AIE-Active Functionalized Acrylonitriles: Structure Tuning by Simple

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AIE-Active Functionalized Acrylonitriles: Structure Tuning by Simple Reaction-Condition Variation, Efficient Red Emission and Two-Photon Bioimaging Guangle Niu, Xiuli Zheng, Zheng Zhao, Haoke Zhang, Jianguo Wang, Xuewen He, Yuncong Chen, Xiujuan Shi, Chao Ma, Ryan T. K. Kwok, Jacky W. Y. Lam, Herman H.-Y. Sung, Ian D. Williams, Kam Sing Wong, Pengfei Wang, and Ben Zhong Tang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06196 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Journal of the American Chemical Society

AIE-Active Functionalized Acrylonitriles: Structure Tuning by Simple Reaction-Condition Variation, Efficient Red Emission and TwoPhoton Bioimaging Guangle Niu,†,‡ Xiuli Zheng,§ Zheng Zhao,‡ Haoke Zhang,‡ Jianguo Wang,‡ Xuewen He,‡ Yuncong Chen,†,‡ Xiujuan Shi,†,‡ Chao Ma,∇ Ryan T. K. Kwok,†,‡ Jacky W. Y. Lam,†,‡ Herman H. Y. Sung,‡ Ian D. Williams,‡ Kam Sing Wong,∇ Pengfei Wang,§ and Ben Zhong Tang*,†,‡,¶ †HKUST-Shenzhen

China

Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen 518057,

‡Department

of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute for Advanced Study, Institute of Molecular Functional Materials and Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China §Key

Laboratory of Photochemical Conversion and Optoelectronic Materials and CityU-CAS Joint Laboratory of Functional Materials and Devices, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ∇Department

of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China ¶Center

for Aggregation-Induced Emission, SCUT-HKUST Joint Research Institute, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China Supporting Information Placeholder

ABSTRACT: Acrylonitriles with aggregation-induced emission (AIE) characteristics have found to show promising applications in two-photon biomedical imaging. Generally, elaborate synthetic efforts are required to achieve different acrylonitriles with distinct functionalities. In this work, we first reported the synthesis of two different group-functionalized AIE-active acrylonitriles (TPAT-AN-XF and 2TPAT-AN) obtained simply by mixing the same reactants at different temperature using a facile and transition metal-free synthetic method. These two AIE luminogens (AIEgens) exhibit unique properties such as bright red emission in the solid state, large Stokes shift and large two-photon absorption cross section. Water-soluble nanoparticles (NPs) of 2TPAT-AN were prepared by nanoprecipitation method. In vitro imaging data show that 2TPAT-AN NPs can selectively stain lysosome in live cells. Besides one-photon imaging, remarkable two-photon imaging of live tumor tissues can be achieved with high resolution and deep tissue penetration. 2TPAT-AN NPs show high biocompatibility and are successfully utilized in in vivo long-term imaging of mouse tumor with high signal-to-noise ratio. Thus, the present work is anticipated to shed light on the preparation of a library of AIE-active functionalized acrylonitriles with intriguing properties for biomedical applications.

INTRODUCTION The past decade has witnessed the extensive developments and significant advances of alkenes as key building blocks for synthesis of diverse functionalized structures in organic chemistry and material science.1-3 Cyano group is one of the best electron-withdrawing groups and its introduction to the π-conjugated structures of alkenes leads to acrylonitriles (Scheme 1) with distinct property changes such as conformation, packing mode, stability, solubility and processability.4-6 On the other hand, acrylonitriles represent a common structural motif and are commonly

found in herbicides, pharmaceuticals, agrochemicals and natural products.7, 8 Of particular importance is that further introduction of donor into the skeleton of acrylonitriles can construct fluorescent materials with donor and acceptor conjugated structures and nonlinear optical characteristics, which are beneficial for two-photon excited imaging in live samples due to minimal background fluorescence, high spatial resolution and deep tissue penetration.9-11 In addition, proper tuning the donor strength and π-conjugation could produce acrylonitriles with bright red emission and large two-photon absorption cross section.9 Therefore, the development of organic

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fluorescent donor and acceptor conjugated materials containing the structure motif of acrylonitriles can potentially realize numerous utilizations especially in biomedical imaging. Scheme 1. Various strategies functionalized acrylonitriles.

to

synthesize

In recent years, considerable efforts have been made to synthesize acrylonitriles with varied substitutions and functions.12-19 Jiao et al. reported that allyl halides or esters reacted with NaN3 or TMSN3 catalysed by Pd(PPh3)4 to form allyl azides, which were transformed to alkenyl nitriles (or acrylonitriles) by subsequent oxidative rearrangement process using DDQ as oxidant (Scheme 1).14 On the other hand, the strategy of direct cyanation of alkenes has also been adopted to develop various acrylonitriles.16-19 A novel oxidative cyanation of terminal and internal alkenes using a homogeneous copper catalyst to access acrylonitriles was reported by Engle et al..16 Based on the above-mentioned elegant organic synthetic methods, various fascinating acrylonitriles have been synthesized. However, these routes involve the use of expensive transition metal complex, hazardous and toxic agent, harsh reaction conditions and low atom economy. Therefore, direct functionalities of acrylonitriles remains a challenging task and there is still room for further improvement. Recently, many research groups discovered that ketones or aldehydes and propane nitriles were reactants for direct production of acrylonitriles using conventional base (NaOH, t-BuOK, etc.) via a transition metal-free, non-hazardous, non-toxic and atom-economic nucleophilic reaction.20-23 Thus, such nucleophilic reaction could offer great potential for the direct and facile synthesis of versatile acrylonitriles with multiple functionalities. Aggregation-induced emission (AIE)24 is a unusual photophysical phenomenon that some luminogens show no or weak fluorescence in solution but highly boosted emissions in the aggregate and solid state.25-40 Previous studies demonstrated that the cyano groups render acrylonitriles with twisted structures to result in AIE properties and avoid aggregation-caused quenching (ACQ) in the aggregate state in aqueous environment.41, 42 Based on acrylonitriles, many multi-color brightly emissive

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AIE luminogens (AIEgens) have been synthesized and applied in various fields such as fluorescence sensing and one- and two-photon bioimaging.43-45 Among these AIEgens, acrylonitriles with bright red emission are particularly preferred in bioimaging because of the reduced photodamage, minimal background autofluorescence and deep tissue penetration.46-48 Unfortunately, highly red-emissive acrylonitrile-based AIEgens are still rare.49-54 In addition, their structures are complicated and their synthesis involves multi-step reaction routes and time-consuming isolation or purification. On the other hand, for specific reactants, the change of reaction condition generally exerts little effect on the reaction type to give only one main product. Thus, is it possible to obtain different group-functionalized fluorescent especially red-emissive acrylonitriles just by simply tuning the reaction condition based on the same reactants? However, to the best of our knowledge, such possibility has seldom been explored. Our group and others have demonstrated that triphenylamine is an excellent donor unit to construct donor and acceptor conjugated fluorescent organic materials with strong two-photon absorption.55-59 Further extension of the π-conjugation of such materials by introduction of thiophene unit could enhance the twophoton absorption and red-shift the fluorescence.9 Based on these considerations, we thus explored the possibility to access donor and acceptor conjugated acrylonitriles with different functionalities by using the facile nucleophilic reaction. In the present work, we indeed synthesized different multi-functionalized acrylonitriles (TPAT-AN-XF and 2TPAT-AN, Scheme 2) with typical donor and acceptor conjugated structures by only tuning the reaction temperature via the transition metal-free, non-hazardous, non-toxic and atom-economic synthetic method. To the best of our knowledge, this is first example to prepare different functionalized acrylonitriles by simple reactioncondition variation. The photophysical properties of these two acrylonitriles have been systemically investigated and they are found to be AIE active. They exhibit highly bright solid-state red emission with high fluorescence quantum yield of up to 37.6%. They also show large two-photon absorption cross sections of up to 508 GM due to their donor and acceptor based structure and high πconjugation. Biocompatible nanoparticles (NPs) of 2TPATAN were prepared by nanoprecipitation method, which were successfully applied in in vitro lysosome specific imaging in live cells, ex vivo two-photon deep-tissue imaging and in vivo tumor imaging in mice with high signal-to-noise ratio. RESULTS AND DISCUSSION Synthesis and characterization. The synthesis routes to 2TPAT-AN and TPAT-AN-XF are shown in Scheme 2. Firstly, the Suzuki coupling reaction of compound 3 and 4 in the presence of catalyst Pd(PPh3)4 resulted in compound 1 (Scheme S1). Then the reaction of compound 1 and 2 in refluxed anhydrous EtOH with t-BuOK didn’t generate TPATAN-XF as our expectation but produce 2TPAT-AN instead,60


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which has been successfully confirmed by the single crystal structure analysis (Scheme 2). The single crystals of 2TPATAN were grown and collected by slow evaporation of the mixed solvents of CH2Cl2 and MeOH (CH2Cl2/MeOH = 3:1, v/v) at room temperature. The details of the X-ray experimental conditions, cell data and refinement data of 2TPAT-AN are summarized in Table S1. It is worthy to note that TPAT-AN-XF was finally prepared by reaction of compound 1 and 2 in the presence of t-BuOK in anhydrous EtOH at room temperature. As far as we know, this is first time to prepare different functionalized acrylonitriles by simply tuning the reaction temperature. The structures of the intermediate compound 1 and the final products (2TPAT-AN and TPAT-AN-XF) were well characterized by 1H NMR, 13C NMR, 19F NMR and HRMS spectroscopy (Figure S1-S10). The detailed synthetic procedures were also summarized in the Supporting Information. It is reasonable that TPAT-AN-XF was formed by direct

nucleophilic reaction at room temperature. Why was the compound 2TPAT-AN obtained under the condition of tBuOK and refluxed anhydrous EtOH? Based on the above experimental results and previously reported report61, a plausible reaction mechanism was suggested to explain the formation of 2TPAT-AN (Scheme S2). Compound 2 was first reacted with compound 1 by nucleophilic attack in the presence of t-BuOK to result in intermediate 6, which was transformed into the negatively charged compound 7. Further nucleophilic reaction of compound 7 and 1 resulted in compound 8. Then the intermediate 9 was probably generated by intramolecular nucleophilic reaction of compound 8 and release of cyano group under heat condition. The released cyano group probably further nucleophilically attacked the newly formed oxirane part of compound 9 to form compound 10. The decomposition of compound 10 under heat condition led to side product 11 and the negatively charged intermediate 12. Finally, 2TPAT-AN was formed by nucleophilic reaction of intermediate 12 and compound 1.

Scheme 2. Synthesis routes to 2TPAT-AN and TPAT-AN-XF and single crystal X-ray structure of 2TPAT-AN (ORTEP drawing at the 50% probability level).

Photophysical property. We first investigated the photophysical properties of 2TPAT-AN and TPAT-AN-XF, the absorption and fluorescence (FL) spectra are shown in Figure 1 and Figure S11-S15 and the corresponding data are summarized in Table 1. 2TPAT-AN showed a red-shifted absorption maximum (λabs) at 482 nm than that of TPAT-ANXF (λabs of 439 nm) in dilute THF solution (Figure 1A). 2TPATAN exhibited an emission maximum (λem) at 572 nm in dilute THF solution, and the emission showed a slight red shift after addition of water to the THF solution, while the FL intensity decreased initially and then increased with the water fraction (fw) exceeding 50% (Figure 1B and 1D). 2TPAT-AN showed a strong FL intensity (λabs of 610 nm) at fw = 70% due to the formation of aggregates, demonstrating the aggregationenhanced emission (AEE) property. Further increasing fw to 90% and 99% cause a slight drop of the FL intensity. This phenomenon is generally observed for AIEgens and probably due to the change of the morphology and size of the aggregates formed at high water fractions in aqueous mixtures.62 It should be pointed out that a shoulder peak of about 650 nm appeared at fw = 90% and 99%, indicating different aggregation modes do exist in such aqueous

mixtures. Similarly, TPAT-AN-XF also shows AEE property as proved by the fluorescence analysis (Figure 1C and 1D). In comparison with the emission in THF, the FL intensity of 2TPAT-AN in aqueous suspensions is only slightly increased in comparison with the several-fold enhancement of TPATAN-XF (Figure 1D), which is possibly ascribed to the loose aggregates of 2TPAT-AN and dense aggregates of TPAT-ANXF in aqueous media. The dynamic light scattering data present the hydrated diameters of 399 nm and 210 nm in aqueous suspensions for 2TPAT-AN and TPAT-AN-XF, respectively, supporting the existence of aggregates (Figure S11). 2TPAT-AN and TPAT-AN-XF exhibited low emission efficiencies in THF but remarkably high fluorescence quantum yields of 34.3% and 37.6%, respectively, in the solid state (Table 1), due to the active intramolecular motion induced energy loss in THF and restriction of intramolecular motion (RIM) in the solid state.62 Remarkably, 2TPAT-AN showed redder shifted fluorescence of 635 nm than that of TPAT-AN-XF (591 nm, Figure 1E), probably due the existence of much stronger interaction in the pristine sample (Figure 1F). Compared with the simulated XRD pattern from X-ray single-crystal data, the XRD pattern of 2TPAT-AN in the



pristine sample clearly showed similar diffraction pattern peaks, also indicating the similar interactions in the pristine sample. Thus, the intermolecular interaction and packing of 2TPAT-AN in the crystal state were further investigated (Figure S12 and 1G). 2TPAT-AN exhibits strong intermolecular π-π interaction and C–H···π interaction to restrain the molecular motion (Figure S12), resulting in low non-radiative energy loss and high fluorescence quantum yield. The existence of multiple π-π and C–H···π interactions between adjacent molecules in the crystal state could contribute to the

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red-shifted emission of 2TPAT-AN in aggregates (the shoulder peak of ~650 nm, Figure 1B) and solid state (630 nm, Figure 1F). It should be noted that the π-π interaction existing in the crystal of 2TPAT-AN is particularly stable even upon strong grinding treatment (Figure S13). In addition, these two AIEgens also exhibit typical positive solvatochromism due to the typical donor and acceptor conjugated structures and intramolecular charge transfer effect, as confirmed by the gradually red-shifted emission in organic solvents with different polarities (Figure S14).

Figure 1. (A) Normalized absorption spectra of 2TPAT-AN and TPAT-AN-XF (10 μM) in THF. FL spectra of (B) 2TPAT-AN and (C) TPAT-AN-XF (10 μM) in THF and THF/water mixtures with different water fractions (fw). (D) Plots of αAIE (fluorescence intensity I/I0) versus the composition of the THF/water mixtures of 2TPAT-AN and TPAT-AN-XF. (E) Normalized FL spectra of 2TPAT-AN and TPAT-AN-XF in the solid state. Inset: Fluorescent photos of solids of 2TPAT-AN and TPAT-AN-XF taken under 365 nm UV irradiation from a hand-hold UV lamp. (F) XRD pattern of the pristine sample of 2TPAT-AN and TPAT-AN-XF and simulated XRD pattern of 2TPAT-AN. (G) Molecular packing in the crystal of 2TPAT-AN at different directions. (H) Two-photon absorption (TPA) cross sections of 2TPAT-AN and TPAT-AN-XF in THF. 1 GM ≡ 10-50 cm4 s/photon.

Table 1. Photophysical properties of 2TPAT-AN and TPAT-AN-XFa THF AIEgen

Solid

λabs (nm)

λem (nm)

2TPAT-AN

482

572

Stokes shift (nm) 90

TPAT-AN-XF

439

599

160

αAIE

ΦF,S (%)

λem (nm)

ΦF,P (%)

3.0

635

34.3

11.4

2.6

591

37.6

14.5


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aAbbreviation:

λabs = absorption maximum; λem = emission maximum; ΦF,S and ΦF,P = fluorescence quantum yield in solution and solid powder, respectively; αAIE = ΦF,S/ΦF,P.

Previous studies have demonstrated that donor and acceptor conjugated fluorescent materials show strong twophoton absorption.55-59 Given their good intramolecular charge transfer effect and conjugated structures, the twophoton absorption properties of 2TPAT-AN and TPAT-AN-XF in THF were investigated via two-photon excited fluorescence by using a femtosecond pulsed laser as excitation source. These two AIEgens obviously show strong two-photon fluorescence signals excited at 800−980 nm (Figure S15). The two-photon absorption cross section was also determined by using Rhodamine B in MeOH as the standard.63 As shown in Figure 1H, they exhibit remarkably high two-photon absorption cross sections especially at 880 nm (508 GM and 366 GM for 2TPAT-AN and TPAT-AN-XF, respectively). Interestingly, 2TPAT-AN shows larger two-photon absorption cross sections especially from 800−880 nm than those of TPAT-AN-XF, probably due to that 2TPAT-AN has longer πconjugation length than that of TPAT-AN-XF.9 These excellent two-photon absorption properties with large twophoton absorption cross sections have tremendous potential in biomedical imaging.

Figure 2. Spatial electron distributions of HOMOs and LUMOs of (A) 2TPAT-AN and (B) TPAT-AN-XF in the optimized ground states and excited states at the B3LYP/631G(d, p) level. Theoretical calculation. The photophysical properties of 2TPAT-AN and TPAT-AN-XF were further studied by using density functional theory (DFT) calculation performed at the B3LYP/6-31G level of theory via the Gaussian 09 program package. The spatial electron distributions of HOMOs and LUMOs in the optimized ground states and excited states are depicted in Figure 2. The electrons of HOMOs of 2TPAT-AN are basically delocalized on the whole molecules, while those of LUMOs are mainly distributed at the part of thiophene substituted acrylonitrile. The electrons of HOMOs of TPATAN-XF are generally delocalized on the conjugated parts of

triphenylamine and thiophene in ground state, while those of HOMOs are mainly distributed at the triphenylamine part in excited state. As for the LUMOs of TPAT-AN-XF, they are mainly located on the other part of molecule except for the triphenylamine moiety. The spatial electron distributions of HOMOs and LUMOs reveal that these two AIEgens especially TPAT-AN-XF show obvious electron separation due to the strong intramolecular charge transfer (ICT) effect. In ground states, 2TPAT-AN displays higher HOMO levels than TPATAN-XF due to its excellent π-conjugation, while TPAT-AN-XF shows slightly lower LUMO levels than 2TPAT-AN owing to its strong ICT effect. Such spatial electron distributions of HOMOs and LUMOs in the optimized ground states lead to narrower energy band gap of 2TPAT-AN than that of TPATAN-XF, resulting in red-shifted absorption of 2TPAT-AN. In excited states, however, the LUMO levels of TPAT-AN-XF are greatly decreased in comparison to those of 2TPAT-AN due to strong ICT effect as well as highly twisted structure (Figure S16), resulting in narrow energy gap in excited states and redshifted emission of TPAT-AN-XF in solution. These DFT data further demonstrate that 2TPAT-AN exhibits red-shifted absorption but blue-shifted emission in solution compared with TPAT-AN-XF, which is in good accordance with the photophysical data measured in THF (Table 1). In vitro imaging in live cells. Encouraged by the excellent photophysical properties of 2TPAT-AN, we then explored its potential for biological imaging. We first prepared watersoluble 2TPAT-AN nanoparticles (NPs) via typical nanoprecipitation method64 by using amphiphilic block copolymer PEG-PLGA (Mw: 1000-1000) as the encapsulation materials65 (Figure 3A). Transmission electron microscopy (TEM) data in Figure 3B shows that the particle diameters of 2TPAT-AN NPs are distributed in the range of 30-65 nm. The 2TPAT-AN NPs have a hydrated diameter of about 102 nm verified by dynamic light scattering (DLS) data (Figure 3C). The transparent solution of 2TPAT-AN NPs shows a comparable absorption maximum at about 479 nm and an emission maximum at 608 nm with a shoulder peak of about 650 nm (Figure 3D), and such fluorescence spectrum is very similar with that of 2TPAT-AN in aggregates in THF with fw = 90% or 99% (Figure 1B), presumably resulting from the formation of different packing modes. In addition, 2TPAT-AN NPs exhibit good colloid stability as the DLS size and absorption almost unchanged after seven days (Figure S17). Before bioimaging performed in live cells, we first evaluated the cytotoxicity of 2TPAT-AN NPs in HeLa cells by standard MTT assay. After incubation of 2TPAT-AN NPs for 24 h, the cell viabilities are still very high (over 85%) even at a concentration of 80 μg/mL (Figure S18), confirming the low cytotoxicity of 2TPAT-AN NPs towards live cells. Then we applied 2TPAT-AN NPs for in vitro live cell imaging by using confocal laser scanning microscopy. After incubation for 30 min, strong red emission was observed in the cytoplasm of HeLa cells, and z-stack imaging data further confirmed the localization of 2TPAT-AN NPs in the cytoplasm rather than being bound or adsorbed on the cell surface (Figure 3E). Generally, fluorescent organic NPs tend to locate in lysosomes due to endocytosis. We thus carried out co-staining imaging



studies by using commercial lysosome dye LysoTraker Green DND-26 to verify the specific localization of 2TPAT-AN NPs in live HeLa cells. As shown in Figure 3F, 2TPAT-AN NPs and LysoTraker Green DND-26 display a very similar staining

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pattern with the Pearson's coefficient of 0.89. These data verified that 2TPAT-AN NPs exhibit good cell permeability and selectively locate in lysosomes in live cells.

Figure 3. (A) Schematic preparation of 2TPAT-AN NPs via nanoprecipitation method by using amphiphilic block copolymer PEGPLGA as the encapsulation materials. (B) TEM image of 2TPAT-AN NPs. (C) DLS data of 2TPAT-AN NPs in water. (D) Normalized absorption and fluorescence spectra of 2TPAT-AN NPs in water. Inset: Photos of 2TPAT-AN NPs in water taken under room light (left) and 365 nm UV irradiation (right) from a hand-hold UV lamp. (E) Confocal laser scanning microscopy images of HeLa cells incubated with 2TPAT-AN NPs (5 μg/mL). Scale bar: 20 μm. (F) Confocal laser scanning microscopy images of HeLa cells incubated with 2TPAT-AN NPs (5 μg/mL) and LysoTraker Green DND-26 (200 nM). Scale bar: 20 μm. 880 nm) fluorescent microscopic images of tumor tissues incubated with 2TPAT-AN NPs. Scale bar: 20 μm.

Figure 4. One-photon (λex = 488 nm) and two-photon (λex =

Ex vivo imaging in live tissues. Compared with onephoton imaging, two-photon imaging excited by near-infrared pulsed laser displays much better performance in terms of lower photodamage, higher signal-to-noise ratio and deeper tissue penetration.9-11 Some recent works also successfully demonstrated the deep-tissue penetration advantage of twophoton bioimaging.66-70 To further confirm such merit of twophoton microscopy, we carried out ex vivo two-photon imaging in live tumor tissues. Given the large two-photon absorption cross section at 880 nm as well as strong twophoton excited fluorescence, two-photon imaging was performed by using a NIR pulsed laser at 880 nm. Compared with one-photon imaging, two-photon excited fluorescence with much better resolution and higher signal-to-noise ratio


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could be distinctly seen in live tumor tissues (Figure 4). Such two-photon fluorescence signals show very similar distribution with that excited by one-photon laser, demonstrating that 2TPAT-AN NPs show high potential in two-photon imaging in live tissues. Then we scanned the fluorescent images at different depths and the fluorescent images were captured every 2 μm along the z-axis (Figure 5). One-photon fluorescent signals excited by 488 nm laser could only be obtained at the depth of less than 40 μm (Figure 5A

and Movie S1), which was distinctly revealed by the reconstructed 3D one-photon fluorescent microscopic image (Figure 5B). In sharp contrast, the two-photon fluorescence signals with high signal-to-noise ration could be clearly detected at the depth of up to 60 μm and 3D two-photon fluorescent microscopic image was successfully reconstructed (Figure 5C, 5D and Movie S2). These excellent ex vivo imaging data revealed that 2TPAT-AN NPs hold great potential in vivo two-photon deep-tissue imaging for cancer diagnosis.

Figure 5. Ex vivo two-photon and one-photon imaging in live deep tissues. (A) One-photon (λex = 488 nm) and (C) two-photon (λex = 880 nm) fluorescent microscopic images of the mouse tumor tissue stained with 2TPAT-AN NPs at different penetration depths along z-axis. Scale bar: 50 μm. Reconstructed 3D (B) one-photon and (D) two-photon fluorescent microscopic images. In vivo imaging in live mice. Considering the wide emission spectrum including deep-red to near-infrared region, we anticipated that 2TPAT-AN NPs could exhibit good imaging performance in live animals. To demonstrate this merit, we further carried out in vivo imaging by intratumor injection of 2TPAT-AN NPs in 4T1 tumor-bearing nude mice and the normalized mean fluorescence intensity from tumor was also recorded at different time points (Figure 6A and S19). After 30 min post-injection, strong in vivo fluorescence of 2TPAT-AN NPs from the 4T1 tumor could be dramatically collected with remarkably high signal-to-noise ratio. However, only some background autofluorescence signal throughout the whole mouse was obtained in the control group without injection of 2TPAT-AN NPs. It should be noted that some of the 2TPAT-AN NPs was already metabolized by mouse, which was verified by the slightly higher fluorescence around the tumor than that from the background autofluorescence. The fluorescence signal from tumor region was gradually decreased with increased time after intratumor injection and reached plateau at about 12 h post-injection, which was obviously confirmed by the normalized mean fluorescence intensity data (Figure S19). It should be noted that the decrease of in vivo fluorescence intensity was due to the metabolism of 2TPAT-AN NPs, because the fluorescence

of 2TPAT-AN NPs in PBS showed negligible changes after imaging for 2 h using the same settings for in vivo imaging (Figure S20). Interestingly, the tumor fluorescence only showed slight decrease even after 72 h post-injection (Figure 6A), indicating the tremendous potential application of 2TPAT-AN NPs for long-term tumor tracking. Furthermore, the cytotoxicity of 2TPAT-AN NPs to live mice was evaluated by hematoxylin and eosin (H&E) staining for histological analysis at 72 h post-injection. In the experimental group treated with 2TPAT-AN NPs, no noticeable abnormality was found in the major organs (heart, liver, spleen, lung, and kidney) of mice (Figure 6B), demonstrating the high biocompatibility of 2TPAT-AN NPs at the tested conditions. We further added blood biochemical assays to evaluated the biocompatibility of 2TPAT-AN NPs to live mouse (Figure S21). All the blood routine indexes of the mouse treated with 2TPAT-AN NPs for 72 h generally showed very weak changes except that the number of white blood cells displayed slight decrease compared with those of the mouse treated only with PBS, basically confirming the biocompatibility of 2TPAT-AN NPs to live mouse. These data indicated that biocompatible 2TPAT-AN NPs exhibit promising potential in in vivo bioimaging.



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Figure 6. In vivo imaging in mice and H&E staining. (A) In vivo imaging in 4T1 tumor-bearing nude mice at different time points after intratumor injection of 2TPAT-AN NPs (2 mg/mL, 100 μL). (B) H&E staining of major organ sections (heart, liver, spleen, lung, and kidney) from mice intratumorally injected with or without 2TPAT-AN NPs. Scale bar: 100 μm. CONCLUSIONS In the present work, we developed a transition metal-free, non-hazardous, non-toxic and atom-economic synthetic method to produce AIE-active acrylonitriles (2TPAT-AN and TPAT-AN-XF) with different functionalities by simple tuning the reaction temperature. This is the first time to realize the synthesis of different functionalized acrylonitriles by simple reaction-condition variation. Of particular importance is that these AIEgens exhibit bright solid-state red emission with high fluorescence quantum yield of up to 37.6%. They also display large two-photon absorption cross section of up to 508 GM because of their donor and acceptor based structure and high π-conjugation. Nanoparticles of TPAT-AN prepared by nanoprecipitation method can serve as a contrast agent for two-photon imaging. The biocompatible TPAT-AN NPs show specific organelle staining in lysosomes in live HeLa cells and two-photon deep-tissue imaging with high resolution in tumor tissues. Additionally, in vivo long-term imaging of mouse tumor with high signal-to-noise ratio was also successfully realized. Thus, these brightly red-emissive AIEgens with strong two-photon absorption are anticipated to show great potential for two-photon deep-tissue bioimaging and long-term dynamic tracking of tumor metastasis. The present work also provides a general strategy to prepare other acrylonitrile-based fluorescent materials with diverse functions and desired properties for biomedical imaging and other applications like luminescent devices and organic fieldeffect transistors.

ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ jacs. Materials and Methods; 1H NMR, 13C NMR, 19F NMR and HRMS spectra of new compounds; Crystallographic data; Photophysical data and Imaging data (PDF) CCDC 1870015 (CIF) One-photon tissue imaging along the z-axis (Movie S1) Two-photon tissue imaging along the z-axis (Movie S2)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (B. Z. Tang)

ORCID Guangle Niu: 0000-0002-5403-6880 Haoke Zhang: 0000-0001-7309-2506 Jianguo Wang: 0000-0003-0984-9716 Xuewen He: 0000-0002-8414-5164 Yuncong Chen: 0000-0002-8406-4866 Xiujuan Shi: 0000-0003-3583-0584 Ryan T. K. Kwok: 0000-0002-6866-3877 Pengfei Wang: 0000-0002-8233-8798 Ben Zhong Tang: 0000-0002-0293-964X

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was partially supported by the National Natural Science Foundation of China (21788102), the International


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Partnership Program of Chinese Academy of Sciences (GJHZ1723), the Instrument Developing Project of the Chinese Academy of Sciences (YJKYYQ20170015), the University Grants Committee of Hong Kong (AoE/P-03/08 and AoE/P02/12), the Research Grants Council of Hong Kong (16305015, A-HKUST 605/16 and C6009-17G), the Innovation and Technology Commission (ITC-CNERC14SC01) and the Science and Technology Plan of Shenzhen (JCYJ20170818113851132, JCYJ20170818113840164, JCYJ20170818113538482, JCYJ20160229205601482 and JCYJ20180507183832744).

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