The quaternized chitosan has promising applications in biomedicine as pharmaceuticals and targeted delivery, in gene therapy, tissue engineering, and cosmetology due to the improved solubility and enhancing many biological functions in comparison to the non-quaternized chitosan. The increased potential for such applications is achieved through the beneficial combination of the inherent properties of chitosan with those of quaternary ammonium units. Since the first publications on the synthesis of quaternary ammonium salts of chitosan, a variety of the synthesis methods have been developed. The each of these methods has its own advantages and disadvantages that affect on the product properties. The aim of this work was to choose the optimal path for quaternization and chemical modification of the polymer. The result of the work was an improvement in the solubility of chitosan in the investigated solutions. Among the various natural polymers, chitosan has attracted the interest of researchers due to its two main properties. This is due to its economic efficiency and the fact that it is the most widespread polysaccharide in the world after cellulose. Chitosan is a D-glucosamine with 5-15% acetamide group and amino acids and up to 1% peptide-related groups.
The molecular weight of artificially derived chitosan ranges from 100, 000 to 1, 200, 000. Chitosan may degradation occur during production. Several factors can affect the breakdown of chitosan. These include dissolved oxygen, high temperatures and voltage fluctuations. Dissolved oxygen slowly decomposes chitosan, but its thermal decomposition takes place at a temperature of 2800C. Fragmentation by hydrodynamic forces can lead to a critical length of a long-chain chitosan molecule.
Chitosan has a number of superior properties, which expand its areas of application. These include unique properties such as non-toxicity, biocompatibility, broad antibacterial and antifungal activity.
Chitosan has a unique biological activity in plants that can resist viral diseases and prevent viral infections. In animals, it is used to prevent the spread of phage infection by infected microorganisms. Positively charged chitosan interacts with negatively charged skin and hair. Due to this feature, it is used in the cosmetics industry. The manifestation of the properties of the sorbent and partially lipophilic emulsifier determines the effect of chitosan. Unlike plant fiber and other natural adsorbents, chitosan is more effective due to its unique molecular structure. Chitosan or its complexes are dozens of times more efficient than ion exchange resins for the effective binding of heavy metal ions and radionuclides of single amine groups.
Chitosan is a linear polysaccharide with good biodegradability, biocompatibility and antimicrobial activity, which allows it to be used alone or in combination with other polymers as an antimicrobial agent in biomedicine. Сhitosans of low molecular weight with high solubility and low viscosity of aqueous solutions are mainly used. The studies carried out confirmed the dependence of the biological activity of chitosan on its molecular weight and the degree of acetylation. These factors also affect the solubility of chitosan. This is due to the fact that the strength of the network of inter- and intramolecular hydrogen bonds depends on the molecular weight, the degree of acetylation / deacetylation and the distribution of the acetyl group in the polymer molecule. Its poor solubility in neutral or high pH in many solvents limits its use. Quaternary ammonium chitosan was obtained by incorporating the quaternary ammonium group into the dissociative hydroxyl or amine group of chitosan.
It can be chemically and / or physically modified to produce chitosan-based derivatives. These processes can be used to control the reactivity of the polymer or to improve the kinetics of sorption.
Chitosan can be easily modified due to the hydroxyl and amine groups located in positions C3-C6, C-2. Chemical modification of chitosan is carried out for two purposes: 1. to prevent the dissolution of the polymer during the sorption or desorption of metals from acidic solutions, and 2. to improve the sorption properties of metals - to increase the sorption capacity or selectivity.
Quartenization of chitosan with methyl iodide in a neutral medium: initially prepared a suspension with 0. 5 g of chitosan and 5 ml of bidistylated water and the resulting solution was kept overnight. 0. 3 ml of CH3J was added to the result solution and mixed in a magnetic stirrer at 300C for 4 h. The resulting product was dried at room temperature after washing several times with acetone for get precipitate.
Quartenization of chitosan with methyl iodide in an acidic medium: Firstly, 1% acetic acid solution was prepared to begin the process. Then 0. 5 g of chitosan was dissolved in 25 ml of acetic acid solution and 0. 3 ml of CH3J was added. After that solution was mixed at 250 C for 5 hours. Finally, the reaction mixture was washed several times with acetone for the same procedure and purpose and dried at room temperature.
Quartization of chitosan with methyl iodide in an alkaline medium: For quaternization in an alkaline environment, a 1% NaOH solution was initially prepared. Then 0. 5 g of chitosan was dispersed in 10 ml of NaOH solution and kept overnight. 0. 3 ml of CH3J was added to the resulting solution and mixed at 250C for 5 h. The reaction mixture was diluted with 10 ml of methanol and mixed again for 2 h, and the product was washed several times with acetone and dried at room temperature. Quaternation of chitosan with benzyl chloride which an average molecular weight of 100-300 kDa was carried out in acidic medium at 45 ° C. To do this, 0. 5 g of natural polymer chitosan is dissolved in 50 ml of 1% acetic acid. Then 0. 2 ml benzyl chloride is added and mixed for 4-5 hours. The resulting product was washed several times with diethyl ether and dried at room temprature.
FTIR study was conducted to determine the success of the quaternization process. Characterization of chitosan FTIR spectrum was obtained by using an infrared spectrometer.
Chitosan has an apparent dissociation constant of conjugate acid pK ̴ 6. 5-6. 7 and does not dissolve at pH above ̴ 6. This is due to the strong network of inter- and intramolecular hydrogen bonds that occured between hydroxyl and amino groups, and depends on the molecular weight, degree deacetylation and distribution of the acetyl group in the polymer molecule. Due to the such disadvantage, the use of chitosan is difficult at physiological pH values in pharmaceutical chemistry and biomedicine.
In order to improve of the solubility of chitosan, at the same time to the enhancement of antimicrobial, anticoagulant, and other properties, it was taken to modify chitosan by converting primary amino groups into quaternary salts with a constant positive charge. The quaternization reaction of chitosan leads to an increasing of the positive charge of chitosan that causes the appearance of electrostatic repulsion between chitosan chains, and also to the grafting of side alkyl chains. In this case, the effect of destruction of intra- and intermolecular networks of hydrogen bonds appears and the polymer chains unfold, so that the chitosan molecule becomes less rigid and, therefore, dissolves better [*+].
In this work, as already mentioned, the quaternization reaction was carried out with methyl iodide in three mediums (alkaline, neutral, and acidic) and with benzyl chloride in an acidic medium. At quaternization in an alkaline and neutral medium, dispersed solutions are obtained with the presence of a curdled precipitate of light yellow and pale yellow, respectively. After drying, products are formed in the form of a powder of the corresponding colors. When quaternization in an acidic medium, a transparent, light yellow gel is obtained. The formation of a gel is apparently associated with the presence of intermolecular hydrophobic interactions between chitosan aggregates, which may be the result of an increase in excess surface energy and attractive forces between particles. After drying, a transparent film with a light yellow tint is formed.
Based on the results of the IR spectra of the obtained samples, we can assume the success of the quaternization process in all four cases. Thus, as shown in the FTIR spectrum of chitosan (Fig. 1), the strong characteristic peak at 1593, 42 sm-1 is due to the N - H vibration of chitosan, and the strong characteristic peaks at 1652, 02 and 2965, 15 sm-1 belong to the C = O and C – H. The absorption bands with a frequency of 3500-3300 sm-1, 1650-1500 sm-1 and 1390-1000 sm-1 are due to the vibrations of the N-H and C-N bonds. The broad medium-intensity absorption band observed in the 1320-1387 sm-1 region indicates the stretching vibration of -OH bonds. The associated amino group corresponds to absorption bands in the region of 3360-3180 sm-1. Absorption in the regions of 1650 1500 sm 1 and 960-650 sm-1 correspond to deformation vibrations of the amino group.
The results of IR spectroscopy at quaternization of chitosan with methyl iodide are shown in Fig. 2-4, at quaternization with benzyl chloride - in Fig. 5. From the analysis of absorption bands belonging to the functional groups of CS and the obtained hydrogels by IR spectroscopy, it was determined that the quarantine of the polymer occurs due to the combination of its iodide groups with the -CH group. The FTIR spectra of the quaternized samples show that the peak characteristic for chitosan at 1593, 42 sm-1 disappears, while new characteristic peaks appear at 1404, 68 in acidic and 1417, 73 in alkaline mediums, as well as narrow weak bands at 1558, 57-1507, 16 - in a neutral medium.
When chitosan is quaternized with benzyl chloride, absorption bands characteristic of an aromatic alkyl derivative of the polymer are expected to appear. The presence of aromatic compounds can be detected by medium-intensity C-H stretching vibrations (≈3000 cm-1), skeletal vibrations of aromatic carbon-carbon bonds, and by intense absorption below 900 sm-1 caused by deformation C H vibrations.
The results of this study confirm that the chitosan quaternization reaction was successful in all cases, as evidenced by the improved solubility of the polymer. Complete quaternization of chitosan is impossible due to the presence of some acetyl groups in chitosan and, probably, due to possible steric effects of attached methyl groups on adjacent quaternary amino groups.
The
quaternized
chitosan has promising applications in biomedicine as pharmaceuticals and targeted delivery, in gene therapy, tissue engineering, and cosmetology
due
to the
improved
solubility and enhancing
many
biological functions
in comparison
to the
non-quaternized
chitosan. The increased potential for such applications
is achieved
through the beneficial combination of the inherent
properties
of chitosan with those of quaternary ammonium units. Since the
first
publications on the synthesis of quaternary ammonium salts of chitosan, a variety of the synthesis methods have
been developed
.
The each
of these methods has its
own
advantages and disadvantages that
affect
on the
product
properties
. The aim of this work was to choose the optimal path for
quaternization
and chemical modification of the polymer.
The
result
of the work was an improvement in the solubility of chitosan in the investigated
solutions
. Among the various natural polymers, chitosan has attracted the interest of researchers
due
to its two main
properties
. This is
due
to its economic efficiency and the fact that it is the most widespread polysaccharide in the world after cellulose. Chitosan is a D-glucosamine with 5-15%
acetamide
group
and amino
acids
and up to 1% peptide-related groups.
The
molecular
weight
of
artificially
derived chitosan ranges from 100, 000 to 1, 200, 000. Chitosan
may degradation
occur during production. Several factors can affect the breakdown of chitosan. These include dissolved oxygen, high
temperatures
and voltage fluctuations. Dissolved oxygen
slowly
decomposes chitosan,
but
its thermal decomposition takes place at a
temperature
of 2800C. Fragmentation by hydrodynamic forces can lead to a critical length of a long-chain chitosan molecule.
Chitosan has a number of superior
properties
, which expand its areas of application. These include unique
properties
such as non-toxicity, biocompatibility, broad antibacterial and antifungal activity.
Chitosan has a unique biological activity in plants that can resist viral diseases and
prevent
viral infections. In animals, it is
used
to
prevent
the spread of phage infection by infected microorganisms.
Positively
charged chitosan interacts with
negatively
charged skin and hair.
Due
to this feature, it is
used
in the cosmetics industry. The manifestation of the
properties
of the
sorbent
and
partially
lipophilic
emulsifier determines the effect of chitosan. Unlike plant fiber and other natural adsorbents, chitosan is more effective
due
to its unique
molecular
structure. Chitosan or its complexes are dozens of
times
more efficient than ion exchange resins for the effective binding of heavy metal ions and radionuclides of single amine groups.
Chitosan is a linear polysaccharide with
good
biodegradability, biocompatibility and antimicrobial activity, which
allows
it to be
used
alone or in combination with other polymers as an antimicrobial agent in biomedicine.
Сhitosans
of low
molecular
weight
with high solubility and low viscosity of aqueous
solutions
are
mainly
used
. The studies carried out confirmed the dependence of the biological activity of chitosan on its
molecular
weight
and the degree of
acetylation
. These factors
also
affect the solubility of chitosan. This is
due
to the fact that the strength of the network of inter- and
intramolecular
hydrogen bonds depends on the
molecular
weight
, the degree of
acetylation
/
deacetylation
and the distribution of the acetyl
group
in the polymer molecule. Its poor solubility in
neutral
or high pH in
many
solvents limits its
use
. Quaternary ammonium chitosan was
obtained
by incorporating the quaternary ammonium
group
into the dissociative hydroxyl or amine
group
of chitosan.
It can be
chemically
and / or
physically
modified to produce chitosan-based derivatives. These
processes
can be
used
to control the reactivity of the polymer or to
improve
the kinetics of
sorption
.
Chitosan can be
easily
modified
due
to the hydroxyl and amine
groups
located in positions C3-C6, C-2. Chemical modification of chitosan
is carried
out for two purposes: 1.
to
prevent
the dissolution of the polymer during the
sorption
or
desorption
of metals from
acidic
solutions
, and 2.
to
improve
the
sorption
properties
of metals
-
to increase the
sorption
capacity or selectivity.
Quartenization
of chitosan with methyl iodide in a
neutral
medium
:
initially
prepared a suspension with 0. 5 g of chitosan and 5 ml of
bidistylated
water and the
resulting
solution
was
kept
overnight. 0. 3 ml of CH3J was
added
to the
result
solution
and
mixed
in a magnetic stirrer at 300C for 4 h. The
resulting
product
was
dried
at
room
temperature
after washing several
times
with acetone for
get
precipitate.
Quartenization
of chitosan with methyl iodide in an
acidic
medium
:
Firstly
, 1% acetic
acid
solution
was prepared
to
begin
the
process
. Then 0. 5 g of chitosan
was dissolved
in 25 ml of acetic
acid
solution
and 0. 3 ml of CH3J was
added
. After that
solution
was
mixed
at 250 C for 5 hours.
Finally
, the
reaction
mixture
was washed
several
times
with acetone for the same procedure and purpose and
dried
at
room
temperature.
Quartization
of chitosan with methyl iodide in an
alkaline
medium
: For
quaternization
in an
alkaline
environment, a 1% NaOH
solution
was
initially
prepared. Then 0. 5 g of chitosan
was dispersed
in 10 ml of NaOH
solution
and
kept
overnight. 0. 3 ml of CH3J was
added
to the
resulting
solution
and
mixed
at 250C for 5 h. The
reaction
mixture
was diluted
with 10 ml of methanol and
mixed
again for 2 h, and the
product
was washed
several
times
with acetone and
dried
at
room
temperature
.
Quaternation
of chitosan with benzyl chloride which an average
molecular
weight
of 100-300
kDa
was carried
out in
acidic
medium
at
45 °
C. To do this, 0. 5 g of natural polymer chitosan
is dissolved
in 50 ml of 1% acetic
acid
. Then 0. 2 ml benzyl chloride is
added
and
mixed
for 4-5 hours. The
resulting
product
was washed
several
times
with
diethyl
ether and
dried
at
room
temprature
.
FTIR
study
was conducted
to determine the success of the
quaternization
process
. Characterization of chitosan
FTIR
spectrum was
obtained
by using an infrared spectrometer.
Chitosan has an apparent dissociation constant of conjugate
acid
pK
̴ 6. 5-6. 7 and does not dissolve at pH above ̴ 6. This is
due
to the strong network of inter- and
intramolecular
hydrogen bonds that
occured
between hydroxyl and amino
groups
, and depends on the
molecular
weight
, degree
deacetylation
and distribution of the acetyl
group
in the polymer molecule.
Due
to the such disadvantage, the
use
of chitosan is difficult at physiological pH values in pharmaceutical chemistry and biomedicine.
In order to
improve
of the solubility of chitosan, at the same
time
to the enhancement of antimicrobial, anticoagulant, and other
properties
, it
was taken
to modify chitosan by converting primary amino
groups
into quaternary salts with a constant
positive
charge. The
quaternization
reaction
of chitosan leads to an increasing of the
positive
charge of chitosan that causes the appearance of electrostatic repulsion between chitosan chains, and
also
to the grafting of side alkyl chains.
In this case
, the effect of destruction of
intra
- and intermolecular networks of hydrogen bonds appears and the polymer chains unfold,
so
that the chitosan molecule becomes less rigid and,
therefore
, dissolves better [*+].
In this work, as already mentioned, the
quaternization
reaction
was carried
out with methyl iodide in three
mediums
(alkaline
,
neutral
, and
acidic)
and with benzyl chloride in an
acidic
medium
. At
quaternization
in an
alkaline
and
neutral
medium
, dispersed
solutions
are
obtained
with the
presence
of a curdled precipitate of light yellow and pale yellow,
respectively
. After drying,
products
are formed
in the form of a powder of the corresponding colors. When
quaternization
in an
acidic
medium
, a transparent, light yellow gel is
obtained
. The formation of a gel is
apparently
associated with the
presence
of intermolecular hydrophobic interactions between chitosan aggregates, which may be the
result
of an increase in excess surface energy and attractive forces between particles. After drying, a transparent film with a light yellow tint
is formed
.
Based on the
results
of the IR spectra of the
obtained
samples, we can assume the success of the
quaternization
process
in all four cases.
Thus
, as shown in the
FTIR
spectrum of chitosan (Fig. 1), the strong
characteristic
peak
at 1593, 42 sm-1 is
due
to the N
-
H
vibration
of chitosan, and the strong
characteristic
peaks
at 1652, 02 and 2965, 15 sm-1 belong to the C = O and C
–
H. The
absorption
bands
with a frequency of 3500-3300 sm-1, 1650-1500 sm-1 and 1390-1000 sm-1 are
due
to the vibrations of the N-H and C-N bonds. The broad medium-intensity
absorption
band
observed in the 1320-1387 sm-1 region indicates the stretching
vibration
of -OH bonds. The associated amino
group
corresponds to
absorption
bands
in the region of 3360-3180 sm-1.
Absorption
in the regions of 1650 1500
sm
1 and 960-650 sm-1 correspond to deformation vibrations of the amino group.
The
results
of IR spectroscopy at
quaternization
of chitosan with methyl iodide
are shown
in Fig. 2-4, at
quaternization
with benzyl chloride
-
in Fig. 5. From the analysis of
absorption
bands
belonging to the functional
groups
of CS and the
obtained
hydrogels by IR spectroscopy, it
was determined
that the quarantine of the polymer occurs
due
to the combination of its iodide
groups
with the -CH
group
. The
FTIR
spectra of the
quaternized
samples
show
that the
peak
characteristic
for chitosan at 1593, 42 sm-1 disappears, while new
characteristic
peaks
appear at 1404, 68 in
acidic
and 1417, 73 in
alkaline
mediums
, as
well
as narrow weak
bands
at 1558, 57-1507, 16
-
in a
neutral
medium.
When chitosan is
quaternized
with benzyl chloride,
absorption
bands
characteristic
of an aromatic alkyl derivative of the polymer are
expected
to appear. The
presence
of aromatic compounds can
be detected
by medium-intensity C-H stretching vibrations (≈3000 cm-1), skeletal vibrations of aromatic carbon-carbon bonds, and by intense
absorption
below 900 sm-1 caused by deformation C H vibrations.
The
results
of this study confirm that the chitosan
quaternization
reaction
was successful in all cases, as evidenced by the
improved
solubility of the polymer. Complete
quaternization
of chitosan is impossible
due
to the
presence
of
some
acetyl
groups
in chitosan and,
probably
,
due
to possible
steric
effects of attached methyl
groups
on adjacent quaternary amino
groups
.
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