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 : 002399 ( Istex/Corpus ); précédent : 002398; suivant : 002400In 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 ; Atsushi Harada ; Yuichi Yamasaki ; Kozo Nakamura ; Hiroshi Kawaguchi ; Kazunori KataokaSource :
- 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
Links to Exploration step
ISTEX:BA4E966450AC096AC778EE500818F9D9BAEF5555Le document en format XML
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Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</mods:affiliation></affiliation></author><author><name sortKey="Harada, Atsushi" sort="Harada, Atsushi" uniqKey="Harada A" first="Atsushi" last="Harada">Atsushi Harada</name><affiliation><mods:affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</mods:affiliation></affiliation></author><author><name sortKey="Yamasaki, Yuichi" sort="Yamasaki, Yuichi" uniqKey="Yamasaki Y" first="Yuichi" last="Yamasaki">Yuichi Yamasaki</name><affiliation><mods:affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</mods:affiliation></affiliation></author><author><name sortKey="Nakamura, Kozo" sort="Nakamura, Kozo" uniqKey="Nakamura K" first="Kozo" last="Nakamura">Kozo Nakamura</name><affiliation><mods:affiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</mods:affiliation></affiliation></author><author><name sortKey="Kawaguchi, Hiroshi" sort="Kawaguchi, Hiroshi" uniqKey="Kawaguchi H" first="Hiroshi" last="Kawaguchi">Hiroshi Kawaguchi</name><affiliation><mods:affiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</mods:affiliation></affiliation></author><author><name sortKey="Kataoka, Kazunori" sort="Kataoka, Kazunori" uniqKey="Kataoka K" first="Kazunori" last="Kataoka">Kazunori Kataoka</name><affiliation><mods:affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: kataoka@bmw.t.u‐tokyo.ac.jp</mods:affiliation></affiliation><affiliation><mods:affiliation>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, Japan.</mods:affiliation></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><istex><corpusName>wiley</corpusName><keywords><teeft><json:string>pdna</json:string><json:string>polyplexes</json:string><json:string>transfection</json:string><json:string>lpei</json:string><json:string>uorescein</json:string><json:string>polyplex</json:string><json:string>bpei</json:string><json:string>intracellular</json:string><json:string>endosomes</json:string><json:string>gene expression</json:string><json:string>decondensation</json:string><json:string>polyethylenimine</json:string><json:string>john wiley sons</json:string><json:string>copyright</json:string><json:string>complexed</json:string><json:string>confocal</json:string><json:string>uorescence</json:string><json:string>pdna decondensation</json:string><json:string>laser</json:string><json:string>software</json:string><json:string>cationic</json:string><json:string>condensed state</json:string><json:string>interexchange reaction</json:string><json:string>gene delivery</json:string><json:string>polymer</json:string><json:string>corresponding phase contrast images</json:string><json:string>complexed pdna</json:string><json:string>molecular weight</json:string><json:string>atomic force microscopy</json:string><json:string>uorescence resonance energy transfer</json:string><json:string>luciferase gene expression</json:string><json:string>gene</json:string><json:string>culture medium</json:string><json:string>intracellular behavior</json:string><json:string>laser confocal microscopy</json:string><json:string>emission intensity ratio</json:string><json:string>linear polyethylenimine</json:string><json:string>confocal microscopy</json:string><json:string>physiological conditions</json:string><json:string>pdna dissociation</json:string><json:string>ratio image</json:string><json:string>cytoplasm</json:string></teeft></keywords><author><json:item><name>Keiji Itaka</name><affiliations><json:string>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</json:string><json:string>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</json:string></affiliations></json:item><json:item><name>Atsushi Harada</name><affiliations><json:string>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</json:string></affiliations></json:item><json:item><name>Yuichi Yamasaki</name><affiliations><json:string>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</json:string></affiliations></json:item><json:item><name>Kozo Nakamura</name><affiliations><json:string>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</json:string></affiliations></json:item><json:item><name>Hiroshi Kawaguchi</name><affiliations><json:string>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</json:string></affiliations></json:item><json:item><name>Kazunori Kataoka</name><affiliations><json:string>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</json:string><json:string>E-mail: kataoka@bmw.t.u‐tokyo.ac.jp</json:string><json:string>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, Japan.</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>polyethylenimine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>fluorescence resonance energy transfer</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>confocal microscopy</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>transfection</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>nucleic acid conformation</value></json:item></subject><articleId><json:string>JGM470</json:string></articleId><arkIstex>ark:/67375/WNG-18CD6L0T-N</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>article</json:string></originalGenre><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.</abstract><qualityIndicators><score>9.927</score><pdfWordCount>4927</pdfWordCount><pdfCharCount>31482</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>9</pdfPageCount><pdfPageSize>595 x 859 pts</pdfPageSize><pdfWordsPerPage>547</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>262</abstractWordCount><abstractCharCount>1882</abstractCharCount><keywordCount>6</keywordCount></qualityIndicators><title>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</title><pmid><json:string>14716679</json:string></pmid><genre><json:string>article</json:string></genre><host><title>The Journal of Gene Medicine</title><language><json:string>unknown</json:string></language><doi><json:string>10.1002/(ISSN)1521-2254</json:string></doi><issn><json:string>1099-498X</json:string></issn><eissn><json:string>1521-2254</json:string></eissn><publisherId><json:string>JGM</json:string></publisherId><volume>6</volume><issue>1</issue><pages><first>76</first><last>84</last><total>9</total></pages><genre><json:string>journal</json:string></genre><subject><json:item><value>Research Article</value></json:item><json:item><value>Research Articles</value></json:item></subject></host><namedEntities><unitex><date><json:string>50 to 1/100</json:string><json:string>7-3-1</json:string><json:string>2004</json:string></date><geogName></geogName><orgName><json:string>Sons, Ltd.</json:string><json:string>Japan Science and Technology</json:string><json:string>Digital Instruments</json:string><json:string>Aldrich Chemical Co.</json:string><json:string>Ministry of Education, Culture, Sports, Science and Technology of Japan</json:string><json:string>CREST</json:string><json:string>Sons, Ltd</json:string><json:string>Beckman Coulter, Inc.</json:string><json:string>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo</json:string><json:string>University of Tokyo</json:string><json:string>Core Research Program for Evolutional Science and Technology</json:string><json:string>USA</json:string><json:string>Department of Materials Science and Engineering</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>John Wiley</json:string><json:string>Carl Zeiss</json:string></persName><placeName><json:string>Germany</json:string><json:string>Japan</json:string><json:string>Tokyo</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>[23]</json:string><json:string>[18]</json:string><json:string>[6]</json:string><json:string>[2,3]</json:string><json:string>[22]</json:string><json:string>[1]</json:string><json:string>Akinc et al.</json:string><json:string>[21]</json:string><json:string>Godbey et al. 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[27]</json:string><json:string>[24]</json:string><json:string>[4,5]</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/WNG-18CD6L0T-N</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - medicine, research & experimental</json:string><json:string>2 - genetics & heredity</json:string><json:string>2 - biotechnology & applied microbiology</json:string></wos><scienceMetrix><json:string>1 - applied sciences</json:string><json:string>2 - enabling & strategic technologies</json:string><json:string>3 - biotechnology</json:string></scienceMetrix><scopus><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Genetics(clinical)</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Drug Discovery</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, 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xml:id="author-0000"><persName><forename type="first">Keiji</forename><surname>Itaka</surname></persName><affiliation><orgName type="division">Department of Materials Science and Engineering</orgName><orgName type="department">Graduate School of Engineering</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8656, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation><affiliation><orgName type="division">Department of Orthopaedic Surgery</orgName><orgName type="department">Faculty of Medicine</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8655, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation></author><author xml:id="author-0001"><persName><forename type="first">Atsushi</forename><surname>Harada</surname></persName><affiliation><orgName type="division">Department of Materials Science and Engineering</orgName><orgName type="department">Graduate School of Engineering</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8656, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation></author><author xml:id="author-0002"><persName><forename type="first">Yuichi</forename><surname>Yamasaki</surname></persName><affiliation><orgName type="division">Department of Materials Science and Engineering</orgName><orgName type="department">Graduate School of Engineering</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8656, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation></author><author xml:id="author-0003"><persName><forename type="first">Kozo</forename><surname>Nakamura</surname></persName><affiliation><orgName type="division">Department of Orthopaedic Surgery</orgName><orgName type="department">Faculty of Medicine</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8655, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation></author><author xml:id="author-0004"><persName><forename type="first">Hiroshi</forename><surname>Kawaguchi</surname></persName><affiliation><orgName type="division">Department of Orthopaedic Surgery</orgName><orgName type="department">Faculty of Medicine</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8655, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation></author><author xml:id="author-0005" role="corresp"><persName><forename type="first">Kazunori</forename><surname>Kataoka</surname></persName><email>kataoka@bmw.t.u‐tokyo.ac.jp</email><affiliation><orgName type="division">Department of Materials Science and Engineering</orgName><orgName type="department">Graduate School of Engineering</orgName><orgName type="institution">The University of Tokyo</orgName><address><addrLine>7‐3‐1 Hongo</addrLine><addrLine>Bunkyo‐ku, Tokyo 113‐ 8656, Japan</addrLine><country key="JP" xml:lang="en">JAPAN</country></address></affiliation></author><idno type="istex">BA4E966450AC096AC778EE500818F9D9BAEF5555</idno><idno type="ark">ark:/67375/WNG-18CD6L0T-N</idno><idno type="DOI">10.1002/jgm.470</idno><idno type="unit">JGM470</idno><idno type="toTypesetVersion">file:JGM.JGM470.pdf</idno></analytic><monogr><title level="j" type="main">The Journal of Gene Medicine</title><title level="j" type="alt">JOURNAL OF GENE MEDICINE, THE</title><idno type="pISSN">1099-498X</idno><idno type="eISSN">1521-2254</idno><idno type="book-DOI">10.1002/(ISSN)1521-2254</idno><idno type="book-part-DOI">10.1002/jgm.v6:1</idno><idno type="product">JGM</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"></date></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.10" when="2019-12-20"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract xml:lang="en" style="main"><head>Abstract</head><head>Background</head><p>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.</p><head>Methods</head><p>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.</p><head>Results</head><p>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.</p><head>Conclusion</head><p>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.</p></abstract><textClass><keywords xml:lang="en"><term xml:id="kwd1">polyethylenimine</term><term xml:id="kwd2">DNA</term><term xml:id="kwd3">fluorescence resonance energy transfer</term><term xml:id="kwd4">confocal microscopy</term><term xml:id="kwd5">transfection</term><term xml:id="kwd6">nucleic acid conformation</term></keywords><keywords rend="articleCategory"><term>Research Article</term></keywords><keywords rend="tocHeading1"><term>Research Articles</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2019-12-20" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/WNG-18CD6L0T-N/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component version="2.0" type="serialArticle" xml:lang="en"><header><publicationMeta level="product"><publisherInfo><publisherName>John Wiley & Sons, Ltd.</publisherName><publisherLoc>Chichester, UK</publisherLoc></publisherInfo><doi registered="yes">10.1002/(ISSN)1521-2254</doi><issn type="print">1099-498X</issn><issn type="electronic">1521-2254</issn><idGroup><id type="product" value="JGM"></id></idGroup><titleGroup><title type="main" xml:lang="en" sort="JOURNAL OF GENE MEDICINE, THE">The Journal of Gene Medicine</title><title type="subtitle">A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications</title><title type="short">J. Gene Med.</title></titleGroup></publicationMeta><publicationMeta level="part" position="10"><doi origin="wiley" registered="yes">10.1002/jgm.v6:1</doi><numberingGroup><numbering type="journalVolume" number="6">6</numbering><numbering type="journalIssue">1</numbering></numberingGroup><coverDate startDate="2004-01">January 2004</coverDate></publicationMeta><publicationMeta level="unit" type="article" position="9" status="forIssue"><doi origin="wiley" registered="yes">10.1002/jgm.470</doi><idGroup><id type="unit" value="JGM470"></id></idGroup><countGroup><count type="pageTotal" number="9"></count></countGroup><titleGroup><title type="articleCategory">Research Article</title><title type="tocHeading1">Research Articles</title></titleGroup><copyright ownership="publisher">Copyright © 2004 John Wiley & Sons, Ltd.</copyright><eventGroup><event type="manuscriptReceived" date="2003-05-09"></event><event type="manuscriptRevised" date="2003-07-11"></event><event type="manuscriptAccepted" date="2003-07-11"></event><event type="firstOnline" date="2004-01-06"></event><event type="publishedOnlineFinalForm" date="2004-01-06"></event><event type="xmlConverted" agent="Converter:JWSART34_TO_WML3G version:2.3.2 mode:FullText source:FullText result:FullText mathml2tex" date="2010-03-04"></event><event type="xmlConverted" agent="Converter:WILEY_ML3G_TO_WILEY_ML3GV2 version:3.8.8" date="2014-01-30"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.1.7 mode:FullText,remove_FC" date="2014-11-04"></event></eventGroup><numberingGroup><numbering type="pageFirst">76</numbering><numbering type="pageLast">84</numbering></numberingGroup><correspondenceTo>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐8656, Japan.</correspondenceTo><linkGroup><link type="toTypesetVersion" href="file:JGM.JGM470.pdf"></link></linkGroup></publicationMeta><contentMeta><countGroup><count type="figureTotal" number="8"></count><count type="tableTotal" number="0"></count><count type="referenceTotal" number="28"></count></countGroup><titleGroup><title type="main" xml:lang="en"><i>In situ</i> single cell observation by fluorescence resonance energy transfer reveals fast intra‐cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine</title><title type="short" xml:lang="en">Intracellular FRET Observation of pDNA</title></titleGroup><creators><creator xml:id="au1" creatorRole="author" affiliationRef="#af1 #af2"><personName><givenNames>Keiji</givenNames><familyName>Itaka</familyName></personName></creator><creator xml:id="au2" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Atsushi</givenNames><familyName>Harada</familyName></personName></creator><creator xml:id="au3" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Yuichi</givenNames><familyName>Yamasaki</familyName></personName></creator><creator xml:id="au4" creatorRole="author" affiliationRef="#af2"><personName><givenNames>Kozo</givenNames><familyName>Nakamura</familyName></personName></creator><creator xml:id="au5" creatorRole="author" affiliationRef="#af2"><personName><givenNames>Hiroshi</givenNames><familyName>Kawaguchi</familyName></personName></creator><creator xml:id="au6" creatorRole="author" affiliationRef="#af1" corresponding="yes"><personName><givenNames>Kazunori</givenNames><familyName>Kataoka</familyName></personName><contactDetails><email normalForm="kataoka@bmw.t.u-tokyo.ac.jp">kataoka@bmw.t.u‐tokyo.ac.jp</email></contactDetails></creator></creators><affiliationGroup><affiliation xml:id="af1" countryCode="JP" type="organization"><unparsedAffiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</unparsedAffiliation></affiliation><affiliation xml:id="af2" countryCode="JP" type="organization"><unparsedAffiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</unparsedAffiliation></affiliation></affiliationGroup><keywordGroup xml:lang="en" type="author"><keyword xml:id="kwd1">polyethylenimine</keyword><keyword xml:id="kwd2">DNA</keyword><keyword xml:id="kwd3">fluorescence resonance energy transfer</keyword><keyword xml:id="kwd4">confocal microscopy</keyword><keyword xml:id="kwd5">transfection</keyword><keyword xml:id="kwd6">nucleic acid conformation</keyword></keywordGroup><fundingInfo><fundingAgency>Grants‐in‐Aid for Scientific Research</fundingAgency><fundingNumber>11167210 and 12877221</fundingNumber></fundingInfo><fundingInfo><fundingAgency>Special Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology of Japan</fundingAgency></fundingInfo><fundingInfo><fundingAgency>Core Research Program for Evolutional Science and Technology, Japan Science and Technology Corporation</fundingAgency></fundingInfo><abstractGroup><abstract type="main" xml:lang="en"><title type="main">Abstract</title><section xml:id="abs1-1"><title type="main">Background</title><p>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.</p></section><section xml:id="abs1-2"><title type="main">Methods</title><p>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.</p></section><section xml:id="abs1-3"><title type="main">Results</title><p>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.</p></section><section xml:id="abs1-4"><title type="main">Conclusion</title><p>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.</p></section></abstract></abstractGroup></contentMeta></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>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</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>Intracellular FRET Observation of pDNA</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>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</title></titleInfo><name type="personal"><namePart type="given">Keiji</namePart><namePart type="family">Itaka</namePart><affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</affiliation><affiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Atsushi</namePart><namePart type="family">Harada</namePart><affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Yuichi</namePart><namePart type="family">Yamasaki</namePart><affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Kozo</namePart><namePart type="family">Nakamura</namePart><affiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Hiroshi</namePart><namePart type="family">Kawaguchi</namePart><affiliation>Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8655, Japan</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Kazunori</namePart><namePart type="family">Kataoka</namePart><affiliation>Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐ 8656, Japan</affiliation><affiliation>E-mail: kataoka@bmw.t.u‐tokyo.ac.jp</affiliation><affiliation>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, Japan.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</genre><originInfo><publisher>John Wiley & Sons, Ltd.</publisher><place><placeTerm type="text">Chichester, UK</placeTerm></place><dateIssued encoding="w3cdtf">2004-01</dateIssued><dateCaptured encoding="w3cdtf">2003-05-09</dateCaptured><dateValid encoding="w3cdtf">2003-07-11</dateValid><copyrightDate encoding="w3cdtf">2004</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><extent unit="figures">8</extent><extent unit="tables">0</extent><extent unit="references">28</extent></physicalDescription><abstract 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.</abstract><note type="funding">Grants‐in‐Aid for Scientific Research - No. 11167210 and 12877221; </note><note type="funding">Special Coordination Funds for Promoting Science and Technology, Ministry of Education, Culture, Sports, Science and Technology of Japan</note><note type="funding">Core Research Program for Evolutional Science and Technology, Japan Science and Technology Corporation</note><subject lang="en"><genre>keywords</genre><topic>polyethylenimine</topic><topic>DNA</topic><topic>fluorescence resonance energy transfer</topic><topic>confocal microscopy</topic><topic>transfection</topic><topic>nucleic acid conformation</topic></subject><relatedItem type="host"><titleInfo><title>The Journal of Gene Medicine</title><subTitle>A cross‐disciplinary journal for research on the science of gene transfer and its clinical applications</subTitle></titleInfo><titleInfo type="abbreviated"><title>J. Gene Med.</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><subject><genre>article-category</genre><topic>Research Article</topic><topic>Research Articles</topic></subject><identifier type="ISSN">1099-498X</identifier><identifier type="eISSN">1521-2254</identifier><identifier type="DOI">10.1002/(ISSN)1521-2254</identifier><identifier type="PublisherID">JGM</identifier><part><date>2004</date><detail type="volume"><caption>vol.</caption><number>6</number></detail><detail type="issue"><caption>no.</caption><number>1</number></detail><extent unit="pages"><start>76</start><end>84</end><total>9</total></extent></part></relatedItem><relatedItem type="references" displayLabel="cit1"><titleInfo><title>Development of biomaterials for gene therapy</title></titleInfo><name type="personal"><namePart type="given">S</namePart><namePart type="family">Han</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">RI</namePart><namePart type="family">Mahato</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">YK</namePart><namePart type="family">Sung</namePart><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">SW</namePart><namePart type="family">Kim</namePart><role><roleTerm type="text">author</roleTerm></role></name><genre>journal-article</genre><note type="citation/reference">Han S, Mahato RI, Sung YK, Kim SW. 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