Here, we synthesise a new isocyanate-based crosslinker with the aim of obtaining novel tailored gels. The newly crosslinked hyaluronic acid (HA) gels were analysed in terms of rheological properties, chemical structure, swelling, biocompatibility and immunological response.
Carbohydrate Polymers 245 (2020) 116501 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Labile crosslinked hyaluronic acid via urethane formation using bis(βisocyanatoethyl) disulphide with tuneable physicochemical and immunomodulatory properties T Fernanda Zambonia,b, Elizabeth Ryanb, Mario Culebrasa, Maurice N Collinsa,b,* a b Bernal Institute, School of Engineering, University of Limerick, Ireland Health Research Institute, University of Limerick, Ireland ARTICLE INFO ABSTRACT Keywords: Hyaluronic acid Diisocyanate Crosslinking Biocompatibility Immunomodulation Here, we synthesise a new isocyanate-based crosslinker with the aim of obtaining novel tailored gels The newly crosslinked hyaluronic acid (HA) gels were analysed in terms of rheological properties, chemical structure, swelling, biocompatibility and immunological response Results show that the synthesised di-isocyanate outperforms other more conventional di-isocyanates due to its alkyl-based which has been shown to be slower to degrade by hydrolysis than aryl isocyanates The alkyl-based isocyanate reacts readily with hydroxyl groups on the HA to form stable carbamate crosslinkages Importantly and uniquely, a centrally located disulphide bond allows versatility with respect to reversible crosslinking and this allows potentially controlled layer-by-layer deposition of the HA for tailored pharmaceutical applications Introduction Hyaluronic acid (HA) is a glycosaminoglycan (GAG) constituted with repeating units of a disaccharide that contains β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine HA is a carbohydrate polymer best known for its intrinsic hydrating properties, which acts as a space filler, lubricant, and osmotic buffer that is found in the synovial fluid, in the vitreous fluid and in the extra cellular matrix (ECM) of many tissues in the body (Zamboni et al., 2017) However, HA also possesses important properties regarding its immunomodulatory activity which is primarily dependent on its molecular size (Zamboni, Vieira, Reis, Oliveira, & Collins, 2018) For these reasons, it is understandable why HA is a material of increasing significance to biomaterials science and why it is finding applications in a wide range of areas that include drug delivery (Liu, Wang, Yang, Zhang, & Gan, 2019) and body repair (Souness, Zamboni, Walker, & Collins, 2018) However, since HA is readily degraded in physiological conditions due to hydrolysis and enzymatic degradation (Collins & Birkinshaw, 2007; Pandey & Weigel, 2014; Valachova et al., 2016), it is necessary to prolong its degradation time through a variety of chemical modifications (Collins & Birkinshaw, 2007; Sigen et al., 2018, 2020; Zamboni et al., 2018) The presence of hydroxyl groups in the N-acetylglucosamine moiety of HA is one of the main domains for crosslinking ⁎ via chemical reaction and covalent bond formation (Schante, Zuber, Herlin, & Vandamme, 2011) Therefore, the development of new covalent crosslinking chemistries is of critical importance to tailor the structural stability of HA Up to date, in the literature only twice HA was crosslinked using molecules bearing isocyanate groups (Chen et al., 2010; Dhanasingh, Salber, Moeller, & Groll, 2010) The main advantage of using isocyanates is their reactivity towards hydroxyl groups to form urethane linkages (Koniev & Wagner, 2015) and there is an increasing interest to crosslink carbohydrate polymers with isocyanates (Sonker et al., 2018b) Herein, it is proposed to use a novel diisocyanate as a crosslinking agent to react with the hydroxyl group present in the N-acetylglucosamine moiety of HA to form a gel through an urethane crosslinkage formation The novel bis(β-isocyanatoethyl) disulphide (BIED) has many advantages over other conventionally used diisocyanates BIED is an alkyl-based isocyanate, whose chemistry is known to be significantly slower to degrade by hydrolysis than aryl isocyanates (Brown, Green, Cedel, & Cairns, 1987) Moreover, alkyl-based isocyanates also produce upon reaction with hydroxyl groups more stable urethane linkages against thermal degradation which transfers to more stable hydrogels at body temperature (Chattopadhyay & Webster, 2009) Alkyl isocyanates are not cytotoxic in contrast to aryl isocyanates (Touchet & CosgriffHernandez, 2016).The incorporation of a disulphide bond centering the Corresponding author at: ER0-003, Engineering Research Building, University of Limerick, Ireland E-mail address: Maurice.collins@ul.ie (M.N Collins) https://doi.org/10.1016/j.carbpol.2020.116501 Received 27 March 2020; Received in revised form May 2020; Accepted 22 May 2020 Available online 02 June 2020 0144-8617/ © 2020 The Author(s) Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/) Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al diisocyanate molecule backbone, gives a versatile and key additional property that can to be exploited for self-healing and reversible crosslinking gels (Bian et al., 2016; Griesser, Hetenyi, & Bernkop-Schnurch, 2018; Pepels, Filot, Klumperman, & Goossens, 2013) The re-oxidation of free thiol groups into disulphide bonds can be performed using oxidizing agents (metal ions and enzymes) or by a disulphide exchange mechanism (Fernandes & Ramos, 2004) Table Formulation of different HA gels HA BIED Formulation (mg/mL) Formulation (mg/mL) Formulation (mg/mL) Formulation (mg/mL) 30 24.6 30 49.3 70 24.6 70 49.3 2.3 HA crosslinking Materials and methods Homogeneous crosslinking of HA was carried for all physico-chemical and mechanical characterization assessments of the gels Homogeneous crosslinking reactions were performed using DMSO as the solvent for the system reaction It is anticipated that this reaction condition is suitable for the encapsulation of drugs, as drugs are normally hydrophobic However, for any biological experimentation, hydrogels must be free of organic solvents To this end, HA films were heterogeneously crosslinked with BIED via a diffusion mechanism This method was deemed a preferred method for all biocompatibility analysis 2.1 Materials HA, with an average molecular weight (Mw) of 1.20 MDa and 0.1 MDa, was kindly supplied by Shanghai Easier Industrial Development Co LTD (Shanghai, China) as dry powder 3,3′-dithiodipropionic acid, hydrazine monohydrate, sodium nitrite, dimethyl sulphoxide (DMSO), dithiothreitol (DTT) were purchased from Lennox Laboratories (Dublin, Ireland) Alamar Blue cell viability reagent was purchased from Invitrogen (Paisley, UK) Mouse granulocyte macrophage colony stimulating factor (GM-CSF) ELISA MAX Deluxe set and human tumour necrosis factor (TNF) alpha ELISA MAX standard set were purchased from BioLegend (San Diego, CA, USA) 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) was purchased from ThermoFisher Scientific PD5 silicon oil was purchased from Momentive The dyalisis kit (Pur-ALyzer™ Mega 1000), deuterium oxide (D2O), Ellman’s Reagent (5,5′dithio-bis-(2-nitrobenzoic acid), fluorescein isothiocyanate (FITC) dextranes, Alamar blue (AB), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), sodium phosphate dibasic dihydrate, Dulbecco’s modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) medium, trypsin-EDTA 0.25 %, foetal bovine serum (FBS), phosphate buffered saline (PBS), L-glutamine and antibiotics and all other reagents used in this work were of analytical grade purchased from Sigma Aldrich (St Louis, MO, USA) L929 fibroblast cell line derived from mouse and THP-1 monocyte cell line derived from human were purchased from commercial sources 2.3.1 Homogeneous crosslinking BIED was added to different concentrations of HA MW 1.20 MDa or 0.1 MDa dissolved in DMSO M HCl was used to adjust the pH to The crosslinking reaction proceeds upon stirring Formulations are shown in Table (below) 2.3.2 Heterogeneous crosslinking HA solutions of 1.2 MDa 3% (w/v) and 0.1 MDa 7% (w/v) in water were prepared overnight at 25 °C and 100 RPM The difference in the solution concentration was based solely on the viscosity of each MW used Concentrations over 3% for 1.2 MDa were much more difficult to prepare homogenous solutions Solutions were poured in petri dishes and dried overnight at 35 °C in a fan oven to form the dried films Dried films were measured using a caliper as follows: 0.1 MDa films had an average thickness of 0.066 mm ( ± 0.013) and 1.2 MDa films had an average thickness of 0.156 mm ( ± 0.123) Films were cut in 1cm² shape and weighted as follows: 0.1 MDa cm² films had an average weight of 11 mg ( ± 1.1) and 1.2 MDa films had an average weight of 6.8 mg ( ± 0.47) Square films were then placed in a mL solution of acetone:water (80:20) Then, 400 mg/mL of BIED were added and pH adjusted to as described previously and allowed to crosslink for 72 h at RT Crosslinked films were washed with deionized water several times before being dried for h at 35 °C in a fan oven Prior to testing, crosslinked films were allowed to re-swell for 30 at 37 °C with deionized water For glutaraldehyde (GLUT)-crosslinked HA, the same heterogeneous crosslinking procedure was adopted HA solutions of 1.2 MDa 3% (w/v) were dried overnight at 35 °C in a fan oven to form the dried films cm2 films were then placed in a mL solution of acetone:water (80:20) with GLUT Films were allowed to crosslink for 72 h at RT Crosslinked films were washed with deionized water several times before being dried for h at 35 °C in a fan oven Prior to testing, crosslinked films were allowed to re-swell for 30 at 37 °C with deionized water 2.2 Bis(β-isocyanatoethyl) disulphide synthesis BIED is syntesised in three steps Firstly, 3,3′-dithiodipropionic acid (10 g) with ethanol in excess (22 g) were dissolved in toluene (100 mL) with ρ-toluene sulfonic acid (2.5 %) as the reaction catalyst The reaction was heated to boiling with temperature constantly monitored using a thermocouple The reaction was performed under reflux for h using a Dean-Stark trap to produce diethyl 3−3’-thiodipropionate The yield of the product was obtained by the evaporation of toluene using a rotary evaporator (Rotavapor R-210, Buchi, Switzerland), giving a yield of 90 % In the second step, diethyl 3−3’-thiodipropionate (10 g) and hydrazine monohydrate in excess (12 g) were dissolved in methanol (13 mL) and heated for h under reflux to obtain dithiodipropionate dihydrazine after crystalization in an ice bath The yield of the product was calculated by the evaporation of methanol using a Gallenkamp vacuum oven at a temperature of 40 °C overnight, obtaining a yield of 97 % In the final step, dithiodipropionate dihydrazine (10 g) was dissolved in HCl 2.5 M (60 mL) and stirred in an ice bath 16 mL of a sodium nitrite aqueous solution (76 %) was added drop wise to avoid an extreme exothermic reaction After, toluene (100 mL) is added to the solution, and this induces a phase separation which is seperated using a separation funnel The toluene phase contained an intermediate product that upon heating produces BIED by curtius rearrangement The toluene is then refluxed for 10 to completely convert acid hydrazine into isocyanate (pale yellow liquid) Toluene (bp: 110 °C) is removed from BIED (bp: 148 °C) by using a rotary evaporator (BUCHI, Switzerland) at 40 °C and under 29 mbar for h (yield of 38 %) The overall yield was calculated to be 33.17 % 2.4 Fourier transform infrared spectroscopy (FT-IR) All spectra were recorded on a Perkin Elmer spectrum 100 FT-IR spectrometer operating in Attenuated Total Reflectance (ATR) mode Each FT-IR spectrum was scanned 20 times with a resolution of cm–1 The ranges used were from 4000 to 650 cm–1 2.5 Rheology The rheological properties of HA gels produced from BIED Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al Fig Stepwise synthesis reaction to produce BIED crosslinking were evaluated by the Discovery Hybrid Rheometer (DHR2) from TA Instruments using parallel plate geometry of 25 mm at 25 °C Prior to oscillatory frequency measurements, a strain sweep analysis was performed using a strain range of 0.01−0.1-1−10-100−1000%, at a fixed frequency of Hz to obtain the linear viscoelstic region of the gels (0.01–1 %) Then, a frequency sweep was conducted in the frequency range of 0.1−10 Hz solvent interaction parameter (0.473) The effective crosslink density, νe, was then calculated as follows: ve = The swollen hydrogel mesh size ξ (nm), was determined with the following equation adapted from Collins et al (Collins & Birkinshaw, 2008a): 2.6 Swelling = 0.1748 Mc + HA gels were prepared as described previously and dried in an oven at 70 °C for 24 h The HA dry samples were weighed and incubated in mL of PBS with pH 7.4 at RT Hydrogels were removed at various time intervals; excess solution was wiped off and sample weights were recorded Swelling ratios (SR) were calculated as follows: SR = 2.6.1 Characterization of the matrix of gels HA hydrogels were dried in an oven at 70 °C, overnight The dried gels were subsequently swollen in PBS until they reached their equilibrium water content Crosslink densities were assessed by measuring volumetric swelling and applying a simplified version of the Flory-Rehner equation as described by Collins et al (Collins & Birkinshaw, 2008a) Mc (QM ( 1) 1) After the crosslinking of HA to produce gels, the disulphide bond in the spacer chain of the crosslinker was reduced to form free thiol groups First, gels had their pH adjusted to using a solution of NaOH M, then the gels were treated with DTT under heat until gels turned into solution, see S1 HA was precipitated from the solution using acetone, re-dissolved in DMSO and dyalised against deionized water HA was freeze dried to obtain a lyophilized powder The lyophilized powder of HA bearing free thiol groups was using the Ellman’s Reagent Briefly, samples and a stardant curve of L-cysteine were prepared and incubated with Ellman’s solution and buffer for 15 and absorvance was read at 412 nm, as described by the manufacturer The absorbance from samples were applied to the standard curve linear equation and concentration of free thiol groups in the HA samples was obtained HA thiolation was confirmed using a Proton Liquid Nuclear Magnetic Resonance (NMR) (Jeol ECX 400 NMR Jeol USA, Inc) Samples were diluted in mL of D2O 128 scans were performed at RT with a 15 Hz sample spinning and 45° tip angle where Wd is the dry weight and Ws is the weight of the sample after the incubation time All swelling measurements were carried out in triplicate s s (QM 2.7 Degree of substitution of HA gels Ws Wd 1+ Mc * V1 )v 2.8 Permeability test where Mc is the average molecular weight between crosslinks, ρp is the density of the dry polymer (1.229 g/cm3) and ρs is the density of PBS QM is the swelling ratio determined experimentally by comparing the mass of the material before and after immersion, ¯ is the specific volume of the dry polymer as the inverse of density, V1 is the molar volume of the solvent (18 cm3/mol for PBS), and χ is the Flory polymer For the permeability test, 0.1 and 1.2 MDa films were heterogeneously crosslinked using BIED The permeability experiment consists of HA-BIED crosslinked films separating the donor solution from the acceptor solution The donor solution contains mg/mL of two Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al distinct FITC-dextranes with molecular weights of 4,000 and 150,000 Da, whilst PBS 1X was the acceptor solution An in-house permeation chamber made of PLA was 3D printed for these experiments using 3D printer MakerGear M3ID (USA) and designed using Simplify3D software as shown in Fig 1B The two 3D-printed parts clip the gel in the middle, separating the top chamber (with the donor solution) from the bottom chamber (with the acceptor solution), see S2 Both chambers hold a volume of 261 μL The detection of FITC-dextran in the acceptor solution is measured at different time intervals using a fluorescence plate reader with an excitation wavelength of 485 nm and emission of 535 nm A standard curve (0.066, 0.0032, 0.0016, 0.0008, 0.0004, 0.0002 mg/mL) for each FITC-dextran was measured and the linear eqution was obtained to convert the fluorescence from the acceptor solution into equivalent concentration All tests were executed at RT (Hsu et al., 2018) The calculation of the permeability coefficient was determined according the equation below: h, with or without LPS stimulation challenge (1:1000) After the incubation period, supernatant was collected by centrifugation and analysed for the production of TNF-alpha, a pro-inflammatory cytokine as described by the ELISA kit manufacturer L929 fibroblast cells at a density of 20.000 cells/well were seeded onto crosslinked films of HA 1.2 and 0.1 MDa for 24, 48 and 72 h in a 96 well-plate After the incubation period, supernatant was collected by centrifugations and analysed for the production of GM-CSF, a cytokine that induces macrophage differentiation as part of the immune response chain, using an ELISA kit as described by the manufacturer 2.10 Statistical analysis Data are presented as mean ± standard deviation (s.d.) and analysed using two-way analysis of variance (ANOVA) p-values < 0.05 (*) were considered to be significant dCA CD = P*A* dt VA Results and discussion Where dCA/dt is the exchange of the concentration of the substance within the acceptor side over time (the slope); CD is the concentration of the donor solution; P is the permeability coefficient; A is the permeation surface; and VA is the volume of the acceptor solution This equation is derived from Fick’s First Law and can only be applied when CD > > CA 3.1 BIED synthesis and HA crosslinking Di-isocyanate was synthesized according to the reaction scheme in Fig In order to chemically verify the product, FTIR analysis was carried after each step Fig shows the expected characteristic vibrational bands for each reaction product For example, in step one the starting material is a carboxylic acid that is converted into an ester The bands for carboxylic acid not appear following the completion of the reaction (OeH broad stretching band between 3,300−2,500 cm−1) and the characteristic band for the ester, i.e the C]O stretch band at 1735 cm−1 becomes apparent In step two, the characteristic ester band 2.9 Biocompatibility For the biocompatibility test, 0.1 and 1.2 MDa films were heterogeneously crosslinked using BIED as described in Section 2.3.2 Prior to the biocompatibility test, these films were sterilized using UV radiation for h and allowed to re-swell for 30 at 37 °C in the incubator using cell growth medium L929 cells were cultured in DMEM and RPMI (respectively) with 10 % FBS, 1% streptomycin-penicillin solution and mM of L-glutamine at 37 °C with 5% CO2 in a humidified atmosphere This medium was replenished every three days until 80 % confluence was reached For the viability study, L929 cells were quantified by resazurin Alamar Blue (AB) -metabolic activity assay Due to AB redox nature, in which the oxidized form of AB (substrate) contained in the medium is reduced (product) by the various components of the cellular respiration chain (when cells are dead, they lose the ability to convert the substrate into product), the reduced form of AB is proportional to the number of viable cells present in the different planes of a 3-D microenvironment, being able to provide a quantitative analysis of cell viability and, assuming that the metabolic activity maintains constant throughout the testing conditions, quantification of cell proliferation is also possible L929 cells were seeded onto HA 1.2 and 0.1 MDa crosslinked films and into the well (control group) of a 24 well-plate at a concentration of 50.000 cells/mL (n = 3) After an initial four hours of incubation to allow cell attachment, AB was added to the media at a concentration of 10 % as specified by the manufacturer The well plates were returned to the incubator and aliquots of the media were taken at h, 24 h and 48 h of incubation to provide a continuous monitoring of cell culture over time Aliquots were measured in a 96 well-plate and fluorescence readings were performed at 540 nm and 590 nm using a SynergyMx plate reader (BioTek, EUA) For cell attachment analysis, L929 cells were seeded onto HA 1.2 and 0.1 MDa crosslinked films for 24 h and 48 h Cell nuclei was stained using DAPI Cell fluorescence imaging was captured using cellSens software and the Olympus BX60 fluorescence microscope (Olympus, Japan) Fig FTIR spectra of the reaction products from the BIED synthesis 3,3′dithiodipropionic acid, diethyl 3-3′-thiodipropionate dithiodipropionate dihydrazine and BIED 2.9.1 Cytokine quantification For the immune activation analysis, THP-1 cells at a density of 50,000 cell/mL were seeded onto HA 1.2 MDa crosslinked films for 24 Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al Fig Schematic reaction between HA and BIED and representative image of the BIED-crosslinked HA gels with MW of 1.2 MDa A) Crosslinked HA shows the formation of urethane bond between isocyanate and the hydroxyl group B) Formulation four retains its shape C) Formulation three exhibits poor structural stability D) Representative FTIR spectra of HA and HA-BIED crosslinked gel dispaying the characterisitic urethane bridges now disappears, and new bands relating to the product appear These bands are related to NeH stretching 3,400−2,800 cm−1 which overlap with primary amine (-NH2) stretch bands at 3,180 cm-1 and 3,320 cm−1 and the carbonyl of the acyl amino at 1,622 (Xu, Guan, Zhou, & Zhu, 2017) Step two (a) involves a reaction between the dithiodipropionate dihydrazine and nitrous acid (which is formed in situ between the reaction of sodium nitrite and hydrochloric acid) producing an intermediate compound called acyl azide The azide (-N3) asymmetric stretching vibration band is shown in Fig and was previously described in the literature at 2,162 nm (Tanver, Huang, Luo, Khalid, & Hussain, 2015) After the acyl azide compound is extracted from the aqueous solution using toluene via phase separation, it is subsequently heated and converted into BIED by the Curtius rearrangement mechanism BIED shows a characteristic broad and strong band at 2,262 cm−1 associated to isocyanates (Beck et al., 2009) The synthesized di-isocyanate compound was then used to crosslink HA in order to improve HA structural stability, as shown in the reaction schematic in Fig 3A The hydroxyl groups of the N-acetylglucosamine moiety of HA is targeted by the isocyanate to form an urethane linkage A representative image of 1.2 MDa HA hydrogels is shown in Fig 3B and C In Fig 3B, formulation shows a stiffer gel that retains its shape whereas in Fig 3C formulation shows a less stiff gel that does not hold its shape This result is expected, as higher crosslinking concentrations react with more functional groups, thereby giving rise to higher crosslinking densities and subsequently stiffer gels In order to Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al chemically confirm that the isocyanate of BIED and HA reacted to form urethane linkages, FTIR was performed as shown in Fig 3D The FTIR spectrum of HA powder, shows the characteristic OH vibration band at 3346 cm−1 and the broad carbonyl band of the acid and the N-acetiylamide group at 1618 cm-1 (Dhanasingh et al., 2010) Once the crosslinking reaction with BIED occurs, the HA hydrogel shows the appearance of the characteristic carbonyl urethane group (C]O stretching) at 1729 cm-1, carbamate group (CeN) at 1564 cm-1, and CeOC at 1228 cm−e1 (Wong & Badri, 2012) The carbonyl band at 1729 cm-1 is regarded to be free of hydrogen bonding (Bahadur, Shoaib, Saeed, & Iqbal, 2016) Importantly, the absence of the characteristic isocyanate band at 2262 cm-1 indicates a fully crosslinked HA, where there was no unreacted isocyanate groups (Sonker et al., 2018a) This is an important finding because unreacted covalent crosslinkers with HA is a common problem for HA based hydrogels and it can lead to biocompatibility issues Furthermore, the increased OH vibration band at 3346 cm−1 showed in the BIED-crosslinked HA hydrogel comes from embedded water indicating they were successfully crosslinked The stiffness of a gel can be modulated and controlled by varying crosslinking concentrations and therefore crosslink densities HA concentration and HA MW also influence stiffness For example, when the HA concentration is increased hydrogel stiffness increases (higher G’) Whilst, the use of higher HA MW G’ (stiffness) decreases With LMW HA gels displaying storage moduli < 30 K Pa, whereas HMW HA gels displaying storage moduli of ∼ 10 K Pa To elucidate this further we examined the degree of substitution of both formulation by Ellman’s assay (shown in Table 2) and confirmed through NMR, as shown in S1 The degree of thiolation for 0.1 MDa was 4.16 μM/mg of HA as opposed to 1.39 μM/mg of HA for 1.2 MDa HA, and is consistent with the difference in crosslink densities, subsequent gel stiffness (∼ 10 kPa for 0.1 MDa and ∼ kPa for 1.2 MDa) and appearance We hypothesise that the thiolation degree decreases with increasing molecular weight possibly due to the formation of hydrogen bonds which result in higher viscosity Also, longer polymeric chains are more likely to become entangled, which makes the sites for crosslinking difficult to reach via diffusion, thus, causing lower reaction efficiency (Cao et al., 2019) Bearing in mind, the main advantage of BIED over other isocyanates is that it has a disulphide bond in the middle of the crosslinker chain, which can be reduced into free thiol groups, and possibly re-oxidized into disulphide bond for reversible or self-healing gel forming properties 3.2 HA hydrogel structure function property relationships The stiffness of HA hydrogels was evaluated through determination of the storage (G’) and loss moduli (G’’), where G’ represents the elastic component of the gel and G” represents the viscous component of the gel as shown in Fig All gels produced in this study display G’ > G”, 3.2.1 Swelling characterization The speed at which gels swell to their equilibrium water content is a function of crosslink density and polymeric concentration, see Fig The comparison between the different HA concentrations in the gel (formulations one and two compared to formulations three and four), shows that 70 mg/mL HA has higher water uptakes than 30 mg/mL HA gels When comparing crosslink densities, it is observed that LMW HA gels show lower swelling ratios than HMW HA gels, this is mainly attributed to the fact that LMW HA gels are more efficiently crosslinked than HMW HA gels The stability of the gels can be estimated by swelling studies Despite lower crosslink densities the HMW gels were more stable than LMW gels and this was attributed to the higher molecular weight of HA Stability of the gels gradually decreases over time as glycosidic bonds in the backbone of HA become more likely to be degraded (Cao et al., 2019) When comparing 1.2 MDa HA gel, the stability of the four formulations progressively increased, of which formulation four is the most stable, presenting no mass loss up to 24 h immersion in PBS at room temperature Overall, hydrogel swelling ratios decrease with decreasing molecular weight, which is due to increased crosslinking density and the formation of tighter network structures with an increasing number of covalent crosslinks (Hegger et al., 2017) However, although low molecular weight hydrogels (0.1 MDa) display higher crosslinking densities, it is also the faster to degrade due to smaller and less entangled polymeric chains where glycosidic bonds are more likely be degraded by hydrolysis (Cao et al., 2019) The gels initially turned white in colour, and then a colourless swelling front moved inwards, separating the highly swollen surface from the less swollen core of the gel and gradually the entire gel turned colourless and was swollen evenly This colour phenomenon was also described by (Collins & Birkinshaw, 2008b) and it is presumed to be due to molecular rearrangement as the water diffuses through the material 3.2.2 Matrix characterization In general, increasing effective crosslinking density decreases mesh size and average MW between crosslinks (Collins & Birkinshaw, 2010) and produces hydrogel matrices with smaller molecular weight cut-off (MWCO) and slower permeation rates (Bal, Kepsutlu, & Kizilel, 2014; Weber, Lopez, & Anseth, 2009) Hence, knowing these matrix parameters (shown in Table 2) is imperative for applications such as drug Fig Rheological profile of BIED-crosslinked HA gels with a) MW of 1.2 MDa and b) 0.1 MDa Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al Table Crosslinking parameters calculated for BIED-crosslinked HA gels HA MW (MDa) Formulation Mesh size (nm) Average MW between crosslinks (g mol−1) Effective crosslinking density (mol cm−3) Thiolation (μM/mg of HA) 1.2 F1 F2 F3 F4 F1 F2 F3 F4 28.9 ± 3.9 32.2 ± 1.8 57.2 ± 5.2 113.9 ± 18.8 14.7 ± 3.1 9.9 ± 3.1 17.0 ± 0.3 17.3 ± 0.9 10,091.1 ± 2611.8 11,695.2 ± 1271.0 26,614.4 ± 4983.8 71,198.1 ± 22452 4983.7 ± 2099.8 2,309 ± 1333.6 5,100 ± 202.5 4,900 ± 520.1 0.000122 ± 3.73754E−05 0.000105 ± 1.22882E−05 0.000046 ± 8.14453E−06 0.000017 ± 6.65833E−06 0.00027 ± 0.00011 0.00069 ± 0.0004 0.00024 ± 6.35085E−06 0.00025 ± 2.6839E−05 – – – 1.39 – – – 4.16 0.1 ± SD dissolves a large variety of polar and non-polar small molecules, making it an important drug solvent for pharmaceutical formulations (Tetko et al., 2013) Furthermore, DMSO enhances drug delivery through tissue and cell membranes causing no ill effects (Marren, 2011) Matrix characterization of gels shown in Table can be translated to the heterogeneous BIED-crosslinking of HA hydrogels LMW HA crosslinked via diffusion also shows slower permeation rates than HMW HA hydrogels, implying higher crosslinking densities and lower mesh size (Fig 6) For cell encapsulation, crosslinking needs to be conducted in physiological conditions, BIED-crosslinked HA gels can produce thiolated HA derivates Due to the fact that BIED contains a centralized disulphide bond, the gel can be reduced into a solution and the disulphide bond can be severed into free thiol groups, that can be re-crosslinked (Black, Sanders, & Stefankiewicz, 2014) Converting these thiol groups into disulphide crosslinks can be achieved by thiol oxidation and disulphide exchange mechanisms Thiol oxidation reactions need strong oxidizing agents and catalysts to occur, which hamper its use for cell encapsulation (Trost & Fleming, 1991; Milheiro et al., 2016; Scharf et al., 2014; Wang et al., 2016; Dewan, Bora, & Kakati, 2012) On the other hand, disulphide exchange mechanism occurs spontaneously via a biomolecular nucleophilic substitution (SN2) reaction This mechanism allows the formation of gel in physiological conditions (aqueous medium and neutral pH) (Fernandes & Ramos, 2004) that can present self-healing properties (Pepels et al., 2013), and are suitable for cell encapsulation As seen in Table 2, the reduction of BIED-crosslinked HA is possible and it produces HA-bearing thiols, which were obtained for the quantification of the degree of substitution of HA which correlates to the crosslinking degree It can be postulated that these HA derivatesbearing thiol groups could be exploited for self-healing and for cell encapsulation by the re-oxidation of thiols into disulphide bonds Therefore, we envisage that for cell encapsulation approaches, the balance between mesh size and permeation rate is imperative to prevent the inflow of molecules like antibodies such as IgG (150 kDa), Fig Swelling profiles of BIED-crosslinked HA gels of a) 1.2 MDa and b) 0.1 MDa delivery and cellular scaffolding Here, the characterization of homogeneous BIED-crosslinked HA gels shows that LMW HA gels display much smaller mesh sizes than HMW HA due to their higher crosslink densities resulting from their higher degrees of thiolation As mentioned before, BIED in HMW HA has lower reaction activity due to the longer polymeric chains that are more likely to become entangled, which makes sites for crosslinking difficult to reach (Cao et al., 2019) It is envisaged that the homogeneous crosslinking carried out in this study can potentially be used to encapsulate drugs Additionally, the use of DMSO can pose little constraint for drug encapsulation, as DMSO Fig Permeation rate of different dextrans through HA gels of different molecular weight Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al Fig Biocompatibility of HA-BIED crosslinked gels A) Metabolic activity analysis of L929 cells over time B) L929 cells fluorescently stained using DAPI Cell attachment on the surface of 0.1 MDa and 1.2 MDa HA films crosslinked with BIED at 24 and 48 h after incubation Magnification 10 × cytokines such as IL-1 (17.5 kDa) and chemokines such as CCL-2 (8 kDa), while allowing the diffusion of oxygen and LMW biological compounds such as glucose (180 Da) and carbon dioxide (44 Da) out of the encapsulate For this reason, when comparing the crosslinking concentration used to produce the gels, higher amounts of crosslinker produced gels with lower mesh sizes The smaller mesh size calculated for these gels, was for LMW HA in Formulation This hydrogel has a mesh size of 9.9 nm ( ± 3.1), which is capable of preventing IgG permeation, the most abundant immunoglobulin (80 %) in the body with a Stokes-Einstein radius of 6.4 nm (Karlsson, Zacchi, & Axelsson, 2002) Previous studies have proven that membrane barriers for IgG are enough to prevent graft rejection in transplants, indicating that blocking cytokines and chemokines may not be a requirement for immunosuppression in allotransplantation (Steele, Halle, Poncelet, & Neufeld, 2014) Thus, it is expected that increasing the concentration of crosslinker to higher amounts could be able to produce gels with much lower mesh size, thus being able to act as an immunoprotective barrier with potential immunomodulatory properties 3.2.3 Permeation study To mimic the penetration rate of IgG (150 kDa) and CCL-2 (8 kDa) through the HA hydrogels, two FITC-labelled dextranes of similar molecular weight were utilized to determine permeation rates through the LMW and HMW HA hydrogel membranes The results shown in Fig demonstrate the delay of permeation of 150 KDa dextran in LMW HA gels (p < 0.05) However, HMW HA gels were inefficient to delay the permeation of both KDa and 150 KDa Due to the high size of its matrix mesh size, both particles were smaller than the mesh, showing equal permeation rates Carbohydrate Polymers 245 (2020) 116501 F Zamboni, et al increase in TNF-alpha secretion is observed for all test groups in comparison to the control group (well) Neither non-crosslinked nor BIEDcrosslinked HA elicited TNF-alpha production Once monocytes are challenged with LPS, a significant (p < 0.05) spike in TNF-alpha is observed in all groups, however, BIED-crosslinked HMW HA shows to protect monocytes from LPS-induced TNF-alpha production This finding correlates well with previous studies which have already established that HMW HA has the ability to suppress inflammation due to LPS exposure in macrophages (Rayahin, Buhrman, Zhang, Koh, & Gemeinhart, 2015) Conclusion In this study, HA of low and high molecular weight are crosslinked using BIED, a di-isocyanate that possesses better characteristics (hydrolytic and thermal degradability, biocompatibility and reversible disulphide bond) than commonly used aryl di-isocyanates, through the formation of urethane linkages between the HA chains Results show that the obtained hydrogels exhibit tailored mechanical and stability properties which depend on HA content, HA molecular weight and degree of crosslinking These parameters have been varied to design hydrogels with predefined structure property relationships HA hydrogels crosslinked with BIED were shown to be biocompatible, and to elicit no cytotoxicity Interestingly, the immunoresponse associated with these hydrogels was due to HA MW solely LMW HA hydrogels increased fibroblast activation overtime, possibly due to a delayed and progressive response, and HMW HA gels decreased fibroblast activation overtime, possibly being related to an acute inflammatory response These results give an insight into how these materials can modulate the immune system For example, cellular grafts of LMW HA-BIED show decreased fibroblast production of GM-CSF, indicating these grafts could be transplanted into tissues abundant in fibroblasts On the other hand, cellular grafts of HMW HA-BIED show no monocyte activation and TNF-alpha production, whilst protecting LPS induction, thus showing the potential of being transplanted in tissues with direct blood contact It is expected that these results will give an insight into the future application of these hydrogels systems For example, pro-inflammatory LMW HA hydrogels could potentially be used for the delivery of vaccines, whilst, HMW HA hydrogels could potentially be used for cell encapsulation to decrease immune rejection of transplanted cells Fig Immunomodulation of HA crosslinked gels A) L929 cell activation and production of GM-CSF after incubation with HA-BIED crosslinked gels of 0.1 MDa and 1.2 MDa B) THP-1 monocyte activation and production of TNF-alpha at 24 h after incubation with 1.2 MDa HA and HA-crosslinked hydrogels using glutaraldehyde and BIED LPS challenge was performed to confirm monocyte activation Biological analysis 4.1 Biocompatibility In Fig 7A, the biocompatibility of HA gels crosslinked with BIED is shown It is observed that crosslinked HA films show similar proliferation profile from non-crosslinked HA films, used as control groups Overtime, the metabolic activity of the cells has a lag time of 24 h before increasing proliferation begins Fig 7B shows cells stained with fluorescent DAPI at 24 and 48 h after being seeded onto crosslinked HA films It is shown that the number of cells increase overtime CRediT authorship contribution statement Fernanda Zamboni: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft Elizabeth Ryan: Investigation Mario Culebras: Data curation, Writing - review & editing Maurice N Collins: Project administration, Supervision, Writing - review & editing 4.2 Immunomodulatory analysis Briefly, fibroblast cells were chosen as they are present in all tissues but predominantly in connective tissues and elicit immunological responses by aiding the immune system by producing proinflammatory cytokines and chemokines to induce and recruit inflammatory cells (Bautista-Hernandez, Gomez-Olivares, Buentello-Volante, & Manuel Bautista-de Lucio, 2017) Fig 8A shows GM-CSF, a cytokine secreted from fibroblasts that induces macrophage differentiation as part of the immune response chain GM-CSF production of 0.1 MDa HA crosslinked film is similar to basal production of GM-CSF demonstrated by the control group (p > 0.05) However, overtime, there is an increasing GM-CSF production trend 1.2 MDa HA was able to elicit a rapid production of GMCSF within 24 h of incubation (p < 0.05), but overtime GM-CSF production decreases Within the first 24 h, acute inflammatory response is observed, but overtime, there isn’t a progression of inflammation, showed by the decrease in production Fig 8B shows the influence of HMW HA on the activation of monocytes to produce TNF-alpha It can be seen that no significant Acknowledgement This work was supported by the Irish Research Council (GOIPG/ 2015/3577) Appendix A Supplementary data Supplementary material related to 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Representative FTIR spectra of HA and HA-BIED crosslinked gel dispaying the characterisitic urethane bridges now disappears, and new bands relating to the product appear These bands are related to NeH... of low and high molecular weight are crosslinked using BIED, a di-isocyanate that possesses better characteristics (hydrolytic and thermal degradability, biocompatibility and reversible disulphide