SYBR Green Ⅰ-荧光法测定载鱼精蛋白缩合基因纳米粒中pDNA的含量和包封率

目的:建立微量 DNA 含量测定方法,用于载基因纳米粒中 DNA 的含量和包封率测定。方法:利用 SYBR Green Ⅰ与 pD-NA 结合后能激发出强烈荧光,荧光分光光度计检测荧光强度建立标准曲线,测定载基因纳米粒提取液中的 pDNA 含量,计算纳米粒的包封率。结果:SYBR Green Ⅰ荧光分光光度法的检测灵敏度为10.188 ng·mL^(-1),线性范围20.375～1062.5 ng·mL^(-1)(r=0.9998),高、中、低3个浓度的回收率分别为101.0%,97.5%,99.6%;RSD 分别为1.26%,1.09%,1.19%(n=3);日内精密度的 RSD 分别为2.13%,1.88%,2.25%(n=5);日间精密度的 RSD 分别为3.86%,4.97%,3.41%(n=5)。结论:本方法准确可靠、灵敏度高、简便快速,可用于载基因纳米粒中 pDNA 的含量和包封率测定。

Hochest染料-荧光分光光度法测定基因治疗非病毒载体中DNA的含量

目的 利用荧光染料Hoechst 332 5 8与DNA结合后 ,荧光强度显著增强的特点 ,建立了基因治疗非病毒载体中DNA的含量测定方法。方法 对Hoechst 332 5 8-DNA溶液进行荧光扫描 ,并建立了标准曲线。分别测定了纳米粒胶体溶液超速冷冻离心后的上清液和用酶消化后的脂质体提取液中DNA的含量。结果 Hoechst332 5 8-DNA溶液的最佳激发波长为 35 3.6nm ,最佳发射波长为 4 5 4 .4nm ,标准曲线的线性范围为 0 .2～ 1.0 μg·ml-1。测得包载DNA纳米粒的平均包封率为 75 .1%± 8.6 %(n =5 ) ,DNA阳离子脂质体的平均抗核酸酶能力为 84 .9%± 7.8% (n =5 )。结论 荧光染料Hoechst332 5 8可用于测定基因治疗非病毒载体中DNA的含量 ,所建立的荧光分光光度法为载基因纳米粒、脂质体制备工艺和质量标准的建立提供了依据。

Effect of Tb/Mg doping on composition and physical properties of hydroxyapatite nanoparticles for gene vector application

Transfection efficiency of hydroxyapatite nanoparticles(HAnps)is relative to the particle size,morphology,surface charge,surface modifier and so on.This study prepared HAnps with doped Tb/Mg by hydrothermal synthesis method(HTSM)and investigated the effects of different Tb/Mg contents on the morphology,particle size,surface charge,composition and cellular endocytosis of HAnps.The results showed that Mg-HAnps possessed better dispersion ability than Tb-HAnps.With increasing doping content of Tb/Mg-HAnps,the granularity of Tb-HAnps increased,while that of Mg-HAnps declined.Both particle size and zeta potential of Mg-HAnps were lower than those of Tb-HAnps.7.5%Mg-doping HAnps presented relatively uniform slender rod morphology with average size of30nm,while10%Mg-doping HAnps were prone to agglomeration.Moreover,Mg-HAnps-GFP(green fluorescent protein)endocytosed by MG63cells was dotted in the perinuclear region,while Tb-HAnps were more likely to aggregate.In conclusion,as gene vectors,Mg-HAnps showed enhanced properties compared to Tb-HAnps.

NANOPARTICLE AS A NEW GENE TRANSFERRING VECTOR IN SPECIFIC EXPRESSION GENE

Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-l), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP-1 (200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-l and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9% , release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-l gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-l mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.

Versatile functionalization of amylopectin for effective biomedical applications

Successful gene vectors should be with high transfection efficiency and minimal cytotoxicity. Natural polysaccharides, due to their good biocompatibility and biodegradability, have been widely studied and applied. Amylopectin is one of polysaccharides with dendritic structure and numerous hydroxyl groups that could be used for subsequent modification. In this work, a series of dendritic cationic gene vectors comprising amylopectin backbones and poly(2-(dimethylamino) ethyl methacrylate)(PDMAEMA) side chains with different lengths(termed as AMY-PDs) were readily prepared by atom transfer radical polymerization(ATRP). The gene condensation ability, cytotoxicity and gene transfection of AMY-PDs carriers were investigated. In comparison with "gold-standard" poly(ethyleneimine)(PEI, 25 k Da), the AMY-PDs exhibited higher transfection efficiency with lower cytotoxicity. AMY-PDs could be further modified with Au nanoparticles(termed as AMY-PD@Au). The potential of the AMY-PD@Au vectors to be utilized as a CT contrast agent for imaging of cancer cells was investigated. Such AMY-PD@Au vectors may realize gene therapy with the ability of real-time imaging.