Hyaluronic-acid based redox-responsive hydrogels using the Diels-Alder reaction for on-demand release of biomacromolecules

Abstract Hydrogels are crosslinked, three-dimensional hydrophilic polymeric networks which possess similarity in structure to human soft tissue, and thus have found a great number of applications as biomaterials. Hydrogels can also act as depot for therapeutic cargo, where small molecule drugs to biomolecules can be encapsulated within their porous structure. The aim of this work is to fabricate redox-responsive hydrogels that can dissolve and release biomacromolecules upon exposure to a reducing environment. In particular, hyaluronic acid (HA), was modified with electron-rich furan groups to yield hydrogels through the Diels-Alder (DA) reaction with a maleimide group containing redox-responsive polyethylene glycol (PEG) based crosslinker. Redox-responsive hydrogels were obtained with high conversions (87–95%) at 37 °C, in a catalyst-free fashion. The stimuli-responsive degradation of hydrogels could be tuned by varying the amount of crosslinker. To examine the passive and on-demand delivery of biomacromolecules, a model protein, fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA), was encapsulated within the hydrogels. While a sustained passive release was observed in PBS, a rapid on-demand release was obtained upon exposure to the reducing agent 1,4-dithiothreitol (DTT). Furthermore, the complete dissolution of the hydrogel was accomplished upon exposure to a solution containing the reducing agent. Graphical Abstract


Introduction
Hydrogels are crosslinked three-dimensional polymeric networks with high water absorption and retain ability. They exhibit porous structure, which can be tuned by controlling the crosslinking density. The porous nature permits the loading of cells, drugs, and proteins into the gel matrix with spatial and temporal control over the release of these agents. This class of soft materials is highly attractive for a range of biomedical applications. [1][2][3] The release of encapsulated cargo can occur via simple diffusion or in a stimuli-responsive fashion, where the latter type has attracted increased attention in recent years. To date, several acid-responsive, [4] thermo-responsive, [5] photo-responsive, [6] or redox-responsive [7] materials have been reported. Hydrogels can be fabricated using synthetic or natural polymers, or using a combination of both. Natural polymers are of high interest as hydrogel precursors, since many of them, such as HA, collagen, and chondroitin sulfate are fundamental constituents of human tissue. These polymers exhibit high swelling capacity and excellent biocompatibility thus have extensively been used to create hydrogel scaffolds in regenerative medicine and wound care applications. [8] On-demand degradable hydrogels are vital for applications where controlled degradation is desired. On-demand degradation could be achieved by incorporating a chemical bond susceptible to cleavage via hydrolysis, or enzymatic degradation within the network structure. [9] In such systems, the release of encapsulated cargo can be enforced, and the hydrogel can be easily washed away from the applied surface. The latter attribute is critical when hydrogels are used to cure burn incidences. Any physical intervention, such as mechanical removal of the dressing, can be circumvented by dissolving it, thus causing minimal discomfort and tissue damage. [10] In this regard, a powerful strategy is the incorporation of redox-responsive disulfide bonds in networks to further allow on-demand degradation. Thiol-based chemistry, such as retro-Michael addition, [11] thiol-thioester, [12] and thiol-disulfide exchange reactions [13][14][15][16][17][18][19] has been employed to fabricate on-demand degradable hydrogels, and in particular thiol-disulfide exchange reaction has widely been used to fabricate mentioned systems. [20] Another widely used tool for cross-linking polymers are the 'click' reactions. The members of this class of reactions are known to proceed with high efficiency under mild conditions. [21] These reactions have not only been used for the fabrication, but also for the functionalization of hydrogels. [22][23][24][25][26][27][28][29] For example, Crescenz and coworkers applied the Huisgen type click reaction to obtain HA hydrogels to act as a reservoir for hydrophobic drugs. [30] HA modified with alkyne and azide functional groups reacted with each other via the dipolar cycloaddition reaction in the presence of a Cu(I) catalyst to give fast gelation. In general, a metal-catalyst free hydrogel synthesis is desirable, considering the possible toxicity of residual metal catalysts in biological applications. To circumvent such concerns, Shoichet et al. synthesized a HA hydrogel system crosslinked via the Diels-Alder cycloaddition reaction which took place between a furan-functionalized HA and a bismaleimide bearing crosslinker. [31] Later, this system was improved by replacing the furan groups with the electron rich methylfuran to accelerate the DA reaction. [32] These hydrogels were not degradable on-demand, and thus we envisioned that the incorporation of disulfide bonds can enable their rapid degradation in a reducing environment. Earlier, we have synthesized a redox responsive, degradable hydrogel via DA reaction for controlled protein release. [33] However, the crosslinker used in that study for crosslinking synthetic polymers was water insoluble, hence could not be used for fabrication of hydrogels in aqueous media that is needed in case of solubilization of most natural hydrophilic polymers.
Herein, we report the direct utilization of the Diels-Alder and disulfide linkages to fabricate HA-based redox-responsive hydrogels (Scheme 1). For this purpose, HA is modified with furan groups and crosslinked with a telechelic PEG-based crosslinker bearing, disulfide-linked maleimide group at the chain ends. Hydrogels with varying crosslinking densities were prepared to observe the morphologies and investigate the effect of crosslinking density on degradation and model protein release behaviors of the systems. Rheological measurements in presence of a reducing agent DTT have demonstrated the change from the gel to the sol state. Finally, FITC-BSA was encapsulated within these gels, and its release profiles in absence and presence of the reducing agent was observed.

Instrumentation
The furan-modified HA and crosslinker characterization were carried out with 1 HNMR spectroscopy (Varian 400 MHz). The microstructures of the hydrogels were investigated using a JEOL Neo Scope JCM-5000 scanning electron microscopy (SEM) instrument run at 10 kV. Scheme 1. Redox-responsive hydrogel containing Diels-Alder and disulfide linkages.
Rheological properties of the hydrogels were investigated with Anton PAAR MCR 302 rheometer. The release of FITC-BSA from hydrogels was analyzed using a Cary Varian 50 Scan UV/Vis spectrophotometer at 494 nm.

Synthesis of maleimide-disulfide-containing PEGbased crosslinker
PEG-bisacid was synthesized through the esterification reaction between 4-4 0 -dithiobutyric acid with PEG (M n ¼ 4 kDa). Anhydrous PEG (5 g, 1.25 mmol) was taken in a dry round bottom flask, and DMAP (90 mg, 0.625 mmol) was added, followed by addition of anhydrous CH 2 Cl 2 (5 mL). 4-4 0 -Dithiobutyric acid (1.19 g, 5 mmol) and DCC (1.17 g, 5.625 mmol) were dissolved in anhydrous THF (2.7 mL) in another flask and the PEG-DMAP mixture was added dropwise to it at 0 C. The reaction was stirred for 24 h at room temperature under N2. For the purification of the PEG-bisacid product, the solid dicyclohexylurea residues were filtered and the reaction solvent was evaporated. The products were dissolved in minimum amount of CH 2 Cl 2 and precipitated in ice cold diethyl ether. This procedure of precipitation was repeated four times. The precipitated PEG-acid product was dried on high vacuum to be obtained as white solid powder.
In a flask, the dried PEG-bisacid (600 mg, 0.134 mmol) was dissolved in anhydrous CH 2 Cl 2 (0.8 mL). DCC was dissolved in anhydrous CH 2 Cl 2 (1 mL) and added to PEG-bisacid linker at 0 C and stirred for 10 min at 0 C under N2.
In a separate flask, furan-protected maleimide containing alcohol (164.1 mg, 0.736 mmol) and DMAP (8.2 mg, 0.067 mmol) were dissolved in anhydrous CH2Cl2 (0.65 mL), and added dropwise to the activated PEG-Bisacid linker at room temperature. The reaction stirred at room temperature for 24 h under N2. For the purification, solid dicyclohexylurea residues were filtered and the reaction solvent was evaporated. The solid residue was precipitated in cold ether. The precipitate was dried under vacuum to yield pure product (72% yield, above 90% coupling). 1  The furan-protected bismaleimide containing PEG polymer (200 mg) was dried by azeotropic removal of toluene and kept on high vacuum for 24 h to remove any residual water. The dried polymer was dissolved in anhydrous toluene (90 mL) and refluxed at 110 C for 8 h to obtain the maleimide-disulfide-containing PEG-based crosslinker as a pale yellow solid (100% yield). 1

Synthesis of hydrogel precursors
3.1.1. Synthesis of furan-containing Hyaluronic Acid As the first step, HA was modified with the electron-rich diene to obtain a polymer that can undergo crosslinking using the DA cycloaddition, using a previously established procedure. [31] Furan-modified HA was synthesized by amidation reaction between the functionalizable carboxylic acid groups on HA and furfuryl amine using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent, using MES buffer (100 mM, pH 5.5) as solvent (Figure 1). After purification through dialysis (12-14 kDa cutoff) in ultrapure water for three days, followed by lyophilization, the product was analyzed using 1 HNMR spectroscopy. The presence of proton resonances from the furan moiety at 6.26, 6.36 and 7.45 ppm indicated successful conjugation of furfuryl amine (Figure 2). The extent of functionalization of HA was estimated as 57 ± 8%, by comparing the protons belonging to the N-acetyl glucosamine group on the polymer backbone with furan protons.

Synthesis of maleimide-disulfide-containing
PEG-based crosslinker To obtain the maleimide-disulfide-containing telechelic PEG polymer, PEG-bisacid was synthesized by coupling the PEG (M n ¼ 4 kDa) with 4-4'dithiobutyric acid via Steglich esterification ( Figure 3). Obtained diacid was reacted with furanprotected maleimide-containing alcohol to obtain a telechelic polymer with masked maleimide groups. The masked maleimide-containing polymer was obtained in pure form by precipitation in cold diethyl ether. The purity of thus obtained polymer was ascertained using 1 HNMR spectroscopy.
Integration of proton resonances of the protons adjacent to the ester oxygen atoms ( Figure 4A, peaks i and e) with the protons belonging to the bicyclic adduct ( Figure 4A, peaks a, b, and c) suggest the quantitative transformation of the end groups. Refluxing this polymer in toluene resulted in removing the furan-protecting group to afford the desired telechelic PEG polymer containing both the disulfide and maleimide functional groups. The clean and quantitative  removal of the furan-protecting groups was evident from the 1 HNMR spectrum of the polymer, where the newly formed maleimide group was visible ( Figure 4B, peak a).

Fabrication of redox-responsive hydrogels
The crosslinking of the redox-responsive HA hydrogels was achieved using DA click reaction. The furan and maleimidecontaining polymers were reacted in MES buffer (pH ¼ 5.5, 100 mM), where HA has enhanced solubility in this acidic media. Notably, as DA reaction is a catalyst-free click reaction, these gels are expected not to contain cytotoxic residues or by-products after removing unreacted species by washings with dH 2 O. Hydrogels with different furan/maleimide ratios, thus with varying amounts of crosslinking were synthesized. Gelation for 15 h at 37 C yielded hydrogels with good conversions ( Table 1). The inverted tube method inferred the end-point detection of the hydrogel formation, where the gels did not exhibit any flow. The gels were washed and allowed to swell in a significant amount of water to obtain them in colorless and fully transparent form ( Figure 5).

Characterization of redox-responsive hydrogels
A variation in the amount of crosslinking is expected to cause a difference in the morphology and pores sizes of the hydrogels. To investigate their microstructures, lyophilized hydrogel samples were observed under SEM ( Figure 6). Since these hydrogels were synthesized using furan-modified HA, a functionalized natural polymer with a high molecular weight, the microstructures of the gels exhibited a porous morphology.
The swelling capacity of hydrogels HG1, HG2 and HG3 were studied. The hydrogels reached their equilibrium swelling points quickly, and stayed constant over time, and HG3 with the least crosslinker exhibited the highest swelling ratio, as expected. Interestingly, in case of HG1 and HG2 swelling ratios were comparable to each other but opposite to expected cross-linking density related trend. (Figure 7A). This profile could be the result of another factor, which is the amount of PEG moieties within the gels which effects the swelling behavior. Since hydrogels HG1 and HG2 did not show a significant difference in their swelling profiles, the higher number of hydrophilic PEG moieties in hydrogel HG1 is believed to result in a higher swelling ratio in comparison to hydrogel HG2. While the hydrogels were opaque in their dry state, upon swelling in water they became quite transparent ( Figure 7B).
The change in viscoelastic properties of the hydrogels according to the change in amount of crosslinker were investigated using rheological analysis. To compare the differences of modulus values of hydrogels with varying amount of crosslinker, frequency sweep and amplitude sweep tests were done. The frequency sweep tests were run to comprehend the time dependent behavior of the gels in terms of their storage modulus (G') abilities in a nondestructive deformation range (0.1-100 rad/s). In rheological analysis, the storage modulus (G') defines the ability of the hydrogel to store the deformation energy in a solid viscoelastic manner. Depending on this, with a higher crosslinker amount, increased storage modulus values were expected. In addition, to be able to define the gel phase, a higher value of storage modulus in comparison to the loss modulus (G") is needed. As expected, hydrogels HG1, HG2 and HG3 all individually demonstrated gel-like properties with G' values greater than their corresponding G 00 values ( Figure S1). Another foreseen trend was that the hydrogels would show an increase in their G 0 values according to increased amount of crosslinker which was also observed via rheological measurements. For HG1 the G 0 value was above 10 3 Pa whereas for the hydrogels HG2 and HG3 this value was under 10 3 Pa.

Degradation of redox-responsive hydrogels
Since this hydrogel system is aimed at as an on-demand degradable material, the redox-responsive degradation profiles and the change in profiles upon an alteration in extent of crosslinking were investigated. To perform the desired on demand removal of the biomaterial during or after the treatment, these dressing were expected to be stable in PBS only but also demonstrate the full dissolution in a reducing environment. The degradation profile of HG1 was observed by visual degradation test where degradation profiles HG1, HG2 and HG3 were all investigated with rheological tests.
For the visual examination of degradation, pre-determined amounts of FITC-BSA was dissolved in gelation media and encapsulated within the hydrogel HG1 real time with gelation. The degradation of gel and release of protein in the presence of DTT (5 mM) was visually followed by shining UV light on the sample media ( Figure 8). To prove that the thiol-rich reducing environment was responsible for the degradation, an additional sample from the same hydrogel was incubated in PBS as a control expriment. By the end of 6 h at room temperature (rt), in DTT the degradation of hydrogel was observed as FITC-BSA was fully dispersed in the solution without any residual hydrogel. However, the sample incubated in PBS did not degrade and only a relatively low intensity fluorescence was observed as a result of the FITC-BSA released through passive diffusion.
The sol-gel transition term stands to define the crossover point of G 0 and G 00 values of a liquid during the  gelation process. The domination of G 0 over G 00 is an indication of the material showing gel-like behavior over its liquid state properties. The reverse of the sol-gel transition suggests the inversed process, that is gel to sol transition, where G 00 crosses over G' and the viscoelastic properties of the material are lost due to a domination of liquid behavior. Using this information, time dependent redox responsive degradation profiles of the hydrogels were inspected via time sweep tests. HG1, HG2 and HG3 were all allowed to stay in PBS (pH ¼ 7.4) to certain swelling points. To prove the stability of HG1, HG2 and HG3 in PBS at 37 C time sweep test was performed first in only PBS for 100 min ( Figure 9A). Following this, the gels were treated with 10 millimolar (mM) disulfide reducing agent DTT and the gel to sol transition points with respect to time were followed as a function of time ( Figure 9B). As expected, HG1 with the highest crosslinker amount, thus involving the highest number of disulfide bonds, displayed the longest interval of degradation with an approximate of 210 min whereas HG2 and HG3 degraded approximately in 110 and 97 min, respectively. This trend proved that these redox responsive HA-hydrogels exhibited tunable redoxresponsive degradation profiles adjusting the disulfide bearing crosslinker amount.   The local delivery of a protein was aimed since this hydrogel system was designed to carry the potential of a local delivery material of tissue regenerative proteins. For this purpose, BSA was chosen as a model protein, and the diffusion and on-demand release profiles were investigated ( Figure 10). To determine the BSA concentration using UVspectroscopy, FITC conjugated BSA was used. First, the passive release of BSA in PBS (1x, pH ¼ 7.4) was investigated. It was observed that hydrogels released 36% of encapsulated FITC-BSA by the end of 6 h and at 55% of the total FITC-BSA after 72 h. At the 72nd hour, 50 mM of DTT was added to the media to investigate the on-demand release of HG1 due to redox-responsive degradation. A rapid BSA release was anticipated since the gels were designed to degrade entirely in the presence of DTT. As expected, upon adding 50 mM DTT the gels degraded rapidly, and a total of another 30% of the encapsulated BSA was released. The release study showed these hydrogels' successful sustained and on-demand release abilities, which acted as a cargo system for protein.

Conclusions
In this work, hyaluronic acid-based redox-responsive hydrogels were synthesized using Diels-Alder 'click' reaction. To this end, the carboxylic acid groups on HA were modified with electron-rich furan groups, and a PEG-based disulfidecontaining bismaleimide-based crosslinker was synthesized. Using these polymers, hydrogels with different amount of crosslinker were synthesized. These hydrogels exhibited higher storage modulus values with increased cross-linking density. Morphological analysis of hydrogels with different amount of crosslinker showed an increase in porosity and decreased crosslinking. The release profiles of physically encapsulated FITC-BSA in the hydrogel with the highest amount of crosslinker was studied in PBS, and also after adding 50 mM DTT. Both sustained and on-demand delivery of the macromolecular cargo, FITC-BSA, was observed. One can envision that the redox-responsive hydrogels disclosed here bear the potential to act as local therapeutic   delivery devices which can be easily removed through ondemand dissolution.

Disclosure statement
No potential conflict of interest was reported by the authors.