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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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Visualization of the gD protein from BoHV-1 by WB.Notes: DC were transfected and after 48 hours, DC lysates were run on an SDS-PAGE/WB with mAb against gD. The de novo synthesized gD (~71 kDa) was detected only in the samples treated with plain-L (lane 6) or TATp-L (lane 7) as the plasmid vehicle (A). The latter had the greater concentration of protein as determined by ImageJ. The control recombinant gD (lane 8) had a lower molecular weight because of the lack of glycosylation (B). The M green band corresponds to 78 kDa. The unspecific band in the duplicate WB was a loading control (C). The M violet band corresponds to 41 kDa and the M orange band corresponds to 32 kDa.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; gD, glycoprotein D; HBS, HEPES-buffered saline; plain-L, plain liposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes; WB, Western blot.
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f7-ijn-9-963: Visualization of the gD protein from BoHV-1 by WB.Notes: DC were transfected and after 48 hours, DC lysates were run on an SDS-PAGE/WB with mAb against gD. The de novo synthesized gD (~71 kDa) was detected only in the samples treated with plain-L (lane 6) or TATp-L (lane 7) as the plasmid vehicle (A). The latter had the greater concentration of protein as determined by ImageJ. The control recombinant gD (lane 8) had a lower molecular weight because of the lack of glycosylation (B). The M green band corresponds to 78 kDa. The unspecific band in the duplicate WB was a loading control (C). The M violet band corresponds to 41 kDa and the M orange band corresponds to 32 kDa.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; gD, glycoprotein D; HBS, HEPES-buffered saline; plain-L, plain liposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes; WB, Western blot.

Mentions: To address the issue of the immunological activity of the de novo synthesized protein, the transfection using an encoding plasmid for the gD of BoHV-1 was performed. After a 48-hour transfection period, DC lysates were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a WB against the gD. Protein was expressed only in DC treated with liposome–plasmid complexes (Figure 7A). However, the expression level achieved with the TATp-L treatment was two-fold higher than the expression level achieved with the plain-L treatment as it was determined by the ImageJ densitometric analysis of the WB bands. As a loading control, a duplicate WB was developed using a BoHV-1 polyclonal Ab that showed an unspecific band in all samples with the same intensity (Figure 7C).


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)

Visualization of the gD protein from BoHV-1 by WB.Notes: DC were transfected and after 48 hours, DC lysates were run on an SDS-PAGE/WB with mAb against gD. The de novo synthesized gD (~71 kDa) was detected only in the samples treated with plain-L (lane 6) or TATp-L (lane 7) as the plasmid vehicle (A). The latter had the greater concentration of protein as determined by ImageJ. The control recombinant gD (lane 8) had a lower molecular weight because of the lack of glycosylation (B). The M green band corresponds to 78 kDa. The unspecific band in the duplicate WB was a loading control (C). The M violet band corresponds to 41 kDa and the M orange band corresponds to 32 kDa.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; gD, glycoprotein D; HBS, HEPES-buffered saline; plain-L, plain liposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes; WB, Western blot.
© Copyright Policy
Related In: Results  -  Collection

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

f7-ijn-9-963: Visualization of the gD protein from BoHV-1 by WB.Notes: DC were transfected and after 48 hours, DC lysates were run on an SDS-PAGE/WB with mAb against gD. The de novo synthesized gD (~71 kDa) was detected only in the samples treated with plain-L (lane 6) or TATp-L (lane 7) as the plasmid vehicle (A). The latter had the greater concentration of protein as determined by ImageJ. The control recombinant gD (lane 8) had a lower molecular weight because of the lack of glycosylation (B). The M green band corresponds to 78 kDa. The unspecific band in the duplicate WB was a loading control (C). The M violet band corresponds to 41 kDa and the M orange band corresponds to 32 kDa.Abbreviations: BoHV-1, bovine herpes virus type 1; DC, dendritic cells; gD, glycoprotein D; HBS, HEPES-buffered saline; plain-L, plain liposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; pCIneo, pCI-neo plasmid not encoding for an antigen; pCIgDA, pCI-neo plasmid encoding for BoHV-1 anchored gD; TATp-L, TAT peptide liposomes; WB, Western blot.
Mentions: To address the issue of the immunological activity of the de novo synthesized protein, the transfection using an encoding plasmid for the gD of BoHV-1 was performed. After a 48-hour transfection period, DC lysates were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a WB against the gD. Protein was expressed only in DC treated with liposome–plasmid complexes (Figure 7A). However, the expression level achieved with the TATp-L treatment was two-fold higher than the expression level achieved with the plain-L treatment as it was determined by the ImageJ densitometric analysis of the WB bands. As a loading control, a duplicate WB was developed using a BoHV-1 polyclonal Ab that showed an unspecific band in all samples with the same intensity (Figure 7C).

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