Tuning the Structure of Galacturonate Hydrogels: External Gelation by

Jun 10, 2019 - We show here how the nature of various divalent cations M2+ (Ca2+, Zn2+, or Fe2+) influences the structure and mechanical properties of...
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Cite This: Biomacromolecules 2019, 20, 2864−2872

Tuning the Structure of Galacturonate Hydrogels: External Gelation by Ca, Zn, or Fe Cationic Cross-Linkers Aline Maire du Poset,†,‡,§ Adrien Lerbret,† François Boue,́ § Andrea Zitolo,‡ Ali Assifaoui,*,† and Fabrice Cousin*,§ †

Université Bourgogne - Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, F-21000 Dijon, France Synchrotron SOLEIL, L’Orme des Merisiers, BP 48 St Aubin, 91192 Gif-sur-Yvette, France § Laboratoire Léon Brillouin, CEA-Saclay, 91191 Gif-sur-Yvette, France Downloaded via BUFFALO STATE on July 25, 2019 at 03:23:22 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: We show here how the nature of various divalent cations M2+ (Ca2+, Zn2+, or Fe2+) influences the structure and mechanical properties of ionotropic polygalacturonate (polyGal) hydrogels designed by the diffusion of cations along one direction (external gelation). All hydrogels exhibit strong gradients of polyGal and cation concentrations, which are similar for all studied cations with a constant ratio R = [M2+]/[Gal] equal to 0.25, showing that every M2+ cation interacts with four galacturonate (Gal) units all along the gels. The regions of the hydrogels formed in the early stages of the gelation process are also similar for all cations and are homogeneous, with the same characteristic mesh size (75 ± 5 Å, as measured by small angle neutron scattering (SANS)) and the same storage modulus G′ (∼5 × 104 Pa). Conversely, in the regions of the gels formed in later stages of the process there exist differences in mechanical properties, turbidity, and local structure from one cation to another. Zn(II)-polyGal and Fe(II)-polyGal hydrogels display mesoscopic heterogeneities, more marked in case of Fe than for Zn, that are not present in Ca(II)-polyGal hydrogels. This comes from the mode and the strength of association between the cation and the Gal unit (bidentate for Ca2+ and monodentate “egg-box” for Zn2+ and Fe2+). Cross-links formed by Zn2+ and Fe2+ have a higher stability (lower ability to untie and reform) that induces the formation of local heterogeneities in the early stages of the gelation process whose size progressively increases during the gel growth, a mechanism that does not occur for cross-links made by Ca2+ that are less stable and enable possible reorganizations between polyGal chains.

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INTRODUCTION Polysaccharide hydrogels have received considerable attention in the biomaterials science because of their broad applications in the drug delivery, tissue engineering, and biosensor fields.1 Such hydrogels can provide spatial and temporal control over the release of various therapeutic agents, including smallmolecule drugs, macromolecular drugs, and cells.2 Drug diffusion is controlled by various network parameters such as the polymer volume fraction in the swollen state, the molecular weight of the polymer chain between two neighboring crosslinks, the cross-link density, and the corresponding mesh size.3,4 Knowledge of the network structure and the mechanical properties of these hydrogels is proven to be an important tool for the design and the control of the drug release. Negatively charged polysaccharides such as pectin or alginate (polyuronates) form ionotropic gels in the presence of divalent cations (e.g., Ca2+, Zn2+, Fe2+), which act as crosslinkers between polysaccharide chains.5−10 Since the associa© 2019 American Chemical Society

tion between cations and polyuronate chains is very fast and efficient, direct mixing generates instantaneously the formation of very heterogeneous gels.11 It is therefore necessary to control the way cations are introduced into the solution. Two strategies called “internal gelation” and “external gelation” are used. Internal gelation consists in two steps: (i) mixing the polyuronate with cross-linking cations strongly associated with other reactants (for instance, CaCO3, CaSO4, or Ca chelated by EDTA12−15) to make a homogeneous mixture, and (ii) inducing the slow release of the cations to allow the formation of a macroscopically homogeneous hydrogel. Such a strategy is very efficient but has the drawback that the other reactants stay within the gels after their formation, which can be detrimental for some applications. Received: May 23, 2019 Published: June 10, 2019 2864

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that is created. It was indeed shown that the network is much more homogeneous in the presence of calcium cations than in the presence of zinc cations.26 These differences were ascribed to the different modes of association of these cations with the carboxylate groups of galacturonate (Gal) units. It was shown by Molecular Dynamics (MD) simulations that Zn2+ cations interact with both carboxylate and hydroxyl groups of Gal units (monodentate mode), in a similar way to that described in the “egg-box” model,6 whereas Ca2+ cations only interact with carboxylate groups (bidentate mode). We recently showed that the “egg-box” model also suitably describes the local association between Fe2+ and Gal units [publication in preparation]. The “egg-box” mode of association is favored by the octahedral geometry of the first hydration shell of the two transition metals (Fe and Zn) in water and by their strong interaction with hydration water. For metal cations whose first water shell is less strongly bound and/or exhibit an irregular geometry, as for calcium and barium, the binding of ions with the Gal units occurs more likely via the bidendate association.9 However, it is worth mentioning at this stage that Mg2+ does not easily induce the gelation of polyuronates, even at C > C*, as observed by many authors,29,30 despite an octahedral first hydration shell structure and a hydration free energy similar to that of Fe2+ in water solution.31 A plausible explanation is that the electronic structure of the valence shell of cations (e.g., 3d6 and 3d10 for Fe2+ and Zn2+, respectively, and 2p6 for Mg2+) also plays a key role on their cross-linking ability. Hence, the local association modes influence the stability and the number of point-like cross-links formed by divalent cations with polyGal chains and, therefore, the structure of the created network.9,13,32 The aim of this paper is to propose a generalized mechanism for the external gelation of polyuronates induced by divalent cations. Based on the corpus of knowledge that we acquired on Fe2+-polyGal hydrogels obtained by external gelation8 and on the influence of cation-Gal association modes on properties of polyGal solutions,9,26 our strategy will be to obtain Ca2+ and Zn2+-based gels using exactly the same protocol that we previously designed for Fe2+. We will elucidate their similarities and differences through the description of their macroscopic and mechanical properties, the determination of the local concentration of polymers and M2+/Gal molar ratios along gels, and the description of the local network structure by SANS.

In case of external gelation, the gel formation is induced by the slow diffusion of divalent cations from an external reservoir into a polyuronate solution either through a dialysis membrane or just through its lower part, which becomes progressively gelled and thus acts like a membrane.10,16−18 The divalent cations diffusion creates a gelation front that moves away from the dialysis membrane.19−21 Such a gelation protocol creates, along the vertical direction z, a large gradient of cross-link concentration: as a consequence, the part near to the cation reservoir expels water, resulting in a higher polymer concentration than in the furthest part.19,20 This induces a much higher elastic modulus since a common trend from all these studies is that the storage modulus G′ of the gel usually scales like ∼ [polyuronate]2, where [polyuronate] is the local concentration of polyuronate within the gel.11,22 Polyuronate hydrogels prepared by external gelation with divalent cations have been thoroughly investigated in the past. While these hydrogels always exhibit concentration gradients, their structures and mechanical properties are cation-dependent.10,18,23 This is due to the way cations interact with the carboxylate groups of the polysaccharide at the molecular level, which is tuned by the affinity of the cation for water and by the geometry of its hydration shell.9 For instance, Morch et al.10 found that Ba-alginate beads showed a higher inhomogeneity than Ca- and Sr-alginate beads, with a concentration of alginate approximately five times higher at the surface (nearest part) of the beads than in the center (furthest part). However, in most studies, the macroscopic heterogeneities were not taken into consideration and the corresponding mechanical properties were averaged along the whole gels.10,18,24 Maki et al.25 have formulated a Ca-alginate gel by the diffusion of calcium ions through a cylindrical dialysis tube containing the alginate solution and have studied the gel structure by using Small Angle X-ray Scattering (SAXS). The SAXS profiles at different positions in the gel indicated the formation of rod-like fibrils consisting of a few tens of alginate chains oriented perpendicular to the direction of the flow of calcium coming from the outer solution. They have also observed a decrease in the degree of fibril orientation at the center of the gel, which was attributed to the reduction of the circular radius of the gelling front in this part. Using an external gelation approach, we recently designed Fe(II)-polygalacturonate hydrogels from the unidirectional diffusion of iron(II) into a polygalacturonate (polyGal) solution,8 perpendicular to a dialysis membrane. In this case, the cation diffusion induces the formation of a planar gelation front that leads to a macroscopic cross-linking gradient along the diffusion axis for hydrogels.8,19,20 In spite of this gradient observed within the gels at the mesoscopic scale, we showed, using Small Angle Neutron Scattering (SANS), that the structure of the network at the local scale was the same in the whole gel: the mesh size showed a constant value of 75 ± 5 Å in any region of the gel. In order to propose a full description of the gelation mechanism, a refined description of the structure of hydrogels at all the relevant length scales thus appears to be necessary. To determine the influence of the nature of divalent cations on the gelation mechanism and therefore on the final structure of the gel, many past studies focused on polyuronate solutions prepared in the diluted regime (chain concentration C < overlap concentration C*) prior to letting them interact with cations.9,10,26−28 Starting from such a diluted regime, it was demonstrated that the nature of the divalent cation plays a huge role on the structure of the network of pectinate chains

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MATERIALS AND METHODS

Materials. D2O (>99.9% of deuterium) was purchased from Eurisotop (Saint-Aubin, France). Polygalacturonate (polyGal; ≥90%, enzymatic, Mw from 25 to 50 kDa), sodium chloride (≥99% of purity), calcium chloride dihydrate (≥99% of purity), zinc chloride tetrahydrate (≥99% of purity), and iron(II) chloride tetrahydrate (≥99% of purity) were purchased from Sigma (St Louis, MO). Preparation of Stock Solutions. Polygalacturonate (polyGal) stock solutions were prepared in 10 mM sodium chloride (NaCl) solution by stirring for 2 h to ensure the optimal dispersion of the polysaccharide. The pH of the polyGal solution was adjusted to 5.5 using sodium hydroxide (NaOH 1 M). In order to remove some of the impurities that may remain after the enzymatic degradation of pectin (small molecules, minerals, etc.), the obtained solution was then dialyzed three times against 10 mM NaCl (2 h) by using a dialysis membrane (Mw cutoff: 3.5 kDa) and a volume ratio of 1:10 between the polyGal solution and the dialysis bath. The divalent cation solutions (100 mM) were also prepared by dissolving the salt powder in NaCl 10 mM. 2865

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Figure 1. Side pictures of Ca(II)-polyGal, Zn(II)-polyGal, and Fe(II)-polyGal hydrogels. The diameter of the gels corresponds to that of the glass tube used to make them. Picture for Fe(II)-polyGal is reprinted with permission from ref 8. Copyright 2018 Elsevier. Preparation of Hydrogels. Hydrogels were prepared by using the protocol described in our previous study.8 Briefly, we poured 5 mL of polyGal solution (20 g/L corresponding to 113.6 mM) in a glass tube (diameter 2.1 cm). The lower part of the glass tube, on which a dialysis membrane was set to separate the solution from the reservoir, was immersed into a 50 mL reservoir of a divalent cation solution (100 mM) for 24 h in order to ensure that the gels were fully formed. The setup was covered during the gelation process with paraffin film and aluminum foil to limit evaporation of water and the oxidation in the case of the iron(II) solution. All the obtained gels called Ca(II)-polyGal, Zn(II)-polyGal, and Fe(II)-polyGal were cut following the protocol described in our previous study8 into slices corresponding to different distances z to the dialysis membrane. The exact thickness of a given slice was systematically measured after the cut. The total heights reached by the different gels were slightly different: 14.5 ± 0.6, 13.2 ± 0.3, and 14.0 ± 1.0 mm for Ca(II)polyGal, Zn(II)-polyGal, and Fe(II)-polyGal, respectively. We have thus normalized the distance z to the total thickness of the hydrogel. The ratio is expressed in %: for example, the nearest slice from the dialysis membrane, called “near”, is defined as the slice 0−25%, and the furthest slice, called “far”, is for a 75−100% ratio. Dry Matter Quantification and Elemental Analysis. Each slice of hydrogels was dried in an oven for 24 h at 100 °C. The final total weight (dry matter) corresponds mainly to the quantity of both polyGal and cation inside the slice (the amount of NaCl is negligible, as shown in Figure S1). The quantity of polyGal thus corresponds to the difference between the total dry matter and the cation content in one slice. The cation content was quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) iCAP 7400. The hydrogels were extensively washed before the experiment in order to eliminate free ions entrapped in the gels. It was considered that Cl− ions introduced by FeCl2, CaCl2, or ZnCl2 were removed from hydrogels by the washing step, since it is known that Cl− does not interact strongly with polyGal. The concentrations of both polyGal and divalent cations were obtained by dividing the corresponding mass in the slice by its initial volume. All measurements were done in triplicate. Error bars were estimated for both the concentration values and the corresponding thicknesses. The standard deviations were calculated from the average polyGal and cation concentrations of the three measurements and the average thickness of the corresponding slices. Mechanical Properties. The viscoelastic moduli G′ (elastic) and G′′ (viscous) for the different slices were measured using a stresscontrolled dynamic rheometer (MCR 302 from Anton Paar) equipped with parallel plates geometry with a diameter of 25 mm at controlled temperature (T = 25 °C). All measurements were done in triplicate. Error bars were estimated for both the concentration values and the corresponding thicknesses. The standard deviations were calculated from the average polyGal and cation concentrations of

the three measurements and the average thickness of the corresponding slices. In order to maintain a good contact between the upper plate and the slices and to reduce potential adverse effects due to slipping, the normal force was adjusted to 1 N. We first performed frequency sweep experiments from 0.001 to 40 Hz at a fixed shear stress of 0.1 Pa (Figure S2). Then shear stress sweep experiments from 0.01% to 10% at a fixed frequency of 1 Hz were performed. These experiments allowed to define the linear viscoelastic region (LVR), where the viscoelastic properties are independent of frequency and shear stress. Small Angle Neutron Scattering Measurements. Small Angle Neutron Scattering (SANS) measurements were performed on the PACE instrument at Laboratoire Léon Brillouin (CEA, Saclay, France). We used four configurations with two different neutron wavelengths and three sample−detector distances (λ = 13 Å, D = 4.7 m; λ = 13 Å, D = 3 m; λ = 4.5 Å, D = 3 m; λ = 4.5 Å, D = 1 m) that cover the 0.0032−0.5 Å−1 wave vector range. We applied standard corrections to the raw signal for sample volume, neutron beam transmission, empty cell signal, and detector efficiency to obtain scattering in absolute units, and then subtracted the signal measured from the blank sample (buffer). D2O was used instead of H2O for the solubilization and dialysis of polyGal and also for the gel preparation procedure in order to obtain a good contrast between the solvent and the polyGal network inside the gels and also to minimize incoherent scattering. After the gelation process, 2 mm thick cylindrical hydrogels slices were placed between two quartz windows separated by a 2 mm spacer, as described in our previous study.8 During measurements, the incoming neutron beam was perpendicular to the faces of the cylinder.

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RESULTS Macroscopic Observations. Figure 1 presents side pictures of the three different formulated hydrogels. The Fe(II)-polyGal hydrogel corresponds to that described in our previous study.8 The three hydrogels showed important macroscopic differences in terms of turbidity, color, and shape. The Ca(II)-polyGal hydrogel is macroscopically homogeneous and totally transparent, that is, it does not present a turbidity gradient. However, the diameter of the bottom part of this gel, that is, the part that was close to the dialysis membrane during the gelation process, is slightly smaller than that of the tube used for the synthesis, showing that the gel has macroscopically shrunk, contrary to the upper part of the gel whose diameter perfectly matches that of the molding tube. In other words, syneresis has occurred in the lower part, as often observed above a cross-linking ratio threshold. The Zn(II)-polyGal hydrogel presents a slight turbidity gradient from the bottom part to the upper part: the 0−50% part of the hydrogel is transparent, whereas the 50− 2866

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and the cation concentrations we fixed). It is remarkable that all hydrogels share the same important features: (i) a strong increase of concentration with respect to the nominal polyGal concentration [polyGal]nom in the 0−25% part of the hydrogel, where the polyGal concentration has more than doubled to reach a concentration close to 250 mM (∼45 g/L); (ii) a continuous decrease from the lower part to the upper part; (iii) a final concentration in the 75−100% part of the hydrogel that reduces down to a value around 50 mM (∼9 g/L), that is, a dilution by a factor of 2 from nominal concentrations. For the Fe(II)-polyGal hydrogels,8 we already showed that the molar ratio R = [M2+]/[Gal] was constant and equal to 0.25 all along the height. The same ratio value clearly applies to other divalent cations. This evidence that all Gal units from the polyGal chains are involved in the cross-linking of the divalent cations and that the density of cross-linking points is constant along the chains in all hydrogels. Determination of the Mechanical Properties. The mechanical properties of hydrogels were characterized by rheological measurements. The evolution of the storage (G′) and loss (G′′) moduli as a function of the position along the hydrogels are reported in Figure 3a. Similarly to what was observed for the polyGal concentration, there is a huge gradient of mechanical properties for the three hydrogels: G′

100% part of the gel is turbid, the furthest part from the dialysis membrane showing the highest turbidity. These trends are much enhanced for the Fe(II)-polyGal hydrogel, for which the height gradient involves very contrasted differences of both color and turbidity, as already described in ref 8. We have shown recently that the macroscopic heterogeneities observed for the Fe(II)-polyGal hydrogel are only due to polyGal and iron concentration gradients that are created by the external gelation protocol used. The high turbidity observed for the 50−100% part of the Fe(II)-polyGal hydrogel can be explained by the formation of mesoscopic heterogeneities that scatter light (typically 1−10 μm size). Such mesoscopic heterogeneities do not appear in Ca2+-based hydrogels; in Zn2+-based hydrogels, their size and density are probably lower than in Fe2+-based hydrogels. It is finally worth noting that the gelation protocol we designed is very robust, because we formulated more than 10 gels per tested cation and obtained highly reproducible samples in all cases. Distribution of PolyGal and Cations along the Hydrogels. Besides the visual aspect, concentration gradients are well-known to be induced by an external gelation protocol, as used in this study. In our former profile in Fe2+-based hydrogels, we obtained a huge concentration gradient from the vicinity of the dialysis membrane to the further parts of the gel. The concentration increases up to ∼45 g/L close to the dialysis membrane,8 much larger than the initial nominal concentration of 20 g/L. Figure 2 compares the concentration

Figure 2. Evolution of both polyGal concentration in the gel (filled symbols) and cation concentration multiplied by a factor 4 (empty symbols) as a function of the distance from the membrane (expressed in % of the total thickness of the hydrogel) for Ca-polyGal hydrogel (square symbols; this study), Zn-polyGal hydrogel (triangle symbols; this study), and Fe(II)-polyGal (circle symbols (Reprinted with permission from ref 8. Copyright 2018 Elsevier.)). The dashed line corresponds to the initial polyGal concentration in the glass tube before the gelation process, [polyGal]nom. We applied a factor 4 to the cation concentration in order to highlight the molar ratio R = [M2+]/ [Gal] = 0.25; the raw values are shown in Figure S3.

profile on Fe2+-based hydrogels with the ones for Ca2+-based hydrogels and Zn2+-based hydrogels. Very nicely, all data merge on the same master curve, in spite of the different macroscopic aspects of the three kinds of hydrogels seen in Figure 1. The polyGal concentration gradient is thus not correlated to the nature of the cation. The gradient is only driven by the protocol we used (which comprises the polyGal

Figure 3. (a) Evolution of the storage (G′) and loss (G′′) moduli as a function of the slice distance to the membrane along the hydrogels (expressed in % of thickness). (b) Evolution of the storage modulus (G′) and of the square of the polyGal concentration (times a constant equal to 35) as a function of the slice distance to the membrane (expressed in % of the thickness). The solid black straight line shows the relationship G′ ∼ 35*[polyGal]2. 2867

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Biomacromolecules and G′′ roughly decrease by two decades along the gel lengths, from respectively ∼5 × 104 Pa and ∼5 × 103 Pa in the 0−25% part of the gels down to ∼5 × 102 Pa and ∼5 × 101 Pa in the 75−100% part. Despite the gradient, tan δ = G′′/G′ remains constant and equal to 0.1 all along the hydrogels. Please note that the value of G′ for the Fe2+-based hydrogels determined from oscillatory measurements are in perfect accordance with the Young modulus, E, obtained in ref 8 from uniaxial compression. Nicely, G′ and G′′ do not depend, within the dispersion, on the three divalent cations considered in the nearest part of the gel (0−25%), but become cation-dependent in the further parts. Especially, between 75 and 100%, the sequence is Ca(II)-polyGal > Zn(II)-polyGal > Fe(II)polyGal. Besides, it has been shown in literature that G′ ∼ [polyGal]2 for gels made by external gelation.11,22 To determine if our gels follow the same phenomenological trend, we plotted the evolution of G′ and of 35*[polyGal]2 as a function of the slice distance (expressed in % of the thickness; Figure 3b). We found that this relationship is true over the whole gel for the Ca(II)-polyGal hydrogel, while G′ decays faster than [polyGal]2 for thicknesses larger than ∼50% for the Zn(II)-polyGal and Fe(II)-polyGal hydrogels. These regions correspond to the most turbid parts of the Zn(II)-polyGal and Fe(II)-polyGal hydrogels (Figure 1), where mesoscopic heterogeneities have been evidenced.8 The larger the deviation from the G′ ∼ [polyGal]2 relationship, the stronger the turbidity due to macroscopic heterogeneities, which become larger and more abundant. Local Structure of the Network. SANS measurements were performed in different slices at various distances from the membrane to determine their local structure. In Figure 4a, the SANS spectra from the 0−25% slice of the Ca(II)-polyGal and Zn(II)-polyGal hydrogels are compared with that reported in ref 8 for the Fe(II)-polyGal hydrogel, while Figure 4b presents the spectra for a further slice from the dialysis membrane. To probe this latter part of the gel, we chose to present slices corresponding to the 60−80% or 50−75% slices of the gel rather than the 75−100% slice, as the polyGal concentration was too low in the uppermost part to obtain a good signal-tonoise ratio (SANS data for all the slices in Ca(II) and Zn(II)polyGal hydrogels are shown in Figure S4). Data were measured in absolute intensities (cm−1) and are shown after normalization by the ratio between the polyGal volume fractions Φsample in each slice (as determined by dry matter measurements after SANS measurements) and in the solution Φsolution (20 g/L). In the nearest part of the hydrogels from the dialysis membrane, it immediately appears that the spectra of the three hydrogels are perfectly superimposed with each other, while they neatly differ from that of the pure polyGal solution. This latter shows a q−1 decay at large q, arising from the rod-like behavior of the polymer at local scale, and a q−2.7 decay at the lowest q, stemming from a limited aggregation of the chains, with a crossover between the two regimes at q* = 0.059 Å−1. The hydrogels show an additional shoulder at intermediate q, which is characteristic from the mesh size of the hydrogel.8 In summary, the local structure of the polyGal network is thus strictly identical for all the divalent cations tested, which is a very robust and striking result. The SANS spectrum for the furthest part of the Ca(II)polyGal hydrogel is distinct from those of the other two hydrogels (Zn(II)-polyGal and Fe(II)-polyGal), which nicely superimpose to each other (Figure 4b). This indicates that

Figure 4. (a) SANS spectra of the 0−25% part of Ca(II)-polyGal and Zn(II)-polyGal hydrogels. (b) SANS spectra of a further part from the dialysis membrane (3rd slice) of Ca(II)-polyGal (50−75% part) and Zn(II)-polyGal (60−80% part) hydrogels. The SANS spectra of the 0−25% and 60−80% parts of the Fe(II)-polyGal hydrogel are also displayed for comparison in (a) and (b), respectively, as well as the SANS spectrum of the polyGal solution. Reprinted with permission from ref 8. Copyright 2018 Elsevier. For both figures, the scattered intensities were normalized by the ratio between the volume fraction of the polyGal inside the slice, Φsample, and the nominal volume fraction of the polyGal solution, Φsolution (20 g/L). The red continuous lines are models of the pure polyGal solution and the purple lines are fits enabling to determine the mesh size of hydrogels (see main text).

there is no change between the nearest and the furthest parts except by a concentration factor suggesting that both Zn(II)polyGal and Fe(II)-polyGal hydrogels have the same network local structure whatever the probed slice, despite the differences in their macroscopic aspect. On the contrary, the scattering for the 50−75% part of the Ca(II)-polyGal hydrogel is different: it almost matches that of the pure polyGal solution over the whole q range (except at intermediate q values (0.03− 0.08 Å−1), where it is slightly higher for the hydrogel). It is likely that the mesh size of the Ca(II)-polyGal network is on average larger than the one formed in the Zn(II)- and Fe(II)polyGal hydrogels. The typical shoulder would thus be shifted 2868

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Figure 5. Schematic representation of the mesoscopic and local structure (insets) of the Ca(II)-polyGal, Zn(II)-polyGal, and Fe(II)-polyGal hydrogels. Picture for Fe(II)-polyGal is reprinted with permission from ref 8. Copyright 2018 Elsevier. The top and bottom panels correspond to the furthest and the nearest parts of the hydrogels, respectively. The scheme for the Fe(II)-polyGal gel is adapted from our previous work. Reprinted with permission from ref 8. Copyright 2018 Elsevier.

toward lower q. It is, however, almost not visible here because its intensity at very low q is masked by that arising from the initial structure of the chains. The additional scattering signal in the Ca(II)-polyGal hydrogel is then probably part of a Lorentzian, but is too low to allow a quantitative modeling, because the Lorentzian variation will stay smaller than the q−2.7 variation. For all other slices of hydrogels, that is, for the three hydrogels in the nearest part of the dialysis membrane and for the upper slices of the Zn(II)- and Fe(II)-polyGal hydrogels, we used the same strategy as in ref 8: we modeled the shoulder with an Ornstein−Zernike (OZ) Lorentzian function IOZ(q), with a correlation length ξ representing the mesh size of the hydrogels. The scattering curve was then perfectly fitted on the whole q range by the sum of this OZ Lorentzian and of the scattering of the solution of pure polyGal chains: I(q)(cm−1) = I(q)polyGal_solution + IOZ(0)

1 1 + (qξ)2

Briefly, the three hydrogels exhibit (i) the same gradient of polyGal concentration and a constant molar ratio (R) of 0.25 all along the gel; (ii) the same increase of concentration from the nominal concentration 20 g/L up to 45 g/L in the nearest part of the gel; (iii) the same local structure in the nearest part of the gel with a mesh size of 75 ± 5 Å; (iv) the same lack of turbidity in the nearest part of the gel; and (v) the same mechanical properties in the nearest part of the gel, since G′ follows the relationship G′ ∼ [polyGal]2 with a maximal value of ∼50 kPa. But the three gels also show strong differences: the Ca(II)-polyGal hydrogels are fully transparent and do not show mesoscopic heterogeneities, whereas their local structure varies as the mesh size increases from the near part to the far part to accommodate the polyGal concentration gradient, so that G′ ∼ [polyGal]2 over the whole gel. On the contrary, Zn(II) and Fe(II) hydrogels show mesoscopic heterogeneities, but, rather surprisingly, their local structure is similar over the whole network in the regions containing polymeric chains. The appearance of turbidity for such gels occurs roughly in the middle of the gels, and interestingly, this occurs when the polyGal concentration becomes of the order of the overlapping concentration C*; this is also where G′ deviates from [polyGal]2. The perfect similarity from one type of gel to another in the nearest part from the dialysis membrane suggests that the initial stages of gel formation are not cation-dependent. When diffusing into the solution of polyGal chains, each cation acts as a cross-linker. Similar to what is observed in dilute regime, there is (i) initial monocomplexation and formation of pointlike cross-links between two Gal chains, when the first cations penetrate into the polymer solution followed by (ii) the formation of dimers, when more cations are present, and (iii) finally, the lateral association of several dimers (multichain junction zones), which exactly gives R = 0.25.13,27,32 In the dilute regime, further formation of these multichain junction zones may occur (0.25 < R < 0.5), but such a step appears too

(1)

where I(q)polyGal_solution is the scattering curve of the pure polyGal chains solution and IOZ(0) is a constant. The result of the fit gives a refined mesh size ξ equal to 75 ± 5 Å.

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DISCUSSION The design and characterization of the three types of polygalacturonate hydrogels prepared with the same external gelation protocol using Ca2+, Zn2+, and Fe2+ as cross-linking agents allow us to propose a full description of the mechanisms at play in the formation of the hydrogels. The suggested structure of the three hydrogels at the different length scales are shown in Figure 5. The three hydrogels share many common features but also display some differences that enable us to decouple the respective influence of the parameters associated with the used protocol (external gelation, polyGal concentration) from those specific to the cations and their association mode with polyGal on the overall gelation process. 2869

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Article

Biomacromolecules

simulations that Zn2+ ions interact with both carboxylate (monodendate mode) and hydroxyl groups of Gal, in a similar way to that described in the so-called “egg-box” model, whereas Ca2+ ions interact only with carboxylate groups (bidentate mode).9,26 We recently combined MD simulations with Extended X-ray Absorption Fine Structure (EXAFS) measurements [publication in preparation] on Fe2+ in a Fe(II)polyGal hydrogel and found that the local association mode corresponds to the “egg-box” model, similar to what was observed for Zn2+. Moreover, after the initial step of formation of monocomplexes and point-like cross-links, the local association mode also controls the step of dimers formation that immediately follows. Indeed, it was proposed in ref 9 that there exists, in diluted regime, a threshold molar ratio R* between these two steps, which is much lower for calcium ions (R* = 0.05) than for zinc (R* = 0.3). The higher R* observed for Zn2+ was attributed to the higher stability of cross-links in comparison to those formed by Ca2+. The following picture thus emerges for the two possible modes of complexation. In the first case of bidentate association with moderate stability of cross-links (low R*, e.g., for Ca2+), the formation of dimers is favored in the early stages. Such cross-links can be broken easily and allow reorganizations in order to form homogeneous structures, which is entropically favorable. In the second case of the “eggbox” association, which occurs for Zn2+ and Fe2+, the difficulty to form dimers induces heterogeneities and the high stability of cross-links limit possible rearrangements within gels: heterogeneities thus propagate and grow as the gel forms. This explains why mesoscopic heterogeneities arise in Zn(II) and Fe(II) hydrogels, but not in Ca(II) hydrogels. The formation of heterogeneities is also enhanced by the progressive reduction of the concentration of polyGal at the gelation front. When such concentration decreases below the overlapping concentration C*, the behavior of the gel mirrors that of solutions in dilute regime. Huynh et al.9 showed by means of turbidity and viscosity measurements that Ca(II)-polyGal solutions in diluted regime (0.5 g/L) are homogeneous, unlike Zn(II)-polyGal solutions, due to the reasons depicted here before. In the case of Zn2+ and Fe2+, when C becomes lower than C* within the hydrogel, the regions the most concentrated in polyGal chains almost behave as individual connected microgels that shrink down to reach the lowest possible mesh size (75 ± 5 Å), explaining why the local structure is similar in every part of Zn(II)-polyGal and Fe(II)polyGal hydrogels (Figure 5). Owing to volume conservation, such a local shrinking thus gives rise to the formation of large zones depleted in polymeric chains that strongly scatter light. This explains why the appearance of turbidity occurs roughly in the middle of the gel, why gels are very turbid in the top part (polyGal concentration of ∼9 g/L) and also why G′ is no longer proportional to [polyGal]2 in the top parts, because hydrogels are very heterogeneous in this region. While the association mode (bidentate versus monodentate “egg-box”) drives the behavior of the gels at first order, a tiny change in the association strength for a given mode of association also influences the ability of hydrogels to let heterogeneities grow during formation: it indeed appears that Fe(II)-polyGal hydrogels are slightly more heterogeneous than Zn(II)-polyGal ones. Given that the rate of hydration water exchange is larger for Zn(II) than for Fe(II),38 the formation of Zn(II)-polyGal cross-links, which implies the loss of hydration water molecules from the cations, has probably a higher

difficult in a fully percolated cross-linked network with a strong modulus, explaining why R is limited to 0.25. Obviously, due to topological constraints, all the galacturonate units cannot be involved in dimers, but such dimers are present in very large majority; however, they must coexist with a few zones with defects (R ≠ 0.25), that lead to a “lack” of bound cations (R < 0.25 for monocomplexes and point-like cross-links) or to an “excess” (0.25 < R < 0.5 for multichain junction zones). Hence, such defects induce the formation of a few heterogeneities in the network, even if the structure stays overall homogeneous. When all potential cross-linking sites for cations are saturated at 0.25, the further cations diffuse within the cross-linked parts of the gels without interacting strongly with polyGal chains until they reach the zones where they encounter some noncross-linked chains. As the nominal concentration of chains is well above C*, the structure of the polyGal chains that was homogeneous before cross-linking stays homogeneous in the vicinity of the dialysis membrane. Since the association of cations with the polyGal chains neutralizes half of the charges of these latter, it changes the chemical potential of water, in other words, the osmotic pressure is decreased for less charged chains, within the crosslinked parts of the gels and water molecules migrate toward non-cross-linked and more charged chains. This can be enhanced by the elastic modulus of the network. This leads to the shrinking of the network down to a mesh size of the order of the persistence length as chains cannot contract any further. Remarkably, we observe that the mesh size ξ ∼ 75 ± 5 Å measured by SANS corresponds to the bare persistence length Lp = 70 Å of polyGal in solution.8,33 The final concentration of 45 g/L in the vicinity of the dialysis membrane is thus set by such a mesh size. The near part of the gel is thus homogeneous; its structure is not cationdependent and its modulus is fixed by the density of crosslinks. Theoretical models of entropic networks made out of rods with flexible cross-links were developed a long time ago.34−36 On a very simplistic basis, the elasticity is first governed by the density of degree of freedom in the material, which can be related to the density of persistence length. Other possible interactions complexify the calculation of the modulus. We used the most simple, that is, to replace the true network by an ideal one made up by a collection of spheres whose diameter coincides with the average network mesh size ξ, as proposed in a more recent paper.37 This gave us a mesh size of the network of about 60 Å in ref 8, that is, a value close to the structural one, for an homogeneous hydrogel having the G′ value we obtained. This initial change of osmotic pressure and shrinking of hydrogel initiates the formation of a gradient of concentration within the gel. Indeed, it creates a depletion zone at the interface between the chains immobilized by cross-links and the free chains. Such depletion drives the diffusion of free chains toward the dialysis membrane in the opposite direction to the diffusion of cations within the hydrogel.17,21 The local association mode of cations with the carboxylate groups of Gal units determines the strength and the stability of point-like cross-links, that is, their ability to untie and reform. It has thus a strong influence on the hydrogels formation, since it drives (i) the density of defects and local heterogeneities formed in the early stages of gel formation and (ii) the possibility for the network to rearrange and, therefore, to either propagate heterogeneities or suppress them during gel formation. Indeed, we have shown previously by MD 2870

DOI: 10.1021/acs.biomac.9b00726 Biomacromolecules 2019, 20, 2864−2872

Article

Biomacromolecules reaction rate than that of Fe(II)-polyGal cross-links. Zn(II)polyGal cross-links are thus probably slightly less stable: from a dynamic point of view, they rearrange more easily. This enables some slight reorganization of the network that are less likely to occur with Fe(II)-polyGal cross-links.

ORCID

CONCLUSION In this study, we have investigated how the nature of divalent cations (Ca2+, Zn2+ or Fe2+) influences the structure of polyGal hydrogels prepared by external gelation. Our results show that polyGal hydrogels exhibit similarities whatever the cation considered, namely, the same significant gradients of polyGal and cation concentrations with a constant ratio R = [M2+]/ [polyGal]. In the lower parts of the gels formed in the vicinity of the dialysis membrane, all hydrogels share also the same mechanical properties and the same structure at the nanometric scale, since a constant mesh size ξ = 7.5 nm was determined in every case. Conversely, in the upper parts of the gels, where water has been expelled leading to C < C*, differences are observed in mechanical properties, in turbidity or in mesh size. In particular, our results evidence that, in this C < C* region, the mesoscopic structure dependence on the cation reflects the different modes of interaction known to take place between the cation and the galacturonate unit: bidentate versus “egg-box” geometry and, to a lower extent, on the capacity of the cation to lose its hydration shell, for a given mode of association. Thus, by choosing a given cation and playing on the initial polyGal concentration C in the glass tube20 (with respect to the overlapping concentration C*), it would be possible to finely tune the structure of hydrogels at different length scales and, therefore, their mechanical for a very wide range of applications. This provides a “tool-box” that would be particularly helpful for dedicated applications such as the encapsulation of large molecules (proteins or probiotics). Note that, for the sake of simplicity, we have considered here a simple geometry of external gelation, where the cations diffusion results in a unidirectional gradient, but the same concepts could be applied for cylindrical or spherical geometries such as pharmaceutical beads or hydrogel implants.24,39 The present results open the way to the design of controlled gels based on pectinate or alginate by playing on the nature of the interaction between the galacturonate and the cation.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Adrien Lerbret: 0000-0003-2085-3767 Andrea Zitolo: 0000-0002-2187-6699 Fabrice Cousin: 0000-0001-7523-5160

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Author Contributions

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Conseil Régional de Bourgogne through the “Plan d’actions régional pour l’innovation (PARI ALIM+)” and by the European Union through the “PO FEDER-FSE Bourgogne 2014/2020 programs”. Mechanical properties were determined at the DIVVA plateform (AgroSup Dijon, Université de Bourgogne Franche-Comté). Laboratoire Léon Brillouin, Synchrotron SOLEIL and Région Bourgogne Franche-Comté are acknowledged for their financial support (cofunding Ph.D. thesis). We also acknowledge Bernadette Rollin for her valuable technical support.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00726. Determination of the sodium concentration in the different slices of the hydrogels; Determination of the linear viscoelastic (LVE) region: Frequency sweep; Evolution of the divalent cations concentrations as a function of the position within the hydrogel; Evolution of the network local structure of Ca(II)-polyGal and Zn(II)-polyGal hydrogels (PDF)

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REFERENCES

(1) Vashist, A.; Vashist, A.; Gupta, Y. K.; Ahmad, S. Recent advances in hydrogel based drug delivery systems for the human body. J. Mater. Chem. B 2014, 2 (2), 147−166. (2) Li, J.; Mooney, D. J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 2016, 1, 16071. (3) Singh, B.; Chauhan, N.; Sharma, V. Design of Molecular Imprinted Hydrogels for Controlled Release of Cisplatin: Evaluation of Network Density of Hydrogels. Ind. Eng. Chem. Res. 2011, 50 (24), 13742−13751. (4) Am Ende, M. T.; Peppas, N. A. Transport of ionizable drugs and proteins in crosslinked poly(acrylic acid) and poly(acrylic acid-co-2hydroxyethyl methacrylate) hydrogels. II. Diffusion and release studies. J. Controlled Release 1997, 48 (1), 47−56. (5) Rees, D. A. Structure, Conformation, and Mechanism in the Formation of Polysaccharide Gels and Networks. Adv. Carbohydr. Chem. Biochem. 1969, 24, 267−332. (6) Grant, G. T.; Morris, E. R.; Rees, D. A.; Smith, P. J. C.; Thom, D. Biological interactions between polysaccharides and divalent cations: the egg-box model. FEBS Lett. 1973, 32, 195−198. (7) Ouwerx, C.; Velings, N.; Mestdagh, M. M.; Axelos, M. A. V. Physico-chemical properties and rheology of alginate gel beads formed with various divalent cations. Polym. Gels Networks 1998, 6, 393−408. (8) Maire du Poset, A.; Lerbret, A.; Zitolo, A.; Cousin, F.; Assifaoui, A. Design of polygalacturonate hydrogels using iron(II) as crosslinkers: A promising route to protect bioavailable iron against oxidation. Carbohydr. Polym. 2018, 188, 276−283. (9) Huynh, U. T. D.; Lerbret, A.; Neiers, F.; Chambin, O.; Assifaoui, A. Binding of divalent cations to polygalacturonate: A mechanism driven by the hydration water. J. Phys. Chem. B 2016, 120, 1021− 1032. (10) Morch, Y. A.; Donati, I.; Strand, B. L. Effect of Ca2+, Ba2+, and Sr2+ on Alginate Microbeads. Biomacromolecules 2006, 7, 1471−1480. (11) Martinsen, A.; Skjak-Braek, G.; Smidsrod, O. Alginate as Immobilization Material: I. Correlation between Chemical and Physical Properties of Alginate Gel Beads. Biotechnol. Bioeng. 1989, 33, 79−89. (12) Khromova, Y. L. The effect of chlorides on alginate gelation in the presence of calcium sulfate. Colloid J. 2006, 68 (1), 115−119.

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Biomacromolecules (13) Ventura, I.; Jammal, J.; Bianco-Peled, H. Insights into the nanostructure of low-methoxyl pectin-calcium gels. Carbohydr. Polym. 2013, 97, 650−658. (14) Bassett, D. C.; Håti, A. G.; Melø, T. B.; Stokke, B. T.; Sikorski, P. Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation. J. Mater. Chem. B 2016, 4 (37), 6175−6182. (15) Draget, K. I.; Østgaard, K.; Smidsrød, O. Homogeneous alginate gels: A technical approach. Carbohydr. Polym. 1990, 14 (2), 159−178. (16) Caccavo, D.; Ström, A.; Larsson, A.; Lamberti, G. Modeling capillary formation in calcium and copper alginate gels. Mater. Sci. Eng., C 2016, 58, 442−449. (17) Thu, B.; Gaserod, O.; Paus, D.; Mikkelsen, A.; Skjak-Braek, G.; Toffanin, R.; Vittur, F.; Rizzo, R. Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiationinduced X-ray emission, magnetic resonance microimaging, and mathematical modeling. Biopolymers 2000, 53 (1), 60−71. (18) Agulhon, P.; Robitzer, M.; David, L.; Quignard, F. Structural Regime Identification in Ionotropic Alginate Gels: Influence of the Cation Nature and Alginate Structure. Biomacromolecules 2012, 13 (1), 215−220. (19) Braschler, T.; Valero, A.; Colella, L.; Pataky, K.; Brugger, J.; Renaud, P. Link between Alginate Reaction Front Propagation and General Reaction Diffusion Theory. Anal. Chem. 2011, 83 (6), 2234− 2242. (20) Skjak-Braek, G.; Grasdalen, H.; Smidsrod, O. Inhomogeneous Polysaccharide Ionic Gels. Carbohydr. Polym. 1989, 10, 31−54. (21) Huynh, U. T. D.; Chambin, O.; Maire du Poset, A.; Assifaoui, A. Insights into gelation kinetics and gel front migration in cationinduced polysaccharide hydrogels by viscoelastic and turbidity measurements: Effect of the nature of divalent cations. Carbohydr. Polym. 2018, 190, 121−128. (22) Smidsrod, O. Molecular Basis for some Physical Properties of Alginates in the Gel State. Faraday Discuss. Chem. Soc. 1974, 57, 263− 274. (23) Agulhon, P.; Robitzer, M.; Habas, J.-P.; Quignard, F. Influence of both cation and alginate nature on the rheological behavior of transition metal alginate gels. Carbohydr. Polym. 2014, 112, 525−531. (24) Huynh, U. T. D.; Assifaoui, A.; Chambin, O. Pellets based on polyuronates: Relationship between gelation and release properties. J. Food Eng. 2017, 199, 27−35. (25) Maki, Y.; Ito, K.; Hosoya, N.; Yoneyama, C.; Furusawa, K.; Yamamoto, T.; Dobashi, T.; Sugimoto, Y.; Wakabayashi, K. Anisotropic Structure of Calcium-Induced Alginate Gels by Optical and Small-Angle X-ray Scattering Measurements. Biomacromolecules 2011, 12 (6), 2145−2152. (26) Assifaoui, A.; Lerbret, A.; Huynh, U. T. D.; Neiers, F.; Chambin, O.; Loupiac, C.; Cousin, F. Structural behaviour differences in low methoxy pectin solutions in the presence of divalent cations (Ca2+ and Zn2+): a process driven by the binding mechanism of the cation with the galacturonate unit. Soft Matter 2015, 11, 551−560. (27) Fang, Y.; Al-Assaf, S.; Phillips, G. O.; Nishinari, K.; Funami, T.; Williams, P. A.; Li, L. Multiple steps and critical behaviors of the binding of calcium to alginate. J. Phys. Chem. B 2007, 111, 2456− 2462. (28) Cescutti, P.; Rizzo, R. Divalent Cation Interactions with Oligogalacturonides. J. Agric. Food Chem. 2001, 49 (7), 3262−3267. (29) Donati, I.; Paoletti, S. Material Properties of Alginates. In Alginates: Biology and Applications; Rehm, B. H. A., Ed.; Springer: Berlin, Heidelberg, 2009; pp 1−53. (30) Manning, G. S. Limiting laws and counterion condensation in polyelectrolyte solutions: IV. The approach to the limit and the extraordinary stability of the charge fraction. Biophys. Chem. 1977, 7 (2), 95−102. (31) Marcus, Y. A simple empirical model describing the thermodynamics of hydration of ions of widely varying charges, sizes, and shapes. Biophys. Chem. 1994, 51 (2), 111−127.

(32) Borgogna, M.; Skjåk-Bræk, G.; Paoletti, S.; Donati, I. On the intial binding of alginate by calcium ions. The tilted egg-box hypothesis. J. Phys. Chem. B 2013, 117, 7277−7282. (33) Ralet, M.-C.; Crépeau, M.-J.; Lefèbvre, J.; Mouille, G.; Höfte, H.; Thibault, J.-F. Reduced Number of Homogalacturonan Domains in Pectins of an Arabidopsis Mutant Enhances the Flexibility of the Polymer. Biomacromolecules 2008, 9 (5), 1454−1460. (34) Boué, F.; Edwards, S. F.; Vilgis, T. A. The entropy of a network of rod molecules. J. Phys. (Paris) 1988, 49 (10), 1635−1645. (35) Jones, J. L.; Marques, C. M. Rigid polymer network models. J. Phys. (Paris) 1990, 51 (11), 1113−1127. (36) Joly-Duhamel, C.; Hellio, D.; Ajdari, A.; Djabourov, M. All Gelatin Networks: 2. The Master Curve for Elasticity. Langmuir 2002, 18 (19), 7158−7166. (37) Pescosolido, L.; Feruglio, L.; Farra, R.; Fiorentino, S.; Colombo, I.; Coviello, T.; Matricardi, P.; Hennink, W. E.; Vermonden, T.; Grassi, M. Mesh size distribution determination of interpenetrating polymer network hydrogels. Soft Matter 2012, 8 (29), 7708−7715. (38) Lincoln, S. F. Mechanistic Studies of Metal Aqua Ions: A SemiHistorical Perspective. Helv. Chim. Acta 2005, 88 (3), 523−545. (39) Bai, X.; Gao, M.; Syed, S.; Zhuang, J.; Xu, X.; Zhang, X.-Q. Bioactive hydrogels for bone regeneration. Bioact. Mater. 2018, 3 (4), 401−417.

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