New antimicrobial chitosan derivatives for wound dressing applications

Article abstract of DOI:10.1016/j.carbpol.2015.12.078

Chitosan is a non-toxic, biocompatible, biodegradable natural cationic polymer known for its low imunogenicity, antimicrobial, antioxidant effects and wound-healing activity. To improve its therapeutic potential, new chitosan-sulfonamide derivatives have

Full text of DOI:10.1016/j.carbpol.2015.12.078

Accepted Manuscript
Title: New antimicrobial chitosan derivatives for wound
dressing applications
Author: Oana Maria Dragostin Sangram Keshari Samal
ˆ
Mamoni Dash Florentina Lupascu Andreea Panzariu Cristina
Tuchilus Nicolae Ghetu Mihai Danciu Peter Dubruel Dragos
Pieptu Cornelia Vasile Rodica Tatia Lenuta Profire
PII:
S0144-8617(15)01256-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.12.078
CARP 10676
To appear in:
Received date:
Revised date:
Accepted date:
13-5-2015
28-12-2015
30-12-2015
Please cite this article as: Dragostin, O. M., Samal, S. K., Dash, M.,
ˆ
Lupascu, F., Panzariu, A., Tuchilus, C., Ghetu, N., Danciu, M., Dubruel, P.,
Pieptu, D., Vasile, C., Tatia, R., and Profire, L.,New antimicrobial chitosan
derivatives for wound dressing applications, Carbohydrate Polymers (2016),
http://dx.doi.org/10.1016/j.carbpol.2015.12.078
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New antimicrobial chitosan derivatives for wound dressing applications
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Oana Maria Dragostina, Sangram Keshari Samalb, Mamoni Dashc, Florentina Lupascua,
Andreea Pânzariua, Cristina Tuchilusd, Nicolae Ghetue, Mihai Danciuf, Peter Dubruelc, Dragos
Pieptue, Cornelia Vasileg, Rodica Tatiah, Lenuta Profirea,*
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aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Medicine and
Pharmacy “Grigore T. Popa” Iasi, 16 University Street, 700115, Iasi, Romania
bLaboratory of General Biochemistry and Physical Pharmacy, Centre for Nano- and
Biophotonics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
cPolymer Chemistry & Biomaterials Research Group, Ghent University, Krijgslaan 281, S4-
Bis, B-9000 Ghent, Belgium
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dDepartment of Microbiology, Faculty of Medicine, University of Medicine and Pharmacy
“Grigore T. Popa” Iasi, 16 University Street, 700115, Iasi, Romania
eDepartment of Plastic Surgery, Faculty of Medicine, University of Medicine and Pharmacy
“Grigore T. Popa” Iasi, 16 University Street, 700115, Iasi, Romania
fDepartment of Morphopathology, Faculty of Medicine, University of Medicine and
Pharmacy “Grigore T. Popa” Iasi, 16 University Street, 700115, Iasi, Romania
gDepartment of Physical Chemistry of Polymers, “Petru Poni” Institute of Macromolecular
Chemistry, 41A Grigore Ghica Voda Alley, 700487, Iasi, Romania
hNational Institute of Research and Development for Biological Sciences, 296 Splaiul
Independentei, 060031, Bucharest, Romania
*Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Medicine and Pharmacy “Grigore T. Popa” Iasi, 16 University Street, 700115,
Iasi, Romania. E-mail: lenuta.profire@umfiasi.ro. Phone: +40 232 301 623. Fax: +40 232 211
820.
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27 Abstract
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Chitosan is a non-toxic, biocompatible, biodegradable natural cationic polymer known for its
low imunogenicity, antimicrobial, antioxidant effects and wound-healing activity. To improve
its therapeutic potential, new chitosan-sulfonamide derivatives have been designed to develop
new wound dressing biomaterials. The structural, morphological and physico-chemical
properties of synthesized chitosan derivatives were analyzed by FT-IR, 1H-NMR
spectroscopy, scanning electron microscopy, swelling ability and porosity. Antimicrobial, in
vivo testing and biodegradation behavior have been also performed. The chitosan derivative
membranes showed improved swelling and biodegradation rate, which are important
characteristics required for the wound healing process. The antimicrobial assay evidenced that
chitosan-based sulfadiazine, sulfadimethoxine and sulfamethoxazole derivatives were the
most active. The MTT assay showed that some of chitosan derivatives are nontoxic.
Furthermore, the in vivo study on burn wound model induced in Wistar rats demonstrated an
improved healing effect and enhanced epithelialization of chitosan-sulfonamide derivatives
compared to neat chitosan. The obtained results strongly recommend the use of some of the
newly developed chitosan derivatives as antimicrobial wound dressing biomaterials.
KEYWORDS: chitosan derivative, membrane, biomaterial, healing activity
Chemical compounds studied in this article:
Chitosan (PubChem CID: 21896651); Sulfadiazine (PubChem CID: 5215);
Sulfamethoxydiazine (PubChem CID: 5326); Sulfamerazine (PubChem CID: 5325);
Sulfadimethoxine (PubChem CID: 5323); Sulfisoxazole (PubChem CID:5344);
Sulfamethoxazole (PubChem CID:5329); Chloroacetyl chloride (PubChem CID: 6577);
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Acetic acid (PubChem CID: 176); Sodium hydroxide (PubChem CID: 14798); Sodium
tripolyphosphate (PubChem CID: 24455).
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54 Abbreviations used
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ANOVA, analysis of variance; CS MMW, chitosan with medium molecular weight; DA,
degree of acetylation; DS, degree of substitution; DMFA, dimethylformamide; FT-IR, Fourier
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transform infrared spectroscopy; H-NMR, Proton Nuclear Magnetic Resonance; MSR,
membrane swelling ratio; PBS, phosphate buffered saline; SEM, scanning electron
microscope; TLC, layer chromatography; TPP, sodium tripolyphosphate.
61 1. Introduction
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The wound is a type of injury in which dermis of the skin is damaged by burn, trauma, and
cut. Often a serious wound can cause death and should be treated with specialized wound
dressing materials. Although there is a high demand for wound dressing materials, wound
dressing technology is still far behind, due to the intrinsic complexity of the wound healing
process. Ideal wound dressing materials need to have good tissue conformity, easy
application, moist environment, low risk of infection, proper removal of exudates and
accelerated tissue healing rate. There are various types of wound dressing biomaterials that
are commercially available, generally manufactured from natural or synthetic polymers or a
combination of both. In recent years, porous membranes are considered as the best wound
dressing materials since they provide a moist and occlusive environment, which significantly
facilitates the wound healing process (Willi & Chandra, 2004). However, the moist
conditions, provided by membranes, are equally necessary for pathogen proliferation and
colonization. The impregnation of the wound dressing material with an antimicrobial agent is
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also highly preferred (Jones, Grey & Harding, 2006; Sudheesh Kumar et al., 2012; Mallick et
al., 2012).
Another desirable function of the wound dressing is to accelerate hemostasis and tissue
regeneration. A cationic dressing is known to fulfill this requirement. Chitosan is a naturally
derived cationic polysaccharide consisting of N-acetyl glucosamine and D-glucosamine
sugars (Yang et al., 2008; Batista, Pinto, Gomes & Gomes, 2006). During chitosan
biodegradation, it produces N-acetyl glucosamine, which is used to accelerate the re-
epithelialization process. Its non-toxicity, versatile biological activities as antimicrobial, low
immunogenicity, wound-healing activity, antioxidant properties, and low cost have provided
ample opportunities for further development of biomaterials for various therapeutic
applications. Due to its polycationic nature the chitosan inhibits the growth of a wide variety
of bacteria and fungi, showing a broad spectrum of antibacterial activity, high killing rate
against bacteria, and low toxicity toward mammalian cells (Xie, Liu & Chen, 2007). Chitosan
and its derivatives received increasing attention in diverse areas such as food preservation
(Aider, 2010), water purification (Kołodyńska, 2012; Wang, Chen, Yuan, Sheng & Yu, 2009;
Gupta, Chauhan & Sankararamakrishnan, 2009), paper industry (Dutta, Ravikumar & Dutta,
2002), pharmacy or medicine (Dash et al., 2011; Gallaher et al., 2000; Xie et al., 2007; Ma et
al., 2008; Pillai, Willi & Chandra, 2009; Yang, Chou & Li, 2005; Li et al., 2012; Dias et al.,
2013; Öztürk, Agalar, Keçeci & Denkbas, 2006; Xing, Lie, Zhengwei & Changyou, 2011).
Chitosan possesses three reactive sites including a primary amine and two primary or
secondary hydroxyl groups per glucosamine unit readily subjected to chemical modification
(Okamoto et al., 2003; Samal et al., 2012). The structural characteristics of chitosan mimic
glycosaminoglycan components of the extracellular matrix, while the biocompatibility,
biodegradability, antibacterial (Samal et al., 2014), antioxidant activities and mucoadhesive
properties impart versatility. Utilization of chitosan in biomedical applications is limited due
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to its low solubility at physiological pH of 7.4, althoughit is soluble in acidic aqueous media.
Moreover, the antimicrobial effects of chitosan are improved in acidic conditions due the
interactions between protonated amino groups of chitosan and anionic components of bacteria
(Martins et al., 2014). In order to overcome these limitations and enhance antimicrobial
properties, various chitosan derivatives have been designed (Yang et al., 2008). Many efforts
have been made to introduce hydrophilic groups by covalent attachment to reactive amino
groups at the C2 position. Various kinds of modification of chitosan have been investigated in
recent years using acylation, alkylation, carboxymethylation and quaternization (Ma et al.,
2008). Recently, the antibacterial and antifungal activities of chitosan have been followed
with great interest. Furthermore incorporation of antibacterial agents into the chitosan
backbone offers excellent antibacterial properties (Samal et al., 2014; Ignatova, Manolova &
Rashkov, 2013; Sashiwa, Yamamori, Ichinose, Sunamoto & Aiba, 2003).
On the other hand, sulfonamides and their different derivatives are extensively used in
medicine due to their pharmacological properties such as antibacterial activity (Kremer et al.,
2006; Zahid, 2009). Sulfonamide derivatives were successfully employed as effective
chemotherapeutic agents for the prevention and cure of bacterial infections in humans. These
drugs act on the bacteria and either prevent their growth (bacteriostatic effect) or act as
germicides (bactericides) and have no effect on the smooth muscles, heart, blood pressure or
respiration (Gomes & Gomes, 2005). Therefore, it is essential to have an ideal membrane
which can combine all the above mentioned features of chitosan and sulfonamides into one
single design, to address the different aspects of a dynamic wound dressing biomaterial.
Herein, we report on the preparation, physico-chemical characterization and biological
evaluation of the novel chitosan-sulfonamide derivatives as membranes which have all
theoretical premises to be useful in the therapy of wounds, focused on burn wounds. New
sulfonamide drug-functionalized chitosan membranes were proposed to reduce bacterial
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infection risks as well as promote re-epithelialization of damaged tissue and thus be effective
as wound dressing biomaterials.
128 2. Material and methods
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2.1. Materials
Chitosan with medium molecular weight (CS MMW, 425 kDa, deacetylation degree of
85%), sulfonamides (sulfamethoxydiazine, sulfadiazine, sulfamerazine, sulfadimethoxine,
sulfisoxazole and sulfamethoxazole), chloroacetyl chloride, acetic acid, sodium hydroxide,
sodium tripolyphosphate (TPP) and organic solvents (p.a.) were purchased from Sigma
Aldrich Company. All solvents and reagents were used without further purification. Thin
layer chromatography (TLC) plates (aluminum foil covered with 0.25 mm thick silica gel 60)
from Merck (VWR International) were used.
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2.2. Chemistry
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2.2.1. Synthesis of sulfonamide-chitosan derivatives (3a-f)
To a stirred solution of chitosan (11 mmol) in 1% acetic acid (100 mL), a solution of N-
chloroacetylsulfonamide derivative (2a-f) (13.2 mmol) in dimethylformamide (DMFA) (50
mL) was added. The reaction mixture was stirred for about 24 h at room temperature and then
the pH was adjusted to 9 with 15% NaOH solution, resulting in a precipitate (Batista, Pinto,
Gomes & Gomes, 2006; Xie, Liu & Chen, 2007; Feng & Xia, 2011). The products were
washed five times with double-distilled water until the pH of the filtrate was 7. The final
compounds were purified by dialysis against deionized water for 5 days and then freeze-dried
on Alpha 1-2 LD Plus Freeze Dryer.
Chitosan-aminoacetyl-sulfamethoxydiazine (3a). IR (KBr, cm-1): 1640 (C=O amide I), 1595
(NH amide II), 1543, 834 (phenyl), 1258, 1165 (SO2-N); 1H-NMR (300 MHz, CD3COOD, δ):
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2.32 (3H, COCH3); 2.32 (3H, OCH3); 3.47 (H-2 chitosan); 4.00-4.18 (H-3,4,5,6 chitosan);
5.15 (H-1 chitosan); 3.15-3.35 (NH), 7.03 (2CH, aromatic), 7.79 (2CH, aromatic), 8.09 (2CH,
pyrimidine).
Chitosan-aminoacetyl-sulfadiazine (3b). IR (KBr, cm-1): 1629 (C=O amide I), 1595 (NH
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amide II), 1546, 839 (phenyl), 1259, 1168 (SO2-N); H-NMR (300 MHz, CD3-COOD, δ):
2.18 (3H, COCH3); 3.67 (H-2 chitosan); 3.85- 4.53 (H-3,4,5,6 chitosan); 5.16 (H-1 chitosan);
3.12 (NH); 6.86-6.88 (2CH, aromatic); 7.01-7.02 (2CH, aromatic); 7.65 (1CH, pyrimidine);
8.42-8.43 (2CH, pyrimidine).
Chitosan-aminoacetyl-sulfadimethoxine (3c). IR (KBr, cm-1): 1656 (C=O amide I), 1592
(NH amide II), 1545, 896 (phenyl), 1262, 1164 (SO2-N); 1H-NMR (300 MHz, CD3-COOD,
δ): 2.18 (3H, COCH3); 1.24-1.38 (6H, OCH3); 3.54 (H-2 chitosan); 3.60- 3.87 (H-3,4,5,6
chitosan); 4.79 (H-1 chitosan); 3.14 (NH); 7.70-7.72 (2CH, aromatic); 7.95-7.98 (2CH,
aromatic); 8.26 (1CH, pyrimidine).
Chitosan-aminoacetyl-sulfamethoxazole (3d). IR (KBr, cm-1): 1650 (C=O amide I), 1594
(NH amide II), 1548, 897 (phenyl), 1261, 1167 (SO2-N); 1H-NMR (300 MHz, CD3-COOD,
δ): 2.68 (3H, COCH3); 2.19 (3H, CH3); 3.13 (H-2 chitosan); 3.52-3.86 (H-3,4,5,6 chitosan);
4.75 (H-1 chitosan); 2.82-2.97 (NH); 6.89-6.91 (2CH, aromatic); 7.67-7.70 (2CH, aromatic);
6.03 (1CH, izoxazole).
Chitosan-aminoacetyl-sulfamerazine (3e). IR (KBr, cm-1): 1654 (C=O amide I), 1596 (NH
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amide II), 1548, 896 (phenyl), 1259, 1165 (SO2-N); H-NMR (300 MHz, CD3-COOD, δ):
2.63 (3H, COCH3); 2.40 (3H, CH3); 3.14 (H-2 chitosan); 3.65-3.87 (H-3,4,5,6 chitosan);
4.88 (H-1 chitosan); 2.82-2.98 (NH); 6.87-6.89 (2CH, aromatic); 7.68-7.71 (2CH, aromatic);
7.96 (1CH, pyrimidine); 8.19 (1CH, pyrimidine).
Chitosan-aminoacetyl-sulfisoxazole (3f). IR (KBr, cm-1): 1654 (C=O amide I), 1565 (NH
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amide II), 1546, 897 (phenyl), 1261, 1164 (SO2-N); H-NMR (300 MHz, CD3-COOD, δ):
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2.50 (3H, COCH3); 1.54 (3H, CH3); 1.61 (3H, CH3); 3.14 (H-2 chitosan); 3.54-3.87 (H-
3,4,5,6 chitosan); 4.77 (H-1 chitosan); 2.79-2.99 (NH); 6.82-6.84 ( 2CH, aromatic); 7.72-7.81
(2CH, aromatic).
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2.3. Preparation of chitosan derivatives membranes
Chitosan (CS MMW) and its sulfonamide derivatives (3a-f) (2%, w/v), in 2% acetic acid
solutions, were kept at -20 °C overnight and after that the frozen samples were lyophilized for
24 h, crosslinked with a solution of 5% TPP and then washed several times with double-
distilled water in order to obtain porous chitosan membranes (Feng & Xia, 2011; Anisha et
al., 2013; Arpornmaeklong, Pripatnanont & Suwatwirote, 2008; Sionkowska & Płanecka,
2013). The resulting membranes were stored in Falcon tubes at 4 °C.
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2.4. Characterization of Chitosan Derivatives
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2.4.1. ATR -FTIR Spectroscopy
Chloroacetylsulfonamide derivatives (2a-f) and chitosan derivatives (3a-f) were
characterized by ATR-FTIR measurements using a Biorad FT-IR spectrometer FTS 575C.
Spectra were recorded in the range of 4000- 500 cm-1 with 32 scans at a resolution of 4 cm−1.
Spectral processing was carried out with Horizon MBTM FTIR Software and GRAMS 32
Software (Galactic Industry Corporation, Salem, NH), Version 6.00 (Samal et al, 2014).
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2.4.2. 1H-NMR Spectroscopy
1H-NMR spectra of chitosan derivatives were recorded in D2O/CD3COOD with a Bruker
Avance 300 MHz instrument. The chemical shifts were expressed in ppm downfield of
tetramethylsilane (TMS) as an internal standard. Nuclear magnetic resonance spectral analysis
(1H-NMR) was used to determine the acetylation degree of chitosan, and the substitution
degrees of various chitosan derivatives.
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Degree of Acetylation (DA) of chitosan it was calculated by using ratio between the integral of
the peak of the three protons from acetyl group and integral of the characteristic peak of
proton from -CH-NH2 group of deacetylated monomer, using the following formula:
DA = ([Ia]/3)/ [Ib] x 100
(1)
where: Ia=integral of the peak of the three protons of acetyl group, Ib = integral of the
characteristic peak of proton from -CH-NH2 group.
Degree of Substitution (DS) of chitosan derivatives (3a-f) was calculated by using the ratio
between the integral of the peak of the four aromatic protons from sulfonamide part and
integral of the characteristic peak of proton from -CH-NH2 group of deacetylated monomer,
using the following formula:
DS = ([Ia]/4)/ [Ib] x 100
(2)
where: Ia = integral of the peak of the four aromatic protons from sulfonamide part, Ib =
integral of the characteristic peak of proton from -CH-NH2 group.
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2.5. Characterization of chitosan derivatives membranes
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2.5.1. Morphology
The morphology of chitosan membrane was examined by using a Fei Quanta 200F (field
emission gun) scanning electron microscope (SEM). The dried samples were coated with gold
before observation in a scanning electron microscope.
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2.5.2. Porosity test
The porosity of prepared crosslinked chitosan derivatives membranes was determined
using the method of immersing in absolute ethanol until saturation. The samples have been
weighed before and after immersion in alcohol and the porosity degree (P) was calculated
according to the following formula (3):
P = (W2-W1)/ρV1
(3)
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where: W1 and W2 = the weight of membrane before and after immersion in absolute ethanol,
V1 and ρ are the volume and density of alcohol.
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2.5.3. In vitro biodegradation
The degradation of the membranes was studied in a phosphate buffered saline (PBS) (pH
7.4) containing lysozyme at 37°C. Membranes were equally weighed and immersed first in
PBS until swelling equilibrium was reached, then the PBS solution was changed to a PBS
medium containing lysozyme (10000 UI/mL) and incubated at 37°C for 7 days. The
membranes were taken out and weighed after 1, 4 and 7 days. The percentage biodegradation
(D%) was calculated using the following formula (Baran, Tuzlakoğlu, Mano & Reis, 2012):
D% = (W0-Wx)/W0 x 100
(4)
where: W0 = wet weight before incubation, WX = wet weight after incubation.
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2.5.4. Swelling ratio
The crosslinked chitosan derivative membranes were cut into small pieces that had equal
weights (Wd) and then were immersed in double-distilled water and acetate buffer solution
respectively. The membranes were taken out at specified periods of time, the excess of water
was removed by gentle wiping with a filter paper and immediately weighing them (Ww). The
membrane swelling ratio (MSR) was calculated using the following formula (Lin, Tan, Marra,
Jan & Liu, 2009):
MSR (%) = (Ww- Wd)/ Wdx100
(5)
where: Wd, Ww are the weight of membranes before and after immersion in double-distilled
water.
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2.5.5. Surface free energy estimation by contact angle measurement
Surface free energy (SFE) can be calculated by measuring contact angles of various fluids
with different known surface tension characteristics. To obtain the components of the surface
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free energy and the total surface free energy of chitosan and chitosan derivative membranes,
the contact angles at equilibrium between the film surface and three pure liquids: double-
distilled water, formamide and diiodomethane were measured. The contact angle was
determined by the sessile drop method, at room temperature and controlled humidity, within
10 s, after placing 1 µL of pure liquids on the film surface, using a CAM-200 instrument from
KSV-Finland. Contact angle was measured at least 10 times on different sites of the surface,
the average value being considered. It was calculated using Young-Laplace equation (Rotta et
al., 2009).
SFE and the components were calculated using the acid-base approach which divides the
total SFE into dispersive Lifshitz–van der Waals interaction (γ sLW ) and polar Lewis acid–base
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interactions (γ sAB ) according to the equation (6) (Shabalovskaya, Siegismund, Heurich &
Rettenmayr, 2013). The acid base interactions are subdivided into electron donor γ s− (Lewis
base) and electron acceptor γ s+ (Lewis acid) parts.
γ TOT = 2
(
γ sLW γ lLW
+
γ s+γl−
+
γ s−γ l+
)
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(1+cosθ)
(6)
s
where: θ is the contact angle, γ lTOT is the liquid’s total surface tension, γ lLW and γ sLW are the
apolar Lifshitz–van der Waals components of the liquid and the solid, respectively, and γ s+γ l−
and γ s−γ l+ are the Lewis acid–base contributions of either the solid or the liquid phase.
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2.6. Biological evaluation
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2.6.1. Antimicrobial assay
Diameter of inhibition area
Antibacterial activity measured as the diameter of inhibition area was evaluated by agar
disc diffusion method (CLSI, 2012) using the following bacterial strains: Staphylococcus
aureus ATCC 25923, Sarcina lutea ATCC 9341, Bacillus cereus ATCC 14579, Bacillus
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subtilis, Escherichia coli ATCC 25922, Pseudomonas aeruginosa CIP 82118.The antifungal
effect was evaluated on Candida albicans ATCC 10231, Candida glabrata and Candida sake.
All antimicrobial strains were obtained from the Culture Collection of the Department of
Microbiology, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and
Pharmacy, Iasi, Romania.
Sterile stainless steel cylinders (50 mm internal diameter; 100 mm height) were applied on the
agar surface in Petri dishes and test samples (3a-f), prepared as disc-shaped membranes (2.5
mg/disc), were added. The Petri dishes were incubated at 37°C for 24 h (for bacteria) and at
24°C for 48 h (for yeasts). After incubation, the diameter of inhibition area was measured.
Commercially available discs containing nitrofurantoin (300 µg/disc) and ciprofloxacin (5
µg/disc) were used as positive controls.
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Minimum inhibitory/bactericidal concentrations (MICs/MBCs)
The MICs and MBCs for two of the most common bacterial strains (Staphylococcus
aureus ATCC 25923, Escherichia coli ATCC 25922) were evaluated according to the the
guidelines of EUCAST Def 3.1 (2012). Briefly stock solutions were prepared by dissolving
the chitosan derivatives (3a-f) in the concentration of 1% in acetic acid 1%. Using these
solutions, series of two-fold dilutions were subsequently obtained. In a 9 cm diameter Petri
dish, one milliliter of each dilution was mixed throughly with Mueller-Hinton agar (19 mL),
sterilized by autoclaving and cooled to 50ºC. After this, the concentrations of the chitosan
derivatives inside the medium were 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.62 mg/mL, 0.31
mg/mL, 0.15 mg/mL, 0.07 mg/mL and 0.03 mg/mL respectively. A blank plate (control of
growth) was also prepared by mixing acetic acid 1% (1 ml) with molten agar (19 mL). For
each bacterial strain, a 0.5 McFarland suspension was prepared in 0.85% saline solution and
after that, the inoculum was standardized in order to assure 104 colony-forming units (CFU)
per spot (5 µL). All inoculated plates were incubated for 18 h at 36°C. The MIC was
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interpreted as the lowest concentration of the membrane that completely inhibits the growth of
bacteria in the spot area and the MBC, as the lowest concentration of the membrane that
completely kill the bacteria in the spot area. Each determination was performed in triplicate.
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2.6.2. MTT cell proliferation assay
Biocompatibility of the polymeric materials was assessed using MTT Cell Proliferation
Assay (ATCC® 30-1010KTM). Mouse fibroblasts (L929) were cultured in T-25 tissue culture
flasks and incubated overnight at 37°C and 95% relative humidity in air atmosphere
containing 5% CO2. The cell suspension was seeded at a density of 5x104 cells/mL and
incubated for 24 h until a monolayer was formed and exposed to polymeric extracts. In order
to prepare the polymeric extract the samples (25 mm2) of chitosan derivative and chitosan (as
negative control) were sealed in polyethylene foils and sterilized by exposure to UV radiation
for 8 h. After that the samples were placed in the culture medium with or without fetal bovine
serum in the closed tubes of 15 mL and incubated at 37oC for 24±2 h. The medium was
filtered and used immediately.
Morphology of cells was assessed using inverted microscope and quantitative
evaluation of cytotoxicity was done using tetrazolium salt (MTT).
The fibroblasts cultured in the presence of polymeric samples were washed with PBS,
fixed in methanol, stained with Hematoxylin and Eosin (H&E) dyes and pictures were taken
using an inverse-phase microscope (Nikon Japan).
After the desired time exposure (24 h, 48 h, 72 h), the culture medium was replaced
with fresh medium containing 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) solution, in a 10:1 (v/v) ratio, and the plates were incubated at 37°C, for 3 h. Then,
500 µL of isopropanol was added to each well to dissolve the formazan crystals by gently
shaking on a platform, for 3 h. The coloured solution was transferred to a 96-well plate and
13
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317
318
319
320
321
322
the optical density (OD) was read at 570 nm using a Sunrise microplate reader (Tecan,
Austria). Cell viability was evaluated using the following formula (7):
Cell viability (%) = ODsample / ODcontrol x 100
(7)
where: ODsample = optical density of the sample (chitosan and chitosan derivatives), ODcontrol
optical density of the cell culture without polymeric materials).
=
The test was performed also in the presence of hydrogen peroxide (0.03%) as positive control.
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
2.6.3. Wound healing assay
The study related to burn wound protocols was approved by the Animal Research
Committee of the “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania.
Eighteen male Wistar rats of 300 grams were placed prone, under deep inhalation anesthesia
(Isoflurane 2L/min), and the dorsal areas were shaved with electric clippers to ensure even
burn wounding. High-pressure steam at 114°C was applied for 2 seconds through controlled
electro-valve to inflict intermediate thickness burn wound in the rat back. After debridement,
the rats were randomly divided in 3 groups (of 6 rats each) and the burn surface was covered
with standard gauze dressing (control rats group), chitosan (chitosan rats group) and chitosan-
sulfadiazine (chitosan-sulfadiazine rats group) membranes. During the test the membranes
were periodically removed and replaced. It is important to replace the membranes for cleaning
the wound of dead cells, damaged tissue, fibrin and excess of exudates. All these aspects lead
to the necessity of changing dressings in 8th, 11th and 14th day. During every dressing renewal,
visual scale analysis, photographs and punch biopsies of the burn surface were performed.
ImageJ software assessed the area of wound surface and the level of wound contraction.
Paraffin-embedded Hematoxylin and Eosin (H&E) staining was used to compare wound
healing processes between groups. At day 14, after the punch biopsy, the rats were euthanized
with 1-2 cc KCl via intracardiac injection, while in deep inhalation anesthesia.
14
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342
343
344
345
346
347
348
349
2.7. Statistical analysis
All tests were performed in triplicate and the data are expressed as means ± SD. The
statistical software package StatView was used for data analysis of biological assays.
Experimental results were analyzed by 3 (groups) x 3 (time sample points) repeated measures
ANOVA and Fisher's post hoc test to compare the burn surface area between control, chitosan
rats group and chitosan-sulfadiazine rats group at Day 8, 11 and 14. The criterion for
significance was P< 0.05.
350 3. Results and Discussion
351
352
353
354
355
3.1.
Chemistry
In order to obtain functionalized chitosan derivatives, several sulfonamides -
sulfamethoxydiazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamerazine and
sulfisoxazole (1a-f) were reacted with chloroacetyl chloride in dry acetone in the presence of
anhydrous potassium carbonate to obtain N-chloroacetyl sulfonamides (Fig. 1a).
15
Page 15 of 36

a) N-chloracetyl-sulfonamides
R:
O
C
NH2
HN
CH2 Cl
N
N
N
OCH3
(a)
(b)
;
;
O
N
+
Cl CH2
C
Cl
CH3
(
H3C
N
N
O
;
(e)
;
(d)
H
H
O2S
N R
O2S
N R
N
N
CH3
1a-f
O
2a-f
CH3
CH3
b) Sulfoanamides-chitosan derivatives
O
C
O
OH
CH3
NH
HN
C
CH2 Cl
HO
O
O
C
O
HO
HO
OH
O
OH
NH
NH
CH2
HO
O
+
OH
O
HO
O
O
HO
O
C
NH2
NH2
H
NH
OH
CS MMW
O2S
N R
2a-f
H
O2S
3a-f
Fig. 1. Two steps synthesis of chitosan derivatives with sulfonamide structures.
N R
356
357
358
359
In the next step by reaction of chitosan with N-chloroacetyl sulfonamides (2a-f) six new
chitosan sulfonamide derivatives were obtained denoted with 3a-f (Fig. 1b).
360
3.2. Characterization of chitosan derivatives
361
362
363
364
365
366
367
3.2.1. Spectral data
In the IR spectra of chitosan derivatives (3a-f), appear characteristic peaks from chitosan
and sulfonamide moieties (Fig 2). New peaks appeared at around 1540 cm-1 and 830 cm-1
attributed to phenyl group and two other peaks attributed to SO2-N sulfonamide group
appeared at around 1250 cm-1 and 1160 cm-1. Increased intensities of the amide I and II bands
of the chitosan are evident due to the new amide moieties arising from the amide-linked
sulfonamide substituents.
16
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368
369
Fig. 2. IR spectra of chitosan (CS MMW) and its sulfonamide derivatives (3a-f).
370
371
372
373
374
375
376
377
1
In H-NMR spectra of chitosan derivatives (3a-f) appeared the characteristic signals for
both units: chitosan and sulfonamide (Batista, Pinto, Gomes & Gomes, 2006), that prove the
substitution took place. Sulfonamide residue appeared in the range of 6.82-7.72 ppm and
7.65-7.95 ppm (aromatic protons); 7.65-8.43 ppm (pyrimidine - 3a, 3b, 3c, 8e) and 6.03 ppm
(isoxazole - 3d).
For physical and spectral characterization of N-chloroacetyl-sulfonamides (2a-f) see
supplementary data (Dragostin et al., 2015).
378
3.2.2. Degree of acetylation (DA) and degree of substitution (DS)
379
380
381
The values of substitution degree of chitosan derivatives (3a-f) was found to vary between
9.61 % and 34.24 % - Table 1. That means that chitosan derivatives still have between
67.95% and 43.32 % free amino groups.
382
383
Table 1
Degree of substitution (DS%) of chitosan derivatives (3a-f).
Compound 3a
3b
3c
3d
3e
3f
17
Page 17 of 36

N
N
R1
OCH
N
N
N
N
N
CH3
O
N
CH
C
CH3
N
O
N
OCH
15.45
31.64
12.50
9.61
34.24
10.27
DS%
384
385
386
387
388
389
390
391
3.3. Characterization of Chitosan Derivative Membranes
Chitosan derivative membranes were prepared using sodium tripolyphosphate(TPP) as a
crosslinking agent. It is known that non crosslinked chitosan membranes have poor chemical
stability and will dissolve in an acid environment and therefore need to be crosslinked. Free
amine groups of chitosan derivatives are protonated in acid conditions and consequently are
positively charged. In the presence of TPP, a network is produced based on electrostatic
interactions between negatively charged crosslinking agent and positively charged
glucosamine chains (Giri, Thakur, Ajazuddin, Badwaik & Tripathi, 2012).
392
393
394
395
396
397
398
399
400
401
402
403
404
3.3.1. Morphology and porosity analysis
The morphology and porosity degree of polymeric membranes as wound dressing
materials are important features, because they influence the absorption capacity of exudates,
the colonization rate and cellular organization but also the process of angiogenesis (Xing, Lie,
Zhengwei & Changyou, 2011). It has been shown that the porosity degree could be influenced
by the concentration of polymeric solution (2%, w/v), by freezing temperature (-20˚C), but
also by the cross-linking method (chemical cross-linking) and cross-linking agent (sodium
tripolyphosphate) (Kumirska, Weinhold, Thöming & Stepnowski, 2011; O’Brien, Harley,
Yannas & Gibson, 2005). It was observed that in the series of chitosan-sulfonamide
derivatives (3a-f), the membrane porosity was comparable with the porosity of the chitosan
membrane (84.42%). The highest porosity degree was recorded for chitosan-sulfadiazine (3b),
for which the value of the porosity degree was 94.49% (Fig. 3a). These results are supported
also by the SEM images - Fig. 4, for chitosan (A and B) and chitosan-sulfadiazine (3b) (C and
18
Page 18 of 36

405
406
D) membranes. For SEM images of other chitosan-sulfonamide membranes see Fig. 2
(Dragostin et al., 2015).
407
408
409
410
Fig. 3. a): porosity degree of chitosan derivatives (3a-f),b): percentage biodegradation of
chitosan derivatives membranes (3a-f), in respect with that of chitosan.
411
412
413
414
Fig. 4. SEM images of chitosan (CSMMW) (A and B) and chitosan-sulfadiazine (3b) (C and
D) membranes.
415
416
417
418
419
420
421
422
423
3.3.2. In vitro biodegradation
The biodegradation study of chitosan, and the chitosan-sulfonamide derivatives under the
action of lysozyme revealed considerable differences between them (Fig. 3b). The percentage
biodegradation of the chitosan derivatives was higher than chitosan, excepting chitosan-
sulfadimethoxine (3c). The highest percentage biodegradation was recorded for chitosan-
sulfisoxazole (3f), for which, at the end of the experiment (7th day), the biodegradation was
52.89% while the biodegradation of chitosan was 38.19%. According to the literature data the
lysozyme biodegradation products of chitosan could be D-glucosamine and
glycosaminoglycan which are nontoxic for cells (Baran, Tuzlakoğlu, Mano & Reis, 2012).
19
Page 19 of 36

424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
3.3.3. Swelling degree
According to Gethin’s study, the pH of intact skin is about 5, while in the case of
wounds this value increases to 7 or even more, depending on the type of wound (Gethin,
2007). In this study, we have performed a comparison between swelling behavior at
physiological environment of intact skin (pH 5) and wounded environment (pH 7), at 37°C
(Fig. 5). Although there are no significant differences between the swelling ratio recorded at
both pH values, it is to be noted that at pH 5 the swelling degree is a little bit higher than at
that of pH 7.
Among the chitosan derivatives the highest swelling ratio was recorded for 3c (chitosan-
sulfadimethoxine) derivative, for which swelling capacity was 2407 % after 60 min, at pH 7
and 2675 % after 30 min, at pH 5. A very good swelling ratio in comparison with chitosan at
both pH values was also recorded for 3b (chitosan-sulfadiazine), 3d (chitosan-
sulfamethoxazole) and 3f (chitosan-sulfisoxazole) derivatives. For example in case of 3b the
thermodynamic equilibrium was reached after 60 min, with an increased swelling capacity of
2070 % at pH 7 and 2217 % at pH 5 respectively.
In the similar conditions for chitosan (CS MMW) thermodynamic equilibrium was
installed after 60 min with an increased uptake capacity of 1823 %, at pH 7 and after 30 min
at pH 5 (2107 %).
These results support that, excepting 3a (chitosan-sulfamethoxidiazine derivative), all
other chitosan derivatives showed a higher swelling ratio in respect with to neat chitosan,
which means they could have a higher absorption capacity of exudates in the wound healing
process.
20
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446
447
Fig. 5. Swelling degree profiles of chitosan and chitosan derivatives (3a-f) membranes at pH
448
449
450
451
452
453
454
455
456
457
458
459
7 (a) and pH 5 (b).
3.3.4. Surface free energy estimation by contact angle measurement
As concerns the biocompatibility several parameters should be considered, such as surface
free energy (SFE). The SFE is an important property for cell attachment which determines the
quality of the material surface and its possible biomedical applications (Yang, Huang, Shen &
Yeh, 2010). It has been demonstrated a strong connection between the total surface energy
and the cell attachment: a higher energy surface supports a greater attachment than a lower
energy surface (Hallab, Bundy, O’Connor, Moses & Jacobs, 2001). The chemical
functionalization of chitosan with different sulfonamides does not negatively impact on total
surface free energy -Table 2, which means that the biocompatibility of chitosan derivatives is
similar to the chitosan one. For data referring to the values of the surface free energy
components see Table 3 (Dragostin et al., 2015).
460
461
462
Table 2
The contact angle and total surface free energy values for chitosan and chitosan-derivatives
(3a-f) membranes.
γ STOT
Contact angle (degree)
Samples
water
formamide diiodomethane
(mN/m)
29.09
23.57
26.49
26.76
28.71
CS MMW
73.28±1.32 69.86±0.72 65.16±1.16
47.81±0.89 40.55±0.75 38.45±1.45
58.45±1.07 60.65±1.71 59.80±0.96
78.96±1.45 73.25±0.80 68.28±0.79
71.58±1.05 66.47±0.99 60.88±1.12
3a
3b
3c
3d
21
Page 21 of 36

32.45
33.13
3e
3f
71.89±1.62 67.43±1.45 61.05±1.03
78.11±1.33 73.98±1.22 67.98±0.78
463
464
3.4. Biological evaluation
3.4.1. Antimicrobial assay
465
466
467
The antimicrobial activity results of chitosan derivatives (3a-f), expressed as diameters
of inhibition area, are presented in Table 3.
468
469
Table 3
Diameter of inhibition area (mm) of chitosan derivatives (3a-f).
Diameter of inhibition area (mm)
Compound
S.a.
S.l.
B.c.
B.s.
E.c.
P.a.
CS MMW
0
0
0
0
0
0
0
0
0
0
0
3a
3b
3c
3d
3e
3f
0
15
26
32
0
0
0
0
11
10
0
15
18
0
12
19
0
11
17
0
12
26
12
9
12
0
0
0
0
0
0
470
471
472
473
S.a. - Staphloccocus auresus ATCC 25923, S.l. - Sarcina lutea ATCC 9341, B.c. -
Bacillus cereus ATCC 14579, B.s. - Bacillus subtilis, E.c. - Escherichia coli ATCC 25922,
P.a. - Pseudomonas aeruginosa CIP 82118.
474
475
476
477
478
479
480
For all bacterial strains, chitosan (CS MMW) was inactive at a concentration of 2.5
mg/disc. In the same experimental conditions, 3b (chitosan-sulfadiazine), 3c (chitosan-
sulfadimethoxine) and 3e (chitosan-sulfamethoxazole) showed a good antimicrobial activity,
for tested bacterial strains, excepting Pseudomonas aeruginosa CIP 82118, to which the
compounds were inactive.
Compared with positive controls (Table 4), the tested compounds showed a higher
activity than ciprofloxacin (5 µg/disc) and similar with that of nitrofurantoin (300 µg/disc).
481
482
Table 4
Diameter of inhibition area (mm) of Nitrofurantoin and Ciprofloxacin.
22
Page 22 of 36

Diameter of inhibition area (mm)
Control
N 300µg/disc 19
S.a.
S.l.
8
B.c.
12
-
B.s.
20
-
E.c.
20
-
P.a.
-
33
C 5 µg/disc
-
-
483
484
485
486
N - nitrofurantoin, C - ciprofloxacin, S.a. - Staphloccocus auresus ATCC 25923, S.l. - Sarcina lutea
ATCC 9341, B.c. - Bacillus cereus ATCC 14579, B.s. - Bacillus subtilis, E.c. - Escherichia coli ATCC
25922, P.a. - Pseudomonas aeruginosa CIP 82118, " - " no effect.
487
488
489
490
491
Evaluation ofantifungal activity of chitosan derivatives (3a-f), showed that all the
compounds are inactive at 2.5 mg/disc to Candida albicans ATCC 10231, Candida glabrata
and Candida sake.
The values of MICs and MBCs for chitosan-derivatives (3a-f) evaluated using the broth
micro dilution method are listed in Table 5.
492
493
Table 5
The MICs and MBCs values for chitosan and chitosan derivatives.
S. aureus ATCC 25923
MIC (mg/ml) MBC (mg/ml) MIC (mg/ml) MBC (mg/ml)
E. coli ATCC 25922
Chitosan derivatives
CS-sulfametoxidiazine (3a)
CS-sulfadiazine (3b)
CS-sulfadimetoxine (3c)
CS-sulfametoxazol (3d)
CS-sulfamerazine (3e)
CS-sulfizoxazol (3f)
CS MMW
5
1.25
2.5
2.5
5
1.25
5
0.25
>5
2.5
5
0.62
0.03
2.5
0.15
2.5
2.5
2.5
0.5
1.25
0.15
5
0.31
5
5
5
1
5
>5
2.5
>5
0.5
Ampicilin(µg/ml)
494
495
496
497
498
499
500
501
According to the results, all tested compounds are more active than chitosan, but less
active than ampicillin used as positive control. It was observed that the activity is closely
related to the sulfonamide moiety that substitute the glucosamine unit of chitosan. The most
active derivative was chitosan-sulfadiazine (3b). It was active on Staphylococcus aureus
ATCC 25922, with MIC value of 1.25 mg/ml and MBC value of 2.5 mg/ml. More than that
this compound was more active on Escherichia coli, a Gram-negative bacterial strain, MIC
and MBC values being less (0.03 and 0.15 mg/ml respectively) than values recorded for
Gram-positive bacterial strain - Staphylococcus aureus.
23
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502
503
504
505
506
507
508
509
510
511
512
In conclusion, the antimicrobial effects of chitosan derivatives are more intense than
chitosan and comparable with other derivatives reported in the literature. For example, the
MIC and MBC of diethyl methyl chitosan (DEMC), against Escherichia coli was lower than
chitosan (Avadi et al, 2004). The antibacterial effects of the acyl thiourea derivatives of
chitosan were also much better than chitosan, the value of MIC and MBC against
Escherichia coli being 15.62 µg/mL and 62.49 µg/mL, respectively (Zhong et al, 2008). In
our research, MIC value of pure chitosan against Escherichia coli was found to be 2.5
mg/mL, while the most active chitosan derivative of sulfadiazine (3b) shows an MIC value of
30 µg/mL.
Considering all obtained results, derivative 3b was selected for further testing in vivo
wound healing test.
513
514
515
516
517
518
3.4.2. MTT cell proliferation assay
It was observed that the cell viability (%) evaluated using MTT assay decreases in
presence of chitosan derivatives in comparasion with chitosan - Table 6. Even if the cell
viability for these derivatives is less than chitosan, some of these compounds are considered
nontoxic because the viability is higher than 70%, according to the literatute data (Lönnroth,
2005).
519
520
Table 6
The cell viability value (%) for chitosan derivatives at 24 h, 48 h and 72 h.
24
Page 24 of 36

521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
Cell viability (%)
Samples
24 h
89.58
48 h
69.25
72 h
62.00
CS-sulfametoxidiazine (3a)
CS-sulfadiazine (3b)
CS-sulfadimetoxine (3c)
CS-sulfametoxazol (3d)
CS-sulfamerazine (3e)
CS-sulfizoxazol (3f)
CS MMW (negative control)
H2O2 0.03% (positive control)
Control
80.08
87.33
82.67
3.45
80.62
124.60
1.49
77.65
74.34
74.34
2.65
56.86
113.94
0.66
96.18
98.73
62.79
0.64
52.78
114.94
0.00
100.00
100.00
100.00
The least toxic are CS-sulfadiazine (3b) and CS-sulfadimetoxine (3c), for which the cell
viability was higher than 70% at all exposed periods (24 h, 48 h and 72 h). After 72 h of
incubation the viability of these derivatives was 96.18% (3b) and 98.73% (3c), the values
being comparable with those of chitosan (CS MMW) (114.94%).
Microscopic images of the cell culture incubated in the presence of chitosan derivatives
are presented in Fig. 6. In comparison with the control, in which the cells have round and
polygonal shape, in the cell culture incubated in the presence of chitosan derivative 3c and 3b,
the cells are elongated, some of them becoming spindly, their shape being similar to the cells
incubated in presence of chitosan (CS MMW).
24 h
48 h
72 h
3a
25
Page 25 of 36

3b
3c
3d
3e
3f
538
539
Fig. 6. Cell cultures incubated in the presence of chitosan derivatives (3a-f) in reference with
chitosan (CS MMW) and control.
26
Page 26 of 36

540
541
542
543
544
545
546
3.4.3. Wound healing assay
Burn degree was evaluated by skin wounds punch-biopsy and Hematoxylin and Eosin
staining protocol of the samples harvested at 48 hours post-infliction. Intermediate burn
wounds resulted at 2 seconds exposure to high-pressure hot steam.
The chitosan-sulfadiazine derivative (3b) was evaluated for its wound healing properties
vs. chitosan (CS) as a reference. The polymeric membranes (CS, 3b) were stapled to gauze
and fixed on top of the burn wound with tie-over suture.
C)
Eschar
H&E staining
547
548
Fig. 7. Burn wounds, histological exam and topical treatment. A: acute burn injury inflicted
by high-pressure steam at 2 seconds exposure (day 0), B: intense edema of the burn wound
27
Page 27 of 36

549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
will subside and the eschar will form, C: at 48 hours the histology shows necrosis and
detachment of the epidermis and the superior half of the dermis together with skin appendages
are necrosed, D: shows CS membrane (D1) and 3b membrane (D2) underneath (of equal
sizes), stapled on gauze, E: tie-over suture fixed the membrane on the burn wound, F: shows
dehydrated, contracted membrane after 4 days, before changing to the new one.
The burn wound healing was evaluated at 8, 11 and 14 days after the start of the
experiment. The average value and standard deviation of burn surface area in each group at
different days during recovery phase are shown in Table 7.
The burning surface in all groups at day 0 is not different, showing the burn infliction
methods consistency. The burning surface in rats group treated with chitosan-sulfadiazine
(3b) was significantly smaller than rats group treated with chitosan (CS) (p<0.01) and control
group (p<0.01) at day 8, 11 and 14, and the burn surface in rats group treated with chitosan
was also significantly smaller than control group (p=0.03) at day 8, 11 and 14. Both CS and
3b dressing materials were more effective for wound healing than the control group (with
standard gauze dressing), and 3b dressing in particular, had the best healing effect among all
the groups. In addition, wound healing progressed with time of all three groups, i.e. the longer
recovery duration, smaller the burn wound area (p<0.01).
Table 7
Burn surface area and standard deviation (mm2) for control, chitosan (CS) and chitosan-
sulfadiazine (3b) rats groups at day 0 (burn infliction) and at 8, 11 and 14 day.
Group
Day
Mean ± SD (mm2)
Day 0
955±16
Control
Day 8
858±10
758±19
742±26
Day 11
Day 14
28
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CS rats group Day 0
Day 8
945±11
742±27
699±20
556±51
951±13
589±52
Day 11
Day 14
3b rats group
P value
Day 0
Day 8
Day 11
Day 14
Group
Day
474±25
374±35
<0.01
<0.01
Interaction 0.2
571
572
573
574
575
576
577
578
579
580
581
582
Macroscopic aspect of the burn wounds was documented by standard photographs shown
in Fig. 8. Macroscopic evaluation of control group shows subsequent biopsies and the surface
of the burn wound after eschar detachment is whitish, pearly appearance with weak signs of
healing even at day 14. CS rats group shows color turning over slightly pink appearance as a
sign of improved healing and better local vascularization, yet it retains raw non-epithelialized
dermis in the upper quadrants of the wound (approx. 40% of the wound surface). Chitosan-
sulfadiazine (3b) rats group shows no more raw appearance, the shiny areas are the new
epithelial cell layers formed due to better wound healing, and fully cover the previous
wounded area at day 14, compared to control and CS rats group, respectively. All burn wound
areas significantly decrease over time.
Day 8
Day 11
Day 14
Control
29
Page 29 of 36