ARTICLE pubs.acs.org/Biomac
Synthesis of Amidic Alginate Derivatives and Their Application in Microencapsulation of λ-Cyhalothrin Ji Sheng Yang,* Hai Bing Ren, and Ying Jian Xie School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China ABSTRACT: 1-Octyl amine was covalently coupled to sodium alginate(NaAlg) in an aqueous-phase reaction via acidamide functions using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) as a coupling reagent to provide octyl-grafted amphiphilic alginate-amide derivative(OAAD) for subsequent use in λ-cyhalothrin (LCH) microcapsule application. The structure of OAAD was confirmed by FT-IR and 1H NMR spectroscopies. The new alginate-amide derivative was used for fabricating microcapsule that can effectively encapsulate LCH by emulsificationgelation technique. The microcapsules were characterized by optical microscopy (OM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and laser particle size analysis. The encapsulation efficiency and drug release behavior of LCH from the microcapsules were investigated. Results showed that the microcapsules were in spherical form with diameter mostly in the range of 0.510 μm and possessed a structure with LCH as core and OAAD as shell. The encapsulation efficiency and the release performance of the microcapsules were influenced by DS of OAAD and amount of CaCl2. The mechanism of LCH release was found to vary from anomalous to Fickian to quasi-Fickian transport with the DS of OAAD varied from 10.8 to 30.3 and the CaCl2/ emulsion ratios varied from 0.09 to 0.03%.
’ INTRODUCTION Since the 1950s, pyrethroids such as λ-cyhalothrin (LCH) have been widely applied as insecticides in households (both indoor and outdoor) as well as for the protection of crops.1 Releases to the air represent the most important emission pathway for pyrethroids. Because of that, inhalation is an important route of exposure for humans, especially just after spraying application in domestic indoors or agricultural close areas. In regards to effects on humans, after exposure to pyrethroids, some reversible symptoms of poisoning and suppressive effects on the immune system have been reported.2 Pyrethroids have been included in a list of suspected endocrine-disrupting chemicals by an EU working group. The use of pyrethroids does involve risk because their inherent properties can make them dangerous to health and the environment if not used properly.3 Therefore, it is very essential to alleviate or minimize these above effects. One way of doing so is to encapsulate pyrethroids with the appropriate polymers.4 The benefits of microencapsulation include improvement of long-time efficiency, ease of handling through solidification of liquid core, and a controlled release of pyrethroids.5 During the microencapsulation process, selecting environmentally friendly wall materials is the principal issue. It is wellknown that alginate is a polyanionic linear polysaccharide of 1,4linked-R-L-guluronic acid (G) and β-D-mannuronic acid (M).6 Because of its unique properties of biocompatibility, gelling capacity, and nontoxicity, alginate has been used to encapsulate a wide variety of proteins such as hemoglobin,7 albumin,8 and r 2011 American Chemical Society
DNA.9 Most recently, amphiphilic alginate derivatives have aroused considerable interest in biomedical areas.10 Dellacherie11,12 reported that the microparticles prepared by amphiphilic derivatives of sodium alginate bearing with long alkyl chains, in aqueous solution, can be used as protein carriers with specific controlled release properties. The research of Hall showed that Ca2+ cross-linked hydrogels of amphiphilic alginate derivative with butyl chains are capable of encapsulating both hydrophobic and hydrophilic materials.13 In these studies, alginate or amphiphilic alginate derivatives were made into beads or microparticles by gelation to offer the controlled release of substances. However, our strategy was to prepare microcapsules with coreshell structure via the use of amphiphilic alginate derivatives. To the best of our knowledge, this is the first report on fabrication of the microcapsule with amphiphilic alginate derivatives to encapsulate LCH. In this study, the octyl-grafted amphiphilic alginate-amide derivative (OAAD) was prepared by amide linkage attachment of octyl amine onto the carboxylate group of alginate. For the sake of concision, the nomenclature used for the amphiphilic alginate-amide derivative is xOAAD, where x is the percent degree of substitution (DS) of OAAD, and defined as the average numbers of octyl groups amide-linked to hexuronic acid residues (Noctyl/Nhexuronic). A series of OAAD then was used to fabricate Received: April 26, 2011 Revised: June 24, 2011 Published: June 27, 2011 2982
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Figure 1. Octylamin coupling to sodium alginate via EDC.
LCH/OAAD microcapsules by emulsificationgelation technique, and the morphology, coreshell structure, particle size, and size distribution of the microcapsules were characterized. The effect of DS of OAAD and Ca2+ on encapsulation efficiency and drug release were investigated in detail. The kinetics of drug release from the microcapsule was discussed. The purpose of this study is to demonstrate the possible applicability of the present release system.
’ EXPERIMENTAL SECTION Materials. Sodium alginate (NaAlg, Mη ≈ 430 kDa), octyl amine ̅ (AR), and toluene (AR) were bought from Sinopharm Chemical Reagent (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, AR) was purchased from Sangon Biotech (Shanghai, China). LCH (C23H19ClF3NO3, >97.0%) was provided by Yangnong Chemical (Jiangsu, China). The distilled water was used throughout the experiments. OAAD Synthesis and Characterization. OAAD was prepared according to the literature1416 with some modifications. NaAlg (4 g) was dissolved in water to a concentration of 3.0 wt %. The pH of the solution was adjusted to 3.4 using 0.4 M HCl, and the solution was then diluted to 2.0 wt %. Next, an aqueous solution of EDC-HCl was added slowly to the system, and the pH of the reaction mixture was maintained at 3.4 by the addition of 0.4 M HCl. To prepare different DS of OAAD, the amount of EDC-HCl (NEDC/Nhexuronic) was 0.69, 0.42, and 0.21, respectively. After 5 min of reaction, octyl amine (Noctylamine/Nhexuronic = 1.14) was added, and the mixture was stirred at 35 °C for 24 h. The product was completely precipitated from the above mixture by adding ethanol, filtrated, and then successively washed with ethanol. The solid product obtained was dissolved in distilled water and dialyzed against water for 5 day to remove low-molecular-weight impurities and then lyophilized. The DS of OAAD was derived from elementary analysis of nitrogen contents. FT-IR spectra of NaAlg and OAAD were recorded with an Tensor 27 FT-IR spectrophotometer (Bruker). The samples were scanned from 500 to 4000 cm1 at a resolution of 4 cm1. Samples were prepared by processing compressed semitransparent KBr disks before measurement. 1 H NMR was performed on an AVANCE 600 nuclear magnetic resonance spectrometer (Bruker) at 80 °C using 5 mm NMR tube. The samples were dissolved in D2O (99.9%) to a concentration of ∼10 mg/mL, and the chemical shift was calculated with respect to 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) as the internal standard. Microcapsule Preparation and Analysis. The preparation of LCH/OAAD microcapsules was adopted from an emulsificationgelation technique17 and briefly described as follows. First, the aqueous phase (300 mg xOAAD dissolved in 120 mL of water) and the organic phase (2.25 g LCH dissolved in 15 mL of toluene) were prepared separately. Then, the organic phase was added to the aqueous phase while homogenizing at 10 000 rpm for 15 min at 40 °C. Finally, in the
system of oil-in-water emulsion, the interfacial membrane between oil and water was gelled by adding different amount of CaCl2 and kept stirring slowly for 30 min. The shape and size of microcapsules were monitored by using DMLP optical microscopy (OM) (Leica, Germany). The formation of macromolecular membrane was confirmed using a Leica DMRXA fluorescence microscope system. Approximately 10 mL of microcapsules suspension was placed in a test tube, and moderate rhodamine 6G aqueous solution (1 mg/mL) was added and mixed for 30 min. The mixture was then dropped on a microscope slide and covered with a coverslip before observed. The surface morphology of the microcapsules under dry state was monitored by S-4800 II SEM (Hitachi High-Technologies). A drop of the microcapsules suspension was placed on a glass slide and dried using an infrared lamp. The samples was coated with a thin gold layer under vacuum at 15 mA and then observed at the accelerating voltage of 15.0 kV. The coreshell structure of microcapsules was observed by a Tecnai12 TEM (Philips) at the acceleration voltage of 100 kV. The suspension of microcapsules was dropped on a carbon-coated copper grid, and stained with phosphotungstic acid solution (∼2 wt %) as a negative staining reagent for 2 to 3 min and then dried using the infrared lamp before observation. The size distribution of the microcapsules was determined in aqueous by a BT-9300H laser particle size analyzer (Bettersize). Determination of Encapsulation Efficiency. It was determined indirectly by measuring the amount of nonencapsulated LCH through an extraction method.18 The LCH concentration was analyzed by a 6890N gas chromatography (GC) (Agilent). Encapsulation efficiency was calculated from the ratio of the amount of LCH encapsulated in microcapsules to the initial amount of LCH. Release Studies. For the release experiment of LCH from the microcapsule, 10 mL of suspension of microcapsule was sprayed onto glass slides. After they were blown dry, they were rinsed with 2 mL of fresh toluene. At predetermined time intervals, repeat the above rinsing process. Each liquor was withdrawn and analyzed for the amount of LCH with GC.
’ RESULTS NaAlg was hydrophobically modified by use of the coupling agent EDC-HCl to form amide linkages between octylamin molecules and the carboxylate moieties on the alginate polymer backbone according to the scheme shown in Figure 1. The structures of samples were confirmed by FT-IR and 1H NMR, as shown in Figures 2 and 3. The microcapsules exhibited spherical morphology and nonaggregated in aqueous suspension (Figure 4a). From the fluorescence microscopy image of LCH/OAAD microcapsules (Figure 4b), we could clearly observe a red ring encompassing a dark spot. To observe further the structure of microcapsules, we applied TEM means. As can be seen from Figure 4c, the coreshell structure of microcapsules was clearly visible. The SEM image of the 2983
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Biomacromolecules microcapsules under dry state (Figure 4d) showed irregular or ruptured spherical morphology, and the size decreased sharply compared with that of the microcapsules in water. The size distribution of the microcapsules in aqueous solution was illustrated in Figure 5. The size distribution showed three peaks, and the microcapsules were mostly in the size range of 0.510 μm. The effects of different DS of OAAD and the amount of CaCl2 on encapsulation efficiency of LCH were evaluated, as shown in Table 1. For the 30.3 OAAD microcapsules, varying the CaCl2/ emulsion ratio from 0.03 to 0.09% caused slightly an increase in encapsulation efficiency of LCH from 94.3 to 96.4%. However, with respect to both 22.7 OAAD and 10.8 OAAD microcapsules, the encapsulation efficiency of LCH increased distinctly from 89.2 to 96.0% and 85.0 to 92.9%, respectively. Under the same CaCl2/ emulsion ratio, increasing the %DS of OAAD from 10.8 to 30.3% resulted in the distinct increase in encapsulation efficiency of LCH. Release profiles of LCH from microcapsules were studied, as shown in Figures 6 and 7. In general, within the first 120 min, a
Figure 2. FT-IR spectra of (a) NaAlg, (b) NaAlg + octyl amine, and (c) 30.3 OAAD.
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rapid drug release process occurred; then, the release rate became slower and displayed prolonged behavior. At a CaCl2/emulsion ratio of 0.09% (Figure 6), preparing microcapsule with the %DS of OAAD varied from 10.8 to 22.7 to 30.3%, the values of percent cumulative release of LCH were 47.7, 56.3, and 70.2%, respectively, within 300 min. Effects of different CaCl2/emulsion ratio on release behavior of LCH from the microcapsules were studied (Figure 7). The values of percent cumulative release of LCH increased from 47.7 to 58.9 to 63.4% with the CaCl2/emulsion ratio varied from 0.09 to 0.06 to 0.03%.
’ DISCUSSION From the FT-IR spectrum of NaAlg (Figure 2a), it was being observed that a broad peak at 3450 cm1 was due to the stretching vibrations of OH, and a small peak at 2930 cm1 was attributed to the CH stretching vibrations of methyne groups. The bands at 1090 and 1030 cm1 were assigned to COC stretching vibrations. It was further noted that two strong peaks at 1609 and 1417 cm1 were assigned to asymmetric and symmetric stretching vibrations of carboxyl groups. In the spectrum of mixture of NaAlg and octyl amine (Figure 2b), compared with NaAlg (Figure 2a), a small peak at 3319 cm1 was attributed to the NH stretching vibrations of amino group, and the peaks of 2929 and 2857 cm1 were assigned to methylene groups of octyl amine. Comparing the spectrum of OAAD (Figure 2c) with that of NaAlg and octyl amine (Figure 2b), the peak of 3319 cm1 disappeared, and the OH stretching band of hydroxyl group and the NH stretching band of amide group overlapped with each other and led to broad band at 3425 cm1. The peak at 1609 cm1 in the spectrum of NaAlg shifts to 1630 cm1 in that of OAAD, and the peaks of 2929 and 2857 cm1 appear. These suggested that octyl groups successfully grafted onto alginate. In Figure 3a, the proton peaks from 3.6 to 5.1 ppm contributed to the H of alginate, in which the anomeric protons were observed in the region 4.5 to 5.1 ppm. In contrast with the spectrum of alginate, additional peaks in Figure 3b were observed from 0.8 to
Figure 3. 1H NMR spectra of (a) NaAlg and (b) 30.3 OAAD. 2984
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Figure 4. (a) Optical photomicrograph, (b) fluorescence photomicrograph, (c) TEM, and (d) SEM of the microcapsules prepared with 30.3 OAAD and CaCl2/emulsion = 0.09%.
Table 1. Effect of DS of OAAD and CaCl2/Emulsion Ratio on Encapsulation Efficiency of LCH
Figure 5. Size distribution of microcapsules prepared with 30.3 OAAD and CaCl2/emulsion = 0.09%.
3.2 ppm, which were assigned to the methyl and methylene protons of octyl chain, respectively. Besides, it exhibited signal relative to the presence of the H of amide group at 4.9 ppm.15,19 On the basis of the above data, it could be obviously concluded that OAAD was synthesized by the EDC-HCl method. Because of opposite charges attracted, the fluorescent dye of Rh6G with positive charges bound to the OAAD with the negative charges, so that the region of OAAD macromolecules appeared red (Figure 4b). Oil droplets (LCH and toluene), however, inside the microcapsules appeared nonfluoresced black area because there was no interaction between Rh6G and the
DS of OAAD (%)
CaCl2/emulsion (%)
encapsulation efficiency (%)
30.3
0.03
94.3
30.3
0.06
95.5
30.3
0.09
96.4
22.7
0.03
89.2
22.7
0.06
93.2
22.7
0.09
96.0
10.8
0.03
85.0
10.8 10.8
0.06 0.09
89.8 92.9
molecules of oily phase. The dark spots inside the microcapsules indicated the presence of LCH. The bright layer showed that the macromolecular membrane formed.20 This suggested that OAAD did adsorb on the interface between oil droplet and water and further confirmed that the microcapsules were successfully fabricated. Compared with OM results (Figure 4a,b), the diameter of microcapsules observed by TEM and SEM decreased sharply. The reasonable explanation was that organic solvent inside the microcapsules and water in the membranes was evaporated during the preparation of the TEM or SEM specimen. Eventually, the microcapsules exhibited spherical shell collapsed morphology. The data of diameter distribution of the microcapsules in aqueous determined by laser particle size analyzer were in accordance with that obtained from microscopic observation. The diameter 2985
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Figure 6. Release profiles of LCH from microcapsules prepared with different %DS of OAAD at a CaCl2/emulsion ratio of 0.09%.
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Figure 8. Fitting curves of power law model data in different %DS of OAAD and CaCl2/emulsion ratio.
Table 2. Comparison of the Power Law Model Data in Different %DS of OAAD and CaCl2/Emulsion Ratio power law batches
a
Figure 7. Release profiles of LCH from microcapsules prepared with 30.3OAAD at a different CaCl2/emulsion ratio.
distribution of microcapsules depended on the size of the emulsion droplets, which were determined by the mixer and means during the emulsification process. As the substitution degree increased, the number of hydrophobic alkyl chains onto the polysaccharide backbone increased. During emulsification, the increase in the number of hydrophobic chains inserted into oil droplets improved the compactness of the membrane, which might be responsible for increasing encapsulation efficiency of LCH. Besides, under the same DS conditions, the increase in the amount of Ca2+ improved the cross-link densities so as to enhance mechanical strength and compactness of the membrane, resulting in an increase in encapsulation efficiency. As can be seen from Figure 6, the lower the DS of OAAD, the faster the release of LCH, within the same period of time. This was explained on the basis of the fact that because of the low substitution degree, the number of hydrophobic alkyl chains was so few that the compactness and mechanical strength of the membrane obtained were little good. Moreover, we can see from Figure 7 that the values of percent cumulative release of LCH increased with the decreasing of CaCl2/emulsion ratio. The results may be explained by the fact that increase in the amount of CaCl2 enhanced the compactness and mechanical strength of the membrane and reduced the mesh sizes of the network. This consequently restrained the diffusion of LCH molecules from the microcapsules into air.
na
R2
mechanism
10.8OAAD+0.09%CaCl2
0.50 ( 0.025
0.99
Fickian
22.7OAAD+0.09%CaCl2
0.39 ( 0.050
0.93
Fickian
30.3OAAD+0.09%CaCl2
0.52 ( 0.053
0.96
anomalous
30.3OAAD+0.06%CaCl2
0.41 ( 0.038
0.96
Fickian
30.3OAAD+0.03%CaCl2
0.34 ( 0.039
0.94
quasi-Fickian
Values for n expressed as mean (95% confidence interval value.
To investigate the effect of DS of OAAD and Ca2+ on the mechanism of LCH release from the microcapsules, a classical model, known as the power law,21 was used M t =M ∞ ¼ kt n where Mt and M∞ are, respectively, the amounts of drug released at time t and at infinite time, k is a constant incorporating structural and geometrical characteristic of the delivery system, and n represents the diffusion exponent indicative of the mechanism of drug release, provided that t is limited to times where Mt/M∞ < 0.6. For swellable release systems, values of n ranging from 0.43 to 0.5 indicate Fickian or diffusion-controlled release, and values of n ranging from 0.85 to 1.0 indicate Case II transport mechanisms. Values of n intermediate between the above limits indicate anomalous or non-Fickian transport. By applying least-squares method to release data, the fitting curves were obtained (Figure 8), and the values of n were estimated (Table 2). When the ratio of CaCl2 to emulsion was 0.09%, the values of n were found to vary from 0.50 to 0.39 with the %DS of OAAD varied from 10.8 to 22.7. This indicates, under such circumstances, that the release of LCH followed Fickian transport mechanism. For 30.3 OAAD, as the CaCl2/emulsion ratio was varied from 0.09 to 0.06 to 0.03%, the value of n was 0.51, 0.41, and 0.34, respectively, indicating that the mechanism of release from anomalous to Fickian to quasi-Fickian transport.
’ CONCLUSIONS In this research, we synthesized an amphiphilic macromolecule OAAD; then, LCH was successfully encapsulated with 2986
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Biomacromolecules OAAD in microcapsules by emulsificationgelation technique. The microcapsules were spherical morphology and nonaggregated in aqueous solution. A high encapsulation efficiency of LCH in the 30.3 OAAD microcapsules was achieved, reaching up 96.4%. The release behavior of LCH from the microcapsules could be controlled by changing the DS of OAAD or amount of CaCl2. The data of LCH release kinetics could be well-fitted by the power law model. This research implied that our strategy would potentially be an effective method to prepare LCH microcapsules and control LCH release.
’ AUTHOR INFORMATION Corresponding Author
*Tel: +86 514 87975568. Fax: +86 514 87975244. E-mail:
[email protected].
’ ACKNOWLEDGMENT We gratefully acknowledge the financial support from the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (08KJB530007) and the Open Project Program of the State Key Laboratory of Food Science and Technology (SKLF-KF-200906). ’ REFERENCES (1) Barro, R.; Garcia-Jares, C.; Llompart, M.; Bollain, M. H.; Cela, R. Rapid and sensitive determination of pyrethroids indoors using active sampling followed by ultrasound-assisted solvent extraction and gas chromatography. J. Chromatogr., A 2006, 1111, 1–10. (2) Mak, S. K.; Shan, G.; Lee, H. J.; Watanabe, T.; Stoutamire, D. W.; Gee, S. J.; Hammock, B. D. Development of a class selective immunoassay for the type II pyrethroid insecticides. Anal. Chim. Acta 2005, 534, 109–120. (3) Fernandez-Alvarez, M.; Llompart, M.; Lamas, J. P.; Lores, M.; Garcia-Jares, C.; Cela, R.; Dagnac, T. Simultaneous determination of traces of pyrethroids, organochlorines and other main plant protection agents in agricultural soils by headspace solid-phase microextraction-gas chromatography. J. Chromatogr., A 2008, 1188, 154–163. (4) Zhang, Q.; Zhang, P. P.; Jiao, Q. Z. Synthesis and characterization of microcapsules with chlorpyrifos cores and polyurea walls. Chem. Res. Chin. Univ.s 2006, 22, 379–382. (5) Jang, I. B.; Sung, J. H.; Choi, H. J. Synthesis of microcapsule containing oil phase via in-situ polymerization. J. Mater. Sci. 2005, 40, 1031–1033. (6) Jang, J. S.; Zhao, J. Y.; Fang, Y. Calorimetric studies of the interaction between sodium alginate and sodium dodecyl sulfate in dilute solutions at different pH values. Carbohydr. Res. 2008, 343, 719–725. (7) Silva, C. M.; Ribeiro, A. J.; Figueiredo, M.; Ferreira, D.; Veiga, F. Microencapsulation of hemoglobin in chitosan-coated alginate microspheres prepared by emulsification/internal gelation. AAPS J. 2006, 7, 903–913. (8) Wang, K.; He., Z. Alginate-konjac glucomannan-chitosan beads as controlled release matrix. Int. J. Pharm. 2002, 244, 117–126. (9) Quong, D.; Neufeld, R. J.; Skjak-Braek, G.; Poncelet, D. External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation. Biotechnol. Bioeng. 1998, 57, 438–446. (10) Burckbuchler, V.; Kjøniksen, A. L.; Galant, C.; Lund, R.; Amiel, C.; Knudsen, K. D.; Nystr€om, B. Rheological and structural characterization of the interactions between cyclodextrin compounds and hydrophobically modified alginate. Biomacromolecules 2006, 7, 1871–1878. (11) Leonard, M.; Rastello de Boisseson, M.; Hubert, P.; Dellacherie, E. Production of microspheres based on hydrophobically associating alginate
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