Plasmid size influences chitosan nanoparticle mediated gene transfer to chondrocytes.
Plasmid size influences chitosan nanoparticle mediated gene transfer to chondrocytes.
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DOI：
10.1002/jbm.a.31479
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发表时间：
2008-03
期刊：
影响因子：
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通讯作者：
Ximing Xu;R. Capito;M. Spector
中科院分区：
文献类型：
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作者：
Ximing Xu;R. Capito;M. Spector
本研究的目的是制备含有相对较大的编码成骨蛋白(OP)-1的质粒的壳聚糖纳米颗粒，并确定这些纳米颗粒在体外转染成年犬关节软骨细胞的能力。壳聚糖的正电荷作用于凝聚较大的带负电荷的OP-1质粒，使其可以被纳入纳米颗粒中。凝胶电泳表明，将质粒掺入壳聚糖纳米颗粒中不会影响质粒的结构完整性。纳米颗粒的形态和大小随壳聚糖与质粒重量比的变化而变化。壳聚糖与质粒比例为10:1的纳米颗粒具有均匀的小尺寸（小于250 nm）和球形。这些纳米粒子的正电荷约为20mv。fitc标记的壳聚糖纳米颗粒与纳米颗粒孵卵24小时后，几乎在所有细胞中都发现了fitc标记的壳聚糖纳米颗粒，共聚焦显微镜显示软骨细胞核中有fitc相关荧光。虽然用含有增强绿色荧光蛋白质粒的壳聚糖纳米颗粒处理的细胞的荧光可以证明转染了软骨细胞，但用含有较大的OP-1质粒的纳米颗粒转染的细胞在培养2周内没有显示OP-1的表达。这些结果表明，尽管大的质粒可以成功地掺入壳聚糖纳米颗粒中，但在纳米颗粒中掺入的质粒的大小仍然可能显著影响基因向细胞的转移。
The objective of the present study was to prepare chitosan nanoparticles incorporating a relatively large plasmid encoding for osteogenic protein (OP)-1 and to determine the ability of these nanoparticles to transfect adult canine articular chondrocytes in vitro. The positive charge of chitosan acted to condense the relatively large negatively-charged OP-1 plasmid such that it could be incorporated into nanoparticles. Incorporation of the plasmid into the chitosan nanoparticles did not affect the structural integrity of the plasmid as demonstrated by gel electrophoresis. The morphology and size of the nanoparticles were found to vary with the chitosan:plasmid weight ratio. Nanoparticles formulated with a chitosan:plasmid ratio of 10:1 were of uniformly small size (less than 250 nm) and spherical shape. These nanoparticles had a positive charge of about 20 mV. FITC-labeled chitosan nanoparticles were found in virtually all of the cells after 24 h of incubation with the nanoparticles, and confocal microscopy revealed FITC-related fluorescence in the nucleus of the chondrocytes. Although transfection of the chondrocytes was demonstrated by the fluorescence of cells treated with chitosan nanoparticles containing the plasmid for the enhanced green fluorescence protein, cells transfected with nanoparticles incorporating the larger OP-1 plasmid did not show OP-1 expression measured by ELISA for up to 2 weeks in culture. These results indicate that although a large plasmid can be successfully incorporated within chitosan nanoparticles, the size of the plasmid incorporated within the nanoparticles may still significantly affect gene transfer to cells.