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Conference Paper: Endocytic DNA and siRNA delivery mediated by pH sensitive peptides

TitleEndocytic DNA and siRNA delivery mediated by pH sensitive peptides
Authors
KeywordsPharmacy and pharmacology biology
Biotechnology
Issue Date2010
PublisherElsevier Ltd, Trends Journals. The Journal's web site is located at http://www.elsevier.com/locate/drugdiscov
Citation
The 3rd International Symposium on Cellular Delivery of Therapeutic Macromolecules (CDTM 2010), Cardiff University, Wales, UK., 26-29 June 2010. In Drug Discovery Today, 2010, v. 15 n. 23-24, p. 1086, abstract no. A15 How to Cite?
AbstractAmphipathic peptides have emerged as promising candidates for both DNA and siRNA delivery. Consisting of both hydrophilic and hydrophobic domains, they bind to nucleic acids and at the same time provide pH dependent membrane destabilising activity, promoting endosomal escape. The aim of this study was to investigate the efficiency of such peptides in delivering siRNA and plasmid DNA, and to improve our understanding of how the structural difference between the peptides could affect the uptake mechanism and intracellular trafficking of the system. A series of structurally related histidine-rich amphipathic peptides (LAH4-L1, LAH6-X1L, LAH6-X1-26 and LAH6-X1-W) were investigated. The LAH peptides are 25–26 amino acids in length and comprise cationic lysines to allow electrostatic interaction and complexation with the negatively charged nucleic acids. Each of the peptides also contains four or six histidine residues. With a starting pKa around 6.0, the imidazole group of histidine may allow buffering and subsequently destabilise endosomes, thus enhancing endosomal escape of the nucleic acids. The LAH peptides demonstrated pH responsive character whish is classically manifested as a conversion from an alpha helical conformation at neutral pH to a disordered conformation at acidic pH. Differences in the number of charges and the hydrophobicity in the four peptides affect the nature and pH dependence of this transformation. Luciferase reporter gene studies showed that the in vitro DNA transfection efficiency of the LAH peptides were comparable to commercially available lipofectamine in both A549 and MCF-7 cells. These peptides, in particular LAH6-X1L, also showed high resistance to serum in MCF-7 cells. In addition, both LAH4-L1 and LAH6-X1L mediated significant knockdown of GAPDH enzyme in siRNA transfection studies in the presence of serum. Live cell confocal imaging was carried out to study the intracellular trafficking of the peptide/nucleic acid complexes. Co-localisation experiments were performed with LAH6-X1L- DNA/siRNA complexes and dextran in A549 cells, with the nucleic acids labelled with rhodamine (red), nucleus labeled with Hoechst (blue) and dextran labelled with Alexa-fluor-488 (green). Dextran is known to be internalised through fluid-endocytosis and end up in endososmes and later lysosomes. At an early stage (within the first hour of post-transfection) there was a high level of co-localisation between LAH6-X1L complexes and dextran (shown as orange in colour). At later stages (300 minutes post-transfection), the degree of co-localisation significantly reduced as the siRNA (red) and dextran (green) was shown to be clearly separated from each other. Our results indicate that the histidinerich peptides offer great promise as siRNA delivery vectors with the ability to promote endosomal/lysosomal escape.
DescriptionOral Presentation
Persistent Identifierhttp://hdl.handle.net/10722/126872
ISSN
2015 Impact Factor: 5.625
2015 SCImago Journal Rankings: 2.084
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorSo, MSWen_HK
dc.contributor.authorWitt, Ken_HK
dc.contributor.authorLan, Yen_HK
dc.contributor.authorMason, AJen_HK
dc.contributor.authorLam, JKWen_HK
dc.date.accessioned2010-10-31T12:53:31Z-
dc.date.available2010-10-31T12:53:31Z-
dc.date.issued2010en_HK
dc.identifier.citationThe 3rd International Symposium on Cellular Delivery of Therapeutic Macromolecules (CDTM 2010), Cardiff University, Wales, UK., 26-29 June 2010. In Drug Discovery Today, 2010, v. 15 n. 23-24, p. 1086, abstract no. A15en_HK
dc.identifier.issn1359-6446-
dc.identifier.urihttp://hdl.handle.net/10722/126872-
dc.descriptionOral Presentation-
dc.description.abstractAmphipathic peptides have emerged as promising candidates for both DNA and siRNA delivery. Consisting of both hydrophilic and hydrophobic domains, they bind to nucleic acids and at the same time provide pH dependent membrane destabilising activity, promoting endosomal escape. The aim of this study was to investigate the efficiency of such peptides in delivering siRNA and plasmid DNA, and to improve our understanding of how the structural difference between the peptides could affect the uptake mechanism and intracellular trafficking of the system. A series of structurally related histidine-rich amphipathic peptides (LAH4-L1, LAH6-X1L, LAH6-X1-26 and LAH6-X1-W) were investigated. The LAH peptides are 25–26 amino acids in length and comprise cationic lysines to allow electrostatic interaction and complexation with the negatively charged nucleic acids. Each of the peptides also contains four or six histidine residues. With a starting pKa around 6.0, the imidazole group of histidine may allow buffering and subsequently destabilise endosomes, thus enhancing endosomal escape of the nucleic acids. The LAH peptides demonstrated pH responsive character whish is classically manifested as a conversion from an alpha helical conformation at neutral pH to a disordered conformation at acidic pH. Differences in the number of charges and the hydrophobicity in the four peptides affect the nature and pH dependence of this transformation. Luciferase reporter gene studies showed that the in vitro DNA transfection efficiency of the LAH peptides were comparable to commercially available lipofectamine in both A549 and MCF-7 cells. These peptides, in particular LAH6-X1L, also showed high resistance to serum in MCF-7 cells. In addition, both LAH4-L1 and LAH6-X1L mediated significant knockdown of GAPDH enzyme in siRNA transfection studies in the presence of serum. Live cell confocal imaging was carried out to study the intracellular trafficking of the peptide/nucleic acid complexes. Co-localisation experiments were performed with LAH6-X1L- DNA/siRNA complexes and dextran in A549 cells, with the nucleic acids labelled with rhodamine (red), nucleus labeled with Hoechst (blue) and dextran labelled with Alexa-fluor-488 (green). Dextran is known to be internalised through fluid-endocytosis and end up in endososmes and later lysosomes. At an early stage (within the first hour of post-transfection) there was a high level of co-localisation between LAH6-X1L complexes and dextran (shown as orange in colour). At later stages (300 minutes post-transfection), the degree of co-localisation significantly reduced as the siRNA (red) and dextran (green) was shown to be clearly separated from each other. Our results indicate that the histidinerich peptides offer great promise as siRNA delivery vectors with the ability to promote endosomal/lysosomal escape.-
dc.languageengen_HK
dc.publisherElsevier Ltd, Trends Journals. The Journal's web site is located at http://www.elsevier.com/locate/drugdiscov-
dc.relation.ispartofDrug Discovery Today-
dc.subjectPharmacy and pharmacology biology-
dc.subjectBiotechnology-
dc.titleEndocytic DNA and siRNA delivery mediated by pH sensitive peptidesen_HK
dc.typeConference_Paperen_HK
dc.identifier.emailMason, AJ: james.mason@kcl.ac.uken_HK
dc.identifier.emailLam, JKW: jkwlam@hku.hk-
dc.identifier.authorityLam, JKW=rp01346en_HK
dc.identifier.doi10.1016/j.drudis.2010.09.368-
dc.identifier.hkuros175310en_HK
dc.identifier.volume15-
dc.identifier.issue23-24-
dc.identifier.spage1086, abstract no. A15-
dc.identifier.epage1086, abstract no. A15-
dc.identifier.isiWOS:000285235700028-
dc.publisher.placeUnited Kingdom-
dc.description.otherThe 3rd International Symposium on Cellular Delivery of Therapeutic Macromolecules (CDTM2010), Cardiff University, Wales, UK., 26-29 June 2010. In Drug Discovery Today, 2010, v. 15 n. 23-24, p. 1086, abstract no. A15-
dc.identifier.citeulike8119520-

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