pH-Sensitive Zwitterionic Polymer as an Antimicrobial Agent with

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pH-Sensitive Zwitterionic Polymer as an Antimicrobial Agent with Effective Bacterial Targeting Peng Liu, Gang Xu, Dicky Pranantyo, LiQun Xu, Koon-Gee Neoh, and En-Tang Kang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00723 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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ACS Biomaterials Science & Engineering

pH-Sensitive Zwitterionic Polymer as an Antimicrobial Agent with Effective Bacterial Targeting

Peng Liu, Gang Xu, Dicky Pranantyo, Li Qun Xu, Koon-Gee Neoh, En-Tang Kang* Department of Chemical and Biomolecular Engineering National University of Singapore Kent Ridge, Singapore 119260

* To whom correspondence should be addressed E-mail: [email protected] (E.T.K) 1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract It is highly desirable to develop new and more potent biocompatible antimicrobial agents to reduce the increasing risk of bacterial infection worldwide. To address this problem, we prepared a

smart

pH-sensitive

polymer,

poly(N’-citraconyl-2-(3-aminopropyl-N,N-dimethyl

ammonium)ethyl methacrylate), or P(CitAPDMAEMA), which can undergo change in functionality from a biocompatible zwitterionic polymer to an antimicrobial cationic polymer at acidic bacterial infection sites. The precursor polymer, poly(2-(3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate) (P(APDMAEMA)), was first prepared by reversible additionfragmentation chain transfer (RAFT) polymerization, and then modified with citraconic anhydride to obtain the zwitterionic P(CitAPDMAEMA). P(CitAPDMAEMA) is zwitterionic at physiological pH and exhibits low hemotoxicity and good biocompatibility. However, P(CitAPDMAEMA) can change from neutral to cationic with decreasing pH because of the hydrolysis of citraconic amide under low pH conditions. This switch leads to pronounced bacteria binding of cationic P(CitAPDMAEMA) under acidic conditions of the infection sites and significantly inhibits the growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). These results indicate that P(CitAPDMAEMA) is potentially a new on-demand antimicrobial agent.

Keywords: pH-sensitive, zwitterionic polymer, biocompatible, charge-convertible, targeting, antimicrobial.

2 ACS Paragon Plus Environment

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1. Introduction Diseases related to microbial infections are of great concern in various areas, and cause severe worldwide health problems and even death every year. Since the discovery of penicillin in 1928, antibiotics have played an important role in fighting bacterial infections by inhibiting the growth of bacteria or killing the bacteria. However, the overuse of antibiotics has led to a worldwide rise in bacterial resistance, a new challenge in infectious disease treatment today.1,2 In order to overcome this problem, considerable efforts have been devoted to the discovery of new antimicrobial agents.3,4

Because of the facile preparation with desired structure, synthetic cationic polymers mimicking natural antimicrobial peptides have been developed as alternative antimicrobial agents.5-9 Polymers with quaternary ammonium groups10-14 are probably the most explored type of polymeric biocides, and they can kill bacteria via a membrane disruption mechanism. Although these cationic polymers showed high antibacterial efficiency and ease of preparation, their high cytotoxic and hemolytic effects on human cells have limited their wide spread applications. Therefore, there is a need to develop new and versatile macromolecular antimicrobial agents which have effective antimicrobial activities with minimal toxicity towards host cells for treatment of bacterial infections.

Zwitterionic polymers have been extensively investigated as stealth materials in drug delivery systems because of their poor interactions with biological proteins. They show an excellent biocompatibility, and can escape from reticuloendothelial system (RES) to achieve long circulation time in bloodstream.15-17 The infection sites usually shows localized acidity as a



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result of the combined actions of bacterial metabolism and host immune response.18-20 Due to massive infiltration of neutrophils and macrophages during the process of inflammatory responses to microorganism infection, local acidosis occurs and the pH of infection sites can decrease to as far as 5.5.21 Staphylococcus aureus, which causes severe clinical infections, can grow over a wide pH range from 4.2 to 9.3.22 Thus, it is desirable to fabricate a pH-sensitive polymers with antimicrobial quaternary ammonium groups, which are zwitterionic under physiological pH and cationic in an acidic environment. This approach can reduce the toxicity of common cationic polymers under physiological conditions and recover their antimicrobial ability “on-demand” at acidic infection sites. Many pH-sensitive charge-conversion polymers23-27 have been developed as drug delivery systems in cancer therapy. These polymers are negatively charged under physiological conditions, whereas they can switch to cationic polymers at acidic tumor sites. Wang et al.28 have fabricated polymers which can switch from zwitterions to cations under acidic conditions for anticancer drug delivery. Although these charge-convertible polymers have been widely investigated for drug delivery in cancer therapy, they were seldom utilized as antimicrobial agents.

In order to fabricate a pH-sensitive charge-convertible polymer with high content of quaternary ammonium groups for antimicrobial therapy, we developed a zwitterionic polymer, poly(N’-citraconyl-2-(3-aminopropyl-N,N-dimethyl

ammonium)ethyl

methacrylate)

(P(CitAPDMAEMA)), with bacterial targeting and antimicrobial ability (Scheme 1). Due to the presence of quaternary ammonium and carboxyl groups in citraconic acid, P(CitAPDMAEMA) is zwitterion and shows good biocompatibility. However, P(CitAPDMAEMA) can switch back to poly(2-(3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate) (P(APDMAEMA)) and


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becomes cationic under acidic conditions because of the hydrolysis of citraconic amide and exposure of quaternary ammonium and primary amine groups. The cationic nature of P(APDMAEMA) makes it easy to bind to bacteria and inhibit bacterial growth effectively in an acidic environment.



2. Experimental Section 2.1. Materials 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 98%), citraconic anhydride (Cit, 98%), fluorescamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%) and 4-cyanopentanoic acid dithiobenzoate (CPADB, 98%) were purchased from Sigma-Aldrich Chemical Co. The dialysis tube membrane with molecular weight cut-off (MWCO) of 1000 was purchased from Spectrum, Inc. The deionized (DI) water was produced from a Water Still Aquatron A4000D. Escherichia coli (E. coli, ATCC®25922TM) and Staphylococcus aureus (S. aureus, ATCC® 25923TM) were purchased from American Type Culture Collection (Manassas, VA). The LIVE/DEAD BacLight Bacterial Viability Kit L131152 was purchased from Thermo Fisher Scientific Inc.. All other regents were purchased from Sigma-Aldrich or Merck Chem. Co., and were used without further purification. N-Boc-3-bromopropylamine was synthesized according to the method reported in the literature.29

2.2. Synthesis of 2-(N-Boc-3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate (BocAPDMAEMA) N-Boc-3-bromopropylamine (9.4 g, 39.5 mmol) was dissolved in acetonitrile (20 mL) and DMAEMA (5.4 mL, 31.8 mmol) was added to the mixture. The solution was stirred at room temperature for 24 h and poured into diethyl ether. The resulting suspension was stirred for 1 h and the ether phase was decanted. The process was repeated three times and the residue was dried under reduced pressure to obtain the BocAPDMAEMA. 1H-NMR (600 MHz, CDCl3): δ 6.12 (s, 1H), 5.65 (d, 1H), 4.67 (d, 2H), 4.03 (d, 2H), 3.86 - 3.69 (m, 2H), 3.42 (d, 6H), 3.32 3.16 (m, 2H), 2.03 - 1.95 (m, 2H), 1.93 (s, 3H), 1.40 (s, 9H).


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2.3. Synthesis of poly(2-(3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate) (P(APDMAEMA)) Homopolymerization of BocAPDMAEMA was conducted via reversible addition-fragmentation chain transfer (RAFT) polymerization in dimethyl sulfoxide (DMSO) at 65 °C, using 2,2’azobis(2-methylpropionitrile) (AIBN) as the initiator and CPADB as the chain transfer agent. Briefly, BocAPDMAEMA (3 g, 9.5 mmol) was dissolved in DMSO (4.5 mL), and then CPADB (23.1 mg, 0.082mmol) and AIBN (5.4 mg, 0.0328 mmol) were added to the solution. The mixture was degassed by purging with argon for 30 min and polymerization was carried out at 65 °C for 20 h. The reaction mixture was dialyzed against pure water utilizing dialysis tube membrane (MWCO: 1000) for 3 days and poly(2-(N-Boc-3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate), P(BocAPDMAEMA), was obtained by freeze drying.

P(BocAPDMAEMA) (1 g) was dissolved in methanol (1 mL), and trifluoroacetic acid (TFA, 2 mL) was added. The solution was stirred at room temperature for 10 h and poured into diethyl ether. The precipitate was collected by centrifugation and dried under reduced pressure to obtain P(APDMAEMA).

2.4.

Synthesis

of


ammonium)ethyl

methacrylate) (P(CitAPDMAEMA)) P(APDMAEMA) (0.2 g) was dissolved in water (2 mL) and citraconic anhydride (0.9 mL) was gradually added to the mixture. The reaction mixture was maintained at pH 8.5 by adjusting with 3 M NaOH solution, and stirred at room temperature overnight. The resulting solution was



dialyzed against water (pH 8-9) utilizing dialysis tube membrane (MWCO: 1000) for 2 days. The product, P(CitAPDMAEMA), was obtained by freeze drying. The molecular weights and polydispersity index (PDI) of the product were measured using a Waters’ gel permeation chromatography (GPC) system equipped with a 2410 refractive index detector and two Ultrahydrogel columns. Millipore ultra-pure water was used as the mobile phase at a flow rate of 1.0 mL/min and 40 °C.

For fluorescein labelling of P(CitAPDMAEMA), P(APDMAEMA) (100 mg) was dissolved in DI water (1 mL) and fluorescein isothiocyanate (FITC, 1 mg) was added. The mixture was stirred for 6 h at room temperature. Citraconic anhydride (0.45 mL) was gradually added to the mixture. The reaction mixture was maintained at pH 8.5 by adjusting with 3 M NaOH solution, and stirred at room temperature overnight. The resulting solution was dialyzed against water (pH 8-9) utilizing a dialysis tube membrane (MWCO: 1000) for 2 days. The product, P(CitAPDMAEMA)-FITC, was obtained by freeze drying.

2.5. Hydrolysis of citraconic amide of P(CitAPDMAEMA) The kinetics of citraconic amide hydrolysis of P(CitAPDMAEMA) was monitored by the fluorescamine method as described previously.30 P(CitAPDMAEMA) was dissolved in deionized (DI) water to a final concentration of 5 mg/mL, and 50 µL of this stock solution was mixed with 450 µL of citric acid-sodium phosphate buffer (pH 7.4). The same process was carried out in pH 6.0 or pH 5.0 buffers. These mixtures were incubated at 37 °C for 24 h, and at each predetermined time interval, 10 µL of each sample was taken out and diluted into 1000 µL of borate buffer (pH 9.3, 0.1 M). This mixture was added by 10 µL of fluorescamine solution in


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N,N-dimethylformamide (2 mg/mL) and incubated at room temperature for 10 min. Its fluorescence at 470 nm was measured under an excitation wavelength of 375 nm. Positive control (100% of exposed amine) was determined from the fluorescence of sample solution after incubating the P(CitAPDMAEMA) in 0.01 M HCl overnight, and negative control (0% of exposed amine) was determined from the fluorescence of fluorescamine in blank buffer solution.

2.6. Hemolysis assay of P(CitAPDMAEMA) Fresh rabbit whole blood was purchased from InVivos Pte Ltd (Singapore). Briefly, fresh rabbit whole blood was diluted in phosphate-buffered saline (PBS) to achieve 8% blood content (by volume). P(CitAPDMAEMA) solutions (100 µL) of different concentration were mixed with equal volume of the diluted blood suspension (100 µL). The mixtures were incubated at 37 °C for 2 h, and then subjected to centrifugation at 1500 rpm for 10 min. After which the supernatant (100 µL) was transferred into a 96-well plate. The hemoglobin release was evaluated by measuring the optical absorbance at 576 nm using a microplate reader. As the reference controls, 0% of hemoglobin release was from the group of blood suspension treated with PBS only, while 100% of hemoglobin release was from the group of blood suspension treated with 0.1% Triton-X 100.

2.7. In vitro cytotoxicity of P(CitAPDMAEMA) and P(APDMAEMA) The cytotoxicity of P(CitAPDMAEMA) and P(APDMAEMA) towards 3T3 mouse fibroblast was determined by MTT assay. Briefly, 3T3 cells were seeded in a 96-well plate at 5×103 cells per well, using 100 µL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubated overnight at 37 ℃. Then the culture medium in each 9 ACS Paragon Plus Environment


well was replaced by the same volume of DMEM medium in the presence of P(CitAPDMAEMA) or P(APDMAEMA) of different concentrations (0.1 mg/mL-3.2 mg/mL), and the cells were incubated for 24 h. Afterwards, the culture medium in each well was replaced by 100 µL of the DMEM medium containing MTT at a concentration of 0.5 mg/mL, and the cells were incubated for an additional 4 h at 37 ℃. After 4 h, the MTT-containing medium was carefully replaced with 100 µL DMSO. After gentle agitation for 10 min, the absorbance at 595 nm of each well was recorded on a microplate reader. Data are expressed as the average ± S.D. (n =3).

2.8. pH dependent P(CitAPDMAEMA)/E. coli binding studies E. coli was used as model bacterial strains for binding studies. In brief, E. coli were cultured overnight in Tryptic soy broth (TSB) at 37 ℃, collected in mid-log phase growth, centrifuged and washed in deionized (DI) water, and then re-suspended in sterile DI water with the final concentration of about 2×108 colony forming units (CFU)/mL. About 250 µL of FITC labeled P(CitAPDMAEMA) solution (1 mg/mL) in different pH buffers (pH 7.4, 6.0 and 5.0) was mixed with equal volume of E. coli suspension (250 µL) in sterile 2 mL tubes. After 3 h of incubation at 37 °C, the suspension was centrifuged at 6000 rpm for 5 min, washed twice and resuspended in 1 mL of DI water. The P(CitAPDMAEMA)/E. coli interaction was characterized by flow cytometry using the FITC signal (Excitation wavelength: 488 nm). For microscopic studies, 20 µL of this suspension was placed on a microscope glass slide and covered with a cover slip. Micrographs were taken on a fluorescence microscope (Nikon Ti-U) using the green channel.

2.9. Antimicrobial activity of P(CitAPDMAEMA) 10 ACS Paragon Plus Environment

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E. coli was used as gram-negative bacterial strain and S. aureus was used as gram-positive bacterial strain for antibacterial assays. Typically, bacteria were cultured in TSB by incubation overnight at 37 ℃, collected in mid-log phase growth and dispersed in Mueller Hinton Broth (MHB) to give rise to a final concentration of 1×106 CFU/mL. P(CitAPDMAEMA) was pretreated in different buffers (pH 7.4 and 5.0) for 24 h, and then the polymer solutions with different concentrations were prepared using PBS. Each well of the 96-well plate was filled with 100 µL of P(CitAPDMAEMA) solution, to which 100 µL of bacterial suspension (1×106 CFU/mL) was added. The plate was incubated at 37 °C for another 18 h and the OD values at 600 nm (OD600) were recorded using a microplate reader. The OD600 of the control group (without polymer) was set as 100%. All experiments were repeated three times.

2.10. Live/Dead Staining For live/dead staining analysis, E. coli was collected in mid-log phase growth, centrifuged, washed and then re-suspended in PBS with the final concentration of about 2×108 CFU/mL. P(CitAPDMAEMA) was pretreated in different buffer (pH 7.4, 5.0) for 24 h, and then diluted in PBS (128 µg/mL). About 250 µL of this stocked solution was mixed with equal volume of bacteria suspension (250 µL) in sterile 2 mL tubes. After incubation for 3 h at 37 °C, the suspension was treated with PI/SYTO Kit following the protocol. About 10 µL of this suspension was placed on a microscope glass slide and covered with a coverslip, and then imaged on a fluorescence microscope (Nikon Ti-U). Each experiment was carried out in triplicate.



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3. Results and Discussion 3.1.

Synthesis

and

characterization

of

poly(N’-citraconyl-3-aminepropyl-2-

(dimethylamino)ethyl methacrylate) (P(CitAPDMAEMA)) There is an urgent need to develop new powerful biocompatible antimicrobial agents for treatment of bacterial infection. As such, we prepared a smart pH-sensitive polymer, P(CitAPDMAEMA), which can undergo functional change from biocompatible zwitterionic polymer to efficient antimicrobial polymer at acidic environment. The approach to the synthesis of P(CitAPDMAEMA) is shown in Figure 1. 2-(N-Boc-3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate (BocAPDMAEMA) was prepared by conjugation of 2(dimethylamino)ethyl methacrylate (DMAEMA) with N-Boc-3-bromopropylamine. Bocprotected

homopolymer,

poly(2-(N-Boc-3-aminopropyl-N,N-dimethyl

ammonium)ethyl

methacrylate) (P(BocAPDMAEMA)), was obtained by reversible addition-fragmentation chain transfer

(RAFT)

polymerization

of

BocAPDMAEMA

using

4-cyanopentanoic

acid

dithiobenzoate (CPADB) as the chain transfer agent. The successful synthesis of P(BocAPDMAEMA) was confirmed by gel permeation chromatography (GPC) and 1H-NMR spectroscopy (Figure 2). P(BocAPDMAEMA) has a weight average molecular weight of 6685 and a polydispersity index of 1.34. The slightly higher PDI suggests that CPADB is not a perfect chain transfer agent for the RAFT polymerization of BocAPDMAEMA. Deprotection of P(BocAPDMAEMA) was achieved by treatment with trifluoroacetic acid, and the obtained polymer,

poly(2-(3-aminopropyl-N,N-dimethyl

ammonium)ethyl

methacrylate)

(P(APDMAEMA)), was characterized by 1H-NMR spectroscopy. Disappearance of the peak of Boc group at 1.4 ppm indicated successful deprotection and exposure of the primary amine. Finally, P(APDMAEMA) was modified using citraconic anhydride to obtain the zwitterionic


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polymer, poly(N’-citraconyl-2-(3-aminopropyl-N,N-dimethyl ammonium)ethyl methacrylate) (P(CitAPDMAEMA)), with 100% of the primary amine groups in P(APDMAEMA) converted into amides, as confirmed by 1H-NMR spectroscopy.

3.2. Hydrolysis of citraconic amide of P(CitAPDMAEMA) Amides with β-carboxylic acid groups are acid labile, and primary amine can be exposed through degradation of amides with β-carboxylic acid groups.31 P(CitAPDMAEMA) is expected to undergo conversion from zwitterion to cationic P(APDMAEMA) due to degradation of citraconic amide under an acidic condition. The process endows P(CitAPDMAEMA) with efficient bacterial targeting and antimicrobial property under acidic conditions. Fluorescamine is an amine reactive fluorescence dye. It can react with primary amine to form fluorescent product. As such, the pH-dependent degradation rate of citraconic amide of P(CitAPDMAEMA) can be monitored using the fluorescamine method by comparison of the fluorescence before and after incubation. The results are shown in Figure 3. About 90% of the citraconic amides were degraded and the primary amine exposed in the pH 5.0 buffer within 24 h, while only about 5% of citraconic amides was degraded in the pH 7.4 buffer within the same time period. In addition, approximately 24% of the primary amine was exposed in the pH 6.0 buffer within 24 h. These results demonstrate that P(CitAPDMAEMA) is stable and retain the zwitterionic property at the physiological environment (pH 7.4), but will expose the primary amine and quaternary ammonium, and thus the positive charge, under acidic condition because of the removed carboxyl group of citraconic acid.

3.3. In vitro toxicity of P(CitAPDMAEMA)



For potential clinical application, the antimicrobial agents should exhibit low cytotoxicity to host cells and should not have any detrimental effect on human body. Hemolysis is a standard assay to evaluate the cytotoxicity of antimicrobial polymers that are particularly relevant to intravenous application.9 Fresh rabbit whole blood was used to determine the hemotoxicity of P(CitAPDMAEMA). The resulting extent of hemolysis versus polymer concentration is shown in Figure 4(a). There are almost no red blood cells that were lysed among the tested concentrations, indicating ultralow hemolytic activity of P(CitAPDMAEMA).

3T3 mouse embryonic fibroblast (3T3 cells) were used as a model cell line to test the cytotoxicity of P(CitAPDMAEMA) in MTT assay. Figure 4(b) shows the viability of 3T3 cells exposed to different P(CitAPDMAEMA) concentrations. It is clear that P(CitAPDMAEMA) shows good biocompatibility with 3T3 cells even at high polymer concentrations. However, P(APDMAEMA) exhibits a high cytotoxicity even at low concentration of 0.1 mg/mL (Supporting Information, Figure S1). These results suggest that P(CitAPDMAEMA) is safe and biocompatible for intravenous injection as an antimicrobial agent.

3.4. pH-dependent binding of P(CitAPDMAEMA) to bacteria The zwitterion and biocompatible P(CitAPDMAEMA) is expected to change to cationic and bind to the surface of bacteria at acidic environment. The binding of P(CitAPDMAEMA) to bacteria under acidic conditions was evaluated by fluorescence microscopy and flow cytometry using Escherichia coli (E. coli) as the model bacteria strain. P(CitAPDMAEMA) was labelled by FITC first and then incubated with E. coli for 3 h in buffers of different pH. The P(CitAPDMAEMA)/E. coli suspensions were subsequently centrifuged, washed and re-


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suspended in pH 7.4 buffer. Fluorescence microscopy was used to characterize the bonding property first. The results are shown in Figure 5(a). Strong fluorescence can be detected on E. coli in the pH 5.0 group while no fluorescent signal was observed in the pH 7.4 group. Flow cytometry was

also

used

to

characterize this interaction

between

FITC

labelled

P(CitAPDMAEMA) and E. coli. E. coli in the pH 5.0 group showed high FITC fluorescent intensity compared to the E. coli in PBS (control group), while there is almost no difference in FITC fluorescent signal between E. coli in the pH 7.4 group and the control group (Figure 5(b)). These results indicate that P(CitAPDMAEMA) can undergo charge conversion and bind to bacteria effectively under acidic conditions.

3.5 Antibacterial activity of P(CitAPDMAEMA) When P(CitAPDMAEMA) bind to bacteria under acidic conditions, it is expected to achieve high antimicrobial efficacy because it can be converted back to catioinc P(CitAPDMAEMA) under acidic conditions. Antibacterial property of P(CitAPDMAEMA) in different pH conditions was investigated to prove this hypothesis. P(CitAPDMAEMA) was first pretreated in different buffer (7.4 and 5.0) for 24 h at 37 °C, and then incubated with E. coli or S. aureus for 20 h in the MHB medium. OD value at 600 nm was measured to characterize its antibacterial properties. From Figure 6(a), it can be observed that P(CitAPDMAEMA) pretreated at pH 7.4 did not have any effect on the growth of E. coli, due to its zwitterion nature at this pH. However, for P(CitAPDMAEMA) pretreated at pH 5.0, the growth of E. coli was significantly inhibited because of the hydrolysis of citraconic amide and the presence of cationic charges. Similar pHdependent antibacterial efficacy of P(CitAPDMAEMA) against gram-positive Staphylococcus aureus (S. aureus) was observed (Figure 6(b)).



Viability of E. coli cells was also analyzed in live/dead assay. The SYTO 9 dye generally labels all bacteria with intact and damaged membranes. In contrast, propidium iodide (PI) can penetrate only bacteria with damaged membranes and cause a reduction in the fluorescence intensity of SYTO 9 dye when both dyes are present. According to the fluorescence microscopy images presented in Figure 7, E. coli cells in the pH 7.4 group show only green fluorescence, suggesting that P(CitAPDMAEMA) (64 µg/mL) under this condition cannot cause any damage to the bacterial cell wall/membrane. However, E. coli in the pH 5.0 group emitted much stronger red fluorescence, indicating that P(CitAPDMAEMA) (64 µg/mL) pretreated in the pH 5.0 buffer solution can disrupt cell wall/membrane of E. coli, allowing the passage of PI dyes into the bacterial cells. These results indicate that P(CitAPDMAEMA) can effectively target the bacteria and disrupt the cell wall/membrane, leading to the death of bacteria under acidic conditions.


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4. Conclusion A

smart

pH-responsive

polymer,


ammonium)ethyl methacrylate) (P(CitAPDMAEMA)), for targeting bacterial cells and effective antimicrobial effect at acidic environment has been developed. P(CitAPDMAEMA) can undergo conversion from zwitterionic to cationic at acidic conditions to allow P(CitAPDMAEMA) to rapidly bind to E. coli under acidic conditions. In addition, it was demonstrated that P(CitAPDMAEMA) can effectively inhibit the growth of bacteria, disrupt the cell wall/membrane and lead to the death of bacteria under acidic conditions. On the basis of these results, this polymer is a potential antimicrobial agent for bacterial targeting and antibacterial effect. The present proof-of-concept work provides a new strategy for developing biocompatible and efficient antimicrobial polymers and agents.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Cytotoxicity of P(APDMAEMA) against 3T3 cells in MTT assay.

Acknowledgements The authors would like to acknowledge the financial support of this study from the Singapore Millennium Foundation under Grant 1123004048 (NUS WBS no. R279-000-428-592).



References 1.

Davies, J.; Davies, D. Origins and Evolution of Antibiotic Resistance. Microbiol. Mol.

Biol. Rev. 2010, 74, 417-433, DOI: 10.1128/MMBR.00016-10. 2.

Chellat, M. F.; Raguž, L.; Riedl, R. Targeting Antibiotic Resistance. Angew. Chem. Int.

Ed. 2016, 55, 6600-6626, DOI: 10.1002/anie.201506818. 3.

Huh, A. J.; Kwon, Y. J. “Nanoantibiotics”: A New Paradigm for Treating Infectious

Diseases Using Nanomaterials in the Antibiotics Resistant Era. J. Control. Release 2011, 156, 128-145, DOI: 10.1016/j.jconrel.2011.07.002. 4.

O'Connell, K. M.; Hodgkinson, J. T.; Sore, H. F.; Welch, M.; Salmond, G. P.; Spring, D.

R. Combating Multidrug‐Resistant Bacteria: Current Strategies for the Discovery of Novel Antibacterials. Angew. Chem. Int. Ed. 2013, 52, 10706-10733, DOI: 10.1002/anie.201209979. 5.

Munoz-Bonilla, A.; Fernández-García, M. Polymeric Materials with Antimicrobial

Activity. Prog. Polym. Sci. 2012, 37, 281-339, DOI: 10.1016/j.progpolymsci.2011.08.005. 6.

Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y.-Y.

Emerging Trends in Macromolecular Antimicrobials to Fight Multi-Drug-Resistant Infections. Nano Today 2012, 7, 201-222, DOI: 10.1016/j.nantod.2012.04.003. 7.

Rawlinson, L.-A. B.; Ryan, S. M.; Mantovani, G.; Syrett, J. A.; Haddleton, D. M.;

Brayden, D. J. Antibacterial Effects of Poly (2-(dimethylamino ethyl) methacrylate) against Selected Gram-Positive and Gram-Negative Bacteria. Biomacromolecules 2009, 11, 443-453, DOI: 10.1021/bm901166y. 8.

Liu, R.; Chen, X.; Chakraborty, S.; Lemke, J. J.; Hayouka, Z.; Chow, C.; Welch, R. A.;

Weisblum, B.; Masters, K. S.; Gellman, S. H. Tuning the Biological Activity Profile of


Page 18 of 32

Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Antibacterial Polymers via Subunit Substitution Pattern. J. Am. Chem. Soc. 2014, 136, 44104418, DOI: 10.1021/ja500367u. 9.

Locock, K. E.; Michl, T. D.; Valentin, J. D.; Vasilev, K.; Hayball, J. D.; Qu, Y.; Traven,

A.; Griesser, H. J.; Meagher, L.; Haeussler, M. Guanylated Polymethacrylates: A Class of Potent Antimicrobial Polymers with Low Hemolytic Activity. Biomacromolecules 2013, 14, 4021-4031, DOI: 10.1021/bm401128r. 10.

Pascual, A.; Tan, J. P.; Yuen, A.; Chan, J. M.; Coady, D. J.; Mecerreyes, D.; Hedrick, J.

L.; Yang, Y. Y.; Sardon, H. Broad-Spectrum Antimicrobial Polycarbonate Hydrogels with Fast Degradability. Biomacromolecules 2015, 16, 1169-1178, DOI: 10.1021/bm501836z. 11.

Cheng, J.; Chin, W.; Dong, H.; Xu, L.; Zhong, G.; Huang, Y.; Li, L.; Xu, K.; Wu, M.;

Hedrick, J. L.; Yang, Y. Y.; Fan, W. Biodegradable Antimicrobial Polycarbonates with in Vivo Efficacy against Multidrug‐Resistant MRSA Systemic Infection. Adv. Healthcare Mater. 2015, 4, 2128-2136, DOI: 10.1002/adhm.201500471. 12.

Engler, A. C.; Shukla, A.; Puranam, S.; Buss, H. G.; Jreige, N.; Hammond, P. T. Effects

of Side Group Functionality and Molecular Weight on the Activity of Synthetic Antimicrobial Polypeptides. Biomacromolecules 2011, 12, 1666-1674, DOI: 10.1021/bm2000583. 13.

Harvey, A. C.; Madsen, J.; Douglas, C. I.; MacNeil, S.; Armes, S. P. Antimicrobial Graft

Copolymer Gels. Biomacromolecules 2016, 17, 2710-2718, DOI: 10.1021/acs.biomac.6b00760. 14.

Engler, A. C.; Tan, J. P.; Ong, Z. Y.; Coady, D. J.; Ng, V. W.; Yang, Y. Y.; Hedrick, J. L.

Antimicrobial

Polycarbonates:

Investigating

the

Impact

of

Balancing

Charge

and

Hydrophobicity Using a Same-Centered Polymer Approach. Biomacromolecules 2013, 14, 43314339, DOI: 10.1021/bm401248t.



15.

Page 20 of 32

Jin, Q.; Chen, Y.; Wang, Y.; Ji, J. Zwitterionic Drug Nanocarriers: A Biomimetic

Strategy

for

Drug

Delivery.

Colloids

Surf.,

B

2014,

124,

80-86,

DOI:

10.1016/j.colsurfb.2014.07.013. 16.

Lin, W.; Ma, G.; Kampf, N.; Yuan, Z.; Chen, S. Development of Long-Circulating

Zwitterionic Cross-Linked Micelles for Active-Targeted Drug Delivery. Biomacromolecules 2016, 17, 2010–2018, DOI: 10.1021/acs.biomac.6b00168. 17.

Shao, Q.; Jiang, S. Molecular Understanding and Design of Zwitterionic Materials. Adv.

Mater. 2015, 27, 15-26, DOI: 10.1002/adma.201404059. 18.

Chu, L.; Gao, H.; Cheng, T.; Zhang, Y.; Liu, J.; Huang, F.; Yang, C.; Shi, L.; Liu, J. A

Charge-Adaptive Nanosystem for Prolonged and Enhanced in Vivo Antibiotic Delivery. Chem. Commun. 2016, 52, 6265-6268, DOI: 10.1039/C6CC01269H. 19.

Korupalli, C.; Huang, C. C.; Lin, W. C.; Pan, W. Y.; Lin, P. Y.; Wan, W. L.; Li, M. J.;

Chang, Y.; Sung, H. W. Acidity-Triggered Charge-Convertible Nanoparticles That Can Cause Bacterium-Specific Aggregation in situ to Enhance Photothermal Ablation of Focal Infection. Biomaterials 2017, 116, 1-9, DOI: 10.1016/j.biomaterials.2016.11.045. 20.

Radovic-Moreno, A. F.; Lu, T. K.; Puscasu, V. A.; Yoon, C. J.; Langer, R.; Farokhzad, O.

C. Surface Charge-Switching Polymeric Nanoparticles for Bacterial Cell Wall-Targeted Delivery of Antibiotics. Acs Nano 2012, 6, 4279-4287, DOI: 10.1021/nn3008383. 21.

Pichavant, L.; Bourget, C.; Durrieu, M. C.; Heroguez, V. Synthesis of pH-Sensitive

Particles for Local Delivery of an Antibiotic via Dispersion ROMP. Macromolecules 2011, 44, 7879-7887, DOI: 10.1021/ma2015479.


Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22.

Halpindohnalek, M. I.; Marth, E. H. Staphylococcus aureus: Production of Extracellular

Compounds and Behavior in Foods - A Review. J. Food Prot. 1989, 52, 267-282, DOI: 10.4315/0362-028X-52.4.267. 23.

Zhou, Z. X.; Shen, Y. Q.; Tang, J. B.; Fan, M. H.; Van Kirk, E. A.; Murdoch, W. J.;

Radosz, M. Charge-Reversal Drug Conjugate for Targeted Cancer Cell Nuclear Drug Delivery. Adv. Funct. Mater. 2009, 19, 3580-3589, DOI: 10.1002/adfm.200900825. 24.

Deng, H. Z.; Zhao, X. F.; Liu, J. J.; Deng, L. D.; Zhang, J. H.; Liu, J. F.; Dong, A. J.

Reactive Oxygen Species (ROS) Responsive PEG-PCL Nanoparticles with pH-Controlled Negative-to-Positive Charge Reversal for Intracellular Delivery of Doxorubicin. J. Mater. Chem. B 2015, 3, 9397-9408, DOI: 10.1039/C5TB01939G. 25.

Wang, Y.; Lv, S. X.; Deng, M. X.; Tang, Z. H.; Chen, X. S. A Charge-Conversional

Intracellular-Activated Polymeric Prodrug for Tumor Therapy. Polym. Chem. 2016, 7, 22532263, DOI: 10.1039/C5PY01618E. 26.

Yi, H. Q.; Liu, P.; Sheng, N.; Gong, P.; Ma, Y. F.; Cai, L. T. In situ Crosslinked Smart

Polypeptide Nanoparticles for Multistage Responsive Tumor-Targeted Drug Delivery. Nanoscale 2016, 8, 5985-5995, DOI: 10.1039/c5nr07348k. 27.

Du, J. Z.; Du, X. J.; Mao, C. Q.; Wang, J. Tailor-Made Dual pH-Sensitive Polymer-

Doxorubicin Nanoparticles for Efficient Anticancer Drug Delivery. J. Am. Chem. Soc. 2011, 133, 17560-17563, DOI: 10.1021/ja207150n. 28.

Yuan, Y. Y.; Mao, C. Q.; Du, X. J.; Du, J. Z.; Wang, F.; Wang, J. Surface Charge

Switchable Nanoparticles Based on Zwitterionic Polymer for Enhanced Drug Delivery to Tumor. Adv. Mater. 2012, 24, 5476-5480, DOI: 10.1002/adma.201202296.



29.

Page 22 of 32

Huang, A. Y.-T.; Tsai, C.-H.; Chen, H.-Y.; Chen, H.-T.; Lu, C.-Y.; Lin, Y.-T.; Kao, C.-L.

Concise Solid-Phase Synthesis of Inverse Poly (Amidoamine) Dendrons Using AB2 Building Blocks. Chem. Commun. 2013, 49, 5784-5786, DOI: 10.1039/C3CC40661J. 30.

Lee, Y.; Fukushima, S.; Bae, Y.; Hiki, S.; Ishii, T.; Kataoka, K. A Protein Nanocarrier

from Charge-Conversion Polymer in Response to Endosomal pH. J. Am. Chem. Soc. 2007, 129, 5362-5363, DOI: 10.1021/ja071090b. 31. Based

Zhou, T.; Zhou, X.; Xing, D. Controlled Release of Doxorubicin from Graphene Oxide Charge-Reversal

Nanocarrier.

Biomaterials

2014,

10.1016/j.biomaterials.2014.01.044.


35,

4185-4194,

DOI:

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Captions for Scheme and Figures Scheme 1. Schematic illustration of the P(CitAPDMAEMA) as a smart antimicrobial agent. Figure 1. Synthetic route for the preparation of BocAPDMAEMA and P(CitAPDMAEMA). Figure 2. 1H-NMR spectra of P(BocAPDMAEMA) in DMSO-d6, P(APDMAEMA) in D2O and P(CitAPDMAEMA) in D2O. Figure 3. The degradation of citraconic amide of P(CitAPDMAEMA) under different pH. Figure 4. (a) Hemolysis as a function of P(CitAPDMAEMA) concentration. (b) Cytotoxicity of P(CitAPDMAEMA) against 3T3 cells in MTT assay. Figure 5. pH-dependent interaction between FITC-labelled P(CitAPDMAEMA) and E. coli. (a) Fluorescence microscopy of E. coli after interaction with FITC-labelled P(DMAEMA-PA-Cit) at different pH levels (pH 7.4 and pH 5.0) (Scale bar: 100 µm). (b) Binding of FITC-labelled P(CitAPDMAEMA) on E.coli was assessed by flow cytometry. Figure 6. Antibacterial ability of P(CitAPDMAEMA) under different pH conditions: (a) E. coli, and (b) S. aureus. Figure 7. Fluorescence microscopy images of E. coli stained with Live/Dead kit showing the presence of live bacteria (green) and dead bacteria (red) with the treatments of P(CitAPDMAEMA) under different pH conditions (Scale bar: 100 µm).



Scheme 1. Schematic illustration of the P(CitAPDMAEMA) as a smart antimicrobial agent.


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Figure 1. Synthetic route for the preparation of BocAPDMAEMA and P(CitAPDMAEMA).



Figure 2. 1H-NMR spectra of P(BocAPDMAEMA) in DMSO-d6, P(APDMAEMA) in D2O and P(CitAPDMAEMA) in D2O.


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Figure 3. The degradation of citraconic amide of P(CitAPDMAEMA) under different pH.



Figure 4. (a) Hemolysis as a function of of P(CitAPDMAEMA) concentration. (b) Cytotoxicity of P(CitAPDMAEMA) against 3T3 cells in MTT assay.


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Figure 5. pH-dependent interaction between FITC-labelled P(CitAPDMAEMA) and E. coli. (a) Fluorescence microscopy of E. coli after interaction with FITC-labelled P(DMAEMA-PA-Cit) at different pH levels (pH 7.4 and pH 5.0) (Scale bar: 100 µm). (b) Binding of FITC-labelled P(CitAPDMAEMA) on E. coli was assessed by flow cytometry.



Figure 6. Antibacterial ability of P(CitAPDMAEMA) under different pH conditions: (a) E. coli, and (b) S. aureus. The data are expressed as the average ± S.D. (n = 3).


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Figure 7. Fluorescence microscopy images of E. coli stained with Live/Dead kit showing the presence of live bacteria (green) and dead bacteria (red) with the treatments of P(CitAPDMAEMA) under different pH conditions (Scale bar: 100 µm).



TOC Graphic

Title: pH-Sensitive Zwitterionic Polymer as an Antimicrobial Agent with Effective Bacterial Targeting

Authors: Peng Liu, Gang Xu, Dicky Pranantyo, Li Qun Xu, Koon-Gee Neoh, En-Tang Kang

Summary/highlights: A smart pH-sensitive zwitterionic polymer as a promising biocompatible antimicrobial agent with effective bacterial targeting.

For Table of Contents Use Only


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pH-Sensitive Zwitterionic Polymer as an Antimicrobial Agent with

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