Electrospun Non-Woven
Mats Based on Chitosan
Dressing Applications
N
Introduction
The process of wound healing is a very complex one and is
affected by a number of factors. Bacteria infection is one of
the factors that exert a significant effect on the healing
process and in some cases even hamperwound healing.[1,2]
One of the approaches for treating the wound infection is
the use of antibacterial agents having a broad-spectrum of
activity and high kill rates. It is well known that natural
polysaccharide chitosan and its quaternized derivatives
possess high intrinsic activity against bacteria and fungi,
structure similar to glycosaminoglycans in the extracel-
lular matrices and possesses haemostatic activity. All
these advantages of (quaternized) chitosan-containing
materials might contribute to faster wound healing
processes, making them good candidates for wound-
dressing applications.
Electrospinning is an attractive approach for the
fabrication of fibers with diameters ranging from a few
nanometers to several micrometers by creating an
electrically charged jet of polymer solution or melt. The
unique properties of electrospun mats – high specific
surface area and small pores are very favorable for the
.
Recently two approaches for preparation of nanofibrous
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hybrid mats were very effective in suppressing the adhesion
of pathogenic bacteria S. aureus. The hybrid nanofibers areelectrospunmats suitable forwound-dressing applications
have been developed – immobilization of drugs or
antibacterial agents in the nanofibers by electrospin
ning[7–14] and electrospinning of polymers with intrinsic
antibacterial and wound-healing properties.[15–19] Nano-
fibrous mats based on chitosan have been prepared
successfully by electrospinning of mixed aqueous solutions
of chitosan/polyoxyethylene,[20,21] chitosan/poly(viny
M. Ignatova, N. Manolova, I. Rashkov
Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian
Academy of Sciences, Acad. G. Bonchev 103A, 1113 Sofia, Bulgaria
Fax: þ359 2 870 0309; E-mail: rashkov@polymer.bas.bg
N. Markova
Institute ofMicrobiology, Bulgarian Academy of Sciences, Acad. G.
Bonchev 26, 1113 Sofia, Bulgarialow toxicity, biodegradability and ability to affect macro-
phage function.[3–6] In addition, chitosan has a chemical
adsorption of liquids and for preventing bacteria penetra-
tion and thus provide good conditions for wound healingpromising for wound-healing applications.Milena Ignatova, Nevena Manolova,
Continuous defect-free nanofibers containin
were successfully prepared by one-step elect
poly[(L-lactide)-co-(D,L-lactide)] in common so
position of the bicomponent electrospun ma
Ch- and QCh-containing nanofibers exhibit
rates against bacteria S. aureus and E. coli t
sponding solvent-cast films. SEM observationMacromol. Biosci. 2008, 8, 000–000

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Early View Publication; these are NOT theNanofibrous Hybrid
and PLA for Wound-
adya Markova, Iliya Rashkov*
chitosan (Ch) or quaternized chitosan (QCh)
pinning of Ch or QCh solutions mixed with
nt. XPS revealed the surface chemical com-
Crosslinked
higher kill
n the corre-
howed thatDOI: 10.1002/mabi.200800189 1
final page numbers, use DOI for citation !! R

polyesters have been successfully prepared by a number
of groups.[27–32] Poly[(L-lactide)-co-(D,L-lactide)] (PLA) is an
(DCM) (Fluka) were of analytical grade of purity and were used as
received. PLA (Resomer1 LR 708 with Mw ¼911000 g mol1, Mw/
Mn ¼2.46, molar ratio L-lactide:D,L-lactide¼69:31) was kindly
provided as a gift from Boehringer-Ingelheim Chemicals Inc
(Germany). Chitosan with molecular weight 380000 g mol1
(Aldrich) with degree of deacetylation 80% was used.
M. Ignatova, N. Manolova, N. Markova, I. Rashkov
2
REaexample of biodegradable and biocompatible aliphatic
polyesters with relatively high strength and an appro-
priate degradation rate for most musculoskeletal applica-
tions. It has been extensively studied for biomedical
applications including surgical sutures, substrates for
tissue regeneration and carriers for drug and gene delivery.
Combination of the advantageous properties of polyesters
and the antibacterial properties of chitosan or its
derivatives is a promising strategy for the preparation
of novel nanofibrous materials suitable for wound-
dressing applications. One of the approaches is surface
coating. Recently, we have proposed one approach to
prepare such materials and poly(L-lactide) and bicompo-
nent poly(L-lactide)/poly(ethylene glycol) electrospun
mats were coated with thin chitosan film.[33]
In the present study a one-step method – electrospin-
ning in common solvent – has been used for the
preparation of novel bicomponent hybrid nanofibrous
materials based on natural polysaccharide chitosan or its
quaternized derivative and synthetic aliphatic polyester
PLA. Further, water-resistant hybrid nanofibers have been
obtained by crosslinking of the nanofibrous mats with
glutaraldehyde vapor. The antibacterial activity of the
hybrid non-wovenmats has been evaluated by both Gram-
positive bacteria Staphylococcus aureus (S. aureus) and
Gram-negative bacteria Escherichia coli (E. coli). The
adhesion of pathogenic bacteria S. aureus onto the surface
of electrospun mats has also been studied as a criterion for
their adequacy as wound dressings.
Experimental Part
Materials
Formaldehyde solution (
36% in water), NaBH4, CH3I, NaI and
glutaraldehyde solution (
50% in water) were purchased from
Fluka. The chemicals were with analytical grade of purity and
were used without further purification. Prior to use, N-methyl-2-
pyrrolidone (NMP) (Fluka) was freshly distilled under reduced
pressure. Dimethylformamide (DMF) (Fluka) and dimethylsulf-
oxide (DMSO) (Fluka) were dried with molecular sieves (4 A˚) prioralcohol)[22,23] and chitosan/silk fibroin,[24] or by electro-
spinning of chitosan solutions in trifluoroacetic acid,[22,25]
trifluoroacetic acid/dichloromethane mixture[22] or con-
centrated acetic acid.[26] Recently, we have reported on the
preparation of quaternized chitosan containingmicro- and
nanofibers by electrospinning of quaternized chitosan in
the presence of poly(vinyl alcohol) or poly(vinyl pyrroli-
done)[15,16] The electrospun fibers exhibited good anti-
bacterial activity.
Electrospun nonwovens of biodegradable aliphaticto use. Trifluoroacetic acid (TFA, Aldrich) and dichloromethane
Macromol. Biosci. 2008, 8, 000–000

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
rly View Publication; these are NOT the finaPreparation and Characterization of
N,N,N-Trimethyl Chitosan Iodide
The quaternized chitosan derivative (QCh, Scheme 1) N,N,N-
trimethyl chitosan iodide was prepared according to known
procedure.[5] Briefly, on the first step N-methyl chitosan was
prepared by reacting chitosan and formaldehyde via Schiff’s base
intermediate and subsequent hydrogenation with NaBH4. The
product was purified from unreacted aldehyde and inorganic
products by Soxhlet-extraction with ethanol and diethyl ether for
4 d. The obtainedN-methyl chitosanwas filtered out and vacuum-
dried at 40 8C for 2 d. Yield: 94%. The resulting N-methyl chitosan
was quaternized using methyl iodide. N,N,N-Trimethyl chitosan
iodide was purified by twice precipitating in acetone and dried
under reduced pressure at 40 8C. Yield: 96%.
Fourier-transform infrared (FT-IR) spectra of QCh (films) were
recorded on a Bruker Vector 22 infrared spectrometer at room
temperature on KBr pellets. 1H NMR spectra were taken on a
Bruker spectrometer (400.13 MHz) at 333 K in D2O. The degree of
quaternization of QChwas determined by the 1HNMRdata and by
potentiometric titration of the iodide form with aqueous silver
nitrate, using working silver electrode and reference calomel
electrode.
IR (film): n¼ 3 417 (N–H stretching vibration), 2 924, 2 879 (C–H
stretching vibration from N,N,N-trimethyl residues and from
polysaccharide structure), 1 654 (amide I), 1 561 (amide II), 1 470
(antisymmetric C–H deformation of the N,N,N-trimethyl group),
1 376 (symmetric C–H deformation), and 1 068 cm1 (C–O–C
stretching vibration) cm1.
1H NMR (D2O): d¼2.07 (s, –NHCOCH3), 3.12 (s, –Nþ–(CH3)3I),
3.38 (s, CH3–O
6), 3.51 (s, CH3–O
3), 3.84 (m, H2 and H6), 4.0 (m, H5),
4.18 (m, H3), 4.33 (m, H4), and 5.21 (m, H1).
The quaternization degree of QCh was determined using two
independent methods: 1H NMR spectroscopy and potentiometric
titration of the iodide form with aqueous silver nitrate. The
quaternization degree was calculated from the intensity ratio of
the signal at 3.12 ppm for –Nþ–(CH3)3I
 to that of H1. This value
(70%) is in very good agreement with the quaternization degree
determined potentiometrically (72%).
The degree of methylation of –OH functions was determined
from the intensity ratios of the signal of CH3–O at 3.51 and
3.38 ppm, for OH at C3 and C6 position, respectively, and the H1
signal. The degree of methylation was 100% for both H3 and H6.Scheme 1. Quaternized chitosan (QCh) chain sequence.
DOI: 10.1002/mabi.200800189
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