Injectable and Self-Healing Dynamic Hydrogels Based on Metal(I

Article pubs.acs.org/Biomac

Injectable and Self-Healing Dynamic Hydrogels Based on Metal(I)Thiolate/Disulfide Exchange as Biomaterials with Tunable Mechanical Properties Pablo Casuso, Ibon Odriozola, Adrián Pérez-San Vicente, Iraida Loinaz, Germán Cabañero, Hans-Jürgen Grande, and Damien Dupin* Materials Division, IK4-CIDETEC Research Centre, Paseo Miramón 196, Donostia-San Sebastián 20009, Spain S Supporting Information *

ABSTRACT: Despite numerous strategies involving dynamic covalent bonds to produce self-healing hydrogels with similar frequency-dependent stiffness to native tissues, it remains challenging to use biologically relevant thiol/disulfide exchange to confer such properties to polymeric networks. Herein, we report a new method based on Metal(I) [Au(I) or Ag(I)] capping to protect thiolates from aerial oxidation without preventing thiolate/disulfide exchange. Dynamic hydrogels were readily prepared by injecting simultaneously aqueous solutions of commercially available HAuCl4 and 4arm thiol-terminated polyethylene glycol [(PEGSH)4], resulting in a network containing a mixture of Au(I)-thiolate (Au−S) and disulfide bonds (SS). While the dynamic properties of the hydrogel were closely dependent on the pH, the mechanical properties could be easily tuned by adjusting (PEGSH)4 concentration and amount of Au−S, as judged by dynamic rheology studies. Permanent Au−S/SS exchange at physiological pH conferred self-healing behavior and frequency-dependent stiffness to the hydrogel. In addition, in vitro studies confirmed that Au-based dynamic material was not cytotoxic to human dermal fibroblasts, demonstrating its potential use as a medical device. Dynamic hydrogels obtained using Ag(I) ions demonstrated that the exchange reaction was not affected by the nature of the Metal(I) capping. Finally, this efficient thiolate capping strategy offers a simple way to produce injectable and self-healing dynamic hydrogels from virtually any thiol-containing polymers.

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INTRODUCTION Hydrogels with increasing functionality and complexity offer excellent promise for regenerative medicine applications.1−4 Their ability to absorb a large amount of water similarly to biological tissues allows biomaterials to be designed for the replacement of damaged tissue such as synovial fluid5 and cartilaginous tissues.6−9 In all cases, hydrogels must show good biocompatibility and similar mechanical properties as the tissue to be replaced, especially in acellular substitutes when no regeneration is considered.6,7 However, some biological tissues can present peculiar properties due to their specific role in the body. For example, the synovial fluid (SF) based on high molecular weight hyaluronic acid provides lubricant and shock absorber properties between the cartilage while walking and running.10 Similarly, nucleus pulposus (NP), mainly composed of collagen type II and proteoglycans located in the intervertebral disc (IVD), allows the dissipation of energy between vertebras, as cushion with shock absorber properties, while sitting down or in movements. 9 Unfortunately, degradation of SF and NP induced by factors such as aging and obesity, among others, can result in painful traumas such as rheumatoid arthritis (RA) and IVD herniation, respectively. Although reasonable recovery could be achieved, biomaterials based on hydrogels could not match the particular mechanical © XXXX American Chemical Society

properties of the native tissue, especially shock absorber properties, which are mechanically defined as a frequencydependent stiffness of the material.8,9 In addition, one can assume that hydrogels with self-healing ability would represent a tremendous advantage to prolong the lifetime of the biomaterials that will inherently suffer damage due to heavy lifting during work or sport activities, for example.11 Recently, dynamic covalent chemistry has proved to be a promising tool to regenerate damaged 3D networks.12 Such bonds can selectively undergo reversible breaking/reformation under equilibrium conditions. Various self-healing polymer networks have been produced based on reversible reactions such as Diels−Alder reaction,13,14 transesterification,15,16 disulfide exchange,17 or metallophilic attractions.18 However, only few examples of hydrogels with dynamic covalent crosslinkers have been reported (e.g., [phenylboronate-salicylhydroxamate] reaction,19 metal−ligand exchange,20,21 boronic ester bonds,22 dual acylhydrazone/disulfide bonds,23 radically reshuffling trithiocarbonate,24 and radical-mediated disulfide fragmentation25). In most cases, dynamic rheology studies Received: July 23, 2015 Revised: September 28, 2015

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DOI: 10.1021/acs.biomac.5b00980 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. (A) Schematic representation of Au(I)−thiolate (Au−S) and disulfide (SS) formed after mixing (PEGSH)4 with 20 mol % of HAuCl4 (Au20) based on a theoretical amount of thiols. (B) Schematic representation describing the cascade reactions of Au(III) with thiols to form Au−S. Digital pictures of (C) the reversible state of Au20 at 5.0 wt % as a free-standing hydrogel at pH 3.2 and a viscous dynamic hydrogel at pH 9.0 and (D) the injection of the dynamic hydrogel by mixing an aqueous solution of (PEGSH)4 and HAuCl4 using a double barrel syringe. Five microliters of NaOH was added to (PEGSH)4 aqueous solution to neutralize HCl and obtain Au20 at pH 7.5. Note that in both cases, phenol red was added as a visual pH indicator, which is yellowish at pH < 6.8 and bright pink at pH > 6.8.

synovial fluid were obtained, as judged by rheological studies. Despite the efficient protection of thiolates by Au(I), one major drawback of this system was the inherent difficulty to prepare aqueous solutions of Au(I) ions, which are likely unstable at room temperature.33 Also, free-standing hydrogels at physiological pH could not be achieved due to the permanent exchange. This flowing behavior under gravity could represent a major issue for tissue engineering applications, where the hydrogel scaffold must remain at the same location, or for tissues exhibiting high stiffness, e.g., NP and cartilage.5,7 In the present study, we report the facile preparation of selfhealing dynamic hydrogels with frequency-dependent stiffness based on Au−S species and disulfide (SS) bonds prepared by simultaneously injecting aqueous solutions of commercially available 4-arm thiol-terminated PEG homopolymer [(PEGSH)4] and HAuCl4. The rheological properties of the resulting hydrogel were investigated as a function of pH, (PEGSH)4 concentration, and gold amount. Dynamic rheology and compression tests were used to estimate the mechanical recovery of dynamic hydrogels after the self-healing process. Preliminary in vitro studies were carried out to assess the cytotoxicity of Au(I)-based dynamic hydrogels for application as medical devices. Finally, Ag(I)-based dynamic hydrogels were prepared to study the effect of the nature of the capping Metal(I) on the exchange reaction.

demonstrated that dynamic covalent bonds conferred frequency-dependent mechanical properties to the resulting hydrogels, in addition to self-healing ability. Thiol/disulfide exchange reaction represents a biologically relevant example of reversible exchange readily occurring in water at physiological conditions.26,27 It is well-known that thiol/disulfide exchange reaction only occurs in the presence of nucleophilic thiolates at neutral and alkaline pH;26,27 thus, the term thiolate/disulfide exchange is more appropriate. In aqueous and normal atmospheric conditions, thiolate/disulfide exchange is limited by the inherent aerial oxidation of thiolate into disulfide. Recently, Barcan et al. reported the synthesis of dynamic hydrogels based on triblock copolymers with both outside blocks containing 1,2-dithiolane pendant groups which were cross-linked with dithiol poly(ethylene glycol).28 1,2Dithiolane units offered disulfide bonds for the cross-linking reaction via thiolate/disulfide exchange. Interestingly, the resulting hydrogel showed dynamic properties at neutral and alkaline pH attributed to thiolate/disulfide exchange reaction. By contrast, the hydrogel behaved like a conventional covalently cross-linked hydrogel at low pH due to the kinetically locked exchange reaction. However, one can assume that aerial oxidation of free thiolates will inevitably occur at long time scales resulting in the loss of the dynamic properties. Thus, easy and stable protection of reactive thiolates from oxidation with the ability to allow thiolate/disulfide exchange to proceed remains the most reasonable solution. Gold(I)−thiolate (Au−S) compounds have been used since the 1920s as the principal compound in chrysotherapy for the treatment of rheumatoid arthritis.29,30 Au−S compounds are currently of prime importance for the preparation of Au nanoparticles.31 Our group has recently prepared a new dynamic hydrogel based on a linear thiolated polymeric poly(ethylene glycol) dithiol [PEG(SH)2] cross-linked via Au−S interactions in a characteristic zigzag conformation by means of aurophilic attractive forces.32 The resulting hydrogel showed pH-dependent mechanical properties due to the dynamic exchange between Au−S species and free thiolate. Materials with similar frequency-dependent stiffness to the

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EXPERIMENTAL SECTION

Materials, preparation of Ag based hydrogels and Au60 hydrogel, 1H NMR and UV visible spectroscopy, as well as the protocol to determine the cytotoxicity of the dynamic hydrogel are detailed in the Supporting Information. Au-Based Hydrogel Preparation. For a typical preparation of Au20 hydrogel, (PEGSH)4 (50 mg, 12.0 μmol thiol group, 20.0 μmol theoretical thiols) was dissolved in water (480 μL). Then 4.16 μL of an aqueous solution of HAuCl4 (1 M) was diluted in 495.86 μL of deionized water. Each aqueous solution was placed in two separate syringes of 1 mL, which were assembled with an M-system applicator from Medmix (Switzerland). A transparent gel was formed after a few seconds. Then, 10 μL of an aqueous solution of phenol red at 0.07 wt % were added as a pH indicator. The resulting pH of the hydrogel was B


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Biomacromolecules measured to be approximately 3.2. Then, the pH was adjusted by addition of an aqueous solution of NaOH 5.0 M (5.0 μL) with vigorous shaking until the pink coloration was persistent. To prepare the hydrogel at pH 7.5, 5 μL of NaOH (5.0 M) was previously added into the aqueous solution containing (PEGSH)4. Hydrogels at different concentrations were prepared by changing the initial amount of water. Therefore, for 20.0, 10.0, and 3.0 wt % (PEGSH)4, the total volume of water was 250 μL, 500 and 1666 μL, respectively. Hydrogels at 5.0 wt % with different amounts of gold were prepared taking into account the desired thiol:gold ratio adding the amount needed in each case from the 1 M HAuCl4 solution, i.e., 2.08 and 1.04 μL of HAuCl4 to prepare Au10 and Au5 respectively. The remaining free thiolates were left oxidized until no variation of the rheological properties was observed after 24 h. Rheology Studies. Rheological measurements were carried out using an AR2000Ex (TA Instruments) rheometer using a parallel plate geometry (20 mm diameter acrylic plate). The pH of the hydrogels was previously adjusted with a Crison GLP22 pH meter. The experiments were conducted at constant temperature, i.e., 20 °C. Shear storage and shear loss moduli (G′ and G′′, respectively) were obtained at constant deformation (1%) with increasing frequency (from 0.01 to 50 Hz). Compression Studies. Compression Strain−Stress tests were carried out using an INSTRON (3365 model) tensometer controlled by Bluehill Lite software. Hydrogel samples were prepared and molded in the form of a cylinder 8.5 Ø × 12 mm. Once formed, samples were placed between 15 cm compression plates and tested under a 10 mm/ min compression speed.

in Supporting Information) exhibited two separate populations of molecular weight corresponding to single and disulfidelinked (PEGSH)4. This bimodal distribution of the molecular weight of (PEGSH)4 only indicated that a significant fraction of the commercial product is oxidized. Thus, the amount of Au(III) required to form a stoichiometric amount of Au−S was calculated based on 0.24 μmol SH group/mg determined by Ellman’s test. Simultaneous injection of aqueous solutions containing (PEGSH)4 and HAuCl4 adjusted at stoichiometric ratio 1:3 [Au(III):free thiols] (Figure 1C, left-hand side pictures) resulted in the spontaneous formation of a free-standing hydrogel based on (PEGSH)4 at 5.0 wt % linked through Au−S interactions and disulfide bonds at around pH 3.2 (Figure 1A).42 This hydrogel was designated Au20, as the relative amount of Au(I) present in the hydrogel compared to the theoretical amount of thiol offered by (PEGSH)4 is around 20 mol %. As expected, excess HAuCl4 led to the formation of Au nanoparticles at pH 8, as judged by the formation of brown or pink stains in the hydrogel and also confirmed by the characteristic absorbance of Au nanoparticles at λ ∼ 600 nm observed by UV−visible spectroscopy (Figure S3).43,44 More importantly, the absence of Au nanoparticles for Au20 hydrogel was also confirmed by UV/vis spectroscopy, where no signal characteristic of Au nanoparticles was observed at λ ∼ 600 nm at pH 3 and pH 9 (Figure S4). In addition, the characteristic shoulder at λ ∼ 320 nm assigned to dσ* → pσ transition of Au(I)−Au(I) interactions could be observed from the same spectra.45 Thus, the characteristic zigzag conformation of Au−S species by means of Au(I)−Au(I) attractive forces is expected to be present in a Au20 dynamic hydrogel, as shown in Figure 1A.32,45 The overlapping spectra at pH 3 and pH 9 confirmed that such metallophilic interactions were pH independent. Highly viscous material was obtained after adjusting the pH of Au20 to pH 9 by adding an aqueous solution of NaOH (1 M) (Figure 1C, right-hand side picture). The pink color observed for the hydrogel at pH 9 was due to the addition of phenol red as a visual pH indicator. Free-standing hydrogel and highly viscous polymer solution states were determined by the capacity of the material to maintain (or not) its dimensions with the inverse tube method over 24 h. Both states were completely reversible by simple pH adjustment, as shown in Figure 1C. It is interesting to point out that, by definition, hydrogels do not flow. Therefore, Au20 at alkaline pH cannot be considered as a conventional hydrogel. In our previous studies,32 the ability of aurophilically cross-linked linear PEG(SH)2 dynamic hydrogel to flow under gravity was attributed to thiolate/Au−S dynamic exchange. In the present work, free thiols are not expected to be present in the final material but only disulfide bonds and Au−S species.32 Thus, a rapid exchange reaction between Au−S interactions and disulfide bonds (Au−S/SS exchange) should be considered responsible for the dynamic properties of Au20 at pH 9. As a control experiment, aerial oxidation of an aqueous solution of (PEGSH)4 adjusted at pH 9 for 5 days resulted in the formation of a free-standing hydrogel (PEGSS). In this case, (PEGSH)4 star-like polymers were linked via disulfide bridges to form a covalent 3D network. The selection of (PEGSH)4 with an existing cross-linking point to strengthen the final hydrogel was justified, as linear PEG(SH)2 only led to chain extension after aerial oxidation, as previously reported.32 Unlike Au20, free-standing PEGSS hydrogel was observed at all

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RESULTS AND DISCUSSION Hydrogel Preparation. The family of hydrogels reported in this work is based on FDA-approved poly(ethylene glycol) (PEG) homopolymer, which is extensively used in biomedical applications due to its biocompatibility and high hydrophilicity.34−36 Specifically, commercially available (PEGSH)4 with star-like architecture and 10 000 g mol−1 molecular weight was used in this work. Compared to our previous study,32 (PEGSH)4 was selected to offer a cross-linking point that is expected to result in stiffer free-standing hydrogels. In addition, the presence of this cross-linking point implies that Au(I)− Au(I) metallophilic attractions are not essential to form the 3D network, unlike our previous studies with linear PEG(SH)2 dynamic hydrogel that required the presence of sufficient aurophilic attractions to cross-link polymer chain ends. Furthermore, more stable gold precursor, such as Au(III), could also be used to generate Au−S species. Here, thiol end groups of (PEGSH)4 were employed first, as a reducing agent of Au(III) to Au(I) and then, to form Au−S species, as shown in the inset in Figure 1B.31 Therefore, a 1:3 molar ratio of HAuCl4:SH was required to convert Au(III) added into Au−S. It is important to notice that 4 equiv. of HCl and 1 equiv. of disulfide were formed during Au−S formation.37−40 Obviously, the final material will contain a mixture of Au−S species (which are expected to self-assemble via Au(I)−Au(I) aurophilic attractions) and disulfide bonds at acidic pH (Figure 1A).33,41 Quantification of the amount of free thiols for (PEGSH)4 was carried out by Ellman’s test and was found to be around 0.24 μmol SH/mg of polymer. This value was much lower than the theoretical amount of free thiols calculated at 0.4 μmol SH/ mg, assuming that all end chains of (PEGSH)4 are functionalized with a thiol group. Thus, around 40 mol % of thiols from the commercial product are already oxidized into disulfide bridges. While 1H NMR could not confirm the degree of oxidation of (PEGSH)4, gel permeation chromatography (GPC) of (PEGSH)4 dissolved in tetrahydrofuran (Figure S2 C


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Figure 2. (left) Unification of PEGSS hydrogel (white) with Au20 dynamic hydrogel (pink), both at pH 8, after simple contact for 2 days. (right) Schematic representation of Au−S/SS exchange between Au20 and PEGSS to form a unique network.

Table 1. Summary of the Relaxation Time (λt) and Shear Elastic Modulus (G′) Obtained at 0.1 and 10 Hz for Dynamic Hydrogels Prepared with Different Amounts of Au(I) or Ag(I) Ions, Concentration of (PEGSH)4 and at Different pH Values name

Metal(I)

amount of Metal(I) (mol %)

[hydrogel] (wt %)

pH

statea

G′b at 0.1 Hz (Pa)

G′b at 10 Hz (Pa)

λtc (s)

PEGSS Au20 Au20 Au20 Au20 Au20 Au20 Au20 Au20 Au20 Au10 Au5 Ag20 Ag60 Au60

Au Au Au Au Au Au Au Au Au Au Au Ag Ag Au

0 20 20 20 20 20 20 20 20 20 10 5 20 60 60

5 5 5 5 5 5 3 7 10 20 5 5 5 5 5

8.0 2.1 4.0 6.0 7.5 10.7 7.5 7.5 7.5 7.5 7.5 7.5 7.0 7.5 7.5

free-standing free-standing free standing flowing flowing flowing flowing free-standing free-standing free-standing free-standing free-standing flowing flowing flowing

670 720 650 210 30 1 1 590 2160 4770 50 570 20 1030 1400

690 730 750 1480 1650 400 175 4530 10 650 14 990 950 1340 1150 4140 5900

∼ 100 4.0 0.18 0.03 0.14 0.20 0.18d 0.18d 0.33d 3.98d 0.56 6.31 6.31

Determined by the inverse tube method as “free-standing” when the material maintained its dimensions and “flowing” when the material reacted with gravity over 24 h. bDetermined by rheology studies. cRelaxation time calculated with λt = 1/ωc, where ωc is the frequency when G′ = G″; d Determined as G″ maximum from dynamic rheology data. a

In order to demonstrate the existence of Au−S/SS exchange, PEGSS hydrogel (5.0 wt %) was placed in contact with Au20 dynamic hydrogel, adjusted at pH 8 in a sealed vial, as shown in Figure 2 (left-hand side pictures). Phenol red was added to Au20 for visual identification of both hydrogels. Within a few hours, both hydrogels were attached, with the pink Au dynamic hydrogel on one side, and PEGSS hydrogel on the other side. After 48 h, homogeneous pink hydrogel was obtained, which demonstrates the formation of a single network via Au−S/SS exchange (Figure 2, right-hand-side scheme). It is also worth mentioning that both hydrogels adjusted at pH 3 remained separated as two independent networks. This result confirms that Au−S/SS exchange, similarly to thiolate/disulfide exchange, is closely dependent on the pH of the hydrogel. The absence of exchange reaction at low pH also explains that Au20 was obtained as a free-standing hydrogel when freshly prepared. Rheology Studies. Au-based hydrogels were prepared at different pH values, (PEGSH)4 concentration and with lower amount of Au−S species (Table 1). The mechanical properties

pHs. Clearly, the presence of Au−S species is responsible for the dynamic character observed for Au20 at alkaline pH. The inherent acidification of the hydrogel, resulting from HCl produced during the formation of Au−S species, could represent a major issue for its application in the field of biomaterials. To solve this problem, a sufficient amount of NaOH to neutralize the quantity of HCl produced during the formation of the hydrogel was added to (PEGSH)4 aqueous solution. By monitoring the aerial oxidation of thiolates with Ellman’s test, the number of free thiolates appeared to be stable for 30 min in the presence of sufficient NaOH (5 μL at 5.0 M) to neutralize HCl (Figure S5). Therefore, dynamic hydrogels could be easily injected at physiological pH within 30 min after adding NaOH to the aqueous solution containing (PEGSH)4 (see Figure 1D and Movie 1 in the Supporting Information). It is also worth mentioning that NaOH could not be added to the aqueous solution containing HAuCl4 due to the low stability of Au(III) at alkaline conditions, resulting in the spontaneous formation of Au nanoparticles. D


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Figure 3. Frequency sweeps (■ G′ and □ G″) of Au20 hydrogels at 5.0 wt % adjusted at different pH values (2.0, 4.0, 6.0, 7.5 and 10.7) as well as the corresponding oxidized PEGSS hydrogel at 5.0 wt % and pH 8. The dashed vertical line indicates the frequency, ωc, at G′ = G″, which corresponds to the liquid-to-gel transition.

mention that PEGSS hydrogel exhibited similar rheological behavior at pH 2 and pH 10 (see Figure S7 in the Supporting Information), unlike the frequency-dependent mechanical properties attributed to disulfide exchange observed by Deng et al.23 At pH 4, G′ and G″ reached similar values at high frequencies compared to Au20 at pH 2 and PEGSS hydrogel. However, a crossover between G′ and G″ is likely to occur at around 0.01 Hz. This crossover between G′ and G″ prefigures the rheological properties observed at higher pH. For instance, Au20 at pH > 6 and 5.0 wt % exhibited frequency-dependent rheological characteristic of dynamic hydrogels.19,23,28,32 In more details, G″ was greater than G′ at low frequencies, which is characteristic of a liquid. This result confirmed the ability of Au20 at 5.0 wt % and pH > 6 to flow under gravity. At increasing frequency, G′ became greater than G″, resulting in a crossover corresponding to a liquid-to-gel transition (G′ = G″) at a frequency ωc, as shown in Figure 3. The relaxation time of the material, λt, was estimated from the relationship ωc = 1/λt. As already reported, λt for dynamic hydrogel represents the time required for the network to rearrange via its dynamic bonds.19,23,28,32,46 Typically, at low frequencies, Au−S/SS exchange was sufficiently rapid for the polymer chains to reorganize, which resulted in a liquid-like behavior of the dynamic hydrogel. In contrast, at frequencies higher than ωc Au−S/SS exchange was slower than the frequency of deformation, which did not allow the network to rearrange, resulting in a solid-like behavior of the hydrogel. From Table 1,

of those hydrogels were studied by dynamic rheology, which depicts shear elastic modulus (G′) and shear loss modulus (G′′) presented as a function of frequency (ω) at fixed strain γ = 1.0%. This fixed strain was determined to be in the viscoelastic region of the material where the network is not damaged, as judged by strain sweep experiments (Figure S6). For easier interpretation, the variations of G′ and G″ resulting from frequency sweep experiments were plotted linearly and not on logarithmic scale as commonly presented. Figure 3 shows the rheological properties for Au20 at 5.0 wt % adjusted at different pH values. The free-standing hydrogel states observed for Au20 at pH 2.0 and 4.0 was confirmed with G′ > G″ at all frequencies, which is characteristic of covalent hydrogels. Nearly identical frequency sweep trends were obtained for Au20 at pH 2 and PEGSS hydrogels at frequencies higher than 0.1 Hz. On the other hand, the slight increase of G′ (and decrease of G″) observed at very low frequencies might indicate that Au−S/SS exchange occurs even at very low pH. However, this exchange reaction was so slow that the hydrogel could not flow and its dimensions were maintained under gravity, as judged by the inverse tube method over 24 h (Table 1). Moreover, similar G′ at high frequencies also indicates that, unlike Au-based PEG(SH)2 dynamic hydrogel, the low amount of Au(I)−Au(I) attractive forces, confirmed by UV−visible spectroscopy, was not sufficient to act as additional cross-linker and did not affect the mechanical rheological properties of the hydrogel. It is important to E


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Figure 4. Frequency sweeps (■ G′ and □ G″) of Au20 dynamic hydrogels at pH = 7.5 prepared at 3.0, 7.0, 10, and 20 wt % concentrations.

it is clear that λt for Au20 at 5.0 wt % was shorter at increasing pH, which corresponds to higher concentration of nucleophilic thiolate anions ([S−]). More surprisingly, Au−S/SS exchange appeared to occur at very low [S−]. For instance, with a pKa located at pH 8.4 (as judged by acid titration of (PEGSH)4, Figure S8), less than 0.01% of the 20 mol % of Au−S were in their reactive thiolate form at pH 4 but appeared to be sufficient to induce Au−S/SS exchange. Plotting λt versus pH showed that the dynamic properties of Au20 hydrogels at 5.0 wt % were kinetically locked below pH 4, where an asymptotic limit is reached due to insufficient [S−] to promote the exchange reaction (Figure S9). In contrast, dynamic hydrogels with frequency-dependent mechanical properties were produced at pH > 4. Surprisingly, stiffer Au20 hydrogels were obtained at pH 6.0 and 7.5 compared to PEGSS hydrogel, with G′ reaching values >1000 Pa at 10 Hz (Table 1). Stiffer dynamic hydrogels at increasing frequency was attributed to higher degree of polymer chains entanglements, which act as additional cross-linking points.10,32 On the other hand, at pH 10.7 high [S−] promoted fast Au−S/SS exchange. The corresponding dynamic hydrogel, with short λt ∼ 0.03 s, behaved mainly like a liquid over the frequency range. To the best of our knowledge, Au(I) capping is the first example that allows thiolate/disulfide exchange to be readily occurring in aqueous media and alkaline pH under ambient aerobic conditions for a long period of time (>1 year, Figure S10 in the Supporting Information). Au−S interactions were somewhat sufficiently strong to protect thiols from aerial oxidation but did not affect the nucleophilic character of thiolates required for the exchange reaction. Further studies would be required to understand this mechanism. Dynamic rheology studies of Au20 dynamic hydrogels prepared at different concentrations (3.0, 7.0, 10.0, and 20.0 wt %) were carried out, as shown in Figure 4. The characteristic liquid-to-gel transition was observed for Au20 prepared at only 3.0 wt % (Figure 4). It is noteworthy that similar λt values were obtained for Au20 at 5.0 and 3.0 wt % adjusted at the same pH. More interestingly, at pH 7.5 free-standing dynamic hydrogels

were achieved at 7.0, 10.0, and 20.0 wt % concentrations, with G′ > G″ at all frequencies, as shown in Figure 4 and confirmed by visual inspection (Table 1). As expected, stiffer hydrogels (higher G′) were obtained at increasing concentrations of (PEGSH)4, as shown in Table 1 and Figure 4. For instance, G′ at 0.1 Hz increased from 590 Pa to 4,770 Pa for Au20 prepared at 7.0 and 20 wt %, respectively (Table 1). The formation of free-standing hydrogels at concentrations greater than 7.0 wt % indicates that sufficient (PEGSH)4 are linked via disulfide bridges to produce a fully cross-linked network. However, the dynamic character was observed at high frequencies as G′ was increasing, especially for Au20 at 10.0 and 20.0 wt %. Hence, Au−S/SS exchange is still dictating the rheological behavior, conferring dynamic properties to the hydrogels despite the formation of a 3D network via disulfide bridges. The rheological properties of dynamic hydrogels prepared at 5.0 wt % and pH 7.5 with 10.0 and 5.0 mol % of Au−S (designated as Au10 and Au5, respectively) were also studied (Figure 5). By using HAuCl4 to form Au−S bonds, only hydrogels with less than 20 mol % Au−S could be prepared

Figure 5. Frequency sweeps (G′: plain symbols; G″: open symbols) of Au10 (circles) and Au5 (squares) dynamic hydrogels prepared at 5.0 wt % and adjusted at pH 7.5. F


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Biomacromolecules without forming Au nanoparticles. It is important to mention that the excess of unreacted thiolates was left to oxidize to form disulfide bridges via aerial oxidation at pH 10. Full oxidation of free thiolates was achieved when overlapped frequency sweep curves were obtained after 24 h for the same dynamic hydrogel, as judged by dynamic rheological studies. Similarly to Au20, liquid-to-gel transition was observed for Au10. As expected, λt for Au10, estimated at 0.25 s, was slightly longer than λt for Au20 at 0.18 s due to lower [S−] at the same pH. In contrast, free-standing hydrogel was obtained for Au5, which exhibits G′ greater than G″ at all frequencies. At frequencies between 0.01 and 0.1 Hz, G′ and G″ appeared to be constant with similar values to the corresponding PEGSS hydrogel. However, an increase of both moduli was observed between 0.1 and 1.5 Hz. Finally, G′ increased dramatically at frequencies higher than 1.5 Hz, while G″ decreased. This inflection point at 1.5 Hz, also observed for Au20 at 10.0 and 20.0 wt % concentrations at around 5 Hz in Figure 4, could be considered as a rough estimation of ωc to determine λt for free-standing dynamic hydrogels (Table 1). Then, λt was estimated to be at around 0.67 s for Au5. Thus, λt(Au5) > λt(Au10) > λt(Au20) confirms that a lower amount of Au−S species at the same pH, corresponding to lower [S−], led to slower Au−S/SS exchange and longer time required for the network to rearrange. Interestingly, the delay of oxidation of free thiols required to obtain Au5 hydrogel could be used to adjust the gelation time of the dynamic hydrogel. For instance, Au5 required around 5 h after mixing for G′ to be greater than G″. Therefore, the gelation time of Au-based dynamic hydrogels could be tuned by adding judicious amounts of Au(III). Optimization of the gelation time of those dynamic hydrogels merits further study; nevertheless, these data illustrate how the properties of Aubased dynamic hydrogels can be easily tuned by adjusting parameters such as the amount of Au−S, (PEGSH) 4 concentration and pH. In addition, judicious adjustment of those parameters would allow injectable dynamic hydrogels to be prepared with the exact same rheological properties as SF or NP to treat patients suffering RA or IVD degeneration, respectively. Self-Healing Properties. Self-healing properties of Au−Sbased dynamic hydrogel could be anticipated due to the permanent Au−S/SS exchange at pH > 4. As shown in the movie in the Supporting Information, Au20 dynamic hydrogel prepared at 10 wt % and pH 7.5 cut in two pieces was recovered after simple contact during a dozen of seconds. It is worth mentioning that the corresponding PEGSS hydrogel at 20 wt % and pH 7.5 did not show self-healing properties. Interestingly, Au−S/SS exchange allowed the form of the dynamic hydrogel to be changed within a couple of hours when the intact dynamic hydrogel was placed in a different mold with slight pressure (Figure 6). The relatively fast self-healing ability of Au20 dynamic hydrogel offers the advantage to inject the preformed material previously shredded into confined space. In addition, sufficient pressure in the injected location would ensure the hydrogel to fill the totality of the cavity. Also, this unconventional injection method could be useful to avoid the diffusion of the biomaterial into living tissues. The recovery of the mechanical properties after self-healing of damaged dynamic hydrogels was evaluated by dynamic rheology and compression studies (Figure 7). First, disc-shaped Au20 dynamic hydrogel required for rheology studies was cut in 2 pieces and recovered by simple contact during 1 min (inset pictures in Figure 7A). Frequency sweep experiments were

Figure 6. Self-healing behavior of Au20 dynamic hydrogel at 10 wt %: the hydrogel cut into small pieces with a scalpel could be reformed as a disc or as a tube depending on the shape of the mold used. The tubular shaped hydrogel could also from a disc by simple pressure into the corresponding mold for a couple of hours.

Figure 7. (A) Dynamic rheology results of G′ (plain symbols) and G″ (open symbols) for Au20 at 10 wt % (▲) before and (■) after cutting the hydrogel in two pieces and put in contact for self-healing process for 1 min. (B) Stress versus strain curves for Au5 at 20 wt % (green line) before and (blue line) after cutting the hydrogel into pieces and placing it in cylindrical mold with slight pressure overnight as shown in inset.

carried out on the pristine and recovered hydrogels. Similar frequency sweep curves were obtained for both materials. This result confirmed that complete recovery of the rheological properties was achieved after putting in contact during 1 min both pieces (Figure 7A). On the other hand, dynamic hydrogel G


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Biomacromolecules with a low amount of Au−S and high concentration of polymer was selected in order to challenge its self-healing ability. Au5 dynamic hydrogel prepared at 20 wt % was first compressed at 10 mm min−1. Interestingly, more than 90% deformation was observed without any rupture or break of the dynamic hydrogel (Figure 7B). After compression, the hydrogel was completely flattened, but no water was expelled from the polymeric network. After cutting the dynamic hydrogel into pieces with a scalpel, Au5 at 20 wt % placed in the same tubular mold under light pressure was completely recovered overnight, as shown in the inset of Figure 7B. The same compression test was carried out on the recovered hydrogel. As shown in Figure 7B, up to 90% of the resistance of the material was recovered. Full recovery could not be achieved as nonhealing C−C and C−O bonds were certainly broken when the hydrogel was cut into pieces. Nevertheless, sufficiently high recovery was obtained for such hydrogel confirming that small pieces of hydrogel could also be injected with no dramatic loss over the mechanical properties of the former hydrogel. Cytotoxicity Studies of the Dynamic Hydrogel. In vitro studies were carried out in order to investigate the cytotoxicity of Au20 dynamic hydrogel at 5.0 wt % on the cell morphology, proliferation and viability of human dermal fibroblasts (HDFs) during 7 days (Figure 8). Cell proliferation was assessed using MTS colorimetric assay on days 1, 3, and 7. MTS results (Figure 8, top) showed that HDFs proliferation was influenced by the dynamic hydrogel. HDFs in the presence of Au20

dynamic hydrogel were growing at a slower rate than cells alone, but a population doubling level of 2.8 ± 1.2 was reached after 7 days. Moreover, the absorbance values increased gradually from day 1 to day 7, confirming the proliferation, albeit slower, of HDFs in the presence of Au20 dynamic hydrogel. Ki67 staining at day 3, which allows the visualization of proliferating nuclei by fluorescence microscopy, demonstrated the effective proliferation of HDFs as shown on the merged images shown in Figure 8 (middle figure). Finally, Live/Dead assay confirmed that in the presence of the hydrogel a large majority of healthy HDFs (green) were observed compared to the high number of dead cells (red) observed for the negative control (Triton X-100) (Figure 8, down). In addition, cultured HDFs exhibited their typical morphology, appearing as elongated, spindle-shaped cells. Thus, despite a slight effect on cell proliferation Au20 dynamic hydrogel did not show cytotoxicity toward HDFs. It is important to point out that cells cannot be seeded into the hydrogel due to the lack of bioactivity of PEG homopolymer. However, one can assume that grafting biomimetic peptides such as RGD peptides, via the reactive thiols, will enhance cell adhesion and proliferation within the dynamic hydrogel, which allows tissue engineering applications to be anticipated, as previously reported by others.47,48 Silver-Based Dynamic Hydrogels. As already studied in previous works, silver(I) ions [Ag(I)] can also be used in combination with low molecular weight compounds having a single thiol group to form supramolecular hydrogels containing silver-thiolate species (Ag−S).49 Here, Ag−S based dynamic hydrogels were prepared by mixing an aqueous solution of (PEGSH)4 and AgNO3. It is noteworthy that only one thiol is consumed to form of Ag−S species, as AgNO3 already provides the required oxidation state I to form the corresponding thiolate. First, Ag20 was prepared at 5.0 wt %, resulting in a highly viscous solution at around pH 3.6. The excess of unreacted thiols were left to oxidize under atmospheric conditions after adjusting Ag20 to pH 9 with an aqueous solution of NaOH (1.0 M). It is important to mention that the pH was further adjusted to pH 7 with an aqueous solution of HNO3 (1 M) because the addition of HCl resulted in the irreversible precipitation of AgCl. Then, Ag20 adjusted at pH 7 exhibited similar dynamic properties as Au20 in the same conditions, as shown in Figure 9A. Here, a liquid-to-gel transition was also observed for the dynamic hydrogel with λt at around 0.56 s (Table 1). This relaxation time is in good agreement with the value obtained for Au20 at pH 6.0 and 7.5, which were estimated at 4 and 0.03 s, respectively (Table 1 and Figure S9). This result illustrates that the nature of Metal(I) capping does not affect the exchange rate responsible for the dynamic behavior of the hydrogel. The direct reaction of Ag(I) with thiols allowed the preparation of Ag60, where all free thiols are capped with Ag(I). As expected, the resulting hydrogel at pH 7.5 exhibited frequency-dependent mechanical properties with a liquid-to-gel transition, as shown in Figure 9B. However, despite higher [S−], Ag60 exhibited λt at around 6.31 s, which is much longer than λt obtained for Ag20. In this case, higher [S−] induced lower [SS], as both species are intimately linked. Thus, slower Ag−S/SS exchange (longer λt) can be attributed to a lower concentration of disulfide available for the exchange reaction. For comparison, Au60 was prepared by using an aqueous solution of Au(I) ions prepared as previously described.33 The liquid-to-gel transition was observed at the exact same

Figure 8. (Top) Proliferation at day 1, 3, and 7 of human dermal fibroblast (HDF) cells alone (black) and in the presence of Au20 dynamic hydrogel prepared at 5.0 wt % and pH 7.4 (gray). (Middle), Ki67 staining of HDF. Nuclei were counterstained with Hoechst 33258 (blue). Scale bars, 50 μm. (Down) Fluorescent microscope images of the Live/Dead assay with healthy cells (green) and dead cells (red) for (left) HDF alone as positive control, (middle) HDF in the presence of Au20 at 5.0 wt %, and (right) HDF treated with Triton X-100 as negative control (scale bar = 100 μm). H


Article

Biomacromolecules

at room temperature. Simultaneous injection of aqueous solutions of commercially available HAuCl4 (or AgNO3) and (PEGSH)4 resulted in a dynamic hydrogel based on Au−S/SS (or Ag−S/SS) exchange. The dynamic character of Au−S/SS exchange could be controlled by adjusting the pH of the hydrogel, resulting in free-standing hydrogels at pH < 4 due to low [S−] or frequency-dependent dynamic hydrogels at pH > 4 due to increasing [S−]. In addition to pH, the mechanical properties of Au-based hydrogels could be tuned by varying the concentration of (PEGSH)4 and the amount of Au−S incorporated into the network. Interestingly, the permanent Au−S/SS exchange conferred self-healing properties and reshaping abilities to the dynamic hydrogel with up to 90% recovery of the original mechanical properties. In vitro studies proved that Au-based dynamic hydrogels were not cytotoxic to HDF, which confirmed that Au-based dynamic hydrogels could be a good candidate for applications as medical device. Finally, Ag(I)-based dynamic hydrogels demonstrated that the nature of the Metal(I) capping did not affect the rate of the exchange reaction. In addition, a high amount of Metal(I) appeared to strengthen the hydrogel due to the presence of metallophilic attractions as additional cross-linking interactions. The facile capping of thiols using Metal(I) offers a new way to produce injectable and self-healing dynamic hydrogels with tunable mechanical properties offering frequency-dependent stiffness, from virtually any thiolated polymers. Such materials would be of interest for application ranging from viscosupplementation material to drug/cell delivery scaffolds, as well as wound healing, depending on the Metal(I) used to cap thiolates.

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Figure 9. Frequency sweep with G′ (plain symbols) and G″ (open symbols) of (A) Ag20 prepared at 5 wt % and pH 7.0 and (B) Ag60 (circles) and Au60 (squares) prepared at 5 wt % and adjusted at pH 7.5. Note that Au(I) was used instead of HAuCl4 to prepare Au60.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00980. Experimental procedures (Ellman’s Test, calibration curve, Ag-based hydrogel, Au60 hydrogel, and in vitro studies) and additional characterization (1H NMR, gel permeation chromatography, and UV−visible spectroscopy) (PDF) Movie of hydrogel injection (AVI) Movie of self-healing ability (The logo appearing in the movie is used with permission from IK4-CIDETEC) (AVI)

frequency for Au60 and Ag60 (as shown in Figure 8B). Identical λt confirms that not only [S−] but also [SS] affect the rate of the exchange reaction responsible for the dynamic properties of the hydrogel. Interestingly, Au60 and Ag60 dynamic hydrogels proved to be much stiffer than any of the hydrogels studied in this work with G′ > 4,000 Pa at 10 Hz. Such high concentration of Metal(I) might result in a nonnegligible amount of metallophilic attractions. Similarly to Aubased PEG(SH)2 dynamic hydrogel, Metal(I)−Metal(I) attractive forces can act as an additional cross-linker, resulting in a stiffer hydrogel.32 In addition, it is well know that Au(I)− Au(I) attractions are stronger than Ag(I)−Ag(I) attractions, due to relativistic effects that considerably strengthen aurophilic attractions.50−52 Hence, the higher stiffness observed for Au60 was attributed to the stronger aurophilic attractions compared to the argentophilic attractions present in Ag60. Finally, applications of Ag-based dynamic hydrogels as antimicrobial coatings for medical devices and wound healing applications could be anticipated, due to the long lasting biocide properties of active Ag(I) toward bacteria including Eschcerichia coli.53−55 Such studies are currently under progress and expected to be published in a near future.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

ETORTEK research program (IE11-301 and IE12-332) from the Basque government and INNPACTO (IPT-2012-0745300000) from the Spanish Ministry of Economy and Competitiveness are acknowledged for funding this work.

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Notes

The authors declare no competing financial interest.

CONCLUSIONS In summary, we report an efficient yet simple method to protect thiols/thiolates from aerial oxidation by Metal(I) capping [Au(I) or Ag(I)] without preventing exchange reaction between nucleophilic thiolates and disulfides that readily occurs

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ACKNOWLEDGMENTS Miss Nati Diaz is acknowledged for her precious help in the experimental work. I


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Biomacromolecules

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Injectable and Self-Healing Dynamic Hydrogels Based on Metal(I

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