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Dynamic cross-linking of an alginate–acrylamide tough hydrogel system: time-resolved in situ mapping of gel self-assembly

Hydrogels are a popular class of biomaterial that are used in a number of commercial applications (e.g.; contact lenses, drug delivery, and prophylactics). Alginate-based tough hydrogel systems, interpenetrated with acrylamide, reportedly form both ionic and covalent cross-links, giving rise to their remarkable mechanical properties. In this work, we explore the nature, onset and extent of such hybrid bonding interactions between the complementary networks in a model double-network alginate–acrylamide system, using a host of characterisation techniques (e.g.; FTIR, Raman, UV-vis, and fluorescence spectroscopies), in a time-resolved manner. Further, due to the similarity of bonding effects across many such complementary, interpenetrating hydrogel networks, the broad bonding interactions and mechanisms observed during gelation in this model system, are thought to be commonly replicated across alginate-based and broader double-network hydrogels, where both physical and chemical bonding effects are present. Analytical techniques followed real-time bond formation, environmental changes and re-organisational processes that occurred. Experiments broadly identified two phases of reaction; phase I where covalent interaction and physical entanglements predominate, and; phase II where ionic cross-linking effects are dominant. Contrary to past reports, ionic cross-linking occurred more favourably via mannuronate blocks of the alginate chain, initially. Evolution of such bonding interactions was also correlated with the developing tensile and compressive properties. These structure–property findings provide mechanistic insights and future synthetic intervention routes to manipulate the chemo-physico-mechanical properties of dynamically-forming tough hydrogel structures according to need (i.e.; durability, biocompatibility, adhesion, etc.), allowing expansion to a broader range of more physically and/or environmentally demanding biomaterials applications.

Resilience (i.e.; ability to recover from elastic deformation), strength (i.e.; ability to bear a mechanical load) and toughness (i.e.; ability to resist fracture) are inherently contradictory material properties and so, hard to combine. High strength requires low mechanical dissipation (i.e.; suppression of dislocation and plastic deformation) while high toughness requires high mechanical dissipation during deformation (i.e.; large amounts of work before fracture). 31 DN tough hydrogels (e.g.; alginate-PAAm networks) can offer both high toughness and resilience via delayed stiffening and mechanical dissipation due to broad physico-chemical bonding and varying but complementary physico-mechanical properties between the gel components. 12,32 Physically cross-linked (i.e.; reversible) gels are bound by attractive non-covalent forces (i.e.; H-bonds, ionic cross-links and protein-ligand associations) between polymer chains. 13 Thus, absent covalent cross-links, linear chains form networks via topological (i.e.; entanglement) interactions, so exhibiting viscoelastic rheology. 21 Physical hydrogels, (e.g.; as formed by alginate), exhibit good toughness, but lack the creep resistance of covalent gels. 13 Ionic cross-links (e.g.; via Ca 2+ ) complexed with polyelectrolyte anions offer strong bonding and cross-link density, but also provide points of detachment/re-attachment, leading to, e.g.; self-healing activity and modied de-/swelling behaviour of hydrogels. For example, Ca 2+ co-ordinates to Mand G-blocks on alginate during gelation, acting as junctions between blocks on adjacent chains (i.e.; egg-box model). [33][34][35][36] Thus, the distribution of M and G units along the alginate chain, as well as the changing M/G ratio value of the alginate system, as preferential ionic cross-linking occurs to one uronic acid block over another, determines many physico-chemical properties of the gel structure that are closely related to their functionality, i.e.; high ratios results in a more elastic, exible (although more fragile) gel whilst low M/G provide brittle, water-insoluble, more rigid gels. 18,37,38 This is because the semirigid chains of G-rich alginates strongly electrostatically interact with Ca 2+ via (G)-COO À groups, leading to either charge neutralisation of a single chain, or cross-linking across separate chains and so, possess greater durability due to the higher shear rate necessary to induce the chain orientation. [39][40][41][42] Conversely, for M-rich alginates, the electrostatic interactions are less signicant, especially for low molecular mass, and the viscosity decrease starts at a lower shear rate. 43 However, there have been reports that low molecular weight and low M/G alginates produce the strongest, most well organised alginate structures. 44 Chemically cross-linked (i.e.; permanent) gels are facilitated through various functional groups in the polymer backbone, (e.g.; hydroxyl, amine and hydrazide) oen via specic chemical cross-linking agents (e.g.; N,N 0 -methylenebisacrylamide). The resultant permanent structure is usually more stable than for physically cross-linked, but may exhibit poor mechanical strength and toughness. 13 The degree of covalent cross-linking is usually the most important factor in determining the resultant macroscopic properties (e.g.; mechanical strength, swelling and encapsulant release). 45,46 Alternative chemical cross-linking methods include enzymatic linking and free-radical polymerisation. 13,47 Both physically-and chemically-cross-linked hybrid hydrogel systems undergo entanglement as well as ionic and covalent cross-linking of multi-component polymer networks; the extent of cross-linking dependent on the polymer functional groups, as well as the size and type(s) of cross-linking agent used. Such systems are the focus of this paper. A systematic exploration into the dynamic formation processes of model DN alginate-PAAm tough hydrogels should yield a fuller understanding of the myriad dynamic bonding interactions that occur within the tough hydrogel polymer structure. While past (e.g.) NMR studies have reported on chemical structure, bonding and internal mobility of constituents, the nature of solution NMR processing, (i.e.; gel dissolution prior to analysis, as well as the fact that sample spectra need to be acquired at high-temperature to decrease the viscosity of the gel) confounds data outputs and may introduce inaccuracies into systems; the various required interventions may affect the bonding environment and so give erroneous interpretations of structural changes and relationships in cross-linking processes. 16,[48][49][50][51] Other methods (e.g.; mass spectrometry, XPS analysis etc.), also suffer from potential process inaccuracies for tough hydrogels.
This paper offers a detailed, time-resolved, investigation into tough hydrogel physico-chemical bonding effects and dynamic changes over a reaction, using myriad complementary analytical techniques (i.e.; spectroscopy (FTIR, Raman, UV-vis and uorescence), microscopy (optical and video uorescence), TGA-DSC, DLS, tensile-and compression-testing). Mechanistic insights into the formation routes and their correlations with resultant viscoelastic properties, are afforded using a nonintrusive continuous monitoring approach to give a clearer understanding of the structural changes over time, removing the need for guesswork or post-rationalisation with snapshot data, in order to highlight routes towards improved versatility and control over the resultant polymer physico-mechanical properties. 48,[51][52][53][54][55] We explored a model DN alginate-PAAm system, with good molecular affinity between the two networks, as aided by both ionic (CaSO 4 ), and covalent (N,N 0 -methylenebisacrylamide) cross-linkers.
Past reports into alginate hydrogels (theoretical and experimental) have explained the favourability of uronic acid block coordination for ionic bonding, where G-block co-ordination has frequently reported as most favourable in accordance with the egg-box model. However, the same such in-depth reports have not yet been made for alginate-based tough hydrogel systems, to the best of our knowledge. We hypothesise that, in line with past reports, G-block alginate co-ordination will be favoured during ionic cross-linking over the course of a gelation process. In such a G-block co-ordination, progress in a dynamically evolving reaction will result in rising M/G (i.e.; a positive correlation of M/G with time). were used for synthesizing polyacrylamide. All materials were used as received without any further purication. [N.B.; It is recommended that a fresh source of TMEDA precursor is used for all polymerisation reactions.] Laser grade Rhodamine 6G (R6G) dye was procured from exciton and used without further purication. Deionized water (DI-H 2 O) was used for all experiments and spectroscopic studies.

Experimental
Double network hybrid alginate-polyacrylamide (Alg-PAAm) tough hydrogels were synthesised using known, free radical copolymerisation protocols. [56][57][58] Sodium alginate polymer (6.76 g) and monomeric AAm (40.54 g) at a 1 : 6 ratio, were dissolved in DI-H 2 O (300 ml) then stirred for at least 2 h to remove all alginate lumps. 1 All experimental characterisation (Raman, FTIR, UV-vis, and uorescence spectroscopies) used 50 mL of this stock solution for free radical polymerisation. The free radical polymerisation was triggered through the introduction of: APS initiator (67.5 mg), TMEDA accelerator/catalyst (0.21 mL), MBA co-monomer/covalent cross-linker (4.05 mg) and CaSO 4 ionic cross-linker (148.5 mg), which were homogenised on a vortex mixer immediately prior to loading and analysis. 34,59 Reactions using R6G as tracker dye (i.e.; UV-vis and uorimetry), used 0.04 M dye, which was fully and homogeneously dispersed in the stock solution, at the initial stage of stock solution preparation. All synthesis and analyses were carried out at room temperature and pressure (RTP), in air ambient. Beyond the initial reaction, no further treatments (e.g.; sealing, silicone oil, etc.) were conducted on samples.

Tough hydrogel materials characterisation
Subsequent to initial gel preparation and loading, there was no further sample movement or exchangeall readings were taken on the same sample, in the same conguration, as the gelation evolved. Time-based readings were initiated as soon as the reaction components were mixed in a single vessel; the time codes corresponding to the time reading at which point each data acquisition was initiated. Tough hydrogel in situ structurebonding evolution effects in real-time, at RTP, were examined by: Raman spectroscopy (BaySpec Nomadic) with a 532 nm laser excitation source (100% intensity), over 200-3200 cm À1 , at 20 s integration, with any cosmic ray artefacts manually removed through data averaging; transmission ATR-FTIR (Perkin-Elmer Spectrum 100) over 4000-650 cm À1 , at 4 cm À1 resolution and 16 averaged scans; UV-visible spectroscopy (UH5300 Hitachi) recorded at 1 nm step, across 200-800 nm, at 400 nm.min À1 against a DI-H 2 O reference standard; uorescence spectroscopy (Edinburgh Instruments Spectrouorometer FS5) excited at 470 nm, recorded over 500-700 nm, at a 1 nm step size and 1 s dwell time.

Tough hydrogel mechanical properties characterisation
Mechanical properties were evaluated using uniaxial tensile and unconned compression tests at RTP, to generate stress-strain curves. Compressive tests were done on an INSTRON 4411 with 5000 N (ASTM D1424-09) at constant crosshead velocity of 10 mm min À1 on cylindrical samples (18 Â 35 mm; h Â d). 22 Tensile tests, on (size 75 Â 40 Â 2 mm; l Â w Â h) rectangular samples with notch and without notch, and (50 Â 4 Â 3 mm; l Â w Â h) dumbbell-shaped samples, were done on an INSTRON 5566 with a 500 N load cell (ASTM D1424-09), and 20 mm gauge length, at a constant extension rate of 100 mm.min-1.61 Because creep, high stretchability, water loss and time consumption were major concerns for data accuracy, a relatively high strain rate of 100 mm min À1 was used. For notched samples, an initial notch of $2 mm was cut using a razor blade and measured using calipers. Tensile strength was taken from the point of maximum stress. The modulus was taken from the average slope over 0-10% of strain ratio from stress-strain curves. 34,61

Reaction overview
A double-network (DN) tough hydrogel system was formed based on a 1 : 6 alginate(Alg)-acrylamide(AAM) ratio, in accordance with Sun et al.'s procedure ( Fig. 1; deviations from conventional mechanisms are based on the experimental data from this paper). APS and TMEDA together combined to overcome the lack of experimental photo-activation, speeding up gelation as well as controlling the structure and uniformity of gels. The APS-initiator undergoes homolytic ssion to produce SO 4 À . Radicals via accelerated conversion from sulde ions in combination with TMEDA, which in turn also produce hydroxyl radicals in contact with water. 62 These active initiator ions help convert cross-linkable C]C to C-C in AAm, as they co-ordinate to Alg, as a result of sulfate addition onto AAm, aiding polymerisation. [62][63][64] Thus, the APS inuences polymerisation speed as well as gel uniformity. The covalent cross-linker MBA, affords random bonding and co-polymerisation, eventually resulting in gel formation.
The room-temperature, free-radical polymerisation process was analysed in situ, in a time-resolved manner, using a host of complementary characterisation techniques. Insights into the evolution of bonding interactions and their potential impacts on gel mechanical properties, were obtained as a result. The CaSO 4 ionic cross-linked system was explored in detail, although similar trends were observed for equivalent molar ratio variations of alternative cationic cross-linker (i.e.; MgSO 4 , Na 2 SO 4 , CaCl 2 , and Ca-lignosulfonate); see ESI. † Tough hydrogel properties (and gel properties in general), can be controlled via the onset and extent of gelation, with the subsequent mechanical properties modied via chemical precursor component presence (including the presence of metal (e.g.; calcium) salts), relative concentrations and relative ratio variation. 65,66 The unstable colloidal system dynamically agglomerates into a non-ergodic gel over time, as facilitated by charged functional groups at the gel surface. In such out-ofequilibrium systems, the DN tough hydrogel undergoes extensive entanglement, increasing interpenetrating bond formation and cross-linking, which in turn increases the likelihood of further intermolecular associations, resulting in increased shear thickening, gel viscosity and elastic characteristics. In general, the macroscopic, rheological properties of polymers arise from microscopic entanglement of dynamic polymer chains and the constraints imposed by gelation, as determined by reptation dynamics, including the resultant elastic properties of polymeric gels. 67,68 3.2 Fluorescence spectroscopy emission data R6G uorescent tracer dye was used as a sensitive stain that coordinates to the dynamically forming tough hydrogel, and allows for the reaction process to be accurately followed in realtime. 69 During the formation process, a visually-detectable change occurs in the R6G from approximately pink-red to an orange coloured emission (Fig. 2). 70 In the time-evolved uorescence emission spectra, from the rst minute onwards, there are two clearly dened signals for the R6G; at l 560 and l 597 . The band splitting and changes are indicative of varying monomer and dimer concentrations. The band at l 597 indicates H-type and J-type uorescent dimer formation, depending on the specic conguration adopted upon interaction. 69,71,72 The band at l 560 is due to fully solvated and relatively well-isolated monomeric dye molecules.
Aer $519 s, there is a uorescence band shi from a doublet to a single, broad band at l 594 , which is attributed to excimer formation and also signies the onset of the main gelation process. 73 Empirically, it arises due to the broad (and continued) conversion of the monomers and dimers of the precursors condensing and cross-linking into longer chain polymers that undergo further entanglement and aggregation, as gelation progresses. 74,75 The high density of subsequent intermolecular interactions results in the formation of electronically excited states; excimer emission. 76 This process is caused by enhancement of the polarisability in the dyesurrounding environment that results from the close packing of dye moieties in dimers and aggregates. 71 The observed spectral band is generally broader because the uorescence occurs between a bound upper state and an unbound ground state, meaning a broader range of vibrational states can be inhabited (due to fewer quantisation selection rule pressures). 77 There is little further change in trace prole apart from decreasing band intensity (i.e.; a static uorescence quenching process), indicating a stable tracer dye with little further change in the R6G-surrounding environment and that chains are broadly in the most condensed state, although further agglomeration and aggregation still continues over the rst hour of reaction and during the course of a week. 71,78-80

UV-vis absorption spectroscopy
UV-vis analyses of the hydrogel formation process were also done in the presence of cationic R6G uorescent tracer dye (Fig. 3A-F), to follow the increase of cross-link density in situ and in real time, as a corollary to the uorimetry data. 81 Seven regions of interest are identied at l 242 ; l 332 ; l 349 ; l 410 ; l 505 ; l 533 , and; l 668 . Analysis indicates differing intensity variation relationships, reecting different reaction regimestentative assignments have been made. The bands at l 410 and l 668 , are primarily attributed to sodium alginate (ESI Fig. S5 †), in contrast to past reports, which asserted that there is no observable signal from sodium alginate. 82,83 The l 411 shows little movement in l max position, but diminishes in intensity during the reaction, indicating consumption of the sodium alginate precursor as it undergoes polymerisation. The strong signal at l 242 , is predominantly attributable to the acrylamide component, although the APS, MBA and TMEDA, are all thought to also contribute. These all undergo a heavy intensity reduction, blue-shi and tailing, over time as the elements undergo conjugation; the largest decrease corresponding to consumption of acrylamide during cross-linking. 84 The band prole change indicates a differential reaction rate for the different components. Two bands at l 349 and l 533 are attributed solely to the R6G dye and decrease in spectral intensity as cross-linking proceeds within the hydrogel. In such reactions, as reected in the uorimetry data, the positively charged dye co-ordinates to the forming hydrogel, likely via hydrogen bonds, such that the spectral features undergo change as the cross-linking reaction proceeds and the dye is consumed. 85,86 The l 349 signal is attributed to p / p* transitions arising from dye co-ordination to the forming hydrogel. 87,88 The l 533 prole arises due to the excellent co-ordination abilities of R6G to Ca 2+ . 89  co-ordination route has marked effect on the resultant gel mechanical properties.
Comparisons of M/G ratio allows a valuable semiquantitative appraisal of the relative composition of alginatebased gels, whose initial composition is based on the original seaweed source, as well as any changes that occur during a reaction. The specic ratios depend on the presence, identity and concentration of the ionic cross-linker (i.e.; Ca 2+ ) and the ability to readily ion-exchange. 93,94 These M/G ratios will change over the course of a reaction. There are broadly three FTIR doublets from which M/G are empirically approximated, of which we utilised two; M $1290 cm À1 /G $1320 cm À1 , and; M $1030 cm À1 /G $1080 cm À1 (no doublet was observed at M 808 cm À1 /G 787 cm À1 ). [95][96][97] For Raman spectroscopy, the characteristic bands were at M $975 cm À1 /G $825 cm À1 . 98,99 Comparison between this complementary data is valuable as IR is more sensitive to side group vibrations and Raman is more sensitive to skeleton vibrations; any difference between the values is thought due to the inherent mechanism of the methods. 98 A baseline method was used and maximum intensities compared, for all datasets (Fig. 4). 41 There is a decrease in M/G and a greater decrease in the intensity ratio of some of the most representative bands associated to M-units, over 60 min, which suggests that the di-    across both Raman and FTIR M/G data, with similar onsets of change, rates of change and trends in M/G ratio, despite the difference in absolute values. It is only over the longer-term curing process (24-168 h), that G-block bonding becomes more favourable and this is thought to arise due to the decreasing number of freely available M-block units for Ca 2+ to easily co-ordinate to. Thus, during the cross-linking analysis period in our tough hydrogel system, an acidic polysaccharide comprising a major fraction of G-block residues (i.e.; G-rich) is formed initially, which in turn explains the relatively high tensile strength of the experimentally obtained tough hydrogel. 17,18,40,100 This unexpected result contradicts the "egg-box model", since G-blocks have previously been considered to have more affinity to Ca 2+ than M blocks, although it may be explained by the presence and relative concentration of the Ca 2+ , which is known to produce stronger gels in high-M alginate and/or above a critical cation concentration. 42,44,101,102 Initially, there is little change in the ratio, as well as little change in the absolute intensitiesthe ionic interactions do not predominate, and where it occurs, it seems to occur relatively evenly between M and G units. From $8 min onwards, the bonding occurs more favourably via the M-units in Alg-PAAm DN tough hydrogels. The largest rate of change seemingly occurs within the rst 10-15 min, aer which a much slower rate of change is observed, (i.e.; both M-and G-block co-ordination are favoured thereaer). ; hydrophilic carboxyl and hydroxyl groups) and cross-linkable C]C bonds, that facilitate hydrogel formation. 45 Peak shis and proles evolve mainly within the rst $60 min, although further changes are observed over $168 h. Furthermore, there are seemingly two broad reaction stages within this rst hour; phase I occurs within $10 min, and phase II spans the next $50 min and beyond. Due to the multiple components involved in the gel and the overlapping signals present, a tentative assignment is offered here, especially in areas where corroborating literature sources are absent. Unsurprisingly, the majority of FTIR peaks and Raman bands correspond to those of acrylamide and alginate.

ATR-FTIR spectroscopy
For the transitions of interest in phase I, there is an increase in signals at 3380 cm À1 (n(OH)), 2918 cm À1 (n s (CH 3 )), 2850 cm À1 (n as (CH 3 )), and 2116 cm À1 (n(CN)). 103 There is likewise, a speedy decrease in signal at 1676 cm À1 (n s (CO) amide I). In phase II, there is an increase in signal at 2957 cm À1 (n(CH)), 2933 cm À1 (n as (CH 2 )), and 1033 cm À1 (M n+ -O), as well as new peaks that grow in intensity at 1458 cm À1 (n as (CH 3 )), 1359 cm À1 (n s (CH 3 )), and 1335 cm À1 (u(CH)), all of which continue to evolve up to 168 h 41,104-106 There is also a relative decrease of peaks at; 1618 cm À1 (n as (COO À )), 1436 cm À1 (r(CH)), 1420 cm À1 (n s (COO À )), 1284 cm À1 (n(CO)), 1125 cm À1 (n(CO)), 1050 cm À1 (n as (C-O-C)), 987 cm À1 (G-block d(C]C)), 972 cm À1 (G-block n(CO)), 836 cm À1 (M-block d(C 1 -H)) and 816 cm À1 (M-block residues). The signals at 1268 cm À1 (n(CO)), 1098 cm À1 (n as (C-O-C)) and 1022 cm À1 (n(CO)), broadly remain unchanged. 107,108 Naturally, because of the precursors used and the nature of the hydrogel itself, all IR spectra are dominated by a strong, broad hydroxyl band spanning 3100-3600 cm À1 . 109 Possibly, there is an N-H stretch also present but drowned out by the strength of the overlapping -OH stretch $3380 cm À1 . As the reaction proceeds, beyond the rst hour and into 24-168 h, the -OH stretch broadens, accompanied by a slight increase in intensity. However, it is difficult to deconvolute from the contribution of the N-H shoulder ($3250 cm À1 ) and so parse whether the contributions are from increased intramolecular and/or intermolecular bonding. 106 Signals associated with alkyl groups increase during the reaction. 15 Many of these changes occur within the rst $10 min, indicating bonding involvement through unsaturated carbons, in the initial stages of reaction. Peaks at 2850 cm À1 and 2918 cm À1 are present and increase with time. There are also two new peaks, at 2933 cm À1 and 2957 cm À1 , not present at the outset, that gradually emerge over the course of the reaction, with increasing speed of onset beyond the $600 seconds of reaction mark, i.e.; they are associated a different phase of the hydrogel formation to the 2850 cm À1 and 2918 cm À1 signals. Similarly, signals related to geminal methyl groups, at 1458 cm À1 and 1335 cm À1 , also not present initially, become apparent in phase II, growing in intensity thereaer. 15 In the double bond stretching region, four peaks can be clearly observed; those at 1618 cm À1 and 1436 cm À1 are attributed to asymmetric and symmetric stretching vibrations of C]O from the -COOÀ groups of alginate respectively. 15,104,[110][111][112] As the reaction proceeds, the signal decreases, broadens and shis slightly, to higher wavenumber, indicating exchange of cross-linking ions at the alginate and the procession of ionic bonding via Ca 2+ . 113 Signals assigned to the bending vibrations of the NH(amide band); -COCand -CO stretch; and -OH angular coupling, indicating the existence of free carboxyl groups, decrease over time 109 In addition, another broad, medium-intensity signal is present throughout the reaction; a -CN stretch at 2116 cm À1 , characteristic of plant gums like alginate that increases over the course of the reaction, which may in turn facilitate acylhydrazone bond formation to AAm. 114,115 The 1033 cm À1 region increases with timethis signal is also associated with metaloxygen bonds; the relative signal increase indicating greater Ca 2+ -binding involvement with reaction time, as calcium displaces sodium. 106 Peaks at 1676 cm À1 and 1436 cm À1 broaden with time and increasing Ca 2+ incorporation. 106,116 As cross-linking proceeds, the characteristic amide I carboxylate group at 1676 cm À1 , grows in prominence while the aldehyde vibrational band diminishes, indicating its critical role in cross-linking to form a conjugated amine system. The change in the singlet at 1436 cm À1 , likely corresponding to a C]O conjugated, symmetric stretch of COO groups, decreases in intensity and converts into a doublet; the new peak found at higher wavenumbers. This peak ssion is likely due to the stretching vibrations of C-O and the O-H angular coupling, which are specic to ionic binding; the Ca 2+ modies the environment around the carbonyl group and also signies that much of the cross-linking occurs via the carbonyl groups. 117 The decrease of a M-block C-O-C pyranose stretch at 1050 cm À1 over the course of the reaction again shows the consumption and binding that occurs via such units, although the 1033 cm À1 peak does not undergo such a marked change. 104,106 No peaks were observed at 1010 cm À1 , which would otherwise be observed, as related to Ca 2+ -oxygen bonds to G-blocks; the absence again supporting the favourability of the M-block co-ordination. 106 This is further supported by the decreasing intensity of two bands at 1284 cm À1 and 1098 cm À1 , as well as the broad, weak signal at 1050 cm À1 which correspond to elongation in the C-O-C asymmetric stretch. 109,110,118 All these peaks shi to lower wavenumbers as calcium content increases, indicating weakening of C-C and C-O bonds due to sharing with Ca 2+ . 106 The weak doublet at 987 cm À1 and 972 cm À1 likely corresponds to a-(1-4)-glycosidic bonds, as observed in the original alginate precursor, and the decreased signal over time is related to the interaction of these binding sites to Ca 2+ . 106 Additionally, a very weak doublet related to the MM bands at 816 cm À1 and 836 cm À1 decreases over the rst 60 min (largely absent aer $20 min), as ionic cross-linking proceeds, until completely absent by the latter stages of the reaction. 113,119,120

Raman spectroscopy
Raman spectroscopy data are in broad agreement with those of FTIR spectroscopy and show that Raman bands shi and undergo changes in peak prole and intensity during gelation, indicating broader evolving structural changes. 121 Tentative assignments of the multicomponent system are offered in Fig. 6 (individual precursor signals are in ESI Fig. S6 †).
Large intensity changes were observed for the -OH stretching vibration, possibly due to consumption of the individual monomers during gelation (i.e.; covalent binding as well as hydrogen bond interactions). New Raman bands evolved in two regions over the reaction; $2930 cm À1 , and $1460 cm À1 , attributed to; acrylamide isopropyl -CH 2 symmetric stretches, and acrylamide -CH 2 bends respectively. The $2930 cm À1 band undergoes a large increase, from near non-existent, to high intensity, which indicates that polymer chains undergo bond saturation (i.e.; sp 3 -C-H stretch) as a result of covalent crosslinking. 15,122,123 In addition, a shoulder starts to develop and increase in intensity aer 24 h, at $2870 cm À1 , which corresponds to aliphatic -CH 2 À. 124,125 Likewise, the $1460 cm À1 signal also becomes increasingly present, as the reaction proceeds.
All other Raman bands diminished signicantly during the reaction process. Bands above 1300 cm À1 are attributed to deformations of -CH x functional groups and stretching vibrations of the carboxylate functional group -COO-. 126 Thus, unsaturated acrylamide carbon bands at $3114 cm À1 vanish over time; the $3040 cm À1 band greatly diminishes in intensity. Furthermore, a band corresponding to N 2 reaction by-products, appears at $2330 cm À1 ; the intensity of the free N 2 vibron increases over the course of an hour but disappears aer 24 h 127 The C-O and COO stretching vibrations present over 1800-1400 cm À1 and can be used to characterise the alginate and identify different cation binding states. The C-O stretch band does not occur near $1730 cm À1 , as would be expected but rather, the asymmetric stretch bands of the carboxylate (COO) appear as a weak, broad peak at $1617 cm À1 , possibly due to the increased mass of the Ca 2+ counter ion, as compared to the proton or sodium. The triplet at $1600 cm À1 /1636 cm À1 / 1676 cm À1 corresponds to carboxylate stretches, including the carbonyl of the non-self-associated amide (1636 cm À1 ) and the carbonyl asymmetric and symmetric stretches of the associated amide (1600 cm À1 and 1680 cm À1 respectively). 93,[128][129][130] All three diminish as cross-linking occurs, as well as broaden and undergo a slight change in their relative ratios, during gelation (i.e.; as a result of the difference between carboxylic acid (-COOH) and calcium carboxylate salt (-COO-Ca 2+ -OOC-)); the Ca 2+ replacing protons effecting shis to higher wavenumbers. 15,94,131 In addition, a band at $1436 due to the symmetric vibration of n(COO À ); initially starting as a shoulder aer 1 h and then becomes an overlapping doublet aer 24 h (i.e.; 1430 cm À1 and 1457 cm À1 ). 15,132 The changes arise from interactions of alginate with Ca 2+ from the Na + conformer, perhaps as a result of binding via both oxygen atoms of the carboxylate function; the relative increase in $1457 cm À1 due to the resultant conformational changes in the polyacrylamide chain. 94,99,128,132,133 The 1400-1200 cm À1 region corresponds to -C-H deformation, as well as -N-H and -C-O stretching vibrations. More specically, alginate vibrations of the polymer backbone are located at wave numbers <1300 cm À1 . 94 At 1300 cm À1 a weak CO vibration band appears. 15 The band at 1287 cm À1 (AAm -CH 2 vibrations) diminishes over 60 min, but then reappears as a stronger band at 1334 cm À1 (-N-H stretch) aer 24 h, which corresponds to copolymerisation and imidisation. 134-136 A small broad band at $1223 cm À1 , corresponds to the -C-O-Cstretch, evolves beyond 24 h; although nothing is seen in the initial 1 h 137 The 1200-1000 cm À1 region corresponds to C-O-H deformation, C-C-H deformation, C-O, and C-C ($1123 cm À1 ) stretching vibrations, as well as symmetric and asymmetric vibration bands of COC bonds typical of polysaccharide rings. 15 The -C-Cband undergoes broadening and a slight intensity decrease over time. 138 The band at 1090 cm À1 is attributed to glycosidic ring breathing of the alginate. 126 A band at $1056 exhibits a large intensity decrease in the initial hour and is completely absent aer 24 h. New bands also arise at 1034-1016 cm À1 (and $850 cm À1 ) related to metal-oxygen-metal bonds, which could correspond to partial bonding between Na + (and subsequently Ca 2+ ) and oxygen atoms in the G-blocks of alginate (at $1025 cm À1 ). 99,139 Skeletal stretching, deformation modes, and ring breathing are identied in the 1000-700 cm À1 region. Three sharp bands at $809 cm À1 , $876 cm À1 and $977 cm À1 are assigned to skeletal -C-C$ and -CO stretching; deformational -C-C-H, and; -C-CO bending modes, respectively. Their relative intensities change characteristically on going from M-rich content alginates to low-M content ones, which is also in agreement with the experimentally observed M/G changes (Fig. 4). 94 The $876 cm À1 band decreases in intensity but there is no further shi, meaning there is no subsequent weakening of the -C-Cand -CO bonds, since there is no sharing of the bonds with the cation. 94 Additionally, the $806 cm À1 spectral band conrms the presence of a-conguration G-units. 99 Another inuential spectral band centred at 955 cm À1 is thought to be a marker for alginate (it corresponds to G-rich, M-rich or mixed parts) and decreases in intensity as polymerisation proceeds through consumption of the alginate precursor, although there is no associated band shi. 99 A weak, broad shoulder at $766 cm À1 arises aer 24 h, although there is nothing present in the rst 60 min of reaction. This is attributed to the y 4 -asymmetric carbonate bending mode, which may correspond to metal carbonate formation. 140,141 The region below 700 cm À1 is attributed to pyranosyl ring deformation, C-O-C glycosidic linkage vibrations, as well as coordination interactions of Ca 2+ , primarily from alginic acid. 93 A band at $627 cm À1 (d(C]O, trans)) diminishes and almost disappears aer 1 h, as polymerisation partly occurs through the carbonyl groups, and then re-develops at -642 cm À1 (d(C] O, cis)). 142 Thus, there is a partial trans to cis transition at the carbonyl sites, perhaps facilitated by unzipping of the copolymer as the co-ordinating metal cation moves away, and hydrogen bonding comes to the fore. Such co-ordination conversions have previously been reported for proteinaceous polymers. 143,144 The band at $502 cm À1 diminishes and almost disappears aer 1 h and then later re-develops at $485 cm À1 , possibly due to changing interactions with the phenyl groups and changes in the ring conformation. [145][146][147] Finally, the diminishment of intensity in the low-frequency <200 cm À1 region, beyond $10 min, indicates increasing restriction placed on localized lattice vibrations as a result of the gelation and densication process. 148,149

Tensile and compression testing
Bonding effects evolved over 168 h, as reected in mechanical properties variation, e.g.; stress-strain curves show the nonlinear viscoelastic behaviour of the tough hydrogel (Fig. 7). There was also a variation in materials properties under compression and tension. Mechanical property data could only be obtained for gels that had been aged a minimum of $4 h. All tensile curves, regardless of conguration, typically show four stages of deformation; (1) elastic behaviour up to $150-250% strain corresponding to the elastic peak stresses; (2) yield point; (3) ultimate tensile strength, and; (4) the failure point. 12 Following application of strain, an instantaneous network of permanent and transient cross-links arise, thus causing the elastic peak stress.
As time progresses, stress relaxation occurs due to unzipping of ionic cross-links, as well as disruption of transient physical crosslinks, enabling topological rearrangement of chains. This is thought to be facilitated by water migration and exudation; well-known to be the predominant mechanism by which stressrelaxation in hydrogels occurs. 150,151 The subtle absence of yieldpoint in the 4 h aged sample shows that the hydrogel is still undergoing reversible, complex breaking and reformation processes with chain undulation, contributing to the initial stress application before the yield point. The high strength and toughness of traditional double gel networks are derived from the rigid yet brittle, ionic sacricial network; the unzipping effect of Ca 2+ cross-links exceed the process of soening commonly encountered in highly stretchable hydrogels. 1,22,152,153 Thus, the increasing strain hardening affords increasing tensile properties with aging, namely, toughness (up to $650 kJ m À3 ; a $117% increase), ultimate tensile strength (up to $78 kPa; a $60% increase) and Young's modulus (up to $21 kPa; a $160% increase). 154 Whilst toughness and tensile strength tend to increase and then plateau aer $48 h of aging, the elastic modulus continues increasing with aging time, in correlation with increasing cross-link density. 155 These properties are in line with the traditional double network gels where the high mechanical properties are derived from the strong entanglement and contrasting network structures. 156,157 The high toughness is due to energy dissipation induced by the unzipping of physical cross-links across alginate networks, allowing high stresses to be shied between adjacent alginate G-blocks, which have a stiffer conguration due to a greater hindered rotation about the glycosidic linkages. 158 This also explains the improvement in experimental mechanical properties over the course of 168 h; there is a greater G-block coordination with time. The conventional trade-off between ultimate strength, stiffness (elastic modulus) and toughness is not encountered here; there is a simultaneous increase in all properties. 60,156,159,160 The partially overlapping nature of the ve-cycle, compressional loading-unloading curves, and presence of loops indicate a small degree of network structure damage in the conventional double network hydrogels. 19,161 However, subsequent to the rst cycle, there is also almost no decrease in maximum stress for the following four cycles, which conrms the highly resilient behaviour; the hydrogel dissipates energy effectively. Even at 80% strain, the hydrogel shows high recoverability of 65-70%; the loss of full recovery attributed to partial rupture of covalent cross-links and disruption in topological entanglements which decreases the overall cross-link density. 162,163 Such disruption cannot be restored under ambient conditions, thus decreasing compressive stress and compressional resilience. 164 Likewise, the maximum energy dissipation by the unzipping calcium cross-links occurs in the rst cycle (Fig. 7C), with no prominent dissipation observed thereaer. During compression, ionic cross-links consume the dissipated energy, yielding good compressional resilience (compressive stress maximum of $145 kPa), and covalent crosslinks help preserve elasticity, simultaneously. 152 This cyclic soening and a simultaneous strain hardening as the compression cycle proceeds, is in accordance with recent reports on sliding gels by Ito and coworkers. [165][166][167]

Discussion
The internal arrangement of the copolymer structure materially impacts the favourability of subsequent co-ordination to the alginate backbone and so, the favoured reaction path in a tough hydrogel formation process. Knowledge of such rearrangements is important, since pre-dominant co-ordination through one backbone unit-type preferentially over the other manifests marked differences in the resultant mechanical properties. Furthermore, the agglomeration route, as facilitated by the cross-linkers, and through electrostatic repulsion minimisation, indicates the mechanism by which aggregation is favoured. [168][169][170][171] Specically, for the model DN Alg-PAAm tough hydrogel system explored in this study, time-resolved investigations identify two broad stages in tough hydrogel formation, where different bonding interactions seem to predominate across the macromolecular chains in solution, which has a critical impact on the resultant mechanical properties. Initially, during phase I, physical entanglement and covalent cross-linking are the major drivers, as monomers and dimers cross-link into longer polymer chains, signifying gelation onset. 73 Covalent bonding is thought to proceed via both the unsaturated carbon bonds on the Alg-PAAm frameworks, as well as the Alg-carbonyl groups which bind to the AAm-amide groups, via a MBA bridge. 14,107,114,172,173 Aer $8-12 min (and not appreciably earlier), in phase II, ionic cross-linking effects come to the fore, as Ca 2+ -mediated cross-linking initially proceeds via both M-block (slightly more favourable) and G-block units. 15,110 M-block co-ordination is thought to proceed via a bidentate bridging co-ordination, with the Alg-carboxylate groups playing a key role. 92,117,172 This also corresponds with increasing stiffness of the structure. The delayed onset of ionic contributions observed experimentally, may be partly due to the slower dissolution of the CaSO 4 precursor, which in turn delays the ionic contributions to bonding. This seemingly results in a more effective tough hydrogel system with more benecial mechanical properties. Past empirical reports indicate that when insufficient durations are spent at each cross-linking stage (e.g.; faster onset of ionic cross-linking contribution that bypasses the dimerization phase), there is incomplete diffusion and entanglement of molecular chains, non-ideal cross-linking effects, and void formation, resulting in weaker and unsatisfactory mechanical properties. 34,74,75,174 Equivalent molar ratio variations of alternative cationic cross-linker (i.e.; CaCl 2 , MgSO 4 , Na 2 SO 4 , and Calignosulfonate) were also explored, in place of CaSO 4 (see ESI †). Thus, ideal mechanical properties (i.e.; ultimate strength and toughness), need optimisation of cross-link density and type in polymer systems, so as to improve (e.g.) the energy dissipation mechanism. 175-178

Conclusions
This study explored mechanistic insights into the stages and factors involved in tough hydrogel formation and its correlation with the subsequent mechanical properties. A model double network alginate-acrylamide polymeric system was explored, in the presence of both ionic (CaSO 4 ), and covalent (N,N 0 -methylenebisacrylamide) cross-linkers. The study offered an alternative reaction route to that predicted in the hypothesis; specic to ionic binding, and contrary to past reports, we observed a particular favouring of M-group co-ordination by Ca 2+ -linkers during gelation. More broadly, in the overall gelation reaction, two formation stages were identied spanning physical entanglement, dimerisation and covalent cross-linking in phase I, prior to the predominance of ionic cross-linking bonding interactions in phase II, which in turn seems to impact the exhibited mechanical properties.
The detailed insights and broader ndings offered in this paper may facilitate practical new synthetic routes to enhance the intrinsic gel chemo-physico-mechanical properties via direct intervention at various bonding stages, during the evolving cross-linking processes. We expect formation mechanisms in similar systems to be governed by the time-dependent bond formation, near-surface viscoelasticity of the bulk material, as well as the molecular architecture and composition of the gel relative to the crosslinking dynamics. These foundational insights will also aid understanding of tissue failure, tissue repair therapies, and design principles for future biomaterials and functional polymer gels. Such investigations will be the focus of future studies.

Author contributions
AP, SM and MWY carried out the lab syntheses and acquired data. AP organised and analysed data, made gures, and composed the mechanical properties analyses. SKP helped with all aspects of experimental data acquisition, feedback on analysis and edited the manuscript. PKS acquired MALDI-TOF MS and analysis. FW and ZZ helped with project planning, feedback on analysis and edited the manuscript. NN planned and funded the study, analysed data, made gures and composed the manuscript.

Conflicts of interest
There are no known conicts to declare.