Different Strategies for Formation of PEGylated EGF-Conjugated PEI/DNA Complexes for Targeted Gene Delivery
Identifieur interne : 000928 ( Istex/Corpus ); précédent : 000927; suivant : 000929Different Strategies for Formation of PEGylated EGF-Conjugated PEI/DNA Complexes for Targeted Gene Delivery
Auteurs : Thomas Blessing ; Malgorzata Kursa ; Robert Holzhauser ; Ralf Kircheis ; Ernst WagnerSource :
- Bioconjugate Chemistry [ 1043-1802 ] ; 2001.
Abstract
With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF−PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.
Url:
DOI: 10.1021/bc0001488
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ISTEX:5BDA273D2BD7C85D0528CE1426C5CDE263EF638CLe document en format XML
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PEI/DNA Complexes for Targeted Gene Delivery</title><author><name sortKey="Blessing, Thomas" sort="Blessing, Thomas" uniqKey="Blessing T" first="Thomas" last="Blessing">Thomas Blessing</name><affiliation><mods:affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and BoehringerIngelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. New address: Faculte de Pharmacie, 15 av. Charles Flahault, F-34060Montpellier. Telephone: ++33-467-418-267. Fax: ++33-467-520-898. 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New address: Faculte de Pharmacie, 15 av. Charles Flahault, F-34060Montpellier. Telephone: ++33-467-418-267. Fax: ++33-467-520-898. 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PEI/DNA Complexes for Targeted Gene Delivery</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2001 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="e-published" when="2001-06-23">2001</date><date when="2001-07-18">2001</date><date type="Copyright" when="2001">2001</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">Different Strategies for Formation of PEGylated EGF-Conjugated
PEI/DNA Complexes for Targeted Gene Delivery</title><author xml:id="author-0000" role="corresp"><persName><surname>Blessing</surname><forename type="first">Thomas</forename></persName><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and Boehringer
Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><affiliation role="corresp"> To whom correspondence should be addressed. New address: Faculte de Pharmacie, 15 av. Charles Flahault, F-34060 Montpellier. Telephone: ++33-467-418-267. Fax: ++33-467-520-898. E-mail: blessing@pharma.univ-montp1.fr.</affiliation></author><author xml:id="author-0001"><persName><surname>Kursa</surname><forename type="first">Malgorzata</forename></persName><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and Boehringer
Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation></author><author xml:id="author-0002"><persName><surname>Holzhauser</surname><forename type="first">Robert</forename></persName><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and Boehringer
Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation></author><author xml:id="author-0003"><persName><surname>Kircheis</surname><forename type="first">Ralf</forename></persName><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and Boehringer
Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation></author><author xml:id="author-0004"><persName><surname>Wagner</surname><forename type="first">Ernst</forename></persName><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and Boehringer
Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><note place="foot"><ref>†</ref><p>
New address: Department of Pharmacy, Ludwig-Maximilians-University, Butenandtstr 5-13, D-81377 Munich, Germany.</p></note></author><idno type="istex">5BDA273D2BD7C85D0528CE1426C5CDE263EF638C</idno><idno type="ark">ark:/67375/TPS-J0F4G1J1-8</idno><idno type="DOI">10.1021/bc0001488</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="2001-06-23">2001</date><date when="2001-07-18">2001</date><biblScope unit="vol">12</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="529">529</biblScope><biblScope unit="page" to="537">537</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><abstract><p>With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate
consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides.
Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor
cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal
carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold
increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF
strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway.
Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface
modification of EGF−PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative
way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was
projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded
particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated
EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without
EGF.
</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/bc0001488</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Different Strategies for Formation of PEGylated EGF-Conjugated
PEI/DNA Complexes for Targeted Gene Delivery</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Blessing</surname><given-names>Thomas</given-names></name><xref rid="bc0001488AF1">*</xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Kursa</surname><given-names>Malgorzata</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Holzhauser</surname><given-names>Robert</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Kircheis</surname><given-names>Ralf</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Wagner</surname><given-names>Ernst</given-names></name><xref rid="bc0001488AF2"><sup>†</sup></xref></contrib><aff>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and Boehringer
Ingelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</aff></contrib-group><author-notes><corresp id="bc0001488AF1">
To whom correspondence should be addressed. New address: Faculte de Pharmacie, 15 av. Charles Flahault, F-34060
Montpellier. Telephone: ++33-467-418-267. Fax: ++33-467-520-898. E-mail: blessing@pharma.univ-montp1.fr.</corresp><fn id="bc0001488AF2"><label>†</label><p>
New address: Department of Pharmacy, Ludwig-Maximilians-University, Butenandtstr 5-13, D-81377 Munich, Germany.</p></fn></author-notes><pub-date pub-type="epub"><day>23</day><month>06</month><year>2001</year></pub-date><pub-date pub-type="ppub"><day>18</day><month>07</month><year>2001</year></pub-date><volume>12</volume><issue>4</issue><fpage>529</fpage><lpage>537</lpage><history><date date-type="received"><day>15</day><month>11</month><year>2000</year></date><date date-type="rev-recd"><day>28</day><month>04</month><year>2001</year></date><date date-type="asap"><day>23</day><month>06</month><year>2001</year></date><date date-type="issue-pub"><day>18</day><month>07</month><year>2001</year></date></history><permissions><copyright-statement>Copyright © 2001 American Chemical Society</copyright-statement><copyright-year>2001</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate
consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides.
Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor
cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal
carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold
increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF
strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway.
Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface
modification of EGF−PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative
way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was
projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded
particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated
EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without
EGF.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bc0001488</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="d7e145"><title>Introduction</title><p>Cancer gene therapy by systemic gene delivery would
be a most attractive way for treatment of cancer. Approaching the tumor via blood circulation should allow
several advantages over the local intratumoral injection
such as reaching multiple distant metastases. Unfortunately, the required tumor-targeted gene delivery system
does not exist yet. An ideal gene delivery system has to
fulfill several criteria to access tumor tissue: (1) ligands
able to mediate cell specific recognition and internalization into target cells must be incorporated; (2) the gene
delivery system must be inert against nonspecific interactions with the biological environment such as blood
components and nontarget cells.
</p><p>Synthetic gene delivery systems have recently attracted much attention particularly those based on
polyethylenimine (PEI) polymers. Native PEI polymers
of various molecular weights and topological isomers
have been shown to be efficient for transfection of
eukaryotic cells (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00001" ref-type="bibr"></xref>−<xref rid="bc0001488b00002" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00003" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00004" ref-type="bibr"></xref></named-content></italic>), and receptor-mediated endocytosis
was achieved by chemical modification of PEI backbone
with ligands such as sugars (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00005" ref-type="bibr"></xref>−<xref rid="bc0001488b00006" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00007" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00008" ref-type="bibr"></xref></named-content></italic>), peptides (<italic toggle="yes"><xref rid="bc0001488b00009" ref-type="bibr"></xref></italic>), and
proteins (<italic toggle="yes"><xref rid="bc0001488b00010" ref-type="bibr"></xref></italic>). PEI polymers have also been used for gene
delivery in vivo (<italic toggle="yes">1, 11−17</italic>) and particularly for systemic
delivery to the lung (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00018" ref-type="bibr"></xref>, <xref rid="bc0001488b00019" ref-type="bibr"></xref></named-content></italic>). However, unspecific interactions of the DNA complexes with blood components and
nontarget cells strongly reduce the applicability of complexes. Recently, our group reported tumor-targeted gene
delivery based on transferrin−PEI conjugate (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00020" ref-type="bibr"></xref>, <xref rid="bc0001488b00021" ref-type="bibr"></xref></named-content></italic>).
Surface modification of transferrin-containing complexes
with hydrophilic poly(ethylene glycol) (PEG) polymers
strongly reduced interactions with blood components and
prolonged circulation in blood after systemic delivery.
Moreover, PEGylated transferrin-containing complexes
mediated gene expression in distant tumor and showed
reduced tropism for the lung compared to nonmodified
PEI/DNA complexes.
</p><p>Epithelial growth factor (EGF) receptor is an attractive
therapeutic target for tumor targeting because it is
overexpressed in a high percentage of human carcinomas
including glioblastoma and lung, liver, breast, head, neck,
and bladder cancers (<italic toggle="yes"><xref rid="bc0001488b00022" ref-type="bibr"></xref></italic>). The natural EGF ligand is a
53-residue polypeptide that binds specifically and with
high affinity (<italic toggle="yes"><xref rid="bc0001488b00023" ref-type="bibr"></xref></italic>) to the EGF receptor. The binding of
EGF triggers the dimerization of the receptor and the
clustering of EGF−EGF receptor into coated pits that are
internalized.
</p><p>With the aim of specific gene delivery to EGF receptor-bearing tumor cells we synthesized a EGF−PEI conjugate and tested its ability to deliver DNA in three
carcinoma cell lines. Keeping in mind future systemic
gene delivery, the effect of PEGylation of preformed EGF-containing complexes was studied. We also describe an
alternative novel way to generate PEGylated ligand-containing complexes where ligands are projected away
from core complexes through a PEG linker.
</p></sec><sec id="d7e202"><title>Experimental Procedures</title><p><bold>Chemicals.</bold> Branched polyethylenimine with an average molecular weight of 25 kDa (PEI 25K) was obtained from Aldrich (Vienna, Austria). For complex
preparation, PEI 25K was diluted with water, neutralized
with HCl solution, and used as a 0.43 mg/mL working
solution (10 mM of amine functions, assuming a MW of
43 Da for the repeating unit). Linear polyethylenimine
(PEI 22K), kindly provided by Jean-Paul Behr (Faculté
de Pharmacie, Illkirch, France) and commercially
available from Euromedex (Exgen 500, Euromedex,
Souffelweyersheim, France), was also used as a neutral
10 mM working solution. Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was purchased from Fluka (Buchs,
Switzerland); chloroquine, 1,4-dithio-<sc>l</sc>-threitol (DTT),
bovine serum albumin (BSA) from Sigma (St. Louis, MO);
α-vinyl sulfone-ω-<italic toggle="yes">N</italic>-hydroxy succinimide ester poly(ethylene glycol) (VS-PEG-NHS, MW 3400), α-maleimide-ω-<italic toggle="yes">N</italic>-hydroxysuccinimide ester poly(ethylene glycol) (MAL-PEG-NHS, MW 3400) from Shearwater Polymers
(Huntsville, AL); mouse epidermal growth factor (mEGF)
from Serotec (Oxford, England); cell culture media,
antibiotics, foetal calf serum (FCS), Geneticin (G418-sulfate) from Life Technologies (Gaithersburg, MD).
Plasmid pCMVLuc (Photinus pyralis luciferase under
control of the CMV enhancer/promoter) described in
Plank et al. (<italic toggle="yes"><xref rid="bc0001488b00024" ref-type="bibr"></xref></italic>) was purified with the EndoFree Plasmid
Kit from Qiagen (Hilden, Germany).
</p><p><bold>Synthesis of Thiol-Functionalized PEI 25K.</bold> PEI
25K was diluted in water, neutralized with 32% aqueous
hydrochloric and subjected to gel filtration as described
previously (<italic toggle="yes"><xref rid="bc0001488b00010" ref-type="bibr"></xref></italic>). To 50 mg of PEI 25K (1.16 mmol of amine
functions, quantified by ninhydrine assay at 570 nm (<italic toggle="yes"><xref rid="bc0001488b00025" ref-type="bibr"></xref></italic>))
in 758 μL was added 394 μL of a 20 mM ethanolic
solution of SPDP (2.5 mg, 7.9 μmol). The reaction was
carried out for 1 h at room temperature. Low molecular
weight products were removed by gel filtration on a
Sephadex G25 superfine (Pharmacia, Uppsala, Sweden;
300 × 10 mm column) in 0.25 M NaCl, 20 mM Hepes,
pH 7.3. After dialysis and concentration under vacuum,
a 720 μL solution containing 0.97 mmol of PEI monomer
(41.5 mg) with 4.2 μmol of pyridyldithiopropionate linker
(PDP; quantified spectrophotometrically at 343 nm by
release of thiopyridone after reduction of an aliquot with
excess DTT (<italic toggle="yes"><xref rid="bc0001488b00026" ref-type="bibr"></xref></italic>)) was obtained (i.e., ca. 2.5 PDP groups
per PEI molecule). Half of this solution (20.7 mg PEI,
0.48 mmol of amine functions) was mixed under argon
with an excess of DTT (73 μmol) and was left to react for
1 h at room temperature. For removal of the low molecular weight products an additional gel filtration on a
Sephadex G25 superfine (0.25 M NaCl, 20 mM Hepes pH
7.3; 100 × 10 mm) was performed yielding 1.5 mL of
thiol-functionalized PEI 25K that contained 242 μmol of
PEI monomer (10.4 mg) modified with 1.25 μmol of thiol
functions (quantified by Ellman assay at 412 nm (<italic toggle="yes"><xref rid="bc0001488b00027" ref-type="bibr"></xref></italic>));
i.e., ca. three thiol groups per PEI molecule.
</p><p><bold>Synthesis of PDP- and Thiol-Functionalized
mEGF.</bold> A solution of 4 mg mEGF (0.66 μmol) in 1 mL of
16 mM Hepes buffer pH 7.9 was mixed with 3.1 mg of
SPDP (10 μmol) in 0.5 mL of ethanol. The reaction and
the purification steps (see below) were carried out in the
presence of ethanol (20−50%) to avoid precipitation of
the PDP-functionalized product. The reaction was left to
react for 3 h at room temperature. The functionalized
peptide was purified by either dialysis (MWCO 1000;
Spectra Por, Spectrum, Houston, TX) against 50% aqueous ethanol or gel filtration on a Sephadex G10 (Pharmacia, Uppsala, Sweden; 100 × 10 mm column) in 20
mM Hepes pH 7.3/20% ethanol.
</p><p>The purified conjugate contained about 0.9 PDP group
per mEGF peptide (±10%, depending on the preparation).
</p><p>Thiol-functionalized mEGF (mEGF-SH) was obtained
by reduction of an aliquot of mEGF-PDP (at a concentration of about 50 μM) with DTT (1 mM) for 1 h under
argon. Low molecular weight products were removed by
gel filtration on a Sephadex G25M PD-10 column (Pharmacia, Uppsala, Sweden) in 20 mM Hepes pH 7.3/30%
ethanol. Quantification of thiol functions with Ellman
assay indicated that the conjugate contained about one
thiol group per mEGF peptide (±15%).
</p><p><bold>Synthesis of mEGF</bold><bold>-</bold><bold>PEI 25K Conjugate.</bold> Two solutions of PDP-functionalized mEGF were pooled, and 2
mL of the resulting solution that contained 4.2 mg EGF
(0.69 μmol) was modified with 0.56 μmol of pyridyldithiopropionate linker was mixed under argon with 7.5 mg of
thiol-functionalized PEI 25K (174 μmol of amine functions, 0.9 μmol thiol functions) in 1.08 mL of 0.25 M NaCl,
20 mM Hepes, pH 7.9. After 1 day at room temperature,
the reaction mixture was loaded on a cation-exchange
column (Macro-Prep High S, BioRad, München, Germany; 100 × 10 mm column) and was fractionated with
a salt gradient from 0.5 to 3.0 M sodium chloride with a
constant content of 20 mM Hepes, pH 7.3. The major
amount of conjugate eluted between 2.7 and 3.0 M salt
and was pooled. After dialysis against HBS (MWCO
6000−8000; Gibco BRL, Gaithersburg, MD) and concentration under vacuum, 6.5 mL of mEGF−PEI solution
was obtained that contained 148 μmol PEI of monomer
(6.4 mg) modified with 0.32 μmol of mEGF peptide (2.0
mg); i.e., approximately 1.3 mEGF peptides per PEI
molecule. Before use, EGF conjugate samples were
diluted to 10 mM PEI monomer.
</p><p><bold>Formation of PEI/DNA, mEGF</bold><bold>−</bold><bold>PEI/DNA, and
PEGylated </bold><bold>mEGF</bold><bold>−</bold><bold>PEI/DNA Complexes.</bold> DNA complexes used for the transfection of KB and Renca-EGFR
cells were prepared as described by Boussif et al. (<italic toggle="yes"><xref rid="bc0001488b00001" ref-type="bibr"></xref></italic>) with
some modifications. Briefly, 4 μg of pCMVLuc plasmid
(12 nmol of phosphate) and 7.2 μL of a 10 mM PEI or
EGF−PEI (72 nmol of amine functions) were each diluted
in 100 μL of the indicated buffer: HBS (Hepes buffered
saline: 145 mM NaCl/20 mM Hepes pH 7.3), HBS 1/2
(HBS twice diluted with water) or 5 mM Hepes pH 7.3,
and mixed together. After 15 min incubation an aliquot
of the complex solution was added to the cells (100 μL/well for example corresponding to 2 μg DNA/well).
</p><p>Complexes that were subsequently PEGylated were
prepared in advance. Fifteen minutes after mixing DNA
and polymer solutions, 0.6 μL of a 6 mM VS-PEG-NHS
(or MAL-PEG-NHS) stock solution in DMSO (3.6 nmol)
was added and left to react overnight.
</p><p>For titration of different N/P ratios on CMT-93 cells
mEGF−PEI/DNA or PEI/DNA complexes were prepared
by mixing 5 μg of pCMVLuc plasmid, prediluted in 250
μL of HBS, with different amounts of mEGF−PEI 25K
or PEI 25K prediluted in 250 μL of HBS (2.25 μg of PEI
corresponding for example at N/P 3.6).
</p><p><bold>Sequential Addition of Bifunctional PEG and
mEGF</bold><bold>−</bold><bold>SH to PEI/DNA </bold><bold>Complexes.</bold> pCMVLuc plasmid (20 μg, 60 nmol of phosphate) and PEI (360 nmol of
amine) were each diluted in 0.5 mL of the indicated buffer
previously saturated with argon. The two solutions were
gathered and mixed with argon stream. The mixture was
kept under argon until the end of the protocol. After 15
min, 3 μL of a 6 mM VS-PEG-NHS (or MAL-PEG-NHS)
stock solution in DMSO (18 nmol) were added. Two hours
later, various amounts of EGF-SH (between 7.2 and 144
pmol; as indicated in the Figure legend) were added to
200 μL aliquots of the complex solution and left to react
overnight.
</p><p><bold>Cells and Cell Culture.</bold> KB human epidermoid
carcinoma cells (ATCC CCL-17) and CMT-93 murine
rectum carcinoma cells (ATCC CCL-223) were cultured
in DMEM medium supplemented with 10% FCS, 2 mM
glutamine, 100 U/ml penicillin, and 100 μg/mL streptomycin. Renca mouse renal carcinoma cells stably cotransfected with the plasmids pLTR-EGFR and pSV2neo (<italic toggle="yes"><xref rid="bc0001488b00028" ref-type="bibr"></xref></italic>)
(kindly provided by Winfried Wels, Georg-Speyer-Haus,
Frankfurt am Main, Germany), named Renca-EGFR,
were grown in RPMI-1640 medium supplemented with
10% FCS, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.5 mg/mL Geneticin. All cell lines
were maintained at 37 °C in a 5% CO<sub>2</sub> humidified
atmosphere.
</p><p><bold>Transfection.</bold> One day before transfection, KB and
Renca-EGFR cells were seeded at 4 × 10<sup>4</sup> cells per well
in 24-well dishes. Prior to transfection, medium was
removed and replaced with 1 mL (or as indicated in
Figure legend) of fresh culture medium complemented
with 10% FCS. Transfection complexes were added to the
cells, and after 4 h incubation, transfection medium was
removed and replaced by 1 mL fresh culture medium.
</p><p>Murine CMT-93 cells were seeded at 5 × 10<sup>5</sup> cells per
25 cm<sup>2</sup> tissue culture flask 1 day before transfection. Prior
to transfection, medium was removed and replaced by 2
mL of fresh culture medium complemented with 10%
FCS. Transfection complexes (in 0.5 mL of HBS) were
added to the cells. After 4 h incubation, transfection
medium was removed and replaced by fresh culture
medium.
</p><p>The cells were grown for additional 20 h and then
washed in PBS and lysed (0.25 M Tris·HCl pH 7.4, 0.1%
Triton X-100). Luciferase activity and protein concentration were determined from aliquot of the lysate using a
luminometer (Lumat LB9507, Berthold, Bad Wildbad,
Germany) and Bio-Rad protein assay (Bio-Rad, Hercules,
CA), respectively. Values are given as light units integrated over 10 s per mg of protein.
</p></sec><sec id="d7e319"><title>Results</title><p><bold>Synthesis of mEGF</bold><bold>−</bold><bold>PEI 25K Conjugate.</bold> mEGF
was covalently coupled to the PEI backbone through a
disulfide bridge as described in Experimental Procedures.
Murine EGF can bind both the murine and human EGF
receptor (<italic toggle="yes"><xref rid="bc0001488b00029" ref-type="bibr"></xref></italic>) and was chosen because it lacks lysine
residues in its primary sequence and can react with
SPDP-activated ester only through the N-terminus. It
has been shown that the derivatization of this free amino
group had little effect on EGF receptor binding compared
to native EGF (<italic toggle="yes"><xref rid="bc0001488b00030" ref-type="bibr"></xref></italic>). Modification of this amino group with
the heterobifunctional cross-linker SPDP (<italic toggle="yes"><xref rid="bc0001488b00026" ref-type="bibr"></xref></italic>) rendered
the peptide poorly soluble, and ethanol had to be present
in the reaction and purification steps. The reaction was
carried out with an excess of SPDP (15 molar equiv
excess) and led to a mEGF−PDP conjugate containing
almost one linker per peptide. PEI with an average
molecular weight of 25 kDa was also modified with SPDP
and then treated with an excess of DTT. A thiol-functionalized PEI conjugate that contained approximately three thiol groups per PEI molecule was obtained.
Reaction between thiol-functionalized PEI and mEGF−PDP followed by purification by cation-exchange chromatography resulted in a mEGF−PEI conjugate that
contained 1.3 mEGF peptides per PEI molecule.
</p><p><bold>EGF Receptor-Mediated Gene Transfer in Various Carcinoma Cell Lines.</bold> Transfection efficiency of
mEGF−PEI 25K-containing DNA complexes was compared with EGF-free PEI 25K/DNA complexes in three
carcinoma cell lines (Figure <xref rid="bc0001488f00001"></xref>). Formulation of complexes
in three different Hepes buffers containing different
concentrations of NaCl (Hepes: 0 mM, HBS 1/2: 75 mM
and HBS: 150 mM) at a PEI-nitrogen to DNA-phosphate
ratio of 6 (N/P = 6) resulted in complexes of various sizes
(Table <xref rid="bc0001488t00001"></xref>) which were tested in transfection experiments
with Renca-EGFR cells (Figure <xref rid="bc0001488f00001"></xref>A) and KB cells (Figure
<xref rid="bc0001488f00001"></xref>B). EGF-containing DNA complexes showed a 5- to 300-fold increase in gene delivery efficiency compared to
unmodified PEI/DNA complexes. This ligand-mediated
enhancement was found for all formulations. Large
complexes formulated in physiological salt concentration
(HBS) were more efficient than small complexes prepared
in salt-free medium (5 mM Hepes pH 7.3), similar as
observed previously with other cell lines using transferrin-modified PEI/DNA complexes (<italic toggle="yes"><xref rid="bc0001488b00031" ref-type="bibr"></xref></italic>). Chemical ligation of mEGF to the PEI 25K backbone did not change
the biophysical properties (size, zeta potential) of resulting complexes generated either in Hepes or HBS buffer
(Table <xref rid="bc0001488t00001"></xref>). This strongly suggests the expected biological
involvement of EGF in the increase of gene expression.
In contrast, in HBS 1/2, the difference of size between
both type of complexes might also contribute to the
improved gene expression.
<fig id="bc0001488f00001" position="float" orientation="portrait"><label>1</label><caption><p>EGF-mediated gene transfer to Renca-EGFR mouse renal, KB human epidermoid and CMT-93 murine rectum carcinoma cells. Renca-EGFR (A) and KB cells (B) were transfected with pCMVLuc plasmid (2 μg/well) complexed with PEI 25K or mEGF−PEI 25K/PEI 25K (1:1) at a PEI (nitrogen)/DNA (phosphate) ratio of 6 (N/P = 6) in either 5 mM Hepes pH 7.3, HBS 1/2 or HBS. After 4 h, transfection medium was replaced with fresh culture medium and cells were grown for additional 20 h. Luciferase activity was measured as described in Experimental Procedures. Values are the mean ± SD of two independent experiments made in duplicate. (C) KB cells were transfected with various amounts of pCMVLuc plasmid (2 to 0.125 μg per well) complexed with PEI 25K or mEGF−PEI 25K/PEI 25K (1:1) at N/P = 6 in HBS. Luciferase activity is the mean ± SD of two independent experiments made in duplicate. (<bold>D</bold>)
CMT-93 cells were transfected with pCMVLuc plasmid (5μg/flask) complexed with various amounts of PEI 25K or EGF−PEI 25K (at indicated N/P ratios) in HBS. Luciferase activity
is shown as mean ± SD of duplicate.</p></caption><graphic xlink:href="bc0001488f00001.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bc0001488t00001" position="float" orientation="portrait"><label>1</label><caption><p>Biophysical Characteristics of DNA Complexes and Their PEGylated Derivatives<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"></oasis:entry><oasis:entry namest="2" nameend="4">size (nm)</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="5"><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:colspec colnum="5" colname="5"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1"></oasis:entry><oasis:entry namest="2" nameend="2">Hepes</oasis:entry><oasis:entry namest="3" nameend="3">HBS 1/2</oasis:entry><oasis:entry namest="4" nameend="4">HBS</oasis:entry><oasis:entry namest="5" nameend="5">zeta (mV) Hepes
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PEI 25K
</oasis:entry><oasis:entry colname="2">57 ± 14
</oasis:entry><oasis:entry colname="3">116 ± 36
</oasis:entry><oasis:entry colname="4">478 ± 111
</oasis:entry><oasis:entry colname="5">+20.0 ± 1.7
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PEI 25K + MAL-PEG-NHS
</oasis:entry><oasis:entry colname="2">47 ± 11
</oasis:entry><oasis:entry colname="3">96 ± 30
</oasis:entry><oasis:entry colname="4">446 ± 118
</oasis:entry><oasis:entry colname="5">−0.7 ± 1.1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PEI 25K + MAL-PEG-NHS + EGF-SH
</oasis:entry><oasis:entry colname="2">49 ± 15
</oasis:entry><oasis:entry colname="3">81 ± 23
</oasis:entry><oasis:entry colname="4">403 ± 118
</oasis:entry><oasis:entry colname="5">−0.6 ± 1.4
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">EGF-PEI 25K/PEI 25K
</oasis:entry><oasis:entry colname="2">53 ± 12
</oasis:entry><oasis:entry colname="3">447 ± 82
</oasis:entry><oasis:entry colname="4">590 ± 132
</oasis:entry><oasis:entry colname="5">+21.5 ± 2.3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">EGF-PEI 25K/PEI 25K + MAL-PEG-NHS
</oasis:entry><oasis:entry colname="2">48 ± 14
</oasis:entry><oasis:entry colname="3">553 ± 128
</oasis:entry><oasis:entry colname="4">> 1 μm
</oasis:entry><oasis:entry colname="5">−0.6 ± 1.0
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PEI 25K/PEI 22K
</oasis:entry><oasis:entry colname="2">43 ± 11
</oasis:entry><oasis:entry colname="3">∼ 1 μm
</oasis:entry><oasis:entry colname="4">∼ 1 μm
</oasis:entry><oasis:entry colname="5">+28.0 ± 2.5
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PEI 25K/PEI 22K + MAL-PEG-NHS
</oasis:entry><oasis:entry colname="2">42 ± 10
</oasis:entry><oasis:entry colname="3">> 1 μm
</oasis:entry><oasis:entry colname="4">> 1 μm
</oasis:entry><oasis:entry colname="5">−0.1 ± 1.4</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Complexes were prepared as described in Experimental Procedures in 1 mL total volume of the indicated buffer containing 20 μg
DNA (for zeta potential measurement of PEI 25K/PEI 22K/DNA complexes DNA concentration was 50 μg/mL). Size and zeta potential of
non-PEGylated and PEGylated complexes were measured 20 min and 18 h after their formation respectively, using a Zetasizer 3000HS
(Malvern Instruments, Worcestershire, England). Three measurements were carried out per sample.</p></table-wrap-foot></table-wrap></p><p>Comparative transfection was also investigated with
sub-micromolar amounts of DNA on KB cells (Figure <xref rid="bc0001488f00001"></xref>C).
Luciferase expression of PEI complexes generated in HBS
dropped rapidly with the decrease of DNA amount while
expression of EGF−PEI complexes remained relatively
high.
</p><p>Luciferase gene expression was examined as a function
of the PEI-nitrogen to DNA-phosphate ratio used to
generate the complexes. Gene expression in CMT-93
murine rectum carcinoma cells was strongly dependent
on the N/P ratio (Figure <xref rid="bc0001488f00001"></xref>D). Increasing the N/P ratio
from 3.6 to 8.4 increased the efficiency of both EGF-containing and EGF-free DNA complexes by more than
three orders of magnitude. There was a 3- to 30-fold
ligand-mediated enhancement at all N/P ratios tested.
With ligand-free complexes a drop in transfection efficacy
was found at high N/P ratio correlating with some
toxicity.
</p><p>Small particles generated in salt-free medium led to
low gene expression in comparison with large particles
generated in HBS (Figures <xref rid="bc0001488f00001"></xref>A and 1B). We tested
whether the addition of an endosomolytic compound during the transfection could enhance the efficiency of small
complexes. Therefore, the lysomotropic drug chloroquine
was added during transfection in cell culture medium of
Renca-EGFR cells at a concentration of 100 and 200 μM
(Figure <xref rid="bc0001488f00002"></xref>A). Addition of chloroquine resulted in a 100-fold increase of gene expression with both EGF-containing DNA complexes and EGF-free DNA complexes while
the ligand-mediated enhancement remained approximately constant.
<fig id="bc0001488f00002" position="float" orientation="portrait"><label>2</label><caption><p>(A) Chloroquine strongly enhances transfection efficiency of small particles generated in salt free medium on Renca-EGFR cells. Renca-EGFR were transfected with pCMVLuc plasmid (2 μg/well) complexed with PEI 25K or mEGF−PEI 25K/PEI 25K (1:1) at N/P = 6 in Hepes. Transfection was carried out in the presence of 0, 100, or 200 μM chloroquine. After 4 h, transfection medium was replaced with fresh culture medium and cells were grown for additional 20 h. Luciferase activity values are the mean ± SD of two independent experiments made in duplicate. (B) Competitive inhibition of EGF receptor-mediated gene transfer on Renca-EGFR cells. Renca-EGFR cells incubated in 0.5 mL of fresh culture medium containing 200 μM chloroquine were transfected with pCMVLuc plasmid (1 μg/well) complexed with PEI 25K or mEGF−PEI 25K/PEI 25K (1:1) at N/P = 6 in Hepes. For the inhibition and the control experiments transfection was carried out in the presence of free mEGF (12 μg/well) and BSA (12 μg/well), respectively. After 4 h, transfection medium was replaced with fresh medium and cells were grown for additional 20 h. Luciferase activity is shown as mean ± SD of duplicate.</p></caption><graphic xlink:href="bc0001488f00002.tif" position="float" orientation="portrait"></graphic></fig></p><p>To test whether the EGF receptor was involved in
EGF−PEI-mediated gene transfer, a competition assay
was performed using small complexes formulated in salt-free medium. Addition of exogenous mEGF in cell culture
medium during transfection of Renca-EGFR did not
interfere with PEI/DNA receptor-independent gene transfer (Figure <xref rid="bc0001488f00002"></xref>B). However, competition of EGF-containing
DNA complexes with free mEGF (100 molar equiv excess)
decreased gene transfer efficacy down to nearly the
values found for the receptor-independent transfection.
On the other hand, addition of BSA, a protein that does
not interact with EGFR, did not significantly affected
gene transfer efficiency of both type of complexes. Taken
together, competitive and comparative transfection
experiments strongly suggested involvement of EGF receptor in gene delivery with EGF−PEI-based systems.
</p><p><bold>Transfection Efficiency of PEGylated EGF-Containing DNA Complexes.</bold> Targeted transfection particles for systemic gene delivery should have uncharged
surface to avoid nonspecific electrostatic interaction with
blood components and nontarget cells. On the other hand,
DNA is usually efficiently condensed using an excess of
polymer, giving rise to cationic particles. One way to
overcome this dilemma is to covalently modify the surface
of ligand-containing DNA complexes with an electro-neutral polymer such PEG (<italic toggle="yes"><xref rid="bc0001488b00020" ref-type="bibr"></xref></italic>) (Figure <xref rid="bc0001488f00003"></xref>, strategy A).
Coating the DNA particle with PEG shields its cationic
surface and prevents interaction with macromolecules by
steric hindrance. On the other hand PEG potentially
might also shield small ligands bound to the polymer and
thus preventing to some extent the binding to the
receptor on the target cell. As described in strategy B
(Figure <xref rid="bc0001488f00003"></xref>), incorporation of the ligand at the distal end
of PEGylated PEI/DNA complexes should allow better
accessibility of the ligand for its receptor and thus should
increase cellular internalization of DNA complexes.
<fig id="bc0001488f00003" position="float" orientation="portrait"><label>3</label><caption><p>Schematic presentation of alternatives for the formation of PEGylated ligand-containing DNA complexes. Strategy A: A ligand is covalently conjugated to a polycation (step 1). Condensation of DNA with this conjugate leads to ligand-containing DNA complexes (step 2) which are subsequently modified with a PEG derivative reacting with free amino groups of the polycation (step 3). Strategy B: DNA is condensed with a polycation (step 1), and the resulting complexes are modified by a heterobifunctional PEG which first reacts with amino groups of the polycation (step 2). Subsequently ligands are incorporated into the complexes by covalent conjugation with the distal end of the PEG (step 3). The latter strategy should allow a better accessibility of the grafted ligand for its receptor.</p></caption><graphic xlink:href="bc0001488f00003.tif" position="float" orientation="portrait"></graphic></fig></p><p>These two strategies using EGF as ligand were evaluated with Renca-EGFR cells (Renca mouse renal carcinoma cells stably transfected with pLTR-EGFR plasmid
carrying human EGF receptor cDNA (<italic toggle="yes"><xref rid="bc0001488b00028" ref-type="bibr"></xref></italic>)) and KB cells
(human epidermoid carcinoma cells that endogenously
express human EGF receptor). Transfection of Renca-EGFR was performed in the presence of chloroquine with
small complexes formulated in salt free medium. It was
previously observed that addition of chloroquine on
Renca-EGFR cells increases considerably PEI-mediated
gene transfection (Figure <xref rid="bc0001488f00002"></xref>A), in contrast to KB cells
where no beneficial effect is observed. PEGylation of PEI/DNA complexes dramatically decreased the zeta potential
(which reflects the surface charge) close to the neutrality
(Table <xref rid="bc0001488t00001"></xref>) and was accompanied by a reduction of gene
expression (Figure <xref rid="bc0001488f00004"></xref>A). Addition of increasing amounts
of thiol-functionalized mEGF to PEGylated complexes
without changing their biophysical properties (size, zeta
potential) increased gene transfection by two-orders of
magnitude. In contrast, addition of the functionalized
ligand to PEI/DNA complexes without reactive PEG
linker had no effect. This finding strongly suggested that
EGF binding at the distal end of the PEG allows the
delivery of PEGylated complexes in a ligand-dependent
manner. We compared this strategy with the one previously developed in our group (<italic toggle="yes"><xref rid="bc0001488b00020" ref-type="bibr"></xref></italic>) (Figure <xref rid="bc0001488f00003"></xref>, strategy A).
As previously observed, EGF−PEI conjugate-containing
DNA complexes were more efficient than those prepared
with native PEI 25K. PEGylation of EGF-containing
DNA complexes decreased the zeta potential close to the
neutrality (Table <xref rid="bc0001488t00001"></xref>) and also gene expression. This
PEGylated complexes were still about 5-fold more effective than EGF-free PEI/DNA complexes but about one
order less efficient than PEGylated complexes obtained
according to strategy B (Figure <xref rid="bc0001488f00003"></xref>).
<fig id="bc0001488f00004" position="float" orientation="portrait"><label>4</label><caption><p>Transfection efficiency of PEGylated EGF-containing DNA complexes on Renca-EGFR and KB cells. (A) Renca-EGFR cells incubated in fresh culture medium containing 200 μM chloroquine were transfected with the following complexes generated as described in Experimental Procedures in Hepes buffer at a N/P ratio of 6 (2 μg DNA/well). Column 1: PEI 25K/DNA; 2: PEI 25K/DNA modified with VS−PEG-NHS; 3, 4, and 5: as in 2 and subsequently modified with 14, 72, and 144 pmol of thiol-functionalized mEGF, respectively; 6: mEGF−PEI 25K/PEI 25K (1:1)/DNA; 7: mEGF−PEI 25K/PEI 25K (1:1)/DNA modified with VS−PEG-NHS; 8: as in 1 in the presence of 144 pmol mEGF in culture medium during the transfection. Luciferase activity values are the mean ± SD of two independent experiments made in duplicate. (B) KB cells incubated in fresh culture medium were transfected with the following complexes prepared in HBS 1/2 at a N/P ratio of 6 (2 μg DNA/well). Column 1: PEI 25K/DNA; 2: PEI 25K/DNA modified with MAL-PEG-NHS; 3 and 4: as in 2 and subsequently modified with 72 pmol and 144 pmol of thiol-functionalized mEGF, respectively; 5: EGF−PEI 25K/PEI 25K (1:1)/DNA; 6: EGF−PEI 25K/PEI 25K (1:1)/DNA modified with MAL-PEG-NHS. Luciferase activity is shown as mean ± SD of duplicate.</p></caption><graphic xlink:href="bc0001488f00004.tif" position="float" orientation="portrait"></graphic></fig></p><p>The two strategies were also used for the delivery of
PEGylated EGF-containing DNA complexes in KB cells
in the absence of chloroquine (Figure <xref rid="bc0001488f00004"></xref>B). Subsequent
addition of PEG and thiol-functionalized EGF to PEI/DNA complexes generated in HBS 1/2 increased their
efficiency by more than one order of magnitude. Complexes resulting from the PEGylation of EGF−PEI
conjugate-containing DNA complexes showed approximately the same level of gene expression.
</p><p><bold>Transfection with Different PEI Polymers Complexes and Their PEGylated </bold><bold>Derivatives.</bold> Among all
available PEI, which differ by their average molecular
weight and topology, linear PEI 22K had shown the most
promising properties to deliver DNA (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00004" ref-type="bibr"></xref>, <xref rid="bc0001488b00018" ref-type="bibr"></xref></named-content></italic>). We were
interested to know whether the combination of PEI 22K
with mEGF−PEI 25K could enhance the ligand-mediated
gene delivery in our in vitro cell models. KB cells were
transfected with various amount of DNA complexed with
PEI 25K, EGF−PEI 25K/PEI 25K (1:1), PEI 25K/PEI
22K (1:1), or EGF−PEI 25K/PEI 22K (1:1) in HBS 1/2 at
N/P = 6 (Figure <xref rid="bc0001488f00005"></xref>A). Incorporation of PEI 22K in the
preparation of ligand and ligand-free PEI 25K/DNA
complexes strongly increased gene expression especially
at low DNA amounts. At 60 ng of DNA, PEI 22K-containing complexes remained efficient (10<sup>7</sup>−10<sup>8</sup> RLU/mg) whereas PEI 22K-free complexes showed only low
activity. Interestingly, a ligand-dependent enhancement
was observed with PEI 22K-containing complexes on KB
cells but also on Renca-EGFR cells independently of the
medium in which the complexes were prepared (Figure
<xref rid="bc0001488f00005"></xref>B).
<fig id="bc0001488f00005" position="float" orientation="portrait"><label>5</label><caption><p>Effect of different PEI polymers (linear PEI 22K versus branched PEI 25K) on transfection efficiency. Complexes were generated from a mixture of mEGF−PEI 25K and different PEIs and tested on KB and Renca-EGFR cells. (A) KB cells were transfected with various amounts of pCMVLuc plasmid (2 to 0.06 μg per well) complexed with PEI 25K, mEGF−PEI 25K/PEI 25K (1:1), PEI 25K/PEI 22K (1:1) or mEGF−PEI 25K/PEI 22K (1:1) at N/P=6 in HBS 1/2. Luciferase activity is shown as mean ± s.d. of duplicate (* is <10<sup>3</sup> RLU/mg of protein). (B)
Renca-EGFR cells were transfected with pCMVLuc plasmid (2
μg/well) complexed with PEI 25K, mEGF−PEI 25K/PEI 25K (1:1), PEI 22K or mEGF−PEI 25K/PEI 22K (1:1) at N/P = 6 in
either Hepes buffer (in the presence of 200 μM chloroquine in
cell culture medium during the transfection), HBS 1/2 or HBS.
Luciferase activity is shown as mean ± SD of duplicate.</p></caption><graphic xlink:href="bc0001488f00005.tif" position="float" orientation="portrait"></graphic></fig></p><p>Transfection efficiency of PEGylated EGF-containing
PEI 25K/PEI 22K complexes was studied in KB cells
(Figure <xref rid="bc0001488f00006"></xref>). As observed previously with PEI 22K-free
complexes (Figure <xref rid="bc0001488f00004"></xref>B), subsequent addition of PEG and
thiol-functionalized EGF to PEI/DNA complexes strongly
increased transfection efficiency to a level comparable to
the one obtained by PEGylation of EGF-containing DNA
complexes.
<fig id="bc0001488f00006" position="float" orientation="portrait"><label>6</label><caption><p>Transfection efficiency of PEGylated EGF-containing PEI 25K/PEI 22K complexes on KB cells. Complexes were prepared as described in Figure <xref rid="bc0001488f00004"></xref>B with PEI 25K/PEI 22K (1:1) instead of PEI 25K (Columns 1<italic toggle="yes">−</italic>4) and EGF−PEI 25K/PEI
22K instead of EGF−PEI 25K/PEI 25K (Columns 5 and 6).
Column 1: PEI 25K/PEI 22K/DNA; 2: PEI 25K/PEI 22K/DNA
modified with MAL-PEG-NHS; 3 and 4: as in 2 and subsequently modified with 72 pmol and 144 pmol of thiol-functionalized mEGF respectively; 5: EGF−PEI 25K/PEI 22K/DNA; 6:
EGF−PEI 25K/PEI 22K/DNA modified with MAL-PEG-NHS.
Luciferase activity is shown as mean ± SD of duplicate.</p></caption><graphic xlink:href="bc0001488f00006.tif" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e583"><title>Discussion</title><p>Our aim is to develop synthetic gene delivery systems
that can be administered by intravenous application. One
important aspect for the construction of such systems is
the use of a convenient ligand able to mediate cell specific
recognition and internalization into target cells. EGF
receptor is overexpressed in many human tumors of
epithelial origin and may therefore serve as a useful cell
surface target (<italic toggle="yes"><xref rid="bc0001488b00022" ref-type="bibr"></xref></italic>). Gene delivery into EGF receptor-expressing cells was previously demonstrated using
polylysine conjugates where receptor ligand (EGF or anti-EGF receptor antibody) and polylysine were covalently
coupled (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00032" ref-type="bibr"></xref>, <xref rid="bc0001488b00033" ref-type="bibr"></xref></named-content></italic>) or bound through a biotin/streptavidin
linkage (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00034" ref-type="bibr"></xref>−<xref rid="bc0001488b00035" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00036" ref-type="bibr"></xref></named-content></italic>). Also other synthetic systems were
described including chimeric TGF-alpha fusion protein
(<italic toggle="yes"><xref rid="bc0001488b00037" ref-type="bibr"></xref></italic>) and EGF-containing liposomes (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00038" ref-type="bibr"></xref>, <xref rid="bc0001488b00039" ref-type="bibr"></xref></named-content></italic>). In these
cases gene delivery in EGF receptor-expressing cells was
effective only in the presence of an endosomolytic agent
such as chloroquine or inactivated adenovirus. Chloroquine would be difficult to apply in vivo at the required
dose due to its toxicity. The use of inactivated adenovirus
could lead to undesired immune problems inherent of the
virus.
</p><p>Recently some new polycations such as PEI (<italic toggle="yes"><xref rid="bc0001488b00001" ref-type="bibr"></xref></italic>) and
polyamidoamine dendrimers (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00040" ref-type="bibr"></xref>, <xref rid="bc0001488b00041" ref-type="bibr"></xref></named-content></italic>) have been described
as particularly efficient gene delivery vectors without the
need for additional endosomolytic agents. These cationic
polymers share with chloroquine a strong buffering
capacity that promotes escape of the complexes out of the
endosomal compartment (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00001" ref-type="bibr"></xref>, <xref rid="bc0001488b00042" ref-type="bibr"></xref></named-content></italic>). We combined the powerful gene delivery activity of PEI with the specificity of
EGF-receptor binding by generating an EGF−PEI conjugate able to deliver DNA into tumor cells with considerably higher efficiency compared to the native PEI
complexes. When EGF was incorporated in polyplexes,
10-fold lower amounts of DNA complexes were required
for similar efficiency. High transfection ability with low
dose of DNA is essential for in vivo application. This
ligand-mediated enhancement and competition experiments performed with free EGF strongly suggest that
EGF-containing DNA complexes are taken up specifically
by the EGF-receptor mediated endocytosis pathway. This
is also supported by recent data from our group by FACS
analysis (Ogris et al., submitted to <italic toggle="yes">AAPS Pharm Sci.</italic>)
where enhanced uptake of EGF-containing complexes
was demonstrated.
</p><p>For in vivo administration, small DNA particles are
required for escape out of normal blood vessels to reach
surrounding tissues such as hepatocytes (<italic toggle="yes"><xref rid="bc0001488b00043" ref-type="bibr"></xref></italic>) and much
effort is applied in this direction (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00044" ref-type="bibr"></xref>−<xref rid="bc0001488b00045" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00046" ref-type="bibr"></xref></named-content></italic>). Preparation
of EGF-polyplexes in salt-free buffer generates small
particles that, however, are relatively inefficient. These
complexes are not limited by the EGF receptor uptake
process, but by ineffective endosomal release, as suggested by the large increase of transfection efficiency in
the presence of chloroquine. This finding is in good
agreement with previous observations using transferrin-PEI particles (<italic toggle="yes"><xref rid="bc0001488b00031" ref-type="bibr"></xref></italic>). For tumor-targeted gene delivery the
DNA particle size is less problematic because of the
leakiness of the tumor vasculature. Indeed accumulation
of large DNA complexes (approximately 500 nm) was
observed recently in our group (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00020" ref-type="bibr"></xref>, <xref rid="bc0001488b00021" ref-type="bibr"></xref></named-content></italic>). Therefore, we
evaluated also EGF−PEI complexes of medium-large
size. Interestingly linear PEI 22K but also in mixture
with PEI 25K (1:1) was more efficient to deliver DNA
than the branched PEI 25K alone, particularly at low
DNA doses. Indeed, gene expression obtained with 60 ng
of DNA complexed with EGF−PEI 25K/PEI 22K was
higher (10<sup>8</sup> RLU/mg protein) even compared to much
higher amounts (2 μg) of EGF−PEI 25K/PEI 25K complexes (Figure <xref rid="bc0001488f00005"></xref>).
</p><p>Gene delivery systems for intravenous application
must be inert against nonspecific interactions with
biological environment such as nontarget cells and blood
components in order to maintain their physical integrity
and to avoid rapid elimination via activation of the
immune system. Covalent surface modification of preformed polyplexes with PEG has been shown to strongly
reduce nonspecific interactions with erythrocytes and
plasma proteins (<italic toggle="yes"><xref rid="bc0001488b00020" ref-type="bibr"></xref></italic>) and it also reduces complement
system activation (<italic toggle="yes"><xref rid="bc0001488b00047" ref-type="bibr"></xref></italic>). Incorporation of PEG in polyplexes was also achieved using block copolymer as
condensing agent such polylysine−polysaccharide (<italic toggle="yes"><xref rid="bc0001488b00048" ref-type="bibr"></xref></italic>),
polylysine−PEG (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00049" ref-type="bibr"></xref>−<xref rid="bc0001488b00050" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00051" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00052" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00053" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0001488b00054" ref-type="bibr"></xref></named-content></italic>), and PEI−PEG (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00055" ref-type="bibr"></xref>, <xref rid="bc0001488b00056" ref-type="bibr"></xref></named-content></italic>). Major
drawback of this latter strategy is the crowding effect of
the neutral polymer that can hinder proper DNA condensation in dense particles (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00049" ref-type="bibr"></xref>, <xref rid="bc0001488b00055" ref-type="bibr"></xref></named-content></italic>).
</p><p>We evaluated two post-PEGylation strategies to generate shielded EGF-containing complexes, where EGF was
either coupled to PEI (Figure <xref rid="bc0001488f00003"></xref>A) or to the distal end of
a PEG spacer arm (Figure <xref rid="bc0001488f00003"></xref>B). In the latter strategy the
ligand is incorporated at the final step of complex
preparation. Optimal amounts of ligand giving best
transfection efficiencies can be rapidly found. The later
strategy should also allow a better accessibility of the
ligand as suggested by studies on liposomes. Indeed, it
was shown that incorporation of PEG in immunoliposomes can substantially weaken antigen/antibody
interaction (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00057" ref-type="bibr"></xref>, <xref rid="bc0001488b00058" ref-type="bibr"></xref></named-content></italic>). In contrast, coupling the ligand to
the distal end of the PEG produced stabilized liposomes
still allowing efficient binding to the target receptor
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0001488b00059" ref-type="bibr"></xref>, <xref rid="bc0001488b00060" ref-type="bibr"></xref></named-content></italic>). We showed that both types of shielded EGF-containing complexes were effective to deliver DNA.
Efficiencies were approximately 10- to 100-fold higher
compared to EGF-free PEGylated complexes. In contrast
to the published liposome work, the beneficial effect of
grafting the ligand at the distal end of PEG compared to
coupling to PEI and PEGylation was less clear with
polyplexes. With small complexes this strategy (Figure
<xref rid="bc0001488f00003"></xref>B) resulted in 8-fold higher gene expression than with
strategy illustrated in Figure <xref rid="bc0001488f00003"></xref>A, but with large complexes efficacies were nearly similar. This strongly suggests that the PEG covalently coupled to the surface of
EGF-containing PEI/DNA complexes only weakly interferes with the binding to the EGF-receptor. This is quite
surprising knowing that a brush of PEG (MW 5000)
extending out of the surface of liposomes has been
described to have a thickness of 10 nm (<italic toggle="yes"><xref rid="bc0001488b00061" ref-type="bibr"></xref></italic>). Nevertheless,
this is in line with a recent finding by Ogris et al.
(submitted to <italic toggle="yes">AAPS Pharm Sci.</italic>) suggesting that PEGylation does not reduce internalization of EGF-containing
complexes.
</p><p>In summary we have generated EGF-containing polyplexes able to efficiently transfect cells in a receptor-dependent fashion. PEGylated EGF-containing complexes maintained high efficiency and are interesting
candidates for in vivo evaluation in tumor models.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Winfried Wels who provided us with Renca-EGFR cells. We are grateful to Sylvia Brunner and Lionel
Wightman for careful review of the manuscript. This
work was supported by grants from the European Community and the Austrian Science Foundation.
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Drug Target.</source><year>1994</year><volume>2</volume><fpage>397</fpage><lpage>403</lpage><pub-id pub-id-type="doi">10.3109/10611869408996815</pub-id></element-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Different Strategies for Formation of PEGylated EGF-Conjugated PEI/DNA Complexes for Targeted Gene Delivery</title></titleInfo><name type="personal" displayLabel="corresp"><namePart type="family">BLESSING</namePart><namePart type="given">Thomas</namePart><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and BoehringerIngelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><affiliation> To whom correspondence should be addressed. New address: Faculte de Pharmacie, 15 av. Charles Flahault, F-34060Montpellier. Telephone: ++33-467-418-267. Fax: ++33-467-520-898. E-mail: blessing@pharma.univ-montp1.fr.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">KURSA</namePart><namePart type="given">Malgorzata</namePart><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and BoehringerIngelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">HOLZHAUSER</namePart><namePart type="given">Robert</namePart><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and BoehringerIngelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">KIRCHEIS</namePart><namePart type="given">Ralf</namePart><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and BoehringerIngelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">WAGNER</namePart><namePart type="given">Ernst</namePart><affiliation>Institute of Medical Biochemistry, University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna and BoehringerIngelheim Austria, Dr. Boehringer Gasse 5-11, A-1121 Vienna, Austria</affiliation><affiliation> New address: Department of Pharmacy, Ludwig-Maximilians-University, Butenandtstr 5-13, D-81377 Munich, Germany.</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">2001-06-23</dateCreated><dateIssued encoding="w3cdtf">2001-07-18</dateIssued><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><abstract>With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF−PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.</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>2001</date><detail type="volume"><caption>vol.</caption><number>12</number></detail><detail type="issue"><caption>no.</caption><number>4</number></detail><extent unit="pages"><start>529</start><end>537</end></extent></part></relatedItem><identifier type="istex">5BDA273D2BD7C85D0528CE1426C5CDE263EF638C</identifier><identifier type="ark">ark:/67375/TPS-J0F4G1J1-8</identifier><identifier type="DOI">10.1021/bc0001488</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2001 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 © 2001 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-J0F4G1J1-8/record.json</uri></json:item></metadata><annexes><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/5BDA273D2BD7C85D0528CE1426C5CDE263EF638C/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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