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In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

Pappalardo JS, Langellotti CA, Di Giacomo S, Olivera V, Quattrocchi V, Zamorano PI, Hartner WC, Levchenko TS, Torchilin VP - Int J Nanomedicine (2014)

Bottom Line: TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane.Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC.The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

View Article: PubMed Central - PubMed

Affiliation: Virology Institute, Center for Research in Veterinary and Agronomic Sciences, National Institute for Agricultural Technology (INTA), Hurlingham, BA, Argentina ; National Council for Scientific and Technical Research (CONICET), Autonomous City of Buenos Aires, Argentina ; Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, USA.

ABSTRACT
Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

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IL-6 secretion by dendritic cells 48 hours post-transfection.Notes: Cytokines were assayed in DC culture supernatants 48 hours post-transfection by ELISA. IL-6 was expressed at a high value only from TATp-L-treated cells. pCIneo: pCIneo plasmid (empty, as control). pCIgDA: pCIgDA plasmid (encoding for BoHV-1 gD anchored version). ***P<0.001 for multiple comparisons in a Bonferroni post hoc test (against all), n=6.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; ELISA, enzyme-linked immunosorbent assay; gD, glycoprotein D; IL-6, interleukin 6; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes.
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f8-ijn-9-963: IL-6 secretion by dendritic cells 48 hours post-transfection.Notes: Cytokines were assayed in DC culture supernatants 48 hours post-transfection by ELISA. IL-6 was expressed at a high value only from TATp-L-treated cells. pCIneo: pCIneo plasmid (empty, as control). pCIgDA: pCIgDA plasmid (encoding for BoHV-1 gD anchored version). ***P<0.001 for multiple comparisons in a Bonferroni post hoc test (against all), n=6.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; ELISA, enzyme-linked immunosorbent assay; gD, glycoprotein D; IL-6, interleukin 6; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes.

Mentions: After the transfection period (48 hours), supernatants of DC cultures were screened for IL-6, IL-10, IL-12, and TNFα. Only IL-6 was found in the supernatants of DC treated with TATp-L plus pCIgDA (the plasmid for BoHV-1 gD). A significant difference in IL-6 secretion (P<0.001) was observed between TATp-L and pCIgDA and the other treatments: TATp-L plus pCIneo (empty plasmid), or plain-L plus pCIneo or pCIgDA (Figure 8). There was also IL-6 secretion with Lipofectamine or Lipofectin alone (without plasmid), and with Lipofectamine and naked plasmid, both with and without the gD encoding gene, suggesting that this response was nonspecific and due to stimulation by the transfection reagent or the naked plasmid (data not shown). In addition, when liposomes were incubated with no cargo (empty liposomes), there was no cytokine detection (data not shown).


In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

Pappalardo JS, Langellotti CA, Di Giacomo S, Olivera V, Quattrocchi V, Zamorano PI, Hartner WC, Levchenko TS, Torchilin VP - Int J Nanomedicine (2014)

IL-6 secretion by dendritic cells 48 hours post-transfection.Notes: Cytokines were assayed in DC culture supernatants 48 hours post-transfection by ELISA. IL-6 was expressed at a high value only from TATp-L-treated cells. pCIneo: pCIneo plasmid (empty, as control). pCIgDA: pCIgDA plasmid (encoding for BoHV-1 gD anchored version). ***P<0.001 for multiple comparisons in a Bonferroni post hoc test (against all), n=6.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; ELISA, enzyme-linked immunosorbent assay; gD, glycoprotein D; IL-6, interleukin 6; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928453&req=5

f8-ijn-9-963: IL-6 secretion by dendritic cells 48 hours post-transfection.Notes: Cytokines were assayed in DC culture supernatants 48 hours post-transfection by ELISA. IL-6 was expressed at a high value only from TATp-L-treated cells. pCIneo: pCIneo plasmid (empty, as control). pCIgDA: pCIgDA plasmid (encoding for BoHV-1 gD anchored version). ***P<0.001 for multiple comparisons in a Bonferroni post hoc test (against all), n=6.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; ELISA, enzyme-linked immunosorbent assay; gD, glycoprotein D; IL-6, interleukin 6; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes.
Mentions: After the transfection period (48 hours), supernatants of DC cultures were screened for IL-6, IL-10, IL-12, and TNFα. Only IL-6 was found in the supernatants of DC treated with TATp-L plus pCIgDA (the plasmid for BoHV-1 gD). A significant difference in IL-6 secretion (P<0.001) was observed between TATp-L and pCIgDA and the other treatments: TATp-L plus pCIneo (empty plasmid), or plain-L plus pCIneo or pCIgDA (Figure 8). There was also IL-6 secretion with Lipofectamine or Lipofectin alone (without plasmid), and with Lipofectamine and naked plasmid, both with and without the gD encoding gene, suggesting that this response was nonspecific and due to stimulation by the transfection reagent or the naked plasmid (data not shown). In addition, when liposomes were incubated with no cargo (empty liposomes), there was no cytokine detection (data not shown).

Bottom Line: TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane.Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC.The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

View Article: PubMed Central - PubMed

Affiliation: Virology Institute, Center for Research in Veterinary and Agronomic Sciences, National Institute for Agricultural Technology (INTA), Hurlingham, BA, Argentina ; National Council for Scientific and Technical Research (CONICET), Autonomous City of Buenos Aires, Argentina ; Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, USA.

ABSTRACT
Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

Show MeSH
Related in: MedlinePlus