中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

邵帅; 崔光华; 周旭; 高钟镐; 黄伟

doi:10.3969/j.issn.1006-0111.2014.06.006

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中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

邵帅, 崔光华, 周旭, 高钟镐, 黄伟

文章导航 > 药学实践与服务 > 2014 > 32(6): 419-424

邵帅, 崔光华, 周旭, 高钟镐, 黄伟. 中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域[J]. 药学实践与服务, 2014, 32(6): 419-424. doi: 10.3969/j.issn.1006-0111.2014.06.006

引用本文:

SHAO Shuai, CUI Guanghua, ZHOU Xu, GAO Zhonggao, HUANG Wei. Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design[J]. Journal of Pharmaceutical Practice and Service, 2014, 32(6): 419-424. doi: 10.3969/j.issn.1006-0111.2014.06.006

Citation:

SHAO Shuai, CUI Guanghua, ZHOU Xu, GAO Zhonggao, HUANG Wei. Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design[J]. Journal of Pharmaceutical Practice and Service, 2014, 32(6): 419-424. doi: 10.3969/j.issn.1006-0111.2014.06.006

中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

doi: 10.3969/j.issn.1006-0111.2014.06.006

邵帅¹,
崔光华²,
周旭³,
高钟镐⁴,
黄伟^4,

1.
中国医学科学院、北京协和医学院药物研究所药物制剂研究室, 北京 100050;延边大学药学院药剂学教研室, 吉林延吉 133000
2.
军事医学科学院毒物药物研究所药物制剂研究室, 北京 100850
3.
中国人民解放军302医院, 北京 100039
4.
中国医学科学院、北京协和医学院药物研究所药物制剂研究室, 北京 100050

基金项目: 北京市自然科学基金资助项目(7142114);中央级公益性科研院所基本科研业务费专项基金(2014ZD02);协和青年科研基金(2012G07).

Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design

1.
Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;Department of Pharmaceutics, Yanbian University, Yanji 133000, China
2.
Department of Pharmaceutics, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
3.
302 Military Hospital of China, Beijing 100039, China
4.
Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

摘要: 目的采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217.6 nm,粒径多分散系数为0.241,表明粒径分布较窄。纳米粒zeta电位为+22.4 mV,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。
- 纳米粒 /
- 质粒基因 /
- 中心组合设计 /
- 壳聚糖 /
- 转染效率
Abstract: Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfection efficency through a central composition design. Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan. We selected the concentrations of chitosan and plasmid as two experimental factors, and a central composite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfection efficency. The concentrations of chitosan and plasmid were selected as the independent variables, respectively. The dependent variables included average particle size and transfection efficiency. The morphology of DNA-chitosan nanoparticles was observed using a transmission electron microscope. The size and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis. The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope. The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry. Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully. Under the optimum preparative conditions, the DNA-chitosan nanoparticles were almost spherical. The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241. The zeta potential was +22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension. The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation. The cell transfection of nanoparticles was very highly efficient. The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level. Conclusion The established mathematic models have the good predictive function. Under the optimum preparative conditions, the DNA-chitosan nanoparticles have the high potential of cell transfection.
- nanoparticles /
- plasmid DNA /
- central composition design /
- chitosan /
- transfection efficency

[1]	Li XW, Lee DK, Chan AS, et al. Sustained expression in mammalian cells with DNA complexed with chitosan nanoparticles[J]. Biochim Biophys Acta, 2003, 1630(1): 7-18.
[2]	Cui Z, Mumper RJ. Chitosan-based nanoparticles for topical genetic immunization[J]. J Control Release, 2001, 75(3): 409-419.
[3]	Mao HQ, Roy K, Troung-Le VL, et al. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency[J]. J Control Release, 2001, 70(3): 399-421.
[4]	蒋挺大. 壳聚糖[M].北京:化学工业出版社,2001:8-12.
[5]	Roy K, Mao HQ, Huang SK, et al. Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy[J]. Nat Med, 1999,5(4): 387-391.
[6]	Hirosue S, Müller BG, Mulligan RC, et al. Pasmid DNA encapsulation and released from solvent diffusion nanospheres[J]. J Control Release, 2001, 70(1-2): 231-242.
[7]	Truong-Le VL, August JT, Leong KW. Controlled gene delivery by DNA-gelatin nanospheres[J]. Hum Gene Ther, 1998, 9(12): 1709-1717.
[8]	Mao S, Sun W, Kissel T. Chitosan-based formulations for delivery of DNA and siRNA[J]. Adv Drug Deliv Rev, 2010, 62(1): 12-27.
[9]	Strand SP, Lelu S, Reitan NK, et al. Molecular design of chitosan gene delivery systems with an optimized balance between polyplex stability and polyplex unpacking[J]. Biomaterials, 2010, 31(5): 975-987.
[10]	马丽杰, 赵静. 壳聚糖/木质素磺酸钠复凝聚法制备生物农药微胶囊[J]. 北京化工大学学报(自然科学版), 2006, 33(6): 51-56.
[11]	吴伟, 崔光华. 星点设计—效应面优化法及其在药学中的应用[J]. 国外医学(药学分册), 2000, 27(5): 292-298.
[12]	Hassan EE, Parish RC, Gallo JM. Optimized formulation of magnetic chitosan microspheres containing the anticancer agent, oxantrazole[J]. Pharm Res, 1992, 9(3): 390-397.

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中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

doi: 10.3969/j.issn.1006-0111.2014.06.006

1. 中国医学科学院、北京协和医学院药物研究所药物制剂研究室, 北京 100050;延边大学药学院药剂学教研室, 吉林延吉 133000

2. 军事医学科学院毒物药物研究所药物制剂研究室, 北京 100850

3. 中国人民解放军302医院, 北京 100039

4. 中国医学科学院、北京协和医学院药物研究所药物制剂研究室, 北京 100050

基金项目: 北京市自然科学基金资助项目(7142114);中央级公益性科研院所基本科研业务费专项基金(2014ZD02);协和青年科研基金(2012G07).

收稿日期: 2013-12-15
修回日期: 2014-06-13

关键词:

纳米粒 /

质粒基因 /

中心组合设计 /

壳聚糖 /

转染效率

摘要: 目的采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217.6 nm,粒径多分散系数为0.241,表明粒径分布较窄。纳米粒zeta电位为+22.4 mV,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。

English Abstract

Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design

1. Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;Department of Pharmaceutics, Yanbian University, Yanji 133000, China

2. Department of Pharmaceutics, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

3. 302 Military Hospital of China, Beijing 100039, China

4. Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

Received Date: 2013-12-15
Rev Recd Date: 2014-06-13

Keywords:

Abstract: Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfection efficency through a central composition design. Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan. We selected the concentrations of chitosan and plasmid as two experimental factors, and a central composite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfection efficency. The concentrations of chitosan and plasmid were selected as the independent variables, respectively. The dependent variables included average particle size and transfection efficiency. The morphology of DNA-chitosan nanoparticles was observed using a transmission electron microscope. The size and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis. The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope. The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry. Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully. Under the optimum preparative conditions, the DNA-chitosan nanoparticles were almost spherical. The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241. The zeta potential was +22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension. The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation. The cell transfection of nanoparticles was very highly efficient. The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level. Conclusion The established mathematic models have the good predictive function. Under the optimum preparative conditions, the DNA-chitosan nanoparticles have the high potential of cell transfection.

引用本文:

Citation:

SHAO Shuai, CUI Guanghua, ZHOU Xu, GAO Zhonggao, HUANG Wei. Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design[J]. Journal of Pharmaceutical Practice and Service, 2014, 32(6): 419-424. doi: 10.3969/j.issn.1006-0111.2014.06.006