Article pubs.acs.org/Macromolecules
Acid-Activated Antimicrobial Random Copolymers: A MechanismGuided Design of Antimicrobial Peptide Mimics Yunjiang Jiang,†,§,‡ Xin Yang,†,‡ Rui Zhu,† Kan Hu,† Wang-Wei Lan,§ Fang Wu,⊥ and Lihua Yang*,†,§ †
CAS Key Laboratory of Soft Matter Chemistry, CAS Key Laboratory of Materials for Energy Conversion, Department of Materials Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026 China § School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610064 China ⊥ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064 China S Supporting Information *
ABSTRACT: How to reduce the off-target adverse effects during antimicrobial administration remains an ongoing challenge. We show a mechanism-guided design of acid-activated antimicrobial peptide mimics (aSMAMPs) that have antibacterial activity triggered by acidic pH, a factor associated with many infected conditions. The cationicity of membrane-active antimicrobials is known to facilitate activity. By reinforcing a membrane-active antimicrobial random copolymer with an extra pH-responsive monomer, we obtain aSMAMP that is net neutral at physiological pH but net cationic at acidic pH. Plate killing assays indicate that Escherichia coli cells at pH 5.0 rather than those at pH 7.4 are susceptible to such aSMAMPs, whereas the opposite is true when challenged with conventional metabolic antibiotics. Comparison between the aSMAMPs and one homologue that is cationic at both pH conditions suggests that the acid-triggered antibacterial activity of aSMAMPs may be attributed to their pH-tunable net cationicity. At normal blood pH, these aSMAMPs demonstrate greatly diminished hemolytic toxicity against human erythrocytes. Taken together, such aSMAMPs show that switching on-or-off the cationic motif of a membrane-active antimicrobial via pH offers a feasible approach toward “smart” antimicrobials with activity triggered by acidic pH associated with many infected conditions, which may have implications in reducing the off-target adverse effects on both microbiota and host cells during antimicrobial administration.
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INTRODUCTION How to reduce the off-target adverse effects during antimicrobial administration remains an ongoing challenge. One proposed approach is the differential release of nanoparticle-encapsulated antibiotics triggered by pathogenic factors such as toxin1 and lipase,2 aiming to reduce toxicity to host cells. Microbiota are as essential to human health as host cells. 3−5 Disturbance to microbiota is another major component of the off-target adverse effects during antimicrobial administration. Elevated local extracellular acidity is associated with many infected conditions such as dental caries6 and airway surface of cystic fibrosis lung,7 and infections in guts8 and women vagina.9 Lowered pH impairs bacterial eradication in vitro10,11 and in vivo.7 It is therefore desirable to develop antimicrobials which have antibacterial activities triggered by acidic pH, as an approach to improve antibacterial efficacy while reduce the off-target adverse effects on both microbiota and host cells during antimicrobial administration. Many antimicrobial peptides (AMPs) from innate immunity kill bacteria by destabilizing the barrier function of bacterial cytoplasmic membranes,12−17 a generic activity mode which appears to be more difficult for bacteria to circumvent than the specific metabolic targeting modes of antibiotics.18,19 Cati© 2013 American Chemical Society
onicity and hydrophobicitytwo structural motifs most AMPs have in commonare vital for the membrane-activity. The cationic moiety of an AMP facilitates its association with the anionic bacterial membrane surface via electrostatic interactions;12,20 the hydrophobic moiety of an AMP facilitates the subsequent membrane destabilization which leads to cell death.13 By being simultaneously cationic and hydrophobic, synthetic mimics of antimicrobial peptides (SMAMPs) including non-natural peptides,21−28 peptoids,29 oligomers,30−34 and polymers,35−43 have demonstrated similar in vitro antimicrobial activities as natural AMPs. Inefficient electrostatic interactions between cationic AMPs and the nearneutral mammalian cell surface may account for the preferential activity of AMPs against bacteria over host cells.12,44,45 Owing to enhanced cationic charges at acidic pH, histidine-rich AMPs such as Clavanins from Styela clava46 and Hepcidin from human liver47 have demonstrated enhanced antibacterial potency at acidic pH than at neutral pH. Inspired by the aforementioned facilitating effects of net cationic charges on Received: March 6, 2013 Revised: April 22, 2013 Published: May 8, 2013 3959
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membrane-activity, we hypothesize that, by rationally switching on-or-off the cationicity motif via pH, we may achieve SMAMPs specifically active at acidic but not physiological pH (Figure 1).
Article
MATERIALS AND METHODS
The copolymers were prepared via AIBN-initiated free radical copolymerizations of N-(tert-butoxycarbonyl)aminoethyl methacrylate (Boc-AEMA), methacrylic acid (MAA), and methacrylate (MA) to give the Boc-protected precursor copolymers which after subsequent TFA-deprotection yielded the desired copolymer products (Scheme S1, Supporting Information). Methyl 3-mercaptopropionate (MMP) was used as chain transfer agent (CTA) when necessary to control the copolymer molecular weight. 1H NMR analysis was performed on the Boc-precursor copolymers in deuterated dimethyl sulfoxide (dDMSO). GPC analysis was performed using acetonitrile-supplemented acetic acid/acetate buffer (100 mM acetate/acetic acid, 20% acetonitrile, pH 5.0) as mobile phase and poly(ethylene glycol) (PEG) as standard for calibration. Copolymer net charge fractions were quantitatively calculated based on 1H NMR data or qualitatively deduced based on ζ-potential measurements. To evaluate the antibacterial activities of the as-prepared copolymers at different pH, plate killing assays were performed against E. coli (ATCC 25922) in either 10 mM acetate buffer (10 mM HAc, 142 mM NaCl, pH 5.0) or 10 mM Tris−HCl buffer (10 mM Tris, 142 mM NaCl, pH 7.4). To evaluate the hemolytic toxicity of the copolymers, we performed hemolysis assays in buffer at normal blood pH (10 mM Tris, 142 mM NaCl, pH 7.4). More experimental and calculation details are described in the Supporting Information.
Figure 1. Membrane-association, the initiating process for membraneactivity many cationic AMPs and SMAMPs exhibit, is facilitated by the electrostatic interactions between the cationic antimicrobial and the anionic bacterial membrane. By rationally switching on-or-off the “cationic” motif of a SMAMP via pH, we aim to achieve membraneactive antimicrobial polymers with differential antibacterial activity at physiological and acidic pH.
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RESULTS AND DISCUSSIONS The as-proposed MAA-co-AEMA-co-MA random copolymers are prepared via AIBN-initiated free radical copolymerizations of N-(tert-butoxycarbonyl)aminoethyl methacrylate (BocAEMA) MAA, and MA to obtain the Boc-protected precursor copolymers which, after subsequent TFA-deprotection, yield the desired final copolymer products (Scheme S1, Supporting Information). Methyl 3-mercaptopropionate (MMP) is used as chain transfer agent (CTA), when necessary, to control the copolymer molecular weight. To achieve neutrality at pH 7.4 but cationicity at pH 5.0, the aSMAMP copolymers are designed to contain equal amounts of MAA and AEMA (∼40%) (Figure 2a). CPB7one homologue which contains significantly more AEMA than MAAis prepared as a control
To test this hypothesis, we develop acid-activated synthetic mimics of antimcirobial peptides (aSMAMPs) by simply reinforcing a family of well-characterized membrane-active copolymeric SMAMPs with a pH-responsive extra component. Specifically, we reinforce random copolymers of cationic aminoethyl methacrylate (AEMA) and hydrophobic methacrylate (MA), a well-characterized family of membrane-active SMAMPs,36,48,49 with methacrylic acid (MAA)a monomer with pH-dependent deprotonation. We recently show that antimicrobial AEMA-co-MA copolymers may act by permeabilizing the cytoplasmic membranes of bacterial cells and, during their membrane-destabilization processes, negative Gaussian curvature (NGC) may be generated.49 As homologues of AEMA-co-MA copolymers, MAA-co-AEMA-co-MA copolymers may inherit the membrane-activity. Cationicity is known to facilitate membrane-activity. Therefore, aSMAMPscopolymers containing equal amounts of MAA and AEMA and thus being net cationic at acidic but not physiological pHare expected to exhibit acid-triggered membrane-activity (Figure 1). Plate killing assays indicate that Escherichia coli cells at pH 5.0 rather than those at pH 7.4 are susceptible to these aSMAMPs, whereas the opposite is true when challenged with conventional metabolic antibiotics. Comparison between these aSMAMPs and one homologue that is cationic at both examined pH indicates that the acid-triggered antibacterial activity of aSMAMPs may be attributed to their pH-tunable cationic charges. At normal blood pH 7.4, these aSMAMPs are barely hemolytic against human erythrocytes, owing to their noncationicity. With acid-triggered antibacterial activity and diminished hemolytic toxicity, these aSMAMPs show that switching on-or-off the cationic motif via pH offers a feasible approach toward “smart” antimicrobials with activity triggered by the acidic pH, a factor locally accompanying many infected conditions. Such acid-activated “smart” antimicrobials may have implications for targeted antibacterial activity at the acidic sites of infections and hence enhanced efficacy while reduced disturbance to both microbiota and host cells during antimicrobial administration.
Figure 2. (a) Structures of our aSMAMP copolymers which contain equal amounts of MAA and AEMA to achieve neutrality at pH 7.4 but cationicity at pH 5.0. Variation in the side-group in methacrylate (R1) and end-group of a chain (R2) leads to a series of aSMAMPs used in this work. (b) Structure of CPB7 which contains significantly more AEMA than MAA is used as a control to examine the effect of cationicity. 3960
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(Figure 2b). 1H NMR spectroscopy and gel permeation chromatography (GPC) are used to characterize the asprepared copolymers, which yield their monomer compositions and molecular weights (Table 1 and Table S3, Supporting Table 1. Monomer Molar Ratios and Average Molecular Weights of the As-Prepared Copolymers copolymer CPH1 CPH2 CPH4 CPB1 CPB2 CPB4 CPE1 CPB7
fexpected (MAA:AEMA:MA)
fa (MAA:AEMA:MA)
Mwb (g/mol)
0.40:0.40:0.20
0.39:0.40:0.21 0.37:0.39:0.24 0.40:0.39:0.21 0.40:0.40:0.20 0.39:0.41:0.20 0.39:0.39:0.22 0.40:0.40:0.20 0.32:0.44:0.24
21 500 6050 1980 13 100 7420 2160 65 600 33 900
0.30:0.45:0.25
a
Monomer molar ratios are calculated based on 1H NMR spectra of the Boc-protected precursor copolymers. bMolecular weights are obtained based on GPC characterizations on the TFA-deprotected copolymers, using acetonitrile-supplemented acetate/acetic acid buffer (100 mM acetate/acetic acid buffer, 20% acetonitrile, pH 5.0) as mobile phase and PEG as standards for calibration.
Information). Because of coexistence of carboxylic and amine groups in a TFA-deprotected final copolymer, we quantify the monomer composition in a copolymer using the 1H NMR spectrum on the corresponding Boc-protected precursor copolymer (Figure S1−S5, Supporting Information), given that TFA deprotection process does not significantly change methacrylate copolymer structure.36 Gel permeation chromatography (GPC) characterizations yield the molecular weights and polydispersities of the as-prepared copolymers (Figure S6, Supporting Information). Despite their heterogeneity, random copolymer SMAMPs have been demonstrated to destabilize membranes in similar ways as do homogeneous natural AMPs.49 The theoretical net charge fraction of a copolymer is quantitatively calculated using the Henderson−Hasselbalch equation based on 1H NMR data. The calculations indicate that all aSMAMPs are cationic at pH 5.0 but almost neutral at pH 7.4, whereas CPB7 is cationic at both pH values (Figure 3a). Net charge property of the as-prepared copolymers can also be qualitatively deduced based on ζ-potential measurements which show that all aSMAMPs exhibit positive ζ-potentials at pH 5.0 but negative ζ-potentials to various extent at pH 7.4 whereas CPB7 exhibited cationic ζ-potentials at both pH (Figure 3b). Therefore, both theoretical charge fraction calculations and ζpotential measurements are consistent on that these aSMAMPs have cationicity “off” at pH 7.4 but “on” at pH 5.0. To assess whether these aSMAMPs have differential antibacterial activity as expected, we perform comparative bactericidal assays against E. coli (ATCC 25922) at pH 7.4 and 5.0. Assay results show that our aSMAMPs demonstrate differential bactericidal activity at pH 5.0 and 7.4 (Figure 4a−c). Challenged by CPH1one of our aSMAMPscells at pH 5.0 survive dose-dependently, with 89% of CPH1-treated cells survive at doses up to 128 μg/mL (Figure 4a). Such differential activity is observed with CPH2 and CPH4, two homologues which compared to CPH1 have same monomer compositions but smaller molecular weights (Figure 4b), as well as CPB1 and CPE1, two homologues which compared to CPH1 have similar
Figure 3. (a) Theoretical charge calculations show that all aSMAMPs are cationic at pH 5.0 but almost neutral at pH 7.4, whereas CPB7 is cationic at both pH. (b) ζ-Potential measurements show that all aSMAMPs exhibit cationic ζ-potentials at pH 5.0 but almost neutral or negative ζ-potentials at pH 7.4, whereas CPB7 exhibits cationic ζpotentials at both pH.
monomer molar ratios but shorter alkyl side-chains (Figure 4c). That our aSMAMPs are bactericidal at pH 5.0 but not pH 7.4 suggests that cationicity may be a necessary condition for polymeric SMAMPs to act against gram-negative bacteria such as E. coli. In an additional set of control experiments, we challenge the E. coli strain with gentamicina conventional metabolic antibiotic that targets the bacterial ribosomal machinery instead of the membrane. Although gentamicintreated cells survive dose-dependently at both pH, those at pH 5.0 survive at significantly higher percentages than those at 7.4. 24.0% of cells at pH 5.0 survive at dose of 128 μg/mL, whereas 90% hemolysis at 500 μg/mL (Figure 5b). The about 10 times lower hemolytic toxicity of our aSMAMPs compared to their cationic homologueCPB7suggests that removal of net cationic charges may help greatly diminish the hemolytic toxicity in future SMAMP development.
Figure 4. (a) Killing assays against E. coli show that CPH1 demonstrate differential bactericidal activity at pH 7.4 and 5.0. At pH 5.0, the survival percentage of CPH1-treated cells decreases dosedependently, with survival percentage 89% of CPH1-treated cells survive at doses up to 128 μg/mL. (b) Similar differential bactericidal activity is observed with CPH2 and CPH4, two homologues which have smaller molecular weights than CPH1. At pH 5.0, CPH2 and CPH4 at 64 μg/mL kill E. coli cells to survival percentages of 21.5% and 28.6%, respectively. In contrast, at pH 7.4, > 99% cells treated at same doses survive. (c) Similar differential activity is also observed with CPE1 and CPB1, another two homologues of CPH1 but with shorter alkyl side-chain in methacrylate monomer. At pH 5.0, CPE1 and CPB1 at 64 μg/mL kill E. coli cells to survival percentages of 69.5% and 21.8%, respectively. In contrast, at pH 7.4, > 98% cells treated at same doses survive. (d) Opposite trend is true when we challenge the E. coli strain with gentamicina conventional metabolic antibiotic. Gentamicin-treated cells at pH 5.0 survive at significantly higher percentages than those at
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CONCLUSIONS In conclusion, we develop “smart” membrane-active antimicrobials with acid-triggered bactericidal activity, as an approach to reduce disturbance to both microbiota and host cells during 3962
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Macromolecules antimicrobial administration. As a proof-of-concept, we develop acid-activated synthetic mimics of antimicrobial peptides (aSMAMPs). Our aSMAMPs which have net cationicity at pH 5.0 but not pH 7.4 demonstrate bactericidal activity at pH 5.0 but not pH 7.4, which suggests that net cationicity may be a necessary condition for membrane-active SMAMP copolymers to act against gram-negative bacteria such as E. coli. In stark contrast, the opposite is true when E. coli cells are challenged with gentamicina conventional metabolic antibiotic. Therefore, acid-activated antimicrobials such as aSMAMPs reported herein may have implications as novel antimicrobial candidates for improved antibacterial efficacy at acidic sites of infection while reduced disturbance to normal microbiota at physiologically nonacidic sites. Comparison between the aSMAMPs and one homologue which is cationic at both pH suggests that the as-observed differential antibacterial activity of aSMAMPs may be attributed to their pH-tunable net cationicity. At normal blood pH, these aSMAMPs demonstrate around 10 times lower hemolytic toxicity compared to their cationic homologue, which suggests that removing cationic charges may help diminish hemolytic toxicity in future SMAMP development. With acid-triggered activity and low hemolytic toxicity, our aSMAMPs show that switching on-or-off the net cationic motif via pH offers a feasible approach toward “smart” antimicrobials with activity triggered by the acidic pH, a factor locally accompanying many infected conditions. Such acid-activated “smart” antimicrobials may have implications for targeted antibacterial activity at the acidic sites of infections and hence enhanced efficacy while reduced disturbance to both microbiota and host cells during antimicrobial administration.
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ASSOCIATED CONTENT
Copolymer synthesis and characterization, including NMR spectra, GPC traces, net charge calculations, ζ-potential measurements, and biological assays. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* (L.Y.) E-mail:
[email protected] Telephone: (86) 551 6360 6960. Author Contributions ‡
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS
AMP, antimicrobial peptide; SMAMP, synthetic mimic for antimicrobial peptide; MAA, methacrylic acid; AEMA, aminoethyl methacrylate; MA, methacrylate; NMR, nuclear magnetic resonance; GPC, gel permeation chromatography; MMP, methyl 3-mercaptopropionate; CTA, chain transfer agent; ATCC, American Type Culture Collection; E. coli, Escherichia coli
S Supporting Information *
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ACKNOWLEDGMENTS
The authors thank Gerard C. L. Wong and Ghee Hwee Lai for helpful discussions, Hongjun Liang for critical reading and comments, Jun Wang and Yanhua Zhu at the University of Science and Technology of China for assistance on ζ-potential measurements, and Yuande Long at Chengdu Institute of Organic Chemistry (Chinese Academy of Sciences) for GPC characterizations. The authors also thank Gang Wei, Senbao Lin, Ge He, and Jing He for technical assistance. This work was supported in part by the National Natural Science Foundation of China (11074178, 21174138, J1030412) and Ministry of Education of China (SRF for ROCS 20091001-9-8, SRF for DPHE 20090181120046, FRF for CU WK2060140008) . 3963
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