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Nucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein : design and mechanism of action

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dc.contributor.authorFreire, João M.
dc.contributor.authorVeiga, Ana Salomé
dc.contributor.authorFigueiredo, Inês Rego de
dc.contributor.authorTorre, Beatriz G. de la
dc.contributor.authorSantos, Nuno C.
dc.contributor.authorAndreu, David
dc.contributor.authorPoian, Andrea T. da
dc.contributor.authorCastanho, Miguel A. R. B.
dc.date.accessioned2016-04-18T16:18:20Z
dc.date.available2016-04-18T16:18:20Z
dc.date.issued2014
dc.description© 2013 FEBSpt_PT
dc.description.abstractCell penetrating peptides (CPPs) can be used as drug delivery systems for different therapeutic molecules. In this work two novel CPPs, pepR and pepM, designed from two domains of the dengue virus (DENV) capsid protein, were studied for their ability to deliver nucleic acids into cells as non-covalently bound cargo. Translocation studies were performed by confocal microscopy in HepG2, BHK and HEK cell lineages, astrocytes and peripheral blood mononuclear cells. Combined studies in HepG2 cells, astrocytes and BHK cells, at 4 and 37 °C or using specific endocytosis inhibitors, revealed that pepR and pepM use distinct internalization routes: pepM translocates lipid membranes directly, while pepR uses an endocytic pathway. To confirm these results, a methodology was developed to monitor the translocation kinetics of both peptides by real-time flow cytometry. Kinetic constants were determined, and the amount of nucleic acids delivered was estimated. Additional studies were performed in order to understand the molecular bases of the peptide-mediated translocation. Peptide–nucleic acid and peptide–lipid membrane interactions were studied quantitatively based on the intrinsic fluorescence of the peptides. pepR and pepM bound ssDNA to the same extent. Partition studies revealed that both peptides bind preferentially to anionic lipid membranes, adopting an α-helical conformation. However, fluorescence quenching studies suggest that pepM is deeply inserted into the lipid bilayer, in contrast with pepR. Moreover, only pepM is able to promote the fusion and aggregation of vesicles composed of zwitterionic lipids. Altogether, the results show that DENV capsid protein derived peptides serve as good templates for novel CPP-based nucleic acid delivery strategies, defining different routes for cell entry.pt_PT
dc.description.sponsorshipThis work was supported by Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) (PTDC/QUI-BIQ/112929/2009), by the European Union [projects FP7-PEOPLEIRSES MEMPEPACROSS and FP7-HEALTH-F3-2008-223414 (LEISHDRUG)], by the Spanish Ministry of Economy and Competitiveness (SAF2011-24899), by the Generalitat de Catalunya (2009 SGR 492), by the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and by the National Institute of Science and Technology in Dengue (INCT-Dengue). JMF also acknowledges FCT-MEC for PhD fellowship SFRH/BD/70423/2010. MC acknowledges a grant from Programa Ciência Sem Fronteiras PVE171/2012 (CAPES and CNPq, Brazil). The authors thank Sónia Sá Santos (IMM, Lisbon, Portugal) for the kind gift of the primary astrocyte cultures.pt_PT
dc.identifier.citationFEBS Journal 281 (2014) 191–215pt_PT
dc.identifier.doi10.1111/febs.12587pt_PT
dc.identifier.issn1742-464X
dc.identifier.urihttp://hdl.handle.net/10451/23432
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherWileypt_PT
dc.relation.publisherversionhttp://febs.onlinelibrary.wiley.com/hub/journal/10.1111/(ISSN)1742-4658/pt_PT
dc.subjectCapsid proteinpt_PT
dc.subjectCell penetrating peptidept_PT
dc.subjectDengue viruspt_PT
dc.subjectGene therapypt_PT
dc.subjectTime-resolvedpt_PT
dc.subjectFlow cytometrypt_PT
dc.titleNucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein : design and mechanism of actionpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FQUI-BIQ%2F112929%2F2009/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBD%2F70423%2F2010/PT
oaire.citation.titleFEBS Journalpt_PT
oaire.fundingStream3599-PPCDT
oaire.fundingStreamSFRH
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
relation.isProjectOfPublication954cb0bb-03f8-45f6-a48d-27c88a2f8690
relation.isProjectOfPublication1a004e63-0ace-4b41-bd0c-db90b919807e
relation.isProjectOfPublication.latestForDiscovery954cb0bb-03f8-45f6-a48d-27c88a2f8690

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