S. POURANVARI et al.: CHEMICAL CROSS-LINKING OF CHITOSAN/POLYVINYL ALCOHOL ...
663–666
CHEMICAL CROSS-LINKING OF CHITOSAN/POLYVINYL
ALCOHOL ELECTROSPUN NANOFIBERS
KEMIJSKO ZAMRE@ENJE ELEKTRO SPREDENIH NANOVLAKEN
IZ HITOSAN/POLIVINIL ALKOHOLA
Sara Pouranvari1, Firouz Ebrahimi2, Gholamreza Javadi1, Bozorgmehr Maddah3
1Islamic Azad University, Department of Biology, Science and Research Branch, Tehran, Iran
2IHU, Basic Sciences Faculty, Biology Research Center, Tehran, Iran
3IHU, Basic Sciences Faculty, Department of Chemistry, Tehran, Iran
febrhimi@ihu.ac.ir
Prejem rokopisa – received: 2015-04-17; sprejem za objavo – accepted for publication: 2015-07-08
doi:10.17222/mit.2015.083
Electrospun nanofibrous scaffolds have great potential for many biomedical applications. In the present study, we fabricated and
characterized chitosan/polyvinyl alcohol (Chi/PVA) nanofibrous scaffolds through electrospinning. Cross-linking was per-
formed using chemically with 5 % glutaraldehyde vapor. The morphology and chemical banding of the electrospun nanofibers
before and after cross-linking were evaluated using scanning electron microscopy (SEM) and Attenuated Total Reflectance-
Fourier Transform InfraRed (ATR-FTIR) spectroscopy. SEM micrographs and FTIR spectra showed that the cross-linking
process was accomplished successfully. With the biocompatibility and non-toxicity of chitosan and PVA, it is expected that this
electrospun nanofibrous scaffold could be an excellent candidate for biomedical applications.
Keywords: electrospinning, chitosan, polyvinyl alcohol, cross-linking
Mre`e iz elektro spredenih nanovlaken imajo velik potencial za uporabo v biomedicini. V {tudiji smo izdelali in karakterizirali
nanovlaknasto mre`o, izdelano z elektro predenjem nanovlaken iz hitosan/polivinil alkohola (Chi/PVA). Zamre`enje je bilo
izdelano s pomo~jo kemijske metode s 5 % glutaraldehidne pare. Morfologija in kemijsko povezovanje elektro predenih
nanovlaken, pred in po zamre`enju, sta bila ocenjena z uporabo vrsti~nega elektronskega mikroskopa (SEM) in z metodo z
oslabljenim odbojem infrarde~e spektroskopije s Fourierjevo transformacijo (ATR-FTIR). SEM-posnetki in FTIR-spekter sta
pokazala, da je bil postopek zamre`enja uspe{no dose`en. Glede na biokompatibilnost in netoksi~nost hitosana in PVA se
pri~akuje, da bodo mre`e iz elektro spredenih nanovlaken odli~en element za uporabo v biomedicini.
Klju~ne besede: elektro-predenje, hitosan, polivinil alkohol, zamre`enje
1 INTRODUCTION
Electrospinning is a simple, versatile and cost effec-
tive method for forming non-woven fibrous scaffolds.
Technically, the electrospinning process uses a high
voltage source to draw a polymer fluid into fine fibers
which are deposited on a collector.1 In recent years, the
use of electrospun nanofibers for biomedical applications
such as tissue engineering2, wound dressing3, protein
immobilization4, materials for artificial blood vessels5,
barriers for the prevention of induced adhesion after
operation6, and vehicles for drug or gene delivery7 has
attracted a great deal of attention from scientists. Elec-
trospinning of synthetic and natural polymers has been
reported for collagen8, gelatin9, silk fibroin10, polygly-
colide (PGA)11, polylactide (PLA)12 and poly(-capro-
lactone) (PCL)13, polyurethane14, poly(vinylalcohol)15,
PEO16, polydioxanone17, and polyphosphazene deriva-
tives.18 Furthermore, the blending of two or more
polymers and copolymerization are effective methods for
the preparation of composites with new and desirable
properties. Obviously, by adjusting the ratio of the
components, structure and morphology of the nanofibers
and the biological properties of the electrospun scaffolds
can be tailored to the desired traits and functions.1 For
example PLGA7, P(LA-CL) copolymers,19 and mixtures
of collagen with elastin,20 gelatin with PCL,9 chitosan
with poly(ethylene oxide) (PEO)21 and chitosan with
PVA22 have all been utilized to fabricate electrospun
nanofibrous scaffolds for biomedical applications.
In biomedical applications, after electrospinning,
different cross-linking methods can be uses to provide
stabilization against aqueous environments for those
scaffolds produced from aqueous soluble polymers (For
example: PVA).
In the present study, electrospinning of a chitosan and
PVA blend was performed. Chitosan was selected due to
its cytocompatibility, biocompatibility, biodegradability
and antibacterial activity.23 PVA was used due to its bio-
compatibility, biodegradability, non-toxicity, chemical
resistance, and good fiber-forming properties.24
2 MATERIALS AND METHODS
2.1 Materials
PVA (average molecular weight of 70000–100000
g/mol) and chitosan (medium molecular weight) were
purchased from Sigma-Aldrich (St. Louis, MO). Acetic
Materiali in tehnologije / Materials and technology 50 (2016) 5, 663–666 663
UDK 620.192.4:660.017:678.744.7 ISSN 1580-2949
Original scientific article/Izvirni znanstveni ~lanek MTAEC9, 50(5)663(2016)
acid and glutaraldehyde were obtained from Merck
(Germany).
2.2 Preparation of the solutions
Chitosan and PVA were dissolved in 50 % aqueous
solution of acetic acid at a concentration of 2 % mass
fraction and 15 % mass fraction, respectively. The
chitosan solution and PVA solution were mixed together
with a weight ratio of 40/60 (Chi/PVA) under magnetic
stirring at 60 °C.
2.3 Preparation of nanofibrous membranes
The optimal conditions for the electrospinning were
as follows: 25 kV applied voltage, 15 cm tip-to-collector
distance, and 1 ml/h flow rate. Moreover, a 5 ml syringe
with a 21 gauge stainless-steel needle was used for the
delivery of the polymer solution via a syringe pump.
2.4 Crosslinking of nanofibrous membranes
Samples were exposed to the 5 % glutaraldehyde
(GA) vapor at room temperature for 48 h for cross-link-
ing to stabilize them against aqueous media solubility
and enhance their biomechanical properties biomedical
applications. After crosslinking, the samples were care-
fully washed several times with 2 % glycine for the inac-
tivation and removal of the GA.25
2.5 Characterization of nanofibrous membranes
The morphology and microstructure of the electro-
spun nanofibers before and after cross-linking were
determined by Scanning Electron Microscopy (SEM).
The average diameter of fibers were calculated using the
ImageJ (US National Institute of Health, Bethesda, MD)
image analysis program by analyzing at least 50 fibers in
ten SEM micrographs. The chemical structures of the
chitosan and PVA powders and Chi/PVA nanofiber mem-
branes before and after cross-linking were investigated
by Attenuated Total Reflectance-Fourier Transform
InfraRed (ATR-FTIR) spectroscopy (Bruker Tensor 27,
USA). FTIR spectra were obtained in the 4000 cm–1 to
400 cm–1 wavenumber range, with the data analyzed
using OPUS software.
RESULTS AND DISCUSSION
3.1 Morphology of the nanofibrous scaffold
SEM images of Chi/PVA nanofibers before and after
GA cross-linking are shown in Figure 1. As seen in
Figure 1a, relatively fine, continuous, uniform fiber-
structures (no bead), and randomly oriented fibers were
obtained. The average fiber diameter was found to be
180±2.28 nm. In accordance with the relatively fine
fibers fabricated, it is expected that a suitable porosity
exists for biomedical applications.
3.2 Crosslinking of nanofibrous scaffold
The SEM micrographs of the cross-linked nanofibers
after immersion in water (at least 48 h) are shown in
Figure 1b. As PVA is a water-soluble polymer, cross-
linking should be performed for the use of Chi/PVA
nanofibers in biomedical applications. Several studies
have been reported on GA cross-linking for medical
application. For example, Jafari et al. used a saturated
vapor of a 25 % GA aqueous solution for cross-linking
of chitosan-gelatin electrospun nanofibers.26 In another
study cross-linking of electrospun water-soluble carbo-
S. POURANVARI et al.: CHEMICAL CROSS-LINKING OF CHITOSAN/POLYVINYL ALCOHOL ...
664 Materiali in tehnologije / Materials and technology 50 (2016) 5, 663–666
Figure 1: SEM micrographs of electrospun Chi/PVA nanofibers, after
48 h immersion in water at 37 °C: a) before GA cross-linking and b)
after GA cross-linking
Slika 1: SEM posnetek elektro spredenih Chi/PVA nanovlaken, po
48 h namakanja v vodi s 37 oC: a) pred GA zamre`enjem in b) po GA
zamre`enju
xyethyl chitosan/poly (vinyl alcohol) nanofibrous mem-
brane towards wound dressings for skin regeneration was
performed using a GA vapor.27 Unlike the previous work,
in the present study, the cross-linking was performed
using a lower concentration of GA vapor (5 %). Despite
using this low concentration, it was proved that the
fabricated membranes’ structure is stable in an aqueous
solution. Moreover, according to the SEM micrographs
shown in Figure 1, the porous structure of the fabricated
membranes remained intact implying that they are insol-
uble in water. The cross-linking mechanism of chitosan
and PVA with GA is shown in Figure 2.28,29
3.3 ATR-FTIR analysis
FTIR spectra were taken of the electrospun nano-
fibers before and after cross-linking, to assess their
chemical groups. The FTIR spectrum of the Chi/PVA
blended nanofibers before cross-linking, is shown in
Figure 3. The two peaks at 1423 cm–1 and 1565 cm–1
arise fromcarboxylic acid and symmetric deformation of
–NH3+ groups due to ionization of primary amino groups
in the acidic medium, respectively. The peak at 1703 cm–1
is attributed to the carboxylic acid dimer.22 In this study,
this peak is due to the acetic acid utilized for dissolving
the chitosan. The peak located at 1244 cm–1 is related to
the C–O of the CH2OH chitosan group forming a hydro-
gen bond with the OH of PVA, confirming the fabrica-
tion of Chi/PVA blend nanofibers.30 The FTIR spectra of
the Chi/PVA blended nanofibers before cross-linking is
given in Figure 3. Chemical crosslinking of the chito-
san/PVA is verified by the peak located at 1586 cm–1
attributed to the C-N band. All chitosan-derived blends
cross-linked with GA, have shown the presence of the
imine (C=N) band. The imine band was formed by the
nucleophilic reaction of the amine from chitosan with the
aldehyde group of GA.31 Due to imine band instability
with temperature and pH, this group can transform to a
C-N group.32
4 CONCLUSION
In this work, nanofibrous Chi/PVA was fabricated via
electrospinning and stabilized by chemical cross-linking
using 5 % GA. The porous structure of the electrospun
scaffolds, antimicrobial properties of the chitosan and
chemical resistant traits of PVA, make our fabricated
electrospun scaffold an excellent candidate for biomedi-
cal applications. However, in vitro and in vivo experi-
ments for evaluation of the biocompatibility of these
Chi/PVA nanofiberous membranes is necessary.
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S. POURANVARI et al.: CHEMICAL CROSS-LINKING OF CHITOSAN/POLYVINYL ALCOHOL ...
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666 Materiali in tehnologije / Materials and technology 50 (2016) 5, 663–666
S. POURANVARI et al.: CHEMICAL CROSS-LINKING OF CHITOSAN/POLYVINYL ALCOHOL ...