A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability

ARTICLES
PUBLISHED ONLINE: 12 DECEMBER 2010 | DOI: 10.1038/NMAT2915

A polycationic antimicrobial and biocompatible
hydrogel with microbe membrane
suctioning ability
Peng Li1† , Yin Fun Poon1† , Weifeng Li2 , Hong-Yuan Zhu3 , Siew Hooi Yeap1 , Ye Cao1 , Xiaobao Qi1 ,
Chuncai Zhou1 , Mouad Lamrani4 , Roger W. Beuerman3,5 , En-Tang Kang6 , Yuguang Mu7 ,
Chang Ming Li1 , Matthew W. Chang1 , Susanna Su Jan Leong1 and Mary B. Chan-Park1 *

Despite advanced sterilization and aseptic techniques, infections associated with medical implants have not been eradicated.
Most present coatings cannot simultaneously fulﬁl the requirements of antibacterial and antifungal activity as well as
biocompatibility and reusability. Here, we report an antimicrobial hydrogel based on dimethyldecylammonium chitosan (with
high quaternization)-graft-poly(ethylene glycol) methacrylate (DMDC-Q-g-EM) and poly(ethylene glycol) diacrylate, which
has excellent antimicrobial efﬁcacy against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Fusarium
solani. The proposed mechanism of the antimicrobial activity of the polycationic hydrogel is by attraction of sections
of anionic microbial membrane into the internal nanopores of the hydrogel, like an ‘anion sponge’, leading to microbial
membrane disruption and then microbe death. We have also demonstrated a thin uniform adherent coating of the hydrogel
by simple ultraviolet immobilization. An animal study shows that DMDC-Q-g-EM hydrogel coating is biocompatible with rabbit
conjunctiva and has no toxicity to the epithelial cells or the underlying stroma.

but these often have high toxicity to mammalian cells16,17 . At present

I
nfections arising in association with medical implants and
devices are a significant problem and, when these happen, there are few reports of coatings that are broadly antimicrobial
surgical removal or other surgical intervention, with attendant to fungi and bacteria (both Gram-negative and Gram-positive)
medical risks and complications, is often inevitable1 . To minimize and that are also non-haemolytic and biocompatible18 . Also, most
alteration of bulk properties (for example mechanical strength reported methods of surface immobilization of polymers are
or transparency) of an implant, coating with an effective an- multistep and post-synthesis3,8,19 , involve organic solvents20,21 and
timicrobial agent seems to be an attractive approach to combat do not result in permanent coatings.
infection1–3 . Earlier generations of antimicrobial coatings based In this work we demonstrate highly antimicrobial surfaces based
on drug/chemical elution have only short-term antimicrobial ef- on in situ ultraviolet immobilization of a protein-/cell-repelling and
fect, and cause cumulative toxicity and/or microbe resistance4,5 . contact-active hydrogel layer made from quaternized ammonium
A contact-active coating with immobilized antimicrobial agent chitosan-graft -poly(ethylene glycol) methacrylate (qC-g -EM;
is generally less likely to lead to the development of microbe Fig. 1a). We chose chitosan, an inherently biocompatible and
resistance. This class of coating disrupts the microbes’ membranes antimicrobial material, for further derivation to add modalities to
without targeting their metabolic activity, which is associated with (1) increase the antibacterial and antifungal activity, (2) achieve
the emergence of resistance6 . excellent biocompatibility and (3) enable easy in situ coating
Antimicrobial polymers have been applied as contact- (that is, surface grafting carried out concurrently with hydrogel
active coatings2 . Cationic polymers such as derivatives of crosslinking). Accordingly, we have modified chitosan to add
polyethylenimine7 , poly(vinyl-N -hexylpyridinum)8 , polynor- (1) a hydrophobic alkyl side chain and cationic charge through
bornene9 , polymethacrylates10 , poly(phenylene ethynylene)11 and quaternization of the amino group, (2) hydrophilic poly(ethylene
so on, in solution form, have been reported to disrupt the pathogen glycol) with six ethylene glycol repeats (PEG6 ) and (3) methacrylate
cytoplasmic membrane, and some have impressive selectivity for functionality (Fig. 1a). Others have shown that quaternized
bacterial over mammalian cells11–13 . However, when these polymers chitosan derivatives are water soluble and more antimicrobial
are immobilized, their antimicrobial activities may be greatly than pristine chitosan22 . PEGylation of chitosan derivatives
reduced because their diffusion into cell membranes is impeded14,15 . has been shown to decrease cytotoxicity and haemolysis23,24 .
Polymers that retain their antimicrobial activities even after However, PEGylated quaternized chitosan derivatives have not
immobilization typically contain dangling hydrophobic polycations been reported for use as antimicrobial agents. Also, hydrogels

1 School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore, 2 School of Physical

and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore, 3 Singapore Eye Research Institute, 11 Third
Hospital Avenue, Singapore 168751, Singapore, 4 Menicon Co. Ltd., Immeuble Espace Cordelier, 2 Président Carnot, Lyon 69002, France, 5 Duke-NUS, SRP
Neuroscience and Behavioral Disorders, 8 College Road, Singapore 169857, Singapore, 6 Department of Chemical and Biomolecular Engineering, National
University of Singapore, 10 Kent Ridge, Singapore 119260, Singapore, 7 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,
Singapore 637551, Singapore. † These two authors contributed equally to the paper. *e-mail: mbechan@ntu.edu.sg.

NATURE MATERIALS | VOL 10 | FEBRUARY 2011 | www.nature.com/naturematerials 149
© 2011 Macmillan Publishers Limited. All rights reserved.

ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT2915

a OH OH OH
O (I) N-alkylation O O
HO HO O O
O HO
NH2
m+n RCHO, NaBH4 NH (R) NH2
r.t., 1.5 h m n

(I) Quaternization
(II) Quaternization
NaOH, Nal, RX with x = Br or I
NaOH, Nal, CH3l, 50 °C, 24 h
50 °C¬70 °C, 24 h

OH OH OH
OH OH
O O O
O O O HO O
HO HO O
HO O HO O
+ + NR'2
+ N (R) N (CH3)3
N R3 NR2
m n (CH3)2
m n p

(III) PEG methacrylation
(II) PEG methacrylation
Cl¬CH2COO¬(CH2CHO2O)6¬CO¬C(CH3)=CH2 Cl¬CH2COO¬(CH2CH2O)6¬CO¬C(CH3)=CH2
r.t., 3 h r.t., 3 h

OH OH OH
OH OH
O O O
O O O HO O HO O
O HO HO
HO O
+ + NR'R"
+ N (R) N (CH3)3
N R3 NRR'
m n (CH3)2 m n p

R= CH3 or H for TMC-g-EM R= CH2(CH2)4CH3 for DMHC-g-EM
TMC-Q-g-EM R= CH2(CH2)8CH3 for DMDC-g-EM
O O R' = CH3 or H DMDC-Q-g-EM
=
=

R' = R or CH2C O (CH2CH2 O)6 C C=CH2
CH3 O O
=

=
R" = R' or CH2 C O (CH2CH2 O)6 C C=CH2
CH3

Series I Series II

b Cytoplasmic side c t = 0 ns d t = 50 ns

Disrupted microbe
_ Lipid bilayer
___ __ _ ___ __
____ outer membrane
_
Anionic
+ + + + + + + + + + lipids
LPS
+ + + + + + + + + +
Pore Cationic antimicrobial
+ + + + + + + + + + hydrogel
qC-g-EM
DMDC-Q
Substrate hydrogel

Figure 1 | qC-g-EM polymers and the antimicrobial killing mechanism of their hydrogels. a, Synthetic scheme for preparation of TMC-g-EM and
TMC-Q-g-EM (series I, left side) and DMHC-g-EM, DMDC-g-EM and DMDC-Q-g-EM (series II, right side). b, Schematic diagram of the ‘anion sponge’
model—parts of the negatively charged bacterial membrane are ‘suctioned’ into the pores of the qC-g-Em hydrogel. c,d, Computer simulation of the killing
mechanism showing the ‘suctioning’ of P. aeruginosa bacterial membrane (lipid bilayer) LPS molecules into the DMDC-Q hydrogel after 50 ns.

based on qC-g -EM have not been reported or explored as entropy maximization will drive anionic outer microbe membrane
antimicrobial coatings. Coulombic attraction would exist between components into the nanopores of the hydrogel, leading to
the cationic quaternized ammonium group of qC-g -EM and the membrane disruption (Fig. 1b).
bacterial or fungal cell membrane, which is typically anionic12,25 . Three qC-g -EMs, specifically trimethylammonium chitosan-
However, unlike other coatings2,3 , hydrogels are porous26 , so g -EM (TMC-g -EM), dimethylhexylammonium chitosan-g -EM
they have interior ‘space’ to receive parts of the microbe (DMHC-g -EM) and dimethyldecylammonium chitosan-g -EM
membrane that may be ‘attracted into’ the hydrogel. When (DMDC-g -EM), with different alkyl chains, were synthesized
the attraction between anionic membrane components and the and tested (Fig. 1a and entries 2–4 in Table 1). Two different
cationic hydrogel is strong enough, energy minimization and degrees of quaternization, that is 20–27% and 51–56% (Table 1),

150 NATURE MATERIALS | VOL 10 | FEBRUARY 2011 | www.nature.com/naturematerials

NATURE MATERIALS DOI: 10.1038/NMAT2915 ARTICLES
Table 1 | Characteristics of the qC-g-EM polymers.

No Name Series Degree of Double bond Mn (×105 g mol−1 ) Mw (×105 g mol−1 ) PID
quaternization (%) content (mmol g−1 )

1 C-g-EM - 0.76 0.74 0.86 1.15
2 TMC-g-EM I 24 0.84 0.75 1.22 1.63
3 DMHC-g-EM II 27 1.02 0.49 0.67 1.38
4 DMDC-g-EM II 20 1.00 0.36 0.52 1.46
5 TMC-Q-g-EM I 51 0.80 0.35 0.41 1.17
6 DMDC-Q-g-EM II 56 0.80 0.24 0.38 1.56
7 DMDC-M II 20 1.13 0.33 0.36 1.06

Mn : number-average molecular weight. Mw : weight-average molecular weight. PID: polydispersity index.

were explored; ‘Q’ is added to the polymer name, for example the alkyl-side-chain length from TMC to DMHC to DMDC
TMC-Q-g -EM, to denote the higher degree of quaternization (entries 2–4, Fig. 2a) increases the killing efficacy against Gram-
(entries 5 and 6). In addition, the effect of a PEG6 side chain positive S. aureus. More interestingly, the two highly quaternized
was also explored: DMDC-M (entry 7, Table 1), which does TMC-Q-g -EM and DMDC-Q-g -EM hydrogels (entries 5 and 6)
not have the PEG6 side chain but is methacrylated, was also have excellent log reductions of above 2.0 (>99% kill) for all
prepared by reacting DMDC with glycidyl methacrylate in the four microbes, which are superior to their counterparts with
presence of triethylamine27 rather than with α-terminated PEG6 lower degrees of quaternization (entries 2 and 4). (More detailed
methacrylate (Cl-PEG6 M). The solutions and hydrogels based antimicrobial activity results of the hydrogels can be found in
on the various qC-g -EMs were tested for efficacy in killing Supplementary S3.)
four clinically significant pathogens: the Gram-negative bacteria The LIVE/DEAD bacterial viability assay was carried out on
Pseudomonas aeruginosa (P. aeruginosa, ATCC9027 isolated from DMDC-Q-g -EM hydrogel with S. aureus, using PEG13 DA hydrogel
ear infection) and Escherichia coli (E. coli, ATCC8739 isolated as control (Fig. 2c,d). Using this assay, bacterial cells that look green
from faeces), the Gram-positive bacterium Staphylococcus aureus are live cells with intact membranes whereas bacterial cells that stain
(S. aureus, ATCC6538 isolated from human lesion) and the red are dead cells that have damaged membranes. Figure 2c,d shows
fungus Fusarium solani (F. solani, ATCC36031 isolated from that S. aureus cells are alive on the PEG13 DA control but dead
human corneal ulcer). on the DMDC-Q-g -EM hydrogel. Field emission scanning electron
NMR analyses confirm the successful syntheses of these microscopy was also carried out to observe the morphologies of
polymers (Supplementary Fig. S1 in Supplementary Section S1). the various microbes on DMDC-Q-g -EM hydrogel after 1 h contact
After quaternization and PEG6 methacrylate (PEG6 M) grafting, (Fig. 2e–h). Distorted and wrinkled membranes were observed in
all chitosan derivatives are soluble in neutral water. Minimum E. coli and F. solani on the hydrogel surfaces (Fig. 2f,h). Lesions and
inhibitory concentration (MIC) determination was carried out with holes were observed in P. aeruginosa and S. aureus (Fig. 2e,g) after
the chitosan derivative solutions to evaluate their antimicrobial contact with the hydrogel. Many of the small pores in P. aeruginosa
activities (Supplementary Section S2). The results (Table 2) confirm cells (arrows in Fig. 2e) were near the edge where the microbe
that the quaternized chitosan derivatives (entries 2–7) have better membrane contacted the hydrogel.
antimicrobial activities than unquaternized chitosan (entry 1). The We postulate that our cationic nanoporous hydrogels act
MICs of qC-g -EMs against fungi are generally low (≤200 µg ml−1 ). like a molecular ‘anion sponge’, suctioning out parts of the
Increasing the alkyl chain length from TMC-g -EM to DMDC- anionic microbe membrane into the gel interior voids to cause
g -EM (entries 2–4, Table 2) alone decreases the MICs against microbe membrane disruption (Fig. 1b). This interpretation is
Gram-positive S. aureus but not against the Gram-negative suggested by computer molecular dynamics simulations conducted
E. coli and P. aeruginosa. However, the more highly quaternized to probe the interaction between DMDC-Q hydrogel and bacterial
TMC-Q-g -EM and DMDC-Q-g -EM (entries 5 and 6, Table 2) membranes. (See Supplementary Section S4 on charges of microbial
have dramatically lower MICs against both bacteria and fungi and mammalian cell membranes.) Figure 1c,d shows a model
(24–200 µg ml−1 ), which are comparable to those of common of Gram-negative P. aeruginosa membrane, consisting of 243
antimicrobial peptides that we also tested (Table 2). Further, zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine and nine
TMC-Q-g -EM and DMDC-Q-g -EM are fairly non-haemolytic, anionic lipopolysaccharide (LPS) molecules. The molecular model
with 0% haemolytic activity at 3,100 µg ml−1 and 1,600 µg ml−1 of LPS of P. aeruginosa PAO1 was generated on the basis of
respectively (Supplementary Fig. S3), which are at least 15 times experimentally sequenced data28 . When the bacterial lipid bilayer
their bacterium/fungus MICs (Table 2). was placed near the DMDC-Q chains for 50 ns (Fig. 1d), it
To form free-standing hydrogel films, the chitosan derivatives, was significantly disturbed; some of the anionic LPSs were even
poly(ethylene glycol) diacrylate (with 13 ethylene glycol repeats, completely pulled out of the bilayer and drawn into the pores
denoted hereafter as PEG13 DA) and water were mixed in the ratio of between the DMDC-Q chains. The negatively charged regions of
1:1:8 (w/w), together with 0.1 wt% photoinitiator (Irgacure 2959), the remaining LPS molecules moved into close proximity to the
and then ultraviolet irradiated. The hydrogels were challenged with hydrogel surface. The suctioning of cell-membrane components
the four pathogens at a concentration of about 2.5 × 106 CFU cm−2 into the porous hydrogel distorts and wrinkles the membrane,
and the cell-count reductions after one hour were recorded. or even produces holes in it, corroborating our field emission
Figure 2a shows the antimicrobial activity of the hydrogel films. scanning electron microscopy results (Fig. 2e). This membrane
The hydrogel based on the non-quaternized C-g -EM (entry 1) disruption eventually kills the microbe. Similar results were
exhibited low activity against these microbes. All the qC-g -EM also found in the simulation of the interaction between a
hydrogels (entries 2–6, Fig. 2a) show outstanding antifungal activity Gram-positive bacterial-membrane model and DMDC-Q hydrogel
against F. solani but differing antibacterial activities. Increasing (Supplementary Section S4.2).



Table 2 | MICs of the qC-g-EM solutions against various pathogens.

No Name MIC (µg ml−1 )
Gram negative Gram positive Fungi
P. aeruginosa E. coli S. aureus F. solani

1 C-g-EM 13,000 13,000 6,300 3,100
2 TMC-g-EM 780 780 1,600 200
3 DMHC-g-EM 780 3,100 200 200
4 DMDC-g-EM 780 1,600 200 200
5 TMC-Q-g-EM 98 200 98 24
6 DMDC-Q-g-EM 98 98 49 24
7 DMDC-M 1,600 1,600 390 200
Melittin 63 63 8 -
LL-37 >250 >250 >250 -
Indolicidin 130 >250 130 -
Magainin I >250 130 130 -
Defensin (HNP-1) >130 >130 >130 -

Our ‘anion sponge’ model of these cationic nanoporous calculated the pore sizes of hydrogels 4, 7 and 8 to be 16.5 nm,
hydrogels differs from previous models of cell-membrane 10.4 nm and 1.6 nm respectively (Supplementary Section S3.6).
disruption based on the penetration of antimicrobial polymer into The zeta potentials of hydrogels 7 and 8 (10.9 ± 0.4 mV and
the microbe membrane or ion exchange between the cell membrane 11.9 ± 0.1 mV, Supplementary Section S3.7) are similar to that of
and antimicrobial polymer6,8 . Previous antimicrobial surfaces have hydrogel 4 (11.4 ± 0.2 mV), ruling out the effect of differences in
typically been based on brushes or pendant chains of hydrophobic charge density in this series. Decreasing the water swelling and
cationic polymers emanating from a monolayer or thin coating. hence pore size in hydrogel 8 significantly decreases its killing
These coatings do not contain pores needed to ‘receive’ disrupted efficacy (Fig. 2a). Conversely, hydrogel 4 has the best killing efficacy
membrane parts6,8 and the antimicrobial polymers may show because of its largest pore size of about 16.5 nm. Each folded LPS
greatly reduced microbe activities after immobilization14,15 . Our molecule is estimated to be about 3.5 nm–4.5 nm in size and so
qC-g -EM hydrogel, however, retains its antimicrobial efficacy after it seems that hydrogel 8 does not have large enough pores to
ultraviolet immobilization because of the presence of the pores. receive these molecules. Computer simulation also confirmed that
The high antifungal activity of all the highly hydrated hydrogels decreased hydrogel pore size causes fewer LPS molecules to be
(entries 2–6, Fig. 2a) may possibly be due to the compatibility pulled into the hydrogel (Supplementary Section S4.4).
of the chitosan derivatives with fungal cell wall, which contains The DMDC-Q-g -EM hydrogel was repeatedly challenged with
a significant amount of chitin, enabling close approach of the S. aureus up to four times. The tested hydrogel films were washed
hydrogel to the cell membrane. with PBS and vortexed before the next use. Figure 2b shows
Increasing the positive charge density and pore size of our that, even after three previous challenges with high concentrations
cationic hydrogels would significantly increase their killing efficacy. of microbes, the dead cells easily washed from the hydrogel,
The measured surface zeta potentials (Supplementary Section S3.7) preserving its antimicrobial activity in the next (fourth) challenge.
confirm that the highly quaternized TMC-Q-g -EM and DMDC- One problem of present contact-active antimicrobial surfaces
Q-g -EM hydrogels have higher charge densities: entries 5 and 6 in application is that they can easily be masked by adsorbed
have zeta potentials of 11.9±0.4 mV and 13.7±0.9 mV respectively, conditioning films from organic compounds such as proteins or
higher than the values (10.4 ± 0.3 mV and 11.4 ± 0.2 mV) for their remnants of dead cells, resulting in loss of effectiveness29 . This
counterparts with lower degrees of quaternization (entries 2 and 4). conditioning film undoubtedly blocks contact-active antimicrobial
The higher antibacterial efficacies of TMC-Q-g -EM and DMDC- surfaces from contact with microbes, causing loss of antimicrobial
Q-g -EM hydrogels compared with TMC-g -EM and DMDC-g -EM activity. In contrast, hydrated PEGylated hydrogels are known
(Fig. 2a) illustrate the significance of hydrogel charge density in to be inherently cell/protein resistant30 , accounting partly for
effective microbe killing by these materials. The effect of charge the observed durable antimicrobial activity. Further, diffusion of
on killing efficacy was corroborated by computer simulation of disrupted membrane molecules into the interior of the gel layer
less-charged DMDC hydrogel, which seems not to interact as is expected to prevent the gel surface from becoming fouled
strongly with the microbe membranes (Supplementary Section after repeated challenges. Large pores are needed to admit the
S4.2) compared with DMDC-Q hydrogel (Fig. 1c,d). microbe membrane fragments. Also, a high total pore volume
To demonstrate the importance of the pores of the cationic fraction guarantees plenty of interior volume for membrane
networks for achieving effective microbe killing, we compared fragments to diffuse into, so that the contact-active surface
DMDC-g -EM hydrogel (entry 4) with two other smaller-pore- possibly cleans itself if it becomes momentarily fouled by absorbed
size hydrogels formulated using DMDC-M (entries 7 and 8, membrane components after contact with a microbe. Our hydrogels
Fig. 2a). Specifically, we replaced (1) DMDC-g -EM with DMDC-M (entries 1–7, not entry 8) have high pore volume, as can be
(without the PEG6 side chain) in entry 7 (Fig. 2a) and (2) PEG13 DA inferred from their high water volume fractions of greater than 90%
crosslinker with a shorter diethylene glycol diacrylate (DEG2 DA) (Supplementary Fig. S6).
in DMDC-M-LS (low swelling) (entry 8 in Fig. 2a). The killing As a demonstration of application of our material and coating, a
efficacies of DMDC-M (entry 7) and, especially, DMDC-M-LS hydrogel layer of DMDC-Q-g -EM was in situ immobilized on a flu-
(entry 8) are lower than that of DMDC-g -EM hydrogel (entry 4). oropolymer substrate (contact lens Z from Menicon). The surface
The water uptakes of hydrogels 4, 7 and 8 were measured to be to be coated was first surface activated with peroxides using argon
1,040 ± 30%, 830 ± 8% and 110 ± 3% respectively (Supplementary plasma followed by air ageing, and the substrate was then placed in
Section S3.5). On the basis of the Peppas–Merill theory26 , we the hydrogel precursor solution and photopolymerized without a


Control DMDC-Q-g-EM hydrogel
a 6
P. aeruginosa
e
E. coli
5 S. aureus 99.999 P. aeruginosa
F. solani
Log reduction

4 99.990

%kill
3 99.900 1 µm 1 µm
1 µm
2 99.000 f
E. coli
1 90.000

0
1 2 3 4 5 6 7 8 9
Coating
C TMC DMHC DMDC TMC-Q DMDC-Q DMDC-M DMDC-M-LS 1 µm 1 µm
Hydrogel (DMDC-Q)
1 µm 1 µm
g
b c S. aureus
6
S. aureus
5 99.99
Log reduction

4 99.99 1 µm 1 µm
%kill

1 µm 1 µm
3 99.90 20 µm
d h
2 99.00 F. solani

1 90.00
0
1 2 3 4 10 µm 10 µm
No. of repeated challenges 20 µm 10 µm 10 µm
on DMDC-Q hydrogel (6)

Figure 2 | Antimicrobial activities of qC-g-EM hydrogels against various bacteria and fungi. a, Log reduction and %kill of four pathogens on various
qC-g-EM hydrogels. b, Multiple-use antimicrobial activities of DMDC-Q-g-EM hydrogel (entry 6) against S. aureus. Error bars represent mean ± standard
deviation of mean for n = 3. c,d, LIVE/DEAD bacterial viability assay of S. aureus on PEG13 DAcontrol (c) and DMDC-Q-g-EM hydrogel (entry 6) (d) after
1 h incubation at 37 ◦ C and washing (scalebar = 20 µm). e–h, Morphology of various pathogens in contact with DMDC-Q-g-EM hydrogel (right, entry 6)
and control (left). Arrows indicate lesions and holes on the cell membrane after contact with DMDC-Q-g-EM hydrogel.

a b

c d

10 µm 10 µm

10 µm 10 µm

Figure 3 | Coating of DMDC-Q-g-EM hydrogel on ﬂuoropolymer substrate. a,b, Photographic image of uncoated (a) and ﬂuorescein-stained
DMDC-Q-g-EM hydrogel-coated (b) substrate. c,d, Scanning electron microscopy images of top view and cross-section (inset) of uncoated (c) and
freeze-dried DMDC-Q-g-EM hydrogel- (entry 6) coated (d) substrate surface.

mould. Under ultraviolet irradiation, a crosslinked hydrogel layer precursor solution. Our method is simpler than typical procedures
emanates from the surface as the acrylate/methacrylate function- for forming antimicrobial coatings.
ality of the precursor solution reacts with the peroxide-decorated Figure 3 shows photographs and scanning electron microscopy
surface and with itself. The coating is concurrently covalently images of the hydrogel-coated fluoropolymer substrate, together
attached to the surface as the hydrogel is crosslinked from the with the control uncoated disc. Fluorescein, a dye that binds to



a 1.2 human primary epidermal keratinocytes with DMDC-Q-g -EM
TCPS control hydrogel films for 7 days, with tissue culture polystyrene dish
1.0 DMDC-Q hydrogel (6)
(TCPS) as control (Supplementary Section S3.8). Methyl tetra-

MTT absorbance
0.8 zolium (MTT) and LIVE/DEAD cell viability assays (Fig. 4a–c)
0.6
show that the human keratinocytes proliferate well in contact with
DMDC-Q-g -EM hydrogel. The MTT assay measures mitochon-
0.4 drial activity and cell viability and proliferation. Figure 4a shows
0.2 that the MTT absorbance increases with culture time, indicating
that the cells proliferate even in the presence of the hydrogel. In
0
1 day 3 days 5 days 7 days Fig. 4b,c, the keratinocytes are mostly stained green, indicating that
b c they are alive on the hydrogels, suggesting that our hydrogel is
in vitro biocompatible.
The in vivo biocompatibility of DMDC-Q-g -EM hydrogel
coating with the epithelial cells of the rabbit ocular surface was
examined. Uncoated and coated lenses were each implanted into
a pocket underneath the rabbit conjunctival epithelium. The
response of the conjunctival tissue was examined clinically by
slit-lamp and in vivo confocal microscopy over 5 days. The bulbar
50 µm 50 µm conjunctiva overlying the control and DMDC-Q-g -EM hydrogel-
coated lenses remained healthy as determined by examination with
d e in vivo confocal microscopy on post-operative (PO) days 3 and
5 (Supplementary Fig. S12). Representative histological images
of rabbit conjunctiva collected at PO day 5 and stained by
haematoxylin and eosin are shown in Fig. 4d–g. Importantly, there
was no indication of epithelial erosion, or of unusual neutrophil
infiltration other than what may be expected after surgery. A few
neutrophils appeared in the conjunctival tissue associated with
the lens (Fig. 4f,g). No erosions of the epithelium, or vascular
100 µm 100 µm
changes, were found to be associated with the presence of the
f g lens. The in vivo results show that the coating has no effect
on the epithelial cells and does not lead to any pathological
changes. The coated lens is compatible with conjunctiva and
the hydrogel coating has no toxicity to the epithelial cells or
the underlying stroma.
Our hydrogel has good in vitro and in vivo biocompatibility.
Unlike microbial membranes, the outer leaflet of the mammalian
cell membrane lacks anionic lipids12,25 . There is therefore much
100 µm 100 µm
less Coulombic attractive ‘suctioning’ force to disrupt mammalian
cells when they are in contact with the ‘anion sponge’, which
Figure 4 | In vitro and in vivo biocompatibility studies. a, MTT activities accounts for its excellent biocompatibility. The lack of Coulom-
(absorbance at 490 nm) of primary epidermal keratinocytes on TCPS bic attraction between DMDC-Q-g -EM and zwitterionic outer
control and DMDC-Q-g-EM hydrogel (entry 6). Error bars represent mean mammalian membrane is also shown by computer simulation
± standard deviation of mean for n = 3. b,c, LIVE/DEAD analysis of primary (Supplementary Section S4.3).
epidermal keratinocytes on TCPS control (b) and DMDC-Q-g-EM hydrogel We have demonstrated simultaneous polymerization and cova-
(entry 6) (c) after 7 days of culture. d–g, Microscopic observations of lent grafting of a qC-g -EM-based hydrogel onto a fluoropolymer
haematoxylin- and eosin-stained frozen sections of conjunctiva. d, Normal substrate surface by means of a simple two-step process to create a
conjunctiva epithelium showing normal epithelium and stromal blood uniform covalent adherent thin coating that effectively reduces the
vessels. e, PO day 5 positive control, tissue overlying the surgically created viability of four tested pathogens by two to more than four orders of
pocket without a lens implant. f, PO day 5, tissue overlying the pocket with magnitude. Specifically, DMDC-Q-g -EM hydrogel exhibits supe-
an uncoated lens. g, PO day 5, tissue overlying the pocket containing a rior antimicrobial activity with an inhibition above 99% for all four
DMDC-Q-g-EM hydrogel-coated lens (entry 9). White arrows indicate the clinically significant pathogens tested, including Gram-negative and
conjunctival epithelium and black arrows indicate blood vessels. Gram-positive bacteria and fungi. Our hydrogel is contact-active
antimicrobial and also in vitro and in vivo biocompatible. The
the N+ present on the quaternized chitosan, was used to stain the coating has very good adhesion and is re-useable. We propose
hydrogel coating. Figure 3a,b shows the gross visual appearance that the polycationic hydrogel acts like an ‘anion sponge’ to draw
of the uncoated and fluorescein-stained hydrogel-coated surfaces. anionic phospholipids out of the bacterial cell membrane into the
Figure 3b shows that the hydrogel layer was fairly uniformly gel pores, and that the hydrogel positive charge density and pore
coated on the substrate. Figure 3d shows that the freeze-dried size determine the killing efficacy of the coating. We believe that
hydrogel layer was a few micrometres thick and had good adhesion this PEGylated quaternized chitosan-based hydrogel coating, which
to the fluoropolymer substrate, although the hydrogel has very is easily ultraviolet immobilized on any surface, will be widely appli-
high water content. The thin layer of hydrogel grafted on the cable for combating infection in many classes of implant/prosthesis
surface showed good antimicrobial action, with log reductions of and in other medical devices.
2.1–4.2, which are comparable to those of free DMDC-Q-g -EM
hydrogel films (Fig. 2a). Methods
The biocompatibility of DMDC-Q-g -EM hydrogel was demon- Synthesis of qC-g -EMs. Quaternized chitosan (qC) was first synthesized by either
strated by both in vitro and in vivo tests. The in vitro test contacted route I or route II (shown in Fig. 1a). For quaternized TMC (route I), chitosan


(1 g, 6.21 mmol) in N -methyl-2-pyrrolidone (50 ml) was added to NaOH solution Received 23 May 2010; accepted 2 November 2010;
(1.5 M, 15 ml). Sodium iodide (1.08 g, 7.23 mmol) and methyl iodide (11.2 g, published online 12 December 2010
78.7 mmol) were then added to the chitosan/ N -methyl-2-pyrrolidone/NaOH
mixture and then reacted for 24 h at 50 ◦ C. For DMDC (route II), chitosan (1 g, References
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29. Cheng, G., Xue, H., Zhang, Z., Chen, S. & Jiang, S. A switchable biocompatible microscopy and atomic force microscopy. The provision of computation time from the
polymer surface with self-sterilizing and nonfouling capabilities. Angew. Chem. NTU HPC centre is gratefully acknowledged.
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30. Ostuni, E., Chapman, R. G., Holmlin, R. E., Takayama, S. & Whitesides, G. M. Author contributions
A survey of structure–property relationships of surfaces that resist the P.L. carried out the testing and coating experiments. Y.F.P., P.L. and S.H.Y. did
adsorption of protein. Langmuir 17, 5605–5620 (2001). the syntheses and characterization of all the polymers. Y.C. carried out the in vitro
biocompatibility studies. X.Q. carried out some early antimicrobial testing. W.L. and
Y.M. did the computer simulation and related writing. H-Y.Z. and R.W.B. did the animal
Acknowledgements study and related writing. C.Z., E-T.K., M.L., M.W.C., S.S.J.L., C.M.L. and M.B.C-P.
This work was funded and supported by Menicon Holdings (Japan), a Singapore advised on the design and interpretation of the experiments. M.B.C-P. directed the overall
Ministry of Education Tier 2 grant (M45120007), Nanyang Technological University project. P.L., Y.F.P. and M.B.C-P. did the main writing of the manuscript.
(Singapore) and a Singapore SingHealth Foundation grant (SHF/09/GMC(1)/012(R)
(R705)). R.W.B. and H-Y.Z. were supported by NMRC/TCR/002-SERI/2008 R618. Y.C. Additional information
was supported by SingHealth Foundation SHF/09/GMC(1)/012(R) (R705). W.L. and The authors declare competing financial interests: details accompany the paper at
Y.M. were supported by a Singapore Ministry of Education Tier 2 grant (T206B3210RS). www.nature.com/naturematerials. Supplementary information accompanies this paper
We acknowledge the Singapore General Hospital (Pathology Department) for carrying on www.nature.com/naturematerials. Reprints and permissions information is available
out some of the early antimicrobial tests. We thank Y. Shucong, W. Xiujuan and F. Ning online at http://npg.nature.com/reprintsandpermissions. Correspondence and requests
for their help in using field emission scanning electron microscopy, scanning electron for materials should be addressed to M.B.C-P.


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