Fish gelatin/Laponite biohybrid elastic coacervates: A complexation kinetics–structure relationship study

https://doi.org/10.1016/j.ijbiomac.2013.06.054Get rights and content

Abstract

Complex coacervation in exfoliated Laponite nanoplatelets and fish gelatin mixtures was studied as a function of four key parameters: pH, ionic strength, gelatin/Laponite weight ratio, and total weight. The aim was to understand how these parameters influence phase separation kinetics, composition, internal structure, and viscoelastic properties of coacervates. By careful experimental design and turbidity measurements, the optimum conditions for coacervation were obtained. Thermogravimetric analysis revealed an outstanding heat-resistance for gelatin/nanoclay coacervates. Finally, structure of the coacervate phase was characterized by oscillatory shear experiments. The storage modulus data was observed to follow a power-law behavior and it was confirmed that under the optimum conditions, the coacervate phase was dense and structured with a characteristic length scale (ξrheol) of ∼8.25 nm. Regardless of the physicochemical condition at which complexation occurred, it was shown that the equilibrium structure of the coacervates is related to the kinetics of intermediate and late stages of phase separation; as the new defined kinetics parameter K was observed to be inversely proportional to ξrheol that quantifies the compactness of the coacervate networks.

Introduction

Nowadays, an increasing number of researchers have focused their attention on a study of new classes of advanced materials, for instance, materials based on interaction of biomacromolecules with nanosized inorganic solids [1], [2], [3].

Coacervation is a process in which a homogenous solution of charged macromolecules can be driven toward a liquid–liquid phase separation in which the stock solution is separated into two coexisting phases that are immiscible, incompatible and in thermodynamic equilibrium. The dense phase that contains a big amount of macromolecules is called coacervate and the dilute, transparent one is called supernatant. The basis of coacervation is a charge-neutralization that can occur in a homogenous solution containing either a polyampholytic macromolecule or two oppositely charged macromolecules (or a polyelectrolyte and an oppositely charged colloid); the former is named simple coacervation and the latter complex coacervation [4], [5], [6].

When complex coacervation takes place between a biopolymer and an oppositely charged nanoparticle, the coacervate phase can be considered as a nano-biohybrid material with improved properties [7]. Nevertheless, most of the studies on complex coacervation have been carried out on the protein/polysaccharide systems [5], [7]. Regarding the biopolymer investigated here, gelatin is widely used in pharmaceutical, food industry and also in photographic applications [8]. It has many merits, including biodegradability, biocompatibility, and nonimmunogenic and noncarcinogenic properties in nature. These advantages make gelatin an appealing biomacromolecule in specialized technical areas and in health care [9]. In addition to its exclusive properties, gelatin molecules exhibit an excellent versatility due to the presence of both positively and negatively charged amino acids. Phase behavior of gelatin chains in dilute and semidilute solutions can be easily organized and tuned by pH, ionic strength, and temperature without the necessity of additional functionalization [10].

In recent years, fish gelatin has gained much attention as an alternative to mammalian gelatin due to several factors. It can be easily extracted from byproducts of fish processing industry and more importantly, there are no ethic- or safety-related consumer concerns [11]. However, fish gelatin possesses lower gelling and melting temperatures and weaker gel strength [12], [13].

In recent years, it has been shown that some nanomaterials can effectively blend with biomacromolecules because of their ability to improve functional properties while preserving their biocompatibility [14], [15]. As a charged inorganic nanomaterial with aforementioned ability, Laponite is used in this study. It is a unique specialty additive, which improves the performance and properties of a wide range of industrial and consumer products. It can be used as a rheology modifier to improve stability and syneresis control, and as a film former to produce electrically conductive, antistatic and barrier coatings [16].

Laponite is an entirely synthetic clay, however its crystal structure and composition is closely similar to the naturally occurring smectite clays–hectorite and bentonite [17]. It has a layered structure with aspect ratio ∼30 (disk diameter ∼30 nm and thickness ∼1 nm) and density of ∼2.53 g cm−3. Laponite discs have a negatively charged face and positively charged edges and can be easily dispersed in water in a form of disk-shaped crystal colloids [16]. Above a critical concentration of ∼2% (w/v), Laponite discs assemble through interparticle electrostatic forces and consecutively, the homogeneous sol transform into a soft gel [17].

Due to presence of negative charges on the Laponite surface, positively charged macromolecules and Laponite mixture can undergo complex coacervation via associative mechanism [17], [18], [19], [20]. Investigations related to the complexation tendency of various organic substances including humic colloid of soil and proteins with clays were developed many years ago [21], [22], [23].

Recently, Pawar and Bohidar have investigated intermolecular binding between Laponite and gelatin (type A or B) under various ionic strength of the solution. They noticed that the strong electrostatic interactions driven by binding of gelatin chains to Laponite nanoclays and different binding strengths resulted in complexes with different sizes. Their results revealed that complex sizes and also intrinsic viscosity was raised by increasing the salt concentration. Moreover, potential energy has been calculated by treating Laponite as a point charge and gelatin as a dipole [17]. They also used depolarized dynamic light scattering experiments for investigating the initial stages of liquid–liquid phase separation in Laponite/gelatin-A systems. Their studies showed that below a distinct temperature, the initial phase separation is governed by spinodal decomposition and the behavior could be described adequately through Cahn–Hilliard theory. Via small amplitude rheological measurements, a network structure with a correlation length of 35 ± 3 nm and a melting temperature of Tm = 312 ± 4 K was determined for the coacervate phase [18]. Furthermore, they showed that phase separation proceeded via the domains formed of soluble complexes, which subsequently underwent time-dependent anisotropic growth. Growth in the equatorial axis of domains with time was observed to follow a power law behavior with exponent of 0.25, while the polar axis shrunk had a smaller exponent of 0.15 [19]. In another work, Pawar and Bohidar [20] formulated a statistical thermodynamic model and applied the Flory–Huggins lattice model to describe phase separation in a ternary system undergoing complex coacervation. Their deductions showed that the coacervate yield was directly related to the intermolecular interactions operating between the soluble complex and the solvent [20].

Just recently, structural and rheological characterization of fish gelatin/sodium montmorillonite coacervates as a function of pH of the gelatin solution before mixing has been studied. Using different experimental methods including atomic force microscopy (AFM), diffusion wave spectroscopy (DWS), small angle X-ray scattering (SAXS) and rheological measurements, the authors introduced pH 3 as the optimum pH, at which the coacervate phase showed tightest structure and highest elastic properties [10].

Although a lot of information on the complexation of gelatin-nanoclay can be found in these studies, a comprehensive study about how various parameters influence each other, study on the phase separation kinetics, and structure and properties of the final material are missing.

Due to ease of preparation and biocompatibility, gelatin/Laponite complex materials can be potentially employed in the production of highly performing barrier films, microcapsules, and high strength porous structures for tissue engineering. Therefore, motivation of the present study is to generate a complete and complementary set of data on kinetics of phase separation and structure–properties relationships. We evaluate the effect of different conditions on the kinetics of complexation, macro- and microscopic structure of gelatin/Laponite coacervates and understanding how rheological and compositional properties of the complexes depend on their network structures.

Section snippets

Materials

Teleostean gelatin (cold water fish skin, Mw  60 kDa) was obtained from Sigma–Aldrich (USA). Laponite RD, nanoclay was donated from Southern Clay products (USA). Hydrochloric acid (HCl, Merck) solutions and sodium chloride (NaCl, Merck) were used for pH and ionic strength regulation, respectively. Milli-Q water was used for all the experiments. All the materials were used as received.

Coacervates preparation

The stock solution of 1.5% (w/v) gelatin was prepared by slow adding of gelatin powder to milli-Q water at 40 °C

Zeta potential measurement

Prior to complex coacervation experiments, the first purpose was to ascertain the most appropriate pH range of gelatin chains before mixing them with nanoclay particles (pH 9.8) for formation of electrostatic complexes. For this objective, zeta potential of gelatin was measured when pH varied from 2.8 to 10.0. The zeta potential data of Laponite was extracted from [18], reported in the same pH range.

As shown in Fig. 1, the zeta potential is negative for Laponite nanoclay in the whole tested pH

Conclusions

The target of this work was to perform a fundamental study on the effect of physiochemical parameters on the kinetics of formation, structure and properties of fish gelatin-Laponite nanoclay coacervate systems. Design expert software was used to determine the optimum conditions under which all governing parameters including pH, ionic strength (I), gelatin/Laponite weight ratio (G/L), and total weight (Wt) are in their best magnitude and kinetics of the phase separation is the fastest. The

Acknowledgments

Partial financial support from the Iranian Nanotechnology Initiative and vice-president for research and technology of University of Tehran is gratefully appreciated. Zeta potential measurements were performed at the Laboratory of Food and Soft Materials, ETH Zurich, Switzerland. The authors also thank Ms. Barbora Ehrlichová for proofreading of the manuscript.

References (46)

  • S. Bolisetty et al.

    Journal of Colloid and Interface Science

    (2011)
  • C.L. Cooper et al.

    Current Opinion in Colloid & Interface Science

    (2005)
  • E. Kizilay et al.

    Advances in Colloid and Interface Science

    (2011)
  • A.A. Karim et al.

    Food Hydrocolloids

    (2009)
  • Y. Yang et al.

    Food Chemistry

    (2012)
  • I.J. Haug et al.

    Food Hydrocolloids

    (2004)
  • N. Pawar et al.

    Advances in Colloid and Interface Science

    (2011)
  • R. Khani et al.

    Desalination

    (2011)
  • J. Eysturskard et al.

    Food Hydrocolloids

    (2009)
  • O. Malay et al.

    International Journal of Biological Macromolecules

    (2007)
  • D.J. Burgess et al.

    Journal of Colloid and Interface Science

    (1984)
  • C.G. De Kruif et al.

    Current Opinion in Colloid & Interface Science

    (2004)
  • S.L. Turgeon et al.

    Current Opinion in Colloid & Interface Science

    (2007)
  • Q. Ru et al.

    Carbohydrate Polymers

    (2012)
  • E. Ruiz-Hitzky et al.

    Advanced Materials

    (2010)
  • M. Darder et al.

    Advanced Materials

    (2007)
  • F. Weinbrreck et al.

    Biomacromolecules

    (2004)
  • F. Carn et al.

    Soft Matter

    (2008)
  • R. Schrieber et al.

    Gelatine Handbook: Theory and Industrial Practice

    (2007)
  • N. Taheri Qazvini et al.

    Biomacromolecules

    (2012)
  • M. Gudmundsson

    Journal of Food Science

    (2002)
  • M. Darder et al.

    Materials Science and Technology

    (2008)
  • E. Ruiz-Hitzky et al.

    Chemical Society Reviews

    (2011)
  • Cited by (30)

    • Interactions between smectites and polyelectrolytes

      2020, Applied Clay Science
      Citation Excerpt :

      The polymer bridging, charge neutralization, and polymer adsorption (Song et al., 2010) between the smectite and amphoteric polyelectrolytes promote flocculation in aqueous smectite-amphoteric polyelectrolytes dispersion, so amphoteric polyelectrolyte has been used in water treatment (Wang et al., 2019), and drilling fluid (Bai et al., 2015; Chu et al., 2013) (Table 3). Gelatin, a natural amphoteric polyelectrolyte, due to biocompatibility, biodegradable properties, and no toxicity and availability, can potentially be a suitable substitute for the existing flocculants (Karimi et al., 2013). Gelatin can be effectively adsorbed on montmorillonite surfaces thereby changing the electrochemical nature of montmorillonite particle surface changes (Nazarzadeh et al., 2017).

    • Physicochemical and rheo-mechanical properties of titanium dioxide reinforced sage seed gum nanohybrid hydrogel

      2018, International Journal of Biological Macromolecules
      Citation Excerpt :

      Considering the number of the charged groups on particle surface commensurate with the specific surface area, the increase in particle size could lead to a decline in the surface charge and the ζ-potential. This result was in good agreement with those reported for gelatin-nanoclay hydrogel [14]. Density and its derivatives are essential intensive factors in fluid mechanics calculations and predicting the material thermodynamic properties and rheo-texture behavior.

    • Characterization of hybrid microparticles/Montmorillonite composite with raspberry-like morphology for Atorvastatin controlled release

      2018, Colloids and Surfaces B: Biointerfaces
      Citation Excerpt :

      These results support pH-dependent elasticity of the MP/MMT composite. At pH = IEP the composite presents a highly elastic structure based on a continuous network (“house-of-cards”) which relaxes as the system moves away from IEP [18,19]. It is important to mention that at pH 3.0 ± 0.1 and 7.0 ± 0.1, the viscous character dominated along the entire range of frequencies (G″ > G′), but at pH 4.8 ± 0.1 the elastic character dominated (G′ > G″́) corresponding to a semi-solid behavior (see Fig. 6D).

    View all citing articles on Scopus
    View full text