壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原的制备及体外释放性能

doi:10.3969/j.issn.2095-4344.2015.21.010

摘要/Abstract

摘要：

背景：包裹幽门螺杆菌全菌蛋白抗原的研究仍处于探索阶段，有关壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原的制备工艺及体外释放性能的文献甚少。
目的：探讨幽门螺杆菌全菌蛋白抗原壳聚糖微球的制备工艺及体外释放特性。
方法：采用沉淀法制备壳聚糖微球，筛选最佳制备工艺及配比、包裹时间，并在电镜下观察微球的形态和粒径。采用壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原，BCA法测定幽门螺杆菌全菌蛋白抗原微球的包裹率、包裹量及体外释放率。
结果与结论：终体积分数为1%的冰醋酸、硫酸钠为交联剂、pH 5.0、滴加交联剂时不粉碎处理为壳聚糖微球最佳制备工艺，电镜观察显示微球表面光滑、形态圆整，具有良好的分散性，多数微球粒径为1.0-5.0 μm。幽门螺杆菌全菌蛋白抗原微球的包裹率为80.4%，包裹量为16.4%，48 h总释放率为19.4%，幽门螺杆菌全菌蛋白抗原微球整体呈缓慢释放状态。结果证实，实验制备的壳聚糖微球对幽门螺样菌全菌蛋白抗原具有良好的包裹率和包裹量，幽门螺杆菌全菌蛋白抗原微球整体呈缓慢释放状态。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 缓释材料, 壳聚糖, 微球, 幽门螺杆菌, 体外释放

Abstract:

BACKGROUND: Studies on encapsulated whole cell protein antigen of Helicobacter pylori are still at the exploration stage. There is limited literature concerning the preparation process and in vitro release characteristics of chitosan microspheres encapsulated with whole cell protein antigen of Helicobacter pylori.
OBJECTIVE: To explore the preparation process and in vitro release characteristics of chitosan microspheres encapsulating whole cell protein antigen of Helicobacter pylori.
METHODS: Precipitation method was used to prepare chitosan microspheres, and the best preparation process, matching and encapsulation time were screened. Under electron microscope, the morphology and particle size of microspheres were observed. Chitosan microspheres were used to encapsulate Helicobacter pylori whole cell protein antigen, and BCA method was used to determine encapsulation efficiency, encapsulation content and release efficiency in vitro of Helicobacter pylori whole cell protein antigen.
RESULTS AND CONCLUSION: Final concentration of 1% glacial acetic acid, sodium sulfate as crosslinking agent, pH=5.0, with no pulverization when the crosslinking agent was added was the best preparation process for chitosan microspheres. Electron microscopy showed the smooth surface morphology of microspheres with roundness and good dispersion, and the majority of the microspheres were 1-5 μm in diameter. The encapsulation efficiency of Helicobacter pylori whole cell protein antigen microspheres was 80.4%, the encapsulated amount was 16.4%, and total 48-hour release rate was 19.4%. Helicobacter pylori whole cell protein antigen microspheres showed an overall slow release status. Chitosan microspheres show good encapsulation efficiency and amount of Helicobacter pylori whole cell protein antigen, and Helicobacter pylori total bacteria protein antigen microspheres show an overall slow release status.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Tissue Engineering, Biocompatible Materials, Chitosan, Vaccines

中图分类号:

R318

李娟，赵清喜. 壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原的制备及体外释放性能[J]. 中国组织工程研究, 2015, 19(21): 3334-3338.

Li Juan, Zhao Qing-xi. Chitosan microspheres loading whole cell protein antigen of Helicobacter pylori: preparation and in vitro release characteristics[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(21): 3334-3338.

图/表（结果） 2

参考文献

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引言

材料方法

MATERIALS AND METHODS
Design
Materiology experiments.

Time and setting
Experiments were completed at the laboratory
of the Medical College of Qingdao University from September 2012 to January 2015.

Materials

Reagents and instruments for preparation of chitosan microspheres loading whole cell protein antigen of Hp:
Instrument and reagent	Source
Chitosan	Weifang Haizhiyuan Biological Products Co., Ltd.
Hp standard strain	American Type Culture Collection (ATCC), USA
BCA method protein content detecting kit	Nanjing Kaiji Biological Technology Development Co., Ltd.
Sterility anticoagulants sheep whole blood	Nanjing Senjiabei Biological Technology Co., Ltd.
Campylobacter jejuni agar	Shanghai Yansheng Biochemical Regent Co., Ltd.
Tri-gas incubator	Tianjin Fuma Science and Technology Co., Ltd.
High speed centrifuge	Beckman, USA
S4800 scanning electron microscopy	Hitachi, Japan
Melvin particle size analyzer	MasterSizer 2000, England
Magnetic stirrers	Shanghai Renhe Scientific Instrument Co., Ltd.
Microplate reader	Bio-rad680, America

Methods
Hp culture method
Hp standard strain was inoculated on campylobacter jejuni agar containing sheep whole blood. Culture condition: 5% O2, 10% CO2, 85% N2 and 37 ℃. After culturing for 2 days, the cultured bacteria were qualified, washed-out, and the absorbance value at 660 nm was determined.

Hp whole cell protein antigen preparation
The cultured Hp standard strain was eluted with PBS (0.01 mol/L) for three times, followed by smashed with ultrasound and centrifuged (4 ℃, 30 minutes). The supernatant was collected and the protein content was investigated with BCA method and saved in a-20 ℃ freezer.

Preparation optimization of chitosan microspheres
Chitosan was dissolved in glacial acetic acid, and the Tween-80 (1%) as dispersing agent, sodium sulfate or sodium tripolyphosphate as cross-linking agent was added to prepare chitosan microsphere precipitate. In this experiment, 1% or 2% glacial acetic acid, sodium sulfate or sodium tripolyphosphate was used as cross-linking agent. The detailed 16 kinds of preparation schemes are listed in Table 1. The prepared microspheres were observed using scanning electron microscopy.

Preparation of chitosan microspheres after optimization
The optimum preparation conditions were confirmed according to scanning electron microscopy results. 1.5 mg chitosan was dissolved in 500 mL distilled water (at a concentration of 3%), which was stirred using magnetic stirrers (500 r/min), and 3 mL of 1% glacial acetic acid was added. When chitosan was totally dissolved, 5 mL Tween-80 (1%) was added to adjust pH to 5.0 and 2% sodium sulfate was added drop by drop until the precipitate occurred, and the stirred time was 2 hours. The resulted product was centrifuged (2 500 r/min×10 minutes) for three times and the precipitate was collected, freeze-dried, and save at 4 ℃.

Table 1 Preparation optimization of chitosan microspheres

Factor	Level
Glacial acetic acid	1%	2%
Cross-linking agent	sodium sulfate	sodium tripolyphosphate
pH	5.0	5.5
Smashed with ultrasound	Yes	No

Optimization of microsphere optimum ratio and encapsulating time
Hp whole cell protein antigen and chitosan microspheres at fixed ratio was dissolved in acetate buffer (10 mL, 0.1 mol/L, pH 6.0) and stirred for 24 hours at room temperature. The encapsulation efficiency of resulted products was determined and the optimum ratio of Hp whole cell protein antigen to chitosan microspheres was obtained (Table 2). The optimum encapsulating time was investigated by determining the encapsulation efficiency at 1, 2, 3, 6, 9, 12, 15, 18, 21, and 24 hours using the optimum preparation method. Encapsulation efficiency=(total protein content-reminded protein content)/total protein content. Encapsulation content=(total protein content-residual protein content)/chitosan microspheres content.

Table 2 Preparation optimization of chitosan microspheres

Hp whole cell protein antigen: chitosan microspheres	Hp whole cell protein antigen (mg)	Chitosan microspheres (mg)
1:1	50	50
1:2.5	50	125
1:5	50	250
1:7.5	50	375

Preparation of Hp whole cell protein antigen microspheres
According to the optimum ratio and encapsulating time mentioned above, 500 mg chitosan microspheres and 100 mg Hp whole cell protein antigen were dissolved in acetate buffer (50 mL, 0.1 mol/L, pH 6.0) and stirred for 2 hours at room temperature, centrifuged (1 000 r/min× 20 minutes), to afford Hp whole cell protein antigen microspheres. 50 mL PBS (pH=6.8) was added to the precipitate, stirred, and 2 mL solution was collected at 1, 3, 6, 9, 12, 24, 48 hours, respectively, to determine the protein content with BCA method. Release efficiency=release content in supernatant/(total encapsulation content-residual protein content). Blank chitosan microspheres were used as controls.

Main outcome measures
Morphological characteristics, particle size, encapsulation efficiency, encapsulation content, release efficiency.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

讨论

Using chitosan as the carrier can avoid the high temperature, organic environment, and decrease the loss of protein. Moreover, chitosan, as natural macromolecular substance, exhibit biodegradable and nontoxic properties, which provides a new idea for microspheres preparation. Li et al [6] reported that chitosan could encapsulate a large amount of antigens and showed controlled-release abilities, and enhanced the bioavailability, prolonged the residence time at the mucous membrane by adhesive action with epithelial cells. Particle size of microspheres, however, is tightly correlated with distribution in vivo, for example, microspheres with a particle size of less than 5 μm mainly distribute in nodi lymphatici mesenterici, spleen and macrophage in the blood system; microspheres with a particle size of over 5 μm mainly stay around the Peyer’s patches, which are induced to produce sIgA antibodies[7-9]. Therefore, controlling the particle size of microspheres is good for playing its immune function extremely.

Nowadays, there are four methods to prepare microspheres: emulsification chemical-crossline method, solvent evaporation method, spray drying process and Berthold precipitation method, among which Berthold precipitation method is simple, without organic solvent, and has low effect on encapsulated medicine, being helpful for maintaining bioactivities of protein. Liu et al [10] demonstrated that compared with the emulsification chemical-crossline method, the precipitation method could decrease the retention time at mouse stomach and increase the distribution at colon. For Hp whole cell protein antigen microspheres, increased distribution at colon could avoid the degradation under low pH circumstance at stomach. When preparing chitosan microspheres, chitosan acetate showed good solubility by adding glacial acetic acid, while decreased solubility by adding sulfate, which was beneficial to microspheres formation. Dilute acid was good for dissolving chitosan using Berthold precipitation method; however, chitosan could also degrade under acid circumstance. Thus, 1% acetic acid was used as acid, tween 80 as dispersing agent, and sodium sulfate as precipitator. During preparing microspheres, the concentration of acetic acid, pH, magnetic stirring, and dripping speed of sodium sulfate all had effect on microspheres quality. The resulted optimum preparation condition was 1% glacial acetic acid, sodium sulfate as cross-linking agent, and non-smashing process in this study, which was proved by electron microscope results. Through detecting the encapsulation efficiency of different ratio and encapsulation time, the optimum ratio was 5:1 and the optimum encapsulating time was 3 hours, under which the encapsulation efficiency was 70%-80%. The encapsulation efficiency of prepared microspheres under the optimum preparing method was 80.4%. Particle size of 80% of the prepared microspheres was 1.0-5.0 μm.

Drug release in vitro is an important index to evaluate the properties of microsphere. In this study, the release rate of Hp whole cell protein antigen microspheres at 1 hour was 17.8%, which may be due to the reason that part of the protein absorbed at the surface of microspheres directly diffused to solution and was proved by Wu et al [11]. Hu et al [12] prepared acyclovir chitosan microspheres with emulsification chemical crosslinking method and the release rate at 1 and 12 hours was 23.4%, and 87%, respectively. Drug release speed depended on the properties of chitosan microspheres, drug characteristic and circumstance[13-22]. Under 37 ℃, 50 mL PBS (pH=6.8), the release rate at 1, 24 and 48 hours was 17.8%, 19.2%, and 19.4%, respectively, demonstrating that chitosan microspheres had controlled-release effect. When the prepared Hp whole cell protein antigen microspheres were orally taken, it could be absorbed by the intestinal mucosa. And when the released antigen was phagocytosed by macrophage, the antigen could execute its immune function and prevent Hp infection function.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程