A Synthetic Fluorescent Nanoplatform Based on Benzoxaborole for

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A Synthetic Fluorescent Nanoplatform Based on Benzoxaborole for Broad-Spectrum Inhibition of Bacterial Adhesion to Host Cells Yunjian Yu, Xijuan Dai, Xiaosong Wei, Xiaomei Dai, Cong Yu, Xiaozhuang Duan, Xinge Zhang, and Chaoxing Li Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b03346 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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Chemistry of Materials

A Synthetic Fluorescent Nanoplatform Based on Benzoxaborole for Broad-Spectrum Inhibition of Bacterial Adhesion to Host Cells Yunjian Yu, Xijuan Dai, Xiaosong Wei, Xiaomei Dai, Cong Yu, Xiaozhuang Duan, Xinge Zhang* and Chaoxing Li Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China ABSTRACT: The rising prevalence of antibiotic-resistant bacteria pathogens has attracted increasing concern in the whole world. The anti-adhesion strategy without triggered bacterial resistance is currently considered a promising alternative to treat bacteria-induced infections. Here, we developed a novel bacteria-binding florescent polymeric nanoplatform for non-lethal anti-adhesion therapy of bacterial infections. This versatile platform will allow simultaneous bacterial agglutination and fluorescent reporting for both Gram-positive and Gram-negative bacteria by taking advantage of strong interaction between the benzoxaborole groups and diol moieties on bacterial surfaces. Furthermore, impressive performance of inhibiting biofilm formation was entirely shown in the generic cell-binding glues. The trapping nanoparticles were capable of taking invasive bacteria pathogens away from the infected host cells with negligible damage to neither bacterial nor host cells, which will not trigger drug resistance, indicating a far-reaching future of the potential application for anti-adhesion therapy of whole-bacterial infection diseases.

INTRODUCTION

glycopolymers,9 glycodendrimers,10,11 glyconanoparticles,12 multivalent nanofibers13 have recently been developed as novel inhibitors to potentially protect the host cells from bacterial infections. For example, Amine et al. synthesized an antiadhesive glycoclusters decorated with galactosides or fucosides against Pseudomonas aeruginosa (P. aeruginosa) lung infection utilizing the specific binding of galactose and fucose to LecA, and LecB in P. aeruginosa, respectively.14 Yan et al. reported on the construction of multivalent n-Heptyl α-D-mannose-based glycopolymers as potent anti-adhesive agents to inhibit E. coli binding to host cells. These multivalent anti-adhesive agents based on specific carbohydrate-protein recognitions in some ways provide a potential treatment for bacteria-induced inflammatory diseases.15 However, there is a great diversity of pathogenic bacteria in clinical practice and the type of microbe may have not been previously identified, so that the exclusive potencies of these carbohydrate-modified scaffolds are acknowledged to confine to specific species of bacteria and fails to meet clinical needs. Additionally, antibiotic-conjugated polymers16,17 and polyvalent cationic polymers18-21 have also been utilized to agglutinate pathogens into clusters so as to sequester them away from infective sites. Unfortunately, the underlying cytotoxicity of antibiotics and positive charges to bacterial strains or host cells extremely limited their clinical application of anti-adhesion therapy. Hence, there is a significant urgency to explore novel cell-binding agents that are

Therapies of bacterial infection are becoming increasingly intractable owing to the rising prevalence of antibiotic-resistant bacteria, and the simultaneous dearth of novel antimicrobial agents.1 A sharp increase annually in global mortality and economic losses resulted from drug-resistant bacterial infections is highlighting the urgent need for new drugs or novel antibacterial strategies. Allowing for the first key step in initiation of colonization, invasion and biofilm formation is the adhesion of bacteria to host cell surfaces, 2 anti-adhesion therapy that using agents to block this process has recently emerged as an attractive alternative for antimicrobial treatment.3-5 This non-lethal approach does not compromise microbial viability － hence reduces the generation of drug resistance, which shows a promising future for the displacement of conventional antibiotic treatment. Since recognition and attachment of pathogenic bacteria to host tissues are generally driven by the interactions between bacterial surface proteins and complementary carbohydrate receptors presented at the host cell periphery, conventional anti-adhesive agents usually acted as glycomimetics are designed to block the microbial adhesion pathway in a competition mechanism.6,7 Considering the low affinity of monosaccharide for bacterial proteins, multivalent glycosylated macromolecules including glycoclusters,8 1

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used first to remove the white precipitate, the obtained solution was washed with the saturated NaHCO3 solution 3 times and dried with anhydrous magnesium sulfate overnight. After filtration and evaporation of the solvent, the white solid was obtained as final product with 78% yield. 1H NMR of TPE(OCH3)4 (400 MHz, CDCl3, 25 °C) δ (ppm): 6.92 (d, J = 5.73Hz, 8H), 6.63 (d, J = 5.74Hz, 8H), 3.74 (s, 12H). 1H NMR of TPEOH4 (400 MHz, DMSO-d6, 25 °C) δ (ppm):9.24 (s, 4H), 6.70 (d, J = 4.38Hz, 8H), 6.48 (d, J = 6.66Hz, 8H). 1H NMR of TPEBr4 (400 MHz, CDCl3, 25 °C) δ (ppm):7.04 (s, 8H), 6.93 (s, 8H), 2.00 (d, J = 35.11Hz, 24H).

capable to adhere a broad spectrum of bacterial pathogens and do not invoke the death of target bacterial cells for anti-adhesion therapy. More importantly, the development of efficient anti-adhesion therapies to treat bacterial infections requires the availability of innovative techniques for the visualized assessment of anti-adhesion activity.22,23 Fluorescence techniques based on general fluoresceins,24 quantum dots,25 conjugated polymers26-27 and aggregation-induced emission fluorogens (AIEgens)28-30 have been extensively utilized to detect or aggregate bacterial cells by antibody-antigen recognition, carbohydrate-protein interaction or other ligand-receptor interaction. Among these, AIEgens have attracted the greatest research interests worldwide inspired by their outstanding advantages in the field of bioimaging, e.g. high signal-to-noise ratio, free of self-quenching, and strong photostability.31 Herein, we developed a novel synthetic fluorescent nanoplatform based on benzoxaborole to realize broad-spectrum anti-adhesion therapy combined with facile visualization of the bacterial aggregation process by naked eyes. As phenylboronic acid and its derivatives can form reversible borate complexes with glycols,32 a general chemical substance found on glycoproteins, such as N-acetylneuraminic acid, galactose, mannose, and fucose residues, benzoxaborole moieties presented in the copolymer construction could act as high-affinity ligands to selectively bind to diol-groups on bacterial cell walls. Furthermore, the 3D spherical composites with surround ligands were expected to agglutinate both Gram-positive and Gram-negative bacterial cells into clusters via strong polyvalent interaction with little bind and damage to the host cell. Synchronously, the distinctive AIE feature of TPE core might endow the nanoplatforms with unique advantage of fluorescence reporting microbial behaviours. Therefore, the versatile bacterial-binding agents were desired to inhibit the biofilm formation by clustering bacterial cells and then take pathogenic bacteria away from infection sites, which holds great promise for whole-bacterial anti-adhesion therapy. This precisely defined nanoplatform based on benzoxaborole is first proposed to selectively bind as well as image various pathogens, thereby promising a more generic approach for alternative bacterial infection treatments.

Synthesis of TPE-Based Star Poly(5-acrylamido-1,2-benzoxaborole-co-di(ethylene glycol) methyl ether methacrylate) (star-PAD). Star-PAD was synthesized by ATRP using AAmBO and DMEMA as monomers. The copolymerization was performed at [TPEBr4]:[AAmBO]:[DMEMA]:[CuBr]:[Me6TREN] = 1:40:120:3.2:3.2. The solution was stirred at 60 °C for 24 h after three standard freeze-pump-thaw cycles. After the reaction, the copolymer was purified by dialysis (MWCO 3500) against distilled water for 3 days, and then obtained by freeze-dried. By altering the feed molar ratios of AAmBO and DMEMA to TPEBr4, three distinguishing star copolymers were obtained abbreviated as star-PA1D3, star-PA1D1, and star-PA3D1, respectively. Formulation of Star-PAD Nanoparticles. Star-PAD nanoparticles were prepared using a modified method from our previous work.33 briefly, amphiphilic star copolymer (10 mg) was dissolved in DMSO (1 mL), and subsequently distilled water (7 mL) was added to the solution under vigorous stirring. Then, the solution was transferred to a dialysis tube (MWCO 3500) and dialyzed against phosphate puffer solution (PBS, pH 7.4, 0.01M) for 1 day. The organic solvent was removed by replacing PBS every 3 h. Bacterial Aggregation Assay. To evaluate the bacterial aggregation effects of star-PAD nanoparticles, Gram-negative Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Gram-positive Bacillus amyloliquefaciens (B. amyloliquefaciens), Staphylococcus aureus (S. aureus) were used as models. Firstly, Bacteria were incubated overnight in lysogeny broth (LB) medium at 37 °C and then 1 mL of bacterial suspension was concentrated at 4000 rpm for 5 min, washed with PBS (pH 7.4, 0.01M) 3 times and redispersed in 1 mL of PBS. A solution of nanoparticles at a concentration of 1.0 mg/mL was prepared with PBS, and 1 mL was withdrawn and co-cultured with the above-prepared bacterial suspension at 37 °C under static conditions for 2 h. Then the suspension was concentrated and performed triple wash with PBS to eliminate the free nanoparticles. The samples were dispersed in a small amount of 70% glycerol solution, mixed evenly before dropping 10 μL solution on the slide and covered with coverslips. The fluorescence photographs of nanoparticles-bacteria clusters were

EXPERIMENTAL SECTION Synthesis of Atom Transfer Radical Polymerization (ATRP) initiator—TPEBr4. For the synthesis of TPEBr4, TPEOH4 (397 mg, 1 mmol) and triethylamine (1.108 mL, 8 mmol) were dissolved in freshly-dried CH2Cl2 (20 mL) in a three-necked flask (50 mL). The mixture was stirred at 0 °C under nitrogen for 0.5 h, and then 2-Bromoisobutyryl bromide (0.989 mL, 8 mmol) in 2 mL of CH2Cl2 was slowly added to the solution at 0 °C with nitrogen bubbling. After being reacted at 0 °C for 1 h, the mixture was stirred at 25 °C for an additional 24 h in the nitrogen atmosphere with reflux. After the reaction, filtration was 2


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obtained by the confocal laser scanning microscope (CLSM, Leica TCS SP8), the maximum excitation and emission wavelength of TPE were 351 and 443nm, respectively.

absorbance of each well at 590 nm using a micro plate reader (384 Plus, American Molecular Instruments). The percent inhibition of biofilm formation (%) was calculated by setting control groups as 100% of biofilm formation. MBIC50 was referred to half maximal inhibitory concentration in biofilm formation compared with the control groups. Each group was tested in five parallel wells, and each independent experiment was carried out in three replicates.

Then the effect of the nanoparticle concentration on the aggregation activity was further studied. The bacterial suspension was diluted to give a working concentration of approximately OD600 (the absorbance at  = 600 nm) = 2.0. The nanoparticles were dispersed in PBS to obtain various concentrations (31.25 - 1000 μg/mL). The copolymer nanoparticles (0.8 mL) were added into 0.8 mL of bacterial suspension. After 2 h of incubation at 37 °C, the supernatant of the mixture was taken out and tested the OD600 by UV-vis spectrophotometer. Control group was incubated with an equal volume of PBS. The OD600 value of the solution was used to determine the number of bacteria. Each experiment was carried out in three replicates.

CLSM 3D Imaging of Biofilms. Bacteria were incubated overnight in LB medium at 37 °C followed by diluted to be OD600 = 0.05. Then, the bacterial diluent was further combined with different nanoparticles (1.0 mg/mL) to give a final OD600 = 0.025 as the seeding solutions. Bacteria treated by equal PBS were set as the control group. 2 mL of the seeding solution was added into the wells of a 12-well PVC microtiter plate with sterile coverslips vertically placed, and then co-cultured at 37 °C for 24 h under static conditions. The planktonic bacteria on coverslips were removed by rinsing with PBS 3 times. Subsequently, the biofilms were fixed with glutaraldehyde (4%, v/v) for 4 h, and stained with ethidium bromide (EB) and fluorescein isothiocyanate conjugated concanavalin A (FITC-ConA) in darkness at 4 °C for 15 min. Finally, the biofilm samples were observed by CLSM 3D imaging.

The effect of the incubation time on the aggregation activity was further evaluated. The bacterial suspension was diluted to give a working concentration of approximately OD600 = 2.0. The nanoparticles were dispersed in PBS, and 0.8 mL of suspension was added to 0.8 mL bacterial suspension (i.e. final concentration was 500 g/mL). After a period of time (from 0 to 120 min) of incubation at 37 °C, the supernatant of mixture was taken out and recorded the optical density at  = 600 nm every 10 minutes by using UV-vis spectrophotometer. Control samples incubated with PBS in different period. Each experiment was carried out in three replicates. The OD600 value of the solution was used to determine the amount of bacteria, and %OD600, the relative optical density at 600 nm of each sample calculated according to following equation: %𝑂𝐷 =

𝑂𝐷0 ― 𝑂𝐷 𝑂𝐷0

Inhibition of Adhesion of the Bacteria to Host Cells. To evaluate the anti-adhesion effectiveness of the nanoparticles, we used S. aureus as model pathogen which can cause a lot of serious infection,35 and co-cultured the strain with NIH 3T3 cells. The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with fetal bovine serum (10%, v/v), penicillin/streptomycin (1%, v/v) and in CO2 (5%, v/v) at 37 °C. Then the cells were seeded in a 12-well PVC microtiter plate at a density of 105 cells per well and incubated for 12 h. Subsequently, the cells were performed triple rinse by sterile PBS (pH 7.4, 0.01M) prior to adding a 150 μL bacterial suspension of FITC labeled S. aureus (OD600 = 1.0). After 1 h of incubation, the nanoparticles (150 μL) were added at a concentration of 1.0 mg/mL. After the cells co-cultured with bacteria (in the presence or absence of the material) for 3 h, the cells were rinsed with sterile PBS 3 times and then fixed with paraformaldehyde (4%, v/v) for 10 min. After that, NIH 3T3 nuclei were dyed with 4',6'-diamidino-2-phenylindole (DAPI). Cellular adhesion was observed by CLSM at the excitation wavelength of 340 nm for DAPI and 490 nm for FITC. For bacteria counting, the cells were performed triple rinse by sterile PBS and lysed with Triton X-100 in deionized water. Samples were diluted and plated onto LB agar plates to determine the number of bacteria.

×100 %

OD0: the initial OD600 of bacteria; OD: the OD600 of bacteria after treated with nanoparticles or PBS at different period.

Biofilm Inhibition. The biofilm inhibition assay was performed as described previously with minor modifications.34 Bacteria were incubated overnight in LB medium at 37 °C and then diluted with LB medium to be OD600 = 0.05. Subsequently, the bacterial diluent was further combined with copolymer nanoparticles (1.0 mg/mL) to give a final OD600 = 0.025 as the seeding solutions. Bacteria treated by equal PBS were set as the control groups. 120 μL of the seeding solution was then added into the wells of a 96-well PVC microtiter plate and then co-cultured at 37 °C for 24 h under static conditions. The formed biofilms were performed triple rinse by sterile PBS to discard planktonic bacteria after the media were removed. After thoroughly dried, the biofilms were fixed with absolute methanol and then stained with crystal violet solution (0.5%, w/v). Subsequently, acetic acid (33%, v/v) was added to each well to release the dye by shaking for 15 min. Biofilms were quantified by measuring the

LIVE / DEAD Bacterial Fluorescence Assay. Bacterial cells were collected by centrifugation and redispersed in PBS with OD600 = 1.0. Then, 1 mL of bacterial suspension was incubated with an equal volume of star-PAD nanoparticles (1.0 mg/mL) for 2 h. The treated bacteria were stained with EB in darkness at 4 °C for 15 min. 3



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band) resulted in an absorption band of 1680 cm−1, while an absorption band at 1599 cm−1 was assigned to N-H bending vibration (amide II band) of a secondary amide. For star-PDMEMA, a peak absorption band located in region of 1200 - 1050 cm−1 was resulted from C-O stretching of alkoxyl bond. In contrast, star-PAD maintained the typical absorption band of homopolymers of star-PDMEMA and star-PAAmBO described above. As a result, the 1H NMR spectra combined with FT-IR spectra results indicated that TPE-based star copolymers are successfully prepared. In addition, thermostability of the obtained materials was also investigated as shown in Figure S4.

Subsequently, bacteria were performed triple rinse by sterile PBS and observed by CLSM. Bacteria that treated with PBS were set as the control group. Specially, the control group was co-stained with AO and EB in darkness at 4 °C for 15 min.

RESULTS AND DISCUSSION Synthesis and Characterization of Star-PAD. To obtain a novel polyvalent synthetic anti-adhesion material, the fluorescent benzoxaborole-based copolymers were prepared as depicted in Scheme 1. Firstly, we prepared the compound TPE(OCH3)4 prior to the synthesis of phenolic hydroxyl species (TPEOH4) according to previously reported method.36 The information of 1H NMR spectra for TPE(OCH3)4 and TPEOH4 was displayed in Figure S1. The ATRP initiator, TPE derivative (TPEBr4) was prepared by the reaction of TPEOH4 with 2-bromo-2-methylpropionyl bromide. As shown in Figure 1A, peaks at 6.93 and 7.05 ppm were assigned to phenyl ring while signal at 2.00 ppm was attributed to methyl, demonstrating the successful synthesis of TPEBr4. The monomer of 5-acrylamido-1,2-benzoxaborole (AAmBO) was prepared from 5-amino-1,2-benzoxaborole by acyl chloride method (Scheme S1, Figure S2). TPE-based star poly(5-acrylamido-1,2-benzoxaborole-co-di(ethylene glycol) methyl ether methacrylate) (Star-PAD) was synthesized by ATRP using TPEBr4 as initiator under the catalyst system of CuBr/Me6TREN. A p00l of copolymers were acquired by varying the feed molar ratios of monomers (1:3, 1:1 and 3:1) to ATRP initiator, which were labeled as star-PA1D3, star-PA1D1 and star-PA3D1, respectively. The obtained star-PAD displayed similar absorption peaks with TPEBr4 (Figure S3). The chemical structure of star-PAD was first confirmed by the 1H NMR spectroscopy. As shown in Figure 1B, peaks at 6.81 and 6.98 ppm were ascribed to protons on phenyl of TPE while signals at 0.75-1.20 ppm were attributed to methyl protons on isobutyryl of TPEBr4. Furthermore, the characteristic resonances of the aromatic protons of phenyl group on AAmBO at 4.17-4.40 ppm and the characteristic peaks of DMEMA at 3.19-3.97 ppm were also found in the 1H NMR spectrum. In addition, the proton resonance signals from main chain of star-PAD appeared in the range of 1.5-2.2 ppm, accompanied by the disappearance of double bond signals from 5.5 to 6.5 ppm. The AAmBO/DMEMA composition ratios in star-PAD copolymers were determined by means of 1H NMR integral intensity of signals between the 3H in benzene ring of AAmBO moieties and 9H in DMEMA segments. The molecular weight of the obtained copolymers measured by GPC in DMF was 6.1, 6.7 and 6.4 kDa for star-PA1D3, star-PA1D1 and star-PA3D1, respectively (Table S1). FT-IR spectra were further performed to analyze the chemical structure of star-PAD as shown in Figure 1C. In the sample of star-PAAmBO, a characteristic peak appeared at 3311 cm-1 was observed, which was assigned to the N-H stretching. In addition, C=O stretching (amide I

AIE Performance of Star-PAD. To determine the AIE performance of obtained TPE-derived copolymer, the fluorescent behaviour was studied in DMSO/water system with different water contents. As shown in Figure S5, the copolymer star-PAD was weakly emissive at 443 nm when dissolved in DMSO. With the increase of water amount in the DMSO solution of star-PAD (with 1 mg/mL of final concentration), the photoluminescence (PL) intensity became significantly strong, allowing for water is a poor solvent for TPE. Especially, the PL spectrum demonstrated an abrupt enhancement when star-PAD was dispersed in pure water (Figure S5A and S5B). In addition, the star-PAD nanoparticles exhibited prominent photostability, and no evident fluorescent quenching was observed after irradiated under UV light irradiation (365 nm) for 30 min (Figure S5C). The increase in PL intensity was ascribed to an AIE effect as a result of the strong aggregation of the TPE moiety in the aqueous medium, in which intramolecular rotations were restricted.37 These above phenomena implied that star-PAD inherits the AIE property, further demonstrating the successful synthesis of our copolymers. Formulation of Star-PAD Nanoparticles. Due to the intrinsic amphiphilicity of the copolymers, the obtained star-PAD was capable of self-assembling to nanoparticles in aqueous solution (Figure 2A). The coronas of the resulting copolymer nanoparticles were decorated with hydrophilic ether groups and benzoxaborole moieties, while the hydrophobic main chain along with conjugated TPE moieties was assembled into the core. Therefore, the AIE effect of TPE components was turned on inside the nanoparticles, endowing the obtained nanoparticles with capability to emit intense fluorescence in aqueous solution. From the results of transmission electron microscope (TEM) analysis as shown in Figure 2B, the star-PAD nanoparticles were uniformly spherical in shape with size distribution of ~200 nm , which was consistent with the results determined by dynamic light scattering (DLS) in aqueous solution (Figure 2C, Table S2). Allowing for the inherent electronegativity of AAmBO moieties and DMEMA segments, star-PAD nanoparticles became overall negatively charged. From the UV-vis absorption spectrum (Figure 2D), it can be observed that there were two absorption peaks located at 211 and 249 nm as well as 4


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obtained nanoparticles prefer bacteria over mammalian cells, NIH 3T3 cells without DAPI staining were incubated with the star-PA3D1 nanoparticles for 3 h, the CLSM results of Figure S7 demonstrated that the nanoparticles scarcely stained NIH 3T3 cells at a concentration of 200 μg/mL, indicating the low affinity of benzoxaborole-based nanoparticles to mammalian cells. The significant difference might be ascribed to the diverse components of out membrane structure between bacteria and mammalian cells. The exterior membrane of most mammalian cells consists mainly of proteins and phospholipids, only containing a small amount of carbohydrates.46 On the contrary, the outer surface of bacterial cells contains a high fraction of peptidoglycan and other related polysaccharide contents (Teichoic acid, long phosphodiester polymers decorated with sugar for Gram-positive bacteria; lipopolysaccharide for Gram-negative bacteria).43,44 As a result, the star-PAD nanoparticles are capable of adhering bacterial cells and promoting aggregation in all of the bacterial species, forming highly fluorescent bacterial clusters with little binding and damage to mammalian cells, which shows great potential for whole-spectrum inhibition of bacteria attachment to host cells.

level-off tails existing in the visible spectral region which are generally featured by nanoparticle suspensions and are ascribed to the light scattering effect of the nanoaggregates.38 The inset of Figure 2D presented the opalescent appearance of star-PAD nanoparticles which had been stood at 4 °C for 3 months. Their size and fluorescence over time in PBS and mammalian cell culture medium were further determined at 25 °C or 37 °C, the results shown in Figure S6 demonstrated the remarkable stability of star-PAD nanoparticles. The fluorescence spectra of copolymer nanoparticles shown in Figure 2E exhibited a well-defined bandwidth with a maximum excitation wavelength at 351 nm and maximum emission wavelength at 443 nm. Owing to the TPE, an AIE chromophore aggregated into the micelle core, the star-PAD nanoparticles exhibited strong blue fluorescence in PBS under UV light irradiating at 365 nm (inset of Figure 2E). Upon this case the obtained nanoparticles with excellent fluorescent features are greatly benefited for their potential application in the field of bacteria imaging. Bacterial Aggregation Property of Star-PAD nanoparticles. Benzoxaboroles can reversibly form stable esters with 1,2or 1,3-diols at physiological pH and have a higher affinity than the corresponding phenylboronic acids.39 Considering the abundant distribution of cis-diols structure on the cell walls of bacteria,40,41 there is inherent ability that the star-PAD nanoparticles can bind to all kinds of bacterial cells. Here, a pool of in vitro bacterial aggregation assays were performed to investigate the interaction of copolymer nanoparticles with P. aeruginosa, E. coli, B. amyloliquefaciens, and S. aureus by co-culture, which was observed by CLSM. As shown in Figure 3A, bacteria treated with star-PDMEMA exhibited no aggregation phenomena despite of its strong blue fluorescence, indicating less binding to bacteria. In contrast, the introduction of benzoxaboroles in star-PAD resulted in the formation of dense bacterial fluorescent clusters whose size increased with the augmented content of AAmBO moieties in the copolymer, confirming that the binding effect with bacteria was ascribed to the benzoxaborole moieties of copolymer nanoparticles. In other words, the benzoxaborole-coated nanoparticles induced multiple binding events with the bacterial-bound saccharides-riched and DNA surface via “multivalent effect”.42 Meanwhile, the nanoparticles generated more and larger aggregates with Gram-positive S. aureus and B. amyloliquefaciens than with Gram-negative E. coli and P. aeruginosa. This might because there is a significant difference in the cell wall structures between Gram-positive and Gram-negative bacteria,43,44 dense cell wall structure of Gram-negative microorganisms resulted in relatively less effective saccharide sites bind with copolymers than Gram-positive strains. Moreover, it is well-known that various bacterial cell walls contain various carbohydrate structures with diverse affinity for benzoxaborole groups.45 In order to confirm whether the

Since the binding of the star-PAD nanoparticles to bacterial cell walls induced the fast sedimentation of bacteria, the aggregation rate was further evaluated by measuring the decrease of OD600 value in the supernatant within 120 min (Figure 3B-E). Compared with PBS and homopolymer star-PDMEMA, the OD600 continued to decline much rapidly in the presence of nanoparticles. Consistent with the results of microscopic observation, star-PA3D1 exhibited the preferable adhesion performance among the four nanoparticles, which induced the decline of OD600 from ~1.3 to ~0.3 in 10 minutes and reached nearly 80% of bacteria sedimentation (Figure S8). It is worth mentioning that there was no significant difference in the settlement property of copolymer nanoparticles to different kinds of bacterial cells, although the more superior capacity of bacterial aggregation for Gram-positive than Gram-negative strains was observed in the CLSM assays. Agar plate method was further used to reaffirm the bacterial sedimentation from the medium. As shown in Figure S9, the number of colonies in the supernatant of four bacterial strains after the treatment with star-PA3D1 was much fewer than that of PBS groups, demonstrating the bacterial sedimentation from the medium when aggregated by the nanoparticles. To determine the minimum concentration of the nanoparticles-bound bacteria, the OD600 value of the bacterial supernatant was measured after the incubation of star-PA3D1 nanoparticles for 2 h. As shown in Figure S10A, when the nanoparticle concentration was 31.25 g/mL, adhesion capacity was up to 50% for any kind of bacteria analyzed. Even if the concentration was as low as 15.6 g/mL, adhesion capacity still reached 35.7% for P. aeruginosa, 38.6% for E. 5



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E. coil, B. amyloliquefaciens and S. aureus. Briefly, the star-PA3D1 nanoparticles with various concentrations (15.6 to 500 μg/mL) were added into the bacteria culture solution and co-cultured for 24 h ， then crystal violet staining assay was employed to assess bacterial cell metabolic activity. The control group was treated with PBS and defined as having a biofilm capacity of 100%. As shown in Figure 4D, the maximum inhibition percentage of biofilm formation by the nanoparticles with a concentration of 500 μg/mL was ~45% for P. aeruginosa, ~65% for E. coli, ~75% for B. amyloliquefaciens and ~65% for S. aureus, respectively. Additionally, MBIC50 was determined to be approximately 500 μg/mL for P. aeruginosa, 250 μg/mL for E. coli, 15.6 μg/mL for B. amyloliquefaciens and 31.25 μg/mL for S. aureus. The variation of copolymeric inhibition capability against different kinds of biofilms was ascribed to the discriminating adhesion efficiency of copolymer nanoparticles toward bacterial strains. Recently, boron was found to play a key role in signal mechanisms for communication among bacteria, so boron-rich natural or synthetic compounds are expected to be used as bacterial quorum sensing (QS) inhibitors, e.g. inhibition of biofilm formation.47,48

coil, 42.8% for S. aureus and 48.5% for B. amyloliquefaciens, which might be caused by the intrinsic fast sedimentation of B. amyloliquefaciens even if in the absence of copolymer nanoparticles. Furthermore, the PL intensity of star-PA3D1 nanoparticle suspensions at a concentration of 500 μg/mL after co-culturing with bacteria all reduced compared to that before being treated (positive control group) (Figure S10B), and the bacterial clusters aggregated by copolymer nanoparticles deposited into the bottom of the pool with highly blue fluorescence (inset in Figure S10B). These results further demonstrated the existed strong interactions existed between star-PA3D1 nanoparticles and the bacteria. Antibacterial Activity. To evaluate the antibacterial activity of star-PAD nanoparticles, bacterial growth inhibitory experiments were carried out by measuring the OD600 of bacteria treated with star-PA3D1 nanoparticles. The results (Figure S11A-D) revealed that there was no antibacterial effect against all the bacterial species analyzed even when the concentration of star-PA3D1 nanoparticles was up to 500 μg/mL. At the same time, the viability of the four bacterial strains in the presence of star-PA3D1 nanoparticles (500 μg/mL) was also monitored during 12 h. As shown in Figure S11E-H, star-PA3D1 did not influence the growth of bacteria. LIVE/DEAD assay was further performed to investigate potential impacts of star-PA3D1 nanoparticles on bacterial cell integrity and viability. Under the CLSM, the blue fluorescence was derived from the star-PAD nanoparticles that adhered to bacteria. The green fluorescent clusters referred to live bacterial cells which were labeled by AO, a dye for intact cell membranes. The red signal belonged to the inactive bacteria dyed by EB with damaged cell membranes. The results observed by CLSM were depicted in Figure 4A, 4B and Figure S12. The untreated group (PBS) displayed bright green fluorescence, demonstrating that the bacteria were alive. After treated with star-PA3D1 nanoparticles, the large clusters with blue and green fluorescence signal were observed in the CLSM images, which were attributed to strong interaction between the benzoxaborole-based nanoparticles and bacterial cells. Besides, the size of bacteria-copolymer aggregates varied from the type of bacteria, while clusters formed by star-PA3D1 nanoparticles with B. amyloliquefaciens and S. aureus were somewhat larger than with P. aeruginosa and E. coil, which was in accordance with the results of bacteria aggregation assays. In addition, there was negligible red fluorescence in the bacterial clusters, suggesting that the adhesion of copolymer nanoparticles to bacteria did not compromise microbial viability and survival, indicating no possibility to induce bacterial resistance.

To visualize the biofilm inhibition effects of star-PA3D1 nanoparticles, biofilm samples were stained with EB and FITC-ConA for CLSM 3D imaging. EB was used for locating dead cells in biofilms, and FITC-ConA acted as a probe to confirm the exopolysaccharides. Under CLSM 3D, the mature bacteria biofilms were densely colonized with hierarchically and three-dimensionally structured formations in the control group as shown in Figure 4C. We found that biofilms had a significant amount of extracellular polysaccharide with dense bacteria cells. By contrast, dramatically reduced bacterial cells and less polysaccharide in the biofilms with a scanty architecture were observed after treated with star-PA3D1 nanoparticles, indicating that the copolymer nanoparticles exhibited excellent biofilms inhibition efficiency. These results confirmed that the nanoparticles can act as autoinducers which are regulated and induced by bacterial QS, leading to biofilm inhibition. Inhibition of Bacteria Binding to Host Cells. That virulent strains of bacteria invade the host cells generally starts with attachment to cell-surface plasma membrane receptors and ends with delivery of the bacteria genome to the host. Anti-virulence methods were utilized as a promising therapeutic concept to target key stages in infection such as prohibiting adhesion of the pathogen to the host. Here, the discovery of a novel nanoplatform with superior bacterial adhesion property makes it necessary to further explore the ability of inhibiting attachment of bacteria to host cells. Using NIH 3T3 as the model cell and S. aureus as the model bacteria, the anti-adhesion behavior of star-PA3D1 nanoparticles was investigated by treating with NIH 3T3 cells which firstly infected by S. aureus and subsequently washed to remove

Biofilm Inhibition. It is noted that the formation of biofilm plays a significant part in producing the drug tolerance and infection progression. The potential effects of the adhesive copolymer nanoparticles on biofilm formation were further investigated toward P. aeruginosa, 6


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uncombined portion. As shown in Figure 4E, in the control group, a great number of bacterial cells with green fluorescence were distributed around the NIH 3T3 cell membranes, cell nuclei displayed shrinking and distorted morphology, indicating that the cells infected by S. aureus were entering a period of apoptosis. On the contrary, after the addition of star-PA3D1 nanoparticles at a concentration of 200 μg/mL, the majority of S. aureus attached to the cell surface was cleared up, and the NIH 3T3 cell nuclei showed intact structure, demonstrating that the nanoparticles possessed predominant capacity of separating bacteria from host cells by disrupting pre-established interactions between bacteria and host cells. Simultaneously, the number of residual adhesion bacteria was determined by using LB agar plates count method. As a result, the amount of colony-forming units (CFU) after treated with the nanoparticles reduced almost 79% compared with the PBS group (Figure 4F). Besides, the IC50 (half maximal inhibitory concentration) value of star-PA3D1 nanoparticles for eliminating invasive S. aureus in NIH 3T3 cell determined by agar plates method was approximately 50 μg/mL. Above results declared that the nanoparticles bearing benzoxaborole were capable of eliminating invasive bacteria in host cell.

ASSOCIATED CONTENT

Cell Viability. To evaluate the biocompatibility of star-PDMEMA and star-PAD in vitro, both hemolysis and 3-(4,5-Dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out on red blood cells and NIH 3T3 cell lines, respectively. As shown in Figure S13, the hemolysis percentage of star-PDMEMA and star-PAD nanoparticles was below the acceptable value of 5% across all material concentrations. Moreover, the results determined by MTT assay displayed that the viability of NIH 3T3 cells all exceed 80% after treated with different concentrations of star-PDMEMA and star-PAD nanoparticles for as long as 48 h (Figure S14 ), confirming that the cell proliferation was not susceptible to the nanoparticles. The introduction of DMEMA moieties effectively improved the biocompatibility of the nanoparticles, suggesting that the obtained materials have a potential application in the biological application.

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Supporting Information. Materials, additional characterization and synthetic methods, Scheme S1, Figures S1−S14, Tables S1-S2. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Xinge Zhang. E-mail address: [email protected]

ORCID Xinge Zhang: 0000-0003-3399-1659

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge the support from National Natural Science Foundation of China (Grant No. 21474055, 21774062 and 51673102) and the Natural Science Foundation of Tianjin City, China (Grant No. 18JCYBJC29300)

REFERENCES

CONCLUSIONS In conclusion, we developed a novel diagnostic nanoplatform for generic bacterial detection and anti-adhesion therapy. An ideal anti-adhesive candidate is required to present potency, selective inhibition of pathogen, cross-clade broad spectrum activity, and biocompatibility. The current approach of developing synthetic nanoplatforms bearing benzoloroxole moieties meets all criteria of an ideal anti-adhesive agent. Additionally, the benzoloroxole moieties can be readily conjugated into various biocompatible polymers which can improve their specificity and activity. Finally, the potential of florescent star-PAD as a new generation of anti-adhesive therapeutic agent can selectively recognize and eliminate various pathogens, thereby promising a new approach to treat diverse bacterial infection diseases. 7



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Scheme 1. Synthetic route of (A) ATRP initiator-TPEBr4 and (B) TPE-based star copolymers star-PAD.



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Figure 1. (A) 1H NMR spectrum of TPEBr4 in CDCl3 at 25 °C; (B) 1H NMR spectra of star-PAD in a mixed solvent of DMSO-d6/D2O (v/v =4:1) at 25 °C; (C) FT-IR spectra of star-PDMEMA, star-PAAmBO, and star-PAD.


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Figure 2. (A) Schematic illustration of star-PAD nanoparticles; (B) TEM micrographs and (C) size distribution of star-PAD nanoparticles measured by DLS; (D) UV-visible absorption spectrum of star-PAD nanoparticles, the inset is the visible image of the nanoparticles in PBS; (E) fluorescence excitation and emission spectra of star-PAD nanoparticles, the inset is the fluorescent graph of the nanoparticles in PBS.



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Figure 3. (A) The aggregation activity to P. aeruginosa (PA), E. coil (EC), B. amyloliquefaciens (BA) and S. aureus (SA) observed by CLSM of after treated with star-PDMEMA, star-PA1D3, star-PA1D1 and star-PA3D1 for 2 h at 37 °C, respectively. The concentration of all copolymer nanoparticles was 1 mg/mL, respectively. Blue fluorescence: TPE molecules in the nanoparticles. The OD600 in the PA (B), EC (C), BA (D) and SA (E) supernatant over time after treated with star-PDMEMA, star-PA1D3, star-PA1D1 and star-PA3D1 nanoparticles, respectively.


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Figure 4. Fluorescence micrographs of (A) B. amyloliquefaciens (BA), and (B) S. aureus (SA) with AO (green fluorescence) and EB (red fluorescence) after incubation with the star-PA3D1 nanoparticles for 2 h at 37 ℃; (C) The CLSM 3D images of P. aeruginosa (PA), E. coil (EC), B. amyloliquefaciens (BA), and S. aureus (SA) biofilms in the presence of PBS or star-PA3D1 nanoparticles. The concentration of nanoparticles was 500 μg/mL. Green fluorescence: the EPS stained by FITC-ConA; Red fluorescence: dead bacterial stained by EB; (D) Inhibition percentage of biofilm formation with various concentrations (from 15.6 to 500 μg/mL) of star-PA3D1 nanoprticles against PA, EC, BA, and SA, respectively. Each sample was analyzed in quintuplicate and the results were reported as mean ± standard deviation (n = 5); (E) CLSM of NIH 3T3 nuclei stained with DAPI, infected by SA with FITC or in the presence of star-PA3D1 nanoparticles (200 μg/mL) after washing off nonadherent bacterial with PBS; (F) Residual adhesion of SA to NIH 3T3 cells after treatment with various concentrations of star-PA3D1 nanoparticles; ** p < 0.01 vs. PBS group.


A Synthetic Fluorescent Nanoplatform Based on Benzoxaborole for

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