Journal Pre-proof

Advancement on modification of chitosan biopolymer and its

potential applications

Nabel A. Negm, Hassan H.H. Hefni, Ali A. Abd-Elaal, Emad A.

Badr, Maram T.H. Abou Kana

PII: S0141-8130(20)30419-0
DOI: https://doi.org/10.1016/j.ijbiomac.2020.02.196
Reference: BIOMAC 14813

To appear in: International Journal of Biological Macromolecules

Received date: 14 January 2020

Revised date: 15 February 2020
Accepted date: 18 February 2020

Please cite this article as: N.A. Negm, H.H.H. Hefni, A.A. Abd-Elaal, et al., Advancement
on modification of chitosan biopolymer and its potential applications, International
Journal of Biological Macromolecules(2018), https://doi.org/10.1016/
j.ijbiomac.2020.02.196

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 Journal Pre-proof

Advancement on modification of chitosan biopolymer and its potential applications

Nabel A. Negm1,2#, Hassan H.H. Hefni1, Ali A. Abd-Elaal1, Emad A. Badr1, Maram T.H. Abou Kana3
1- Egyptian Petroleum Research Institute (EPRI), Nasr City, Cairo, 11727, EGYPT.
2- Egypt Nanotechnology Center (EGNC), Cairo University, El-Shiekh Zayed, 12588, EGYPT.
3- National Institute of Laser Enhanced Sciences, Cairo University, Giza, EGYPT.
# Corresponding author: nabelnegm@hotmail.com
Abstract
Chitosan is the second abundant biopolymer present on the earth after cellulose. Chitosan is
extracted from the shells of shrimp and other crustaceans. Several methods were reported for
its extraction, but the most commercial is the deacetylation of chitin. Chitosan as a
biopolymer has numerous applications and uses. But, its mechanical, chemical and biological
characteristics can be enhanced by modification of its chemical structures. Several
modification methods and derivatives were reviewed in the literatures, and several were

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collected in this review. The reviewed modified chitosan derivatives herein were five types of

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derivatives. The first is substituted chitosan derivatives including thiolated, phosphorylated,
and N-phthaloylated derivatives. The second is crosslinked chitosan derivatives including
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chitosan-glutaraldehyde, chitosan-ethylene diamine tetraacetic acid, and chitosan-
epichlorohydrin derivatives. The third is carboxylic acid derivatives of chitosan obtained from
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Carboxyalkylation, acrylation, methacrylation, and benzoylation of chitosan. The fourth is
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ionic chitosan derivatives including highly cationic and sulfated derivatives. The last is
bounded chitosan to specific molecules including cyclodextrin, thiosemicarbazone, dioxime,
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and crown ether precursors. The review also highlights the reported advantages and
applications of the modified chitosan and the synthetic routes of the biopolymer modification.
Keywords: Chitosan; modification; nonionic chitosan; ionic chitosan; drug delivery; metal
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uptake
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1. Introduction
Chitosan is a copolymer formed of repeated units of 2-amino-2-deoxy-d-glucopyranose units,
and residual 2-acetamido-2-deoxy-d-glucopyranose units (Figure 1).
Chitosan is obtained from alkaline hydrolysis (deacetylation) of chitin, and it is also found
naturally in some fungal cell walls. Chitosan has amenable functional groups such as primary
amine (NH), primary as well as secondary hydroxyl group (OH) in its monomer (Figure 2),
so it can be chemically modified without disturbing its degree of polymerization (Mourya and
Inamdar, 2008). The chemical modification (Croisier and Jerome, 2013) of chitosan is a
powerful technique to control the interaction of the polymer with other components, such as
metal ions, organic compounds, e.g., drugs. Chemical modification of chitosan improves its
bulk properties for the preparation of sustained drug release system.

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2. Substituted chitosan derivatives

2.1. Thiolated chitosan
The amino group in 2-position of glucosamine units of chitosan is the central position for the
immobilization of thiol groups. The thiolated chitosan derivatives includes different
compounds such as: chitosan-cysteine, chitosan-thiolactic acid, chitosan-thioglycolic acid,
chitosan-homocystenine, chitosan-4-thiobutylamidine, chitosan-thioethylamidine, chitosan-
glutathione, chitosan-N-acetyl cysteine, and chitosan-6-mercaptonicotinic acid conjugates
(Figure 3).
Anticancer drugs have several drawbacks which were solved by employing thiolated chitosan
which offers efficient mucoadhesivity, membrane permeation enhancing capability and

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improved inhibition for P-glycoprotein. Ciro et al., (2017) represented the synthesis and
characterization of thiolated chitosan (Scheme 1) designed in transfer structures such as films,

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hydrogels, and nanoparticles were reported for anticancer drugs.
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The thiolated chitosan compounds were promising as drug carriers and efficient as nano-
medicine for enhancing the ocular bioavailability. Shastri (2017) described several
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applications of thiolated chitosan as nano-carriers for ophthalmic drug delivery.
Yong et al., (2013) enhanced the removal efficiency of Cu2+ and Cd2+ from wastewater by
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modification of chitosan-beads throughout introduction of thiol groups to their skeleton to

obtain thiolated chitosan-beads, ETB (Figure 4). The removal enhancement was accredited to
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the theory of Hard-Soft Acid-Base.

Au(III) ion-imprinted thiol-modified chitosan was created by Monier and Abdel-Latif (2017)
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using epichlorohydrin as the cross-linker. The polymer presented greater uptake capacity of
370 mg/g than the non-imprinted polymer which has the uptake capacity of 195 mg/g. Chang
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et al. (2010) synthesized molecular imprinted chitosan using dibenzothiophene as the template
and studied the influence of crosslinking and its adsorption for gasoline. The removal of
mercury from aqueous solutions using thiol modified chitosan in the cysteine as -SH was
investigated by Merrifield et al. (2004), where the maximum uptake capacity was 8 mmol/g in
neutral medium. Zhu et al., (2012) introduced thiol group on chitosan skeleton using xanthate.
The competitive uptake of lead (II), copper (II), and zinc (II) were in the order of 79.9 mg/g,
34.5 mg/g, and 20.8 mg/g, respectively.
Al-Ghamdi et al., (2018) were prepared novel type of sulfone-modified chitosan (SMC)
derivatives in reasonable yield by the reaction of chitosan polymer and p-bromo-β-
ketosulfone derivative (Figure 5) under mild acidic condition. The obtained sulfone-modified
chitosan biopolymer was used as efficient adsorbent for Hg(II) metal ions from wastewater.

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2.2. Phosphorylated chitosan

Phosphorylated chitosan and phosphorylated chitosan derivatives acquired their importance
due to their characteristics such as: high water solubility, and metal chelating tendency. These
types of biopolymers had important applications during regeneration of tissues, drug delivery
intermediates, fuel cells and in food industries (Jayakumar et al., 2006).
Phosphorylation of chitosan was performed using phosphorous pentaoxide in methane
sulphonic acid as a solvent at low temperature, and produced water soluble products with high
substitution extent. Methane sulphonic acid acts also during the reaction as a catalyst (Scheme
2) (Jayakumar et al., 2008). Also, phosphorylated chitosan synthesized by reaction of chitosan
and orthophosphoric acid at 150 °C in the presence of urea as a catalyst and DMF as a solvent

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(Scheme 3) (Jayakumar et al., 2008). The phosphorylated chitosan was also synthesized
during grafting copolymerization reaction of and mono-(2-methacryloyl oxyethyl) phosphoric

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acid. The product exhibited zwitterionic properties and enriched antimicrobial activities
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(Scheme 4) (Jayakumar et al., 2008).
Ramos et al., (2003) were synthesized N-methylene phosphonic chitosan derivative using
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chitosan, phosphoric and formaldehyde. The combination of methylene phosphonic groups
and chitosan enhances its solubility in aqueous medium under mild conditions (Scheme 5).
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In another report (Heras et al., 2001), the functionalization of chitosan by phosphorous acid
and formaldehyde instantaneously in acidic medium gives water soluble N-mono- and N-di-
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phosphonic methylene derivatives (Scheme 6).

Cellulose nano-fibrils (CNF) were modified (Mautner et al., 2016) with phosphate groups by
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reacting CNF derived from cellulose sludge, a waste stream from paper industries, with
phosphoric acid (Scheme 7). It was found that nano-papers were able to adsorb copper from
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aqueous solutions up to 200 mg/m2.

In Valmikinathan et al., 2018 study, new photocrosslinkable chitosan derivatives were
innovated (Scheme 8) in a methodology allows construction of degradable, in-situ gelling
hydrogels with reasonable gelling times and governable physical characters.
2.3. N-phthaloylated chitosan
Chitosan polymer is poorly soluble in organic solvents, while N-phthaloylation of chitosan is
efficient for solubilization. N-phthaloyl chitosan derivatives have high reactivity than N,O-
phthaloylated chitosan derivatives (Kurita et al., 2007). Completely deacetylated chitosan
polymer reacted by phthalic anhydride yields N-phthaloyl chitosan derivative which has high
solubility in polar-organic solvents (Scheme 9) (Dutta et al., 2004).

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Huh (2001) was synthesized two chitosan derivatives: chitosan-grafted poly(ethylene

terephthalate) (C-PET) and quaternized C-PET to obtain antibacterial textile agents to prevent
the growth of Staphylococcus aureus. C-PET and quaternized C-PET exhibited high efficacy
against the bacterial growth within 75%–86%.
The removal of the hydrogen atoms of amino groups of chitosan and introduction of some
hydrophobic nature will result the destruction of its inherent crystalline structure and improve
the solubility in general organic solvents. This has been initially demonstrated by the partial
N-phthaloylation reaction of water-soluble chitin with phthalic anhydride and by the complete
N,O-poly acylation of chitosan with an excess of long chain acid chlorides (Scheme 10)
(Nishimura et al., 1991).

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3. Crosslinked chitosan derivatives
3.1. Chitosan-glutaraldehyde crosslinked polymers

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Biosorbents, particularly crosslinked chitosan, are widely utilized during heavy metal uptake.
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Cross-linker type and degree of crosslinking are largely influence the metal uptake behavior
of chitosan derived biosorbents.
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The study of Wang and Zhuang (2017) presented the synthesis of cross-linked
chitosan/sporopollenin microcapsules using various concentrations of glutaraldehyde as
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crosslinking agent. The uptake efficiency of copper (II) at different concentration, contact
time, amount of adsorbent, temperature and pH was studied.
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The equilibrium conditions of biosorption of manganese (II) from aqueous solution by

chitosan biopolymer crosslinked by glutaraldehyde (GCC) were studied (Suguna et al., 2010),
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and the determined Langmuir capacity of GCC at 25 oC was 278 mg/g.

Nagireddi et al., (2017) reported the ability of crosslinked gluteraldehyde-chitosan (GCC)
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copolymer towards the uptake and recovery of lead (II) ions from electroplating solutions.
GCC adsorbent (Scheme 11) was prepared by grafting of chitosan and glutaraldheyde in 1:17
ratio.
Chitosan in forms of membranes and beads were chemically crosslinked using gluteraldehyde
(Adarsh and Madhu, 2014) and the adsorption tendencies of the produced adsorbents were
represented that cross-linked chitosan have higher adsorption tendency for several metal ions
as follows: Cd > Cu > Ni > Ag > Pb > Zn.
Fe/Mn oxides were loaded on chitosan biopolymer (Ocinski, 2019) to obtain a hybrid
adsorbent for removal of arsenic ions from the aqueous solution. The obtained adsorbents
were effective toward arsenic ions adsorption with high durability as fixed-bed systems.

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Chitosan-citric acid modified flakes crosslinked by gluteraldehyde (Suc and Ly, 2012)
showed enhanced capacity towards lead (II) ions uptake. Chitosan-citric acid modified flakes
can be applied as effective modified biosorbent during lead ions uptake from contaminated
water. Modified chitosan-citric acid flakes represented maximum lead (II) capacity at
101.7 mg/g.
Sobahi et al., (2014) reported the modification of chitosan with aldehydes and organic acids.
Furthermore, some chitosan-carbohydrates blends were synthesized to produce modified
chitosan biopolymers with certain physical and chemical characteristics.
Beppu et al., (2007) reported the crosslinking process of chitosan biopolymer with
gluteraldehyde, and showed that macro-properties and micro-properties were improved such

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as: permeability, wetting, mechanical properties and chemical resistance. Crosslinked
chitosan biopolymer membranes had higher hydrophobic characters than the un-crosslinked.

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Pokhrel et al. (2017) reported the preparation of poly(vinyl alcohol)-chitosan (PVA-CS)
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biofilm using solution casting protocol in the presence of gluteraldehyde as cross-linker. The
degradation rate of PVA-CS biofilm was increased by increasing the chitosan content.
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3.2. Ethylene diamine tetraacetic acid chitosan polymer
Several chemical modification methods were tried to increase uptake capacity of cross-linked
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chitosan beads. Aminated chitosan beads formed through the chemical reaction using ethylene
diamine and carbodiimide (Jeon and Holl, 2003) (Scheme 12) showed the highest uptake
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capacity for mercury ions.

The chitosan biopolymer derivatives are more potent than the unmodified biopolymer.
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Grafting of ethylene diamine tetraacetic acid (EDTA) on chitosan biopolymer (Scheme 13)
(Schnurch et al., 1998) enhances the antibacterial potency due to the chelating the magnesium
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ions which stabilizes the outer cellular membrane of gram-negative bacteria. Chitosan-EDTA
modified biopolymer utilized as carrier matrix in the process of controlled drugs release. The
controlling function of the drugs release came from the ionic crosslinking of the biopolymer
by dicationic species as 1,8-diaminooctane or lysine.
Chitosan-ethylene diamine tetraacetic acid (EDTA) sodium salt biopolymer (NaCN-EDTA)
was examined (Valenta et al., 1998) in numerous topical uses. The chemical, physical,
mechanical and microbial properties of NaCN-EDTA compared several hydrophilic
biopolymers including: hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl
cellulose (NaCMC), sodium carbopol-980 (NaC980) and sodium polycarbophil (NaPCP).
NaCN-EDTA hydrogels is chemically stable, colorless at 0.5% polymer concentration and

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highly compatible with multivalent metal ions. General approaches for synthesis of muco-
adhesive chitosan biopolymer derivatives were reported (Ways et al., 2018).
Chitosan-graphene oxide (CS-GO) and chitosan/ethylene diamine tetraacetic acid-graphene
oxide (CS-EDTA-GO) nanocomposite thin films were prepared (Syuhada et al., 2014)
throughout environmental friendly technique. Particle analysis of the products showed fine
dispersion of GO and EDTA-GO (Scheme 14) in chitosan matrix. Some interactions were
occurred between the filler (GO) and the chitosan segments leads to well distribution of stress
property. CS-GO and CS-EDTA-GO showed maximum tensile stress at 0.5% of GO by 51%
and 71% compared to chitosan, respectively.
Zhao et al., (2017) reported the fabrication of chitosan-ethylene diamine tetraacetic acid-p-

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cyclodextrin (CS-EDTA-CD) trifunctional biosorbent (Scheme 15) using a facile and green
one-pot reaction. The obtained biosorbent was examined in remediation of some toxic metal

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ions and organic pollutants from contaminated wastewater. The adsorption capacities of
-p
bisphenol-S, ciprofloxacin, procaine, and imipramine were: 0.177, 0.142, 0.203, and 0.149
mmol/g, respectively.
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3.3. Chitosan-epichlorohydrin crosslinked polymers
Chen and coworkers (2008) synthesized the crosslinked chitosan by the homogeneous
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reaction of chitosan in aqueous acetic acid solution with the epichlorohydrin (Scheme 16) and
investigated the adsorptions of Cu (II), Zn (II), and Pb (II) ions. Adsorption of these metal
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ions in aqueous solution followed monolayer coverage of adsorbents through physical

adsorption.
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Tirtom et al. (2012) prepared epichlorohydrin crosslinked chitosan-clay composite beads as

biosorbent for the removal of Ni (II) and Cd (II) ions from aqueous solution.
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Cross-linked chemically modified chitosan namely epichlorohydrin cross-linked xanthate

chitosan (ECXCs) (Figure 6) was prepared by crosslinking of xanthate-chitosan with
epichlorohydrin and the prepared modified polymer was applied in uptake of copper (II) metal
ions from aqueous medium (Kannamba et al., 2010).
4. Carboxylic acid chitosan derivatives
4.1. Chitosan carboxyalkylate derivatives
Carboxyalkyl (mainly carboxymethyl) chitosan is one of the most significant water soluble,
amphoteric chitosan derivative, which had a great potential in medical applications as it is
excellent water soluble, nontoxic, biocompatible and biodegradable (Zargar et al., 2015; Jiang
et al., 2015). Carboxymethyl chitosan has attracted great interests in various fields such as

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antimicrobial activity, biosensor, wound healing, food industry and bio-imaging. Sun et al.
(2006) reported the preparation of quaternized carboxymethyl chitosan (Scheme 17).
The solid-phase interaction of native mono- or dicarboxylic acids such as phthalic, succinic,
and maleic ones with chitosan in a screw extruder and in absence of solvents and condensing
agents was carried out (Demina et al., 2011) (Scheme 18).
Acylation of chitosan was performed by reaction of chitosan with acetic, propionic, butanoic,
valeric, and hexanoic acids anhydrides and yielded grafted amides with the modification
degree of 20–50% to achieve biodegradable compounds with high compatibility with blood
(Lee et al., 1995) (Scheme 19).
The modification of chitosan with linear poly(anhydrides) of dicarboxylic acids was carried

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out in toluene medium, and yielded 60.3% and 51.1% of poly(adipine) and poly(sebacine)
anhydrides, respectively. The derivatives were promising carriers for DNA and genes

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(Mutasher et al., 2016) (Figure 7). -p
Succinic, maleic, and phthalic anhydrides were used in a solid phase modification of chitosan
biopolymer to obtain the carboxyalkyl chitosan bio-derivatives (Rogovina et al., 1998)
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(Scheme 20).
4.2. Chitosan methacrylate derivatives
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Water soluble chitin-methacrylate (CM) was synthesized by Khor et al., (2011) via reaction of
methacrylic acid and chitin in 5% lithium chloride/dimethyl acetamide and N,N’-
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dicyclocarbodiimide and dimethyl aminopyridine (Scheme 21). The obtained hydrogel was
prepared from chitin-methacrylate by photochemical crosslinking reaction in the presence of
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Irgacure-2959 photo initiator. CM-hydrogels were assessed in in-vitro cytotoxicity.

Chitosan (CN)-methacrylate (M) product was prepared (Cankaya, 2019) by esterification of
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CN biopolymer and M in 1:0.25 ratio, respectively. Grafting reaction was performed on the
prepared CN/M polymer using different monomers (Scheme 22) such as: 1-vinylimidazole,
methacrylamide and 2-acrylamido-2-methyl-1-propanesulfonic acid throughout free radical
polymerization. The graft copolymers exhibited higher resistance to bacteria than the
chitosan-methacrylate biopolymer.
For the first time, Savin et al., (2019) were prepared polymeric nano-carriers for drugs
delivery based on chitosan grafted-poly(ethylene glycol) methacrylate copolymer. The high
soluble chitosan grafted-poly(ethylene glycol) methacrylate in aqueous medium was obtained
from Michael addition reaction to synthesize nontoxic micro/nanoparticles (MNPs) (Scheme
23). The chemical modification of chitosan was enhanced its solubility in aqueous media.

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In the study of Kyzas et al., (2008), three chitosan microsphere derivatives were synthesized
and evaluated in removal of basic dye (Basic blue 3G). The biosorbents were prepared by
cross-linking of chitosan biopolymer and ionic gelation with tripoly phosphate followed by
cross-linking using glutaraldheyde, to obtain the maximum swelling for the powdered form.
In another study, Kyzas et al., (2008) modified chitosan derivatives via grafting of chitosan
biopolymer by poly(acrylic acid) and poly(acrylamide) using free radical polymerization.
4.3. Chitosan Benzoylate derivatives
Chitosan biopolymer was chemically functionalized by 3-nitro-4-amino benzoic acid moiety
and the fabricated modified biopolymer was applied in collection and concentrating traces of
molybdenum. The modification process of the chitosan biopolymer was preceded via

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attachment of carboxyl group of 3-nitro-4-amino benzoic acid to -NH2 groups through amide
linkage (Scheme 24). The adsorption tendencies of molybdenum, vanadium, gallium, arsenic,

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selenium, silver, bismuth, thorium, tungsten, tin, tellurium, and copper metal ions on the
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modified biopolymer were tested. The highest selectivity and adsorption tendency was
observed for molybdenum in acidic medium of pH 3-4 with uptake capacity of 380 mg.g-1
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resin (Sabarudin et al., 2007).
A new industrial scale approach was used for synthesis of o-benzoyl chitosan derivatives of
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benzoic acid and p-methoxybenzoic acid by using tetrafluoroacetic acid anhydride/phosphoric

acid mediated acylation (Scheme 25). Benzoyl chitosan biopolymers play significant role
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during drug delivery and cosmetics, wound healing preparation, and chromatographic
separation technologies. The modified chitosan biopolymers were soluble in organic solvents
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such as dimethyl formamide, dimethyl sulfoxide, acetone, and not in tetrahydrofurn and ethyl
alcohol. Scanning electron microscopic pictures of native and modified biopolymers showed
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significant structural changes, which was interpreted due to hydrogen bonds breakdown and
the interaction between the introduced phenyl groups (Lee et al., 2012).
5. Ionic chitosan derivatives
5.1. Cationic chitosan derivatives
The cationic nature of the chitosan is essential during several applications such as absorption
enhancement, bio-adhesion, and transfection efficiency as well as to biological activities such
as antitumor, antimicrobial, anti-inflammatory, and anti-hypercholesterolemia effect.
Therefore, highly cationic derivatives of chitosan have been prepared. They can be prepared
by reaction of chitosan and dialkyl aminoalkyl chloride in alkaline condition (Je and Kim,
2006). Chitosan dialkyl aminoalkyl modified by N-aminoethyl, N-dimethyl aminoethyl, N-

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diethyl aminoethyl, N-dimethyl aminoisopropyl displayed substantial b-site APP-cleaving

enzyme inhibition (BACE1) property and cytotoxic activity (Je and Kim, 2005).
A novel water soluble trimethyl quaternary derivative contains quaternary and amino
functional groups on the glycoside units, of chitosan, was prepared through protection-
deprotection protocol (Seidi et al., 2016). Trimethyl chitosan derivative was prepared by
multi-step reaction pathway (Scheme 26).
N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC) was prepared
(Mivehi et al., 2008) in water soluble form by reaction of chitosan with glycidyl trimethyl
ammonium chloride under neutral condition.
In the study of Tan et al., (2016), some chitosan ammonium salts such as: chitosan-

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bromoacetate, chitosan-(mono, di, tri)chloroacetate, and chitosan-trifluoroacetate were
effectively prepared by one-step reaction. The prepared chitosan salts were water soluble and

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exhibited high antifungal activities. -p
A simple and green method of functionalizing chitosan was effectively performed by
dissolving chitosan in a new solvent formed of alkali and urea (Song et al., 2018). Afterward,
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quaternized chitosan was prepared by reaction of the dissolved chitosan and 3-chloro-2-
hydroxypropyl trimethyl ammonium chloride as quaternizing agent at different reaction times
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and temperatures.
Tan et al., (2017) were innovated two cationic chitosan derivatives modified by quaternary
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phosphonium salts throughout trimethylation, chloroacetylation, and reacted with

tricyclohexyl and triphenyl phosphine (Scheme 27). Antifungal activities of the derivatives
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showed significantly improved antifungal efficiency compared to chitosan.

N,N,N-trimethyl chitosan biopolymer and highly substituted N-alkyl-N,N-dimethyl chitosan
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were synthesized (Scheme 28)(Benediktsdottir et al., 2011) in efficient selectivity using di-
tert-butyldimethylsilyl-3,6-O-chitosan (di-TBDMS chitosan).
Chitosan and glycidyl trimethyl ammonium chloride were used (Bu et al., 2012) to synthesize
water soluble N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC)
(Scheme 29). The cationic chitosan derivative was used in modification of cotton fabrics to
improve their aqueous pigment based inkjet printing and to act as antibacterial agent. HTCC-
treated textile was performed superior crocking and laundering fastness than the untreated
cotton textiles and antibacterial potential against Staphylococcus aureus Escherichia coli.
N-trimethyl chitosan-poly(vinyl alcohol) (TMC-PVA) copolymers were synthesized (Scheme
30) in water (Martins et al., 2013) by variation PVA-succinate-TMC ratio. The effect of

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PVA/TMC biopolymer at different polymerization ratios on the cytotoxicity and CC50 values
was tested.
Quaternized N-alkyl chitosan biopolymers with diverse alkyl substituents were synthesized
(Vallapa et al., 2011) and evaluated as antibacterial agents against S. aureus and E. coli. The
modified chitosan biopolymers showed enhanced efficacy during antibacterial activity.
5.2. Sulfated chitosan derivatives
Sulfation of chitosan is preceded using different sulfating reagents, including: concentrated
H2SO4 (Nagasawa et al., 1971), oleum (Vikhoreva et al., 2005), SO3, SO3/pyridine
(Gamzazade et al., 1997), SO3/trimethyl amine (Je et al., 2005), SO3/SO2, and chlorosulfonic
acid-sulfuric acid (Huang et al., 2003). Chemical modification of sulfated chitosan is

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fascinating due to its improvement of the structural similarity of chitosan salt to heparin
(Paulo et al., 1999). The anticoagulant activity of sulfated chitosan is produced by the

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interaction of –SO42- groups and positively charged peptide segments. N-acetyl groups are
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shown to improve the anticoagulant activity. Sulfated chitosan showed anticoagulant as well
as inhibition heama-agglutination activities due to the structural similarity to heparin (Cao et
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al., 2014). Other biological activities of chitosan sulfates including: antisclerotic, antioxidant,
antibacterial, anti-HIV, antiviral, and enzyme inhibition were established (Xing et al., 2005).
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6. Bounded chitosan to specific molecules

6.1. Cyclodextrin (CD) linked chitosan
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Chitosan bearing cyclodextrin derivatives were developed in order to combine the exceptional
potential features of chitosan and cyclodextrin to form non-covalent attachment complexes
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with a number of species that alters the physicochemical properties for improved applications
as medical delivery, and cosmetics (Prabaharan and Mano, 2006; Tanida et al., 1998).
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Cyclodextrin-linked chitosan derivatives (Scheme 31) have gained interests in numerous

applications as: drug delivery, cosmetics and in analytical chemistry (Martel et al., 2001).
There are different mechanisms for linking cyclodextrin and chitosan (Scheme 32).
Several reports (Tanida et al., 1998) were reported the preparation of α-CD-linked chitosan
using 2-O-formylmethyl-α-CD in reduction reaction with p-nitrophenol (Figure 8).
Auzely-Velty and Rinaudo (2002) described analogous preparation of chitosan-cyclodextrin
derivatives via reductive amination reaction by 4-tert-butyl benzoic acid (Figure 9), or by
adamantyl groups.
Cyclodextrin-chitosan derivative monoaldehyde was prepared and evaluated for muco-
adhesion (Venter et al., 2006).

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Chen and Wang, (2001) prepared cyclodextrin-chitosan biopolymer using tosylated β-

cyclodextrin (Scheme 33) and evaluated the product in the release of radioactive iodine (I-
131).
The cyclodextrin-chitosan derivative was also synthesized by reacting monochlorotriazinyl
cyclodextrin with chitosan biopolymer (Figure 10), in the presence of a spacer of triazinyl
(Martel et al., 2001). The product was acted as adsorbent for textile dyes from wastewater.
El-Tahlawy et al. (2006) used a novel technique for preparation of β-cyclodextrin-chitosan by
the reaction of β-cyclodextrin citrate and chitosan biopolymer using formic acid solution as a
reaction medium (Scheme 34), and the product was evaluated as antimicrobial agents. In
another report, the reaction between β-cyclodextrin-itaconate and chitosan (Scheme 35)

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yielded a modified biopolymer utilized as ion exchange resin (Gaffar et al., 2006).
β-cyclodextrin-chitosan modified by 1,6-hexamethylene diisocyanate was prepared (Chen et

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al., 2007) and evaluated as cholesterol adsorbent. The spacer can be 2-hydroxypropyl moiety
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introduced by grafting β-CD on chitosan using epoxy-activated chitosan (Scheme 36) (Zhang
et al., 2004), reducing sugar derivative, and maleic spacer (Scheme 37) (Aime et al., 2006).
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An insoluble cross-linked chitosan bearing β-CD was prepared using N-succinyl chitosan and
aminated-β-CD via amide bond formation in the presence of the water soluble 1-ethyl-3-(3-
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dimethylaminopropyl) carbodiimide (Scheme 38) (Aoki et al., 2003).

6.2. Thiosemicarbazone linked chitosan
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Thiosemicarbazone-o-carboxymethyl chitosan derivatives were prepared (Mohamed et al.,

2014) via by condensing thiosemicarbazide-o-carboxymethyl chitosan by: p-chloro
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benzaldehyde, o-hydroxy benzaldehyde, and p-methoxy benzaldehyde (Scheme 39). The

prepared derivative showed improved antimicrobial action.
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Tailoring of chitosan through involvement of amino and hydroxyl groups by

thiosemicarbazone derivatives (Scheme 40) gave derivatives with improved solubility and
anticancer efficacy (Adhikari et al., 2018).
Chitosan-oligosaccharide-thiosemicarbazide compound was synthesized in one-pot (Jinping et
al., 2019) by reaction of chitosan-thiosemicarbazide and 2-pyridine carboxaldehyde (Figure
11). Antifungal behaviors evaluation of the products against P. capsici, P. nicotianae, F.
graminearum was showed inhibiting efficacy at 74.19% and 66.60%, respectively.
6.3. Dioxime linked chitosan
A new vic-dioxime-chitosan derivative was prepared (Demetgul and Serin, 2008) by
heterogeneous reaction of chitosan and monochloro-glyoxime at room temperature (Scheme
41).

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New crosslinked derivative of chitosan was prepared (Timur, 2019) by the condensation
reaction of chitosan and dichloro-glyoxime (Scheme 42). Metal ion uptake capacity of the
obtained modified biopolymer was studied towards some selected transition metals ions of:
Cu(II), Ni(II), Co(II), Fe(III) and Cd(II) in aqueous medium. According to the results of the
analyses; water retention capacity and metal uptake capacity of new cross-linked chitosan
derivative was higher than chitosan, and metal uptake sequence was Cu(II) > Fe(II) > Cd(II) >
Co(II) > Ni(II) respectively.
6.4. Crown ether linked chitosan
The literature showed new synthetic polymers carrying out both types of structures as well as
general properties of both chitosan and crown ethers as well. Crown ethers have good

of
complexing selectivity for metal ions due to their specific molecular structures. Crown ether
bound chitosan were synthesized with Schiff’s base type reaction (Scheme 43) (Tang et al.,

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2002). -p
Wan et al. first prepared N-benzylidene chitosan (CNB) from chitosan and benzaldehyde.
Chitosan-dibenzo-18-crown-6 crown ether (CNBD) and chitosan-dibenzo-18-crown-6 crown
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ether (CND) (Scheme 44) were prepared from 4,4-dibromodibenzo-18-crown-6 crown ether
with CNB and CTN, respectively. Their adsorption and selectivity for Ag, Cu, Pb, and Ni ions
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showed that CNBD had better adsorption properties (Wan et al., 2002).
Two kinds of novel chitosan-crown ether resins, Schiff base type chitosan-benzo-15-crown-5
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(CTS-B15) and chitosan-benzo-18-crown-6 (CTS-B18) were synthesized through the reaction

between-NH2 and -CHO (Scheme 45). Adsorption properties of CTS-B15 and CTS-B18 for
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Pd2+, Cu2+ and Hg2+ were studied and showed good adsorption characteristics and selectivity
for Pd2+ when Cu2+ and Hg2+ were existed (Peng et al., 2003).
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Chemically stable porous aza-crown ether-crosslinked chitosan films were prepared (Toeri et
al., 2017) by reacting different molar amounts of N,N-diallyl-7,16-diaza-1,4,10,13-tetraoxa-
dibenzo-18-crown-6 (molar equivalents ranging from 0, 0.125, 0.167, 0.25 and 0.5) with
chitosan (Figure 12). The crosslinking chemistry between allyl and amine groups of the
reactants was further evidenced with solution NMR studies on model compound of
glucosamine with the aza-crown. X-ray diffraction analysis of the chitosan/aza-crown films
using wide angle X-ray scattering, revealed that the crystalline arrangement of chitosan was
partially destroyed with increasing grafting of aza-crown ether proportion on chitosan
polymer chain.
7. Conclusions

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As a result of the presence of various reactive functional groups on chitosan biopolymer, it

can be functionalized by numerous function groups. The functionalization of chitosan
biopolymer can be addition, coupling, cross-linking or bounded to small molecules. The
modification of chitosan biopolymer can be undergoes through substitution reaction according
to thiolation, phosphorylation, N-phthaloylation. Crosslinking of chitosan can be performed
using different crosslinking agents such as: glutaraldehyde, ethylene diamine tetraacetic acid
and epichlorohydrin. Carboxylation of chitosan can be performed by attachment of different
carboxylic acids by chitosan such as: free carboxylic acids, alkylcarboxylate, methacrylic
acid, and benzoic acid. Ionic chitosan can be formed in different forms such as cationic and
sulfated forms. Chitosan can be bounded to specific molecules such as cyclodextrin,

of
thiosemicarbazones, dioxime, and crown ethers. The functionalized chitosan biopolymers
have upgrade characteristics in drug delivery, gene delivery, antimicrobial activity against

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different germs, and enhanced metal uptake efficiency with high selectivity than the un-
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functionalized chitosan.
Acknowledgement
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This work was supported and funded by Science & Technology Development Fund (STDF),
Project No. 26370/2019.
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Figure 1: Chemical structure of chitosan.

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Figure 2: Functional groups in chitosan unit.

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Figure 3: Thiolated chitosan derivatives.
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Figure 4: Modified chitosan-beads by thiol groups.

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Figure 5: p-Bromo-β-ketosulfone chitosan derivative.

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Figure 6: Chemical structure of epichlorohydrin cross-linked xanthate chitosan.
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Figure 7: Chemical structure of chitosan modified by modified by linear poly

anhydrides.

Figure 8: α-CD-linked chitosan using 2-o-formylmethyl-α-CD.

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Figure 9: Chitosan-cyclodextrin derivative obtained by reductive amination reaction.
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Figure 10: Chemical structure of monochlorotriazinyl cyclodextrin-chitosan biopolymer.

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Figure 11: Condensation product of chitosan-thiosemicarbazide and 2-pyridine

carboxaldehyde.

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Figure 12: Chemical structure of aza-crown ether crosslinked chitosan.

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Scheme 1: Synthesis of different thiolated chitosan derivatives.

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Scheme 2: Phosphorylation of chitosan using phosphorus pentaoxide.

Scheme 3: Phosphorylation of chitosan using orthophosphoric acid.

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Scheme 4: Phosphorylation of chitosan using mono-(2-methacryloyl oxyethyl)
phosphoric acid.

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Scheme 5: Phosphorylation of chitosan using phosphoric and formaldehyde.

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Scheme 6: Functionalization of chitosan by phosphorous acid and formaldehyde.

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Scheme 7: Reaction of cellulose nano-fibrils with phosphoric acid.

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Scheme 8: Formation of photocrosslinkable chitosan derivatives.

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Scheme 9: N-phthaloyl chitosan derivative.

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Scheme 10: N-phthaloylation reaction of water-soluble chitin with phthalic anhydride.

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Scheme 11: Grafting of chitosan by glutaraldheyde.

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Scheme 12: Formation of aminated chitosan.

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Scheme 13: Grafting of ethylene diamine tetraacetic acid (EDTA) on chitosan

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Scheme 14: Formation of chitosan/ethylene diamine tetraacetic acid-graphene oxide

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Scheme 15: Fabrication of chitosan-ethylene diamine tetraacetic acid-p-cyclodextrin.

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Scheme 16: Chitosan-epichlorohydrin modified biopolymer.
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Scheme 17: Preparation of carboxymethyl chitosan in its quaternized form.

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Scheme 18: Interaction of mono- and dicarboxylic acids with chitosan in screw extruder.

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Scheme 19: Interaction of mono-carboxylic acids with chitosan.

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Scheme 20: Modification of chitosan by succinic, maleic, and phthalic anhydrides.

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Scheme 21: Formation of Water soluble chitin-methacrylate.
-p
re
lP
na
ur
Jo

Scheme 22: Esterification of chitosan and methacrylic acid.

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Scheme 23: Formation of chitosan grafted-poly(ethylene glycol) methacrylate

copolymer.

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-p
re
lP
na
ur
Jo

Scheme 24: Chemically functionalization of chitosan by 3-nitro-4-amino benzoic acid.

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Scheme 25: Modification of chitosan by benzoic acid and p-methoxybenzoic acid.

of
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na

Scheme 26: Formation of trimethyl quaternary chitosan using protection-deprotection

protocol.
ur
Jo

Scheme 27: Formation of cationic chitosan derivatives modified by quaternary

phosphonium salts.

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Scheme 28: Formation of highly substituted N-alkyl-N,N-dimethyl chitosan.
lP
na
ur
Jo

Scheme 29: Synthesis of N-(2-hydroxy)propyl-3-trimethyl chitosan ammonium chloride.

Scheme 30: Formation of N-trimethyl chitosan- poly(vinyl alcohol) (TMC-PVA)

copolymers.

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Scheme 31: Modification of chitosan by cyclodextrin derivatives.

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na

Scheme 32: Mechanisms of linking cyclodextrin and chitosan.

ur
Jo

Scheme 33: Preparation of cyclodextrin-chitosan using tosylated β-cyclodextrin.

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na
ur

Scheme 34: Reaction of β-cyclodextrin citrate and chitosan biopolymer.

Jo

Scheme 35: Reaction of β-cyclodextrin-itaconate and chitosan.

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Scheme 36: Grafting of β-CD on chitosan in the presence of 2-hydroxypropyl spacer.

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Scheme 37: Grafting of β-CD on chitosan in the presence of sugar and maleic spacer.
re
lP
na
ur
Jo

Scheme 38: Preparation of chitosan-β-cyclodextrin via amide bond formation.

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Scheme 39: Condensation of thiosemicarbazide-o-carboxymethyl chitosan and
aldehydes.
lP
na
ur
Jo

Scheme 40: Preparation of chitosan thiosemicarbazide modified biopolymer.

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Scheme 41: Synthesis of monochloro-glyoxime chitosan modified polymer.
lP
na
ur
Jo

Scheme 42: condensation of chitosan and dichloro-glyoxime.

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Scheme 43: Formation of crown ether-chitosan derivative via Schiff’s base type reaction.

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na
ur
Jo

Scheme 44: Preparation of chitosan-dibenzo-18-crown-6 crown ether (CNBD) and

chitosan-dibenzo-18-crown-6 crown ether (CND).

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Scheme 45: Preparation of chitosan-benzo-15-crown-5 (CTS-B15) and chitosan-benzo-

18-crown-6 (CTS-B18) derivatives.

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CRediT author statement
Nabel A. Negm: Conceptualization, Methodology, Writing - Review & Editing, Project

ro
administration. -p
Hassan H. Hefni, Maram T.H. Abou Kana: Conceptualization, Methodology, Writing.
Ali A. Abd-Elaal, Emad A. Badr: Validation and Investigation
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lP

Highlights

Chitosan biopolymer can be functionalized by numerous function groups.

na

Functionalization can be addition, coupling, and crosslinking.

Modification of chitosan upgrades its potential applications.
ur
Jo

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