Antimicrobial Organometallic Dendrimers with Tunable Activity against

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Antimicrobial Organometallic Dendrimers with Tunable Activity against Multidrug-resistant Bacteria Alaa S. Abd-El-Aziz, Christian Agatemor, Nola Etkin, David P. Overy, Martin Lanteigne, Katherine McQuillan, and Russell G. Kerr Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01207 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015

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Antimicrobial Organometallic Dendrimers with Tunable Activity against Multidrug-resistant Bacteria Alaa S. Abd-El-Aziz,*a Christian Agatemor,a Nola Etkin,a David P. Overy,a,b Martin Lanteigne,a Katherine McQuillan,a Russell G. Kerr,a,c a

Department of Chemistry, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3, Canada

b

Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3, Canada

c

Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3, Canada.

ACS Paragon Plus Environment

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Abstract

Multidrug-resistant pathogens are an increasing threat to public health. In an effort to curb the virulence of these pathogens, new antimicrobial agents are sought. Here, we report a new class of antimicrobial organometallic dendrimers with tunable activity against multidrug-resistant Grampositive bacteria that included methicillin-resistant Staphylococcus aureus and vancomycinresistant Enterococcus faecium. Mechanistically, these redox-active, cationic organometallic dendrimers induced oxidative stress on bacteria and also disrupted the microbial cell membrane. The minimum inhibitory concentrations, which provide a quantitative measure of the antimicrobial activity of these dendrimers, were in the low micromolar range. AlamarBlue cell viability assay also confirms the antimicrobial activity of these dendrimers. Interestingly, these dendrimers were non-cytotoxic to epidermal cell lines and to mammalian red blood cells, making them potential antimicrobial platforms for topical applications.


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Introduction Multidrug-resistant infections are a global burden with an annual loss in human life in the hundreds of thousands, and a projected annual loss of 10 million USD by 2050.1 This projection is probable given that microbes rapidly evolve resistance to antimicrobial chemotherapies and humans continuously exert discriminating pressure on microbes through abuse of these chemotherapies.2 While prudent use of chemotherapies provides a solution, the discovery of new ones with new mechanism of action is critical to gaining an edge in the fight against the rise in multidrug-resistant infections. Synthetic cationic polymers are an example of molecules currently investigated as new antimicrobial agents, where their mechanism of action differs from those of conventional antimicrobial agents.3 While conventional antimicrobial agents target biosynthesis of proteins, RNA, DNA, peptidoglycan, and folic acid; cationic polymers interact with the negatively charged bacterial cell membrane, disrupting membrane integrity and its dependent functions, eventually killing the bacteria.3 This mechanism of action has positioned cationic polymers as potential platforms for combating resistance from a broad spectrum of bacteria.4-7 However, clinical application of this class of antimicrobial agents is restricted since they interact in a similar manner with mammalian cells.3 In contrast, some organometallic compounds are evaluated as chemotherapeutics for a number of diseases, such as cancer8 and malaria,9 and are therefore,

considered

as

potential

antimicrobial

agents.10-14

Synthetic organometallic

antimicrobial agents are structurally unique, leading to new mechanism of action that bypass resistance mechanism, ultimately resulting in potent activity against multidrug-resistant microbes. Regrettably, research on this class of antimicrobial is still at the early stage,15 focusing


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on small molecules or conventional macromolecular scaffolds,11 and consequently, the unexplored potential of this class still needs to be evaluated. A macromolecular scaffold yet to be explored in the quest for new organometallic-based antimicrobial agents is that of the dendrimer. The dendritic scaffold is a three-dimensional macromolecule composed of a central core from which several perfectly branched monomers stem, yielding multiple surface terminal groups.16 Their uniform dispersity, nanoscale size, and controlled core, branch, and surface functionalities position dendrimers as attractive platform in biomedical applications.17 The ability to control functionality is a powerful synthetic strategy that allows property tunability and leads to the discovery of new functional materials. Thus, the dendritic scaffold is potentially useful in the design of antimicrobial agents with new mechanism of action. Indeed, numerous organic dendrimers that include glycodendrimers, peptide-based dendrimers, cationic and anionic dendrimers have been described as effective antimicrobial agents;18-22 however, to our knowledge, an organometallic dendrimer with antimicrobial activity is yet to be reported. Expanding the frontiers of antimicrobial dendrimers to organometallics is, therefore, desirable and represents a strategy towards the design of new antimicrobial agents with new mechanism of action. In this study, we designed antimicrobial organometallic dendrimers based on the cationic, redox-active sandwich complex, η6-arene-η5-cyclopentadienyliron(II) (Cp-FeII-arene) complex. Previously, the conjugation of redox-active sandwich complexes, such as ferrocene, to a peptide backbone enhanced antimicrobial activity against Gram-positive bacteria.23 Mechanistically, these redox-active organometallic complexes generate reactive oxygen species (ROS), which induces oxidative damage on microbes.23 Thus, we expected that the incorporation of redoxactive Cp-FeII-arene into the dendritic scaffold would impact antimicrobial activity in a similar


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manner. In addition, the cationic charge on Cp-FeII-arene would disrupt the microbial cell membrane, further enhancing the antimicrobial activity. The presence of two different mechanism of action in a single antimicrobial agent is rare but attractive as combining antimicrobial agents with different mechanism of action is recommended for the effective treatment of multidrug-resistant infections.1 Recently, a linear organometallic polymer with pendent cationic, redox-active cobaltocenium moieties acted in synergy with conventional antibiotics against the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) by protecting the antibiotic from β–lactamase hydrolysis.15 Although the mechanism of action of this linear organometallic polymer is far from the one presented in this work, it exemplifies the emerging efforts toward combating multidrug-resistant infections with cationic, redox-active sandwich complexes-based macromolecules. In this work, we opted for the dendritic scaffold due to its amenability to property tunability via a structure–activity relationship approach as well as its uniform dispersity and nanoscale sizes, which are attractive features for the design of biomaterials. Thus, we synthesized a series of cationic, redox-active dendrimers and screened their activity against a panel of infection-causing microbes that included Candida albicans, Pseudomonas aeruginosa, Proteus vulgaris, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and Staphylococcus warneri. To tune the antimicrobial activity, the counteranion as well as the structure of the arene nucleus was altered. The structure of the arene nucleus affects the lipophilicity and the redox chemistry of the dendrimer, which are important parameters that tune the antimicrobial activity.23-25 We also examined the biocompatibility of the dendrimers by screening their activity against human epidermal keratinocytes cells (HEka), human foreskin BJ fibroblast cells, human breast adenocarcinoma cells (HTB-26), and mammalian, specifically, sheep red blood cells.


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2. MATERIALS AND METHODS All chemicals were purchased from Sigma-Aldrich or Alfa Aesar. Unless otherwise stated, the chemicals were used without further purification. Deuterated solvents, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were dried over molecular sieves before use. The syntheses of 3, 4, 5, DEN3, and DEN9 were reported previously.26 Instrumentation A Bruker Avance NMR spectrometer (1H, 300 MHz;

13

C, 75 MHz;

11

B, 96 MHz; and

31

P, 122

MHz) was used to record the 1H, 13C, 11B, and 31P NMR spectra of all synthesized compounds. All spectra were acquired using DMSO-d6. For 1H and referenced to the DMSO-d6 residue peak while for

13

11

C, the chemical shifts were internally

B and

31

P, the chemical shifts were

externally referenced to boron trifluoride diethyl etherate in CDCl3 and phosphoric acid in D2O, respectively. Elemental, specifically carbon and hydrogen (CH), analyses were performed on CE-440 Elemental Analyzer, Exeter Analytical, Inc. Glass transition temperatures were obtained on TA Instruments differential scanning calorimeter (DSC) Q100. The samples were sealed in aluminium pans, and were heated and cooled at 10 °C/min under nitrogen atmosphere. Cyclic voltammetry (CV) was performed on Princeton Applied Research/EG&G Model 263A potentiostat/galvanostat equipped with glassy carbon working electrode, Pt counter electrode, and Ag reference electrode. General Synthetic Procedure for Zeroth Generation Dendrimers (DEN1–DEN6): The syntheses of the dendrimers were carried out using a general procedure described in a previous report26 but with slight modification. Briefly, in a 50 mL round-bottom flask, was added pentaerythritol (0.170 g, 1.25 mmo), 4 (5.00 mmol), 4-(dimethylamino)pyridine (DMAP) (0.470


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g, 3.85 mmol), 10 mL of 3:1 CH2Cl2:DMSO solution. The resulting solution was cooled to 0 °C using ice bath, and stirred under nitrogen atmosphere while N,N'-dicyclohexylcarboiimide (DCC) (1.13 g, 5.50 mmol) was added over 5 minute. Next, the ice bath was removed, and the mixture was stirred at room temperature for 12 hours under nitrogen. Afterwards, precipitated dicyclohexylurea was removed by filtration. The filtrate was added to 50 mL of ice water, extracted twice with 20-mL portions of CH2Cl2/acetone mixture, and the organic extract was washed twice with 50-mL portions of 5% HCl. The organic extract was added to 10 mL of water to which NH4PF6 or NH4BF4 (10.0 mmol) was dissolved, and was allowed to stand for 30 minutes. The aqueous layer was separated, the organic portion dried over MgSO4, filtered under gravity, and solvent removed using rotary evaporator. The residue was dissolved in acetone, cooled to −10 °C in a freezer for 1 hour, filtered to remove precipitated dicyclohexylurea, and precipitated from diethyl ether. The yellow-green solid was collected, and dried at room temperature to give corresponding dendrimer. Characterization of DEN1 and DEN4: Yield: DEN1, 74%; DEN4, 49%; 1H NMR (DMSO-d6, 300 MHz): δ 7.41 (16 H, br s, Ar H), 7.28 (16 H, br s, Ar H), 6.33 (32 H, br s, iron-complexed Ar H), 6.19 (8 H, br s, iron-complexed Ar H), 5.16 (40 H, s, cyclopentadienyl H), 4.04 (8 H, br s, CH2), 2.45 (8 H, br s, CH2), 2.16 (8 H, br s, CH2), 1.69 (12 H, br s, CH3);

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C{1H} NMR

(DMSO-d6, 75 MHz): δ 172.6, 151.0, 145.9, 132.5, 129.1, 120.2, 118.6, 86.6, 76.8, 76.9, 64.8, 45.0, 35.9, 29.8, 27.0; elemental analysis: DEN1: calcd for C:49.01; H: 3.78; found: C:49.67; H:3.96; DEN4: calcd for C: 55.57; H: 4.29; found: C: 55.86; H: 4.65. Characterization of DEN2 and DEN5: Yield: DEN2 77%; DEN5, 56%; 1H NMR (DMSO-d6, 300 MHz): δ 7.34 (16 H, br s, Ar H), 7.23 (16 H, br s, Ar H), 6.23 (32 H, s, iron-complexed Ar H), 5.12 (40 H, s, cyclopentadienyl H), 4.10 (8 H, br s, CH2), 2.36, (32 H, br s, CH2 and CH3


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(CH2 overlapped CH3), 2.15 (8H, br s, CH2), 1.65 (12 H, br s, CH3); 13C{1H} NMR (DMSO-d6, 75 MHz): δ 172.5, 151.4, 145.8, 131.3, 129.1, 120.2, 100.7, 86.9, 75.7, 77.4, 64.9, 44.4, 35.9, 30.6, 26.7, 20.1; elemental analysis: DEN2, calcd for C: 50.03; H: 4.07; found: C: 50.31; H: 4.13; DEN5, calcd for C:56.51; H: 4.60; found: C: 57.03; H: 4.83. Characterization of DEN6: Yield: 57%; 1H NMR (DMSO-d6, 300 MHz): δ 7.35 (16 H, br s, Ar H), 7.24 (16 H, br s, Ar H), 6.77 (16 H, br, s, iron-complexed Ar H) 6.39 (16 H, br s, ironcomplexed Ar H), 5.25 (40 H, s, cyclopentadienyl H), 4.01 (8 H, br s, CH2), 2.40 (8 H, br s, CH2), 2.13 (8 H, br s, CH2), 1.64 (12 H, br s, CH3);

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C{1H} NMR (DMSO-d6, 75 MHz): δ

172.9, 151.0, 146.5, 132.3, 129.6, 120.5, 118.8, 86.6, 76.7, 79.7, 65.3, 45.4, 36.3, 30.1, 27.3; elemental analysis: calcd for C: 51.49; H: 3.76; found: C: 51.87; H: 4.07. General Synthetic Procedure for First Generation Dendrimers DEN7–DEN9: The syntheses of these dendrimers followed a general synthetic procedure reported previously.26 These dendrimers were synthesized from the appropriate 4 (1.22 mmol), 5 (0.750 g, 0.152 mmol), DMAP (0.115 g, 0.940 mmol), and DCC (0.276 g, 1.34 mmol) in 10 mL of 1:3 CH2Cl2/DMSO mixture. Characterization of DEN7: Yield: 1.30 g, 68%; 1H NMR (DMSO-d6, 300 MHz): δ 7.39 (48 H, br s, Ar H), 7.30 (48 H, br s, Ar H), 7.00 (32 H, br s, Bz H), 6.33 (96 H, br s, iron-complexed Ar H), 6.19 (16 H, br s, iron-complexed Ar H), 5.20 (40 H, s, cyclopentadienyl H), 5.16 (80 H, s, cyclopentadienyl H), 4.55 (16 H, br s, CH2), 4.03 (8H, br s, CH2), 2.43 (24 H, br s, CH2), 2.11 (24 H, br s, CH2), 1.68 (36 H, s, CH3);

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C{1H} NMR (DMSO-d6, 75 MHz): δ 174.2, 172.7,

152.1, 151.6, 151.1, 146.1, 145.9, 145.8, 133.9, 132.5, 132.4, 129.9, 129.1, 128.5, 120.5, 120.3,


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120.1, 119.7, 86.6, 84.7, 74.9, 74.4, 77.7, 76.7, 64.8, 62.1, 45.0, 44.9, 44.7, 36.0, 35.8, 29.7, 29.6, 27.1, 26.8; elemental analysis: calcd for C:50.66; H: 3.76; found: C:50.38; H:3.67. Characterization of DEN8: 1.38 g, 71%; 1H NMR (DMSO-d6, 300 MHz): δ 7.35 (48 H, br s, Ar H), 7.26 (48 H, br s, Ar H), 6.98 (32 H, br s, Bz H), 6.25 (96 H, br s, iron-complexed Ar H), 5.21 (40 H, s, cyclopentadienyl H), 5.12 (80 H, s, cyclopentadienyl H), 4.56 (16 H, br s, CH2), 4.03 (8 H, br s, CH2), 2.37, (64 H, br s, CH3 and CH2 (the CH2 overlapped with CH3), 1.67 (36 H, br s, CH3); 13C{1H} NMR (DMSO-d6, 75 MHz): δ 174.3, 172.6, 151.7, 151.5, 151.2, 146.1, 145.9, 145.8, 131.3, 130.3, 129.8, 129.7, 129.1, 128.5, 127.9 120.3, 120.1, 100.1, 86.6, 76.1, 75.0, 74.4, 77.7, 77.2, 64.8, 62.1, 45.0, 44.9, 44.6, 36.7, 35.8, 29.7, 29.6, 27.1, 26.9, 19.1; elemental analysis: calcd for C:51.28; H: 3.95; found: C:51.60; H:4.18. DOSY NMR Spectroscopy: Diffusion-order spectroscopy (DOSY) measurements were carried using a Bruker Avance III 600 MHz NMR spectrometer (Bruker Corporation, East Milton, ON) equipped with a 1.7 mm Bruker gradient triple resonance inverse (TXI) probe. Dendrimer samples were dissolved in DMSO-d6 (ηs = 1.99 × 10−3 Pa s at 25 °C) at 2.5 mM and the DOSY experiments conducted at 25.0 ± 0.1 °C. Each sample was auto-locked on DMSO-d6, auto-tuned at 600.28 MHz and shimmed. The Bruker DOSY pulse program “dstebpgp3s”, which included bipolar gradients and double stimulated echo for compensating for gradient errors and convection, respectively, was used. The pulsed gradient strength of 56 G/cm in the z-direction (GPZ) was calibrated using water. For all spectra, the diffusion time (d20) and the eddy current delay (d21) were set to 200 ms and 5 ms, respectively. The duration of the pulse gradient (p30) was optimized for each sample (ranging from 1550-2050 µs) in order to ensure that about 10% of the original signal remained after linear ramping from 5–90% of the maximum gradient strength GPZ (Bruker gradient shaped pulse SMSQ10.100) with 18 data points (gradient ramp in 5%


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increments). Other operating parameters were 16k/18 F2/F1 time domain sizes, 6s relaxation delay, 8 dummy scans, and 16 or 64 acquisition scans. All peaks were referenced to DMSO-d6 residual peak at 2.50 ppm. After acquisition, the data was zero filled to 64 K, Fourier transformed and baseline corrected in F2. The Bruker Dynamics Center Software was used to fit the data and to provide diffusion coefficients. For each sample, well-resolved, strong peaks, such as the cyclopentadienyl peak ~ 5.20 ppm, were used to extract individual diffusion coefficients. The hydrodynamic radius was calculated from the diffusion coefficient using the Stokes-Einstein relationship:27

=

6

where D0 is the diffusion coefficient, kB the Boltzmann constant, T the absolute temperature, ηs the viscosity, and Rh the hydrodynamic radius. Antimicrobial Activity. Antimicrobial activity assays of the dendrimers against methicillinresistant Staphylococcus aureus ATCC 33591 (MRSA), Staphylococcus warneri ATCC 17917, vancomycin-resistant Enterococcus faecium EF379 (VRE), Pseudomonas aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and Candida albicans ATCC 14035 were carried out in 96-well plates using the Clinical Laboratory Standards Institute (CLSI) microbroth antimicrobial testing protocol.28,29 The microorganisms were grown according to the CLSI protocol29 and overnight seed cultures of assay microorganisms were diluted in their respective growth medium to a concentration of 6.5 x 105 cfu/mL and dispensed into assay plates. Assays were carried out in triplicate against each microorganism at twelve different dendrimer concentrations obtained by serial dilution of the initial concentration, 128 µg/mL, to give the final concentration, 0.0625 µg/mL, in 2% DMSO. Each plate also contained eight uninoculated positive controls (media +


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2% DMSO), eight untreated negative controls (media + 2% aqueous DMSO + microorganism), and one column containing a concentration range of a control antibiotic (vancomycin for MRSA, and S. warneri; rifampicin for VRE; gentamycin for P. aeruginosa; ciprofloxacin for P. vulgaris; or nystatin for C. albicans). The optical density of the plate was recorded using a Thermo Scientific Varioskan Flash plate reader at 600 nm before and after incubation of the plates at 37 °C for 22 hours. After subtracting the initial OD600 reading from the final, the percentages of microorganisms’ survival relative to the positive control wells were calculated, the minimum inhibitory concentration (MIC) as well as the half maximal inhibitory concentration (IC50) was determined and reported in µM. For MRSA, bactericidal activity was inferred using AlamarBlue cell viability assay. Twenty-four hours after treatment, AlamarBlue was added to each well at 10% of the culture volume (11 µL in 100 µL). Fluorescent emission at 590 nm was monitored using a Thermo Scientific Varioskan Flash plate reader after excitation at 560 nm. The emission was monitored before AlamarBlue was added and 4 hours later. Oxidative Stress Assay. Using a previously reported procedure,30 the oxidative stress assay was carried out in 96 well plates with the same inoculum density generated using the antimicrobial assay protocol described above. Prior to plate inoculation, the MRSA inoculum was split by transferring equal volumes into two 50 mL conical centrifuge tubes and both tubes were centrifuged at 19,040 g-force for 5 minutes. The supernatant was discarded and the bacterial pellets resuspended in 10 mL of assay buffer (4.2 g MOPS, 80 mg NH4NO3, 4 mg K2HPO4 in 1L sterile deionized H2O), one of which included dichlorodihydrofluorescein (H2DCF, 4.87 mg/L), and incubated for 30 minutes at room temperature. Bacterial cells were then centrifuged at 19,040 g-force, the cell pellet washed twice with assay buffer, and then resuspended to the original volume in pre-warmed CAMHB media (37°C), of which 90 µL of the H2DCF treated


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and untreated bacterial cells were dispensed into 96 well assay plates containing test compounds, a vancomycin dilution series (as per antimicrobial assays), and a 200–25 µM dilution series of H2O2. Fluorescent emission at 535 nm was monitored using a Thermo Scientific Varioskan Flash plate reader after excitation at 485 nm. Plates were incubated at 37°C and fluorescence measurements were taken at 0, 0.5, 4, and 24 hrs. The assay results were corrected for baseline fluorescence (measurements of untreated controls were subtracted from treated controls) and expressed as a percentage of maximal oxidative stress response relative to H2O2 controls. Cytotoxicity Assay. The toxicity of the dendrimers against adult human epidermal keratinocytes (HEKa) (Invitrogen#C-005-5C), human foreskin BJ fibroblast cells (ATCC CRL-2522), and human breast adenocarcinoma cells (ATCC HTB-26) was carried out as previously reported.28 Prior to the cytotoxicity assays, the cells were grown as follows: human foreskin BJ fibroblast cells were grown and maintained in 15 mL of Eagle’s minimal essential medium supplemented with 10% fetal bovine serum and 100 µU penicillin and 0.1 mg/mL streptomycin in T75 cm2 cell culture flasks at 37 oC in a humidified atmosphere of 5% CO2. Culture medium was refreshed every 2-3 days and cells were not allowed to exceed 80% confluency. The adult human epidermal keratinocytes (HEKa) isolated from skin were grown in 15 mL of EpiLife medium supplemented with human keratinocyte growth supplements in T75 cm2 cell culture flasks, and incubated at 37 oC in a humidified atmosphere of 5% CO2. Growth medium was refreshed every 2 days until the cells reached 50% confluency and then the medium was refreshed everyday until 80% confluency was obtained. The human breast adenocarcinoma cells were grown and maintained in 15 mL of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham supplemented with 10% fetal bovine serum and 100 µU penicillin and 0.1 mg/mL streptomycin


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in T75 cm2 cell culture flasks at 37 oC in a humidified atmosphere of 5% CO2. Culture medium was refreshed every two to three days and cells were not allowed to exceed 80% confluency. At 80% confluency, the cells were counted, diluted and plated into 96 well-treated cell culture plates. The BJ fibroblast and HEKa cells were plated at a cell density of 10,000 cells per well and the HTB-26 cells were plated at cell density of 5000 cells per well in 90 µL of respective growth medium, which were the same as those described above except antibiotics were not added. The plates were incubated at 37 oC in a humidified atmosphere of 5% CO2 to allow cells to adhere to the plates for 24 hrs before treatment. DMSO was used as the vehicle at a final concentration of 1% in the wells. The dendrimers were resolublized in sterile DMSO and a dilution series was prepared for each cell line using the respective cell culture growth medium of which 10 µL were added to the respective assay plate well to give eight final concentrations that ranged from 128 µg/mL to 1 µg/mL per well that had a final volume of 100 µL. Each plate also contained four uninoculated positive controls (media + 1% DMSO), four untreated negative controls (Media + 1% DMSO + cells), and one column containing a concentration range of zinc pyrithione or doxorubicin as standard. Next, the plate that contained the BJ fibroblast or HEKa cells were incubated at 37 oC in a humidified atmosphere of 5% CO2 for 24 hrs, while those that contained the HTB-26 cells were incubated at 37 oC in a humidified atmosphere of 5% CO2 for 72 hrs. AlamarBlue was added, 24 hours after treatment, to each well at 10% of the culture volume (11 µL in 100 µL). Fluorescent emission at 590 nm was monitored using a Thermo Scientific Varioskan Flash plate reader after excitation at 560 nm. The emission was monitored before AlamarBlue was added and 4 hrs later. After subtracting the initial emission reading from the final, the inferred percentage of cell viability relative to positive control wells were calculated, the IC50 was determined and reported in µM. Assays were conducted in triplicate.


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Haemolysis Assay: The haemolytic activity of the dendrimers was tested using defibrinated sheep whole blood. To pellet the blood cells, 1 mL of defibrinated sheep whole blood was centrifuged at 1680 g-force for 5 min and the pellet was washed four times with 0.9% saline. The blood cell pellet was re-suspended in red blood cell (RBC) buffer that consisted of 0.5999 g of 5 mM sodium phosphate, and 8.766 g of 150 mM NaCl in dH2O maintained at pH 7.4 to make it up to 1 mL. A 20 µL aliquot of dendrimer (6.40 mg/mL) in 2% sterilized DMSO was added to 980 µL of RBC buffer. Then, 25 µL of blood cell suspension was added to 1 mL of dendrimer solution prepared in RBC buffer. A vehicle control (20 µL of 2% DMSO in 980 µL RBC buffer), positive control (25 µL of cell suspension in 1 mL of dH2O), and negative control (25 µL of cell suspension in 1 mL RBC buffer) were also assayed. Tubes that contained test samples and assay controls were incubated for 2 hrs at room temperature. Afterwards, the tubes were centrifuged at 1680 g-force for 5 min, and 200 µL of the supernatant was transferred into a 96 well plate. The absorbance was measured at 540 nm after shaking for 10 seconds. All measurements were blanked with the negative and vehicle control and were taken in triplicate. The haemolysis percentage was determined relative to the positive control. Field Emission Scanning Electron Microscopy (FE-SEM): A 20 µL of MRSA was cultured in 20 mL of cation adjusted Mueller Hinton (CAMH) broth supplemented with 12.5 µg/mL of penicillin G. The microbe was inoculated at 37 oC for 18 hours under constant shaking at 3.25 gforce. The inoculum was transferred into a 50 mL flacon tube with beads, vortexed for 1 minute and allowed to sit for 5 minutes to allow aerosols to settle. Then, the inoculum was diluted to give 6.5 × 105 or 6.5 × 107 cfu/mL. Using the same antibacterial testing protocol described above, the diluted inoculum was treated with the dendrimer diluted in DMSO at expected IC50 concentration, and incubated for 22 hrs. A negative control, which consisted of untreated


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inoculum, was also prepared using the above protocol. Afterwards, the inoculum was centrifuged at 1075 g-force for 5 minutes. The supernatant was collected, washed twice with 1 mL of 2.5% glutaraldehyde/phosphate-buffered saline, centrifuged at 1075 g-force, and the supernatant removed. The bacterial cells were fixed with 500 µL of 2.5% glutaraldehyde/phosphate-buffered saline for 60 minutes, pelleted and washed twice with phosphate-buffered saline. Dehydration was carried out in a series of ethanol solutions (25%, 35%, 50%, 75%, 90%, 95%, and 100%). The bacterial cells were re-suspended in 100% ethanol, dripped on copper tape, and dried at room temperature for 2 days. The dried samples were sputtered coated with gold/palladium layer before SEM imaging using Hitachi S-4700 FE-SEM. Confocal Laser Fluorescence Microscopy: The cells were grown to 80% confluency using the cytotoxicity assay protocol.28 At 80% confluency, the cells were counted, diluted, and plated into 4-chamber culture slides. The BJ fibroblast cells were plated at a cell density of 50,000 cells per chamber and the HTB-26 cells were plated at cell density of 25,000 cells per chamber in 540 µL of respective growth medium. All media used in the preparation of the slides was the same as those used to grow the cells except antibiotics were absent. The culture slides were incubated at 37 oC in a humidified atmosphere of 5% CO2 to allow cells to adhere to the slides for 24 hrs before treatment. Dendrimers were resolublized in sterile DMSO and were diluted to 1.28 mg/mL using the respective cell culture growth medium. The diluted dendrimer solution (60 µL) was added to the respective chamber well to give a final concentration of 128 µg/mL with a final DMSO concentration of 1% per chamber. Each culture slide included an untreated negative control chamber that contained cells, growth media and 1% DMSO. The BJ fibroblast cells were incubated at 37 oC in a humidified atmosphere of 5% CO2 for 24 hours and the HTB-26 cells were incubated at 37 oC in a humidified atmosphere of 5% CO2 for 72 hours. Next, the growth


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media was removed from the culture slide chambers by vacuum. The chambers were washed twice with a pH 7.5, phosphate-buffered saline solution. The upper plastic chambers of the slides were removed. The slides were transferred to a glass-staining dish, fixed with cold acetone for 10 minutes at 4 oC, air-dried in a fumehood for 2 minutes, and washed twice with phosphatebuffered saline solution. A fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI), diluted to 1:500 in dH2O was added to the slides to stain the nuclei of the cells. The slides were then incubated at room temperature for 1 minute, and were washed twice with phosphate-buffered saline solution. A mounting medium of 30% glycerol and 70% phosphate-buffered saline was prepared and was used to mount coverslips onto the slides before confocal laser fluorescence microscopic imaging using Carl Zeiss Confocal Laser Fluorescence Microscope.

Results and Discussion Syntheses and Characterization of Dendrimers. The objective of this work was the design of an organometallic dendrimer with tunable antimicrobial activity. To achieve this, redox-active, cationic η6-arene-η5-cyclopentadienyliron(II) (Cp-FeII-arene) complexes of PF6− or BF4− counteranions, obtained via the well-established ligand exchange reaction,31-33 were used as functional building blocks in syntheses of the dendrimers. The dendrimers were synthesized from these complexes using a general synthetic route previously described (Schemes 1 and 2).26 The synthetic route is versatile, allowing modification of the arene nucleus and the counteranions. For instance, we altered the structure of the arene nucleus by substituting the hydrido groups with electron-donating methyl or electron-withdrawing chloro groups. This substitution tuned the electron density at the iron centre, eventually affecting its redox chemistry. The substitution also affected the lipophilicity of the dendrimers, as alkyl groups are known to


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enhance lipophilicity.24,25 Using a previously established synthetic method,31-33 we tuned the availability of the cationic charge on the dendrimers by replacing the counteranion, hexafluorophosphate (PF6−), in DEN1–DEN3, with a relatively more coordinating counteranion, Scheme 1. Schematic representation of the synthesis of zeroth generation dendrimers


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Scheme 2. Schematic representation of the syntheses of first generation dendrimer


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Table 1. Reduction potential (Epc)a, glass transition temperature (Tg), diffusion coefficient (D0) and hydrodynamic radii (Rh) of dendrimers Dendrimer

Epc (V)

Tg (ºC)

D0 (× 10−11 m2/s)

Rh (nm)

Den1

−1.49

122

8.60

1.28

Den2

−1.49

126

7.47

1.46

Den3

−1.25

136

8.55

1.28

Den4

−1.48

118

9.40

1.18

Den5

−1.50

116

9.00

1.22

Den6

−1.19

122

ndb

ndb

Den7

−1.41

144

6.50

1.69

Den8

−1.41

142

5.80

1.89

Den9

−1.23

178

6.20

1.77

a

Cyclic voltammetry was carried out on nitrogen purged, 6 mM solution of dendrimer in propylene carbonate at room temperature. Scan rate, 0.1 V/s; supporting electrolyte, 0.1 M [n-Bu4N][PF6] for PF6− series of dendrimers or 0.1 M [n-Bu4N][BF4] for BF4− series of dendrimers; E vs Fc0/+ (external). bNot determined.

tetrafluoroborate (BF4−) 34,35 in DEN4–DEN6. Moreover, this switch in counteranion also altered the aqueous solubility of the dendrimers as previously confirmed.34 First generation dendritic analogues (DEN7–DEN9) of DEN1–DEN3 were also synthesized to gain insight into the influence of dendritic effect on antimicrobial activity. We employed 1H, 13C, 11B, and 31P NMR as well as CH analyses to characterize these dendrimers. The expected characteristic NMR peaks (see Materials and Methods and Supporting Information) were found and served as spectroscopic tools to confirm the successful synthesis of the dendrimers. Results from CH analyses further


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confirm the successful syntheses as the experimental percentages of carbons and hydrogens in the dendrimers were in agreement with calculated values. The redox activity of Cp-FeII-arene complexes is well documented36-40 and we confirmed this activity in these dendrimers using cyclic voltammetry (Table 1). The cyclic voltammetry was carried out at room temperature in propylene carbonate solution of the dendrimer. Substitution of the hydrido groups with electron-donating methyl groups or electron-withdrawing chloro groups affected the redox activity of the dendrimers (Table 1). For instance, an appreciable change in reduction potential (Epc) was observed when chloro groups substituted the hydrido groups. Of course, the chloro group is electron withdrawing, decreasing the electron density at the iron centre, and ultimately increasing susceptibility to reduction. As previously confirmed,41 exchanging PF6− with BF4− did not change reduction potential of the dendrimers (Table 1); however, the counteranion exchange affected the aqueous solubility of the dendrimers. Indeed, counteranion exchange is a demonstrated strategy for controlling the aqueous solubility of cationic macromolecules.34 In this work, the BF4− series of dendrimers were relatively more water-soluble than their PF6− congeners, as attested by their lower percent yield (see Materials and Methods section). It is also worth noting that the BF4− series were tedious to work with due to their enhanced solubility. In agreement with previous reports,42,43 the counteranion exchange affected the glass transition temperature (Tg) of the dendrimers with the PF6− series exhibiting higher Tg than the BF4− series (Table 1). In these previous reports,42,43 this trend in Tg was attributed to the presence of hydrogen-bonding interactions in the PF6− polymer; however, this is unlikely with these present dendrimers. Thus, we attributed the lower Tg of the BF4 dendrimers to the relatively more coordinating ability of its counteranion, which could lead to an embedding


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of the anion within the dendrimer, resulting in an increase in free volume and flexibility of the dendrimer, and ultimately lowering of Tg. Further, using diffusion-order spectroscopy (DOSY) NMR, we determined the size of the dendrimers, as this is an important parameter that influences not just solubility, but most importantly, the diffusion of chemotherapeutics through the cell wall.44 The diffusion coefficient obtained from the DOSY NMR experiments is related to the hydrodynamic radii of the dendrimers by the Stokes–Einstein equation.27 Typical of dendrimers,45,46 all our dendrimers were nanoscopic with the hydrodynamic radii increasing with generation (Table 1). The results also show that the chloro- and hydrido-substituted dendrimers had similar but smaller radii than their methyl-substituted congeners. In addition, the counteranion also affected the radii of the dendrimers with the BF4− series being smaller than the PF6− series (Table 1). Antimicrobial Activity. Using the microbroth dilution protocol28,29 we assayed the dendrimers against a broad spectrum of infection-causing microbes that included Gram-positive bacteria methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and Staphylococcus warneri; Gram-negative bacteria Pseudomonas aeruginosa, and Proteus vulgaris; and fungus Candida albicans. At the tested concentrations all dendrimers were inactive against the Gram-negative bacteria and the fungus. Most of the dendrimers were active against the Gram-positive bacteria with the minimum inhibitory concentration (MIC) in the low micromolar range (Table 2). In addition, AlamarBlue cell viability assays estimating cellular respiration confirm antibacterial activity. Although most membrane-disrupting antimicrobial agents such as peptides exhibits activity against Gram-positive and Gram-negative bacteria, some reports12,23 that corroborate our results shows that some cationic, membranedisrupting organometallic antimicrobial agents are active only against Gram-positive bacteria.


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Table 2. Antimicrobial activity and cytotoxicity of dendrimersa IC50/MIC (µM) MRSA

VRE

S. warnerii

HTB-26 IC50 (µM)

Den1

3.3/4.1

2.5/4.1

3.5/4.1

31

Den2

1.8/3.9

2.2/3.9

2.5/3.9

20

Den3

3.6/7.6

6.6/15

6.9/7.6

ia

Den4

6.1/9.2

22/37

9.6/18

ia

Den5

10/18

13/36

11/18

ia

Den6

15/17

16/34

12/17

30

Den7

3.5/5.1

5.9/ia

2.2/2.6

ia

Den8

2.2/5.0

3.3/5.0

2.1/2.5

ia

Den9

ia

ia

ia

ia

Dendrimer

aThe

compounds were tested at twelve different concentrations obtained by serial dilution of the initial concentration, 128 µg/mL, to a final concentration, 0.0625 µg/mL, in 2% DMSO. Inactive compounds (ia) did not show activity at ≤ 128 µg/mL. The dendrimers were also inactive against human epidermal keratinocytes (HEka) and BJ fibroblast cell lines at ≤ 128 µg/mL.

The inactivity towards Gram-negative bacteria may be due to presence of an outer membrane structure, which is absent in Gram-positive bacteria.34 Another probable reason for the narrow spectrum of activity of these organometallic antimicrobial agents may be their reduced lipophilicity compare to their organic counterparts such as the antimicrobial peptides.


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The results further show that the nature of counteranion tuned the activity of the dendrimers with the PF6− dendrimers being more active than their BF4− congener (Table 2). Although the result is explicable, it is still paradoxical, as the BF4− analogues had smaller hydrodynamic radii and enhanced aqueous solubility, suggesting better systemic availability of the antimicrobial agents, and ultimately, better activity.24,44 Actually, it was previously demonstrated that BF4−-containing cationic polymers are more active than their PF6− congener and this activity correlates with the solubility product of the polymer.34 While other factors may play a role in the observed contrasting trend in this present studies, we attributed our findings to the relatively more coordinating ability of BF4−, which will less likely facilitate the formation of free cations.30 Indeed, our findings agreed with those of others47-49 where more coordinating counteranions decease antimicrobial activity of cationic antimicrobial agents. The results also show the absence of dendritic effect in the hydrido and methyl-substituted PF6− dendrimers as no appreciable difference in antimicrobial activity was found between the zeroth (DEN1 and DEN2) and the first generation (DEN7 and DEN8) (Table 2). However, with their chloro-substituted congeners, a noticeable dendritic effect on activity was observed with the first generation being inactive against all microbes (Table 2). Moreover, with the PF6− series, the activity of the dendrimers was tuned by substituting the hydrido group with an electron-donating methyl group or electron-withdrawing chloro group (Table 2). For instance, generally, the methyl-substituted dendrimers (DEN2 and DEN8) were more active than their hydrido (DEN1 and DEN7) or chloro (DEN3 and DEN9) congeners. This is plausible as the methyl group increases the lipophilicity of the dendrimers,24,25 possibly enhancing cell permeability, and ultimately resulting in an increase in biological activity.


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The antimicrobial activity of the macromolecules is impacted by interplay of several parameters that include redox activity.23 As these dendrimers are redox active, they are likely to generate reactive oxygen species (ROS) via electron transfer to oxygen as previously confirmed.23 The presence of ROS induces oxidative stress, a cellular defence strategy employed against a broad spectrum of microbes.50-53 Indeed, a number of redox-active organometallic

% Oxidative Stress Relative to Hydrogen Peroxide

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100

80

60

40

20

0 DEN3

DEN2

DEN1

Vancomycin Hydrogen peoxide

Figure 1. Relative percentage of oxidative stress induced by zeroth generation PF6− dendrimers on MRSA. Hydrogen peroxide was the positive control, while vancomycin, known to induce oxidative stress,50,51 was used as reference.

Figure 2. Field Emission scanning electron micrographs of MRSA (a, without treatment with DEN2; b, treated with DEN2 at the antimicrobial IC50)


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compounds enhance antimicrobial activity via the induction of cellular oxidative stress.23 Using the dichlorodihydrofluorescein (H2DCF) oxidative assay,30,54 we probed the induction of cellular oxidative stress by DEN1–DEN3 on MRSA. Compared with a negative control, DEN1 and DEN2 induced oxidative stress on MRSA with the chloro-substituted dendrimer (DEN3) being less efficient in inducing oxidative stress (Figure 1). The reduced efficiency of DEN3 in inducing oxidative stress may be attributed to its higher Epc (Table 1) as the Epc drives the generation of ROS that induces the oxidative stress.55 The oxidative stress induced by DEN1 and DEN2 were comparable to that of vancomycin, which is known to enhance its antimicrobial activity by inducing cellular oxidative stress on bacteria.56,57 Therefore, it is reasonable to implicate oxidative stress as a contributing mechanism to the antimicrobial activity of the DEN1 and DEN2. In addition to the induction of oxidative stress, the dendrimers are also cationic. Cationic polymers function as antibacterial agents by interacting with the negatively charged bacterial cell membrane, disrupting its integrity, and eventually leading to fatal processes that include depolarization of membrane, disruption of cellular processes, modification of membrane lipid composition and/or leakage of cell content.58 To confirm the membrane-interacting property of the our cationic dendrimers, we used field emission scanning electron microscope (FE-SEM) to visually assess the integrity of the cell membrane of MRSA before and after treatment with the most active dendrimer, DEN2, at its antimicrobial IC50 concentrations. This approach qualitatively confirms a membrane-disrupting mechanism of action of cationic antimicrobial agents.5 The micrographs revealed that the untreated MRSA had intact morphology (Figure 2a) whereas evidence of ruptured and shrunken membranes was found in DEN2-treated MRSA cells at the antimicrobial IC50 (Figure 2b).


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Figure 3. Confocal laser fluorescence micrographs showing autofluorescence of cell membrane and DAPI-stained nuclei of BJ fibroblast cells. a) Without treatment with DEN2; b) treated with DEN2 at 128 µg/mL)

Figure 4. Percentage haemolysis of sheep red blood cells treated with 128 µg/mL of zeroth generation dendrimers.


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Cytotoxicity and Interaction of Dendrimers with Red Blood Cells. Membrane-active antimicrobial agents also interact with mammalian cell membranes, thereby precluding their clinical applications.3,15 We investigated the toxicity of these cationic dendrimers against human epidermal keratinocytes cells (HEka), human foreskin BJ fibroblast cells, and human breast adenocarcinoma cells (HTB-26). The dendrimers were cytocompatible with HEka and BJ fibroblast cells under assay conditions. For instance, DEN2 was cytocompatible with cells even at concentration 8 times the antimicrobial MIC against MRSA (Table 2). Indeed, examination of the cells using confocal laser fluorescence microscopy revealed no treatment-induced changes in morphology of the cell membrane as visualized from intracellular autoflouresence of the cell membrane as well as from the DAPI-stained cell nuclei (Figures 3a and b). Further, DEN1, DEN2 and DEN6 exhibited mild activity against the cancer cells (HTB-26) with DEN2 being most active (Table 2). To gain deeper insight into the cytocompatibility of these dendrimers with mammalian cells, their haemolytic activity was assayed using defibrinated sheep whole blood. Specifically, we treated harvested sheep blood cells with 128 µg/mL of DEN1, DEN2, or DEN3, incubated them at pH = 7.4 for 2 hours, and measured the absorbance of heme released due to haemolysis. Compared to a positive control, these dendrimers did not remarkably disrupt red blood cells membrane under these conditions (Figure 4), which include concentrations that are higher than their MIC.

Conclusion In summary, cationic, redox-active organometallic dendrimers were synthesized and assayed as antimicrobial agents. These antimicrobial agents are rare, as they are based on an organometallic dendritic scaffold and function via two mechanisms, which include interaction with the cell membrane and induction of oxidative stress on bacteria. Our results suggested that both


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mechanisms contributed to the antimicrobial activity of these dendrimers against Gram-positive bacteria including multidrug-resistant strains, methicillin-resistant S. aureus and vancomycinresistant E. faecium. These dendrimers were cytocompatible with human epidermal keratinocytes and human foreskin BJ fibroblast cell lines, as well as mammalian red blood cells at concentrations greater than their antibacterial MIC. Further, the antimicrobial activity was tunable via control of parameters such as lipophilicity, and cationic charge availability. In future work, we plan to exploit the fundamental knowledge gain from this study to build a new library of highly efficacious, cytocompatible antimicrobials. We also plan to investigate the effect of these dendrimers on depolarization of cell membrane, disruption of cellular processes, modification of membrane lipid composition and leakage of cell content. ACKNOWLEDGMENTS The Natural Sciences and Engineering Research Council of Canada is thanked by AA, RK and CA for financial support. We also thank Professor Rabin Bissessur for assistance with thermal and elemental analyses. Dr. Chris Kirby, Stephan Scully, and Maike Fischer are thanked for assistance with NMR analyses. Dorota Wadowska and Patricia Scallion are also thanked for their assistance with confocal laser and scanning electron microscopic imaging, respectively. REFERENCES

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An organometallic dendrimer with activity against multi-‐drug resistant Gram-‐positive via membrane-‐ disrupting and oxidative stress-‐inducing modes of action.


Antimicrobial Organometallic Dendrimers with Tunable Activity against

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