Aggregation‐induced emission luminogens for in vivo molecular imaging and theranostics in cancer

Cancer is one of the most fatal diseases for decades. Aggregation‐induced emission luminogens (AIEgens) have been recently used as molecular imaging or therapeutic agents in cancers, due to the advantages of large Stokes shift, high quantum yield, great biocompatibility, and strong photostability. AIEgens can specifically target different types of cancer via diverse targeting strategies. AIEgen‐based fluorescence imaging, especially near‐infrared imaging, demonstrated deep penetration and suitable signal‐to‐noise ratio, which allows reliable in vivo cancer imaging. Combined with other imaging modalities, AIEgen‐based multimodal imaging could provide multidimensional cancer hallmarks from different perspectives. In addition, AIEgen‐based phototherapy can be used for photodynamic therapy and photothermal therapy, which facilitate ablation of cancer cells with good biosafety and high therapeutic effects in vivo. AIEgens nanoparticles fabricated with some specific chemicals, drugs, or siRNA, could display synergistic therapeutic effects for cancers. This paper comprehensively describes the current status and future perspectives of AIEgens, which have showed a great potential for the future preclinical and clinical translation on in vivo molecular imaging and theranostics in cancer.

][7] Cancer diagnosis always contains epidemiologic history, clinical situation, laboratory tests, and imaging examinations.10][11] As for cancer therapy, traditional therapeutic approaches like chemotherapy, radiotherapy, and recently developing gene therapy, immunotherapy, and so on, have been already utilized to save people from the threat of cancers. [12,13]16] Real-time molecular imaging-based evaluation of cancer therapy is essential to precisely learn about the whole process of cancer treatment.
[19] To solve the main health threat, cancer imaging and theranostics have become a research hotspot in the development of organic dyes that can realize visualization and localization of cancer cells by fluorescence imaging and kill them through different therapies.][22] However, traditional organic materials for fluorescence imaging and theranostics are always limited by the notorious aggregationcaused quenching (ACQ) effect due to π-π stacking, which makes easy photobleaching and low signal-to-noise ratio (SNR) as well as hampers further photodynamic therapy (PDT) and photothermal therapy (PTT) for cancer. [23]Opposite to the ACQ effect, the aggregation-induced emission (AIE) phenomenon was discovered by Tang's group in 2001 with large Stokes shift and strong photostability. [24]The AIE luminogens (AIEgens) exhibit almost no fluorescence in the dispersed state, but have a significant emission in the aggregated or solid-state, benefiting from the restriction of intramolecular motions. [25]And many other processes like J-aggregation formation, twisted intramolecular charge transfer (TICT), and excited-state intramolecular proton transfer, and so on are also contributed to the AIE phenomenon. [26,27]istinctly different from the large planar structures of conventional ACQ dyes, most of the AIEgens show highly twisted propeller-like structures that quench the emission in dilute solution by high nonradiative decay rate and restrict intermolecular π-π stacking and intramolecular motions when aggregated.][31] Based on those above, numerous AIEgens have been designed, synthesized, and reported for biomedical applications based on several classic AIE structures, such as siloles, tetraphenylethylene (TPE), triphenylamine (TPA), and so on.The unique property of AIEgens vigorously promotes the development of both fluorescence imaging and phototherapy.[34] To realize deep penetration and high SNR, the emissive wavelength of AIE NPs is expected to expand into the near-infrared (NIR) region, especially the NIR-II region.40] In this review, we comprehensively summarize the AIEgen-based in vivo molecular imaging and theranostics for cancers.Considering the numerous research reports, we focus on the clearly characterized features and high-quality efficacy of imaging and theranostics (Table 1).First, we introduce the common and typical cancer targeting strategies clarified by the process of entering tumor tissues.Second, we summarize in vivo molecular imaging for cancers modalities guided by AIEgens and sort them by imaging modalities.Then, we focus on the theranostics based on AIEgens, containing phototherapy and other therapies based on the fabricated chemicals/drugs.Finally, we conclude the achievement of AIEgen-based in vivo molecular imaging and theranostics for cancers and give several new insights to promote future work in the development of AIEgens for cancer applications.

CANCER TARGETING STRATEGIES OF AIEGENS
To achieve satisfactory imaging or therapeutic efficacy, AIEgens are modified with different ligands for fabricating AIE NPs to enhance the accumulation in tumor regions.Generally, after intravenously injected into the body, the AIE NPs circulate in the bloodstream and part of them is accumulated in the reticuloendothelial system and cleared by the urinary system.The escaped AIE NPs enter the tumor tissues via four parts (Figure 1): first, they pass through the blood-tumor barrier to transport into tumors by enhanced permeability and retention (EPR) effect or endothelial transcytosis; then, they are activated in the tumor extracellular microenvironment; or they interact with the noncancer cells (stromal cells) or cancer cells and internalized by active targeting; finally, they are effectively localized on the subcellular organelles due to specific ligands. [41]

2.1
Transporting through the blood-tumor barrier

2.1.1
Targeting AIE NPs based on EPR effect AIE NPs are able to be passively entrapped in the tumor tissues through EPR effect, due to the high vascular density, defective vascular structure, and deficient lymphatic circulation in tumor tissues. [42,43]And the size, shape, and surface modification of AIE NPs are the pivotal factors that determine their utilization and accumulation in tumor microenvironment.
The size of AIEgens plays a crucial role in their passive targeting abilities, which should be designed appropriately to avert from renal elimination or hepatic phagocytosis. [44]47] A variety of AIEgen-based probes with suitable sizes have been developed for tumor-targeting imaging or theranostics on account of the EPR effect, such as AIE quantum dots

TA B L E 1
Representative AIEgen-based nanoparticles for in vivo cancer imaging and theranostics.(QDs) (∼10 nm), [48] doxorubicin (DOX)-based DOX/mPEGss-Tripp (117 nm) [49] and M-NPAPF-Au (∼120 nm). [50]ntriguingly, sub-10 nm AIE QDs were reported to enhance tumor-targeting ability and reduce liver retention compared with AIE dots larger than 25 nm. [48]The AIE QDs assembled by microfluidics realized better cellular uptake and NIR-II fluorescence tumor imaging with no assistance from other target molecules or peptides.The ratio of fluorescence intensity of the tumor to that of the liver in the TTB QDs-treated mice was two to three times that in TTB dots-treated mice, which could distinguish tumors as small as 80 mm 3 .

Nanoparticles
It is noted that the shape of AIEgens has a great impact on their uptake and accumulation in tumors.[53][54] However, there was no consistent conclusion on the best shape, probably owing to differences in tumor models and AIEgens properties.For instance, spherical QM-5 nanoaggregates demonstrated a more conspicuous tumor-targeting fluorescence imaging than rod-like aggregates of QM-2 in U87MG tumor-bearing BALB/c nude mice. [51]Conversely, TPETPAFN nanorods exhibited a better fluorescent contrast of tumors than nanodots in SKBR-3 tumor-bearing BALB/c nude mice, after the transition of TPETPAFN nanodots to nanorods through ultrasound sonication. [54]Another systematic research assessed fluorescence imaging of bare nanoparticle, nanosphere, nanorod, and microrod of Dex-b-PLA-ED particles in SMMC-7721 cells-transplanted zebrafish embryos, manifesting that the nanorods with a length-width ratio of about 5 performed best. [52]On balance, the optimal shape of AIEgens for passive tumor targeting may rely on the specific application and objective.
To enhance the tumor passive targeting efficiency, a series of strategies have been adopted to modify AIEgens, including polyethylene glycol (PEG), [55][56][57] micelles, [58][59][60] and liposomes. [61]PEG is used widely in the modifica-tion of nanoparticles to prolong their systemic circulation time by protecting them from aggregation, opsonization, and phagocytosis. [62]TPD NPs with AIE features were coated with 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 to disperse well in phosphate buffer solution, making in vivo application possible.Fluorescence imaging illustrated that TPD NPs could passively accumulate in tumors within 6 h by EPR effect, obtaining a satisfactory photodynamic therapeutic effect.Micelles are self-assembled by amphiphilic block copolymers in an aqueous solution to form nanostructures with a hydrophobic core shield by a hydrophilic shell, which are regarded as promising candidates for drug delivery vehicles. [63,64]Moreover, the copolymers are often connected by stimuli-responsive linkage, making it activated in certain circumstances.P(TPMA-co-AEMA)-PEI(DA)-Blink-PEG micelles were reported with high-resolution two-photon fluorescence bioimaging and efficient drug delivery. [60]To fabricate them, a two-photon AIE fluorophore was conjugated to the triblock copolymer, and DOX was encapsulated into the core of these micelles, which exhibited excellent accumulation and retention in tumor tissues with long circulation time and acid-triggered drug release.Liposomes are sphere-shaped vesicles composed of one or multiple phospholipid bilayers, delivering drugs by endocytosis or fusion with the cell membrane. [65,66]otably, liposomes can load hydrophilic molecules into their aqueous core or trap hydrophobic drugs between their bilayers. [65]TPE, one of the hydrophobic AIEgens, was encapsulated into the lipid bilayers of liposomes to construct TPE-liposomes for in vivo tumor fluorescence imaging. [61]PE was kept in a nonrestricted rotation state in the bilayers and showed no fluorescence when circulating in the blood.While in tumor regions, the liposomal membranes were ruptured, and TPE monomers were released and aggregated into AIE probes with fluorescent signal amplification for tumor imaging.

Targeting AIE NPs based on endothelial transcytosis
Transcytosis through endothelial layers is another important way to cross the blood-tumor barrier for highly efficient accumulation of AIE NPs.
It was reported that human serum albumin-bound nanoparticles (BPBBT-HSA NPs) was able to realize great tumor accumulation dominantly by means of endothelial transcytosis.Biodistribution studies exhibited a 7.2-fold of the concentration of BPBBT-HSA NPs in tumor, compared with BPBBT micelles with similar size of 110 nm.And intravital NIR-II fluorescence microscopy further validated that BPBBT-HSA NPs could enter tumor parenchyma from the tumor vasculature efficiently and be endocytosed within 3 h-injection in vivo. [67]ore importantly, tumor endothelial cells express various angiogenic markers, which can be regarded as the binding sites for the tumor-targeted nanoparticles, such as integrin α v β 3 , nucleolin, vascular endothelial growth factor receptors, VE-cadherin, and E-selectin. [68]Arginine-glycine-aspartic acid (RGD) peptide is one of the most investigated tumor targeting peptides, which can bind to integrin α v β 3 on tumor endothelial cells, facilitating the transcytosis of NPs into tumor tissues. [69]Recent studies have reported a large amount of AIEgens decorated with RGD peptide to target tumor regions positively, such as TPE-red-PEG-RGD NPs, [70] TB1-RGD dots, [35] RGD-4R-MPD/TTB NPs, [71] and cRGD-TPETS nanodots. [72]For example, TB1-RGD dots was constructed by covalently grafting c-RGD peptides onto the AIE dots for NIR-II fluorescence and NIR-I photoacoustic (PA) imaging of brain tumors. [35]And these dots showed stronger fluorescence and higher PA intensity of brain tumors than those of the TB1 dots treated group on the mice model, which was attributed to the specific tumor affinity of RGD peptides.AS1411 DNA aptamer, binding with nucleolin expressed in the tumor endothelial cells, was also applied to functionalize gold nanoprisms together with cell membrane-targeted TPE@Zn to synthesize dualtargeted Au-Apt-TPE@Zn. [73]Au-Apt-TPE@Zn showed great photothermal efficacy in vivo guided by real-time PAI and fluorescence imaging of tumors, manifesting its excellent targeting ability to tumors.Bradykinin ligand-modified AIE NPs (BK@AIE NPs) was another example of active transporting through endothelial cells. [74]Bradykinin, a type of kinin B1 receptor (B1R) agonist, could bind to B1R overexpressed on the capillary endothelial cells and activate it on the blood-tumor barrier, facilitating the photothermal treatment of deep tumors and further inducing the local immune responses.

Targeting AIE NPs triggered in tumor microenvironment
Tumor microenvironment is a complex structure characterized by acidic pH, hypoxia, elevated glutathione (GSH), and several upregulated enzymes, which can be utilized as incentives to activate AIE NPs after entering the tumor tissues.
The acidic pH of extracellular tumor microenvironment is attributed to the increased glycolysis and lactate produc-tion, often regarded as a distinctive tumor target. [75]A novel water-soluble AIEgen (WAPS) was studied for targeted PDT of tumors, triggered by the acidic tumor microenvironment via host-guest interaction with Pillar [5]arene (WP5). [76]The WAPS-WP5 complex was constructed in the neutral media with a low photodynamic activity, but produced considerable reactive oxygen species (ROS) at an acid pH of 5.2 due to the binding interface shift of the complex.Therefore, it realized a remarkable tumor-targeting PDT effect with negligible damage to other normal tissues.
The hypoxic microenvironment is one of the typical characters of cancer growth, which has been adopted to design cancer-targeting bioprobes including AIEgens.Hypoxiaresponsive AIEgens recently reported are mainly produced based on azo, nitro, and N + -O + moieties. [77]For example, PEG-azo-PS4 probe was synthesized by linking AIEgen (PS4) to PEG chain with the assistance of hypoxia-responsive group (azo). [78]Under hypoxia conditions, the azo linker was cleaved, and the product (AAPS) was generated to aggregate for fluorescence emission and 1 O 2 generation.In vitro and in vivo experiments demonstrated its excellent fluorescence imaging of hypoxic cells and antineoplastic effect with light irradiation.
It is well known that intracellular GSH level is higher in tumor tissues compared with normal tissues, generally employed as a stimulus for responsive tumor-targeting AIE probes.And the disulfide linkage was the most widely used for GSH-sensitive cleavage. [79]TPE-SS-PLAsp-b-PMPC was an amphiphilic copolymer comprising disulfide bond, which could transform into micelles and encapsulate DOX by self-assembly. [80]In vitro drug release study illustrated that the DOX-loaded micelles released much more DOX in the solution with 10 mM GSH than without GSH, over 90% after 48 h.Also, the DOX-loaded micelles showed great antitumor efficacy with fewer side effects and AIE active imaging in vitro and in vivo.
There are various enzymes overexpressed in tumor tissues such as caspase, matrix metalloproteinases and cathepsin, laying a foundation for the design of enzyme-triggered AIE probes in tumor targeting. [81]Enzyme-activatable probe (QM-HSP-CPP) was developed for intraoperative clinicopathological diagnosis of human pancreatic cancer sections and AIE-active fluorescence imaging of tumors. [82]AIEgen (QM-COOH), cathepsin E (CTSE)-sensitive peptide, and cell penetrating peptide (CPP) were integrated to synthesize QM-HSP-CPP.Remarkably, the AIE probe realized a distinct difference between tumor and paratumor tissues in human pancreatic cancer sections owing to the overexpression of CTSE in tumors, more significant than the immunofluorescence staining of Alexa Fluor 488.Furthermore, QM-HSP-CPP probe could visualize the endogenous CTSE with obvious AIE signal in xenograft-tumor-bearing mice model, thus achieving a real-time tumor-targeting fluorescent imaging.

Cellular uptake and internalization
Transportation of AIE NPs from extracellular to intracellular generally relies on the active targeting, which is based on the interactions between targeted AIE NPs and specific tumor receptors to obtain an enhanced uptake efficiency.Here, the active cellular targeting strategies of AIE NPs are divided into binding of noncancer cells and cancer cells.

Targeting AIE NPs based on binding to noncancer cells
Tumor-associated fibroblasts, the most abundant stromal cells in tumors, have the functions of promoting proliferation by secretion of multiple cytokines and growth factors. [83]Typically, the sigma receptor is employed to promote the uptake of nanoparticles by cancer cells and stromal cells, particularly tumor-associated fibroblasts. [84]nisamide (AA), a sigma receptor targeting ligand, was grafted with Pluronic F127 to stabilize the hexagonal nanoliquid crystalline (NLC) nanoparticles coloaded with TPE and anticancer drug formononetin. [85]The as-prepared targeted AA-NLC-TF demonstrated a brighter tumor fluorescence imaging and better anticancer activity in vitro and in vivo, compared with the nontargeted nanoparticles NLC-TF.
Immune cells mainly consist of macrophages, dendritic cells (DC), and several lymphocytes.And immune response is always suppressed in tumor tissues for the tumor growth and metastasis.DC-coated nanoparticles (DC@BPBBT dots) were fabricated by coating AIEgens BPBBT dots with DC membrane, endowing their ability for presenting antigen and high affinity for T cells. [86]The DC membrane-assisted hitchhiking strategy obtained high tumor delivering efficiency onto endogenous T cells, further realizing in vivo NIR-II fluorescence-guided photothermal immunotherapy for tumors.

2.3.2
Targeting AIE NPs based on binding to cancer cells Distinguished from normal cells, cancer cells always overexpress a wide range of receptors, which can be specifically bound with small molecules, peptides, and antibodies, and so on.
Folic acid (FA), a small molecule participating in the synthesis of nucleotide bases, has been extensively implemented in targeting tumor cells.FA possesses a high affinity with folate receptor (FR) upregulated in cancer tissues of the ovary, cervix, breast, lung, kidney, and colon. [87]It was reported that DTPEPBI conjugated with DSPE-PEG-folate displayed a superior targeting ability to FR-overexpressed tumor cells, compared with AIEgens linked with DSPE-PEG. [88]Cell experiments showed more Folate-AIE dots were internalized into the MCF-7 breast cancer cells than AIE dots.Further, in vivo fluorescence imaging demonstrated that the fluorescence intensity ratio of tumor to the liver of Folate-AIE dots was much higher than that of AIE dots in H 22 tumor-bearing mice.
Biotin, also named vitamin H, has become a mainstream of tumor-targeting groups owing to its precise binding to biotin receptors (sodium-dependent multivitamin transporters; SMVT) overexpressed on the cancer cell surface. [89]oreover, there is a much higher expression of SMVT than other transporters (e.g., FR) in several cancer cells such as breast, lung, colon, and renal, making it a more suitable candidate for tumor targeting. [90]A tumor-targeting polymer TPE-bi(SS-CS-Bio) was developed grounded on AIEgen (TPE), chitosan, and biotin, which could selfassemble into micelles and load paclitaxel (PTX) into the core in an aqueous environment. [91]In cellular experiments, TPE-bi(SS-CS-Bio) micelles exhibited stronger fluorescent intensity in human breast cancer cells (MCF-7 cells) than that of TPE-bi(SS-CS) micelles.And no evident fluorescence was observed in healthy human breast cells (MCF-10A cells) after incubation with TPE-bi(SS-CS-Bio), further proving their selective tumor-targeting capability via biotin.Also, PTXloaded micelles manifested excellent antitumor efficacy in vitro and in vivo.
Hyaluronic acid (HA), a biocompatible natural anionic polysaccharide, can function as a ligand of CD44 receptors upregulated on many kinds of tumor cells, which is frequently used to construct tumor-targeting nanoparticles. [92]uNSs-BD3@HA nanocomposites (ABH) were synthesized by combining photothermal agent (AuNSs) with AIEgen (berberine dimers BD3), then coated with HA to anchor tumor cell surface via CD44 receptors.[93] 4T1 cells incubated with ABH displayed more obvious yellow fluorescence compared with BD3, and a sharp decrease in fluorescence intensity was observed after preincubation with HA.The HAblocked fluorescence confirmed it was HA that improved the targeting ability through CD44 receptor-mediated endocytosis.In vivo experiments also showed satisfactory tumor growth inhibition in 4T1 tumor-bearing mice, achieving a synergistic therapeutic effect of PTT and PDT.
Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed on the surface of prostate cancer cells and recognized as a specific target for PSMA ligands.Urea-based PSMA ligands are small-molecule agents with strong affinity and rapid elimination, which are the most widely used in clinics. [94]PCP-2 was constructed by Gd-DTPA, AIEgen (TPE), and a urea-based PSMA ligand connected with a disulfide bond. [95]It could aim at the PSMA+ prostate cancer cells, respond to the intracellular GSH, and self-assemble into aggregates to achieve turn-on fluorescence imaging and MR imaging with high contrast.
T7 (sequenced HAIYPRH), another representative tumortargeting peptide, has a superior affinity to transferrin receptors (TfR) overexpressed on cancer cells.T7 is similar to transferrin (Tf) but its cellular uptake would not be inhibited by endogenous Tf. [96] PLA-PEG-T7/TMZ/TPE was reported as a hopeful platform for fluorescence imaging and drug delivery of nasopharyngeal cancer. [97]In vivo fluorescence imaging of PLA-PEG-T7-Cy5 and antitumor efficacy of PLA-PEG-T7/TMZ/TPE was much better than probes undecorated with T7, because T7 could penetrate the blood-brain barrier and aim at the brain tumors through TfR-mediated endocytosis.
Antibodies are widely explored in tumor-targeting applications, depending on the specific binding of tumor antigens and antibodies. [98]Her-2, one of the epidermal growth factor receptors family, is mainly overexpressed on the breast tumor cells and related to tumorigenesis. [66]AIEPS5-NPs-NB was synthesized by modification of the AIEPS5 with PEG chain and anti-Her-2 nanobody and applied for PDT of oral cancer in patient-derived tumor xenograft. [99]It was found that there was a high expression of Her-2 in one of the gingival cancer patients, and the tumor tissues were used for developing PDX models.AIEPS5-NPs-NB demonstrated superb far-red/ NIR imaging and PDT effect in PDX models, significantly better than those of AIEPS5-NPs.Additionally, programmed death-ligand 1 antibody (αPD-L1) is a novel checkpoint blockade inhibitor for immunotherapy and tumor targeting conjugating with PD-L1 overexpressed on tumor cells. [100]PD-L1@TPE-BT-BBTD NPs were reported with stronger NIR-II fluorescence imaging and more outstanding photoimmunotherapy ability compared with TPE-BT-BBTD NPs because of the tumor binding moiety of αPD-L1.[101] Interestingly, to expand the type of targeted tumor cells and not count on the existing cell surface receptors, artificial receptors were developed by bio-orthogonal reaction.[102] It was reported that the AIEgen (BCN-TPET-TEG) showed good dispersity and low fluorescence intensity in biological media.Once it arrived on the tumor cell membrane with azide expression introduced by metabolic engineering, bio-orthogonal reaction began between BCN and azide groups, thus achieving a high SNR for in vivo turn-on tumor fluorescence imaging.[103] Besides, a similar tactic was applied between AIE dots (DBCO-AIE dots) and artificially produced azide-modified sialic acids on the cancer cell membranes, which amplified the fluorescence signal.[104] Homologous targeting plays an important role in the tumor-targeted system, which deploys cell membranes or vesicles derived from cancer cells to coat the delivering molecules. [105]he high tumor-targeting specificity benefits from the cell adhesion molecules aberrantly expressed on cancer membranes, such as the cadherin superfamily and immunoglobulin superfamily.[106] It was reported that AIEgens could be enveloped into tumor cell-derived "exosome caps", [107] microvesicles, [108] or cell membranes [109] to induce homologous adhesion.All of them displayed high-efficiency tumor-targeting imaging or therapy based on various AIEgens.

Subcellular organelles location
Mitochondria are the most common target organelle in the design of cancer-targeting AIEgens, which is contributed to their more obvious negative membrane potentials.Lots of cationic AIEgens are investigated for mitochondria targeting owing to the electrostatic interactions between AIEgens and membrane. [110]Several moieties have been utilized in the AIEgens design to selectively target mitochondria, such as triphenylphosphine (TPP), pyridinium, isoquinolinium, and indolium. [111]TPP-TPEDCH was reported for intracellular mitochondrial fluorescence imaging and PDT, which could even trace the movement of mitochondria dynamically in living cells due to TPP moiety. [112]MeTTPy was developed with outstanding far-red/NIR fluorescence imaging and efficient ROS generation for PDT. [113]It was able to selectively target mitochondria in tumor cells, giving credit to its pyridinium group with positive charge.Under the guidance of isoquinolinium, TPE-IQ-2O was synthesized with the mitochondrial-targeted capability to differentiate tumor cells from normal cells, and it could kill cancer cells through PDT in retinoblastoma. [114]MeO-TPE-indo was designed to target mitochondria with the aid of indolium moiety, and obtained superb PDT effect based on the structure of D-π-A conjugate. [115]And it was further integrated with polydopamine nanoparticles to construct PDA-MeO-TPE-indo by π-π stacking and hydrogen bond, first combining AIEgens with polydopamine for image-guided synergistic efficacy of PDT and PTT.TPP group is also employed for lysosome binding, not only for mitochondria targeting.Red AIE conjugated polyelectrolytes were developed for fluorescence imaging-guided PDT of cancers, and TPP moiety linked on the side chains facilitated the targeting to lysosomes. [116]Additionally, lysosomes-targeting morpholine groups were used to synthesize three AIEgens (MPAT, MPAN, and MPAA), enabling them track lysosomes in living cells and zebrafish. [117]nd it was worth noting that the MPAT exhibited better photostability and higher contrast than the commercially LysoTracker.
The building blocks of TPA and thiophene are utilized to construct lipid droplets (LD)-targeted AIEgens. [118]A series of AIEgens (TPMN, TTMN, MeTTMN, and MeOTTMN) were simply synthesized via a one-pot method for LD targeting imaging in tumor cells and zebrafish, accompanied with producing 1 O 2 for precise PDT. [119]They made a clear visualization of LDs at low working concentrations, nearly equivalent to that of commercial BODIPY493/503 Green, and meanwhile displayed better photostability.
1,4-Dihydropyridine-based AIE probe (TPA-DHPy) was developed to imaging LDs and endoplasmic reticulum (ER) after transformation into pyridine-bearing TPA-Py. [120]TPA-DHPy was activated by white light and went through photo-oxidative dehydrogenation to form TPA-Py after endocytosis by cancer cells, making satisfactory retention in the lipid structure of LDs and ER.TPA-Py was not only capable to light up LDs and ER with self-monitoring the changes, but also induce cell apoptosis for excellent in vivo PDT efficacy.
Two AIEgens (TTFMN and TPE-TTMN-TPA) targeting towards nuclei were developed by Tang's group. [121,122]Both were designed mainly on the TPA as electron donor (D), furan or thiophene as D and π-bridge, carbon-carbon double bond as π-bridge, and two cyano units as electron acceptor (A), together with TPE for extending intermolecular distance and reducing π-π interaction.The two acid-activated AIEgens were applied and achieved great efficacy in nuclear targeting NIR fluorescence imaging-guided PDT for precise cancer treatment.
Golgi apparatus-targeted TPE-PyT-CPS was reported for stimulating oxidative stress and apoptosis by light irradiation in PDT of tumors. [123]Subcellular colocalization experiments verified that the TPE-PyT-CPS-targeted Golgi apparatus through the caveolin/raft mediated endocytosis.The Pearson correlation coefficient was as high as 0.98, which may be on the strength of molecular rod-like stacking induced by cyano moiety according to the structure-property relationship studies.

CANCER IMAGING BASED ON AIEGENS
AIEgens-based cancer imaging in the NIR region has significant advantages of deep penetration and high SNR in vivo due to the intrinsic features of AIEgens.And combining with other imaging modalities, the formed multimodal imaging agents can provide multidimensional information of cancers from different aspects (Figure 2).

NIR imaging based on AIEgens
Many AIEgens with NIR emission have been reported for in vivo cancer imaging with strong brightness, low autofluorescence, and good photostability.For example, BTPETQ had long-wavelength absorption and 700 nm-peak NIR emission that was employed for in vivo tumor vasculature imaging (Figure 3A). [124]BTPETQ dots were fabricated through nanoprecipitation by encapsulating hydrophobic BTPETQ molecules with amphiphilic DSPE-PEG copolymer and exhibited enhanced fluorescence signal in tumor vasculatures through leakage with high contrast even in the 900 μm depth of the tumor.The emission of BTPETQ could expand to the NIR-II wavelength region.Thus, the penetration and SNR of NIR-II imaging based on BTPETQ dots were further enhanced, leading to better imaging qualities than NIR-I imaging.NIR-II cancer imaging in vivo could be also accomplished by L897 NPs, which were designed and manufactured to extend the emission wavelength over 1200 nm.The L897 NPs showed a high QY of 5.8% and were employed as contrast agents for tumor imaging and image-guided surgery with the high SNR peaking at 9.0. [125]Moreover, much longer emission was reported by a series of Pdots based on SP with AIE structures with the QYs of up to 14% in the NIR-II region. [126]Among them, IR-TPE Pdots emitting longer than 1400 nm were further adopted for in vivo tumor imaging and mapping in mice models (Figure 3B).Among recent reports of AIEgens for NIR-II cancer imaging, XA1 NPs exhibited nearly the highest QY of 14.8%. [127]XA1 had a typical D-π-A-π-D structure with AIE characteristics and was encapsulated into F127 to prepare NIR-II nanoprobes.The formed NPs were used for high-resolution imaging of tumor blood vessels and precise detection of tumors with good contrast based on the EPR mechanism.NIR imaging by AIEgens can not only detect the tumor locations but also guide surgery of tumors.Highly photostable PTZ-TQ-AIE dots were prepared by nanoprecipitation using DSPE-PEG 3400 -NH 2 as the encapsulation matrix and showed strong NIR-II emission at 1250 nm which could extend to 1600 nm.Compared with indocyanine green used in the clinic practice, PTZ-TQ-AIE dots were adopted for NIR-II imaging-guided surgery of orthotopic tumors and exhibited tumor bioimaging with higher resolution and deeper penetration (Figure 3C). [128]

Fluorescence imaging and CT
Dual fluorescence imaging and CT have been reported in the applications of silver@AIEgen core-shell NPs (AAC-SNs).AACSNs were produced via a redox reaction by the synergistic formation of the silver core and the self-assembly of the redox-active AIEgens outsides.These NPs showed both enhanced fluorescence signal of the AIE moieties and distinctive plasmonic scattering of silver NPs for enhanced CT (Figure 4A). [129]In vivo studies proved their ability of high-quality fluorescence imaging and enhanced X-ray CT for multimodal imaging on 4T1 breast tumor-bearing mice.Moreover, gold NPs were also utilized to design CT contrast agents and further offset the low sensitivity of CT by combining with fluorescence imaging.M-NPAPF-Au coloaded with an AIEgen and gold NPs to form micelles was also applied for dual fluorescence/CT imaging. [50]It was prepared based on a one-pot ultrasonic emulsification method and could image tumors with specific tumor-targeting ability, enhanced fluorescence signal, and CT imaging.

Fluorescence imaging and MRI
A dual AIE/MRI agent called NGd was reported and the formed NGd-albumin aggregates (NGd-AAs) showed enhanced superior fluorescence QY and MRI contrast over NGd, as well as Gd-DOTA for clinical usage (Figure 4B). [130]hey were designed by utilizing AAs to enhance both the fluorescence intensity of AIE and the MRI contrast, which contained the prolonged rotational correlation time of Gd(III) chelates and the diffusion correlation time of surrounding water molecules.In vivo study proved that NGd-AAs were capable of eliciting high-performance dual-modal imaging of fluorescence imaging and MRI.And it could be observed that high contrast MR signals were in the liver and tumor sites after intravenous administration.Another common MRI agent, superparamagnetic iron oxide (SPIO) was reported to be combined with TB, a fluorescent dye with AIE features and NIR emission, to form high-quality multimodal imaging agents named TSP NPs. [131]The NPs were fabricated by a one-pot self-assembly method via using PS-PEG to encapsulate TB and SPIO to afford nanoparticles with surface hydroxyl groups for improved biocompatibility and possessed good superparamagnetism and relaxivity used as novel magnetic contrast agents for MRI and magnetic particle imaging (MPI).In dual-modal tumor imaging, these NPs were capable of monitoring the growth of subcutaneous tumor over 24 days for real time.Moreover, the TSP NPs could also monitor a liver tumor in situ with high spatiotemporal resolution and nearly no limitation of penetration by multimodal imaging consisting of fluorescence imaging, MRI, and MPI.

Fluorescence imaging and PA imaging
As a burgeoning imaging modality, PA imaging attracted the inspiring interest of investigators in cancer imaging.An AIEgen with tailored donor-acceptor structure, DTPA-TBZ was proposed with NIR-II fluorescence that exhibited intense fluorescence signals in the NIR-II region and PA signals in the NIR-I region. [132]DTPA-TBZ showed a significant QY of 8.98% in the solid state and the formed AIE dots were prepared via a nanoprecipitation strategy by encapsulating DTPA-TBZ into FA-DSPE-PEG 2000 to further enhance its water dispersity and biocompatibility.The dots exhibited a high QY of up to 11.1%.These features of the dots contributed to good performance in both fluorescence imaging and PA imaging which visualized tumors clearly in vivo.Others like DTPA-BBTD dots could also be capable of NIR-II fluorescence imaging and NIR-I PAI for in vivo visualization of tumors on mice models, as well as in the report of DPBTA-DPTQ NPs. [133,134]PA-TQ3 NPs were prepared via nanoprecipitation by adopting DSPE-PEG 2000 as the encapsulation matrix and TPE-TQ3 self-assembled in the core.The formed NPs with typical AIE features showed high thermal-to-acoustic conversion efficiency due to intense intramolecular motions. [135]nd the strong fluorescence signal and PA signal of TPA-TQ3 NPs were capable of precisely locating orthotopic breast tumors and tiny residual tumors with high SNR.And in vivo dual-modality imaging (fluorescent/PA imaging) was also realized by H10@FSH dots. [136]The H10 was an AIE dye with NIR-II emission and the formed H10@FSH dots were prepared by encapsulating H10 with DSPE-mPEG 3400 -FSH as the matrix with ovarian-specific targeting ability.The dots showed superior optical properties and PA signals, as well as enhanced ovarian-specific targeting ability to achieve dual-mode imaging and image-guided surgery in xenograft tumor model and tiny metastatic lesions of ovarian cancer in the abdomen.Moreover, orthotopic brain tumors under intact skull and scalp could also be detected and imaged by fluorescent/PA imaging (Figure 4C). [35]TB1 dots were prepared via nanoprecipitation with DSPE-PEG 2000 matrix and showed a high QY of 6.2% and an emission maximum near 1000 nm.After being decorated with c-RGD, the dots with AIE features exhibited high tumor uptake with good specificity and selectivity, showing a high SNR of 4.4 and resolution of up to 38 μm, realizing precise orthotopic brain tumor diagnosis through the intact scalp and scalp by dual NIR-II/PA imaging.
Based on traditional PA techniques, multispectral optoacoustic tomography (MSOT) is a functional one that has been combined with NIR-I fluorescence imaging in the report of NP-Q-NO 2 . [137]NP-Q-NO 2 was prepared by dispersing DMSO solution of Q-NO 2 into pH 7.4 PBS, composed of a dihydroxanthene moiety as the donor, quinolinium as the acceptor, and nitrobenzyloxydiphenylamino as the recognition element.It could specifically respond to nitroreductase and then be transformed into a D-π-A structure with NIR emission extending over 900 nm.This probe exhibited enhanced NIR emission by AIE and strong PA signal that successfully detect and image orthotopic breast tumors and the metastases in both the lymph nodes and the lung using different breast cancer mouse models.

Other imaging modalities based on AIEgens
Other imaging technologies except for fluorescence also have been realized by AIEgens for cancer imaging and detection.NIR chemiluminescence (CL) emission investigated in biomedical applications of AIEgens has been achieved by TBL dots (Figure 5A). [138]TBL was synthesized through conjugation of a luminol unit with benzothiadiazole as the acceptor and TPA as the donor.And the corresponding TBL dots were prepared by modifying TBL with F127 on the surface.The CL emission of these dots could last continuously beyond 60 min and be employed for quantitative and qualitative detection of 1 O 2 .And the TBL dots were proved to image deep tissues over 3 cm with good penetration by NIR CL which was better than NIR fluorescence imaging and blue CL imaging.Moreover, guided by TBL-based CL imaging, it could successfully differentiate tumor and normal tissues in vivo with good SNR.Another report for CL was QM-B-CF [139] which obtained an analyte-triggered accumulation of stable prechemilluminophore with AIE features.It exhibited a rapid photooxidation process (1,2-dioxetane generation) through TICT-based free-radical addition which achieved an enhanced CL signal with high brightness.And it was capable of accurately sensing and tracing biomolecules with high resolution, dual-mode imaging of CL and fluorescence imaging, and 3D visualization in animal models.
Afterglow luminogens based on semiconducting polymer (SP) have been proved with an increasing potential of in vivo imaging benefiting from their luminescence with long lifetime and the relevant advantages including no background and high SNR.The AIE-featured SP lumino-gens (P-TNPs) [140] for afterglow imaging were reported to enhance afterglow intensity and prolong afterglow duration by AIE characteristics of TTMN, an AIE dye (Figure 5B).P-TNPs were prepared by coencapsulating PFPV and TTMN with F127.TTMN could provide sufficient 1 O 2 to excite SPs (PFPV) and form large amounts of intermediates with high energy.PFPV intermediated emitted photons that could activate TTMN to generate 1 O 2 which simultaneously trigger the energy transfer process between PFPV and TTMN.It resulted in a deep-red emission and showed good performance of tumor imaging in vivo.

CANCER THERANOSTICS BASED ON AIEGENS
Various AIE NPs used for in vivo cancer theranostics have been widely investigated and reported, containing phototherapy (PDT and PTT) of the intrinsic molecular properties and combined therapies of the carried chemicals/drugs (Figure 6).

PDT for cancer theranostics
PDT based on photosensitizers (PSs) can produce a high concentration of ROS to induce cancer cell death with good therapeutic effects and low side effects and overcome traditional drug resistance of cancer therapy.Benefiting from the advantages of AIEgens, PSs with AIE features have been widely explored for image-guided PDT of cancer in vivo.
As a common AIE structure, TPE has been adopted to design PSs for PDT.A typical TPE-based AIE PS, TPE-IQ-2O, was mentioned above and employed for photodynamic ophthalmic therapy for retinoblastoma. [60]TPE-IQ-2O was an efficient mitochondria-targeting agent for PDT against cancer cells.It could inhibit the expression of inflammatory factors and thus efficiently reduce tissue inflammation in mice models of retinoblastoma with good biocompatibility.The TPE-IQ-2O maximized the damage to cancer with high specificity and selectivity and minimized side effects on normal tissue in the process of PDT for cancer and suppression of metastasis for a long time.With the same TPE core, TPE-Py-OH was another TPE derivative PS with AIE features. [141]t was designed to target LD and mitochondria and had long-term intracellular retention over one week.After multiple irradiations, the TPE-Py-OH could significantly ablate the tumors with sustained PDT effect for hepatocellular carcinoma in mice models.With mitochondria-targeted ability and TPE core, TPE-4QL + was reported with highly efficient photosensitivity and high tumor cell specificity. [142]Under a low dose of radiation, TPE-4QL + could realize the high efficiency of PDT on the 4T1-xenograft mouse model.Besides, not targeting mitochondria, TPE-MEM was reported to target cell membrane that also showed efficient PDT for cancer in vivo. [143]TPE-MEM exhibited high fluorescence yield, good biocompatibility, and specific cell membrane-targeted ability.After irradiation by natural light with low energy, it could destroy the cell membrane and then leading to cell necrosis.In vivo study proved that the PDT effect of TPE-MEM could efficiently inhibit tumor growth in different mice models.Without TPE structure, other AIE PSs like TTB were also reported with strong fluorescence and efficient PDT for cancers.TTB showed enhanced NIR emission and generation of ROS under white light irradiation. [144]After being encapsulated into the polymeric matrix (MDP) and modified with RGD-4R peptide to target tumors, the formed RGD-4R-MPD/TTB NPs showed NIR emission peaked at 730 nm, high photostability, and low dark cytotoxicity.When investigated in tumor models in vivo, the NPs selectively accumulated in tumor sites for real-time and long-term imaging.Upon laser irradiation, the growth of multiple xenograft tumors including cervical, prostate, and ovarian cancers was efficiently inhibited by RGD-4R-MPD/TTB NPs-based PDT treatment (Figure 7).TBMPEI with AIE features showed strong NIR fluorescence and excellent ROS generation capability with cellular membranes-targeted ability like TPE-MEM. [145]It could light up and ablate cancer cells through necroptosis causing the rupture of cell membrane and DNA degradation by light irradiation, which endowed TBMPEI with high performance for imaging-guided PDT in vitro and in vivo.
Mitochondria targeting DCQu was reported to efficiently generate 1 O 2 more than commercial PSs, and the corresponding PDT excited by two-photon laser could selectively distinguish cancer cells from normal ones without the help of any other targeting moieties. [146]With lysosome-targeted ability, TBTDC NPs were reported based on an AIE-active Schiff base TBTDC with D-A-π-A skeleton and encapsulated by Pluronic F-127. [147]The TBTDC NPs showed bright NIR emission at 825 nm and imaged tissue with deep penetration over 300 um, high 1 O 2 generation, lysosometargeting capability, good biocompatibility and photostability.In vivo study proved that the NPs had remarkable cytotoxicity for cancer cells and efficiently inhibited tumor growth in tumor-bearing mice models through ROS generation upon irradiation of white light.Besides, quinoxalinone CN (QCN) was also reported as a good AIE PS.It was synthesized through optimization of the quinoxalinone scaffold which could exhibit effective 1 O 2 generation under 530 nmwavelength laser irradiation and AIE characteristics in the NIR region. [148]By encapsulating with DSPE-PEGs, the formed NPs exhibited 1 O 2 generation with high efficiency and NIR fluorescence (800 nm) with almost no dark toxicity, which contributed to the promising applications for image-guided PDT of tumors in vivo.

Strategies to enhance PDT for cancer theranostics
Increasing EPR capability, active targeting ability and other strategies have been mentioned before to improve targeting ability.Other strategies in designing AIE PS in molecular structures and conjugated process are also essential to develop ideal AIE PSs from different perspectives.
The polymerization [149][150][151] of small molecule AIEgens has been reported as an efficient strategy to enhance photosensitization and thus improve the PDT effect on tumor treatment.Compared with the small molecular counterparts, conjugated polymers could exhibit much higher 1 O 2 generation efficiency. [149]The polymer PSs showed an enhanced intersystem crossing efficiency and were used as PSs for image-guided PDT of cancer.Another conjugated polymer PSs with AIE characteristics also showed good performance for cancer cell ablation by two-photon excited PDT, as well as in zebrafish liver cancer treatment. [150]Moreover, intracellular polymerization can further promote the accumulation and retention of NPs in the targeted cells that significantly enhances the imaging and theranostics for cancers.Guided by this strategy, an AIEgen mediated by cathepsin protease reaction was reported for prolonged imaging and enhanced tumor growth inhibition. [151]It was composed of an AIEgenpeptide conjugate (D2P1) and cyanobenzothiazole-cysteine through a rapid condensation reaction.When the cathepsin protease reaction happened in the tumor, condensate polymerization was induced by the cleavage of peptides between the exposed cysteine and 2-cyanobenzothiazole on cyanobenzothiazole-cysteine.It triggered the accumulation of D2P1 into the tumor and made fluorescence light-up which could remarkably enhance the PDT effect of the tumor in mice models upon light irradiation.
A cationization molecular engineering strategy was also adopted in designing AIE PSs with enhanced both 1 O 2 and radical generation to promote the efficiency of PDT.For instance, cationization of DTPAN and DTPAPy could convert their neutral D-A type to A-D-A typed cationic counterparts. [152]It revealed that such cationization strategy could not only enhance the AIE characteristics of molecules and promote their intersystem crossing processes but also increase the capability of charge transfer and separation.All these properties above worked synergistically to enhance the efficiency of ROS generation, especially hydroxyl and superoxide radicals in the aggregated state.And these cationic PSs with AIE features possessed mitochondria-targeting ability in cancer cells and performed high PDT efficacy in vitro and in vivo.The similar strategy was also utilized in the report of TPE-TeV-PPh 3 , which exhibited effective PDT toward solid tumors as type-I PSs. [153]ransition metal complexes can highly generate 1 O 2 that make them effective PSs.A series of Ir(III) complexes with AIE characteristics containing a different number of Ir centers were reported to enhance PDT by increasing 1 O 2 generation ability benefiting from transition metal complexes. [154]These NPs were prepared by using the iridium complexes as the core, biocompatible DSPE-PEG-MAL as the encapsulation matrix, and the HIV-I transactivator as the surface functionalization group.And the formed AIE NPs showed high 1 O 2 generation positively related to the number of Ir centers.Compared with the pure Ir(III) complexes, the AIE NPs were endowed with brighter emission, longer lifetime, higher generation of 1 O 2 , and superior cellular uptake.The AIE NPs of trinuclear Ir(III) complexes efficiently achieved cytotoxicity to cancer cells and remarkable suppression of tumor growth.Except Ir(III) complexes, transition metal complexes like Cu(II) were also utilized to promote the PDT effect of AIE PSs.MOF-199 was a typical Cu(II) carboxylate-based metal-organic framework. [155]As an inert carrier, MOF-199 could prohibit photosensitization during PSs delivery.The Cu(II) in the MOF-199 could effectively decrease intercellular endogenous GSH and simultaneously induce the release of encapsulated PSs which recovered ROS generation.And MOF-199 was further employed to ablate cancer cell with high efficiency and inhibit tumor growth by PDT with almost no side effects.To further minimize the side effects of AIE PSs, TPE-PHO was constructed through electrostatic complexation of pyridium-functionalized TPE and water-soluble calixarene. [156]This cascaded substation-activated phototheranostics using the host-guest strategy could dramatically inhibit the dark cytotoxicity of TPE-PHO and achieve cytoplasm-mitochondria translocation to display the PDT effect upon light irradiation.It was also reported with significant inhibition by PDT on tumor models in vivo.This enzyme-mediated intracellular polymerization system was also proved with the advantages of enhancing the accumu-lation of theranostic agents in tumors and thus achieving effective cancer imaging and therapy with minimized dosage and negligible damage to normal tissues.
Although PDT is considered an effective treatment for tumors, hypoxia in the tumor microenvironment is an obstructive problem in the PDT effect.To overcome the hypoxia in solid tumors, it is essential to develop highly efficient type-I PSs with less oxygen consumption and unveil their structure-function relationship.Thus, AQPO and AQPI were reported with AIE features and boosted ROS generation through reducing singlet-triplet splitting and further fabricated via a facile nanoprecipitation method by using DSPE-PEG 2000 for assembly. [157]After being incorporated with electron-rich anisole, the production of AIE PSs were changed from 1 O 2 to superoxide anion radical and hydroxyl radical.Compared with the AQPI NPs without anisole, the AQPO NPs showed a remarkable increase (3.2-and 2.9-fold, respectively) in the superoxide anion radical and hydroxyl radical generation efficiencies, whereas the 1 O 2 generation was much lower (0.4-fold).The small singlet-triplet splitting and anisole group endowed AQPO with capacity for highly efficient type-I ROS generation.Moreover, AQPO NPs were proved to exhibit the hypoxia-overcoming PDT effect through efficient elimination of tumor cells and good biocompatibility upon white light irradiation both in vitro and in vivo.Another report of both alleviating hypoxia and promoting PDT effect introduced a multistage drug delivery system. [158]Herein, the system was composed of TPE-Py, Rubioncolin C, an oxidative phosphorylation blocker, and an NF-κB inhibitor.It efficiently suppressed O 2 consumption of oxidative phosphorylation and thus relieved hypoxia.And in vitro and in vivo experiments confirmed the promoted PDT efficiency in triple-negative breast cancer models.Moreover, this system with Rubioncolin C and NF-κB inhibitors synergistically induced a burst of ROS and enhanced ROS-mediated apoptosis, as well as inhibiting the NF-κB signaling pathway.Another facile strategy to convert type I PSs from type II PSs to enhance PDT effect was reported as acceptor planarization and donor rotation. [159]he acceptor planarization enforced intramolecular charge transfer that redshifted NIR absorption and transferred the type of PSs by photochemical pathways.2TPAVDPP, TPAT-PEVDPP, and 2TPEVDPP with a different number of rotors were prepared guided by this strategy which exhibited good performance as type I PSs.Among them, 2TPEVDPP had the most rotors and was further encapsulated with DSPE-PEG 2000 to enhance water solubility and biocompatibility.The formed NPs realized NIR fluorescence imaging-guided PDT in mice models.

PTT for cancer theranostics
PTT to convert NIR light into the heat for cancer treatment has been widely investigated, as a minimally invasive strategy due to its favorable biosafety.Except AIEgens reported in vitro, AIEgens used for in vivo PTT should be focused with more attention from researchers.Small molecules like DTPA-BBTD with strong fluorescence signal in the NIR-II region were prepared for PTT on tumor therapy. [134]The DTPA-BBTD had the D-A-D structure with AIE features and exhibited high absorption in the NIR-I region and intense brightness in the NIR-II region, and showed 1.51% photoluminescence QY using IR26 as the reference.Due to the efficient TICT characteristics and loose molecular packing when aggregated, DTPA-BBTD was endowed with efficient photothermal conversion efficiency (PCE).The DTPA-BBTD-based AIE dots were prepared via the nanoprecipitation strategy by using FA-DSPE-PEG 2000 to modify DTPA-BBTD.In vivo study proved that these AIE dots could realize NIR-II fluorescence imaging and NIR-I PAI to visualize the tumors in mice models, and further hinder tumor growth through PTT under irradiation.NIR-II imaging-guided PTT was also implemented by BPBBT NPs in orthotopic and metastatic tumors (Figure 8A). [160]This lipophilic BPBBT possessed both TICT and AIE features.After combing with human serum albumin, the planarity of BPBBT was changed and the intramolecular rotation was restricted, which further tailored the fluorescence and photothermal efficiency.The BPBBT NPs were prepared using a modified nab-technology and could clearly delineate primary orthotopic tumors in mice models, as well as tiny metastatic lesions of 0.5 mm × 0.3 mm.And guided by intraoperative NIR-II fluorescence, these NPs could provide efficient photothermal ablation for cancer treatment.Another single molecular AIEgen reported as an efficient photothermal platform was BPN-BBTD. [161]After being encapsulated with amphiphilic polymers, the BPN-BBTD NPs realized NIR-II fluorescence imaging and efficient PTT for bladder tumors in tumor-bearing mice models.Both subcutaneous tumor and orthotopic tumors were both eradicated and inhibited under the 785 nm laser irradiation.Moreover, the BPN-BBTD NPs could monitor both subcutaneous and orthotopic tumors for 32 days by NIR-II fluorescence.
AIEgen-based PTT has been adopted to overcome the most malignant brain tumor, glioblastoma.NK@AIEdots were developed for PTT of glioblastoma in the brain, with ideal through-skull imaging and therapy. [162]The NK@AIEdots were designed by coating NK cell membrane on the PBPTV, a NIR-II conjugated polymer with AIE features.The formed NK@AIEdots possessed an intense NIR-I brightness (7.9% QY) and good biocompatibility.And these dots were capable of crossing the blood-brain barrier by triggering a signaling cascade that disrupted tight junction and reorganized actin cytoskeleton to form an intercellular channel.In vivo study showed that NK@AIEdots could remarkably suppress tumor growth upon NIR light irradiation.BK@AIE NPs could also induce PTT of glioblastoma (Figure 8B). [74]BK@AIE NPs were prepared via a nanoprecipitation method using DSPE-PEG 2000 as the doping matrix and fabricated with BK ligand on the surface.The BK ligand could prompt adenosine receptor activation on the blood-tumor barrier that enhanced transportation and accumulation into tumors.The NPs showed high PCE upon 980 nm NIR laser irradiation that facilitated glioblastoma treatment in the deep location of brain.In vivo studies proved that BK@AIE NPs could efficiently inhibit tumor progression after spatiotemporal PTT and the survival span of mice was significantly extended.As for the further mechanism of BK@AIE NPs PTT, it activated NK cells, CD3 + cells, CD8 + cells, and M1 macrophages in the tumor lesion to increase the immune response and thus therapeutic efficacy.

Strategies to enhance PTT for cancer theranostics
To enhance the PTT effect, relatively higher photothermal efficiency by increasing nonradiative decay is the main approach in designing ideal AIEgens for PTT on tumor therapy.The strategies of acceptor planarization and donor rotation mentioned above efficiently enhanced not only the radical generation of PDT but also the PCE of PTT. [159]Besides, stabilizing dark TICT state or restricting radiative decay could also help construct a photothermal conversion-boosted NIR-II theranostics. [163]A series of AIEgens (NIRb14, NIRb10, NIRb6, and NIR6) with different substituent groups were synthesized with typical TICT and AIE properties that had bulky alkyl chains in the planar D-A-D skeleton and molecular rotors as the branches.Among them, NIRb14 displayed the highest PCE due to its larger bulky chains for shielding that restricted intramolecular interactions and motions when aggregated.And NIRb14 was further used to form NPs by integration with PAE-b-PCL and PEG-b-PCL.After being injected into the 4T1 tumor-bearing mouse model, the formed NIRb14 PAE/PEG-NP showed good PA imaging capabilities and PTT performance for in vivo PA imaging-guided PTT ability, in which the injected mice also showed thermal imaging with significant temperature variation in the tumor after irradiation.And the in vivo anticancer efficacy proved that NIRb14-PAE/PEG NPs could significantly inhibit tumor growth and even decrease the tumor volume after laser irradiation.
Dual PTT could obtain a better therapeutic effect than single PTT with higher PCE in the report of DTPR. [164]DTPR was reported for image-guided dual PTT for optimized cancer therapy upon 808 nm laser irradiation.It was constructed through coencapsulating TPA-BDTO and PDPPP by DSPE-PEG 2000 -Mal and then conjugated RGD by click reaction.TPA-BDTO could emit NIR fluorescence which was partially converted into thermal energy through fluorescence resonance energy transfer between TPA-BDTO and PDPPP.After coupling with the original heat energy generated by PDPPP itself, image-guided dual PTT was implemented.The PCE of DTPR reached 60.3% by dual PTT which was much higher than its inherent single PTT of only 31.5%.Moreover, it was further proved that DTPR caused more severe photothermal ablation both in vitro and in vivo.
More than one targets can also realize higher efficiency of PTT for cancers.Au-Apt-TPE@Zn showed dual-targeted ability synthesized by conjugating bare gold nanoprisms to the functionalized TPE and further stabilized with aptamers for targeting via Au-S bonds, and then chelated with Zn 2+ ions. [73]The zinc conjunction brought cell membranetargeted ability to Au-Apt-TPE@Zn that could selectively recognize apoptotic cells at the early stage for monitoring therapeutic efficacy.And the AS1411 DNA aptamer decorated on the surface could realize specifically nuclear-targeted ability.Upon NIR irradiation, Au-Apt-TPE@Zn exhibited highly efficient PTT against cancer cells in vitro, which induced apoptosis by triggering ROS generation and regulating multiple signal crosstalk.In vivo studies further proved that Au-Apt-TPE@Zn could exhibit strong PTT against gastric carcinoma xenograft growth with deep penetration and negligible side effects guided by NIR-II fluorescence imaging and PAI.
TA B L E 2 Comparisons of advantages and disadvantages between AIEgen-based nanoparticle drug delivery system and other conventional nanoparticle drug delivery systems.

Drug delivery system Advantages Disadvantages
AIEgen

Drug delivery for cancer theranostics
AIEgen-based drug delivery system integrates luminescent AIEgens and therapeutic drugs, realizing a fluorescence imaging-guided drug delivery for cancer theranostics and monitoring the drug kinetics with high SNR in comparison with other delivery systems.Comparing AIEgen-based nanoparticle delivery system and other conventional nanoparticle delivery systems, there are several advantages and disadvantages between them (Table 2).

AIEgens as the drug delivery carrier
AIEgens, as hydrophobic molecules, can themselves be employed as carrier for hydrophobic drug due to their hydrophobic interaction. [39]n AIEgen PS and an acidic pH-activatable DOX were linked by a caspase-3-responsive peptide to prepare a prodrug AIE-Pep-DOX. [165]AIE-Pep-DOX was a hydrophilic molecule when circulating in the physiological environment.While in acidic tumor microenvironment, the engineered prodrug released DOX responsively, further activating the caspase-3 for triggering the release and aggregation of AIEgens.The aggregated AIEgens enhanced the tumor retention for long-term fluorescence imaging and repeatable PDT.And the tumor-detained PDT induced immunogenic cell death of triple-negative breast cancer cells and accelerated the maturation of DCs, achieving a chemo-PDT boosted immunotherapy in vitro and in vivo.Moreover, AIEgenbased gene carrier was fabricated on the basis of large π-conjugation TPA derivatives modified with two polar [28] aneN3-triazole heads and long hydrophobic tail. [166]The transfection efficiency of this gene vector was more than six times higher than that of commercial transfection agent Lipo-fectamine 2000 in HEK293T cell lines.AIEgen-based gene carrier showed bright NIR fluorescence signal for depicting transfection process in vitro and visualizing tumor in vivo, and collaborative under the laser irradiation.At the same time, it generated sufficient 1 O 2 for PDT under laser irradiation, collaborated with gene therapy for cancer treatment in vivo.

AIE self-assembled as the drug delivery carrier
AIE polymer can self-assemble into drug delivery nanoparticles with great biocompatible, which is composed of a hydrophobic core of AIEgens and a hydrophilic shell of polymer.And it is often designed with stimulus-triggered linker to enhance the drug delivery efficiency to tumors.
The anticancer drug, DOX, was loaded into a TBP-based delivery system with the self-indicating ability. [167]The TBP had H 2 O 2 -responsive amphiphilic polymers with H 2 O 2sensitive phenylboronic and PEG moieties.And it could self-assemble into micelles with an encapsulation efficiency of 74.9% for DOX.And nearly 80% DOX would be released from TBP@DOX micelles benefiting from the decomposition of TBP by oxidation, hydrolysis, and rearrangement.In vivo experiments proved that TBP@DOX micelles could accumulate in the tumor of mice models and remarkably inhibit tumor growth.Combining Pt(IV) prodrug and DOX, dual-drug therapeutic AIE NPs (PtAIECP@DOX NPs) were self-assembled through copolymerizing Pt(IV) prodrug and an AIEgen embedded in the main chain of PtAIECP and subsequently encapsulated with DOX. [168]The PtAIECP@DOX NPs were capable of monitoring the light-activation of Pt(IV) prodrug and the intracellular DOX release by "turnon" fluorescence.Moreover, these NPs could maximize therapeutic efficiency by the spatiotemporal control of light irradiation.

Organic-inorganic AIE nanocarrier for drug delivery
AIEgen-functionalized organic-inorganic hybrid nanoparticles were also used as drug delivery carriers, due to their outstanding biocompatibility, strong fluorescence and easy surface functionalization. [39]IE-based organosilica nanoparticles (AIE-ONs) was fabricated through a facile bottom-up coassembled strategy, harnessing an amphiphilic AIEgen (MeOTTVP) as the directing template.[169] AIE-ONs not only possessed the inherent unique functions of AIE PS, but also demonstrated outstanding drug loading ability.After assembly of DOX and HA, the hybrid AIE-ONs demonstrated a combined effect of PDT and chemotherapy for tumors in vivo, monitored by NIR fluorescence imaging.Also, AIE-ONs could load antibiotic rifampicin efficiently for killing microbes, manifesting their versatility as drug carrier.Ag@AIE core@shell nanocarriers with tunable diameter and morphology was developed to deliver siRNA for cancer therapeutics.[170] The as-prepared nanocarrier with a high siRNA efficiency exhibited prominent anticancer efficacy in vivo with negligible toxicity.More importantly, the inherent properties of AIE made them achieve a real-time intracellular tracking and in vivo tumor imaging, prospective for RNA interference-related therapeutic applications.
AIEgen-based drug delivery system with NIR fluorescence can trace and monitor the systemic distribution and tumor distribution using in vivo fluorescence imaging.However, they also accumulated in liver and kidney for clearance of the AIE NPs.Delivery efficiency to tumor and the accumulation and retention of AIEgens in tumor always depend on their physicochemical structures, targeting ability, biocompatibility, in vivo circulation, and so on, which follow the same principles as other drug delivery systems.And the off-target effect of AIEgens-based system is similar with other nanoparticle delivery system.After injected and circulating in the blood, the formation of protein corona on their surface blocks a certain number of NPs to their intended target, making undesired interactions with off-target cells. [171]Some receptors are not merely expressed in the cancer cells, but also in the normal cells, which reduces the specificity and increases the side effect. [172]And the affinity of ligands and receptors are not very high, making insufficient uptake into target cells. [173]

PDT-PTT for cancer theranostics
Attributed to the hypoxic microenvironment in the tumor for PDT and the heat shock effect of PTT, single treatment for cancer in vivo via PDT or PTT cannot always achieve satisfactory efficacy.Besides enhancing PDT through improving ROS generation and PTT through increasing PCE, combining PDT and PTT to realize synergistic phototherapy has the potential to effectively enhance the tumor treatment effect.
A TPE-based AIEgen named Meo-TPE-indo (MTi) targeting mitochondria was reported to have a typical D-π-A conjugated structure for image-guided PDT and PTT in tumor treatment. [115]After being modified with PDA NPs on the surface by π-π and hydrogen interactions, the formed PMTi exhibited lasting retention in the tumor with good PCE.Combining MTi and PDA NPs realized mitochondria-targeting PDT-PTT in vivo, while single MTi could just ablate cancer cells in vitro, not in vivo.And HJS and DHJS were also designed and synthesized for mitochondria-targeting PDT-PTT of tumors in vivo. [174]Interestingly, these two AIEgens showed dual-channel fluorescence imaging (red and NIR) targeting mitochondria and could decrease mitochondria membrane potentials and induce apoptosis for tumor growth inhibition.
Multimodal phototherapy was also achieved by TPA-BTZ, which showed NIR-I emission at 800 nm and could generate ROS via mechanisms of both type I and type II for enhanced PDT upon irradiation. [175]TPA-BTZ had the propeller-like TPA rotators that facilitated heat generation for PTT by intramolecular rotation.After being encapsulated with DSPE-mPEG 2000 via a nanoprecipitation method, the intermolecular spatial isolation created by alkyl chainsin the formed TPA-BTZ@PEG 2000 NPs allowed a sufficient intramolecular motion for PCE.In vivo studies proved these formed NPs had long-time NIR-imaging capability, good antitumor efficacy, and efficient suppression of tumor growth in the PDT-PTT synergistic therapy.
Metal materials have been also utilized to synthesize PDT-PTT AIEgens for tumor treatment.AuNSs-BD3@HA (ABH) was introduced to exhibit efficient PDT-PTT capability. [93]he decoration of HA endowed ABH with CD44-targeting ability that improved the tumor-targeting specificity.The ABH showed a significant therapeutic effect on breast cancer by PDT and PTT from BD3 and AuNSs, respectively.After laser irradiation, it further induced remarkable cancer apoptosis and necrosis both in vitro and in vivo.Another report used two metals, Pt and Ag, to design efficient synergistic phototherapeutic materials.Pt 1 Ag 28 @ACD made oil-soluble bimetallic nanoclusters with precise structure for nicely enhanced PDT-PTT on the tumor in vivo. [176]After loaded by amphiphilic chitosan derivate micelles via hydrophobic interaction, the formed nanoclusters could efficiently accumulate in the tumor area by passive targeted delivery and negative charge of cancer cells.Another Pt-based AIEgen, C-DTPP was constructed as a prism-like metallacage with intense NIR-II fluorescence emission by assembly of fourarm ligand with Pt(Pet 3 ) 2 (OTf) 2 . [177]The C-DTTP exhibited the maximum emission wavelength at 1005 nm and both a high PCE of 39.3% and efficiency of ROS generation.
Additionally, PDT-PTT synergistic therapy was utilized as a compensated alternative for tumor resection.An AIE NIR-II fluorophore (A1) was reported with D1-π-A-D2-π-R structure and the corresponding A1 NPs were prepared by encapsulating with PEG-PPG-PEG via a nanoprecipitation method, showing high absolute QY of 1.23%, excellent PCE of 55.3% and moderate ROS generation efficiency. [178]uided by NIR-II imaging based on A1 NPs, 2 mm-diameter tumors and metastatic lymph nodes could be precisely resected and other invisible lesions to the naked eye could be ablated by PDT-PTT.

Multiple therapies for cancer theranostics
Many different therapies like chemotherapy, radiotherapy, gene therapy, and immunotherapy have been widely explored and successfully proved with remarkable therapeutic effects for cancer in the past several decades.Considering the advantages of phototherapy, multiple therapies will realize higher therapeutic effects and lower side effects than single ones.

Combining chemotherapy and phototherapy
Chemotherapy is conventional cancer therapy that adopting chemical drugs to kill cancer cells and has been widely utilized for combining therapy in cancer.TACQ was a typical AIE-active probe that combined chemotherapeutic effect with phototherapy for cancer in vivo. [179]The TACQ had the emission extended into the NIR-II region benefiting from the strengthened push-pull interaction, and exhibited high PCE (55%) and efficient generation of ROS.After selectively accumulating in cancer cells and targeting mitochondria, TACQ could induce mitophagy and block mitophagic flux for cancer cell apoptosis to perform an efficient therapeutic effect which combined chemo-photothermal and PDT guided by NIR fluorescence and photothermal imaging (Figure 9A).
BITT was another one designed for photoenhanced cancer chemotherapy. [180]After added into BSA solution in PBS buffer, BITT could induce the self-assembly of BSA to form nanoparticles (BITT@BSA-DSP NPs) without precipitation.The BITT@BSA-DSP NPs with multiple functions were used as nanocarriers based on albumin that were loaded with cisplatin(IV) prodrug to exhibit intense NIR fluorescence imaging ability and enhanced therapeutic efficacy by other PDT and PTT.It was proved that BITT@BSA-DSP NPs could be taken up with high efficiency by cancer cells and release Pt(II) reacted with reductase for chemotherapy.Moreover, in vitro and in vivo studies demonstrated that it efficiently promoted the sensitivity of bladder cancer to cisplatin chemotherapy with negligible side effects.
Resistance of metallodrug in the cancer treatment has attracted much attention.To overcome Pt resistance, two mitochondria-targeted AIEgens, DP-PPh3 and TPE-PPh3 were synthesized with good abilities of overcoming the Pt resistance in the treatment of lung cancer and exhibited good anticancer efficacy both in vitro and in vivo. [181]These two AIEgens could alter drug metabolism by upregulating influx and downregulating efflux, as well as block autophagic flux by failing to degrade autophagosomes.Moreover, these two AIEgens could also promote ROS generation to disrupt the mitochondria and impair glycolytic metabolism.

Combining radiotherapy and phototherapy
Radiotherapy uses radiation to eliminate and eradicate the primary or metastatic lesions of local tumors.Although there are fewer reports in combining with phototherapy during the development of AIE materials, it is still essential to be regarded as an alternative therapeutic approach in multiple theranostics, which focuses more on precise spatial localization of tumor lesions.Gold nanoparticles have been used as radiosensitizers for boosting the efficacy of radiotherapy that remarkably reduce the given dose of X-ray and potential side effects.For instance, AIE-Au was reported with good capabilities of efficient X-ray-induced PDT with low dose and negligible side effects (Figure 9B). [182]The glutathioneprotected gold clusters were assembled through a cationic polymer to enhance the luminescence signal excited by Xray.And the therapeutic mechanism of AIE-Au was that it could absorb X-rays and then generate much hydroxyl radicals with high efficiency upon low-dose X-ray irradiation to promote the radiotherapy effect.Moreover, the conjugated PSs in AIE-Au could be excited by X-ray-induced luminescence to facilitate additional PDT effect.Both in vitro and in vivo studies confirmed that AIE-Au could effectively trigger ROS generation with remarkable reduction of X-ray dose and high anticancer efficacy.

Combing gene therapy and phototherapy
Gene therapy is recently developed based on the delivery of therapeutic genes into the target cells through vectors.Different from conventional vectors in gene therapy, such as virus and plasmid, delivery by AIE nanomaterials can enhance the therapeutic effect for cancer with good biocompatibility.Gene therapy was also adopted to be combined with PDT to improve the therapeutic effect.MnO 2 -DNAzyme-TB nanocomposite (MDT) was reported to employ GSH-responsive MnO 2 to deliver both TB and DNAzyme for cancer imaging and PDT-gene combining therapy. [183]In MDT, TB was adopted for PDT and DNAzyme was employed for gene silencing by catalyzing EGR-1 mRNA degradation.Thus, MDT could efficiently reduce the expression of EGR-1 and thus inhibit cancer cell growth that simultaneously suppress the growth by ROS generation from the aggregated TB.Moreover, under illumination, MDT could effectively inhibit the growth of tumor in MCF-7 tumor-bearing mice model by combining PDT and gene-silencing therapy.The combination of PDT and gene therapy was also reported in the applications of four large-π-conjugation TPA derivates with multiple functions of gene delivery, NIR fluorescence imaging, and combined PDT and gene therapy for cancers. [166]These AIEgens possessed typical NIR AIE properties, large Stokes shift, intense two-photon-excited fluorescence signal, and superior DNA condensation abilities.Among them, vector 4, was proven to successfully trace the transfection process and image tumor in vivo with long-term retention, high resolution, strong brightness, deep tissue penetration, and good biocompatibility.The DNA complex formed by vector 4 could efficiently produce 1 O 2 for effective PDT, and combined with anticancer gene therapy, achieving a dramatically enhanced anticancer effect.

Combining immunotherapy and phototherapy
Cancer immunotherapy is a powerful strategy to manipulate the immune system to recognize and attack cancer cells, including checkpoint inhibitors and adoptive cell therapy. [184]Cs are specialized antigen-presenting cells with essential roles in the initiation and regulation of innate and adaptive immune responses.The maturation of DCs occurs within tumors but transports tumor antigens to draining lymph nodes and cross-present antigens to activate cytotoxic T lymphocytes. [185]A biomimetic AIE PS (DC@AIEdots) was reported with antigen-presenting and hitchhiking abilities coated by the membranes of DCs on the AIE NPs (Figure 9C). [186]The AIE molecules in the inner cavity could selectively accumulate in the LD, and DC membrane outsides could facilitate the delivery of DC@AIEdots by T cells and enhance the accumulation in the tumor.In vivo studies demonstrated that DC@AIEdots could stimulate the proliferation and activation of T cells and then trigger the immune response for cancer treatment.The same strategy to endow NPs with DC membrane was adopted in the reports of DC@BPBBT dots and saDC@Fs-NPs. [86,187] membrane-targeted PS (TBD-3C) could trigger cancer immunotherapy via PDT that stimulated polarization of macrophages, caused maturation of DCs, and activated CD8 + cytotoxic T-lymphocytes.It not only inhibited primary cancer growth but attacked the distant metastatic tumor. [188]Cs maturation was also induced by TP-IS1@M1, an AIE PS to induce photodynamic immunotherapy for cancer with good hypoxia-tolerance.It was demonstrated to cause an effective immune response by releasing damage-associated molecular patterns, maturating DCs, and vanishing the distant tumors. [189]Benefiting from the phototherapy from the AIE parts, synergistic photodynamic immunotherapy showed the great potential for AIEgen-based cancer therapy with efficient activation of DCs and further stimulation of cytotoxic T lymphocytes. [187,190,191]mmune checkpoint inhibitors can enhance immunotherapy efficacy by inducing cytotoxic T lymphocytes to kill cancer cells, especially anticytotoxic T lymphocyteassociated protein 4 and antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1). [192]PMRA was reported to increase the response rate of immune checkpoint blockade by a cascade amplification with the immune adjuvant. [193]It was composed of (D)PPA-1 peptide, an immune checkpoint inhibitor, an MMP-2-responsive sequence and PyTPA.As an AIE PS, PyTPA could promote the release of tumor-associated antigens and primed T cells.After the stimulation of PDT and the immune adjuvant, the induced cytokines could promote the activation of T cells and further the migration and infiltration of immune cells into the tumor.Thus, through immune checkpoint blockade with (D)PPA-1 peptide, T cells efficiently enhanced the recognition and elimination of cancer cells.Moreover, immunogenic cell death reinforcing the release of tumor-associated antigens that completed a cycle could further achieve an almost 100% objective response rate in the animal models.Combining immune checkpoint inhibitors for immunotherapy can also regulate immune system.SPSS NPs were reported with surface-mimicking protein secondary structure for self-synergistic cancer immunotherapy through the combination of immunogenic cell death and immune checkpoint blockade therapy (Figure 10A). [194]They exhibited superior photodynamic properties and the induction of immunogenic cell death.Moreover, peptide antagonists were self-assembled on the surface of NPs as efficient lysosometargeting moieties to mediate the degradation of PD-L1.And in vivo experiments demonstrated that SPSS NPs could elicit superior anticancer immunity to inhibit both primary and metastatic tumors, as well as evoke long-term immune memory against tumor recurrence.Anti-PD-1 was used in the report of a biomimetic immune metabolic nanoplatform designed by encapsulating type I AIE PS and a glutamine antagonist into cancer cell membranes for cancer-targeting delivery in vivo. [195]It fully satisfied the glucose and glutamine consumption of T cells, obviously improved the hypoxia, enabled the metabolism reprogramming of tumor and immune cells, induced immunogenic cell death, promoted DC maturation, and effectively suppressed tumor growth.And by reducing immunosuppressive cells, it could trigger strong tumor-specific immune responses and modulate the tumor immune-suppressing microenvironment.And combining anti-PD-1, it could prevent tumor metastasis and form long-term immune memory against tumor recurrence.
TB/PTX@RTK with the light-triggered drug release capability was designed by loading TB and PTX for synergistic chemo-PDT by inducing immunogenic cell death and eliciting immune response (Figure 10B). [196]The TB/PTX@RTK micelles were prepared by dialysis method using cRGD-PEG-TK-PLGA and PEG-TK-PLGA as carries material.The formed micelles could selectively accumulate in the tumor by cRGD-mediated active targeting and promote image-guided PDT to ablate cancer cells.Upon light irradiation, the TB could generate massive ROS for PDT, and the generated ROS could cleave the thioketal linker to control the precise release of PTX in the targeted cancer cells.It synergistically inhibited tumor growth, induced immunogenic cell death, and elicited the immune response for anticancer.Moreover, it could significantly upregulate the PD-L1 expression on the cell membranes of tumor for immune modulation.

CONCLUSION AND PERSPECTIVES
The concept of AIE has become a highly promising linkage, theoretically and practically, between chemical/materials science and medicine, especially for exploring new avenues in the field of cancer investigations and treatment.Due to the highly emissive feature from intramolecularly restricted states of aggregated AIEgens, the corresponding NPs exhibit bright fluorescence, as well as excellent ROS generation and high PCE for in vivo cancer phototherapy guided by AIEgenbased molecular imaging.Moreover, upon combining with other imaging agents and therapeutic drugs, the resultant multifunctional NPs show better performance in molecular imaging and theranostics for cancer.The structure of AIEgens directly influence the properties.[199] The aspects like π-conjugation, conformation, and packing, influence the light-emitting behaviors of AIEgens by adjusting planarity and rotatability, intramolecular restrictions, intermolecular interactions, and even ACQ-to-AIE transformation.The structure of AIEgens, including the molecular skeleton, substituents, and functional groups, plays a significant role in determining their optical properties.For example, the rigidity of the molecular skeleton, the type and position of substituents, and the strength and direction of intermolecular interactions can all affect the efficiency and wavelength of fluorescence emission, as well as the stability and sensitivity of AIEgens to environmental factors such as temperature, pH, and polarity. [200]Moreover, physical state and morphology of AIEgens can also influence the optical properties, in which the size and shape of aggregates, the degree of molecular packing, and the presence of intermolecular interactions can all affect the efficiency and intensity of fluorescence emission, as well as the performance in further biomedical applications. [201]or the further development of AIE materials in cancer studies and clinical translation to realize multimodal imaging,  [194].Copyright © 2022 Wiley-VCH GmbH.(B) Schematic illustration of TB/PTX@RTK micelles synthesis and effects of chemo-PDT combined with anti-PD-L1 (G1, PBS; G2, anti-PD-L1; G3, TB/PTX@RTK + L; G4, TB/PTX@RTK + L + anti-PD-L1).Primary and distant tumor images in the different groups after the indicated treatments.Representative plots of flow cytometric analysis and statistical analysis of the infiltration of CTLs (CD45 + CD3 + CD8 + ) and Treg cells (CD3 + CD4 + Foxp3 + ) in the distant tumors.Adapted permission from Ref. [196].Copyright © 2021.The Author(s).
combining the advantages of AIEgens and clinical imaging modalities have broad prospects.Clinical imaging modalities contain ultrasound, CT, MRI, SPECT, and PET, as well as fluorescence imaging like using indocyanine green in surgery.Imaging modalities such as ultrasound, CT, and MRI, may have some limitations that are not appropriate for clinical translation.For instance, ultrasound is always limited by penetration for cancer detection and disturbed by the bone, gastrointestinal tract, and lung.CT has large exposure of radiation, especially using agents to enhance contrast, and the imaging information focuses on the structure of tissues and organs.MRI have many contrast agents in both preclinical and clinical use and can get both structural and functional imaging information, but the lack the precise binding pathways and targeting specificity of MRI agents.Additionally, the contrast agents of CT and MRI may cause anaphylaxis.PET molecular imaging should be considered to combine with AIEgens to achieve multimodal imaging of cancer in vivo and promote clinical translation.It has the advantages of various imaging agents with excellent binding specificity that can precisely target different proteins, receptors, and other molecules in the pathophysiological processes of cancer, guided by a new theory, that is, transpathology. [202]Unlike other imaging agents, PET molecular imaging agents can be produced by radiolabeling AIEgens with different radionuclides without forming nanoparticles, especially 11 C and 18 F labeling by nucleophilic substitution reaction and electrophilic substitution reaction. [31]This process has not significantly changed the molecular and spatial structure that minimizes the influence of AIE features, guarantees the original biodistribution, accumulation, and metabolism of the precursors, and further supports subsequent fabrication.Considering the achievement of AIEgen-based fluorescence imaging and advantages of PET agents, dual AIE/PET imaging modality agents are necessary to be developed for in vivo cancer imaging and theranostics.It has been already reported that some studies designed and synthesized AIE/PET imaging agents through 11 C-and 18 F-radiolabeling of AIE core, [203] as well as other radionuclides labeling like 68 Ga chelation. [204]However, these reports were limited in vitro or by poor PET imaging quality that lacked the potential for in vivo investigations.Moreover, after combined with other radionuclides like 131 I, the formed agents can also realize therapeutic effects.However, there remains some limitations.The AIE feature needs specific structures to induce restriction of intramolecular motions when aggregated, but it is usually difficult to realize direct substitution reaction of radionuclides like 11 C and 18 F on these structures.Other radionuclides like 68 Ga have simplified process of radiolabeling but should introduce coordination compound on the AIE structure that may significantly change the molecular structure and influence the biodistribution and metabolism when applied in vivo.Benefiting from the rapid development in aromatic ring radiofluorination, [191,205,206] such as the development of iodoethylidene precursors, phenylboronic acid pinacol precursors, and copper-based catalysis recent years, the difficulty of using 18 F for radioactive labeling of AIE has decreased.For in vivo applications, AIE/PET lacks precise structure imaging information compared with CT and MRI, which can be solved by simultaneous scanning on PET/CT or PET/MR to easily get complementary structure imaging.It still has great potential to develop dual AIE/PET agents for in vivo molecular imaging and theranostics for both materials design and biomedical applications.
Different from common programmed cell death like apoptosis and autophagy, new cell death patterns including ferrotopsis, [207] cuproptosis, [208] and pyroptosis [209] have been recently investigated for cancer treatment.Ferroptosis is an iron-dependent and ROS-reliant cell death induced by the loss of selective permeability of the plasma membrane due to intense membrane lipid peroxidation and the occurrence of oxidative stress. [207]The ROS generation of AIEgen-based PDT and the generated photothermal effect of AIEgens can enhance the efficacy of ferroptosis inducers and also directly expedite ferroptosis to promote therapeutic function for cancer. [210,211]And ferroptosis can conversely enhance the efficacy of phototherapy based on AIEgens. [212][215][216] Cuproptosis is a copper-dependent death that occurs employing direct binding of copper to lipoylated components of the tricarboxylic acid cycle resulting in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss and further leading to proteotoxic stress and ultimately cell death. [217] cuproptosis-based synergistic cancer therapy, the depletion of glucose and glutathione sensitizes cancer cells to cuproptosis by producing aggregation of lipoylated mitochondrial proteins, and also enhance the PDT efficacy due to the oxidation of glucose. [218]It can also effectively kill cancer cells and inhibit tumor growth with high selectivity and cytotoxicity that combining cuproptosis and PTT for synergistic therapy. [219]AIEgens have been confirmed with excellent performance in both fluorescence imaging and phototherapy that can not only realize synergistic therapy when inducing cuproptosis but visualize and monitor the whole process of cuproptosis-based cancer therapy.Pyroptosis is a lytic and inflammatory form of cell death leading to the cleavage of gasdermin D and activation of inactive cytokines like IL-18 and IL-1β. [209]The specific cancer targeting ability and selective phototherapy of AIE NPs promote photo-activated pyroptosis to become the dominant cell death pathway and realize light-controlled antitumor immunity and solid tumor immunotherapy aroused by cell pyroptosis. [188,220]Therefore, new cell death patterns provide a promising perspective for potential synergistic cancer therapy, and inspire the design of advanced AIEgen-based nanoplatforms.And AIEgens can also help investigate the unclear process and mechanism of these cell death patterns in cancer.
Metastatic cancer remains an obstruction in cancer therapy that impairs prognosis, lifespan, and clinical decision.Many reports confirmed the good performance of AIEgen-based NPs applied in metastatic cancer imaging and theranostics.It is essential to image metastatic cancer foci as tiny as possible before tumor surgery.BPBBT mentioned above could image tiny metastatic lesions of 0.5 mm × 0.3 mm with high SNR and guide the further resection surgery under intraoperative NIR-II fluorescence. [160]The abdominal metastatic foci of ovarian cancer could be facilely eliminated through resection surgery guided by NIR-II fluorescence imaging and further NIR-II fluorescence imaging and PAI. [136,221]And distant metastatic cancer like pulmonary metastasis could be also detected by AIEgen-based fluorescence and luminescence imaging. [222,223]Lymph nodes play important roles in tumor grading and staging, and lymph node metastasis is always the first event in cancer metastasis.As mentioned, A1 NPs could precisely detect metastatic lymph nodes by NIR-II fluorescence imaging, achieving complete tumor eradication with no local recurrence and metastasis after surgery. [178]etastases from the orthotopic breast tumors to lymph nodes and then to lung could be also detected and imaged by an AIEgen for NIR-I/NIR-II fluorescence and MSOT imaging with intense signal, high SNR, and deep penetration. [137]In the synergistic cancer therapy, the growth of metastatic cancer could be obviously suppressed in different cancer models, that showed great potential in the treatment of metastatic cancer.Some studies used AIEgen-labeled cancer cells for metastatic cancer, which are meaningful to explore and monitor their biological behaviors. [224]To extend applications of AIEgens in cancer, increasing accumulation and retention of AIEgens in primary cancer cells can help investigate and monitor the whole process and mechanism of cancer metastasis from the primary tumor to lymph nodes and even to distant organs, as well as achieving metastatic cancer theranostics.
There are no AIEgens and AIEgen-based nanomaterials for cancer imaging and therapy that have yet progressed to clinical trials.However, the development of AIE materials for clinical translation is a promising area of research, and it is possible that clinical trials will be initiated in the future.The main hurdles in the preclinical study and clinical translation of AIE materials are their limited stability and biocompatibility.AIE materials can be sensitive to environmental factors such as temperature, pH, and light, which can affect their stability and performance over time.In addition, AIE materials need to be biocompatible and nontoxic to be used in vivo, which requires rigorous testing in preclinical studies.Compared with conventional delivery carriers, AIE materials can be more expensive to produce and less stable over time.[227] However, AIE materials have the advantage of high fluorescence efficiency and can be designed to target specific cancer cells, which make them more effective for imaging and therapy in cancer.Additionally, the synthesis and manufacturing of AIEgen-based nanomaterials can be more complex and costly than conventional carriers, which may limit their scalability and cost-effectiveness.Moreover, it is essential to testing biocompatibility, toxicity, and efficacy in the evaluation of AIE materials in further clinical studies, which can assess whether the AIE materials elicit an immune response or cause toxicity, the effects on healthy tissues and organs, and the ability of cancer detection and treatment in the human body.Several reports have successfully applied AIEgen-based materials in the detection and diagnosis of clinical samples, including diagnosis of human papillomavirus infection and screening of human germ cell quality. [228,229]n the field of AIEgen-based in vivo cancer imaging and theranostics, some challenges must be addressed.Tumor heterogeneity is a major challenge for in vivo cancer imaging and theranostics, as cancer cells can differ in their genetic, epigenetic, and phenotypic characteristics, even within the same tumor. [230]Common targeting strategies endow AIE materials with specific targeting ability but may limit their recognition of tumor heterogeneity.Tumor microenvironment is highly complex and dynamic that affects AIEgen targeting, uptake, and response at different times.And AIEgens cannot efficiently aggregate in the nonsolid tumors like leukemia, which weakens the fluorescence emission and further imaging and theranostics, as well as influenced by complex localization of nonsolid tumors.The imaging depth and resolution of tumor in the deep site is still a challenge without invasive operations due to the limited penetration depth of light, although many efforts have been made to solve it. [138,231]Additionally, others like clearance, toxicity, stability, side effects, and cost, should be also fully considered when applying AIEgens for in vivo imaging and theranostics in cancer.Moreover, the developing future of AIEgens in the field of cancer should not be limited by the reports mentioned above.New AIE core structures with multiple and synergistic responsive functions, such as thermal, sound, electric, magnetic, mechanical, photo-radiation of various frequencies, nanotribological, chemical and biological stimuli/reagents, nanoenvironment of cancer cells, and so on, should be continuously encouraged to explore.New fabricating and targeting strategies, imaging modalities, and therapeutic approaches should be promoted to the final goal of clinical translation and effective treatment of cancers.

A C K N O W L E D G M E N T S
Abdominal vessels, hind limb vasculature, cerebral vessels, and tumor NIR-II imaging and NIR-NIR imaging and MSOT imaging on orthotopic tumor, to lymph nodes and then to lung metastatic tumor on 4T1 breast tumor

F I G U R E 1
In vivo cancer-targeting strategies of AIE NPs contain four parts: first, they pass through the blood-tumor barrier (BTB) to transport into tumors by enhanced permeability and retention (EPR) effect or endothelial transcytosis; then, they are activated in the tumor extracellular microenvironment; or they interact with the noncancer cells (stromal cells) or cancer cells and internalized by active targeting; finally, they are effectively localized on the subcellular organelles due to specific ligands.

F I G U R E 2
Near-infrared (NIR) fluorescence imaging (FLI) is the most common modality for cancer imaging in vivo by AIE NPs.And combined with other imaging agents including CT, MRI, PET, and PAI, multimodal imaging of cancer can be realized by AIE NPs.

F I G U R E 3
In vivo near-infrared fluorescence imaging based on AIEgens for cancers.(A) Chemical structure of BTPETQ, schematic illustration of the BTPETQ dots synthesis, 3D reconstructed images of tumor at different time points after injection, the overlay 2PF image of tumor blood vessels labeled by LuminiCell Tracker 540 nm at 24 h postinjection and the 3D 2PF images of BTPETQ dots accumulated in tumor under NIR-I and NIR-II excitation.Adapted permission from Ref. [124].Copyright © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.(B) Whole-body imaging of 4T1 tumors in living mice intravenously injected by IR-TPE Pdots in prone position at certain time intervals from 2 to 8 h, the enlarged view of the area around the spinal cord in the blue square at 10 min and 8 h postinjection of IR-TPE Pdots compared with 1 min of ICG, and reconstructed 3D mapping of whole-body mouse at 6 h postinjection of IR-TPE Pdots.Adapted permission from Ref. [126].Copyright © 2021 Wiley-VCH GmbH.(C) Representative in vivo long-term NIR-II fluorescence images of the orthotopic liver cancer at different time points after tail vein injection of the PTZ-TQ-AIE dots, the pictures of liver and tumor, and the NIR-II and bioluminescence imaging in surgery.Adapted permission from Ref. [128].Copyright © 2021, The Author(s).

F I G U R E 4
In vivo multimodal imaging based on AIEgens for cancers.(A) FL images and CT images of 4T1 tumor-bearing nude mice at different time points after intratumoral injection of AACSNs.The tumor site is highlighted with a red dotted circle.Adapted permission from Ref. [129].Copyright © 2018, American Chemical Society.(B) Schematic illustration of the synthesis of NGd-Albumin aggregates (NGd-AAs) and the mechanism of synergistic FLI/MRI enhancement, and dual-modal in vivo and ex vivo T1-weighted MRI and FLI and analysis of mice after intravenous injection of NGd-AAs or Gd-DOTA.Adapted permission from Ref. [130].Copyright © 2021, American Chemical Society.(C) Synthetic route of TB1-RGD dots, noninvasive NIR-II fluorescence imaging (1000 LP, 100 ms) results of mouse brain through intact scalp and skull under 808 illumination (60 mW cm −2 ) with TB1-RGD dots and TB1, and noninvasive PA imaging of orthotopic brain tumor through intact scalp and skull at different time points upon intravenous administration (0.5 mg kg −1 ) with TB1 dots and TB1-RGD dots.Adapted permission from Ref. [35].Copyright © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

F I G U R E 5
In vivo chemiluminescence imaging and afterglow imaging based on AIEgens for cancers.(A) The proposed CL generation mechanism of TBL oxidized by 1 O 2 , schematic illustration of the preparation of TBL dots and the generation of CL, in vivo images in hair-shaved mice after subcutaneous injection of TBL dots with H 2 O 2 and NaClO for 5, 10, 30, and 60 min, and in vivo CL images and SNR ratio of normal tissue and tumor area after injection of TBL dots.Adapted permission from Ref. [138].Copyright © 2020 Wiley-VCH GmbH.B) The mechanism of amplified afterglow luminescence of P-TNPs, and fluorescence images and afterglow images of 4T1 tumor-bearing mice at corresponding periods after intravenous injection of PNPs and P-TNPs.Adapted permission from Ref. [140].Copyright © The Royal Society of Chemistry 2020.F I G U R E 6 Cancer theranostics based on AIE NPs mainly contain phototherapy (PDT and PTT), AIE NPs-guided drug delivery, PDT-PTT synergistic therapy and combining phototherapy with other therapies (chemotherapy, radiotherapy, gene therapy, and immunotherapy).

F I G U R E 7
In vivo image-guided photodynamic therapy based on AIEgens for cancers.Chemical structure of TTB, MPD, RGD-4R, schematic illustration of the preparation of RGD-4R-MPD/TTB NPs, in vivo fluorescence images of PC3 tumor-bearing mice after intratumoral administration of Nile red, TTB, RGD-MPD/TTB NPs, and RGD-4R-MPD/TTB NPs for designated time intervals, and RGD-4R-MPD/TTB NPs mediated PDT for multiple xenograft tumors of HeLa, PC3, and SKOV3.Adapted permission from Ref. [144].Copyright © 2019 American Chemical Society.

F I G U R E 8
In vivo image-guided photothermal therapy based on AIEgens for cancers.(A) MR images of BK@AIE NPs for mice brains in different groups, in vivo fluorescence imaging in mice at different times after AIE NPs and BK@AIE NPs injections, quantitative fluorescence analysis of the tumor site at different time points after AIE NPs and BK@AIE NPs treatment, and infrared thermographic maps of tumors of mice measured 5 min after continuous laser irradiation and representative photos of U87-MG-glioma-bearing mice treated with PBS, AIE NPs, and BK@AIE NPs followed by NIR-II laser irradiation.Adapted permission from Ref.[74].Copyright © 2019, The Author(s).(B) Infrared thermal images before (0 min) and at the end of PTT of the tumor-bearing mice at 30 h following tail vein injection with BPBBT NPs or PBS.And in vivo bioluminescence imaging of BALB/c mice bearing orthotopic CT26 colon cancer before or after PBS or BPBBT NPs treatment.Adapted permission from Ref.[160].Copyright © 2021 Wiley-VCH GmbH.

F I G U R E 9
In vivo image-guided combined therapies of phototherapy and chemo-/radio-immuno-therapy based on AIEgens for cancers.(A) Photothermal images and heating temperatures at the tumor sites and tumor growth curves, and photos of the tumors harvested at day 21 after different treatments.Adapted permission from Ref. [179].Copyright © 2021 American Chemical Society.(B) In vivo fluorescent images and CT imaging of tumor-bearing mice at 2, 4, 8, 12, and 24 h postinjection of R-AIE-Au, and in vivo antitumor treatment with tumor growth curves and representative images of different groups of tumor-bearing mice after various treatments.Adapted permission from Ref. [182].Copyright © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.(C) In vivo and ex vivo distribution of DC@AIEdots and in vivo antitumor effect of DC@AIE dots.Adapted permission from Ref. [186].Copyright © 2021 Wiley-VCH GmbH.

F I G U R E 1 0
In vivo image-guided combined therapies based on AIEgens for cancers.(A) Chemical structures of D F D FG D PPA peptide and D PPA peptide and schematic illustration of the preparation procedure of SP3 NPs-D F D FG D PPA and the proposed mechanism of SPSS NPs-mediated lysosomal degradation of PD-L1.Adapted permission from Ref.
This work was funded by the National Natural Science Foundation of China (21788102, 32027802), Key R&D Program of Zhejiang (2022C03071), and the Fundamental Research Funds for the Central Universities.C O N F L I C T O F I N T E R E S T S TAT E M E N T The authors declare no conflict of interest.O R C I D Peili Cen https://orcid.org/0000-0003-1497-2209Hong Zhang https://orcid.org/0000-0002-1892-3335Mei Tian https://orcid.org/0000-0002-1587-2114R E F E R E N C E S How to cite this article: P. Cen, J. Huang, C. Jin, J. Wang, Y. Wei, H. Zhang, M. Tian, Aggregate 2023, 4, e352.https://doi.org/10.1002/agt2.352A U T H O R B I O G R A P H I E S Peili Cen received his bachelor degree from Zhejiang University in 2019 and is currently pursuing PhD, under the supervision of Prof. Hong Zhang and Prof. Mei Tian, in Zhejiang University School of Medicine.His research work focuses on the development and applications of multimodal molecular imaging and theranostics based on aggregation-induced emission materials and PET agents.