Pathogen-Specific Polymeric Antimicrobials with Significant

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Pathogen-Specific Polymeric Antimicrobials with Significant Membrane Disruption and Enhanced Photodynamic Damage to Inhibit Highly Opportunistic Bacteria Fengfeng Xiao, Bing Cao, Congyu Wang, Xujuan Guo, Mengge Li, Da Xing, and Xianglong Hu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07251 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Pathogen-Specific Polymeric Antimicrobials with Significant Membrane Disruption and Enhanced Photodynamic Damage to Inhibit Highly Opportunistic Bacteria Fengfeng Xiao,†,‡,§ Bing Cao,†,‡ ,§ Congyu Wang, †,‡ Xujuan Guo, †,‡ Mengge Li, †,‡ Da Xing†,‡ and Xianglong Hu*,†,‡

†

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China

Normal University, Guangzhou 510631, China

‡



College of Biophotonics, South China Normal University, Guangzhou 510631, China

To whom correspondence should be addressed:

E-mail: [email protected], [email protected]

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ABSTRACT

Highly pathogenic Gram-negative bacteria and the drug resistance are severe public health threat with high mortality. Gram-negative bacteria are hard to kill due to the complex cell envelopes with low permeability and extra defensive mechanisms. It is challenging to treat them by current strategies, mainly including antibiotics, peptides, polymers, and some hybrid materials, still facing the issue of drug resistance, limited antibacterial selectivity, and severe side effects. Together with precise bacteria targeting, synergistic therapeutic modalities including physical membrane damage and photodynamic eradication are promising to combat Gram-negative bacteria. Herein, pathogen-specific polymeric antimicrobials were formulated from amphiphilic block copolymers, poly(butylmethacrylate)-b-poly(2-(dimethylamino) ethylmethacrylate-co-Eosin)-b-ubiquicidin, PBMA-b-P(DMAEMA-co-EoS)-UBI, in which pathogen-targeting peptide ubiquicidin (UBI) was tethered in the hydrophilic chain terminal, and Eosin-Y was copolymerized in the hydrophilic block. The micelles could selectively adhere to bacteria instead of mammalian cells, inserting into the bacteria membrane to induce physical membrane damage and out-diffusion of intracellular milieu. Furthermore, significant in-situ generation of reactive oxygen species was observed upon light irradiation, achieving further photodynamic eradication. Broad-spectrum bacterial inhibition was demonstrated for the polymeric antimicrobials, especially highly opportunistic gram-negative bacteria, such as Pseudomona aeruginosa (P. aeruginosa) based on the synergy of physical destruction and photodynamic therapy, without detectable resistance. In vivo P. aeruginosa-infected knife injury model and burn model both proved the good potency of bacteria eradication and promoted wound healing, which was comparable with commercial antibiotics, yet no risk of


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drug resistance. It is promising to hurdle the infection and resistance suffered from highly opportunistic bacteria.

KEYWORDS: pathogen targeting, physical destruction, photodynamic therapy, selective recognition, polymeric antimicrobials, wound healing, drug resistance

The emergence of antibiotic resistant bacteria in both nosocomial and community settings is very stressful towards public healthcare systems around the world.1, 2 Much attention has been focused on Gram-positive bacteria in the past decades, expecially methicillin-resistant Staphylococcus aureus (MRSA);3 however, Gram-negative microbes have been increasingly recognized as much more critical issues,4-7 because they have extra outer membrane and additional defensive mechanisms,8-10 however few antimicrobials have been developed to target and kill Gram-negative bacteria efficiently.11-13 Gram-negative Pseudomona aeruginosa (P. aeruginosa) is one of the leading causes of nosocomial infections.14-16 The infection of P. aeruginosa is very hard to eradicate thoroughly, which is a severe therapeutic challenge in medicine due to the increasingly acquired resistance against different antibiotics.17-19 Herein it is imperative to develop high-efficient antibacterial agents with distinct inhibition mechanisms to combat Gram-negative bacteria without inducing resistance.20, 21 Antimicrobial peptides (AMPs), which have positive charge and proper hydrophobic moiety, have been regarded as a promising strategy due to the physical damage towards bacteria by pore-forming and non-pore forming mechanisms.22-27 However, AMPs have had limited success in clinical settings, primarily due to their high toxicity towards mammalian cells,28-31


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high cost and susceptibility to enzymatic degradation.32,33 To overcome these limitations of AMPs, polymeric antimicrobials are developed to simulate AMPs,34 exhibiting facile synthesis, scaling up, multiple functionalization as well as low toxicity towards mammalian cells.35, 36 There are also many new antimicrobial systems adopting photodynamic or photothermal therapy which could efficiently kill bacteria and handle the problem of drug resistance.37-40 Among them, photodynamic therapy (PDT) is promising to treat persistant bacteria.22, 41-43 Photodynamic therapy requires three simultaneously present factors: photosensitizer (PS), light source and molecular oxygen. Fundamentally, excited photosensitizer is able to transform molecular oxygen into toxic reactive oxygen species (ROS) such as singlet oxygen (1O2), thus achieving apoptosis-mediated PDT. Most photosensitizers show good antibacterial ability against Gram-positive bacteria, but they are insufficient to kill Gram-negative bacteria because of their distinct membrane structure.44, 45 Moreover, the PDT efficiency has been limited by the low water solubility of most photosensitizers,46, 47 and the lack of selectivity for bacteria over mammalian cells often results in serious side effects.48, 49 Furthermore, some affinitive ligands have been developed to target bacteria, including sugar moieties and peptides.50-53 Among these, ubiquicidin (UBI29-41, TGRAKRRMQYNRR) was an antimicrobial peptide with six positively charged residues (5 Arg +1 Lys),54 which demonstrated high affinity and specificity against bacteria,55, 56 and the conjugated product of UBI with imaging agents was even clinically investigated.57, 58 UBI could bind to the negatively charged groups on the bacterial cell membrane by electrostatic interaction, exhibiting high accumulation in bacterial infection sites but not the inﬂammation locations.59-62 Herein, the conjugation of UBI with other theranostic moieties was expected to provide distinct potency


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of broad-spectrum bacteria targeting and differentiation from inflammation. In this work, pathogen-specific polymeric antimicrobials were fabricated from PBMA-bP(DMAEMA-co-EoS)-UBI (noted as PAM-UBI) to exert dual-modality bacterial inhibition, including physical membrane damage and concurrent photodynamic eradication (Scheme 1). Based on the simulation of AMPs, high bacterial affinity and polymer amphiphilicity endowed PAM-UBI with significant potency of membrance insertion, fusion, leakage of intracellular milieus, and the resulting physical damage. The polymeric antimicrobials could selectively adhere to Gram-negative bacteria but not mammalian cells, resulting in physical damage to the bacteria membrane to some extent. Furthermore, upon light irradiation, in-situ photodynamic activation of ROS formation would result in further disruption of bacterial membrane, thus eliminating remnant persistent bacteria. The combined therapeutic profile could solve the bacterial resistance issue. Systematic explorations have been demonstrated, including selective recognition of bacteria, antibacterial evaluation, biocompatibility test, inhibition of antibiotic resistance, mechanism exploration of bacteria inhibition, and in vivo evaluation towards two distinct P. aeruginosa infected models, all suggesting the excellent potency.

RESULTS AND DISCUSSIONS Well-defined polymeric antimicrobials were synthesized via facile reversible addition fragmentation chain transfer (RAFT) polymerization and facile post-functionalization (Scheme S1). Typically, the copolymerization of DMAEMA and Eosin Y monomer afforded hydrophilic P(DMAEMA-co-EoS),63, 64 which performed as macro RAFT agent to polymerize BMA to give the resulting amphiphilic PBMA-b-P(DMAEMA-co-EoS), which was characterized by


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1

H NMR spectra (Figure S1). Bacteria targeting peptide, UBI, was tethered in the hydrophilic

chain end by well-accepted amidation reaction, generating the resultant pathogen-targeting PBMA-b-P(DMAEMA-co-EoS)-UBI. Upon self-assembly in water, polymeric antimicrobial micelles were fabricated from PBMA-b-P(DMAEMA-co-EoS)-UBI, and noted as PAM-UBI. For comparison, non- targeting polymeric antimicrobial micelles were fabricated from PBMAb-P(DMAEMA-co-EoS), and shortened as PAM. The zeta potential of PAM-UBI was determined to be ~ +13.6 mV, compared with that of PAM without UBI moieties to be ~ +4.62 mV, respectively, suggesting the successful conjugation of UBI with many positively-charged amine acid residues. Then, the aqueous size distribution and structural morphology of two kinds of polymeric antimicrobial micelles were characterized by DLS and TEM analysis (Figure 1a, 1b). The hydrodynamic diameter of PAM-UBI was slightly larger than that of PAM, ~43.6 nm vs ~28.2 nm, respectively. Spherical morphology was observed for PAM-UBI and PAM in dry state by TEM analysis, with the diameter to be ~25 nm and ~15 nm, respectively. UV-vis absorbance spectra and fluorescence emission spectra of P(DMAEMA-co-EoS), PAM, and PAM-UBI exhibited typical absorbance at ~530 nm and emission peak at ~560 nm, which was red-shifted compared with these of eosin-Y due to the extended conjugation effect (Figure 1c, Figure S2), further confirming the successful polymerization of eosin moieties in the polymer. Eosin-Y was a typical photosensitizer for photodynamic therapy, thus the 1O2 generation potency of the polymeric antimicrobials was evaluated using 1,3-diphenylisobenzofuran (DPBF) as a 1O2 detector.65 Upon exposure to a green light lamp (520 ± 10 nm), obvcious absorption decrease of DPBF at ~410 nm was observed, suggesting increased 1O2 content for the groups of EoS,


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P(DMAEMA-co-EoS), PAM and PAM-UBI (Figure 1d, Figure S3). In contrast, less than 10% absorbance decrease of DPBF was determined for the blank group and polymeric antimicrobials without light irradiation.23, 66 Due to the presence of bacteria-targeting UBI within PAM-UBI, the possibility of selective bacteria recognition and imaging was examined against the mixture of bacteria and mammalian cells.61 RAW 267.4 cells were chosen as model mammalian cells, and co-cultured with P. aeruginosa, followed by incubation with PAM or PAM-UBI at 37 °C for 30 min, respectively. The mixture of RAW 267.4 cells and P. aeruginosa was observed by CLSM imaing (Figure 2a). Bacteria-targeting PAM-UBI could only adhere to the bacteria, exhibiting strong red fluorescence, whereas for the arrow indicated RAW 264.7 cells, unobvious fluorescence was found around or inside the cells. For non-targeted PAM, inapparent fluorescence was detected for both P. aeruginosa and RAW 264.7 cells. Furthermore, similar targeting potency of PAMUBI was also demonstrated for Gram-positive S. aureus over RAW 264.7 cells (Figure S4). These results further verified that, in spite of conjugation with PAM, the broad-spectrum bacteria targeting property of UBI within PAM-UBI still worked well for typical Gramnegative bacteria and Gram-positive bacteria.61 The intense fluorescence of bacteria clearly indicated the selectively recognizing ability of PAM-UBI towards bacterial strains over mammalian cells. For the component of bacterial membrane, negatively charged phosphatidylcholine (PC) and phosphatidylserine (PS) are common lipid components at the exoplasmic leaflet of bacteria, whereas for mammalian cells, PC and PS are the compositions of inner leaflet of the plasma membrane.67 Furthermore, lipopolysaccharides (LPS) which constitute the main component of the outer membranes of


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Gam-negative bacteria would also contribute the selective recognition of PAM-UBI to bacterial strains instead of mammalian cells.68 To quantify the binding efficiency of PAM-UBI and PAM with P. aeruginosa, flow cytometry assays were conducted respectively (Figure 2b, 2c). Upon incubating with P. aeruginosa, the group of PAM-UBI exhibited much significant fluorescence than that of PAM, ~3.72-fold determined by flow cytometry analysis. Together with CLSM imaging and flow cytometry analysis, the polymeric antimicrobial micelles of PAM-UBI could selectively recognize and adhere to bacteria, which was favorable for selective bacteria inhibition to alleviate potential side effects. Based on the standard broth microdilution method, the antimicrobial activities of current polymeric antimicrobials were performed against Gram-negative E. coli, P. aeruginosa, and Gram-positive S. aureus and MRSA, respectively (Figure 3, Table 1). Minimal inhibition concentration (MIC) values were employed to define the lowest content that could prevent visible growth of a microorganism at diverse defined conditions.69 The antibacterial ability of PAM-UBI was determined to be better than PAM, which demonstrated that the conjugation of UBI strengthened the inhibition capability due to enhanced adhesion to bacteria. The MIC values of PAM-UBI for P. aeruginosa under light irradiation was ~1.8 M, which was much lower than P(DMAEMA-co-EoS) under light (>12.8M) and PAM-UBI under dark (~6.0M). What’s more, PAM-UBI under dark only could physically damage bacteria to some extent, which was moderately effective to inhibit bacteria. As the main hydrophilic block of PAM and PAM-UBI, P(DMAEMA-co-EoS) was nearly fully hydrophilic, thus it didn’t possess efficient hydrophobic portion to penetrate and fuse into bacterial membrane, exhibiting low physical cdamage towards bacteria at dark.70 Even upon light irradiation to activate the photodynamic


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process, the inhibition ability of P(DMAEMA-co-EoS) was still limited, because the generated ROS could not access and attack the bacteria membrane due to the short life time and action range of ROS. Hence, efficient bacteria recognition and membrane fusion to induce physical membrane damage as well as followed in-situ ROS attack were all necessary for the effective inhibition of Gram-negative bacteria. To further explore the synergistic inhibition of physical damage and photodynamic action for the polymeric antimicrobials, combination index (CI) was utilized as a parameter via the Chou–Talalay method,71 a widely used approach for assessment of pharmacodynamic drug interactions,72 as shown in Equation (1), where DA/A+B and DB/A+B is the MIC for PAM or PAMUBI under light, DA is the MIC for P(DMAEMA-co-EoS) under light, and DB is the MIC for PAM or PAM-UBI under dark. The CI value reflects the interaction effect of the dual functional machanisms, and less value suggests much more efficient synergy (0.3 < CI < 0.7 indicates obvious synergism).73 As shown in Table 1, the CI value of PAM under light for tested microbial was ~0.42, indicating moderate synergism. Furthermore, the CI value of PAM-UBI with light irradiation was determined to be 80%, even at 51.2 M, exhibiting good biocompatibility towards mammalian cells, whereas relatively high selectivity and toxicity to bacteria. Herein, current polymeric antimicrobials were quite promising with minimal side effects. To demonstrate the potential clinical application of current polymeric antimicrobials, P. aeruginosa-infected mouse models, including knife injury and burn, were investigated in vivo respectively.84, 85 The polymeric antimicrobial micelles were applied to the infected knife injury sites, followed by light irradiation to finish one treatment (Figure 6a). Two commercially available antibiotics, ciprofloxacin and colistin, were examined in parallel. For the infected knife injury model after 8 days’ healing process, the woud size in the group of PAM-UBI with light irradiation was in comparable with the groups of commercial antibiotics, suggesting accelerated wound healing (Figure 6b). The bacteria content in each mouse was evaluated by agar plate dilution method during the treating process (Figure 6c, 6d, 6e, 6f). The PAM-UBI group could accelerate the healing significantly upon light irradiation. Herein, in vivo P. aeruginosa-infected knife injury model demonstrated the moderate physical inhibition and significant combination inhibition based on physical damage and photodynamic eradication from PAM-UBI. In vivo therapeutic bacterial inhibition and injury recovering by PAM-UBI


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were similar with those of ciprofloxacin and colistin, but the polymeric antimicrobials had much lower possibility to develop resistance than commercial available ones. Furthermore, histological analysis of the infected area was performed during the treatment (Figure 7). At early infection stage, there were many neutrophils (indicated by white arrow), and the skin had damaged epidermis or even lacked epidermis. Upon treating with PAM-UBI and light irradiation, obvious re-epithelialization was found in the infected sites (indicated by black arrow), exhibiting much more fibroblasts (indicated by red arrow), which was related to the formation of epidermis and more hair follicles and sweat glands when compared with other groups. Thus, after total three treatments in 8 days, PAM-UBI could inhibit in vivo infected P. aeruginosa in knife injury mouse model and efficiently accelerate the recovering. Finally, in vivo evaluation was performed against P. aeruginosa infected burn model (Figure 8a). After two weeks’ treatment, the PAM-UBI group upon light treatment exhibited most efficient burn recovering, the eschar sites was the smallest among all tested groups (Figure 8b-d). Two commercial available antibiotics exhibited comparable therapeutic efficacy, which agreed well with the burn model (Figure 8e). Thus, two kinds of mouse models were examined for the in vivo therapy and infection inhibition of P. aeruginosa based on the synergy of physical and photodynamic inhibition of pathogen-targeting PAM-UBI, both exhibiting excellent efficiency.

CONCLUSION In conclusion, pathogen-targeting polymeric antimicrobials, PAM-UBI, were fabricated to conduct synergistic bacterial inhibition towards highly opportunistic bacteria based on the physical membrane and photodynamic eradication under light irradiation. The inhibition


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mechanism was demonstrated to possess following process: (1) polymeric antimicrobial micelles adhere to bacteria based on targeting recognition and electrostatic interaction; (2) The fusion and assembly of PAM-UBI can depolarize the bacterial membrane to result in physical damage of Gram-negative bacterial membrane; (3) Upon light irradiation, in-situ produced ROS can further kill bacteria. Overall, pathogen-specific membrane insertion micelles are promising to combat bacteria infections by synergistic potency without resistance acquirement. Current polymeric antimicrobials hold great potential as precise antimicrobial agents for biomedical applications.


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EXPERIMENTAL SECTION Materials. 2-(Dimethylamino) ethyl methacrylate (DMAEMA) was purchased from Aldrich and passed through a neutral alumina column to remove the inhibitor, then stored at -20 °C prior to use. Butylmethacrylate (BMA) was purchased from Aldrich and distilled under vacuum and then stored at -20 °C prior to use. Dioxane, tetrahydrofuran (THF), dimethylfomamide (DMF), and all other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Eosin Y, 4-chloromethylstyrene, Dulbecco’s modified Eagle medium (DMEM) were purchased from Aldrich and used as received. Chain transfer agent (CTA), 4-Cyano-4(phenylcarbonothioylthio) pentanoic acid was synthesized according to literature procedures.86 Synthesis of P(DMAEMA-co-EoS). CTA (13.9 mg, 0.05 mmo1), DMAEMA (353.7 mg, 2.25 mmol), EoS monomer (191 mg, 0.25 mmol), AIBN (1.64 mg, 0.01 mmol), and 1,4-dioxane (1.5 mL) were charged into a glass ampoule. The mixture was degassed by three freeze-pump-thaw cycles and flame-sealed under vacuum. After stirring for 20 h at 70 °C, the reaction tube was quenched into liquid N2, opened and exposed to air, and precipitated into an excess of diethyl ether. The above dissolutionprecipitation cycle was repeated three times. The final product was dried in a vacuum oven overnight at room temperature, yielding a red solid (366 mg, yield: 67.2%). The DP of DMAEMA and EoS was determined by 1H NMR analysis (Figure S1), and determined to be P(DMAEMA20-co-EoS0.8). Synthesis of PBMA-b-P(DMAEMA-co-EoS). P(DMAEMA20-co-EoS0.8) (302 mg, 0.075 mmol), BMA (213 mg, 1.5 mmol), AIBN (2.46 mg,


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0.015 mmol), 1,4-dioxane (1 mL) and DMSO (1 mL) were charged into a glass ampule. The mixture was degassed by three freeze-pump-thaw cycles and flame-sealed under vacuum. After stirring for 19 h at 70 oC, the reaction tube was quenched into liquid N2, opened, and exposed to air. The mixture was precipitated into an excess of petroleum ether. The above dissolutionprecipitation cycle was repeated for three times. The final product was dried in a vacuum oven overnight at room temperature to afford a red solid (251 mg, yield: 48.7%). The DP of PBMA block was determined to be 16 by 1H NMR analysis in CDCl3 (Figure S1). Thus, the polymer was denoted as PBMA16-b-P(DMAEMA20-co-EoS0.8). Synthesis of PBMA-b-P(DMAEMA-co-EoS)-UBI. PBMA16-b-P(DMAEMA20-co-EoS0.8) (94.5 mg, 0.005 mmol) was first reacted with EDC·HCl (1.9 mg, 0.01 mmol) and NHS (1.2 mg, 0.01 mmol) in anhydrous N,N-dimethylformamide (DMF, 0.2 mL). After stirring in the dark for 6 h at room temperature,the solution was mixed with UBI (6.76 mg, 0.004 mmol) in anhydrous N,N-dimethylformamide (DMF, 0.2 mL). The mixture was stirred in the dark overnight at room temperature. The mixture was finally precipitated into an excess of diethyl ether to afford the resultant product. ROS detection. The ROS production of micelles was determined by the DPBF bleaching method. An ethanol solution of DPBF (40 µL, 2 mM) was added to the aqueous solution of different samples. The samples were continuously irradiated with green light (520 ± 10 nm) for 2 min, and the absorbance of DPBF at 410 nm was collected every 2 min. As a control, the solution of micelles with an equal amount of DPBF was measured in the dark. Selective recognition of bacteria over RAW 264.7 cells.


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The selective recognition capability of micelles was examined using RAW 264.7 cells. The micelles of PAM and PAM-UBI with same content (10 μM) were incubated with RAW 264.7 cells (1×105 cells cm-2), E. coli (1×108 CFU mL-1) in a PBS buffer (pH 7.4, 10M) at 37 °C for 30 min, respectively. After incubation with pre-determined time, the cells were washed with PBS buffer (pH 7.4, 10 M) three times. The collected suspensions were added to confocal dishes with covers lips for immobilization. The specimens were observed by confocal laser scanning microscopy with a ×63 oil immersion objective lens using a 514 nm laser. Minimum inhibitory concentration (MIC) measurements. The MICs of the antibacterial materials were determined by the broth microdilution method as described previously. Briefly, a 2-fold serial dilution of the micelles (0.4, 0.8, 1.6, 3.2, 6.0, 12.8 and 25.6 M) was prepared and added to an equal volume of bacterial solution (100 L) containing approximately 5ｘ105 CFU/mL in each well of a 96-well plate. The plates were incubated at 37 °C and read every 2 hours. The MIC value was defined as the lowest micelle concentration at which no microbial growth was observed visually or spectrophotometrically via readings of optical density (OD) at 600 nm using a microplate reader (Infinite M200, TECAN). Growth media containing only microbial cells was used as the negative control. The OD600 values recorded at different time were used as time-killing kinetics results. Each MIC test was carried out in 3 replicates and repeated 3 times. Evaluation of dual antibacterial efficiency of micelles by viable cell counting method. P. aeruginosa solution (~105 CFU/mL, 100 L) was incubated with 100 L micelles solution (0.4, 0.8, 1.6, 3.2, 6.0, 12.8 and 25.6 M), respectively. Each sample was prepared in triplicate. After 20 min incubation at 37 oC with shaking, the samples were irradiated green light (0.4


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W/cm2) for 15 min. Then, 100 µL of each sample was placed on MH solid medium with appropriately dilution and cultured at 37 oC for 24 h, followed by counting bacterial colonies. Bacterial colonies of P. aeruginosa treated with micelles in the absence of light were counted following the aforementioned procedures. Inhibition zone assays. Inhibition zone assays of polymeric antimicrobial micelles were performed according to a previously reported method. 59 Briefly, after culture bacteria into Mueller-Hinton broth (5 mL) at 37 °C, plate with appropriately dilution were conducted. Polymeric antimicrobial micells with identical content at 12.8 µM were placed in Oxford cup and the plates were incubated at 37 °C for 18 h. The antibacterial activity was then evaluated by measuring the inhibition zones. Membrane Potential Analysis. To corroborate the postulated mechanism, membrane potential was determined by flow cytometry using a BacLight Bacterial Membrane Potential Kit (Invitrogen). DIOC2(3) exhibits green fluorescence in all bacteria cells, but the fluorescence emission shifts towards red as the dye molecules self-associate at higher cytosolic concentrations caused by large membrane potential. P. aeruginosa cells were inoculated to mid-log phase. Viable cells were then diluted to 2.5 × 107 CFU mL−1 in PBS and added with micelles at concentration (10× MIC). A fully depolarized control was provided by the addition to untreated cells of the proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at a final concentration of 5 M. After 1 h incubation at 37 oC, DiOC2(3) at 30 M was added to all samples. Membrane potential analysis was determined by a flow cytometer as the ratio of cells that exhibited red fluorescence to those that displayed green fluorescence. Gates were drawn based on the untreated (polarized)


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and CCCP-treated (fully depolarized) controls. Data are representative of two independent assays completed in duplicate. Outer Membrane Permeabilization Assay. Outer membrane permeabilization of Gram-negative bacteria was studied by the polarity sensitive dye, N-phenyl napthylamine (NPN). Freshly grown bacteria (P. aeruginosa) were harvested, washed, and resuspended in PBS buffer. The suspension (108 CFU/mL, 150 L) was then transferred to a black 96-well plate. The bacteria suspension was pretreated with diverse samples for 30 min, then added with NPN dye (10 μM, 50 L). The fluorescence intensity was recorded every 2 min in the following 20 min (excitation wavelength = 350 nm, emission wavelength = 420 nm). A control experiment was performed by treating the bacterial suspension with Millipore water. Fluorescence Microscopic observation (LIVE/DEAD). Logarithmic-phase bacteria (1.5 mL, OD600 nm at 0.1) were centrifuged at 5000 rpm for 5 min and washed with phosphate buffer solution (PBS, 0.01 mol/L, pH 7.4) for three times. The supernatant was discarded and the remaining bacteria precipitates were resuspended in 1.5 mL PBS. Bacteria were treated with 100 µL 6.8 µM PAM and PAM-UBI, followed by green irradiation for 20 min. Then 100 µL solution of fluorescent dyes was added and stained in the dark for 20 min. The fluorescent dyes were mixed, by SYTO 9 and propidium iodide (PI), then the mixture were centrifuged at 5000 rpm for 5 min. Individual mixed suspensions were added to slides with covers lips for immobilization. The bacteria cells were imaged using laser scanning confocal microscope (Carl Zeiss LSM 510 META). The control assay was added equal amount of PBS.


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Transmittance electron microscopy (TEM) and scanning electron microscope (SEM). The morphologies of the microorganisms before and after treatment with the micelles were observed under a JEM-2100 transmittance electron microscope (JEOL, Japan). The microorganism solution (0.5 mL) was incubated with 0.5 mL of PAM-UBI micelles at a final of 10 M for diverse total durations. For the light treating groups, continuous 30 min light irradiation was performed during the incubation using a green OLED light. After different treatment, several drops of the micelle solution were placed on a formvar/carbon coated 200 mesh copper grid and left to dry under room temperature, then observed by TEM. Similarly, after these treatments, the bacteria were washed three times with PBS and then fixed with 2.5% glutaraldehyde for 4 h. After fixation, the samples were washed three times with PBS and dehydrated in an alcohol series. Finally, the samples were disposed by metal spraying and observed by SEM. Antibacterial Resistance Test. A standard MIC test of micelles was conducted with E. coli or P. aeruginosa using the protocol mentioned above and beginning with the original strain of bacteria (passage 0). The spread plate used for colony forming unit (CFU) counting in the MH solid medium was used to subculture bacterial cells for this study. Bacterial colonies were observed on the spread plate that was inoculated with the mixture of bacterial cells and polymer micelles at a content of the half MIC value, i.e., the polymer concentration was chosen as 3.2 μM if MIC was identified as 6.0 μM. One bacteria colony, representing a surviving cell from the previous polymer treatment, was carefully picked from this MH-agar plate and designated as passage 1 cells. The colony of passage 1 cells was transferred to a centrifuge tube containing 3 mL sterile MH medium and


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dispersed under vortex mixing for 20 s. This cell suspension was subcultured by inoculating on a MH-agar plate and incubating at 37 °C overnight. The cultured cells at passage 1 on a MHagar plate were suspended in MH medium and used for the next round of standard MIC test. This operation was repeated to evaluate the impact of micelles on E. coli and P. aeruginosa for 10 successive passages. Hemolysis assays. Hemolytic behavior of the polymers was tested using mouse red blood cells (RBCs) . 4% v/v of RBCs was obtained by subjecting the freshly drawn RBCs to a 25× dilution with phosphate buffer saline (PBS), then washed with PBS for three times. 50 μL of red blood cell suspension in PBS (4% in volume) was placed in each well of 96-well plates and 50 μL of polymer solution was added to each well. The plates were incubated for one hour at 37 °C. The cell suspensions were taken out and centrifuged at 1000 rpm for 5 minutes. Aliquots (50 μL) of supernatant were transferred to 96-well plates, and hemoglobin release was monitored at 576 nm using a microplate reader (infinite M200, TECAN). The red blood cell suspension in PBS was used as negative control. Absorbance of wells with red blood cells lysed with 0.5% Triton X-100 was taken as 100% hemolysis. Percentage of hemolysis was calculated using the following formula: Hemolysis (%) = [(OD.576 nm in the nanoparticle solution-OD.576nm in PBS)/(OD.576nm in 0.5% Triton X-100- OD.576nm in PBS)]×100. Cell viability assays MTT assay was used to measure the cytotoxicityof polymeric antimicrobial. Mouse skin fibroblast cells were used to evaluate the cytotoxicity of the materials. Fibroblast cells were cultured in DMEM/F 12 media containing 10% calf serum, 100 U mL−1 penicillin, and 100 µg


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mL−1 streptomycin. Fibroblast cells were seeded under the down chamber of 96-well roundbottom microplate with a density of 5 × 104 cells per well and incubated in a 37 °C humidified incubator (5% CO2) for 24 h. After that, cell medium was replaced by fresh cell medium containing polymeric antimicrobial with diverse contents and incubated for another 24 h. Then, MTT agent (20 µL, 5 mg mL−1) was added into each well and co-incubated for another 4 h. The cultured medium in each well was replaced with DMSO (100 µL). The relative cell viability was obtained using a microplate reader (infnite M200, Tecan). In vivo infection Animal Models and Histological Analysis. To evaluate the antibacterial efficacy in vivo, two kinds of infection models were built. All the animal experiments were performed following a protocol approved by the Institutional Animal Care and Use Committee (South China Normal University, Guangzhou, China). Ten groups of 50 male mice (4-6 weeks, five mice per group) were divided into saline, P(DMAEMA-co-EoS), PAM and PAM-UBI at 320 M under dark or light irradiation. Two commercial antibiotics, ciprofloxacin (0.8 mg/mL), and colistin (0.8 mg/mL), were also evaluated for comparison. When the injury model was built, diverse bacteria suspensions (108 CFU/mL, 50 L) were added, respectively. For each therapeutic treatment, 20 L of these samples was treated, respectively. 30 min light irradiation was given for the groups with ligh treating. The mice in these different groups with different gauze bandage on their wound were observed and photographed and gauze bandage were changed with 24 h interval. During the treatment process, aliquots of diluted homogenized skin tissues were placed on agar, on which the grown colonies were counted for analysis. For histological analysis, the wound tissues from each group of mice were taken for further analysis. The tissues were set in 4% paraformaldehyde


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solution for further staining and H&E analysis. Characterization. All nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend 500 HD NMR spectrometer. The UV-Vis absorption spectra were acquired by Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, USA). Hydrodynamic diameter distribution and zeta potential were determined by a Malvern Zetasizer Nano ZS90 instrument (Malvern, UK). Transmission electron microscopy analysis was operated on JEOL JEM-2100 at an accelerating voltage of 80 kV. Confocal laser scanning microscopy (CLSM) images were acquired using Carl Zeiss LSM 510 META.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.XXX. Synthetic routes, 1H-NMR analysis, fluorescence spectra, in vitro ROS detection, the evaluation of targeting adhesion against S. aureus observed by CLSM imaging, in vitro inhibition evaluation of polymeric antimicrobials against E. coli, CLSM images of S. aureus after diverse treatments and stained with SYTO 9 and PI, resistance evaluation of E. coli after different treatments, hemolysis analysis, and MTT assays.

AUTHOR INFORMATION

Corresponding Authors


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

mail: [email protected], [email protected]

ORCID Xianglong Hu: 0000-0001-9202-1543 Author Contributions §

F. F. Xiao and B. Cao contributed equally to this work.

ACKNOWLEDGMENTS The authors thank for the helpful discussion and kind support from Dr. Xin Ding at School of Pharmacy (Shen Zhen Campus), Sun Yat-Sen University, and Dr. Dingqiang Chen at Department of Laboratory Medicine, First Affiliated Hospital of Guangzhou Medical University. This work was supported by the National Natural Scientific Foundation of China (21674040, 81630046), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2016A030306013), the Guangdong Program for Support of Top-notch Young Professionals (2015TQ01R604), the Scientific Research Projects of Guangzhou (201805010002), the National Key Research and Development Program of China (2018YFA0209800),

and

the

Guangdong-HongKong

Joint

Innovation

Project

(2014B050504009).

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2018, 14, 1800201. 67. Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y. Y. Emerging Trends in Macromolecular Antimicrobials to Fight Multi-Drug-Resistant Infections. Nano Today 2012, 7, 201-222. 68. Yan, S.; Luan, S.; Shi, H.; Xu, X.; Zhang, J.; Yuan, S.; Yang, Y.; Yin, J. Hierarchical Polymer Brushes with Dominant Antibacterial Mechanisms Switching from Bactericidal to Bacteria Repellent. Biomacromolecules 2016, 17, 1696-1704. 69. Wiegand, I.; Hilpert, K.; Hancock, R. E. W. Agar and Broth Dilution Methods to Determine the Minimal Inhibitory Concentration (Mic) of Antimicrobial Substances. Nat. Protoc. 2008, 3, 163-175. 70. Liu, Y.; Busscher, H. J.; Zhao, B.; Li, Y.; Zhang, Z.; van der Mei, H. C.; Ren, Y.; Shi, L. Surface-Adaptive, Antimicrobially Loaded, Micellar Nanocarriers with Enhanced Penetration and Killing Efficiency in Staphylococcal Biofilms. ACS Nano 2016, 10, 4779-4789. 71. Xing, C.; Yang, G.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Conjugated Polymers for LightActivated Antifungal Activity. Small 2012, 8, 524-529. 72. Zhu, Y.; Xu, C.; Zhang, N.; Ding, X.; Yu, B.; Xu, F.-J. Polycationic Synergistic Antibacterial Agents with Multiple Functional Components for Efficient Anti-Infective Therapy. Adv. Funct. Mater. 2018, 1706709. 73. Wu, C.; He, Q.; Zhu, A.; Li, D.; Xu, M.; Yang, H.; Liu, Y. Synergistic Anticancer Activity of Photo- and Chemoresponsive Nanoformulation Based on Polylysine-Functionalized Graphene. ACS Appl. Mater. Interfaces 2014, 6, 21615-21623. 74. Fernandes, M. M.; Francesko, A.; Torrent-Burgues, J.; Carrion-Fite, F. J.; Heinze, T.; Tzanov, T. Sonochemically Processed Cationic Nanocapsules: Efficient Antimicrobials with Membrane Disturbing Capacity. Biomacromolecules 2014, 15, 1365-1374. 75. Liu, S. Q.; Venkataraman, S.; Ong, Z. Y.; Chan, J. M.; Yang, C.; Hedrick, J. L.; Yang, Y. Y. Overcoming Multidrug Resistance in Microbials Using Nanostructures Self-Assembled from Cationic Bent-Core Oligomers. Small 2014, 10, 4130-4135. 76. Zou, X.; Zhang, L.; Wang, Z.; Luo, Y. Mechanisms of the Antimicrobial Activities of Graphene Materials. J. Am. Chem. Soc. 2016, 138, 2064-2077. 77. Wang, J.; Zhuo, L.; Liao, W.; Yang, X.; Tang, Z.; Chen, Y.; Luo, S.; Zhou, Z. Assessing


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ACS Nano

2018, 176, 1-12.

Scheme 1. (a) Schematic illustration for fabrication of pathogen-targeting polymeric antimicrobial micelles. (b) The polymeric antimicrobials can significantly inhibit highly opportunistic gram-negative bacteria via selective recognition to bacteria but not mammalian cells, fusing into bacterial membrane through the quadruple barriers, including lipopolysaccharide (LPS), out membrane (OM), peptidoglycan (PG), cytoplasmic membrane (CM). The polymeric antimicrobial micelles can physically destroy the integrity of bacterial membrane resulting in the leakage of intracellular milieu, synergistically strengthening the


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photodynamic bacteria eradication upon light irradiation.

Figure 1. Characterization of the polymeric antimicrobial micelles. Hydrodynamic diameter distributions and TEM images recorded for the polymeric antimicrobial micelles of (a) nontargeted PBMA-b-P(DMAEMA-co-EoS) (noted as PAM), and (b) bacteria-targeted PBMA-bP(DMAEMA-co-EoS)-UBI (noted as PAM-UBI), respectively. (c) Absorbance spectra of eosin-Y (Eos), P(DMAEMA-co-EoS), PAM and PAM-UBI. (d) Decay curves of the absorption intensities of DPBF at 410 nm upon treating with PAM and PAM-UBI in the presence of green light irradiation with diverse duration.


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ACS Nano

Figure 2. Selective recognition of bacteria over host mammalian cells. (a) CLSM imaging for the mixture of RAW 264.7 cells and P. aeruginosa upon incubation with PAM and PAM-UBI for 30 min at 37 °C, respectively. RAW 264.7 cells are marked with the blue arrows, scale bar: 10 m. (b) Flow cytometer analysis of P. aeruginosa upon incubating with 6.0 µM PAM and PAM-UBI for 30 min in the dark, respectively. (c) Statistical analysis of the fluorescence intensity in (b), n=3. p12.8

>12.8

P(DMAEM A-co-EoS)

(HC50/MIC)

Index) d

P. aeruginosa

E. coli

P. aeruginosa

E. coli

Pathogen-Specific Polymeric Antimicrobials with Significant

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