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Broad-Spectrum Antimicrobial Polycarbonate Hydrogels with Fast Degradability Ana Pascual, Jeremy PK Tan, Alexander Y. Yuen, Julian M. W. Chan, Daniel J. Coady, David Mecerreyes, James L Hedrick, Yi Yan Yang, and Haritz Sardón Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501836z • Publication Date (Web): 12 Mar 2015 Downloaded from http://pubs.acs.org on March 22, 2015

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Broad-Spectrum Antimicrobial Polycarbonate Hydrogels with Fast Degradability Ana Pascual,1 Jeremy P. K. Tan,2 Alex Yuen,1 Julian M. W. Chan,3 Daniel J. Coady3, David Mecerreyes,4 James L. Hedrick3,* Yi Yan Yang,2,* and Haritz Sardon1,*

1

POLYMAT, University of the Basque Country UPV/EHU Joxe Mari Korta Center, Avda. Tolosa 72,

20018 Donostia-San Sebastián, Spain. 2

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore

3

IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA

4

Ikerbasque, Basque Foundation for Science, E-48011 Bilbao, Spain

Abstract In this study, a new family of broad-spectrum antimicrobial polycarbonate hydrogels has been successfully synthesized and characterized. Tertiary amine-containing eightmembered monofunctional and difunctional cyclic carbonates were synthesized, and chemically cross-linked polycarbonate hydrogels were obtained by copolymerizing these monomers with a poly(ethylene glycol)-based bifunctional initiator via organocatalyzed ring-opening polymerization using 1,8-diazabicyclo[5.4.0]undec-7-ene catalyst. The gels were quaternized using methyl iodide to confer antimicrobial properties. Stable hydrogels were obtained only when the bifunctional monomer concentration was equal or higher than 12 mol %. In vitro antimicrobial studies revealed that all quaternized hydrogels exhibited broad-spectrum antimicrobial activity against Staphylococcus

aureus

(Gram-positive),

Escherichia

coli

(Gram-negative), 1

ACS Paragon Plus Environment

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Pseudomonas aeruginosa (Gram-negative), and Candida albicans (fungus), while the antimicrobial activity of the non-quaternized hydrogels was negligible. Moreover, the gels showed fast degradation at room temperature (4-6 days), making them ideal candidates for wound healing and implantable biomaterials.

Introduction Wound treatment is an important issue in healthcare, and progress in this area can have a major impact on patients’ quality of life. It is believed that the treatment of chronic wounds will become an important challenge to healthcare systems worldwide due to an aging society.1 According to the United Nations, six to eight million Europeans were affected by wound care in 2009.2 Bearing in mind the significant impact of wounds on patient health, appropriate diagnosis and treatment are essential.3 Despite these urgent needs, wound dressing technology still has a long way to go probably due to the intrinsic complexity of the wound healing process.4, 5 In the past, scientists had focused on drying the wound site with absorptive gauzes, though the value of gauze dressings is heavily debated because of the pain and damage they cause to the neo-epithelium during removal.6, 7 In past years, different polymer-based technologies have been developed to improve the care of conventional wounds. For instance, Winter et al. found that by covering the wound with a polymer dressing, the rate of epithelialization was significantly accelerated.8 In the last decade, hydrogels have been studied and used as alternatives to gauze dressing materials in wound dressing applications.9-15 Hydrogels have the ability 2


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to maintain wound occlusion, creating a more conducive environment for tissue regeneration than exposed wounds.16

4, 11, 17, 18

In addition, hydrogels offer a more

versatile platform that allows for incorporation of growth factors to accelerate wound healing as compared to traditional gauze dressings.19, 20 The main problem in using hydrogels for wound care is the high risk of infections, as the moist environment of the hydrogels promotes pathogen proliferation and colonization. In order to overcome this issue, antibiotics13, 21, 22 or silver11 were loaded into the hydrogels. Although the killing efficacy and biocompatibility of conventional antibiotics have been demonstrated, the inability to combat multidrug resistant infections is their major drawback.10,

23, 24

Therefore, several attempts have been made to develop antimicrobial hydrogels that are less susceptible to the development of multi drug resistance (MDR) by virtue of their different mechanism of action.3, 5, 7, 13, 14 Advances in synthetic chemistry have offered the ability to tailor the molecular structure and functionality of polymers to impart broad-spectrum antimicrobial activity25,

26

,

predictable mechanical and rheological properties to hydrogels. Hence, natural biomaterials, synthetic polymers, or their blends have been explored for wound dressing applications.27 Hydrogels based on natural materials like chitosan28, 29 or gelatin30, 31 suffer from certain limitations such as batch-to-batch variations in molecular weight and immunogenicity. In order to sidestep these issues, synthetic polymers including polyacrylates32, polyurethanes33 and polyesters17 have been explored for wound dressing applications.6,

7

Recently, attention has been directed towards the development of

antimicrobial aliphatic polycarbonates with broad-spectrum activity and the ability to circumvent MDR

12, 24, 26, 34, 35

. Aliphatic polycarbonates are ideal candidates for 3


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biomedical applications because of their low toxicity and ease of incorporating functionality.

36, 37

The ability of polycarbonates to eradicate Gram-positive and Gram-

negative bacteria by incorporating quaternary ammoniums in the polymer backbone has been shown23, 26, 34. The ideal hydrogels used in wound healing applications should be degradable and able to prevent infection. Feng et al. showed that linear polycarbonates functionalized with tertiary amines could degrade in three months without creating any significant toxicity upon degradation but without antimicrobial properties.38

The goal of this study is to design an ideal hydrogel material for wound healing applications, featuring a combination of antimicrobial properties and biodegradability. In particular, eight-membered cyclic and dicyclic carbonate monomers containing tertiary amines were synthesized, and subsequently converted into hydrogels via organocatalyzed ring-opening polymerization using 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) as catalyst and poly(ethylene glycol) diol as the initiator. Rheological behavior, swelling degree, and gel content were studied to demonstrate gel formation. In addition, the antimicrobial behavior of these gels and their degradation profiles were also investigated.

4


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Materials and Methods Materials N-methyldiethanolamine (≥99%), N,N,N′,N′-Tetrakis(2-hydroxyethyl)ethylenediamine (≥99%),

triphosgene

(98

%),

triethylamine

(TEA)

(≥99%),

1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU) (98 %), poly(ethylene glycol) end-capped diol (PEG1500, Mn = 1500 g mol−1), poly(ethylene glycol) end-capped diol (PEG4000, Mn = 4000 g mol−1), poly(ethylene glycol) end-capped diol (PEG8000, Mn = 8000 g mol−1) and methyl iodide (CH3I) (≥99%),

were purchased from Sigma-Aldrich and used as

received. Dichloromethane (DCM) ((≥99%) and tetrahydrofuran (THF) (≥99%) were dried using activated alumina columns and stored over molecular sieves (3 Å). Monomer and Polymer Synthesis Synthesis of 6-methyl-1,3,6-dioxazocan-2-one (Monomer 1, Scheme 1a) A flask was charged with N-methyl diethanolamine (5.00 g, 42.0 mmol), triethylamine (9.30 g, 92.4 mmol) and THF (400 mL). The reaction mixture was stirred for 30 min under N2 at -80 ºC. Next, triphosgene (4.60 g, 15.0 mmol) dissolved in 50.0 mL of THF was added drop-wise to the reaction mixture, and stirred for 3 h. The reaction mixture was treated with cold diethyl ether (2 L), in which the triethylammonium chloride salt was precipitated and recovered. The product-containing filtrate was evaporated to dryness to afford monomer 1 as a liquid (5.1 g, 84%). Monomer 1: 1H NMR (400 MHz, CDCl3, 22 °C): δ = 4.14 (t, OCOOCH2, 4H), 2.72 (t, CH2, 4H), 2.43 (s, CH3, 3H) (Figure SI 1). 13C NMR (400 MHz, CDCl3, 22 °C): δ = 156.5 (C=O) 69.2 (CH2), 56.7 (CH2), 44.9 (CH3) (Figure SI 2).

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Synthesis of 6,6'-(ethane-1,2-diyl)bis(1,3,6-dioxazocan-2-one) (Monomer 2, Scheme 1b) A flask was charged with N,N,N′,N′-Tetrakis(2-hydroxyethyl)ethylenediamine (5.00 g, 21.1 mmol), triethylamine (17.8 g, 180 mmol) and THF (400 mL). The reaction mixture was stirred for 30 min under N2 at -80 ºC, to which Triphosgene (9.20 g, 30.0 mmol) dissolved in 50.0 mL of THF was then added drop-wise, and stirred for 3 h. The reaction mixture was treated with cold diethyl ether (2 L), and the product was precipitated together with triethylammonium chloride salt. The precipitate was redissolved in cold dichloromethane and rinsed sequentially with saturated aqueous NaHCO3 and water, dried over MgSO4 and concentrated in vacuo. The crude product was recrystallized from hexanes to afford Monomer 2 (Yield: 3.2 g, 52%). Monomer 2: 1

H NMR (400 MHz, CDCl3, 22 °C): δ = 4.21 (t, OCOOCH2, 4H), 2.85 (t, CH2, 4H),

2.80 (2, CH2, 2H) (Figure SI 3). 13C NMR (400 MHz, CDCl3, 22 °C): δ = 156.6 (C=O) 69.5 (CH2), 55.6 (CH2), 55.3 (CH2) (Figure SI 4).

6


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Scheme 1. General synthetic route of eight-membered cyclic (a) and dicyclic (b) carbonate monomers.

a) O OH

HO

TEA

O Cl 3C

N

O

O

O

O

CCl 3

DCM, -80 C

Et3NHCl N

6-methyl-1,3,6-dioxazocan-2-one N-methyl diethanolamine

triphosghene

Monomer 1

O

b) OH HO

TEA

O Cl 3C

N

O

O

O

CCl 3

O

Et3NHCl

N

DCM, -80 C

N

N

triphosghene

OH HO

O

O O

N,N,N,´N,´Tetrakis(2-hydroxyethyl) ethylenediamine

6,6'-(ethane-1,2-diyl)bis(1,3,6-dioxazocan-2-one) Monomer 2

Synthesis of linear polymer Monomer 1 (0.310 g, 2.2 mmol) and PEG1500 end-capped diol (0.0300 g, 0.022 mmol) were dissolved in 2 mL of DCM. DBU (0.0180 g, 0.11 mmol) in DCM (0.1 mL) was added and conversion to product was monitored using 1H NMR until completion (approximately 24 h). The catalyst was quenched with an excess of benzoic acid and the crude polymer solution was precipitated in ether and dried under vacuum (Yield: 0.32 g,

7


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94 %). 1H NMR (CDCl3, 400 MHz): 4.23 (t, CH2, 8H) 3.75-3.35 (s, CH2, 136H) 2.76 (t, CH2, 8H) 2.37 (s, CH3, 6H) (Supporting Information Figure SI 5). Synthesis of polycarbonate hydrogels A 20 mL glass vial containing a magnetic stir-bar was charged with monomer 1 (0.310 g, 2.2 mmol), monomer 2 (0.120 g, 0.42 mmol), PEG1500 (0.0300 g, 0.022 mmol), and 2.0 mL of DCM. DBU (0.0180 g, 0.11 mmol) in DCM (0.1 mL) was added and the solution was stirred for 8 h. Then, the stir bar was removed and the mixture was kept at room temperature for 24 h. DBU was quenched with an excess of benzoic acid. The resulting hydrogel was washed by immersion in DCM to remove catalyst and soluble fractions, and dried under vacuum. (Yield: 0.40 g, 89 %). Synthesis of other hydrogels with different compositions is described in the Supporting Information. Quaternization of polycarbonate gels. In a 20.0 mL glass vial the gel was immersed in methyl iodide for 48 h at room temperature under N2. Methyl iodide excess was removed by drying in vacuo. Methods 1

H NMR spectra were obtained on a Bruker Avance 400 instrument. Chemical shifts

are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard (CDCl3: δ 7.26 ppm).

13

C NMR spectra were recorded on a Bruker Avance

400 spectrometer with complete proton decoupling. Chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard (CDCl3: δ 77.16 ppm). Gas Permeation Chromatography (GPC) was performed in THF at 30 °C using a Waters chromatograph equipped with four 5-mm Waters columns (300 mm x 7.7 mm) 8


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connected in series with increasing pore size (100, 1000, 105, 106 Å). Fourier Transform Infrared (FTIR) spectroscopy analysis was conducted on a Nicolet Magna 560 spectrometer at a resolution of 2 cm−1, and a total of 64 interferograms were signalaveraged. Samples were prepared by solution casting the reaction mixture onto a KBr window. Rheological analysis Rheology measurements on the dry gels were conducted on a Thermo-Haake Rheostress I viscoelastometer using oscillatory tests. Angular frequency sweeps from 0.1 to 10 rad/s at constant strain amplitude (γ = 0.005) were applied at 25°C. G' and G'' values were plotted versus frequency. Gel fraction The gel fraction (Fg) was determined by extraction with DCM after drying the samples for 24 h at 45 ºC. The process consisted of a 24 h continuous extraction with DCM under reflux in a 250 mL round bottom flask39. After the extraction, the samples were dried and the gel content was calculated as the ratio between the dry polymer remaining after the extraction (mt) and the initial amount of dry polymer (m0). The molecular weight of the soluble part was determined by GPC.

F! (%) =

m0 - mt × 100 m0

Swelling Dry hydrogels (diameter of 4 mm and weight 10– 15 mg) were immersed in aqueous solution at ambient temperature. After keeping the gel for the desired time interval the 9


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gel was withdrawn. Excess solution was gently removed from the hydrogel surface with tissue paper and the mass increase was determined gravimetrically. The swelling degree (St) was calculated as follows: St = (mt-m0)/m0 x 100 % where mt = mass of the hydrogel after time t and m0 = initial mass of the dry hydrogel.40 Antibacterial activity Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 29737) were reconstituted from their lyophilized forms according to the manufacturer's protocol, and cultured in Tryptic Soy Broth (TSB) at 37 °C under constant shaking of 300 rpm, while Candida albicans (ATCC 10231) was cultured in Yeast Media Broth (YMB) at room temperature under constant shaking of 50 rpm. Prior to treatment, the microbes were first inoculated overnight to enter into log growth phase. The quaternized hydrogel was cut into a 5 x 5 mm square and placed into a 1.7 mL micro-centrifuge tube. TSB or YMB (100 µL) was added into the tube before an equal volume of microbe suspension (3 × 105 CFU/mL) was added. Prior to this, the concentration of microbe solution was adjusted to give an initial optical density (O.D.) reading of approximately 0.07 at 600 nm wavelength on a microplate reader (TECAN, Switzerland), which corresponds to the concentration of McFarland 1 solution (3 × 108 CFU/mL). The tube was kept either at 37 °C for bacterial samples or room temperature for C. albicans under constant shaking (300 and 50 rpm for bacteria and fungi, respectively) for 24 and 42 h, respectively. After the hydrogel treatment, the samples were taken for a series of tenfold dilution, and plated onto agar plates. The plates were incubated for 24 h at 37 °C for bacterial samples or 42 h at room 10


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temperature for C. albicans before the number of colony-forming units (CFU) was counted. Microbes treated with hydrogel without cationic polycarbonates were used as negative control, and each test was carried out in 3 replicates.

Kinetics study E. coil were inoculated and prepared according to the procedure described in the above section. The bacteria were treated with Gel 20%_8000 and incubated at 37 oC under constant shaking of 100 rpm. At regular time intervals (15 min, 30 min, 1 h, 2 h, 4 h and 8 h), bacterial samples were taken for a series of tenfold dilution and plated onto agar plates. The plates were incubated for 24 h at 37 oC before the number of CFU was counted. Bacteria without hydrogel treatment were used as negative controls and each test was carried out in 3 replicates.

Field emission scanning electron microscopy (FE-SEM) E. coli grown in TSB alone or after treatment with Gel 20%_8000 were prepared using the same protocol as the antibacterial testing but with a shorter incubation time of 2 h. The bacteria were collected into microfuge tubes, pelleted at 4000 rpm for 5 min and washed with phosphate-buffered saline (PBS) twice. The samples were fixed with 2.5% glutaraldehyde for 60 min followed by washing with DI water twice. Dehydration was performed with a series of ethanol solution (35%, 50%, 75%, 90%, 95% and 100%) before the samples were dripped onto copper tape and left to dry for 2 days. The dried samples were coated with platinum before SEM imaging under the FE-SEM (JEOL JSM-7400, Japan). 11


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Hemolysis assay The undesired activity of the gel against red blood cells (RBC) was tested with freshly drawn rat RBC (rRBC) obtained from Animal Holding Unit of Biomedical Research Center, Singapore. rRBCs were subjected to a 25X volumetric dilution in PBS to achieve a 4% blood content. The diluted rRBCs were added to hydrogels and incubated in 37 oC for an hour. After incubation, the samples were pelleted under 3000 g for 5 min. The supernatant (100 µL) was transferred to a 96 well plate and hemoglobin released was analysed spectrophotometrically by measuring absorbance at 576 nm using a microplate reader (TECAN, Switzerland). Two control groups were used in this assay: untreated RBC suspended in PBS (negative control) and RBC treated with 0.1% TritonX (positive control). Each assay was performed in 4 replicates and repeated 3 times. Percentage of hemolysis was calculated as follows: %ℎ𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 =

!"!"# !" !"#$!#% !"#$%!! !"!"# !" !"#$%&'" !"#$%"& !"!"# !" !"#$%$&' !"#$%"&! !"!"# !" !"#$%&'" !"#$%"&

𝑥 100%

In vitro cytotoxicity Cytotoxicity of the hydrogels and the degraded products of the hydrogels was investigated by MTT assay. HEK 293 human embryonic kidney cell line was obtained from ATCC (U.S.A.) and cultured jn DMEM supplemented with 10% fetal bovine serum and 5% penicillin-streptomycin. HEK 293 cells were seeded at a density of 10000 cells/well on 96 well plates. The cells were incubated overnight at 37 oC with 5% CO2. Cell culture media was replaced with fresh DMEM (100 µL) and to each well, the hydrogel was added. Each hydrogel was tested in 3 replicates. Gel 20%_8000 was 12


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soaked in DMEM for 7 days to ensure complete degradation before 100 µL of the degraded solution was added into the wells to check on the cytotoxicity of the degraded product. The control used was DMEM media that was left at room temperature for 7 days. The plates were placed back into the incubator at 37 oC with 5% CO2 for 48 h. Next, hydrogels were fished out of the cell and media was replaced with 100 µL fresh media and 20 µL MTT solution (5 mg/mL) and returned to the incubator for 4 h. The mixture was carefully removed and the purple formazan crystals internalized by live cells were dissolved with dimethyl sulfoxide (150 µL). The absorbance of the purple formazan crystals in each well was calculated as that at 550 nm deducted by that at 690 nm. Cell viability was calculated as a percentage of absorbance of the non-treated control.

Degradation Dry hydrogels (diameter of 4 mm and weight 10–15 mg) were immersed in aqueous solution at ambient temperature. At specific time points, the gel was withdrawn. The samples were subsequently dried under vacuum and analyzed by FTIR and NMR spectroscopy.

13


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Results and Discussion Monomer Synthesis and Polymerization The 6-methyl-1,3,6-dioxazocan-2-one cyclic carbonate (Monomer 1) was synthesized via a strategy previously described by Hedrick and coworkers.41 Here, reaction between the N-methyldiethanolamine with triphosgene resulted in a one-pot ring-closure of the diol to generate an eight-membered cyclic carbonate in the presence of triethylamine (Scheme 1a). The monomer structure was confirmed by 1H and 13C NMR spectroscopy. Similarly,

6,6'-(ethane-1,2-diyl)bis(1,3,6-dioxazocan-2-one)

dicyclic

carbonate

(Monomer 2) was synthesized using the same strategy (Scheme 1b), and its successful synthesis was confirmed by 1H and 13C NMR spectroscopy.

The ability to promote the controlled polymerization of functionalized monomer 1 by ring-opening polymerization (ROP) in the presence of DBU was evaluated (Scheme S1). DBU was chosen as the catalyst as it has been demonstrated to be effective in the synthesis of cyclic carbonates.42 Polymerization was successfully initiated using PEG1500 end-capped diol at ambient temperature in DCM for a targeted DP of 100 as evidenced by 1H NMR (Supporting Information Figure SI 5). The polymerization was carried out in the presence of air and also under an inert N2 atmosphere. It was observed that the polymerization conditions did not affect the conversion, and full conversion was achieved after 24 h. However, when the polymerization was performed in the presence of air, the molecular weight and dispersity were substantially affected. Specifically, The number molecular weight (Mn) was reduced from 12 kDa to 6.5 kDa and the polydispersity index was broadened from 1.2 to 1.8 (Table S1). It is known that water 14


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traces can facilitate the formation of diols coming from carbonate monomers.43 These diols may act as initiator in the ring-opening polymerization process, reducing the molecular weight and increasing the polydispersity index. Nevertheless, these results suggest that the polymerization of monomer 1 could be carried out in air although the control of the polymerization was compromised.

Synthesis of cross-linked polycarbonates and hydrogel preparation Monofunctional monomer 1 was copolymerized with different amounts of difunctional monomer 2 in the presence of PEG diol initiator to obtain cross-linked hydrogels. Due to the bifunctional nature of monomer 2, it generates crosslinking points, forming a polymer network (Figure 1). The hydrogels were synthesized by ROP initiated by PEG diol in DCM and catalyzed by DBU at ambient temperature under air atmosphere. An initial monomer concentration of 1 M was applied and different monomer 1/monomer 2 ratios were used, while the initiator and catalyst concentrations were kept constant as shown in Table 1. Hydrogels with different PEG molecular weights (Mn 1500, 4000 and 8000 g mol-1) used as initiator were synthesized to study their effect on the rheological, antimicrobial and swelling properties.

15


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Monomer 1

N

O O

N

O

DBU 5 % mol

N

O

+

O

HO

O O

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34

O N

N

N

O O

O

O 34

x

H

O O

N

O

N

O x

O

O

O

34

y

n

DCM RT

O

N

O

O

O O

O 34

y

N

O

N

O

O y

O y

O

m

O

Quaternization

CH3I

Monomer 2

I-

N O CH3

CH3

CH3

O O

x

O 34

O O

N

N

O I-

x

O

I-

n

O CH3

N I-

O

O

O

34

y

O O

y

O O

O 34

O O

N

N I H 3C

O y

O y

m

Figure 1. Synthesis of quaternized polycarbonate hydrogels.

Hydrogels were prepared by immersing the resulting cross-linked materials in water for 1 h. As shown in Table 1, at low difunctional monomer concentrations, the hydrogels were not able to maintain a homogeneous structure after swelling in aqueous media (Figure SI 6). When the concentration of bifunctional monomer 2 was raised above 8 mol %, an easy-to-handle hydrogel was obtained (Figure SI 7). It was also observed that when monomer 2 concentration is higher than 12 %, a gel fraction around 100 % was achieved due to the presence of a significant number of crosslinking points.

One particular characteristic of hydrogels is their ability to swell in water without losing their three-dimensional structure. The level of swelling, responsiveness and degradability are important features to take into account when designing hydrogels for wound dressing applications. High swelling capacity helps to maintain wound occlusion and a conducive environment that favors the regeneration process. Although all 16


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materials were able to swell in water, the polymers with low bifunctional concentrations were not able to maintain the three-dimensional structure. Swelling ability and behavior of the hydrogels were adversely affected when the concentration of bifunctional monomer was raised. As increasing bifunctional monomer 2 concentration increased the number of cross-linking points per chain for a given DP, a higher concentration considerably reduced the swelling behavior.39, 44, 45 In addition, swelling ratio increased significantly when PEG with a greater molecular length was used (Table 1). The results showed that the degree of swelling is a function of the cross-linking density and PEG length. This phenomenon was also observed in the work reported by Dubois et al. 40

Table 1. Conversion rate (gel fraction) and swelling degree for different hydrogels synthesized using a molar ratio of PEG/catalyst/monomers at 1/5/100 in DCM (monomer concentration: 1 M) at 20 °C.

Data corresponds to mean ± standard

deviation (n = 3). Monomer1

Monome2

PEG Mn

Fga

St (1h)

Mw(sol)c

[mol %]

[mol %]

[KDa]

[%]

[%]

[kDa]

Gel4%_1500

96

4

1.5

71± 4

-*

10.7

Gel8%_1500

92

8

1.5

77 ± 6

50 ± 3

6.5

Gel12%_1500

88

12

1.5

98 ±1

47 ± 4

11.1

Gel16%_1500

84

16

1.5

97 ± 2

40 ± 2

8.9

Gel20%_1500

80

20

1.5

98 ± 1

35 ± 1

7.8

Gel20%_4000

80

20

4.0

97 ± 1

55 ± 2

7.4

Entry

17


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80

20

8.0

98 ± 1

77 ± 3

7.2

80

20

1.5

98 ± 1

20 ± 2

7.8

80

20

8.0

98 ± 1

68 ± 3

7.2

Gel20%_1500qua ternized Gel20%_8000qua ternized a

calculated as (mass of recovered dry gel/mass of the co-monomers used for the synthesis) 100 %. b Swelling degree was calculated after immersing the gels for 1 h in H2O. cMw was calculated by GPC after extracting the soluble part in DCM.* Not possible to measure. Quaternization of tertiary amine-containing polycarbonate hydrogels

From the work reported by Hedrick et al., positively charged polycarbonates containing pendant quaternary amines are highly active towards Gram-positive and Gram-negative bacteria. 24, 46 A simple and facile quaternization reaction was carried out by immersing the previous synthesized hydrogels in pure methyl iodide (Scheme 1). Again, we found that the materials with low cross-linking degree such as Gel 4% and Gel 8% did not remain robust and consistent when they were immersed in methyl iodide. Therefore, they were excluded from further analysis. The rheological behavior of the nonquaternized and quaternized cross-linked materials was investigated by measuring the elastic (G´) and viscous (G´´) moduli in the dry state as a function of frequency at room temperature (Figure SI 8). In all cases, G´ was greater than G´´ in all the frequency range suggesting that the materials remained chemically cross-linked after the quaternization step.

18


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Swelling behavior for Gel 20% with PEG1500 and PEG8000 before and after quaternization was investigated (Table 1 and Figure 2). The swelling ratio of hydrogels synthesized at the monomer 1/monomer 2 molar ratio of 4:1 increased from 210 ± 20 wt% to 705 ±15 wt% when PEG8000 diol was used as macroinitiator. Nevertheless, when hydrogels were quaternized with methyl iodide, the swelling ratio was substantially reduced. Although the quaternization considerably reduced the water uptake, the swelling ratio was still above 200 wt % when PEG8000 diol was used as macroinitiator, which is ideal for keeping the wound in an occluded state. It should be pointed out that that due to the fast degradation process in the non-quaternized gel, the gels are not able to reach a plateau before degradation. Meanwhile, the quaternized gels were able to reach the plateau giving a more realistic value of the maximum water-uptake. This could explain the higher water uptakes observed for the non-quaternized gels compared to the quaternized gels. Another important parameter when dealing with hydrogels is the gel fraction. The gel fraction for most of the gels listed in Table 1 is close to 100 wt %, indicating that the molar composition of the hydrogels is very close to the feed ratio. In addition, quaternization did not affect gel fraction (Gel20%_1500quaternized vs. Gel20%_1500; Gel20%_8000quaternized vs. Gel20%_8000). These results are in good agreement with similar systems studied by Nederberg

45

and Kawalec et al.40 for

polycarbonate based systems.

19


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PCH20%_1500 Gel20%_1500* Gel20%_1500quaternized* PCH20%_1500_quaternized Gel20%_8000* PCH20%_8000 Gel20%_8000quaternized* PCH20%_8000_quaternized 800 700 600

S (%)

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

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500 400 300 200 100 0 0

10

20

30

40

50

60

70

80

time (h)

Figure 2. Swelling behavior of hydrogels before and after quaternization in Milli-Q water at ambient temperature. Data corresponds to mean (n = 3).

Degradation behavior of polycarbonate hydrogels

Aliphatic polycarbonates are known to be biodegradable. Thus, the degradation process at 25ºC of the non-quaternized gel containing 20 mol % of monomer 2 initiated with PEG8000 diol was monitored by FTIR as shown in Figure 3.

As the degradation

proceeded, there was an intensity decrease and complete disappearance of the carbonate (C=O) stretch at 1740 cm−1 after 4 and 6 days of incubation in phosphate-buffered saline (PBS, pH 7.4), respectively. Meanwhile, two new broad bands appear at 3300 cm-1 and 1640 cm-1 that are attributed to alcohol groups and carboxylic acid generated as a 20


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consequence of the hydrolysis of the polycarbonate hydrogel. From the photographs of the hydrogels as shown in Figure 3, the mechanical strength of hydrogels decreased as a function of incubation time. After 6 days of incubation in PBS, the hydrogel completely PCH20%_8000_quaternized_initial gel PCH20%_8000_quaternized_4 days PCH20%_8000_quaternized_6 days

decomposed.

Ini,al"gel" C=O"(carbonate)" 1740cm21"

1"day"

4"days" C2O""(carbonate)" 1250cm21" O2H"strech""

5"days"

C=O"(carboxylic"acid)" 1640cm21"

6"days"

4000

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

Figure 3. Infrared spectra of non-quaternized gel containing 20 mol % of monomer 2 initiated with PEG8000 diol after incubation in PBS (pH 7.4) for various periods of time.

To further verify the degradation process, 1H NMR studies were carried out at t=0 (initial gel) and after 6 days (Figure 4). The characteristic signal attributed to the methylene groups located next to carbonate at 4.26 ppm and the adjacent methylene at 2.77 ppm disappeared, while three new signals at 3.66, 2.60 and 2.32 ppm corresponding to the signals of pure N-methyldiethanolamine and/or N,N,N,N′21


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tetrakis(2-hydroxyethyl) were seen after 6 days of degradation. Furthermore, after 3 days a new signal at 9.90 ppm was observed, which is assigned to carboxylic acids. To further verify the presence of carboxylic acids, (Supporting Information). In

13

13

C NMR analysis was carried out

C NMR spectrum, a new signal at 170 ppm associated

with carboxylic acid groups was clearly observed (Supporting Information Figure SI 9).

Initial gel

3 days immersed

6 days immersed

Figure 4. 1H NMR spectra of the gel containing 20 mol % of monomer 2 initiated with PEG8000 diol after incubation in water at room temperature for 3 and 6 days. From NMR and FTIR analyses, a degradation mechanism is proposed as shown in Figure 5. Hydrolysis of the polycarbonate backbone occurred, forming the branched 22


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polycarbonate copolymers with carboxylic groups as end groups. Decarboxylation may then occur, followed by transesterification reactions to yield the monomers. This behavior was also observed in similar systems.

47

It is worth to mention that the

degradation process was really fast. In our opinion, this occurred because the amines presented in the polymer backbone promoted the degradation process by catalyzing the hydrolysis and decarboxylation reactions. Similar behavior was observed in quaternized gels although the degradation time was slightly increased from 6 days to 8 days. Similar degradation times were observed by Mespouille et al. in a poly(N,N-dimethylamino-2ethyl methacrylate-graft-poly[e-caprolactone]). It is worth mentioning, although they were using highly acidic conditions (dioxane/aqueous HCL mixture (85:15)), only partial degradation was observed.48

N

O O

O

x

O 34

O O

N

O

N

O x

O

O

O

34

y

n O

N

O

O

O O

O 34

y

O O

N

N

O y

O y

m

1. hydrolysis 2. decarboxylation

OH

HO

OH

HO

N

HO

O 34

OH N

N

OH

H

Figure 5. Schematic representation of polycarbonate hydrogel degradation in aqueous media. 23


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Antimicrobial properties of polycarbonate hydrogels

The antimicrobial activity of different hydrogels was evaluated against Gram-positive bacteria S. aureus, Gram-negative bacteria E. coli and P. aeruginosa, and fungi C. albicans. These microbes are common pathogens that often manifest on dermal wounds, and are typically treated by topical application of antibiotics to the infected areas.49, 50 The number of colony forming unit (CFU) for the bacteria or fungi was determined through the agar gel assay with a series of ten-fold dilution. After treating the microbes with the quaternized hydrogels, the growth of the microbes was completely suppressed with killing efficiency of at least 99.999% (Figure SI 10). Furthermore, the CFU counts after 18 h of treatment (Figure 6) showed that the quaternized hydrogels were bactericidal to a wide range of microbes. There was a 6 – 10 log reduction in colony counts. Meanwhile, the unquaternized gels did not show any significant activity towards the microbes. All hydrogels demonstrated similar antimicrobial activity against all microbes tested, independent from the length of PEG diol and monomer 2 concentration. These results proved that the polycarbonate hydrogels must be quaternized to offer antimicrobial efficacy. The ability of the Gel 20%_8000 to kill bacterial cells upon exposure for various time intervals was investigated using E. coli as a model microbe. After exposing the bacteria to the hydrogel for 15 min, 30% of the bacteria were killed by the hydrogel and after 2 h, 99.99% killing efficiency was achieved (Figure SI 11). The longer time was needed to

24


Page 25 of 33

achieve 99.9% kill because the bacteria had to come into contact with the hydrogel, which was settled at the bottom of the well. (a) 1.00E+16

Unquaternized

(b) 1.00E+16

Quaternized

Unquaternized

Quaternized

1.00E+12

CFU/mL

CFU/mL

1.00E+12

1.00E+08

1.00E+08

1.00E+04

1.00E+04

1.00E+00

1.00E+00

Gel 12% _1500 (c) 1.00E+18

Gel 16% _1500

Gel 20% _1500

Unquaternized

Gel 20% _4000

Gel 20% _8000

Gel 12% _1500 (d) 1.00E+10

Quaternized

1.00E+15

Gel 16% _1500

Gel 20% _1500

Unquaternized

Gel 20% _4000

Gel 20% _8000

Quaternized

1.00E+08

CFU/mL

1.00E+12

CFU/mL

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

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1.00E+09

1.00E+06

1.00E+04

1.00E+06 1.00E+02

1.00E+03

1.00E+00

1.00E+00

Gel 12% _1500

Gel 16% _1500

Gel 20% _1500

Gel 20% _4000

Gel 20% _8000

Gel 12% _1500

Gel 16% _1500

Gel 20% _1500

Gel 20% _4000

Gel 20% _8000

Figure 6. Antimicrobial efficacy of the gels synthesized with varying cross-linked degrees (i.e. monomer 2) and different lengths of PEG diol before and after postquaternization against S. aureus (a), E. coli (b), P. aeruginosa (c) and C. albicans (d). (Initial bacterial counts: 1 x 105 CFU/mL, incubated at 37°C for 18 h). Data corresponds to mean ± standard deviation (n = 3)

Antimicrobial mechanism

From the earlier section, the hydrogels have a bactericidal property. Observation of morphological changes of microbes before and after hydrogel treatment gives further 25


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Page 26 of 33

insights into the antimicrobial mechanism. As shown in Figure 7, the surfaces of E. coli cells after hydrogel treatment for 2 h are highly distorted and corrugated. This suggested that the hydrogels killed the bacteria via disrupting the bacterial membrane. (a)

(b)

1 µm

1 µm (c)

(d)

1 µm

1 µm

Figure 7. Field emission scanning electron microscopic images of E. coli before (a, c) and after (b, d) 2 h treatment with Gel 20%_8000. images c and d are at higher magnification. Scale bar: 1 µm. Hemolytic and cytotoxicity activities Hydrogel toxicity was evaluated via hemolysis and MTT assays using rRBCs and HEK 293 cells. As seen in Figure SI12, both the unquaternized and quaternized hydrogels were non-hemolytic to rRBCs after an hour of incubation. From the MTT assay done on HEK 293 cells (Figure SI13), all the unquaternized and quaternized hydrogels showed 26


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no toxicity towards the cells, and the degraded byproducts from the hydrogel were also compatible with the cells. These results demonstrated that the hydrogels were non-toxic towards rRBC and HEK 293cells. These hydrogels were chemically cross-linked as thin transparent films, making them desirable for wound healing applications.

Conclusions

A new family of fast biodegradable broad-spectrum antimicrobial polycarbonate hydrogels has been successfully synthesized from tertiary amine-functionalized eightmembered cyclic carbonate monomers. The use of 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) as an organocatalyst efficiently catalyzed the synthesis of cross-linked polycarbonates from new mono- and di-functional cyclic carbonate monomers. The swelling of the cross-linked polycarbonates depended on the cross-linking degree, the length of the PEG macroinitiator and the quaternization of the amine. The cross-linked polycarbonates were successfully quaternized with methyl iodide, and the resulting quaternized polycarbonate hydrogels showed excellent ability to kill Gram-positive and Gram-negative bacteria as well as fungi. Both the quaternized and non-quaternized hydrogels showed rapid biodegradability on the order of 4 to 6 days. Importantly, the hydrogels and their degradation products are non-tocis to mammalian cells. In summary, these materials show great potential for applications in the areas of wound care and medical implants for the prevention of infections.

Author Information 27


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Corresponding Authors E-mail: Haritz Sardon [email protected]; James L. Hedrick [email protected]; Yi Yan Yang: [email protected] Notes

The authors declare no competing financial interest.

28


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Acknowledgments H.S. and A.P gratefully acknowledge financial support through a postdoctoral grant (DKR) from the Basque Government. Financial support from the Basque Government and MINECO through project number FDI 16507 and the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) through SERC Personal Care Programme Grant No: 1325400028 is also acknowledged.

Supporting Information Available Details of gel preparation protocol, 1H and

13

C NMR spectra, rheological data and

antimicrobial data. This information is available free of charge via the Internet at http://pubs.acs.org/.

References 1. Sen, C. K.; Gordillo, G. M.; Roy, S.; Kirsner, R.; Lambert, L.; Hunt, T. K.; Gottrup, F.; Gurtner, G. C.; Longaker, M. T., Human skin wounds: A major and snowballing threat to public health and the economy. Wound. Repair. Regen. 2009, 17, (6), 763-‐771. 2. http://www.eucomed.be/blog/108/144/blog/2012/02/23/Challenges-‐in-‐ chronic-‐wound-‐care-‐the-‐need-‐for-‐interdisciplinary-‐collaboration 3. Zahedi, P.; Rezaeian, I.; Ranaei-‐Siadat, S.-‐O.; Jafari, S.-‐H.; Supaphol, P., A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages. Polym. Adv. Technol. 2010, 21, (2), 77-‐95. 4. Sarabahi, S., Recent advances in topical wound care. Indian J Plast Surg 2012, 45, (2), 379-‐87. 5. Murphy, P. S.; Evans, G. R. D., Advances in wound healing: a review of current wound healing products. Plast Surg Int 2012, 2012, 190436. 6. Lalani, R.; Liu, L., Electrospun Zwitterionic Poly(Sulfobetaine Methacrylate) for Nonadherent, Superabsorbent, and Antimicrobial Wound Dressing Applications. Biomacromolecules 2012, 13, (6), 1853-‐1863. 7. Mogoşanu, G. D.; Grumezescu, A. M., Natural and synthetic polymers for wounds and burns dressing. Int. J. Pharm. 2014, 463, (2), 127-‐136. 8. Winter, G. D., Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig. Nature 1962, 193, 293-‐4.

29 ACS Paragon Plus Environment

Biomacromolecules

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

Page 30 of 33

9. Du, H.; Zha, G.; Gao, L.; Wang, H.; Li, X.; Shen, Z.; Zhu, W., Fully biodegradable antibacterial hydrogels via thiol-‐ene "click" chemistry. Polym. Chem. 2014, 5, 4002-‐ 4008. 10. 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, (3), 201-‐222. 11. Fan, Z.; Liu, B.; Wang, J.; Zhang, S.; Lin, Q.; Gong, P.; Ma, L.; Yang, S., A Novel Wound Dressing Based on Ag/Graphene Polymer Hydrogel: Effectively Kill Bacteria and Accelerate Wound Healing. Adv. Funct. Mater. 2014, 24, (25), 3933-‐ 3943. 12. Liu, S. Q.; Yang, C.; Huang, Y.; Ding, X.; Li, Y.; Fan, W. M.; Hedrick, J. L.; Yang, Y.-‐Y., Antimicrobial and Antifouling Hydrogels Formed In Situ from Polycarbonate and Poly(ethylene glycol) via Michael Addition. Adv. Mater. (Weinheim, Ger.) 2012, 24, (48), 6484-‐6489. 13. Malmsten, M., Antimicrobial and antiviral hydrogels. Soft Matter 2011, 7, (19), 8725-‐8736. 14. Salomé Veiga, A.; Schneider, J. P., Antimicrobial hydrogels for the treatment of infection. Pept. Sci. 2013, 100, (6), 637-‐644. 15. Nyanhongo, G. S.; Sygmund, C.; Ludwig, R.; Prasetyo, E. N.; Guebitz, G. M., Synthesis of multifunctional bioresponsive polymers for the management of chronic wounds. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2013, 101B, (5), 882-‐891. 16. Hoffman, A. S., Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2002, 54, (1), 3-‐12. 17. Mi, L.; Xue, H.; Li, Y.; Jiang, S., A Thermoresponsive Antimicrobial Wound Dressing Hydrogel Based on a Cationic Betaine Ester. Adv. Funct. Mater. 2011, 21, (21), 4028-‐4034. 18. Stojadinovic, A.; Carlson, J. W.; Schultz, G. S.; Davis, T. A.; Elster, E. A., Topical advances in wound care. Gynecologic Oncology 2008, 111, (2, Supplement), S70-‐ S80. 19. Zhao, C.; Li, X.; Li, L.; Cheng, G.; Gong, X.; Zheng, J., Dual Functionality of Antimicrobial and Antifouling of Poly(N-‐hydroxyethylacrylamide)/Salicylate Hydrogels. Langmuir 2013, 29, (5), 1517-‐1524. 20. Parra, F.; Vázquez, B.; Benito, L.; Barcenilla, J.; San Román, J., Foldable Antibacterial Acrylic Intraocular Lenses of High Refractive Index. Biomacromolecules 2009, 10, (11), 3055-‐3061. 21. Du, A. W.; Stenzel, M. H., Drug Carriers for the Delivery of Therapeutic Peptides. Biomacromolecules 2014, 15, (4), 1097-‐1114. 22. Zhang, X.; Soontornworajit, B.; Zhang, Z.; Chen, N.; Wang, Y., Enhanced Loading and Controlled Release of Antibiotics Using Nucleic Acids As an Antibiotic-‐ Binding Effector in Hydrogels. Biomacromolecules 2012, 13, (7), 2202-‐2210. 23. Li, Y.; Fukushima, K.; Coady, D. J.; Engler, A. C.; Liu, S.; Huang, Y.; Cho, J. S.; Guo, Y.; Miller, L. S.; Tan, J. P. K.; Ee, P. L. R.; Fan, W.; Yang, Y. Y.; Hedrick, J. L., Broad-‐ Spectrum Antimicrobial and Biofilm-‐Disrupting Hydrogels: Stereocomplex-‐Driven Supramolecular Assemblies. Angew. Chem., Int. Ed. 2013, 52, (2), 674-‐678. 24. Nederberg, F.; Zhang, Y.; Tan, J. P. K.; Xu, K.; Wang, H.; Yang, C.; Gao, S.; Guo, X. D.; Fukushima, K.; Li, L.; Hedrick, J. L.; Yang, Y.-‐Y., Biodegradable nanostructures with selective lysis of microbial membranes. Nat. Chem. 2011, 3, (5), 409-‐414.


Page 31 of 33

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

Biomacromolecules

25. Chin, W.; Yang, C.; Ng, V. W. L.; Huang, Y.; Cheng, J.; Tong, Y. W.; Coady, D. J.; Fan, W.; Hedrick, J. L.; Yang, Y. Y., Biodegradable Broad-‐Spectrum Antimicrobial Polycarbonates: Investigating the Role of Chemical Structure on Activity and Selectivity. Macromolecules (Washington, DC, U. S.) 2013, 46, (22), 8797-‐8807. 26. Engler, A. C.; Tan, J. P. K.; Ong, Z. Y.; Coady, D. J.; Ng, V. W. L.; 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, (12), 4331-‐4339. 27. Ng, V. W. L.; Chan, J. M. W.; Sardon, H.; Ono, R. J.; Garc a, J. M.; Yang, Y. Y.; Hedrick, J. L., Antimicrobial hydrogels: A new weapon in the arsenal against multidrug-‐resistant infections. Adv. Drug Delivery Rev. 2014, 78, (0), 46-‐62. 28. Ribeiro, M. P.; Espiga, A.; Silva, D.; Baptista, P.; Henriques, J.; Ferreira, C.; Silva, J. C.; Borges, J. P.; Pires, E.; Chaves, P.; Correia, I. J., Development of a new chitosan hydrogel for wound dressing. Wound. Repair. Regen. 2009, 17, (6), 817-‐ 824. 29. Jayakumar, R.; Prabaharan, M.; Sudheesh Kumar, P. T.; Nair, S. V.; Tamura, H., Biomaterials based on chitin and chitosan in wound dressing applications. Biotechnol. Adv. 2011, 29, (3), 322-‐337. 30. Wang, T.; Zhu, X.-‐K.; Xue, X.-‐T.; Wu, D.-‐Y., Hydrogel sheets of chitosan, honey and gelatin as burn wound dressings. Carbohydr. Polym. 2012, 88, (1), 75-‐83. 31. Rattanaruengsrikul, V.; Pimpha, N.; Supaphol, P., Development of Gelatin Hydrogel Pads as Antibacterial Wound Dressings. Macromol. Biosci. 2009, 9, (10), 1004-‐1015. 32. Dragan, E. S., Design and applications of interpenetrating polymer network hydrogels. A review. Chem. Eng. J. 2014, 243, (0), 572-‐590. 33. Hong, Y.; Fujimoto, K.; Hashizume, R.; Guan, J.; Stankus, J. J.; Tobita, K.; Wagner, W. R., Generating Elastic, Biodegradable Polyurethane/Poly(lactide-‐co-‐ glycolide) Fibrous Sheets with Controlled Antibiotic Release via Two-‐Stream Electrospinning. Biomacromolecules 2008, 9, (4), 1200-‐1207. 34. Ng, V. W. L.; Tan, J. P. K.; Leong, J.; Voo, Z. X.; Hedrick, J. L.; Yang, Y. Y., Antimicrobial Polycarbonates: Investigating the Impact of Nitrogen-‐Containing Heterocycles as Quaternizing Agents. Macromolecules (Washington, DC, U. S.) 2014, 47, (4), 1285-‐1291. 35. Qiao, Y.; Yang, C.; Coady, D. J.; Ong, Z. Y.; Hedrick, J. L.; Yang, Y.-‐Y., Highly dynamic biodegradable micelles capable of lysing Gram-‐positive and Gram-‐ negative bacterial membrane. Biomaterials 2012, 33, (4), 1146-‐1153. 36. Feng, J.; Zhuo, R.-‐X.; Zhang, X.-‐Z., Construction of functional aliphatic polycarbonates for biomedical applications. Prog. Polym. Sci. 2012, 37, (2), 211-‐ 236. 37. Mespouille, L.; Coulembier, O.; Kawalec, M.; Dove, A. P.; Dubois, P., Implementation of metal-‐free ring-‐opening polymerization in the preparation of aliphatic polycarbonate materials. Prog. Polym. Sci. 2014, 39, (6), 1144-‐1164. 38. Wang, H.-‐F.; Su, W.; Zhang, C.; Luo, X.-‐h.; Feng, J., Biocatalytic Fabrication of Fast-‐Degradable, Water-‐Soluble Polycarbonate Functionalized with Tertiary Amine Groups in Backbone. Biomacromolecules 2010, 11, (10), 2550-‐2557. 39. Sardon, H.; Irusta, L.; Fernández-‐Berridi, M. J.; Lansalot, M.; Bourgeat-‐Lami, E., Synthesis of room temperature self-‐curable waterborne hybrid polyurethanes functionalized with (3-‐aminopropyl)triethoxysilane (APTES). Polymer 2010, 51, (22), 5051-‐5057.


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40. Kawalec, M.; Dove, A. P.; Mespouille, L.; Dubois, P., Morpholine-‐ functionalized polycarbonate hydrogels for heavy metal ion sequestration. Polym. Chem. 2013, 4, (4), 1260-‐1270. 41. Sanders, D. P.; Fukushima, K.; Coady, D. J.; Nelson, A.; Fujiwara, M.; Yasumoto, M.; Hedrick, J. L., A Simple and Efficient Synthesis of Functionalized Cyclic Carbonate Monomers Using a Versatile Pentafluorophenyl Ester Intermediate. J. Am. Chem. Soc. 2010, 132, (42), 14724-‐14726. 42. Kiesewetter, M. K.; Shin, E. J.; Hedrick, J. L.; Waymouth, R. M., Organocatalysis: Opportunities and Challenges for Polymer Synthesis. Macromolecules 2010, 43, (5), 2093-‐2107. 43. Coady, D. J.; Horn, H. W.; Jones, G. O.; Sardon, H.; Engler, A. C.; Waymouth, R. M.; Rice, J. E.; Yang, Y. Y.; Hedrick, J. L., Polymerizing Base Sensitive Cyclic Carbonates Using Acid Catalysis. ACS Macro Letters 2013, 2, (4), 306-‐312. 44. Sardon, H.; Irusta, L.; Santamaría, P.; Fernández-‐Berridi, M. J., Thermal and mechanical behaviour of self-‐curable waterborne hybrid polyurethanes functionalized with (3-‐aminopropyl)triethoxysilane (APTES). J. Polym. Res. 2012, 19, (9), 1-‐9. 45. Nederberg, F.; Trang, V.; Pratt, R. C.; Kim, S.-‐H.; Colson, J.; Nelson, A.; Frank, C. W.; Hedrick, J. L.; Dubois, P.; Mespouille, L., Exploring the versatility of hydrogels derived from living organocatalytic ring-‐opening polymerization. Soft Matter 2010, 6, (9), 2006-‐2012. 46. Li, Y.; Fukushima, K.; Coady, D. J.; Engler, A. C.; Liu, S.; Huang, Y.; Cho, J. S.; Guo, Y.; Miller, L. S.; Tan, J. P. K.; Ee, P. L. R.; Fan, W.; Yang, Y. Y.; Hedrick, J. L., Broad-‐ Spectrum Antimicrobial and Biofilm-‐Disrupting Hydrogels: Stereocomplex-‐Driven Supramolecular Assemblies. Angew. Chem. 2013, 125, (2), 702-‐706. 47. Wang, L.-‐S.; Cheng, S.-‐X.; Zhuo, R.-‐X., Synthesis and hydrolytic degradation of aliphatic polycarbonate based on dihydroxyacetone. Polym. Sci. Ser. B 2013, 55, (11-‐12), 604-‐610. 48. Mespouille, L.; Coulembier, O.; Paneva, D.; Degée, P.; Rashkov, I.; Dubois, P., Novel Biodegradable Adaptive Hydrogels: Controlled Synthesis and Full Characterization of the Amphiphilic Co-‐Networks. Chem. Eur. J. 2008, 14, (21), 6369-‐6378. 49. Howell-‐Jones, R. S.; Wilson, M. J.; Hill, K. E.; Howard, A. J.; Price, P. E.; Thomas, D. W., A review of the microbiology, antibiotic usage and resistance in chronic skin wounds. J. Antimicrob. Chemother. 2005, 55, (2), 143-‐149. 50. Lee, A. L. Z.; Ng, V. W. L.; Wang, W.; Hedrick, J. L.; Yang, Y. Y., Block copolymer mixtures as antimicrobial hydrogels for biofilm eradication. Biomaterials 2013, 34, (38), 10278-‐10286.


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