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.
Carbohydrate Polymers 242 (2020) 116395 Contents lists available at ScienceDirect 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 h, whereas CS-NAC-NAC adhered even for h on this mucosa Accordingly, the Cys-Cys substructure could be identified as highly potent ligand for the design of mucoadhesive polymers 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 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: andreas.bernkop@uibk.ac.at, a.bernkop@thiomatrix.co (A Bernkop-Schnürch) https://doi.org/10.1016/j.carbpol.2020.116395 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 Subsequently, activated ligand solution was slowly added to chitosan solution (Fig 1D) The reaction mixture was stirred at room temperature for 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 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 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 using M sodium hydroxide solution followed by the addition of 1.75 mL of 50 % v/v hydrogen peroxide solution and it was stirred for h Afterwards, pH was decreased to with 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 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 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 h Then, the mixture was dialyzed against 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 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, 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 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 M NaOH The mixture was kept under continuous stirring at room temperature for 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, mL) was dropwisely added to the suspension until the yellow suspension turned off-white The off-white suspension was continuously stirred at pH for 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 mL of dimethyl sulfoxide NAC (320 mg) was dissolved in 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 Thereafter, pH of chitosan solution was slowly adjusted to 5.5 by addition of 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 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 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 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 Then 500 μL of Ellman’s reagent was added The mixture was further incubated at room temperature for 90 protected from light Afterwards, the mixture was centrifuged The absorbance of supernatant was measured at the wavelength of 405 nm Carbohydrate Polymers 242 (2020) 116395 K Netsomboon, et al Fig 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 Then, mL of freshly prepared M sodium borohydride solution was added to each sample The mixtures were incubated at 37 °C for 60 and 250 μL of M hydrochloric acid was slowly added followed by mL of M phosphate buffer pH 8.0 Subsequently, Ellman’s reagent (100 μL) was added and the mixtures were further incubated for 90 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, 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 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 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 Carbohydrate Polymers 242 (2020) 116395 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 °C for h Then, the mixture was centrifuged at 10,400 rpm at °C for 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 h at °C and centrifuged at the same condition described previously 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 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 CS-NAC-MNA and CS-NAC-NAC were obtained by amide bond formation between chitosan and respective ligands as illustrated in Figs and Fig 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 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, 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 × 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 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 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 ± and 77 ± 14 μmol MNA/g polymer were released from the polymer The increase in viscosity of CS-NACMNA is depicted in Fig 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 Then, the cylinder was rotated with a speed of 200 rpm The time of minitablet detachment from the mucosa was observed and recorded Carbohydrate Polymers 242 (2020) 116395 K Netsomboon, et al Fig 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 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 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 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 1H NMR spectra of NAC-NAC ligand in deuterated DMSO Carbohydrate Polymers 242 (2020) 116395 K Netsomboon, et al Fig IR spectra recorded from 4000 to 400 cm−1 using 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 Thiol group contents on S-protected chitosans and amount of conjugated MNA on CS-NAC-MNA Data are shown as means ± SE, n = 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 During the observation period, minitablets of unmodified chitosan detached from porcine intestinal mucosa after h CS-NAC-MNA minitablets adhered up to h, whereas CSNAC-NAC minitablets attached for 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 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 3.4 Adhesivity on intestinal mucosa 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 Carbohydrate Polymers 242 (2020) 116395 K Netsomboon, et al Fig 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 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 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 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 Carbohydrate Polymers 242 (2020) 116395 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 (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) 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 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 = *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 Carbohydrate Polymers 242 (2020) 116395 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 hydrophobic and hydrophilic drugs Carbohydrate Polymers, 193, 163–172 https:// doi.org/10.1016/j.carbpol.2018.03.080 Hauptstein, S., Bonengel, S., Rohrer, J., & Bernkop-Schnürch, A (2014) Preactivated thiolated poly(methacrylic acid-co-ethyl acrylate): synthesis and evaluation of mucoadhesive potential European Journal of Pharmaceutical Sciences, 63, 132–139 https://doi.org/10.1016/j.ejps.2014.07.002 Kast, C E., & Bernkop-Schnürch, A (2001) Thiolated polymers–Thiomers: Development and in vitro evaluation of chitosan-thioglycolic acid conjugates Biomaterials, 22(17), 2345–2352 Laffleur, F., & Röttges, S (2019) Mucoadhesive approach for buccal application: Preactivated chitosan European Polymer Journal, 113, 60–66 https://doi.org/10 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varied etiology International Ophthalmology https://doi.org/10.1007/s10792-018-0843-0 Miles, K B., Ball, R L., & Matthew, H W (2016) Chitosan films with improved tensile strength and toughness from N-acetyl-cysteine mediated disulfide bonds Carbohydrate Polymers, 139, 1–9 https://doi.org/10.1016/j.carbpol.2015.11.052 Moreno, J A S., Mendes, A C., Stephansen, K., Engwer, C., Goycoolea, F M., Boisen, A., Chronakis, I S (2018) Development of electrosprayed mucoadhesive chitosan microparticles Carbohydrate Polymers, 190, 240–247 https://doi.org/10.1016/j 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 References Bae, I H., Jeong, B C., Kook, M S., Kim, S H., & Koh, J T (2013) Evaluation of a thiolated chitosan scaffold for local delivery of BMP-2 for osteogenic differentiation and ectopic bone formation BioMed Research International, 2013, 878930 https:// doi.org/10.1155/2013/878930 Chen, C H., Lin, Y S., 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synthesized by a multi-step process before attaching to chitosan... mucosa was cut into × 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... respectively Then, the thiourea suspension was slowly added to the 6-chloronicotinamide suspension The mixture was brought to reflux under nitrogen for h At the end of the reaction, the suspension