中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (2): 218-225.doi: 10.3969/j.issn.2095-4344.1510

• 药物控释材料 drug delivery materials • 上一篇    下一篇

超顺磁性壳聚糖明胶微球作为缓释基因载体的磁转染及释放

岑超德1,张 永2,罗 聪3,杨晓兰4,邬 均3,吴声忠1,刘福尧1   

  1. 1贵州省骨科医院骨科,贵州省贵阳市 550000;2贵阳市第一人民医院妇产科,贵州省贵阳市 550000;3重庆医科大学附属儿童医院骨科,重庆市 400014;4重庆医科大学临床诊断学重点实验室化学系,重庆市 400016
  • 收稿日期:2018-07-21 出版日期:2019-01-18 发布日期:2019-01-18
  • 通讯作者: 罗聪,主任医师,重庆医科大学附属儿童医院骨科,重庆市 400014
  • 作者简介:岑超德,男,1989年生,贵州省惠水县人,布依族,2016年重庆医科大学毕业,硕士,医师,主要从事磁靶向、磁性纳米材料及骨组织工程的研究。
  • 基金资助:

    重庆市科技攻关计划项目(CSTC2011ggB1004,项目负责人:罗聪);国家临床重点专科建设项目(国卫办医函[2013]544)

Superparamagnetic chitosan gelatin microspheres as sustained-release gene carrier: magnetofection and release in vitro

Cen Chaode1, Zhang Yong2, Luo Cong3, Yang Xiaolan4, Wu Jun3, Wu Shengzhong1, Liu Fuyao1   

  • Received:2018-07-21 Online:2019-01-18 Published:2019-01-18
  • Contact: Luo Cong, Chief physician, Department of Orthopaedics, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China
  • About author:Cen Chaode, Master, Physician, Department of Orthopaedics, Guizhou Provincial Orthopedics Hospital, Guiyang 550000, Guizhou Province, China
  • Supported by:

    Chongqing Science and Technology Research Project, No. CSTC2011ggB1004 (to LC); the National Clinical Key Specialty Construction Project, No. [2013]544

摘要:

文章快速阅读:

 


文题释义:
超顺磁性
:指铁磁性物质的颗粒小于临界尺寸时,颗粒间的热振动能足以克服磁吸引力,在磁场中有较强的磁性,没有磁场时磁性很快消失,不会被永久磁化及团聚。
磁转染:是一种将目的基因与磁性纳米微粒结合形成磁性复合物,利用外加磁场力的导向作用,使磁性复合物高效靶向地转运至细胞内发挥预期作用的方法。

背景:利用缓释技术将成血管基因转染至细胞是组织工程骨充分血管化的关键,而选择安全有效的基因载体至关重要。
目的:制备超顺磁性壳聚糖纳米粒(superparamagnetic iron oxide nanoparticles,SPIOCN)与超顺磁场壳聚糖质粒明胶微球(superparamagnetic chitosan plasmid gelatin microspheres,SPCPGM),并对其进行表征。
方法:利用脱水缩合反应制备SPIOCN,对其分子结构、形态和粒径、饱和磁化强度、ζ电位及DNA结合能力进行表征。将SPIOCN溶液与成骨肉瘤细胞MG-63共孵育24 h,采用透射电镜观察细胞吞噬SPIOCN的过程。制备SPCPGM与非磁性壳聚糖明胶微球,将其填入人工多孔骨植入体中,在37 ℃的PBS(pH=7.4)中进行体外药物溶出实验,采用振荡磁场干预,测定质粒释放量。取成骨肉瘤细胞MG-63及人脐静脉内皮细胞HUVEC-1,分4组转染,PolyMag200(商业化磁转染试剂)/pDNA+静磁场组、SPIOCN/pDNA+静磁场组、SPIOCN/pDNA组及裸pDNA质粒组(对照组),转染24 h后,利用倒置荧光显微镜与流式细胞仪检测转染效果。将人脐静脉内皮细胞HUVEC-1分4组培养,PolyMag200/pDNA+静磁场组、SPIOCN/pDNA+静磁场组、SPIOCN/pDNA组及裸pDNA质粒组(对照组),培养24,48,72 h后检测细胞存活率。
结果与结论:①SPIOCN的平均粒径为(187±24) nm,饱和磁化强度为(20.3±4.5) emu/g,ζ电位为(9.5±2.4) mV,具有结合保护质粒DNA转染MG-63细胞及HUVEC-1细胞的能力;②SPIOCN贴附细胞膜后,通过胞膜内吞作用进入细胞,细胞浆内可见散布吞噬SPIOCN的内涵体;③在磁场干预下,SPCPGM多孔骨植入体的质粒释放量明显高于非磁性壳聚糖明胶微球多孔骨植入体(P < 0.05);④转染MG-63或HUVEC-1细胞后,PolyMag200/pDNA+静磁场组转染效率高于其余3组(P < 0.05),SPIOCN/pDNA+静磁场组高于SPIOCN/pDNA组及对照组(P < 0.05);⑤与对照组比较,SPIOCN/ pDNA+静磁场组、PolyMag200/pDNA+静磁场组不同时间点的细胞存活率降低(P < 0.05),而SPIOCN/pDNA组细胞存活率无明显变化;SPIOCN/pDNA+静磁场组不同时间点的细胞存活率高于PolyMag200/pDNA+静磁场组(P < 0.05);⑥结果表明,SPIOCN具有粒径小、分散性好、毒性低、超顺磁性及结合保护DNA转染细胞的特点,振荡磁场联合SPCPGM是一种较理想的缓释基因载体系统。 

关键词: 非病毒基因载体, 病毒基因载体, 纳米技术, 微球, 超顺磁性氧化铁纳米粒, 缓释, 控释, 血管形成, 静磁场, 骨组织工程, 生物材料

Abstract:

BACKGROUND: The transfection of angiogenic genes into cells by slow-release technology is the key to adequate vascularization of tissue engineered bone, and it is most important to select the safe and effective gene carrier.

OBJECTIVE: To prepare and characterize superparamagnetic iron oxide chitosan nanoparticles (SPIOCN) and superparamagnetic chitosan plasmid gelatin microspheres (SPCPGM).
METHODS: SPIOCN was prepared by dehydration condensation reaction, and the molecular structure, morphology and particles size, saturation magnetization, ζ potential and DNA binding ability were respectively characterized. SPIOCN solution was incubated with MG-63 cells for 24 hours, and cell phagocytosis of SPIOCN was observed by transmission electron microscope. SPCPGM and non-magnetic chitosan gelatin microspheres were prepared, which were filled in the porous cage, and the plasmid release of SPCPGM intended for implantation in the porous cage in the presence of and absence of oscillating magnetic fields was carried out in 0.01 mol/L phosphate buffer at 37 oC (pH=7.4). MG-63 cells and human umbilical vein endothelial cells were selected as target cells for transfection, which were divided into four groups and respectively intervened by PolyMag200 (commercial magnetic transfection reagent)/Pdna+the static magnetic field, SPIOCN/pDNA+the static magnetic field, SPIOCN/pDNA and naked pDNA. The transfection efficiency was then detected by inverted fluorescence microscope and flow cytometry after 24 hours. Human umbilical vein endothelial cells were cultured in four groups: PolyMag200/pDNA+the static magnetic field group, SPIOCN/pDNA+the magnetic field group, SPIOCN/pDNA group and naked pDNA group (control group). Cell viability was detected after 24, 48 and 72 hours of culture.
RESULTS AND CONCLUSION: (1) The average particle size of SPIOCN was (187±24) nm, the saturation magnetization was (20.3±4.5) emu/g and the zeta potential was (9.5±2.4) mV, indicating that SPIOCN endows the combination ability with plasmid DNA transfected into MG-63 cells and human umbilical vein endothelial cells. (2) SPIOCN was attached to the cell membrane and entered into the cells through intracellular endocytosis pathway. SPIOCN swallowed as endosomes were dispersed in the cytoplasm. (3) The releasing plasmid amount of SPCPGM implanted in the porous cage in the presence of magnetic field was significantly higher than that of non-magnetic chitosan gelatin microspheres implanted in the porosity cage (P < 0.05). (4) The transfection efficiency of PolyMag200/pDNA+the static magnetic field group was higher than that of the other three groups (P < 0.05), and the transfection efficiency of SPIOCN/pDNA+the static magnetic field group was higher than that of SPIOCN/pDNA group and naked pDNA group (P < 0.05). (5) Compared with the naked pDNA group, the cell survival rate of SPIOCN/pDNA+the static magnetic field group and PolyMag200/pDNA+the static magnetic field group decreased at different time points  (P < 0.05), while the cell survival rate of SPIOCN/pDNA group showed no significant changes. The cell survival rate of SPIOCN/ pDNA+ static magnetic field group at different time points was higher than that of PolyMag200/pDNA+the static magnetic field group (P < 0.05). To conclude, SPIOCN has the characteristics of small particle size, good dispersibility, low toxicity, superparamagnetism and combined protection of DNA transfected cells. The oscillating magnetic field integrated with SPCPGM is an ideal system of slow-release gene carriers.  

Key words: Chitosan, Electromagnetic Fields, Nanoparticles, Tissue Engineering

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