UNIVERSITY OF PARDUBICE, CZECH REPUBLIC
FACULTY OF CHEMICAL TECHNOLOGY
INSTITUTE OF ORGANIC CHEMISTRY AND TECHNOLOGY
New Wound Dressing Based on
Chitosan/Hyaluronan Biopolymers
Ph.D. Thesis
Rasha Abdel-Rahman
2016
TABLE OF CONTENTS
Author’s declaration…………………………………………………………………...
Title………………………………………………………………………………………
Název……………………………………………………...……………………………..
Aims of work……………………………………………………………………………
Acknowledgments……………………………………………………………………..
Contents…………………………………………………………………………………
List of Figures…………………………………………………………...………………
List of Tables……………………………………………………………………………
Abbreviations……………………………………………………………………………
Chapter 1: Theoretical part………………………………………………...…………
1. Biomaterials………………………………………………………...…………………
1.1. Polymers…………………………………………………………………………
1.2. Chitin…………………………………………………………..………………..
1.3. Chitosan…………………………………………………………………………
1.4. Hyaluronic acid………………………………………………………………...…
2. Wound healing process………………………………………………………...………
3. Wound dressing……………………………………………………………..…………
Reference…………………………………………………………………………………
Chapter 2: Chitin and Chitosan from Brazilian Atlantic Coast: Isolation,
Characterization and Antibacterial Activity………………………………………….
Abstract…………………………………………………………………………………..
Keywords………………………………………………………………………………...
1. Introduction……………………………………………………………………………
2. Experimental…………………………………………………………………………
2.1. Materials…………………………………………………………………………
2.1.1. Sample preparation……………………….………………………………...
2.1.2. Sieving shrimp shell to different particle sizes………………………….….
2.2. Isolation of chitin and chitosan from shrimp shells……………………………..
2.3. Characterization of chitin and chitosan………………………………………….
2.3.1. Inductively coupled plasma optical emission spectrometry (ICP-OES) …..
2.3.2. Scanning electron microscopy (SEM) ……………………………………..
2.3.3. Elemental analysis………………………………………………………….
2.3.4. UV-Vis spectroscopy……………………………………………………….
2.3.5. Fourier transform infrared spectroscopy-attenuated total reflection (FTIRATR) ……………………………………………………………………….
2.3.6. Determinations of ash content………………………….……………..……
2.3.7. Solid NMR spectroscopy……………………………………………...……
2.3.8. Diffusion NMR spectroscopy………………………………………………
2.3.9. Acid-base titration…………………………………………………………..
2.3.10. X-Ray diffraction (XRD) ………………………………………………..
2.3.11. Thermal analysis measurements……………………………………….
2.3.12. Antibacterial activity of chitin and chitosan……………………………..
2.3.13. Statistical analysis
3. Results and discussion……………………………………………………………….
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3.1. Isolation of chitin………………………………………………………………
3.1.1. Demineralization step (DM) …………………………………………….
3.1.2. Deproteinization step (DP) ………………………………………………
3.1.2.1.1. Scanning electron microscopy of pure chitin……………...…
3.1.3. Isolation of chitosan from chitin (Deacetylation step) ……..……………
3.1.4. Isolation sequences of chitin and chitosan……………….………………
3.1.5. Thermal stability of chitin and chitosan………………………………….
3.1.6. ATR-FTIR of chitin and chitosan………………………………………..
3.1.7. Solid NMR of chitin and chitosan…………………………..……………
3.1.8. Residual of metals on chitin and chitosan after isolation process……..…
3.1.9. X-ray diffraction of chitin and chitosan………………………………….
3.1.10. Scanning electron microscopy of chitosan………………………….……
3.1.11. Deacetylation degree (DDA) measurements of chitosan by different
techniques……………………………………………………………….….
3.1.12. Antibacterial activity of chitin and chitosan……………………………..
4. Conclusion……………………………………………………………………………
References………………………………………………………………………………..
Chapter 3: Wound Dressing based on Chitosan/hyaluronan/Nonwoven
Fabrics: Preparation, Characterization and Medical Applications……………
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Abstract…………………………………………………………………………………...
Keywords………………………………………………………………………………….
1. Introduction……………………………………………………………………………
2. Experimental…………………………………………………………………………..
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2.1. Materials and methods……………………………………………..……………
2.2. Isolation of chitosan from shrimp shell………………………………….………
2.2.1. Immobilization of chitosan citrate/ hyaluronan onto nonwoven cotton
fabrics……………………………………………………………………..
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2.3. Wound dressing characterization ……………………………………………………………
67
3. Results and discussion…………………………………………………..……………
3.1. Immobilization of chitosan citrate/ hyaluronan onto nonwoven cotton fabrics…
3.2. Physical characterization of the wound dressing……………………………….
3.3. Antibacterial activity of chitosan/hyaluronan wound dressing………………….
3.4. Cytotoxicity of the wound dressing……………………………………………..
3.5. Wound healing measurements…………………………………………………………..
4. Conclusions…………………………………………………………………………..
References………………………………………………………………………………..
Chapter 4: Conclusion and future work
Dissertation work conclusion ……………………………………………………………
Future work ……………………………………………………………………………...
Supporting information 1……………………………………………………..…………
Supporting information 2………………………………………………………….……..
Curriculum vitae……………………………………………………………………….…
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LIST OF FIGURES
Chapter 1
Figure 1
Figure 2
Figure 3
Figure 4
Chapter 2
Figure 1
Figure 2A
Figure 2B
Figure 2C
Figure 2D
Figure 2E
Figure 3A
Figure 3B
Figure 4
Figure 5A
Figure 5B
Figure 6
Figure 7A
Figure 7B
Figure 8A
Figure 8B
Figure 9
Figure 10
Figure 11
Figure 12
Theoretical part
Chemical structure of cellulose and chitin…………………………
Different forms of chitin……………………………………………
Chemical structure of chitosan……………………………………..
Chemical structure of hyaluronic acid………………………………
Chitin and Chitosan from Brazilian Atlantic Coast: Isolation,
Characterization and Antibacterial Activity
Isolation process of chitin and chitosan from shrimp shell…………
Effect of particle size of shrimp shell on the yield percent after
sieving step …………………………………………………………..
Demineralization step-effect of hydrochloric acid on nitrogen percent
of dry shrimp shell with particle size 800-1000 µm ……………..
Demineralization step-effect of hydrochloric acid on yield percent of
shrimp shell with……………………………………………………
Demineralization step - effect of hydrochloric acid on ash content of
shrimp shell with particle size 800-1000 µm ………………………..
Effect of liquor ratio of hydrochloric acid on yield percent and
nitrogen percent of shrimp shell with particle size 800-1000 µm……
Effect of particle size of demineralized shell on protein percent of
demineralized shell………………………………………………….
Relation between time of treatment of demineralized shell and protein
percentages of demineralized shell…………………………..
Scanning electron microscope of shrimp shell and different particle
sizes (sieving process) of chitin after deproteinization and
demineralization steps……………………………………………..
DA-deacetylation step - effect of the sodium hydroxide on the degree
of deacetylation of formed chitosan (DDA)………………….
DA-deacetylation step - effect of time on the degree of deacetylation
of chitosan……………………………………………………………
Isolation sequence of chitin and chitosan from shrimp shell of
particle size (800-1000 µm)………………………………………….
TGA and DTG of chitin……………………………………………
TGA and DTG of chitosan…………………………………………...
FTIR-ATR of chitin………………………………………………..
FTIR-ATR of chitosan……………………………………………….
Solid 13C CP/MAS NMR spectra of chitin and chitosan with
different degree of deacetylation after various time…………………
Residual metals in the extracted chitin and chitosan in the comparison
with shrimp shell……………………………….…………………..
XRD of chitin and chitosan…………………………………………
Scan electron microscope of pure chitosan extracted from shrimp
shells with particle size 800-1000 µm………………………………..
viii
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8
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Figure 13
Kinetic curve of the bioluminescence of E-coli within sixty minutes
in the presence of extracted chitin and chitosan……………………..
Chapter 3
Wound Dressing based on Chitosan/hyaluronan/Nonwoven
Fabrics:
Preparation,
Characterization
and
Medical
Applications
Figure 1
Immobilization of chitosan citrate Layers, sodium hyaluronate layers
onto nonwoven fabrics………………………………………….…..
Proposed mechanism of layer by layer assembled of chitosan citrate
(CSCTA), sodium hyaluronate (HA) onto nonwoven fabrics………
FTIR-ATR of chitosan citrate /nonwoven fabrics; chitosan/sodium
hyaluronate/nonwoven, nonwovens (CTA/NWFs), respectively….
TGA-DTG of chitosan citrate /nonwoven fabrics; chitosan/sodium
hyaluronate/nonwoven, nonwovens (CTA/NWFs), respectively……
Swelling percentage of wound dressing fabricated from chitosan
citrate/nonwoven fabrics without/with hyaluronan;, and nonwovens
(CTA/NWFs)…………………………………………………………
Scanning electron microscopy of layer by layer wound dressing
sheet………………………………………………………………
Antibacterial activity of nonwoven wound dressing fabrics…………
Cytotoxicity measurements of plain chitosan (CS), plain hyaluronan
(HA) and chitosan/hyaluronan treated nonwoven fabrics
(CS1CTA/HA/NWFs) ……………………………………………..
Fluorescence microscopy of nonwoven cotton fabrics (CTA/NWFs)
immobilized by chitosan citrate (CS1CTA/NWFs), chitosan
citrate/hyaluronan layer (CS1CTA/HA/NWFs)……………………...
Effect of chitosan citrate (CS/CTA/NWFs), sodium hyaluronate
(HA/NWFs) and chitosan/hyaluronan nonwoven fabric sheets
(CS1CTA/HA/NWFs) on the spontaneous and zymosan-activated
oxidative burst of blood phagocytes……………………………………
Visual observation of the surface healing dressed with nonwoven
sheet (control-non-diabetic) at 0th, 3th, 7th and 14th days after
operation…………………………………………………………….
76
Wound contraction as a function of postoperative time for (non-diabetic and
diabetics) wounds treated with chitosan/hyaluronan nonwoven fabric
(CS1CTA/HA/NWFs) sheets and control (nonwoven cellulosic fabricsgauze), indicated by the percentage of wound size vs healing
dates……………………………………………………………….………
96
FTIR-ATR (A); TGA-STG (B); H-NMR (C); XRD (D) of the
isolated chitosan from shrimp shell…………………………………
107
Figure 1B
Figure 2A
Figure 2B
Figure 2C
Figure 3
Figure 4
Figure 5A
Figure 5B
Figure 6
Figure 7A
Figure 7B
Supporting
information 2
Figure S1
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LIST OF TABLES
Chapter 2
Chitin and Chitosan from Brazilian Atlantic Coast: Isolation,
Characterization and Antibacterial Activity
Table 1
Determination of degree of deacetylation (DDA) by different analytical
techniques……………………………………………………………
Supporting
Information 1
Table S1
Comparison among the components of shrimp shell and other
sources…...…………………………………………………………
55
104
Supporting
Information 2
Table S1
Table S2
Code of treated and untreated cotton fabrics with different chitosan
citrate/hyaluronan concentrations…………………………………..
Reduction
rate
percentage
of
chitosan
and
chitosan/hyaluronan/nonwoven cotton fabrics………………………...
x
105
106
LIST OF ABBREVIATIONS
PVC
PVB
GlcNAc
GlcN
CS
DDA
MW
GPC
SLS
TGF-β1
PDGF
HA
CD44
RHAMM
DM
DP
DA
ICP-OES
SEM
FTIR-ATR
NMR
BL
RLU
NWFs
CTA/NWFs
CSCTA/NWFs
CSCTA/HA/NWFs
S. aureus
E. coli
NIH 3T3
DMEM
FBS
HaCaT
MTT
DTG
TGA
XRD
LBL
Polyvinyl chloride
Poly acrylonitrile
acetyl glucosamine
Glucosamine
Chitosan
The degree of deacetylation
the molecular weight
gel Permeation Chromatography
Static Light Scattering
transforming growth factor-β-1
platelet-derived growth factor
Hyaluronic acid
cell surface glycoprotein on most cell types
Hyaluronan-mediated motility receptor
Demineralization
Deproteinization
Deacetylation
Inductively coupled plasma optical emission spectrometry
Scanning electron microscopy
Fourier transform infrared spectroscopy-attenuated total reflection
Nuclear magnetic renounce
Bioluminescent
relative light units
nonwoven fabrics
nonwoven fabrics (NWFs) treated with citrate
nonwoven fabrics (NWFs) treated with chitosan citrate
CSCTA/NWFs immersed in sodium hyaluronate solution
Staphylococcus aureus
Escherichia coli
Mouse fibroblast cell line
Dulbecco's Modified Eagle Medium
Fetal bovine serum
keratinocytes cell lines
3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide
differenational thermal analysis
thermal gravimetric analysis
X-ray diffraction
layer by layer
xi
DECLARATION
I developed this work alone. All literary sources and information that I used are listed in
the bibliography – reference list. I was aware that my work is to apply the rights and obligations
under Act. No. 121/2000 coll. – Copyright law, especially with the fact the University of Pardubice
is entitled to the concession contract for this work such as school work according to § 60 par. 1
Copyright law, and that if this work will be used by me or be licensed to use another entity,
University of Pardubice entitled to require from me a reasonable allowance to cover costs incurred
to create this work, until their actual amount, according to circumstances.
I agree with full disclosure of this work in the Library of University of Pardubice.
Title of Thesis/Dissertation:
New Wound Dressing Based on Chitosan/Hyaluronan Biopolymers
Signature Author
Rasha Abdel-Rahman
TITLE
New Wound Dressing Based on Chitosan/Hyaluronan Biopolymers
ANNOTATION
This dissertation explores strategies for preparing new wound dressings from chitosan /
hyaluronan polysaccharides. These wound dressings have excellent healing properties and can be
used in different medical application.
This dissertation is organized as follows. Chapter 1 presents an in-depth literature review.
Chapter 2 presents the isolation and characterization of chitin and chitosan from Brazilian Atlantic
Coast. Chapter 3 focuses on the preparation and characterization of new wound dressing based on
hyaluronan and chitosan with using non-woven fabric. Chapter 4 focuses on the conclusion and
future work.
Keywords: Polysaccharides, wound dressing, chitosan, hyaluronan, diabetic and non-diabetic
case.
NÁZEV
Nové kryty ran založené na chitosanovém/hyaluronovém biopolymeru
ANOTACE
Tato disertační práce popisuje strategie pro přípravu nových krytů ran založených na
polysacharidech – chitosanu a hyaluronanu. Tyto kryty ran mají vynikající hojivé vlastnosti a
mohou být použity v různých lékařských aplikacích.
Tato disertační práce je organizována následovně. Kapitola 1 je hluboká literární rešerše.
Kapitola 2 se zabývá izolací a charakterizací chitinu a chitosanu připraveného ze skořápek krevet
z atlantického pobřeží Brazílie. Kapitola 3 je zaměřena na přípravu a charakterizaci nových krytů
ran založených na hyaluronových a chitosanových netkaných textiliích. Kapitola 4 se zabývá se
závěrem a budoucí práce.
Klíčová slova: chitin, chitosan, hyaluronan, kryt rány, netkaná textílie
AIMS OF DISSERTATION WORK
The present work aims at research and development of isolation pure chitin and chitosan
with high quality in order to use these biopolymers to prepare new wound dressing with high
healing properties that can be used in the field of medical textiles. The main objectives of this
study can be summarized as follows.
Isolation of extra pure chitin from new source and preparation of chitosan from it.
Full characterization of isolated chitin and prepared chitosan.
Evaluation of biological activity and cell viability of chitin and chitosan and healing properties of
chitosan.
Preparation and characterization of new wound dressing material from non-woven fabrics treated
with chitosan and blend of chitosan and hyaluronan.
Evaluation of biological activity, cell viability, fluorescence, phagocytosis, and healing properties
of prepared wound dressing on diabetic and non-diabetic rats.
BIOGRAPHY
My name is Rasha Mahmoud Mohammed Abdel-Rahman. I was born in Fayoum, Egypt.
I completed my B.Sc. in Faculty of Science-Al-Azhar University at Cairo in Special Organic
Chemistry. In 2011, I got my master degree of Organic Chemistry under supervision of Prof. A.
Y. El-Kady, Prof. F.K.Mohammed and Prof. R.M.Abdel-Motelab. University of Pardubice,
Faculty of Chemical Technology, Czech Republic I joined in December 2011 to pursue the Ph.D.
program “Chemistry and Chemical Technologies”, study field “Organic Technology” at Institute
of Organic Chemistry and Technology. My dissertation work was on the area of functional
materials based on biopolymers under the supervision of Prof. Ing. Radim Hrdina, CSc.
ACKNOWLEDGMENTS
First of all, thanks to ALLAH to whom I relate my success achieving in my work. I would like to
sincerely and gratefully thank to my supervisor prof. Ing. Radim Hrdina, CSc. for all of his support,
valuable supervision, patience, beneficial discussions, understanding, careful reading and facilities
provided for producing this work.
My deep thanks to all my colleagues who cooperated with me in this work.
Additionally, I extend my deep thanks to my brother, my sisters, my children and especial thanks
to my husband for all his efforts, encouragement, helps and loving me that much.
Chapter 1
1. Biomaterials
It can be defined as any substance natural or synthetic can be interact with biological
systems in order to use in medical field [1]. The first biomaterials used were gold and ivory and
this was done by Egyptians and Romans. In the past, all types of natural materials including wood,
glue, rubber, tissues from living forms, and manufactured materials such as iron, gold, zinc and
glass were used as biomaterials; but nowadays, a great progress has been made in understanding
the interactions between the tissues and the materials. It has been acknowledged that there are
differences between living (vital) and non-living (avital) materials. A Biomaterial used for implant
should possess some important properties to can be used for long time in the body without
rejection, these properties as the Biocompatibility, which can be described as the ability of
materials to perform their function with an appropriate host response and the ability to exist in
contact with tissues without causing any harm to the body [2]. Also, the biomaterial must not be
toxic and must not react with any tissue in the body. The most commonly used biomaterials are
biopolymers, composite, ceramics, metals and their alloys. Biomaterials are used in various
medical devices and system such as joint replacements, bone plates, artificial ligaments and
tendons, heart valves, contact lenses and skin repair devices.
In the last few decades, research has started to move in the direction of renewable raw
materials that are environmentally friendly and sustainable. Biopolymers, such as cellulose, chitin,
chitosan and starch have been assessed, not only as sustainable resources, but also as attractive
materials with interesting properties and functionalities [3]. Biopolymers or their derivatives can
be blended with other polymers resulting in a number of new composite materials with enhanced
properties and applications in several fields.
1
Chapter 1
1.1. Polymers
Polymers are the most widely attractive materials for biomedical applications [4] as
cardiovascular devices, replacement and proliferation of various soft tissues and also used in drug
delivery [5]. Polymers are organic materials that form large chains made up of many repeating
units. Polymers may be natural or synthetic [6]. Synthetic polymers can be prepared in laboratory
by polymerization method such as polyvinyl chloride (PVC or vinyl), polystyrene, polyethylene,
polypropylene, poly acrylonitrile, PVB, silicone and many more [2]; natural polymers are derived
from sources within the body such as hyaluronic acid, collagen and fibrin (from carbohydrates) or
outside such as chitin, chitosan (from exoskeletons of crawfish and shrimp shells) or alginate (from
seaweed). There are three main classes of biopolymers: polysaccharides, polypeptides, and
polynucleotides. In living cells, they may be synthesized by enzyme-mediated processes, such as
the formation of DNA catalyzed by DNA polymerase
1.2. Chitin (Ch)
Chitin is the second most abundant biopolymers found in nature next to cellulose. It is
primarily distributed in the exoskeleton of animals such as insects; crustaceans (crab, shrimp,
lobsters and etc.) [7, 8]; and also found in microorganisms, e.g. in the cell walls and structural
membranes of mycelia of fungi, yeast and green algae in varying proportions from species to
species and from region to region [9-11]. Chitin is composed of linear repeating units of N-acetylD-glucosamine (GlcNAc), which are linked by β-(1-4)-glycosidic bonds [12, 13]. It is used as a
raw material for the industrial production of many products as chitosans, oligosaccharides and
glucosamine (GlcN)[14]. The chemical structure of chitin (C8H13O5N)n is similar to cellulose,
having one hydroxyl group on each monomer substituted with an acetyl amine group [15-17].
2
Chapter 1
Figure 1: Chemical structure of cellulose and chitin
Due to the hydrogen bond, chitin has been found in three polymorphic forms α, β and γ chitin [18,
19], which differ in the arrangement of the chains within the crystalline regions as shown below.
Figure 2: Different forms of chitin
The three chitin types can be differentiated by IR and solid-state NMR spectroscopy
together with X-ray diffraction. These types of chitin are mainly differ in the degree of hydration,
in the size of the unit cell and in the number of chitin chains per unit cell [20-22]. In α-chitin, the
chains are arranged in an antiparallel direction and it is mostly found in crabs, shrimp, insects,
fungi and yeast. In β-chitin, all chains are parallel, while in γ-chitin two out of three chains are
3
Chapter 1
parallel with the third oriented in the opposite direction and both β and γ forms are found in squid
pens and cocoons [23].
Like cellulose, chitin is insoluble in water and most organic solvents[24]; the compact
structure and the crystallinity of chitin are responsible of this character and this owing to the strong
intra- and inter-molecular hydrogen bonds. The solubility of chitin is the main problem inherent
with operating difficulties [25, 26]. Chitins exist in nature associated with proteins, minerals, and
pigments [27], all these components have to be removed to reach to the highest purity which is
necessary for using in bio-medical applications. There are two methods to extract chitin chemical
method and biological method [28]. The chemical method performed by using acids and bases[28],
while the biological method by using enzymatic or fermentation treatment [29, 30]. In the skeletal
tissue, chitin combine with protein and form protein-chitin matrix, which is yield hard shells [31].
Chitin can be isolated through two major steps demineralization and deproteinization; in the first
step minerals as calcium carbonates are dissolved by acidic treatment of the shell, the most acidic
reagent used in this demineralization process is hydrochloric acid while in the second step the
alkaline treatment is used in order to cleavage the covalent chemical bonds between the chitin–
protein complexes, alkaline solution from sodium hydroxide is mostly used to remove the proteins,
lipids and pigments from the shell [28, 32, 33]. One of the main reactions carried out on chitin is
the deacetylation, strong concentration of sodium hydroxide is used to remove the acetyl groups
from chitin chain in order to obtain chitosan, the main derivative of chitin[34, 35]. While Chitin
has been confirmed as attractive biopolymer with various properties, its applications are limited
due to its hydrophobic character [36]. Although, it can be used in different fields such as textiles,
water treatment, cosmetics and in biomedical applications [37, 38] .
4
Chapter 1
1.3. Chitosan (Cs)
Chitosan, the N-deacetylated form of chitin can be prepared by cleavage of N-acetyl groups
of chitin from alkaline or enzymatic deacetylation process [39-41]. It can be described as a
copolymer structure comprised of glucosamine and N-acetyl glucosamine. Chitosan is only present
in some fungi with small quantities [42]. It exhibits various physicochemical and biological
characteristics as antimicrobial [43], antioxidant[44], antitumor [3] and anticancer leading to use
in different applications as cosmetics, biotechnology, water purification [45], pharmaceuticals,
food additives and agriculture[46, 47].
Figure 3: Chemical structure of chitosan
Study of chitin and chitosan structure showed that these polysaccharides are linear semicrystalline polymers; and this crystallinity is a function of the degree of deacetylation. Both chitin
and chitosan tend to form micro-fibrils that are joined by hydrogen bonds between (NH2 group
and C=O group) in order to form supramolecular structure]48 ,17[. Chitosan can be obtained from
chitin using high concentrated sodium hydroxide solution [49]; The degree of deacetylation (DDA)
and the molecular weight (MW) are the fundamental parameters in characterizing chitosan [50].
The DDA represents the molar % of monomeric unit that have free amino groups, it can be
determined by various methods like Infrared spectroscopy, NMR spectroscopy, elemental
analysis, and acid-base titration, if the DDA is increased than 40%, the term chitosan is used.
Also, different techniques can be used to determine the molecular weight of chitosan like gel
5
Chapter 1
Permeation Chromatography (GPC), Static Light Scattering (SLS), and intrinsic viscosity
measurement. The molecular weight can be range from 300 kDa to over 1000 kDa varies from
source to source and from place to place [51].
Since chitosan has three functional groups along its chain (hydroxyl group, primary and
secondary amino group) this make it highly reactive material which can be used in different
application. Also, the cationic character of chitosan (protonation of amino groups NH3+) is
demonstrated highly reactivity of this polysaccharide; the chitosan surfaces support adhesion,
proliferation and growth types of cells and this is known as electrostatic interactions [52]. Chitosan
is soluble in organic and inorganic acids below pH 6 like hydrochloric, acetic and lactic acid this
is because this polymer can be regarded as strong base since it has amino group with pKa 6.3 [36].
Also the DDA and the cationic character of chitosan effect on its solubility, because the protonated
amine groups contribute to destroy the hydrogen bonds [53, 54]. On other hand, Chitosan can be
used in water treatment [55], in the last years it has gained wide attention as effective bio-sorbent
due to low cost and high contents of amino and hydroxyl functional groups which show significant
adsorption potential for the removal of various aquatic pollutants [56] Natural chitosan has been
modified by several methods in order to enhance the adsorption capacity for various types of
pollutants. Different shapes of chitosan, such as membranes, microspheres, gel beads and films
have been prepared and examined for the removal of various pollutants from water and wastewater
[57, 58].
Chitosan has many advantages for developing micro/nanoparticles, which can be used for
preparing scaffolds and for gene and drug delivery [4]. Chitosan’s solubility in aqueous acidic
solutions avoids the need for the use of organic solvents when fabricating particulate systems [59].
In addition, the free amino groups become protonated at low pH values which allow the formation
6
Chapter 1
of ionic cross-linking with multivalent anions [60]. Moreover, chitosan surfaces support the
attachment and the subsequent proliferation and growth of different types of cells, which have
been attributed to the high cationic charge density of chitosan [61]. Also, chitosan promotes the
production of transforming growth factor-β-1 (TGF-β1) and platelet-derived growth factor
(PDGF) [62, 63]and maintains the chondrogenic phenotype [64]. Due to their attractive properties,
including non-toxicity, biodegradability, biocompatibility and their natural source [65], Chitin and
chitosan have been investigated thoroughly for different applications, including medical [66],
pharmaceutical [67], textile [68], cosmetics, chemotherapy, wound healing [69], wound dressing
[66], gene delivery [46], tissue engineering and enzyme immobilization [70]. And they can be
easily processed into different products as nanofibers [71], membranes, hydrogels [72], beads,
micro/nanoparticles [73], scaffolds and sponges.
In the past decades, chitosan has been shown highly reactivity in wound healing process;
it can be used in large open skin because chitosan can enhance the functions of inflammatory cells
and promotes granulation and organization [74].
1.4. Hyaluronic acid (HA)
Hyaluronic acid (also known as hyaluronan or hyaluronate) is a naturally occurring
polysaccharide has a linear structure composed of repeat unit of disaccharides N-acetyl
glucosamine and D-glucuronic acid [75-77]. Firstly HA was isolated from the vitreous humor of
bovine ; nowadays it can be isolated from many organs and microorganisms [78]. It is the main
component of the extracellular matrix (ECM) of vertebrate tissues, also it present in all blood
vessels, eyes, ligament, umbilical cord, main artery, tendon and skin [79-81], this due to its vital
role in the body as bind water and lubricate movable parts of the body like muscles and joints [82].
7
Chapter 1
Hyaluronan is a high molecular weight (105-107 Da) and exists as polyanion salt with bioavailable
salt as sodium, potassium, calcium and magnesium [83, 84].
Figure 4: Chemical structure of hyaluronic acid
Hyaluronic acid is a promising biopolymer , it has multiple functions in bacteria and animal
including human [85]; HA is a biocompatible, biodegradable, anti-inflammatory and non-toxic
biopolymer [86, 87]. Also, the oligomer sugars, the degradation products of hyaluronan are
considered nontoxic [88]. HA is viscoelastic and its solution is viscous at a low shear rate but
become elastic at a high shear rate[86]. In this way, HA keeps skin which contains the major
amount of HA, moist and elastic and this elastic properties decrease with age [89, 90]. HA is a
hydrophilic biopolymer and has high rate of elimination and turnover depending on its molecular
weight and location in the body[91]. When it hydrated it can be swollen to 1,000 times the size of
the dry molecule. It can be degraded into smaller oligosaccharides by the action of enzymes such
as hyaluronidase (hyase), β-D-glucuronidase [92]. Hyaluronic acid can be prepared with various
molecular weights (low and high molecular weight) and can be bind to several other materials in
order to use in different field [93]. The mechanical properties of hyaluronic acid could be improved
by forming the hydrogel cross-linking of the hydroxyl group and or the carboxyl group of HA
8
Chapter 1
chain; this crosslinking bond has great effect on its properties and on its uses [94, 95]. Furthermore,
hyaluronic acid is able to transform into different physical forms as electro-spun fiber [96], fibrillar
sponges [97, 98], non-woven meshes [97], scaffolds [99], flexible sheets [100] and nanoparticles
fluids [101].
Hyaluronic acid has a diversified in its functions including cell growth, differentiation
and migration [102]. The high reactivity of hyaluronan can be attributed to hyaluronan binding
receptors as cell surface glycoprotein CD44 and the receptor for hyaluronic acid-mediated motility
(RHAMM) [103]. And many tumors like ovarial, colon over express HA-binding receptors CD44
and RHMM and these tumor cells are characterized by enhance by binding and internalization of
HA [104]. CD44-Ha interactions play very important physiological roles, including mediation or
promotion of macrophage aggregation, cell migration, chondrocyte pericellular matrix assembly,
and leukocyte activation [105].
Hyaluronic acid has a wide range of different applications, it can be used in cosmetic field,
medical devices as Guidewires, medical and esthetic applications as dermal filling, tissue
augmentation and eye surgery [106]. Also HA has found success in nanoparticles as drug delivery
agent for different fields including ophthalmic, pulmonary and nasal [107, 108]. Hyaluronic acid
plays a vital role in healing process, it helps in formation of connective tissue which is known as
a Glycosaminoglycan and it’s able to act as an anti-inflammatory agent by scavenging
prostaglandin, metalloproteinases and other bio-active molecules, thus reducing the level of
inflammatory mediator’s [107].
9
Chapter 1
2. Wound healing process
Wounds can be defined as any injury, cut, blow, surgery in the protective layer of skin or
in the other words it’s a damage in epithelium with or without loss the connective tissue [109,
110]. Some studies have been done on understanding the whole process of the wound healing that
has been used by the human body itself [111]. All wounds can be classified into one of the
following categories: mechanical, for instance surgical/traumatic wounds; chronic such as leg
ulcers or diabetic foot ulcers; burns, which may be chemical or thermal injuries and further
classified by depth of injury. Wounds are generally divided into two major types depending on the
time of healing: acute and chronic [112]. The acute wounds is a wound that can be healed in the
expected time without any problem, passing through all phases of healing process; but the acute
wound can be turned into a chronic wound if the healing process is interrupted; so a chronic wound
can be defined as any wound fail to heal in the expected time [113, 114]. The failure to heal can
be return to different factors as impaired venous drainage; metabolic abnormalities; and genetic
disorder, a lack in the oxygen, nutrients and clean environment and there are several types of
chronic wounds such as infection wounds, radiation poisoning wounds, surgical wounds, diabetic
ulcers and pressure ulcer [115].
Healing of wounds is a complicated process of restoring cellular structures and epidermal
tissue in damaged part to its normal condition as closely as possible [116]. The main function of
this process is to prevent the body from being infected through wounds, promote wounds to heal
with a minimum scar, and return skin functions quickly [117]. This dynamic process consisting of
three continuous, overlapping phases, inflammation, proliferation, and remodeling; during these
phases a series of reactions are done among the cells and mediators [118, 119]. The first phase is
inflammatory phase, which occurs immediately after wounding and continue for 24 to 48 h; the
10
Chapter 1
initial response of the wound is Hemostasis (clotting) which prevents further bleeding, where the
damaged blood vessels technically must be sealed[110]. The damaged vessels are sealed off by
platelet cells that act as “utility workers”. The platelets secrete vaso-constructive substances to
provide stable clot sealing of the damaged vessels, epithelial cells have been migrated during a
day and new tissue formation happens over two weeks [120]. Platelet-derived growth factors are
released into the wound that promote the chemotaxis and proliferation of neutrophils in order to
clean the wounds from bacteria, dead tissues or debris [121]. The proliferation phase is defined as
the regenerative phase where the fibroblasts activity produce collagen (the main component of
connective tissue it forms about 30 % of the total protein in mammal) which provides structure to
the wound and replaces the fibronectin–fibrin matrix [122, 123]. This stage can be characterized
by the presence of red tissue in the wound base, replacement of dermal tissues and sometimes subdermal tissues in the deeper wound [124]. The last phase is the remodeling phase which begins at
about 2 to 3 weeks and can take more than 2 years [125]. In this phase, new collagen forms and
wound strength gradually increases [121]. Many factor affecting on healing process as the type of
damage, the ability of the tissue to repair and the general state of the host’s health [126-128].
3. Wound dressing
In the past, health care faced with an increasing number of patients suffering from wounds
and burns difficult to treat and heal. During the healing process, the dressing protects the injury
and contributes to the recovery of dermal and epidermal tissues [129]. Because their
biocompatibility, biodegradability and similarity to macromolecules recognized by the human
body, some natural polymers as polysaccharides (alginates, chitin, chitosan, heparin),
proteoglycans and proteins (collagen, gelatin, fibrin, keratin, silk fibroin, eggshell membrane) are
extensively used in wounds and burns management[130,131]. The wound dressing can be regarded
11
Chapter 1
as a medical means of cleaning and protecting of wounds against physical, chemical, and
microbiological stresses in order to facilitate, accelerate the healing process, decrease the time
needed to heal , and reduce the pain and absorb the drainage [132]. Wound dressing products
should provide the optimum conditions of wound healing, which has many diverse and interrelated
aspects [133]. It should preserve a moist environment at the wound interface, act as a barrier to
microorganisms and allow gaseous exchange. Also, the wound dressing material should be
nontoxic, non-allergenic, and non-adherent; and made from suitable biomaterial that possesses
antimicrobial properties, and promotes wound healing. Achieving this condition has been
approached by using either passive or interactive products. Passive products which protect and
cover wounds have limited use wherein they impair healing by drying out the wound or adhering
to the wound surface [134]. Such products are made of conventional gauzes (yarn-based products
consist of viscose rayon, cotton or polyester); paraffin tulle dressings, non-paraffin non-tulle
products, and/or a combination of all of them [135]. The main drawback for passive products is
that the dressing sticks to the wound resulting in trauma upon removing it. The main focus at this
time is on interactive dressings (3rd generation biomaterials) and highly healing materials on
nonwoven fabrics have recently been of increasing interest to both the academic and industrial
sector [132, 133]. Nowadays many research have been developed in order to make a progress in
wound healing agents[136]. Plants with bioactive constituents have been used in treating burns
and wounds [137]. When the dermis layer is penterated and blood vessels are destroyed after any
cut or injury or etc… wound care and direct treatment of this site of infection is required [138].
Gauze, Films, Hydrogels , Foams , Alginates, Composites, Hydrocolloids and Bioactive/biological
dressings are the common types of wound dressings , some of them are made from woven or
nonwoven material as gauze [135, 139].
12
Chapter 1
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20
Chapter 2
Abstract
Chitin and chitosan were obtained by chemical treatments of shrimp shells. Different
particle sizes (50-1000 µm) of the raw material were used to study its effect on size distribution,
demineralization, deproteinization and deacetylation of chitin and chitosan extraction process. The
particle size in the range of 800-1000 µm was selected to isolate chitin, which was achieved by
measuring nitrogen, protein, ash, and yield %. Hydrochloric acid (5 % v/v) was optimized in
demineralization step to remove the minerals from the starting material. Aqueous solution of
sodium hydroxide (5 % w/v) at 90 °C for (20 h) was used in deproteinization step to remove the
protein. Pure chitin was consequently impregnated into high concentration of sodium hydroxide
(50 %) for 3.5 h at 90 °C to remove the acetyl groups under the formation of high pure chitosan.
The degree of deacetylation (DDA) of chitosan was controlled and evaluated by different tools.
The chemical structure of chitin and chitosan was confirmed by elemental analysis, ATR-FTIR,
H/C NMR, XRD, SEM, UV-Vis spectroscopy, TGA, and acid-base titration. The isolated chitin
and chitosan from shrimp shell showed excellent antibacterial activity against gram (-ve) bacteria
(E.coli) comparing with commercial biopolymers.
Keywords: shrimp shells, chitin, chitosan, isolation, antibacterial activity.
Rasha M. Abdel-Rahman, Radim Hrdina, A. M. Abdel-Mohsen, A. Y. Soliman, F. K. Mohamed, Kazi
Mohsin , Tiago Dinis Pinto. Chitin and Chitosan from Brazilian Atlantic Coast: Isolation, Characterization
and Antibacterial Activity. International Journal of Biological Macromolecules, 2015, 80, 107-120.
21
Chapter 2
1. Introduction
Chitin is one of the most abundant amino polysaccharides found in nature next to cellulose;
it has a wide variety of sources as exoskeleton of crustacean (crab, shrimp crawfish and Oniscus
asellus) [1-3], insect cuticles (Melolontha melolontha) [4], Orthoptera species[5,6], wings of
cockroach [5], Grasshopper Species [7], Medicinal Fungus [8], Larvae and adult Colorado potato
beetle [9], Aquatic Invertebrates [10], Bat guano [11], Resting Eggs of Daphnia longispina [12],
spider species [13], Daphnia magna resting eggs [14], and cell wall of fungi and in the green
algae[15]. The chemical structure of chitin (C8H13O5N)n is similar to cellulose, having one
hydroxyl group on each monomer substituted with an acetyl amine groups. Chitin is a linear chain
composed of (1-4) linked 2-acetamido-2-deoxy-D-glucosamine while chitosan (the main
derivative of chitin) is obtained by removing enough acetyl groups from chitin, the actual
difference between chitin and chitosan is the acetyl content of the polymer [1,2,16-21].
Both chitin and chitosan have high nitrogen content varies from (2-8 %) that makes them
more attractive to several industrial applications. Chitin is insoluble in water and most organic
solvents, this due to its rigid structure and strong intra and inters molecular hydrogen bonds. In
contrast, chitosan is readily soluble in diluted acids (as hydrochloric acid, citric acid and acetic
acid) with pH below 6 [22-27]. Chitin and chitosan have great economic impact due to their
biological activities and their industrial and biomedical applications. Due to the excellent
properties of chitosan such as adsorption [28], film-forming and antimicrobial properties [29]. It is
used in food preservation [30], cosmetics [31], agriculture [32], biotechnology [33], textiles [1,3,3437] as well as medical fields [38-40]. Shrimp shell wastes contain about 20–30 % chitin which can
be isolated to produce many products such as chitosan, glucosamine and their derivatives, but since
chitin is closely associated with proteins, minerals, and pigments, all these components have to be
22
Chapter 2
removed to reach to the highest purity necessary for bio-medical applications [41-53]. The medical
applications of chitin and chitosan were limited due to the allergic properties of the both
biopolymers [54,56], the phenomenon due to the presence of some impurities like proteins,
pigments, and heavy metals attached into the chitin/chitosan chains. In this thesis, I tried to study
and optimize all conditions to remove the maximum amount of the hazard material interacted with
chitin/chitosan chains.
In this chapter, the acid and base treatments of chitin was fully investigated and optimized,
the particle size effect, obtained by sieving of chitin, was evaluated in term of yield (%) of chitin
and the residual of protein (%) too. The extraction sequencing of chitin and chitosan was
extensively studied which is considered one of the novelty of this work. In addition, the
antibacterial assessment of both commercially and isolated chitin and chitosan was evaluated using
a novel bioluminescence technique.
2. Experimental
2.1. Materials
Shrimp shells obtained from VCI BrasilIndústriae Commerce de Embalagens Ltda.
(Brazil) were selected as a source of raw material used for the extraction of chitin/chitosan.
Shrimps were captured in the Atlantic coast of São Paulo, Brazil. Sodium hydroxide, hydrochloric
acid, ethanol, isopropyl alcohol and acetone were purchased from Lach-Ner, s.r.o., Czech
Republic.
2.1.1. Samples preparation
In order to remove the water-soluble impurities and soluble impurities from the raw material
of shrimp, the shrimp shell wastes were washed with tap water and acetone and then dried at 60
°C overnight in dry air oven. The dried material was boiled for 5 h in distilled water and then
23
Chapter 2
refluxed with isopropyl alcohol for 2 h to remove impurities attached into the shrimp surface. Thus,
the sample mass decreased after purification by 10-15 %. The last step before the extraction
process was sieved the shell to different particle sizes from (less 50, 100-200, 200-300, 300400,400-500, 500-700, 800-1000 µm).
2.1.2. Sieving shrimp shell to different particle sizes
Particle size was determined on a duplicate 100 g sample with a Bühler laboratory siever
MLU 300 (Bühler-Miag, 9240 Uzwil, Switzerland) using a set of woven-wire cloth sieves having
a diameter of 26 cm (12 sieves maximum). The sieve openings were chosen according to AFNOR
specifications NF X11-501 (AFNOR NF X11-501, 1970) which recommend a geometrical
progression of screen sizes in a 50–1000 μm range. The sieving time was 30 min.
2.2. Isolation of chitin and chitosan from shrimp shells
The isolation of chitin and chitosan from shrimp shell begun with the preparation of the
samples followed by three classical steps: demineralization (DM), deproteinization (DP) and
deacetylation (DA) as described below. Demineralization was carried out by using hydrochloric
acid solution. This treatment was done at ambient temperature (25 ± 2 ºC) with varying times (0.55 h). The emission of CO2 gas was observed during the reaction of carbonates with diluted
hydrochloric acid [6]. The resulting solid fraction of crude chitin was washed with distilled water
until neutral pH was achieved, then the chitin samples were dried at 60°C for 24 h. During this
process, the effect of hydrochloric acid with different concentration on the yield, nitrogen and ash
content in the product was studied.
Deproteinization (DP) was done by using aqueous sodium hydroxide. Dry crude chitin was
dispersed into aqueous solution of sodium hydroxide to remove the non-bounded material like
proteins, dyes, lipids and pigments [6]. Thus, crude chitin was stirred in 5 % of sodium hydroxide
24
Chapter 2
at 90 ºC for 20 h, where the alkaline solution was exchanged every 2 h with fresh solution of
sodium hydroxide. The product was filtered off, washed with demineralized water until
neutralization after which the sample was dried at 60 °C for 24 h. Deacetylation step was achieved
by impeded the chitin sample under reflux with 20 – 60 % of sodium hydroxide [7] at 90 0C with
(1/30 w/w) solid to solvent in the time range 0.5-5 h. The product; chitosan was collected, washed
to neutrality using demineralized water, rinsed with acetone, and vacuum filtered off and dried at
60 °C for 5 h to remove moisture.
2.3. Characterization of chitin and chitosan
2.3.1. Inductively coupled plasma optical emission spectrometry (ICP-OES)
Elemental analysis was carried out using the sequential, radically observed inductively
coupled plasma atomic emission spectrometer INTEGRA XL-2 (GBC, Dandenong Australia),
furnished with the ceramic V-groove nebulizers and the glass cyclonic spray chamber (both Glass
expansion, Australia). The limits of detection (the concentration equivalent to three times standard
deviation of the blank sample in the place of background correction) were 10 μg/L for lines 338,
289 and 328 nm, for analysis of real samples, results from both analytical lines used were averaged.
2.3.2. Scanning electron microscopy (SEM)
All sample images were taken by scanning electron microscope; Pardubice University., Tescan
VEGA II LSU electron microscope (Tescan USA Inc.) under the following conditions: high
voltage 5 kV, working distance 4.4 mm, display mode secondary electrons, high vacuum room
temperature. SC7620 Mini Sputter Coater (Quorum Technologies, UK) applied 15 nm layers of
gold particles on the sample. The samples were dusted for 120 s with the current of 18 mA. The
pictures were made at these conditions: voltage 2.44–10 kV, detector-SE. the magnification 300–
25
Chapter 2
20000 times, vacuum high, the distance between sample and objective: 4 - 5 mm. The nonwoven
dressing sheets were cut with a razor scalpel after being frozen in liquid nitrogen for 10 min.
2.3.3. Elemental analysis
The micro- analyses were performed on an apparatus of FISONS. Instruments EA 1108 C;
H; N. The content of proteins (%) was calculated from the nitrogen content by using the following
equation 1[6,42],
P% = [N%- 6.9] * 6.25
Eq. 1
where P % represents the percentage of proteins remaining in the deproteinized shell, N %
represents the percentage of nitrogen measured by elemental analysis, 6.9 corresponds to the
theoretical percentage of nitrogen in fully acetylated chitin (this value was adjusted as a function
of DA, the degree of acetylation), and 6.25 corresponds to the theoretical percentage of nitrogen
in proteins. All the determinations were done in quadruplicate [37].
2.3.4. UV-Vis spectroscopy
UV/Vis spectroscopy was also used to determine the P %. The measurements were carried
out on UV-visible spectrophotometer UV-160A, Shimadzu, Japan using quartz cuvette with an
optical path of 1 cm. The concentration of the measured solutions was kept at 0.59 mg.ml-1. The
protein content in supernatant was calculated from the following equation 2[43,57],
P % = 2.37 (A564/W)
Eq.2
where the A564 was the absorbance value at 564 nm and W was the weight of sample in mg.
2.3.5. Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR)
Fourier transform infrared spectroscopy (FTIR) was performed by using a Nicolet Impact
400 D FTIR-ATR spectrophotometer (Nicolet CZ, Prague, Czech Republic) equipped with a ZnSe
crystal for the FTIR spectroscopy. Transmittance was measured as a function of the wavenumber
26
Chapter 2
between 4000 cm−1 and 600 cm−1 with the resolution of 8 cm−1 and the number of scans equal to
32. The degree of deacetylation of chitosan was calculated from equation 3 [6,34],
DA% = 100 - [(A1655/A3450)* 100/ 1.33]
Eq.3
where A1655 and A 3450 were the absorbance at 1655 cm-1 of the amide I. The factor 1.33 denoted
the value of the ratio of A1655/A3450 for fully N-acetylated chitosan.
2.3.6. Determinations of ash content
Chitin and chitosan ash content were determined by combustion using a constant weight
crucible. Chitosan (2–5 g) was subjected to combustion at constant weight crucible in an oven at
550 °C ± 20 °C for 3 h. The crucible was removed, cooled in desiccators for 30 min, and reweighed (W1). The heating and cooling process were repeated every 1.5 h until a constant weight
was established (W2). The ash percentage was calculated by the equation 4 [58],
Ash%=
W2-W0
W1-W0
* 100
Eq.4
where W0 was the constant weight of crucible, W1 was the weight of sample and crucible, W2 was
the weight of ash and crucible.
2.3.7. Solid NMR spectroscopy
1
D solid-state NMR spectra were measured using a Bruker Avance 500 NMR
spectrometer. Magic angle spinning (MAS) frequency of the sample was 11 kHz. Amplitude
modulated cross-polarization (CP) with duration 1 ms was used to obtain
13
C CP/MAS NMR
spectra with 5 s recycle delay. 13C scale was calibrated with glycine as external standard (176.03
ppm low-field carbonyl signal).
27
Chapter 2
The degree of deacetylation of chitin and chitosan were calculated from equation 5 [59],
ICH3
mol(%)=
*100
(IC1 + IC2 + IC3 + IC4 + IC5 +IC6 )/6
Eq.5
where ICH3 and IC1 corresponds to the integral of the hydrogen atom in NHCOCH3 groups and to
the hydrogen atom of C1 in glucosamine units respectively.
2.3.8. Diffusion NMR spectroscopy
NMR spectra were measured on BRUKER AV 500 MHz Ultra-shield plus with the probe
BBOF plus. For all tests except for accuracy, the solutions of chitosan were prepared by stirring at
room temperature; 10 mg of chitosan in a solution composed of 1.96 ml of D2O and 0.04 ml of
DCl and waiting about 1h to ensure complete dissolution of the polymer. The degree of
deacetylation of chitosan was determined according to the following equation 6 [60,61],
DD(%)=
HID
HID+HAc/3 *100
Eq.6
where H1D was H-NMR signal for proton at C-1 at deacetylation monomer; HAc was signal of
acetyl group.
2.3.9. Acid-base titration
Chitosan sample (0.1 g) was dissolved in 25 ml of 0.1 M HCl aqueous solution. The sample
was titrated with 0.1 M NaOH. pH meter was used for pH measurements under continues stirring
at ambient temperature (25 ± 2 ºC). The titrant was added until the pH value reached to 1.5. The
standard solution of sodium hydroxide was added drop-wise and the pH values of the solutions
were recorded and a curve with two inflection points was obtained. The degree of deacetylation of
chitosan was determined by acid-base titration according to the following equation 7 [62,63],
DDA (%) = CNaOH× (V2-V1) ×161/m
28
Eq.7
Chapter 2
Where, CNaOH was the concentration of sodium hydroxide; (V2-V1) was the difference between two
volume values of sodium hydroxide between the two inflection points; 161 was the molecular mass
unit of chitosan; m was the mass of chitosan sample. The average degree of deacetylation (DDA)
of chitosan samples could be determined from elemental analysis by using the following equation
8 [7,10,64],
DDA (%) = 6.857-C/N/ 1.7143
Eq. 8
Where, C/N was the carbon/nitrogen ratio measured from the elemental composition of the
chitosan samples.
2.3.10. X-Ray diffraction (XRD)
X-ray diffraction was collected on D-8 Advance diffracto-meter (Bruker AXS, Germany)
with Bragg-Brentano θ-θ goniometer (radius 217.5 mm) equipped with a secondary beam curved
graphite mono-chromator and Na (Tl) I scintillation detector. The generator was operated at 40 kV
and 30 mA. The scan was completed at room temperature from 5 to 100° (2θ) in 0.02° step with a
counting time of 8 s per step.
2.3.11. Thermal analysis measurements
Samples were studied with regard to the kinetics of thermal decomposition, using different
heating rate thermo-gravimeter (TG, Netzsch 209F3 instrument, Al2O3 crucible) and under heating
rates of 5 (with data collecting rate of 40 points/K) and 10 °C.min-1 (collecting rate data of 60
points/K). The test temperature range for TG was 25– 600 °C with the sample mass of about 10.35–
1.45 mg under 30 ml.min-1 dynamic nitrogen atmosphere.
2.3.12. Antibacterial activity of chitin and chitosan
Antibacterial effect was examined via bioluminescent (BL) bacteria. Genetically modified
E. coli K-12 capable of bioluminescence were exposed to three different concentrations of the
29
Chapter 2
isolated chitin and chitosan (5, 10,15 mg) which led to diminishment of bacterial viability
visualized by real time BL measurement [6]. Concentrated stock bacterial suspension was prepared
[6], and stored at temperature –80 °C. Final concentration of bacterial cells in applied suspension
was set to approximately 380 000 cells per 100 μL. This suspension (200 μL) was mixed with 5,
10 or 15 mg of isolated chitin and chitosan partly suspended in phosphate buffer (100 μL) in wells
of a white flat-bottom 96-well µl plates (Thermo Scientific, Czech Republic). MC cellulose
samples (5 to15 mg) were used as an appropriate control. Light (490 nm) generated by enzymatic
reaction in bioluminescent bacteria was measured continuously during 60 minutes by luminometer
LM-01T (Immunotech, Czech Republic) at laboratory temperature (25 ± 2 ºC). Results are
expressed in relative light units (RLU). Average integral under the kinetic curve was compared
with control and the percentage of bacterial killing was calculated. Experiments were repeated
independently three times.
2.3. 13. Statistical analysis
All experiments were carried out in triplicate and the results were expressed with ± S.D
(standard deviation).
30
Chapter 2
3. Results and discussion
The present data represents the first attempt to investigate various physicochemical and
functional properties of chitin and chitosan from shrimp shell. The shrimp shells were collected
from Brazilian Atlantic Coast, washing with water, acetone and isopropyl alcohol then dried at
oven at 60 °C overnight (as mentioned before in sample preparation) followed by sieving the
shrimp shell to different particle sizes from (less 50, 100-200, 200-300, 300-400,400-500, 500700, 800-1000 µm) using sieving machine. The variation in chemical, physical, physicochemical
and functional properties of isolated biopolymers in the three sequential processes (DP, DM, DA)
of the isolation process were investigated.
3.1. Isolation of chitin
In shrimp shell, chitin is found as a part of a complex based on proteins and minerals as
calcium carbonate and calcium phosphate deposit to form the rigid shell [65, 66].Thus The
isolation of chitin from Atlantic shrimp shell is summarized in two major steps: removal of calcium
salts in demineralization step using diluted hydrochloric acid and second step: removal of proteins
in deproteinization process using sodium hydroxide [66].
31
Chapter 2
Figure 1: Isolation process of chitin and chitosan from shrimp shell
3.1.1. Demineralization step (DM)
Shrimp shell contains about 25-40 % of minerals and the huge amount of these minerals
are calcium carbonate and calcium phosphate [8, 47]. Hydrochloric acid was used as a reagent in
demineralization step in order to remove the minerals from the shell. In this process, minerals were
hydrolysed into highly water soluble salts which could be separated by filtration and washing with
deionized water for several times. Various concentrations of HCl were used at room temperature
with agitation to detect their effects on nitrogen percent, the yield and ash content [67].
In the beginning, studying on the best yield that obtained from different particle sizes (less 501000 µm) of shrimp shell by using aqueous solution of hydrochloric acid (5 %) was performed as
32
Chapter 2
in Figure 2A; as expected the yield increased with increasing the size of shell. It was improved
slightly with increasing the size of shell from less 50 to 1000 µm, and it could be returned to the
bigger size bonded lower impurities. So, the particle size 800-1000 µm, was chosen to study the
influence of the isolated chitin and chitosan through isolation sequence process. Figure 2B reveals
the effect of different concentration of hydrochloric acid (0.5-5 %) on nitrogen percentage. The
treatments were carried out at room temperature with various time intervals (0.5-5 h). At zero time
treatment, the nitrogen percent was about 4, and increased slowly by increasing the concentration
of HCl (0.5 to 2 %) and there was no significant differences in the treatment time (Figure 2B). By
increasing the concentration of hydrochloric acid from 2-5 %, the nitrogen percent value was raised
from 6 to 8.5 and this could be confirm the presence of large amount of chitin. Thus, the 5 % of
hydrochloric acid produced higher percent of chitin.
Yield percent (%)
80
60
40
20
0
less 50
100-200
200-300
300-400
400-500
500-700
800-1000
Particle size (µm)
Figure 2A: Effect of particle size of shrimp shell on the yield percent after sieving step.
Experimental conditions: different sizes of dry shrimp shell, 5 % aqueous hydrochloric acid, RT (22±2 oC), for 2h.
33
Chapter 2
10
Nitrogen (%)
8
6
4
2
0.50%
0
0
0.5
1%
1
1.5
2%
2
2.5
3%
3
4%
3.5
5%
4
4.5
5
Time (h)
Figure 2B: Demineralization step-effect of hydrochloric acid on nitrogen percent of dry shrimp
shell with particle size 800-1000 µm. Experimental conditions: shrimp shell with particle size 800-1000 µm, ratio
of solid (shrimp shell) to aqueous solution of hydrochloric acid 1:20 (w/v), RT (22± 2 oC).
Figure 2C shows the effect of concentrations of hydrochloric acid on the yield percent
during the treatment of the shrimp shell under different time. It’s obviously clear that, the yield
percentage decreased with increasing the concentration of hydrochloric acid; this is due to the
removal higher percentage of mineral salts (calcium carbonate and/or phosphate) during the
treatment [8]. The yield percentage decreased rapidly in the first 1 h then the rate became constant
as the composition of minerals decreased with demineralization time.
34
Chapter 2
100
90
Yield percent (%)
80
70
60
50
40
30
20
10
0.50%
1%
2%
3%
4%
5%
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Time (h)
Figure 2C: Demineralization step-effect of hydrochloric acid on yield percent of shrimp shell with
particle size (800-1000 µm). Experimental conditions: shrimp shell with particle size 800-1000
µm, ratio of solid (shrimp shell) to aqueous solution of hydrochloric acid 1:20 (w/v), RT (22±2
o
C).
Figure 2D shows the effect of different concentrations of hydrochloric acid on ash content
varying with treatment time. Based on the obtained results, the ash content was decreased rapidly
with increasing the concentration of hydrochloric acid, then it started to be more constant, this
could be explain in term the fast treatment and fast removal of the impurities and the metal salts
in the first hour. The decrease of ash content percentage reflect the purity of chitin. The ratio of
solid to solvent was an important factor affecting in yield percent and nitrogen percent of shrimp
shell.
35
Chapter 2
50
0.50%
1%
2%
3%
4%
5%
45
Ash content (%)
40
35
30
25
20
15
10
5
0
0
1
2
3
4
5
Time (h)
Figure 2D: Demineralization step - effect of hydrochloric acid on ash content of shrimp shell
with particle size 800-1000 µm. Experimental conditions: shrimp shell with particle size 800-1000 µm, ratio
of solid (shrimp shell) to aqueous solution of hydrochloric acid 1:20 (w/v), RT (22±2 oC).
In Figure 2E, the demineralization of shrimp shell with 5 % hydrochloric acid for 2 h at
room temperature with different liquor ratio between solid to solvent 1:5, 1:10, 1:15, 1:20, 10; 30:
1:40, and 1:50 (w/v) were effective in decreasing the yield % and nitrogen % by increasing the
liquor ratio between solid to solvent. According to our result the mineral content obtained from
Atlantic Ocean shrimp shell had lower percentage yield (8-10) which was lower when comparing
with the different sources of chitin in the previous literature as shown in Table S2 [24, 68-71].
36
Chapter 2
Y%
N%
10
80
8
60
6
40
4
20
2
0
0
0
5
10
15
20
Liquor ratio (S/L)
30
40
Nitrogen (%)
Yield (%)
100
50
Figure 2E: Effect of liquor ratio of hydrochloric acid on yield percent and nitrogen percent of
shrimp shell with particle size 800-1000 µm. Experimental conditions: 5 % hydrochloric acid,
shrimp shell with particle size 800-1000 µm, RT (22± 2 oC).
3.1.2. Deproteinization step (DP)
In deproteinization step the chemical treatment is used to destroy the covalent chemical
bonds between the chitin-protein complexes (Proteins are bound by covalent bonds to the chitin
through aspartyl or histidyl residues or both forming stable complexes such as glycoproteins) [72,
73]. Alkaline solution from sodium hydroxide was used to remove the proteins, lipids and
pigments from the shrimp shell [8, 74]. Effect of particle sizes and the time of chemical
deproteinization process influence the properties of the chitin product. Figure 3A shows the effect
of different particle sizes of the shell on the yield percentage and the residual protein percent. The
particle sizes play an important role in the purity of chitin. (Figure 3A) reveals the relation between
particle size and protein content. The amount of protein percent decreased sharply at higher particle
37
Chapter 2
size of the shell. In addition; bigger particle size effect the reaction efficiency of shell with
hydrochloric acid (demineralization step) and/or with sodium hydroxide (deproteinization step).
Based on figure 3A the protein content decreased to 0.005 for particle size 800-1000 µm in
comparing with 0.317 for particle size less 50 µm.
Protien (%)
0.4
0.3
0.2
0.1
0
less 50
100-200
200-300
300-400
400-500
500-700 800-1000
Particle size (µm)
Figure 3A: Effect of particle size of demineralized shell on protein percent of demineralized shell.
Experimental conditions: ratio of solid of demineralized shell to solvent (aqueous NaOH) 1:20
(w/v), RT (22±2 oC), 2h.
Figure 3B shows the relation of treatment time with the protein percent measured by
various two methods. In Figure 3B, the protein percent decreased in the shell by increasing the
time of treatments from (2-20 h) confirmed by elemental analysis. In the same time the percentage
of protein increased in the supernatant by increasing the time of treatment from (2-20 h) confirmed
by UV/Vis spectroscopy. The reason of protein decrease, was the reaction of sodium hydroxide
with the residual impurities [75]; the protein percent was very low comparing with different
sources of shrimp shell in previous literature as shown in table S1[24, 65, 69-71].
38
35
40
30
35
30
25
25
20
20
15
15
10
10
5
Protien (%) by UV spectra
Protien (%) by EA
Chapter 2
5
0
0
2
6
10
14
18
20
Time (h)
Figure 3B: Relation between time of treatment of demineralized shell and protein percentages of
demineralized shell. Experimental conditions: DM-demineralization: 5 % hydrochloric acid, 2 h,
RT (22±2 oC), solid to liquid (1: 20 w/v); DP-deproteinization: 5 % sodium hydroxide, 90 °C, solid
to liquid (1:20 w/v).
3.1.2.1. Scanning electron microscopy of pure chitin
To better understand of chitin morphology, some chitin flakes were isolated from different
places of the exoskeleton and were observed by SEM. Figure 4A shows scanning electron
microscopy of the raw material of shrimp shell after washing and drying process. As shown (Figure
4A), there was heterogeneous in particle size, beside some impurities are attached on the shrimp
surface. Also, a roughness surface of the raw material without porosity was observed which might
be related to their high molecular packing, with inter- or intramolecular hydrogen bonds, imparting
a high crystalline degree to chitin. Figure (4B-H) shows the different particle sizes (less 50-1000
µm) of chitin after deproteinization and demineralization treatments. It was observed that,
independent of part of the shrimp exoskeleton, the isolated chitin retains its fibrils character. After
39
Chapter 2
separation, different particle sizes (Figure 4B-H), the surface morphology of the chitin was
changed due to the treatment with acid and basic agents. The mobility or diffusion of the acid or
basic inside chitin structure was crucial and inhabited by low porosity and high crystallinity of
chitin. Due to the sieving process, each sample has approximately the same particle size, and it
easy to detect the surface changes due to the treatment process. Different sizes of chitin has porous
structure after the extraction process which somehow could significantly reduce the time of
treatments. The surface of the isolated chitin from shrimp shell was found to have smooth surface
structure similar to morphology of chitin isolated from spider species [14]. It was very clear that
the particle size 800-1000 µm gave the best clear morphology, smooth, and high porosity than
other sizes. Therefore to perform all experiments in our study.
40
Chapter 2
Figure 4: Scanning electron microscope of shrimp shell and different particle sizes (sieving
process) of chitin after deproteinization and demineralization steps. Experimental conditions:
DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22±2 oC); DP-deproteinization: 5 % sodium
hydroxide; 20 h, 90 °C; (a) Raw material of shrimp shell bar 500 µm; (b) less 50 (bar 20 µm); (c)
100-200 (bar 20 µm); (d) 200-300 (bar 200 µm); (e) 300-400 (bar 200 µm); (f) 400-500 (bar 200
µm); (g) 500-700 (bar 200 µm); (h) 800-1000 µm (bar 200 µm).
3.2. Isolation of chitosan from chitin (Deacetylation step)
Chitosan was isolated from chitin by removing the acetyl groups from the chain of
chitin using strong concentration of sodium hydroxide in process called deacetylation. Different
parameters could be effect the deacetylation step like concentration of sodium hydroxide, time and
temperature of the reaction medium. In order to determine the effect of sodium hydroxide on the
degree of deacetylation of chitosan (DDA % calculated by Solid NMR), different concentrations
of sodium hydroxide solution (30 %, 40 %. 50 %, 60 %) were used to replace the acetyl group of
chitin with free amino groups. Figure 5A, demonstrated the great influence of NaOH in removing
the acetyl groups along the chitin Chain. The DDA values of chitosan increased gradually as the
41
Chapter 2
sodium hydroxide concentration increased up to 30 % and increased rapidly when the
concentration was higher than 50 %. The DDA of chitosan was lower than 45 % when the
concentration of NaOH was lower than 30 %. However, the DDA of chitosan increased
dramatically up to 95.5 when the concentration of NaOH was between 50-60 %. Figure 5B shows
the influence of time on the DDA of chitosan (calculated by elemental analysis). Chitin was
refluxed with 50 % of sodium hydroxide at 90 °C for different treatment time (0.5-4 h). In the first
hour of the treatment the degree of DDA reached to 40 %; then increased gradually with the time
till 3.5 h and then the DDA became 95 %.
100
DDA (%)
80
60
40
20
0
20
30
40
50
60
NaOH (%)
Figure 5A: DA-deacetylation step - effect of the sodium hydroxide on the degree of deacetylation
of formed chitosan (DDA). Experimental conditions :DM-demineralization: 5 % hydrochloric
acid, 2 h, RT (22±2 oC), solid to liquid (1:20 w/v); DP-deproteinization: 5 % sodium hydroxide,
20 h, 90 °C, solid to liquid (1:20 w/v); DA-deacetylation: 2 h, 90 °C, chitin to liquid (1:30 (w/v).
42
Chapter 2
100
DDA (%)
80
60
40
20
0
1
2
3
4
5
Time (h)
Figure 5B: DA-deacetylation step - effect of time on the degree of deacetylation of chitosan.
Experimental conditions: DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22±2 oC), solid
to liquid (1:20 w/v); DP-deproteinization: 5 % sodium hydroxide, 20 h, 90 °C, solid to liquid
(1:20 w/v); DA-deacetylation: 50 % sodium hydroxide, 90 °C, solid to liquid (1:30 w/v).
3.3. Isolation sequences of chitin and chitosan
Figure 6 shows the extraction sequence process of the isolated chitin and chitosan by
demineralization (DM), deproteinization (DP) and deacetylayion steps (DA); in obviously clear
the sequence treatment steps effect on the yield percent of chitin and chitosan. The yield percent
of chitin and chitosan was higher by the following sequence DMPA>DPMA>DAPM>DAMP,
respectively. Based on Figure 6, the demineralization, deproteinization, and deacetylayion
sequence were the best sequence for isolation of pure and high yield percent of chitin and chitosan
from shrimp shell.[76, 77].
43
Chapter 2
Chitin
60
chitosan
Yield percent (%)
50
40
30
20
10
0
DMPA
DPMA
DAMP
DMAP
Isolation process
Figure 6: Isolation sequence of chitin and chitosan from shrimp shell of particle size (800-1000
µm). Experimental conditions for chitin; DM-demineralization: 5 % hydrochloric acid, 2 h, RT
(22±2 oC), solid to liquid (1:20 w/v); DP-deproteinization: 5 % sodium hydroxide, 20 h, 90 °C,
solid to liquid (1:20 w/v). Experimental conditions for chitosan; DM-demineralization: 5 %
hydrochloric acid, 2 h, RT (22±2 oC), solid to liquid (1:20 w/v); DP-deproteinization: 5 % sodium
hydroxide, 20 h, 90 °C, solid to liquid (1:20 w/v); DA-deacetylation: 50 % sodium hydroxide, 3.5
h, 90 °C, solid to liquid (1:30 w/v), degree of deacetylation DDA (95%, measured by NMR ).
3.4. Thermal stability of chitin and chitosan
Thermal gravimetric analysis of chitin and chitosan has been used to investigate the thermal
degradation and crystallization of the polymers. Figure 7A shows the TGA curves and the
corresponding derivate-grams (DTG) of the isolated chitin and chitosan. The isolated chitin
(Figure 7A) shows two major peaks on the DTG curves. The first, which appeared around 50-100
°C, could be explain as the evaporation of physically adsorbed and strongly hydrogen boned water
to the complex and alcohol solvent, respectively [14, 15, 78]. Second peaks appeared at 330 °C
was related to cleavage of glyosidic amine units by dehydration or deamination. Commercial chitin
44
Chapter 2
had three peaks on the DTG curve. At 50-100 °C, could be explained as the evaporation of
physically adsorbed and strongly hydrogen boned water to the chitin [78]. The second peaks were
appeared at 230 °C, which may corresponding to the residual of proteins, pigments encored to
chitin chains. The third peak appeared at 300 °C, related to cleavage of glyosidic amine units by
dehydration or deamination [79].
Based on the TGA and DTG data of both isolated and
commercial chitin, the isolated chitin from Brazilian Atlantic Ocean was more pure and more
thermal stable than the commercial chitin. Figure 7B showed the TGA and DTG of isolated and
commercial chitosan. The isolated chitosan had two different peaks appeared in DTG curve. The
first peak appeared at 50-90 °C, which was related to the evaporation of residual of water[12]. The
second decomposition peak was appeared at 290 °C, may be related to decomposition of
glucosamine and residual acetyl glucosamine in chitosan chain[80]. In commercial chitosan, there
was two different peaks appear at 50-80 °C and at 270 °C for evaporated water and decomposition
of the glucosamine and residual of acetyl glucose amine units in chitosan chains [80]. From the
comparison data between isolated chitin, chitosan and commercial product chitin, chitosan, the
isolated chitin and chitosan were more thermal stable, pure than commercial products.
45
Chapter 2
120
3
TG of isolated chitin
TG of commerical chitin
Weight (%)
DTG of commerical chitin
2.5
80
2
60
1.5
40
1
20
0.5
0
Deriv.weight (°C/%)
DTG of isolated chitin
100
0
0
100
200
300
400
500
600
Temperature °C
Figure 7A: TGA and DTG of chitin
Experimental conditions; DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22±2 oC); DP deproteinization: 5
% sodium hydroxide, 20 h, 90 °C. Degree of deacetylation DDA (5 %, measured by FTIR).
46
Chapter 2
1
TG of commerical chitosan
TG of isolated chitosan
DTG of commerical chitosan
DTG of isoalted chitosan
100
0.9
0.8
0.7
80
Weight (%)
0.6
60
0.5
0.4
40
0.3
0.2
20
Deriv.weight (°C/%)
120
0.1
0
0
0
100
200
300
400
500
600
Temperature (°C)
Figure 7B: TGA and DTG of chitosan
Experimental conditions; DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22±2 oC); DPdeproteinization: 5 % sodium hydroxide, 20 h, 90 °C; DA-deacetylation: 50 % sodium hydroxide,
3.5 h, 90 °C, degree of deacetylation DDA (95 %, measured by NMR).
3.5. ATR-FTIR of chitin and chitosan
Figure 8 shows the FTIR-ATR of the isolated biopolymers (chitin, chitosan) comparing
with the commercial one. Figure 8A shows the FTIR-ATR of chitin isolated from shrimp shell and
commercial chitin, respectively; both commercial and isolated chitin showed similar peaks there
were not identical. Different peaks appeared at 3470-3390 cm-1 for stretching vibration of hydroxyl
and secondary amine groups and at 2880 cm-1 for methylene groups in chitin. Within the FTIRATR spectrum of isolated β-chitin, bands were observed at 1650 and 1550 cm-1 [6], the peaks were
appeared at 1620 cm-1 and 1590 cm-1 for amide I and amide II, respectively [6, 7]. Beak at 1050
47
Chapter 2
cm-1 correspond to the C-O-C in chitin chain. isolated chitosan (Figure 8B) has a broad band about
3200-3374 cm-1 for stretching vibration of OH and NH groups, 1661 cm-1 for (amide I), 1595 cm1
for (amide II), 1380 cm-1 for (amide III)[5, 7]. The intensity of the absorption band at 1595 cm-1
for very short due to the highly degree of deacetylation of the isolated chitosan (95 %) comparing
with the commercial chitosan. Asymmetric stretching of (C-O-C bridge) and 1080 cm-1 for (C-O
stretching) [11] characteristic of its polysaccharide structure (Figure 8B) in comparing with the
commercial chitosan. Higher similarity was recorded between the commercial and the chitin and
chitosan isolated from Brazilian Atlantic Ocean.
0.1
Isolated chitin
Commercial chitin
0.09
Absorbance (a.u.)
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
3700
3200
2700
2200
1700
1200
700
Wavelength (cm-1)
Figure 8A: FTIR-ATR of chitin Experimental conditions; DM-demineralization: 5 %
hydrochloric acid, 2 h, RT (22±2 oC); DP-deproteinization: 5 %sodium hydroxide, 20 h, 90 °C,
Degree of deacetylation DDA (5 %, measured by FTIR).
48
Chapter 2
0.09
Isolated Chitosan
Commercial chitosan
0.08
Absorbance (a.u.)
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
3700
3200
2700
2200
1700
1200
700
Wavenumber (cm-1)
Figure 8B: FTIR-ATR of chitosan
Experimental conditions; DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22±2 oC); DPdeproteinization: 5 % sodium hydroxide, 20 h, 90 °C; DA-deacetylation: 50 % sodium hydroxide,
3.5 h, 90 °C, degree of deacetylation DDA (95%, measured by FTIR).
3.6. Solid NMR of chitin and chitosan
Solid-state NMR spectroscopy has been identified as the most useful and accurate tool
for structurally analyzing insoluble complex biomaterial [81]. Figure 9 shows solid-NMR spectra
of different deacetylated products prepared from chitin with various time using sodium hydroxide.
The deacetylation temperature and concentration of sodium hydroxide were consistently set at 90
o
C 50 %, respectively. Each spectrum included seven well-defined resonances of C1-C6 and
acetyl. They were ε=173.7 ppm for C=O, ε=102.8 ppm for C1, ε= 82.5 ppm for C4, ε = 74.5 ppm
for C5, ε = 61.3 ppm for C6, ε= 56.1 ppm for C2, and ε=23.2 ppm for CH3. In highly deacetylated
49
Chapter 2
chitosan, there was no any appearance for ε=73.7, and 23.2 for carbonyl and acetyl groups in chitin,
respectively. The DDA increased by increasing the treatment times, DDA (%) = 30 %, 67 %, 93
% and 95 % after 1, 2,3,and 4 h. treated with 50 % sodium hydroxide at 90 °C, respectively, were
illustrated in Figure 9. Different peaks were related to protein residual appeared at ∼ 181.5, 128.7,
32.9, 30.0 and 14.2 ppm [82-84] which were not appeared in our products spectra this indicated
that there was no any residual of protein interconnected to Atlantic source of purified chitin and
chitosan compared with different other sources [82, 85-87].
Figure 9: Solid 13C CP/MAS NMR spectra of chitin and chitosan with different degree of
deacetylation after various time. (a) DDA 30 % after 1h ; (b) DDA 67 % after 2h ; (c) DDA 93 %
after 3h; (d) DDA 95 % after 4h. Experimental conditions; DM-demineralization: 5 %
hydrochloric acid, 2 h, RT (22±2 oC); DP-deproteinization: 5 % sodium hydroxide, 20 h, 90 °C.
50
Chapter 2
3.7. Residual of metals on chitin and chitosan after isolation process
Figure 10 shows the amount of minerals of the isolated chitin and chitosan in comparison
to their source (shrimp shell). The concentrations of calcium, potassium, sodium, magnesium,
aluminum, copper, manganese and titanium was decreased after the treatment with hydrochloric
acid in demineralization step and sodium hydroxide in deproteinization and deacetylation steps of
the extraction chitin and chitosan from the shrimp shell. Comparing with commercial different
sources of chitin and chitosan, the metal residual in commercial products were 20 times higher
than the isolated chitin and chitosan, indicated that the Brazilian Atlantic Ocean biopolymers were
more pure than the commercial biopolymers (Table S1).
Mass of metals* 103 (mg/Kg)
20
Shrimp shell
Chitin
Chitosan
18
16
14
12
10
8
6
4
2
0
Ca
K
Na
Zn
Mg
Mn
Al
Cu
Ti
Minerals Contents
Figure 10: Residual metals in the extracted chitin and chitosan in the comparison with shrimp shell. Experimental
conditions; DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22 oC±2); DP-deproteinization: 5 % sodium
hydroxide, 20 h, 90 °C, DA-deacetylation: 50 % sodium hydroxide, 3.5 h, 90 °C, degree of deacetylation DDA (95
%, measured by NMR).
51
Chapter 2
3.8. X-ray diffraction of chitin and chitosan
The XRD spectra of the isolated chitin and chitosan comparing with commercial
biopolymers were shown in Figure 11. Diffraction pattern of isolated chitin (Figure 11a) exhibits
ten markable diffraction peaks on angle 2θ= 9.3°, 19.3°, 20.9°, 23.5°, 26.5°, 34.8°, 38,9°, positions
of peaks corresponds to inter-planar distances 9.5 Å, 4.6 Å, 4.2 Å, 3.8 Å, 3.4 Å, 2.3 Å which in
accordance with the literature data from different sources of chitin and chitosan [70, 88-90]. The
commercial chitin exhibits also 10 remarkable diffraction peaks on the same position peaks
comparing with isolated one. Only one more beaks at 2θ=12.7° corresponds to inter-planar
distances 7 Å which was corresponding to residual of impurities in commercial chitin like protein
and pigments [91]. Some recent studies also reported similar crystalline peaks in the XRD
measurements of β-chitin [76, 92]. From comparable intensities of corresponding peaks concluded
that the crystallinity of isolated chitin and commercial chitin were similar. The Diffraction patterns
of the isolated and commercial chitosan (Figure 11b) shows comparable data. Isolated chitosan
and commercial chitosan had two broad beaks at 2θ = 9.5°, and 21.5° corresponded to inter-lattice
distances 4.6 Å and 2.6 Å [6]. From XRD data both isolated and commercial chitin and chitosan
(shrimp shell sources) had the same crystalline and amorphous properties.
52
Chapter 2
Figure 11:
XRD of chitin and chitosan
(a) Experimental conditions; DM-demineralization: 5 % hydrochloric acid, 2 h, RT (22 oC±2); DP-deproteinization:
5 % sodium hydroxide, 20 h, 90 °C. (b) Experimental conditions: DM-demineralization: 5 % hydrochloric acid, 2
h, RT (22 oC±2; DP-deproteinization: 5 % sodium hydroxide, 20 h, 90 °C; DA-deacetylation: 50 % sodium hydroxide,
3.5 h, 90 °C; degree of deacetylation DDA (95 %, measured by NMR).
53
Chapter 2
3.9. Scanning electron microscopy of chitosan
The morphology of the isolated chitosan sample was studied using a scanning electron
microscope. SEM images with different magnifications 1000, 500, 200, 50 µm (Figure 12A-D),
respectively; of chitosan sample were presented in Figure 12. It was observed that the biopolymer
surface became very smooth as comparing with the chitin sample before deacetylation step [93,
94].
Figure 12: Scan electron microscope of pure chitosan extracted from shrimp shells with particle
size 800-1000 µm at different magnification (A) 1000 µm; (B) 500 µm; (C) 200 µm; (D) 50 µm,
respectivelly. Experimental conditions; DM-demineralization: 5 % hydrochloric acid, 2 h, RT
(22±2 oC); DP-deproteinization: 5 % sodium hydroxide, 20 h, 90 °C; DA-deacetylation: 50 %
sodium hydroxide, 3.5 h, 90 °C, degree of deacetylation DDA (95 %, measured by NMR).
54
Chapter 2
3.10. Deacetylation degree (DDA) measurements of chitosan by different techniques
Chitosan was obtained from chitin using high concentrated sodium hydroxide solution. The
degree of deacetylation (DDA), was an important parameter that indicated the molar % of
monomeric units that have amino groups and vary from zero (chitin) to 100 (fully deacetylated)
chitosan. DDA of chitosan was measured and compared between the data as described in Table 1.
As shown in Table 1 the DDA of chitin was very low after treatment with 5 % of sodium hydroxide
for 20 h. DDA was 35, 30, 23, and 25 by ATIR-FTIR, H/C-NMR, elemental analysis and acidbase titration, respectively. After removing the acetyl groups from chitin by using high
concentration of sodium hydroxide for 3.5 h at 90 °C, the degree of deacetylation was measured
and confirmed by ATR-FTIR, H/C-NMR, elemental analysis, and acid-base titration and it was
65, 95, 85, 75, respectively. From the data in Table 1, we could conclude that, the DDA of chitosan
have different variable values due to the different techniques used.
Table 1: Determination of degree of deacetylation (DDA) by different analytical techniques
Degree of deacetylation (%)
FTIR
NMR
Elemental analysis
Acid-base titration
Chitin
35±2
30
23±5
25±1.5
Chitosan
65±3
95
85±3
75±2.5
*mean (standard deviation)
3.11. Antibacterial activity of chitin and chitosan
Figure 13, shows the antibacterial activity of chitin and chitosan isolated from shrimp shell
by chemical treatments. The biological activity of chitin and chitosan was evaluated against gram
negative bacteria (E. coli) by chemo-luminescence technique. This technique is more accurate
55
Chapter 2
comparing with the other techniques like disc diffusion method. As described in (Figure 13), three
different concentrations (5, 10, 15 mg) of chitin, chitosan, isolated from Atlantic Ocean and
commercial were measured and evaluated to compare the biological activity of both material. As
shown in Figure 13, the antibacterial activity of the isolated chitin increased by increasing the
concentration of chitin (5, 10, 15 mg). However, with lower reduction rate value (10, 15, and 20
%, respectively). This could be explain by the blocking of the amino groups (low deacetylation
degree, DDA = 5 % confirmed by NMR data). On the other hand the antibacterial activity of
isolated chitosan increased (reduction rate; 60, 80, 90 %) dramatically by increasing the
concentration of chitosan (5, 10 15 mg), since in this case chitosan had highly DDA=95 %
confirmed by NMR data. There was relations between the antibacterial activity of chitosan and the
free amino groups (DDA). The mechanism of the antibacterial activity of chitin and chitosan are
still well unknown. The antibacterial activity of chitin and chitosan has been correlated with its
degree of deacetylation (DDA), purity and molecular weight [1, 13, 18, 21, 95-97]. The
antibacterial or antifungal activity of chitosan depends on the presence of positive charge numbers
created on chitosan chains by DDA step, could be expected to have a more potent antibacterial
activity. From the obtained results, this concluded that the biological activity of both chitin and
chitosan isolated from Atlantic ocean as a source after DM, DP, DA steps had very high
antibacterial activity in comparison with biopolymers (chitin and chitosan) from different other
sources [98-101].
56
Chapter 2
20
18
16
RLU *103
14
12
10
8
6
4
Control
Chitin 15 mg
Chitosan 15 mg
2
0
0
5
10
Chitin 5 mg
Chitosan 5 mg
15
20
25
Chitin 10 mg
Chitosan 10 mg
30
35
40
Time (s)*102
Figure 13: Kinetic curve of the bioluminescence of E-coli within sixty minutes in the presence
of extracted chitin and chitosan. Experimental conditions for chitin; DM-demineralization: 5
% hydrochloric acid, 2 h, RT (22±2 oC); DP-deproteinization: 5 % sodium hydroxide, 20 h, 90
°C. Experimental conditions for chitosan; DM-demineralization: 5 % hydrochloric acid, 2 h,
RT (22 oC±2); DP-deproteinization: 5 % sodium hydroxide, 20 h, 90 °C; DA-deacetylation: 50
% sodium hydroxide, 3.5 h, 90 °C, degree of deacetylation DDA (95 %, measured by NMR).
57
Chapter 2
4. Conclusions
Highly pure chitin and chitosan were isolated from Brazilian Atlantic Coast shrimp shell
by the chemical treatment method. Furthermore, chitosan of different DDA and zero % of protein
is obtained by alkali treatment. The isolated biopolymers were characterized and confirmed by
different analytical tools like ATR-FTIR, TGA, XRD, elemental analysis, NMR, SEM, acid-base
titration
and
compared
with
the
commercial
available
chitin
and
chitosan.
DMPA>DPMA>DAPM>DAMP had the heights value of chitin and chitosan. The isolated chitin
and chitosan showed excellent antibacterial activity against gram-negative bacteria (E. coli). In
addition, the high pure chitin and highly DDA of chitosan were obtained by Demineralization,
deproteinization and deacetylation sequences steps. Finally we can conclude that, chitin and
chitosan from Brazilian Atlantic Ocean had low protein, minerals percentage, high DDA, thermal
stable as well as excellent antibacterial activity compared with the other sources of chitin and
chitosan. According to this finding, Atlantic chitin and chitosan can be used for different medial
application especially for tissue engineering and drug carrier purposes.
58
Chapter 2
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62
Chapter 3
Abstract
Thin layers of chitosan (positively charged) / sodium hyaluronate (negatively
charged) / nonwoven fabrics were constructed by polyelectrolyte multilayer pad-dry-cure
technique. Pure chitosan (CS) was isolated from shrimp shell and immobilized onto
nonwoven fabrics (NWFs) using citric acid (CTA) as cross linker and solvent agents through
a pad-dry-cure method. The prepared thin layer of chitosan citrate/ nonwoven fabrics
(CSCTA/NWFs) were consequently impregnated with hyaluronan (CSCTA/HA/NWFs) in
the second path through a pad-dry-cure method. Chitosan/hyaluronan/nonwoven fabrics
wound dressing were characterized by different tools such as FTIR-ATR, TGA and SEM.
The antibacterial activity and the cytotoxicity of the dressing sheets were evaluated against
Escherichia coli (E.coli) and Streptococcus aureus (St. aureus), mouse fibroblast (NIH-3T3)
and keratinocytes (HaCaT) cell lines, respectively. The cell-fabrics interaction was also
investigated using fluorescence microscope, based on live/dead staining assay of 3T3 cells.
The healing properties of the new wound dressing were evaluated and compared with the
control sample.
Keywords: Hyaluronan, chitosan, nonwoven fabrics, cytotoxicity, healing properties
Rasha M. Abdel-Rahman, A. M. Abdel-Mohsen, R. Hrdina, L. Burgert, Z. Fohlerova, D.
Pavliňák, O. N. Sayed,J. Jancar; Wound Dressing based on Chitosan/hyaluronan/Nonwoven
Fabrics: Preparation, Characterization and Medical Applications. International Journal of
Biological Macromolecules, 89 (2016) 725–736.
63
Chapter 3
1. Introduction
Healing of wound is a complicated process of restoring cellular structures and
epidermal tissue in damaged part to its normal condition as closely as possible [1]. This
dynamic process consisting of three continuous, overlapping phases, the first phase is the
inflammation phase which occurs immediately after wounding and continue for 24 to 48 h,
hemostasis is the beginning of this phase which leads to the inflammation. Platelet-derived
growth factors are released into the wound that promote the chemotaxis and proliferation of
neutrophils in order to clean the wounds from bacteria, dead tissues or debris [2-6]. The
second phase is fibroblast proliferation; in this phase, collagen is produced in order to supply
the structure to wounds and replaces the fibronectin–fibrin matrix. The last phase is the
remodeling which approximately starts after two or three weeks and can take more than two
years. In this phase, new collagen forms and wound strength gradually increases. Many
factors affecting on healing process as the type of damage, the ability of the tissue to repair
and the general state of the host’s health [7-9]. Layer-by-layer (LbL) assembly is a
technique that was used in large scale due to its extraordinary advantages in the preparation
of multilayer films [10-14]. Multilayer films were formed as the result of impregnation
procedure, different alternating polyelectrolyte layers of oppositely charged were
successfully assembled on flat surfaces. Also, these films were easy synthesized on different
substrates. On other hand, these layers had varies features as uniform, continuous, easily
tailored, and resistant to protein adsorption in especial cases [15-27].
Chitosan is a cationic biopolymer, owing to its characteristic as nontoxicity,
biodegradability, biocompatibility, antimicrobial activity and improving the healing of
wounds; chitosan has been widely used as topical dressing in wound management [28-35].
64
Chapter 3
Chitosan is characterized by three functional groups, an amino, acetamido and hydroxyl
groups. Cationic character of amino group (after its potentization) gives good opportunities
for it to react with other compounds, and the key properties of chitosan includes bactericidal,
fungicidal, and it has immune-enhancing properties [28, 36, 37]. Also, chitosan is water
soluble in acidic media due to the protonation of amino group on the second carbon of
glucosamine [37]. Recently, chitosan has received extensive attention because it is a
promising natural substances used in the biomedical, food, and chemical industries [36].
Chitosan and its derivatives have been shown an excellent antimicrobial activities towards
most of living microorganisms as yeast, fungi and bacteria, this make many scientists be
attention by this biopolymers [38, 39]. The anti-bactericidal activity of chitosan is related
to its cationic character with high affinity for the microbial cell wall [28, 38, 40-42].
Hyaluronic acid (HA) is a natural biopolymer found in all living organisms; the major
component in the extracellular of many organs in all body fluids as eyes, skin, joints and
hyaline cartilage [43, 44]. HA has various applications especially in medical field, it can be
used as tumor marker in different types of cancer such as prostate and breast cancer. Also,
it’s used in wound healing repair and improve the biomechanical properties of tissues. HA
interact with many cell surface receptor as CD44, and ICAM-1 causing cellular processess
including morghogensis, wound repair, inflammation and metastasis [45-47]. Furthermore,
HA has a main role in the viscoelasticity of bio-fluids, it can control the hydration of tissue
and transport of water [48-50]. The characteristic of hyaluronic acid as the biocompatibility
and the hydrophobicity makes it an excellent component in cosmetics[50]. Nowadays HA
has been used as drug delivery agent in various fields as ophthalmic, topical and parental
[51].
65
Chapter 3
This chapter describes the preparation of novel wound dressing sheets based on
chitosan/hyaluronan/nonwoven fabrics and their physical, chemical and biological
properties were evaluated.
2. Experimental Section
2.1. Materials
Chitosan was isolated from shrimp shell of Brazilian Atlantic Ocean obtained from
VCI Brasil Indústria e Comércio de Embalagens Ltda. (Brazil). Citric acid was purchased
from Sigma Aldrich (Germany) and hyaluronic acid (sodium salts) high molecular mass
(1.5-1.7 MDa, determined by SEC-MAALS) was purchased from Contipro Biotech Ltd.,
Dolni Dobrouč, Czech Republic. Viscose (100 %, 40 g/m2) nonwoven cotton fabrics and
samples (produced by random distribution for web and spun bonded thermally) were used
in this study and kindly provided from El-Nasr Company for Spinning, Weaving and
Dyeing–El-Mahalla El-Kubra, Egypt. These fabrics were cut into identical sheets (10 × 10
cm) and used for all experiments as described below.
2.2. Isolation of chitosan from shrimp shell
Pure chitosan (CS) was isolated from Brazilian Atlantic ocean as described in
chapter 2 [28]. Briefly, shrimp shells were washed with water, acetone and isopropyl alcohol
to remove sand, salts and other impurities. Washing was followed by three classical steps:
the first step was demineralization which was carried out by 5 % hydrochloric acid solution
for 2 h; the second step was deproteinization step (DP) which was done by the use of 5 %
aqueous sodium hydroxide for 20 h; finally, the crude chitin was stirred in 50 % sodium
hydroxide for 3.5 h to obtain pure chitosan. The isolated chitosan was fully characterized
66
Chapter 3
by FTIR, X-ray diffraction, NMR, TGA-DTG and elemental analysis. The deacetylation
degree of chitosan was 95 % (determined by NMR and FTIR-ATR techniques).
2.2.1. Immobilization of chitosan citrate/hyaluronan onto nonwoven cotton fabrics
Cotton nonwoven fabrics (NWFs) were used as substrates for immobilization of
chitosan citrate (CSCTA) and sodium hyaluronate (HA). Firstly, cotton nonwoven fabrics
were washed with hot water for 30 min then with 0.1 % sodium bicarbonate to activate the
fabric surface. The nonwoven fabrics (10 ×10 cm) were immersed in chitosan citrate (CTA
was 5 wt. %) of 0.5, 1, 2 % solutions for 30 min, then squeezed at constant pressure using
padding machine. The CSCTA/NWFs (coded as CS0.5CTA/NWFs, CS1CTA/NWFs and
CS2CTA/NWFs) were dried at 80 oC for 10 min in thermal fixation machine. The
CSCTA/NWFs were rinsed for three times with ultrapure water to remove nonattached
(non-bonded) materials from the surface of nonwoven sheets and then dried at 80 oC for
10 min. The washing step was repeated three times to obtain layer of chitosan citrate on
cotton layer (CSCTA/NWFs).
The CSCTA/NWFs (coded as CS0.5CTA/NWFs, CS1CTA/NWFs, CS2CTA/NWFs)
were immersed in the second bath contained 0.5 wt. % of sodium hyaluronate, then
CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs, CS2CTA/HA/NWFs were dried at 80 oC for 10
min, rinsed with ultrapure water to remove nonattached (non-bonded) materials. The
washing step was repeated three times to obtain layer of hyaluronan layer onto chitosan
citrate layers. Keep the wet pick up 100 % in immersing step in each step. All the prepared
layers assembled nonwoven fabrics were dried at 150 oC for 5 min. The CSCTA/HA/NWFs
were code as CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs and CS2CTA/HA/NWFs.
67
Chapter 3
2.3. Wound dressing characterization
Attenuated total reflectance Fourier transforms infrared spectroscopy (ATR-FTIR)
was performed by the spectrophotometer Nicolet Impact 400 D FTIR-ATR (Nicolet CZ,
Prague, Czech Republic) equipped with a ZnSe crystal for the ATR-FTIR spectroscopy.
Transmittance was measured as a function of the wavenumber (cm-1) between 4000 cm−1
and 650 cm−1 with the resolution of 8 cm−1 and the number of scans equal to 128. Thermal
decomposition was measured on thermogravimetric TG, Netzsch 209F3 instrument (Al2O3
crucible) with the heating rate 10 °C min-1 (with data collecting rate 40 points per Kelvin),
which was performed in the dynamic nitrogen atmosphere with the pressure of 0.1 MPa.
The sample mass for TGA was about 0.9 mg, TGA temperature range 25-800 °C. The
morphology of nonwoven fabrics was studied by the scanning electron microscopy (SEM).
The nonwoven dressing sheets were cut with a razor scalpel after being frozen in liquid
nitrogen for 10 min. X-Ray diffraction (XRD) data were collected utilizing the D8 Advance
diffractometer (Bruker AXS, Germany) with Bragg-Brentano θ-θ goniometer (radius 217.5
mm) equipped with a secondary beam curved graphite mono-chromator and Na(Tl) I
scintillation detector. The generator was operated at 40 kV and 30 mA and the scan was
performed at room temperature from 2 to 50° (2θ) in 0.02° steps with a counting time of 8s
per step.
The antibacterial activity of the wound dressing sheets was evaluated by the following way:
Muller-Hinton agar (which consists of 3 g beef extract powder, 17.5 g hydrolyzed casein,
1.5 g starch and 17 g agar) was suspended in one liter of distilled water and heated to the
formation of clear solution. The prepared medium was sterilized by autoclaving at 121 ºC
for 15 min [52, 53]. The nonwoven fabrics were divided into three groups. The first group
68
Chapter 3
was control group (CTA/NWFs; treated with 5 % of citric acid). The second group were
nonwoven fabrics (NWFs) treated with 0.5 %, 1 % and 2 % of chitosan citrate forming
CSCTA/NWFs coded as (CS0.5CTA/NWFs, CS1CTA/NWFs, CS2CTA/NWFs). The third
group were CSCTA/NWFs immersed in 0.5 % sodium hyaluronate solution forming
CSCTA/HA/NWFs assembly coded as (CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs, and
CS2CTA/HA/NWFs). The all prepared nonwoven fabrics (untreated/treated) were sterilized
by autoclaving at 121 °C for 20 min. All treated and non-treated wound dressing code as in
table S1.
The antibacterial activity of the wound dressing nonwoven fabrics was performed
as follow [54, 55]; 1 cm of each wound dressing nonwoven fabric were prepared in sterilize
manner. Each sample was placed in a sterile vial and the dressing nonwoven fabrics were
pre-treated with 5 ml distilled water for 30 min. Tryptone soy broth (3 ml). 10 µl of
Escherichia coli (E. coli) and Staphylococcus aureus (St. aureus) suspension was added to
each nonwoven dressing fabric-containing vial (1.6 × 103/ml). Control agar with/without
bacterial inoculation was also included. The vials were incubated with agitation at 37 ◦C at
500 rpm. Aliquots of 10 µl broth were sampled at 24 h and serial dilution for the aliquots
was prepared in broth. Duplicate aliquots (50 µl) of the serially diluted samples were spread
onto plates. The plates were incubated at 37 ◦C and bacterial counts were performed. The
bactericidal activity was evaluated after 24 h and the Antibacterial efficacy of bacteria was
calculated by the following equation 1:
Antibacterial efficacy (%) = (A − B) A × 100
(1)
Where, A= the number of bacterial colonies from untreated nonwoven fabrics, and B = the
number of bacterial colonies from treated wound dressing nonwoven fabrics. Each
69
Chapter 3
measurement was carried out in triplicate and the arithmetic means are reported for all
experiments.
The swelling ratio of nonwoven fabrics (NWFs), fabrics immobilized with chitosan
citrate (CSCTA/NWFs), and fabrics immobilized with chitosan citrate/sodium hyaluronate
(CSCTA/HA/NWFs) was measured under the physiological conditions. Thus, the samples
were immersed in phosphate buffer solution (pH 7.5) at 37 oC for different times (0.5 - 72
h). The swelling percentage of the wound dressing was calculated by the following equation
2;
Swelling percentage (%) = (Wf-Wi)/Wi ×100
(1)
where, Wf was weight of sample after certain swelling time and Wi was weight of sample
in the dry state.
Cell viability assays are commonly used to measure the response of cells to toxic
substances. Mouse fibroblast cell line (NIH 3T3) was cultured till 20th passage. Cells of 5th20th passage were used in the following experiments. NIH-3T3 were grown in DMEM
(Dulbecco's Modified Eagle Medium) supplemented with 10 % FBS, glutamine (0.3
mg/ml), penicillin (100 µ/ml) and streptomycin (0.1 mg/ml) in 7.5 % CO 2 at 37 °C in 75
cm2 culture flask as recommended by the supplier. Spontaneously immortalized human
keratinocyte cell line (HaCaT) was grown in DMEM supplemented with 10% FBS,
glutamine, and gentamycin in 5% CO2 at 37 °C as described previously [56-59]. All
nonwoven cotton fabric (NWFs) treated with citric acid 5 % (CTA/NWFs), nonwoven
cotton fabric (NWFs) treated with chitosan citrate 1 % (CS1CTA/NWFs) and nonwoven
cotton fabric treated with 1 % chitosan citrate then treated with 0.5 % hayluronan
(CS1CTA/HA/NWFs) samples were sterilized by autoclave at 120 oC for 20 min.
70
Chapter 3
3000 (3T3) and 4000 (HaCaT) cells/well were seeded to wells of 96-well test plates. The
cells were cultured for 24 h before treated with the suspended solutions. Then, the suspended
solutions were added to each well using as diluent medium. Cell viability was measured 0,
24, 48, 72 h after treatment using the 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium
bromide (MTT) assay. In the assay, MTT was reduced by viable cells to a colored formazan
salt, which was later released from the cells and determined spectrophotometric [59, 60].
When cells die, they lose the ability to convert MTT into formazan, thus color formation
express of only the viable cells [61]. MTT stock solution was added to the cell culture
medium and plates were incubated at 37°C for 2.5 h. The supernatant was discarded and
cells were lysed in lysis solution for 30 min on a shaker. The optical density was measured
in 96-well plate in triplicate by a versamax microplate reader at a wavelength of 570 nm.
All experiments were carried out at least in four independent repeats.
In vitro qualitative analysis of cell viability as performed with live/dead assay. The
samples of differently treated nonwoven fabrics (0.5 x 0.5 cm) were sterilized using UV
light for 30 min. Before seeding with NIH-3T3 cells, the fabrics were incubated in culture
medium (DMEM with L-glutamine, 10 % FBS, 1 % penicillin/streptomycin) one hour at 37
°C. Cells were seeded on fabrics at a density of 105 /area in 24-well plate. Samples with
seeded 3T3 cells were cultivated at 37 °C and 5% CO2 for 3, 24, 48 and 72 h. Fluorescence
microscopy and live/dead staining (calcein-AM/ propidium iodide) were used to determine
cell viability 3, 24, 48 and 72 h after seeding. The mix of calcein-AM (2 μM) and propidium
(1,5 μM) in PBS was added to fabrics containing seeded cells and incubated for 15 min at
37 °C and 5 % CO2 for live/dead cell detection. Afterwards, fabrics were rinsed twice in
PBS and visualized using a Zeiss Axio Imager 2 microscope.
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Chapter 3
The healing properties of control sample (CTA/NWFs), CS0.5CTA/NWFs, CS1CTA/NWFs,
CS2CTA/NWFs, CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs, and CS2CTA/HA/NWFs
were measured by using phagocytosis test [62,63]. Untreated/treated wound dressing fabrics
were dispersed in phosphate buffer HBSS (pH 7.4) and each sample was analyzed in the
following concentrations: 1.6, 0.8 and 0.4 mg/100 μl. PBS (178 μl) in case of spontaneous
phagocytosis and 158 μl of HBSS in case of activated phagocytosis were added into microwells. The content of micro-wells was mixed with 20 μl of 0.01 M luminophor and 2 μl of
whole human blood. Zymosan (20 μl) particles suspension (2.5 mg/ml) were added into
micro-wells with activated phagocytosis and the control micro-wells did not contain
analyzed substances, otherwise the contents was identical. Light (490 nm) emitted during
reaction was measured continuously (1 h) by luminometer LM-01T. Results were expressed
in relative light units (RLU). Experiments were repeated independently twice with the same
results. The images of samples were done at the scanning-electron microscope. Tuscan
VEGA II LSU electron microscope (Tuscan USA Inc.) under the following conditions: high
voltage 5 kV, working distance 4.4 mm, display mode secondary electrons, high vacuum
room temperature. SC7620 Mini Sputter Coater (Quorum Technologies, UK) applied 15 nm
layers of gold particles on the sample. The samples were dusted for 120 s with the current
of 18 mA. The pictures were made at these conditions: voltage 2.44–10 kV, detector: SE.
the magnification 300–20000 times, vacuum high, the distance between sample and
objective: 4 - 5 mm.
Fifty male rats (Division of veterinary medicine, National Research Centre, Cairo,
Egypt) weighing (150 ± 20 g) were used as the test animals. The animals were maintained
at normal room temperature (25–29 °C) on a 12 h. Light/dark cycle, with free access to a
72
Chapter 3
commercial pellet diet and water. After the wound procedure, the animals were kept in
individual cages. All experiments were conducted according to the National Communities
Council directives on animal care. The dorsal hair of control and treated rats were shaved
and (2.5 × 2.5 cm) diameter full thickness wounds were created with a biopsy punch. The
wounds were cleaned with normal saline and disinfected by 75 % ethanol. The test wounds
(n=3) were then covered with the sterile chitosan/hyaluronan nonwoven fabrics and fixed
with sterile gauze and plasters. Similarly, control wounds (n=3) were covered with sterile
nonwoven cotton fabrics and fixed with plasters. On 0, 3rd, 7th, and 14th, postoperative days,
the dressings were removed and the appearance of the wound was photographed. The wound
area was estimated by outlining the wound area. New dressings were applied to the wound
sites after they were cleaned with normal saline. Photographs of wounds were taken every
one to three days. If the bandage was applied, photographs were taken at each bandage
change. When photographing the wound, a millimeter ruler was placed approximately 5 mm
from the wound edge. Wound surface and ruler were in one plane. The images of the wound
were open in Image J software. The image calibrated using a known length (2–4 cm): The
rate of wound closure was determined by the following equation 2;
Amount of wound healing (%) = wound area on A0-AX/ A0 ×100 (2)
Where, A0- wound area at day 0, Ax wound area at specified time of surgery,
respectively.The Student (– t) test for two samples was used to analyze the significance of
the experimental data (p≤ 0.05). IFS Services Statistical tests on-line service was utilized.
73
Chapter 3
3. Results and discussion
3.1. Immobilization of chitosan citrate/ hyaluronan onto nonwoven cotton fabrics
Pure chitosan (particle size 800-1000 µm) was used in this work by the method described
in previous chapter [28]. Thus, the isolation of chitosan (CS) was done by four consequent
steps. Firstly, shrimp shells were washed for many hours under reflux with hot water to kill
some microorganisms and to remove the organic and inorganic impurities. In
demineralization process (second step) the shrimp shell was treated with aqueous
hydrochloric acid (5 % v/v) for 2 h at room temperature (22 oC) to remove the minerals
(mainly CaCO3) from the shells. The third step was deproteinization step which was done
by aqueous solution of sodium hydroxide (5 % w/v) at 90 °C for 20 h in order to remove
the proteins. The last step was deacetylation, where highly concentrated sodium hydroxide
(50 %) was used for 3.5 h at 90 °C in order to hydrolyze the acetamido groups under the
formation of high pure chitosan. The chemical structure and degree of deacetylation of
chitosan were measured and confirmed by different techniques like ATR-FTIR, TG-DTG,
XRD, NMR (Fig.S1). The degree of deacetylation of chitosan was found 95 %. Citric acid
was used as dual functional material for dissolution of chitosan and as cross-linker agent
between nonwoven fabrics (NWFs)/chitosan (CS) and chitosan/nonwoven fabrics
(CSCTA/NWFs)/ hyaluronan (HA). Citric acid have three carboxylic groups which can
prepare anhydride cycle under higher temperature [30]. Layer by layer (LbL) assembly is
mainly occurred by the electrostatic interactions. This process consist of adsorption and
deposition of different charged constituents and washing step removed the non-reacted
specimens [62]. Citrate was improved the solubility of chitosan compared with other
organic solvent for LbL assembled with sodium hyaluronate. Layer by layer technique itself
74
Chapter 3
was an effective way produced functionalized materials. In fact, both CSCTA and HA are
pH-sensitive. It can be expected that the multilayers agglomerated of CSCTA–HA also
responsive to the change of environmental pHs.
Nonwoven cotton fabrics (NWFs; 10×10 cm, 0.45 g) were first impregnated in a
solution of chitosan citrate solutions (CS0.5CTA, CS1CTA, CS2CTA) prepared different
layer of chitosan citrate onto NWFs surface coded as CS0.5CTA/NWFs, CS1CTA/NWFs,
CS2CTA/NWFs and the molar ratio between CS and CTA was (1:8;1:4; 1:2), respectively
(Fig.1A). During the activated step of NWFs surface the hydroxyl groups in cellulosic
fabrics convert to salt forms (ONa+) can easily interact with carboxylic anhydride in the
citric acid generated during the increased the reaction temperature in curing step. Citric
acid/anhydride citric acid was used as cross-linker between NWFs and CS by amidation or
esterification reactions. CS0.5CTA/NWFs, CS1CTA/NWFs, and CS2CTA/NWFs were dried
at 80 oC for 10 min in thermal fixation machine. The samples were rinsed for three times
with ultrapure water to remove nonattached (non-bonded) materials from the surface of
nonwoven sheets, then dried at 80 oC for 10 min. this step was repeated three times to obtain
LBL (multi-layer) of
chitosan citrate. The CS0.5CTA/NWFs,
CS1CTA/NWFs,
CS2CTA/NWFs were immersed in the second bath contained 0.5 wt. % of sodium
hyaluronate, coded as CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs, CS2CTA/HA/NWFs
(molar ratio between CS0, 5, 1, 2 %) and HA was 2:1, 4:1, 8:1, respectively. The dressing
materials were dried, washed with water to remove the non-bonded hyaluronan then
immersed again in HA solution, then dried at 80 oC for 10 min. All the prepared nonwoven
fabrics were dried at 150 oC for 5 min. Different layers of hyaluronan were generated on
the surface of CSCTA/NWFs by pad-dry cure technique. Figure 1B shows the proposed
75
Chapter 3
mechanism of interactions between NWFs, CS and CTA. CTA could interact with hydroxyl
groups on NWFs surface formed ester bond as well as with CS formed ester/and or amide
bonds with hydroxyl and amino groups of chitosan chains under higher temperature. During
the layers preparations efficiency of interactions was improved. Hyaluronan layer was
generated during the impregnated process of CS0.5CTA/NWFs, CS1CTA/NWFs, and
CS2CTA/NWFs into sodium hyaluronan solution. Hyaluronan have different functional
groups like coo- and -OH which could interact and/or assembled with the chitosan layers
on the surface of nonwoven fabrics formed new ester or amide bonds between
CSCTA/NWFs and HA (Fig.1B). During dissolution of hyaluronan in alkaline solution, the
deacetylation process was partially done and few free amino groups [57, 63] were generated,
and the pH of final product was 4.7.
A
Padder machine Drying
Chitosan citrate solution
Padder machine
washing
Hyaluronan solution
Drying machine
OH
NH2
CONH
washing
NWFs/CSCTA/HA
COONa
Fig.1A: Immobilization of chitosan citrate Layers, sodium hyaluronate layers onto
nonwoven fabrics.
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Chapter 3
NH2
OH
1)
NH2 OH
CS
+
CH2COOH
HO C COOH
CH2COOH
CS
H2O
OH
NH3
CH2COO
HO C COOH
CH2COOH
NH2 OH
2)
OH
OH
ONa OH
NWFs
OH
NaHCO3
+
NWFs
OH
OH
3)
OH
NWFs
ONa
NH2
+
ONa
ONa OH
CS
ONa OH
OH
H2O
NWFs
OH
NH3
CH2COO
HO C COOH
CH2COOH
O
Ester bond
C O
HO C COOH
H2C C NH OH
O
OH
Amide bond
CS
NH2 OH
ONa OH
OH
COONa
OH
COONa
H3COCHN
NWFs
HA
O
Ester bond
C O
HO C COOH
C NH OH
H2C
O
CS
Amide bond
OH
H3COCHN
OH
HN
C
O
OH
H3COCHN
HA
COONa
H3COCHN
HO
CS=chitosan; HA=hyaluronan; NWFs=nonwoven fabrics
Figure 1B: Proposed mechanism of layer by layer assembled of chitosan citrate (CSCTA),
sodium hyaluronate (HA) onto nonwoven fabrics.
77
Chapter 3
3.2. Physical characterization of the wound dressing
Figure 2A shows ATR-FTIR spectra of nonwoven fabrics (NWFs), chitosan
citrate/nonwoven
(CS0.5CTA/NWFs,
chitosan/hyaluronan/nonwoven
fabrics
CS1CTA/NWFs,
CS2CTA/NWFs),
(CS0.5CTA/HA/NWFs,
and
CS1CTA/HA/NWFs,
CS2CTA/HA/NWFs) sheets prepared by layer by layer assembled technique.
Polycarboxylic acid such as citric acid (CTA), butane-tetrcarboxylic acid (BUTCA) could
form a five member cyclic anhydride as reactive intermediate by dehydrate of two
carboxylic groups bound to adjacent carbons in its backbone, which could reacted with a
hydroxyl groups in nonwoven cellulosic fabrics and/or hydroxyl/ amino groups in the
backbone of chitosan chains[30, 32, 64-67]. New band was appeared at 1715-1720 cm-1 due
to the overlapping band between carbonyl, carboxyl and ester bonds formed between
cellulosic nonwoven fabrics (hydroxyl groups) and chitosan (hydroxyl groups). The
intensity of the carbonyl/carboxylic/ ester decreased by increase the concentrations of
chitosan citrate [68-70]. Due to the presence of chitosan (free hydroxyl, amino groups), it
could easy interact with a cyclic anhydride of citric acid and prevented appearance of
anhydride cyclic ester band at 1852 and 1785 cm-1, respectively[64]. It was seen in figure
2Aa that the intensity of amide bond at 1650 cm-1 was slightly increased by increase of
chitosan concentrations (0.5-2 wt. %). by addition of hyaluronan layers interacted with
chitosan/nonwoven fabrics layers (CSCTA/CNFs), the intensity of the band 1715 cm-1 was
slightly increased due to the formation of ester bonds between chitosan citrate and
hyaluronan (carboxyl, Hydroxyl) groups. (Fig. 2A). It was seen in figure 2Aa that the
intensity of amide bond at 1650 cm-1 was slightly increased by increase of chitosan
concentrations (0.5-2 wt. %) after addition of hyaluronan layer generated. From figure 2,
78
Chapter 3
we could conclude that citric acid can work as cross-linking agent between nonwoven
cellulosic fabrics/chitosan, and sodium hyaluronate generated ester amide, and/or
electrostatic hydrogen bonds between the different functional groups of the biomaterial and
fabric matrix.
Figure 2A: FTIR-ATR of chitosan citrate/nonwoven fabrics (CS0.5CTA/NWFs,
CS1CTA/NWFs,
CS2CTA/NWFs);
chitosan/sodium
hyaluronate/nonwoven
(CS0.5CTA/HA/NWFs,
CS1CTA/HA/NWFs,
CS2CTA/HA/NWFs),
nonwovens
(CTA/NWFs), respectively.
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Chapter 3
Figure 2B shows the thermogravimetric analysis (TGA-DTG) of wound dressing
chitosan/hyaluronan/ nonwoven fabrics prepared by layer by layer technique. Three
different concentrations of chitosan citrate were used (CS0.5CTA/NWFs, CS1CTA/NWFs,
CS2CTA/NWFs) for preparation different layers on the surface of nonwoven fabrics.
Chitosan citrate/nonwoven fabrics layers had three different stage were appeared. The first
stage was related to evaporate the non-bonded and/or bonded water/solvent solution at
decomposition at 75 oC and the second stage around 250 oC corresponded to decomposition
stage of chitosan citrate. The last stage around 320 oC was associated with the oxidative
degradation of chitosan backbone [30, 32, 71].
Hyaluronan layers were generated onto chitosan citrate/nonwoven fabrics in (Figure
2B) only two different stages were appeared after CSCTA/NWFs impregnated with HA
solution
and
formed
HA
layers
(CS0.5CTA/HA/NWFs,
CS1CTA/HA/NWFs,
CS2CTA/HA/NWFs). There were two different stages distinguished, the first stage was
appeared in the same area of chitosan citrate layers at 75 oC related to the evaporations of
non/bonded water and solvent from the dressing sheets. The stage was appeared when
NWFs was treated with CSCTA solution, it means the hyaluronan assembled on the surface
of chitosan layers improved the stability of wound dressing layers. Furthermore, the second
stage (appeared at 330 oC) was related to decomposition of chitosan/hyaluronan layers , it
was shifted comparing with only chitosan layers. Thus, a assembled of chitosan citrate and
hyaluronan layers provided strong chemical interactions reflected in improved thermal
stability of the new wound dressing sheet.
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Chapter 3
Figure 2B: TGA-DTG of chitosan citrate /nonwoven fabrics (CS0.5CTA/NWFs,
CS1CTA/NWFs,
CS2CTA/NWFs);
(CS0.5CTA/HA/NWFs,
chitosan/sodium
CS1CTA/HA/NWFs,
hyaluronate/nonwoven
CS2CTA/HA/NWFs),
nonwovens
(CTA/NWFs), respectively.
Figure 2C shows the swelling percentage of nonwoven fabrics (NWFs), chitosan
citrate/nonwoven fabrics with different concentrations of chitosan (CS0.5CTA/NWFs,
CS1CTA/NWFs, CS2CTA/NWFs) and chitosan citrate/sodium hyaluronate/nonwoven
fabrics with different chitosan concentrations (CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs,
CS2CTA/HA/NWFs). The layers of chitosan citrate (CSCTA) improved the swelling
percentage of nonwoven fabrics (NWFs) compared with the non-treated sample. The
swelling percentage of nonwoven fabrics was increased with the decrease the concentration
of chitosan from CS0.5CTA/NWFs to CS2CTA/NWFs. This decreasing may be due to the
hydrophobicity of chitosan chains, affecting the swelling of NWFs. After layers of sodium
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Chapter 3
hyaluronate were immobilized onto chitosan citrate/nonwoven fabrics (CSCTA/NWFs)
surface, the swelling percentage (Figure 2C) was increased comparing with CSCTA/NWFs
and plain NWFs and the percentage of swelling was raised by the increase the
concentrations of chitosan (CS0.5CTA/HA/NWFs to CS2CTA/HA/NWFs). Due to the
amphiphilic properties (hydrophobic of chitosan and hydrophilic of hyaluronan) of the
immobilized layers onto NWFs, the swelling percentage was improved.
Swelling percentage (%) *10
8
6
4
2
CS0.5CTA/NWFs
CS2CTA/NWFs
CS1CTA/HA/NWFs
CTA/NWFs
0
0
10
CS1CTA/NWFs
CS0.5CTA/HA/NWFs
CS2CTA/HA/NWFs
20
30
40
Time (h)
Figure 2C: Swelling percentage of wound dressing fabricated from chitosan
citrate/nonwoven fabrics without/with hyaluronan (CS0.5CTA/NWFs, CS1CTA/NWFs,
CS2CTA/NWFs); (CS0.5CTA/HA/NWFs, CS1CTA/HA/NWFs, CS2CTA/HA/NWFs), and
nonwovens (CTA/NWFs) under physiological conditions (PBS, pH 7.5; 37 oC).
82
50
Chapter 3
Figure 3 shows scanning electron microscopy of wound dressing nonwoven fabrics
impregnated with chitosan citrate different concentrations (0.5, 1, 2 wt.%) and sodium
hyaluronate solution (0.5
wt.%) . Layer thickness of chitosan citrate onto the
nonwoven fabrics surface was increased by increase the chitosan concentration for 0.5 %
(a-c), 1 0.5 1 2 % for (d-f) and 2 wt. % for (g-h). From the cross-section (Figure 3 c,f,i),
the chitosan citrate coated
the nonwoven fabrics and
homogenous
layer
with
aggregations appeared on the monofilament surface and between the fabrics matrix. The
hyaluronan layer was assembled on the chitosan/nonwoven fabrics surface by chemical /
physical interactions (Figure 3k-s). The hyaluronan solution was coated the
citrate layer onto nonwoven fabrics and the coated layers thickens was
chitosan
increased
and
CSCTA concentration increase. From the cross-section of CS CTA/HA/NWFs (Figure 3m),
CS CTA / HA/NWFs (Figure 3p), CS CTA/HA/NWFs (Figure 3s), the layers of chitosan /
hyaluronan were surrounded the 0.5 1 2 monofilament formed coated layer on fiber surface.
Figure 3t-z, the plain nonwoven fabrics with clean surface and circular shape from crosssection, respectively.
83
Chapter 3
Figure 3: Scanning electron microscopy of layer by layer wound dressing sheet
CS0.5CTA/NWFs (a,b,c); CS1CTA/NWFs (d,e,f); CS2CTA/NWFs (g,h,i); CS0.5CTA/HA/NWFs (k,l,n);
CS1CTA/HA/NWFs (m,o,p); CS2CTA/NWFs (q,r,s); CTA/NWFs (t,x,z). (a, d,g,k,m,q,t) scale bars were 100
µm; (b,e,h,l,o,r,z) scale bars were 10 µm; (c,f,i,n,p,s,z) scale bars were 5 µm.
84
Chapter 3
Fibers and fabrics materials were carriers of different microorganism like pathogenic
bacteria, odor-producing bacteria, and fungi due to the adhesion of these microorganisms
to the fibers/fabric surface. Most nonwoven fabrics recently used in operation rooms and
hotels were good environment to cross-infection or transfer of diseases occurred by
microorganisms. Bacteria attached on fabrics tend to pass through fabric, especially the
fabric is wet [32]. Research revealed that ordinary hospital textiles could not prevent
bacterial transmission from person in surgical rooms [30]. The innovate of new wound
healing dressing products (dressing) fabricated from immobilization of antibacterial and
highly healing materials on nonwoven fabrics have recently increasing the importance to
both the research and production sectors. Nowadays, a huge range of biological materials
with different chemical structures were immobilized on the fibers/fabrics, bringing new
unique performance to the final fibers/fabrics products [72, 73]. Chitosan, was used as an
antimicrobial agent, have recently received renewed interest [72, 73].
3.3. Antibacterial activity of chitosan/hyaluronan wound dressing
The antibacterial activity of chitosan, chitosan/hyaluronan immobilized onto
nonwoven cotton fabrics against different types of bacteria was evaluated as shown in figure
4. The antibacterial activities of chitosan were remarkably affected by the concentration of
it, (CS0.5CTA/NWFs, CS1CTA/NWFs, and CS2CTA/NWFs) and the type of bacteria.
CS1CTA/NWFs has high reduction rate (RD % = 95, 92 for – G and +G) than
CS0.5CTA/NWFs (RD % = 85, 80 for -G and +G) and for CS2CTA/NWFs (RD (%) = 90,
75 for –G and + G) on the growth of both types of bacteria Staphylococcus aureus (S.
aureus) as Gram positive bacteria (+G) and Escherichia coli (E.coli)as Gram negative
bacteria (-G), respectively. The antibacterial activity of chitosan clearly decreased with low
85
Chapter 3
concentration of its solution and it higher concentrations of chitosan (CS2CTA/NWFs) the
diffusion of chitosan inside the medium was hard and inhibited the interaction of chitosan
and bacteria. Chitosan/hyaluronan/nonwoven cotton fabrics have also highly antibacterial
activity (Table S2) against E.coli and St. aureus compared with plain chitosan coated
nonwoven fabrics. The reduction rate was 88, 84 % for CS0.5CTA/HA/NWFs, 98, 92 % for
CS1CTA/HA/NWFs, and 80, 70 % for CS2CTA/HA/NWFs against E.coli and St. aureus,
respectively. The antibacterial activity of chitosan/hyaluronan was higher than plain
chitosan against both types of bacteria due to hyaluronan has antibacterial activity against
different types of microorganisms [72, 74].
In figure 4, chitosan possess high antibacterial activity against both types of bacteria
especially with gram-negative bacteria; and the mechanisms were proposed that, the
chemical interaction between positively charged chitosan and negatively charged microbial
cell membranes this could be explain as the electrostatic forces between the protonated
+
NH3 groups and the negative residues or the positively charged +NH3 in glucosamine unit
inhibit the growth of bacteria and this was occurred by binding the negatively charged cell
wall [9, 75, 76].
86
Chapter 3
Figure 4: Antibacterial activity of nonwoven wound dressing fabrics
CTA/NWFs (a); CS0.5CTA/NWFs (b); CS1CTA/NWFs (c); CS2CTA/NWFs (d) against
(E.coli),.
CTA/NWFs
CS2CTA/HA/NWFs
CS1CTA/NWFs
(k);
(e)
(h)
CS0.5CTA/HA/NWFs
against
(f);
(E.coli).CTA/NWFs
CS2CTA/NWFs
(i)
against
CS1CTA/HA/NWFs
(i);
(St.
CS0.5CTA/NWFs
aureus),
(g);
(j),
CTA/NWFs
(m);CS0.5CTA/HA/NWFs (n); CS1CTA/HA/NWFs (b); CS2CTA/HA/NWFs (q) against (St.
aureus).
3.4. Cytotoxicity of the wound dressing
Viability assays were studies aimed to detect the cellular response to a toxic materials.
From the evaluation of cytotoxicity, the information about metabolic activity and the death
87
Chapter 3
of cell can be obtained from it [77]. There were many of helpful assay technologies that use
to evaluate metabolic markers to estimate the number of viable cells. The MTT 3-(4.5dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide has been widely used to detect the
viable cells; and this assay is based on the reduction of tetrazolium salt (yellow color) to
insoluble formazan crystals (purple color) by metabolically viable cells [60, 61]. The MTT
viability test was carried out to obtain the main information about the metabolism and
proliferation influenced by CS1CTA/NWFs and CS1CTA/HA/NWFs wound dressing. This
assay is rapid and very effective method for testing for both NIH 3T3 and HaCaT cell line
activity [78, 79]. On the basis of obtained results in figure 5A (a), CS1CTA/NWF did not
induce any changes in the cell growth for both NIH-3T3 and HaCaT cell line and the cell
viability of tested biopolymers demonstrating the nontoxic effect (Fig. 5Aa,d). The viability
of cells after the treatment with chitosan had small increases changed in whole tested time
0-72 hours but still in safe values. Figure 5A (b,e) shows the toxicity of sodium hyaluronate
immobilized into nonwoven fabrics with different concentrations (100-1000 µg/ml) under
different
time
treatments.
Interestingly,
chitosan/hyaluronan
nonwoven
fabrics
(CS1CTA/HA/NWFs) enhanced and accelerated the cell growth with all concentrations
ranges (100-1000 µg/ml) and with different treatment times (0-3 days). This result agrees
very well with the good biocompatibility properties of chitosan/hyaluronan with different
types of cells.
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Chapter 3
Figure 5A: Cytotoxicity measurements of plain chitosan (CS), plain hyaluronan (HA) and
chitosan/hyaluronan treated nonwoven fabrics (CS1CTA/HA/NWFs) with different
concentrations (100-1000 µg/ml), 3T3 viability, MTT, n=6; nonwoven complex sheets with
different concentrations (100-1000 µg/ml), 3T3 viability, MTT, n=4 (a, b, c) respectively.
Toxicity measurement plain chitosan (CS), plain hyaluronan (HA) and chitosan/hyaluronan
treated nonwoven fabrics (CS1CTA/HA/NWFs) with different concentrations (100-1000
µg/ml) on the keratinocytes cell line (HaCaT), (d, e, f) respectively. Cell viability was
measured at 0-3 days. The data were expressed as mean ± SEM (n= 6). Statistically
significant differences (Student’s t-test, p≤0.05) to the non-treated sample (control).
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Chapter 3
Figure 5B shows fluorescence images of CS1CTA/NWFS; a-d, CS1CTA/HA/NWFS; e-h
and cell-wound dressing fabrics interaction (V,VI) after 3 and 72 h of treatments with mouse
fibroblast cell lines (NIH-3T3). From our observation the cells prefer treated fabrics without
any evident differences between CS1CTA/NWFs and CS1CTA/HA/NWFs treated surfaces;
that could be explain as the more biocompatible treatment of fabrics for cell adhesion.
However, it seems that the cells did not show the character of well spread cells. By contrast,
they were round, captured in the fibers and just a couple of cells were spread and interact
with the surface of fabrics. This interaction was observed only for CS1CTA/HA/NWFs
wound dressing treated fabrics (Figure 5Bm) higher magnification of interacting cells in
Figure 5Bp,q marked with red arrows in CS1CTA/HA/NWFs and detailed cell-fiber
interaction in Figure 5Bn). The low spreading density could be the reason why the cells did
not exhibit extensive proliferation as just a slight increasing number of cells is evident for
treated fabrics and a constant number for the control. Further, for all samples the decreasing
number of cells was observed 72 h of cultivation. This could imply the washing cells out
from the fabrics due to the poorer adhesion in time. In summary , the treatment of nonwoven
fabrics with different concentrations of chitosan and chitosan/hyaluronan ratio were
promising as new wound dressing/healing materials and correspond with our results
obtained from the cytotoxicity assay.
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Chapter 3
Figure 5B: Fluorescence microscopy of nonwoven cotton fabrics (CTA/NWFs)
immobilized by chitosan citrate (CS1CTA/NWFs), chitosan citrate/hyaluronan layer
(CS1CTA/HA/NWFs) and 3T3 cells after staining with calcein and propidium iodide. (a-d)
nonwoven cotton fabrics treated by chitosan citrate (CS1CTA/NWFs); layered hyaluronan
(HA) assembled on the chitosan/nonwoven fabrics layers (e-h; CS1CTA/HA/NWFs);
nonwoven fabrics (control; i-l) and cell dressing interaction (m-q) after different seeding
time (3, 24, 84, 72 h).
91
Chapter 3
Phagocytosis measurements were performed to measure the level of reactive oxygen species
(ROS) in blood due to the increase consumption of oxygen by phagocytic cells [80]. During
phagocytosis process, oxygen is converted into different types as hydrogen peroxide,
monomolecular oxygen and hydroxyl radicals by several kinds of phagocytic cells [81].
These reactive oxygen species (ROS) cause different biological effects as parasites and
tumor cells, destruction of bacterial cells, promoting inflammation and modulating the
immune reaction [82]. The generation processes of reactive oxygen species can be
monitored using luminescence analysis.
Figure 6 shows that chitosan did not change the spontaneous production of ROS by
intact phagocytes it means that the isolated chitosan was safe for cells while hyaluronan
immobilized nonwoven fabrics demonstrated slightly release of free radical but still safe
and there was no damages on cells (Figure 6). On the other hand zymosan-induced ROS
production was significantly decreased by all concentrations of chitosan/hyaluronan
nonwoven fabrics (1.6, 0.8 and 0.4 mg/µl) in comparison with control. It was suggested that
the presence of chitosan/hyaluronan composite couldn’t be responsible for the inhibition of
zymosan-activated oxidative burst of phagocytes and this is very important since phagocyte
derived ROS has harmful effect in healing wound.
92
Chapter 3
140
1.6 mg/100 μl
0.8 mg/100 μl
0.4 mg/100 μl
CTA/NWFs
Chemilumenscence (RLU)
120
100
80
60
40
20
0
Spontaneous
Activated
Spontaneous
CS1CTA/NWFs (mg/100µ)
Activated
HA/NWFs (mg/100µ)
Spontaneous
Activated
CS1CTA/HA/NWFs (mg/100µ)
Figure 6: Effect of chitosan citrate (CS/CTA/NWFs), sodium hyaluronate (HA/NWFs) and
chitosan/hyaluronan nonwoven fabric sheets (CS1CTA/HA/NWFs) on the spontaneous and
zymosan-activated oxidative burst of blood phagocytes. Oxidative burst of phagocytes was analysed
chemiluminometrically. Light emission expressed as relative light units (RLU) was recorded
continuously for 90 min at 37°C and corrected to the number of polymorph nuclear cells.
3.5. Wound healing measurements
Wound healing is stepwise process which consists of various phases as
inflammation, proliferative and remodeling [7, 9, 80]. The genetic response regulating
(normal state) cellular resistance mechanisms contributes to the wound and its repair. The
damaged tissues requires biocompatible materials like chitosan on which cells may adhere
and proliferate. Chitosan prepared from natural biopolymer chitin has been tested as wound
dressing at the skin-shaving site in rats [7, 81-83]. Figure 7Aa induced the results of
nondiabetics rats wounds treated with control nonwoven fabric (without any treatment with
chitosan or chitosan/hyaluronan) at 0, 3, 7, and 14 days and for wounds treated. There was
93
Chapter 3
slightly evident effect of non-treated nonwoven fabrics (control sample) onto nondiabetic
rats after two weeks of treatments and the wound closer percentage was 40 % (Fig. 7B).
Comparing with chitosan/hyaluronan composite wound dressing, the healing properties of
nondiabetic rates (Fig. 7Ab) was enhanced and accelerated after wound treated with
biomaterials and the wound closer rate after two week was 95 % (Fig.7B).
In figure 7Ac control diabetic’s rat treated with nonwoven cotton fabrics (treated
with 5 wt. % CTA). The closer percentage after two week of treatments was 25 % comparing
with control diabetic rats. Figure 7Ad present the CS1CTA/HA/NWFs treated the diabetic
rats. The wound closure was 60 % after 14 days of the treatment. It was suggested that their
wound-healing properties of chitosan were due to their ability to stimulate fibroblast
production by affecting the fibroblast growth factor [39, 84-88]. The results from figure
7A, B that, chitosan/hyaluronan was highly efficient accelerated and enhancement the
healing properties of diabetics and nondiabetics rats comparing with control sample.
Chitosan/hyaluronan composite had very high healing properties compared between plain
chitosan or plain hyaluronan (data not shown), due to healing properties of both chitosan
and hyaluronan together. Enhanced tissue repair ability of CS/HA immobilized into
nonwoven cotton fabrics in the early phase of inflammatory response could be reasonably
ascribed to the recapitulation of induction signals exerted by HA. Indeed, HA naturally
interacts with signaling receptors (primarily CD44) to initiate inflammatory response, to
maintain structural cell integrity and to promote recovery from tissue injury [89].
Furthermore, a possible activity of HA on peroxisome proliferator-activated receptors
(PPARs) during wound healing could not be excluded [90]. The formation of a highly
hydrated HA-rich matrix could be supposed to facilitate cell migration into the provisional
94
Chapter 3
wound matrix fostering proliferation and in turn regeneration process [91]. Although wound
contraction is the primary mechanism underlying healing process of closure in the murine
model adopted, re-epithelialization of the wound site, which is prevalent in human skin, and
suggested by in vitro results, can well take place and contribute to wound closure as
observed elsewhere [92].
Figure 7A: Visual observation of the surface healing dressed with nonwoven sheet (control-non-diabetic)
at 0th, 3th, 7th and 14th days after operation (a-d), respectively; (one of three same cases in each group was
shown); healing dressed with chitosan/hyaluronan nonwoven dressing sheet (non-diabetic) at 0th, 3th, 7th and
14th days after operation (e-h), respectively; surface healing dressed with nonwoven sheet (gauzy) from
cellulosic material (control diabetic) at 0th, 3th, 7th and 14th days after operation (i-m), respectively; (one of
three same cases in each group was shown); healing dressed with chitosan/hyaluronan/nonwoven fabrics sheet
(diabetic) at 0th, 3th, 7th and 14th days after operation (n-r), respectively.
95
Chapter 3
120
Control diabetics
Control nondiabetics
Treated diabetic by CS1CTA/HA/NWFS
Treated nondiabetic by CS1CTA/HA/NWFS
100
Wound area (%)
80
60
40
20
**
0
0
3
7
Treatment time (Day)
14
Figure 7B: Wound contraction as a function of postoperative time for (non-diabetic and diabetics)
wounds treated with chitosan/hyaluronan nonwoven fabric (CS1CTA/HA/NWFs) sheets and control
(nonwoven cellulosic fabrics- gauze), indicated by the percentage of wound size vs healing dates.
96
Chapter 3
4. Conclusions
New wound dressing materials based on chitosan/hyaluronan composite/
nonwoven fabrics were prepared and evaluated. Pure chitosan was isolated from shrimp
shell with high degree of deacetylation (95 %) and non-protein residual percent. Nonwoven
viscose fabrics were used for immobilization of biologically active biopolymers (chitosan,
hyaluronan) by pad-dry-cure method. Chitosan/hyaluronan/composite showed well
distribution of chitosan and hyaluronan on the surface of viscose fibers confirmed by
scanning electron microscope. The prepared wound dressing material showed excellent
antibacterial activity against two types of bacterial E. coli and
S.
aureus
as
gram
negative and positive, respectively. The cytotoxicity of the new dressing materials was
revealed by using mouse fibroblast cell line (NIH-3T3) and keratinocytes cell lines (HaCaT)
with different times and concentration of treatments and showed that there was not any
toxicity of the prepared dressing material. From the healing measurements,
chitosan/hyaluronan viscose nonwoven fabrics composite accelerated and enhanced the
healing properties of diabetics and nondiabetics rats compared with control sample. In
summary, this novel chitosan/hyaluronan/nonwoven fabrics wound dressing will open new
doors for realizing potential using the dressing as carrier of drugs to better treat wound
defects and promote the wound healing.
97
Chapter 3
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101
Chapter 4
Dissertation work conclusion
At the end of this thesis can be stated that despite all doubts can chitin and chitosan from
shrimp shells to obtain such purity to be used in medicine and in textile applications where the
fabric comes into contact with the human skin and possibly infected wounds.
Based on the positive results presented in this work the following research and development will
be done in the future:
Chitin, the second most abundant biopolymers found in nature next to cellulose and
chitosan (the N-deacetylated form of chitin) could be isolated from Brazilian Atlantic Coast shrimp
shell by the chemical treatment method in highly pure form. Demineralization, deproteinization
and deacetylation processes were the main steps in the isolation process. The particle size in the
range of 800-1000 µm was selected to isolate chitin, which was achieved by measuring nitrogen,
protein, ash, and yield %. Pure chitosan was isolated from shrimp shell with high degree of
deacetylation (95 %) and non-protein residual percent.
The isolated biopolymers were
characterized and confirmed by different analytical tools like ATR-FTIR, TGA, XRD, elemental
analysis, NMR, SEM, acid-base titration and compared with the commercial available chitin and
chitosan.
The
best
sequences
in
isolation
chitin
and
chitosan
was
DMPA>DPMA>DAPM>DAMP. The isolated chitin and chitosan showed excellent antibacterial
activity against gram-negative bacteria (E. coli). New wound dressing materials based on
chitosan/hyaluronan composite/ nonwoven fabrics were prepared and evaluated. Thin layers of
chitosan (positively charged) / sodium hyaluronate (negatively charged) / nonwoven fabrics were
constructed by polyelectrolyte multilayer pad-dry-cure technique. Chitosan/hyaluronan/composite
showed well distribution of chitosan and hyaluronan on the surface of viscose fibers confirmed by
102
Chapter 4
scanning electron microscope. The prepared wound dressing material showed excellent
antibacterial activity against two types of bacterial E. coli and S. aureus as gram negative and
positive, respectively. The cytotoxicity of the new dressing materials was revealed by using mouse
fibroblast cell line (NIH-3T3) and keratinocytes cell lines (HaCaT) with different times and
concentration of treatments and showed that there was not any toxicity of the prepared dressing
material. From the healing measurements, chitosan/hyaluronan viscose nonwoven fabrics
composite accelerated and enhanced the healing properties of diabetics and nondiabetics rats
compared with control sample. Finally this novel chitosan/hyaluronan/nonwoven fabrics wound
dressing will open new doors for realizing potential using the dressing as carrier of drugs to better
treat wound defects and promote the wound healing.
Future work

Isolation of chitin and chitosan in nano-fibrilis structure with control size and length of the
nano-fibrous.

Preparation of hydrogel from chitin/chitosan/hyaluronan and study the physical and
chemical properties of the hydrogel.

The chitin and chitosan nano-fibrous will be used as blood clotting in 3D scaffold shape.
103
Supporting Information 1
Chitin and Chitosan from Brazilian Atlantic Coast: Isolation, Characterization and
Antibacterial Activity
Components
Compositions of
Shrimp shell from
Atlantic cost
Compositions of Shrimp shell from
other sources(Ref.1-5)
Protein
30
52.03[162];48.97[215]; 40.6[216];
48.1[217]; 48.8[218]
Mineral
8-10
28.84[162]; 29.5[215];
20.6[216];14[217];29.93[218]
Chitin
40-53
19.1[162]; 21.53[215]; 16.2[216];38[217];
20[218]
Chitosan
30-43
10[162]; 7.11[215]; 9[216]; ,20[217];-[218]
Table S1: Comparison among the components of shrimp shell and other sources
References
[1] M.S. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M.F.A. Goosen, N. Mameri, Food
Hydrocolloids, 29 (2012) 48-56.
[2] S.-L. Wang, T.-W. Liang, Y.-H. Yen, Carbohydrate Polymers, 84 (2011) 732-742.
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International Journal of Biological Macromolecules, 65 (2014) 298-306.
[4] F.A.A. Sagheer, M.A. Al-Sughayer, S. Muslim, M.Z. Elsabee, Carbohydrate Polymers,
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104
Supporting Information 2
Wound Dressing via Layer by Layer Assembly of Chitosan Citrate /Hyaluronan/
/Nonwoven Fabrics: Preparation, Characterization, In Vitro and In Vivo Evaluations
Sample codes
Treatment code
CTA/NWFs
Nonwoven cotton fabric (NWFs) treated with citric acid (5%).
CS0.5CTA/NWFs
Nonwoven cotton fabric (NWFs) treated with chitosan citrate
(0.5 %).
CS1CTA/NWFs
Nonwoven cotton fabric (NWFs) treated with chitosan citrate (1
%).
CS2CTA/NWFs
Nonwoven cotton fabric (NWFs) treated with chitosan citrate (2
%).
CS0.5CTA/HA/NWFs Chitosan citrate (0.5 %) nonwoven cotton fabric (NWFs) treated
with hayluronan (0.5 %).
CS1CTA/HA/NWFs
Chitosan citrate (1 %) nonwoven cotton fabric (NWFs) treated
with hayluronan (0.5 %).
CS2CTA/HA/NWFs
Chitosan citrate (2 %) nonwoven cotton fabric (NWFs) treated
with hayluronan (0.5 %).
Table S1: Code of treated and untreated cotton fabrics with different chitosan
citrate/hyaluronan concentrations.
105
NONWOVEN TREATMENTS REDUCTION RATE (%)
E. coli
St. aureus
CTA/NWFS
0
0
CS0.5CTA/NWFS
85
80
CS1CTA/NWFS
95
92
CS2CTA/NWFS
90
75
CS0.5CTA/HA/NWFS
88
84
CS1CTA/HA/NWFS
98
92
CS2CTA/HA/NWFS
80
70
Table S2. Reduction rate percentage of chitosan and chitosan/hyaluronan/nonwoven
cotton fabrics
106
Fig.S1. FTIR-ATR (A); TGA-DTG (B); H-NMR (C); XRD (D) of the isolated chitosan
from shrimp shell (our work).
107
Curriculum vitae
Name: Rasha Abdel-Rahman
Nationality: Egyptian
Gender: Female
Marital status: Married
E- Mail: [email protected]
Academic Degree

M.Sc. in Organic Chemistry
Faculty of Science, Fayoum University,
In titled "Synthesis and Some Reactions of Heterocyclic Compounds Containing Coumarin
Moiety"

Bachelor of Science
Faculty of Science, Al-Azhar University, [Very Good with Class Honor].
Current Research
 Extraction processes of biopolymers (chitin, chitosan).
 Biological activity of biopolymers.
 Preparation of different metal nanoparticles
List of Patent
 Abdel-Mohsen Abdel-Lattif, Hrdina Radim, Burgert Ladislav, Rasha Abdel-Rahman, Velebný
Vladimír, Šuláková Romana, Smirnou Dzianis, Hrdinová. Process for preparing fibers of
chitin/chitosan-glucan complex, fibers and wound cover.CZ 304564, B6- 2014.
List of Publications
1. Rasha M. Abdel-Rahman, Radim Hrdina, A. M. Abdel-Mohsen, A. Y. Soliman, F. K. Mohamed,
Kazi Mohsin , Tiago Dinis Pinto. Chitin and Chitosan from Brazilian Atlantic Coast: Isolation,
Characterization and Antibacterial Activity. International Journal of Biological Macromolecules,
2015, 80, 107-120.
2. Rasha M. Abdel-Rahman, A. M. Abdel-Mohsen, R. Hrdina, L. Burgert, Z. Fohlerova, D.
Pavliňák, O. N. Sayed,J. Jancar; Wound Dressing based on Chitosan/hyaluronan/Nonwoven
Fabrics: Preparation, Characterization and Medical Applications. International Journal of
Biological Macromolecules, 89 (2016) 725–736.
3. A. M. Abdel-Mohsen, Rasha M. Abdel-Rahman, Moustafa. M. G. Fouda, L. Vojtova, L.
Uhrova, A. F. Hassan, Salem S. Al-Deyab, I. E. El-Shamy, J. Jancar. Preparation, characterization
and cytotoxicity of schizophyllan / silver nanoparticle composite. Carbohydrate Polymers,
2014,102, 238-245.
4. Hana Přichystalová, Numan Almonasy, A. M. Abdel-Mohsen, Rasha. M. Abdel-Rahman, Libor
Kobera, Moustafa M. G. Fouda, Zdenek Spotz, L.Vojtova, Ladislav Burgert, J. Jancarb,Synthesis,
characterization and antibacterial activity of new fluorescent chitosan derivatives . International
Journal of Biological Macromolecules. 2014, 65 234–240.
5. A. M. Abdel-Mohsen, Rasha M. Abdel-Rahman, Moustafa. M. G. Fouda, L. Vojtova, L. Uhrova,
A. F. Hassan, Salem S. Al-Deyab, I. E. El-Shamy, J. Jancar. Preparation, characterization and
cytotoxicity of schizophyllan/silver nanoparticle composite. Carbohydrate Polymers, 2014,102,
238-245.(highly cited article)
6. Rasha. M. Abdel-Rahman, A. M. Abdel-Mohsen, Moustafa M. G. Fouda, Salem S. Al Deyab,
Asmaa S. Mohamed. Finishing of cellulosic fabrics with Chitosan/polyethylene glycol-siloxane to
improve their Performance and antibacterial properties. Life Science Journal, 2013, 10, 4 834839.
7. A. M. Abdel-Mohsen, Radim Hrdina, Ladislav Burgert, Rasha M. Abdel-Rahman, Martina
Hašová, Daniela Šmejkalová, Michal Kolář, M. Pekar , & A. S. Aly. Antibacterial activity and cell
viability of hyaluronan fiber with silver nanoparticles .Carbohydrate Polymers Journal. 92
(2013) 1177– 1187.
8. F. K. Mohamed, A. Y. Soliman, Ramadan M. Abdel-Motaleb, Rasha M. Abdel-Rahman, A.M.
Abdel-Mohsen, Moustafa M. G. Fouda, N. Almonasy, Asmaa S. Mohamed, Synthesis and
antibacterial activity of 3-arylidene chromen-2, 4-dione derivatives. Life Science Journal, 2013,
10, 4 840-845.
9. A. Y. Soliman, F. K. Mohamed, Ramadan M. Abdel-Motaleb, Rasha M. Abdel-Rahman, A.M.
Abdel-Mohsen, Moustafa M. G. Fouda, Asmaa S. Mohamed. Synthesis of new coumarin
derivatives using Diels-Alder reaction. Life Science Journal, 201310, 4, 846-850.
10. A. Y. Soliman, F. K. Mohamed, Ramadan M. Abdel-Motaleb, Rasha M. Abdel-Rahman, A. M.
Abdel-Mohsen, Moustafa M. G. Fouda, Salem S. Al-Deyab, Asmaa S. Mohamed, Reaction and
Antibacterial efficacy of active methylene compounds with coumarin derivatives. Journal of
Pure and Applied Microbiology, 7, Special Edition 2013, 435-439.
11. F. K. Mohamed, A. Y. Soliman, Ramadan M. Abdel-Motaleb, Rasha. M. Abdel-Rahman, A.
M. Abdel-Mohsen, Moustafa M. G. Fouda, Salem S. Al-Deyab, Radim Hrdina Synthesis and
antibacterial activity of new quinoline derivatives started from coumarin compounds,. Journal of
Pure and Applied Microbiology, 7, Special Edition. 2013, 453-458.
12. A. M. Abdel-Mohsen, Radim Hrdina, Ladislav Burgert, Gabriela Krylova, Rasha M. AbdelRaman, Anna Krylova, Milos Stienhart & Ludvik Benes. Green synthesis of hyaluronan fibers
with silver nanoparticles. Carbohydrate Polymers Journal, 2012, 89,411– 422.
13. A.M. Abdel-Mohsen, Rasha M. Abdel-Rahman, R. Hrdina, Aleš Imramovský, Ladislav Burgert,
& A. S. Aly. Antibacterial cotton fabrics treated with core shell nanoparticles. International
Journal of Biological Macromolecules. 2012, 50, 1245-1253.
Poster papers in international and national conferences
1. A. M. Abdel-Mohsen, Radim Hrdina, Ladislav Burgert, Gabriela Krylova, Rasha M. AbdelRaman, Anna Krylova, Milos Steinhart, & Ludvík Beneš. Green synthesis of hyaluronan fibers
with silver nanoparticles. 1st World Textile Conference SMARTEX, 22-24 November 2011,
Egypt. ISSN (2090-7109).
2. A. M. Abdel-Mohsen, Radim Hrdina. Rasha M. Abdel-Rahman. Antibacterial and cytotoxicity
of hyaluronic-silver nanoparticles. 1st international conference of natural fibers 8-12 June 2013.
Gumaries, Portugal.
3. A.M. Abdel-Mohsen, L.Vojtova, Rasha M. Abdel-Rahman, J. Jancar, Antibacterial bacterial
cotton fabrics treated with core shell nanoparticles.7st Aachen-dresden textile international
conference. Aachen –Germany, 27-29, November 2013.
4. A.M. Abdel-Mohsen, L.Vojtova, R. M. Abdel-Rahman, J. Jancar. Preparation, characterization
and cytotoxicity of silver nanoparticles using schizophyllan .Third Euro-Med conference on
Natural Products from Biotechnology to NanoMedicine (Bio-Nat III) 4-6, January, 2014, Cairo,
Egypt.
5. A.M. Abdel-Mohsen, R. M. Abdel-Rahman, L.Vojtova, J. Jancar. Preparation, characterization
and antibacterial activity of new florescent chitosan derivatives. 15th international conference of
polymer and organic chemistry, 10-13 June,2014 Timisoara, Romania. ISBN: 978-606-554-481
6. Rasha M. Abdel-Rahman, Radim Hrdina; Finishing of cellulosic fabrics with
Chitosan/polyethylene glycol-siloxane to improve their Performance and antibacterial
properties;5th EUCHEMS Chemistry Congress 31 August-4 September 2014 in Istanbul, Turkey.
ISSN: 1614-7499.
7. Rasha M. Abdel-Rahman, A.M.Abdel-Mohsen, R. Hrdina, L.Burgert. An Evaluation of Chitin
and Chitosan Extracted from the Shrimp Shell. Third international conference of chemical
technology, 13-15.04.2015, Mikulov, Czech Republic.
8. A.M.Abdel-Mohsen, Rasha M. Abdel-Rahman, R. Hrdina, H. Nejezchlebová H.
Chitin/Chitosan-glucan Complex: Extraction, Characterization, and Bio- medical Applications.
Third international conference of chemical technology, 13-15.04.2015, Mikulov, Czech Republic.