polymers

Review
Chitosan: An Overview of Its Properties and Applications
Inmaculada Aranaz 1,2 , Andrés R. Alcántara 1 , Maria Concepción Civera 1 , Concepción Arias 1 ,
Begoña Elorza 1 , Angeles Heras Caballero 1,2 and Niuris Acosta 1,2, *

1 Departmento de Química en Ciencias Farmacéuticas, Universidad Complutense de Madrid,

28040 Madrid, Spain; iaranaz@ucm.es (I.A.); andalcan@ucm.es (A.R.A.); mccivera@ucm.es (M.C.C.);
carias@ucm.es (C.A.); belorza@ucm.es (B.E.); aheras@farm.ucm.es (A.H.C.)
2 Instituto Pluridisciplinar, Universidad Complutense de Madrid, Paseo Juan XXIII, n 1, 28040 Madrid, Spain
* Correspondence: facosta@ucm.es; Tel.: +34-913-943-284

Abstract: Chitosan has garnered much interest due to its properties and possible applications. Every
year the number of publications and patents based on this polymer increase. Chitosan exhibits poor
solubility in neutral and basic media, limiting its use in such conditions. Another serious obstacle is
directly related to its natural origin. Chitosan is not a single polymer with a defined structure but a
family of molecules with differences in their composition, size, and monomer distribution. These
properties have a fundamental effect on the biological and technological performance of the polymer.
Moreover, some of the biological properties claimed are discrete. In this review, we discuss how





 chitosan chemistry can solve the problems related to its poor solubility and can boost the polymer
properties. We focus on some of the main biological properties of chitosan and the relationship with
Citation: Aranaz, I.; Alcántara, A.R.;
the physicochemical properties of the polymer. Then, we review two polymer applications related to
Civera, M.C.; Arias, C.; Elorza, B.;
green processes: the use of chitosan in the green synthesis of metallic nanoparticles and its use as
Heras Caballero, A.; Acosta, N.
Chitosan: An Overview of Its
support for biocatalysts. Finally, we briefly describe how making use of the technological properties
Properties and Applications. Polymers of chitosan makes it possible to develop a variety of systems for drug delivery.
2021, 13, 3256. https://doi.org/
10.3390/polym13193256 Keywords: chitosan; chitin; biological activity; drug delivery; antioxidant; antimicrobial; metallic
nanoparticles; biocatalysis
Academic Editors: Rebeca
Hernandez Velasco,
David Mecerreyes, Rafael Antonio
Balart Gimeno, Ana 1. Introduction
María Díez-Pascual, Vicente
Chitin and its deacetylated derivative, chitosan, are a family of linear polysaccharides
Compañ Moreno and Angels Serra
composed of varying amounts of (β1→4) linked residues of N-acetyl-2 amino-2-deoxy-
D-glucose (glucosamine, GlcN) and 2-amino-2-deoxy-D-glucose (N-acetyl-glucosamine,
Received: 30 August 2021
Accepted: 22 September 2021
GlcNAc) residues. Chitosan is soluble in aqueous acidic media via primary amine protona-
Published: 24 September 2021
tion. In contrast, in chitin, the number of acetylated residues is high enough to prevent the
polymer for dissolving in aqueous acidic media.
Publisher’s Note: MDPI stays neutral
Chitin is a very abundant biopolymer that can be found in the exoskeleton of crustacea,
with regard to jurisdictional claims in
insect’s cuticles, algae and in the cell wall of fungi. Chitosan is less frequent in nature
published maps and institutional affil- occurring in some fungi (Mucoraceae). Historically, commercial chitosan samples were
iations. mainly produced from chemical deacetylation of chitin from crustacean sources. More
recently, chitosan from fungi is gaining interest in the market, driven by vegan demands.
Moreover, these samples are better controlled in terms of low viscosity and exhibit a very
high deacetylation degree [1]. Production from insect cuticles is also gaining interest,
Copyright: © 2021 by the authors.
driven by the increased interest in protein production from these sources.
Licensee MDPI, Basel, Switzerland.
The interest in chitin and chitosan relies on the myriad biological and technological
This article is an open access article
properties exhibited by these polymers (Table 1). However, these properties are tightly
distributed under the terms and related to the physicochemical properties of the polymers (mainly molecular weight and
conditions of the Creative Commons acetylation degree) [2]. Therefore, when working with chitin and chitosan a good and com-
Attribution (CC BY) license (https:// pleted polymer characterization is mandatory. Several methodologies have been described
creativecommons.org/licenses/by/ to characterize chitin, chitosan and chitooligosaccharides, a description of which is far from
4.0/). the objective of this paper—but for interested readers, we recommend publications [3,4].

Polymers 2021, 13, 3256. https://doi.org/10.3390/polym13193256 https://www.mdpi.com/journal/polymers

Polymers 2021, 13, 3256 2 of 27

Table 1. The main properties of chitin and chitosan.

Property/Activity Reference
Mucoadhesive [5,6]
Anti-inflammatory [7]
Antioxidant [8]
Antimicrobial [9]
Antifungal [10]
Antihyperglycemic [11]
Antitumoral [7–12]
Wound healing [13]

Chitosan is the only polycation in nature and its charge density depends on the degree
of acetylation and pH of the media. The solubility of the polymer depends on the acetylation
degree and molecular weight. Chitosan oligomers are soluble over a wide pH range, from
acidic to basic ones (i.e., physiological pH 7.4). On the contrary, chitosan samples with
higher Mw are only soluble in acidic aqueous media even at high deacetylation degrees.
This lack of solubility at neutral and basic pH has hindered the use of chitosan in some
applications under neutral physiological conditions (i.e., pH 7.4). This is the reason why a
great number of chitosan derivatives with enhanced solubility have been synthetized.
In 2019, the global chitosan market size was valued at USD 6.8 billion, and it is ex-
pected to expand at a revenue based CAGR of 24.7% between 2020 and 2027. The drivers for
the market’s growth are the increasing application of the polymer in water treatment and
several high-value industries such as the pharmaceutical, biomedical, cosmetics and food
industries [14]. Some of the interest areas identified include the modification of the poly-
mers to extend their applicability; knowledge of the mechanisms involved in the biological
activity of chitosan, chitosan derivatives and chitooligosaccharides; and the in-depth study
of chitosanolytic and chitinolytic enzymes presented in different microorganisms [15].
This review aims to provide readers with a general overview of the state of the art
of chitosan science, covering different aspects such as polymer chemistry, biological and
technological properties and applications in drug delivery and as a biocatalyst.

2. Technological Chitosan Properties

2.1. Solubility
Chitosan is produced by deacetylation of chitin; in this process, some N-acetylglucosamine
moieties are converted into glucosamine units. The presence of large amounts of protonated
-NH2 groups on the chitosan structure accounts for its solubility in acid aqueous media since
its pKa value is approximately 6.5 [16]. When around 50% of all amino groups are protonated,
chitosan becomes soluble [17].
Chitosan solubility depends on different factors such as polymer molecular weight,
degree of acetylation, pH, temperature, and polymer crystallinity. Homogeneous deacetyla-
tion (alkali treatment, 0 ◦ C) of chitin permits the production of polymers soluble in aqueous
acetic acid solutions with DD as low as 28%, with this value never being reached under
heterogeneous deacetylation (alkali treatment, high temperatures). Moreover, with a DD
of 49%, the samples are soluble in water. This behaviour is explained by the fact that
homogeneous deacetylation leads to an increase in the number of glucosamine units and
a modification in the crystalline structure of the polymer. Depending on polymer DD,
these modifications range from a reduction in crystal size and crystal perfection to the
presence of a new crystal structure close to β-chitin [18]. Sogias et al. [19] studied the role
of crystallinity and inter- or intramolecular forces on chitosan solubility; in this work, a
parent chitosan sample was half re-acetylated with anhydride acetic or fully N-deacetylated
under homogeneous conditions. After reacetylation, the solubility of the polymer was
expanded until pH 7.4, while a slight reduction in the solubility range of the fully deacety-
lated chitosan was determined. The lower solubility was explained due to the increase
in the polymer crystallinity after deacetylation, which offsets the effect of the increase in
Polymers 2021, 13, 3256 3 of 27

glucosamine moieties. On the contrary, a reduction in the crystallinity was observed in the
half-acetylated sample. The use of hydrogen bond disruptors such as urea or guanidine
hydrochloride also alters the solubility window of chitosan. In fact, by a combination of
chemical and physical disruption of the hydrogen bonds, broad solubility is achieved.

2.2. Viscosity
The viscosity of polymers is a parametre of great interest from the technological point
of view since highly viscous solutions are difficult to manage. Moreover, viscometry is
a powerful tool for determining chitosan’s molecular weight, as it is a simple and rapid
method even though it is not an absolute method, therefore requiring the determination of
constants that are specific to the solvent. The average molecular weight is determined by the
Mark–Houwink–Sakurada equation, which relates this parametre with the intrinsic viscosity:

η = KMv α (1)

where K and α are constants that must be determined experimentally. Several values
of K and α have been reported depending on the solvent composition, pH, and ionic
strength [20]. Chitosan viscosity depends on the molecular weight of the polymer and
deacetylation degree and decreases as the molecular weight of chitosan is reduced. In fact,
viscosity can be used to determine the stability of the polymer in solution, as a reduction
is observed during polymer storage due to polymer degradation [21]. Shear viscosity
increases with chitosan deacetylation degree. The shear viscosity at the same rate was
studied in two samples with different deacetylation degrees (91% vs. 75%) and represented
versus intrinsic viscosity [22]; it was reported that shear viscosity was larger for those
samples with the highest deacetylation degree; when the curves were evaluated, straight
lines were observed in both chitosan samples This is explained due to the nature of chitosan,
as this polymer is a cationic polyelectrolyte because of the amine protonation in acidic
media. Therefore, the higher the DD, the larger chain expansion is expected, as more
glucosamine units are found in the polymer chain, leading to a greater charge density in
this sample. In order to modulate chitosan viscosity, the addition of different co-solvents
has been evaluated; in this sense, Kassai et al. [20] studied the effect of the addition of
isopropanol and ethanol to a chitosan solution in 1% acetic acid, reporting that the presence
of the cosolvents decreased the intrinsic viscosity of the polymer.

3. Chemistry of Chitosan
As seen in Figure 1, the reactive groups found in chitosan are a primary amino
group (C2) and primary and secondary hydroxyl groups (C6, C3). Glycosidic bonds and
the acetamide group can also be considered functional groups. These functional groups
allow for a great number of modifications, producing polymers with new properties
and behaviours.
Chitosan derivatives have been produced, aiming to improve chitosan’s properties,
such as solubility or biodegradability, or to introduce new functions or properties. For
instance, solubility has been improved in water aqueous media by deacetylation, de-
polymerization, or quaternisation among other processes [23]. New chitosan activities
have been reported after its modification, for example, 6-O-sulphated chitosan promotes
neuronal differentiation while phosphorylated chitosan inhibits corrosion [24,25].
Polymers 2021, 13, 3256 4 of 27
Polymers 2021, 13, 3256 4 of 28

Figure 1. Functional groups in chitosan’s structure that are able to be chemically modified.

Chitosan derivatives have been produced, aiming to improve chitosan’s properties,

such as solubility or biodegradability, or to introduce new functions or properties. For
instance, solubility has been improved in water aqueous media by deacetylation, depoly-
merization, or quaternisation among other processes [23]. New chitosan activities have
been reported
Figure
Figure 1. aftergroups
1. Functional its modification,
groups in chitosan’s
in for example,
chitosan’s structure
structure 6-O-sulphated
that are
that are ableto
able tobe chitosan
bechemically
chemically promotes neu-
modified.
modified.
ronal differentiation while phosphorylated chitosan inhibits corrosion [24,25].
The
The field
field of
Chitosan of chitosan
chitosan chemistry
derivatives have beenis
chemistry wide,
wide, and
isproduced, in
in this
andaiming review,
thisto we
improve
review, wewant
wanttotofocus
chitosan’s on
ontwo
properties,
focus two
types
such
typesasof processes,
ofsolubility
processes,or chitosan phosphorylation
biodegradability,
chitosan and
and chitosan
or to introduce
phosphorylation newdegradation.
chitosan functions or Our
degradation. Our group
group has
properties. For
has
participated
instance,
participated in the
solubility development
in the has of
been improved
development a phosphorylated
in water aqueous
of a phosphorylated derivative via
media byvia
derivative a simple method
deacetylation, in
depoly-
a simple method in
which chitosan
merization,
which chitosan and phosphorus
or quaternisation
and phosphorus acid
among are
acid other mixed at
processes
are mixed the same
at the[23]. New
same ratio and
chitosan
ratio formaldehyde
activities have
and formaldehyde isis
added at ◦ C [26] (Figure 2).
been
added at 70
reported
70 °C after its modification,
[26] (Figure 2). for example, 6-O-sulphated chitosan promotes neu-
ronal differentiation while phosphorylated chitosan inhibits corrosion [24,25].
The field of chitosan chemistry is wide, and in this review, we want to focus on two
types of processes, chitosan phosphorylation and chitosan degradation. Our group has
participated in the development of a phosphorylated derivative via a simple method in
which chitosan and phosphorus acid are mixed at the same ratio and formaldehyde is
added at 70 °C [26] (Figure 2).

Figure 2.
Figure 2. Scheme
Scheme of
of phosphorylated
phosphorylated chitosan
chitosan derivative
derivative synthesis.
synthesis.

This N-methylene
This N-methylene phosphonic
phosphonic chitosan
chitosan isis soluble
soluble inin water
water and
and keeps
keeps the
thefilmogenic
filmogenic
properties
properties of thethe parent
parentchitosan.
chitosan.With With a similar
a similar methodology,
methodology, a soluble
a soluble in water
in water N-
N‐meth‐
methylenephenyl
ylenephenyl phosphonicphosphonic chitosan
chitosan hashas been
been produced[27].
produced [27].Additionally,
Additionally, the surfactant
surfactant
derivative N-lauryl-N-methylene
derivative N-lauryl-N-methylene phosphonic
phosphonic chitosan
chitosan waswas produced
produced via viaN-alkylation
N-alkylationof of
N-methylene
N-methylene
Figure 2. Scheme phosphonic
phosphonic chitosan
chitosan
of phosphorylated [28]. This
[28]. This
chitosan derivative
derivative
derivative has a lower solubility in aqueous
has a lower solubility in aqueous
synthesis.
media
media compared to N-methylenephosphonic
to N-methylene phosphonicchitosan
chitosan butbut better
better solubility
solubility in organic
in organic me-
media
dia and and forms
Thisforms
N-methylene micelles. N-methylene
micelles.phosphonic
N-methylene chitosanphosphonic
is soluble
phosphonic N-methylene
in water and
N-methylene carboxylic
keeps the
carboxylic chitosan
filmogenic
chitosan has
has
beenbeen
properties obtained
of the
obtained in water-soluble
inparent chitosan.
water-soluble With
form form using
a similar
using N-methylene
methodology,
N-methylene aphosphonic
phosphonicsoluble chitosan
in water
chitosan and and
N‐meth‐
glyox-
glyoxylic
ylenephenyl acid.
ylic acid. The The polymer
phosphonic
polymer maintains
chitosan
maintains thehas the filmogenic
been
filmogenicproduced properties
properties[27]. ofchitosan
parent the
Additionally,
of parent chitosan
and, and,
surfactant
because
because
derivative of the presence of
N-lauryl-N-methylene
of the presence multidentate ligands,
phosphonic
of multidentate ligands, its use
its usechitosan as a bivalent
was produced
as a bivalent metal chelating agent
via N-alkylation
metal chelating agent is pro-of
is proposed
[29]. [29].
N-methylene
posed phosphonic chitosan [28]. This derivative has a lower solubility in aqueous
media Although
compared thetouse of chitosan
N-methylene as a gene chitosan
phosphonic carrier hasbutbeen
betterreported,
solubilitythe use of this
in organic me-
biopolymer for this application is limited due to a relatively low
dia and forms micelles. N-methylene phosphonic N-methylene carboxylic chitosan transgenic efficacy. Phos-
has
phorylated
been obtained derivatives have shown
in water-soluble formbetter
usingperformance
N-methylene(transfection was improved
phosphonic chitosan 100-
and glyox-
fold) and The
ylic acid. therefore
polymer are maintains
more suitable than chitosan
the filmogenic to this end.
properties Moreover,
of parent phosphorylated
chitosan and, because
derivatives also exhibit and improve metal ion chelating activity when
of the presence of multidentate ligands, its use as a bivalent metal chelating agent is compared topro-
the
parent chitosan
posed [29]. [30,31].
Due to the presence of cleavage glycosidic bonds, it is possible to degrade chitosan,
thus reducing its molecular weight. As previously mentioned, the control of chitosan
Polymers 2021, 13, 3256 5 of 27

depolymerization (polymer size) permits us to control some properties such as solubility

or viscosity. Moreover, the biological and technological properties of chitosan are related to
size, among other properties as previously reviewed [2]. Chitosan degradation can occur
through different mechanisms such as acid hydrolysis, oxidative–reductive or nitrous
acid depolymerization, ultrasonic degradation, or enzymatic degradation using specific
and non-specific enzymes. Chitosan has four types of glycosidic linkages -D-D-, -A-A-,
-A-D- and -D-A- (where A and D denote N-acetylglucosamine and glucosamine monomers,
respectively). Depending on the process, there is a prevalence in the breakage of certain
linkages and therefore different samples can be produced from the same parent chitosan by
selecting different methodologies. Chemical and physical methods are less selective than
enzymatic ones for producing specific patterns due to enzyme-specific recognition but by
controlling the parametres of the process some control over the composition can be gained.
Ultrasonic degradation of chitosan does not affect the degree of acetylation or poly-
dispersity of the recovered polymers allowing for the moderate degradation of the poly-
mer [32]. The rate of degradation depends on the acetylation degree of the parent chitosan
and not on the initial molecular weight [33].
Hydrogen peroxide produces random degradation of chitosan in a faster manner
than ultrasonic methodologies, producing a significant number of monomers and chi-
tooligosaccharides, the composition of which depends on the temperature and H2 O2
concentration [34]. Nitrous acid depolymerization can be considered somewhat specific
since HNO2 attacks the primary amine in glucosamine and subsequently the cleavage of
the glycosidic bonds occurs. That is, only the glycosidic linkage following a D-unit can be
cleaved [35]. The chemical processes yield large amounts of monomers (D-glucosamine)
and when the intended final products are chitooligosaccharides rather than low molecular
weight chitosan, the yields are low [36]. HNO2 provokes the formation of 2,5-anhydro-D-
mannose at the new reducing end, which may be considered a disadvantage of this acid.
When chitosan is degraded by HCl, the polymer not only suffers the hydrolysis of the
O-glycosidic linkage between residues but also the N-acetyl linkage can be hydrolyzed
but at a lower rate. The hydrolysis rate of D-D and D-A glycosidic linkages is lower than
the hydrolysis of A-A and A-D, therefore the reducing ends are dominated by acetylated
units [37]. By using a controlled precipitation method with methanol, it has been possible to
obtain chitooligosaccharides with DPs up to 16 with few low molecular weight oligomers
with a good yield [38].
The specific enzymatic degradation of chitosan occurs with a family of enzymes
named chitosanases (EC 3.2.1.132) and chitinases (EC 3.2.1.14). Chitosanases are glycosyl
hydrolases that catalyse the endo hydrolysis of β-1,4-glycosidic bonds of partially acetylated
chitosan to release chitosan oligosaccharides (COS) with little monomer release [39]. Chi-
tosanase specifically hydrolyses chitosan by cleavage of glycosidic bonds with a -DD·DA-
pattern or a -DD·DD-pattern. Chitinases, which occur in families GH18 and GH 19, are
glycosyl hydrolases that can degrade both A-A and A-D linkages and show no activity
against D-D linkages. Chitinases can be classified into two major categories (endochitinases
and exochitinases) according to their mode of action [40].
Non-specific enzymes, also called promiscuous enzymes, are also able to degrade
chitosan. These enzymes belong to the protease, lipase, cellulase, and hemicellulase
families, among others. Lysozyme is one of the most studied due to its relationship with
polymer biodegradation. this enzyme is a protease that hydrolyses chitosan by cleavage
of glycosidic bonds with A-A-A-A- pattern or A-A-A-D-pattern, while A-D-A-pattern or
D-D-A-A are not or very slowly hydrolysed by lysozyme [14,17]. Apart from the previously
mentioned lysozyme, other proteolytic enzymes such as pepsin, papain and pronase caused
chitosan depolymerization, rendering low molecular chitosans (4–10 kDa) as the main
products and chitooligosaccharides and monomers in smaller amounts. Results indicated
that papain and pepsin had a similar action pattern. Both enzymes decreased LMWC
acetylation degree when compared to the parent chitosan; DP 2–6 were detected in the
supernatant monomers (D and A) and oligomers. Pronase showed different behaviour
Polymers 2021, 13, 3256 6 of 27

since no glucosamine was detected. It showed selectivity through A-A and A-D, resulting
in products having A monomers at the reducing end [41].
Neutral protease degraded chitosan in a manner dependent on the deacetylation
degree. The higher the DD, the higher the Km and the lower the Vmax. During degradation,
a reduction in the DD of the recovered LMW chitosans was observed. An analysis of the
partially hydrolysed chitosan revealed that the enzyme degraded D-D and A-D β-1,4-
glycosidic linkages, producing a mixture of hetero oligosaccharides carrying an A residue
at the reducing end [42]. The same authors have studied the effect of the chitosan molecular
weight in the enzymatic activity since this parametre affects its chain flexibility in solution,
which in turn may affect its affinity for the enzyme in hydrolysis reactions. Their results
showed a lower affinity of the enzyme with a slower degradation rate when high molecular
weight chitosan samples were tested [43].
Hemicellulase, an enzyme related to the degradation of hemicellulose, has proven its
ability to reduce chitosan molecular weight in a manner that depends on the deacetylation
degree of the chitosan, rendering lower molecular weight samples when a chitosan sample
with a DD of 85% was tested. Dimers, trimers, tetramers, pentamers and hexamers were
observed after 4 hours of reaction, and the enzyme was considered endo-acting since no
N-acetylglucosamine was detected [44].
Lipases have also proved their ability to hydrolysate chitosan, although the degra-
dation rates are slower than the ones reported by other enzymes such as proteases or
hemicellulose. Controlling reaction temperature, a commercial lipase rendered low molec-
ular weight samples or chitooligosaccharides [45,46]. This lipase acted following both exo
and endo cleavage mode. The presence of D end products indicates that it acted on chitosan
in an exo-type mode while the sharp reduction in viscosity during the hydrolysis indicates
that an endo splitting occurred in the initial hydrolysis stage. Therefore, by controlling the
reaction time the final products can be led to oligomers with high DP or monomers. The
polymer polydispersity depended on the used enzyme, lipase from wheat germ rendered
samples with very wide molecular weight while lipase from R. japonicus exhibited better
control over polydispersity [47].
The data previously showed that it is possible to somehow select the degradation
products (LMW chitosans or oligosaccharides) by selecting the appropriate methodology
(Table 2). As we can see in some sections of this review, specific biological and technological
behaviour of chitosan degradation products depends not only on the method (physical,
chemical, or enzymatic) selected to degrade the chitosan but also on the type of chemical
or enzyme used for these processes. This effect is more related to the degraded polymer
pattern rather than to the size or acetylation degree of the samples.

Table 2. The main products produced in the enzymatic degradation of chitosan.

Enzyme Main Product

Chitosanase Oligomers DP 2–3
Hemicellulase Dimers, trimers, tetramers, pentamers and hexamers
Pepsine Glucosamine, N-acetylglucosamine oligomers with DP 2–6
Pronase 4–10 kDa
Papain Glucosamine, N-acetylglucosamine oligomers with DP 2–6
Lipase High DP
DP: depolymerization degree.

4. Biological Properties
Chitin, chitosan, oligosaccharides, and derivatives exert many biological activities
including antitumoral, antimicrobial, antioxidant, and anti-inflammatory activities, which
could be used as therapeutic polymers. It is remarkable that up today chitosan and chitosan
hydrochloride are only accepted as excipients by the regulatory agencies and not as a drug
for the treatment of diseases.
Polymers 2021, 13, 3256 7 of 27

4.1. Antimicrobial Activity

Bacterial resistance to antibiotics is a critical public health concern and, therefore,
there is an urgency to find alternatives to antibiotics. Chitosan, chitosan derivatives
and chitooligosaccharides exert antimicrobial activity against different microorganisms,
including bacteria, filamentous fungi, and yeast [48]; some examples of the different
microorganisms sensible to chitosan are shown in Table 3. Chitosan seems to have a
growth-inhibitory activity since bacteria is able to grow after the polymer is removed from
the media. This is of importance since resistant populations might emerge if the cells adapt
to chitosan [49].

Table 3. Antimicrobial and antifungal activity of chitosan.

System Target Inhibition References

Aeromonas hydrophila Complete
Chitosan Edwardsiella ictalurid 0.4% (E I, F C) [50]
Flavobacterium columnare 0.8% (A. H)
Candida albicans
Gram-positive bacteria (such as
Bacillus cereus, S. aureus, Bacillus megaterium,
Lactobacillus plantarum, Listeria
monocytogenes, Lactobacillus brevis, and
Chitosan Lactobacillus bulgaricus) Strong and safe effect [51,52]
Gram-negative bacteria (such as Salmonella
typhimurium, E. coli, Pseudomonas aeruginosa,
Pseudomonas fluorescens, Vibrio
parahaemolyticus, Enterobacter aerogenes, and
Vibrio cholera)
No effect: chitosan oligosaccharide
Chitosan hydrochloride
and N-acetyl-D-glucosamine.
Carboxymethyl chitosan
Candida krusei, C. albicans, C. glabrata Weak effect: Carboxymethyl chitosan. [53]
Chitosan oligosaccharide
Strong effect: Chitosan
N-acetyl-D-glucosamine
hydrochlorides.
Strong effect:
wound management due to their
Chitosan wound dressing P. aeruginosa, B. cereus, L. monocytogenes antimicrobial nature, ability to [54–57]
accelerate wound contraction and
healing, haemostatic and analgesic
Chitosan sponges S. aureus, E. coli [58,59]
E. coli, Vibrio cholerae, S. enterica,
Chitosan microparticles Streptococcus uberis, S. uberis, S. enterica, K.
Strong effect [60–62]
and nanoparticles pneumonia, S. aureus, V. cholerae, Salmonella
choleraesuis, S. typhimurium

Due to chitosan’s poor solubility above pH 6.5, the use of chitooligosaccharides is

under consideration as polycationic biocides since they are soluble in water. Chitosan
soluble derivatives such as sulphated chitosan, N-trimethyl chitosan, N-diethylmethyl
chitosan or 2,6-diamino chitosan also avoid the use of acidic environments and exert
antimicrobial activity [63–65]. This antimicrobial activity has applications in different
fields such as the food, textile, or cosmetic industry, among others. Thus, due to the
ability of chitosan to form shift bases, some new chitosan derivatives based on heterocyclic
moieties have been developed, including pyrazole ring and furanyl, pyridyl, or thiophenyl
moieties. Although these derivatives do not show higher solubility in aqueous media, their
performance against gram-positive microorganism was improved when compared with
the parent chitosan [66].
How these polymers (chitosan, chitooligosaccharides and derivatives) exert their
antimicrobial activity is still under discussion. This fact can be explained by taking into
account the lack of appropriate polymer characterization, purity issues, the use of different
microorganisms, and the lack of methodological uniformity. Some studies point to the
Polymers 2021, 13, 3256 8 of 27

reduction in cell membrane permeability due to polymer coating on the surface of the cells
that blocks cell access to nutrients. This process occurs due to the interaction of -NH2 groups
from chitosan chains with -COO- groups on the external cell membranes of microorganisms.
Therefore, the antimicrobial activity depends on the acetylation degree. It has also been
hypothesized that chitosan can penetrate the cells and block RNA transcription as a result
of adsorption with bacterial DNA [9]. Most likely, these mechanisms are not mutually
exclusive, and several events are related to cell growth inhibition.
Intrinsic factors affecting the antimicrobial chitosan activity are due to the polymer
characteristics such as Mw, acetylation degree, polymer viscosity, or polymer concentration.
The solvent used to dissolve the polymer also affects its behaviour. We have observed that
typical solvents used to dissolve chitosan such as acetic acid, citric acid, or buffers such as
AcOH-NaAc exert some antimicrobial activity per se (unpublished results). Other factors
with great impact on the antimicrobial activity are related to the tested microorganism,
growth media, pH, temperature, ionic strength, or physiological state of the cells.
The effect of polymer size is controversial. Some studies claim that the antimicrobial
activity of chitosan improves with the polymer size and have found that oligosaccharides
have lower antimicrobial activity [67–69]. When comparing chitooligosaccharides, those
showing higher DP exhibited higher antimicrobial activity [70]. Moreover, Tokura and
co-workers reported that chemically produced chitooligosaccharides of 2200 Da not only
had no antimicrobial activity but also served as growth accelerators of E. coli, while a
sample with 9300 Da inhibited bacterial growth [71]. On the contrary, other studies showed
better antimicrobial activity for a lower molecular weight chitosan sample (55 kDa) than a
higher one (155 kDa); in the same study when a sample of 90 kDa was tested a promotion
of bacterial growth was observed [72]. In another study, different tendencies were observed
depending on the pH of the media. In acidic pH conditions, the antimicrobial activity
increased with increasing MW. However, at neutral pH, antimicrobial activity increased as
the MW decreased [73]. Even so, no trend on the effect of chitosan Mw on antimicrobial
activity has been reported [74]. Regarding acetylation degree, it seems that the lower the
acetylation degree, the better the antimicrobial activity [69,74,75].
After depolimerization of a chitosan sample (400 kDa, DD~85%) with hemicellulose,
a set of chitosan samples with similar DD and Mw ranging from 130 to 2.8 kDa and a
chitooligosacharide sample with Mw 1.4 were produced. Some of these samples were
also half-acetylated, furnishing two chitosan samples with Mw of 53 and 18 kDa, and
some chitoligosaccharides with Mw of 1.4 kDa. Both chitooligosacharides samples and the
half-acetylated samples were water soluble, while the others were not soluble in water. All
samples were tested against Staphylococcus aureus, Escherichia coli, and Candida albicans.
In this study, water soluble chitosans and oligosaccharides did not exhibit antimicrobial
activity; in fact, they promoted the growth of C. albicans. Insoluble chitosan samples
exhibited antimicrobial activity with the most pronounced effect when medium molecular
weight samples were tested (Mw 78–48 kDa) [76].
Our group has studied the antimicrobial activity of low molecular weight chitosans
and oligosaccharides produced by enzymatic degradation in order to determine if the poly-
mer pattern has some effect on this activity. Chitooligosaccharides were produced by two
different processes; thus, in process P1 chitosan was enzymatically depolymerized with
chitosanase, while in process P2 the sample was depolymerized in a two-step process with
HNO2 and chitosanase. The samples were tested against E coli and L. monocytogenes. COS
from P1 showed a higher capability to inhibit bacterial growth than COS from P2. In both
cases, COS were more effective at inhibiting E. coli (Gram-negative) than the Gram-positive
L. monocytogenes. Antimicrobial activity depended on the production process and com-
position and structure of COS. COS produced in a one-step enzymatic procedure showed
better antimicrobial activity than those produced in the two-step chemical–enzymatic
process even when the samples exhibited similar DA and MW [77].
 P2. In both cases, COS were more effective at inhibiting E. coli (Gram-negative) than the
Gram-positive L. monocytogenes. Antimicrobial activity depended on the production
process and composition and structure of COS. COS produced in a one-step enzymatic
procedure showed better antimicrobial activity than those produced in the two-step
Polymers 2021, 13, 3256 chemical–enzymatic process even when the samples exhibited similar DA and MW [77]. 9 of 27

4.2. Antioxidant Activity

Antioxidants
4.2. Antioxidant are gaining interest due to the relationship between oxidative stress and
Activity
several diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease,
Antioxidants are gaining interest due to the relationship between oxidative stress and
amyotrophic lateral
several diseases suchsclerosis, and cancer.
as Alzheimer’s Moreover,
disease, it is related
Parkinson’s to complications
disease, Huntington’s in other
disease,
diseases such as diabetes [78–80].
amyotrophic lateral sclerosis, and cancer. Moreover, it is related to complications in other
Chitosan
diseases contains
such as diabetesan[78–80].
amino and several hydroxyl groups, which can react with free
radicals exhibiting scavenging
Chitosan contains an amino ability.
and Some
severalchitosan
hydroxyl derivatives such ascan
groups, which chitosan sul-
react with
phates or N-2 carboxyethyl chitosan exhibited improved antioxidant
free radicals exhibiting scavenging ability. Some chitosan derivatives such as chitosanactivity [81–83].
Chitooligosaccharides have alsochitosan
sulphates or N-2 carboxyethyl been chemically
exhibitedmodified
improvedtoantioxidant
improve their antioxidant
activity [81–83].
activity, for instance by modification of the polymers with gallic acid [84,85]
Chitooligosaccharides have also been chemically modified to improve their antioxidant or phenolic
compounds [86].
activity, for instance by modification of the polymers with gallic acid [84,85] or phenolic
Different
compounds [86]. methodologies have been used to determine chitosan and its derivatives’
antioxidant
Differentassays, which includes
methodologies have been DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate),
used to determine chitosan and its derivatives’
ABTS (2,2-azinobis
antioxidant (3-ethylbenzothiazoline-6-sulphonic
assays, which acid), and FRAP (ferric antioxi-
includes DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), ABTS
dant power) assays,
(2,2-azinobis peroxide and hydroxyl radical acid),
(3-ethylbenzothiazoline-6-sulphonic scavenging assays(ferric
and FRAP or the use of mac-
antioxidant
rophage models.
power) assays, DPPH and
peroxide andABTS assays
hydroxyl are based
radical on electron
scavenging and
assays or Htheatom transfer,
use of while
macrophage
the FRAP assay is based on electron transfer reaction, as depicted in
models. DPPH and ABTS assays are based on electron and H atom transfer, while the Figure 3. The ORAC
(oxygen
FRAP assayradical absorbance
is based capacity
on electron assay)reaction,
transfer is also widely used tointest
as depicted antioxidant
Figure activi-
3. The ORAC
ties.
(oxygen radical absorbance capacity assay) is also widely used to test antioxidant activities.

Figure 3. Methodologies used to determine antioxidant activities.

Figure 3. Methodologies used to determine antioxidant activities.
The disparity between the polymers tested and the methodologies used to test the
The disparity between the polymers tested and the methodologies used to test the
activity produces considerable differences in the polymer concentrations that range from
activity produces considerable differences in the polymer concentrations that range from
50 µg/mL to 400 mg/mL [83]. Antioxidant activity is more remarkable for low molecular
50 µ g/mL to 400 mg/mL [83]. Antioxidant activity is more remarkable for low molecular
weight samples rather than for high molecular weight ones since shorter chains form fewer
weight sampleshydrogen
intramolecular rather than forand
bonds high molecular
therefore the weight
reactive ones since
groups are shorter chains form
more accessible, con-
tributing to the radical scavenging activity [87,88]. Regarding the effect of the acetylation
degree, the antioxidant activity seems to decrease when this parametre increases [88].

4.3. Anti-Inflammatory Properties

The inflammatory process is an automatic physiological response of the body related
to tissue damage. The main goal of the inflammatory response is to bring circulating
leukocytes and plasma proteins to the site of the infection or tissue damage, to eliminate
the causative agent, when possible, and to start the healing process. Although inflamma-
tion is necessary for survival, when it is very severe, unable to eradicate the causative
agent, or is directed against the host, the inflammatory process may cause damage. The
inflammatory process is strongly related to the generation of free radicals. Again, this
activity seems to be more remarkable when the molecular weight of the chitosan is reduced
and chitooligosaccharides exhibit higher activity.
After chitosan (300 kDa) depolymerization with cellulose, the activity of degraded
polymers with medium molecular weight, low molecular weight and chitooligosaccharides
(156, 72, 7 and 3.3 kDa) were tested in terms of NO secretion, cytokine production, and
mitogen-activated protein kinase pathways in a model of lipopolysaccharide (LPS)-induced
Polymers 2021, 13, 3256 10 of 27

murine RAW 264.7 macrophages. Chitosan samples (parent, medium, and low) signifi-
cantly inhibited NO production. On the contrary, the opposite effect was observed with the
COS. The mechanism followed by the medium and low Mw chitosan to inhibited NF-κB
activation and iNOS expression differed. For medium chitosan (156 kDa) the process oc-
curred via the binding to CR3 while for low molecular weight chitosan the process occurred
via the binding to CR3 and TLR4 receptors. On the contrary, the lower molecular weight
chitosans activated NF-κB and enhanced iNOS expression by binding to CD14, TLR4, and
CR3 receptors to activate JNK signalling proteins [89]. In general, chitooligosaccharides
are studied in more detail for this application compared to chitosan, due to their better
solubility in aqueous media and better performance.
The effect of acetylation degree on the anti-inflammatory activities of COS has also
been studied. Chitooligosaccharides with MW between 0.2 and 1.2 kDa were enzymati-
cally depolymerized, depending on the enzyme, fully deacetylated (fdCOS, mainly GlcN,
(GlcN)2, (GlcN)3, and (GlcN)4), partially acetylated (paCOS: a mixture of at least 11 Cos
with different proportions of GlcNAc and GlcN), and fully acetylated (faCOS, mainly
GlcNAc, (GlcNAc)2 and (GlcNAc)3) were produced. The anti-inflammatory activity of the
three COS mixtures was studied by measuring their ability to reduce the level of TNF-α in
stimulated LPS murine macrophages (RAW 264.7). Only fdCOS and faCOS were able to
significantly reduce this factor [90,91]. The inhibition of NO secretion by COSs revealed
that 10% acetylated COS inhibited NO secretion significantly more than those with 50%
acetylation [92]. Citronellol grafted chitosan oligosaccharide derivatives have been pro-
duced to improve the anti-inflammatory activity of the oligosaccharides with degrees of
substitution of 0.165, 0.199 and 0.182, respectively. In all cases, the derivatives showed
better performance than the parent COS. These derivatives reduced the expression levels of
TNF-α by promoting the secretion of IL-4 and IL-10 and inactivated the NF-κB signalling
pathway via inhibiting the phosphorylation of p65, IKBα, and IKKβ [93].
Using the same chitosan as a starting material to produce chitooligosaccharides ren-
dered samples with different anti-inflammatory behaviour. Chitooligosaccharides (5–10 kDa,
DD: 87%) composed mainly of 42% fully deacetylated oligomers (A1-A3) plus 54% monoacety-
lated oligomers, produced by enzymatic degradation with chitosanase, attenuated the in-
flammation in lipopolysaccharide-induced mice and in RAW264.7 macrophages. On the
contrary, chitooligosaccharides (5–10 kDa, DD: 89%) from a two-step preparation (chemical
degradation followed by enzymatic degradation with chitosanase) were composed of 50%
fully deacetylated oligomers plus 27% monoacetylated oligomers (A1-A3) promoted the
inflammatory response in both in vivo and in vitro models [94]. This result shows how
small differences in the COS mixture have a strong effect on the mixture behaviour.

5. Metallic Nanoparticles and Chitosan

Metallic nanoparticles are usually defined as particles of metal atoms with sizes
ranging between 1 nm to a few hundred nanometres [95]. These particles exhibit optical,
chemical, and electronic properties that differ from individual atoms or bulk materials.
These unique properties are highly appreciated for different applications such as catalysis,
photonics, or biomedicine [96].
Metallic nanoparticles can be prepared using myriad physical or chemical methods.
Metal ions can be reduced using chemicals (NaBH4 , vitamin C and others) [97,98], plant
extracts (due to their phenolic compounds) [99], using polymers such as chondroitin
sulphate or heparin [100,101], or using microorganisms containing specific enzymes such
as nitrate reductase [102,103]. Other authors have proposed the use of sonochemical
reduction [104], radiation [105], electrochemical reduction [106] or heat evaporation [107].
Once the formed metallic nanoparticles aggregate, the addition of stabilizers is needed [108]
(Figure 4).
 extracts (due to their phenolic compounds) [99], using polymers such as chondroitin sul-
phate or heparin [100,101], or using microorganisms containing specific enzymes such as
nitrate reductase [102,103]. Other authors have proposed the use of sonochemical reduc-
tion [104], radiation [105], electrochemical reduction [106] or heat evaporation [107]. Once
Polymers 2021, 13, 3256
the formed metallic nanoparticles aggregate, the addition of stabilizers is needed11[108]
of 27

(Figure 4).

Figure 4. Scheme of metallic nanoparticle production and stabilization with chitosan.

Figure 4. Scheme of metallic nanoparticle production and stabilization with chitosan.
The synthesis of metallic nanoparticles using chitosan as a reducing agent and/or
The synthesis
stabilizing of metallic
agent is well nanoparticles
described. usinghave
Some authors chitosan as a reducing
also proposed that agent and/or
chitosan playssta-
a
role in the control of nanoparticle nucleation, thus controlling nanoparticle size to somea
bilizing agent is well described. Some authors have also proposed that chitosan plays
role insince
extent the control of nanoparticle
metal concentration nucleation,
also affects thethus controlling
nanoparticle sizenanoparticle
[97,109]. size to some
extentThesince metaland
reducing concentration
stabilizing also affectsof
properties the nanoparticle
chitosan seems size
to be[97,109].
related to the presence
of CHThe2 OH,reducing
CHO, and
and stabilizing
NH 2 groups properties
in the of chitosan
polymeric seems
chain. to
Changesbe related
in theto the pres-
molecular
ence of CH OH, CHO, and NH groups in the polymeric chain. Changes in
weight or deacetylation degrees not only alter the number of these reactive groups but also
2 2 the molecular
weight the
modify or deacetylation degrees not
interactions (hydrogen only electrostatic
bonds, alter the number of theseorreactive
interactions, groups but
steric interactions)
also modify the
present in the system.interactions (hydrogen bonds, electrostatic interactions, or steric interac-
tions)Inpresent
Table 4,insome
the system.
examples of the usage of chitosan in metallic nanoparticle synthe-
Inreviewed,
sis are Table 4, some examples
including of the usage
information of chitosan
about in metallic
the molecules used nanoparticle
as reducingsynthesis
agents,
are reviewed,
properties including
of the chitosaninformation
used when data aboutare
thegiven,
molecules used assize,
nanoparticle reducing agents, prop-
and morphology.
erties of the chitosan used when data are given, nanoparticle size, and morphology.
Table 4. Metallic nanoparticle based on chitosan.
Table 4. Metallic nanoparticle based on chitosan.
Stabilizer
Metal Reducing Agent Chitosan
Stabilizer NPs Size Morphology Ref.
Mw and DD
Metal Reducing Agent Chitosan NPs Size Morphology Ref.
Ascorbic acid Cs 180 kDa,
Mw 75–85%
and DDDD 5–20 Spherical [98]
Ascorbic acid Cs 50 to 190 kDa, 75–85% 50–70 Flower-spherical [110]
Ascorbic
Ascorbic acid acid Cs,Cs
50 180 kDa,
to 190 75–85%
kDa, 75–85%DD 5–2030–150 Spherical
Flower [98]
[111]
Ascorbic acid acid
Ascorbic TMCs
Cs 50 to 19020kDa,
kDa 75–85% 55–120
50–70 Spherical
Flower-spherical [112]
[110]
Palladium NaBH 4
Ascorbic acid Cs,
Cs, 400
50 tokDa
190DD 100%
kDa, 75–85% 30–150nd nd
Flower [109]
[111]
NaBH4 Cs, (~400 kDa) 2 Spherical [113]
Ascorbic acid TMCs 20 kDa 55–120 Spherical
Spherical, large aggregate [112]
MeOH Cs, (~400 kDa) 2–5 [113]
Palladium NaBH4 Cs, 400 kDa DD 100% nd nd
(Pd:MeOH 10:1) [109]
Hydrazine
NaBH4 Cs,Cs, (∼400
(~400 kDa)
kDa) 2 20 * Spherical
Highly aggregate [113]
[113]
N2 H4
MeOH Cs, (∼400 kDa) 2–5 Spherical, large aggregate (Pd:MeOH 10:1) [113]
NaBH 4
Hydrazine Cs, 400 kDa DD 100% 2–5 spherical [109]
NaBH4 Cs,Cs,
(~400
(∼400kDa)
kDa) 20* 2–3 Highlyspherical
aggregate [113]
[113]
Platinum MeOH N 2 H4 Cs, (~400 kDa) 2 spherical [113]
Hydrazine
NaBH4 Cs,
Cs,400 kDakDa)
(~400 DD 100% 2–517–25 * spherical
aggregates [109]
[113]
Platinum N2 HNaBH
4 4 Cs, (∼400 kDa) 2–3 spherical [113]
Cs, 1278MeOH
kDa Cs,
Cs,1278
(∼400kDa
kDa) 2 16 spherical [114]
[113]
Cs 817 KDa Cs, 817 KDa 5 Spherical [115]
NaBH4 Cs, 400 kDa DD 100% [109]
Gold Cs DD > 85%; >200,000 cps Cs, DD > 85%; >200,000 5–20 Spherical [101]
NaBH4 Cs n.c. 6–20 Spherical; polyhedral [97]
COS 5 kDa COS 5 kDa 7–15 Spherical [116]
Cs, Cs, DD 53–95%, Mw 2.6–490 kDa 5–200 nm Spherical, triangles, polyhedral [117]
Spherical
Cs Cs 1240 kDa, DA 0.13 10–150 [118]
Triangles in long storage
Cs Cs, high Mw, DA 0.25 5 Spherical [119]
Cs DD > 85%; >200,000 cps Cs DD > 85%; >200,000 cps 20–200 Spherical, fractal [101]
Ascorbic acid Cs 180 kDa, 75–85% DD 5–20 Spherical [98]
Silver NaBH4 Cs 400 kDa DD 100% 30–200 Spherical clusters [109]
Gamma radiation Cs n.c. 4–5 Spherical [101]
Cs n.c. Cs n.c. 10–60 Spherical [120]
Ascorbic acid/Cs 1278 kDa Cs 1278 kDa 8 [114]
Cs n.c. Cs n.c. [121]
Cs Cs (50–190 kDa DD 75–85%) Fractal patterns [122]
Cs: chitosan; TMCs: trimethyl chitosan; n.c.: non-characterized; nd: non-determined; * aggregate size.

Data from Table 4 clearly show that the characteristics of the produced nanoparticles
depend on the method used to produce the nanoparticles and the characteristics of the
chitosan used to reduce and stabilize the metal ions. In general, due to the lack of a proper
characterization of the chitosan samples and the variety of reaction conditions used it
Polymers 2021, 13, 3256 12 of 27

is very difficult to relate chitosan properties with the characteristics of the nanoparticles.
Recently, the effect of chitosan Mw and acetylation degree on the preparation of AuNPs
both as reducing and stabilizing agents has been analysed in detail [117]. The authors
also took into consideration the effect of polymer and gold concentration, temperature,
and reaction time. Their results showed that the chitosan acetylation degree and polymer
concentration are the main parameters affecting the size and shape of the nanoparticles.
Polymer molecular weight is related to the reductive efficiency since the reduction of the
polymer size increases the amount of reducing sugars in the media. Our group has focused
its research on the production of AgNPs using low molecular weight chitosan samples.
As previously described in this review, the characteristics of these low molecular weight
chitosan samples depend on the enzyme used to produce the samples. We hypothesised
that samples with similar Mw and acetylation degrees may exhibit different behaviour
due to the monomer pattern. Our results showed that pattern is a key parameter in
the stabilization of the AgNPs, corroborating this hypothesis [123] A chitosan sample
(538 kDa, DD 52%) with little ability to stabilize AgNPs was depolymerized with lysozyme
(fraction L) and chitosanase (fraction Q) and the resulting reaction mixture was separated
into three fractions by tangential ultrafiltration (fraction F1 (Mw > 30 kDa), fraction F2
(Mw 30–10 kDa), and fraction F3 (Mw 10–5 kDa). After depolymerization, an increase in the
DD was observed with values between 62–74%). All fractions were able to reduce the silver
ion, but relevant differences were observed in terms of stabilization (Figure 5). AgNPs
produced with chitosan samples depolymerized with chitosanase (FQ2 and FQ3) were
larger, poorly stabilized, and tended to form large aggregates visible with the naked eye. On
the contrary, AgNPs produced with chitosan depolymerized with lysozyme were smaller
and more stable in all cases. As the Mw of the fraction was reduced, the polydispersity
was also lowered. After one month, the stability of the AgNPs was evaluated and results
Polymers 2021, 13, 3256 showed that AgNPs produced with the fractions F1Q and F1L were the most appropriate 13 of 28
for nanoparticle stabilization.

Figure
Figure 5.
5.Visual
Visualevaluation
evaluation of
of AgNP–polymer
AgNP–polymer solutions after 5 h at 90 °C.◦ C. (A)
(A) F1Q,
F1Q, (B)
(B)F2Q,
F2Q,(C)
(C)F3Q,
F3Q,
(D)
(D) F1L, (E) F2L,
F1L, (E) F2L,(F)
(F)F3L,
F3L,and
and(G)
(G) parent
parent chitosan.
chitosan. Arrows
Arrows indicate
indicate the the presence
presence of aggregates.
of aggregates. ©
© 2021
2021 by the authors. Licensee MDPI, Basel, Switzerland (CC BY) license
by the authors. Licensee MDPI, Basel, Switzerland (CC BY) license [123]. [123].

The AgNPs
The AgNPs produced
produced with
with lysozyme
lysozyme fractions
fractions and
and the
the higher
higher Mw
Mw fraction
fraction of
of chi-
chi-
tosanase were
tosanase were tested
tested in
in the catalytic reduction of TBO [124]. AgNPs produced through
chitosan depolymerization with lysozyme showed better performance than the sample
produced using chitosanase. Moreover, AgNPs produced with fraction F1L exhibited the
best performance in the reaction. That is, the effect of the polymer pattern goes further
than affecting optical properties and stability and differences in the catalytical behaviour
Polymers 2021, 13, 3256 13 of 27

chitosan depolymerization with lysozyme showed better performance than the sample
produced using chitosanase. Moreover, AgNPs produced with fraction F1L exhibited the
best performance in the reaction. That is, the effect of the polymer pattern goes further than
affecting optical properties and stability and differences in the catalytical behaviour was
also observed. This difference is not due to the polymer, since control reactions showed
that the polymeric fractions were not able to catalyse the reduction in TBO and therefore
the effect is solely ascribed to the AgNPs.

6. Chitosan in Biocatalysis
The use of immobilized enzymes for catalysing chemo-, regio- and/or stereoselective
chemical reactions is a very useful and well-known technique [125–142]. In this sense, the
use of chitosan for immobilizing enzymes, either as a carrier for covalent linking or as an
encapsulation vehicle, is well reported [143–149]. Our group described the production
of enantiopure D-p-hydroxyphenylglycine (D-p-HPG, Figure 6) using a multi-enzyme
Polymers 2021, 13, 3256 system containing D-hydantoinase and D-carbamoylase encapsulated in chitosan-based 14 of 28
materials [150–153].

Schematicrepresentation
6. Schematic
Figure 6. representationofofthe
theproduction
productionofof p-hydroxyphenylglycine
p-hydroxyphenylglycine (p-HPG)
(p-HPG) starting
starting from
from a racemic
a racemic mix-
mixture
of p-hydroxyphenyl
ture hydantoin
of p-hydroxyphenyl (HPH)(HPH)
hydantoin using using
a multi-enzyme systemsystem
a multi-enzyme containing immobilized
containing D-hydantoinase
immobilized and D-car-
D-hydantoinase and
bamoylase.
D-carbamoylase.

D-p-HPG
D-p-HPG (or(or simply
simply D-HPG,
D-HPG, aa D-amino
D-amino acid)
acid) is
is aa very
very useful
useful chiral
chiral synthon,
synthon, mainly
mainly
used for the preparation of different semi-synthetic antibiotics, such as amoxicillin,
used for the preparation of different semi-synthetic antibiotics, such as amoxicillin, ce-
cefadroxil, cefprozil,ororcefoperazone
fadroxil, cefprozil, cefoperazone[154–156]
[154–156](Figure
(Figure6), 6),but
but also
also anticancer
anticancer drugs
drugs [157]
[157]
and
and some
some heterocyclic
heterocyclic compounds
compounds [158–161].
[158–161].
For preparing D-HPG, one of the most efficient processes is the so-called “hydan‐
toinase process”, depicted in Figure 6. This cascade of enzymatic reactions, aiming to pro‐
duce optically pure amino acids [162,163], requires an initial step catalyzed by a D-specific
hydantoinase [E.C. 3.5.2.2.] to transform D-p-hydroxyphenyl hydantoin (D-HPH) into N-
carbamoyl-D-p-hydroxyphenylglycine (C-p-HPG), which should be subsequently hydro-
lyzed by a second enzyme, a highly enantiospecific N-carbamoyl amino acid amidohy-
Polymers 2021, 13, 3256 14 of 27

For preparing D-HPG, one of the most efficient processes is the so-called “hydan-
toinase process”, depicted in Figure 6. This cascade of enzymatic reactions, aiming to
produce optically pure amino acids [162,163], requires an initial step catalyzed by a D-
specific hydantoinase [E.C. 3.5.2.2.] to transform D-p-hydroxyphenyl hydantoin (D-HPH)
into N-carbamoyl-D-p-hydroxyphenylglycine (C-p-HPG), which should be subsequently
hydrolyzed by a second enzyme, a highly enantiospecific N-carbamoyl amino acid amido-
hydrolase (also termed D-carbamoylase; E.C.3.5.1.77), to furnish the free amino acid. One of
the main features of the hydantoinase process derives from the spontaneous racemization
of D-HPH at pH values higher than pH 8, caused by the acidic hydrogen at position 5 of
the imidazolidine-2,4-dione ring, which allows for oxo-enol-tautomerism. This leads to a
dynamic-kinetic resolution (DKR), allowing for the use of a mixture of L-and D-HPH as
the initial substrate and a theoretical 100% conversion and 100% optically pure D-amino
acid production (Figure 6).
Both enzymes have been reported to be present in different microorganisms, such
as Agrobacterium sp., Pseudomonas sp., Arthrobacter crystallopoites, or Sinorhizobium
morelense [151], and can be used either as whole cells, crude cell extracts, or purified
enzymes (see Aranaz et al. [151] and references therein). If using isolated enzymes, im-
mobilization is an excellent strategy for stabilizing the enzymatic cocktail due to the fact
that D-hydantoinases are quite stable but D-carbamoylases display low thermostability
and are prone to suffer oxidative degradations. In this sense, different protocols have
been described (see Aranaz et al. [151] and references therein), and our group described
how a multi-enzyme extract from Agrobacterium radiobacter rich in D-hydantoinase and
N-carbamoyl-D-amino acid amidohydrolase was easily immobilized via adsorption on
chitin and chitosan for its application in the synthesis of p-hydroxyphenylglycine [153]. In
fact, this adsorption derivative on chitin showed higher activity compared to the covalent
one, and much greater pH stability compared to the soluble multi-enzymatic extract; on
the other hand, the adsorption derivative exhibited greater pH-stability in the pH range
under study, showing higher activity at low temperatures. Anyhow, as the immobilized
derivatives could not be properly reused, we developed a new strategy based on the
encapsulation of a crude cell extract from the same microorganism, containing both en-
zymes, in alginate beads [164]. This biocatalyst could be reused six times in the presence of
solid HPH particles in a stirred batch reactor without losing any activity until the beads
started to burst. Anyhow, as these alginate-based catalysts showed low stability in calcium
chelating buffers (i.e. phosphate buffers) and easy microbial contamination during storage
at 4 ◦ C, another immobilization matrix, alginate–chitosan polyelectrolyte complexes, was
assessed [150,152]. Thus, alginate mixed chitosan capsules were prepared in one step (by
simply dropping an alginate solution containing the extract into a chitosan solution con-
taining calcium ions) or in a two-step process (preformed calcium–alginate capsules loaded
with the crude cell extract were subsequently coated with chitosan). The encapsulation
yields were around 60% and independent of the characteristics of the different chitosans
used. However, p-HPG production was indeed affected by chitosan acylation degree
D-D (the lower D-D, the lower p-HPG) but not by chitosan molecular weight. Generally
speaking, the best biocatalyst allowed for a p-HPG production yield of around 60% without
any significant protein release to the reaction media. Interestingly, this encapsulation pro-
cedure improved the stability of D-carbamoylase against oxidative damage during storage,
particularly after freeze-drying. In addition, the alginate coated chitosan capsules could be
reused eight times without enzymatic activity loss before D-carbamoylase started losing its
activity and alginate–chitosan beads suffered burst problems contaminating the reaction.
In a collaboration with the group of Dr. Fernández-Lucas, we described the covalent
immobilization of a recombinant nucleoside 20 -deoxyribosyltransferase from Lactobacil-
lus reuteri (LrNDT) on cross-linked magnetic chitosan beads via epichlorohydrin activa-
tion under alkaline conditions, and subsequent incubation with glutaraldehyde [165], as
schematized in Figure 7.
Polymers 2021, 13, 3256 15 of 27
Polymers 2021, 13, 3256 16 of 28

Figure 7.
Figure 7. Schematic representation of the immobilization
immobilization of aa recombinant
recombinant nucleoside 0 -deoxyribosyltransferase from
nucleoside 22’-deoxyribosyltransferase
Lactobacillus reuteri (LrNDT) on cross-linked magnetic chitosan beads. Adapted from Fernández-Lucas
Lactobacillus reuteri (LrNDT) on cross-linked magnetic chitosan beads. Adapted from Fernández-Lucas et et al. [165].
al.[165].

Hence, by varying the amount of magnetite (Fe33O4)

Hence, O4) and
and epichlorohydrin
epichlorohydrin (EPI),
(EPI), dif-
dif-
macroscopic beads were prepared and fully characterized (by scanning electron
ferent macroscopic
microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM))
before being used as supports. Once activated with glutaraldehyde, the best support was
chosen after
after assessment
assessmentofofimmobilization
immobilization yield and
yield product
and yield
product using
yield as a as
using standard reac-
a standard
tion for the synthesis of thymidine (dThd) from 2 0 -deoxyuridine (dUrd) and thymine (Thy),
reaction for the synthesis of thymidine (dThd) from 2’-deoxyuridine (dUrd) and thymine
as depicted
(Thy), in Figure
as depicted 7. Additionally,
in Figure optimal
7. Additionally, conditions
optimal for chitooligosaccharides
conditions with
for chitooligosaccharides
the highest activity of immobilized LrNDT on magnetic chitosan
with the highest activity of immobilized LrNDT on magnetic chitosan were carried out were carried out using
response
using surfacesurface
response methodology (RSM). Thus,
methodology (RSM). theThus,
best-immobilized biocatalyst
the best-immobilized retained 50%
biocatalyst re-
of its maximal
tained 50% of itsactivity
maximalafteractivity 60 ◦ C56.3
56.3 h atafter andhnoat lost
60 °Cactivity
and nowas observed
lost activityafter
was storage
observedat
40 ◦ C for 144 h. Subsequently, this innovative immobilized biocatalyst was employed in the
after storage at 40 °C for 144 h. Subsequently, this innovative immobilized biocatalyst was
enzymatic in
employed synthesis of 20 -deoxyribonucleoside
the enzymatic analogues and arabinosyl
synthesis of 2’-deoxyribonucleoside analogues nucleosides such
and arabinosyl
as vidarabine (ara-A) and cytarabine (ara-C), as depicted in Figure
nucleosides such as vidarabine (ara-A) and cytarabine (ara-C), as depicted in Figure 8, 8, leading to moderate
or good to
leading yields at 2 hor
moderate reaction time. Remarkably,
good yields at 2 h reaction thetime.
immobilized
Remarkably,derivatives could be
the immobilized
easily recovered and recycled for 30 consecutive batch reactions without
derivatives could be easily recovered and recycled for 30 consecutive batch0 reactions with- any significant
decrease
out in the catalytic
any significant decreaseactivity
in theincatalytic
the synthesis
activityofin2,6-diaminopurine-2 -deoxyriboside
the synthesis of 2,6-diaminopurine-
(2,6-DAPdRib) and 5-trifluorothymidine (5-tFThd).
2’-deoxyriboside (2,6-DAPdRib) and 5-trifluorothymidine (5-tFThd).
Polymers 2021, 13, 3256 16 of 27
Polymers 2021, 13, 3256 17 of 28

Figure 8.8.Synthesis
Figure of different
Synthesis natural and
of different non-natural
natural nucleosides nucleosides
and non-natural using a recombinant
using nucleoside nucleoside 20 -
2’-deoxyribosyltrans-
a recombinant
ferase from Lactobacillus reuteri
deoxyribosyltransferase (LrNDT) immobilized
from Lactobacillus on cross-linked
reuteri (LrNDT) immobilizedmagnetic chitosan beads
on cross-linked [165]chitosan
magnetic . Commission
beads on Bi-
[165].
ochemical Nomenclature:
Commission on Biochemical adenine (Ade), uracil
Nomenclature: (Ura),
adenine cytosine
(Ade), (Cyt),
uracil thymine
(Ura), cytosine(Thy),
(Cyt),2,6-diaminopurine (2,6-DAP), 5-tri-
thymine (Thy), 2,6-diaminopurine
fluorothymine
(2,6-DAP), (5-tFThy), 2’-deoxyuridine
5-trifluorothymine (5-tFThy), 2(dUrd), 2’-deoxyadenosine
0 -deoxyuridine (dAdo), 2’-deoxycytidine
(dUrd), 20 -deoxyadenosine (dAdo),(dCyd), thymidine(dCyd),
20 -deoxycytidine (dThd),
2,6-diaminopurine-2’-deoxyriboside (2,6-DAPdRib),
0 5-trifluorothymidine (5-tFdThd),2’-fluoro-20-deoxyuridine 0 (2’-
thymidine (dThd), 2,6-diaminopurine-2 -deoxyriboside (2,6-DAPdRib), 5-trifluorothymidine (5-tFdThd), 2 -fluoro-20-
FdUrd), 2’-fluoro-2’-deoxycitydine (2’-FdCyd), ara-uracil (ara-U), ara-adenine (ara-A).
deoxyuridine (20 -FdUrd), 20 -fluoro-20 -deoxycitydine (20 -FdCyd), ara-uracil (ara-U), ara-adenine (ara-A).

7. Chitosan in Drug Delivery

7. Chitosan in Drug Delivery
Since the introduction of the first polymers in drug delivery, chitosan has shown su-
Since the introduction of the first polymers in drug delivery, chitosan has shown
perior biological and physiochemical properties for a wide variety of biomedical and in-
superior biological and physiochemical properties for a wide variety of biomedical and
dustrial applications. The main feature of this biopolymer is its cationic character due to
industrial applications. The main feature of this biopolymer is its cationic character due to
amino groups. These amino groups are also responsible for properties such as controlled
amino groups. These amino groups are also responsible for properties such as controlled
drug release,
drug release, mucoadhesion,
mucoadhesion, inin situ
situ gelation,
gelation, transfection,
transfection, permeation
permeation enhancement,
enhancement, and
and
efflux pump inhibitory properties [166]. Moreover, interest in this biomaterial due
efflux pump inhibitory properties [166]. Moreover, interest in this biomaterial due to its to its
central nervous system (CNS) bio-medical implementation has increased because of its
ability to cross the blood brain barrier (BBB) [167].
 Polymers 2021, 13, 3256 17 of 27
Polymers 2021, 13, 3256 18 of 28

central nervous
Therefore, system
chitosan (CNS) used
is widely bio-medical
in drugimplementation hastechnological
delivery due to its increased because of its
proper-
ability to cross the blood brain barrier (BBB) [167].
ties, which allow us to process the polymer in different ways (Table 5).
Therefore, chitosan is widely used in drug delivery due to its technological properties,
which
Table 5. Someallow us to process
examples thepresentations
of chitosan polymer in different ways (Table 5).
in drug delivery.

Table 5.Presentation
Some examples of chitosan presentations in drug References
delivery.
Films [168–171]
SpongesPresentation [172,173]References
Scaffolds Films [174,175] [168–171]
Sponges
Nanoparticles [176] [172,173]
Scaffolds [174,175]
Microspheres [177–179]
Nanoparticles [176]
HydrogelsMicrospheres [180–182][177–179]
AerogelsHydrogels [183–185][180–182]
Fibers Aerogels [186,187] [183–185]
MicroneedlesFibers [188,189] [186,187]
Microneedles [188,189]
Coated Liposomes
Coated Liposomes [190,191] [190,191]
Nanocomposites
Nanocomposites [192,193] [192,193]
Composites
Composites [194] [194]

Initially, a chitosan
Initially, salt salt
a chitosan (chitosan hydrochloride)
(chitosan hydrochloride) waswasapproved
approved in 2002 by the
in 2002 by Phar-
the Phar-
macopeia. Chitosan was first introduced as an excipient into the European
macopeia. Chitosan was first introduced as an excipient into the European Pharmacopeia Pharmacopeia
6.0 and the the
6.0 and 29th29th
edition of the
edition United
of the States
United Pharmacopeia
States Pharmacopeia (USP) 34-NF
(USP) almost
34-NF ten ten
almost years
years
later. These monographs contain the assays and establish limits to be observed
later. These monographs contain the assays and establish limits to be observed when when the the
polymer
polymeris used as aaspharmaceutical
is used a pharmaceutical excipient
excipient[195,196]. TheThe
[195,196]. increase in the
increase number
in the numberof of
publications
publicationsregarding the the
regarding use use
of this polymer
of this polymer in drug delivery
in drug is shown
delivery in Figure
is shown 9 and
in Figure 9 and
reveals a strong
reveals increase
a strong since
increase 20022002
since thatthat
is still maintained
is still maintainedtoday.
today.

Figure 9. Publications about chitosan drug delivery in Scopus (1987–2020).

Figure 9. Publications about chitosan drug delivery in Scopus (1987–2020).
 Polymers 2021, 13, 3256 19 of 28
Polymers 2021, 13, 3256 18 of 27

Chitosan films are easily produced by solvent-casting methodologies, but more com-
plexChitosan
systems films
can beare produced by blending
easily produced the polymer with
by solvent-casting others suchbut
methodologies, as pectin
more com-[197]
or by
plex producing
systems can belayer-by-layer films with
produced by blending thenegatively
polymer with charged
otherspolymers like polyacid
such as pectin [197] or
by[198], poly (lactic-co-glycolic
producing layer-by-layer films acid)with
[199] or polylactic
negatively charged[200],polymers
among others. Besides[198],
like polyacid their
safety,
poly biocompatibility,
(lactic-co-glycolic and[199]
acid) biodegradability, biopolymer-based
or polylactic [200], among others. films havetheir
Besides beensafety,
draw-
ing increasing interest
biocompatibility, as excellent candidates
and biodegradability, not only films
biopolymer-based as controlled-drug
have been drawing delivery sys-
increas-
ing
temsinterest as excellent
but also as materials candidates not only
to produce as controlled-drug
contact delivery systems
lenses, wound dressings, but engi-
and tissue also
as materials
neering to produce contact lenses, wound dressings, and tissue engineering matrices.
matrices.
Particulate
Particulate chitosan-based
chitosan-based systems (micro (microand andnano nanosystems)
systems)are are widely
widely used
used forfor
the
the encapsulation
encapsulation of aoflarge
a large variety
variety of molecules
of molecules suchsuch as growth
as growth factors
factors [178],
[178], antimicro-
antimicrobials
bials
[201],[201], painkillers
painkillers [202],[202], anti-tumoral
anti-tumoral [203][203] or anti-inflammatory
or anti-inflammatory drugs drugs [204].
[204].
Recently,
Recently, chitosan has been used for the fabrication of microneedles (MNs)due
chitosan has been used for the fabrication of microneedles (MNs) duetotoitsits
film-forming
film-forming ability, biodegradability, and biocompatibility, making it suitable fortopical
ability, biodegradability, and biocompatibility, making it suitable for topical
and
andtransdermal
transdermaldrug drugdelivery
delivery[188].
[188].InInparticular,
particular,the theuse
useofofchitosan
chitosanMNs MNsininvaccination
vaccination
isisaahot
hottopic
topicofofdiscussion
discussion[205–207].
[205–207]. TheTheuse useof ofchitosan
chitosanMNs MNsininwound woundhealing
healingand and
point-of-care testing is revolutionary and gives hope of more useful
point-of-care testing is revolutionary and gives hope of more useful developments in developments in these
areas. However,
these areas. some some
However, drawbacks still need
drawbacks further
still need investigation.
further investigation.The The
development
development of
MNs devices with adequate mechanical strength to penetrate the skin
of MNs devices with adequate mechanical strength to penetrate the skin without causing without causing pain
and
painskin
anddamage and theand
skin damage development of efficient
the development methodsmethods
of efficient for theirforsterilization remain
their sterilization
challenging [208].
remain challenging [208].
AAcomparison
comparisonof ofthe
thenumber
numberof ofpublications
publicationscontaining
containing“Chitosan
”Chitosan++drug drugdelivery”
delivery“
ininScopus
Scopus andand patents
patents in in Lens portal (free,
Lens portal (free, open
openpatent,
patent,and andscholarly
scholarlysearch)
search)isisshown
shownin
in Figure 10. As observed, the number of patents is almost four
Figure 10. As observed, the number of patents is almost four times the number of publi- times the number of
publications, showing the increasing application of this polymer in the
cations, showing the increasing application of this polymer in the drug delivery field. An drug delivery field.
An interesting article by Kurakula and Raghavendra summarizes the chitosan biomedical
interesting article by Kurakula and Raghavendra summarizes the chitosan biomedical
trends and the related patents [209].
trends and the related patents [209].

Figure10.
Figure 10.Publications
Publicationsabout
aboutchitosan
chitosandrug
drugdelivery
deliveryininScopus
Scopusand
andpatents
patentsininLens
Lens(1987–2020).
(1987–2020).

8.8.Conclusions
Conclusionsand
andPrognosis
Prognosis
Chitosan
Chitosanand
anditsits
derivatives
derivatives have been
have used
been in ain
used myriad of applications
a myriad for a long
of applications for atime.
long
The potential
time. interest
The potential of these
interest polymers
of these is clear
polymers whenwhen
is clear observing the number
observing the numberof articles
of arti-
and
clespatents that appear
and patents every every
that appear year and
yearthe
andgrowing marketmarket
the growing perspective. In some
perspective. Inof theseof
some
applications such assuch
these applications agriculture or the or
as agriculture food
theindustry, the use
food industry, ofuse
the chitosan in thein
of chitosan market
the mar-is
well established. The use of chitosan has extended to a large number of research
ket is well established. The use of chitosan has extended to a large number of research areas from
Materials Science
areas from to Arts
Materials and the
Science Humanities
to Arts (Figure 11). (Figure 11).
and the Humanities
Polymers 2021, 13, 3256 19 of 27
Polymers 2021, 13, 3256 20 of 28

Figure11.
Figure 11. Number
Number of
of publications
publications andanddistribution
distributionby
byarea
areaininthe period
the 2011–2021.
period Search
2011–2021. of chi-
Search of
tosan word in Scopus (abstract, title, keywords).
chitosan word in Scopus (abstract, title, keywords).

However,chitosan
However, chitosanpotentiality
potentialityisissomehow
somehowhindered
hinderedby bythe
theinconsistency
inconsistencyin inthe
there-
re-
search data and the lack of knowledge in the ultimate mechanism
search data and the lack of knowledge in the ultimate mechanism underlying the properties underlying the proper-
ties
of of chitosan.
chitosan. Between
Between 2011–2020,
2011–2020, thethe number
number of of publications
publications onon chitosanhas
chitosan hasdisplayed
displayed
aasteady
steadygrowth.
growth. In In 2021,
2021, aa drop
drop isis observed,
observed,whichwhichisisascribed
ascribedininpart
parttotothethelarge
large number
num-
of reviews
ber of reviews published
published in 2020,
in 2020, probably
probably duedueto to
thetheCOVID-19
COVID-19 pandemic,
pandemic, which
which hashas
af-
fected normal
affected normallaboratory
laboratory work work worldwide.
worldwide. Regardless, we we consider
consider that
thatthisthisgrowth
growthwill will
continuein
continue inthe
thefollowing
followingyears,years,driven
drivenby bythe
thestrong
strongeffort
effortthat
thathas
hasbeen
beencarried
carriedout outbyby
the Chitin Science Scientific Community in the systematic research on this polymer. InIn
the Chitin Science Scientific Community in the systematic research on this polymer.
fact,its
fact, itsapproval
approvalby bydifferent
differentagencies
agencieshas hasboosted
boostedthe theinterest
interestininthis
thispolymer
polymerboth bothby bythe
the
industrialand
industrial andscientific
scientificcommunities.
communities.
Chitosan specifications
Chitosan specifications are are ultimately
ultimatelyrelated
relatedtotoitsits
final application.
final application. Thus, highhigh
Thus, qual-
ity chitosans
quality withwith
chitosans low heavy
low heavymetalmetal
and lowandendotoxin contents
low endotoxin are required
contents for biomed-
are required for
biomedical and pharmaceutical
ical and pharmaceutical uses. Moreover,
uses. Moreover, strict of
strict control control of production
production is needed is needed
to avoid
to avoid uncontrollable
uncontrollable hydrolysis hydrolysis and chemical
and chemical modifications
modifications during during
polymer polymer
isolation. isolation.
There-
Therefore, chitosan
fore, chitosan production
production is notisanot a trivial
trivial issue.issue. To date,
To date, chitosan
chitosan production
production cannot cannot be
be con-
considered
sidered fully fully sustainable
sustainable duedue to the
to the large
large amount
amount of acid
of acid andand basic
basic reagents
reagents neededneeded
and
and the high
the high temperatures
temperatures required.
required. Unfortunately,
Unfortunately, biotechnological
biotechnological processes processes using
using biocata-
biocatalysts are currently
lysts are currently limitedlimited to the laboratory
to the laboratory scale soscale
that so that implementation
implementation of theseofgreener
these
greener processes at a large scale is certainly one of the milestones we
processes at a large scale is certainly one of the milestones we want to see being achieved want to see being
achieved
in the nextin decade.
the next decade.

Author Contributions:Conceptualization:
AuthorContributions: Conceptualization:I.A.
I.A.and
andN.A.;
N.A.;writing—original
writing—originaldraft
draftpreparation,
preparation,I.A.,
I.A.,
A.R.A.,
A.R.A., N.A., M.C.C., and C.A.; writing—review and editing, I.A., A.R.A., N.A., M.C.C.,C.A.
N.A., M.C.C. and C.A.; writing—review and editing, I.A., A.R.A., N.A., M.C.C., C.A.,and
and
B.E.;
B.E.;funding
fundingacquisition, A.H.C.,
acquisition, N.A.
A.H.C., andand
N.A., A.R.A. All authors
A.R.A. havehave
All authors read read
and agreed to the to
and agreed published
the pub-
version of the manuscript.
lished version of the manuscript.
Funding: This
Funding: This research
research was
was funded
funded by
by Spanish
Spanish Ministry
Ministry of
of Science
Science and
and Innovation
Innovation (PID2019-
(PID2019-
105337RB-C22) and Banco de Santander-Complutense Research Projects (PR87/19-22676).
105337RB-C22) and Banco de Santander-Complutense Research Projects (PR87/19-22676).
Institutional
InstitutionalReview
ReviewBoard Statement:Not
BoardStatement: Notapplicable.
applicable.
Informed
InformedConsent Statement:Not
ConsentStatement: Notapplicable.
applicable.
Data
DataAvailability Statement:Not
AvailabilityStatement: Notapplicable.
applicable.
Conflicts Interest:The
ConflictsofofInterest: Theauthors
authorsdeclare
declareno
noconflict of of
conflicts interest.
interest.

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