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

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.

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

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

Kesinee Netsomboona, Aamir Jalilb, Flavia La ffleurb, Andrea Hupfaufc, Ronald Gustc,

a Division of Pharmaceutical Sciences, Faculty of Pharmacy, Thammasat University (Rangsit Campus), Khlong Luang, Pathumthani 12120, Thailand

b Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innsbruck 6020, Austria

c 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 L E I N F O

Keywords:

Thiolated

Mucoadhesive

Mucosal drug delivery

Polymer

Thiomers

S-protected thiomers

Thiolated polymer

Chitosan

A B S T R A C T The potential of Cys-Cys ligands for the development of a novel type of protected thiomers was evaluated S-protected thiomers chitosan-N-acetylcysteine-mercaptonicotinamide (CS-NAC-MNA) and chitosan-N-acet-ylcysteine-N-acetylcysteine (CS-NAC-NAC) were synthesized and characterized Viscosity of polymers in pre-sence 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 550μmol/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

glyco-proteins (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

gen-eration The formation of disulfide bonds between the thiomer and

mercaptopyridine analogues such as mercaptonicotinic acid or

2-mercaptonicotinamide 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 morefirmly 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 re-gions According to this working hypothesis, less reactive S-protected thiomers might be even higher mucoadhesive than preactivated thio-mers

It was therefore the aim of this study to synthesize less reactive S-protected 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 chitosan-N-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

https://doi.org/10.1016/j.carbpol.2020.116395

Received 28 February 2020; Received in revised form 22 April 2020; Accepted 28 April 2020

⁎Corresponding author

E-mail addresses:andreas.bernkop@uibk.ac.at,a.bernkop@thiomatrix.co(A Bernkop-Schnürch)

Available online 23 May 2020

0144-8617/ © 2020 Elsevier Ltd All rights reserved

T

substructure that can be regarded as safe This novel low reactive

S-protected 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

sulf-oxide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

(EDAC), 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent),

N-acet-ylcysteine (NAC), 6-chloronicotinamide, reduced glutathione, L

-cy-steine, methionine, taurine, thiourea and Tris HCl were purchased from

Sigma-Aldrich, Gumpoldskirchen, Austria Hydrogen peroxide was

ob-tained from Herba Chemosan Apotheker—AG, Vienna, Austria All

other chemicals were of analytical grade and obtained from commercial

sources

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

6-chloronicotinamide as starting material (Fig 1A) Briefly, 5 g of

6-chloronicotinamide 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 inFig 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

di-methyl sulfoxide NAC (320 mg) was dissolved in 2 mL of didi-methyl

sulfoxide Then, the NAC solution was slowly added to the dimer

so-lution 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

demi-neralized water Hydrochloric acid (5 M) was added to chitosan

dis-persion 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-6-MNA solution to activate the carboxylic acid moiety of the ligand The

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

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 afinal 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 section2.2.1.1 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

2.2.2 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

DMSO-d6was used as solvent for recording1H NMR spectra The TMS signal was used as internal standard In addition, molecular mass of the NAC-NAC 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 withflow rate of 2 μL min−1in 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−1resolution

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 500μL of 0.5 M phosphate buffer pH 8.0 and incubated at room temperature for 30 min Then 500μL 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

K Netsomboon, et al. Carbohydrate Polymers 242 (2020) 116395

(Tecan infinite® M200 spectrophotometer, Grödig, 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 500μL 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 250μL 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

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

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 includingL-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

visc-osity (η *) were calculated using the equations given below (1)-(3).

=

(max)cos

max

'

(1)

=

( max)sin

max

''

(2)

=

′′

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−1G 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 andG′′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

toG′′at the crossover point, the polymer has as many elastic as viscous components (Sakloetsakun, Hombach, & Bernkop-Schnürch, 2009) 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

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

2.2.4.2 Rheological studies Viscosity of MNA and

CS-NAC-NAC 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

Hy-drated minitablets were removed from the buffer at predetermined time

points After having removed excess of water, water uptake was

de-termined gravimetrically The measurement was done in triplicate

Water uptake percentage was calculated regarding to the following Eq

(4):

W

Water uptake (%) ( t 0) 100

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

compac-tion 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 tofix 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)

2.2.6.2 Rotating cylinder studies Serosal side of freshly excised porcine

intestinal mucosa was fixed on a rotating cylinder (apparatus

4-cylinder, USP XXIII) by cyanoacrylate adhesive Minitablets of CS,

CS-NAC-MNA and CS-NAC-NAC were applied on the mucosa, respectively

The cylinder wasfixed 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

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

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 by1H NMR and mass spectrometry After purification, the yield of NAC-NAC was 40.0 %.Fig 3 shows the1H NMR spectrum and chemical shift on the NAC-NAC ligand The sym-metrical 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 signalsδ

= 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 NAC-NAC ligand was confirmed by mass spectrometry showing a mass of 324

Da as depicted in SupplementaryFig 1 CS-NAC-MNA and CS-NAC-NAC were obtained by amide bond for-mation between chitosan and respective ligands as illustrated in Figs.1 andFig 2 By lyophilization, an off-white fibrous structure was ob-tained in case of both thiomers Yields of MNA and CS-NAC-NAC 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−1confirmed disulfide bonds of the ligands attached on chitosan backbone Intensity increase of peaks at∼1630 and ∼1530

cm−1which 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 inTable 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

3.2 Rheological studies

3.2.1 Rheological behavior in the presence ofL-cysteine and GSH Results of rheological studies of CS-NAC-MNA and CS-NAC-NAC are depicted inFig 5 In the presence ofL-cysteine and GSH, viscosity of CS-NAC-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 ofL-cysteine was determined photometrically In the presence of 0.25, 0.50 and 1.00 % w/v ofL-cysteine, 55 ± 3, 54 ± 9 and 77 ± 14μmol MNA/g polymer were released from the polymer The increase in viscosity of CS-NAC-MNA is depicted inFig 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

K Netsomboon, et al. Carbohydrate Polymers 242 (2020) 116395

shown inTable 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

be-tween the increase in viscosity of mucoadhesive polymer/mucus

mix-tures 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 inFig 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)

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 mu-coadhesive properties of the less reactive S-protected thiomer

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 mu-coadhesive properties As depicted inFig 7, CS-NAC-MNA minitablets swelled and started to disintegrate after 75 min due to overhydration while water uptake of unmodified chitosan minitablets was compara-tively low and no disintegration process at all could be seen It was

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

Fig 3.1H NMR spectra of NAC-NAC ligand in deuterated DMSO

observed that unmodified chitosan minitablets were not completely

hydrated even until the end of experiment CS-NAC-NAC showed

con-stant water uptake and neither erosion nor disintegration was observed

during 120 min

3.4 Adhesivity on intestinal mucosa

3.4.1 Tensile strength of polymers

As shown in Fig 8, MDF and TWA of CS-NAC-NAC were

sig-nificantly 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

2.7-fold 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

reactive Cys-Cys ligand could be identified as comparatively more po-tent ligand to provide high mucoadhesive properties

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 inFig 9 During the observation period, mini-tablets of unmodified chitosan detached from porcine intestinal mucosa after 2 h NAC-MNA minitablets adhered up to 3 h, whereas CS-NAC-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)

4 Discussion

The type of mucus has a great impact on the performance of mu-coadhesive polymers Generally, mucus can be divided into two types: loose andfirm mucus Loose mucus layer is composed of poorly inter-connected 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 adheringfirmly to the epithelial surface and being resistant to removal by suction and shear In order to provide

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

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 Not detectable 566.7 ± 32.2 549.5 ± 14.4

CS-NAC-NAC Not detectable 610.0 ± 91.3 Not available

K Netsomboon, et al. Carbohydrate Polymers 242 (2020) 116395

strong mucoadhesion, mucoadhesive polymers have to deeply inter-penetrate the loose mucus and preferably also thefirm 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 mucusfirst 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 suf-ficient 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 5 Viscosity of 10 mg/mL CS-NAC-MNA (black bars) and CS-NAC NAC (grey bars) in the presence ofL -cysteine 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 re-spective 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

com-pounds

Test substance Improvement ratio

CS-NAC-MNA CS-NAC-NAC L-Cysteine 0.25 % 7.5 40.5

0.50 % 8.2 147.4 1.00 % 12.8 165.8 Glutathione 0.25 % 2.4 51.1

0.50 % 3.3 110.6 1.00 % 4.3 152.5 Methionine 1.00 % 0.8 1.2

Taurine 1.00 % 0.6 1.2

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-NAC-MNA at the same test condition

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)

between the thiomers and mucus glycoproteins is also much greater.

Taken all, less is obviously more The validity of this working

hypoth-esis 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

much less pronounced than that having been achieved with CS-NAC-NAC

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 inFig 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 moreL-cysteine and glutathione was added to these thiomers, the more pronounced was the increase in viscosity The increase in viscosity of S-protected thio-mers in the presence of mucus is in agreement with thesefindings 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 toL -cysteine and glutathione This observation can be explained by the huge amount of thiol moieties of cysteine-rich subdomains of mucins cross-linking 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 ofL-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 con-tributing to the formation of additional intra- and inter- polymer chain disulfide bonds This extensive crosslinking of even less reactive S-protected 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 cross-linking 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 elim-ination 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 en-dogenous amino acid that can be regarded as safe In contrast to mer-captonicotinamide, whose side effects have not been investigated in detail yet, the safety profile of cysteine and NAC is well-established

5 Conclusion

So far, thiolated chitosans were S-protected with mercaptopyridine analogues resulting in highly reactive asymmetric disulfides Such S-protected 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 NAC-MNA In addition, CS-NAC-NAC showed comparatively much more pronounced gelling properties in the presence of endogenous thiols than CS-NAC-MNA

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)

Fig 9 Mucoadhesion time of minitablets containing 30 mg of unmodified

chitosan (control), CS-NAC-MNA and CS-NAC-NAC performed by rotating

cy-linder method Data are shown as means ± SEM (n = 3 *p < 0.05)

K Netsomboon, et al. Carbohydrate Polymers 242 (2020) 116395

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

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

online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116395

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