Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes
Identifieur interne : 000719 ( Istex/Corpus ); précédent : 000718; suivant : 000720Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes
Auteurs : Patrick Midoux ; Michel MonsignySource :
- Bioconjugate Chemistry [ 1043-1802 ] ; 1999.
Abstract
Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ± 5% of the ε-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.
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DOI: 10.1021/bc9801070
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ISTEX:78F597C5553A58D20F360CE80E382DEB873465D7Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</title><author><name sortKey="Midoux, Patrick" sort="Midoux, Patrick" uniqKey="Midoux P" first="Patrick" last="Midoux">Patrick Midoux</name><affiliation><mods:affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Fax: 33 (0)238 69 00 94. Phone: 38 (0)2 38 25 55 95. E-mail: midoux@cnrs-orleans.fr.</mods:affiliation></affiliation></author><author><name sortKey="Monsigny, Michel" sort="Monsigny, Michel" uniqKey="Monsigny M" first="Michel" last="Monsigny">Michel Monsigny</name><affiliation><mods:affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:78F597C5553A58D20F360CE80E382DEB873465D7</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1021/bc9801070</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000719</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000719</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</title><author><name sortKey="Midoux, Patrick" sort="Midoux, Patrick" uniqKey="Midoux P" first="Patrick" last="Midoux">Patrick Midoux</name><affiliation><mods:affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Fax: 33 (0)238 69 00 94. Phone: 38 (0)2 38 25 55 95. E-mail: midoux@cnrs-orleans.fr.</mods:affiliation></affiliation></author><author><name sortKey="Monsigny, Michel" sort="Monsigny, Michel" uniqKey="Monsigny M" first="Michel" last="Monsigny">Michel Monsigny</name><affiliation><mods:affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Bioconjugate Chemistry</title><title level="j" type="abbrev">Bioconjugate Chem.</title><idno type="ISSN">1043-1802</idno><idno type="eISSN">1520-4812</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published" when="1999-03-11">1999</date><date when="1999-05-17">1999</date><biblScope unit="vol">10</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="406">406</biblScope><biblScope unit="page" to="411">411</biblScope></imprint><idno type="ISSN">1043-1802</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1043-1802</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ± 5% of the ε-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>polylysine</json:string><json:string>luciferase</json:string><json:string>histidylated</json:string><json:string>polyplexes</json:string><json:string>plasmid</json:string><json:string>luciferase activity</json:string><json:string>transfection</json:string><json:string>histidylated polylysine</json:string><json:string>acidic</json:string><json:string>pdna</json:string><json:string>polyfection</json:string><json:string>dmem</json:string><json:string>chloroquine</json:string><json:string>histidyl</json:string><json:string>vesicle</json:string><json:string>histidyl residue</json:string><json:string>fusogenic</json:string><json:string>peptide</json:string><json:string>bafilomycin</json:string><json:string>midoux</json:string><json:string>monsigny</json:string><json:string>chem</json:string><json:string>bioconjugate</json:string><json:string>cell lysates</json:string><json:string>lysates</json:string><json:string>bioconjugate chem</json:string><json:string>imidazole</json:string><json:string>fusogenic peptide</json:string><json:string>cationic</json:string><json:string>imidazole group</json:string><json:string>protonation</json:string><json:string>cytosol</json:string><json:string>acidic medium</json:string><json:string>atcc</json:string><json:string>relative light unit</json:string><json:string>hepg2</json:string><json:string>complete culture medium</json:string><json:string>endosomal</json:string><json:string>gene expression</json:string><json:string>gene transfer</json:string><json:string>transfection efficiency</json:string><json:string>polymer</json:string><json:string>mammalian cell</json:string><json:string>humidified atmosphere</json:string><json:string>acidic vesicle</json:string><json:string>hepg2 cell</json:string><json:string>molar ratio</json:string><json:string>cationic polymer</json:string><json:string>average degree</json:string><json:string>various cell line</json:string><json:string>form complex</json:string><json:string>cell culture</json:string><json:string>culture medium</json:string><json:string>firefly luciferase gene</json:string><json:string>plasmid molecule</json:string><json:string>amino group</json:string><json:string>efficient gene transfer</json:string><json:string>cell line</json:string><json:string>membrane destabilization</json:string><json:string>acidic compartment</json:string><json:string>drug delivery</json:string></teeft></keywords><author><json:item><name>MIDOUX Patrick</name><affiliations><json:string>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</json:string><json:string>To whom correspondence should be addressed. 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We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ± 5% of the ε-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.</abstract><qualityIndicators><score>9.198</score><pdfWordCount>4990</pdfWordCount><pdfCharCount>30206</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>6</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>832</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>184</abstractWordCount><abstractCharCount>1229</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</title><genre><json:string>research-article</json:string></genre><host><title>Bioconjugate Chemistry</title><language><json:string>unknown</json:string></language><issn><json:string>1043-1802</json:string></issn><eissn><json:string>1520-4812</json:string></eissn><volume>10</volume><issue>3</issue><pages><first>406</first><last>411</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-N3J84NW4-S</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - chemistry, organic</json:string><json:string>2 - chemistry, multidisciplinary</json:string><json:string>2 - biochemistry & molecular biology</json:string><json:string>2 - biochemical research methods</json:string></wos><scienceMetrix><json:string>1 - natural sciences</json:string><json:string>2 - chemistry</json:string><json:string>3 - organic chemistry</json:string></scienceMetrix><scopus><json:string>1 - Physical Sciences</json:string><json:string>2 - Chemistry</json:string><json:string>3 - Organic Chemistry</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Pharmaceutical Science</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Pharmacology</json:string><json:string>1 - Physical Sciences</json:string><json:string>2 - Engineering</json:string><json:string>3 - Biomedical Engineering</json:string><json:string>1 - Physical Sciences</json:string><json:string>2 - Chemical Engineering</json:string><json:string>3 - Bioengineering</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biotechnology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1021/bc9801070</json:string></doi><id>78F597C5553A58D20F360CE80E382DEB873465D7</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 1999 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="e-published" when="1999-03-11">1999</date><date when="1999-05-17">1999</date><date type="Copyright" when="1999">1999</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main" xml:lang="en">Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</title><author xml:id="author-0000" role="corresp"><persName><surname>Midoux</surname><forename type="first">Patrick</forename></persName><affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,
rue Charles-Sadron, F-45071 Orléans Cedex 02, France</affiliation><affiliation role="corresp"> To whom correspondence should be addressed. Fax: 33 (0)2 38 69 00 94. Phone: 38 (0)2 38 25 55 95. E-mail: midoux@cnrs-orleans.fr.</affiliation></author><author xml:id="author-0001"><persName><surname>Monsigny</surname><forename type="first">Michel</forename></persName><affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,
rue Charles-Sadron, F-45071 Orléans Cedex 02, France</affiliation></author><idno type="istex">78F597C5553A58D20F360CE80E382DEB873465D7</idno><idno type="ark">ark:/67375/TPS-N3J84NW4-S</idno><idno type="DOI">10.1021/bc9801070</idno></analytic><monogr><title level="j" type="main">Bioconjugate Chemistry</title><title level="j" type="abbrev">Bioconjugate Chem.</title><idno type="acspubs">bc</idno><idno type="coden">bcches</idno><idno type="pISSN">1043-1802</idno><idno type="eISSN">1520-4812</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published" when="1999-03-11">1999</date><date when="1999-05-17">1999</date><biblScope unit="vol">10</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="406">406</biblScope><biblScope unit="page" to="411">411</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><abstract><p>Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of
DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the
nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents
such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the
delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a
plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine
or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially
substituted with histidyl residues which become cationic upon protonation of the imidazole groups at
pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ± 5% of the ε-amino
groups substituted with histidyl residues; it was not significantly impaired in the presence of serum
in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A<hi rend="subscript">1</hi>,
indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery
of pDNA into the cytosol.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="zxx"></language></langUsage></profileDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bc</journal-id><journal-id journal-id-type="coden">bcches</journal-id><journal-title-group><journal-title>Bioconjugate Chemistry</journal-title><abbrev-journal-title>Bioconjugate Chem.</abbrev-journal-title></journal-title-group><issn pub-type="ppub">1043-1802</issn><issn pub-type="epub">1520-4812</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/bc</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bc9801070</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Midoux</surname><given-names>Patrick</given-names></name><xref rid="bc9801070AF1">*</xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Monsigny</surname><given-names>Michel</given-names></name></contrib><aff>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,
rue Charles-Sadron, F-45071 Orléans Cedex 02, France</aff></contrib-group><author-notes><corresp id="bc9801070AF1">
To whom correspondence should be addressed. Fax: 33 (0)2
38 69 00 94. Phone: 38 (0)2 38 25 55 95. E-mail: midoux@cnrs-orleans.fr.</corresp></author-notes><pub-date pub-type="epub"><day>11</day><month>03</month><year>1999</year></pub-date><pub-date pub-type="ppub"><day>17</day><month>05</month><year>1999</year></pub-date><volume>10</volume><issue>3</issue><fpage>406</fpage><lpage>411</lpage><history><date date-type="received"><day>14</day><month>09</month><year>1998</year></date><date date-type="rev-recd"><day>22</day><month>12</month><year>1998</year></date><date date-type="asap"><day>11</day><month>03</month><year>1999</year></date><date date-type="issue-pub"><day>17</day><month>05</month><year>1999</year></date></history><permissions><copyright-statement>Copyright © 1999 American Chemical Society</copyright-statement><copyright-year>1999</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of
DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the
nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents
such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the
delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a
plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine
or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially
substituted with histidyl residues which become cationic upon protonation of the imidazole groups at
pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ± 5% of the ε-amino
groups substituted with histidyl residues; it was not significantly impaired in the presence of serum
in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A<sub>1</sub>,
indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery
of pDNA into the cytosol.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bc9801070</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="d7e112"><title>Introduction</title><p>Poly-<sc>l</sc>-lysine, which strongly interacts with DNA, may
be used to compact plasmid DNA (pDNA) in order to
increase the uptake of foreign genes in mammalian cells
with the aim to transfect them. The resulting polyplexesa polyplex is a cationic polymer/DNA complex (<italic toggle="yes"><xref rid="bc9801070b00001" ref-type="bibr"></xref></italic>)are
taken up by the cells via either a nonspecific endocytosis
or a receptor-mediated endocytosis after polylysine substitution with a recognition signal (for reviews, see refs<italic toggle="yes">
2−5</italic>). However, one of the main limiting factors for the
transfection is the inefficacy of the delivery of plasmids
into the cytosol from the endocytotic vesicles. Indeed,
upon internalization, most of the plasmid molecules are
sequestered for several hours into vesicles from where
very few plasmid molecules can escape to reach the
nuclear machinery. Polyfectionpolyfection is a polyplex-mediated transfectionwas shown to be much more
efficient when cells were incubated in the presence of
membrane-disrupting agents such as chloroquine (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc9801070b00006" ref-type="bibr"></xref>−<xref rid="bc9801070b00007" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc9801070b00008" ref-type="bibr"></xref></named-content></italic>),
glycerol (<italic toggle="yes"><xref rid="bc9801070b00009" ref-type="bibr"></xref></italic>), or fusogenic peptides which destabilize the
membrane of acidic vesicles containing polyplexes (<italic toggle="yes">7</italic>,<italic toggle="yes"> 10−17</italic>). Therefore, the designing of cationic polymers inducing pDNA compaction and membrane disruption in a
slightly acidic medium will be good candidates to help
the pDNA to enter the cells and to escape the endocytotic
pathway leading to the delivery to lysosomes. Knowing
that (i) the imidazole group of histidine has a p<italic toggle="yes">K</italic> around
6.0 and thus becomes cationic in a slightly acidic medium
and (ii) poly-<sc>l</sc>-histidine mediates an acid-dependent
fusion and leakage of negative charged liposomes (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc9801070b00018" ref-type="bibr"></xref>, <xref rid="bc9801070b00019" ref-type="bibr"></xref></named-content></italic>),
we planned to use a histidylated cationic polymer as a
vector for transfecting cells. Because poly-<sc>l</sc>-histidine is
not charged at pH 7.4 and does not form complexes with
pDNA at neutral pH, polylysine was partially substituted
with histidyl residues.
</p><p>In the present work, we show that (i) histidylated poly-<sc>l</sc>-lysine was suitable to make complexes with pDNA and
to efficiently transfect various cell lines in the absence
of chloroquine or fusogenic peptides; (ii) the highest gene
transfer efficiency was obtained with a polylysine (average degree of polymerization of 190) having 38 ± 5% of
the ε-amino groups substituted with histidyl residues; (iii)
the transfection was rather insensitive to the presence
of serum in the culture medium; and (iv) the transfection
efficiency depended on the protonation of the imidazole
groups in the acid lumen of intracellular vesicles.
</p></sec><sec id="d7e184"><title>Materials and Methods</title><p><bold>Preparation of Partially Histidylated Polylysine
(His-pLK)</bold>. Poly-<sc>l</sc>-lysine and HBr 30000−50000 (pLK,<xref rid="bc9801070b00001" ref-type="bibr"></xref>
DP = 190) (Bachem Feinchemikalien, Bubendorf, Switzerland) (1 g in 200 mL H<sub>2</sub>O) were passed through an
anion-exchange column (Dowex 2 × 8, <sup>-</sup>OH form, 20−50
mesh) in order to remove bromide ions (<italic toggle="yes"><xref rid="bc9801070b00020" ref-type="bibr"></xref></italic>). The eluate
was neutralized with a 10% <italic toggle="yes">p</italic>-toluenesulfonic acid solution in water and freezed-dried. pLK <italic toggle="yes">p</italic>-toluenesulfonate
salt (50 mg; 0.86 μmol) in 3 mL of dimethyl sulfoxide
(Aldrich, Strasbourg, France) in the presence of diisopropylethylamine (42 μL; 288 μmol) (Aldrich) was reacted
for 24 h at 20 °C with (Boc)His(Boc)-OH (32 mg; 96 μmol)
(Novabiochem, Bad Soden, Germany) in the presence of
benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphonium
hexafluorophosphate (Richelieu Biotechnologies, Saint
Hyacinthe, Canada) (43 mg; 97 μmol). The N-protecting
Boc groups were removed by acidic treatment by adding
20 mL of H<sub>2</sub>O/trifluoroacetic acid mixture (1:1; v/v) for
24 h at 20 °C. Water and trifluoroacetic acid were
removed under reduced pressure. The polymer was
precipitated by adding 10 vol of 2-propanol and spun
down by centrifugation (1800<italic toggle="yes">g</italic> for 15 min). The pellet was
washed with 2-propanol, collected by centrifugation
(1800<italic toggle="yes">g</italic> for 15 min), solubilized in distilled water, and
freeze-dried. The average number of His molecules bound
per pLK molecule was determined by <sup>1</sup>H NMR spectroscopy at 300 MHz in D<sub>2</sub>O according to <italic toggle="yes">x</italic> = 6(<italic toggle="yes">h</italic><sub>8.7</sub>/<italic toggle="yes">h</italic><sub>Lys</sub>)DP,
where <italic toggle="yes">h</italic><sub>8.7</sub> was the value of the integration of the signal
at 8.7 ppm corresponding to the proton (1H C<sub>12</sub>) of His
(Figure <xref rid="bc9801070f00001"></xref>), <italic toggle="yes">h</italic><sub>Lys</sub> was in the range 1.3−1.9 ppm corresponding to the 6 methylene protons (C<sub>3</sub>, C<sub>4</sub> and C<sub>5</sub>) of
lysine residues (Figure <xref rid="bc9801070f00001"></xref>), and DP was the degree of
polymerization of pLK. The number of His residues
bound per pLK molecule was 70. Histidylated polylysine
was fluoresceinylated by using fluoresceinyl isothiocyanate (FITC isomer I, Molecular Probe, La Jolla, CA) as
previously described (<italic toggle="yes"><xref rid="bc9801070b00007" ref-type="bibr"></xref></italic>).
<fig id="bc9801070f00001" position="float" orientation="portrait"><label>1</label><caption><p>Schematic structure of histidylated polylysine. i = DP; R = NH<sub>3</sub><sup>+</sup> or His.</p></caption><graphic xlink:href="bc9801070f00001.eps" position="float" orientation="portrait"></graphic></fig></p><p><bold>Cells and Cell Culture</bold>. COS-7 (SV40 transformed
kidney cells of African green monkey, ATCC CRL 1651,
ATCC, Rockville, MA), 293-T7 (human embryonic kidney
producing T7 RNA polymerase) (<italic toggle="yes"><xref rid="bc9801070b00021" ref-type="bibr"></xref></italic>), and Rb1 (rabbit
smooth muscle cells) (<italic toggle="yes"><xref rid="bc9801070b00022" ref-type="bibr"></xref></italic>) cells were cultured in DMEM
(Gibco, Renfrewshire, U.K.) supplemented with 10% heat
inactivated fetal bovine serum (FBS, Gibco); HeLa cells
(ATCC CCL 2.1) in DMEM with 5% FBS; B16 cells
(murine melanoma cells, ATCC CRL 6322) in DMEM
with 10% heat-inactivated new borne calf serum (Gibco);
HepG2 (human hepatoma cells, ATCC HB 8065), MCF-7
cells (human breast adenocarcinoma cells, ATCC HTB22),
and HOS cells (human osteosarcoma cells, ATCC CRL
1543) in MEM (Gibco) with 10% heat inactivated FBS;
16HBE cells (human airway epithelial cells) in DMEM
with 10% heat-inactivated FBS in fibronectin-coated
culture plates; A549 cells (human nonsmall cell lung
carcinoma cells, ATCC CCL 185) in RPMI with 10% heat-inactivated FBS. All culture media were supplemented
with 2 mM <sc>l</sc>-glutamine (Merck, Darmstadt, Germany)
and antibiotics (100 units/mL penicillin and 100 μg/mL
streptomycin, Eurobio, France). Cells were grown at 37
°C in a humidified atmosphere containing 5% CO<sub>2</sub> and
95% air. Cells were harvested by treatment with PET
[PBS with 0.02% (w/v) EDTA and 2.5 μg/mL trypsin] at
37 °C for 5 min. Cells were mycoplasma free as evidenced
by bisbenzimidazole (Hoechst 33258) (<italic toggle="yes"><xref rid="bc9801070b00023" ref-type="bibr"></xref></italic>).
</p><p><bold>Plasmids</bold>. pSV2<italic toggle="yes">LUC</italic> plasmid (5 kb) was an expression
vector encoding the firefly luciferase gene under the
control of the SV40 T large antigen promoter (<italic toggle="yes"><xref rid="bc9801070b00024" ref-type="bibr"></xref></italic>).
pUT650 plasmid (5.15 kb) (CAYLA, Toulouse, France)
was an expression vector encoding the firefly luciferase
gene under the control of the human cytomegalovirus
promoter. Supercoiled DNA plasmids were isolated by a
standard alkaline lysis method followed by CsCl gradient
centrifugation in the presence of ethidium bromide,
extensive extraction with <italic toggle="yes">n</italic>-butanol, and precipitation
with ethanol.
</p><p><bold>Polyfection</bold>. Polyplexes (histidylated polylysine or
polylysine/pDNA complexes) were prepared by adding,
under agitation, polymers in 0.3 mL of serum-free
DMEM, pH 7.4 to 5 μg (1.5 pmol) of plasmid in 0.7 mL of
serum-free DMEM. The mixed solution was kept for 30
min at 20 °C before use. Adherent cell lines [(2−4) × 10<sup>5</sup>
cells] were plated (day 0) into 4 cm<sup>2</sup> culture dishes (12-well culture plates). On day 1, the medium was removed
and 1 mL of a solution containing a polyplex supplemented with 1% FBS, unless otherwise specified, was
added into each well. After 4 h of incubation at 37 °C in
a humidified atmosphere (95% air, 5% CO<sub>2</sub>), the medium
was removed and cells were further incubated at 37 °C
in 2 mL of the relevant complete culture medium in a
humidified atmosphere (95% air, 5% CO<sub>2</sub>). When used,
chloroquine (100 μM) was added to the transfection solution, which was immediately supplemented with 1% FBS
and put into each well. When used, the fusogenic peptide
(E5CA, GLFEAIAEFIGGWEGLIEGCA) (<italic toggle="yes"><xref rid="bc9801070b00007" ref-type="bibr"></xref></italic>) (10 μM) was
added and the transfection solution was immediately
supplemented with 1% FBS and put into each well.
</p><p>Polyethylenimine (PEI 25 kDa from Aldrich) (4.5 mg)
was dissolved in 10 mL of H<sub>2</sub>O, neutralized with HCl,
and sterilized by filtration. Polyplexes were prepared by
mixing 10 μL of PEI in 50 μL of 0.15 M NaCl with 5 μg
(1.5 pmol) plasmid in 50 μL 0.15 M NaCl. The mixed
solution was kept for 30 min at 20 °C. Then, the solution
supplemented with 1 mL of DMEM containing 10% FBS
was added into each well. After 4 h of incubation at 37
°C in a humidified atmosphere (95% air, 5% CO<sub>2</sub>), the
medium was removed and cells were further incubated
at 37 °C in 2 mL of the relevant complete culture medium
in a humidified atmosphere (95% air, 5% CO<sub>2</sub>).
</p><p><bold>Luciferase Assay</bold>. Luciferase gene expression was
measured by monitoring its luminescence activity according to De Wet et al. (<italic toggle="yes"><xref rid="bc9801070b00025" ref-type="bibr"></xref></italic>). The medium was discarded,
and the cells were washed three times with PBS. The
homogenization buffer (200 μL of 8 mM MgCl<sub>2</sub>, 1 mM
DTT, 1 mM EDTA, 1% Triton X100, 15% glycerol, 25 mM
Tris-phosphate buffer, pH 7.8) was poured into each well,
and the tissue culture plates were kept for 15 min at 20
°C. The solution was recovered and further spun down
(5 min at 800<italic toggle="yes">g</italic>). A total of 95 μL of a 2 mM ATP solution
in the homogenization buffer without Triton X100 was
added to 60 μL of supernatant, and the solution was
shaken with a vortex. The luminescence was recorded
for 4 s in a Lumat LB 9501 luminometer (Berthold,
Wildbach, Germany) upon addition of 150 μL of a 167
mM luciferin solution in water. Measurements were done
in duplicate. The data shown correspond to the number
of relative light units (RLU) from 10<sup>6</sup> cells. The number
of RLU of 1 pg/mL of luciferase was 2000 under these
assay conditions.
</p><p><bold>ζ</bold><bold> Potential Measurement</bold>. Polyplexes were prepared
in 10 mM NaCl, 1 mM MOPS, and 0.1 mM EDTA, at pH
7.2 to a pDNA final concentration of 10 μg/mL. ζ potential
was measured by using a ZetaSizer 3000 (Malvern
Instruments, Orsay, France) with the following parameters: viscosity, 1.014 cP; dielectric constant, 79; temp,
25 °C; <italic toggle="yes">F</italic>(Ka), 1.50 (Smoluchowsky); current, 10 mA.
</p></sec><sec id="d7e379"><title>Results</title><p><bold>Polyfection by Using Histidylated Polylysine</bold>.
Histidylated polylysine containing 84 histidyl residues
(His<sub>84</sub>-pLK) (Figure <xref rid="bc9801070f00001"></xref>) was complexed in 1 mL of serum-free DMEM at pH 7.4 with pUT650 plasmid encoding
the luciferase gene, and the polyplexes were used to
transfect HepG2 cells. The luciferase activity in the cell
lysates was compared to that obtained after polyfection
with pLK/pUT650 complexes in the absence and in the
presence of either chloroquine or E5CA, a fusogenic
peptide. Regarding to the luciferase activity, the polyfection was very efficient with His<sub>84</sub>-pLK/pDNA complexes and very inefficient with pLK/pDNA complexes.
The luciferase activity was 4.5 orders of magnitude (10<sup>7</sup>
RLU/10<sup>6</sup> cells) higher than with pLK/pDNA complexes
(200 RLU/10<sup>6</sup> cells); it was 3.5 (2 × 10<sup>7</sup> RLU/10<sup>6</sup> cells)
and 3 (10<sup>7</sup> RLU/10<sup>6</sup> cells) orders of magnitude higher than
with pLK/pDNA complexes in the presence of chloroquine
and the fusogenic peptide, respectively (Figure <xref rid="bc9801070f00002"></xref>). The
luciferase activity was maximal when the cells were
incubated for about 3−4 h in the presence of His<sub>84</sub>-pLK/pDNA complexes (data not shown); increasing the incubation period (up to 24 h) did not increase the luciferase
activity. It is noteworthy to emphasize that no cytoxicity
was observed whatever the incubation duration (from 4
to 24 h) in the presence of the polyplexes. The amount of
plasmid was varied between 0.05 and 10 μg, keeping
constant the histidylated polylysine/pDNA ratio equal to
4:1 (weight by weight), the number of cells per well [(2−3) × 10<sup>5</sup> cells in a 4 cm<sup>2</sup> well] and the volume (1 mL) of
the solution containing the polyplexes. The polyfection
was maximal in the presence of 5 μg of plasmid. Increasing the amount of plasmid did not increase the luciferase
activity (Figure <xref rid="bc9801070f00002"></xref> inset). Increasing the number of cells
per well did not increase the luciferase activity.
<fig id="bc9801070f00002" position="float" orientation="portrait"><label>2</label><caption><p>Histidylated polylysine mediated polyfection. Polyplexes were prepared by mixing either His<sub>84</sub>-pLK (40 μg) or pLK
(5 μg) in 0.3 mL of serum-free DMEM with 10 μg (3 pmol) of
pUT650 plasmid in 0.7 mL of serum-free DMEM. The solution
was kept for 30 min at 20 °C. When it is relevant the solution
was made 100 μM in chloroquine or 10 μM in E5CA. HepG2
cells (3 × 10<sup>5</sup> cells plated in a 4 cm<sup>2</sup> well) were incubated for 4
h at 37 °C with polyplexes in the presence of 5% FBS. Then
cells were washed and incubated in complete culture medium
containing 10% FBS. The gene expression was determined 48
h later by assaying the luciferase activity in cell lysates. RLU,
the number of relative light units, represents the luciferase
activity in 10<sup>6</sup> cells. (Inset) Concentration dependence of polyfection with His<sub>84</sub>-pLK/pDNA complexes.</p></caption><graphic xlink:href="bc9801070f00002.tif" position="float" orientation="portrait"></graphic></fig></p><p><bold>Optimal Number of Histidyl Residues on One
Polylysine Molecule</bold>. HepG2 cells were transfected by
using polyplexes made with pUT650 and polylysine
substituted with different number of histidyl residues.
The luciferase activity in cell lysates was maximal with
polylysine substituted with 72 ± 9 histidyl residues/pLK
molecule corresponding to 38 ± 5% substitution of the
polylysine ε-amino groups (Figure <xref rid="bc9801070f00003"></xref>). When polyfection
was performed with pLK containing 20 histidyl residues,
the luciferase activity was 10-fold lower than with pLK
substituted with 70 histidyl residues.
<fig id="bc9801070f00003" position="float" orientation="portrait"><label>3</label><caption><p>Histidylated polylysine mediated polyfection:influence of the His/pLK ratio. Polyplexes were prepared by mixing pLK substituted with histidyl residues in 0.3 mL of serum-free DMEM with 10 μg (3 pmol) of pUT650 plasmid in 0.7 mL of serum-free DMEM. The solution was kept for 30 min at 20 °C. HepG2 cells (3 × 10<sup>5</sup> cells plated in a 4 cm<sup>2</sup> well) were incubated
for 4 h at 37 °C with polyplexes in the presence of 1% FBS.
Then, cells were washed and incubated in complete culture
medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell
lysates. RLU, the number of relative light units, represents the
luciferase activity in 10<sup>6</sup> cells.</p></caption><graphic xlink:href="bc9801070f00003.tif" position="float" orientation="portrait"></graphic></fig></p><p><bold>Influence of the Histidylated Polylysine/pDNA
Molar Ratio</bold>. The luciferase activity was maximal with
polyplexes made by complexing 10 μg of plasmid with 30
(polymer/pDNA molar ratio of 183) to 40 μg (polymer/pDNA molar ratio of 244) of His<sub>84</sub>-pLK (Figure <xref rid="bc9801070f00004"></xref>).
Increasing the molar ratio did not increase the luciferase
activity. The efficiency decreased 10-fold when the molar
ratio was down to 122 (20 μg of His<sub>84</sub>-pLK) and was
completely inefficient when the molar ratio was lower
than 60 (10 μg of His<sub>84</sub>-pLK). Under conditions optimal
for transfection, polyplexes were positively charged with
a ζ potential of +24 mV (Figure <xref rid="bc9801070f00004"></xref>).
<fig id="bc9801070f00004" position="float" orientation="portrait"><label>4</label><caption><p>Histidylated polylysine mediated polyfection:influence of the polymer/pDNA ratio and ζ potentials of polyplexes. Polyplexes were prepared by mixing 10 μg (3 pmol) of pUT650 plasmid in 0.7 mL of serum-free DMEM with different amounts of histidylated polylysine (His<sub>84</sub>-pLK) in 0.3 mL of serum-free
DMEM. The solution was kept for 30 min at 20 °C. HOS cells
(2 × 10<sup>5</sup> cells/4 cm<sup>2</sup>) were incubated for 4 h at 37 °C with
polyplexes in the presence of 5% FBS. Then cells were washed
and incubated in complete culture medium containing 10% FBS.
The gene expression was determined 48 h later by assaying the
luciferase activity in cell lysates. RLU, the number of relative
light units, represents the luciferase activity in 10<sup>6</sup> cells.</p></caption><graphic xlink:href="bc9801070f00004.tif" position="float" orientation="portrait"></graphic></fig></p><p><bold>Influence of the Serum Concentration</bold>. Polyfection
with His<sub>70</sub>-pLK was not impaired in the presence of
serum. The luciferase activity was 2-fold greater in the
presence of 5 and 10% FBS than in the presence of 1%
FBS, and it was as large in the presence of 20% FBS as
in the presence of 1% FBS (Figure <xref rid="bc9801070f00005"></xref>).
<fig id="bc9801070f00005" position="float" orientation="portrait"><label>5</label><caption><p>Histidylated polylysine mediated polyfection:influence of the serum. HepG2 cells (3 × 10<sup>5</sup> cells/4 cm<sup>2</sup> well) were
incubated for 4 h at 37 °C with 5 μg/mL of the polyplexes (His<sub>70</sub>-pLK/pUT650) in the presence of various amount of FBS. Then,
cells were washed and incubated in complete culture medium
containing 10% FBS. The gene expression was determined 48
h later by assaying the luciferase activity in cell lysates. RLU,
the number of relative light units, represents the luciferase
activity in 10<sup>6</sup> cells.</p></caption><graphic xlink:href="bc9801070f00005.tif" position="float" orientation="portrait"></graphic></fig></p><p><bold>Gene Transfer into Various Cell Lines</bold>. The transfection of different mammalian cell lines including human hepatoma cells (HepG2), human breast adenocarcinoma cells (MCF-7), human epidermoid carcinoma cells
(HeLa), human osteosarcoma cells (HOS), human airway
epithelial cells (16HBE), murine melanoma cells (B16),
and simian kidney cells (COS) was assessed by using
His<sub>70</sub>-pLK/pUT650 complexes and rabbit smooth muscle
cells (Rb-1) by using His<sub>70</sub>-pLK/pSV2<italic toggle="yes">LUC</italic> complexes. All
the cell lines (except COS cells) were efficiently transfected with luciferase activity ranging from 2 × 10<sup>6</sup> to
10<sup>7</sup> RLU/10<sup>6</sup> cells (Figure <xref rid="bc9801070f00006"></xref>). The transfection was
compared with that obtained with PEI/pDNA complexes
(Table <xref rid="bc9801070t00001"></xref>). Depending on the cell line, the transfection by
His-pLK/pDNA complexes was as effective or less effective (up to 20-fold) than with PEI/pDNA complexes.
<fig id="bc9801070f00006" position="float" orientation="portrait"><label>6</label><caption><p>Histidylated polylysine mediated polyfection of various cell lines. Polyplexes were prepared by mixing His<sub>70</sub>-pLK (30 μg) in 0.3 mL serum-free DMEM with 10 μg (3 pmol)
of plasmid (pUT650 or pSV2<italic toggle="yes">LUC</italic> for Rb-1 cells) in 0.7 mL of
serum-free DMEM. The mixed solution was kept for 30 min at
20 °C. Cell lines [(2−3) × 10<sup>5</sup> cells/4 cm<sup>2</sup> well] were incubated
for 4 h at 37 °C with polyplexes in the presence of 10% FBS.
Then, cells were washed and incubated in complete culture
medium containing 10% FBS. The gene expression was determined 48 h later by assaying the luciferase activity in cell
lysates. RLU, the number of relative light units, represents the
luciferase activity in 10<sup>6</sup> cells.</p></caption><graphic xlink:href="bc9801070f00006.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bc9801070t00001" position="float" orientation="portrait"><label>1</label><caption><p>Comparison of the Transfection Efficiency by Using Either Histidylated Polylysine or Polyethyleni- mine as DNA Carriers<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">cells</oasis:entry><oasis:entry colname="2">PEI/His-pLK</oasis:entry><oasis:entry colname="3">cells</oasis:entry><oasis:entry colname="4">PEI/His-pLK
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">B16
</oasis:entry><oasis:entry colname="2">5.8
</oasis:entry><oasis:entry colname="3">A549
</oasis:entry><oasis:entry colname="4">21
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">293
</oasis:entry><oasis:entry colname="2">0.6
</oasis:entry><oasis:entry colname="3">Rb-1
</oasis:entry><oasis:entry colname="4">17</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Cells (2 × 10<sup>5</sup> cells/4 cm<sup>2</sup> well) were transfected for 4 h at 37
°C with 5 μg/mL (pUT650 or pSV2<italic toggle="yes">LUC</italic> for Rb-1 cells) complexed
with either His-pLK or PEI in the presence of 10% FBS. Then,
cells were washed and incubated in complete culture medium
containing 10% FBS. The gene expression was determined 48 h
later by assaying the luciferase activity in cell lysates. PEI/His-pLK represents the ratio of the luciferase activity into cells
transfected by PEI polyplexes to the luciferase activity into cells
transfected by His-pLK polyplexes.</p></table-wrap-foot></table-wrap></p><p><bold>Influence of the Endosomal pH</bold>. Histidylated polylysine is supposed to increase the delivery of pDNA into
the cytosol via membrane destabilization of acidic endocytotic vesicles containing His<sub>70</sub>-pLK/pDNA complexes
following the protonation of the imidazole groups. This
was assessed by transfecting cells in the absence or in
the presence of bafilomycin A<sub>1</sub>, an inhibitor of vacuolar
ATPase endosomal proton pump (<italic toggle="yes"><xref rid="bc9801070b00026" ref-type="bibr"></xref></italic>). When the polyfection of Rb-1 cells by His<sub>70</sub>-pLK/pSV2<italic toggle="yes">LUC</italic> complexes was
performed in the presence of 200 nM bafilomycin A<sub>1</sub>, the
luciferase activity into cells was about 3 order of magnitude lower than in the absence of bafilomycin A<sub>1</sub>
(Figure <xref rid="bc9801070f00007"></xref>). A flow cytometry analysis showed that, in the
absence of bafilomycin A<sub>1</sub> , the polyplexes were in acidic
compartment as evidenced by a 1.8-fold increase of the
cell fluorescence intensity after a postincubation in the
presence of monensin (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc9801070b00027" ref-type="bibr"></xref>, <xref rid="bc9801070b00028" ref-type="bibr"></xref></named-content></italic>); conversely, the fluorescence intensity of cells incubated in the presence of
bafilomycin A<sub>1</sub> did not increase upon a postincubation
in the presence of monensin (data not shown). Therefore,
the decrease of the transfection efficiency of histidylated
polylysine/pDNA complexes in the presence of bafilomycin A<sub>1</sub> , indicated that the protonation of the imidazole
groups in acidic medium was involved in the polyfection
process.
<fig id="bc9801070f00007" position="float" orientation="portrait"><label>7</label><caption><p>Influence of bafilomycin A1 on the polyfection efficiency. Rb-1 cells (2 × 10<sup>5</sup> cells/4 cm<sup>2</sup> well) were incubated
at 37 °C in the presence of 1 mL His<sub>70</sub>-pLK/pSV2<italic toggle="yes">LUC</italic> complexes
(5 μg of pDNA) and in the absence or in the presence of 200 nM
bafilomycin A<sub>1</sub>. After 4 h, the cells were washed and incubated
in complete culture medium containing 10%. The gene expression was determined 48 h later by assaying the luciferase
activity in cell lysates. RLU, the number of relative light units,
represents the luciferase activity in 10<sup>6</sup> cells.</p></caption><graphic xlink:href="bc9801070f00007.tif" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e709"><title>Discussion</title><p>The introduction in mammalian cells of a foreign gene
as a plasmid DNA by nonviral vectors requires the entry
of the plasmid into the cell and then into the nucleus.
The compaction of a pDNA in the presence of polylysine
increases its uptake by the cells via making the endocytotic process more easy. Once complexed and taken up
by cells, plasmids are sequestered for several hours in
vesicular compartments from where they cannot escape
to reach the nucleus. The delivery of the pDNA into the
cytosol should be obtained from early acidic vesicles
formed during polyplex uptake under the condition that
the endosomal membrane is disrupted by devices bound
to the plasmid-associated carrier working at a weakly
acidic medium. In this study, we show that partial
substitution of poly-<sc>l</sc>-lysine with histidyl residues results
in a cationic polymer that forms complexes with a
plasmid DNA at pH 7.4. These complexes actively transfect cells in culture in the absence of any helper compounds such as chloroquine or a fusogenic peptide.
Optimal conditions for transfection were found when
polylysine was substituted with 72 ± 9 histidyl residues,
corresponding to a polylysine substitution level of 38 ±
5%; the efficiency was 10-fold lower when polylysine
substitution was down to 10%. The polyfection was
efficient when the global charge of the polyplexes was
slightly positive (ζ potential = +24 mV), which likely
increased their binding on the cell surface and facilitated
their uptake by the cells.
</p><p>The polyplexes are internalized by the cells in acidic
compartments. The mechanism of polyfection mediated
by histidylated polyplexes is not yet known, but it is
supposed that a subsequent delivery of pDNA into the
cytosol occurs by the membrane destabilization of acidic
vesicles containing polyplexes, upon the protonation of
imidazole groups. Although, there is no evidence that the
DNA leaves endocytotic vesicles, this statement is supported by (i) the fact that poly-<sc>l</sc>-histidine induces fusion
of lipid bilayers (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc9801070b00018" ref-type="bibr"></xref>, <xref rid="bc9801070b00019" ref-type="bibr"></xref></named-content></italic>) and (ii) the fact that bafilomycin
A<sub>1</sub> which is known to impair the acidification of the
lumen of the endosomes, made polyfection inefficient.
Poly-<sc>l</sc>-histidine destabilizes lipid bilayers in a slightly
acidic medium and induces fusion upon protonation of
the imidazole groups by increasing interactions between
this polymeric polycation and the membrane phospholipids (<italic toggle="yes"><xref rid="bc9801070b00018" ref-type="bibr"></xref></italic>). In acidic medium, poly-<sc>l</sc>-histidine is more
fusogenic than polylysine: efficient fusion of negatively
charged liposomes (phosphatidylserine liposomes) occurs
at a ratio of the positive charge of poly-<sc>l</sc>-histidine to the
negative charge of the liposomes equal to 0.2 while it
occurs at a ratio equal to 1 in the case polylysine.
Recently, we have reported that the amphipatic peptide
H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) containing five histidyl residues, which does not adopt a helical
structure in acidic medium, permeabilizes cell membranes in such an acidic medium (pH 6.4) (<italic toggle="yes"><xref rid="bc9801070b00017" ref-type="bibr"></xref></italic>). In addition, this peptide increased the expression efficiency
when a gene is transferred into cells as glycosylated
polylysine/pDNA complexes; the transfection was also
inhibited in the presence of bafilomycin A<sub>1</sub> (<italic toggle="yes"><xref rid="bc9801070b00017" ref-type="bibr"></xref></italic>). These
results suggest that the generation of cationic charges
via the protonation of several imidazole groups bound to
pDNA inside the endosomes consecutively to the acidification of their lumen is a suitable approach to modify
the intracellular routing of pDNA, especially its delivery
into cytosol. Therefore, the mechanism involved for
polyfection with histidylated polylysine could be different
from that of polyethylenimine which is supposed to act
as a buffer, preventing acidification of the endosomal
lumen and inducing the swelling of these vesicles, leading
to a membrane destabilization (<italic toggle="yes"><xref rid="bc9801070b00029" ref-type="bibr"></xref></italic>).
</p><p>The transfection efficiency by using histidylated polylysine/pDNA complexes was greater than by using polylysine/pDNA complexes in the presence of either chloroquine or the fusogenic peptide E5CA. Chloroquine
concentrates in the acidic compartments and prevents
the delivery of the endosomal content to lysosomes; it
induces the formation of large endosome-derived vacuoles. In addition, it allows the dissociation of pDNA/polylysine conjugate complexes (<italic toggle="yes"><xref rid="bc9801070b00008" ref-type="bibr"></xref></italic>). Anionic permeabilizing peptides, adopting an α-helical structure at pH <5.5,
may destabilize the membrane of weakly acidic vesicles
containing the polyplexes leading to the delivery of pDNA
into the cytosol. Histidylated polyplexes were efficient for
all cell types tested while the efficiency of anionic
peptides such as E5CA depends on several parameters
in relation with the cell type. These parameters include
the number of vesicles containing both polylysine/pDNA
complexes and peptides separately taken up, the peptide
concentration, and the luminal pH of the vesicles. In
addition, histidylated polylysine may destabilize membranes as well as H5WYG peptide at a pH less acidic
than E5CA peptide and thus may be effective in early
endosomes at pH around 6.4; at this pH, anionic peptides
were inefficient.
</p><p>Chloroquine and fusogenic peptides cannot be used in
vivo. Indeed, (i) they are small molecules which are
cleared soon after their injection; (ii) chloroquine must
concentrate in acidic vesicles and reach a high concentration before being able to induce an efficient transfection
(<italic toggle="yes"><xref rid="bc9801070b00008" ref-type="bibr"></xref></italic>); (iii) polyplex solution to be injected cannot contain a
high concentration of chloroquine, because above 1 mM
chloroquine, polyplexes start to dissociate (<italic toggle="yes"><xref rid="bc9801070b00008" ref-type="bibr"></xref></italic>); (iv) the
effect of anionic peptides bearing negative charges at
neutral pH is inhibited in the presence of serum probably
because they bind to serum proteins, and in addition, the
effect is diminished when they are chemically linked to
polyplexes. With histidylated polylysine, the membrane-disrupting device is bound to the plasmid and the
transfection efficiency was not significantly reduced in
the presence of serum up to 20%.
</p><p>In conclusion, histidylated polylysine, a synthetic
compound, easy to prepare and to purify, is a good
nonviral vector for gene transfer into mammalian cells.
Histidylated polylysine gives polyplex formulation suitable for transferring gene in vivo. In addition, histidylated polylysine may be substituted with recognition
signal molecules in order to deliver the gene into cells
which specifically express a related receptor; this aspect
is currently under development in our laboratory.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We express our gratitude to Dr. A. B. Brasier (Massachusetts General Hospital, Boston, MA) for pSV2<italic toggle="yes">LUC</italic>
plasmid, Dr. D. C. Gruenert (Gene Therapy Core Center,
University of California, San Francisco), Dr. M. Nachtigal
(University of South Carolina, Columbia, SC), and Dr.
L. Huang (University of Pittsburgh, Pittburgh, PA) for
16HBE, Rb-1, and 293-T7 cells, respectively. We thank
Mahajoub Bello-Roufaï for ζ potential measurements and
Sophie Fauvin and Suzanne Nuques for their skillful
technical helps in cell culture and transfections. We
thank Françoise Fargette for the preparation of plasmids
and Henri Labbé for his skillfull assistance in NMR
spectroscopy. This work was partly supported by grants
from ANRS (ANRS No. 97003), Ministère de la Recherche
et de la Technologie (ACCSV14 No. 9514104), Ligue
Nationale Contre le Cancer, Biotechnocentre and EU
(BIO4-CT97-2216). Michel Monsigny is Professor at the
University of Orléans. Patrick Midoux is Research Director at INSERM.
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U.S.A.</source><year>1995</year><volume>92</volume><fpage>7297</fpage><lpage>72301</lpage><pub-id pub-id-type="doi">10.1073/pnas.92.16.7297</pub-id></element-citation></ref><ref id="bc9801070n00001"><mixed-citation><comment>Abbreviations: DP, average degree of polymerization; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; FBS, fetal bovine serum; His, histidyl residue; PBS, phosphate-buffered saline; pLK, poly-<sc>l</sc>-lysine.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes</title></titleInfo><name type="personal" displayLabel="corresp"><namePart type="family">MIDOUX</namePart><namePart type="given">Patrick</namePart><affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</affiliation><affiliation> To whom correspondence should be addressed. Fax: 33 (0)238 69 00 94. Phone: 38 (0)2 38 25 55 95. E-mail: midoux@cnrs-orleans.fr.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">MONSIGNY</namePart><namePart type="given">Michel</namePart><affiliation>Centre de Biophysique Moléculaire, Glycobiologie CNRS UPR4301 and University of Orléans,rue Charles-Sadron, F-45071 Orléans Cedex 02, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">1999-03-11</dateCreated><dateIssued encoding="w3cdtf">1999-05-17</dateIssued><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><abstract>Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ± 5% of the ε-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.</abstract><relatedItem type="host"><titleInfo><title>Bioconjugate Chemistry</title></titleInfo><titleInfo type="abbreviated"><title>Bioconjugate Chem.</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><identifier type="ISSN">1043-1802</identifier><identifier type="eISSN">1520-4812</identifier><identifier type="acspubs">bc</identifier><identifier type="coden">BCCHES</identifier><identifier type="uri">pubs.acs.org/bc</identifier><part><date>1999</date><detail type="volume"><caption>vol.</caption><number>10</number></detail><detail type="issue"><caption>no.</caption><number>3</number></detail><extent unit="pages"><start>406</start><end>411</end></extent></part></relatedItem><identifier type="istex">78F597C5553A58D20F360CE80E382DEB873465D7</identifier><identifier type="ark">ark:/67375/TPS-N3J84NW4-S</identifier><identifier type="DOI">10.1021/bc9801070</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 1999 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">ACS</recordContentSource><recordOrigin>Copyright © 1999 American Chemical Society</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/record.json</uri></json:item></metadata><annexes><json:item><extension>eps</extension><original>true</original><mimetype>image/x-eps</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-N3J84NW4-S/annexes.eps</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/78F597C5553A58D20F360CE80E382DEB873465D7/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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