Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components
| Autor: | G Thurston, Lisa S. Uyechi, Francis C. Szoka, Lucie Gagne |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Cytoplasm
Genetic Vectors Green Fluorescent Proteins Oligonucleotides Gene Expression Mice Inbred Strains Gene delivery Biology Green fluorescent protein law.invention Mice Confocal microscopy law Lectins Gene expression Genetics Fluorescence microscope Animals Molecular Biology Lung Fluorescent Dyes Cell Nucleus Microscopy Confocal Oligonucleotide Genetic transfer Gene Transfer Techniques Genetic Therapy Molecular biology Trachea Luminescent Proteins Injections Intravenous Liposomes Nucleic acid Molecular Medicine lipids (amino acids peptides and proteins) Female |
| Zdroj: | Gene therapy. 8(11) |
| ISSN: | 0969-7128 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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