Emerging Applications of Aggregation‐Induced Emission Luminogens in Bacterial Biofilm Imaging and Antibiofilm Theranostics

Threats posed by recalcitrant bacterial biofilms have been continuously challenging the public health due to the dramatically magnified antibiotic resistance resulted from the complicated biofilm microenvironment. Urgent demands on effective biofilm combating have propelled the rapid exploration of high‐performance antibiofilm systems. Thanks to the distinguished features of aggregation‐intensified fluorescence and aggregation‐enhanced generation of reactive oxygen species, aggregation‐induced emission luminogens (AIEgens) are becoming increasingly eye‐catching in the field of biofilm combating by serving as excellent fluorescence imaging probes or theranostic agents. This review aims to, for the first time, outline the current progress of AIEgens in bacterial biofilm imaging and antibiofilm theranostics. The up‐to‐date advancements of AIEgens in enzyme‐responsive biofilm imaging, discriminative imaging of Gram‐positive bacterial biofilm, as well as biofilm viability monitoring are summarized at first. Subsequently, the antiadhesion intervention‐mediated prevention of biofilm formation and photodynamic therapy‐involved eradication of preexisting biofilms are detailedly elucidated. Finally, a brief conclusion as well as a discussion on the current challenges and future expectations is presented.


Introduction
Here is an inescapable fact that is worthy to be reiterated, pathogenic bacterial infection is still seriously threatening the public health and has been regarded as one of the major health security challenges worldwide due to its high infectivity and mortality. [1,2] The statistics of the World Health Organization (WHO) manifested that bacterial infection caused the highest mortality in less developed countries over the last 15 years. [3] Despite the antibiotics, we termed "magic bullet", being discovered in the 20th century, the fast-development antimicrobial resistance induced by the abuse or overprescription of antibiotics during the harsh treatments has made the battle against pathogenic bacteria more and more troublesome. [4,5] In addition to the acquisition of genetic mutations to develop drug-resistant bacterial strain or species, forming a stubborn fortress known as biofilm to defend attacks from antibiotics and immune systems is another important trick that most sly pathogenic bacteria frequently utilize to survive. [6][7][8] Bacterial biofilms are multicellular 3D assemblages with diverse surface-attached bacterial cells encased by the self-secreted extracellular polymeric substances (EPS), and it can completely establish after going through four main stages in terms of initial adhesion, early biofilm formation, biofilm maturation, and dispersal. [9,10] As an emergent form of bacterial life, the obstinate biofilms are extensively existed and have significant impacts on a range of application scenarios, for instance, it has been estimated that up to an approximated 80% of all microbial infections are associated with the formation of disturbing biofilms in the realm of healthcare. [11,12] Shielded by the EPS, the bacteria swathed in the biofilm inherently exhibit more resistance (up to 1000 fold) to the antibiotic agents in comparison with their independent, free-swimming planktonic counterparts, making it exceptionally difficult to eradicate or cure a bacterial biofilm infection once it formed. [6,13,14] This extremely high tolerance is attributable to not only the weakened antibiotics penetration restricted by the EPS but also the low bacterial growth rate caused by the oxygen-lacking and nutrient-deprived microenvironment within biofilm. As most of the antibiotics are designed DOI: 10.1002/sstr.202200329 Threats posed by recalcitrant bacterial biofilms have been continuously challenging the public health due to the dramatically magnified antibiotic resistance resulted from the complicated biofilm microenvironment. Urgent demands on effective biofilm combating have propelled the rapid exploration of highperformance antibiofilm systems. Thanks to the distinguished features of aggregation-intensified fluorescence and aggregation-enhanced generation of reactive oxygen species, aggregation-induced emission luminogens (AIEgens) are becoming increasingly eye-catching in the field of biofilm combating by serving as excellent fluorescence imaging probes or theranostic agents. This review aims to, for the first time, outline the current progress of AIEgens in bacterial biofilm imaging and antibiofilm theranostics. The up-to-date advancements of AIEgens in enzyme-responsive biofilm imaging, discriminative imaging of Gram-positive bacterial biofilm, as well as biofilm viability monitoring are summarized at first. Subsequently, the antiadhesion intervention-mediated prevention of biofilm formation and photodynamic therapy-involved eradication of preexisting biofilms are detailedly elucidated. Finally, a brief conclusion as well as a discussion on the current challenges and future expectations is presented.
to target the rapidly replicating bacteria, the low bacterial growth rate would inevitably lead to the antibiotic failure. [15][16][17] This actuality, together with the increasingly growing formation of stubborn biofilms in clinics, jointly exacerbates the health risk of bacterial biofilm infections, which is doomed to impose a substantial economic and social burden. [6,[18][19][20] Accordingly, treatment of biofilm infections becomes therefore utmost significant, yet rather challenging in the bumpy road of combating pathogens.
To tackle this dilemma, lots of endeavors have been devoted to seeking after alternatively spiffy antibacterial strategies with distinct inhibition mechanisms. Thereinto, photodynamic therapy (PDT), as an important category of phototherapy, has attracted special attention with promising progress. [21][22][23][24] PDT exploits the inherent destruction of highly toxic reactive oxygen species (ROS) produced by photosensitizers (PSs) to trigger bacteria injury, biofilm degradation, and probably immunity response simultaneously. [25][26][27] As ROS can directly attack bacteria aggressively, even though the PSs just anchor on the bacterial envelop and could not completely enter the bacteria, the generation of specific resistant strains could be significantly held back. [28] Apart from that, PDT has also been proved to possess the prominent advantages of minimal invasiveness, favorable spatiotemporal controllability, and negligible systemic toxicity in comparison with traditional antibiotics. [29][30][31][32][33] Aiming to obtain desired photodynamic antibacterial efficacy, the exploration of high-performance PSs has been intensively propelled. Benefitting from the unparalleled fluorescence properties and the consequent excellent performances in fluorescence imaging (FLI)-guided PDT, the emergence of PSs with aggregation-induced emission (AIE) property made the development of organic PSs enter upon a new phase by enabling the concomitant diagnostic FLI. [34][35][36] To be specific, AIE refers to a unique phenomenon that some conformation-twisted fluorophores emit immensely enhanced fluorescence in aggregate state than that of their dissolved molecule state owing to the mechanism of restriction of motion (RIM) (Figure 1a). [28,[37][38][39] After aggregating, the excited energy of AIE luminogens (AIEgens), which originally consumed by the active intramolecular motions in solution, is switched to undergo radiative consumption pathway because the intramolecular motions-involved nonradiative thermal deactivation is largely restrained, thus elucidating the aggregation-intensified emission (Figure 1b). [28] Apart from the radiative decay, the ROS generation-involved intersystem crossing (ISC) from the excited singlet state to the excited triplet state can also be promoted due to the suppression of nonradiative thermal dissipation in the aggregate state. [28] Beyond that, the energy splitting is inclined to occur upon aggregates formation, and the much denser singlet and triplet states can cause more energy levels overlapping, thus producing more available channels with diminished energy gaps for ISC (known as aggregationinduced intersystem crossing [AI-ISC]) and therefore facilitating the yield of excited triplet state as well as the subsequent generation of cytotoxic ROS (Figure 1b). [28,40,41] Compared with the traditional PSs that encounter considerably diminished fluorescence and ROS generation caused by detrimental π-π stacking at aggregate state, these distinct advantages of aggregationenhanced emission and aggregation-induced generation of ROS (AIG-ROS) definitely empower AIEgens with great superiorities in diagnostic biofilm detection and FLI-guided PDT for bacterial biofilm theranostics.
Profiting from the bloom developments of AIE PSs as well as their intrinsic merits of satisfactory biocompatibility, facile Figure 1. Schematic illustration of AIE phenomenon and aggregation-induced superiorities of AIEgens. a) The fluorescence turn-on phenomenon of the typical AIE luminogen, tetraphenylethene (TPE), from solution state to aggregate state. Reproduced with permission. [28] Copyright 2020, Wiley-VCH GmbH. b) Jablonski diagrammatic explanation of the aggregation-induced superiorities of AIEgens in terms of aggregation-enhanced emission and aggregation-enhanced generation of ROS. The width of the lines indicates the energy strength.
processability, easy functionalization, high fluorescence quantum efficiency in aggregates, large Stokes shift, and outperformed ROS-generated features, significant breakthroughs and advancements of AIEgens in pathogenic bacteria combating have been witnessed in the last few years. [42][43][44][45][46][47][48][49] Although there have related reviews summarizing the corresponding progress in terms of AIEgen-involved antimicrobial application ever been reported, they generally focused on the planktonic bacteria rather than bacterial biofilms. [50][51][52][53] In view of the increasing developments of AIEgen-based biofilm imaging and antibiofilm theranostics, [54] herein, we intend to give a systematical overview specifically emphasizing this promising research field for the first time. The breakthroughs of AIEgens in bacterial biofilm detection and differentiation, as well as biofilm forming inhibition and eradication during the last 5 years are highlighted in this review. For the former part, the up-to-date signs of progress in biofilm imaging are primarily elaborated; thereinto, enzymeresponsive biofilm imaging, Gram-positive bacterial biofilm discriminative imaging, and biofilm viability monitoring are introduced successively. For the latter part, comprehensive AIEgen-based antibiofilm theranostics, including imagingguided antiadhesion therapy, imaging-guided PDT, as well as imaging-guided combination therapy based on PDT, are showcased in detail. In the end, limitations and challenges that exist in current works are briefly discussed, and perspectives for further exploration in the future are given. We are in hope that this mini-review will provide valuable insights into AIEgen-based bacterial biofilm imaging, inhibition, and eradication and offer an inspiration for devising integrated multifunctional systems, thereby fostering more meaningful studies in this research frontier.

Bacterial Biofilm Imaging
The battle to fight against the enemy that lurks in dark is vague and inefficient. Similar predicament occurs in the antibiofilm combat. Ambiguous infected sites pose challenges for drug administration position and dose; thus, precise biofilm detection and discrimination are needed. Biofilm imaging reveals the microbial dynamic in the biological process, thus providing meaningful insights, information, and strategies to fight against bacteria inside the biofilm. [55,56] As a type of noninvasive approach employing the spatial distribution of luminogens to visualize the location of biofilm, FLI has captivated intense interest on account of its simple operation, rapid response, high sensitivity, real-time detection, and preferable biosafety, especially for organic fluorophores. Despite appreciable brightness in an isolated molecular state, conventional organic fluorophores incline to aggregate in a physiological environment composed of water due to their hydrophobic structure and always suffer from vanished fluorescence in aggregate state ascribing to the π-π interaction, which is known as the notorious aggregation-caused quenching (ACQ) effect. [28,[37][38][39] As the aggregation (especially in the complex biofilm microenvironments with water-rich matrixes) and the resulted fluorescence quenching are inevitable because of the inherent aromatic rings and conjugated chains in ACQ fluorophores, there is no alternative but to dilute the dye's working concentrations. Nevertheless, photobleaching resistance declines dramatically upon dilution, eliciting unsatisfactory imaging outcomes, especially for the investigation of in vivo bacterial biofilms. Bearing virtues of the unique aggregationintensified fluorescence with brilliant photostability, large Stokes shift, high brightness, and profitable wavelength adjustability, AIEgens exhibit outstanding potential as the qualified fluorescent agents for biofilm imaging or even selectively discrimination because multiple functional substituents could be readily introduced according to the requirements. [49,[57][58][59][60][61][62][63] In this part, the recent advancements in biofilm microenvironment (e.g., enzyme)-responsive imaging, [64] discriminative imaging of Gram-positive bacterial biofilms, [65] and biofilm viability monitoring [66] based on AIEgens are summarized.

Enzyme-Responsive Imaging of Biofilm
EPS, the substances that capsulated biofilms, are composed of proteins, exopolysaccharides, lipids, enzymes, and extracellular bacterial DNA, playing a significant role in protecting bacteria from external environmental interference, including immune system attacks and antibiotics interventions. [2,7,12] With EPS serving as barriers, bacteria in biofilms are difficult to reach. However, behind every cloud lies a silver lining, the pathogenic microenvironment generated in biofilm is commonly hypoxia, acidic, and full of activated enzymes, [15,67,68] which can be utilized to design biofilm microenvironment-responsive fluorescent probes for specific biofilm detection. In view of the characteristics of biofilms as well as the aggregation-enhanced fluorescence feature of AIEgens, skillfully manipulating the molecular structure could endow AIEgens with superior biofilm sensitivity and advantageous fluorescence turn-on capacity upon responding the stimulus, which facilitates light-up imaging of biofilms. The actualization of stimuli-responsive imaging facilely accommodates precise and efficient identification and visualization of biofilm, thus offering a promising future for quick diagnosis and real-time monitoring.
Among the biomarkers qualified as identification targets in biofilm imaging, alkaline phosphatase (ALP) is a particularly suitable enzyme stimulus. As an indispensable element in phosphate metabolism, ALP catalyzes the dephosphorylation of various substrates such as nucleic acids, proteins, and other small molecules. [69][70][71] In the well-known pathogens Staphylococcus aureus (S. aureus, SA) and Escherichia coli (E. coli, EC), ALP is expressed both on the membrane surface and in the periplasmic space, respectively, indicating its essential role in phosphate metabolism and hydrolysis of DNA molecules. [69,72] More importantly, ALP has been proven to appear in dephosphorylate teichoic acid of prokaryotes, which is a crucial molecule in bacteria colonization on artificial surfaces. [69,73] Collectively, the ALP is closely involved in biofilm formation and bacteria reproduction, offering a compatible stimulus target for biofilm imaging. Based on this, Sessler and co-workers designed an ALP-responsive ExPhos by modifying the FDA-approved iron chelator deferasirox and successfully achieved ALP-responsive biofilm imaging (Figure 2a). [64] In this case, ExPhos could be initially hydrolyzed into the AIE-active ExBT with the presence of ALP, thus enabling the light-up imaging of biofilms. Subsequently, due to its inherent chelator characteristic, ExBT inside the biofilms would chelate with Fe(III), which also plays a critical role in biofilm formation and development, accompanying with the disappearance of the fluorescence.  (Figure 2c) verified the typical AIE property of ExBT. In order to illustrate the ALP-triggered fluorescence response ability of ExPhos, the authors investigated the fluorescence spectra of ExPhos after exposure to Fe(III), ALP, as well as ALP plus Fe(III), respectively. As shown in Figure 2d,e, apparent dose-and time-dependent emission amplification was seen as the addition of ALP. Negligible change in emission appeared when ExPhos exposed to Fe(III) solely, as depicted in Figure 2f. However, after preincubated with ALP, a declined fluorescence emission with the . e) Time-dependent fluorescence intensity changes of ExPhos after incubation with ALP at the concentrations of 32 or 64 U, respectively. f ) Fluorescence emission spectra of ExPhos when exposed to different concentrations of Fe(III). g) Fluorescence spectra of ExPhos preincubated with 64 U of ALP followed by exposure to different concentrations of Fe(III). h) Confocal laser scanning microscopy (CLSM) images of biofilms formed by PA or MRSA bacteria after treatments with ExBT or ExPhos at different times. i) Normalized fluorescence intensities of above CLSM pictures. Reproduced with permission. [64] Copyright 2021, American Chemical Society.
www.advancedsciencenews.com www.small-structures.com rising of Fe(III) concentration was observed in Figure 2g. These results solidly demonstrated the ALP-mediated hydrolysis of ExPhos to AIE-active ExBT, and the subsequent fluorescence quenching caused by chelation with Fe(III) (Figure 2a), which facilitated the dual-responsive biofilm imaging. Similar phenomenon was also observed in biofilm imaging. As displayed in Figure 2h,i, the ExBT-stained biofilms of Pseudomonas aeruginosa (P. aeruginosa, PA) and methicillin-resistant S. aureus (methicillin-resistant S. aureus, MRSA) showed an intense fluorescence emission initially and then a time-dependent decline ascribing to Fe(III) chelation. By contrast, the emission intensity of ExPhos-stained biofilms was feeble initially, which then underwent climbing up due to the dephosphorylation of ExPhos and finally descended owing to the subsequent Fe(III) chelation of ExBT. This vivid fluorescence on-off process fully demonstrated the biofilm-responsiveness of ExPhos. In short, this report successfully provided a protocol to develop an ALP-responsive AIEgen precursor, which can be applied to implement lightup imaging of biofilms.

Discriminative Imaging of Gram-Positive Bacterial Biofilm
According to the Gram-staining method, bacteria can be divided into two main groups, including Gram-positive bacteria (e.g., S. aureus, S. pneumonia, M. diphtheria, M. tuberculosis) and Gramnegative bacteria (e.g., Gonococci, Meningococci, E. coli, P. aeruginosa) on the basis of the different cell surface components. [74,75] To be specific, the outer envelope of the Gram-positive bacteria is a thick and porous peptidoglycan layer with lipoteichoic acid (LTA) attached on it, whereas that of Gram-negative ones consists of a thin peptidoglycan layer as well as a phospholipid membrane outside. And that, there is lipopolysaccharide (LPS) decorated on the outer phospholipid membrane of Gram-negative bacteria. [76,77] Apart from distinct cell envelopes, these two kinds of bacteria have been proved to differ significantly in terms of their sensitivity to the particular antibiotic. For instance, most Gram-positive bacteria are sensitive to penicillin, whereas Gram-negative bacteria, which are susceptive to streptomycin and chloramphenicol, are insensitive to penicillin except for Neisseria spp. in epidemic meningitis and Neisseria gonorrhoeae. [78] Therefore, in order to avoid the overuse of antibiotics and slow down the acquisition of drug resistance, accurate and rapid discrimination of Gram-positive and Gram-negative bacterial biofilm is of utmost significance in clinical practice, and it represents an important precondition for the clinical practitioners to choose pertinent therapeutic strategy for addressing microbial infection-related problems.
In view of the serious threat that Gram-positive bacteria posed to the mankind, Sayed et al. designed two novel AIEgens named tetraphenylethylene-naphthalimide (TPE-NIM) and triphenylamine-naphthalimide (TPA-NIM) for selectively identifying Gram-positive bacteria and the corresponding biofilm ( Figure 3a). [65] In this research, the AIE effect of TPE-NIM and TPA-NIM was first substantiated in DMSO/H 2 O mixture with H 2 O serving as the poor solvent, as shown in Figure 3b Figure 3g). This highly selective targeting ability toward Gram-positive bacteria was attributed to the strong affinity between the tertiary amine groups of the AIEgens and the acid components (e.g., LTA and teichuronic acid) on the Gram-positive bacteria driven by electrostatic attraction. Besides, due to the hydrophobicity of these two AIEgens, the hydrophobic interactions also contributed to this staining process. Further experiments suggested that the two probes also successfully stained the S. aureus biofilm over E. coli biofilm (Figure 3d), exhibiting an excellent ability to visualize biofilms formed by Gram-positive bacteria. Overall, the two AIEgens with powerful differentiation ability of Gram-positive bacteria and the corresponding biofilm make it possible to provide efficient strategy for discriminative imaging of Gram-positive bacterial biofilms from Gram-negative ones.

Biofilm Viability Monitoring
As a critical parameter to appraise the functionality, virulence factor secretion, drug resistance generation, and other characteristics, [79,80] it is of great importance to monitor the bacterial viability and metabolic status. Discriminative imaging of live and dead bacteria is a universal method to directly know about the activity of bacteria. [79,81,82] However, the most commonly used Live/Dead kit (e.g., SYTO 9/PI) usually requires washing steps, after which the morphology and structure of biofilms might be inevitably changed. Moreover, unavoidable system errors could be introduced when using SYTO 9/PI fluorescent probe on account of the spectrum overlapping of SYTO 9 and PI. [83,84] Taking advantage of the easy modification, tunable absorption/emission wavelength, and wash-free characteristic, AIEgens have been recognized as promising candidates for the biofilm viability monitoring.
Recently, a dual-AIEgen system was reported by He et al. for morphological imaging and metabolic state detection of microbes and their biofilm aggregates ( Figure 4a). [66] This system consisted of a newly synthesized AIEgen, termed DCAQ with near-infrared (NIR) emission pearked at 729 nm ( Figure 4b,c). Owing to the AIE feature, large Stokes shift, NIR emission, and two positive charges, DCAQ was able to stain all kinds of microbes, including Gram-positive bacteria, Gramnegative bacteria, and fungi in both live and dead forms. Additionally, aiming to efficiently indicate dead bacteria and avoid the result inaccuracies, a previously reported AIEgen TPE-2BA with blue fluorescent emission was employed to minimize the spectral overlap. TPE-2BA has been reported to be capable of selectively staining dead bacteria because it can enter the membrane-damaged bacteria and bind to the groove of doublestranded DNA, thus generating bright blue emission. [85] Profiting from the negligible overlapping bands between DCAQ and TPE-2BA (Figure 4c), the monitoring of bacterial viability could be successfully realized. As shown in Figure 4d, taking Gram-positive S. aureus, B. subtilis, Gram-negative E. coli, S. marcescens, and fungi (C. albicans, S. cerevisiae) as typical representatives, DCAQ achieved all kinds of microbial staining for testing the total amount of microbes, either live or dead, and TPE-2BA determined the number of dead microbes at the same time owing to its characteristic of only probing dead ones.
Apart from planktonic bacterial imaging and sensing, the dual-AIEgen system also worked efficiently in biofilm imaging. Figure 4e demonstrated that DCAQ was able to stain all the microbes of biofilms formed by S. aureus, E. coli, and C. albicans through electrostatic interaction, thus describing the overall morphology of the biofilm. Meanwhile, the distinctions of the three kinds of biofilms could be revealed by observing the quantity and distribution of dead microbes indicated by the blue-emissive TPE-2BA. Therefore, the dual-AIEgen system is expected to be used as a promising morphological imaging and activity visualization kit for both planktonic bacteria and their biofilm counterparts.

Antibiofilm Theranostics
AIEgens-based biofilm imaging systems make it possible to facilely actualize light-up biofilm imaging based on the biomarker in the biofilm microenvironment, selectively discriminate biofilms formed by Gram-positive bacteria over Gram-negative ones, and vividly reveal the biofilm viability. In addition to FLI ability, AIEgens could also be tactfully endowed with additional therapeutic functionalities, such as photodynamic property, yielding theranostic AIEgens. [31,60,61,[86][87][88][89] Benefitting from the adjustable molecular structures and the increasing efforts that researchers devoted in this field, a series of theranostic AIEgens have been explored and successfully applied in the imaging-guided biofilm elimination, in these years. [54,[90][91][92][93][94][95] In comparison with the traditional protocols such as antibiotics, theranostics could enable the precise diagnostic imaging and efficient treatment concurrently, thus significantly enhancing the accuracy and efficiency of biofilm elimination.
The life cycle of biofilm is a complex process, and the classic model includes four stages. [11,12,[96][97][98] Prior to the initial attachment on the surface, there is a conditioning layer forming for  [65] Copyright 2020, Elsevier.
www.advancedsciencenews.com www.small-structures.com altering the properties of the surface and assisting the microbial attachment. Then the microbes deposit on the surface either actively or passively and attach to it. After the generation of the surface-adhered microcolonies, they gradually proliferate and secret EPS as a biofilm matrix to form the initial biofilm.
In the subsequent period, biofilm is restructured to form a 3D morphology, the mature state. At the end of the process, the release of the planktonic microbes occurs to colonize new regions. Depending on the different characteristics at different states of the biofilm's life cycle, the antibiofilm strategies are diverse. [66,99] For instance, antiadhesion intervention is employed to block the adhesion of microbes to the surfaces in the preliminary stage, which is an effective way to prevent the biofilm's formation with less drug resistance caused. [97][98][99] Regarding to the mature biofilm that has been formed, the therapeutic approaches focus on the degradation of EPS and the eradication of the internal microcolonies. [67] Given this circumstance, the recent advances of AIEgen-based theranostic systems in combating biofilms at different states of the biofilm's life cycle will be introduced in this section.

Imaging-Guided Antiadhesion Therapy for Preventing Biofilm Formation
As mentioned before, the attachment and aggregation of microbes to the host cell surfaces are the prerequisite for biofilm formation. The initial step is always triggered by the interactions between the bacterial surface proteins and the carbohydrate receptors on the periphery of host tissues. [99,100] Based on this recognition mechanism, traditional antiadhesion agents usually hinder this attachment process through disguising as glycomimetics that can bind with microbes. [100][101][102] Unfortunately, these kinds of antiadhesion agents are specially synthesized for specific species of bacteria because the proteins on the microbial surface are diverse, which usually fail to meet the requirements of clinical treatment with mixed unknown microcolonies. Moreover, the designing principles of antiadhesion agents for abiotic surfaces and biotic surfaces are different to some extent, which needs more moderate strategies to adapt to different surfaces. In recent years, boron-rich natural or synthetic compounds have emerged as potential materials for inhibition of biofilm formation because  boron was found to affect signal mechanisms for communication among bacteria significantly. [103,104] Meanwhile, the supportable techniques are also demanded to monitor the process of antiadhesion, which is beneficial to appraise the efficacy of antiadhesion therapy. Therefore, exploring efficient antiadhesion agents with broad spectrum antiadhesion properties, good biocompatibility, as well as excellent visualization ability is urgent. Under this demand, early in 2018, Zhang et al. proposed a strategy of constructing 3D spherical AIE nanoparticles (NPs) based on benzoxaborole groups for nonlethal antiadhesion therapy of bacterial infections as well as facile visualization of the bacterial aggregation process. [54] In this work, TPEBr 4 was initially prepared as the initiator of the atom transfer radical polymerization (ATRP) to synthesize the TPE-based star-poly(5acrylamido-1,2-benzoxaborole-co-di(ethylene glycol) methyl ether methacrylate) (star-PAD) by using 5-acrylamido-1,2-benzoxaborole (AAmBO) and dimethylaminoethyl methacrylate (DMEMA) as monomers. Then the star-PAD NPs were obtained by virtue of the inherent self-assembly ability of the amphipathic copolymers (Figure 5a, upper). As shown in Figure 5a (lower), the star-PAD NPs could move away both Gram-positive and Gram-negative bacteria through agglutinating them into clusters via strong polyvalent interaction but do not inflict visible harm to the host cell. To be specific, this broad-spectrum antibacterial property relied on the reversible reaction between benzoxaborole moieties surrounding on the surfaces of the star-PAD NPs and the abundant distribution of diol moieties on the bacterial cell wall. Additionally, they found that the size of dense bacterial fluorescent clusters was related to the species of bacteria and it increased along with the augmented content of AAmBO moieties in the copolymer. Thus, the star-PA 3 D 1 NPs with higher content of AAmBO moieties were employed for the following biological experiments. After adding the star-PA 3 D 1 NPs into the PBS suspension of bacteria, distinct clusters of both B. amyloliquefaciens (BA) and S. aureus were observed with blue fluorescence signal emitted by star-PA 3 D 1 NPs, which merged well with the green fluorescence of the live bacteria sensor acridine orange (AO) (Figure 5b). This result manifested that the star-PA 3 D 1 NPs could adhere to bacteria and agglutinate them into clusters. Moreover, the weak red fluorescence of ethidium bromide (EB) definitely indicated the negligible impacts of star-PA 3 D 1 NPs on bacterial cell integrity and viability, thus suggesting minimal possibility of inducing bacterial resistance. Furtherly, the biofilm forming inhibition effect of star-PA 3 D 1 was evaluated by employing EB (acting as the dead bacteria probe) and fluorescein isothiocyanate (FITC) conjugated concanavalin A (ConA) (FITC-ConA, acting as the exopolysaccharides probe) for CLSM imaging. As depicted in Figure 5c, in contrast to the biofilms that presented mature  structures with abundant extracellular polysaccharide and dense bacteria in the control group, there were less polysaccharide and bacteria in the biofilms with a scanty architecture in the star-PA 3 D 1 NPs-treated group, suggesting that the star-PA 3 D 1 could effectively inhibit biofilm formation. The maximum inhibition percentage of biofilm formation was determined to be %45% for P. aeruginosa, %65% for E. coli, %75% for B. amyloliquefaciens, and %65% for S. aureus with a concentration of 500 μg mL À1 . Subsequently, the ability of star-PA 3 D 1 in inhibiting bacteria attachment to host cells was further explored by using NIH 3T3 and S. aureus as the model host cell and bacterium, respectively. Compared with the control group, in which a large scale of green FITC-labeled bacteria was observed around the NIH 3T3 cells with shrinking and distorted cell nuclei, in the star-PA 3 D 1 group, fewer bacteria were distributed around the NIH 3T3 cells with intact cell nuclei (Figure 5d), evidently substantiating the predominant capacity of star-PA 3 D 1 in prohibiting adhesion of bacteria to host cells. This result was also supported by the significantly reduced number of residual adhesion bacteria determined by using LB agar plates count method. Finally, the excellent biocompatibility of star-PDMEMA and star-PAD was verified by hemolysis and 3-(4,5-Dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. This work provided a meaningful inspiration on developing functional antiadhesion agents with broad-spectrum antibacterial properties and negligible damage to host cells.

Imaging-Guided PDT
Once the biofilm matures, it will be more challenged to eradicate owing to the existence of EPS which could withstand the attack of the immune response or antimicrobial drugs and lead to the increased resistance of bacteria. [6,13,14,20] As an emerging antibiofilm strategy, PDT is a noninvasive therapeutic modality with the merits of high spatiotemporal precision, negligible drug resistance, and low systemic toxicity. [22][23][24]28] And that, it mainly relies on the oxidative ROS generated by the phototriggered reactions between the PSs and surrounding substrates to disrupt the bacterial integrity through oxidizing the biotic components such as proteins, EPS, DNA, and lipids in the biofilm matrix as well as bacterial cells. [25][26][27][47][48][49] Owing to the unique aggregationenhanced emission and aggregation-induced enhancement of ROS generation capacities, a handful of AIE-active PSs have been successfully explored [45,48,60,61] and their breakthroughs in the imaging-guided photodynamic elimination of biofilms have been witnessed. Water-soluble AIE PSs have been intensively demanded in the practical bioapplications due to the aqueous physiological environment of the life entry. Regarding to the antibiofilm application, the advantages of water-soluble AIE PSs are apparent. For instance, the good water solubility of AIE PSs can not only facilitate their penetration into the interior area of biofilm through water-filled channels but also can enable turn-on theranostics of bacterial biofilm. To be specific, the water-soluble AIE PSs are usually dispersed in molecular state in the aqueous physiological environment showing rather faint fluorescence emission and low ROS generation, both of which however can be boosted upon the water-soluble AIEgens adhering to the bacteria owing to the RIM mechanism. These considerably enhanced fluorescence and ROS generation triggered by the interaction between AIE PSs with bacteria as well as the favorable biofilm penetration ability endow the water-soluble AIE PSs with competitive superiorities in eradicating bacterial biofilm.
For example, Hu et al. reported a water-soluble AIE PS, namely TPA-PyOH, and successfully achieved fast FLI of broad-spectrum pathogens with minimal background and biofilm inhibition of pathogenic microorganisms. [92] In this work, the quaternary ammonium group and terminal hydroxyl unit were introduced into the molecular structure to endow TPA-PyOH with excellent water solubility (Figure 6a). Owing to its good hydrophilicity and AIE feature (Figure 6b), TPA-PyOH emitted no fluorescence in water, but unique light-up AIE-active fluorescence could be observed when the positively charged molecules contacted with the negatively charged pathogenic membrane, thus facilitating the FLI of pathogenic microorganisms. Just as the CLSM images shown in Figure 6h, obvious fluorescence signals could be found in Gram-positive bacteria (S. aureus, L. monocytogenes), Gram-negative bacteria (E. coli), and fungi (C. albicans) after incubation with TPA-PyOH for only 5 min. These results elucidated that TPA-PyOH could achieve fast broad-spectrum imaging for different kinds of pathogens despite the permeability barrier of Gram-negative bacteria and the protective cell wall with complex eukaryotic cell structure of fungi. Then, the outstanding single oxygen ( 1 O 2 ) generation ability of TPA-PyOH was determined by the commonly used 1 O 2 indicator, 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) (Figure 6c). On the basis of its efficient ROS production, TPA-PyOH exhibited excellent killing effect in various pathogens, including S. aureus, MRSA, E. coli, and C. albicans (Figure 6d-g). Additionally, the inhibition ability of TPA-PyOH on S. aureus biofilm was further evaluated by using a Live/Dead staining assay. After different treatments, the group of TPA-PyOH plus white light irradiation exhibited the strongest red fluorescence of PI among all the groups, suggesting the favorable photodynamic antibiofilm efficiency of TPA-PyOH ( Figure 6i). Besides, the relatively weaker red fluorescence showed that the TPA-PyOH alone could exert moderate native inhibition effect on the S. aureus biofilm. It was worth noting that visible green fluorescence of SYTO 9 can still be seen in the group of TPA-PyOH plus white light irradiation, indicating the existence of residual live bacteria inside the biofilm which evaded from the attack of ROS. The subsequent quantitative examination via broth dilution method supported the apparent biofilm inhibition effect of TPA-PyOH under white light irradiation demonstrated in Figure 6i. Taken together, this work provided a feasible strategy to design a water-soluble AIE PS, which could be employed for the observation and inhibition of different kinds of pathogenic microorganisms as well as the biofilms by virtue of in situ FLI and efficient PDT.
Nowadays, most reported AIE PSs were prepared through tedious multistep organic synthesis and subsequent complicated purification, which are always high-cost and time-consuming, and may raise the concerns of environmental destruction. From this perspective, as many naturally occurring compounds with good biocompatibility could be easily obtained in large scale from abundant natural sources, employing AIE-active natural resources as PSs becomes a new direction. [105] www.advancedsciencenews.com www.small-structures.com In 2022, Tang and co-workers reported a naturally occurring red-emissive AIE PS, Tanshinone IIA, which is capable of promoting bacteria aggregation, as well as targeting and prohibiting the bacterial clusters or biofilms in the aqueous environment. [93] Tanshinone IIA is a natural compound from the herb Salvia miltiorrhiza, exhibiting bright red emission at solid state, which benefits from the electron donor-acceptor (D-A) interaction and extended π-conjugation with good electron delocalization ( Figure 7a). Additionally, the presence of heteroatom and the incredible separation of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) distribution jointly contributed to the significant ROS generation of Tanshinone IIA through reducing the singlettriplet energy gap (Figure 7b). At first, CLSM imaging was employed to investigate the targeting behavior of Tanshinone IIA. As Figure 7c illustrated, Tanshinone IIA could label densely grown bacteria in the existence of biofilm with red emission. This result could be interpreted that Tanshinone IIA was trapped in the sticky matrix layer consisting of EPS and thus initiated RIM to emit bright fluorescence. To further explain this phenomenon, the authors next explored the interaction between Tanshinone IIA and individual bacteria. It was shown that with the increase of Tanshinone IIA dose, the degree of bacterial aggregation gradually intensified (Figure 7d). Accordingly, Tanshinone IIA was believed to serve as an external substance that could provide a hydrophobic surface for bacterial adhesion and thus promote the bacterial auto-aggregation process. The process, in turn, trapped more Tanshinone IIA into the bacteria clusters to further exacerbate the bacterial auto-aggregation. This phenomenon implied that Tanshinone IIA could act as a "fishnet" to collect and settle the dispersing bacteria in solution effectively. Thanks to the excellent ROS generation capacity, distinct photodynamic killing effect on both S. aureus and S. epidermidis was observed after light irradiation (Figure 7e). Of particular interest was that the biofilm formation of S. aureus could be significantly inhibited after photodynamic treatment. As Figure 7f,g demonstrated, along with the increasing of Tanshinone IIA dose, a distinct decline tendency in the biofilm formation rate of S. aureus was observed after light irradiation, and only less than 20% biofilm formed in the group with treatment of Tanshinone IIA (100 μM) and light irradiation in comparison with the control groups. Moreover, the excellent biocompatibility of Tanshinone IIA to normal cells (HLF cells) further suggested the great application potentials of Tanshinone IIA in in vivo bacterial infection model as an antibacterial agent (Figure 7h). This work provided an alternative direction to explore effective bactericides via utilizing natural resources as a simple and cost-effective agent.  It is acknowledged that the combination of AIEgens with various drug delivery systems could render AIEgens diverse application potentials, as well as promote the theranostic ability of AIEgens. [28] In this regard, the injectable hydrogels have been proposed to be a promising delivery system in the antibiofilm research, owing to the advantageous merits of unique shearthinning injectability, outstanding biocompatibility, minimal invasion, high water swelling ability, and excellent oxygen permeability. [106,107] As the injectable hydrogels exhibit dynamic and diffusive nature under sheer stress, they can be readily delivered to various sites of the body via a needle and syringe and subsequently in situ recover their original mechanical properties to maintain their injected volume and sustain repetitive load, which enables the injectable hydrogels to conform to the deform application sites without causing obvious discomfort. [108] Besides, bearing similarity to the natural extracellular matrix (ECM), the hydrogels can maintain the good moisture environment for wounds and absorb the extensive exudate effectively, thus fulfilling the critical wound dressing requirements. [109][110][111] On the basis of these excellent superiorities, integrating highperformance AIE PSs with injectable hydrogels to obtain AIE PSs-loaded injectable hydrogels would significantly benefit the antibiofilm outcomes and profitably promote the wound healing by taking full advantages of their mutual outstanding properties. Furthermore, the in situ gelling property of injectable hydrogels makes the formed gels firmly bond to the tissues, which effectively confines the diffusion of AIE PSs and raises the concentration of AIE PSs within the infectious sites, thus boosting  the antibiofilm effect and minimizing the undesired toxicity of AIE PSs to the surrounding tissues. Inspired by this, Xie et al. prepared a novel AIE-active hybrid hydrogel with enhanced photodynamic antibacteria and antibiofilm ability via the self-assembly of commercial N-(9fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-F) and berberine chloride (BBR). [90] As illustrated in Figure 8a, BBR, the natural isoquinoline alkaloid with AIE feature, could first self-assembled into AIE-active NPs driven by the intermolecular electrostatic interaction and π-π stacking and then disperse throughout the gel network of the amino acid-based Fmoc-F hydrogels due to their weak, noncovalent bonding to the amino acids in the hydrogel. Therein, BBR served as an AIE-active PS, which presented excellent 1 O 2 production ability under light irradiation (Figure 8b). Benefitting from this, significantly enhanced antibacteria effect of Fmoc-F/BBR hydrogel could be observed on both Gram-positive S. aureus and Gram-negative E. coli under light irradiation (Figure 8c). Before evaluating the antibiofilm Figure 8. Imaging-guided photodynamic therapy of biofilm based on AIE PSs-loaded injectable hydrogels. a) The chemical structures of Fmoc-F and BBR, and the schematic illustration displaying the self-assembly of AIE-active Fmoc-F/BBR hydrogel as well as its application in bacteria killing, biofilm disruption, and wound dressing. b) Variation in the relative absorbance of ABDA for 1 O 2 detection. ΔA represented the difference between origin absorbance value and the absorbance value at different time at 378 nm. c) The bacteria viability of E. coli with different treatments. d) CLSM 3D images of biofilms forming from red fluorescence protein expressing E. coli DH5α incubated with Fmoc-F/BBR hydrogel (green). e) The bacteria viability of E. coli biofilm with different treatments. f ) Variation of in vivo wound size upon different treatments at different days. g) The bacteria colony-forming units from mice skin wound upon different treatments at different days. Reproduced with permission. [90] Copyright 2020, Elsevier.
www.advancedsciencenews.com www.small-structures.com capacity, the biofilm penetration ability of Fmoc-F/BBR hydrogel, which was the precondition of biofilm combating, was preferentially assessed. The CLSM images in Figure 8d showed that the Fmoc-F/BBR hydrogel (indicated by green fluorescence) could penetrate and disperse throughout the E. coli biofilms (indicated by red fluorescence), reasonably suggesting the great potential for further antibiofilm applications. Subsequently, the Crystal Violet assay was employed to appraise the antibiofilm activity of Fmoc-F/BBR hydrogel as a PDT agent. It can be seen from Figure 8e that the photodynamic eradication rates of E. coli biofilm increased to 46.7% and 59.3% under light irradiation after incubation with Fmoc-F/BBR hydrogel for 30 or 60 min, respectively. Similar antibiofilm tendency and better antibiofilm efficiency were also observed in the S. aureus biofilm. Then, the in vivo photodynamic antibacteria and wound healing efficiency of the Fmoc-F/BBR hydrogel were assessed through a fullthickness skin infection model on the backs of mice. Compared with the control group without any treatments as well as the Fmoc-F/BBR hydrogel-only group, the group with treatment of Fmoc-F/BBR hydrogel and white light irradiation exhibited excellent wound repair efficiency (Figure 8f,g). Besides, the bacterial growth in the skin wounds of different groups counted on days 1, 3, and 7 also indicated that the Fmoc-F/BBR hydrogel with white light irradiation could significantly inhibit the growth of bacteria, showing outstanding antibacterial properties. This work represented a typical example of integrating AIE PSs with amino acid-based hydrogels, thus achieving broad-spectrum antibacterial and antibiofilm activity as well as wound healing ability.

Imaging-Guided Combination Therapy
Although some progress of AIEgens have been witnessed in the field of imaging-guided photodynamic antibiofilm, it cannot be denied that the antibiofilm outcomes are not very satisfactory. We should also note that, considering the complicated local microenvironments that formed in biofilm systems, the single PDT is not always competent for offering thorough eradication of the bacteria in deep biofilms, that is, because the PDT efficacy is always limited by many external conditions. To be specific, the unsatisfactory penetration depth of general excitation light and the accumulated bacteria-resulted light scattering both lead to insufficient light density and thus diminish the generation of ROS under the same concentration of PSs. [23] Besides, the hypoxic microenvironment inside the biofilm is deem to inevitably impair the generation of Type II ROS, which is recognized to be highly oxygen dependent. [89,112,113] Furthermore, the PDT effect could also be compromised by the antioxidant glutathione (GSH) existing in biofilm EPS due to its counteracting capacity to ROS. [114,115] Facing the currently challenging situation, researchers have devoted more efforts to improve the biofilm combating results. On the basis of PDT, integrating other therapeutic modalities or therapeutic agents to implement combination therapy represents a promising strategy to efficiently eradiate the bacteria that slyly lurk in deep biofilms. Here, the combination therapy of AIEgenbased PDT with dark toxicity or antimicrobials was summarized in this part.

Imaging-Guided Photodynamic and Dark Toxicity Combination Therapy
Antimicrobial peptides (AMPs) have been acknowledged to play an important role in bacteria combating, especially for the drugresistant bacteria. [116] The antibacterial mechanism of AMPs mainly relies on their cationic portion that can interact with the negatively charged bacterial surface driven by electrostatic interaction, thus disrupting bacterial membranes, disordering the membrane permeability, and irritating the leakage of intracellular components. [117] Inspired by the significant antibacterial effect of cations in AMPs, AIE PSs have been rationally designed with cations to achieve the synergistic therapy of phototoxicity and dark toxicity in pursuit of improved antibacterial efficiency. Moreover, it is envisioned that cationic AIE PSs possess enhanced bacteria-targeting capacity, improved biofilm-penetration potential, and elevated imaging ability benefitting from their positive charges.
On the basis of this conception, Zhao et al. designed and synthesized a cationic AIE PS termed β-PM-phosphindole oxide (PIO), exhibiting the synergistic effect of phototoxicity and dark toxicity (Figure 9a). [91] By employing TPA and thiophene as electron donors, PIO and pyridinium as electron acceptors, the typical D-A structured β-PM-PIO was endowed with distinct aggregation-induced NIR fluorescence peaked at 715 nm and enhanced ROS generation ability (Figure 9b). Besides, the ingenious introduction of hydrophilic pyridinium empowered β-PM-PIO with better bacteria-targeting ability by virtue of the electrostatic interaction with negatively charged bacterial membranes, as well as higher biofilm-penetrating capacity. The planktonic bacteria imaging experiments showed that β-PM-PIO could selectively stain bacteria over mammalian cells, particularly Gram-positive bacteria (S. aureus, MRSA) with a staining efficiency nearly 100% after 10 min incubation. The 3D CLSM images of S. aureus and MRSA biofilms in Figure 9e certified the outstanding bacterial affinity and prominent biofilm-penetrating capacity, demonstrating that β-PM-PIO could serve as an excellent imaging agent for both planktonic bacteria and biofilm. Possessing satisfactory ROS generation ability, distinct cationic portion, and selective bacteria-targeting capacity, β-PM-PIO was observed to show excellent antibacterial efficiency due to the synergistic effect of phototoxicity and dark toxicity; as shown in Figure 9c,d, the bactericidal concentration of β-PM-PIO on S. aureus and MRSA could be decreased to 1 μM after white light irradiation from 5 μM under dark condition. Subsequently, SYTOX blue was employed as the dead bacterial fluorescence probe to explore the ability of antibiofilm formation and mature biofilm eradication. After going through a series of treatments involving incubation with β-PM-PIO (10 μM) for 30 min, white light (40 mW cm À2 ) irradiation for 10 min, and static culture for 24 h at 37°C, only a few red clusters indicated by the redemissive β-PM-PIO with no sign of forming biofilm were observed in the light irradiation group. Similar phenomenon in the dark group was also seen (Figure 9f ). Compared with the densely arranged biofilm in the control group without any treatment, the significantly decreased bacterial clusters in the β-PM-PIO treated group with/without light irradiation solidly demonstrated that β-PM-PIO can efficiently prevent biofilm formation. On the other hand, the eradication capacity on mature biofilm was also substantiated by the obviously brighter cyan fluorescence signal of SYTOX blue in the light irradiation group of Figure 9g, evidently suggesting the remarkable biofilm elimination effect under the synergistic effect of phototoxicity and dark toxicity. Further in vivo wound healing experiment also demonstrated the superior antibacterial performance of β-PM-PIO on MRSA. Overall, this work offered a new perspective in the construction of FLI-guided antibiofilm agents equipped with synergistic phototoxicity and dark toxicity.

Imaging-Guided Photodynamic and Antimicrobial Combination Therapy
As another treatment modality, antimicrobial therapy has long been the frontline and most frequently used protocol for the prevention of bacterial infection and biofilm formation. [4,5] Without suffering from the limitation of light penetration depth, antimicrobial agents can effectively destroy deep-seated bacteria in biofilms. Nevertheless, the long-term sole utilization of antimicrobials would easily give the risk of multidrug resistance generation of bacteria. In contrast, the ROS produced by PDT is able to directly inactivate multidrug-resistant bacteria or sensitize the bacteria to make them be easily killed by antimicrobials. From this, the union of PDT and antimicrobial therapy will unambiguously result in synergistic actions with improved therapeutic outcomes. On the other hand, a major cause of multidrug resistance is the production of related enzymes that can efficiently break down antimicrobials before they can interact with bacterial components or interfere with bacterial vitalities. [118][119][120] Thereout, the further integration of multidrug resistanceassociated enzyme inhibitors is capable of powerfully inhibiting www.advancedsciencenews.com www.small-structures.com enzymatic activity and reviving the antibacterial ability of antimicrobials. In addition, considering the special pathological characteristic of biofilms, the design of stimuli-responsive theranostic systems by making the best of biofilm microenvironments to realize efficient drug delivery and controllable turn-on theranostics in biofilms will be highly desired for safe and effective treatment. Under these circumstances, Li et al. developed a lighttriggerable and pH/lipase-sensitive supramolecular nanotheranostic platform, enabling high drug penetration, stimuli-responsive synchronous release of antibiotics and β-lactamase inhibitors as well as simultaneous PDT to defeat MRSA biofilms. [94] To be specific, as shown in Figure 10a, the supramolecular nanotheranostics were constructed based on host-guest interaction, where β-cyclodextrin-bonded phenylboronic acid-TPE (PBA-TPE) conjugating with ampicillin (Amp) via a ROS-cleavable thioketal linker (cd-PTTA) serve as the host and adamantane-linked poly(ethylene glycol)-poly(ε-caprolactone) (PECL-ad) act as the guest. Benefitting from the glorious AIE nature of the introduced TPE unit, the host Reproduced with permission. [94] Copyright 2021, Elsevier.
www.advancedsciencenews.com www.small-structures.com segment of cd-PTTA showed significantly enhanced fluorescence emission at aggregate states both in the poor solvent of water and self-assembled host-guest complexation of PECL@PTTA ( Figure 10b). Moreover, the light exposure to TPE moieties of PECL@PTTA could effectively induce ROS generation as demonstrated by 1 O 2 indicator of ABDA in different pH buffers (Figure 10c). Compared with the group of pH 7.4 buffer plus light irradiation, the elevated 1 O 2 production ability of PECL@PTTA at pH 5.5 buffer and light illumination could be attributed to the acid-triggered removal of β-cyclodextrin and guest corona from the supramolecular micelles which facilitated the diffusion of surrounding oxygen and ABDA into the hydrophobic core of TPE derivatives. Apart from combating biofilms, the produced ROS could also break the thioketal linker to liberate Amp antibiotics. Using phenylboronate ester as the pH-sensitive linkage between β-cyclodextrin and PBA-TPE, the PECL@PTTA could controllably release and activate PBA β-lactamase inhibitors in responding to biofilm acidic microenvironments. Furthermore, the overexpressed lipase secreted by bacteria in biofilms was expected to digest the poly(ε-caprolactone) segments of PECL-ad and destabilize the structure of micelles to accelerate the release of encapsulated drugs. Hence, the light irradiation and inherent dual stimulation of pH and lipase would afford mutual promotions of Amp release, micelle destabilization, and β-lactamase inhibition, exhibiting synergistic actions on MRSA. Figure 10d showed that PECL@PTTA could induce excellent antibacterial capability against planktonic MSRA after light exposure in pH 5.5 buffer.
In vitro biofilm experiments revealed that the designed PECL@PTTA nanotheranostics could effectively destruct the MRSA biofilms and kill the embedded bacteria under the acid condition and white light irradiation, indicated by Crystal Violet and Live/Dead staining assays (Figure 10e). The same experimental results could also be further confirmed by a quantified test of biofilm residuals (Figure 10f ). In sharp contrast, the free Amp treatment displayed no obvious bactericidal efficacy. Eventually, the in vivo antibiofilm potential of ECL@PTTA was investigated in an MRSA-infected subcutaneous abscess model of mice (Figure 10g). It turned out that the PECL@PTTA with light exposure enabled severe biofilm destruction, bacterial eradication, and complete wound healing, allowing for re-epithelialization and normal skin morphology after 12 day treatment. The efficient woundrecovery promotion was also manifested by monitoring the wound area changes (Figure 10h). Taken together, this work provided a useful perspective for overcoming biofilms and resistant bacteria by tactfully integrating stimuli-responsive design and synergistic photodynamic-antibiotic therapy. Apart from natural or semisynthetic antibiotics, AIE PSs could also combine with fully synthetic antimicrobials to offer FLI-guided synergistic therapy. In this respect, Chen et al. reported a highly effective dental treatment strategy by synergizing PDT and antimicrobial therapy for tooth biofilm elimination and tooth whitening. [95] As shown in Figure 11a, a D-π-A structural AIE PS named DTTPB consisting of TPA unit (serving as electron donor), bithiophene fragments (acting as donor and π-bridge), a carbon-carbon double bond (functioning as π-bridge), and a pyridinium moiety (working as electron acceptor) was reasonably designed and synthesized. Bearing a positively charged head and two hydrophobic tails, DTTPB was engineered to simulate the biomembrane phospholipid architectures and anticipated to interact with bacterial envelopes through both electrostatic and hydrophobic interactions. With the structural superiority, DTTPB was indicated to possess good targeted imaging capability both in planktonic S. mutans bacteria and their biofilms (Figure 11b). Upon the white light irradiation, DTTPB could effectively kill the bacteria and inhibit biofilm formation, which was visualized by the NucGreen staining (Figure 11c). The underlying mechanism of DTTPB-mediated photodynamic biofilm suppression was further analyzed by detecting the biofilm-related gene expression. As depicted in Figure 11d, the expressions of EPS synthesis-related genes (gtfB, gtfC, and gtfD), bacteria adhesion gene (srtA), and bacteria virulence-associated genes (sodA and Nox) all presented remarkable reduction with the presence of DTTPB and white light illumination. It was implied that besides directly destroying biomolecules of S. mutans, the produced ROS by DTTPB could also simultaneously downregulate biofilm growth-associated gene expression, which could synergistically boost antibiofilm efficacy. Moreover, DTTPB with light irradiation exhibited outstanding tooth whitening performance with negligible harm to teeth structure as compared with 30% H 2 O 2 and other groups (Figure 11e). As a major ingredient of most commercially available oral care products, chlorhexidine (CHX) was selected as the fully synthetic antimicrobial to explore the cooperative effect with DTTPB. As elucidated in Figure 11f, a synergistic antibiofilm function of CHX and DTTPB under light irradiation was distinctly observed by the Live/Dead staining assay. Scanning electron microscope (SEM) images further confirmed that the combination treatment of DTTPB-mediated PDT and CHX could not only damage the S. mutans but also degrade the EPS. Last but not least, DTTPB also exhibited favorable biocompatibility and biosecurity, enabling its potential application in clinical dentistry.

Conclusion and Perspectives
As summarized in this review, AIEgen-based bacterial biofilm imaging and antibiofilm theranostic agents have been emerging as powerful weapons in fighting against pathogenic biofilms when facing the currently challenging situation of pathogenic bacterial infection. Owing to their prestigious advantages of unparalleled aggregation-intensified fluorescence with robust brightness, large Stokes shift, outstanding photobleaching resistance, real-time responsiveness, and tunable wavelength even to NIR region, several examples of success in AIEgen-based bacterial biofilm imaging have been reported. For example, the achievement of biofilm enzyme-responsive detection, the accomplishment of discriminative imaging of Gram-positive bacterial biofilm, and the realization of biofilm viability monitoring collectively validated the competence of AIEgens. Apart from the unique fluorescence property, AIEgens could be facilely endowed with therapeutic functions, such as ROS generation-involved phototoxicity, benefitting from its structure flexibility and the reputable AI-ISC. This merit empowers AIEgens with favorable performance in imaging-guided photodynamic inhibition of biofilms by serving as theranostic agents. Besides, by introducing cationic group into AIEgens or integrating AIEgens with other therapeutic agents, such as antibiotics or fully synthetic antimicrobials, imaging-guided synergistic biofilm eradiation could be achieved for improved antibiofilm outcomes. On the other hand, in addition to combating the preexisting biofilms, well-designed AIE NPs could also prohibit the biofilm formation by means of triggering the bacteria agglutination and subsequently preventing the adhesion of the bacterial clusters to the surfaces.
Despite recent progress in this field, the following challenges or perhaps research opportunities should be taken into consideration in the future outlook of AIEgen-based biofilm combating. For the diagnostic imaging part, for the purpose of superior biofilm-targeting ability, the exploration of various biofilm stimulus-responsive detection systems should be strengthened on the basis of the complicated biofilm microenvironment, such as acidic pH, over-expressed GSH, and multiple enzymes. [2,67,69,114] In addition, although discriminative imaging system of Gram-positive bacterial biofilm has been constructed based on AIEgens, there is still a long way to go through in consideration of satisfying the practical clinical needs in which the specific bacterial species should be precisely distinguished. From this point of view, how to furnish AIEgens with more accurate identification ability to specific biofilms is worthy to be carefully considered. Modifying AIEgens with specific targeting units (e.g., nucleotide sequence, antibodies, peptides, etc.) and applying these engineered AIEgens into the traditional fluorescenceinvolved identification methods (e.g., immunofluorescent staining, enzyme linked immunosorbent assay, fluorescence in situ hybridization, etc.) [121,122] point out one possible direction. Besides, elaborate AIEgens-based fluorescence sensor arrays might be another alternative identification approach. [123] For the antibiofilm theranostics part, AIEgen-based multimodal theranostic systems, not only including FLI and PDT but also involving photoacoustic imaging (PAI) and photothermal therapy (PTT), are expected to provide much superior antibiofilm results. As an excellent template for balancing the excited energy dispassion, multimodal AIEgens have been intensively designed and used in the cancer theranostics [28,39,[124][125][126][127][128][129][130][131][132] but rarely applied for antibiofilm applications. In view of the outstanding Figure 11. AIE PSs-based imaging-guided photodynamic therapy combined with synthetic antibacterial agents for biofilm eradication. a) The chemical structure of AIE PS named DTTPB and the schematic illustration of tooth biofilm elimination and tooth whitening. b) In vitro CLSM 3D image of S. mutans biofilm stained with DTTPB. c) CLSM images of the biofilm derived from S. mutans incubated with DTTPB at different concentrations in the presence of light irradiation or not. The bacteria in the biofilms were labeled with NucGreen. d) The biofilm formation-related gene expression levels after different treatments. e) Photographs of clinical teeth (upper row) and corresponding SEM images of tooth surfaces (lower row) upon various treatments. f ) The CLSM 3D images and g) SEM images of S. mutans biofilms with different treatments. Reproduced with permission. [95] Copyright 2022, Wiley-VCH GmbH.
www.advancedsciencenews.com www.small-structures.com performance of AIE-active multimodal theranostic agents in tumor eradication, it is reasonable to expect the future perspectives of multimodal AIEgens on antibiofilm theranostics. Furthermore, effective as the theranostic agents are, how to actualize their deeper penetration and effective delivery into the interior biofilm to further build a microenvironment with sufficient antibacterial concentration is another critical issue that can affect the final consequence. In this regard, some polymeric nanodelivery systems, such as surface charge adaptive nanocarriers, [27,133,134] may point out a promising direction. With these issues being detailedly and appropriately handled, even bigger breakthroughs of AIEgens in pathogenic biofilm combating even clinical trials could be anticipated under the intensive collaboration of researchers from multiple related interdisciplinary areas.