Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)

Nadia Bendifallah; Frank Winther Rasmussen; Vladimir Zachar; Peter Ebbesen; Uffe Koppelhus; Peter E. Nielsen

doi:10.1021/bc050283q

Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)

Nadia Bendifallah, Frank Winther Rasmussen, Vladimir Zachar, Peter Ebbesen, Uffe Koppelhus, Peter E. Nielsen

Transcription, RNA, and Gene Medicine Program

Abstract

Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.

Originalsprog	Engelsk
Tidsskrift	Bioconjugate Chemistry
Vol/bind	17
Udgave nummer	3
Sider (fra-til)	750-8
Antal sider	9
ISSN	1043-1802
DOI	https://doi.org/10.1021/bc050283q
Status	Udgivet - 18 maj 2006

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10.1021/bc050283q

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title = "Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)",

abstract = "Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.",

keywords = "Arginine/chemistry, Cell Membrane Permeability/drug effects, Cross-Linking Reagents/chemistry, Culture Media, Serum-Free, Esters/chemistry, Galanin/toxicity, HeLa Cells, Humans, Molecular Structure, Oligonucleotides, Antisense/chemistry, Peptide Nucleic Acids/chemistry, Recombinant Fusion Proteins/toxicity, Serum, Wasp Venoms/toxicity",

author = "Nadia Bendifallah and Rasmussen, {Frank Winther} and Vladimir Zachar and Peter Ebbesen and Uffe Koppelhus and Nielsen, {Peter E.}",

year = "2006",

month = may,

day = "18",

doi = "10.1021/bc050283q",

language = "English",

volume = "17",

pages = "750--8",

journal = "Bioconjugate Chemistry",

issn = "1043-1802",

publisher = "American Chemical Society",

number = "3",

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TY - JOUR

T1 - Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)

AU - Bendifallah, Nadia

AU - Rasmussen, Frank Winther

AU - Zachar, Vladimir

AU - Ebbesen, Peter

AU - Koppelhus, Uffe

AU - Nielsen, Peter E.

PY - 2006/5/18

Y1 - 2006/5/18

N2 - Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.

AB - Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.

KW - Arginine/chemistry

KW - Cell Membrane Permeability/drug effects

KW - Cross-Linking Reagents/chemistry

KW - Culture Media, Serum-Free

KW - Esters/chemistry

KW - Galanin/toxicity

KW - HeLa Cells

KW - Humans

KW - Molecular Structure

KW - Oligonucleotides, Antisense/chemistry

KW - Peptide Nucleic Acids/chemistry

KW - Recombinant Fusion Proteins/toxicity

KW - Serum

KW - Wasp Venoms/toxicity

U2 - 10.1021/bc050283q

DO - 10.1021/bc050283q

M3 - Journal article

C2 - 16704214

SN - 1043-1802

VL - 17

SP - 750

EP - 758

JO - Bioconjugate Chemistry

JF - Bioconjugate Chemistry

IS - 3

ER -

Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)

Abstract

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