular motion but high emission in aggregates formed with gradually increasing water fractions, indicating typical AIE properties. The αAIE values of NDA-PDTPE (8.2) and NDA-PTPE (6.7) are both higher than that of NDA-TPE (2.3). To further demonstrate the AIE nature, we measured PL spectra in 2-methyltetrahydrofuran at varying temperatures. As shown in Figures 1f and S20, the compounds exhibit weak emission at 298 K and strong emission at 77 K. The enhanced emission when molecular motion is frozen can be attributed to suppressed non-radiative decay. The compounds exhibit weak fluorescence in polar solvents, with luminescence increasing as polarity decreases. This is due to the active intramolecular motion of the excited state in polar solvents being suppressed in low-polarity solvents. It limits the conformational change of the molecule to polarized forms, revealing the significant role of molecular motion in emission behavior.
Theoretical Calculations
To gain deeper insights into the mechanism, density functional theory (DFT) calculations were performed. The dihedral angles between D and A in NDA-PTPE and NDA-PDTPE are 43.7°, smaller than 45.0° in NDA-TPE. Planar molecular conjugation favors the delocalization of electrons. Due to strong electron-withdrawing properties, the LUMO distribution is similar on NDA. Their HOMOs exhibit different characteristics. The HOMOs of NDA-TPE are confined to TPE, while the HOMOs of NDA-PTPE and NDA-PDTPE are more extended (Figure 2a). Therefore, the higher kr values of NDA-PTPE and NDA-PDTPE are related to the extended HOMOs. To reveal the reasons for different knr values, we calculated the reorganization energy (ΔE), reflecting the intrinsic structural changes after excitation. The ΔE value of NDA-PTPE is 0.35 eV, lower than 0.37 eV for NDA-TPE, indicating a more rigid structure (Figure 2b). Simultaneously, we determined the root mean square deviation (RMSD) between the ground state and excited state of NDA-TPE and NDA-PTPE, providing an intuitive method to observe the deformation between molecular ground state and excited state. The RMSD of NDA-PTPE is 0.41, far less than 0.65 for NDA-TPE, indicating that NDA-PTPE enhances molecular rigidity, suppressing molecular deformation (Figure 2c). Molecular motion is considered the main cause of non-radiative decay, and the increased molecular rigidity is the reason for NDA-PTPE’s slower decay rate compared to NDA-TPE. Additionally, Figure 2d shows simulated FT-IR spectra, demonstrating that after deuteration, some high-frequency C−H stretching modes shift from 3208 cm-1 to 2375 cm-1. Experimental results in Figure S24 record the same phenomenon, proving that deuteration can effectively suppress high-frequency oscillations. The zero-point energy (ZPE) values of NDA-PTPE and NDA-PDTPE also demonstrate the alleviation of high-frequency oscillations. The ZPE value of NDA-PDTPE is lower than that of NDA-PTPE. By analyzing the contributions of different normal vibration modes to ZPE for NDA-PTPE and NDA-PDTPE using the Shermo program (Figure 1e), it is found that deuteration has little effect on the vibrations of carbon-carbon (C−C), carbon-oxygen (C−O), carbon-nitrogen (C−N), and nitrogen-oxygen (N−O) bonds in aromatic segments, thus the smaller ZPE value of NDA-PDTPE is mainly due to the lower vibration frequency of C−D compared to C−H. Analysis indicates that compared to NDA-PTPE, deuteration can effectively suppress NDA-PDTPE’s knr by reducing high-frequency oscillations.
Fluorescent Properties of Aggregates
First, UV-vis-NIR absorption in the film state was obtained. Compared to the spectra in solution, NDA-PTPE and NDA-PDTPE show significant shoulders in the film state, indicating enhanced intermolecular interactions after aggregation. In addition, the powder X-ray diffraction (XRD) spectrum in Figure S26 shows that the interplanar crystal spacing of NDA-PTPE and NDA-PDTPE is smaller than that of NDA-TPE according to the Bragg equation, which is beneficial for increasing intermolecular interactions and restricting intramolecular motion. Then, PL spectra in the solid state were measured, showing NIR-II emission (Figure S27). From Figure S28, the emission intensity of NDA-PDTPE solid is enhanced, indicating that the emission characteristics of the molecules can indeed be inherited by the aggregates. Next, PL spectra were obtained as a function of temperature. The PL intensities of NDA-PTPE and NDA-PDTPE at 77 K are also higher than that of NDA-TPE, indicating superior performance after employing this strategy (Figure S29). Therefore, NDA-PTPE and NDA-PDTPE exhibit higher αAIE values compared to NDA-TPE, likely due to suppressed intramolecular motion within the aggregates. In addition to experiments, molecular dynamics (MD) simulations were also performed to better understand the intermolecular interactions in the aggregated state. The dihedral angles between the innermost molecules of NDA-TPE, NDA-PTPE, and NDA-PDTPE were calculated, which play an important role in molecular rotation (Figures 3a−e, Figures S30 and S31a−c). In the molecular state, NDA-TPE, NDA-PTPE, and NDA-PDTPE exhibit a wide distribution of dihedral angles, indicating free rotation. However, the dihedral angle distribution of the aggregates shows significant differences. The dihedral angle distribution of the innermost molecules of NDA-TPE is broad, similar to the molecular state, consistent with its active intramolecular motion in the aggregates. In contrast, NDA-PTPE and NDA-PDTPE exhibit a significantly narrowed dihedral angle distribution during aggregate formation, indicating effective suppression of intramolecular motion. Further calculations of the atomic contact ratios of the innermost NDA-TPE, NDA-PTPE, and NDA-PDTPE molecules show that NDA-PTPE and NDA-PDTPE exhibit higher contact ratios compared to NDA-TPE, indicating a higher proportion of atomic interactions with surrounding molecules, thereby suppressing intramolecular motion. Overall, the aggregates of NDA-PTPE and NDA-PDTPE not only inherit the improved emission characteristics of the molecules but also suppress intramolecular motion, which is precisely the goal of the proposed molecular roadmap.
Preparation, Characterization, and Properties of NPs
To ensure good water solubility and biocompatibility of AIEgens, we encapsulated them in NPs using F127 (Figure 5a). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) show (Figure 5b) that NDA-PDTPE NPs exhibit good uniformity, with a dry size of about 125 nm and an average hydrodynamic size of about 156 nm, with a polydispersity index (PDI) of 0.056. Negative surface charge (-13.7 mV) was also detected, ensuring prolonged blood circulation and tumor targeting. Their size and fluorescent stability were evaluated under different conditions (phosphate-buffered saline (PBS), fetal bovine serum (FBS), cysteine (Cys), and blood). As shown in Figures S34 and S35, NPs exhibit good colloidal stability and fluorescent stability. From Figures 5c – e, it can be seen that the fluorescent performance of NDA-TPE, NDA-PTPE, and NDA-PDTPE NPs gradually improves. The good stability and high QY of NPs favor their application in biological imaging (Figures S36 and S37). The maximum absorption and emission of NDA-PDTPE NPs are located at 673 nm and 1018 nm, respectively. Impressively, NDA-PDTPE NPs exhibit a large Stokes shift of 345 nm, which can effectively reduce the overlap between the absorption and emission spectra, avoiding harmful self-absorption effects (Figure S38). The emission spectrum of NDA-PDTPE NPs has a tail extending to 1400 nm, which is more favorable for NIR-IIa (1300−1400) fluorescence imaging. Fluorescence imaging in the NIR-IIa window can provide better imaging contrast, higher resolution, and clearer image output due to reduced photon scattering. Furthermore, the fluorescence intensity of NDA-PDTPE NPs increases linearly with concentration, providing a platform for quantitative analysis. Continuous monitoring scenarios in biological imaging, such as stem cell tracking, long-term inflammation monitoring, and image-guided tumor surgeries, are significant yet challenging. In long-term monitoring, photostability is crucial for obtaining reliable imaging information. By monitoring the fluctuation of absorbance intensity of DMF under light irradiation, we first studied the photostability of DMF in the molecular state. As shown in Figure S40, under continuous irradiation of near-infrared light (808 nm, 0.6 W/cm2) for 60 min, the absorbance intensity remains nearly unchanged, demonstrating good photostability in the molecular state. Subsequently, the photostability of NPs was further studied by recording NIR-II PL intensity (Figures S41 and 5f). Under continuous irradiation, the NIR-II PL intensity of NPs remains almost unchanged, while the intensity of ICG decreases rapidly. Moreover, the biocompatibility of NDA-PDTPE NPs was evaluated through 4T1 cells, showing good biocompatibility even at a concentration of 50 μg mL-1 (Figure 5g).
Photothermal and Photoacoustic Properties
Light and heat are competing forms of excited-state energy dissipation. To study the photothermal effect, the photothermal performance of NPs in a 100 μM aqueous solution was measured under 808 nm (0.6 W cm-2) laser irradiation. As shown in Figures 6a, b, NDA-PTPE and NDA-PDTPE NPs exhibit relatively low platform photothermal temperatures, which may be due to the restricted intramolecular motion within the NPs. This result further confirms that π-extension and deuteration are effective methods to suppress non-radiative decay. For image-guided surgery, the combination of different imaging modalities can provide comprehensive information about lesions, thereby improving surgical outcomes. Therefore, considering the moderate photothermal performance of NDA-PDTPE NPs, their photoacoustic (PA) performance was evaluated as dependent on the photothermal effect. As shown in Figure 6c, with increasing concentration, the PA intensity gradually increases, indicating the potential application of NDA-PDTPE NPs in PA imaging. In addition, to study the photothermal treatment capacity of NDA-PDTPE NPs, we assessed the viability of 4T1 cancer cells cultured with different concentrations of NDA-PDTPE NPs under 808 nm laser irradiation and non-irradiation. As shown in Figure S43, NDA-PDTPE NPs exhibit low cytotoxicity in the dark. However, under 808 nm laser irradiation, NDA-PDTPE NPs demonstrate significant cytotoxicity towards 4T1 cells, with a killing rate of about 80% at 100 μg mL-1. The results suggest the potential of NDA-PDTPE NPs in multifunctional therapeutic systems.
Lymph Node and Blood Vessel Imaging
First, the imaging ability was evaluated in vitro. Compared to NDA-TPE NPs and NDA-PTPE NPs, NDA-PDTPE NPs exhibit higher signal-to-background ratios (SBR) at different coverage depths, indicating that the combination of π-extension and deuteration indeed favors obtaining high-contrast imaging results. Before in vivo imaging, the blood circulation time was studied by measuring the fluorescent intensity in plasma at different time points. NDA-PDTPE NPs exhibit good blood retention, attributed to their good stability. Subsequently, to demonstrate the significance of this strategy, in vivo performance was also evaluated through vascular imaging. As shown in Figure S46, NDA-PTPE NPs can improve the SBR of blood vessels compared to NDA-TPE. NDA-PDTPE NPs can further enhance SBR. In vivo imaging indicates that the hybrid strategy is beneficial for achieving high-contrast live images. Dissection imaging analyzed the biodistribution of NDA-PDTPE NPs (Figure S47). The fluorescent signals from different organs indicate that NPs are mainly distributed in the liver and spleen, suggesting that NDA-PDTPE NPs are primarily captured by the reticuloendothelial system. Subsequently, lymph nodes were imaged through footpad injection (Figure S48). High contrast and low background indicate that NDA-PDTPE NPs possess good fluorescent imaging capability. Due to their excellent fluorescence and PA properties, we employed NDA-PDTPE NPs for imaging subcutaneously implanted tumor-bearing mice. NDA-PDTPE NPs gradually accumulate within the tumor, with clear boundaries (Figure S49), which may be related to the enhanced permeability and retention (EPR) effect of tumors. These results demonstrate that NDA-PDTPE NPs are suitable for dual-mode NIR-II and PA imaging.
Dual-Mode Image-Guided Surgery and SLN Dissection
After in vivo testing of good NIR-II and PA performance, it is worthwhile to explore whether biomedical treatments, such as image-guided tumor surgery, are feasible. NPs were injected via the tail vein into mice with implanted breast tumors, recording PA and fluorescent images at different time intervals. As shown in Figures 6d – f, after intravenous injection of NDA-PDTPE NPs, the PA signal and fluorescent intensity at the tumor site gradually increase over time, consistent with the premise of surgical resection. Sentinel lymph nodes are the primary pathways for tumor metastasis, closely related to tumor prognosis, staging, and treatment decisions. Therefore, observing tumor metastasis in sentinel lymph nodes during tumor surgery is crucial. With the assistance of PA and fluorescent imaging, successful tumor resection was achieved while excising sentinel lymph nodes (Figure 7a). The bioluminescence from luciferase-expressing 4T1 tumors and LNs overlaps well with the NIR-II fluorescent signals, revealing the excellent performance of NDA-PDTPE NPs in imaging-guided surgeries (Figure 7b). Hematoxylin and eosin (H&E) staining of the excised tumors and LNs confirmed the significant tumor metastasis in LNs. The results further demonstrate the completion of precise resection under dual-mode image guidance, enabling rapid confirmation of tumor metastasis.
Conclusion
This paper proposes a hybrid strategy of π-extension and deuteration, ingeniously synchronizing the construction of high-efficiency NIR-II AIEgens with accelerated radiative decay and restricted non-radiative decay. Through in-depth experiments and theoretical calculations, its potential mechanisms are revealed. The results indicate that the f improvement caused by HOMO delocalization accelerates radiative decay, while suppressed deformation and high-frequency oscillations lead to restricted non-radiative decay, ultimately enhancing luminescence efficiency. Moreover, the extension of aromatic segments increases intermolecular interactions, effectively limiting intramolecular motion, allowing the excellent emission characteristics of NDA-PDTPE in the molecular state to be inherited by the aggregates. Given the outstanding brightness, photostability, and large Stokes shift of NDA-PDTPE NPs, we successfully performed dual-mode image-guided tumor and sentinel LN surgeries in live mice. This work provides a reasonable approach to developing high-luminescence efficiency NIR-II light sources.
References
Boosting Luminescence Efficiency of NearInfrared-II Aggregation-Induced Emission Luminogens via a Mash-Up Strategy of π‑Extension and Deuteration for Dual-Model Image-Guided Surgery Fulong Ma, Qian Jia,# Ziwei Deng, Bingzhe Wang, Siwei Zhang, Jinhui Jiang, Guichuan Xing, Zhongliang Wang, Zijie Qiu, Zheng Zhao,* and Ben Zhong Tang,* ACS Nano , https://doi.org/10.1021/acsnano.3c11078
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