Biotechnology Advances 66 (2023) 108172

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Current and emerging applications of saccharide-modified chitosan: a

critical review
Hamed Kazemi Shariat Panahi a, b, Mona Dehhaghi b, Hamid Amiri c, d, Gilles J. Guillemin b,
Vijai Kumar Gupta e, f, Ahmad Rajaei g, Yadong Yang h, Wanxi Peng a, **, Junting Pan h, **,
Mortaza Aghbashlo i, **, Meisam Tabatabaei j, a, k, *
a
Henan Province Engineering Research Center for Forest Biomass Value-added Products, School of Forestry, Henan Agricultural University, Zhengzhou 450002, China
b
Neuroinflammation Group, Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, NSW, Australia
c
Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan 81746-73441, Iran
d
Environmental Research Institute, University of Isfahan, Isfahan 81746-73441, Iran
e
Centre for Safe and Improved Food, Scotland’s Rural College (SRUC), Kings Buildings, West Mains Road, Edinburgh EH9 3JG, UK
f
Biorefining and Advanced Materials Research Center, Scotland’s Rural College (SRUC), Kings Buildings, West Mains Road, Edinburgh EH9 3JG, UK
g
Department of Food Science and Technology, Faculty of Agriculture, Shahrood University of Technology, Shahrood, Iran
h
State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China, Institute of Agricultural Resources and Regional Planning, Chinese
Academy of Agricultural Sciences, Beijing 100081, China
i
Department of Mechanical Engineering of Agricultural Machinery, Faculty of Agricultural Engineering and Technology, College of Agriculture and Natural Resources,
University of Tehran, Karaj, Iran
j
Higher Institution Centre of Excellence (HICoE), Institute of Tropical Aquaculture and Fisheries (AKUATROP), Universiti Malaysia Terengganu, Kuala Nerus,
Terengganu 21030, Malaysia
k
Department of Biomaterials, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Chennai 600 077, India

A R T I C L E I N F O A B S T R A C T

Keywords: Chitin, as the main component of the exoskeleton of Arthropoda, is a highly available natural polymer that can
Drug delivery be processed into various value-added products. Its most important derivative, i.e., chitosan, comprising β-1,4-
galactosylated chitosan linked 2-amino-2-deoxy-β-d-glucose (deacetylated d-glucosamine) and N-acetyl-d-glucosamine units, can be
hydrogel
prepared via alkaline deacetylation process. Chitosan has been used as a biodegradable, biocompatible, non-
lactose
wound dressing
antigenic, and nontoxic polymer in some in-vitro applications, but the recently found potentials of chitosan for
in-vivo applications based on its biological activities, especially antimicrobial, antioxidant, and anticancer ac­
tivities, have upgraded the chitosan roles in biomaterials. Chitosan approval, generally recognized as a safe
compound by the United States Food and Drug Administration, has attracted much attention toward its possible
applications in diverse fields, especially biomedicine and agriculture. Despite some favorable characteristics, the
chitosan’s structure should be customized for advanced applications, especially due to its drawbacks, such as low
drug-load capacity, low solubility, high viscosity, lack of elastic properties, and pH sensitivity. In this context,
derivatization with relatively inexpensive and highly available mono- and di-saccharides to soluble branched
chitosan has been considered a "game changer". This review critically scrutinizes the emerging technologies
based on the synthesis and application of lactose- and galactose-modified chitosan as two important chitosan
derivatives. Some characteristics of chitosan derivatives and biological activities have been detailed first to
understand the value of these natural polymers. Second, the saccharide modification of chitosan has been dis­
cussed briefly. Finally, the applications of lactose- and galactose-modified chitosan have been scrutinized and
compared to native chitosan to provide an insight into the current state-of-the research for stimulating new ideas
with the potential of filling research gaps.

* Corresponding author at: Henan Province Engineering Research Center for Forest Biomass Value-added Products, School of Forestry, Henan Agricultural Uni­
versity, Zhengzhou 450002, China.
** Corresponding authors.
E-mail addresses: [email protected] (W. Peng), [email protected] (J. Pan), [email protected] (M. Aghbashlo), [email protected],
[email protected] (M. Tabatabaei).

https://doi.org/10.1016/j.biotechadv.2023.108172
Received 30 October 2022; Received in revised form 15 April 2023; Accepted 6 May 2023
Available online 9 May 2023
0734-9750/© 2023 Elsevier Inc. All rights reserved.
H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

1. Introduction natural polymers and polysaccharides. Due to its biocompatibility,

biodegradability, and suitable reactive functional groups, chitosan can
Microbial biomass or plant and animal residues can be utilized in a be applied as fibers or nanofibers, films, membranes, sponges, beads,
wide range of applications ranging from bioremediation and toxification micro or nanoparticles, gels or hydrogels, suspensions, and solutions.
(Panahi et al., 2020; Hamedi et al., 2015a) to the production of value- Besides biodegradability and biocompatibility, chitosan shows desirable
added bioproducts such as biofuels (Tabatabaei et al., 2020; Kazemi biological properties, including wound healing, antioxidant, antimi­
Shariat Panahi et al., 2019; Tabatabaei et al., 2019). Despite the great crobial, antifungal, antitumor, and immunoenhancing activities. These
potential of natural products, petroleum-based raw products are the characteristics, together with non-antigenic, non-toxicity, and low cost,
common substrate for main commercial polymers. Being environmen­ make chitosan very promising for different extensive pharmaceutical
tally unfriendly and toxic, attention has shifted in recent decades to applications such as wound healing (Zhang et al., 2018), protein binding
replace synthetic polymers with biopolymers such as alginates, chitin (Chang et al., 2016), gene delivery (Cupino et al., 2018), contact lenses
and its derivatives, cellulose, and starch. These natural polymers can be (Anirudhan et al., 2016), implants (Nawrotek et al., 2016), bioimaging
synthesized from renewable resources or living organisms. In addition to (Agrawal et al., 2010), tissue engineering (Ahsan et al., 2018),
their high availability, biopolymers are biodegradable and nontoxic. biomedicine (Khan et al., 2017), and drug delivery systems (Shariatinia
The most important derivative of chitin is chitosan, which can be and Zahraee, 2017; Fazli and Shariatinia, 2017).
prepared via an alkaline deacetylation process (Fan et al., 2018). Chi­ On the downside, native chitosan shows poor solubility in organic
tosan comprises β-1,4-linked 2-amino-2-deoxy-β-d-glucose (deacety­ solvents and water, which limits its application. More specifically, the
lated d-glucosamine) and N-acetyl-d-glucosamine units (Moreno et al., rigid crystalline structure of unmodified chitosan almost completely
2018). Chitosan has received much attention in numerous diverse fields, prevents its solubility in aqueous solutions above pH 6.5–7 (Ma et al.,
including but not limited to nanotechnology, biotechnology, medicine 2009; Roy et al., 2017). Similarly, native chitosan is almost insoluble in
and biomedicine, agriculture, wastewater treatment and sludge dew­ various organic solvents such as N,N dimethylformamide, dichloro­
atering, and food industry (Amiri et al., 2022). methane, tetrahydrofuran, cyclohexane, N-methyl-2-pyrrolidone, ben­
Physical, chemical, and enzymatic processes are used for extracting zene, methylbenzene, acetone, -n-hexane, trichloromethane, and
chitin and chitosan from their natural sources (Fig. 1). Depending on the ethanol. A linear decrease in the solubility of chitosan in water-ethanol
sources from which chitin is extracted, the deacetylation process (10–15 mixtures with increasing ethanol concentration was observed, and only
M NaOH, 40–100◦ C) to obtain chitosan may take several hours to 0.1% chitosan could be dissolved at 50% ethanol (Sano et al., 1999).
several days (Muxika et al., 2017; Vasconcelos, 2014). Another important factor affecting the solubility of chitosan is the de­
The approval of chitosan as generally recognized as a safe (GRAS) gree of acetylation, which is calculated by the deduction of the degree of
compound by the United States Food and Drug Administration intro­ deacetylation (the molar ratio of N-amino-d-glucosamine to N-acetyl-d-
duced a wide range of possibilities for its applications in the pharma­ glucosamine) from 100. When the degree of acetylation is between 10
ceutical and biomedical industry (Ma et al., 2017). Compared to and 40%, the chitosan is soluble in organic acids such as formic acid
synthetically modified cellulose, chitosan is a more commercially (Roy et al., 2017). Due to large charge density, protonated charge
feasible and effective chelating and complexing agent because of its high condensation occurs in the solution of chitosan with a low degree of
percentage of nitrogen (1.25 vs. 6–7%). Some other advantages of chi­ acetylation (<25%), leading to electrostatic repulsion and increasing
tosan are excellent polyelectrolyte characteristics at acidic pH and bio­ solubility (Popa-Nita et al., 2010). The molecular weight of chitosan is
logical properties. Chitosan cationic nature is unique among many another factor that has an inverse relationship with chitosan solubility

Fig. 1. Schematic presentation of chitin and chitosan extraction from natural sources along with chemical structure of chitin molecule, showing N-acetyl glucos­
amine unit and randomly distributed β-(1 → 4)-linked d-glucosamine (deacetylated unit) of completely deacetylated chitosan. Adapted from (Naveed et al., 2019).

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

(Tian et al., 2015). Overall, the solubility issue can be addressed by 2. Main chitosan characteristics affecting its applications
modifying the chemical structure of chitosan. Concerning chemical
structure, three types of reactive functional groups are present in the Variables such as source, polydispersity, molecular weight, and
chitosan molecule. Each repeating unit consists of two hydroxyl groups deacetylation must be considered for the commercial production of
on C-3 and C-6, while each deacetylated unit has one amino group on C- chitosan and the target application. Chitosan can be produced from
2 (Crini and Lichtfouse, 2019). The presence of these reactive functional animals, plants, or microorganisms containing chitin. Chitin is the main
groups would allow the conjugation of many substituents forming component of the exoskeleton of Arthropoda, contributing to a signifi­
different chitosan derivatives with various physical and chemical cant proportion of the body weight. For example, the shell makes up to
properties (Fig. 2). 80% of the whole crab and shrimp body weight (Hamedi et al., 2015b)
Several chemical modifications such as acylation, N-alkylation, sul­ and may comprise up to 46% chitin, depending on the species (El Knidri
fonation, carboxylation, quaternarization, thiolation, phosphorylation, et al., 2018). Insect cuticles such as Cicada slough contain comparable
and graft copolymerization can be used to endow chitosan with specific amounts of chitin to shellfish (Gonil and Sajomsang, 2012).
properties, e.g., light, thermos-, or pH-sensitivity as well as molecular It is worth mentioning that chitin may also be found in significant
design (Sacco et al., 2020; El Knidri et al., 2018). However, these amounts in some filamentous fungal cell walls, such as ruminal fungi
modifications may be toxic or generate toxic by-products (Andreica (phylum Neocallimastigomycota) (Panahi et al., 2022). Mucor rouxii and
et al., 2020), be expensive (Loganathan et al., 2020), or forms a chitosan Aspergillus niger comprise up to 45% chitin in their cell wall, whereas the
structure lacking desired specificity for binding to and being uptaken by amount of chitin in the cell wall of Penicillium notatum is about 20%
target cells and organs. Therefore, the knowledge of the potentials and (Kaur and Dhillon, 2014; Darwesh et al., 2018). Compared to crustacean
limitations of saccharide-modified chitosans can have a key role in their shells, chitosan derived from fungal sources has no seasonal variations in
further development. To this end, this review first presents some char­ amounts, no heavy metal (e.g., copper, nickel), no animal-source aller­
acteristics of chitosan derivatives, including saccharide-modified chi­ gens contaminations, lower molecular weight, and superior particle
tosans, with a separate section on biological activities, i.e., particles size uniformity (Huq et al., 2022). Fungal chitosan has clus­
antimicrobial, antioxidant, and anticancer activities. The next section tered acetylation pattern rather than evenly distributed acetyl groups
briefly discusses different technologies for synthesizing two important observed in chitosan obtained from shellfish (Huq et al., 2022).
saccharide-modified chitosans, viz., lactose- and galactose-modified Generally, fungal source chitosan is more promising for technical
chitosan. The last section of the manuscript scrutinizes the applica­ applications, as both degrees of deacetylation and molecular weight can
tions of the above-mentioned saccharide-modified chitosans in gene be controlled during production. Respectively, the activity of two en­
delivery, drug delivery, transcutaneous immunization, hydrogel, tissue zymes, i.e., chitin deacetylase and chitin synthase, can determine the
engineering, anti-inflammation, anti-skin aging, and wound dressing degree of acetylation and chain length and, in turn, the molecular
and healing. The chemical and biological advantages of lactose- and weight of chitosan (Huq et al., 2022). In contrast, fungal chitosan has
galactose-modified chitosan have been compared to native chitosan significantly lower availability and quantity and higher production cost
where possible to provide an insight into the current state-of-the than animal-derived chitosan (Huq et al., 2022). To improve the eco­
research for stimulating new ideas with the potential of filling nomic feasibility, fungal chitosan can be produced as a by-product of the
research gaps. fungi-based industry.
A commercial method for producing chitosan from crustacean shells
has already been established, and the commercial product is available in

Fig. 2. Some examples of chitosan derivatives. Adapted from (Lima et al., 2022).

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

the market. However, there are still some constraints for this commer­ to 28 d) to lysozyme (1.5 μg/mL in phosphate-buffered saline, 37◦ C)
cial method, including high consumption of acids and bases, time- (Freier et al., 2005). Unlike chitosan, with a 99.2% degree of deacety­
consuming process, and highly energy-intensive process. Moreover, lation, 0.5% showed the highest cell adhesion capability and viability
the production process requires a demineralization step to remove a (Freier et al., 2005). Chitosan with a 50% degree of deacetylation
large amount of calcium carbonate. The main drawback of this process is showed poor cell adhesion capability but was rapidly degradable (Freier
the complexity of producing highly deacetylated chitosan, which is et al., 2005).
suitable for biomedical applications (Kaur and Dhillon, 2014). Unlike a membrane made of chitosan with 65–80% degree of
Molecular weight is another important characteristic that decides the deacetylation, those made of chitosan with 94% degree of deacetylation
chitosan application. Chitosan is categorized based on molecular weight exhibited mild inflammation, low degradation, but minimal osteo­
into low (<100 kDa), medium (<100–1000 kDa), and high (>1000 kDa) genesis in Sprague–Dawley rats with calvaria defects (Hidaka et al.,
molecular weight chitosan (Gonçalves et al., 2021). Increasing the mo­ 1999). The water-holding capacity and the water solubility of chitosan
lecular weight decreases permeability. The molecular weight of com­ can be boosted by increasing its degree of deacetylation (Nam et al.,
mercial chitosan varies from 50 to 2000 kDa, with a common degree of 2010).
acetylation of 80–90% (Mourya et al., 2011). Chitosan with a degree of deacetylations of 55–70%, 70–85%,
Concerning the production of chitosan nanoparticles, highly mono­ 85–95%, and above 95% are defined as low, middle, high, and ultrahigh
disperse particles can be obtained using parent chitosan sources with deacetylation degrees of chitosan (Lv, 2016). Based on this classifica­
narrow molecular weight (Zhang et al., 2004). Moreover, particle tion, low deacetylated chitosan is completely insoluble in water,
polydispersity has an inverse relationship with chitosan molecular whereas middle or high deacetylation degree makes chitosan partly or
weight (Sawtarie et al., 2017). The effect of chitosan molecular weight completely water soluble (Lv, 2016). It is worth mentioning that degree
on the size and other properties of nanoparticles is controversial. One of deacetylation shows an inverse relationship with the molecular
study found that nanoparticle size increases (~190 vs. ~300 nm) with weight of chitosan (Yuan et al., 2011). Therefore, the degree of deace­
decreasing molecular weight of chitosan (10–17 vs. 48–213 kDa) while tylation of the chitosan must be tailored along with other physico­
keeping the degree of deacetylation constant at 88% (Huang et al., chemical properties (e.g., molecular weight, viscosity) depending on its
2005). A lower transfection efficiency on the A549 cell line was final application. Alkaline processing of the native chitosan, extending
observed by larger nanoparticles (Huang et al., 2005). In contrast, high- the processing time, and elevating the process temperature, can increase
molecular-weight chitosan and larger nanoparticles were found to the degree of deacetylation.
maximize target gene expression in the HEK293 cell line (Lavertu et al.,
2006). 3. Biological activity of chitosan and its derivatives
It is worth mentioning that the molecular weight of chitosan particles
can determine their mode of biological activity (Aghbashlo et al., 2022). 3.1. Antimicrobial activity
More specifically, low-molecular-weight chitosan should be used if
penetration into the target cells is desired. In contrast, Antimicrobial activity is one of the most extensively studied char­
high-molecular-weight chitosan can be used in the case of cell surface acteristics of chitosan and its derivatives polymer (Shahbaz et al., 2022;
interaction. Through this interaction, chitosan can exert biological ac­ Rahayu et al., 2022; Li et al., 2022). In 1979, Allan and Hadwiger (Allan
tivity, for example, antibacterial activity, by modifying cell permeability and Hadwiger, 1979) reported the biological activity of chitosan against
and blocking the transport of essential elements into the cell (Leuba and broad-spectrum fungi for the first time. Since then, the antimicrobial
Stossel, 1986). Regarding antioxidant activity, low-molecular-weight activity of this polymer or its derivatives against other microorganisms,
chitosan has more scavenging activities for different radicals than chi­ including yeast and bacteria, has been reported (Jain et al., 2022; Xing
tosan with higher molecular weight (Park et al., 2003; Younes and et al., 2021; Shih et al., 2019). Compared to gram-negative bacteria,
Rinaudo, 2015). Some studies also reported that low-molecular-weight gram-positive bacteria are more susceptible to chitosan (No et al., 2002).
chitosan exhibit higher immunostimulating and antitumor activities Besides microorganism type and cell growth phase, the chitosan’s
than higher-molecular-weight chitosan (Jeon and Kim, 2002; Suzuki antimicrobial activity and magnitude depend on the solvent type, pH,
et al., 1986). deacetylation degree, concentration, and molecular weight. The pres­
It should be noted that chitosan with higher molecular weight may ence of other molecules and their interaction with chitosan also affect
be more suitable for certain biomedical applications. Molecular weight the antimicrobial response (Hosseinnejad and Jafari, 2016). More spe­
increases the impact of mechanical and chemical resistance (Wei et al., cifically, protonated amino groups are formed in the chitosan chain
2012), both of which may be critical for successful tissue engineering. when the pH of the solution is less than the pKa of chitosan. With that
On the other hand, as molecular weight increases, the number of en­ being said, the polycationic structure of chitosan, developed at pH < 6.5,
tanglements of the polymer chain in solution and, in turn, solution can increase membrane permeability and induce its lysis following
viscosity increase. This physicochemical property of chitosan can interaction with negatively charged molecules on the microbial cell
negatively or positively affect its application. On the bright side, even a surface (Tan et al., 2013; Follmann et al., 2012; Carmona-Ribeiro and de
low concentration of high-molecular-weight chitosan can be suitably Melo Carrasco, 2013). However, this is not the only mechanism of chi­
used to form uniform fibers web during electrospinning due to providing tosan’s antibacterial activity. Chitosan with a positive charge can bind to
an adequate number of entanglements of polymer chains and restraining microbial DNA and kill cells by seizing the replication and transcription
effects of surface tension. This property would be suitable if chitosan is (Hosseinnejad and Jafari, 2016). Through the flocculation of electro­
meant to be used for transcutaneous immunization or wound dressing negative particles in the cytoplasm, chitosan can interfere with the
and healing applications. On the downside, the high viscosity of chitosan physiological activities of bacterial cells (El-Tahlawy et al., 2005).
decreases the pharmaceutical characteristics of chitosan by lowering its Furthermore, chitosan may inhibit microbial growth by chelating the
dispersion, diffusion, and permeability. Therefore, the molecular weight trace elements that are essential nutrients for microorganisms (Chien
of chitosan must be considered and tailored for exploiting the best mode et al., 2016; Li et al., 2010a; Li et al., 2010b). For example, chitosan
of activity depending on the target. chelation of various metal ions can prevent microorganisms from
Many biological and physicochemical parameters of chitosan, such accessing them for synthesizing intact cell walls. Fig. 3 schematically
as cell response, degradation, solubility, and crystallinity, are deter­ represents the antibacterial mechanism of chitosan and its derivatives.
mined by its deacetylation degree (Yuan et al., 2011). In this context, Chitosan can be modified to boost its antibacterial activity signifi­
chitosan with either the highest (99.2%) or lowest (0.5%) degree of cantly. For example, Escherichia coli is more significantly inhibited by
deacetylation showed very low mass loss upon prolonged exposure (up chitosan quaternary ammonium salt derivative (N-trimethyl chitosan,

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

Fig. 3. Schematic representation of the antibacterial mechanism of chitosan and its derivatives.

N-propyl-N, N-dimethyl chitosan, and N-furfuryl-N, N-dimethylforma­ and the reaction can contribute to the antibiotic activity of the
mide), compared to native chitosan (Jia and Xu, 2001). Functionalizing saccharide-modified chitosan. For example, the antibacterial activity of
chitosan with arginine can significantly increase the growth-inhibiting the Millard reaction products of chitosan oligosaccharide (MRPCO) and
activity against E. coli and Pseudomonas fluorescens by increasing the glucose or maltose were compared to that of the chitosan oligosaccha­
interaction of chitosan with cell membranes for more efficient alteration ride alone (Sun et al., 2017). The glucose derivative increased both
of cell membrane permeability (Tang et al., 2010). antibacterial activities against Vibrio parahaemolyticus and Pseudomonas
Chitosan’s protonated amino groups are responsible for the anti­ aeruginosa and preservative activities against Penaeus vannamei, whereas
bacterial activity, particularly based on the electrostatic interactions. In the maltose derivative decreased them (Sun et al., 2017).
that case, modifying chitosan with saccharides is expected to decrease
the antibacterial activity following decreasing the number of protonated 3.2. Antioxidant activity
amino groups. In contrast, the chitosan-glucose complex exhibited
similar antibacterial activities (minimum inhibitory concentration, Chitosan and its derivatives show remarkable redox regulatory ac­
0.05%) to native chitosan against Bacillus cereus, Staphylococcus aureus, tivity owing to a high scavenging capacity for reactive oxygen species
Pseudomonas sp., and Escherichia coli. Adding chitosan or chitosan- (ROS). Consequently, chitosan can inhibit lipid peroxidation, oxidative
glucose complex as a food preservative to pork cocktail salami and stress, and disruption in cell function (Ivanova and Yaneva, 2020).
chilled-stored lamb meat increased their shelf-life by about 28 and 14 d, Excellent scavenging activities of chitosan for free radicals are due to the
respectively (Kanatt et al., 2008). Interestingly, compared to native presence of active hydroxyl and amino groups. By removing free radi­
chitosan, galactose-modified chitosan showed greater corresponding cals, the activity of cellular antioxidant enzymes is increased. The level
antibacterial and antifungal activities against pathogenic bacteria from of antioxidant activity of chitosan is significantly determined by its
tilapia fillets (8–9 vs. 12 mm inhibition zone) and pathogenic fungi from molecular weight and degree of deacetylation (Samar et al., 2013; Kim,
smoked fish (8–12 vs. 18 mm inhibition zone) (Sulistijowati et al., 2019). 2018; Avelelas et al., 2019; Zheng et al., 2022). The lower the molecular
In another study (Il’ina et al., 2008), chitosan’s molecular weight and weight of chitosan, the more its antioxidant activity will be (Kim, 2018).
chemical modification were found significant for its antibacterial ac­ In contrast, the degree of deacetylation generally shows, with some
tivities. Modifying chitosan with galactose and mannose improved the exceptions (Zheng et al., 2022), a direct correlation with the antioxidant
antibacterial activity of the compound (molecular weight 5 kDa. 0.01% activity of chitosan (Kim, 2018).
concentration) against B. subtilis and E. coli. Increasing the molecular The antioxidant activity of chitosan can be engineered through
weight of the chitosan or its saccharide-modified derivatives from 5 kDa chemical modifications. In this context, N-alkylated chitosan derivatives
to 24 kDa improved the antibacterial activity against the tested bacteria exhibit a concentration-depended antioxidant activity against Cu2+
(92–98% vs. 99.92–99.98% lethality) (Il’ina et al., 2008). Therefore, it chelating activity, superoxide anion radicals, hydrogen peroxide, and
can be concluded that the antibacterial effect of chitosan is not solely DPPH radicals. It is worth mentioning that increasing the degree of
due to electrostatic interaction. Moreover, the type of the reducing sugar substitution of N-alkylated chitosan derivatives from 20% to 70%

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

decreases the scavenging activity (Lin and Chou, 2004). The gold- and induce apoptosis while activating the immune system for targeting
chitosan nanocomposite is another chemically modified chitosan tumor cells (Fig. 4) (Shi et al., 2017). The extent of antitumor activity
showing excellent antioxidant activity equivalent to 80 times that of depends on several parameters, including tumor type, chitosan source,
ascorbic acid (Esumi et al., 2003). Active oxygen radicals (-OH, acetylation degree, and molecular weight. Chitosan extracted from
hydrogen peroxide, O2-) can be effectively scavenged by chitosan sulfate shiitake mushrooms exhibits higher antitumor activity than crab chito­
in a dose-dependent manner (Esumi et al., 2003). san (Chien et al., 2012). Compared to high molecular weight commer­
Chitosan-glucose complex showed superior antioxidant activity than cial chitosan, May fruit or commercially available shrimp low-molecular
glucose or chitosan alone. The scavenging activity was dose-dependent, chitosan more significantly induced apoptosis in cancer cells (Tan et al.,
and the IC50 value of the chitosan-glucose complex for DPPH radical 2018).
scavenging was 51.1 μg/mL. Although the compound showed high Song et al. (2020) found that nanoparticles stabilized with low-
scavenging activity for superoxide anion and hydroxyl radicals, it had a molecular-weight chitosan have less efficient anti-proliferative activity
low, reducing power (Kanatt et al., 2008). Similarly, galactose-modified against HepG2 than those stabilized with high-molecular-weight chito­
chitosan delivered the highest antioxidant activity among all the treat­ san. In contrast, another study reported an inverse correlation between
ments, viz., chitosan, chitosan-glucose complex, fructose chitosan com­ the molecular weight of chitosan and its anti-tumor activity on cancer
plex, and galactose chitosan complex (Sari et al., 2013). The intensity of cell lines (Tan et al., 2018). This observation can be attributed to the
brownish color, antioxidant activity (determined using the DPPH lower diffusion of high-molecular-weight chitosan samples because of
method), and the reducing power of the chitosan and its derivatives higher viscosity, limiting their penetration into the cell membrane. A
ranged from 0.031–0.224, 92–131 ppm, and 1059–1274, respectively direct correlation has been reported concerning the effect of acetylation
(Sari et al., 2013). (Sulistijowati et al. (2019) found a superior antioxi­ degree on antitumor activity (Younes et al., 2016).
dant activity of 1 g galactose-chitosan complex than native chitosan Exposing human gastric cancer BGC-823 to seleno-short-chain-
using the DPPH method (IC50 value of 43.2 vs. 73.1 ppm). In summary, chitosan-induced apoptosis via the mitochondrial signaling pathway
the type of groups in chitosan structure determines its antioxidant ca­ (Dong et al., 2019). More specifically, mitochondrial film potential is
pacity and the type of scavenged free radical. For example, OH radicals destroyed, reactive oxygen species are accumulated, and the Bax-to-Bcl-
can be removed by reacting with hydroxyl groups in chitosan via 2 ratio, caspases 3 and 9, and Cytochrome C are over-activated activa­
substituting hydrogen (Wang et al., 2020). tion (Dong et al., 2019). Hyaluronic acid-coated chitosan nanoparticles
can bind to the CD44 receptor, a receptor highly expressed in many
cancer cells, improving drug delivery efficiency. Apart from acting as a
3.3. Anticancer activity
drug carrier, the synthesized nanoparticles were found distributed in
mitochondria, activating the mitochondrial apoptotic pathway by
Anticancer activity is another promising characteristic of chitosan
increasing reactive oxygen species (Wang et al., 2017). Tumor metas­
and its derivatives that can be exploited in chemotherapy. In this
tasis can be inhibited, and apoptosis can be induced by
context, chitosan and its derivatives can inhibit tumor cell proliferation

Fig. 4. A schematic representation of the antitumor mechanism of chitosan and its derivatives.

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

norbornin@carboxymethyl chitosan. The polymer-drug conjugates inhibited in mice by CD73-specific small interfering RNA@lactate chi­
down-regulated the expression of matrix metalloproteinase-2 and 9, B- tosan. When administered along with dendritic cell vaccines, a syner­
cell lymphoma 2, and vascular endothelial growth factor while up- gistic anti-tumor response was observed. More specifically, the presence
regulating the expression of Bcl-2-associated X protein and TNF-α (Chi of immunosuppressive cells in the tumor site was decreased, and tumor
et al., 2019). growth and metastasis were prevented, extending mouse survival time
Alternatively, chitosan and derivatives may indirectly destroy tumor (Jadidi-Niaragh et al., 2017). A summary of the chitosan derivatives,
cells. By regulating the biological activity of immune cells, these poly­ their properties, and applications is tabulated in Table 1.
mers can increase the chance for the host immune system’s recognition
and destruction of tumor cells. Chitosan nanoparticles with surface 4. Characteristics and synthesis of saccharide-modified chitosan
mannose moieties loaded with tumor cell lysates generated from B16
melanoma cells with a nanoparticles size of 120±10 nm, dry powder As discussed in Section 2, efforts should be put into selecting a chi­
inhaler (DPI) of 0.121±0.05, and Zeta potentials of -12.07±1.36 mV tosan source with matching physicochemical characteristics (i.e., mo­
were synthesized (Shi et al., 2017). This chitosan nanoparticle could lecular weight, degree of deacetylation) to the target application. The
specifically target dendritic cells, elevating interleukin 4 and interferon- next step would be tailoring and customizing the native chitosan to
gamma titers in serum and evoking cytotoxic T lymphocyte responses. address its shortcomings that impede the therapeutic and cell experi­
The mice vaccinated with this nanoparticle showed immunity to cancer mental applications. For example, despite its potential for expanding
for some time (Shi et al., 2017). Hydrophilic cationic polymer (N,N,N- hepatic cells due to its resemblance to glycosaminoglycan, the pH-
trimethyl chitosan, TMC) based nano-complexes (FTCD/ recombinant sensitive nature of native chitosan limits its cell culture application
human interleukin-2) is another chitosan derivative with antitumor (Aghdam et al., 2020). The modification of the chitosan backbone can
growth activity (Wu et al., 2017). CD73 expression in tumor cells was change the physical-chemical properties in the obtained derivative.

Table 1
A summary of applications of chitosan derivatives.
Chitosan derivatives Properties Applications Main findings Ref.

- DS: 0.260 to 0.283

Antibacterial activity against Dose-dependent anti-bacterial activity
Geraniol-conjugated chitosan oligosaccharide - Yields: up to 78% (Bi et al.,
Escherichia coli and with more sensitivity on
(COS-N-Ger) - Solubility: water-soluble 2021)
Staphylococcus aureus S. aureus
in the pH range of 1–7
- DS: 0.255 to 0.366 Antibacterial activity against (Yue et al.,
Cinnamyl conjugated chitosan (COS-N-Cin) DS-dependent anti-bacterial activity
- Yields: up to 82% E. coli and Staphylococcus aureus 2021)
Pyridine-4-Aldehyde Schiff Bases Grafted - DS: 50% Antibacterial activity against (Zhang et al.,
MIC and MBC: >16 mg/mL
Chloracetyl Chitosan (CACS) - Yields: 30.25% E. coli 2023)
Pyridine-4-Aldehyde Schiff Bases Grafted - DS: 50% (Zhang et al.,
Antioxidant activity Inhibitory index: 7.92% at 1.6 mg/mL
Chloracetyl Chitosan (CACS) - Yields: 30.25% 2023)
- 99.95% antibacterial activity against
Quaternized chitosan-modified nanofiber - DQ: 60–72% Antibacterial activity against E. coli (Cheah et al.,
membrane - Water-stable E. coli - Antibacterial activity was connected to 2019)
acidic and alkali during synthesis
Antibacterial activity against
- Swelling degree: 193% - MIC against E. coli, S. aureus, and
Glutaraldehyde cross-linked chitosan quaternary E. coli, (Li et al.,
- Stable in acidic, neutral, P. aeruginosa was 40, 320, and 640 μg/
ammonium salt film (CHACC) S. aureus, Pseudomonas 2018)
and alkaline conditions mL, respectively
aeruginosa
- Soluble in a wide range
of pH (up to 8).
- Size: 200 nm
Drug delivery Enhanced transfection rate in cancer cell (Jaiswal
Methyl methacrylate-modified chitosan - Zeta potential: 52.0
(curcumin) lines (A549, HeLa, HepG2) et al., 2019)
± 4.38 mV.
- Drug encapsulation
efficiency: 68%
- Size: 153.8 ± 12.54 nm
Poly-β-amino ester/thiolated O-carboxymethyl Drug delivery - Increased cellular uptake (Tang et al.,
- Stable in the presence of
chitosan nanoparticles (siRNA) - Enhanced transfection rate 2020)
RNase A and serum
- Size: 31.6 ± 4.5nm
Drug delivery - Increased cellular uptake (Wen et al.,
Polyethylene glycol grafted chitosan nanoparticles - Zeta potential: 21.6 ± 1.5
(P53) - Enhanced transfection rate 2019)
mV.
- Water-soluble
Trans transcriptional activator/poly(N-3- - Size: 397 ± 19 nm Drug delivery (Kamra et al.,
Increased drug delivery efficiency
benzyloxycarbonyl-lysine) chitosan - Zeta potential: 22 ± 2 (P53, doxorubicin) 2019)
mV
- Cell compatibility
- Microporous structure In situ tissue regrowth
Hydroxypropyltrimethylammonium chloride (Cui et al.,
- Pore size: 115 ± 40 μm- Tissue engineering
chitosan 2019)
150 ± 50 μm - Inducing differentiation of
mesenchymal stem cells
- Porous structure - Suitable for cell infiltration/
- Pore size: 150 and 200 colonization (Teimouri
silk fibroin-chitosan/Nano ZrO2 Tissue engineering
μm - Non-toxicity to the Human Gingival et al., 2015)
- Porosity: 79.4 ± 1.20 Fibroblast
- Effective loading and release of ions
- Size: 1–10 μm (Gritsch et al.,
Chitosan/hydroxyapatite composite Tissue engineering - Dual ion delivery
- Porosity: 90% 2019)
- Cytocompatible

Degree of substitution (DS).

Degree of quaternization (DQ).

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

Solubility at neutral pH is one of the most significant physicochemical calculated to be 6.69, while the secondary amino group of lactose-
properties for formulating chitosan derivatives with novel applications. modified chitosan had a pKa of 5.87. The possible formation of
Upon increasing the pH of the medium, chitosan shows a linear decrease hydrogen bonds within the chitosan structure, which modifies the
in its linear charge density. Chitosan has a low charge density at phys­ dissociation equilibrium, can explain the differences between the pKa of
iological pH, reducing chain-chain electrostatic repulsion. These char­ the modified amino functionality and the primary amine (Sacco et al.,
acteristics lower the chitosan solubility and eventually decrease the 2020).
stability of chitosan-based formulations (Sacco et al., 2020). The lower viscosity of saccharide-modified chitosan is attributed to
Because native chitosan is almost insoluble at pH above 6.5–7, the the more compact structure of chitosan following the introduction of
solubility or similar constraints can be addressed by forming chitosan branches on its backbone (Tømmeraas et al., 2002). The Millard reaction
derivatives such as phenolic compounds, galactosylated, acrylated, was used to synthesize low-molecular-weight chitosan derivatives (5
thiolated, or carboxylated chitosan (Khoshfetrat et al., 2016; Chopra and 25 kDa) with mannose, galactose, N-acetyl galactosamine, and
et al., 2006). The attachment of such compounds can also increase glucosamine (Il’ina et al., 2008). The resulting yield and substitution
mechanical properties and the dissolution rate (Aghdam et al., 2020). degrees were 60–80% and 4–14%, respectively. The viscosities of 24-
For example, oligosaccharides can be added to the primary amino kDa chitosan and its derivatives were in the following order (lowest to
groups in the backbone of chitosan to increase its aqueous solubility. highest): galactose-chitosan (0.33 dl/g), mannose-chitosan (0.36 dl/g),
From this perspective, cheap and highly available saccharides, particu­ N-acetyl-glucosamine-chitosan (0.38 dl/g), chitosan (0.76 dl/g) (Il’ina
larly mono- and di-saccharides, have been extensively used to synthesize et al., 2008). Decreasing the molecular weight to 5 kDa kept the chitosan
soluble branched chitosans. Under physiological conditions, saccharide- as the most viscous compound (0.20 dl/g), whereas rendering
modified chitosan is hydrolyzed with lower viscosity and better solu­ glucosamine-chitosan as the least viscous derivative (0.12 dl/g) fol­
bility than native chitosan (Termsarasab et al., 2013). This behavior is lowed by galactose- and mannose-chitosan with the corresponding vis­
related to more free amino groups in a D-glucosamine unit as saccharide- cosities of 0.14 and 0.15 dl/g (Il’ina et al., 2008). Other advantages of
modified chitosan has a higher deacetylation degree and shorter chain saccharide-modified chitosan are their non-cytotoxic nature, easy in­
lengths than native chitosan (Hamed et al., 2020; Sun et al., 2017). testinal epithelium uptake, and the eventual fate of excretion in the
Modifying chitosan with galactose or mannose with a corresponding urine. The presence of reactive functional groups on saccharide-
substitution degree of 14% or 11% can improve the pH solubility range modified chitosan allows its chemical modification. These reactive
from ≤5.3 to 5.3–8 or 5.3–7.5 (Il’ina et al., 2008). functional groups include a glycosidic link between the N-glucosamine
Typically, the introduction of oligosaccharides side chains into chi­ units, the amine/acetamide group, primary hydroxyl groups at C3, and
tosan structure (≥40%) can solubilize the compound at all pH values secondary hydroxyl groups at C6 (Lodhi et al., 2014; Riva et al., 2011).
(Tømmeraas et al., 2002). Despite the ionic strength, lactose-modified Millard reaction, amide bond formation, and reductive N-alkylation
chitosans are completely soluble at neutral pH (D’Amelio et al., 2013). are possibly the most popular chemical modification reactions to syn­
Glycosylation of chitosan not only changes its solubility at non-acidic pH thesize saccharide-modified chitosan (Sacco et al., 2020). Based on the
values but also provides better interactions with polyanions such as type of saccharide used and the operating conditions, a wide range of
DNA. Compared to linear chitosan, branched chitosan increased the substitution degrees can be obtained using the mentioned reactions. The
transfection efficiency of HeLa cells from almost 0 to 70% (Malmo et al., Maillard reaction is particularly employed for the synthesis of chitosan-
2011). Additionally, more stable complexes with DNA could be achieved glucose conjugates. Chung et al. (Chung et al., 2005) and Gullon et al.
in Hanks Balanced Solution and Phosphate Buffered Saline by intro­ (Gullón et al., 2016) have extensively characterized glucose-modified
ducing oligosaccharides on the chitosan backbone (Issa et al., 2006). chitosan with the latter group optimizing the reactive conditions for
Depending on the ionic strength used, treating lactose-modified chitosan synthesizing chitosan-glucose derivative with a substitution degree of
with a highly charged polyanionic polysaccharide (e.g., alginate) forms around 65%. In 1984, the Millard reaction was used to conjugate a range
soluble complexes, a phase-separated polyanion-polycation aggregate, of saccharides to the free amino function at C2 of chitosan with substi­
or a completely non-interacting system (Sacco et al., 2020). Upon tution degrees of 1–97% of free amino acids (Yalpani and Hall, 1984). N-
decreasing the ionic strength, the transition from a non-interacting alkylation reaction was used by Sashiwa and Shigemasa (1999) to syn­
system to soluble complexes with phase separated system as the inter­ thesize various glycosylated chitosan with substitution degrees of
mediate can be observed at a polyanion/polycation ratio from 0.75 to 37–100% and 7–74% for mono- and di-saccharide-modified chitosan,
0.25. In contrast, no formation of soluble complexes could be observed respectively. Amide bond formation reaction delivered the lowest
at the polyanion/polycation ratio of 0.15 (Donati et al., 2007a). condensation degree between the acidic group introduced in the
The formation and stability of complex coacervates between lactose- saccharide and the free amino groups of chitosan, compared to the
modified chitosan and hyaluronic acid as radical-scavenging drug car­ Millard and N-alkylation reactions (Sacco et al., 2020). A substitution
riers were studied (Vecchies et al., 2018). At acidic pH, the positive degree of 8–23% was obtained during the chemical modification of
charges on lactose-modified chitosan bind to negative charges on hya­ chitosan with lactobionic acid (Il’ina and Varlamov, 2007).
luronan through electrostatic interactions. Increasing pH significantly Lactose is one of the disaccharides used in engineering chitosan
changes the colloidal stability, and completely dissolved lactose- structures for desired applications. Lactose-modified chitosan,
modified chitosan and hyaluronic acid coacervates at pH>6 by commercially known as Chitlac, is synthesized by N-alkylation of chi­
removing positive charges on the chitosan (Vecchies et al., 2018). By tosan in the presence of lactose, which involves the amino group of
manipulating the primary-to-secondary amine ratio, a 13% decrease in chitosan (Fig. 5). Chitlac is a novel modified polymer that has advan­
the degree of glycosylated chitosan substitution can increase the disso­ tages over chitosan due to its solubility at neutral pH and high misci­
lution stability. Concerning mechanical properties, a synergistic inter­ bility with polyanions (e.g., alginate, hyaluronan) (Donati et al., 2007a;
action between lactose-modified chitosan and highly charged Pizzolitto et al., 2022). Interaction between Chitlac and polyanions
polyanions such as alginate results in higher strength over the polymers forms a soluble binary polysaccharide mixture. The advantage of this
alone (Donati et al., 2007b). mixture is that a synergistic effect in dilute and semi-dilute conditions
1
H-NMR titrations were used to calculate the apparent pKa values of occurs, which increases the viscosity of the solution and facilitates the
the primary and secondary amino groups of saccharide-modified chi­ formation of a temporary network. Chitlac has also been applied to form
tosan. The values of 6.9 and 5.2 were reported for the primary amino a ternary complex with alginate and hyaluronan (Marsich et al., 2013b;
group of chitosan and the secondary amino group of glycosylated chi­ Sacco et al., 2018). Chitlac has recently gained attention due to its po­
tosan (Tømmeraas et al., 2002). In another study (D’Amelio et al., tential applications in tissue engineering and drug delivery (Pizzolitto
2013), the apparent pKa of the primary amino group of chitosan was et al., 2022; Medelin et al., 2018). Outstanding biological applications of

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

Fig. 5. Synthesis of Chitlac through N-alkylation of chitosan in the presence of lactose. Adapted from (Sacco et al., 2018).

Chitlac have been studied both in vitro and in vivo. However, the effect of based gene carriers to obtain cell-specificity (Hashida et al., 2001; Molas
lactose release by hydrolysis of Chitlac must be studied as lactose (pKa, et al., 2003). Moreover, sugar can enhance chitosan intracellular traf­
~11.3) is strongly acidic. In this context, the effect of lactose release in ficking and nuclear import (Monsigny et al., 2004; Satoh et al., 2007). A
the surrounding tissue may be irritating and potentially ulcerative. reductive alkylation technique was used to galactosylate a chitosan
Additionally, the application of Chitlac in lactose intolerance people as derivative, i.e., 6-amino-6-deoxy chitosan (6ACT) with 3–50% substi­
well as those allergic to milk may cause serious health complications. tution degrees per pyranose in the presence of lactose (Satoh et al.,
Such potential issues must be studied to better understand the possible 2007). This modification did not change the electrostatic interaction
side-effects of using Chitlac for biomedical applications. between plasmid DNA and galactose-modified 6ACT. Compared to
6ACT, galactose-modified 6ACT with substitution degrees of 18–50%
5. Application of saccharide-modified chitosan and at N/P values of 1.5–2.5 showed significantly higher transfection
efficiency for Hep G2 (Satoh et al., 2007). The transfection efficiency
5.1. Gene delivery increased with increasing the substitution range, despite the size limi­
tations that impeded the inclusion of aggregated galactose-mediated
The presence of primary amino groups in the chitosan backbone and 6ACT complexes into clathrin-mediated endocytosis. Probably, facili­
its ability to form spontaneous complexes with nucleic acids by elec­ tated intracellular trafficking was the reason for the higher transfection
trostatic interaction have made chitosan an interesting non-viral gene efficiency (Satoh et al., 2007).
carrier (Satoh et al., 2007). However, one of the main constraints of A cationic plasmid DNA/chitosan (pDNA/Cs) complex coated with
chitosan-based gene delivery is the insufficient transfection efficiency of lactose-/maltose-modified chitosan/polyethylene glycol (sugar-PEG-A/
the system. To address this issue, chitosan must be chemically modified. Cs) was synthesized to deliver the desired gene in vitro efficiently.
For example, the solubility of chitosan under physiological conditions PDNA/Cs-sugar-PEG-A/Cs showed spherical shapes with diameters of
can be increased by hydrophilic polymer graft on or trimethylation of 500 nm and 200 nm (at N:P:C = 1:5:10) and pDNA/Cs (at P:N > 3),
the chitosan backbone (Kean et al., 2005; Wong et al., 2006). To increase respectively. Both complexes had significant degradation resistance
the binding specificity to cell receptors, transferrin (Leong et al., 1998), against DNaseI compared to the uncoated plasmid DNA. Moreover, 20-
folate (Mansouri et al., 2006), and glycosides (Hashimoto et al., 2006; and 5-time higher transfection efficiencies were achieved using pDNA/
Kim et al., 2004) can be linked to chitosan. Endosomal vesicles can be Cs-maltose-PEG-A/Cs and pDNA/Cs-lactose-PEG-A/Cs in B16 mouse
ruptured via the proton sponge mechanism by urocanic acid-modified melanoma cells, respectively (Hashimoto et al., 2008).
chitosan (Kim et al., 2003).
Among different modification methods, saccharide modification has 5.2. Drug delivery
gained much attraction because it is cost-effective, nontoxic, easily
available, and can enhance the binding specificity of chitosan to the Treating some diseases, such as neurodegenerative diseases and
target cells or organ (Hashida et al., 2001; Molas et al., 2003; Monsigny cancers, is still underdeveloped. The global cancer-related deaths in
et al., 2004; Satoh et al., 2007). Compared to proteins and peptides, 2018 were estimated at 9.5 million cases, which is expected to surge to
sugar moieties are chemically more stable. Through sugar modification, 16.4 million by 2040 (GCO, 2022). The causative agents of cancers or
receptor-mediated recognition capability can be introduced to chitosan- neurodegenerative diseases cannot be deliberately removed or even

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

reduced. For example, traffic-related air pollution (Dehhaghi et al., Despite overall nontoxicity and biocompatibility, the minute size of
2021; Panahi et al., 2021), other environmental pollution such as heavy carbon dots (<10 nm) prevents the passive targeting of tumor tissues
metal (Kazemi Shariat Panahi et al., 2020), insects such as ticks (Deh­ through enhanced permeability and retention effect. Mesoporous silica
haghi et al., 2019a), and even some microorganisms or their metabolites is another interesting compound for drug delivery due to its efficient
such as BMAA (Kazemi Shariat Panahi et al., 2022) may be associated loading capacity of some drugs (e.g., doxorubicin) (Atabaev et al., 2014;
with the development of cancers or neurodegenerative diseases. Gai et al., 2012). Despite sustained drug release characteristics, the
Therefore, drug companies must develop and commercialize efficient, application of mesoporous silica in the drug delivery system is limited as
intelligent drugs to alleviate such diseases. On the other hand, systemic it may cause severe hemolysis.
drug administration may trigger side effects due to unwanted drug- Saccharide-modified chitosan, such as galactose-modified chitosan,
tissue interactions. To address these issues, the release of therapeutic is a promising drug delivery carrier with good biocompatibility char­
agents in the body must be controlled or targeted at specific locations by acteristics. However, galactose-modified chitosan lacks enough drug-
engineering appropriate drug delivery systems. loading capacity for efficient therapy. This constraint can be removed
Carbon dots and other carbon nanomaterials have been purposed as by grafting galactose-modified chitosan on some potent carriers, such as
great compounds for bioimaging and loading anti-cancer drugs (D’souza liposomes and nanocomposites (graphene oxide), which have high drug
et al., 2016b; D’souza et al., 2016a; Mehta et al., 2017). Unlike metallic- loading capacity. Liposomes are small artificial spherical nanoshells
engineered nanomaterial, carbon-based nanoparticles do not release with a vesicle shell and an aqueous core. This compound has a structural
free radicals inside cells, triggering oxidative stress, inflammation, and resemblance to the cell membrane and is formed by the spontaneous
DNA damage (Dehhaghi et al., 2019b). Generally, carbon dots are closure of phospholipid molecules in an aqueous solution (Wei et al.,
considered nontoxic and biocompatible nanoparticles. A comprehensive 2021). Liposomes can encapsulate both lipophilic and hydrophilic drugs
toxicity study using a zebrafish embryo acute toxicity test calculated the in lipid-soluble moiety between phospholipid bilayers and the internal
LD50 of two carbon dots prepared by spermidine or ammonia citrate at cavity, respectively. Besides high loading capacity, liposomes can
100 and 500 ppm, respectively (Chung et al., 2021). The corresponding improve drug delivery efficiency by eliciting mononuclear phagocytosis
LD50 for these carbon dots dropped to 55 and 340 ppm in the later stage (Che et al., 2020; Pattni et al., 2015). However, some disadvantages of
of the embryonic phase, i.e., eleutheroembryos (Chung et al., 2021). No liposomes include their high production cost and low solubility. More­
bioaccumulation behavior was observed, as carbon dots were quickly over, thermodynamically unstable lysosome structures can lead to size,
eliminated from the embryo and eleutheroembryo. Long oral exposure residual surface charge, and structural reorganization over time.
of adult zebra fish to carbon dots for up to 12 weeks caused no adverse Graphene oxide, the other type of potent carrier to be matched with
effects, including on reproduction and offspring (Chung et al., 2021). galactose-modified chitosan, can load a significant amount of drugs by
Mouse models also supported that nitrogen-doped carbon dots have Van der Waals interaction and pi stacking (also called π–π stacking). The
no toxicity after 30 d (Lee et al., 2016). However, it should be noted that large loading capacity of this biocompatible compound arises from its
size, dosage, charge, and surface chemistry can affect carbon dots large surface area and many functional groups (Wang et al., 2018).
toxicity. Concerning size, carbon dots <6 nm are easily and quickly However, the toxicity of graphene oxide depends on its route of
excreted via renal routes (i.e., kidneys) and have negligible toxicity administration, size and other physicochemical properties, and its non-
(Truskewycz et al., 2022). However, those larger than 6 nm circulate in targeted cellular uptake. Briefly, orally administered graphene oxide in
the body for an extended time, increasing the chance for interactions adult mice was absorbed by the intestine and quickly excreted (Fu et al.,
with different molecules and organs. 2015). Following subcutaneous injection, the smaller graphene oxide
The increase in carbon dots size beyond 8 nm does not necessarily particles (2 μm) summoned more mononuclear cells than larger ones
cause toxicity (Truskewycz et al., 2022). More specifically, the toxicity (350 μm) to infiltrate the adipose tissue. High accumulation of graphene
rather depends on dosage, charge, and surface chemistry than the size of oxide in mice organs, including bone marrow, liver, lung, and spleen,
carbon dots. On this basis, polyethylene glycol-modified carbon dots was observed after its intravenous administration (10 mg/kg body
with a neutral charge (up to 300 mg/L) did not trigger any cell abnor­ weight) (Liu et al., 2012). The accumulation of graphene oxide in mice’s
malities regarding cell cycle, intracellular trafficking, and cell lungs triggered granuloma formation and pulmonary edema (Liu et al.,
morphology (Havrdova et al., 2016). In contrast, pristine carbon dots 2012). Therefore, the physiological consequences (inflammation, ag­
with negative charge due to carboxylic groups increased oxidative gregation, etc.) of administrating carbonaceous nanoparticles such as
stress, arrested the G2/M phase of the cell cycle, and stimulated pro­ graphene oxide to an organism via different routes (e.g., oral, intrave­
liferation (Havrdova et al., 2016). Positively charged polyethylenimine- nous, inhalation) must be carefully studied, considering nanoparticle
coated carbon dots could enter the cell nucleus, manipulating the G0/G1 physicochemical properties.
phase of the cell cycle at ~100 mg/L (Havrdova et al., 2016). Among various saccharides, galactose has been extensively studied
Besides toxicity, surface functionalities of carbon dots determine as a modifying material in developing drug-delivery systems for the
nanoparticle retention and organ distribution. Therefore, the surface liver. More specifically, hepatocytes primarily express asialoglycopro­
functionality of carbon dots is an important criterion that must be tein receptors on their surface that can specifically bind to galactose
considered to minimize the health consequences of carbon dots residues coupled with either positively or negatively charged molecules.
adsorption by non-target cells (Truskewycz et al., 2022). Typically, Galactosylated positively charged molecules such as galactosylated
carbon dots, in comparison with larger carbon nanomaterials (e.g., chitosan can be further coupled with anionic liposomes to develop a
graphene oxide, carbon nanotubes), show significantly lower retention viable targeted delivery system. Lin and Chen (2007) synthesized chi­
in the lung (2 vs. 43–55% ID/g) (Chen et al., 2015). The retention of tosan-O-poly(ethylene,glycol)-galactose through hydroxyl groups of
carbon dots in the spleen and liver was 5% ID/g and 12% ID/g, chitosan. The pegylated chitosan shows solubility in organic solvents
respectively (Chen et al., 2015). Opsonization, the binding of plasma and neutral aqueous solutions with a critical micelle concentration and
proteins to the surface of carbon dots, is another influential biological size of 0.56 mg/mL and 295 nm, respectively (Lin and Chen, 2007). The
barrier to drug delivery, eventually leading to the enzymatic degrada­ degree of O-substitution of chitosan by poly(ethylene, glycol) was
tion of carbon dots by phagocytes (Blanco et al., 2015). Additionally, 177.7%, indicating the conjugation of both C3-OH and C6-OH of chi­
carbon dots and other carbonaceous nanoparticles can be degraded by tosan with the polymer chains. The final product consisted of up to 3.1%
other enzymes, including eosinophil peroxidase and myeloperoxidase galactose with an 86.7% degree of substitution of poly(ethylene, glycol)
(Martín et al., 2019; Kagan et al., 2010; Lu et al., 2014). This enzymatic by galactose (Lin and Chen, 2007). The attachment of galactose to the
degradation can decrease the bioaccumulation of carbon dots in the hydroxyl group of poly(ethylene, glycol) provides an appreciable tar­
body. geting moiety for asialoglycoprotein receptors of hepatocytes. It is worth

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

mentioning that the biocompatibility and stealth properties of poly­ expensive drug. The drug delivery system had the corresponding
ethylene glycol are the main reasons for its wide application in drug entrapment efficiency, poly-dispersity index, zeta potential, and particle
delivery (Fang et al., 2006). These properties can support the prolonged size of ~54.7%, 0.28, 27.15 mV, and 218.2 nm (Fatouh et al., 2021).
circulation of the drug delivery system. Controversially, some studies Compared to ledipasvir dispersion, the galactosylated chitosan-coated
have reported that polyethylene glycol can induce opsonization (Ver­ one showed significantly improved pharmaceutical characteristics.
hoef and Anchordoquy, 2013), promote the production of anti- The liver peak concentration of the drug (11.4 vs. 34.2 μg/g), liver ter­
polyethylene glycol antibodies by the immune system (Ishida et al., minal half-life (32 vs. 19 h) and mean residence time (18.11 vs. 13.45 h),
2007; Wang et al., 2007), and reduce uptake and delivery of the drug, and the area under the liver concentration vs. time curve AUC(0-72 h)
especially intracellular delivery, to the target tissues (Harvie et al., 2000; (~88.86 vs. 26 μg*h/g) were significantly improved. The maximum time
Bao et al., 2013). Therefore, more studies are required to confirm the the drug needed to reach the liver cells was the same for both coated and
safety and effectiveness of drug delivery system based on pegylated uncoated drugs, i.e., 3 h (Fatouh et al., 2021).
chitosan. Galactose-modified chitosan can also be used to improve the
Concerning liver cancer, galactose-modified chitosan can target he­ bioavailability of water-insoluble drugs, such as quercetin. Quercetin is
patocellular carcinoma cells by interacting with the asialoglycoprotein a natural antioxidant, anti-inflammatory polyphenolic flavonoid
receptor (Wang et al., 2018). Using this characteristic, the delivery of a showing promising liver protection activity (Pingili et al., 2020). To
novel hepatitis C direct-acting antiviral agent, known as ledipasvir, to address the low bioavailability of quercetin (Boots et al., 2007), the drug
hepatocytes was enhanced after coating its liposome formulation on was loaded on anionic liposomes and then coated with galactosylated
galactosylated chitosan (Fatouh et al., 2021). chitosan to specifically bind to the asialoglycoprotein receptor on the
Among various advantages, such as decreasing the drug side effects liver cells (Wei et al., 2021). Following targeting the receptor, liposomes
and enhancing drug efficacy, the system could significantly lower the fused with the membrane and entered the cell via endocytosis. The drug
therapeutic regimen costs by decreasing the recommended dose of this delivery system injected into the tail vein of mice could enrich the liver

Fig. 6. (a) Chemical synthesis of galactosylated chitosan (GC) using chitosan (CS) and lactobionic acid (LA), (b) liposomes preparation and their coating with
galactosylated chitosan, and (c) the possible protective mechanism of quercetin liposomes against ALI in mice (Wei et al., 2021). With Permission from Elsevier.
Copyright© 2021. LN: 5411670590615.

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

with a sustained drug release, promoting M2 polarization of macro­ deliver anti-inflammatory agents specifically to the organ of desire while
phages. Thus, the M1 polarization of macrophages was inhibited, and lowering the medication’s side effects. For example, biological treat­
the inflammatory response was decreased because of blocking NF-κB, ments (e.g., golimumab pegol, adalimumab, infliximab) of inflammatory
JNK-STAT, and ERK pathways and lowering the secretion of pro- bowel disease can increase the risk of autoimmunity, infusion reactions,
inflammatory cytokines (e.g., interleukins 6 and 8, TNF-α) (Li et al., lymphoma, and opportunistic infections (Magro and Portela, 2010;
2016). Quercetin@liposoms coated with glycosylated-chitosan pro­ Huang et al., 2018; Guo et al., 2016). Concerning inflammatory bowel
tected the liver from damage in the acute liver injury model by mini­ disease, Crohn’s disease and ulcerative colitis are its main types (Larsen
mizing lipid oxidation and expression levels of alkaline phosphatase, et al., 2018). One approach to suppress ulcerative colitis is to decrease
alanine aminotransferase, and aspartate aminotransferase while maxi­ the secretion of tumor necrosis factor-alpha (TNF-α) from monocytes
mizing glutathione (Wei et al., 2021). Consequently, the expression of and macrophages in the intestine using RNAi technology either through
caspase 3, 6, 9, and other pro-apoptotic caspase family proteins was short-interfering RNA (siRNA) (Cheng et al., 2015; Chiu and Rana,
inhibited while anti-apoptotic Bcl-2 protein expression was activated 2003) or microRNA method (Olesen et al., 2014; Cucchiara et al., 2012).
(Hartman and Czyz, 2020; Zhang et al., 2020). Fig. 6 schematically Following synthesis through in vitro technique, siRNA can be deliber­
presents the chemical synthesis of galactosylated chitosan, liposomes ately introduced into the body by transfection to precisely regulate the
preparation and their coating with galactosylated chitosan, and the desired genes owing to its complete complementarity with the target
possible protective mechanism of quercetin liposomes against ALI in mRNA sequence (Cheng et al., 2015). Concerning ulcerative colitis, a
mice. drug-loaded carrier with TNF-α siRNA can be orally administered to
Through the fabrication of galactosylated chitosan/graphene oxide, form an RNA-induced silencing complex (RISC) in the cytoplasm of
hepatocellular carcinoma can be specifically and efficiently targeted. macrophages. RISC uses the TNF-α siRNA as a template for recognizing
Wang et al. (2018) used a carbamide reaction to couple chitosan with the complementary TNF-α mRNA, resulting in RNase activation, cleav­
lactobionic acid (Fig. 7). Then, doxorubicin (1.08 mg/mg polymer) and age of the RNA, and eventually, down-regulation of TNF-α (Xiao et al.,
galactosylated chitosan were loaded on a graphene-oxide carrier 2016). The drug prescription and side effects can be minimized if TNF-α
(Fig. 7). siRNA is positioned and specifically released in inflamed tissues within
The drug delivery system showed stability under physiological con­ the colon.
ditions and released doxorubicin within the tumor environment (i.e., In this context, TNF-α siRNA was loaded on poly(lactic-co-glycolic
low pH). Compared to chitosan/graphene oxide/doxorubicin nano­ acid) nanoparticles with galactosylated chitosan grafted on its surface
particles, galactosylated one delivered higher cytotoxicity against (Huang et al., 2018). The galactosylated chitosan shell could specifically
SMMC-7721 and Hep G2 cells due to its better uptake (Fig. 8) (Wang bind to the macrophage galacto-type lectin (MGL), a type II trans­
et al., 2018). Although the authors did not study the uptake mechanism; membrane CLR containing a single carbohydrate recognition domain
however, it might be attributed to asialoglycoprotein receptors on the specific for galactose and N-acetylgalactosamine. MGL is highly
liver, capable of specific interaction with galactose moiety on the expressed on the surface of macrophages during inflammation (McCar­
modified chitosan. thy et al., 2013). Apart from the specific recognition, the uptake of the
Apart from the liver, galactosylated chitosan can be engineered to drug delivery system was also increased owing to its nanoparticle

Fig. 7. Schematic chemical synthesis of galactosylated chitosan and galactosylated chitosan/graphene oxide/doxorubicin (GC–GO–DOX) nanoparticles based on the
method explained by (Wang et al., 2018).

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

Fig. 8. Intracellular uptake and localization of two different nanoparticles, viz., chitosan/graphene oxide/doxorubicin and (CS–GO–DOX) and galactosylated chi­
tosan/graphene oxide/doxorubicin nanoparticles (GC–GO–DOX) in different hepatoma cell lines after 2 h incubation. With Permission from Elsevier. Copyright©
2018. LN: 5411701386136.

nature. Compared to conventional delivery systems, polylactic-co-gly­ drug after freeze-drying in the presence of a cryoprotectant (Vecchies
colic acid nanoparticles could conveniently evade phagocytosis and pass et al., 2018).
through the physiological barrier (Huang et al., 2018). TNF-α siRNA To offer a controlled release drug delivery system for tenofovir, an
galactosylated-chitosan-modified nanoparticles formulated with antiviral drug that selectively inhibits the reverse transcriptase DNA
different concentrations (1–3 mg/mL) of poly(lactic-co-glycolic acid) polymerase of hepatitis B virus, to the liver cells, Chitlac-based nano­
showed corresponding particle size, peak particle size, Zeta potential, particles were used. The encapsulation of tenofovir on the carriers
poly-dispersity index, and encapsulation efficiency of 236.8–322.1 nm, (named F2 and F3) was performed using ionic gelation of Chitlac with
285.2–371 nm, 20–22 mV, 0.170–0.186, and 77.7–90.1% (Huang et al., tripolyphosphate. Tenofovir-loaded chitosan was also synthesized as the
2018). Galactosylated-chitosan modified and unmodified nanoparticles control (named F1). The size of nanoparticles F1 and F3 were 226.4 and
were compared using a murine leukemia monocyte-macrophage cell line 176.5 nm with polydispersity index of 0.26 and 0.1 and electrical charge
(RAW 264.7 cells) and a mouse animal model (Male C57BL/6 mice). The of +28.4 and +11.7 mV, respectively (Avinash et al., 2015). The
former formulation improved over unmodified nanoparticles (Huang encapsulation efficiency percentage was increased from 39.3% (F2) in
et al., 2018). Only a low therapeutic dosage of 66 μg/kg was required for optimal aqueous conditions to 65.9% (F3) in the presence of isopropyl
treating a mice model suffering from ulcerative colitis. The developed alcohol/acetic acid solution. Compared to the control, the in vitro studies
drug was made from biodegradable materials, i.e., chitosan and revealed that Chitlac nanoparticles loaded with tenofovir had zero-order
polylactic-co-glycolic acid. Interestingly, the FDA has already approved kinetics with sustained drug release up to 48 h and higher liver distri­
polylactic-co-glycolic acid as a pharmaceutical excipient in United bution (Avinash et al., 2015).
States Pharmacopeia (Hoda et al., 2017). A novel multi-functionalized polymeric micelle for cell imaging and
In summary, galactose-modified chitosan can improve the carried Taxol (anticancer drug) delivery was synthesized by polymerizing
drug’s bioavailability, target specificity, and stability. Lactose is another phenylboronic acid conjugated pluronic and Chitlac (Lee et al., 2014).
saccharide that can be used for modifying chitosan for drug delivery To initiate the drug release and optical cells imaging, pH-responsive
purposes, providing relatively similar advantages to galactose. Howev­ boronate ester and redox-sensitive disulfide bonds were involved,
er, Chitlac has less binding specificity for liver cells than galactose- respectively. The low pH in cancer cells was employed to promote the
modified chitosan. drug release from the micelles, and the reducing activity of the intra­
Chitlac and hyaluronan coacervate could be introduced as free cellular environment helped to break down the disulfide bonds (Lee
radical scavengers and carriers of different therapeutic molecules et al., 2014). The efficiency of Taxol loading in micelles was about 83.6,
(Fig. 9). The presence of NaCl decreased the size distribution of the of which more than 90% was released within 48 h at pH 5 in the pres­
coacervates. Concerning biological activity, spheroidal nano/micro­ ence of 10 mM dithiothreitol. Dithiothreitol contributed to a higher drug
particles of the coacervates showed an efficient capsulation capacity and release rate by cleaving more disulfide bonds at acidic pH. Importantly,
preserved the physical and biological characteristics of the encapsulated Taxol-loaded micelles showed less cytotoxicity than free Taxol on KB

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

Fig. 9. A schematic representation of the interaction between Hyaluronan and Chitlac.

cells due to the lower amount and slow release of the active agent (Lee stability upon freeze-drying with no cryoprotectant or upon maintaining
et al., 2014). at 4◦ C for three weeks. However, increasing pH and ionic strength could
Furlani et al. (2017) synthesized highly monodisperse colloidal co­ dissolve the coacervates. At 0.5 μg/mL fluorophore concentration, 60%
acervates based on a bioactive Chitlac, i.e., 1-deoxylactit-1-yl chitosan encapsulation efficiency and about 0.3 μg/mg loading capacity were
(CTL60) and the multivalent anion tripolyphosphate in nano-size range. achieved. The amount of payload showed a negative correlation with
At pH 4.5, colloidal coacervates were formed at specific values of the encapsulation efficiency, dropping it to below 40% (Furlani et al.,
molar ratio of TPP to the CTL60 repeating unit. The fact that coacer­ 2017).
vation could only be obtained at pH 4.5 and not at pH 7.4 demonstrated
that electrostatic interactions played a vital role in stabilizing the
compound (Furlani et al., 2017). Contrary to chitosan, CTL60 induced 5.3. Transcutaneous immunization
the formation of highly homogeneous coacervates with very low poly­
dispersity index values. Additionally, CTL60 coacervates showed high Transcutaneous immunization can be used as a safe and effective
substitute for conventional vaccination methods, including oral

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

administration, intramuscular injection, or subcutaneous injection. The adsorption capacity and mechanical resistance of chitosan can be
Unlike the conventional method, antigen delivered through trans­ improved when it is chemically modified in the presence of glycine,
cutaneous immunization does not get degraded in the liver and/or amine-functionalized magnetic nanoparticles, aromatic polyimides,
gastrointestinal routes (Ita, 2016; Engelke et al., 2015). This advantage, carbodiimide, ethylenediamine, pyridyl, oxo-2-glutaric acid, and amino
together with the presence of abundant antigen-presenting cells (APCs) acid esters (Jeon and Höll, 2003; Kavianinia et al., 2012; Bahram et al.,
on the skin, decreases the vaccine doses required to achieve similar ef­ 2016).
fects to the conventional vaccination methods (Engelke et al., 2015; To improve the functional behavior of hepatocytes, collagen, and
Yang et al., 2020). The other advantage of transcutaneous immunization galactosylated chitosan can be introduced to the alginate microcapsule
over injection methods is its painlessness. However, the efficiency of backbone with an extra chitosan layer. Briefly, the alginate provides
transcutaneous immunization can be limited by the outermost layer of excellent cell encapsulation but only allows weak hepatocyte cultivation
the epidermis, i.e., the stratum corneum. This layer is almost completely because of inefficient cell-matrix and cell-cell interactions. This short­
impermeable to pathogens and antigens (molecular weight> 500 Da) coming can be addressed by combining alginate with natural substrates,
(Chen et al., 2018). This issue can be overcome by introducing etho­ such as chitosan and collagen (Dvir-Ginzberg et al., 2003). Compared to
somes, a special liposome with high encapsulation ability and deform­ the systems with different hydrogel groups of alginate/chitosan,
ability. The efficiency of transcutaneous immunization can be increased alginate-galactosylated chitosan-collagen/chitosan (AGCCol/C) could
further by modifying the antigen carriers to target dendritic cells, the enhance the proliferation of microencapsulated hepatocyte model cells,
main APCs on the skin. The antigen-carrier scaffolds containing gal­ i.e., HepG2 cells. AGCCol/C could also favorably affect the secretion of
actosylated chitosan can effectively target dendritic cells in their urea and albumin (Aghdam et al., 2020).
galactosyl groups, i.e., galactose-type C-type lectin (Park et al., 2003; Coating alginate with chitosan increased the microcapsule consis­
Feng et al., 2009). This type II transmembrane galactosyl group can bind tency and integrity from 72 h (complete degradation of uncoated algi­
oligosaccharides, Lewis X (e.g., monosaccharides), N-acetyl galactos­ nate) to at least 192 h at pH 7.4 (Aghdam et al., 2020). In contrast,
amines, and galactose (Ebner et al., 2004; Higashi et al., 2002). adding galactosylated chitosan to alginate decreased the swelling and
To improve stability and skin permeation efficiency, galactosylated degradation rates, perhaps due to strong cross-linking between proton­
chitosan can be supplemented with hyaluronic acid as the transdermal ated amino groups of glycosylated chitosan and carboxyl groups of al­
carrier because it is found in high concentrations in the skin (Xie et al., ginates. Polyelectrolyte complex membrane of alginate/chitosan is
2018). On this basis, layer-by-layer self-assembly method and green formed due to the simultaneous outward migration of these two com­
electrospinning were used to prepare the hyaluronic acid and gal­ pounds, hence improving the microcapsule stability further (Lou et al.,
actosylated chitosan modified ethosome on a silk fibroin nanofibrous 2016)
mats, loaded with ovalbumin (OVA@Eth-HA-GC/SF) (Yang et al., Another important property for engineering suitable hydrogels to
2020). The transdermal immunological efficiency of the mat on a mu­ tolerate stresses during in-vitro and in-vivo applications is mechanical
rine model was studied. Following transdermal administration, the mat strength, regulating cell-extracellular-matrix interaction and cell fate
surged the serum titer for anti-OVA-specific IgG and induced the decision. This property is determined by the configuration and structure
expression of interleukins 2 and 6, and interferon-gamma by spleen cells of the porous scaffold and cross-linking between different hydrogel
(Yang et al., 2020). The mat could also suppress the growth of EG7 tu­ components (Kim et al., 2015; Kim et al., 2017). Compared to arginine/
mors in the animal model. chitosan, arginine-galactosylated chitosan/chitosan (AGC/C) hydrogel
has significantly lower bulk and wall Young’s modulus (42.8 and 4703
5.4. Hydrogel vs. 10.3 and 1546 kPa) (Aghdam et al., 2020). Therefore, extra supple­
ments should be added to enhance the mechanical strength of the system
Hydrogel is a three-dimensional cross-linked polymeric system with if galactosylated chitosan must be used. Adding collagen to AGC/C
a significantly large capacity to entrap water in its intermolecular space hydrogel can significantly boost the bulk Young’s modulus to 48.6 kPa
(at least 10% w/v or w/w). This hydrophilicity trait gives natural me­ while only moderately improving the wall Young’s modulus to 2118 kPa
chanical flexibility to hydrogels. -SO3H, -CONH-, -CONH2, -OH, -COOH, (Aghdam et al., 2020).
-NH2 are some hydrophilic groups within the hydrogel network (Bahram It is worth mentioning that high mechanical strength is not always
et al., 2016). However, the physical or chemical cross-links and/or chain satisfactory. More specifically, the mechanical strength must suit well
entanglements typically prevent their dissolution in water. The high the purpose of application. For example, a healthy liver tissue has a
versatility in their synthesis methods, as well as their tunable properties, modulus range of 5–10 kPa. Therefore, the chitosan hydrogels engi­
make hydrogels very interesting materials for various engineering and neered for liver injury treatment must have a similar mechanical
biomedical applications. These applications can range from wastewater strength to that of healthy liver cells. This parameter is extremely
treatment and regenerative medicine to tissue engineering. important in preventing liver fibrosis and damage to healthy tissue
In response to stimuli, hydrogens often show a major reversible (Huang et al., 2013). Considering this parameter, Chen et al. (2020)
conformational change, i.e., sol-gel phase transition (also known as designed a gelatine methacrylate (GelMA)/oxidized hyaluronic acid
gelation) or volume phase transition. The chemical stimuli include ions, (OHA)/galactosylated chitosan (Gal-CS)/Fe (III)@TA@IGF-2 200
pH, and media or solution compositions, whereas the physical ones (TA200) hydrogel loaded with insulin-like growth factor 2 (IGF-2),
include pressure, light intensity, solvent composition, magnetic or showing modulus of ~2 kPa. This mechanical strength was formulated
electric fields, and temperature. The degree and type of response to these by forming an interpenetrating polymer network with the addition of
stimuli significantly depend on pendant chains, charge density, the na­ oxidized hyaluronic acid to dissipate the external energy. IGF-2 was
ture of the monument, and the degree of cross-linkage (Bahram et al., gradually released (up to 90% within 14 days) from its carrier, i.e., Fe
2016). Hydrogels can be classified based on the preparation method (III)@TA microspheres, to stimulate the regeneration of damaged he­
(homopolymeric, copolymeric, interpenetrating, semi- patocytes. Hepatocytes could conveniently migrate into the prepared
interpenetrating), ionic charge (cationic, anionic, non-ionic), source hydrogel owing to its storage modulus of 1.1 kPa. The hydrogel speci­
(natural, synthetic, hybrid), crosslinking (physical, chemical), and their ficity for binding asialoglycoprotein receptors of hepatocytes was due to
structure (hydrocolloid aggregates, crystalline, semi-crystalline, amor­ galactosylated chitosan. The hydrogel could stimulate the expression of
phous) (Bahram et al., 2016). transferrin and hepatocyte nuclear factor-1 alpha from human hepatic
Chitosan can be defined as an interpenetrating thermosensitive stellate cell line LX2 damaged with carbon tetrachloride (Chen et al.,
biopolymeric hydrogel. This hydrogel can adsorb heavy metal ions from 2020). Therefore, the hydrogels not only improve the survival of LX2
aqueous media by multiple hydroxyl and amino groups in its structure. cells but also restore their specific function. Therefore, the prepared

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

hydrogel has the potential for future application either as a liver shapes (Wang et al., 2016). After attachment for 6 hours, most cells on
regenerator or in constructing artificial livers for drug screening. Chitlac prepared with higher concentrations of NaBH4 recovered normal
Donati et al. (2005) reported that Chitlac could induce cell aggre­ morphology (Wang et al., 2016). It can be concluded that galactose
gation in the primary culture of chondrocytes and stimulate the pro­ moieties improve the recovery of original cell morphology and biolog­
duction of type II collagen and glycosaminoglycans (Donati et al., 2005). ical properties.
The beneficial effects of Chitlac in the treatment of osteoarthritis have In medical implants, metals can be replaced with biostable fiber-
been recently studied (Scognamiglio et al., 2020). Accordingly, a reinforced composites (Evans and Gregson, 1998). These composites
hydrogel based on Chitlac reticulated with boric acid was an effective were originally developed for dental applications and were based on
complex for viscosupplementation. The lactose-boric acid complex triethyleneglycoldimethacrylate and bisphenol-A-dimethacrylate ther­
showed high capability as a free radical scavenger with notable moset in combination with non-cross-linked polymer with E-glass fibers
biocompatibility in vitro with no stimulating activity on cytokine pro­ (Vallittu, 1999; Vallittu and Sevelius, 2000). To meet the demands of
duction by macrophages (Scognamiglio et al., 2020). The interaction cranial bone reconstructions, fiber-reinforced composites were
between Chitlac and a transient cross-linker molecule, such as boric customized to synthesize load-bearing implants and got approval for
acid, provides a structure with transient nature and geometric stiffening clinical use (Aitasalo et al., 2012; Zhao et al., 2009). Like other medical
feature. Boric acid plays important roles, including network nucleator, implants and autologous bone flaps, one of the main constraints for the
network reorganizer, and network disassembling molecule (Furlani successful medical application of these composites is their health
et al., 2019) and can mimic the role of myosin II or actin-binding pro­ complication because of the adherence of antimicrobial-resistant bac­
teins on actin (Ideses et al., 2013). teria leading to infectious diseases (Widmer, 2001; Sundseth et al.,
2014). This issue makes the patient visit clinics after some times to
5.5. Tissue-engineering remove the infected implant (Nganga et al., 2014).
A promising solution for implant-associated infections is to coat
Tissue engineering is a new interdisciplinary field in biomedical implants with bacteriocidic or bacteriostatic materials. In this context,
engineering aiming to regenerate new living tissue for organ trans­ the Chitlac solution was used to reduce silver ions with ascorbic acid
plantation (Wang et al., 2016). Appropriate artificial or natural 3D (Marsich et al., 2013a). The resulting Chitlac-silver nanoparticles were
biodegradable matrices must be easily available for tissue-engineering used for coating a nano/composite scaffold as bone grafts, i.e., alginate-
development. As one of the three essential factors, this factor not only nanohydroxyapatite (average diameter, 1.6 cm; thickness, 1 cm) (Mar­
functions as an adhesive substrate but also acts as the payload and sich et al., 2013a). The interaction occurred due to electrostatic forces
storage of growth factor (Feng et al., 2009; Cho et al., 2006). While the between the polyanion alginate and the polycation Chitlac, which was
selected compound must be cytologically compatible, it must establish soluble at neutral pH. Morphological and structural analyses using
biological interactions with cells to achieve desired tasks (e.g., induce scanning electron microscopy showed the scaffold has an isotropic
tissue repair and growth). Cellular responses, including attachment, porous interconnected homogenous structure with a pore size distribu­
viability, growth, and differentiation into the desired organ, can be tion range of 150–450 μm. Inductively coupled plasma atomic emission
monitored by developing engineered artificial scaffolds. However, un­ spectroscopy showed about 32 μg silver has been deposited on each
like natural polymers, synthetic polymer-based scaffolds may not be alginate-nanohydroxyapatite scaffold, equivalent to 500 μg of silver per
biocompatible. Therefore, natural biologically active polymers such as each gram of scaffold (Marsich et al., 2013a). Compared to uncoated
extracellular matrix can be used for tissue engineering to obtain excel­ scaffolds, the coated ones showed significant antibacterial activity
lent cell adhesion and growth. The extracellular matrix is the natural 3D against four tested bacteria, i.e., Staphylococcus aureus, Pseudomonas
microenvironment scaffold composing fiber-forming proteins (e.g., aeruginosa, E. coli, and a clinical strain of Streptococcus epidermidis with
elastic fibers, collagen) and non-fiber-forming proteins (e.g., soluble very fast killing kinetics (within 300 min of exposure) (Marsich et al.,
factors, glycosaminoglycans, proteoglycans (Hinderer et al., 2016). The 2013a).
structural organization, components, and biomechanics of the extra­
cellular matrix vary from tissue to tissue and are crucial for designing 5.6. Anti-inflammation and anti-skin aging
biomaterials for tissue engineering. More specifically, cells attach to the
extracellular matrix through cell surface receptors, whereby cellular Skin aging is a slow natural process in which changes in the dermal
responses, including migration, proliferation, and differentiation, are compartment lead to degradation or lower formation of the extracellular
activated (Hinderer et al., 2016). One promising extracellular matrix for matrix. Consequently, skin hydration, elasticity, and strength dramati­
a three-dimensional scaffold is chitosan. When used as an implant, the cally change (Wlaschek et al., 2021). Besides age, intrinsic and extrinsic
native structure of chitosan consisting of 2-amino-2-deoxy-d-glucose factors, including the human microbiome, air pollution, and ultraviolet
(GlcNH2) residues with a variable number of randomly located N- radiation, can enhance the accumulation of senescent cells, causing
acetyl-glucosamine groups (GlcNAc) causes no pathological and almost structural changes to dermal extracellular matrix composition (Boyajian
no immunological inflammatory responses. et al., 2021; Pillai et al., 2005). This process also increases the produc­
Through biopolymer engineering, oligosaccharides, peptides, other tion of pro-inflammatory cytokines and reactive oxygen species that
extracellular signaling molecules, or cell-specific ligands can be intro­ changes the micro-environment of the skin. As a result, a chronic in­
duced into the chitosan chain. To investigate the effect of galactose flammatory state is triggered, further disrupting the extracellular matrix
moieties in chitosan scaffold on cell attachment, mechanical properties, (Xia et al., 2016). In summary, inflammation and reactive oxygen spe­
and biocompatibility, Chitlac, with various degrees of substitution of cies are two main causes of skin aging by bringing subtle functional and
galactose moieties, was synthesized (Wang et al., 2016). The chemical structural modifications to the skin.
process included a Schiff base reaction between the amino group of Macrophage is one of the primary immune cells mediating chronic
chitosan and the glucose group of lactose in different concentrations skin inflammation, subsequently causing skin aging by releasing pro-
(0.2–1.6 g) of reducing agent NaBH4. This reaction was followed by the inflammatory mediators and reactive oxygen species and degrading
formation of the porous scaffolds through the freezing-drying method. the extracellular matrix (Guimarães et al., 2021; Kammeyer and Luiten,
The attachment and biocompatibility of hepatocytes (LO2 cells) in the 2015). In turn, the degradation rate of the extracellular matrix is
engineered chitosan scaffold were studied using a phase contrast mi­ enhanced by fibroblasts, i.e., the most abundant cellular component of
croscope and hemolysis and cell growth assays. The increasing con­ the dermis). These cells target collagen types I and III, and elastin with
centration of the reducing agent during chitosan preparation could shift extracellular matrix by producing cytokines (Interleukin-1β, tumor ne­
the attachment shape of LO2 cells on the scaffold from round to spheroid crosis factor α) and catabolic enzymes (metalloproteinase 3) (Cole et al.,

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

2018; Gruber et al., 2020). To slow down skin aging, Chitlac and hyal­ surgery (Millner et al., 2009). On the downside, native chitosan fibers
uronic acid mixture was produced. Chitlac improved the sustained lack elastic properties, limiting their application in wound dressings. To
therapeutic activity of hyaluronic acid to inhibit macrophage-induced improve the dissolution behaviors and mechanical properties, addi­
inflammation, reactive oxygen species production, and metal­ tional polymers must be blended with chitosan before producing
loproteinase expression (Donato et al., 2022). (Tarricone et al., 2020) chitosan-based fibrous membranes using electrospinning. This is
reported that a binary solution of Chitlac and hyaluronic acid could because extended rigid blocks of molecular chains exist in the chitosan
attenuate the macrophages-induced inflammation and decrease the backbones, following dissolution in the aqueous acetic acid solution
expression of metalloproteinases (MMP-3 and MMP-13), suggesting (Sapkota and Chou, 2020). Together with the formation of strong
potential anti-inflammatory and antioxidant activities towards the intramolecular hydrogen bonds, it is impossible to obtain a continuous
treatment of inflammatory diseases (Tarricone et al., 2020). It is well fiber formation during electrospinning, and the process is rather an
known that the cytokines IL-1β and TNF-α which are released by acti­ electrospraying due to jet break up (Barchuk et al., 2016; Nie et al.,
vated synoviocytes or chondrocytes, can inhibit the synthesis of extra­ 2008). Adding synthetic polymers (e.g., polyesters, polyethylene tere­
cellular matrix and increase the expression of metalloproteinases phthalate, polyvinyl alcohol, polyethylene oxide) or natural polymers (e.
(Funato et al., 2017; McDermott et al., 2019; Weinmann et al., 2016). g., cellulose, fibroin, silk, gelatin) to chitosan can facilitate electro­
Decreasing the expression of metalloproteinases and suppressing the spinning by causing chain entanglement and increasing chain flexibility
production of IL-1β, TNF-α, and galectin-1 and 3 in human dermal fi­ (Sapkota and Chou, 2020).
broblasts by Chitlac/hyaluronic acid complex may be therapeutically Polysaccharide-membranes-based electrospun wound dressing was
applicable to reduce chronic skin inflammation and, consequently, prepared by dissolving polyethylene oxide in deionized water and
prevent skin aging (Donato et al., 2022). The effects on galectin-1 and 3 adding hyaluronic acid and Chitlac to it (Gruppuso et al., 2022). An
expressions are attributed to the direct interaction of lactose residues of optimum bead-free fiber morphology with thin and uniform membranes
Chitlac inhibiting the binding of galectins to the cell surface. was obtained after 90 min of electrospinning. The average fiber diam­
eter from 100 randomly selected fibers on the sample was 442±117 nm.
5.7. Wound dressing and healing In the presence of Tween 20 as the surfactant, Chitlac and hyaluronic
acid were used in a ternary mixture with polyethylene oxide (Gruppuso
Chronic non-healing wounds are defined by an impaired wound- et al., 2022). Through this strategy, a Chitlac-based nanofibrous mesh
healing cycle, mainly due to dysregulated inflammatory response and with defect-free, thin, and homogeneous nanofibers was synthesized for
disrupted angiogenesis. Some special therapies, such as surgical the first time. The mesh diameter range mimicked that of the natural
debridement, oxygen therapy, negative pressure therapy, ultrasound collagen nanofibers (50–500 nm) (Gruppuso et al., 2022). The high
treatment, and skin grafts, can be used to treat non-healing wounds. hydrophilicity nature of Chitlac and hyaluronic acid led to the quick
After wound cleaning, an important step is dressing, eliminating the dissolution of the synthesized membranes upon contact with water. To
excess fluid in the wound area, and providing a protective layer against address this issue, carbonyldiimidazole was used as a crosslinking agent
infectious agents. Some key characteristics of an ideal wound dressing to provide water resistance. The resulting electrospun product showed
that can affect the performance of the medical device include high excellent swelling and degradation behavior and liquid absorbance ca­
flexibility to the wound shape, gas permeability, water adsorption ca­ pacity while maintaining the balance between gas permeability and
pacity, anti-scarring potential, antimicrobial activity, biocompatibility water retention (Gruppuso et al., 2022).
(Fu et al., 2021; Kraskouski et al., 2021). It should also be cost-effective
and mimic extracellular matrix structure and mechanical properties 6. Conclusions
(Gruppuso et al., 2022). The electrospinning technique can be effec­
tively and conveniently used for synthesizing nanofibrous wound Chitosan is an abundant polysaccharide with great potential to fill
dressings like extracellular matrix. This type of dressing is highly the increasing demand for natural polymeric materials. As antimicro­
interconnected porous structures with high surface area, improving bial, antioxidant, and non-immunogenic, with biodegradable, biocom­
drainage of excess fluid, exchange of gases, and hemostasis (Li et al., patible, and bio-based properties, chitosan is a promising lead
2021; Tonda-Turo et al., 2018). Because of these characteristics, the compound that can be exploited in many fields. These fields include
electrospun mats accelerate wound repair and closure while inhibiting gene delivery, drug delivery, transcutaneous immunization, hydrogel,
scar formation (Ekambaram and Dharmalingam, 2020). tissue engineering, anti-inflammation, anti-skin aging, and wound
Besides FDA approval, good biocompatibility, mechanical strength, dressing/healing. The physicochemical characteristics of native chito­
thermal stability, and degradation profile have recently favored syn­ san can be varied based on its source. Fungi-based chitosan has lower
thetic polymers as bulk materials for electrospun wound dressings. In molecular weight and polydispersity than shellfish-based chitosan.
contrast, hydrophobicity and lack of intrinsic bioactivity and biological Despite higher technical applicability, the current production of chito­
signal directly recognizable by cells are some disadvantages of synthetic san from fungi is not economically feasible. To improve the technical
polymers (Wang et al., 2021; Zhou et al., 2021). A promising alternative and economic feasibility of chitosan for commercial application, native
is natural polymers, including polysaccharides (e.g., dextran, cellulose, chitosan must be obtained from the source providing the right physi­
hyaluronic acid, chitosan, alginate) or proteins (e.g., fibroin, fibrinogen, cochemical properties (e.g., molecular weight, degree of deacetylation)
collagen). Natural polymers have similar advantages to synthetic poly­ for the target application. As the molecular weight increases, the
mers as a substrate for electrospun wound dressing. Additionally, they permeability, diffusibility, dispersibility, and scavenging activity of
are hydrophilic and provide appropriate regenerative substrate owning chitosan decrease. In contrast, chemical and mechanical resistance and
to their recognition signals for cells, even at unmatched mechanical the viscosity of chitosan increase with increasing molecular weight.
strength to the final product (Dodero et al., 2021; Aghmiuni et al., 2021; Therefore, high-molecular-weight chitosans are more likely suitable for
Memic et al., 2019). Of them, chitosan and hyaluronic acid are exten­ tissue engineering, transcutaneous immunization, and wound dressing
sively used for synthesizing wound dressing devices (Suo et al., 2021; and healing application than low-molecular-weight ones. On the con­
Gruppuso et al., 2022). trary, low-molecular-weight chitosans may be more appropriate for
Besides biocompatibility and high availability, chitosan has excellent some applications, such as drug delivery, anti-inflammation, and anti-
cationic polyelectrolyte activity under typical wound conditions, sup­ aging skin.
pressing inflammation and microbial growth. Positively charged chito­ Like molecular weight, the deacetylation degree of chitosan must fit
san bonded to negatively charged red blood cell membranes via direct the desired chitosan application. These two physicochemical charac­
electrostatic interaction to clot heparinized blood during cardiovascular teristics have an inverse relationship with each other. Generally, the

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H. Kazemi Shariat Panahi et al. Biotechnology Advances 66 (2023) 108172

degree of deacetylation can affect cell response, degradation, solubility, of Henan Province (No. 21IRTSTHN020) and Central Plain Scholar
and crystallinity of chitosan. Therefore, first, one should try sourcing Funding Project of Henan Province (No. 212101510005). The authors
native chitosan with the right molecular weight and degree of deace­ would like to acknowledge that this work, in part, has been conducted
tylation for the target application. Then, chemical modification of chi­ under the umbrella of the MoU between the Scotland’s Rural College
tosan can be used as a promising approach for addressing the existing (SRUC) (UK) and Universiti Malaysia Terengganu (Malaysia). V.K.G.
weak points of chitosan and providing an additional degree of freedom would like to acknowledge the institutional funding sources supported
for designing efficient structures. Among various modification methods, by the SRUC, UK, for research and development. M.T. and V.K.G. would
saccharide modification of chitosan is easy, eco-friendly, and cost- like to acknowledge that this work has been conducted in part under the
effective. This modification technique, in some instances, introduces a umbrella of the MoU between UMT and SRUC. M.A. would also like to
binding specificity to chitosan structure for specific cells or organs. For extend their sincere appreciation to the University of Tehran for their
example, galactosylated chitosan can specifically be taken up by hepa­ support throughout this project. The authors would like to acknowledge
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Y.Yang and J.Pan would like to acknowledge the financial support Bi, R., Yue, L., Niazi, S., Khan, I.M., Sun, D., Wang, B., Wang, Z., Jiang, Q., Xia, W., 2021.
Facile synthesis and antibacterial activity of geraniol conjugated chitosan
from the National Natural Science Foundation of China (42271401) and oligosaccharide derivatives. Carbohydr. Polym. 251, 117099.
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Partnership Research Grant (UMT/CRIM/2-2/2/23 (23), Vot 55302) for Boyajian, J.L., Ghebretatios, M., Schaly, S., Islam, P., Prakash, S., 2021. Microbiome and
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and Fisheries (AKUATROP) program (Vot. No. 56052, UMT/CRIM/2-2/
Che, J., Najer, A., Blakney, A.K., McKay, P.F., Bellahcene, M., Winter, C.W., Sintou, A.,
5 Jilid 2 (11)). The manuscript is also supported by the Program for Tang, J., Keane, T.J., Schneider, M.D., 2020. Neutrophils enable local and non-
Innovative Research Team (in Science and Technology) in the University

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