Amino-Acid Conjugated Polymers: Antibacterial Agents Effective

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Biological and Medical Applications of Materials and Interfaces

Amino-Acid Conjugated Polymers: Antibacterial Agents Effective against Drug-resistant A. baumannii with no Detectable Resistance Swagatam Barman, Mohini Mohan Konai, Sandip Samaddar, and Jayanta Haldar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09016 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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ACS Applied Materials & Interfaces

Amino-Acid Conjugated Polymers: Antibacterial Agents Effective against Drug-resistant A. baumannii with no Detectable Resistance Swagatam Barman, Mohini Mohan Konai, Sandip Samaddar and Jayanta Haldar*

Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India. Address correspondence to Jayanta Haldar, [email protected]

ABSTRACT An optimum hydrophilic/hydrophobic balance has been recognised as a crucial parameter in designing cationic polymers that mimic antimicrobial peptides (AMPs). Till date, this balance was achieved either by hydrophilicity variation through altering the nature and the number of cationic charges or by hydrophobicity modulation through incorporation of alkyl group of different chain lengths. However, how does the hydrophobicity variation through AMP’s building block, amino acids influence antibacterial efficacy of AMP mimicking cationic polymers has rarely been explored. Towards this goal, herein we report a class of amino-acid conjugated polymers (ACPs) with tunable antibacterial activity through a simple post polymer functionalization strategy. Our polymeric design comprised a permanent cationic charge in every repeating unit, whereas the hydrophobicity was tuned through incorporation of different amino acids. Our results revealed that the amino acid alteration has a strong influence on antibacterial 1 ACS Paragon Plus Environment

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efficacy. Upon increasing the amino acid side chain hydrophobicity, both the antibacterial activity (against broad spectrum of bacteria) as well as toxicity increases. However, the distinct feature of this class of polymers was their good activity against Acinetobacter baumannii (topmost critical pathogen according to WHO) which has created an alarming situation worldwide causing majority of the infections in human. The non-toxic (no hemolysis even at 1000 µg/mL) ACP comprising of glycine residue (ACP-1 (Gly)) showed very good activity (MIC = 8-16 µg/mL) against both drug-sensitive and drug-resistant strains of A. baumannii including clinical isolates. This polymer was not only rapidly bactericidal against growing planktonic A. baumannii, but also killed non-dividing stationary phase cells instantaneously (< 2 min). Moreover, it eradicated the established biofilm formed by drug-resistant A. baumannii clinical isolates. No propensity of bacterial resistance development was seen against this polymer even after 14 continuous passages. Taken together, the results highlighted the role of hydrophobicity modulation through incorporation of amino acids in cationic polymers will provide a significant insight in designing new Amino-acid Conjugated Polymers (ACPs) with potent antibacterial activity and minimum toxicity towards mammalian cells. More importantly, the excellent anti-A. baumannii efficacy of the optimised lead polymer indicates its immense potential for being developed as therapeutic agent. KEYWORDS. Antibiotic resistance, Biofilms, AMP mimics, Antibacterial polymers and AntiA. baumannii activity. INTRODUCTION Rapid emergence of drug-resistant bacteria and their biofilm forming capability have created an enormous threat towards human health worldwide.1-4 Recently, the World Health Organization (WHO) has published a list of priority pathogens, where Gram-negative bacteria (Acinetobacter 2 ACS Paragon Plus Environment

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baumannii topped the list) are considered as the most critical ones causing majority of infections which are difficult-to-treat with conventional antibiotics.1,

5, 6

Hence, the current situation

demands an urgent development of new class of antibacterial agents with a novel mechanism of action. A plethora of membrane-active polymers have been developed along with small molecular agents offering some hope to tackle this crisis.7-26 Design of this class of molecules was inspired by naturally occurring antimicrobial peptides (AMPs), which serve as the first line of defence as a component of innate immune system.27,28 The peptide sequence of AMPs is primarily enriched with hydrophobic and cationic amino acid residues.27,29 The presence of cationic charges ensure the selective interaction with negatively charged bacterial membrane over zwitterionic mammalian membrane and hydrophobicity is also imperative to achieve high selectivity towards bacterial cells.27 Thus, an optimum amphiphilicity (hydrophilic/hydrophobic balance) has been identified as an important parameter in designing AMP-mimicking antimicrobial polymers.7Till date, various classes of antibacterial polymers such as polynorbornenes,8

polymethacrylates,9,10

poly-β-lactams,11,12

polycarbonates,13-15

peptidopolysaccharides,16 polymalemide,17-18 pyridinium co-polymers,20 and many more30-38 have been developed to address the limitations associated with naturally occurring AMPs, which include mainly synthetic complexity, high manufacturing cost, toxicity and stability. So far, the optimum hydrophilic/hydrophobic balance has been achieved either by varying the number and nature of cationic charges or by alteration of alkyl chain hydrophobicity in the design. However, the hydrophobicity variation could be achieved easily through incorporation of various amino acids (building block of AMPs) in the side chain of AMP-mimicking cationic polymers. It is noteworthy that the amino acid-based peptidomimetic small molecular designs are well explored in literature, where the role of amino acids are distinctly recognized in regulating selective

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antibacterial activity.21,39 However, the effect of amino acid incorporation in AMP-mimicking polymeric design has rarely been reported. In one report, a class of amino acid (such as Ala, Leu and Phe) bearing polymers was synthesized to understand the morphological switching of bacterial cell envelope.40

However in order to understand the role of hydrophobicity in

regulating antibacterial activity through amino acid variation, herein we report a new class of amino-acid conjugated polymers (ACPs) with potent antibacterial activity. Amino acids with different side chain hydrophobicity (such as Gly, L-Ala, L-Val, L-Leu, L-Ile, L-Phe and L-Tyr) including isomeric variation (D-Ala) as well as functionality (such as side chain methyl ester derivatives of L-Asp and L-Glu) were introduced in our polymeric design. An additional interest of developing amino acid based antibacterial polymers was due to the high abundance and biocompatible nature of amino acids.19 A permanent cationic charge (contributed by quaternary ammonium moiety) was introduced in the polymer design to achieve selective interaction towards negatively charged bacterial membrane over zwitterionic mammalian membrane alike AMPs which attain such interaction through positively charged amino acids. By keeping the number of charges constant, the hydrophilic/hydrophobic balance of this class of polymers was modulated through amino acids with different hydrophobicity. By employing post functional modification of a commercially available precursor polymer, a series of amino acid tunable polymers was prepared through simple synthetic steps and detailed characterization was performed by using 2D proton-carbon correlation HSQC (DEPT edited) NMR spectroscopy. Initially, the antibacterial activity of these newly synthesized polymers was evaluated against various drug-sensitive, drug-resistant bacteria and toxicity was determined against human erythrocytes. A thorough structure-activity-relationship (SAR) was then carried out by varying the amino acid residues keeping methyl group constant in the ester functionality. The most


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interesting feature of this class of amino acid bearing polymers was their potent antibacterial efficacy against drug-resistant A. baumannii, which is considered as the topmost priority pathogen. This result therefore motivated us to further investigate the anti-A. baumannii efficacy in details. Towards this direction, bactericidal kinetics of the optimized polymer was evaluated against both metabolically active (growing planktonic) and inactive (stationary phase)33 cells of drug-sensitive as well as drug-resistant A. baumannii. The mode of membrane activity was also investigated against these metabolically distinct bacterial cells. Furthermore, anti-biofilm efficacy and propensity of bacterial resistance development were evaluated for the optimized antibacterial polymer. EXPERIMENTAL SECTION Materials and Instrumentations. Dichloromethane (DCM), methanol (MeOH), ethanol (EtOH), thionyl chloride were obtained from Spectrochem (India) and all the solvents were of reagent grade and dried prior to use wherever required. Poly(isobutylene-alt-maleic anhydride) (Mw ~ 6000 Da, PDI ~1.2, catalogue no. 531278),44 bromoacetyl bromide ( catalogue no. B56412-500G), N, Nˈ-dimethyl-1, 3-propanediamine (catalogue no. D145009-25ML), N-phenyl naphthylamine (catalogue no. 104043-500G), propidium iodide (catalogue no. 11348639001), 3, 3′-dipropylthiadicarbocyanine iodide [DiSC3 (5)] (catalogue no. 43608-100MG) were procured from Sigma-Aldrich. L/D- Amino acids (Glycine, L-Alanine, D-Alanine, L-Valine, L-Leucine, L-Isoleucine, L-Phenylalanine, L-Tyrosine, L-Aspartic acid, and L-Glutamic acid) were obtained from Spectrochem (India). These chemicals were used for reaction directly. Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 precoated E. Merck TLC plates and 5 ACS Paragon Plus Environment


visualized by using iodine or ninhydrin. 800 MHz Bruker AscendTM and Bruker AMX-400 spectrometer were used to record nuclear magnetic resonance (NMR) spectra in deuterated solvents. 6538-UHD Accurate Mass Q-TOF LC-MS instrument was used to record mass spectra. PerkinElmer LS-55 luminescence spectrometer was used to perform static light scattering (SLS) measurements. Tecan Infinite M200 PRO Microplate Reader was used for optical density (O.D.) measurement. Bacterial strains, S. aureus MTCC737, E. coli MTCC443 and A. baumannii MTCC1425 were procured from MTCC (Chandigarh, India). A. baumannii ATCC19606 & A. baumannii BAA1605 were obtained from Anthem Bioscience (Bangalore, India). A. baumannii R674 & A. baumannii R676 were collected from NIMHANS hospital (Bangalore, India). MRSA ATCC33591, E. faecium ATCC19634, and K. pneumoniae ATCC700603 were purchased from ATCC (Rockville, MD, USA). S. aureus, E. coli, MRSA, K. pneumoniae and A. baumannii were grown in Mueller Hinton Broth (MHB-HIMEDIA-M391). Brain heart infusion broth (BHI) was to inoculate E. faecium. MacConkey Agar and Nutrient agar were used as a solid media for Gram negative and gram positive bacteria. 96 well plates, 6 well plates and transparent black 96 well plates were obtained from Vasa Scientific (Bangalore, India). General Synthesis Procedure of Alkyl Ester Bromide of Amino Acids (1a-10a). To a suspension of individual amino acid (Gly, L-Ala, D-Ala, L-Val, L-Leu, L-Ile, L-Phe, L-Tyr, LAsp and L-Glu; (0.5 g, 6.7 mmol for Gly and 0.5-1g scale for other amino acids) in 15-20 mL methanol, thionyl chloride (1.46 mL, 20.2 mmol) was added drop wise at 5 °C. The reaction was refluxed for 12h with stirring. The solvent and excess thionyl chloride were removed by using rotary evaporator. The solid residue was washed with dry diethyl ether and dried to get the crude product as white solid. This crude white solid was dissolved in 10 mL of dichloromethane and potassium carbonate (2.3 g, 16.65 mmol) was added to the organic solution after dissolving it in 6 ACS Paragon Plus Environment

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10 mL of distilled water. A solution of bromoacetyl bromide (0.87 mL, 10 mmol) in dichloromethane (10 mL) was then added dropwise to the reaction mixture at 5 °C for 1h. The reaction mixture was stirred at room temperature for another 12h. The aqueous solution was separated and washed with dichloromethane. The organic solution was washed with water and passed over the anhydrous Na2SO4 and concentrated to yield a white or yellowish white solid product with 60-95% yield. The detail characterizations of 1a-10a are provided in Supporting Information. Synthesis of poly(isobutylene-alt-N-(N', N'-dimethylaminopropyl)-maleimide) (1b). This intermediate polymer was synthesized by following our previously published protocol.18 In brief, to a solution of poly(isobutylene-alt-maleic anhydride) (5 g, Avg. Mw = 6000 g/mol, PDI ~1.2) 44

in DMF, N,N-Dimethyl-1,3-propanediamine (4.9 mL, 40 mmol considering the monomeric

weight of the polymer (154 g/mol)) was added and stirred at 120 °C for 48h in a screw-top pressure tube. The reaction mixture was precipitated with 100 mL of distilled water and was centrifuged for 15 min at 10,000 rpm. Lastly, polymer was dried at 45 °C for 24h under vacuum to get a pale yellow solid with quantitative yield. The characterizations of 1b are provided in Supporting Information. General Synthesis Procedure of Amino-Acid Conjugated Polymers (ACPs 1-10). Intermediate 1a-10a (0.5 g, 2 equivalent according to the monomeric weight of one repeating unit of 1b (238 g/mol) were reacted individually with poly (isobutylene-alt-N-(N', N'dimethylaminopropyl)-maleimide) (0.4 g, 1 equivalent) in dry chloroform in a screw-top pressure tube at 65 °C. At the end of 96h, solvent was evaporated by using rotary-evaporator and the residue was dissolved in minimum amount of CHCl3. The product was precipitated by adding excess dry diethyl ether and the white residue was washed repeatedly with dry diethyl ether to 7 ACS Paragon Plus Environment


achieve cationic polymers with quantitative yields. Finally, all the polymers (ACPs 1-10) were characterized by 1H-NMR,

13C-NMR, 1H-13C

HSQC (DEPT edited) and FT-IR (Supporting

Figure S1−S30) Nuclear Magnetic Resonance Studies. 1H and 2D HSQC (DEPT edited) NMR spectra were recorded using 800 MHz Bruker AscendTM and Bruker AMX-400 spectrometer. The pulse program for proton and carbon NMR spectroscopy were zg30 and zgpg30 respectively. 2D HSQC spectra were recorded using “hsqcedetgp” pulse program which is linked with DEPT 135 pulse sequence. CDCl3 and D2O were used for the NMR experiments for all the intermediates and final polymers respectively. The residual solvent peaks were calibrated at δ 7.26 ppm and 4.79 ppm for CDCl3 and D2O respectively. Trimethylsilyl propionate (TSP) was used as an external reference in 3mm tube for peak was calibrated at δ 0.0 ppm..

13C

and HSQC NMR experiments and the residual solvent

13C-NMR

spectra of ACP-6 (Phe) and ACP-7 (Tyr) were

recorded using DMSO-d6 where residual solvent peak was calibrated at 39.5ppm. Bruker TOPSPIN 3.0 software were used to process all the NMR data. Hydrolytic Stability Study. In brief, 4 mg/mL solution of ACP-1 (Gly) was prepared in D2O and 1H-NMR were recorded every day for 7 days.. This measurement was executed at room temperature. The 1H-NMR data were analysed by using TOPSPIN 3.0 software. Critical Aggregation Concentration (CAC) Measurement. This study was performed through static light scattering (SLS) on a photoluminescence spectrometer. Briefly, 256 µg/mL aqueous solution of the polymers (ACP 1-9) were prepared and scattering intensity of these polymer solution (2 mL) was measured upon two fold serial dilution upto 2 µg/mL. The scattered light intensity was measured at 90 ˚ angle by fixing the excitation and emission wavelength at 400 nm.


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The excitation and emission slit widths were kept constant at 10 nm and 2.5 nm, respectively throughout the experiment. The scattered intensity was plotted against the concentration of the polymers. In general, CAC value is determined form an inflection point (abscissa where the intensity rises steeply and decreases after reaching a local maximum). However, in this case no inflection point were found for any of the polymers. This observation indicated that none of the polymer form aggregate even at 256 µg/mL. Antibacterial Assay.18,21 The antibacterial assay was executed by following our previously reported protocol. Briefly, at first bacteria from the frozen stock (at -80°C) were streaked either on nutrient broth (for Gram-positive bacteria) or Macconky agar plate (for Gram-negative bacteria). The stricked plates were then incubated overnight at 37°C for bacterial growth. A single bacterial colony was next inoculated for 6h (midlog phase) in 3 mL of nutrient broth to produce about 108 to 109 CFU/mL cells depending upon the nature of the bacteria. The 6 h grown culture was diluted to ~105 CFU/mL which was then used for antibacterial assay determination. Compound were 2-fold serially diluted in a 96-well plate from the starting concentration using sterile millipore water. Afterwards, 150 μL of ~105 CFU/mL bacterial solution were added in each wells containing 50 µL aqueous solution of test compound. The plates were then incubated for 16-18 h at 37°C in shaking condition. The O.D at 600 nm was recorded by using TECAN (Infinite series, M200 PRO) plate reader. Each concentration were triplicate and the experiment was performed at least twice and the antibacterial activity (MIC) was evaluated based on visual turbidity. Hemolytic Assay.18,21,22 Briefly, aqueous solution of individual polymers (ACPs 1−9) were serially diluted by two fold in triplicate in a 96-well plate. Freshly collected human blood (heparinized) was then centrifuged down and supernatant was thrown away to collect the human 9 ACS Paragon Plus Environment


red blood cells (hRBCs). Later, collected hRBCs (5 vol %) was slowly suspended using 1 ×PBS (pH = 7.4). Next, 150μL of this suspension was added to the wells of 96 well plate containing 50 μL compound’s solution and plate was allowed to incubate at 37°C for 1 h. Then centrifugation at 3500 rpm for 5 min was performed and the supernatant (100 μL) was then transferred to another 96-well plate for recording the absorbance at 540 nm by using Tecan Infinite M200 PRO microplate reader. In this study, same volume of 1 x PBS without compound served as a negative control whereas same volume of Triton X-100 (1 vol% solution in 1X PBS) was used as a positive control. The percentage of hemolysis was determined by using the following formula: (Atret –Anontret) /(ATX ‑ tret –Anontret ) ×100, where Atret corresponds to the absorbance of the compound-treated well, Anontret stands for the absorbance of the negative controls (without compound), and ATX ‑ tret is the absorbance of the Triton X-100 treated well. Each concentration had triplicate values and the HC50 was determined by considering the average of triplicate O.D. Kinetics of Bacterial Killing.21 Against Planktonic Cell. Briefly, a single colony of A. baumannii was inoculated in nutrient broth for 6 h at 37 °C to produce 108 to 109 CFU/mL cells. Next, this mid log phase bacterial solution was further diluted to ~5× 105 CFU/mL and 150 µL of this diluted bacterial solution in Mueller Hinton Broth was added to the 50 µL aqueous solution of test polymer, ACP-1 (Gly) with the concentration of about 16 µg/mL and 32 µg/mL. Same volume of autoclaved water without test compound was used as a control. Afterwards, 20 μL of aliquots from the individual mixture of bacteria and compound were serially diluted by 10-fold in sterile saline at different time points (0 min , 60 min, 120 min, 240 min, and 360 min). Then, spot plating on MacConkey agar plates was executed with 20 μL solution from each dilution and allowed to incubate for 24h


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at 37°C. Finally, the number of bacterial colonies were counted and results were presented in logarithmic scale, i.e., Log (CFU/mL) vs time. Against Stationary Cell. A mid-log phase (6h grown culture) A. baumannii culture was diluted to 1:1000 ratio in nutrient broth and incubated at 37°C for 16h in shaking condition to achieve stationary phase cells. Later on, the bacterial suspension was centrifuged (9000 rpm, 2 min) and resuspended in 1X PBS (pH = 7.4). Then 150 μL of the stationary phase bacteria (~5× 105 CFU/mL) was added to 50 μL of ACP-1 (Gly) solution with the concentrations of 8 μg/mL and 16 μg/mL. Similarly for meropenem antibiotic concentration was 64 µg/mL and same volume of water without compound was considered as an untreated control. At different time intervals (< 2 min, 5 min, 15 min & 30 min) 20 μL aliquots from that solution were serially diluted 10-fold in sterile saline. Then 20 μL solution from each dilutions was spot plated on MacConkey agar plates and after 24 h of incubation at 370C, the number of bacterial colonies were counted. The results were presented in a bar plot in logarithmic scale, i.e. log10 (CFU/mL) at different time points. Cytotoxicity Assay.21,41 LDH assay. CytoTox 96 Non-Radioactive (Promega) kit had been used to determine the cytotoxicity of the optimized compound, ACP-1 (Gly) against human embryo kidney (HEK 293) cell line through LDH assay. Concisely, the cells were seeded in a 96-well plate at a concentration of ~104 cells per well using DMEM media, supplemented with 5% penicillinstreptomycin and 10% fetal bovine serum. The cells of each well were then incubated with 100 μL of serially diluted polymer in DMEM media. Same volume of media without compound (untreated cells) and 0.1 vol % Triton-X solution were considered as negative and positive control respectively. Finally, the plate was allowed to incubate at 37 °C for 24 h by maintaining



5% CO2 atmosphere at 95% relative humidity and then centrifugation was done at 1100 rpm for 5 min. After transferring supernatants from respective wells to a fresh 96-well plate, the cytotoxicity assay was executed by following the manufacturer’s protocol. To the end, absorbance at 490 nm was measured with Tecan Infinite M200 PRO microplate reader. Cell viability was determined by following this formula, [1-{(A-A0)/ (Atotal -A0)}] ×100, where A corresponds to the absorbance of the test well, A0 stands for the absorbance of the untreated wells (negative controls), and Atotal is the absorbance of the positive control (Triton-X treated wells). At the end, EC50 value (compound’s concentration corresponding to 50% LDH release relative to the positive control) was determined from the plot of percentage of cell survival vs concentration of the test compound. Fluorescence Microscopy. In brief, ∼104 cells (HEK 293) were seeded into the individual wells of a 96-well plate. Then 100 µL of 250 μg/mL and 500 μg/mL of ACP-1 (Gly) was added over the seeded cells. 0.1% Triton X treated and untreated cells were considered as positive and negative controls respectively. After single time washing with 1X PBS the untreated and treated cells were then stained with 50 μL of 1:1 calcein AM (2 μM) and propidium iodide (PI) (4.5 μM) for 15 min under 5% CO2 atmosphere at 37°C. Finally, the excess dyes were removed by washing the cells with 1X PBS, and images were captured at 40×objective with the help of a Leica DM2500 fluorescence microscope. During imaging a band-pass filter for calcein-AM (at 500−550 nm) and a long-pass filter for PI (at 590−800 nm) were used. Biofilm Disruption Assay.21, 41 Crystal violet staining. Biofilm disruption study was conducted over cover slips of diameter 13 mm. First, 6h grown culture of A. baumannii (mid-log phase) was diluted to 105 CFU/mL in BM2 media (0.07 M (NH4)2SO4 and potassium phosphate buffer (0.4 M K2HPO4 and 0.2 M


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KH2PO4) (pH =7), 0.5% glucose with the supplements of 200 mM FeCl3 and 0.5% casamino acids). Then this diluted bacterial suspension (2 mL/well) was added to wells containing the sterilized cover slips and the plate was allowed to incubate at static condition at 300 C for 48 h. Then, after removal of media, biofilm containing cover slips were cautiously washed with 1X PBS (pH = 7.4) to remove the planktonic bacteria and cover slips were then placed into the well of a new 6-well plate. Afterward, two solutions (2 mL) of ACP-1 (Gly) with two different concentrations (64 µg/mL and 128 µg/mL) were added to the wells containing biofilm coated coverslip incubated for 24 h. As an untreated control, 2 mL of fresh media without compound was added to the well. For this study, meropenem and colistin at 64 µg/mL were used as the antibiotic control. After 24 h, 1X PBS was used to wash the planktonic cells from the coverslips. Later on, all the compound treated and untreated cover slips were carefully positioned into another 6-well plate. To visualize biofilm disruption, those coverslips were incubated with 1 mL of 0.1% of crystal violet (CV) dye in the 6-well plate and allowed to incubate for 10 min. After washing with 1X PBS, the crystal violet associated with the biofilm containing coverslip was dissolved in 95% ethanol and absorbance was recorded at 520 nm. The amount of biomass left on the cover slips was indicated by the absorbance of CV dye. Confocal Laser-Scanning Microscope (CLSM) of Biofilms. Like the previous studies, the optimized polymer, ACP-1 (Gly) had been used for this experiment. The treated and untreated cover slips (previously described in biofilm disruption assay section) were placed on glass slides after washing with 1X PBS. The biofilms staining were performed with 10 mL of SYTO9 (3 mM) and images were captured with the help of a Zeiss 510 Meta confocal laser-scanning microscope. LSM 5 Image examiner was used to process the images.



Membrane Active Mechanism of Action.18,21,41 Cytoplasmic Outer Membrane Permeabilization assay. Mid log phase and stationary phase A. baumannii cells were independently harvested, washed with 1:1 mixture of 5 mM HEPES buffer and glucose and resuspended with the same. The working concentration of mid log phase and stationary phase bacteria were ~108 CFU/mL. This study was performed in a Corning 96 black well plate with clear bottom containing 10 μM of N-Phenyl naphthylamine (NPN) dye and 190 μL of bacterial suspension. Then, the fluorescence was monitored for first 4 min at excitation wavelength of 350 nm and emission wavelength of 420 nm. After that, bacterial suspension with dye at each well were treated with 10 μL of test compound, ACP-1 (Gly) at working concentration of 5 μg/mL, 10 μg/mL and 20 μg/mL. Same volume of water without compound was used as the control for this experiment. Increase in fluorescence intensity was monitored for another 25 min with Tecan Infinite M200 PRO microplate reader. Cytoplasmic Membrane Depolarization Assay. Similar to the membrane permeabilization assay, mid log phase (working concentration: ~108 CFU/mL) and stationary phase (working concentration: ~108 CFU/mL) of A. baumannii cells were collected separately (centrifugation at 3500 rpm for 5 min), washed with 1:1 ratio of 5 mM glucose and HEPES buffer (pH = 7.4). Next, the bacterial plate was resuspended in 1:1:1 ratio of 5 mM HEPES buffer, 100 mM KCl solution supplemented with 0.2 mM EDTA and 5 mM glucose. For this study EDTA was used to allow the dye uptake by permeabilizing outer membrane of A. baumannii. This study was performed in a Corning 96 black well plate with clear bottom containing 2 μM of 3,3′dipropylthiadicarbocyanine iodide [DiSC3(5)] and 190 μL of bacterial suspension. After 60 min incubation of the plate, fluorescence intensity was measured at 622 nm excitation wavelength and 670 nm emission wavelength for 4 min. After that, 10 μL of ACP-1 (Gly) (at working


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concentration of 5 μg/mL, 10 μg/mL and 20 μg/mL) was mixed with the suspension of bacteria and dye of each well. Same volume of water without compound was used as the control for this experiment. Increment in fluorescence intensity was measured for another 25 min using Tecan Infinite M200 PRO microplate reader. Cytoplasmic Inner Membrane (IM) Permeabilization Assay. Briefly, mid log phase and stationary phase of A. baumannii were separately centrifuged (3500 rpm, 5 min), washed and resuspended in 1:1 ratio

of 5 mM glucose and HEPES buffer (pH = 7.4). The working

concentration of mid lpg phase and stationary phase bacteria were ~108 CFU/mL. After that, 190 μL of bacterial suspension containing 10 μM of propidium iodide (PI) were added to the well of a corning 96 black well plate with clear bottom. Excitation wavelength of 535 nm and emission wavelength of 617 nm were used to monitor the fluorescence of the PI for 4 min. Next, 10 μL of ACP-1 (Gly) (at working concentration of 5 μg/mL, 10 μg/mL and 20 μg/mL) was added to the wells containing dye and bacterial suspension. Same volume of water without compound was used as the control for this experiment. The increment in fluorescence intensity of PI was monitored as a measure of membrane permeabilization for another 25 min using Tecan Infinite M200 PRO microplate reader. Resistance Development Study.21,42 The MIC of ACP-1 (Gly) meropenem and colistin against A. baumannii were determined by following the mentioned protocol in the antibacterial assay section. For this experiment, positive control was meropenem and colistin antibiotic for drugsensitive and drug-resistant (except colistin resistance) A. baumannii respectively. Bacterial suspension from sub-MIC (MIC/2) concentration of ACP-1 (Gly) and control antibiotics were diluted approximately 105 CFU/mL for the MIC experiment on the next day. Similarly MIC experiments on the subsequent days (up to 14 days) were performed. The fold of increase in MIC



for the test ACP-1 (Gly) and the control antibiotics were plotted against the number of passages or days.

RESULTS AND DISCUSSION Synthesis and Characterization. The synthesis of the amino-acid conjugated polymers (ACPs 1-10) was carried out through a simple three synthetic steps as outlined in Scheme 1. At first, the carboxylic acid group of different L/D amino acids were esterified by using methanol at refluxing condition. The free amine functionality of the esterified amino acids present in the crude reaction mixture was then reacted with bromoacetyl bromide in presence of potassium carbonate to prepare activated alkyl ester bromide derivative of amino acids (1a-10a). On the other hand, the precursor polymer, poly(isobutylene-alt-maleic anhydride, PDI ~ 1.2)44 was reacted with N,N-dimethyl-1,3-propanediamine at 120 0C for 48 h. This resulted in complete conversion of anhydride to imide, leading to intermediate polymer derivative poly(isobutylenealt-N-(N',N'-dimethyl aminopropylmaleimide) (1b). The complete conversion of anhydride to imide was supported by the appearance of two new infrared peaks at 1767 cm-1 (imide C=O asym. str.) and 1690 cm-1 (imide C=O sym. str.). At the end, the quaternization of the tertiary nitrogen groups of imide intermediate 1b was achieved by using 1a-10a to yield the final cationic polymers (ACPs 1-10). All the newly synthesized polymers including the intermediates were characterized by various spectroscopic technique such as FT-IR, HR-MS and 1H-NMR, 13C-NMR

and DEPT edited 1H-13C HSQC NMR (Supporting Figure S1-S30).

The degree of quaternization (DQ) of the cationic polymers has been determined by 1H-


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Scheme 1. General synthetic scheme for amino-acid conjugated polymers (ACPs)



NMR and DEPT edited two dimensional proton-carbon correlation NMR spectroscopy (1H-13C HSQC). The NMR spectroscopy experiment showed 100% degree of quaternization of the final polymers (ACPs 1-10). Upon the quaterinization of the pendent tertiary nitrogen of imide intermediate (1b), Chemical shift of -CH2N(CH3)2 protons have been altered from 2.2-2.6 ppm to 3.3 ppm (-CH2N(CH3)2 ) and 3.6 ppm (-CH2N(CH3)2) respectively. (Figure 1) Therefore, no signature peaks corresponding to unreacted part (-CH2N(CH3)2) have been found in the region

Figure 1. Stacked 1H-NMR spectra of (A) ACP-1 (Gly) (B) Poly(isobutylene-alt-N-(N', N'dimethylaminopropyl)-maleimide),1b (C) Poly(isobutylene-alt-maleic anhydride) of2.2-2.6 ppm. This indicated the complete conversion of every repeating pendent tertiary nitrogen group to quaternary nitrogen group. In addition, we further confirmed the absence of any unreacted pendent tertiary nitrogen group in 2.2-2.6 ppm region for the final cationic polymers through DEPT edited two dimensional proton-carbon correlation NMR spectroscopy (1H-13CHSQC) in Figure 2. This 2D proton-carbon correlation NMR is associated with DEPT135 pulse sequence where methyl (-CH3) or methine (-CH-) proton gives positive signal


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(indicated by blue contour) and methylene (-CH2-) proton gives negative signal (indicated by green contour). We observed the correlations for Cd, Hd (green contour) and Cb, Hb (green contour) in the region of 1.8-2.6 ppm in case of 1H-13C HSQC spectra of ACP-1 (Gly) whereas no correlations corresponding to the unreacted portion (i.e. Cf, Hf and Cf ', Hf ' ) have been found in this region. This therefore suggested that no unreacted pendent tertiary nitrogen functionality is present in the final polymers. Hence DQ of the cationic amino acid based polymers had been considered as 100%. Similarly, the 2D NMR of all other cationic polymers (ACPs 1-9) are depicted in the supporting information. The molecular weight of these polymers was calculated

Figure 2. 1H-13C HSQC (DEPT edited) spectra of ACP-1 (Gly).The NMR was taken in D2O. 19 ACS Paragon Plus Environment


based on the DQ and was observed in the range of 17.4-20.8 kDa (Supporting Table S1). In order to examine the hydrolytic stability of this class of polymer, 1H-NMR of ACP-1 (Gly) was recorded over a week. No change in the chemical shift of the existing peaks or appearance of additional peaks was observed even at seventh day (Supporting Figure S31). The results therefore confirmed hydrolytic stability of the polymer. In order to understand a detailed structure-activity relationship (SAR), hydrophobicity variation in the polymeric design was achieved through incorporation of amino acids such as Gly (ACP-1), L-Ala (ACP-2), L-Val (ACP-3), L-Leu (ACP-4), L-Ile(ACP-5), L-Phe (ACP-6) and LTyr (ACP-7), respectively by keeping the number of cationic charges constant. The polymers consisted of the methyl ester protected acidic side chain bearing amino acids; L-Asp (ACP-8) and L-Glu (ACP-9) were also synthesized. Furthermore, to understand the effect of chirality, Disomeric analogue of alanine conjugated polymer, ACP-10 was prepared. Antibacterial Activity. The antibacterial activity was determined against both Gram-positive (S. aureus and E. faecium) as well as Gram-negative (E. coli, and A. baumannii) bacteria including drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA), β-lactam resistant K pneumoniae and various carbepenem-resistant A. baumannii. The antibacterial activity was tested in Muller Hinton Broth (MHB) and activity was expressed in terms of MIC, the minimum concentration of the polymers required for bacterial growth inhibition. The details of antibacterial activity (MIC) are summarized in Table 1. Overall, the polymers displayed appreciable antibacterial activity against both Gram-positive as well as Gram-negative bacteria. A general observation suggested that antibacterial activity is dependent on hydrophobicity variation originated from alteration of the amino acids in the polymer design. The lowest


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Table 1. Antibacterial and hemolytic activities of amino-acid conjugated polymers (ACPs)

aMethicillin-

resistant S. aureus, bND stands for “not determined”

hydrophobic polymer, ACP-1 (Gly) consisting of glycine residue (where hydrogen present in the side chain) was found to be moderately active against S. aureus (MIC = 64 µg/mL), however a potent activity was observed against A. baumannii with MIC value of 8-16 µg/mL. Increasing a slight hydrophobicity in the polymer through incorporating alanine, wherein methyl group present in the side chain did not display much difference in antibacterial activity profile. ACP-2 (Ala) displayed similar antibacterial activity compared to ACP-1 (Gly). However, further increment in the hydrophobicity by introducing valine (bearing isopropyl moiety in the side chain), there was a significant enhancement in the antibacterial potency. ACP-3 (Val) was active against all the tested bacterial strains with MIC in the range between 4-64 µg/mL. This polymer was active against S. aureus with the MIC value of 8-16 µg/mL, whereas a value of 4 µg/mL was observed against A. baumannii. The antibacterial activity was found to be improved further upon increasing the hydrophobicity through incorporation of more hydrophobic amino acid, leucine (consisting of isobutyl side chain). ACP-4 (Leu) showed MIC values in the concentration ranged between 4-32 µg/mL. This leucine bearing polymer was active against drug-resistant Gram21 ACS Paragon Plus Environment


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positive bacteria MRSA with the MIC value of 16 µg/mL, whereas valine comprising polymer, ACP-3 (Val) displayed comparatively a higher value, 64 µg/mL. The next polymer in the series, which consisting of isoleucine (bearing sec-butyl side chain), corresponding isomeric analogue of leucine, ACP-5 (Ile) also showed antibacterial activity in the similar concentration range (Table 1). Thus, the results suggested that overall hydrophobicity rather positional isomerization in the amino acid side chain plays the major role in regulating antibacterial activity. However, the polymers consisted of aromatic amino acids in the side chain displayed compromised antibacterial activity profile. ACP-6 (Phe) and ACP-7 (Tyr) bearing phenylalanine and tyrosine in the structure displayed MIC in the concentration ranged between 8-32 µg/mL and 16-128 µg/mL, respectively. The compromised activity possibly resulted due to lesser interaction of aryl group with the bacterial cell envelope over the polymers bearing aliphatic amino acids. The polymers bearing dimethyl ester protected aspartic acid ACP-8 (Asp) and glutamic acid ACP-9 (Glu) residues also displayed compromised activity. This is possibly due to the presence of additional ester moiety in the amino acid side chain leads to compromised interaction towards bacterial cell. Both the polymers displayed MIC in the higher concentration ranged of

≥128

µg/mL against all the bacteria tested, except the MIC value of 16-32 µg/mL was observed for A. baumannii. In order to investigate the effect of amino acid chirality towards antibacterial efficacy, Disomeric analogue of alanine conjugated polymer, ACP-10 (D-Ala) was synthetized and antibacterial activity was evaluated against A. baumannii. The results suggested that there was no difference in antibacterial activity (MIC equal to 16 µg/mL) compared to L-isomeric analogue polymer, ACP-2 (Ala). This indicated that amino acid chirality does not have any influence on the antibacterial activity. 22 ACS Paragon Plus Environment

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Furthermore, to understand the effect of aggregation of the polymers towards antibacterial activity, the critical aggregation concentration (CAC) of the polymers was determined through static light scattering (SLS) study. It was found that this class of cationic polymers did not aggregate even at much high concentration of 256 µg/mL (Supporting Figure S32). On the contrary, the polymers displayed antibacterial activity at much lower concentration (MIC values ranged mostly between 4-32 µg/mL). Therefore, the antibacterial activity of this class of polymer is independent on their aggregated state. A special characteristic of this class of polymers was their good activity against A. baumannii. It is clear from Table 1 that all the polymers displayed anti-A. baumannii activity with the MIC values in the concentration ranged of 4-32 µg/mL against drug-sensitive bacterial strains. Interestingly, the anti-A. baumannii efficacy was hardly affected on amino acid variation in this molecular design. However, WHO has categorized A. baumannii as the topmost notorious pathogen that poses a serious threat in hospital settings.1,6 As a result, there is a pressing need for

Table 2. Anti-A. baumannii activity and selectivity of amino-acid conjugated polymers (ACPs)

bSelectivity

Index = HC50/MIC A. baumannii R674, cND stands for “not determined” 23 ACS Paragon Plus Environment


the development of potent anti-A. baumannii agent to tackle this urgent situation. Thus, we focused our interest to evaluate the efficacy of the polymers as an anti-A. baumannii agents. Firstly, the anti-A. baumannii activity of these polymers was investigated against drug-resistant strains including clinical isolates. The MIC values are presented in Table 2 which further confirmed potent anti-A. baumannii activity of this new class of polymers (MIC values in the range of 4-32 µg/mL). Hemolytic Activity. In order to have a general idea about the toxicity profile of this new class of amino-acids conjugated polymers (ACPs 1-9), their effect on human red blood cells (hRBCs) were studied. The hemolytic activity of the compounds was expressed in terms of HC50, which is defined as the concentration of the polymer corresponds to the lysis of 50% hRBCs. The HC50 values of the polymers (presented in Table 1) were in the concentration range of 26 µg/mL to >1000 µg/mL. Figure 3A demonstrated the dose-dependent hemolysis of hRBCs upon treatment of this class of polymers. Gradual increment of hydrophobicity from glycine to isoleucine, the HC50 value of the polymers decreased from >1000 µg/mL to 36 µg/mL. ACP-4 (Leu) and ACP-5 (Ile) although exhibited the broad spectrum antibacterial activity, were found to be relatively toxic that displayed the HC50 values of 36 µg/mL and 38 µg/mL, respectively. The compound ACP-3 (Val) that showed moderate antibacterial activity profile, exhibited lesser toxicity with the HC50 value of 170 µg/mL. The polymers bearing aromatic amino acids, ACP-6 (Phe) and ACP-7 (Tyr) were also toxic towards hRBCs that displayed HC50 values of 24 µg/mL and 68 µg/mL respectively. However, the polymers ACP-1 (Gly) and ACP-2 (Ala) which displayed decent activity against A. baumannii were completely nontoxic even at 1000 µg/mL with minimal amount of hemolysis (1-3%) of hRBCs. The polymers, ACP-8 (Asp) and ACP-9 (Glu) also exhibited higher HC50 value of >1000 µg/mL with negligible lysis (less than 1%) of human 24 ACS Paragon Plus Environment

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erythrocytes (Figure 3A). The selectivity index (HC50/MIC) of the polymers was determined by considering the MIC against A. baumannii R674 (Table 2). ACP-1 (Gly) and ACP-2 (Ala) were found to be most selective over other compounds, which possessed admirable selectivity in the range of >62.5-125. Therefore, further investigation was executed with ACP-1 (Gly) one of best selective compounds resulted from the detailed structure-activity studies. Cytotoxicity. Further toxicity of ACP-1 (Gly) was evaluated against human embryonic kidney (HEK 293) cell line by performing both LDH assay as well as fluorescence microscopy (Figure

Figure 3. (A) Hemolysis percentage upon treatment of ACPs 1-9; (B) HEK 293 Cell viability upon treatment with ACP-1 (Gly) [EC50 >500 µg/mL]; (C) Fluorescence microscopy images of HEK 293 cells upon 24 h treatment with ACP-1 (Gly), Panel (i): untreated cells (negative control); panel (ii): cells treated with ACP-1 (Gly) (250 µg/mL); panel (iii): cells treated with ACP-1 (Gly) (500 µg/mL); panel (iv): cells treated with 0.1% Triton X (positive control); The scale bar is 50 μm.



3B and 3C). The optimized polymer displayed complete nontoxic nature (~100% cell viability) at its MIC concentration. The bar plot (Figure 3B) demonstrated ~80% cell viability even at highest tested concentration (500 µg/mL) of ACP-1 (Gly). The EC50 values (the concentration of the polymer corresponds to 50% cell viability) of this glycine bearing amphiphilic polymer was found to be > 500 µg/mL. The compound therefore displayed >31.2-62.5 fold selectivity towards killing A. baumannii over HEK cell line. Fluorescence microscopy experiment was performed by simultaneous staining of HEK cells with calcein-AM and propidium iodide (PI). It can be clearly visualized from Figure 3C that most of the cells were alive even after treatment with high concentration of 250 µg/mL and 500 µg/mL of compound. Very few cells had been seen which were labelled with red fluorescence corresponds to dead cells. Thus, the results suggested that the lead polymer, ACP-1 (Gly) is not only non-toxic towards hRBCs, it is also non-toxic against other mammalian cells. Bactericidal Kinetics against Planktonic A. baumannii. Time-kill kinetics for ACP-1 (Gly) was performed against both drug-sensitive (A. baumannii MTCC1425) as well as drug-resistant clinical (A. baumannii R674, A. baumannii R676) strains. The compound displayed rapid bactericidal kinetics. The complete killing (~5 Log CFU/mL reduction) of A. baumanni MTCC1425 was observed within 2 h at a concentration of 16 μg/mL (Figure 4A). At the same concentration, the compound was also capable to completely kill the drug-resistant strains as well (~ 5 Log CFU/mL reduction) within 2 h (A. baumannii R676) and 4 h (A. baumannii R674) respectively (Figure 4B and 4C). At the higher concentration of 32 μg/mL, the compound displayed even faster bactericidal kinetics with complete killing of all the A. baumannii strains within 1h. The results therefore demonstrated rapid bactericidal kinetics of this polymer.


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Figure 4. Bactericidal kinetics of ACP-1 (Gly) against planktonic bacteria (A) A. baumannii MTCC1425; (B) A. baumannii R676; (C) A. baumannii R674; and against stationary phase bacteria (D) A. baumannii MTCC1425; (E) A. baumannii R676; (F) A. baumannii R674; (Asterisks correspond to

Amino-Acid Conjugated Polymers: Antibacterial Agents Effective

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