荧光载体CS-Qdots的构建及生物相容性分析

发布时间：2018-05-21 10:01

本文选题：量子点 + 荧光纳米颗粒　；参考：《南京医科大学学报(自然科学版)》2017年10期

【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。
[Abstract]:Aim: to construct a novel in vivo and in vitro tracer gene vector and evaluate its biocompatibility. Methods: the fluorescent nanoparticles CS-Qdotswere formed on the surface of Qdots. The morphology, optical characteristics, surface charge and Fourier transform near infrared spectroscopy (FT-NIR) of CS-Qdotswere measured, and then injected into nude mice to observe the imaging signals in vivo. The ability of CS-Qdots to carry plasmid DNA was detected by gel retardation electrophoresis, and the expression of green fluorescent protein (GFP) was observed by confocal laser microscopy. The relative cell proliferation rate was measured by MTT assay, and the biocompatibility of CS-Qdots was evaluated by hemolysis rate and acute toxicity test in mice. Results: the surface potential of CS-Qdots nanoparticles was determined to be 28.02 卤1.15m ~ (-1) m ~ (-1) V ~ (-1). The characteristic band of chitosan was found in FTIR spectra. The maximum emission peak value of CS-Qdots was 630nm ~ (-1) nm. Gel block electrophoresis showed that the ratio of nanoparticles to DNA was more than 10:1, and the ratio of nanoparticles to DNA did not move towards the positive electrode after mixing. Laser confocal observation showed that CS-Qdots could carry plasmid p EGFP-C1 to express green fluorescent protein in Hep G2 cells. In vivo imaging of mice, CS-Qdots showed strong fluorescence imaging signal in nude mice xenografts. MTT test showed that the RGR of cells incubated with CS-Qdots of 50100200 and 400 渭 g / mL were 1.0000.0000.000,0.917 and 0.875respectively, while the corresponding concentrations of Qdots incubated cells RGR were 1.000,0.8500.0.621 and 0.326, respectively, and the concentrations were 100 渭 g / mL. The hemolysis rates of Qdots QDs on the above QDs are all greater than 5, while the hemolysis rates of CS-Qdots nanoparticles are less than 5 within 400 渭 g / mL. The acute toxicity test of 72 h CS-Qdots injection in mice showed that there was no obvious organ pathological injury, normal liver and kidney function and normal blood cell count compared with normal saline control group. Conclusion: the fluorescent nanoparticles CS-Qdotswere successfully constructed, which can effectively transfect gene expression in cells, have high biocompatibility, and be able to carry out fluorescence imaging in vivo and in vitro, which can be used as a tracer gene delivery nano-carrier.
【作者单位】：东南大学附属中大医院检验科;
【基金】：国家自然科学基金青年基金项目(81501525)
【分类号】：R450

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