AIE Luminogens for Bioimaging and Theranostics: From Organelles to Animals

Review

AIE Luminogens for Bioimaging and Theranostics: from Organelles to Animals Jun Qian1,2 and Ben Zhong Tang2,3,*

High-quality fluorescence bioimaging requires organic exogenous contrast agents with biocompatibility, brightness, and photostability. The recently discovered and rapidly developed aggregation-induced emission luminogens (AIEgens) are excellent candidates because they exhibit enhanced emission upon the restriction of intramolecular motions (RIM) effect. AIEgens are individually lit up once bound with biomolecules or influenced by the surrounding bio-environment, and abundant biological information can be acquired on the basis of high-sensitivity fluorescence imaging. In addition, AIEgen-incorporated nanoparticles possess bright fluorescence and improved photostability, which is beneficial to long-term bioimaging with high contrast and spatial resolution. In this review, we summarize the latest advances in AIEgen-based fluorescence bioimaging, as well as the relevant applications in theranostics.

INTRODUCTION As a branch of bioimaging, fluorescence imaging with high sensitivity and spatial and temporal resolution is a very powerful tool for noninvasive, on-site, and realtime monitoring of biosamples of interest, revealing information on biological structures and processes, and it has attracted increasing attention in biological and (pre-) clinical studies.1,2 To facilitate high-quality fluorescence imaging, well-designed exogenous contrast agents are often used, including organic dyes,3 organic-dyedoped nanoparticles (NPs),4 and inorganic NPs (e.g., quantum dots, rare-earth ion-doped upconversion NPs, metallic NPs).5–7 In terms of biocompatibility and synthetic processes, organic dyes and organic-dye-doped NPs are better alternatives for fluorescence bioimaging. However, for most conventional organic dyes, fluorescence is quenched in aggregate state or at high concentration, which is known as ACQ or concentration quenching due to p-p stacking.8 This effect seriously limits their application in fluorescence imaging. Hydrophobic organic dyes are incompatible with water and naturally form aggregates in a hydrophilic bio-environment, resulting in a distinct self-quenching phenomenon. In addition, they can only be used in very diluted concentrations, and such small numbers of molecules are easily photobleached under excitation light. Furthermore, the ACQ effect also introduces problems in the design of dye-doped NPs because the brightness of NPs cannot be improved by simply increasing the doping concentration (aggregation degree) of dyes in each NP. The concept of aggregation-induced emission (AIE) was coined by our group in 2001.9 Distinct from ACQ fluorophores, AIE luminogens (AIEgens) are weakly or nonemissive when molecules are uniformly dispersed in solution, but are ‘‘lit up’’

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The Bigger Picture For mankind, good health is the basis of well-being; thus, frontier biomedical and (pre-)clinical studies always draw great attention from scientists. Aggregation-induced emission (AIE) is an opposite phenomenon to that documented in classic textbooks, called aggregationcaused quenching (ACQ). AIE luminogens (AIEgens) are weak or nonemissive molecules with free intramolecular motions, but they ‘‘light up’’ when they form aggregates and are excellent candidates as fluorescent bioprobes. In this review, we summarize the latest advances in AIEgen-based fluorescence bioimaging and theranostics. The working mechanisms of specific AIE light-up bioprobes and their biomedical applications are the key learning points. In the future, more exciting and practical ideas will be triggered to promote AIEgens for a wide range of biomedical and (pre-)clinical applications. For further information on AIE, readers are directed to our series of recently published review articles.

Figure 1. Aggregation-Induced Emission Effect (A) Fluorescence photographs of HPS in tetrahydrofuran/water mixtures with different water content upon UV illumination. (B) Confocal images of TPE-TPA-FN (TTF) NPs with different particle sizes with 543 nm continuouswave laser excitation. Scale bars, 100 mm. Adapted with permission from Wang et al. 10 Copyright 2014, Nature Publishing Group. 1State

when forming (nano)aggregates (Figure 1A). Restriction of intramolecular motion (RIM, including restriction of intramolecular rotations and restriction of intramolecular vibrations),11–14 which promotes radiative decay, has been rationalized as the cause of AIE. The nonplanar conformations of AIEgens also effectively prevent p-p stacking. AIEgens are excellent candidates as exogenous contrast agents for fluorescence imaging: (1) once bound with biomolecules or influenced by the surrounding bio-environment, intramolecular motion in ‘‘free’’ AIE molecules is restricted; the fluorescence from the ‘‘lit up’’ AIE molecules can therefore trace biomolecules and certain biological status,15 (2) AIEgens are ‘‘turned on’’ when spontaneously aggregated in a hydrophilic bio-environment or artificially synthesized to fluorescent NPs (Figure 1B).16,17 The brightness of nanoaggregates or NPs can be easily enhanced by increasing the amount of AIE molecules inside, which is helpful for improving the contrast and spatial resolution of imaging. In addition, they are

Key Laboratory of Modern Optical Instrumentations, Centre for Optical and Electromagnetic Research, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310058, China

2Department

of Chemistry, Institute of Molecular Functional Materials, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China

3Guangdong

Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chempr.2017.05.010

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Figure 2. Typical Examples of AIEgen-Based Bioimaging and Theranostics Adapted with permission from Shi et al. 18 (copyright 2012, American Chemical Society), Zhu et al. 19 (copyright 2013, Springer), Zhang et al. 20 (copyright 2016, American Chemical Society), Yuan et al. 21 (copyright 2015, John Wiley), Zhao et al. 22 (copyright 2013, John Wiley), Wang et al. 23 (copyright 2014, Royal Society of Chemistry), Chen et al. 24 (copyright 2013, American Chemical Society), Zhao et al. 25 (copyright 2015, Royal Society of Chemistry), Shi et al. 26 (copyright 2012, American Chemical Society), Zhao et al. 27 (copyright 2013, Royal Society of Chemistry), Zhang et al. 28 (copyright 2014, American Chemical Society), Zhao et al.29 (copyright 2016, Royal Society of Chemistry), Qin et al.30 (copyright 2011, John Wiley), Wang et al.31 (copyright 2015, Optical Society of America), and Li et al. 32 (copyright 2016, Springer).

very resistant to photobleaching under laser excitation, facilitating long-term tracing of dynamic biological processes.10 Hexaphenylsilole (HPS) was reported as the first AIEgen by our group.9 Since then, a substantial number of AIEgens (e.g., tetraphenylethane [TPE], tetraphenylsilole [TPS], and distyrylanthracene [DSA] derivatives) with high luminescence quantum yield have been developed, with emission colors covering the visible range,18,33 even extending to the near-infrared (NIR) range.34 Sufficient species and unique optical properties increase the availability of AIEgens as exogenous contrast agents and promote their wide applications in fluorescence bioimaging. In this review, we focus on the latest advances in AIEgen-based bioimaging, as well as the relevant applications in theranostics, and the biosamples of interest range from organelles to living animals. The review is arranged in three sections: cellular imaging, cancer cell therapy, and in vivo imaging and theranostics. Some typical examples (Figure 2), such as nucleus targeting,19 photodynamic therapy,20 gene delivery,21 bacteria detection and killing,22,35 and monitoring of in vivo inflammation,36 are highlighted. Finally, we give our perspectives regarding the challenges and future opportunities for AIEgens in potential clinical diagnostic and disease therapeutic applications.

CELLULAR IMAGING The cell is the basic structural and functional unit of the organism, and studies on cells are critical. Fluorescence microscopy is an important tool for high-contrast

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imaging of transparent cell samples. As promising contrast agents, AIEgens with ‘‘turned on’’ properties and negligible cytotoxicity have been successfully utilized for organelle targeting,19,23,37–44 intracellular environment and macromolecule mapping,24,25,45–48 and cell process tracing.26,49–54 Organelle Targeting Mitochondria Mitochondria, whose major function is to generate energy in the cell, exist in almost all eukaryotic cells. They play a vital role in the life and death of cells. Tracking the morphology, functions, and dynamics of mitochondria has attracted much research interest. However, almost all commercially available mitochondria trackers are ACQ dyes with poor photostability, which cannot be improved by increasing the dye concentration. To overcome this limitation, a series of AIEgens have been designed as photostable and specific fluorescent probes for mitochondrial targeting and functional imaging.27,37–41 Mitochondria are one of the major targets in autophagy, and mitophagy is a process of selective autophagic mitochondrial degradation. As shown in Figure 3A, a yellow emission AIEgen named TPE-Py-NCS has been synthesized, and it can target mitochondria specificity in live cells with superior photostability.39 In addition, the covalent conjugation of the isothiocyanate moiety of TPE-Py-NCS to mitochondrial proteins endows the AIEgen with high resistance to microenvironmental changes. Combining the above unique advantages, TPE-Py-NCS has been successfully utilized for real-time monitoring of mitophagy. Mitochondrial membrane potential (DJm) is a vital parameter reflecting the mitochondrial functional status, and is thus closely related to cell health, injury, and function. As shown in Figure 3B, a positively charged AIEgen named TPE-Ph-In has been synthesized and utilized to specifically target the mitochondria in living cells.40 TPEPh-In emits red fluorescence and exhibits high photobleaching resistance under long-term laser excitation. More importantly, when oligomycin is used as a stimulant to add DJm in TPE-Ph-In-treated cells, red fluorescence is significantly enhanced. When carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is used to reduce DJm, a decrease in fluorescence is observed as a result of less accumulation of TPE-PhIn in the mitochondria. TPE-Ph-In holds great potential for probing and tracing the change in intracellular DJm, which is difficult to achieve for traditional dyes with the ACQ effect. Traditional fluorescence microscopy, as well as organic dyes, is not suitable for monitoring the morphology and dynamics of mitochondria with higher spatial resolution (e.g., nanoscale level). A class of mitochondria-specific AIEgens called o-TPE-ON+ has been developed (Figure 3C). It is almost non-emissive in aqueous solution as a result of active intramolecular rotations and the twisted intramolecular charge-transfer effect.41 Upon UV light irradiation, o-TPE-ON+ is photoactivated to a fluorophore c-TPE-ON+ with high emission efficiency, and c-TPE-ON+ can be excited to blink spontaneously and further photobleached by a strong 561 nm laser beam. Together, these features make the probe well suited for stochastic optical reconstruction microscopic imaging (STORM,55 a typical class of super-resolution imaging method) of mitochondria in living cells. Nanoscale structures of the mitochondria are visualized, and the spatial resolution is much higher than that of total internal reflection fluorescence (TIRF) microscopy. The fission and fusion behaviors of mitochondria are also clearly identified.

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Figure 3. AIEgens for Functional Imaging of Mitochondria (A) TPE-Py-NCS for mitochondria targeting and real-time monitoring of the mitophagy process. The appearance of the red fluorescent spot (white arrows) implies the initiation of the mitophagy process, as acidic autophagosome (red) forms and then moves to mitochondria (yellow). The disappearance of the red fluorescent spot suggests that the mitophagy process has completed. Scale bars, 2 mm. Reproduced with permission from Zhang et al. 39 Copyright 2015, Royal Society of Chemistry. (B) TPE-Ph-In for sensing mitochondrial membrane potential. Scale bar, 20 mm. Reproduced with permission from Zhao et al. 40 Copyright 2015, Royal Society of Chemistry. (C) Photoactivatable o-TPE-ON+ for super-resolution nanoscopic imaging of mitochondria. Transverse profiles of the single mitochondrion along the yellow dashed line are marked in the TIRF (left) and STORM (right) images. Fission (green arrowheads) and fusion (red arrowheads) events are captured by a time series of 2.5 s. Scale bars, 500 nm. Reproduced with permission from Gu et al. 41 Copyright 2016, John Wiley.

Nucleus The nucleus, the biggest and most important organelle in eukaryotic cells, is the control center of cell genetics and metabolism. Although some commercially available dyes (e.g., Hoechst, 40 ,6-diamidino-2-phenylindole [DAPI]) have been widely utilized for specific nucleus targeting, reports regarding photostable AIEgens with nucleus staining capability are very rare.19,42,56 As shown in Figure 4A, AIE-featured TPE-TPA-FN (TTF) molecules are encapsulated with organically modified silica (ORMOSIL) NPs.19 Imaged with a confocal microscope, the fluorescent NPs are observed to stain the cytoplasm of HeLa cells effectively.

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Figure 4. AIEgens for Nucleus Staining (A) TTF-doped silica NPs for nucleus staining. Reproduced with permission from Zhu et al. 19 Copyright 2013, Springer. (B) ASCP for simultaneous labeling and dual-color imaging of mitochondria and nucleolus in live cells. Reproduced with permission from Yu et al. 56 Copyright 2016, Royal Society of Chemistry.

Surprisingly, many red fluorescent spots are found in the cell nucleus (co-stained with DAPI, emits blue fluorescence), illustrating that TTF NPs can also enter the cell nucleus and stain it. The results can be attributed to the high penetrating capacity of TTF NPs into the nucleus membrane. The nanoplatform can be used for delivery of biomolecules or drugs to the cell nucleus, performing further biological functions. In addition to doping into NPs, it is anticipated that AIE molecules will allow imaging of the nucleus directly. In addition, it will be exciting if a single class of AIEgens can target two or more organelles simultaneously. ASCP, an a-cyanostilbene derivative with the AIE feature, is utilized as a dual-color organelle-specific probe with superior photostability for simultaneous targeting of the mitochondria and nucleolus (Figure 4B).56 Under a fluorescence microscope, distinct emission colors from the mitochondria (orange) and nucleolus (red) are observed, as a result of ASCP’s different interactions with the mitochondrial membrane and nucleic acids.

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Figure 5. An AIEgen for Functional Imaging of Lysosomes (A) AIE-LysoY for lysosome labeling. (B) AIE-LysoY for long-term autophagy visualization. Scale bars, 30 mm (first five images) and 10 mm (last image). Reproduced with permission from Leung et al. 44 Copyright 2015, John Wiley.

Lysosome The lysosome is another important organelle in eukaryotic cells. Lysosomes are known as the waste-disposal sites of cells, because they are the leading players in autophagy. Thus, a photostable and targeting-specific probe facilitates not only visualization of lysosomes but also the long-term tracing of lysosome-involved autophagy.43 A class of AIEgens, namely AIE-LysoY, has been developed.44 Guided by its morpholine functionality, AIE-LysoY specifically accumulates and forms nanoaggregates in the lysosomes of cells, which activates RIM and excited-state intramolecular proton-transfer effects, and further lights up the lysosomes (Figure 5A). Cells stained with AIE-LysoY are then treated with rapamycin for induction of autophagy (Figure 5B). Lysosomes increase in amount and fuse with autophagosomes to form autolysosomes during the autophagy process. Thus, the amount of yellow spots, which correspond to the AIE-LysoY-targeted lysosomes, increases with prolonged rapamycin treatment. Thanks to the photobleaching resistance of AIE-LysoY, the 60 min autophagy process is completely recorded with negligible loss of fluorescence signal. Even when the excess AIE-LysoY is washed away before rapamycin treatment, the newly formed lysosomes are lit up, which strengthens the occurrence of fusion between the autophagic compartment and primitive lysosomes during autophagy. AIE-LysoY can serve as a promising lysosome-selective bioprobe for the study of autophagy. Cellular Mapping and Tracing Intracellular pH and Viscosity Monitoring of intracellular environments can also be realized with AIEgens. Intracellular pH (pHi) plays a critical role in regulating many cellular behaviors, and its normal range is usually from 4.7 to 8.0 in a typical mammalian cell. Mutation in pHi can lead to dysfunction of the organelles, and abnormal pHi can lead to many well-known diseases. As shown in Figure 6A, AIEgen TPE-Cy is pH sensitive, and its fluorescence changes from red to blue with increasing pH.45 Once TPE-Cy diffuses into cells, the color transition point of its red-to-blue emission is shifted from extracellular pH 10 to intracellular pH 6.5, making its dynamic range for pHi sensing cover the whole pHi window (4.7–8.0). Considering TPE-Cy also possesses excellent biocompatibility, its ratiometric fluorescence signal in blue and red channels (I489/ I615, the lower the ratio, the more acidic) is further utilized as an indicator for pHi

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Figure 6. AIEgen for Mapping of Intracellular pH and Viscosity (A) TPE-Cy for intracellular pH sensing based on the ratiometric fluorescence signal in red and blue channels. Reproduced with permission from Chen et al. 24 (copyright 2013, American Chemical Society) and Chen et al. 45 (copyright 2012, Royal Society of Chemistry). (B) TPE-Cy for intracellular viscosity detection by means of two-photon fluorescence lifetime microscopy. Scale bar, 30 mm. Reproduced with permission from Chen et al. 46 (copyright 2015, John Wiley) and Chen et al. 45 (copyright 2012, Royal Society of Chemistry).

imaging.24 Living cells stained by TPE-Cy are incubated with acetic acid (a cellpermeable weak acid) and observed with a confocal microscope. Upon treatment, the acidic area with pseudo red color (low I489/I615) expands greatly in the cells, which clearly demonstrates the acidification effect. On the contrary, when the cells are treated with DMEM (pH 8.5), regions with pseudo blue color (high I489/I615) dominate the intracellular area. Similar results can also be observed from flow cytometry. TPE-Cy holds great potential for high-resolution and high-throughput analysis of intracellular environments. In addition to intracellular pH imaging, TPE-Cy can be also used for mapping intracellular viscosity,46 which is a crucial parameter that controls signal transduction and the speed of biomass transportation in the living system. TPE-Cy was first utilized for extracellular viscosity sensing. With increasing viscosity of the solution, the intensity of TPE-Cy fluorescence is enhanced and its lifetime becomes longer. According to the AIE mechanism, the free rotation of the peripheral phenyl rings of the TPE in TPE-Cy serves as a nonradiative channel to decay excited species. In a viscous medium, the intramolecular motion of TPE-Cy is restricted, thus inhibiting nonradiative decay and populating the radiative decay species, which endows TPE-Cy with enhanced fluorescence intensity and longer lifetime. On the basis of fluorescence lifetime imaging microscopy (FLIM) with the two-photon excitation at 600 nm, TPE-Cy is also used for the detection of intracellular viscosity (Figure 6B). The lifetime of TPE-Cy is distributed over a broad range from 300 to 1,500 ps, in the cellular environment. The long lifetime (1 ns) mostly distributes in membrane-bound organelles such as mitochondria, because the ordered packing of the lipid bilayers of most organelles results in high viscosity. The short lifetime (500 ps) is observed in lipid droplets, because the loose packing of the lipid molecules in lipid droplets offers a low-viscosity environment. AIEgen-assisted FLIM is applicable in biological systems and drug screening, which are related to intracellular viscosity.

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Figure 7. AIEgens for Sensitive Detection of S-Phase DNA Synthesis (A) Scheme depicting DNA synthesis detection by AIEgens via the EdU assay. (B) Detection of S-phase DNA synthesis in HeLa cells with TPE-Py-N3 (top) and Cy-Py-N3 (bottom) under a fluorescence microscope. (C) Normalized fluorescence intensity of HeLa cells incubated at different concentrations of EdU/dye. (D) Change in fluorescence of dye-labeled HeLa cells with increasing irradiation time. Reproduced with permission from Zhao et al.25 Copyright 2015, Royal Society of Chemistry.

Intracellular Biomacromolecules Biomacromolecules, including proteins, nucleic acids, starch, and lipids, constitute the basic material of life. Because of their biological importance, scientists have put much effort into developing techniques for monitoring and better understanding biomacromolecules. With the use of fluorescence microscopy, AIEgens have been successfully employed as probes for the detection of intracellular DNA,25 microRNA,57 and cancer-related protein.48 Because of space limitations, here we just give one typical sample. 5-Ethynyl-20 -deoxyuridine (EdU) assay is an effective way for detecting S-phase DNA synthesis and cell proliferation. In this method, EdU with a terminal alkyne group is first incorporated into newly synthesized DNA. The click reaction of the alkyne unit with an azide group (–N3) in a fluorescent dye is then catalyzed by Cu(I) complex and generates dye-labeled double-stranded DNA (Figure 7A). AIEgens TPE-Py-N3 and Cy-Py-N3 are synthesized.25 Cells pre-labeled with EdU are treated with TPEPy-N3/Cy-Py-N3, and the fluorescence from their nuclei is discernible (Figure 7B). In contrast, no fluorescence is observed from the cells incubated with TPE-Py-N3/ Cy-Py-N3, but without EdU labeling. The results prove that the origin of the fluorescence in the nuclei indeed comes from the DNA synthesis, which occurs at S phase in cell proliferation. In addition, because of the features of AIE, TPE-Py-N3 and CyPy-N3 possess brighter emission, a much broader working concentration range (Figure 7C), and higher photostability than traditional ACQ dyes (e.g., Alexa 647-azide) in the EdU assay (Figure 7D). Coupled with their synthetic accessibility and low cost, they can serve as promising candidates for many high-throughput studies. Apoptosis Apoptosis is a mode of programmed cell death in multicellular organisms. Realtime imaging and monitoring of apoptosis in living organisms is important for early

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Figure 8. An AIEgen for Real-Time Apoptosis Imaging in Target Cancer Cells (A) The principle of apoptosis imaging in a target cancer cell based on Ac-DEVD-TPS-cRGD. (B) Confocal laser scanning microscopic images of cells treated with Ac-DEVD-TPS-cRGD. Scale bars, 30 mm. Reproduced with permission from Ding et al. 49 Copyright 2013, Royal Society of Chemistry.

diagnosis of diseases, monitoring cancer progress, and efficacy estimation of new anticancer agents. To achieve this, an asymmetric and hydrophilic bioprobe, by conjugation of Asp-Glu-Val-Asp (Ac-DEVD) and cyclic Arg-Gly-Asp (cRGD) peptides onto a typical AIEgen TPS, has been developed (Figure 8A).49 The probe shows specific targeting capability to integrin avb3 receptor overexpressing U87MG human glioblastoma cancer cells by virtue of the efficient binding between cRGD and integrin avb3 receptors. In contrast, almost no signal is detected in Ac-DEVD-TPS-cRGDtreated MCF-7 human breast cancer cells (with low integrin avb3 receptor expression, right column in Figure 8B). For probe-treated U87MG cells, the cell apoptosis induced by staurosporine (STS) can ‘‘turn on’’ the fluorescence inside the cells, giving high-contrast fluorescence imaging (left column in Figure 8B). The result is due to the cleavage of the DEVD moiety by caspase-3 (one of the key mediators of cell apoptosis). The hydrophobic TPS-cRGD residues released subsequently aggregate in the hydrophilic intracellular environment, which activates the RIM process of TPS and populates the radiative relaxation channels. In contrast, the fluorescent signals are not ‘‘lit up’’ when STS-induced U87MG cells are pretreated with the specific caspase inhibitor, before probe incubation (middle column in Figure 8B). Ac-DEVDTPS-cRGD has both cancer-targeting and apoptosis-specific imaging capabilities

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and provides guidance for the molecular design of more apoptosis imaging agents based on AIEgens. Long-Term Tracing Long-term noninvasive fluorescence cell tracing is critical to the life sciences and biomedical engineering; e.g., understanding the genesis, development, invasion, and metastasis of cancerous cells and monitoring tissue regeneration after stem cell transplantation and therapy. GFPs, quantum dots, and organic NPs have been adopted as fluorescent probes for long-term cell tracing. However, they each have some drawbacks, such as susceptibility to proteolytic enzymes, poor photostability, cytotoxicity, short cellular retention time, and the ACQ effect. AIE NPs possess high biocompatibility, bright fluorescence, super photostability, and excellent cellular retention, and they have been successfully utilized as promising probes for long-term noninvasive and fluorescent tracing of cells,51–54 which opens new opportunities for oncology studies and cell-based therapy. Here, we take one typical example.53 As shown in Figure 9A, a mixture of lipidpoly(ethylene glycol) (lipid-PEG) and lipid-PEG-maleimide is utilized as the encapsulation matrix to endow AIEgen BTPEBT into NPs with surface functionality. A cell-penetrating peptide (Tat) is further conjugated to the surface of NPs, rendering BTPEBT-Tat NPs with high cellular internalization efficiency. BTPEBT-Tat NPs show successful internalization into six different human cell lines tested (including stem cells), all with 100% labeling efficiency, superior to the commonly used GFP transfection method where only HEK293T cells show a high GFP-labeling rate of 70%. The cell-tracing experiments further demonstrate that the internalized BTPEBT-Tat NPs can be efficiently transferred to daughter HEK293T cells during cell proliferation (Figure 9B, top). Within the first 5 days, the cell-labeling rate is almost 100%. Even up to 10 days, the cell-labeling rate maintains at 20%, and clear fluorescence signals from untreated cells are still observable. BTPEBT-Tat NP-based long-time fluorescent cell tracing outperforms the GFP plasmid transfection method (Figure 9B, bottom).

CANCER CELL THERAPY In addition to functional cellular imaging, AIEgens can also be incorporated into drug delivery systems as promising candidates for imaging-guided therapy of cancer cells; they can provide real-time and precise information about the location and function of drugs inside cells. In addition, some specially designed AIEgens simultaneously possess chemo- and photon-induced cytotoxicity and fluorescence properties, and it is beneficial to realize imaging-guided therapy in a facile single system. So far, chemotherapy,28,29,58–62 photodynamic therapy,20,63–69 gene therapy,21,70 and multimodal synergistic therapy toward cancer cells,71–73 which are based on AIEgen-assisted fluorescence imaging, have been carried out, and some exciting results have been accomplished. Chemotherapy Chemotherapy is still one of the most important clinical treatment methods for cancer.74 However, it is often difficult to eradicate all cancer cells with a single chemo-drug, because of drug resistance.75 To overcome this problem, non-crossresistant chemo-drugs have been widely utilized for efficient cancer therapy. Some studies have reported that the co-administration of cisplatin (Pt(II)) and doxorubicin (DOX), two of the most effective anticancer drugs used in clinics, shows a synergistic anticancer effect.76 To further realize fluorescence imaging-guided synergistic chemotherapy, a targeted theranostic delivery system has been constructed

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Figure 9. AIE NPs for Long-Term Cell Tracking (A) Schematic illustration of BTPEBT-Tat NP formation. (B) Confocal laser scanning microscopic images of HEK293T cells labeled by BTPEBT-Tat NPs (top) or pMAX-GFP (bottom) at different days after incubation. Reproduced with permission from Feng et al. 53 Copyright 2014, Elsevier.

composed of a targeted cRGD moiety, a TPE derivative with AIE features, an anticancer drug DOX with fluorescence, and a chemotherapeutic Pt(IV) prodrug as the linker (Figure 10A).60 In the initial TPE-DOX pair, because the fluorescence of TPE is quenched as a result of energy transfer to DOX before drug activation, the resultant red fluorescence of DOX can be utilized for prodrug tracking. The whole theranostic system can specifically target avb3 integrin overexpressed MDA-MB-231 cells through receptor-mediated endocytosis. Upon cellular internalization, the Pt(IV) prodrug is reduced to the active Pt (II) drug. Accompanied by drug activation, TPE and DOX molecules are separated and the blue fluorescence of TPE is recovered, which can be used for drug activation reporting. Furthermore, the subcellular behavior of DOX in MDA-MB-231 cells can also be monitored with its red fluorescence under 488 nm excitation. Moreover, the simultaneous activation of DOX and cisplatin shows superb synergistic anticancer effects. The theranostic system capable of

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Figure 10. AIEgens for Imaging-Guided Chemotherapy (A) Schematic illustration of the targeted theranostic dual-acting prodrug for real-time drug tracking and activation monitoring. Reproduced with permission from Yuan et al. 60 Copyright 2014, Royal Society of Chemistry. (B) Molecular structures of TMX, TPE, and TPE-TMX. (C–F) Bright-field (C) and fluorescent (D and E) images of MCF-7 breast cancer cells stained with LysoTracker Red DND-99 (50 nM, E) for 15 min after being treated with TPE-TMX (2 mM, D). (F) The merged image of (D) and (E). Excitation wavelength: (D) 330–385 nm and (E) 520–560 nm. (G) Left: viability of MCF-7 cells incubated with different concentrations of TPE-TMX and TMX. Right: viability of different cells in the presence of TPETMX with different concentrations. (H) Bright-field (top) and fluorescent (bottom) images of MCF-7 cells taken at different times after being treated with TPE-TMX (2 mM). Excitation wavelength: 330–385 nm. (B–H) Reproduced with permission from Zhao et al. 29 Copyright 2016, Royal Society of Chemistry.

prodrug tracking and real-time monitoring of drug activation can minimize side effects, enhance therapeutic efficiency, and is beneficial for cancer therapy. As a selective modulator of estrogen receptor (ER), tamoxifen (TMX; Figure 10B) is usually adopted as the chemotherapeutic agent for the treatment of 70% patients with ER+ breast cancer. TPE is a typical AIEgen, and it is found to have an analogous structure to TMX (Figure 10B). TMX has been modified by replacing its ethyl group with a phenyl group, and the capability of the resulting fluorogen (TPE-TMX; Figure 10B) for simultaneous imaging and treatment of breast cancer has been explored.29 Like other TPE derivatives, TPE-TMX exhibits AIE characteristics. Unsurprisingly, TPE-TMX can also stain ER+ MCF-7 breast cancer cells, and further experiments have illustrated that it selectively stains the autolysosomes in live MCF-7 cells (Figures 10C–10F). The cytotoxicity of TPE-TMX to different cell lines has also been studied. As shown in Figure 10G, TPE-TMX shows only a therapeutic response toward ER + MCF-7 cells with evident dose-dependent efficacy, and the response is similar to TMX. In contrast, TPE-TMX has no therapeutic response to other cell lines, including ER– MDA-MB-231 breast cancer cells, even when the dosage of TPE-TMX is very high. After treatment with TPE-TMX, swelling of lysosomes and population loss are observed in MCF-7 cells as time elapses. Almost the whole cells are emissive (except the nuclei) 192 hr after the staining process (Figure 10H). The results illustrate that the therapeutic effect of TPE-TMX on breast cancer cells might be caused by the

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Figure 11. AIE NPs for Delivery of Chemo-Agents to Drug-Resistant Cancer Cells (A) Molecular structure of TK-bridged TPETP-TK-PEG polymer. (B) Schematic illustration of light-responsive AIE NPs self-assembled from TPETP-TK-PEG polymer in aqueous media with the encapsulation of DOX and visible-light-triggered drug release (top). The itinerary of AIE NPs/DOX to overcome drug resistance in cancer cells (bottom). (C) Viability of MDA-MB-231/DOX cells after treatment with free DOX, AIE NPs/DOX with and without light irradiation (2 min, 0.1 W cm 2 ) for 72 hr. Reproduced with permission from Yuan et al. 62 Copyright 2016, Royal Society of Chemistry.

induction of cell autophagy by TPE-TMX. Thus, the present work is anticipated to open a new avenue for the development of drugs with theranostic function. Drug resistance in cancer cells is a major obstacle for successful cancer chemotherapy. To overcome DOX resistance in breast cancer cells, AIEgen-based new light-responsive NPs with cytosolic drug release upon light irradiation have been developed.62 In this nano-system, an amphiphilic polymer (TPETP-TK-PEG), composed of a hydrophobic photosensitizer (PS) named TPETP with AIE features and a hydrophilic PEG chain, is the core component. TPETP and PEG are conjugated via a reactive oxygen species (ROS) cleavable thioketal (TK) linker (Figure 11A). As shown in Figure 11B, the TPETP-TK-PEG polymer can self-assemble into AIE NPs and encapsulate DOX molecules inside with high loading capacity in aqueous media. With the help of AIE NPs, DOX can be effectively delivered into a DOX-resistant breast cancer cell line MDA-MB-231 (MDA-MB-231/DOX). Guided by the fluorescence signals from TPETP, AIE NPs/DOX mainly locates in endosomes and lysosomes after endocytosis by the cancer cells. Upon white light irradiation, the ROS generated from TPETP not only induces endo-lysosomal membrane rupture but also breaks the AIE NPs. This facilitates endo-lysosomal escape and triggers cytosol release of DOX, which can significantly improve intracellular DOX accumulation and retention in MDA-MB-231/DOX cells and significantly inhibit the growth of the cells. In contrast, free DOX and AIE NPs/DOX without light irradiation produce less cytotoxicity toward MDA-MB-231/DOX cells than AIE NPs/DOX with light irradiation (Figure 11C). AIEgen-based NPs offer a potentially effective way to delivery certain chemo-agents to drug-resistant cancer cells. Photodynamic Therapy As a typical light-mediated therapy strategy, photodynamic therapy (PDT) uses a photosensitizer (PS) to transfer photon energy to neighboring oxygen molecules,

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producing ROS, mainly singlet oxygen, 1O to kill cancer cells. Thus, three key components are essential in most PDT systems: a PS, a light source, and tissue oxygen.1 Because most PSs are fluorophores, fluorescence imaging-guided PDT is straightforward but attractive to improve the accuracy of treatment.77 However, PS fluorophores are hydrophobic and they also suffer from ACQ, which leads to evident reduction in the efficacy of ROS generation and intensity of fluorescence when they aggregate in aqueous media. Thus, the development of efficient AIE-active PSs with both ROS generation capacity and fluorescence even in the (nano)aggregation state is very promising. So far, several related exciting studies have been carried out,20,63–69 and here we give two examples. An AIEgen-based probe, namely TPETP-SS-DEVD-TPS-cRGD, has been developed (Figure 12A) for specific cancer cell targeting, activatable and traceable cell PDT, and self-reporting of therapeutic responses upon a single-wavelength excitation.67 TPETP is a red-emissive PS with AIE characteristics whereas TPS is a green-emissive AIEgen, and both can be excited with a 405 nm laser. The probe is initially nonemissive. After selective uptake by avb3 integrin overexpressed MDA-MB-231 cells through c-RGD-mediated endocytosis, the –S–S– bond in the probe is cleaved by intracellular glutathione (GSH), leading to release of the apoptosis sensor (DEVD-TPS-cRGD) and activated PS (TPETP). Upon 405 nm laser excitation, red fluorescence is emitted from TPETP, reporting its activation. Meanwhile, ROS generated by TPETP induces cell apoptosis and activates caspase-3/-7, which further cleaves the DEVD substrate on the apoptosis sensor (DEVD-TPS-cRGD) and releases the hydrophobic TPS residue. Consequently, the TPS nanoaggregates formed in the hydrophilic intracellular environment emit intense green fluorescence to report the therapeutic effect under the same 405 nm excitation. The AIEgen-based probe with theranostic function shows great potential in imagingguided PDT. AIEgen-based PSs can also be designed as NPs for targeted and imaging-guided PDT.63,65 TPE-TPA-DCM (TTD, as both AIEgen and PS) molecules are encapsulated with DSPE-PEG-Mal to form AIE NPs, and cRGD tripeptides are further reacted with Mal groups and functionalize the NPs (Figure 12B).63 TTD NPs are specifically taken up by MDA-MB-231 cells via integrin avb3 receptor-mediated endocytosis and label the cellular cytoplasm, with the guidance of bright red fluorescence from TTD. TTD NPs can efficiently generate ROS upon light irradiation and display evident cytotoxicity to MDA-MB-231 cells with a half-maximal inhibitory concentration (IC50) of 1.1 mg mL1. In contrast, TTD NPs show very little cytotoxicity to MCF-7 and MIH 3T3 cells with low avb3 integrin expression. This PDT nanoplatform is facile and flexible in terms of design and preparation, providing a new idea for image-guided PDT. Gene Therapy Gene therapy has emerged as a valuable medicinal strategy for cancer treatment.78 In this approach, negatively charged genetic materials (e.g., DNA or RNA) are used as molecular medicine and delivered to cancer cells to either inhibit some undesirable gene expression or express therapeutic proteins. Viral vectors are commonly used as carriers for gene delivery cause of their high transfection efficiency.78 However, some studies have illustrated that viral vectors can induce harmful immune responses,79 and it is therefore necessary to develop some positively charged NPs as nonviral vectors for gene delivery. Previously, NPs doped with organic dyes (with ACQ features) have been utilized for fluorescence-imaging-guided gene delivery.80 However, the design and synthesis of NPs require careful control, because the

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Figure 12. AIEgens for Imaging-Guided PDT (A) Schematic illustration of the TPETP-SS-DEVD-TPS-cRGD probe for real-time and in situ monitoring of PS activation and the therapeutic responses. Reproduced with permission from Yuan et al. 67 Copyright 2015, John Wiley. (B) Schematic illustration of TTD NPs and surface modification with the target moiety of cRGD (top). Inhibition of growth of MDA-MB-231, MCF-7, and NIH 3T3 cells in the presence of different concentrations of TTD NPs with light irradiation (0.25 W cm 2 , 2 min) followed by further incubation of the cells for 24 hr (bottom). Data represent mean values G standard deviation (n = 3). Reproduced with permission from Yuan et al. 63 Copyright 2014, Royal Society of Chemistry.

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Figure 13. AIE NPs for Imaging-Guided Gene Therapy (A) Schematic illustration of the construction of TTD NPs. (B) Schematic illustration of the construction of siRNA-TTD NPs and fluorescence images of MiaPaCa-2 cells treated with siRNA-TTD NPs. K-ras mRNA relative expression levels were detected by quantitative real-time PCR in MiaPaCa-2 cells treated with two types of siRNA-TTD NPs. A non-treated control is shown for comparison. According to the quantitative real-time PCR results, the two types of siRNA-TTD NPs show effective knockdown of the mutant K-ras gene with efficiencies of 83.52% G 2.45% and 74.96% G 1.41%, respectively. (A and B) Reproduced with permission from Hu et al. 70 Copyright 2014, Springer. (C) ROS-responsive polymer P(TPECM-AA-OEI)-g-mPEG for effective light-controlled transgene expression. Reproduced with permission from Yuan et al. 21 Copyright 2015, John Wiley.

overall brightness of NPs cannot be enhanced by simply increasing the loading concentration of the dyes inside each NP. Hu et al.70 first adopted AIE NPs for imaging-guided gene therapy of cancer cells. In their work, AIEgen TTD was encapsulated with DSPE-PEG and tri-block copolymer Pluronic F127, respectively, and the two types of TTD NPs were further coated with poly(allylamine) hydrochloride (PAH), which is a type of positively charged polymer. After conjugation with negatively charged siRNA molecules through electrostatic adsorption, both the siRNA-TTD NPs were used for the transfection of pancreatic cancer cells MiaPaCa-2 (Figure 13A). The successful delivery of the NPs to the MiaPaCa-2 cells was confirmed with fluorescence imaging by co-location of the fluorescence from the TTD (red)- and the FAM (green)-labeled siRNA molecules (Figure 13B), as well as flow cytometry. Furthermore, real-time PCR experiments illustrate that both types of siRNA-TTD NPs are capable of suppressing the gene expression of K-ras (reported to have the highest alteration frequency in pancreatic cancers), with efficiency as high as 70% (Figure 13B). Endosomal and lysosomal escape of gene vectors and the subsequent unpacking of nucleic acids in the cytosol are usually considered as two barriers for efficient gene delivery.81 To overcome them, Yuan et al.21 utilized a photoactivatable AIE polymer for effective light-controlled gene delivery. As shown in Figure 13C, the polymer consists of PSs with AIE characteristics (TPECM) and oligoethylenimine (OEI), which are conjugated via ROS cleavable linkers (aminoacrylate [AA]). A hydrophilic PEG chain is linked with each OEI unit, and the functional polymer can therefore self-assemble into bright red fluorescent NPs in aqueous media. The NPs are further efficiently bound to DNA on the basis of the electrostatic interaction between DNA

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Figure 14. AIEgens for Imaging-Guided multimodal Synergistic Therapy (A) TPECM-2TPP for chemotherapy alone (top) and synergistic chemotherapy and PDT (bottom). Reproduced with permission from Zhang et al. 71 Copyright 2015, Royal Society of Chemistry. (B) Illustration of the formation of cRGD-siVEGF-TTD NPs and the mechanism for synergistic gene therapy and PDT. (C) Left: the relative VEGF protein level in culture medium of cRGD-siVEGF-TTD NPs (5 mg mL 1 of TTD)-treated MDA-MB-231 cells and the VEGF mRNA level from the lysate of MDA-MB-231 cells. Controls were set at 100%. Right: viability of MDA-MB-231, MCF-7, and SK-BR-3 cells after incubation with cRGD-siVEGF-TTD NPs (5 mg mL 1 of TTD) for 4 hr followed by light irradiation (0.20 W cm 2 , 10 min) and further incubation in fresh medium for 24 and 48 hr. Data represent mean values G SD, n = 3. (B and C) Reproduced with permission from Jin et al. 72 Copyright 2016, Royal Society of Chemistry.

molecules and OEI units. When incubated with cells, the complex NPs are endocytosed by them and then entrapped in the endosomes and lysosomes. Upon visible light irradiation, ROS is generated from TPECM, which can break both the endosomal and lysosomal membrane and the polymer. This results in light-activated endosomal and lysosomal escape of NPs, as well as unpacking of DNA for gene delivery. The ROS-responsive polymer shows a significant increase in transfection efficiency in comparison with the commercial PEI25k, and the same design strategy can also be applied for general cytosolic drug delivery. Multimodal Synergistic Therapy In recent years, the development of multimodal synergistic therapy has attracted considerable attention because of its enhanced therapeutic efficiency over any single therapy modality.82 AIEgen is promising to play an important role in imagingguided multimodal synergistic therapy, and here we provide some typical examples of application.71–73 A type of AIEgen named TPECM-2TPP, which can target the mitochondria of cells, has been synthesized.71 The cytotoxicity of TPECM-2TPP on cancer cells was tested by staining with propidium iodide (PI), which is a well-known cell-impermeable dye that only stains dead cells or late apoptotic cells with a damaged membrane. As shown in Figure 14A, part of the TPECM-2TPP-treated cells incubated in the dark has been stained by PI, illustrating the good chemotherapy capability of

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TPECM-2TPP. Furthermore, nearly all the TPECM-2TPP-treated cells were stained by PI after they were exposed to white-light irradiation (8 min, 0.10 W cm2). In the control experiment, the cells were treated with both TPECM-2TPP and a singlet oxygen scavenger (vitamin C), and very few cells were stained by PI even after whitelight irradiation (not shown). This indicates that TPECM-2TPP can efficiently generate ROS with distinct phototoxicity under light illumination, which further enhances its anticancer effect. Collectively, the combination of chemotherapy and PDT provides a better anticancer effect with minimized side effects. PDT is an attractive approach for cancer treatment. However, cancer cells might respond to ROS stress by upregulating the level of vascular endothelial growth factor (VEGF) during PDT treatment, which suppresses cellular senescence by inducing angiogenesis and further attenuates the therapeutic effect.83 To overcome this obstacle, small interfering RNA-VEGF (siVEGF) has been widely adopted to suppress VEGF expression and weaken the resistance process in PDT-treated tumors. Unfortunately, the delivery of short small interfering RNA (siRNA) remains a key challenge in gene therapy because of the poor stability of siRNA in a biological environment.84 TTD (as both an AIEgen and a PS) molecules are encapsulated with a mixture of DSPE-PEG-Mal and DSPE-PEG-siVEGF (synthesized via the conjugation of DSPEPEG-NH2 with modified siVEGF).72 Cyclic arginine-glycine-aspartic acid (cRGD) peptides are further conjugated with the Mal-group decorated TTD NPs, facilitating the targeting ability of NPs to cancer cells with a high expression level of avb3 integrin (Figure 14B). As a glutathione (GSH)-cleavable disulfide bond exists in DSPEPEG-siVEGF, siVEGF can be released from the NP surface by the increased level of GSH in the cell cytoplasm. cRGD-siVEGF-TTD NPs can effectively downregulate the expression levels of VEGF protein (by 50%) and VEGF mRNA (by 64%) in comparison with control cells (Figure 14C, left). Cell viability studies further show that cRGDsiVEGF-TTD NPs can selectively and efficiently kill avb3 integrin overexpressed MDA-MB-231 cells upon light irradiation (Figure 14C, right). The AIEgen-based all-in-one nanoplatform successfully realizes imaging-guided and gene-interference-assisted PDT of cancer cells. In addition to PDT, photothermal therapy (PTT) is another typical method of lightmediated therapy, which usually utilizes photothermal agents to convert light energy into hyperthermia, leading to the ablation of adjacent cancer cells.82 Very recently, Wang et al.73 fabricated a targeted theranostic nanoplatform based on AIEgen PhENH2 (as a fluorescent agent), paclitaxel (as a chemotherapy agent), and modified polypyrrole (as a photothermal agent), achieving effective fluorescence-imaging-guided cancer treatment with combined chemotherapy and PTT, both in vitro and in vivo.

IN VIVO IMAGING AND THERANOSTICS In addition to the applications in cell imaging and therapy, AIEgens also play an important role in real-time and in vivo imaging and the detection and therapy of microorganisms and animals, such as the detection and killing of bacteria and fungi,22,35,85,86 tumor targeting and treatment,30,87–90 and monitoring of apoptosis and inflammation.36,91 Combined with advanced microscopy, AIEgens act as high-performance contrast agents for deep-tissue 3D angiography of mouse brain.10,31,92,93 Fluorescent AIEgens can be further conjugated with MRI contrast agents, realizing multimodal biomedical functional imaging.94,95 We introduce these exciting studies in this section.

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Microorganism Imaging and Therapy Bacteria Studies on bacteria have attracted increasing attention from the areas of environmental monitoring, medical hygiene, food processing, and the pharmaceutical industry. In these applications, long-term tracking of bacterial viability is critical. However, most conventional dyes suffer from high toxicity and/or poor photostability, making them unsuitable for long-term viability studies. AIEgen TPE-2BA (Figure 15A) can effectively differentiate dead and living bacteria.22 Dead bacteria with compromised membrane provide access for cell-impermeable TPE-2BA to stain the DNA inside, endowing it with bright fluorescence because of the RIM effect (Figure 15A). In addition, TPE-2BA is highly photostable and has negligible toxicity toward bacteria; it can therefore serve as an excellent probe for a long-term bacterial viability assay. Another AIEgen (4) is amphiphilic and positively charged, and it can bind to a negatively charged bacterial cell envelop via electrostatic interaction.85 After lighting up because of its AIE characteristics, 4 can image both Gram-positive and -negative bacteria (Figure 15B). The unbound amphiphilic molecules of 4 remain weakly fluorescent, because they are mono-dispersed in medium and do not have a RIM effect. Thus, the background emission is extremely low even without the washing process, which simplifies the whole bacteria imaging process. In addition, as loss of bacteria during washing is avoided, the accuracy of bacterial quantification can be increased. In biosensing, zero offset and linearity are also significant. A solution of 4 alone (zero bacteria) shows very weak fluorescence, with only 15th of the emission intensity of a solution containing both 4 and Staphylococcus epidermidis (108 colony-forming units [CFU] mL1). Furthermore, the emission intensity of 4 is linearly dependent on bacteria concentration in the range of 5 3 106 to 2 3 108 CFU mL1. 4 is further utilized for facile and fast bacterial susceptibility evaluation and high-throughput antibiotics. Inspired by the success of AIEgens in PDT of cancer cells, another AIEgen, TPE-Bac, with a similar molecular structure to 4, has been utilized for imaging and lightenhanced killing of bacteria (Figure 15C).35 As a result of good aqueous dispersity and AIE features, positively charged TPE-Bac can image both Gram-positive and -negative bacteria without the washing process, greatly simplifying the imaging process. TPE-Bac can be used for bacteria elimination, as it produces certain dark toxicity toward bacteria. This could be attributed to the fact that the amphiphilic TPE-Bac with two long alkyl chains and positively charged amine groups intercalates into the bacteria membrane and destroys the membrane integrity. TPE-Bac can also serve as a PS for ROS generation, which poses additional toxicity to the bacteria. After irradiation with room light for 1 hr, less than 1% of both Gram-positive and -negative bacteria can survive. Fungus In addition to killing bacteria, AIEgen also shows antifungal activity.86 A group of imidazole derivatives with facile synthesis and wide color tunability have been designed. All the compounds show typical AIE characteristics because they are based on imidazole-cored molecular rotors. Moreover, they exhibit the capability for mitochondria-specific imaging. All these imidazole derivatives have also been utilized for antifungal testing. However, only DPI-In inhibits the growth of yeasts potently. As shown in Figure 16A, 5 3 106 M DPI-In and 10 3 106 M miconazole (a commercially available antifungal drug) exhibit a similar inhibition effect on proliferation of yeasts. The antifungal activity of DPI-In has also been evaluated by the disc diffusion method. A much larger inhibition zone around DPI-In is observed

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Figure 15. AIEgens for Bacteria Imaging and Tracking (A) TPE-2BA lights up dead bacteria. Reproduced with permission from Zhao et al.22 Copyright 2013, John Wiley. (B) Top: bright-field and fluorescence images of S. epidermidis incubated with 10 3 10 6 M 4 for 10 min. Excitation wavelength: 400–440 nm. Bottom left: fluorescence spectra of a MOPS/ethanol (8:2, v/v) mixture of 4 without and with 10 8 CFU mL 1 of S. epidermidis. Bottom right: change in fluorescence intensity of 4 with the concentration of S. epidermidis in a MOPS/ethanol (8:2, v/v) mixture. Excitation wavelength: 430 nm. Reproduced with permission from Zhao et al.85 Copyright 2015, John Wiley. (C) Top: TPE-Bac for imaging and light-enhanced killing of bacteria. Bottom left: bright-field and fluorescence images of S. epidermidis and E. coli incubated with 10 mM TPE-Bac for 10 min. Excitation wavelength: 460–490 nm. Bottom right: killing efficiency of TPE-Bac (10 mM) on E. coli and S. epidermidis in the absence and presence of room-light irradiation for different times. Reproduced with permission from Zhao et al.35 Copyright 2015, American Chemical Society.

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Figure 16. An AIEgen as the Antifungal Agent (A) Optical density change of yeast incubated with different antifungal agents with different incubation times. Inset: molecular structure of DPI-In. (B) Antifungal activity of different antifungal agents evaluated by the disc diffusion method. Reproduced with permission from Song et al. 86 Copyright 2015, John Wiley.

than around miconazole and another imidazole derivative (DPI-BP) under the same conditions (Figure 16B), illustrating that DPI-In is a more effective antifungal agent. Tumor Targeting and Therapy As noninvasive fluorescence imaging technology has opened new avenues for the development of in vivo tumor diagnosis and therapeutics, efficient, biocompatible, and stable exogenous optical agents are now required. The utilization of AIEgen for in vivo tumor targeting and imaging was first realized in 2011.87 AIEgen StCN was encapsulated with DSPE-PEG to form StCN NPs (<30 nm), which gave much brighter fluorescence than mono-StCN and were very stable in various bio-environments. StCN NPs with Mal groups have been fabricated by encapsulating StCN molecules with both DSPE-PEG-Mal and DSPE-PEG, and further bioconjugated with arginine-glycine-aspartic acid (RGD) peptides. On the basis of observations on in vivo fluorescence imaging, both StCN NPs and StCN-RGD NPs are able to target subcutaneously xenografted tumors in mice (Figure 17A). For StCN NPs, the targeting is passive and time consuming (48 hr), via the enhanced permeability and retention (EPR) effect. However, the targeting of StCN-RGD NPs is positive and relatively time saving (24 hr), because RGD peptides have high binding affinity to the avb3 integrin receptor, which is a type of cell-surface receptor overexpressing at the endothelium of growing blood vessels associated with tumor growth. In addition to amphiphilic DSPE-PEG, BSA can also serve as the polymer matrix to formulate AIE NPs for tumor targeting (Figure 17B),30 because albumin is biocompatible, nonantigenic, and clinically utilized. TTD NPs have been fabricated in this way and intravenously injected into H22-tumor-bearing mice. Bright TTD NPs with a uniform size of 100 nm can illuminate the tumor tissue selectively with high contrast, which is attributed to the passive tumor-targeting ability from the EPR effect. As a control, accumulation of bare TTD NPs (300 nm, without BSA coating) in the tumors was limited. AIEgens can be further utilized for in vivo fluorescence-imaging-guided PDT of tumors.90 Han et al.90 designed a polymer consisting of protoporphyrin IX (PpIX, a PS approved by the US Food and Drug Administration) and TPE (AIEgen) by using

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Figure 17. AIE NPs for In Vivo Tumor Targeting (A) Top: schematic illustration of the preparation of StCN NPs. Bottom: in vivo imaging of mice bearing subcutaneous tumor xenografts, injected with StCN NPs and StCN-RGD NPs. Adapted with permission from Wang et al. 87 Copyright 2011, Elsevier. (B) Top: schematic illustration of the fabrication of BSA-coated TTD NPs. Bottom: in vivo fluorescence imaging of an H22-tumor-bearing mouse after intravenous injection of BSA-coated TTD NPs. The tumor sites are marked with white circles. Reproduced with permission from Qin et al.30 Copyright 2011, John Wiley.

the PEGylated Pro-Leu-Gly-Val-Arg (PLGVR) peptide sequence as a linker (Figure 18A). In TPE-PLGVR2-(PEG8)2-K(PpIX) (TPPP), PpIX serves as a PS for ROS generation and provides fluorescence signals during in vivo fluorescence imaging. TPE is initially nonemissive in the polymer. The amphiphilic TPPP can self-assemble to NPs in PBS. After they were injected intravenously into tumor-bearing mice, TPPP NPs effectively accumulated in the tumor region via the EPR effect. The overexpressed MMP-2 in the tumor hydrolyzed the PLGVR sequence, leading to detachment of TPE and PEGylated PpIX. The red fluorescence from PpIX highlights the tumor tissue accurately. In addition, the two-photon fluorescence image shows bright blue fluorescence in the PpIX fluorescence-accumulated region, from the recovered fluorescence of detached TPE (Figure 18B). PDT was further performed under the guidance of dual fluorescence imaging. The growth of the tumor was significantly retarded once the mice received certain light irradiation, suggesting good in vivo PDT efficacy of TPPP (Figure 18C). This study will have great potential in practical treatment. Monitoring of In Vivo Apoptosis and Inflammation Detection of apoptosis in cells in vitro has been realized with AIEgens. However, in vivo imaging of apoptosis generally requires AIEgens with long-wavelength emission to minimize the autofluorescence and increase imaging depth. A bioprobe has been designed by conjugating an orange AIEgen named PyTPE with Asp-Glu-ValAsp (Ac-DEVD), as shown in Figure 19A.91 Hydrophilic Ac-DEVD-PyTPE disperses

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Figure 18. An AIEgen for In Vivo Imaging-Guided Tumor Therapy (A) Working principle of TPPP for in vivo fluorescence imaging-guided PDT of a tumor. (B) Two-photon fluorescence imaging of the tumor tissue; the wavelength is 720 nm for TPE excitation. (C) In vivo antitumor study via intravenous injection of TPPP: representative tumor images on the 11 th day. Reproduced with permission from Han et al. 90 Copyright 2015, American Chemical Society.

well and shows weak fluorescence in aqueous media, but PyTPE becomes highly emissive when separated from the bioprobe via the cleavage of caspase-3/-7 (the key mediators of cell apoptosis). To demonstrate the capability of Ac-DEVD-PyTPE for in vivo apoptosis monitoring, subcutaneous tumor-bearing mice, injected in advance with and without STS via the tail vein, were further injected intratumorally with Ac-DEVD-PyTPE. As shown in Figure 19B, STS-induced apoptosis produced Ac-DEVD-cleavable caspase-3/-7 and gave rise to a gradual increase in PyTPE fluorescence from the tumors. In contrast, the signals from tumors without STS treatment and the normal tissues were very weak. Figure 19C shows the fluorescence changes in tumor and normal tissues as a function of time. A more obvious increase in fluorescence in the apoptotic tumor tissues is observed as early as 5 min after the injection of Ac-DEVD-PyTPE. The increased generation of ONOO is a key feature of acute and chronic inflammation, as well as indicative of many major diseases. Utilizing fluorescence imaging for specific detection of increased ONOO production at the inflammatory level is definitely beneficial for early diagnosis of diseases. Song et al.36 designed and synthesized a fluorescence light-up nanoprobe by encapsulating an AIE-active molecule (TPE-IPB) with a lipid-PEG matrix. TPE-IPB NPs are non-fluorescent in aqueous solution. After reacting with ONOO at pH 7.4, they turn to TPE-IPH NPs, which can emit bright yellow fluorescence (Figure 20A). On the basis of this mechanism, TPE-IPB NPs have been utilized for selective detection of in vivo inflammation with increased ONOO production. TPE-IPB NPs preferentially accumulate in the inflammatory region via the EPR effect, because the blood vessels are permeable in that area. Substantial amounts of ONOO are released under the stimulation of the immune cells in the inflammatory site, which further activates the fluorescence of TPE-IPB NPs (Figure 20B). TPE-IPB NPs also have the capability for noninvasive monitoring of in vivo therapeutic efficacy of anti-inflammatory agents. Intravenously injected TPE-IPB NPs targeted and lit up two inflammation sites on the left and right back of nude mice, where methicillin-resistant Staphylococcus aureus (MRSA,

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Figure 19. An AIEgen for In Vivo Apoptosis Monitoring (A) The molecular structure of Ac-DEVD-PyTPE. (B) In vivo fluorescence images of a subcutaneous tumor-bearing mouse after intratumoral injection of Ac-DEVD-PyTPE, with pre-treatment of STS for 12 hr. (C) Quantitative image analysis of Ac-DEVD-PyTPE-treated tissues at different times. Reproduced with permission from Shi et al. 91 Copyright 2013, Royal Society of Chemistry.

a Gram-positive bacterium) and Escherichia coli (a Gram-negative bacterium) were subcutaneously inoculated, respectively. After antibiotic treatments for 14 days, TPE-IPB NPs were intravenously injected into the mice again. The fluorescence signals from MRSA-infected foci in the vancomycin (antibiotic for Gram-positive bacteria)-treated mice and E. coli-infected foci in the penicillin (antibiotic for Gram-negative bacteria)-treated mice both vanished. In contrast, the fluorescence intensity from the inflammatory region injected with the bacteria-resistant antibiotic remained intense (Figure 20C). Multimodal Imaging MRI has been widely used in clinical diagnosis, because it is noninvasive without limitation of penetration depth and no harmful radiation. To achieve the dual functionalities of both MRI and fluorescence imaging, a contrast agent, TPE-2Gd, has been synthesized.94 Amphiphilic TPE-2Gd consists of a hydrophobic TPE molecule and two hydrophilic gadolinium (Gd) diethylenetriaminepentaacetic acid moieties (Figure 21A), and shows AIE characteristics. TPE-2Gd molecules aggregate into nanomicelles with negligible cytotoxicity and excellent photostability at a high concentration in aqueous medium, and they are used for fluorescence imaging of cells. As an MRI contrast agent, TPE-2Gd (in water) exhibits similar longitudinal relaxivity as a commercial agent (Magnevist), suggesting that it can provide enough signals during MRI imaging. The MRI results demonstrate that the circulation lifetime of intravenously injected TPE-2Gd in living rats is 1 hr, much longer than that of

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Figure 20. AIE NPs for Selective Detection of In Vivo Inflammation (A) Schematic illustration of TPE-IPB and TPE-IPH and the performance of TPE-IPB NPs after incubation with ONOO . (B) Left: schematic illustration of TPE-IPB NPs utilized for in vivo inflammation targeting via the EPR effect. Right: an in vivo fluorescence image of the inflammation-bearing mouse after intravenous injection of TPE-IPB NPs for 3 hr. (C) In vivo fluorescence images of MRSA- and E. coli-infected mice before and after vancomycin and penicillin treatment for 14 days. Reproduced with permission from Song et al. 36 Copyright 2016, John Wiley.

Magnevist (only 10 min); this is due to the formation of nanomicelles. With relatively high specificity of TPE-2Gd to the liver, the MR imaging signal in the liver remains hyperintense up to 150 min after administration (Figure 21A). In addition, TPE2Gd is excreted gradually via renal filtration as a result of disassembly of the nanomicelles into small molecules during circulation. This implies that TPE-2Gd can be used as a liver-specific MRI contrast agent for clinical diagnosis. Yan et al.95 proposed another dual-modal (MRI and fluorescence) contrast agent, with an AIEgen DPPBPA and magnetic Fe3O4 NPs as the core and a biocompatible polymer Pluronic F-127 as the encapsulation matrix (Figure 21B, top). With very little fluorescence quenching of the intraparticle DPPBPA molecules, the Fe3O4/ DPPBPA@F-127 NPs possess bright-red and near-infrared fluorescence and strong magnetism (Figure 21B, bottom left). In vitro cell imaging demonstrates that the

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Figure 21. AIE NPs for Dual-Modal MRI and Fluorescence Imaging (A) Coronal T1-weighted MR images of a rat after intravenous injection of TPE-2Gd and Magnevist with a concentration of 0.1 mmol/kg Gd3+ . Reproduced with permission from Chen et al. 94 Copyright 2014, American Chemical Society. (B) Top: schematic illustration of the chemical formation of Fe 3 O 4 /DPPBPA@F-127 NPs. Bottom left: photographs of aqueous dispersion of Fe3 O 4 / DPPBPA@F-127 NPs taken under normal room lighting and UV illumination, in an external magnetic field from a bar magnet. Bottom right: MRI results of water (left) and an aqueous dispersion of Fe 3 O4 /DPPBPA@F-127 NPs (right). Reproduced with permission from Yan et al. 95 Copyright 2016, Royal Society of Chemistry.

biocompatible NPs are stained in the cellular cytoplasm. The Fe3O4/DPPBPA@F-127 NPs also possess effective MRI ability. Figure 21B (bottom right) compares T1 contrast MRI images of water without and with the presence of NPs. The contrast of pure water is fairly poor, whereas that of an aqueous dispersion of Fe3O4/ DPPBPA@F-127 NPs is much better, suggesting the potential of NPs for future biological application. Multiphoton Fluorescence Imaging Two-Photon Fluorescence Imaging Two-photon fluorescence microscopy (2PFM) is a powerful tool for realizing accurate in vivo bioimaging.31 Relying on simultaneous absorption of two near-infrared photons by a fluorophore, 2PFM is capable of achieving deep-tissue penetration and efficient light detection noninvasively. The 2PF intensity has a square power dependence on the intensity of excitation light, endowing 2PFM with both inherent tissuesectioning capability and high imaging quality. Contrast agents with bright 2PF are needed for 2PFM. The 2PF of a fluorophore is determined by its two-photon action cross-section (Fs2) value, which is a product of the two-photon absorption (2PA) cross-section value (s2) and the fluorescence quantum yield (F).96 AIE NPs are promising candidates for high sensitivity with 2PFM. First, as a result of the RIM effect, the F of AIE NPs can be easily enhanced by increasing the amount of AIE molecules inside the NPs. Second, the s2 of an individual NP is roughly a product of the s2 of an individual AIE molecule and the loading amount of the encapsulated AIE molecules (N). Thus, the s2 of each NP could be maximized by increasing the AIEgen content inside. These two independent positive factors endow AIE NPs with large Fs2 values, enabling them to perform better than NPs doped with ACQ chromophores. Neuron imaging is of great significance in brain science research. Qian et al.97 realized deep-tissue in vivo neuron imaging with an AIEgen named TPE-TPP

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Figure 22. AIEgens for In Vivo 2PFM Imaging (A) Left: chemical structure of TPE-TPP. Middle: A reconstructed 3D image illustrating the staining of TPE-TPP in the microglia of a mouse brain (30 min after treatment) via 2PFM. Right: a magnified 3D 2PFM image of the brain of a mouse. Reproduced with permission from Qian et al. 97 (copyright ª 2015, Optical Society of America). (B) Top: a scheme illustrating the synthesis of BT NPs. Bottom: 3D reconstructed in vivo 2PFM images of BT NP stained mouse brain blood vessels with different visual angles. l ex = 1,040 nm. Reproduced with permission from Wang et al. 31 Copyright 2015, Optical Society of America.

(Figure 22A, left) on the basis of 2PFM. The TPE-TPP molecules can spontaneously form NPs by mixing a DMSO solution of TPE-TPP with water. Under femtosecond (fs) laser excitation of 740 nm, bright and cyan 2PF of TPE-TPP NPs was observed, with its peak wavelength at 480 nm. 2PFM imaging illustrates that TPE-TPP NPs specifically stain primary neurons in vitro. TPE-TPP was further microinjected into mouse brain at a depth of 300 mm. The spontaneously formed TPE-TPP NPs in the hydrophilic biological environment have net positive charges on the surface, rendering them to stain microglia in vivo. A typical 3D 2PFM image shows the distribution of TPE-TPP NPs (the cyan spots) inside the mouse brain, which can be discriminated easily from the background (Figure 22A, middle). As a result of the bright 2PF from TPE-TPP NPs, the morphology of the microglia is vividly demonstrated, and the NPs are widely distributed in the somas, dendrites, and axons of the microglia (Figure 22A, right). In addition, as a result of the

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photobleaching resistance of TPE-TPP NPs, these microglia can be observed for a long period. In addition to neuron imaging, AIE NPs can be also utilized for 2PFM-assisted in vivo brain angiography.31,92 The excitation wavelength in conventional 2PFM is usually from 770 to 860 nm. However, by studying the propagation of laser beams in biological tissue with Monte Caro simulation, a 1,040 nm laser beam was found to have better penetrating and focusing capability than an 800 nm laser beam because of less tissue scattering. Wang et al.31 fabricated red-emissive AIE NPs by utilizing DSPE-PEG to encapsulate BODIPY-TPE (BT) molecules (Figure 22B, top). The 2PA cross-section value of BT NPs was 2.9 3 106 GM at 1,040 nm, much larger than those at wavelengths ranging from 770 to 860 nm. Thus, biocompatible BT NPs are further applied in 2PFM in vivo imaging of brain blood vessels of mice by utilizing a 1,040 nm fs laser as the excitation source. High-contrast 3D 2PFM images are constructed (Figure 22B, bottom), and the major blood vessels and small capillaries can be visualized clearly. The 2PFM imaging depth reaches 700 mm in the mouse brain, much deeper than with 770–860 nm fs excitation and based on other AIE NPs. The combination of red emission, high 2PA cross-section from AIE NPs, and efficient 1,040 nm fs excitation will be helpful for functional in vivo 2PFM in the future. Three-Photon Fluorescence Imaging Three-photon fluorescence microscopy (3PFM) is a more powerful tool for highresolution and large-depth in vivo bioimaging, and has been developing rapidly in the past few years.98 Compared with 2PF, 3PF is a higher-order nonlinear optical effect. 3PF intensity has cubic dependence on the intensity of fs excitation laser, and the emitted 3PF signal at the focal point (very small) is much brighter than those out of the focus. Therefore, 3PFM dramatically reduces the out-of-focus background in regions far from the focal plane, improving the signal-to-background ratio (SBR) by orders of magnitude in comparison with 2PFM. The high SBR is helpful to improve spatial resolution and imaging contrast, as well as the imaging depth of 3PFM. In addition, the fs excitation wavelength of 3PFM is usually in the 1,000–1700 nm region, where light has less attenuation caused by tissue scattering, endowing the fs excitation of 3PFM with deeper-tissue penetration and better focusing capability.98 The 3PF of a fluorophore is determined by its three-photon action cross-section (Fs3) value, which is a product of the three-photon absorption (3PA) cross-section value (s3) and the fluorescence quantum yield (F).96 Similarly, AIE NPs are also a good choice as contrast agents for 3PFM, because both the F and s3 of AIE NPs can be enhanced by simply increasing the amount of AIE molecules inside the NPs. However, not all AIEgens have a large value for s3, and that is why reports on AIE NP-assisted in vivo 3PFM are still rare. TTF is an AIEgen possessing a typical donor-p-acceptor-p-donor structure with a large p-conjugation length, endowing it with rich nonlinear optical effects. Qian et al.93 systematically studied the higher-order nonlinear optical effects of TTF. They demonstrated 2PF, 3PF, and 4PF of molecular-state TTF in organic solution under excitation of 1,560 nm. In the solid state of TTF, both 3PF and third harmonic generation (THG) can be observed, and the 3PF signal is induced via one-photon absorption of the THG signal. TTF NPs were further fabricated by encapsulating TTF molecules with DSPE-PEG. Very interestingly, increasing THG and 3PF intensities were observed in the TTF NPs as the loading ratio (or aggregation degree) of TTF increased, according to both the spectra measurements and microscopic imaging

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Figure 23. AIE NPs for 3PFM Imaging (A) Left: quantitative comparison of 3PF and THG from TTF NPs with various TTF loading ratios. Top left: absorption and 3PF and THG spectra of aqueous dispersion of TTF NPs with various TTF loading ratios. Bottom left: 3PF and THG images of aqueous dispersion of TTF NPs in a capillary glass tube, with various TTF loading ratios (from left to right: 9 wt %, 14 wt %, and 20 wt %) under 1,560 nm fs excitation. Right: 3PFM images of brain blood vessels of a mouse at different depths under 1,560 nm fs excitation. Scale bar, 100 mm. Reproduced with permission from Qian et al. 93 Copyright 2015, John Wiley. (B) Left: bright-field and 3PFM images of zebrafish at 96 hr after microinjection of TTF NPs under 1,560 nm fs excitation. Scale bar, 200 mm. Right: 3PFM images of the heart and blood vessels of zebrafish under 1,560 nm fs excitation. Scale bar, 200 mm. Reproduced with permission from Li et al. 32 Copyright 2016, Springer.

results (Figure 23A, left). The two new phenomena can be therefore named as ‘‘aggregation-induced THG enhancement’’ and ‘‘aggregation-induced 3PF00 , respectively. TTF NPs were utilized for in vivo 3PFM imaging under 1,560 nm fs excitation, because this wavelength is very close to an optical tissue window ranging from 1,600 to 1800 nm. Because of the distinct 3PF signals from intravenously injected TTF NPs, the vascular architecture in the mouse brain at different vertical depths is vividly revealed. In addition, some tiny capillary vessels can be also observed very clearly. The 3PFM imaging depth in the mouse brain reaches 550 mm. Li et al.32 further utilized TTF NPs (encapsulated with DSPE-PEG2000) for 3PFM imaging of zebrafish, because TTF NPs showed no in vivo toxicity toward zebrafish according to a series of biological tests. TTF NPs were microinjected into zebrafish embryos, and the stained embryos at different growth stages were monitored with 3PFM under 1,560 nm fs excitation. As a result of the bright 3PF and long-time retention capability of TTF NPs inside zebrafish, the fish can be observed for as long as 120 hr. In addition, TTF NPs have been used for angiography of zebrafish. The zebrafish were imaged by 3PFM 2 hr after microinjection of TTF NPs. The morphologies of the heart and blood vessels can be clearly observed and distinguished. Furthermore,

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Figure 24. Four Main Working Mechanisms of AIEgens in Bioimaging and Theranostics

TTF NPs are highly photostable under 1,560 nm fs excitation, and long-term imaging and observation of the zebrafish are feasible.

SUMMARY AND PERSPECTIVES As the rational cause of AIE characteristics, the RIM effect promotes radiative decay in AIEgens, endowing them with a unique ‘‘light-up’’ feature. This review summarizes some recent advances in AIEgen-based bioimaging and theranostics. The working mechanism of novel AIEgens can be mainly classified into four categories (Figure 24). (1) turning ‘‘free’’ AIE molecules with intramolecular motion into AIE molecules with RIM. In this case, the RIM of AIE molecules is induced by specific targeting or binding with biomolecules or the influence of the bio-environment, rather than aggregation. Some applications of organelle imaging,18 intracellular pH mapping,24 and microorganism imaging and therapy are based on this mechanism.35,85 (2) AIE NPs spontaneously form on the basis of the hydrophobic AIE molecules, which are inside the functional polymers. As a result of cleavage of some specific molecules (e.g., caspase-3/-7, ROS, and GSH), the AIE molecules can be separated from the ‘‘welldesigned’’ polymers, and form nanoaggregates (NPs) in the hydrophilic bio-environment. The ‘‘lit up’’ AIE NPs can play a role in signaling and/or self-reporting of certain biological conditions or dynamics. As a result of this working principle, real-time apoptosis,49,91 imaging-guided chemotherapy, and imaging-guided PDT have been implemented.60,67 (3) Artificially synthesizing AIE NPs by encapsulating AIE molecules with a certain matrix. This is the most straightforward and widely used method in bioapplications, because AIE NPs have a host of inherent advantages (discussed below). Cell imaging and long-term tracing,19,53 imaging-guided therapy,21,62,63,70 in vivo imaging and monitoring,30,87 in vivo multimodal imaging,94,95 and MPFM imaging31,32,93 have been performed with the help of AIE NPs. (4) Activating ‘‘dark’’ AIE NPs to ‘‘bright’’ AIE NPs upon external stimuli (e.g., increased generation of ONOO) can

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simultaneously take advantage of the inherent features of AIE NPs and the ‘‘light-up’’based sensing characteristics. Selective in vivo detection of inflammation has been demonstrated with this effective mechanism.36 AIEgens are very promising in bio-applications. During fluorescence bioimaging and theranostics, through ingenious design of bioprobes, the ‘‘light-up’’ response of AIEgens is suitable for recognizing a wide variety of bioanalytes, including nucleic acids, proteins, and other macromolecules, as well as intracellular status (e.g., pH and viscosity), with high signal-to-noise ratio. In addition, if the AIEgens utilized in these applications are designed to by hydrophilic, wash-free detection can also be realized, which is of significance for real-time monitoring of biological processes with high accuracy and good reliability. In imaging-guided therapy applications, the ‘‘lit up’’ AIE NPs can signal and/or self-report the delivery and activation of drugs, as well as therapeutic responses. Because AIEgens are highly emissive in the aggregated state, AIE NPs are perfectly suited as bioprobes, which provides a series of advantages. AIE NPs have intense fluorescence, and biosamples stained with them can be clearly differentiated from the background, which is beneficial to high-contrast and high-resolution bioimaging. The AIE phenomenon usually refers to one-photon fluorescence, which is a linear optical effect. Recently, some aggregation-induced enhancement of nonlinear optical effects, including ‘‘aggregation-induced 2PF/ 3PF00 and ‘‘aggregation-induced THG enhancement’’, have also been discovered.97 Therefore, AIE NPs are also promising candidates for highly sensitive 2PFM, 3PFM, and other nonlinear optical bioimaging. As a result of the AIE effect, the loading concentration of AIE molecules inside each NP can be very high. This makes the bioprobes photobleaching resistant, even upon continuous irradiation by (fs) laser beams, which facilitates long-term imaging or monitoring of biological dynamics with good stability and reliability. Through certain matrix encapsulation, AIE NPs can possess excellent biocompatibility, which has been verified in cell, zebrafish, and mouse samples. Surface modification of AIE NPs with certain molecules would further enhance their selectivity toward biosamples, as well as prolong their blood circulation time inside a live animal’s body. AIE NPs possess excellent cellular retention capability (even better than quantum dots), and they can be utilized for long-term and noninvasive tracing of cells both in vitro and in vivo, which has great potential for oncology studies. Another unique advantage of an AIE NP over an individual ACQ molecule is that the former can realize extremely high drug or biomolecule loading efficiency, which is very helpful to high-performance selective targeting and imaging-guided therapy. Furthermore, AIE NPs can also be combined with other biomedical imaging methods, achieving multimodal functional imaging. In addition to the examples given in this review, the direction of future potential development of AIEgen-based bioimaging and theranostics includes but is not limited to the following: (1) Improving the optical performance of AIEgens. (a) Brightness and emission quantum yield: highly bright AIEgens or AIE NPs are always desirable, and more effort still need to be made to achieve this. (b) Long-wavelength absorption and emission: long-wavelength light has better penetrating capability as a result of less tissue scattering, which is helpful to realize deeper-tissue bioimaging. Long-wavelength excitation on AIEgen-treated biosamples also produces less autofluorescence, which can improve the contrast of bioimaging. So far, the emission maxima of reported AIEgens are still lower than 900 nm. Methods for synthesizing AIEgens with a peak emission wavelength beyond 900 nm, as well as certain brightness, are therefore highly anticipated.

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(c) High 2PF/3PF efficiency: 2PFM and 3PFM imaging are another alternative to achieve in vivo deep-tissue bioimaging, because fs excitation beams with long wavelength penetrate deep and focus well inside biological tissues. Furthermore, the spatial resolution of 2PFM and 3PFM imaging is higher than that of whole-body imaging. Thus, AIEgens with large 2PA and 3PA cross-section and far-red and NIR emission are favored. (d) Super-resolution imaging capability: Photoswitchable AIEgens have been adopted in STORM. AIEgens with a stimulated emission effect, as well as photobleaching resistance, have great potential in another class of super-resolution bioimaging,99 called ‘‘stimulated emission-depletion’’ nanoscopy. (e) Lifetime: recently developed AIEgens with room-temperature phosphorescence (RTP) are very promising for ‘‘zero background’’ lifetime bioimaging, because the lifetime of autofluorescence from biosamples is much shorter than that of AIEgens with RTP, which can be easily eliminated through ‘‘time-gate’’ technology. (2) Making AIEgen-based bioprobes multifunctional. (a) Specificity and selectivity: AIEgens or AIE NPs with highly targeting molecules are of significance for the specificity and selectivity toward biosamples. One of our recent studies has demonstrated the successful linking of antibodies with AIE NPs.100 Bioconjugated AIE NPs would greatly broaden their academic and clinical applications. (b) Controllability: so far, selective labeling or drug release in imaging-guided therapy is mainly achieved via bio- or photo-activation. It is anticipated that more stimuli-responsive (e.g., thermo-, piezo-, and magnetism-activatable) AIE NPs will be developed to enhance controllability. (c) All-in-one function: bioprobes designed on the basis of AIEgens have realized multifunctional applications in imaging-guided therapy. However, the structure and synthesis routes of some bioprobes are still complicated. AIEgens and AIE NPs with a simple molecular structure, as well as an all-in-one function (e.g., TMX for imaging-guided chemotherapy of breast cancer29), will be most favored. (d) Functional bioimaging and tracing: for AIEgens, there is plenty of scope for biomedical and clinical applications. For example, excellent tumor targeting and long-term cellular retention capabilities make AIE NPs very suitable for the study of cancer metastasis in vivo. AIEgens with high 2PF or 3PF efficiency, as well as calcium ion and oxygen sensitivity, have great potential in functional brain and neuron imaging. (e) Multimodal bioimaging: in the near future, AIEgens will be combined with some clinical imaging methods (e.g., computed tomography, MRI, and ultrasonography), and applied for multimodal clinical diagnosis. (3) Toxicity and clearance. With the increasing number of studies of AIEgens and AIE NPs at the cellular and organism levels, their biocompatibility, distribution, clearance, and final fate are required to be clarified, which will rely on the joint efforts of a broad spectrum of participants, including synthetic chemists, physicists, biologists, biomedical engineers, as well as clinical doctors. During the preparation of this review, many excellent studies on biomedical applications of AIEgens have been published, which illustrates the rapid development of AIE. We believe that, in the future, more researchers will be involved in this exciting platform and more interesting ideas will be triggered to promote AIEgens for various biomedical and clinical applications.

AUTHOR CONTRIBUTIONS B.T. proposed the topic of the review. J.Q. investigated the literature and wrote the manuscript. B.T. and J.Q. discussed and revised the manuscript.

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ACKNOWLEDGMENTS We are grateful to the editor for the kind invitation. The authors are pleased to acknowledge all the people who have worked with us in this area and whose names can be found in the references. This work was supported by National Basic Research Program of China (973 Program; 2013CB834701 and 2013CB834704), the Zhejiang Provincial Natural Science Foundation of China (LR17F050001), the National Natural Science Foundation of China (61275190), the Research Grants Council of Hong Kong (16301614 and 16305015), the Innovation and Technology Commission (ITC-CNERC14SC01), and the Shenzhen Science and Technology Program (JCYJ20160509170535223). B.T. thanks the Guangdong Innovative Research Team Program (201101C0105067115) for financial support.

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