Broad-Spectrum Antimicrobial Polycarbonate Hydrogels with FastDegradabilityAna Pascual,† Jeremy P. K. Tan,‡ Alex Yuen,† Julian M. W. Chan,§ Daniel J. Coady,§ David Mecerreyes,⊥

James L. Hedrick,*,§ Yi Yan Yang,*,‡ and Haritz Sardon*,†

†POLYMAT, University of the Basque Country UPV/EHU Joxe Mari Korta Center, Avda. Tolosa 72, 20018 Donostia-San Sebastian,Spain‡Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore§IBM Almaden Research Center, 650 Harry Road, San Jose, California 95120, United States⊥Ikerbasque, Basque Foundation for Science, E-48011 Bilbao, Spain

*S Supporting Information

ABSTRACT: In this study, a new family of broad-spectrumantimicrobial polycarbonate hydrogels has been successfullysynthesized and characterized. Tertiary amine-containingeight-membered monofunctional and difunctional cycliccarbonates were synthesized, and chemically cross-linkedpolycarbonate hydrogels were obtained by copolymerizingthese monomers with a poly(ethylene glycol)-based bifunc-tional initiator via organocatalyzed ring-opening polymer-ization using 1,8-diazabicyclo[5.4.0]undec-7-ene catalyst. Thegels were quaternized using methyl iodide to conferantimicrobial properties. Stable hydrogels were obtained onlywhen the bifunctional monomer concentration was equal to orhigher than 12 mol %. In vitro antimicrobial studies revealed that all quaternized hydrogels exhibited broad-spectrumantimicrobial activity against Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative), Pseudomonas aeruginosa(Gram-negative), and Candida albicans (fungus), while the antimicrobial activity of the nonquaternized hydrogels was negligible.Moreover, the gels showed fast degradation at room temperature (4−6 days), which makes them ideal candidates for woundhealing and implantable biomaterials.

■ INTRODUCTION

Wound treatment is an important issue in healthcare, andprogress in this area can have a major impact on patients’quality of life. It is believed that the treatment of chronicwounds will become an important challenge to healthcaresystems worldwide due to an aging society.1 According to theUnited Nations, six to eight million Europeans were affected bychronic wounds in 2009.2 Bearing in mind the significantimpact of wound care on patient health, appropriate diagnosisand treatment are essential.3 Despite these urgent needs,wound dressing technology still has a long way to go probablydue to the intrinsic complexity of the wound healing process.4,5

In the past, scientists had focused on drying the wound sitewith absorptive gauzes, though the value of gauze dressings isheavily debated because of the pain and damage they cause tothe neo-epithelium during removal.6,7

In past years, different polymer-based technologies have beendeveloped to improve the care of conventional wounds. Forinstance, Winter et al. found that by covering the wound with apolymer dressing, the rate of epithelialization was significantlyaccelerated.8 In the past decade, hydrogels have been studiedand used as alternatives to gauze dressing materials in wound

dressing applications.9−15 Hydrogels have the ability tomaintain wound occlusion by creating a more conduciveenvironment for tissue regeneration than exposedwounds.4,11,16−18 In addition, hydrogels offer a more versatileplatform that allows for incorporation of growth factors toaccelerate wound healing as compared to traditional gauzedressings.19,20 The main problem in using hydrogels for woundcare is the high risk of infections, as the moist environment ofthe hydrogels promotes pathogen proliferation and coloniza-tion. To overcome this issue, antibiotics13,21,22 or silver11 wereloaded into the hydrogels. Although the killing efficacy andbiocompatibility of conventional antibiotics have been demon-strated, the inability to combat multidrug resistant infections istheir major drawback.10,23,24 Therefore, several attempts havebeen made to develop antimicrobial hydrogels that are lesssusceptible to the development of multidrug resistance (MDR)by virtue of their different mechanism of action.3,5,7,13,14

Received: December 18, 2014Revised: March 10, 2015

Article

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© XXXX American Chemical Society A DOI: 10.1021/bm501836zBiomacromolecules XXXX, XXX, XXX−XXX

Advances in synthetic chemistry have offered the ability totailor the molecular structure and functionality of polymers toimpart broad-spectrum antimicrobial activity and25,26 predict-able mechanical and rheological properties to hydrogels. Hence,natural biomaterials, synthetic polymers, or their blends havebeen explored for wound dressing applications.27 Hydrogelsbased on natural materials like chitosan28,29 or gelatin30,31 sufferfrom certain limitations such as batch-to-batch variations inmolecular weight and immunogenicity. To sidestep theseissues, synthetic polymers including polyacrylates,32 polyur-ethanes,33 and polyesters17 have been explored for wounddressing applications.6,7 Recently, attention has been directedtoward the development of antimicrobial aliphatic polycar-bonates with broad-spectrum activity and the ability tocircumvent MDR.12,24,26,34,35 Aliphatic polycarbonates areideal candidates for biomedical applications because of theirlow toxicity and ease of incorporating functionality.36,37 Theability of polycarbonates to eradicate Gram-positive and Gram-negative bacteria by incorporating quaternary ammoniums inthe polymer backbone has been shown.23,26,34 The idealhydrogels used in wound healing applications should bedegradable and able to prevent infection. Feng et al. showedthat linear polycarbonates functionalized with tertiary aminescould degrade in three months without creating any significanttoxicity upon degradation but without antimicrobial proper-ties.38

The goal of this study is to design an ideal hydrogel materialfor wound healing applications, featuring a combination ofantimicrobial properties and biodegradability. In particular,eight-membered cyclic and dicyclic carbonate monomerscontaining tertiary amines were synthesized and subsequentlyconverted 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 theinitiator. Rheological behavior, swelling degree, and gel contentwere studied to demonstrate gel formation. In addition, theantimicrobial behavior of these gels and their degradationprofiles were also investigated.

■ MATERIALS AND METHODSMaterials. N-methyldiethanolamine (≥99%), N,N,N′,N′-tetrakis-

(2-hydroxyethyl)ethylenediamine (≥99%), triphosgene (98%), trie-thylamine (TEA) (≥99%), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)(98%), poly(ethylene glycol) end-capped diol (PEG1500, Mn = 1500 gmol−1), poly(ethylene glycol) end-capped diol (PEG4000, Mn = 4000 gmol−1), poly(ethylene glycol) end-capped diol (PEG8000, Mn = 8000 gmol−1), and methyl iodide (CH3I) (≥99%) were purchased fromSigma-Aldrich and used as received. Dichloromethane (DCM)(≥99%) and tetrahydrofuran (THF) (≥99%) were dried usingactivated 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 chargedwith N-methyl diethanolamine (5.00 g, 42.0 mmol), triethylamine(9.30 g, 92.4 mmol), and THF (400 mL). The reaction mixture wasstirred for 30 min under N2 at −80 °C. Next, triphosgene (4.60 g, 15.0mmol) dissolved in 50.0 mL of THF was added dropwise to thereaction mixture and stirred for 3 h. The reaction mixture was treatedwith cold diethyl ether (2 L), in which the triethylammonium chloridesalt was precipitated and recovered. The product-containing filtratewas 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,Supporting Information). 13C NMR (400 MHz, CDCl3, 22 °C): δ =156.5 (CO), 69.2 (CH2), 56.7 (CH2), 44.9 (CH3) (Figure SI 2).

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), triethyl-

Scheme 1. General Synthetic Route of Eight-Membered Cyclic (a) and Dicyclic (b) Carbonate Monomers

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amine (17.8 g, 180 mmol), and THF (400 mL). The reaction mixturewas stirred for 30 min under N2 at −80 °C, to which triphosgene (9.20g, 30.0 mmol) dissolved in 50.0 mL of THF was then added dropwiseand stirred for 3 h. The reaction mixture was treated with cold diethylether (2 L), and the product was precipitated together withtriethylammonium chloride salt. The precipitate was redissolved incold dichloromethane and rinsed sequentially with saturated aqueousNaHCO3 and water, dried over MgSO4, and concentrated in vacuo.The crude product was recrystallized from hexanes to afford Monomer2 (Yield: 3.2 g, 52%). Monomer 2: 1H 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(CO), 69.5 (CH2), 55.6 (CH2), 55.3 (CH2) (Figure SI 4).Synthesis of Linear Polymer. Monomer 1 (0.310 g, 2.2 mmol)

and PEG1500 end-capped diol (0.0300 g, 0.022 mmol) were dissolvedin 2 mL of DCM. DBU (0.0180 g, 0.11 mmol) in DCM (0.1 mL) wasadded, and conversion to product was monitored using 1H NMR untilcompletion (approximately 24 h). The catalyst was quenched with anexcess of benzoic acid, and the crude polymer solution wasprecipitated in ether and dried under vacuum (Yield: 0.32 g, 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) (Figure SI 5, SupportingInformation).Synthesis of Polycarbonate Hydrogels. A 20 mL glass vial

containing a magnetic stir-bar was charged with monomer 1 (0.310g, 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) inDCM (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 roomtemperature for 24 h. DBU was quenched with an excess of benzoicacid. The resulting hydrogel was washed by immersion in DCM toremove catalyst and soluble fractions, and dried under vacuum. (Yield:0.40 g, 89%). Synthesis of other hydrogels with different compositionsis 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 temperatureunder N2. Methyl iodide excess was removed by drying in vacuo.Methods. NMR Spectra. 1H NMR spectra were obtained on a

Bruker Avance 400 instrument. Chemical shifts are reported in ppmfrom tetramethylsilane with the solvent resonance as the internalstandard (CDCl3: δ 7.26 ppm). 13C NMR spectra were recorded on aBruker Avance 400 spectrometer with complete proton decoupling.Chemical shifts are reported in ppm from tetramethylsilane with thesolvent resonance as the internal standard (CDCl3: δ 77.16 ppm).Gas Permeation Chromatography. Gas Permeation Chromatog-

raphy (GPC) was performed in THF at 30 °C using a Waterschromatograph equipped with four 5 mm Waters columns (300 mm ×7.7 mm) connected in series with increasing pore size (100, 1000, 105,106 Å).Fourier Transform Infrared Spectroscopy. Fourier Transform

Infrared (FTIR) spectroscopy analysis was conducted on a NicoletMagna 560 spectrometer at a resolution of 2 cm−1, and a total of 64interferograms were signal-averaged. Samples were prepared bysolution casting the reaction mixture onto a KBr window.Rheological Analysis. Rheology measurements on the dry gels

were conducted on a Thermo-Haake Rheostress I viscoelastometerusing oscillatory tests. Angular frequency sweeps from 0.1−10 rad/s atconstant strain amplitude (γ = 0.005) were applied at 25 °C. G′ andG″ values were plotted versus frequency.Gel Fraction. The gel fraction (Fg) was determined by extraction

with DCM after the samples were dried for 24 h at 45 °C. The processconsisted of a 24 h continuous extraction with DCM under reflux in a250 mL round-bottom flask.39 After the extraction, the samples weredried, and the gel content was calculated as the ratio between the drypolymer remaining after the extraction (mt) and the initial amount ofdry polymer (m0). The molecular weight of the soluble part wasdetermined by GPC.

=−

×Fm m

m(%) 100g

0 t

0

Swelling. Dry hydrogels (diameter of 4 mm and weight 10−15mg) were immersed in aqueous solution at ambient temperature. Afterthe gel was kept for the desired time interval, the gel was withdrawn.Excess solution was gently removed from the hydrogel surface withtissue paper, and the mass increase was determined gravimetrically.The swelling degree (St) was calculated as follows:

= − ×S m m m(%) ( )/ 100t t 0 0

where mt = mass of the hydrogel after time t, and m0 = initial mass ofthe dry hydrogel.40

Antibacterial Activity. Escherichia coli (ATCC 25922), Pseudo-monas aeruginosa (ATCC 9027), and Staphylococcus aureus (ATCC29737) were reconstituted from their lyophilized forms according tothe manufacturer’s protocol and cultured in tryptic soy broth (TSB) at37 °C under constant shaking of 300 rpm, while Candida albicans(ATCC 10231) was cultured in yeast media broth (YMB) at roomtemperature under constant shaking of 50 rpm. Prior to treatment, themicrobes were first inoculated overnight to enter into log growthphase. The quaternized hydrogel was cut into a 5 mm × 5 mm squareand placed into a 1.7 mL microcentrifuge 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 ofmicrobe solution was adjusted to give an initial optical density (O.D.)reading of approximately 0.07 at 600 nm wavelength on a microplatereader (TECAN, Switzerland), which corresponds to the concen-tration of McFarland 1 solution (3 × 108 CFU/mL). The tube waskept either at 37 °C for bacterial samples or room temperature for C.albicans under constant shaking (300 and 50 rpm for bacteria andfungi, respectively) for 24 and 42 h, respectively. After the hydrogeltreatment, the samples were taken for a series of 10-fold dilution andplated onto agar plates. The plates were incubated for 24 h at 37 °Cfor bacterial samples or 42 h at room temperature for C. albicansbefore the number of colony-forming units (CFU) was counted.Microbes treated with hydrogel without cationic polycarbonates wereused as negative control, and each test was carried out in threereplicates.

Kinetics Study. E. coli were inoculated and prepared according tothe procedure described in the previous section. The bacteria weretreated with Gel 20%_8000 and incubated at 37 °C under constantshaking 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 10-folddilution and plated onto agar plates. The plates were incubated for 24h at 37 °C before the number of CFU was counted. Bacteria withouthydrogel treatment were used as negative controls, and each test wascarried out in three replicates.

Field Emission Scanning Electron Microscopy (FE-SEM). E.coli grown in TSB alone or after treatment with Gel 20%_8000 wereprepared using the same protocol as the antibacterial testing but with ashorter incubation time of 2 h. The bacteria were collected intomicrofuge tubes, pelleted at 4000 rpm for 5 min, and washed withphosphate-buffered saline (PBS) twice. The samples were fixed with2.5% glutaraldehyde for 60 min followed by washing with DI watertwice. Dehydration was performed with a series of ethanol solutions(35%, 50%, 75%, 90%, 95%, and 100%) before the samples weredripped onto copper tape and left to dry for 2 days. The dried sampleswere coated with platinum before SEM imaging under the FE-SEM(JEOL JSM-7400, Japan).

Hemolysis Assay. The undesired activity of the gel against redblood 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 25× volumetric dilution in PBSto achieve a 4% blood content. The diluted rRBCs were added tohydrogels and incubated in 37 °C for an hour. After incubation, thesamples were pelleted under 3000 g for 5 min. The supernatant (100μL) was transferred to a 96-well plate, and hemoglobin release wasanalyzed spectrophotometrically by measuring absorbance at 576 nmusing a microplate reader (TECAN, Switzerland). Two control groupswere used in this assay: untreated RBC suspended in PBS (negativecontrol) and RBC treated with 0.1% Triton-X (positive control). Each

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assay was performed in four replicates and repeated three times.Percentage of hemolysis was calculated as follows:

=−−

×

⎡⎣⎢

⎤⎦⎥

OD ODOD OD

%hemolysisof treated sample of negative controlof positive control of negative control

100

576 576

576 576

In Vitro Cytotoxicity. Cytotoxicity of the hydrogels and thedegraded products of the hydrogels was investigated by MTT assay.HEK 293 human embryonic kidney cell line was obtained from ATCC(USA) and cultured in DMEM supplemented with 10% fetal bovineserum and 5% penicillin−streptomycin. HEK 293 cells were seeded ata density of 10 000 cells/well on 96-well plates. The cells wereincubated overnight at 37 °C with 5% CO2. Cell culture media wasreplaced with fresh DMEM (100 μL), and to each well, the hydrogelwas added. Each hydrogel was tested in three replicates. Gel 20%_8000 was soaked in DMEM for 7 days to ensure completedegradation before 100 μL of the degraded solution was added intothe wells to check on the cytotoxicity of the degraded product. Thecontrol used was DMEM media that was left at room temperature for7 days. The plates were placed back into the incubator at 37 °C with5% CO2 for 48 h. Next, hydrogels were fished out of the cell, andmedia was replaced with 100 μL of fresh media and 20 μL of MTTsolution (5 mg/mL) and returned to the incubator for 4 h. Themixture was carefully removed, and the purple formazan crystalsinternalized by live cells were dissolved with dimethyl sulfoxide (150μL). The absorbance of the purple formazan crystals in each well was

calculated as that at 550 nm deducted by that at 690 nm. Cell viabilitywas calculated as a percentage of absorbance of the nontreated 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 weresubsequently dried under vacuum and analyzed by FTIR and NMRspectroscopy.

■ RESULTS AND DISCUSSION

Monomer Synthesis and Polymerization. The 6-methyl-1,3,6-dioxazocan-2-one cyclic carbonate (monomer 1) wassynthesized via a strategy previously described by Hedrick andco-workers.41 Here, reaction between the N-methyldiethanol-amine with triphosgene resulted in a one-pot ring-closure of thediol to generate an eight-membered cyclic carbonate in thepresence of triethylamine (Scheme 1a). The monomerstructure was confirmed by 1H and 13C NMR spectroscopy.Similarly, 6,6′-(ethane-1,2-diyl)bis(1,3,6-dioxazocan-2-one) di-cyclic carbonate (monomer 2) was synthesized using the samestrategy (Scheme 1b), and its successful synthesis wasconfirmed 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 SI 1).DBU was chosen as the catalyst as it has been demonstrated to

Figure 1. Synthesis of quaternized polycarbonate hydrogels.

Table 1. Conversion Rate (Gel Fraction) and Swelling Degree for Different Hydrogels Synthesized Using a Molar Ratio ofPEG/Catalyst/Monomers at 1:5:100 in DCM (Monomer Concentration: 1 M) at 20 °C. Data Correspond to Mean ± StandardDeviation (n = 3)

entry monomer 1 [mol %] monomer 2 [mol %] PEG Mn [KDa] Fga [%] St (1 h)b [%] Mw(sol)

c [kDa]

Gel4%_1500 96 4 1.5 71 ± 4 -d 10.7Gel8%_1500 92 8 1.5 77 ± 6 50 ± 3 6.5Gel12%_1500 88 12 1.5 98 ± 1 47 ± 4 11.1Gel16%_1500 84 16 1.5 97 ± 2 40 ± 2 8.9Gel20%_1500 80 20 1.5 98 ± 1 35 ± 1 7.8Gel20%_4000 80 20 4.0 97 ± 1 55 ± 2 7.4Gel20%_8000 80 20 8.0 98 ± 1 77 ± 3 7.2Gel20%_1500quaternized 80 20 1.5 98 ± 1 20 ± 2 7.8Gel20%_8000quaternized 80 20 8.0 98 ± 1 68 ± 3 7.2

aCalculated as (mass of recovered dry gel)/(mass of the comonomers used for the synthesis) × 100. bSwelling degree was calculated after the gelswere immersed in H2O for 1 h. cMw was calculated by GPC after the soluble part was extracted in DCM. dNot possible to measure.

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be effective in the synthesis of cyclic carbonates.42 Polymer-ization was successfully initiated using PEG1500 end-capped diolat ambient temperature in DCM for a targeted DP of 100 asevidenced by 1H NMR (Figure SI 5, Supporting Information).The polymerization was carried out in the presence of air andalso under an inert N2 atmosphere. It was observed that thepolymerization conditions did not affect the conversion, andfull conversion was achieved after 24 h. However, when thepolymerization was performed in the presence of air, themolecular weight and dispersity were substantially affected.Specifically, the number molecular weight (Mn) was reducedfrom 12 kDa to 6.5 kDa, and the polydispersity index wasbroadened from 1.2 to 1.8 (Table SI 1). It is known that watertraces can facilitate the formation of diols coming fromcarbonate monomers.43 These diols may act as initiator in thering-opening polymerization process, reducing the molecularweight and increasing the polydispersity index. Nevertheless,

these results suggest that the polymerization of monomer 1could be carried out in air, although the control of thepolymerization was compromised.

Synthesis of Cross-Linked Polycarbonates and Hydro-gel Preparation. Monofunctional monomer 1 was copoly-merized with different amounts of difunctional monomer 2 inthe presence of PEG diol initiator to obtain cross-linkedhydrogels. Because of the bifunctional nature of monomer 2, itgenerates cross-linking points, forming a polymer network(Figure 1). The hydrogels were synthesized by ROP initiatedby PEG diol in DCM and catalyzed by DBU at ambienttemperature under air atmosphere. An initial monomerconcentration of 1 M was applied and different monomer 1/monomer 2 ratios were used, while the initiator and catalystconcentrations were kept constant as shown in Table 1.Hydrogels with different PEG molecular weights (Mn 1500,4000, and 8000 g mol−1) used as initiators were synthesized tostudy their effect on the rheological, antimicrobial, and swellingproperties.Hydrogels were prepared by immersing the resulting cross-

linked materials in water for 1 h. As shown in Table 1, at lowdifunctional monomer concentrations, the hydrogels were notable to maintain a homogeneous structure after swelling inaqueous media (Figure SI 6). When the concentration ofbifunctional monomer 2 was raised above 8 mol %, an easy-to-handle hydrogel was obtained (Figure SI 7). It was alsoobserved that when monomer 2 concentration is higher than12%, a gel fraction around 100% was achieved due to thepresence of a significant number of cross-linking 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 areimportant features to take into account when designinghydrogels for wound dressing applications. High swellingcapacity helps to maintain wound occlusion and a conduciveenvironment that favors the regeneration process. Although allmaterials were able to swell in water, the polymers with lowbifunctional concentrations were not able to maintain the three-dimensional structure. Swelling ability and behavior of thehydrogels were adversely affected when the concentration ofbifunctional monomer was raised. As bifunctional monomer 2concentration increased, the number of cross-linking points perchain for a given DP increased; a higher concentrationconsiderably reduced the swelling behavior.39,44,45 In addition,swelling ratio increased significantly when PEG with a greatermolecular length was used (Table 1). The results showed thatthe degree of swelling is a function of the cross-linking densityand PEG length. This phenomenon was also observed in thework reported by Dubois et al.40

Quaternization of Tertiary Amine-Containing Poly-carbonate Hydrogels. From the work reported by Hedrick etal., positively charged polycarbonates containing pendantquaternary amines are highly active toward Gram-positive andGram-negative bacteria.24,46 A simple and facile quaternizationreaction was carried out by immersing the previous synthesizedhydrogels in pure methyl iodide (Scheme 1). Again, we foundthat the materials with low cross-linking degree such as Gel 4%and Gel 8% did not remain robust and consistent when theywere immersed in methyl iodide. Therefore, they were excludedfrom further analysis. The rheological behavior of thenonquaternized and quaternized cross-linked materials wasinvestigated by measuring the elastic (G′) and viscous (G″)moduli in the dry state as a function of frequency at room

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

Figure 3. Infrared spectra of nonquaternized gel containing 20 mol %of monomer 2 initiated with PEG8000 diol after incubation in PBS (pH7.4) for various periods of time.

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temperature (Figure SI 8). In all cases, G′ was greater than G″in the entire frequency range, which suggests that the materialsremained chemically cross-linked after the quaternization step.Swelling behavior for Gel 20% with PEG1500 and PEG8000

before and after quaternization was investigated (Table 1 andFigure 2). The swelling ratio of hydrogels synthesized at themonomer 1/monomer 2 molar ratio of 4:1 increased from 210± 20 wt % to 705 ± 15 wt % when PEG8000 diol was used asmacroinitiator. Nevertheless, when hydrogels were quaternizedwith methyl iodide, the swelling ratio was substantially reduced.Although the quaternization considerably reduced the water

uptake, the swelling ratio was still above 200 wt % whenPEG8000 diol was used as macroinitiator, which is ideal forkeeping the wound in an occluded state. It should be pointedout that that because of the fast degradation process in thenonquaternized gel, the gels are not able to reach a plateaubefore degradation. Meanwhile, the quaternized gels were ableto reach the plateau giving a more realistic value of themaximum water-uptake. This could explain the higher wateruptakes observed for the nonquaternized gels compared to thequaternized gels. Another important parameter when dealingwith hydrogels is the gel fraction. The gel fraction for most ofthe gels listed in Table 1 is close to 100 wt %, which indicatesthat the molar composition of the hydrogels is very close to thefeed ratio. In addition, quaternization did not affect gel fraction(Gel20%_1500quaternized vs Gel20%_1500; Gel20%_8000quaternized vs Gel20%_8000). These results are ingood agreement with similar systems studied by Nederberg45

and Kawalec et al.40 for polycarbonate-based systems.Degradation Behavior of Polycarbonate Hydrogels.

Aliphatic polycarbonates are known to be biodegradable. Thus,the degradation process at 25 °C of the nonquaternized gelcontaining 20 mol % of monomer 2 initiated with PEG8000 diolwas monitored by FTIR as shown in Figure 3. As thedegradation proceeded, there was an intensity decrease andcomplete disappearance of the carbonate (CO) stretch at1740 cm−1 after 4 and 6 days of incubation in PBS (pH 7.4),respectively. Meanwhile, two new broad bands appear at 3300and 1640 cm−1 that are attributed to alcohol groups andcarboxylic acid generated as a consequence of the hydrolysis ofthe polycarbonate hydrogel. From the photographs of thehydrogels as shown in Figure 3, the mechanical strength of

Figure 4. 1H NMR spectra of the gel containing 20 mol % of monomer 2 initiated with PEG8000 diol after incubation in water at room temperaturefor 3 and 6 days.

Figure 5. Schematic representation of polycarbonate hydrogeldegradation in aqueous media.

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hydrogels decreased as a function of incubation time. After 6

days of incubation in PBS, the hydrogel completely

decomposed.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 groupslocated next to carbonate at 4.26 ppm and the adjacentmethylene at 2.77 ppm disappeared, while three new signals at3.66, 2.60, and 2.32 ppm corresponding to the signals of pureN-methyldiethanolamine or N,N,N,N′-tetrakis(2-hydroxyethyl)

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

Figure 7. FE-SEM images of E. coli before (a, c) and after (b, d) 2 h treatment with Gel 20%_8000. Panels c and d are at higher magnification. Scalebar: 1 μm.

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were seen after 6 days of degradation. Furthermore, after 3days, a new signal at 9.90 ppm was observed, which wasassigned to carboxylic acids. To further verify the presence ofcarboxylic acids, 13C NMR analysis was carried out (SupportingInformation). In 13C NMR spectrum, a new signal at 170 ppmassociated with carboxylic acid groups was clearly observed(Figure SI 9, Supporting Information).From NMR and FTIR analyses, a degradation mechanism is

proposed as shown in Figure 5. Hydrolysis of the polycarbonatebackbone occurred, forming the branched polycarbonatecopolymers with carboxylic groups as end groups. Decarbox-ylation may then occur, followed by transesterification reactionsto yield the monomers. This behavior was also observed insimilar systems.47 It is worth to mention that the degradationprocess was really fast. In our opinion, this occurred becausethe amines presented in the polymer backbone promoted thedegradation process by catalyzing the hydrolysis and decarbox-ylation reactions. Similar behavior was observed in quaternizedgels, although the degradation time was slightly increased from6 days to 8 days. Similar degradation times were observed byMespouille et al. in a poly(N,N-dimethylamino-2-ethylmethacrylate-graf t-poly[e-caprolactone]). It is worth mention-ing that although they were using highly acidic conditions(dioxane/aqueous HCL mixture (85:15)), only partialdegradation was observed.48

Antimicrobial Properties of Polycarbonate Hydrogels.The antimicrobial activity of different hydrogels was evaluatedagainst Gram-positive bacteria S. aureus, Gram-negative bacteriaE. coli and P. aeruginosa, and fungi C. albicans. These microbesare common pathogens that often manifest on dermal woundsand are typically treated by topical application of antibiotics tothe infected areas.49,50 The number of CFU for the bacteria orfungi was determined through the agar gel assay with a series of10-fold dilution. After the microbes were treated with thequaternized hydrogels, the growth of the microbes wascompletely suppressed with killing efficiency of at least99.999% (Figure SI 10). Furthermore, the CFU counts after18 h of treatment (Figure 6) showed that the quaternizedhydrogels were bactericidal to a wide range of microbes. Therewas a 6−10 log reduction in colony counts. Meanwhile, theunquaternized gels did not show any significant activity towardthe microbes. All hydrogels demonstrated similar antimicrobialactivity against all microbes tested, independent from the lengthof PEG diol and monomer 2 concentration. These resultsproved that the polycarbonate hydrogels must be quaternizedto offer antimicrobial efficacy. The ability of the Gel 20%_8000to kill bacterial cells upon exposure for various time intervalswas investigated using E. coli as a model microbe. After thebacteria was exposed to the hydrogel for 15 min, 30% of thebacteria was killed by the hydrogel, and after 2 h, 99.99% killingefficiency was achieved (Figure SI 11). The longer time wasneeded to achieve 99.9% kill because the bacteria had to comeinto contact with the hydrogel, which was settled at the bottomof the well.Antimicrobial Mechanism. From the earlier section, the

hydrogels have a bactericidal property. Observation ofmorphological changes of microbes before and after hydrogeltreatment gives further insights into the antimicrobialmechanism. As shown in Figure 7, the surfaces of E. coli cellsafter hydrogel treatment for 2 h are highly distorted andcorrugated. This suggested that the hydrogels killed the bacteriavia disrupting the bacterial membrane.

Hemolytic and Cytotoxicity Activities. Hydrogel toxicitywas evaluated via hemolysis and MTT assays using rRBC andHEK 293 cells. As seen in Figure SI 12, both the unquaternizedand quaternized hydrogels were nonhemolytic to rRBC after anhour of incubation. From the MTT assay done on HEK 293cells (Figure SI 13), all the unquaternized and quaternizedhydrogels showed no toxicity toward the cells, and thedegraded byproducts from the hydrogel were also compatiblewith the cells. These results demonstrated that the hydrogelswere nontoxic toward rRBC and HEK 293 cells. Thesehydrogels were chemically cross-linked as thin transparentfilms, which makes them desirable for wound healingapplications.

■ CONCLUSIONSA new family of fast biodegradable broad-spectrum antimicro-bial polycarbonate hydrogels has been successfully synthesizedfrom tertiary amine-functionalized eight-membered cycliccarbonate monomers. The use of DBU as an organocatalystefficiently catalyzed the synthesis of cross-linked polycarbonatesfrom new mono- and di-functional cyclic carbonate monomers.The swelling of the cross-linked polycarbonates depended onthe cross-linking degree, the length of the PEG macroinitiator,and the quaternization of the amine. The cross-linkedpolycarbonates were successfully quaternized with methyliodide, and the resulting quaternized polycarbonate hydrogelsshowed excellent ability to kill Gram-positive and Gram-negative bacteria as well as fungi. Both the quaternized andnonquaternized hydrogels showed rapid biodegradability on theorder of 4−6 days. Importantly, the hydrogels and theirdegradation products are nontoxic to mammalian cells. Insummary, these materials show great potential for applicationsin the areas of wound care and medical implants for theprevention of infections.

■ ASSOCIATED CONTENT*S Supporting InformationDetails of gel preparation protocol, 1H and 13C NMR spectra,rheological data, and antimicrobial data. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: haritz.sardon@ehu.es.*E-mail: hedrick@us.ibm.com.*E-mail: yyyang@ibn.a-star.edu.sg.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSH.S. and A.P. gratefully acknowledge financial support througha postdoctoral grant (DKR) from the Basque Government.Financial support from the Basque Government and MINECOthrough Project No. FDI 16507 and the Institute ofBioengineering and Nanotechnology (Biomedical ResearchCouncil, Agency for Science, Technology and Research,Singapore) through SERC Personal Care Programme GrantNo: 1325400028 is also acknowledged.

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