Bio-Medical Materials and Engineering 24 (2014) 1837–1849 DOI 10.3233/BME-140994 IOS Press
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Effects of gamma irradiation and moist heat for sterilization on sodium alginate Tingzhang Hu, Yongwei Yang, Lili Tan, Tieying Yin, Yazhou Wang and Guixue Wang ∗ Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China Received 23 May 2013 Accepted 22 April 2014 Abstract. Polysaccharides, such as alginates, are already being used as carriers for drug delivery. The physicochemical and biological properties of alginates may be affected via irradiation and thermal treatments. To explore and compare effects of two kinds of sterilization methods, gamma irradiation and moist heat, on sodium alginate (SA), physicochemical and biological properties of SA powder and solutions were investigated after sterilization. Human umbilical vein endothelial cells (HUVEC) was used to assess the cytotoxicity of the SA after sterilization. The research showed that 25 kGy gamma ray can effectively sterilize microorganism. Both gamma irradiation and moist heat hardly affect the native pH of SA. Compared to irradiation sterilization, moist heat sterilization showed smaller changes in intrinsic viscosity for all SA samples and lead to less glycosidic bond breaking of SA powders. The moist heat sterilization can cause the main chain scission and double bonds formation of the SA solutions. Cytotoxicity studies demonstrated that sterilized SA powers and SA solutions treated by gamma ray sterilization can increase the viability of HUVEC. However, SA solutions treated by moist heat sterilization were found to present severe cytotoxicity. The research results may provide interesting future advancements toward the development of SA-based products for biomedical applications. Keywords: Sodium alginate, sterilization, gamma ray, moist heat
1. Introduction Polysaccharidic polymers are already being used as thickeners, stabilizers, suspension agents, gelling agents, film-formers, food packaging, emulsifiers, lubricants, plus in medicine as scaffold materials in tissue engineering and as carriers for drug delivery, owing to their unique structure, distinctive properties, safety and biodegradability [1]. By changing the physicochemical and biological properties, such as concentrations, pH value and viscosities, it is possible that the alginate is used to control drug release. Among polysaccharides, sodium alginate (SA) is a biocompatible, non-toxic, non-immunogenic and biodegradable natural linear polysaccharide [2–5], which is widely applied in the fields of medical and industrial. SA is a natural biopolymer typically obtained from the seaweed of brown algae (Phaeophyta) such as genera Macrocystis, Laminaria and Ascophyllum. Alginate is the only polysaccharide that naturally * Address for correspondence: Guixue Wang, Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China. Tel.: +86 23 65112675; Fax: +86 23 65112675; E-mail: [email protected].
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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contains carboxyl groups in each constituent residue of macromolecule, and possesses various abilities for application as a functional material [6,7]. From the molecular point of view, alginate is composed mainly of (1-4)-linked β-D-mannuronic (M) acid units and α-L-guluronic (G) acid units, forming regions of M-blocks, G-blocks and blocks of alternating sequence (MG-blocks). The relative proportions of these sequential organizations depends on the source [8,9]. Most applications of alginate is based on its gel-forming ability through cations binding; for example, the transition from water-soluble SA to water insoluble calcium alginate. Divalent cations preferentially bind toward the G-block rather than the M-block [10]. The macromolecules of polysaccharides are linked together by hydrophilic groups, such as hydroxyl, carboxyl and amino groups that are able to form non-covalent bonds with biological tissues, thus promoting bio-adhesion [11]. Their physical properties depend on their monomer compositions, chain-shapes, viscosities and molecular weights, which influence solubility, gelation and surface properties. Polysaccharides can be cross-linked by chemical or physical interactions. Chemical cross-linking enhances good mechanical stability, however, cross-linking agents are often toxic compounds. For that reason, traces of non-reactive agents should be extracted from the gel before application. Thus preference is given to the use of physically cross-linked gels. Different methods are used for physical cross-linking. One of them is the polysaccharides is cross-linked by ionic interaction. Alginates are able to undergo reversible gelation within aqueous solutions through interaction with divalent cations such as Ca2+ that create ionic inter-chain bridges [12]. SA is a polysaccharide with mannuronic and guluronic acid residues. The gel is formed by binding the guluronic segments of SA to cations and creating three-dimensional networks. Owing to unique physicochemical properties, such as good solubility and high viscosity in aqueous solution as well as gel forming ability in the presence of divalent cations (such as Ca2+ ), SA is used in a wide range of commercial enterprises, including textile, paper, food, and health care industries [13,14]. SA has been tested to prepare environmentally friendly packaging materials such as films and coatings and added to other biopolymers such as pectin or hydroxyl ethyl cellulose to improve their mechanical and water vapor barrier properties [7,15–17]. Alginate gels are pH-sensitive because of the presence of an acidic carboxylic group. Alginate gels shrink at low pH, therefore reducing the release of the drug. This phenomenon may prove advantageous for drug delivery systems. At higher pH, alginate dissolves rapidly and causes a bursting of released drugs, which is usually undesirable. However, by changing the concentrations and viscosities of the alginate solution during synthesis of the gel, it is possible to control drug release [18]. Sterilization refers to the intense processes inactivating spores resulting in products that are shelf stable. It is usually achieved by applying heat, chemicals, irradiation, pressure and filtration [19,20]. Sterilization, however, may affect the surface or bulk properties of the sterilized material, inducing chemical or physical modifications that may alter the characteristics of the biomedical devices in terms of physicochemical, morphological, mechanical and biological behavior. Natural polymers are susceptible to high temperatures and ionizing radiations, which may induce depolymerization, oxidation or the formation of free radicals [21], thus affecting the properties of the gels they may form, such as mechanical strength, swelling and stability behavior [22,23]. In this paper, in order to explore and compare effects of two kinds of traditional physical sterilization methods, gamma irradiation and moist heat, on SA, physicochemical and biological properties of SA powders and solutions were investigated after sterilization. Human umbilical vein endothelial cells (HUVEC) was used to assess the cytotoxicity of the SA after sterilization.
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2. Materials and methods 2.1. Sterilization methods SA was purchased from Dongsheng chemical plant (Wenzhou, China). Gamma irradiation and heat were employed to treat SA samples, according to the recommendations of The International Pharmacopeia (WHO Department of Essential Medicine and Pharmaceutical Policies, 2011). SA powder and 2% and 5% (w/v) SA aqueous solution were used to study effects of damma irradiation and moist heat for sterilization. For moist heat sterilization, SA samples were sterilized by saturated steam (100% humidity) at T = 121◦ C, P = 2 atm for 20 min, then SA solutions were rapidly refrigerated to 4◦ C. For gamma rays sterilization, SA samples were irradiated using gamma radiation from a cobalt-60 source (Biotechnology and Nuclear Technology Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China) with the sterilization doses of 5 kGy, 15 kGy, 25 kGy and 35 kGy (standard dose) at temperature of 20◦ C. After sterilization, the solutions were kept at −30◦ C, whereas powders were stored at room temperature. 2.2. Assay of sterilization effect In order to enumerate the total bacterial populations in the SA solutions and powders sterilized by gamma rays and heat, the sterilized and non-sterilized powders were dissolved in sterile water at 2% (w/v) concentration, respectively, and 5% (w/v) SA aqueous solution also were diluted with sterile water to 2% (w/v). One hundred microlitre samples were placed on Petri plates with 20 ml of solid agar medium. The plates were incubated at 37◦ C for 48 h, and the colonies on the plates were manually counted. Non-sterilized samples were used as control. 2.3. pH variations of sterilized SA samples The pH of the sterilized samples was measured with a pH meter (Beckman, USA). The sterilized and non-sterilized SA samples were dissolved or diluted in sterile water at 0.2% (w/v) concentration before measuring pH. The pH of the samples were analyzed as described by the instruction. 2.4. Intrinsic viscosity of sterilized SA samples The intrinsic viscosity of SA samples was measured using an Ubbelohde viscometer (Dongfanghuabo, China). After dissolving the polymers in water with 0.01, 0.03, 0.05, 0.10 and 0.20% (w/v) concentrations, the measurements were carried out at temperature of 25◦ C. The experiment were repeated three times, and the efflux time of the solutions for each concentration was considered as the average of three measurements. 2.5. Structure of sterilized SA samples In order to perform UV/Vis spectrophotometry analysis, sterilized and non-sterilized SA samples were prepared in aqueous 1% (w/v) solutions, respectively. 100 µl of each sample were analyzed with DU800 UV/Vis spectrophotometer (Beckman, USA). Spectra were acquired over the wavelength range, 190– 1100 nm, at T = 20◦ C.
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Fourier transform infrared spectroscopy (FTIR) was used to analyse the samples in transmission mode, pellets with 0.5 mg of sterilized or non-sterilized SA powders in 100 mg of anhydrous potassium bromide (KBr) were prepared. A Jasco FT-IR-460 plus spectrometer (Jasco, Japan) was used for the analysis. The sum of 128 scans with a resolution of 2 cm−1 was used to obtain the spectra, which are collected from 4000 to 400 cm−1 as set up in previous works [24]. 2.6. Cell culture and cytotoxicity assay HUVEC was purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. The cells were cultured at 37◦ C with 5% CO2 in RPMI-1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% antibiotics (Beyotime, China) [25]. The cultured cells were routinely trypsinized after confluency, counted, and seeded at a density of 1 × 104 cells/well in 96 multi-well culture plates. After 24 h of incubation, the culture medium was replaced by 100 µl of culture medium with 0.1% (w/v) SA was added in each well. Cells seeded with culture medium without SA represented the positive control. To evaluate cell adhesion and proliferation, Olympus IX50 optical microscope (Olympus, Japan) was used to observed the cells after 24, 72 and 120 h of incubation, respectively. To assess the mitochondrial activity of the seeded cells, that is, the cell viability during the culture period, a test with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich, USA) was curried out at 24, 72 and 120 h, respectively. At each time point, the culture medium was removed and replaced by 100 µl of fresh culture medium without serum, and 20 µl of MTT solution (5 mg/ml in phosphate-buffered saline) was added to each well. Viable cells are able to reduce MTT into formazan crystals. After the cell cultures were incubated for 4 h in a humidified 5% CO2 incubator at 37◦ C, MTT solution was removed and 100 µl of dimethyl sulphoxide (DMSO) was added to solubilize the formazan products. The absorbance values were measured at 490 nm using ELx800 microplate reader (BioTek, USA). 2.7. Statistical analysis All measurements were tested in triplicate. The data obtained were averaged and expressed as mean ± SD. Student’s t-test was used to compare the data, differences were considered statistically significant at p < 0.05.
3. Results and discussion 3.1. Assay of sterilization effect In order to make certain minimum effective dose of gamma irradiation sterilization, the sterilization effect was analyzed by counting viable microorganism based on counting of colonies grown in Petri plates. The status of viable microorganism in 2% (w/v) sterilized SA sample is showed in Fig. 1 and Table 1. The total aerobic bacteria was 3.33 CFU/mg before sterilization. After irradiation at 5 and 15 kGy, the populations in the SA sample were 2.17 and 1.00 CFU/mg, respectively. Whereas populations in the sample decreased to below detection limit (1.00 CFU/mg) after sterilization at 25 and 35 kGy gamma irradiation or moist heat sterilization. The yeast and molds was 1.33 CFU/mg in non-sterilized SA, but
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Fig. 1. The status of the colony growth from 2% (w/v) SA solutions. Non-sterilized (a); gamma ray at the dose of 5 kGy (b), 15 kGy (c), 25 kGy (d), and 35 kGy (e); moist heat sterilization (f). (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140994.) Table 1 Inactivation of microbial population in 2% (w/v) SA aqueous solution by gamma irradiation and moist heat Microorganism (CFU/mg)
Not-sterilized
Gamma irradiation
Moist heat
Total aerobic bacteria Yeast/molds
3.33 ± 0.85 1.33 ± 0.24
5 kGy 2.17 ± 0.24 ND
15 kGy 1.00 ± 0.41 ND
25 kGy ND ND
35 kGy ND ND
ND ND
Total
4.67 ± 0.62
2.17 ± 0.24
1.00 ± 0.41
ND
ND
ND
Note: ND: not detectable within a detection limit < 1.00 CFU/mg.
populations in the sterilized samples reduced to below detection limit (1.00 CFU/mg) after gamma irradiation or moist heat sterilization. SA powders and 5% (w/v) SA is showed in Tables 2 and 3, respectively, which also presented the similar results as 2% (w/v) SA. The findings suggested that the irradiation dose of 25 kGy or more can effectively sterilize microorganism. Gamma ray irradiation is a rapid, convenient and extensive sterilization treatment, commonly used for medical devices [26]. Because ionizing energy rapidly penetrates through the polysaccharide granules, destroying the DNA or RNA of pathogen agents [27]. Gamma ray irradiation has also been proposed for the sterilization of polysaccharides [28–31]. Our research suggests that the irradiation dose of 25 kGy can effectively sterilize microorganism, which can achieve the same sterilization effect with moist heat sterilization. Then, the sterilized SA via 25 kGy gamma irradiation was selected for further analysis.
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Microorganism (CFU/mg) Total aerobic bacteria Yeast/molds Total
Not-sterilized 3.33 ± 0.85 1.33 ± 0.24 4.67 ± 0.62
Gamma irradiation 5 kGy 2.33 ± 0.62 ND 2.33 ± 0.62
15 kGy 1.17 ± 0.24 ND 1.33 ± 0.47
25 kGy ND ND ND
Moist heat 35 kGy ND ND ND
ND ND ND
Note: ND: not detectable within a detection limit < 1.00 CFU/mg.
Table 3 Inactivation of microbial population in SA powders by gamma irradiation and moist heat Microorganism (CFU/mg) Total aerobic bacteria Yeast/molds Total
Not-sterilized 3.33 ± 0.85 1.33 ± 0.24 4.67 ± 0.62
Gamma irradiation 5 kGy 2.33 ± 0.24 ND 2.50 ± 0.41
15 kGy 0.83 ± 0.47 ND 0.83 ± 0.47
25 kGy ND ND ND
Moist heat 35 kGy ND ND ND
ND ND ND
Note: ND: not detectable within a detection limit < 1.00 CFU/mg.
Fig. 2. pH variations induced by sterilization of SA samples after moist heat and 25 kGy gamma ray sterilization. The data were the averages ± standard deviations of three samples.
3.2. The sterilization did not affect the native pH of SA polysaccharide The pH of SA samples measured before and after sterilization. The pH of non-sterilized samples was 7.69. After sterilization, The pH of sterilized samples were ranging from 7.71 to 7.75 for both sterilization techniques. The maximum variation of the pH was observed for sterilized SA (initial pH + 0.06) (Fig. 2). The data represent pH ± standard deviations (SD) with three replicates. Statistical analysis showed there were not any significative difference related to the pH of samples found. Therefore, it is possible to conclude that both of the considered sterilization procedures were not affecting the native pH of SA polysaccharide.
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Fig. 3. Percentage intrinsic viscosity of SA samples after moist heat and 25 kGy gamma ray sterilization. The data were the averages ± standard deviations of three samples. ∗∗ p < 0.01 compared with the non-sterilized SA sample.
3.3. The sterilization decreased intrinsic viscosity of SA The intrinsic viscosity of SA samples was measured. The intrinsic viscosity of non-sterilized samples was taken as reference (100%). After gamma ray and moist heat sterilization, percentage intrinsic viscosity of SA powders were 47.2% and 75.5%, respectively; percentage intrinsic viscosity of 5% (w/v) sterilized SA were 24.0% and 51.6%, respectively; whereas percentage intrinsic viscosity of 2% (w/v) sterilized SA were only 17.4% and 50.3%, respectively (Fig. 3). These showed that both of the considered sterilization techniques had significant effects on the intrinsic viscosity for the SA sample, and the lower the concentration of SA sample is, the lower intrinsic viscosity is after sterilization. For the same concentration of SA, intrinsic viscosity retain better by moist heat sterilization than that by gamma ray sterilization (Fig. 3). Gamma ray irradiation and heat can result in chain scission. Mainly, it is caused by the enhanced –OH mobility rising with dilution of the solution, due to reduced viscosity [32]. Also, effect of chain scission can be followed by a degradation of the polymer. It has been pointed out previously that a viscosity of the solution increases the energy required for cavitation to occur, decrease in the viscosity of the solution must result in the increase of the degradation rate [33,34]. 3.4. The change of UV/Vis spectroscopy In organic chemistry, spectroscopic methods are used to determine and confirm molecular structures, to monitor reactions and to control the purity of compounds. In this work, the absorption UV/Vis spectroscopy is one of the most suitable methods for studying the structures and properties of sterilized and non-sterilized SA. In order to detect possible modifications occurred on SA structure after sterilization, spectra of non-sterilized samples were compared with spectra of sterilized aqueous solutions. UV/Vis spectroscopy of SA samples with various treatment under air is showed in Fig. 4. The peak at 265 nm showed a very weak intensity for SA solutions treated by gamma ray and SA powers treated by gamma ray and heat, which had no significant difference compared with that of non-sterilized SA. Whereas the absorbance increase of SA solutions treated by moist heat sterilization at 265 nm was especially obvious. It was explained that the peak at 265 nm can be ascribed to double bonds formed after the main chain scission of the polymer followed by the ring opening [21,32]. Therefore, the results suggested gamma
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Fig. 4. UV/Vis spectra of 1% (w/v) SA solutions before and after sterilization. The red dashed line marked point to 265 nm. (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140994.)
ray sterilization hardly lead to the main chain scission and double bonds formation of SA powers and SA solutions; moist heat sterilization also can hardly cause the main chain scission and double bonds formation of the polymer SA powers, but can lead to the main chain scission and double bonds formation of the polymer SA solutions. Therefore, only moist heat sterilization can lead to the main chain scission and double bonds formation of the polymer SA solutions. 3.5. The change of FTIR spectroscopy The FTIR spectrum provides information through band properties, frequencies and intensities, which can be used to predict chemical processes, identify species and determine the increase in the number of certain entities from the increase in the area of the band. In recent years, FTIR has attracted attention as a method for characterization and identification of polymer. FTIR spectra of SA powders were taken before and after sterilization. Peaks of SA at 3440, 1610 and 1089 cm−1 are attributed to hydroxyl (–OH), carboxyl (–COOH), glycosidic bond (C–O–C–), respectively [35,36]. Peak at 1610 cm−1 for SA was taken as the reference peaks due to the fact that carboxyl and amine groups do not change after degradation. The scission of glycosidic bonds form hydroxyl group, which leads to decrease the peak ratio of glycosidic bonds to carboxyl group and increasing the peak ratio of hydroxyl group to carboxyl group [37]. FTIR spectra analysis showed peak ratio of glycosidic bonds (peak at 1089 cm−1 ) to carboxyl groups (peak at 1610 cm−1 ) decreased and that of hydroxyl groups (peak at 3440 cm−1 ) to carboxyl groups increased, which suggested SA powders undergone mainly the breakage of glycosidic bonds during sterilization (Fig. 5). Comparing with moist heat sterilization, gamma irradiation sterilization led to more glycosidic bond breaking (Fig. 5(b) and (c)). 3.6. Cytotoxicity The color of sterilized samples did not change appreciably in the radiation dose of 25 kGy and moist heat sterilization. MTT test showed SA powers and solutions sterilized with gamma rays increased the
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Fig. 5. FT-IR spectra of SA powders treated by non-sterilized (a); moist heat sterilization (b) and gamma irradiation doses of 25 kGy (c).
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viability of HUVEC cells with respect of the cells not in contact with SA samples, the relative cell viability of HUVEC after 5 days of incubation with SA were more than 107% of the control (Fig. 6). The SA powder with moist heat sterilization hardly affected the viability of the cells, the relative cell
Fig. 6. Relative cell viability of SA samples with respect of the control at different times of incubation. (a) SA powers; (b) 5% (v/v) SA solutions; (c) 2% (v/v) SA solutions. The data were the averages ± standard deviations of three samples. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the controls (without SA), respectively.
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Fig. 7. HUVEC cells after 5 days of culture. Untreated (a); in 5% (w/v) SA solution not sterilized (b); sterilized gamma rays at 25 kGy (c) and moist heat (d). Scale bars = 50 µm.
viability of HUVEC after 5 days of incubation with SA was 103% of the control (Fig. 6(a)). However, the relative cell viability of HUVEC after 5 days of incubation with SA solutions was less than 77% of the control, which suggested moist heat sterilization of SA solutions resulted in a dramatic decrease of viability of HUVEC cells (Fig. 6(b) and (c)). As observed at the optical microscope, the HUVEC cells treated with non-sterilized SA and sterilized SA by gamma rays at 25 kGy exhibited more proliferation than the untreated cells. Whereas the HUVEC cells seeded in moist heat treated SA resulted round-shaped and with a reduced number (Fig. 7). The findings suggested that SA solution with gamma rays sterilization had no cytotoxicity, which are used as nutrient substance. While SA solutions with moist heat sterilization had a large amount of cytotoxicity, which caused cytotoxic effects. It is proved that heating SA solutions led to methanol accumulation due to ester hydrolysis, which produced cytotoxic effects [36].
4. Conclusions Polysaccharides, such as alginates, are already being used as carriers for drug delivery. By changing the physicochemical and biological properties, it is possible that the alginate is used to control drug release. SA is a biocompatible, non-toxic, non-immunogenic and biodegradable natural linear polysaccharide. The two kinds of physical sterilization techniques, gamma irradiation and moist heat sterilization,
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hardly affect the native pH of SA polysaccharide, but deeply affected the intrinsic viscosity and structure of SA samples. The gamma irradiation was a suitable treatment to sterilize SA, avoiding the side effects caused by moist heat sterilization and retaining cell viability. The moist heat sterilization seems only appropriate for SA powers because of the severe side effects produced by heating. The research results may provide interesting future advancements toward the development of SA-based products for biomedical applications. However, further studies are required to investigate the mechanical and gelling properties of sterilized powders and solutions. Acknowledgements The author would like to thank Yufei Ma for her technical support on FT-IR spectroscopy analyses. This work was supported by the National Key Technology R&D Program of China (2012BAI18B02) and Public Experiment Center of State Bioindustrial Base (Chongqing, China). References [1] T.Y. Wong, L.A. Preston and N.L. Schiller, Alginate lyase: Review of major sources and enzyme characteristics, structurefunction analysis, biological roles, and applications, Annu. Rev. Microbiol. 54 (2000), 289–340. [2] R. Jayakumar, M. Rajkumar, H. Freitas, N. Selvamurugan, S.V. Nair, T. Furuike and H. Tamura, Preparation, characterization, bioactive and metal uptake studies of alginate/phosphorylated chitin blend films, Int. J. Biol. Macromol. 44 (2009), 107–111. [3] M. Yadav, D.K. Mishra, A. Sand and K. Behari, Modification of alginate through the grafting of 2-acrylamidoglycolic acid and study of physicochemical properties in terms of swelling capacity, metal ion sorption, flocculation and biodegradability, Carbohyd. Polym. 84 (2011), 83–89. [4] J.S. Yang, Y.J. Xie and W. He, Research progress on chemical modification of alginate: A review, Carbohyd. Polym. 84 (2011), 33–39. [5] T. Coviello, P. Matricardi, C. Marianecci and F. Alhaique, Polysaccharide hydrogels for the modified release of formulations, J. Control Release 119 (2007), 5–24. [6] A. Ikeda, A. Takemura and H. Ono, Preparation of low-molecular weight alginic acid by acid hydrolysis, Carbohyd. Polym. 42 (2000), 421–425. [7] J.W. Rhim, Physical and mechanical properties of water resistant sodium alginate films, LWT-Food Sci. Technol. 37 (2004), 323–330. [8] K. Norajit, K.M. Kim and G.H. Ryu, Comparative studies on the characterization and antioxidant properties of biodegradable alginate films containing ginseng extract, J. Food Eng. 98 (2010), 377–384. [9] H. Ertesvag and S. Valla, Biosynthesis and applications of alginates, Polym. Degrad. Stabil. 59 (1998), 85–91. [10] I. Braccini and S. Perez, Molecular basis of Ca2+ -induced gelation in alginates and pectins: the egg-box model revisited, Biomacromolecules 2 (2001), 1089–1096. [11] Z. Liu, Y. Jiao and Y. Wang, Polysaccharides-based nanoparticles as drug delivery systems, Adv. Drug Deliver Rev. 60 (2008), 1650–1662. [12] S. Patil, Cross-linking of polysaccharides: methods and applications, Pharmaceutical Reviews E-Journal (2008). [13] Z. Mohamadnia, M.J. Zohuriaan-Mehr, K. Kabiri, A. Jamshidi and H. Mobedi, pH-sensitive IPN hydrogel beads of carrageenan-alginate for controlled drug delivery, J. Bioact. Compat. Pol. 22 (2007), 342–356. [14] L. Yang, G. Liang, Z. Zhang and S. He, Sodium alginate/Na-rectorite composite films: preparation, characterization, and properties, J. Appl. Polym. Sci. 114 (2009), 1235–1240. [15] S. Benavides, R. Villalobos-Carvajal and J.E. Reyes, Physical, mechanical and antibacterial properties of alginate film: effect of the crosslinking degree and oregano essential oil concentration, J. Food. Eng. 110 (2011), 232–239. [16] R. Russo, M. Abbate, M. Malinconico and G. Santagata, Effect of polyglycerol and the crosslinking on the physical properties of a blend alginate–hydroxy ethyl cellulose, Carbohyd. Polym. 82 (2010), 1061–1067. [17] E.M. Zactiti and T.G. Kieckbusch, Release of potassium sorbate from active films of sodium alginate crosslinked with calcium chloride, Packag. Technol. Sci. 22 (2009), 349–358. [18] P. Malafaya, G. Silva and R. Reis, Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications, Adv. Drug Deliver Rev. 59 (2007), 207–233.
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Effects of gamma irradiation and moist heat for sterilization on sodium alginate Tingzhang Hu, Yongwei Yang, Lili Tan, Tieying Yin, Yazhou Wang and Guixue Wang ∗ Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China Received 23 May 2013 Accepted 22 April 2014 Abstract. Polysaccharides, such as alginates, are already being used as carriers for drug delivery. The physicochemical and biological properties of alginates may be affected via irradiation and thermal treatments. To explore and compare effects of two kinds of sterilization methods, gamma irradiation and moist heat, on sodium alginate (SA), physicochemical and biological properties of SA powder and solutions were investigated after sterilization. Human umbilical vein endothelial cells (HUVEC) was used to assess the cytotoxicity of the SA after sterilization. The research showed that 25 kGy gamma ray can effectively sterilize microorganism. Both gamma irradiation and moist heat hardly affect the native pH of SA. Compared to irradiation sterilization, moist heat sterilization showed smaller changes in intrinsic viscosity for all SA samples and lead to less glycosidic bond breaking of SA powders. The moist heat sterilization can cause the main chain scission and double bonds formation of the SA solutions. Cytotoxicity studies demonstrated that sterilized SA powers and SA solutions treated by gamma ray sterilization can increase the viability of HUVEC. However, SA solutions treated by moist heat sterilization were found to present severe cytotoxicity. The research results may provide interesting future advancements toward the development of SA-based products for biomedical applications. Keywords: Sodium alginate, sterilization, gamma ray, moist heat
1. Introduction Polysaccharidic polymers are already being used as thickeners, stabilizers, suspension agents, gelling agents, film-formers, food packaging, emulsifiers, lubricants, plus in medicine as scaffold materials in tissue engineering and as carriers for drug delivery, owing to their unique structure, distinctive properties, safety and biodegradability [1]. By changing the physicochemical and biological properties, such as concentrations, pH value and viscosities, it is possible that the alginate is used to control drug release. Among polysaccharides, sodium alginate (SA) is a biocompatible, non-toxic, non-immunogenic and biodegradable natural linear polysaccharide [2–5], which is widely applied in the fields of medical and industrial. SA is a natural biopolymer typically obtained from the seaweed of brown algae (Phaeophyta) such as genera Macrocystis, Laminaria and Ascophyllum. Alginate is the only polysaccharide that naturally * Address for correspondence: Guixue Wang, Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China. Tel.: +86 23 65112675; Fax: +86 23 65112675; E-mail: [email protected].
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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contains carboxyl groups in each constituent residue of macromolecule, and possesses various abilities for application as a functional material [6,7]. From the molecular point of view, alginate is composed mainly of (1-4)-linked β-D-mannuronic (M) acid units and α-L-guluronic (G) acid units, forming regions of M-blocks, G-blocks and blocks of alternating sequence (MG-blocks). The relative proportions of these sequential organizations depends on the source [8,9]. Most applications of alginate is based on its gel-forming ability through cations binding; for example, the transition from water-soluble SA to water insoluble calcium alginate. Divalent cations preferentially bind toward the G-block rather than the M-block [10]. The macromolecules of polysaccharides are linked together by hydrophilic groups, such as hydroxyl, carboxyl and amino groups that are able to form non-covalent bonds with biological tissues, thus promoting bio-adhesion [11]. Their physical properties depend on their monomer compositions, chain-shapes, viscosities and molecular weights, which influence solubility, gelation and surface properties. Polysaccharides can be cross-linked by chemical or physical interactions. Chemical cross-linking enhances good mechanical stability, however, cross-linking agents are often toxic compounds. For that reason, traces of non-reactive agents should be extracted from the gel before application. Thus preference is given to the use of physically cross-linked gels. Different methods are used for physical cross-linking. One of them is the polysaccharides is cross-linked by ionic interaction. Alginates are able to undergo reversible gelation within aqueous solutions through interaction with divalent cations such as Ca2+ that create ionic inter-chain bridges [12]. SA is a polysaccharide with mannuronic and guluronic acid residues. The gel is formed by binding the guluronic segments of SA to cations and creating three-dimensional networks. Owing to unique physicochemical properties, such as good solubility and high viscosity in aqueous solution as well as gel forming ability in the presence of divalent cations (such as Ca2+ ), SA is used in a wide range of commercial enterprises, including textile, paper, food, and health care industries [13,14]. SA has been tested to prepare environmentally friendly packaging materials such as films and coatings and added to other biopolymers such as pectin or hydroxyl ethyl cellulose to improve their mechanical and water vapor barrier properties [7,15–17]. Alginate gels are pH-sensitive because of the presence of an acidic carboxylic group. Alginate gels shrink at low pH, therefore reducing the release of the drug. This phenomenon may prove advantageous for drug delivery systems. At higher pH, alginate dissolves rapidly and causes a bursting of released drugs, which is usually undesirable. However, by changing the concentrations and viscosities of the alginate solution during synthesis of the gel, it is possible to control drug release [18]. Sterilization refers to the intense processes inactivating spores resulting in products that are shelf stable. It is usually achieved by applying heat, chemicals, irradiation, pressure and filtration [19,20]. Sterilization, however, may affect the surface or bulk properties of the sterilized material, inducing chemical or physical modifications that may alter the characteristics of the biomedical devices in terms of physicochemical, morphological, mechanical and biological behavior. Natural polymers are susceptible to high temperatures and ionizing radiations, which may induce depolymerization, oxidation or the formation of free radicals [21], thus affecting the properties of the gels they may form, such as mechanical strength, swelling and stability behavior [22,23]. In this paper, in order to explore and compare effects of two kinds of traditional physical sterilization methods, gamma irradiation and moist heat, on SA, physicochemical and biological properties of SA powders and solutions were investigated after sterilization. Human umbilical vein endothelial cells (HUVEC) was used to assess the cytotoxicity of the SA after sterilization.
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2. Materials and methods 2.1. Sterilization methods SA was purchased from Dongsheng chemical plant (Wenzhou, China). Gamma irradiation and heat were employed to treat SA samples, according to the recommendations of The International Pharmacopeia (WHO Department of Essential Medicine and Pharmaceutical Policies, 2011). SA powder and 2% and 5% (w/v) SA aqueous solution were used to study effects of damma irradiation and moist heat for sterilization. For moist heat sterilization, SA samples were sterilized by saturated steam (100% humidity) at T = 121◦ C, P = 2 atm for 20 min, then SA solutions were rapidly refrigerated to 4◦ C. For gamma rays sterilization, SA samples were irradiated using gamma radiation from a cobalt-60 source (Biotechnology and Nuclear Technology Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China) with the sterilization doses of 5 kGy, 15 kGy, 25 kGy and 35 kGy (standard dose) at temperature of 20◦ C. After sterilization, the solutions were kept at −30◦ C, whereas powders were stored at room temperature. 2.2. Assay of sterilization effect In order to enumerate the total bacterial populations in the SA solutions and powders sterilized by gamma rays and heat, the sterilized and non-sterilized powders were dissolved in sterile water at 2% (w/v) concentration, respectively, and 5% (w/v) SA aqueous solution also were diluted with sterile water to 2% (w/v). One hundred microlitre samples were placed on Petri plates with 20 ml of solid agar medium. The plates were incubated at 37◦ C for 48 h, and the colonies on the plates were manually counted. Non-sterilized samples were used as control. 2.3. pH variations of sterilized SA samples The pH of the sterilized samples was measured with a pH meter (Beckman, USA). The sterilized and non-sterilized SA samples were dissolved or diluted in sterile water at 0.2% (w/v) concentration before measuring pH. The pH of the samples were analyzed as described by the instruction. 2.4. Intrinsic viscosity of sterilized SA samples The intrinsic viscosity of SA samples was measured using an Ubbelohde viscometer (Dongfanghuabo, China). After dissolving the polymers in water with 0.01, 0.03, 0.05, 0.10 and 0.20% (w/v) concentrations, the measurements were carried out at temperature of 25◦ C. The experiment were repeated three times, and the efflux time of the solutions for each concentration was considered as the average of three measurements. 2.5. Structure of sterilized SA samples In order to perform UV/Vis spectrophotometry analysis, sterilized and non-sterilized SA samples were prepared in aqueous 1% (w/v) solutions, respectively. 100 µl of each sample were analyzed with DU800 UV/Vis spectrophotometer (Beckman, USA). Spectra were acquired over the wavelength range, 190– 1100 nm, at T = 20◦ C.
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Fourier transform infrared spectroscopy (FTIR) was used to analyse the samples in transmission mode, pellets with 0.5 mg of sterilized or non-sterilized SA powders in 100 mg of anhydrous potassium bromide (KBr) were prepared. A Jasco FT-IR-460 plus spectrometer (Jasco, Japan) was used for the analysis. The sum of 128 scans with a resolution of 2 cm−1 was used to obtain the spectra, which are collected from 4000 to 400 cm−1 as set up in previous works [24]. 2.6. Cell culture and cytotoxicity assay HUVEC was purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. The cells were cultured at 37◦ C with 5% CO2 in RPMI-1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% antibiotics (Beyotime, China) [25]. The cultured cells were routinely trypsinized after confluency, counted, and seeded at a density of 1 × 104 cells/well in 96 multi-well culture plates. After 24 h of incubation, the culture medium was replaced by 100 µl of culture medium with 0.1% (w/v) SA was added in each well. Cells seeded with culture medium without SA represented the positive control. To evaluate cell adhesion and proliferation, Olympus IX50 optical microscope (Olympus, Japan) was used to observed the cells after 24, 72 and 120 h of incubation, respectively. To assess the mitochondrial activity of the seeded cells, that is, the cell viability during the culture period, a test with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich, USA) was curried out at 24, 72 and 120 h, respectively. At each time point, the culture medium was removed and replaced by 100 µl of fresh culture medium without serum, and 20 µl of MTT solution (5 mg/ml in phosphate-buffered saline) was added to each well. Viable cells are able to reduce MTT into formazan crystals. After the cell cultures were incubated for 4 h in a humidified 5% CO2 incubator at 37◦ C, MTT solution was removed and 100 µl of dimethyl sulphoxide (DMSO) was added to solubilize the formazan products. The absorbance values were measured at 490 nm using ELx800 microplate reader (BioTek, USA). 2.7. Statistical analysis All measurements were tested in triplicate. The data obtained were averaged and expressed as mean ± SD. Student’s t-test was used to compare the data, differences were considered statistically significant at p < 0.05.
3. Results and discussion 3.1. Assay of sterilization effect In order to make certain minimum effective dose of gamma irradiation sterilization, the sterilization effect was analyzed by counting viable microorganism based on counting of colonies grown in Petri plates. The status of viable microorganism in 2% (w/v) sterilized SA sample is showed in Fig. 1 and Table 1. The total aerobic bacteria was 3.33 CFU/mg before sterilization. After irradiation at 5 and 15 kGy, the populations in the SA sample were 2.17 and 1.00 CFU/mg, respectively. Whereas populations in the sample decreased to below detection limit (1.00 CFU/mg) after sterilization at 25 and 35 kGy gamma irradiation or moist heat sterilization. The yeast and molds was 1.33 CFU/mg in non-sterilized SA, but
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Fig. 1. The status of the colony growth from 2% (w/v) SA solutions. Non-sterilized (a); gamma ray at the dose of 5 kGy (b), 15 kGy (c), 25 kGy (d), and 35 kGy (e); moist heat sterilization (f). (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140994.) Table 1 Inactivation of microbial population in 2% (w/v) SA aqueous solution by gamma irradiation and moist heat Microorganism (CFU/mg)
Not-sterilized
Gamma irradiation
Moist heat
Total aerobic bacteria Yeast/molds
3.33 ± 0.85 1.33 ± 0.24
5 kGy 2.17 ± 0.24 ND
15 kGy 1.00 ± 0.41 ND
25 kGy ND ND
35 kGy ND ND
ND ND
Total
4.67 ± 0.62
2.17 ± 0.24
1.00 ± 0.41
ND
ND
ND
Note: ND: not detectable within a detection limit < 1.00 CFU/mg.
populations in the sterilized samples reduced to below detection limit (1.00 CFU/mg) after gamma irradiation or moist heat sterilization. SA powders and 5% (w/v) SA is showed in Tables 2 and 3, respectively, which also presented the similar results as 2% (w/v) SA. The findings suggested that the irradiation dose of 25 kGy or more can effectively sterilize microorganism. Gamma ray irradiation is a rapid, convenient and extensive sterilization treatment, commonly used for medical devices [26]. Because ionizing energy rapidly penetrates through the polysaccharide granules, destroying the DNA or RNA of pathogen agents [27]. Gamma ray irradiation has also been proposed for the sterilization of polysaccharides [28–31]. Our research suggests that the irradiation dose of 25 kGy can effectively sterilize microorganism, which can achieve the same sterilization effect with moist heat sterilization. Then, the sterilized SA via 25 kGy gamma irradiation was selected for further analysis.
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Microorganism (CFU/mg) Total aerobic bacteria Yeast/molds Total
Not-sterilized 3.33 ± 0.85 1.33 ± 0.24 4.67 ± 0.62
Gamma irradiation 5 kGy 2.33 ± 0.62 ND 2.33 ± 0.62
15 kGy 1.17 ± 0.24 ND 1.33 ± 0.47
25 kGy ND ND ND
Moist heat 35 kGy ND ND ND
ND ND ND
Note: ND: not detectable within a detection limit < 1.00 CFU/mg.
Table 3 Inactivation of microbial population in SA powders by gamma irradiation and moist heat Microorganism (CFU/mg) Total aerobic bacteria Yeast/molds Total
Not-sterilized 3.33 ± 0.85 1.33 ± 0.24 4.67 ± 0.62
Gamma irradiation 5 kGy 2.33 ± 0.24 ND 2.50 ± 0.41
15 kGy 0.83 ± 0.47 ND 0.83 ± 0.47
25 kGy ND ND ND
Moist heat 35 kGy ND ND ND
ND ND ND
Note: ND: not detectable within a detection limit < 1.00 CFU/mg.
Fig. 2. pH variations induced by sterilization of SA samples after moist heat and 25 kGy gamma ray sterilization. The data were the averages ± standard deviations of three samples.
3.2. The sterilization did not affect the native pH of SA polysaccharide The pH of SA samples measured before and after sterilization. The pH of non-sterilized samples was 7.69. After sterilization, The pH of sterilized samples were ranging from 7.71 to 7.75 for both sterilization techniques. The maximum variation of the pH was observed for sterilized SA (initial pH + 0.06) (Fig. 2). The data represent pH ± standard deviations (SD) with three replicates. Statistical analysis showed there were not any significative difference related to the pH of samples found. Therefore, it is possible to conclude that both of the considered sterilization procedures were not affecting the native pH of SA polysaccharide.
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Fig. 3. Percentage intrinsic viscosity of SA samples after moist heat and 25 kGy gamma ray sterilization. The data were the averages ± standard deviations of three samples. ∗∗ p < 0.01 compared with the non-sterilized SA sample.
3.3. The sterilization decreased intrinsic viscosity of SA The intrinsic viscosity of SA samples was measured. The intrinsic viscosity of non-sterilized samples was taken as reference (100%). After gamma ray and moist heat sterilization, percentage intrinsic viscosity of SA powders were 47.2% and 75.5%, respectively; percentage intrinsic viscosity of 5% (w/v) sterilized SA were 24.0% and 51.6%, respectively; whereas percentage intrinsic viscosity of 2% (w/v) sterilized SA were only 17.4% and 50.3%, respectively (Fig. 3). These showed that both of the considered sterilization techniques had significant effects on the intrinsic viscosity for the SA sample, and the lower the concentration of SA sample is, the lower intrinsic viscosity is after sterilization. For the same concentration of SA, intrinsic viscosity retain better by moist heat sterilization than that by gamma ray sterilization (Fig. 3). Gamma ray irradiation and heat can result in chain scission. Mainly, it is caused by the enhanced –OH mobility rising with dilution of the solution, due to reduced viscosity [32]. Also, effect of chain scission can be followed by a degradation of the polymer. It has been pointed out previously that a viscosity of the solution increases the energy required for cavitation to occur, decrease in the viscosity of the solution must result in the increase of the degradation rate [33,34]. 3.4. The change of UV/Vis spectroscopy In organic chemistry, spectroscopic methods are used to determine and confirm molecular structures, to monitor reactions and to control the purity of compounds. In this work, the absorption UV/Vis spectroscopy is one of the most suitable methods for studying the structures and properties of sterilized and non-sterilized SA. In order to detect possible modifications occurred on SA structure after sterilization, spectra of non-sterilized samples were compared with spectra of sterilized aqueous solutions. UV/Vis spectroscopy of SA samples with various treatment under air is showed in Fig. 4. The peak at 265 nm showed a very weak intensity for SA solutions treated by gamma ray and SA powers treated by gamma ray and heat, which had no significant difference compared with that of non-sterilized SA. Whereas the absorbance increase of SA solutions treated by moist heat sterilization at 265 nm was especially obvious. It was explained that the peak at 265 nm can be ascribed to double bonds formed after the main chain scission of the polymer followed by the ring opening [21,32]. Therefore, the results suggested gamma
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Fig. 4. UV/Vis spectra of 1% (w/v) SA solutions before and after sterilization. The red dashed line marked point to 265 nm. (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140994.)
ray sterilization hardly lead to the main chain scission and double bonds formation of SA powers and SA solutions; moist heat sterilization also can hardly cause the main chain scission and double bonds formation of the polymer SA powers, but can lead to the main chain scission and double bonds formation of the polymer SA solutions. Therefore, only moist heat sterilization can lead to the main chain scission and double bonds formation of the polymer SA solutions. 3.5. The change of FTIR spectroscopy The FTIR spectrum provides information through band properties, frequencies and intensities, which can be used to predict chemical processes, identify species and determine the increase in the number of certain entities from the increase in the area of the band. In recent years, FTIR has attracted attention as a method for characterization and identification of polymer. FTIR spectra of SA powders were taken before and after sterilization. Peaks of SA at 3440, 1610 and 1089 cm−1 are attributed to hydroxyl (–OH), carboxyl (–COOH), glycosidic bond (C–O–C–), respectively [35,36]. Peak at 1610 cm−1 for SA was taken as the reference peaks due to the fact that carboxyl and amine groups do not change after degradation. The scission of glycosidic bonds form hydroxyl group, which leads to decrease the peak ratio of glycosidic bonds to carboxyl group and increasing the peak ratio of hydroxyl group to carboxyl group [37]. FTIR spectra analysis showed peak ratio of glycosidic bonds (peak at 1089 cm−1 ) to carboxyl groups (peak at 1610 cm−1 ) decreased and that of hydroxyl groups (peak at 3440 cm−1 ) to carboxyl groups increased, which suggested SA powders undergone mainly the breakage of glycosidic bonds during sterilization (Fig. 5). Comparing with moist heat sterilization, gamma irradiation sterilization led to more glycosidic bond breaking (Fig. 5(b) and (c)). 3.6. Cytotoxicity The color of sterilized samples did not change appreciably in the radiation dose of 25 kGy and moist heat sterilization. MTT test showed SA powers and solutions sterilized with gamma rays increased the
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Fig. 5. FT-IR spectra of SA powders treated by non-sterilized (a); moist heat sterilization (b) and gamma irradiation doses of 25 kGy (c).
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viability of HUVEC cells with respect of the cells not in contact with SA samples, the relative cell viability of HUVEC after 5 days of incubation with SA were more than 107% of the control (Fig. 6). The SA powder with moist heat sterilization hardly affected the viability of the cells, the relative cell
Fig. 6. Relative cell viability of SA samples with respect of the control at different times of incubation. (a) SA powers; (b) 5% (v/v) SA solutions; (c) 2% (v/v) SA solutions. The data were the averages ± standard deviations of three samples. ∗ p < 0.05 and ∗∗ p < 0.01 compared with the controls (without SA), respectively.
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Fig. 7. HUVEC cells after 5 days of culture. Untreated (a); in 5% (w/v) SA solution not sterilized (b); sterilized gamma rays at 25 kGy (c) and moist heat (d). Scale bars = 50 µm.
viability of HUVEC after 5 days of incubation with SA was 103% of the control (Fig. 6(a)). However, the relative cell viability of HUVEC after 5 days of incubation with SA solutions was less than 77% of the control, which suggested moist heat sterilization of SA solutions resulted in a dramatic decrease of viability of HUVEC cells (Fig. 6(b) and (c)). As observed at the optical microscope, the HUVEC cells treated with non-sterilized SA and sterilized SA by gamma rays at 25 kGy exhibited more proliferation than the untreated cells. Whereas the HUVEC cells seeded in moist heat treated SA resulted round-shaped and with a reduced number (Fig. 7). The findings suggested that SA solution with gamma rays sterilization had no cytotoxicity, which are used as nutrient substance. While SA solutions with moist heat sterilization had a large amount of cytotoxicity, which caused cytotoxic effects. It is proved that heating SA solutions led to methanol accumulation due to ester hydrolysis, which produced cytotoxic effects [36].
4. Conclusions Polysaccharides, such as alginates, are already being used as carriers for drug delivery. By changing the physicochemical and biological properties, it is possible that the alginate is used to control drug release. SA is a biocompatible, non-toxic, non-immunogenic and biodegradable natural linear polysaccharide. The two kinds of physical sterilization techniques, gamma irradiation and moist heat sterilization,
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hardly affect the native pH of SA polysaccharide, but deeply affected the intrinsic viscosity and structure of SA samples. The gamma irradiation was a suitable treatment to sterilize SA, avoiding the side effects caused by moist heat sterilization and retaining cell viability. The moist heat sterilization seems only appropriate for SA powers because of the severe side effects produced by heating. The research results may provide interesting future advancements toward the development of SA-based products for biomedical applications. However, further studies are required to investigate the mechanical and gelling properties of sterilized powders and solutions. Acknowledgements The author would like to thank Yufei Ma for her technical support on FT-IR spectroscopy analyses. This work was supported by the National Key Technology R&D Program of China (2012BAI18B02) and Public Experiment Center of State Bioindustrial Base (Chongqing, China). References [1] T.Y. Wong, L.A. Preston and N.L. Schiller, Alginate lyase: Review of major sources and enzyme characteristics, structurefunction analysis, biological roles, and applications, Annu. Rev. Microbiol. 54 (2000), 289–340. [2] R. Jayakumar, M. Rajkumar, H. Freitas, N. Selvamurugan, S.V. Nair, T. Furuike and H. Tamura, Preparation, characterization, bioactive and metal uptake studies of alginate/phosphorylated chitin blend films, Int. J. Biol. Macromol. 44 (2009), 107–111. [3] M. Yadav, D.K. Mishra, A. Sand and K. Behari, Modification of alginate through the grafting of 2-acrylamidoglycolic acid and study of physicochemical properties in terms of swelling capacity, metal ion sorption, flocculation and biodegradability, Carbohyd. Polym. 84 (2011), 83–89. [4] J.S. Yang, Y.J. Xie and W. He, Research progress on chemical modification of alginate: A review, Carbohyd. Polym. 84 (2011), 33–39. [5] T. Coviello, P. Matricardi, C. Marianecci and F. Alhaique, Polysaccharide hydrogels for the modified release of formulations, J. Control Release 119 (2007), 5–24. [6] A. Ikeda, A. Takemura and H. Ono, Preparation of low-molecular weight alginic acid by acid hydrolysis, Carbohyd. Polym. 42 (2000), 421–425. [7] J.W. Rhim, Physical and mechanical properties of water resistant sodium alginate films, LWT-Food Sci. Technol. 37 (2004), 323–330. [8] K. Norajit, K.M. Kim and G.H. Ryu, Comparative studies on the characterization and antioxidant properties of biodegradable alginate films containing ginseng extract, J. Food Eng. 98 (2010), 377–384. [9] H. Ertesvag and S. Valla, Biosynthesis and applications of alginates, Polym. Degrad. Stabil. 59 (1998), 85–91. [10] I. Braccini and S. Perez, Molecular basis of Ca2+ -induced gelation in alginates and pectins: the egg-box model revisited, Biomacromolecules 2 (2001), 1089–1096. [11] Z. Liu, Y. Jiao and Y. Wang, Polysaccharides-based nanoparticles as drug delivery systems, Adv. Drug Deliver Rev. 60 (2008), 1650–1662. [12] S. Patil, Cross-linking of polysaccharides: methods and applications, Pharmaceutical Reviews E-Journal (2008). [13] Z. Mohamadnia, M.J. Zohuriaan-Mehr, K. Kabiri, A. Jamshidi and H. Mobedi, pH-sensitive IPN hydrogel beads of carrageenan-alginate for controlled drug delivery, J. Bioact. Compat. Pol. 22 (2007), 342–356. [14] L. Yang, G. Liang, Z. Zhang and S. He, Sodium alginate/Na-rectorite composite films: preparation, characterization, and properties, J. Appl. Polym. Sci. 114 (2009), 1235–1240. [15] S. Benavides, R. Villalobos-Carvajal and J.E. Reyes, Physical, mechanical and antibacterial properties of alginate film: effect of the crosslinking degree and oregano essential oil concentration, J. Food. Eng. 110 (2011), 232–239. [16] R. Russo, M. Abbate, M. Malinconico and G. Santagata, Effect of polyglycerol and the crosslinking on the physical properties of a blend alginate–hydroxy ethyl cellulose, Carbohyd. Polym. 82 (2010), 1061–1067. [17] E.M. Zactiti and T.G. Kieckbusch, Release of potassium sorbate from active films of sodium alginate crosslinked with calcium chloride, Packag. Technol. Sci. 22 (2009), 349–358. [18] P. Malafaya, G. Silva and R. Reis, Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications, Adv. Drug Deliver Rev. 59 (2007), 207–233.
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