Ishii T, Okahata Y, Sato T
Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Japan.
Biochim Biophys Acta. 2001 Sep 3;1514(1):51-64. doi: 10.1016/s0005-2736(01)00362-5.
Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.
壳聚糖作为一种用于基因递送的非病毒载体很有用。尽管有几份报告支持使用壳聚糖进行基因递送，但关于其对转染的影响以及壳聚糖特异性转染机制的研究仍然不足。在本报告中，观察到在SOJ细胞中，质粒/壳聚糖的表达水平不低于质粒/脂质体复合物的表达水平。通过使用异硫氰酸荧光素标记的质粒和德克萨斯红标记的壳聚糖，分析了质粒/壳聚糖复合物的转染机制以及转染活性与细胞摄取之间的关系。关于对转染的影响，有几个因素会影响转染活性和细胞摄取，例如：壳聚糖的分子量、复合物的化学计量比，以及转染培养基的血清浓度和pH值。当壳聚糖的分子量为40或84 kDa、壳聚糖氮与DNA磷酸的比例（N/P比）为5且转染培养基在pH 7.0时含有10%血清时，发现质粒/壳聚糖复合物的转染水平最高。我们还研究了转染机制，发现质粒/壳聚糖复合物很可能凝聚形成大聚集体（5-8微米），这些聚集体吸附到细胞表面。此后，质粒/壳聚糖复合物被内吞，并且除了质粒/壳聚糖复合物肿胀外，还可能由于溶酶体肿胀而从内体释放，导致内体破裂。最后，使用共聚焦激光扫描显微镜还观察到复合物在细胞核中积累。
