Li Xiong Wei, Lee Daniel Ken Lem, Chan Albert Sun Chi, Alpar H Oya
The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK.
Biochim Biophys Acta. 2003 Oct 20;1630(1):7-18. doi: 10.1016/j.bbaexp.2003.08.011.
This work investigates the preparation and in vitro efficiency of chitosan gene transfection systems. Chitosan was used to prepare nanoparticles with a size range of 40-200 nm as determined using photon correlation spectroscopy (PCS) and 40-80 nm as determined using transmission electron microscopy (TEM). The ability of particles to complex DNA was investigated using gel retardation. Plasmid DNA pGL3-Control encoding firefly luciferase and pCH110 encoding beta-galactosidase were used as reporter genes. For transfection 293 human embryonal kidney cells and Chinese hamster ovary (CHO-K1) cells were used. The expression of luciferase was assayed and expressed as relative light units per milligram of protein (RLU/mg protein). Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection by the chitosan-pGL3-Control particles showed a sustained expression of the luciferase gene for about 10 days. Commercial transfection reagents, SuperFect and Lipofectin were also used. In contrast to chitosan particles, the duration of expression for both SuperFect and Lipofectin was only about 2 days. Agarose gel electrophoresis and displacement experiments using polyaspartic acid indicated a probable multiple interaction between DNA and chitosan whilst the interaction between DNA and the polyamidoamine dendrimer appears to be only ionic interaction. No toxic effect on the mammalian cells was seen with chitosan. SuperFect and Lipofectin however, were observed to engender marked cytotoxicity. Poly-D,L-lactide (PLA) nanoparticles (40-80 nm) and poly-L-lactide (PLLA) lamellae (2-6 microm) were also used to load DNA by an adsorption procedure, but these failed to give good expression data.
本研究探讨了壳聚糖基因转染系统的制备及其体外转染效率。采用光子相关光谱法（PCS）测定，壳聚糖制备的纳米颗粒尺寸范围为40 - 200 nm，采用透射电子显微镜（TEM）测定则为40 - 80 nm。通过凝胶阻滞实验研究了颗粒与DNA的复合能力。使用编码萤火虫荧光素酶的质粒DNA pGL3-Control和编码β-半乳糖苷酶的pCH110作为报告基因。转染实验使用了293人胚肾细胞和中国仓鼠卵巢（CHO-K1）细胞。通过测定荧光素酶的表达，并以每毫克蛋白质的相对光单位（RLU/mg蛋白质）表示。结果表明，这些壳聚糖颗粒具有作为将DNA导入哺乳动物细胞载体的潜力。壳聚糖-pGL3-Control颗粒介导的细胞转染显示荧光素酶基因持续表达约10天。还使用了商业化转染试剂SuperFect和Lipofectin。与壳聚糖颗粒相比，SuperFect和Lipofectin的表达持续时间仅约2天。琼脂糖凝胶电泳和使用聚天冬氨酸的置换实验表明，DNA与壳聚糖之间可能存在多重相互作用，而DNA与聚酰胺胺树枝状大分子之间的相互作用似乎仅是离子相互作用。壳聚糖对哺乳动物细胞未见毒性作用。然而，观察到SuperFect和Lipofectin具有明显的细胞毒性。还通过吸附法使用聚-D,L-丙交酯（PLA）纳米颗粒（40 - 80 nm）和聚-L-丙交酯（PLLA）薄片（2 - 6微米）负载DNA，但这些未能给出良好的表达数据。
