Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Aggregation-Induced Emission (AIE) Dots: Emerging Theranostic Nanolights Guangxue Feng and Bin Liu* Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore CONSPECTUS: Theranostic nanolights refer to luminescent nanoparticles possessing both imaging and therapeutic functions. Their shape, size, surface functions, and optical properties can be precisely manipulated through integrated efforts of chemistry, materials, and nanotechnology for customized applications. When localized photons are used to activate both imaging and therapeutic functions such as photodynamic or photothermal therapy, these theranostic nanolights increase treatment efficacy with minimized damage to surrounding healthy tissues, which represents a promising noninvasive nanomedicine as compared to conventional theranostic approaches. As one of the most promising theranostic nanolights, organic dots with aggregation-induced emission (AIE dots) are biocompatible nanoparticles with a dense core of AIE fluorogens (AIEgens) and protective shells, whose sizes are in the range of a few to tens of nanometers. Different from conventional fluorophores that suffer from aggregation-caused quenching (ACQ) due to π−π stacking interaction in the aggregate state, AIEgens emit strongly as nanoaggregates due to the restriction of intramolecular motions. Through precise molecular engineering, AIEgens could also be designed to show efficient photosensitizing or photothermal abilities in the aggregate state. Different from ACQ dyes, AIEgens allow high loading in nanoparticles without compromised performance, which makes them the ideal cores for theranostic nanolights to offer high brightness for imaging and strong photoactivities for theranostic applications. In this Account, we summarize the recent advance of AIE dots and highlight their great potential as theranostic nanolights in biomedical applications. Starting from the design of AIEgens, the fabrication of AIE dots and their bioimaging applications are discussed. The exceptional advantages of superbrightness, high resistance to photobleaching, lack of emission intermittency, and excellent biocompatibility have made them reliable cross platform contrast agents for different imaging techniques such as confocal microscopy, multiphoton fluorescence microscopy, super-resolution nanoscopy, and light-sheet ultramicroscopy, which have been successfully applied for cell tracking, vascular disease diagnosis, and image-guided surgery. The integration of therapeutic functions with customized AIEgens has further empowered AIE dots as an excellent theranostic platform for imageguided phototherapy. Of particular interest is AIE photosensitizer dots, which simultaneously show bright fluorescence and high photosensitization, yielding superior performance to commercial photosensitizer nanoparticles in image-guided therapy. Further development in multiphoton excited photodynamic therapy has offered precise treatment with up to 5 μm resolution at 200 μm depth, while chemiexcited photodynamic therapy has completely eliminated the limitation of penetration depth to realize powerfree imaging and therapy. With this Account, we hope to stimulate more collaborative research interests from different fields of chemistry, materials, biology, and medicine to promote translational research of AIE dots as the theranostic nanolights.
1. INTRODUCTION Nanolights are small size luminescent nanoparticles and are delightful tools for bioimaging and disease diagnosis.1,2 When introduced with therapeutic functions such as reactive oxygen species (ROS) generation or light-to-heat conversion, these nanolights demonstrate great potential in photodynamic therapy (PDT), photoacoustic (PA) imaging, and photothermal therapy (PTT).3 Different from traditional nanomedicine, both imaging and therapeutic functions of nanolights can be controlled by localized photons to generate minimal damage to surrounding tissues.4 Inorganic nanolights such as quantum dots exhibit high brightness and size-dependent © XXXX American Chemical Society
emission, but the toxic heavy metal components make them less desirable for theranostic construction.5 Organic nanolights with excellent biocompatibility and easy modification appear to be a more promising choice, but conventional luminophores suffer from aggregation-caused quenching (ACQ) due to strong π−π stacking in aggregate states (Figure 1A), which yield quenched fluorescence and reduced photosensitization.6 This phenomenon restricts fluorophore loading in nanoparticles, leading to limited theranostic performance. Received: February 2, 2018
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DOI: 10.1021/acs.accounts.8b00060 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research
Figure 1. Fluorescence photographs of (A) DDPD and (B) TPE in THF/water mixtures under UV illustration. (C) Brightness and (D) photosensitization of ACQ and AIE dots as a function of loading percentage. Reproduced with permission from refs 10 and 14. Copyright 2013 American Chemical Society and Copyright 2016 Wiley-VCH.
2. FABRICATION AND CHARACTERIZATION OF AIE DOTS
Aggregation-induced emission (AIE) dots are small-sized nanoparticles with AIE fluorogens (AIEgens) as the core, and biocompatible matrices as the protective shells.7 Different from ACQ fluorophores, AIEgens such as tetraphenylethene (TPE) are not emissive as isolated molecules in solution but emit strongly in solid or aggregate state (Figure 1B).8−10 The dense packing of AIEgens in confined space yields high brightness for AIE dots,11 which can be 10−40 times brighter than quantum dots.12 AIE dots represent promising contrast agents across different imaging techniques and animal models. Moreover, through precise molecular design, AIEgens could be realized with efficient photosensitization in the aggregate state, superior to many commercial photosensitizers.13 Most impressively, AIE dots show exceptionally linear loading percentage-dependent increase in brightness and photosensitization, which distinguishes them from ACQ dots that exhibit compromised brightness and photosensitization at high loadings (Figures 1C,D). Different from traditional systems where multiple theranostic components are introduced into one nanoparticle, the integration of different functions within a single AIEgen represents a facile strategy for theranostic nanolights with great simplicity and reproducibility.14 In this Account, we summarize the recent development of AIE dots with highlights on their biological and biomedical applications. We start with AIEgen design, followed by AIE dot fabrication. The great potential of AIE dots as theranostic nanolights is then illustrated through selected examples. Due to space limitation, we apologize that the AIE dots discussed here only include small size organic nanoparticles directly synthesized from nanoprecipitation. With this Account, we hope to stimulate more collaborative work to promote the translational research on AIE dots.
2.1. Design of AIEgens
The function and performance of AIE dots are largely dependent on the AIEgens. The simple structure and facile synthesis have made TPE one of the elementary units for construction of AIEgens with different emissions. TPE also serves as a magic stick to transform many ACQ molecules to be AIE-active.8 Alternatively, linking AIEgens with another AIE moiety generates new AIEgens (e.g., 2,3-bis(4-(phenyl(4(1,2,2-triphenylvinyl)-phenyl)amino)phenyl)-fumaronitrile, TPETPAFN) with elongated conjugation length and redshifted emission.12 Another approach is through introducing electron donating (e.g., methoxy) or withdrawing (e.g., benzothiadiazole, benzobisthiadiazole) units to TPE to form donor-acceptor structures. This yields AIEgens with emission fine-tuned from the visible to the near-infrared (NIR) region (Figure 2).15−17 The advancement of AIE mechanisms from restriction of intramolecular rotations to restriction of intramolecular motions,18 and the recent anti-Kasha effect,19 has further broadened the guidelines for new AIEgen design, providing more AIEgens for AIE dot fabrication. 2.2. Design of AIE Dots
Most AIEgens are only soluble in organic solvents, which makes them not suitable for biological applications. Covalently conjugating AIEgens with ionic or hydrophilic chains generates water-soluble AIE analogues or amphiphilic AIE macromolecules, which readily self-assemble into AIE nanoparticles. However, they require chemical modification on each AIEgen, and the obtained nanoparticles generally lack proper size control. A more general and promising approach is physical encapsulation of AIEgens with biocompatible matrices. A generalized strategy also helps standardize AIE dot fabrication B
DOI: 10.1021/acs.accounts.8b00060 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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more widely studied class of encapsulation matrices is amphiphilic copolymers, such as 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(poly(ethylene glycol))] (DSPE-PEG) derivatives.11,12 The sizes of DSPE-PEG encapsulated AIE dots can be precisely controlled from a few to tens of nanometers via controlling ultrasound sonication, PEG chain lengths, AIEgen to matrix ratios, etc.11 The simple fabrication strategy also allows coloading of other components, such as magnetic nanoparticles, conjugated polymers, or conventional NIR fluorophores for multimodal imaging or amplified optical imaging.24,25 One key challenge for nanoparticle fabrication is the reproducibility of large scale production. An automated millifluidic system is therefore developed to eliminate the batch-dependent variation for AIE dots. This is achieved by a jet mixer system; the mixing of organic solvent and water in a small tube generates vortex and turbulence to induce AIE dot formation in a continuous manner (Figure 3C). AIE dots with well-controlled sizes and high uniformity can be easily modulated by Reynolds numbers, flow rates, and solvent ratios.26 With the jet-mixer system, uniform AIE dots with a low polydispersity index of less than 0.1 could be obtained in a high production rate of up to 200 g/day, which provides a reliable platform to evaluate the biomedical applications of AIE dots.
Figure 2. Examples of AIEgens and their representative emission spectra in aggregates.
with well-controlled size, high reproducibility, and precise surface functionalization.20 In this Account, we focus on these physically encapsulated AIE dots. AIE dot fabrication involves transferring AIEgens and matrices from organic solvents to water to induce the encapsulation process via nanoprecipitation followed by surface conjugation (Figure 3A).7 Alternatively, prefunctionalized matrices could directly yield surface functionalized AIE dots via a single step.21 The selection of matrices and methods controls their sizes and properties (Figure 3B). For example, desolvation of bovine serum albumin (BSA) could load AIEgens into BSA dots with sizes around 100 nm.22 Silicashelled AIE dots with sizes of 12 nm were synthesized by a sol− gel procedure.23 Moreover, the aggregation patterns of AIEgens inside the dots could also be modulated.21 Through adjusting emulsifier amount used during nanoemulsion, eccentrically aggregated TPETPAFN inside poly(DL-lactide-co-glycolide) (PLGA) dots shows a higher fluorescence quantum yield (η) (34%) than those with homogeneous distribution (23%).21 A
2.3. Properties of AIE Dots
To highlight the excellent properties of AIE dots, DSPE-PEG encapsulated TPETPAFN dots are selected as an example.12,15,27 The emission maximum of TPETPAFN dots is centered at 670 nm with an η of 25%. Their brightness is over 26-fold higher than that of commercial Qtracker 655 dots.12 Their sizes and η remain unchanged even after storage for over 30 days at room temperature (Figure 4A,B). Different from Qtracker 655, TPETPAFN dots show stable brightness upon incubation in cell culture medium (Figure 4C). Moreover, they demonstrate high resistance to photobleaching (Figure 4D). Unlike quantum dots, the blinking and intermittency are absent in TPETPAFN dot emission (Figure 4E), which makes them promising for real-time tracking. In addition, TPETPAFN dots show excellent biocompatibility. They do not cause alteration
Figure 3. (A) Schematic illustration of AIE dot formation by nanoprecipitation, where AIEgens and matrices are transferred from organic solvent to water under sonication to induce encapsulation. (B) Examples of AIE dots with different shells and sizes. (C) Millifluidic automation of AIE dot fabrication. Reproduced with permission from refs 22, 21, 16, 23. Copyright 2012 and 2013 Wiley-VCH, Copyright 2014 Elsevier, and Copyright 2016 American Chemical Society. C
DOI: 10.1021/acs.accounts.8b00060 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research
Figure 4. (A) Size and (B) η changes of TPETPAFN dots upon 30 day storage at room temperature. Fluorescence changes of TPETPAFN dots and Qtracker 655 upon (C) incubation in cell culture medium at 37 °C or (D) continuous laser scanning. I0 and I represent the fluorescence intensities before and after incubation or laser scanning. (E) Fluorescence time-traces of representative TPETPAFN dots and Qtracker 655. (F) Secretome analysis of stem cells treated with TPETPAFN dots. (G) Mouse weight changes after injection with TPETPAFN dots. (H) Size and polydispersity index for TPETPAFN dots fabricated from different batches. Reproduced with permission from refs 12, 15, and 27. Copyright 2013 Nature Publishing Groups, Copyright 2014 American Chemical Society, and Copyright 2017 Elsevier.
Figure 5. Confocal images of (A) cytoplasm and (B) mitochondria of MCF-7 cancer cells labeled by AIE dots. (C) Confocal and (D) STED images of cellular microtubule structures labeled by Red-AIE-OXE dots. (E) The line profile of position indicated by the arrow heads (i) in panels C and D. Reproduced with permission from refs 21, 28, and 31. Copyright 2013, 2015, and 2017 Wiley-VCH.
mitochondria of MCF-7 cancer cells (Figure 5B).28 To provide sufficient resolution for organelle structure mapping, stimulated emission depletion (STED) nanoscopy was recently applied.29 It utilizes a secondary laser to deplete fluorophore emission to increase lateral resolution, which requires strong photoresistance for fluorophores. In this regard, bright AIE dots with smaller sizes (400 μm) in the brain could be clearly visualized in real-time (Figure 6D). In addition, some AIE dots also exhibit excellent three-photon fluorescence where the focal-point emission under NIR-II (1000−1700 nm) laser further promotes deep-tissue imaging with high signal-to-noise ratios (Figure 6E).34 Recently, more AIE dots with red and NIR emission have been developed for vascular network mapping in different organs, such as bone
tubulin antibodies, these AIE dots can clearly image subcellular microtubules with a spatial resolution of ∼95 nm under STED, which is much better than confocal imaging (∼190 nm) (Figures 5C−E). 3.2. In Vivo Imaging
AIE dots are also compatible with different in vivo imaging systems. For example, red-emissive AIE dots with an η of 32% and an emission maximum at 660 nm are able to track cancer cell progression in xenografted zebrafish (Figure 6A).32 To avoid strong tissue absorption and autofluorescence in the visible light region, AIE dots with long wavelength excitation and high NIR fluorescence were developed for mouse tumor imaging, where they show excellent tumor accumulation via enhanced permeability and retention effect (Figure 6B).22 Further decorating the AIE dots with targeting moieties offered active tumor targeting.24 As compared to one-photon imaging, two-photon fluorescence imaging (TPFI) provides a clearer optical window for E
DOI: 10.1021/acs.accounts.8b00060 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research 3.5. Image-Guided Tumor Resection
marrow and muscle, offering a useful tool to study vascular related diseases.35
After demonstrating their strong compatibility with cells, vascular and tumor imaging, one straightforward practical application of AIE dots is image-guided tumor surgery. This is achieved using 4,8-bis(4-(1-(4-butoxyphenyl)-2,2diphenylvinyl)phenyl)benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (DTPEBBTD) dots (λmax = 820 nm, η = 4.8% in water) as an example (Figure 2).17 The bright NIR fluorescence from DTPEBBTD dots can distinctly delineate tumor nodules over normal tissues with a high tumor-to-normal tissue ratio of ∼7.2. With DTPEBBTD dot-guided surgery, nearly all the tumor nodules were removed, especially for small nodules (
