AIE Nanoparticles for in Vitro and in Vivo Imaging - American

Chapter 9

AIE Nanoparticles for in Vitro and in Vivo Imaging Downloaded by CORNELL UNIV on October 9, 2016 | http://pubs.acs.org Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch009

Duo Mao and Dan Ding* State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, P. R. China *E-mail: [email protected]

In recent years, fluorogens with aggregation-induced emission (AIE) characteristics have attracted considerable research interest in design and preparation of fluorescent organic nanoparticles (NPs) for bioimaging. So far, plenty of AIE NPs have been successfully utilized for in vitro and in vivo fluorescence imaging applications, which show outstanding performances by virtue of their unique advantages in terms of high brightness, excellent photostability, free of random blinking, facile cell internalization and superb cellular retention as well as negligible cytotoxicity and in vivo toxicity. In this chapter, the recent status of the development of AIE NPs for in vitro fluorescence imaging was summarized according to their utilizations, including non-specific cell imaging, targeted cancer cell imaging, specific organelle imaging, in vitro long-term cell tracking, and bacterial imaging. Furthermore, we also discuss the in vivo applications of AIE NPs in in vivo fluorescence imaging of tumors, intravital two-photon fluorescence imaging, in vivo long-term cell tracking as well as in vivo dual-modality imaging. The perspectives for the future investigation of advanced AIE NPs for bioimaging are discussed in this chapter as well.

© 2016 American Chemical Society Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Downloaded by CORNELL UNIV on October 9, 2016 | http://pubs.acs.org Publication Date (Web): September 27, 2016 | doi: 10.1021/bk-2016-1227.ch009

Introduction In recent years, considerable research interest has focused on the biocompatible nanostructured materials for in vitro and in vivo imaging (1–3). Versatile imaging modalities, including fluorescence imaging, magnetic resonance imaging (MRI) and nuclear imaging (such as positron emission tomography (PET) and single photon emission computed tomography (SPECT)), et al., along with the corresponding imaging contrast agents have been becoming increasingly prosperous (4–6). Among them, fluorescence imaging has been extensively investigated and attracted great attentions as this technique holds the advantages of high sensitivity, excellent temporal resolution, good safety, large in vitro and in vivo throughputs and manoeuvrable imaging instruments (7). To date, various fluorescent materials, which include organic dyes (8), fluorescent proteins (9) and inorganic semiconducting quantum dots (QDs) (10), have been widely employed for the purpose of in vitro and in vivo bioimaging. However, each material suffers its own limitations, such as low molar absorptivity, small Stokes shift, limited photobleaching thresholds or high cytotoxicity (11, 12). Exploration of alternative fluorescent materials with improved properties is highly desirable. Fluorescent organic nanoparticles (NPs) have recently emerged as a new generation of nanoprobes for bioimaging, which show such merits as flexible synthetic approaches, good biocompatibility and facile surface functionalization (13, 14). Besides, the NP probes are able to passively accumulate into many disease regions (i.e., tumor or inflammatory sites) by the enhanced permeability and retention (EPR) effect (15). As highly emissive probes are always desirable for high contrast imaging, it would be ideal that the brightness of the fluorescent organic NP is proportional to the amount of its doped fluorophores. Unfortunately, due to the well-known aggregation-caused quenching (ACQ) effect, conventional fluorophores that often possess planar aromatic structures suffer from severe emission quenching at high dye loading contents in NPs. This is because the doped dye molecules are prone to aggregation in NPs when a high fluorophore loading is achieved, which leads to π-π stacking as well as other non-radiative pathways and thus quenches the light emission (16). Tang’s group has recently developed a novel class of organic fluorogens with aggregation-induced emission (AIE) signature, which is exactly opposite to the ACQ effect (17, 18). The AIE fluorogens (AIEgens) are brightly emissive in the aggregate state by virtue of the restriction of intramolecular motion (RIM) mechanism (19). Consequently, the emergence of AIEgens has well addressed the concerns of emission quenching resulting from the high fluorogen loading in NPs, which has also opened up new opportunities for fabricating super-bright fluorescent organic NPs applied for bioimaging. In this chapter, we sum up the recent status and advances in the development of AIE NPs for in vitro and in vivo imaging. Considering that there are vast amounts of successful examples of AIE NPs for bioimaging, we divide them into two sections: 1) in vitro and 2) in vivo imaging applications. We then summarize each of the two sections in detail according to the utilizations of AIE NPs. Finally, the perspectives for the future exploration of advanced AIE NPs for bioimaging are discussed. 218 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

AIE NPs for in Vitro Imaging So far, AIE NPs have been extensively investigated for in vitro fluorescence imaging, as AIE-involved imaging systems exhibit such features as high fluorescence, excellent photostability, superb biocompatibility, and free of random blinking (20). In this section, the in vitro imaging applications of AIE NPs will be summarized in terms of non-specific cell imaging, targeted cancer cell imaging, specific organelle imaging, in vitro long-term cell tracking, and bacterial imaging.


Non-Specific Cell Imaging Cell imaging with fluorescent probes is of great importance for providing the scientists and clinicians with sights and insights into the function and mechanism of biological species and processes in cells (21). The development of AIE NPs offers a new avenue for cell imaging with high brightness and excellent photostability. To date, a variety of AIE NPs including pure AIE fluorogen nanoaggregates (NPs formed by only small AIE molecules without using any encapsulation matrix) (22), AIEgen-based silica NPs (23–25), AIEgen-based block co-polymer NPs (26) and in situ AIEgen-polymerized NPs (27–36) et al., were utilized for non-specific cell imaging. For example, several research groups focused on the design and synthesis of AIEgen-based silica NPs, as silica NPs are known to be hydrophilic, biocompatible and size-tunable (23–25). AIEgen-based silica NPs that are colloidally stable could be prepared via the reactions between tetraphenylethene (TPE)- (compound 1) or silole-functionalized siloxane (compound 2) and tetraethoxysilane (TEOS) (23). The size of the obtained NPs is uniform and can be tunable by altering the reaction conditions (45-295 nm). The fluorescent AIE-based silica NPs have been demonstrated to be non-toxic against the cancer cells and internalized into the cell cytoplasm for imaging (Figure 1). Moreover, in situ AIEgen-polymerized NPs refer to covalently bonded AIE-polymer NPs, which were prepared by in situ polymerizing AIEgen into a polymer chain. Wei’s group has made a great effort on such NPs. Through synthesizing different polymerizable AIEgens, they employed a variety of polymerization methods such as emulsion polymerization (27, 28), reversible addition-fragmentation chain transfer (RAFT) polymerization (29–31), anhydride ring-opening polymerization (32–35), and cross-linked polymerization (36) to generate a library of amphiphilic AIE-polymers, which were able to self-assemble into fluorescent NPs in aqueous solution. These in situ AIEgen-polymerized NPs show the advantages of compact structure without dye leaking and surface coating detachment in harsh biological environments as well as good cytocompatibility and high cell uptake efficiency in cellular imaging application.

219 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 1. (A) Chemical structures of Compounds 1 and 2, as well as the synthetic route to 1- or 2-based silica NPs. Fluorescence images of HeLa cancer cells stained with (B) 1- and (C) 2-based silica NPs. [1] = 8 μM; [2] = 6 μM. Adapted with permission from ref (23). Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Targeted Imaging of Cancer Cells Targeted imaging of cancer cells is highly valuable for recognition of the class and subclass of cancers, diagnosis and treatment of cancers at early stage, and evaluation of the anticancer efficacy of drugs (2). One of the most efficient strategies to promote the selectivity and targeting effect of the fluorescent NPs toward cancer cells is to modify the NP surfaces with specific targeting ligands. Generally, the targeting ligands possess selective interaction with receptors that are overexpressed in the cell membrane (11, 37). In the field of AIE NPs, many a ligand, including folic acid (38–40), RGD peptide (41) and biotin (42) et al. have been widely utilized for targeted cancer cell imaging. For instance, several groups selected folic acid as the targeting ligand to functionalize the surface of AIE NPs, as there is high binding affinity between folic acid and folate receptors. The folate receptors have been well established to be overexpressed in many kinds of cancer cells, whereas at a low expression level in normal cells (43). Liu and co-workers reported the design and preparation of compound 3-based AIE NPs with folic acid-functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethyleneglycol) (DSPE-PEG5000-folic acid) and DSPE-PEG2000 as the encapsulation matrix (Figure 2A) (38). The 3-encapsulated AIE NPs have a spherical morphology with 220 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


mean hydrodynamic size of around 85 nm, high quantum yield of 24%, and low cytotoxicity against MCF-7 breast cancer cells. Besides, such AIE NPs exhibit much superior selectivity toward MCF-7 cancer cells to NIH/3T3 normal cells. The fluorescence intensity of 3-encapsulated AIE NP-stained MCF-7 cancer cells can be further enhanced with the increase of folic acid density on the NP surface, which saturates when the feed ratio of DSPE-PEG5000-folic acid in the matrix is 40% (Figure 2B).

Figure 2. (A) Chemical structures of Compound 3, DSPE-PEG2000 and DSPE-PEG5000-folic acid, as well as the schematic illustration of the formation of 3-encapsulated AIE NPs. (B) Confocal images of MCF-7 cancer cells after incubation with 3-encapsulated AIE NPs with various folic acid densities on the NP surfaces for 2 h at 37 °C. The feed ratio of DSPE-PEG5000-folic acid in the matrix is 0%, 20%, 40% and 60%, respectively. Adapted with permission from ref (38). Copyright 2011 Royal Society of Chemistry. Specific Organelle Imaging Fluorescent probes that are capable of visualizing specific organelles have been attracting considerable research interest, as the organelles play key roles on cell function, growth and death (44, 45). In particular, mitochondrion is an important organelle that is found in nearly all eukaryotes and generates the energy currency of the cells (46). As mitochondria are involved in plenty of human 221 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


diseases, which include mitochondrial disorders, cardiac dysfunction, and heart failure (47), specific mitochondrion imaging is particularly valuable. Recently, a series of AIEgens with different emission colors have been explored for specific mitochondrial imaging (48–52). In general, these AIEgens can form NPs in aqueous solution without any encapsulation matrix. Taking compound 4 as an example, 4 is composed of one TPE and two triphenyphosphonium (TPP) moieties, and TPP has been extensively reported as a mitochondria-specific targeting ligand (48). Additionally, 4 appears as AIE NPs in aqueous solution with an average diameter of about 144 nm. These blue color emissive NPs of 4 have been demonstrated to be able to clearly stain the mitochondria (reticulum structures) of live HeLa cells, which was further confirmed by the good co-localization of NPs with commercial mitochondria probe MitoTracker red FM (Figure 3A).

Figure 3. Chemical structures of 4-6. (A) Fluorescent images of 4 (5 μM) and MitoTracker red FM (50 nM) co-stained HeLa cells. Reprinted with permission from ref (48). Copyright 2013 American Chemical Society. (B) Fluorescent images of 5 (5 μM) and MitoTracker red FM (100 nM) co-stained HeLa cells. Reprinted with permission from ref (49). Copyright 2013 Royal Society of Chemistry. (C) Confocal images of 6 (5 μM) and Mito-GFP (CellLight® Mitochondria-green fluorescent protein) co-stained HeLa cells. Reprinted with permission from ref (50). Copyright 2015 Royal Society of Chemistry. 222 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Subsequently, Tang’s group developed AIEgens of 5 and 6 with yellow and red fluorescence, respectively (49, 50). Each of the two AIEgens consists of a large hydrophobic moiety donated by TPE motif and other π–conjugated groups as well as a positively charged head. Thanks to such intelligent molecular design, both the NPs of 5 and 6 can vividly visualize the mitochondria with extremely high resolution, using MitoTracker red FM and CellLight® Mitochondria-green fluorescent protein (Mito-GFP) as the co-staining agent, respectively (Figures 3B and 3C). It is important to note that all of the aforementioned mitochondria-specific AIE NPs show excellent photostability with high photobleaching resistence, making them ideal probes for long-term monitoring. Noteworthy is that in addition to mitochondria-staining AIE NP probes, the ones that can selectively visualize other organelles such as lysosome (53) and lipid droplets (54) have also been developed with bright emission, high resolution and large photobleaching threshold. Given these facts, AIEgens can serve as promising fluorescent materials in creating photostable fluorescent NP probes for specific organelle imaging.

In Vitro Long-Term Cell Tracking Fluorescent probes that have the ability in continuous tracking the cells over a long period of time have received great attention (7). GFP and its variants have been extensively reported as genetic cell tagging for cell tracing (55); however, the GFP labeling method suffers from high cost and safety issues owing to the introduction of random insertional mutation at integration sites (56). In addition to GFP, inorganic semiconducting QDs have been widely utilized as cell trackers, as they hold the advantages of high brightness, good photostability and nanoscale sizes that enable superb retention in living cells without leaking out from cytoplasm (57, 58). So far, several QD-based cell tracing reagents have been commercialized. Nevertheless, the oxidative degradation of the heavy metal components followed by heavy metal ions releasing makes QDs rather toxic against cells (12). As a result, only relative low concentrations of QDs can be safely used to label cells, which undoubtedly influence the long-term tracking effect. Moreover, as compared to organic NPs, inorganic QDs are more inclined to aggregate in biological systems, leading to fluorescence quenching (59). These concerns significantly limit the practical application of QDs in long-term cell tracking. Recently, a series of AIE NPs have been explored for in vitro cell tracking application, which exhibit such merits as bright emission, high cell labeling efficiency, excellent photostability, low cytotoxicity, superb retention in live cells without leaking during proliferation, as well as long cell tracking period (60–64). For instance, Tang’s group reported an AIE-active chitosan-TPE conjugate (compound 7) by covalent conjugation of isothiocyanate-bearing TPE molecules onto chitosan chains (60). The chitosan-TPE conjugates can be readily internalized by HeLa cancer cells, which have been testified to be capable of rooting within the cells and efficiently tracing the living HeLa cells for 15 passages (Figure 4). The remarkable long-term cell tracking capability 223 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


of chitosan-TPE conjugates is much superior to that of commercial CellTracker Green CMFDA, which was reported to track the cells for no more than 3 passages. For another example, Liu, Tang and co-workers synthesized an AIEgen (8) with high far-red/near-infrared (FR/NIR) fluorescence in the aggregate state (61). Then 8 was encapsulated using DSPE-PEG2000 and its amine end-capped DSPEPEG2000-NH2 as the matrix, affording 8-doped AIE NPs. The surfaces of NPs were further functionalized by cell penetrating peptide HIV-1 Tat via carbodiimidemediated coupling. The resulted 8-doped AIE-Tat NPs have spherical morphology with a mean size of around 30 nm, high quantum yield of 24% as well as strong photobleaching resistance. Both the flow cytometry and confocal imaging studies indicate that the 8-doped AIE-Tat NPs can efficiently track MCF-7 cancer cells up to 12 generations, whereas the commercial Qtracker® 655 is only able to trace 5-6 generations of the cells, revealing the far superior in vitro cell tracking capacity of 8-doped AIE-Tat NPs over Qtracker® 655 that is well-known as a good long-term cell tracker (Figures 5A and 5B). Furthermore, the retention of 8-doped AIE-Tat NPs in the labeled cells was also investigated. The NP-stained MCF-7 cells were mixed with untreated cells at 1:1 ratio, which were subsequently co-cultured for 24 h. Flow cytometry analysis indicates that the ratio of MCF-7 cells with and without fluorescence is still nearly 1:1, revealing that 8-doped AIE-Tat NPs would not easily leak out from the labeled cells (Figure 5C). Given these facts, AIE NPs hold great promise as a new generation of fluorescent cell trackers for long-term reporting the fate of cells.

Figure 4. Chemical structure of 7. Fluorescent images of HeLa cells stained by 7 at various passages. Adapted with permission from ref (60). Copyright 2013 American Chemical Society. 224 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 5. Chemical structure of 8. Flow cytometry histograms and confocal images of (A) 8-doped AIE-Tat NPs-stained and (B) Qtracker® 655-stained MCF-7 cells, followed by subculture for designated generations. Control: untreated MCF-7 cells. (C) Flow cytometry histograms and confocal images of a mixture of 8-doped AIE-Tat NPs-stained MCF-7 cells and unlabeled cells, which were co-cultured for 24 h. Adapted with permission from ref (61). Copyright 2013 Rights Managed by Nature Publishing Group.

In Vitro Bacterial Imaging In addition to cellular imaging, AIE NPs also show good performance in bacterial imaging and tracking (65–67). Tang’s group developed a highly emissive and photostable AIEgen (9) that can differentiate dead and live bacteria (65). As shown in Figures 6A-L, upon incubation of the live as well as dead bacteria with 9 (formed NPs in aqueous solution), it is found that only dead bacteria are visualized, while there is almost no detectable fluorescence in the live bacteria. This is because the AIE NPs of 9 is live bacterial cell-impermeable, 225 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


which is not capable of entering and lighting up the live bacteria. On the other hand, as the membranes of dead bacteria are broken, the AIE NPs of 9 would readily penetrate into the bacterial protoplasm and interact with the intracellular DNA strands, which significantly activate the fluorescence of 9 by virtue of the restriction of intramolecular rotation mechanism. Moreover, thanks to the unique capability in discrimination of live and dead bacteria, 9 can serve as an efficient probe on evaluating the effectiveness of different bactericides. To date, besides 9 that emits blue fluorescence, the AIEgens with yellow, orange and red emission colors have also been explored for bacterial imaging (66, 67).

Figure 6. Chemical structure of 9. Images of 9-stained live and dead (A-D) E. coli, (E-H) S. epidermidis and (I-L) B. subtilis. (A, C, E, G, I, K): bright-field images; (B, D, F, H, J, L): fluroescent images. Adapted with permission from ref (65). Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

AIE NPs for in Vivo Imaging Besides in vitro fluorescence imaging, great efforts have also been focused on the in vivo imaging of AIE NPs (68). Actually, many AIE NPs give excellent performances in a variety of in vivo imaging applications, evolving as an alternative fluorescence probe for in vivo use. In this section, the in vivo imaging of AIE NPs will be summarized in terms of in vivo fluorescence imaging of tumors, intravital two-photon fluorescence imaging, in vivo long-term cell tracking as well as in vivo dual-modality imaging. In Vivo Fluorescence Imaging of Tumors In vivo fluorescence imaging of tumors is of undoubtedly vital importance for cancer diagnosis and therapeutics (10). As for such use, fluorescent probes would possess several necessary qualities to meet the requirements: 1) intense emission in the FR/NIR region (>650 nm) that enables bioimaging with low biological background fluorescence and high tissue penetration (69); 2) optimal nanoscale size, as it has been well established that the nanomaterials are capable 226 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


of preferentially accumulating into tumors via the enhanced permeability and retention (EPR) effect, known as "passive" targeting (15); 3) low cytotoxicity and in vivo toxicity; and 4) "active" tumor-targeting ability through functionalizing the surfaces of the probes with tumor-targeting ligands (5). The emergence of AIEgens sweeps away our concerns of emission quenching caused by a high fluorogen loading in NPs, which endows AIEgens with great potential in fabricating NP probes with extremely high fluorescence and photostability. Consequently, AIE NPs become ideal probes applied for in vivo fluorescence imaging.

Figure 7. Chemical structure of 10. (A) Schematic illustration of the preparation of 10-loaded BSA NPs. (B) Time-dependent in vivo non-invasive fluorescence images of H22 tumor-bearing mice post intravenous injection of 10-loaded BSA NPs. To set up the tumor-bearing mouse model, H22 cancer cells were subcutaneously inoculated into the left axillae of the ICR mice. The circle indicate the location of tumor. Adapted with permission from ref (70). Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA. 227 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Taking 10 for example, Tang and co-workers reported a 10-loaded bovine serum albumin (BSA) NP with intense FR/NIR fluorescence (emission maximum at 668 nm) (70). To prepare the NPs, tetrahydrofuran (THF) solution containing 10 was added into the aqueous solution of BSA. During NP formation, the molecules of 10 aggregate and entangle with the hydrophobic sections of BSA biopolymers, becoming the inner part of NPs. On the other hand, the ionized carboxylic groups of BSA serve as the outer layers and stabilize the NPs in aqueous solution, which was confirmed by the negative zeta potential of -29 mV. Finally, the aqueous suspension of 10-loaded BSA NPs were obtained by cross-linking of BSA matrix with glutaraldehyde and removal of THF (Figure 7A). Through tuning the encapsulation amounts of 10 in NPs, the resulted NPs have particle sizes ranging from about 100-150 nm and high quantum yields up to 12% (utilizing Rhodamine 6G in ethanol as the standard). Noteworthy, 10-loaded BSA NPs can selectively visualize tumor tissues at about 28 h after intravenous injection of the NPs into murine hepatoma-22 (H22) tumor-bearing mice (Figure 7B), by virtue of the suitable NP size that enables prolonged blood circulation and prominent EPR effect in vivo. It is also important to note that 10-loaded BSA NPs are able to be easily excreted from the body through biliary pathway, that is, from liver, bile duct, intestine, to feces, indicating that the NPs would not long-term reside in the mice, causing possible in vivo side toxicity. This was testified by the obvious fluorescent signals in the collected feces from tumor-bearing mice post intravenous administration of 10-loaded BSA NPs. For another instance, to further enhance the brightness of 10-loaded BSA NPs in FR/NIR region, a fluorescence resonance energy transfer (FRET) strategy was employed (41). A conjugated polymer, poly[9,9bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorenyldivinylene] (PFV), was selected as the FRET donor owing to its π-conjugated backbones and large absorption coefficients. PFV and 10 (FRET acceptor) were co-encapsulated utilizing BSA as the polymer matrix, affording PFV/10 co-loaded BSA NPs (Figure 8A). Subsequently, the surfaces of the NPs were functionalized by arginine-glycine-aspartic acid (RGD) peptide (Figure 8A), endowing the NPs with active targeting ability, as RGD peptide is a well-acceptable targeting ligand to selectively bind to integrin receptors overexpressed in many a cancer cell (5). The resulted PFV/10 co-loaded BSA-RGD NPs have a large Stokes shift of about 223 nm as well as low cytotoxicity against HT-29 colon cancer cells. More importantly, as compared to 10-loaded BSA NPs, the FR/NIR fluorescence of PFV/10 co-loaded BSA-RGD NPs is amplified for 5.3 times (Figure 8B), thanks to the good spectral overlap and close proximity between PFV and 10 within BSA matrix, which achieve efficient FRET. Using H22 tumor-bearing mice as model animals, PFV/10 co-loaded BSA-RGD NPs and PFV/10 co-loaded BSA NPs (used as a control) were intravenously injected into the mice, respectively, followed by non-invasive in vivo fluorescence imaging. By virtue of the amplified FR/NIR signal and good EPR effect, both NPs with and without RGD functionalization can passively target and clearly visualize the tumors in vivo (Figures 8C and 8D). Noteworthy, the fluorescence signals of the tumors from PFV/10 co-loaded BSA-RGD NP-injected mice are significantly higher as compared to that of tumors from PFV/10 co-loaded BSA NP-injected 228 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


mice at all tested time points after administration (Figures 8C and 8D). As H22 tumors have been reported to overexpress integrin receptors, the in vivo imaging data reasonably illustrate that the PFV/10 co-loaded BSA-RGD NPs can image tumors in vivo in a high-contrast and specific manner due to both active and passive targeting effects.

Figure 8. (A) Chemical structure of PFV and schematic illustration of the fabrication of PFV/10 co-loaded BSA-RGD NPs. (B) Photoluminescence (PL) spectra of various NPs in water. Time-dependent in vivo non-invasive fluorescence images of H22 tumor-bearing mice after intravenous injection of (C) PFV/10 co-loaded BSA NPs and (D) PFV/10 co-loaded BSA-RGD NPs, respectively. The circles in (C) and (D) indicate the locations of tumors. Adapted with permission from ref (41). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Intravital Two-Photon Fluorescence Imaging Two-photon fluorescence imaging is a powerful technique that generates high energy visible fluorescence from low energy irradiation in the NIR region, which allows much deeper imaging of living tissues (up to around 1 millimeter in depth) as compared to conventional one-photon fluorescence imaging (71). For effective two-photon fluorescence imaging, high two-photon absorption (TPA) cross-section (δ) and large two-photon action cross-section (ηδ, η is the fluorescence quantum yield) are needed. So far, many AIE NPs have been 229 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


demonstrated as TPA materials (72–75). Compared with commercially available two-photon imaging agents such as QD-based probes and Evans Blue, several AIE NPs have been proved to possess higher δ and ηδ as well as show better performance in intravital two-photon fluorescence imaging.

Figure 9. Chemical structure of 11. (A) Two-photon absorption spectra of different agents in aqueous solution. (B) Intravital two-photon fluorescence imaging of blood vessels in various tissues from mice after administration of 11-encapsulated DSPE-PEG2000 NPs. Scale bar is 50 μm. Excitation: 800 nm; signal collected at 542±27 nm. Adapted with permission from ref (72). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Taking 11 for example, 11-encapsulated DSPE-PEG2000 NPs were prepared with spherical morphology and a mean hydrodynamic diameter of about 33 nm (72). Thanks to the AIE signature of 11, such AIE NPs show a high η of 62% (using Rhodamine 6G in ethanol as the standard), large δ and ηδ values (higher 230 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


than QD655 and Evans Blue (Figure 9A)), free of random blinking and strong photobleaching resistance. The utility of 11-encapsulated DSPE-PEG2000 NPs in real-time two-photon fluorescence imaging of blood vasculature in the brain, bone marrow and ear skin tissues of living mice was then investigated. From the results depicted in Figure 9B, it is reasonable to conclude that the 11-encapsulated DSPE-PEG2000 NPs can serve as an outstanding two-photon fluorescence imaging agent for in vivo deep-tissue blood vessels imaging application. Finally, the 11-encapsulated DSPE-PEG2000 NPs have been testified to show negligible in vivo toxicity to the mice after intravenous injection, indicating that they are safe probes for in vivo use. Considering these unique advantages, AIE NPs are very promising materials for next generation of intravital two-photon fluorescence imaging probes. In Vivo Long-Term Cell Tracking In addition to good performance in in vitro cancer cell tracing, 8-doped AIE-Tat NPs also show a unique merit in in vivo long-term cancer cell tracking (61). The 8-doped AIE-Tat NP-stained C6 glioma cells were subcutaneously injected into the flank of live mice. As a reference, another group of mice were subcutaneously inoculated with Qtracker® 655-stained C6 cells (C6 cells were pre-incubated with 2 nM 8-doped AIE-Tat NPs or Qtracker® 655 for 4 h). As displayed in Figure 10, 8-doped AIE-Tat NPs can continuously monitor the labeled C6 glioma cells for 21 days, whereas there is almost no detectable fluorescence signal in the Qtracker® 655-stained C6 cells at day 7 post-injection, revealing that 8-doped AIE-Tat NPs possess much superior in vivo cell tracking ability to the most popular commercial cell tracker, Qtracker® 655.

Figure 10. Representative in vivo fluorescence images of the mice after subcutaneously injected with (A) 8-doped AIE-Tat NP-stained and (B) Qtracker® 655-stained C6 glioma cells for designated time intervals. Adapted with permission from ref (61). Copyright 2013 Rights Managed by Nature Publishing Group. 231 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Besides, Liu and co-workers employed 8-doped AIE-Tat NPs to track the fate of stem cells in vivo as well (76). Stem cells are undifferentiated biological cells that can differentiate into specialized cells and possess paracrine effects. In the field of stem cells, one of the most urgent issues is to develop efficient stem cell trackers, which are capable of precisely reporting the fate of transplanted stem cells over a long period of time. Generally, reporter gene labeling and exogenous imaging agent labeling are the main strategies for stem cell tracking (77). The reporter gene labeling method needs to transfect genetic material and modify DNA, which holds the advantage of being able to precisely and quantitatively report the distribution and proliferation of the labeled stem cells. However, this strategy is hard to implement clinically because of several factors in terms of complex cell manipulation, concern of insertional mutagenesis and safety issues, etc.. On the other hand, exogenous imaging agents as stem cell trackers may be more promising for clinical translation. To date, iron oxide nanoparticles have been extensively studied for stem cell tracking through MRI technique. However, they have been reported to suffer from low sensitivity and decreased MRI signal owing to cell proliferation and cell exocytosis (78). As compared to MRI, fluorescence imaging technique shows the advantages of high sensitivity, excellent temporal resolution as well as maneuverable instruments. Considering the unique virtues of AIE NPs in cell tracing application, Liu and co-workers investigated the feasibility of 8-doped AIE-Tat NPs in tracking of the transplanted adipose-derived stem cells (ADSCs) (76). Encouragingly, the 8-doped AIE-Tat NPs exhibit very low cytotoxicity and in vivo side toxicity, excellent retention in living ADSCs as well as negligible interference on ADSC plurpotency (Figure 11A), paracrine (Figure 11B) and in vivo treatment efficacy (Figure 11C). Using ischemic hind limb bearing mice as model animals, the 8-doped AIE-Tat NPs show an excellent performance in precisely and quantitatively reporting the fate of the transplanted ADSCs for 6 weeks (Figure 11D), which represents the longest in vivo cell tracking duration among the currently available exogenous fluorescent cell trackers. Furthermore, through the labeling of 8-doped AIE-Tat NPs, it is found that in the ischemic tissues, the transplanted ADSCs can secrete angiogenic factors and differentiate into necessary cells to participate in neovascularisation, which thus provides important information on how ADSCs contribute to ischemia therapy. Based on these exciting results, it is reasonable to say that AIE NP-based cell trackers hold great promise for potential clinical translation.



Figure 11. (A) Chondrogenic, adipogenic, and osteogenic differentiation capacities and (B) paracrine analyses of ADSCs labeled with and without 8-doped AIE-Tat NPs. (C) Representative photographs of ischemic hind limb-bearing mice after 30 days post treatments with saline, ADSCs labeled with and without 8-doped AIE-Tat NPs, respectively. (D) Representative time-dependent in vivo fluorescence images of the ischemic hind limb-bearing mouse that was intramuscularly injected with 8-doped AIE-Tat NP-labeled ADSCs. Adapted with permission from ref (76). Copyright 2014 American Chemical Society.



Dual-Modality Imaging In recent years, great research interest has been focused on dual-modality imaging, which can overcome the limitations of either imaging modality when used alone (79). The NPs with the capabilities for both fluorescence imaging and MRI are particularly valuable. The main advantages of fluorescence imaging is the high sensitivity, temporal resolution as well as large in vitro and in vivo throughputs even at cellular level; however, the poor anatomical resolution makes fluorescence imaging hard to display the exact spatial location of a three-dimensional (3D) target (80). On the other hand, MRI holds the advantages of excellent spatial resolution that clearly offers 3D information, but MRI suffers from low sensitivity toward contrast agents (81). Therefore, as mentioned above, these two imaging techniques are complementary in principle. Recently, AIE systems have also been extended to enjoy dual functions of fluorescence imaging and MRI. Tang and co-workers developed an AIEgen 12 that consists of a hydrophobic TPE and two hydrophilic gadolinium (Gd) diethylenetriaminepentaacetic acid moieties (Figure 12) (82). It has been well acceptable that Gd-based agents provide positive contrast information under MRI. The amphiphilic 12 self-assembles into highly fluorescent AIE NPs in aqueous solution with a mean size of about 165 nm. Besides fluorescent cell imaging, such dual-functional AIE NPs shows similar longitudinal relaxivity in water as commercial Magnevist®. The in vivo MRI imaging study reveals that the dual-functional AIE NPs can act as a liver specific MRI contrast agent to continuously image liver tissue for at least 150 min (Figure 12).

Figure 12. Chemical structure of 12 and Axial T1-weighted MR images through the liver of rat after intravenous injection of the NPs of 12 (0.1 mmol/kg Gd3+) for designated time intervals. Adapted with permission from ref (82). Copyright 2014 American Chemical Society. 234 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 13. Chemical structure of 13. (A) Schematic illustration of the formation of 13/gold NPs co-loaded DSPE-PEG2000 NPs. (B) In vivo fluorescence images ex vivo fluorescence images of CT26 tumor-bearing mice after intravenous injection of 13/gold NPs co-loaded DSPE-PEG2000 NPs for 6, 12, and 24 h. The arrows and circles indicate tumor regions. 1: Liver; 2: Spleen; 3: Kidney; 4: Heart; 5: Lung; 6: Tumor; 7: Brain; 8: Intestine. (C) CT images (top row: stereo images; bottom row: sectional images) of CT26 tumor-bearing mice before and after intravenous injection of 13/gold NPs co-loaded DSPE-PEG2000 NPs for 6, 12, and 24 h. The circles indicate tumor regions. Adapted with permission from ref (84). Copyright 2014 Elsevier Ltd. 235 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Similarly, X-ray computed tomography (CT) is an imaging modality with high spatial resolution and unlimited penetration depth, but possesses an inherent limitation of low sensitivity (83). Liang and co-workers thus designed and synthesized a complementary dual-modal fluorescence/CT imaging NP probe via co-encapsulating a red emissive AIEgen 13 and gold NPs employing DSPE-PEG2000 as the matrix (Figure 13A) (84). It is known that conventional fluorescent dyes would be significantly quenched by gold NPs; however, thanks to the AIE feature, 13 can effectively overcome the strong fluorescence quenching of shielding-free gold NPs. In vivo imaging studies reveal that the 13/gold NPs co-loaded DSPE-PEG2000 NPs display good tumor-targeting ability and are able to act as a safe and efficient probe for dual-modal fluorescence/CT imaging (Figures 13B and 13C). On the basis of these successful examples, AIEgens can serve as ideal fluorescent materials to be the fluorescent component of dual-modal imaging nanoprobes.

Conclusions and Perspectives In this chapter, recent progress of AIE NPs for in vitro and in vivo imaging has been summarized and discussed. A vast amount of successful in vitro and in vivo examples verify that AIE NPs are highly promising materials for next generation fluorescent probes. As compared to the nanoprobes based on conventional fluorescent materials, AIE NPs show such advantages as remarkably high brightness due to the opposite ACQ effect, strong resistance to photobleaching, nonblinking signature, excellent cellular retention ability, large two-photon cross-section as well as negligible cytotoxicity and in vivo toxicity. In addition, AIE NPs also exhibit merits of facile synthesis, flexible surface functionalization, effective cell uptake and permeability, long blood circulation time as well as passive and active tumor-targeting ability, making them accessible to various bioimaging applications. Future work of AIE NPs for bioimaging will mainly focus on the development of smart and multifunctional AIE NPs. For example, many AIEgens can efficiently generate reactive oxygen species (ROS) under light irradiation (85–87). These AIEgen-based NPs can serve as both emitters and photosensitizers, which would be used for tumor tissue detection and subsequent imaging-guided photodynamic therapy. Moreover, exploration of smart stimuli-responsive (i.e., enzyme, pH, or ROS) AIE NPs that can selectively activate the fluorescence in the disease sites is also highly desirable. Although some efforts have been devoted to develop AIE-active dual-modal imaging NPs, there is still large room for creating advanced nanoprobes. We believe AIE NPs have been, are, and will provide the scientists and clinicians with new sights and insights in the areas of life science and biomedical engineering.

Acknowledgments The authors gratefully acknowledge financial support provided by the National Natural Science Foundation of China (31571011). 236 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

References 1. 2. 3.


4.

5.

6.

7.

8.

9.

10.

11.

12. 13. 14. 15. 16.

Chen, M.; Yin, M. Design and development of fluorescent nanostructures for bioimaging. Prog. Polym. Sci. 2014, 39, 365–395. Li, K.; Liu, B. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging. Chem. Soc. Rev. 2014, 43, 6570–6597. Peng, H. S.; Chiu, D. T. Soft fluorescent nanomaterials for biological and biomedical imaging. Chem. Soc. Rev. 2015, 44, 4699–4722. Olson, E. S.; Jiang, T.; Aguilera, T. A.; Nguyen, Q. T.; Ellies, L. G.; Scadeng, M.; Tsien, R. Y. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4311–4316. Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotechnol. 2007, 2, 47–52. Wu, W.; Li, R.; Bian, X.; Zhu, Z.; Ding, D.; Li, X.; Jia, Z.; Jiang, X.; Hu, Y. Covalently combining carbon nanotubes with anticancer agent: preparation and antitumor activity. ACS Nano 2009, 3, 2740–2750. Wu, T. J.; Tzeng, Y. K.; Chang, W. W.; Cheng, C. A.; Kuo, Y.; Chien, C. H.; Chang, H. C.; Yu, J. Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds. Nat. Nanotechnol. 2013, 8, 682–689. Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 2010, 110, 2620–2640. Shu, X. K.; Royant, A.; Lin, M. Z.; Aguilera, T. A.; Lev-Ram, V.; Steinbach, P. A.; Tsien, R. Y. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 2009, 324, 804–807. Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Nanotechnol. 2004, 22, 969–976. Li, K.; Pan, J.; Feng, S. S.; Wu, A. W.; Pu, K. Y.; Liu, Y.; Liu, B. Generic strategy of preparing fluorescent conjugated polymer-loaded poly (DL-lactide-co-glycolide) nanoparticles for targeted cell imaging. Adv. Funct. Mater. 2009, 19, 3535–3542. Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 2004, 4, 11–18. Fery-Forgues, S. Fluorescent organic nanocrystals and non-doped nanoparticles for biological applications. Nanoscale 2013, 5, 8428–8442. Li, K.; Liu, B. Polymer encapsulated conjugated polymer nanoparticles for fluorescence bioimaging. J. Mater. Chem. 2012, 22, 1257–1264. Farokhzad, O. C.; Langer, R. Impact of nanotechnology on drug delivery. ACS Nano 2009, 3, 16–20. Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Aggregation-induced emission: Phenomenon, mechanism and applications. Chem. Commun. 2009, 4332–4353. 237 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


17. Luo, J. D.; Xie, Z. L.; Lam, J. W. Y.; Cheng, L.; Chen, H. Y.; Qiu, C. F.; Kwok, H. S.; Zhan, X. W.; Liu, Y. Q.; Zhu, D. B.; Tang, B. Z. Aggregationinduced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. 2001, 1740–1741. 18. Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Aggregation-induced emission. Chem. Soc. Rev. 2011, 40, 5361–5388. 19. Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-induced emission: Together we shine, united we soar. Chem. Rev. 2015, 115, 11718–11940. 20. Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregationinduced emission: The whole is more brilliant than the parts. Adv. Mater. 2014, 26, 5429–5479. 21. Ferrari, M. Cancer nanotechnology: Opportunities and challenges. Nat. Rev. Cancer 2005, 5, 161–171. 22. Li, Z.; Zheng, M.; Guan, X.; Xie, Z.; Huang, Y.; Jing, X. Unadulterated BODIPY-dimer nanoparticles with high stability and good biocompatibility for cellular imaging. Nanoscale 2014, 6, 5662–5665. 23. Faisal, M.; Hong, Y.; Liu, J.; Yu, Y.; Lam, J. W. Y.; Qin, A.; Lu, P.; Tang, B. Z. Fabrication of fluorescent silica nanoparticles hybridized with AIE luminogens and exploration of their applications as nanobiosensors in intracellular imaging. Chem. Eur. J. 2010, 16, 4266–4272. 24. Mahtab, F.; Lam, J. W. Y.; Yu, Y.; Liu, J.; Yuan, W. Z.; Lu, P.; Tang, B. Z. Covalent immobilization of aggregation‐induced emission luminogens in silica nanoparticles through click reaction. Small 2011, 7, 1448–1455. 25. Zhang, X.; Zhang, X.; Yang, B.; Liu, L.; Hui, J.; Liu, M.; Chen, Y.; Wei, Y. Aggregation-induced emission dye based luminescent silica nanoparticles: Facile preparation, biocompatibility evaluation and cell imaging applications. RSC Adv. 2014, 4, 10060–10066. 26. Wu, W. C.; Chen, C. Y.; Tian, Y.; Jang, S. H.; Hong, Y.; Liu, Y.; Hu, R.; Tang, B. Z.; Lee, Y. T.; Chen, C. T.; Chen, W. C.; Jen, A. K. Y. Enhancement of aggregation-induced emission in dye-encapsulating polymeric micelles for bioimaging. Adv. Funct. Mater. 2010, 20, 1413–1423. 27. Zhang, X.; Zhang, X.; Yang, B.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Fabrication of aggregation induced emission dye-based fluorescent organic nanoparticles via emulsion polymerization and their cell imaging applications. Polym. Chem. 2014, 5, 399–404. 28. Liu, M.; Zhang, X.; Yang, B.; Liu, L.; Deng, F.; Zhang, X.; Wei, Y. Polylysine crosslinked AIE dye based fluorescent organic nanoparticles for biological imaging applications. Macromol. Biosci. 2014, 14, 1260–1267. 29. Li, H.; Zhang, X.; Zhang, X.; Yang, B.; Yang, Y.; Wei, Y. Ultra-stable biocompatible cross-linked fluorescent polymeric nanoparticles using AIE chain transfer agent. Polym. Chem. 2014, 5, 3758–3762. 30. Li, H.; Zhang, X.; Zhang, X.; Yang, B.; Yang, Y.; Wei, Y. Stable cross-linked fluorescent polymeric nanoparticles for cell imaging. Macromol. Rapid Commun. 2014, 35, 1661–1667.



31. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. A novel method for preparing AIE dye based cross-linked fluorescent polymeric nanoparticles for cell imaging. Polym. Chem. 2014, 5, 683–688. 32. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. Facile preparation and cell imaging applications of fluorescent organic nanoparticles that combine AIE dye and ring-opening polymerization. Polym. Chem. 2014, 5, 318–322. 33. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. PEGylation and cell imaging applications of AIE based fluorescent organic nanoparticles via ring-opening reaction. Polym. Chem. 2014, 5, 689–693. 34. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Wei, Y. Facile fabrication of AIE-based stable cross-linked fluorescent organic nanoparticles for cell imaging. Colloids Surf., B 2014, 116, 739–744. 35. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. Novel biocompatible cross-linked fluorescent polymeric nanoparticles based on an AIE monomer. J. Mater. Chem. C 2014, 2, 816–820. 36. Zhang, X.; Zhang, X.; Yang, B.; Yang, Y.; Wei, Y. Renewable itaconic acid based cross-linked fluorescent polymeric nanoparticles for cell imaging. Polym. Chem. 2014, 5, 5885–5889. 37. Ding, D.; Pu, K. Y.; Li, K.; Liu, B. Conjugated oligoelectrolyte-polyhedral oligomeric silsesquioxane loaded pH-responsive nanoparticles for targeted fluorescence imaging of cancer cell nucleus. Chem. Commun. 2011, 47, 9837–9839. 38. Li, K.; Jiang, Y.; Ding, D.; Zhang, X.; Liu, Y.; Hua, J.; Feng, S. S.; Liu, B. Folic acid-functionalized two-photon absorbing nanoparticles for targeted MCF-7 cancer cell imaging. Chem. Commun. 2011, 47, 7323–7325. 39. Geng, J.; Li, K.; Qin, W.; Ma, L.; Gurzadyan, G. G.; Tang, B. Z.; Liu, B. Eccentric loading of fluorogen with aggregation-induced emission in PLGA matrix increases nanoparticle fluorescence quantum yield for targeted cellular imaging. Small 2013, 9, 2012–2019. 40. Lu, H.; Zhao, X.; Tian, W.; Wang, Q.; Shi, J. Pluronic F127–folic acid encapsulated nanoparticles with aggregation-induced emission characteristics for targeted cellular imaging. RSC Adv. 2014, 4, 18460–18466. 41. Ding, D.; Li, K.; Qin, W.; Zhan, R.; Hu, Y.; Liu, J.; Tang, B. Z.; Liu, B. Conjugated polymer amplified far-red/near-infrared fluorescence from nanoparticles with aggregation-induced emission characteristics for targeted in vivo imaging. Adv. Healthcare Mater. 2013, 2, 500–507. 42. Li, M.; Lam, J. W. Y.; Mahtab, F.; Chen, S.; Zhang, W.; Hong, Y.; Xiong, J.; Zheng, Q.; Tang, B. Z. Biotin-decorated fluorescent silica nanoparticles with aggregation-induced emission characteristics: Fabrication, cytotoxicity and biological applications. J. Mater. Chem. B 2013, 1, 676–684. 43. Leamon, C. P.; Reddy, J. A. Folate-targeted chemotherapy. Adv. Drug Delivery Rev. 2004, 56, 1127–1141. 44. Hoye, A. T.; Davoren, J. E.; Wipf, P.; Fink, M. P.; Kagan, V. E. Targeting mitochondria. Acc. Chem. Res. 2008, 41, 87–97. 239 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


45. Settembre, C.; Fraldi, A.; Medina, D. L.; Ballabio, A. Signals from the lysosome: A control centre for cellular clearance and energy metabolism. Nat. Rev. Mol. Cell Biol. 2013, 14, 283–296. 46. Ow, Y. L. P.; Green, D. R.; Hao, Z.; Mak, T. W. Cytochrome c: Functions beyond respiration. Nat. Rev. Mol. Cell Biol. 2008, 9, 532–542. 47. Gardner, A.; Boles, R. G. Is a “mitochondrial psychiatry” in the future? A review. Curr. Psychiatry Rev. 2005, 1, 255–271. 48. Leung, C. W. T.; Hong, Y.; Chen, S.; Zhao, E.; Lam, J. W. Y.; Tang, B. Z. A photostable AIE luminogen for specific mitochondrial imaging and tracking. J. Am. Chem. Soc. 2013, 135, 62–65. 49. Zhao, N.; Li, M.; Yan, Y.; Lam, J. W. Y.; Zhang, Y. L.; Zhao, Y. S.; Wong, K. S.; Tang, B. Z. A tetraphenylethene-substituted pyridinium salt with multiple functionalities: synthesis, stimuli-responsive emission, optical waveguide and specific mitochondrion imaging. J. Mater. Chem. C 2013, 1, 4640–4646. 50. Zhao, N.; Chen, S.; Hong, Y.; Tang, B. Z. A red emitting mitochondriatargeted AIE probe as an indicator for membrane potential and mouse sperm activity. Chem. Commun. 2015, 51, 13599–13602. 51. Zhang, W.; Kwok, R. T. K.; Chen, Y.; Chen, S.; Zhao, E.; Yu, C. Y. Y.; Lam, J. W. Y.; Zheng, Q.; Tang, B. Z. Synthesis and aggregation-induced emission properties of pyridine and pyridinium analogues of tetraphenylethylene. Chem. Commun. 2015, 51, 9022–9025. 52. Gao, M.; Sim, C. K.; Leung, C. W. T.; Hu, Q.; Feng, G.; Xu, F.; Tang, B. Z.; Liu, B. A fluorescent light-up probe with AIE characteristics for specific mitochondrial imaging to identify differentiating brown adipose cells. Chem. Commun. 2015, 51, 8312–8315. 53. Gao, M.; Hu, Q.; Feng, G.; Tang, B. Z.; Liu, B. A fluorescent light-up probe with “AIE+ ESIPT” characteristics for specific detection of lysosomal esterase. J. Mater. Chem. B 2014, 2, 3438–3442. 54. Wang, E.; Zhao, E.; Hong, Y.; Lam, J. W. Y.; Tang, B. Z. A highly selective AIE fluorogen for lipid droplet imaging in live cells and green algae. J. Mater. Chem. B 2014, 2, 2013–2019. 55. Daly, C. J.; McGrath, J. C. Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol. Ther. 2003, 100, 101–118. 56. Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M. Use of quantum dots for live cell imaging. Nat. Methods 2004, 1, 73–78. 57. Muller-Borer, B. J.; Collins, M. C.; Gunst, P. R.; Cascio, W. E.; Kypson, A. P. Quantum dot labeling of mesenchymal stem cells. J. Nanobiotechnol. 2007, 5, 9. 58. Yang, K.; Li, Z.; Cao, Y.; Yu, X.; Mei, J. Effect of peptide-conjugated near-infrared fluorescent quantum dots (NIRF-QDs) on the invasion and metastasis of human tongue squamous cell carcinoma cell line Tca8113 in vitro. Int. J. Mol. Sci. 2009, 10, 4418–4427. 59. Smith, A. M.; Duan, H.; Mohs, A. M.; Nie, S. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Delivery Rev. 2008, 60, 1226–1240.



60. Wang, Z.; Chen, S.; Lam, J. W. Y.; Qin, W.; Kwok, R. T. K.; Xie, N.; Hu, Q.; Tang, B. Z. Long-term fluorescent cellular tracing by the aggregates of AIE bioconjugates. J. Am. Chem. Soc. 2013, 135, 8238–8245. 61. Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J.; Liu, R.; Liu, J.; Zhang, X.; Liu, H.; Liu, B.; Tang, B. Z. Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing. Sci. Rep. 2013, 3, 1150. 62. Feng, G.; Tay, C. Y.; Chui, Q. X.; Liu, R.; Tomczak, N.; Liu, J.; Tang, B. Z.; Leong, D. T.; Liu, B. Ultrabright organic dots with aggregation-induced emission characteristics for cell tracking. Biomaterials 2014, 35, 8669–8677. 63. Li, K.; Zhu, Z.; Cai, P.; Liu, R.; Tomczak, N.; Ding, D.; Liu, J.; Qin, W.; Zhao, Z.; Hu, Y.; Chen, X.; Tang, B. Z.; Liu, B. Organic dots with aggregation-induced emission (AIE dots) characteristics for dual-color cell tracing. Chem. Mater. 2013, 25, 4181–4187. 64. Qin, W.; Li, K.; Feng, G.; Li, M.; Yang, Z.; Liu, B.; Tang, B. Z. Bright and photostable organic fluorescent dots with aggregation-induced emission characteristics for noninvasive long-term cell imaging. Adv. Funct. Mater. 2014, 24, 635–643. 65. Zhao, E.; Hong, Y.; Chen, S.; Leung, C. W. T.; Chan, C. Y. K.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Highly fluorescent and photostable probe for long-term bacterial viability assay based on aggregation-induced emission. Adv. Healthcare Mater. 2014, 3, 88–96. 66. Zhao, E.; Chen, Y.; Chen, S.; Deng, H.; Gui, C.; Leung, C. W. T.; Hong, Y.; Lam, J. W. Y.; Tang, B. Z. A luminogen with aggregation-induced emission characteristics for wash-free bacterial imaging, high-throughput antibiotics screening and bacterial susceptibility evaluation. Adv. Mater. 2015, 27, 4931–4937. 67. Zhao, E.; Chen, Y.; Wang, H.; Chen, S.; Lam, J. W. Y.; Leung, C. W. T.; Hong, Y.; Tang, B. Z. Light-enhanced bacterial killing and wash-free imaging based on AIE fluorogen. ACS Appl. Mater. Interfaces 2015, 7, 7180–7188. 68. Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes based on AIE fluorogens. Acc. Chem. Res. 2013, 46, 2441–2453. 69. Frangioni, J. V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 2003, 7, 626–634. 70. Qin, W.; Ding, D.; Liu, J.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang, B. Z. Biocompatible nanoparticles with aggregation-induced emission characteristics as far-red/near-infrared fluorescent bioprobes for in vitro and in vivo imaging applications. Adv. Funct. Mater. 2012, 22, 771–779. 71. Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 2003, 300, 1434–1436. 72. Ding, D.; Goh, C. C.; Feng, G.; Zhao, Z.; Liu, J.; Liu, R.; Tomczak, N.; Geng, J.; Tang, B. Z.; Ng, L. G.; Liu, B. Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging. Adv. Mater. 2013, 25, 6083–6088. 73. Gao, Y.; Feng, G.; Jiang, T.; Goh, C. C.; Ng, L. G.; Liu, B.; Li, B.; Yang, L.; Hua, J.; Tian, H. Biocompatible nanoparticles based on 241 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

74.

75.


76.

77.

78.

79.

80.

81.

82.

83. 84.

diketo-pyrrolo-pyrrole (DPP) with aggregation-induced Red/NIR emission for in vivo two-photon fluorescence imaging. Adv. Funct. Mater. 2015, 25, 2857–2866. Geng, J.; Goh, C. C.; Qin, W.; Liu, R.; Tomczak, N.; Ng, L. G.; Tang, B. Z.; Liu, B. Silica shelled and block copolymer encapsulated red-emissive AIE nanoparticles with 50% quantum yield for two-photon excited vascular imaging. Chem. Commun. 2015, 51, 13416–13419. Qian, J.; Zhu, Z.; Qin, A.; Qin, W.; Chu, L.; Cai, F.; Zhang, H.; Wu, Q.; Hu, R.; Tang, B. Z.; He, S. High-order non-linear optical effects in organic luminogens with aggregation-induced emission. Adv. Mater. 2015, 27, 2332–2339. Ding, D.; Mao, D.; Li, K.; Wang, X.; Qin, W.; Liu, R.; Chiam, D. S.; Tomczak, N.; Yang, Z.; Tang, B. Z.; Kong, D.; Liu, B. Precise and long-term tracking of adipose-derived stem cells and their regenerative capacity via superb bright and stable organic nanodots. ACS Nano 2014, 8, 12620–12631. Jokerst, J. V.; Thangaraj, M.; Kempen, P. J.; Sinclair, R.; Gambhir, S. S. Photoacoustic imaging of mesenchymal stem cells in living mice via silicacoated gold nanorods. ACS Nano 2012, 6, 5920–5930. Xu, C.; Miranda-Nieves, D.; Ankrum, J. A.; Matthiesen, M. E.; Phillips, J. A.; Roes, I.; Wojtkiewicz, G. R.; Juneja, V.; Kultima, J. R.; Zhao, W.; Vemula, P. K.; Lin, C. P.; Nahrendorf, M.; Karp, J. M. Tracking mesenchymal stem cells with iron oxide nanoparticle loaded poly (lactide-co-glycolide) microparticles. Nano Lett. 2012, 12, 4131–4139. Nam, T.; Park, S.; Lee, S. Y.; Park, K.; Choi, K.; Song, I. C.; Han, M. H.; Leary, J. J.; Yuk, S. A.; Kwon, I. C.; Kim, K.; Jeong, S. Y. Tumor targeting chitosan nanoparticles for dual-modality optical/MR cancer imaging. Bioconjugate Chem. 2010, 21, 578–582. Ding, D.; Wang, G.; Liu, J.; Li, K.; Pu, K. Y.; Hu, Y.; Ng, J. C. Y.; Tang, B. Z.; Liu, B. Hyperbranched conjugated polyelectrolyte for dual-modality fluorescence and magnetic resonance cancer imaging. Small 2012, 8, 3523–3530. Howes, P.; Green, M.; Bowers, A.; Parker, D.; Varma, G.; Kallumadil, M.; Hughes, M.; Warley, A.; Brain, A.; Botnar, R. Magnetic conjugated polymer nanoparticles as bimodal imaging agents. J. Am. Chem. Soc. 2010, 132, 9833–9842. Chen, Y.; Li, M.; Hong, Y.; Lam, J. W. Y.; Zheng, Q.; Tang, B. Z. Dualmodal MRI contrast agent with aggregation-induced emission characteristic for liver specific imaging with long circulation lifetime. ACS Appl. Mater. Interfaces 2014, 6, 10783–10791. Lee, N.; Choi, S. H.; Hyeon, T. Nano-sized CT contrast agents. Adv. Mater. 2013, 25, 2641–2660. Zhang, J.; Li, C.; Zhang, X.; Huo, S.; Jin, S.; An, F. F.; Wang, X.; Xue, X.; Okeke, C. I.; Duan, G.; Guo, F.; Zhang, X.; Hao, J.; Wang, P. C.; Zhang, J.; Liang, X. J. In vivo tumor-targeted dual-modal fluorescence/CT imaging using a nanoprobe co-loaded with an aggregation-induced emission dye and gold nanoparticles. Biomaterials 2015, 42, 103–111. 242 Fujiki et al.; Aggregation-Induced Emission: Materials and Applications Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


85. Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B. Targeted and image-guided photodynamic cancer therapy based on organic nanoparticles with aggregation-induced emission characteristics. Chem. Commun. 2014, 50, 8757–8760. 86. Yuan, Y.; Zhang, C. J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific lightup bioprobe with aggregation-induced emission and activatable photoactivity for the targeted and image-guided photodynamic ablation of cancer cells. Angew. Chem., Int. Ed. 2015, 54, 1780–1786. 87. Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Targeted bioimaging and photodynamic therapy of cancer cells with an activatable red fluorescent bioprobe. Anal. Chem. 2014, 86, 7987–7995.


AIE Nanoparticles for in Vitro and in Vivo Imaging - American

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