A novel gene carrier based on amino-modified silica nanoparticles

Uniform-sized amino-modified silica nanopar-ticles have been prepared by the controlled synchronous hydrolysis of tetraethoxysilane and N-(?amimoethyl)-- aminopropyltriethoxysilane in water nanodroplet of the wa-ter-in-oil microemulsion. These nanoparticles display posi-tive charge potential at definited pH. This is due to the pres-ence of amino groups on the surface of the nanoparticles. Nanoparticles-plasmid DNA complexes can easily form through electrostatical binding between the positive charges of the amino-modified silica nanoparticles and the negative charges of the plasmid DNA. The complexes can be also dis-sociated under alkaline pH or high ionic strength conditions. And enzymatic digestion of the plasmid DNA is almost in-hibited by these nanoparticles complexes. A novel non-viral gene carrier based on the amino-modified silica nanoparti-cles is proposed under the combination of nanotechnology, biotechnology and gene engineering technology. The plasmid DNA can successfully cross various systemic barriers to COS-7 cells as well as mediate high expression of Green Fluorescence Protein (GFP) gene in cells by use of this novel gene carrier.

Silica Nanoparticles-mediated Stable Genetic Transformation in Nicotiana tabacum

Nanoparticles as gene carriers become popular in the mammalian cells, whereas the application of them in plant cells is still very limited. Herein lies a report on silica nanoparticles（SiNPs） modified with positively charged poly-L-lysine（PLL） successfully delivering plasmid-encoded β-glucuronidase（GUS） gene into tobacco with the help of gene gun. The stable transgenic tobacco plants mediated by SiNPs can be obtained. Furthermore, we revealed the quantity of gene and types of receptor materials could affect the expression efficiency. In comparison to conven- tional gold particles-mediated transformation, the silica nanoparticles-mediated stable genetic transformation enhances transformation efficiency, potentially overcoming transgenic silencing. Our results demonstrate the great potential of SiNPs as gene carrier in plant genetic transformation and prove a novel approach for plant genetic decoration.

Silica nanoparticle is a possible safe carrier for gene therapy

In order to develop a safe and effective gene therapy carrier, some toxicological and biodynamical ex- periments were carried out on silica nanoparticles (SiNPs). First we prepared SiNPs with appropriate portions of cyclo- hexane, deionized water and ethyl silicate, and then trans- fected the modified SiNPs and GFP plasmid DNA complex into the HT1080 cells to test the effectiveness of transfection for gene therapy. At the same time, we injected the SiNPs into a number of mice through tail vein. Then we made the mice crossed to evaluate the acute, long-term and reproduc- tive toxicity. In vivo distribution analysis and pathological examination were made on both adult mice and their off- spring. SiNPs were uniform and had an average diameter of 40 nm, and the modified SiNPs carried exogenous DNA molecules into target cells and the transferred GFP fusion gene was effectively expressed in the cells. The SiNPs injected via tail vein were widely distributed in almost all of tissues, and the injected mice had the ability to reproduce normally. The in vivo and in vitro results of this study clearly show that SiNPs can be used as a safe and effective carrier for gene transfection and gene therapy.

硅纳米颗粒作为基因转染载体的研究

通过不同浓度的NaCl、NaI修饰硅纳米颗粒 ,用琼脂糖凝胶电泳分析硅纳米颗粒与DNA结合力及对DNA的保护作用 ,同时用绿色荧光蛋白基因作报告基因 ,以硅纳米颗粒作为基因转染的载体 ,转染HT10 80细胞。经电镜观察证实硅纳米颗粒进入细胞内 ;硅纳米颗粒与DNA结合后 ,能对DNA起保护作用 ;并且硅颗粒作为基因转染的载体 ,将绿色荧光蛋白基因导入HT10 80细胞 ,用荧光显微镜观察到发绿色荧光的细胞。结果表明 。

用于基因治疗的硅纳米颗粒的制备及生物安全性评价

采用化学合成法制备用于基因治疗的易修饰的氨基化硅纳米颗粒和荧光氨基化硅纳米颗粒。用透射电镜和Zeta电位仪对纳米颗粒进行分析,并参照ISO7406技术报告中的相关标准,对纳米颗粒的生物相容性进行一系列体内及体外实验,包括细胞毒性实验、动物急性毒性实验、动物生殖毒性实验。结果表明:新研制的氨基化硅纳米颗粒的粒径为40 nm左右;在中性条件下,氨基化硅纳米颗粒的表面净正电荷约为16 mV;纳米颗粒对细胞生长无明显影响,对实验动物无生长和生殖毒性,与对照组相比无显著性差异。

介孔硅纳米粒作为植物细胞转基因载体的研究

随着纳米生物技术的快速发展,以纳米材料作为基因载体在植物细胞转导研究中取得了初步进展。本研究制备了两种尺寸的介孔硅纳米颗粒,氨基修饰后对其进行表征,并负载含smGFP基因的质粒DNA对拟南芥原生质体进行细胞转导。结果表明,直径约20 nm的氨基化介孔硅纳米颗粒(Am-MSN-20)呈类球形,花形结构的氨基化介孔硅纳米颗粒(Am-MSN-50)直径约50 nm,两者均带正电荷。Am-MSN-50结合DNA的能力高于Am-MSN-20,两种纳米载体都表现出了良好的稳定性,能够保护负载的pDNA不被细胞核酸酶降解,并且对原生质体没有毒害作用。与Am-MSN-20相比较,Am-MSN-50具备更高的转导效率。本研究表明,氨基修饰的MSNs有望成为一种安全高效的新型植物基因载体。

硅纳米载体转染成人角质形成细胞的初步研究

目的探讨硅纳米颗粒作为基因转染载体转染成人角质形成细胞的可行性,为后续的基因治疗和创面修复研究打下基础.方法自制硅纳米颗粒,与增强型绿色荧光蛋白基因(质粒pEGFP-N1)结合后,在氯化钠修饰下转染成人离体角质形成细胞,观察转染后细胞的绿色荧光蛋白表达,计算转染率.结果硅纳米颗粒能有效地与pEGFP-N1 DNA结合后转染成人离体角质形成细胞,转染率约为20%～30%.结论硅纳米颗粒可作为基因转染载体,能有效地转染成人离体角质形成细胞.

介孔二氧化硅纳米粒子的合成及其在生物医学中的应用

介孔二氧化硅纳米粒子具有均一孔径、稳定的骨架结构、易于修饰的内外表面等特点,在药物缓/控释及靶向释放和基因输送等方面的应用日益得到重视。虽然国内外许多研究组使用不同的方法进行了尝试,但是制备粒径均一的、单分散的、尺寸可控的介孔二氧化硅纳米粒子仍然是一大难点。纳米生物医学技术的安全性问题已经成为纳米技术应用于医学临床的主要障碍之一。为了能够更好地指导纳米医药技术的发展,这些问题都将是今后我们研究的焦点。

氨基改性磁性介孔二氧化硅纳米颗粒的细胞毒性和体内外分布研究

目的研究新型氨基修饰的磁性介孔二氧化硅纳米颗粒的生物相容性以及跨膜转运能力,并观察该纳米颗粒的体内代谢分布,以此评价该纳米颗粒在基因或药物载体方面的应用前景。方法制备新型介孔二氧化硅纳米颗粒,以20 nm Fe3O4为核心包裹的二氧化硅颗粒,并且进行修饰使之表面氨基化,对此氨基修饰的磁性介孔二氧化硅纳米颗粒细胞毒性研究,携带N-甲基-D-天冬氨酸(NMDA)受体NR2B亚单位基因小干扰RNA(siRNA)质粒细胞转染实验,以及白鼠活体注射靶向模拟实验研究其在体内的生物分布。采用单因素方差分析。结果该新型氨基修饰的磁性介孔二氧化硅纳米颗粒是一种直径在80~100 nm之间,孔径在2~4 nm之间的均一球体型纳米颗粒。在5~125μg/ml浓度范围内,介孔二氧化硅纳米颗粒的对于细胞活性影响差异无统计学意义(P>0.05),始终保持在6%以下。在姜黄素染色的装载有NR2B siRNA质粒的该纳米颗粒转染实验中,可以在荧光显微镜下观察到细胞内的荧光现象。在体外红外测定仪观察红外激发荧光染料标记的纳米颗粒实验中,可见该纳米颗粒在小鼠体内不会有蓄积作用,其最终会通过肾脏随尿液汇聚至膀胱,并最终排出体外。同时在外加磁场的情况下,会有大量荧光颗粒向磁场方向聚集,并且在撤出磁场后不会蓄积,浓度会持续下降。结论氨基化磁性介孔二氧化硅颗粒具有较好的装载能力以及生物安全性,可以携带NR2B基因siRNA质粒通过胞吞作用进入细胞,在外加磁场诱导下可靶向到达病灶区域,为靶向基因治疗奠定基础。