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DOI 10.1007/s10570-014-0371-7
ORIGINAL PAPER
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.
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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
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
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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
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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.
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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
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)
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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)
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
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.
Conclusions References
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efficiency of copolymerization to prepare cotton-g- K, Carr W, McNamee G, McKeague A, Govindaraj K,
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