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

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


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

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3768 Cellulose (2014) 21:3767–3779

Introduction To carry out the work, inexpensive cotton gauzes and a


grafting procedure suitable for large-scale production
Wound healing is delayed, or even hampered, by were chosen. Several wet chemistry modification
hypoxia and infection (Stadelmann et al. 1998). techniques have been previously reported for modifi-
Hypoxia leads to necrotic tissue and also predisposes cation of cotton fabrics with different biomedical
the wound to invasion by microorganisms (Hopf et al. purposes, such as phosphorylation to reduce elastase
1997; Sen 2009). Bacterial growth is accompanied by and collagenase activity (Edwards and Howley 2007;
release of enzymes and metalloproteinases that may Edwards et al. 2009), grafting of antibacterial chitosan
degrade fibrin as well as wound growth factors. (Fouda et al. 2009), impregnation with antioxidants
Although wound sterility is an unrealistic goal, the and bacteriostatic plant extracts (Hong 2014) or
number of microorganisms should be kept as low as functionalization with cyclodextrins for loading/
possible, and biofilm formation has to be avoided release of antiseptic drugs (Garcia-Fernandez et al.
(Kirketerp-Møller et al. 2011). Wound healing 2013). Less attention has been paid to the functional-
depends on a delicate balance between the host ization of cotton cellulose by means of radiation
defenses and the pathologic effects of microorgan- grafting techniques, which can provide greater yields
isms. For most bacteria, the critical threshold is and require minimal use of initiators, catalysts and
considered to be 105 CFU per gram tissue, above solvents (Alvarez-Lorenzo et al. 2010; Desmet et al.
which complications are likely to arise (Robson et al. 2011; Verma and Kaur 2012). c-Ray grafting is
1990). If foreign solid materials, including dressings particularly useful for the surface modification of
and sutures, are present, this threshold can be notably medical devices, including cotton gauzes, with poly-
lower (Campoccia et al. 2013). mers able to host and release drugs in a controlled way
Wound dressings containing proteolytic enzymes (Hiriart-Ramı́rez et al. 2012) or with cationic moieties
(e.g. collagenase) have been extensively used for that exhibit antimicrobial properties (Kumar et al.
pressure ulcer healing as a way to improve the cleaning 2005; Goel et al. 2011). Quaternary ammonium salts
of necrotic tissue, which may also prevent the growth are expected to disrupt the negatively charged mem-
of biofilm (Reddy et al. 2008). Certain chronic wounds brane of microorganisms, leading to cell death (Se-
are associated with prolonged inflammation, which numa et al. 1993) and functionalization of cotton with
results in excessively high protease levels and free quaternary ammonium salts was shown to be useful in
radical generating enzymes that degrade extracellular avoiding growth of various gram-positive bacteria
matrix proteins and growth factors required for healing (Gottenbos et al. 2002; Goel et al. 2011). Nevertheless,
and also inactivate endogenous protease inhibitors, since the nature of the cationic monomer and the
resulting in uncontrolled protease activity (Bank et al. experimental parameters strongly determine the extent
2000). These wounds may benefit from down-regula- of the radiation-grafting, the antibacterial performance
tion of proteolytic enzymes by means of dressings that of the resulting material is hard to predict. Moreover,
incorporate sacrificial collagen substrate or sequester effective grafting of cationic monomers (e.g. vinyl
the enzyme (Edwards and Howley 2007; Wiegand and benzyl trimethyl ammonium chloride, or 2-(acryloyl-
White 2013). As an alternative, some dressings incor- oxyethyl)trimethyl ammonium chloride), requires
porate growth factors that favor the healing process. combination with non-ionic monomers (e.g.
However, there is still insufficient evidence to show 2-hydroxyethyl methacrylate) that act as facilitators
that these expensive products are better than those of the process (Kumar et al. 2005; Goel et al. 2011).
simply containing an antiseptic agent (Reddy et al. In the present work, functionalized gauzes were
2008). In fact, addressing high protease and cytokine prepared following a two-step procedure that avoids the
concentrations and low growth factor levels are two use of facilitator comonomers: (1) grafting of a mono-
main challenges of wound bed preparation (Reddy mer that contains amino groups, namely 2-(dimethyl-
et al. 2008; Edwards et al. 2009). amino)ethyl methacrylate (DMAEMA), and (2)
The aim of this work was to implement a cost- transformation of the amino groups into quaternary
effective method to prepare multifunctional gauzes ammonium groups having iodide as counterion. Poly[2-
that can regulate the activity of proteolytic enzymes (dimethylamino)ethyl methacrylate] (PDMAEMA)
while simultaneously offering antimicrobial features.

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Cellulose (2014) 21:3767–3779 3769

itself exhibits a critical point at pH around 8.5 (Burillo Ethanol, methanol, toluene, tetrahydrofuran HPLC
et al. 2007; Titaux et al. 2009); thus, it is partially degree (THF), and diethyl ketone were from Baker
protonized at physiological pH. PDMAEMA has pre- (D.F. Mexico). MeI, collagenase from Clostridium
viously been grafted to low density polyethylene and histolyticum (C0130), heparin and N-(3-[2-furyl]acry-
silicone rubber using a direct c-ray irradiation method loyl)-Leu-Gly-Pro-Ala (FALGPA) were from Sigma-
with the purpose of providing binding points for the Aldrich Co. (St. Louis, MO, USA).
antimicrobial agent nalidixic acid (Contreras-Garcı́a
et al. 2011). Quaternization of the amine group has been Radiation grafting procedure
shown to improve the antimicrobial activity of PDMA-
EMA (Gottenbos et al. 2002; De Prijck et al. 2010; Grafted cotton gauzes (cotton-g-DMAEMA) were
Rawlinson et al. 2010), although the alkyl halides used prepared applying the direct method (mutual irradia-
for the quaternization should bear a short alkyl chain to tion, Scheme 1). Cotton gauze portions (6 cm 9 9 cm)
not compromise the biocompatibility (particularly with were washed with ethanol, dried at room temperature
erythrocytes) of the functionalized material (Venkatar- and weighted. The gauzes were placed in glass
aman et al. 2010). In the present work, the quaternization ampoules containing DMAEMA/methanol solutions,
was carried out with methyl iodide (MeI) in order to with the monomer concentration ranging from 5 to
balance antimicrobial effect and biocompatibility (Con- 50 % (v/v). The ampoules were bubbled with argon for
treras-Garcı́a et al. 2011). In addition to the antimicro- 20 min to remove air, and then sealed and exposed to
60
bial features, we hypothesize that DMAEMA- Co c-source (Gammabeam 651 PT, MDS Nordion
functionalized gauzes may have an impact on the International, Ottawa, Canada) 40,000 Ci, at dose rates
hemostasis and the activity of wound proteolytic of 6.5 and 14.5 kGy/h and irradiation exposure doses
enzymes. Ethylamino-ether cellulose derivatives have ranging from 1 to 25 kGy. DMAEMA homopolymer
been shown to promote clotting in hemorrhagic wounds and residual monomer were extracted from the func-
(Edwards et al. 2009). On the other hand, gauzes tionalized gauzes by immersion in ethanol for 24 h,
containing cadexomer iodine (a water-soluble modified exchanging the medium three times. Finally, the gauzes
dextrin with 0.9 % iodine) have been shown to inacti- were dried under vacuum at 40 °C for 24 h. The
vate proteolytic enzymes (Shi et al. 2010). Conversely, grafting percentage (Yg) in the cotton-g-DMAEMA
gauzes impregnated with iodoform exhibit fibrinolytic gauzes was calculated as follows:
activity and remove necrotic tissue from pressure ulcer  
ðW2  W1 Þ
wounds, with the advantage of being more stable than Yg ð%Þ ¼ 100  ð1Þ
W1
enzyme-loaded dressings (Mizokami et al. 2012).
Therefore, functionalization of gauzes with DMAEMA In this equation, W1 and W2 represent the weight of
and subsequent quaternization with MeI may have a the gauze before and after grafting, respectively.
strong impact on the proteolytic activity during healing.
The information gathered in this study may help to Quaternization of the grafted cotton gauzes
identify suitable functionalized gauzes able to modulate
events in the wound, namely proteolytic activity, Cotton-g-DMAEMA gauzes were immersed in MeI
hemorrhage hemostasis, and microbial growth, in a solution (10 % w/v in THF) and kept for 4 h under
cost-effective way. stirring at room temperature. Then, the solution was
removed and the unreacted MeI was extracted from
the quaternized samples by immersion in diethyl
Experimental ketone for 24 h. The gauzes were washed with
bidistilled water and dried under vacuum to constant
Materials weight. The degree of quaternization was estimated
as
DMAEMA was from Aldrich Chemical Co. (St. Louis,  
ðW3  W2 Þ
MO, USA) and distilled under reduced pressure DQ ð%Þ ¼ 100 
½ðW2  W1 Þð157:22=141:94Þ
before use. Commercial cotton fiber gauzes (sterilized
100 % cotton) were from Dimacu S.A. (D.F. Mexico). ð2Þ

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3770 Cellulose (2014) 21:3767–3779

Scheme 1 Reaction mechanisms of grafting of DMAEMA onto cotton gauze

where W1 and W2 mean the same as in Eq. 1, W3 collagenase (1 mL, 250 lg/mL in tricine buffer) and
represents the weight of the quaternized gauze, and incubated at 37 °C. Samples of collagenase medium
157.22 and 141.94 are the molecular weights of (100 lL) were taken after 30 min and 12 h. The
DMAEMA and MeI, respectively. samples were immediately transferred to 96-well
microplates and mixed with FALGPA solution
Characterization of the grafted gauzes (5 mM, 150 lL in tricine buffer) (Shi et al. 2010).
The reaction was monitored for 30 min by recording
FTIR-ATR spectra of cotton gauze and cotton-g- the absorbance at 350 nm (BP 10 nm) in a FLUOstar
DMAEMA before and after quaternization were Optima microplate reader (BMG Labtech, Ortenberg,
recorded using a Perkin-Elmer Spectrum 100 (Perkin Germany). Collagenase solutions (controls without
Elmer Cetus Instruments, Norwalk, CT, USA) with 16 gauze) kept at 20 and 37 °C were processed similarly.
sample scans. Thermal decomposition was monitored All experiments were carried out in triplicate for each
in nitrogen atmosphere between 25 and 700 °C at a data point and temperature.
heating rate of 10 °C/min using a TGA Q50 (TA
Instruments, New Castle, DE, USA). The samples for Blood absorption
TGA analysis were previously dried during 24 h at
40 °C. Scanning electron microscopy (SEM) micro- Pieces of pristine, DMAEMA-grafted and quaternized
graphs of freeze-dried gauzes (Genesis 25ES pilot DMAEMA-grafted gauzes (ca. 30 mg) were placed, in
lyophilizer, VirTis, UK) were taken using a LEO triplicate, in EppendorfÒ LoBind tubes containing human
435VP scanning electron microscope (Leica, Cam- blood [2 mL pre-treated with citrate phosphate dextrose
bridge, UK) at various magnifications. These exper- (CPD), anticoagulant solution 14 % v/v; healthy volun-
iments were carried out in duplicate. teers; Centro de Transfusión de Galicia, Spain]. The
systems were incubated at room temperature and the
Collagenase activity assay weight of the gauzes was recorded at 30 min and 3 h. To
do that, the gauzes were transferred to 5 mL syringe and
Pieces of pristine, DMAEMA-grafted and quaternized squeezed to remove unbound blood. Absorption factor
DMAEMA-grafted gauzes (30–40 mg) were directly was quantified gravimetrically as the amount of blood
placed in EppendorfÒ LoBind tubes containing absorbed per weight unit of the gauze.

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Cellulose (2014) 21:3767–3779 3771

Biofilm inhibition and eradication assays Results and discussion

The biofilm inhibiting and eradicating activity of Grafting of DMAEMA


the gauzes was evaluated as previously described
(Brackman et al. 2013). In brief, an overnight Gauzes grafted with DMAEMA (cotton-g-DMA-
culture of Staphylococcus aureus Mu50 was pel- EMA) were prepared by placing pieces of cotton
leted, washed, resuspended in physiological saline gauzes in glass ampoules containing a DMAEMA/
(PS) and diluted to 106 CFU/mL. Medical grade MeOH solution (direct method; Scheme 2). The
silicone disks (Q7-4735, Dow Corning, Midland, irradiation doses ranged from 1 to 25 kGy at two
MI, USA) were placed in the wells of a 24-well different dose rates. The efficiency of the grafting
microtiter plate (SPL life sciences, Novolab, Ger- polymerization was gravimetrically measured after
aardsbergen, Belgium) and 10 lL aliquots of the extensive washing with ethanol and drying of the
cell suspension were spotted on each disk. About grafted cotton gauzes. The dependence of the grafting
1 mL of Bolton Broth with 50 % plasma (Sigma- percentage on monomer concentration was firstly
Aldrich, St. Louis, MO, USA), 10 U/mL heparine examined at a fixed radiation dose (Fig. 1). The
and 5 % freeze–thaw laked horse blood was added. grafting percentage increased from 20 to 70 % w/w as
To evaluate inhibition of biofilm formation, 1 cm2 the concentration of DMAEMA in the reaction
gauzes were placed over each silicone disk imme- solution increased from 5 to 50 % v/v. Homopolymer
diately after inoculation. To assess the biofilm formation was promoted as monomer concentration
eradicating activity, the plate was first incubated for increased. The increase in grafting percentage as a
24 h at 37 °C to allow biofilm formation on the function of the monomer concentration was due to the
silicone disks, after which the medium was availability of more molecules of monomer for
removed, the biofilms were washed with PS, fresh reaction with the radicals generated on the backbone
medium was added, the gauzes were placed on top of cotton gauze. Nevertheless, as more monomer
of the biofilm and the plates were incubated at radicals were generated in the bulk, homo-polymer-
37 °C for another 24 h. After this, the gauzes were ization was equally favored.
removed and placed in 10 mL PS, and biofilms In the direct grafting method, the number of grafted
were rinsed once with PS. The silicone disks chains and their length depend on the total radiation
containing the biofilm cells were also placed in a dose and the dose rate. While the total exposure dose
separate falcon containing 10 mL PS. The sessile determines the number of free radicals generated on
cells present on the silicone disks and on the the cotton substrate, the dose rate determines the rate
gauzes were removed from the disks/gauzes apply- of initiation of polymerization (Bucio et al. 2002). In
ing three cycles of vortexing (30 s) and sonication the present case, the grafting augmented with the dose
(30 s; Branson 3510; Branson Ultrasonics Corp., of radiation more rapidly in the low dose range. As
Danbury, CT, USA) and the number of CFU/ shown in the Online Resource (Figure S1), the grafting
biofilm or CFU/cm2 gauze was determined by percentages obtained when the process was carried out
plating the resulting suspensions. Three samples of in DMAEMA 50 % v/v solution in methanol, with
each gauze were analyzed in the biofilm inhibition irradiation dose from 1 to 20 kGy, and dose rate of 6.5
assay. Pristine, DMAEMA-modified (76.7 % graft) and 14.5 kGy/h. The smaller increase in grafting
and quaternized DMAEMA-modified (74.0 % graft) extent at doses above 10 kGy may be due to either
gauzes were analyzed in the biofilm eradication monomer exhaustion or viscosity increase in the
assay. reaction solution, which restricts the monomer diffu-
sion during the propagation step of the grafted chains.
Statistical analysis The gauzes irradiated at a dose rate of 6.5 kGy/h
showed higher grafting percentages because low dose
The data were analyzed by means of a one-way rate favors the monomer diffusion (due to little
ANOVA and Dunnet test. Statistical analysis and homopolymer formation) towards the active sites in
linear regression analysis were carried out using SPSS the cotton gauze backbone. The grafting percentages
software, version 21.0 (SPSS, Chicago, IL, USA). of gauzes irradiated with an exposure dose of 20 kGy

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3772 Cellulose (2014) 21:3767–3779

Scheme 2 Schematic representation of preparation of cotton-g-DMAEMA and subsequent quaternization with methyl iodide

80
80
70

60
60
Graft (%)

50

Graft (%)
40
40

30
20

20

0 10 20 30 40 50 0
Monomer concentration (% v/v) 0 5 10 15 20 25

Dose (kGy)
Fig. 1 Effect of monomer concentration on grafting percentage
of cotton-g-DMAEMA. Direct method, dose 10 kGy, and dose Fig. 2 Effect of irradiation dose on grafting percentage of
rate 6.5 kGy/h cotton-g-DMAEMA, for 50 % (square) and 60 % (triangle)
monomer concentration in methanol. Direct method, dose rate
14.5 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 grafting percentage reached a plateau. It has previously
gauze because of minor interference of the homopol- been reported that an increase in monomer concentra-
ymer formation. Thus, lower grafting percentages tion usually leads to an amplification of the dose effect;
obtained at 14.5 kGy/h are explained by the produc- the polymerization rate increases at higher monomer
tion of more radicals per unit area of cotton, which concentration since more monomer units are available
favors recombination of free radicals generated in the for the propagation of polymerization. However, using
close vicinity, as well as by faster reaction of 50 and 60 % v/v monomer concentration similar
monomers in solution which leads to homopolymer grafting percentages were obtained, probably because
synthesis. of the large excess of monomer in the medium.
To gain an insight into the effect of the irradiation Cotton-g-DMAEMA gauzes covering a wide range
dose (in the 2.5–25 kGy range) on the grafting of grafting percentages were quaternized by means of
percentage, graft polymerization was carried out at a soaking in a MeI solution in THF. The DQ values were
fixed dose rate placing the gauzes in solutions of between 94 and 98 % disregarding the initial degree of
different monomer concentration (50 and 60 % v/v). grafting (Table 1). Therefore, the method applied led
The number of latent initiating sites was expected to to an almost complete quaternization of all amine
increase as the radiation dose became higher, although groups available in PDMAEMA chains. The quatern-
not necessarily in a linear manner. Up to 15 kGy, the ized cotton gauzes acquired a yellow-brownish colour
higher the irradiation dose, the greater the amount of of intensity proportional to the content of DMAEMA,
PDMAEMA grafted (Fig. 2). Above 15 kGy, the which is typical for iodide-containing products.

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Cellulose (2014) 21:3767–3779 3773

Table 1 Degree of quaternization of cotton-g-DMAEMA 3,000, 1,626, and 950 cm-1 associated to –NR3?
gauzes treated with MeI groups in the quaternized cotton-g-DMAEMA gauze
Material Grafting Quaternization confirmed methylation of DMAEMA amine groups.
percentage degree
Thermogravimetric analysis
cotton-g-DMAEMA 24 98 (1)
50 94 (4)
Pristine cotton gauze exhibited a single degradation
75 97 (2)
step in the 290–400 °C interval, and a char residue of
80 95 (1)
13 % at 450 °C (Online Resource, Figure S2), as
Mean values and, in parenthesis, standard deviations (n = 3) 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
Fig. 3 FTIR-ATR of pristine cotton gauze (a), PDMAEMA subsequent quaternization could have caused in the
(b), cotton-g-DMAEMA (c), and quaternized cotton-g-DMA- texture or conformation of the cotton fibers (Fig. 4).
EMA (d) 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
Infrared spectroscopy thickness of single fibers after grafting was similar to
that of unmodified cotton gauze, and untangled fibers
The FTIR-ATR spectra confirmed that the graft also appeared in the cotton-g-DMAEMA gauze.
polymerization of DMAEMA was successful Quaternization of the amine groups of DMAEMA
(Fig. 3). The spectrum of the pristine gauze showed did not cause further changes in the structure of the
the absorption bands of –OH groups at 3,343 cm-1, fabric.
methyl and methylene groups at 2,899 cm-1, and C–O
bonds at 1,024 cm-1. In the PDMAEMA homopoly- Collagenase activity
mer spectrum, peaks assigned to methyl and methylene
groups at 2,930 and 2,821 cm-1, carbonyl group at The effect of the gauzes on the activity of crude
1,721 cm-1, –N–CH2– bond at 2,767 and 1,149 cm-1, collagenase after direct incubation at 37 °C for 30 min
and –OC–O group at 1,239 and 1,099 cm-1 were or 12 h was compared to that of control collagenase
observed. The spectra of cotton-g-DMAEMA gauze solutions stored at 20 and 37 °C (Fig. 5). Crude
exhibited the characteristic bands of DMAEMA collagenase from C. histolyticum is a mixture of
(–C=O at 1,721 cm-1, –N–CH2– at 2,769 and proteases, mainly two collagenases, clostripain and a
1,149 cm-1, and –OC–O at 1,239 cm-1). Peaks at neutral protease. Collagenase solution was prepared in

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3774 Cellulose (2014) 21:3767–3779

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 In contrast, quaternized gauzes strongly inhibited
enzymatic activity. No differences were observed collagenase activity, reducing the activity to 34 % (SD
between the activity of collagenase solely solutions 5) and to 7.5 % (SD 2.9) when incubated for 30 min
stored at 20 or 37 °C. Pristine gauzes did not modify and 12 h, respectively. These later results are in
the enzyme activity in the first 30 min, but decreased it agreement with those reported for Iodoflex and
to the half after 12 h incubation at 37 °C, while cotton- Iodosorb-medicated dressings, which consist of mac-
g-DMAEMA gauzes increased collagenase activity up rogol ointments containing cadexomer iodine (Shi
to 171 % (SD 2) after 30 min incubation and up to et al. 2010). It has previously been observed that
146 % (SD 1) after 12 h. Such an increase in iodine inhibits the activity of proteases in wounds
enzymatic activity can be related to the increase in more efficiently than silver, because the stronger
hydrophilicity and in positive charges of the gauzes oxidant character of the former causes denaturation
after grafting of DMAEMA, which in turn prevents and inactivation of enzymes (Eming et al. 2006).
non-specific adsorption of the enzyme also positively Previous studies focused on extracts of the semisolid
charged at physiological pH (Edwards and Howley dressings (Shi et al. 2010). In our study the gauzes
2007). were directly placed into contact with collagenase

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

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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%

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Cellulose (2014) 21:3767–3779 3777

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

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