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Synthesis, evolution and chemorheology studies of a novel biocopolymer based on protein: collagen-g-poly (AMPS).

Introduction

The market for superabsorbent polymers (SAPs) has increased by a factor of 5 over the past 10 years. These materials are crosslinked hydrophilic polymers, capable of absorbing large quantities of water, saline or physiological solutions [1,2]. The absorbed fluids are hardly removable even under some pressure. They are widely used in various applications such as hygienic, foods, cosmetics, and agriculture [2-4]. This accounts for increase in the worldwide production of superabsorbent polymers (SAPs) from 6000 tons in 1983 to 450000 tons in 1996 [1]. Nowadays, the worldwide production of SAPs is more than one million tons in year. Hence, synthesis and investigation of specific and new superabsorbent hydrogels with high absorbency, mechanical strength and initial absorption rate, is the main goal of the several research groups in the world [5-10].

The properties of the swelling medium (e.g. pH, ionic strength and the counter ion and its valency) affect the swelling characteristics. SAPs responding to external stimuli such as heat, pH, electric field, chemical environments, etc, are often referred to as "intelligent" or "smart" polymers. Among these, pH-sensitive hydrogels have been extensively investigated for potential use in site-specific delivery of drugs to specific regions of the gastrointestinal tract and have been prepared for delivery of low molecular weight protein drugs. Therefore, these hydrogels have important applications in the field of medicine, pharmacy, and biotechnology [11,12].

Natural-based superabsorbent hydrogels have attracted much interest from the viewpoint of improving the tissue tolerance of synthetic polymers and the mechanical properties of natural polymers. The presence of the natural parts guarantees biodegradability of the superabsorbing materials. Because of their biocompatibility, biodegradability and non-toxicity, natural polymers, i.e. polysaccharides and proteins, are the main part of these biopolymers. One of the best methods for the synthesis of these superabsorbent hydrogels is graft copolymerization of vinylic monomers onto natural polymers. Monomers such as acrylonitrile (AN), acrylic acid (AA), acrylamide (AAm) have been graft copolymerized onto polysaccharides such as starch, cellulose and their derivatives [13-15]. The first industrial superabsorbent hydrogel was synthesized using this method via ceric-induced graft copolymerization of acrylonitrile onto starch followed by alkaline hydrolysis of the resulted graft copolymer [16].

Proteins are widely distributed in nature and are synthesized mainly in animals, i.e. collagen, keratin, gelatin, and etc., and in a few plants such as Soya. In general, proteins are high molecular weight polymers and their solubility in aqueous solutions is difficult. Two efficient methods for preparation of aqueous soluble proteins are alkaline and enzymatic hydrolysis. According to the literature survey based on Chemical Abstract Service, a few studies have been reported in the case of Synthesis of hydrogels based on protein and study of rheology them [17-19]. The chemical reaction has a pronounced effect on the molecular structure of polymers. In addition, rheological properties of polymers depend on the molecular mobility. Consequently, it is possible to monitor rheological properties of a reacting system, i.e., viscosity or modulus (storage and loss modulus) by using a chemorheological approach due to following the reaction. Chemorheology is a powerful tool to study chemical cross-linking reactions at which a transition from the liquid to the solid state takes place. A reactive thermosetting resin can be used in an industrial production process with respect to its curing performance. The question of interest for a thermosetting polymer is determination of its gelation temperature and gelation time. These points of gelation are the basic parameters characterizing processability of a thermosetting resin [13,14].

Hence, the objective of the present paper is to describe the preparation and evelution of a collagen-g-poly (2-acrylamido-2-methylpropanesulfonic acid) hydrogel as a new natural-based polymer and chemorheology properties it.

Experimental:

Materials:

Hydrolyzed collagen (Parvar Novin-E Tehran Co.) was industrial grade which is available in market and has nearly 25% insoluble phosphate salt. 2-acrylamido-2-methylpropanesulfonic acid (Merck, Darmstadt, Germany), N',N'-methylene bisacrylamide and ammonium persulfate (Fluka, Buchs, Switzerland) were of analytical grade and used without further purification. Double distilled water was used for the hydrogel preparation and swelling measurements.

Preparation of hydrogel:

A pre-weighed amount of hydrolyzed collagen (1.0-4.0 g) was dissolved in 40 mL degassed distilled water and filtered to remove its insoluble salt. The solution was added to a 1-L three-neck reactor equipped with a mechanical stirrer (RZR 2021, a three-blade propeller type, Heidolph, Schwabach, Germany) and the reactor was immersed in a thermostated water bath preset at a desired temperature (80[degrees]C). Then 2-acrylamido-2-methylpropanesulfonic acid (2.0-8.0 g) was added to the reactor. After stirring for 10 min, ammonium persulfate (0.01-0.40 g APS in 5 mL [H.sub.2]O) and methylene bisacrylamide (0.05-0.20 g in 5 mL [H.sub.2]O) were added simultaneously to the reaction mixture. The temperature was maintained at 80[degrees]C and the reaction mixture was stirred continuously (300 rpm) for 1 h. At the end of the propagation reaction, the gel product was poured into ethanol (200 mL) and was dewatered for 12 h. Then, the product was cut into small pieces, washed with 200 mL ethanol and filtered. The particles were dried in an oven at 50[degrees]C for 12 h. After grinding, the powdered superabsorbent hydrogel was stored in absence of moisture, heat and light.

Instrumental analysis:

Fourier transform infrared (FTIR) spectroscopy absorption spectra of samples were taken in KBr pellets, using an ABB Bomem MB-100 FTIR spectrophotometer (Quebec, Canada), at room temperature. To study the morphology of the hydrogel, the surface and cross-sectioned area of the hydrogel were examined using scanning electron microscopy (SEM). After Soxhlet extraction with methanol for 24 h and drying in an oven, superabsorbent powder was coated with a thin layer of gold and imaged in a SEM instrument (Leo, 1455 VP). Brunauer-Emmett-Teller (BET) analysis was used to determine the pore size of the hydrogels. Thermogravimetric analyses (TGA) were performed on a Universal V4.1D TA Instruments (SDT Q600) with 8-10 mg samples on a platinum pan under nitrogen atmosphere. Experiments were performed at a heating rate of 20[degrees]C/min until 550[degrees]C.

Results and discussion

Synthesis and spectral characterization:

The mechanism for crosslinking graft copolymerization of AMPS onto collagen backbones in the presence of APS and MBA is shown in Scheme 1. In the first step, the thermally dissociating initiator, i.e. APS, is decomposed under heating (80[degrees]C) to produce sulfate anion-radicals. Then, the anion-radicals abstract hydrogen from one of the functional groups (i.e. COOH, SH, OH, and N[H.sub.2]) in side chains of the collagen backbones to form corresponding macro-initiators. These macroradicals initiate grafting of AMPS onto collagen backbones leading to a graft copolymer. Crosslinking reaction also occurred in the presence of the crosslinker, i.e. MBA.

FTIR spectroscopy was used for identification of the hydrogel. Figure 1 shows the IR spectra of the collagen and the resulted hydrogel. The band observed at 1658 [cm.sup.-1] can be attributed to C=O stretching in carboxamide functional groups of substrate backbone (Figure 1-a). The broad band at 3200-3600 [cm.sup.-1] is due to stretching of -OH groups of the collagen. The collagen-g-AMPS hydrogel comprises a collagen backbone with side chains that carry sulfate groups that are evidenced by a new characteristic absorption band at 1221 [cm.sup.-1] (Figure 1b). This peak attributed to ester sulfate stretching of AMPS. The stretching band of -NH overlapped with the OH stretching band of the collagen portion of the copolymer.

To obtain additional evidence of grafting, a similar polymerization was conducted in the absence of the crosslinker. After extracting the homopolymer and unreacted monomers using a cellophane membrane dialysis bag (D9402, Sigma-Aldrich), an appreciable amount of grafted collagen (87%) was observed. The graft copolymer spectrum was very similar to Figure 1-b. Also according to preliminary measurements, the sol (soluble) content of the hydrogel networks was as little as 1.8 %. This fact practically proves that all AMPS are almost involved in the polymer network. So, the monomers percent in the network will be very similar to that of the initial feed of reaction.

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[FIGURE 1 OMITTED]

Scanning electron microscopy:

One of the most important properties that must be considered is hydrogel microstructure morphologies. Figure 2 shows the scanning electron microscope (SEM) photographs of the surface (Fig. 2A) and the cross-sectional area (Fig. 2B) of the hydrogel with interconnected pores. These pictures verify that the synthesized polymer in this work have a porous structure, where the pores might be induced into the hydrogel by water evaporation resulting from reaction heat. It is supposed that these pores are the regions of water permeation and interaction sites of external stimuli with the hydrophilic groups of the graft copolymers. The cross-sectional view of hydrogels (Fig. 2B) also exhibited large, open, channel-like structure.

The results of BET analysis showed that the average pore diameter of the synthesized hydrogel was 16.7 nm. In general, the size of the pores can be controlled by adjusting the various factors such as the type and amount of surfactant, porosigens and gas forming agent during crosslinking polymerization, and the amount of diluent in the monomer mixture (i.e., monomer-diluent ratio) [9]. For example, as the amount of diluent (usually water) in the monomer mixture increases, the pore size also increases up to the micrometer (um) range [11].

The porosity plays the multiple role of enhancing the total water sorption capability and the rate of response by reducing the transport resistance [14,17]. Therefore, creation of porosity in hydrogels has been considered as an important process in many ways. The phase-separation technique, [10] the water-soluble porogens [25] and the foaming technique [12,13] are three different methods for preparing porous hydrogel structures. In this paper, as mentioned above, however, the pores were simply produced from water evaporation resulting from reaction medium heat.

Thermal analysis:

Thermogravimetric analysis (TGA) was employed to thermally characterize the hydrogel in comparison with the intact collagen (Figure 3). The thermal stability of the grafted collagen is improved as is obvious from the TGA curve. TGA of collagen (Figure 3-a) shows a weight loss in two distinct stages. The first stage ranges between 10 and 130[degrees]C and shows about 17% loss in weight. This may correspond to the loss of adsorbed and bound water [28]. No such inflexion was observed in the TGA curve of collagen-g-poly (AMPS) hydrogel (Figure 3-b). This indicated that the grafted copolymers were resistant to moisture absorption. The second stage of weight loss starts at 230[degrees]C and continues up to 300[degrees]C during which there was 52% weight loss due to the degradation of collagen. In general, degradation of native collagen is faster than that of grafted collagen. About 60% weight loss takes place in the temperature range of 220-370[degrees]C for collagen. In the collagen-g-poly (AMPS) sample, a residual weight of 77% was observed at 310[degrees]C. The appearance of these stages indicates the structure of collagen backbones has been changed, which might be due to the grafting of poly (AMPS) chains. In general, the copolymer had lower weight loss than collagen. This means that the grafting of collagen increases the thermal stability of collagen in some extent.

Rheological Behaviors of hydrogel:

The rheological test was carried out with UDS 200 Parr Physica rheometer with two oscillation and rotation modes of measurement. Dynamic oscillatory measurements allow accurate determination of the systems gel time. In these experiments, the evolution of the modulus G(t) is measured in small amplitude oscillatory shear as a function of cross-linking time while, frequency is kept constant throughout the experiment [16]. As an example, a plot of the G(t) versus time at 120[degrees]C is presented in Figure 4. Trends in changes modules at different isothermal temperatures are the same. Fig 4 shows that the prepared gel is a structural gel.

Rheological properties such as G(t) is very sensitive to changes in molecular structure and phase transitions occurring in thermosetting polymer systems [17]. Cross-linking of thermosetting polymers can be modeled as a cluster formation process. During the initial period of reaction, microgels are formed with branched and partially crosslinked molecules of colloidal sizes [18]. The polymer continues reactivity to form larger clusters of various sizes distributed randomly in the system [19]. At final when the gel is formed completely, we have a broad distribution of macro molecules.

Before gelation time, the thermosetting resin at the early stage of curing is in liquid state and the viscous behavior dominates the initial part of the curing process. Both of the dynamic moduli increase as a result of increasing cross-link density and molecular weight of the curing polymer system. At the gel point an infinitely large cluster extends throughout the whole system, and a three-dimensional continuous network is formed [16].

The creep compliance test were carried out with applying of shear stress in 30 sec and measuring of continuous deformation in 4000 sec. The result is shown in fig 5. It is show that the gel is a structural and the gel is formed completely after a certain time [20].

The loss factor is high at the beginning of the test and decreases with time [16].

[FIGURE 2 OMITTED]

Figure 4 shows the viscosity change during isothermal curing of the gelatin reactive formulation at the strain amplitude of 1% and dynamic frequency of 10 [s.sup.-1]. As seen in Figure 4, at isothermal curing temperature a steep decrease of the value of compliance is observed, reflecting a phase transition from liquid to solid. In some reports, the gel time has been determined as the time when the viscosity of the reacting system tends to infinity [21].

Effects of shear thinning for Gelatin reactive formulation is shown in fig. 6. As seen, at low shear rates the viscosity increases and with the increase of the shear rate the viscosity decreases. With the progression of the reaction, the gel network is formed. Consequently, the application of shear rate can induce a shear thinning behavior in long chain entangled macromolecules.

By applying of the shear rate to the gel, it causes an orientation of molecular chains which leads to shear thinning behavior and the dilution effect of shear rate on viscosity seems to be dominant. Hence, the viscosity of the reactive formulation at higher shear rates is lower than that at lower shear rates [21].

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Conclusion:

A novel protein-based superabsorbent hydrogel was synthesized via graft copolymerization of 2acrylamido-2-methylpropanesulfonic acid (AMPS) onto collagen backbones in an aqueous solution using a persulfate initiator and a hydrophilic crosslinker. The study of FTIR spectra and thermogravimetric analysis provide the graft copolymerization do takes place. In this study, the rheometry results showed that trend of the change in gelation times lower temperature sensitivity above the gelation temperature.

By propagating of the gel reaction and formation of long chain macromolecules shear thinning behavior was observed in viscoelastic regions. The shear thinning phenomenon of long chain gelatin molecules counters the influence of the shear rate on the increase of the mobility of functional groups. The changes of the modulus, G(t) the loss modulus with time measured in small amplitude oscillatory shear as a function of cross-linking time while, frequency is kept constant throughout. The creep compliance test shows the formation of a structural gel.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

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Mohammad Sadeghi, Sadegh Morady

Department of Chemistry, Science Faculty, Islamic Azad University, Arak Bra nch, Arak, Iran.

Corresponding Author

Mohammad Sadeghi, Department of Chemistry, Science Faculty, Islamic Azad University, Arak Branch, Arak, Iran.

Tel: 861-3670017, Fax: 861-3670017, P.O. Box 38135-567 E-mail: m-sadeghi@iau-arak.ac.ir
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Title Annotation:Original Article
Author:Sadeghi, Mohammad; Morady, Sadegh
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Feb 1, 2012
Words:2896
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