Antimicrobial and Hemolytic Activities of Copolymers with Cationic and ...

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Antimicrobial and Hemolytic Activities of Copolymers with Cationic and Hydrophobic Groups: A Comparison of Block and Random Copolymersa Yanqiang Wang, Junjuan Xu, Yueheng Zhang, Husheng Yan,* Keliang Liu*

Random and diblock copolymers of 2-(N,N-dimethylamino)ethyl methacrylate and butyl methacrylate are prepared by ATRP. As mimics of cationic antimicrobial peptides, the random and diblock copolymers show similar antimicrobial activities. In contrast, the diblock copolymers have much lower hemolytic activities than the random copolymers. The cell selectivity (HC50/MIC, where HC50 is the concentration to lyse 50% of human red blood cells and MIC is the minimum concentration to inhibit bacterial growth) of the diblock copolymers are 150 to 27 500 times higher than that of random copolymers with similar compositions.

Introduction The ever-increasing prevalence of bacterial resistance to traditional antibiotics has reached alarming levels.[1] We are now facing the threat of so-called ‘‘superbugs,’’ pathogenic bacteria resistant to most or all available antibiotics. Thus, there is great interest in the development of new classes of antimicrobial agents with fundamentally Y. Wang, Y. Zhang, Prof. H. Yan Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China E-mail: [email protected] Prof. J. Xu Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Prof. K. Liu Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China E-mail: [email protected] a

Supporting Information is available from the Wiley Online Library or from the author.

Macromol. Biosci. 2011, 11, 1499–1504 ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

different modes of action than those of traditional antibiotics. Cationic antimicrobial peptides, which are distributed widely in many organisms from invertebrates to humans, are a possible new class of antibiotics.[2–4] Most cationic antimicrobial peptides interact with and permeabilize microbial membranes,[2–4] although a few such peptides have other mechanisms of action.[5,6] The exact mechanism of membrane permeabilization is still not completely understood. However it is generally accepted that positively charged residues such as Lys and Arg in cationic antimicrobial peptides attach to the negatively charged bacterial cell membrane; and the hydrophobic residues trigger membrane permeation and the disruption of the pathogen cells, which leads to a breakdown of the membrane potential, plasma leakage, and cell death.[2–4] The development of resistance to these membrane-active peptides is not expected, as it would require substantial changes in the lipid composition of the cell membranes. However, the application of cationic antimicrobial peptides as drugs has been hindered by their facile degradation by proteolytic enzymes in the blood, high manufacturing costs, and poor pharmacokinetics.[4,7]

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DOI: 10.1002/mabi.201100196

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Y. Wang, J. Xu, Y. Zhang, H. Yan, K. Liu www.mbs-journal.de

In recent years, considerable attention has been focused on synthetic polymers with cationic groups and hydrophobic moieties that mimic the essential features of cationic antimicrobial peptides.[8–25] These antimicrobial polymers have many advantages over antimicrobial peptides, including easy synthesis and tailoring and stability toward enzymatic degradation. These polymers are becoming increasingly important for a wide range of applications, including clinical therapeutic agents, medical devices, and healthcare products. Similar to antimicrobial peptides, some antimicrobial polymers show toxicity to mammalian cells,[8–13,20–23] which would limit their utility in medical applications. Among the antimicrobial polymers, those prepared by radical (co)polymerization of vinyl monomers are particularly interesting, because of their advantages of easy preparation and low cost.[8–19] However, vinyl polymers often suffer from low selectivity to cell types or high levels of toxicity to human cells, although the cell selectivity of these polymers can be improved (i.e., such that the polymers lyse bacterial rather than mammalian cells) by tuning the composition, cations (primary and tertiary amines and quaternary ammonium groups), hydrophobic groups, and spatial relations between the positive charge and the alkyl tail in the random copolymers.[8–13] There are only a few reports on block copolymers as antimicrobial polymers,[16–18] and to our knowledge no study has compared the cell selectivity of random and block copolymers. In this report, the arranged forms of repeating units along block or random copolymer chains with tertiary amine groups and hydrophobic side chains were examined. In particular, we investigated the effect of the arranged forms on the antimicrobial and hemolytic activities and cell selectivity.

Deoxygenated cyclohexanone (4.4 mL), DMAEMA (1.18 g, 7.5 mmol), and BMA (1.08 g, 7.5 mmol) were added to the flask. PMDETA (0.260 g, 1.5 mmol) was added, and the mixture in the flask was stirred until the solution changed from cloudy and colorless to clear and light green (about 20 min). MBrP (0.251 g, 1.5 mmol) was added, and the flask was immersed into a water bath preheated to 70 8C. Polymerization was carried out at 70 8C for 12 h with stirring. The resulting mixture was diluted with dichloromethane (10 mL) and filtered through a column packed with neutral alumina to remove the catalyst. The filtrate was concentrated by rotary evaporation under reduced pressure. The polymer was precipitated into petroleum ether (30–60) and dried to give the random copolymer P(BMA-co-DMAEMA) (R1). Figure S1 shows the 1H NMR spectrum of the copolymer. The degrees of polymerization of the BMA and DMAEMA repeat units, which were calculated from an analysis of the peak integration of the 1H NMR spectrum based on the characteristic peaks at d ¼ 3.6 (a), 4.6 (d, e), and 2.7 (f), were 8.4 and 7.1, respectively. Random copolymers R2–R6 were prepared similarly, and their preparation conditions and structures are shown in Table S1 (see the Supporting Information) and Table 1.

Experimental Section

Synthesis of PDMAEMA-block-PBMA Diblock Copolymers

Materials 1,1,4,7,7-Pentamethyldiethylenetriamine (PMDETA) and methyl 2bromopropionate (MBrP) were purchased from Aldrich. 2-(N,Ndimethylamino)ethyl methacrylate (DMAEMA, Acros) and butyl methacrylate (BMA, Aldrich) were distilled over CaH2 under reduced pressure before use. The Escherichia coli and Staphylococcus aureus bacterial strains were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Deionized water was used to prepare all solutions.

Synthesis of Random P(BMA-co-DMAEMA) Copolymers Typically, CuBr (0.215 g, 1.5 mmol) was added to a Schlenk flask equipped with a stir bar. After sealing with a rubber septum, the flask was evacuated and purged with nitrogen (3 cycles).

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Synthesis of Bromo-Terminated Poly(butyl methacrylate) (PBMA) Macroinitiators Typically, CuBr (0.287 g, 2 mmol) was added to a Schlenk flask equipped with a stir bar. After sealing with a rubber septum, the flask was evacuated and purged with nitrogen (3 cycles). Deoxygenated cyclohexanone (5 mL) and BMA (2.58 g, 18 mmol) were added to the flask. PMDETA (0.346 g, 2 mmol) was added, and the mixture in the flask was stirred until the solution changed from cloudy and colorless to clear and light green (about 20 min). MBrP (0.334 g, 2 mmol) was added, and the flask was immersed into a water bath preheated to 40 8C. Polymerization was carried out for 12 h with stirring. The resulting mixture was diluted with dichloromethane (10 mL) and filtered through a column packed with neutral alumina to remove the catalyst. The filtrate was concentrated by rotary evaporation under reduced pressure. The polymer was precipitated into methanol-water (1/4 v/v) and dried to give PBMA-Br.

CuBr (72 mg, 0.5 mmol) and the above-prepared PBMA-Br (0.64 g) were added to a Schlenk flask equipped with a stir bar. After sealing with a rubber septum, the flask was evacuated and purged with nitrogen (3 cycles). Deoxygenated cyclohexanone (2 mL) was added to the flask, and the mixture was stirred until the polymer was dissolved. Deoxygenated PMDETA (87 mg, 0.5 mmol) was added to the flask, and the mixture was stirred until the solution changed from cloudy and colorless to clear and light green. Deoxygenated DMAEMA (1.57 g, 10 mmol) was added to the flask, and the mixture was stirred for 10 min. The flask was immersed into a water bath preheated to 60 8C. Polymerization was carried out for 12 h with stirring. The resulting mixture was diluted with dichloromethane (10 mL) and filtered through a column packed with neutral alumina to remove the catalyst. The filtrate was concentrated by rotary evaporation under reduced pressure. The polymer was precipitated into petroleum ether (30–60) and dried to give the diblock


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Antimicrobial and Hemolytic Activities of Copolymers with Cationic . . . www.mbs-journal.de

Table 1. Characterization and biological activities of copolymers.

Polymer

DRDMAEMA/DRBMAa)

fDMAEMAa)

Mn a) [kDa]

Mw /Mn b)

MIC [mg mL–1] S. aureus

HC50 [mg mL–1]

E. coli

Selectivity (HC50/MIC) S. aureus

E. coli

R1

7.1/8.4

0.46

2.49

1.16

31

40

0.25

0.0081

0.0063

R2

17.6 /18.1

0.49

5.53

1.31

100

125

0.1

0.001

0.0008

R3

11.5/10.7

0.52

3.51

1.25

100

100

0.5

0.005

0.005

R4

18.4/12.8

0.59

4.89

1.31

40

36

0.6

0.015

0.017

R5

9.9/5.6

0.64

2.53

1.21

28

28

0.13

0.0046

0.0046

R6

17.8/6.3

0.74

3.87

1.19

26

26

0.07

0.0027

0.0027

B1

15.5/11.8

0.57

4.29

1.32

67

97

250

3.7

2.6

B2

15.3/10.0

0.60

4.00

1.25

51

54

300

5.9

5.6

B3

19.6/12.3

0.61

5.01

1.31

65

70

380

5.8

5.4

B4

10.2/6.1

0.63

2.64

1.37

34

73

360

11

4.9

B5

15.4/8.2

0.65

3.76

1.30

55

55

590

11

11

B6

14.9/6.4

0.70

3.43

1.41

58

58

1 290

22

22

a)

DRDMAEMA and DRBMA (the degree of polymerization of DMAEMA and BMA repeat units, respectively), fDMAEMA (molar fraction of DMAEMA in the polymer chains), and Mn were calculated from analysis of the peak integration of the 1H NMR spectra; b)Determined from gel permeation chromatography. copolymer poly(butyl methacrylate)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (PBMA-b-PDMAEMA, B2). Figure S8 shows the 1H NMR spectrum of the copolymer. The degrees of polymerization of the BMA and DMAEMA repeat units, which were calculated from an analysis of the peak integration of the 1H NMR spectrum based on the characteristic peaks at d ¼ 3.6 (a), 4.6 (d,e), and 2.7 (f), were 10.0 and 15.3, respectively. Diblock copolymers B1 and B3–B6 were prepared similarly, and their preparation conditions and structures are shown in Table S2 (see the Supporting Information) and Table 1.

Measurement of Antimicrobial Activity Stock solutions of copolymers were dissolved in 0.01 M hydrochloric acid (final pH ¼ 5.0–7.0). Bacterial strains of E. coli and S. aureus were individually grown in nutrient broth (10 g of peptone, 3 g of beef powder, and 5 g of NaCl per L of medium) overnight at 37 8C. The medium was diluted with physiological saline to an absorbance at 600 nm of 0.001 [105 colony-forming units (CFU)]. Twofold serial dilutions of the copolymer solution were added to 1 mL of the medium containing the inocula of the test strains. Growth inhibition was determined by measuring the absorbance at 600 nm after incubation at 37 8C for 24 h. The antibacterial activity is expressed as the minimal inhibitory concentration (MIC).

remove plasma. The erythrocytes were rinsed three times with phosphate-buffered saline (PBS) and diluted into PBS to get the stock suspension (5% RBC v/v). Twofold serial stock solutions of the copolymer were added to 1 mL of medium containing the stock suspension. The final concentration of RBC was 2.5%. The mixture was kept at 37 8C for 60 min and then centrifuged (4 000 rpm, 10 min). The absorbance of the supernatant at 414 nm was detected. A control solution that contained only PBS was used as a reference for 0% hemolysis. A control solution that contained 1% Triton X-100 was used as a reference for 100% hemolysis. Hemolytic activity is expressed as the concentration to lyse 50% of human RBCs (HC50).

Results and Discussion Random copolymers P(BMA-co-DMAEMA) (R1–R6, Figure 1) were prepared by initiating a mixture of BMA and DMAEMA with different feed ratios (using MBrP as an initiator) via atom-transfer radical polymerization (ATRP),

Measurement of Hemolytic Activity Toxicity to human red blood cells (RBCs) was assessed by a hemoglobin release assay using the same polymer stock solutions used for the MIC measurement. Fresh human erythrocytes were obtained by centrifuging whole blood (3 000 rpm, 10 min) to


Figure 1. Structures of the random copolymer series P(BMA-coDMAEMA) and the block copolymer series PBMA-b-PDMAEMA.


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similar to a process reported previously.[26] Predetermined molecular weights and narrow molecular weight distributions are the characteristics of (co)polymers prepared by ATRP. In the preparation of the random copolymers, the numbers of the repeating units of both of BMA and DMAEMA were greater than those expected based on the feed ratios. For example, the numbers of the repeating units of BMA and DMAEMA were 8.4 and 7.1 in R1 while the [MBrP]/[BMA]/[DMAEMA] was 1/5/5 (molar ratio). This may be explained by high bimolecular determination of polymer radicals or even initiator radicals due to the high initiator concentration because of the high initiator/ monomer ratios (low molecular weights of these polymers). Bimolecular determination predominates in the initial stages of the polymerization.[27] In the precipitation process, the lower molecular component was removed and thus the numbers of the repeating units were greater than those expected. ATRP is also a facile method to prepare block copolymers.[28] Diblock copolymers PBMA-b-PDMAEMA (B1–B6, Figure 1) with similar compositions to the random copolymers were prepared by ATRP. The PBMA macroinitiator was prepared by the initiation of BMA (using MBrP as the initiator) via ATRP, and the initiation of DMAEMA by the PBMA macroinitiator via ATRP gave diblock copolymers. For the random and diblock polymer series, the number of repeating units (degree of polymerization) of each segment and the average molecular weights were calculated from an analysis of the peak integration of the 1 H NMR spectra of the copolymers, as shown in Table 1. Antimicrobial activities (MIC) against the Gram-positive bacterium S. aureus and Gram-negative bacterium E. coli and the hemolytic activity (HC50) of the copolymers were determined (Table 1). The random and diblock copolymers both showed antimicrobial activities against S. aureus and E. coli. These copolymers are mimics of cationic antimicrobial peptides. The amine groups of the repeating units of PDMAEMA in these copolymers would be protonated in physiological conditions, which would render the polymers as cationic. The PBMA would provide hydrophobic moieties. These structural features are similar to those of cationic antimicrobial peptides, and the antimicrobial activities of the copolymers are comparable to or slightly lower than those of most natural occurring cationic antimicrobial peptides (e.g., host-defense peptides[29]). In contrast to the similar antimicrobial activities of the copolymer series, the hemolytic activity of the random copolymers was much higher than that of the diblock copolymers (Table 1). The random copolymers showed a very high toxicity to human RBCs, with HC50 values of 60– 1 250 times less than the MIC values. The cell selectivity (HC50/MIC) of the random copolymers ranged from 0.0008 to 0.017 (Table 1). Conversely, the diblock copolymers had higher HC50 values than MIC values, with HC50 values that

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were 410–18 400 times higher than those of the random copolymers (Table 1). The selectivity of the diblock copolymers ranged 2.6–22 (i.e., 150–27 500 times higher than those of the random copolymers with similar compositions and molecular weights). The distinct properties of the block and random copolymers may be exploited in the design of antimicrobial polymers with high antimicrobial activity coupled with low hemolytic activity or high cell selectivity. The block copolymers are much better materials than the random copolymers for biomedical applications. The hemolytic activity of the block copolymers decreased with increases in the number of positive charges (i.e., the mole fraction of DMAEMA) within the range studied or with decreases in the hydrophobicity (i.e., the mole fraction of BMA) (Figure 2a). The cell selectivity of the block copolymers increased with increases in the number of positive charges or with decreases in the hydrophobicity (Figure 2b). It has been believed that facially amphiphilicity is a key structural feature for the membrane-activity of most of antimicrobial peptides and polymers.[30–35] Kuroda and DeGrado[8] and Mowery et al.[22] proposed that random copolymers containing hydrophobic groups and amino groups can form ideal facially amphiphilic structures in the induction of a membrane surface. However, for block

Figure 2. Plots of (a) hemolytic activity (HC50) and (b) selectivity (HC50/MICS.aureus) against the mole fraction of DMAEMA ( fDMAEMA) for the block copolymers.



Antimicrobial and Hemolytic Activities of Copolymers with Cationic . . . www.mbs-journal.de

structure that is crucial for hemolysis, due to flexibility of the polymer backbone. In contrast, hydrophobicity rather than amphiphilicity is important for antimicrobial activity. These results may improve our understanding of the different mechanisms of lysis between bacterial and mammalian cells.

Acknowledgements: We thank the National Natural Science Foundation of China (20774048) and the National Key Technologies R&D Program for New Drugs of China (2009ZX09301-002) for support of this study.

Received: May 27, 2011; Revised: June 23, 2011; Published online: August 4, 2011; DOI: 10.1002/mabi.201100196 Keywords: antimicrobial; atom-transfer radical polymerization (ATRP); cationic polymers; diblock copolymers; hemolysis Figure 3. Schematic illustration of the induction of facially amphiphilic structures for copolymers upon binding to mammalian cell membranes.

copolymers such as those shown in this paper, such facially amphiphilic structure may only be formed in the region adjacent to the joint of two blocks and no such structure can be formed along most of the chains, due to the block structural characteristic (Figure 3). The facially amphiphilic structure may be crucial for hemolysis, but less important for antimicrobial activity. This mechanism is supported by several previous studies on cationic antimicrobial peptides.[36–38] Using analogs of the cationic antimicrobial peptide melittin, we previously observed that the amphiphilicity is crucial for hemolysis, while hydrophobicity is important for antimicrobial activity.[36] Dathe et al.[37] and Oren and Shai.[38] showed that partial destabilization of the a-helical conformation by the incorporation of a few Dresidues into cationic antimicrobial peptides abrogates the hemolytic activity, but not the antimicrobial activity.

Conclusion Random and diblock copolymers of methacrylates with tertiary amine and butyl groups were prepared by ATRP. The random and diblock copolymer series showed similar antimicrobial activities. The random copolymers showed very high hemolytic activity and very low cell selectivity, while the diblock copolymers showed much lower hemolytic activity and much higher cell selectivity than random copolymers with similar compositions and molecular weights. The different behaviors of these copolymer series may be attributed to the fact that the random copolymers, but not block copolymers, can form the facially amphiphilic


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