Cellulose (2014) 21:3767–3779

DOI 10.1007/s10570-014-0371-7

ORIGINAL PAPER

Wound debridement and antibiofilm properties

of gamma-ray DMAEMA-grafted onto cotton gauzes
Marco A. Luna-Straffon • Angel Contreras-Garcı́a •
Gilles Brackman • Tom Coenye • Angel Concheiro •
Carmen Alvarez-Lorenzo • Emilio Bucio

Received: 17 March 2014 / Accepted: 22 July 2014 / Published online: 30 July 2014
Ó Springer Science+Business Media Dordrecht 2014

Abstract Cotton gauze fabric was functionalized
with 2-(dimethylamino)ethyl methacrylate (DMA-
EMA) with the aim of developing wound dressings
with antibiofilm activity and tunable debriding activ-
ity. Cotton-g-DMAEMA gauzes were prepared via
one-step grafting (direct method) using 60Co c-rays as
source to initiate the polymerization process. The
effects of absorbed dose, dose rate, and monomer
concentration on the degree of grafting were evaluated
in detail. Some cotton-g-DMAEMA gauzes were
subsequently quaternized with methyl iodide. Grafting
of DMAEMA and sequential quaternization were
confirmed by FTIR-ATR spectroscopy; thermal prop-
erties were analyzed using TGA, and morphology by
scanning electron microscopy. Grafting of DMAEMA
gauzes enhanced blood absorption and collagenase
Electronic supplementary material The online version of
this article (doi:10.1007/s10570-014-0371-7) contains supple-
mentary material, which is available to authorized users.
activity, while further quaternization led to a remark-
able inhibition of the proteinase activity. Their anti-
microbial features were analyzed by evaluating their
biofilm inhibiting and biofilm eradicating properties in
an in vitro chronic wound model. Although the non-
quaternized gauzes only displayed moderate biofilm
inhibitory properties at best, the quaternized cotton-g-
DMAEMA bearing the highest content of DMAEMA
displayed strong biofilm inhibiting and biofilm erad-
icating properties. This indicates that quaternized
cotton-g-DMAEMA gauzes are less prone to be
colonized by bacteria and can notably reduce the
number of colonies in an infected wound.
Keywords Poly[2-(dimethylamino)ethyl
methacrylate]  c-Ray irradiation  Collagenase
inhibition  Blood absorption  Antimicrobial gauze 
Biofilm inhibition and eradication

M. A. Luna-Straffon  E. Bucio (&) G. Brackman  T. Coenye

Departamento de Quı́mica de Radiaciones y
Radioquı́mica, Instituto de Ciencias Nucleares,
Universidad Nacional Autónoma de México, Circuito
Exterior, Ciudad Universitaria, 04510 Mexico, D.F.,
Mexico
e-mail: ebucio@nucleares.unam.mx
A. Contreras-Garcı́a
Laboratorio de Investigación y Desarrollo, Signa S.A. de
C.V., Av. Industrial Automotriz 301, Zona Industrial,
50071 Toluca, Estado de México, Mexico
Laboratory of Pharmaceutical Microbiology, Faculty of
Pharmaceutical Sciences, Ghent University,
Harelbekestraat 72, 9000 Ghent, Belgium
A. Concheiro  C. Alvarez-Lorenzo
Departamento de Farmacia y Tecnologı́a Farmacéutica,
Universidad de Santiago de Compostela,
15782 Santiago de Compostela, Spain

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3768
Introduction
Wound healing is delayed, or even hampered, by
hypoxia and infection (Stadelmann et al. 1998).
Hypoxia leads to necrotic tissue and also predisposes
the wound to invasion by microorganisms (Hopf et al.
1997; Sen 2009). Bacterial growth is accompanied by
release of enzymes and metalloproteinases that may
degrade fibrin as well as wound growth factors.
Although wound sterility is an unrealistic goal, the
number of microorganisms should be kept as low as
possible, and biofilm formation has to be avoided
(Kirketerp-Møller et al. 2011). Wound healing
depends on a delicate balance between the host
defenses and the pathologic effects of microorgan-
isms. For most bacteria, the critical threshold is
considered to be 105 CFU per gram tissue, above
which complications are likely to arise (Robson et al.
1990). If foreign solid materials, including dressings
and sutures, are present, this threshold can be notably
lower (Campoccia et al. 2013).
Wound dressings containing proteolytic enzymes
(e.g. collagenase) have been extensively used for
pressure ulcer healing as a way to improve the cleaning
of necrotic tissue, which may also prevent the growth
of biofilm (Reddy et al. 2008). Certain chronic wounds
are associated with prolonged inflammation, which
results in excessively high protease levels and free
radical generating enzymes that degrade extracellular
matrix proteins and growth factors required for healing
and also inactivate endogenous protease inhibitors,
resulting in uncontrolled protease activity (Bank et al.
2000). These wounds may benefit from down-regula-
tion of proteolytic enzymes by means of dressings that
incorporate sacrificial collagen substrate or sequester
the enzyme (Edwards and Howley 2007; Wiegand and
White 2013). As an alternative, some dressings incor-
porate growth factors that favor the healing process.
However, there is still insufficient evidence to show
that these expensive products are better than those
simply containing an antiseptic agent (Reddy et al.
2008). In fact, addressing high protease and cytokine
concentrations and low growth factor levels are two
main challenges of wound bed preparation (Reddy
et al. 2008; Edwards et al. 2009).
The aim of this work was to implement a cost-
effective method to prepare multifunctional gauzes
that can regulate the activity of proteolytic enzymes
while simultaneously offering antimicrobial features.
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Cellulose (2014) 21:3767–3779
To carry out the work, inexpensive cotton gauzes and a
grafting procedure suitable for large-scale production
were chosen. Several wet chemistry modification
techniques have been previously reported for modifi-
cation of cotton fabrics with different biomedical
purposes, such as phosphorylation to reduce elastase
and collagenase activity (Edwards and Howley 2007;
Edwards et al. 2009), grafting of antibacterial chitosan
(Fouda et al. 2009), impregnation with antioxidants
and bacteriostatic plant extracts (Hong 2014) or
functionalization with cyclodextrins for loading/
release of antiseptic drugs (Garcia-Fernandez et al.
2013). Less attention has been paid to the functional-
ization of cotton cellulose by means of radiation
grafting techniques, which can provide greater yields
and require minimal use of initiators, catalysts and
solvents (Alvarez-Lorenzo et al. 2010; Desmet et al.
2011; Verma and Kaur 2012). c-Ray grafting is
particularly useful for the surface modification of
medical devices, including cotton gauzes, with poly-
mers able to host and release drugs in a controlled way
(Hiriart-Ramı́rez et al. 2012) or with cationic moieties
that exhibit antimicrobial properties (Kumar et al.
2005; Goel et al. 2011). Quaternary ammonium salts
are expected to disrupt the negatively charged mem-
brane of microorganisms, leading to cell death (Se-
numa et al. 1993) and functionalization of cotton with
quaternary ammonium salts was shown to be useful in
avoiding growth of various gram-positive bacteria
(Gottenbos et al. 2002; Goel et al. 2011). Nevertheless,
since the nature of the cationic monomer and the
experimental parameters strongly determine the extent
of the radiation-grafting, the antibacterial performance
of the resulting material is hard to predict. Moreover,
effective grafting of cationic monomers (e.g. vinyl
benzyl trimethyl ammonium chloride, or 2-(acryloyl-
oxyethyl)trimethyl ammonium chloride), requires
combination with non-ionic monomers (e.g.
2-hydroxyethyl methacrylate) that act as facilitators
of the process (Kumar et al. 2005; Goel et al. 2011).
In the present work, functionalized gauzes were
prepared following a two-step procedure that avoids the
use of facilitator comonomers: (1) grafting of a mono-
mer that contains amino groups, namely 2-(dimethyl-
amino)ethyl methacrylate (DMAEMA), and (2)
transformation of the amino groups into quaternary
ammonium groups having iodide as counterion. Poly[2-
(dimethylamino)ethyl methacrylate] (PDMAEMA)
Cellulose (2014) 21:3767–3779
itself exhibits a critical point at pH around 8.5 (Burillo
et al. 2007; Titaux et al. 2009); thus, it is partially
protonized at physiological pH. PDMAEMA has pre-
viously been grafted to low density polyethylene and
silicone rubber using a direct c-ray irradiation method
with the purpose of providing binding points for the
antimicrobial agent nalidixic acid (Contreras-Garcı́a
et al. 2011). Quaternization of the amine group has been
shown to improve the antimicrobial activity of PDMA-
EMA (Gottenbos et al. 2002; De Prijck et al. 2010;
Rawlinson et al. 2010), although the alkyl halides used
for the quaternization should bear a short alkyl chain to
not compromise the biocompatibility (particularly with
erythrocytes) of the functionalized material (Venkatar-
aman et al. 2010). In the present work, the quaternization
was carried out with methyl iodide (MeI) in order to
balance antimicrobial effect and biocompatibility (Con-
treras-Garcı́a et al. 2011). In addition to the antimicro-
bial features, we hypothesize that DMAEMA-
functionalized gauzes may have an impact on the
hemostasis and the activity of wound proteolytic
enzymes. Ethylamino-ether cellulose derivatives have
been shown to promote clotting in hemorrhagic wounds
(Edwards et al. 2009). On the other hand, gauzes
containing cadexomer iodine (a water-soluble modified
dextrin with 0.9 % iodine) have been shown to inacti-
vate proteolytic enzymes (Shi et al. 2010). Conversely,
gauzes impregnated with iodoform exhibit fibrinolytic
activity and remove necrotic tissue from pressure ulcer
wounds, with the advantage of being more stable than
enzyme-loaded dressings (Mizokami et al. 2012).
Therefore, functionalization of gauzes with DMAEMA
and subsequent quaternization with MeI may have a
strong impact on the proteolytic activity during healing.
The information gathered in this study may help to
identify suitable functionalized gauzes able to modulate
events in the wound, namely proteolytic activity,
hemorrhage hemostasis, and microbial growth, in a
cost-effective way.
Experimental
Materials
DMAEMA was from Aldrich Chemical Co. (St. Louis,
MO, USA) and distilled under reduced pressure
before use. Commercial cotton fiber gauzes (sterilized
100 % cotton) were from Dimacu S.A. (D.F. Mexico).
3769
Ethanol, methanol, toluene, tetrahydrofuran HPLC
degree (THF), and diethyl ketone were from Baker
(D.F. Mexico). MeI, collagenase from Clostridium
histolyticum (C0130), heparin and N-(3-[2-furyl]acry-
loyl)-Leu-Gly-Pro-Ala (FALGPA) were from Sigma-
Aldrich Co. (St. Louis, MO, USA).
Radiation grafting procedure
Grafted cotton gauzes (cotton-g-DMAEMA) were
prepared applying the direct method (mutual irradia-
tion, Scheme 1). Cotton gauze portions (6 cm 9 9 cm)
were washed with ethanol, dried at room temperature
and weighted. The gauzes were placed in glass
ampoules containing DMAEMA/methanol solutions,
with the monomer concentration ranging from 5 to
50 % (v/v). The ampoules were bubbled with argon for
20 min to remove air, and then sealed and exposed to
60
Co c-source (Gammabeam 651 PT, MDS Nordion
International, Ottawa, Canada) 40,000 Ci, at dose rates
of 6.5 and 14.5 kGy/h and irradiation exposure doses
ranging from 1 to 25 kGy. DMAEMA homopolymer
and residual monomer were extracted from the func-
tionalized gauzes by immersion in ethanol for 24 h,
exchanging the medium three times. Finally, the gauzes
were dried under vacuum at 40 °C for 24 h. The
grafting percentage (Yg) in the cotton-g-DMAEMA
gauzes was calculated as follows:
 
ðW2  W1 Þ
Yg ð%Þ ¼ 100  ð1Þ
W1
In this equation, W1 and W2 represent the weight of
the gauze before and after grafting, respectively.
Quaternization of the grafted cotton gauzes
Cotton-g-DMAEMA gauzes were immersed in MeI
solution (10 % w/v in THF) and kept for 4 h under
stirring at room temperature. Then, the solution was
removed and the unreacted MeI was extracted from
the quaternized samples by immersion in diethyl
ketone for 24 h. The gauzes were washed with
bidistilled water and dried under vacuum to constant
weight. The degree of quaternization was estimated
as
 
ðW3  W2 Þ
DQ ð%Þ ¼ 100 
½ðW2  W1 Þð157:22=141:94Þ
ð2Þ
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3770
Scheme 1 Reaction mechanisms of grafting of DMAEMA onto cotton gauze
where W1 and W2 mean the same as in Eq. 1, W3
represents the weight of the quaternized gauze, and
157.22 and 141.94 are the molecular weights of
DMAEMA and MeI, respectively.
Characterization of the grafted gauzes
FTIR-ATR spectra of cotton gauze and cotton-g-
DMAEMA before and after quaternization were
recorded using a Perkin-Elmer Spectrum 100 (Perkin
Elmer Cetus Instruments, Norwalk, CT, USA) with 16
sample scans. Thermal decomposition was monitored
in nitrogen atmosphere between 25 and 700 °C at a
heating rate of 10 °C/min using a TGA Q50 (TA
Instruments, New Castle, DE, USA). The samples for
TGA analysis were previously dried during 24 h at
40 °C. Scanning electron microscopy (SEM) micro-
graphs of freeze-dried gauzes (Genesis 25ES pilot
lyophilizer, VirTis, UK) were taken using a LEO
435VP scanning electron microscope (Leica, Cam-
bridge, UK) at various magnifications. These exper-
iments were carried out in duplicate.
Collagenase activity assay
Pieces of pristine, DMAEMA-grafted and quaternized
DMAEMA-grafted gauzes (30–40 mg) were directly
placed in EppendorfÒ LoBind tubes containing
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Cellulose (2014) 21:3767–3779
collagenase (1 mL, 250 lg/mL in tricine buffer) and
incubated at 37 °C. Samples of collagenase medium
(100 lL) were taken after 30 min and 12 h. The
samples were immediately transferred to 96-well
microplates and mixed with FALGPA solution
(5 mM, 150 lL in tricine buffer) (Shi et al. 2010).
The reaction was monitored for 30 min by recording
the absorbance at 350 nm (BP 10 nm) in a FLUOstar
Optima microplate reader (BMG Labtech, Ortenberg,
Germany). Collagenase solutions (controls without
gauze) kept at 20 and 37 °C were processed similarly.
All experiments were carried out in triplicate for each
data point and temperature.
Blood absorption
Pieces of pristine, DMAEMA-grafted and quaternized
DMAEMA-grafted gauzes (ca. 30 mg) were placed, in
triplicate, in EppendorfÒ LoBind tubes containing human
blood [2 mL pre-treated with citrate phosphate dextrose
(CPD), anticoagulant solution 14 % v/v; healthy volun-
teers; Centro de Transfusión de Galicia, Spain]. The
systems were incubated at room temperature and the
weight of the gauzes was recorded at 30 min and 3 h. To
do that, the gauzes were transferred to 5 mL syringe and
squeezed to remove unbound blood. Absorption factor
was quantified gravimetrically as the amount of blood
absorbed per weight unit of the gauze.
Cellulose (2014) 21:3767–3779
Biofilm inhibition and eradication assays
The biofilm inhibiting and eradicating activity of
the gauzes was evaluated as previously described
(Brackman et al. 2013). In brief, an overnight
culture of Staphylococcus aureus Mu50 was pel-
leted, washed, resuspended in physiological saline
(PS) and diluted to 106 CFU/mL. Medical grade
silicone disks (Q7-4735, Dow Corning, Midland,
MI, USA) were placed in the wells of a 24-well
microtiter plate (SPL life sciences, Novolab, Ger-
aardsbergen, Belgium) and 10 lL aliquots of the
cell suspension were spotted on each disk. About
1 mL of Bolton Broth with 50 % plasma (Sigma-
Aldrich, St. Louis, MO, USA), 10 U/mL heparine
and 5 % freeze–thaw laked horse blood was added.
To evaluate inhibition of biofilm formation, 1 cm2
gauzes were placed over each silicone disk imme-
diately after inoculation. To assess the biofilm
eradicating activity, the plate was first incubated for
24 h at 37 °C to allow biofilm formation on the
silicone disks, after which the medium was
removed, the biofilms were washed with PS, fresh
medium was added, the gauzes were placed on top
of the biofilm and the plates were incubated at
37 °C for another 24 h. After this, the gauzes were
removed and placed in 10 mL PS, and biofilms
were rinsed once with PS. The silicone disks
containing the biofilm cells were also placed in a
separate falcon containing 10 mL PS. The sessile
cells present on the silicone disks and on the
gauzes were removed from the disks/gauzes apply-
ing three cycles of vortexing (30 s) and sonication
(30 s; Branson 3510; Branson Ultrasonics Corp.,
Danbury, CT, USA) and the number of CFU/
biofilm or CFU/cm2 gauze was determined by
plating the resulting suspensions. Three samples of
each gauze were analyzed in the biofilm inhibition
assay. Pristine, DMAEMA-modified (76.7 % graft)
and quaternized DMAEMA-modified (74.0 % graft)
gauzes were analyzed in the biofilm eradication
assay.
Statistical analysis
The data were analyzed by means of a one-way
ANOVA and Dunnet test. Statistical analysis and
linear regression analysis were carried out using SPSS
software, version 21.0 (SPSS, Chicago, IL, USA).
3771
Results and discussion
Grafting of DMAEMA
Gauzes grafted with DMAEMA (cotton-g-DMA-
EMA) were prepared by placing pieces of cotton
gauzes in glass ampoules containing a DMAEMA/
MeOH solution (direct method; Scheme 2). The
irradiation doses ranged from 1 to 25 kGy at two
different dose rates. The efficiency of the grafting
polymerization was gravimetrically measured after
extensive washing with ethanol and drying of the
grafted cotton gauzes. The dependence of the grafting
percentage on monomer concentration was firstly
examined at a fixed radiation dose (Fig. 1). The
grafting percentage increased from 20 to 70 % w/w as
the concentration of DMAEMA in the reaction
solution increased from 5 to 50 % v/v. Homopolymer
formation was promoted as monomer concentration
increased. The increase in grafting percentage as a
function of the monomer concentration was due to the
availability of more molecules of monomer for
reaction with the radicals generated on the backbone
of cotton gauze. Nevertheless, as more monomer
radicals were generated in the bulk, homo-polymer-
ization was equally favored.
In the direct grafting method, the number of grafted
chains and their length depend on the total radiation
dose and the dose rate. While the total exposure dose
determines the number of free radicals generated on
the cotton substrate, the dose rate determines the rate
of initiation of polymerization (Bucio et al. 2002). In
the present case, the grafting augmented with the dose
of radiation more rapidly in the low dose range. As
shown in the Online Resource (Figure S1), the grafting
percentages obtained when the process was carried out
in DMAEMA 50 % v/v solution in methanol, with
irradiation dose from 1 to 20 kGy, and dose rate of 6.5
and 14.5 kGy/h. The smaller increase in grafting
extent at doses above 10 kGy may be due to either
monomer exhaustion or viscosity increase in the
reaction solution, which restricts the monomer diffu-
sion during the propagation step of the grafted chains.
The gauzes irradiated at a dose rate of 6.5 kGy/h
showed higher grafting percentages because low dose
rate favors the monomer diffusion (due to little
homopolymer formation) towards the active sites in
the cotton gauze backbone. The grafting percentages
of gauzes irradiated with an exposure dose of 20 kGy
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3772
Scheme 2 Schematic representation of preparation of cotton-g-DMAEMA and subsequent quaternization with methyl iodide
80
70
60
Graft (%)
50
40
30
20
0 10 20 30 40 50
Monomer concentration (% v/v)
Fig. 1 Effect of monomer concentration on grafting percentage
of cotton-g-DMAEMA. Direct method, dose 10 kGy, and dose
rate 6.5 kGy/h
at dose rates of 6.5 and 14.5 kGy/h were 140 and
80 %, respectively. Thus, 6.5 kGy/h appeared to be
more adequate for grafting of DMAEMA onto cotton
gauze because of minor interference of the homopol-
ymer formation. Thus, lower grafting percentages
obtained at 14.5 kGy/h are explained by the produc-
tion of more radicals per unit area of cotton, which
favors recombination of free radicals generated in the
close vicinity, as well as by faster reaction of
monomers in solution which leads to homopolymer
synthesis.
To gain an insight into the effect of the irradiation
dose (in the 2.5–25 kGy range) on the grafting
percentage, graft polymerization was carried out at a
fixed dose rate placing the gauzes in solutions of
different monomer concentration (50 and 60 % v/v).
The number of latent initiating sites was expected to
increase as the radiation dose became higher, although
not necessarily in a linear manner. Up to 15 kGy, the
higher the irradiation dose, the greater the amount of
PDMAEMA grafted (Fig. 2). Above 15 kGy, the
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Cellulose (2014) 21:3767–3779
80
60
Graft (%)
40
20
0
0 5 10 15 20 25
Dose (kGy)
Fig. 2 Effect of irradiation dose on grafting percentage of
cotton-g-DMAEMA, for 50 % (square) and 60 % (triangle)
monomer concentration in methanol. Direct method, dose rate
14.5 kGy/h
grafting percentage reached a plateau. It has previously
been reported that an increase in monomer concentra-
tion usually leads to an amplification of the dose effect;
the polymerization rate increases at higher monomer
concentration since more monomer units are available
for the propagation of polymerization. However, using
50 and 60 % v/v monomer concentration similar
grafting percentages were obtained, probably because
of the large excess of monomer in the medium.
Cotton-g-DMAEMA gauzes covering a wide range
of grafting percentages were quaternized by means of
soaking in a MeI solution in THF. The DQ values were
between 94 and 98 % disregarding the initial degree of
grafting (Table 1). Therefore, the method applied led
to an almost complete quaternization of all amine
groups available in PDMAEMA chains. The quatern-
ized cotton gauzes acquired a yellow-brownish colour
of intensity proportional to the content of DMAEMA,
which is typical for iodide-containing products.
Cellulose (2014) 21:3767–3779
Table 1 Degree of quaternization of cotton-g-DMAEMA
gauzes treated with MeI
Material Grafting Quaternization
percentage degree
cotton-g-DMAEMA 24 98 (1)
50 94 (4)
75 97 (2)
80 95 (1)
Mean values and, in parenthesis, standard deviations (n = 3)
Fig. 3 FTIR-ATR of pristine cotton gauze (a), PDMAEMA
(b), cotton-g-DMAEMA (c), and quaternized cotton-g-DMA-
EMA (d)
Infrared spectroscopy
The FTIR-ATR spectra confirmed that the graft
polymerization of DMAEMA was successful
(Fig. 3). The spectrum of the pristine gauze showed
the absorption bands of –OH groups at 3,343 cm-1,
methyl and methylene groups at 2,899 cm-1, and C–O
bonds at 1,024 cm-1. In the PDMAEMA homopoly-
mer spectrum, peaks assigned to methyl and methylene
groups at 2,930 and 2,821 cm-1, carbonyl group at
1,721 cm-1, –N–CH2– bond at 2,767 and 1,149 cm-1,
and –OC–O group at 1,239 and 1,099 cm-1 were
observed. The spectra of cotton-g-DMAEMA gauze
exhibited the characteristic bands of DMAEMA
(–C=O at 1,721 cm-1, –N–CH2– at 2,769 and
1,149 cm-1, and –OC–O at 1,239 cm-1). Peaks at
3773
3,000, 1,626, and 950 cm-1 associated to –NR3?
groups in the quaternized cotton-g-DMAEMA gauze
confirmed methylation of DMAEMA amine groups.
Thermogravimetric analysis
Pristine cotton gauze exhibited a single degradation
step in the 290–400 °C interval, and a char residue of
13 % at 450 °C (Online Resource, Figure S2), as
typically observed for cellulose polymers (Hosoya
et al. 2007; Shen et al. 2010). PDMAEMA homopol-
ymer showed a two-step degradation pattern, with a
char residue of 5 % after 450 °C. Cotton-g-DMA-
EMA gauzes decomposed in three steps: the two first
ones are related to degradation of PDMAEMA and the
last one is caused by decomposition of cotton gauze.
Decomposition of quaternized gauzes was initiated at
around 200 °C and occurred in four steps. Overall,
these findings indicate that the methyl group attached
to the amine of DMAEMA affects the thermal stability
of the gauze, but from temperature values well above
those of common handle conditions.
Scanning electron microscopy
SEM micrographs were obtained to visualize the
changes that the grafting of DMAEMA and the
subsequent quaternization could have caused in the
texture or conformation of the cotton fibers (Fig. 4).
Pristine cotton gauze consisted of tight fibers in a well-
defined network, with some fibers untangled. DMA-
EMA grafting led to some shrinking, with fibers
adopting a more compact structure. Nevertheless, the
thickness of single fibers after grafting was similar to
that of unmodified cotton gauze, and untangled fibers
also appeared in the cotton-g-DMAEMA gauze.
Quaternization of the amine groups of DMAEMA
did not cause further changes in the structure of the
fabric.
Collagenase activity
The effect of the gauzes on the activity of crude
collagenase after direct incubation at 37 °C for 30 min
or 12 h was compared to that of control collagenase
solutions stored at 20 and 37 °C (Fig. 5). Crude
collagenase from C. histolyticum is a mixture of
proteases, mainly two collagenases, clostripain and a
neutral protease. Collagenase solution was prepared in
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3774
Fig. 4 SEM images of unmodified cotton gauze (a), cotton-g-DMAEMA (b), and quaternized cotton-g-DMAEMA (c) at two different
magnifications
tricine buffer containing Ca2? for activation of the
enzymatic activity. No differences were observed
between the activity of collagenase solely solutions
stored at 20 or 37 °C. Pristine gauzes did not modify
the enzyme activity in the first 30 min, but decreased it
to the half after 12 h incubation at 37 °C, while cotton-
g-DMAEMA gauzes increased collagenase activity up
to 171 % (SD 2) after 30 min incubation and up to
146 % (SD 1) after 12 h. Such an increase in
enzymatic activity can be related to the increase in
hydrophilicity and in positive charges of the gauzes
after grafting of DMAEMA, which in turn prevents
non-specific adsorption of the enzyme also positively
charged at physiological pH (Edwards and Howley
2007).
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Cellulose (2014) 21:3767–3779
In contrast, quaternized gauzes strongly inhibited
collagenase activity, reducing the activity to 34 % (SD
5) and to 7.5 % (SD 2.9) when incubated for 30 min
and 12 h, respectively. These later results are in
agreement with those reported for Iodoflex and
Iodosorb-medicated dressings, which consist of mac-
rogol ointments containing cadexomer iodine (Shi
et al. 2010). It has previously been observed that
iodine inhibits the activity of proteases in wounds
more efficiently than silver, because the stronger
oxidant character of the former causes denaturation
and inactivation of enzymes (Eming et al. 2006).
Previous studies focused on extracts of the semisolid
dressings (Shi et al. 2010). In our study the gauzes
were directly placed into contact with collagenase
Cellulose (2014) 21:3767–3779 3775

Fig. 5 Activity of crude

30 min 12 h
collagenase after being 1.0
incubated with various
cotton gauzes recorded as

Relative absorbance
the decrease in absorbance 0.9
at 350 nm of FALGPA-
containing solutions. Values
were referred to the 0.8
absorbance values
registered after 5 min of
mixing collagenase solution Control
0.7
and FALGPA solution Pristine gauze
Cotton-g-DMAEMA 50%
Cotton-g-DMAEMA 75%
0.6

30 min 12 h
1.0
Relative absorbance

0.9

0.8

0.7 Control
Q cotton-g-DMAEMA 23%
Q cotton-g-DMAEMA 75%
0.6
0 15 30 45 60 75 90 0 15 30 45 60 75 90
Time (min) Time (min)

medium in order to discern the inhibitory effects due to Blood absorption

simple adsorption of the enzyme to pristine gauzes
(i.e., commercially available cotton gauzes) and due to
the presence of iodine forming part of the quaternized
DMAEMA groups grafted to the functionalized
gauzes. Results indicate that pristine gauzes may
reduce collagenase activity after prolonged contact
time, probably because adsorption of the enzyme to
the cotton decreases the amount available for reaction
with the substrate. On the other hand, quaternization of
cotton-g-DMAEMA gauzes strongly inhibited colla-
genase activity, even in the case of the gauzes with a
grafting percentage as low as 23 %. Compared to
semisolid dressings, quaternized cotton-g-DMAEMA
gauzes may offer the possibility of an easier handling
for a more precise regulation of over-expressed
proteases in chronic non-healing wounds. Quaternized
gauzes also compare favorably with those gauzes
functionalized with negatively charged groups
designed to act as protease sequestrants, in terms of
faster response attained for similar gauze weight
(Edwards et al. 2009).
Depending on the etiology of the wound, the gauzes
can be exposed to large volumes of exudates or
blood. To gain an insight into the effects of grafting
and quaternization on these issues, the capability of
the gauzes to absorb fresh whole blood was
measured. All gauzes rapidly absorbed blood
although some differences were observed (Fig. 6).
Compared to pristine cotton gauze that absorbed ca.
4.05 g/g in 180 min, the absorption factor was
higher for cotton-g-DMAEMA, which incorporated
nearly 4.72 g/g in 180 min (statistically significant
differences, p \ 0.05; ANOVA and rank test). This
finding confirms that grafting of DMAEMA makes
the cotton gauzes to be more hydrophilic. Quatern-
ization led to smaller absorption factors, ca. 3.6 g/g
in 180 min (statistically significant differences,
p \ 0.05; ANOVA and rank test). Nevertheless,
compared to other dressings, the absorption factors
of all gauzes were in the range of those recorded for
efficient hemostatic dressings (Arnaud et al. 2009).

123
3776 Cellulose (2014) 21:3767–3779

Thus, grafting of DMAEMA and further quatern- Biofilm inhibition and eradication assays
ization may have minor practical effects on blood
absorptive capacity of the gauzes. Finally, the gauzes were challenged against a wound
infection model (Brackman et al. 2013) in order to test
both their capability to inhibit the formation of biofilm
6 immediately after microbial contamination, and their
30 min
180 min ability to eradicate the biofilm once this has been
5
developed in the wound. In either case, the number of
Blood sorption (g/g)

4 microorganisms in the wound (i.e., the contaminated

silicon disk model) and in the gauzes was determined
3
24 h after the treatment with the gauzes.
2 Staphylococcus aureus Mu50 formed biofilm on the
1
1 cm2 slabs treated with pristine gauze and cotton-g-
DMAEMA with 50 or 75 % grafting. Conversely,
0 significantly less S. aureus biofilm cells were recov-
ze 0% 5% 3% 5%
au A5 A7 A2 A7
ntr
olg
A EM A EM A EM A EM ered on quaternized cotton-g-DMAEMA with 23 and
Co DM DM DM DM
n-g
-
n-g
-
n-g
-
n-g
- 50 % grafting, and S. aureus Mu50 was incapable of
tto tto tto tto
Co Co co co
Q Q forming a biofilm on quaternized gauzes bearing the
highest content in DMAEMA tested (75 %) (Fig. 7a).
Fig. 6 Absorption factor expressed as weigh of blood absorbed
per weight unit of gauze (g/g) in 30 min (black columns) and Regarding the biofilm formed in the chronic wound
180 min (grey columns) (i.e., on the silicone disks), quaternized cotton-g-

Fig. 7 a Biofilm formation (a) Fresh biofilm growth on gauze (b) Biofilm inhibition in wound model
of S. aureus Mu50 on
different gauzes. b Biofilm 1010 1010

formation of S. aureus Mu50 109 109

** **
in the chronic wound biofilm 108 108
*
model in the presence of the 10 7 107 * *
CFU/biofilm
CFU/cm2

different gauzes. 106 106

c Eradication of S. aureus 5
10 105
Mu50 biofilm formed on the 4
10 104
different gauzes when they
3 103
were placed on a mature 10
biofilm. d Biofilm 10 2 102 **
**
eradicating effect of the 101 101
different gauzes on mature 100 100
biofilms of S. aureus Mu50. ol % 5% 3% 0% 5% ol % % % % %
ntr 50 7 2 5 7 ntr 1-50 2-75 1-23 2-50 3-75
*p \ 0.05; **p \ 0.005 Co N1- N2- Q1- Q2- Q3- Co N N Q Q Q
compared to control pristine
gauze. Codes: control refers
to pristine cotton gauze; N1 (c) Mature biofilm growth on gauze (d) Biofilm eradication in wound model
and N2 refer to cotton-g- 1010 1010
DMAEMA gauzes with 50 109 109
and 75 % grafting; and Q1, 108 108
Q2 and Q3 refer to 107 107
*
CFU/biofilm

quaternized cotton-g- **
CFU/cm2

6 106
10
DMAEMA gauzes with 23,
50 and 75 % grafting 105 105
4 104
10
3 103
10
2 102
10
1 101
10
100 100
Control N2-75% Q3-75% Control N2-75% Q3-75%

123
Cellulose (2014) 21:3767–3779
DMAEMA with 23 % grafting did not inhibit biofilm
formation. In contrast, biofilm formation on the
silicone disks was significantly inhibited in the
presence of the cotton-g-DMAEMA gauzes with 50
and 75 % grafting percentage either quaternized or not
(Fig. 7b). Once again, S. aureus Mu50 biofilm
formation was almost completely inhibited in the
wound model in the presence of quaternized gauzes
bearing the highest content in DMAEMA tested
(75 %) (Fig. 7b). This biofilm inhibitory activity is
higher than what is observed for most of the commer-
cially available wound care dressings (Brackman et al.
2013).
In the next step we allowed the S. aureus Mu50
strain to form a 24 h old biofilm which was then
treated with the gauzes. When gauzes were placed on
mature biofilms for 24 h, significantly less biofilm
cells were recovered from the quaternized cotton-g-
DMAEMA 75 % grafting in comparison to non-
quaternized and pristine gauze (Fig. 7c). In addition,
although no biofilm eradicating activity was observed
for non-quaternized gauzes, a significant eradicating
activity was confirmed for the quaternized cotton-g-
DMAEMA 75 % grafting (Fig. 7d). Overall, these
findings indicate that quaternized cotton-g-DMA-
EMA are less prone to be colonized by bacteria and
can even notably reduce the number of colonies
infecting a wound. Moreover, quaternized cotton-g-
DMAEMA gauzes may help to prevent cross-con-
tamination. Removal of conventional gauzes from
infected wounds has been demonstrated to spread
bacteria into the air (Lawrence 1994). Functionaliza-
tion with DMAEMA followed by quaternization
might contribute to reduce airborne bacteria and thus
to a better control of the infection and a lower
incidence of complications.
Conclusions
The direct irradiation method using gamma radiation
has shown to be adequate to induce the grafting of
DMAEMA onto cotton gauze (cotton-g-DMAEMA).
The irradiation exposure dose affects directly the
efficiency of copolymerization to prepare cotton-g-
DMAEMA, however, the rate dose is also important to
take in count, since high dose rate of 14.6 kGy/h
promotes the formation of the DMAEMA homopol-
ymer, reducing the grafting yield. The changes in the
3777
functional groups and in thermal stability after mod-
ification of the cotton gauze by copolymerization with
DMAEMA and subsequent quaternization of amine
groups were confirmed by infrared spectroscopy and
thermogravimetric analysis, respectively. DMAEMA
grafting makes gauzes to be more hydrophilic with
enhanced blood absorption capability and that favor
collagenase activity. Oppositely, quaternization with
MeI led to gauzes able to strongly inhibit protease
activity. Although the non-quaternized gauzes only
display moderate biofilm inhibitory properties at best,
the quaternized cotton-g-DMAEMA bearing the high-
est content of DMAEMA displays strong biofilm
inhibiting and biofilm eradicating properties both on
the wound-bed surface as on the gauze itself. This
indicates that quaternized cotton-g-DMAEMA are
less prone to be colonized by bacteria and can even
notably reduce the number of colonies infecting a
wound either as fresh or mature biofilm thereby
contributing to a better control of the infection and a
lower incidence of complications. Overall the results
point out this functionalization approach as a suitable
cost-effective method to develop gauzes able to
modulate events in the wound, namely proteolytic
activity, blood sorption, and microbial growth.
Acknowledgments The authors thank to M. Cruz, F. Garcı́a and
B. Leal from ICN-UNAM for technical assistance. This work was
supported by DGAPA-UNAM Grant IN200714 and CONACYT-
CNPq Project 174378 Mexico, MICINN (SAF2011-22771) Spain
and FEDER. Authors also thank ‘‘Red iberoamericana de nuevos
materiales para el diseño de sistemas avanzados de liberación de
fármacos en enfermedades de alto impacto socioeconómico’’
(RIMADEL) of the Ibero-American Programme for Science,
Technology and Development (CYTED) and funding by the
Institute for the Promotion of Innovation through Science and
Technology in Flanders (IWT-Vlaanderen, SBO programme).
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