properties and potential applications of carboxymethyl

Charito Tranquilan-Aranilla1, Bin Jeremiah D. Barba1, Lorna S. Relleve1, and Naotsugu Nagasawa2

1Philippine Nuclear Research InstituteDepartment of Science and Technology

Commonwealth Avenue, Diliman, Quezon City 1101 Philippines2Takasaki Advanced Radiation Research Institute

National Institutes for Quantum and Radiological Science and TechnologyWatanuki 1233, Takasaki, Gunma 370-1292 Japan

Properties and Potential Applications of Carboxymethyl-kappa-carrageenan Hydrogels

Crosslinked by Gamma Radiation

Keywords: carboxymethyl-k-carrageenan, hydrogel, radiation crosslinking, wound dressing, water retention, metal adsorbent

Carboxymethyl κ-carrageenan (CMKC), with different degrees of substitution (DSs), were gamma-irradiated in viscous or paste solutions. Successfully synthezised chemically crosslinked hydrogels showed dependence on the DS, concentration, and radiation dose. The highest gel fraction was 76% exhibited by CMkC-3s hydrogel with a DS of 1.58. The hydrogels showed different swelling degrees in water and saline. Swelling behavior vs. time, in both solvents, corresponded to 2nd-order kinetics. The CMkC-3s at 20% concentration irradiated at 15 kGy had the highest water absorption of 334 g water/g dry gel. Selected hydrogels were evaluated for applications as wound dressing, as water retainer in sandy soil, and as metal adsorbent. As a wound dressing, CMkC-2s and CMkC-3s hydrogels exhibited considerable tensile strengths, abilities to absorb pseudo extracellular fluid, and extractables with pH/conductivity conducive for healing promotion. Also, the CMkC-3s hydrogel had no cytotoxic potential based on the MTT test. As water retainer in sandy soil, test samples with 0.1, 0.3, and 0.5% CMkC-3s granules initially retained 25.1%, 32.2%, and 42.6% water, respectively, compared to 19.2% of the sandy soil alone. On Day 7, the three sandy soil-CMkC groups still had 13.7–29.3% water, while the control had only 3.85%. In the batch adsorption studies, the hydrogels adsorbed Cu2+, Zn2+, Cd2+, and Pb2+ heavy metals in the solution at different capacities, with Cd2+ as the highly adsorbed and Pb2+ as the least. The CMkC-3s hydrogel showed the highest metal uptake and adsorption efficiency, followed by CMkC-2s, then CMkC-1s. The CMkC-3s hydrogel, further tested on pH effect, exhibited optimum metal uptake at neutral pH.

*Corresponding Author: [email protected]

INTRODUCTIONHydrogels are macromolecular-based materials characterized by hydrophilicity, stimuli-sensitivity, soft-tissue likeness, and flexibility. These materials

are extensively studied for vast applications. In the biomedical field, hydrogels are developed into wound dressings, injectables, drug delivery systems, self-healing materials, and cell culture matrices (Chai et al. 2017). Hydrogels with a water-absorbing capacity of 100–1000 g water per g of dry gel are considered

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Philippine Journal of Science150 (1): 85-97, February 2021ISSN 0031 - 7683Date Received: 27 Jul 2020

superabsorbent (Zohuriaan and Kabiri 2008) and used in commercial products like diapers and pads (Dey et al. 2016). Also, superabsorbent hydrogels are utilized to retain water in sandy soil, as a nutrient carrier, as a bed to protect seedlings during transplant, and many more (Guilherme et al. 2015). In the development of hydrogels, primary raw materials that gained the most attention are polysaccharides – known for their biocompatibility, renewability, environment-friendliness, and competitive properties (Lindblad et al. 2007).

Carrageenan is a high-molecular-weight, anionic polysaccharide that occurs as structural matrix material in several species of red seaweeds. It is a linear sulfated galactan containing alternating (1->3)-α- and (1->4)-β-d-galactopyranosyl linkages (Sudhakar et al. 2018). K-carrageenan, which has one sulfate per disaccharide, is one of the three most important commercial forms of carrageenans capable of forming a strong thermo-reversible gel. It is a good candidate material for developing hydrogels for various applications. K-carrageenan hydrogels can be formed by ionic crosslinking using salts of potassium, sodium, and calcium, but have poor stability issues (Berton et al. 2020). Covalent bonding is typically preferred for the formation of stable gels, and this is usually achieved by using chemical crosslinkers such as glutaraldehyde (Distantina et al. 2013). However, glutaraldehyde has known risks associated with its toxicity (Zeiger et al. 2005). Genipin has also been used as an alternative non-toxic crosslinker, but results so far still exhibit limited swelling capabilities (Hezaveh and Muhamad 2013).

Radiation-induced crosslinking is an attractive processing method that eliminates the use of toxic initiators and can be carried out in aqueous systems at ambient temperatures (Woods and Pikaev 1993). Moreover, it has the unique ability to simultaneously synthesize hydrogels and sterilize the product (Caló and Khutoryanskiy 2015). Polysaccharide derivatives – such as carboxymethyl cellulose (Wach et al. 2003a), carboxymethyl starch (Pant et al. 2011; Nagasawa et al. 2004), carboxymethyl hyaluronic acid (Relleve et al. 2018), carboxymethyl chitosan, and carboxymethyl chitin (Wasikiewicz et al. 2006; Zhao et al. 2005) – have been successfully cross-linked by radiation. The common denominator is irradiation of aqueous polymer solution in high concentration, above 10% up to as high as 60%, where homogeneity is still maintained. At these working concentrations, polymer chains are near each other which facilitated the crosslinking.

K-carrageenan is highly susceptible to depolymerization when exposed to high-energy radiation (Abad et al. 2009). Hence, in this work, KC is carboxymethylated to attempt radiation crosslinking of its aqueous solution.

The properties of synthesized hydrogels, assessed in response to radiation dose, are reported. Investigations on the potential applications of selected CMkC hydrogels as wound dressing, super water-absorbent for sandy soil amelioration, and metal adsorbent gel are also presented. While CMkC has been studied for biomedical applications like hemostatic agent (Tranquilan-Aranilla et al. 2016) and wound dressing (Fan et al. 2011), there has there been no published work on extending its application as soil ameliorant or as metal adsorbent.

MATERIALS AND METHODSDetailed information on the materials used, synthesis of CMkC, and characterization methods are given in the Appendices section.

RESULTS AND DISCUSSIONCarboxymethyl k-carrageenan derivatives, with different DS were obtained via multistep conversions and the yield for each step was > 90%. The DS and molecular weights were determined by nuclear magnetic resonance spectroscopy and gel permeation chromatography, respectively, as reported in our previous publication (Tranquilan-Aranilla et al. 2016) and summarized in Appendix Table I.

Radiation Crosslinking of CMkC HydrogelsThe formation of hydrogels via radiation-induced crosslinking is a result of mutual recombination of macroradicals. Irradiation of polysaccharide derivatives homogenously dispersed in water in high concentrations or paste-like conditions favors crosslinking (Relleve et al. 2018; Wach et al. 2014). The presence of water contributes to the crosslinking process in two ways. First, a high concentration of macroradicals is formed because of indirect effect due to reactions of OH radicals (water radiolysis product) with macromolecules. Second, water enhances the mobility of rigid polymer chains, allowing the diffusion of the macroradicals to proximate distance favorable for recombination (Olejniczak et al. 1993).

Irradiation successfully transformed CMkC paste solutions into crosslinked hydrogels, as indicated by the percent insoluble gels shown in Figure 1. Gel fractions of 20–40% CMkC-2s and CMkC-3s based hydrogels ranged 6–54% and 18–76%, respectively. For CMkC-1s hydrogels, insoluble gels were retrieved only at concentrations 30–40% ranging from 6–39%. There were no gels that formed in all irradiated hydrogels containing 10% polymer

Aranilla et al.: Properties and Applications of CMkC Hydrogels

Philippine Journal of ScienceVol. 150 No. 1, February 2021

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and in 20% CMKC-1s. The crosslinking of CMkC showed dependence on the polymer concentration and absorbed dose. An increase in absorbed dose results in a substantial radical formation and an increase in concentration leads to a high probability of recombination of radicals due to the entanglement of polymer chains (Wach et al. 2003a). The effect of concentration on crosslinking dose was also evident, with an increase in concentration leading to a decrease in crosslinking dose. Considering CMkC-1s hydrogels, at 30% concentration, the GF was about 30% at 50 kGy while the same GF value formed at 30 kGy at 40% concentration. The DS is another factor that had a major influence on the crosslinking of CMkC hydrogels. The CMkC-3s of DS 1.58 exhibited the highest gel fraction (76%, 100 kGy), while CMkC-1s of DS 1.15 had the lowest gel content (39.0%, 100kGy). Higher substituents in the KC chain enhanced its crosslinking ability, and this supports the assumption that crosslinking of the derivatives is due to the carboxymethyl side chains. The same was observed in the radiation-induced crosslinking for carboxymethyl cellulose. The gel fraction of CMC with a DS of 2.2 was higher compared to CMC with a DS of 1.32 at lower doses with the same concentration (Fei et al. 2001).

The most probable crosslinking sites for CMkC involve the carboxymethyl side chains (Appendix Figure I). Electron spin resonance (ESR) studies on an irradiated aqueous solution of carboxymethyl cellulose revealed a significant number of radicals generated in the carboxymethyl substituents by reaction with OH radicals. The stability of the radicals formed at the α-carbon or secondary carbon of the carboxymethyl side chain was favorable to participate in intermolecular crosslinking (Saiki et al. 2011; Wach et al. 2003b). ESR studies on other polysaccharide derivatives such as carboxymethyl chitosan (Saiki et al. 2010), methylcellulose, and hydroxypropyl cellulose (Wach et al. 2003b) also reported the generation of

radicals on the side chains that may be responsible for recombination reactions leading to crosslinking.

Swelling Behavior of CMkC HydrogelsA hydrogel can absorb and hold a significant amount of water without being dissolved. This ability comes from the hydrophilic functional groups present in the polymer backbone, while the resistance to dissolution arises from the crosslinks between network chains (Okay 2010). The swelling capacity of hydrogels is one important consideration for potential applications and is usually expressed as the fractional increase in the weight of the hydrogel due to water absorption (Park et al. 2009).

Figures 2a and 2b depict the swelling curves of the different hydrogels in deionized water at various radiation doses. The common trend observed for all hydrogels was decreasing swelling capacity with increasing radiation dose, as well as with increasing polymer concentration. This behavior is due to the dependence of swelling on the cross-link densities of the hydrogels. High cross-linking makes the hydrogel network rigid and less pliable and, hence, less accessible for water to diffuse (Kolodynska et al. 2016). The CMkC-3s hydrogel at 20% concentration exhibited the highest amount of absorbed water, 334 g/g dried gel at 15 kGy, while CMkC-3s hydrogel at 40% absorbed only 6 g/g dried gel at 100 kGy.

The swelling behavior of CMkC hydrogels in saline showed the same dependence in dose and concentration, but with a drastic decrease (more than 50%) in the swelling capacity (Figures 2c and 2d). Since CMkC hydrogels are polyelectrolyte in nature, due to the presence of COO– and SO3

– functionalities, they can be sensitive to even a small amount of salt or variation in pH value (Arndt and Sadowski 2014). Generally, the swelling capacity of ionic hydrogels in salt solutions is significantly decreased compared to

Figure 1. Gel fractions of crosslinked hydrogels at different doses: (a) CMkc-1s, (b) CMkC-2s, and (c) CMkC-3.



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the absorbency in water due to the charge screening effect of the cations, causing a marked reduction in the repulsive forces of charged segments of cross-linked polysaccharide. Therefore, the osmotic swelling pressure difference between the gel and aqueous phase decreases and, consequently, leads to gel contraction (Relleve et al. 2018).

Swelling vs. time of 30% CMkC hydrogels irradiated at 30 kGy was also studied, showing a similar trend in both media. The results are presented in Figures 3a and 3b. Solvent absorption increased quickly during the first two hours because, in this stage, the swelling was controlled

mainly by diffusion of the solvent into the gel network. Then, absorption slowed down and reached equilibrium in eight hours. During this period, the relaxation of the polymer chain mainly controlled the swelling process. In the presence of salt ions, rearrangement of weak secondary interactions such as electrostatic repulsion and neutralization of charges occur (Lu et al. 2015). The linear variation of t/St against time revealed second order kinetics of swelling, given by the equation (Li et al. 2019):

tSt

1 ++kSeq2

tSeq

Figure 2. Degree of swelling in deionized water (a*, b**) and saline (c*, d**) at different doses (*CMkC hydrogels at 30% concentration; **CMkC-3s hydrogels at different concentrations).



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The St and Seq denotes the degree of swelling at time t and at equilibrium, and k is the swelling rate constant. The parameters of swelling kinetics are listed in Appendix Table II.

Preliminary Investigation on Potential Applications of CMkC Hydrogels as Wound DressingNatural skin is recognized as the ideal wound dressing and so the development of “moist dressings” was based on the desire to replicate skin with its 85% water content and inherent permeability (Lloyd et al. 1998). Polysaccharide-based hydrogels are focal materials for investigation as potential wound management aids. Alginic acid/alginate, hyaluronic acid/hyaluronate, cellulose, dextran, chitin and chitosan, carrageenan, and heparin are used in wound management as dressing materials (Aramwit 2016). Some characteristics of a wound dressing that would fulfill optimum healing environment are: 1) maintain high humidity at the wound-dressing interface while removing through adsorption excess wound exudates and associated toxic compounds; 2) permit exchange of gases but impermeable to microorganisms preventing secondary infections; 3) provide thermal insulation; 4) biocompatible (all components of the dressing) and does not provoke any allergic reactions through prolonged contact with the tissue; 5) minimal adhesion to the surface of the wound and can be removed without trauma; 6) physically strong when wet; 7) can be produced in sterile form; and 8) easy to dispose of when removed at the end of the use (Ghomi et al. 2019; Dhivya et al. 2015).

For wound dressing application, hydrogels of 30% CMkC-2s and CMkC-3s irradiated at doses 20, 30, and 50 kGy were evaluated for mechanical strength, swelling in simulated wound fluid, and pH and conductivity of extracts. A hydrogel wound dressing must have an appreciable mechanical strength and should not disintegrate during and after use to prevent small gel debris to remain in the wound area (Sen and Avci 2005). The tensile strengths of both hydrogel formulations showed increasing value from 20–50 kGy (Figure 4a). However, CMkC-3s exhibited higher strengths than CMkC-2s due to more crosslinking in its network.

An ideal wound dressing can absorb wound fluid to prevent secondary infection (Dhivya et al. 2015). The accumulation of wound exudates often causes maceration and bacterial overgrowth on the wound site. Swelling studies of hydrogels in simulated physiological fluid give a good indicator of performance as wound dressing, in absorbing wound exudates, and in providing an advantage to design the hydrogel from a scientific point of view (Sen and Avci 2005). Fresh CMkC hydrogels, irradiated at 30 kGy, absorbed simulated exudates more than 30 times its weight after 8 h. Although CMkC-2s continued to absorb PECF after 48 h and CMkC-3s almost reached equilibrium, the difference in absorption capacities was not significant (Figure 4b).

Wound healing is promoted when the pH of the wound environment is maintained within the skin pH range of 4–6.8. Moreover, creating an acidic environment in a wound bed positively influences the wound healing

Figure 3. (a) Degree of swelling as a function of time of 30% CMkC hydrogels irradiated at 30 kGy; (b) swelling behavior corresponding to 2nd order kinetics.



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process by controlling wound infections, increasing antimicrobial activity, altering protease activity, releasing oxygen, reducing the toxicity of bacterial end products, and enhancing epithelization and angiogenesis (Nagoba et al. 2015). The pH range of extracts from CMkC-2s was 6.68–6.96, while for CMkC-3s, the range was 6.26–6.31 (Figure 4c). Between the two test samples, CMkC-3s had more acidic extracts due to the presence of more ionizable carboxylic groups (higher DS) and higher free sulfates present in the soluble portion due to desulfation during irradiation (Tranquilan-Aranilla et al. 2012).

The conductivity of hydrogels is also considered an important property for application as a wound dressing. Conductive hydrogels were observed to give additional analgesic effects or acute pain reduction (Sen and Avci 2005). The extracts from CMkC-3s have higher conductivities compared to CMkC-2s extracts due to more ionic groups in the sol fraction (Figure 4c).

In Vitro Cytotoxicity Test of Hydrogel ExtractsCytotoxicity investigation is the first step for the screening of biocompatibility and safety of materials intended for biomedical use. In vitro methods measure the adverse biological effect of extractable from test materials on cultured cells (Li et al. 2015). The MTT (methyl thiazolyl tetrazolium) assay principally determines cell viability based on the mitochondrial function of cells. The water-soluble MTT converts to purple crystalline formazan (Aslantürk 2017), and the quantified colorimetric value (optical density) indicates the number of surviving cells (Vidal and Granjeiro 2017). Rejection criteria set to < 70% cell viability compared to the blank, rates a material with cytotoxic potential. The cell viability value for the CMkC-3s hydrogel was > 99%, indicating it has no cytotoxic potential.

From these results, CMkC-3s hydrogel, irradiated at 30 kGy, has the ideal properties to be developed further as a

wound dressing. In previous work, the hemostatic properties of CMkC hydrogels were also studied. Blood coagulation efficiency was comparable to commercial counterparts, based on in vitro simulations of clotting and platelet adhesion (Tranquilan-Aranilla et al. 2016). Hence, CMkC hydrogels have promising properties for biomedical applications.

As Water Retainer in Sandy SoilHydrogels are capable of absorbing irrigation, thus increasing the water retention capacity of soils and reduce deep percolation. The water held in the swollen hydrogel serves as a soil reservoir for maximizing the efficiency of plant water uptake (Abdallah 2019). The CMkC-3s hydrogel with a concentration of 20% irradiated at 15 kGy (has the highest swelling capacity) was applied as dried granules and mixed with the sandy soil. The water holding capacity of the sandy soil improved with the addition of CMkC-3s granules. Soil with 0.1, 0.3, and 0.5% granules had 42.6%, 32.2%, and 25.1% water contents, respectively, while the control (0% hydrogel) had 19.2%. After 7 d, the soil-hydrogel system still has a substantial amount of water, ranging from 10–28%, while the control had a remaining 3% (Figure 5). These results show that even with a small amount of the hydrogel improves the water retention capacity of the sandy soil.

Biodegradability. The microbial biodegradability of the CMkC-3s hydrogel, as well as the polymer form, was evaluated by measuring released carbon dioxide using MODA. The results show that both samples were biodegradable, but the rate of degradation was lower compared to the control cellulose. Degradation values of CMkC-3s polymer, CMkC-3s hydrogel, and cellulose were 60%, 32%, and 95%, respectively (Figure 6). The KC backbone is specifically degraded by carrageenases. Due to this specificity, the soil microbes present in the test mixture were not able to utilize the CMkC polymer as a carbon source as effectively as cellulose. Also, the

Figure 4. Characteristics of CMkC hydrogels for wound dressing application: a) tensile strength, b) swelling in PECF of hydrogels irradiated at 30 kGy, and c) pH and conductivity of extracts. Mean values were statistically significant at P < 0.05, except for swelling capacity.



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crosslinked network in the CMkC hydrogel slowed down access of microbes to the inner portion of the hydrogel. The slower rate of degradation of the hydrogel is advantageous for application in agriculture since retained water will be available during the crop/plant growth and development period.

As Metal AdsorbentDiverse industries – such as metal plating facilities, mining operations, leather tanning, and pesticide manufacturing – generate large discharge of effluents

containing toxic substances (heavy metal ions, dyes, etc.) into the environment (Kanamarlapudi et al. 2018). Recently, hydrogels with high sorption capacity and high functionality are considered good contenders for the removal of various aquatic pollutants, including heavy metal ions (Shalla et al. 2018). The chemical or physical interactions between heavy metals and functional groups of adsorbents contribute significantly to the adsorption process. Studies reported hydrogels with oxygen-containing functional groups (such as -OH, -SO3H, and -COOH) could remove heavy metals in aqueous solutions through physical adsorption, electrostatic interactions, ion exchange, or complexation mechanisms (Tran et al. 2018).

Low swelling CMkC hydrogels (40%, 75 kGy, dry state) were tested for adsorption of divalent heavy metals –

Figure 5. Water retention of sandy soil without (control) and with different concentrations of CMkC-3s hydrogel.

Figure 6. Microbial degradation of CMkC-3s polymer and hydrogel in 60 days.

Figure 7. Metal adsorption capacities of:a) CMkC hydrogels (40%, 75 kGy) at constant pH (4.03) and (b) CMkC-3s hydrogel at different pH.



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Cu2+, Zn2+, Cd2+, and Pb2+ – at constant pH of 4.03. The concentrations of the metals in the solution were 94.8, 94.2, 91.6, and 84.8 mg/L, respectively. As seen in Figure 7a, the hydrogels adsorb all four metals in the solution at different capacities, with Cd2+ (81.7 mg/g by CMkC-3s) as the highly adsorbed and Pb2+ (62.8mg/g by CMkC-3s) as the least. The CMkC-3s hydrogel showed the highest metal uptake and adsorption efficiency, followed by CMkC-2s, then CMkc-1s. The varying number of functional groups present in the network is responsible for the adsorption capacity and efficiency performance of each hydrogel.

The metal uptake of CMkC-3s further studied for the effect of pH, showed increased adsorption capacity from pH 1–7, then decreased at pH 9 (Figure 7b). The pH of the solution has a direct effect on the metal chemistry in the solution and the ionization state of the functional groups in the adsorbent, leading to variation in adsorption capacity (Farghali et al. 2013). At pH 1, the adsorption capacity for all metals was almost none, as large quantities of H+ compete with the metal cations for the adsorption sites. Since the adsorbent holds mainly protonated sites, it maintains a net positive charge surface making it difficult for the metal ions to access the functional groups (Rahangdale et al. 2020). The maximum adsorption was at the neutral pH. As the pH increased to 7, the concentration of H+ decreased thus, making more adsorption sites available for metal ions. Above pH 7, adsorption capacity decreased due to the tendency of metal ions to precipitate as hydroxides (Zhang et al. 2020). A study on the sorption behavior of radiation crosslinked carboxymethyl KC for Cr6+ showed that CMkC best adsorbs Cr6+ at pH 6, removing 41.59% of the metal ions present in the solution (Antonio et al. 2009). Further studies to characterize the adsorption kinetics of CMkC-3s are planned.

CONCLUSIONRadiation-induced crosslinking of CMkC paste solutions successfully synthesizes hydrogels showing dependence on the DS, concentration, and absorbed dose. The highest gel content is 76%, exhibited by CMkC-3s hydrogel with a DS of 1.58. The hydrogels show different swelling degrees in water and saline. The highest water absorption of 334 g water/g dry gel is exhibited by 20% CMkC-3s irradiated at 15 kGy. Results of the preliminary evaluation of selected CMkC hydrogels as wound dressing, water retainer in sandy soil, and metal adsorbent prove very promising. Further studies need to optimize the properties of the hydrogels for the intended applications.

ACKNOWLEDGMENTSThis study was made possible through the MEXT Nuclear Research Exchange Program hosted by the Takasaki Advance Radiation Research Institute. The authors would like to thank the Philippine Nuclear Research Institute and the Department of Science and Technology for the support.

STATEMENT ON CONFLICT OF INTEREST The authors declare no potential conflict of interest related to this publication.

NOTES ON APPENDICESThe complete appendices section of the study is accessible at http://philjournsci.dost.gov.ph

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APPENDICES

MATERIALS AND METHODS

Commercial grade refined KC with a molecular weight of 916 kDa was purchased from Shemberg, Philippines, and used “as received” in the carboxymethylation reaction. All reagents were prepared from analytical grade chemicals. Purified or deionized water was utilized for all reagent preparations.

Synthesis of CMkCCMkC was synthesized according to the established method from previous work (Aranilla et al. 2012). Briefly, KC powder (80 g) was suspended in isopropyl alcohol (80%, 720 mL) and added dropwise with NaOH (10 M, 93.5 mL) while mixing the slurry vigorously for 1 h. Then, monochloroacetic acid (49.1 g) was added slowly, and the synthesis proceeded for 3 h at 40 °C. The product was vacuum filtered, suspended in IPA (80%, 500 mL), and then neutralized with acetic acid to pH 6.5. After three washes with 80% IPA, followed by pure IPA (300 mL), the product was oven-dried at 50 °C. Up to three conversion steps were performed to obtain derivatives with different degrees of substitution.

Preparation and Crosslinking of CMkC Hydrogels Pre-determined amounts of CMkC were soaked in deionized water for 1 h then loaded in a centrifugal planetary mixer with degassing capability (Thinky ARE-250, Keyence Co. Ltd., Japan), to prepare 10–40% (w/v) viscous or paste solutions. All mixtures were kept overnight at ambient temperature before irradiation to ensure complete dissolution and homogeneity. Samples packed flat in plastic pouches and vacuum-sealed were gamma-irradiated at doses of 0, 5, 10, 15, 20, 30, 50, 75, and 100 kGy. The hydrogels were relaxed overnight at ambient temperature before characterization

Characterization of CMkC HydrogelsGel fraction. The hydrogels were cut into square slabs and dried to constant weight in a vacuum oven at 40 °C. About 0.2 g of dried gels were soaked in 500 mL of purified water at ambient temperature, with occasional stirring to effectively extract the soluble portion. After 72 h, the gels were collected in stainless steel net then dried again at 40 °C. Gel fraction was calculated as follows:

(1)

Swelling behavior. Swelling behavior was studied in both purified water and saline solution (0.9% NaCl). About 0.2 g of dried gels were placed in stainless steel net pouches and then immersed in the given solvents at ambient temperature. After 48 h, the swollen gels were collected, dried superficially with lint-free tissue and weighed. Degree of swelling was calculated as follows:

(2)

The swelling kinetics of the hydrogels in both solvents was also measured in the first 24 h. Swollen gels retrieved at different swelling times were dried superficially with lint-free tissue, weighed, and returned into the same bath. Degree of swelling was calculated using Equation 2. Average of three replicates was reported.

Potential Applications of Selected Hydrogels Evaluation as wound dressing. Based on gel content and swelling properties, selected hydrogel formulations were further characterized. Hydrogels of 30% CMkC-2s and CMkC-3s irradiated at 20, 30, and 50 kGy were evaluated as wound dressing.

Tensile test. Tensile strength was measured using a Strograph R1 Testing Machine (Toyo Seiki, Japan) according to JIS K-6301 method, with crosshead speed of 50 mm/min. Hydrogels films (1 mm thickness) in relaxed state were cut into dumbbell shapes with central cross section of 5 x 0.5 mm2. At least five measurements for each sample were recorded.



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Swelling behavior in PECF. PECF or simulated wound exudate was prepared by mixing 0.68 g NaCl, 0.22 g KCl, 2.5 g NaHCO3, and 0.35 g NaH2PO4 in 100 mL deionized water (Sen and Avci 2005). Fresh hydrogels, irradiated at 30 kGy, were left to swell in PECF solution at 37 °C. Swollen gels were removed from the medium at regular intervals, dried superficially with lint-free tissue, weighed, and placed in the same bath. Swelling was monitored for 72 h.

pH and conductivity of hydrogel extract. The pH and conductivity of the extracts were measured using HM-60G pH meter and CM-60G EC meter (DKK-TOA, Japan). Ten grams (10 g) of fresh hydrogels were soaked in 50 mL deionized water and kept in a thermostat bath at 37 °C for 72 h. Water level was maintained during the incubation period. Results were reported as average of three trials.

Cytotoxicity of hydrogel extract. To evaluate the cellular response, MTT cytotoxicity assay based on ISO 10993-5 using L-929 mouse fibroblast cells (ATCC, CCL1, NCTC 929, Strain L) was done for CMkC-3s. The sample was extracted using MEM at 37 °C for 24 h with a mass-to-volume extraction ratio of 0.05 g/mL. Medium without test specimen was used as blank.

Evaluation as metal adsorbent. Hydrogels (40% composition) irradiated at 75 kGy were dried and evaluated for adsorption of common metals present in wastewater effluents such as Cu, Zn, Cd, and Pb. Competitive sorption experiments were carried out at 25 °C using 50 mL of mixed metal solution (pH adjusted to 4.0) containing about 100 ppm of each metal and 0.050 g dried hydrogels. The adsorption capacities were measured after 24 h contact time using an inductively coupled plasma mass spectrometer (Hewlett Packard 4500 Series) and expressed as mg of metal adsorbed per g hydrogel.

As Superwater Absorbent for Sandy Soil AmeliorationWater retention. Dried CMkC-3s gels were granulated and applied in the concentration range of 0.1–0.5% when mixed with the sandy soil. One-hundred grams (100 g) of the sandy soil-CMkC system were placed in plastic pots. Set-ups without hydrogel were used as control. Each pot was poured with 300 mL of deionized water and allowed to drain completely. The weight of the system was monitored for 7 d. Water content was calculated as:

(3)

Biodegradation test. The microbial biodegradability of the CMkC hydrogels, as well as polymer form, was evaluated by measuring released carbon dioxide using a specially designed apparatus called microbial oxidative degradation analyzer (Saida Ironworks Co., Ltd.). Ten grams (10 g) of sample was mixed well with sea sand (450 g) rinsed with water and compost (130 g), then placed in the heated reaction column and incubated at 35 °C for 60 d. CO2-free moisturized air flowed into the reaction column at a rate of 30 mL/min. The air with CO2 (product of sample decay) that flowed out of the reactor passed through a series of columns filled with silica gel, calcium chloride, soda lime, and calcium chloride. The CO2 was collected quantitatively by soda lime, while water produced during the reaction caught up in the last column containing calcium chloride. The amount of evolved CO2 was calculated as the difference in the weight of the last two columns, in the beginning, during, and at the end of the testing period. Pure compost mixed with sea sand was blank and cellulose powder was the reference (Nagasawa et al. 2004). Biodegradation was calculated as follows:

(4)

Statistical AnalysisData were presented as means ± SD. Means of different treatment groups/parameters/samples were analyzed using ANOVA, when applicable. Values were considered significant at p < 0.05.



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Appendix Table I. Characteristics of CMkC.

Samplecode

Conversion steps Degree of substitution Molecular weight(kDa)

CMkC-1s 1 1.15 538

CMkC-2s 2 1.47 504

CMkC-3s 3 1.58 496

Appendix Table II. Kinetic parameters of CMkC-hydrogels (30%, 30 kGy).

SampleSeq (g/g) k x 10–5 [g / (g*min)]

H2O Saline H2O Saline

CMkC-1s 149 89 2.02 1.48

CMkC-2s 124 47 2.61 5.38

CMkC-3s 76 31 3.02 17.2

Appendix Figure I. Most probable crosslinking sites in CMkC polymer.



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properties and potential applications of carboxymethyl

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