Twisted Intramolecular Charge Transfer—Aggregation-Induced Emission Fluorogen with Polymer Encapsulation-Enhanced Near-Infrared Emission for Bioimaging

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Mar 2021Twisted Intramolecular Charge Transfer—Aggregation-Induced Emission Fluorogen with Polymer Encapsulation-Enhanced Near-Infrared Emission for Bioimaging Lingchen Meng†, Xibo Ma†, Shan Jiang, Song Zhang, Zhiyuan Wu, Bin Xu, Zhen Lei, Leijing Liu and Wenjing Tian Lingchen Meng† State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 †L. Meng and X. Ma contributed equally to this work.Google Scholar More articles by this author , Xibo Ma† CBSR&NLPR, Institute of Automation, Chinese Academy of Sciences, Beijing 100049 School of Artificial Intelligence, University of Chinese Academy of Sciences, Beijing 100049 †L. Meng and X. Ma contributed equally to this work.Google Scholar More articles by this author , Shan Jiang State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 Google Scholar More articles by this author , Song Zhang State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 Google Scholar More articles by this author , Zhiyuan Wu State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 Google Scholar More articles by this author , Bin Xu State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 Google Scholar More articles by this author , Zhen Lei CBSR&NLPR, Institute of Automation, Chinese Academy of Sciences, Beijing 100049 School of Artificial Intelligence, University of Chinese Academy of Sciences, Beijing 100049 Google Scholar More articles by this author , Leijing Liu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 Google Scholar More articles by this author and Wenjing Tian *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Supramolecular Structure and Materials, Department of Chemistry, Jilin University, Changchun 130012 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000420 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Fluorescence probes with strong near-infrared (NIR) emission and water solubility are considered useful visualization tools for localization marking as well as investigating cell migration and transplantation. Here, we designed and synthesized a new donor–π–acceptor (D–π–A) fluorogen, 2-(4-[(E)-4-(diphenylamino)styryl]phenyl)-3-(4′-[1,2,2-triphenylvinyl]-[1,1′-biphenyl]-4-yl) fumaronitrile (TB-TPE). TB-TPE exhibits twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) in the NIR region, with an emission peak at 714 nm and a fluorescence quantum yield (Qy) of 6.6% in the solid state. By encapsulating TB-TPE with polystyrene–polyethylene glycol (PS-PEG), water-soluble TB-TPE-PS-PEG nanoparticles (TP NPs) are fabricated, which display polymer encapsulation-enhanced emission with a Qy of 46.5% due to the strong restriction effect on the TICT process and the destruction of H aggregation for TB-TPE by the polymer matrix. Au-coated Fe3O4 (Fe3O4@Au) nanocrystals were then embedded in the TP NPs to form highly fluorescent TB-TPE-Fe3O4-Au-PS-PEG nanoparticles (TFAP NPs) with a Qy of 39.7%. Our demonstration of successful cellular imaging of TP NPs for Hep-G2 cells and multimodality imaging of TFAP NPs in mouse liver tumors indicates that polymer-encapsulated TB-TPE offers great prospects as a multifunctional fluorescence probe for bioimaging. Download figure Download PowerPoint Introduction Ultrahigh-sensitive, low-cost, noninvasive, on-site, and real-time fluorescence molecular imaging (FMI) has attracted considerable interest in biological and preclinical studies for the monitoring of biosamples and the acquisition of information on biological structures.1–3 Organic fluorescent dyes have been developed and extensively utilized in FMI because of their good biocompatibility and low toxicity.4,5 One issue in the application of conventional organic molecular probes is the aggregation-caused quenching (ACQ) effect in the aggregate state, due to a planar aromatic conformation.6,7 Exploiting twisted structural organic molecular probes will avoid the ACQ problem because the twisted molecular conformation can reduce intermolecular π–π interactions and thus lead to aggregation-induced emission (AIE) in the aggregate state.8–13 Water-soluble AIE luminogens (AIEgens) have attracted much attention because they are beneficial for biological probing and imaging. In general, encapsulation media, such as inorganic substrates of silica,14,15 organic substrates of surfactants,16–18 and amphiphilic copolymers,19–21 are used as matrices to improve the water solubility of lipophilic AIE dyes. AIE dyes encapsulated by polymer matrices are widely used in FMI due to their good biocompatibility, photostability, and water solubility; polymer-encapsulated AIEgens with a far-red/near-infrared (NIR) wavelength range are especially attractive because of their superior performance due to high imaging penetration depth and enhanced signal-to-noise ratio.22–25 However, NIR-emissive AIEgens with polymer-coated twisted donor (D)–acceptor (A) structures may cause undesirable twisted intramolecular charge transfer (TICT) effects in polar solvents, which promote an efficient channel for the excited state to decay nonradiatively, thereby leading to a substantial decrease in the fluorescence quantum yield (Qy).26–30 For example, Li et al.31 developed an NIR-emissive TPETPAFN (2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl) phenylamino) phenyl) fumaronitrile) AIEgen with a high Qy in the solid state (Qysolid) of 52.5%, but when TPETPAFN was embedded in a DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol)) polymer matrix to prepare AIE nanoparticles, the fluorescence Qy decreased to 24%. Likewise, Zong et al.32 synthesized a perylene diimide fluorophore (Qysolid: 12.3%) with AIE and TICT features for three-photon fluorescence bioimaging of brain blood vessels and found that the Qy of nanoparticles prepared using Pluronic F-127 as the matrix decreased to 2.37%. Che et al.33 prepared efficient NIR-emissive nanoparticles for long-term cell tracing by coating an AIE-active BODIPY (Boron Dipyrromethene)-derivative with DSPE-PEG2000 amphiphilic polymer, whose Qy (26%) is lower than that of BODIPY AIEgen (30%). As reported in the previous studies, the fluorescence efficiency of nanoparticles prepared by polymer encapsulating NIR-emissive AIE dye with the TICT feature is generally lower than that of contained fluorescent dyes in the solid state. Herein, to acquire an NIR-emissive fluorescent probe, we chose an electron-donating triphenylamine group as the donor, an electron-accepting fumaric acid nitrile unit as the acceptor, and a vinyl bond as the π-bridge to synthesize 2-(4-[(E)-4-(diphenylamino)styryl]phenyl)-3-(4′-[1,2,2-triphenylvinyl]-[1,1′-biphenyl]-4yl) fumaronitrile (TB-TPE). The introduction of AIE active tetraphenylethylene with twisted conformation increased the steric hindrance, leading to enhanced fluorescence emission of the molecule. TB-TPE possessed TICT and AIE as well as polymer encapsulation-enhanced emission features. The Qy of TB-TPE was 6.6% in the solid state, while that of nanoparticles prepared by encapsulating TB-TPE with polystyrene–polyethylene glycol (PS-PEG) increased to 46.5%, due to the restriction of the TICT process and the destruction of H-type aggregation for TB-TPE in the aggregate state. The Qy remained as high as 39.7% even when Au-coated Fe3O4 (Fe3O4@Au) nanocrystals were embedded into PS-PEG-encapsulated nanoparticles. The cellular imaging of Hep-G2 cells and the multimodality imaging of mouse liver tumors demonstrated here indicate that nanoparticles prepared by polymer-encapsulating TB-TPE hold great potential in biological fluorescence imaging. Experimental Methods Materials All reagents and starting materials are commercially available and were used without further purification, unless otherwise stated. Sodium methanolate, 4-bromophenylacetonitrile, Ph3P+CH3Br−, t-BuOK, 1-(4-phenylboronic acid pinacol ester)-1,2,2-triphenylethene, AgCO3, tetrakis(triphenylphosphine)palladium [Pd(PPh3)4], iodine, 4-(diphenylamino)-benzaldehyde, and Pd(OAc)2 were purchased from Aladdin Reagent Co. (Shanghai, China). Tetrahydrofuran (THF), methanol, diethyl ether, and toluene were purchased from Energy Chemical Co. (Shanghai, China). PS-PEG was purchased from RUIXIBIO Co. (Xi'an, Shanxi, China). The ultrapure deionized water used had a resistivity of 18.2 MΩ·cm. Instrumentations 1H NMR spectra were acquired in dimethyl sulfoxide-d6 on a Bruker AVANCE III 500-MHz spectrometer and mass spectra on a Bruker Autoflex Speed TOF. Absorption spectra were obtained on a SPECORD 210 PLUS UV–Vis spectrophotometer. Transmission electron microscopy (TEM) images were obtained using a JEM-2100F system. Fluorescence lifetime and Qy measurements were conducted on an Edinburgh FLS920 spectrometer. Cell fluorescence images were obtained using LEICA confocal laser scanning microscopy (CLSM) (TCS SP8 STED 3X), and hydrodynamic size distribution was measured with a Malvern Zetasizer Nano ZS apparatus at 37 °C. Fluorescence imaging in vivo was performed using an IVIS spectrum imaging system. Magnetic resonance imaging (MRI) was performed using the Bruker BioSpec 70/20 USR MRI, and Computerized Tomography (CT) imaging was performed using a PE Quantum FX device. Synthesis of TB-TPE TB molecules were prepared per the previous literature synthesis.34 Under continuous N2 flow, 100 mL toluene, 10 mL THF, and 10 mL water were added to a mixture of TB (1.16 g, 2 mmol), Pd(PPh3)4 (100 mg, 0.1 mmol), pinacol ester-1,2,2-triphenylethene (2.8 g, 3 mmol), and potassium carbonate (4.14 g, 30 mmol) in a 250-mL Shrek tube eggplant-shaped reaction bottle. The solution was heated to reflux over 12 h with stirring and then cooled to room temperature and extracted three times with dichloromethane. The red organic phase of the solution was collected; the crude product was purified by rapid ultraperformance liquid chromatography using dichloromethane:n-hexane (v/v = 5∶1) as eluent to furnish a red solid. Yield: 70%. 1H NMR (500 MHz, DMSO-d6, δ): 7.88 (d, J = 21.6 Hz, 6H), 7.81 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 16.3 Hz, 1H), 7.34 (t, J = 7.8 Hz, 4H), 7.25–6.95 (m, 26 H). 13C NMR (126 MHz, CDCl3, δ): 148.15, 147.32, 144.06, 143.85, 143.54, 141.61, 141.31, 140.21, 137.03, 132.02, 131.37, 130.87, 130.38, 129.35, 129.16, 127.80, 127.65, 127.43, 126.77, 126.65, 126.30, 125.12, 124.80, 124.36, 123.45, 123.38, 123.00, 117.05, 116.89. High-resolution mass spectrometry (HRMS) (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF]) (m/z): 828.261. Single-crystal structure X-ray crystallographic diffraction intensity data for TB-TPE were collected on a Rigaku RAXIS-PRID diffractometer with graphite-monochromator Mo/Cu Kα radiation. The structure was solved with direct methods using the SHELXTL and Olex2 1.2 programs and refined with least-squares methods. The Cambridge Crystallographic Data Centre (CCDC) number for TB-TPE is 2010997.35,36 Computational methods Geometry optimizations for TB-TPE were acquired using time-dependent density functional theory (TDDFT) calculations with the CAM-B3LYP functional. Solvent effects in THF solution were considered through a solvation model based on density (SMD).37–39 [email protected] nanoparticles for cellular imaging One million Hep-G2 cells were incubated with [email protected] nanoparticles (TP NPs) and Hoechst 33258 for 3 h in a confocal dish containing 1 mL Dulbecco’s modified Eagle’s medium (DMEM) culture medium. After three washes with phosphate-buffered saline (PBS) solution, 1 mL PBS solution was added to the confocal dish and immediately followed by CLSM imaging. The experimental procedures regarding cell cytotoxicity are described in detail in the Supporting Information. TB-TPE&Fe3O4@[email protected] nanoparticles for FMI/MRI/CT multimodal imaging All animal experiments were carried out following the guidelines of the institutional ethics committee of animal experimentation of Peking University (Beijing, China). Hep-G2 cells incubated with TB-TPE&Fe3O4@[email protected] nanoparticles (TFAP NPs) were administered through subcutaneous injection into the abdomen of 6-week-old male BALB/c mice. In vivo imaging of the mice was conducted using an IVIS instrument with laser excitation at 465 nm. The fluorescence signals were filtered using a 660-nm filter. MRI was conducted using a Bruker 7.0T small-animal MRI system equipped with a commercial coil with the following scan parameters: Repetition Time/Echo Time = 3000/40 ms, 1-mm slice thickness, Field of View 35 × 35 mm2, 200 × 200 matrix. CT imaging in vivo was conducted using a PE Quantum FX system and processed with Analyze 11.0 software. Results and Discussion Synthesis and characterization of TB-TPE TB-TPE was synthesized via a typical Suzuki coupling reaction using TB and [4-(1,2,2-triphenylethenyl)phenyl]boronic acid; the synthesis procedure is depicted in Figure 1a. The molecular structure of the TB-TPE compound was confirmed by 1H NMR and 13C NMR spectroscopy and further validated by HRMS ( Supporting Information Figures S1–S3). The normalized photoluminescence (PL) spectra of TB-TPE in n-hexane and THF solution are shown in Supporting Information Figure S4, exhibiting a significant red shift in emissions from 577 to 784 nm with the increase in solvent polarity. To fully understand the solvatochromic behavior of TB-TPE, we assessed the photophysical properties of TB-TPE in mixed solvents of THF/n-hexane, including absorption and emission maximum, fluorescence Qy, lifetime, and radiative (kr) and nonradiative (knr) decay rates; the parameters are listed in Supporting Information Table S1. The emission spectra of TB-TPE in solvent mixtures with different THF fractions (fTHF) (Figure 1b) show that increasing the solvent polarity considerably reduced the PL intensity of the TB-TPE solution, decreased the Qy significantly from 92.3% to 0.8%, and red-shifted the emission maximum (Figure 1c). We further investigated the solvent effect on the emission characteristics using the Lippert–Mataga equation by assessing the relationship between the solvent polarity parameter (Δf) and the Stokes shift of the absorption and emission maximum. The linearity of the Lippert–Mataga plot in Supporting Information Figure S5a suggests a strong intramolecular charge transfer (ICT) process during the TB-TPE excitation process, thereby leading to a large Stokes shift of TB-TPE in polar solvents.40–42 When the fTHF in the mixed solvents increased from 0% to 100%, the knr of the TB-TPE solution gradually increased from 3.02 × 107 to 7.43 × 108 s−1, while kr decreased from 3.64 × 108 to 2.96 × 107 ( Supporting Information Figure S5b). This indicates that high solvent polarity promoted the nonradiative relaxation process, resulting in a sharp decline in fluorescence Qy and in almost complete loss of fluorescence. Figure 1 | (a) Synthetic procedures and chemical structure of TB-TPE; (b) PL spectra; (c) fluorescence Qy in THF/n-hexane solvent mixture with different fTHF; (d) PL spectra of TB-TPE in THF/water mixtures with different water fractions (fw), and (e) plots of PL maximum and relative PL intensity (I/I0) versus the composition of the THF/water mixture of TB-TPE, where I0 was the PL intensity at 50%. Download figure Download PowerPoint The PL spectra of TB-TPE in water/THF solution with different water fractions (fw) were measured to investigate the photophysical properties of TB-TPE molecules in the aggregate state. As shown in Figures 1d and 1e, when the fw increased from 0% to 50%, the PL intensity of TB-TPE gradually decreased, accompanied by a red-shifted emission due to an enhanced ICT effect in the presence of the more polar solvent (water) in the surrounding environment. We recorded a significant (57-fold) enhancement in the fluorescence intensity, with a blue shift in emissions from 807 to 691 nm, when fw increased from 50% to 99%, indicating the dominant role of the AIE over the ICT effect. Concurrently, TB-TPE powder displayed an obvious gain in Qy (6.6%) and a blue-shifted emission (709 nm) peak compared with the TB-TPE in THF solution (Qy of 0.8% and emission peak at 771 nm) ( Supporting Information Figure S6). To investigate the AIE properties of TB-TPE, we studied the viscosity dependence of TB-TPE monomer emissions in a mixture of glycerol and ethanol with varied volume fractions ( Supporting Information Figure S7a). When viscosity increased, fluorescence Qy of TB-TPE likewise increased ( Supporting Information Figure S7b). Additionally, as the glycerol/ethanol mixture became more viscous, the TB-TPE kr increased and knr decreased ( Supporting Information Figure S7c). These results suggest that the AIE properties of TB-TPE in the aggregate state originated from a restricted intramolecular motion (RIM) mechanism, which could suppress the molecular motion and effectively improve the fluorescence probe efficiency. Construction of highly efficient fluorescent TP NPs for cellular imaging TB-TPE was encapsulated by PS-PEG to form water-soluble TP NPs as a potential candidate for bioimaging contrast agents. We investigated the influence of different concentrations of PS-PEG on the fluorescence performance of TB-TPE. Figure 2 shows a gradual blue shift in the PL maximum of TP NPs (Figure 2a) and an obvious enhancement in the fluorescent Qy of TP NPs with an increase in the concentration of PS-PEG (Figure 2b). When the concentration of PS-PEG reached 10 μg/mL, the highest fluorescence Qy (46.5%) was observed, seven times higher than that of TB-TPE powder, followed by a slight abatement in fluorescence Qy with the sequential increase in PS-PEG concentration. When the concentration of PS-PEG approached 20 μg/mL, the self-absorption of excessive amphiphilic polymers in fluorescence nanoparticles blocked the excitation light and the encapsulated TB-TPE inside nanoparticles were not effectively excited, resulting in a slight decline in the fluorescence Qy of TP NPs.43,44 Figure 2 | (a) PL spectra and (b) fluorescence Qy of TP NPs with different concentration of PS-PEG. Download figure Download PowerPoint To understand the unique polymer encapsulation-enhanced emission properties of TP NPs, we investigated and compared TB-TPE as a solution and in crystal. The photophysical properties of TB-TPE in THF solution, in crystal form, and in TP NPs are depicted in Table 1. We found that TB-TPE is nearly nonemissive in THF solution, with the lowest luminous efficiency performance (i.e., a Qy of 0.8%) of the three. To gain further insight into this photophysical behavior of TB-TPE in polar solvent, we computationally simulated the ground state and the excited state conformation of TB-TPE in THF solution using TDDFT (Figure 3a). The optimized ground state conformation is characterized by a highly planar dicyanoethylene core with a dihedral angle of 2.3°, and the corresponding excited state conformation is more twisted, with a large dihedral angle of 55.10°. The calculation shows that TB-TPE molecules in polar solvent undergo a molecular geometry change from a near-planar configuration in the ground state to a nonplanar (twisted) configuration in the excited state to form the TICT state, thereby promoting the nonradiative relaxation process with significantly increased knr and resulting in nearly nonemissive fluorescence.45–47 Furthermore, the emission spectra of TB-TPE in THF solution were obtained at various temperatures from 77 to 273 K. As shown in Figure 3b, TB-TPE exhibits a strong orange emission at 77 K because the TICT process is restricted in the THF solution at low temperatures. As temperatures rise, the PL intensity of TB-TPE gradually decreases and the emission is red-shifted and broadened because the TB-TPE molecules have undergone a transition from a restricted intramolecular rotation to a free rotation, thereby leading to the promotion of the TICT effect.48–50 Table 1 | Photophysical Parameters of TB-TPE in THF Solution, TB-TPE Crystals, and TP NPs λem,max (nm) τ (ns) Φ (%) kr (105 s−1) knr (107 s−1) TB-TPE in THF solution 770 1.23 0.8 7.96 61.1 TB-TPE crystal 710 3.23 2.1 22.1 19.3 TP NPs 650 5.63 46.5 825.8 9.5 Figure 3 | (a) The optimized geometry of TB-TPE in the ground state and the excited state in THF solution. (b) PL spectra of TB-TPE in THF solution (concentration: 1 × 10−5 M) at 77–273 K. Download figure Download PowerPoint We then investigated the luminescence performance of TB-TPE in crystal form. Single-crystal TB-TPE (CCDC number: 2010997) was obtained by solvent evaporation in chloroform/petroleum ether mixed solution (see Figure 4a). The powder X-ray diffraction (PXRD) patterns of the crystalline powders of TB-TPE show sharp and intense peaks, indicating good crystalline structures ( Supporting Information Figure S8). The Qy of TB-TPE increased to 2.1% in the crystal state from 0.8% observed in THF solution, with kr increasing from 7.96 × 105 to 2.21 × 106. It is worth noting that the Qy of crystal TB-TPE was significantly reduced compared with that of TB-TPE powder; this surprising finding encouraged us to further study the intermolecular interactions and molecular packing of TB-TPE in the crystalline state. Abundant intermolecular interactions such as C–H–π (distance: 3.830, 3.812, 3.775, 3.643, and 2.938 Å) and C–H–N (distance: 2.938 Å) were found in the crystal lattice (see Figure 4b). The crystal packing of TB-TPE indicated the existence of H-type aggregation in the crystal lattice, which ultimately resulted in a lower Qy in the aggregate state (Figure 4c). Although the intermolecular interactions of TB-TPE in the aggregate state may rigidify the molecular conformation and restrict intramolecular motions, which are beneficial to enhance the fluorescence, the packing mode of TB-TPE in the aggregate state greatly limits its Qy for further enhancement. Figure 4 | (a) Fluorescence image, (b) intermolecular interactions, and (c) stacking mode of TB-TPE single crystal. Download figure Download PowerPoint TP NPs in which TB-TPE was encapsulated by a polymer matrix showed the highest fluorescence performance, compared with the TB-TPE solution and the crystal form. We found that in TP NPs, the nonradiative transition process of TB-TPE was restricted, which prompted fluorescence emission through radiation transitions and led to the largest reduction in knr (9.5 × 10−7) and the highest increase in kr (8.2 × 10−7). Based on our comparison between TB-TPE in THF solution and crystal TB-TPE, we speculate that the polymer matrix of TB-TPE plays a key role in the remarkably enhanced Qy of TB-TPE in aqueous solution. According to previous studies, hydrophobic dyes encapsulated by amphiphilic copolymers can enter the hydrophobic segment of the copolymer via hydrophobic–hydrophobic interactions.51–53 Therefore, we employed a molecular simulation approach to investigate the interaction between TB-TPE and the polymer matrix, using TB-TPE/PS as a model system. As shown in Figure 5a, the PS segment provides a hydrophobic nonpolar environment, and TB-TPE molecules are easily dispersed into the PS matrix, based on atomistic molecular dynamics (MD) simulations (Figure 5b). Our results indicate that the PS matrix not only provided a nonpolar environment for the encapsulated dye to inhibit the TICT process but also dispersed the aggregated dye to disrupt H-type aggregation in the aggregate state, resulting in significant enhancement in fluorescence efficiency. This explains why the Qy of polymer-encapsulated TB-TPE is more than seven times higher than that of TB-TPE powder. Figure 5 | (a) Three-dimensional images showing the lowest energy conformation of TB-TPE/PS complex. Blue color indicates hydrophobic area and green color indicates mild polar area. (b) MD simulations model for TB-TPE/PS complex. Download figure Download PowerPoint Because of their good water solubility and highly efficient fluorescence, TP NPs can be used for cellular imaging. We obtained fluorescence images of Hep-G2 cells with CLSM after incubation with TP NPs for 3 h. Flow cytometry was conducted to characterize the Hep-G2 cells upon uptake of TP NPs ( Supporting Information Figure S9). Emissions in the red channel upon excitation by a 488-nm laser light are shown in Supporting Information Figure S10, indicating that TP NPs passed across the cell membrane and entered the cell cytoplasm. Our cytotoxicity experiments indicate good cellular biocompatibility of TP NPs (see Supporting Information Figure S10e). Construction of efficient fluorescence TFAP NPs for FMI/MRI/CT multimodal imaging Multimodal imaging probes were prepared by co-encapsulating TB-TPE NIR dye and Fe3O4@Au nanocrystal into a PS-PEG polymer matrix to form TFAP NPs, following a one-pot self-assembly method ( Supporting Information Figure S11a). Under 365-nm excitation, TFAP NPs displayed strong red emission (with a maximum at 650 nm) ( Supporting Information Figure S11b), a high Qy (39.7%), and a small size distribution (72 nm) ( Supporting Information Figure S11c). The application of TFAP NP multimodal probes for FMI/MRI/CT triple-modality imaging was demonstrated by subcutaneous liver tumor imaging. Upon injecting TFAP NPs labeled Hep-G2 cells into a nude mouse, as shown in Figure 6a, a strong fluorescence signal was detected. Three-dimensional (3D) fluorescence images of tumor-bearing mice were reconstructed, and their 3D anatomical information could be clearly visualized (see Figure 6c). In vivo MRI results (Figure 6b) were compared with the contrast MRI of tumor-bearing mice without TFAP NPs injection ( Supporting Information Figure S12). T2-weighted images of tumor-bearing mice labeled by TFAP NPs showed enhanced negative-contrast signal in the tumor region. A significant tumor contrast is visible in the postinjection CT image in Figure 6d. These results show that TFAP NPs are effective contrast-enhancing multimodal agents and suggest TFAP NPs are useful multimodal imaging agents with good depth penetration and spatiotemporal resolution. Figure 6 | (a) Representative in vivo fluorescence images from day 0 to day 20, (b) MR image, (c) 3D reconstruction fluorescence image, and (d) CT image of the mouse subcutaneously injected with 1 × 104 of TAFP NPs labeled Hep-G2 cells. Download figure Download PowerPoint Conclusion We rationally designed and synthesized a NIR-emissive D–π–A structure fluorescent probe TB-TPE with TICT and AIE duo-features. TP NPs were prepared by encapsulating TB-TPE with a PS-PEG polymer matrix and exhibited unusual polymer encapsulation-enhanced emission. The fluorescence Qy (46.5%) was seven times higher than that of TB-TPE powder (6.6%) due to the interaction between TB-TPE molecules and the PS-PEG polymer matrix that suppressed the TICT process in polar solvent and destroyed the H-type aggregation in the aggregate state of TB-TPE. The Qy remained as high as 39.7% even when TB-TPE and Fe3O4@Au nanocrystals were co-embedded into PS-PEG to form TFAP NPs. The good performance of the TP NPs in cellular imaging of Hep-G2 cells and in multimodality imaging of mouse liver tumors indicates that a strategy based on polymer encapsulation-enhanced emission holds great potential for the application of AIEgens in biological fluorescence imaging. Supporting Information Supporting Information is available. Conflict of Interest The authors declare no conflict of interest regarding the publication of this article. Funding Information This work was financially supported by the National Natural Science Foundation of China (nos. 21835001, 51773080, and 21674041), Program for Changbaishan Scholars of Jilin Province, and the “Talents Cultivation Program” of Jilin University.