Antibiotic Activity of Iron-Sequestering Polymers | Biomacromolecules

Apr 14, 2015 - Zhou, T.; Neubert, H.; Liu, D. Y.; Liu, Z. D.; Ma, Y. M.; Kong, X. L.; Luo, W.; Mark, S.; Hider, R. C. Iron binding dendrimers: a novel...
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Antibiotic Activity of Iron-Sequestering Polymers Nashwa El-Gendy,†,∥ Jian Qian,† Kate Eshelman,‡ Mario Rivera,‡ and Cory Berkland*,†,§ †

Department of Pharmaceutical Chemistry and ‡Department of Chemistry, The University of Kansas, Multidisciplinary Research Building, 2030 Becker Drive, Lawrence, Kansas 66047, United States § Department of Chemical and Petroleum Engineering, The University of Kansas, Learned Hall, 1530 West 15th, Lawrence, Kansas 66045, United States ∥ Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt ABSTRACT: Increasing antibiotic resistance has compelled the development of novel antibiotics and adjuvant therapies that enhance the efficacy of existing antibiotics. Iron plays a critical role in bacterial infections, yet the use of iron chelators as adjuvant therapy with antibiotics has yielded highly variable outcomes. Multivalent polymeric materials offer an alternative approach to bind and sequester iron via high avidity interactions. Here, a biomimetic ironsequestering polymer (PAI-DHBA) was synthesized by modifying side chains of cross-linked polyallylamine (cPAI) with 2,3-dihydroxybenzoic acid (DHBA). PAI-DHBA polymer gels with various DHBA contents showed high iron affinity indices and high selectivity for iron. The polymers showed mild antibiotic properties when used to treat established bacterial cultures. Pretreating culture media with PAI-DHBA polymer, however, removed all detectable iron from media and effectively inhibited the growth of Pseudomonas aeruginosa. In addition, bacterial growth was more susceptible to antibiotics combined with PAI-DHBA. Multivalent polymers that bind and sequester iron, such as PAI-DHBA, offer a promising early intervention or adjuvant to antibiotics.

1. INTRODUCTION Pseudomonas aeruginosa is a quintessential example of a problematic Gram-negative pathogen that can cause a wide range of human infections.1−4 It is a frequent cause of acute lifethreatening infections in burn wounds and of chronic infections, for example in the lungs of cystic fibrosis (CF) patients. P. aeruginosa is also notorious for developing resistance to antimicrobial agents and continues to cause serious public health problems worldwide.5−7 Iron is an essential nutrient needed as a cofactor in bacterial respiration, nitrogen fixation, photosynthesis, and DNA synthesis and repair.8,9 Sequestration of iron from the local environment or depletion from bacterial iron storage represents a feasible antimicrobial strategy. Iron depletion has been shown to weaken bacteria and produce an adjuvant effect if combined with antibiotics.10,11 Some small molecular iron chelators have shown an antimicrobial effect.12,13 Deferasirox (marketed as Exjade), an FDA approved iron chelator for the treatment of chronic iron overload, showed a synergistic effect against Vibrio vulnificus infections combined with standard antibiotics such as ciprofloxacin.14 Deferasirox can cause serious damage to the kidneys or liver or severe bleeding in the stomach or intestines and was the second drug on the list of “Most frequent suspected drugs in reported patient deaths” compiled by the Institute for Safe Medical Practices in 2009.15−17 In some studies, EDTA exhibited activity against Gram-positive bacteria but was much less effective against Gram-negative bacteria.18 © 2015 American Chemical Society

Conversely, EDTA was also found to increase P. aeruginosa biofilm formation when used alone at certain concentrations.4 Other low-molecular-weight iron chelators have also demonstrated evidence of toxicity at near therapeutic doses, but results tend to be highly variable and can depend on testing conditions.16,17 For the topical treatment of bacterial infections, smallmolecule iron chelators might have a marginal effect against common Gram-negative infections, and the small molecular size could possibly allow absorption through skin tissue. A nonabsorbable chelator with the ability to bind and sequester iron may be an ideal material for a safe topical treatment of bacterial infections, such as P. aeruginosa infections on burns and wounds. Polymers, especially cross-linked polymeric materials, cannot be absorbed through skin, thereby limiting concerns about toxicity. Moreover, the combination of ironsequestering materials with traditional antibiotics may produce a synergistic therapeutic effect and reduce the minimum inhibitory concentrations (MICs) of antibiotics. Here, polymeric iron sequestrants (i.e., polymers that bind and retain iron) were synthesized and tested as antibiotics and adjuvants. Effective polymeric iron sequestrants were designed with several key features such as high affinity, a large binding capacity, and selectivity for iron. Polymers mimicking the Received: November 10, 2014 Revised: April 13, 2015 Published: April 14, 2015 1480

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weight gain, indicative of complete swelling. The swelling ratio was defined as the fractional increase in the weight of the cross-linked polymer because of water absorption.23 The swelling ratio of the crosslinked polymers was determined by the following equation:

structure of high-affinity iron-chelating siderophores produced by bacteria (e.g., enterobactin) were synthesized. Primary amine groups on polyallylamine (PAI) were simultaneously cross-linked by N,N′-methylenebis(acrylamide) (MBA) and conjugated with 2,3-dihydroxybenzoic acid (DHBA) molecules, which serve as iron-chelation sites. The resultant ironsequestering polymer (PAI-DHBA) showed strong affinity and high selectivity for iron. This polymer was studied as an antibiotic against P. aeruginosa alone and in combination with the antibiotics ciprofloxacin and gentamycin, which are commonly used for treatment of bacterial infections, especially those caused by P. aeruginosa.19−21

Swelling ratio =

Ws − Wd Wd

(1)

where Ws and Wd represent the weight of polymer after full swelling in PBS and the weight of dried polymer, respectively. 2.5. Determination of the Iron Affinity Index. The iron affinity index of the PAI-DHBA polymers was measured using a ligand competition assay. The competitive chelation of iron by the polymers in equilibrium with EDTA (a water-soluble chelator) was used to determine the affinity index. Briefly, 1.5 mL of 10 mM EDTA solution, 2 mL of 5 mM FeCl3 solution, 21.5 mL of PBS, and 10 mg of polymer were mixed together and rotated at 25 °C for 5 days. Then, the concentration of the soluble iron complex was determined by inductively coupled plasma optical emission spectrometry (ICPOES; Optima 2000 DV, PerkinElmer, USA). The affinity index of the polymer was determined following the procedure reported in literature.24 In the equilibrium situation, the system could be represented in the following way (for brevity, all charges have been omitted):

2. MATERIALS AND METHODS 2.1. Materials. PAI (56 kDa), DHBA, triethylamine (TEA), MBA, N-(3 dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich. Ciprofloxacin hydrochloride (Cipro) was purchased from MP Biomedicals, Inc. Gentamicin sulfate (Gent), sodium ethylene diamine tetraacetate (EDTA), agar, and potassium chloride (KCl) were purchased from Fisher Scientific. Luria broth media (LB, pH 7.1) was purchased from Teknova. N,N-bis (2-hydroxybenzyl) ethylenediamine-N,N-diacetic acid (HBED) was purchased from Strem Chemicals, Inc. Sodium chloride (NaCl) was purchased from Acros Organics. Potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) were purchased from Santa Cruz Biotechnology, Inc. 2.2. Bacterial Strains and Growth Conditions. P. aeruginosa strain PA01 wild type (WT) purchased from the University of Washington Genome Sciences was used in all studies. P. aeruginosa was grown in M63 minimal media (2 g of KH2PO4, 13.6 g of (NH4)2SO4, 2 g of glucose, 5 g of casamino acids, 0.25 g of tryptophan, 4 g of citric acid, 3 μM FeSO4·7H2O, and 1 mM MgSO4 per 1 L water, pH 7.0) modified after O’Toole and Kolter.22 All glassware was acid-washed by soaking in nitric acid and rinsed five times with deionized water. 2.3. Preparation and Characterization of Iron-Sequestering Polymer. NHS-activated DHBA was synthesized before preparing the polymer. A solution of DHBA (770 mg, 5 mmol) and NHS (690 mg, 6 mmol) in 5 mL of DMF was mixed with a solution of EDC (1200 mg, 6.2 mmol) in 5 mL of DMF. The mixture was stirred at room temperature for 8 h and used for the next step without any purification. The PAI cross-linking and DHBA conjugation were conducted in a single step. Briefly, a 15% w/w PAI hydrochloride (56 kDa) solution containing a predetermined amount of MBA (5%, molar ratio of cross-linker to total amines) was prepared in H2O/DMF (50:50 v/v) mixture. Then, the NHS-activated DHBA solution with a desired DHBA/amine molar ratio (5−40%) was added to the solution. After sonication for 2 min to get a transparent solution, TEA was added and the solution was mixed thoroughly. Then, the solution was incubated at room temperature for 48 h. The cross-linked polymer gels were first washed with 0.1 M sodium hydroxide and then deionized water for several days under the protection of nitrogen and then lyophilized. The polymer gels were ground to powder for subsequent studies. The polymer samples with 0, 5, 10, 15, 20, 25, 30, 35, and 40% of DHBA/amine molar ratios were denoted as G0, G5, G10, G15, G20, G25, G30, G35, and G40, respectively. The particle size of the ground powder was ∼100 μm, as measured by optical microscopy. Because the polymers were cross-linked particles, the DHBA conjugation ratios could not be characterized directly by NMR analysis. Instead, the unconjugated DHBA left in the solution after the reaction was determined by NMR. The real conjugated DHBA ratio was calculated by deducting the unconjugated DHBA ratio from the feed DHBA ratio. 2.4. Swelling Studies. The swelling behavior of PAI-DHBA was studied in PBS buffer (pH 7.4). Dried polymer samples were placed in PBS buffer at room temperature. The weight of the swollen polymer samples was determined at different time points until there was no

Fe(EDTA) + 3DHBA ⇌ Fe(DHBA)3 + EDTA where

Keq =

[Fe(DHBA)3 ][EDTA]

[Fe(EDTA)][DHBA]3 [Fe(DHBA)3 ] [Fe][EDTA] = × [Fe(EDTA)] [Fe][DHBA]3

(2)

The iron stability constant of DHBA (Q) could be defined as follows:

Q=

[Fe(DHBA)3 ] [Fe][DHBA]3

(3)

The iron stability constant of EDTA (K) could be defined as

K=

[Fe(EDTA)] [Fe][EDTA]

(4)

Herein, the iron affinity index was defined as log Q. On the basis of eqs 2−4 iron affinity index = log Q = log(Keq × K ) = log Keq + log K (5) The value of K was known, and Keq could be easily calculated on the basis of eq 2. As the total EDTA concentration was higher than the total Fe concentration, we assumed that there is no free iron in solution. The iron concentration detected by ICP-OES could be considered as the concentration of Fe(EDTA). The concentration of EDTA in the solution after competition could be calculated as the total EDTA concentration subtracted by the concentration of Fe(EDTA). The concentration of Fe(DHBA)3 could be calculated as the total concentration of iron subtracted by the concentration of Fe(EDTA). The concentration of DHBA could be calculated as the total concentration of DHBA subtracted by three times of the concentration of Fe(DHBA)3. The iron affinity index shows that the polymer chelates iron more strongly than EDTA. 2.6. Determination of the Iron Sequestration Capacity. A known mass of different PAI-DHBA polymers was incubated in a 5 mM FeCl3 solution in the presence of 5 mM EDTA as a stabilizer at 25 °C for a week. The remaining iron concentration was determined by ICP-OES. 2.7. Selectivity study. The iron selectivity of PAI-DHBA polymers was determined in the presence of copper, zinc, manganese, calcium, nickel, and potassium. A solution containing all these metal ions, each at a concentration of 0.4 mM, was prepared in a phosphate buffer at pH 7.2 containing 2 mM EDTA. A predetermined amount of 1481

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Biomacromolecules polymers was added into the solution and incubated at 25 °C for 5 days. The concentration of each metal ion remaining in solution was determined by ICP-OES. For the selectivity in M63 media, a predetermined amount of PAI-DHBA polymer (G25) was added into the M63 solution and incubated at 25 °C for 3 days. The concentrations of the metals (Fe3+ and Mg2+) remaining in the solution were determined by ICP-OES. 2.8. Bacterial Growth Using PAI-DHBA-Treated Media. The iron-sequestering polymer PAI-DHBA (G25) was selected for studies with P. aeruginosa. Prior to bacterial growth, cross-linked polymer cPAI (G0) or PAI-DHBA (G25) powder was washed twice with phosphate buffer saline (PBS at pH 7.4) and once with deionized water, then lyophilized. A 50 mL aliquot of M63 media containing 1, 10, or 20 mg/mL insoluble G25 was incubated for 20 min with shaking (230 rpm, 37 °C). In addition, 50 mL of M63 media was also incubated with G0 (13.9 mg/mL) for 20 min to control for the absence of DHBA moieties in the polymer; 13.9 mg/mL G0 was used because this mass is equivalent to 20 mg of G25, which is composed of 13.9 mg of cPAI and 6.1 mg of DHBA. After a 20 min incubation, the polymer was separated by centrifugation (4000 rpm, 4 °C, and 15 min), and the supernatant was transferred to an acid-washed glass Erlenmeyer flask (250 mL). A 10 mL aliquot of media was removed to analyze iron content by ICP-OES. Bacterial growth was also examined using M63 medium prepared without the addition of FeSO4·7H2O. To initiate bacterial growth, a single colony of P. aeruginosa was inoculated into 5 mL of LB (25 g/L, pH 7.1) and grown overnight with shaking at 230 rpm and 37 °C. The overnight inoculum was centrifuged for 12 min at 4000 rpm and 4 °C and then resuspended in 5 mL of fresh M63 media. Resuspended cells were transferred to 40 mL of polymer-treated or untreated M63 media, which resulted in an OD600 = 0.01. The cells were cultured at 37 °C and 230 rpm. Samples (1−2 mL) were removed from cultures every hour to measure OD600. At 2, 4, 6, 8, and 10 h of growth, 100 μL of culture was serially diluted in PBS and plated on LB agar plates using a previously described dropplate method.25 The LB/agar plates were incubated between 16 and 18 h at 37 °C. Single colonies were enumerated and colony-forming units per milliliter (CFU/mL) were determined. 2.9. Bacterial Growth in the Presence of PAI-DHBA (G25). A culture grown overnight in LB was diluted with LB to achieve an OD600 = 0.3. The diluted culture was then added to 1 mL of M63 media in clear flat-bottomed 24-well plates (Midsci) for a starting OD600 = 0.003. Then, the appropriate amount (1, 5, 10, 15, or 20 mg/ mL) of G25 was added to each well in the 24-well plate to compare different concentrations of the polymer. The plate was wrapped with Parafilm to avoid evaporation. After incubating at 37 °C and 40 rpm for 12 h, the entire content of each well was serially diluted in PBS (pH 7.4) and plated on LB agar. The plates were incubated between 16 and 18 h at 37 °C, and colonies were enumerated to determine CFU/mL. 2.10. Bacterial Growth in the Presence of PAI-DHBA Compared to Traditional Iron Chelating Agents. The experiments were set up in 24-well plates as previously described. Chelators, including 20 mg of PAI-DHBA (G25) or 208 mg of EDTA (equivalent to 500 μM), were added to the media immediately after inoculation with P. aeruginosa. CFU/mL values were determined after 5, 6, and 12 h of incubation. 2.11. PAI-DHBA as an Adjuvant to Conventional Antibiotics on Bacterial Growth. Ciprofloxacin (1 μg/mL) or gentamicin (24 μg/mL) was added to 24-well plate cultures after 5 h of incubation. G25 (20 mg/mL) was added to M63 media immediately after inoculation with P. aeruginosa or together with the antibiotics after 5 h of growth. At different time intervals of incubation (5, 6, 7, and 9 h), the entire content of each well was serially diluted and plated on LB agar to determine CFU/mL. cPAI (G0) was also tested with ciprofloxacin by adding polymer immediately after inoculation with bacteria. Ciprofloxacin was also added to 1 mL cultures after 12 h of incubation. G25 (20 mg/mL) was added to media either immediately after inoculation or simultaneously with ciprofloxacin. CFU/mL were obtained 12, 13, 14, 16, and 24 h after starting the incubation.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PAI-DHBA Polymer. Previous preparations of PAI-DHBA polymer used a two-step synthesis strategy.26 PAI hydrochloride was first cross-linked with MBA by a Michael-type addition reaction and then the formed PAI hydrogel was further conjugated to DHBA via EDC/NHS conjugation chemistry. This two-step strategy was time-consuming, and in the second step, DHBA conjugation may be favored near the particle surface. In this report, the polymer cross-linking and DHBA conjugation were conducted in a single step. DHBA conjugation was controlled by adjusting the DHBA/polymer feed ratios. Several PAIDHBA polymers with varied DHBA content (5−40% of total amines) but the same cross-linking density (5%) were prepared via this one-step strategy (Scheme 1). DHBA conjugation ratios Scheme 1. Synthesis of Cross-Linked PAI-DHBA Polymer

Table 1. Synthesis and Characterization of PAI-DHBA Polymers sample

cross-linking densitya

feed DHBA/ amineb

found DHBA/ aminec

G0 G5 G10 G15 G20 G25 G30 G35 G40

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

0 0.0311 0.0700 0.1117 0.1488 0.1743 0.2216 0.2689 0.3216

swelling ratio at pH 7.4 17.2 11.8 8.2 6.9 5.6 5.4 5.3

± ± ± ± ± ± ±

2.1 1.9 1.1 0.8 1.2 0.6 0.4

a

Feed molar ratio of cross-linker to total amines. bFeed molar ratio of DHBA to total amines. cFound molar ratio of DHBA to total amines by a modified NMR analysis.

(Table 1) were determined by NMR analysis. As the DHBA content increased from 5 to 30%, the swelling ratios decreased from 11.8 to 5.3, indicating that the gel became more hydrophobic as DHBA conjugation increased. When incubated with Fe3+ solution, all the PAI-DHBA samples exhibited a dark color, indicating chelation with Fe3+, whereas the PAI gel did not show a color change. The strength of iron chelation is an important parameter for iron-chelating materials; however, affinity cannot be calculated for materials in the conventional sense. Because the polymers are cross-linked particles, the chelation between the polymer materials and iron ions presents a heterogeneous system, and direct equilibrium constants are not obtainable. Thus, the term “iron affinity index” was used to assess how strongly the polymers bind and trap iron relative to a reference iron chelator with a documented stability constant. The iron affinity index was determined by a ligand competition method in equilibrium with EDTA (iron stability constant = 1025). The iron affinity 1482

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DHBA content increased from 5 to 30%, the affinity indexes of polymers decreased from 32.2 to 28.1. It should be noted that, theoretically, all the samples should have almost the same affinity indices, because the intrinsic affinity indexes of the DHBA groups in different samples were the same. As the DHBA content increased, however, the increased hydrophobicity of the polymers may have hindered Fe3+ access or coordination, hence reducing the apparent iron affinity indexes based on the calculation. In comparison, cPAI (G0) did not bind iron in the presence of EDTA at pH 7.4, suggesting that the affinity of G0 for iron is lower than that of EDTA. The iron sequestration capacity, which describes the maximum iron adsorption by the polymers, was also investigated. To reach the maximum iron sequestration, all the samples were incubated in a Fe3+ solution for 1 week. The theoretical and experimental iron sequestration capacities of the polymers with various DHBA contents were determined (Figure 1B). As the DHBA content increased, the experimental iron sequestration capacities also went up for low DHBA conjugation (5−20%) and reached a plateau (20−30%) at around 20 mg Fe per gram of polymer. For all the samples tested, only the samples with low DHBA content achieved the theoretical iron sequestration capacities. The increased hydrophobicity of the polymers at higher DHBA conjugation percentages probably limited Fe3+ access to the gel-particle interior. Another possible reason may be that the positive surface of the particle and the increased density of the polymer network after iron chelation prevent the further diffusion of iron ions to the deep particle interior. It is noteworthy that after the polymers containing chelated iron were incubated with fresh PBS containing 2 mM EDTA for 1 week, iron was not detectable in the medium by ICP-OES (data not shown), indicating that iron sequestration by the polymers is not reversible. 3.2. PAI-DHBA Exhibited High Selectivity for Iron. Selectivity to iron is especially important for the application of iron-sequestering polymers in the biological field. Poor selectivity may affect the bioavailability or the balance of essential metal ions such as Cu2+, Zn2+, Ca2+, Mn2+, Ni2+, or K+. The influence of other metals on the sequestration of Fe3+ by the polymers was investigated using a multimetal system. The concentration of each metal was fixed at 0.4 mM, and the metal/polymer ratio was fixed at 0.2 mmol per gram of polymer. All the samples absorbed almost 100% of the iron present in the media, whereas typically the absorption for other essential metals was considerably lower, demonstrating high selectivity for iron (Figure 1C). PAI-DHBA polymer G25 showed the highest selectivity with optimal iron-sequestering capacity and stability constant. Accordingly, it was selected for testing its effectiveness in inhibiting bacterial growth. The selectivity of the PAI-DHBA polymer (G25) was tested in the M63 media used in the P. aeruginosa studies. The M63 media only contained Fe3+ and Mg2+ metals. All of the Fe3+ and only about 12% of the Mg2+ in the solution were sequestered by the polymer (Figure 2). When considering swelling of the polymer, Mg2+ is likely primarily physically absorbed with imbibed water, rather than specifically chelated. 3.3. Treating Growth Media with PAI-DHBA Suppresses Bacterial Growth. M63 media was treated with 1, 10, or 20 mg/mL of G25 as described in the Materials and Methods section. The effect of treating the media with G25 on P. aeruginosa growth was assessed by enumerating CFU/mL as

index was calculated on the basis of the equation for the calculation of stability constant as described in section 2.5. All the polymers with various DHBA contents showed higher iron affinity indices than EDTA (Figure 1A). The G10 sample had the highest iron affinity index (32.2), which indicated that the iron affinity of G10 polymer is 107 times stronger than EDTA (log stability constant = 25.1, also shown in Figure 1A for comparison). For all the other samples tested, they all showed at least 103 times stronger iron affinity than EDTA. As the

Figure 1. (A) Iron affinity indexes of PAI-DHBA polymers. Affinity indexes were measured using a ligand competition assay. Competitive chelation of iron by PAI-DHBA hydrogels in equilibrium with a watersoluble iron chelator EDTA. (B) Iron sequestration capacities of PAIDHBA polymers (mg Fe/g PAI-DHBA): ■ , theoretical Fe sequestration capacity, ▼, experimental Fe sequestration capacity. (C) Metal selectivity studies for essential metals (mmol metals/g PAIDHBA). All the metals have the same initial concentration (0.4 mM) for the study. EDTA (2 mM) was added to maintain the stability of Fe3+. 1483

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treated with G0 was nearly identical to growth in untreated media (Figure 3B, ● and ▼). These observations are consistent with the fact that G0 does not sequester iron from the medium (Table 2). In fact, growing P. aeruginosa in M63 Table 2. Analysis of Iron Content Left in Treated M63 Media Using ICP-OES treatments untreated media untreated media prepared without adding iron G0-treated media G25-treated media (1 mg/mL) G25-treated media (10 mg/mL) G25-treated media (20 mg/mL)

Figure 2. Metal selectivity studies for PAI-DHBA polymers in M63 media. One milliliter of media was incubated with 20 mg G25.

a function of time (Figure 3A). Small differences in cell growth were observed in cultures treated with 1 or 10 mg/mL of G25,

iron content left in media (μM) ± S.D. 4.9 ± 1 2.7 ± 0.4 4.6 ± 0.3 1.2 ± 0.02 0.5 ± 0.04 undetectable

prepared by omitting iron (FeSO4·7H2O) showed that the low iron content in the medium slowed bacterial growth significantly (Table 2 and Figure 3B, ○). However, depleting iron to undetectable levels with G25 treatment resulted in arrested growth and bacterial death. We also investigated the effect of treating the M63 media with polymer immediately after inoculation with P. aeruginosa. In these experiments, bacterial cultures in 24-well plates were treated with 0, 1, 5, 10, 15, and 20 mg/mL G25. As observed in the 50 mL cultures, treatment with 20 mg/mL G25 caused severe growth retardation (Figure 4).

Figure 4. Effect of varying G25 concentrations (0, 1, 5, 10, 15, and 20 mg/mL) in 1 mL cultures using 24-well plates. Bacterial growth was assessed by enumerating CFU/mL 12 h after inoculation. Figure 3. (A) Effects of treating M63 media with G25 on P. aeruginosa growth. Treating media with 1 mg/mL (○) or 10 mg/mL (▼) G25 slows P. aeruginosa growth relative to growth in untreated media (●). Treating media with 20 mg/mL G25 not only arrests growth but also shows bactericidal effects (△). (B) Treating M63 media with 13.9 mg/ mL G0 (▼) does not affect bacterial growth relative to P. aeruginosa growth in untreated media (●). Growth in untreated M63 medium prepared with omission of iron is significantly slower (○). Growth in M63 media treated with 20 mg/mL G25 shows bacterial death (△).

3.4. PAI-DHBA Is a More Potent Inhibitor of Bacterial Growth than Traditional Iron Chelators. Currently, the most commonly used drug for systemic iron chelation therapy is deferoxamine (DFO).27 Despite the efficacy of DFO as a chelator, the utility of this drug is limited because of high toxicity and a very short plasma half-life (∼5.5 min).28−30 Other low-molecular-weight iron chelators have also been explored, but they have also demonstrated evidence of toxicity at near-therapeutic doses.16,17 High-molecular-weight or crosslinked iron-chelating polymers have emerged as a promising mode of topical or nonabsorbed iron chelation therapy.30−32 In this study, the effect of a well-known low-molecularweight iron chelator was studied on the growth of P. aeruginosa and compared to the effect of G25. A 208 mg aliquot of EDTA (equivalent to 500 μM) was added to the M63 media

relative to the cell growth in untreated media. In contrast, cell growth in media treated with 20 mg/mL G25 was arrested, and in fact, cell death was observed (△ in Figure 3A). Similar experiments, in which M63 was treated with G0, that is, polymer devoid of the iron-chelating DHBA moieties, were also conducted. The results showed that growth in media 1484

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Biomacromolecules immediately after inoculation and the CFU/mL determined after 5, 6, and 12 h of incubation (Figure 5). A small, transient

Figure 5. G25 is more effective at chelating the iron and keeping it from P. aeruginosa compared to EDTA. At 500 μM, EDTA causes a slight delay in bacterial growth compared to growth in untreated M63 medium. In comparison, treating media with 20 mg/mL G25 has a strong retardation effect.

difference between the control (untreated) and EDTA-treated media was observed after 6 h of incubation, but it disappeared after 12 h, with values resembling those of untreated media. At pH 7.0, the EDTA affinity constant for Fe (III) is 1025.33 In comparison, pyoverdin binds Fe(III) with an affinity constant of 1025;34 thus, the reversible chelation of iron by EDTA13,35 may have facilitated iron uptake via siderophores secreted by P. aeruginosa. In contrast, nearly irreversible sequestration of iron by PAI-DHBA efficiently depletes the medium from iron and inhibits bacterial growth. 3.5. Adjuvant Effect of Iron-Sequestering Polymer on the Antimicrobial Activity of Ciprofloxacin and Gentamicin against P. aeruginosa. Iron depletion has been shown to enhance the bactericidal effect of antibiotics,11,36 such that the combination of an iron chelator and antibiotics may have synergistic therapeutic effects. Aminoglycosides such as gentamicin and fluoroquinolones such as ciprofloxacin are commonly used to treat P. aeruginosa infections in clinical practice.3 In this study, the potential additive or synergistic effect of these antibiotics and PAI-DHBA (G25) was investigated (i) on cultures where media was treated with G25 (20 mg/mL) at the time of inoculation and supplemented with either ciprofloxacin (1 μg/mL) or gentamicin (24 μg/mL) after 5 h and (ii) on cultures in log phase (5 h after inoculation), treated simultaneously with polymer and antibiotic at the indicated concentrations. The concentrations of antibiotics reflect reported MICs against P. aeruginosa20,37 Results obtained from treating media at the time of inoculation are shown in Figure 6A. Compared to control, G25(−) and Cipro(−), cultures treated with antibiotic alone, G25(−) and Cipro(+), showed significant growth inhibition, with an overall reduction in CFU/mL of approximately 4 log units at 9 h. Treatment with polymer alone, G25(+) and Cipro(−), caused similar growth inhibition at the 9 h as treatment with ciprofloxacin, except that the growth inhibition was more noticeable in the earlier hours when the polymer was present. Treatment with polymer and ciprofloxacin, G25(+) and Cipro(+), caused even larger growth inhibition than treatment with either antibiotic alone or polymer alone, strongly suggesting synergistic action.

Figure 6. (A) Addition of 20 mg/mL G25 to M63 media immediately after inoculation with P. aeruginosa causes growth retardation relative to untreated media. Treatment with G25 (20 mg/mL) immediately after inoculation and with ciprofloxacin (1 μg/mL) 5 h after inoculation results in significantly lower CFU/mL values relative to treatment with only Ciprofolxacin 5 h after inoculation. (B) G25 (20 mg/mL) and ciprofloxacin (1 μg/mL) were added simultaneously 5 h after inoculation with P. aeruginosa. Treatment with G25 alone results in modest retardation compared to treatment at the time of bacterial inoculation (see Figure 6A). Simultaneous treatment with G25 and ciprofloxacin is more effective in reducing viable cells than treatment with only ciprofloxacin. (C) Addition of G0 (13.9 mg/mL) to M63 media immediately after inoculation with P. aeruginosa does not significantly affect bacterial growth relative to untreated media. Simultaneous treatment with G0 and ciprofloxacin (1 μg/mL) is less effective than treatment with ciprofloxacin alone. G0 may lower the efficiency of the antibiotic by sequestration or by deactivation.

Results from treating cultures with polymer and antibiotic during log phase are shown in Figure 6B. Control and 1485

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G25 by treating cell cultures in stationary phase (12 h, Figure 8). The observations suggest that bacteria in growing cultures

ciprofloxacin treated cultures were very similar to those in Figure 6A. Note, however, that polymer exerted a significantly lower growth retardation effect when added to growing cultures, and although there was a synergistic effect of adding polymer and ciprfloxacin to growing cultures, the inhibitory effect was less pronounced than treating media with polymer at the time of inoculations. Results obtained from treating media with cross-linked polymer cPAI (G0) at the time of inoculation are shown in Figure 6C. Compared to control, G0(−) and Cipro(−),cultures treated with the cross-linked polymer alone, G0(+) and Cipro(−), showed growth similar to that observed with the untreated media. Treatment with cPAI and ciprofloxacin caused significantly less growth inhibition than treatment with antibiotic alone, suggesting that the polymer either absorbed or deactivated ciprofloxacin. Similar experiments were carried out to study possible synergistic effects with gentamicin (Figure 7). When polymer

Figure 8. (A) To study the effect of ciprofloxacin on cells grown to stationary phase in the presence of G25 (20 mg/mL), the antibiotic (1 μg/mL) was added 12 h after inoculation. Treatment with polymer and ciprofloxacin causes significant reduction in cell viability relative to treatment with Ciprofolxacin alone. (B) Simultaneous treatment with G25 (20 mg/mL) and ciprofloxacin (1 μg/mL) 12 h after inoculation with P. aeruginosa is more effective at decreasing cell viability than treatment with ciprofloxacin alone.

or in stationary phase may have accumulated iron in storage proteins, such as bacterioferritn,38 which enables growth and survival even if the medium is depleted of iron by treatment with polymer. In contrast, when the medium is iron-depleted before active cell division, bacterial cells cannot store iron and become more susceptible to iron starvation. Figure 7. (A) Treatment with G25 (20 mg/mL) immediately after inoculation with P. aeruginosa, followed by treatment with Gentamycin (24 μg/mL) 5 h after bacterial inoculation causes a mild reduction in cell viability relative to treatment with only G25. (B) Simultaneous treatment with G25 (20 mg/mL) and Gentamycin (24 μg/mL) 5 h after inoculation with P. aeruginosa also causes only a mild reduction in cell viability relative to treatment with only Gentamycin.

4. CONCLUSIONS A novel iron-sequestring polymer, PAI-DHBA, which mimics the structure of a high affinity iron-chelating siderophore, was prepared using a new single-step synthesis. The antibacterial activity of a PAI-DHBA (G25) against P. aeruginosa was examined alone and in combination with the conventional antibiotics ciprofloxacin or gentamicin. Treating the growth media with G25 reduced iron to undetectable levels and resulted in arrested growth and bacterial death. The combination of the polymer and selected antibiotics demonstrated a high degree of synergism against P. aeruginosa. Synergistic combinations of iron-sequestering polymers and existing drugs may significantly reduce the MICs of antibiotics and provide a promising way to combat multidrug resistance.

was added at the time of inoculation, there was significant growth retardation (Figure 7A), whereas when polymer and antibiotic were added simultaneously to growing cultures, the effect was much less pronounced (Figure 7B). It is noteworthy that iron sequestration from the media by polymer was less efficient at inhibiting bacterial growth when carried out mid log phase. Similar observations were made with ciprofloxacin and 1486

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AUTHOR INFORMATION

Corresponding Author

*The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, United States. Phone: (785) 864-1455. Fax: (785) 864-1454. E-mail: [email protected]. Author Contributions

N.E, J.Q., and K.E. contributed equally to this work. Notes

The authors declare no competing financial interest

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ACKNOWLEDGMENTS This study was supported by grants from the Institute for Advancing Medical Innovation to C.B. and from the National Science Foundation to M.R. (MCB 1158469). We also thank Karla Leslie and Karenin Peltier (Tertiary Oil Recovery Program; TORP) for ICP-OES assistance.

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