Thiolated chitosans: Are Cys-Cys ligands key to the next generation?

Carbohydrate Polymers 242 (2020) 116395

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Thiolated chitosans: Are Cys-Cys ligands key to the next generation?
a

b

b

c

T

c

Kesinee Netsomboon , Aamir Jalil , Flavia Laffleur , Andrea Hupfauf , Ronald Gust ,
Andreas Bernkop-Schnürchb,*
a
b
c

Division of Pharmaceutical Sciences, Faculty of Pharmacy, Thammasat University (Rangsit Campus), Khlong Luang, Pathumthani 12120, Thailand
Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innsbruck 6020, Austria
Center for Chemistry and Biomedicine, Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria

A R T I C LE I N FO

A B S T R A C T

Keywords:
Thiolated
Mucoadhesive
Mucosal drug delivery
Polymer
Thiomers
S-protected thiomers
Thiolated polymer
Chitosan

The potential of Cys-Cys ligands for the development of a novel type of S-protected thiomers was evaluated. Sprotected thiomers chitosan-N-acetylcysteine-mercaptonicotinamide (CS-NAC-MNA) and chitosan-N-acetylcysteine-N-acetylcysteine (CS-NAC-NAC) were synthesized and characterized. Viscosity of polymers in presence of various concentrations of S-amino acids was monitored. Mucoadhesive properties were evaluated. FT-IR
characterization confirmed the covalent attachment of NAC-MNA and NAC-NAC. Attached sulfhydryl groups
were found in the range of 550 μmol/g. In the presence of amino acids bearing a free thiol group viscosity of both
polymers increased. This increase in viscosity depended on the amount of added free thiols. Maximum force
required to detach CS-NAC-MNA and CS-NAC-NAC from porcine intestinal mucosa was 1.4- and 2.7-fold higher
than that required for chitosan, respectively. CS-NAC-MNA adhered up to 3 h, whereas CS-NAC-NAC adhered
even for 8 h on this mucosa. Accordingly, the Cys-Cys substructure could be identified as highly potent ligand for
the design of mucoadhesive polymers.

1. Introduction
Among mucoadhesive polymers, thiomers are by far those of highest
potential as they are able to form disulfide bonds with mucus glycoproteins (Leitner, Walker, & Bernkop-Schnürch, 2003). Their superior
mucoadhesive properties have been shown in numerous studies(Chen,
Lin, Wu, & Mi, 2018; Laffleur et al., 2017; Leichner, Jelkmann, &
Bernkop-Schnurch, 2019; Palazzo, Trapani, Ponchel, Trapani, &
Vauthier, 2017; Suchaoin et al., 2016). The shortcoming of a limited
stability in solution due to thiol oxidation at pH above 5 unless sealed

under inert conditions (Kast & Bernkop-Schnürch, 2001) led to the
development of S-protected thiomers being regarded as second generation. The formation of disulfide bonds between the thiomer and
mercaptopyridine analogues such as 2-mercaptonicotinic acid or 2mercaptonicotinamide provides on the one hand protection towards
oxidation and on the other hand raises even the reactivity of thiol
groups for thiol/disulfide exchange reactions. And in fact, S-protected
thiomers that are also referred as preactivated thiomers were shown to
exhibit higher mucoadhesive properties than thiomers with just free
thiols (Netsomboon et al., 2016; Perrone et al., 2018). Taking the
crucial role of interpenetration of the mucoadhesive polymer into the
mucus gel layer into account generating a huge interface for thiol/

⁎

disulfide exchange reactions and anchoring the thiomer in deeper
mucus regions that are more firmly bound to the mucosa, however,
highly reactive thiomers are likely disadvantageous. As preactivated
thiomers react already extensively with thiols on the surface of the
mucus gel layer, they are hindered to penetrate into deeper mucus regions. According to this working hypothesis, less reactive S-protected
thiomers might be even higher mucoadhesive than preactivated thiomers.
It was therefore the aim of this study to synthesize less reactive Sprotected thiomers and to compare their mucoadhesive properties with
those of a preactivated thiomer. As model polymer backbone chitosan
was chosen as it exhibits per se high mucoadhesive properties and its
thiolation (Makhlof, Werle, Tozuka, & Takeuchi, 2010; Miles, Ball, &
Matthew, 2016; Zambito & Di Colo, 2010; Zambito et al., 2009) and
preactivation are well-described in previous studies (Laffleur & Röttges,
2019; Moreno et al., 2018; Netsomboon, Suchaoin, Laffleur, Prüfert, &
Bernkop-Schnürch, 2017; Zambito, Felice, Fabiano, Di Stefano, & Di
Colo, 2013). Furthermore, the great potential of in particular chitosanN-acetylcysteine conjugates as superior mucoadhesives has already
been demonstrated in numerous clinical trials (Lorenz et al., 2018;
Messina & Dua, 2018; Schmidl et al., 2017). A Cys-Cys substructure was

chosen as less reactive disulfide ligand, as it is an endogenous

Corresponding author.
E-mail addresses: , (A. Bernkop-Schnürch).

/>Received 28 February 2020; Received in revised form 22 April 2020; Accepted 28 April 2020
Available online 23 May 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 242 (2020) 116395

K. Netsomboon, et al.

mixture was stirred at room temperature for 30 min. Subsequently,
activated ligand solution was slowly added to chitosan solution
(Fig. 1D). The reaction mixture was stirred at room temperature for 6 h
and pH was kept constant at 5.5. The mixture was dialyzed (Nadir®
membrane, MWCO: 10–20 kDa) to remove unbound compounds. The
purified CS-NAC-MNA solution was frozen and lyophilized (Gamma
1–16 LSC, Martin Christ Gefriertrocknungsanlagen GmbH, Germany)
for 3 days at -80 °C. CS-NAC-MNA was kept at room temperature until
further use.

substructure that can be regarded as safe. This novel low reactive Sprotected thiomer was compared with a corresponding highly reactive
thiomer in its mucoadhesive properties in terms of rheological analysis,
tensile studies and rotating cylinder studies.
2. Materials and methods
2.1. Materials
Low molecular weight chitosan (100−300 kDa) was purchased

from Acros Organics (Belgium). 6-Chloronicotinamide, dimethyl sulfoxide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDAC), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent), N-acetylcysteine (NAC), 6-chloronicotinamide, reduced glutathione, L-cysteine, methionine, taurine, thiourea and Tris HCl were purchased from
Sigma-Aldrich, Gumpoldskirchen, Austria. Hydrogen peroxide was obtained from Herba Chemosan Apotheker—AG, Vienna, Austria. All
other chemicals were of analytical grade and obtained from commercial
sources.

2.2.1.2. N-Acetyl cysteine disulfide (NAC-NAC). 2.638 g (16.17 mmol)
of N-acetyl cysteine was dissolved in 50 mL of deionized water. The pH
was adjusted to 7 using 2 M sodium hydroxide solution followed by the
addition of 1.75 mL of 50 % v/v hydrogen peroxide solution and it was
stirred for 1 h. Afterwards, pH was decreased to 4 with 1 M
hydrochloric acid. The solvent was evaporated and the product was
dried by lyophilization. Thereafter, 500 mg of this product was purified
by column chromatography on silica gel with 90 % dichloromethane
and 10 % of methanol as mobile phase.
2.2.1.3. Synthesis of CS-NAC-NAC. 1 g of NAC-NAC dimer was
dissolved in deionized water and EDAC was added to the dimer
solution in a final concentration of 150 mM and pH was adjusted to
5.5. The mixture was further incubated at room temperature under
stirring for 30 min. Chitosan (1 g) was hydrated under the same
conditions described in section 2.2.1.1. NAC dimer was slowly added to
chitosan solution and the pH was kept constant at 5.5. The reaction
mixture was stirred at room temperature at pH 5.5 for 6 h. Then, the
mixture was dialyzed against 1 mM hydrochloric acid. Thin layer
chromatography was conducted during dialysis process to monitor the
removal of unbound compounds. CS-NAC-NAC was frozen and
lyophilized for 3 days at -80 °C. CS-NAC-NAC was stored at room
temperature until use.

2.2. Methods

2.2.1. Synthesis of S-protected chitosan
In this study, two types of S-protected chitosans were synthesized by
using either 6-mecaptonicotinamide (6-MNA) or NAC as leaving group,
respectively. To ensure entire S-protection, preactivated ligands were
prepared and subsequently attached to the chitosan backbone.
2.2.1.1. Synthesis
of
CS-NAC-MNA. NAC-MNA
ligands
were
synthesized by a multi-step process before attaching to chitosan via
amide bond formation. Synthesis was modified from a previously
established method (Laffleur & Röttges, 2019; Lupo et al., 2017).
Firstly, 6-MNA monomer was synthesized by using 6chloronicotinamide as starting material (Fig. 1A). Briefly, 5 g of 6chloronicotinamide was suspended in 40 mL of ethanol and 2.92 g of
thiourea was suspended in 30 mL of ethanol, respectively. Then, the
thiourea suspension was slowly added to the 6-chloronicotinamide
suspension. The mixture was brought to reflux under nitrogen for 6 h.
At the end of the reaction, the suspension was allowed to cool down.
Ethanol was removed by rotary evaporator resulting in a yellow salt of
S-(5-carbamyl-2-pyridyl)thiouranium chloride, that was decomposed
by addition of 50 mL of 3 M NaOH. The mixture was kept under
continuous stirring at room temperature for 1 h. Then pH of the mixture
was adjusted to 4.9. Subsequently, the mixture was filtrated and
brought to dryness by lyophilization for obtaining the 6-MNA
monomer.
The oxidation step forming the dimer was initiated by dispersion of
6-MNA monomer in 100 mL of demineralized water and addition of
hydrogen peroxide as illustrated in Fig. 1B. Hydrogen peroxide (50 %
v/v, 3 mL) was dropwisely added to the suspension until the yellow
suspension turned off-white. The off-white suspension was continuously

stirred at pH 7 for 1 h. At the end of the reaction, the suspension was
filtrated and brought to dryness by lyophilization. Off-white powder of
the dimer namely 6,6′-dithionicotinamide was obtained. This dimer
was conjugated with NAC resulting in NAC-6-MNA ligand (Fig. 1C).
Briefly, 6,6′-dithionicotimide (250 mg) was dissolved in 8 mL of dimethyl sulfoxide. NAC (320 mg) was dissolved in 2 mL of dimethyl
sulfoxide. Then, the NAC solution was slowly added to the dimer solution. The resulting yellow solution was stirred at room temperature
for 24 h.
In the next step, chitosan (1 g) was dispersed in 400 mL of demineralized water. Hydrochloric acid (5 M) was added to chitosan dispersion to dissolve chitosan at pH 2. Thereafter, pH of chitosan solution
was slowly adjusted to 5.5 by addition of 5 M sodium hydroxide. EDAC
in a final concentration of 150 mM was slowly added to the NAC-6MNA solution to activate the carboxylic acid moiety of the ligand. The

2.2.2. Characterization of S-protected chitosan
2.2.2.1. NAC-NAC ligand characterization. 1H NMR spectra were
recorded by Varian Bruker NMR spectrometer (Bruker Advance 4 Neo
spectrometer 400 MHz) to confirm NAC-NAC ligand formation. DMSOd6 was used as solvent for recording 1H NMR spectra. The TMS signal
was used as internal standard. In addition, molecular mass of the NACNAC ligand was recorded on a Thermo Fisher Orbitrap Elite via direct
infusion and electrospray ionization. The conditions used for measuring
molecular mass of NAC-NAC ligand were as follow: ionization potential:
2000 V, ion injection: 2.0 eV, counter gas flow: 1.0 (L/min), AIF
temperature: 140 °C and ion source temperature: 80 °C. Methanol was
used as mobile phase. NAC-NAC ligand was dissolved in methanol in a
concentration of 500 ng/mL. Mass range of m/z 150 → 2000 negatively
electrospray ionization (ESI) mode was run to determine molecular
mass of NAC-NAC ligand. The sample was directly loaded using a
syringe pump with flow rate of 2 μL min−1 in order to obtain a clear
mass spectrum without any background noise.
2.2.2.2. Polymer characterization via FT-IR. To characterize the
modification of chitosan, IR spectra were recorded by Spectrum Two
FT-IR spectrometer (Perkin Elmer, Beaconsfield, United Kingdom).
Spectra were typically recorded from 4000 to 400 cm−1 using four

scans at 1-cm−1 resolution.
2.2.2.3. Determination of free thiol group contents. Thiol groups were
determined by a previously established method (Netsomboon et al.,
2017). First, each 1 mg of CS-NAC-MNA and of CS-NAC-NAC were
hydrated in 500 μL of 0.5 M phosphate buffer pH 8.0 and incubated at
room temperature for 30 min. Then 500 μL of Ellman’s reagent was
added. The mixture was further incubated at room temperature for 90
min protected from light. Afterwards, the mixture was centrifuged. The
absorbance of supernatant was measured at the wavelength of 405 nm
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Carbohydrate Polymers 242 (2020) 116395

K. Netsomboon, et al.

Fig. 1. Synthetic pathway for CS-NAC-MNA starting with (A) synthesis of 6-MNA from 6-chloronicotinamide, (B) formation of 6-MNA dimer, (C) coupling of 6-MNA
with NAC resulting in NAC-MNA ligand and (D) attachment of the ligand to chitosan backbone.

The temperature was kept at 37 ± 0.1 °C. The gap between two plates
was 0.5 mm.
Sample was prepared by hydrating thiomer with 50 mM Tris buffer
pH 7.4. Then, various concentrations of thiols in 50 mM Tris buffer pH
7.4 including L-cysteine and glutathione were added to the hydrated
thiomers (10 mg/mL) and mixed thoroughly. Methionine and taurine
served as negative controls. Viscosity of thiomers and endogenous
thiols was measured. Each experiment was performed in triplicate.
Parameters obtained from oscillating measurement were the phase
shift angle (δ ), the shear stress (τ ) and the shear deformation (γ ). The
elastic modulus (G′), the viscous modulus (G′′) and the dynamic viscosity (η *) were calculated using the equations given below (1)-(3).

(Tecan infinite® M200 spectrophotometer, Grưdig, Austria). NAC was
used for a calibration curve. All samples were measured in triplicate.
2.2.2.4. Determination of disulfide bonds. Quantification of thiol groups
and disulfide bonds was carried out as described previously
(Netsomboon et al., 2017). Each modified polymer (1 mg) was
hydrated in 500 μL of 50 mM Tris buffer pH 7.6 at room temperature
for 30 min. Then, 1 mL of freshly prepared 1 M sodium borohydride
solution was added to each sample. The mixtures were incubated at 37
°C for 60 min and 250 μL of 5 M hydrochloric acid was slowly added
followed by 1 mL of 1 M phosphate buffer pH 8.0. Subsequently,
Ellman’s reagent (100 μL) was added and the mixtures were further
incubated for 90 min at room temperature. NAC was also used in order
to establish a calibration curve. Absorbance of the mixture was
measured at 405 nm. All samples were tested in triplicate.
2.2.2.5. Determination of conjugated MNA. The amount of MNA
attached to CS-NAC was determined photometrically (Netsomboon
et al., 2017). In brief, 1 mg of CS-NAC-MNA was hydrated in 0.5 M
phosphate buffer pH 6.8 containing 65 mM reduced glutathione. The
mixture was incubated in the dark at room temperature for 1 h.
Absorbance was measured at 354 nm. MNA was used for a
calibration curve. The test was performed in triplicate.

G' = (

τmax
)cos δ
γmax

(1)

G '' = (

τmax
)sin δ
γmax

(2)

η*=

G′′
ω

(3)

where ω is the angular frequency which was kept constant at 6.283 rad/
s and for the frequency sweep vice versa, the ω was varied from 0.6283
to 62.83 rad/s. The phase shift angle (δ ) is defined by δ = tan−1 G′′/ G′
and indicates whether a material is solid-like component or liquid-like
component. For instance, a gel is defined in rheological terms where the
G′ and G′′ are frequency independent and tan δ is less than 1, in contrast
to a liquid-like material where tan δ is greater than 1. When G′ is equal
to G′′ at the crossover point, the polymer has as many elastic as viscous
components (Sakloetsakun, Hombach, & Bernkop-Schnürch, 2009).

2.2.3. In vitro rheological studies
Viscosity of polymers in the presence of endogenous thiols were
determined by a plate-plate rheometer (Haake Mars Rheometer,
379−0200, Therma Electron GmbH, Karlsruhe, Germany; Rotor: PP 35

Ti, D =35 mm). The shear stress was setup at a range of 0.5−500 Pa.
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K. Netsomboon, et al.

2.2.7. Statistical data analysis
IBM SPSS statistics 21 (SPSS Inc., Chicago, IL) was used for data
analysis. Independent t-test was used for two groups comparison. The
analysis of variance (ANOVA) was used to compare means (p = 0.05)
and Scheffe’s test was used as the post hoc multiple comparison test.
When violation of ANOVA assumption was observed, Welch’s ANOVA
was used to compare the means followed by Dunnett’s T3 for the post
hoc analysis.

2.2.4. In situ rheological studies
2.2.4.1. Mucus isolation. In this study, purified mucus was used for
experiments. Freshly excised porcine small intestine was used for
collection of mucus. The intestine was obtained from a local slaughter
house (Josef Mayr, Natters, Austria). Mucus was purified by an
established procedure (Wilcox, Van Rooij, Chater, Pereira de Sousa, &
Pearson, 2015). Firstly, intestinal segments containing no visible chyme
were selected. Mucus collection was carried out by gentle scratching of
the intestine with a spatula. The obtained mucus was subjoined with
sodium chloride. The mixture was gently stirred (< 100 rpm) at 4 °C for
1 h. Then, the mixture was centrifuged at 10,400 rpm at 4 °C for 2 h.
The supernatant and granular materials were discarded. Sodium
chloride (0.1 M) was added to the clean portion of the mucus and

stirred (< 100 rpm) for another 1 h at 4 °C and centrifuged at the same
condition described previously.

3. Results
3.1. Synthesis and characterization of S-protected chitosans
To obtain entirely S-protected chitosan, NAC-MNA and NAC-NAC
ligands were covalently attached to the chitosan backbone. NAC-MNA
ligand was synthesized by an already established method (Lupo et al.,
2017). The yields of 6-MNA monomer and dimer were 40.7 % and 64.9
%, respectively. In addition, NAC-NAC ligand was synthesized by new
method and characterized by 1H NMR and mass spectrometry. After
purification, the yield of NAC-NAC was 40.0 %. Fig. 3 shows the 1H
NMR spectrum and chemical shift on the NAC-NAC ligand. The symmetrical NAC-NAC showed a broad signal at 12.9 ppm (OH) and a
sharper one at 8.28 ppm (NH). The methylene protons give the signals δ
= 2.87–2.93 ppm and 3.09–3.17 ppm and the methine proton was at
4.43–4.50 ppm (CH). In addition, the chemical structure of the NACNAC ligand was confirmed by mass spectrometry showing a mass of 324
Da as depicted in Supplementary Fig. 1.
CS-NAC-MNA and CS-NAC-NAC were obtained by amide bond formation between chitosan and respective ligands as illustrated in Figs. 1
and Fig. 2. By lyophilization, an off-white fibrous structure was obtained in case of both thiomers. Yields of CS-NAC-MNA and CS-NACNAC were 85.4 % and 64.7 %, respectively. Unmodified chitosan being
subject of the same synthesis process but omitting EDAC served as
control. IR spectra of CS-NAC-MNA and CS-NAC-NAC are shown in
Fig. 4A and 4B, respectively.
As there was no peak in the frequency range of 2600−2540 cm−1,
remaining traces of free thiol groups could be excluded in case of both
thiomers. Furthermore, the SeS stretching peaks in the frequency range
of 560−570 cm−1 confirmed disulfide bonds of the ligands attached on
chitosan backbone. Intensity increase of peaks at ∼1630 and ∼1530
cm−1 which are the frequency of C]O and NHe bending, respectively,
showed that there is a raised amount of amide bonds on the modified
polymers. According to these results the covalent attachment of the two

ligands could be qualitatively confirmed.
The quantity of thiol groups immobilized on chitosan backbone is
shown in Table 1. There was no significant difference in the amount of
the two covalently attached S-protected NAC ligands allowing a direct
comparison in their properties.

2.2.4.2. Rheological studies. Viscosity of CS-NAC-MNA and CS-NACNAC was measured in the presence of various concentrations of
porcine intestinal mucus in 50 mM Tris buffer pH 7.4 ranging from
0.25 to 1.00% v/v. Measurements of thiomer viscosity in the presence
of mucus were performed in triplicate.
2.2.5. Swelling behavior
Swelling behavior of polymers was determined by a gravimetric
method (Peh & Wong, 1999). Polymer minitablets were fixed to a
needle and immersed in 0.1 M phosphate buffer pH 6.8 at 37 °C. Hydrated minitablets were removed from the buffer at predetermined time
points. After having removed excess of water, water uptake was determined gravimetrically. The measurement was done in triplicate.
Water uptake percentage was calculated regarding to the following Eq.
(4):

Water uptake (%) = (

Wt − W0
) × 100
W0

(4)

2.2.6. Mucoadhesion studies
Mucoadhesion studies were carried out in a similar manner to a
method having been described previously (Netsomboon et al., 2017).
CS, CS-NAC-MNA and CS-NAC-NAC were compressed with a compaction pressure of 10 kN into minitablets (30 mg, 5 mm diameter) with a

single punch eccentric press (Paul Weber, Germany).
2.2.6.1. Tensile studies. Tensile studies were performed with a texture
analyzer (TA.XTPLUS, Texture Technologies, Surrey, England). Freshly
excised porcine intestinal mucosa was cut into 3 × 3 cm pieces. The
serosal side of mucosa was put on the lower stand. Then, the upper
stand with the 2-cm diameter hole in the center was put over the lower
stand to fix the mucosa. Minitablets were attached to the flat surface of
the cylindrical probe by double-sided adhesive tape. For the
measurement, each minitablet was placed on the mucosa and
incubated for 15 min with applied force of 0.1 N. At the end of
incubation time, the probe was detached from the mucosa with the rate
of 0.1 mm/sec. The maximum detachment force (MDF) and the total
work of adhesion (TWA) were calculated. The experiments were
performed in quadruplicate (n = 4).

3.2. Rheological studies
3.2.1. Rheological behavior in the presence of L-cysteine and GSH
Results of rheological studies of CS-NAC-MNA and CS-NAC-NAC are
depicted in Fig. 5. In the presence of L-cysteine and GSH, viscosity of CSNAC-MNA and CS-NAC-NAC was significantly increased compared to
the corresponding thiomers without the addition of these thiols
(p < 0.05).
MNA release from CS-NAC-MNA in the presence of L-cysteine was
determined photometrically. In the presence of 0.25, 0.50 and 1.00 %
w/v of L-cysteine, 55 ± 3, 54 ± 9 and 77 ± 14 μmol MNA/g polymer
were released from the polymer. The increase in viscosity of CS-NACMNA is depicted in Fig. 5. The viscosity of both thiomers increased with
higher concentrations of free thiol groups whereas no effect on viscosity
could be observed in case of both controls - methionine and taurine.
Considering the type of ligands attached to thiomers, NAC-NAC led to a
more pronounced increase in viscosity compared to NAC-MNA as

2.2.6.2. Rotating cylinder studies. Serosal side of freshly excised porcine
intestinal mucosa was fixed on a rotating cylinder (apparatus 4cylinder, USP XXIII) by cyanoacrylate adhesive. Minitablets of CS, CSNAC-MNA and CS-NAC-NAC were applied on the mucosa, respectively.
The cylinder was fixed with the dissolution apparatus and incubated in
100 mM phosphate buffer pH 6.8 at 37 °C for 15 min. Then, the cylinder
was rotated with a speed of 200 rpm. The time of minitablet
detachment from the mucosa was observed and recorded.
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K. Netsomboon, et al.

Fig. 2. Synthetic pathway for CS-NAC-NAC. In the first step, NAC dimer was formed (A). Then, NAC dimer was attached to the chitosan backbone (B) via amide bond
formation.

In case of CS-NAC-NAC this increase in viscosity was even much more
pronounced. In the presence of 0.25, 0.50 and 1.00 % v/v mucus
viscosity of CS-NAC-NAC was 105-, 45- and 5-fold higher compared to
that of CS-NAC-MNA, respectively (p < 0.05) showing higher mucoadhesive properties of the less reactive S-protected thiomer.

shown in Table 2.
3.2.2. Rheological behavior in the presence of mucus
Rheological studies of mucoadhesive polymers with mucus provide
valuable data about the mucoadhesive properties of these polymers.
The higher the increase in viscosity of mucoadhesive polymer/mucus
mixtures are, the more they are obviously interacting with each other.
Mortazavi and Smart could even demonstrate a direct correlation between the increase in viscosity of mucoadhesive polymer/mucus mixtures and the mucoadhesive properties of the tested polymer (Mortazavi
& Smart, 1994). Increase in viscosity of CS-NAC-MNA and CS-NAC-NAC
in the presence of mucus is illustrated in Fig. 6. CS-NAC-MNA showed

2.63- and 33.3-fold higher viscosity compared to unmodified chitosan
in the presence of 0.50 and 1.00 % v/v mucus, respectively (p < 0.05).

3.3. Swelling behavior of thiomers
When polymers are applied in dry form to mucosal membranes,
their swelling behavior can have a substantial impact on their mucoadhesive properties. As depicted in Fig. 7, CS-NAC-MNA minitablets
swelled and started to disintegrate after 75 min due to overhydration
while water uptake of unmodified chitosan minitablets was comparatively low and no disintegration process at all could be seen. It was

Fig. 3. 1H NMR spectra of NAC-NAC ligand in deuterated DMSO.
5


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K. Netsomboon, et al.

Fig. 4. IR spectra recorded from 4000 to 400 cm−1 using 4 scans at 1-cm−1 resolution of CS-NAC-MNA (A) and CS-NAC-NAC (B) compared with unmodified chitosan
(grey).

reactive Cys-Cys ligand could be identified as comparatively more potent ligand to provide high mucoadhesive properties.

Table 1
Thiol group contents on S-protected chitosans and amount of conjugated MNA
on CS-NAC-MNA. Data are shown as means ± SE, n = 3.
Polymer

SH (μmol/g of
polymer)

S-S (μmol/g of
polymer)

MNA (μmol/g of
polymer)

CS-NAC-MNA
CS-NAC-NAC

Not detectable
Not detectable

566.7 ± 32.2
610.0 ± 91.3

549.5 ± 14.4
Not available

3.4.2. Adhesive behavior of polymers on the rotating cylinder
Rotating cylinder study was performed by using a USP dissolution
apparatus (Hauptstein, Bonengel, Rohrer, & Bernkop-Schnürch, 2014).
Results are highlighted in Fig. 9. During the observation period, minitablets of unmodified chitosan detached from porcine intestinal mucosa
after 2 h. CS-NAC-MNA minitablets adhered up to 3 h, whereas CSNAC-NAC minitablets attached for 8 h before falling off. The shorter
mucoadhesion time of CS-NAC-MNA is at least to some extent also a
result of its rapid swelling and overhydration behavior as shown in
Fig. 7. Residence time of CS-NAC-MNA and CS-NAC-NAC minitablets
was 1.6- and 3.9-fold prolonged compared to control (p < 0.05).

observed that unmodified chitosan minitablets were not completely
hydrated even until the end of experiment. CS-NAC-NAC showed constant water uptake and neither erosion nor disintegration was observed

during 120 min.

3.4. Adhesivity on intestinal mucosa

4. Discussion

3.4.1. Tensile strength of polymers
As shown in Fig. 8, MDF and TWA of CS-NAC-NAC were significantly higher than those of CS-NAC-MNA and control (p < 0.05),
respectively. MDF of CS-NAC-MNA and CS-NAC-NAC was 1.7- and 2.7fold higher compared with unmodified chitosan, respectively. TWA of
CS-NAC-MNA and CS-NAC-NAC were also 1.7- and 3.1-fold higher than
the control, respectively (p < 0.05). According to these results, the less

The type of mucus has a great impact on the performance of mucoadhesive polymers. Generally, mucus can be divided into two types:
loose and firm mucus. Loose mucus layer is composed of poorly interconnected mucins binding water to a high extent. This layer can be
easily removed by suction and shear. Firm mucus is typically composed
of highly crosslinked mucins adhering firmly to the epithelial surface
and being resistant to removal by suction and shear. In order to provide
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Carbohydrate Polymers 242 (2020) 116395

K. Netsomboon, et al.

Fig. 5. Viscosity of 10 mg/mL CS-NAC-MNA (black
bars) and CS-NAC NAC (grey bars) in the presence of Lcysteine and GSH (0.25-1.00 % w/v). Methionine and
taurine served as negative control. Data are shown as
mean ± SEM, n = 3; *p < 0.05, compared with respective polymer alone; ** p < 0.05, compared with
CS-NAC-MNA at the same test condition.

Table 2
Viscosity improvement ratio (viscosity of polymer with indicated endogenous
compound / viscosity of polymer without indicated endogenous compound) of
CS-NAC-MNA and CS-NAC-NAC in the presence of listed endogenous compounds.
Test substance

L-Cysteine

Glutathione

Methionine
Taurine

Improvement ratio

0.25
0.50
1.00
0.25
0.50
1.00
1.00
1.00

%
%
%
%
%
%

%
%

CS-NAC-MNA

CS-NAC-NAC

7.5
8.2
12.8
2.4
3.3
4.3
0.8
0.6

40.5
147.4
165.8
51.1
110.6
152.5
1.2
1.2

Fig. 7. Swelling behavior of unmodified chitosan (close circle), CS-NAC-MNA
(open circle) and CS-NAC-NAC (close triangle). Water uptake study was carried
out in 0.1 M phosphate buffer pH 6.8 at 37 °C. Arrow indicates disintegration of
minitablets. Data are shown as mean ± SEM (n = 3, *p < 0.05, compared with
control; ** p < 0.05, compared with CS-NAC-MNA).

strong mucoadhesion, mucoadhesive polymers have to deeply interpenetrate the loose mucus and preferably also the firm mucus getting in
this way anchored on a solid base. Utilizing highly reactive preactivated
thiomers is therefore likely not the best strategy to provide strong
mucoadhesion as such polymers form already on the surface of loose
mucus first disulfide bonds with mucins hindering these polymers to
penetrate into deeper mucus regions. Taking also the mucus turn over
into account, attachment of such systems on the mucosa will likely last
comparatively short. In contrast, less reactive S-protected thiomers will
penetrate much deeper into the mucus layer forming nevertheless sufficient new disulfide bonds with mucins. Because of a more intensive
interpenetration more stabilizing polymer chain entanglements can
take place and the interface for thiol/disulfide exchange reactions

Fig. 6. Viscosity of 10 mg/mL CS-NAC-MNA (black bars) and CS-NAC NAC
(grey bars) in the presence of mucus (%v/v) (mean ± SEM, n = 3; *p < 0.05,
compared with respective polymer alone; ** p < 0.05, compared with CS-NACMNA at the same test condition.
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K. Netsomboon, et al.

much less pronounced than that having been achieved with CS-NACNAC.
Another interesting aspect of this study is the observation that an
extensive crosslinking of both S-protected thiomers can be achieved due
to the addition of comparatively low amounts of endogenous thiols. In a
first step these thiols react with CS-NAC-MNA or CS-NAC-NAC partially
de-protecting thiol groups on these polymers that in a second step
crosslink via thiol/disulfide exchange reactions as outlined in Fig. 10.

The presence of L-cysteine and glutathione increased viscosity of
both S-protected chitosan significantly (p ≤ 0.05), whereas methionine
and taurine had no significant impact on viscosity. The more L-cysteine
and glutathione was added to these thiomers, the more pronounced was
the increase in viscosity. The increase in viscosity of S-protected thiomers in the presence of mucus is in agreement with these findings. It
was noticed that viscosity of both CS-NAC-MNA and CS-NAC-NAC was
to a higher extent increased in the presence of mucus compared to Lcysteine and glutathione. This observation can be explained by the huge
amount of thiol moieties of cysteine-rich subdomains of mucins crosslinking with numerous NAC-MNA and NAC-NAC ligands of thiomers,
whereas monovalent thiols can just trigger disulfide bond formation
within thiomers. The increase in viscosity was in case of CS-NAC-NAC
much higher than in case of CS-NAC-MNA. These results are in good
agreement with theoretical considerations. As MNA being released by
the reaction of L-cysteine or glutathione with CS-NAC-MNA can attack
further NAC-MNA substructures just to a very low extent, a polymer
crosslinking being additionally mediated by released MNA is of minor
relevance. In contrast, NAC being released from CS-NAC-NAC can
mediate further NAC/NAC-NAC exchange reactions strongly contributing to the formation of additional intra- and inter- polymer chain
disulfide bonds. This extensive crosslinking of even less reactive Sprotected thiomers in the presence of a low amount of free thiols is
highly beneficial for various applications. For instance in regenerative
medicine where among various other thiomers also thiolated chitosan
have already shown great potential(Bae, Jeong, Kook, Kim, & Koh,
2013; Zahir-Jouzdani et al., 2018), thiomers being stable during storage
due to S-protection can be injected at low viscosity crosslinking in situ
at the target site due to endogenous thiols. The addition of oxidizing
agents (Sakloetsakun et al., 2009) or other auxiliary agents such as
oxidized glutathione (Zarembinski et al., 2014) to initiate the crosslinking process in situ is not anymore necessary. In case of nasal sprays,
eye drops or vaginal gels third generation thiomers can be administered
at low viscosity strongly increasing their viscosity in the presence of
endogenous thiols and avoiding subsequently unintended rapid elimination via an outflow.
A further advantage of Cys-Cys ligands is that the protective group

being released in vivo by thiol/disulfide exchange reactions is an endogenous amino acid that can be regarded as safe. In contrast to mercaptonicotinamide, whose side effects have not been investigated in
detail yet, the safety profile of cysteine and NAC is well-established.

Fig. 8. (A) Maximum detachment force (MDF) and (B) total work of adhesion
(TWA) of chitosan, CS-NAC-MNA and CS-NAC-NAC. Data are shown as
means ± SEM (n = 4, *p < 0.05).

5. Conclusion
So far, thiolated chitosans were S-protected with mercaptopyridine
analogues resulting in highly reactive asymmetric disulfides. Such Sprotected thiolated chitosans react rapidly with thiols found on mucus
glycoproteins forming new disulfides. Because of this rapid reaction
with mucus glycoproteins, however, the mucoadhesive polymer cannot
penetrate in deeper mucus regions in order to get firmly anchored
there. Less reactive S-protected thiolated chitosans might consequently
be higher mucoadhesive than highly reactive ones. In this study, the
high reactive CS-NAC-MNA and the low reactive CS-NAC-NAC were
compared in their mucoadhesive properties. Results from rheology and
mucoadhesion studies indicated that CS-NAC-NAC possesses superior
mucoadhesive properties compared to CS-NAC-MNA. In addition, CSNAC-NAC showed comparatively much more pronounced gelling
properties in the presence of endogenous thiols than CS-NAC-MNA.

Fig. 9. Mucoadhesion time of minitablets containing 30 mg of unmodified
chitosan (control), CS-NAC-MNA and CS-NAC-NAC performed by rotating cylinder method. Data are shown as means ± SEM (n = 3 *p < 0.05).

between the thiomers and mucus glycoproteins is also much greater.
Taken all, less is obviously more. The validity of this working hypothesis could be confirmed in this study as the less reactive CS-NAC-NAC
showed much higher mucoadhesive properties than the highly reactive
CS-NAC-MNA. Menzel and co-workers designed an even more reactive
thiolated chitosan than CS-NAC-NAC showing improved mucoadhesive
properties (Menzel et al., 2016). This improvement, however, was

8


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K. Netsomboon, et al.

Fig. 10. Schematic presentation of mediated thiol/disulfide exchange reactions taking place in CS-NAC-NAC and CS-NAC-MNA.

According to these results, the less reactive Cys-Cys substructure could
be identified as highly potent ligand for the design of mucoadhesive and
in situ gelling chitosans.

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CRediT authorship contribution statement
Kesinee
Netsomboon:
Investigation,
Formal
analysis,
Visualization, Writing - original draft. Aamir Jalil: Investigation,
Visualization, Writing - original draft. Flavia Laffleur: Investigation,
Visualization, Writing - original draft. Andrea Hupfauf: Investigation.
Ronald Gust: Investigation, Validation. Andreas Bernkop-Schnürch:
Conceptualization, Methodology, Resources, Writing - review &,

Writing - review & editing, Supervision.
Acknowledgement
This publication has been written during a scholarship supported
stay within the Ernst Mach Grants scholarship, financed by the Austrian
Federal Ministry for Education, Science and Research (BMBWF) via
ASEAN-European Academic University Network (ASEA-UNINET) and
implemented/administered by the Austrian Agency for International
Cooperation in Education and Research (OeAD). The Austrian Research
Promotion Agency FFG (West Austrian BioNMR 858017) is also kindly
acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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