Organic Mitoprobes based on Fluorogens with Aggregation...

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DOI: 10.1002/ijch.201800031

Organic Mitoprobes based on Fluorogens withAggregation-Induced EmissionXiaolei Cai,[a] Fang Hu,[a] Guangxue Feng,[a] Ryan Tsz Kin Kwok,[b] Bin Liu,*[a] and Ben Zhong Tang*[b]

Abstract: Fluorescence imaging of mitochondria is of greatinterest to understand its functions and roles in variouscritical bioprocesses. Conventional fluorescence reagents formitochondrial imaging and tracking suffer from variousproblems such as poor photostability and high cytotoxicity.The emerging of fluorogens with aggregation-induced emis-sion (AIE) property provides great opportunities to develop

mitoprobes with high specificity, good photostability andmultiple functions for mitochondrial imaging and tracking.This review summarizes the recent advance of AIE mitop-robes, including the design principle for AIE mitoprobes, theapplications of AIE mitoprobes for super-resolution mito-chondrial imaging and tracking, as well as the therapeuticfunctions of AIE mitoprobes for cancer cell ablation.

Keywords: aggregation-induced emission · mitochondria · fluorescence imaging · super-resolution imaging · image-guided therapy

1. Introduction

The origin of extant mitochondria can be traced to symbioticarrangements involving a-proteobacteria invasion to host cellsthat give rise to eukaryotic cells, which is supported bygenome analysis.[1,2] Although this interesting endosymbionthypothesis has not been totally confirmed yet, mitochondriado play critical roles in eukaryotic cells. The most well-knownfunction of mitochondria is to produce adenosine triphosphate(ATP) through citric cycle[3,4] to supply chemical energy forcomplicated metabolic processes.[5] Recent studies also re-vealed that mitochondria are also involved in reactive oxygenspecies (ROS) regulation,[6–8] calcium homeostasis[9–11] and cellapoptosis.[2,12] It is expected that the mitochondrial dysfunc-tions would give rise to a convenient and predictable set ofdefects in all tissues. However, mitochondrial dysfunction hasdiversiform effects in multicellular organelles.[13] As a con-sequence, tremendous efforts have been made to monitor andstudy them. In the early stage, the properties and functions ofmitochondria have been elaborately studied as separatedorganelles.[14–16] However, mitochondria are highly dynamicorganelles with fission-fusion events and frequent communica-tion with other intracellular organelles,[17] posing severechallenges for further exploration. Fortunately, advancedmicroscopies allow us to visualize cellular organelles in livingcells with spatial resolution of micrometer[18] or even nano-meter,[19,20] which offers good opportunity to understand theirfunctions and relationships with other cell components easily.

Amongst numerous imaging modalities, fluorescenceimaging is one of the most powerful tools for visualizing andanalysing the distribution and dynamics of mitochondria. Itutilizes fluorescent agents to label mitochondria with real-timeoperation, non-invasive testing and cost-effective perform-ance.[21] A variety of fluorescent agents, including quantumdots (QDs),[22] organometallic compounds,[23] fluorescent pro-teins (FPs),[24] organic dyes,[25] and so on, have been developed

and used in mitochondrial dynamism tracking and monitoring.Amongst them, QDs are highly emissive and photostable,surface-modified QDs with target peptides/proteins are effec-tive fluorescent markers,[22] but most of them (e. g., CdSe andPbS) are inherently cytotoxic and they often blink.[26] Organo-metallic compounds also suffer from cytotoxicity.[27] FPs havereceived considerable attention because they can be genet-ically encoded to mitochondria,[28] but the use of FPs requirestransfection processes and their labelling sometimes candisrupt normal cell functions.[29] Therefore, organic dyes aremost widely used and easily accessible with good biocompat-ibility for mitochondria imaging. Simple combination offluorophores with mitochondria specific ligands can lead tomitochondria probes (mitoprobes). Some other mito-probeswere designed to be selectively oxidized for light-upmitochondria imaging. The absorption and emission wave-lengths could also be easily regulated through altering theluminescent organic dyes.[30] However, typical organic dyesexhibit serious fluorescence quenching at high concentrationsdue to aggregation-caused quenching (ACQ) and show poorphotostability at low concentrations. Meanwhile, most oftraditional fluorophores show narrow Stokes shifts. All thesedisadvantages greatly limited their applications for bio-imaging.[31]

[a] X. Cai, F. Hu, G. Feng, B. LiuDepartment of Chemical and Biomolecular Engineering, NationalUniversity of Singapore, 4 Engineering Drive 4, Singapore 117585E-mail: [email protected]

[b] R. T. K. Kwok, B. Z. TangDepartment of Chemistry, Hong Kong Branch of Chinese NationalEngineering Research Center for Tissue Restoration and Re-construction, Institute of Molecular Functional Materials, Institutefor Advanced Study and Division of Life Science, The Hong KongUniversity of Science and Technology, Clear Water Bay, Kowloon,Hong Kong, ChinaE-mail: [email protected]

Isr. J. Chem. 2018, 58, 860 –873 © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 860

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Recently, the rapid development of fluorogens withaggregation-induced emission (AIE) characteristics (AIEgens)creates new expectations and opportunities in the biomedicalfield due to their outstanding fluorescent property that isopposite to ACQ dyes.[31–35] Their fluorescence is negligible inmolecularly dissolved state, but booms dramatically inaggregate state or at high concentrations. This abnormalphenomenon is ascribed to the intrinsic propeller-shapestructures of AIEgens.[36] For example, tetraphenylethene(TPE) is an iconic AIEgen (Figure 1A), in which the freerotation and vibration of the phenyl rings consume energyfrom excited states via radiationless decay. When TPE is inaggregate state or at high concentration, its intramolecularmotions are more or less inhibited, resulting in the eliminationof non-radiative decay and enhanced fluorescence. In addition,some specially designed AIEgens may also exhibit excited-state intramolecular proton transfer (ESIPT) phenomenon.[37]

For example, (2-hydroxy-4-methoxyphenyl)(phenyl)-metha-none azine (HMPA) is weakly emissive in dilute solution, inwhich the intramolecular motions are not severely restricted.In aggregate state or at high concentrations, the formation ofintramolecular H-bonds in HMPA greatly inhibit the intra-molecular motions and thus promote radiative decay to givestrong green fluorescence (Figure 1B).[38] Benefiting from the

strong aggregate-state fluorescence, AIEgens enable to beused at high concentrations to ensure reliable signals and highphotostability. Meanwhile, AIEgens also enjoy tunable emis-sion colors[39] and large Stokes shift[37] which can avoid self-absorption when used as luminescent agents. So far, AIEgenshave been widely applied for chemical and biologicalsensing[40,41] and imaging[35] including mitochondria trackingand mitochondria-related bioprocess monitoring.[42] In addi-tion, the accumulation of some AIEgens on mitochondria caninduce mitochondrial dysfunctions to ablate cancer cells.[43]

Furthermore, rational design of AIEgens can endow them withsinglet oxygen generation ability in the presence of oxygenupon light irradiation, which makes them qualified to serve asphotosensitizers for photodynamic cancer cell ablation.[44–49]

Since mitochondria play an important role in cellularmetabolism and are directly related to cell apoptosis, thechemo-toxicity and photo-toxicity of properly designedAIEgens to mitochondria make them highly efficient therapeu-tic agents for cancer cell ablation.[50]

In this review, we summarize the applications of mitochon-dria-targeting probes (mitoprobes) based on AIEgen formitochondria tracking, mitochondria-related process monitor-ing and cancer cell ablation. Firstly, we discuss the emissioncolor tuning and design principles of AIE mitoprobes.

Dr. Xiaolei Cai received his PhD degree in 2017from National University of Singapore (NUS).He is currently a postdoctoral research fellowin Prof. Bin Liu’s group at the Department ofChemical and Biomolecular Engineering(ChBE) in NUS. His research focuses ondeveloping organic nanomaterials forfluorescence imaging, photoacoustic imaging,and image-guided therapy.

Dr. Fang Hu received his PhD degree from theInstitute of Chemistry, Chinese Academy ofSciences in 2015. Now he is a postdoctoralresearch fellow in Professor Bin Liu’s group atthe Department of ChBE in NUS. His researchfocuses on the development of fluorogens withaggregation-induced emission characteristicsfor monitoring of biological processes andimage-guided therapy.

Dr. Guangxue Feng received his Ph.D. degreein 2015 from NUS. He is currently a postdoc-toral research fellow in Professor Bin Liu’sgroup at the Department of ChBE in NUS. Hisresearch focuses on the design of organicnanoparticles for biological and biomedicalapplications.

Dr. Ryan T. K. Kwok received his PhD degree in2009 from the Hong Kong University of Scienceand Technology (HKUST). He conducted hispostdoctoral work in Professor Ben ZhongTang’s group. He is currently Research Assis-tant Professor of the Department of Chemistryand Junior Fellow of HKUST Institute forAdvanced Study. His research focuses ondevelopment of AIE luminescent materials andexploration of their biological applications.

Prof. Bin Liu received her Ph.D. degree fromNUS and conducted her postdoctoral work atUniversity of California, Santa Barbara. She iscurrently the Head and Chair Professor ofChemical and Biomolecular Engineering De-partment of NUS. She focuses on organicnanomaterials for biomedical and energy appli-cations. She is a Fellow of Royal Society ofChemistry and Singapore Academy of Engineer-ing, and serves as an associate editor forPolymer Chemistry.

Prof. Ben Zhong Tang received his PhD degreefrom Kyoto University and conducted hispostdoctoral work at the University of Toronto.He is Chair Professor in the Department ofChemistry and Chemical and Biological Engi-neering of HKUST. He was elected to theChinese Academy of Sciences in 2009 and theRoyal Society of Chemistry in 2013. Hisresearch interests include macromolecularchemistry, materials science, and biomedicaltheranostics. He is now serving as Editor-in-Chief of Materials Chemistry Frontiers.

Isr. J. Chem. 2018, 58, 860 –873 © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.ijc.wiley-vch.de 861

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Secondly, we demonstrate the utilization of mitoprobes formitochondria dynamism tracking with high- and super-resolution imaging, mitochondria-related bioprocess such asmitophagy monitoring and mitochondrial membrane potentialsensing. Lastly, we summarize the mitochondria-specificimage-guided cancer cell ablation including chemotherapy andphotodynamic therapy (PDT). Considering the importance ofmitochondria in cellular metabolism, aging and diseasedevelopment, the emergence of AIE mitoprobes with highperformance not only helps to visualize mitochondrialstructures, but also provides great opportunities for diseasediagnosis and therapy.

2. Design of AIE Mitoprobes

Resulting from the redox process for ATP production, there isa constant membrane potential of around �180 mV acrossouter and inner mitochondrial membranes.[51] This negativepotential is much higher than that in other organelles andprovides an opportunity to design positive chemical ligandsfor specific targeting mitochondria through electrostaticinteractions. Triphenylphosphonium (TPP)[52] and pyridi-nium[53] groups bearing positive charges are generally deco-rated on fluorescent dyes for probing mitochondria. Mean-while, ammonium,[54] isoquinolinium[55] and indolium[56] canalso serve as mitochondrial delivery vehicles following asimilar mechanism. The chemical structures of these positivelycharged ligands are shown in Figure 2A. A number of AIEmitoprobes with high photostability and specificity have beendeveloped by integrating AIEgens with the targeting ligands.

As the emission color of AIEgens can be fine-tuned with awide range from blue to the second near-infrared win-dow,[39,57,58] so far, AIE mitoprobes with different emission

colors have been developed. Among them, TPE,[37] salicylal-dehyde azine (SAA) are typical AIEgens with greenish blueand green emission colors, respectively. The red-shift inemission of AIE mitoprobes can be accomplished by introduc-ing electron donating/withdrawing groups to AIE units.Electron donating groups can increase the highest occupiedmolecular orbital (HOMO) levels of AIEgens while electronwithdrawing groups can decrease the lowest unoccupiedmolecular orbital (LUMO) levels of AIEgens, both can reducethe HOMO-LUMO gaps to result in red-shifted absorption andemission spectra.[59] As shown in Figure 2B, TPE is greenishblue emissive with a emission peak at 460 nm. Afterdecoration with two electron donating methoxy groups, theemission peak of obtained 2MeOTPE is slightly red-shifted to480 nm. The decoration of electron withdrawing groups canalso red shift the emissions of TPE-based AIEgens. Forexample, conjugation of isoquinolinium makes the emission ofTPE-IQ red-shifted to green color with a peak at 502 nm.[55]

Stronger electron withdrawing groups can result in further red-shifted emission. By introducing electron-withdrawing vinyldicyano,[39] vinyl pyridinium,[60] and indolium groups,[61] theobtained TPECN, TPE-Py, and TPE-In exhibit graduallyred-shifted emissions at 564 nm, 600 nm, and 650 nm,respectively. Furthermore, the integration of both electrondonating and withdrawing groups can lead to more red-shiftedAIEgens due to the formation of donor-acceptor structures.For example, after introducing two electron donating methoxygroups, TPE-IQ, TPECN, TPE-Py were turned to TPE-IQ-2O,[62] TPECM,[63] AIE-Red,[64] respectively. And theiremission peaks are red-shifted from 502 nm, 564 nm, 600 nmto 620 nm, 600 nm, 660 nm, respectively. Therefore, theemission color of AIE mitoprobes can be easily tuned from460 nm to 660 nm, almost covering the whole visible region.

Figure 1. Chemical structures of TPE (A) and HMPA (B), and their AIE-active mechanisms.


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3. Mitochondrial Imaging and Tracking by AIEMitoprobes

Based on the design principles illustrated in the previoussection, numerous AIE mitoprobes with tunable emissioncolors have been developed, which provide great opportunitiesto realize polychromatic imaging and tracking of mitochondriawith high and super-resolution and to achieve real-timemonitoring of mitochondrial activities. In the followingsection, we summarize the recent development and imagingapplications of AIE mitoprobes in detail with selectedexamples.

3.1 Polychromatic Imaging of Mitochondria

The first design and utilization of AIEgens for mitochondrialtargeted imaging and tracking was reported in 2012.[65] Byincorporating the mitochondrial targeting ligand, TPP, to TPE,the first AIE mitoprobe called TPE-TPP was synthesized,showing typical AIE property with bright blue emissionpeaked at 466 nm in solid state. TPE-TPP could target themitochondria of HeLa cells and light them up with bright bluefluorescence (Figure 3A). After co-staining the cells usingcommercial MitoTracker® Red FM, a perfect overlap could be

observed with a Pearson’s correlation coefficient of 0.96,indicating the high targeting specificity of TPE-TPP tomitochondria. More importantly, TPE-TPP obviously excelsMitoTracker® Red FM in terms of photostability. Undercontinuous laser scanning by the confocal microscope, TPE-TPP exhibits less than 20% signal loss even after 50 scans,while MitoTracker® red FM already losses more than 75 % ofits fluorescent signals after 6 scans. These observationsindicate that AIE mitoprobes could serve as betterfluorescence trackers for mitochondrial imaging and monitor-ing as compared to conventional dyes.

Subsequently, AIE mitoprobes with red-shifted excitationand emission wavelengths were developed, attributing to theadvance in the molecular design of AIEgens, which realizedthe polychromatic imaging of mitochondria. In 2014, wereported a new AIE mitoprobe with green emission formitochondrial imaging.[66] AIE-MitoGreen-1 was obtained byincorporating SAA with two positively charged pyridiniumgroups as the mitochondrial targeting ligands, which exhibitstypical AIE property due to both the restriction of intra-molecular rotation and ESIPT in aggregate state. The emissionmaximum of AIE-MitoGreen-1 locates at 532 nm, yielding alarge Stokes shift of 176 nm. AIE-MitoGreen-1 couldspecifically accumulate at the mitochondria of brown pre-adipose cells and light-up the mitochondria with bright green

Figure 2. Chemical structures of different positively charged mitochondria-targeting ligands (A) and AIEgens with different emission peaks (B).


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fluorescence without washing steps (Figure 3B), which real-ized the in situ monitoring of mitochondria morphology duringthe differentiation of brown pre-adipose cells.

To further decrease the influence of auto-fluorescencegenerated from the background during mitochondria imagingprocess, AIE mitoprobes with yellow and red emission weredeveloped. Tang’s group reported a yellow-emissive AIEmitoprobe named TPE-Py by simply introducing an vinylpyridinium to TPE.[60] TPE-Py shows excellent mitochondriastaining capability attributing to the positively charged vinylpyridinium group (Figure 3C). Meanwhile, it exhibits greaterphotobleaching resistance as compared to the commercialMitoTracker® Red CMXRos. The signal loss of TPE-Py isless than 20% after continuous excitation for 180 s, whileMitoTracker® Red CMXRos loses more than 80 % of theinitial signal due to photobleaching. Jiang et al. furthersynthesized a red-emissive AIE mitoprobe by replacing thevinyl pyridinium with an positively charged indolium moi-ety.[56] The obtained AIE mitoprobe, TPE-indo, also demon-strates good mitochondrial targeting efficiency (Figure 3D)and better photostability as compared to commercial mitop-robes such as JC-1, MitoTracker®, and Rhod123. Moreover,TPE-indo could selectively response to both human andmouse cancerous cells but not non-cancerous cells, providinggreat opportunities for the targeted mitochondrial imaging ofcancer cells and subsequent therapeutic applications.

Although one-photon fluorescence imaging of mitochon-dria was successfully achieved by various AIE mitoprobes, theshort excitation wavelength of these probes limits thepenetration depth and could easily lead to intrinsic backgroundautofluorescence and photo-damage to the cells. To overcomethese limitations, AIE mitoprobes with multiphoton absorp-tion, which are suitable for multiphoton fluorescence imagingof mitochondria, were developed. The AIE mitoprobe of IQ-TPA has been successfully used to realize two-photonfluorescence imaging of mitochondria.[67] IQ-TPA has largetwo-photon absorption cross section of ~215 GM at 900 nm,which ensures bright two-photon fluorescence imaging ofmitochondria when excited at 900 nm to achieve low auto-

fluorescence signal generation and low photodamage. Threephoton fluorescence imaging of mitochondria were alsoexplored by He and Qian using TPE-TPP, the first AIEmitoprobe, upon 1020 nm three-photon excitation.[68] TPE-TPP also exhibits better photostability than commercialMitoTracker® Red FM even upon 1020 nm excitation. Ascompared to one-photon excitation at 405 nm and two-photonexcitation at 810 nm, three-photon excitation of TPE-TPP at1020 nm could almost completely remove the autofluores-cence generated by the intrinsic endogenous fluorophoresinside the cells since these fluorophores have negligible multi-photon absorption compared to that of TPE-TPP, furtherincreasing the signal to noise ratio to image and monitor themitochondria. Meanwhile, the photodamage to cells could alsobe significantly reduced. Furthermore, the fact that theannoying autofluorescence problem faced by the AIE mitop-robes with absorption and emission spectra in short wave-length range could be overcome by the multi-photon imagingtechnique further broaden their application for mitochondrialimaging and monitoring.

3.2 Super-Resolution Imaging of Mitochondria by AIEMitoprobes

The monitoring of mitochondrial dynamics is of particularimportance to understand the function and activity ofmitochondria as well as their interaction with other cellularstructures. However, the resolution of visible light microscopeis limited to 200 nm due to the diffraction barrier,[69] whichcould not provide precise characterization of mitochondria.The emerging of super-resolved fluorescence imaging such asphotoactivated localization microscopy (PALM),[70] stochasticoptical reconstruction microscopy (STORM)[71] could breakdown the barrier and lead to higher resolution up to nanometerscale. However, the operation of such super-resolution micro-scope relies on the photoactivatable fluorogens, while theconventional mitochondria targeting fluorogens suffer from

Figure 3. Polychromatic imaging of mitochondria by AIE mitoprobes. (A) Blue;[65] (B) Green;[66] (C) Yellow;[60] and (D) Red.[56] Scale bars: 30 mm.


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high background signal, high phototoxicity and unstablephotoswitching.

The first fluorescence turn-on photoactivatable AIE mitop-robe was developed in 2015.[72] The AIE mitoprobe in the pre-activated state was in a form called o-TPP3M, whichcontained TPE, electron-donating methoxy groups and cati-onic, electron-withdrawing 1-methylpyridinium unit and wasnon-emissive as the twisted intramolecular charge-transfer(TICT) effect quenched the light emission. Upon UV lightirradiation, o-TPP3M was transformed into c-TPP3M due tothe photocyclodehydrogenation reaction, which resulted in thedominance of the AIE effect over the TICT effect and theturn-on of blue fluorescence. This mitoprobe could specifi-cally stain and light-up the mitochondria upon UV lightirradiation.

Subsequently, another fluorescence turn-on photoactivat-able AIE mitoprobe of o-TPE-ON+ with yellow emissionwas reported,[73] which could be photoactivated after trans-forming into c-TPE-ON+ through the same mechanism asprevious one (Figure 4A). The probe could efficiently light upthe mitochondria in living and fixed HeLa cells upon UV lightirradiation. Furthermore, the fluorescence of c-TPE-ON+could be photobleached by applying appropriate light (Fig-ure 4B), making this AIE mitoprobe suitable for super-resolution imaging. Furthermore, it could exhibit spontaneousblink under physiological conditions with higher photoncounts, faster fluorescence switching on-off rate, and higherlocalization precision as compared to the widely usedcommercial MitoTracker® Orange CMTMRos. Attributing tothese merits, real-time super-resolution imaging of mitochon-drial dynamics was successfully achieved under physiologicalconditions on STORM without adding external additives toenhance the blink. The imaging quality could be significantlyimproved as indicated by the comparison between the blurryepifluorescent image and the clear STORM image ofmitochondria as shown in Figure 4C, in which the resolutionwas increased from 697.1 nm (epifluorescent image) to104.5 nm (STROM image). Attributing to the excellentcapability of o-TPE-ON+ for super-resolution mitochondrialimaging, the fission and fusion process of mitochondria couldbe clearly monitored as indicated by the green and red arrowsshown in Figure 4D, demonstrating the promising potential ofAIE mitoprobes for super-resolution imaging applications.

3.3 Monitoring of Mitochondria Related Bioprocess by AIEMitoprobes

As illustrated in the introduction, mitochondria are highlydynamic organelles which are involved in various bioprocessby communicating or interacting with other intracellularorganelles.[17] Thus, apart from imaging mitochondria alone,one of the most important applications of AIE mitoprobes is tomonitor the mitochondria related bioprocess. Recently, wereported a red-emissive AIE mitoprobe, AIE-Red (Figure 5A,lem =665 nm), which could cooperate with another green-

emissive AIE lysosome tracker, AIE-Green (Figure 5A, lem =538 nm), to monitor the mitophagy process.[64] AIE-Red couldefficiently stain mitochondria without interfering the stainingof lysosome by AIE-Green, which excels the interferenceproblem that commercial dyes sometimes encounter duringco-stain of cells. Meanwhile, it exhibits extremely highphotostability and low cytotoxicity towards mitochondria uponlaser irradiation. Real-time monitoring of mitophagy processwas achieved in HeLa cells by observing the overlap betweenthe red fluorescence from mitochondria and the greenfluorescence from lysosome (Figure 5C), which provideddirect visual information with high accuracy. Tang’s groupalso achieved similar process monitoring using an AIE mitop-robe, TPE-Py-NCS, and the commercial LysoTracker® RedDND-99.[74] Mitochondria and lysosome of HeLa cells werestained by TPE-Py-NCS and LysoTracker® Red DND-99,

Figure 4. (A) Illustration of photocyclodehydrogenation process of o-TPE-ON+ . (B) Photoactivation and photobleaching behaviors ofmitochondria in fixed HeLa cells after incubation with o-TPE-ON+ .Scale bar: 20 mm. (C) Comparison of epifluorescent image andsuper-resolution image of mitochondria. Scale bars: 500 nm. (D)Real-time monitoring of the mitochondrial dynamics in HeLa cellsstained by o-TPE-ON+ using super-resolution microscope.[73] Scalebars: 500 nm.


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respectively. The mitophagy process were indicated by theweaker green emission from TPE-Py-NCS stained mitochon-dria when the mitophagy was induced by rapamycin and themitochondria were digested by the LysoTracker® Red DND-99labeled autophagosome. Furthermore, as reported by Tang’sgroup in 2016, AIE mitoprobe could also help to monitor thecell behavior during the cell population using a yellowish AIEmitoprobe TPE-PyN3 as the fluorescent indicator.[75] TPE-PyN3 could retain in living HeLa cells to 5th generation duringin vitro cell proliferation and monitor the cell morphology andbehavior in living zebrafish embryos up to 60 hours,demonstrating good photostability and high biocompatibility,which is a promising candidate for non-invasive imaging ofcell behavior in living organisms.

3.4 Biosensing of Mitochondria by AIE Mitoprobes

Since mitochondria play critical roles in various cellularprocesses, the sensing and detection of its membrane potentialand the analytes inside it is highly desirable to understand itsfunction status. TPE-Ph-In is the first non-self-quenchingprobe that could monitor the membrane potential change ofmitochondria.[61] TPE-Ph-In is AIE-active with an absorptionpeak located at ~445 nm and an emission peak located at~670 nm (Figure 6A), which could stain the mitochondria ofHeLa cells specifically with stable fluorescent signal (Fig-ure 6B) and avoid the self-quenching problem. Since thetargeting of TPE-Ph-In to mitochondria depends on itsmembrane potential, which promotes the accumulation of dyes

in normal condition, after incubating HeLa cell with TPE-Ph-In, the membrane potential change could be monitored by itsfluorescence variation when the cells were treated witholigomycin or carbonyl cyanide 3-chlorophenylhydrazone toincrease or decrease the membrane potential, respectively(Figure 6C). Furthermore, light-up sensing of esterase accu-mulating inside the mitochondria was realized by Tong et al.by using an AIE mitoprobe named Probe 4 in 2017.[76] Probe4 contains an esterase responsive acetoxyl group and exhibitsvery weak fluorescence in aqueous solutions. The presence ofesterase could cleave the acetoxyl and release the hydroxylgroup, resulting in the activation of ESIPT and AIE to inducestrong red fluorescence (lem =650 nm), achieving a promisingdetection limit of 0.005 U/mL for in vitro esterase detection.Attributing to the donor-acceptor structure, including the SAAcore, the electron-donor diethylamine and the electron-accept-or maleonitrile, the emission of ESIPT dye was firstly shiftedto the red range, which is more suitable for fluorescenceimaging applications. Probe 4 could specifically stain andlight-up the mitochondria after reacting with esterase in livingMCF-7 cells, which further extend the application of AIEmitoprobes to the analytes detection related to mitochondrialactivities.

4. Theranostics with AIE Mitoprobe

As the powerhouse of cells, mitochondria dysfunctions arerelated to many diseases such as tumorigenesis. Therefore,mitochondria targeting has emerged as a promising strategy

Figure 5. (A) Chemical structure of AIE-Red and AIE-Green. (B) Real-time monitoring of mitophagy process after staining the mitochondriaand lysosome using AIE-Red and AIE-Green.[17]


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for enhanced therapy outputs with minimized side effects. Inthe previous section, we have introduced the application ofnontoxic AIE mitoprobes for visualization of mitochondriastructures or related progresses. In this section, we focus onthe theranostic applications of AIE mitoprobes.

4.1 Chemotherapy with AIE Mitoprobes

The pioneer work of AIE mitoprobes for theranostics wasreported in 2014.[43] AIE-2TPP was synthesized by conjugat-ing two triphenylphosphonium (TPP) segments to an AIEgenexhibiting ESIPT feature (Figure 7A). Benefiting from theAIE and ESIPT characteristics, AIE-2TPP could inducefluorescence change through intramolecular hydrogen bond-ing, which further promotes high image contrast and largeStokes shift. Due to the higher plasma membrane potentialand mitochondria membrane potential of cancerous cells overnormal epithelial cells, AIE-2TPP exhibited selective cellularuptake towards cancer cells, where they showed predominateaccumulation and specific fluorescence turn-on in mitochon-dria. The accumulation of the bi-armed AIE-2TPP inmitochondria could cause depolarized membrane, increasedintracellular reactive oxygen species (ROS) level, and reducedATP generation (Figure 7B), leading to specific cell apoptosisand death to cancer cells but not normal cells.

By linking positively charged pyridinium group to TPEunits and with careful selection of counter anions, the resultantAIE mitoprobes also exhibited selective cytotoxicity towardscancer cells.[77] Upon changing the counter anions fromtoluenesulfonate (TPE-Py-2O-1) to tetraphenyl borate (TPE-

Py-2O-2) or tetra(4-chlorophenyl) borate (TPE-Py-2O-3)(Figure 7A), the resultant TPE-Py-2O-X mitoprobes showedred-shifted emission maximum from 560 to 605 and 620 nm,while the mitochondrial targeting ability remain inherited.Different from TPE-Py-2O-1, the latter two TPE-Py-2Oderivatives showed selective cytotoxicity towards cancer cellsvia inhibiting the oxidative phosphorylation process and ATPproduction in mitochondria. It is further demonstrated that thecationic TPE-Py-2O contributes to the cytotoxicity, while thecounter anions are used to fine-tune the selectivity, where thecationic TPE-Py-2O and tetraphenyl borate (2) or tetra(4-chlorophenyl) borate (3) worked cooperatively to selectivelytargeting cancer cell mitochondria and kill them. Moreover,TPE-Py-2O-2 and TPE-Py-2O-3 also demonstrated excellenttherapeutic performance in in vivo tumor models with signifi-cant tumor volume shrinkage and minimal systemic toxicity.

Kim and co-workers also reported a responsive AIEmitoprobe to achieve in situ activation for selective cancer cellablation.[78] The AIE mitoprobe (TPE-TPP-a) contains a NAD(P)H:quinone oxidoreductase-1 (NQO1) responsive segment toquench TPE fluorescence via photon-induced electron transfer(PET) effects (Figure 8A).[79] The fluorescence could only beactivated by sodium dithionite (an enzyme free model ofNQO1) among various analytes, with an enhancement factorof over 200-fold (Figure 8B). As NQO1 is overexpressed incancer tissues or cells, TPE-TPP-a showed selective light-upmitochondria only in cancer cell with high NQO1 level.Different from TPE-TPP-b which could kill all the testedcells, TPE-TPP-a only showed cytotoxicity towards NQO1overexpressed cancer cells such as A549 cells (Figure 8C).The selective toxicity was further confirmed with in vivo

Figure 6. (A) Chemical structure and PL spectra of TPE-Ph-In in solvent and aggregation state. (B) Mitochondrial staining using MitotrackerGreen and TPE-Ph-In. Scale bar: 20 mm. (C) Monitoring of membrane potential change using TPE-Ph-In.[61]


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tumor model, where TPE-TPP-a showed low therapeuticefficiency towards siNQO1-transfected tumor with NQO1knockdown to reduce the NQO1 expression (Figure 8D). Veryrecently, the same group further conjugated an chlorambucilprodrug to the TPE-TPP system to achieve mitochondriatargeted combinational chemotherapy, where chlorambucilcould be activated by the high glutathione concentration incancer cells and the accumulation of TPE-TPP promote their

cancer cell ablation performance, which further expanded thescope of AIE mitoprobes for chemotherapy.[80]

4.2 Photodynamic Therapy with AIE Mitoprobes

Photodynamic therapy (PDT) as a non-invasive cancer treat-ment approach has attracted great research interest re-cently.[81,82] PDT relies on a photosensitizer to absorb localized

Figure 7. (A) Chemical structures of AIE-2TPP and TPE-Py-2O derivatives. (B) Schematic illustration of chemotherapeutic function of AIEmitoprobes.[43]

Figure 8. (A) Reaction of TPE-TPP-a with NQO1 to light-up fluorescence. (B) Fluorescence response assay of TPE-TPP-a with various biologicalreductant. (C) Effects of TPE-TPP-a on the viability of different cells. (D) Effects of TPE-TPP-a on in vivo tumor growth (A549 cells).[78]


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photons to produce toxic ROS to kill cancer cells. As ROS hasshort lifetime and small radius of action, PDT showedminimized side toxicity towards healthy tissue, which requiresthe precise subcellular localization and efficient intracellularROS generation of photosensitizers to achieve maximumlesion towards cancer cells.[83,84] Mitochondria are reported tobe the primary target of ROS, whose dysfunctions alwaysoccurs as early stage of PDT.[85] However, conventionalphotosensitizers exhibited largely decreased ROS productionin aggregates when they were bound to mitochondria. In thisregard, AIEgens with efficient ROS production in aggregatestate are beneficial for the design of AIE mitoprobes for PDT.

The earliest report of AIE mitoprobes for PDT is based onTPE-IQ (Figure 9A), which exhibited absorption and emis-sion maxima of 400 nm and 502 nm, respectively.[86] The ROSgeneration ability of TPE-IQ under UV light irradiation isverified by H2DCF-DA, which emits green emission at530 nm after reaction with ROS (Figure 9B). Together with itsexcellent mitochondria targeting ability, TPE-IQ couldeffectively kill cancer cells upon UV irradiation while showingexcellent dark biocompatibility (Figure 9C). Moreover, themitochondrial morphology changes under PDT could also bereal-time visualized by its green emission, where themitochondria networks swelled from long-tubular-like networkto dispersed fragments.

Recently, two methoxy groups were introduced as theelectron donating units to TPE-IQ to render TPE-IQ-2O withred-shifted absorption and emission spectra. TPE-IQ-2Oshowed an absorption maximum at 430 nm with a molarabsorptivity of 15000 M�1 cm�1, and an emission maximum at620 nm. TPE-IQ-2O could be internalized into HeLa cellsand show strong fluorescence in mitochondria with sharpcontrast, while it exhibited poor uptake towards normal cells,e. g. COS-7 cells (Figure 9D). Incubation with six differentcancerous cells and four normal cells further demonstrate thatTPE-IQ-2O could differentiate cancer cells from normal cells(Figure 9E), making it a promising fluorescent probe forcancer cell targeting. The high cancer cell uptake of TPE-IQ-2O is believed to be associated with the high plasma andmitochondrial membrane potentials of cancer cells.[87] Theefficient ROS production of TPE-IQ-2O under white lightirradiation further makes it an excellent PDT reagent toselective kill cancer cells over normal cells (Figure 9F).

4.3 Combination Therapy of AIE Mitoprobes

Despite the great advances in cancer therapy, single modaltreatment approach could not provide sufficient therapeuticoutput owing to their intrinsic drawbacks, e. g. PDT suffers

Figure 9. (A) Chemical structures of TPE-IQ and TPE-IQ-2O. (B) Detection of ROS generation of TPE-IQ by H2DCF-DA. (C) Viability of TPE-IQtreated HeLa cells with or without UV irradiation. (D) Fluorescence images of HeLa and COS-7 cells labelled with TPE-IQ-2O. (E) Relativefluorescence intensity of various cells after treatment with TPE-IQ-2O. (F) Viabilities of TPE-IQ-2O treated HeLa and COS-7 cells with orwithout light irradiation.[86]


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from the low oxygen level in tumor microenvironment. Therehas been increasing research interests to develop combina-tional cancer therapy that through incorporation of two ormore therapeutic modalities to achieve therapy outcome thatcannot be obtained by individual therapy approach.[48,88–90] Onesimple approach is to introduce photosensitizing ability to theAIE mitoprobes that already exhibit chemotherapeutic effects.Based on this concept, Liu and coworkers synthesizedTPECM-2TPP by conjugating two TPP segments to thecentral core of TPECM for image-guided chemo- and photo-dynamic combination cancer cell ablation (Figure 10A).[63]

TPECM is a typical AIEgen, which not only showed brightemission in aggregate, but also demonstrate efficient ROSgeneration under light irradiation, as accessed by the ABDAabsorbance changes in response to its reaction with ROS toshow decreased absorbance (Figure 10B). As predicted, theTPECM-2TPP showed excellent mitochondria targeting abil-ity. Moreover, it exhibited a very high dark toxicity (chemo-therapy) towards cancer cells with a half maximum inhibitionconcentration (IC50) of 6.31 mM, while TPECM-1TPP withonly one TPP arm exhibited no obvious toxicity towards HeLacells (Figure 10C). Upon light irradiation, the generated ROSat mitochondria site could quickly oxidize and damage themitochondria membrane, leading largely improve cancerablation effects, where IC50 value of TPECM-2TPP is furtherreduced to 3.13 mM (Figure 10D). The development of photo-sensitizing AIE mitoprobes demonstrates a simple and facileapproach, where introducing mitochondria targeting ligands toAIE photosensitizers could readily generate a molecular probe

with four functions: mitochondria targeting, fluorescenceimaging, chemotherapy, and PDT.

Very recently, a theranostic probe composed of a photo-sensitizing AIE mitoprobe and a natural product of Artemisi-nin (ART) was reported.[91] TPETH-Mito-1ART consiststhree components: 1) an AIE core as the fluorescence reporterand ROS generator; 2) two cationic ammonium salt arms asmitochondria targeting moieties, and 3) ART on one arm(Figure 11A). The TPETH-Mito-1ART could not onlyprovide selectivity between cancer cells and normal cells, butalso aid to deliver ART and AIE photosensitizer to themitochondria at which site the fresh produced heme couldquickly active the anticancer ability of ART to generatechemotoxicity to cancer cells, while further light irradiationcould promote its lesion to cancer cells by ROS generation(Figure 11A). Delivery of ART to mitochondria could largelyenhance its lesion towards cancer cells, where the IC50 valuedrops from 88.8 mM for ART to 11.1 mM for TPETH-Mito-1ART, and to 3.1 mM for two ART armed AIE mitoprobe(TPETH-Mito-2ART). However, TPETH-Mito-2ART alsoexhibits severe toxicity (IC50 =12.6 mM) to normal cells due tohigh ART loading. In this regard, TPETH-Mito-1ART thatshows low toxicity toward normal cells (IC50>100 mM)represents a non-invasive probe for cancer cell ablation.Further light irradiation could reduce the IC50 value to 8.1 mM.Moreover, the light irradiation leads to more severe damage tomitochondria which not only depolarizes mitochondrial mem-brane potential but also inhibits cancer cell migration. Thecombination of ART and PDT leads to synergistic therapeutic

Figure 10. (A) Chemical structure of TPECM-1TPP and TPECM-2TPP. (B) ROS generation ability of TPECM access by ABDA decompositionunder light irradiation. Cell viabilities of HeLa cells after treatment with TPECM-1TPP or TPCM-2TPP under dark (C) or light irradiation (D).[63]


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effects, which opens new opportunities of AIEgen conjugatedfor theranostic applications.

4. Conclusion

In summary, we have demonstrated the great advantages ofAIEgen based mitochondria probes in the bioimaging andtheranostic applications. Different from conventional fluoro-phores, AIEgens show very weak emission as isolatedmolecules and are able to turn on their fluorescence uponaggregate formation. This unique feature has led to design ofAIE bioprobes for specific imaging of mitochondria networksor monitoring progresses in mitochondria with very highsignal-to-noise ratios. Moreover, the unique light-up featuresand excellent photostability of AIE mitoprobes also provideopportunities for real-time visualization of the mitochondrialprogresses without washing steps or concerns of photobleach-ing. With facile molecular design and synthesis, AIEgen basedmitoprobes with different emission colors and targeting groupshave been reported, which show promising imaging perform-ance. The recent development of AIE mitoprobes with cancercell selective cytotoxicity further expands their scopes totheranostics. When photosensitizing ability is introduced tothese AIE mitoprobes, they provide excellent PDT perform-ance as mitochondria is the main target of ROS. Moreover,drug delivery capability of AIE mitoprobes also provides newwindows for combination cancer therapy. With this review, we

hope to stimulate more research interests from chemistry,materials and biology to further promote the advancement ofAIE mitoprobes in the biological field.

Acknowledgement

We thank the Singapore National Research Foundation (R279-000-444-281, R279-000-483-281, R-719-000-018-281), theNational University of Singapore (R279-000-433-281), Inno-vation and Technology Commission (ITC-CNERC14SC01 andITS/254117) and the Research Grants Council of Hong Kong(16305015, 16308016, N-HKUST604/14, A-HKUST605/16and C6009-17G) for financial support. The authors declare nocompeting financial interest.

References

[1] M. W. Gray, Cold Spring Harbor Perspect. Biol. 2012, 4,a011403.

[2] D. R. Green, J. C. Reed, Science 1998, 281, 1309.[3] J. E. Baldwin, H. Krebs, Nature 1981, 291, 381.[4] B. P. Tu, A. Kudlicki, M. Rowicka, S. L. McKnight, Science

2005, 310, 1152.[5] B. Levy, S. Gibot, P. Franck, A. Cravoisy, P.-E. Bollaert, The

Lancet 2005, 365, 871.

Figure 11. (A) Schematic illustration of combination cancer cell ablation based on of TPETH-Mito-1ART. (B) Viabilities of HeLa cells treatedwith different probes for 24 h in dark. (C) Viability of HeLa cells and NIH-3T3 cells treated with TPETH-Mito-1ART probe for 4 h followed withor without light irradiation.[91]


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123456789

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[6] H.-U. Simon, A. Haj-Yehia, F. Levi-Schaffer, Apoptosis 2000, 5,415.

[7] J. F. Turrens, J. Physiol. 2003, 552, 335.[8] L. A. Sena, N. S. Chandel, Mol. Cell 2012, 48, 158.[9] D. G. Nicholls, Biochem. J. 1978, 176, 463.

[10] F. Celsi, P. Pizzo, M. Brini, S. Leo, C. Fotino, P. Pinton, R.Rizzuto, Biochim. Biophys. Acta 2009, 1787, 335.

[11] S. L. Budd, D. G. Nicholls, J. Neurochem. 1996, 67, 2282.[12] R. S. Balaban, S. Nemoto, T. Finkel, Cell 2005, 120, 483.[13] D. C. Chan, Cell 2006, 125, 1241.[14] S. Passarella, E. Casamassima, S. Molinari, D. Pastore, E.

Quagliariello, I. Catalano, A. Cingolani, FEBS Lett. 1984, 175,95.

[15] D. Johnson, H. Lardy, in Methods Enzymol., Vol. 10, Elsevier,1967, 94.

[16] J. F. Turrens, A. Boveris, Biochem. J. 1980, 191, 421.[17] R. J. Youle, A. M. Van Der Bliek, Science 2012, 337, 1062.[18] S. W. Hell, S. Lindek, C. Cremer, E. H. Stelzer, Appl. Phys. Lett.

1994, 64, 1335.[19] S.-H. Shim, C. Xia, G. Zhong, H. P. Babcock, J. C. Vaughan, B.

Huang, X. Wang, C. Xu, G.-Q. Bi, X. Zhuang, Proc. Natl. Acad.Sci. 2012, 109, 13978.

[20] B. Huang, M. Bates, X. Zhuang, Annu. Rev. Biochem. 2009, 78,993.

[21] G. Themelis, J. S. Yoo, K. Soh, R. B. Schulz, V. Ntziachristos, J.Biomed. Opt. 2009, 14, 064012.

[22] A. M. Derfus, W. C. Chan, S. N. Bhatia, Adv. Mater. 2004, 16,961.

[23] S. Imstepf, V. Pierroz, R. Rubbiani, M. Felber, T. Fox, G. Gasser,R. Alberto, Angew. Chem. Int. Ed. 2016, 55, 2792.

[24] B. P. Cormack, R. H. Valdivia, S. Falkow, Gene 1996, 173, 33.[25] S. Zhang, T. Wu, J. Fan, Z. Li, N. Jiang, J. Wang, B. Dou, S.

Sun, F. Song, X. Peng, Org. Biomol. Chem. 2013, 11, 555.[26] U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke,

T. Nann, Nat. Methods 2008, 5, 763.[27] G. M. Gadd, A. J. Griffiths, Microb. Ecol. 1977, 4, 303.[28] S. A. McKinney, C. S. Murphy, K. L. Hazelwood, M. W.

Davidson, L. L. Looger, Nat. Methods 2009, 6, 131.[29] S. Ramakrishna, A.-B. K. Dad, J. Beloor, R. Gopalappa, S.-K.

Lee, H. Kim, Genome Res. 2014, 24, 1020.[30] M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, L. B. Chen, Cell

1984, 38, 101.[31] J. Mei, Y. Hong, J. W. Y. Lam, A. Qin, Y. Tang, B. Z. Tang, Adv.

Mater. 2014, 26, 5429.[32] J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang,

Chem. Rev. 2015, 115, 11718.[33] G. Feng, R. T. K. Kwok, B. Z. Tang, B. Liu, Appl. Phys. Rev.

2017, 4, 021307.[34] X. Gu, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang, Biomaterials

2017, 146, 115.[35] J. Liang, B. Z. Tang, B. Liu, Chem. Soc. Rev. 2015, 44, 2798.[36] Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2011, 40,

5361.[37] Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Commun. 2009, 4332.[38] R. Wei, P. Song, A. Tong, J. Phys. Chem. C 2013, 117, 3467.[39] X. Gu, J. Yao, G. Zhang, C. Zhang, Y. Yan, Y. Zhao, D. Zhang,

Chem. Asian J. 2013, 8, 2362.[40] M. Wang, G. Zhang, D. Zhang, D. Zhu, B. Z. Tang, J. Mater.

Chem. 2010, 20, 1858.[41] G. Zhang, F. Hu, D. Zhang, Langmuir 2014, 31, 4593.[42] F. Hu, B. Liu, Org. Biomol. Chem. 2016, 14, 9931.[43] Q. Hu, M. Gao, G. Feng, B. Liu, Angew. Chem. Int. Ed. 2014, 53,

14225.

[44] F. Hu, Y. Huang, G. Zhang, R. Zhao, H. Yang, D. Zhang, Anal.Chem. 2014, 86, 7987.

[45] W. Wu, D. Mao, S. Xu, S. Ji, F. Hu, D. Ding, D. Kong, B. Liu,Mater. Horiz. 2017, 4, 1110.

[46] W. Wu, D. Mao, F. Hu, S. Xu, C. Chen, C. J. Zhang, X. Cheng,Y. Yuan, D. Ding, D. Kong, Adv. Mater. 2017, 33, 29.

[47] S. Xu, Y. Yuan, X. Cai, C.-J. Zhang, F. Hu, J. Liang, G. Zhang,D. Zhang, B. Liu, Chem. Sci. 2015, 6, 5824.

[48] G. Feng, B. Liu, Small 2016, 12, 6528.[49] F. Hu, S. Xu, B. Liu, Adv. Mater. 2018, DOI: 10.1002/

adma.201801350.[50] S. E. Weinberg, N. S. Chandel, Nat. Chem. Biol. 2015, 11, 9.[51] J. D. Ly, D. Grubb, A. Lawen, Apoptosis 2003, 8, 115.[52] M. Ross, G. Kelso, F. Blaikie, A. James, H. Cocheme, A.

Filipovska, T. Da Ros, T. Hurd, R. Smith, M. Murphy,Biochemistry (Moscow) 2005, 70, 222.

[53] J. Rafael, D. Nicholls, FEBS Lett. 1984, 170, 181.[54] G. Bai, K. Rama Rao, C. R. Murthy, K. Panickar, A. Jayakumar,

M. Norenberg, J. Neurosci. Res. 2001, 66, 981.[55] C. W. T. Leung, J. W. Y. Lam, B. Z. Tang, Chem. Commun.

2014, 50, 14451.[56] L. Zhang, W. Liu, X. Huang, G. Zhang, X. Wang, Z. Wang, D.

Zhang, X. Jiang, Analyst 2015, 140, 5849.[57] G. Lin, P. N. Manghnani, D. Mao, C. Teh, Y. Li, Z. Zhao, B. Liu,

B. Z. Tang, Adv. Funct. Mater. 2017, 27, 1701418.[58] J. Qi, C. Sun, A. Zebibula, H. Zhang, R. T. K. Kwok, X. Zhao,

W. Xi, J. W. Y. Lam, J. Qian, B. Z. Tang, Adv. Mater. 2018, 30,1706856.

[59] E. Havinga, W. Ten Hoeve, H. Wynberg, Synth. Met. 1993, 55,299.

[60] N. Zhao, M. Li, Y. Yan, J. W. Y. Lam, Y. L. Zhang, Y. S. Zhao,K. S. Wong, B. Z. Tang, J. Mater. Chem. C 2013, 1, 4640.

[61] N. Zhao, S. Chen, Y. Hong, B. Z. Tang, Chem. Commun. 2015,51, 13599.

[62] C. Gui, E. Zhao, R. T. K. Kwok, A. C. S. Leung, J. W. Y. Lam,M. Jiang, H. Deng, Y. Cai, W. Zhang, H. Su, Chem. Sci. 2017, 8,1822.

[63] C.-J. Zhang, Q. Hu, G. Feng, R. Zhang, Y. Yuan, X. Lu, B. Liu,Chem. Sci. 2015, 6, 4580.

[64] F. Hu, X. Cai, P. N. Manghnani, W. Wu, B. Liu, Chem. Sci. 2018,9, 2756.

[65] C. W. T. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Y. Lam, B. Z.Tang, J. Am. Chem. Soc. 2013, 135, 62.

[66] M. Gao, C. K. Sim, C. W. T. Leung, Q. Hu, G. Feng, F. Xu, B. Z.Tang, B. Liu, Chem. Commun. 2014, 50, 8312.

[67] M. Jiang, R. T. K. Kwok, X. Li, C. Gui, J. W. Y. Lam, J. Qu,B. Z. Tang, J. Mater. Chem. B 2018, 6, 2557.

[68] Z. Zhu, C. W. T. Leung, X. Zhao, Y. Wang, J. Qian, B. Z. Tang,S. He, Sci. Rep. 2015, 5, 15189.

[69] S. W. Hell, Nat. Biotechnol. 2003, 21, 1347.[70] E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S.

Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, Science 2006, 313, 1642.

[71] M. J. Rust, M. Bates, X. Zhuang, Nat. Methods 2006, 3, 793.[72] X. Gu, E. Zhao, J. W. Y. Lam, Q. Peng, Y. Xie, Y. Zhang, K. S.

Wong, H. H. Y. Sung, I. D. Williams, B. Z. Tang, Adv. Mater.2015, 27, 7093.

[73] X. Gu, E. Zhao, T. Zhao, M. Kang, C. Gui, J. W. Y. Lam, S. Du,M. M. T. Loy, B. Z. Tang, Adv. Mater. 2016, 28, 5064.

[74] W. Zhang, R. T. K. Kwok, Y. Chen, S. Chen, E. Zhao, C. Y. Y.Yu, J. W. Y. Lam, Q. Zheng, B. Z. Tang, Chem. Commun. 2015,51, 9022.


Review


123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556

[75] B. Situ, S. Chen, E. Zhao, C. W. T. Leung, Y. Chen, Y. Hong,J. W. Y. Lam, Z. Wen, W. Liu, W. Zhang, L. Zheng, B. Z. Tang,Adv. Funct. Mater. 2016, 26, 7132.

[76] L. Peng, S. Xu, X. Zheng, X. Cheng, R. Zhang, J. Liu, B. Liu, A.Tong, Anal. Chem. 2017, 89, 3162.

[77] Y. Huang, G. Zhang, F. Hu, Y. Jin, R. Zhao, D. Zhang, Chem.Sci. 2016, 7, 7013.

[78] W. S. Shin, M.-G. Lee, P. Verwilst, J. H. Lee, S.-G. Chi, J. S.Kim, Chem. Sci. 2016, 7, 6050.

[79] W. C. Silvers, B. Prasai, D. H. Burk, M. L. Brown, R. L.McCarley, J. Am. Chem. Soc. 2013, 135, 309.

[80] W. S. Shin, S. K. Park, P. Verwilst, S. Koo, J. H. Lee, S.-G. Chi,J. S. Kim, Chem. Commun. 2017, 53, 1281.

[81] A. P. Castano, P. Mroz, M. R. Hamblin, Nat. Rev. Cancer 2006,6, 535.

[82] D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev.Cancer 2003, 3, 380.

[83] E. Skovsen, J. W. Snyder, J. D. C. Lambert, P. R. Ogilby, J. Phys.Chem. B 2005, 109, 8570.

[84] S. Kim, T. Tachikawa, M. Fujitsuka, T. Majima, J. Am. Chem.Soc. 2014, 136, 11707.

[85] E. J. Ngen, P. Rajaputra, Y. You, Bioorg. Med. Chem. 2009, 17,6631.

[86] E. Zhao, H. Deng, S. Chen, Y. Hong, C. W. T. Leung, J. W. Y.Lam, B. Z. Tang, Chem. Commun. 2014, 50, 14451.

[87] L. B. Chen, Annu. Rev. Cell Dev. Biol. 1988, 4, 155.[88] L. Brannon-Peppas, J. O. Blanchette, Adv. Drug Delivery Rev.

2012, 64, 206.[89] P. Huang, D. Wang, Y. Su, W. Huang, Y. Zhou, D. Cui, X. Zhu,

D. Yan, J. Am. Chem. Soc. 2014, 136, 11748.[90] G. Feng, Y. Fang, J. Liu, J. Geng, D. Ding, B. Liu, Small 2017,

13, 1602807.[91] G. Feng, J. Liu, C.-J. Zhang, B. Liu, ACS Appl. Mater. Interfaces

2018, 14, 11546.

Received: April 13, 2018Accepted: June 14, 2018

Published online on July 4, 2018


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