A Water-Soluble Galactose-Decorated Cationic Photodynamic

Subscriber access provided by READING UNIV

Article

A Water-Soluble Galactose-Decorated Cationic Photodynamic Therapy Agent Based on BODIPY to Selectively Eliminate Biofilm Xiaomei Dai, Xuelei Chen, Yu Zhao, Yunjian Yu, Xiaosong Wei, Xinge Zhang, and Chaoxing Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01316 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

A Water-Soluble Galactose-Decorated Cationic Photodynamic Therapy Agent Based on BODIPY to Selectively Eliminate Biofilm

Xiaomei Dai, Xuelei Chen, Yu Zhao, Yunjian Yu, Xiaosong Wei, Xinge Zhang,∗ Chaoxing Li

a

Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute

of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China

∗

Corresponding author:

The Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Weijin Road 94, Tianjin 300071, China

E-mail: [email protected]

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A multitude of serious chronic infections involved in bacterial biofilms that are difficult to eradicate. Here, a water-soluble galactose functionalized cationic 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based photodynamic therapy agent was synthesized for selectively eliminating the bacterial biofilm. These conjugates can capture bacteria to form aggregations through electrostatic interaction and then generate a large number of reactive oxygen species (ROS) under visible light irradiation to kill the bacteria without the emergence of bacterial resistance. Simultaneously, this agent could effectively inhibit and eradicate both Gram-positive and Gram-negative bacterial biofilms. The in-depth analysis of antimicrobial mechanism confirmed that the conjugates can quickly bind on the bacterial surface, irreversibly disrupt the bacterial membrane, distinctly inhibit intracellular enzyme activity, ultimately leading to the bacterial death. Importantly, these conjugates are highly selectivity toward bacterial cells over mammalian cells as well as no cytotoxicity to A549 cells and no discernible hemolytic activity. Collectively, this water-soluble galactose-decorated cationic BODIPY-based photodynamic therapy agent design provides promising insights for the development of therapy for antibiotic-resistant bacteria.

Keywords: photodynamic therapy, biofilm, antimicrobial mechanism, bacterial selectivity, bacterial resistance


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Introduction The majority of bacterial infections are accompanied by biofilms formation.1 Biofilms are composed of one or more species of microorganisms that are encased in extracellular polymeric substances (EPS) and attached to both a solid surface and to each other.2,3 By existing in a quiescent state, biofilms are responsible for causing persistent infections such as burn wound infections, catheter infections, chronic wound infections, and lung infection of cystic fibrosis patients.4,5 Since biofilms lead to a dramatic enhancement in resistance to the commercial antibiotics, there has been an increasing attention in developing novel strategies to eradicate the bacterial biofilms. Based on the understanding of features and signals during the development of biofilm, the novel strategies against bacterial biofilm have been designed in the past years. One strategy is usage of biofilm self-generated components to only disrupt biofilms by targeting EPS, such as D-amino acid, diguanylate cyclase, glycosyl hydrolase, and so on.6-9 The other strategy is to exploit nanocarriers to deliver antibiotics into biofilm to eradicate the bacterial biofilm.10-12 These strategies are utilized to disperse the bacterial biofilms, but not kill the bacteria or depend on antibiotic to ablate bacteria, which cannot overcome the onset of acquired resistance. Antimicrobial agents that can selectively eradicate biofilm as well as kill the bacteria without bacterial resistance have become attractive candidates for biofilm elimination. Photodynamic therapy (PDT), which uses a photosensitizer to generate toxic ROS, was thus proposed and applied to resistant bacteria without rapid development of resistance.13 The application of PDT was limited for their low solubility and short


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

effective distance to bacteria.14 Cationic polymer functionalized PDT agents can be considered as a viable option as they are likely to penetrate through the biofilms matrix, access the encased cells owing to their property to interact with the negatively charged bacterial membrane, generate ROS and render irreversibly membrane damage.15 However, the application of cationic polymers is restricted by their poor biocompatibility. Galactose is widely found in avocados, sugar beets, dairy products, other mucilages and gums. It is also synthesized by the body, where it forms part of glycoproteins and glycolipids in several tissues.16 Hence, galactose is used to modify PDT agents to improve their biocompatibility, water-solubility as well as their antibacterial activities. Here, BODIPY core was used as PDT agent due to their excellent fluorescence quantum yield, photostability and extinction coefficient.18,19 Thiazole ring as a common structural component in a large number of biological agents can effectively inhibit the activities of enzymes and proteins.20 Hence, thiazole derivatives containing quarternary ammonium groups (PATA-C4) was designed to capture as well as kill bacteria. In this work, BODIPY agent with different molar ratios of thiazole (TA) to galactose (GAL) [P(ATA-C4)-r-GAL-I2] was synthesized, and their selective antimicrobial activity, antibiofilm activity and biocompatibility were systematically studied (Scheme 1). The novel copolymer as an efficient antibacterial agent can be widely used in the treatment of bacterial infections.


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Scheme 1. Presentation of P(ATA-C4)-r-GAL-I2 for selective eradication of bacteria and bacterial biofilm.

EXPERIMENTAL SECTION Materials: 2,4-Dimethylpyrrole, 4-(chloromethyl)benzoyl chloride and pyrrole were purchased from Aladdin (Shanghai, China). 5-(2-Hydroxyethyl)-4-methylthiazole (98%),

O-nitrophenyl-β-D-galactopyranoside

(ONPG)

and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) were available from Tianjin Heowns Biochem Technologies LLC (Tianjin, China). Acridine orange (AO) and ethidium bromide (EB) were gained from Alfa Aesar (Beijing, China). FITC-labeled concanavalin A (FITC-ConA) was purchased from Sigma-Aldrich (Shanghai, China). Levofloxacin-resistant Staphylococcus aureus (S. aureus) ATCC 6538 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027 strains were provided by the Department of Microbiology of Nankai University (Tianjin, China). The other chemicals were analytical reagents with desiccation before use.

Synthesis of BODIPY-Conjugated Chain Transfer Agent


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The compound was prepared as previously reported with slight modifications.21,22 Briefly, 2,4-dimethylpyrrole (4 mL, 38.9 mmol) and 4-(chloromethyl)benzoyl chloride (2.27 mL, 19.5 mmol) were added to dried dichloromethane (150 mL) and sealed under nitrogen. After the mixture was stirred at 25 °C for 2 h, 80 mL of triethylamine was added to the mixture. After 1 h, 80 mL of boron trifluoride ethyl ether was added and stirred at 25 °C for 6 h. The product was purified and a red solid was obtained in a yield of 40%. 1H NMR (400 mHz, TMS, CDCl3, ppm): 7.32, 7.52 (d, 4H, -C6H4-), 5.98 (s, 2H, pyrrole), 4.68 (s, 2H, -CH2Cl-), 2.55, 1.38 (s, 12H, 4 × -CH3). Pyrrole (0.03 mL, 0.48 mmol) and sodium hydride (11.4 mg, 0.48 mmol) was added to dried dimethyl sulfoxide (DMSO) (5 mL). After the mixture was stirred for 30 min at 25 °C, carbon disulfide (0.028 mL, 0.48 mmol) was added to the mixture. After the mixture was stirred for further 30 min, the above prepared red solid was added, and the reaction was heated to 50 °C. After the mixture was stirred for 12 h, 50 mL of deionized water was added, and the mixture was extracted with dichloromethane (20 mL × 3). The crude product was purified by a silica gel eluted with petroleum ether/ethyl acetate (v/v, 10:1) with about 30% yield. 1H NMR (400 mHz, TMS, CDCl3, ppm): 7.71 (m, 2H, pyrrole), 7.33, 7.53 (m, 4H, -C6H4-), 6.34 (m, 2H, pyrrole), 5.99 (s, 2H, 2,4-dimethylpyrrole), 4.14 (s, 2H, -CH2-C6H4), 2.55, 1.38 (s, 12H, 4 × -CH3). Synthesis of 2-O-Acryloyloxyethyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (AcGAL).


Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Monomer AcGAL was synthesized as previously reported.22 α-D-Galactose pentaacetate (1.95 g, 5.0 mmol), hydroxyethyl acrylate (0.79 mL, 6.1 mmol) and dried dichloromethane (10 mL) were added into three-necked round-bottomed sealed under nitrogen. Boron trifluoride ethylether (3.5 mL, 22.0 mmol) was added to the mixture and stirred for 1 h at 0 °C, then heated to 25 °C for 3 h. The reaction was quenched with chloroform (30 mL), washed with deionized water (30 mL) and brine (30 mL), followed by drying over anhydrous sodium sulfate. The product was obtained as colorless oil with 50% yield. 1H NMR (400 mHz, TMS, CDCl3, ppm): 6.41-6.46 (d, 1H, -CH2=CH-), 6.11-6.17 (q, 1H, -CH2=CH-), 5.83-5.86 (d, 1H, -CH2=CH-), 5.37-5.41 (d, 1H, galactose), 5.19-5.23 (t, 1H, galactose), 4.97-5.03 (q, 1H, galactose), 4.54-4.56 (d, 1H, galactose), 4.27-4.32 (m, 2H, galactose), 4.09-4.21 (m, 2H, -OCOCH2-), 4.02-4.06 (m, 1H, galactose), 3.89-3.94 (t, 2H, -OCOCH2-CH2O-), 1.96-2.16 (q, 12H, -COCH3). Synthesis of Polymer

The polymer was prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization. The radical polymerization of ATA and AcGAL monomers at total conversion was carried out in dry DMSO solution at 70 °C using AIBN as an initiator. The polymer (PATA-r-AcGAL) was purified by precipitation in deionized water and dried under vacuum.

Synthesis of PATA-r-GAL Copolymer PATA-r-AcGAL copolymer (1 equiv) was added to DMSO (8 mL) solution with


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

hydrazine (10 equiv) at 25 °C under nitrogen.22 After 24 h, acetone was dropped to quench the reaction. The mixture was dialyzed against deionized water (3.5 kDa cutoff membrane) for 3 days and dried using a Flexi-dry freeze dry system.

Modification of Polymer P(ATA-C4)-r-GAL-I2

PATA-r-GAL (1.0 equiv), alkyl bromide (10.0 equiv. ethyl bromide, butyl bromide or hexyl bromide) and anhydrous DMF (10 mL) were added into a sealed tube containing a magnetic stirring bar. The mixture was purged with nitrogen for 30 min and heated at 70 °C for 72 h. The reaction was monitored by 1H-NMR. Finally, the mixture was dialyzed using a dialysis tube (3.5 kDa cutoff membrane) against deionized water for 3 days with the replacement of the water every 6 h and dried using a Flexi-dry freeze-dry system.

Iodic acid in deionized water was used as an iodination agent to modify BODIPY molecule. The product [P(ATA-C4)-r-GAL-I2] was obtained as a deep red solid.

Generation of ROS

To assess whether the copolymer could generate ROS rapidly in aqueous solution, the test was carried out as previously reported.23 p-Nitrosodimethylaniline (RNO) and histidine was serve as ROS scavengers. A decrease in absorption at 440 nm will be observed due to RNO react with the product formed by the reaction of ROS with histidine. A mixture of histidine (100 µg/mL), RNO (5 µg/mL) and copolymer (31.3 µg/mL) was prepared. The mixture was irradiated under visible light (1.5 mW/cm2, 400-800 nm) at 25 °C. The rate of ROS generation was evaluated by monitoring the


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

decrease in the absorbance of RNO at 440 nm at fixed intervals. The solution without copolymer was used as negative control. The solution with Rose Bengal was used as positive control.

Antimicrobial Assay

Levofloxacin-resistant S. aureus and P. aeruginosa bacteria were chosen as the bacterial model to explore the antimicrobial activity of P(ATA-C4)-r-GAL-I2. Bacteria were cultured in Lysogeny Broth (LB) medium at 37 °C overnight. The copolymers solutions (250, 125, 62.5, 31.3, 15.7, 7.9, 4 and 2 µg/mL) were mixed with the same volume of bacterial suspension (2.0 × 106 CFU/mL), and then incubated for 8 h at 37 °C. The minimum inhibitory concentration (MIC) was evaluated by measuring the optical density at 600 nm (OD600) using UV-vis spectroscopy. All experiments were carried out in triplicates.

Hemotoxicity Activity To investigate the selectivity of the copolymers, the copolymers were incubated with the human red blood cells, and the HC10 for the copolymers was calculated. HC10 is defined as the concentration that causes 10% hemolysis of the human red blood cells (hRBCs).24,25 The blood was obtained from Tianjin Medical University (Tianjin, China). Erythrocytes were separated from blood plasma and leukocytes by centrifugation (3000 rpm, 3 min) at 4 °C and washed with phosphate buffer solution (PBS) three times. Then, copolymers were added to erythrocytes of 1% hematocrit and incubated at 37 °C to study the effect of copolymers on erythrocyte hemolysis


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 40

activity. After 45 min incubation, suspensions were centrifuged (3000 rpm, 3 min) and supernatant obtained for analysis. To reference, red blood cells were treated with Triton X-100, which corresponds to 100% hemolysis activity. The blood treated with PBS was used as negative control. The percentage of hemolysis activity was determined by released hemoglobin in supernatant and evaluated by measuring the optical density at 540 nm (OD540) using UV-vis spectroscopy. The percentage of hemolysis was calculated as follows:

Hemolysis% =

ODହସ଴ ሺPolymerሻ − ODହସ଴ ሺPBSሻ × 100% ODହସ଴ ሺTritonX − 100ሻ − ODହସ଴ ሺPBSሻ

Zone of Inhibition Assay The zone of inhibition of bacteria was used to determine the antibacterial activity of P(ATA-C4)-r-GAL-I2. P. aeruginosa and S. aureus were incubated in LB broth at 37 °C overnight. Then, 50 µL of the bacterial suspension (5 × 107 CFU/mL) was inoculated evenly on an LB agar plate. The disk with the diameter of 8 mm containing P(ATA-C4)-r-GAL-I2 solution (31.3 µg/mL) was gently placed at the centre of the LB agar plate and incubated for 10 h at 37 °C. The antimicrobial activity was evaluated by determining the diameter of the zone of inhibition around the disks in triplicates.

Live/Dead Assay The Live/Dead assay was used to further determine the antimicrobial activity of P(ATA-C4)-r-GAL-I2.

Levofloxacin-resistant

P.

aeruginosa

and

S.

aureus

suspensions (1 mL, 5 × 107 CFU/mL) were obtained by washed three times with PBS


Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

and resuspended in 1.0 mL of PBS. Then, 1.0 mL of P(ATA-C4)-r-GAL-I2 (31.3 µg/mL) was added and incubated at 37 °C for 2 h. All bacteria were collected by centrifugation, and then stained with 10 µL of dyes (10 mg AO and 10 mg EB dissolve in 1 mL PBS) for 15 min in the dark, finally imaged using an inverted fluorescence microscope (Leica DMI 4000B).

The Inhibition of β–Galactosidase Activity

The solution containing the cytoplasm was extracted from E. coli by ultrasound at 0 °C for 10 h. Then, P(ATA-C4)-r-GAL-I2 with different concentrations and ultrapure water were added to the filtrate (1 mL). After incubation at 25 °C for 30 min, ONPG (1.0 mL, 10 mg/mL) was added. The generated ortho-nitrophenol (ONP) was assayed by determining the optical density at 420 nm (OD420). The addition of PBS was used as a control. The percentage of β–galactosidase inhibition was calculated as follows:

Inhibition% =

ODସଶ଴ ሺcontrolሻ − ODସଶ଴ ሺsampleሻ × 100% ODସଶ଴ ሺcontrolሻ

The Detection of ROS in Bacteria The generation of ROS in bacteria was confirmed via dichlorofluorescein diacetate (DCFH-DA), which could be oxidized by ROS to fluorescent 2,7-dichlorofluorescein (DCF).26 All bacterial cells medium was replaced with DCFH-DA solution, and incubated with P(ATA-C4)-r-GAL-I2 (7.9 µg/mL) for 10 min in the dark. The group treated with PBS was used as control. All cells were irradiated in the visible light for 5 min, and imaged using an inverted fluorescence microscope (Leica DMI 4000B).


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Morphological Characterization of Bacteria Levofloxacin-resistant P. aeruginosa and S. aureus suspension (1 mL, 5.0 × 107 CFU/mL) were collected and washed with PBS. The appropriate amount of P(ATA-C4)-r-GAL-I2 (31.3 µg/mL) was added into the bacterial suspension and incubated at 37 °C for 1 h. Then, all cells were centrifuged (5000 rpm) at 4 °C for 5 min. All bacterial suspensions were placed on the slide, and fixed with glutaraldehyde for 12 h, and dried in a freeze dryer (Flexi-Dry TM MP), gold sputter-coated, and imaged using a scanning electron microscope (SEM; Shimadzu SS-550).

Induction of Bacterial Resistance To evaluate whether the copolymer could induce bacterial resistance, the experiment was performed as previously reported.27,28 P. aeruginosa and S. aureus suspensions (1 × 107 CFU/mL) were cultured with 1/2 MIC value of P(ATA-C4)-r-GAL-I2 or levofloxacin at 37 °C until the concentrations were up to 2 × 107 CFU/mL. Then, bacteria were cultured on agar plates containing P(ATA-C4)-r-GAL-I2 or levofloxacin for 12 h, and the MIC values of polymers and levofloxacin were investigated. The incubation was repeated 30 times to obtain the resistant variants, and the MIC value was evaluated after each passage.

Selective Interaction between Mammalian Cells and Bacteria A549 cells were seeded in a 24-well plate at a density of 3 × 104 cells per well. After incubation at 37 °C for 24 h, the medium was changed to the fresh medium without antibiotics. Bacterial suspensions (150 µL, 5 × 107 CFU/mL) were added to the


Page 12 of 40

Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

24-well plate and cultured at 37 °C for further 30 min. Then, MIC value of P(ATA-C4)-r-GAL-I2 was added into the 24-well plate and incubated at 37 °C for 1 h. After washing with PBS three times, all cells were observed using optical microscope.

Inhibition of Bacterial Biofilm Formation

The capability of P(ATA-C4)-r-GAL-I2 to prevent the formation of P. aeruginosa, S. aureus, E.coli and B. amyloloquefaciens biofilm was investigated via crystal violet (CV) staining method. All bacteria were diluted into 1 × 106 CFU/mL with LB medium. Then, 100 µL of bacteria was added to 96-well microtiter plates and incubated with different concentrations of P(ATA-C4)-r-GAL-I2 at 37 °C for 24 h without shaking. Subsequently, the media was removed from the 96-well microtiter plates and washed with 150 µL of PBS three times. Then, 100 µL of CV (0.25%) was added to each well of the microtiter plate and incubated at 37 °C for 20 min. After washing with PBS three times, 150 µL of acetic acid (33%) was added. Following 10 min incubation at room temperature with brief mixing, the absorbance of the solution was determined at a wavelength of 590 nm using a microtiter plate reader. The bacterial biofilms treated with PBS was used as control. All samples were performed five times. The percentage inhibition of bacterial biofilm formation was calculated as follows:

Inhibition% =

ODହଽ଴ ሺcontrolሻ − ODହଽ଴ሺsampleሻ × 100% ODହଽ଴ ሺcontrolሻ

Static Biofilm Assays Static biofilm assays were performed in 96-well microtiter plates as previously


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

described with slight modifications.29-32 P. aeruginosa, S. aureus, E.coli and B. amyloloquefaciens were prepared by transferring frozen stock cultures into an LB medium under shaking at 37 °C overnight. All bacteria were diluted into 1 × 106 CFU/mL with LB medium. Then, 100 µL of bacteria were added to 96-well microtiter plates and incubated at 37 °C for 24 h without shaking. After formation of the bacterial biofilms, 100 µL of fresh growth media with different concentrations of P(ATA-C4)-r-GAL-I2 was added into the 96-well microtiter plates and cultured at 37 °C for further 24 h. Subsequently, the media were removed from the 96-well microtiter plates and washed with 150 µL of PBS three times. Then, 100 µL of CV (0.25%) was added to each well of the microtiter plate and incubated at 37 °C for 20 min. After washing with PBS three times, 150 µL of acetic acid (33%) was added. Following 10 min incubation at room temperature with brief mixing, the absorbance of the solution was determined at a wavelength of 590 nm using a microtiter plate reader. The bacterial biofilms treated with PBS was used as control. All samples were performed five times. The percentage of bacterial biofilm inhibition was calculated as follows:

Inhibition% =

ODହଽ଴ ሺcontrolሻ − ODହଽ଴ሺsampleሻ × 100% ODହଽ଴ ሺcontrolሻ

Confocal Laser Scanning Microscope Analysis Biofilms of P. aeruginosa, S. aureus, E. coli and B. amyloloquefaciens were grown on glass coverslips placed in the bottom of 24-well microtitre plate as previously described.33 After cultivation for 24 h, culture medium was removed, and fresh


Page 14 of 40

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

growth media with P(ATA-C4)-r-GAL-I2 (11 µg/mL) were added into the 24-well microtitre plate. After 24 h incubation, the biofilms were washed with PBS three times and finally immersed in PBS. Then, 10 µL of FITC-ConA and EB was added into the 24-well microtitre plate and cultured at 37 °C for 10 min. After washing with PBS three times, the coverslip was placed on the glass slide and imaged using a confocal laser scanning microscope (CLSM, TCS SP8).

Cytotoxicity Tests

The cytotoxicity of P(ATA-C4)-r-GAL-I2 was evaluated by MTT assay. 100 µL of A549 cells (1.0 × 104) was seeded into each well of a 96-well plate and cultured at 37 °C for 24 h. After that, the media was replaced with the fresh media containing different concentrations of P(ATA-C4)-r-GAL-I2. After incubation for 24 h, 20 µL of MTT was added and incubated for further 4 h. Media in each well of a 96-well plate was completely removed before the next experiment, and 150 µL of DMSO was added and the optical density was read on a microplate reader at a wavelength of 490 nm.

Result and Discussion

Synthesis, Characterization and Optimization of P(ATA-C4)-r-GAL-I2

Although cationic PDT agents are likely to penetrate through the biofilm matrix, access the encased cells owing to their property to interact with the negatively charged bacterial membrane, generate ROS and render irreversibly membrane damage, nevertheless, their application is restricted by their poor biocompatibility. Based on


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

galactoses

significant

recognition

due

to

their

Page 16 of 40

bacterial

protein-targeting

properties,34,35 galactose was used to improve the biocompatibility and antibacterial activity of the cationic polymer. A series of PATA-r-AcGAL with different molecular weights

(PATA27-r-AcGAL31,

PATA24-r-AcGAL62,

PATA21-r-AcGAL93

and

PATA18-r-AcGAL124) were prepared by changing the free ratio of monomer to chain transfer agent in DMSO at 70 °C. According to the results of gel permeation chromatography (GPC), PATA-r-AcGAL copolymers showed narrow polydispersity index (PDI) (approximately 1.01) with molecular weights of 5421, 6632, 7310 and 8100 Da, respectively (Table 1). The structure of PATA-r-AcGAL was further analyzed using 1H NMR spectroscopy. As shown in Figure 1, peaks at 8.8 and 4.6-5.1 ppm were assigned to ATA and AcGAL unit appeared in the spectrum, certifying successful synthesis of PATA-r-AcGAL.

Figure 1. 1H NMR spectrum of copolymer PATA-r-AcGAL in DMSO-d6.


Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

After being treated with hydrazine hydrate, PATA-r-GAL was obtained, the peaks at 1.5-2.5 ppm of the acetyl group vanished, and the stronger peaks at 3.5-4.5 ppm of PATA-r-GAL were assigned to galactose residues (Figure 2), indicating the successful deacetylation reaction.

Figure 2. 1H NMR spectrum of copolymer PATA-r-GAL in DMSO-d6.

Then, PATA-r-GAL was quaternized with butyl bromide, the strong peaks at 1.0-1.6 ppm of P(ATA-C4)-r-GAL was assigned to the butyl bromide, demonstrating successful

quaternization

of

PATA-r-GAL.

The

emission

spectra

of

P(ATA-C4)-r-GAL displayed a well-defined band width with emission maximum at 512 nm (Figure S1, Supporting Information). After being introduced the iodine atom into BODIPY molecule, the fluorescence of P(ATA-C4)-r-GAL in aqueous solution was quenched (Figure 3 inside). The peaks at 6.1 ppm of the Ar-H in the 1H-NMR spectrum vanished (Figure 3). These results collectively confirmed that the copolymer


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[P(ATA-C4)-r-GAL-I2] was successfully synthesized.

Figure 3. 1H NMR spectrum of copolymer P(ATA-C4)-r-GAL-I2 in D2O.

As shown in Table 1, copolymers with different compositions were synthesized, and their antibacterial activity was evaluated using levofloxacin-resistant S. aureus and P. aeruginosa as model bacteria, and the result showed that P(ATA-C4)-r-GAL2-I2 performed the excellent antibacterial activity. Cationic polymers possess common characteristics of amphiphilic nature and cationic charge. These two characteristics allow cationic polymers to be electrostatically attracted to the negatively charged bacterial membrane and then to penetrate into the hydrophobic lipid bilayer interior.36,37 We hypothesized that P(ATA-C4)-r-GAL2-I2 obtained the balance of hydrophily/hydrophobicity to damage the membrane of bacteria. Compared with P(ATA-C4)-r-GAL without BODIPY, P(ATA-C4)-r-GAL2-I2 showed the stronger antibacterial activity against both P. aeruginosa and S. aureus. It is attribute to the


Page 18 of 40

Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

ROS release from P(ATA-C4)-r-GAL2-I2 under visible light. The hematotoxicity of these copolymers was investigated, and the result displayed that all copolymers did not show any cytotoxicity to blood cells even if the concentration was up to 1 mg/mL. The HC10 values of copolymers were 63 fold for MIC value and increased with increasing the content of galactose. The results suggested that these copolymers are highly selectivity toward bacteria and thus hold great promise for the design of potent antimicrobial agents. Taken together, P(ATA-C4)-r-GAL2-I2 (abbreviated as P(ATA-C4)-r-GAL-I2) was applied for the following work.

Table 1. Physicochemical and biological properties of the polymers. MIC (µg/mL)

Mn Sample

PDI

DS (%)

(Da)

HC10

P.aeruginosa

S.aureus

(mg/mL)

P(ATA-C4)-r-GAL1-I2

5421

1.01

81.3

15.7 ± 0.4

7.9 ± 0.1

> 2.0

P(ATA-C2)-r-GAL2-I2

6632

1.01

82.5

7.9 ± 0.2

7.9 ± 0.2

> 4.0

P(ATA-C4)-r-GAL2-I2

6632

1.01

80.5

7.9 ± 0.1

4.0 ± 0.1

> 4.0

P(ATA-C4)-r-GAL2

6632

1.01

80.5

31.4 ± 1.5

15.7 ± 0.5

> 4.0

P(ATA-C6)-r-GAL2-I2

6632

1.01

78.9

7.9 ± 0.3

15.7 ± 0.4

> 1.0

P(ATA-C4)-r-GAL3-I2

7310

1.01

79.5

15.7 ± 0.5

7.9 ± 0.2

> 5.0

P(ATA-C4)-r-GAL4-I2

8100

1.05

82.4

15.7 ± 0.5

7.9 ± 0.2

> 8.0

Generation of ROS The generation of ROS is an important parameter to be considered in identifying an ideal PDT agent. The rate of generation of ROS by the copolymer was measured


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

using UV-vis spectroscopy. Rose bengal was used as the reference. As depicted in Figure 4, the copolymer could generate ROS more rapidly than rose bengal in aqueous solution. Yogo et al. found that iodine-BODIPY could generate high efficiency of ROS because of the triplet excited state.38 The results indicated that the copolymer could rapidly generate ROS in aqueous solution and had the potential to eradicate bacteria as a PDT agent.

Figure 4. The RNO absorption at 440 nm under visible light irradiation (400-800 nm, 1.5 mW/cm2) for different time in the aqueous solution.

The Antibacterial Mechanism of P(ATA-C4)-r-GAL-I2 The zone of inhibition was tested to confirm that P(ATA-C4)-r-GAL-I2 had excellent antibacterial activity. As depicted in Figure S2 (Supporting Information), the zone of inhibition of P(ATA-C4)-r-GAL-I2 was obviously exhibited on the agar medium. The zone of inhibition of S. aureus was larger than that of P. aeruginosa, indicating that P(ATA-C4)-r-GAL-I2 was more sensitive to S. aureus. This is possibly because of the difference of membrane component between Gram-positive (S. aureus) and


Page 20 of 40

Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Gram-negative (P. aeruginosa) bacteria. The components of Gram-positive bacteria cell wall are peptidoglycan, well-associated protein, and teichoic acid.39 Among them, teichoic acid as a special ingredient in Gram-positive bacteria cell wall is charged negatively, and positively charged P(ATA-C4)-r-GAL-I2 can rapidly attach to the bacterial surface through electrostatic interactions, ultimately kill bacteria.

To further confirm the antimicrobial activity of P(ATA-C4)-r-GAL-I2, the live/dead assay was carried out using levofloxacin-resistant S. aureus and P. aeruginosa as the research model. Under the fluorescence microscope, the live/dead bacterial cells appeared as green/red points with intact/damage membrane. As displayed in Figure 5A, the control groups exhibited green fluorescence, suggesting that all bacteria are alive. However, after being treated with P(ATA-C4)-r-GAL-I2 for 1 h, all bacterial displayed the red fluorescence, signifying that all bacteria are dead. We found that most

of

the

bacteria

were

bound

together

after

being

treated

with

P(ATA-C4)-r-GAL-I2. To further confirm that P(ATA-C4)-r-GAL-I2 can quickly bind on the bacterial surface. P(ATA-C4)-r-GAL with green fluorescence was used to incubate with P. aeruginosa and S. aureus for 30 min. As shown in Figure S3 (Supporting Information), after being treated with P(ATA-C4)-r-GAL, all cells showed the green point, indicating that P(ATA-C4)-r-GAL have a highly accumulate in bacterial membrane, which implies that P(ATA-C4)-r-GAL could be a potential membrane-specific imaging material. The result confirmed that P(ATA-C4)-r-GAL-I2 can provide polyvalent interactions with bacteria and help to form tight bacteria-polymer agglomerates, then generated ROS under visible light irradiation to


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

kill the bacteria.

To eliminate bacteria, P(ATA-C4)-r-GAL-I2 must be in close proximity to the outer surfaces of bacterial cells, which allows the generated ROS to diffuse into the cytoplasmic membrane40 or to directly penetrate into the bacterial cells.41 Hence, the intracellular ROS levels were evaluated using fluorescence microscopy. As depicted in

Figure

S4

(Supporting

Information),

the

bacterial cells treated

with

P(ATA-C4)-r-GAL-I2 (10 min) displayed high-intensity green fluorescence compared to the control group, indicating that bacterial cells internalized with copolymers generate a large amount of ROS upon visible light irradiation. Here, the aggregation of bacteria was not observed due to insufficient incubation time.

To further explore whether P(ATA-C4)-r-GAL-I2 could interfere with bacterial cell membrane integrity, morphological changes in P. aeruginosa and S. aureus isolates were observed by SEM after being treated with P(ATA-C4)-r-GAL-I2. As shown in Figure 5B, untreated P. aeruginosa and S. aureus were rod- and round-shaped with smooth and intact cell wells. After treatment of P(ATA-C4)-r-GAL-I2 for 1 h, holes, widened lesions and amorphous membrane were observed, indicating that the bacteria were dead. This is possibly because P(ATA-C4)-r-GAL-I2 can anchor bacterial membrane through electrostatic interaction, and then generate ROS under visible light, irreversibly disrupt the membrane structure of the bacteria. The result confirmed that P(ATA-C4)-r-GAL-I2 could damage bacterial membrane and lead to the bacterial death.


Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 5. Fluorescence micrographs of P. aeruginosa and S. aureus stained with AO&EB before and after being treated with P(ATA-C4)-r-GAL-I2 (A); SEM images of P. aeruginosa and S. aureus before and after being treated with P(ATA-C4)-r-GAL-I2 (B).

β-Galactosidase

was

used

as

the

research

model

to

evaluate

whether

P(ATA-C4)-r-GAL-I2 can inhibit the activity of vital intracellular substances. β-Galactosidase catalyzes the enzymatic hydrolysis of ONPG in solution to generate ONP, which can be evaluated by UV-vis spectroscopy. In this work, β-galactosidase was obtained from E. coli suspension by ultrasound for 10 h in an ice-water bath and filtrated through a 0.22 µm cellulose membrane. As shown in Figure 6, the inhibitory activity of β-galactosidase was increased with increasing the concentration of


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

P(ATA-C4)-r-GAL-I2. When the concentrations of P(ATA-C4)-r-GAL-I2 were 125, 15.7 and 2 µg/mL, 88.1%, 54.6% and 7.9% inhibition of β-galactosidase was observed. This is possibly because P(ATA-C4)-r-GAL-I2 could generate ROS under visible light irradiation, and ROS can effectively inhibit the activity of β-galactosidase. The result confirmed that P(ATA-C4)-r-GAL-I2 can inhibit the activity of the intracellular enzyme, lead to the bacterial death.

Figure 6. Absorption of ONP treated with P(ATA-C4)-r-GAL-I2 at different concentrations.

Bacterial Resistance by P(ATA-C4)-r-GAL-I2 The potential bacterial resistance against P(ATA-C4)-r-GAL-I2 and levofloxacin was evaluated using P. aeruginosa and S. aureus as research model. After 30 batches, the MIC values of P(ATA-C4)-r-GAL-I2 against S. aureus and P. aeruginosa were similar to the original values, illustrating no emergence of bacterial resistance. In contrast, the MIC values of levofloxacin increased from 0.7 to 89.6 µg/mL against S.


Page 24 of 40

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

aureus and from 4.0 to 128 µg/mL against P. aeruginosa during the 30 passages, suggesting generation of bacterial resistance to the antibiotic. The result confirmed that P(ATA-C4)-r-GAL-I2 with double mechanism of antimicrobial action, as well as destruction of bacterial membrane and inhibition of enzymatic activity, which can efficiently suppress the emergence of bacterial resistance.

Selective Interaction between Mammalian Cells and Bacteria

To further evaluate the possibility for selective interaction of P(ATA-C4)-r-GAL-I2 with bacteria compared to mammalian cells, optical microscope was used to detect the morphology of A549 cells after the copolymer was cultured with mammalian cells for 1 h in the presence of bacteria. As shown in Figure 7A, after being treated with P(ATA-C4)-r-GAL-I2, few bacteria could be observed and all A549 cells displayed intact morphology. However, in the control group, a large amount of bacteria could be observed, and A549 cells were destroyed. As shown in Figure 7B, after being treated with P(ATA-C4)-r-GAL-I2, a lot of A549 cells were alive. In contrast, a small number of A549 cells were alive in the control group. As depicted in Figure 7C, after being treated with P(ATA-C4)-r-GAL-I2, bits of bacteria was existed while a large number of bacteria was alive in the control group. In a short period of time, P(ATA-C4)-r-GAL-I2 bond to bacteria by strong hydrophobic and electrostatic interactions, but did not adhere to the surface of the A549 cells. Hence, the selective interaction towards mammalian cells and bacteria can be applied to acquire the selective killing of bacteria over mammalian cells.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Images of A549 cells infected with bacteria (A), A549 cells number (B) and bacterial load (C) before and after treatment of P(ATA-C4)-r-GAL-I2 for 1 h. (The scale bar 20 µm)

Inhibition of Bacterial Biofilm Formation


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The capability of P(ATA-C4)-r-GAL-I2 to prevent the formation of bacterial biofilm was investigated via CV staining method. As displayed in Figure 8, the formation of bacterial biofilm was obviously inhibited in a concentration-dependent manner. The inhibited biofilm formation > 90% when the concentration of P(ATA-C4)-r-GAL-I2 was up to 22 µg/mL. The above study confirmed that P(ATA-C4)-r-GAL-I2 could efficiently kill the bacteria at a low concentration (5.5 µg/mL). Based on these findings, we can conclude that the inhibition of copolymer-mediated biofilm is owing to the inhibition of bacterial growth.

Figure 8. Inhibition of biofilm formation by P(ATA-C4)-r-GAL-I2.

Static Biofilm Assays To examine the capacity of P(ATA-C4)-r-GAL-I2 to eradicate mature biofilms. Four kinds of bacteria were used to evaluate the eradication degree of bacterial biofilm. As shown in Figure 9, P(ATA-C4)-r-GAL-I2 could eradicate over 70% of mature bacterial biofilms at the concentration of 22 µg/mL. Furthermore, the eradication


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

degree

of

biofilm

was

increased

with

Page 28 of 40

increasing

the

concentration

of

P(ATA-C4)-r-GAL-I2. Alginate is the major component of EPS in bacterial strains that can bind to antibacterial agents to prevent them from penetrating into the biofilm.42 Due to poor penetration of the EPS, conventional antibiotic therapies are ineffective against bacterial biofilm. A simple experiment was designed to investigate the binding capability between alginate and antibacterial agents. The alginate solutions were mixed with PBS and P(ATA-C4)-r-GAL-I2, respectively, and the average

diameters

of

each

samples

was

determined

using

DLS.

If

P(ATA-C4)-r-GAL-I2 was bound to the alginate, the average diameter would increase compared to the control group (PBS). The results displayed that the diameter of P(ATA-C4)-r-GAL-I2

was

similar

to

the

control

group,

indicated

that

P(ATA-C4)-r-GAL-I2 could obtain some level of shielding from binding to the alginate. Hence, P(ATA-C4)-r-GAL-I2 can penetrate into the bacterial biofilms and then release ROS under visible light irradiation to disrupt the biofilms matrix. The eradication degree of Gram-positive bacterial biofilms was higher than that of Gram-negative bacterial biofilms. This is possibly because Gram-positive bacteria are more

sensitive

to

P(ATA-C4)-r-GAL-I2

P(ATA-C4)-r-GAL-I2. could

effectively

The

eradicate

result both

Gram-negative bacterial biofilms.


confirmed Gram-positive

that and

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 9. The eradication degree of biofilms with P(ATA-C4)-r-GAL-I2 at different concentrations. CLSM analysis was also carried out to confirm whether P(ATA-C4)-r-GAL-I2 could effectively eradicate the bacterial biofilms. As displayed in Figure 10, after being treated with P(ATA-C4)-r-GAL-I2 for 24 h, the biofilms exhibited only a few isolated bacterial colonies instead of a recognizable biofilms structure. However, the biofilms treated with PBS showed a certain degree of preservation of the three-dimensional architecture and extracellular matrix. This phenomenon was consistent with previous report.3,43

Thus,

these

qualitative

findings

further

confirmed

P(ATA-C4)-r-GAL-I2 could effectively eradicate the bacterial biofilms.


that

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10. Composite 3-D micrographs of bacterial biofilms before and after being treated with P(ATA-C4)-r-GAL-I2.

Cytotoxicity Assay The cell viability is important features that should be determined for in vivo applications. The effect of P(ATA-C4)-r-GAL-I2 on cell viability was investigated using the MTT assay. As shown in Figure S5 (Supporting Information), after being treated with P(ATA-C4)-r-GAL-I2 for 24 h, even if the concentration was up to 4 mg/mL that exceeded 506-fold MIC levels against P. aeruginosa and S. aureus, the cells maintained a viability of 80%, indicating low toxicity to cells. The selective cytotoxicity of P(ATA-C4)-r-GAL-I2 may be ascribed to the difference in the surface membrane of bacteria and mammalian cells. Bacteria have more negative surface charges, which quickly absorb positively charged polymers.44 These results confirmed that P(ATA-C4)-r-GAL-I2 have good biocompatibility and can be used in vivo.

Conclusion


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

In this work, a water-soluble galactose-functionalized cationic photosensitizer based on BODIPY was developed for antibiofilm. This photosensitizer could selectively bind on the bacterial surface, produce a large amount of ROS, irreversible disrupt the bacterial membrane, effectively inhibit intracellular enzyme activity, ultimately leading to the bacterial death. These conjugates are highly selectivity toward bacterial cells over mammalian cells as well as no cytotoxicity to A549 cells and no discernible hemolytic activity. Importantly, the photosensitizer could effectively inhibit and eradicate over 70% of bacterial biofilm at very low concentration (22 µg/mL) without the emergence of bacterial resistance. The novel photosensitizer as an efficient antibacterial agent has a potential for application in bacterial infectious diseases.

ASSOCIATED CONTENT

Supporting Information Fluorescence emission spectra of P(ATA-C4)-r-GAL, the zone of inhibition of P. aeruginosa and S. aureus treated with P(ATA-C4)-r-GAL-I2, fluorescence micrographs of bacteria after treatment of P(ATA-C4)-r-GAL, the levels of the ROS generation based on fluorescence images before and after being treated with P(ATA-C4)-r-GAL-I2, and cell viability percentage after being treated with different dosages of P(ATA-C4)-r-GAL-I2 (PDF).

AUTHOR INFORMATION

ORCID


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Xinge Zhang: 0000-0003-3399-1659

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 21474055, 51673102 and 21774062).

REFERENCES [1] Joseph, R.; Naugolny, A.; Feldman, M.; Herzog, I. M.; Fridman, M.; Cohen, Y. Cationic Pillararenes Potently Inhibit Biofilm Formation without Affecting Bacterial Growth and Viability. J. Am. Chem. Soc. 2016, 138, 754-757.

[2] Vu, B.; Chen, M.; Crawford, R. J.; Ivanova, E. P. Bacterial Extracellular Polysaccharides Involved in Biofilm Formation. Molecules 2009, 14, 2535-2554.

[3] Du, J.; Bandara, H. M. H. N.; Du, P.; Huang, H.; Hoang, K.; Nguyen, D.; Mogarala, S. V.; Smyth, H. D. C. Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms. Mol. Pharmaceutics 2015, 12, 1544-1533.

[4] Stewart, P. S.; Costerton, J. W. Antibiotic Resistance of Bacteria in Biofilms. Lancet 2001, 358, 135-138.

[5] Konai, M. M.; Adhikary, U.; Samaddar, S.; Ghosh, C.; Haldar, J.


Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Structure-Activity Relationship of Amino Acid Tunable Lipidated Norspermidine Conjugates: Disrupting Biofilms with Potent Activity against Bacterial Persisters. Bioconjugate Chem. 2015, 26, 2442-2453.

[6] Kolodkin-Gal, I.; Romero, D.; Cao, S. J.; Clardy, J.; Kolter, R.; Losick, R. D-Amino Acid Trigger Biofilm Disassembly. Science 2010, 328, 627-629.

[7] Kolodkin-Gal, I.; Cao, S. G.; Chai, L.; Böttcher, T.; Kolter, R.; Clardy, J.; Losick, R. A Self-Produced Trigger for Biofilm Disassembly That Targets Exopolysaccharide. Cell 2012, 149, 684-692.

[8] Petrova, O. E.; Cherny, K. E.; Sauer, K. The Diguanylate Cyclase GcbA Facilitate Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA. J. Bacteriol. 2015, 197, 174-187.

[9] Sanchez, C. J. J.; Prieto, E. M.; Krueger, C. A.; Zienkiewicz, K. J.; Romano, D. R.; Ward, C. L.; Akers, K. S.; Guelcher, S. A.; Wenke, J. C. Effects of Local Delivery of D-Amino Acids from Biofilm-Dispersive Scaffolds on Infection in Contaminated Rat Segmental Defects. Biomaterials 2013, 34, 7533-7543.

[10] Zhou, J. Y.; Horev, B.; Hwang, G.; Klein, M. I.; Koo, H.; Benoit, D. S. W. Characterization and Optimization of pH-Responsive Polymer Nanoparticles for Drug Delivery to Oral Biofilms. J. Mater. Chem. B 2016, 4, 3075-3085.

[11] Liu, Y.; Busscher, H. J.; Zhao, B.; Li, Y.; Zhang, Z.; Mei, H. C.; Ren, Y.; Shi, L. Surface-Adaptive, Antimicrobially Loaded, Micellar Nanocarriers with Enhanced


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Penetration and Killing Efficiency in Staphylococcal Biofilms. ACS Nano 2016, 10, 4779-4789.

[12] Horev, B.; Klein, M. I.; Hwang, G.; Li, Y.; Kim, D.; Koo, H.; Benoit, D. S. W. pH-Activated Nanoparticles for Controlled Topical Delivery of Farnesol to Disrupt Oral Biofilm Virulence. ACS Nano 2015, 9, 2390-2404.

[13] Ballatore, M. B.; Durantini, J.; Gsponer, N. S.; Suarez, M. B.; Gervaldo, M.; Otero, L.; Spesia, M. B.; Milanesio, M. E.; Durantini, E. N. Photodynamic Inactivation of Bacteria Using Novel Electrogenerated Porphyrin-Fullerene C60 Polymeric Films. Environ. Sci. Technol. 2015, 49, 7456-7463.

[14] Shibua, E. S.; Hamadaa, M.; Murasea, N.; Biju, V. Nanomaterials Formulations for Photothermal and Photodynamic Therapy of Cancer. J. Photoch. Photobio. C: Photoch Rev. 2013, 15, 53-72.

[15] Huang, Y.; Pappas, H. C.; Zhang, L.; Wang, S.; Cai, R.; Tan, W.; Wang, S.; Whitten, D. G.; Schanze, K. S. Selective Imaging and Inactivation of Bacteria over Mammalian Cells by Imidazolium-Substituted Polythiophene. Chem. Mater. 2017, 29, 6389-6395.

[16] Sunehag, A. L.; Louie, K.; Bier, J. L.; Tigas, S.; Haymond, M. W. Hexoneogenesis in the Human Breast during Lactation. J. Clin. Endocr. Metab. 2002, 87, 297-301.

[17] Joachim, I.; Rikker, S.; Hauck, D.; Ponader, D.; Boden, S.; Sommer, R.;


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Hartmann, L.; Titz, A. Development and Optimization of a Competitive Binding Assay for the Galactophilic Low Affinity Lectin LecA from Pseudomonas aeruginosa. Org. Biomol. Chem. 2016, 14, 7933-7948.

[18] Kamkaew, A.; Burgess, K. Double-Targeting Using a TrkC Ligand Conjugated to Dipyrrometheneboron Difluoride (BODIPY) Based Photodynamic Therapy (PDT) Agent. J. Med. Chem. 2013, 56, 7608-7614.

[19] Sun, T.; Guan, X.; Zheng, M.; Jing, X.; Xie, Z. Mitochondria-Localized Fluorescent BODIPY-Platinum Conjugate. ACS Med. Chem. Lett. 2015, 6, 430-433.

[20] Kashyap, S. J.; Garg, V. K.; Sharma, P. K.; Kumar, N.; Dudhe, R.; Gupta, J. K. Thiazoles: Having Diverse Biological Activities. Med. Chem. Res. 2012, 21, 2123-2132.

[21] Lu, Z.; Mei, L.; Zhang, X.; Wang, Y.; Zhao, Y.; Li, C. Water-Soluble BODIPY-Conjugated Glycopolymers as Fluorescent Probes for Live Cell Imaging. Polym. Chem. 2013, 4, 5743-5750.

[22] An, J.; Dai, X.; Wu, Z.; Zhao, Y.; Lu, Z.; Guo, Q.; Zhang, X.; Li, C. An Acid-Triggered Degradable and Fluorescent Nanoscale Drug Delivery System with Enhanced Cytotoxicity to Cancer Cells. Biomacromolecules 2015, 16, 2444-2454.

[23] Kochevar, I. E.; Redmond, R. W. Photosensitized Production of Singlet Oxygen. Methods Enzymol. 2000, 391, 20.

[24] Locock, K. E. S.; Michl, T. D.; Valentin, J. D. P.; Vasilev, K.; Hayball, J. D.; Qu,


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Y.; Traven, A.; Griesser, H. J.; Meagher, L.; Haeussler, M. Guanylated Polymethacrylates: A Class of Potent Antimicrobial Polymers with Low Hemolytic Activity. Biomacromolecules 2013, 14, 4021-4031.

[25] Niu, Y.; Padhee, S.; Wu, H.; Bai, G.; Qiao, Q.; Hu, Y.; Harrington, L.; Burda, W. N.; Shaw, L. N.; Cao, C.; Cai, J. Lipo-γ-AApeptides as a New Class of Potent and Broad-Spectrum Antimicrobial Agents. J. Med. Chem. 2012, 55, 4003-4009.

[26] Liu, Z.; Liu, X.; Du, Y.; Ren, J.; Qu, X. Using Plasmonic Copper Sulfide Nanocrystals as Smart Light-Driven Sterilants. ACS Nano 2015, 9, 10335-10346.

[27] Zhao, Y.; Tian, Y.; Cui, Y.; Liu, W.; Ma, W.; Jiang, X. Small Molecule-Capped Gold Nanoparticles as Potent Antibacterial Agents That Target Gram-Negative Bacteria. J. Am. Chem. Soc. 2010, 132, 12349-12356.

[28] Chopra, I. The Increasing Use of Silver-Based Products as Antimicrobial Agents: a Useful Development or a Cause for Concern? J. Antimicrob. Chemother. 2007, 59, 587-590.

[29] Loo, C.-Y.; Rohanizadeh, R.; Young, P. M.; Traini, D.; Cavaliere, R.; Whitchurch, C. B.; Lee, W.-H. Combination of Silver Nanoparticles and Curcumin Nanoparticles for Enhanced Anti-biofilm Activities. J. Agric. Food. Chem. 2016, 64, 2513-2522.

[30] O’Toole, G. A.; Kolter, R. Initiation of Biofilm Formation in Pseudomonas fluorescens WCS365 Proceeds via Multiple, Convergent Signalling Pathways: a Genetic Analysis. Mol. Microbiol. 1998, 28, 449-461.


Page 36 of 40

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

[31] O’Toole, G. A.; Kolter, R. Flagellar and Twitching Motility Are Necessary for Pseudomonas fluorescens Biofilm Development. Mol. Microbiol. 1998, 30, 295-304.

[32] Geske, G. D.; Wezeman, R. J.; Siegel, A. P.; Blackwell, H. E. Small Molecule Inhibitors of Bacterial Quorum Sensing and Biofilm Formation. J. Am. Chem. Soc. 2005, 127, 12762-12763.

[33] Bandara, H. M.; Yau, J. Y.; Watt, R. M.; Jin, L. J.; Samaranayake, L. P. Pseudomonas aeruginosa Inhibits in-vitro Candida Biofilm Development. BMC Microbiol. 2010, 10, 125.

[34] Richards, S.-J.; Jones, M. W.; Hunaban, M.; Haddleton, D. M.; Gibson, M. I. Probing Bacterial-Toxin Inhibition with Synthetic Glycopolymers Prepared by Tandem Post-Polymerization Modification: Role of Linker Length and Carbohydrate Density. Angew. Chem. Int. Ed. 2012, 51, 7812-7816.

[35] Novoa, A.; Eierhoff, T.; Topin, J.; Varrot, A.; Barluenga, S.; Imberty, A.; Römer, W.; Winssinger, N.; A LecA Ligand Identified from a Galactoside-Conjugate Array Inhibits Host Cell Invasion by Pseudomonas Aeruginosa. Angew. Chem. Int. Ed. 2014, 53, 8885-8889.

[36] Yogo, T.; Urano, Y.; Ishitsuka, Y.; Maniwa, F.; Nagano, T. Highly Efficient and Photostable Photosensitizer Based on BODIPY Chromophore. J. Am. Chem. Soc. 2005, 127, 12162-12163.

[37] Takahashi, H.; Nadres, E. T.; Kuroda, K. Cationic Amphiphilic Polymers with


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Antimicrobial Activity for Oral Care Applications: Eradication of S. mutans Biofilm. Biomacromolecules 2017, 18, 257-265.

[38] Nimmagadda, A.; Liu, X.; Teng, P.; Su, M.; Li, Y.; Qiao, Q.; Khadka, N. K.; Sun, X.; Pan, J.; Xu, H.; Li, Q.; Cai, J. Polycarbonates with Potent and Selective Antimicrobial Activity toward Gram-Positive Bacteria. Biomacromolecules 2017, 18, 87-95.

[39] Thoma, L. M.; Boles, B. R.; Kuroda, K. Cationic Methacrylate Polymers as Topical Antimicrobial Agents against Staphylococcus aureus Nasal Colonization. Biomacromolecules 2014, 15, 2933-2943.

[40] Hamblin, M. R.; O’Donnell, D. A.; Murthy, N.; Rajagopalan, K.; Michaud, N.; Sherwood, M. E.; Hasan, T. Polycationic Photosensitizer Conjugates: Effects of Chain Length and Gram Classification on the Photodynamic Inactivation of Bacteria. J. Antimicrob. Chemother. 2002, 49, 941-951.

[41] Hamblin, M. R.; Hasan, T. Photodynamic Therapy: a New Antimicrobial Approach to Infectious Disease? Photochem. Photobiol. Sci. 2004, 3, 436-450.

[42] Toyofuku, M.; Roschitzki, B.; Riedel, K.; Eberl, L. Identification of Proteins Associated with the Pseudomonas aeruginosa Biofilm Extracellular Matrix. J. Proteome Res. 2012, 11, 4906-4915.

[43] Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial Biofilms: from the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2, 95-108.


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

[44] Matsuzaki, K. Control of Cell Selectivity of Antimicrobial Peptides. Biophys. Acta Biomembr. 2009, 1788, 1687-1692.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only


Page 40 of 40

A Water-Soluble Galactose-Decorated Cationic Photodynamic

Recommend Documents