- Email: [email protected]
Self-healing gelatin ionogels
International Journal of Biological Macromolecules 95 (2017) 603–607
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Review
Self-healing gelatin ionogels Anshu Sharma a,b , Kamla Rawat b,c,∗ , Pratima R. Solanki b , H.B. Bohidar a,b,∗ a
School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi, India c Inter University Accelerator Centre, New Delhi 110067, India b
a r t i c l e
i n f o
Article history: Received 21 October 2016 Accepted 27 November 2016 Available online 28 November 2016 Keywords: Self-healing gel Ionogel Rheology Recovery Storage modulus
a b s t r a c t We demonstrate room temperature (20 ◦ C) self-healing, and substantial recovery (68–96%) of gel rigidity of gelatin, a polypeptide, ionogels (made in 1-ethyl-3-methylimidazolium chloride ionic liquid (IL) solutions via thermal treatment, IL ≤ 5% (w/v)) after they were cut using a surgical blade. The recovery process did not require any stimuli, and the complete healing under ambient condition required about 10 h.The self-healing owed its origin to the reformation of network structures via imidazolium ion mediated charge quenching of deprotonated residues, and hydrophobic interaction between neighbouring alkyl tails of IL molecules. The rate of healing determined from the growth of rigidity modulus was 20 ± 5 mPa/s independent of ionic liquid content of the gel. This was true regardless of the fact that ionogels containing more IL had a lower gel modulus due to propensity of hydrophobic linkages, but these were agile enough to recover their network structures to a higher degree during the healing process. These features indicate that the gelatin ionogel being biocompatibile, and biodegradable holds great potential for applications in the field of biomedical engineering. © 2016 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 3.1. Time-dependent microscope imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 3.2. Time-dependent rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 3.3. Dye diffusion studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605 3.4. Phenomenology of healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
1. Introduction Self-healing is a key property of living tissues that allows them to sustain repeated damage. Physical hydrogels are solventfilled networks, formed by physically crosslinked biopolymers, in which secondary interactions such as hydrogen bonds, electrostatic interactions, hydrophobic interactions or van der Waals forces
∗ Corresponding authors at: Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi, India. E-mail addresses: [email protected] (K. Rawat), [email protected] (H.B. Bohidar). http://dx.doi.org/10.1016/j.ijbiomac.2016.11.103 0141-8130/© 2016 Elsevier B.V. All rights reserved.
are responsible for formation of reversible crosslinks (entanglements). Hydrogels are crosslinked hydrophilic polymer assemblies classically made from high molecular weight biopolymers such as gelatin, fibrin, chitosan, pectin, carrageenan, cellulose etc to name a few. Gelatin, in particular is an interesting biomaterial that is available in plenty in the biosphere, and this material has been extensively studied in the past. Chemical composition of this biopolymer depends on its source of origin. Hydrophobic amino acids like proline (Pro), hydroxyproline (Hyp), and glycine (Gly) are in propensity in gelatin. The general primary sequence is given by of (Gly-X-Pro) and (Gly-X-Hyp), in which X represents other amino acids [1]. Due to the self-assembled origin of these gels, the gel strength quickly recovers after the material experiences mechani-
604
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
cal deformation [2]. Thus, physical polymer networks offer unique advantages, over the chemical gels that are difficult to manipulate. In mammals self-healing is provided by fibrous tissues, like cartilage that are structured by non-covalent crosslinked biopolymers. In the recent past, major attempts have been made to introduce non-covalently crosslinked biopolymers and their supramolecular assembly to develop self-healing materials. Sometimes, an external trigger in the form of energy or healing moiety is needed for a self-healing to occur [2]. Dynamic-reversible materials (efficiency in detecting and “autonomically” healing damage) displays a wide range of responses from self-healing to mechanical work for applications such as sensors, actuators and various other biomedical applications. Supramolecular chemistry [3] uses non-covalent forces such as hydrogen bonding and – stacking giving rise to a variety of stimuli-responsive self-healing materials [4–10]. Macroscopic self-assembly uses the common host–guest molecular recognition [11]. Often external stimuli thermal, radiation, catalytic, and mechanical actions may cause transformations in these structures by thermodynamic reorganization [12–14]. Deng et al have synthesized crosslinked polymer gels with reversible covalent bonds under acidic conditions that showed healable properties [15]. Synthesis of healable polymer gels with trithiocarbonate units in their structures has been proposed by Matyjaszewski and coworkers [16,17]. Similarly, reversible Diels–Alder units have been studied for thermally healable potential[18–20]. It is reported that Diarylbibenzofuranone (DABBF) can reach a state of thermodynamic equilibrium in the absence of any stimuli and the radical species arising from cleaved DABBF was found to tolerant to oxygen [21–26]. Imato et al. prepared crosslinked DABBF containing polymer gels with cleavable properties under ambient conditions [27]. All the above mentioned examples involve chemically crosslinked network gels formed of synthetic polymers that often use toxic crosslinkers. Not many self-healing physical gels made of biopolymers are reported in the literature. Herein, we report the gel recovery process (self-healing) of gelatin ionogels under ambient conditions after their mechanical incision. The extensive physical characterization of gelatin ionogels has been reported by us earlier [28]. The enormous application potential of healable biocompatible gels makes the current report timely and significant, it is more so because these gels are green materials.
2. Materials and methods Gelatin (GB) (225 bloom, pI ∼ 4.9 ± 0.2) having nominal molecular weight of 50 kDa and ionic liquid (IL) 1-ethyl-3methylimidazolium chloride were purchased from Sigma-Aldrich, USA, and used as received. The molecular structure of gelatin and the ionic liquid used are shown in Fig. 1. Gelatin ionogel was prepared by dissolving 5% (w/v) of GB in IL solution with varying percentage of IL [0–5% (w/v)] made in deionized water maintained at 60 ◦ C. Continuous stirring for 30 min produced a clear solution, which was then cooled to room temperature (20 ◦ C). After a lapse of gelation time, the contents turned into a rigid gel which was checked by, inversion of the sample holder, and the observation of a non-flowing meniscus implying that it could support the weight of the gel. Concentrations are in (% w/v) unless otherwise stated. The dynamic rheological profiling of the samples were done on a stress controlled rheometer (AR-500, T.A. Instruments, UK). A2◦ cone-plate geometry of 20 mm radius and a truncation gap of 500 m was used, and the oscillatory stress value was fixed at 4.775 Pa. About, 100 mL of the sample was placed on the peltier plate and it was to equilibrate for 5 min before proceeding with measurements. Silicone oil was applied to the outer circular edge of the paltier plate and wet sponge was used as solvent trap to
prevent loss of solvent due to evaporation. Bright field biological microscope (Leedzmicro-imaging LTD, U.K.) was used for imaging of the samples. 3. Results and discussion Gelatin, a biopolymer, is produced by denaturation of collagen through either alkaline or acid processing, during which the interconnected triple-helix units are melted into three distinct single strand gelatin chains [29]. When gelatin is dissolved in warm water these random-coil chains form a homogeneous sol. Upon cooling it to room temperature, these molecules undergo a coil–helix transition, and gradually get physically crosslinked by entanglements to form thermoreversible physical gels with a typical gelation tem◦ perature of Tgel ≈ 28 C [30]. 3.1. Time-dependent microscope imaging First we prepared thin films of gels on cover slip glass plates by pouring about 0.5 mL of the sol on the plate surface. The plate was stored in a constant temperature incubator over night at a temperature of 20 ◦ C. A transparent gel was set during this period. The ionogel film was then cut into two pieces by using a surgical blade. These samples were placed in the incubator maintained at the same temperature for about 10 h without disturbance. Their recovery pattern was recorded using an optical microscope. These images are depicted in Fig. 2 for two representative ionogels(IL = 1% and 5%). It is clearly seen from the time-sequence of these images that the incision scar disappears typically over a period of 10 h, after which the two pieces are visually unidentifiable and the gel pieces fuse to become one unit again. 3.2. Time-dependent rheology The healing process was monitored by following the time dependent rigidity modulus of the gels. For the recovery analysis of the ionogel samples, a four-step program on stress controlled rheometer was used. In the first step, the hot sol samples were poured onto the peltier plate maintainedat 10 ◦ C, and kept isothermal at the room temperature for 3 min, and then time sweep measurement was performed at an angular frequency of 1 rad s−1 at the oscillatory stress value of 4.775 Pa to observe the temporal growth of low frequency storage modulus, G0 . When storage modulus became constant, we stopped the measurement (generally after 4–5 h). This characterized the ionogel sample. In the second step, the sample was cut into two pieces by using a surgical blade and these pieces were placed touching face to face for 10 h without disturbance. In the third step, we repeated the time sweep measurement on the healed gel sample until its G0 value reached a plateau. For this measurement, the reference to initiation of healing was defined as the time when the sample wasloaded onto the geometry of the rheometer, which was set as t = 0. Fig. 3(A) shows the data of isochronal time sweep experiment at constant strain during which the temporal growth of the low frequency storage modulus was monitored for the gel samples. Rheology data shows the self-assembly of the ionogels progressed, and the formation of networks continued to reach a matured state typically after tsat = 4 h of gelation. The saturation values of G0 are depicted in Fig. 3(A) for various ionogels samples. The time sweep experiment was halted after when saturation in storage modulus values was observed, this equilibrium G0max values are clearly shown in Fig. 3(B). Next the gel was cut into two pieces using a surgical blade (tcut in Fig. 3(A)). A sharp drop in storage modulus after incision resulted from the disruption of physical crosslinks is clearly seen in this figure. For instance, the saturation modulus of
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
605
Fig. 1. The molecular structure of gelatin and ionic liquid used.
Fig. 2. Hourly time sequence images of ionogels, (a) top panel, IL = 1%, and (b) bottom panel, IL = 5%, indicating healing under ambient conditions. Arrows indicate the incision line which fades with time as the gel heals. Note the complete healing after 10 h.
the ionogel was G0max = 585 Pa for 5% IL sample, which after incision dropped to G0cut = 512 Pa. The two pieces were placed touching each other and rested for theal = 5.5 h to heal under ambient condition. Rheology measurement was initiated and the full recovery of gel modulus was followed starting at trs (Fig. 3(A)). After recovery for 5% IL ionogel, the restored modulus was G0recov = 562 Pa This procedure was followed for all the ionogels samples. The quantitative recovery profile is shown as a bar diagram in Fig. 3(B). The complete recovery process continued over a period of several hours with corresponding increase in gel modulus. The% recovery and the complete healing time were dependent on IL concentration. For example, 96% of the gel modulus was restored for 5% IL ionogels after healing making this an excellent healable material. In contrast, for gelatin hydrogel (IL = 0) the recovery of gel rigidity was limited to 45% only. The rate of recovery, defined as (dG0 /dt was 20 ± 5 mPa/s independent of IL content of the gel. Here, G0 = (G0recov - G0cut ). The best recovery was noticed for [IL] = 5% ionogels.
3.3. Dye diffusion studies
Fig. 3. (A) Shows variation of low frequency dynamic storage modulus G0 as a function of time for different concentration of IL. (B) Shows dynamic storage modulusG0 and% recovery as a function of IL concentration. Arrow indicates the time when incision was made.
In the next step, we wanted to demonstrate that a diffusion path existed between the rejoined gel pieces. For this the two cut gel pieces were colored separately. For preparing the red gel block, aqueous solution of rhodamine B dye (0.1 mg/mL) was kept in a beaker in which one of the two gel pieces was kept immersed for 30 min which turned the gel red in color due to osmosis. Other piece was similarly prepared by dipping it in aqueous solution of orange G dye (0.1 mg/mL) for the same duration of time, which produced a yellow gel (Fig. 4(c)).The two blocks were placed touching each ◦ other, and rested in an incubator at 10 C temperature without any intervention (Fig. 4(d)). After 10 h, they were observed to have fused into one piece with a sharp boundary clearly visible between the two pieces. After 24 h, the healed block appeared red indicating the diffusion of rhodamine G dye into the entire gel due to higher diffusivity of this dye(Fig. 4(e)).
606
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
Fig. 4. Self-healing of a 5% IL ionogel. (a) the original gel, (b) the gel cut into two pieces, (c) one piece was soaked in rhodamine B (0.1 mg/mL) and another in orange G ◦ (0.1 mg/mL) dye solution for 30 min each, (d) the two pieces were kept in physical contact face to face for 12 h without at 10 C temperature in the incubator, (e) the rhodamine G dye diffused into the gel piece containing orange G dye due to its higher diffusion coefficient. The boundary line between the two halves is clearly seen in (e, arrow) butthe two gel pieces have fused as a whole.
3.4. Phenomenology of healing Physical gels due to the transient character of the entanglements are in a position to sustain the external stress by dislocation, dissociation, and re-orientation of the entangled junctions. This unique relaxation property enables them to exhibit healable property, which in the case of chemical gels is impossible [1,31]. It can be conjectured that the self-healing after mechanical damage was due to the renewal of their network structure via hydrophobic interactions between the aliphatic hydrocarbon chains of the two gel pieces. Based on the data in hand it is possible to offer the following phenomenology of the healing process. In the sol state, the gelatin molecules assume random coil conformation, and under normal pH conditions there is presence of almost a 1:1 ratio of protonated and deprotonated residues in its composition [29]. The positively charged imidazolium head group of the IL will selectively bind to the deprotonated residues while the free Cl− ions will bind to the protonated sites. Further, the hydrophobic alkyl tail of ILs must assemble locally to minimize water contact. Because, of this there is reduced possibility of formation of triple helices in the sol. Thus, what gets formed is a heterogeneous physical network gel. With increased IL content this heterogeneity increases. This is the reason why G0 decreases with IL concentration (Fig. 3 data). Recall, that a homogeneous gelatin hydrogel has a propensity of inter twined triple helix networks which provides it with a high gel rigidity value. When such a hydrogel is subjected to mechanical incision millions of these networks are ruptured. During healing only a fraction of these are restored and the rest are trapped in a dangling state with no binding partner. Note that there is no force that enables their restoration (See Scheme 1). On the contrary, in ionogels plenty of inter gelatin chain binding is facilitated by hydrophobic interactions. When ionogels are exposed to mechanical incision, these hydrophobic interlink (bridges) between gelatin chains get ruptured in abundance, thereby exposing the alkyl tails of IL molecules to interstitial water contact which is energetically unfavourable. When the two gel pieces are placed touching each other, these dangling hydrophobic tails immediately form an overlapped junction to create a water depletion region which facilitates gel healing (See Scheme 1). Thus, the healing is mostly governed by hydrophobic interactions. More is the IL content of the gel, stronger are the hydrophobic forces, faster will be the recovery, and higher will be the restoration of gel
Scheme 1. Gel healing mechanism.
rigidity after healing. This is clearly manifested in our experimental data. 4. Conclusion We have described a biopolymer thermo-reversibleionogel prepared in an ionic liquid solution. The presence of ionic liquid molecules changes the assembly of individual gelatin chains with formation of triple helices mostly suppressed. The IL clad gelatin chains organize under hydrophobic forces to form a physical gel network. After rupture, these gels heal under a driving force which is the hydrophobic interaction between alkyl tails of IL molecules. Ionogelsexhibited healing behaviourat temperatures lower than their gelation temperature. These observations may provide basic clues to the design of self-healing biomaterials that were stabilized via secondary forces. The self-healing ionogels were found to formbonds strong enough to withstand repeated damage. These results could give adequate design directions to the development of self-healing biomaterials. Acknowledgments AS acknowledges University Grants Commission, Government of India for a Junior Research Fellowship. This work was supported
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
by the Department of Science and Technology (DST), India. KR is very thankful to Department of Science and Technology, Government of India, for DST-Inspire Faculty Award. References [1] S.K. Kundu, T. Mztsunaga, M. Yoshida, M. Shibayama, Rheological study on rapid recovery of hydrogel based on oligomeric electrolyte, J. Phys. Chem. B 112 (2008) 11537–11541. [2] L. Yan, S. Gou, Z. Ye, S. Zhang, L. Ma, Self-healing and moldable material with the deformation recovery ability from self-assembled supramolecularmetallogels, Chem. Commun. 50 (2014) 12847–12850. [3] L. Brunsveld, B.J.B. Folmer, E.W. Meijer, R.P. Sijbesma, Supramolecular polymers, Chem. Rev. 101 (2001) 4071–4098. [4] M. Burnworth, L. Tang, J.R. Kumpfer, A.J. Duncan, F.L. Beyer, G.L. Fiore, S.J. Rowan, C. Weder, Optically healable supramolecular polymers, Nature 472 (2011) 334–338. [5] S. Burattini, H.M. Colquhoun, D. Fox, B.W. Greenland, W. Harris, M.E. Mackay, S.J. Rowan, A Self-repairing, supramolecular polymer system: healability as a consequence of donor–acceptor – stacking interactions, Chem. Commun. (2009) 6717–6719. [6] Y. Xu, Q. Wu, Y. Sun, H. Bai, G. Shi, Three-dimensional self-assembly of graphene oxide and DNA into multifunctional hydrogels, ACS Nano 4 (2010) 7358–7362. [7] P. Cordier, F. Tournilhac, C. Soulie-Ziakovic, L. Leibler, Self-healing and thermoreversible rubber from supramolecular assembly, Nature 451 (2008) 977–980. [8] S. Burattini, B.W. Greenland, W. Merino, J. Weng, H.M. Colquhoun, W. Hayes, M.E. Mackay, I.W. Hamley, S.J. Rowan, A healable supramolecular polymer blend based on aromatic - stacking and hydrogen-bonding interactions, J. Am. Chem. Soc. 132 (2001) 12051–12058. [9] P. Mukhopadhyay, N. Fujita, A. Takada, T. Kishida, M. Shirakawa, S. Shinkai, Regulation of a real – time self – healing process in organogel tissues by molecular adhesives, Angew. Chem. 122 (2010) 6482–6486. [10] Q. Wang, J.L. Mynar, M. Yoshida, E. Lee, M. Lee, K. Okuro, K. Kinbara, T. Aida, High-water-content mouldable hydrogels by mixing clay and a dendritic molecular binder, Nature 463 (2010) 339–343. [11] R. Sun, C. Xue, X. Ma, M. Gao, H. Tian, Q. Li, Light-driven linear helical supramolecular polymer formed by molecular-recognition-directed self-assembly of bis (p-sulfonatocalix [4] arene) and pseudorotaxane, J. Am. Chem. Soc. 135 (2013) 5990–5993. [12] S.J. Rowan, S.J. Cantrill, G.R.L. Cousins, J.K.M. Sanders, J.F. Stoddart, Dynamic covalent chemistry, Angew. Chem. Int. Ed. 41 (2002) 898–952. [13] H. Otsuka, K. Aotani, Y. Higaki, A. Takahara, Polymer scrambling: macromolecular radical crossover reaction between the main chains of alkoxyamine-based dynamic covalent polymers, J. Am. Chem. Soc 125 (2003) 4064–4065. [14] X. Chen, M.A. Dam, K. Ono, A. Mal, H. Shen, S.R. Nutt, K. Sheran, F. Wudl, A thermally re-mendable cross-linked polymeric material, Science 295 (2002) 1698–1702.
607
[15] G. Deng, C. Tang, F. Li, H. Jiang, Y. Chen, Covalent cross-linked polymer gels with reversible sol-gel transition and self-healing properties, Macromolecules 43 (2010) 1191–1194. [16] R. Nicolay, J. Kamada, A.V. Wassen, K. Matyjaszewski, Responsive gels based on a dynamic covalent trithiocarbonate cross-linker, Macromolecules 43 (2010) 4355–4361. [17] Y. Amamoto, J. Kamada, H. Otsuka, A. Takahara, K. Matyjaszewski, Repeatable photoinduced self-healing of covalently cross-linked polymers through reshuffling of trithiocarbonate units, Angew. Chem. 123 (2011) 1698–1701. [18] E.B. Murphy, E. Bolanos, C. Schaffner-Hamann, F. Wudl, S.R. Nutt, M.L. Auad, Synthesis and characterization of a single-component thermally remendable polymer network: Staudinger and Stille revisited, Macromolecules 41 (2008) 5203–5209. [19] X. Chen, M.A. Dam, K. Ono, A. Mal, H. Shen, S.R. Nutt, K. Sheran, F. Wudl, A thermally re-mendable cross-linked polymeric material, Science 295 (2002) 1698–1702. [20] K. Ishida, N. Yoshie, Synthesis of readily recyclable biobased plastics by Diels-Alder reaction, Macromol. Biosci. 8 (2008) 916–922. [21] J.C. Scaiano, A. Martin, G.P.A. Yap, K.U. Ingold, A carbon-centered radical unreactive toward oxygen: unusual radical stabilization by a lactone ring, Org. Lett. 2 (2000) 899–901. [22] E.V. Bejan, E.F. Sanchis, J.C. Scaiano, Lactone-derived carbon-centered radicals: formation and reactivity with oxygen, Org. Lett. 3 (2001) 4059–4062. [23] M. Frenette, C. Aliaga, E.F. Sanchis, J.C. Scaiano, Bond dissociation energies for radical dimers derived from highly stabilized carbon-centered radicals, Org. Lett. 6 (2004) 2579–2582. [24] M. Frenette, P.D. MacLean, L.R.C. Barclay, J.C. Scaiano, Radically different antioxidants: thermally generated carbon-centered radicals as chain-breaking antioxidants, J. Am. Chem. Soc. 128 (2006) 16432–16433. [25] K.S. Focsaneanu, J.C. Scaiano, The persistent radical effect: from mechanistic curiosity to synthetic tool, Helv. Chim. Acta 89 (2006) 2473–2482. [26] H.-G. Korth, Carbon radicals of low reactivity against oxygen: radically different antioxidants, Angew. Chem. Int. Ed. 46 (2007) 5274–5276. [27] K. Imato, M. Nishihara, T. Kanehara, Y. Amamoto, A. Takahara, H. Otsuka, Self-healing of chemical gels cross-linked by Diarylbibenzofuranone-based trigger-free dynamic covalent bonds at room temperature, Angew. Chem. Int. Ed. 51 (2012) 1138–1142. [28] K. Rawat, J. Pathak, H.B. Bohidar, Effect of solvent hydrophobicity on gelation kinetics and phase diagram of gelatinionogels, Soft Matter 10 (2014) 862–872. [29] A. Veis, Macromolecular Chemistry of Gelatin, Academic Press, New York, 1964. [30] H.B. Bohidar, S.S. Jena, Study of sol-state properties of aqueous gelatin solutions, J. Chem. Phys. 100 (1994) 6888–6895. [31] P.J. Skrzeszewska, J. Sprakel, F.A. de Wolf, R. Fokkink, M.A. Cohen Stuart, J. van der Gucht, Fracture and self-healing in a well-defined self-assembled polymer network, Macromolecules 43 (2010) 3542–3548.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Review
Self-healing gelatin ionogels Anshu Sharma a,b , Kamla Rawat b,c,∗ , Pratima R. Solanki b , H.B. Bohidar a,b,∗ a
School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi, India c Inter University Accelerator Centre, New Delhi 110067, India b
a r t i c l e
i n f o
Article history: Received 21 October 2016 Accepted 27 November 2016 Available online 28 November 2016 Keywords: Self-healing gel Ionogel Rheology Recovery Storage modulus
a b s t r a c t We demonstrate room temperature (20 ◦ C) self-healing, and substantial recovery (68–96%) of gel rigidity of gelatin, a polypeptide, ionogels (made in 1-ethyl-3-methylimidazolium chloride ionic liquid (IL) solutions via thermal treatment, IL ≤ 5% (w/v)) after they were cut using a surgical blade. The recovery process did not require any stimuli, and the complete healing under ambient condition required about 10 h.The self-healing owed its origin to the reformation of network structures via imidazolium ion mediated charge quenching of deprotonated residues, and hydrophobic interaction between neighbouring alkyl tails of IL molecules. The rate of healing determined from the growth of rigidity modulus was 20 ± 5 mPa/s independent of ionic liquid content of the gel. This was true regardless of the fact that ionogels containing more IL had a lower gel modulus due to propensity of hydrophobic linkages, but these were agile enough to recover their network structures to a higher degree during the healing process. These features indicate that the gelatin ionogel being biocompatibile, and biodegradable holds great potential for applications in the field of biomedical engineering. © 2016 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 3.1. Time-dependent microscope imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 3.2. Time-dependent rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604 3.3. Dye diffusion studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605 3.4. Phenomenology of healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
1. Introduction Self-healing is a key property of living tissues that allows them to sustain repeated damage. Physical hydrogels are solventfilled networks, formed by physically crosslinked biopolymers, in which secondary interactions such as hydrogen bonds, electrostatic interactions, hydrophobic interactions or van der Waals forces
∗ Corresponding authors at: Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi, India. E-mail addresses: [email protected] (K. Rawat), [email protected] (H.B. Bohidar). http://dx.doi.org/10.1016/j.ijbiomac.2016.11.103 0141-8130/© 2016 Elsevier B.V. All rights reserved.
are responsible for formation of reversible crosslinks (entanglements). Hydrogels are crosslinked hydrophilic polymer assemblies classically made from high molecular weight biopolymers such as gelatin, fibrin, chitosan, pectin, carrageenan, cellulose etc to name a few. Gelatin, in particular is an interesting biomaterial that is available in plenty in the biosphere, and this material has been extensively studied in the past. Chemical composition of this biopolymer depends on its source of origin. Hydrophobic amino acids like proline (Pro), hydroxyproline (Hyp), and glycine (Gly) are in propensity in gelatin. The general primary sequence is given by of (Gly-X-Pro) and (Gly-X-Hyp), in which X represents other amino acids [1]. Due to the self-assembled origin of these gels, the gel strength quickly recovers after the material experiences mechani-
604
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
cal deformation [2]. Thus, physical polymer networks offer unique advantages, over the chemical gels that are difficult to manipulate. In mammals self-healing is provided by fibrous tissues, like cartilage that are structured by non-covalent crosslinked biopolymers. In the recent past, major attempts have been made to introduce non-covalently crosslinked biopolymers and their supramolecular assembly to develop self-healing materials. Sometimes, an external trigger in the form of energy or healing moiety is needed for a self-healing to occur [2]. Dynamic-reversible materials (efficiency in detecting and “autonomically” healing damage) displays a wide range of responses from self-healing to mechanical work for applications such as sensors, actuators and various other biomedical applications. Supramolecular chemistry [3] uses non-covalent forces such as hydrogen bonding and – stacking giving rise to a variety of stimuli-responsive self-healing materials [4–10]. Macroscopic self-assembly uses the common host–guest molecular recognition [11]. Often external stimuli thermal, radiation, catalytic, and mechanical actions may cause transformations in these structures by thermodynamic reorganization [12–14]. Deng et al have synthesized crosslinked polymer gels with reversible covalent bonds under acidic conditions that showed healable properties [15]. Synthesis of healable polymer gels with trithiocarbonate units in their structures has been proposed by Matyjaszewski and coworkers [16,17]. Similarly, reversible Diels–Alder units have been studied for thermally healable potential[18–20]. It is reported that Diarylbibenzofuranone (DABBF) can reach a state of thermodynamic equilibrium in the absence of any stimuli and the radical species arising from cleaved DABBF was found to tolerant to oxygen [21–26]. Imato et al. prepared crosslinked DABBF containing polymer gels with cleavable properties under ambient conditions [27]. All the above mentioned examples involve chemically crosslinked network gels formed of synthetic polymers that often use toxic crosslinkers. Not many self-healing physical gels made of biopolymers are reported in the literature. Herein, we report the gel recovery process (self-healing) of gelatin ionogels under ambient conditions after their mechanical incision. The extensive physical characterization of gelatin ionogels has been reported by us earlier [28]. The enormous application potential of healable biocompatible gels makes the current report timely and significant, it is more so because these gels are green materials.
2. Materials and methods Gelatin (GB) (225 bloom, pI ∼ 4.9 ± 0.2) having nominal molecular weight of 50 kDa and ionic liquid (IL) 1-ethyl-3methylimidazolium chloride were purchased from Sigma-Aldrich, USA, and used as received. The molecular structure of gelatin and the ionic liquid used are shown in Fig. 1. Gelatin ionogel was prepared by dissolving 5% (w/v) of GB in IL solution with varying percentage of IL [0–5% (w/v)] made in deionized water maintained at 60 ◦ C. Continuous stirring for 30 min produced a clear solution, which was then cooled to room temperature (20 ◦ C). After a lapse of gelation time, the contents turned into a rigid gel which was checked by, inversion of the sample holder, and the observation of a non-flowing meniscus implying that it could support the weight of the gel. Concentrations are in (% w/v) unless otherwise stated. The dynamic rheological profiling of the samples were done on a stress controlled rheometer (AR-500, T.A. Instruments, UK). A2◦ cone-plate geometry of 20 mm radius and a truncation gap of 500 m was used, and the oscillatory stress value was fixed at 4.775 Pa. About, 100 mL of the sample was placed on the peltier plate and it was to equilibrate for 5 min before proceeding with measurements. Silicone oil was applied to the outer circular edge of the paltier plate and wet sponge was used as solvent trap to
prevent loss of solvent due to evaporation. Bright field biological microscope (Leedzmicro-imaging LTD, U.K.) was used for imaging of the samples. 3. Results and discussion Gelatin, a biopolymer, is produced by denaturation of collagen through either alkaline or acid processing, during which the interconnected triple-helix units are melted into three distinct single strand gelatin chains [29]. When gelatin is dissolved in warm water these random-coil chains form a homogeneous sol. Upon cooling it to room temperature, these molecules undergo a coil–helix transition, and gradually get physically crosslinked by entanglements to form thermoreversible physical gels with a typical gelation tem◦ perature of Tgel ≈ 28 C [30]. 3.1. Time-dependent microscope imaging First we prepared thin films of gels on cover slip glass plates by pouring about 0.5 mL of the sol on the plate surface. The plate was stored in a constant temperature incubator over night at a temperature of 20 ◦ C. A transparent gel was set during this period. The ionogel film was then cut into two pieces by using a surgical blade. These samples were placed in the incubator maintained at the same temperature for about 10 h without disturbance. Their recovery pattern was recorded using an optical microscope. These images are depicted in Fig. 2 for two representative ionogels(IL = 1% and 5%). It is clearly seen from the time-sequence of these images that the incision scar disappears typically over a period of 10 h, after which the two pieces are visually unidentifiable and the gel pieces fuse to become one unit again. 3.2. Time-dependent rheology The healing process was monitored by following the time dependent rigidity modulus of the gels. For the recovery analysis of the ionogel samples, a four-step program on stress controlled rheometer was used. In the first step, the hot sol samples were poured onto the peltier plate maintainedat 10 ◦ C, and kept isothermal at the room temperature for 3 min, and then time sweep measurement was performed at an angular frequency of 1 rad s−1 at the oscillatory stress value of 4.775 Pa to observe the temporal growth of low frequency storage modulus, G0 . When storage modulus became constant, we stopped the measurement (generally after 4–5 h). This characterized the ionogel sample. In the second step, the sample was cut into two pieces by using a surgical blade and these pieces were placed touching face to face for 10 h without disturbance. In the third step, we repeated the time sweep measurement on the healed gel sample until its G0 value reached a plateau. For this measurement, the reference to initiation of healing was defined as the time when the sample wasloaded onto the geometry of the rheometer, which was set as t = 0. Fig. 3(A) shows the data of isochronal time sweep experiment at constant strain during which the temporal growth of the low frequency storage modulus was monitored for the gel samples. Rheology data shows the self-assembly of the ionogels progressed, and the formation of networks continued to reach a matured state typically after tsat = 4 h of gelation. The saturation values of G0 are depicted in Fig. 3(A) for various ionogels samples. The time sweep experiment was halted after when saturation in storage modulus values was observed, this equilibrium G0max values are clearly shown in Fig. 3(B). Next the gel was cut into two pieces using a surgical blade (tcut in Fig. 3(A)). A sharp drop in storage modulus after incision resulted from the disruption of physical crosslinks is clearly seen in this figure. For instance, the saturation modulus of
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
605
Fig. 1. The molecular structure of gelatin and ionic liquid used.
Fig. 2. Hourly time sequence images of ionogels, (a) top panel, IL = 1%, and (b) bottom panel, IL = 5%, indicating healing under ambient conditions. Arrows indicate the incision line which fades with time as the gel heals. Note the complete healing after 10 h.
the ionogel was G0max = 585 Pa for 5% IL sample, which after incision dropped to G0cut = 512 Pa. The two pieces were placed touching each other and rested for theal = 5.5 h to heal under ambient condition. Rheology measurement was initiated and the full recovery of gel modulus was followed starting at trs (Fig. 3(A)). After recovery for 5% IL ionogel, the restored modulus was G0recov = 562 Pa This procedure was followed for all the ionogels samples. The quantitative recovery profile is shown as a bar diagram in Fig. 3(B). The complete recovery process continued over a period of several hours with corresponding increase in gel modulus. The% recovery and the complete healing time were dependent on IL concentration. For example, 96% of the gel modulus was restored for 5% IL ionogels after healing making this an excellent healable material. In contrast, for gelatin hydrogel (IL = 0) the recovery of gel rigidity was limited to 45% only. The rate of recovery, defined as (dG0 /dt was 20 ± 5 mPa/s independent of IL content of the gel. Here, G0 = (G0recov - G0cut ). The best recovery was noticed for [IL] = 5% ionogels.
3.3. Dye diffusion studies
Fig. 3. (A) Shows variation of low frequency dynamic storage modulus G0 as a function of time for different concentration of IL. (B) Shows dynamic storage modulusG0 and% recovery as a function of IL concentration. Arrow indicates the time when incision was made.
In the next step, we wanted to demonstrate that a diffusion path existed between the rejoined gel pieces. For this the two cut gel pieces were colored separately. For preparing the red gel block, aqueous solution of rhodamine B dye (0.1 mg/mL) was kept in a beaker in which one of the two gel pieces was kept immersed for 30 min which turned the gel red in color due to osmosis. Other piece was similarly prepared by dipping it in aqueous solution of orange G dye (0.1 mg/mL) for the same duration of time, which produced a yellow gel (Fig. 4(c)).The two blocks were placed touching each ◦ other, and rested in an incubator at 10 C temperature without any intervention (Fig. 4(d)). After 10 h, they were observed to have fused into one piece with a sharp boundary clearly visible between the two pieces. After 24 h, the healed block appeared red indicating the diffusion of rhodamine G dye into the entire gel due to higher diffusivity of this dye(Fig. 4(e)).
606
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
Fig. 4. Self-healing of a 5% IL ionogel. (a) the original gel, (b) the gel cut into two pieces, (c) one piece was soaked in rhodamine B (0.1 mg/mL) and another in orange G ◦ (0.1 mg/mL) dye solution for 30 min each, (d) the two pieces were kept in physical contact face to face for 12 h without at 10 C temperature in the incubator, (e) the rhodamine G dye diffused into the gel piece containing orange G dye due to its higher diffusion coefficient. The boundary line between the two halves is clearly seen in (e, arrow) butthe two gel pieces have fused as a whole.
3.4. Phenomenology of healing Physical gels due to the transient character of the entanglements are in a position to sustain the external stress by dislocation, dissociation, and re-orientation of the entangled junctions. This unique relaxation property enables them to exhibit healable property, which in the case of chemical gels is impossible [1,31]. It can be conjectured that the self-healing after mechanical damage was due to the renewal of their network structure via hydrophobic interactions between the aliphatic hydrocarbon chains of the two gel pieces. Based on the data in hand it is possible to offer the following phenomenology of the healing process. In the sol state, the gelatin molecules assume random coil conformation, and under normal pH conditions there is presence of almost a 1:1 ratio of protonated and deprotonated residues in its composition [29]. The positively charged imidazolium head group of the IL will selectively bind to the deprotonated residues while the free Cl− ions will bind to the protonated sites. Further, the hydrophobic alkyl tail of ILs must assemble locally to minimize water contact. Because, of this there is reduced possibility of formation of triple helices in the sol. Thus, what gets formed is a heterogeneous physical network gel. With increased IL content this heterogeneity increases. This is the reason why G0 decreases with IL concentration (Fig. 3 data). Recall, that a homogeneous gelatin hydrogel has a propensity of inter twined triple helix networks which provides it with a high gel rigidity value. When such a hydrogel is subjected to mechanical incision millions of these networks are ruptured. During healing only a fraction of these are restored and the rest are trapped in a dangling state with no binding partner. Note that there is no force that enables their restoration (See Scheme 1). On the contrary, in ionogels plenty of inter gelatin chain binding is facilitated by hydrophobic interactions. When ionogels are exposed to mechanical incision, these hydrophobic interlink (bridges) between gelatin chains get ruptured in abundance, thereby exposing the alkyl tails of IL molecules to interstitial water contact which is energetically unfavourable. When the two gel pieces are placed touching each other, these dangling hydrophobic tails immediately form an overlapped junction to create a water depletion region which facilitates gel healing (See Scheme 1). Thus, the healing is mostly governed by hydrophobic interactions. More is the IL content of the gel, stronger are the hydrophobic forces, faster will be the recovery, and higher will be the restoration of gel
Scheme 1. Gel healing mechanism.
rigidity after healing. This is clearly manifested in our experimental data. 4. Conclusion We have described a biopolymer thermo-reversibleionogel prepared in an ionic liquid solution. The presence of ionic liquid molecules changes the assembly of individual gelatin chains with formation of triple helices mostly suppressed. The IL clad gelatin chains organize under hydrophobic forces to form a physical gel network. After rupture, these gels heal under a driving force which is the hydrophobic interaction between alkyl tails of IL molecules. Ionogelsexhibited healing behaviourat temperatures lower than their gelation temperature. These observations may provide basic clues to the design of self-healing biomaterials that were stabilized via secondary forces. The self-healing ionogels were found to formbonds strong enough to withstand repeated damage. These results could give adequate design directions to the development of self-healing biomaterials. Acknowledgments AS acknowledges University Grants Commission, Government of India for a Junior Research Fellowship. This work was supported
A. Sharma et al. / International Journal of Biological Macromolecules 95 (2017) 603–607
by the Department of Science and Technology (DST), India. KR is very thankful to Department of Science and Technology, Government of India, for DST-Inspire Faculty Award. References [1] S.K. Kundu, T. Mztsunaga, M. Yoshida, M. Shibayama, Rheological study on rapid recovery of hydrogel based on oligomeric electrolyte, J. Phys. Chem. B 112 (2008) 11537–11541. [2] L. Yan, S. Gou, Z. Ye, S. Zhang, L. Ma, Self-healing and moldable material with the deformation recovery ability from self-assembled supramolecularmetallogels, Chem. Commun. 50 (2014) 12847–12850. [3] L. Brunsveld, B.J.B. Folmer, E.W. Meijer, R.P. Sijbesma, Supramolecular polymers, Chem. Rev. 101 (2001) 4071–4098. [4] M. Burnworth, L. Tang, J.R. Kumpfer, A.J. Duncan, F.L. Beyer, G.L. Fiore, S.J. Rowan, C. Weder, Optically healable supramolecular polymers, Nature 472 (2011) 334–338. [5] S. Burattini, H.M. Colquhoun, D. Fox, B.W. Greenland, W. Harris, M.E. Mackay, S.J. Rowan, A Self-repairing, supramolecular polymer system: healability as a consequence of donor–acceptor – stacking interactions, Chem. Commun. (2009) 6717–6719. [6] Y. Xu, Q. Wu, Y. Sun, H. Bai, G. Shi, Three-dimensional self-assembly of graphene oxide and DNA into multifunctional hydrogels, ACS Nano 4 (2010) 7358–7362. [7] P. Cordier, F. Tournilhac, C. Soulie-Ziakovic, L. Leibler, Self-healing and thermoreversible rubber from supramolecular assembly, Nature 451 (2008) 977–980. [8] S. Burattini, B.W. Greenland, W. Merino, J. Weng, H.M. Colquhoun, W. Hayes, M.E. Mackay, I.W. Hamley, S.J. Rowan, A healable supramolecular polymer blend based on aromatic - stacking and hydrogen-bonding interactions, J. Am. Chem. Soc. 132 (2001) 12051–12058. [9] P. Mukhopadhyay, N. Fujita, A. Takada, T. Kishida, M. Shirakawa, S. Shinkai, Regulation of a real – time self – healing process in organogel tissues by molecular adhesives, Angew. Chem. 122 (2010) 6482–6486. [10] Q. Wang, J.L. Mynar, M. Yoshida, E. Lee, M. Lee, K. Okuro, K. Kinbara, T. Aida, High-water-content mouldable hydrogels by mixing clay and a dendritic molecular binder, Nature 463 (2010) 339–343. [11] R. Sun, C. Xue, X. Ma, M. Gao, H. Tian, Q. Li, Light-driven linear helical supramolecular polymer formed by molecular-recognition-directed self-assembly of bis (p-sulfonatocalix [4] arene) and pseudorotaxane, J. Am. Chem. Soc. 135 (2013) 5990–5993. [12] S.J. Rowan, S.J. Cantrill, G.R.L. Cousins, J.K.M. Sanders, J.F. Stoddart, Dynamic covalent chemistry, Angew. Chem. Int. Ed. 41 (2002) 898–952. [13] H. Otsuka, K. Aotani, Y. Higaki, A. Takahara, Polymer scrambling: macromolecular radical crossover reaction between the main chains of alkoxyamine-based dynamic covalent polymers, J. Am. Chem. Soc 125 (2003) 4064–4065. [14] X. Chen, M.A. Dam, K. Ono, A. Mal, H. Shen, S.R. Nutt, K. Sheran, F. Wudl, A thermally re-mendable cross-linked polymeric material, Science 295 (2002) 1698–1702.
607
[15] G. Deng, C. Tang, F. Li, H. Jiang, Y. Chen, Covalent cross-linked polymer gels with reversible sol-gel transition and self-healing properties, Macromolecules 43 (2010) 1191–1194. [16] R. Nicolay, J. Kamada, A.V. Wassen, K. Matyjaszewski, Responsive gels based on a dynamic covalent trithiocarbonate cross-linker, Macromolecules 43 (2010) 4355–4361. [17] Y. Amamoto, J. Kamada, H. Otsuka, A. Takahara, K. Matyjaszewski, Repeatable photoinduced self-healing of covalently cross-linked polymers through reshuffling of trithiocarbonate units, Angew. Chem. 123 (2011) 1698–1701. [18] E.B. Murphy, E. Bolanos, C. Schaffner-Hamann, F. Wudl, S.R. Nutt, M.L. Auad, Synthesis and characterization of a single-component thermally remendable polymer network: Staudinger and Stille revisited, Macromolecules 41 (2008) 5203–5209. [19] X. Chen, M.A. Dam, K. Ono, A. Mal, H. Shen, S.R. Nutt, K. Sheran, F. Wudl, A thermally re-mendable cross-linked polymeric material, Science 295 (2002) 1698–1702. [20] K. Ishida, N. Yoshie, Synthesis of readily recyclable biobased plastics by Diels-Alder reaction, Macromol. Biosci. 8 (2008) 916–922. [21] J.C. Scaiano, A. Martin, G.P.A. Yap, K.U. Ingold, A carbon-centered radical unreactive toward oxygen: unusual radical stabilization by a lactone ring, Org. Lett. 2 (2000) 899–901. [22] E.V. Bejan, E.F. Sanchis, J.C. Scaiano, Lactone-derived carbon-centered radicals: formation and reactivity with oxygen, Org. Lett. 3 (2001) 4059–4062. [23] M. Frenette, C. Aliaga, E.F. Sanchis, J.C. Scaiano, Bond dissociation energies for radical dimers derived from highly stabilized carbon-centered radicals, Org. Lett. 6 (2004) 2579–2582. [24] M. Frenette, P.D. MacLean, L.R.C. Barclay, J.C. Scaiano, Radically different antioxidants: thermally generated carbon-centered radicals as chain-breaking antioxidants, J. Am. Chem. Soc. 128 (2006) 16432–16433. [25] K.S. Focsaneanu, J.C. Scaiano, The persistent radical effect: from mechanistic curiosity to synthetic tool, Helv. Chim. Acta 89 (2006) 2473–2482. [26] H.-G. Korth, Carbon radicals of low reactivity against oxygen: radically different antioxidants, Angew. Chem. Int. Ed. 46 (2007) 5274–5276. [27] K. Imato, M. Nishihara, T. Kanehara, Y. Amamoto, A. Takahara, H. Otsuka, Self-healing of chemical gels cross-linked by Diarylbibenzofuranone-based trigger-free dynamic covalent bonds at room temperature, Angew. Chem. Int. Ed. 51 (2012) 1138–1142. [28] K. Rawat, J. Pathak, H.B. Bohidar, Effect of solvent hydrophobicity on gelation kinetics and phase diagram of gelatinionogels, Soft Matter 10 (2014) 862–872. [29] A. Veis, Macromolecular Chemistry of Gelatin, Academic Press, New York, 1964. [30] H.B. Bohidar, S.S. Jena, Study of sol-state properties of aqueous gelatin solutions, J. Chem. Phys. 100 (1994) 6888–6895. [31] P.J. Skrzeszewska, J. Sprakel, F.A. de Wolf, R. Fokkink, M.A. Cohen Stuart, J. van der Gucht, Fracture and self-healing in a well-defined self-assembled polymer network, Macromolecules 43 (2010) 3542–3548.