Exploring the Kinetics of Gelation and Final Architecture of

15 Mar 2015 - Enzymatically Cross-Linked Chitosan/Gelatin Gels ... King's College London, Institute of Pharmaceutical Science, 150 Stamford Street, Lo...
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Exploring the Kinetics of Gelation and Final Architecture of Enzymatically Cross-Linked Chitosan/Gelatin Gels Marcelo A. da Silva,*,†,‡ Franziska Bode,† Isabelle Grillo,§ and Cécile A. Dreiss† †

King’s College London, Institute of Pharmaceutical Science, 150 Stamford Street, London, SE1 9NH, United Kingdom LSS Group, Institut Laue-Langevin, 6 rue Jules Horowitz BP 156, F-38042 Grenoble, Cedex 9, France

§

S Supporting Information *

ABSTRACT: Small-angle neutron scattering (SANS) was used to characterize the nanoscale structure of enzymatically cross-linked chitosan/gelatin hydrogels obtained from two protocols: a pure chemical cross-linking process (C), which uses the natural enzyme microbial transglutaminase, and a physical-co-chemical (PC) hybrid process, where covalent cross-linking is combined with the temperature-triggered gelation of gelatin, occurring through the formation of triple-helices. SANS measurements on the final and evolving networks provide a correlation length (ξ), which reflects the average size of expanding clusters. Their growth in PC gels is restricted by the triple-helices (ξ ∼ 10s of Å), while ξ in pure chemical gels increases with cross-linker concentration (∼100s of Å). In addition, the shear elastic modulus in PC gels is higher than in pure C gels. Our results thus demonstrate that gelatin triple helices provide a template to guide the cross-linking process; overall, this work provides important structural insight to improve the design of biopolymer-based gels.

1. INTRODUCTION Soft matter covers a range of ubiquitous materials in modern life. From polymers to colloids, they are present in food, personal care products, construction, and a myriad of other important aspects of life, making them a prime research interest. The macroscopic properties of soft matter are a complex interplay of both microscopic and mesoscopic supramolecular organization. Thus, both its dynamics and structure are not defined solely by the added properties of its components, but their spatial organization over different length scales. The focus of this work is on biopolymer-based hydrogels, which are both of academic and practical interest. Due to their biological origin, biopolymers are appealing starting materials for biomedical applications, for instance, as skin substitutes, adhesives, or drug delivery matrices.1−3 For tissue engineering applications, for instance, the main goal is to artificially induce and guide cell proliferation to replace tissue lost to diseases.4,5 One approach is to design materials that mimic the cellular microenvironment to induce cellular growth and direct cellular specification. Crucially, it is now known that the elasticity of the extracellular matrix can guide stem cell differentiation,6 in addition to nanoscale features being key in dictating cell behavior.7 Therefore, not only chemical signaling needs to be addressed, but also mechanical and spatial properties of the hydrogel network needs to be understood in order to design better materials. Biopolymers offer a renewable source of raw materials from which to design functional products, which is attractive both from an economic and environmental viewpoints. The biopolymers used in this study, gelatin (extracted from fish) and chitosan, obtained from the partial deacetylation of chitin (found in the exoskeleton of crustaceans), offer not only intrinsic biological signaling to cells, © 2015 American Chemical Society

but also the potential of cheap, value-added products from waste recycling. The goal of this work is therefore to provide new insights into the nanoscopic-level molecular organization of biopolymer-based gels, namely, chitosan/gelatin hydrogels crosslinked with the enzyme transglutaminase. Pure gelatin gels have been studied before in our group,8,9 but it is of interest to explore the use of mixtures due to the added possibility of combining or improving properties. The rheological properties and biological potential have been discussed in previous work;10 this paper provides a detailed characterization of the gels architecture and how it is affected by the gelation protocol and the addition of a second biopolymer (chitosan) and how, in turn, this dictates the mechanical properties on the macroscopic scale. Gelatin is the denatured form of the most abundant animal protein, collagen,11 and its biological properties derived from it: it promotes cell-adhesion, is biocompatible, and biodegradable.3,12 Gelatin also offers a useful tool in the form of a thermally induced sol−gel transition: below the melting temperature, gelatin acquires a conformation resembling the native helical structure of collagen: three chains join together, thus, acting as junction points of a 3D network stabilized by intramolecular hydrogen-bonding,13 and forming a macroscopic gel. The second component, chitosan, has a structure very similar to glycosaminoglycans, a major component of the extracellular matrix. Chitosan is known for its nontoxicity, biocompatibility, biodegradability, wound-healing, and hemoReceived: February 11, 2015 Revised: March 11, 2015 Published: March 15, 2015 1401

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Biomacromolecules static capabilities14,15 and, thus, adds further attributes to the potential of gelatin as a scaffold for cell regeneration. The crosslinking reaction was achieved by an enzymatic route, using the enzyme transglutaminase, a calcium-dependent, naturally occurring enzyme found in almost all living organisms. It catalyzes acyl-transferase reactions between a γ-amine group of a glutamine residue and a primary amino group (e.g., lysine) resulting in a ε-(γ-glutamyl) lysine isopeptide bond. Microbial transglutaminase (mTGase) offers the advantage of being calcium-independent.16 In this work, we use SANS to study and contrast the structure of the enzymatically cross-linked chitosan/gelatin gels obtained from two protocols: the unrestricted cross-linking process, and a hybrid process, in which the thermally triggered triple-helical network of gelatin is present and impacts the chemical cross-linking process. SANS covers the ideal lengthscale range to explore the nanoscale architecture of these macromolecular gels17,18 and, thus, brings precious insight into these multicomponent biopolymer gels, on which structural knowledge is currently very limited.19−24 In particular, we show how the improved elastic properties of the gels obtained from the hybrid gelation protocol10 are reflected by a more homogeneous network on the nanoscale and how the removal of the physical network, which leads to an irreversible, macroscopic transition (decrease of the elastic modulus), is reflected, on the nanoscale, by an also irreversible expansion of the cross-linked clusters, which are no longer constrained by the triple-helical networks. The manuscript first provides a description of the architecture of the gels in order of increasing complexity of the gelation protocol: (3.1) physical, (3.2) chemical (C), and (3.3) physical-co-chemical (PC) gels, both at equilibrium and over time. This is followed (section 3.4) by a comparison of the rheological properties of C gels versus PC gels, which is linked to the nanostructure of the gels just established. Finally, the effect of adding chitosan to the pure gelatin gels is summarized and discussed in the final section (3.5).

Microbial transglutaminase (mTGase) solutions were prepared by solubilization of the solid in D2O. On average, 20 μL of mTGase solution were added to 1.000 g of chitosan/gelatin or gelatin solutions. mTGase concentrations of 10, 20, 30, and 40 U/ggelatin were studied. For rheological experiments, ultrapure water (18.2 MΩ·cm, Milliporefiltered) was used. In this work, the concentrations of biopolymers are fixed at 1.0% chitosan (Cs1.0%) and 10% tilapia gelatin (Gel10%), in order to facilitate comparison with previous work conducted in our group, where pure tilapia gels were studied.8,9 To produce the gels, three different gelation protocols were employed: Physical Gelation (P). For pure physical gels, Cs1.0%/Gel10% or Gel10% solutions were cooled for 2 h at 12 or 21 °C (below tilapia gelatin gelation temperature of ∼23 °C). Chemical Gelation (C). Chemical gelation was performed at 37 °C, that is, above gelatin melting temperature. A fixed amount of mTGase, from 10 to 40 U/ggelatin, was added to Cs1.0%/Gel10% or Gel10% solutions, vortex-mixed and left to gel for a fixed amount of time before thermal deactivation of mTGase by a temperature jump to 70 °C. For SANS experiments, the gelation was conducted for 30 min, 60 min, and 24 h before the enzyme was deactivated. For rheological experiments, the gelation was conducted for 3.5 h. Physical-co-chemical Gelation (PC). Physical and chemical gelation were performed simultaneously. To achieve this, selected amounts of mTGase (10−40 U/ggelatin) were added to Cs1.0%/Gel10% or Gel10% solutions and the system was quickly cooled down to 21 °C (below gelation temperature). The gelation was conducted for 3.5 h for the rheological experiments, following which mTGase was thermally deactivated. For SANS, the gelation was conducted for 24 h, SANS data acquired without enzyme deactivation; then, the enzyme was thermally deactivated and data was acquired at 37 °C and at 21 °C after 24 h of physical gelation. 2.2. Methods. Small-Angle Neutron Scattering. Small-angle neutron scattering (SANS) experiments were performed on the instrument D11 at the Institut Laue-Langevin (ILL), Grenoble, France. Incidental wavelengths of 4.5 and 10 Å were used with sample detector distances of 1.2, 8.0, and 39 m, corresponding to a total scattering vector range q from 1.5 × 10−3 to 0.7 Å−1. The sample temperature was controlled by an external circulating thermal bath. The data set is available at 10.5291/ILL-DATA.9−11−1654.27 SANS Data Fitting. All data were fitted using a power law combined to a Lorentzian equation17,28−30 using SasView software.31 This equation has been used to describe the scattering from cross-linked gels in the dilute regime and has provided good results for pure gelatin gels.9

2. MATERIALS AND METHODS 2.1. Materials. Chitosan (Cs), derived from crab shell with a minimum deacetylation degree of 85%, acetic acid (99%), anhydrous sodium acetate, and D2O (99.9% purity) were purchased from SigmaAldrich. Chitosan molecular weight was found to be 900 kDa ± 5%, as determined by viscometric methods.25 Type A tilapia fish skin gelatin (Gel) was kindly donated by Rousselot (France). The average molecular weight was determined to be about 36 kDa ± 12% using GPC (Smithers Rapra) with a melting temperature of about 23 °C.8 Bacterial transglutaminase (mTGase) was purchased from N-Zyme BioTec GmbH (Darmstadt, Germany). mTGase has a specific activity of 1.6 units/mg solid, molecular weight of 38 kDa, and a purity superior to 80% (SDS-PAGE), as indicated by the supplier. All compounds were used as received. Preparation of the Solutions and Gelation Protocols. Chitosan samples were prepared by solubilization in 2% (w/w) D2O solutions of acetic acid overnight. After complete dissolution, the pH was raised to 5 by adding suitable amounts of anhydrous sodium acetate, forming an acetate buffer (ca. 250 mM of acetic acid/550 mM sodium acetate). This pH is still within the activity range of mTGase.26 Chitosan/ gelatin solutions were prepared by adding gelatin to the chitosan solutions. Gelatin samples were prepared by adding gelatin to the acetate buffer in D2O. Both solutions were left to swell overnight at 4 °C. Before use, the samples were heated up to 37 °C for 30 min to ensure complete melting of tilapia gelatin and system homogenization.

Il(q) =

Il(0) A + + BKG qm 1 + (qξ)n

(1)

The first term describes Porod scattering from clusters (exponent = m) in the low-q range and the second term characterizes the polymer chains behavior detected in the high-q region. The exponent n relates to the chain thermodynamics.21,29 A and Il(0) are constants, and ξ is the correlation length, which describes the size of the scattering centers. The analysis will be focused on n and ξ. For this analysis, the important values of n are for a highly swollen chain in a good solvent (chain with excluded volume), n = 1.66, and for randomly branched Gaussian chains, n = 2.28. The errors stated are not the errors calculated by SasView31 but an estimate from repeated fits using different input parameters. Rheology. All rheological measurements were conducted on a strain-controlled ARES rheometer (TA Instruments) equipped with a titanium parallel plate geometry (25 mm diameter). Temperature was controlled by a Peltier unit (±0.1 °C). To prevent evaporation, a thin layer of low viscosity paraffin oil was applied on the edge of the plate. The tests were repeated three times, with a standard deviation typically below 20%. The results presented in this work are examples of typical data obtained, not averages, except when indicated otherwise. 1402

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Biomacromolecules Time sweep experiments (curing curves) were conducted at a fixed frequency (6.28 rad·s−1) and strain amplitude (1.0% strain) for 3.5 h at 37 °C (chemical gelation) and 21 °C (physical-co-chemical gelation). Oscillatory frequency sweep tests covering the range of 100 to 0.1 rad· s−1 were performed in the linear viscoelastic regime (1.0% strain), as determined by dynamic strain sweep measurements (SI.1). Frequency sweep tests were run after gelation was completed and the enzyme thermally deactivated (70 °C, 5 min).

systems above and below gelatin melting temperature are compared. The scattering curves in the sol versus gel state are very similar, differing slightly in the mid-q range. The data were suitably fitted with a combination of a power-law model and a Lorentzian function9,29,30,33 (cf. Methods section), giving a correlation length (ξ) of about 31 ± 3 Å for the physical Cs1.0%/Gel10% gel and about 22 ± 2 Å for the solution of the same composition. This behavior departs from the pure gelatin systems (both systems at pH 5), where the correlation length in the solution and gel states were nearly equivalent, 23 ± 2 and 26 ± 2 Å, respectively,9 showing that the transition from single strands to the helical network did not induce a major change in the configuration of the gelatin chains. In Figure 1B, mixed Cs1.0%/Gel10% gels are compared to pure gelatin physical gels. Over the mid to high q range, no significant changes are observed, and a correlation length (ξ) of about 31 ± 3 Å is associated with both scattering curves in these conditions (pH 5). Both chitosan and gelatin show similar neutron scattering length densities, 1.69 × 1010 cm−2 and 2.47 × 1010 cm−2 for gelatin34 and chitosan,35 respectively. Therefore, the individual contribution from chitosan and gelatin cannot be distinguished, and the scattering data reflects the global mixture. In physical gels, the correlation length can be interpreted as an average mesh size of the network,9,36 that is, the average distance between the topological constraints that supports the gel network, which is maintained by the gelatin physical network in both Cs/Gel and Gel. In the low q range, however, both curves diverge, Cs1.0%/Gel10% showing a power law dependence with a higher exponent than the pure gelatin gel: 1.6 ± 0.2 (closer to the value expected for a chain in a good solvent37) against 2.0 ± 0.2 (a value associated with a chain in a θ solvent or Gaussian chain37). This divergence may arise from differences in solvation between chitosan and gelatin. Physical Cs1.0%/Gel10% gels consist of a physical 3D network built from gelatin chains and interpenetrating but unbound chitosan chains. At the experimental pH (pH 5) both biopolymers are positively charged (chitosan amino group have a pKa of 6.5 and type A gelatin a pI around 8−9); thus, a certain level of repulsion between the species and in-between segments of the chains themselves is expected, which would affect the flexibility of the chains, making the Gaussian chain a poor approximation of the chains dynamic behavior. 3.2. Chemical Gels: Evolving Structure and Effect of Cross-Linker Concentration. The second type of gel studied is the chemical network generated by the covalent bonding of gelatin and chitosan strands via the action of the enzyme mTransglutaminase (mTGase). Cross-linking between gelatin strands and chitosan/gelatin strands is expected to take place during the enzymatic reaction. mTGase catalyzes acyl-transferase reactions between the NH2 part of the γ-amine group of a glutamine residue and a primary amino group (e.g., lysine), resulting in an ε-(γ-glutamyl) lysine isopeptide bond. As reported in the literature,26,38 chitosan free amines (from the deacetylated units) are viable reaction sites for mTGase. The chitosan used in this work has a degree of deacetylation of 85%, while the lysine residue is present at ca. 2.5% and the glutamine residue at around 6.9% in tilapia fish gelatin.39 Thus, the limiting step for the cross-linking reaction is the availability of glutamine residues in gelatin, since chitosan should provide a large excess of free amines. The chemical network is obtained by conducting the enzymatic cross-linking at 37 °C. Here, both gelatin and

3. RESULTS AND DISCUSSION 3.1. Structure of the Physical Gels: The Gelatin TripleHelices Network. Gelatin naturally undergoes a thermally reversible sol−gel transition. Upon decreasing the temperature, gelatin single-strands self-associate to reform the collagen triplehelix based on the left-handed, PII conformation of the individual strands.32 The transition temperature is dependent on the animal’s body temperature: fish gelatin gels at a lower temperature, due to the lower amount of proline and hydroxyproline content in the native collagen sequence; tilapia gelatin gels around 23 °C.8 Our analysis therefore starts by studying the first type of network employed in the gel preparation: the gelatin physical networks, which are induced here at 21 °C. Figure 1 shows the scattering curve for mixed

Figure 1. Scattering curves and fits for (A) chitosan (Cs) 1%/gelatin (Gel) 10% solutions (at 37 °C), and physical gels (P; at 21 °C) and (B) Cs 1%/Gel 10% and Gel10% physical gels at 21 °C.

chitosan (Cs) 1.0%/gelatin (Gel) 10% and pure gelatin (Gel) 10% physical gels. Scattering curves for both Cs1.0%/Gel10% and Gel10% (Figure 1B) physical gels show a sigmoidal shape. Gel10% presents a well-defined shoulder around 0.02 Å−1 (Figure 1B), while the shoulder for Cs1.0%/Gel10% gels is much less prominent, although appearing in the same q-region. In Figure 1A, SANS scattering curves for Cs1.0%/Gel10% 1403

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Biomacromolecules chitosan are in the sol state, and thus access to binding sites is only limited by the diffusion of the enzyme and the biopolymer strands. The kinetics of the cross-linking process can be investigated by deactivating the enzyme at specific time points (through a temperature jump to 70 °C for 5 min), thus, freezing the pattern in time and providing a unique opportunity to monitor the evolving structure of the gels by SANS with very good statistics. The effect of both enzyme concentration (10−40 U/ ggelatin) and reaction time was followed and scattering curves collected after 30 min, 60 min, and 24 h (Figure 2A, B, and C, respectively). For the first 30 min, gels at 10 U enzyme concentration show a correlation length (ξ) of about 57 ± 6 Å, while the other concentrations (20, 30, and 40 U) reach much higher values of about 300 ± 30 Å. At the next time point (60 min, Figure 2B), divergences between the four enzyme concentrations become much clearer. The correlation length increases with enzyme concentration: about 52 ± 6, 400 ± 40, 614 ± 62, and 625 ± 63 Å for 10, 20, 30, and 40 U, respectively. At this stage, however, the shapes of the scattering curves are still quite similar for all samples studied. After 24 h, large disparities in the curves are evidenced (Figure 2C), clearly showing that enzyme concentrations dictate different final structures at the nanoscale. At this long cross-linking time, a drop in ξ is observed from 20 U mTGase concentration and above to 108 ± 11, 380 ± 40, 330 ± 33, and 290 ± 29 Å (for 10, 20, 30, and 40 U, respectively). In chemical gels, ξ does not reflect an average mesh size as in physical gels (see previous section), but the size of growing regions of highly cross-linked density, which act as scattering centers.9 Thus, a larger availability of enzyme leads to larger clusters of highly connected chains from the very beginning of the reaction (Figure 2A), reflecting a faster process with higher enzyme concentration (10 U vs 20, 30, and 40 U). As the cross-linking process continues, the expansion of the clusters proceeds (t = 60 min, Figure 2B). At “infinite” times (24 h, Figure 2C), well after the sample has reached a macroscopic equilibrium (as confirmed by rheology time-sweep measurements, Figure 3A), the decrease in cluster size observed at higher enzyme concentrations (20−40 U) reflects the further growth of clusters to sizes reaching length-scales beyond those accessible by SANS, thus making lower correlation lengths in the network “visible” to neutrons. The scaling of ξ with enzyme concentration, and thus, increasing cluster size, is also reflected by the increasing elastic shear modulus of the networks10 (see also, Figure 7A). Previous experiments10 have shown that Cs1.0%/Gel10% chemical gels are highly turbid in appearance, and the turbidity increases with enzyme concentration. The turbidity arises from the presence of large domains dispersed through a transparent continuous medium, thus suggesting highly inhomogeneous, highly segregated networks. Therefore, these gels are expected to present a large distribution of length scales, some of them large enough to scatter visible light and, therefore, beyond the size range of neutrons. 3.3. Physical-co-chemical Gels: Cross-Linking in the Presence of the Triple-Helices Network. The third type of network studied is the hybrid process, which results from the combination of the previous two networks. It consists in conducting the enzymatic cross-linking (chemical network) within the confines of a developing gelatin physical network: the so-called physical-co-chemical gel or PC gel. In order to achieve this, all components are quickly mixed at 37 °C, followed by rapid cooling to 21 °C. From a rheological point of

Figure 2. Kinetics of chitosan (Cs) 1%/gelatin (Gel) 10% chemical cross-linking by mTGase monitored by small-angle neutron scattering after (A) 30 min, (B) 60 min, and (C) 24 h of gelation as a function of enzyme (mTGase) concentration. Solid lines are fits to the model described in the text.

view, we have shown previously10 that PC gels have higher moduli than their pure chemical counterparts; this is shown in Figure 3A,B for a 1% chitosan composition. While this could easily be attributed to the added elasticity from the physical network, melting the helices reveals that the remaining chemical networks still display higher shear moduli than the equivalent chemical gels grown with a free cross-linking process10 (Figure 4A,B). In this section, we explore the nanostructure of these hybrid gels and contrast them with the 1404

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Figure 3. Curing curves for chitosan (Cs) 1%/gelatin (Gel) 10% chemical gels (A), and physical-co-chemical gels (B). Curves in (A) were measured at 37 °C, above the gelatin melting temperature (no physical network present). Curves in (B) were measured at 21 °C, below the gelatin gelation temperature (both physical and chemical gelation present). mTGase concentrations of 10 (pink square), 20 (red circle), 30 (brown triangle), and 40 (black diamond) U/ggelatin were used. G′ are shown by filled symbols, and G″ are shown by empty symbols.

Figure 4. Shear frequency sweep curves for chitosan (Cs) 1%/gelatin (Gel) 10% chemical gels (A), and physical-co-chemical gels (B). Curves were measured at 37 °C, above the gelatin melting temperature (no physical network present). mTGase concentrations of 10 (pink square), 20 (red circle), 30 (brown triangle), and 40 (black diamond) U/ggelatin were used. G′ are shown by filled symbols, and G″ are shown by empty symbols.

sizable amount of physical network is present before any extensive chemical bonding takes place.8,10 Therefore, it is expected that the physical network is able to spatially constrain the growth of the chemical network due to either the burying of binding sites within the triple-helices junctions or the restricted diffusion of the gelatin chains participating to the triple-helices junctions. According to the equilibrium swelling theory,40 the swelling of a cross-linking polymer is controlled by two forces: the elastic pressure and the osmotic pressure. The osmotic pressure leads to coil expansion, increasing solvent content within the coil and reducing the polymer local concentration; this pushes binding sites further away. The elastic pressure instead leads to coil contraction, toward a more favorable entropic state; this increases the local polymer concentration and brings binding sites spatially closer. Thus, the elastic pressure favors the crosslinking reaction, and the cross-linking reaction offsets the expansion caused by the osmotic pressure through intermolecular bonding. The sum of both of these contributions results in a positive feedback, which leads to the formation of very localized clusters of cross-linked bundles. Baumberger and coworkers presented a quantitative study of the development of inhomogeneities in pure chemical gels, due to this positive feedback, showing how the cross-linking in the sol state leads to

chemical gels just described, before we come back to discussing the rheology in the next section, in the light of the structural findings. Figure 5A shows the scattering curves for Cs1.0%/Gel10% PC gels after 24 h of gelation at 21 °C for different mTGase concentrations. Correlation lengths obtained at 10, 20, 30, and 40 U of mTGase are 61 ± 6, 56 ± 6, 63 ± 6, and 75 ± 8 Å, respectively. These values are significantly lower than those observed for chemical gels in the same conditions (108 ± 11, 380 ± 40, 330 ± 33, and 290 ± 29 Å for 10, 20, 30, and 40 U, respectively). In addition, there is a more limited dependence on enzyme concentration, compared to the pure chemical gels. In Figure 5B the scattering curves for the same samples are shown, following the melting of the physical networks by raising the temperature to 37 °C. This results in an increase of the correlation length to hundreds of angstroms: 1271 ± 130, 602 ± 60, 446 ± 50 and 361 ± 40 Å for Cs1.0%/Gel10% with 10, 20, 30, and 40 U of mTGase, respectively. This shows that, without the triple-helices holding the chemical network in place, a substantial reorganization of the gels occurs, leading to the emergence of large aggregates. Previous rheological and optical rotational studies have shown that gelatin physical gelation is a much faster process than the cross-linking reaction,8,10 hence, in this simultaneous gelling protocol, a 1405

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Figure 6. Schematic representation of (A) chitosan/gelatin PC gels, where the size of the cross-linked clusters is limited by the presence of the triple-helices, acting as a template to direct the reticulation process. (B) Upon removal of the helices, an elastic pressure-driven reorganization of the chemical networks occurs, allowing the clusters to come closer together, reaching a size-range similar to the pure chemical (C) gels.

give cluster sizes smaller than in pure chemical gels; this is followed by an irreversible expansion of the coils, triggered by the melting of the physical network (Figure 6B). In Figure 5C, the same PC gels are cooled back to the original temperature of 21 °C, allowing the regrowth of the triple-helices on top of the reorganized chemical network. The final networks (Figure 5C) clearly depart from the original PC networks (Figure 5A). In the mid- and high-q range, the curves are now nearly superimposable, independent of enzyme concentration, in contrast to Figure 5A. A drop in the correlation length values is observed, but ξ still falls within the 100s of Å range: 712 ± 70, 437 ± 50, 370 ± 40, and 333 ± 40 Å for 10, 20, 30, and 40 U of mTGase, respectively. This shows that, once removed, the initial structure of the physicalco-chemical gels is irreversibly lost, that is, the regrown physical network (Figure 5C) does not match the initial physical network (Figure 5A). A similar behavior was recently observed by Kim and co-workers with gelatin nanogels (using a different cross-linker): the nanoparticles volume was drastically reduced after thermally removing the physical network due to the irreversible collapse of the chemical network.42 Our results here clearly show that the gelatin triple-helices act as a “template” by constraining the cross-linking process (smaller cluster sizes) and that, upon their removal, the clusters reorganize and expand (Figure 6) but are necessarily looser, as the process is accompanied by a decrease in the elasticity of the networks.8,10 This interesting transition could thus find applications in the development of thermally triggered smart materials, for instance, where irreversible nanoscale rearrangements are sought. In terms of the Lorentz exponent (n), values around 1.6 ± 0.2 to 1.8 ± 0.2 were obtained for all mTGase concentrations and all three conditions evaluated: both physical and chemical networks present (gelling at 21 °C, Figure 5A), chemical network only (melting at 37 °C, Figure 5B), and physical network regrown on top of the chemical network (cooling back to 21 °C, Figure 5C), a value similar to the pure physical networks of Cs1.0%/Gel10% (n = 1.6 ± 0.2). This suggests that the ordering imposed by the physical network persists to some extent, even after its removal, and it is not altered by the amount of mTGase in the system. For the Porod exponent, m, values of about 2.2 ± 0.2 to 2.3 ± 0.2 were obtained for 20 and 30 U, whereas for 10 and 40 U, m = 2.8 ± 0.2 to 3.3 ± 0.2 at all

Figure 5. SANS curves and fits to the Porod/Lorentzian model (solid lines, see text) of physical-co-chemical gels of chitosan (Cs) 1%/gelatin (Gel) 10% as a function of enzyme (mTGase) concentration: (A) after 24 h gelation at 21 °C, (B) after melting of the physical network at 37 °C, (C) after rebuilding of the physical network on top of the chemical network at 21 °C.

the formation of inhomogeneous gels.41 The physical-cochemical gelation adds a third element, the constraints of the physical triple-helices, which both hinder the elastic pressuredriven coil contraction and the osmotic pressure-driven coil expansion. Therefore, the presence of the physical network counteracts the positive feedback loop observed in chemical gels that leads to the formation of highly localized clusters, enabling the formation of more homogeneous networks. Once the cross-linking reaction is stopped (step 2, Figure 5B), the melting of the physical network by heating removes the constraints imposed by the triple-helices; the macromolecular chains are then free to undergo an elastic pressure-driven reorganization, leading to the large values of the correlation length observed. The proposed structure of the PC gels is schematically represented in Figure 6: the original networks, constrained by the presence of the triple helices (Figure 6A), 1406

DOI: 10.1021/acs.biomac.5b00205 Biomacromolecules 2015, 16, 1401−1409

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Biomacromolecules three conditions evaluated. This shows that the dimensionality of the largest scatterers (low-q region) is set in the starting networks by the enzyme concentration and preserved even after the removal of the physical networks. 3.4. Comparing Gelation Protocols: The Key Role of Triple-Helices. One striking difference between the chemical and physical-co-chemical gelation (PC) is the overall gel strength. In Figure 4, oscillatory shear frequency sweeps are presented for both Cs1.0%/Gel10% chemical (Figure 4A) and PC gels (Figure 4B) above gelatin melting temperature, that is, only chemical networks are present. When comparing the chemical networks in both types of gels, PC gels clearly show higher values of the storage modulus (G′), that is, the chemical networks grown within the framework of the physical network show higher elasticity compared to the chemical networks grown freely. The strongest gels are obtained at the highest enzyme concentration (40 U mTGase/ggelatin): for PC gels, G′ = 13.2 kPa against G′ = 5.6 kPa for chemical gels. There is clearly a positive influence of the physical networks in inducing a cross-linking process that produces stronger gels,8,10,43 that is, the triple-helices are able to direct covalent bonding into a more efficient process. The natural question is then how are these macroscopic differences between the chemical and PC gels reflected in the nanoscopic structure of the biopolymer networks? In Figure 7A−C, the fittings parameters of chitosan/ gelatin gels obtained from either a pure chemical gelation (37 °C) or a physical-co-chemical gelation process (21 °C) are presented. Strikingly, ξ values are smaller and fluctuate less in PC gels than in the correspondent chemical gels (Figure 7A). The two exponents (Porod and Lorentzian) can help better understand the differences between the two types of networks. Both exponents carry information about the dimensionality of the aggregates over different length scales. The exponent n in the Lorentzian function reflects the length scale of the macromolecular strands, while the exponent m in the Porod law describes larger structures made-up of macromolecular chains bundled together by topological interactions (physical gels) or covalent bonds (chemical gels).29,37 Figure 7B,C shows the dependence of these two exponents for chemical and PC gels after 24 h gelation as a function of mTGase concentration. The n exponent (Figure 7C) shows similar values for both PC and chemical gels, within the estimated error; therefore, it is possible to say that between PC and chemical gels no large differences can be observed by SANS on the scale of the polymer chains. Instead, the Porod exponent, m, shows consistently larger values in chemical gels compared to PC gels (Figure 7B). Thus, large differences in aggregate morphology are seen between the two types of gels at large length scales. Indeed, PC gels have also been observed to be notably less turbid than the equivalent chemical gels,10 also reflecting the formation of larger structures in chemical gels, which are constrained in PC gels. Overall, it thus appears that triple-helices induce the formation of cross-linked networks where the “clusters” are smaller in size than in “freely” crosslinked gels (pure chemical gels), however, these clusters are more efficient in achieving a higher elasticity of the networks. Positive synergy can thus be achieved by using triple-helices as a framework to guide cross-linking in a hybrid gelation process, and this has important consequences in improving the design of biopolymer gels. 3.5. Single (Gelatin) Gels versus Binary (Chitosan/ Gelatin) Gels. When comparing the single- (Gel10%) and mixed-component (Cs1.0%/Gel10%) systems, large differences

Figure 7. Dependence of the fitting parameters: (A) correlation length (ξ), (B) the Porod exponent m, and (C) the Lorentzian exponent n, as a function of transglutaminase (mTGase) concentration for chitosan (Cs) 1%/gelatin (Gel) 10% chemical, and physical-co-chemical gels measured at 37 °C (chemical gelation) and 21 °C (physical-cochemical gelation).

in ξ and m are observed, while similar values are obtained for n. In Figure 8A,B, the scattering curves for both Gel and Cs/Gel, PC and chemical gels, are presented, all systems at an intermediate mTGase concentration of 20 U. Simple visual inspection reveals that the curves are very similar in the mid to mid-q regions. The n values are the same for both Cs/Gel and Gel PC gels, n = 1.8 ± 0.2, while for chemical gels, n are also very similar, n = 1.9 ± 0.2 and n = 2.0 ± 0.2 for Cs/Gel and Gel, respectively, reflecting the similarity of both systems at this length scale. At larger length scales instead (low q range), large differences, are observed between single and mixed gels, both in PC and chemical gels. The power law exponent values for chemical gels are m = 4.0 ± 0.2 and 2.7 ± 0.2, for Cs/Gel and pure gelatin gels, respectively, and for PC gels, m = 2.3 ± 0.2 1407

DOI: 10.1021/acs.biomac.5b00205 Biomacromolecules 2015, 16, 1401−1409

Article

Biomacromolecules

4. CONCLUSIONS In this work, the nanoscale architecture of biopolymer gels cross-linked by a natural enzymatic process was investigated by means of small-angle neutron scattering (SANS), and this understanding was linked to the macroscopic properties of the gels. Namely, mixtures of gelatin and chitosan were studied, where an enzymatic process with microbial transglutaminase was conducted in two different microenvironments: a conventional chemical (C) process, where access to the binding sites is limited by diffusion of the components; and a hybrid physicalco-chemical (PC) process, where a thermally triggered, simultaneous, but faster, physical gelation process takes place, thus, ensuring that a network of triple-helices is present before extensive chemical cross-linking takes place, creating a spatially restricted environment. For the pure chemical process, the correlation length (ξ) obtained from SANS measurements, which can be understood as the average size of expanding crosslinked domains, increases with the concentration of cross-linker and increases over time (faster at higher cross-linker concentrations); larger clusters at higher enzyme concentration directly correlate with a higher elasticity of the networks. At “infinite” time, the original clusters have grown beyond the accessible SANS length scales, and clusters of smaller size become detectable; there is clearly a distribution of length scales in the networks, some well within the range of light scattering. For PC gels, a more complex picture emerges. When both physical and chemical networks are present, ξ values for PC gels are less sensitive to cross-linker concentration and are much lower than in the equivalent chemical gels: 10s of Å versus 100s of Å. After the removal of the physical networks, the elasticity of the network is reduced, and ξ jumps to 100s of Å, showing a possible collapse of the network structure driven by elastic pressure. Reintroducing the physical network does not recover the initial network. Overall, it thus appears that the triple-helices induce the formation of cross-linked networks where the clusters are smaller in size than in “freely” crosslinked gels (pure chemical gels); however, these clusters are more efficient at achieving a higher elasticity of the networks, suggesting a better ratio between elastically active and inactive chains. Positive synergy can thus be achieved by using triplehelices as a template to guide the cross-linking process, both in terms of mechanical properties (higher shear modulus for the same composition), but also a more homogeneous structure at the nanoscale. The comparison of pure gelatin gels (single system) and chitosan/gelatin gels (binary system) also confirms the large influence of the triple-helices in dictating the nanostructure of the gels. Both single (Gel) and mixed (Cs/Gel) PC gels are rather similar in nature. Both Lorentzian and Porod exponents are in the same range, about 1.8 and 2.4, respectively, and ξ are very similar, 50 and 56 Å for the single and binary systems, respectively. Instead, the chemical single and binary gels are rather dissimilar, showing that in a freely cross-linking system, the differences in composition become more relevant, while they are smeared in the “constrained” cross-linking system (PC). This type of detailed, nanoscale structural investigation of biopolymer gels is scarce in the literature; it is however crucial to understand the impact of composition and gelation processes to provide a rationale for the design of hydrogels with controlled functional properties. The gels presented in this work, based on abundant natural products available from waste

Figure 8. Comparison between the structure of single system gels (pure gelatin: Gel 10%), and mixed gels (chitosan (Cs) 1%/gelatin (Gel) 10%): SANS curves of the (A) chemical (C) and (B) physicalco-chemical gels (PC) obtained after 24 h gelation and fixed mTGase of 20 U/ggelatin. (C) Correlation length (ξ) for physical (21 °C), physical-co-chemical (21 °C) and chemical (37 °C) gels of gelatin 10% and chitosan 1%/gelatin 10%.

and 2.4 ± 0.2, for Cs/Gel and pure gelatin gels, respectively. This suggests that in PC gels, the gelatin physical network largely defines the dimensionality of the scatterer centers at larger length scales, therefore both single (gelatin alone) and mixed (chitosan/gelatin) gels show similar structural parameters. The physical network forms faster than the chemical one; the size of the scattering centers is thus largely defined by the spatial constrains imposed by the triple helices. Instead, in chemical gels, no physical network is present to impose any type of spatial constraints. The differences between single and mixed systems and the impact of the gelatin physical network can better be noticed when comparing ξ values (Figure 8C). In pure chemical gels, ξ values are much larger for Cs/Gel gels than for pure gelatin gels. Instead, the PC gels show very similar ξ values for both single and mixed gels, reinforcing the dominating role of the physical network in dictating the gel nanostructure. 1408

DOI: 10.1021/acs.biomac.5b00205 Biomacromolecules 2015, 16, 1401−1409

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Biomacromolecules

(17) Shibayama, M. Polym. J. 2011, 43, 18−34. (18) Shibayama, M. Macromol. Chem. Phys. 1998, 199, 1−30. (19) Crompton, K. E.; Forsythe, J. S.; Horne, M. K.; Finkelstein, D. I.; Knott, R. B. Soft Matter 2009, 5, 4704−4711. (20) Guilbauda, J. B.; Saiani, A. Chem. Soc. Rev. 2011, 40, 1200− 1210. (21) Hyland, L. L.; Taraban, M. B.; Feng, Y.; Hammouda, B.; Bruce, Y. Y. Biopolymers 2012, 97, 177−188. (22) Chatterjee, T.; Nakatani, A. I.; Adden, R.; Brackhagen, M.; Redwine, D.; Shen, H.; Li, Y.; Wilson, T.; Sammler, R. L. Biomacromolecules 2012, 13, 3355−3369. (23) Horkay, F. J. Polym. Sci., Part B: Polym. Phys. 2012, 50, 1699− 1705. (24) White, J. C.; Saffer, E. M.; Bhatia, S. R. Biomacromolecules 2013, 14, 4456−4464. (25) Wang, W.; Bo, S.; Li, S.; Qin, W. Int. J. Biol. Macromol. 1991, 13, 281−285. (26) Sang, L.-Y.; Zhou, X.-H.; Yun, F.; Zhang, G.-L. J. Sci. Food. Agric. 2009, 90, 58−64. (27) da Silva, M. A.; Dreiss, C. A.; Grillo, I.; Sekhar, G. Chitosan/ gelatin enzymatically cross-linked hydrogels: Composition and temperature effects on the gel’s molecular structure. Institut LaueLangevin (ILL) 2013, DOI: 10.5291/ILL-DATA.9-11-1654. (28) Hule, R. A.; Nagarkar, R. P.; Hammouda, B.; Schneider, J. P.; Pochan, D. J. Macromolecules 2009, 42, 7137−7145. (29) Hammouda, B.; Ho, D. L.; Kline, S. R. Macromolecules 2004, 37, 6932−6937. (30) Bastide, J.; Leibler, L. Macromolecules 1988, 21, 2647−2649. (31) SasView, http://www.sasview.org/. (32) Drake, A. F.; Siligardi, G.; Gibbons, W. A. Biophys. Chem. 1988, 31, 143−146. (33) Horkay, F.; Hammouda, B. Colloid Polym. Sci. 2008, 286, 611− 620. (34) Carn, F.; Boué, F.; Djabourov, M.; Steunou, N.; Coradin, T.; Livage, J.; Floquet, S.; Cadot, E.; Buhler, E. Soft Matter 2012, 8, 2930− 2944. (35) Shi, L.; Carn, F.; Boué, F.; Mosser, G.; Buhler, E. Soft Matter 2013, 9, 5004−5015. (36) Daoud, M.; Cotton, J. P. Macromolecules 1975, 8, 804−818. (37) King, S. M. In Modern Techniques for Polymer Characterization; Pethrick, R. A., Dawkins, J. V., Eds.; John Wiley & Sons Ltd: Chichester, U.K., 1999; p 171. (38) Kolodziejska, I.; Piotrowska, B.; Bulge, M.; Tylingo, R. Carbohydr. Polym. 2006, 65, 404−409. (39) Sarabia, A. I.; Gómez-Guillén, M. C.; Montero, P. Food Chem. 2000, 70, 71−76. (40) Sperling, L. H. In Introduction to Physical Polymer Science, 4th; John Wiley & Sons, Inc: Hoboken, NJ, U.S.A., 2006; p 76. (41) Souguir, H.; Ronsin, O.; Larreta-Garde, V.; Narita, T.; Caroli, C.; Baumberger, T. Soft Matter 2012, 8, 3363−3373. (42) Gandhi, S. S.; Yan, H.; Kim, C. ACS Macro Lett. 2014, 3, 1210− 1214. (43) Coimbra, P.; Gil, M. H.; Figueiredo, M. Int. J. Biol. Macromol. 2014, 70, 10−19.

recycling and a natural reticulation process, are attractive materials for biomedical applications. In particular, both the guiding of the cross-linking process by the triple-helices and the collapse of the chemical networks after removal of the physical scaffold are interesting processes to exploit for the development of thermally responsive nanomaterials for drug delivery, tissue engineering or biosensors, and could be extended to other biopolymers presenting a natural, thermal gelation process, such as gellan gum.

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ASSOCIATED CONTENT

S Supporting Information *

Shear strain amplitude sweep curves for chitosan (Cs) 1%/ gelatin (Gel) 10% chemical gels, and physical-co-chemical gels above and below gelatin melting temperature. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ‡

MNP, School of Physics and Astronomy, University of Leeds, 8.61 E.C. Stoner Building, Leeds, LS2 9JT, U.K. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by a Leverhulme Trust Research Grant (F/07 040/AR). F.B. thanks the EPSRC (Grant EP/F037902/ 1) for financial support. ILL is acknowledged for the provision of beam time on D11 (#9-11-1654). JCNS is acknowledged for the provision of beam time at KWS1 instrument at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany (Proposal #5246), and Aurel Rădulescu for help with the experiments (data not published). Dr. Rafael Bini is acknowledged for the freeze-dried SEM image of a gelatin gel used in the TOC.

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REFERENCES

(1) Zhang, S. G. Nat. Biotechnol. 2003, 21, 1171−1178. (2) Peppas, N.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1−17. (3) Vlierberghe, S. V.; Dubruel, P.; Schacht, E. Biomacromolecules 2011, 12, 1387−1408. (4) Tabata, Y. J. R. Soc., Interface 2009, 6, S311−S324. (5) Place, E. S.; Nicholas, D. E.; Stevens, M. M. Nature 2009, 8, 457− 470. (6) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Cell 2006, 126, 677−689. (7) Stevens, M. M.; George, J. H. Science 2005, 310, 1135−1138. (8) Bode, F.; da Silva, M. A.; Drake, A. F.; Ross-Murphy, S. B.; Dreiss, C. A. Biomacromolecules 2011, 12, 3741−3752. (9) Bode, F.; da Silva, M. A.; Smith, P.; Lorenz, C.; McCullen, S.; Stevens, M. M.; Dreiss, C. A. Soft Matter 2013, 9, 6986−6999. (10) da Silva, M. A.; Bode, F.; Drake, A. F.; Goldoni, S.; Stevens, M. M.; Dreiss, C. A. Macromol. Biosci. 2014, 14, 817−830. (11) Ward, A. G. J. Appl. Phys. 1954, 5, 85−90. (12) Fan, Z. P.; Zhang, Y. M.; Fang, S.; Xu, C.; Li, X. S. RSC Adv. 2015, 5, 1929−1936. (13) Djabourov, M. Contemp. Phys. 1988, 29, 273−297. (14) Tchemtchoua, V. T.; Atanasova, G.; Aqil, A.; Fil, P.; Garbacki, N.; Vanhooteghem, O.; Deroanne, C.; Noel, A.; Jerome, C.; Nusgens, B.; Poumay, Y.; Colige, A. Biomacromolecules 2011, 12, 3194−3204. (15) Khora, E.; Limb, L. Y. Biomaterials 2003, 24, 2339−2349. (16) Yang, X.; Liu, Y.; Payne, G. F. J. Adhes. 2009, 85, 576−589. 1409

DOI: 10.1021/acs.biomac.5b00205 Biomacromolecules 2015, 16, 1401−1409