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Mussel inspired self-healing double cross-link hydrogels by controlled combination of metal coordination and covalent cross-linking Amanda Andersen, Marie Krogsgaard, and Henrik Birkedal Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01249 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017
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Biomacromolecules
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Mussel inspired self-healing double cross-link
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hydrogels by controlled combination of metal
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coordination and covalent cross-linking
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Amanda Andersen, Marie Krogsgaard, Henrik Birkedal*.
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Department of Chemistry & iNANO, Aarhus University, Gustav Wieds Vej 14, DK-8000
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Aarhus, Denmark.
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KEYWORDS self-healing; hydrogels; mussel-inspired materials; coordination chemistry; double
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crosslink hydrogels.
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Abstract
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Mussel-inspired hydrogels held together by reversible catecholato–metal coordination bonds
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have recently drawn great attention owing to their attractive self-healing, viscoelastic and
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adhesive properties together with their pH-responsive nature. A major challenge in these systems
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is to orchestrate the degree of catechol oxidation that occurs under alkaline conditions in air and
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has a great impact on the aforementioned properties because it introduces irreversible covalent
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cross-links to the system, which stiffens the hydrogels but consume catechols needed for self-
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healing. Herein, we present a catechol-based hydrogel design that allows for the degree of
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oxidative covalent cross-linking to be controlled. Double cross-linked hydrogels with tunable
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stiffness are constructed by adding the oxidizable catechol analogue, tannic acid, to an oxidation
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resistant hydrogel construct, held together by coordination of the dihydroxy functionality of 1-
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(2’-carboxyethyl)-2-methyl-3-hydroxy-4-pyridinone to trivalent metal ions. By varying the
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amount of tannic acid, the hydrogel stiffness can be customized to a given application while
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retaining the self-healing capabilities of the hydrogel’s coordination chemical component.
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Introduction
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The use of orthogonal cross-linking mechanisms in hydrogel material design has been an idea
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based on double network (DN) hydrogels, which were first introduced by Gong et al. in 20031. In
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these designs, the hydrogel network consists of two different cross-linked polymers; one is
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highly cross-linked, rigid and fragile, while the other is loosely cross-linked and thus soft and
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ductile. The first network is responsible for high stiffness and the second is responsible for
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dissipating energy during deformations by flowing (enabled by loose cross-linking). Hydrogels
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designed using the DN principle have shown promising mechanical properties2-4. Such DN
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hydrogels exhibit a large degree of hysteresis during the first cycle of loading, which has been
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attributed to the breaking of irreversible bonds in the first network5. In 2016, Rodell et al.
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introduced dynamic (self-healing) cross-links in the first network to reduce the degree of
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hysteresis
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adamantine/cyclodextrine host-guest chemistry. These authors showed that DN hydrogels can be
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constructed by mixing polymers containing built-in cross-linking sites; the two-step
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polymerization technique used by Gong et al. is thus not a necessity6. Furthermore, they
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demonstrated the importance of covalent interconnections between the two networks for the
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mechanical performance of the DN hydrogels, i.e. they observed improved mechanical
by introducing
a
reversible
sacrificial
mechanism
through
the
use
of
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performance when the two networks were covalently crosslinked to form a double crosslinked
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(DC) double network (DN) combined hydrogel. We recently combined genipin covalent
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crosslinking of chitosan with coordination chemical crosslinking of g-DOPA-chitosan (DOPA =
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3,4-dihydroxyphenylalanine) to yield DN hydrogels that were self-healing through the use of
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metal coordination to the DOPA catechol50. Indeed, Materials with self-healing capabilities are
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of interest for a wide range of applications. Several routes to self-healing abilities have been
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proposed7-9. Especially implementations of self-healing through reversible bonds are interesting
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as it allows materials to heal repeatedly and without external energy input. Systems inspired by
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the self-healing coatings of the blue mussel adhesive threads (byssi) have solicited great recent
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interest10-17. They are based on the reversible coordination bonds of DOPA whose catechol side
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chain binds hard cations such as FeIII strongly and reversibly. This has been harnessed to make
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several classes of self-healing hydrogel materials14, 15. The blue mussel utilizes the exceptionally
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versatile reaction chemistry of the catechol15,
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environments using its DOPA-containing mussel foot proteins (mfp’s)14. The catechols not only
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mediate attachment to solid substrates but also play a key role in establishing the cohesive
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network between the mfp’s through non-covalent and covalent interactions. Above neutral pH,
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catechols are readily oxidized to reactive o-quinones, which then participate in a wide range of
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secondary reactions19. Among others, they have the ability to react with catechol-, amine- and
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thiol-groups; resulting in irreversible covalent cross-links18. The ability of catechols to
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participate in oxidative covalent cross-linking has been exploited in the design of a range of
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materials, including polydopamine (PDA)10, 20.
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, when sticking to various substrates in wet-
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Scheme 1. Catechol chemistry (A) cHOPO-PAH (polyallylamine-graft-1-(2’-carboxyethyl)-2-
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methyl-3-hydroxy-4-pyridinon, grafting density ~ 7%) (B) Chemical structure of tannic acid,
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TA. (C) pH-dependent coordination between cHOPO-PAH and hard metal ions, MIII. (D) TA’s
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ability to react with hard metal ions, MIII (left) and to form covalent bonds (right). TA reacts with
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backbone amines through i) Schiff base reaction and ii) Michael-type addition. Additionally, it
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reacts with iii) other pyrogallol groups to form diphenol cross-links. R represents the remainder
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of the polymer in (C) and the TA molecule in (D).
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Biomacromolecules
In general, great efforts have been put into creating mussel-inspired constructs10,
13, 14, 21-32
.
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Similarly, other polyphenols, such as tannic acid (TA, Scheme 1B) have been shown to be useful
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in this regard33-35. Another branch of this field revolves around emulating the catechol-metal
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coordination chemistry of the mfp’s in the design of self-healing hydrogels11, 12, 15-17, 36-40. The
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blue mussel utilizes these quite strong yet reversible coordination bonds in the creation of a self-
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healing protective byssal coating41-43 and in the adhesive plaque39. Commonly, mussel-inspired
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hydrogels held together by reversible catecholato-metal coordination bonds are formed upon a
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pH increase from acidic to alkaline conditions, thus benefiting from the pH-dependent speciation
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of the catechol-metal bond (Scheme 1C) and circumventing metal oxide precipitation at high
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pH11,
12, 15-17, 36, 37
, with similar chemistry employable with polyphenols such as TA44-47. The
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increased pH values required for hydrogel formation induce catechol oxidation and
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consequently, over time the reversible and rapidly formed catechol-metal cross-links are
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accompanied by covalent cross-links formed as a result of catechol oxidation12, 17, 18. Oxidative
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covalent cross-links can be formed in various ways. If the catechol-functionalized polymers
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contain primary amine groups, the generated quinones can react to the amines e.g. through
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Michael-type additions forming irreversible cross-links, Scheme 1D. In contrast to reversible
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catecholato-MIII cross-links (M = metal), which impart self-healing properties to the gels, the
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covalent network contributes with increased hydrogel stiffness and tanned materials potentially
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at the expense of the self-healing and adhesive properties. Unwanted catechol oxidation and
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coloring can be prevented by inhibiting oxidation via the removal of molecular oxygen12;
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however, for most applications this is impractical, necessitating other methods. Alternatively, the
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gels can be designed to resist oxidation, thereby preserving their dynamic mechanical properties
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since they are completely held together by reversible pH-responsive bonds. Catechols with
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electron-withdrawing substituents such as -Cl, -NO2, -CN, -CF3 are more difficult to oxidize,
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whereas it is the opposite for catechols with electron-donation substituents such as -OMe and –
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Me26, 48. Recently, Menyo et al. demonstrated how to prevent oxidation in mussel-inspired gels
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by the use of intelligently designed polymers equipped with a chelating HOPO functionality
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instead of DOPA (the structure of HOPO, 3-hydroxy-4-pyridinone, is shown in Scheme 1A)37.
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HOPO has a lower susceptibility to oxidation than DOPA as electron density is withdrawn from
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the aromatic ring through inductive and resonance effects, resulting in reduced phenolic pKa
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values (HOPO: pKa1 = 3.6, pKa2 = 9.9, DOPA: pKa1 = 9.1, pKa2 = 14)37. HOPO is classified as a
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“softer” ligand than DOPA but it still displays high affinity towards hard metal ions such as FeIII
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and AlIII, making it an excellent replacement for DOPA in mussel-inspired gels49. Thus, in
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contrast to traditional DOPA-gels, these oxidation-resistant gels are solely held together by
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reversible HOPO-metal coordination bond, rendering the materials fully pH-responsive and
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reversible. Nevertheless, the omission of covalent cross-links may result in limited mechanical
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stiffness, making them unsuited for certain applications.
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The goal of the present study was to design a polyphenol-based DC hydrogel system that
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allows for the degree of oxidative covalent cross-linking, and thus the mechanical stiffness, to be
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adjusted to a desired level while retaining full self-healing ability. This calls for orthogonal
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coupling chemistries. In our recent chitosan system, we employed genipin covalent crosslinking
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of chitosan and coordination chemical crosslinking of g-DOPA-chitosan to yield DN hydrogels
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that were self-healing50. However, this system, while very effective, did not afford fully
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independent crosslinking systems since genipin could cross-link both chitosan components and
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(slow) oxidative covalent crosslinking by DOPA could not be excluded. To ensure control over
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cross-linking mechanisms, we here use two different catechol analogues: one resistant to
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Biomacromolecules
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oxidation (HOPO) and one sensitive to oxidation (tannic acid, TA) to obtain double crosslink
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(DC) hydrogels. By controlling the relative amounts of HOPO, MIII and TA the equilibrium state
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of the hydrogels can be tuned to contain all MIII ions coordinated by HOPO, leaving TA free to
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oxidize and form covalent cross-link. This way, we aim to turn the twofold cross-linking
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properties of catechols to a highly controllable advantage instead of an unassessed problem, as is
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the case in previous studies. By varying the amount of TA added, the mechanical stiffness can be
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adjusted while retaining the reversible nature of the HOPO-metal bond.
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Materials and Methods
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To synthesize HOPO functionalized poly(allylamine hydrochloride) (PAH), a HOPO analogue
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that included a carboxylic acid (cHOPO, 1-(2’-carboxyethyl)-2-methyl-3-hydroxy-4(1H)-
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pyridinone) was made because this would later facilitate conjugation to the PAH polymer
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through standard EDC/NHS coupling. In brief, cHOPO was synthesized from 3-hydroxy-2-
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methyl-4-pyrone through a procedure inspired by the synthesis of 1-carboxymethyl-3-hydroxy-2-
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methyl-4-pyridinone described by Mawani et al. and Zhang et al.51, 52. At 80 oC, 4.98 g (39.5
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mmol) maltol (3-hydroxy-2-methyl-4-pyrone, 99%, Sigma Aldrich) and 7.08 g (79.5 mmol) β-
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alanine (99%, Sigma Aldrich) was dissolved in 100 mL demineralized water by stirring and pH
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was adjusted to 9 by addition of 6 M NaOH. Under continuous stirring, the mixture was heated
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to reflux for 24 h, decolorized with activated carbon at 60 oC for 30 min, filtered with suction
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and then allowed to cool to room temperature. The solution volume was reduced to half by rotary
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evaporation and pH was adjusted to 3 by addition of 6 M HCl. The brown precipitate was
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isolated by suction filtration and recrystallized from water to yield cHOPO as off-white crystals.
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The reaction was confirmed using 1H and
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of cHOPO together with the assignment of the different peaks is shown in Figure S1A.
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C NMR and FT-IR spectroscopy. The 1H-spectrum
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Subsequently, cHOPO was covalently attached to PAH (Poly(Allylamine Hydrochloride),
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40wt% (aq.), MW: 120000-200000, PolySciences.com) through standard EDC/NHS coupling
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chemistry (Scheme S1C) and the reaction was confirmed using 1H NMR spectroscopy (Figure
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S1B, bottom). The cHOPO grafting density (݃ =
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UV/VIS absorption measurements using pure cHOPO as a standard. DOPA-PAH was
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synthesized as in reference our previous works
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hydrogels and/or coordination complexes, AlCl3·6H2O (puriss, Sigma Aldrich) or FeCl3·6H2O
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(>98%,
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(ethylenediaminetetraacetic acid, >98.5%) were purchased from Sigma Aldrich. All chemicals
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Sigma
Aldrich)
was
used.
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ಹೀುೀ ೝ
) was determined to ~ 7% from
and had a grafting density of 9.5%. To form
Tannic
acid
(ACS
reagent)
and
EDTA
were used as received.
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AlIII:cHOPO-PAH hydrogels were made following our previously published 3-step recipe 16, 17
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inspired by using the mussel-mimetic acidic-to-alkaline pH change of Holten-Andersen et al.11:
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17 mg cHOPO-PAH was dissolved in 75 µL 0.1 M HCl to a starting concentration of 226.7 mg/mL,
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corresponding to a cHOPO concentration of 173 mM. 25 µL 0.173 M AlCl3· 6H2O was added to the
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polymer solution. This will result in an cHOPO:AlIII ration of 3:1. Then, 50 µL NaOH was added to
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adjust pH to the targeted value and the hydrogel was formed by mixing. For the TA containing
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AlIII:cHOPO-PAH hydrogels, TA was dissolved with the polymer (amounts adjusted to fit the
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desired molar ratio between pyrogallol groups on TA and monomers). Photographs of the steps
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are provided in Figure S2 (ESI).
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Figure 1. pH-dependent absorbance. UV-Vis absorption profiles of (A) cHOPO (B) cHOPO-
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PAH. Integrated absorbance (C) and fitted positions (D) of the three cHOPO peaks. Black lines
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in (C) shows the fit to a double Boltzmann model.
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Results and Discussion
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We first investigated the acid/base and coordination abilities of cHOPO alone and grafted onto
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PAH. The behavior of cHOPO was seen to change upon grafting onto the polymer as compared
8
to free cHOPO. The pKa was determined from UV/VIS absorption spectroscopy, Figure 1 (See
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ESI Figure S3 for full spectra); due to the dependence of absorbance on the electronic structure
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of light-absorbing molecules, the pKa of the cHOPO diphenol could be observed as pH-
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dependent shifts in the position of the absorption peaks. For pure cHOPO, the shift starts at pH
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~9.5 (Figure 1A) whereas when cHOPO is grafted onto PAH (Figure 1B), the peaks start shifting
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at pH ~7.5. To quantify this shift in cHOPO pKa, we fitted the full UV-Vis spectra (Figure S3A
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& B, ESI). The integrated absorbance (Figure 1C) of the spectra above the minimum at 220-240
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nm (depending on pH) was extracted from these fits and used as a measure of the total oscillator
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strength of cHOPO. The oscillator strength shows steps at pH values equal to the two pKa values
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of cHOPO. The position (pKa) and width of the steps were extracted by fitting the integrated
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absorbance to a double Boltzmann model (fitting parameters are provided in Table S1, ESI). For
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pure cHOPO, the first transition occurs at pH 3.52(3) whereas for cHOPO-PAH the value is
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shifted down to 2.60(4), showing that the first pKa value of HOPO is shifted down by a full pH
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unit when grafted onto PAH. The second transition is also shifted down from pH 9.89(2) to
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9.65(3); but here, the shift is much smaller. The width of the first transition (see ESI for details)
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is only changed by a small amount: 3.2 versus 2.9 pH units. However, the apparent width of the
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transition is broadened from 2.9 to 5.5 pH units due to overlap with the pKa of the amines on the
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PAH backbone. Changing pH not only changes that absorbance (oscillator strength) but also the
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wavelength of the absorption peaks, Figures 1A & B. The peaks originating from cHOPO
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(Figure 1A & B) can be described by three peaks, hereafter referred to as HOPOa, HOPOb,
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HOPOc, respectively. The peaks positions were extracted from the peak fits for pH
