Targeted antibacterial photodynamic therapy with aggregation‐induced emission photosensitizers

With the increasing prevalence of infectious diseases caused by drug‐resistant bacteria, there is an urgent need to develop innovative therapies alternative to antibiotics. Among these alternatives, the aggregation‐induced emission (AIE) photosensitizers (PSs) stand out with their integrated imaging and therapeutic functionalities, allowing for early monitoring and image‐guided ablation of bacteria. AIE fluorescent probes with unique optical properties excel in selective bacterial imaging. Furthermore, AIE‐enabled reactive oxygen species (ROS)‐mediated antibacterial photodynamic therapy can operate on multiple targets to oxidize bacteria. Also, as they are able to specifically target bacteria, AIE PSs can ameliorate the limitations of the small‐scale action of ROS. This review methodically discusses the different strategies that can be employed using AIE PSs for targeting bacteria, including sheltered bacteria. The challenges and future opportunities of using AIE PSs in this emerging field are also briefly discussed.


| INTRODUCTION
Since humans can serve as the hosts to bacterial communities, the relationships between humans and bacteria are intricate.On the one hand, humans benefit from probiotics due to their active metabolic and immune mechanisms. 1 On the other hand, humans suffer from many diseases caused by pathogenic bacteria as a result of the limited ability of the human body's resident flora and immune system in resisting the invasion of these bacteria. 2Bacterial infections may seriously endanger human health, and even threaten life.It is estimated that nearly a quarter of the world's population is at risk of tuberculosis as a result of the chronically latent infection with Mycobacterium tuberculosis.Separetely, Staphylococcus aureus is a popular pathogen that usually colonizes the nares, skin, or mucous membranes, where it may penetrate the skin or mucosal barrier to enter adjacent tissues or the bloodstream leading to infectious diseases such as pneumonia and sepsis. 3Antibacterial agents are supposed to have high selectivity for bacteria and be relatively non-toxic to mammalian cells.Fortunately, many antibiotics can induce damage to bacteria through diverse mechanisms, including destruction of cell wall, nucleic acid, protein synthesis, etc. 4 The successful development of antibiotics is a milestone in our ongoing fight against infectious bacteria.However, antibiotics usually aim high-affinity bacterial targets such as particular proteins that can easily mutate. 5The overuse of antibiotics accelerates the process of bacterial evolution following natural laws, which leads to the emergence of multidrug-resistant (MDR) bacterial strains.Therefore, the battle against infectious diseases caused by MDR bacteria and polybacteria continues. 6acteria have evolved cell envelope as an exterior barrier to protect themselves.Based on the fundamental structural differences in the membrane, bacteria can be classified into Gram-positive and Gram-negative bacteria.Both bacteria possess peptidoglycan layer intercalated with teichoic acids, as well as inner membranes made up of a phospholipid bilayer and proteins.The Gram-positive bacteria have a thick layer of peptidoglycan in their cell wall while the Gram-negative bacteria have a thin layer of peptidoglycan.The Gram-negative bacteria have an outer membrane made of lipopolysaccharides (LPS) while the Gram-positive bacteria have no outer LPS.Teichoic acid, peptidoglycan, and LPS in the bacterial cell envelope are negatively charged.Because of that, cationic molecules can bind to bacteria.Antimicrobial peptides (AMPs), which typically have cationic and amphipathic properties, can selectively bind to anionic bacterial membranes via electrostatic and hydrophobic interactions.Such lowaffinity interactions with general non-protein targets make it difficult for bacteria to develop resistance.Although AMPs have broad-spectrum antibacterial and immunomodulatory effects, they have many drawbacks such as poor stability and high cost.Thus, some peptidomimetic compounds with similar structures to AMPs have been synthesized to achieve good antibacterial potency as well as stability.However, the design and prediction of the properties of these compounds appeared to be arduous. 5,7eactive oxygen species (ROS) have been actively explored to induce lethal oxidative damage to bacteria by disrupting their DNA and cytoplasmic membranes.Even undergoing mutations, it is hard for bacteria to evade ROS-mediated ablation because ROS oxidation can act on multitargets, leading to resistance loss. 8Benefiting from the role of ROS in the immune defense mechanism against bacteria, bacterial infections can be potentially treated by improving ROS generation or interfering with ROS defense mechanisms. 9Photosensitizers (PSs) can produce ROS under appropriate light stimulation for broad-spectrum treatment of bacteria and reduce bacterial resistance concerns, making antibacterial photodynamic therapy (APDT) a potential alternative antibacterial modality.
Limited by the intrinsic short lifespan and diffusion distance of ROS, the oxidative effect of ROS on bacteria is largely determined by the localization of PSs to bacteria. 8,10Since PSs often exhibit poor selectivity toward infectious bacteria in APDT, it is worthwhile to improve the selectivity of PSs toward bacteria, and the targeting process can be detected by fluorescence imaging. 11The importance of monitoring bacteria in situ through visualization technology is recognized widely, and fluorescence imaging has the advantages of easy operation, realtime response, and high sensitivity.Many organic PSs with π-π stacking structures can emit fluorescence in dilute solutions when activated, but they tend to form aggregates when they interact with bacteria or when their concentration is increased, leading to quenched fluorescence and decreased ROS generation.4c,12 In contrast to the aggregation-caused quenching molecules, the aggregation-induced emission (AIE) PSs can be activated to turn on fluorescence and photosensitization in an aggregate state rather than in a unimolecular state, mainly owing to the restriction of intramolecular motions and non-radiative decay pathway. 13Taking advantage of the unique properties of AIE PSs, we can assess the selectivity and phototherapeutic effect of AIE PSs on bacteria by detecting emission and photosensitization at bacterial infection sites.
Much effort has gone into winning the tug-of-war against bacteria.The foremost requirement for AIE PS design is bacterial localization and specific targeting.Fortunately, the surface structural characteristics and life activities of bacteria (e.g., membrane charge, expressed protein shape, unique cell wall compositions, and metabolic pathways) offer useful information to guide the design of AIE PSs.4b Exploiting the predator-prey relationship between phages or macrophages and bacteria is also a reliable strategy to design AIE PSs to target bacteria.In this review, we summarize the bacteria-targeting strategies based on AIE PSs and categorize them into five types depending on the modes of interaction, which are schematically shown in Scheme 1: (A) AIE PSs with cationic group and a rather proportion of hydrophobic groups can interact with the negatively charged bacterial membranes via cations and integrate into the hydrophobic core of the membrane according to the similarityintermiscibility theory. 14 Hence, AMPs with positive charge and amphipathic properties can attach to bacterial membranes and insert themselves into lipid membranes, leading to membrane destabilization and membrane integrity disruption. 16ost PSs are hydrophobic structures.Inspired by the structure of AMPs, a fairly common targeting strategy is to introduce cations to AIE PSs to obtain amphiphilic molecules.Highly hydrophilic AIE PSs may simply attach to bacterial membranes rather than penetrate through them.Therefore, the cationic charge and hydrophobic ratio of AIE PSs need to be properly tuned to realize bacterial targeting through electrostatic and hydrophobic interactions.14a Furthermore, AIE PSs with amphiphilic properties show good water solubility in physiological hydrophilic conditions, ensuring a low fluorescence background signal.
Numerous AIE probes have been fabricated with pyridine salts or quaternary ammonium salts as hydrophilic terminal groups to endow the probes with antibacterial properties. 17Considering the characteristics of AIE and the limited range of ROS in biological systems, organelle-specific targeting AIE PSs would have enhanced photostability and photodynamic therapy (PDT) efficacy. 18Liu et al. constructed an AIE PS named TBD-anchor with long alkyl chains and three quaternary ammonium salts (Figure 1A). 19Fluorescence imaging visually verified that the TBD-anchor was capable of targeting bacterial cell membranes, and the isothermal titration calorimetry further confirmed its binding mode as electrostatic and hydrophobic interactions.The TBDanchor with a singlet oxygen ( 1 O 2 ) quantum yield of 0.48 has broad-spectrum antibacterial activity against Escherichia coli and S. aureus.It displayed a better inhibitory effect on methicillin-resistant S. aureus (MRSA) than antibiotics under light illumination (Figure 1B).Upon 15 J/cm 2 irradiation, the inhibition rate of 800 nM TBD-anchor against MRSA exceeded 99.8% (Figure 1C), indicating that the membraneanchored PS with efficient 1 O 2 generation is promising to kill MDR bacteria through specific membrane damage.
Motivated by the membrance structure of amphipathic phospholipids, a group of phospholipid-mimetic AIE PSs with pyridine salt and extended length of substituted carbon chains were designed with selectivity toward the two types of bacteria.20c It was reported that the phospholipid-mimetic AIE PSs with short alkyl chains manifested specific antibacterial activity against Gram-positive bacteria.On the other hand, those with long alkyl chains exhibited a killing effect on Gram-negative bacteria.Fluorescence imaging showed that the phospholipid-mimetic molecules with short carbon chains could diffuse from the bacterial membrane into the cytoplasm, while those with long carbon chains were primarily inserted into the membranes.In contrast to Gram-positive bacteria, Gram-negative bacteria have a much thinner peptidoglycan layer and outer membranes containing lipids and LPS.The thick peptidoglycan layer of Gram-positive bacteria limits the access of large-size nanoparticles (NPs) (>10 nm). 20By analyzing the mechanism of regulating the length of the alkyl chain to realize antimicrobial selectivity, it was hypothesized that long alkyl chains could contribute to the ablation of Gram-negative bacterial membranes, which was ascribed to the increased hydrophobicity.On the other hand, amphiphilic molecules with short alkyl chain could selfassemble to form small-sized NPs to penetrate Grampositive bacterial membranes and exert antibacterial properties (Figure 2A).Synthetic molecules with pyridine salt connected to methyl or dodecyl both executed negligible toxicity to normal cells, leading to a minimal pathological change to major organs after treatment.Taking into consideration the different characteristics of the two types of bacterial membranes, there is an opportunity to control the interactions between molecules and bacterial membranes for specific targeting by simply modulating the structures of molecules.This unique molecular design provides a way to develop antibacterial medicines to achieve selective killing.Nevertheless, the mechanism and application of fine-tuning molecular hydrophobicity to damage the outer membrane instead of the thick peptidoglycan layer remains worth exploring.For example, a previous study reported that in a series of synthetic AIE PSs containing quinolinium with different alkyl chain lengths, the one with n-hexane displayed the best inhibitory effect on both S. aureus and E. coli, and its performance was enhanced when connected with quaternary ammonium salt.Research also found that the increase in the basicity of molecular end groups might enhance the antibacterial activity against Gram-negative bacteria, but decreases the antibacterial activity against Gram-positive bacteria.With the increase in alkyl chain length of molecules containing basic amine groups such as amidines, guanidines and quaternary bases, the antibacterial activity increases up to a maximum and then decreases. 21nspired by the antibacterial activity and facile cationization of azole groups, the non-PS AIE motifs can be converted into AIE PS by connecting with selected antibacterial agents.The antibacterial agents should be able to receive electrons transported from the AIE donor segment, which then contributes to the intersystem crossing process that generates ROS.Azole drugs have been frequently used as antifungals for selectively targeting fungal 14αsterol demethylase, whose homologs also exist in the cytochrome P450 monooxygenase (P450s) of some bacteria; thus, azole drugs are promising as antibiotics. 22Both naphthylimide and triazole groups can act easily on receptors in biological systems through non-covalent forces, and naphthylimide triazoles are regarded as potential antimicrobial agents for their good antibacterial activity against both Gram-positive and Gram-negative bacteria. 23aking advantage of the antibacterial activity of common antibacterial azoles-substituted naphthalimide and the charge transfer between AIE element triphenylethylene and naphthalimide triazole, Tang et al. synthesized an AIE PS named triphenylethylene-naphthalimide triazole (TriPE-NT) with ROS production ability and antibacterial activity which were more superior than the antibiotics polymyxin. 12TriPE-NT showed no obvious cytotoxicity to normal cells, and superior targeting of Gram-positive bacteria over Gram-negative bacteria, which might be due to electrostatic and ligand-targeted interactions.TriPE-NT at the micromolar level could exert antibacterial activity under dark conditions to kill almost all Staphylococcus epidermidis and MDR S. epidermidis due to the antibiotic effect.It showed much better performance under white light irradiation (4 mW/cm 2 ) to kill nearly all E. coli, MDR E. coli, Klebsiella pneumoniae, S. aureus, MDR S. aureus, and 61% MDR K. pneumoniae with the support of PDT (Figure 2E).Developing such drugs with dual functionalities of antibiotic and PS to selectively inhibit bacterial infections and promote wound healing is an attractive antibacterial strategy.
The amphiphilic AIE PSs containing cations are easy to prepare and their properties can be adjusted by molecular engineering.However, they may be rapidly metabolized in vivo or bound by potentially interfering substances in complex physiological environments due to the low molecular mass and non-specifically physical interaction between molecular probes and biological targets. 24To improve specific recognition and treatment, approaches capitalizing on ligand-receptor recognition interaction can be employed to bind macromolecular biological targets on bacterial surfaces.

| Ligand-receptor interaction
Cellular activities cannot be divorced from the membrane surface receptors (mostly proteins) that can bind smallmolecule ligands covalently or noncovalently to mediate signal transduction. 25Ligand and receptor must fit precisely together like a key and a lock, where the binding must exhibit biological activity consistent with high affinity. 26With this in mind, AIE PSs with targeting ligands have been developed to bind to high-affinity receptors on bacterial membranes, intending to distinguish the different types of bacteria and reduce side effects.

| AIE PS-antibiotic
Based on the lock and key principle, the geometric structural and chemical complementarity of the ligand and receptor are the main factors determining the specific binding.The essential contribution of hydrogen bonds in structural recognition and binding affinity of receptors and ligands has been reported previously. 26Vancomycin (Van) can form complementary hydrogen bonds with D-Ala-D-Ala C-terminal dipeptides of peptidoglycan precursor, hence blocking the peptidoglycan synthetic pathway. 27Since the D-Ala-D-Ala C-terminus is universal in bacterial peptidoglycan, Van has been used for targeted therapy of Gram-positive bacteria surrounded by thick peptidoglycan layer, until the emergence of vancomycinresistant enterococcus (VRE).VRE has evolved resistance to Van such as the synthesis of D-Lac or D-Ser to substitute the C-terminal D-Ala residue to diminish the binding affinity (over 1000-fold) between the peptidoglycan precursors and Van, or the elimination of the highaffinity precursors to prevent the binding. 28Combining the advantages of Van in bacterial recognition and PDT in antimicrobial resistance, AIE-2Van comprising an AIE PS and two Van glycopeptides was devised for selective identification, wash-free imaging, and photodynamic disruption of Gram-positive bacteria, including VRE (Figure 3A). 29AIE-2Van could be used for wash-free imaging due to its AIE property, which exhibited almost no fluorescence in dilute aqueous solution and its fluorescence intensity primarily corresponded to its state of interaction with bacteria.With the support of graphene oxide to pre-quench background noise, the activated lightup fluorescence level of bacteria visualized with the naked eye could credibly reveal the degree of binding.The varying fluorescence intensity indicated that the developed probe had a high binding affinity for Gram-positive strains, a medium binding affinity for VRE, and a low binding preference for Gram-negative bacteria.The therapeutic effect of AIE-2Van on different kinds of bacteria as reflected by the minimum inhibitory concentration, showed the same trend as their recognition ability.Under dark conditions, the therapeutic efficiency of 10 μM AIE-2Van inheriting cell membrane damage by Van against Bacillus subtilis, Enterococcus faecium (Van A), and E. faecium (Van B) were about 100%, 20%, and 30%, respectively.Upon light irradiation, PDT significantly boosted the killing effect of AIE-2Van on VRE at the same concentration (70% for Van A, 90% for Van B) (Figure 3B).The efficacy of AIE PSs conjugated with targeting ligands in tackling drug-resistant bacteria makes them excellent candidates for antimicrobial therapy.
Apart from direct attachment to Van, AIE PSs can integrate with Van-modified self-assembling peptides to improve delivery efficiency.Liu and colleagues developed E-probe combining Van-modified self-assembling peptide with AIE PS, which could assemble in situ on the bacterial surface to sensitively detect and ablate Grampositive bacteria (Figure 3C,D). 30In vitro experiments suggested that the self-assembly ability of the E-probe was beneficial to enhance the emission and 1 O 2 generation of AIE PSs.In vivo imaging and inhibition of E-probe on a myositis-bearing BALB/c nude mice model showed that E-probe was useful for sensitive diagnosis and therapy of Gram-positive bacterial infection.This could be attributed to the role of assembly in causing a stronger multivalent interaction between Van and biotargets to induce good accumulation of E-probe at the infection site.In virtue of the blessings of self-assembly to AIE PSs and targeting, AIE PSs combined with targeting structures modified self-assembling peptide can be regarded as a potential strategy to overcome VRE.Such conjugates of antibacterial compounds with bacteria-targeting capabilities and self-assembling peptides can act as AMPs, and in situ self-assembly within bacteria further amplifies the fluorescence and therapeutic effects of AIE PSs.

| AIE PS-phage
Phages are bacterial viruses that can specifically recognize and lyse bacteria.They have viral genomes in the form of DNA or RNA encased in a protein capsid, which can be injected into the bacterial host through numerous ways and proliferate in a replicating manner.Tailed phages adhere to receptors on the bacterial surface using receptor-binding proteins at the distal end of their tails, whereas tailless phages have other attachment mechanisms.Phages have the advantage of specifically targeting bacteria. 31Although phages can evolve to overcome bacterial resistance to a certain extent, the evolutionary arms race between phages and resistant bacteria is probably won by bacteria at the expense of virulence and fitness.Fortunately, phages can synergize with other antimicrobial therapies to reduce the probability of the evolution of bacterial resistance.To compensate for the inability of phages to diagnose bacterial infection, phages can be conjugated with AIE PSs to achieve enhanced antimicrobial treatment with monitoring functionality.Tang et al. constructed an AIE-PAP bioconjugate equipped with bacteriophage (PAP), which inherited the targeting function of phage and could perform AIE imaging to guide PDT to cooperate with phage therapy (Figure 4A). 32The synthesized PS TVP-S with a 1 O 2 generation quantum yield of 72.3% could be conjugated with phage through an esterification reaction to obtain TVP-PAP.It was ascertained that normally 8200 AIE PS bound to one PAP entity, which was calculated by the ratio of molar extinction coefficients between the two moieties.Since TVP-PAP could recognize the corresponding host bacteria in 30 min, TVP-PAP with the inherited targeting capability from phage had better selectivity than AIE PSs relying on electrostatic and hydrophobic interactions.TVP-PAP showed selectivity and eradication of its target bacteria, and it could kill MDR Pseudomonas aeruginosa with almost 100% efficiency based on the combined therapeutic effects of phage and APDT.P. aeruginosa treated with TVP-PAP irradiated by white light experienced roughened or even ruptured cell walls, and the perforation effect of phage on the bacterial surface could facilitate further penetration of 1 O 2 , demonstrating the superiority of synergistic therapy.AIE in combination with phages mapped to bacteria can be an attractive solution to tackle various MDR bacteria, which becomes a general way to fight against various MDR bacteria by changing the species of phages.
In addition, the phage's host specificity leads to a narrow host range, which can be improved using phage cocktail therapy that can treat the bacterial infection with a wide range of hosts such as sepsis.Gu's group developed AIE-PS-engineered phages for early sepsis diagnosis and extracorporeal phage cocktail-photodynamic blood disinfection (Figure 4B). 33The prepared PS TBTCP-PMB with benzyl bromide group could undergo nucleophilic reaction with plentiful sulfhydryl functional groups of proteins on four phages, which have specificity toward major sepsis pathogens, to obtain a functional F I G U R E 4 (A) Schematic illustration of AIE PS conjugated phage for specific bacterial targeting, fluorescent imaging, and synergistic killing.Reproduced with permission.32c Copyright 2020, American Chemical Society.(B) Schematic representation of the cocktail therapy based on the specific targeting of AIE-PS engineered phage for recognition and destruction of bacterial species in sepsis.Reproduced with permission. 33Copyright 2022, John Wiley and Sons.AIE, aggregation-induced emission; PS, photosensitizer.phage-engineering system based on AIE-PS.TBTCP-PMB-engineered phages could identify pathogen species of sepsis quickly and accurately and exhibit nearly 100% antibacterial efficacy at a low concentration of 80 nM while causing negligible damage to phages.The blood in the established mouse sepsis model was effectively disinfected in vitro by AIE-PS-engineered phages.This, could then improve various sepsis symptoms, including multiple organ dysfunction, inflammation, and tissue fibrosis, and boost rehabilitation containing angiogenesis and tissue repair by hemodialysis treatment to cure sepsis.This approach is expected to be applied for detecting bacterial species in clinical samples in a short time (≈30 min).However, phage therapy in vivo also faces many challenges such as being quickly cleared by the immune system or antibody responses, so there is still a long way to go.

| AIE PS-glycomimetic
Many bacteria produce lectins, carbohydrate-binding proteins exposed in the form of fimbriae expanding from the bacterial surface, to bind oligosaccharide-containing glycosphingolipids on the surface of the host cells at the initial stage of infection for the sake of mediating bacterium-cell recognition and adhesion. 34Multivalent sugar ligands can attach to lectins with high affinity through C-H•••π interaction and cooperative hydrogen-bonding motifs, 35 resulting in bacterial agglutination and interference with lectin-mediated bacterial adhesion. 36lectrospun polystyrene comaleic anhydride fibers bound to mannose and AIE element have been designed to sensitively detect E. coli based on sugar-binding properties of lectin FimH overexpressed on bacterial membrane and turn-on fluorescent of AIE structure, while they were incompetent to eliminate E. coli. 37Many glycolactosefunctionalized glycomimetics have been used to interact multivalently with lectin LecA to prevent P. aeruginosa adhesion, and cause bacterial agglutination that may affect the antibacterial therapeutic efficacy. 38Glycomimetics can only exert limited antibacterial effects by acting on lectin sites to inhibit virulence factors and biofilm formation.Therefore, glycomimetics combined with AIE PSs have been resorted to compensate for their roles in bacterial imaging and treatment.Qiao et al. developed TPyGal containing cationic AIE PS and galactosyl groups, which could self-assemble into spherical aggregates with multiple galactosyl groups on the surface to monitor and damage P. aeruginosa (Figure 5A). 39Supported by the synergistic interactions of polyvalent sugar-lectin and ancillary electrostatics, TPyGal could target and cluster P. aeruginosa, and then insert into the bacterial membrane for PDT in situ (Figure 5B).Under light irradiation (20 mW/cm 2 ), TPyGal almost killed all P. aeruginosa at the concentration of 20 μM while caused less damage to normal liver cells LO 2 up to 128 μM, which proved the selective recognition of bacteria by TpyGal.In vivo evaluations demonstrated that TPyGal could reliably and biocompatibly cure P. aeruginosa-infected wounds in mice models (Figure 5C).Therefore, the AIE PS-glycomimetic strategy offers a prospective platform for preventing MDR bacteria invasion into host cells and healing infected wounds.
AIE PSs conjugated with targeting ligands can interact with receptors on the bacterial membrane with high specificity.However, the lock-and-key pairings can be influenced by various interferents similar to target analytes in the physiological environment.To avoid compromising targeting, which may be caused by nonanalytes interference, a bacteria-targeting strategy based on reactive chemical reactions to bind excess markers on bacteria was developed.

| Responsive targeting
The overproduction of acidic metabolites, bacterial proteins, and free radicals in infected tissues creates a unique environment with specific stimulations (acidity, highly expressed enzymes, and abundant ROS).4b,8 AIE PSs with reactive cleavable groups were exploited to specifically respond to endogenous stimuli for selective targeting of bacteria based on intrinsic differences in chemical reactivity. 15

| pH response
Due to the diffusional limitation of the biofilm matrix, the acidic metabolites of bacteria accumulate at the site of infection, resulting in acidic microenvironment (pH 4.5-6.5). 8,40Bibo et al. prepared zwitterionic pH-responsive nano-micelles with AIE PS polyurethane (PU) as the core and carboxybetaine as the shell, which could gain positive charges through protonation under acidic condition to realize enhanced APDT. 41Under the acidic condition, the zeta potential of PU nano-micelles increased significantly because of the protonation of the carboxybetaine moiety imposing the increase in positive charge, which proved the binding of nano-micelles to bacteria was driven by electrostatic interaction.The pH-responsive AIE PS nano-micelles dramatically improved biocompatibility and antimicrobial efficiency under acidic conditions.However, the excessive exposure duration in in vitro antibacterial test limited its clinical application.

| Enzyme response
Phagocytes, including macrophages, are the front-line guard in the host defense mechanism against pathogenic bacteria by engulfing and removing invading organisms.Neutrophils are pioneer cells recruited to the site of inflammation via chemokines to phagocytose bacteria and tissue fragments.Macrophages play an important role in host defense mechanisms against pathogenic bacteria.Macrophages can phagocytose pathogens and induce the activation of Caspase-1 to initiate an innate immune response, and then an adaptive response. 40,42Active Caspase-1 can act as a protease to cleave proteins such as pro-IL-1β and be specifically recruited into bacteriacontaining phagosomes, 43 on the basis of this, Liu's group proposed a molecular probe (PyTPE-CRP) with an AIE PS (PyTPE) and an enzyme-responsive peptide linker (NEAYVHDAP) specific to Caspase-1 in phagosomes for bacterial infection detection and killing. 44It was noted that viable bacteria could induce the activation of Caspase-1 enzyme to specifically cleave PyTPE-CRP, and the resultant hydrophobic residues would self-assemble into aggregates on the bacterial phagosomes (Figure 6A).As such, the fluorescence of PyTPE-CRP was significantly enhanced in bacterial phagosomes, in which the ROS generation was about 2.7 times higher than that in the cytoplasm after incubation with bacteria-infected macrophages (Figure 6B).Furthermore, under white light irradiation (40 mW/cm 2 , 10 min), 20 μM PyTPE-CRP could destroy almost all intracellular and extracellular S. aureus with negligible cytotoxicity to macrophages.This study showed that the target-specific activatable AIE PS strategy can detect bacteria hidden in macrophages to guide antibiotic treatment, hence avoiding damage to macrophages and abuse of antibiotics.

| ROS response
Phagocytes like macrophages can stimulate the release of ROS such as hypochlorous (ClO − ) and peroxynitrite (ONOO − ) to destroy bacteria, which are suitable as specific markers of bacterial infection in response to the cleavable probe.However, certain bacterial species have evolved multiple strategies to block immunity or survive in macrophage phagosomes, and the survival and proliferation of the bacteria hidden in host phagocytes may lead to repeated infections. 45It is therefore worthwhile to develop tools that are responsive to endogenous ROS produced by phagocytes and kill the in situ intracellular bacteria.Based on the Förster resonance energy transfer (FRET) between PS TBD and near-infrared (NIR) IR786S, and the specific response between IR786S and ONOO − or ClO − , IR786S can function as an intermediate to indicate a bacterial infection and regulate the activity of TBD.In 2018, Liu's group reported 33% IRTP NPs through the self-assembly of an amphiphilic TBD-PEG and IR786S, which could be activated against bacterial infection in phagocytes for highly specific bacterial diagnosis and treatment (Figure 6C). 46IRTP NPs containing 33% IR786S could quench the fluorescence and 1 O 2 generation of TBD-PEG by FRET, instead only exhibiting NIR emission of IR786S without producing 1 O 2 by TBD.Once IR786S in 33% IRTP NPs was decomposed by ONOO − or ClO − overexpressed at the infection sites, TBD-PEG could be activated to restore red emission and 1 O 2 production for bacterial imaging and APDT (Figure 6D,E).
In a subsequent study, Liu's group developed HClOactivatable DTF-FFP NPs by encapsulating AIE PS DTF and NIR FFP with Pluronic F127 for selective imaging and precise APDT.45c Normal cells treated with DTF-FFP NPs only showed NIR fluorescence of FFP, while the emission and ROS generation of DTP were blocked due to FRET between DTF and FFP.Once DTF-FFP NPs accumulated at the infection sites, the FFP in DTF-FFP was decomposed by the excessive release of HClO from bacteria-containing macrophages.This then resulted in the weakening of NIR emission but activation of the red emission and ROS production of DTF for imaging-guided precise eradication of bacteria.This ROS-responsive theranostic nanoplatform provides a way to detect and eliminate bacteria by selectively switching on the fluorescent and photosensitive properties of AIE PSs.
AIE PSs containing reactive groups (e.g., cleavable/ decomposable quenchers) can chemically respond to special chemical or biological stimuli at infected sites, while avoiding interference from other factors.However, stimulus-responsive AIE PSs are cumbersome to fabricate and characterize, and their effectiveness and safety in clinical application against bacteria infection remain to be validated. 8Inspired by the natural metabolic reactions in organisms, AIE PSs-based metabolic labeling techniques to target bacteria have been actively explored.

| Metabolic labeling
Metabolic labeling strategy is a chemical labeling technique that emerges at the historic moment, capitalizing on metabolic precursors such as D-amino acid analogs or trehalose analogs to integrate into the peptidoglycan wall or mycomembrane of corynebacterial through natural metabolic processes. 47 2.4.1 | Direct metabolic labeling AIE PS modified with metabolic precursors can conveniently and one-step metabolically label bacteria in line with the need of practical applications. B The good water dispersibility and very small size (~0.5 nm) of TPACN-D-Ala ensured that the probe could diffuse and accumulate in the host cells or biofilms (Figure 7B,C).TPACN-D-Ala could label bacteria via the D-amino aciddependent metabolic pathway and destroy invasive drugresistant bacteria in host cells without affecting the host cell viability.Importantly, the PDT effect of this probe was more potent than that of methylene blue.TPACN-D-Ala could penetrate biofilms with ease and be metabolically absorbed by sheltered bacteria, thereby successfully lighting up and ablating bacteria through PDT.After intravenous (i.v.) injection, TPACN-D-Ala could specifically label bacteria in infected tissues and realize precise APDT (Figure 7D).This bacterial metabolic probe could be embedded in the bacterial peptidoglycan and could precisely label and ablate hidden bacteria by photoactive AIE PS.
However, the structural discrepancies between different species of bacteria present additional challenges.The peptidoglycan cell wall of Gram-negative bacteria is additionally surrounded by an outer membrane containing dense LPS, which acts as a barrier to molecules with a molecular weight >650 Da.Interestingly, 3-deoxyd-mannosuccinate (Kdo) can be metabolically absorbed to specifically bind to LPS, similar to exogenous D-amino acids which can substitute D-Ala residues to be incorporated into peptidoglycan. 49Based on the fact that D-Ala and Kdo can bound to the peptidoglycan of Gram-positive bacteria and the LPS of Gram-negative bacteria, respectively, Tang's group prepared AIE PSs bridging the metabolic precursors TPEPy-Ala and TPAPy-Kdo that could label and destroy intracellular Gram-positive and Gram-negative bacteria with high specificity through their respective metabolic pathways (Figure 7E).14a Possibly due to the positive charge of TPAPy, the destruction of bacteria by TPAPy-Ala and TPAPy-Kdo came at the expense of certain interference to the host cell to some extent, while a slight flaw could not obscure the luster of jade.Therefore, it can be reasonably predicted that the one-step metabolic strategy combining AIE PS with metabolic precursors has broad prospects in the absence of endogenous metabolic precursors.

| Bioorthogonal metabolic labeling
The combination of bioorthogonal chemistry and metabolic engineering broadens the scope of the applications of metabolic labeling.Generally, bioorthogonal covalent labeling introduces biomolecules containing bioorthogonal functional groups and metabolic precursors to establish connections between probes with reactive functional groups and biological targets.The bioorthogonal metabolic labeling process to target bacteria is carried out in two steps.First, metabolic precursors (e.g., D-amino acids, N-acetylmuramic acid) can be functionalized by chemical groups (biorthogonal groups) to form bioorthogonal chemical reporters.Such chemical reports could integrate into bacteria through metabolic processes to be employed as an artificial marker.Next, once the chemical reporter is modified on the bacterial surface, probes with complementary bioorthogonal tags can label bacteria through click reaction. 50ased on the copper-catalyzed azide-alkyne cycloaddition, click reaction applicable to living systems, Liu et al. established an AIE PS TPEPA linked to alkyne groups, which could metabolically label bacteria modified with azide group for photo-guided bacterial discrimination and therapy (Figure 7F,G). 51The rate of ABDA decomposed by TPEPA (8.4 nmol/min) was higher than that by Ce6 (5.5 nmol/min).Fluorescence imaging revealed that in the presence of copper catalyst, a much shorter time (30 min) than traditional culturingbased techniques was sufficient for TPEPA to selectively label and identify Kdo-N 3 -pretreated gram-negative bacteria and D-Ala-N 3 -pretreated Gram-positive bacteria (Figure 7H).TPEPA also exhibited highly selective killing effects on bacteria and exhibited good biocompatibility with mammalian cells.This bioorthogonal fluorescent turn-on probe enables fast imaging and precise ablation of pathogenic bacteria.Since rapid rates are required for successful bioorthogonal chemical reactions under physiological conditions, it is preferable to label bacteria with biocompatible copper-free bioorthogonal methods. 52s simple metabolic precursors modified with bioorthogonal groups can be easily inserted into bacterial structures, the two-step metabolic labeling method can avoid the interference of macromolecular fluorescent probes on bioorthogonal reactions and biological functions if the complex operation, time-consuming, and challenging carrier can be tolerated.
Overall, AIE PSs with metabolic precursors or functionalized groups that are complementary to chemical reporters can be used for highly precise targeting, luminescence imaging, and antibacterial PDT via direct labeling or bioorthogonal covalent labeling.Aside from the strategies mentioned before, many other approaches based on AIE PS targeting bacteria that are not yet systematically summarized but are novel ideas have also been proposed.

| Other ways
Considering that a single targeting strategy may be constrained by the physiological environment, AIE PS with two or more targeting elements can potentially combine the strengths of different targeting strategies complementing each other to ultimately improve the targeting of bacteria.In addition, to improve target identification accuracy, AIE PS-loaded macrophages have been developed for precise bacterial eradication in combination with APDT and adoptive transfer therapy.

| Multiple targeting functionalities
Responsive elements can serve as carriers for transporting metabolic molecules to inflammatory lesions, thus improving drug delivery efficiency.Although bioorthogonal metabolic labeling has shown great potential in targeting bacteria in vitro, its application for in vivo bacterial detection and therapy still faces problems such as the easy metabolism of D-Ala due to its good water solubility.Therefore, carriers encapsulating metabolic precursors modified with bioorthogonal functional groups that can reach and dissociate only at inflammatory lesions are highly attractive.Metal-organic frameworks stand out for their low toxicity and stimulus responsiveness.Similar to most solid tumors, inflammation sites exhibit enhanced vascular permeability mediated by multiple vascular factors such as nitric oxide and vascular permeability factor. 53Based on MIL-100 can be degraded by oversecreted H 2 O 2 at the infection site and NPs can passively target the bacterial infection area through enhanced permeation and retention effect (EPR), MIL-100 was used to encapsulate D-AzAla, and then wrapped within polymer pluronic F-127 to form D-AzAla @MIL100 (Fe) NPs for in vivo metabolic labeling in bacteria (Figure 8A). 54After i.v.injection of D-AzAla@MIL-100 (Fe) NPs into the bacteria-bearing mice, MIL-100 (Fe) could preferentially accumulate in the infected tissue because of the EPR effect and be activated by the overexpressed For the sake of selectively matching specific bacteria, a targeting strategy equivalent to a high-scoring answer sheet toward bacterial questionnaire that antibacterial agent can bind specific bacteria through metabolic pathways, electrostatic and ligand-receptor interactions is proposed.The detection of specific bacterial species with existing methods requires "cold chain" reagents such as antibodies and aptamers that are relatively unstable and costly, which have hindered their practical applications.Although AIE PS with targeting ligands can bind to bacteria through highly specific receptor-ligand interactions, it is not yet viable to construct a platform for receptorbased selective recognition of specific bacterial species due to the limited access to microbial surface information.Fortunately, almost all types of microorganisms form extracellular matrix (ECM) to defend themselves and participate in life activities such as signaling. 55With the knowledge of bacterial metabolic pathways, it is possible for bacteria to guide synthetic monomers to synthesize ECM via copper-catalyzed free radical polymerization. 56urther study showed that it was feasible to synthesize biomimetic extracellular polymeric substances on the surface of templating bacteria by copper-mediated atom transfer radical polymerization (ATRP) for selective templating bacteria recognition. 57Liu's group recently utilized bacteria as a template to synthesize AIE acrylic polymer via copper-catalyzed ATRP for selective binding and killing of the templating bacteria. 58The synthesis of templated polymers took advantage of the bacterial metabolic pathway of ECM formation and intermolecular electrostatic and ligand-receptor interactions, to screen the sequences of three monomers bound to bacterial surfaces, including a cationic molecule, an AIE PS, and an amphiphilic polymer (Figure 8B).The templated polymers could bind to the templating bacteria at a low concentration of 600 ng/mL but had a low binding affinity to the mismatched bacteria because the monomeric sequence was encoded by the surface components of the templating bacteria (Figure 8C).Due to AIE PS monomer TMAEMC-TPAPy with ROS generation ability, 2.4 μg/mL templated polymer could selectively deactivate the corresponding bacteria, even the MDR strain, under light irradiation (20 min, 40 mW/cm 2 ).This bacterium-templated method has the potential to be widely used for the diagnosis and treatment of specific bacteria if the current production yields (0.5%-0.7%) can be increased.

| Adoptive macrophage transfer
Macrophages can recognize the receptors targeting the "pathogen-associated molecular patterns" of bacteria to initiate phagocytosis and enclose bacteria in phagosomes.42a Phagosomes can then recruit and fuse with lysosomes to form phagolysosomes with the ability to degrade and kill bacteria.However, some bacteria have evolved mechanisms to resist this digestion and survive in phagolysosomes. 59Relying on the self-directed capture of pathogenic bacteria by macrophages, Liu et al. constructed AIE PS TTD-loaded macrophages (TLMs) to achieve accurate APDT through an adoptive transfer pathway. 11By modulating electron acceptors to improve PS efficiency, the synthesized AIE PS TTD with three acceptor units exhibited strong emission and ROS generation.TTDm NPs were then formed with encapsulating TTD in the core of polymer matrix NPs and modifying lysosome-targeting morpholine on the NP surface (Figure 9A).TLMs were established to realize precise recognition of bacteria because TTDm NPs could accumulate in the lysosomes of macrophages, and then encountered the engulfed infectious bacteria in the phagolysosomes (Figure 9B,C).Transferring the adoptive TLMs into infected tissues could accomplish specific capture, recognition, and APDT of bacteria.It was illustrated that the adoptive TLMs were induced into the pro-inflammatory M1 phenotype in the early stage of infection without impacting wound repair during the post-infection.This APDT strategy based on TLMs demonstrates the precise combination of bacteria and AIE PS through adoptive transfer, which may be used to combat severe bacterial infections if the long-term survival of TLMs in bacterial infection can be ensured.
As it is recognized that AIE PSs can generate ROS to act on multiple targets for broad-spectrum bactericidal action, ensuring their effective accumulation within bacteria is a key factor for their efficacy.Since bacterial resistance mechanisms include preventing the binding of drug to its target and altering the structure of drug targets or drugs, the combination of multiple targeting elements and the recognition of pattern recognition receptors on the bacterial surface by macrophages can avoid resistance interference to a greater extent. 60

| CONCLUSION AND PERSPECTIVES
To date, a wide range of AIE PSs developed using chemical or physical methods, such as molecular engineering, polymerization, and carrier encapsulation, have been explored for different biomedical applications, although the development of AIE PS targeting bacteria is still in its early stage and holds broad prospects.AIE PSs have several advantages in terms of bacterial detection and treatment: (1) luminescence and ROS generation of AIE PSs enables simultaneous optical imaging and treatment of bacteria, (2) spatiotemporal selectivity of PDT induces negligible toxicity to normal tissues, (3) APDT is a broadspectrum bactericidal treatment modality, (4) APDTbased treatment toward bacteria does not cause drug resistance, (5) AIE PSs can synergize with other antibacterial medications to realize a better therapeutic effect.Despite these unique advantages, the clinical translation of AIE PSs for antibacterial therapy still faces many challenges: (1) The potential biotoxicity of AIE PSs has not (3) As the optimal spectral window for in vivo APDT is around 1 μm, developing NIR or multiphoton AIE PSs capable of absorbing long-wavelength light is an avenue to overcome the issue of limited penetration depth. 61AIE PSs based on chemical, mechanical, or bioenergetic excitation can fundamentally address the drawback of restricted depth of excitation light to penetrate tissue.Compared with light-mediated treatment, sonodynamic therapy based on low-intensity ultrasound performed higher treatment efficiency and fewer side effects by virtue of the local focusing characteristics and deeper tissue penetration. 62(4) Multimodal theranostic systems outperform individual-modality treatment.Therefore, it is preferable to develop multifunctional AIE PSs with PDT and photothermal therapy capabilities or to combine AIE PSs with other imaging or therapeutic approaches.(5) Poor delivery of AIE PSs to the infected tissues will cause inferior imaging and therapeutic outcomes, so the bacterial targeting ability of AIE PSs is a prerequisite to highly specific illumination and photoinactivation of bacteria.Overall, the bacteria-targeting strategies of AIE PSs discussed in this review revolve around the information provided by the surface characteristics and cellular activities of bacteria, ranging from simple chemical modification of AIE PSs including attachment of cationic groups, targeted ligands, or responsive groups to physiological functionalization of AIE PSs guided by special biological principles, including metabolic processes and inter-organismal predatory relationships.
Although there are some challenges ahead, AIE PSs are expected to carve out a place in the field of antibacterial therapy with the continuous improvement of cognition level, scientific technology, and research methods.The interactions of bacteria with AIE PSs are mainly dominated by the bacterial surface structure and properties.Through a series of methods such as advanced microscopy, microbiology, immunology, and molecular biology, more bacterial surface information can be gained, which can provide guidance to explore new bacteria-targeting strategies. 63Besides, artificial intelligence can assist in analyzing the interactions of AIE PS with bacteria through mathematical models to sift through large datasets and further forecast potential antiinfection candidates. 64The antibacterial action mechanism of existing antibiotics also offers many possibilities for designing novel antibacterial AIE PSs.Moreover, the behavioral activities and interorganismal connections of bacteria provide additional clues for the development of novel targeting strategies.For example, engineered macrophages or phages, and biomimetic macrophage membrane-camouflaged NPs, have been used to target bacteria.The multimodal-targeting method has the potential to integrate the advantages of various targeting strategies, which deserves further investigation.With all these anticipated explorations and future developments, the antibacterial applications of AIE PSs are promising.

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(B) AIE PSs conjugated with targeting ligands or organisms like bacteriophages can specifically recognize and combine with the highly expressed receptors on the bacterial membrane.(C) AIE PSs modified with specific active groups can chemically react with excess intracellular chemical or biological markers in bacteria, including biological analytes, ROS, and enzymes.(D) AIE PSs connected with natural metabolic moieties can label bacteria by biological metabolic processes.Bioorthogonal labeling technology makes AIE PSs more available to marker bacteria through metabolism, further expanding its scope of application.4b,4c,15 (E) AIE PSs can also improve the selectivity toward bacteria in other ways, such as functionalization with two or more targeted elements and adaptive AIE PSs-engineered macrophages.Many representative examples are elaborated in the next sections to illustrate the above strategies.Finally, we discuss the current limitations of AIE PSs and propose future directions based on latest advances.Electrostatic and hydrophobic interactions Bacterial surfaces are negatively charged and bacterial cell membranes are composed of amphipathic lipids.

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I G U R E 2 (A) Possible schematic diagram of selective enrichment of phospholipid-mimetic AIE PSs based on different bacterial structures.(B) Molecular structures of C1 and C12.(C) Antibacterial selectivity of C1 and C12 to Gram-positive and Gram-negative bacteria.Reproduced with permission.20c Copyright 2023, American Chemical Society.(D) Chemical structure of TriPE-NT.(E) Antibacterial activity of TriPE-NT (10 μM) against different species of bacteria with 4 mW/cm 2 white-light irradiation for 0, 2, 30 min.Reproduced with permission. 12Copyright 2018, John Wiley and Sons.AIE, aggregation-induced emission; PSs, photosensitizers; TriPE-NT, triphenylethylene-naphthalimide triazole.WU ET AL.

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I G U R E 3 (A) Chemical structure of AIE-2Van.(B) Cytotoxicity of AIE-2Van at various concentrations to Bacillus subtilis, Escherichia coli, Van A, and Van B in darkness or upon light irradiation (100 mW/cm 2 ).Inset images are pictures of bacterial suspension treated with AIE-2Van (20 μM) and GO (1.6 μg) under a 365 nm lamp.Reproduced with permission. 29Copyright 2015, Royal Society of Chemistry.(C) Chemical structure of E-probe.(D) Schematic representation of sensitive detection and elimination of Gram-positive bacteria using in situ self-assembly of E-probes attached with self-assembling peptides on bacterial surface.Reproduced with permission.30Copyright 2020, Elsevier.

F I G U R E 6
Bacteria targeting based on reactivity to endogenous stimulation.(A) Schematic representation for intracellular bacterial detection and elimination based on the reactivity of Caspase-1 enzymes in macrophages and PyTPE-CRP containing enzyme-responsive peptide linker.(B) CLSM images showing localization of PyTPE-CRP residues on Staphylococcus aureus-invaded phagosomes inside macrophages, followed by ROS detection.Reproduced with permission. 44Copyright 2019, John Wiley and Sons.(C) Preparation of ONOO − and ClO − -responsive IRTP NPs.(D) Jablonski diagram illustrating the response mechanism of IRTP NPs.(E) Fluorescence images of skin slices from GFP transgenic Escherichia coli-infected mice treated with saline (left) or 33% IRTP NPs (right) under white light irradiation (300 mW/cm 2 , 10 min).Reproduced with permission.46Copyright 2018, American Chemical Society.CLSM, confocal laser scanning microscope; GFP, green fluorescent protein; NPs, nanoparticles; ROS, reactive oxygen species.

F I G U R E 7
Bacterial targeting based on natural metabolic processes.(A) Schematic illustration of the incorporation of TPACN-D-Ala into the bacterial wall via one-step in vivo metabolism to illuminate and destroy bacteria.(B) Infiltration of bacterial metabolic probe TPACN-D-Ala into MRSA biofilms.(C) CLSM images of MRSA-infected RAW264.7 cells treated with TPACN-D-Ala.(D) Fluorescence imaging of infected sites in mice after i.v.injection with TPACN-D-Ala for 0.5, 3, and 6 h and distinct ex vivo tissues in mice after treatment with TPACN-D-Ala for 12 h.Reproduced with permission. 48Copyright 2020, Royal Society of Chemistry.(E) Schematic illustration of AIE PS based on metabolic precursors D-Ala and Kdo for selective recognition and killing of Gram-positive and Gram-negative bacteria.Reproduced with permission.14a Copyright 2022, Royal Society of Chemistry.(F) Molecular structures of TPEPA, D-Ala-N 3 , and KDO-N 3 .(G) Schematic diagram based on labeling of TPEPA to azide-modified engineered bacteria for detection and eradication of bacteria.(H) Selective labeling of Kdo-N 3 -pretreated Gram-negative or D-Ala-N 3 -pretreated Gram-positive bacteria by TPEPA.Reproduced with permission. 51Copyright 2019, American Chemical Society.CLSM, confocal laser scanning microscope; MRSA, methicillin-resistant Staphylococcus aureus.
H 2 O 2 to release D-AzAla.Subsequently, D-AzAla labeled bacteria through intrinsic metabolism, resulting in the expression of azide groups on the bacterial surface that could be illuminated by dibenzocyclooctyne (DBCO)-modified fluorescent probes for bacterial imaging.Separately, far-red/near-infrared AIE PS 2-(1-(5-(4-(1,2,2-tris(4-methoxyphenyl) vinyl) phenyl) thiophen2-yl)ethylidene)malononitrile (TPETM) was wrapped in DBCO-DSPE-PEG 2000 to manufacture ultrasmall US-TPETM NPs modified with DBCO, which could reach the infected site and combine with the metabolically engineered bacteria through a copper-free click reaction to realize bacterial detection and destruction.This strategy of compensating the targeting deficiency of bioorthogonal metabolic labeling with activatable carriers opens new opportunities for broadening the in vivo application of APDT based on bioorthogonal covalent labeling.

F I G U R E 8
Multimodal targeting system.(A) Schematic diagram of H 2 O 2 -responsive MIL-100 assisting bacterial intrinsic metabolic labeling for bacterial imaging and therapy.Reproduced with permission. 54Copyright 2018, John Wiley and Sons.(B) Preparation of bacterial template polymers.(C) CLSM images illustrate the binding proneness of bacterium-templated polymers to matched and mismatched bacteria.Reproduced with permission. 58Copyright 2020, John Wiley and Sons.CLSM, confocal laser scanning microscope.
WU ET AL. been fully elucidated.The long-term biosafety and biocompatibility of AIE PSs should be clinically proven.(2) AIE PSs are required to possess much improved water solubility to reduce the background fluorescence interference.

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I G U R E 9 (A) Chemical structure of TTD and diagram of TTD NPs.(B) Schematic representation of the APDT process based on adoptive macrophage transfer triggering precise targeting of bacteria.(C) CLSM image showing close coupling between TTD NPs (red) loaded in TLMs and Staphylococcus aureus (green) captured by TLMs.Reproduced with permission. 11Copyright 2023, American Chemical Society.APDT, antibacterial photodynamic therapy; CLSM, confocal laser scanning microscope; NPs, nanoparticles; TLMs, TTD-loaded macrophages.