Smart Hydrogels: Application in bioethanol production
Lucinda Mulko1, Edith Yslas2, Silvestre Bongiovanni Abel1, Claudia Rivarola1, Cesar Barbero*1, Diego Acevedo*1,3
1Department of Chemistry, National University of Rio Cuarto, Ruta 36 Km 601, Rio Cuarto, Cordoba (5800) Argentina.
2Department of Molecular Biology, National University of Rio Cuarto, Ruta 36 Km 601, Rio Cuarto, Cordoba (5800) Argentina.
3Department of Chemical Technology, National University of Rio Cuarto, Ruta 36 Km 601, Rio Cuarto, Cordoba (5800) Argentina.
Abstract
Hydrogels are crosslinked macromolecular materials formed by hydrophilic polymers that absorb water or similar fluids. Some kinds of hydrogels are known as “smart hydrogels”.They are stimuli responsivematerials, which are able to suffer a volume phase transition in response to changing different environmental conditions such as temperature, pH, solvent composition, ionic strength or electrical-magnetical stimuli. In this chapter, the synthesis and characterization of smart hydrogels with different porosity (nanopores, macropores, superpores) are described. The application ofsmart hydrogels in bioethanol production is discussed, taking into account the advantage ofthesematerialsto generate a safe and biocompatible environment to the biocatalyst.
Hydrogels
Over the last few decades, significant progress has been made in the research and development of hydrogels as biomaterials with application in the field of biology, biomedicine, biosensors, agriculture and water purification (Davidson et al. 2013; Ma et al. 2015; Liang et al. 2015; Hickey et al. 2015; Keller et al. 2015; Li et al. 2015).
Hydrogels are defined as three-dimensional crosslinked polymeric structures which can absorb and retain large quantity of water and other biological fluids (Kopeček 2007). These materials can swell several hundred times respect to the dry network mass, when are in contact with water or other solutions. Moreover, they can be highly porous materials and to trap small molecules and cells into its cavities. The particular kind of gels stimulus responsive are also called “smart” or “intelligent hydrogels” (Shi et al. 2008; Ramos et al. 2012; Jiang et al. 2013; Chen et al. 2015). Many chemical and physical stimuli have been applied to induce several responses of the intelligent hydrogel. These materials can respond to environmental changes by a modification in their volume, depending of the chemical structure, as shown in Figure 1. Changes could occur in response to various types of stimuli include pH, temperature, ionic strength, nature and composition of solvent, chemical or enzymatic reactions and electrical or magnetic stimulus. The nature of these behaviors is reversible because hydrogels are capable of returning to its initial state. Another class of these materials, results from a combination of two stimuli responsive mechanisms in one unique hydrogel system which is called dual responsive.
Figure 1: Stimuli responsive reversible-hydrogels.
Several materials based on smart hydrogels with biocompatible and biodegradable characteristics could be used in tissue engineering. Because of these special properties, they can be widely applied in biomaterials such as controlled drug-release and drug-delivery systems, on–off switching materials, artificial muscles, biosensors, chemical separations and adsorptive materials.
History of hydrogels
The term “hydrogel” appeared in 1894 in scientific reports when it was used to refer a colloidal gel based on inorganic salts. In 1960, Wichterle and Lim reported the first work on poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels. This materials can be used as soft contact lenses (Wichterle and Lím 1960). In the 1970s appeared Pluronic® hydrogels for the controlled release of several pharmaceutical agents. The research of Lin and Sun in 1980 showed the successful application of calcium alginate microcapsules for cell encapsulation (Lim and Sun 1980). Furthermore, in 1980s, hydrogels were modified for other applications such as contact lens materials or as possible matrix for drug release (Harris et al. 1980; Graham and McNeill 1984). In 1989, Bae et al, described the synthetic process and the thermo sensitivity of a hydrogel and investigated the effects of temperature on the permeation rates of insulin (Bae et al. 1989). Brøndsted and Kopecek described the synthesis of different kinds of hydrogels based on hydrophilic N-substituted methacrylamides, N-tert-butylacrylamide and acrylic acid crosslinked with 4,4'-di(methacryloylamino)azobenzene and demonstrated that they have a great potential use in localized specific drug delivery to the colon (Brøndsted and Kopecek 1991). Past research showed that hydrogels could be used in various research fields such as biomaterials (Burczak et al. 1996; Kang et al. 1999; Miyazaki et al. 2000). Moreover, a large number of authors have reported that hydrogels show interesting properties that make them excellent candidates for biocompatible materials (Kwon et al. 1991; Lee et al. 2013).
The water in hydrogels
Gibas et al. reported that the swelling of hydrogels is a complex process comprising different steps (Gibas and Janik 2010). The moment when a piece of dry hydrogel begins to absorb water, the polar hydrophilic groups of the hydrogel matrix are hydrated which appears in the form of primary bound water. Formerly, in the next step, when the polar groups are hydrated, the exposes hydrophobic groups also interact with water molecules, leading secondary bound water. Primary and secondary bound water are often combined and both constitute the total bound water. In a third step, the osmotic driving force of the network towards infinite dilution is kept by the physical or chemical crosslinks, consequently additional water is absorbed. At this time, the hydrogel will achieve the equilibrium swelling state. The additional swelling water become saturated with bound water is called bulk water or free water, and to fill the space between the network chains, and the center of larger pores called macropores. The Flory-Huggins theory explains the fact that the amount of water absorbed by a hydrogel depends of different factors as the temperature and the specific interaction between water molecules and the chains of the polymer (Ganji et al. 2010).
Classifications of hydrogels
The hydrogels are classified in several ways (Kopeček and Yang 2007). The simplest classification is hydrogel obtained from products natural sources, hydrogels made synthetic materials or hybrid hydrogels which are a mixture of synthetic and natural molecules. These are composed of a large variety of natural and synthetic polymers that held together using different physical or chemical crosslinks. For example, natural polymers widely used in this materials it is possible to list some polysaccharides (e.g., cellulose, hyaluronic acid (HA), chitosan, chondroitin sulfate, dextran sulfate, alginate, etc.) or different proteins such as collagen, gelatin, fibrin, heparin, etc. The most typical synthetic polymers are polyacrylamide or polyvinyl alcohol (PVA), etc. The advantages of natural polymers include inherent biocompatibility, biodegradability, bacteriostatic and wound healing properties. According to polymeric composition can be classified like homopolymeric hydrogels, copolymeric hydrogels, multipolymer and interpenetrating polymeric hydrogel (IPN). Another way to classify hydrogels is based on configuration as amorphous (non-crystalline), semicrystalline (regions of partially ordered structure), crystalline. classification based on type of cross-linking is very usual: hydrogels can be separated into two categories depending of the chemical or physical nature of the cross-link junctions. Based on physical appearance they are classified as films, matrix or microspheres gels depend on the technique involved in the preparation process. Hydrogels might also be classified according to network electrical charge: they can be neutral (noionic), ionic (anionic or cationic) or ampholytic (amphoteric electrolyte that containing either acidic and basic groups or zwitterionic groups).These materials can be classified as well by its morphology as macroporous, microporous and nonporous. Other differentiation depends of their final destination in the organism; it is possible classified the hydrogels as degradable and non-degradable. If these materials are biodegradables, provide additional advantages as the fact that can be degrade in an aqueous environment after been used. Also can be classified by response to external stimuli in three groups: chemically responsive (pH, ionic factors and chemical agents), physically responsive (temperature, pressure, light, electric field and magnetic field) and biochemical responsive (antigen, enzymes ligands and other biochemical agents).
Synthesis
Hydrogels can be synthesized by simultaneous copolymerization of one or more mono and multifunctional monomers or by crosslinking of a homopolymer or copolymer in solution. The latter could involve two steps in which in the first step the linear polymer could be synthesized in the absence of a crosslinking agent and in the second step the polymer could be crosslinked using chemical reagents or irradiation. Also, hydrogels can be synthesized by different procedures: (a) polymerization and simultaneous post polymerization cross-linking of hydrophilic monomers, and (b) modification of hydrophilization of existing polymers or functionalization of existing polymers with potential hydrogel properties. Polymer networks can be synthesized using various chemical methods (e.g., photo- and thermal-initiated polymerization. In both of procedure the hydrogels are most commonly synthesized by free-radical polymerizations of hydrophilic monomers(Jeong et al. 2007). Cross-linked polymeric structures may be produced by including small quantities of crosslinking agent, which may produce a crosslink between two radical chainsduring the propagation.
Is possible generate active centers by different methods, depending on the monomers, solvents and the reaction conditions. The most used solvents include water, organic solvents (e.g. ethanol, benzyl alcohol and others) or mixtures in different proportions such as a water/ethanol. In general, reaction conditions of polymerization initiation require heat (in the case of thermal initiators), light (if a photo-initiator is used), irradiation, electron beams and others.
Cryo-polymerization [DFA1]process, as shown in Figure 2, is a novel freeze-thaw technique has recently been used to generate macroporous matrix by the polymerizations of hydrophilic monomers around the ice crystals formed (Ivanov and Lozinsky 2006). The procedure allows increase the effective pore size of the hydrogel, whereas the polymerization process occurs, due to the existence of the crystalline matrix.
Figure 2: Cryopolymerization hydrogeltechnique
Hydrogels synthesizedby free radical polymerization
The more widespread hydrogels synthesis is the one which occurs by radical polymerization(Jeong et al. 2007; Ozaydin Ince et al. 2010; Ahmed 2013). This synthesis is essentially a process of free radical polymerization with a series of peculiarities that give specific characteristics of these materials. Besides, the solvent (generally water or water solutions)the vinylmonomer and the initiation system, is necessary introducing another element called cross-linker or cross linking agent. Free radical polymerization is a kind of chain-growth polymerization in which the active species are free radicals. Radicals are organic molecules containing an unpaired electron, are unstable and highly reactive species. The polymerization process consists of three stages: initiation, propagation and termination(Odian 2004), as it can be seen in Figure 3.
Figure 3: Radical polymerization.
Initiation step usually splits into two reactions: first, a molecule called initiator (I2) leads to free radical (R*) by different mechanisms, the most common is the homolytic dissociationin two radical generating species. The radicals are formed providing to the system enough thermal, electrical or photochemical energy. The second step of the initiation reactionthe radical is added to a vinyl monomer molecule yield an active radical species that already contains a monomeric unit. This active specie (true initiator of the chain reaction)reacts with more monomeric molecules, during the propagation stage, causing a chain growth. Finally, the completion of the reaction can be given by combination reactions, disproportionation or both.
The chain transfer reactions also suspend the process chain addition reaction, resulting in inert polymer chains(Heatley et al. 2001). In this case, the growing radical suffers a premature deactivation by a process of atom transfer, generally a proton, to other molecule presents in the reaction medium. This molecule is called transfer agent, and might be the monomer itself, the initiator, the polymer or the reaction solvent. Transfer reactions decrease the chain length without changing the reaction rate.
Monomers
Hydrogels might be synthesized from a large variety of precursor monomers. Most of vinyl monomers used are based on acrylamide and their derivatives. Same chemical structures of vinyl monomersare shown in Figure 4.
Figure 4: Chemical structures of vinyl monomers used as precursor of hydrogels
Initiators
Regardless of the type of polymerization and the monomers used, it is necessary to employ a trigger agent or initiator to start the reaction by free radical initiation. For instance, a redox initiation system commonly used is sodium persulfate (APS) and N,N,N',N'-tetramethyletilenediamine (TEMED). TEMED functions as an accelerating agent of homolytic cleavage in ammonium peroxydisulfate (APS), generating the initiator radical (Figure 5).
Figure 5: Chemical structures of initiatorTEMED
Cross-linkers
The choice of cross-linker is substantial to defining the hydrogel properties. The crosslinking forms the three-dimensional architecture network which gives specific characteristics of gel. These cross-linkers (typically tetra or hexa-functionalized) have several reactive groups in their molecular structure to weave cross-polymer chains between the classical linear-polymer chains(Best et al. 2013).Generally, crosslinking agents are used in a small percentage compared to the total monomer used in the polymerization reaction. A high degree of crosslinking - number of crossovers that exist per unit volume - generates fragile polymers of low elasticity. On the other hand, a low degree of crosslinking allows displacement between different polymer chains and leads to high elasticity, but usually low mechanical strengths. It has been reported several times use of N,N-methylene-bis-acrylamide (BIS) as cross-linking agent (Suedee et al. 2006), the percentage of cross-linker was usually between 2% to 5% mol respect of monomer. The chemical structure of the crosslinker is show in Figure 6:
Figure 6: Chemical structure of cross-linking agent, N,N-methylene-bis-acrylamide (BIS)
Hydrogel Properties
Hydrogels are crosslinked materials with the capacity to store water due to their chemically or physically crosslinked network.In addition, the crosslinks between network chainscause their resistance to dissolution. The characteristic property of hydrogels is their ability to swell in the presence of water and to shrink in the absence of it. Swelling capacity properties of hydrogelsdepend on the degree of crosslinking, the chemical composition of the polymer chains, and the interactions of network and surrounding liquid(Tse and Engler 2010). The ability of swelling isdue to the hydrophilicity of polymer chains for the presence of hydrophilic groups, such as alcohols, carboxylic acids, amides, etc., and the crosslink density. The crosslinking can be provided by covalent bonds, hydrogen bonding, Van der Waals interactions, or physical entanglements(Vácha et al. 2008). Responsive smart hydrogel exhibit rapid changes in their swelling behavior as responses to changes in environmental conditions lend these materials as carriers for the delivery of drugs, peptides and proteins.Properties as swelling-mechanical behavior and toxicity studiesshould be evaluated to allow hydrogels could be successfully used in biomedical and pharmaceutical fields(Deligkaris et al. 2010).
A small change in environmental condition might trigger fast and reversible changes in hydrogel(Ganji et al. 2010). Exhibit drastic volume changes in response to specific external stimuli, such as the temperature, pH, irradiation, etc. For instance, there is a kind of hydrogels that respond to the fluctuations in the external environmental pH due the presence of acidic or basic functional groups(Gupta et al. 2002); other materials are thermo-responsive (Tokarev and Minko 2009)whenlineal polymers undergo a thermally reversible phase transitioninduced are soluble in a solvent (water) at low temperatures but become insoluble as the temperature rises above the critical solution temperature (CST).
The swelling capacity of hydrogel is mainly governed by the polarity of the formulations, which is dependent on the nature of the polymers, the concentration of the cross-linking agent, and the reaction conditions(Hoare and Kohane 2008).The swelling capacity is a simple but powerful measure, because it brings valuable information about the physical properties (such as porosity, hydrophobicity-hydrophilicity, diffusion, etc.) of hydrogel. Moreover, swelling kinetics show the behavior of hydrogel relating to changes on different variables of their environment. Regarding to that, different gravimetric and volumetric methods can be distinguished to study thepercentswelling (%Sw) as a function of time, and modifying the environment:temperature, pH or co-solvent concentration(Xia et al. 2013).Forthat propose, a sample of dry hydrogel, previously weighed, is immerse in water solution and the swelling capacity is being calculated withEquation 1
Equation 1
Where:, corresponding to the initial mass of the gel and mi is the mass after a time.
Figure 7: Swelling kinetic of PAAm1M, PNIPAM 0.5M hydrogels and co-polymerhydrogels.
Figure 8: Swelling kinetic of PNIPAM 0.5 M hydrogels co-polymers with 1%,2% and 5% MAA.
Mechanical properties
Studies related to mechanicalbehavior are essential in biomedical applications such as ligament and tendon repair, matrix for drug delivery, tissue engineering and cartilage replacement material(Kim and Park 2004; Kopeček 2007). It is known that a hard hydrogel could be achieved through the higher degree of crosslinking.Hence, it is possible obtain an optimaldegree of crosslinking to achieve a relatively strong and yet elastic hydrogel.
Biocompatible properties
This is a key property to be biocompatible and nontoxic material for applicable in biomedical field(Hoffman 2002; Khan et al. 2009).For this reason, all residues of initiators, solvents, stabilizers, emulsifiers, unreacted monomers and cross-linkers used in polymerization should be eliminated for not caused toxicity to host cells.It is important to remove hazardous chemicals from preformed gels, through of various purification processes such as solvent washing or dialysis. Cell viability on these materials could be estimated by the classic MTT assay.
Hydrogels biomedical applications
Recently hydrogels have gained considerable attention due to their several favorable properties such as hydrophilicity, tissue-mimicking consistency and high permeability to metabolites and oxygen(Brøndsted and Kopecek 1991; Jiang et al. 2013; Sato et al. 2014). These biocompatible materials are used in different field aspharmaceutical and biomedical engineering(Brøndsted and Kopecek 1991; Khademhosseini and Langer 2007; Burdick 2012). One of the most intense areas of interest from 1960 was the study of these kind ofmaterials used in drug delivery formulations. Most common drug release mechanism for hydrogel is diffusion controlled. Intelligent hydrogels are a kind of hydrogel, which can change their network structures in response to any stimulus liketemperature, pH, light, electric fields, etc(Hoare and Kohane 2008). After drug encapsulation into hydrogel, the release of drug could be controlled or activated through an applied stimulus. Actually, temperature and/or pH responsive hydrogel are the mostinvestigated for drug delivery applications(Cuggino et al. 2014). For Helicobacter pylori (H. pylori) eradication were also clinically evaluated the hydrogel of chitosan and polyacrylic acids containing amoxicillin and clarithromycin(Gisbert et al. 2006). Chang et al. developed other system consisting of amoxicillin loaded pH-sensitive hydrogels composed of chitosan and poly(g-glutamic acid) for the treatment of H. pylori infection in the peptic ulcer disease(Chang et al. 2010). The hydrogels delivers drugs to specific site in the gastrointestinal tract showing tissue specificity and the change in the pH causes local delivery of drug(Jain et al. 2007). Addition, the properties of hydrogels are important for tissue engineering and other areas of biomedical field.A wide range of hydrogels has been proposed for cartilage regeneration but few studies have been studied on chondrocyte behavior within the hydrogels(Mirahmadi et al. 2013). The hydrogels should not only be able to encapsulate cells but also maintain both viability and cell phenotype and furthermore, they have the potential to supportneocartilage formation. The properties of hydrogels, including chemical composition, stiffness and network porosity and permeability, have been shown to have a significant impact on the survival and differentiation of encapsulated mesenchymal stem cells. Park et al. reported a novel thermosensitive hydrogel composed of chitosan and pluronic developed as an injectable cell delivery carrier for cartilage regeneration(Park et al. 2009). The literature have reported the potential applications of modified chitosan hydrogel due that is a non-toxic, biocompatible and biodegradable polymer and has attracted considerable interest for biomedical and pharmaceutical uses, particularly to drug delivery and tissue engineering(G. Tahrir et al.; Ji et al. 2010; Giri et al. 2012; Mahajan et al. 2015). Alginates have been used as injectable cell transplantation vehicles due to their biocompatibility, low toxicity, and relatively low cost. Cells could be encapsulated during the cross-linking process to create cell-hydrogel constructs(Lambricht et al. 2014). For tissue regeneration the hyaluronic acid have also been widely used and become commercially available under the name CorgelTM(Hilborn 2011; Burdick 2012). Besides dental pulp regeneration, the results of the work reported by Lambricht et al.mightalso be beneficial to other applications involving dental stem cells in regenerative medicine(Lambricht et al. 2014). The bacterial cellulose hydrogels a potential material for various bioengineering applications e.g. implant replacement of human tissues, encasement for tissue regeneration, etc.(Bäckdahl et al. 2006; Gibas and Janik 2010; Nimeskern et al. 2013; Gao et al. 2016).On the other hand, the enzymes are very promising alternatives to conventional industrialcatalysts in industrial chemistry. In the field of enzyme immobilization, polysaccharide hydrogels such as alginate, chitosan, starch, and agarose have been employed for entrapping a large number of enzymes such as lipase, lactase, invertaseand peroxidase. Immobilized enzymes can be used repeatedly or continuously in a variety of reactors decreasing the cost by reuse of costly enzymes(Sato et al. 2014).