摘要
目的:构建一种可通过荧光成像进行体内外示踪的纳米基因载体,并对其生物相容性进行初步评价。方法:在量子点Qdots表面修饰壳聚糖后形成荧光纳米颗粒CS-Qdots,检测其电镜形态、光学特征、表面电荷和傅里叶转换近红外光谱(FTIR),并将其注射入裸鼠移植瘤内观察体内成像信号;以凝胶阻滞电泳检测CS-Qdots携带质粒DNA的能力,并利用激光共聚焦显微镜观察其转染报告基因绿色荧光蛋白在细胞内的表达情况。通过MTT试验检测细胞相对增殖率(RGR)、测定溶血率和小鼠急性毒性试验评价CS-Qdots的生物相容性。结果:电镜观察显示CS-Qdots纳米颗粒粒径为20~30 nm,zeta电位分析其表面电位为(28.02±1.15)m V,FTIR图谱显示出壳聚糖的特征谱带,发射光谱分析CS-Qdots最大发射峰值在630 nm。凝胶阻滞电泳显示纳米颗粒和DNA的比例大于10∶1混合以后不再向正极泳动,激光共聚焦观察CS-Qdots能携带质粒p EGFP-C1在Hep G2细胞内表达绿色荧光蛋白,小鼠活体成像中CS-Qdots在裸鼠移植瘤内有较强荧光成像信号。MTT试验显示,50、100、200和400μg/m L的CS-Qdots共孵育的细胞RGR分别为1.000、1.000、0.917和0.875,而相应浓度的Qdots孵育细胞RGR为1.000、0.850、0.621和0.326;浓度在100μg/m L以上的Qdots量子点溶血率均大于5%,而CS-Qdots纳米颗粒在400μg/m L以内溶血率均小于5%。小鼠尾静脉注射CS-Qdots 72 h急性毒性试验显示,与生理盐水对照组相比,没有明显的脏器病理损伤,肝肾功能正常,血细胞计数正常。结论:成功构建了荧光纳米颗粒CS-Qdots,它能有效转染基因在细胞内表达,具有较高的生物相容性,并可在体内外进行荧光成像,是可示踪基因的运送纳米载体。
Objective： To prepare fluorescent nano gene vectors for traceable gene delivery and evaluate the biocompatibility of the nanoparticles. Methods： The fluorescent nanopaticles were constructed by quantum dots （Qdots） encapsulated by chitosan, then characterized by transmission electron microscopy （TEM）, fluorospectrophotometer, zeta potential and Fourier transform infrared spectrometry （FTIR）. The imaging of tumor-injected xenograft nude mouse was taken to evaluate the in vivo fluorescent signal of CS-Qdots nanoparticles. Gel retardation assays were used to test the DNA binding affinity of CS-Qdots. The expression of green fluorescent protein （GFP） gene delivered in cells by CS-Qdots was observed by confocal laser microscope. The biocompatibility of CS-Qdots was evaluated by cells relative grow rate （RGR） of MTT test, hemolysis rate and acute toxicity tests in mice. Results： The mean particle size of the CS-Qdots nanoparticles was 20-30 nm in the TEM image and the surface charge of them was （28.02 ± 1.15） mV. The characteristic peaks of chitosan were observed on the CS-Qdots FTIR spectra. The light emission peak of CS-Qdots was at 630 nm. Complete retardation was observed for particle-DNA weight ratios over 10∶1, for which the DNA was well packed in the gene-CS-Qdots complexes with a positive surface charge. High level expression of GFP genes delivered into HepG2 cells by CS-Qdots was detected by a confocal laser microscopy. The strong fluorescence signal of CS-Qdots in vivo was observed in xenograft nude mouse imaging. The MTT results indicated that RGRs of cells co-incubated with CS-Qdots at concentrations of 50, 100, 200 and 400 μg/mL were 1.000, 1.000, 0.917, and 0.875, respectively. The RGRs of cells co-incubated with Qdots at the corresponding concentrations were 1.000, 0.850, 0.621 and 0.326. The hemolysis rates of Qdots over the concentration of 100 μg/mL were all above 5%. Oppositely, the hemolysis rates of CS-Qdots were all below 5% at all the concentrations tested. The
出处
《南京医科大学学报（自然科学版）》
CSCD
北大核心
2017年第10期1227-1233,1250,共8页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金青年基金项目(81501525)
关键词
量子点
荧光纳米颗粒
可示踪基因运送
生物相容性
quantum dots
fluorescent nanoparticles
traceable gene delivery
biocompatibility
