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Preparation and characterization of iron–cobalt composite microcapsules
Journal of Alloys and Compounds 653 (2015) 570e576
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Journal of Alloys and Compounds journal homepage: http://www.elsevier.com/locate/jalcom
Review
Preparation and characterization of ironecobalt composite microcapsules Yan Qiao, Yanchun Hu*, Jiahui An, Dongmei Wu, Gang Shu, Hualin Fu, Guangneng Peng, Junliang Deng Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Sichuan Province, Wenjiang 611130, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2015 Received in revised form 25 July 2015 Accepted 26 August 2015 Available online 1 September 2015
Abstract: Iron and cobalt as trace elements can promote the growth and development of the body. In our experiments, they were added as a drug for treatment of animal anemia. Ironecobalt composite microcapsules were prepared by emulsion solvent diffusion method (ESDM) can meet the requirements of the body to trace elements, thus promoting the body's normal hematopoietic function. The optimum condition was investigated by response surface methodology (RSM) in order to improve the bioavailability of oral ironecobalt preparations. By using the analysis of flame atomic absorption spectrophotometry and scanning electronic microscope (SEM), the characteristics of the microcapsules were investigated respectively. The results showed that the optimum preparation conditions for ironecobalt composite microcapsules were: 0.48 g of ferric citrate, 0.25 g cobalt chloride, 0.2 ml of twain-80 and 1.8 ml of span-80. The microcapsules were successfully prepared with 90.61% of encapsulation efficiency (EE) and 45.32% of drug loading (DL). The particles of the microcapsule conformed to a normal distribution with the average diameter of 180 ± 9.54 mm. The average accumulated dissolution is lower than 10.00% in dissolution medium of pH 1.0 within 120 min but that is more than 75.00% in dissolution medium of pH 6.8 within 120 min. © 2015 Elsevier B.V. All rights reserved.
Keywords: Ironecobalt composite microcapsules preparation Response surface methodology (RSM) Encapsulation efficiency (EE) In vitro release studies
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 2.2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.1. Preparation of ironecobalt composite microcapsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.2. Analysis of microcapsule size and shell morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.3. Optimization of preparation conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.4. In vitro release studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.1. Results of RSM design experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.1.1. The establishment of the model and the significance test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.1.2. The RSM results of factors' interaction on EE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.2. Surface morphology and size distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.3. Determination of EE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
* Corresponding author. E-mail address: [email protected] (Y. Hu). http://dx.doi.org/10.1016/j.jallcom.2015.08.211 0925-8388/© 2015 Elsevier B.V. All rights reserved.
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
1. Introduction Iron and cobalt are essential trace elements in animals, and lack of these elements can result in animal anemia. Iron is essential for almost all living organisms, which not only participate in the metabolism of the body, but also helps regulate the immune function [1]. Studies have shown that iron is involved in liver deoxyribonucleic acid synthesis, promote liver cell growth, promote synthesis of liver cells and other cell mitochondria, reduce energy consumption, and in a certain extent can treat anemia, severe atypical hyperplasia etc.. The sufficient evidence showed that the metabolism, differentiation of lymphocytes and bactericidal effect of neutrophils need to have enough iron [2]. Cobalt has hematopoietic function, cobalt deficiency may easily lead to a series of diseases such as malignant anemia and nerve degeneration [3]. Cobalt can promote gastrointestinal absorption of iron, and using combined with iron is obvious than adding cobalt alone on the effect of treatment for anemia. On the other hand, cobalt poisoning can be caused by a regular injection of cobalt in cobalt or exposure to an excess of the original cobalt environment. Therefore, we must strictly control the amount of cobalt. In ferric citrate and cobalt chloride, they are present as trivalent iron and two valence cobalt. This ion is relatively stable and easy to absorb, therefore enhance ironecobalt microcapsules' stability. There has been a growing interest in research, development and commercialization of trace elements over the past decade [4]. So the purpose of this study is to develop the composite microparticle of ferric citrate and cobalt chloride so as to prevent and cure anemia of animals. Iron and cobalt increasingly complex properties from food ingredients and such complex properties can oftentimes only be provided by microencapsulation [5]. Early study showed that microcapsules can not only cover the odor and taste of volatile materials but also improve the stability of volatile drugs [6]. Emulsion solvent diffusion method, also called drying in liquid, is a kind of microencapsulation technology. In the process of this method, the organic solvents that dissolve capsule materials and core of capsules are diffused, separated and condensed to form microcapsule. The method satisfies the safety requirements in drug and food industry, and it is able to control the factors that affect the properties of microcapsules. With carefully finetuned controlled release properties, microencapsulation is not just an added value, but is also the source of totally new ingredients with matchless properties. Recently, the most common coating materials used for microcapsules are arabic gum, poly lactose and the composite of poly (ethylene glycol)-poly (lactic acid) copolymers (PLA-PEG), etc. Since
Table 1 The four factors and three levels of the RSM optimization analysis experiments Code
Factors
Levels 1
0
1
A B C D
ferric citrate dosage (g) cobalt chloride dosage (g) Span80 dosage (ml) twain-80dosage (ml)
0.38 0.15 0.9 0.1
0.48 0.20 1.8 0.2
0.58 0.25 2.7 0.3
polypropylene resin has stable chemical properties, good bioavailability and is easy to form membrane, it is always used as the capsule shell material in the preparation of microcapsule. In the present study, polypropylene resin was chosen as capsule shell material to prepare the ironecobalt composite microcapsules because of it will not be dissolved in pH1. Intestinal pH is acidic environment which close to 1, polypropylene resin's function of avoid drugs to dissolution release in pH1, effectively enhances the stability of the drug in the intestinal tract, play good role of target orientation, improve the biological utilization rate, enhance the oral bioavailability. The factors, such as the ratio of coating material mass (or the concentration of emulsion) to core material volume, the volume ratio of organic phase to aqueous phase, stirring speed, time and water amount, which influence the DL (the ratio of the amount of encapsulated into microcapsules and the weight of the microcapsules) and EE (the ratio of package into dosage and dosage of microcapsule) of microcapsules, were investigated. Different core materials and microencapsulation methods will cause different optimum conditions. Thus the preparation conditions of ironecobalt composite microcapsules by ESDM were optimized by orthogonal assay in the present study. 2. Materials and methods 2.1. Materials Ferric citrate, cobalt chloride, liquid paraffin, methanol, twain80, span-80 and n-hexane were purchased from Chengdu Kelong Table 2 BBD experiment design and results Serial number
A (g)
B (g)
C (ml)
D (ml)
EE (%)
DL (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
0.58 0.58 0.48 0.48 0.48 0.48 0.48 0.38 0.58 0.38 0.38 0.48 0.58 0.48 0.48 0.48 0.48 0.48 0.38 0.48 0.58 0.48 0.48 0.48 0.48 0.58 0.38 0.38 0.48
0.20 0.15 0.25 0.15 0.20 0.20 0.15 0.20 0.20 0.15 0.25 0.20 0.20 0.25 0.20 0.20 0.20 0.25 0.20 0.15 0.25 0.20 0.15 0.20 0.25 0.20 0.20 0.20 0.20
1.80 1.80 0.90 1.80 2.70 1.80 0.90 1.80 0.90 1.80 1.80 2.70 1.80 1.80 1.80 1.80 0.90 1.80 0.90 2.70 1.80 1.80 1.80 1.80 2.70 2.70 1.80 2.70 0.90
0.10 0.20 0.20 0.10 0.10 0.20 0.20 0.30 0.20 0.20 0.20 0.30 0.30 0.30 0.20 0.20 0.10 0.10 0.20 0.20 0.20 0.20 0.30 0.20 0.20 0.20 0.10 0.20 0.30
89.19 83.87 83.47 82.40 65.46 90.61 81.03 71.67 70.51 75.25 65.57 77.10 80.40 75.46 90.61 90.61 76.72 81.14 75.88 87.25 81.03 90.61 78.30 90.61 72.72 74.06 67.35 68.51 73.56
39.52 27.31 27.06 25.14 13.54 45.32 23.41 16.46 15.13 19.37 12.48 20.06 22.31 18.49 45.32 45.32 19.51 23.16 17.58 36.69 22.03 45.32 18.64 45.32 16.93 18.75 13.62 14.96 17.12
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Table 3 ANOVA for response surface quadratic model. Source
Sum of squares
df
Mean squares
F Value
P-value (Prob > F)
Significance
A B C D AB AC AD BC BD CD A2 B2 C2 D2 Model Residual Total
250.53 68.69 21.52 2.77 11.70 29.81 42.97 72.00 0.62 54.76 461.61 54.96 421.74 274.34 1402.61 290.75 1693.36
1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 14 28
250.53 68.69 21.52 2.77 11.70 29.81 42.97 72.00 0.62 54.76 461.61 54.96 421.74 274.34 100.19 20.77 e
12.06 3.31 1.04 0.13 0.56 1.44 2.07 3.47 0.030 2.64 22.23 2.65 20.31 13.21 4.82 e e
<0.005 0.0090 <0.005 0.0072 0.004 0.0025 0.0015 <0.0001 0.2649 0.0126 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 e e
** ** ** * * * ** * ** ** ** ** ** e e
Notes:* indicates significant difference(P < 0.05); ** indicates high significant different(P < 0.01).
Chemical Reagent Factory (Chengdu, China). Polypropylene resin was obtained from Cosmos Pharmaceutical Material Co., Ltd., Lianyungang (Batch number: 20120906). Acetone was purchased from West Long Chemical Co., Ltd., Sichuan. Talcum powder was supplied by Anhui Sunward Pharmaceutical Excipients Co., Ltd.. Standard solution of iron and cobalt were supplied by National Steel Materials Testing Center CISRI (Batch number: 10012632). All other chemicals and reagents were of analytical grade. 2.2. Methods 2.2.1. Preparation of ironecobalt composite microcapsules Microcapsules were prepared by emulsion solvent diffusion method according to the literature [7]. On the basis of encapsulation efficiency (EE) and drug loading (DL), the RSM optimization analysis [8e10] was used to determine the optimum conditions for preparing ironecobalt composite microcapsules loaded with ferric citrate and cobalt chloride. The factors and levels used in the RSM optimization analysis are shown in Table 1 and twenty-nine test runs were conducted in Table 2. Entrapment efficiency and drug loading can be expressed as: EE ¼ Approximate mass of ferric citrateecobalt chloride in microcapsule/Total mass of ironecobalt used to prepare microcapsule 100% DL ¼ Approximate mass of ferric citrateecobalt chloride in microcapsule/Total mass of ironecobalt microcapsule 100% The
preparation
process
of
ironecobalt
composite
microcapsules is briefly described as follows. Firstly, ferric citrateecobalt chloride and polypropylene resin (PR) were dissolved in acetoneemethanol (3:1) mixture solution with constant stirring using a mixing apparatus [11]. Secondly, emulsifiers (appropriate amount of span-80, twain-80 and enough talcum powder was added to the 80 ml of liquid paraffin) were added into the aqueous solution saturated with acetoneemethanol (3:1) mixture solution to form the aqueous phase. Thirdly, the aqueous phase was constantly stirred; meanwhile, the organic phase containing ferric citrateecobalt chloride prepared in the first step was dropwise added into the aqueous phase to develop a homogeneous and stable water-in-oil (W/O) emulsion [12]. After mixing for 30 min, the liquid drops distributed throughout aqueous phase evenly. Finally, superfluous deionized water was loaded to the emulsion to remove the organic solvent, and then the emulsion was continuously stirred up to form microcapsule. The ironecobalt composite microcapsule was obtained after filtration by qualitative filter paper, cleaning by n-hexane and desiccation after drying by constant temperature oven [13,14].
2.2.2. Analysis of microcapsule size and shell morphology Microcapsule surface morphology, shell thickness and the size of microcapsules were determined by scanning electron microscopy (Motic China Group Co., China).
2.2.3. Optimization of preparation conditions (a) Flame atomic absorption spectrometry Determinations of iron and cobalt concentrations in microcapsule were flame atomic absorption spectrometry with atomic absorption spectrophotometer (Beijing Analytical Instrument Co. Ltd., China), which used acetylene gas as fuel gas, used air as assistant gas [15,16]. In order to ensure all data accurate, it is necessary to prepare a certain gradient concentration of standard solutions as a reference to create standard curve [17]. The standard solutions of 1000 mg ml1 iron and cobalt were step-by-step diluted to 100 mg ml1 and 10 mg ml1, respectively, then a series of standard solutions (0.3, 0.5, 1, 3 and 5 mg ml1) were obtained. (b) Recovery and precision test Before digestion treatment, the standard solutions 0.2, 0.6 and 0.8 mg ml1 were respectively added to 10 mg of microcapsules samples [18]. After digestion the contents of iron/cobalt concentration were determined for six times under instrument operating conditions, then relative standard deviation was calculated and standard addition test was carried out for reliability [19]. The recovery results were calculated (Table 4).
Table 4 Results of recovery and precision test. Number
Iron concentration (mg ml1)
Added (mg ml1)
Determined (mg ml1)
Average recovery (%)
RSD (%) (n ¼ 3)
Sample 1 Sample 2 Sample 3
0.3258 0.5056 0.5291
0.20 0.60 0.80
0.5227 1.0936 1.3319
100.6 101.4 99.79
1.86 2.32 1.07
Number Sample 1 Sample 2 Sample 3
Cobalt concentration (mg ml1) 0.3240 0.5036 0.5198
Added (mg ml1) 0.20 0.60 0.80
Determined (mg ml1) 0.5231 1.0932 1.3298
Average recovery (%) 100.7 101.1 99.65
RSD (%) (n ¼ 3) 1.89 2.24 1.05
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Fig. 1. Response surface figure of factors AB, AC, AD, BC, BD and CD on EE.
(c) The pretreatment of the microcapsules samples and determination of iron/cobalt concentration 10 mg microcapsules were weighed accurately in 50 ml of erlenmeyer flask and were washed using hot ultra-pure water for
five times, then the residual microcapsules were dried at 60 C. Afterward, 1 ml of methanol was added to ironecobalt composite microcapsules and the five times volume of acid mixture for sample digestion is a 4:1 mixture of reagent grade nitric and perchloric acids was added with 4e6 glass beads, then parafilm was for seal.
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The system was retained for 24 h at room temperature. Erlenmeyer flask has been heated with electric slow from producing mass brown gas to the solution's becoming clear and yellowish green, until the erlenmeyer flask remained 1 ml colorless liquid, which indicated complete digestion. After cooling, the liquid would be filter and washed with 1% (w/v) of HNO3 solution into 100 ml volumetric flask, moved to 10 ml numbered eppendorf tubes for determination.
dosage) 2and (twain-80 dosage) 2 were highly significant at the level of p < 0.01, while factors of ferric citrate dosage and cobalt chloride dosage, ferric citrate dosage and span-80 dosage, ferric citrate dosage and with twain-80 dosage, span-80 dosage and twain-80 dosage were significant at the level of 0.01 < p < 0.05, others were of no significance. After square regression analysis, a polynomial regression model equation was fitted as follows:
2.2.4. In vitro release studies All samples were studied by the method of Chinese Pharmacopoeia 2010 (for enteric preparations) using 500 ml of phosphate buffer at a constant pH 6.8 and 500 ml of hydrochloric acid solution at a constant pH 1.0 as dissolution medium [20]. The medium were maintained at 37 ± 0.5 C, rotation speed was 75 rpm. Each medium had six parallel samples to determine, respectively in the first 10, 20, 40, 60, 90, 120 min sampled 5 ml of solution (while adding the same volume of fresh synthermal medium) [21]. All solvents were pre-filtered with 0.22 nm microporous membrane and subsequent filtrate was used for determination. The cumulative dissolution percentage and dissolution curve of the average accumulated dissolution were calculated [22].
Y ¼ 89:54 þ 4:57A 2:39B 1:34C 0:48D þ 1:71AB
3. Results and discussions 3.1. Results of RSM design experiment 3.1.1. The establishment of the model and the significance test The results of preliminary experiments had determine the main factors significantly affecting the EE of microcapsules: coreeshell ratio, emulsifier concentration. Based on EE, RSM design experiment of four factors and three levels was obtained (Table 1), the four factors are ferric citrate dosage(A), cobalt chloride dosage(B), Span-80 dosage(C), twain-80 dosage(D). As shown in Table 2, the EE of ironecobalt composite microcapsules ranged from 65.46% to 90.61%, and the DL of ironecobalt composite microcapsules ranged from 12.48% to 45.32%. To determine the optimal condition of EE, the relationship between the response (EE) and the significant variables, ANOVA for response surface quadratic model was performed (Table 3). Among the linear, quadratic, and cross-product forms of independent variables, ferric citrate dosage, cobalt chloride dosage, span-80 dosage, cobalt chloride dosage and span-80 dosage, (ferric citrate dosage) 2, (cobalt chloride dosage) 2, (span-80
þ 2:73AC 3:28AD 4:24BC 0:39BD þ 3:70CD 8:44 A2 2:91 B2 8:06 C2 6:50 D2 The correlation coefficient, R-squared was 0.8283 and adjust Rsquared was 0.8566, showed a highly significant relativity for regression equation. Within the levels of selected factors, the impact order on the response value was ferric citrate dosage > span-80 dosage > twain-80dosage > cobalt chloride dosage. 3.1.2. The RSM results of factors' interaction on EE Through data analysis of Design-Expert, the response surface figures of the interaction between factors on EE were built as shown in Fig. 1, that can observe visually the effect of every factor on EE and the interaction between factors [23]. The response surface figures between ferric citrate dosage and twain-80 dosage appeared to be at its roundest and at the biggest change, which indicated the interaction between factor ferric citrate dosage and twain-80 dosage had the greatest effect on EE. Followed by the interaction between factor ferric citrate dosage and span-80 dosage, next was the interaction between factor span-80 dosage and twain-80dosage. The flatter response surface figures, the less interaction factor was between cobalt chloride dosage and span-80 dosage. 3.2. Surface morphology and size distribution The surface photographs of the ironecobalt composite microcapsules were characterized by binocular microscope (B203LED,10 4) illustrate that under optimum conditions are spherical in shape and have a smooth surface with good liquidity properties (Fig. 2(A)). Fig. 2(B) shoot by light microscope (ZOOM730,10 4)displays light tight microcapsule core material inside the microcapsules but void microcapsules with little microcapsule
Fig. 2. (A) Ironecobalt composite microcapsules shot by binocular microscope (10 4); (B) void microcapsules shot by light microscope (10 4).
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Fig. 3. Iron/Cobalt standard curve.
Fig. 4. The dissolution curve in vitro under pH 1.0 and pH 6.8.
core material are approximately transparent to light. The particles of the microcapsule conformed to a normal distribution with the average diameter of 180 ± 9.54 mm. 3.3. Determination of EE The standard curve between absorbance and iron/cobalt concentration (Fig. 3) shows that linear correlation coefficient and regression equation. The determination results of select three
samples (Table 4) is reliable, displays good accuracy and precision of measurement in line with the conditions of quantitative analysis due to 99.65%~101.4% average recovery with RSD of 1.05%~2.32%. The accuracy test and the recovery test showed that the method of flame atomic absorption spectrometry presented in this study was accurate and reliable. The dissolution curve (Fig. 4) shows the changes of average accumulated dissolution along with time [24]. It turns out that the average accumulated dissolution is lower than 10% in
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dissolution medium of pH 1.0 within 120 min but that is more than 75.00% in dissolution medium of pH 6.8 within 120 min. Therefore IroneCobalt composite microcapsules can achieve the goal of enteric effect and conform to the requirement of the pharmacopoeia. 4. Conclusion Emulsion solvent diffusion method was convenient and feasible to prepare ironecobalt composite microcapsules. In this study, the optimum condition for ironecobalt composite microcapsule was obtained by considering the EE of microcapsule with the use of RSM analysis. The optimum condition was 0.48 g of ferric citrate, 0.20 g of cobalt chloride, 80 ml of liquid paraffin, 0.32 g of talcum powder, 1.8 ml of span-80, 0.2 ml of twain-80. The level of importance based on the RSM analysis was in the order: ferric citrate dosage > span80 dosage > twain-80 dosage > cobalt chloride dosage. The microcapsules prepared under the optimum condition not only had regular surface morphology and suitable particle size but also displayed good controlled release over a long period of time in vitro. The determination results indicated that was successfully microencapsulated with 90.61% of EE, which conforms to enteric preparation standards and provides reliable basis for the further study of trace element formulation and clinical use.
[6]
[7]
[8]
[9] [10]
[11]
[12] [13]
[14]
[15]
[16]
Acknowledgments This work was supported by Science and Technology Support Program of Sichuan Province (Grant No. 2015SZ0201), Special Fund for Agroscientific Research in the Public Interest (Grant No. 201203062) and Cultivated Funds for Academic and Technical Leaders of Sichuan Province (Grant No.03109107).We would like to thank the classmates who done work with me. We also give our sincere thanks to the reviewers.
[17]
[18] [19]
[20]
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Journal of Alloys and Compounds journal homepage: http://www.elsevier.com/locate/jalcom
Review
Preparation and characterization of ironecobalt composite microcapsules Yan Qiao, Yanchun Hu*, Jiahui An, Dongmei Wu, Gang Shu, Hualin Fu, Guangneng Peng, Junliang Deng Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Sichuan Province, Wenjiang 611130, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2015 Received in revised form 25 July 2015 Accepted 26 August 2015 Available online 1 September 2015
Abstract: Iron and cobalt as trace elements can promote the growth and development of the body. In our experiments, they were added as a drug for treatment of animal anemia. Ironecobalt composite microcapsules were prepared by emulsion solvent diffusion method (ESDM) can meet the requirements of the body to trace elements, thus promoting the body's normal hematopoietic function. The optimum condition was investigated by response surface methodology (RSM) in order to improve the bioavailability of oral ironecobalt preparations. By using the analysis of flame atomic absorption spectrophotometry and scanning electronic microscope (SEM), the characteristics of the microcapsules were investigated respectively. The results showed that the optimum preparation conditions for ironecobalt composite microcapsules were: 0.48 g of ferric citrate, 0.25 g cobalt chloride, 0.2 ml of twain-80 and 1.8 ml of span-80. The microcapsules were successfully prepared with 90.61% of encapsulation efficiency (EE) and 45.32% of drug loading (DL). The particles of the microcapsule conformed to a normal distribution with the average diameter of 180 ± 9.54 mm. The average accumulated dissolution is lower than 10.00% in dissolution medium of pH 1.0 within 120 min but that is more than 75.00% in dissolution medium of pH 6.8 within 120 min. © 2015 Elsevier B.V. All rights reserved.
Keywords: Ironecobalt composite microcapsules preparation Response surface methodology (RSM) Encapsulation efficiency (EE) In vitro release studies
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571 2.2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.1. Preparation of ironecobalt composite microcapsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.2. Analysis of microcapsule size and shell morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.3. Optimization of preparation conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 2.2.4. In vitro release studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.1. Results of RSM design experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.1.1. The establishment of the model and the significance test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.1.2. The RSM results of factors' interaction on EE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.2. Surface morphology and size distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 3.3. Determination of EE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
* Corresponding author. E-mail address: [email protected] (Y. Hu). http://dx.doi.org/10.1016/j.jallcom.2015.08.211 0925-8388/© 2015 Elsevier B.V. All rights reserved.
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
1. Introduction Iron and cobalt are essential trace elements in animals, and lack of these elements can result in animal anemia. Iron is essential for almost all living organisms, which not only participate in the metabolism of the body, but also helps regulate the immune function [1]. Studies have shown that iron is involved in liver deoxyribonucleic acid synthesis, promote liver cell growth, promote synthesis of liver cells and other cell mitochondria, reduce energy consumption, and in a certain extent can treat anemia, severe atypical hyperplasia etc.. The sufficient evidence showed that the metabolism, differentiation of lymphocytes and bactericidal effect of neutrophils need to have enough iron [2]. Cobalt has hematopoietic function, cobalt deficiency may easily lead to a series of diseases such as malignant anemia and nerve degeneration [3]. Cobalt can promote gastrointestinal absorption of iron, and using combined with iron is obvious than adding cobalt alone on the effect of treatment for anemia. On the other hand, cobalt poisoning can be caused by a regular injection of cobalt in cobalt or exposure to an excess of the original cobalt environment. Therefore, we must strictly control the amount of cobalt. In ferric citrate and cobalt chloride, they are present as trivalent iron and two valence cobalt. This ion is relatively stable and easy to absorb, therefore enhance ironecobalt microcapsules' stability. There has been a growing interest in research, development and commercialization of trace elements over the past decade [4]. So the purpose of this study is to develop the composite microparticle of ferric citrate and cobalt chloride so as to prevent and cure anemia of animals. Iron and cobalt increasingly complex properties from food ingredients and such complex properties can oftentimes only be provided by microencapsulation [5]. Early study showed that microcapsules can not only cover the odor and taste of volatile materials but also improve the stability of volatile drugs [6]. Emulsion solvent diffusion method, also called drying in liquid, is a kind of microencapsulation technology. In the process of this method, the organic solvents that dissolve capsule materials and core of capsules are diffused, separated and condensed to form microcapsule. The method satisfies the safety requirements in drug and food industry, and it is able to control the factors that affect the properties of microcapsules. With carefully finetuned controlled release properties, microencapsulation is not just an added value, but is also the source of totally new ingredients with matchless properties. Recently, the most common coating materials used for microcapsules are arabic gum, poly lactose and the composite of poly (ethylene glycol)-poly (lactic acid) copolymers (PLA-PEG), etc. Since
Table 1 The four factors and three levels of the RSM optimization analysis experiments Code
Factors
Levels 1
0
1
A B C D
ferric citrate dosage (g) cobalt chloride dosage (g) Span80 dosage (ml) twain-80dosage (ml)
0.38 0.15 0.9 0.1
0.48 0.20 1.8 0.2
0.58 0.25 2.7 0.3
polypropylene resin has stable chemical properties, good bioavailability and is easy to form membrane, it is always used as the capsule shell material in the preparation of microcapsule. In the present study, polypropylene resin was chosen as capsule shell material to prepare the ironecobalt composite microcapsules because of it will not be dissolved in pH1. Intestinal pH is acidic environment which close to 1, polypropylene resin's function of avoid drugs to dissolution release in pH1, effectively enhances the stability of the drug in the intestinal tract, play good role of target orientation, improve the biological utilization rate, enhance the oral bioavailability. The factors, such as the ratio of coating material mass (or the concentration of emulsion) to core material volume, the volume ratio of organic phase to aqueous phase, stirring speed, time and water amount, which influence the DL (the ratio of the amount of encapsulated into microcapsules and the weight of the microcapsules) and EE (the ratio of package into dosage and dosage of microcapsule) of microcapsules, were investigated. Different core materials and microencapsulation methods will cause different optimum conditions. Thus the preparation conditions of ironecobalt composite microcapsules by ESDM were optimized by orthogonal assay in the present study. 2. Materials and methods 2.1. Materials Ferric citrate, cobalt chloride, liquid paraffin, methanol, twain80, span-80 and n-hexane were purchased from Chengdu Kelong Table 2 BBD experiment design and results Serial number
A (g)
B (g)
C (ml)
D (ml)
EE (%)
DL (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
0.58 0.58 0.48 0.48 0.48 0.48 0.48 0.38 0.58 0.38 0.38 0.48 0.58 0.48 0.48 0.48 0.48 0.48 0.38 0.48 0.58 0.48 0.48 0.48 0.48 0.58 0.38 0.38 0.48
0.20 0.15 0.25 0.15 0.20 0.20 0.15 0.20 0.20 0.15 0.25 0.20 0.20 0.25 0.20 0.20 0.20 0.25 0.20 0.15 0.25 0.20 0.15 0.20 0.25 0.20 0.20 0.20 0.20
1.80 1.80 0.90 1.80 2.70 1.80 0.90 1.80 0.90 1.80 1.80 2.70 1.80 1.80 1.80 1.80 0.90 1.80 0.90 2.70 1.80 1.80 1.80 1.80 2.70 2.70 1.80 2.70 0.90
0.10 0.20 0.20 0.10 0.10 0.20 0.20 0.30 0.20 0.20 0.20 0.30 0.30 0.30 0.20 0.20 0.10 0.10 0.20 0.20 0.20 0.20 0.30 0.20 0.20 0.20 0.10 0.20 0.30
89.19 83.87 83.47 82.40 65.46 90.61 81.03 71.67 70.51 75.25 65.57 77.10 80.40 75.46 90.61 90.61 76.72 81.14 75.88 87.25 81.03 90.61 78.30 90.61 72.72 74.06 67.35 68.51 73.56
39.52 27.31 27.06 25.14 13.54 45.32 23.41 16.46 15.13 19.37 12.48 20.06 22.31 18.49 45.32 45.32 19.51 23.16 17.58 36.69 22.03 45.32 18.64 45.32 16.93 18.75 13.62 14.96 17.12
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Table 3 ANOVA for response surface quadratic model. Source
Sum of squares
df
Mean squares
F Value
P-value (Prob > F)
Significance
A B C D AB AC AD BC BD CD A2 B2 C2 D2 Model Residual Total
250.53 68.69 21.52 2.77 11.70 29.81 42.97 72.00 0.62 54.76 461.61 54.96 421.74 274.34 1402.61 290.75 1693.36
1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 14 28
250.53 68.69 21.52 2.77 11.70 29.81 42.97 72.00 0.62 54.76 461.61 54.96 421.74 274.34 100.19 20.77 e
12.06 3.31 1.04 0.13 0.56 1.44 2.07 3.47 0.030 2.64 22.23 2.65 20.31 13.21 4.82 e e
<0.005 0.0090 <0.005 0.0072 0.004 0.0025 0.0015 <0.0001 0.2649 0.0126 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 e e
** ** ** * * * ** * ** ** ** ** ** e e
Notes:* indicates significant difference(P < 0.05); ** indicates high significant different(P < 0.01).
Chemical Reagent Factory (Chengdu, China). Polypropylene resin was obtained from Cosmos Pharmaceutical Material Co., Ltd., Lianyungang (Batch number: 20120906). Acetone was purchased from West Long Chemical Co., Ltd., Sichuan. Talcum powder was supplied by Anhui Sunward Pharmaceutical Excipients Co., Ltd.. Standard solution of iron and cobalt were supplied by National Steel Materials Testing Center CISRI (Batch number: 10012632). All other chemicals and reagents were of analytical grade. 2.2. Methods 2.2.1. Preparation of ironecobalt composite microcapsules Microcapsules were prepared by emulsion solvent diffusion method according to the literature [7]. On the basis of encapsulation efficiency (EE) and drug loading (DL), the RSM optimization analysis [8e10] was used to determine the optimum conditions for preparing ironecobalt composite microcapsules loaded with ferric citrate and cobalt chloride. The factors and levels used in the RSM optimization analysis are shown in Table 1 and twenty-nine test runs were conducted in Table 2. Entrapment efficiency and drug loading can be expressed as: EE ¼ Approximate mass of ferric citrateecobalt chloride in microcapsule/Total mass of ironecobalt used to prepare microcapsule 100% DL ¼ Approximate mass of ferric citrateecobalt chloride in microcapsule/Total mass of ironecobalt microcapsule 100% The
preparation
process
of
ironecobalt
composite
microcapsules is briefly described as follows. Firstly, ferric citrateecobalt chloride and polypropylene resin (PR) were dissolved in acetoneemethanol (3:1) mixture solution with constant stirring using a mixing apparatus [11]. Secondly, emulsifiers (appropriate amount of span-80, twain-80 and enough talcum powder was added to the 80 ml of liquid paraffin) were added into the aqueous solution saturated with acetoneemethanol (3:1) mixture solution to form the aqueous phase. Thirdly, the aqueous phase was constantly stirred; meanwhile, the organic phase containing ferric citrateecobalt chloride prepared in the first step was dropwise added into the aqueous phase to develop a homogeneous and stable water-in-oil (W/O) emulsion [12]. After mixing for 30 min, the liquid drops distributed throughout aqueous phase evenly. Finally, superfluous deionized water was loaded to the emulsion to remove the organic solvent, and then the emulsion was continuously stirred up to form microcapsule. The ironecobalt composite microcapsule was obtained after filtration by qualitative filter paper, cleaning by n-hexane and desiccation after drying by constant temperature oven [13,14].
2.2.2. Analysis of microcapsule size and shell morphology Microcapsule surface morphology, shell thickness and the size of microcapsules were determined by scanning electron microscopy (Motic China Group Co., China).
2.2.3. Optimization of preparation conditions (a) Flame atomic absorption spectrometry Determinations of iron and cobalt concentrations in microcapsule were flame atomic absorption spectrometry with atomic absorption spectrophotometer (Beijing Analytical Instrument Co. Ltd., China), which used acetylene gas as fuel gas, used air as assistant gas [15,16]. In order to ensure all data accurate, it is necessary to prepare a certain gradient concentration of standard solutions as a reference to create standard curve [17]. The standard solutions of 1000 mg ml1 iron and cobalt were step-by-step diluted to 100 mg ml1 and 10 mg ml1, respectively, then a series of standard solutions (0.3, 0.5, 1, 3 and 5 mg ml1) were obtained. (b) Recovery and precision test Before digestion treatment, the standard solutions 0.2, 0.6 and 0.8 mg ml1 were respectively added to 10 mg of microcapsules samples [18]. After digestion the contents of iron/cobalt concentration were determined for six times under instrument operating conditions, then relative standard deviation was calculated and standard addition test was carried out for reliability [19]. The recovery results were calculated (Table 4).
Table 4 Results of recovery and precision test. Number
Iron concentration (mg ml1)
Added (mg ml1)
Determined (mg ml1)
Average recovery (%)
RSD (%) (n ¼ 3)
Sample 1 Sample 2 Sample 3
0.3258 0.5056 0.5291
0.20 0.60 0.80
0.5227 1.0936 1.3319
100.6 101.4 99.79
1.86 2.32 1.07
Number Sample 1 Sample 2 Sample 3
Cobalt concentration (mg ml1) 0.3240 0.5036 0.5198
Added (mg ml1) 0.20 0.60 0.80
Determined (mg ml1) 0.5231 1.0932 1.3298
Average recovery (%) 100.7 101.1 99.65
RSD (%) (n ¼ 3) 1.89 2.24 1.05
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Fig. 1. Response surface figure of factors AB, AC, AD, BC, BD and CD on EE.
(c) The pretreatment of the microcapsules samples and determination of iron/cobalt concentration 10 mg microcapsules were weighed accurately in 50 ml of erlenmeyer flask and were washed using hot ultra-pure water for
five times, then the residual microcapsules were dried at 60 C. Afterward, 1 ml of methanol was added to ironecobalt composite microcapsules and the five times volume of acid mixture for sample digestion is a 4:1 mixture of reagent grade nitric and perchloric acids was added with 4e6 glass beads, then parafilm was for seal.
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The system was retained for 24 h at room temperature. Erlenmeyer flask has been heated with electric slow from producing mass brown gas to the solution's becoming clear and yellowish green, until the erlenmeyer flask remained 1 ml colorless liquid, which indicated complete digestion. After cooling, the liquid would be filter and washed with 1% (w/v) of HNO3 solution into 100 ml volumetric flask, moved to 10 ml numbered eppendorf tubes for determination.
dosage) 2and (twain-80 dosage) 2 were highly significant at the level of p < 0.01, while factors of ferric citrate dosage and cobalt chloride dosage, ferric citrate dosage and span-80 dosage, ferric citrate dosage and with twain-80 dosage, span-80 dosage and twain-80 dosage were significant at the level of 0.01 < p < 0.05, others were of no significance. After square regression analysis, a polynomial regression model equation was fitted as follows:
2.2.4. In vitro release studies All samples were studied by the method of Chinese Pharmacopoeia 2010 (for enteric preparations) using 500 ml of phosphate buffer at a constant pH 6.8 and 500 ml of hydrochloric acid solution at a constant pH 1.0 as dissolution medium [20]. The medium were maintained at 37 ± 0.5 C, rotation speed was 75 rpm. Each medium had six parallel samples to determine, respectively in the first 10, 20, 40, 60, 90, 120 min sampled 5 ml of solution (while adding the same volume of fresh synthermal medium) [21]. All solvents were pre-filtered with 0.22 nm microporous membrane and subsequent filtrate was used for determination. The cumulative dissolution percentage and dissolution curve of the average accumulated dissolution were calculated [22].
Y ¼ 89:54 þ 4:57A 2:39B 1:34C 0:48D þ 1:71AB
3. Results and discussions 3.1. Results of RSM design experiment 3.1.1. The establishment of the model and the significance test The results of preliminary experiments had determine the main factors significantly affecting the EE of microcapsules: coreeshell ratio, emulsifier concentration. Based on EE, RSM design experiment of four factors and three levels was obtained (Table 1), the four factors are ferric citrate dosage(A), cobalt chloride dosage(B), Span-80 dosage(C), twain-80 dosage(D). As shown in Table 2, the EE of ironecobalt composite microcapsules ranged from 65.46% to 90.61%, and the DL of ironecobalt composite microcapsules ranged from 12.48% to 45.32%. To determine the optimal condition of EE, the relationship between the response (EE) and the significant variables, ANOVA for response surface quadratic model was performed (Table 3). Among the linear, quadratic, and cross-product forms of independent variables, ferric citrate dosage, cobalt chloride dosage, span-80 dosage, cobalt chloride dosage and span-80 dosage, (ferric citrate dosage) 2, (cobalt chloride dosage) 2, (span-80
þ 2:73AC 3:28AD 4:24BC 0:39BD þ 3:70CD 8:44 A2 2:91 B2 8:06 C2 6:50 D2 The correlation coefficient, R-squared was 0.8283 and adjust Rsquared was 0.8566, showed a highly significant relativity for regression equation. Within the levels of selected factors, the impact order on the response value was ferric citrate dosage > span-80 dosage > twain-80dosage > cobalt chloride dosage. 3.1.2. The RSM results of factors' interaction on EE Through data analysis of Design-Expert, the response surface figures of the interaction between factors on EE were built as shown in Fig. 1, that can observe visually the effect of every factor on EE and the interaction between factors [23]. The response surface figures between ferric citrate dosage and twain-80 dosage appeared to be at its roundest and at the biggest change, which indicated the interaction between factor ferric citrate dosage and twain-80 dosage had the greatest effect on EE. Followed by the interaction between factor ferric citrate dosage and span-80 dosage, next was the interaction between factor span-80 dosage and twain-80dosage. The flatter response surface figures, the less interaction factor was between cobalt chloride dosage and span-80 dosage. 3.2. Surface morphology and size distribution The surface photographs of the ironecobalt composite microcapsules were characterized by binocular microscope (B203LED,10 4) illustrate that under optimum conditions are spherical in shape and have a smooth surface with good liquidity properties (Fig. 2(A)). Fig. 2(B) shoot by light microscope (ZOOM730,10 4)displays light tight microcapsule core material inside the microcapsules but void microcapsules with little microcapsule
Fig. 2. (A) Ironecobalt composite microcapsules shot by binocular microscope (10 4); (B) void microcapsules shot by light microscope (10 4).
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Fig. 3. Iron/Cobalt standard curve.
Fig. 4. The dissolution curve in vitro under pH 1.0 and pH 6.8.
core material are approximately transparent to light. The particles of the microcapsule conformed to a normal distribution with the average diameter of 180 ± 9.54 mm. 3.3. Determination of EE The standard curve between absorbance and iron/cobalt concentration (Fig. 3) shows that linear correlation coefficient and regression equation. The determination results of select three
samples (Table 4) is reliable, displays good accuracy and precision of measurement in line with the conditions of quantitative analysis due to 99.65%~101.4% average recovery with RSD of 1.05%~2.32%. The accuracy test and the recovery test showed that the method of flame atomic absorption spectrometry presented in this study was accurate and reliable. The dissolution curve (Fig. 4) shows the changes of average accumulated dissolution along with time [24]. It turns out that the average accumulated dissolution is lower than 10% in
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dissolution medium of pH 1.0 within 120 min but that is more than 75.00% in dissolution medium of pH 6.8 within 120 min. Therefore IroneCobalt composite microcapsules can achieve the goal of enteric effect and conform to the requirement of the pharmacopoeia. 4. Conclusion Emulsion solvent diffusion method was convenient and feasible to prepare ironecobalt composite microcapsules. In this study, the optimum condition for ironecobalt composite microcapsule was obtained by considering the EE of microcapsule with the use of RSM analysis. The optimum condition was 0.48 g of ferric citrate, 0.20 g of cobalt chloride, 80 ml of liquid paraffin, 0.32 g of talcum powder, 1.8 ml of span-80, 0.2 ml of twain-80. The level of importance based on the RSM analysis was in the order: ferric citrate dosage > span80 dosage > twain-80 dosage > cobalt chloride dosage. The microcapsules prepared under the optimum condition not only had regular surface morphology and suitable particle size but also displayed good controlled release over a long period of time in vitro. The determination results indicated that was successfully microencapsulated with 90.61% of EE, which conforms to enteric preparation standards and provides reliable basis for the further study of trace element formulation and clinical use.
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Acknowledgments This work was supported by Science and Technology Support Program of Sichuan Province (Grant No. 2015SZ0201), Special Fund for Agroscientific Research in the Public Interest (Grant No. 201203062) and Cultivated Funds for Academic and Technical Leaders of Sichuan Province (Grant No.03109107).We would like to thank the classmates who done work with me. We also give our sincere thanks to the reviewers.
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