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

Abstract

Cell 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 a-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.

siRNAs encapsulated in recombinant capsid protein derived from Dengue serotype 2 virus inhibits the four serotypes of the virus and proliferation of cancer cells

“…The intracellular distribution of Cy3 labelled siRNA delivered using our D2C protein preparation, in C6/36 cells and BHK-21 cells, appeared to be comparable to the observations of Freire et al (2014) using the same protein derived from a different DENV2 strain (Freire et al, 2013), and those of Choi et al (2011) using the Hepatitis B virus capsid protein, as the siRNA nanocarrier. We have successfully demonstrated that DENV2 targeted siRNA, encapsulated in D2C, can inhibit DENV2 infection with comparable efficiency as that of a widely used commercial siRNA transfection reagent, HiPerfect, in the human liver cell line, Huh-7.…”

“…pH shift assembly of adenoviral serotype 5 capsid protein nanosystems has been used for macromolecular delivery . Recently, E. coli expressed D2C protein and a D2C based peptide, designated Pep M, were shown to encapsulate and deliver siRNA into cultured cell lines, but target knockdown data was restricted to TLR-3 in endothelial cells (Freire et al, 2013(Freire et al, , 2014. The utility of D2C protein or its peptide derivatives for siRNA delivery in the context of any particular disease indication is not known at present.…”

“…A Clustal W alignment of D2C based peptides with the full length D2C protein and previously reported D2C based peptides Pep M and Pep R (Freire et al, 2013(Freire et al, , 2014) is shown as Supporting information II.…”

“…Chimeric capsid protein of Hepatitis B Virus (HBV) was shown to assemble into 50 nM diameter particles in the presence of siRNA, and serve as nanocarriers for siRNA (Choi et al, 2013). Recently, D2C protein and a D2C based peptide, Pep M, were demonstrated to encapsulate and deliver fluorescently labelled siRNA into a variety of cell lines and inhibit TLR-3 in endothelial cells (Freire et al, 2013(Freire et al, , 2014. To the best of our knowledge, the potential of either D2C protein, or its peptide derivatives, for functional siRNA delivery in the context of any specific disease model, has not been reported.…”

Receptors and routes of dengue virus entry into the host cells

“…In this context, recent data support an additional participation of DENV C protein in the virus genome internalization, a role that would be shared by all flaviviruses' capsid proteins. It has been found that recombinant DENV C protein as well as two synthetic peptides comprising C protein residues 45-72 and 67-100 were able to translocate short oligonucleotide sequences (ssDNA and siRNA) across cellular membranes (Freire et al, 2013b, Freire et al, 2014. More interestingly, large nucleic acids, such as a GFP-encoding plasmid, were not only transported into the cells but also expressed at levels comparable to those obtained when traditional transfection agents, such as lipofectamine, were used (Freire et al, 2013b).…”