Exploiting macromolecular design to optimize the antibacterial activity

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Exploiting macromolecular design to optimize the antibacterial activity of alkylated cationic oligomers James L. Grace, Elena K Schneider-Futschik, Alysha G Elliott, Maite Amado, Nghia P. Truong, Matthew A. Cooper, Jian Li, Thomas P. Davis, John F. Quinn, Tony Velkov, and Michael R. Whittaker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01317 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Biomacromolecules

Exploiting macromolecular design to optimize the antibacterial activity of alkylated cationic oligomers

James L. Grace,ab Elena K. Schneider-Futschik,b,c Alysha G. Elliott,d Maite Amado,d Nghia P. Truong,ab Matthew A. Cooper,d Jian Li,e Thomas P. Davis,abf John F. Quinn,*ab Tony Velkov*b,c and Michael R. Whittaker*ab a

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash

University, 381 Royal Pde, Parkville, VIC, 3052, Australia. b Drug

Delivery, Disposition and Dynamics Theme, Monash Institute of Pharmaceutical Sciences,

Monash University, 381 Royal Pde, Parkville, VIC, 3052, Australia. c

Department of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of

Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia d

Institute of Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072,

Australia e

Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University,

Clayton, Victoria 3800, Australia f Department

of Chemistry, Warwick University, Gibbet Hill, Coventry, CV4 7AL, UK

ABSTRACT: There is growing interest in synthetic polymers which co-opt the structural features of naturally occurring antimicrobial peptides. However, our understanding of how macromolecular

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architecture affects antibacterial activity remains limited. To address this, we investigated whether varying architectures of a series of block and statistical co-oligomers influenced antibacterial and haemolytic activity. Cu(0)-mediated polymerization was used to synthesize oligomers constituting 2-(Boc-amino)ethyl acrylate units and either diethylene glycol ethyl ether acrylate (DEGEEA) or poly(ethylene glycol) methyl ether acrylate units with varying macromolecular architecture: subsequent deprotection produced primary amine functional oligomers. Further guanylation provided an additional series of antimicrobial candidates. Both chemical composition and macromolecular architecture were shown to affect antimicrobial activity. A broad spectrum antibacterial oligomer (containing guanidine moieties and DEGEEA units) was identified that possessed promising activity (MIC = 2 µg mL-1) towards both Gram-negative and Gram-positive bacteria. Bacterial membrane permeabilization was identified as an important contributor to the mechanism of action.

Introduction Antibiotic-resistant bacterial infections are becoming increasingly widespread across the globe.1, 2 This increased prevalence increases patient morbidity and mortality, preventable deaths, and the associated economic burden.3, 4 Therefore, there is a need to find new classes of antibiotics that can be manufactured simply and inexpensively, and which can evade the development of resistance. Natural antimicrobial peptides (AMPs) occur in all classes of life with the primary function of defending against pathogens. As such, AMPs have recently gained interest as potential candidates in the fight against bacterial resistance.5,

6

These materials generally act by binding to the

negatively charged phosphate head-groups present in the bacterial membrane, and from there


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facilitate membrane permeabilization and cell death.7 AMPs display a high selectivity for bacteria over mammalian cells, due to the net neutral charge at the surface of mammalian cells. Importantly, bacteria do not as easily develop resistance to AMPs as other antimicrobial drugs as such induction of resistance would require extensive alteration of their membrane composition via highly complexed evolutionary changes.8 Although it is not entirely uncommon with recent studies having shown AMP resistant bacteria,9,

10

with bacterial resistance to cationic AMPs arising

through continual passage of the bacteria in the laboratory.11, 12 Despite their considerable potential as antimicrobial agents, the cost of manufacturing peptides on an industrial scale and degradation of many AMPs by proteases within the body introduce significant barriers to commercialization.1315

Therefore, numerous researchers have turned their attention to synthetic polymer mimics as

AMP analogues to overcome these downfalls. To this end, a wide range of structures have been synthesized including: polyacrylates,16, 17 polymethacrylates,14, 18-21 polynorbornene derivatives,22 polycarbonates,23 polyacrylamides,24 poly(maleimide) analogues,25 and cationic poly(benzyl ether)s26. For further structures and applications readers are directed to reviews published.27, 28 Cu(0)-mediated polymerization has been employed to synthesize architecturally controlled polymers at room temperature at rapid rates, and narrow molecular weight distributions.29-31 In previous work we reported the use of Cu(0)-mediated polymerization to synthesize a series of well-defined antimicrobial polyacrylates, and studied the effects of varying components within the polymer (e.g., end-group, side chain).16 We found that using polymers with a low degree of polymerization (DP ≤27) containing guanidine pendant groups increased the antimicrobial activity, compared to polymers with primary or tertiary amine-pendant groups and at an equivalent DP. However, these studies also revealed a concomitant increase in mammalian cell toxicity for the best performing materials.32 As such, it was hypothesized that if ethoxy-containing repeat units



were incorporated into the guanidine-pendant polymers, cytotoxicity might be reduced, and therefore the selectivity would increase. Antimicrobial polymers typically incorporate both hydrophobic and cationic moieties. While higher hydrophobicity enhances the antibacterial activity of the polymer, it is also the main factor that influences toxicity towards mammalian cells.33 Cationicity, on the other hand, enables binding to the outer membrane of bacteria, but in excessive amounts can render the polymer unable to permeabilize the bacterial membrane. Therefore, a suitable balance between the cationicity and hydrophobicity of the polymer must be maintained since both parameters influence the activity and selectivity of the polymer.34, 35 Recent work by Perrier and co-workers demonstrated that the antibacterial activity of large molecular weight block copolymers could be manipulated by variation of the cationicity and block order structure.36 Following on from this work Judzewitsch et al. also synthesized high molecular weight block copolymers and demonstrated similar structure-property relationships.37 Within the literature, there has been a focus on the incorporation of poly(ethylene glycol) (PEG) into polymers due to its ability to lower mammalian cell toxicity. PEG and PEG methacrylate units have previously been incorporated into antibacterial polymers, where they lowered the haemolysis caused by the polymer and, in some cases, improved antibacterial activity.38-41 Recent work by Wong and co-workers employed oligo ethylene side chains to maintain antibacterial activity and copolymerized with amine and hydrophobic containing monomers and allowing the formation of single chain polymer nanoparticles allowed for the death of both planktonic and bacterial biofilms.42 In this study, we report the antimicrobial activity of a series of novel oligomeric species which combine, for the first time, (i) low molecular weight; (ii) an alkyl group at the chain end; (iii) PEG/DEG sidechains; (iv) different cationic groups; and (v) variable block order. Specifically, we


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employed Cu(0)-mediated polymerization to synthesize amine and guanidine functional cooligomers with low DP that additionally include ethoxy-containing repeat units and a C12 hydrophobic tail incorporated courtesy of an alkylated initiator. A series of co-oligomers of 2(Boc-amino)ethyl acrylate (2-BocAEA) with either diethylene glycol ethyl ether acrylate (DEGEEA) or poly(ethylene glycol) methyl ether acrylate Mn ~480 g mol-1 (PEGMEA) were synthesized with block DP ~5. Three different co-oligomer architectures were employed as illustrated in Scheme 1: (i) co-oligomers with the ethoxy-containing repeat units incorporated first followed by the 2-BocAEA units; (ii) co-oligomers with the 2-BocAEA units incorporated first followed by the ethoxy-containing repeat units; and (iii) statistical co-oligomers formed by direct copolymerization of the 2-BocAEA and ethoxy-containing monomer targeting low DP. These oligomers were then modified by removal of the Boc protecting group to afford a primary amine, which was subsequently converted to a guanidine group. The minimum inhibitory concentration (MIC) was determined against reference strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus. The oligomers were further tested against an extended panel of drug-resistant bacteria (see Supplementary Information Table SI 2). Additional studies were undertaken against human red blood cells, and Human Embryonic Kidney 293 (HEK 293) cells, to examine toxicity levels towards mammalian cells. The mechanism of cell death associated with the most promising active oligomer identified in this study was examined using inner membrane and outer membrane permeabilization assays with E. coli ATCC 25922 and inner membrane assay with S. aureus MRSA ATCC 43300. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were also employed to identify any changes to cell morphology caused by exposure to this same active oligomer.



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O

O Br

O 11

n O

O O

O 11

(ii)

O O

NH O

(i)

O O

Br

O

O O R

O 11

(iv)

O R

m O

O

O

(v)

O

z

O Br m O

O 11

(iii)

O

NH O

O

Br m

n O

O O R

z

O O

Br n O NH

O z

O Br

O 11

m O

O O O R

n O

O O

R=

CH3

z = 8-9 (PEGMEA)

CH2CH3 z = 1

(DEGEEA)

n = m = ~5

NH O

z

Scheme 1. Representative scheme of the co-oligomers block order structure using DBiB as the initiator; (i) 2-BocAEA (5 eq.), Me6TREN (0.16 eq.), CuBr2 (0.05 eq.), DMSO; (ii) DEGEEA/PEGMEA (5 eq.), DMSO; (iii) DEGEEA/PEGMEA (5 eq.), Me6TREN (0.16 eq.), CuBr2 (0.05 eq.), DMSO; (iv) 2-BocAEA (5 eq.), DMSO; (v) 2-BocAEA (5 eq.), DEGEEA/PEGMEA (5 eq.), Me6TREN (0.16 eq.), CuBr2 (0.05 eq.), DMSO.

Materials and Methods Materials Tris(2-(dimethylamino)ethyl)amine

(Me6TREN)43

and

(2-Boc-amino)ethyl

acrylate

(2-

BocAEA)44, 45 were synthesized according to literature procedures with modifications. Copper wire (Sigma-Aldrich) was activated by washing in sulphuric acid for 10 min, followed by washing with deionized water and drying. Poly(ethylene glycol) methyl ether acrylate Mn ~480 g/mol (PEGMEA, Sigma-Aldrich), and di(ethylene glycol) ethyl ether acrylate were de-inhibited by percolating over a column of basic alumina.


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Dimethyl sulfoxide (DMSO, Sigma Aldrich), dodecyl 2-bromoisobutyrate (DBiB, SigmaAldrich), copper (II) bromide (CuBr2, Sigma-Aldrich), tetrahydrofuran (THF, Sigma Aldrich), trifluoroacetic acid (TFA, Sigma Aldrich), 1H-pyrazole-1-carboxamidine hydrochloride (Sigma Aldrich), anhydrous ethanol (EtOH), N,N-diisopropylethylamine (DIEA, Sigma Aldrich) were used as received. The cell line used in the cytotoxicity assay was HEK 293 ATCC CRL-1573. The bacteria strains used in the primary panel are shown in Table SI 1, and the bacteria used in the extended panel are shown in Table SI2. Deuterated solvents CDCl3 and CD3OD were purchased from Sigma-Aldrich.

Oligomer Synthesis Synthesis of block co-oligomers DEGEEA (0.5 mL, 2.70 mmol, 5 eq.), DMSO (1.0 mL), DBiB (0.171 mL, 0.540 mmol, 1 eq.), Me6TREN (0.0230 mL, 0.0864 mmol, 0.16 eq.), CuBr2 (6 mg, 0.0270 mmol, 0.05 eq.) and a magnetic stir bar were charged to a polymerization flask fitted with a rubber septum and the mixture degassed via nitrogen sparging for 10 min. A slight positive pressure of nitrogen was then applied and the pre-activated copper wire was carefully added under a nitrogen blanket. The polymerization flask was then resealed, deoxygenated for a further five minutes and then allowed to polymerize at room temperature for 24 hours. After 24 hours a sample was taken for 1H NMR and gel permeation chromatography (GPC) to confirm full conversion. The 1H NMR sample was diluted with CDCl3, while the sample for GPC was first diluted with THF then passed over a neutral aluminium oxide column to remove salts.



Upon confirmation of complete conversion, 2-BocAEA (0.5806 g, 2.70 mmol, 5 eq.) was dissolved in DMSO (1.16 mL) and added to the reaction mixture after deoxygenation via a deoxygenated syringe. After 24 hours the reaction was stopped and samples were taken and prepared for 1H NMR and GPC. The oligomers were purified by dialysis in acetone, with the acetone being removed by evaporation under a stream of air. The polymerization was then repeated for the other cooligomers, method is located in the Supplementary Information. Synthesis of random co-oligomers DEGEEA (0.35 mL, 1.89 mmol, 5 eq.), 2-BocAEA (0.4064 g, 1.89 mmol, 5 eq.), (DMSO (1.0 mL), DBiB (0.1198 mL, 0.378 mmol, 1 eq.), Me6TREN (0.0161 mL, 0.00605 mmol, 0.16 eq.), CuBr2 (4.2 mg, 0.0189 mmol, 0.05 eq.) and a magnetic stir bar were charged to a polymerization flask fitted with a rubber septum and the mixture degassed via nitrogen sparging for 10 min. A slight positive pressure of nitrogen was then applied and the pre-activated copper wire was carefully added under a nitrogen blanket. The polymerization flask was then resealed, deoxygenated for a further 5 minutes and then polymerized at room temperature for 24 hours. After 24 hours the reaction was stopped by exposing to the air and samples were prepared for 1H NMR and GPC. The sample for 1H NMR was diluted into CDCl3, while the sample for GPC was first diluted with THF and the metal ions removed by passing the sample through a column of neutral aluminium oxide. The oligomers were purified by dialysis in acetone and the solvent was removed with a stream of air. This oligomerization was then repeated replacing DEGEEA with PEGMEA, method is located in the Supplementary Information. Deprotection of Boc-protected oligomers C12-DEGEEA5-b-2-BocAEA7 was dissolved in DCM (2.0 mL) and 1.0 mL was transferred to a 20 mL glass vial and 1.0 mL of DCM was added. TFA (2.0 mL) was added and allowed to react


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overnight. The resulting deprotected oligomer solution was then evaporated to dryness under a stream of air, 5.0 mL of acetone was added and evaporated to dryness under a stream of air, and this was repeated three times. Complete deprotection was confirmed by 1H NMR spectroscopy. Guanylation of the deprotected oligomers 1H-pyrazole-1-carboxamidine hydrochloride (55.76 mg, 0.38 mmol) in anhydrous ethanol (2.00 mL) was added to C12-DEGEEA5-b-AEA7 (0.1008 g). Then, N,N-diisopropylethylamine base (0.133 mL, 0.761 mmol) was added to the vial, the reaction was heated to 55 oC overnight under positive nitrogen pressure. Solvent was removed by evaporation under a stream of air, and purified by trituration by adding diethyl ether to the vial and allowing it to sit for three hours, and removing the solvent by pipette, and repeating five times. Successful guanylation was confirmed by 1H NMR spectroscopy and ATR-FTIR.

Antibacterial and Haemolysis Testing Minimum inhibitory concentration (MIC) assay CaMHB media The oligomers were tested against four bacteria: A. baumannii ATCC 19606, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and K. pneumoniae ATCC 13883. Experiments were performed with cation adjusted Mueller Hinton broth (CaMHB) in 96-well polystyrene microtiter plates. The oligomers were tested against the selected bacteria over a concentration range of 128 to 0.125 μg mL-1 in a 96-well plate. A positive control (bacteria and no drug) and a negative control (no bacteria and no drug) were also performed. Polymyxin B and ciprofloxacin were used as control compounds against Gram-negative and Gram-positive bacteria, respectively. The 96-well plate was then incubated for 20 h at 37 °C in a Contherm Biocell 1000 Incubator, and the MIC was determined as the lowest concentration at which no visible growth was observed.



Oligomer 3G was tested using this method against E. coli ATCC 25922 to calculate an MIC for the SEM and TEM. Minimum Inhibitory Concentration (MIC) Assay MHB media All strains (cf. Tables SI1 and SI2) were cultured in Mueller Hinton broth (MHB) at 37 °C overnight. A sample of each culture was then diluted 40-fold in fresh MHB broth and incubated at 37 °C for 1.5-3 h. The compounds were serially diluted across the wells of polystyrene 384-well plates (Corning; Cat. No 3680). Mid-log phase cultures were added to each well of the compoundcontaining 384-well plates giving a final cell density of 5×105 CFU/mL, and a final compound concentration range of 0.004 μg mL-1 to 128 μg mL-1 in 50 μL assay volume. All the plates were covered and incubated at 37 °C for 18 h. Inhibition of bacterial growth was determined by reading measuring the absorbance at OD600 after 18 h, where the MIC was recorded as the lowest compound concentration with ≥85% growth inhibition compared to the positive growth control after incubation. Haemolysis Testing Human red blood cells are acquired from the Australian Red Cross Blood Service. In a 96-well plate 100 µL of the oligomer solution was added into each well in the following order with the aid of multichannel reservoirs: PBS only, PBS, 30 μg mL-1, 50, 100, 250, 500, 1000, 1500, 2000, 3000 μg mL-1, and 2% Triton-X in PBS. To the wells containing PBS to 2% Triton-X, 100 μg mL-1 of an 8% red blood cell solution in PBS. The plates were then incubated for 1 hour at 37 °C, followed by centrifugation at 1000 g for 5 minutes with the lid off. The supernatant (100 µL) was transferred into a new, sterile flat-bottom 96-well plate, and the absorbance at 450 nm was measured with a PerkinElmer EnVision™ 2101 Multilabel Reader.


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Cytotoxicity Oligomers were prepared in a stock solution of 100% DMSO then diluted to 20% final DMSO. HEK293 cells suspended in DMEM medium supplemented with 10% FBS were seeded at 6000 cells per well in a volume of 20 μL. The compound was serially diluted two-fold for concentrations ranging from 2 to 512 μg mL-1 and then plated in duplicate (20 μL). The cell plates were incubated for 20 h at 37 oC, 5% CO2. After the incubation, 5 μL of 100 μM resazurin in PBS was added to each well for a final concentration of approx. 11 μM. The plates were then incubated for 3 hours at 37 oC, 5% CO2. The fluorescence intensity was read using the TECAN Infinite M1000 PRO with excitation/emission 560/590 nm. Time-kill assay On the afternoon before the time-kill assay commences, a sterile swab is used to add one isolated colony of A. baumannii ATCC 19606 to 10 mL of CaMHB in a 50 mL centrifuge tube. The inoculum is incubated overnight in the shaking water bath at 35-37 °C. On the day of the experiment, 100 µL of overnight culture is inoculated into 10 mL of CaMHB and returned to the shaking water bath for 2-3 h. This incubation period allows for the bacteria to reach exponential (log) growth phase. After 2 h, 200 µL of the log phase bacteria broth is added to 20 mL of CaMHB, followed by the 0 h sample being taken, quickly followed by the broth being spiked with the previously prepared antimicrobial stock solution to make the concentrations of interest, refer to Section 1.3.1. At specified time intervals, samples are taken from both the control and antibiotic-containing culture. The time points chosen were: 0, 0.5, 1, 3, 6 and 24 hours.



Dilutions of the sample were prepared (Table SI3) that were appropriate for the appearance of the inoculum (Table SI4). The samples were plated using a Whitley automated spiral plater (Wasp). Afterwards the plates were incubated at 35-37 °C overnight, and read using a Synbiosis ProtoCOL 3. The limit for detection is 20 CFU/mL. Transmission/Scanning Electron Microscopy Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed as described previously.46, 47 In brief, one colony of E. coli ATCC 25922 and S. aureus MRSA ATCC 43300 was randomly selected and used to prepare an overnight culture of which 10 mL of log-phase cultures (at ~108 CFU/mL) in CaMHB were obtained. The S. aureus containing tubes were treated with Oligomer 3G at 16 and 32 μg mL-1, and E. coli containing tubes were treated with Oligomer 3G at 32 and 64 μg mL-1 and incubated for 1 h at 37 °C followed by centrifugation at 3220 ×g for 10 min. Bacterial cells were fixed with 2.5% glutaraldehyde, washed and imaged as described in detail.46, 47 Permeabilization assays Stock solutions of the oligomers were prepared at 10 mg mL-1 in water and subsequently diluted for the final test concentration of each assay. The compound concentration range tested was 64 to 0.5 μg mL-1. Outer Membrane Permeabilization

1-N-phenylnaphthylamine (NPN) uptake was measured as previously described.48 Briefly, midlogarithmic phase cells were pelleted and washed twice in 5 mM HEPES buffer (pH 7.4) containing 20 mM Glucose, then re-suspended in the same buffer (4 x108 CFU/mL). NPN was added to washed cells to a final concentration of 10 μM. Fluorescence was measured with the excitation and emission wavelengths 350 and 420 nm, respectively, with slit widths of 10 nm. The


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initial baseline was recorded after NPN addition before treatment. Upon the addition of compounds, the increase in NPN fluorescence intensity was monitored over 900 s. Triton X-100 0.1% and meropenem served as positive and negative controls, respectively. The fluorescence results were converted to percentage of NPN uptake using the equation:

% 𝑁𝑃𝑁 𝑢𝑝𝑡𝑎𝑘𝑒 =

(𝐹𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 ― 𝐹0) ― (𝐹𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 ― 𝐹0) 𝐹𝑇𝑟𝑖𝑡𝑜𝑛 ― 𝐹0

× 100

where Ftreatment is the observed fluorescence at a given treatment concentration, Fo is the fluorescence of cells with NPN before treatment, Fcompound is the compounds fluorescence in the presence of NPN, and FTriton is the fluorescence of cells with NPN and 0.1% of Triton X-100. Permeabilization of cytoplasmic membrane

Mid-logarithmic phase cells were pelleted, washed twice and resuspended in PBS to a final OD600 of 0.5. Cells were incubated with compounds at 37 °C for 1 h in a black 384-well plate. Propidium iodide (PI) was dissolved in distilled sterile water to a stock concentration of 0.125 mg mL-1, and added to the bacteria to give a final concentration of 5 μg mL-1. Isopropanol-treated cells were used as positive control, indicating maximal fluorescence. PI fluorescence was measured at 544/620 nm for an hour. The fluorescence results were converted to percentage of PI binding using the equation:

% 𝑃𝐼 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 =

𝐹𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 ― 𝐹𝑃𝐵𝑆 𝐹𝑖𝑠𝑜𝑝𝑟𝑜𝑝 ― 𝐹𝑃𝐵𝑆

× 100

where Ftreatment is the observed fluorescence at a given treatment concentration, FPBS is the fluorescence of cells with PI and PBS, and FIsoprop is the fluorescence of isopropanol-treated cells.



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Results and Discussion Oligomer Synthesis and Characterization. Synthesis of the co-oligomers. The effect of varying co-oligomer block order on bacterial and mammalian cell toxicity was examined using block co-oligomers comprising ethoxy-containing repeat units and 2-Bocprotected amine units. Two monomers with different length ethoxy-side chains (DEGEEA and PEGMEA) were selected to determine their efficacy in lowering mammalian cytotoxicity. Cooligomerization of the DEGEEA and PEGMEA with 2-BocAEA gave co- oligomers with 2-Bocprotected amine units, which enabled subsequent incorporation of primary amine or guanidine pendant groups into the structure. The different block order structures were prepared using dodecyl 2-bromoisobutyrate as the initiator to provide an alkyl chain on the oligomer end-group, as this was previously shown to increase the efficacy of similar materials.16 Co- oligomers with two different block orders were synthesized: (i) with the 2-Boc-protected units in the first segment (i.e., adjacent to the alkyl tail); or (ii) with the ethoxy-containing units in the first block. A third, statistical, co-oligomer was also prepared. A small library of co-oligomers were prepared by Cu(0)-mediated polymerization with low polydispersity indexes (PDIs) (1.08-1.13) and block DPs of ~5 (see Table 1). Table 1. Oligomer characterization of diblock and statistical co-oligomers by 1H NMR and GPC

Oligomer 1 2 3 4 5

DEGEEA 6 6 -

DPa PEGMEA 6 7 5

2-BocAEA 7 8 6 6 6

1st block

Mna

Mnb

PDIb

DEG PEG Boc Boc -

2800 4700 2800 4500 4000

3600 5400 3700 5700 4800

1.08 1.09 1.08 1.13 1.10


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6 aM

5

n (number

-

5

-

2200

2900

1.10

average molecular weight) and DP (degree of polymerization) values were determined by 1H

NMR peak integration analysis. bPDI and Mn (number average molecular weight) were determined in DMAc against polystyrene standards.

Modification of the oligomer Deprotection of the Boc-group using an excess of trifluoroacetic acid revealed the primary amine functional group in quantitative yield. Deprotection was confirmed by 1H NMR spectroscopy (see Figures SI 16 to SI 21), by the loss of the Boc-group hydrogens at 1.45 ppm as demonstrated in our previous work.32 Guanylation was conducted by reacting the exposed primary amine groups with 1.5 equiv. of 1Hpyrazole-1-carboxamidine and 3 equiv. of N,N-diisopropylethylamine base in anhydrous ethanol (Scheme 2). The successful guanylation was confirmed by 1H NMR (see Figures SI 22 to SI 33) through the movement of the peak at ~3.10 ppm downfield to underneath the protons in the PEGMEA/DEGEEA ether chain. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was also utilized to confirm guanylation, with substantial spectral changes evident in the region 1600-1700 cm-1 (consistent with the inclusion of C=N bonds), as well as the appearance of a new peak in the region of 3000 and 3500 cm-1 consistent with a N-H stretch (see Figures SI 34 to SI 39). O O m

O

n O O

Br n O

O O m

TFA DCM, r.t, overnight

m= 11 n= ~5

HN

O O O O

O

Br DIEA, EtOH n 55oC, overnight O

n O O

NH3 CF3COO O

NH O

NN

H Cl NH2

O O m

O HN

Br n O

n O O NH

NH2

O

O

Scheme 2. Representative scheme of a deprotection of Boc-groups by TFA and subsequent guanylation of the deprotected oligomer using 1H-pyrazole-1-carboxamidine hydrochloride.



Effect of PEG length and order of co-oligomer on activity of the oligomer. Minimum inhibitory concentration (MIC) assay. The effect of the ethoxy-containing repeat unit segment length and block order structure on antibacterial activity was assessed by determining the MIC for the various materials. Incorporation of ethoxy-containing repeat units into the oligomer was shown to influence the activity of the cooligomer (Table 2). Specifically, PEGMEA repeat units decreased the antibacterial activity, with each oligomer possessing an MIC greater than the maximum concentration tested. In contrast, incorporation of DEGEEA repeat units led to an increase in the antibacterial activity. For example, O(DEGEEA4-co-AEA5) had an MIC of 2 µg mL-1 against A. baumannii, whereas the control amine oligomer (O(AEA7)) exhibited no activity over the concentration range tested (up to 128 µg mL1).

While oligomers containing primary amine and DEGEEA repeat units showed an increase in activity against Gram-negative bacteria (compared to the control amine oligomer), substitution of the primary amine with guanidine led to the opposite effect: the incorporation of ethoxy-containing units generally decreased the antibacterial activity (compared to a control guanidine oligomer). It has been proposed in literature that polymers containing amine or guanidine groups exhibit different antibacterial mechanisms.14 Moreover, it has been observed that the antibacterial activity of certain amine-containing polymers is independent of molecular weight, whereas the MIC values of guanidine containing polymers have been shown to have a strong dependence on the polymer molecular weight.14, 49 The block order structure was also shown to significantly influence the antibacterial activity. For instance, for oligomers containing primary amine and DEGEEA repeat units, those oligomers with the ethoxy-containing segment adjacent to the alkyl tail were shown to have similar activity to the


Page 16 of 34

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Biomacromolecules

statistical co-oligomer, and lower antibacterial activity compared to the block co-oligomers with the opposite segment order. In the case of oligomers containing guanidine and DEGEEA repeat units, oligomers with the guanidine segment adjacent to the alkyl tail were considerably more potent than both statistical co-oligomers and block co-oligomers with the opposite segment order. As the oligomers were synthesized by a radical oligomerization, they are of course heterogeneous samples comprised of discrete polymer chains with varying number of repeat units. In principle, these discrete oligomers may each have a different antimicrobial activity. As such the determined MICs represent the combined effect of all discrete oligomers in the sample. Altogether, these results indicate that controlling the oligomer structure can have a profound impact on the antibacterial properties of the material. The oligomers were further assayed against an extended panel of drug-resistant bacteria (Table 3 and 4) including: E. coli (ESBL), K. pneumoniae (NDM-1), P. aeruginosa (XDR, polymyxin resistant), A. baumannii (XDR, polymyxin resistant), Streptococcus aureus (MDR), and S. aureus (VRSA). The activity of the oligomers against the extended panel of bacteria showed similar trends to the primary panel, with those oligomers with DEGEEA repeat units generally possessing (i) greater antibacterial activity than PEGMEA containing oligomers, and (ii) limited activity against Gram-positive bacteria beyond that obtained with amine- or guanidine-functional oligomers with no ethoxy-containing segment. Table 2. Antibacterial testing of oligomer compounds against reference bacteria E. coli ATCC 25922, K. quasipneumoniae K6 ATCC 700603, A. baumannii ATCC 19606, P. aeruginosa ATCC 27853, and S. aureus ATCC 43300.

MIC [µg mL-1] Oligomer no. -

Vancomycin

>64

K. quasipneumoniae ESBL >64

-

Polymyxin B

0.25

0.5

Compound

E. coli

P. aeruginosa

S. aureus MRSA

>64

>64

0.5

0.25

0.25-0.5

32

A. baumannii



0D

O(AEA7)

128

>128

Page 18 of 34

>128

>128

32

1D

O(DEGEEA6-b-AEA7)

32

>128

32

64

64

2D

O(PEGMEA6-b-AEA8)

>128

>128

>128

>128

>128

3D

O(AEA6-b-DEGEEA6)

16

32

2

16

64

4D

O(AEA6-b-PEGMEA7)

>128

>128

>128

>128

>128

5D

O(PEGMEA5-co-AEA6)

>128

>128

>128

>128

>128

6D

O(DEGEEA5-co-AEA5)

8

32-64

2

16

32

0G

O(GEA7)

16-64

16-64

16-32

16

2-4

1G

O(DEGEEA6-b-GEA7)

16-64

64

16

64

32

2G

O(PEGMEA6-b-GEA8)

>128

>128

>128

>128

>128

3G

O(GEA6-b-DEGEEA6)

8-16

16

2

2

2

4G

O(GEA6-b-PEGMEA7)

128

>128

>128

>128

32

5G

O(PEGMEA5-co-GEA6)

>128

>128

128

>128

128

6G O(DEGEEA5-co-GEA5) 32 32 32 32 16 Table 3. Antibacterial testing of oligomer compounds against drug-resistant Gram-negative bacteria: clinical isolates of E. coli, P. aeruginosa, A. baumannii, and K. pneumoniae ATCC BAA-2146.

Oligomer no.

Compound

0D

Colistin sulfate Polymyxin B O(AEA7)

E. coli ESBL 0.25-0.5 0.25-0.5 >128

MIC [µg mL-1] K. pneumoniae P. aeruginosa NDM-1 positive XDR, PmxR 0.5 >32 1 8-16 >128 >128

A. baumannii XDR, PmxR 16-32 8 128

1D

O(DEGEEA6-b-AEA7)

32

>128

>128

32

2D

O(PEGMEA6-b-AEA8)

>64

>64

>64

>64

3D

O(AEA6-b-DEGEEA6)

16

64

>128

16

4D

O(AEA6-b-PEGMEA7)

>128

>128

>128

>128

5D

O(PEGMEA5-co-AEA6)

>128

>128

>128

>128

6D

O(DEGEEA5-co-AEA5)

16

32

>128

16

0G

O(GEA7)

16

128

>128

32

1G

O(DEGEEA6-b-GEA7)

16-64

64->128

>128

32

2G

O(PEGMEA6-b-GEA8)

>128

>128

>128

>128

3G

O(GEA6-b-DEGEEA6)

8

32

64-128

8

4G

O(GEA6-b-PEGMEA7)

128

>128

>128

128

5G

O(PEGMEA5-co-GEA6)

>128

>128

>128

128

6G

O(DEGEEA5-co-GEA5)

32

64

>128

32

Table 4. Antibacterial testing of oligomer compounds against drug-resistant Gram-positive bacteria: S. pneumoniae ATCC 700677, and S. aureus NARSA-VRS 1.

Oligomer no.

Compound

0D

Vancomycin O(AEA7)

MIC [µg mL-1] S. pneumoniae MDR S. aureus VRSA 1-2 >32 64 64


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1D 2D 3D 4D 5D 6D 0G 1G 2G 3G 4G 5G 6G

O(DEGEEA6-b-AEA7) O(PEGMEA6-b-AEA8) O(AEA6-b-DEGEEA6) O(AEA6-b-PEGMEA7) O(PEGMEA5-co-AEA6) O(DEGEEA5-co-AEA5) O(GEA7) O(DEGEEA6-b-GEA7) O(PEGMEA6-b-GEA8) O(GEA6-b-DEGEEA6) O(GEA6-b-PEGMEA7) O(PEGMEA5-co-GEA6) O(DEGEEA5-co-GEA5)

>128 >64 >128 >128 >128 64 32 64 >128 16-64 64 >128 32

>128 >64 64 >128 >128 32 32 64 >128 16 32 128 32

Time-kill assay. Time-kill assays were performed using A. baumannii to differentiate between bacteriostatic activity (inhibition of growth) and bacteriocidal activity (cell death) of the most promising oligomer. This assay was done using a cation adjusted broth as opposed to the non-adjusted used for the MIC assay, this potentially explains the reason for the higher MICs (see Table SI 5) used compared to the MICs shown in Table 2. The MIC increases as the cation adjusted broth has more cationic ions than non-adjusted broth, which requires a higher concentration of the oligomer to displace the ions from the bacterial surface and cause death. Cells were either untreated or treated with one of two oligomer (the lead compound (Oligomer 3G) or its amine functional analogue (Oligomer 3D)) at differing concentrations and then agitated at 37 °C. Aliquots were removed periodically (t = 0, 0.5, 1, 3, 6 and 24 hours) and placed back at 37 °C with agitation. Serial 10fold dilutions were spread on agar plates and incubated at 37 °C for 16 h to determine viable colony counts. Viable cell counts were plotted on a log-scale graph (CFU/mL vs time) to determine logreduction of bacterial growth compared to the untreated control.


Biomacromolecules

Both oligomers induced quick inhibition of A. baumannii growth with complete killing observed after 30 minutes at 32 µg mL-1. Incubation of the bacteria at 32 µg mL-1 of the lead compound (Oligomer 3G) led to no observable regrowth of bacteria throughout the study (Figure 1B). In contrast, using Oligomer 3D (Figure SI 40) or adding only 16 µg mL-1 of Oligomer 3G (Figure 1A) allowed for full regrowth of the bacteria by the end of the study, indicating 32 µg mL-1 to be the bacteriocidal concentration and 16 µg mL-1 to be the bacteriostatic concentration of Oligomer 3G.

A

12

B

Control

10 8 6 4 2 0

12

Control

10

Oligomer 3G 16 g mL-1

log10 CFU/mL

log10 CFU/mL

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 20 of 34

Oligomer 3G 32 g mL-1

8 6 4 2

0

2

4

6

20

25

0

0

2

Time (hr)

4

6

20

25

Time (hr)

Figure 1. Time-kill assay of Oligomer 3G against A. baumannii ATCC 19606 at A) 16 µg mL-1; and B) 32 µg mL-1over 24 hours. Data are presented as Mean ± Standard Deviation.

Haemolysis study. The oligomers were subsequently investigated against human red blood cells to evaluate their haemolytic activity over a concentration range of 15 – 1500 µg mL-1 (see Figure 2A). We postulated that the addition of ethoxy containing repeat units would enhance the biosafety of the oligomer. The primary amine containing oligomers generally exhibited lower levels of haemolysis compared to the oligomers containing guanidine repeat units, particularly at the higher concentrations tested. The effect on haemolytic activity of substituting a primary amine with guanidine in the oligomer side chain has previously been reported to exhibit both positive and negative effects, depending on the specific constituents of the studied oligomer. Locock et al.


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reported that when the polymer contained methyl methacrylate units, and the primary amine was substituted with guanidine, there was a decrease in the induced haemolysis.14 In contrast, in a separate study the same authors reported that when using a polymer with 2-(1H-indol-3-yl)ethyl methacrylate units, the guanidine containing materials exhibited higher haemolytic activity than those with amine containing units.21 Herein we noted that the incorporation of DEGEEA repeat units generally increased haemolysis compared to oligomer with no ethoxy-containing repeat unit. In contrast, the addition of PEGMEA repeat units produced a noticeable decrease in the haemolytic activity of the oligomer, with no significant haemolysis observed until higher concentrations (i.e. ≥1000 µg mL-1). We hypothesize that the drastic difference in effect between the two ethoxycontaining repeat units was due to the shorter DEGEEA chains being sufficiently hydrophobic to facilitate interactions between the oligomer and human red blood cells. The effect of changing the segment order on haemolysis appears to be dependent on the particular substituents of the oligomer. While Oligomer 1D (O(DEGEEA5-b-AEA7)), showed minimal haemolysis, reversing the block order (i.e., Oligomer 3D (O(AEA6-b-DEGEEA6))) led to substantially increased haemolysis. Moreover, guanylation of the oligomer further increased the haemolytic activity, as has been noted in our previous work.32 A key indicator of haemolytic toxicity is the HC50 value, which is the concentration at which 50% of the red blood cells have been lysed, and these values are given in Table 5. Those oligomers containing PEGMEA repeat units were essentially non-toxic over the concentration range tested and therefore we have not reported HC50 values. Cytotoxicity. Mammalian cell toxicity was further characterized by conducting a cell viability study against HEK 293 cells using the library of oligomers synthesized (see Figures 2B and SI 42). This study


Biomacromolecules

showed similar trends to those observed in the haemolysis experiments. Specifically, the inclusion of PEGMEA repeat units in the oligomer resulted in limited observed toxicity until the concentrations were substantially increased (>100 µg mL-1). Further, oligomers containing DEGEEA repeat units exhibited significant toxicity at lower concentrations than oligomers with no ethoxy-containing repeat units. As was observed for the haemolysis experiments, the substitution of the primary amine units with guanidine led to a general increase in toxicity. A

100

O(AEA6-b-DEGEEA6) O(DEGEEA6-b-AEA7)

% Haemolysis

80

O(DEGEEA6-b-GEA7) O(GEA6-b-DEGEEA6)

60

O(GEA6-b-PEGMEA7) 40

O(AEA7) O(GEA7)

20 0 10

100

1000

Concentration (g mL-1)

B

150

O(AEA6-b-DEGEEA6) O(GEA6-b-DEGEEA6)

Cell survival (%)

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

O(GEA6-b-PEGMEA7)

100

O(DEGEEA6-b-GEA7) O(AEA7) O(GEA7)

50

0 1

10

100

1000


Figure 2. Oligomer toxicity against mammalian cells; A) Haemolytic toxicity of primary amine and guanidine functionalized oligomers after 1 hour exposure to human red blood cells.; B) Cell viability vs concentration for amine and guanidinefunctionalized oligomers against HEK293 cells. Data are presented as Mean ± Standard Deviation.


Page 22 of 34

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Biomacromolecules

Selectivity. For human therapeutic applications, an antimicrobial compound should possess both potent antimicrobial activity (i.e., low MIC) and low haemolytic activity. To quantify this, selectivity is calculated using the haemolytic activity and the MIC for a given bacterium (i.e., by dividing the HC50 value by the MIC for the bacterium): these values are given in Table 5. For the majority of PEGMEA containing oligomers selectivity could not be computed as they exhibited no activity within the tested concentration range. While the control guanidine oligomer (O(GEA7)) exhibited the greatest selectivity for S. aureus (with a selectivity index of 142-283), the incorporation of either ethoxy-containing repeat unit lowered the selectivity of the co- oligomer against this bacterial species. In contrast, the addition of DEGEEA repeat units to the control amine oligomer (O(AEA7)) led to an increase in the selectivity towards E. coli, A. baumannii and P. aeruginosa. These results compare favourably with MSI-78, a potent magainin analogue which possesses a selectivity index of 9.6 when tested against E. coli, and which has been used in other studies as a benchmark for selectivity.50 Moreover, the block co-polymers synthesized by Perrier and coworkers did not exhibit such broad spectrum activity as those presented in this study, however it should be noted that their polymers were more selective for bacteria. In particular, the 30% cationic multiblock polymer, which exhibited higher selectivity indexes for P. aeruginosa and S. epidermidis than other polymers studied >128 and >256, respectively.36 Table 5. Selectivity index valuesa for the oligomer library. MIC values are shown in Table 2. Oligomer no.

HC50 (µg mL-1)b E. coli K. quasipneumoniae ESBL A. baumannii

P. aeruginosa S. aureus MRSA

0D

935

7

47

23

23

2D

>1500

-

-

-

-

-

3D

501

31

16

251

31

8

4D

>1500

-

-

-

-

-

5D

>1500

-

-

-

-

-



aThe

Page 24 of 34

6D

162

20

3-5

81

10

5

0G

566

9-35

9-35

18-35

35

142-283

1G

242

4-15

4

15

4

8

2G

>1500

-

-

-

-

-

3G

123

8-15

8

62

62

62

4G

>1500

>12

-

-

-

>47

5G

>1500

-

-

>12

-

>12

6G

525

16

16

16

16

33

selectivity index is calculated by dividing the HC50 by the MIC value for a given bacterium. The

selectivity index is used routinely51, 52 to describe the selectivity of the compound towards a particular bacterium. bHC50 values were calculated by interpolation of results in Figure 2A.

Mechanism of action of co-oligomers. Membrane perturbation assays. The mechanism of action against a small selection of the synthesized library was elucidated by investigating membrane disruption of a Gram-negative and Gram-positive bacterial species: E. coli ATCC 25922 and S. aureus MRSA ATCC 43300, respectively. Polymyxin B was used as a comparator for outer membrane (OM) assay, as it is a commercially available and clinically relevant antibiotic that acts by the perturbation of the Gram-negative bacterial membrane. Melittin, an AMP from honeybee venom, was used as a positive control for the inner membrane permeabilization assay, as it efficiently permeates both Gram-negative and Gram-positive membranes. Outer membrane permeabilization.

The ability of the oligomers to disrupt bacterial membrane integrity was assessed by an outer membrane permeabilization assay using N-phenyl-1-naphthylamine (NPN). NPN is an uncharged lipophilic probe that exhibits fluorescence intensity upon partitioning into the hydrophobic phospholipid leaflet of the OM of bacteria. The observed increase in fluorescence following the addition of the oligomer to a solution of bacterial cells and NPN, confirms that the oligomer has


Page 25 of 34

permeabilized the outer membrane of the bacteria to some extent, as NPN would be excluded from intact bacterial cells and remain non-fluorescent. All oligomers tested against E. coli, showed outer membrane disruption (Figure 3). The observation of NPN fluorescence for all compounds tested, irrespective of MIC activity, indicates that the main mechanism of action of the active compounds is potentially membrane permeabilization. It should be noted that there were interactions between the oligomer and the dye at high concentrations (32-64 µg mL-1) as determined by testing the fluorescence of compound and dye without cells, which has been compensated for in Figure 3.

100

NPN uptake (%)

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

80 60 40

O(AEA6-b-DEGEEA6) O(AEA5-co-DEGEEA5) O(GEA6-b-DEGEEA6) O(GEA7) Polymyxin B

20 0 0.1

1

10

100


Figure 3. NPN uptake assay using E. coli over a range of oligomer concentrations. Data are presented as Mean ± Standard Deviation.

Cytoplasmic membrane permeabilization.

Propidium iodide (PI) was employed as a molecular probe to investigate the interaction of the compounds with the cytoplasmic (inner) membrane of E. coli and bacterial cell membrane of S. aureus. PI is a DNA intercalating dye that enters cells with compromised membranes and exhibits fluorescence when bound to nucleic acids. Disruption of the cytoplasmic membrane of E. coli was reported for all antimicrobial oligomers tested (Figure 4A). In the case of S. aureus, only those oligomers containing amine pendant groups were able to permeabilize the peptidoglycan layer, and in this case, also the phospholipid leaflet of the cell membrane at 2× MIC (Figure SI 44). As


Biomacromolecules

has been noted in the literature, polymers containing guanidine repeat units exert a different mechanism of action compared to those containing amine repeat units,14 as they exhibited significantly higher antibacterial activity without disrupting the S. aureus cell membrane. None of the compounds tested interfere with the fluorescence of the dye as was observed with NPN. A

B

100

100

O(AEA6-b-DEGEEA6)

60

80

O(AEA5-co-DEGEEA5)

PI binding (%)

80

PI binding (%)

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 26 of 34

O(GEA6-b-DEGEEA6) O(GEA7)

40

Melittin

20

O(AEA5-co-DEGEEA5) O(GEA6-b-DEGEEA6) O(GEA7)

40

Melittin

20 0

0 0.1

60

O(AEA6-b-DEGEEA6)

1

10

100

Concentration (g ml-1)

0.1

1

10

100

Concentration (g ml-1)

Figure 4. PI binding (%) assay using A) E. coli; and B) S. aureus over a range of oligomer concentrations. Data are presented as Mean ± Standard Deviation.

Scanning and Transmission Electron Microscopy. SEM and TEM were performed using the MICs determined with cationic adjusted broth. SEM and TEM imaging of E. coli ATCC 25922 (Figures 5 and 7) and S. aureus MRSA ATCC 43300 (Figures 6 and 8) revealed that the bacterial outer membrane structure was drastically disrupted due to the treatment with O(GEA6-b-DEGEEA6). The disruption to the membrane shown in the SEM and TEM figures confirms the membrane assay observations. The untreated cells exhibited no membrane disruption however no difference in damage was observed if concentrations were increased from 16 µg mL-1 to 32 µg mL-1 for E. coli ATCC 25922. While testing against MRSA ATCC 43300 showed that 32 µg mL-1 shows milder damage (potentially peptidoglycan deformity) compared to when the concentration was increased to 64 µg mL-1 already showing the loss of cell contents potentially due to leaking of the cells (hence the light structure of the cell). The figures show that the concentrations used are of a bactericidal concentration. In either treatment group,


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Biomacromolecules

wide-spread blebbing of the outer membrane and in particular leaking, of cellular contents was observed. B

A

C

Figure 5. TEM images of E. coli ATCC 25922 incubated with O(GEA6-b-DEGEEA6) at various concentrations. A) Control; B) 16 µg mL-1; and C) 32 µg mL-1. Blue circles highlight membrane disruption.

B

A

C

Figure 6. TEM images of S. aureus MRSA ATCC 43300 incubated with O(GEA6-b-DEGEEA6) at various concentrations. A) Control; B) 32 µg mL-1; and C) 64 µg mL-1.

A

B

C

Figure 7. SEM images of E. coli ATCC 25922 incubated with O(GEA6-b-DEGEEA6) at various concentrations. A) Control; B) 16 µg mL-1; C) 32 µg mL-1.



A

B

Page 28 of 34

C

Figure 8. SEM images of S. aureus MRSA ATCC 43300 incubated with O(GEA6-b-DEGEEA6) at various concentrations. A) Control; B) 32 µg mL-1; C) 64 µg mL-1.

Conclusions The aim of the present study was to synthesize a library of antimicrobial peptide analogues capable of mimicking the function of natural antimicrobial peptides. To this end, a series of block and statistical co-oligomers containing 2-BocAEA and either DEGEEA or PEGMEA were synthesized. The resulting oligomers were characterized using GPC and 1H NMR spectroscopy, and exhibited a high degree of control over the oligomerization. Those oligomers incorporating DEGEEA repeat units possessed both more potent antibacterial and haemolytic activity than oligomers containing PEGMEA repeat units. Substitution of the primary amine groups with guanidine generally increased the antibacterial activity of the co-oligomer against Gram-negative bacteria, but in contrast led to lower MIC values against Gram-positive bacteria. However, the substitution of guanidine groups increased the haemolytic activity of the oligomer, compared to primary amine containing oligomers. A broad spectrum antibacterial oligomer (Oligomer 3G) was selected for further study as it exhibited significant antimicrobial potential towards both Gramnegative and Gram-positive bacteria. Incubation of this material with E. coli and S. aureus led to distinct outer membrane damage as observed by electron microscopy. The oligomer appeared to exhibit its effect over S. aureus and E. coli through considerable cell damage that caused intracellular components to leak out. We hypothesise that the mode of action is due to outer


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Biomacromolecules

membrane damage, however there may be another mechanism due to the observation that only amine-containing oligomers showing permeabilization of the membrane, therefore further studies to elucidate the mechanism of action are underway. ASSOCIATED CONTENT Supporting Information. Experimental details, materials, 1H NMR confirmation of deprotection, MIC, haemolysis and cell viability testing, ANOVA of membrane permeabilization at 2× MIC or 64 µg mL-1. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] *Email: [email protected] *Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS



This collaborative research was conducted by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (project number CE140100036). JLG wishes to acknowledge receiving an APA scholarship. TPD wishes to acknowledge the award of an Australian Laureate Fellowship and JFQ a Future Fellowship from the Australian Research Council. JL and TV are supported by a research grant from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (RO1 AI111965). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health. JL is an Australian NHMRC Senior Research Fellow. TV is an Australian NHMRC Industry Career Development Research Fellow. Matthew Cooper is an Australian NHMRC Principal Research Fellow (APP1059354). We thank Janet C. Reid for conducting cytotoxicity assays.

References 1. Ventola, C. L., The Antibiotic Resistance Crisis: Part 1: Causes and Threats Pharmacy and Therapeutics 2015, 40, (4), 277-283. 2. Organization, W. H., Antimicrobial Resistance: Global Report on Surveillance. World Health Organization: 2014. 3. Stone, P. W., Economic burden of healthcare-associated infections: an American perspective. Expert review of pharmacoeconomics & outcomes research 2009, 9, (5), 417-422. 4. MacVane, S. H., Antimicrobial Resistance in the Intensive Care Unit: A Focus on Gram-Negative Bacterial Infections. Journal of Intensive Care Medicine 2017, 32, (1), 25-37. 5. Hamley, I. W., Lipopeptides: from self-assembly to bioactivity. Chem. Commun. 2015, 51, (41), 8574-8583. 6. Hancock, R. E. W.; Diamond, G., The role of cationic antimicrobial peptides in innate host defences. Trends in Microbiology 2000, 8, (9), 402-410. 7. Hancock, R. E. W.; Chapple, D. S., Peptide antibiotics. Antimicrob. Agents Chemother. 1999, 43, (6), 1317-1323. 8. Zasloff, M., Antimicrobial peptides of multicellular organisms. Nature 2002, 415, (6870), 389395. 9. Andersson, D. I.; Hughes, D.; Kubicek-Sutherland, J. Z., Mechanisms and consequences of bacterial resistance to antimicrobial peptides. Drug Resistance Updates 2016, 26, 43-57.


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Exploiting macromolecular design to optimize the antibacterial activity

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