Biocompatible Hydrogel Nanocomposite with Covalently Embedded

Article pubs.acs.org/Biomac

Biocompatible Hydrogel Nanocomposite with Covalently Embedded Silver Nanoparticles Clara García-Astrain,† Cheng Chen,‡ María Burón,§ Teodoro Palomares,§ Arantxa Eceiza,† Ljiljana Fruk,‡ M. Á ngeles Corcuera,† and Nagore Gabilondo*,† †

Materials + Technologies Group, Department of Chemical and Environmental Engineering, Polytechnic School, University of the Basque Country, Plaza Europa 1, 20018 San Sebastián, Spain ‡ DGF-Centre for Functional Nanostructures, Karlsruhe Institute of Technology (KIT), Wofgang Gaede Str. 1a, 76131 Karlsruhe, Germany § Faculty of Medicine and Dentistry, University of the Basque Country, Barrio Sarriena s/n, 48940 Leioa, Spain ABSTRACT: Bionanocomposite materials, combining the properties of biopolymers and nanostructured materials, are attracting interest of the wider scientific community due to their potential application in design of implants, drug delivery systems, and tissue design platforms. Herein, we report on the use of maleimidecoated silver nanoparticles (Ag NPs) as cocross-linkers for the preparation of a bionanocomposite gelatin based hydrogel. Diels− Alder cycloaddition of benzotriazole maleimide (BTM) functionalized Ag NPs and furan containing gelatin in combination with additional amide coupling resulted in stable and biocompatible hybrid nanocomposite. The storage moduli values for the hydrogel are nearly three times higher than that of control hydrogel without NPs indicating a stabilizing role of the covalently bound NPs. Finally, the swelling and drug release properties of the materials as well as the biocompatibility and toxicity tests indicate the biomedical potential of this type of material.

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materials,20 as well as the surface enhanced Raman scattering (SERS) substrates. 21 The most common methods of introducing Ag NPs to the polymer matrix were based on the physical incorporation either through chemical17 or microwaveassisted reduction22 methods or photoinduced nanoparticle growth.23 However, such physical incorporation of the NPs into the network can lead to their continuous release to the surrounding environment, resulting in unwanted agglomerations, polymer collapse, or toxicity problems.24 Moreover, despite the advances in the use of NPs in bioapplications, the potential adverse health effects of Ag NPs have not yet been thoroughly investigated, and the toxicity reports are often contradictory.25 To overcome the aforementioned drawbacks, in particular, NP leakage and subsequent polymer collapse, it is desirable for NPs to be immobilized and, at the same time, stabilized by irreversible attachment to the polymeric chains. In such way, NPs become an active structural, cross-linking element and, at the same time, act both as scaffold stabilizers and function carriers.8,26 Several strategies have been used for the preparation of chemically cross-linked hydrogel networks to prevent the leaking of NPs during the hydrogel’s use.27−29 For example, printable and extrudable hydrogels were prepared after cross-linking of gold NPs with thiolated biopolymers16 and cobalt ferrite NPs functionalized with amine groups have

INTRODUCTION Bionanocomposite (BNC) hydrogels have recently attracted interest due to new properties introduced by synergetic effects between their organic and inorganic components.1 Indeed, nanocomponents can introduce changes to the classic hydrogels or lead to entirely new properties. Nanocomponents such as metallic nanoparticles (NPs) have already found numerous applications in catalysis,2 electronics,3 design of sensors,4 and luminescence devices,5 in photonics,6 biotechnology, and medicine.7 The combination of inorganic nanospecies, such as NPs and soft and elastic three-dimensional hydrogel networks, can result in materials with enhanced performance and new properties, as already demonstrated in the case of magnetically guided drug delivery systems or improvement of the electrically controlled tissue growth.8−10 Taking into account the range of possible biomedical applications, in particular in view of new material design for drug delivery or tissue/bone replacement, nanocomposites stemming from biopolymeric hydrogels are of particular interest.1 They have already been shown to exhibit improved mechanical, optical, and swelling/deswelling properties when compared to conventional hydrogel homologues.11,12 In the past few years, a significant effort has been put into the development of nanocomposite hydrogels for preparation of drug delivery systems,13 scaffolds,14 biosensors,15 and in tissue engineering.16 Recently, there have been reports of silver nanoparticle (Ag NP) containing hybrid hydrogels and their potential applications as antimicrobial,17 optoelectronic18,19 or catalytic © XXXX American Chemical Society

Received: January 25, 2015 Revised: March 17, 2015

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DOI: 10.1021/acs.biomac.5b00101 Biomacromolecules XXXX, XXX, XXX−XXX

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Gelatin was modified with furan units (G-FGE) as described in our previous work.42 Typically, gelatin was reacted with FGE in aqueous solution at basic pH at 55 °C for 24 h. The solution was then neutralized and purified by dialysis against water. After freeze-drying, G-FGE was recovered as a yellowish solid. Synthesis of BTM-Coated Silver Nanoparticles (BTM-Ag NPs). The synthesis of maleimide-coated silver nanoparticles was carried out following a procedure reported in the literature.41 In short, benzotriazole-maleimide was prepared by the reaction of 5-aminobenzotriazole with maleic anhydride in DCM at room temperature overnight to afford maleic acid derivative. The as-prepared washed and dried compound was mixed with anhydrous sodium acetate in acetic anhydride and refluxed for 4 h. After removal of the acetic anhydride, benzotriazole-maleimide (BTM) was purified by column chromatography. For nanoparticle synthesis, BTM and AgNO3 were dissolved in a mixture of MeCN and H2O (v/v = 2:3) in a 50:1 molar ratio (Scheme 1). The mixture was stirred in an ice bath for 1 h to achieve a chelating

been used as cross-linkers for carboxymethylcellulose to prepare magnetic hybrid hydrogels.30 Gelatin is one of the most used precursors for preparation of a range of biohydrogels either alone or in combination with other biopolymers, such as chitosan or hyaluronic acid.31,32 Gelatin is a nontoxic and biodegradable protein substance obtained after partial denaturation of collagen and it displays a triple-helical structure, which easily forms physical hydrogels.33 Cross-linking is often used in order to improve both the thermal and the mechanical stability of gelatin-based hydrogels.31 Traditionally, chemically cross-linked gelatin-based hydrogels have been synthesized using glutaraldehyde as cross-linker.34 However, due to the toxicity concerns, in recent years, friendlier cross-linkers such as genipin or D , Lglyceraldehyde have been investigated,35,36 but the field of the gelatin-based hydrogels has been suffering from the lack of methodology to prepare stable nanocomposites. Gelatin-based bionanocomposites embedding Ag NPs have been previously prepared, resulting in materials with interesting properties such as thermo-reversible optical shifts37 or antibacterial activity.38 Gelatin was also used as stabilizing agent for the synthesis of Ag NPs for the preparation of thermo-responsive silver nanocomposite hydrogels composed of gelatin and N-isopropylacrylamide.39 Ag NPs were also used in combination with hydroxyapatite for the preparation of gelatin and poly(acrylic acid)-based hydrogels for the substitution of bone tissue and the influence of Ag NPs on the degradation behavior of the hybrid hydrogels was studied.40 Nevertheless, none of these strategies involves the binding of Ag NPs to gelatin chains and its use as multifunctional crosslinkers. Herewith, we present a new method to prepare bionanocomposite hydrogels based on the use of a furan modified gelatin with chondroitin sulfate (CS) and surface modified Ag NPs as multifunctional cross-linkers. Diels−Alder (DA) cycloaddition of benzotriazole maleimide capped Ag NPs was employed as a mild covalent strategy for covalent binding to the furan modified gelatin.41,42 The DA reaction is one of the common types of “click” chemistry and represents a suitable methodology for the preparation of chemically cross-linked hydrogels.43−46 This [4 + 2] cycloaddition can be performed under mild reactions conditions using water as a solvent and is characterized by the absence of secondary products.47,48 To the best of our knowledge, the DA reaction has not yet been employed for the formation of a silver NP-cross-linked biohydrogel. Herein we describe a methodology of nanocomposite hydrogel preparation and the effect of Ag NPs on the cross-linking and structural, swelling, and viscoelastic properties of hybrid hydrogels as well as their potential use as drug delivery systems.

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Scheme 1. Synthesis of BTM-Coated Silver Nanoparticles

balance. After that time, 66 μL of NaBH4 solution (37.8 mM) were added and the reaction was stirred in the ice bath for 1 h. Nanoparticles were then centrifugated and washed several times to obtain BTM-Ag NPs. Hydrogel Formation. Furfuryl-gelatin was dissolved in 800 μL of BTM-Ag NPs solution and CS was added in a 1:2 weight ratio with respect to G-FGE. EDC (5.2 × 10−4 mol, 80.0 mg) and NHS (4.6 × 10−4 mol, 53.2 mg) were added to the mixture, which was allowed to gel for 1 h at room temperature (Scheme 2) to obtain G-CS-BTM-Ag nanocomposite hydrogel. A control hydrogel without Ag NPs (G-CS) was prepared following the same procedure but using 800 μL of deionized water instead of the NPs solution. Characterization. Scanning Electron Microscopy (SEM). SEM experiments were performed by a JEOL JSM-6400 with a wolframium filament operating at an accelerated voltage of 20 kV and at a working distance of 15 mm. Freeze-dried samples were coated with approximately 20 nm of chromium using a Quorum Q150 TES metallizer. Transmission Electron Microscopy (TEM). TEM analysis of Ag NPs solution was performed using a transmission electron microscope TECNAI G2 20 TWIN (FEI), operating at an accelerating voltage of 200 keV in a bright-field image mode. A solution drop was deposited on a carbon film copper grid and the sample was spin coated. In order to perform the TEM analysis of the bionanocomposite hydrogel, samples were prepared by casting the hydrogel precursor solution using the same procedure as described for nanoparticles solution. The samples were allowed to react for 1 h at room temperature, in the same way as that for the prepared BNC hydrogels. Rheology. Dynamic rheological behavior of the hydrogels was measured with a Rheometric Scientific Advanced Rheometric Expansion System (ARES), using parallel plate geometry (25 mm diameter). Frequency sweep measurements were performed at 37 °C from 0.1 to 500 rad s−1 at a fixed strain in the linear viscoelastic region, previously assessed by strain sweep experiments for each sample. Hydrogel samples were prepared in the form of disks of 25 mm diameter. Swelling and Degradation Studies. Swelling capacity of freezedried hydrogels was studied by a general gravimetric method. Samples (n = 3) were incubated at 37 °C in deionized water, physiological solution (NaCl 0.9% w/v), simulated gastric fluids (HCl 0.1 M), and simulated intestinal fluid (phosphate buffer (PBS), pH = 7.4). At

EXPERIMENTAL SECTION

Materials. Gelatin (from porcine skin Type A ≈ 300 Bloom), furfuryl glycidyl ether (FGE, 96%), chondroitin sulfate A sodium salt from bovine trachea (CS, ≥ 60.0%), N-hydroxysuccinimide (NHS, 98.0%), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, 99.0%), sodium acetate (99.0%), silver nitrate (AgNO3, ≥99%), sodium borohydride (NaBH4, ≥99%), trifluoroacetic acid, dichlorometane (DCM), and acetic anhydride were purchased from Sigma-Aldrich. Phosphate buffered saline (PBS) solution was prepared with tablets from Panreac (pH = 7.4). 5-Aminobenzotriazole was purchased from Alfa Aesar and acetonitrile (MeCN) from Scharlau. Deionized water was employed as solvent. All reagents and solvents were employed as received. B


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Scheme 2. Diels−Alder Cycloaddition-Based Cross-Linking between Furan-Modified Gelatin (G-FGE) and Maleimid-Coated Silver Nanoparticles (BTM-Ag NPs)

antibiotic-antimycotic solution (Sigma-Aldrich) at 37 °C for 24 h to obtain the extracted culture media. L-929 murine fibroblasts were seeded in 96-well microplates at a density of 4 × 103 cells well−1 and allowed to grow in the presence of standard culture medium for 24 h before the experiments. Then, for the cytotoxicity assays, cultures were incubated for 24, 48, and 72 h with the extracted media. High-density polyethylene (HDPE, USP Rockville, U.S.A.) and polyvinyl chloride (PVC, Portex, U.K.) were used as negative and positive controls, respectively. The metabolic activity of viable cells was determined using the colorimetric MTT assay (Sigma-Aldrich), in which 3-(4,5dimethyltriazol-2-yl)-2,5 diphenyltetra-sodium bromide is reduced to formazan by the mitochondria of living cells. The cell number per well was proportional to the amount of formazan crystals and was determined by measuring the absorbance at 540 nm using a microplate reader (ELISA). Viability (%) was calculated as following: [A]test/ [A]control × 100, where [A]test is the absorbance of the sample cells and [A]control is the absorbance of the negative control cells. All assays were conducted in triplicate and mean values and their standard deviations were calculated. Second, we evaluated cell adhesion and viability of L929 murine fibroblasts on biomaterial by performing a Live/Dead assay. Sterilized G-CS-BTM-Ag hydrogel shaped as discs of 0.5 cm3 were placed on 24well ultralow attachment culture plates (Sarstedt, Germany) and fibroblasts were seeded onto these structures at a density of 5 × 104 cells mL−1 in droplets of 15 μL for 1 h. When the cells were attached, 1 mL of the complete media was added to each well, and regular media changes were made. After 7 days, cells were incubated with 3.9 μM calcein-AM (Sigma-Aldrich) and 5.9 μM propidium iodide (Life Technologies, Carlsbad, U.S.A.) following manufacturer’s indications. Samples were then examined under a confocal microscope (Olympus LV500, Japan). Adhered viable cells stained green, while dead cells appeared red.

selected time intervals of 0.5, 1, 1.5, 2, 24, and 48 h, the swollen hydrogels were removed, the excess of water absorbed with a filter paper, and weighed. Hydrogels were then replenished with fresh solution. The swelling ratio (SR) was calculated using eq 1:

SR(%) =

(Ws − Wd) ·100 Wd

(1)

where Ws and Wd are the weight of the swollen and dried hydrogel samples, respectively. The equilibrium swelling was considered to be achieved when the weight of the hydrogels no longer increased. For degradation studies weighted hydrogel freeze-dried samples were immersed in PBS and incubated at 37 °C for 21 days. The weight of the samples was recorded at different time intervals and the swelling ratio was calculated as described before using eq 1. Drug Delivery Experiments. Antibiotic drug, chloramphenicol (ClPh), was used as a model drug namely because its water-solubility and its wide antibacterial spectrum.49 Freeze-dried samples of the hydrogels were loaded by soaking a 0.25 mg mL−1 aqueous solution of ClPh at 25 °C. Hydrogels were maintained in the drug solution for 3 h. After that time, the gel sample was removed from the solution, dried at ambient temperature and weighed. The amount of drug loaded was determined by the difference between the initial dried weight of the sample and the final dried weight after loading. The release experiments were carried out by shaking the dried hydrogel samples (kept in a metallic perforated container) in 80 mL of PBS 0.01 M, pH = 7.4 at 37 °C. After predetermined time intervals, 1.0 mL of the release medium was withdrawn and analyzed by UV−vis spectroscopy using a UV-3600/3100 from Shimadzu to determine the amount of drug released at each time point and the aliquot was returned to the beaker. The amount of ClPh released was quantified by comparing the absorbance at 275 nm with a standard calibration curve prepared for pure drug solutions in the appropriate concentration regions. The cumulative drug release was calculated using eq 2: m cumulative release(%) = t ·100 m0 (2)

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RESULTS AND DISCUSSION Synthesis and Characterization of BTM-Ag-NPs. Dienophile containing Ag NPs were prepared using benzotriazole maleimide (BTM) as a capping agent in a one-pot reaction, as previously reported (Scheme 1).41 Benzotriazole was chosen as surface capping agent by virtue of its strong interactions with metallic surfaces and its well-known silver anticorrosion effect.51 We have also recently shown that maleimide coated Ag NPs can further be used for Diels− Alder (DA) cycloaddition to furan-modified DNA strands,

where mt is the cumulative mass of ClPh released at time t and m0 is the total amount of ClPh loaded. Cell Culture Studies. To assess in vitro cell response to G-CS-BTMAg hydrogel, first we performed a short-term cytotoxicity assay, following ISO 10993 recommendations.50 Briefly, sterilized samples were immersed into standard cell culture medium (Dulbecco’s modified Eagle’s medium; Sigma-Aldrich St. Louis, MO, U.S.A.) plus 10% fetal calf serum (Gibco, Life Technologies, Carlsbad, U.S.A.) and C


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Figure 1. TEM image of the maleimide-coated BTM-Ag nanoparticles at different magnifications.

affording stable bionano hybrids.41 NPs were characterized by TEM, indicating that small, 2.2 ± 0.5 nm, Ag NPs were obtained (Figure 1). The concentration of the obtained BTMAg NPs was in the range of 2.1 nM, estimated from the amount of silver precursor and the average diameter of the final nanoparticles calculated from TEM.52 Hydrogel Synthesis and Characterization. The bionanocomposite hydrogel was synthesized by combining two different cross-linking reactions with furfuryl modified gelatin (G-FGE; Figure 2A). Maleimide-coated Ag NPs acted as multifunctional elements for covalent cross-linking of furan containing gelatin through DA cycloaddition (Scheme 2). In addition, to ensure the formation of stable hydrogels, furfurylgelatin and chondroitin sulfate (CS) were coupled through amide coupling between remaining free ε-amino groups of GFGE and the CS carboxylic groups in the presence of EDC and NHS. CS is a water-soluble glycosaminoglycan, and one of the main components of the extracellular matrix, which has previously been successfully used for the cross-linking of gelatin.53 Upon completion of both reactions, the formation of a hydrogel containing covalently bound Ag NPs (G-CS-BTM Ag) was achieved (Figure 2B). In addition, a control hydrogel (G-CS) without NPs was also prepared and it is shown in Figure 2C. The preparation of a bionanocomposite hydrogel using only BTM-Ag NPs as the cross-linking agent was also attempted. However, only resinous solutions were obtained, probably due to the reduced number of binding sites available on small Ag NPs present in a low concentration, which does not allow for an efficient cross-linking. TEM image of G-CS-BTM Ag hydrogel (Figure 2D) shows that BTM-Ag NPs were successfully dispersed into the hydrogel matrix without noticeable particle agglomeration, which might impair the properties and stability of the formed gel. The microstructure of hydrogels directly influences their final properties as drug delivery systems, whereas the kinetics will be affected, among other factors, by the porosity, density, and surface area of the hydrogel. Therefore, the microstructure of G-CS-BTM-Ag and G-CS hydrogels, swollen in PBS for 24 h and freeze-dried, was analyzed by scanning electron microscopy

(SEM). When compared, the microstructure of G-CS-BTM-Ag and G-CS hydrogels differs (Figure 3). G-CS-BTM-Ag showed a wrinkled surface with homogeneously distributed pits, while a combination of smooth and wrinkled areas as well as some randomly distributed holes were observed in the case of G-CS. Taking this observation into account, it could be expected that, when employed as drug delivery systems, G-CS-BTM-Ag hydrogel would have more interactions with the drug moieties due to the higher contact area. In contrast, in the case of control G-CS hydrogel, a collapsed and reduced surface area is obtained, which could, in turn, have an impact on the release properties of the system (discussed later in the paper). Rheological Behavior of Hybrid Hydrogels Influenced by BTM-Ag NPs. Before the drug absorption/release studies, rheological characterization of the hydrogels was performed to study the viscoelastic properties of both nanocomposite G-CSBTM-Ag and the control, G-CS hydrogels at 37 °C using parallel plate geometry. The frequency sweep test performed for both samples at the constant strain is shown in Figure 4. Three samples were analyzed in each case and the calculated mean values of the storage (G′) and the loss modulus (G″) are presented in Table 1. Several conclusions could be extracted from the obtained rheological data. First of all, for both systems, G″ was smaller than G′, indicating the formation of an elastic network. Furthermore, the presence of a stable crosslinked network was confirmed since G′ was constant within the used frequency range,54 confirming the efficacy of the crosslinking strategy. When both hydrogels are compared, G-CSBTM-Ag showed higher (almost three times, 19927 vs 7381 Pa) G′ value than G-CS sample. This result confirmed the significant role played by Ag NPs within the hydrogel network. The storage moduli of this bionanocomposite hydrogel was close to that of liver, fat, relaxed muscle, and breast gland tissue (103−104 Pa), which is one of the key parameters for potential biomedical applications.55 Effect of Bionanocomposite Network over Swelling Properties and Stability. Another parameter to be evaluated for the development of materials such as drug delivery systems or muscle-like actuators is the swelling capacity of hydrogels.56,57 The ability of the hydrogel to absorb and retain liquids would be decisive for its later use as drug delivery system. For D


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Figure 2. (A) Schematic representation of G-CS-BTM-Ag hydrogel, (B) picture of G-CS-BTM-Ag hydrogel, (C) picture of the control G-CS hydrogel, and (D) TEM image of G-CS-BTM-Ag hydrogel.

that purpose, swelling experiments were performed at 37 °C in different media; water, acidic solution simulating gastric fluids (HCl 0.1 M), physiological solution (NaCl 0.9% w/v), and simulated intestinal fluid (phosphate buffer solution (PBS), pH = 7.4). The equilibrium swelling ratios as a function of the swelling media for G-CS-BTM-Ag and G-CS hydrogels are shown in Figure 5. The swelling ratios (SR) of the bionanocomposite hydrogel in all the solvents were smaller than that of the control, indicating that the maleimide-coated NPs did play the expected role since cross-linked hydrogels

usually show low swelling ratio.43 Furthermore, the equilibrium swelling was reached faster in the case of G-CS-BTM-Ag than for G-CS, especially in water and PBS, indicating that G-CSBTM-Ag hydrogel represents the more stable system. The highest swelling ratios were observed in water and PBS as solvents. Upon immersion in physiological solution (NaCl in Figure 5), hydrogels displayed intermediate swelling ratios. The presence of salts such as NaCl can produce a screening effect thus, lowering the repulsion between polymer chains and, consequently, leading to the reduction of the swelling E


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Figure 3. SEM images of (A) G-CS-BTM-Ag and (B) G-CS hydrogels.

hydrogen bonding between gelatin chains as reported previously.59 The stability of bionanocomposite hydrogels over time was also explored (PBS solution at 37 °C, Figure 6). Again, G-CS-

Figure 4. Frequency sweep of hydrogels at 37 °C: G′ (filled symbols) and G″ (empty symbols); ■ G-CS-BTM-Ag and ● G-CS.

Table 1. Storage Moduli (G′) and Loss Moduli (G″) Values for G-CS-BTM-Ag and G-CS Hydrogels at 37 °Ca

a

sample

G′ (Pa)

G″ (Pa)

G-CS-BTM-Ag G-CS

19927.3 ± 6371.6 7381.8 ± 1421.2

5068.1 ± 837.1 1564.0 ± 567.4

Figure 6. Swelling ratio (SR) of G-CS-BTM-Ag and G-CS in PBS solution at 37 °C.

BTM-Ag displayed lower swelling ratio (SR) than the control hydrogel, however there was no significant mass loss over the period of 21 days. The swelling ratio for G-CS-BTM-Ag remained constant during the time range of the study, whereas the SR of G-CS hydrogel samples first increased and then it started to decrease after 10 days of incubation. It has to be taken into account that during the study, the mass loss could have started earlier, even though a weight increase was recorded, since both the weight gaining and the mass loss phenomena take place concurrently.60 Furthermore, under the studied conditions, the degradation could only be attributed to the formation of a more swelled and disbanded structure, which resulted in partial solubilization of the hydrolyzed sample. Under physiological conditions, where reagents or enzymes are present, which can damage the hydrogel network, the rate and extent of degradation would probably increase.53 However, the obtained results indicated that the reduced swelling capacity of the bionanocomposite hydrogel resulted in its stability, preventing the early decomposition of the sample, making it a suitable candidate material for further applications. Following the study of the nanocomposite hydrogel properties, we were interested in exploring its drug delivery potential. Controlled Drug Release from Bionanocomposite Hydrogel. The drug release properties of the hydrogels were assessed in PBS at 37 °C using antibiotic chloramphenicol

Average ± standard deviation, n = 3.

Figure 5. Equilibrium swelling data for G-CS-BTM-Ag and G-CS hydrogels after incubation in water, HCl (0.1 M), NaCl (0.9 % wt) and PBS (pH = 7.4) solutions at 37 °C.

capacity.58 At acidic pH, lower swelling ratios were detected, which could be attributed to the inter- and intramolecular F


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the cumulative release of ClPh varied. G-CS released up to 27% of the loaded drug, whereas a significantly higher (78%) drug release was recorded for G-CS-BTM-Ag hydrogel. It should also be noted that the release profiles differed from one formulation to another. G-CS showed a fast initial release, the steady concentration was achieved almost immediately and the resulted drug release was low. In contrast, for G-CS-BTM-Ag hydrogel, a more sustained drug release was recorded. One possible explanation to these different behaviors could be related to the fact that G-CS hydrogel displayed higher degree of swelling resulting in a deeper penetration of the drug moieties into the bulk of the hydrogel and the released drug molecules would predominantly be those, which are surface associated. The difference in the release profiles could also be a result of the chemical structure of the networks, in particular the contact area of nanocomposite hydrogel as observed in the SEM images of the microstructure (Figure 3). Finally, we used a method proposed by Peppas et al. to further explore the drug release.61 Namely, more light onto the possible mechanism of drug release can be deduced from a simple expression (eq 3):

(ClPh) as a model drug. The amount of ClPh released was monitored by UV−vis spectroscopy allowing also for the quantification of the cumulative release (Figure 7). The drug

Figure 7. Drug release profile of G-CS and G-CS-BTM-Ag hydrogels at 37 °C in PBS using chloramphenicol (ClhP) as a model drug.

loading for each case was related to the swelling properties of the two different samples, and thus, higher drug loadings were recorded for G-CS hydrogel (nearly 9 mg ClPh g−1 hydrogel for G-CS and 0.7 mg ClPh g−1 for G-CS-BTM-Ag). However,

Mt = k·t n M∞

(3)

Figure 8. Cytotoxicity assays. (A) Cell viability of L-929 murine fibroblast cultured with extracted medium obtained from PVC (positive control) and G-CS-BTM-Ag hydrogel with respect to that obtained from HDPE (negative control); (B) and (C) Live/Dead assay for G-CS-BTM-Ag hydrogel, where alive cells appear in green and dead cells in red (bar scale 100 μm). G


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where Mt and M∞ are the amount of drug released at time t and infinite time, respectively, k is a constant incorporating structural and geometric characteristics of the device, and n is the release exponent, indicative of the mechanism of drug release. For the as-prepared bionanocomposite hydrogel, the obtained value of n was 0.64, indicating that the drug release is a case of anomalous transport where two apparently independent mechanisms are superposed, diffusion controlled drug release (Fickian transport) and swelling controlled drug release (case II transport).59 In Vitro Cell Response Evaluation of Hybrid Hydrogel. Finally, taking into account the drug release properties and potential toxicity of incorporated Ag NPs, preliminary in vitro cytotoxicity assays were performed by investigating material extracts and direct cell-material interactions with L-929 murine fibroblast cells. Cytotoxicity assays based on DIN EN ISO 10993−1−12 were carried out and confirmed the favorable cytocompatibility of G-CS-BTM-Ag hydrogel. Figure 8A shows the cell viability at 24 and 48 h for positive control (polyvinyl chloride, PVC) and for G-CS-BTM-Ag hydrogel sample in comparison with negative control (high-density polyethylene, HDPE). As expected, the positive control showed a toxic effect, as the L-929 murine fibroblasts were not able to proliferate. GCS-BTM-Ag hydrogel was significantly less toxic, with cell growth being similar to that of the negative control. Thus, cell viability was higher than 70% for the first 24 h and close to 100% after 48 h of incubation. Cytotoxicity was also additionally evaluated by direct cellmaterial contact. For this assay, cells were seeded onto the hydrogel and cell viability was evaluated using a Live/Dead assay, where live cells are stained in green and dead cells in red. G-CS-BTM-Ag hydrogel showed an adequate level of cell survival as indicated by large number of fluorescently green cells, and only few red stained dead cells (Figure 8B,C). Therefore, cytotoxicity and cell viability studies revealed that the as-prepared bionanocomposite hydrogel is a good candidate for biomedical applications.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Basque Country Government in the frame of Saiotek S-PE12UN036 and Grupos Consolidados (IT776-13) and from the University of the Basque Country (UPV/ EHU) in the frame of EHUA12/19 is gratefully acknowledged. C.G.-A. wishes to acknowledge the Universidad del Paiś Vasco/ Euskal Herriko Unibertsitatea (Ayudas para la Formación de Personal Investigador) for its Ph.D. grant PIFUPV10/034. C.C. acknowledges the DAAD Ph.D. scholarship and L.F. DFGCFN A5.7 Project. Moreover, technical and human support provided by SGIker (UPV/EHU, MINECO, GV/EJ, ERDF, and ESF) is also gratefully acknowledged.

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CONCLUSIONS A bionanocomposite hydrogel based on gelatin and chondroitin sulfate containing covalently bound silver nanoparticles was prepared. Maleimide-coated silver nanoparticles were used as cross-linkers via Diels−Alder cycloaddition to furan-modified gelatin. The as-prepared hydrogel was characterized by SEM and TEM and its swelling and degradation properties were studied. The role of silver nanoparticles as cross-linkers of the gelatin gels was confirmed by rheological and swelling measurements. Higher storage modulus and significantly lower swelling ratios were observed for bionanocomposite hydrogels when comparing with nanoparticle-free control, indicating the formation of more stable cross-linked networks when maleimide-coated silver nanoparticles were present. Finally, drug release and biocompatibility studies were performed and the as-prepared bionanocomposite hydrogel displayed potential for design of drug delivery systems and low toxicity, opening a new route to the number of potential biomedical applications such as controlled therapeutic delivery or tissue engineering.

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REFERENCES

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