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Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components

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 distri... Full description

Journal Title: Gene Therapy Jun 2001, Vol.8(11), pp.828-36
Main Author: Uyechi, L
Other Authors: Gagné, L , Thurston, G , Szoka, F
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: ISSN: 09697128 ; DOI: 10.1038/sj.gt.3301461
Link: http://search.proquest.com/docview/218711049/?pq-origsite=primo
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title: Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components
format: Article
creator:
  • Uyechi, L
  • Gagné, L
  • Thurston, G
  • Szoka, F
subjects:
  • Animals–Metabolism
  • Cell Nucleus–Metabolism
  • Cytoplasm–Methods
  • Female–Administration & Dosage
  • Fluorescent Dyes–Metabolism
  • Gene Expression–Genetics
  • Gene Transfer Techniques–Genetics
  • Genetic Therapy–Genetics
  • Genetic Vectors–Genetics
  • Green Fluorescent Proteins–Genetics
  • Injections, Intravenous–Genetics
  • Lectins–Genetics
  • Liposomes–Genetics
  • Luminescent Proteins–Genetics
  • Lung–Genetics
  • Mice–Genetics
  • Mice, Inbred Strains–Genetics
  • Microscopy, Confocal–Genetics
  • Oligonucleotides–Genetics
  • Trachea–Genetics
  • Fluorescent Dyes
  • Lectins
  • Liposomes
  • Luminescent Proteins
  • Oligonucleotides
  • Green Fluorescent Proteins
ispartof: Gene Therapy, Jun 2001, Vol.8(11), pp.828-36
description: 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.
language: eng
source:
identifier: ISSN: 09697128 ; DOI: 10.1038/sj.gt.3301461
fulltext: fulltext
issn:
  • 09697128
  • 0969-7128
url: Link


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titleMechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components
creatorUyechi, L ; Gagné, L ; Thurston, G ; Szoka, F
ispartofGene Therapy, Jun 2001, Vol.8(11), pp.828-36
identifierISSN: 09697128 ; DOI: 10.1038/sj.gt.3301461
subjectAnimals–Metabolism ; Cell Nucleus–Metabolism ; Cytoplasm–Methods ; Female–Administration & Dosage ; Fluorescent Dyes–Metabolism ; Gene Expression–Genetics ; Gene Transfer Techniques–Genetics ; Genetic Therapy–Genetics ; Genetic Vectors–Genetics ; Green Fluorescent Proteins–Genetics ; Injections, Intravenous–Genetics ; Lectins–Genetics ; Liposomes–Genetics ; Luminescent Proteins–Genetics ; Lung–Genetics ; Mice–Genetics ; Mice, Inbred Strains–Genetics ; Microscopy, Confocal–Genetics ; Oligonucleotides–Genetics ; Trachea–Genetics ; Fluorescent Dyes ; Lectins ; Liposomes ; Luminescent Proteins ; Oligonucleotides ; Green Fluorescent Proteins
descriptionWe 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.
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titleMechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components
descriptionWe 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.
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titleMechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components
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6Gene Transfer Techniques–Genetics
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8Genetic Vectors–Genetics
9Green Fluorescent Proteins–Genetics
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abstractWe 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.
copHoundmills
pubNature Publishing Group
doi10.1038/sj.gt.3301461
urlhttp://search.proquest.com/docview/218711049/
eissn14765462
date2001-06-01