AIE-based energy transfer systems for biosensing, imaging, and therapeutics

Journal Pre-proof AIE-Based Energy Transfer Systems for Biosensing, Imaging, and Therapeutics Xuewen He, Ling-Hong Xiong, Yalan Huang, Zheng Zhao, Zaiyu Wang, Jacky Wing Yip Lam, Ryan Tsz Kin Kwok, Ben Zhong Tang PII:

S0165-9936(19)30507-2

DOI:

https://doi.org/10.1016/j.trac.2019.115743

Reference:

TRAC 115743

To appear in:

Trends in Analytical Chemistry

Received Date: 31 August 2019 Revised Date:

21 October 2019

Accepted Date: 17 November 2019

Please cite this article as: X. He, L.-H. Xiong, Y. Huang, Z. Zhao, Z. Wang, J.W. Yip Lam, R.T. Kin Kwok, B.Z. Tang, AIE-Based Energy Transfer Systems for Biosensing, Imaging, and Therapeutics, Trends in Analytical Chemistry, https://doi.org/10.1016/j.trac.2019.115743. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

AIE-Based Energy Transfer Systems for Biosensing, Imaging, and Therapeutics Xuewen He1,3#, Ling-Hong Xiong2#, Yalan Huang2, Zheng Zhao1,3, Zaiyu Wang1,3, Jacky Wing Yip Lam1,3, Ryan Tsz Kin Kwok1,3*, Ben Zhong Tang1,3,4* 1. Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Centre for Tissue Restoration and Reconstruction, Institute for Advanced Study and Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China 2. Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China 3. HKUST-Shenzhen Research Institute, Shenzhen 518057, China 4. NSFC Center for Luminescence from Molecular Aggregates, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China # These authors contribute equally. * Corresponding authors: E-mail: [email protected] and [email protected]

Abstract: With the rapid development of optical tools, biosensing and imaging have deepened to analyze the molecule-level substrates in living systems. As a molecular-ruler, Förster resonance energy transfer (FRET)-based analytical methods could sensitively response to the tiny fluctuation of biomolecules and events with ultrahigh spatiotemporal resolution in living cells, tissues and animals. Luminogens with aggregation-induced emission (AIE) characteristics and their derived nanostructures display unique photophysical properties, including tunable emission wavelength, robust brightness, broad absorption, large Stokes shift, excellent photostability, flexible molecular design and versatile nanostructural fabrication, etc., and have been widely applied in building FRET probes for bioanalytes sensing, imaging and drug delivery monitoring with high sensitivity and specificity. In addition, AIE-based energy transfer systems exhibit unique advantages in photodynamic therapy, holding great potential in analytical research and biomedical applications.

Keywords: Förster resonance energy transfer (FRET), aggregation-induced emission (AIE), fluorescence, bioimaging, therapeutics Förster resonance energy transfer (FRET) is a photophysical process in which an excited fluorescent (FL) donor transfers its excitation energy to an acceptor through a long-range dipole-dipole interaction[1]. The energy transfer between the donor and the acceptor is strongly dependent on the center-to-center separation distance. And its efficiency is inversely proportional to the sixth power of the donor to acceptor distance. Since Stryer and Haugland first defined the FRET as a “spectroscopic ruler’’ for distance measurement in 1967[2], a new era was initiated with the use of FRET rulers to address a wide range of biological problems. Till now, FRET still serves a powerful tool for determining conformational changes and biomolecular interactions, as well as the inter and intramolecular distance measurements in biochemical processes[3-5]. Particularly, when combining FRET with microscopy imaging, spatial- and temporal-resolvable information of bioactivities, such as the distribution, association and disassembly of biomolecules in living cells, could be achieved with ultrahigh sensitivity, superior specificity, fast responsiveness, as well as molecular-level precision[6].

When designing a FRET system, the following requirements are indispensable: ( ) a substantial spectrum overlap of the donor emission with acceptor absorption; ( ) an appropriate linker and distance between the donor and the acceptor; and ( ) the proper orientation of the dipole-dipole interaction[7] (Fig. 1). Ideally, there should be extensive overlap between the donor emission spectrum and the acceptor excitation spectrum, but no overlap in the excitation spectra or emission spectra of the FRET pair. Otherwise, the acceptor can be excited directly by the donor excitation light, which would lead to contaminating FL noise in the FRET measurements, forming a type of error called crosstalk[8]. And the crosstalk of spectra between the donor and the acceptor is a major challenge for quantitative FRET analysis. Unfortunately, it is unavoidable as the Stokes shifts of most organic fluorophores are comparable or even smaller than the width of the spectra[9]. Therefore, numerous efforts have been devoted to design large Stokes shift fluorophores, such as inorganic quantum dots (QDs) and upconversion nanocrystals, to eliminate or reduce the crosstalk problems. These inorganic nanoparticles enjoy many advantages, such as excellent photostability, broad absorption spectra, narrow/symmetrical emission spectra, exceptional brightness, large absorbance cross-section and saturation intensity[10-17]. But their inherent toxicity has limited their further translations in biological applications. Also, their blinking behavior, rapidly alternating between emitting and dark state, brings another difficulty for single-molecule imaging and tracking[18]. Unlike conventional organic fluorophores, luminogens with aggregation-induced emission (AIEgens) with propeller-shaped structures emerged as promising alternatives for building sensing and imaging systems[19-29]. As isolated molecule, the rotor-like AIEgen undergoes low-frequency motions and dissipate exciton energy, leading to fast nonradiative decay and weak emission. Whereas, in the aggregated state, the radiative pathway dominates with robust emission via the restriction of intramolecular motion. AIE nanoaggregates and nanoparticles exhibit large Stokes shift, robust luminosity, strong photobleaching resistance, no random blinking and excellent biocompatibility, endowing their great potential in the construction of FRET systems for in vitro and in vivo applications. What’s more, using FRET could overcome many limitations existed in traditional FL measurements. Because the FRET signal is activated by analyte, background from probe itself is mitigated, improving the signal to noise ratio as well as the sensitivity. Further, it could compensate for the inhomogeneous distribution of probes in the cells. FRET probes that producing ratiometric signals could function as an internal standard and allow for a correction to interference from probe degradation or attenuation of luminescence in the cellular environment[9]. In this review, the powerful AIE-based energy transfer systems for biosensing, imaging and therapy will be discussed in the following two aspects: ( ) strategies for AIE-based energy transfer systems were first capitalized, with divisions, such as non-conjugate covalent bond energy transfer, through-bond energy transfer, co-encapsulation in a nanoplatform for FRET, host-guest interaction bridged FRET, chemiluminescence resonance energy transfer (CRET) and energy transfer from triplet state to oxygen (Fig. 2); ( ) typical application examples of AIE-based energy transfer systems, in which, ion sensing, detection of microenvironmental factors and small molecules, biomacromolecular assay, bioimaging, drug delivery monitoring and therapeutics via energy transfer, will be detailed discussed, with a short perspective for the future development of

AIE-FRET systems in bio-applications.

1. Strategies for AIE-based energy transfer systems 1.1 Non-conjugate covalent bond energy transfer Employing non-conjugate linker to import AIEgen into FRET pair is a widely applied strategy for construction of AIE-based energy transfer systems. The linker could be alkyl chain [30] or peptide sequence[31]. Liang’s group chose an alkyl chain, pH-sensitive hydrazone bond, as the linker between blue-emissive AIEgen, tetraphenylethene (TPE) and aggregation-caused quenching (ACQ)-featured doxorubicin (Dox) (Fig. 3)[32]. And stable nanoparticles formed in aqueous solution with e cient FRET between the TPE donor and the Dox acceptor. However, Dox quenched itself by ACQ in the constrained space of the nanoparticle. Therefore, both the donor and acceptor were quenched. Under intracellular low pH condition, the hydrazone bond broke, resulting the separation of TPE and Dox and the disappearance of FRET effect. As a result, the emission of TPE and Dox were both lighten up from the dark state. And the drug-releasing site and time, destinations of the carrier, as well as the executing site of the drug at subcellular level could be real-time monitored through dual-color FL signals from TPE and Dox, respectively. In another example, a caspase-3 specific peptide was employed to covalently link the FRET pair with green-color coumarin as donor and red-color AIEgen as acceptor[31]. As shown in Fig. 4, the water-soluble FRET probe was non-emissive due to the energy transfer as well as the free molecular motion of AIEgen in aqueous solution. Upon interaction with caspase-3, the probe displayed strong green and red FL signals synchronously due to the separation of the donor– acceptor and aggregation of the released AIEgen. The turn-on and dual-color FL signals allowed the sensitive detection of caspase-3 activity and real-time imaging of living cell apoptosis. 1.2 Through-bond energy transfer Instead of the flexible spacer in the above intramolecular FRET system, the donor and acceptor in through-bond energy transfer (TBET) systems were connected by a rigid and conjugated linker[33]. It can solve the paradox between the large pseudo-Stokes shift and the spectral overlapping between donor emission and acceptor absorption in the FRET system. The energy transfer rate in a TBET system was up to 100-fold faster than that in the conventional FRET system, making it just requiring that the energy gap (the difference between HOMO and LUMO energy level) of the donor be higher than that of the acceptor and less dependent on the overlapping of their emission and absorption spectra, respectively. Therefore, the emission leakage from the donor due to the limited spectra overlap in the FRET system could be well avoided, and the background noise could be effectively reduced. It is expectable that hybridizing AIEgen with TBET was another promising strategy to realize sensitive sensing and imaging. Tang et al. developed a TPE-rhodamine based ratiometric probe for Hg2+ sensing (Fig. 5)[34]. TPE derivative with AIE characteristics was selected as a dark donor to eliminate emission leakage. As a result, the energy transfer efficiency to rhodamine moiety was up to 99%. In solution state, no emission from the donor was observed and the pseudo-Stokes shift was larger than 280 nm. And more than 6000-fold ratiometric FL enhancement in 0-25 µM concentration range of Hg2+ was achieved with

detection limit as low as 0.3 ppb. 1.3 Co-encapsulation in a nanoplatform Fluorescent nanoparticles have showed brilliance in the construction of optoelectronics, biomedical actuators, cell imaging, diagnostic sensors and targeted drug delivery[19]. In general, encapsulation or chemical modification of fluorescent dyes into self-assembled skeleton was the common methodology, to construct emissive nanoparticles with desirable features in spatial dimension, stealthy surface, water solubility and colloidal stability in blood circulation. In contrast to the traditional organic dyes suffering ACQ problem, the constrained space in the nanoparticles was naturally suitable for AIEgens to aggregate and emit out strong FL signal. What’s more, intelligent theranostic systems could be facially constructed, as the nanoscale distance in the inner space just provided a harbor to construct the FRET systems. For an instance, Zhang’s group designed a type of smart and photo-switchable fluorescent nanoparticles (PFPNs), based on the AIE and FRET effect via encapsulation with a photochromic spiropyran-linked amphiphilic copolymer[35]. As shown in Fig. 6, the emission of the AIE donor (p-DTPACO) can be e ciently and reversibly switched on/o by the acceptor (photochromic spiropyran) upon irradiation with UV/visible light. Moreover, PFPNs could be applied for dual-color FL imaging in living cell, due to their outstanding reversibility and rapid photo-responsiveness. 1.4 Host-guest interaction In contrast to the complicated organic/polymeric synthesis and post modifications demanded by the nanoplatforms to incorporate multiple functions or stimuli-responsive capability, the dynamic and flexible nature of the noncovalent bonds grants the resultant supramolecular nanomaterials with tunable mechanical, chemical and biological characteristics[36]. Among diverse noncovalent forces, host−guest interactions have showed unique advantages and attracted great attention owing to their a uent stimuli-responsive behaviors to fabricate non-covalent FRET system[37]. Among them, crown ethers, cyclodextrins, pillararenes and amphiphilic compounds were frequently employed as building scaffolds[38]. For example, He’s group reported an efficient strategy to achieve the coupling of AIE and ACQ fluorophores for the construction of high-efficiency FRET system via host-guest interaction [39]. A shown in Fig. 7, a crown ether modified TPE served as the host and energy donor, a BODIPY derivative was chosen as the guest and energy acceptor. As a result, due to the high spectra overlap, an energy transfer efficiency of up to 93% from the AIEgen to BODIPY was achieved, with excellent tolerance to acidic and basic environments. 1.5 Chemiluminescence resonance energy transfer (CRET) CRET involves a nonradiative dipole−dipole energy transfer from a chemiluminescence (CL) donor to a suitable acceptor when they are located in close proximity (<10 nm). It occurs via the specific oxidation of a luminescent substrate during CL reaction that then exciting the fluorescent acceptor without the need for an external excitation source, and thus it can minimize nonspecific signals observed in FRET by simultaneously utilizing external excitation of both donor and acceptor[40]. However, the concentration of fluorescent dyes employed as CL energy acceptors in solution is limited to a low level to avoid the ACQ at high concentrations. In contrast, AIEgens

have showed great potential to contribute as acceptor in CRET systems since the first CL-excitable AIEgen (NSTPE) was reported by Tang’s group (Fig.8a)[41]. Moreover, Lv’s group developed a robust CRET system for sensitive detection of H2O2 with the utilization of AIE-active gold nanocluster aggregates instead of pure organic molecules [42]. As shown in Fig. 8b,c, gold precursors in the diluted solution were not able to boost the CL signal of the TCPO−H2O2 system, but their aggregates displayed a strongly enhanced CL emission. In the absence of any catalyst, the detection limit of H2O2 by this gold nanocluster aggregate-amplified CL probe was as low as 2.0 µM, which was even better than that with aid of imidazole catalyst. 1.6 Energy transfer from photosensitizers (PSs) to oxygen As a treatment strategy that is both minimally invasive and minimally toxic, photodynamic therapy (PDT) has proven ability to kill microbial cells, including bacteria, fungi and viruses, as well as to elicit cancer cell death[43, 44]. In PDT system, photosensitizers (PSs) were conjugated with oxygen. After irradiation, excited triplet-state photosensitizers can react with oxygen via Type-I and Type-II processes. Type-I process involves the transfer of electron or hydrogen atom to the excited photosensitizer, generating radicals to react with oxygen and produce a mixture of reactive oxygen intermediates. In contrast, direct energy transfer occurred in Type-II process between the excited triplet photosensitizer and the triplet oxygen (3O2). This energy transfer dependent process predominates in the induction of cell damage. Compared with traditional ACQ fluorophores, whose binding interaction with biomolecules could inevitably lead to aggregation and cause FL quenching with reduced reactive oxygen species (ROS) generation, aggregated AIEgens and their nanosized structures possessing robust brightness, excellent photostability and colloidal stability can generate efficient ROS. Thus, energy transfer from the triplet state of AIE aggregates to oxygen could offer a great potential for PDT therapy. As a prototype of nanoparticles, AIE nanoaggregates could be prepared easily through solvent polarity tuning and self-assembly of AIEgens, with robust ROS generation capability. As reported by Tang’s group, a series of dipolar AIEgens were employed to prepare nanoaggregates in aqueous solution with bright far-red/NIR emission (Fig. 9a)[45]. Their quantum yields could reach 30% and Stokes shifts were up to 244 nm, with two-photon absorption cross-sections as large as 887 GM. As a result, deep penetration of up to 150 µm could be achieved in living tissues imaging. Furthermore, singlet oxygen could be generated with efficiency even higher than the commercial Ce6, indicating efficient energy transfer from AIE aggregates to oxygen. Another widely employed strategy was using stabilizing matrix to encapsulate AIEgens into nanoparticles for high-efficiency of triplet state energy transfer and ROS generation [44]. For example, Liu and Zheng’s group reported a class of AIE dots for image-guided PDT via simple encapsulation of AIE PSs by amphiphilic DSPE-PEG polymer[44]. As shown in Fig. 9b, with cRGD peptide decorated, the obtained AIE dots could specifically target to integrin ανβ3 over-expressed cholangiocarcinoma cells with excellent PDT outcome in vitro and in vivo.

2

AIE coupling energy transfer in biosensing

Sensitive and specific detection of chemical and biological analytes, such as heavy metal ion,

microenvironmental factor, small molecule, nucleic acid, biopolymer, protein and enzyme, etc. is of great significance to the environmental protection and human health. Especially for the biomarkers of diseases, their early diagnoses usually decide the final outcomes of medical treatments. And nowadays, some chemical factors, such as redox, pH, hypoxia, ROS, etc, existing in the in vivo microenvironment have been verified to be specific hallmarks in the occurrence and development of various diseases. And they are employed as recognition targets in designing of FL probes for disease accurate diagnosis, smart drug delivery and advanced image-guided therapy. 2.1 Heavy metal ions Although trace heavy metal elements are fundamental to living organisms for a normal and healthy life, excessive levels of heavy metals are threating the human body, causing cute or chronic toxicities and leading to serious diseases or death. As one of the most important toxic heavy metal ions, mercury ion pollution increasingly damaged environment and biological system. More seriously, mercury ion can be transformed into methylmercury by microorganisms and subsequently get into food chain to cause serious disorders and diseases[46]. Qian’s group reported a ratiometric AIE probe, namely REPN, for Hg2+ detection[47]. As shown in Fig. 10a, the modified 1,8-naphthalimide was brought into rhodamine B with a hydrazine hydrate linker. REPN exhibited typical FRET signal from pyridine-naphthalimide to rhodamine moiety toward Hg2+, with sensitivity up to 2.72 µM. Similarly, Kumar’s group designed a HPB-rhodamine probe for Hg2+ detection[48]. The probe exhibited aggregation-induced emission enhancement (AIEE) property due to the HPB moiety in aqueous media. In the presence of Hg2+, energy transfer was triggered from HPB donor to rhodamine acceptor, with two emission colors from AIEE and FRET, respectively. And the detection limit was determined to be 100 nM, with excellent specificity toward other metal ions. Further, FL imaging of Hg2+ in MCF-7 cell were also achieved by this HPB-rhodamine probe. Sun’s group reported another ratiometric probe (TR-Hg) for Hg2+ detection[49]. TR-Hg was constructed by linking TPE and rhodamine B thiolactone. In the water absent of Hg2+, the probe showed AIE emission from TPE. After reacting with Hg2+, the acceptor became positively charged and TPE became non-emissive. On the other hand, due to DTBET effect, the dark donor of TPE transferred 100% of its energy to the rhodamine. And the FL intensity from rhodamine was augmented by more than 30 000-fold. Thus, the TR-Hg probe showed ultrahigh sensitivity with detection limit lower to 43 pM, enabling the sensitive detection of Hg2+ in living tissues. Copper is third in abundance among the essential heavy metal ions in the human body and plays an important role in the physiological processes of microorganisms and mammals. Excessive copper, however, is related to cellular damage, triggering in series of diseases. Thus, it is necessary to develop analytical methods for detecting Cu2+ with high selectivity and sensitivity. For this, Liu’s group reported a highly efficient FRET system based on AIEgens and Nile red dyes[50]. As shown in Fig. 10b, AIEgen acted as energy donor and Nile red as acceptor in the FRET system with the optimum concentrations ratio [AIEgen]/[Nile red] = 100. The AIEgen itself displayed excellent selectivity to Cu2+ with a detection limit of 132 nM. Through combination with Nile red, the detection limit was further decreased to 9.12 nM by the FRET process.

2.2 Microenvironmental factors Microenvironmental factors, such as pH value, ROS generation, hypoxia level, are essential indicators of our physiological conditions. Their balances play a central role in maintenance of the normal bioactivity and health of all kinds of living things. It is well known that pH plays crucial roles in cell growth, osmotic equilibrium, enzymatic activity and oncogenesis, etc[51]. Liu’s group designed a cyanine-based TBET cassette to sensitively image pH changes in live cells [52]. The TPE unit acted as donor and the cyanine acted as acceptor, with a large pseudo-Stokes shift up to 484 nm. In addition, the cassette exhibited unique AIE property under both neutral and acidic conditions. However, an increase of pH from 5.0 to 10.7 resulted in significant FL quenching of both the TPE donor and cyanine acceptor, as its increased solubility in basic solution with charge repulsion. Further, a ratiometric NIR probe was reported by the same group for intracellular pH sensing[53]. The probe consisted of a TPE donor and a NIR hemicyanine acceptor via TBET strategy. The lysosome targeting was achieved by introducing morpholine residue to the hemicyanine moiety. It showed typical AIE property at neutral or basic pH. When pH decreased from 7.0 to 3.0, ratiometric signals generated from FL signals of AIEgen and hemicyanine would decrease and increase, respectively, with a up to 238-fold increase in the FL ratio. In addition, the probe offered dual Stokes shifts including a large pseudo-Stokes shift up to 361 nm and well-separated dual emissions, enabling the precisely double-check assay of pH in living cell. Similarly, Yang’s group prepared a FRET-based pH sensor by bridging cyano-diphenylethylene with BODIPY unit[54]. It exhibited strong emission in all fractions of THF/H2O solutions due to the AIE-activity of cyano-diphenylethylene unite and FRET effect. As its pKa value was 9.79, sensitive response to subtle change of pH between 9 and 10 was achieved in living cells. Hypoxia is among the most important features of malignant tumors[55, 56]. He’s group reported a hypoxia-responsive probe with amphiphilic PEGylated azobenzene caged TPE[57]. It possessed excellent solubility in aqueous medium due to the easy formation of micelles. The FRET process caused FL quenching of the caged AIEgen. When cultured with tumor cells, the azo-bond was reduced under hypoxia conditions and the FL emission of the AIEgen recovered dramatically, as the upregulated azoreductase in tumor hypoxia catalyzed the cleavage of the azobenzene linkage. As a minimally invasive approach for cancer treatment, PDT utilized excited photosensitizers to generate singlet oxygen (1O2). Considering 1O2 is the ultimate cytotoxic agent required for e ective PDT, it is of great significance to monitor its formation dynamics to screen out e cient photosensitizers. Thus, Lv’s group designed an AIE-CRET based probe with this capability [58]. The AIE-active sulfonate surfactant (TPE-SDS) remarkably amplified the intrinsic CL emission from 1O2 by integrating its micellar structure with the CL energy acceptor (TPE). And rose bengal, rhodamine 101 and riboflavin were then screened. Results showed that the 1O2 production was nearly stopped in rhodamine 101 and riboflavin after 10 min, but abundant 1O2 was continuously produced by rose Bengal even after 20 min. Zhao’s group further extended the CL sensor for monitoring of the 1O2 in living organisms [59]. Due to its µs-level lifetime and the high possibility to be quenched by omnipresent reductants in tissues, real time sensing of non-irradiation-induced 1 O2 in whole-animal is of great significance. Taking advantage of the efficient intramolecular

energy transfer and aggregation-induced CL amplification, the detection limit could reach 4.6 nM, with high selectivity toward 1O2 among other reactive oxygen species. And finally, real-time mapping of ultratrace 1O2 in whole animal of rat during acute and chronic inflammations was successfully demonstrated. Another type of reactive oxygen species, hypochlorite acid (HOCl) and its conjugate base (OCl-) are implicated in various physiological and pathological processes, whose misregulation is associated with various diseases [60]. Thus, Sun’s group reported an ultra-sensitive and ratiometric probe, namely TR-HOCl, for HOCl detection in living cell[61]. As shown in Fig. 11, the probe consisted of an energy transfer cassette, in which, TPE acted as dark donor and rhodamine B thiohydrazide served as acceptor. In the absence of HOCl, the probe was aggregated and emitted out blue AIE emission. After reacting with HOCl, the rhodamine moiety became positively charged, resulting in enhanced water solubility of the probe. Thus, emission from rhodamine was activated due to the efficient DTBET process. What’s more, emission leakage from the dark donor was eliminated, resulting in an ultra-high sensitivity towards HOCl with an over 7000-fold FL ratio enhancement. Lastly, the successful endogenous HOCl imaging indicated the potential of the probe in living system application. 2.3 Small molecules In the biochemical reactions in living system, many types of small molecules are participating and playing significant influences. Endogenous hydrogen sulfide (H2S) derived from L-cysteine in lysosomes, can disturb the lysosomal membrane and induce cell apoptosis. For its specific detection, Chen’s group designed a photo-switchable AIE nanoprobe (DNBS-DCM-SP) with H2S recognizing group anchored to photochromic spiropyran moiety (Fig. 12a)[62]. In the presence of H2S, the DNBS moiety deviated from DNBS-DCM-SP via O-S bond cleavage, inducing a turn-on FL signal. High specificity to H2S over other thiols was achieved, with excellent sensitivity (∼5.0 nM). In addition, via alternating UV/visible light irradiation, the FL of H2S-activated nanoprobe (DCM-SP) could be reversibly switched by energy transferred from AIEgen (DCM) to ring-opened spiropyran. The monitoring of endogenous H2S was then realized in living cell with high spatiotemporal resolution and imaging contrast. In another example, Li’s group designed an AIE dot for detection of anticancer drug, bleomycin (BLM)[63]. BLM works by preventing the replication of DNA and inducing breaks in DNA strands, and its therapeutic effect is highly depended to its blood concentration. As shown in Fig. 12b, quencher-labeled DNA (Q-DNA) were first coated outside the anti-charged AIE dots with FL quenched via efficient FRET. The recognition and cleavage of Q-DNA by BLM resulted in the formation of three-mer quencher-linked fragments (Q-DNA-1), which significantly decreased the amounts of quenchers on the surface of AIE dot and therefore relieved the FRET e ect. Thus, ultrasensitive detection of BLM was achieved, with detection limit down to 3.4 fM for patients’ serum samples. As the abuse of food dye could cause hyperactivity in children, thyroid toxicity or cancer in animal, Patil’s group reported an strategy for detection of erythrosine (ETS) food dye[64].

AIEE-active nanoprobe (PHNNPs) with negative-charge was responsible to adsorb positive-charge analyte on its surface and further permit efficient FRET to take place from PHNNPs to ETS. The FL of PHNNPs was stepwise quenched with successive addition of ETS, making it applicable for sensitive detection of food dyes as the detection limit could be as low as 3.6 nM (3.1 ng/mL). 2.4 Nucleic acids As the sequence of nucleotides represents the genetic information in living organisms, it is of significance to selectively detect nucleic acids with special sequences. Yang’s group reported a chemo-sensing assemble for specific detection of thymine-rich ssDNA (Fig. 13a,b)[65]. Compared to the sole AIE probe (TPECyZn) which could response FL enhancement upon binding to thymine-rich DNA, the ensemble of TPECyZn with phenol red could eliminate the background noise due to the energy transfer between them and the competitively replacement of phenol red by thymine-rich ssDNA. More importantly, this ensemble probe displayed high selectivity toward thymine-rich ssDNA over other DNA sequences, with ability to discern polyT sequence as short as 2 nt. Accurate detection of mRNA in living cells is of great significance for tumor diagnosis. Thus, Zhu’s group established a molecular beacon (AIE-MB) based strategy for mRNA detection in living cell [66]. As shown in Fig. 13c, green-emissive AIEgen, TPEQ, was labeled on one end of the MB as FRET acceptor. And a blue-emitting ACQ fluorophore, AMCA, was labelled on the other end as FRET donor. The AIE-MB exhibited weak emission at primary hairpin state with efficient FRET between AMCA and TPEQ. In tumor cells, blue-color FL (specific signal) generated after pairing with target mRNA, in which the FRET effect broke down but FL of TPEQ was still inactive due to the good water solubility of DNA. On the other hand, both blue and green FL (false-positive signal) appeared due to the endogenous degradation in normal cells. The AIE-MB, thus, could precisely diagnose the cancer through the di erent FL responses. 2.5 Biomacromolecules Detection of biomacromolecules with high sensitivity and selectivity is a fascinating research field because of its huge potential in biological science and engineering applications.The functions of protein are influenced by particular fibrillation and conformation change. As a signature of Alzheimer’s disease (AD), a sensitive detection method for amyloid β (Aβ) peptides and fibrils was reported by He’s group by AIE-featured glyconanoparticle (AIE-GNP) [67]. Supramolecular complexation between the AIE-GNP and glycoprobe (glycoligand coupled to a red-emitting fluorophore) enhanced the emission of the latter as FRET effect between them. Subsequently, the interaction of AIE-GNP with Aβ dissembled the nanoparticles, with recovery of its FL signal for Aβ detection. Another example to study the conformation transitions of human serum albumin (HSA), was presented by Tang’s group using an specific AIEgen, BSPOTPE[68]. After adding denaturant, the FRET effect between the tryptophan residue in HSA and the bound BSPOTPE unveiled that the HSA unfolding occurred in a stepwise fashion with intermediate states involved in the denaturing process.

The hydrolysis of acetylcholine by acetylcholinesterase (AChE) can accelerate the aggregation of amyloid peptide and plays a causative role in the development of Alzheimer’s disease (AD). Zhang’s group reported a fluorometric assay of AChE activity with core–shell silica containing AIEgens[69]. Precursor containing TPE and one silane moiety first hydrolyzed to form silica nanoparticles, with bright AIE fluorescence. The substrate of AChE, dabcyl chromophores, was then adsorbed onto its surfaces via electrostatic interaction. As a result, the fluorescence of the silica nanoparticles was quenched as the FRET between TPE and dabcyl chromophores. Upon AChE, the hydrolysis of the substrate departed the dabcyl chromophores away from the nanoparticle surfaces, with FL of the TPE restored. In this way, AChE was sensitively analyzed with a limit of detection of 0.2 U mL-1. Similarly, Wu’s group designed a ratiometric system for carboxylesterase (CaE) assay[70](Fig. 14a). CaE is a group of enzymes that catalyze the hydrolysis of fatty acid esters into acids and alcohols. It was discovered that human plasma CaE could be a novel serologic biomarker for hepatocellular carcinoma diagnosis. In the report, fluorescein diacetate was catalyzed from neutral to negative-charge fluorescein in the presence of CaE. As a result, the electrostatic interaction between the positive-charge AIE dot and fluorescein induced the FRET happening. And ratiometric FL signal was achieved for sensitive detection of CaE activity, with detection limit as low as 0.26 U L-1 for human serum sample. Based on the same principle, another essential biomarker in diagnosis of many diseases, alkaline phosphatase (ALP), was sensitively detected by a FL and colorimetric dual-readout platform[71]. Anti-charged DCIP and AIE-active gold nanocluster (PAH-AuNC) were first assembled together with FL of PAH-AuNC quenched via FRET effect. In the presence of ALP, 2-phospho-L-ascorbic acid was transferred to L-ascorbic acid, which could then reduce DCIP from blue to colorless. As a result, FRET was inhibited with FL recovery of PAH-AuNCs. And the distinct colorimetric signal enabled visual distinction of ALP. A wide linear detection range from 0.5 to 100 U/L) was achieved, with detection limits of 0.2 and 0.5 U/L from FL and colorimetric mode, respectively. Due to the high activity in cancer cells and low activity in normal cells, telomerase plays important roles in early detection and diagnosis of cancers. Lou’s group developed a high-specificity strategy for telomerase detection in cancer patients’ urine by combining AIEgen with quencher[72]. Before telomerase extension, Silole-R was e ciently quenched by the FRET effect with the quencher. After the specifically elongation of TS primer catalyzed by telomerase, Silole-R that binding to extension repeat units could be therefore estranged away from the quencher. And the signal was increased by 1424%, which was much higher than the case without quencher labelling (signal increase by 586%). Furthermore, the distinguishing of telomerase extracted from 38 cancer and 15 normal urine specimens verified the reliability of this protocol. They extended the telomerase detection into the living cell[73]. As shown in Fig. 14b, before telomerase extension reaction, emission from AIEgen, TPE-Py, was e ciently quenched because of the FRET effect with the quencher. After transfected into living cells, the quencher-labeled TS primer could be extended by telomerase. TPE-Py then bound to the extension repeated units and was far away from the quencher, producing a turn-on FL signal. This strategy performed well in in-situ tracking of telomerase in various types of living cancer cells.

3

AIE-based FRET probe for enhanced performance in bioimaging

FL-based optical bioimaging provides a direct visual tool for observing biological processes[74]. For optimum imaging of biological species, it is desirable to have the probe fluorescence above the window of the cellular autofluorescence (> 650 nm) to help reduce interference from the substrates. Moreover, large Stokes shift of FL probes can be utilized to improve the imaging sensitivity and avoid the interference between excitation and emission lights. Thus, the AIE-constituted FRET systems possessing red/NIR emission wavelength, large Stokes shift and high signal to noise ratio present unique advantage in the in vitro and in vivo bioimaging. 3.1 Co-encapsulation in nanoplatform for NIR bioimaging By encapsulating two types of AIEgens, including green-emitting 1,1,2,3,4,5-hexaphenylsilole (HPS) and red-emitting bis(4-(N-(1-naphthyl) phenylamino)-phenyl)fumaronitrile (NPAFN), into polymeric micelles, Jen’s group reported a FRET system with large Stokes shift and red emission [75]. The spatial confinement in the micelles induced efficient FRET effect between those AIEgens. As a result, enhancement as much as 8 times in acceptor emission was achieved when the HPS/NPAFN molar ratio was adjusted to be 16:1. Accompanying with large Stokes shift, a great potential was presented in living cell imaging. Liu’s group extended this encapsulation strategy to animal imaging by taking advantages of the conjugated polymer (CP) to further enhance the AIE fluorescence[76]. Green-emitting CP (PFV) was used as a FRET donor. And TPE-TPA-DCM, a far-red/NIR AIE-emitter, served as an acceptor. The donor-acceptor pairs were co-encapsulated into BSA nanoparticles. Due to their conjugated backbones and large absorption coefficiency, the CP could efficiently enhance the fluorescence intensity of AIE acceptors via energy transfer within the BSA nanoparticles. And an over 5-fold amplification of TPE-TPA-DCM FL intensity was achieved. After further functionalization of cRGD peptide, targeted imaging in vitro and in vivo with ultrahigh contrast was realized. To further deepen the penetration depth of bioimaging, Tian and Qian’s group constructed an AIE probe with two-photon NIR excitation and NIR emission for the mouse brain vasculature imaging[77]. A red emissive AIEgen, TB, was chosen as the FRET donor, and a commercial NIR dye, NIR-775, was selected as the acceptor. FRET pairs were co-encapsulated into amphiphilic polymer to construct the NIR FRET NPs. Under 1040 nm femtosecond laser excitation, NIR-775 was excited via energy transfer from TB. The strong NIR (780 nm) fluorescence realized the imaging of mouse brain vasculature with depth up to 150 µm. Similarly, Qian’s group further extended to a three-photon excitation system[78]. As shown in Fig. 15, a three-photon AIEgen, TPATCN, serving as FRET donor, with NIR-775 adopted as the acceptor, were co-encapsulated in polymeric NPs. Under the excitation of a 1550 nm femtosecond laser, TPATCN–NIR-775 FRET pair displayed a bright FL signal at 785 nm, and the FRET e ciency was as high as 90%. Finally, a vivid 3D reconstruction of mouse brain vasculature was obtained with even small blood vessels were clearly visualized. 3.2 CRET-based long-term bioimaging and tracking Taking the advantage of chemiluminescence resonance energy transfer, Ding’s group designed a

NIR afterglow luminescent AIE nanoparticle (AGL AIE dots) for promoted image-guided cancer surgery (Fig. 16)[79]. An enol ether precursor and NIR emissive AIEgen, named TPE-Ph-DCM were co-encapsulated by amphiphilic copolymer to form the afterglow luminescent AIE dots. TPE-Ph-DCM possessed strong 1O2 production capacity. Through a series of cyclic processes occurring in the AIE dots, including 1O2 production, dioxetane formation, chemiexcitation and energy transfer back to TPE-Ph-DCM, the AGL AIE dots emitted persistent luminescence more than 10 days after triggering by single light excitation. In vivo, the NIR afterglow signal of AGL AIE dots could be detected persistently in cancerous tissues but rapidly quenched in normal tissues, leading to an ultrahigh tumor-to-liver signal ratio, indicating its potential in precise image-guided cancer surgery. 3.3 Covalent bond linked FRET system for NIR bioimaging Beyond these co-encapsulation methods for FRET construction, intramolecular system with both AIE and energy transfer properties was also developed for bioimaging. Qian’s group designed a dual-channel dye consisting of electron donor coumarin and electron acceptor BODIPY through TBET effect [80]. The FL intensities increased apparently in water due to the AIE-active coumarin after binding with BSA and the energy transfer from coumarin to the red-emissive BODIPY, presenting a dual-channel FL signals in living cell imaging. Similarly, Liang’s group reported a TBET cassette, TRc, to realize precise cell imaging. The TBET is constructed by covalently conjugated AIE-active TPE unit (donor) and rhodamine (acceptor) through an acetylene bond[81]. It exhibited a large pseudo-Stokes shift (up to 260 nm) from the donor’s absorption to the acceptor’s emission caused by energy transfer, and a small Stokes shift of the acceptor itself. Thus, specific “dual-excitation, single-emission” properties could be achieved, with precisely “double checked” imaging outcome in living cells by colocalization of these two-channel signals. Different to this intramolecular TBET systems, Yang’s group reported a porphyrin derivative with NIR emission via intramolecular FRET effect[82]. Four AIE-active diphenylacrylonitrile units were covalently introduced onto the porphyrin skeleton with non-conjugation bonds, resulting in efficient energy transfer from AIE moieties to porphyrin unit. Interesting, this reformed porphyrin derivative exhibited not only strong fluorescence in solution state, but also in solid/aggregated state due to the FRET effect. Finally, excellent performance in FL imaging of living cells was realized by this NIR agent.

4

Integration of AIE and FRET for real-time monitoring of drug delivery

The intracellular release and activation rate decide the therapeutic outcome of drug. Development of drug delivery system that can self-monitor and offer real-time information of its release behavior, therefore, is of great significance to unveil the pharmacodynamic mechanism and guide the further optimization of the delivery system. The sensitive response of FRET pair to spatial and distance thus provide a powerful tool to realize these real-time monitoring tasks. 4.1 Polymers and nanoparticles-based delivery systems Polymeric micelles with core–shell structure from amphiphilic block copolymers are of great

interest in drug delivery. The hydrophobic core of micelle facilitates the encapsulation of water-insoluble materials, such as anticancer drugs and imaging contrast agents, and the hydrophilic shell ensures good dispersion in the bioenvironment. The incorporation of fluorescent characteristics into polymeric micelles has been fulfilled by physically encapsulation or chemically attachment of fluorophores. And FRET-based probes that constructed with suitable pairs of fluorophores showed unique advantages by harnessing the constrained space of polymeric micelles. For example, Wu’s group developed a type of fluorescent polymeric micelles by self-assembly from a series of amphiphilic block copolymers, PEG-b-P(S-co-PPSEMA)[83]. It contained a hydrophilic PEG block and a hydrophobic block synthesized by copolymerization of an AIE-active monomer (PPSEMA) with styrene. In the micelles, efficient energy transfer from PPSEMA (donor) to Dox (acceptor) happened due to FRET effect between them. The occurrence and decrement of FRET for the Dox-loaded micelles was utilized as an indication for the encapsulation of Dox and its subsequent release, respectively. As a result, the real-time monitoring the drug delivery process of Dox-loaded micelles in living cellular was realized. In addition, He’s group extended the drug delivery strategy into in vivo level with micelles self-assembled by an amphiphilic conjugate of 1H-pyrrole-1-propanoicacid (MAL)−PEG−Tripp-bearing AIEgens[84]. As shown in Fig. 17a, the micelles showed high drug-loading capacity (10.4%) and encapsulation e ciency (86%). The in vivo anticancer activity tests revealed that the Dox-loaded micelles exhibited promising therapeutic e cacy to cancer with low systematic toxicity. More advanced, a dual FRET strategy was reported by Yuan’s group based on an AIE-active polymer (FTP) with upload of Dox for self-indicating cancer therapy[85]. The FTP polymer could self-assemble into nanoparticles (NPs) in aqueous solutions to give strong fluorescence emission via the first intramolecular FRET process. The Dox loaded FTP NPs (drug loading content: 21.77%) were homogeneous particles with size around 50 nm and neutral surface charge. In particularly, the second intermolecular FRET process between FTP (donor) and Dox (acceptor) could serve as indicator for monitoring the in vitro and in vivo drug release profile. Further, with AIE-active conjugated polymers, Cheng’s group provided a color-tunable strategy for self-indicating cancer therapy via adjusting the FRET pairs.[86]. Attributing to intramolecular FRET between TPE and DTBT moiety, the FL color could be tuned from green to red, with Stokes shift increasing from 125 nm to 255 nm. Furthermore, the polymers could form stable nanoparticles in an aqueous solution for paclitaxel (PTX) delivery, and strong inhibition in the growth of cancer cells was achieved. Especially, intracellular locations of the drug carries could be tracked via the two-color FL signal. In addition to the chemotherapy, gene delivery is also monitored via AIE-based FRET systems. Gao’s group[87] reported an example that delivery DNA payload labelled by a green-emissive fluorophore through a blue-emissive AIEgen modified cationic polymer. Real-time observation of the endocytosis of DNA was realized by monitoring the FRET signal between them, with dramatic gene interference effect to the living cell. Without aid from polymer, Tang’s group developed a dual-organelle-targeted nanoparticles (NPs) for synergistic chemo-photodynamic therapy with FRET-based self-monitoring function[88]. Mitochondrion-targeted AIE-Mito-TPP was self-assembled with lysosome-targeted ACQ photosensitizer AlPcSNa4. As shown in Fig. 17b, the fluorescence of both AIE-Mito-TPP/AlPcSNa4 was almost quenched due to the FRET process, and the ACQ effect of

AlPcSNa4 constrained in the NPs. The endocytosis, intracellular release and target accumulation of the drugs can be self-monitored by the turn-on green-emission of AIE-Mito-TPP and red-emission of AlPcSNa4. With synergistic destroy of mitochondria and lysosomes, a remarkable inhibition of the in vitro and in vivo cancer growth was realized. 4.2 Stimuli responsive delivery systems Controlled release of drugs in the polymer-based nanocarriers could be accomplished via tailoring different ‘‘smart’’ moieties, which can be triggered by microenvironmental stimuli, such as pH, redox reagents, enzymatic responsiveness, etc[89, 90]. Gao’s group reported a pH-response release system based on cationic amino poly(glycerol methacrylate) (PGMA) nanoparticulate formed by aggregation of AIE-active para-carboxyl-functionalized TPE and Dox via electrostatic interaction[91]. The Dox could be controlled released under the acidic condition. Two-photon excitable TPE derivatives served as FRET donor to promote emission of Dox acceptor, allowing the identification of Dox in the first mode. And the overall Dox was captured via the second direct single-photon excitation, consequently providing double-checked information of Dox release. What’s more, after functionalizing with folic acid, the vehicle could serve as targeting delivery of drug with surveillance capacity in the whole body and subcellular level. With Schiff bases in polymer structure, Wu’s group developed another type of pH-responsive delivery system[92]. Amphiphilic polymer, PEG–POSS–(TPE)7, was first assembled into fluorescent vesicles in aqueous solutions, with TPE units linked via Schiff bases. Its emission was apparently enhanced in the rigidified cage-shaped polymer structure and formed a FRET pair with the encapsulated Dox. The Schiff bases maintained their structural integrity under neutral conditions but rendered vesicle disassembly under acidic conditions. As a result, intracellular behave of the pH-responsive Dox release was tracked along with the disassembly, accompanying efficient inhibitions to cancer cell proliferation. Besides these pH-responsive systems, redox-responsive polymer micelles are also used as versatile platforms for drug delivery. Since the concentration of GSH in cytosol is 1000-fold higher than extracellular environment, the covalent drug loading could efficiently avoid the premature drug release during systemic circulation. Zhao’s group developed a redox-responsive micelle by united FRET and AIE effects for in situ probing intracellular drug release process[93]. AIE-active TPE was selected as a donor and curcumin was chosen as the model drug as well as FRET receptor. The ACQ-active drug was covalently linked to a block copolymer (mPEG-PLys) via a disulfide bond linker and TPE was chemically linked to the polymer via an amide bond, and the obtained amphiphilic polymer self-assembled into micelles. Upon endocytosis, the drug release triggered by the endogenous GSH, resulted in the fluorescence increase of both TPE and curcumin with disruption of the FRET effect. 4.3 Host-guest bridged smart delivery systems As an important type of noncovalent force, host−guest interactions bridged supramolecular were also widely applied in smart drug delivery owing to their a uent stimuli-responsive behaviors. Huang’s group reported a pillar[5]arene(P5)-based [2] rotaxane supramolecular structure (R1) for

pH-response drug delivery system with AIE-active TPE and mitochondria-targeting triphenylphosphonium (TPP) as stoppers[94]. R1 was further transformed to prodrug by conjugating with anticancer drugs containing amine groups via an imine bond. Thus, a dual-FL-quenched FRET system was constructed, with TPE-based axle served as a donor and the drug, Dox, acted as the acceptor. Upon hydrolysis of the pH-responsive imine bonds in endo/lysosomes, both FL signals from the carrier and drug recovered, enabling real-time monitoring of the drug delivery in living cells. Surpassing the above single stimulus-response system, Huang’s and Chen’s groups further developed a reductase-pH dual stimuli-response systems (P5-PEG-Biotin⊃PTPE and CB[8]⊃(PEG-Np·PTPE), respectively), with introducing of reductase-response moiety(viologen salt) (Fig. 18)[95]. Encapsulation of Dox in the supramolecular nanoparticles formed by self-assembly of P5-PEG-Biotin⊃PTPE or CB[8]⊃(PEG-Np·PTPE), caused the fluorescence quenching of both TPE component and Dox due to the FRET and ACQ e ects. The intracellular low pH and reductase (such as NADPH and NADH) could led to the dissociation of the host−guest complexation and trigger the release of loaded Dox, with recovery of the FL signal via interruption of the FRET e ect. Therefore, in situ visualization of the drug release process by observing the energy transfer-dependent FL signals was achieved, with excellent antitumor e cacy with negligible systemic toxicity in vivo after further decoration of biotin ligands on the nanoparticles. Similarly, e ective accumulation in tumor tissues through the enhanced EPR e ect and a long blood circulation time was also achieved in Chen’s supramolecular system in vivo, indicating their excellent self-imaging and controllable drug release abilities[96].

5

Therapeutics via energy transfer from excited AIE PSs to oxygen

The singlet oxygen generated in PDT process can damage the cell membrane and intracellular nucleic acid, amino acid residues and subsequently lead to cell destruction and death. The efficiency of PDT process is dependent upon the energy transfer efficiency from the triplet state of PS to oxygen. For which, the long triplet state lifetime and the triplet quantum yield of the AIE PSs as well as high diffusion concentration of oxygen were the prerequisites. And optimization of these parameters to enhance energy transfer efficiency and thus to improve the phototherapeutic effectiveness was the target in designing of powerful PSs and PDT systems. Several strategies are developed regarding to this target, from tuning the photophysical properties of PSs to refining the nanostructures, from single-route energy transfer to dual-mode energy transfer, and from photo-irradiation to chemical excitation, etc. 5.1 Regulation of the single-triplet energy gap of PSs Tuning the single-triplet energy gap (∆Est) of AIE PSs to enhance the intersystem crossing (ISC) process from the lowest excited singlet state (S1) to the lowest triplet state (T1) is one strategy to optimize the PDT efficiency. Liu’s group reported a series of AIEgens (TPEDC1, TPEDC2 and TPETCAQ) and tuned their ∆Est values from 0.350 eV to 0.067 eV to enhance the performance in NIR fluorescent image-guided PDT [97]. The D-A structure led to HOMO-LUMO separation with ∆Est value of 0.350 eV in TPEDC1. After introducing a phenyl ring in TPEDC1, the conjugation between the donor and acceptor in TPEDC2 was further separated, leading to the decrease in ∆Est

value to 0.230 eV. Further addition of one more dicyanovinyl group produced TPETCAQ, leading to an extremely low ∆Est value of 0.067 eV. TPETCAQ showed the longest emission wavelength centered at 820 nm, and the most efficient 1O2 generation capability in these three types of AIE PSs, which was even greater than commercial PSs (Ce6). Meanwhile, the energy levels in both the singlet and the triplet states of conjugated polymers with π-conjugated backbone are usually much denser than those of their small molecule analogues, which is beneficial to ISC process for 1O2 generation. Thus, polymerization of AIE PSs is another way to enhance the PDT performance[98]. Liu’s group recently reported a conjugated polymeric PS, PTPEDC2, based on a small molecule AIE PS (TPEDC)[98]. After encapsulating by amphiphilic polymer, the 1O2 generation efficiency of PTPEDC2 dots was 5.48- and 6.37-fold than those of TPEDC dots and Ce6 dots, respectively. Theory calculation showed that the gaps between di erent energy levels become much smaller after polymerization, indicating more channels available for energy transfer to 3O2. Further, Liu’s group demonstrated that nanocrystallization could enable significant enhancement in 1O2 generation with simultaneous increase in the brightness of AIE PSs[99]. The absorption and emission intensities were simultaneously enhanced, with quantum yield of nanocrystals increase more than 2 times compared to the amorphous nanoaggregates, due to the stronger intermolecular electron coupling. Together with the restricted molecular motions, improved energy transfer efficiency to oxygen happened with a 50% increase of 1O2 production rate compared to the nanoaggregates. It solved the problem in traditional AIE PS systems that the increase in 1O2 generation always led to the loss of fluorescence, due to the fundamental competition between FL emission and 1O2 generation. 5.2 Refining the nanostructures of PSs As the donor in the energy transfer process, increasing the oxygen diffusion rate is also an important target to realize efficient generation of 1O2. In a recent study, Liu and co-author reported a strategy to enhance the oxygen diffusion by integration of AIEgen with liposome to form a type of AIEgen–lipid conjugate, AIEsome[100]. The AIEsome not only overcame the ACQ problem of conventional fluorescent liposome with bright red fluorescence, but also offered better oxygen exposure than that of the AIE NPs constructed by DSPE-PEG amphiphilic polymer. As a result, ROS quantum yield of the AIEsomes (0.88) was much higher than that from AIE NPs (0.51). It is believed that the higher surface of AIEsome and the looser packing of the AIE PSs could induce better oxygen diffusion inside the thin lipid bilayer, while the large and solid aggregated NPs caused the PSs inside the core hardly touched oxygen. Moreover, a dual energy transfer strategy was employed by Xu and Lou’s group to enhance the PDT performance in deep-seated tumor by encapsulating inorganic upconversion nanoparticles (UCNPs) and organic AIEgens (TTD) using an amphiphilic polymer (Fig. 19)[101]. Through conjugating with cRGD peptide, UCNP@TTD-cRGD NPs with targeting capability to cancer cells were generated. The dense packing and hydrophobic interaction within the NPs made the first route energy transfer efficiently occurred between TTD and UCNPs, resulting in the green-color emission of UCNPs changing to the red emission of the UCNP@TTD-cRGD NPs upon 980 nm laser illumination. Under the coverage of a 3 mm thickness tissue, the 1O2 quantum yield of

UCNP@TTD-cRGD NPs under 980 nm excitation was up to 18.2%, which was much higher than white light excitation (10.4%). And a significant amount of ROS could even be generated with the coverage thickness increasing to 6 mm, verifying the efficient second-type energy transfer from excited TTD to oxygen. Attributing to this dual-type energy transfer, the growth of deep tumor model was significantly restricted, implying a powerful PDT tool for treatment of deep-seated tumor. 5.3 Harnessing chemical excitation for efficient 1O2 generation Beside the above photo triggered energy transfer systems, Liu’s group developed a novel system with chemiexcited far-red/NIR emission and 1O2 generation for precise cancer theranostics[102]. As shown in Fig. 20, CPPO and photosensitizer TBD were co-encapsulated by F-127 to form C-TBD NPs. TBD molecule was intensively designed with capabilities for effective 1O2 generation and aggregation-induced far-red /NIR emission. More importantly, it was endowed with a suitable energy level to match the chemical excitation source for direct excitation by chemical energy. As this process did not require any external excitation sources, it overcame the limitation of penetration depth in traditional FL imaging and PDT. In addition, the in situ and effective 1O2 generation by energy transfer from the chemiexcited C-TBD NPs could efficiently induce the tumor cells apoptosis and inhibit the tumor growth. With the aid of b-phenylethyl isothiocyanate (FEITC) to enhance the H2O2 production at the tumor site, both the chemiluminescence response and the therapeutic function were further enhanced, indicating a powerful strategy for intelligent, accurate, and non-invasive tumor therapy.

6

Perspective

6.1 Introducing novel components to FRET pairs As the spectrum crosstalk between the donor and acceptor, in which the donor emission spectrum can overlap with the acceptor emission spectrum and the acceptor may be directly excited at the donor excitation wavelength, is the major challenge for quantitative FRET analysis, many experimental efforts have attempted to circumvent these challenges over the years. Besides the above mentioned AIEgens with large Stokes shift, time-resolved FRET assays with fluorophores that exhibit long half-lives, alternatively have proven effective in eliminating interfering FL from unwanted sources and reducing the crosstalk. AIE-based pure organic room temperature phosphorescent (RTP) luminogens, such as dibenzothiophene, carbazole, borate and polyacid derivatives, etc., with versatility in molecular design and exceptional RTP photophysical properties, have sprung out and been extensively explored [103]. They have showed immense potentials in applications of highly sensitive sensing and high-contrast time-gated phosphorescent bioimaging. In this sense, AIE-based RTP materials with ultralong luminescence life-time could be a promising candidate for construction of this class of crosstalk-free FRET systems to contribute the background-free biosensing and imaging. 6.2 Beyond molecules: measuring force, tension, voltage in biosystems In modulating cell activities, three types of signaling systems including biochemical, mechanical

and electrical, play the fundamental roles[104]. Almost all biological processes are responsive to modulation by mechanical or electrical forces that trigger dispersive downstream biochemical pathways. However, due to the lack of real time measuring methods at cellular and subcellular levels, the mechanical and electrical signaling systems are still elusive. Cellular response to physical cues from adjacent cells and the extracellular matrix leads to a dynamic cycle in which cells respond by remodeling their local microenvironment, fine-tuning cellular adhesion forces, stiffness, polarity, viscoelastic, shape and so on[105]. Traditional techniques, including atomic force microscopy, laser tweezers, and DNA calibrated force sensors were used to study cell mechanics[106]. However, none of them can be used to measure intracellular forces in real time. Fortunately, molecular tension probes based on FRET that could report on piconewton scale tension events in live cells are developed, allowing to correlate these events with intracellular biochemical processes. To estimate the forces and their spatial-temporal distribution in live cells, genetically coded FRET probes emerged as suitable techniques and can be integrated into individual proteins, turning structural forces into optical signals to estimate the intracellular force in specific proteins, such as the structural proteins, in live cells[107]. In this sense, the protein and organelle targeting or biorthogonal labeling AIEgens could be integrated into the genetic-encode FRET systems, and the unique photophysical properties of AIE molecules or nanoparticles would certainly bring great progress to the measurement of mechanical signals in living biosystems. On the other hand, electrical signals in nerve and muscle are crucial for learning, memory, movement and many other physiological processes. The tiny electrical signal is produced by voltage-gated sodium channel in living cells, which form a voltage-regulated pore that allows rapid passage of positively charged sodium atoms across the cell membrane. Voltage-sensitive dyes-based FRET are developed to sense these electrical signals in living cells[108]. However, due to the limitations of synthetic membrane potential dyes in tissue penetration and indiscriminate cellular staining, poor signal-to-noise ratio almost accompanied the voltage sensing[108]. In particular, labeling target neurons among non-neuronal cells, such as glia, is full of challenge. Thus, utilizing genetically encoded method by expressing and anchoring fluorescent protein (FP) between the voltage-gated ion channels in living cell showed great potential[109]. Considering the availability of AIE-based sensing systems for physiologic pH and ions involved in electrical signaling pathway (e.g. calcium), approaches that integrating genetically encoded membrane-bound FP with multifunctional AIEgen, would create versatile hybrid FRET systems for voltage monitoring in living cells and tissues. What’s more, most FPs show some tendency of self-association, especially when they are fused to other proteins and induce artificial clustering. As a result, the introducing of AIEgens would be extremely suitable to further improve the performance of hybrid FRET systems as their natural aggregation-induced luminescence signal change.

Conclusion In this review, we discussed various types of AIE-based energy transfer systems and their versatile applications in biosensing, imaging and therapeutics. They were constructed via non-conjugate

covalent bond energy transfer, through-bond energy transfer, co-encapsulation in nanoplatform for FRET, host-guest interaction bridged FRET, chemiluminescence resonance energy transfer and energy transfer from triplet state to oxygen and so on. The unique photophysical properties of AIE molecules and nanoaggregates with large Stokes shift, robust luminosity, strong photobleaching resistance, no random blinking and tunability in emission wavelength, make them to be superior choices for construction of efficient FRET systems. And the AIE-based FRET probes had exhibited great potential in applications from in vitro to in vivo, such as ion sensing, microenvironmental factor/small molecule analysis, biomacromolecular assay, drug delivery monitoring and energy transfer in PDT. We expect that introducing of novel types of AIEgen into FRET pairs and building multicomponent hybrid FRET systems, would further provide promising progress for analytical chemistry, biological and biomedical applications.

Acknowledgments The authors acknowledge funding to B.Z.T. from the Research Grants Council of Hong Kong (C6009-17G), the Innovation and Technology Commission (ITC-CNERC14SC01), National Natural Science Foundation of China (21788102), the Natural Science Foundation of Guangdong Province (2019B030301003), and the National Key Research and Development program of China (2018YFE0190200). B.Z.T. is also grateful for the support from the Science and Technology Plan of Shenzhen (JCYJ20160229205601482). Funding to R.T.K.K. from the Science and Technology Plan of Shenzhen (JCYJ20180507183832744). And funding to L.-H. X. from the National Science Foundation of China (21705111).

Fig. 1. Schematic illustration of the construction of FRET pairs. The spectra of donor emission (Em) should be overlapped with the spectra of acceptor excitation (Ex). The efficiency of FRET tended to be high when the distance between donor and acceptor was within 10 nm. Reprinted with permission from Ref.[5]

Fig. 2. Schematic illustrate of various strategies for constructions of energy transfer systems. (a) Non-conjugated covalent bond energy transfer. (b) Through-bond energy transfer, (c) Co-encapsulation in a nanoplatform. (d) Host-guest interaction bridged FRET. (e) Chemiluminescence resonance energy transfer. (f) Energy transfer from the triplet state of a

fluorophore to oxygen.

Fig. 3. (a) Schematic illustration of the design of AIE-based FRET probe-inspired nano-prodrug, Energy transfer happened between F and R and the acceptor (Dox) quenched itself by ACQ effect. Thus, the FL of both the donor and acceptor was quenched; once FRET was disrupted, both FL reappeared. The hydrazone bond was employed as spacer responding to the low pH in cells, with woken of both FL from TPE and Dox. F: fluorophore group; S: spacer; R: receptor group; G: guidance group; D: drug moiety. (b) Confocal laser scanning microscopy (CLSM) images of nano-prodrug in the MCF-7 cell line co-stained with LysoTracker Deep Red (scale bar: 20 µm). Reprinted with permission from Ref. [32].

Fig. 4. (a) Schematic illustration of the FRET probe using AIEgen as energy quencher with dual signal output for caspase-3 detection. (b) PL spectra of Cou–DEVD–TPETP upon incubation with different concentrations of caspase-3. (c) PL intensities monitored at both 465 and 665 nm for Cou–DEVD–TPETP upon treatment with various proteins. Reprinted with permission from Ref. [31].

Fig. 5. (a) Schematic illustration of the ratiometric Hg2+ sensing mechanism of p/m-TPE–RNS via DTBET. (b) PL spectra of m-TPE–RNS in the presence of different amounts of Hg2+. (c) PL spectra of m-TPE–RNS in the mixture of different metal ions. Reprinted with permission from Ref.

[34].

Fig. 6. Schematic illustration of the preparation and photo-switchable behavior of PFPNs under UV/visible light irradiation. Below: FL images of HeLa cells treated with PFPNs under visible/UV illumination. Reprinted with permission from Ref. [35].

Fig. 7. FRET donor M1 and acceptor M2 and proposed host-guest interaction mechanism. Reprinted with permission from Ref. [39].

Fig. 8. (a) Molecular structure of AIEgen (NSTPE) and its solid-state CL after NSTPE coated on glass with time elapse. H2O2 was first injected on the surface of NSTPE-deposited slices followed by addition of oxalyl chloride. The generated 1,2-dioxetane-3,4-dione encountered the AIEgens and induced them to emit. Reprinted with permission from Ref. [41]. (b) Schematic illustration of gold nanocluster aggregate-amplified TCPO–H2O2 CL. (c) FL spectra of the Au(I)–thiolate complexes in water (red line) and in a 95% ethanol–5% water mixture (blue line). Insets: AIE curve with fraction of ethanol and photographs in water (left) and in a 95% ethanol–5% water mixture (right) under 365 nm UV light. (d) CL amplified by Au(I)–thiolate complexes in water and in a 95% ethanol–5% water mixture. Inset: CL spectrum in a 95% ethanol–5% water mixture. Reprinted with permission from Ref. [42].

Fig. 9. (a) A series of dipolar far-red/NIR AIE aggregates for two-photon imaging and 1O2 generation. Reprinted with permission from Ref. [45]. (b) Schematic illustration of T-TTD dots preparation and 1O2 generation. Reprinted with permission from Ref. [44].

Fig. 10. (a) Proposed binding mode of REPN with Hg2+ and its detection mechanism via intramolecular FRET effect. Reprinted with permission from Ref. [47]. (b) Schematic illustration of the detection of Cu2+ based on FRET strategy in aqueous solution. Reprinted with permission from Ref. [50].

Fig. 11. (a) Proposed reaction mechanism of TR-OCl with HOCl via TBET effect. (b) Specificity test of TR-OCl. (c) Sensitivity test of TR-OCl. Reprinted with permission from Ref. [61].

Fig. 12. (a) Schematic illustration of the strategy for sensing H2S by DNBS-DCM-SP with reversible dual-color FL imaging. Reprinted with permission from Ref. [62]. (b) Schematic illustration of AIE dots preparation (I) and the detection for BLM (II). Reprinted with permission

from Ref. [63].

Fig. 13. (a) Schematic detection of ploy dT by the chemo-sensing ensemble of TPECyZn with phenol red. (b) Detection performance of poly dT by TPECyZn-phenol red, from left to right were FL spectra of TPECyZn upon addition of phenol red and then addition of T10 and FL intensity versus concentration of various kinds of DNA. Reprinted with permission from Ref. [65]. (c) Structure of AIE-MB and schematic illustration of the preparation of AIE-MB nanoparticles and their working mechanism in living cell. Reprinted with permission from Ref. [66].

Fig. 14. (a) Schematic illustration for the CaE assay system and corresponding FL photographs taken under 365 nm UV light. Reprinted with permission from Ref. [70]. (b) Schematic illustration of the AIE-based in-situ and real-time telomerase imaging in living HeLa cells. Reprinted with permission from Ref. [73].

Fig. 15. (a) Structures of TPATCN, NIR775, and F127. (b) Schematic illustration of the fabrication of TPATCN and NIR775 co-encapsulated NPs. (b)Schematic illustration of the FRET process, including the energy overlap between donor fluorescence and acceptor absorption and the energy level and relaxation process between the donor and the acceptor. (c) Three-photon FL images of a mouse's brain vessels at different vertical depths (scale bar 100 µm). Reprinted with permission from Ref. [78].

Fig. 16. (a) Structure of AIEgen (TPE-Ph-DCM) and reaction route of Schaap’s dioxetane to generate afterglow luminescence. (b) Schematic illustration of the mechanism for amplified NIR afterglow luminescence from AGL AIE Dot. (c) Afterglow luminescence decay behaviors over time of AGL AIE dots in different tissue homogenates. (d) Ex vivo NIR afterglow imaging and FL imaging of various tissues from peritoneal carcinomatosis-bearing mice received intravenous injection of AGL AIE dots for 2 h. Reprinted with permission from Ref. [79].

Fig. 17. (a) Schematic diagram of the self-assembly of MAL–PEG–Tripp polymeric micelles with FRET effect for drug delivery. (b) CLSM images of distributions of Dox loaded MAL–PEG–Tripp micelles co-localized with Lysotracker Green in 4T1 cells. Reprinted with permission from Ref. [84]. (c) Schematic illustration of dual organelle targeted NPs with synergistic chemo photodynamic therapy functions through self assembly of mitochondria targeted chemotherapeutic agent AIE Mito TPP and lysosomes targeted photosensitizer AlPcSNa4. Reprinted with permission from Ref. [88].

Fig. 18. (a) Chemical structures of M, P5, P5-PEG-Biotin and PTPE. (b) Schematic illustration of the formation of SNPs self-assembled from the amphiphilic supramolecular brush copolymer P5-PEG-Biotin⊃PTPE and their use as drug delivery vehicles. Reprinted with permission from Ref. [95].

Fig. 19. (a) Schematic illustration of preparation of UCNP@TTD-cRGD nanoparticles and their applications in bioimaging and PDT upon NIR laser illumination. (b) FL spectra of UCNPs and UCNP@TTD-cRGD NPs. (c) Relative ROS generation. (d) Relative ROS generation of UCNP@TTD-cRGD with coverage of different thickness of tissues. Reprinted with permission from Ref. [101].

Fig. 20. (a) Preparation of C-TBD NPs and the principle for chemiluminescence (CL) and 1O2 generation of C-TBD NPs in the presence of H2O2. (b) Time-dependent in vivo CL and FL images of mice (with tumor region marked by yellow circle). (c) Tumor growth curves with different therapies. Reprinted with permission from Ref. [102].

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Ben Zhong Tang, Ph.D. Chair Professor Polymer Research Lab Department of Chemistry

Hong Kong University of Science & Technology Clear Water Bay, Kowloon Hong Kong, China

Tel. (852) 2358 7375 Fax. (852) 2358 1594 E-mail: [email protected] http://home.ust.hk/~tangbenz

 Oct. 21, 2019

Subject:

Invited Review for the special issue on “Resonance energy transfer techniques in analytical chemistry”

Type of Article:

Review

Title:

AIE-Based Energy Transfer Systems for Biosensing, Imaging, and Therapeutics

Authors:

Xuewen He, Ling-Hong Xiong, Yanlan Huang, Zheng Zhao, Zaiyu Wang, Jacky Wing Yip Lam, Ryan Tsz Kin Kwok, Ben Zhong Tang

The highlights of this review article are as following: 1. Unlike conventional organic fluorophores, AIE nanoaggregates and nanoparticles exhibit large Stokes shift, robust luminosity, strong photobleaching resistance, no random blinking and excellent biocompatibility, indicating their great potential in construction of FRET systems for application in vitro and in vivo;

2. The flexible molecular design and diversified nanostructural fabrication of AIE based materials, and have been widely applied in building FRET probes for bioanalytes sensing, imaging and drug delivery monitoring with high sensitivity and specificity;

3. Versatile strategies for AIE-based energy transfer systems, with divisions, such as nonconjugate covalent bond energy transfer, through-bond energy transfer, coencapsulation in nanoplatform for FRET, host-guest interaction bridged FRET, binding with graphene oxide quencher, chemiluminescence resonance energy transfer as well as energy transfer from triplet stated photosensitizer to oxygen, are discussed detailly with several vivid application examples.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.