ARTICLE pubs.acs.org/Biomac
Enzymatically Cross-Linked Tilapia Gelatin Hydrogels: Physical, Chemical, and Hybrid Networks Franziska Bode,† Marcelo Alves da Silva,† Alex F. Drake,‡ Simon B. Ross-Murphy,§ and Cecile A. Dreiss*,† †
Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom ‡ Biomolecular Spectroscopy Centre, King’s College London, The Wolfson Wing, Hodgkin Building, London SE1 1UL, United Kingdom § Materials Science Centre, University of Manchester, Grosvenor Street, Manchester M1 7HS, United Kingdom
bS Supporting Information ABSTRACT: This Article investigates different types of networks formed from tilapia fish gelatin (10% w/w) in the presence and absence of the enzymatic crosslinker microbial transglutaminase. The influence of the temperature protocol and cross-linker concentration (055 U mTGase/g gelatin) was examined in physical, chemical, and hybrid gels, where physical gels arise from the formation of triple helices that act as junction points when the gels are cooled below the gelation point. A combination of rheology and optical rotation was used to study the evolution of the storage modulus (G0 ) over time and the number of triple helices formed for each type of gel. We attempted to separate the final storage modulus of the gels into its chemical and physical contributions to examine the existence or otherwise of synergism between the two types of networks. Our experiments show that the gel characteristics vary widely with the thermal protocol. The final storage modulus in chemical gels increased with enzyme concentration, possibly due to the preferential formation of closed loops at low cross-linker amount. In chemicalphysical gels, where the physical network (helices) was formed consecutively to the covalent one, we found that below a critical enzyme concentration the more extensive the chemical network is (as measured by G0 ), the weaker the final gel is. The storage modulus attributed to the physical network decreased exponentially as a function of G0 from the chemical network, but both networks were found to be purely additive. Helices were not thermally stabilized. The simultaneous formation of physical and chemical networks (physical-co-chemical) resulted in G0 values higher than the individual networks formed under the same conditions. Two regimes were distinguished: at low enzyme concentration (1020 U mTGase/g gelatin), the networks were formed in series, but the storage modulus from the chemical network was higher in the presence of helices (compared to pure chemical gels); at higher enzyme concentration (3040 U mTGase/g gelatin), strong synergistic effects were found as a large part of the covalent network became ineffective upon melting of the helices.
’ INTRODUCTION Gelatin, a product of heat-denaturated collagen,1 is widely used for applications in the food and cosmetic industries as a stabilizer and to improve elasticity and consistency. The need for gelatin in biomedicine is increasing for applications such as encapsulation, plasma expanders, microspheres, injectable drug formulations, and matrices for implants.27 The rising demand for gelatin has recently led to the search for alternatives to mammalian sources of gelatin, particularly in response to health concerns or religious issues. Fish skin and bone are rich in collagen and represent up to 75% of the total fish weight that is caught.8 Therefore, fish gelatin provides a value-added product from waste recycling. The major difference between gelatin from different species is the gelation and melting temperature, which is a consequence of the animal’s body temperature.9 For homeothermic animals, small variations in melting temperature are observed. However, in the case of poikilothermic animals, their body temperature matches the r 2011 American Chemical Society
surrounding habitat. Fish gelatin reflects a larger biodiversity in relation to its environment and therefore displays a wider range of gelation temperatures.1215 The proline and hydroxyproline content in the collagen sequence determines the gelation temperature. Lower amounts of these imino acids result in lower thermal stability and therefore a lower gelation temperature.10,11 Physical gels of gelatin form through the single-strand-totriple-helix transition of gelatin chains. In the sol state, the singlestranded form of gelatin prevails and does not display any extensive, classical elements of secondary structure (R-helix, β-sheet, or turns). The gelatin single strands are in a dynamic conformational state with a marked tendency to adopt the PI or PII extended conformations (left-handed helices), which is a prerequisite for triple-helix formation.16,17 During the gelation Received: July 17, 2011 Revised: August 5, 2011 Published: August 07, 2011 3741
dx.doi.org/10.1021/bm2009894 | Biomacromolecules 2011, 12, 3741–3752
Biomacromolecules Scheme 1. Enzymatic Cross-Linking of Glutamine and Lysine Residues Leading to a New Peptide Bond
process, the gelatin single-strands self-associate to form the collagen triple-helix based on the left-handed, PII conformation of the individual strands. The triple helices form the junction zones of the 3D network.18 This gelation process is reversible.19 Triple-helix formation is a complex process, which has attracted the interest of many research groups for years; however, the mechanism of formation is still not fully understood.14,15,2024 Djabourov et al.15 established a master curve showing that the rheological properties (storage and loss modulus) of gelatin gels are directly related to the fraction of amino acids in the triplehelix conformation in the network, independent of the thermal history, source of the gelatin, concentration, or molecular weight. Whereas the “melt-in-the-mouth” behavior of gelatin makes it an interesting material in the food industry, thermal instability is a drawback for medical applications (such as tissue engineering), which require the gel to be stable at physiological temperatures. A variety of reagents have been used to cross-link gelatin chains permanently. These chemically cross-linked gelatin networks, however, have been barely characterized compared with their physical counterparts. Synthetic cross-linkers, such as glutaraldehyde2529 or carbodiimide27,3032 improve mechanical strength and proteolytic stability but induce cytotoxic effects or immunological responses in the body.33,34 In contrast, naturally occurring compounds such as genipin2,26,27,29,31,35 or the enzyme transglutaminase3641 have been used more successfully for the cross-linking of biomatrices. They are biocompatible and nonimmunogenic and show lower toxicity in cell culture studies.33 Transglutaminase (TGase, EC 2.3.2.13, amine-γ-glutamyl transferase), a naturally occurring enzyme found in almost all living organisms, functions as a blood clotting and liver detoxification agent and is commonly used in industry.42,43 TGase catalyzes acyl-transferase reactions between the side chain of a glutamine residue and a primary amino group (e.g., lysine), resulting in an ε-(γ-glutamyl)lysine isopeptide bond (Scheme 1).44 Microbial transglutaminase (mTGase) is produced by a variety of microorganisms and shows a catalytic activity independent of Ca(II) ions, differing in this way from human or animal transglutaminase. Previous studies have shown that this enzyme can covalently cross-link gelatin to create a chemical gel.3638 The combination of physical and chemical networks can be an advantage to tune the final properties of the gel and gelation kinetics. There is also a fundamental interest in understanding the competing mechanisms in place when both types of networks form either simultaneously or one after the other and how this affects the final structure of the gels. However, there is very little account in the literature of this type of “hybrid” process.38,40,41,45,46 One of the few examples is the work by Djabourov et al.,46 who have studied the combination of the cross-linker BVSM (bisvinylsulfonyl-methane) with physical networks of gelatin (type B) from dematerialized ossein and reported that the presence of a covalent network does not
ARTICLE
Scheme 2. Formation of the Four Different Types of Networks Using the Protocols Described in the Methods Section: Physical (P), Chemical (C), ChemicalPhysical (CP), and Physical-co-Chemical (CP) Gelsa
a
(P) Triple-helix formation without mTGase at temperature below 23°C. (C) Covalent bond formation by mTGase at 37°C. (PC) Simultaneous formation of chemical and physical networks at 21°C in the presence of mTGase. (CP) Formation of helices after prior formation of the covalent network (C) at 21°C.
thermally stabilize the triple helices. In physicalchemical gels, they concluded that a more ordered covalent network was formed, directed by the preformed helical network.46 Furthermore, the presence of triple helices seemed to increase the amount of cross-links formed. The literature is particularly scarce on the characterization of fish gelatin networks produced using a catalytic cross-linker.47,48 The objective of the study reported here is to understand the gelation process in fish (tilapia) gelatin gels when both a physical network (triple helices) and covalent bonds form one after the other (chemicalphysical (CP) gels) or simultaneously (physicalco-chemical (PC) gels), using mTGase to catalyze the covalent cross-linking of the gelatin strands. The gelation process is simply tuned by setting the temperature to below or above the gelation temperature (T ≈ 23 °C), in the absence or presence of mTGase, as depicted in Scheme 2. Depending on the sequence of events, it is expected that a different distribution of cross-links could be present in the formed network (Scheme 2), therefore leading to different rheological properties of the final gels.
’ EXPERIMENTAL SECTION Materials. Gelatin. Fish gelatin was obtained from Rousselot, France. All experiments were carried out using a single batch of gelatin (purchased in 2008). The gelatin was extracted from tilapia fish skin by an acidic process possessing an isoelectric point of pI 8 to 9, bloom strength of 275, and a gelation temperature of 23 °C. The average molecular weight was determined to be ∼36 kDa ( 12% using GPC (Smithers Rapra). Cold-water fish gelatin was obtained from Healan Ingredients (York, U.K.). Enzyme. The cross-linker used in this study was bacterial transglutaminase (mTGase) obtained from N-Zyme BioTec (Darmstadt, Germany) (specific activity, 1.6 U/mg solid; molecular weight, 38 kDa; and purity, >80%). The enzyme is stable between pH 59 and 040 °C. 3742
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Biomacromolecules
ARTICLE
Dyes. The dyes, used to color the chemical gels were safranin (CAS: 477-73-6) and methyl orange (CAS: 547-58-0) obtained from SigmaAldrich. Methods. Rheology. Rheological measurements were carried out on a strain-controlled ARES rheometer from TA Instruments using titanium parallel plate geometry (0.8 mm gap) of 50 mm diameter with the temperature controlled by a Peltier unit ((0.1 °C). To prevent loss of solvent due to evaporation, we applied a thin layer of paraffin oil (1 mL) around the sample, and geometry-covers were placed around the plates. To study the evolution of the gel networks, we performed timesweep/cure experiments where the storage modulus (G0 ) and loss modulus (G00 ) were followed at a fixed frequency of 1 Hz and strain of 1% (within the viscoelastic region, as determined by amplitude sweeps, Supporting Information SI 1). Several criteria to determine the gelation point (temperature or time) are described in the literature. The crossover point of G0 and G00 has been widely used.49 It assumes that in the sol state the unassociated strands have no elastic contribution and at the point of formation of the spanning cluster, there is a sudden increase in the system’s elasticity. For some systems, a non-negligible elastic contribution exists even in the sol state, where G0 could be higher than G00 , and this criteria therefore cannot be used. The WinterChambon criterion50 seems to be most rigorous. They propose that G0 and G00 follow a power-law dependency with oscillatory frequency and the exponents for G0 and G00 are equal at the gel point. However, for some systems, it is difficult to acquire good quality pregel data.51 For the systems studied in the work here, this criterion could not be applied. Instead, we found it a more reliable and straightforward method in our case to define the gel point as the point where G0 reaches the arbitrarily defined value of 1 Pa, as used previously.52 Optical Rotation. Optical rotation (OR) measurements were performed on an Applied Photophysics (Leatherhead, U.K.) Chirascan spectrometer, equipped with a Quantum NorthWest TC125 Peltier unit. The instrument was flushed continuously with pure evaporated nitrogen throughout the measurements. Temperatures were measured directly with a thermocouple probe in the sample with an accuracy of (0.2 °C. All samples were measured at a wavelength of 436 nm. Values for solvent, air, water, and a sucrose solution (2 g/10 mL) were taken at 20 °C to normalize and calibrate. The calibration sucrose solution was measured in a 10 mm path length cell. The gelatin sample, single strand, and buffer were measured in a 1 mm path length strain-free fused-silica cell supplied by Hellma. The optical path length is set by the high sample concentration (10% w/w gelatin) and to guarantee good temperature control. The optical rotation angle for the gelatin single-strand state, Rcoil (10% w/w), was measured at 37 °C. The gelation kinetics measurements were made directly after transferring about 300 μL of a 10% w/w gelatin solution (incubated at 35 °C) to an empty 1 mm cuvette placed in the spectrometer and prethermostated at the appropriate temperature. The gelatin solution in the Chirascan took approximately 1.5 min to reach the target temperature. The gelatin conformation change from single strand to triple helix is accompanied by a change in optical rotation angle R from which it is possible to derive the fraction of triple helices χ.53 The procedure is explained in detail below. The literature specific rotation value of a standard sucrose solution is = 128.38° for a 1 g/100 mL sucrose solution in a 1 dm cell at [R]lit.sucrose 436nm λ = 436 nm.54 The specific rotation for the theoretical optical rotation angle Rsucrose calcd under the experimental concentration and path length is given by eq 1 ¼ ½Rlit:sucrose 436nm
Rsucrose calcd 100 cs l
ð1Þ
where Rsucrose calcd is in degrees, cs is the experimental sucrose concentration in g/100 mL, and l is the experimental cell path length in dm (l = 0.1 dm). The specific optical rotation angle of the sample [R]helix 436nm is
obtained after subtracting the background Rbkg and correcting by the sample concentration c (g/mL), path length (dm), and calibration factor (eq 2) ½Rhelix 436nm ¼
R Rbkg cl
ð2Þ
[R]helix 436nm is then used to calculate the fraction of triple helices in the gel, according to eq 355,56 χ¼
coil ½Rhelix 436nm ½R436nm collagen
½R436nm ½Rcoil 436nm
ð3Þ
is the specific optical rotation of native soluble collagen where [R]collagen λ (χ = 1, 100% triple helices) and [R]coil λ is the specific optical rotation of the single strands (χ = 0): 3 [R]coil 436nm = 237 deg cm /(g dm) for tilapia gelatin, determined experimentally 3 14 [R]collagen 436nm = 800 deg cm /(g dm) for calf skin collagen All sample compositions were measured three times. Preparation of the Gels and Temperature Protocols. Typically, 15% w/w gelatin was left to swell overnight in phosphate buffer (100 mM, pH 7.2) at 4 °C. Before use, the solutions were incubated for ca. 30 min at 35 °C until transparent (slightly yellowish) and then diluted to 10% w/w stock solutions. The enzyme mTGase was also dissolved in phosphate buffer (100 mM, pH 7.2) to make up a stock solution of typically 20 mg/mL. Any undissolved particles present were separated by centrifugation (7000 rpm, 3 min), and the supernatant was stored in the fridge until needed (for a maximum of 3 days). The protocols used to prepare the different network structures are described below. Protocol 1, Physical Gels. To form the physical gels, we cooled the pure 10% w/w gelatin solutions below their gelation temperature to 12, 18, and 21 °C. The samples were loaded on the rheometer at 25 °C, and the temperature was set to the target temperature. The Peltier unit has a cooling rate of ca. 30 °C/min. Protocol 2, Chemical Gels. Chemical networks were formed in the presence of the enzyme mTGase. Adequate volumes (∼100 μL) of enzyme stock solution (giving concentrations of 1040 U mTGase/g gelatin) were added to 10% w/w gelatin samples, vortex-mixed, and loaded onto the rheometer plate preheated to 37 °C. It is estimated that
