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chitosan polyelectrolyte complex hydrogels and multilayers
European Polymer Journal 120 (2019) 109268
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Self-healable hyaluronic acid/chitosan polyelectrolyte complex hydrogels and multilayers
T
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Nagore Barrosoa, Olatz Guarestic, Leyre Pérez-Álvareza,b, , Leire Ruiz-Rubioa,b, Nagore Gabilondoc, José Luis Vilas-Vilelaa,b a
Macromolecular Chemistry Group (LABQUIMAC), Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country, UPV/EHU, Barrio Sarriena, s/n, 48940 Leioa, Spain b BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain c Materials + Technologieś Group, Department of Chemical and Environmental Engineering, Engineering College of Gipuzkoa, University of the Basque Country (UPV/ EHU), Plaza Europa 1, 20018 Donostia–San Sebastián, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Self-healing Polyelectrolyte complexes Multilayers Hydrogels Hyaluronic acid Chitosan
In this work, polyelectrolyte complexes (PEC) were obtained in form of nanometric polyelectrolyte multilayers (PEMs) and macroscopic hydrogels by electrostatic interactions between two natural polysaccharides: hyaluronic acid (HA) and chitosan (CHI). PEM was developed onto poly(ethylene terephthalate) (PET) surface by layer-by-layer approach. The characterization of both systems was carried out simultaneously by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X ray diffraction (XRD), thermogravimetric analysis (TGA) and transmission and scanning electronic microscopy (TEM and SEM). The rheological properties of PEC hydrogels were also analysed. The formation of the complexes was demonstrated due to electrostatic interactions, which in turn resulted being responsible for intrinsic self-healing ability. This is a highly demanded property in reducing replacements costs. Both, PEC hydrogels and multilayers showed selfhealing properties within few minutes, this fact proves the versatility of HA/CHI complexes to easily obtain different forms of self-healable materials interesting as functional biomedical supports and coatings.
1. Introduction Self-healing is the ability showed by some materials, which are able to repair themselves after being damaged by restoring broken bonds or interactions [1]. When an implantable and biodegradable material is designed, self-healing is an essential property since materials suffer from mechanical stress derived from movement, cell growth or proliferation, which could damage and even degrade materials before completing its objective. Thus, self-healing property improves biomaterial’s safety, reduces replacement costs and increases its lifetime, as well as, allows recovering their original shape [2,3]. Materials showing this ability can be classified in extrinsic or intrinsic. The extrinsic implies the release of a self-healing agent embedded in the polymeric matrix by crack propagation; the intrinsic, repairs itself owing to the reversibility of chemical bonds or non-covalent bonds, such as, hydrogen bonds, electrostatic interactions and metal-ligand coordination. Lately, especial attention in intrinsic self-healing materials has been paid, owing to the fact that they do not require the use of healing-
agents, which can cause side interaction and biocompatibility problems associated [4]. Autonomous self-healing materials repair themselves by a two-step mechanism, which is independent of the interactions between damaged parts. Firstly, interdiffusion of polymer chains between injured zones occurs; subsequently, bonds are restored. Diffusion rate, which depends on the temperature and the length of the free chain, has to be considered when designing self-healing materials. In this way, augmenting chain length will produce a greater interpenetration; just as increasing temperature will enhance diffusion [5,6]. Regarding to non-covalent bonds, systems based on hydrogen bonds and electrostatic interactions are the most widely studied. Few works, which based their healability only on hydrogen bonds are found. This is due to the fact that water can also form these interactions with polymer chains weakening polymer-polymer interactions and subsequently, the healing process. Even so, it can be found self-healing of polyvinyl acetate (PVA) hydrogels by hydrogen bonds reported by Zhang et al. [7]. On the other hand, ionic bonds formed between oppositely charged
⁎ Corresponding author at: Macromolecular Chemistry Group (LABQUIMAC), Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country, UPV/EHU, Barrio Sarriena, s/n, 48940 Leioa, Spain. E-mail address: [email protected] (L. Pérez-Álvarez).
https://doi.org/10.1016/j.eurpolymj.2019.109268 Received 17 April 2019; Received in revised form 31 July 2019; Accepted 23 September 2019 Available online 24 September 2019 0014-3057/ © 2019 Published by Elsevier Ltd.
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[25]. PEMs could be constructed by layer-by-layer (LbL) method, developed by Decher et al. [26] in 1992, which allows the control of layers thickness, molecular architecture and surface chemistry. The use of this technique is widely spread nowadays due to the simplicity to adsorb different layers on materials surface as well as being versatile and cost-effective [25]. Within all the types of substrates that can be used for PEMs construction, polymers have attracted great interest for being ideal candidates to develop biomaterials as they show great versatility comparing to other materials, such as metals or ceramics. Among them, a commercial low-cost polymer with great mechanical properties and biocompatibility is found: poly(ethylene terephthalate) (PET) [27], which can be used in catheters, heart-valves and implants [18]. PET surface modification allows to change its hydrophobic nature, providing the surface with good wettability, lubrication and biocompatibility [28]. Tough many attempts of surface modifications have been reported for PET with the aim of improving its features, nowadays layer-by-layer approach is the most widely used procedure. In the last decades, polyelectrolyte multilayers based on interactions between hyaluronic acid and chitosan has been widely studied on different substrates like titanium [21] or polydimethylsiloxane [29]. Nevertheless, few attempts have been made onto PET using layer-by-layer approach [28,30], which will provide PET with the above mentioned characteristics, as well as with an additional advantage: self-healing. Surface self-healing ability has been studied after modifying the surface of different materials. For example, Zu et al. [31] constructed chitosan and polyacrylic acid layers onto a glass substrate using different pH values for the chitosan solution in order to study the influence of the pH in the self-healing. Wang et al. constructed different layers of poly(ethylenimine) and polyacrilic acid onto a silicon substrate and [4] showed that self-healing depends on the pH of polyelectrolyte solutions, thickness of the layers and the width of the cuts made to the substrate. This work aims to synthesize and characterize polyelectrolyte complex hydrogels based on the interaction of HA/CHI polysaccharides to develop a self-healing material suitable for medical applications. Although few works have studied the complexation between these biopolymers [23,32], to the best of our knowledge, self-healing ability has not been studied yet. Furthermore, HA/CHI complexation was also carried out onto PET surface by the built-up of a multilayered system with a well known biocompatibility and antibacterial properties. Additionally, surface self-healing provided by HA/CHI multilayers was also studied, to design a long-lasting and secure biomaterial.
ions, also showed repairing process; for example iron(III)/poly(acrylic acid) (Fe3+/PAA) hydrogel, which heals due to the diffusion of Fe3+ and the following interaction with carboxylic groups of PAA [8,9] Concerning dynamic covalent bonds, many types are found, to mention imine bonds. These are also known as Schiff base and are formed between aldehyde and amine groups. Yang et al. [10] reported healability resulting from the association between benzaldehyde groups of difunctional poly(ethylene glycol) and amine groups of glycol chitosan. Interpolymer complexes are formed as a result of polymers association, which can be driven by an specific interaction between polymer chains, such as hydrogen bond or electrostatic interactions [11]. The latter are known as polyelectrolyte complexes (PECs) and could give rise to self-healing materials due to the dynamic nature of electrostatic interactions between polyions. In order to form polyelectrolyte complexes, compounds must present functional groups capable of interacting with each other by means of electrostatic interactions. Their formation depends on many factors, such as, molecular weight and concentration of polyelectrolytes and mixing ratio [12]. Moreover, it is also pH dependent, due to the nature of the polyions; strong polyions dissociate in all pH values, whereas, weak ones only dissociate in specific range of pH [13]. Among their applications, it is worth mentioning the use of most PECs obtained from natural polymers as biomaterials, such as wound-dressing, drug delivery or bioadhesives [14]. Biopolymers, rather than synthetic polymers, are a suitable option to develop self-healing polycomplexes because they provide materials with biocompatibility and non-toxicity. Many combinations have been reported, such as alginate-chitosan complexes for drug or gene delivery [15], chitosan and γ-poly(glutamic acid) for wound healing [14] or chitosan-xanthan for controlled delivery of encapsulated products [16]. In this work, hyaluronic acid (HA) and chitosan (CHI) based complexes were studied, since their combination has been proven to enhance antimicrobial ability [14]. Hyaluronic acid (HA), a linear glycosaminoglycan with high molecular weight, is composed of repeating disaccharide molecules of β-(1,4)-D-glucuronic acid and β-(1,3)-N-acetilD-glucosamine. It is a weak polyanion with pKa = 2.9, biocompatible, viscoelastic and non-toxic; it also shows a high hydrophilicity, lubricant and moisturising nature which helps in enhancing biocompatibility and avoiding biofilm formation [17]. In fact, some research has shown the ability of hydrophilic surfaces to reduce biofilm formation due to hydrophobic nature of bacteria [18]. Chitosan (CHI) is a natural and linear polysaccharide, formed by two randomly distributed units, Nacetyl-2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose. It derives from the partial deacetylation of chitin, which is founded in crustaceans’ exoskeletons. CHI is a weak polycation with pKa = 6.5, biocompatible, biodegradable and non-toxic; its cationic nature causes the disruption of negatively charged cell membranes of bacteria, showing great antibacterial properties [19,20]. Although electrostatic interactions between HA and CHI has been well-studied [21,22], few works have reported polyelectrolyte complexes based on this system. Among them, Lee et al. [23] synthetized hyaluronic acid and chitosan sponges in different pH in order to prepare a wound healing material. On the other hand, Ma et al. [24] prepared HA-CHI nanofibers as a suitable material for tissue engineering. Although systems based on these natural polymers have been prepared in the last years with the aim of developing suitable materials for medical applications, to the best of our knowledge, self-healing ability of HA/ CHI polyelectrolyte complexes has not been studied yet. These electrostatic interactions between HA and CHI have also been reviewed to develop biodegradable coatings that provide synthetic materials with enhanced biocompatibility and valuable properties, such as, wound dressing or antibacterials. For that, polyelectrolyte multilayers (PEMs) could be constructed, formed by the alternate adsorption of polycations and polyanions on a substrate surface. As a result, a very stable coating can be formed due to the crosslinking of the polyelectrolytes which can be controlled changing pH values; so, according to pH values, polyelectrolyte charges change and so do the crosslinking
2. Experimental part 2.1. Materials and chemicals PET films (75 µm) were obtained from HIFI industry. Hyaluronic acid (low and high molecular weight: 0.7–1.2 MDa and 1.9–2.2 MDa, respectively) was obtained from Contipro. Methanol (99.5%), ethylendiamine (99%) and hydrochloride acid (37%) were purchased from Panreac. Sodium hydroxide (99%), acetic acid (> 99%) and chitosan (medium molecular weight, 190–310 KDa and highly viscous, > 400 mPa·s 20 °C in 1% acetic acid) were purchased from Sigma Aldrich and potassium phosphate dibasic (99%) from Acros Organics. 2.2. Synthesis of HA-CHI complexes 1 wt% solutions of high molecular weight hyaluronic acid (HMWHA) and highly viscous (Hvisc) CHI were prepared separately in 1% (v/v) acetic acid solution. The solutions were mixed at 1:1 wt ratios and stirred. The precipitates formed by mixing both polymer solutions were left 2 h at room temperature and then the remaining solution was separated from the complex. 2
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2.5.6. Scanning electron microscopy (SEM) The surface of lyophilized polyelectrolyte complex hydrogels were coated with a thin gold overlay and analysed with a Hitachi S-4800 microscope (150 s, 20 mA, 15 kV, zoom at ×30,000), in order to determine pore size.
2.3. Construction of HA-CHI multilayers PET films were cut into 4 × 2 cm2 sections and subsequently, washed in ethanol and water in an ultrasonic bath and then, they were aminolyzed following a reported procedure [28]. Dried films were immersed in ethylendiamine/methanol 40:60 (v/v) at 50 °C for 1.5 h and activated in a 0.1 M solution of HCl for 3 h, at room temperature. Amiloyzed films were dipped in 1 g/L of low molecular weight hyaluronic acid (LMWHA) solution for 15 min, followed by an immersion in acetic buffer. Having adsorbed the first HA layer, films were dipped in 1 g/L medium molecular weight chitosan (MMWCHI) for 15 min followed by buffer rinsing. This process was carried out in order to obtain different numbers of layers (40, 100, and 200).
2.5.7. Rheological measurements Frequency sweep measurements of HA/CHI complex were performed in a Haake Viscotester IQ (Thermo Fisher Scientific), using parallel plate geometry (titanium upper plate of 35 mm and steel lower plate of 60 mm) with a gap distance of 1 mm at 25 °C. The viscometer was equipped with a Peltier system for the temperature control and a solvent trap. The storage (G′) and loss (G″) modulus values were recorded from 0.62 to 62 rad/s at a fixed strain of 0.1% previously assessed strain sweep tests. The dynamic rheological properties of three replicates were studied. Moreover, the self-healing capacity of the complex was also analysed by conducting the frequency sweep measurements after submitting the complexes to several cut-recovery cycles at 25 °C. Both moduli values were measured in triplicate.
2.4. Self-healing Self-healing process was studied in polyelectrolyte complexes (PECs) and polyelectrolyte multilayers (PEMs). Due to the fact that this ability is based on electrostatic interactions between polyelectrolytes, pH value was adjusted at 5, in order to form ions and consequently, favour interactions between ions. The study was done following the next procedure: PECs were split in two, pieces were held together and deionized water was added in order to see the healing process. On the other hand, in order to analyse self-healing of multilayers a scratch was done on coated PET films and after water immersion, healing process was observed using Leica DM2500 M optical microscope, with an augmentation of 5×.
2.5.8. Mechanical tests Unconfined compression tests were carried out by using an instrument (Metrotec FTM-50) equipped with a 500 N load cell, at room temperature. Hydrogels were measured for size, and then a progressive compression at a rate of 10 mm/min was performed until breaking. Hydrogels were subjected to five cycles of self-healing. Compression modules were calculated from the slope of the linear portion (50–75% strain range) of the stress–strain plot.
2.5. Characterization methods
3. Results
2.5.1. Fourier transform infrared spectroscopy (FT-IR) Infrared spectra of pure components (HA and CHI), polyelectrolyte complex and multilayers were recorded using Nicolet Nexus FT-IR spectrometer (Thermo Scientific, Loughborough, UK) in KBr pellets, at a resolution of 4 cm−1 and 32 scans per spectrum.
3.1. Synthesis and characterization of HA/CHI polyelectrolyte complexes and multilayers In this work, polyelectrolyte complexes were synthesized basing on the electrostatic interactions between hyaluronic acid and chitosan. The use of these polysaccharides allowed the design of biocompatible, biodegradable and non-toxic materials suitable for biomedical applications. In order to favour the electrostatic interactions between the carboxylate (eCOO−) groups of hyaluronic acid and the amine (eNH3+) groups of chitosan, the pH value was adjusted at 5 in which the ionization of both polyelectrolytes is maximum. The electrostatic interactions between the two biopolymers allow the formation of the complex on the one hand, but also the self-healing ability, due to the reversibility of these interactions. In Fig. 1 TEM and SEM images of the polyeletrolyte multilayers and complex hydrogels, respectively, are shown. Although both systems are formed by hyaluronic acid and chitosan, there is a great difference between their morphology as a consequence of the synthetic procedure, even though both are guided by the same interactions. PEMs are materials formed by the assembly of polysaccharides with nanometric width, while the complexes are macroscopic networks with micrometric pore size (164 ± 14 μm). The difference in scale is a key factor in their swelling behaviour and therefore, in the healability of the materials. Fig. 2 shows the FT-IR spectra of pure chitosan and hyaluronic acid, as well as their polyelectrolyte complex hydrogels and multilayers. The characteristic peaks of chitosan appear at 1654 cm−1 and 1596 cm−1 corresponding to amide I and amide II bands, respectively. It also shows the twisting of eCH2 group at 1419 cm−1 and CeN stretching at 1321 cm−1. The amide I and amide II bands of hyaluronic acid are observed at 1670 cm−1 and 1554 cm−1 respectively, the asymmetric and symmetric stretching of carboxylate group appears at 1625 cm−1 and 1414 cm−1, as well as CeN stretching at 1322 cm−1. Regarding to FTIR spectra of complex and multilayers, the following bands were identified: a wide band was displayed in both HA/CHI systems,
2.5.2. Differential scanning calorimetry (DSC) DSC was used to determine the glass transition temperature (Tg) of polyeletrolyte complexes and multilayers. Mettler Toledo Differential Scanning Calorimeter, DSC 822e calorimeter (Gießen, Germany), was used and samples were heated from 25 °C to 200 °C at a heating rate of 10 °C/min, under constant purging of nitrogen (20 mL/min). Each measurement was done three times and Tg was taken as the extrapolated onset of the baseline shift. 2.5.3. X ray diffraction (XRD) The X-ray powder diffraction patterns were collected by using a Malvern Panalytical (Almelo, Netherlands) X’PERT PRO automatic diffractometer operating at 40 kV and 40 mA, in theta-theta configuration, secondary monochromator with Cu-Kα radiation (λ = 1.5418 Å) and a PIXcel solid state detector (active length in 2θ 3.347°). Data were collected from 5 to 70° 2θ (step size = 0.02626 and time per step = 200 s) at RT. 2.5.4. Thermogravimetric analysis (TGA) TGA was used to determine the swelling behaviour of polyelectrolyte complexes and PET films coated with different number of HA/ CHI layers (40, 100 and 200) after hydrating samples in PBS solution. Measurements were carried out in Mettler Toledo TGA/SDTA 851e and samples were heated from 25 °C to 600 °C at a heating rate of 10 °C/min under 20 mL/min nitrogen flux. 2.5.5. Transmission electron microscopy (TEM) Polyelectrolyte multilayers thickness was examined with a Philips CM120 TEM instrument operating at 120 kW. The images were obtained with a magnification of ×56,000. 3
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Fig. 1. (a) TEM image of polyelectrolyte multilayers (20 HA/CHI bilayers) (b) SEM images of polycomplex hydrogel and pore distribution.
Fig. 3. Glass transition temperature of the samples: high and low molecular weight hyaluronic acid (HMW HA and LMW HA), high viscosity and medium molecular weight chitosan (HVisc CHI and MMW CHI), polyelectrolyte complex (PEC) hydrogel and polyelectrolyte multilayer (PEM).
polysaccharides with different molecular weight or viscosities were studied. High molecular weight hyaluronic acid (HMWHA) shows its transition at 134 °C while low molecular weight (LMWHA) shows at 120 °C. Similar observations were done for chitosan; highly viscous chitosan (HVCHI) shows the transition at 127 °C and medium molecular weight (MMWCHI) shows at 118 °C. The glass transition temperature of polymeric materials is highly dependent on the structure of the polymer; if polymer chains have a reduced mobility derived from a higher molecular weight, the transition would required more energy and therefore, the glass transition temperature will increase. Finally, the Tg of the multilayers and PEC hydrogels were studied. In both cases, a single Tg with a value lower than those of the constituting polymers was observed, at 113.35 °C in the hydrogels and 111 °C in the multilayers. The appearance of a unique glass transition temperature corroborated that complexation between hyaluronic acid and chitosan took place. XRD patterns of the samples are presented in Fig. 4. Chitosan showed two peaks at 10° and 20°, corresponding to hydrated and anhydrous crystals, respectively [36]. These peaks suggest an ordered structure formed as a consequence of the hydrogen bonds between amine and hydroxyls groups and that is responsible for hindering chains movement. In the case of hyaluronic acid, two peaks at 10° and 20° are also shown. After complexing, these peaks disappear forming an
Fig. 2. FTIR spectra of the samples: chitosan (CHI), hyaluronic acid (HA), polyelectrolyte complex hydrogel and polyelectrolyte multilayer (PEM).
1670–1598 cm−1 for the complex and 1630–1590 cm−1 for the multilayers, corresponding to the above described overlapping of amide I bands from chitosan and hyaluronic acid. The slight shifting of amide bands could be ascribed to the interactions between polysaccharides, confirming complexation [33]. Furthermore, two other bands were identified in each spectra, 1412 cm−1 and 1317 cm−1 for PEC hydrogels and 1410 cm−1 and 1322 cm−1 for PEMs, which coincide with the symmetric stretching of hyaluronic acid carboxylate and chitosan CeN, respectively [34,35]. The described overlapping of CHI and HA bands suggests the contribution of both polysaccharides in HA/CHI systems. If we compare the grade of overlapping of spectra of both systems, we could conclude that the interpenetration of polysaccharides is greater in PEMs that can be related to the higher interpenetration on the layer-bylayer deposition, which allows a better assembly of the components than in the macroscopic complexes formation. On the other hand, DSC was used to determine the glass-transition temperature (Tg) of the samples, shown in Fig. 3. Firstly, 4
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Fig. 4. XRD of pure CHI and HA and after their interaction forming hydrogels and PEMs.
Fig. 6. Storage modulus (G′, filled symbol) and loss modulus (G″, empty symbol) of the HA/CHI complex in a frequency scan.
amorphous structure in hydrogels and PEMs. The latest show peaks, which do not appear in PEC hydrogels; they correspond to the diffraction pattern of sodium salts from the PBS solution used in the detachment of polysaccharide layers from PET for characterization. Overall, CHI and HA undergo a loss of order when complexing, which may be the cause of the higher glass transition temperature of pristine polysaccharides in comparison with the single and lower glass transition temperatures measured for the corresponding hydrogels and PEMs. Due to the nanometric nature of HA/CHI PEMs, their capacity to absorb water was determined by means of thermogravimetry. The swelling behaviour of these polyelectrolyte multilayers is a crucial factor to the self-healing ability of studied materials, thus, the swelling behaviour of samples with different layers at different time of immersion in a PBS solution (pH = 7) was determined. All samples showed two main loss of weight; the first one is given around 100 °C, which corresponds to water loss of the samples. The other change of slope of TGA curves was displayed at 400 °C and corresponds to the degradation of the substrate PET. Quantification of water loss could be done determining the weight loss around 100 °C in each sample at different immersion times. As can be seen in Fig. 5, water content of PEMs increased with immersing time, up to a point where the maximum absorption was reached. In addition, the more layers deposited on substrate surface the greater water absorption was observed, due to the increasing amount of polysaccharide
on the substrate surface. It is worth to mention that LbL methodology leads to heterogeneously constructed multilayers, in which a mayor content of polysaccharides was accumulated in specific areas of PET films. This justifies the high standard deviations (0.6–11%) obtained in the swelling measurement of the samples despite the high accuracy of TGA technique. On the other hand, swelling behaviour of polyelectrolyte complexes could be studied by gravimetry immersing dried samples in PBS solution. The swelling degree was 24 ± 3 times higher than in multilayered system. As mentioned before, PEMs showed a nanometric width and higher interpenetration of the polymeric chains, while hydrogels are macroscopic networks with a micrometric pore size, which provides the complex with a higher capacity to absorb water. Rheological properties of the HA/CHI hydrogels were studied by frequency sweep test at 25 °C. As it could be appreciated in Fig. 6, the G′ value was maintained constant (mean value 75.4 ± 36.8 Pa) during the test and independent of the applied frequency, and always higher than the loss modulus. G′ represents the elastic part of the hydrogel, whereas G″ corresponds to the viscous part. The observed behaviour indicated that the hydrogel responded as elastic solid in the studied frequency range.
3.2. Self-healing Healing mechanism of HA/CHI PEC hydrogels and multilayers should happen due to the association-dissociation process between ionized carboxylate groups of hyaluronic acid and amine groups of chitosan. In order to study healing capacity on PEC hydrogels, the following procedure was carried out: after cutting the samples, water was added gently so as to facilitate the healing. Then, the two pieces were kept together and after 2 min polycomplexes were healed. The addition of water favoured the healing because it enhanced polymeric chains movement and therefore, the regeneration of the physical interactions between carboxylates of hyaluronic acid and protonated amines of chitosan chains. The healing process of polyelectrolyte complexes can be seen in Fig. 7. Above observed macroscopically regeneration process was also studied on a coated form on PET surface with different amount of HA/ CHI layers, via microscope. The healing process shown in Fig. 8, was carried out in a similar way as the above described; scratched films were dipped in water and within 2 min all the samples were healed. In this case, the self-healing ability was observed regardless the number of deposited polysaccharide bilayers (40, 100 and 200 HA/CHI).
Fig. 5. (a) Swelling behaviour and (b) TGA curves of HA/CHI polyelectrolyte multilayers onto PET determined by TGA. 5
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Fig. 7. (a) Self-healing process of polyelectrolyte complex (b) polyelectrolyte complex after healing.
Nevertheless, Wang et al. [4] deposited 30 and 300 poly(ethylenimine)/poly(acrylic acid) layers on a substrate, and demonstrated that the higher the layers number the lower the healing ability, that was ascribed to the fact that polyelectrolytes content hinders chain mobility and thus, the healing. Therefore, it could be concluded that the healing process happened in a similar way in the nanometric multilayered and in the macroscopic hydrogel form by the reversibility of the electrostatic interactions between employed polyanion and polycation. Furthermore, rheological oscillatory analyses were also carried out in order to investigate the self-healing process of the HA/CHI complex hydrogels. With that purpose, samples were cut into two halves as mentioned previously (Fig. 7). In this case, the process was repeated for two consecutive cycles and the rheological measurements were conducted after each one. As illustrated in Fig. 9, G′ was higher than G″ after every cut-recovery cycle (Table 1) thus, demonstrating the elastic property of the complex. The obtained data demonstrated a quick recovery of the viscoelastic properties of the broken complexes due to the physical interactions based on the formation of a polyanion-polycation bond between the oppositely charged polymers. The similarity between the values of mean storage and loss modulus of HA/CHI original and self-healable complexes indicated the formation of a new material with adequate self-healing capacity, which is in accordance with reported literature [37].
Fig. 9. Frequency sweep measurement (storage modulus (filled symbol) and loss modulus (empty symbol)) at a constant strain (0.1%) for ■ original HA/ CHI complex, ▲ HA/CHI complex after one cut-recovery cycle and ▾ HA/CHI complex after two cut-recovery cycles.
Results of unconfined compression testing on HA-CHI fresh hydrogel (0) and self-healed hydrogel (1–5) are shown in Fig. 10. The compression modulus increased with the deformation, reflecting a
Fig. 8. Self-healing process of polyelectrolyte multilayers: (a–c) 40 HA/CHI, (d–f) 100 HA/CHI (g–i) 200 HA/CHI. 6
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Grant PIF15/092. Authors thank for technical and human support provided by SGIker of UPV/EHU and European funding (ERDF and ESF).
Table 1 Mean storage and loss modulus of HA/CHI complex at 25 °C (average ± standard deviation, n = 3). HA/CHI complex hydrogels
G′ (Pa)
G″ (Pa)
Original After one cut-recovery cycle After two cut-recovery cycle
75.4 ± 36.8 74.0 ± 14.0 133.4 ± 50.3
40.1 ± 23.3 34.6 ± 10.3 55.1 ± 15.4
Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.109268. References [1] M.D. Hager, P. Greil, C. Leyens, S. Van Der Zwaag, U.S. Schubert, Self-healing materials, Adv. Mater. 22 (2010) 5424–5430, https://doi.org/10.1002/adma. 201003036. [2] A.B. South, L.A. Lyon, Autonomic self-healing of hydrogel thin films, Angew. Chemie – Int. Ed. 49 (2010) 767–771, https://doi.org/10.1002/anie.200906040. [3] L. Li, B. Yan, J. Yang, L. Chen, H. Zeng, Novel mussel-inspired injectable selfhealing hydrogel with anti-biofouling property, Adv. Mater. (2015) 1294–1299, https://doi.org/10.1002/adma.201405166. [4] X. Wang, F. Liu, X. Zheng, J. Sun, Water-enabled self-healing of polyelectrolyte multilayer coatings, Angew. Chemie - Int. Ed. 50 (2011) 11378–11381, https://doi. org/10.1002/anie.201105822. [5] F. Herbst, D. Döhler, P. Michael, W.H. Binder, Self-healing polymers via supramolecular forces, Macromol. Rapid Commun. 34 (2013) 203–220, https://doi.org/10. 1002/marc.201200675. [6] B. Gyarmati, B.Á. Szilágyi, A. Szilágyi, Reversible interactions in self-healing and shape memory hydrogels, Eur. Polym. J. 93 (2017) 642–669, https://doi.org/10. 1016/j.eurpolymj.2017.05.020. [7] H. Zhang, H. Xia, Y. Zhao, Poly(vinyl alcohol) hydrogel can autonomously self-heal, ACS Macro Lett. 1 (2012) 1233–1236, https://doi.org/10.1021/mz300451r. [8] Y. Guo, X. Zhou, Q. Tang, H. Bao, G. Wang, P. Saha, A self-healable and easily recyclable supramolecular hydrogel electrolyte for flexible supercapacitors, J. Mater. Chem. A. 4 (2016) 8769–8776, https://doi.org/10.1039/c6ta01441k. [9] Z. Wei, J. He, T. Liang, H. Oh, J. Athas, Z. Tong, C. Wang, Z. Nie, Autonomous selfhealing of poly(acrylic acid) hydrogels induced by the migration of ferric ions, Polym. Chem. 4 (2013) 4601–4605, https://doi.org/10.1039/c3py00692a. [10] B. Yang, Y. Zhang, X. Zhang, L. Tao, S. Li, Y. Wei, Facilely prepared inexpensive and biocompatible self-healing hydrogel: a new injectable cell therapy carrier, Polym. Chem. 3 (2012) 3235–3238, https://doi.org/10.1039/c2py20627g. [11] E. Tsuchida, K. Abe, Interactions Between Macromolecules in Solution and Intermacromolecular Complexes, Adv. Polym. Sci. Springer, Berlin, Heidelberg, 1982, , https://doi.org/10.1007/BFB0017548. [12] A. Shovsky, I. Varga, R. Makuška, P.M. Claesson, Formation and stability of watersoluble, molecular polyelectrolyte complexes: effects of charge density, mixing ratio, and polyelectrolyte concentration, Langmuir 25 (2009) 6113–6121, https:// doi.org/10.1021/la804189w. [13] S.J. Kim, S.G. Yoon, K.B. Lee, Y.D. Park, S.I. Kim, Electrical sensitive behavior of a polyelectrolyte complex composed of chitosan/hyaluronic acid, Solid State Ionics 164 (2003) 199–204, https://doi.org/10.1016/j.ssi.2003.08.005. [14] M. Buriuli, D. Verma, Advances in biomaterials for biomedical applications, 2017. < https://doi.org/10.1007/978-981-10-3328-5 > . [15] H.V. Sæther, H.K. Holme, G. Maurstad, O. Smidsrød, B.T. Stokke, Polyelectrolyte complex formation using alginate and chitosan, Carbohydr. Polym. 74 (2008) 813–821, https://doi.org/10.1016/j.carbpol.2008.04.048. [16] H. Chen, Y. Song, N. Liu, H. Wan, G. Shu, N. Liao, Effect of complexation conditions on microcapsulation of Lactobacillus acidophilus in xanthan-chitosan polyelectrolyte complex gels, Acta Sci. Pol. Technol. Aliment. 14 (2015) 207–213, https:// doi.org/10.17306/J.AFS.2015.3.22. [17] C.L. Romanò, E. De Vecchi, M. Bortolin, I. Morelli, L. Drago, Hyaluronic acid and its composites as a local antimicrobial/antiadhesive barrier, J. Bone Jt. Infect. 2 (2017) 63–72, https://doi.org/10.7150/jbji.17705. [18] L. Pérez-Álvarez, E. Lizundia, S. del Hoyo, A. Sagasti, L.R. Rubio, J.L. Vilas, Polysaccharide polyelectrolyte multilayer coating on poly(ethylene terephthalate), Polym. Int. 65 (2016) 915–920, https://doi.org/10.1002/pi.5116. [19] R. Logithkumar, A. Keshavnarayan, S. Dhivya, A. Chawla, S. Saravanan, N. Selvamurugan, A review of chitosan and its derivatives in bone tissue engineering, Carbohydr. Polym. 151 (2016) 172–188, https://doi.org/10.1016/j. carbpol.2016.05.049. [20] R.C.F. Cheung, T.B. Ng, J.H. Wong, W.Y. Chan, Chitosan: an update on potential biomedical and pharmaceutical applications, Mar. Drugs. 13 (2015) 5156–5186, https://doi.org/10.3390/md13085156. [21] P.H. Chua, K.G. Neoh, E.T. Kang, W. Wang, Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion, Biomaterials 29 (2008) 1412–1421, https://doi.org/10.1016/j.biomaterials.2007.12.019. [22] K. Mulligan, Z.J. Jakubek, L.J. Johnston, Supported lipid bilayers on biocompatible polysaccharide multilayers, Langmuir 27 (2011) 14352–14359, https://doi.org/10.
Fig. 10. Stress–strain representation from compression testing of fresh hydrogel (0) and hydrogel after successive (1–5) self-healing cycles and calculated compression modules (at 50–75% deformation).
typical non-linear behaviour of hydrogels even after 5 cycles of selfhealing. In addition, similar mechanical properties were showed after self-healing processes, regardless the number of the cycles (1–5) and there was no significant difference, neither in modulus nor in the breaking-up. Obtained data showed that fresh gels exhibited a uniformly linear strain–stress relationship from 0% to 60% strain, while healed hydrogels from 0 to 20%. These last ones were approximately 9 times stiffer than initial hydrogels and broken up at 60% lower strain. This fact can be ascribed to the loss of water during compression testing in self-healing cycling. 4. Conclusions Hyaluronic acid and chitosan based systems were successfully developed by electrostatic interactions, in order to form polyelectrolyte complexes. The assembly between the polysaccharides was held macroscopically in form of hydrogels, but also as a multilayered coating onto PET surface. This last one led to a nanometric thickness that resulted in a 24 times lower swelling capacity but an enhanced polymeric interpenetrability in comparison with bulk hydrogels. FTIR and DSC demonstrated the interactions and the consequent formation of polyelectrolyte complexes on multilayers and hydrogels. In addition, selfhealing ability of both systems was proved due to the association-dissociation process of ionic bonds between employed biopolymers. Overall, biocompatible, biodegradable and cost-effective materials had been developed for potential biomedical applications. Acknowledgments This work was funded by Ministerio de Economía Industria y Competitividad (grant MAT2017-89553-P). Financial support from the Basque Country Government in the frame of Grupos Consolidados (IT–776–13) is gratefully acknowledged. O. Guaresti wishes to acknowledge the University of the Basque Country (UPV/EHU) for its PhD 7
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1021/la203207p. [23] S.B. Lee, Y.M. Lee, K.W. Song, M.H. Park, Preparation and properties of polyelectrolyte complex sponges composed of hyaluronic acid and chitosan and their biological behaviors, J. Appl. Polym. Sci. 90 (2003) 925–932, https://doi.org/10. 1002/app.12668. [24] G. Ma, Y. Liu, D. Fang, J. Chen, C. Peng, X. Fei, J. Nie, Hyaluronic acid/chitosan polyelectrolyte complexes nanofibers prepared by electrospinning, Mater. Lett. 74 (2012) 78–80, https://doi.org/10.1016/j.matlet.2012.01.012. [25] J.A. Lichter, K.J. Van Vlietpa, M.F. Rubner, Design of antibacterial surfaces and interfaces: Polyelectrolyte multilayers as a multifunctional platform, Macromolecules 42 (2009) 8573–8586, https://doi.org/10.1021/ma901356s. [26] G. Decher, J.D. Hong, J. Schmitt, Buildup of ultrathin multilayer films by a selfassembly process: III. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces, Thin Solid Films 210–211 (1992) 831–835, https://doi.org/10.1016/0040-6090(92)90417-A. [27] M.G. Carneiro-da-Cunha, M.A. Cerqueira, B.W.S. Souza, S. Carvalho, M.A.C. Quintas, J.A. Teixeira, A.A. Vicente, Physical and thermal properties of a chitosan/alginate nanolayered PET film, Carbohydr. Polym. 82 (2010) 153–159, https://doi.org/10.1016/j.carbpol.2010.04.043. [28] S. Del, H.-G. Leyre, P. Alvarez, F. Gómez-Galván, E. Lizundia, I. Kuritka, V. Sedlarik, J.M. Laza, J.L. Vila-Vilela, Construction of antibacterial poly(ethylene terephthalate) films via layer by layer assembly of Chitosan and hyaluronic acid, Carbohydr. Polym. 143 (2016) 35–43, https://doi.org/10.1016/j.carbpol.2016.02. 008. [29] J.H.H. Bongaerts, J.J. Cooper-White, J.R. Stokes, Low biofouling chitosan-hyaluronic acid multilayers with ultra-low friction coefficients, Biomacromolecules 10 (2009) 1287–1294, https://doi.org/10.1021/bm801079a. [30] H. Li, Y. Ge, P. Zhang, L. Wu, S. Chen, The effect of layer-by-layer chitosan-
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Self-healable hyaluronic acid/chitosan polyelectrolyte complex hydrogels and multilayers
T
⁎
Nagore Barrosoa, Olatz Guarestic, Leyre Pérez-Álvareza,b, , Leire Ruiz-Rubioa,b, Nagore Gabilondoc, José Luis Vilas-Vilelaa,b a
Macromolecular Chemistry Group (LABQUIMAC), Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country, UPV/EHU, Barrio Sarriena, s/n, 48940 Leioa, Spain b BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain c Materials + Technologieś Group, Department of Chemical and Environmental Engineering, Engineering College of Gipuzkoa, University of the Basque Country (UPV/ EHU), Plaza Europa 1, 20018 Donostia–San Sebastián, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Self-healing Polyelectrolyte complexes Multilayers Hydrogels Hyaluronic acid Chitosan
In this work, polyelectrolyte complexes (PEC) were obtained in form of nanometric polyelectrolyte multilayers (PEMs) and macroscopic hydrogels by electrostatic interactions between two natural polysaccharides: hyaluronic acid (HA) and chitosan (CHI). PEM was developed onto poly(ethylene terephthalate) (PET) surface by layer-by-layer approach. The characterization of both systems was carried out simultaneously by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X ray diffraction (XRD), thermogravimetric analysis (TGA) and transmission and scanning electronic microscopy (TEM and SEM). The rheological properties of PEC hydrogels were also analysed. The formation of the complexes was demonstrated due to electrostatic interactions, which in turn resulted being responsible for intrinsic self-healing ability. This is a highly demanded property in reducing replacements costs. Both, PEC hydrogels and multilayers showed selfhealing properties within few minutes, this fact proves the versatility of HA/CHI complexes to easily obtain different forms of self-healable materials interesting as functional biomedical supports and coatings.
1. Introduction Self-healing is the ability showed by some materials, which are able to repair themselves after being damaged by restoring broken bonds or interactions [1]. When an implantable and biodegradable material is designed, self-healing is an essential property since materials suffer from mechanical stress derived from movement, cell growth or proliferation, which could damage and even degrade materials before completing its objective. Thus, self-healing property improves biomaterial’s safety, reduces replacement costs and increases its lifetime, as well as, allows recovering their original shape [2,3]. Materials showing this ability can be classified in extrinsic or intrinsic. The extrinsic implies the release of a self-healing agent embedded in the polymeric matrix by crack propagation; the intrinsic, repairs itself owing to the reversibility of chemical bonds or non-covalent bonds, such as, hydrogen bonds, electrostatic interactions and metal-ligand coordination. Lately, especial attention in intrinsic self-healing materials has been paid, owing to the fact that they do not require the use of healing-
agents, which can cause side interaction and biocompatibility problems associated [4]. Autonomous self-healing materials repair themselves by a two-step mechanism, which is independent of the interactions between damaged parts. Firstly, interdiffusion of polymer chains between injured zones occurs; subsequently, bonds are restored. Diffusion rate, which depends on the temperature and the length of the free chain, has to be considered when designing self-healing materials. In this way, augmenting chain length will produce a greater interpenetration; just as increasing temperature will enhance diffusion [5,6]. Regarding to non-covalent bonds, systems based on hydrogen bonds and electrostatic interactions are the most widely studied. Few works, which based their healability only on hydrogen bonds are found. This is due to the fact that water can also form these interactions with polymer chains weakening polymer-polymer interactions and subsequently, the healing process. Even so, it can be found self-healing of polyvinyl acetate (PVA) hydrogels by hydrogen bonds reported by Zhang et al. [7]. On the other hand, ionic bonds formed between oppositely charged
⁎ Corresponding author at: Macromolecular Chemistry Group (LABQUIMAC), Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country, UPV/EHU, Barrio Sarriena, s/n, 48940 Leioa, Spain. E-mail address: [email protected] (L. Pérez-Álvarez).
https://doi.org/10.1016/j.eurpolymj.2019.109268 Received 17 April 2019; Received in revised form 31 July 2019; Accepted 23 September 2019 Available online 24 September 2019 0014-3057/ © 2019 Published by Elsevier Ltd.
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[25]. PEMs could be constructed by layer-by-layer (LbL) method, developed by Decher et al. [26] in 1992, which allows the control of layers thickness, molecular architecture and surface chemistry. The use of this technique is widely spread nowadays due to the simplicity to adsorb different layers on materials surface as well as being versatile and cost-effective [25]. Within all the types of substrates that can be used for PEMs construction, polymers have attracted great interest for being ideal candidates to develop biomaterials as they show great versatility comparing to other materials, such as metals or ceramics. Among them, a commercial low-cost polymer with great mechanical properties and biocompatibility is found: poly(ethylene terephthalate) (PET) [27], which can be used in catheters, heart-valves and implants [18]. PET surface modification allows to change its hydrophobic nature, providing the surface with good wettability, lubrication and biocompatibility [28]. Tough many attempts of surface modifications have been reported for PET with the aim of improving its features, nowadays layer-by-layer approach is the most widely used procedure. In the last decades, polyelectrolyte multilayers based on interactions between hyaluronic acid and chitosan has been widely studied on different substrates like titanium [21] or polydimethylsiloxane [29]. Nevertheless, few attempts have been made onto PET using layer-by-layer approach [28,30], which will provide PET with the above mentioned characteristics, as well as with an additional advantage: self-healing. Surface self-healing ability has been studied after modifying the surface of different materials. For example, Zu et al. [31] constructed chitosan and polyacrylic acid layers onto a glass substrate using different pH values for the chitosan solution in order to study the influence of the pH in the self-healing. Wang et al. constructed different layers of poly(ethylenimine) and polyacrilic acid onto a silicon substrate and [4] showed that self-healing depends on the pH of polyelectrolyte solutions, thickness of the layers and the width of the cuts made to the substrate. This work aims to synthesize and characterize polyelectrolyte complex hydrogels based on the interaction of HA/CHI polysaccharides to develop a self-healing material suitable for medical applications. Although few works have studied the complexation between these biopolymers [23,32], to the best of our knowledge, self-healing ability has not been studied yet. Furthermore, HA/CHI complexation was also carried out onto PET surface by the built-up of a multilayered system with a well known biocompatibility and antibacterial properties. Additionally, surface self-healing provided by HA/CHI multilayers was also studied, to design a long-lasting and secure biomaterial.
ions, also showed repairing process; for example iron(III)/poly(acrylic acid) (Fe3+/PAA) hydrogel, which heals due to the diffusion of Fe3+ and the following interaction with carboxylic groups of PAA [8,9] Concerning dynamic covalent bonds, many types are found, to mention imine bonds. These are also known as Schiff base and are formed between aldehyde and amine groups. Yang et al. [10] reported healability resulting from the association between benzaldehyde groups of difunctional poly(ethylene glycol) and amine groups of glycol chitosan. Interpolymer complexes are formed as a result of polymers association, which can be driven by an specific interaction between polymer chains, such as hydrogen bond or electrostatic interactions [11]. The latter are known as polyelectrolyte complexes (PECs) and could give rise to self-healing materials due to the dynamic nature of electrostatic interactions between polyions. In order to form polyelectrolyte complexes, compounds must present functional groups capable of interacting with each other by means of electrostatic interactions. Their formation depends on many factors, such as, molecular weight and concentration of polyelectrolytes and mixing ratio [12]. Moreover, it is also pH dependent, due to the nature of the polyions; strong polyions dissociate in all pH values, whereas, weak ones only dissociate in specific range of pH [13]. Among their applications, it is worth mentioning the use of most PECs obtained from natural polymers as biomaterials, such as wound-dressing, drug delivery or bioadhesives [14]. Biopolymers, rather than synthetic polymers, are a suitable option to develop self-healing polycomplexes because they provide materials with biocompatibility and non-toxicity. Many combinations have been reported, such as alginate-chitosan complexes for drug or gene delivery [15], chitosan and γ-poly(glutamic acid) for wound healing [14] or chitosan-xanthan for controlled delivery of encapsulated products [16]. In this work, hyaluronic acid (HA) and chitosan (CHI) based complexes were studied, since their combination has been proven to enhance antimicrobial ability [14]. Hyaluronic acid (HA), a linear glycosaminoglycan with high molecular weight, is composed of repeating disaccharide molecules of β-(1,4)-D-glucuronic acid and β-(1,3)-N-acetilD-glucosamine. It is a weak polyanion with pKa = 2.9, biocompatible, viscoelastic and non-toxic; it also shows a high hydrophilicity, lubricant and moisturising nature which helps in enhancing biocompatibility and avoiding biofilm formation [17]. In fact, some research has shown the ability of hydrophilic surfaces to reduce biofilm formation due to hydrophobic nature of bacteria [18]. Chitosan (CHI) is a natural and linear polysaccharide, formed by two randomly distributed units, Nacetyl-2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose. It derives from the partial deacetylation of chitin, which is founded in crustaceans’ exoskeletons. CHI is a weak polycation with pKa = 6.5, biocompatible, biodegradable and non-toxic; its cationic nature causes the disruption of negatively charged cell membranes of bacteria, showing great antibacterial properties [19,20]. Although electrostatic interactions between HA and CHI has been well-studied [21,22], few works have reported polyelectrolyte complexes based on this system. Among them, Lee et al. [23] synthetized hyaluronic acid and chitosan sponges in different pH in order to prepare a wound healing material. On the other hand, Ma et al. [24] prepared HA-CHI nanofibers as a suitable material for tissue engineering. Although systems based on these natural polymers have been prepared in the last years with the aim of developing suitable materials for medical applications, to the best of our knowledge, self-healing ability of HA/ CHI polyelectrolyte complexes has not been studied yet. These electrostatic interactions between HA and CHI have also been reviewed to develop biodegradable coatings that provide synthetic materials with enhanced biocompatibility and valuable properties, such as, wound dressing or antibacterials. For that, polyelectrolyte multilayers (PEMs) could be constructed, formed by the alternate adsorption of polycations and polyanions on a substrate surface. As a result, a very stable coating can be formed due to the crosslinking of the polyelectrolytes which can be controlled changing pH values; so, according to pH values, polyelectrolyte charges change and so do the crosslinking
2. Experimental part 2.1. Materials and chemicals PET films (75 µm) were obtained from HIFI industry. Hyaluronic acid (low and high molecular weight: 0.7–1.2 MDa and 1.9–2.2 MDa, respectively) was obtained from Contipro. Methanol (99.5%), ethylendiamine (99%) and hydrochloride acid (37%) were purchased from Panreac. Sodium hydroxide (99%), acetic acid (> 99%) and chitosan (medium molecular weight, 190–310 KDa and highly viscous, > 400 mPa·s 20 °C in 1% acetic acid) were purchased from Sigma Aldrich and potassium phosphate dibasic (99%) from Acros Organics. 2.2. Synthesis of HA-CHI complexes 1 wt% solutions of high molecular weight hyaluronic acid (HMWHA) and highly viscous (Hvisc) CHI were prepared separately in 1% (v/v) acetic acid solution. The solutions were mixed at 1:1 wt ratios and stirred. The precipitates formed by mixing both polymer solutions were left 2 h at room temperature and then the remaining solution was separated from the complex. 2
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2.5.6. Scanning electron microscopy (SEM) The surface of lyophilized polyelectrolyte complex hydrogels were coated with a thin gold overlay and analysed with a Hitachi S-4800 microscope (150 s, 20 mA, 15 kV, zoom at ×30,000), in order to determine pore size.
2.3. Construction of HA-CHI multilayers PET films were cut into 4 × 2 cm2 sections and subsequently, washed in ethanol and water in an ultrasonic bath and then, they were aminolyzed following a reported procedure [28]. Dried films were immersed in ethylendiamine/methanol 40:60 (v/v) at 50 °C for 1.5 h and activated in a 0.1 M solution of HCl for 3 h, at room temperature. Amiloyzed films were dipped in 1 g/L of low molecular weight hyaluronic acid (LMWHA) solution for 15 min, followed by an immersion in acetic buffer. Having adsorbed the first HA layer, films were dipped in 1 g/L medium molecular weight chitosan (MMWCHI) for 15 min followed by buffer rinsing. This process was carried out in order to obtain different numbers of layers (40, 100, and 200).
2.5.7. Rheological measurements Frequency sweep measurements of HA/CHI complex were performed in a Haake Viscotester IQ (Thermo Fisher Scientific), using parallel plate geometry (titanium upper plate of 35 mm and steel lower plate of 60 mm) with a gap distance of 1 mm at 25 °C. The viscometer was equipped with a Peltier system for the temperature control and a solvent trap. The storage (G′) and loss (G″) modulus values were recorded from 0.62 to 62 rad/s at a fixed strain of 0.1% previously assessed strain sweep tests. The dynamic rheological properties of three replicates were studied. Moreover, the self-healing capacity of the complex was also analysed by conducting the frequency sweep measurements after submitting the complexes to several cut-recovery cycles at 25 °C. Both moduli values were measured in triplicate.
2.4. Self-healing Self-healing process was studied in polyelectrolyte complexes (PECs) and polyelectrolyte multilayers (PEMs). Due to the fact that this ability is based on electrostatic interactions between polyelectrolytes, pH value was adjusted at 5, in order to form ions and consequently, favour interactions between ions. The study was done following the next procedure: PECs were split in two, pieces were held together and deionized water was added in order to see the healing process. On the other hand, in order to analyse self-healing of multilayers a scratch was done on coated PET films and after water immersion, healing process was observed using Leica DM2500 M optical microscope, with an augmentation of 5×.
2.5.8. Mechanical tests Unconfined compression tests were carried out by using an instrument (Metrotec FTM-50) equipped with a 500 N load cell, at room temperature. Hydrogels were measured for size, and then a progressive compression at a rate of 10 mm/min was performed until breaking. Hydrogels were subjected to five cycles of self-healing. Compression modules were calculated from the slope of the linear portion (50–75% strain range) of the stress–strain plot.
2.5. Characterization methods
3. Results
2.5.1. Fourier transform infrared spectroscopy (FT-IR) Infrared spectra of pure components (HA and CHI), polyelectrolyte complex and multilayers were recorded using Nicolet Nexus FT-IR spectrometer (Thermo Scientific, Loughborough, UK) in KBr pellets, at a resolution of 4 cm−1 and 32 scans per spectrum.
3.1. Synthesis and characterization of HA/CHI polyelectrolyte complexes and multilayers In this work, polyelectrolyte complexes were synthesized basing on the electrostatic interactions between hyaluronic acid and chitosan. The use of these polysaccharides allowed the design of biocompatible, biodegradable and non-toxic materials suitable for biomedical applications. In order to favour the electrostatic interactions between the carboxylate (eCOO−) groups of hyaluronic acid and the amine (eNH3+) groups of chitosan, the pH value was adjusted at 5 in which the ionization of both polyelectrolytes is maximum. The electrostatic interactions between the two biopolymers allow the formation of the complex on the one hand, but also the self-healing ability, due to the reversibility of these interactions. In Fig. 1 TEM and SEM images of the polyeletrolyte multilayers and complex hydrogels, respectively, are shown. Although both systems are formed by hyaluronic acid and chitosan, there is a great difference between their morphology as a consequence of the synthetic procedure, even though both are guided by the same interactions. PEMs are materials formed by the assembly of polysaccharides with nanometric width, while the complexes are macroscopic networks with micrometric pore size (164 ± 14 μm). The difference in scale is a key factor in their swelling behaviour and therefore, in the healability of the materials. Fig. 2 shows the FT-IR spectra of pure chitosan and hyaluronic acid, as well as their polyelectrolyte complex hydrogels and multilayers. The characteristic peaks of chitosan appear at 1654 cm−1 and 1596 cm−1 corresponding to amide I and amide II bands, respectively. It also shows the twisting of eCH2 group at 1419 cm−1 and CeN stretching at 1321 cm−1. The amide I and amide II bands of hyaluronic acid are observed at 1670 cm−1 and 1554 cm−1 respectively, the asymmetric and symmetric stretching of carboxylate group appears at 1625 cm−1 and 1414 cm−1, as well as CeN stretching at 1322 cm−1. Regarding to FTIR spectra of complex and multilayers, the following bands were identified: a wide band was displayed in both HA/CHI systems,
2.5.2. Differential scanning calorimetry (DSC) DSC was used to determine the glass transition temperature (Tg) of polyeletrolyte complexes and multilayers. Mettler Toledo Differential Scanning Calorimeter, DSC 822e calorimeter (Gießen, Germany), was used and samples were heated from 25 °C to 200 °C at a heating rate of 10 °C/min, under constant purging of nitrogen (20 mL/min). Each measurement was done three times and Tg was taken as the extrapolated onset of the baseline shift. 2.5.3. X ray diffraction (XRD) The X-ray powder diffraction patterns were collected by using a Malvern Panalytical (Almelo, Netherlands) X’PERT PRO automatic diffractometer operating at 40 kV and 40 mA, in theta-theta configuration, secondary monochromator with Cu-Kα radiation (λ = 1.5418 Å) and a PIXcel solid state detector (active length in 2θ 3.347°). Data were collected from 5 to 70° 2θ (step size = 0.02626 and time per step = 200 s) at RT. 2.5.4. Thermogravimetric analysis (TGA) TGA was used to determine the swelling behaviour of polyelectrolyte complexes and PET films coated with different number of HA/ CHI layers (40, 100 and 200) after hydrating samples in PBS solution. Measurements were carried out in Mettler Toledo TGA/SDTA 851e and samples were heated from 25 °C to 600 °C at a heating rate of 10 °C/min under 20 mL/min nitrogen flux. 2.5.5. Transmission electron microscopy (TEM) Polyelectrolyte multilayers thickness was examined with a Philips CM120 TEM instrument operating at 120 kW. The images were obtained with a magnification of ×56,000. 3
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Fig. 1. (a) TEM image of polyelectrolyte multilayers (20 HA/CHI bilayers) (b) SEM images of polycomplex hydrogel and pore distribution.
Fig. 3. Glass transition temperature of the samples: high and low molecular weight hyaluronic acid (HMW HA and LMW HA), high viscosity and medium molecular weight chitosan (HVisc CHI and MMW CHI), polyelectrolyte complex (PEC) hydrogel and polyelectrolyte multilayer (PEM).
polysaccharides with different molecular weight or viscosities were studied. High molecular weight hyaluronic acid (HMWHA) shows its transition at 134 °C while low molecular weight (LMWHA) shows at 120 °C. Similar observations were done for chitosan; highly viscous chitosan (HVCHI) shows the transition at 127 °C and medium molecular weight (MMWCHI) shows at 118 °C. The glass transition temperature of polymeric materials is highly dependent on the structure of the polymer; if polymer chains have a reduced mobility derived from a higher molecular weight, the transition would required more energy and therefore, the glass transition temperature will increase. Finally, the Tg of the multilayers and PEC hydrogels were studied. In both cases, a single Tg with a value lower than those of the constituting polymers was observed, at 113.35 °C in the hydrogels and 111 °C in the multilayers. The appearance of a unique glass transition temperature corroborated that complexation between hyaluronic acid and chitosan took place. XRD patterns of the samples are presented in Fig. 4. Chitosan showed two peaks at 10° and 20°, corresponding to hydrated and anhydrous crystals, respectively [36]. These peaks suggest an ordered structure formed as a consequence of the hydrogen bonds between amine and hydroxyls groups and that is responsible for hindering chains movement. In the case of hyaluronic acid, two peaks at 10° and 20° are also shown. After complexing, these peaks disappear forming an
Fig. 2. FTIR spectra of the samples: chitosan (CHI), hyaluronic acid (HA), polyelectrolyte complex hydrogel and polyelectrolyte multilayer (PEM).
1670–1598 cm−1 for the complex and 1630–1590 cm−1 for the multilayers, corresponding to the above described overlapping of amide I bands from chitosan and hyaluronic acid. The slight shifting of amide bands could be ascribed to the interactions between polysaccharides, confirming complexation [33]. Furthermore, two other bands were identified in each spectra, 1412 cm−1 and 1317 cm−1 for PEC hydrogels and 1410 cm−1 and 1322 cm−1 for PEMs, which coincide with the symmetric stretching of hyaluronic acid carboxylate and chitosan CeN, respectively [34,35]. The described overlapping of CHI and HA bands suggests the contribution of both polysaccharides in HA/CHI systems. If we compare the grade of overlapping of spectra of both systems, we could conclude that the interpenetration of polysaccharides is greater in PEMs that can be related to the higher interpenetration on the layer-bylayer deposition, which allows a better assembly of the components than in the macroscopic complexes formation. On the other hand, DSC was used to determine the glass-transition temperature (Tg) of the samples, shown in Fig. 3. Firstly, 4
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Fig. 4. XRD of pure CHI and HA and after their interaction forming hydrogels and PEMs.
Fig. 6. Storage modulus (G′, filled symbol) and loss modulus (G″, empty symbol) of the HA/CHI complex in a frequency scan.
amorphous structure in hydrogels and PEMs. The latest show peaks, which do not appear in PEC hydrogels; they correspond to the diffraction pattern of sodium salts from the PBS solution used in the detachment of polysaccharide layers from PET for characterization. Overall, CHI and HA undergo a loss of order when complexing, which may be the cause of the higher glass transition temperature of pristine polysaccharides in comparison with the single and lower glass transition temperatures measured for the corresponding hydrogels and PEMs. Due to the nanometric nature of HA/CHI PEMs, their capacity to absorb water was determined by means of thermogravimetry. The swelling behaviour of these polyelectrolyte multilayers is a crucial factor to the self-healing ability of studied materials, thus, the swelling behaviour of samples with different layers at different time of immersion in a PBS solution (pH = 7) was determined. All samples showed two main loss of weight; the first one is given around 100 °C, which corresponds to water loss of the samples. The other change of slope of TGA curves was displayed at 400 °C and corresponds to the degradation of the substrate PET. Quantification of water loss could be done determining the weight loss around 100 °C in each sample at different immersion times. As can be seen in Fig. 5, water content of PEMs increased with immersing time, up to a point where the maximum absorption was reached. In addition, the more layers deposited on substrate surface the greater water absorption was observed, due to the increasing amount of polysaccharide
on the substrate surface. It is worth to mention that LbL methodology leads to heterogeneously constructed multilayers, in which a mayor content of polysaccharides was accumulated in specific areas of PET films. This justifies the high standard deviations (0.6–11%) obtained in the swelling measurement of the samples despite the high accuracy of TGA technique. On the other hand, swelling behaviour of polyelectrolyte complexes could be studied by gravimetry immersing dried samples in PBS solution. The swelling degree was 24 ± 3 times higher than in multilayered system. As mentioned before, PEMs showed a nanometric width and higher interpenetration of the polymeric chains, while hydrogels are macroscopic networks with a micrometric pore size, which provides the complex with a higher capacity to absorb water. Rheological properties of the HA/CHI hydrogels were studied by frequency sweep test at 25 °C. As it could be appreciated in Fig. 6, the G′ value was maintained constant (mean value 75.4 ± 36.8 Pa) during the test and independent of the applied frequency, and always higher than the loss modulus. G′ represents the elastic part of the hydrogel, whereas G″ corresponds to the viscous part. The observed behaviour indicated that the hydrogel responded as elastic solid in the studied frequency range.
3.2. Self-healing Healing mechanism of HA/CHI PEC hydrogels and multilayers should happen due to the association-dissociation process between ionized carboxylate groups of hyaluronic acid and amine groups of chitosan. In order to study healing capacity on PEC hydrogels, the following procedure was carried out: after cutting the samples, water was added gently so as to facilitate the healing. Then, the two pieces were kept together and after 2 min polycomplexes were healed. The addition of water favoured the healing because it enhanced polymeric chains movement and therefore, the regeneration of the physical interactions between carboxylates of hyaluronic acid and protonated amines of chitosan chains. The healing process of polyelectrolyte complexes can be seen in Fig. 7. Above observed macroscopically regeneration process was also studied on a coated form on PET surface with different amount of HA/ CHI layers, via microscope. The healing process shown in Fig. 8, was carried out in a similar way as the above described; scratched films were dipped in water and within 2 min all the samples were healed. In this case, the self-healing ability was observed regardless the number of deposited polysaccharide bilayers (40, 100 and 200 HA/CHI).
Fig. 5. (a) Swelling behaviour and (b) TGA curves of HA/CHI polyelectrolyte multilayers onto PET determined by TGA. 5
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Fig. 7. (a) Self-healing process of polyelectrolyte complex (b) polyelectrolyte complex after healing.
Nevertheless, Wang et al. [4] deposited 30 and 300 poly(ethylenimine)/poly(acrylic acid) layers on a substrate, and demonstrated that the higher the layers number the lower the healing ability, that was ascribed to the fact that polyelectrolytes content hinders chain mobility and thus, the healing. Therefore, it could be concluded that the healing process happened in a similar way in the nanometric multilayered and in the macroscopic hydrogel form by the reversibility of the electrostatic interactions between employed polyanion and polycation. Furthermore, rheological oscillatory analyses were also carried out in order to investigate the self-healing process of the HA/CHI complex hydrogels. With that purpose, samples were cut into two halves as mentioned previously (Fig. 7). In this case, the process was repeated for two consecutive cycles and the rheological measurements were conducted after each one. As illustrated in Fig. 9, G′ was higher than G″ after every cut-recovery cycle (Table 1) thus, demonstrating the elastic property of the complex. The obtained data demonstrated a quick recovery of the viscoelastic properties of the broken complexes due to the physical interactions based on the formation of a polyanion-polycation bond between the oppositely charged polymers. The similarity between the values of mean storage and loss modulus of HA/CHI original and self-healable complexes indicated the formation of a new material with adequate self-healing capacity, which is in accordance with reported literature [37].
Fig. 9. Frequency sweep measurement (storage modulus (filled symbol) and loss modulus (empty symbol)) at a constant strain (0.1%) for ■ original HA/ CHI complex, ▲ HA/CHI complex after one cut-recovery cycle and ▾ HA/CHI complex after two cut-recovery cycles.
Results of unconfined compression testing on HA-CHI fresh hydrogel (0) and self-healed hydrogel (1–5) are shown in Fig. 10. The compression modulus increased with the deformation, reflecting a
Fig. 8. Self-healing process of polyelectrolyte multilayers: (a–c) 40 HA/CHI, (d–f) 100 HA/CHI (g–i) 200 HA/CHI. 6
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Grant PIF15/092. Authors thank for technical and human support provided by SGIker of UPV/EHU and European funding (ERDF and ESF).
Table 1 Mean storage and loss modulus of HA/CHI complex at 25 °C (average ± standard deviation, n = 3). HA/CHI complex hydrogels
G′ (Pa)
G″ (Pa)
Original After one cut-recovery cycle After two cut-recovery cycle
75.4 ± 36.8 74.0 ± 14.0 133.4 ± 50.3
40.1 ± 23.3 34.6 ± 10.3 55.1 ± 15.4
Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.eurpolymj.2019.109268. References [1] M.D. Hager, P. Greil, C. Leyens, S. Van Der Zwaag, U.S. Schubert, Self-healing materials, Adv. Mater. 22 (2010) 5424–5430, https://doi.org/10.1002/adma. 201003036. [2] A.B. South, L.A. Lyon, Autonomic self-healing of hydrogel thin films, Angew. Chemie – Int. Ed. 49 (2010) 767–771, https://doi.org/10.1002/anie.200906040. [3] L. Li, B. Yan, J. Yang, L. Chen, H. Zeng, Novel mussel-inspired injectable selfhealing hydrogel with anti-biofouling property, Adv. Mater. (2015) 1294–1299, https://doi.org/10.1002/adma.201405166. [4] X. Wang, F. Liu, X. Zheng, J. Sun, Water-enabled self-healing of polyelectrolyte multilayer coatings, Angew. Chemie - Int. Ed. 50 (2011) 11378–11381, https://doi. org/10.1002/anie.201105822. [5] F. Herbst, D. Döhler, P. Michael, W.H. Binder, Self-healing polymers via supramolecular forces, Macromol. Rapid Commun. 34 (2013) 203–220, https://doi.org/10. 1002/marc.201200675. [6] B. Gyarmati, B.Á. Szilágyi, A. Szilágyi, Reversible interactions in self-healing and shape memory hydrogels, Eur. Polym. J. 93 (2017) 642–669, https://doi.org/10. 1016/j.eurpolymj.2017.05.020. [7] H. Zhang, H. Xia, Y. Zhao, Poly(vinyl alcohol) hydrogel can autonomously self-heal, ACS Macro Lett. 1 (2012) 1233–1236, https://doi.org/10.1021/mz300451r. [8] Y. Guo, X. Zhou, Q. Tang, H. Bao, G. Wang, P. Saha, A self-healable and easily recyclable supramolecular hydrogel electrolyte for flexible supercapacitors, J. Mater. Chem. A. 4 (2016) 8769–8776, https://doi.org/10.1039/c6ta01441k. [9] Z. Wei, J. He, T. Liang, H. Oh, J. Athas, Z. Tong, C. Wang, Z. Nie, Autonomous selfhealing of poly(acrylic acid) hydrogels induced by the migration of ferric ions, Polym. Chem. 4 (2013) 4601–4605, https://doi.org/10.1039/c3py00692a. [10] B. Yang, Y. Zhang, X. Zhang, L. Tao, S. Li, Y. Wei, Facilely prepared inexpensive and biocompatible self-healing hydrogel: a new injectable cell therapy carrier, Polym. Chem. 3 (2012) 3235–3238, https://doi.org/10.1039/c2py20627g. [11] E. Tsuchida, K. Abe, Interactions Between Macromolecules in Solution and Intermacromolecular Complexes, Adv. Polym. Sci. Springer, Berlin, Heidelberg, 1982, , https://doi.org/10.1007/BFB0017548. [12] A. Shovsky, I. Varga, R. Makuška, P.M. Claesson, Formation and stability of watersoluble, molecular polyelectrolyte complexes: effects of charge density, mixing ratio, and polyelectrolyte concentration, Langmuir 25 (2009) 6113–6121, https:// doi.org/10.1021/la804189w. [13] S.J. Kim, S.G. Yoon, K.B. Lee, Y.D. Park, S.I. Kim, Electrical sensitive behavior of a polyelectrolyte complex composed of chitosan/hyaluronic acid, Solid State Ionics 164 (2003) 199–204, https://doi.org/10.1016/j.ssi.2003.08.005. [14] M. Buriuli, D. Verma, Advances in biomaterials for biomedical applications, 2017. < https://doi.org/10.1007/978-981-10-3328-5 > . [15] H.V. Sæther, H.K. Holme, G. Maurstad, O. Smidsrød, B.T. Stokke, Polyelectrolyte complex formation using alginate and chitosan, Carbohydr. Polym. 74 (2008) 813–821, https://doi.org/10.1016/j.carbpol.2008.04.048. [16] H. Chen, Y. Song, N. Liu, H. Wan, G. Shu, N. Liao, Effect of complexation conditions on microcapsulation of Lactobacillus acidophilus in xanthan-chitosan polyelectrolyte complex gels, Acta Sci. Pol. Technol. Aliment. 14 (2015) 207–213, https:// doi.org/10.17306/J.AFS.2015.3.22. [17] C.L. Romanò, E. De Vecchi, M. Bortolin, I. Morelli, L. Drago, Hyaluronic acid and its composites as a local antimicrobial/antiadhesive barrier, J. Bone Jt. Infect. 2 (2017) 63–72, https://doi.org/10.7150/jbji.17705. [18] L. Pérez-Álvarez, E. Lizundia, S. del Hoyo, A. Sagasti, L.R. Rubio, J.L. Vilas, Polysaccharide polyelectrolyte multilayer coating on poly(ethylene terephthalate), Polym. Int. 65 (2016) 915–920, https://doi.org/10.1002/pi.5116. [19] R. Logithkumar, A. Keshavnarayan, S. Dhivya, A. Chawla, S. Saravanan, N. Selvamurugan, A review of chitosan and its derivatives in bone tissue engineering, Carbohydr. Polym. 151 (2016) 172–188, https://doi.org/10.1016/j. carbpol.2016.05.049. [20] R.C.F. Cheung, T.B. Ng, J.H. Wong, W.Y. Chan, Chitosan: an update on potential biomedical and pharmaceutical applications, Mar. Drugs. 13 (2015) 5156–5186, https://doi.org/10.3390/md13085156. [21] P.H. Chua, K.G. Neoh, E.T. Kang, W. Wang, Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion, Biomaterials 29 (2008) 1412–1421, https://doi.org/10.1016/j.biomaterials.2007.12.019. [22] K. Mulligan, Z.J. Jakubek, L.J. Johnston, Supported lipid bilayers on biocompatible polysaccharide multilayers, Langmuir 27 (2011) 14352–14359, https://doi.org/10.
Fig. 10. Stress–strain representation from compression testing of fresh hydrogel (0) and hydrogel after successive (1–5) self-healing cycles and calculated compression modules (at 50–75% deformation).
typical non-linear behaviour of hydrogels even after 5 cycles of selfhealing. In addition, similar mechanical properties were showed after self-healing processes, regardless the number of the cycles (1–5) and there was no significant difference, neither in modulus nor in the breaking-up. Obtained data showed that fresh gels exhibited a uniformly linear strain–stress relationship from 0% to 60% strain, while healed hydrogels from 0 to 20%. These last ones were approximately 9 times stiffer than initial hydrogels and broken up at 60% lower strain. This fact can be ascribed to the loss of water during compression testing in self-healing cycling. 4. Conclusions Hyaluronic acid and chitosan based systems were successfully developed by electrostatic interactions, in order to form polyelectrolyte complexes. The assembly between the polysaccharides was held macroscopically in form of hydrogels, but also as a multilayered coating onto PET surface. This last one led to a nanometric thickness that resulted in a 24 times lower swelling capacity but an enhanced polymeric interpenetrability in comparison with bulk hydrogels. FTIR and DSC demonstrated the interactions and the consequent formation of polyelectrolyte complexes on multilayers and hydrogels. In addition, selfhealing ability of both systems was proved due to the association-dissociation process of ionic bonds between employed biopolymers. Overall, biocompatible, biodegradable and cost-effective materials had been developed for potential biomedical applications. Acknowledgments This work was funded by Ministerio de Economía Industria y Competitividad (grant MAT2017-89553-P). Financial support from the Basque Country Government in the frame of Grupos Consolidados (IT–776–13) is gratefully acknowledged. O. Guaresti wishes to acknowledge the University of the Basque Country (UPV/EHU) for its PhD 7
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