Multivalent LecA/LecB Inhibitors based on the Co-assemblies of Perylene Monoimide-carbohydrate Conjugates for Antibiotic-free Antibacterial and Wound Healing

Multivalent LecA/LecB Inhibitors based on the Co-assemblies of Perylene Monoimide-carbohydrate Conjugates for Antibiotic-free Antibacterial and Wound Healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multivalent LecA/LecB Inhibitors based on the Co-assemblies of Perylene Monoimide-carbohydrate Conjugates for Antibiotic-free Antibacterial and Wound Healing Ke-Rang Wang, Jian-Xing Yang, Hai-Qing Li, Fangqian Yin, Wen-Juan Yin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4641881/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pathogenic infection is becoming a global health threat to human health. Especially for the treatment of P. aeruginosa remains particularly challenging. Fortunately, it is interestingly found that the LecA and LecB lectins of P. aeruginosa played crucial roles in bacterial adhesion, biofilm formation, virulence, and host cell invasion. Herein, a co-assemble strategy to prepare antibiotic-free antibacterial and antibiofilm agents by using two kinds of perylene-carbohydrate conjugates ( PMI-3Gal and PMI-3Fuc ) with synergistic targeting for two lectins of P. aeruginosa LecA and LecB was developed. Due to the strong multivalent carbohydrate-lectin interactions both for LecA and LecB lectins, the co-assembly PMI-3Gal @ PMI-3Fuc showed selective adhesion effects, inhibition activity of biofilm formation and potent photothermal antibacterial activities for P. aeruginosa and a clinical-isolated P. aeruginosa strain , and showed the acceleration effect for the wound healing in mice. This result opens a supramolecular principle for antibiotic-free antibacterial and antibiofilm effects based on multivalent glycoconjugates. Physical sciences/Chemistry/Supramolecular chemistry/Self-assembly Physical sciences/Chemistry/Materials chemistry/Biomaterials/Biomedical materials self-assembly carbohydrate-lectin interactions antimicrobial photothermal therapy multivalent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Pathogenic infection is becoming a global health threat to human health in recent years. There are approximately 700000 deaths per year worldwide, and the death toll caused by pathogenic infection will increase to 10 million per year by 2050 [1] , resulting a cumulative financial burden of $ 100 trillion from the year 2014 to 2050 [2] . According to the World Health Organization (WHO), Gram-negative bacteria, such as Acinetobacter baumannii , Pseudomonas aeruginosa ( P. aeruginosa ), Enterobacteriaceae , rank as priority list 1 due to its complex Gram-negative bacterial cell envelope, high antimicrobial resistance, and lack of new antibiotics. Compared to the other Gram-negative bacteria, the development of new antibacterials for P. aeruginosa is particularly challenging due to the absence of non-specific porins for the drugs across the outer membrane, the presence of tripartite efflux pumps, and substrate-specific outer-membrane porins for antibiotic efflux in P. aeruginosa [3] . To date, only four classes of antibiotics are available to treat P. aeruginosa infections in clinic, such as β-lactams, fluoroquinolones, aminoglycosides, and polymyxins [3] . As a result, there is an urgent need for the development of new antibacterials for P. aeruginosa infections with novel mechanisms of action and therapeutic strategies [4] . The antimicrobial resistance in most pathogenic bacteria and chronic bacterial infections are related to the formation of bacterial biofilm [5] . This is especially true for chronic wound infections of P. aeruginosa , where the thickness of bacterial biofilm can reach 200 µm and 1400 µm above and below the wound surface, respectively. Both are thicker than those (150 µm and 190 µm) of Staphylococcus aureus ( S. aureus ) [5] . Furthermore, treatments of biofilm bacteria require 10 to 1000 times higher antibiotic concentrations than that of planktonic bacteria [6] , which further lead to antimicrobial resistance and persistent infections. Therefore, biofilm infection microenvironment (BIM) targeted therapeutic strategy [7] would be an effective path to overcome antimicrobial resistance and kill biofilm bacteria. It is interestingly found that the tetravalent lectins [8] LecA and LecB of P. aeruginosa , which were the carbohydrate-binding proteins exhibiting selectively recognition interactions with D-galactose and L-fucose/D-mannose [9] with multivalency [10] , respectively, played crucial roles in bacterial adhesion, biofilm formation, virulence, and host cell invasion [11,12] . Thus, small LecA/LecB inhibitors [13] and multivalent LecA/LecB inhibitors [14] paved a promising way to develop antibiotic-free antibacterial and antibiofilm agents as an alternative to antibiotic treatments. Based on the structural characteristics of two adjacent binding sites for simultaneous binding of two galactose moieties, a series of divalent LecA inhibitors conjugated two galactoses were developed, which exhibited ca. 500-fold binding enhancements for LecA lectin [15] and a potent LecA inhibition activity with K d values of 11–81 nM [16] . In order to enhance the water solubility, a divalent sulfonate LecA inhibitor was synthesized, showing potently inhibited LecA binding to lung epithelial cells and reduced invasion of P. aeruginosa into host cells [17] . Multivalent glycoclusters and multimodal antibacterial therapy system based on calix[4]arene derivatives [18,19] targeting LecA or LecB lectins were constructed, which showed anti-adhesive property and biofilm dispersion effect. Furthermore, a series of supramolecular rotaxanes [20] based on pillar[5]arene derivatives were reported, exhibited high antibiofilm activity through simultaneous targeting the two lectins LecA and LecB. However, it is surprising to find that supramolecular rotaxanes exhibited antibiofilm activity, but showed no bacteria killing activity. In order to enhance the bactericidal activity and to reduce the potential immunogenic effect, several biofilm-targeted drugs based on monovalent D-galactose or L-fucose modified glycoconjugates of sulfonamides [21,22] and ciprofloxacin [23] were developed, which could decrease the systemic side effects. However, the antibiotic activity was significantly reduced. In an effort to improve the antibiotic activity, a series of activated prodrugs based on fluoroquinolones-carbohydrate derivatives were constructed [24] , which showed the biofilm-targeted effects and LasB (Zn(II)-dependent metalloprotease in P. aeruginosa )-mediated release of antibiotic. Multivalent carbohydrate-drug systems targeting LecA, LecB, or LecA/LecB based on series of glycopolymers conjugated with a BODIPY photosensitizer [25] , copper sulfide nanocrystals [26] , gold nanorods [27] and gold nanoparticles [28] were also reported, exhibiting efficient biofilm inhibition, photothermal or photodynamic killing capability. On the other hand, biofilm-targeted drug delivery system was another way to improve antibiotic activity [29] . Recently, hypoxia-responsive delivery of lactose-modified azocalix[4]arene and ciprofloxacin by us and our collaborators [30] was constructed, showed the inhibition effect in biofilm formation and hypoxia-responsive delivery antibiotic for killing P. aeruginosa . Due to the hetero-multivalent effect in carbohydrate-lectin interactions, hetero-multivalent targeting drug delivery systems of ciprofloxacin-loaded liposomes [31] and ICG-loaded co-assembled nanoparticles [32] were developed, showed the treatment of infections caused by P. aeruginosa and enhanced photothermal and photodynamic therapy of antibiotic-resistant bacterial pneumonia. Antibiofilm agents [33] based on glycoconjugates targeting for LecA and LecB lectins in P. aeruginosa are efficient approaches to treat antimicrobial resistance through inhibiting bacterial biofilm formation, disrupting mature biofilm, and removing biofilm. However, the killing efficiency for P. aeruginosa is limited, so the combined therapeutic functional molecules as the key therapeutic agents can efficiently enhance the bactericidal activity. As a result, multivalent glycoconjugates with combination of the recognition function of carbohydrates and the therapeutic function of drug would be a powerful strategy for the treatment of bacterial infections. One of the inherent problems in the multivalent glycoconjugates is the efficient recognition of P. aeruginosa with controllable and suitable carbohydrate types and numbers for simultaneous targeting of LecA and LecB. Another problem is the efficiently therapeutic agents. Supramolecular assembly strategy [34,35] is an effective way to form multivalent glycoclusters with different types and number of carbohydrates. Moreover, photothermal therapy (PTT) as an alternative strategy for bacterial infections has attracted much more attentions due to its causing of cell membrane damages, protein denaturation, cell membrane damages, protein denaturation and heat stress [36] . In them, perylene monoamide (PMI) derivatives exhibited strong near infrared (NIR) absorption [37] , controllable self-assembly behaviors [37] , potent photothermal stability, and phototherapy effect [38] . In this paper, multivalent lectin inhibitors for LecA, LecB, and LecA/LecB based on the self-assemblies and co-assemblies of perylene monoamide (PMI)-glycoconjugates ( PMI-3Gal and PMI-3Fuc , Fig. 1 , Scheme S1) were developed, which showed selective adhesion effects and inhibition activity of biofilm formation for P. aeruginosa . In particular, the co-assemblies of PMI-3Gal@PMI-3Fuc demonstrated enhanced photothermal therapy effects and excellent bacteria killing activity in vitro and in vivo. Moreover, the effective photothermal and inherent antimicrobial synergistic therapy of the co-assemblies of PMI-3Gal@PMI-3Fuc showed the acceleration effect for the wound healing in mice. This result opens a supramolecular principle for antibiotic-free antibacterial and antibiofilm effects based on multivalent glycoconjugates. 2. Results and discussion 2.1 Synthesis of perylene monoamide-glycoconjugates (PMI-3Gal and PMI-3Fuc) As shown in Scheme S1, compounds PMI-3Gal and PMI-3Fuc were synthesized by a click reaction of the intermediate PMI-1 with the azide group modified D-galactose and L-fucoside, respectively, and followed by deprotection of the acetyl groups. The intermediates and the target molecules ( PMI-3Gal and PMI-3Fuc ) were fully characterized by 1 H and 13 C nuclear magnetic resonance ( 1 H NMR and 13 C NMR) as well as high resolution mass (HRMS) spectra. (Figure S1 - S1 2, Supporting information). 2.2 Assembly properties of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc Due to the strong π-π stacking interactions of the perylene backbones, PMI-3Gal and PMI-3Fuc (Fig. 2 a) could form the self-assemblies and the co-assemblies in water. Firstly, solvent-dependent UV-vis spectra of PMI-3Gal and PMI-3Fuc were used to study the self-assembly and co-assembly behaviors. As shown in Fig. 2 b and 2 c, PMI-3Gal and PMI-3Fuc showed the maximum absorption band at 630 nm and 636 nm in DMSO solution, respectively, indicating that different types of the carbohydrate modification influenced the optical property of PMI. These optical properties of PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc (1:1) in DMSO solution remained a non-aggregated state [37] . As shown in Fig. 2 d, the maximum absorption band of the co-assembly PMI-3Gal @ PMI-3Fuc was at 633 nm, which was lower than the maximum absorption of PMI-3Fuc and higher than the maximum absorption of PMI-3Gal . Upon increasing of the water ratio, the intensities of the maximum bands decreased, and the maximum bands underwent a hypsochromic shift to 566 nm, 569 nm and 566 nm for PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc , respectively, indicating the formation of H-aggregates due to strong intermolecular π-π stacking interactions in water [37] . Fluorescence spectra of PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc showed a similar result. As shown in Figure S13, strong fluorescence emission bands at 736 nm, 736 nm and 738 nm of PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc were found in DMSO solution, and decreased along with the increasing of water due to the enhanced π − π stacking interactions. The self-assembly and co-assembly behaviors were further investigated by the dynamic light scattering (DLS) measurements. As shown in Figure S14a-c, the assemblies with the mean diameters of 259.85 nm, 259.33 nm and 261.98 nm for PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc were observed, and no obvious differences in the self-assemblies and the co-assemblies were found. Different morphologies were characterized by the SEM images, as shown in Figure S14 d-f, which showed silk ribbon for PMI-3Gal and PMI-3Gal @ PMI-3Fuc , and flocculent aggregates for PMI-3Fuc . In addition, the co-assembly model of PMI-3Gal and PMI-3Fuc was investigated by a turbidity assay (Fig. 2 e). Two possible assembly models can be postulated, that is, the complexes (Model I, Fig. 2 f) of the assemblies of PMI-3Gal and the assemblies of PMI-3Fuc or the co-assemblies (Model II, Fig. 2 f) of PMI-3Gal and PMI-3Fuc with an interlaced mode. It is well known that lectin-carbohydrate interactions are specific and selective [39] , and the self-assembled glycoclusters show enhanced binding interactions [40] with unique lectin through multivalent effect. As shown in Fig. 2 e, the turbidity of the assemblies of PMI-3Gal showed a sharp increase upon addition of peanut agglutinin (PNA) lectin because PNA selectively bound to the terminal-galactosyl residues [40] . However, the turbidity of the mixture of the self-assemblies of PMI-3Fuc with PNA showed no obvious change, indicating no binding to PNA. When adding co-assemblies of PMI-3Gal and PMI-3Fuc , the turbidity increased, but was lower than that of the self-assemblies of PMI-3Gal . This result was due to that the insertion of non-recognizable carbohydrate in the assemblies weakened the specific and selective binding interactions of PNA with the terminal-galactosyl residues. These results indicated that the possible assembly model was the co-assemblies (Model II, Fig. 2 e) of PMI-3Gal and PMI-3Fuc with an interlaced mode. 2.3 Photothermal properties of PMI-3Gal and PMI-3Fuc Benefited from the strong π-π stacking interactions, the assemblies of PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc showed very weak fluorescence in water, which was advantageous to the photothermal effect [41] . The photothermal effects of the assemblies and the co-assemblies of PMI-3Gal and PMI-3Fuc were studied under different laser powers. Under the concentration of 100 µM, as shown in Fig. 2 g-i, the temperatures of PMI-3Gal at the laser powers of 0.2, 0.4 and 0.6 W/cm 2 increased to 48.2°C, 65.6°C and 72.6°C, respectively, which were higher than that of PMI-3Fuc and lower than that of PMI-3Gal @ PMI-3Fuc . It is well known that photothermal therapy for tumor needs sufficient light and intense power to penetrate the tissues of the organism [36] . However, it is dangerous to the surrounding healthy tissue, especially for wound infections, which are not as deep as tumors [42] . Therefore, concentration-dependent photothermal effects under a power of 0.4 W/cm 2 were studied. As shown in Fig. 2 j-i, the temperatures of PMI-3Gal increased to 48.2°C,56.2°C, 61.0°C, and 65.6°C under the concentrations of 25 µM, 50 µM, 75 µM and 100 µM, respectively. The co-assemblies of PMI-3Gal @ PMI-3Fuc also showed the best photothermal effect with maximum temperatures of 49.1°C, 56.4°C, 62.7°C, and 66.3°C. PMI-3Fuc showed the low temperatures of 46.2°C, 54.8°C, 59.5°C, and 64.2°C. This result indicated that different types of carbohydrate modification in PMI influenced the photothermal effect. As a control experiment, the temperature of the water solution only increased from 27.0°C to 29.0°C under laser irradiation. It is very interesting to note that the co-assemblies of PMI-3Gal @ PMI-3Fuc exhibited the best photothermal effect. Furthermore, the quantitative photothermal-conversion efficiency (𝜂) was calculated by warming/cooling curves with a Roper’s method [43] . As shown in Figure S15, the co-assemblies of PMI-3Gal @ PMI-3Fuc showed a high photothermal conversion efficiency value of 63%, which was higher than that of PMI-3Gal (55%) and PMI-3Fuc (48%). Combined with the optical properties and the morphologies, we can conclude that different types of carbohydrate modification in PMI influence the optical properties, assembly behaviors, and the photothermal effect. In addition, the photostability of the self-assemblies of PMI-3Gal and PMI-3Fuc , and the co-assemblies of PMI-3Gal @ PMI-3Fuc was evaluated upon five cycles under laser irradiation. A cycle was performed by irradiation of the sample for 10 min with laser, and then remove the laser, and the temperature reached a high point and then decreased to the room temperature. There were no obvious changes for the high temperature during five cycles of laser irradiation, indicating that PMI-3Gal , PMI-3Fuc and PMI-3Gal @ PMI-3Fuc possessed high photostability. 2.4 Lectin-targeted antimicrobial activity of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc for P. aeruginosa The tetravalent lectins LecA and LecB of P. aeruginosa exhibited selective recognition with D-galactose and L-fucose and played crucial roles in bacterial adhesion, biofilm formation, and virulence. Firstly, the adhesion actions of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc toward P. aeruginosa were performed. As shown in Figure S16 and Table S1 , P. aeruginosa with a working concentration of 9 × 10 8 colony forming units (CFU) mL − 1 was incubated for 6 h at 37°C with various concentrations (50 µM, 75 µM, 100 µM, 125 µM, 150 µM, and 200 µM) of the assemblies. The adhesion concentrations, determined by the Lambert-Beer law, were 16 µM, 29 µM, 56 µM, 92 µM, and 140 µM for PMI-3Gal , 12 µM, 24 µM, 45 µM, 68 µM, 79 µM, and 125 µM for PMI-3Fuc , and 17 µM, 30 µM, 56 µM, 80 µM, 93 µM, and 140 µM for PMI-3Gal @ PMI-3Fuc . Compared with PMI-3Fuc , PMI-3Gal showed a better adhesion interaction for P. aeruginosa . Considering the result that PMI-3Fuc showed a weak adhesion interaction for P. aeruginosa , the co-assemblies of PMI-3Gal @ PMI-3Fuc should exhibit weaker adhesion effect for P. aeruginosa than that of PMI-3Gal in theory. However, it is interestingly found that the co-assemblies of PMI-3Gal @ PMI-3Fuc showed the best adhesion effect for P. aeruginosa , which might due to a hetero-multivalent effect [44] that enhances the affinity towards the target lectin by cooperative recognition interactions with different saccharides. Benefited from the strong adhesion effect for P. aeruginosa and high photothermal effect of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc , their antibacterial activities were further studied. The antibacterial performances of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc with and without laser irradiation against P. aeruginosa in vitro were evaluated by observing the counts of colonies growing on the agar plate (Fig. 3 a-c). As shown in Fig. 3 d-f, there were no “dark” therapeutic effects of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc against P. aeruginosa with the viabilities of 99.75 ± 0.08%, 99.38 ± 0.41% and 99.95 ± 0.03% at high concentrations of 140 µM, 125 µM, and 140 µM, respectively. Under laser irradiation of 0.40 W/cm 2 for 10 minutes, the remarkably photothermal antibacterial activities were observed. Along with increasing of the concentrations, PMI-3Fuc exhibited enhanced photothermal antibacterial activities for P. aeruginosa with the antibacterial ratios of 23.19 ± 0.90%, 34.57 ± 1.77%, 55.87 ± 1.06%, 66.23 ± 1.38%, 74.46 ± 0.69%, and 85.75 ± 0.43%, respectively. As expected, PMI-3Gal showed a better photothermal antibacterial activity than PMI-3Fuc due to stronger adhesion effect for P. aeruginosa and higher photothermal effect. The antibacterial ratios were 39.90 ± 0.21%, 57.47 ± 0.48%, 70.79 ± 0.33%, 77.15 ± 2.70%, 84.07 ± 0.34%, and 99.62 ± 0.12% under the concentration of 16 µM, 29 µM, 56 µM, 92 µM, and 140 µM, respectively. Moreover, the co-assemblies of PMI-3Gal@PMI-3Fuc demonstrated a significant cooperative therapeutic effect with simultaneous targeting to LecA and LecB lectins, resulting in concentration-dependent photothermal antibacterial activities of 39.07 ± 2.24%, 61.62 ± 1.64%, 80.46 ± 0.11%, 88.19 ± 0.10%, 98.14 ± 0.21%, and 100% under the concentration of 16 µM, 29 µM, 56 µM, 92 µM and 140 µM, respectively. Full eradication of P. aeruginosa was observed under the concentration of 140 µM for the assemblies of PMI-3Gal and PMI-3Gal@PMI-3Fuc , indicated that LecA lectin showed a main adhesion effect for P. aeruginosa . Under the same concentration of 93 (92) µM, the photothermal antibacterial activity of PMI-3Gal@PMI-3Fuc was 98.14%, larger than that of PMI-3Gal (84.07%), suggesting a cooperative therapeutic effect for targeting of LecB lectin. Furthermore, the antibacterial performances of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc against multidrug-resistant P. aeruginosa isolated from mucus samples of Neurocritical care patient (WE3101, that is resistant to Imipenem and Meropenem) were evaluated (Figure S17). Along with increasing of the concentrations, as expected, PMI-3Gal showed a better photothermal antibacterial activities for multidrug-resistant P. aeruginosa than that of PMI-3Fuc .The bacterial survival rate was calculated as 90.15 ± 1.24%, 79.58 ± 1.51%, 65.26 ± 1.36%, 48.90 ± 3.72%, 33.98 ± 1.38% and 12.43 ± 1.15% under the concentration of 43 µM, 76 µM, 100µM, 126 µM, 158 µM and 165 µM (Fig. 3 g). The antibacterial activities were observed for PMI-3Fuc , which exhibited the bacterial survival rate of 97.81 ± 1.92%, 92.43 ± 2.79%, 84.13 ± 2.08%, 73.77 ± 2.40%, 65.54 ± 1.71% and 50.25 ± 1.45% under the concentration of 35 µM, 65 µM, 83 µM, 120 µM, 145 µM and 155 µM (Fig. 3 h), respectively. Moreover, the co-assemblies of PMI-3Gal@PMI-3Fuc became more significant after NIR irradiation. The bacterial survival rate decreased to 82.02 ± 3.05%, 65.46 ± 2.45%, 48.63 ± 0.93%, 35.90 ± 0.91%, 16.96 ± 1.02% and 2.09 ± 0.81% under the concentration of 52 µM, 88 µM, 112 µM, 149 µM, 164 µM and 172 µM (Fig. 3 i), respectively. Moreover, the PTT antibacterial efficacy of the co-assemblies of PMI-3Gal and PMI-3Fuc with different assembling ratios were investigated. As shown in Figure S18, the best antibacterial effect was observed for the 1:1 co-assembly of PMI-3Gal and PMI-3Fuc ( PMI-3Gal@PMI-3Fuc ), which exhibited nearly 95% inhibition ratio for P. aeruginosa after coincubation with the bacteria for 6 h under 635 nm laser irradiation for 10 min. When the assemble ratios of PMI-3Gal and PMI-3Fuc changed to 1:2 and 2:1, the PTT antibacterial inhibition ratios for P. aeruginosa were 83% and 86%, lower than that of PMI-3Gal@PMI-3Fuc . Moreover, the selectivity of the co-assemblies of PMI-3Gal@PMI-3Fuc was also studied against E. coli and S. aureus , which showed the antibacterial activities of 72% and 48%, as shown in Figure S18b-c, lower than the PTT antibacterial activity for P. aeruginosa . These results indicated that PMI-3Gal@PMI-3Fuc with 1:1 ratio demonstrated a high PTT antibacterial effect and exhibited selectively killing effect for P. aeruginosa . In addition, the bacterial live/dead staining assay was further performed to investigate the photothermal antibacterial effect for P. aeruginosa with SYTO 9 and PI staining live and dead bacteria, respectively. As shown in Fig. 3 j and S19, abundant green fluorescence labeled live bacteria (indicating no damage effect to the bacteria) for five groups: PBS groups regardless of laser irradiation, and the assemblies of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc at high concentrations of 140 µM (125 µM) without irradiation. Under 635nm (0.40 W/cm 2 ) NIR irradiation for 10 min, live bacteria with green fluorescence decreased and dead bacteria with red fluorescence increased, both showed a concentration-dependent antibacterial activity. As shown in Fig. 3 k-m, the antibacterial effects of PMI-3Fuc were calculated to be 7.81 ± 1.50%, 24.37 ± 2.11%, 44.97 ± 2.19%, and 77.48 ± 2.05% at 12, 45, 79, and 125 µM using Image J. Enhanced antibacterial activities were observed for PMI-3Gal , which exhibited antibacterial ratios of 25.57 ± 4.52%, 46.73 ± 2.59%, 72.26 ± 2.33%, and 90.96 ± 0.34% under the concentrations of 16, 56, 92, and 140 µM, respectively. As expected, the co-assemblies of PMI-3Gal@PMI-3Fuc showed the best antibacterial activities with the antibacterial ratios of 38.26 ± 0.03%, 55.63 ± 0.73%, 89.23 ± 1.45%, and 99.92 ± 0.03% at 17, 56, 93 and 140 µM, respectively. These results were consistent with colony growth on the LB agar plates. The morphological changes of bacteria treated with PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc with and without laser irradiation were further explored. As shown in Fig. 3 n, an integrated cell membrane with clear edges and smooth surface of bacteria were observed for the PBS group and the unirradiated PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc . Under laser treatment, the collapsed cell membranes for P. aeruginosa were found, and almost all the bacterial membranes were destroyed due to the PTT effects. 2.5 Lectin-targeted antibiofilm activity of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc for P. aeruginosa Formation of bacterial biofilm is one of the most critical factors leading to drug resistance, which further prevents antimicrobial drugs from contacting bacteria, thereby delaying the healing of infected wounds. Fortunately, the formation of biofilm is related with lectins LecA and LecB presenting on the outer membrane of P. aeruginosa , which brings opportunity for the development of antibiotic-free antibacterial agents (Fig. 4 a). The antibiofilm performances of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc against P. aeruginosa biofilms were evaluated by crystal violet (CV) staining assay. Due to strong adhesion interactions of galactose with LecA and fuctose with LecB, the co-assemblies of PMI-3Gal@PMI-3Fuc showed the highest biofilm inhibition effects with the ratios of 25.08%, 34.96%, 45.94%, and 58.14% (Fig. 4 b) under the concentrations of 10 µM, 20 µM, 40 µM, and 80 µM, respectively. PMI-3Gal displayed a better antibacterial effect than PMI-3Fuc . These results suggested that lectin-targeted agents showed a potential application for antibiotic-free antibiofilm effects for P. aeruginosa . It is well known that removal of mature biofilms is more difficult than the inhibition of biofilm formation due to the formation of a microbial community with polysaccharides, extracellular DNA (eDNA), lipids, and proteins. Upon increasing of the concentrations to 20 µM, 40 µM, 80 µM and 160 µM, the assemblies of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc also exhibited dispersion effects on the mature biofilms for P. aeruginosa , as shown in Fig. 4 c, the trends were PMI-3Gal@PMI-3Fuc > PMI-3Gal > PMI-3Fuc . PMI-3Gal@PMI-3Fuc displayed the dispersion ratios of 28.83%, 33.06%, 39.97%, and 49.41%. PMI-3Gal with the dispersion ratios of 25.22%, 33.18%, 39.96%, and 46.80%; and PMI-3Fuc with the dispersion ratios of 24.69%, 33.05%, 38.49%, and 43.93% were observed. These results indicated that lectin-targeted agents showed the application for antibiotic-free antibiofilm effects both for mature biofilms and immature biofilms. Moreover, antibiofilm effects were further examined with fluorescence imaging by live/dead bacterial staining. Intense green fluorescence for the control group was observed, indicated a very intact biofilm structure. When addition of 50 µM and 100 µM of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc , green fluorescence on biofilm was decreased, suggesting inhibition biofilm effect (Fig. 4 d). In addition, removal effects of biofilm based on PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc with and without laser irradiation were also studied by a live/dead bacterial staining (Fig. 4 e-g). With laser irradiation, the removal ratios of biofilm based on PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc increased from 38.53%, 27.14%, and 44.18–51.10%, 42.22%, and 55.83% at the concentration of 100 µM, respectively. Increasing the concentration to 200 µM, the removal ratios of biofilm based on PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc with laser irradiation were 89.73%, 75.97%, and 99.60%, which were larger than that (55.38%, 45.17% and 59.14%) of the group without laser irradiation. These results demonstrated that PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc could effectively inhibited and destroyed the formation of bacterial biofilms, showing the potential to eliminate bacteria. 2.6 In vitro cell migration and in vivo wound repair of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc Cell migration and proliferation are critical processes for wound healing [45] . The biocompatibility of PMI-3Gal, PMI-3Fuc , and PMI-3Gal@PMI-3Fuc with L929 cells was studied by MTT assay under various concentrations. As shown in Figure S20 Near 100% cell viability was observed even at a high concentration of 100 µM, suggesting no toxicity to L929 cells. Moreover, the effects of PMI-3Gal, PMI-3Fuc , and PMI-3Gal@PMI-3Fuc on cell migration were studied by cell scratch assay at different time. The L929 cells were used to mimic wound infection in vitro. As illustrated in Figure S21, PMI-3Gal, PMI-3Fuc , and PMI-3Gal@PMI-3Fuc all showed promoting effect on cell migration. PMI-3Gal and PMI-3Gal@PMI-3Fuc exhibited a relatively high ability to promote cell migration compared with the other groups, which showed the healing rates of 65.92 ± 0.32% and 65.69 ± 3.33% at 24 h through calculating the scratched area, both higher than that of PMI-3Fuc (51.02 ± 0.37%). Increasing of the cell incubation time to 48 h, the cell scratch coverages increased to 96.21 ± 1.06%, 82.03 ± 2.03% and 99.56 ± 0.16% for PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc , respectively. These results suggested that galactose residue was advantageous to promote cell migration, and the combination of galactose residue and fuctose residue exhibited a collaborative promotion effect. Hemolysis is a key evaluation factor of the further application in vivo for multivalent glycoclusters. A hemolysis rate over 5% is adverse according to the standard of International Organization for Standardization (ISO) [46] . As shown in Figure S22, bright red supernatant of the Triton X-100 group was observed, indicated a serious hemolysis effect for the control group. However, the hemolysis rates of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc were lower than 3% even at a high concentration of 200 µM, suggesting good biocompatibility. Furthermore, the wound healing effects of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc were investigated. Firstly, P. aeruginosa infected mouse whole skin wound models based on BALB/c mice were successfully established after P. aeruginosa infection, and the bacterial colonies was observed on Day 2. These mice were randomly divided into eight groups, including: PBS groups without (I) and with laser irradiation (II), PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc groups without (III-V) and with laser irradiation (VI-VIII). Treatments with PMI-3Gal , P MI-3Fuc , and PMI-3Gal@PMI-3Fuc were implemented on the infected sites. Upon 635 nm laser irradiation, the temperatures of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc on wounds were recorded by an IR camera. As shown in Figure S23, the temperatures increased to 49.2℃, 44.7℃, 52.0℃ for PMI-3Gal , P MI-3Fuc and PMI-3Gal@PMI-3Fuc after irradiation for 10 min, which were higher than that of PBS group (37.9℃). Dynamic wound healing process was photographed on dyes 2, 4 and 7. All wound sizes progressively reduced during the treatment period. As shown in Fig. 5 a, it was obvious found that the wounds of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc with and without laser irradiation were smaller than those of the PBS groups with and without laser irradiation. The average unhealing areas (Fig. 5 b) in PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc without laser irradiation were 46.72 ± 0.51%, 56.27 ± 2.54%, and 30.58 ± 1.25% on the 7th day, respectively, indicating that carbohydrate-lectin interactions can promote wound healing due to lectin-targeted antibacterial effects. With laser irradiation, the average unhealing areas in PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc increased to 27.79 ± 0.14%, 37.82 ± 1.84% and 16.79 ± 0.95% on the 7th day, respectively, suggesting a photothermal promoted killing bacteria activity. As the control groups, the average unhealing areas in PBS groups were 55.59 ± 0.89% (without laser irradiation) and 63.93 ± 1.15% (with laser irradiation), respectively. From the photographic images, the wound closure simulation plots (Fig. 5 c) suggested that the skin regeneration of the assemblies with laser irradiation groups is obvious faster than the assemblies without laser irradiation groups, and which all faster than the control. The trend of the skin regeneration is PMI-3Gal@PMI-3Fuc > PMI-3Gal > PMI-3Fuc . In addition, the antibacterial effects of PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc during infected wound healing process were investigated. As shown in Fig. 5 d, tissue fluids were collected from the infected wound surface after treatment on dyes 2, 4 and 7. The extracted P. aeruginosa was incubated on agar plates, and the bacterial colonies were evaluated using Image J. After treated for 4 days, the bacteria colonies in PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc groups decreased to 67.68 ± 2.48%, 74.40 ± 1.71% and 57.30 ± 0.36% (Fig. 5 e), respectively, which were lower than that (88.69 ± 1.09%) of PBS group. After treated for 7 days, the bacteria colonies continuously decreased to 57.03 ± 1.48%, 65.56 ± 0.17%% and 38.15 ± 0.71% (Fig. 5 e) for PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc , respectively. With laser irradiation, significantly bacteria killing effects were observed. The bacteria colonies decreased to 30.37 ± 1.14%, 37.02 ± 1.81%, and 1.68 ± 0.04% (Fig. 5 e) for PMI-3Gal , PMI-3Fuc, and PMI-3Gal@PMI-3Fuc after treatment of 7 days, respectively. These results demonstrated that synergistic therapy combined with inherent antimicrobial effect and photothermal killing activity (Fig. 5 f) based on PMI-3Gal , PMI-3Fuc and PMI-3Gal@PMI-3Fuc in vivo showed potent wound healing in mice. 2.7 Histopathologic evaluations of collagen deposition, neovascularization, and inflammation microenvironment Histopathologic evaluations of the regenerated skin after 7 d treatment provided insight into the wound healing effect. The skin sections of Hematoxylin & Eosin (H&E) staining (Fig. 6 a) showed severe infection in tissues for the control groups. After 7 d treatment with the assemblies of the perylene-carbohydrate conjugates without laser irradiation, the infiltrated inflammatory cells decreased, suggesting an antibacterial effect through a mechanism of carbohydrate-lectin interactions. Moreover, an obvious reduction of the infiltrated inflammatory cells was found based on the assemblies of the perylene-carbohydrate conjugates with laser irradiation, indicating a remarkable antibacterial effect by a synergistic therapy combined with inherent antimicrobial effect and photothermal killing activity. In addition, an intact epidermis with a thick granulation tissue (Fig. 6 b) was observed in the treatment groups of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc (with laser irradiation) with the thickness of 233.80 ± 18.16, 153.53 ± 7.79, and 252.05 ± 8.94 µ m, respectively. The granulation tissues of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc (without laser irradiation) showed a thickness of 132.23 ± 7.63, 113.21 ± 10.22, and 164.23 ± 5.75 µm, lower than that of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc (with laser irradiation) groups. On the other hand, the thickness of granulation tissue for the control was only 90.11 ± 2.40 and 95.60 ± 3.86 µ m for PBS groups with and without laser irradiation. In addition, the organs in mice (heart, liver, spleen, lung, and kidney) were isolated and collected to investigate the biocompatibility. H&E staining images (Figure S24) showed no significant toxic side effects, and the body weight (Figure S25) of mice under the treatment process has no significant changes. Furthermore, the collagen deposition and arrangement of the healing skin were employed by a Masson staining assay (Fig. 6 c and 6 d). As shown in Fig. 6 c, abundant collagen with dense and organized structures were observed for the groups of PMI-3Gal , PMI-3Fuc , and PMI-3Gal@PMI-3Fuc (with laser irradiation), which were larger than that of the other groups. In them, the groups of PMI-3Gal@PMI-3Fuc exhibited a higher content of collagen in the dermis. Angiogenesis is an essential parameter for wound regeneration [47] . CD31 is a classical marker expressed in vascular endothelial cells [30] . The neovascularization for the regenerated tissue after 7 d treatment was investigated by an immunofluorescence staining method (Fig. 6 e and 6 f). As shown in Fig. 6 e, marked red fluorescence was observed, which exhibited a relative capillary intensity of 31.22 ± 4.12 for PMI-3Gal@PMI-3Fuc group with laser irradiation. This result was higher than the groups of PMI-3Gal (16.27 ± 0.69) and PMI-3Fuc (14.26 ± 0.78) under laser irradiation. Without laser irradiation, the treatment groups of PMI-3Gal, PMI-3Fuc , and PMI-3Gal@PMI-3Fuc also showed a proangiogenic effect with the relative capillary intensities of 9.25 ± 0.44, 2.80 ± 0.10, and 10.24 ± 0.16, respectively, higher than the control group (1.00 ± 0.11). These results clearly demonstrated that the co-assemblies of PMI-3Gal@PMI-3Fuc with laser irradiation considerably promoted P. aeruginosa infected wound healing with an intact epidermis and regeneration of other appendages via abundant collagen deposition and prominent angiogenesis. Moreover, the level of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) at the wound site can reflect the level of tissue inflammation to some extent [48] . The cytokines of IL-6 and TNF- α were studied to evaluate the activation and termination of many cellular activities related to repair during wound healing. The IL-6 levels (Fig. 6 g and 6 h) of PMI-3Gal, PMI-3Fuc , and PMI-3Gal@PMI-3Fuc with laser irradiation were 0.05 ± 0.002, 0.19 ± 0.03 and 0.02 ± 0.002, lower than the levels (0.32 ± 0.02, 0.65 ± 0.02, and 0.23 ± 0.02) of PMI-3Gal, PMI-3Fuc , and PMI-3Gal@PMI-3Fuc without laser irradiation. These results all outperformed the control group. Similar result was also observed for the TNF- α expression. With laser irradiation, the TNF- α level (Fig. 6 i and 6 j) in PMI-3Gal@PMI-3Fuc group was 0.01 ± 0.002, lower than PMI-3Gal (0.05 ± 0.004), PMI-3Fuc (0.20 ± 0.10), and control (1.00 ± 0.07). These results indicated that the co-assemblies of PMI-3Gal@PMI-3Fuc with laser irradiation significantly showed a higher repair effect to promote wound healing by reducing the levels of IL-6 and TNF- α [49] . 3. Conclusion In this paper, we have reported a co-assembling strategy to prepare antibiotic-free antibacterial and antibiofilm agents for wound healing using two perylene-carbohydrate conjugates ( PMI-3Gal and PMI-3Fuc ) with synergistic targeting for two lectins of P. aeruginosa LecA and LecB. The self-assembled results and carbohydrate-lentin recognition effect indicated that the co-assembly of PMI-3Gal @ PMI-3Fuc might be an interlaced mode, which exhibited a higher photothermal conversion efficiency with a value of 63% than that of PMI-3Gal and PMI-3Fuc . Due to the strong multivalent carbohydrate-lectin interactions, the co-assembly PMI-3Gal @ PMI-3Fuc showed selectively adhesion effects and the inhibition activity of biofilm formation for P. aeruginosa. Furthermore, the co-assemblies of PMI-3Gal@PMI-3Fuc showed the enhanced photothermal bacteria killing activity by a synergistic therapy combined with inherent antimicrobial effect through carbohydrate-lectin interactions and photothermal killing activity. What is more exciting is that PMI-3Gal @ PMI-3Fuc showed a better photothermal antibacterial activities for a clinical-isolated P. aeruginosa strain that is resistant to different antibiotics. Moreover, PMI-3Gal@PMI-3Fuc exhibited the acceleration effect for the wound healing in mice by simultaneously inhibiting the inflammatory response and promoting angiogenesis and tissues remodeling. This work provides a supramolecular principle for antibiotic-free antibacterial and antibiofilm effects based on multivalent glycoconjugates. Declarations Conflict of interest There are no conflicts to declare. Acknowledgements The authors thank the National Natural Science Foundation of China (U20A20259), the Natural Science Foundation of Hebei Province (B2024201007, B2023201108 and 22567635H), and the Foundation of Hebei Education Department (JZX2024018) for financial support. Supporting Information Supplementary data associated with this article can be found in the Web products. References Bali, A.; Kamal, M. A. M.; Mulla, G.; Loretz, B.; & Lehr, C. M. Functional materials to overcome bacterial barriers and models to advance their development. Adv. Funct. Mater. 33 , 2304370 (2023). Doolan, J. A. et al. Hiscock. Advancements in antimicrobial nanoscale materials and self-assembling systems. Chem. Soc. Rev. 51 , 8696-8755 (2022). Geddes, E. J. et al. Porin-independent accumulation in Pseudomonas enables antibiotic discovery. 2023 , 624 , 145-153 (2023). Wagner, S. et al. Novel strategies for the treatment of Pseudomonas aeruginosa infections. J. Med. Chem. 59 , 5929-5969 (2016). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4641881","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":320885034,"identity":"7d95590a-ff9f-47e5-b4fa-9357db69c98c","order_by":0,"name":"Ke-Rang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACCQST+RgzmD5AvBa2NJK18JgRp0Wyvffh54Jfh+XN+dd8e1zYxiDHdyOB8XMBHi3SPMeNpWf2HTbcOePtduOZbQzGkjcSmKVn4NEiJ5HGIM3bc5txw42z26R52xgSN9xIYGPmwadF/hnzb6AW+w03zjwDaaknqEVago1NmufH7cQN53vYQFoSDAhpkexJY7PmbfifvOEGm5n0jHMShjPPPGyWxqdF4vgx5ts8f9JsN5w//Ey6oMxGnu948sHP+LSAAWMbSHMC2AgQt4GQBiD4A8T8B4hQOApGwSgYBSMSAAAObkwvDbqtfQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5607-6552","institution":"Hebei University","correspondingAuthor":true,"prefix":"","firstName":"Ke-Rang","middleName":"","lastName":"Wang","suffix":""},{"id":320885035,"identity":"cf6fc905-63a2-43ca-86dd-2634a5152090","order_by":1,"name":"Jian-Xing Yang","email":"","orcid":"","institution":"Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Jian-Xing","middleName":"","lastName":"Yang","suffix":""},{"id":320885036,"identity":"7b0b4f48-2016-44f6-bf96-4264d7719a0b","order_by":2,"name":"Hai-Qing Li","email":"","orcid":"","institution":"Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Hai-Qing","middleName":"","lastName":"Li","suffix":""},{"id":320885037,"identity":"a9381ca2-714a-4e6b-999f-7093e9bd4558","order_by":3,"name":"Fangqian Yin","email":"","orcid":"","institution":"Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Fangqian","middleName":"","lastName":"Yin","suffix":""},{"id":320885038,"identity":"f7e6c590-3b09-4342-9f13-58a3769f9600","order_by":4,"name":"Wen-Juan Yin","email":"","orcid":"","institution":"Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Juan","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2024-06-26 10:10:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4641881/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4641881/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59493151,"identity":"487491dc-ca29-46c0-97e5-c3bda7ee795b","added_by":"auto","created_at":"2024-07-02 12:35:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":718407,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the self-assemblies and co-assemblies of PMI-3Gal and PMI-3Fuc targeting for LecA, LecB and LecA/LecB, and the applications of antibiotic-free antibacterial, antibiofilm effects and accelerated wound healing.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/d8c4308720cc9b0d392e9f52.png"},{"id":59493153,"identity":"228c42e4-463b-48ae-9430-00005981b630","added_by":"auto","created_at":"2024-07-02 12:35:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":446626,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The molecular structures of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e and \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e; Solvent-dependent UV–vis spectra of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e (b), \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e (c), \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e (d); (e) Turbidity assay of \u003cstrong\u003ePMI-3Gal \u003c/strong\u003e(2 ´ 10\u003csup\u003e-5\u003c/sup\u003e M), \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e (2 ´ 10\u003csup\u003e-5\u003c/sup\u003e M) and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e (2 ´ 10\u003csup\u003e-5\u003c/sup\u003e M) with PNA (2 mg/mL) and in PBS buffer (pH = 7.4, 10 mM) containing 0.1 mM NcCl\u003csub\u003e2\u003c/sub\u003e and 0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e; (f) Two possible assembly models of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e and \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e; photothermal temperature changes of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e (g), \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e (h) and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e (i) with the laser powers of 0.2 W/cm\u003csup\u003e2\u003c/sup\u003e, 0.4 W/cm\u003csup\u003e2\u003c/sup\u003e and 0.6 W/cm\u003csup\u003e2\u003c/sup\u003e under the concentration of 100 μM; and photothermal temperature changes of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e (d), \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e (e) and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e (f) with 635 nm laser irradiation (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e) at the concentrations of 25 μM, 50 μM, 75 μM and 100 μM.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/5d810f8cf1692a94c3176abb.png"},{"id":59493152,"identity":"b186f678-0ca8-46a1-93b3-620c9c72885a","added_by":"auto","created_at":"2024-07-02 12:35:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1321299,"visible":true,"origin":"","legend":"\u003cp\u003ePhotographs of bacterial colonies formed by \u003cem\u003eP. aeruginosa\u003c/em\u003e after exposed to \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e(a), \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e (b) and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e (c) with and without a laser irradiation, and the relevant quantitative analysis of bacterial viability (d-f); The quantitative determination of antibacterial efficacy of multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e after being treated the targeted photosensitizer with and without a laser irradiation, Data are presented as the mean ± SD (n = 3) (g-i); Data are presented as the mean ± SD (n = 3). Live/dead fluorescent staining images (j) and relevant quantitative analysis (k-m) of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003ewith and without a laser irradiation. Scale bar = 20 μm. Data are presented as mean values ± standard deviation (n = 3 independent experiments); (n) SEM images (scale bar = 1μm) of \u003cem\u003eP. aeruginosa\u003c/em\u003e after PTT treatments.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/0c7d5160ba55e3c3f48f33ae.png"},{"id":59493155,"identity":"c660de65-f819-4745-b476-ca4aeaa84047","added_by":"auto","created_at":"2024-07-02 12:35:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":771551,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The schematic mechanism of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e for eradicate the bacterial biofilm; (b) Biofilm inhibition by using crystal violet staining assay and absorbance values at 590 nm after treatment with different concentration of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e; (c) OD\u003csub\u003e590\u003c/sub\u003e values to evaluate the efficacy of eliminating \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms after treatment with different concentrations of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e, (n = 5 independent samples; mean ± SD); (d) CLSM images of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e for inhibiting the formations of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms. Representative CLSM images recorded for \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm after incubation with (e) \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, (f) \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and (g) \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e by photoirradiation with or without a 635 nm laser (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 min. (h) Inhibition percentage of \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms formation with various concentrations of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e. Data are presented as mean ± SD, n = 3; (i) The quantitative determination of the biofilm elimination without or with NIR irradiation. Data are presented as mean ± SD, n = 3.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/ca645c98e59b56615dc9ed5d.png"},{"id":59493156,"identity":"e7f70fa9-b980-4ac5-9040-0a070740b298","added_by":"auto","created_at":"2024-07-02 12:35:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":822872,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Evaluation of \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e, \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e in accelerating wound healing in vivo; (b) Photographs of the infected wound in different treatment groups on days 0, 2, 4, and 7; (b) The relative wound area among different groups within 7 days. Data are presented as mean ± SD (n = 3) and analyzed using a one-way ANOVA test Significances are presented by ***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05; (c) The simulation analysis of the wound trace for 7 days; (d) Representative photographs of the colony growth from wound sites in different treatment groups; (e) Corresponding quantitative results of the colony growth from wound sites in different treatment groups. Data are presented as mean ± SD (n = 3) and analyzed using a one-way ANOVA test Significances are presented by ***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05; (f) Schematic illustration of building model of \u003cem\u003eP. aeruginosa\u003c/em\u003e -infected wound and treatments to the wound.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/7afaf775d2dda747cff021fa.png"},{"id":59493157,"identity":"58b3542a-d716-461e-8d0c-2f749ea726b8","added_by":"auto","created_at":"2024-07-02 12:35:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1295322,"visible":true,"origin":"","legend":"\u003cp\u003e(a-b) H\u0026amp;E staining and Masson trichrome staining of healed skin after 7 days treatment in different groups. (c) The thickness of granulation tissue after various treatments on day 7. (d) The collagen volume fraction of tissue after various treatments on day 7. Dadas are presented as mean values ± SD (n = 3 independent experiments). (f-g) Immunofluorescence images of CD31, IL-6 and TNF-α (red) in the infected tissue on day 7. Scale bar is 100 μm. (h) Quantitative analysis of CD31 of wound tissues on day 7; (i) Quantitative analysis of IL-6 of wound tissues on day 7; (j) Quantitative analysis of TNF-α of wound tissues on day 7; (h-j) Statistical significance was analyzed via one-way ANOVA with a Tukey post-hoc test. Control; \u003cstrong\u003ePMI-3Gal\u003c/strong\u003e; \u003cstrong\u003ePMI-3Fuc\u003c/strong\u003e and \u003cstrong\u003ePMI-3Gal@PMI-3Fuc\u003c/strong\u003e with or without 635 nm irradiation (0.4 W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 min.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/bc094124e8545c774cad197a.png"},{"id":62129987,"identity":"ceb33449-e198-46b7-a800-75b53c99c355","added_by":"auto","created_at":"2024-08-09 15:33:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1516579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/e58bec76-618b-4d83-8100-5abeb5a0107d.pdf"},{"id":59493158,"identity":"75920374-da64-4fc9-9ef4-cbdc8f25b84e","added_by":"auto","created_at":"2024-07-02 12:35:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41112328,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationsubmit.docx","url":"https://assets-eu.researchsquare.com/files/rs-4641881/v1/92fb803d9b4fff312fbddb98.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Multivalent LecA/LecB Inhibitors based on the Co-assemblies of Perylene Monoimide-carbohydrate Conjugates for Antibiotic-free Antibacterial and Wound Healing","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e Pathogenic infection is becoming a global health threat to human health in recent years. There are approximately 700000 deaths per year worldwide, and the death toll caused by pathogenic infection will increase to 10\u0026nbsp;million per year by 2050 \u003csup\u003e[1]\u003c/sup\u003e, resulting a cumulative financial burden of \u003cspan\u003e$\u003c/span\u003e100 trillion from the year 2014 to 2050 \u003csup\u003e[2]\u003c/sup\u003e. According to the World Health Organization (WHO), Gram-negative bacteria, such as \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e), \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, rank as priority list 1 due to its complex Gram-negative bacterial cell envelope, high antimicrobial resistance, and lack of new antibiotics. Compared to the other Gram-negative bacteria, the development of new antibacterials for \u003cem\u003eP. aeruginosa\u003c/em\u003e is particularly challenging due to the absence of non-specific porins for the drugs across the outer membrane, the presence of tripartite efflux pumps, and substrate-specific outer-membrane porins for antibiotic efflux in \u003cem\u003eP. aeruginosa\u003c/em\u003e \u003csup\u003e[3]\u003c/sup\u003e. To date, only four classes of antibiotics are available to treat \u003cem\u003eP. aeruginosa\u003c/em\u003e infections in clinic, such as β-lactams, fluoroquinolones, aminoglycosides, and polymyxins \u003csup\u003e[3]\u003c/sup\u003e. As a result, there is an urgent need for the development of new antibacterials for \u003cem\u003eP. aeruginosa\u003c/em\u003e infections with novel mechanisms of action and therapeutic strategies \u003csup\u003e[4]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe antimicrobial resistance in most pathogenic bacteria and chronic bacterial infections are related to the formation of bacterial biofilm \u003csup\u003e[5]\u003c/sup\u003e. This is especially true for chronic wound infections of \u003cem\u003eP. aeruginosa\u003c/em\u003e, where the thickness of bacterial biofilm can reach 200 \u0026micro;m and 1400 \u0026micro;m above and below the wound surface, respectively. Both are thicker than those (150 \u0026micro;m and 190 \u0026micro;m) of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) \u003csup\u003e[5]\u003c/sup\u003e. Furthermore, treatments of biofilm bacteria require 10 to 1000 times higher antibiotic concentrations than that of planktonic bacteria \u003csup\u003e[6]\u003c/sup\u003e, which further lead to antimicrobial resistance and persistent infections. Therefore, biofilm infection microenvironment (BIM) targeted therapeutic strategy \u003csup\u003e[7]\u003c/sup\u003e would be an effective path to overcome antimicrobial resistance and kill biofilm bacteria.\u003c/p\u003e \u003cp\u003eIt is interestingly found that the tetravalent lectins \u003csup\u003e[8]\u003c/sup\u003e LecA and LecB of \u003cem\u003eP. aeruginosa\u003c/em\u003e, which were the carbohydrate-binding proteins exhibiting selectively recognition interactions with D-galactose and L-fucose/D-mannose \u003csup\u003e[9]\u003c/sup\u003e with multivalency \u003csup\u003e[10]\u003c/sup\u003e, respectively, played crucial roles in bacterial adhesion, biofilm formation, virulence, and host cell invasion \u003csup\u003e[11,12]\u003c/sup\u003e. Thus, small LecA/LecB inhibitors \u003csup\u003e[13]\u003c/sup\u003e and multivalent LecA/LecB inhibitors \u003csup\u003e[14]\u003c/sup\u003e paved a promising way to develop antibiotic-free antibacterial and antibiofilm agents as an alternative to antibiotic treatments. Based on the structural characteristics of two adjacent binding sites for simultaneous binding of two galactose moieties, a series of divalent LecA inhibitors conjugated two galactoses were developed, which exhibited ca. 500-fold binding enhancements for LecA lectin \u003csup\u003e[15]\u003c/sup\u003e and a potent LecA inhibition activity with \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e values of 11\u0026ndash;81 nM \u003csup\u003e[16]\u003c/sup\u003e. In order to enhance the water solubility, a divalent sulfonate LecA inhibitor was synthesized, showing potently inhibited LecA binding to lung epithelial cells and reduced invasion of \u003cem\u003eP. aeruginosa\u003c/em\u003e into host cells \u003csup\u003e[17]\u003c/sup\u003e. Multivalent glycoclusters and multimodal antibacterial therapy system based on calix[4]arene derivatives \u003csup\u003e[18,19]\u003c/sup\u003e targeting LecA or LecB lectins were constructed, which showed anti-adhesive property and biofilm dispersion effect. Furthermore, a series of supramolecular rotaxanes \u003csup\u003e[20]\u003c/sup\u003e based on pillar[5]arene derivatives were reported, exhibited high antibiofilm activity through simultaneous targeting the two lectins LecA and LecB. However, it is surprising to find that supramolecular rotaxanes exhibited antibiofilm activity, but showed no bacteria killing activity. In order to enhance the bactericidal activity and to reduce the potential immunogenic effect, several biofilm-targeted drugs based on monovalent D-galactose or L-fucose modified glycoconjugates of sulfonamides \u003csup\u003e[21,22]\u003c/sup\u003e and ciprofloxacin \u003csup\u003e[23]\u003c/sup\u003e were developed, which could decrease the systemic side effects. However, the antibiotic activity was significantly reduced. In an effort to improve the antibiotic activity, a series of activated prodrugs based on fluoroquinolones-carbohydrate derivatives were constructed \u003csup\u003e[24]\u003c/sup\u003e, which showed the biofilm-targeted effects and LasB (Zn(II)-dependent metalloprotease in \u003cem\u003eP. aeruginosa\u003c/em\u003e)-mediated release of antibiotic. Multivalent carbohydrate-drug systems targeting LecA, LecB, or LecA/LecB based on series of glycopolymers conjugated with a BODIPY photosensitizer \u003csup\u003e[25]\u003c/sup\u003e, copper sulfide nanocrystals \u003csup\u003e[26]\u003c/sup\u003e, gold nanorods \u003csup\u003e[27]\u003c/sup\u003e and gold nanoparticles \u003csup\u003e[28]\u003c/sup\u003e were also reported, exhibiting efficient biofilm inhibition, photothermal or photodynamic killing capability. On the other hand, biofilm-targeted drug delivery system was another way to improve antibiotic activity \u003csup\u003e[29]\u003c/sup\u003e. Recently, hypoxia-responsive delivery of lactose-modified azocalix[4]arene and ciprofloxacin by us and our collaborators \u003csup\u003e[30]\u003c/sup\u003e was constructed, showed the inhibition effect in biofilm formation and hypoxia-responsive delivery antibiotic for killing \u003cem\u003eP. aeruginosa\u003c/em\u003e. Due to the hetero-multivalent effect in carbohydrate-lectin interactions, hetero-multivalent targeting drug delivery systems of ciprofloxacin-loaded liposomes \u003csup\u003e[31]\u003c/sup\u003e and ICG-loaded co-assembled nanoparticles \u003csup\u003e[32]\u003c/sup\u003e were developed, showed the treatment of infections caused by \u003cem\u003eP. aeruginosa\u003c/em\u003e and enhanced photothermal and photodynamic therapy of antibiotic-resistant bacterial pneumonia.\u003c/p\u003e \u003cp\u003eAntibiofilm agents \u003csup\u003e[33]\u003c/sup\u003e based on glycoconjugates targeting for LecA and LecB lectins in \u003cem\u003eP. aeruginosa\u003c/em\u003e are efficient approaches to treat antimicrobial resistance through inhibiting bacterial biofilm formation, disrupting mature biofilm, and removing biofilm. However, the killing efficiency for \u003cem\u003eP. aeruginosa\u003c/em\u003e is limited, so the combined therapeutic functional molecules as the key therapeutic agents can efficiently enhance the bactericidal activity. As a result, multivalent glycoconjugates with combination of the recognition function of carbohydrates and the therapeutic function of drug would be a powerful strategy for the treatment of bacterial infections. One of the inherent problems in the multivalent glycoconjugates is the efficient recognition of \u003cem\u003eP. aeruginosa\u003c/em\u003e with controllable and suitable carbohydrate types and numbers for simultaneous targeting of LecA and LecB. Another problem is the efficiently therapeutic agents. Supramolecular assembly strategy \u003csup\u003e[34,35]\u003c/sup\u003e is an effective way to form multivalent glycoclusters with different types and number of carbohydrates. Moreover, photothermal therapy (PTT) as an alternative strategy for bacterial infections has attracted much more attentions due to its causing of cell membrane damages, protein denaturation, cell membrane damages, protein denaturation and heat stress \u003csup\u003e[36]\u003c/sup\u003e. In them, perylene monoamide (PMI) derivatives exhibited strong near infrared (NIR) absorption \u003csup\u003e[37]\u003c/sup\u003e, controllable self-assembly behaviors \u003csup\u003e[37]\u003c/sup\u003e, potent photothermal stability, and phototherapy effect \u003csup\u003e[38]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this paper, multivalent lectin inhibitors for LecA, LecB, and LecA/LecB based on the self-assemblies and co-assemblies of perylene monoamide (PMI)-glycoconjugates (\u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Scheme S1) were developed, which showed selective adhesion effects and inhibition activity of biofilm formation for \u003cem\u003eP. aeruginosa\u003c/em\u003e. In particular, the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e demonstrated enhanced photothermal therapy effects and excellent bacteria killing activity in vitro and in vivo. Moreover, the effective photothermal and inherent antimicrobial synergistic therapy of the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e showed the acceleration effect for the wound healing in mice. This result opens a supramolecular principle for antibiotic-free antibacterial and antibiofilm effects based on multivalent glycoconjugates.\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Synthesis of perylene monoamide-glycoconjugates (PMI-3Gal and PMI-3Fuc)\u003c/h2\u003e \u003cp\u003eAs shown in Scheme S1, compounds \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e were synthesized by a click reaction of the intermediate \u003cb\u003ePMI-1\u003c/b\u003e with the azide group modified D-galactose and L-fucoside, respectively, and followed by deprotection of the acetyl groups. The intermediates and the target molecules (\u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e) were fully characterized by \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR) as well as high resolution mass (HRMS) spectra. (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e2, Supporting information).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Assembly properties of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/h2\u003e \u003cp\u003eDue to the strong π-π stacking interactions of the perylene backbones, \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) could form the self-assemblies and the co-assemblies in water. Firstly, solvent-dependent UV-vis spectra of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e were used to study the self-assembly and co-assembly behaviors. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e showed the maximum absorption band at 630 nm and 636 nm in DMSO solution, respectively, indicating that different types of the carbohydrate modification influenced the optical property of PMI. These optical properties of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e (1:1) in DMSO solution remained a non-aggregated state \u003csup\u003e[37]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, the maximum absorption band of the co-assembly \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e was at 633 nm, which was lower than the maximum absorption of \u003cb\u003ePMI-3Fuc\u003c/b\u003e and higher than the maximum absorption of \u003cb\u003ePMI-3Gal\u003c/b\u003e. Upon increasing of the water ratio, the intensities of the maximum bands decreased, and the maximum bands underwent a hypsochromic shift to 566 nm, 569 nm and 566 nm for \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e, respectively, indicating the formation of H-aggregates due to strong intermolecular π-π stacking interactions in water \u003csup\u003e[37]\u003c/sup\u003e. Fluorescence spectra of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed a similar result. As shown in Figure S13, strong fluorescence emission bands at 736 nm, 736 nm and 738 nm of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e were found in DMSO solution, and decreased along with the increasing of water due to the enhanced π\u0026thinsp;\u0026minus;\u0026thinsp;π stacking interactions.\u003c/p\u003e \u003cp\u003eThe self-assembly and co-assembly behaviors were further investigated by the dynamic light scattering (DLS) measurements. As shown in Figure S14a-c, the assemblies with the mean diameters of 259.85 nm, 259.33 nm and 261.98 nm for \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e were observed, and no obvious differences in the self-assemblies and the co-assemblies were found. Different morphologies were characterized by the SEM images, as shown in Figure S14 d-f, which showed silk ribbon for \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e, and flocculent aggregates for \u003cb\u003ePMI-3Fuc\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn addition, the co-assembly model of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e was investigated by a turbidity assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Two possible assembly models can be postulated, that is, the complexes (Model I, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) of the assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e and the assemblies of \u003cb\u003ePMI-3Fuc\u003c/b\u003e or the co-assemblies (Model II, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e with an interlaced mode. It is well known that lectin-carbohydrate interactions are specific and selective \u003csup\u003e[39]\u003c/sup\u003e, and the self-assembled glycoclusters show enhanced binding interactions \u003csup\u003e[40]\u003c/sup\u003e with unique lectin through multivalent effect. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, the turbidity of the assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e showed a sharp increase upon addition of peanut agglutinin (PNA) lectin because PNA selectively bound to the terminal-galactosyl residues \u003csup\u003e[40]\u003c/sup\u003e. However, the turbidity of the mixture of the self-assemblies of \u003cb\u003ePMI-3Fuc\u003c/b\u003e with PNA showed no obvious change, indicating no binding to PNA. When adding co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e, the turbidity increased, but was lower than that of the self-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e. This result was due to that the insertion of non-recognizable carbohydrate in the assemblies weakened the specific and selective binding interactions of PNA with the terminal-galactosyl residues. These results indicated that the possible assembly model was the co-assemblies (Model II, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e with an interlaced mode.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Photothermal properties of PMI-3Gal and PMI-3Fuc\u003c/h2\u003e \u003cp\u003eBenefited from the strong π-π stacking interactions, the assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed very weak fluorescence in water, which was advantageous to the photothermal effect \u003csup\u003e[41]\u003c/sup\u003e. The photothermal effects of the assemblies and the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e were studied under different laser powers. Under the concentration of 100 \u0026micro;M, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-i, the temperatures of \u003cb\u003ePMI-3Gal\u003c/b\u003e at the laser powers of 0.2, 0.4 and 0.6 W/cm\u003csup\u003e2\u003c/sup\u003e increased to 48.2\u0026deg;C, 65.6\u0026deg;C and 72.6\u0026deg;C, respectively, which were higher than that of \u003cb\u003ePMI-3Fuc\u003c/b\u003e and lower than that of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIt is well known that photothermal therapy for tumor needs sufficient light and intense power to penetrate the tissues of the organism \u003csup\u003e[36]\u003c/sup\u003e. However, it is dangerous to the surrounding healthy tissue, especially for wound infections, which are not as deep as tumors \u003csup\u003e[42]\u003c/sup\u003e. Therefore, concentration-dependent photothermal effects under a power of 0.4 W/cm\u003csup\u003e2\u003c/sup\u003e were studied. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-i, the temperatures of \u003cb\u003ePMI-3Gal\u003c/b\u003e increased to 48.2\u0026deg;C,56.2\u0026deg;C, 61.0\u0026deg;C, and 65.6\u0026deg;C under the concentrations of 25 \u0026micro;M, 50 \u0026micro;M, 75 \u0026micro;M and 100 \u0026micro;M, respectively. The co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e also showed the best photothermal effect with maximum temperatures of 49.1\u0026deg;C, 56.4\u0026deg;C, 62.7\u0026deg;C, and 66.3\u0026deg;C. \u003cb\u003ePMI-3Fuc\u003c/b\u003e showed the low temperatures of 46.2\u0026deg;C, 54.8\u0026deg;C, 59.5\u0026deg;C, and 64.2\u0026deg;C. This result indicated that different types of carbohydrate modification in PMI influenced the photothermal effect. As a control experiment, the temperature of the water solution only increased from 27.0\u0026deg;C to 29.0\u0026deg;C under laser irradiation. It is very interesting to note that the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e exhibited the best photothermal effect.\u003c/p\u003e \u003cp\u003eFurthermore, the quantitative photothermal-conversion efficiency (\u0026#120578;) was calculated by warming/cooling curves with a Roper\u0026rsquo;s method \u003csup\u003e[43]\u003c/sup\u003e. As shown in Figure S15, the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed a high photothermal conversion efficiency value of 63%, which was higher than that of \u003cb\u003ePMI-3Gal\u003c/b\u003e (55%) and \u003cb\u003ePMI-3Fuc\u003c/b\u003e (48%). Combined with the optical properties and the morphologies, we can conclude that different types of carbohydrate modification in PMI influence the optical properties, assembly behaviors, and the photothermal effect. In addition, the photostability of the self-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e was evaluated upon five cycles under laser irradiation. A cycle was performed by irradiation of the sample for 10 min with laser, and then remove the laser, and the temperature reached a high point and then decreased to the room temperature. There were no obvious changes for the high temperature during five cycles of laser irradiation, indicating that \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e possessed high photostability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Lectin-targeted antimicrobial activity of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc for \u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe tetravalent lectins LecA and LecB of \u003cem\u003eP. aeruginosa\u003c/em\u003e exhibited selective recognition with D-galactose and L-fucose and played crucial roles in bacterial adhesion, biofilm formation, and virulence. Firstly, the adhesion actions of \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e toward \u003cem\u003eP. aeruginosa\u003c/em\u003e were performed.\u003c/p\u003e \u003cp\u003eAs shown in Figure S16 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e with a working concentration of 9 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e colony forming units (CFU) mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was incubated for 6 h at 37\u0026deg;C with various concentrations (50 \u0026micro;M, 75 \u0026micro;M, 100 \u0026micro;M, 125 \u0026micro;M, 150 \u0026micro;M, and 200 \u0026micro;M) of the assemblies. The adhesion concentrations, determined by the Lambert-Beer law, were 16 \u0026micro;M, 29 \u0026micro;M, 56 \u0026micro;M, 92 \u0026micro;M, and 140 \u0026micro;M for \u003cb\u003ePMI-3Gal\u003c/b\u003e, 12 \u0026micro;M, 24 \u0026micro;M, 45 \u0026micro;M, 68 \u0026micro;M, 79 \u0026micro;M, and 125 \u0026micro;M for \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and 17 \u0026micro;M, 30 \u0026micro;M, 56 \u0026micro;M, 80 \u0026micro;M, 93 \u0026micro;M, and 140 \u0026micro;M for \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e. Compared with \u003cb\u003ePMI-3Fuc\u003c/b\u003e, \u003cb\u003ePMI-3Gal\u003c/b\u003e showed a better adhesion interaction for \u003cem\u003eP. aeruginosa\u003c/em\u003e. Considering the result that \u003cb\u003ePMI-3Fuc\u003c/b\u003e showed a weak adhesion interaction for \u003cem\u003eP. aeruginosa\u003c/em\u003e, the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e should exhibit weaker adhesion effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e than that of \u003cb\u003ePMI-3Gal\u003c/b\u003e in theory. However, it is interestingly found that the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed the best adhesion effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e, which might due to a hetero-multivalent effect \u003csup\u003e[44]\u003c/sup\u003e that enhances the affinity towards the target lectin by cooperative recognition interactions with different saccharides.\u003c/p\u003e \u003cp\u003eBenefited from the strong adhesion effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e and high photothermal effect of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e, their antibacterial activities were further studied. The antibacterial performances of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with and without laser irradiation against \u003cem\u003eP. aeruginosa\u003c/em\u003e in vitro were evaluated by observing the counts of colonies growing on the agar plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f, there were no \u0026ldquo;dark\u0026rdquo; therapeutic effects of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e against \u003cem\u003eP. aeruginosa\u003c/em\u003e with the viabilities of 99.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08%, 99.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41% and 99.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% at high concentrations of 140 \u0026micro;M, 125 \u0026micro;M, and 140 \u0026micro;M, respectively. Under laser irradiation of 0.40 W/cm\u003csup\u003e2\u003c/sup\u003e for 10 minutes, the remarkably photothermal antibacterial activities were observed. Along with increasing of the concentrations, \u003cb\u003ePMI-3Fuc\u003c/b\u003e exhibited enhanced photothermal antibacterial activities for \u003cem\u003eP. aeruginosa\u003c/em\u003e with the antibacterial ratios of 23.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90%, 34.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77%, 55.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06%, 66.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38%, 74.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69%, and 85.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%, respectively. As expected, \u003cb\u003ePMI-3Gal\u003c/b\u003e showed a better photothermal antibacterial activity than \u003cb\u003ePMI-3Fuc\u003c/b\u003e due to stronger adhesion effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e and higher photothermal effect. The antibacterial ratios were 39.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21%, 57.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48%, 70.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33%, 77.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.70%, 84.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34%, and 99.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12% under the concentration of 16 \u0026micro;M, 29 \u0026micro;M, 56 \u0026micro;M, 92 \u0026micro;M, and 140 \u0026micro;M, respectively. Moreover, the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e demonstrated a significant cooperative therapeutic effect with simultaneous targeting to LecA and LecB lectins, resulting in concentration-dependent photothermal antibacterial activities of 39.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24%, 61.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64%, 80.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%, 88.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10%, 98.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21%, and 100% under the concentration of 16 \u0026micro;M, 29 \u0026micro;M, 56 \u0026micro;M, 92 \u0026micro;M and 140 \u0026micro;M, respectively. Full eradication of \u003cem\u003eP. aeruginosa\u003c/em\u003e was observed under the concentration of 140 \u0026micro;M for the assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e, indicated that LecA lectin showed a main adhesion effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e. Under the same concentration of 93 (92) \u0026micro;M, the photothermal antibacterial activity of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e was 98.14%, larger than that of \u003cb\u003ePMI-3Gal\u003c/b\u003e (84.07%), suggesting a cooperative therapeutic effect for targeting of LecB lectin.\u003c/p\u003e \u003cp\u003eFurthermore, the antibacterial performances of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e against multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolated from mucus samples of Neurocritical care patient (WE3101, that is resistant to Imipenem and Meropenem) were evaluated (Figure S17). Along with increasing of the concentrations, as expected, \u003cb\u003ePMI-3Gal\u003c/b\u003e showed a better photothermal antibacterial activities for multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e than that of \u003cb\u003ePMI-3Fuc\u003c/b\u003e.The bacterial survival rate was calculated as 90.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24%, 79.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51%, 65.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36%, 48.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.72%, 33.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38% and 12.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15% under the concentration of 43 \u0026micro;M, 76 \u0026micro;M, 100\u0026micro;M, 126 \u0026micro;M, 158 \u0026micro;M and 165 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). The antibacterial activities were observed for \u003cb\u003ePMI-3Fuc\u003c/b\u003e, which exhibited the bacterial survival rate of 97.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92%, 92.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79%, 84.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08%, 73.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40%, 65.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71% and 50.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45% under the concentration of 35 \u0026micro;M, 65 \u0026micro;M, 83 \u0026micro;M, 120 \u0026micro;M, 145 \u0026micro;M and 155 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), respectively. Moreover, the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e became more significant after NIR irradiation. The bacterial survival rate decreased to 82.02\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05%, 65.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45%, 48.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93%, 35.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91%, 16.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02% and 2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81% under the concentration of 52 \u0026micro;M, 88 \u0026micro;M, 112 \u0026micro;M, 149 \u0026micro;M, 164 \u0026micro;M and 172 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the PTT antibacterial efficacy of the co-assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e with different assembling ratios were investigated. As shown in Figure S18, the best antibacterial effect was observed for the 1:1 co-assembly of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e (\u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e), which exhibited nearly 95% inhibition ratio for \u003cem\u003eP. aeruginosa\u003c/em\u003e after coincubation with the bacteria for 6 h under 635 nm laser irradiation for 10 min. When the assemble ratios of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e changed to 1:2 and 2:1, the PTT antibacterial inhibition ratios for \u003cem\u003eP. aeruginosa\u003c/em\u003e were 83% and 86%, lower than that of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e. Moreover, the selectivity of the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e was also studied against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, which showed the antibacterial activities of 72% and 48%, as shown in Figure S18b-c, lower than the PTT antibacterial activity for \u003cem\u003eP. aeruginosa\u003c/em\u003e. These results indicated that \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with 1:1 ratio demonstrated a high PTT antibacterial effect and exhibited selectively killing effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn addition, the bacterial live/dead staining assay was further performed to investigate the photothermal antibacterial effect for \u003cem\u003eP. aeruginosa\u003c/em\u003e with SYTO 9 and PI staining live and dead bacteria, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej and S19, abundant green fluorescence labeled live bacteria (indicating no damage effect to the bacteria) for five groups: PBS groups regardless of laser irradiation, and the assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e at high concentrations of 140 \u0026micro;M (125 \u0026micro;M) without irradiation. Under 635nm (0.40 W/cm\u003csup\u003e2\u003c/sup\u003e) NIR irradiation for 10 min, live bacteria with green fluorescence decreased and dead bacteria with red fluorescence increased, both showed a concentration-dependent antibacterial activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek-m, the antibacterial effects of \u003cb\u003ePMI-3Fuc\u003c/b\u003e were calculated to be 7.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50%, 24.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11%, 44.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19%, and 77.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05% at 12, 45, 79, and 125 \u0026micro;M using Image J. Enhanced antibacterial activities were observed for \u003cb\u003ePMI-3Gal\u003c/b\u003e, which exhibited antibacterial ratios of 25.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.52%, 46.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59%, 72.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33%, and 90.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34% under the concentrations of 16, 56, 92, and 140 \u0026micro;M, respectively. As expected, the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e showed the best antibacterial activities with the antibacterial ratios of 38.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%, 55.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73%, 89.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45%, and 99.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% at 17, 56, 93 and 140 \u0026micro;M, respectively. These results were consistent with colony growth on the LB agar plates.\u003c/p\u003e \u003cp\u003eThe morphological changes of bacteria treated with \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with and without laser irradiation were further explored. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en, an integrated cell membrane with clear edges and smooth surface of bacteria were observed for the PBS group and the unirradiated \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e. Under laser treatment, the collapsed cell membranes for \u003cem\u003eP. aeruginosa\u003c/em\u003e were found, and almost all the bacterial membranes were destroyed due to the PTT effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Lectin-targeted antibiofilm activity of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc for \u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eFormation of bacterial biofilm is one of the most critical factors leading to drug resistance, which further prevents antimicrobial drugs from contacting bacteria, thereby delaying the healing of infected wounds. Fortunately, the formation of biofilm is related with lectins LecA and LecB presenting on the outer membrane of \u003cem\u003eP. aeruginosa\u003c/em\u003e, which brings opportunity for the development of antibiotic-free antibacterial agents (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe antibiofilm performances of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e against \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms were evaluated by crystal violet (CV) staining assay. Due to strong adhesion interactions of galactose with LecA and fuctose with LecB, the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e showed the highest biofilm inhibition effects with the ratios of 25.08%, 34.96%, 45.94%, and 58.14% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) under the concentrations of 10 \u0026micro;M, 20 \u0026micro;M, 40 \u0026micro;M, and 80 \u0026micro;M, respectively. \u003cb\u003ePMI-3Gal\u003c/b\u003e displayed a better antibacterial effect than \u003cb\u003ePMI-3Fuc\u003c/b\u003e. These results suggested that lectin-targeted agents showed a potential application for antibiotic-free antibiofilm effects for \u003cem\u003eP. aeruginosa\u003c/em\u003e. It is well known that removal of mature biofilms is more difficult than the inhibition of biofilm formation due to the formation of a microbial community with polysaccharides, extracellular DNA (eDNA), lipids, and proteins. Upon increasing of the concentrations to 20 \u0026micro;M, 40 \u0026micro;M, 80 \u0026micro;M and 160 \u0026micro;M, the assemblies of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e also exhibited dispersion effects on the mature biofilms for \u003cem\u003eP. aeruginosa\u003c/em\u003e, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, the trends were \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003ePMI-3Gal\u003c/b\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003ePMI-3Fuc\u003c/b\u003e. \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e displayed the dispersion ratios of 28.83%, 33.06%, 39.97%, and 49.41%. \u003cb\u003ePMI-3Gal\u003c/b\u003e with the dispersion ratios of 25.22%, 33.18%, 39.96%, and 46.80%; and \u003cb\u003ePMI-3Fuc\u003c/b\u003e with the dispersion ratios of 24.69%, 33.05%, 38.49%, and 43.93% were observed. These results indicated that lectin-targeted agents showed the application for antibiotic-free antibiofilm effects both for mature biofilms and immature biofilms.\u003c/p\u003e \u003cp\u003eMoreover, antibiofilm effects were further examined with fluorescence imaging by live/dead bacterial staining. Intense green fluorescence for the control group was observed, indicated a very intact biofilm structure. When addition of 50 \u0026micro;M and 100 \u0026micro;M of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e, green fluorescence on biofilm was decreased, suggesting inhibition biofilm effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In addition, removal effects of biofilm based on \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with and without laser irradiation were also studied by a live/dead bacterial staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-g). With laser irradiation, the removal ratios of biofilm based on \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e increased from 38.53%, 27.14%, and 44.18\u0026ndash;51.10%, 42.22%, and 55.83% at the concentration of 100 \u0026micro;M, respectively. Increasing the concentration to 200 \u0026micro;M, the removal ratios of biofilm based on \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with laser irradiation were 89.73%, 75.97%, and 99.60%, which were larger than that (55.38%, 45.17% and 59.14%) of the group without laser irradiation. These results demonstrated that \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e could effectively inhibited and destroyed the formation of bacterial biofilms, showing the potential to eliminate bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 In vitro cell migration and in vivo wound repair of PMI-3Gal, PMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/h2\u003e \u003cp\u003eCell migration and proliferation are critical processes for wound healing \u003csup\u003e[45]\u003c/sup\u003e. The biocompatibility of \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with L929 cells was studied by MTT assay under various concentrations. As shown in Figure S20 Near 100% cell viability was observed even at a high concentration of 100 \u0026micro;M, suggesting no toxicity to L929 cells. Moreover, the effects of \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e on cell migration were studied by cell scratch assay at different time. The L929 cells were used to mimic wound infection in vitro. As illustrated in Figure S21, \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e all showed promoting effect on cell migration. \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e exhibited a relatively high ability to promote cell migration compared with the other groups, which showed the healing rates of 65.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32% and 65.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33% at 24 h through calculating the scratched area, both higher than that of \u003cb\u003ePMI-3Fuc\u003c/b\u003e (51.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37%). Increasing of the cell incubation time to 48 h, the cell scratch coverages increased to 96.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06%, 82.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03% and 99.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16% for \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e, respectively. These results suggested that galactose residue was advantageous to promote cell migration, and the combination of galactose residue and fuctose residue exhibited a collaborative promotion effect.\u003c/p\u003e \u003cp\u003eHemolysis is a key evaluation factor of the further application in vivo for multivalent glycoclusters. A hemolysis rate over 5% is adverse according to the standard of International Organization for Standardization (ISO) \u003csup\u003e[46]\u003c/sup\u003e. As shown in Figure S22, bright red supernatant of the Triton X-100 group was observed, indicated a serious hemolysis effect for the control group. However, the hemolysis rates of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e were lower than 3% even at a high concentration of 200 \u0026micro;M, suggesting good biocompatibility.\u003c/p\u003e \u003cp\u003eFurthermore, the wound healing effects of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e were investigated. Firstly, \u003cem\u003eP. aeruginosa\u003c/em\u003e infected mouse whole skin wound models based on BALB/c mice were successfully established after \u003cem\u003eP. aeruginosa\u003c/em\u003e infection, and the bacterial colonies was observed on Day 2. These mice were randomly divided into eight groups, including: \u003cb\u003ePBS\u003c/b\u003e groups without (I) and with laser irradiation (II), \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e groups without (III-V) and with laser irradiation (VI-VIII). Treatments with \u003cb\u003ePMI-3Gal\u003c/b\u003e, P\u003cb\u003eMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e were implemented on the infected sites. Upon 635 nm laser irradiation, the temperatures of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/b\u003e on wounds were recorded by an IR camera. As shown in Figure S23, the temperatures increased to 49.2℃, 44.7℃, 52.0℃ for \u003cb\u003ePMI-3Gal\u003c/b\u003e, P\u003cb\u003eMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/b\u003e after irradiation for 10 min, which were higher than that of PBS group (37.9℃).\u003c/p\u003e \u003cp\u003eDynamic wound healing process was photographed on dyes 2, 4 and 7. All wound sizes progressively reduced during the treatment period. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, it was obvious found that the wounds of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with and without laser irradiation were smaller than those of the PBS groups with and without laser irradiation. The average unhealing areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) in \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/b\u003e without laser irradiation were 46.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51%, 56.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54%, and 30.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25% on the 7th day, respectively, indicating that carbohydrate-lectin interactions can promote wound healing due to lectin-targeted antibacterial effects. With laser irradiation, the average unhealing areas in \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/b\u003e increased to 27.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14%, 37.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84% and 16.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95% on the 7th day, respectively, suggesting a photothermal promoted killing bacteria activity. As the control groups, the average unhealing areas in PBS groups were 55.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89% (without laser irradiation) and 63.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15% (with laser irradiation), respectively. From the photographic images, the wound closure simulation plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) suggested that the skin regeneration of the assemblies with laser irradiation groups is obvious faster than the assemblies without laser irradiation groups, and which all faster than the control. The trend of the skin regeneration is \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003ePMI-3Gal\u003c/b\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cb\u003ePMI-3Fuc\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the antibacterial effects of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/b\u003e during infected wound healing process were investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, tissue fluids were collected from the infected wound surface after treatment on dyes 2, 4 and 7. The extracted \u003cem\u003eP. aeruginosa\u003c/em\u003e was incubated on agar plates, and the bacterial colonies were evaluated using Image J. After treated for 4 days, the bacteria colonies in \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e groups decreased to 67.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48%, 74.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71% and 57.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), respectively, which were lower than that (88.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09%) of PBS group. After treated for 7 days, the bacteria colonies continuously decreased to 57.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48%, 65.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17%% and 38.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) for \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc and PMI-3Gal@PMI-3Fuc\u003c/b\u003e, respectively.\u003c/p\u003e \u003cp\u003eWith laser irradiation, significantly bacteria killing effects were observed. The bacteria colonies decreased to 30.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14%, 37.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81%, and 1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) for \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc, and PMI-3Gal@PMI-3Fuc\u003c/b\u003e after treatment of 7 days, respectively. These results demonstrated that synergistic therapy combined with inherent antimicrobial effect and photothermal killing activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) based on \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e in vivo showed potent wound healing in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histopathologic evaluations of collagen deposition, neovascularization, and inflammation microenvironment\u003c/h2\u003e \u003cp\u003eHistopathologic evaluations of the regenerated skin after 7 d treatment provided insight into the wound healing effect. The skin sections of Hematoxylin \u0026amp; Eosin (H\u0026amp;E) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) showed severe infection in tissues for the control groups. After 7 d treatment with the assemblies of the perylene-carbohydrate conjugates without laser irradiation, the infiltrated inflammatory cells decreased, suggesting an antibacterial effect through a mechanism of carbohydrate-lectin interactions. Moreover, an obvious reduction of the infiltrated inflammatory cells was found based on the assemblies of the perylene-carbohydrate conjugates with laser irradiation, indicating a remarkable antibacterial effect by a synergistic therapy combined with inherent antimicrobial effect and photothermal killing activity. In addition, an intact epidermis with a thick granulation tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) was observed in the treatment groups of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e (with laser irradiation) with the thickness of 233.80\u0026thinsp;\u0026plusmn;\u0026thinsp;18.16, 153.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.79, and 252.05\u0026thinsp;\u0026plusmn;\u0026thinsp;8.94 \u003cem\u003e\u0026micro;\u003c/em\u003em, respectively. The granulation tissues of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e (without laser irradiation) showed a thickness of 132.23\u0026thinsp;\u0026plusmn;\u0026thinsp;7.63, 113.21\u0026thinsp;\u0026plusmn;\u0026thinsp;10.22, and 164.23\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75 \u0026micro;m, lower than that of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e (with laser irradiation) groups. On the other hand, the thickness of granulation tissue for the control was only 90.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40 and 95.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86 \u003cem\u003e\u0026micro;\u003c/em\u003em for PBS groups with and without laser irradiation.\u003c/p\u003e \u003cp\u003eIn addition, the organs in mice (heart, liver, spleen, lung, and kidney) were isolated and collected to investigate the biocompatibility. H\u0026amp;E staining images (Figure S24) showed no significant toxic side effects, and the body weight (Figure S25) of mice under the treatment process has no significant changes.\u003c/p\u003e \u003cp\u003eFurthermore, the collagen deposition and arrangement of the healing skin were employed by a Masson staining assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, abundant collagen with dense and organized structures were observed for the groups of \u003cb\u003ePMI-3Gal\u003c/b\u003e, \u003cb\u003ePMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e (with laser irradiation), which were larger than that of the other groups. In them, the groups of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e exhibited a higher content of collagen in the dermis. Angiogenesis is an essential parameter for wound regeneration \u003csup\u003e[47]\u003c/sup\u003e. CD31 is a classical marker expressed in vascular endothelial cells \u003csup\u003e[30]\u003c/sup\u003e. The neovascularization for the regenerated tissue after 7 d treatment was investigated by an immunofluorescence staining method (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, marked red fluorescence was observed, which exhibited a relative capillary intensity of 31.22\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12 for \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e group with laser irradiation. This result was higher than the groups of \u003cb\u003ePMI-3Gal\u003c/b\u003e (16.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69) and \u003cb\u003ePMI-3Fuc\u003c/b\u003e (14.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78) under laser irradiation. Without laser irradiation, the treatment groups of \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e also showed a proangiogenic effect with the relative capillary intensities of 9.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44, 2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, and 10.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, respectively, higher than the control group (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11). These results clearly demonstrated that the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with laser irradiation considerably promoted \u003cem\u003eP. aeruginosa\u003c/em\u003e infected wound healing with an intact epidermis and regeneration of other appendages via abundant collagen deposition and prominent angiogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the level of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) at the wound site can reflect the level of tissue inflammation to some extent \u003csup\u003e[48]\u003c/sup\u003e. The cytokines of IL-6 and TNF-\u003cem\u003eα\u003c/em\u003e were studied to evaluate the activation and termination of many cellular activities related to repair during wound healing. The IL-6 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh) of \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with laser irradiation were 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, lower than the levels (0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, and 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) of \u003cb\u003ePMI-3Gal, PMI-3Fuc\u003c/b\u003e, and \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e without laser irradiation. These results all outperformed the control group. Similar result was also observed for the TNF-\u003cem\u003eα\u003c/em\u003e expression. With laser irradiation, the TNF-\u003cem\u003eα\u003c/em\u003e level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej) in \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e group was 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, lower than \u003cb\u003ePMI-3Gal\u003c/b\u003e (0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004), \u003cb\u003ePMI-3Fuc\u003c/b\u003e (0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10), and control (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07). These results indicated that the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e with laser irradiation significantly showed a higher repair effect to promote wound healing by reducing the levels of IL-6 and TNF-\u003cem\u003eα\u003c/em\u003e \u003csup\u003e[49]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn this paper, we have reported a co-assembling strategy to prepare antibiotic-free antibacterial and antibiofilm agents for wound healing using two perylene-carbohydrate conjugates (\u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e) with synergistic targeting for two lectins of \u003cem\u003eP. aeruginosa\u003c/em\u003e LecA and LecB. The self-assembled results and carbohydrate-lentin recognition effect indicated that the co-assembly of \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e might be an interlaced mode, which exhibited a higher photothermal conversion efficiency with a value of 63% than that of \u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e. Due to the strong multivalent carbohydrate-lectin interactions, the co-assembly \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed selectively adhesion effects and the inhibition activity of biofilm formation for \u003cem\u003eP. aeruginosa.\u003c/em\u003e Furthermore, the co-assemblies of \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e showed the enhanced photothermal bacteria killing activity by a synergistic therapy combined with inherent antimicrobial effect through carbohydrate-lectin interactions and photothermal killing activity. What is more exciting is that \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed a better photothermal antibacterial activities for a clinical-isolated \u003cem\u003eP. aeruginosa strain\u003c/em\u003e that is resistant to different antibiotics. Moreover, \u003cb\u003ePMI-3Gal@PMI-3Fuc\u003c/b\u003e exhibited the acceleration effect for the wound healing in mice by simultaneously inhibiting the inflammatory response and promoting angiogenesis and tissues remodeling. This work provides a supramolecular principle for antibiotic-free antibacterial and antibiofilm effects based on multivalent glycoconjugates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e \u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank the National Natural Science Foundation of China (U20A20259), the Natural Science Foundation of Hebei Province (B2024201007, B2023201108 and 22567635H), and the Foundation of Hebei Education Department (JZX2024018) for financial support.\u003c/p\u003e\n\u003ch2\u003eSupporting Information\u003c/h2\u003e \u003cp\u003eSupplementary data associated with this article can be found in the Web products.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBali, A.; Kamal, M. A. M.; Mulla, G.; Loretz, B.; \u0026amp; Lehr, C. M. 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Photothermal hydrogel encapsulating intelligently bacteria-capturing bio-MOF for infectious wound healing. \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 19491-19508 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"self-assembly, carbohydrate-lectin interactions, antimicrobial, photothermal therapy, multivalent","lastPublishedDoi":"10.21203/rs.3.rs-4641881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4641881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathogenic infection is becoming a global health threat to human health. Especially for the treatment of \u003cem\u003eP. aeruginosa\u003c/em\u003e remains particularly challenging. Fortunately, it is interestingly found that the LecA and LecB lectins of \u003cem\u003eP. aeruginosa\u003c/em\u003e played crucial roles in bacterial adhesion, biofilm formation, virulence, and host cell invasion. Herein, a co-assemble strategy to prepare antibiotic-free antibacterial and antibiofilm agents by using two kinds of perylene-carbohydrate conjugates (\u003cb\u003ePMI-3Gal\u003c/b\u003e and \u003cb\u003ePMI-3Fuc\u003c/b\u003e) with synergistic targeting for two lectins of \u003cem\u003eP. aeruginosa\u003c/em\u003e LecA and LecB was developed. Due to the strong multivalent carbohydrate-lectin interactions both for LecA and LecB lectins, the co-assembly \u003cb\u003ePMI-3Gal\u003c/b\u003e@\u003cb\u003ePMI-3Fuc\u003c/b\u003e showed selective adhesion effects, inhibition activity of biofilm formation and potent photothermal antibacterial activities for \u003cem\u003eP. aeruginosa\u003c/em\u003e and a clinical-isolated \u003cem\u003eP. aeruginosa strain\u003c/em\u003e, and showed the acceleration effect for the wound healing in mice. This result opens a supramolecular principle for antibiotic-free antibacterial and antibiofilm effects based on multivalent glycoconjugates.\u003c/p\u003e","manuscriptTitle":"Multivalent LecA/LecB Inhibitors based on the Co-assemblies of Perylene Monoimide-carbohydrate Conjugates for Antibiotic-free Antibacterial and Wound Healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 12:35:27","doi":"10.21203/rs.3.rs-4641881/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"19b1ef26-c7c8-444f-b7a6-1a8d784e07db","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33923011,"name":"Physical sciences/Chemistry/Supramolecular chemistry/Self-assembly"},{"id":33923012,"name":"Physical sciences/Chemistry/Materials chemistry/Biomaterials/Biomedical materials"}],"tags":[],"updatedAt":"2024-08-09T15:25:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-02 12:35:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4641881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4641881","identity":"rs-4641881","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}