Vectors based on reducible polycations facilitate intracellular release of nucleic acids
Identifieur interne : 002048 ( Main/Curation ); précédent : 002047; suivant : 002049Vectors based on reducible polycations facilitate intracellular release of nucleic acids
Auteurs : Martin L. Read [Royaume-Uni] ; K. Helen Bremner [Royaume-Uni] ; David Oupick [Royaume-Uni] ; Nicola K. Green [Royaume-Uni] ; Peter F. Searle [Royaume-Uni] ; Leonard W. Seymour [Royaume-Uni]Source :
- The Journal of Gene Medicine [ 1099-498X ] ; 2003-03.
English descriptors
- Teeft :
- Assay, Bioconjug chem, Cancer cells, Cancer gene, Cancer gene therapy, Cationic, Cationic lipid, Cationic lipids, Cell lines, Cells transfected, Cellular uptake, Chem, Chloroquine, Copyright, Cytometry analysis, Cytosolic nucleases, Dotap, Endosomal, Ethidium, Ethidium bromide, Expression plasmid, Fresh media, Gene, Gene delivery, Gene expression, Gene therapy, Gene transfer, Grey bars, Hela, Hela cells, Hepes buffer, High levels, Higher level, Imagequanttm software, Initial experiments, Intracellular, Intracellular activation, Intracellular delivery, Intracellular release, John wiley sons, Lipid, Lipoplexes, Lncap, Lncap cells, Luciferase, Luciferase activity, Molecular weight, Mrna, Nacl, Nitroreductase, Nuclease, Nuclease degradation, Nucleic, Nucleic acids, Oxidative polycondensation, Pegfpn1, Peptide, Photon correlation spectroscopy, Physiological conditions, Plasmid, Polycation, Polycations, Polyplexes, Prodrug, Reducible, Reducible polycations, Reporter gene expression, Similar levels, Standard deviation, Surface charge, Synthetic vectors, Toxicity, Transfected, Transfected cells, Transfection, Transfection activity, Uorescence, White bars.
Abstract
Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys10‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N‐(1‐(2,3‐dioleoyloxy)propyl)‐N, N, N‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors. Results: A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions: These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/jgm.331
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<term>Cancer gene therapy</term>
<term>Cationic</term>
<term>Cationic lipid</term>
<term>Cationic lipids</term>
<term>Cell lines</term>
<term>Cells transfected</term>
<term>Cellular uptake</term>
<term>Chem</term>
<term>Chloroquine</term>
<term>Copyright</term>
<term>Cytometry analysis</term>
<term>Cytosolic nucleases</term>
<term>Dotap</term>
<term>Endosomal</term>
<term>Ethidium</term>
<term>Ethidium bromide</term>
<term>Expression plasmid</term>
<term>Fresh media</term>
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<term>Gene delivery</term>
<term>Gene expression</term>
<term>Gene therapy</term>
<term>Gene transfer</term>
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<term>Hela</term>
<term>Hela cells</term>
<term>Hepes buffer</term>
<term>High levels</term>
<term>Higher level</term>
<term>Imagequanttm software</term>
<term>Initial experiments</term>
<term>Intracellular</term>
<term>Intracellular activation</term>
<term>Intracellular delivery</term>
<term>Intracellular release</term>
<term>John wiley sons</term>
<term>Lipid</term>
<term>Lipoplexes</term>
<term>Lncap</term>
<term>Lncap cells</term>
<term>Luciferase</term>
<term>Luciferase activity</term>
<term>Molecular weight</term>
<term>Mrna</term>
<term>Nacl</term>
<term>Nitroreductase</term>
<term>Nuclease</term>
<term>Nuclease degradation</term>
<term>Nucleic</term>
<term>Nucleic acids</term>
<term>Oxidative polycondensation</term>
<term>Pegfpn1</term>
<term>Peptide</term>
<term>Photon correlation spectroscopy</term>
<term>Physiological conditions</term>
<term>Plasmid</term>
<term>Polycation</term>
<term>Polycations</term>
<term>Polyplexes</term>
<term>Prodrug</term>
<term>Reducible</term>
<term>Reducible polycations</term>
<term>Reporter gene expression</term>
<term>Similar levels</term>
<term>Standard deviation</term>
<term>Surface charge</term>
<term>Synthetic vectors</term>
<term>Toxicity</term>
<term>Transfected</term>
<term>Transfected cells</term>
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<front><div type="abstract" xml:lang="en">Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L‐lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys‐Lys10‐Cys and used to condense nucleic acids. The stability of RPC‐based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC‐3 and B16‐F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N‐(1‐(2,3‐dioleoyloxy)propyl)‐N, N, N‐trimethylammonium chloride) (DOTAP). A CMV‐driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC‐based delivery vectors. Results: A 187‐fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non‐reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC‐3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20–75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions: These results demonstrate that lipid‐mediated activation of RPC‐based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity. Copyright © 2002 John Wiley & Sons, Ltd.</div>
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