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chondroitin sulfate
Reactive & Functional Polymers 73 (2013) 1662–1671
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Dual-network hydrogels based on chemically and physically crosslinked chitosan/chondroitin sulfate André R. Fajardo ⇑, Silvia L. Fávaro, Adley F. Rubira, Edvani C. Muniz Grupo de Materiais Poliméricos e Compósitos (GMPC), Chemistry Department, Maringá State University, Av. Colombo, 5790, 87020-900 Maringá, Paraná, Brazil
a r t i c l e
i n f o
Article history: Received 2 August 2013 Received in revised form 2 October 2013 Accepted 7 October 2013 Available online 19 October 2013 Keywords: Soft materials Hydrogels Dual-network Chitosan Chondroitin sulfate
a b s t r a c t The formation of a novel type of hydrogel that combines chemically and physically crosslinked networks in a dual-network approach is presented here. Chitosan (CHT) and chondroitin sulfate (CS) were chemically modified with glycidyl methacrylate (GMA) and then crosslinked. The chemical hydrogels (CHT- and CS-gel) were deposited in different vials filled with CS or CHT stock solutions to form the dual-network hydrogels. FTIR, TGA and XRD analyses were used to characterize the chemical and the dual-network hydrogels. The percentages of CS or CHT complexed to the CHT- and CS-gel networks were calculated from the HPLC data. SEM images and swelling assays indicated that the formation of a secondary network by polyelectrolyte complexation changes the morphologies and liquid uptake capacities of the chemical hydrogels. Hence, the data and discussion presented here enable the formation of dual-network hydrogels with very interesting properties, such as the ability to interact with charged specimens (i.e., drugs, proteins or metal ions), a desirable feature for a wide range of applications. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Hydrogels are very attractive soft materials that have been applied in several fields due to their interesting and unique properties [1–3]. The three-dimensional (3D) hydrogel network is responsible for absorbing and retaining large amounts of water without dissolving or losing its structural integrity [4]. Chemical or physical crosslinking process can form this 3D network [5–8]. Chemically crosslinked networks (or chemical hydrogels) are usually formed by polymerization and parallel crosslinking of multifunctional monomers. This involves the synthesis of polymers with reactive groups and their subsequent crosslinking by reacting them with suitable crosslinking agents. On the other hand, in physically crosslinked networks (or physical hydrogels) the polymer chains are held together by electrostatic forces (polyelectrolytes), hydrogen bonds, hydrophobic interactions or chain entanglements. Over decades of research, chemical and physical hydrogels have been developed to provide different advantages/properties for applications in different fields (agriculture, hygienic industry, pharmaceutical and tissue engineering, for instance). Despite the constant enhancements in this area of research, both hydrogel types still show some disadvantages that restrict their application. In general, the crosslinking agents utilized to form chemical hydrogels show some level of toxicity, which restricts the use of the ob-
⇑ Corresponding author. Tel./fax: +55 44 3261 4215. E-mail address: [email protected] (A.R. Fajardo). 1381-5148/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.reactfunctpolym.2013.10.003
tained hydrogel as a biomaterial. Furthermore, physical hydrogels may demonstrate poor mechanical properties in their swollen state and eventually disintegrate. Considering the different approaches used to overcome these disadvantages, a strategy focused on the formation of the hydrogel network may be a promising solution. Hence, we developed an original hydrogel formed by both chemical and physical crosslinked networks – a dual-network hydrogel. Firstly, a conventional chemical hydrogel based on methacrylated chitosan (CHT-methacrylate) is formed. In a second step, a physically crosslinked network is formed between the CHT-gel network and chondroitin sulfate (CS) due to the electrostatic interaction among the charged functional groups in the CHT and CS backbones [9]. CS, a sulfated glycosaminoglycan (GAG) component of the extracellular matrix (ECM), is a polyelectrolyte specimen with anionic charge density that can associate with polycationic moieties through electrostatic interactions [10–13]. This methodology was also tested in reverse: raw CS was chemically modified first and then chemically crosslinked (resulting in CS-gel). Then, a physically crosslinked network was formed between the CS-gel and CHT. The complete characterization of these novel types of hydrogels was performed in this work and demonstrates that the formation of such a dual-network system combines, in the same material, the advantages of chemically and physically crosslinked networks. Additionally, the use of natural polymers such as CHT and CS broadens the range of applicability of the as-prepared hydrogels due to their low toxicity, biodegradability and biocompatibility. All of these features are desirable for applications in many fields (i.e., pharmacological, biological, environmental, etc.) [14].
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2. Experimental 2.1. Materials Chitosan (CHT, CAS 90-77-7), 85% deacetylated and with a viscometric mass (MV) of 87,000 g/mol, was purchased from Golden-Shell Biochemical (China) [12]. Chondroitin sulfate (CS, CAS 9007-28-7) with Mw of 20,000 g/mol determined by GPC/SEC was kindly supplied by Solabia, Brazil. Note that the CS utilized in this work is a mixture of CS sulfated in the C4 and C6 positions. Glycidyl methacrylate (GMA, 97% CAS: 106-91-2) and N,N-methylenebisacrylamide (MBA, CAS 110-26-9) were purchased from Sigma– Aldrich (St. Louis, USA). All materials and reagents were used as received. 2.2. Characterization techniques Gel permeation chromatography (GPC) analyses were performed at 25 °C in a Waters 150C system (Waters, Milford, USA) equipped with a multiangle laser light scattering detector (DAWN DSP-F, Wyatt, Hollister, USA) and two Shodex columns in series (OHPack 802 and 803) using water as a solvent. Infrared (IR) spectra were recorded with a Bomem (Model MB 100-C26) spectrometer, operating in the region from 4000 to 500 cm1, with a resolution of 4 cm1. 1H nuclear magnetic resonance (NMR) and solid-state 13C NMR spectra were recorded using a Varian spectrometer (Model Oxford 300, Illinois, USA). High performance liquid chromatography (HPLC) analyses were performed at 25 °C in a Surveyor Plus chromatographic workstation (Thermo Fisher Scientific, Illinois, USA) equipped with a PolySep-GFC-P 6000 chromatographic column (300 7.8 mm, Phenomenex) and a Surveyor Plus PDA Detector with a diode array, controlled by ChromChest™ software. An acetic acid solution (1.5 vol.%) was used as a mobile phase at a flow rate of 0.5 ml/min. The eluted analytes were monitored at k = 212 nm. Thermogravimetry (TGA) was carried out in a Simultaneous Thermal Analyses System, Netzsch (Model STA 409 PG/4/G Luxx) at a scanning rate of 10 °Cmin1 under N2 gas flowing at 20 ml/min in a range of temperatures from 22 to 800 °C. Xray diffraction (XRD) measurements were performed using a DMAXB diffractometer (Rigaku, Japan) equipped with a Cu Ka radiation source in a scattering angle (2h) from 5° to 70°, with a resolution of 0.02°, at a scanning speed of 2° min1. Scanning electron microscopy (SEM) images were recorded with a Shimadzu (Model SS550 Superscan, Kyoto, Japan) microscope coupled with an energy-dispersive X-ray (EDX) analyzer. Prior to the analyses, the samples were immersed in distilled water at room temperature up to the equilibrium swelling (24 h). Then, the samples were frozen using liquid nitrogen. Thereafter, the frozen hydrogel samples were lyophilized on a freeze dryer (Christ, Alpha 1-2 LD Plus, Germany) at 55 °C for 24 h. The dried samples were carefully fractured and then gold-coated by sputtering before SEM visualization. 2.3. Synthesis of CHT- and CS-methacrylate The strategy to prepare CHT- and CS-methacrylate was adapted from the literature [15,16]. Briefly, CHT (2 g; 23 lmol) was solubilized in an aqueous acid acetic solution (1.5 vol.%, 200 ml) at room temperature for 4 h. Afterwards, GMA (2.1 ml; 15.4 mmol) was added to the reaction system, which was then heated to 60 °C. The reaction was maintained under magnetic stirring for 12 h. The reaction system was cooled, and the material of interest was precipitated by the addition of ethanol (200 ml) and then recovered by filtration. The precipitate was washed several times with ethanol portions to remove all the unreacted chemicals. CHT-meth-
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acrylate was lyophilized at 55 °C for 24 h, yielding a white powder. Yield: 1.75 g (87.5%). FTIR (cm1): 3431 (OAH and NAH); 2928 and 2876 (CAH); 1728 (C@Oester); 1657 (C@C); 1554 (NAH, bending); 1418 and 1384 (CAH, bending); 1316 (CAN); and 1162, 1054, and 1034 (CAO). 1H NMR (300 MHz, D2O, 298 K, d, ppm): 6.02 (d, C@CH2); 5.62 (d, C@CH2); 4.79 (m, H-1GlcNAc and H-1GlcN); 4.52 (d, OACH2); 3.90–3.30 (m, CH, 5 CH2, NH and OH); 3.77 (m, H-3,4,6GlcNAc and H-3,4,6GlcN); 3.57 (m, H-5,6GlcNAc and H-5,6GlcN); 3.40 (m, OACH); 3.03 (m, H-2GlcN and H-2GlcN); 1.93 (s, CH3); and 1.73 (s, CH3) (Note: GlcN = D-glucosamine unit and GlcNAc = N-acetyl-D-glucosamine unit). CS-methacrylate was synthesized using a similar methodology. CS (10 g; 500 lmol) was solubilized in water (100 ml) at room temperature for 4 h. The pH was adjusted to 3.5 by HCl (0.1 M) addition. GMA (2.1 ml; 15.4 mmol) was then added to the reaction system. The reaction system was heated to 60 °C and maintained under magnetic stirring for 12 h. The reaction system was cooled, and the CS-methacrylate was precipitated by the addition of ethanol (100 ml) and then recovered by filtration. The precipitate was washed several times with ethanol portions to remove all the unreacted chemicals. CS-methacrylate was lyophilized at 55 °C for 24 h to yield a white powder. Yield: 9.23 g (92.3%). FTIR (cm1): 3417 (OAH and NAH); 2972 and 2915 (CAH); 1719 (C@Oester); 1633 (C@Oamide and C@C); 1574 (NAH, bending); 1421 and 1378 (CAH, bending); 1315 (CAN); 1250 (SO2); and 1131 and 1035 (CAO). 1H NMR (300 MHz, D2O, 298 K, d, ppm): 6.11 (d, C@CH2); 5.70 (d, C@CH2); 4.69 (d, OACH2); 4.48 (m, H-1GlcUa and H-1GalNAc); 4.30–3.20 (m, H-2,3,4,5,6GlcUA and H-2,3,4,5,6GalNAc, OACH, CH, 5 CH2, NH, and OH); 1.94 (s, CH3); and 1.89 (s, CH3) (Note: GlcUa = Glucuronic acid unit and GlcNAc = N-acetylgalactosamine unit).
2.4. Dual-network hydrogels formation Initially, chemical hydrogels were formed. For this purpose, CHT-methacrylate (1.50 g) was solubilized in an acetic acid solution (1.5 vol.%, 50 ml) at room temperature for 4 h. MBA (45 mg; 29 lmol) and sodium persulfate (Na2S2O8, 30 mg; 12.6 lmol) were added to the reaction system. The temperature was increased to 70 °C. The solution was maintained at 70 °C for 1 h to complete the crosslinking process. The as-formed CHT hydrogel (CHT-gel) was washed with distilled water for 24 h to achieve neutral conditions (ca. pH 7) and to remove the unreacted materials. The CHTgel was cut into cubes (1 1 1 cm) and then oven-dried (50 °C) for 24 h. Yield: 1.323 g (dry weight). Immersing the dried CHT-gel samples in a CS stock solution formed the physically crosslinked network. The CS stock solution was prepared by the addition of CS (2.5 g) in an acetic acid solution (1.5 vol.%, 50 ml). The CHT-gel samples were immersed in different vials filled with CS stock solution for different time intervals (6, 12, and 24 h). After each time interval, the CHT-gels physically crosslinked with CS (CHT-gel/CS) were removed and washed several times with distilled water and then oven-dried (50 °C) for 24 h. Aliquots of each vial were collected and stored after the formation of CHT-gel/CS to quantify the amount of CS complexed to the CHT-gel network. Using a similar methodology, CS-gel/CHT hydrogels were formed. Initially, CS-methacrylate (7.50 g) was solubilized in distilled water (50 ml) at room temperature for 4 h. MBA (225 mg; 94 lmol) and Na2S2O8 (225 mg; 145 lmol) were added, and the reaction system was maintained at 70 °C for 1 h to complete the hydrogel crosslinking. The as-formed CS hydrogel (CS-gel) was washed with distilled water for 24 h and then cut into cubes (1 1 1 cm) and oven-dried (50 °C) for 24 h. Yield: 5.890 g (dry weight).
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The CS-gel samples were immersed in a CHT stock solution [0.75 g of CHT in aqueous acetic acid solution (1.5 vol.%, 50 ml)] to form the physically crosslinked network with the CS-gel. The CS-gel samples were immersed in different vials filled with CHT stock solution and left for different time intervals (6, 12 and 24 h). Afterwards, they were removed and washed with distilled water and then oven-dried (50 °C) for 24 h. Aliquots of each vial were collected and stored following the CS-gel/CHT formation to quantify the amount of CHT complexed to the CS-gel network.
2.5. Liquid uptake capacity The liquid uptake capacities of the chemical and dual-network hydrogels were investigated by determining the equilibrium swelling ratio (ESR) parameter using a classical gravimetric method [17]. The hydrogel samples [CHT- and CS-gel, CHT-gel/CS(24 h) and CS-gel/CHT(24 h)] (approximately 1.0 g dry mass) were deposited in several vials filled with different swelling media (distilled water and buffer solutions of pH 2, 4, 7, 10 and 12) to reach swelling equilibrium (24 h). The volume of swelling media was 100 ml, and the swelling assays were performed at room temperature. Afterwards, the swollen samples were collected and then filtered in a 100-mesh sieve to remove the excess liquid. The swollen samples were weighed, and the ESR parameter was calculated using the following equation:
ESR ¼ ðW s W d Þ=W d
ð1Þ
where Ws is the swollen sample mass (in grams) and Wd is the dry sample mass (in grams). The ESR parameter is given in grams of liquid (gliquid) by grams of dried sample (gsample), and it is an average value calculated from triplicate measurements.
3. Results and discussion 3.1. Synthesis of CHT- and CS-methacrylate The chemical modification of raw polysaccharides with GMA is an attractive and oft-used strategy to form chemically crosslinked networks from methacrylated products [18,19]. According to Reis et al., the reaction between the polysaccharide’s functional groups (hydroxyl and carboxyl) and GMA could occur by two mechanistic routes [20]. The first is a transesterification reaction that forms methacrylated products and glycidol as a byproduct [20]. Transesterification is a fast and reversible reaction that predominates when the solvent shows aprotic character, e.g., dimethylsulfoxide (DMSO) [20]. The second mechanistic route is an epoxide ringopening reaction that could result in two different products depending on the position at which the ring is opened [20]. The latter mechanism is slow and irreversible and is more efficient when carried out in a protic solvent, such as water [20]. These characteristics lead to the epoxide ring-opening reaction route being predominant over the transesterification route. In view of these considerations, Fig. 1 gives a general idea of the strategy utilized here to modify the raw polysaccharides (CHT and CS) with GMA. The structural characterization of the methacrylated polysaccharides (CHT- and CS-methacrylate) was performed by FTIR and 1 H NMR spectroscopy. According to the literature, under these reaction conditions (protic solvent and acidic conditions), GMA reacts with both carboxyl and hydroxyl groups (the functional groups present in the CHT and CS backbones) by epoxide ring opening in a forward and irreversible reaction [21,22]. As stated above, this mechanistic route allows the insertion of two different methacrylate compounds into the polysaccharide backbone, 3-methacryloyl-1-glycerol ester and 3-methacryloyl-2-glyceryl ester [21]. This occurs due to the ability of hydroxyl and carboxyl groups to attach to the GMA at different positions of the epoxide ring. Fig. 2a shows
Fig. 1. Synthesis of CHT- and CS-methacrylate.
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Fig. 2. (a) FTIR spectra of raw CHT, GMA, and CHT-methacrylate and (b) FTIR spectra of raw CS, GMA, and CS-methacrylate.
Fig. 3. 1H NMR spectra of raw CHT, GMA, and CHT-methacrylate (300 MHz, D2O for raw CHT and CHT-methacrylate and CDCl3 for GMA, 298 K).
the FTIR spectra related to the synthesis of the CHT-methacrylate. The FTIR spectrum of CHT-methacrylate showed the characteristic bands of CHT, as well bands assigned to GMA, which are observed at 1728 and 1633 cm1. These bands are assigned to the carbonyl group (C@O) from the ester functionality and to the carbon–carbon double bonds (C@C) of the conjugated system, respectively [21,23]. In addition, the CHT-methacrylate spectrum shows a slight
decrease in the intensity of the broad band assigned to the hydroxyl group of raw CHT due to the reaction with GMA via epoxide ring opening. Furthermore, the intensity of the band assigned to carbon–oxygen (CAO) stretching (ca. 1086 cm1) increased due to the presence of the GMA moiety. The occurrence of these bands indicates the insertion of methacrylated compounds into the CHT backbone. A similar analysis could be performed with the FTIR
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spectra of the CS-methacrylate (see Fig. 2b). The CS-methacrylate spectrum showed the characteristic bands of raw CS and GMA, confirming the CS functionalization. The CS-methacrylate FTIR spectrum shows bands at 1719 and 1633 cm1, which are assigned to the carbonyl group (C@O) from the ester functionality and the carbon–carbon double bonds (C@C) of the conjugated system, respectively. These FTIR analyses are in agreement with other similar studies of the chemical modification of polysaccharides with GMA [21,23]. The 1H NMR spectra recorded for raw CHT, GMA, and CHTmethacrylate are presented in Fig. 3. After the insertion of the methacrylated compounds into the CHT backbone, the appearance of two signals with chemical shifts (d) at 6.02 and 5.62 ppm (denoted as 13 and 130 ) was observed. These signals correspond to the hydrogen atoms linked to the vinyl carbons (vinyl groups). Additionally, the intense signal observed at d 1.78 ppm in the CHT-methacrylate spectra (denoted as 14) is assigned to the methyl-linked hydrogen of the methacrylated compounds. Similar to other studies, the signals assigned to the glyceryl spacer from the methacrylated compounds were observed at d 4.69 and 3.40 ppm in the CHT-methacrylate spectrum [20,22]. The appearance of these signals confirms the reaction between the raw CHT and GMA. As a result, two different methacrylated compounds were attached to the CHT backbone. From the CHT-methacrylate spectrum, it is possible to determinate the degree of methacrylation (DM) of CHT using Eq. (2) [24].
DM ¼ ½ðAd6:02 þ Ad5:62 Þ=2=Ad3:03
ð2Þ
This equation correlates the areas of the two signals assigned to the hydrogen atoms from the vinyl groups (d 6.02 and 5.62 ppm) observed in the CHT-methacrylate spectrum to the signal at d 3.03 ppm observed in the spectrum of the raw CHT, which is assigned to the H-2 GlcNAc and H-2 GlcN protons. The signal assigned to the a-carbon-linked hydrogen of the GlcNAc unit (at d 4.79 ppm) was not utilized as a reference because it overlaps with the deuterium oxide resonance peak. Therefore, the amount of methacrylate groups introduced into the CHT backbone cannot be calculated accurately. The respective signal areas were measured after a careful baseline treatment, and the values were determined to be Ad6.02 0.12; Ad5.62 0.14; and Ad3.03 1.35. The DM value calculated for CHT-methacrylate is 0.096 (ca. 10%). Poon et al. [25] obtained a DM close to 13% for O-carboxymethylchitosan, allowing the formation of an interesting photo-crosslinkable cytocompatible hydrogel. The low DM value obtained from the chemical modification of raw CHT with GMA is desirable because it allows a reduced number of crosslinkable points, forming a handling material with a higher swelling ability. The CS-methacrylate spectrum (Fig. 4), compared to the raw CS spectrum, shows three new signals at d 6.11, 5.70 and 1.89 ppm (denoted as 12, 120 and 13) (Fig. 4). These signals are assigned to the vinyl and methyl groups of the methacrylate compounds from the GMA. Additionally, the signals corresponding to the glyceryl spacer were observed at d 4.69 ppm and d 3.45 ppm, confirming the insertion of the methacrylated compounds into the CS backbone [20,22]. The DM value for CS-methacrylate was calculated from the ratio between the areas of the signals at d 6.11 and 5.70 ppm observed in the CS-methacrylate spectrum (denoted as
Fig. 4. 1H NMR spectra of raw CS, GMA, and CS-methacrylate (300 MHz, D2O for raw CS and CS-methacrylate, and CDCl3 for GMA, 298 K).
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Fig. 5. (a) FTIR spectra of MBA, CHT-gel, and CHT* (CHT-methacrylate) and (b) FTIR spectra of MBA, CS-gel, and CS* (CS-methacrylate).
12 and 120 in Fig. 4) and the area of the signal observed at d 3.34 ppm in the raw CS spectrum. This signal, which is denoted as 7 in Fig. 4, is assigned to the H-2 proton of the GlcUa unit. The values calculated for the area ratios were 0.068 (Ad6.11/Ad3.34) and 0.065 (Ad5.70/Ad3.34). Comparing these results with the theoretical values (1 for both), it is possible to infer that the DM was ca. 7%. Although several articles report the chemical modification of CS with GMA, the degree of methacrylation was not reported, which makes any comparison difficult. However, note that the DM obtained for CS-methacrylate was lower than that calculated for CHT-methacrylate. In acidic media, the epoxy ring of GMA is protonated, which allows the attack of weak nucleophiles, such as alcohols (AOH) or carboxylic acids (ACOOH). Comparing the reactivity of these functional groups, AOH (present in the CHT backbone) is more reactive than ACOOH (present in the CS backbone). Consequently, the epoxy ring-opening reaction, performed under acidic conditions, was more efficient with CHT than with CS. 3.2. Hydrogel characterization The first step to forming the dual-network hydrogels was the chemical crosslinking of the methacrylated polysaccharides. For this purpose, CHT- or CS-methacrylate was crosslinked with MBA (as a crosslinker) using K2S2O8 (as a thermal initiator). In the next step, the as-formed chemical hydrogels (labeled CHT- and CS-gel) were immersed in CS or CHT stock solutions to perform the physical crosslinking process. The physical crosslinking occurs under acidic conditions due to the polyelectrolyte complexation between the CHT and CS chains. Under acidic conditions, the CHT amine groups are in their protonated form (ANHþ 3 ). Therefore, these charged groups strongly interact with the CS carboxylate and sulfate (ACOO and AOSO3) groups by electrostatic forces [26,27]. The structural characterization of the chemical hydrogels (CHTand CS-gel) was performed by FTIR spectroscopy. The FTIR spectrum of MBA (Fig. 5a and b) shows a broad and intense band at 3308 cm1, assigned to NAH stretching; the bands at 1658 and 1652 cm1 are assigned to conjugated C@O and C@C stretching; the band at 1545 cm1 is assigned to NAH bending, and the band at 1380 cm1 is assigned to CAN of the amine. For both hydrogels, CHT- and CS-gel, the FTIR spectra show intense bands assigned to CAH stretching close to 3000 cm1, resulting from the MBA alkyl (ACH2A) groups. Furthermore, it was observed in these spectra
that the C@O band was slightly shifted to a lower wavenumber due to a loss of conjugation of the carboxyl groups after the crosslinking reaction. Moreover, two bands, assigned to NAH bending and CAN stretching of the amine bonds from the MBA, are observed in these FTIR spectra. It is worthwhile to mention that the low degree of methacrylation limits the number of crosslinkable points in the polysaccharide backbone. For this reason, there is a low amount of bound MBA. The gel formation of both the CHT- and CS-gels was investigated by 13C-CP/MAS NMR spectroscopy (Fig. S1a and b, Supporting Information). Both 13C-CP/MAS NMR spectra showed signals assigned to saturated alkyl groups (ACH2ACA) (ca. d 42–24 ppm), which indicates the consumption of the vinyl groups (C@C) and the consequent alkyl formation in the CHT- and CS-gel network due to the radical reaction with MBA. Additionally, this reaction binds the MBA network to the polymer chains, forming the chemical network. Consequently, signals assigned to the MBA alkyl groups are observed in the 13C-CP/MAS NMR spectra recorded for the CHT- and CS-gels. The absence of any signals in the spectral range of 140–125 ppm in both NMR gel spectra due to the consumption of the vinyl groups (C@C) confirms the crosslinking among the methacrylated polysaccharides and MBA by radical reaction. Additionally, quite intense signals are observed at d 171–168 ppm, assigned to carbonyl groups (>C@O) of a non-conjugated system that arise due to the crosslinking reaction; also, signals at d 179–175 ppm due to carbonyl groups (NAC@O) from MBA are present. In the NMR spectrum of the CS-gel, such a signal overlaps the C@O signal due to the presence of carbonyl groups in the CS backbone. Physically crosslinked networks were formed along with the chemical hydrogel networks. The scheme presented in Fig. 6 illustrates the dual-network hydrogel formation. HPLC analyses were performed to quantify the percentages of each polysaccharide (CHT or CS) complexed to the chemical hydrogels after different immersion intervals (6, 12 and 24 h). The percentages of CHT or CS were calculated using the following equation:
% complexed ¼ ½ðC o C F Þ=C o 100
ð3Þ
where C0 is the initial concentration of the stock solution (CHT or CS stock solution) in which the chemical hydrogels were immersed, and CF is the final concentration of the stock solution after the different immersion intervals (Fig. S2, Supporting Information).
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Fig. 6. Dual-network hydrogel formation schematic (Step 1 is the chemical crosslinking; Step 2 is the physical crosslinking).
According to the results, for both cases, the immersion time did not greatly influence the amount of polysaccharide complexed to the CHT- or CS-gel network. For the CHT-gel, the percentage of complexed CS varied from 65% (6 h) to 69% (24 h). This means that approximately 1.61–1.72 g of CS was complexed to the CHT-gel network. The formation of polyelectrolyte complexes between CHT and CS is a well-known strategy to form physically crosslinked networks [27,28]; this process is pH-sensitive, as stated in previous studies. Furthermore, these studies showed that when CHT and CS are immersed together in an aqueous solution at low pH, the immersion time has little effect on their complexation capacity [27]. Similarly, the percentages of polysaccharide complexed to the chemical hydrogels showed only a slight change as a function of the immersion interval. The results determined for the CS-gel were very similar, although the percentages of the polysaccharide complexed to the hydrogel network [53% (6 h) and 59% (24 h)] were lower than those for the CHT-gel. Some of the CS carboxyl groups reacted with GMA, and for this reason, the ability of the CS-gel network to form polyelectrolyte complexes with CHT was limited. Therefore, control of the degree of methacrylation (DM) of CS is a key factor in preserving its complexation ability. The methodology presented here allows control over the DM of CS; as a consequence, its complexation ability was not compromised. After the physical crosslinking reaction, the as-obtained dualnetwork hydrogels were labeled as CHT-gel/CS(6–24 h) and CS-gel/CHT(6–24 h). The structural characterization of the dual-
network hydrogels was performed by FTIR spectroscopy and TGA and XRD analyses. The FTIR spectra of the dual-network hydrogels (Fig. 7a and b) did not show significant differences between the CHT- and CS-gels. However, some features should be noted. The band close to 1560 cm1, assigned to the CHT primary amine groups, was shifted to a lower wavenumber as the polyelectrolyte complexation between CHT and CS became more intense [29–31]. Moreover, the CHT-gel/CS(6–24 h) FTIR spectra (Fig. 7a) show a band assigned to S@O stretching (at 1238 cm1) and a more intense band assigned to C@O stretching (at 1733 cm1) of the CS carboxyl groups. The CS-gel/CHT(6–24 h) FTIR spectra (Fig. 7b) show a decrease in the intensity of the band assigned to S@O stretching (at 1238 cm1) due to the electrostatic interactions among the sulfate groups present in the CS-gel network and the protonated amine groups of CHT [27]. On the other hand, the bands assigned to the CAN bonds of the CHT amine groups increased in intensity. The FTIR data confirm that the polysaccharides (CS and CHT) were complexed to the CHT- and CS-gel networks. XRD patterns for the dual-network hydrogels were recorded to characterize any structural modifications resulting from the physical crosslinking (Fig. 3S, Supporting Information). It is known that raw CHT shows some crystallinity due to its interchain interactions (H-bonds, for instance) [32]. A decrease in crystallinity was observed due to the chemical modification of CHT with GMA and subsequent gel formation (Fig. 3Sa, Supporting Information). According to the XRD data, the physical crosslinking of the CHT-gel network with the CS chains did not change its diffraction pattern.
Fig. 7. FTIR spectra of (a) CHT-gel/CS(6–24 h) and (b) CS-gel/CHT(6–24 h) hydrogels.
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In other words, no significant structural reorganization due to the polyelectrolyte complexation occurs, and the amorphous pattern remains after the dual-network formation. For the CS-gel/CHT(6– 24 h) hydrogels, a similar behavior was observed. The XRD patterns of the CS-gel/CHT(6–24 h) hydrogels did not show any crystallinity. Despite the fact that raw CHT exhibited some crystallinity, after its physical crosslinking with the CS-gel network, that crystallinity was lost (Fig. 3Sb, Supporting Information). In both cases, the dualnetwork approach increased the density of the hydrogels. Generally, dense hydrogel networks show amorphous features because the crystalline arrangement of the macromolecules is hindered. The low amount of crosslinker utilized in the chemical crosslinking step also contributed to the lack of crystallite formation. From an applications point of view, amorphous hydrogels are advantageous for wound dressing and tissue engineering. Fajardo et al. [12,27] observed by XRD measurements that polyelectrolyte complexes based on CHT/CS showed some crystallinity. The diffraction patterns for those complexes, which are in fact physical hydrogels, showed two diffraction peaks close to 2h = 44° and 64°. Comparing these previous results with the diffraction patterns recorded for the dual-network hydrogels [CHTgel/CS(6–24 h) and CS-gel/CHT(6–24 h)] it may be inferred that the presence of two different types of networks precludes the formation of crystalline regions. The formation of chemical crosslinked networks between CHT- or CS-methacrylate and MBA in the first step restricts the mobility of the CHT and CS chains. This configuration prevents the rearrangement of the chains to form crystalline regions during physical crosslinking. The dual-network hydrogels remained amorphous, as observed for the chemical crosslinked hydrogels. The TGA curves obtained for CHT-gel/CS(6–24 h) and CS-gel/ CHT(6–24 h), the CHT- and CS-gels, and raw CS and CHT showed different stages of weight loss (Fig. 4Sa and 4Sc, Supporting Information). The first one at ca. 50–150 °C can be attributed to the loss of water and other volatile compounds. In this stage, the weight
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loss was approximately 10–15%. The second and third stages occur in the range of 180–400 °C, in which thermal degradation is observed for the dual-network hydrogels. In the second stage (ca. 180–250 °C), the chemically crosslinked network of both hydrogels and the physically crosslinked CS are thermally degraded. It must be noted that according to the NMR data, the DMs calculated for the modified polysaccharides were ca. 7% (for CS-methacrylate) and 10% (for CHT-methacrylate), which limits the number of crosslinking points in the CHT- and CS-gel networks. For this reason, the thermal energy necessary to degrade such a network was not high. The third stage (ca. 250–400 °C) involves the thermal degradation of CHT. This inference can be confirmed by evaluating the first derivative (DTG) (Fig. 4Sb and 4Sd, Supporting Information) of the TGA data. According to the DTG data, the CHT-gel shows two stages during its thermal degradation. On the other hand, the CSgel shows just one stage in its thermal degradation. After the complexation of the CS-gel network with the raw CHT, the second stage of the thermal degradation is observed. The higher temperature necessary to degrade CHT can explain this. Fajardo et al. [12] showed that the thermal degradation of raw CHT occurs close to 300 °C. The TGA data showed that the formation of a dual-network did not considerably affect the thermal stability of the CH- and CSgels. Additionally, increased immersion time in the polysaccharide stock solution did not result in an enhancement of the thermal properties of the dual-network hydrogels. The morphological characterization of the chemically crosslinked and dual-network hydrogels was performed using SEM microscopy (Fig. 8). The SEM images were recorded from the fractured surface of the hydrogel samples. The CHT- and CS-gels (Fig. 8a and c) showed a porous and irregular network that corresponds to the morphological characteristics of hydrogels. After the polyelectrolyte complexation of the chemically crosslinked networks with the polysaccharides CS or CHT, the dual-network hydrogels showed the same porous and very irregular network. However, analyzing the SEM images of CHT-gel/CS(24 h) and
Fig. 8. SEM images of (a) CHT-gel, (b) CHT-gel/CS(24 h), (c) CS-gel, and (d) CS-gel/CHT(24 h) (fracture images; magnification 100; scale bar 100 lm).
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CS-gel/CHT(24 h) (Fig. 8b and d), some morphological changes and the presence of some peculiar new networks were observed. As confirmed by the FTIR and HPLC data, in both cases, the polysaccharides (CS and CHT) are complexed to the chemical hydrogel networks. The formation of these polyelectrolyte complexes contributes slightly to the morphologies of CHT-gel/CS(24 h) and CS-gel/CHT(24 h). Comparing the SEM images of the chemical hydrogels (see Fig. 8a and c) with those of the dual-network hydrogels (see Fig. 8b and d), the appearance of some non-ordered morphologies in the fractured surfaces of these hydrogels could be observed. We associate the appearance of these non-ordered networks with the polyelectrolyte complexes formed between the chemical hydrogel networks and the polysaccharides (CS or CHT). Furthermore, the SEM images show that the physical crosslinking of the polysaccharides to the CHT- and CS-gels is restricted to their surface and porous walls. Additionally, as seen in the amplified insert in the SEM images (Fig. 8b and d), in certain positions of the hydrogel network, the complexed polysaccharides are projected outward from the fractured surface. These projections form very interesting networks, such as a ‘‘fringe’’ type network. EDX analyses of the chemical and dual-network hydrogels (Fig. S5) were utilized to confirm the physical crosslinking of the polysaccharides (CS or CHT) to the chemical hydrogels. Comparing the EDX spectrum obtained for CHT-gel/CS(24 h) with that from the CHT-gel, it is possible to visualize the appearance of a sulfur peak and a slight increase of the nitrogen peak after the physical crosslinking process (Fig. 5Sa, Supporting Information). The EDX quantitative analysis confirms that the CHT-gel/CS(24 h) network contains ca. 5.2 wt.% sulfur and 7.2 wt.% nitrogen (1.2 wt.% more than those found in the CHT-gel network) (see Table S1, Supporting Information). Sulfur and nitrogen are provided by the CS (AOSO3H and ANHCOCH3) functional groups. Furthermore, the EDX spectrum obtained for CS-gel/CHT(24 h) showed a slight increase of the nitrogen peak after the physical crosslinking of CHT to the CS-gel network (Fig. 5Sb, Supporting Information). The quantitative analysis confirms that the CS-gel/CHT(24 h) network contains approximately 6.6 wt.% nitrogen (2.3 wt.% more than in the CS-gel network) (see Table S1, Supporting Information). In this case, nitrogen is provided by the CHT (ANH2 and ANHCOCH3) functional groups. Taking into account these considerations, it is possible to form a chemically crosslinked material and keep its ‘‘chargeable’’ feature to enable a subsequent physical crosslinking with oppositely charged specimens, such as drugs, cancer cell markers, proteins, and metal ions. Note that changing some external stimuli (i.e., pH, ionic strength, temperature) could disintegrate the physical hydrogels [33–35]. This is a very desirable property from an applications perspective because some active substances (e.g., drugs) could be complexed to the conventional hydrogel network and be released in response to changes in the surrounding medium [36]. Furthermore, the chemically crosslinked network could act as a protective barrier for some solutes during transport across certain media (e.g., stomach region). Therefore, this technique could be utilized to enhance the properties of the chemically crosslinked material (e.g., mechanical properties, biodegradability, biocompatibility) and to tailor it for some specific applications (e.g., to make it viable for cell culture and skin/bone regeneration). The liquid uptake capacity was determined by the ESR parameter for both the chemical and dual-network hydrogels in different swelling media. The ESR values determined for each swelling media are presented in Table 1. According to the ESR values, the pH of the swelling medium affects the liquid uptake capacity of all the hydrogel samples tested. Comparing the ESR values determined for the chemical hydrogels, it is clear that the liquid uptake capacity of the CHT-gel was lower than that of the CS-gel under all swelling conditions. There are two
Table 1 ESR values determined for the chemical and the dual-network hydrogels in different swelling media. Sample
CHT-gel CHT-gel/CS(24 h) CS-gel CS-gel/CHT(24 h)
ESR (gwater/gsample) Water
pH 2
pH 4
pH 7
pH 10
pH 12
89 ± 1 63 ± 4 89 ± 3 90 ± 1
97 ± 8 57 ± 3 86 ± 3 74 ± 2
94 ± 14 55 ± 6 105 ± 4 62 ± 6
93 ± 5 54 ± 1 119 ± 1 96 ± 2
100 ± 2 87 ± 5 125 ± 1 101 ± 4
108 ± 8 96 ± 7 121 ± 3 110 ± 3
reasons for this result: the first one is the higher hydrophilicity of CS in relation to CHT; the second one is the higher DM showed by CHT-methacrylate compared to CS-methacrylate. As a consequence, the CHT-gel shows higher crosslinking density than the CS-gel, which restricts its swelling performance. Another hypothesis that explains these results is based on the CHT and CS functional groups. CS contains two functional groups that could be deprotonated, especially at pH > 5, while CHT shows only one functional group that could be deprotonated at pH > 7. These characteristics promote increased anion-anion repulsive forces in the CS-gel network compared to the CHT-gel network. The repulsive forces in the CS-gel network cause its expansion, and as result, there is an increase in the liquid absorption. Comparing the ESR values of the chemical hydrogels with those for the dual-network hydrogels, the physical crosslinking appears to affect the liquid uptake capacity of both the CHT- and CS-gels. The CHT-gel showed higher ESR values than CHT-gel/CS(24 h) in all the swelling media. In this case, the physical network formed between the CHT-gel network and the CS chains limits the dualnetwork expansion, resulting in low swelling. This is more evident in buffers of pH 2–4, where the amino groups in the CHT-gel network are protonated and interact by electrostatic interaction with the CS functional groups. Additionally, the chain density inside the CHT-gel/CS(24 h) network is higher than in the CHT-gel network, which reduces the empty spaces available for liquid storage. When the CHT-gel/CS(24 h) sample is swelled in buffers with pH P 7, an increase in the ESR values was observed. Above this pH, the strong electrostatic interactions between the CHT-gel network and the CS became weak, which allowed an expansion of the matrix and resulted in a higher content of absorbed liquid. From the ESR values determined for the CS-gel and CS-gel/CHT(24 h) hydrogel, a similar analysis could be performed. The results show that the dualnetwork hydrogels formed in this work have a high liquid uptake capacity over a wide pH range with a great sensitivity to changes in the pH. Wang et al. [35] showed in their work that pH-sensitive hydrogels from chitosan and poly(acrylic acid) interpolymer complexes could show sensitivity to salts as well. These features are desirable in a great number of potential applications, such as in drug delivery systems, water remediation, and dye and metal removal. 4. Conclusions The innovative methodology presented here was efficient in forming a new type of dual-network hydrogel. CHT and CS were chemically modified with GMA and then characterized using FTIR and 1H NMR spectroscopy. The CHT- and CS-methacrylates were chemically crosslinked by a free radical reaction with MBA. Subsequently, physically crosslinked networks were formed by immersion of the chemical hydrogels (CHT- and CS-gel) in CS or CHT stock solutions. The dual-network hydrogels were characterized by FTIR spectroscopy and thermogravimetric and XRD analyses. From the HPLC data, the percentages of CS or CHT complexed to the CHT- and CS-gel networks were calculated. SEM images of the chemical and dual-network hydrogels showed a clear difference in their morphologies. The dual-network gels possessed
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surfaces that projected outwards, which are assigned to the physically crosslinked polysaccharides. This inference corroborates the data obtained from the EDX analyses. The ESR parameter showed that the dual-network hydrogels possessed different liquid uptake capacities than the chemical hydrogels. Despite this, the dualnetwork hydrogels showed a high liquid uptake capacity over a wide pH range, and they also exhibited great sensitivity towards changes in the pH. This original approach could be useful to enhance certain properties of chemically crosslinked materials and tailor them for applications in different research fields (i.e., pharmaceutics, biological, environmental, etc.). Acknowledgments The authors thank CAPES for the A.R. Fajardo doctoral fellowship, CNPq for their financial support and COMCAP-UEM for the XRD analyses and SEM images. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reactfunctpolym. 2013.10.003. References [1] M.J. Zohuriaan-Mehr, H. Omidian, S. Doroudiani, K. Kabiri, J. Mater. Sci. 45 (2010) 5711–5735. [2] P. Schexnailder, G. Schmidt, Colloid Polym. Sci. 287 (2009) 1–11. [3] W. Wu, D.-S. Wang, React. Funct. Polym. 70 (2010) 684–691. [4] H. Omidian, J.G. Rocca, K. Park, J. Control. Rel. 102 (2005) 3–12. [5] Y. Li, C. Yang, M. Khan, S. Liu, J.L. Hedrick, Y.-Y. Yang, P.-L.R. Ee, Biomaterials 33 (2012) 6533–6541. [6] Z.-C. Wang, X.-D. Xu, C.-S. Chen, G.-R. Wang, S.-X. Cheng, X.-Z. Zhang, R.-X. Zhuo, React. Funct. Polym. 69 (2009) 14–19. [7] Y. Bae, K. Huh, Y. Kim, K. Park, J. Control. Rel. 64 (2000) 3–13.
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Reactive & Functional Polymers journal homepage: www.elsevier.com/locate/react
Dual-network hydrogels based on chemically and physically crosslinked chitosan/chondroitin sulfate André R. Fajardo ⇑, Silvia L. Fávaro, Adley F. Rubira, Edvani C. Muniz Grupo de Materiais Poliméricos e Compósitos (GMPC), Chemistry Department, Maringá State University, Av. Colombo, 5790, 87020-900 Maringá, Paraná, Brazil
a r t i c l e
i n f o
Article history: Received 2 August 2013 Received in revised form 2 October 2013 Accepted 7 October 2013 Available online 19 October 2013 Keywords: Soft materials Hydrogels Dual-network Chitosan Chondroitin sulfate
a b s t r a c t The formation of a novel type of hydrogel that combines chemically and physically crosslinked networks in a dual-network approach is presented here. Chitosan (CHT) and chondroitin sulfate (CS) were chemically modified with glycidyl methacrylate (GMA) and then crosslinked. The chemical hydrogels (CHT- and CS-gel) were deposited in different vials filled with CS or CHT stock solutions to form the dual-network hydrogels. FTIR, TGA and XRD analyses were used to characterize the chemical and the dual-network hydrogels. The percentages of CS or CHT complexed to the CHT- and CS-gel networks were calculated from the HPLC data. SEM images and swelling assays indicated that the formation of a secondary network by polyelectrolyte complexation changes the morphologies and liquid uptake capacities of the chemical hydrogels. Hence, the data and discussion presented here enable the formation of dual-network hydrogels with very interesting properties, such as the ability to interact with charged specimens (i.e., drugs, proteins or metal ions), a desirable feature for a wide range of applications. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Hydrogels are very attractive soft materials that have been applied in several fields due to their interesting and unique properties [1–3]. The three-dimensional (3D) hydrogel network is responsible for absorbing and retaining large amounts of water without dissolving or losing its structural integrity [4]. Chemical or physical crosslinking process can form this 3D network [5–8]. Chemically crosslinked networks (or chemical hydrogels) are usually formed by polymerization and parallel crosslinking of multifunctional monomers. This involves the synthesis of polymers with reactive groups and their subsequent crosslinking by reacting them with suitable crosslinking agents. On the other hand, in physically crosslinked networks (or physical hydrogels) the polymer chains are held together by electrostatic forces (polyelectrolytes), hydrogen bonds, hydrophobic interactions or chain entanglements. Over decades of research, chemical and physical hydrogels have been developed to provide different advantages/properties for applications in different fields (agriculture, hygienic industry, pharmaceutical and tissue engineering, for instance). Despite the constant enhancements in this area of research, both hydrogel types still show some disadvantages that restrict their application. In general, the crosslinking agents utilized to form chemical hydrogels show some level of toxicity, which restricts the use of the ob-
⇑ Corresponding author. Tel./fax: +55 44 3261 4215. E-mail address: [email protected] (A.R. Fajardo). 1381-5148/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.reactfunctpolym.2013.10.003
tained hydrogel as a biomaterial. Furthermore, physical hydrogels may demonstrate poor mechanical properties in their swollen state and eventually disintegrate. Considering the different approaches used to overcome these disadvantages, a strategy focused on the formation of the hydrogel network may be a promising solution. Hence, we developed an original hydrogel formed by both chemical and physical crosslinked networks – a dual-network hydrogel. Firstly, a conventional chemical hydrogel based on methacrylated chitosan (CHT-methacrylate) is formed. In a second step, a physically crosslinked network is formed between the CHT-gel network and chondroitin sulfate (CS) due to the electrostatic interaction among the charged functional groups in the CHT and CS backbones [9]. CS, a sulfated glycosaminoglycan (GAG) component of the extracellular matrix (ECM), is a polyelectrolyte specimen with anionic charge density that can associate with polycationic moieties through electrostatic interactions [10–13]. This methodology was also tested in reverse: raw CS was chemically modified first and then chemically crosslinked (resulting in CS-gel). Then, a physically crosslinked network was formed between the CS-gel and CHT. The complete characterization of these novel types of hydrogels was performed in this work and demonstrates that the formation of such a dual-network system combines, in the same material, the advantages of chemically and physically crosslinked networks. Additionally, the use of natural polymers such as CHT and CS broadens the range of applicability of the as-prepared hydrogels due to their low toxicity, biodegradability and biocompatibility. All of these features are desirable for applications in many fields (i.e., pharmacological, biological, environmental, etc.) [14].
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2. Experimental 2.1. Materials Chitosan (CHT, CAS 90-77-7), 85% deacetylated and with a viscometric mass (MV) of 87,000 g/mol, was purchased from Golden-Shell Biochemical (China) [12]. Chondroitin sulfate (CS, CAS 9007-28-7) with Mw of 20,000 g/mol determined by GPC/SEC was kindly supplied by Solabia, Brazil. Note that the CS utilized in this work is a mixture of CS sulfated in the C4 and C6 positions. Glycidyl methacrylate (GMA, 97% CAS: 106-91-2) and N,N-methylenebisacrylamide (MBA, CAS 110-26-9) were purchased from Sigma– Aldrich (St. Louis, USA). All materials and reagents were used as received. 2.2. Characterization techniques Gel permeation chromatography (GPC) analyses were performed at 25 °C in a Waters 150C system (Waters, Milford, USA) equipped with a multiangle laser light scattering detector (DAWN DSP-F, Wyatt, Hollister, USA) and two Shodex columns in series (OHPack 802 and 803) using water as a solvent. Infrared (IR) spectra were recorded with a Bomem (Model MB 100-C26) spectrometer, operating in the region from 4000 to 500 cm1, with a resolution of 4 cm1. 1H nuclear magnetic resonance (NMR) and solid-state 13C NMR spectra were recorded using a Varian spectrometer (Model Oxford 300, Illinois, USA). High performance liquid chromatography (HPLC) analyses were performed at 25 °C in a Surveyor Plus chromatographic workstation (Thermo Fisher Scientific, Illinois, USA) equipped with a PolySep-GFC-P 6000 chromatographic column (300 7.8 mm, Phenomenex) and a Surveyor Plus PDA Detector with a diode array, controlled by ChromChest™ software. An acetic acid solution (1.5 vol.%) was used as a mobile phase at a flow rate of 0.5 ml/min. The eluted analytes were monitored at k = 212 nm. Thermogravimetry (TGA) was carried out in a Simultaneous Thermal Analyses System, Netzsch (Model STA 409 PG/4/G Luxx) at a scanning rate of 10 °Cmin1 under N2 gas flowing at 20 ml/min in a range of temperatures from 22 to 800 °C. Xray diffraction (XRD) measurements were performed using a DMAXB diffractometer (Rigaku, Japan) equipped with a Cu Ka radiation source in a scattering angle (2h) from 5° to 70°, with a resolution of 0.02°, at a scanning speed of 2° min1. Scanning electron microscopy (SEM) images were recorded with a Shimadzu (Model SS550 Superscan, Kyoto, Japan) microscope coupled with an energy-dispersive X-ray (EDX) analyzer. Prior to the analyses, the samples were immersed in distilled water at room temperature up to the equilibrium swelling (24 h). Then, the samples were frozen using liquid nitrogen. Thereafter, the frozen hydrogel samples were lyophilized on a freeze dryer (Christ, Alpha 1-2 LD Plus, Germany) at 55 °C for 24 h. The dried samples were carefully fractured and then gold-coated by sputtering before SEM visualization. 2.3. Synthesis of CHT- and CS-methacrylate The strategy to prepare CHT- and CS-methacrylate was adapted from the literature [15,16]. Briefly, CHT (2 g; 23 lmol) was solubilized in an aqueous acid acetic solution (1.5 vol.%, 200 ml) at room temperature for 4 h. Afterwards, GMA (2.1 ml; 15.4 mmol) was added to the reaction system, which was then heated to 60 °C. The reaction was maintained under magnetic stirring for 12 h. The reaction system was cooled, and the material of interest was precipitated by the addition of ethanol (200 ml) and then recovered by filtration. The precipitate was washed several times with ethanol portions to remove all the unreacted chemicals. CHT-meth-
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acrylate was lyophilized at 55 °C for 24 h, yielding a white powder. Yield: 1.75 g (87.5%). FTIR (cm1): 3431 (OAH and NAH); 2928 and 2876 (CAH); 1728 (C@Oester); 1657 (C@C); 1554 (NAH, bending); 1418 and 1384 (CAH, bending); 1316 (CAN); and 1162, 1054, and 1034 (CAO). 1H NMR (300 MHz, D2O, 298 K, d, ppm): 6.02 (d, C@CH2); 5.62 (d, C@CH2); 4.79 (m, H-1GlcNAc and H-1GlcN); 4.52 (d, OACH2); 3.90–3.30 (m, CH, 5 CH2, NH and OH); 3.77 (m, H-3,4,6GlcNAc and H-3,4,6GlcN); 3.57 (m, H-5,6GlcNAc and H-5,6GlcN); 3.40 (m, OACH); 3.03 (m, H-2GlcN and H-2GlcN); 1.93 (s, CH3); and 1.73 (s, CH3) (Note: GlcN = D-glucosamine unit and GlcNAc = N-acetyl-D-glucosamine unit). CS-methacrylate was synthesized using a similar methodology. CS (10 g; 500 lmol) was solubilized in water (100 ml) at room temperature for 4 h. The pH was adjusted to 3.5 by HCl (0.1 M) addition. GMA (2.1 ml; 15.4 mmol) was then added to the reaction system. The reaction system was heated to 60 °C and maintained under magnetic stirring for 12 h. The reaction system was cooled, and the CS-methacrylate was precipitated by the addition of ethanol (100 ml) and then recovered by filtration. The precipitate was washed several times with ethanol portions to remove all the unreacted chemicals. CS-methacrylate was lyophilized at 55 °C for 24 h to yield a white powder. Yield: 9.23 g (92.3%). FTIR (cm1): 3417 (OAH and NAH); 2972 and 2915 (CAH); 1719 (C@Oester); 1633 (C@Oamide and C@C); 1574 (NAH, bending); 1421 and 1378 (CAH, bending); 1315 (CAN); 1250 (SO2); and 1131 and 1035 (CAO). 1H NMR (300 MHz, D2O, 298 K, d, ppm): 6.11 (d, C@CH2); 5.70 (d, C@CH2); 4.69 (d, OACH2); 4.48 (m, H-1GlcUa and H-1GalNAc); 4.30–3.20 (m, H-2,3,4,5,6GlcUA and H-2,3,4,5,6GalNAc, OACH, CH, 5 CH2, NH, and OH); 1.94 (s, CH3); and 1.89 (s, CH3) (Note: GlcUa = Glucuronic acid unit and GlcNAc = N-acetylgalactosamine unit).
2.4. Dual-network hydrogels formation Initially, chemical hydrogels were formed. For this purpose, CHT-methacrylate (1.50 g) was solubilized in an acetic acid solution (1.5 vol.%, 50 ml) at room temperature for 4 h. MBA (45 mg; 29 lmol) and sodium persulfate (Na2S2O8, 30 mg; 12.6 lmol) were added to the reaction system. The temperature was increased to 70 °C. The solution was maintained at 70 °C for 1 h to complete the crosslinking process. The as-formed CHT hydrogel (CHT-gel) was washed with distilled water for 24 h to achieve neutral conditions (ca. pH 7) and to remove the unreacted materials. The CHTgel was cut into cubes (1 1 1 cm) and then oven-dried (50 °C) for 24 h. Yield: 1.323 g (dry weight). Immersing the dried CHT-gel samples in a CS stock solution formed the physically crosslinked network. The CS stock solution was prepared by the addition of CS (2.5 g) in an acetic acid solution (1.5 vol.%, 50 ml). The CHT-gel samples were immersed in different vials filled with CS stock solution for different time intervals (6, 12, and 24 h). After each time interval, the CHT-gels physically crosslinked with CS (CHT-gel/CS) were removed and washed several times with distilled water and then oven-dried (50 °C) for 24 h. Aliquots of each vial were collected and stored after the formation of CHT-gel/CS to quantify the amount of CS complexed to the CHT-gel network. Using a similar methodology, CS-gel/CHT hydrogels were formed. Initially, CS-methacrylate (7.50 g) was solubilized in distilled water (50 ml) at room temperature for 4 h. MBA (225 mg; 94 lmol) and Na2S2O8 (225 mg; 145 lmol) were added, and the reaction system was maintained at 70 °C for 1 h to complete the hydrogel crosslinking. The as-formed CS hydrogel (CS-gel) was washed with distilled water for 24 h and then cut into cubes (1 1 1 cm) and oven-dried (50 °C) for 24 h. Yield: 5.890 g (dry weight).
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The CS-gel samples were immersed in a CHT stock solution [0.75 g of CHT in aqueous acetic acid solution (1.5 vol.%, 50 ml)] to form the physically crosslinked network with the CS-gel. The CS-gel samples were immersed in different vials filled with CHT stock solution and left for different time intervals (6, 12 and 24 h). Afterwards, they were removed and washed with distilled water and then oven-dried (50 °C) for 24 h. Aliquots of each vial were collected and stored following the CS-gel/CHT formation to quantify the amount of CHT complexed to the CS-gel network.
2.5. Liquid uptake capacity The liquid uptake capacities of the chemical and dual-network hydrogels were investigated by determining the equilibrium swelling ratio (ESR) parameter using a classical gravimetric method [17]. The hydrogel samples [CHT- and CS-gel, CHT-gel/CS(24 h) and CS-gel/CHT(24 h)] (approximately 1.0 g dry mass) were deposited in several vials filled with different swelling media (distilled water and buffer solutions of pH 2, 4, 7, 10 and 12) to reach swelling equilibrium (24 h). The volume of swelling media was 100 ml, and the swelling assays were performed at room temperature. Afterwards, the swollen samples were collected and then filtered in a 100-mesh sieve to remove the excess liquid. The swollen samples were weighed, and the ESR parameter was calculated using the following equation:
ESR ¼ ðW s W d Þ=W d
ð1Þ
where Ws is the swollen sample mass (in grams) and Wd is the dry sample mass (in grams). The ESR parameter is given in grams of liquid (gliquid) by grams of dried sample (gsample), and it is an average value calculated from triplicate measurements.
3. Results and discussion 3.1. Synthesis of CHT- and CS-methacrylate The chemical modification of raw polysaccharides with GMA is an attractive and oft-used strategy to form chemically crosslinked networks from methacrylated products [18,19]. According to Reis et al., the reaction between the polysaccharide’s functional groups (hydroxyl and carboxyl) and GMA could occur by two mechanistic routes [20]. The first is a transesterification reaction that forms methacrylated products and glycidol as a byproduct [20]. Transesterification is a fast and reversible reaction that predominates when the solvent shows aprotic character, e.g., dimethylsulfoxide (DMSO) [20]. The second mechanistic route is an epoxide ringopening reaction that could result in two different products depending on the position at which the ring is opened [20]. The latter mechanism is slow and irreversible and is more efficient when carried out in a protic solvent, such as water [20]. These characteristics lead to the epoxide ring-opening reaction route being predominant over the transesterification route. In view of these considerations, Fig. 1 gives a general idea of the strategy utilized here to modify the raw polysaccharides (CHT and CS) with GMA. The structural characterization of the methacrylated polysaccharides (CHT- and CS-methacrylate) was performed by FTIR and 1 H NMR spectroscopy. According to the literature, under these reaction conditions (protic solvent and acidic conditions), GMA reacts with both carboxyl and hydroxyl groups (the functional groups present in the CHT and CS backbones) by epoxide ring opening in a forward and irreversible reaction [21,22]. As stated above, this mechanistic route allows the insertion of two different methacrylate compounds into the polysaccharide backbone, 3-methacryloyl-1-glycerol ester and 3-methacryloyl-2-glyceryl ester [21]. This occurs due to the ability of hydroxyl and carboxyl groups to attach to the GMA at different positions of the epoxide ring. Fig. 2a shows
Fig. 1. Synthesis of CHT- and CS-methacrylate.
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Fig. 2. (a) FTIR spectra of raw CHT, GMA, and CHT-methacrylate and (b) FTIR spectra of raw CS, GMA, and CS-methacrylate.
Fig. 3. 1H NMR spectra of raw CHT, GMA, and CHT-methacrylate (300 MHz, D2O for raw CHT and CHT-methacrylate and CDCl3 for GMA, 298 K).
the FTIR spectra related to the synthesis of the CHT-methacrylate. The FTIR spectrum of CHT-methacrylate showed the characteristic bands of CHT, as well bands assigned to GMA, which are observed at 1728 and 1633 cm1. These bands are assigned to the carbonyl group (C@O) from the ester functionality and to the carbon–carbon double bonds (C@C) of the conjugated system, respectively [21,23]. In addition, the CHT-methacrylate spectrum shows a slight
decrease in the intensity of the broad band assigned to the hydroxyl group of raw CHT due to the reaction with GMA via epoxide ring opening. Furthermore, the intensity of the band assigned to carbon–oxygen (CAO) stretching (ca. 1086 cm1) increased due to the presence of the GMA moiety. The occurrence of these bands indicates the insertion of methacrylated compounds into the CHT backbone. A similar analysis could be performed with the FTIR
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spectra of the CS-methacrylate (see Fig. 2b). The CS-methacrylate spectrum showed the characteristic bands of raw CS and GMA, confirming the CS functionalization. The CS-methacrylate FTIR spectrum shows bands at 1719 and 1633 cm1, which are assigned to the carbonyl group (C@O) from the ester functionality and the carbon–carbon double bonds (C@C) of the conjugated system, respectively. These FTIR analyses are in agreement with other similar studies of the chemical modification of polysaccharides with GMA [21,23]. The 1H NMR spectra recorded for raw CHT, GMA, and CHTmethacrylate are presented in Fig. 3. After the insertion of the methacrylated compounds into the CHT backbone, the appearance of two signals with chemical shifts (d) at 6.02 and 5.62 ppm (denoted as 13 and 130 ) was observed. These signals correspond to the hydrogen atoms linked to the vinyl carbons (vinyl groups). Additionally, the intense signal observed at d 1.78 ppm in the CHT-methacrylate spectra (denoted as 14) is assigned to the methyl-linked hydrogen of the methacrylated compounds. Similar to other studies, the signals assigned to the glyceryl spacer from the methacrylated compounds were observed at d 4.69 and 3.40 ppm in the CHT-methacrylate spectrum [20,22]. The appearance of these signals confirms the reaction between the raw CHT and GMA. As a result, two different methacrylated compounds were attached to the CHT backbone. From the CHT-methacrylate spectrum, it is possible to determinate the degree of methacrylation (DM) of CHT using Eq. (2) [24].
DM ¼ ½ðAd6:02 þ Ad5:62 Þ=2=Ad3:03
ð2Þ
This equation correlates the areas of the two signals assigned to the hydrogen atoms from the vinyl groups (d 6.02 and 5.62 ppm) observed in the CHT-methacrylate spectrum to the signal at d 3.03 ppm observed in the spectrum of the raw CHT, which is assigned to the H-2 GlcNAc and H-2 GlcN protons. The signal assigned to the a-carbon-linked hydrogen of the GlcNAc unit (at d 4.79 ppm) was not utilized as a reference because it overlaps with the deuterium oxide resonance peak. Therefore, the amount of methacrylate groups introduced into the CHT backbone cannot be calculated accurately. The respective signal areas were measured after a careful baseline treatment, and the values were determined to be Ad6.02 0.12; Ad5.62 0.14; and Ad3.03 1.35. The DM value calculated for CHT-methacrylate is 0.096 (ca. 10%). Poon et al. [25] obtained a DM close to 13% for O-carboxymethylchitosan, allowing the formation of an interesting photo-crosslinkable cytocompatible hydrogel. The low DM value obtained from the chemical modification of raw CHT with GMA is desirable because it allows a reduced number of crosslinkable points, forming a handling material with a higher swelling ability. The CS-methacrylate spectrum (Fig. 4), compared to the raw CS spectrum, shows three new signals at d 6.11, 5.70 and 1.89 ppm (denoted as 12, 120 and 13) (Fig. 4). These signals are assigned to the vinyl and methyl groups of the methacrylate compounds from the GMA. Additionally, the signals corresponding to the glyceryl spacer were observed at d 4.69 ppm and d 3.45 ppm, confirming the insertion of the methacrylated compounds into the CS backbone [20,22]. The DM value for CS-methacrylate was calculated from the ratio between the areas of the signals at d 6.11 and 5.70 ppm observed in the CS-methacrylate spectrum (denoted as
Fig. 4. 1H NMR spectra of raw CS, GMA, and CS-methacrylate (300 MHz, D2O for raw CS and CS-methacrylate, and CDCl3 for GMA, 298 K).
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Fig. 5. (a) FTIR spectra of MBA, CHT-gel, and CHT* (CHT-methacrylate) and (b) FTIR spectra of MBA, CS-gel, and CS* (CS-methacrylate).
12 and 120 in Fig. 4) and the area of the signal observed at d 3.34 ppm in the raw CS spectrum. This signal, which is denoted as 7 in Fig. 4, is assigned to the H-2 proton of the GlcUa unit. The values calculated for the area ratios were 0.068 (Ad6.11/Ad3.34) and 0.065 (Ad5.70/Ad3.34). Comparing these results with the theoretical values (1 for both), it is possible to infer that the DM was ca. 7%. Although several articles report the chemical modification of CS with GMA, the degree of methacrylation was not reported, which makes any comparison difficult. However, note that the DM obtained for CS-methacrylate was lower than that calculated for CHT-methacrylate. In acidic media, the epoxy ring of GMA is protonated, which allows the attack of weak nucleophiles, such as alcohols (AOH) or carboxylic acids (ACOOH). Comparing the reactivity of these functional groups, AOH (present in the CHT backbone) is more reactive than ACOOH (present in the CS backbone). Consequently, the epoxy ring-opening reaction, performed under acidic conditions, was more efficient with CHT than with CS. 3.2. Hydrogel characterization The first step to forming the dual-network hydrogels was the chemical crosslinking of the methacrylated polysaccharides. For this purpose, CHT- or CS-methacrylate was crosslinked with MBA (as a crosslinker) using K2S2O8 (as a thermal initiator). In the next step, the as-formed chemical hydrogels (labeled CHT- and CS-gel) were immersed in CS or CHT stock solutions to perform the physical crosslinking process. The physical crosslinking occurs under acidic conditions due to the polyelectrolyte complexation between the CHT and CS chains. Under acidic conditions, the CHT amine groups are in their protonated form (ANHþ 3 ). Therefore, these charged groups strongly interact with the CS carboxylate and sulfate (ACOO and AOSO3) groups by electrostatic forces [26,27]. The structural characterization of the chemical hydrogels (CHTand CS-gel) was performed by FTIR spectroscopy. The FTIR spectrum of MBA (Fig. 5a and b) shows a broad and intense band at 3308 cm1, assigned to NAH stretching; the bands at 1658 and 1652 cm1 are assigned to conjugated C@O and C@C stretching; the band at 1545 cm1 is assigned to NAH bending, and the band at 1380 cm1 is assigned to CAN of the amine. For both hydrogels, CHT- and CS-gel, the FTIR spectra show intense bands assigned to CAH stretching close to 3000 cm1, resulting from the MBA alkyl (ACH2A) groups. Furthermore, it was observed in these spectra
that the C@O band was slightly shifted to a lower wavenumber due to a loss of conjugation of the carboxyl groups after the crosslinking reaction. Moreover, two bands, assigned to NAH bending and CAN stretching of the amine bonds from the MBA, are observed in these FTIR spectra. It is worthwhile to mention that the low degree of methacrylation limits the number of crosslinkable points in the polysaccharide backbone. For this reason, there is a low amount of bound MBA. The gel formation of both the CHT- and CS-gels was investigated by 13C-CP/MAS NMR spectroscopy (Fig. S1a and b, Supporting Information). Both 13C-CP/MAS NMR spectra showed signals assigned to saturated alkyl groups (ACH2ACA) (ca. d 42–24 ppm), which indicates the consumption of the vinyl groups (C@C) and the consequent alkyl formation in the CHT- and CS-gel network due to the radical reaction with MBA. Additionally, this reaction binds the MBA network to the polymer chains, forming the chemical network. Consequently, signals assigned to the MBA alkyl groups are observed in the 13C-CP/MAS NMR spectra recorded for the CHT- and CS-gels. The absence of any signals in the spectral range of 140–125 ppm in both NMR gel spectra due to the consumption of the vinyl groups (C@C) confirms the crosslinking among the methacrylated polysaccharides and MBA by radical reaction. Additionally, quite intense signals are observed at d 171–168 ppm, assigned to carbonyl groups (>C@O) of a non-conjugated system that arise due to the crosslinking reaction; also, signals at d 179–175 ppm due to carbonyl groups (NAC@O) from MBA are present. In the NMR spectrum of the CS-gel, such a signal overlaps the C@O signal due to the presence of carbonyl groups in the CS backbone. Physically crosslinked networks were formed along with the chemical hydrogel networks. The scheme presented in Fig. 6 illustrates the dual-network hydrogel formation. HPLC analyses were performed to quantify the percentages of each polysaccharide (CHT or CS) complexed to the chemical hydrogels after different immersion intervals (6, 12 and 24 h). The percentages of CHT or CS were calculated using the following equation:
% complexed ¼ ½ðC o C F Þ=C o 100
ð3Þ
where C0 is the initial concentration of the stock solution (CHT or CS stock solution) in which the chemical hydrogels were immersed, and CF is the final concentration of the stock solution after the different immersion intervals (Fig. S2, Supporting Information).
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Fig. 6. Dual-network hydrogel formation schematic (Step 1 is the chemical crosslinking; Step 2 is the physical crosslinking).
According to the results, for both cases, the immersion time did not greatly influence the amount of polysaccharide complexed to the CHT- or CS-gel network. For the CHT-gel, the percentage of complexed CS varied from 65% (6 h) to 69% (24 h). This means that approximately 1.61–1.72 g of CS was complexed to the CHT-gel network. The formation of polyelectrolyte complexes between CHT and CS is a well-known strategy to form physically crosslinked networks [27,28]; this process is pH-sensitive, as stated in previous studies. Furthermore, these studies showed that when CHT and CS are immersed together in an aqueous solution at low pH, the immersion time has little effect on their complexation capacity [27]. Similarly, the percentages of polysaccharide complexed to the chemical hydrogels showed only a slight change as a function of the immersion interval. The results determined for the CS-gel were very similar, although the percentages of the polysaccharide complexed to the hydrogel network [53% (6 h) and 59% (24 h)] were lower than those for the CHT-gel. Some of the CS carboxyl groups reacted with GMA, and for this reason, the ability of the CS-gel network to form polyelectrolyte complexes with CHT was limited. Therefore, control of the degree of methacrylation (DM) of CS is a key factor in preserving its complexation ability. The methodology presented here allows control over the DM of CS; as a consequence, its complexation ability was not compromised. After the physical crosslinking reaction, the as-obtained dualnetwork hydrogels were labeled as CHT-gel/CS(6–24 h) and CS-gel/CHT(6–24 h). The structural characterization of the dual-
network hydrogels was performed by FTIR spectroscopy and TGA and XRD analyses. The FTIR spectra of the dual-network hydrogels (Fig. 7a and b) did not show significant differences between the CHT- and CS-gels. However, some features should be noted. The band close to 1560 cm1, assigned to the CHT primary amine groups, was shifted to a lower wavenumber as the polyelectrolyte complexation between CHT and CS became more intense [29–31]. Moreover, the CHT-gel/CS(6–24 h) FTIR spectra (Fig. 7a) show a band assigned to S@O stretching (at 1238 cm1) and a more intense band assigned to C@O stretching (at 1733 cm1) of the CS carboxyl groups. The CS-gel/CHT(6–24 h) FTIR spectra (Fig. 7b) show a decrease in the intensity of the band assigned to S@O stretching (at 1238 cm1) due to the electrostatic interactions among the sulfate groups present in the CS-gel network and the protonated amine groups of CHT [27]. On the other hand, the bands assigned to the CAN bonds of the CHT amine groups increased in intensity. The FTIR data confirm that the polysaccharides (CS and CHT) were complexed to the CHT- and CS-gel networks. XRD patterns for the dual-network hydrogels were recorded to characterize any structural modifications resulting from the physical crosslinking (Fig. 3S, Supporting Information). It is known that raw CHT shows some crystallinity due to its interchain interactions (H-bonds, for instance) [32]. A decrease in crystallinity was observed due to the chemical modification of CHT with GMA and subsequent gel formation (Fig. 3Sa, Supporting Information). According to the XRD data, the physical crosslinking of the CHT-gel network with the CS chains did not change its diffraction pattern.
Fig. 7. FTIR spectra of (a) CHT-gel/CS(6–24 h) and (b) CS-gel/CHT(6–24 h) hydrogels.
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In other words, no significant structural reorganization due to the polyelectrolyte complexation occurs, and the amorphous pattern remains after the dual-network formation. For the CS-gel/CHT(6– 24 h) hydrogels, a similar behavior was observed. The XRD patterns of the CS-gel/CHT(6–24 h) hydrogels did not show any crystallinity. Despite the fact that raw CHT exhibited some crystallinity, after its physical crosslinking with the CS-gel network, that crystallinity was lost (Fig. 3Sb, Supporting Information). In both cases, the dualnetwork approach increased the density of the hydrogels. Generally, dense hydrogel networks show amorphous features because the crystalline arrangement of the macromolecules is hindered. The low amount of crosslinker utilized in the chemical crosslinking step also contributed to the lack of crystallite formation. From an applications point of view, amorphous hydrogels are advantageous for wound dressing and tissue engineering. Fajardo et al. [12,27] observed by XRD measurements that polyelectrolyte complexes based on CHT/CS showed some crystallinity. The diffraction patterns for those complexes, which are in fact physical hydrogels, showed two diffraction peaks close to 2h = 44° and 64°. Comparing these previous results with the diffraction patterns recorded for the dual-network hydrogels [CHTgel/CS(6–24 h) and CS-gel/CHT(6–24 h)] it may be inferred that the presence of two different types of networks precludes the formation of crystalline regions. The formation of chemical crosslinked networks between CHT- or CS-methacrylate and MBA in the first step restricts the mobility of the CHT and CS chains. This configuration prevents the rearrangement of the chains to form crystalline regions during physical crosslinking. The dual-network hydrogels remained amorphous, as observed for the chemical crosslinked hydrogels. The TGA curves obtained for CHT-gel/CS(6–24 h) and CS-gel/ CHT(6–24 h), the CHT- and CS-gels, and raw CS and CHT showed different stages of weight loss (Fig. 4Sa and 4Sc, Supporting Information). The first one at ca. 50–150 °C can be attributed to the loss of water and other volatile compounds. In this stage, the weight
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loss was approximately 10–15%. The second and third stages occur in the range of 180–400 °C, in which thermal degradation is observed for the dual-network hydrogels. In the second stage (ca. 180–250 °C), the chemically crosslinked network of both hydrogels and the physically crosslinked CS are thermally degraded. It must be noted that according to the NMR data, the DMs calculated for the modified polysaccharides were ca. 7% (for CS-methacrylate) and 10% (for CHT-methacrylate), which limits the number of crosslinking points in the CHT- and CS-gel networks. For this reason, the thermal energy necessary to degrade such a network was not high. The third stage (ca. 250–400 °C) involves the thermal degradation of CHT. This inference can be confirmed by evaluating the first derivative (DTG) (Fig. 4Sb and 4Sd, Supporting Information) of the TGA data. According to the DTG data, the CHT-gel shows two stages during its thermal degradation. On the other hand, the CSgel shows just one stage in its thermal degradation. After the complexation of the CS-gel network with the raw CHT, the second stage of the thermal degradation is observed. The higher temperature necessary to degrade CHT can explain this. Fajardo et al. [12] showed that the thermal degradation of raw CHT occurs close to 300 °C. The TGA data showed that the formation of a dual-network did not considerably affect the thermal stability of the CH- and CSgels. Additionally, increased immersion time in the polysaccharide stock solution did not result in an enhancement of the thermal properties of the dual-network hydrogels. The morphological characterization of the chemically crosslinked and dual-network hydrogels was performed using SEM microscopy (Fig. 8). The SEM images were recorded from the fractured surface of the hydrogel samples. The CHT- and CS-gels (Fig. 8a and c) showed a porous and irregular network that corresponds to the morphological characteristics of hydrogels. After the polyelectrolyte complexation of the chemically crosslinked networks with the polysaccharides CS or CHT, the dual-network hydrogels showed the same porous and very irregular network. However, analyzing the SEM images of CHT-gel/CS(24 h) and
Fig. 8. SEM images of (a) CHT-gel, (b) CHT-gel/CS(24 h), (c) CS-gel, and (d) CS-gel/CHT(24 h) (fracture images; magnification 100; scale bar 100 lm).
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CS-gel/CHT(24 h) (Fig. 8b and d), some morphological changes and the presence of some peculiar new networks were observed. As confirmed by the FTIR and HPLC data, in both cases, the polysaccharides (CS and CHT) are complexed to the chemical hydrogel networks. The formation of these polyelectrolyte complexes contributes slightly to the morphologies of CHT-gel/CS(24 h) and CS-gel/CHT(24 h). Comparing the SEM images of the chemical hydrogels (see Fig. 8a and c) with those of the dual-network hydrogels (see Fig. 8b and d), the appearance of some non-ordered morphologies in the fractured surfaces of these hydrogels could be observed. We associate the appearance of these non-ordered networks with the polyelectrolyte complexes formed between the chemical hydrogel networks and the polysaccharides (CS or CHT). Furthermore, the SEM images show that the physical crosslinking of the polysaccharides to the CHT- and CS-gels is restricted to their surface and porous walls. Additionally, as seen in the amplified insert in the SEM images (Fig. 8b and d), in certain positions of the hydrogel network, the complexed polysaccharides are projected outward from the fractured surface. These projections form very interesting networks, such as a ‘‘fringe’’ type network. EDX analyses of the chemical and dual-network hydrogels (Fig. S5) were utilized to confirm the physical crosslinking of the polysaccharides (CS or CHT) to the chemical hydrogels. Comparing the EDX spectrum obtained for CHT-gel/CS(24 h) with that from the CHT-gel, it is possible to visualize the appearance of a sulfur peak and a slight increase of the nitrogen peak after the physical crosslinking process (Fig. 5Sa, Supporting Information). The EDX quantitative analysis confirms that the CHT-gel/CS(24 h) network contains ca. 5.2 wt.% sulfur and 7.2 wt.% nitrogen (1.2 wt.% more than those found in the CHT-gel network) (see Table S1, Supporting Information). Sulfur and nitrogen are provided by the CS (AOSO3H and ANHCOCH3) functional groups. Furthermore, the EDX spectrum obtained for CS-gel/CHT(24 h) showed a slight increase of the nitrogen peak after the physical crosslinking of CHT to the CS-gel network (Fig. 5Sb, Supporting Information). The quantitative analysis confirms that the CS-gel/CHT(24 h) network contains approximately 6.6 wt.% nitrogen (2.3 wt.% more than in the CS-gel network) (see Table S1, Supporting Information). In this case, nitrogen is provided by the CHT (ANH2 and ANHCOCH3) functional groups. Taking into account these considerations, it is possible to form a chemically crosslinked material and keep its ‘‘chargeable’’ feature to enable a subsequent physical crosslinking with oppositely charged specimens, such as drugs, cancer cell markers, proteins, and metal ions. Note that changing some external stimuli (i.e., pH, ionic strength, temperature) could disintegrate the physical hydrogels [33–35]. This is a very desirable property from an applications perspective because some active substances (e.g., drugs) could be complexed to the conventional hydrogel network and be released in response to changes in the surrounding medium [36]. Furthermore, the chemically crosslinked network could act as a protective barrier for some solutes during transport across certain media (e.g., stomach region). Therefore, this technique could be utilized to enhance the properties of the chemically crosslinked material (e.g., mechanical properties, biodegradability, biocompatibility) and to tailor it for some specific applications (e.g., to make it viable for cell culture and skin/bone regeneration). The liquid uptake capacity was determined by the ESR parameter for both the chemical and dual-network hydrogels in different swelling media. The ESR values determined for each swelling media are presented in Table 1. According to the ESR values, the pH of the swelling medium affects the liquid uptake capacity of all the hydrogel samples tested. Comparing the ESR values determined for the chemical hydrogels, it is clear that the liquid uptake capacity of the CHT-gel was lower than that of the CS-gel under all swelling conditions. There are two
Table 1 ESR values determined for the chemical and the dual-network hydrogels in different swelling media. Sample
CHT-gel CHT-gel/CS(24 h) CS-gel CS-gel/CHT(24 h)
ESR (gwater/gsample) Water
pH 2
pH 4
pH 7
pH 10
pH 12
89 ± 1 63 ± 4 89 ± 3 90 ± 1
97 ± 8 57 ± 3 86 ± 3 74 ± 2
94 ± 14 55 ± 6 105 ± 4 62 ± 6
93 ± 5 54 ± 1 119 ± 1 96 ± 2
100 ± 2 87 ± 5 125 ± 1 101 ± 4
108 ± 8 96 ± 7 121 ± 3 110 ± 3
reasons for this result: the first one is the higher hydrophilicity of CS in relation to CHT; the second one is the higher DM showed by CHT-methacrylate compared to CS-methacrylate. As a consequence, the CHT-gel shows higher crosslinking density than the CS-gel, which restricts its swelling performance. Another hypothesis that explains these results is based on the CHT and CS functional groups. CS contains two functional groups that could be deprotonated, especially at pH > 5, while CHT shows only one functional group that could be deprotonated at pH > 7. These characteristics promote increased anion-anion repulsive forces in the CS-gel network compared to the CHT-gel network. The repulsive forces in the CS-gel network cause its expansion, and as result, there is an increase in the liquid absorption. Comparing the ESR values of the chemical hydrogels with those for the dual-network hydrogels, the physical crosslinking appears to affect the liquid uptake capacity of both the CHT- and CS-gels. The CHT-gel showed higher ESR values than CHT-gel/CS(24 h) in all the swelling media. In this case, the physical network formed between the CHT-gel network and the CS chains limits the dualnetwork expansion, resulting in low swelling. This is more evident in buffers of pH 2–4, where the amino groups in the CHT-gel network are protonated and interact by electrostatic interaction with the CS functional groups. Additionally, the chain density inside the CHT-gel/CS(24 h) network is higher than in the CHT-gel network, which reduces the empty spaces available for liquid storage. When the CHT-gel/CS(24 h) sample is swelled in buffers with pH P 7, an increase in the ESR values was observed. Above this pH, the strong electrostatic interactions between the CHT-gel network and the CS became weak, which allowed an expansion of the matrix and resulted in a higher content of absorbed liquid. From the ESR values determined for the CS-gel and CS-gel/CHT(24 h) hydrogel, a similar analysis could be performed. The results show that the dualnetwork hydrogels formed in this work have a high liquid uptake capacity over a wide pH range with a great sensitivity to changes in the pH. Wang et al. [35] showed in their work that pH-sensitive hydrogels from chitosan and poly(acrylic acid) interpolymer complexes could show sensitivity to salts as well. These features are desirable in a great number of potential applications, such as in drug delivery systems, water remediation, and dye and metal removal. 4. Conclusions The innovative methodology presented here was efficient in forming a new type of dual-network hydrogel. CHT and CS were chemically modified with GMA and then characterized using FTIR and 1H NMR spectroscopy. The CHT- and CS-methacrylates were chemically crosslinked by a free radical reaction with MBA. Subsequently, physically crosslinked networks were formed by immersion of the chemical hydrogels (CHT- and CS-gel) in CS or CHT stock solutions. The dual-network hydrogels were characterized by FTIR spectroscopy and thermogravimetric and XRD analyses. From the HPLC data, the percentages of CS or CHT complexed to the CHT- and CS-gel networks were calculated. SEM images of the chemical and dual-network hydrogels showed a clear difference in their morphologies. The dual-network gels possessed
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surfaces that projected outwards, which are assigned to the physically crosslinked polysaccharides. This inference corroborates the data obtained from the EDX analyses. The ESR parameter showed that the dual-network hydrogels possessed different liquid uptake capacities than the chemical hydrogels. Despite this, the dualnetwork hydrogels showed a high liquid uptake capacity over a wide pH range, and they also exhibited great sensitivity towards changes in the pH. This original approach could be useful to enhance certain properties of chemically crosslinked materials and tailor them for applications in different research fields (i.e., pharmaceutics, biological, environmental, etc.). Acknowledgments The authors thank CAPES for the A.R. Fajardo doctoral fellowship, CNPq for their financial support and COMCAP-UEM for the XRD analyses and SEM images. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reactfunctpolym. 2013.10.003. References [1] M.J. Zohuriaan-Mehr, H. Omidian, S. Doroudiani, K. Kabiri, J. Mater. Sci. 45 (2010) 5711–5735. [2] P. Schexnailder, G. Schmidt, Colloid Polym. Sci. 287 (2009) 1–11. [3] W. Wu, D.-S. Wang, React. Funct. Polym. 70 (2010) 684–691. [4] H. Omidian, J.G. Rocca, K. Park, J. Control. Rel. 102 (2005) 3–12. [5] Y. Li, C. Yang, M. Khan, S. Liu, J.L. Hedrick, Y.-Y. Yang, P.-L.R. Ee, Biomaterials 33 (2012) 6533–6541. [6] Z.-C. Wang, X.-D. Xu, C.-S. Chen, G.-R. Wang, S.-X. Cheng, X.-Z. Zhang, R.-X. Zhuo, React. Funct. Polym. 69 (2009) 14–19. [7] Y. Bae, K. Huh, Y. Kim, K. Park, J. Control. Rel. 64 (2000) 3–13.
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