Abstract
BACKGROUND:Most of the natural chitosan are shaped as powder and have small specific surface area. It is difficult to reclaim both vector and adsorbate by chitosan, thus limiting its application as an adsorbing vector.
OBJECTIVE: To analyze the optimal preparation technology of porous chitosan microspheres.
DESIGN, TIME AND SETTING: Repeated-measures experiment was carried out in the laboratory of Applied Chemistry, Shaanxi University of Technology from November 2006 to May 2007.
MATERIALS: Chitosan with a deacetylation degree of 95.62%.
METHODS: The reverse liquid phase suspension polymerization method was adopted in this study using cyclohexane as porogenic agent and glutaraldehyde as crosslinking agent. Cyclohexane and cutaraldehyde were added continuously, with the alterations of reaction temperature, stirring speed and extraction time.
MAIN OUTCOME MEASURES: The influence on the preparation of porous chitosan microspheres by the dosage of cyclohexane and glutaraldehyde, reaction temperature, stirring speed and extraction time was observed.
RESULTS: The optimal conditions of preparing porous chitosan microspheres, in which 90% particle diameters were 3-12 μm, included: reaction temperature 60-70 ℃, medium stirring speed 100-120 r/min, extraction time no less than 48 hours.
CONCLUSION: The preparation method used in this study has short time-consuming, the prepared chitosan microspheres are characteristic as micron-grade diameter, narrow distributing particle size and porosity.





INTRODUCTION

Chitosan is the only basic polysaccharide in nature. It is greatly dominant to be used as a drug carrier due to the non-toxicity[1]. Chitosan with good biocompatibility and biodegradation has widely application in many fields, such as medicine, drug, environmental protection, light industry and agriculture[2-3]. Most of the natural chitosan are shaped as powder and have small specific surface areas. It is difficult to reclaim both vector and adsorbate by chitosan. Currently, the preparation methods of chitosan include crosslinking method[4], emulsion-crosslinking method[5-6], and O/W/O multiple emulsification[7], etc., but few reports are observed in terms of porous chitosan microspheres.
Porogenic agent can yield to the isoporous and macroporous structure of adsorption resin[8]. Therefore, porogenic agent plays an important role in the preparation of chitosan microspheres. Generally speaking, porogenic agent should be inert, such as chloroform, acetonitrile, methyl benzene, dimethyl benzene, and cyclohexane (CHA)[8]. In this study, the suspension cross-linking method was adopted by using CHA as porogenic agent, liquid paraffin as dispersed medium and glutaraldehyde (GA) as crosslinking agent. The prepared nano-grade chitosan microspheres serve as a good carrier for the adsorbates, due to the large surface area, strong absorbability, good microsphere performance, monodisperse and narrow distribution. This study was designed to choose the optimal conditions for the preparation of porous chitosan microspheres.

METERIALS AND METHODS

Materials
The experiment was carried out in the laboratory of Applied Chemistry, Shaanxi University of Technology from November 2006 to May 2007. Experimental materials were all analytical pure, including chitosan (95.62% deacetylation degree), liquid paraffin, CHA, acetic ether, GA, acetic acid, petroleum benzine (DAB-6) and alcohol. Experimental instruments was consisted of DZKW-4 thermostatic water bath oscillator (Huanghua Bohai Electrical Appliance Factory), SHZ-3 vacuum drawing and filtering pump of water circling (Gongyi Instrument Factory, Henan), and Axioskop 40 biological microscope (Shanghai Precision Instruments Co., Ltd.).

Methods
Preparations of chitosan microsphere and porous microsphere
Chitosan (2.5 g) was dissolved in 100 mL acetic acid solution at the mass fraction of 0.02, and vacuum pump was used for the air bubble exhaustion 2 hours later. Liquid paraffin (50 mL), CHA, acetic ether (5 mL) and few span80 were added into a 250-mL conical flask by turns, which was equipped with the agitator and thermometer. After 0.5 hours of mixture, the above-mentioned chitosan solution was added into the mixture, and was heated to 55 ℃, stirring for one hour. Then constant pressure funnel was used for the dropwise of GA at 0.5 mass fraction; the pH value was adjusted as 9 by means of 1 mol/L NaOH solution, after 1.5 hours of the reaction, the microspheres were filtered and rinsed with distilled water. DAB-6 of 150 mL was added into the round
bottom flask, and the extraction was performed with Soxhlet's extractor, then DAB-6 was substituted by dehydrated alcohol at 0.95 volume fraction, followed by the same extraction to prepare chitosan microspheres after drying at vacuum.

Preparation of porous chitosan microspheres
Adopting the identical method as above, CHA and GA were added continuously, with the alterations of the reaction temperature (T/℃), stirring speed and extraction time (t/h). The influence of these factors on the preparation of porous chitosan microspheres was observed (Table 1).

Characteristics of chitosan microspheres and porous chitosan microspheres
The prepared chitosan microspheres and porous chitosan microspheres were sieved to detect the distribution of particle diameter; their particle size distribution and hole character were assayed under Axioskop 40 biological microscope (5×10)[9-12].

RESULTS

Structure of chitosan microspheres and porous chitosan microspheres
The prepared chitosan microspheres and porous chitosan microspheres were sieve into a particle size of 160 mesh, accounting for over 80% of total microspheres. Determined by Axioskop 40 biological microscope (5×10), 90% of chitosan microspheres and porous chitosan microspheres had even distribution of particle diameter, ranged 3-12 μm, and these microspheres were colored as marron.

Influence of reaction temperature on the preparation of chitosan microspheres
The result of this experiment showed that, the reaction temperature should be controlled as 60-70 ℃ (Figure 1a); a great quantify of CHA would volatilize when the temperature exceeded 80 ℃ (CHA boiling point is 81 ℃), leading to the ineffective hole formation; The reaction should not be completed below 60 ℃, and the microsphere and hole formations were not satisfactory (Figure 1b).

Influence of stirring speed on the preparation of chitosan microspheres
Stirring speed is extremely important for the formation of chitosan microspheres and the size of particle diameter. The microsphere formation was satisfactory and the distribution was even at the medium speed (100-120 r/min) (Figure 2a). However, these outcomes were converse at a low speed (< 80 r/min) (Figure 2b); the microsphere formation was good, but the distribution was severely uneven at a high speed (> 150 r/min).

Influence of extraction time on the preparation of chitosan microspheres
Excellent dispersibility was observed in the porous chitosan microspheres, which were extracted with DAB-6 and dehydrated alcohol at 0.95 volume fraction respectively, each for no less than 24 hours and totally for no less than 48 hours (Figure 3a). The dispersibility would be changed greatly if the extraction time was shortened to a half (Figure 3b).

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Influence of porogenic agent CHA on the preparation of porous chitosan microspheres
With 10 mL CHA as porogenic agent, the polymerization proceeded successfully at 60 ℃ for 2 hours, but the obtained porous chitosan microspheres were not satisfactory due to the larger size (Figure 4a); and 10 mL CHA would volatilize at 80 ℃, indicating no hole formation (Figure 4b); With 15 mL CHA, the polymerization was completed successfully at 60 ℃ for 2 hours, and the obtained porous chitosan microspheres were small and well-distributed (Figure 3a), so CHA dosage was defined as 15 mL.

Influence of cross-linking agent GA on the preparation of porous chitosan microspheres
The dosage of GA played an important role in the microsphere formation of porous chitosan microspheres. Constantly, polymerization was conducted at 60 ℃ for 2 hours to prepare the microspheres. With 2 mL GA, the moderate color and even particle diameter were observed (Figure 5a); With 3 mL GA, the microspheres were darkly colored, which was unbeneficial for the observation (Figure 5b).

 
DISCUSSION

By means of reverse liquid phase suspension polymerization method, porous chitosan microspheres should be prepared at 60-70 ℃ and medium stirring speed, showing good microsphere formation. Stirring speed is extremely important for the formation and diameter of chitosan microspheres. The microspheres form well and distribute well at a medium speed. Over 48 hours of total extraction time may give rise to the excellent dispersibility of porous chitosan microspheres. As for the choice of porogenic agent, the boiling point of toxic methyl benzene is 111 ℃, whereas that of CHA is only 81 ℃, which is helpful for the drying. Therefore CHA is adopted as porogenic agent. The result of this experiment reveals that satisfactory outcome of porous chitosan microspheres will be achieved with 15 mL CHA after polymerization reaction was completed at 60 ℃ for 2 hours. Porous chitosan microspheres show suitable color and well distribution when prepared with 2 mL GA after the polymerization at 60 ℃ for 2 hours.

REFERENCES

1 Jiang TD. Chitosan. Beijing: Huaxue Gongye Chubanshe 2001:256-258
2 Li XB, Zhu H. Research on Preparation Methods of Chitosan Microspheres. Yaoxue Jinzhan 2005;29(4):166-169
3 Ding SM, Feng XH, Wang YT, et al. Equilibrium and Kinetic Analysis of Adsorption for Dyestuff by Cross-linked Chitosan Porous Microbeads. Fenxi Kexue Xuebao 2005;21(2):127-130
4 Ren DW, Yi HF, Bao DC, et al. A novel method to prepare porous chitosan membrane in situ pore formation by selective enzymatic degradation. Gongneng Cailiao 2006;37(3):462-464
5 Zeng MF, Sun XD, Yao XD, et al. Progress in preparation techniques of chitosan-base porous membrane. Gaofenzi Cailiao yu Gongcheng 2005;21(2):52-55
6 Guo JM, Xu XJ, Li L. Preparation and application of the chitin and chitosan. Huagong Huanbao 2004;24(4):126-128
7 Ma XL, Yao ZH, Xu WW, et al. Preparation and Characterization of Biomedical Porous Chitosan Membranes. Gongneng Gaofenzi Xuebao 2005;18(3):434-440
8 Xu XX, Xia LZ, Gao JR. Application of macroporous absorption resin in the separation and purification of asterosaponins. Zhongguo Zhongyiyao Xinxi Zazhi 2003;10(1):79-80
9 Bai C, Fang Y, Zhang Y, et al. Synthesis of novel metal sulfide-polymer composite microspheres exhibiting patterned surface structures. Langmuir 2004;20(1):263-265
10 Fang Y, Bai C, Zhang Y. Preparation of metal sulfide-polymer composite microspheres with patterned surface structures. Chem Commun (Camb) 2004;(7):804-805
11 Zhang Y, Fang Y, Wang S, et al. Preparation of spherical nanostructured poly(methacrylic acid)/PbS composites by a microgel template method. J Colloid Interface Sci 2004;272(2):321-325
12 Majeti NV, Ravi K. A review of chitin and chitosan applications. React Funct Polym 2000;46(1):1-27

多孔壳聚糖微球的最佳制备条件*★

李志洲
陕西理工学院化学与环境科学学院，陕西省汉中市 723000
李志洲★，男，1969年生，陕西省汉中市人，汉族，陕西科技大学在读硕士，副教授，主要从事化工工艺开发与应用的研究。
陕西理工学院科研资助项目（SLGQD0605）*
摘要
背景：天然状态的壳聚糖大部分为粉末状，比表面积小，作为吸附载体，使载体和吸附物都难以回收，从而限制其应用。
目的：分析制备多孔壳聚糖微球的最佳条件。
设计、时间及地点：重复测量设计，于2006-11/2007-05 在陕西理工学院应用化学实验室完成。
材料：壳聚糖脱乙酰度为95.62%。
方法：以环己烷作致孔剂，戊二醛作交联剂，采用反液相悬浮交联法，改变加入环己烷和戊二醛用量，调节反应温度，改变搅拌速度和提取时间。
主要观察指标：观察反应温度、提取时间、搅拌速度、环己烷用量和戊二醛用量对制备多孔壳聚糖微球的影响。
结果：多孔壳聚糖微球最佳制备条件为:添加10 mL环己烷，戊二醛2 mL，反应温度60~70 ℃，搅拌速度中速（100~120 r/min）时，提取时间不小于48 h，可得到90%粒径大小为3~12 μm的多孔壳聚糖微球。
结论：此制备方法具有耗时短，制备的壳聚糖微球粒径小、粒度分布窄、多孔性等特点。
关键词：多孔壳聚糖；微球；制备；生物材料
中图分类号: R318.08 文献标识码: B 文章编号: 1673-8225(2008)14-02770-03
李志洲.多孔壳聚糖微球的最佳制备条件[J].中国组织工程研究与临床康复，2008，12(14):2770-2772
[www.zglckf.com/zglckf/ejournal/upfiles/08-14/14k-2770(ps).pdf]
(Edited by: Nie J/Yang Y/Wang L)