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1.
Gene Ther ; 8(11): 828-36, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11423930

RESUMO

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.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Pulmão/metabolismo , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Corantes Fluorescentes , Expressão Gênica , Proteínas de Fluorescência Verde , Injeções Intravenosas , Lectinas , Lipossomos , Proteínas Luminescentes , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , Oligonucleotídeos/genética , Traqueia
2.
Gene Ther ; 6(6): 1179-83, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10455423

RESUMO

It was recently suggested that intravenously administered lipoplexes serve as a depot for the extracellular release of naked DNA and it is the naked DNA that mediates gene delivery in the lung. If this is the mechanism responsible for gene expression, we reasoned that continuous infusion of plasmid DNA should also result in significant lung expression in the absence of lipoplexes. Moreover, the infusion of non-coding plasmid DNA should inhibit gene delivery by lipoplexes. Infusion of plasmid DNA at a rate of 80 microg/min into the tail vein of a mouse resulted in a DNA serum concentration of 800 microg/ml. This was equivalent to a transcriptionally active DNA concentration of 120 microg/ml plasma as determined by an in vitro transfection assay. In spite of this high level of transcriptionally active DNA, there was no significant gene expression in the lung or any other organ tested. In addition, when lipoplex containing a reporter gene was injected, followed by an infusion of non-coding plasmid DNA as a potential competing molecule for DNA released from the lipoplex there was no effect on gene expression. These experiments indicate that the cationic lipid component of the lipoplex functions in an active capacity beyond that of a simple passive release matrix for plasmid DNA.


Assuntos
Técnicas de Transferência de Genes , Lipídeos , Vacinas de DNA/administração & dosagem , Animais , Genes Reporter/genética , Infusões Intravenosas , Camundongos , Plasmídeos
3.
Biochim Biophys Acta ; 1390(2): 119-33, 1998 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-9507083

RESUMO

The interactions among serum components and cationic lipid-nucleic acid complexes are central to the understanding of how serum inhibits cellular delivery of oligonucleotides in vitro and in vivo. In this study, we show that several serum proteins, in particular bovine serum albumin (BSA), lipoproteins (HDL and LDL) and macroglobulin, interact with cationic lipid/oligonucleotide complexes, alter the complex diameter and zeta potential (from positive to negative values), and significantly interfere with the ability of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) to deliver phosphorothioate oligonucleotides (ODN) into cells. Serum and BSA do not dissociate the ODN and lipid components, therefore inhibition of delivery cannot be attributed to a displacement of cationic lipid from the ODN. Rather BSA at 2.5 mg/ml, comparable to the amount found in 10% serum, decreases the cell association of ODN by about 5-fold and nuclear uptake of ODN by greater than 20-fold. In contrast, immunoglobulin G, the other major serum component, alters the zeta potential from positive to near neutral, has a modest effect on the diameter of the complex but does not affect cell association or nuclear delivery of the ODN at amounts found in 10% serum. Other molecules found in serum, specifically oleic acid and heparin, displace the ODN from the complex and thus interfere with delivery. This displacement is attenuated by first incubating the complex with BSA. Another manifestation of serum-complex interactions is that ODN significantly and cationic liposomes slightly, activate complement. However, formation of the complex markedly reduces the complement activation of the ODN. Finally, the effect of serum can be partially counteracted by the selection of the helper lipid (DOPE or cholesterol). Inclusion of a helper lipid reduces the effective charge ratio (cationic groups/anionic thioates) required to deliver ODN into cells and permits delivery in the presence of greater percentages of serum in the culture medium. These results support the current view that the binding of serum proteins to the complex is a significant factor in modulating the activity of cationic lipid-ODN complexes in culture and after intravenous administration.


Assuntos
Proteínas Sanguíneas/farmacologia , Lipídeos/química , Oligonucleotídeos/química , Fosfatidiletanolaminas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Heparina/metabolismo , Heparina/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Microscopia de Fluorescência , Ácido Oleico/metabolismo , Ácido Oleico/farmacologia , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacocinética , Compostos de Amônio Quaternário/metabolismo , Albumina Sérica/metabolismo , Albumina Sérica/farmacologia
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