КОЛЛОИДНЫЙ ЖУРНАЛ, 2014, том 76, № 5, с. 673-678
PREPARATION AND CHARACTERIZATION OF BIOSURFACTANT BASED ON HYDROPHOBICALLY MODIFIED ALGINATE © 2014 г. Yueqin Yu1, Caifeng Leng, Zhe Liu, Fengjun Jia, Yi Zheng, Kunshan Yuan, Shaopeng Yan
College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology
Qingdao, 266042, P. R. China Поступила в редакцию 10.12.2013 г.
Hydrophobic modified alginate (HM-alginate) was synthesized using a low-energy, environment-friendly process in aqueous solution, with sodium alginate and dodecyl glycidyl ether as starting materials. The HM-alginate was characterized using 1H NMR, and the reaction efficiency was about 40%. The HM-alginate aggregated in solution; the critical micelle concentration (CMC) was determined using surface tension and dynamic light scattering methods. We observed reasonable agreement between the CMC values obtained by the different techniques, and the CMC was 4.0 x 10-4 g/mL. The zeta-potential of the HM-alginate in aqueous solution was about —82 mV, which is higher than —51 mV of the sodium alginate. Rheology measurements showed that the HM-alginate solution exhibits a Newtonian behavior at all shear rates, whereas the apparent viscosity is very low. The solubility of the liposoluble substance Sudan IV increased significantly with HM-alginate concentration. This result is promising for potential applications of HM-alginate as an ecology-safe material to encapsulate lipophilic substances.
Alginate is an anionic natural linear polysaccharide, presenting two kinds of hexuronic acid residues including P-D-mannuronic acid (M) and a-L-gulu-ronic acid (G) residues, which are arranged in repeating GG (MM) blocks or alternating MG blocks. Alginate is commonly isolated from brown algae such as Laminaria hyperborea, Ascophyllum nodosum, and Macrocystis pyrifera. It is also available from red seaweed (Corallinaceae) and is found in some bacteria, such as Azotobacter vinelandii, and several Pseudomonas species [1—3]. Alginate is widely used in the food industry as a thickener, emulsifier, and stabilizer. It has also been used for the formation of particles (100 nm-2 mm in size) for drug delivery due to the biocompati-bility, bioadhesiveness, and pH sensitivity of this biomaterial. However, the pure sodium alginate is not suitable for pharmaceutical or other applications because of its hydrophilicity and instability. Modification of alginate is an effective way to synthesize hydrophobic alginate derivative [2-4]. Qian Li et al. have synthesized a hydrophobic alginate derivative using oleoyl chloride in water and studied the physicochemical properties of its aqueous solutions . There is also considerable interest in chemical modification of alginate to form a range of special chemicals, and recent studies have involved synthesis of hydrophobically modified alginates for their potential application as surfactants . Hydrophobically modified alginate
1 Corresponding author. E-mail: firstname.lastname@example.org.
(HM-alginate) is a commercially promising polyelec-trolyte because of its biodegradability and is expected to find an application as an encapsulation material for fabrication of drugs and fertilizers [6—8].
The attachment of the alkyl chains proceeds through the interaction of the epoxy group of dodecyl glycidyl ether with the hydroxyl groups of sodium alginate units. The product is a carboxylate with hydroxyl group and ether, which salt form is negatively charged. The reaction is non-specific, and the alkyl chains are attached randomly along the polymer backbone. The resulting structure differs from a typical polymeric surfactant, in which the hydrophobic groups are bonded to one or both ends of the polymer chain. Nevertheless, the surface tension measurements have shown that the molecules of HM-alginate form aggregates at a critical concentration. The aggregation behavior of this type of polymer makes them suitable candidates for the dissolution and encapsulation of active compounds. For instance, Qian Li et al.  have recently reported a potential application of HM-alginate micelles in drug delivery.
The goal of this study was to synthesize HM-algi-nate by chemical modification of the backbone of alginate by dodecyl glycidyl ether (DGE) in aqueous solution. The physicochemical characteristics were studied using surface tension, dynamic light scattering (DLS), zeta-potential, and viscosity measurements. The HM-alginate can undergo self-assembly to form micelles when dissolved in water, and the hydrophobic substances may be entrapped within these micelles.
Using Sudan IV as a model lipophilic substance, the solubilization of the dye was studied in HM-alginate solution.
2. MATERIALS AND METHODS
Sodium alginate (molecular mass, 2 x 104 Da) was purchased from Qingdao Bright Moon Seaweed Group Co., China, and was used as received. DGE was supplied by Maya Reagent, China. Sudan IV, a water insoluble diazo dye, was obtained from Tianjin Damao Chemical Reagent Factory, China. All other chemicals used in this study were of analytical grade.
Synthesis of HM-alginate. The hydrophobically modified alginate (HM-alginate) was synthesized from sodium alginate and DGE. Alginate (5 g) and distilled water (150 mL) were added into a 250 mL three-necked round-bottom flask fitted with a condenser, thermometer, and a mechanical stirrer. The mixture was stirred at ambient temperature to obtain a homogeneous solution. The pH was adjusted to 9.0 ± 0.1 using 0.1 M NaOH solution. Then, 5 mL of DGE was added to the mixture and heated at 80°C for 8 h. Once the reaction was complete, the resulting product was neutralized with acetic acid solution (1 : 1) to a pH of 4.5. The product was completely precipitated from the mixture by adding acetone, then filtrated, and dried in a vacuum oven at 50°C overnight.
NMR spectroscopy. 1H NMR spectra were performed on an AVANCE 500 nuclear magnetic resonance spectrometer (Bruker) at 25°C using 5 mm NMR tube. The samples were dissolved in D2O (99.9%) .
2.3. Characterization of Micelles of HM-Alginate
Surface tension. The surface tension of solutions was measured using a JK99C Tensiometer (Shanghai Zhongchen Instruments, China) in the concentration range of 9 x 10—6—1.5 x 10-2 g/mL at 20 °C . The solutions were equilibrated for 24 h prior to surface tension measurements. The break point on the plot of surface tension versus surfactant concentration was used to determine the critical micelle concentration (CMC) of the surfactant [10-12]. All measurements were repeated at least three times.
Dynamic light scattering. The DLS experiments were performed at 25°C using a Zetasizer Nano ZS (Malvern, UK). Three milliliters of HM-alginate solution were placed in polymer cells and measured at a detector angle of 90° and wavelength of 633 nm. Experimental solution samples were prepared by diluting of a 0.01 g/mL of stock solution with deionized water [13, 14]. The CMC was determined from the change
in the slope of the plot of the mean particle size as a function of concentration.
Zeta potential. The zeta potentials of the alginate and HM-alginate species in deionized water were measured using the same Zetasizer at 25 °C [13, 14]. The samples were diluted to achieve concentrations within the range 4 x 10-4-6 x 10-4 g/mL.
2.4. Viscosity Measurements
Kinematic viscosity measurements. The kinematic viscosities of sodium alginate and HM-alginate solutions (2 x 10-5—6 x 10-4 g/mL in deionized water) were measured at the atmospheric pressure with an Ubbelohde capillary viscometer (Shanghai Shen Yi Glass Products Co., China) [15, 16]. During calibration and measurements, the viscometer was immersed in an insulated bath under continuous stirring. Each measurement was repeated four times.
Rheology studies. The rheological properties of sodium alginate and HM-alginate solutions (5 x 10-4 g/mL) were measured using a Stabinger viscometer RheolabQC (Anton Paar GmbH, Austria) at 25°C [17—19].
2.5. Dye Solubilization
The solubilization properties of HM-alginate were determined using Sudan IV as a dye . 10 milligrams of the dye were added to 10 mL of the solutions with varied concentrations of sodium alginate or HM-algi-nate in deionized water. The samples were mixed at 30°C overnight. Then, these solutions were centri-fuged at 3000 rpm for 10 min to separate the undissolved Sudan IV and the supernatant was added into UV-grade 10 mm path length cuvette. The absorbance of the resulting solutions was measured at 510 nm using a SP 723 UV/VIS Spectrometer (Shanghai Spectrum Co., Ltd). Each measurement was performed at least three times.
3. RESULTS AND DISCUSSION 3.1. Synthesis of HM-Alginate
HM-alginate was synthesized by introducing a long linear alkyl chain into alginate. As shown in Scheme 1, the HM-alginate was formed from by nucleophilic substitution reaction between sodium alginate and DGE at 80°C. The non-specific attachment of the alkyl chains to the polymer backbone proceeds through the interactions of the epoxy groups of the ether with the sugar hydroxyls.
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1 i. 1 1
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Fig. 1. XH NMR spectra of (1) alginate and (2) HM-algi-nate in D2O.
Surface tension (mN/m) 75 70 65 60 55 50 45 40
0 2 4 6 8 10 12 14 16 Concentration (10-3 g/mL)
Fig. 2. Concentration dependences of the surface tension of (1) alginate and (2) HM-alginate solutions.
NMR spectra of the native alginate and HM-al-ginate samples are shown in Fig. 1. The prominent peaks at 2.0 ppm and 4.7 ppm are from the solvent, i.e. D2O. The XH NMR signal at 3.27 ppm indicates the presence of carbons 3—5 of the native alginate (see spectrum 1) . XH NMR signals at 0.88 ppm, 1.26 ppm and 1.51 ppm correspond to the hydrogen of methyl and methylene groups of DGE (spectrum 2). New peaks with high intensit
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