Review on recent trends and prospects in π‐conjugated luminescent aggregates for biomedical applications

Metal‐free organic molecules with structurally diverse aggregation‐induced emission (AIE) behavior and new functional photophysical properties reveal unique structure–property relationships, especially bright luminescence, and have attracted extensive attention in the past few decades. Despite tremendous progress on fluorescent molecules development, the extended π‐conjugated organic AIE probes with their nanorange self‐assembly, coassembly, unique morphology, high biocompatibility, and light‐harvesting capabilities enable them as potential candidates in numerous translational application perspectives. In particular, a few important classes of AIE light up small molecules, supramolecules, oligomers, polymers, including nanoparticles and photosensitizer molecules, with their emerging properties of thermally activated delayed fluorescence, room temperature phosphorescence, including emission switching stimuli‐responsive behavior and multifunctional properties, have been boosted by the rare features of aggregation at their condensed or solid states. This review highlights salient features of AIE‐based emitters, encompassing molecular design strategies, stimulating photophysical properties, mechanistic aspects, and their efficacy in various electronic and biomedical applications, broadly covering properties of small molecules to oligomers, macromolecules to polymers.


INTRODUCTION
Discovery of luminescent materials has emerged as an indispensable research area and remains a principle focus in the scientific community as they promote extensive to hightech applications in materials and biomedical science and technology. [1,2] Efficient luminescent emitters, such as metalorganic framework, [3] lanthanide metal-based nanoparticles (NPs), [4] inorganic quantum dots, [5] and so on, have been developed over a few decades. Nonetheless, pure organic molecules containing high-order π-conjugation and heteroatoms in their molecular frameworks have become superior alternatives to realize high fluorescence quantum yields, excellent biocompatibility, good photostability, and low phototoxicity during biological application. [6,7] Moreover, a large number of π-conjugated luminescent molecules have been explored to exhibit fluorescence in dilute solution, thin governed by multiple noncovalent interactions, thereby, ensuing versatile molecular conformations, which in turn control their condensed state luminescence behavior without any structural perturbation. Although significant efforts have been devoted to exploring luminescence in organic AIEgens those mostly rely on singlet excitons only; however, by harnessing triplet at the same time, the efficiency of the luminogens could be enhanced manifold. [12,13] This includes recently developed AIE active thermally activated delayed fluorescence (TADF), room temperature phosphorescence (RTP), and mechanoresponsive properties. [14][15][16] Unlike the sensitivity of triplet state by oxygen and moisture of this particular type of materials, the long-lived phosphorescence and delayed fluorescence resulting from the enhanced intersystem crossing (ISC) and reverse intersystem crossing (RISC) from excited triplet state (T 1 ) to excited singlet state (S 1 ) in these luminophores gives rise to long lifetimes ranging from nanoseconds to milliseconds even up to seconds. [17] Subsequently, aggregation is a key link between AIE and TADF/RTP property, which enables them as excellent chromophores for versatile applications. As compared to the solution, in aggregated state, chromophores are induced by supramolecular noncovalent interactions, which increases the number of ISC channels and facilitates the overlapping of excitons from singlet state to triplet excited state excitons and this further reduces the energy gap (ΔE ST ) between S 1 and T 1 , thereby, enhancing delayed luminescence pathway. [18] Thus, the aggregation is a key phenomenon that furnishes enhanced delayed fluorescence or phosphorescence lifetime for these classes of new fluorescent organic materials. These advanced AIE probes show superior optoelectronic device performances and high-contrast imaging, including deeper penetration depth over traditional fluorescent molecules. [19] Likewise, AIE-based supramolecular frameworks, including macrocycle, macromolecules, oligomers, and selfassembly systems with their intrinsic structure-property, are very promising. [20,21] These classes of AIEgenic materials possess encouraging and superior luminescence efficiency while exhibiting stable self-assembled architecture in the condensed state. They have extended π-conjugated molecules that improves the structural order and controls the morphology of the materials, which play a crucial role in a diverse range of applications like optical devices, biological cell permeability, and so on. [22] Importantly, high luminescence property also enables the real-time visualization of molecules to assembly transition using confocal laser fluorescence microscopy. [23] Moreover, these typical advantages have opened up many strategies to design AIEgenic supramolecular materials through anchoring long hydrophobic alkyl chains, large biomolecules, and so on, and doping of AIEgens into a supramolecular frame. Especially, AIEgens may bind with different biomolecules present in the biological system and due to the influence of the surroundings, the free intramolecular motion of the molecules is constrained, which enhances the radiative decay of the luminophores. [24] By analyzing the nature and intensity of the aggregate emission, some biomolecules and certain crucial interactions inside the system can be traced. [25] The spontaneous aggregation of hydrophobic dyes inside the hydrophilic bioenvironment enables AIEgens to easily "turn-on" followed by impressive fluorescence quantum yield, which is beneficial for improving the spatial resolution of imaging with better contrast. [26] Additionally, AIE polymers are another new class of luminescent materials explored very recently. AIE polymers exhibit properties of both functional materials, such as high solid-state brightness of AIEgen and tunable structure, morphology, and processibility of the polymer. [27] Through the combination of AIE property and polymer physiognomies, a unique functional material can be generated. AIE polymers possess additional advantages compared to small molecule AIEgens as the polymer's backbone or side chains cause steric crowding and render free rotation of AIE fragment and result in boosted emission. Nonetheless, conjugated AIE polymers display amplified sensitivity toward external stimuli (heat, light, chemical species, etc.) as they exhibit efficient exciton migration along the conjugated backbone. [28] Besides, incorporating ionic AIEgen into nonconjugated amphiphilic polymer bestows them with highly emissive functional materials for long-term cellular tracking, drug delivery, and image-guided therapy. Moreover, AIE polymers exhibit excellent solid-state optical properties, charge carrier transport, and remarkable processability, allowing large area manufacturing of optoelectronic devices. [29] In this way, significant efforts have been devoted to the advancement of efficient design principles and realizing new functional properties in various AIE-based small molecules, supramolecules, macromolecules, oligomers, and polymers, respectively. In particular, simple and smart small AIE molecules exhibiting multifunctional properties, which include TADF, RTP, and SR, respectively, have drawn much attention in recent years. [30] The collective properties of such family of molecules enable the probe to be more potential for eliminating tissue autofluorescence and improving signalto-noise ratio by facilitating a proper delay time between the long-lived emission of dyes and the pulsed excitation light. [31,32] Thus, this review highlights the recent developments and key principles of the efficient design strategy, important multifunctional photophysical properties evolved along with newer strategies for various translational applications. Additionally, this review presents various aspects of molecular to electronic properties in aggregated or solid state for most of the important classes of AIE-based π-conjugated luminescent small molecules, supramolecules, oligomers, polymers, AIEnanoparticles (AIE-NPs), and AIE-photosensitizers (AIE-PSs), respectively. In particular, these classes of organic materials have recently drawn wide attention as they offer high photoluminescence quantum yield (PLQY, Φ f ), uniform nanomorphology, good reproducibility, smooth thin filmforming ability, high photo/thermal stability, and high lightharvesting ability, which are the most desirable parameters to overcome all the obstacles associated with conventional fluorophore. Such organic materials evolving with exceptional condensed state photophysical properties have drawn potential interest in the extended field of pharmaceuticals, agrochemicals, pigments, dyestuffs, foods, explosives, fluorescence imaging, photoacoustic imaging, and image-guided photodynamic/photothermal therapy (PDT/PTT), including future opportunities for AIEgens to advance optoelectronic device applications. [33][34][35] F I G U R E 1 (A) Illustration of various photophysical processes and reactive oxygen species (ROS) generation mechanism shown in a typical Jablonski diagram. (B-i, ii) Examples of TADF and AIE-TADF chemical structure. (C) AIE supramolecular architecture (Adapted with permission from ref [112] Copyright 2017 American Chemical Society). (D) Demonstration of AIE-polymers (Adapted with permission from ref [116] Copyright 2015 Royal Society of Chemistry)

AIE-TADF
TADF materials are an emerging class of fluorescent small molecules and considered as an alternative to inorganic phosphorescent emitters, pioneered by Adachi and coworkers in 2012. These materials displayed slightly delayed emission and have achieved higher lifetime of up to several microseconds due to the enhancement of ISC and RISC processes simultaneously. [36] The basic aspects of AIE-TADF materials are favorable to confer the common feature of the molecular design strategy. The most important features of both AIE and TADF molecules are present in the twisted molecular skeleton with twisted intramolecular charge transfer (TICT) states. A typical Jablonski diagram could explain the phosphorescence phenomenon, wherein, by irradiating light on organic conjugated molecules, the generated excitons can go to the singlet excited state S 1 or even at higher vibrational levels (S n ) ( Figure 1A). Through internal conversion and vibrational relaxation, these excitons come to S 1 state and return to S 0 by releasing fluorescence emission. Alternatively, they can move to triplet excited state via ISC and return to ground state by radiative process, which is called phosphorescence. Whereby the radiative decay of the singlet excitons, which results from the upconversion of triplet excitons, gives a delayed fluorescence with thermal activation, hence, termed as TADF. [37] ISC is easily feasible in case of inorganic complexes because they comprise transition metals and heavy metals, which are very much susceptible to spin-orbit coupling (SOC). However, achieving sufficient ISC from metal-free organic compound is a challenging task due to the lesser viability of SOC. [38] Nevertheless, only realizing efficient ISC is not enough to accomplish TADF; yet, the key process of upconversion of triplet excitons via RISC is highly desirable. A small energy difference between the triplet and singlet state (∆E ST ) will make the process easier, whereas a large ∆E ST value will offer a higher energy requirement, which acquires the available thermal energy in the system. Therefore, it is necessary to have a twisted molecular geometry to achieve sufficiently small ∆E ST value for the thermal activation of RISC process, which is generally less than 0.3 eV, although TADF molecules with ∆E ST value of nearly up to 0.5 eV are reported at their spatially separated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), respectively ( Figure 1B). [39,40] The rate of RISC can be calculated by using the following Equations (1) and (2). As per the selection rules, ISC is a spin forbidden transition, but these transitions can be partially allowed if energy gap (ΔE ST ) is small, while their respective HOMO and LUMO are spatially separated.
Here, in Equation (1), k RISC , k ISC k F , and k TADF represent rate constant of RISC, ISC, fluorescence, and TADF, respectively, and Φ F , Φ TADF represent quantum yields of fluorescence and TADF, respectively. In Equation (2), H SO is the Hamiltonian for the spin-orbit perturbations and ΔE ST is energy gap of S 1 and T 1 states. It displays the inversely proportional relation between ΔE ST and ISC.
Although TADF emitters have shown great potential to harvest both singlet and triplet excitons with high PLQY, however, they often suffer notorious aggregation-caused emission quenching (ACQ) effect and/or exciton annihilation. Hence, they generally need to be doped in the host matrix in thin film device application. AIE-TADF luminogens are favored to attain enhanced fluorescence with greatly increased delayed component in their condensed and thin film solid states. [41] While aggregation was found to be a crucial phenomenon accompanying with TADF, this generally restricts the intramolecular rotation and enhances intermolecular noncovalent interaction to realize high brightness PLQY and long-lifetime, thereby, generating AIE-TADF probes. Subsequently, conventional AIE probes face difficulties to overcome low internal quantum efficiency (IQE), unlike AIE-TADF emitters that offer 100% IQE with high-performance efficacy. Therefore, integrating TADF emitters with the AIEcharacteristics could be a feasible strategy to develop efficient solution-processed nondoped OLEDs. [42,43] Aside from the electronic device applications, recently, AIE-TADF emitters are found to be superior for biological fields also owing to their emission tunability, structural diversity, low toxicity, and large stoke shifts and long lifetime. Usually, fluorophores with higher emission lifetimes can be a promising alternative for eliminating the comparatively short-lived background fluorescence by time-resolved fluorescence imaging (TRFI). [44] As AIE-TADF (or TADF only) luminogens have higher lifetime (μs) compared to typical prompt fluorescence (ns), they are expected to have the potential capability of eliminating tissue autofluorescence with improved signal-to-noise ratio by introducing a proper delay time between the long-lived emission of dyes and the pulsed excitation light. [45]

AIE-RTP
Similarly, phosphorescence is another delayed luminescence phenomenon of organic molecules related to TADF, mostly found in small molecular architecture that exhibit much higher lifetime from microseconds to several hours for ultra-long phosphorescence. [46] Owing to their longer lifetime decay, these molecules have been employed in many advanced research fields, such as organic electronics, security, sensing, memory devices, information inscription, and bioimaging. [47][48][49] Nonetheless, efficient phosphorescence molecules have been reportedly found with heavy metal con-taining inorganic complexes. However, their rare availability, high cost, difficult processing, and nonenvironment friendly nature fall-off their utility in real-world applications. [50] To overcome all these obstacles, pure organic phosphorescence small molecules are currently being researched extensively. Phosphorescence is a spin forbidden process; hence, phosphorescence is a slow process and has shown higher lifetime than other fluorescence processes as mentioned above. Moreover, nonemissive triplet can turn on the bright phosphorescence emission under enhanced ISC and uplifted SOC process. A widely used tactic to boost SOC is the inclusion of nonmetallic heavy atoms like bromine (Br), thioesters, carbonyl moiety, and so on in the aromatic cores, assistance of polymer matrix, which facilitate the SOC process, and thereby enhance the viability of ISC. [51,52] Additionally, robust intermolecular and intramolecular nonbonding interactions (H-bonding, π-π interaction, etc.) associated with AIE phenomenon also result in enhancement of the ISC process. In aggregated states, molecules realize greater intermolecular interaction via well-defined packing as compared to monomeric form in solution state. Hence, aggregation helps to rigidify the overall conformation of the molecule and also creates a rigid environment to suppress the nonradiative decay, stabilize triplet state, and facilitate enhanced SOC, which promotes ISC process, thereby, bright emission occurred. [53,54] Thus, aggregation can affect the excited state emission properties and the collective AIE as well as the RTP properties reveal long-lived triplet utilization in water. Hence, important multifunctional properties are generated in organic luminogens for various translational applications alike to TADF materials. [55]

AIE-supramolecules
Molecular assembly is the most spontaneous process occurring widely in nature and helps in the growth of every living system and regulating multicomponent complex biological reactions. [56,57] Taking inspiration from this natural process, scientists have strategically utilized various aspects of supramolecular chemistry to build artificial biomimics that benefit efficient application in the biomedical areas. [58,59] Since the first Nobel Prize on supramolecules was awarded to Pedersen, Cram, and Lehn in 1987, several supramolecular systems were established encompassing guest-host materials, metallacycles, hydrogels, and so on, regulated by weak noncovalent interactions. [60] Among all the noncovalent interactions, π-π interaction is one of the intriguing factors that plays a pivotal role to generate supramolecules with advanced luminescent properties ( Figure 1C). However, this interaction became a curse to planar organic molecules due to the notorious ACQ phenomena until the coining of AIE in 2001. [61] This discovery has revolutionized a new era in the research world by developing supramolecules evolving with macrocycles appended mainly via large rotor units that result in amplified emission in the condensed state due to the restriction in molecular motion. Since its discovery, several new AIEgens have been established and modified with different functionalities to achieve excellent selectivity and sensitivity in diverse fields. The unique "turn on" emission in the condensed state and the spontaneous aggregation in biological medium has endowed AIEgens as potential candidates in bioimaging field. [62] Additionally, AIEgens are also eligible for further advanced level therapeutic application via reactive oxygen species (ROS) generation. [63,64]

AIE-oligomers
In contrast to supramolecular architecture, oligomers are low molecular weight intermediates between the monomers and polymers comprising of a few small or identical number of repeating units, whose molecular properties are proficiently dependent on the chain length. [65,66] The molecular weight of oligomers should be in the range of <10 kDa. The structure of the oligomers can be linear or star-shaped based on their appended conjugated chain length substituted into the aromatic core, that is, the repeating units in the continuous form result in linear structure, whereas if the repeating unit is located around the central core, it leads to starshaped structure. Thus, the oligomers can be developed via the "chain-insertion" linkage pattern, which generally results in linear oligomers or "core-side" linkage pattern that subsequently led to star-shaped oligomers. [67,68] These classes of molecules are found to be employed in wide applications in biology and fabrication of various electronic devices, including solar cells, transistors, and light-emitting diodes (LEDs), respectively. [69] Because of the diversified structure and tunable electronic properties of the oligomer, it could be a promising pathway to develop a smart AIEgenic oligomer to investigate its structure-property relationships and to explore several simple design strategies to fine-tune its molecular properties. Oligomers comprising of identical structures and molecular weights are considered to be more proficient models for studying the structure-property relationships. [70] Thus, AIE active oligomers have emerged as a potential tool for the real-world applications both in solution and condensed states. These include various optoelectronic devices with huge potential in diagnostics and therapeutics field as well due to their condensed state emissive characteristics compared to the traditional fluorophores, which impedes these applications owing to the ACQ behaviors. Hence, the future of broad investigations involving AIE/AIEE-active oligomers at this stage appears very rich and bright.

AIE-polymers
Polymers are macromolecules consisting of several repeating units with unique photophysical and optoelectronic properties that are greatly influenced by the monomer's molecular structure and polymerization conditions. In particular, AIE polymers have been endowed with potential advantages over the conventional polymers and low molecular weight luminogens owing to their solid-state luminescence behavior ( Figure 1D). [71] They have been employed in extensive real-world applications, such as in sensing, biomedical, and optoelectronic devices, due to their structural diversity, easily functional modulations, good thin film-forming ability, and photostability, including high thermal stability. Strategically, various classes of AIE polymers have been developed, such as conjugated, nonconjugated, star-shaped, and hyperbranched polymers, by simply incorporating the low molecular weight AIE luminogens into the main backbone, on the side chains, and center or the periphery unit of the polymers through the covalent linkage. The conjugated polymers can be usually constructed through the Suzuki, Sonogashira, and Yamamoto coupling, while the nonconjugated AIE polymers can be developed via free radical polymerization, such as atom transfer radical polymerization and reversible addition fragmentation chain transfer (RAFT) polymerizations. [72] Moreover, AIE polymers can also be built through the ring-opening metathesis and tandem polymerizations synthetic approaches. Thus, the modulations of the monomers, initiators, and polymerization chain reactions are the key features for the development of the different architectures of the polymers. Moreover, the important property of AIE polymers, including efficient energy transfer, is the high singlet oxygen ( 1 O 2 ) generation ability. These materials exhibit unique electrochemical and morphological properties, and can easily be fine-tuned by simply manipulating the structural modulations via altering the compositions, or influencing the insertion pattern of the AIEgen moieties, which potentially imparts them with various biomedical functions in real-world applications. [73,74]

AIE-NPs
The area of organic nanoparticles has gained tremendous interest in recent years due to their biocompatibility, size tunability, optical properties, and cell-penetration ability. [75][76][77] However, many fluorescent organic NPs suffer from ACQ effect. To prevent ACQ and enhance luminescence quantum efficiency, AIE nanoparticles came into the focus. AIEgens showed enhanced emission in their condensed or aggregated state due to the restriction of intramolecular rotation (RIR) in NP forms. [78] Mainly, AIEgens are encapsulated with hydrophobic/hydrophilic long-chain moieties to produce AIE-NPs, and their fluorescence can be enhanced by increasing the number of π-conjugated units in AIE probe. AIE NPs are inherently found to exhibit very high PLQY, biocompatibility, large stroke shift, and high photostability. By holding these key features, AIE-NPs enable easy cell internalization with high cellular uptake and show bright bioimaging. Thus, AIE-NPs are considered highly potential materials for realtime biomedical applications. [79,80]

AIE-PSs
In general, conventional conjugated small molecules and polymers suffer from low ROS generation and ACQ effect in condensed state, which greatly impede the biomedical application prospective. [81] AIE-PSs offer a significant and straightforward approach to solve these issues, as they exhibit intense emission in aggregated state due to the RIM, which inhibits nonradiative decays. These materials have often shown high light-harvesting ability, which is highly essential for PDT therapy. [82,83] More importantly, the photosensitizing ability relies on a few photophysical processes, which include ISC and SOC. By attaching heavy atoms to the fluorophore, the robust SOC between singlet and triplet state favors enhanced ISC rate that resulted in varied range of efficient PSs at reduced ΔE ST . [84] To reduce the ΔE ST , it is essential to lower the electron repulsion in the S 1 state. [85] Well separation of the LUMO and HOMO distribution is the most important criterion to get reduced ΔE ST . For enhancing the ISC of organic materials, incorporation of the heavy atom is a typical well-known method, whereas this strategy has suffered from many drawbacks, such as high cost of synthesis, higher toxicity, and so on. However, purely organic molecules have solved the toxicity issue by introducing TICT with improved spin-orbit coupling (SOC-TICT) system in orthogonally connected donor-acceptor architecture. During the charge recombination, the angular momentum of these organic materials conserves this phenomenon, leading to the enhancement of ISC. [86] Since the ISC efficiency is related to the extent of torsion present in the molecule accompanying the AIE materials, the PSs having a twisted π-conjugated system are highly capable to generate efficient PSs. The SOC of nonplanar conjugated aromatic molecule is greater than similar planar molecules. [87] Hence, compared to classical fluorophores, AIE congeners showed potential advantages to generate new PSs owing to their higher ISC and lower ΔE ST .
Thus, collectively the current progress in the versatile design of AIEgen with advanced optical properties and fundamental aspects of structure-functionality relationship is summarized. These include emerging AIE probes that exhibit highly tunable emission in aggregated/solid state with large quantum efficiencies of AIE-TADF, AIE-RTP-based small molecules, and AIE-macromolecule-based supramolecular frameworks, including AIE-oligomers, AIE-polymers, and AIE-PSs. Unlike the classical fluorophores with limited applications, this section highlights a few special classes of the smart AIEgens, possessing considerably high photosensitization capability, unique NP-forming ability, and improved photophysical properties that are exclusively devoted to advanced biomedical and electronic research.

AIE-TADF emitters and their impact
Molecules with high fluorescence quantum efficiency and long average lifetime in aggregated/solid states are known to be AIE-TADF, with broad applications over conventional fluorophores. [41] Aside from the electronic device applications, recently AIE-TADF emitters are found to be superior in biological field owing to their emission tunability, structural diversity, low toxicity, and large stoke shifts, offering a new strategy for (time-resolved luminescence imaging) TRLI studies, which resolved the issue of high tissue autofluorescence, low brightness imaging, and short lifetime efficacy. [45] Recently, the enhanced aggregated state luminescence of organic dots (Odots) has attracted intense attention, featuring matrix encapsulation strategy and offering additional advantages like easy synthesis and purification, monodispensiblity, and good biodegradability, minimizing triplet quenching for in vitro and in vivo cell tracking applications. On this view, unique Odots were prepared based on TADF dye 2,3,5,6-tetracarbazole-4-cyano-pyridine (CPy) encapsulated in distearoyl-synglycero-3-phosphoethanolaminepoly(ethylene glycol) (DESPE-PEG2000) matrix. CPy-based organic dots (CPy-Odots) are small in size, stable, and biocompatible offering long-lived fluorescence up to 9.3 μs and high brightness and PLQY upto 33% (Figure 2A). [88] The nanoprobes of CPy-Odots exhibited green emission, which readily differentiated from the background autofluorescence with highly desirable time-resolved and confocal fluorescence imaging in living cells and zebrafish.
Furthermore, another report solved the issue of matrix encapsulation to prevent triplet state quenching in oxygen atmosphere. By embedding a zinc metal ion coordination strategy into a D-A type TADF probe PXZT, the in situ aggregation-induced TADF turn-on response was achieved in oxygen environment, where phenoxazine (PXZ) was a donor and terpyridine was an acceptor ( Figure 2B). [89] By increasing water fraction or PXZT concentration, the lifetime values of delayed component increased, while prompt lifetime remained unchanged, demonstrating AIE-TADF feature of the PXZT probe. Moreover, the delayed fluorescence was quenched by zinc ion coordination with terpyridine unit of PXZT through enhanced intramolecular charge transfer (ICT) mechanism and formed ZnPXZT1. Further, the efficient detection of EDTA in vitro was observed by dissociating the complex, thereby the release of PXZT and subsequent hydrophobic aggregation resulted in 2000-fold enhanced fluorescence intensity due to aggregation-induced TADF turn-on emission. This approach was further utilized for the efficient TRLI imaging of Hela and 3T3 cells, where the background signals were eliminated by this newly proposed dissociation-aggregation strategy in oxygen-containing atmosphere.
A new acceptor aromatic imide (AI) and donor carbazole (Cz)-based TADF emitters AI-Cz were reported. To suppress the triplet quenching due to the oxygen present in aggregated state, long hydrophobic functional groups were incorporated into a TADF core to generate organelle-specific TADF small molecule. [90] The molecule exhibited longest fluorescence lifetime and bright TRLI of the cells. The AI-Cz-MT and AI-Cz-LT are the two biocompatible organelle-specific TADF probes designed by conjugating triphenylphosphonium (TPP) as mitochondria targeting and 2-morpholinoethylamine as lysosome targeting moiety, respectively ( Figure 2C). In the AI-Cz-MT probe, TPP the cationic charge containing moiety carries to the mitochondria, and the hydrophobic region of the mitochondrial membrane was embedded by lipophilic TADF fluorophore. Similarly, AI-Cz-LT was trapped into the lysosomal membrane by 2-morpholinoethylamine carrier. After organelle-targeting attachment, the probes became highly fluorescence with high PLQY and small ΔE ST value of 0.12 and 0.11 eV, which further showed oxygen-dependent delayed fluorescence in toluene and depicted an excellent TADF behavior.
Nonetheless, unlike TRLI imaging, an efficient AIE-TADF probe could be utilized as PS for PDT theranostic application owing to their triplet sensitivity feature. However, an appropriate guideline of TADF design by adjusting ΔE ST and oscillator strength (f) are the crucial elements for an efficient PS and reactive oxygen generator. In this regard, two TADF molecules PT and AT were designed based on two different donor 13,13-dimethyl-5-phenyl-11,13-dihydro-5H-indolo[2,3-b]acridine (A) and 12-phenyl-5,12-dihydroindolo[3,2-b]phenoxazine (P), while 3-bromothianthrene 5,5,10,10-tetraoxide (T) has been chosen as an acceptor ( Figure 2D). [91] Both the emitters exhibited AIE-TADF characteristics; however, due to the presence of different donor segment, a small ΔE ST and f value of 60 meV and 0.03 for PT was observed, while AT showed relatively large ΔE ST and f value of 100 meV and 0.07, respectively. Thus, small ΔE ST and f resulted in excitons control for ROS generation via efficient ISC, which is beneficial for PDT, while larger values are beneficial for radiative transition and fluorescence imaging.
At the same time, the in situ aggregation and oxygen quenching was eliminated in living cells by introducing steric hindrance effect. Two AIE-TADF molecules, CzPOTCF and tBCzPOTCF, were designed by composing electron withdrawing diphenylphophine oxide (DPPO) moiety substituted at orthoposition of the carbazole group. Moreover, by substituting DPEPO groups into the carbazole moiety, the dihedral angles were found to be enlarged between TCF and the carbazole groups in CzPOTCF and tBPOTCF ( Figure 2E). [92] Besides, in DPEPO, the P═O involved with intermolecular H-bonding interaction with the adjacent styryl bridge molecules facilitating the dimeric assembly of the emitters, which effectively mitigate water/oxygen-induced quenching, whereas the bulky tert-butyls in tBCzPOTCF dimer further reduces concentration quenching, which collectively resulting in enhanced aggregation-induced DF without matrix encapsulations. Thus, sterically hindered CzPOTCF and tBCzPOTCF exhibited well-separated HOMO-LUMO and reduced ΔE ST with increased lifetime value of 12.2 and 8.37 μs as compared to DPEPO-free CzTCF (7.56 μs). The long-lived fluorescence of these materials was further employed for TRLI studies and successfully observed with elongated average lifetime of 6.69 and 7.41 μs for CzPOTCF and tBCzPOTCF, respectively.
Although there were a series of TRLI-based AIE-TADF reported emitters, however, most of the developed TADF probes exhibited single emission signal only. This one emission signal may affect the imaging information integrality, which sometimes do not show TR signal if there was a low emission intensity due to some local microenvironments. This issue was further overcome by dual emission strategy in single TADF probe. Thus, to generate dual-emission in single TADF emitter, anti-Kasha's rule has been introduced by employing the first excited state (S 1 and T 1 ) and higher-lying excited state (S 2 and T 2 ) obtained from an unsymmetrical (D-A-D') molecular structure. The integrated emitter featured two different donors phenothiazine (D) and N-(1hindole-5-yl) acetamide (D'), while diphenylsulphone (A) as an acceptor ( Figure 2F). [93] The hypothesis is further justified by investigating a wider spectral wavelength distribution, where distinct emission sources evolved with locally excited triplet ( 3 LE) and charge-transfer triplet ( 3 CT) to chargetransfer singlet (ICT). Thus, the dual TADF was observed in both diluted and aggregated state, which controlled the oxygen sensitivity and quenching effect. Besides, this strategy produced a balanced dual-channel TADF lifetime mapping with an excellent time-resolved imaging and channel selectivity, which was exploited for intracellular local imaging integration. The summary of photophysical properties and applications of AIE-based TADF molecules are discussed in Table 1.

Reported AIE-RTP emitters
Most of the fluorophores in organic small molecules suffer from ACQ effect. Generally, ACQ fluorophores have planar confirmation, which leads to strong π-π interaction or intermolecular interactions. These interactions facilitate thermal or nonradiative decay. Yet, placing two cores at proximate distance to facilitate π-π and noncovalent intermolecular interaction between them results in RIM of the fluorophore in aggregate state. [94] However, aggregation behavior of organic chromophores was found to be a key approach to realize phosphorescence in condensed state owing to their large number of intermolecular interactions. Crystallization is an important prerequisite for most of the RTP molecules as it provides molecular rigidity and prevents nonradiative degeneration of triplet excitons. So, crystallization and molecular assembly of AIE molecules could be a simple strategy to activate RTP. [95] Both these types of interactions could also lead to phosphorescence with requisite molecular structure. Hence, AIE-RTP is a seldom observed phenomenon for purely organic molecules; however, by applying a few key strategies, it can be realized at room temperature. Yet, no clear guidelines and mechanisms exist to develop AIE-RTP molecules. However, a few reports with versatile mechanism of AIE-RTP-based small molecules have made great advances in recent years. The first report on crystallization-induced phosphorescence was investigated for benzophenone (BP) and 4,4′-difluoro benzophenone (DFBF) molecules. [96] In both the molecules, crystallization facilitates phosphorescence in their aggregated state.
Many facile strategies for AIE-RTP were reported where energy gap between singlet and triplet energy state and SOC played an important role in tuning luminescence in solid state. Modulation of ΔE ST and steric constrains in AIE active molecules can tune phosphorescence in donor-TPA-based molecules. On this view, phosphorescence was detected in TPA donor-based o-TPA-3TPE-p-PhCN and o-TPA-3TPEo-PhCN ( Figure 3A). [97] Steric hindrance of ortho substituent renders twisted confirmation to the molecules. AIE active molecular system can lead to other luminescence phenomenon as RTP and delayed fluorescence via crystallization and molecular design with requisite ΔE ST and n-π* tranistion. Phosphorescence peak of ortho position substituent on TPA has emission at 500 nm and for para substituent at 495 nm. Also  Figures 3B, 3Ba, and 3Bb). [98] Inspired by the mechanism behind AIE, a general design principle was evolved to develop AIE-RTP luminogens. By introducing sp 3 spacer between donor acceptor moieties and AIE activating TPA group, AIE active room temperature dual  [93] phosphorescence materials have been explored. To accumulate both properties in a single molecule, the important role of sp 3 spacer group has been proposed. Further, AIE-RTP phenomenon was studied in five TPA-based molecules (TPA1, TPA2, TPA3, TPA4, and TPA5) ( Figure 3C). [99] The design concept was that TPA is used as donor as well as AIE activator and sp 3 spacer was introduced between donor and acceptor to achieve RTP. The sp 3 spacer separates HOMO and LUMO, which helps improve charge separation, promote ISC, and facilitate RTP. The dual phosphorescence as well as color of TPA1-5 was tuned by different acceptor groups. Further investigation with TD-DFT and SC-XRD revealed that TPA derivatives adapt a highly twisted confirmation of 100-120 • , due to the presence of sp 3 spacer group. Twisted confirmation between donor and acceptor renders strong π-π interaction; however, other intermolecular interaction collectively facilitates phosphorescence in these TPA derivatives. Other intermolecular interaction in the condensed state helps to suppress intermolecular motion, which leads to restricted molecular motion, thereby, AIE is realized. All the five TPA substituents exhibit dual phosphorescence with significant QE ( Figure 3D). Phosphorescence lifetimes of 306 ms for TPA1 at 525 nm, 340 ms for TPA2 at 568 nm, 7.6 ms for TPA3 at 545 nm, 77.8 ms for TPA4 at 525 nm, and 31.1 ms for TPA5 at 565 nm were observed. Furthermore, "Tetrahedron like" donor-acceptor imide derivatives (i) and (ii) ( Figure 3E) were reported, [100] where both derivatives have twist angles of 111.7 • and 114.6 • . The twisted confirmation originated because of the methylene linker between donor and acceptor. Owing to the tetrahedron molecular geometry, the methylene linker provides advantage of free molecular rotation between donor and acceptor, which allows luminescence in solution state and prohibits fluorescence charge transfer between donor and acceptor. The tetrahedron geometry permits controlled or limited intermolecular interaction between two cores and behaves opposite to ACQ molecular effect and allows for radiative luminescence. Phosphorescence lifetimes of 243 ms (RT) and 301 ms (77K) for (i) at 553 nm phosphorescence emission and 371 ms (RT) and 523 ms (77K) for (ii) at 548 nm phosphorescence emission, respectively ( Figure 3F,G), were observed.
Aforesaid basic design strategy for RTP behavior is observed by enhancing ISC and SOC by introducing carbonyl group, halogen atoms, and heteroatoms, including crystallization and other rigidification process. This also includes the intermolecular hydrogen bonding between multiple N-H⋯C=O interaction between anthraquinone and amide group that enhance the triplet excitons population and facilitate phosphorescence in its aggregate state. Enhancing intermolecular interaction in AIE molecules via controlled aggregation can be another great strategy to develop AIE-RTP molecules. Aggregation phenomenon was observed in anthraquinone-based AqC6 molecule ( Figure 3H). [101] Phosphorescence was realized by increasing concentration of the luminophore in concentrated solution or in aggregated state via crystallization or solvent/antisolvent combination strategy. While increasing the concentration, the molecule is aggregated, thereby, intermolecular interactions greatly increased and RTP activated. PL emission of AqC6 in DMF was observed at 410 nm, while in crystal state, broad phosphorescence emission was observed at 550 nm. These results confirm that the phosphorescence was facilitated by aggregation and intermolecular interactions. Phosphorescence lifetime was found to be 9 ms at RT and 112.3 ms at 77K for AqC6 ( Figure 3I). Subsequently, SC-XRD analysis depicted that multiple intermolecular interactions facilitated SOC and phosphorescence ( Figure 3J).
As the AIE effect has an advantage of high solid-state luminescence unlike the ACQ and is highly favorable for OLED processing, yet the AIE molecules exhibited strong fluorescence within the 25% electrogenerated singlet excitons. Hence, utilizing 75% of the remaining triplet excitons via ISC could enhance OLED efficiency. In this direction, Zhang et al. prepared a series of aggregation-induced phosphorescent molecular materials with quantum efficiency upto 64%. They introduced substituent in bis-carbazole and prepared a series of carbazole-based aggregation-induced phosphorescence (AIP) molecules, BCZ1, BCZ2, BCZ3, and BCZ4 ( Figure 3K). [102] AIE molecules are popular for nondoped OLED fabrication for homogenous solid-state emission. RTP molecules on the other hand are able to populate triplet excited state population via ISC, which leads to the enhancement of overall quantum efficiency (Φ E ). By combining these concepts of AIE and RTP, AIP molecules have been constructed. While introducing acceptor molecules in bis-carbazole, the molecules retain AIE property as well as exhibit two distinct phosphorescence emissions. Phosphorescence lifetime for all three BCZ1, BCZ2, and BCZ3 (Figures 3La, 3Lb, and 3Lc) was observed up to micro-second range, while nanosecond fluorescence lifetime was observed for BCZ4. OLEDs were fabricated with these integrated AIP molecules with external electroluminescence quantum efficiency of 5.8%, which is more than the theoretical limit for OLED devices with pure fluorescence. The summary of photophysical properties of AIE-based RTP molecules is discussed in Table 2.

3.3
Reported organic AIE-supramolecules Guest-host assembly involving macrocycle entities AIEgenic macrocyclic systems are generally constructed on the basic guest-host principle via inclusion of one or more units into the typical macrocycles, viz. crown ether, [103,104] pillararenes, [105,106] calixarenes, [107] cyclodextrins (CDs), [108,109] cucurbit[n]urils (CB[n]s), [110,111] and so on. The development of a perfect guest-host pair generally depends upon the precise matching of guests with the cavity size of the host and the particular functional groups for various noncovalent interactions, which also provides TA B L E 2 Photophysical properties of AIE-based RTP molecules excellent selectivity toward different substrates. These noncovalent interactions can be triggered via several external stimuli like pH, redox potential, different light, and so on, which decides the dynamic reversibility in assembly-disassembly processes. However, for AIEgens, the seminal approach to restrict their molecular motion has become successful via its encapsulation into the cavity of macrocycles, where the limited area results in twisted configuration with restricted motion and display outstanding emission. Cucurbit [10]urils (CB [10]) is a typical example of a host material that contains a large cavity size of 9.5-10.6 Å with a volume of ∼870 Å. CB [10] was reported as a host material embedding large dumbbell-like TPE-based cationic AIEgen (2TPEV) in its big hydrophobic cavity ( Figure 4A). [112] This bis-cationic guest formed a well-matched complex with CB [10] via ion-dipole interaction between cavity fringed C=O and pyridinium cationic charge, which was further supported by NMR spectroscopy. In DMSO solution, 2TPEV showed no emission, but the introduction of CB [10] at 95 • C leads to amplification in the emission property, endowing RIR mechanism through encapsulation of the AIEgen into the CB [10]. The resultant complex also lost its solubility in several solvents, specifically in THF or CHCl 3 . Due to the loss of solubility, 2TPEV-CB [10] complex exhibited subsequent aggregation, which further displayed intensified emission than earlier. Interestingly, the large TPE unit offered an excellent selectivity toward CB [10], whereas with other members of CB[n] series (where n = 6-8), no enhanced emission was observed. In another report, different numbers of pyridinium-armed TPE-based AIEgens were proposed that can exhibit guest-host complex with Cucurbit[8]urils (CB [8]) and contributed different emission, shapes, and cell affinity ( Figure 4B). [113] TPE group anchored with two (TPE-2EP), three (TPE-3EP), and four (TPE-4EP) pyridinium-arm were developed and converted into nano assembly via complexation with CB [8]. It was speculated that in the complexation, two intermolecular pyridium arms were embedded in a single CB [8] unit and constructed well-ordered linear or hyperbranched nanostructures. After complexation, the constrained molecular structure displayed intensified emission for all three AIEgens and acquired different morphologies. However, different shapes and sizes of the nano assembly also achieved different priorities for cell permeation. Depending upon the affinity towards normal and cancer cells, all the complexes generated different fluorescence signals, which were further utilized in linear discriminant analysis to distinguish between normal and cancer cells, and to identify the amount of cell contamination. Although TPE is ideally more favorable for guest-host encapsulation having propeller-like structure, others are also not far from competition. Furthermore, benzene cored pyridinium fringed cationic AIEgen (3TPBPy) was developed, which helped to construct a twodimensional supramolecular organic framework via guesthost self-assembly with CB [8] ( Figure 4C). [114] In the complex, pyridinium wings were encapsulated into the CB [8] cavity following a head-to-tail packing pattern. The restricted F I G U R E 4 (A) Depicts pictorial presentation of supramolecular assembly of 2TPEV in the presence of CB [10] in different solvents (Adapted with permission from ref [112] Copyright 2017 American Chemical Society). (B) Represents the supramolecular complex of TPE-2EP, TPE-3EP, and TPE-4EP with CB [8] and their corresponding morphology (Adapted with permission from ref [113] Copyright 2020 American Chemical Society). (C) Chemical structure of 3TPBPy, mode of assembly with CB [8], and corresponding morphology (Adapted with permission from ref [114] Copyright 2019 Royal Society of Chemistry). (D) Illustrates the molecular structure of TPE-β-CD, graphical mode of trapping of TPE inside β-CD, and fluorescence spectra in the presence of different CD (Adapted with permission from ref [115] Copyright 2014 Royal Society of Chemistry). (E) Represents AIE nature of TPE-DBA and TPE-DDBC in different water fraction and their reversible assembly in the presence of acid and base (Adapted with permission from ref [116] Copyright 2015 Royal Society of Chemistry). (F) Pictorial representation of supramolecular assembly of TPE-based AIEgen into polymeric network (Adapted with permission from ref [117] Copyright 2019 WILEY-VCH). (G) Polymeric assembly of DSAPB in addition of CB [7] and CB [8] simultaneously (Adapted with permission from ref [118] Copyright 2021 Royal Society of Chemistry) internal rotation consequenced 25-fold increment in the fluorescence emission along with a red shift from 520 to 600 nm with the gradual addition of CB [8] in water. The formation of coassembly in water was further characterized by TEM and SEM images. The enhancement in the emission of the coassembly in the presence of DNA was applied for DNA imaging in HeLa cells.
In addition to noncovalent interactions, AIEgens can be connected with the host materials via covalent bonding. A typical host material CD was linked with TPE unit (TPEβ-CD) and resulted in a new single molecular AIEgen via guest-host encapsulation ( Figure 4D). [115] CDs are consist of glucopyranose joined through glycosidic linkage and provide a toroidal shape structure. The edges of the structure contain hydrophilic hydroxyl groups that decide its solubility in aqueous environment. But the inner side of the toroid is considerably hydrophobic, which allows hosting hydrophobic organic materials inside the cavity. Accordingly, the encapsulation of the TPE unit in β-CD displayed intense emission at 410 nm as dissipation of energy was strictly prohibited due to the RIR mechanism.
Moreover, the presence of physical interaction was further proved by using smaller cavity size α-CD and larger cavity size γ-CD. It was observed that for α-CD the emission intensity was stronger than γ-CD, which clearly indicated the insufficient space of α-CD hindered the internal rotation and exhibited high quantum yield compared to the other two. This outstanding emissive nature and excellent biocompatibility of TPE-CD offered quality performance in cell imaging. Another new AIEgen was established by connecting TPE unit with crown ether (TPE-DDBC) through a covalent bond ( Figure 4E). [116] Although the covalently linked TPE unit was not playing the role of a guest material here, the hydrophobic nature of the probe aided to form the assembly in water and activate the RIR process. However, here another guest AIEgen (TPE-DBA) was introduced, where TPE was anchored with benzylamine groups. These benzylamine groups acted as guest material to host crown ether that resulted in microstructure assembly in the presence of acid. Both the AIEgens were nonemissive in THF, but in the presence of acid, protonated amino group was capable to recognize the guest crown ether and their encapsulation consequenced amplified emission activating the RIR. Moreover, the treatment with a base triggered the deassembly process via deprotonation and provided a dynamic reversible assembly and deassembly phenomena.
Apart from small organic molecules, incorporation of macromolecules also plays an eminent role in developing superior emissive AIEgenic materials. Recently, a copolymer material, anchoring with pillar [5]-arenes on the side chain (poly(MMA-coMMAP [5]A), was introduced as a host against a tetra armed TPE-based (TPE-(CN) 4 ) guest opponent ( Figure 4F). [117] The involvement of noncovalent crosslinking interaction between these two units promoted the fluorescence quantum yield up to 98.22% in polar THF solvent and provided a stable, ordered assembly structure. It was envisioned that the gradual addition of guest TPE-(CN) 4 into the polymer solution exhibited regular enhancement in the emission endowing RIR mechanism of the TPE unit. However, the guest-host interaction was further confirmed by NMR spectroscopy along with a competitive study in the presence of adiponitrile where the higher binding affinity of adiponi-trile replaced the embedded (TPE-(CN) 4 ), leading to a drastic fluorescence quenching. Furthermore, the additional impact of poor solvents like water helped to form tight aggregation and resulted in more enhancement in the emission by forming supramolecular NPs of spherical shape. Next, an excellent example of in situ generated supramolecular polymer was demonstrated via encapsulation of distyrylanthracene cored guest (DSAPB) into host CB [7] and CB [8] subsequently ( Figure 4G). [118] Initially, two benzyl pyridinium of DSAPB were treated with CB [7], leading to the complex (DSAPB@2CB [7]) formation with a ratio of 1:2. The resultant complex perceived a huge jump in fluorescence quantum yield from 3.2% to 17.1% via formation of restricted conformation. However, the prevention of face-to-face stacking also played the leading role in this emission enhancement in the aggregated state. Later on, the DSAPB@2CB [7] complex was further encapsulated with CB [8] where two benzyl groups were embedded into CB [8] cavity and regulated the linear assembly of supramolecular monomer into supramolecular polymer through in situ crystallization.

3.3.2
Self-assembly-based system Manipulation of photophysical and morphological characteristics via self-assembly of the organic molecules has gained potential interest in multidisciplinary application. Supramolecular self-assembly of AIEgenic materials maximized its luminescence efficacy and played a pivotal role mainly monitoring complex biological reactions. Although the nonplanar conformation of the organic AIEgens prevents close stacking into the well-ordered supramolecular assembly, some noncovalent interactions like hydrophobic interaction, hydrogen bonding, van der Waal force, and so on drive it to forward direction. Among them, hydrogen bonding was found to be a more feasible strategy to develop a supramolecular assembly. [119] One unique strategy was implied by designing a Z/E isomer of ureidopyrimidinone linked TPE-based AIEgen, where hydrogen bonding performed a crucial aspect in their aggregated state and as an outcome of supramolecular polymeric assembly ( Figure 5A). [120] Both the isomers showed intense emission in their aggregated and solid-state. Meanwhile, the emission of the Z isomer was more red shifted compared to the E isomer, suggesting different aggregation patterns in the condensed state. In-depth study of the aggregation state confirmed the formation of intermolecular hydrogen bonds between N─H and C=O groups for both the isomers. For Z isomer, the location of two arms in the same side caused steric hindrance in dimerization and restricted the assembly up to low energy dimer configuration, whereas for E isomer, oppositely anchored two arms facilitate the polymer formation. However, viscosity and morphology study concreted their assumption, where it was observed that E isomer resulted in a highly viscous solution than Z isomer and Z/E shaped in spherical/cable network structure, respectively, after aggregation.
In another report, a BODIPY-based luminogen was developed where two covalently linked uracil groups regulate the intermolecular hydrogen bonding leading to supramolecular polymerization through J-type aggregation ( Figure 5B). [121] The incorporation of alkyl functionality in the meso position and the boron atom provided better solubility in an aliphatic solvent and also introduced AIEE feature in place of ACQ behavior preventing π-π stacking in the aggregated state. As hydrogen bonding favorably formed in nonpolar aliphatic solvent, hexane was selected as a poor solvent for aggregation study, and the probe (BODP) acquired superior emission with a mirror like shift in both absorption and emission spectrum. However, the concentration-dependent study in hexane presented a drastic increment in the florescence yield at higher concentrations along with a blue shift, suggesting the J-type aggregation growth. The formation of aggregation was further validated by the temperature-dependent study, where the disassembly of the aggregate into monomer was observed at higher temperature yielding red-shifted monomeric emission. Later, the NMR spectrum, molecular modeling, and the nanowire morphology offered conclusive evidence for the supramolecular polymerization via hydrogen bonding.
Another TPE cored stereogenic AIEgens [(Z)-TPE-OEG and (E)-TPE-UPy] was proposed, anchoring amphiphilic chain in place of ureidopyrimidinone, which had a huge impact on its aggregation and stimuli-responsive properties ( Figure 5C). [122] Both Z, E-isomers exhibited intensified emission-forming vesicles and micellar structure in their aggregated state. An exciting photophysical change was diagnosed for vesicle-shaped Z-isomer, where a morphological transfer was observed, varying temperature from 37 to 45 • C. Initially, the emission was lowered to 41 • C, indicating the dissociation of vesicle form due to dehydration from the amphiphilic chain. Whereas, further increment in the temperature resulted in more exposure of the hydrophobic bare phenyl rings (present on the same side) toward water and formed more tightly packed, less surface energy globule shape morphology. More dense packing in the globule shape directly affected the molecular motion, which is reflected from their bright-induced emission. In contrast, the E-cousin showed a gradual decrement in the emission intensity with temperature increment and it was assumed that the oppositely located amphiphilic chain hampered the exposure of the bare phenyl ring. Overall, the reversible temperature-dependent phase transition of [(Z)-TPE-OEG could be highly beneficial on fluorescence-based thermometer applications. A classic example of self-assembly of a cationic AIEgen (TPE-BPA) into a vesicle structure was proposed where the ionic interaction governs the assembly process along with its AIE phenomena ( Figure 5D). [123] Since the AIEgen contained eight negative charges at the tip of the ligands, it experienced excellent solubility in water medium and emitted weakly, endowing intermolecular rotation. However, the strategical introduction of cationic surfactant lowered the probes negative potential and induced hydrophobic effect, which directly affected the probes solubility and produced highly emissive supramolecular vesicles via RIR activation. It was speculated that after adding eight equivalent of surfactant, the emission of the solution reached maximum level, indicating the generation of a neutral complex between TPE-BPA and CTAB. It was assumed that each negative charge was neutralized by single surfactant unit and produced a multilamellar vesicle structure having two TPE-BPA units as a building block. Further, it was observed that the gradual addition of Zn 2+ into the neutral complex reduced the emission intensity attributing fission of the large vesicles into smaller ones. Finally, the electrical charge-induced fission was utilized for the drug delivery, where the entrapped drug was released with increasing electrostatic potential. Moreover, due to the high charge potential in the cancer cell membrane, this system could be more beneficial for selective cancer cell ablation. The summary of photophysical properties and applications of AIE supramolecules are discussed in Table 3.

Reported organic AIE-oligomer
In this section, the structure-property relationships of the AIEgenic oligomers (linear and star-shaped) (Fig-ure 6A), [63][64][65] along with their distinct luminescence properties, supramolecular self-assembly behaviors in aggregated or solid states are presented. Further, the various design strategies to develop and manipulate potentially novel AIEgenic oligomers from well-known ACQ fluorophores are also discussed. Although TPE-based AIE active derivatives have been extensively established as a standard and an efficient luminogenic core and have been widely studied in the literature, various research groups have actively demonstrated many novel AIE/AIEE-active oligomers rather than TPE. We have summarized here the up-to-date progress and advancement of the AIEgenic oligomers. Carbazole incorporated conjugated oligomers CZ2, CZ4, and CZ6, respectively, have been reported, which exhibited distinct photophysical and morphological properties by simply tuning the conjugation length into the aromatic DVA backbone ( Figure 6B). [124] By increasing the carbazole congeners into the DVA units consequently leads to the transition of the AIE-to-AIEE behaviors of the CZ2 to CZ6 oligomers. CZ2 oligomer exhibits AIE characteristics that were attributed to its shorter conjugated carbazole chain, which led the intramolecular motion of the central DVA rotor by reducing its bulkiness around the DVA unit and leads to total quenching emission intensity (fluorescence quantum yield, Φ f = 0.5%) of the oligomer in solution state. However, CZ4 and CZ6 oligomers showed AIEE behavior due to its increasing conjugated carbazole chain, which restricts the intramolecular motion of the DVA rotor by creating molecular rigidity, which subsequently leads to slight increase in emission intensity of the oligomers (Φ f = 1.7% and 3.6% for CZ4 and CZ6, respectively) in solution state. The enhanced emission intensity of the CZ2, CZ4, and CZ6 oligomers in the aggregated state was due to the RIR effect. The Φ f of the F I G U R E 6 (A) Schematic diagram of the oligomers linkage pattern. (B) Chemical structure (above), digital photographs from 0% to 90% f W in THF (below) of CZ2, CZ4, and CZ6. Reproduced with permission from ref [124] Copyright 2011, The Royal Society of Chemistry. (C) Chemical structure (above), photographs from 0% to 90% f W in THF and thin-film of FF and FM. Adapted with permission from ref [125] Copyright 2013, The Royal Society of Chemistry. (D) Chemical structure, the photophysical properties of the oligo-anilines, and the crystal structure of B2-A2. Reprinted with permission from ref [127] Copyright 2019, The Royal Society of Chemistry. (E) Widely tunable emissive characteristics and the crystal structure of TPA-oligothiophenes at their aggregated state. Adapted with permission from ref [128] Copyright 2019, The Royal Society of Chemistry. (F) Chemical structure (above), mechanoluminescence behavior, and the corresponding blue-shifted PL spectra of the ground oTPETP (below). Reproduced with permission from ref [129] Copyright 2018, The Royal Society of Chemistry. (G) Chemical structure of the TTPE and cyclized CTTPE, piezochromic characteristics, and the corresponding red-shifted PL spectra of the TTPE ground oligomer. [122] Reprinted with permission from ref [130] Copyright 2014, American Chemical Society CZ2, CZ4, and CZ6 oligomers in their thin film state was calculated to be 18.5%, 30.2%, and 12.7%, respectively. More importantly, CZ6 oligomer demonstrated blue-shifted emission maxima and lowered Φ f in comparison to others. The blue-shifted emission maxima of CZ6 were due to the steric hindrance around the DVA core created by the increasing conjugation carbazole unit, whereas the strong intermolecular stacking interaction and twisted conformation of it led to decrease in Φ f . Most interestingly, CZ2 and CZ6 oligomers exhibited nanoaggregates at 90% water fraction (f w ) in THF solution, whereas CZ4 oligomer showed nanorings at this f w . The nanorings comprising of high Φ f (30%) could be a promising design strategy for the application in photonic and electronic devices. Further, TPE-based star-shaped AIE oligomers have been reported, which exhibited similar features of the transition of AIE to AIEE behaviors upon increasing the TPE units around the TPE aromatic backbone. The AIE to AIEE transformation of the oligomers was ascribed to the RIR effect of the aromatic core.
Recently, fluorenone appended into the fluorene aromatic backbone fluorene-fluorenone (FF) oligomer with AIEE characteristics has been demonstrtated, which showed good thin film-forming properties and subsequently employed in OLED applications ( Figure 6C). [125] The fluorenone congener in FF oligomeric system triggers the AIEE behavior of the system. Hence, to demonstrate the primary role of the fluorenone in the generation of the AIE/AIEE characteristics into the FF oligomer, another fluorenone malonitrile-induced fluorenefluorene malonitrile (FM) oligomer was synthesized by simply incorporation of the cyano group into the fluorenone unit appended into the fluorene conjugated aromatic backbone. Herein, the FF oligomer exhibits weak emission intensity in solution state that was attributed to its low transition dipole moment and vibronic coupling of the excitons, whereas Jaggregate formation in FF oligomer in aggregated-state and thin film state correspondingly leads to AIEE behavior. On the other hand, the FM oligomer showed totally quenched fluorescence intensity both in solution and thin film state due to its associated vibronic relaxation and higher transition dipole moments in the excited state. Further, the FF oligomer displayed high quantum yield (Φ f = 79%) in thin film state, which suggested the condensed state optoelectronic device applications. The thin film state Φ f of FM oligomers was found to be 1.3%. Thus, incorporation of the functional group engineering design strategy could emerge as a promising pathway to manipulate the photophysical properties in the condensed state with ACQ-to-AIEE transformation, which leads to various futuristic applications.
Another fluorenyl functionalized AIE homo and triblock oligomers was synthesized and the AIE effect of triblock oligomers was found to be higher compared to that of the homo oligomer due to its more compact structures. [126] The current study provides a new design strategy for the development of luminogens based on homo and triblock oligomers. Moreover, a series of linear oligo-aniline (a conjugated aromatic oligomer) substituted with the diphenyl enamine into the conjugated frameworks have also been developed, which transforms the ACQ to AIE characteristics in aniline oligomers via new "chain-insertion" linkage pattern rather than conventional "core-side pattern" classification and leads to distinct photophysical properties ( Figure 6D). [127] The fully substituted oligoanilines with precise chain inser-tion, such as B2-A2, B3-A3, and B4-A4, respectively, exhibited typical AIE characteristics, while partially substituted derivatives, such as B1-A1, B1-A2, and B2-A4, respectively, were AIE-inactive. Prominently, the solid-state fluorescent lifetime (F T ) and Φ f of the AIE active oligomers were high compared to their solution state. Whereas, the AIE-inactive oligomers exhibited lower F T in the solid state compared to their solution state it was attributed to the inadequate number and uncontrolled degree of chain insertions, respectively. Interestingly, the Φ f increased on increasing the number of chain insertions from 0% to 0.5%, respectively. Thus, the above results suggest that, for linear system, increasing the chain length leads to more twisted conformation, which correspondingly prevents the detrimental π-π stacking interaction in the condensed state and increases its potential for ACQ-to-AIE conversion.
The single crystal study (SCXRD) of B2-A2 crystal showed that the S-shaped chain of the oligomer where the higher torsional angle between the adjacent aniline moieties was of 67.21 • suggested the strongly twisted conformation, which confines the diphenyl enamine and phenyl free rotation during the aggregation. Moreover, the C-H⋯π interactions between the phenyl rings and hydrogen atom on the vinyl functional groups with 3.4-3.7 Å distances and with 2.8-3.4 Å lengths between the neighboring aniline derivatives further contributed in confining the molecular motion, and strengthen its AIE effect. Moreover, fully substituted aniline oligomers showed turn-on fluorescence response toward bovine serum albumin under physiological conditions.
Another example of linear oligothiophene (TTV to TTNIR) consisting of thiophene repeating units into the triphenyl amine (TPA) core has been recently reported. The linear incorporation of the thiophene unit into the TPA aromatic backbone resulted in widely tunable emission wavelength covering the whole visible light region and extending to near infrared (NIR) region ( Figure 6E). [128] The SC-XRD showed that the extended intermolecular distances (>3.2 Å) between two parallel planes of the TPA oligomer backbone prevented the intermolecular π-π stacking interactions and subsequently enhanced the fluorescence intensity in its aggregated and solid state. Moreover, the abundant C-H⋯O, C-H⋯C, and S⋯C interactions strongly rigidify the molecular conformations, which restricted the intramolecular motions by these intermolecular interactions, and leads to solid-state enhanced emission efficiency. These TPA-oligothiophenes exhibited enhancement of Φ f up to 40.79%. The fluorescent colors of these systems were tuned by simply varying the HOMO and LUMO energy levels of these D-A systems. Thus, increasing the chain lengths and strengthening the donor groups by the incorporation of thiophene units, or introduction of the dicyano groups by replacing the carbonyl group, dramatically shifted the emission maxima from 402 to 724 nm. These oligothiophenes could be employed as efficient fluorescent probes for lipid droplet-specific bioimaging and cell fusion assessment, exhibiting excellent image contrast and high photostability.
TPE incorporated donor-acceptor (D-π-A)-based novel AIEE conjugated oTPETP oligomer was developed ( Figure 6F). [129] The introduction of TPE unit into the oligomer backbone resulted in the AIEE characteristics of the oligomer, which showed weak emission in the solution state, whereas the enhanced emission was observed in the Piezochromism picric acid detection, and cell imaging [130] aggregated or solid state. The bithiophene congener has been chosen as a donor, whereas the TPE unit acted as an acceptor in D-A conjugated framework. The orange emission of AIEE active oTPETP oligomer displayed mechanoluminescence (ML) characteristics with abnormal blue-shifted emission maxima (yellow emissive) after grinding by an external stimuli like mechanical forces. However, the original orange emission of the pristine oligomer sample was restored by solvent (DCM) treatment and followed by the precipitation methods of the ground oligomer samples several times. The powder X-ray diffraction (PXRD) pattern and differential scanning calorimetry supported the blue-shifted emission characteristics of the ground oligomer samples. The PXRD pattern demonstrated lower intense peak of the ground samples compared to the pristine oligomer samples that was attributed to the collapse of the twisted conformation of the D-A oTPETP oligomer, which was subsequently triggered by an external mechanical force. Shifting of the phase transition temperature suggested the transformation of the crystalline states presented into the amorphous phases to the fully amorphous states. Further, the blue-shifted emission maxima of the oligomer comprising of the TPE unit was ascribed to the conformational changes of the molecule from more planar to less planar. Thus, this oligomer opens up a new design strategy to develop smart materials with AIEE characteristics and ML activity. Additionally, this AIEE material has been applied for solid-state and aggregated-state picric acid detection. Thus, this novel design strategy could be employed as a powerful tool for the chemosensors, such as explosives detection. Another TPE-induced oligomer (TTPE) has been reported, which showed piezochromic luminescence with redshifted emission wavelengths under the external pressure ( Figure 6G). [130] It also exhibited tunable photoluminescence behavior, such as blue, cyan, and green fluorescence after being cyclized into the oligomer (CTTPE). The red-shifted emission maxima in PL process, lower intense PXRD pattern, and shifting of the phase transition temperature of the ground TTPE samples subsequently confirmed the piezochromic effect of the oligomer. Furthermore, these probes have been employed as chemosonsors and for cell-imaging applications. The summary of photophysical properties and applications of AIE oligomers are discussed in Table 4.

Reported organic AIE-polymer
Polymers with AIE features could be strategically constructed through various synthetic routes. The fundamental principle is to introduce classical AIEgen, such as DSA, TPE, TPA, and HSP into polymer framework. Figure 7 illustrates three general design strategies to furnish AIE polymer: (A) by direct homopolymerization or copolymerization of monofunctional AIE monomer to bestow linear polymer with AIEgen as pendant group, (B) by direct homo or copolymerization of bifunctional AIE monomer to furnish linear polymer with AIEgen at the backbone, and (C) by direct polymerization or copolymerization of multifunctional AIE monomer to yield hyperbranched polymer. [131] Generally, chain polymerization is employed to prepare nonconjugated AIE polymers with AIEgen as pendant group, while step chain polymerization is used to furnish conjugated AIE polymers with AIEgen at backbone. Alternatively, it could also be prepared by modifying the structural framework of as-prepared AIE inactive polymers with AIEgen. The resulting polymers turn into AIE F I G U R E 7 Schematic illustration of general design strategies to furnish AIE polymers active with linear, star, or hyperbranched structure having AIE moieties at main chain, side chain, or center or terminal of the polymer. The following section discusses various types of AIE polymers as well as their synthetic routes through some specific examples. [132,133] 3.

Linear AIE polymer
Linear polymer is the simplest polymer architecture with repeating units interconnected in one direction. Linear AIE polymers could be generated by integration of AIEgens on its main chain or side chain. Conventional free radical polymerization is largely used for the synthesis of well-defined side-chain AIE polymer with desired molecular weight. The resulting polymer emits weak and bright fluorescence in dissolved and aggregate states, respectively. A linear AIE nonconjugated polymer was furnished via free radical polymerization of catonic anhydride monomer and styrene conjugated AIE active phenothiazine molecule. Due to the presence of AIEgen at the side chain of amphiphilic polymer P1, it formed highly emissive NPs in aqueous solution with good biocompatibility and cellular efficacy as shown in Figure 8A. [134] Apart from conventional free radical RAFT polymerization technique, the reversible deactivation radical polymerization is also expanded to generate linear AIE polymers with targeted molecular weight, diverse polymer architectures, high end fidelity, and low dispersity. Similarly, a rapid and cost-effective self-reporting polymerization method was developed for real-time monitoring polymer growth during precipitation polymerization. By taking AIEgen as a monomer of precipitation polymerization, the polymer growth could be easily monitored as the polymerization proceeds the oligomer or polymeric chain starts precipitation in the solution inducing increased emission, which can be observed by the naked eye under daylight or UV lamp ( Figure 8B). [135] It was observed that the number and type of side chain affects the brightness or emission of AIE polymer aggregates. By increasing the number of side chains, the polymer's hydrophobicity increases, endorsing polymer aggregation in water and ultimately enhancing the fluorescence quantum yield by severely constraining the intramolecular motions. [136] The hydrophobic or hydrophilic nature of side chain also affects the photophysical properties of the AIE polymer. For instance, AIE polymer P2a with amide side chain has high solid state Φ f than its alkyl substituted amide P2b containing polymer ( Figure 8C). This is because of the difference in hydrogen bonding sites as higher hydrogen bond network would make the polymer rigid and restrain the intramolecular motion, thereby, fortifying AIE phenomenon. [137] The main chain linear AIE polymers can be obtained by polycondensation or polycoupling reactions, viz. Suzuki, Stille, and Knoevenagel reactions, which have AIE units on the polymer backbone. A series of AIE active conjugated polymers with phenothiazine as core were synthesized via Suzuki cross-coupling reaction. The phenothiazine was taken as donor and nonplanar building block to prevent π-π stacking, and different side-chain groups were installed at phenothiazine donor (P3a, P3b) to further constrain inter or intrachain π-π stacking ( Figure 8D). Moreover, different acceptors were linked with phenothiazine to furnish NIR emissive polymers. By introducing bulkier diphenyl anthracene group at phenothiazine donor, a 21 times fluorescence enhancement of AIE polymer was achieved in aqueous solution (aggregate state) compared to THF solution (dissolved state). [138] It is well known that polymeric backbone influences photophysical properties of main chain polymers. In case of AIE  [144] Copyright 2020 American Chemical Society conjugated polymers, backbone controls the photophysical properties, such as quantum yield, brightness, emission wavelength, and AIE properties. It is to be noted that while incorporating AIE monomer in polymer architecture might not necessarily result in AIE active polymer. For instance, two structurally similar pyrazine conjugated polytriazole P4a-P4b exhibit different emission characteristics even though they both are constructed from AIE active monomers. The cyano substituted pyrazine containing polytriazole suffers from ACQ effect due to inter and intramolecular π-π stacking of planar cyano substituted pyrazine, phenyl, and triazole unit, which leads to nonradiative relaxation. However, no such π-π stacking was experienced by other polymer derivatives and thus, they show AIE property ( Figure 8E). [139] Moreover, a small mole fraction of AIE active monomer unit might be enough to generate an AIE active main chain polymer. For example, a polyflurorene derivative without TPE AIEgen displays ACQ behavior. However, by increasing TPE content in polyfluorene derivative, dual emission of flurorene and TPE moiety is achieved. On further raising the ratio of TPE to 0.3, the resultant polymer exhibited single emission peak of TPE AIEgen in the aggregate state. [140] Along similar lines, another AIE active conjugated polyelectrolyte was reported for visualization of latent fingerprints. [141] Donor acceptor-type AIE conjugated polymers with various structures and red-shifted emission spectra, good brightness, high quantum yield, and advance photophysical properties are also newly explored. [142,143] A series of donor-acceptor AIE conjugated polymers have been reported for high brightness NIR II fluorescence imaging with triphenylamine conjugated alkylthiophene as donor and benzobisthiadiazole as acceptor. To attain high brightness, steric hindrance or twisting was generated by merely changing alkyl group positions at the thiophene spacer. The orthosubstituted alkyl chains result in twisted structure, while the metasubstituted one gives coplanar structure. Such AIE polymer, P5 with both planar and twisted structure, displayed high brightness in NIR region as planar unit assures high absorptivity, while twisted structure affords high quantum yield, the prerequisite for high brightness ( Figure 8F). [144] Moreover, AIE conjugated polymers are also explored for chiral optoelectronic devices as exciton migration along the conjugated polymer backbone, which amplifies circular polarized luminescence (CPL) compared to small organic material. A chiral and aggregation-induced delayed fluorescence (AIDF) active conjugated polymer was designed by strapping chiral alanine pendant and delayed fluorescence unit, dibenzothiophen-2-yl(phenyl)methanone onto the poly(carbazole-ran-acridine) framework to introduce CPL characteristics. The polymers with different fraction of chiral center displayed both CPL and AIDF properties with different solid-state quantum yield as well as glum values, which may be due to the variation in their chiral arrangements and molecular packing. Such type of chiral polymeric material with AIE and TADF features could be potentially useful for the development of photonic devices. [145] 3.5.2 Branched AIE polymer Besides above discussed linear AIE polymers, researchers have also explored branched AIE polymers mainly in two ways, that is, AIE active dendrimers and AIE active hyperbranched polymers. Branched polymers are macromolecules with multiple branches or secondary chains emanating from primary backbone. Branched macromolecular systems exhibit unique features, such as three-dimensional and multifunctional architectures, one pot synthesis, low viscosity, good solubility, and processibility. Through fusion of branched polymer and AIEgen in one system, advance functional materials with diverse applications, such as sensors, organic LEDs, and artificial light-harvesting systems, are greatly appealing. The branched AIE polymers are nonemissive or merely emissive in solution state but exhibit bright emission in aggregate state. The rigid and intrinsically crowded molecular structure of branched polymers restricts the intramolecular motion of AIE unit in isolated state, bestowing the polymer emissive and fostering overall AIE effect. Three main types of AIEgen-based dendrimers are explored so far. [146] Notably, (1) AIE core-dendrimers with AIEgen at core, (2) AIE branch-dendrimers with AIE moiety at branches or branching points, and (3) AIE peripherydendrimers with AIE unit at periphery. A series of AIE conjugated dendrimers were designed with twisted 9,10-divinylanthracene (DVA) AIEgen as the central core, through which multibranched amino building blocks were connected via alkene linker. The DVA unit exhibits AIE behavior, while amine dendrons serve as electron donating group, responsible for interbranch coupling and two-photon augmentation. The AIE dendrimers emit weak emission in solution due to TICT and unrestricted intramolecular rotation. However, all generation of dendrimers P6(G0-G2) exhibit high Φ f in solid and nanoaggregate state owing to restraint of intramolecular motion as well as planar conformation in solid state. It was remarked that Φ f values and emission wavelength increase with rise in the generation number ( Figure 9A). [147] Dendrimers are generally synthesized via two different strategies, divergent and convergent synthetic approaches. The AIE branched dendrimers were constructed via controllable divergent strategy. In TPE-functionalized [2]rotaxane, bulky TPE moiety was incorporated as one of the axle stopper, while monosubstituted platinum-acetylide group not only acts as second axle stopper but also as reactive site for dendrimer growth. The rotaxane wheel, pillar [5]arene macrocycle, was substituted with two TIPS-protected alkynes that generate reactive alkyne group for dendrimer growth. All generation of rotaxane dendrimers P7(G1-G3) are weakly emissive in good solvent DCM; however, all of them showed enhanced emission in 98% methanol ( Figure 9B). Ultimately, the AIEgen branched dendrimers are utilized for the development of artificial light-harvesting systems with impressive photocatalytic performances. [148] Among third type of AIE active dendrimers, there are reports where TPE moieties have been installed at the periphery of the dendrimers. A series of dendrimers with ethylene oxide core and TPE decorated dendritic periphery were constructed via divergent synthetic route by means of click chemistry. The AIEgen decorated dendrimers are strategically designed to probe dendritic structure and steric crowding at the dendritic periphery. Decoration of AIEgenic moiety at the dendritic periphery helps in unraveling the architectural changes with each generation. All dendrimer generations from G0 to G3 are AIE active and show decrease in critical f w , that is, minimum water fraction required for AIE emission is reduced with increase in dendrimer generation. As generation grows, the steric crowding is increased, resulting in severe restriction of intramolecular motions of TPE units and fostering overall AIE effect. [149] Hyperbranched polymers exhibit similar molecular architecture and feature as dendrimers but involve much easier synthesis. However, the rigidity and steric crowding of hyperbranched polymers always leads to some restriction of intramolecular motion of AIE unit even in dissolved state, and gives rise to the polymeric emission in dissolved state and further slumping the overall AIE effect. [150] Two similar hyperbranched polymers, P8a-P8b, were designed via Sonagashira polycoupling of trifunctional triphenylamine monomer with bifunctional TPE or silole unit. Moreover, they have good solubility in organic solvents without any side chain in their framework, which may be because of hyperbranched architecture and propeller-like structure of TPE or silole. Both the hyperbranched polymers behave differently in isolated and aggregate state. The TPE incorporated hyperbranched polymer, P8a, exhibited high Φ f in aggregate state than solution state ( Figure 9C). However, silole incorporated hyperbranched polymer, P8b, displays almost same low Φ f values in isolated and aggregated state, which may be due to its more rigid and crowded structure compared to the former one. [151] Another TPE-based hyperbranched polymers, P9a-P9b, were constructed via A 2 + B 4 type copolymerization of bifunctional fluorene/carbazole/phenylene (A 2 ) monomer with tetrafunctional TPE monomer (B 4 ). To avoid the insoluble gel formation in A 2 + B 4 type polymerization, TPE-4Br concentration was set at 0.04 mmol mL -1 and end capping group was added. Nevertheless, the TPE unit had been integrated into hyperbranched polymer architecture, yet, its phenyl ring still rotates in solution state in spite of more steric hindrance in hyperbranched architecture compared to small molecule. The emission intensity of all three hyperbranched polymers, viz. P9a-P9c, increases with methanol fraction, reflecting AIEE behavior with αAIE (AIE factor = Φaggr/Φsoln) of 2310, 16, and 5.4, respectively ( Figure 9D). [152] The summary of photophysical properties and applications of AIE polymers are discussed in Table 5.

Organic AIE-nanoparticles
There are several highly efficient AIE NPs reported, which have been constructed on TPA, TPE (tetraphenyl amine), and TTF structures. TPA molecule was found to be a strong donor. Combining TPA with strong acceptor molecule with a π-spacer could lead to longer wavelength. Many new and advanced TPAbased AIE NPs have been reported over decades. However, AIE NPs with deep red NIR-I (∼700-900 nm) and NIR-II (1000-1700 nm) emission are highly desirable in biomedical applications owing to their unique properties, such as deep tissue penetration, reduced photodamage, avoid autofluorescence, and high S/N ratio, which enable real-time in vivo, ex vivo bioimaging, two-photon imaging applications. [153,154] In this regard, two-photon bioimaging has gained tremendous interest in tissue imaging due to their aforesaid advanced features. This also enhanced the pursuit for fluorescent probe with high two-photon absorption property. Acrylonitrilebased AIE probe with deep red NIR-II emission range is very potential option for two-photon imaging. Two acrylonitrile TPAT-AN-XF and 2TPAT-AN two-photon fluorescent probes were reported ( Figure 10A). [155] TPA is strong donor and can also act as acceptor molecule in donor-π-spacer-acceptor molecular structure. Introduction of thiophene as spacer could enhance the conjugation, which leads to red-shift in emission and increases two-photon absorbance as well. Interestingly, both probes were prepared by same reactant at different temperature. These water-soluble AIE NPs were prepared by nanoprecipitation method. 2TPAT-AN nanoparticles showed hydrodynamic diameter of 102 nm. Absorbance maxima of 2TPAT-AN and TPAT-AN-XF was 482 and 439 nm in THF, respectively. Emission for both AIEgen NPs was red-shifted at 572 nm for 2TPAT-AN and 599 nm for TPAT-AN-XF, respectively. 2TPAT-AN NPs were prepared by nanoprecipitation method with amphiphilic block copolymer PEG-PLGA (Mw = 100-1000) (Figures 10B and 10Ba). Both AIE probes have high quantum yield up to 37% and solid-state red emission, large twophoton cross section, and large stroke shift ( Figures 10B,  10Bb, 10Bc, and 10Bd). They were employed for in vivo tumor cell imaging with high signal-to-noise ratio and ex vivo two-photon deep tissue imaging and live cell organelle lysosome in Hela cells. Two-photon imaging was performed using NIR laser of 880 nm with large cross section up to 580 GSM. In this way, the acrylonitrile-based AIE NPs TPAT-AN-XF and 2TPAT-AN have been shown as potential candidates for tumor cell tracking and deep tissue two-photon imaging.
Organelle-specific cellular and subcellular bioimaging is highly desirable due to real-life biomedical application. But most of the reported nanoparticles fluorescent probes compromise in targeted delivery and efficiency. TPA-based red emitting AIE-TICT active NPs DTPA ( Figure 10C) were fabricated with amphiphilic triblock copolymer pluorinic F127 composed of polypropylene oxide and polyethylene oxide. [156] F127 was used for encapsulation, which provides biocompatibility and stability to the AIE NPs. To make target specific for subcellular imaging of mitochondria and lysosome, these AIE NPs surface were further conjugated with TPP and morpholinyl (MP) chemoagent, respectively. After fabrication, DTPA NPs as control and DTPA-TPP and DTPA-MP were used in subcellular in vivo imaging. Absorption maxima for DTPA NPs was 510 nm and emission was red shifted to longer wavelength to 800 nm and showed max- and there was only a negligible difference in DTPA-TPP and DTPA-MP compared to the DTPA NPs. These DTPA NPs and fabricated NPs were further conjugated with CDs to facilitate cell penetration. These fluorescent NPs due to their biocompatibility, small size, chemo, and photostability implied for mitochondria and lysosome imaging and tracking dynamic change during autophagy.
TPE with its tetra phenyl propeller-like structure is well explored for its AIE property. It acts as a donor, has a twisted structure and after conjugation with π-spacer donor, it enhances the intermolecular charge transfer and extends the absorption of AIEgen to longer wavelength and emission range upto NIR wavelength depending on the molecular structure.
D-A-D molecular system (BTPETQ AIE NPs) ( Figure 10D) [157] has been reported, where donor TPE group and acceptor TQ [1,2,5-thiadiazolo(3,5-g)-quinoxaline] helped to achieve NIR emission. Deep penetration capacity, high S/N ratio vascular morphology, and dynamic imaging of brain are highly required criteria for vascular-related disease theranostics. BTPETQ NPs, having aggregation-induced NIR emission and long wavelength absorption, were reported for two-photon intravital imaging of mouse brain and tumor vasculatures. BTPETQ showed absorption in visible region with absorption maxima at 450 and 550 nm in THF, which favors NIR-II two-photon excitation. It also showed emission from 600 to 1000 nm in THF. BTPETQ dots were prepared by nanoprecipitation method with DSPE-PEG polymer that had average size of 42 nm and high quantum yield. BTPETQ in 40-90% water fraction showed remarkable increase in fluorescence intensity due to AIE characteristics ( Figure 10E). The BTPETQ dots with highly efficient two-photon fluorescence can differentiate between normal blood vessels and tumor blood vessels in deep tumor tissue due to their leaky structure. Additionally, imaging of blood vessel network of mouse brain with depth of 924 μm was demonstrated. NIR-II excitation is responsible for deeper penetration of BTPETQ dots. This study showed the potential need of NIR-II fluorescent probes for deeper penetration level for vasculature imaging of tumor and high SNB.
NIR-II window in molecular materials is highly attractive due to low cytotoxicity, deep penetration, and photostability, which render them excellent choice for bioimaging and biomedical threaptics. However, NIR-II fluorescent probes suffer from low quantum yield and complicated design strategy. Thus, design of aggregation-induced NIR-II fluorescent probe would solve this issue. A smartly engineered molecule BPST having aggregation-induced NIR-II emission was reported ( Figure 10F). [158] BPST was prepared by just replacing selenium in AIE luminogen BPBT, which has emission in NIR-I window. By replacing sulphur of BPBT, the molecule BPST could extend to NIR-II window. BPST was further encapsulated with DSPE-PEG by nanoprecipitation, which is called L897 NPs. The absorbance maxima of L897 NPs were centered at 347 and 711 nm and also emission maxima at 897 nm, which are 67 and 60 nm red shifted from BPST NPs. QY for L87 NPs was 5.8%. AIE NPs were applied for in vivo lymphatic imaging and noninvasive vessel imaging.
TTF is AIE active luminogen (2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumero-nitrile) with TPE and TPA, which has gained much interest in recent years due to its extended conjugated structure, and TTF-based AIE PS nanoparticle has shown efficient application in nonlinear optical process, blood-brain barrier evaluation, and photodynamic therapy of type-II and type-I. Molecular size tuning has thus expanded the application at multiple levels. AIE-NPs have also shown promising results in bioimaging and multiphoton photonluminescence. TTF molecular system has an extended π-conjugation and donor-π-acceptor-π molecular structure, which enables it for NIR-II emission, multiphoton absorption cross section, and nonlinear optical process, and has been well explored in many other AIE-based applications. One TTF luminogen TPETPAFN ( Figure 10E) [159] having absorption maxima at 515 nm in chloroform and 495 nm in THF and emission ranges from 550 to 750 nm was proposed. It exhibited high-order nonlinear optical properties as nanoaggregates in aqueous solution. Besides aggregation-induced 3PL enhancement, TTF also exhibited third harmonic generation (TGH), which is also called aggregation enhanced TGH as the intensity of TGH is proportional to aggregation. 2PL was observed for 1060 nmfs, while 3PL and TGF were observed for 1550 nm-fs laser ( Figure 10H). TTF doped nanoparticles showed applications in TGH imaging in tumor cells and multimodal 3PL and preliminary in vivo imaging of mouse brain.
PDT is of two types, Type-I and Type-II. Type-II PDT is based on energy transfer and oxygen for singlet oxygen generation and is very less efficient. Type-I PSs (photosensitizers) on the other hand could be more auspicious strategy to treat cancer. Type-I PS is based on electron transfer and generates high ROS, which is more efficient than type-II. Also, type-I is less depended on oxygen concentration, which makes it a better option for PDT. Most of the PDT systems reported are type-II but new PS with type-I PDT, TTFMN was developed ( Figure 10F). [160] TPA of TTF acts as donor unit and furan as acceptor with π-spacer, which facilitates type-I ROS generation. Also, twisted confirmation activates AIE property by suppressing π-π intermolecular interaction. TTFMN PSs are AIE nanoparticles and generate very high type-I ROS. TTFMN NPs were prepared by nanoprecipitation with pH activated TAT peptide-modified amphiphilic polymer ( Figure 10J). TTFMN AIE nanoparticles have shown broader emission wavelength, and red shift emission on introducing TPE group. Absorption maxima for TTFMN was exhibited 490 nm in acetonitrile. pH activated TAT peptide-modified polymer has been introduced as encapsulating matrix to target delivery of PSs AIE nanoparticles into cancer cells and enhance the PDT effect of TTFMN. The summary of photophysical properties and applications of AIE NPs are discussed in Table 6.

Organic AIE-photosensitizers
AIEgens have been also utilized as ROS probes and PSs due to their remarkable properties, such as high quantum yield, excellent photostability, wash-free imaging, and biocompatibility. Several turn-off/turn-on AIE probes have been reported for ROS detection based on various sensing mechanisms, such as photoinduced electron transfer, [161,162] excited state intramolecular proton transfer, [163,164] and Förster resonance energy transfer. [165,166] Moreover, the advances in AIE-based ROS probes provided a detail understanding of ROS dynamics in complex living cells that has sparked  [160] research interest in targeted therapy and potential biomedical applications.
As mention earlier, PDT is considered as a noninvasive treatment for antimicrobial, cancer, and various diseases. Traditional PSs, such as phthalocyanines and porphyrins, form aggregates in aqueous medium that leads to reduction in 1 O 2 . Typical PSs core, such as tetrapyrrole, generally have tendency to form aggregates that affect its photophysical properties and ROS generation ability. Hence, it is desirable to synthesize novel tetrapyrrole-based PSs having AIE properties to enhance its potential performance in PDT. However, PSs with AIE properties exhibit robust photosensitization and higher ROS generation ability in the aggregated state. [167] These characteristic properties make AIE PSs best candidates for PDT applications.
ISC between excited singlet states (S 1 ) to triplet state (T 1 ) is most significant for ROS generation ability of PSs. ISC is improved by lowering the energy gap (ΔE ST ) between S 1 and T 1 state. A rational simple approach, namely, modulation of donor-acceptor system comprising a carbazole moiety as donor and pyridinium fragment as electron acceptor group, was employed for engineering of CPy, Cqu, DCPy, and DCQu AIE active PSs ( Figure 11A). [168] Theoretically, calculation of Cpy, Cqu, DCPy, and DCQu was performed with the help of density functional theory, where the HOMO electron clouds were situated mainly in carbazole part and LUMO electron clouds were found more toward the electron acceptor moiety pyridine and quinoline. The energy band I and ΔE ST of CPy, CQu, DCPy, and DCQu were calculated and found 2.18, 2.04, 1.57, 1.43 eV and 0.586, 0.533, 0.476, 0.445 eV, respectively. These results confirmed that enhancement of the ICT effect leads to gradual lowering in the ΔE ST . The generation of ROS was studied by commercial 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA, commercial ROS generation indicator) and white light irradiation (4.2 mW cm -2 ) ( Figure 11B). ROS generation ability of PSs was found in the order CPy, CQu, DCPy, and DCQu, respectively ( Figure 11C). The ROS generation ability was least in CPy and most in the DCQu, which is also well matched with the ΔE ST energy order, thus, ROS generation can be predicted by calculating ΔE ST .
Fluorescent materials with far-red emission and aggregation-induced emission materials have much sought for imaging and therapeutic applications. Four new NIR AIE fluorescent materials, DCPy, DCFu, DCIs, and DCMa with push-pull system were reported. [168] They were synthesized by incorporation of different electron acceptor, such as malonitrile, isophorone, 3-cyano-4-phenyl-2(5H)-furanone), and methyl pyridinium salt, through ingenious molecular engineering with the strong electron donating diphenyl moiety ( Figure 11D). The ROS generation from AIEgen was evaluated by ROS indicator H2DCF-DA under white light irradiation. As shown in Figure 11E, green fluorescence was generated due to synthesized product from the mixture of H2DCF-DA and AIEgen upon white light irradiation. The ROS efficiency was found by fluorescence titration study and was in the order DCMa < DCIs < DCFu < DCPy ( Figure 11F). Among the different types of ROS, such as superoxide radical, hydroxyl radicals, hydrogen peroxide, and singlet oxygen 1 O 2 , the singlet oxygen is a primary cytotoxic agent to trigger cell death via PDT. [170] Therefore, oxidation of ABDA was measured in the presence of white light by each AIEgens, and the ABDA decomposition rate for DCMa, DCIs, DCFu, and DCPy was 0.49, 0.85, 1.26, and 11.13 nmol min −1 , respectively. This result showed that synthesized AIE DCPy PSs generated more ROS compared to Chlorin e6 (Ce6) commercial PSs.
Recently, triphenylamine-azafluorenone-based PSs were synthesized and its structure-dependent properties were systematically studied. In this work, four PSs TPAN, TPAPy, TPANPF 6 , and TPAPyPF 6 were synthesized using electron withdrawing (azafluorenone) and electron donating (triphenylamine) architecture ( Figure 11G). [171] The molar absorption coefficients of TPAN and TPAPy were higher than their corresponding ionized counterparts (TPANPR6 and TPA-PyPF6) and by comparing all four materials, TPAPyPF6 exhibited least light absorption. The synthesized materials displayed similar fluorescence spectra (500-750 nm) in solution but their solid-state emission spectra were completely different. TPAN behaved as ACQ material, showed prominent solvatochromism, and displayed no fluorescence in polar solvents and solid state because of strong TICT. [172] However, molecules TPAPy, TPANPF6, and TPAPyPF6 showed AIE properties. The ROS production by PSs was evaluated using the H2DCF-DA under white light irradiation. The order of ROS generation is found to be TPANPF6 > TPAPyPF6 > TPAPy > TPAN, respectively. For the monitoring of 1 O 2 generation, ABDA is used with the synthesized materials with white light irradiation, and was found in the order TPANPF6 > TPAPy > TPA-PyPF6 > TPAN ( Figure 11H,I). The calculated ΔE ST for TPANPF6, TPAPyPF6, TPAPy, and TPAN was 0.1831, 0.5155, 0.1645, and 0.0909 eV, respectively. Generally, ΔE ST is considered as the main factor for enhancing efficiency of ROS generation, but many other factors, such as molar absorbance, SOC, and so on, play very important roles in ROS generation. The ROS generation efficiency was also studied and compared with the rationally designed materials and found to be more in aggregate state than the molecular dispersed state.
Materials integrated with fluorophore and photosensitizer have great interest for the therapeutic and imaging applications. A pH sensitive polymer PLL-g-PEG/DPA/TPS/PheA was incorporated with tetraphenylsilole (TPS) as AIEgen and pheophorbideA (PheA) used as photosensitizer ( Figure 12A). [173] Due to mutual energy transfer, generation of singlet oxygen was prevented in the aggregated state but efficiently generated in the dissolved state. [174] In the normal physiological condition (pH 7.4), the nanoparticles self-assembled with low fluorescence and toxicity but upon uptake by cancer cells (pH 5.0), NPS disassembled and lead to strong green fluorescence from free TPS which upon light irradiating, generated ROS that caused lysosomal disruption to trigger cell apoptosis ( Figure 12B, C).
For selective cell killing, a controlled and reversible singlet oxygen generation is the key factor. Spiropyrans show two photoswitchable states and they have opposite fluorescence properties. A photochromic spiropyrans polymer P 1 -P 3 was reported, and these polymers can control fluorescence and generation of singlet oxygen by its photoswitching properties ( Figure 12D). [175] The reversible and efficient PSs allowed multiple photoswitching cycles that induced efficient cellular apoptosis via repeated ROS generation ( Figure 12E, F).

CONCLUSION
In this review, progress in the versatile design of AIEgen with their advanced optical properties and fundamental aspects of structure-functionality relationship have been systematically summarized. Especially, this review covers emerging AIE probes, such as AIE-TADF, AIE-RTP-based small molecules, and AIE-macromolecules-based supramolecular frameworks, including AIE-oligomer and polymers-based purely organic molecules. Unlike the classical fluorophores with limited applications, this review discusses a few special class of the smart AIEgens, those are considerably possessing high photosensitization capability, unique nanoparticle-forming ability, and improved photophysical properties exclusively devoted to advanced biomedical and electronic research.
In addition, precise structural engineering can harvest both  [176] electrogenerated singlet and triplet excitons, thereby, furnishing long-lived delayed fluorescence and phosphorescence, which offers high-contrast TRLI and removes the typical tissue background fluorescence. Further, emission amplification in condensed state is illustrated by selectively choosing a few supramolecular frameworks. The unique "turn on" emission on coassembly of host-guest moieties restricts rotation in the condensed state, and the spontaneous aggregation in biological medium has endowed AIEgens as a potential candidate for subcellular bioimaging and biomedical therapeutics. Additionally, this review also highlights the AIE-oligomers akin to macrocycles, comprising of identical structures, and molecular weights are consider to be more proficient models for studying both in solution and condensed states. Alternatively, by incorporating small AIE molecule into polymer's backbone or side chains, the structural framework modifies and causes steric crowding and render-free rotation, resulting in bright emission on aggregate. Thus, AIE polymers exhibit additional advantages, such as high solid-state brightness and tunable structure, and morphology and processibility of the polymer offers new opportunities in real-world applications. While, classical small and polymeric system was suffering from weak ROS generation, this obstacle is overcome by a special class of light-harvesting luminophores known to be AIE-PSs also discussed herein.

FUTURE OUTLOOK
Rapid development of AIE luminogens has occurred since 2001, and has shown potential activity in versatile fields, such as organic LEDs, biosensors, and chemosensors, including image-guided therapeutic applications. However, some lim-itations impede the efficiency improvement of these materials for practical applications. A few crucial limitations include: (1) the presence of sterically hindered rotor groups in AIE probes, mostly furnish weakly emissive TICT state, thereby low PLQY was observed.
(2) Exact mechanism of AIE phenomenon is relatively not well-established to explore newer functionality-based mechanistic design of AIE materials. (3) Hydrophobic nature of the AIE luminogens showed lower internalization ability into the cellular system due to its longer accumulation time into the cellular environment.
Most of the luminogens are not biocompatible and need quick body clearance after their intended activity. Thus, to specifically evaluate the imaging and therapeautic efficacy of the novel AIEgens, a precise functionalization and design strategy of AIE probes is needed before execution. (4) Along with these, to further improve the biocompatibility, the development of biodegradable AIE probes, which comprises of low molecular weight and smaller backbone or employment of the biodegradable congeners into the AIE nanoparticles, is highly essential. (5) Incorporation of multiple functional groups into one single AIEgen system is facilely more advantageous toward the diagnosis of the disease, treatment, and prognosis of the disease. While classical AIE probes produce low ROS for PDT and PTT applications, recently developed AIE-TADF and AIE-RTP probes produce efficient ROS due to their high triplet yield at reduced ∆E ST , enabling them as potential candidate for clinical practice. Most of the PDT and PTT AIEgen probes are based on TPE core; hence, more efforts are needed to develop non-TPE-based PDT and PTT AIEgen probes. Additionally, the AIE polymers area also has immense possibility to be explored with newer molecular design and functionality. Thus, polymerization methods can be developed, which have more control over repeating units and molecular weight and which will help to develop new varieties of luminescent materials. Therefore, current advancement and prospects of AIEgens have expedited improved functional properties, including optical, morphological, and ML as well as piezochromic characteristics.
In addition, AIEgens have also demonstrated low nonspecific cytotoxicity, targeted therapy, sensing, security, information encryption, and use in multiple organic electronic devices. These have resulted in the emergence of practical devicebased applications for disease diagnosis, ablation of cancer and their biomarkers, drug screening, as well as monitoring of toxic components, which are noticeably evident as part of the bright future and perspectives of these AIE probes. Finally, studies of triplet harvesting AIE probe development are at an early stage, and plenty of new design possibilities with high-precision structural characterization and comprehensive properties with more in-depth exploration still remain as the direction of future efforts.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.