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

doi:10.3969/j.issn.2095-4344.1510

Abstract

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.

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: Chitosan, Electromagnetic Fields, Nanoparticles, Tissue Engineering

CLC Number:

R459.9

R318

Cen Chaode, Zhang Yong, Luo Cong, Yang Xiaolan, Wu Jun, Wu Shengzhong, Liu Fuyao. Superparamagnetic chitosan gelatin microspheres as sustained-release gene carrier: magnetofection and release in vitro[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(2): 218-225.

Figures/Tables 8

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