In situ single cell observation by fluorescence resonance energy transfer reveals fast intra‐cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine
Identifieur interne : 001F86 ( Main/Exploration ); précédent : 001F85; suivant : 001F87In situ single cell observation by fluorescence resonance energy transfer reveals fast intra‐cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine
Auteurs : Keiji Itaka [Japon] ; Atsushi Harada [Japon] ; Yuichi Yamasaki [Japon] ; Kozo Nakamura [Japon] ; Hiroshi Kawaguchi [Japon] ; Kazunori Kataoka [Japon]Source :
- The Journal of Gene Medicine [ 1099-498X ] ; 2004-01.
English descriptors
- Teeft :
- Atomic force microscopy, Bpei, Cationic, Complexed, Complexed pdna, Condensed state, Confocal, Confocal microscopy, Copyright, Corresponding phase contrast images, Culture medium, Cytoplasm, Decondensation, Emission intensity ratio, Endosomes, Gene, Gene delivery, Gene expression, Interexchange reaction, Intracellular, Intracellular behavior, John wiley sons, Laser, Laser confocal microscopy, Linear polyethylenimine, Lpei, Luciferase gene expression, Molecular weight, Pdna, Pdna decondensation, Pdna dissociation, Physiological conditions, Polyethylenimine, Polymer, Polyplex, Polyplexes, Ratio image, Software, Transfection, Uorescein, Uorescence, Uorescence resonance energy transfer.
Abstract
Background: The investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non‐viral gene delivery system. Methods: Intracellular trafficking as well as release of plasmid DNA (pDNA) complexed with polycations, including linear and branched polyethylenimine (LPEI, BPEI) and poly(L‐lysine) (PLL), were explored under confocal microscopy using fluorescence resonance energy transfer (FRET) between a pair of donor–acceptor fluorescent dyes (fluorescein and Cy3) tagged on a single pDNA molecule. Results: pDNA complexed with LPEI underwent a rapid escape from the endosomes, spreading uniformly into the cytoplasm with a substantial decrease in FRET efficiency due to the disintegration of LPEI/pDNA polyplex structure. pDNA complexed with BPEI also achieved a rapid escape from the endosomes. Nevertheless, the pDNA retained high FRET efficiency even after 24 h, indicating an appreciable stability of the BPEI/pDNA polyplex to keep pDNA in a condensed state. In the PLL/pDNA polyplexes, neither endosome escape nor pDNA decondensation was observed. These intracellular characteristics of polyplexes showed a clear correlation to their gene transfection efficiency: The LPEI/pDNA revealed a considerably higher and faster gene expression compared with BPEI/pDNA. Atomic force microscopy revealed that BPEI induced more effective condensation of pDNA than LPEI, being consistent with restricted cytoplasmic release of complexed pDNA. Conclusion: Fast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system. These properties may be particularly beneficial to achieve appreciably high gene expression in a prompt manner. Copyright © 2003 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/jgm.470
Affiliations:
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Kawaguchi</name></author><author><name sortKey="Kataoka, Kazunori" sort="Kataoka, Kazunori" uniqKey="Kataoka K" first="Kazunori" last="Kataoka">Kazunori Kataoka</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:BA4E966450AC096AC778EE500818F9D9BAEF5555</idno><date when="2004" year="2004">2004</date><idno type="doi">10.1002/jgm.470</idno><idno type="url">https://api.istex.fr/ark:/67375/WNG-18CD6L0T-N/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002399</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002399</idno><idno type="wicri:Area/Istex/Curation">002399</idno><idno type="wicri:Area/Istex/Checkpoint">000E27</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">000E27</idno><idno type="wicri:doubleKey">1099-498X:2004:Itaka K:in:situ:single</idno><idno type="wicri:Area/Main/Merge">002001</idno><idno 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sort="Yamasaki, Yuichi" uniqKey="Yamasaki Y" first="Yuichi" last="Yamasaki">Yuichi Yamasaki</name><affiliation wicri:level="4"><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656</wicri:regionArea><orgName type="university">Université de Tokyo</orgName><placeName><settlement type="city">Tokyo</settlement><region type="province">Région de Kantō</region></placeName></affiliation></author><author><name sortKey="Nakamura, Kozo" sort="Nakamura, Kozo" uniqKey="Nakamura K" first="Kozo" last="Nakamura">Kozo Nakamura</name><affiliation wicri:level="4"><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655</wicri:regionArea><orgName type="university">Université de Tokyo</orgName><placeName><settlement type="city">Tokyo</settlement><region type="province">Région de Kantō</region></placeName></affiliation></author><author><name sortKey="Kawaguchi, Hiroshi" sort="Kawaguchi, Hiroshi" uniqKey="Kawaguchi H" first="Hiroshi" last="Kawaguchi">Hiroshi Kawaguchi</name><affiliation wicri:level="4"><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655</wicri:regionArea><orgName type="university">Université de Tokyo</orgName><placeName><settlement type="city">Tokyo</settlement><region type="province">Région de Kantō</region></placeName></affiliation></author><author><name sortKey="Kataoka, Kazunori" sort="Kataoka, Kazunori" uniqKey="Kataoka K" first="Kazunori" last="Kataoka">Kazunori Kataoka</name><affiliation wicri:level="4"><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656</wicri:regionArea><orgName type="university">Université de Tokyo</orgName><placeName><settlement type="city">Tokyo</settlement><region type="province">Région de Kantō</region></placeName></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Japon</country></affiliation><affiliation wicri:level="4"><country xml:lang="fr">Japon</country><wicri:regionArea>Correspondence address: Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐8656</wicri:regionArea><placeName><settlement type="city">Tokyo</settlement><region type="région">Région de Kantō</region></placeName><orgName type="university">Université de Tokyo</orgName><placeName><settlement type="city">Tokyo</settlement><region type="province">Région de Kantō</region></placeName></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">The Journal of Gene Medicine</title><title level="j" type="alt">JOURNAL OF GENE MEDICINE, THE</title><idno type="ISSN">1099-498X</idno><idno type="eISSN">1521-2254</idno><imprint><biblScope unit="vol">6</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="76">76</biblScope><biblScope unit="page" to="84">84</biblScope><biblScope unit="page-count">9</biblScope><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><date type="published" when="2004-01">2004-01</date></imprint><idno type="ISSN">1099-498X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1099-498X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Atomic force microscopy</term><term>Bpei</term><term>Cationic</term><term>Complexed</term><term>Complexed pdna</term><term>Condensed state</term><term>Confocal</term><term>Confocal microscopy</term><term>Copyright</term><term>Corresponding phase contrast images</term><term>Culture medium</term><term>Cytoplasm</term><term>Decondensation</term><term>Emission intensity ratio</term><term>Endosomes</term><term>Gene</term><term>Gene delivery</term><term>Gene expression</term><term>Interexchange reaction</term><term>Intracellular</term><term>Intracellular behavior</term><term>John wiley sons</term><term>Laser</term><term>Laser confocal microscopy</term><term>Linear polyethylenimine</term><term>Lpei</term><term>Luciferase gene expression</term><term>Molecular weight</term><term>Pdna</term><term>Pdna decondensation</term><term>Pdna dissociation</term><term>Physiological conditions</term><term>Polyethylenimine</term><term>Polymer</term><term>Polyplex</term><term>Polyplexes</term><term>Ratio image</term><term>Software</term><term>Transfection</term><term>Uorescein</term><term>Uorescence</term><term>Uorescence resonance energy transfer</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Background: The investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non‐viral gene delivery system. Methods: Intracellular trafficking as well as release of plasmid DNA (pDNA) complexed with polycations, including linear and branched polyethylenimine (LPEI, BPEI) and poly(L‐lysine) (PLL), were explored under confocal microscopy using fluorescence resonance energy transfer (FRET) between a pair of donor–acceptor fluorescent dyes (fluorescein and Cy3) tagged on a single pDNA molecule. Results: pDNA complexed with LPEI underwent a rapid escape from the endosomes, spreading uniformly into the cytoplasm with a substantial decrease in FRET efficiency due to the disintegration of LPEI/pDNA polyplex structure. pDNA complexed with BPEI also achieved a rapid escape from the endosomes. Nevertheless, the pDNA retained high FRET efficiency even after 24 h, indicating an appreciable stability of the BPEI/pDNA polyplex to keep pDNA in a condensed state. In the PLL/pDNA polyplexes, neither endosome escape nor pDNA decondensation was observed. These intracellular characteristics of polyplexes showed a clear correlation to their gene transfection efficiency: The LPEI/pDNA revealed a considerably higher and faster gene expression compared with BPEI/pDNA. Atomic force microscopy revealed that BPEI induced more effective condensation of pDNA than LPEI, being consistent with restricted cytoplasmic release of complexed pDNA. Conclusion: Fast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system. These properties may be particularly beneficial to achieve appreciably high gene expression in a prompt manner. Copyright © 2003 John Wiley & Sons, Ltd.</div></front></TEI><affiliations><list><country><li>Japon</li></country><region><li>Région de Kantō</li></region><settlement><li>Tokyo</li></settlement><orgName><li>Université de Tokyo</li></orgName></list><tree><country name="Japon"><region name="Région de Kantō"><name sortKey="Itaka, Keiji" sort="Itaka, Keiji" uniqKey="Itaka K" first="Keiji" last="Itaka">Keiji Itaka</name></region><name sortKey="Harada, Atsushi" sort="Harada, Atsushi" uniqKey="Harada A" first="Atsushi" last="Harada">Atsushi Harada</name><name sortKey="Itaka, Keiji" sort="Itaka, Keiji" uniqKey="Itaka K" first="Keiji" last="Itaka">Keiji Itaka</name><name sortKey="Kataoka, Kazunori" sort="Kataoka, Kazunori" uniqKey="Kataoka K" first="Kazunori" last="Kataoka">Kazunori Kataoka</name><name sortKey="Kataoka, Kazunori" sort="Kataoka, Kazunori" uniqKey="Kataoka K" first="Kazunori" last="Kataoka">Kazunori Kataoka</name><name sortKey="Kataoka, Kazunori" sort="Kataoka, Kazunori" uniqKey="Kataoka K" first="Kazunori" last="Kataoka">Kazunori Kataoka</name><name sortKey="Kawaguchi, Hiroshi" sort="Kawaguchi, Hiroshi" uniqKey="Kawaguchi H" first="Hiroshi" last="Kawaguchi">Hiroshi Kawaguchi</name><name sortKey="Nakamura, Kozo" sort="Nakamura, Kozo" uniqKey="Nakamura K" first="Kozo" last="Nakamura">Kozo Nakamura</name><name sortKey="Yamasaki, Yuichi" sort="Yamasaki, Yuichi" uniqKey="Yamasaki Y" first="Yuichi" last="Yamasaki">Yuichi Yamasaki</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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