Biomaterials 34 (2013) 10278e10286

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Block copolymer mixtures as antimicrobial hydrogels for biolm

eradication
Ashlynn L.Z. Lee a, Victor W.L. Ng a, Weixin Wang a, James L. Hedrick b, **, Yi Yan Yang a, *
a
b

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore
IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 June 2013
Accepted 8 September 2013
Available online 30 September 2013

Current antimicrobial strategies have mostly been developed to manage infections due to planktonic
cells. However, microbes in their nature state will tend to exist by attaching to and growing on living and
inanimate surfaces that result in the formation of biolms. Conventional therapies for treating biolmrelated infections are likely to be insufcient due to the lower susceptibility of microbes that are
embedded in the biolm matrix. In this study, we report the development of biodegradable hydrogels
from vitamin E-functionalized polycarbonates for antimicrobial applications. These hydrogels were
formed by incorporating positively-charged polycarbonates containing propyl and benzyl side chains
with vitamin E moiety into physically cross-linked networks of ABA-type polycarbonate and poly(ethylene glycol) triblock copolymers. Investigations of the mechanical properties of the hydrogels
showed that the G0 values ranged from 1400 to 1600 Pa and the presence of cationic polycarbonate did
not affect the stiffness of the hydrogels. Shear-thinning behavior was observed as the hydrogels displayed high viscosity at low shear rates that dramatically decreased as the shear rate increased. In vitro
antimicrobial studies revealed that the more hydrophobic VE/BnCl(1:30)-loaded hydrogels generally
exhibited better antimicrobial/antifungal effects compared to the VE/PrBr(1:30) counterpart as lower
minimum biocidal concentrations (MBC) were observed in Staphylococcus aureus (Gram-positive),
Escherichia coli (Gram-negative) and Candida albicans (fungus) (156.2, 312.5, 312.5 mg/L for VE/
BnCl(1:30) and 312.5, 2500 and 625 mg/L for VE/PrBr(1:30) respectively). Similar trends were observed
for the treatment of biolms where VE/BnCl(1:30)-loaded hydrogels displayed better efciency with
regards to eradication of biomass and reduction of microbe viability of the biolms. Furthermore, a high
degree of synergistic antimicrobial effects was also observed through the co-delivery of antimicrobial
polycarbonates with a conventionally-used antifungal agent, uconazole. These hydrogels also displayed
excellent compatibility with human dermal broblasts with cell viability >80% after treatment with
hydrogels loaded with cationic polymers and/or uconazole at minimum biocidal concentrations (MBC).
2013 Elsevier Ltd. All rights reserved.

Keywords:
Block copolymers
Polycarbonate
Poly(ethylene glycol)
Hydrogel
Biolm
Antimicrobial

1. Introduction
In the on-going battle against microbial infections, the risks of
acquiring drug resistance has not ceased despite a considerable
number of strategies that have been devised for new cellular targets. Bearing this in mind, there is an increasing need for more
effective antimicrobial formulations that can be applied broadly in
a variety of biomedical-associated contexts. Antimicrobial hydrogels, which are heavily hydrated networks of polymers possessing

* Corresponding author. Tel.: 65 6824 7106; fax: 65 6478 9084.

** Corresponding author. Tel.: 1 408 927 1632; fax: 1 408 927 3310.
E-mail addresses: hedrick@almaden.ibm.com (J.L. Hedrick), yyyang@ibn.astar.edu.sg (Y.Y. Yang).
0142-9612/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2013.09.029

the ability to eliminate infectious microbes, are being exploited in

many pharmaceutical applications, including medication, disinfectants, sanitizers and personal care products [1]. Ideally, these
materials should exert antimicrobial actions effectively, eliminating
and preventing the recurrence of both planktonic as well as biolm
organisms. This is essentially important for limiting the emergence
of resistant subpopulation since antimicrobial resistance has
become a major global healthcare concern.
The development of an effective antimicrobial hydrogel requires
the understanding of the different growth behavior and treatment
susceptibility between planktonic cells and those embedded within
biolms. The formation of biolms is initiated by the deposition of
planktonic cells onto living tissue or an inanimate surface, such as
those of medical devices. These cells adhere and anchor themselves
to the surfaces via the production of exopolymers. As proliferation

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

occurs, microcolonies appear and the thickening of polymer matrix

around the microcolonies results in the growth of the biolm [2,3].
Biolm formation has been recognized as one of the leading causes
of a signicant amount of human infections [4,5]. Pathogens that
are commonly associated with biolm-induced chronic infections
include Staphylococcus aureus in chronic rhinosinusitis [6],
enteropathogenic Escherichia coli in recurrent urethritis [7,8] and
Candida albicans in candiasis [9].
Microbes that grow in biolms have been known to exhibit
dramatically higher resistance against antimicrobial agents
compared to free-oating cells [10e12]. Several factors contribute
to the intrinsically lower susceptibility to antimicrobials, including
restricted penetration of antimicrobials into a biolm due to
limited diffusion within the cell polymer matrix, decreased growth
rate which minimizes the uptake of biocides into the cells, as well
as the expression of possible resistance genes [13,14]. The minimal
inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of antimicrobials to biolm-associated microbes vary
widely depending on the strains and drugs used, and in some case,
it may be up to 1000-fold higher compared with planktonic bacteria [2]. For instance, Stoodley et al. reported that biolmassociated S. aureus required 3.5 times higher than the MBC of
oxacillin to provide a 3.0-log reduction in bacterial counts [15], and
Ceri et al. reported that E. coli required >500 times the MIC of
ampicillin for a 3.0-log reduction in bacteria growth [16].
With the emergence of antibiotic-resistant superbugs, the
development of newer, more potent antibiotics with antimicrobial
actions that are different from the conventional small molecule
drugs is crucial [17]. Polymeric biocides are a class of materials that
are engineered as synthetic mimics of naturally occurring hostdefense antimicrobial peptides. These amphiphilic, cationic polymers are able to selectively target and bind to microbes by electrostatic interactions and disintegrate the bacterial membranes by
insertion into the membrane lipid bilayer [18,19]. Such membrane
disruption mechanism causes rupture and lysis of the microbes,
thereby decreasing the potential of resistance development.
When used in vivo, selectively killing of microbes generally
comes from enhanced long-range electrostatic interaction between
the polymeric biocides and microbes in comparison to mammalian
cells [19,20]. The design of these polymers is dependent of several
factors that can greatly affect the antimicrobial activity and selectivity, such as the molecular weight, hydrophobic/hydrophilic balance, cationic chemical functionality [21,22]. Chan-Park et al.
recently reported the antimicrobial effects of UV-crosslinked chitosan-based hydrogels and found that the degree of quaternization
of the polymers played a signicant role in inuencing their
biocidal effects [23]. At a high cationic chitosan concentration
(10 wt.%), the hydrogels gave rise to >2.0-log reduction in microbial
counts (i.e. >99.0% killing efciency) for Pseudomonas aeruginosa,
E. coli, S. aureus and Fusarium solani after 1 h exposure, but >3.0-log
reduction (i.e. >99.9% killing efciency) was seen only in S. aureus
and F. solani. More recently, the same group reported a photopolymerized hydrogel system based on the antimicrobial peptide
-poly-L-lysine (EPL) grafted to methacrylic acid (MA) [24]. Antimicrobial activities of EPL-MA were retained after polymerization
and the hydrogels were able to reduce the counts of S. aureus and
P. aeruginosa by more than 99.9% (3-log reduction) at high EPL-MA
concentrations (21e25 wt.%) after 2 h exposure, while the hydrogels were less effective against C. albicans and F. solani (fungi) (<2log reduction in the fungal count). We have previously reported
physically crosslinked antimicrobial hydrogels prepared based on
the stereocomplexation between poly(L-lactide)-PEG-poly(L-lactide) and poly(D-lactide)-cationic polycarbonate-poly(D-lactide)
[25]. Physically crosslinked hydrogel is advantageous when used as
a topically applied antimicrobial formulation because it can be

10279

easily spread onto surfaces of different conrmation. However,

these polymers required a high content of hydrophobic poly(lactide) blocks for gelation and lactide blocks that were longer than
1 kDa prevented complete aqueous dissolution. In addition, the
polymers that were water soluble required high polymer concentration for gelation (13.2 wt.%) [25].
Herein, we report an antimicrobial hydrogel system based on
vitamin E-containing polycarbonate copolymers synthesized by
organocatalytic ring-opening polymerization (ROP). These hydrogels contain two parts e rst being the ABA-type triblock copolymer, consisting of a hydrophilic PEG middle block anked on both
ends by hydrophobic vitamin E-functionalized polycarbonate
blocks and the other is biocidal cationic polycarbonates that
possess vitamin E moieties. The hydrogels were formed based on
hydrophobic interactions between vitamin E-functionalized polycarbonate blocks, and their mechanical properties were characterized. The incorporation of vitamin E in the cationic
polycarbonates could potentially enhance the antimicrobial efcacy as a result of increased hydrophobicity of the polymers [26]. aTocopherol was chosen as it is the most biologically active form of
vitamin E and extensively studied amongst the other variants in the
family. In the body, it functions as an important lipid-soluble
antioxidant and assists in the process of wound healing [27,28].
Antimicrobial efcacy of these physically crosslinked hydrogels
was investigated against S. aureus (Gram-positive) and E. coli
(Gram-negative) bacteria as well as C. albicans (fungus). The
hydrogels were also evaluated for their efciency in eradicating
biolms through metabolism assessment, biomass removal and
scanning electron microscopy. In addition, synergism studies were
also performed on the co-delivery of cationic antimicrobial polycarbonates with conventionally-used antifungal agent, uconazole,
on C. albicans. The cytotoxicity of the hydrogels loaded with
cationic polymers and/or uconazole at MBCs was evaluated
against human dermal broblasts.
2. Materials and methods
2.1. Materials
MTC-VE (5-methyl-5-(a-tocopheryl)carboxyl-1,3-dioxan-2-one), MTC-BnCl (5methyl-5-(4-chloromethyl)benzylcarboxyl-1,3-dioxan-2-one) and MTC-PrBr (5methyl-5-bromopropylcarboxyl-1,3-dioxan-2-one) and N-(3,5-triuoromethyl)
phenyl-N0 -cyclohexylthiourea (TU) were synthesized by adapting a protocol previously reported by Pratt et al. (See the Supplementary Information for full experimental and characterization details) [29]. All reagents were bought from Sigmae
Aldrich and used as received unless otherwise mentioned. 1,8-Diazabicyclo[5,4,0]
undec-7-ene (DBU) and benzyl alcohol were dried over calcium hydride and vacuum
distilled twice before being transferred to the glove box. Methanol and ethanol (ACS
grade) were purchased from Tee Hai (Singapore). Glacial acetic acid was obtained
from VWR (U.S.A.). Ultra pure (HPLC grade) water was obtained from J.T. Baker
(U.S.A.). Tryptic soy broth (TSB) powder and yeast mould broth (YMB) powder were
purchased from BD Diagnostics (Singapore) and used to prepare the microbial
growth media according to the manufacturers instructions. S. aureus (ATCC No.
29737), E. coli (ATCC No. 25922) and C. albicans (ATCC No. 10231) were obtained from
ATCC (U.S.A), and re-constituted according to the suggested protocols. Fluconazole,
formalin solution, crystal violet, menadione and 2,3-bis (2-methoxy-4-nitro-5sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) were
purchased from Sigma, U.S.A.
2.2. Nuclear magnetic resonance (NMR) spectroscopy
The 1H NMR spectra of monomers and polymers were recorded using a Bruker
Avance 400 spectrometer, and operated at 400 MHz, with the solvent proton signal
as the internal reference standard.
2.3. Molecular weight determination by size exclusion chromatography (SEC)
SEC was conducted using THF as the eluent for monitoring the polymer conversion and also for the determination of polystyrene equivalent molecular weights
of the polymers. THF-SEC was recorded on a Waters 2695D (Waters Corporation,
U.S.A.) Separation Module equipped with an Optilab rEX differential refractometer

10280

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

(Wyatt Technology Corporation, U.S.A.) and Waters HR-4E as well as HR 1 columns

(Waters Corporation, U.S.A.). The system was equilibrated at 30  C in THF, which
served as the polymer solvent and eluent with a ow rate of 1.0 mL/min. Polymer
solutions were prepared at a known concentration (ca. 3 mg/mL) and an injection
volume of 100 mL was used. Data collection and analysis were performed using the
Astra software (Wyatt Technology Corporation, U.S.A.; version 5.3.4.14). The columns were calibrated with series of polystyrene standards ranging from
Mp 360 Da to Mp 778 kDa (Polymer Standard Service, U.S.A.).
2.4. General procedure for polymer synthesis
Triblock copolymers of vitamin E-functionalized polycarbonates and poly(ethylene glycol), and vitamin E-containing cationic polycarbonates were synthesized for preparation of antimicrobial hydrogels.
Triblock copolymer ((MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25): In a 20-mL vial containing a magnetic stir bar in the glove box, MTC-VE (58.9 mg, 100 mmol, 4.0 equiv.),
HO-PEG-OH (20 kDa, 500 mg, 25 mmol, 1.0 equiv.) and TU (9.3 mg, 25 mmol,
1.0 equiv.) were dissolved in dichloromethane (4 mL). To this solution, DBU (3.7 mL,
25 mmol, 1.0 equiv.) was added to initiate polymerization. The reaction mixture was
allowed to stir at room temperature and aliquots of samples were taken to monitor
the monomer conversion and evolution of molecular weight by 1H NMR spectroscopy and SEC. After 120 min, the reaction mixture was quenched by the addition of
excess (w20 mg) of benzoic acid and was precipitated into ice-cold diethyl ether
(2  50 mL). The resultant polymer was dried in a vial for about 1e2 days until a
constant sample mass was obtained, as white powder. Selected 1H NMR (400 MHz,
CDCl3): d 3.40e4.00 (s, 1815H, OCH2CH2 PEG), 1.00e2.00 (overlapping peaks, VitE),
0.75e0.95 (m, 30H, overlapping CH3 on VitE). PDI (GPC): 1.07.
Vitamin E-containing cationic polycarbonates (VE/BnCl(1:30)): In a 20-mL vial
containing a magnetic stir bar in the glove box, MTC-BnCl (608.8 mg, 2.04 mmol, 30
equiv.), MTC-VE (40.0 mg, 68 mmol, 1.0 equiv.) and TU (25.2 mg, 68 mmol, 1.0 equiv.)
were dissolved in dichloromethane (3 mL). To this solution, BnOH (7.0 mL, 68 mmol,
1.0 equiv.) followed by DBU (10.2 mL, 68 mmol, 1.0 equiv.) were added to initiate
polymerization. The reaction mixture was allowed to stir at room temperature for
20 min and quenched by the addition of excess (w20 mg) of benzoic acid. The
mixture was then precipitated into ice-cold methanol (50 mL) and centrifuged
at 5  C for 30 min. The resultant semi-transparent oil was dried under vacuo until a
foamy white solid was obtained. GPC analysis of the intermediate was carried out
and the polymer was used without further purication. The polymer was subsequently dissolved in acetonitrile, transferred to a Teon-plug sealable tube and
chilled to 0  C. Trimethylamine was added to start the quarternization process. The
reaction mixture was stirred at room temperature for 18 h in the sealed tube. Precipitation of an oily material was observed during the course of reaction. The mixture
was evacuated to dryness under vacuo and freeze-dried to nally yield a white crispfoamy solid. The desired polymer was characterized by 1H NMR (some of the integral
values are approximated to the nearest DP values). VE/PrBr(1:30) was obtained in a
similar manner using MTC-PrBr as the monomer. The 1H NMR spectra for all the
cationic polycarbonates are provided in the Supplementary Information (Fig. S1).
VE/BnCl(1:30): 1H NMR (400 MHz, CDCl3) d 7.20e7.70 (m, 109H, overlapping
peaks of initiator Ph and Bn), 5.00e5.40 (m, 54H, overlapping peaks of initiator
CH2Ph and Bn), 4.67 (m, 52H), 4.30 (m, 104H), 3.07 (m, 234H, N(CH3)), 1.00e2.00
(overlapping peaks, VitE), 0.70e0.90 (m, 12H, overlapping CH3 on VitE); PDI of intermediate (GPC): 1.21; Actual DP of VE/BnCl(1:30) (from 1H NMR) w 1:26.
VE/PrBr(1:30): 1H NMR (400 MHz, CDCl3) d 7.10e7.60 (m, 5H, initiator Ph), 5.15
(m, 2H, initiator CH2Ph), 4.00e4.50 (m, 144H), 3.46 (m, 48H), 3.30e3.20 (m, 216H,
N(CH3)), 2.10 (m, 48H), 1.00e2.00 (overlapping peaks, VitE), 0.70e0.90 (m, 12H,
overlapping CH3 on VitE). PDI of intermediate (GPC): 1.13. Actual DP of VE/PrBr(1:30)
(from 1H NMR) w 1:24.

performed by placing the hydrogel (1 mL) containing 4 mg/L of uconazole in a

dialysis membrane tube with MWCO of 1000 Da (Spectrum Laboratories, U.S.A.),
which was then immersed in 40 mL of the release medium phosphate-buffered
saline (PBS, pH 7.4). To study if the presence of the antimicrobial polycarbonate
has any effect on drug release, 4 mg/L of VE/BnCl(1:30) was added to the hydrogel
during the gelation process. The samples were kept shaking on an orbital shaker at
100 rpm at 37  C. At designated time intervals, 0.5 mL of the release medium was
removed and replaced with fresh medium. The removed medium was analyzed for
its drug content. To do this, the deposited uconazole was dissolved in 1.5 mL of
mobile phase (A e 10 mM sodium acetate buffer adjusted to pH 5.0, B e Methanol;
65%A/35%B) and analyzed using HPLC at 210 nm.
2.7. Killing efciency tests
E. coli and S. aureus were reconstituted from its lyophilized form according to the
manufacturers protocol, and cultured in TSB at 37  C under constant shaking of
300 rpm, while C. albicans was cultured in YMB at room temperature under constant
shaking of 50 rpm. Prior to treatment, the microbes were rst inoculated overnight
to enter into log growth phase. Cationic polycarbonate (VE/BnCl(1:30) or VE/
PrBr(1:30))-containing hydrogels were prepared using 4 wt.% of the triblock
copolymer (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25 and varying contents of VE/
BnCl(1:30), VE/PrBr(1:30) and/or uconazole. Hydrogels (50 mL) were placed into
each well of a 96-well microplate containing an equal volume of microbe suspension
(3  105 CFU/mL). 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 culture plate was kept
either at 37  C for bacterial samples or room temperature for C. albicans under
constant shaking of 300 or 50 rpm respectively for 24 h. After 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 and 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. Minimum bactericidal concentration, MBC, is dened as the concentration of the cationic
polycarbonate that eliminates >99.9% of the microbes.
2.8. Analysis of synergism between antimicrobial polycarbonate and uconazole
To assess the antifungal effects of the polymereuconazole combination, the
checkerboard and isobologram methods of analyzing drug interactions were used.
For the checkerboard method, the fractional inhibitory concentration (FBC) was
calculated for each component in each combination dose [30,31]. The types and
extent of interaction was determined by calculating the FBC index, which is the ratio
of the MBC of a drug in combination and MBC of the drug alone. For two interacting
drugs, A and B, the sum of the FBCs indicates the extent of the interaction. Synergy is
dened as SFIC index 0.5. Indifference was dened as SFIC index of >0.5 but 4
and antagonism as a SFIC index of >4.0 [32]. As for the isobologram method, evaluation of drug interaction was performed at the MBC level. Using graphical analysis,
the concentrations required to produce the effect of >99.9% killing efciency were
determined for each component and plotted on the x and y axes of a two-coordinate
plot. A line is drawn to connect these two points and this is dened as the line of
additivity. After that, treatment was then performed with the drugs in combination
at varying concentrations. The concentrations of uconazole and polycation in the
combination that provided the same effect were placed in the same plot. Effect of the
drug interaction was determined according to the position of the points with respect
to the line of additivity. Synergy, additivity, and antagonism are represented when
the point is located below, on, and above the line, respectively [33].

2.5. Rheological experiments

2.9. Biolm formation and treatment

Blank hydrogel and antimicrobial polymer-loaded hydrogels were prepared by

dissolving the triblock copolymer or a mixture of the triblock copolymer and a
vitamin E-containing cationic polycarbonate in HPLC grade water at 25  C and 4 wt%.
The rheological analysis of the hydrogels was performed on an ARES-G2 rheometer
(TA Instruments, U.S.A.) equipped with a plateeplate geometry of 8 mm diameter.
Measurements were taken by equilibrating the gels at 25  C between the plates at a
gap of 1.0 mm. The data were collected under a controlled strain of 0.2% and a
frequency scan of 1.0e100 rad/s. Gelation properties of the polymer solutions was
monitored by measuring the shear storage modulus (G0 ), as well as the loss modulus
(G00 ), at each point. For shear-thinning studies, the viscosity of the hydrogels was
monitored as function of shear rate from 0.1 to 10 s1.

S. aureus and E. coli were grown overnight in TSB at 37  C and diluted in TSB to
3  106 and 3  108 CFU/mL, respectively, before use. C. albicans was grown overnight
in YMB at room temperature and diluted to 3  105 CFU/ml before use. The diluted cell
suspension (100 mL) was then inoculated into each well of 96-well plate and cultured
for 7e10 days depending on their growth rates. Due to differences in the rate of biolm
formation, S. aureus and C. albicans were kept shaking at 100 rpm, 37  C and 50 rpm,
25  C respectively, while E. Coli was incubated without shaking at 37  C. The culture
medium was changed everyday with PBS being added to wash off the planktonic and
loosely adhered cells before it was replaced with fresh medium. Treatment was carried
out by rst removing the spent medium and the biolm was washed gently with PBS
to remove the planktonic and loosely adhered cells. After that, the biolm was incubated with 50 mL of polycation-loaded hydrogel at MBCs for 24 h.

2.6. In vitro uconazole release

2.10. Biomass assay
The release of uconazole from the 4 wt.% (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25
hydrogel was studied. Fluconazole-containing hydrogel was prepared by rst dissolving the antifungal drug in HPLC grade water at room temperature. This solution
was then used to dissolve (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25 and the mixture was
left to stand overnight for the formation of hydrogel. The release study was

The biomass left after treatment was analyzed using crystal violet (CV) staining
assay. The spent medium and hydrogel was gently removed and the biolm was
gently washed with PBS to remove the planktonic cells. Fixation was carried out by
adding 100 mL of methanol to the biolm and removing it after 15 min. Crystal violet

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

10281

Scheme 1. Syntheses of (MTC-VE)n-PEG-(MTC-VE)n and vitamin E-containing polycationic polymers. Schematic illustration of incorporating polycationic polymers into hydrogel
system (inset).
staining (0.1 w/v %, 100 mL) was added to the xed biolm and incubated for 10 min.
Excess crystal violet was washed off thoroughly using water of HPLC grade. The
remaining crystal violet bound to the biolm was extracted using 200 mL of 33%
glacial acetic acid. An aliquot of 150 mL was then taken from each well and transferred to a fresh 96-well plate. The absorbance was then measured at 570 nm using a
microplate reader (Tecan, Switzerland) and the biomass of the remaining biolm
was expressed as a percentage of control group.

2.11. XTT reduction assay

XTT assay was used for quantifying viable cells in the biolms after hydrogel
treatment by measuring the mitochondrial enzyme activity of the cells. It is based on
the reduction of 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)
carbonyl]-2H-tetrazolium hydroxide (XTT) in the metabolically active microbial cells
to a water soluble formazan. Briey, XTT solution (1 mg/mL) and menadione

B
Viscosity (Pa.s)

G' (Pa)

1500
1000
500

1000

VE/BnCl(1:30) Gel

800

VE/PrBr(1:30) Gel

600
400
200
0

0
Blank Gel

VE/BnCl(1:30)
Gel

VE/PrBr(1:30)
Gel

4
6
Shear rate (s-1)

10

Fig. 1. Mechanical properties of hydrogels. (A) G0 values of blank and cationic polycarbonate-containing hydrogels. (B) Viscosity of hydrogels as a function of shear rate. The
polycation-loaded hydrogels were prepared by dissolving (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25 (4wt.%) and cationic polycarbonates (0.1 wt.%) in HPLC grade water.

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

solution (0.4 mM) were individually prepared by dissolution in de-ionized (DI) water.
Right before the assay, the two components were mixed together at a volume ratio
of XTT:menadione 5:1. During the assay, the medium was rst removed and biolm
was carefully washed with PBS to remove planktonic cells. PBS (120 mL) and the XTTmenadione mixture (14.4 mL) were then added to each well and incubated for 3 h. An
aliquot of 100 mL was then taken from each well and transferred to a fresh 96-well
plate. The absorbance was then measured at 490 nm using the microplate reader
(Tecan, Switzerland) and cell viability of the remaining biolm was expressed as a
percentage of the control group.
2.12. Field emission-scanning electron microscopy (FE-SEM)
After treatment with the hydrogels, the biolm was gently washed with PBS and
xed with 4% formaldehyde for 30 min. The xed biolm was washed with DI water
to remove formaldehyde and a series of ethanol washes (35, 50, 75, 90, 95 and 100%)
was carried for dehydration of the sample. After two days of air-drying, the samples
were mounted onto carbon tape and coated with platinum for SEM analysis under a
eld emission-scanning electron microscope (JEOL JSM-7400F, Japan).
2.13. Cytotoxicity test
Human dermal broblasts were seeded onto a 96 well plate at a density of
20,000 cells per well and incubated overnight at 37  C. The medium was removed
and 50 mL of colorless DMEM was added to each well, followed by 50 mL of the
hydrogels containing different concentrations of cationic polycarbonates and/or
uconazole. The plate was then incubated for 24 h at 37  C. CellTitre-blue (Promega,
USA) and DMEM were then mixed at a ratio of 2:5 by volume. After 24 h of treatment, 100 mL of this mixture was then added to each well and the cells were left to
incubate in the dark at 37  C for 4 h. Cells that were untreated were used as control.
Subsequently, the absorbance at 570 nm was measured. The readings were then
expressed as a percentage of cell viability of the control group.

3. Results and discussion

cross-links between the polymer chains with the application of

shear stress. Thereby, this indicates that the hydrogels can be wellspread over the skin for topical treatment of dermal infections.
3.3. Antimicrobial activities of cationic polycarbonate-containing
hydrogels
Antimicrobial activities of two different polycation-loaded
hydrogels were evaluated against S. aureus, E. coli and C. albicans
as representative models of Gram-positive and Gram-negative
bacteria, and fungus respectively. These microbes are common
pathogens that often manifest on dermal wounds [35e37], and are
typically treated via topical delivery of antibiotics to infected areas
[38,39].

A
100.0
Killing Efficiency (%)

10282

99.4
99.2
99.0

The triblock copolymer formed hydrogels at 4 wt% through

physical cross-links between ower-like micellar networks
(Scheme 1). The G0 value of the hydrogel was around 1500 Pa
(Fig. 1A). The addition of the cationic polycarbonates VE/PrBr(1:30)
and VE/BnCl(1:30) at 0.1 wt.% (equivalent to 1000 mg/L) to the
hydrogel did not cause signicant difference in stiffness. In addition, as shown in Fig. 1B, the polycation-loaded hydrogels displayed
high viscosity at low shear rates, indicating a rm and well-bodied
structure. As the shear rate increased, the viscosity of the gels fell
drastically, and eventually became a thin liquid. This shear-thinning
behaviour of the hydrogels resulted from the disruption of physical

Killing Efficiency (%)

78.1

156.2
312.5
625
Conc. of Polymer (mg/L)

100.00

100.0

100.00

100.00

100.00

99.88

99.9
99.8
99.7
99.63

99.6
156.2 312.5 625 1250 2500
Conc. of Polymer (mg/L)

C
Killing Efficiency (%)

3.2. Mechanical properties of hydrogel

99.34

99.6

98.8

99.80

99.8

3.1. Polymer synthesis

The syntheses of (MTC-VE)n-PEG-(MTC-VE)n triblock copolymers and vitamin E-containing cationic polycarbonates are
summarized in Scheme 1. The organocatalytic ring opening polymerizations (ROP) of MTC-VE, initiated by HO-PEG-OH and benzyl
alcohol, respectively, were achieved using DBU/thiourea as catalysts [34]. The polycationic polymers were obtained subsequently
by quaternization with trimethylamine. The nal compositions of
the polymers were analyzed and conrmed by GPC and/or proton
NMR. The triblock copolymer had an average number of 2.5 VE
molecules in each chain, i.e. (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25.
The nal cationic polycarbonates, VE/BnCl(1:30) and VE/
BnCl(1:30), contain one MTC-VE molecule each, 24 units of MTCPrBr and 26 units of MTC-BnCl, respectively. There was a slight
decrease in the degree of polymerization (DP) for both polymers
when compared to the pre-quaternized precursors (For VE/
BnCl(1:30), DP 1:30 (before), 1:26 (after); For VE/PrBr(1:30),
DP 1:27 (before), 1:24 (after)). While trimethylamine is basic, it is
not strongly nucleophilic. Under the mild reaction condition,
degradation is minimal. From NMR analysis, all quaternization reactions were complete (Fig. S1).

100.00
100.00

100.00

100.00
99.87

105.0

BnCl

100.0

PrBr

99.94
98.97

100.00

5000

99.92

95.0
90.0

85.98
82.22

85.0
80.0

156.2
312.5
625.0
(mg/L)
Conc. of Polymer

Fig. 2. Antimicrobial activity of polycation-loaded (MTC-VE)1.25-PEG(20k)-(MTCVE)1.25 (4wt.%) hydrogels against (A) S. aureus, (B) E. coli and (C) C. albicans.
VE/
BnCl(1:30) and VE/PrBr(1:30).

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

100.5
100.0

99.22

99.99

300

99.96
99.57

VE/BnCl (mg/L)

Killing Efficiency (%)

10283

99.5
99.0
98.5

98.0
Fluc (mg/L)
VE/BnCl (mg/L)

250
200
150
100
50
0

10
78

10
156

40
39

40
78

100

200
300
400 500
Fluconazole (mg/L)

600

Fig. 3. Synergism between uconazole (Fluc) and VE/BnCl(1:30) in (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25 (4wt.%) hydrogel against C. albicans. (A) Killing efcacy of uconazole and
VE/BnCl(1:30) combination. (B) An isobologram analysis representing the synergy between uconazole (10 mg/L) and VE/BnCl(1:30) (156 mg/L, MBC) or between uconazole
(40 mg/L) and VE/BnCl(1:30) (78 mg/L, MBC). Synergy between the two compounds is demonstrated as the drug combination dose falls to the left of the line of additivity.

B
100
Biomass (%)

Cell Viability (%)

100
80
60
40
20

20
Ctrl

Blank Gel

VE/BnCl(1:30) VE/PrBr(1:30)
Gel
Gel

Ctrl

Blank Gel

VE/BnCl(1:30) VE/PrBr(1:30)
Gel
Gel

D
100
Biomass (%)

100
Cell Viability (%)

60
40

80
60

80
60
40

40
20

Ctrl

Blank Gel

20

VE/BnCl(1:30) VE/PrBr(1:30)
Gel
Gel

Ctrl

Blank Gel

VE/BnCl(1:30) VE/PrBr(1:30)
Gel
Gel

F
100

100

80

Biomass (%)

Cell Viability (%)

80

60
40

60
40

20
0

80

Ctrl

Blank Gel

VE/BnCl(1:30)
Gel

VE/PrBr(1:30)
Gel

20

Ctrl

Blank Gel

VE/BnCl(1:30) VE/PrBr(1:30)
Gel
Gel

Fig. 4. Biolm eradication by polycation-loaded (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25 (4wt.%) hydrogels against (A and B) S. aureus, (C and D) E. coli and (E and F) C. albicans.
Reduction in metabolic activities and biomass of the various biolms is shown in (A, C and E) and (B, D and F) respectively.

10284

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

Two vitamin E-containing cationic polycarbonates, VE/BnCl(1:30)

and VE/PrBr(1:30), were loaded into 4 wt.% (MTC-VE)1.25-PEG(20k)(MTC-VE)1.25 hydrogel in different concentrations. As the concentration of the polycations increased, a greater density of cationic
charge was expected to be displayed on the hydrogel surface and this
would give rise to better inhibition effects. The hydrogels were
challenged with an inoculum of 3  105 CFU/ml and proliferation
capacity of the survived cells was assessed 24 h later via the spread
plate technique. This method of examining the antimicrobial activity
is akin to measuring the minimum inhibitory concentration (MIC) of
the polycations in solution [40]. Our study showed that the polycations were broadly antimicrobial towards the tested bacteria and
fungi. As seen from Fig. 2(AeC), the inhibition efcacy of VE/
BnCl(1:30)- and VE/PrBr(1:30)-loaded hydrogels on the proliferation
of the different microbe species varied. Amongst the tested microbes,
the hydrogels are most effective in preventing Gram-positive
S. aureus proliferation as it required the lowest MBCs for both
polycation-loaded gels (156.2 and 625 mg/L for VE/BnCl(1:30) and
VE/PrBr(1:30), respectively). On the other hand, the hydrogels
inhibited the proliferation of E. coli (Gram-negative) less effectively
(MBCs: 312.5 and 2500 mg/L for VE/BnCl(1:30)and VE/PrBr(1:30)
respectively), and this was likely due to the presence of additional
lipopolysaccharide-containing outer membrane that could impede
the penetration of the polycations. A similar phenomenon was also
observed in other antimicrobial systems [23,40]. The large difference
in MBC values of the two polycations against E. coli indicates that the
Gram-negative bacteria possess greater sensitivity towards the
amphiphilic balance [41] of these vitamin E-functionalized polycations compared to the Gram-positive counterpart. Antimicrobial
effect on C. albicans was intermediary to that of the two bacteria
species with MBC values of 312.5 and 625 mg/L for VE-BnCl(1:30)
and VE/PrBr(1:30), respectively. This further illustrates that the
more hydrophobic polycation VE/BnCl(1:30) generally exhibits
greater antimicrobial/antifungal effects compared to VE/PrBr(1:30).
3.4. Synergism analysis of co-delivery of uconazole and cationic
polycarbonate using hydrogels
Another strategy to enhance the antimicrobial efcacy of the
polycation-loaded hydrogels is the co-delivery with clinically-

relevant antimicrobial agents. The benets of combination therapy have been long-established with appealing attributes such as
the evasion of drug resistance and minimized drug concentrations
for desired therapeutic actions [32,42]. However, it should be noted
that augmentation in drug interaction is both system- and targetdependent and it is vital to utilize appropriate therapeutic partners for favorable effects. In this study, antifungal drug uconazole
was added together with polycation during the preparation of the
hydrogels for combination treatment against C. albicans. The
presence of cationic polycarbonate did not affect the drug release
prole signicantly, and most drug molecules were released from
the hydrogels at 2 h (Fig. S2). Fluconazole is a member of the azole
family of antifungal agents that possesses good activity against
C. albicans and exhibits low toxicity. While azole-based drugs are
considered safe for clinical applications, the main disadvantage is
that they are only fungistatic and resistance can develop easily with
extended usage [43]. Our results showed that even at a high concentration of 500 mg/L, uconazole only exerted fungistatic properties with less than 3.0log reduction in total microbial count
(Fig. S3). By combining this fungistatic drug and polycation in a
hydrogel matrix, signicant improvement in the therapeutic efcacy was observed at two combination doses (10 mg/L uconazole
and MBC of VE/BnCl(1:30) (156 mg/L); 40 mg/L uconazole and
MBC of VE/BnCl(1:30) (78 mg/L)), and the hydrogels were
fungicidal (> 99.9% eradication) (Fig. 3A). In addition, FBC index
of the combination doses were w0.5 and w0.25 respectively,
indicating synergistic interaction between uconazole and
VE/BnCl(1:30). Furthermore, the isobologram method of analyzing
drug interactions further illustrated the strong synergism between
uconazole and VE-BnCl(1:30) (Fig. 3B).
3.5. Biolm eradication
To explore efcacy of the antimicrobial hydrogels in biolm
elimination, various microbes (S. aureus, E. coli and C. albicans) were
cultured for several days to develop biolms prior to the treatment.
Polycations were loaded at MBC concentrations into (MTC-VE)1.25PEG(20k)-(MTC-VE)1.25 (4wt.%) hydrogels and placed onto the
biolms. After 24 h, the biomass and cell viability of these biolms
were analyzed. From Fig. 4A, C and E, hydrogels loaded with

Fig. 5. SEM images of biolms treated with polycation-loaded (MTC-VE)1.25-PEG(20k)-(MTC-VE)1.25 (4wt.%) hydrogels.

A.L.Z. Lee et al. / Biomaterials 34 (2013) 10278e10286

VE/BnCl(1:30) were as efcient as those loaded with VE/PrBr(1:30)

in reducing the proliferation and viability of S. aureus (Gram-positive) and C. albicans (fungus). The major difference was observed in
E. coli (Gram-negative) where cells treated with hydrogels loaded
with the more hydrophobic VE/BnCl(1:30) had signicantly lower
viability compared to those treated with VE/PrBr(1:30)-loaded
hydrogels. In addition, the ability of the polycation-loaded hydrogels to eradicate the biomass displayed a similar trend to reduction
of cell viability residing in the biolms where VE/BnCl(1:30) had
stronger activity in removing E. coli biomass and similar efciency
in the treatment of S. aureus and C. albicans biolms compared to
VE/PrBr(1:30) (Fig. 4B, D and F). Furthermore, SEM images
demonstrated that biolms treated with VE/BnCl(1:30)-loaded
hydrogels showed extensive cell destruction and clearance

10285

(Fig. 5). Only ruptured cell fragments remained for S. aureus and
C. albicans. The images also showed good correlation with the
quantication assays of cell viability and biomass where VE/
BnCl(1:30) hydrogel was signicantly more effective in eradicating
E. coli biolm compared to VE/PrBr(1:30) hydrogel.
3.6. Cytotoxicity studies
The effects of the hydrogels on mammalian cells were evaluated
by treating human dermal broblasts with hydrogels containing
MBC concentrations of the cationic polycarbonates and/or uconazole. As shown in Fig. 6AeC, the blank hydrogels resulted in slight
retardation of cell proliferation as the cell viability was w88%
compared to the untreated group. This is possibly due to the
slowing down of nutrient and metabolite diffusion as a result of the
hydrogels present on the cell surface. Importantly, no signicant
cytotoxicity was observed from the treatment as the cell viability
remained > 80% for all treatment groups.
4. Conclusion
Biodegradable antimicrobial hydrogels have been successfully
prepared by incorporating vitamin E-functionalized cationic polycarbonates into physically cross-linked hydrogels fabricated from
ABA-type triblock copolymer (MTC-VE)1.25-PEG(20k)-(MTCVE)1.25. These hydrogels exhibit thixotropic property, which is ideal
for topical applications. They are effective against both Grampositive and Gram-negative bacteria as well as fungus with more
than 99.9% killing efciency upon contact. In addition, these
physically cross-linked hydrogels are able to eradicate the biomass
and greatly reduce viability of microbes residing as S. aureus, E. coli
and C. albicans biolms. The co-delivery of antimicrobial polycarbonates with the conventionally-used antifungal drug uconazole using the hydrogel provides a high degree of synergistic
antifungal effect on C. albicans. Importantly, the hydrogels containing the cationic polycarbonates and/or uconazole at their
minimum biocidal concentrations do not induce signicant cytotoxicity towards human dermal broblasts. Therefore, these antimicrobial polycarbonate-loaded hydrogels may be used to
eliminate both planktonic microbes and their biolms, and represent a promising approach for the prevention and treatment of skin
infections.
Acknowledgments
This work was funded by the Institute of Bioengineering and
Nanotechnology (Biomedical Research Council, Agency for Science,
Technology and Research, Singapore) and IBM Almaden Research
Center, USA.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.biomaterials.2013.09.029.
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Fig. 6. Viability of human dermal broblasts after 24 h treatment with (MTC-VE)1.25PEG(20k)-(MTC-VE)1.25 (4wt.%) hydrogels loaded with (A) VE/BnCl(1:30), (B) VE/
PrBr(1:30) and (C) VE/BnCl(1:30) and uconazole at different concentrations.

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