Article pubs.acs.org/Biomac
Self-Healing Fish Gelatin/Sodium Montmorillonite Biohybrid Coacervates: Structural and Rheological Characterization Nader Taheri Qazvini,*,† Sreenath Bolisetty,‡ Jozef Adamcik,‡ and Raffaele Mezzenga*,‡ †
Polymer Division, School of Chemistry, College of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran Food and Soft Materials Laboratory, Department of Health Science and Technology, ETH Zurich, Schmelzbergstr. 9, LFO E22, 8092 Zurich, Switzerland
‡
ABSTRACT: Complex coacervation driven by associative electrostatic interactions was studied in mixtures of exfoliated sodium-montmorillonite (Na+MMT) nanoplatelets and fish gelatin, at a specific mixing ratio and room temperature. Structural and viscoelastic properties of the coacervate phase were investigated as a function of pH by means of different complementary techniques. Independent of the technique used, the results consistently showed that there is an optimum pH value at which the coacervate phase shows the tightest structure with highest elasticity. The solid-like coacervates showed an obvious shearthinning behavior and network fracture but immediately recovered back into their original elastic character upon removal of the shear strain. The nonlinear mechanical response characterized by single step stress relaxation experiments revealed the same trend for the yield stress and isochronal shear modulus of the coacervates as a function of pH with a maximum at pH 3.0 and lower values at 2.5 and 3.5 pHs, followed by a very sharp drop at pH 4.0. Finally, small-angle X-ray scattering (SAXS) data confirmed that at pHs lower than 4.0 the coacervate phases were dense and structured with a characteristic length scale (ξSAXS) of ∼7−9 nm. Comparing the ξSAXS with rheological characteristic length (ξrheol) estimated from low-frequency linear viscoelastic data and network theory, it was concluded that both the strength of the electrostatic interactions and the conformation of the gelatin chains before and during of the coacervation process are responsible for the structure and rigidity of the coacervates.
1. INTRODUCTION In recent years, new classes of advanced materials have been developed through the assembly of biomacromolecules with nanosized inorganic solids.1−3 These nanostructured biohybrids show improved properties, which can be tuned by both selecting the appropriate constituents as well as controlling the procedures by which these are brought together. Complex coacervation, arising from electrostatic interactions between two oppositely charged macromolecules or a polyelectrolyte and an oppositely charged colloid, is also a general route to prepare complex fluids.4−6 During complex coacervation, insoluble bound complexes are formed, which then coalesce and demix from the solution to form two incompatible and immiscible phases. The dilute liquid phase, which is usually the supernatant, remains in equilibrium with the coacervates phase. When prepared via complex coacervation between a biopolymer and an oppositely charged nanoparticle, the coacervate phase can be considered as bio nanohybrid material.7 Coacervation has been observed in a wide range of charged macromolecular systems, for instance systems containing a protein and a polysaccharide.5,8 Gelatin, the macromolecule investigated here as the organic component in the coacervation process, is well-known for its excellent biocompatibility and controllable biodegradability. It is nonimmunogenic and noncarcinogenic in nature and possesses a relatively low © 2012 American Chemical Society
antigenicity. Due to these advantages, gelatin is becoming a focal point of interest for new uses in health care and in specialized technical areas.9 Moreover, because of a unique sequence of amino acid groups and the denatured state of this protein, its phase behavior in dilute and semidilute solutions can be easily controlled and tuned by pH, ionic strength, and temperature without the necessity of additional functionalization.9 Among charged inorganic nanomaterials, layered silicates, for example, montmorillonite (MMT), are of particular interest when blended to biomacromolecules, due to their ability to improve functional properties of biopolymers while preserving their biocompatibility.10−12 Sodium montmorillonite (Na+MMT), one of the most commonly used clay minerals, consists of a lamellar stack of crystalline, 1 nm thick aluminosilicate sheets. MMT is also a medicinal material. Owing to their ability to form associative interactions with clotting factors, layered silicate clays such as kaolin and MMT have been shown to induce blood coagulation.13 Apart from the widespread applications in traditional medicine, layered silicates have gained attention also in other advanced biomedical uses such as, for example, tissue engineering.14 Furthermore, as a strong Received: April 4, 2012 Revised: May 22, 2012 Published: May 29, 2012 2136
dx.doi.org/10.1021/bm3005319 | Biomacromolecules 2012, 13, 2136−2147
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a correlation length of about 35 ± 3 nm and a melting temperature, Tm = 312 ± 4 K, was determined for Laponite/ gelatin-A complexes at the spinodal point.28 Pawar and Bohidar29 discussed theoretically the kinetics of phase separation and complex formation in a ternary system containing oppositely charged polyion and macroions. They assumed that the phases are in thermodynamic equilibrium and used lattice model in Flory formalism. The physical condition for phase separation were deduced based on the Flory interaction parameter between the solvent and the complex, ionic strength, and surface charge on the complex. Although, these studies have brought new valuable insights into the complexity of gelatin/clay coacervate systems, to date, no work has tackled the final material properties of the resulting materials and attempted to elucidate systematically the structure−properties relationship. From practical point of view, the gelatin/MMT coacervate materials can potentially be employed in a wide range of applications, in addition to the current use of coacervates for microencapsulation purposes. As novel biocompatible materials, these systems can be used in the production of highly performing barrier films and microcapsules or high-strength scaffolds for tissue engineering. However, to obtain truly engineered, designed, and functional biohybrid coacervates, efforts should be undertaken to understand the fundamental aspects of the structure−property−processing relationship of these materials.30 In the present work, we have combined for the first time atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), diffusing wave spectroscopy (DWS), and linear and nonlinear rheology to gain insights into the microscopic structure of the gelatin-Na+-MMT coacervates prepared at different conditions and to understand how the mechanical properties of the coacervates depend on their structure.
detoxifier, MMT could adsorb dietary, bacterial, and metabolic toxins, which are believed to be responsible for nausea, vomiting, and diarrhea15 and which are typical symptoms of the side effects of anticancer drugs.16 Charge-wise, for montmorillonite clay, the sum of all the oxide anions is in weak excess compared to the total positive charge contributed by structural cations (i.e., Mg2+, Al3+, Fe2+, Fe3+, Si4+), and therefore, the surfaces of the clay sheets bear an overall negative charge. Exchangeable Na+ cations occupy the space between the sheets to balance this overall negative surface charge of the MMT.15 Therefore, positively charged macromolecules/Na+-MMT mixtures can undergo complex coacervation via associative mechanism. Complexes based on the combination of clay minerals and proteins have been studied for decades.17 As early as 1939, Ensminger and Gieseking18 prepared complexes of montmorillonite with gelatin by acidifying an alkaline (pH 10) dispersion of the Na+-MMT containing different amounts of protein. In their work, X-ray diffraction of the dry complexes showed that intercalation had occurred and, for a given clay/protein ratio, the intergallery distance of clay increased with a decrease in suspension pH (from 7 to 2.7). The complex formation was primarily attributed to an exchange of the gelatin and the Na+ ions occupying exchange sites at the MMT surface by the cationic (−NH3+) groups on the amino acid side chains. More detailed studies on the intercalation of montmorillonite by gelatin were made by Talibudeen,19 Pinck et al.,20 McLaren et al.,21 and Weiss22 (1969) using X-ray diffraction techniques. At larger polycations concentrations, however, the electrostatic complexation may result in the destruction of the lamellar stacks, and when combined with mechanical or ultrasonic treatment of the aqueous clay suspensions, this can generate fully exfoliated, electrostatically stabilized dispersions of nanoplatelets. Under these conditions, the high density of ionic charges on thin MMT platelet surfaces is anticipated to show unusual properties. A rough calculation based on surface specific areas of about 720 m2/g and cation exchange capacity of 120 meq/100 g suggests numbers of the orders of 20000 ions per platelet, corresponding to the very high surface charge density of 0.9 nm2 area per ion, and reveal the high capability of the material to interact with polar molecules.23 Furthermore, a highly charged MMT surface is able to polarize the gelatin molecules, thus, enabling favorable associative interactions even when both possess the same charge sign.24 A scaling theory description of this phenomenon has been successfully developed by Dobrynin et al.25 Recently, Pawar and Bohidar26 have investigated the intermolecular binding and the initial stage of liquid−liquid phase separation in Laponite/gelatin solutions under various conditions. Depending on gelatin type (A or B) and the ionic strength, they noticed that different binding strength resulted in complexes with different sizes. Moreover, considering Laponite and gelatin as a point charge and a dipole, respectively, they could model the binding problem.27 Using depolarized dynamic light scattering experiments, they also showed that below a distinct temperature, the early stage of liquid−liquid phase separation in their system could be described adequately through Cahn−Hilliard theory of spinodal decomposition.28 They deduced that coacervation transition in gelatin/Laponite system proceeded via the formation of anisotropic ellipsoidal soluble complexes which grow in time along their equatorial axis, while they shrink in their polar direction.26 Via small amplitude rheological measurements, a network structure with
2. EXPERIMENTAL SECTION 2.1. Materials. Teleostean gelatin (cold water fish skin, Mw ∼ 60 KDa) was obtained from Sigma-Aldrich (U.S.A.). Gelatin powder was used as received. Pure water was obtained from an on-site filtration unit (Millipore, QPOD, 18.2 MΩ.cm, Millipore, Billerica, MA). Sodium montmorillonite (Na+-MMT), Kunipia-F (KF), with a cation exchange capacity (CEC) of 119 meq/100 g and basal spacing of 1.18 nm was supplied by Kunimine Co., Japan, and used without further purification or surface treatment. Hydrochloric acid solutions, for pH regulation, were diluted from a concentrated (∼37% w/v) HCl−water solution (Sigma Aldrich). 2.2. Coacervates Preparation. Typically, 1% (w/v) gelatin solution was prepared by dissolving gelatin powder in the solvent at 40 °C and mixing it for 2 h using a magnetic stirrer. Then, the stock solution was aliquoted into 20 mL precleaned glass vials and the pHs of the solutions were adjusted to 2.5, 3.0, 3.5, 4.0, respectively. A homogeneous aqueous dispersion of MMT was prepared by mixing of Na+-MMT with pure water, followed by ultrasonication for 10 min using a Sonifier 450 (Branson, Danbury, CT) working at duty cycle of 60% and power 6.0. The pH of Na+-MMT dispersion was 9.2. The coacervates were prepared by mixing equal volumes of gelatin solutions (1% w/v) at the desired pH and Na+-MMT dispersion (0.75% w/v, pH 9.2) for 1 min using a magnetic stirrer at 300 rpm. Depending on the initial pH of the gelatin solution, the average pH in the coacervation media was between 3.6 and 5.25 (see Figure 1). The coacervate samples were then designated as pH X, where X stands for the pH of gelatin solution before coacervation. Sodium azide (0.02% w/v) was added to prevent bacterial growth. The ratio of gelatin to nanoclay was kept 4/3 in all samples. This optimum ratio was obtained after a series of preliminary coacervation experiments at different ratios and pH values. Characterization of the composition of the elastic 2137
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controlled rheometer (AR2000, TA Instruments). All measurements were performed using stainless steel cone and plate geometry (20 mm diameter and angle 2°) at 25 °C with a truncation gap of 59 μm. A solvent trap provided by the manufacturer for liquid samples was used to prevent loss of solvent. First, oscillatory stress and strain sweeps were performed to determine the linear viscoelastic region for each sample. Subsequently, the dynamic moduli (G′ and G″) were determined using an oscillatory time sweep test for 10 min at a constant strain of 1% and constant frequency of 1 Hz. Frequency sweep experiments were performed at a low amplitude of strain (in linear regime) in the frequency range of 0.1−100 rad/s. To investigate the recovery kinetics of gelatin/MMT coacervates after network destruction, viscoelastic properties of gel-like samples (pH 2.5, 3, and 3.5) were measured as a function of time in an oscillatory time sweep (5 min, 1% strain, 1 Hz frequency) before and after severe destruction of the gel network (100% strain, 1 min, 1 Hz frequency). All measurements were performed in triplicate (n = 3) to ensure data reproducibility (relative standard deviation less than 5%). The nonlinear viscoelastic response of the coacervates were characterized by adopting a rheological method, as described by McKenna et al.33 for determining the stress−strain and stress relaxation responses of soft materials. Briefly, single-step stress relaxation experiments were performed for approximately 80 s and at increasing deformation magnitudes applied clockwise and counter clockwise, alternately. The just prior strain history was diminished by considering a 800 s waiting time before measurement at the next larger deformation. This sequence was continued to the maximum strain of 20%. The single step stress relaxation data were used to plot the modulus vs time data at specific strain levels as well as stress vs strain isochrones. 2.7. Small-Angle X-ray Scattering (SAXS). Small-angle X-ray scattering experiments were performed using a microfocused Nifiltered Cu Kα X-rays radiation (Rigaku, wavelength λ = 1.54 Å). The applied voltage and filament current were 45 kV and 88 mA, respectively. The samples were held in 1.5 mm quartz X-ray capillaries and exposed to the radiation collimated with a set of three pinholes. The data were collected in a two-dimensional argon-filled detector and the scattering vector q = (4π/λ)sin θ, with 2θ being the scattering angle, was calibrated using silver behenate. The sample−detector distance was chosen to be 1 m, which provided a q range from 0.005 to 0.2 Å−1. Data were collected and azimuthally averaged to yield 1D intensity versus scattering vector q. All measurements were performed at 20 °C, and scattered intensities were collected over 720 min.
Figure 1. Electrophoretic mobility of gelatin solutions and Na+-MMT dispersions in pure water as a function of pH. The region of the final pH of coacervation in the range 3.6 and 5.25 is highlighted. The four vertical lines in the coacervation region correspond to the final mixture pHs after mixing Na+-MMT dispersions with gelation at pH 2.5, 3, 3.5, and 4, respectively. coacervate phases by freeze-drying indicated that all elastic coacervates contained ∼98 wt % water. The coacervation vials were sealed and stored at room temperature (25 °C) for a week and were then subjected to a mild centrifuge at 3000 rpm for 2 min. The mixtures separated into two different liquid phases, namely, the dense coacervates at the bottom and the supernatant at the top. 2.3. Electrophoretic Mobility Measurements. Electrophoretic mobility of gelatin, nanoclay dispersion and the coacervates was determined by the Zetasizer Nano ZS dynamic light scattering device (Malvern Instruments, Worcestershire, U.K.). Samples were placed in capillary cells (DTS 1061, Malvern Instruments) and the electrophoretic mobility was measured using a combination of the electrophoresis and laser Doppler velocimetry techniques. All measurements were carried out at 25 °C and at least in three replicates. The results are presented as averages with error bars representing the standard deviation between measurements. 2.4. DWS Measurements. Theoretical background of the technique has been described in detail elsewhere.31 The measurements were carried out in a backscattering mode with a commercial DWS apparatus (LS Instruments, Fribourg, Switzerland) equipped with the multispeckle echo technique for long correlation times.32 The coacervating systems were contained in a flat glass cell (Hellma 110OS) with an optical path length of 10 mm and placed in the sample holder. A 683 nm laser was used as the light source (40 mW) and backscattered light was collected on a photomultiplier. The detected intensity autocorrelation function was determined with a digital correlator and the electric field autocorrelation function (g2(τ) − 1) was determined. The temperature of the samples was kept at 25 ± 0.2 °C by a Peltier temperature controller. 2.5. Atomic Force Microscopy Measurements. Coacervate and pristine gelatin and nanoclay morphologies were investigated in a dry state using atomic force microscopy. In the case of coacervates, the measurements were performed on noncentrifuged samples prepared with 10 times diluted gelatin and clay dispersions. A 20 μL aliquot of all samples was deposited onto freshly cleaved mica, incubated for 2 min, rinsed with Milli-Q water, and dried by air. Images were collected using a MultiMode VIII (Bruker, U.S.A.) operated in intermittent mode under ambient conditions. The microscope was covered with an acoustic hood to minimize vibrational noise. Aluminum coated silicon cantilevers (Bruker) with a nominal tip radius of
