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maleimide Diels-Alder click reaction in water
International Journal of Biological Macromolecules 141 (2019) 493–498
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Thermally reversible nanocellulose hydrogels synthesized via the furan/ maleimide Diels-Alder click reaction in water Ricardo Klaus Kramer a, Mohamed Naceur Belgacem b, Antonio José Felix Carvalho a,⁎, Alessandro Gandini a,b a b
Department of Materials Engineering, São Carlos School of Engineering, University of São Paulo, Av. João Dagnone n° 1100, São Carlos, São Paulo 13563-120, Brazil Ecole Française de Papeterie et des Industries Graphiques, LGP2 - 5518 Grenoble INP, 461 rue de la papeterie, Saint Martin d'Hères F-38400, France
a r t i c l e
i n f o
Article history: Received 29 March 2019 Received in revised form 25 August 2019 Accepted 4 September 2019 Available online 05 September 2019 Keywords: Diels-Alder Thermoreversible hydrogel Nanofiber cellulose
a b s t r a c t The study deals with the synthesis of thermally reversible hydrogels from modified cellulose nanofibers via the Diels-Alder “click” reaction in an aqueous medium. “Never-dried” cellulose fibres derived from hardwood were submitted to shearing and surface TEMPO-oxidation before being modified with furfurylamine. The ensuing pendant furan moieties were reacted with a water-soluble bismaleimide via Diels-Alder coupling at 65 °C to produce a hydrogel, whose deconstruction was induced by the corresponding retro-Diels-Alder reaction carried out at 95 °C. Differential scanning calorimetry and rheological measurement were used to characterize the hydrogels. These aqueous cellulosic materials should provide original applications in such areas as strong paper-based artefacts and biocompatible gels. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels constitute a family of macromolecular materials possessing crosslinked hydrophilic structures, which can absorb and hold large amount of water [1]. Due the similarity between the hydrated body tissues and hydrogel structure, they have been widely used in biomedical applications [2] such as controlled drug release [3] and biosensors [4]. Hydrogels can be classified as physically or chemically crosslinked polymers [5], whose syntheses include Michael-type [6] and click reactions [7], notably the Diels-Alder (DA) thermallyreversible coupling [1]. The application of the DA reaction to hydrogel synthesis and polymerization systems has attracted growing attention over the last decade, normally involving reactions between the diene/dienophile furan/maleimide combination [8], which displays thermal reversibility within a useful temperature range (Scheme 1). The temperature dependence of this equilibrium is such that below about 60 °C, the forward reaction (formation of the adduct) dominates, whereas above 100 °C the equilibrium is predominantly shifted to its precursors (deconstruction of the adduct). The use of water as a solvent in DA reactions has attracted growing attention because of the significant increase in the reaction rate, mainly due to the hydrophobic effect, and because of the sustainable advantage related to its green character [8]. The investigation of thermoreversible gel based on the click DA reaction could makes it possible the use of such gels in new emerging applications such as additive manufacturing (3D printing) of complex gel structures ⁎ Corresponding author. E-mail address: [email protected] (A.J.F. Carvalho).
https://doi.org/10.1016/j.ijbiomac.2019.09.027 0141-8130/© 2019 Elsevier B.V. All rights reserved.
for use in several applications in special for in biomedical and pharmaceutical uses. In 2009, Wei and co-authors reported the synthesis of thermosensitive hydrogel prepared by N-4-(chlorocarbonyl)phenyl] maleimide with polyethylene glycol (PEG) via the DA reaction in aqueous media and observed that the gelation time decreased with an increase in temperature [1]. In another paper by the same group, a copolymer of N-vinyl-2-pyrrolidone and furfuryl methacrylate was crosslinked with a polymeric dienophile [9]. Gárcia-Astrain et al. prepared a crosslinked hydrogel by the functionalization of poly(2-aminoethyl methacrylate) hydrochloride with furfural and a Jeffamine®-based bismaleimide [10]. Nimmo et al. copolymerized hyaluronic acid with PEG in one-step in aqueous media and prepared a hydrogel for tissue application [11]. Shao and collaborators reported a self-healing hydrogel prepared by cellulose nanocrystals with PEG via DA reaction, using modified CNCs as a reinforcing phase and chemical cross-linker. They obtained a nanocomposite with good mechanical properties and gelation time varying in function of different components [12]. Here, we describe the synthesis of thermally-reversible hydrogels obtained via DA/retro-DA (rDA) reactions in an aqueous medium using cellulose nanofibers as substrates. Refined “never-dried” cellulose fibres were submitted to surface TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) oxidation before grafting furfuryl amine onto the ensuing surface carboxylic groups. A water-soluble crosslinker was synthetized from exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride and 4,7,10trioxa-1,13-tridecanediamine to obtain an oligoether bismaleimide. Both reagents were then brought together to prepare a cellulose hydrogel via the furan/maleimide DA reaction. This study was prompted by
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R.K. Kramer et al. / International Journal of Biological Macromolecules 141 (2019) 493–498
O
O
Δ N
O
N
O O
O Scheme 1. The Diels–Alder equilibrium between furan and maleimide end group.
the growing interest in chemically crosslinked pulp fibres, aimed at widening the range of materials from the pulp and paper industry, including the preparation of papers with improved mechanical properties for special applications, which moreover can be recycled following a simple thermal treatment. New applications in 3D printing can also use such kind of thermoreversible material. The vast majority of nanocellulose surface chemical modifications published to date required the use of organic media and rather cumbersome procedures to revert to aqueous conditions [13]. In the present approach, all operations were conducted in water, thus giving the process a “greener” connotation. 2. Experimental 2.1. Materials Refined “never-dried” cellulose pulp from eucalyptus species was kindly provided by Suzano S.A. Brazil. The reagents and solvents used for this investigation included N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC, pure, Sigma-Aldrich), N-
hydroxysuccinimide (NHS, 98%, Sigma-Aldrich), furfurylamine (99%, Sigma-Aldrich), dry methanol (Sigma-Aldrich), 4,7,10-trioxa-1,13tridecanediamine (97%, Sigma-Aldrich), exo-3,6-epoxy-1,2,3,6tetrahydrophthalic anhydride (Sigma-Aldrich 98%), 2,2,6,6tetramethyl-1-piperidinyloxy (TEMPO, 98%, Sigma-Aldrich), sodium bromide (≥99,5%, Sigma-Aldrich), and sodium hypochlorite solution (10–15%, Sigma-Aldrich). 2.2. TEMPO-mediated oxidation of cellulose [14] In order to prepare nanofibrillated cellulose (NFC), the cellulose fibres (5 g) were suspended in water (300 mL) and dispersed for 5 min using an ultra-turrax homogenizer (Ika-Laboratechnik, Germany). Then, a solution (50 mL) containing TEMPO (0.1 mmol/g of cellulose) and sodium bromide (1 mmol/g of cellulose) was added to the fibre suspension under mechanical stirring at room temperature. The TEMPOmediated oxidation of the fibres started by the dropwise addition of a 50 mL NaClO solution (10–15%, 5 mmol). The pH was maintained at 10 by adding drops of a 0.5 M NaOH solution, until no more variation was observed, and the reaction was protracted for 4 h. The pH was
Scheme 2. Grafting of furfurylamine onto the ToNFC carboxylic groups.
R.K. Kramer et al. / International Journal of Biological Macromolecules 141 (2019) 493–498
495
Scheme 3. Thermally reversible cellulose hydrogel formation through the DA/rDA reaction. The black lines represent the cellulose nanofibers.
then adjusted to 7 with 0.1 M HCl and the modified fibres thoroughly washed with deionized water by filtration and stored at 4 °C. The carboxylate content of TEMPO-oxidized nanofibrillated cellulose (ToNFC) were determined by conductimetric titration. 2.3. Cellulose-furan grafting A 66.6 mL ToNFC aqueous suspension (0.1 g, 1.5 wt%) was mixed with the catalytic couple EDC (3.2 mmol) and NHS (2.4 mmol) and stirred for 30 min. Furfurylamine (3.2 mmol) was added and the pH adjusted to 5 with 3 M HCl before heating the suspension to 40 °C under magnetic stirring for 24 h. After the reaction (Scheme 2), the nanofiber suspension was acidified to pH 1.5 (HCl 3.0 M) and purified by thoroughly dialyzing it against deionized water for 7 days, to remove the remaining excess chemicals and salts. The water suspension was then sonicated at 70% output control for 5 min. Finally, the ensuing furan-modified ToNFC were stored at 4 °C [15].
2.6. Conductometric titration The fibres, ToNFC and ToNFC-furfurylamine samples (0.1 g, 0.1 wt%) were suspended into a 100 mL aqueous solution with 5 mL of 0.1 M hydrochloric acid and stirred for 5 min with the ultraturrax. The suspension was then titrated with 0.1 M NaOH. The degree of oxidation (Eq. (1)) (DO) [17] and the carboxyl group content (Eq. (2)) (mmol/g) were calculated using the following equations: DO ¼ ð162 C ðV 2 −V 1 ÞÞ=ðw−½36 C ðV 2 −V 1 ÞÞ
ð1Þ
X ¼ ðC ½V 2 −V 1 Þ=w
ð2Þ
where C is the NaOH concentration (mol/L), V1 and V2 are the volumes of the NaOH in litres used to neutralize and determine the carboxyl groups in a sample and w is the weight of the dry ToNFC sample (g).
2.4. Synthesis of the bismaleimide (BM) A solution of 10 g of exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride (ETHPA) in 50 mL of methanol, previously dried with 3 Å molecular sieves, was added to 6.83 g of 4,7,10-trioxa-1,13tridecanediamine (TTDDA) (molar ratio of 2:1), and the mixture kept refluxing at 65 °C under magnetic stirring for 24 h. Methanol was then removed under vacuum and the product was maintained at 95 °C for 24 h to remove the furan released from the rDA reaction, which restored the maleimide function. As described previously [16]. 2.5. Preparation of the thermally reversible hydrogel (DA and rDA reactions) The DA/rDA reactions of the hydrogel were conducted, on the one hand, by mixing the furan-modified ToNFC and the BM in water and stirring the suspension at 60 °C under a nitrogen atmosphere, and, on the other hand, by heating the ensuing gel at 95 °C, as shown in Scheme 3.
Fig. 1. FTIR spectra of the initial refined fibres (green line), ToNFC (blue line) and ToNFC modified with furfurylamine (red line).
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Table 1 Degree of oxidation per anhydroglucose unit of cellulose, carboxyl group content and yield of reaction steps.
Pulp ToNFC ToNFC-Furfurylamine
Degree of oxidation (DO)
Carboxyl groups content (mmol/g)
Oxidation yield (%)
Grafting yield (%)
0.088 0.334 0.040
0.543 1.90 0.246
– 73.6 –
– – 87.0
2.7. FTIR spectroscopy Infrared spectra were recorded on a FTIR Perkin-Elmer SpectrumOne spectrometer. All the samples were analysed at room temperature in the ATR mode, using the spectral range 4000 to 600 cm−1 at a resolution of 4 cm−1, by the accumulation of 20 scans. 2.8. Differential scanning calorimetry (DSC) DSC was performed on a Perkin-Elmer differential scanning calorimeter Pyris 1 under a nitrogen atmosphere. The heating/cooling rates were set to 20 °C/min, and the sample was weighed (approximately 11 μL) in a sealed aluminium pan for aqueous samples. The sample was first heated and kept in 55 °C for 20 min, followed by heating to 90 °C and an isotherm of 20 min, followed by cooling to 55 °C. Four cycles were performed. 2.9. Rheological characterization of hydrogels The rheological behaviour was analysed with a Brookfield (DV3TLV) rheometer, using a cone-plate geometry (12 mm cone radius and 3° cone angle). The applied frequency was constant, 0.1 rpm for 1 min at 25 °C. 3. Results and discussion The surface chemical modification of the nanocellulose fibres, obtained by the mechanical fibrillation of a commercial pulp, was carried out in water by the TEMPO-mediated oxidation of their primary hydroxyl groups following a well-established protocol [13,14]. Scheme 2 illustrates the condensation of furfurylamine onto the ensuing carboxylic cellulose functions, using the EDC/NHS-assisted amidation reaction in an aqueous medium. The success of the oxidation of the surface primary cellulose hydroxyl groups to carboxylate moieties was confirmed by FTIR (Fig. 1). The band at 1603 cm−1, characteristic of the carboxylate ion stretching, showed a substantially increase after the oxidation of the nanofibers. When the TEMPO-oxidized nanofibrillated cellulose (ToNFC) was modified with furfurylamine, the band at 1603 cm−1 decreased (Fig. 1) and a band related to the amide C_O stretching appeared at
Fig. 3. Thermal behaviour of the DA and r-DA.
1730 cm−1. Moreover, the band at 1648 cm−1 corresponding to N\\H stretching increased, although the band overlapped with the residual carboxylate peak, making this second assessment less explicit. Additionally, the typical furan-ring breathing band around 1000 cm−1 was completely masked by the strong cellulose ether band. It was therefore necessary to confirm the occurrence of both oxidation and grafting by conductimetric titrations of the carboxylic groups at the various stages of the process. Table 1 summarizes the results of this analytical approach. After the cellulose TEMPO-oxidation, there was a substantial increase in carboxylic groups at the surface of the ToNFC, confirming the success of the reaction. The ToNFC was then used for the surface functionalization with furfurylamine, which led to a very pronounced decrease in the carboxyl group content, thus confirming the occurrence of the furan grafting reaction with a good yield (Table 1). The DA/rDA reaction between the furan-modified nanofibers and the bismaleimide (Scheme 3) was conducted by adding an aqueous solution of the latter to a suspension of the former and stirring the ensuing mixture at 60 °C under a nitrogen atmosphere (Fig. 2a). A strong viscosity increase was observed, followed by the formation of a hydrogel, suggesting that the fibres had crosslinked through the furan/maleimide DA couplings (Scheme 3 and Fig. 2b). The crosslinking reaction finished in 30 min, approximately, reaching the gelation time. The decrosslinking reaction took place when the system was heated at 95 °C. The medium viscosity decreased within an hour to reach the value of the initial mixture (Fig. 2c). The system was then cooled to 60 °C and it displayed the same phenomenology as with the first DA reaction (Fig. 2d), thus regenerating the hydrogel and proving the reproducibility of the DA/rDA cycle, which was in fact successfully repeated three more times. It is important to mention that despite the fact that retroDiels-Alder reactions usually take place at higher temperatures, when furans and maleimides are used lower temperatures are observed such as 80–90 °C as described in the literature [8].
Fig. 2. Illustration of the DA/rDA cycle involving the maleimide/furan system in the process of hydrogel formation and deconstruction. (a) Initial mixture; (b) after the first DA crosslinking (formation of the hydrogel); (c) after the r-DA process (deconstruction of the crosslinked hydrogel); (d) after the second DA crosslinking reaction.
R.K. Kramer et al. / International Journal of Biological Macromolecules 141 (2019) 493–498
497
Fig. 4. Rheological behaviour of (a) DA and (b) r-DA hydrogels.
The efficiency of the DA and rDA reactions was confirmed by the thermal behaviour of cellulose hydrogels. The DSC thermograms (Fig. 3) showed an exothermic peak around 56 °C, during the heating process, corresponding to the energy released during the adduct formation between furan and maleimide. During the cooling process, the endothermic peak around 87 °C was associated with occurrence of the rDA reaction that deconstructed the furan/maleimide adduct. The slight shift of the endothermic and exothermic peak can be attributed to the short duration of the experiments, which did not allow a full crosslink in the DA coupling, nor the completion of the rDA reactions. The successive thermal cycles shown in Fig. 3 proved the reproducibility of the DA/rDA cycle, i.e. of the thermal reversibility of the hydrogel. The viscosity behaviour of the thermally-reversible hydrogel was evaluated at a 0.1 rpm frequency. The hydrogel was kept at 60 °C for 30 min to complete the DA reaction and then tested at ambient temperature, as shown in Fig. 4a. The viscosity value of the hydrogel was 84,200 ± 4400 cP. When the system was heated at 90 °C for 30 min, the occurrence of the retro-DA reaction was observed by the corresponding decrease in viscosity. “Point 1” in Fig. 4b shows the maximum decrosslinking reaction, when the value of viscosity was the lowest, at about 380 cP. Then, as the temperature of the hydrogel was allowed to decrease, the medium viscosity increased progressively, ending at “point 2” (Fig. 4b), at ambient temperature, reaching the value of the initial hydrogel viscosity. These results confirmed that the Diels-Alder click chemistry was quite efficient in promoting the formation of thermally-reversible hydrogels. These results shows the potential of use of the MFC hydrogels for 3D printing of thermoreversible soft systems, in which the printing specimen can be produced at 80–90 °C and just after cooling to 50–60 °C can stabilize its structure based on covalent thermoreversible bonds.
4. Conclusions This preliminary study of the DA/rDA reaction cycle applied to aqueous furan-bearing nanocellulose fibres reacting with a water-soluble bismaleimide to give a thermally reversible hydrogel, validated the aimed strategy and produced a valuable novel material. Work is in progress to extend this investigation to other water-soluble readilypurifiable bismaleimides and assess the physical properties and possible applications of the hydrogels and their dried form in such materials as biocompatible aids and highly strengthened paper artefacts. Among these possible applications is noteworthy the production of biomedical devices using additive manufacturing or 3D printing of the hydrogel at temperatures where retro-DA dominates with subsequent cooling leading to a stable material which can be reprocessed or modified to
accomplish the desired application. The intervals of direct Diels-Alder reaction and its reversion allow hydrogel application in conditions that other added substances such as pharmacological substances and several biologic active molecules can be used.
Declaration of competing interest There are no conflicts to declare. Acknowledgements The authors gratefully acknowledge National Council for Scientific and Technological Development (CNPq) - Program “Ciência sem Fronteiras” program (CNPq 401656/2013-6) for the financial support, the Suzano Celulose e Papel S.A. for providing refined “never-dry” cellulose fibres and thank the Laboratoire de Génie des Procédés Papetiers (LGP2) – Grenoble INP, FRANCE where the work was carried out. R. K. K. thanks National Council for Scientific and Technological Development (CNPq) for a doctorate grant (CNPq 208033/2015-7). The authors also thank Debora T. Balogh for the viscosity measurements. References [1] H.-L. Wei, Z. Yang, L.-M. Zheng, Y.-M. Shen, Thermosensitive hydrogels synthesized by fast Diels–Alder reaction in water, Polymer (Guildf) 50 (2009) 2836–2840, https://doi.org/10.1016/j.polymer.2009.04.032. [2] J.M. Saul, D.F. Williams, Hydrogels in Regenerative Medicine, Handb. Polym. Appl. Med. Med. Devices, Elsevier 2011, pp. 279–302, https://doi.org/10.1016/B978-0323-22805-3.00012-8. [3] C. Chen, T. Zhang, B. Dai, H. Zhang, X. Chen, J. Yang, J. Liu, D. Sun, Rapid fabrication of composite hydrogel microfibers for Weavable and sustainable antibacterial applications, ACS Sustain. Chem. Eng. 4 (2016) 6534–6542, https://doi.org/10.1021/ acssuschemeng.6b01351. [4] E.A. Appel, J. del Barrio, X.J. Loh, O.A. Scherman, Supramolecular polymeric hydrogels, Chem. Soc. Rev. 41 (2012) 6195, https://doi.org/10.1039/c2cs35264h. [5] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug Deliv. Rev. 64 (2012) 18–23, https://doi.org/10.1016/j.addr.2012.09.010. [6] M.P. Lutolf, G.P. Raeber, A.H. Zisch, N. Tirelli, J.A. Hubbell, Cell-responsive synthetic hydrogels, Adv. Mater. 15 (2003) 888–892, https://doi.org/10.1002/adma. 200304621. [7] V. Crescenzi, L. Cornelio, C. Di Meo, S. Nardecchia, R. Lamanna, Novel hydrogels via click chemistry: synthesis and potential biomedical applications, Biomacromolecules 8 (2007) 1844–1850, https://doi.org/10.1021/bm0700800. [8] A. Gandini, The furan/maleimide Diels–Alder reaction: a versatile click–unclick tool in macromolecular synthesis, Prog. Polym. Sci. 38 (2013) 1–29, https://doi.org/10. 1016/j.progpolymsci.2012.04.002. [9] H.-L. Wei, Z. Yang, Y. Chen, H.-J. Chu, J. Zhu, Z.-C. Li, Characterisation of N-vinyl-2pyrrolidone-based hydrogels prepared by a Diels–Alder click reaction in water, Eur. Polym. J. 46 (2010) 1032–1039, https://doi.org/10.1016/j.eurpolymj.2010.01.025. [10] C. García-Astrain, I. Algar, A. Gandini, A. Eceiza, M.Á. Corcuera, N. Gabilondo, Hydrogel synthesis by aqueous Diels-Alder reaction between furan modified methacrylate and polyetheramine-based bismaleimides, J. Polym. Sci. Part A Polym. Chem. 53 (2015) 699–708, https://doi.org/10.1002/pola.27495.
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[11] C.M. Nimmo, S.C. Owen, M.S. Shoichet, Diels−Alder click cross-linked hyaluronic acid hydrogels for tissue engineering, Biomacromolecules 12 (2011) 824–830, https://doi.org/10.1021/bm101446k. [12] C. Shao, M. Wang, H. Chang, F. Xu, J. Yang, A self-healing cellulose nanocrystal-poly (ethylene glycol) nanocomposite hydrogel via Diels-Alder click reaction, ACS Sustain. Chem. Eng. 5 (2017) 6167–6174, https://doi.org/10.1021/acssuschemeng. 7b01060. [13] A. Gandini, M.N. Belgacem, The surface and in-depth modification of cellulose fibers, Adv. Polym. Sci. (2015) 349–359, https://doi.org/10.1007/12_2015_305. [14] T. Saito, A. Isogai, TEMPO-mediated oxidation of native cellulose. The effect of oxidation conditions on chemical and crystal structures of the water-insoluble fractions, Biomacromolecules 5 (2004) 1983–1989, https://doi.org/10.1021/bm0497769.
[15] H. Orelma, I. Filpponen, L.-S. Johansson, M. Österberg, O.J. Rojas, J. Laine, Surface functionalized Nanofibrillar cellulose (NFC) film as a platform for immunoassays and diagnostics, Biointerphases 7 (2012) 61, https://doi.org/10.1007/s13758-0120061-7. [16] M. Montiel-Herrera, A. Gandini, F.M. Goycoolea, N.E. Jacobsen, J. Lizardi-Mendoza, M.T. Recillas-Mota, W.M. Argüelles-Monal, Furan–chitosan hydrogels based on click chemistry, Iran. Polym. J. 24 (2015) 349–357, https://doi.org/10.1007/ s13726-015-0325-4. [17] D. da Silva Perez, S. Montanari, M.R. Vignon, TEMPO-mediated oxidation of cellulose III, Biomacromolecules 4 (2003) 1417–1425, https://doi.org/10.1021/bm034144s.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Thermally reversible nanocellulose hydrogels synthesized via the furan/ maleimide Diels-Alder click reaction in water Ricardo Klaus Kramer a, Mohamed Naceur Belgacem b, Antonio José Felix Carvalho a,⁎, Alessandro Gandini a,b a b
Department of Materials Engineering, São Carlos School of Engineering, University of São Paulo, Av. João Dagnone n° 1100, São Carlos, São Paulo 13563-120, Brazil Ecole Française de Papeterie et des Industries Graphiques, LGP2 - 5518 Grenoble INP, 461 rue de la papeterie, Saint Martin d'Hères F-38400, France
a r t i c l e
i n f o
Article history: Received 29 March 2019 Received in revised form 25 August 2019 Accepted 4 September 2019 Available online 05 September 2019 Keywords: Diels-Alder Thermoreversible hydrogel Nanofiber cellulose
a b s t r a c t The study deals with the synthesis of thermally reversible hydrogels from modified cellulose nanofibers via the Diels-Alder “click” reaction in an aqueous medium. “Never-dried” cellulose fibres derived from hardwood were submitted to shearing and surface TEMPO-oxidation before being modified with furfurylamine. The ensuing pendant furan moieties were reacted with a water-soluble bismaleimide via Diels-Alder coupling at 65 °C to produce a hydrogel, whose deconstruction was induced by the corresponding retro-Diels-Alder reaction carried out at 95 °C. Differential scanning calorimetry and rheological measurement were used to characterize the hydrogels. These aqueous cellulosic materials should provide original applications in such areas as strong paper-based artefacts and biocompatible gels. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels constitute a family of macromolecular materials possessing crosslinked hydrophilic structures, which can absorb and hold large amount of water [1]. Due the similarity between the hydrated body tissues and hydrogel structure, they have been widely used in biomedical applications [2] such as controlled drug release [3] and biosensors [4]. Hydrogels can be classified as physically or chemically crosslinked polymers [5], whose syntheses include Michael-type [6] and click reactions [7], notably the Diels-Alder (DA) thermallyreversible coupling [1]. The application of the DA reaction to hydrogel synthesis and polymerization systems has attracted growing attention over the last decade, normally involving reactions between the diene/dienophile furan/maleimide combination [8], which displays thermal reversibility within a useful temperature range (Scheme 1). The temperature dependence of this equilibrium is such that below about 60 °C, the forward reaction (formation of the adduct) dominates, whereas above 100 °C the equilibrium is predominantly shifted to its precursors (deconstruction of the adduct). The use of water as a solvent in DA reactions has attracted growing attention because of the significant increase in the reaction rate, mainly due to the hydrophobic effect, and because of the sustainable advantage related to its green character [8]. The investigation of thermoreversible gel based on the click DA reaction could makes it possible the use of such gels in new emerging applications such as additive manufacturing (3D printing) of complex gel structures ⁎ Corresponding author. E-mail address: [email protected] (A.J.F. Carvalho).
https://doi.org/10.1016/j.ijbiomac.2019.09.027 0141-8130/© 2019 Elsevier B.V. All rights reserved.
for use in several applications in special for in biomedical and pharmaceutical uses. In 2009, Wei and co-authors reported the synthesis of thermosensitive hydrogel prepared by N-4-(chlorocarbonyl)phenyl] maleimide with polyethylene glycol (PEG) via the DA reaction in aqueous media and observed that the gelation time decreased with an increase in temperature [1]. In another paper by the same group, a copolymer of N-vinyl-2-pyrrolidone and furfuryl methacrylate was crosslinked with a polymeric dienophile [9]. Gárcia-Astrain et al. prepared a crosslinked hydrogel by the functionalization of poly(2-aminoethyl methacrylate) hydrochloride with furfural and a Jeffamine®-based bismaleimide [10]. Nimmo et al. copolymerized hyaluronic acid with PEG in one-step in aqueous media and prepared a hydrogel for tissue application [11]. Shao and collaborators reported a self-healing hydrogel prepared by cellulose nanocrystals with PEG via DA reaction, using modified CNCs as a reinforcing phase and chemical cross-linker. They obtained a nanocomposite with good mechanical properties and gelation time varying in function of different components [12]. Here, we describe the synthesis of thermally-reversible hydrogels obtained via DA/retro-DA (rDA) reactions in an aqueous medium using cellulose nanofibers as substrates. Refined “never-dried” cellulose fibres were submitted to surface TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) oxidation before grafting furfuryl amine onto the ensuing surface carboxylic groups. A water-soluble crosslinker was synthetized from exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride and 4,7,10trioxa-1,13-tridecanediamine to obtain an oligoether bismaleimide. Both reagents were then brought together to prepare a cellulose hydrogel via the furan/maleimide DA reaction. This study was prompted by
494
R.K. Kramer et al. / International Journal of Biological Macromolecules 141 (2019) 493–498
O
O
Δ N
O
N
O O
O Scheme 1. The Diels–Alder equilibrium between furan and maleimide end group.
the growing interest in chemically crosslinked pulp fibres, aimed at widening the range of materials from the pulp and paper industry, including the preparation of papers with improved mechanical properties for special applications, which moreover can be recycled following a simple thermal treatment. New applications in 3D printing can also use such kind of thermoreversible material. The vast majority of nanocellulose surface chemical modifications published to date required the use of organic media and rather cumbersome procedures to revert to aqueous conditions [13]. In the present approach, all operations were conducted in water, thus giving the process a “greener” connotation. 2. Experimental 2.1. Materials Refined “never-dried” cellulose pulp from eucalyptus species was kindly provided by Suzano S.A. Brazil. The reagents and solvents used for this investigation included N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC, pure, Sigma-Aldrich), N-
hydroxysuccinimide (NHS, 98%, Sigma-Aldrich), furfurylamine (99%, Sigma-Aldrich), dry methanol (Sigma-Aldrich), 4,7,10-trioxa-1,13tridecanediamine (97%, Sigma-Aldrich), exo-3,6-epoxy-1,2,3,6tetrahydrophthalic anhydride (Sigma-Aldrich 98%), 2,2,6,6tetramethyl-1-piperidinyloxy (TEMPO, 98%, Sigma-Aldrich), sodium bromide (≥99,5%, Sigma-Aldrich), and sodium hypochlorite solution (10–15%, Sigma-Aldrich). 2.2. TEMPO-mediated oxidation of cellulose [14] In order to prepare nanofibrillated cellulose (NFC), the cellulose fibres (5 g) were suspended in water (300 mL) and dispersed for 5 min using an ultra-turrax homogenizer (Ika-Laboratechnik, Germany). Then, a solution (50 mL) containing TEMPO (0.1 mmol/g of cellulose) and sodium bromide (1 mmol/g of cellulose) was added to the fibre suspension under mechanical stirring at room temperature. The TEMPOmediated oxidation of the fibres started by the dropwise addition of a 50 mL NaClO solution (10–15%, 5 mmol). The pH was maintained at 10 by adding drops of a 0.5 M NaOH solution, until no more variation was observed, and the reaction was protracted for 4 h. The pH was
Scheme 2. Grafting of furfurylamine onto the ToNFC carboxylic groups.
R.K. Kramer et al. / International Journal of Biological Macromolecules 141 (2019) 493–498
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Scheme 3. Thermally reversible cellulose hydrogel formation through the DA/rDA reaction. The black lines represent the cellulose nanofibers.
then adjusted to 7 with 0.1 M HCl and the modified fibres thoroughly washed with deionized water by filtration and stored at 4 °C. The carboxylate content of TEMPO-oxidized nanofibrillated cellulose (ToNFC) were determined by conductimetric titration. 2.3. Cellulose-furan grafting A 66.6 mL ToNFC aqueous suspension (0.1 g, 1.5 wt%) was mixed with the catalytic couple EDC (3.2 mmol) and NHS (2.4 mmol) and stirred for 30 min. Furfurylamine (3.2 mmol) was added and the pH adjusted to 5 with 3 M HCl before heating the suspension to 40 °C under magnetic stirring for 24 h. After the reaction (Scheme 2), the nanofiber suspension was acidified to pH 1.5 (HCl 3.0 M) and purified by thoroughly dialyzing it against deionized water for 7 days, to remove the remaining excess chemicals and salts. The water suspension was then sonicated at 70% output control for 5 min. Finally, the ensuing furan-modified ToNFC were stored at 4 °C [15].
2.6. Conductometric titration The fibres, ToNFC and ToNFC-furfurylamine samples (0.1 g, 0.1 wt%) were suspended into a 100 mL aqueous solution with 5 mL of 0.1 M hydrochloric acid and stirred for 5 min with the ultraturrax. The suspension was then titrated with 0.1 M NaOH. The degree of oxidation (Eq. (1)) (DO) [17] and the carboxyl group content (Eq. (2)) (mmol/g) were calculated using the following equations: DO ¼ ð162 C ðV 2 −V 1 ÞÞ=ðw−½36 C ðV 2 −V 1 ÞÞ
ð1Þ
X ¼ ðC ½V 2 −V 1 Þ=w
ð2Þ
where C is the NaOH concentration (mol/L), V1 and V2 are the volumes of the NaOH in litres used to neutralize and determine the carboxyl groups in a sample and w is the weight of the dry ToNFC sample (g).
2.4. Synthesis of the bismaleimide (BM) A solution of 10 g of exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride (ETHPA) in 50 mL of methanol, previously dried with 3 Å molecular sieves, was added to 6.83 g of 4,7,10-trioxa-1,13tridecanediamine (TTDDA) (molar ratio of 2:1), and the mixture kept refluxing at 65 °C under magnetic stirring for 24 h. Methanol was then removed under vacuum and the product was maintained at 95 °C for 24 h to remove the furan released from the rDA reaction, which restored the maleimide function. As described previously [16]. 2.5. Preparation of the thermally reversible hydrogel (DA and rDA reactions) The DA/rDA reactions of the hydrogel were conducted, on the one hand, by mixing the furan-modified ToNFC and the BM in water and stirring the suspension at 60 °C under a nitrogen atmosphere, and, on the other hand, by heating the ensuing gel at 95 °C, as shown in Scheme 3.
Fig. 1. FTIR spectra of the initial refined fibres (green line), ToNFC (blue line) and ToNFC modified with furfurylamine (red line).
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Table 1 Degree of oxidation per anhydroglucose unit of cellulose, carboxyl group content and yield of reaction steps.
Pulp ToNFC ToNFC-Furfurylamine
Degree of oxidation (DO)
Carboxyl groups content (mmol/g)
Oxidation yield (%)
Grafting yield (%)
0.088 0.334 0.040
0.543 1.90 0.246
– 73.6 –
– – 87.0
2.7. FTIR spectroscopy Infrared spectra were recorded on a FTIR Perkin-Elmer SpectrumOne spectrometer. All the samples were analysed at room temperature in the ATR mode, using the spectral range 4000 to 600 cm−1 at a resolution of 4 cm−1, by the accumulation of 20 scans. 2.8. Differential scanning calorimetry (DSC) DSC was performed on a Perkin-Elmer differential scanning calorimeter Pyris 1 under a nitrogen atmosphere. The heating/cooling rates were set to 20 °C/min, and the sample was weighed (approximately 11 μL) in a sealed aluminium pan for aqueous samples. The sample was first heated and kept in 55 °C for 20 min, followed by heating to 90 °C and an isotherm of 20 min, followed by cooling to 55 °C. Four cycles were performed. 2.9. Rheological characterization of hydrogels The rheological behaviour was analysed with a Brookfield (DV3TLV) rheometer, using a cone-plate geometry (12 mm cone radius and 3° cone angle). The applied frequency was constant, 0.1 rpm for 1 min at 25 °C. 3. Results and discussion The surface chemical modification of the nanocellulose fibres, obtained by the mechanical fibrillation of a commercial pulp, was carried out in water by the TEMPO-mediated oxidation of their primary hydroxyl groups following a well-established protocol [13,14]. Scheme 2 illustrates the condensation of furfurylamine onto the ensuing carboxylic cellulose functions, using the EDC/NHS-assisted amidation reaction in an aqueous medium. The success of the oxidation of the surface primary cellulose hydroxyl groups to carboxylate moieties was confirmed by FTIR (Fig. 1). The band at 1603 cm−1, characteristic of the carboxylate ion stretching, showed a substantially increase after the oxidation of the nanofibers. When the TEMPO-oxidized nanofibrillated cellulose (ToNFC) was modified with furfurylamine, the band at 1603 cm−1 decreased (Fig. 1) and a band related to the amide C_O stretching appeared at
Fig. 3. Thermal behaviour of the DA and r-DA.
1730 cm−1. Moreover, the band at 1648 cm−1 corresponding to N\\H stretching increased, although the band overlapped with the residual carboxylate peak, making this second assessment less explicit. Additionally, the typical furan-ring breathing band around 1000 cm−1 was completely masked by the strong cellulose ether band. It was therefore necessary to confirm the occurrence of both oxidation and grafting by conductimetric titrations of the carboxylic groups at the various stages of the process. Table 1 summarizes the results of this analytical approach. After the cellulose TEMPO-oxidation, there was a substantial increase in carboxylic groups at the surface of the ToNFC, confirming the success of the reaction. The ToNFC was then used for the surface functionalization with furfurylamine, which led to a very pronounced decrease in the carboxyl group content, thus confirming the occurrence of the furan grafting reaction with a good yield (Table 1). The DA/rDA reaction between the furan-modified nanofibers and the bismaleimide (Scheme 3) was conducted by adding an aqueous solution of the latter to a suspension of the former and stirring the ensuing mixture at 60 °C under a nitrogen atmosphere (Fig. 2a). A strong viscosity increase was observed, followed by the formation of a hydrogel, suggesting that the fibres had crosslinked through the furan/maleimide DA couplings (Scheme 3 and Fig. 2b). The crosslinking reaction finished in 30 min, approximately, reaching the gelation time. The decrosslinking reaction took place when the system was heated at 95 °C. The medium viscosity decreased within an hour to reach the value of the initial mixture (Fig. 2c). The system was then cooled to 60 °C and it displayed the same phenomenology as with the first DA reaction (Fig. 2d), thus regenerating the hydrogel and proving the reproducibility of the DA/rDA cycle, which was in fact successfully repeated three more times. It is important to mention that despite the fact that retroDiels-Alder reactions usually take place at higher temperatures, when furans and maleimides are used lower temperatures are observed such as 80–90 °C as described in the literature [8].
Fig. 2. Illustration of the DA/rDA cycle involving the maleimide/furan system in the process of hydrogel formation and deconstruction. (a) Initial mixture; (b) after the first DA crosslinking (formation of the hydrogel); (c) after the r-DA process (deconstruction of the crosslinked hydrogel); (d) after the second DA crosslinking reaction.
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Fig. 4. Rheological behaviour of (a) DA and (b) r-DA hydrogels.
The efficiency of the DA and rDA reactions was confirmed by the thermal behaviour of cellulose hydrogels. The DSC thermograms (Fig. 3) showed an exothermic peak around 56 °C, during the heating process, corresponding to the energy released during the adduct formation between furan and maleimide. During the cooling process, the endothermic peak around 87 °C was associated with occurrence of the rDA reaction that deconstructed the furan/maleimide adduct. The slight shift of the endothermic and exothermic peak can be attributed to the short duration of the experiments, which did not allow a full crosslink in the DA coupling, nor the completion of the rDA reactions. The successive thermal cycles shown in Fig. 3 proved the reproducibility of the DA/rDA cycle, i.e. of the thermal reversibility of the hydrogel. The viscosity behaviour of the thermally-reversible hydrogel was evaluated at a 0.1 rpm frequency. The hydrogel was kept at 60 °C for 30 min to complete the DA reaction and then tested at ambient temperature, as shown in Fig. 4a. The viscosity value of the hydrogel was 84,200 ± 4400 cP. When the system was heated at 90 °C for 30 min, the occurrence of the retro-DA reaction was observed by the corresponding decrease in viscosity. “Point 1” in Fig. 4b shows the maximum decrosslinking reaction, when the value of viscosity was the lowest, at about 380 cP. Then, as the temperature of the hydrogel was allowed to decrease, the medium viscosity increased progressively, ending at “point 2” (Fig. 4b), at ambient temperature, reaching the value of the initial hydrogel viscosity. These results confirmed that the Diels-Alder click chemistry was quite efficient in promoting the formation of thermally-reversible hydrogels. These results shows the potential of use of the MFC hydrogels for 3D printing of thermoreversible soft systems, in which the printing specimen can be produced at 80–90 °C and just after cooling to 50–60 °C can stabilize its structure based on covalent thermoreversible bonds.
4. Conclusions This preliminary study of the DA/rDA reaction cycle applied to aqueous furan-bearing nanocellulose fibres reacting with a water-soluble bismaleimide to give a thermally reversible hydrogel, validated the aimed strategy and produced a valuable novel material. Work is in progress to extend this investigation to other water-soluble readilypurifiable bismaleimides and assess the physical properties and possible applications of the hydrogels and their dried form in such materials as biocompatible aids and highly strengthened paper artefacts. Among these possible applications is noteworthy the production of biomedical devices using additive manufacturing or 3D printing of the hydrogel at temperatures where retro-DA dominates with subsequent cooling leading to a stable material which can be reprocessed or modified to
accomplish the desired application. The intervals of direct Diels-Alder reaction and its reversion allow hydrogel application in conditions that other added substances such as pharmacological substances and several biologic active molecules can be used.
Declaration of competing interest There are no conflicts to declare. Acknowledgements The authors gratefully acknowledge National Council for Scientific and Technological Development (CNPq) - Program “Ciência sem Fronteiras” program (CNPq 401656/2013-6) for the financial support, the Suzano Celulose e Papel S.A. for providing refined “never-dry” cellulose fibres and thank the Laboratoire de Génie des Procédés Papetiers (LGP2) – Grenoble INP, FRANCE where the work was carried out. R. K. K. thanks National Council for Scientific and Technological Development (CNPq) for a doctorate grant (CNPq 208033/2015-7). The authors also thank Debora T. Balogh for the viscosity measurements. References [1] H.-L. Wei, Z. Yang, L.-M. Zheng, Y.-M. Shen, Thermosensitive hydrogels synthesized by fast Diels–Alder reaction in water, Polymer (Guildf) 50 (2009) 2836–2840, https://doi.org/10.1016/j.polymer.2009.04.032. [2] J.M. Saul, D.F. Williams, Hydrogels in Regenerative Medicine, Handb. Polym. Appl. Med. Med. Devices, Elsevier 2011, pp. 279–302, https://doi.org/10.1016/B978-0323-22805-3.00012-8. [3] C. Chen, T. Zhang, B. Dai, H. Zhang, X. Chen, J. Yang, J. Liu, D. Sun, Rapid fabrication of composite hydrogel microfibers for Weavable and sustainable antibacterial applications, ACS Sustain. Chem. Eng. 4 (2016) 6534–6542, https://doi.org/10.1021/ acssuschemeng.6b01351. [4] E.A. Appel, J. del Barrio, X.J. Loh, O.A. Scherman, Supramolecular polymeric hydrogels, Chem. Soc. Rev. 41 (2012) 6195, https://doi.org/10.1039/c2cs35264h. [5] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug Deliv. Rev. 64 (2012) 18–23, https://doi.org/10.1016/j.addr.2012.09.010. [6] M.P. Lutolf, G.P. Raeber, A.H. Zisch, N. Tirelli, J.A. Hubbell, Cell-responsive synthetic hydrogels, Adv. Mater. 15 (2003) 888–892, https://doi.org/10.1002/adma. 200304621. [7] V. Crescenzi, L. Cornelio, C. Di Meo, S. Nardecchia, R. Lamanna, Novel hydrogels via click chemistry: synthesis and potential biomedical applications, Biomacromolecules 8 (2007) 1844–1850, https://doi.org/10.1021/bm0700800. [8] A. Gandini, The furan/maleimide Diels–Alder reaction: a versatile click–unclick tool in macromolecular synthesis, Prog. Polym. Sci. 38 (2013) 1–29, https://doi.org/10. 1016/j.progpolymsci.2012.04.002. [9] H.-L. Wei, Z. Yang, Y. Chen, H.-J. Chu, J. Zhu, Z.-C. Li, Characterisation of N-vinyl-2pyrrolidone-based hydrogels prepared by a Diels–Alder click reaction in water, Eur. Polym. J. 46 (2010) 1032–1039, https://doi.org/10.1016/j.eurpolymj.2010.01.025. [10] C. García-Astrain, I. Algar, A. Gandini, A. Eceiza, M.Á. Corcuera, N. Gabilondo, Hydrogel synthesis by aqueous Diels-Alder reaction between furan modified methacrylate and polyetheramine-based bismaleimides, J. Polym. Sci. Part A Polym. Chem. 53 (2015) 699–708, https://doi.org/10.1002/pola.27495.
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