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Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment

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ABSTRACT

15123453333: Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues – [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 ()] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide may be a useful candidate as a gene delivery carrier.

No MeSH data available.


Transfection efficiency of 1–3 and R9/pDNA complexes at (a) 24-hr and (b) 48-hr post-incubation in HeLa cells. Data are shown as the mean ± standard deviation values of three independent cultures.
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f6: Transfection efficiency of 1–3 and R9/pDNA complexes at (a) 24-hr and (b) 48-hr post-incubation in HeLa cells. Data are shown as the mean ± standard deviation values of three independent cultures.

Mentions: Finally, we conducted pDNA intracellular transport experiments using peptides 1–3, R9, and HeLa cells. Peptide/pDNA complexes were prepared at charge ratios of 2/1, 4/1, and 8/1 because the residual molar ratios of the amino and/or guanidino groups in the peptides have to correspond to the number of phosphate groups in the pDNA. The transfection efficiency of peptides 1–3 and R9 was assessed using a luciferase-based assay. Figure 6 shows the transfection efficiencies of these peptides. pDNA transfection efficiency of the synthesized peptides were lower than that of commercially available transfection reagent TurboFect at 24-hr post-incubation, and peptide 3 transported the pDNA into both types of cells more efficiently than other peptides (Fig. 6a). However, peptide 3/pDNA (8/1) complex reached the higher transfection efficiency at 48-hr post-incubation than TurboFect/pDNA (4/1) complex did (Fig. 6b), indicating that peptide 3 was resistant to enzymatic degradation by proteases in cells, and therefore, appeared to have prolonged transfection abilities due to the protection of encapsulated pDNA in complexes for a longer time (Detailed physicochemical properties and transfection mechanism of peptide/pDNA complexes were most recently reported by our group)45.


Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment
Transfection efficiency of 1–3 and R9/pDNA complexes at (a) 24-hr and (b) 48-hr post-incubation in HeLa cells. Data are shown as the mean ± standard deviation values of three independent cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5016780&req=5

f6: Transfection efficiency of 1–3 and R9/pDNA complexes at (a) 24-hr and (b) 48-hr post-incubation in HeLa cells. Data are shown as the mean ± standard deviation values of three independent cultures.
Mentions: Finally, we conducted pDNA intracellular transport experiments using peptides 1–3, R9, and HeLa cells. Peptide/pDNA complexes were prepared at charge ratios of 2/1, 4/1, and 8/1 because the residual molar ratios of the amino and/or guanidino groups in the peptides have to correspond to the number of phosphate groups in the pDNA. The transfection efficiency of peptides 1–3 and R9 was assessed using a luciferase-based assay. Figure 6 shows the transfection efficiencies of these peptides. pDNA transfection efficiency of the synthesized peptides were lower than that of commercially available transfection reagent TurboFect at 24-hr post-incubation, and peptide 3 transported the pDNA into both types of cells more efficiently than other peptides (Fig. 6a). However, peptide 3/pDNA (8/1) complex reached the higher transfection efficiency at 48-hr post-incubation than TurboFect/pDNA (4/1) complex did (Fig. 6b), indicating that peptide 3 was resistant to enzymatic degradation by proteases in cells, and therefore, appeared to have prolonged transfection abilities due to the protection of encapsulated pDNA in complexes for a longer time (Detailed physicochemical properties and transfection mechanism of peptide/pDNA complexes were most recently reported by our group)45.

View Article: PubMed Central - PubMed

ABSTRACT

15123453333: Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues – [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 ()] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide may be a useful candidate as a gene delivery carrier.

No MeSH data available.