AIEgens for microorganism‐related visualization and therapy

Humans and bacteria have always been closely related. However, pathogenic bacteria and drug‐resistant bacteria pose a certain threat to human life and health. On the other hand, probiotics, such as intestinal flora, also affect our daily life. Understanding the microstructure of microorganisms is important for effective treatment of bacterial infections. Luminogens with aggregation‐induced emission properties are now recognized as potent fluorescent agents for the diagnosis and treatment of microorganisms. In this review, we summarized the most recent developments of AIEgen‐based biomaterials and discussed the advantages of AIE fluorescent probes for imaging. Their applications for rapid imaging of bacteria, differentiation of Gram‐ negative and positive bacteria, and specific imaging of intracellular bacteria and fungi are presented. The monitoring of bacteria‐cell interaction was also introduced. Finally, the design strategy of engineered bacteria‐AIEgen hybrid system and their role in anti‐cancer applications was discussed.

addition to the pathogenic bacteria, there are also many probiotics that can coexist harmoniously with human beings and have made indelible contributions to human health. 2c, 4 For example, researchers discovered that intestinal flora could regulate intestinal motility and secretion, decompose macromolecular complex polysaccharides in food, participate in nutrient digestion and absorption, maintain the integrity of the intestinal epithelial barrier, promote and maintain normal immune development and activity, and so on. 5 For microbial infections, 6 early diagnosis and precise treatment are essential for reducing disease morbidity and death. 7 To achieve a better treatment effect, it is important for visualization of microbes, and their infection processes into host cells, and specifically labeling and precise killing of them in bacteria-cell hybrid environments. Meanwhile, utilizing the nature of non-pathogenic bacteria or opportunistic pathogens to treat other diseases are promising but challenging. 8 Because germs are too small to be seen with naked eyes, asymmetric information is always the most difficult hurdle for humans in conflict. 8d,9 Several imaging technologies for biological research or clinical diagnosis of microbes, such as Gram stanning methods, genetic sequencing, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Cryo-electron microscopy (Cryo-EM), have been developed. 10 However, due to the single scene and sophisticated operation, they encounter limitations. Detection techniques based on fluorescent probes are progressively gaining prominence among existing detection and treatment modalities. 11 Fluorescent techniques have boosted the development of microbiology and antimicrobial material areas. One of the common methods to visualize microorganisms is fluorescent protein labeling. Fluorescent proteins have the advantage of being nontoxic and do not affect the function of the organism while maintaining fluorescence and are now widely used in the study of microorganisms. 12 By labeling with fluorescent proteins, the migration and colonization of the strain can be directly observed, laying the foundation for the study of the mechanism of action of the strain. 13 However, there are also many problems in fluorescent protein labeling methods, such as complicated operations, limited applying scenarios, and limitations in clinical translations. Fluorescent probe is another common method adopted in microbial labeling. For example, commercial fluorescent probes, SYTO 9 14 and propidium iodide (PI), are widely used for microbial viability visualization and relevant investigations. 15 However, traditional fluorescent probes suffer from the intrinsic defect of aggregation caused quenching (ACQ) effect. So far, most of the commercial fluorescent probes are in hydrophobic nature, so they tend to form aggregates in an aqueous phase. 16 The resulting multiple intermolecular π-π interactions lead fluorescence to decrease or even complete quenching. Additionally, this drawback is further exacerbated in complex biological systems and prevents these traditional fluorescent probes from being incorporated into intracellular bacteria and relevant bioprocesses.
Fortunately, the emergence of aggregation-induced emission (AIE) has brought direct solutions for addressing the problems encountered by ACQ fluorophores. 1b,16b AIE luminogens (AIEgens) are weakly emissive or nonemissive in dilute solutions (i.e., isolated molecular species) but intensively emissive in the aggregation or solid states, which demonstrate opposite emission behavior to that of ACQ fluorophores. AIEgens have lots of advantages, such as tunable chemical structure, adjustable emission spectrum, good biocompatibility, and high potential to be used as "wash-free" and "light-up" probes. 17 So far, the restriction of molecular motions (RMM), including restriction of intramolecular motions (RIM intra ) and restriction of intermolecular motions (RIM inter ), is regarded as the primary mechanism to interpret the AIE mechanism ( Figure 1). 16b,17c And the unfavorable close and strong intermolecular π-π stacking interaction in aggregates is prevented by the highly twisted molecular structures of AIEgens. Since AIEgens typically exhibit high fluorescence quantum yield and extraordinary photostability in an aggregate state, they always result in extraordinary performances in high-quality fluorescence imaging and long-term fluorescence tracking. 17a,17c In addition, AIEgens could be smartly designed to exhibit excellent photosensitization, which further broadens their potential applications in photodynamic therapy (PDT) of various diseases. 18 As a result, AIEgens make fluorescence technology more viable for on-demand biological applications. Two major studies are now being conducted in the field of microbiological research. 1b,17a Microbe detection, on one hand, primarily comprises bacterial imaging, specific staining, sensitive identification, and successful categorization. Antibacterial therapy, 19 on the other hand, delves further into multidrug-resistant (MDR) bacteria and intracellular bacteria in the context of successful PDT tactics. 8d Several reviews related to bacterial imaging and therapy have been summarized by other groups, such as AIEgens for microbial detection and antimicrobial therapy 1b ; activatable AIE bioprobes for bacteria imaging 20 ; and AIEgens for the treatment of pathogenic bacteria, fungi, and viruses. 21 When compared with these published works, this review focuses more on "bacteria-cell interaction", which has not been discussed. In the first part "The main design strategy of AIEgens" and "AIEgens for selectively bacterial microbial imaging", we introduce several specific AIEgens currently mainly used for bacterial imaging. In addition, after introducing the research progress of bacteria-cell interaction in recent years, we also explain how to use AIEgens to specifically kill intracellular bacteria and the application of engineered bacteria in specifically targeting cancer cells. Therefore, the whole article focuses more on the interaction of bacteria and cells, which is also the characteristic of the whole article. 22 In this review, we will highlight the most exciting achievements that the AIEgens recently made in the field of microbe-related visualization and therapy. First, we will briefly summarize and discuss three strategies in bacterial labeling and imaging, and then we will describe the functional AIEgens in discriminating different types of bacteria. Next, visualization of phagocytosis processes will be described. Then, selective PDT toward intracellular bacteria will be presented. In addition, engineered bacteria-AIEgen hybrid systems for anticancer applications will be introduced. 23 In these individual sections, the general working principles and design strategies with comprehensive cases and clear-cut images would be shown in detail for further study. A perspective on future directions of AIE research in microbial area would be presented.

| Main design strategies of AIEgens
Microbial labeling and visualization are the priority processes in microbial detection and antibacterial therapy. Plenty of AIEgens have been employed for bacteria imaging and sensing. 1b, 24 Here, three common strategies for developing AIEgen probes for microbial labeling were introduced (shown in Figure 2). As demonstrated in Figure 2A, the first strategy was to introduce positive charges into AIEgens since the cell envelope of bacteria is negatively charged. Specifically, lipopolysaccharides (LPS) in Gram-negative bacteria and the thick peptidoglycan layer in Gram-positive bacteria were all negatively charged structures. 25 After encountering bacteria with negatively charged surfaces, the positively charged AIEgens gradually approached the bacterial due to electrostatic interactions, resulting in efficient bacterial labeling. 24a Because the electrostatic interactions were general interactions between positive charges with negative charges, this kind of AIEgens usually exhibited universal staining ability toward microbes. Furthermore, the molecular design of charged AIEgens is easy and the imaging operation is feasible. To obtain more stable labeling effect, covalently bonding abilities could be incorporated into AIEgens. 26 Consequently, AIEgens that were able to react with bacteria via click reactions have been developed and presented as the second strategy ( Figure 2B). This strategy primarily achieves bacterial lighting by incorporating clickable groups, such as activated alkyne (AA) groups 27 or thiocyanate (NCS) groups, 28 into AIEgens. AA groups and NCS groups could react with amino groups or thiol groups on bacterial cell envelopes. With the rapid development of AIEgens, these AIEgens with clickable groups were increasingly being used in biological imaging due to their advantages, such as simple operation procedures, high labeling efficiency, good biocompatibility, and long-term tracking ability. In addition to these two strategies, metabolic AIEgens were developed for highly specific bacterial imaging. 29 Peptidoglycan was a network of short peptides (L-Ala-D-Glu-mDap-D-Ala-D-Ala) cross-linking the repeated units of N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc). 30 In considering the bacterial cell envelope constituents, the D-Alanine (D-Ala) residue at the peptidoglycan cell wall was a promising target as it could be substituted by exogenous D-amino acids and still undergoes ordinary biosynthesis. Similarly, the LPS in Gram-negative bacteria was tolerant enough to incorporate a modified sugar, 3-deoxy-Dmanno-octulosonic acid (Kdo). Metabolic AIEgens could be incorporated into the bacterial cell walls after incubation with metabolic precursors, that is, D-Ala and Kdo ( Figure 2C). 31 This series of AIEgens possessed high specificity to the bacteria instead of eukaryotic cells. Therefore, metabolic AIEgens were suitable for intracellular bacteria labeling and imaging. AIEgens developed based on these three strategies have been widely applied for bacterial imaging, sensing, detection, and monitoring. Advanced applications in microorganism-related area, such as selectively bacterial imaging, monitoring of bacteria-cell interactions, and subsequent therapy, could be realized by using these basic units. In addition to the above three methods, there is also a staining method, which can target bacterial surface receptors. This staining method specifically recognizes between biological target and hydrophilic target that bind to the AIE core. Ligands, such as proteins, peptides, aptamers, and targeted drugs, can be conjugated to the AIE core to stain bacteria. 19c

| AIEgens for selectively microbial imaging
After effective labeling of bacteria, more information about these tiny organisms were required to be visualized in order to investigate and treat them thoroughly. A variety of AIEgens for the discrimination of various bacterial have been reported. Identification of Gram-positive bacteria and Gram-negative bacteria was a key procedure in clinical examination. It was thus significant to discriminate Gram-positive and Gram-negative bacteria through simple and fast methods. Tang and Wang et al. designed and synthesized a water-soluble, NIR-emissive AIEgen, TTVP, for rapidly distinguishing gram-negative bacteria from positive bacteria, because the cell membranes of Gram-positive bacteria are thinner than those of negative bacteria, so Gram-positive bacteria can be molecularly stained faster than negative bacteria. 32 Ascribing to the water-soluble characteristic of TTVP, it was soluble in aqueous buffer solutions and emitted no light and it could provide an ultra-high signal-tobackground ratio (SBR) in bio-imaging without washing. 36 As shown in Figure 3A, TTVP was reported to selectively target Gram-positive bacteria from Gramnegative bacteria after only 3 s of incubation, which is at least 100 times faster than previously reported methods.
At present, the staining time of common commercial dyes and reported molecules for bacterial imaging is about 5-10 min. Ultrafast bacterial imaging could result from excellent dispersion of TTVP in culture media, strong electrostatic interaction between positively charged TTVP and negatively charged bacterial, and extremely sensitive fluorescence enhancement of AIEgen.
In addition, the viability of bacteria was an important parameter in microbiological study and antibacterial material development. By tuning the bacterial cellular penetration ability and charges of AIEgens, differentiation between live and dead bacteria could be achieved. Tang and coworkers reported TPE-2BA, an AIE molecule that could distinguish dead bacteria from a live one. 33 TPE-2BA was a cell-impermeable and DNA staining molecule that bound to the groove area of doublestranded DNA. 34,37 Bacteria with damaged membranes allowed TPE-2BA to penetrate cell membrane to stain  DNA, endowing it with high emission ( Figure 3B). TPE-2BA showed great potential in high throughput screening of antibiotics efficiency by indicating bacterial viability. In addition, combining TPE-2BA with an AIEgen possessing universal staining ability, a dual channel fluorescence kit for microbial viability visualization has been developed.
Third, intracellular bacteria showed more resistance toward traditional antibiotics than normal pathogens, and their specific labeling was still challenging. Metabolic AIEgens could be a solution for this dilemma. Liu and coworkers reported an AIEgen with pyridiniumsubstituted tetraphenyl ethylene (TPEPy) and D-Ala functional group (TPEPy-D-Ala), which was applied to perform real-time imaging of intracellular bacteria. 34 As shown in Figure 3C, methicillin-resistant Staphylococcus aureus (MRSA) engulfed in RAW 264.7 cells were lighted up specifically. And because of the hydrophilic moieties of D-Ala and pyridinium, TPEPy-D-Ala was mildly emissive in aqueous conditions. Even inside macrophage cells, this ensured a minimal background signal. 24c, 38 In contrast, TPEPy-L-Ala could not stain intracellular MRSA. This work demonstrated that metabolic AIEgens could be applied for intracellular bacterial imaging and relevant study. Furthermore, by rational molecular engineering of AIEgens, more functions, such as PDT, could be introduced into such metabolic systems.
Moreover, fungi possessed more complicated structures than bacteria; typically, they were like mammalian cells. It was more difficult to discriminate fungi from host cells; therefore, it was very important to develop efficient AIEgens for intracellular fungal imaging. Zhou et al. reported a cationic AIEgens (IQ-TPA), which was composed of a cationic isoquinolinium (IQ) moiety and a suitable hydrophobicity range (ClogP values of about 5.5-6.0). 35 Because the intrinsic difference in surface membrane potential between fungal and mammalian cells and mitochondrial membrane potential (MMP) was highly negative, IQ-TPA was reported to preferentially light up the mitochondria of fungi. As demonstrated in Figure 3D, after incubating combinations of human corneal epithelial (HCE) cells and C. albicans with IQ-TPA, strong fluorescence was seen in C. albicans with low fluorescence signals in HCE cells. Such the AIE system is promising for in vivo PDT of fungal infections.

| Visualization of bacteria-cell interactions by AIEgens
Visualization of immunocyte-microbe interaction is of great importance to reveal the physiological role and working mechanism of innate and adaptive immune systems. On the other hand, investigation of the immunocytemicrobe interaction at a subcellular level may provide alternative ways to discover new antimicrobial methods and agents, overcoming the shortage of antibiotic pipelines. 40 Lee et al. reported a new application of TTVP, where TTVP was adopted to trace intracellular Grampositive bacteria. 39 The results indicated that TTVP was capable of monitoring both the engulfing and the commencement of digestive processes of Gram-positive bacteria by macrophages. 36 As illustrated in Figure 4A, the merged image of bright-field and fluorescence signal indicated that B. subtilis was engulfed by the Raw264.7 cell and was then further entrapped into the macrophage. This work also reported that the first step of phagocytosis and engulfing processes occurs between B. subtilis and macrophages that were visualized by using TTVP. In addition, phagolysosomes formed by the fusion of lysosomes with phagosomes were also visualized and investigated.
Furthermore, interactions between organelles with phagosomes were highly dynamic and sophisticated; thus, the powerful and stable labeling fluorescent probes were urgently needed. Recently, Zhao and Tang reported a clickable AIEgen, CDPP-NCS, containing a cationic pyridinium moiety for targeting bacteria and an isothiocyanate (NCS) moiety for covalently bonding with amine groups. 28 CDPP-NCS could stain bacteria through covalent bonding and allowed for more accurate positioning of bacteria. In addition to the successful visualization and monitoring of phagocytosis by use of bacteria that have been labeled with CDPP-NCS to interact with macrophages, the behaviors of lysosomes and mitochondria toward phagosomes were studied ( Figure 4B). 41 In Figure 4C, CDPP-NCS-labeled bacteria were incubated with macrophages for different times. Consequently, the dynamic interactions between phagosomes (red) and lysosomes (green) could be visualized via fluorescence imaging, where lysosomes gradually approached and eventually fused with phagosomes after 6 h. Moreover, another crucial organelle, mitochondria (green), was observed to accumulate around the engulfed bacteria (red) in this work ( Figure 4D). The clickable AIEgens demonstrate a promising avenue to offer researchers direct and vivid imaging information to study sophisticated processes in living cells.

| Selectively killing of intracellular microbial with AIEgens
Theranostic medicine integrating diagnosis and therapy is a promising pathway to fight against pathogens. However, antibacterial theranostic materials reported were disclosed to be trapped in the excessive invasiveness to living mammal cells, leading to unexpected side effects, such as inflammation, allergy, and other biosafety issues. 41,43 Therefore, there still exist demands for improvement in biosafety, accuracy, and efficiency. Zhuang and Zhao et al. designed and synthesized a series of cationic pyridiniumsubstituted phosphindole oxide (PIO) derivatives, which exhibited typical AIE properties. 42 Specifically, their YE ET AL. bioaffinity preference for pathogens over living mammalian cells was tuned by a rational alkyl chain engineering ( Figure 5A). The results turned out that PyBu-PIO was free of living cell invasiveness, had negligible cytotoxicity, and had a high affinity toward Gram-positive bacteria, including drug-resistant strains. 44 In Figure 5B, the bacterial killing ability of these cationic PIO derivatives at various concentrations was shown and the results indicated that PyBu-PIO achieved efficient bacteriostatic effects with a minimum inhibitory concentration (MIC) measured between 5 and 10 μM, for which the survival rates of S. aureus were maintained above 95% at 1 and 2 μM, but diminished by about half at 5 μM and fell to zero at over 10 μM. To demonstrate that the molecule did not affect normal cells, researchers further performed cytotoxicity tests as shown in Figure 5C. PyBu-PIO exhibited no cytotoxic effects with cell viability remaining above 80% even at high concentrations of 40 μM, demonstrating good biocompatibility. This study indicated living cell invasiveness as a criterion for antibacterial theranostic materials and provided a successful example of addressing this issue at the molecular level using a simple strategy.
Moreover, precise targeting and killing of intracellular fungi were considered to be more difficult than intracellular bacteria because fungi possessed much similar structure with mammalian cells. As shown in Figure 5D, E Zhou and coworkers designed and reported three AIEgens, IQTPE-2O, IQ-Cm, and IQ-TPA, with cationic isoquinolinium (IQ) moiety and proper hydrophobicity, could preferentially accumulate at the fungal mitochondria over the mammalian cells. 35 Upon white light irradiation, these AIEgens could generate reactive 1 O 2 efficiently, which caused irreversible damage to fungal mitochondria and further triggered the fungal death. Among them, IQ-TPA showed the highest PDT efficiency against fungi and negligible toxicity to mammalian cells, achieving the selective and highly efficient killing of fungi. Furthermore, they tested the clinical utility of this PDT strategy by treating fungal keratitis on a fungusinfected rabbit model.

| Precise drug delivery system based on AIEgen engineered bacteria system
Live bacteria have drawn a widespread interest as carriers to deliver genes and proteins into eukaryotic cells for the treatment of various cancer types owing to their multiple advantages over artificially synthetic carriers. First, their unique ability to preferentially colonized tumors in an active motility by an aerotaxis or chemotaxis pathway. 46 Secondly, their intrinsic genetic system, which allowed live bacteria to be genetically engineered to deliver tumoricidal agents, such as genes or proteins. 47 Thirdly,  because they were a natural protein-making factory, bacteria vectors were cost-effective compared with most artificial synthetic carriers. 48 However, how to realize effective gene and protein release remains an issue and whether the bacteria could efficiently deliver therapeutic agents remains a big problem. Wu et al. reported a biohybrid system TDNPP-coated Escherichia coli (TDNPP-E. coli), which was constituted by furnishing AIE photosensitizer nanoparticles (TDNP) with cationic polymer and further binding on the surface of bacteria to serve as an AIE photosensitizer delivery vector for precise targeting, effective imaging, and ablation of tumors ( Figure 6A). 45 In addition, the AIEgens coating layer on the surface of E. coli were found to facilitate bacteria to invade cancer cells and efficiently release protein through the production of ROS upon light irradiation ( Figure 6B). Meanwhile, compared to the same photosensitizer NPs without the bacteria carrier, multifunctional TDNPPs delivered by bacteria achieved improved cancer cell imaging and effective light-mediated cancer killing in vitro. The nucleus, as the center of a cell, has been identified as the most appropriate and sensitive subcellular device for an effective PDT treatment outcome. As a result, PSs delivery and accumulation in nuclei should improve photodynamic efficiency for cancer therapy. As shown in Figure 6C, this work demonstrated that the entry of TDNPP-E. coli into the nucleus was caused by active bacteria. This AIE-bacteria biohybrid system presented an alternative strategy to optimize bacteria-mediated cancer therapy and intracellular protein delivery.

| SUMMARY AND OUTLOOK
The extensive applications of AIEgens open a new era of interdisciplinary research. Two application scenarios have been anticipated as shown in Figure 7. On one hand, AIEgens are promising platforms to the inspection of bacterial residues in clinical scenarios. As shown in Figure 7A, specific AIEgens can be used to determine whether the residual number of bacteria on the hand meets the surgical requirements. Through this detection method, bacteria that cannot be eliminated on the doctor's hand can be selectively illuminated instead of normal hand cells. This detection method is not only faster in detection speed, but also very simple and easy to operate, which can provide a convenient method for the elimination of pre-operation and assist in the effective management of smooth operation. On the other hand, bacterial surveillance array to rapidly detect clinical pathogens and evaluate drug resistance could be constructed from AIEgens ( Figure 7B). 49 This array aims at the rapid differentiation, identification, and killing of pathogenic microorganisms, such as bacteria and fungi, in clinical practice. The development of corresponding reagents can fill the shortcomings of pathogenic microorganisms with long growing cycle and high false negative rate, which can help us timely guide the clinical formulation of treatment plans, and improve the accuracy and effect of the treatment of infectious diseases. In addition, AIEgens can also be applied to the detection and treatment of another microbial, virus. For a common virus, influenza virus, its infection of human cells depends on the binding of the trimeric hemagglutinin (HA) molecule on the viral membrane to the sialic acid sugar chain receptor on the host cell membrane, so the detection of influenza virus can be realized by recognizing HA molecules.
In general, the high luminescence efficiency, unique turn-on luminescence property, and superior photostability enable AIEgens excellent probe to detect pathogens and monitoring the microbe-cell interaction process. Additionally, the enhanced ROS generation efficiency of AIEgens in the aggregate state further extends their applications in pathogen killing and microorganism-related therapy. AIEgens can be combined with pathogens to become engineered bacteria, which play an unprecedented role in the diagnosis and treatment of cancer. We hope that this short review about microorganism-related visualization and therapy based on AIEgens could help interdisciplinary researchers to better understand the promising applications of AIEgens in microorganismrelated research, including the fundamental and application research. We also hope that AIEgens could really