Uyechi L S, Gagné L, Thurston G, Szoka F C
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, San Francisco, CA 94143-0446, USA.
Gene Ther. 2001 Jun;8(11):828-36. doi: 10.1038/sj.gt.3301461.
We introduce a lung inflation-fixation protocol to examine the distribution and gene transfer efficiency of fluorescently tagged lipoplexes using fluorescence confocal microscopy within thick lung tissue sections. Using this technique, we tested the hypothesis that factors related to lipoplex distribution were the predominant reason that intravenous (i.v.) administration of lipoplex was superior to intratracheal (i.t.) administration for gene transfer in the murine lung. Lipoplex distribution was analyzed using digitized images of overlapping fields, reconstructed to view an entire lung lobe. Intravenously administered lipoplexes were confined to the capillary network and homogenously distributed throughout the lung lobe. In contrast, i.t. administration resulted in regional distribution of lipoplex, concentrated around bronchioles and distal airways. Not all the bronchioles were stained with lipoplex, suggesting that the airway-administered solution became channeled through certain bronchiolar pathways. A fluorescent oligonucleotide was used as a marker for cytoplasmic release of nucleic acids. Quantification of the resulting fluorescent nuclei was used to define the relationship between cytoplasmic release of nucleic acids and gene expression. Endothelial cells were stained after i.v. administration, and epithelial cells were stained after i.t. administration. The delivery of nucleic acids was also more homogeneous with i.v. administration of lipoplex than with i.t. administration. After i.t. administration, it was notable that high concentrations of fluorescent nuclei correlated with low GFP expression. This suggested that toxicity was associated with high local concentrations of cationic lipoplexes. The ratio of GFP-expressing cells to fluorescent nuclei indicated that capillary endothelial cells were more efficient in gene expression per delivery event than were pulmonary epithelial cells. Thus, the greater gene expression efficiency of i.v. administered lipoplexes was due not only to the initial distribution but also to the greater efficiency of the vascular endothelial cells to appropriately traffic and express the foreign gene.
我们引入一种肺膨胀固定方案，以使用荧光共聚焦显微镜在厚肺组织切片内检查荧光标记脂质体的分布和基因转移效率。利用该技术，我们检验了以下假设：与脂质体分布相关的因素是静脉内（i.v.）给药的脂质体在小鼠肺中进行基因转移优于气管内（i.t.）给药的主要原因。使用重叠视野的数字化图像分析脂质体分布，重建图像以观察整个肺叶。静脉内给药的脂质体局限于毛细血管网络，并均匀分布于整个肺叶。相比之下，i.t.给药导致脂质体呈区域分布，集中在细支气管和远端气道周围。并非所有细支气管都被脂质体染色，这表明气道给药溶液通过某些细支气管途径形成通道。一种荧光寡核苷酸用作核酸胞质释放的标志物。对所得荧光细胞核进行定量，以确定核酸胞质释放与基因表达之间的关系。静脉内给药后内皮细胞被染色，i.t.给药后上皮细胞被染色。脂质体静脉内给药时核酸的递送也比i.t.给药更均匀。i.t.给药后，值得注意的是高浓度荧光细胞核与低绿色荧光蛋白（GFP）表达相关。这表明毒性与阳离子脂质体的高局部浓度有关。表达GFP的细胞与荧光细胞核的比率表明，每次递送事件中毛细血管内皮细胞在基因表达方面比肺上皮细胞更有效。因此，静脉内给药的脂质体具有更高的基因表达效率，这不仅归因于初始分布，还归因于血管内皮细胞更有效地转运和表达外源基因。
