S-protected thiolated hyaluronic acid: In-situ crosslinking hydrogels for 3D cell culture scaffold

The purpose of this study was to synthesize S-protected thiolated hyaluronic acid (HA) and to evaluate its potential for 3D cell culture scaffold. S-protected thiolated HA was synthesized by the covalent attachment of Nacetyl-S-((3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)thio)cysteine hydrazide ligand to the HA.

Carbohydrate Polymers 237 (2020) 116092 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol S-protected thiolated hyaluronic acid: In-situ crosslinking hydrogels for 3D cell culture scaffold T Mulazim Hussain Asima,b, Stefanie Silberhumera, Iram Shahzadia, Aamir Jalila, Barbara Matuszczakc, Andreas Bernkop-Schnürcha,* a Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria b Department of Pharmaceutics, Faculty of Pharmacy, University of Sargodha, 40100 Sargodha, Pakistan 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 LE I N FO A B S T R A C T Keywords: Thiolated Disulfide-crosslinked hydrogels S-protected thiolated hyaluronic acid Hyaluronic acid Cell proliferation Tissue engineering 3D scaffold Thiol-disulfide exchange Rheological properties The purpose of this study was to synthesize S-protected thiolated hyaluronic acid (HA) and to evaluate its potential for 3D cell culture scaffold S-protected thiolated HA was synthesized by the covalent attachment of Nacetyl-S-((3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)thio)cysteine hydrazide ligand to the HA Hydrogels were characterized for texture, swelling behavior and rheological properties Furthermore, the potential of Sprotected thiolated HA hydrogels as a scaffold for tissue engineering was evaluated by cell proliferation studies with Caco-2 and NIH 3T3 cells It showed enhanced cohesion upon addition of N-acetyl cysteine (NAC) Dynamic viscosity of S-protected thiolated HA hydrogel was increased up to 19.5-fold by addition of NAC and 10.1-fold after mixing with mucus Furthermore, Caco-2 and NIH 3T3 cells encapsulated into hydrogels proliferated invitro As this novel S-protected thiolated HA is stable towards oxidation and forms highly cohesive gels when getting into contact with endogenous thiols due to disulfide-crosslinking, it is a promising tool for 3D cell culture scaffold Introduction Hyaluronic acid (HA), a non-sulfated, glycosaminoglycan (GAG) is found in the extracellular matrix (ECM) of many soft connective tissues (Prestwich, 2011; Shu, Liu, Luo, Roberts, & Prestwich, 2002) HA is ubiquitous in the body with many beneficial properties such as hydrophilicity and viscoelasticity (He, Zhao, Yin, Tang, & Yin, 2009) Due to these characteristics, HA has become an important building block for new biomaterials with utility in tissue engineering and regenerative medicine (Bian et al., 2016; Burdick & Prestwich, 2011) As only low viscous HA hydrogels can be injected into the target tissue providing insufficient stiffness to control scaffold architecture and being rapidly diluted in-situ, however, its application in regenerative medicine is still limited (Xu, Jha, Harrington, Farach-Carson, & Jia, 2012) Chemically modified HAs addressing this material design challenge are therefore in focus of many research groups and are used in hydrogels with improved mechanical properties In particular, hydrogels that are able to increase their viscosity after injection are of interest for 3D cell culture scaffold A lot of methods such as Michael-type addition reaction (Jin et al., 2010), Schiff-base reaction (Li et al., 2014), photo polymerization ⁎ (Gramlich, Kim, & Burdick, 2013; Lee & Park, 2009), thiol-ene reaction (Yu et al., 2014) and oxidizing reaction of tyramine (Burdick & Prestwich, 2011) are employed to achieve that goal Although these methods show potential, there are still a lot of shortcomings such as poor gelation efficiency, uncontrollable gelation processes (Shoham et al., 2013) and safety issues (Lin & Stern, 2001) More recently, disulfide-cross-linked HA hydrogels have been introduced in regenerative medicine as they are easy to synthesize with convenient gelation properties and minor safety concerns due to biodegradability (Lee et al., 2010) Thiolated HA hydrogels increase their viscosity by a crosslinking of free –SH groups mediated by oxygen or catalyzed by strong oxidants (Khutoryanskiy, 2011; Shu et al., 2002) These auto-oxidation reactions, however, are hard to control Furthermore, these thiol oxidation reactions are often taking place too slowly in the target tissue, as oxidizing conditions are there insufficient to cause a rapid crosslinking These shortcomings limit the use of such hydrogels, especially when used for in-situ gelation and encapsulation of cells To overcome above-mentioned limitations, a new type of thiolated HA, S-protected thiolated HA was synthesized using SPDP-NAC Corresponding author E-mail address: andreas.bernkop@uibk.ac.at (A Bernkop-Schnürch) https://doi.org/10.1016/j.carbpol.2020.116092 Received 22 January 2020; Received in revised form 27 February 2020; Accepted 28 February 2020 Available online 05 March 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al Fig -A Synthesis of S-protected thiolated hyaluronic acid: First N-acetyl cysteine (NAC) was linked to succinimidyl 3-(2-pyridyldithio) propionate (SPDP) via disulfide bond formation to generate SPDP-NAC ligand that was conjugated with hydrazine to hyaluronic acid (HA) in order to obtain S-protected thiolated HA under inert conditions B Schematic representation of mediated thiol/ disulfide exchange reactions taking place in Sprotected thiolated HA hydrogel upon addition of N-acetyl cysteine (NAC) or endogenous thiol (Endog-SH) Austria hydrazides attached to the HA backbone as outlined in Fig 1-A In contrast to already established thiolated HAs, the crosslinking of this novel polymer is not triggered by an oxidation process but by endogenous thiols as illustrated in Fig 1-B Subsequently, this newly developed S-protected thiolated HA was formulated to a hydrogel and tested for texture, swelling ratio, cytotoxicity and viscoelastic behavior Furthermore, the potential of S-protected thiolated HA hydrogel for 3D cell culture scaffold was evaluated using different cell lines 2.2 Methods 2.2.1 Synthesis of SPDP-NAC hydrazide ligand First, 50 mg of SPDP was dissolved in mL of anhydrous THF and 30 mg of NAC dissolved in mL of anhydrous THF was added drop-wise under constant stirring The reaction was carried out under nitrogen to avoid oxidation of NAC Within 10 reaction, the mixture turned yellow as the pyridine-2-thione (PDT) group of SPDP was replaced by NAC The concentration of the dye was determined by measuring absorbance at 343 nm The reaction mixture was further stirred for 18 h under inert conditions at room temperature in dark (Sharma et al., 2018) The reaction product was analyzed by thin layer chromatography (TLC) to confirm the entire derivatization of SPDP The solvent was removed by evaporation and the crude target compound was dissolved in anhydrous DCM PDT and other impurities were removed by column chromatography using silica gel with a mobile phase of DCM, ethyl acetate and methanol in a ratio of 7:2:1 The purified SPDP-NAC ligand was further reacted with hydrazine (NH2–NH2) to introduce a primary amino group on the ligand for amide bond formation with hyaluronic acid Briefly, 40 mg of SPDP-NAC ligand was dissolved in mL of THF and 0.2 mL of M hydrazine solution in THF was added drop-wise After 30 of stirring at °C, the solvent was evaporated and SPDP-NAC hydrazide was obtained as a white powder that was stored at −20 °C until further use Materials and methods 2.1 Materials Hyaluronic acid sodium salt (low molecular mass 10−50 kDa), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-acetyl- L-cysteine (NAC), tetrahydrofuran anhydrous (THF), ethyl acetate, dichloromethane (DCM), methanol, hydrazine (NH2–NH2), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC), Nhydroxysuccinimide (NHS), N,N-dimethylformamide anhydrous (DMF), sodium chloride (NaCl), L-cysteine hydrochloride hydrate, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, Ellman’s reagent), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), tris (hydroxymethyl)aminomethane hydrochloride (Tris HCl), sodium borohydride (NaBH4), potassium phosphate dibasic (K2HPO4), hydrochloric acid (HCl), silica gel (high purity grade), dialysis tubing MWCO (molecular weight cut-off) 3.5 kDa, resazurin (7-hydroxy-3H-phenoxazin-3-one sodium salt), minimum essential medium eagle (MEM) and Triton™ X-100 were all purchased from Merck, Austria Human colorectal carcinoma cells (Caco-2) were acquired by the European Collection of Cell Cultures (Salisbury, United Kingdom) and embryonic fibroblast cells (NIH 3T3) were donated by the Universitätsklinik für Dermatologie, Venerologie und Allergologie, Innsbruck, Austria The cell culture medium was made of MEM powder 9.66 g/L (modified with Earle’s salts, nineteen amino acids and the non-essential amino acids L-asp, L-asn, L-glu, L-ser, L-ala, L-gly, and L-pro), 2.2 g/L sodium bicarbonate, phenol red, mM L-glutamine, 10 % fetal bovine serum (FBS) and % penicillin-streptomycin solution 100 mM phosphate-buffered saline (PBS) and FBS were purchased from Invitrogen, 2.2.2 Synthesis of S-protected thiolated hyaluronic acid S-protected thiolated HA was synthesized by the covalent attachment of the ligand described above to the carboxyl groups of hyaluronic acid according to a previously described method with minor modifications (de Sousa, Suchaoin, Zupančič, Leichner, & BernkopSchnürch, 2016) In detail, 50 mg of HA was dissolved in 10 mL of demineralized water under permanent shaking The pH of the gel was adjusted at 4.5 with M HCl To activate the carboxyl groups of hyaluronic acid, 233 mg of EDAC and 46 mg of NHS were added and pH was re-adjusted to 4.5 The reaction mixture was stirred for h and then neutralized with M NaOH 50 mg of SPDP-NAC hydrazide ligand Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al and removed immediately with the same velocity The study was conducted at the acquisition rate of 500 points/s with a relaxation period of 30 s between compression cycles From force-distance plots, results were calculated employing Texture Exponent software (Version 6, Stable Micro Systems Ltd) (Tai, Bianchini, & Jachowicz, 2014) Stickiness and work of shear of S-protected thiolated HA hydrogel was measured using the same apparatus equipped with a TTC Spreadability rig (HDP/SR, Stable Micro Systems Ltd) at room temperature The female sample holder was filled with S-protected thiolated HA hydrogel samples and the male probe was brought downwards at a speed of mm/s with an angle of 45° up to a distance of mm above the bottom and then raised upwards with speed of 10 mm/s Unmodified HA served as a control The stickiness was determined as the maximum negative force and work of shear as the positive area under the force-distance curve (Tai et al., 2014) dissolved in 10 mL of anhydrous DCM was drop-wise added to the HAsolution under stirring After 24 h of stirring at room temperature in the dark under nitrogen, the mixture was evaporated by using Hei-VAP Value Digital Rotary Evaporator (Heidolph Instruments GmbH & CO Schwabach, Germany In last, the mixture was transferred into dialysis tubes (MWCO: 3.5 kDa) and dialyzed for 24 h against % NaCl (m/v) and then for further 48 h against demineralized water Finally, the dialyzed solution was freeze-dried to obtain pure S-protected thiolated HA 2.3 Characterization of S-protected thiolated HA by FT-IR and 1H NMR IR spectra of S-protected thiolated HA were recorded using Bruker ALPHA FT-IR apparatus (Billerica, MA, USA) FT-IR spectra of test compounds were collected by placing these compounds on the tip of platinum ART (attenuated total reflection) module Results were obtained by 32 scans at a scanning speed of cm−1 between 4000 cm−1 and 500 cm−1 FT-IR spectra were presented as the average of these 32 scans H NMR spectra were recorded using a Bruker-400 spectrometer (1H :400 MHz) at 30 °C in mm tubes containing the compounds dissolved in DMSO-d6 As internal standard served the center of the DMSO-d6 multiplet that was correlated to TMS (tetramethylsilane) with δ 2.49 ppm (1H) 2.7 Swelling ratio of S-protected thiolated hyaluronic acid hydrogels To study the swelling capabilities of the S-protected thiolated HA hydrogels, equilibrium swell experiments were performed (Ding, He, Zhou, Tang, & Yin, 2012) The S-protected thiolated HA hydrogel (1 % m/v) was washed with demineralized water, freeze-dried and weighed (Wd) Then 25 mg of freeze-dried hydrogel immersed in 500 μL of 10 mM PBS was mixed with 0.1 and % (m/v) NAC, incubated at 37 °C and shaken at 300 rpm After defined time intervals the medium was carefully removed and swollen hydrogel was weighed again (Ws) Then, 500 μL of buffer was added and the hydrogel was incubated again This process was repeated after 5, 10, 20, 30, 60, and 120 The swelling ratio was calculated using the following equation: 2.4 Quantification of the degree of modification The amount of free thiol groups immobilized on the backbone of Sprotected thiolated HA was determined photometrically with Ellman’s reagent as already described by our research group (Asim et al., 2019) The amount of disulfide substructures on the polymer was quantified via Ellman’s reagent as described above but after reduction with NaBH4 as described before (Asim et al., 2018) Furthermore, to confirm the lack of unbound SPDP-NAC hydrazide ligand, free primary amines were determined using TNBS assay as previously described (Suchaoin et al., 2016) All experiments were performed in triplicate Swelling ratio= (Ws − Wd ) Wd 2.8 Safety screening To ensure that the S-protected thiolated HA is non-cytotoxic, a resazurin assay was performed (Shahzadi et al., 2019) Cell viability was evaluated on human colorectal carcinoma cells (Caco-2) Cells were cultivated in a 24-well plate at a density of 2.5 × 104 cells/well for 10 days in 500 μL of MEM with Earle's salts supplemented with 10 % (v/v) fetal bovine serum (FBS), 2.0 mM L-glutamine and % penicillinstreptomycin solution (100 units/0.1 mg/L) at 37 °C in an environment of 5% CO2 and 95 % humidity Every second day, the growing medium was changed When the cells were approximately 80 % confluent, the red MEM was removed and the cells were washed twice with pre-warmed PBS at pH 7.4 Thereafter, 500 μL of unmodified and S-protected thiolated HA (0.5 % m/v) in white MEM without phenol red, 500 μL of Triton™ X-100 (4 % v/v) as negative control and 500 μL of white MEM without phenol red as positive control were added to the cell culture in triplicate The treated cells were incubated at 37 °C for further and 24 h at an atmosphere of % CO2 and 95 % relative humidity (Heracell™ 150i CO2 Incubator, Thermo Fisher Scientific Inc USA) Afterwards, the test solutions were removed and the cells were washed again with prewarmed PBS pH 7.4 and finally 500 μL of a 2.2 μM resazurin solution was added to each well After h of incubation at 37 °C, 100 μL of supernatant was transferred to a black 96-well plate and the fluorescence intensity was measured using Spark® multifunctional microplate reader (Tecan Austria, GmbH) at a wavelength of 540 nm with background subtraction at 590 nm Cell viability was calculated following the under-mentioned equation: 2.5 Molecular weight determination of S-protected hyaluronic acid by gel permeation chromatography The molecular weight (MW) of S-protected thiolated HA were calculated by GPC using Merck Hitachi LaChrom Elite® HPLC-System equipped with Merck Hitachi L-2130 Pump, Merck Hitachi L-2200 Autosampler, Merck Hitachi L-2450 Diode Array Detector (DAD) and VWR Hitachi Refractive Index (RI) detector by following previous established methods with minor modification (Yang et al., 2012; Yang, Guo, Zang, & Liu, 2015) A chromatographic analytical column PSS SUPREMA 3000 (8 × 300, 10 μ) (PSS-Polymer Standards Service-USA Inc.) with an injection volume of 20 μL was used The mobile phase consisted of phosphate buffer saline (PBS) pH 7.4 filtered through 0.2 μm Millipore filter was used with flow rate of 0.5 mL/min The detection wavelength was 220 nm and the column was maintained at 35 °C For calibration curve, HAs of different molecular weights (10–50 KDa, 1,500–1,800 KDa and 2,000–2,400 KDa) were used 2.6 Surface properties and spreadability of S-protected thiolated hyaluronic acid hydrogels The texture properties of hydrogels were analyzed with a TA.XTplus texture analyser with a kg load cell (Stable Micro Systems Ltd, Surrey, UK) To evaluate the effect of NAC on hydrogel cohesion and stickiness increasing concentrations of NAC (0–1 % m/v) in a vial were added to Sprotected thiolated and unmodified HA (1 % m/v) for half an hour at 25 °C (Rupenthal, Green, & Alany, 2011) A 10 mm cylindrical probe (SMSP/10, Stable Micro Systems Ltd) was pushed down into hydrogel samples at room temperature to a depth of mm at a rate of mm/s Cell Viability [%] = Experimental value− Negative control × 100 Positive control− Negative control Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al 2.9 Rheological studies of S-protected thiolated hyaluronic acid hydrogels Results and discussion S-protected thiolated HA (1.5 % m/v) was dissolved in sterile demineralized water Then, 0.1, 0.3, 0.5, 0.8 and % (m/v) solutions of NAC dissolved in 0.01 M PBS pH 7.4 were added to the above solutions in 1:2 ratio The viscosity of all samples was measured after incubation at 37 °C for h The same procedure was followed for unmodified HA serving as a control Moreover, the gelation time of S-protected thiolated HA with increasing concentrations of NAC was noted Rheological studies such as dynamic viscosity (η), apparent elastic modulus (G′) and viscous modulus (G″) were measured on a thermostatically controlled plate-plate rheometer (Thermo Scientific™ HAAKE™ MARS™ rheometer, Thermo Fisher Scientific, Karlsruhe, Germany) Besides, the phase angle (δ), calculated by δ = tan−1 G″/G′, was used for differentiation between gel and solution, while tan δ < is characterizing as gel and tan δ > as a solution The linear viscoelastic region of all samples was determined through initial oscillatory strain sweep measurements at a frequency of Hz at 37 °C with a shear stress of 0.5 Pa Furthermore, the dynamic viscosity of S-protected thiolated HA hydrogel in the presence of freshly collected porcine mucus was evaluated The mucus was collected and purified as previously described (Asim, Nazir, Jalil, Matuszczak, & Bernkop-Schnürch, 2020) Samples of unmodified and S-protected thiolated HA (1 % m/v) in 200 mM phosphate buffer pH 7.4 were mixed with porcine intestinal mucus in 1:2 ratio Mucus mixed with phosphate buffer in the same ratio served as control Samples were incubated for 1, and h at 37 °C and dynamic viscosity (η), apparent elastic modulus (G′) and viscous modulus (G″) of hydrogels were determined as a function of frequency using HAAKE RheoWin software 3.1 Synthesis and characterization of S-protected thiolated hyaluronic acid HA served as a polymeric backbone for the immobilization of thiol moieties via amide bond formation For the synthesis of S-protected thiolated HA, the SPDP-NAC ligand was synthesized by a thiol-disulfide exchange reaction Then, it was reacted with hydrazine to introduce an eNH2 group for amide bond formation with hyaluronic acid to generate SPDP-NAC-azide with 77.8 % yield A carbodiimide was used for activation ofeCOOH groups of HA under acidic conditions for protonation of the carbodiimide nitrogens (Shu et al., 2002) The activated eCOOH groups of HA reacted with SPDP-NAC hydrazide ligand via amide bond formation to gain S-protected thiolated HA with 81.4 % yield as displayed in Fig 1-A The successful immobilization of the ligand was confirmed via FT-IR spectroscopy The FT-IR spectrum of S-protected thiolated HA are given in Supplementary Fig S1 The IR spectrum of the S-protected thiolated HA significantly differed from that of the unmodified HA In detail, the characteristic peak of eNH group at 1556 cm−1 confirmed amide bond formation (Du, Fu, Shi, & Yin, 2015), whereas peak located at 3260 cm−1 is associated with stretching vibration of eOH groups Moreover, the SeS stretching peaks in the frequency range of 560−610 cm−1 confirmed disulfide bonds of the ligand attached to the hyaluronic acid backbone Furthermore, the chemical structure of Sprotected thiolated HA was confirmed via 1H NMR spectral analysis The 1H NMR spectrum of S-protected thiolated HA showed signals at 1.9 ppm that are assigned to eCH3 protons of NAC and amide protons at 9.3 ppm as shown in Supplementary Fig S2 Moreover, the eCH3 protons of the N-acetyl group of HA merged with eCH3 protons of NAC resulting in just one peak for both sub-structures (Li, Yu, Jin, & Yin, 2012; Shu et al., 2002) For molecular weight determination of modified HA, the retention time of unmodified HA was 35 with MW of 10–50 KDa that was shorten with S-protected thiolated HA to 31 indicating increase in MW of modified HA The MW of modified HA was calculated to 15–69 KDa S-protected thiolated HA showed a negligible amount of free eSH groups and eNH2 groups as confirmed by Ellman’s test and TNBS test, respectively These results confirm the purification of the product from unbound ligands and the successful protection of eSH groups In total, 320.55 μmol/g disulfide bonds were detected in the product that corresponds to 7.4 % of the total moles of carboxylic acid groups 2.10 In-situ cell encapsulation S-protected thiolated HA was sterilized under UV radiation for 30 under N2 protection and hydrated in a final concentration of 1.5 % (m/v) in mL of sterile red MEM (3 % m/v) at pH 7.4 To this Sprotected thiolated HA hydrogel, 100 μL of Caco-2 and NIH 3T3 cell suspensions were added to obtain a final concentration of × 106 cells/ mL, respectively Due to the addition of 100 μL of NAC solution (1 % m/ v) viscosity of the S-protected thiolated HA hydrogels was increased via thiol-disulfide exchange reactions encapsulating the dispersed cells The hydrogels were then immersed into 500 μL red MEM with Earle's salts supplemented with 10 % (v/v) fetal bovine serum (FBS), 2.0 mM Lglutamine and % penicillin-streptomycin solution (100 units/0.1 mg/ L) in an environment of % CO2 and 95 % humidity (Heracell™ 150i CO2 Incubator, Thermo Fisher Scientific Inc USA) at 37 °C S-protected thiolated HA hydrogels containing Caco-2 and NIH 3T3 cells were cultured at 37 °C and % CO2 and observed after 3, 7, 12 and 15 days using Motic AE31 inverted microscope (Motic Deutschland GmbH, Wetzlar, Germany) equipped with the ProgRes® series camera (JENOPTIK, Goeschwitzer, Germany) Pictures were taken by using ProgRes® CapturePro 2.7 software 3.2 Texture analysis and spreadability of S-protected thiolated HA hydrogels For the effective application of hydrogels suitable texture properties are essential (Kong, Kim, & Park, 2016) as cells can adapt to substrate mechanics by adjusting their proliferation and migration (Ghosh et al., 2007) Moreover, stiffness of scaffolds plays a key role as it does not only modulate biological processes but has even an impact on the fate of cells (Shi et al., 2012) Surface properties of S-protected thiolated HA hydrogels were investigated by exposure to increasing concentrations of NAC serving as a representative model for endogenous thiols at the target tissue The resulting work of cohesion is shown in Fig S-protected thiolated HA hydrogel remained cohesive over a broad range of added NAC concentrations These textural properties revealed also a high resistance to shear for S-protected thiolated HA hydrogels, which was in good agreement with their high stickiness The stiffness of hydrogels can control scaffold architecture and can facilitate cell growth and survival within the three dimensional network Changes in rheological behavior by altering the storage modulus lead to a change in the morphology of cells during proliferation Therefore, the mechanical properties of hydrogels could potentially be adjusted to meet the required stiffness to aid repairment of injured tissues (da Cunha et al., 2014) High cohesiveness of hydrogels corresponds enhanced 2.11 Statistical analyses Statistical analyses of all data were performed by using the student's t-test A confidence interval of p < 0.05 was used for the analysis of two groups One-way ANOVA was applied to compare different groups difference with 95 % CI (confident interval) (GraphPad Prism®, GraphPad Software, Inc.) All results were expressed as the mean of at least three experiments ± SD Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al Fig The swelling ratio of S-protected thiolated HA with NAC (0.1 % m/v) (black bar) in comparison to S-protected thiolated HA with NAC (1 % m/v) (white bar) in 0.01 M PBS pH 7.4 at 37 °C Indicated values represent an average of at least three experiments ± SD (***P < 0.001) shown poly(propylene fumarate) crosslinked hydrogels and Arg-GlyAsp (RGD)-modified PEG hydrogels (Burdick & Anseth, 2002; He, Yaszemski, Yasko, Engel, & Mikos, 2000) Furthermore, these previously synthesized hydrogels have free –SH groups that form disulfide bonds upon exposure to air This crosslinking process, however, is difficult to control and proceeding too slowly so that a required fast increase in viscosity cannot be achieved (Shu et al., 2002) In contrast, the crosslinking process of S-protected thiolated HA hydrogels can be controlled to a higher extent 3.4 Cytotoxic investigation Fig Analysis of gel texture and spreadabiliy [A] Work of cohesion determined as the negative area under the force/distance curve of mixtures of Sprotected thiolated HA and increasing concentrations of N-acetyl cysteine (NAC) at room temperature All values are means of at least three experiments ± SD [B] Work of shear (light grey bars) determined as positive area under the force/distance curve and stickiness (dark grey bars) determined as maximum negative force of S-protected thiolated HA hydrogels All values are means of at least three experiments ± SD Cytotoxicity of S-protected thiolated HA was quantified by resazurin assay Resazurin is a redox indicator that provides information about the viability of cells Cytotoxic compounds reduce the redox potential of cells, whereby no reduction takes place with non-toxic compounds (Borra, Lotufo, Gagioti, Barros, & Andrade, 2009) Caco-2 cells were treated with unmodified HA and S-protected thiolated HA in a concentration of 0.5 % (m/v) After and 24 h of incubation, cells showed viability over 80 % on Caco-2 cells as illustrated in Fig interactions in the polymer network and an increased area for possible interactions with cells for improved proliferation 3.3 Swelling behavior and stability of S-protected thiolated hyaluronic acid hydrogels The ability of hydrogels to held water in their hydrophilic matrix is influenced by the chemical structure, crosslinking density and molecular weight of polymers (Collins & Birkinshaw, 2013) The swelling ratio plays an important role in maintaining the structure of hydrogels The swelling ratio of S-protected thiolated HA was compared after addition of 0.1 and % (m/v) NAC Consequently, both polymers were crosslinked during the swelling process explaining why they not simply dissolve The initial increase of weight could be due to the polymeric network stretching that allowed an increasing influx of fluid Maximum fluid uptake took place within 30 and then no further increase took place as shown in Fig These results correlate with previously shown results by Bian et al for self-crosslinking smart hyaluronic acid hydrogels exhibiting swelling equilibrium within 30 (Bian et al., 2016) For use in tissue engineering, HA-based hydrogels have a great advantage due to their high water content as shown by swelling properties Moreover, these hydrogels have high permeability for oxygen and other water-soluble compounds, as compare to previously Fig Cell viability in the presence of S-protected thiolated HA (0.5 % m/v) compared to unmodified HA (0.5 % m/v) after and 24 h Cytotoxicity studies were performed using resazurin on Caco-2 cells MEM without phenol red was used as negative control, and Triton™ X-100 (4% m/v) were used as positive control Indicated values represent an average of at least three experiments ± SD (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article) Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al The minor cytotoxic effect of unmodified HA as shown in Fig is in agreement with previous studies demonstrating a similar effect of HA on various cultured cells (Bodo et al., 2002; Boeckel, Shinkai, Grossi, & Teixeira, 2014; Wiig, Abrahamsson, & Lundborg, 1996) Moreover, HAbased thiomers are considered safe for in-vivo application as various thiolated HAs are already subject to clinical trials showing no safety concerns (Safety of Hyaluronan Thiomer i.o, 2020) and even some are already on the market such as HyStem™ hyaluronic acid based-hydrogels from Merck, Glycosil® from ESI BIO (Zarembinski et al., 2014) Furthermore, even the S-protecting leaving group NAC does not seem to reach toxic levels of concern as marketed products containing high amounts of NAC are generally regarded as safe 3.5 Viscoelastic behavior of S-protected thiolated hyaluronic acid hydrogels As the stiffness of hydrogels has a substantial impact on cell migration and proliferation, the characterization of rheological properties of hydrogels used as a scaffold material for tissue engineering is of high relevance (da Cunha et al., 2014) As thiol-disulfide exchange reactions triggered by endogenous free thiols will cause a crosslinking of HA via disulfide bond formation and consequently gelation, increasing concentrations of NAC were added to S-protected thiolated HA to simulate this process Results of this study are listed in Table The gelation process in S-protected thiolated HA occurred via thioldisulfide exchange reaction It was confirmed by mixing S-protected thiolated HA with freshly collected mucus serving as a model for extracellular proteins such as mucins building up the glycocalyx of cells or cysteine-rich membrane bound keratins Mucus consists of glycoproteins with cysteine-rich side chains These thiol groups allow mucin monomers to form disulfide bonds with the S-protected thiomer as shown in Fig Moreover, as NAC concentration in hydrogel increases, dynamic viscosity (η) increases from 1.2 Pa (0.1 % m/v NAC) to a maximum of 23.5 Pa (1 % m/v NAC) confirming gel formation as shown in Fig Elastic modulus (G′) was also increased with increasing concentration of NAC (Fig 6A) characterizing energy recovery stored in the system (Valenta, Kast, Harich, & Bernkop-Schnürch, 2001) A high elastic modulus (G′) of S-protected thiolated HA hydrogel indicates high mechanical strength and rigidity and further denotes more solid-like properties Moreover, a high elastic modulus (G′) shows the polymer’s ability to store deformation energy in an elastic manner The higher the degree of cross-linking was, the greater was the storage modulus Furthermore, the viscous modulus (G″) of the S-protected thiolated HA was also higher than the one of unmodified HA as depicted in Fig 6B In the presence of NAC, S-protected thiolated HA hydrogels showed a tan δ value of 0.2 indicating gel formation, whereas unmodified HA hydrogel did not show any gel formation Similarly, in rheological studies with mucus elastic modulus (G′) and viscous modulus (G″) of the S-protected thiolated HA increased over time as shown in Fig 6-C and D Mucus consists of glycoproteins with cysteine-rich side chains These thiol groups allow mucin monomers to form disulfide bonds with the S-protected thiomer (Kaldybekov, Filippov, Radulescu, & Fig Dynamic viscosity (Pas) [A] of unmodified and S-protected thiolated HA (1.5 % m/v) in the presence of indicated concentrations of N-acetyl cysteine (NAC) and dynamic viscosity [B] of unmodified HA (1 % m/v) and S-protected thiolated HA (1 % m/v) in the presence of 500 μL of mucus in 200 mM phosphate buffer at pH at a frequency of Hz on a plate-plate viscometer at 37 °C for h Indicated values represent an average of at least three experiments ± SD Khutoryanskiy, 2019; Kolawole, Lau, & Khutoryanskiy, 2018) Unmodified hyaluronic acid/mucus mixture showed almost the same viscosity as mucus, whereas the viscosity of S-protected thiolated HA was 10.1-fold higher 3.6 In-vitro cell proliferation and morphology After 3, 7, 12 and 15 days proliferation of Caco-2 and NIH 3T3 cells were monitored as shown in Figs and Results showed that both cell lines having been encapsulated in the hydrogel proliferated within the culture medium After days of culture, cells bilayer was observed whereas after 12 days cell clusters appeared Due to the hydrophilic surface of HA, no cell attachment was observed Spherical cell clusters of Caco-2 cells and elongated, spindlelike morphology of NIH 3T3 cells reflect proper gel stiffness as high hydrogel stiffness leads to change in morphology of cells during proliferation (da Cunha et al., 2014) Moreover, the number and size of cell clusters increased with the increase of incubation time For cell proliferation, Cys/CySS redox potential (Eh) has an important role in redox exchange between cells and organs Ramirez et al explored that in lung fibroblasts the more oxidizing Eh values of Cys/ CySS produced intracellular signals that stimulate cell proliferation These oxidizing Eh values increased extracellular fibronectin production in NIH 3T3 fibroblasts via the protein kinase C pathway and induction of transforming growth factor-β1 (Ramirez et al., 2007) S-protected thiolated HAs having NAC are also predicted to follow the same Table Gelation time of S-protected thiolated HA (1.5 % m/v) in the presence of indicated concentrations of NAC at 37 °C Indicated values are mean ± SD (n = 3) NAC concentration (% m/v) Gelation time (min) 0.1 0.3 0.5 0.8 1.0 20 ± 18 ± 11 ± 9±2 5±1 Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al Fig Rheological measurements of S-protected thiolated HA in terms of [A] elastic modulus (G´) and [B] viscous modulus (G″) after incubation with indicated concentrations of NAC and [C] elastic modulus (G´) and [D] viscous modulus (G″) after incubation with 500 μL of mucus in 200 mM phosphate buffer at 37 °C for h at a frequency of Hz on a plate-plate viscometer All the results are expressed as the mean of at least three experiments ± SD, (***P < 0.001) endogenous thiols on the surface of surrounding cells The intracellular concentration of thiols is in the millimolar range, whereas free thiols in the extracellular environment are in the micromolar range depending on the target compartment (Hu, 1994; Yi & Khosla, 2016) The thiol/ disulfide balance within the extracellular environment is kept constant via various mechanisms As due to the addition of S-protected HA this balance is strongly shifted towards disulfides the concentration of free mechanism for proliferation of cells S-protected thiolated HAs are advantageous over just thiolated HAs that are unstable in aqueous solutions of pH ≥ due to oxidation of thiol groups (de Sousa et al., 2016) On contrary, S-protected thiolated HA hydrogels react in a pHindependent manner being stable towards oxidation Moreover, S-protected thiolated HA hydrogels will not just crosslink but will also be anchored in the target tissue by disulfide bond formation with Fig The S-protected thiolated HA hydrogels (1.5 % m/v)) cultured with Caco-2 cells (5 × 106 cells/mL) incubated at 37 °C in an environment of 95 % humidity and 5% CO2 Inverted phase contrast micrographs of Caco-2 cells encapsulated in S-protected thiolated HA hydrogel after co-culture for days (A1), days (B1), 12 days (C1) and 15 days (D1) All the micrographs are of 10 μM magnification Carbohydrate Polymers 237 (2020) 116092 M.H Asim, et al Fig The S-protected thiolated HA hydrogels (1.5 % m/v)) cultured with NIH 3T3 (5 × 106 cells/mL) incubated at 37 °C in an environment of 95 % humidity and 5% CO2 Inverted phase contrast micrographs of NIH 3T3 cells encapsulated in S-protected thiolated HA hydrogel after co-culture for days (A2), days (B2), 12 days (C2) and 15 days (D2) All the micrographs are of 10 μM magnification thiols will likely rapidly raise in order to compensate this imbalance Furthermore, as the crosslinking of S-protected thiolated HA is triggered by free thiols, whereas the crosslinking of thiolated HA is triggered by oxygen, a direct comparison in their in-situ gelling properties is not feasible The concentration of available free thiols and oxygen is highly variable depending on the type of target tissue and disorders such as inflammation or necrosis In case of S-protected thiolated HA, however, an unintended gelation during production, storage and application can be excluded, as this type of thiolated HA is stable towards oxidation These results confirm that S-protected thiolated HA hydrogels enhance the proliferation of encapsulated cells and can be helpful in 3D cell culture scaffold analysis Andreas Bernkop-Schnürch: Conceptualization, Resources, Funding acquisition, Supervision, Project administration Conclusion This research work was supported by the Higher Education Commission (HEC), Pakistan and the Austrian Agency for International Cooperation in Education and Research (OeAD), Austria Declaration of Competing Interest The authors report no conflicts of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed Acknowledgments In this study, S-protected thiolated HA was synthesized by conjugating a SPDP-NAC hydrazide ligand to the backbone of HA S-protected thiolated HA showed enhanced cohesion and stickiness upon addition of N-acetyl cysteine (NAC) The crosslinking process of previously synthesized thiolated HA hydrogels was slow and un-controlled In contrast, this novel S-protected thiolated HA is stable towards oxidation and forms highly cohesive gels when getting into contact with endogenous thiols due to disulfide-crosslinking In addition, S-protected thiolated HA hydrogels were non-toxic and showed enhanced dynamic viscosity in the presence of extracellular model proteins via thiol-disulfide exchange reactions Furthermore, S-protected thiolated HA hydrogel showed proliferation of Caco-2 and NIH 3T3 cells encapsulated in hydrogels Because of all these properties, it could be concluded that these S-protected thiolated HA hydrogels have potential in the area of 3D cell culture scaffold and tissue engineering as matrices for repairing and regenerating tissues and organs 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.2020.116092 References Asim, M H., Moghadam, A., Ijaz, M., Mahmood, A., Götz, R X., Matuszczak, B., Bernkop-Schnürch, A (2018) S-protected thiolated cyclodextrins as mucoadhesive oligomers for drug delivery Journal of Colloid and Interface Science, 531, 261–268 Asim, M H., Jalil, A., Shahzadi, I., Khan, M., Matuszczak, B., & Bernkop-Schnürch, A (2019) Mucoadhesive S-protected thiolated cyclodextrin-iodine 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Swelling ratio of S-protected thiolated hyaluronic acid hydrogels To study the swelling capabilities of the S-protected thiolated HA hydrogels, equilibrium swell experiments were performed (Ding,... characterization of S-protected thiolated hyaluronic acid HA served as a polymeric backbone for the immobilization of thiol moieties via amide bond formation For the synthesis of S-protected thiolated. .. S-protected thiolated HA was formulated to a hydrogel and tested for texture, swelling ratio, cytotoxicity and viscoelastic behavior Furthermore, the potential of S-protected thiolated HA hydrogel for 3D