Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles
Identifieur interne : 000737 ( Istex/Corpus ); précédent : 000736; suivant : 000738Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles
Auteurs : Ester J. Kwon ; Jamie M. Bergen ; Suzie H. PunSource :
- Bioconjugate Chemistry [ 1043-1802 ] ; 2008.
Abstract
Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.
Url:
DOI: 10.1021/bc700448h
Links to Exploration step
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles</title><author><name sortKey="Kwon, Ester J" sort="Kwon, Ester J" uniqKey="Kwon E" first="Ester J." last="Kwon">Ester J. Kwon</name><affiliation><mods:affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</mods:affiliation></affiliation></author><author><name sortKey="Bergen, Jamie M" sort="Bergen, Jamie M" uniqKey="Bergen J" first="Jamie M." last="Bergen">Jamie M. Bergen</name><affiliation><mods:affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</mods:affiliation></affiliation></author><author><name sortKey="Pun, Suzie H" sort="Pun, Suzie H" uniqKey="Pun S" first="Suzie H." last="Pun">Suzie H. Pun</name><affiliation><mods:affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: spun@u.washington.edu</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:89252D5CD138018F537D1067BA88CEBF7154E631</idno><date when="2008" year="2008">2008</date><idno type="doi">10.1021/bc700448h</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-T4NWZ521-F/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000737</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000737</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles</title><author><name sortKey="Kwon, Ester J" sort="Kwon, Ester J" uniqKey="Kwon E" first="Ester J." last="Kwon">Ester J. Kwon</name><affiliation><mods:affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</mods:affiliation></affiliation></author><author><name sortKey="Bergen, Jamie M" sort="Bergen, Jamie M" uniqKey="Bergen J" first="Jamie M." last="Bergen">Jamie M. Bergen</name><affiliation><mods:affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</mods:affiliation></affiliation></author><author><name sortKey="Pun, Suzie H" sort="Pun, Suzie H" uniqKey="Pun S" first="Suzie H." last="Pun">Suzie H. Pun</name><affiliation><mods:affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: spun@u.washington.edu</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="2008-04-01">2008</date><date when="2008-04-16">2008</date><biblScope unit="vol">19</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="920">920</biblScope><biblScope unit="page" to="927">927</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">Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>polyplexes</json:string><json:string>transfection</json:string><json:string>peptide</json:string><json:string>lytic</json:string><json:string>sirna</json:string><json:string>gapdh</json:string><json:string>hela</json:string><json:string>chloroquine</json:string><json:string>hela cell</json:string><json:string>plasmid</json:string><json:string>endosomal</json:string><json:string>chem</json:string><json:string>polyplex</json:string><json:string>transgene</json:string><json:string>nonviral</json:string><json:string>confocal</json:string><json:string>polymer</json:string><json:string>transgene expression</json:string><json:string>transfection efficiency</json:string><json:string>bioconjugate</json:string><json:string>cytometry</json:string><json:string>flow cytometry</json:string><json:string>intracellular</json:string><json:string>internalization</json:string><json:string>bioconjugate chem</json:string><json:string>triplicate</json:string><json:string>lytic activity</json:string><json:string>trafficking</json:string><json:string>luciferase</json:string><json:string>egfp</json:string><json:string>liposome</json:string><json:string>gene delivery</json:string><json:string>vesicle</json:string><json:string>endosomes</json:string><json:string>endocytic</json:string><json:string>peihgp</json:string><json:string>biol</json:string><json:string>polyethylenimine</json:string><json:string>assay</json:string><json:string>endosomal release</json:string><json:string>nucleic acid</json:string><json:string>confocal imaging</json:string><json:string>gray bar</json:string><json:string>sirna delivery</json:string><json:string>white bar</json:string><json:string>triplicate sample</json:string><json:string>gene transfer</json:string><json:string>endocytic vesicle</json:string><json:string>complete growth medium</json:string><json:string>nucleic</json:string><json:string>xxxx</json:string><json:string>drug delivery</json:string><json:string>charge ratio</json:string><json:string>gapdh activity</json:string><json:string>lytic peptide</json:string><json:string>egfp positive cell</json:string><json:string>confocal microscopy</json:string><json:string>intracellular trafficking</json:string><json:string>unmodified</json:string><json:string>luciferase activity</json:string><json:string>transfected cell</json:string><json:string>hydrodynamic size</json:string><json:string>molecular weight</json:string><json:string>unmodified polyplexes</json:string><json:string>free peptide</json:string><json:string>green fluorescent protein</json:string><json:string>nonviral gene delivery vehicle</json:string><json:string>peihgp polyplexes</json:string><json:string>fluorescence value</json:string><json:string>promega corp</json:string><json:string>membrane disruption</json:string><json:string>intracellular distribution</json:string><json:string>fluorescence microscopy</json:string><json:string>gene</json:string></teeft></keywords><author><json:item><name>KWON Ester J.</name><affiliations><json:string>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</json:string></affiliations></json:item><json:item><name>BERGEN Jamie M.</name><affiliations><json:string>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</json:string></affiliations></json:item><json:item><name>PUN Suzie H.</name><affiliations><json:string>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</json:string><json:string>E-mail: spun@u.washington.edu</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-T4NWZ521-F</arkIstex><language><json:string>unknown</json:string></language><originalGenre><json:string>Article</json:string></originalGenre><abstract>Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. 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In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.</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 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></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bc700448h</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles</article-title><alt-title alt-title-type="short">HIV-Derived Peptide for Gene and siRNA Delivery</alt-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name name-style="western"><surname>Kwon</surname><given-names>Ester J.</given-names></name></contrib><contrib contrib-type="author" id="ath2"><name name-style="western"><surname>Bergen</surname><given-names>Jamie M.</given-names></name></contrib><contrib contrib-type="author" corresp="yes" id="ath3"><name name-style="western"><surname>Pun</surname><given-names>Suzie H.</given-names></name><xref rid="cor1"></xref></contrib><aff>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>
To whom correspondence should be addressed. Phone:
<phone>(206) 685−3488</phone>
. Fax: <fax>(206) 616−3928</fax>. E-mail: <email>spun@u.washington.edu</email>.</corresp></author-notes><pub-date pub-type="epub"><day>01</day><month>04</month><year>2008</year></pub-date><pub-date pub-type="ppub"><day>16</day><month>04</month><year>2008</year></pub-date><volume>19</volume><issue>4</issue><fpage>920</fpage><lpage>927</lpage><supplementary-material xlink:href="bc700448h-File002.pdf" id="si10" orientation="portrait" position="float"></supplementary-material><history><date date-type="issue-pub"><day>16</day><month>04</month><year>2008</year></date><date date-type="asap"><day>01</day><month>04</month><year>2008</year></date><date date-type="received"><day>05</day><month>12</month><year>2007</year></date><date date-type="rev-recd"><day>25</day><month>01</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008 American Chemical Society</copyright-statement><copyright-year>2008</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bc700448h</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>bc-2007-00448h</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value>40.75</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="sec1"><title>Introduction</title><p>The development of safe and efficient gene delivery vehicles is necessary for clinical gene therapy. Synthetic vehicles offer potential advantages over viral-based vectors including safety and low immunogenicity, but transfection efficiencies from many of these systems remain suboptimal <named-content content-type="bibref-group"><xref rid="ref1" ref-type="bibr"></xref>,<xref rid="ref2" ref-type="bibr"></xref></named-content>. An effective nucleic acid delivery vehicle must mediate either cytoplasmic or nuclear delivery, depending on its therapeutic agent. Upon internalization, the majority of carriers accumulate within endocytic vesicles <xref rid="ref3" ref-type="bibr"></xref>. Efficient release from these vehicles before lysosomal degradation has been shown to be a major barrier for many of these systems. Thus, it would be advantageous to develop methods to improve the endosomal release of new and existing vectors for future applications of nonviral gene delivery.</p><p>Polyethylenimine (PEI) has been used widely as a nonviral vector and is among the most efficient of cationic polymer delivery vehicles <xref rid="ref4" ref-type="bibr"></xref>. The efficacies of PEI and other protonatable cationic polymers as gene delivery agents has been attributed to their buffering properties, which have been hypothesized to mediate endosomal escape via the proton sponge mechanism <named-content content-type="bibref-group"><xref rid="ref5" ref-type="bibr"></xref>,<xref rid="ref6" ref-type="bibr"></xref></named-content>. In brief, pH-sensitive polymers buffer endosomes during the natural acidification process, resulting in counterion and water accumulation in the vesicles, eventually leading to osmolysis. However, the buffering capacity of PEI is correlated to the amount of PEI that is present <named-content content-type="bibref-group"><xref rid="ref6" ref-type="bibr"></xref>,<xref rid="ref7" ref-type="bibr"></xref></named-content>. Purification of free PEI from polyplexes after formulation results in polyplexes with only a slight charge excess of PEI. These polyplexes are much less efficient at mediating gene transfer than unpurified polyplexes <named-content content-type="bibref-group"><xref rid="ref8" ref-type="bibr"></xref>,<xref rid="ref9" ref-type="bibr"></xref></named-content>. This has implications for <italic toggle="yes">in vivo</italic> delivery where unassociated polymer can be separated from DNA before endocytosis occurs. The development of agents that have more potent endosomal escape properties is therefore desirable.</p><p>In an effort to develop more potent mediators of endosomal escape, research has looked to nature for inspiration. Peptides taken from the membrane-disrupting domains of proteins, such as melittin <named-content content-type="bibref-group"><xref rid="ref10" ref-type="bibr"></xref>−<xref rid="ref11" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="ref12" ref-type="bibr"></xref></named-content>, Tat <named-content content-type="bibref-group"><xref rid="ref13" ref-type="bibr"></xref>−<xref rid="ref14" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="ref15" ref-type="bibr"></xref></named-content>, and hemagglutinin <named-content content-type="bibref-group"><xref rid="ref16" ref-type="bibr"></xref>,<xref rid="ref17" ref-type="bibr"></xref></named-content>, have been incorporated into nonviral gene delivery vectors, as reviewed elsewhere <named-content content-type="bibref-group"><xref rid="ref18" ref-type="bibr"></xref>,<xref rid="ref19" ref-type="bibr"></xref></named-content>. In this paper, we report the application of a peptide from the endodomain of HIV gp41 envelope glycoprotein found to have lytic properties from a peptide screen recently reported in the literature <xref rid="ref20" ref-type="bibr"></xref>. This sequence of HIV gp41, corresponding to residues 783−806 of gp160, has been shown to have high membrane association and the potential to form multimers <xref rid="ref21" ref-type="bibr"></xref>. Peptides from this region of gp41 have been shown to adopt amphipathic α-helical structures and have the potential to form pores in membranes <named-content content-type="bibref-group"><xref rid="ref22" ref-type="bibr"></xref>,<xref rid="ref23" ref-type="bibr"></xref></named-content>. The goal of this work was to evaluate the lytic peptide from the endodomain of HIV gp41, HGP, as a mediator of enhanced intracellular trafficking of nonviral gene delivery vehicles. Our results demonstrate that modification of PEI with HGP increases transgene expression and siRNA knockdown, likely by increasing the efficiency of endosomal release.</p></sec><sec id="sec2"><title>Methods</title><sec id="sec2.1"><title>Synthesis of HGP-Modified PEI</title><p>C-terminal cysteine-terminated HGP peptide (LLGRRGWEVLKYWWNLLQYWSQELC) was synthesized and HPLC purified by GenScript Corporation (Piscataway, NJ). Branched PEI with molecular weight 25 000 (Sigma, St. Louis, MO) was modified with succinimidyl 4-[<italic toggle="yes">p</italic>-maleimidophenyl]butyrate (SMPB) purchased from Pierce (Rockford, IL) at a molar excess of 5 according to manufacturer’s protocol. SMPB-modified PEI was purified using a PD-10 column (GE Healthcare, Piscataway, NJ) and lyophilized. For peptide conjugation, SMPB-modified PEI was dissolved in DMF and reacted with 5 equiv HGP peptide in the presence of triethylamine (TEA) for 72 h. Unreacted peptide and TEA were removed by dialysis against a MWCO 10 000 membrane (Pierce) with water over 3 days. PEI concentration was determined by monitoring cuprammonium complexes formed by PEI and copper (II) acetate at 630 nm as described previously <xref rid="ref24" ref-type="bibr"></xref>. HGP conjugation efficiency was determined by quantifying absorbance at 280 nm using UV/vis spectrophotometry.</p></sec><sec id="sec2.2"><title>Polyplex Formulation and Physicochemical Characterization</title><p>Polyplexes, complexes of polycations with nucleic acids, were formulated by adding equal volumes of polymer to nucleic acid at the desired charge ratio. The charge ratio is calculated on the basis of molar amount of polymer nitrogen to nucleic acid phosphate (N/P) ratio. Polyplexes were allowed to incubate for 10 min at room temperature to allow for complexation.</p><p>Complexation of DNA was monitored by agarose gel electrophoresis. Polyplexes were loaded on a 0.8% agarose gel containing 0.3 µg/mL ethidium bromide. Hydrodynamic size was measured in triplicate using a ZetaPALS zeta potential and particle size analyzer (Brookhaven Instruments Corp., Holtsville, NY) as described previously <xref rid="ref25" ref-type="bibr"></xref>.</p></sec><sec id="sec2.3"><title>Dye Release Assay</title><p>Liposomes composed of <sc>l</sc>-α-phosphotidylcholine and cholesterol (Avanti Polar Lipids, Alabaster, AL) encapsulating 50 mM sulforhodamine B (Invitrogen, Carlsbad, CA) were prepared by extrusion through 100 nm polycarbonate membranes (Whatman, Florham Park, NJ) using the Avanti Mini-Extruder. Unencapsulated sulforhodamine B was removed by dialysis into 10 mM Tris pH 7.4, 20 mM NaCl, 0.1 mM EDTA.</p><p>Liposome lysis was monitored by dequenching of sulforhodamine B fluorescence upon release from liposomes. Fluorescence intensity was monitored using a Tecan Safire<sup>2</sup> microplate reader (Tecan Systems, Inc., San Jose, CA) by excitation at 565 nm and reading emission at 586 nm. Dye release was calculated as a percentage according to the equation
<disp-formula id="eq1"><label>1</label><alternatives><graphic xlink:href="bc-2007-00448h_m001.gif" orientation="portrait" position="float"></graphic><mml:math display="block" overflow="scroll"><mml:mrow><mml:mtext>%</mml:mtext><mml:mtext> </mml:mtext><mml:mtext>dye</mml:mtext><mml:mtext> </mml:mtext><mml:mtext>release</mml:mtext><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mtext mathvariant="italic">F</mml:mtext></mml:mrow><mml:mrow><mml:mtext>m</mml:mtext></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mtext mathvariant="italic">F</mml:mtext></mml:mrow><mml:mrow><mml:mtext>0</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mtext mathvariant="italic">F</mml:mtext></mml:mrow><mml:mrow><mml:mtext>100</mml:mtext></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mtext mathvariant="italic">F</mml:mtext></mml:mrow><mml:mrow><mml:mtext>0</mml:mtext></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mo></mml:mo><mml:mi>×</mml:mi><mml:mtext> </mml:mtext><mml:mn>100</mml:mn></mml:mrow></mml:math></alternatives></disp-formula>
where <italic toggle="yes">F</italic><sub>m</sub> is the fluorescence value after 15 min of incubation with peptide or polyplex at 37 °C, <italic toggle="yes">F</italic><sub>0</sub> is the fluorescence value of the initial liposome suspension, and <italic toggle="yes">F</italic><sub>100</sub> is the fluorescence value after incubation with a 0.05% Triton X-100 solution.</p></sec><sec id="sec2.4"><title>Cell Culture</title><p>HeLa cells purchased from ATCC (Manassas, VA) were cultured in complete growth medium (minimum essential medium with 10% fetal bovine serum and antibiotics). Cells were passaged every 2−3 days.</p></sec><sec id="sec2.5"><title>Plasmid Delivery to HeLa Cells, Evaluation of Transfection Efficiencies, and Cytotoxicity Assay</title><p>Transfection experiments were performed in triplicate. HeLa cells suspended in complete growth medium were seeded at 30 000 cells/well in 24-well plates and allowed to attach overnight. Polyplexes were formulated as described above using 1 µg of gWiz-Luciferase plasmid DNA (Aldevron, Fargo, ND) and then diluted in OptiMEM medium (Invitrogen). Cells were rinsed once with phosphate buffered saline, pH 7.4 (PBS), and incubated with polyplexes for 4 h at 37 °C in a 5% CO<sub>2</sub> atmosphere. Cells were rinsed with PBS and medium was replaced with fresh complete growth medium. Cells were returned to a 37 °C, 5% CO<sub>2</sub> atmosphere and luciferase expression was quantified after 48 h using a luciferase assay kit (Promega Corp., Madison, WI). Cells were washed with PBS, lysed with 200 µL of reagent lysis buffer (Promega Corp.), and frozen at −20 °C. 100 µL of luciferase substrate was added to 20 µL of lysate and luminescence was measured using a TECAN Safire<sup>2</sup> microplate reader. Luminescence was integrated for 1 s and recorded in relative light units (RLU). Luciferase activity is reported in luminescence normalized by protein content (RLU/mg), as measured by a BCA Protein Assay Kit (Pierce). Cytotoxity was measured by incubating polymer or polyplexes with HeLa cells as described above and adding the reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2<italic toggle="yes">H</italic>-tetrazolium (MTS) (Promega Corp.) after 24 h. Cells were incubated in a 37 °C, 5% CO<sub>2</sub> atmosphere for 4 h and absorbance measurements of lysate at 490 nm were recorded.</p></sec><sec id="sec2.6"><title>siRNA Delivery to HeLa Cells and Expression Analysis by Quantitative PCR and GAPDH Activity Assay</title><p>siRNA targeting endogenous glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) was purchased from Ambion (Silencer GAPDH siRNA; Austin, TX), and control siRNA targeting green fluorescent protein (siGFP) was synthesized by Dharmacon (Lafayette, CO) with the following sequence: 5′-GACGUAAACGGCCACAAGUUC-3′ (sense) and 5′-ACUUGUGGCCGUUUACGUCGC-3′ (antisense). HeLa cells were seeded on a 24-well plate as described for the plasmid transfection studies. For transfection, polyplexes were formulated by condensing either siGAPDH or siGFP with either PEI or PEI-HGP at an N/P ratio of 10 (60 pmol siRNA/well, triplicate samples). Polyplexes were diluted in 400 µL OptiMEM and incubated with cells for 5 h, after which the polyplex solution was removed and replaced with complete growth medium. 48 h after transfection, total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA). 500 ng of RNA from each sample was reverse-transcribed using Omniscript RT (Qiagen) and random hexamers as primers (Operon, Huntsville, AB). Quantitative PCR was performed using a model 7300 Real Time PCR system (Applied Biosystems, Foster City, CA) following universal thermal cycling parameters. GAPDH expression levels were determined in 20 µL reactions using TaqMan Universal PCR Master Mix (Applied Biosystems) and a TaqMan gene expression assay for human GAPDH, and were normalized by expression levels for β-actin (TaqMan gene expression assay for human ACTB). Relative GAPDH expression levels for each sample were determined on the basis of a comparison with untreated control samples, and were calculated by the 2<sup>−ΔΔCT</sup> method <xref rid="ref26" ref-type="bibr"></xref>. To measure GAPDH activity, an identical transfection experiment was performed, and cell lysate was collected 48 h after transfection. GAPDH activity was measured in the lysate using the fluorescence-based KDalert GAPDH Assay Kit (Ambion). For each formulation, the degree of reduction in GAPDH activity mediated by siGAPDH was assessed by comparing GAPDH activity levels in siGAPDH- versus siGFP-treated samples.</p></sec><sec id="sec2.7"><title>Internalization and Binding Studies</title><p>Uptake and binding of PEI-HGP polyplexes relative to PEI polyplexes was investigated by flow cytometry. Nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled oligonucleotides were prepared as described previously <xref rid="ref27" ref-type="bibr"></xref>. HeLa cells were seeded at 300 000 cells/well in 6-well plates and allowed to attach overnight. One hour before transfection, cells were washed with PBS, supplemented with Gibco OptiMEM medium, and then equilibrated to either 37 or 4 °C. Polyplexes were formulated at an N/P ratio of 3 as described above using 4 µg of NBD-labeled oligo. Polyplex solution was added to cells and allowed to incubate for 3 h at either 37 or 4 °C. Cells were then washed twice with cold PBS, trypsinized, and resuspended in complete growth medium. Cell-associated fluorescence was quantified by flow cytometry using a BD FACScan (BD Biosciences, Franklin Lakes, NJ). Live cell populations were identified using forward and side-scattering profiles, and polyplex uptake was assessed by NBD fluorescence using <italic toggle="yes">FlowJo</italic> flow cytometry analysis software. Cell association was reported as mean fluorescence in relative fluorescence units (RFU).</p></sec><sec id="sec2.8"><title>Confocal Imaging</title><p>Confocal imaging was performed to determine the intracellular distribution of polyplexes. gWiz-Luciferase plasmid DNA was labeled with TOTO-3 (Invitrogen, Eugene, OR) at 1 dye per 100 base pairs and dialyzed into 10 mM Tris pH 7.4, 1 mM EDTA overnight at 4 °C. Glass coverslips were precoated with poly(<sc>l</sc>-lysine) in a 24-well plate and HeLa cells were seeded at 75 000 cells/well in complete medium. 3 µg of TOTO-3 labeled DNA was complexed with PEI or PEI-HGP at N/P ratio 3 as described above and added to cells with 2 mg/mL Alexa Fluor 488-labeled dextran (MW 10 000, Invitrogen) for 1 or 3 h in a 37 °C, 5% CO<sub>2</sub> environment. Cells were then washed with PBS and Cell Scrub Buffer (Gene Therapy Systems, San Diego, CA), and then fixed for 7 min in 4% paraformaldehyde. Coverglasses were mounted onto a coverslip with Fluoromount-G (Southern Biotech, Birmingham, AL) and imaged with a Zeiss LSM 510 confocal microscope.</p></sec><sec id="sec2.9"><title>Chloroquine-Mediated Transfection Studies</title><p>HeLa cells were seeded at 120 000 cells/well in 6-well plates and allowed to attach overnight. Polyplexes were formulated at an N/P ratio of 3 as described above using 4 µg of pEGFP-C1 (Clontech Laboratories, Inc., Mountain View, CA) plasmid DNA that codes for the enhanced green fluorescent protein (EGFP) reporter. Polyplex was diluted in OptiMEM and added to cells. A 5 mM chloroquine stock solution was diluted to a final concentration of 0.1 mM and cells were allowed to incubate for 4 h in a 37 °C, 5% CO<sub>2</sub> atmosphere. Cells were rinsed with PBS and incubated for an additional 48 h in complete growth medium. Cells were imaged by brightfield and fluorescence microscopy. Transgene expression was quantified by EGFP fluorescence using flow cytometry as described above. EGFP expression is reported as the percent of EGFP positive cells.</p></sec><sec id="sec2.10"><title>Tail-Vein Injection</title><p>Female C57/Bl6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Polyplexes were formulated with 50 µg gWiz Luciferase plasmid DNA in 250 μL of 20 mM HEPES pH 7.4, 5% glucose. Polyplexes were delivered via tail-vein injections in 12−16 week old mice and lung tissue was excised 24 h after injection. Lungs were collected in reagent lysis buffer (Promega) supplemented with protease inhibitors (Roche) and underwent three freeze−thaw cycles. Tissue was mechanically homogenized using an IKA T8 Disperser (Wilmington, NC) and cell debris was spun down at 14 000 <italic toggle="yes">g</italic> for 15 min at 4 °C. Luciferase activity and protein content were measured as described above.</p></sec></sec><sec id="sec3"><title>Results</title><sec id="sec3.1"><title>Synthesis of PEI-HGP and Polyplex Characterization</title><p>Moreno et al. recently screened 15 amino acid sequences from the HIV surface glycoprotein, gp41, in order to identify regions in the protein that interact with membranes. The sequence of HGP was chosen on the basis of its ability to mediate membrane rupture and is from the C-terminal tail region of HIV gp41 designated as the lentivirus peptide sequences (LLP-2/3) <xref rid="ref20" ref-type="bibr"></xref>. PEI-HGP was synthesized by conjugating a cysteine-terminated HGP to PEI with the heterobifunctional cross-linker SMPB. The concentration of peptide was determined by absorbance measurements at 280 nm and labeling efficiency was found to be 2−3 HGP per PEI on average.</p><p>Polyplexes with plasmid DNA were formed by addition of an equal volume of polymer to DNA followed by rapid mixing and incubation at room temperature for 10 min. Complex formation was examined by agarose gel electrophoresis (Figure <xref rid="fig1"></xref>). Uncomplexed DNA migrates into the gel, whereas complexed DNA is occluded and retained in the loading well. Uncomplexed gWiz-Luciferase plasmid is shown in lanes marked as N/P ratio 0. For both PEI and PEI-HGP, DNA is fully complexed at N/P ratio of 2. Therefore, HGP conjugation does not interfere with complex formation. This is expected, as the percentage of modification is low.</p><fig id="fig1" position="float" orientation="portrait"><label>1</label><caption><p>Gel retardation assay to monitor DNA complexation in PEI-HGP and PEI polyplexes at various N/P ratios. M: New England Biolabs 1kb ladder.</p></caption><graphic id="gr1" xlink:href="bc-2007-00448h_0002.tif" position="float" orientation="portrait"></graphic></fig><p>Hydrodynamic sizes of PEI and PEI-HGP polyplexes were measured using dynamic light scattering (DLS) in triplicate samples (Figure <xref rid="fig2"></xref>). For N/P ratios 2, 3, and 4, sizes of PEI-HGP polyplexes were 153 ± 14.7 nm, 144.8 ± 10.6 nm, and 134.4 ± 7.1 nm, respectively. Sizes of PEI polyplexes were 139.7 ± 7.7 nm, 106.7 ± 4.9 nm, and 107.0 ± 6.7 nm. PEI-HGP polyplexes are slightly larger in size.</p><fig id="fig2" position="float" orientation="portrait"><label>2</label><caption><p>Hydrodynamic size of PEI-HGP (gray bars) and PEI (white bars) polyplexes were measured at N/P ratios 2, 3, and 4 using dynamic light scattering. Triplicate formulations were characterized at each charge ratio. Results are reported as mean diameter ± SD.</p></caption><graphic id="gr2" xlink:href="bc-2007-00448h_0003.tif" position="float" orientation="portrait"></graphic></fig></sec><sec id="sec3.2"><title>Lytic Activity of HGP</title><p>The lytic activity of free HGP peptide and PEI-HGP polyplexes was determined by monitoring the release of sulforhodamine B dye from liposomes after incubation for 15 min at various peptide concentrations <xref rid="ref28" ref-type="bibr"></xref>. Figure <xref rid="fig3"></xref>A shows that HGP displayed concentration-dependent lytic activity when incubated with liposomes. At a concentration of 0.63 µM, HGP was able to mediate approximately 50% dye release. The lytic activities of PEI and PEI-HGP polyplexes formulated at various N/P ratios were also evaluated (Figure <xref rid="fig3"></xref>B). The lytic activity of the equivalent amount of HGP incorporated in the PEI-HGP polyplexes was also determined for comparison. The concentrations of HGP peptide present in polyplex formulations of N/P ratios of 2, 3, and 4 were 0.13 µM, 0.19 µM, and 0.26 µM, respectively. Free peptides at these concentrations resulted in dye release percentages of 1.0%, 2.2%, and 3.0%. The dye release percentages for N/P ratios 2, 3, and 4 were 0.5%, 4.4%, and 50.3% for PEI and 6.5%, 55.3%, and 76.6% for PEI-HGP.</p><fig id="fig3" position="float" orientation="portrait"><label>3</label><caption><p>(A) Lytic activity of HGP was tested as free peptide using a fluorescence-based, liposomal leakage assay. (B) Lytic activities of HGP peptide (black bars), PEI polyplexes (gray bars), and PEI-HGP polyplexes (white bars) were compared. 100% dye release corresponded to fluorescence levels of liposomes treated with Triton X-100 surfactant for complete lysis. Results are reported as mean % dye release ± SD for triplicate samples.</p></caption><graphic id="gr3" xlink:href="bc-2007-00448h_0004.tif" position="float" orientation="portrait"></graphic></fig></sec><sec id="sec3.3"><title>Evaluation of PEI-HGP for Plasmid DNA Delivery</title><p>Transfection efficiency of PEI and PEI-HGP polyplexes in HeLa cells as a function of N/P ratio was determined using the luciferase reporter gene system. PEI-HGP polyplexes showed increased expression profiles over unmodified polyplexes at N/P ratios of 2 and 3 (Figure <xref rid="fig4"></xref>A). At an N/P ratio of 2, transgene expression from PEI-HGP polyplexes (8.9 × 10<sup>6</sup> ± 3.8 × 10<sup>6</sup> RLU/mg) was 38-fold higher than PEI polyplexes (2.3 × 10<sup>5</sup> ± 4.0 × 10<sup>4</sup> RLU/mg) with a significance value of <italic toggle="yes">p</italic> < 0.05. At an N/P ratio of 3, transgene expression from PEI-HGP polyplexes (1.7 × 10<sup>9</sup> ± 2.3 × 10<sup>8</sup> RLU/mg) was 30-fold higher than PEI polyplexes (5.5 × 10<sup>7</sup> ± 3.7 × 10<sup>7</sup> RLU/mg) with a significance value of <italic toggle="yes">p</italic> < 0.001. At an N/P ratio of 4, transgene expression between PEI-HGP polyplexes (4.5 × 10<sup>8</sup> ± 4.3 × 10<sup>8</sup> RLU/mg) was not statistically different than PEI polyplexes (1.7 × 10<sup>8</sup> ± 2.0 × 10<sup>8</sup> RLU/mg). Increases in transfection efficiency at N/P ratios of 2 and 3 were confirmed in several separate experiments.</p><fig id="fig4" position="float" orientation="portrait"><label>4</label><caption><p>(A) Luciferase activity of PEI-HGP (gray bars) and PEI polyplexes (white bars) were compared in HeLa cells at N/P ratios 2, 3, and 4. (B) Cell viability of HeLa cells after transfection with PEI-HGP (gray bars) and PEI (white bars) polyplexes at N/P ratios 2, 3, and 4 as measured by MTS. Results are reported as mean RLU/mg ± SD for triplicate samples. (Student’s <italic toggle="yes">t</italic>-test, *<italic toggle="yes">p</italic> < 0.05, **<italic toggle="yes">p</italic> < 0.001).</p></caption><graphic id="gr4" xlink:href="bc-2007-00448h_0005.tif" position="float" orientation="portrait"></graphic></fig><p>Cellular viability after transfection with PEI and PEI-HGP polyplexes was determined using an MTS assay and compared to untreated cells (Figure <xref rid="fig4"></xref>B). No toxicity was observed with these materials at N/P ratios 2, 3, and 4. At charge ratios greater than an N/P ratio of 15, the PEI-HGP polyplexes were more toxic than PEI polyplexes (data not shown).</p></sec><sec id="sec3.4"><title>Evaluation of PEI-HGP as an siRNA Carrier</title><p>The ability of HGP to enhance PEI-mediated siRNA delivery was evaluated in HeLa cells using siRNA targeting the endogenous GAPDH gene. While both PEI and PEI-HGP incorporating siGAPDH mediated significant GAPDH knockdown compared to identical polymer formulations incorporating siGFP, PEI-HGP was responsible for a significant enhancement in specific GAPDH knockdown activity compared to unmodified PEI (53.4 ± 2.2% reduction in GAPDH expression for PEI vs 82.3 ± 1.6% reduction for PEI-HGP, <italic toggle="yes">p</italic> < 0.001) (Figure <xref rid="fig5"></xref>). In a similar experiment, cell lysates were collected and analyzed for GAPDH protein activity. Compared to the corresponding siGFP control, PEI/siGAPDH reduced GAPDH activity by 24.9 ± 9.5%, while PEI-HGP/siGAPDH reduced GAPDH activity by 57.0 ± 8.4%.</p><fig id="fig5" position="float" orientation="portrait"><label>5</label><caption><p>Quantification of GAPDH mRNA levels in HeLa cells following treatment with PEI or PEI-HGP polyplexes incorporating siRNA. Cells were transfected with polyplexes incorporating either siRNA against GAPDH (siGAPDH, gray bars) or siRNA against GFP (siGFP, white bars) as a negative control (N/P = 10). Total RNA was isolated 48 h after transfection, and relative GAPDH expression levels were measured by quantitative PCR. Values are normalized to an untreated control sample and are reported as the mean relative GAPDH expression ± SD for triplicate samples. (Student’s <italic toggle="yes">t</italic> test, *<italic toggle="yes">p</italic> < 0.001).</p></caption><graphic id="gr5" xlink:href="bc-2007-00448h_0006.tif" position="float" orientation="portrait"></graphic></fig></sec><sec id="sec3.5"><title>Binding and Internalization of PEI-HGP Polyplexes</title><p>Cellular binding and uptake of NBD-labeled PEI and PEI-HGP polyplexes was investigated using flow cytometry (Figure <xref rid="fig6"></xref>). Fluorescently labeled polyplexes were incubated with HeLa cells at 4 °C (for binding) and 37 °C (for binding and uptake) for three hours before analysis. At 4 °C, association of PEI polyplexes was greater than PEI-HGP polyplexes (<italic toggle="yes">p</italic> < 0.005). At 37 °C, there was no significant difference in total associated fluorescence between PEI and PEI-HGP polyplexes.</p><fig id="fig6" position="float" orientation="portrait"><label>6</label><caption><p>Binding and internalization of fluorescently labeled PEI and PEI-HGP polyplexes in HeLa cells. Cells were treated with polyplexes formulated at an N/P ratio 3 for 3 h at 4 °C (binding studies) and 37 °C (internalization studies) and analyzed by flow cytometry. Results are reported as mean fluorescence ± SD for triplicate samples. (Student’s <italic toggle="yes">t</italic>-test, *<italic toggle="yes">p</italic> < 0.005).</p></caption><graphic id="gr6" xlink:href="bc-2007-00448h_0007.tif" position="float" orientation="portrait"></graphic></fig></sec><sec id="sec3.6"><title>Confocal Imaging</title><p>In order to determine the intracellular distribution of carriers, PEI and PEI-HGP polyplexes containing TOTO-3-labeled plasmid DNA were incubated with cells for either 1 or 3 h, fixed, and imaged using confocal microscopy. Endosomes were labeled with Alexa488-dextran, a fluid-phase uptake marker. At 1 h, both PEI and PEI-hgp polyplexes exhibited punctate staining (data not shown). When PEI polyplexes were delivered for 3 h, DNA exhibited punctate intracellular staining that was colocalized with dextran (Figure <xref rid="fig7"></xref>A). There were few examples of dextran staining that were not colocalized with DNA. When PEI-HGP polyplexes were delivered for 3 h, both punctate and diffuse DNA staining were observed (Figure <xref rid="fig7"></xref>B). Incidences of punctate DNA were colocalized with dextran, although there were examples of endocytic vesicles that were not colocalized with DNA. A substantial fraction (∼30%) of cells had diffuse DNA fluorescence throughout the cells (white arrows).</p><fig id="fig7" position="float" orientation="portrait"><label>7</label><caption><p>Confocal microscopy of HeLa cells incubated with PEI (A) and PEI-HGP (B) condensed TOTO-3 labeled gWiz-Luciferase DNA at N/P ratio 3. Vesicles are labeled with Alexa Fluor 488-labeled 10 000 Mw dextran. DNA/dextran (left panel), DNA (top right panel), and differential interference contrast (bottom right) images are shown. Arrows show cells exhibiting diffuse DNA fluorescence.</p></caption><graphic id="gr7" xlink:href="bc-2007-00448h_0008.tif" position="float" orientation="portrait"></graphic></fig></sec><sec id="sec3.7"><title>Chloroquine-Mediated Transfection</title><p>In order to investigate the effects of HGP as a mediator of endosomal escape, PEI and PEI-HGP polyplex transfections were performed in the presence of chloroquine. Chloroquine is a small molecule buffering agent used to mediate endosomal escape of gene delivery vehicles <named-content content-type="bibref-group"><xref rid="ref29" ref-type="bibr"></xref>−<xref rid="ref30" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="ref31" ref-type="bibr"></xref></named-content>. Polyplex transfections to HeLa cells were conducted in the presence of 100 µM chloroquine using an enhanced green fluorescent protein (EGFP) reporter gene. The percent of EGFP positive cells was measured by flow cytometry (Figure <xref rid="fig8"></xref>A). The percentage of EGFP positive cells after PEI-HGP transfection (20.1% ± 1.8%) was increased 16.1-fold when compared to PEI transfection (1.3% ± 0.3%, <italic toggle="yes">p</italic> < 0.001). Chloroquine increased the percentage of EGFP positive cells for both PEI and PEI-HGP; PEI transfection was increased 11.7-fold in the presence of chloroquine (14.6% ± 4.0%, <italic toggle="yes">p</italic> < 0.01), and PEI-HGP transfection was increased 3.8-fold in the presence of chloroquine (76.8% ± 3.7%, <italic toggle="yes">p</italic> < 0.001). Results from flow cytometry were verified by fluorescence microscopy (Figure <xref rid="fig8"></xref>B).</p><fig id="fig8" position="float" orientation="portrait"><label>8</label><caption><p>(A) Percentage of GFP positive HeLa cells after PEI or PEI-HGP polyplex transfection with and without chloroquine (CQ) with analysis by flow cytometry. (B) Fluorescence microscopy of representative EGFP-transfected cells.</p></caption><graphic id="gr8" xlink:href="bc-2007-00448h_0001.tif" position="float" orientation="portrait"></graphic></fig></sec></sec><sec id="sec4"><title>Discussion</title><p>Escape from endocytic vesicles after internalization is necessary for efficient intracellular trafficking of nonviral gene therapy vehicles. For polyplex delivery, several strategies have been implemented to overcome this challenge, including pH-sensitive polymers <xref rid="ref32" ref-type="bibr"></xref>, endosomal buffering polymers <named-content content-type="bibref-group"><xref rid="ref5" ref-type="bibr"></xref>,<xref rid="ref33" ref-type="bibr"></xref></named-content>, and photosensitive systems <xref rid="ref34" ref-type="bibr"></xref>. Membrane-interacting peptides, both natural and synthetic, have been utilized in gene delivery carriers <named-content content-type="bibref-group"><xref rid="ref15" ref-type="bibr"></xref>,<xref rid="ref35" ref-type="bibr"></xref>,<xref rid="ref36" ref-type="bibr"></xref></named-content>. The use of these peptides has been shown to increase the efficiencies of delivery vehicles; however, delivery efficiencies from nonviral vectors remain suboptimal for most clinical gene therapy applications.</p><p>In this work, a lytic peptide was conjugated to a polymeric gene carrier and evaluated for its effect on gene and siRNA delivery. In a screen of HIV gp41 to identify peptides with potent lytic activity, the sequence of HGP was demonstrated to be the most lytic <xref rid="ref20" ref-type="bibr"></xref>. Gp41, together with gp120, forms the monomer unit of the trimer HIV Env, the protein responsible for viral fusion of HIV. The sequence of HGP is from the lentiviral lytic peptide (LLP) domains 2 and 3 of the gp41 cytoplasmic tail. Another delivery system, MPG, is derived from the fusion domain of HIV gp41 and was shown to deliver oligonucleotides into the cytosol of 90% of cells in an endocytic independent pathway <xref rid="ref37" ref-type="bibr"></xref>. However, peptides from the LLP regions of gp41 have not to our knowledge been applied in gene delivery systems.</p><p>In initial studies, the HGP peptide was compared with a recently identified peptide from adenovirus for relative lytic ability <xref rid="ref28" ref-type="bibr"></xref>. A 10-fold higher concentration of peptide from adenovirus was needed to mediate 50% liposome disruption when compared to HGP (data not shown). Thus, we hypothesized that HGP may mediate efficient endosomal rupture at low peptide concentrations when incorporated in polycation delivery vectors.</p><p>PEI-HGP was synthesized by SMPB conjugation, and complete packaging of DNA at N/P ratio of 2 was confirmed by agarose gel electrophoresis. The same N/P ratio was needed to complex DNA with unmodified PEI (Figure <xref rid="fig1"></xref>), indicating that the ability for PEI to complex DNA is retained with peptide conjugation. This was expected, as the percentage of modified amines is very low (<0.5%). PEI-HGP polyplexes formed slightly larger polyplexes compared to PEI polyplexes (Figure <xref rid="fig2"></xref>). The difference in particle sizes of PEI-HGP polyplexes compared to PEI polyplexes is likely due to steric hindrance of peptides interfering with DNA complexation. Because polyplexes were of similar size, differences in transfection efficiencies between PEI-HGP and PEI polyplexes were most likely due to differences in the activity of HGP and not physical differences between polyplexes.</p><p>PEI and PEI-HGP polyplexes showed lytic activity that was dependent on the N/P ratio of formulation (Figure <xref rid="fig3"></xref>B). PEI-HGP polyplexes formulated at N/P ratios of 2, 3, and 4 used in the membrane lysis assay correspond to HGP concentrations of 0.13 µM, 0.19 µM, and 0.26 µM, respectively. PEI-HGP polyplexes showed increases in lytic activity over PEI polyplexes for each N/P ratio tested. The observed increase in lytic ability was much higher than what would be expected on the basis of the presence of HGP; free HGP at these low concentrations have minimal lytic activity (Figure <xref rid="fig3"></xref>A). The higher leakage values of PEI-HGP polyplexes when compared with HGP peptide at matched peptide concentrations may instead be attributed to increased local concentration of peptide tethered to the polyplex surface. During membrane fusion, HIV gp41 has been shown to adopt a six-helix bundle configuration <xref rid="ref38" ref-type="bibr"></xref> and the endodomain of HIV gp41 has shown high potential to form multimers <xref rid="ref21" ref-type="bibr"></xref>.</p><p>PEI-HGP was shown to be significantly more efficient than unmodified PEI for delivery of plasmids at low charge ratios (Figure <xref rid="fig4"></xref>A). Because polymer concentration (N/P ratio) also affects transfection efficiency, the polymer concentrations were carefully matched for PEI and PEI-HGP formulations by quantification of polymer concentration using copper (II) acetate and by gel retardation assay, which confirmed complete DNA condensation at similar charge ratios between the two formulations. At higher charge ratios, the difference between PEI-HGP and PEI transfection efficiency for plasmids is less significant, perhaps because the uncomplexed PEI polymer also exhibits membrane disruption ability (data not shown). Interestingly, siRNA delivery efficiency is enhanced by HGP at polymer charge ratios of 10 N/P, suggesting that the uncomplexed PEI polymer and HGP may act synergistically to increase siRNA availability in the cytoplasm (Figure <xref rid="fig5"></xref>).</p><p>In order to elucidate the mechanism for HGP-mediated increase in transfection, the efficiencies of polyplex binding, internalization, and endosomal release were investigated. The most likely route for the entry of PEI-HGP polyplexes is through endocytosis, which has been shown to be true for other endosomolytic peptide-modified delivery vehicles <named-content content-type="bibref-group"><xref rid="ref39" ref-type="bibr"></xref>,<xref rid="ref40" ref-type="bibr"></xref></named-content>. Cellular internalization and binding was examined at 4 and 37 °C using flow cytometry (Figure <xref rid="fig6"></xref>). At 4 °C, a temperature at which endocytosis is inhibited <xref rid="ref41" ref-type="bibr"></xref>, there is less cell-associated NBD fluorescence of PEI-HGP polyplexes than PEI polyplexes, indicating that there is less binding affinity of PEI-HGP polyplexes to the cellular membrane. Similar cell-associated NBD fluorescence of PEI-HGP polyplexes and PEI polyplexes at 37 °C suggests that the uptake efficiencies of PEI-HGP and PEI polyplexes are similar. Thus, the increase in transfection efficiency of PEI-HGP polyplexes is likely not due to more efficient uptake.</p><p>Confocal imaging is a technique that has been utilized to determine the intracellular distribution of nanoparticulate carriers <xref rid="ref3" ref-type="bibr"></xref>. Cells incubated with PEI polyplexes showed punctate plasmid DNA staining that largely colocalized with endosomes, indicating that polyplexes were most likely trapped within endosomes (Figure <xref rid="fig7"></xref>A). Cells incubated with PEI-HGP polyplexes showed both punctate and diffuse plasmid DNA staining, which is markedly different than the staining observed with PEI polyplexes and indicative of endosomal release of vectors (Figure <xref rid="fig7"></xref>B). Increases in transgene expression mediated by HGP may therefore be explained by changes in intracellular trafficking, most likely escape from endocytic vesicles, since HGP modification of vehicles does not significantly alter uptake.</p><p>The HGP peptide is derived from the LLP-2 and -3 domains of gp41, which have previously been demonstrated to disrupt lipid membranes <xref rid="ref22" ref-type="bibr"></xref>. These peptide regions have been shown to have amphipathic α-helical structure and are proposed to mediate membrane disruption by either spanning the membrane to form ion channels or by lying along the membrane surface <xref rid="ref22" ref-type="bibr"></xref>. Formation of ion channels by viruses, called “viroporins”, is a well-studied mechanism for membrane disruption based on a similar osmolytic mechanism to that proposed for PEI <named-content content-type="bibref-group"><xref rid="ref5" ref-type="bibr"></xref>,<xref rid="ref42" ref-type="bibr"></xref></named-content>. However, unlike PEI, membrane disruption by viroporins is not necessarily pH-sensitive. This raises the intriguing possibility that HGP may not only mediate enhanced endosomal release when coupled to PEI but may also change the release kinetics by relying on a critical peptide concentration in the endosomes instead of proton accumulation during endosomal maturation.</p><p>To further understand the intracellular trafficking pathway of PEI-HGP polyplexes, transfection studies were done in the presence of chloroquine. Chloroquine is a small molecule that is widely used to increase the transfection efficiency of gene delivery vehicles due to its ability to buffer endocytic compartments <xref rid="ref31" ref-type="bibr"></xref>. In addition, chloroquine has been proposed to have auxiliary functions such as increasing the unpackaging of nucleic acids from their carriers and altering intracellular processing by changing the physical and biological characteristics of the released nucleic acids <xref rid="ref31" ref-type="bibr"></xref>. When PEI and PEI-HGP polyplexes were coincubated with chloroquine, the transgene expression of both formulations was increased. These results indicate that HGP may be inadequate for complete release of carriers from endocytic vesicles or that the added increase in gene expression is due to the auxiliary functions of chloroquine. The observation that chloroquine increases the transgene expression of PEI-HGP polyplexes formulated at N/P ratio of 3 to greater than 75% of cells also implies that the vector can be further optimized to yield high transfection efficiencies. In addition, confocal microscopy images demonstrating the endosomal escape of PEI-HGP are supported by the observation that the effect of chloroquine on transgene expression is three times greater for PEI polyplexes over PEI-HGP polyplexes (Figure <xref rid="fig7"></xref>). Furthermore, the diffuse DNA staining observed in PEI-HGP transfected cells is also observed when PEI transfection was done in the presence of chloroquine (<xref rid="si1">Supporting Information</xref>).</p><p>Pulmonary gene therapy has important applications such as cystic fibrosis and lung cancer. PEI has been one of the most effective cationic polymeric carriers used in lung gene therapy <xref rid="ref43" ref-type="bibr"></xref>. When 50 µg of gWiz-Luciferase DNA condensed with PEI-HGP was injected into the tail vein of mice, statistically significant higher expression was found in the lung (∼3-fold higher) compared to PEI (data not shown).</p><p>For the application of gene delivery <italic toggle="yes">in vivo</italic>, it is of particular importance to develop particles that can be efficient at low polymer to DNA ratios. PEI polyplex delivery has been shown to be quite efficient when there is excess PEI present; however, the removal of excess polymer results in polyplexes with N/P ratios between 2 and 3 <xref rid="ref8" ref-type="bibr"></xref>. This has implications for <italic toggle="yes">in vivo</italic> delivery, as free polymer can be separated from complexes in the body. An additional benefit of achieving efficient transgene expression at low polymer to DNA ratios is improved cell viability, as increasing amounts of PEI can cause significant toxicity <xref rid="ref44" ref-type="bibr"></xref>. We were therefore interested in increasing transgene expression at these low N/P ratios.</p><p>In conclusion, a lytic peptide conjugated to the cationic polymer PEI was able to significantly enhance plasmid delivery efficiency over unmodified PEI at N/P ratios of 2 and 3 in cultured cells and in the lung tissue of mice. In addition, PEI-HGP efficiently delivered siRNA to cultured cells. Investigation into the mechanism of HGP indicated that increases in delivery efficiency were not due to increases in uptake. Studies employing the small molecule chloroquine and confocal microscopy supported the hypothesis that increased transfection efficiency was most likely due to endosomal escape. The chloroquine-mediated increase of PEI-HGP polyplex transgene expression indicated that vehicle properties can be further optimized, perhaps by engineering materials that improve vector unpackaging or cytosolic trafficking. This work is applicable for the development of nonviral delivery vehicles that are biologically active for efficient nucleic acid transfer <italic toggle="yes">in vivo</italic>.</p></sec></body><back><ack><title>Acknowledgments</title><p>The authors thank Leonard Meuse for his assistance with tail-vein injections in <italic toggle="yes">in vivo</italic> transfection studies. This work was funded by the National Science Foundation (CBET 0448547), NIH/NINDS (5R21NS052030), and the National Hemophilia Foundation. Flow cytometry was conducted at the Cell Analysis Facility at the Immunology Department of the University of Washington. Confocal imaging was conducted at the Nanotech User Facility at the University of Washington. The Nanotech User Facility is a member of the National Nanotechnology Infrastructure Network (NNIN) which is supported by the National Science Foundation and the Center for Nanotechnology at the University of Washington.</p></ack><notes id="si1" notes-type="si"><p>Confocal microscopy of PEI-HGP and PEI transfected cells in the presence of chloroquine. This material is available free of charge via the Internet at <uri>http://pubs.acs.org/BC</uri>.</p></notes><ref-list><title>References</title><ref id="ref1"><element-citation id="cit1" publication-type="journal"><name name-style="western"><surname>Pack</surname><given-names>D. 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Res.</source><volume>16</volume><fpage>1273</fpage><lpage>9</lpage></element-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles</title></titleInfo><titleInfo type="abbreviated"><title>HIV-Derived Peptide for Gene and siRNA Delivery</title></titleInfo><name type="personal"><namePart type="family">KWON</namePart><namePart type="given">Ester J.</namePart><affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">BERGEN</namePart><namePart type="given">Jamie M.</namePart><affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">PUN</namePart><namePart type="given">Suzie H.</namePart><affiliation>Department of Bioengineering, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195</affiliation><affiliation>E-mail: spun@u.washington.edu</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="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">2008-04-01</dateCreated><dateIssued encoding="w3cdtf">2008-04-16</dateIssued><copyrightDate encoding="w3cdtf">2008</copyrightDate></originInfo><abstract>Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.</abstract><note type="si" ID="si1">Confocal microscopy of PEI-HGP and PEI transfected cells in the presence of chloroquine. This material is available free of charge via the Internet at http://pubs.acs.org/BC.</note><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><part><date>2008</date><detail type="volume"><caption>vol.</caption><number>19</number></detail><detail type="issue"><caption>no.</caption><number>4</number></detail><extent unit="pages"><start>920</start><end>927</end></extent></part></relatedItem><identifier type="istex">89252D5CD138018F537D1067BA88CEBF7154E631</identifier><identifier type="ark">ark:/67375/TPS-T4NWZ521-F</identifier><identifier type="DOI">10.1021/bc700448h</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2008 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 © 2008 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-T4NWZ521-F/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/89252D5CD138018F537D1067BA88CEBF7154E631/annexes/tiff</uri></json:item><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-T4NWZ521-F/annexes.pdf</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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