Structural and formulation factors influencing pyridinium lipid-based gene transfer

Lin Zhu, Yan Lu, Duane D. Miller, Ram I Mahato

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

A series of pyridinium lipids containing a heterocyclic ring and a nitrogen atom were synthesized to determine the structure-activity relationship for gene delivery. Pyrylium chloroaluminate was synthesized by monoacylation of mesityl oxide and converted into pyrylium hexafluorophosphate, which was used as the key intermediate for reaction with different primary amines, to yield hydroxyethylpyridinium hexafluorophosphate and aminoethylpyridinium hexafluorophosphate. Acylation of these pyridinium salts with different types of fatty acid chlorides afforded the final pyridinium lipids, which were mixed with a co-lipid, such as L-α-dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) to prepare cationic liposomes by sonication. These liposomes were mixed with plasmid DNA encoding enhanced green fluorescent protein (pCMS-EGFP) or luciferase (pcDNA3-Luc) and transfected into Chinese hamster ovary (CHO) cells. Several factors including hydrophobic anchor chain length, anchor chain type, configuration of double bond, linker type, co-lipid type, cationic lipid/co-lipid molar ratio, charge ratio (N/P), and cell type had significant influence on transfection efficiency and cytotoxicity. Pyridinium lipids with amide linker showed significantly higher transfection efficiency compared to their ester counterparts. Liposomes prepared at a 1:1 molar ratio of pyridinium lipid and co-lipid showed higher transfection efficiency when either DOPE or cholesterol was used as a co-lipid to prepare cationic liposomes for complex formation with plasmid DNA at 3:1(+/-) charge ratio. Pyridinium liposomes based on a hydrophobic anchor chain length of 16 showed higher transfection efficiency and lower cytotoxicity. The pyridinium lipid with a trans-configuration of the double bond in the fatty acid chain showed higher transfection efficiency than its counterpart with cis-configuration at the same fatty acid chain length. In the presence of serum, C16:0 and Lipofectamine significantly decreased their transfection efficiencies, which were completely lost at a serum concentration of 30% and higher, while C16:1 trans-isomer still had high transfection efficiency under these conditions. In conclusion, pyridinium lipids showed high transfection efficiency and have the potential to be used as transfection reagents in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)2499-2512
Number of pages14
JournalBioconjugate Chemistry
Volume19
Issue number12
DOIs
StatePublished - Dec 17 2008

Fingerprint

Gene transfer
Lipids
Transfection
Liposomes
Genes
Anchors
Chain length
Fatty acids
Fatty Acids
Cholesterol
Cytotoxicity
Plasmids
DNA
Acylation
Sonication
Structure-Activity Relationship
Cricetulus
Luciferases
Serum
Amides

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Structural and formulation factors influencing pyridinium lipid-based gene transfer. / Zhu, Lin; Lu, Yan; Miller, Duane D.; Mahato, Ram I.

In: Bioconjugate Chemistry, Vol. 19, No. 12, 17.12.2008, p. 2499-2512.

Research output: Contribution to journalArticle

Zhu, Lin ; Lu, Yan ; Miller, Duane D. ; Mahato, Ram I. / Structural and formulation factors influencing pyridinium lipid-based gene transfer. In: Bioconjugate Chemistry. 2008 ; Vol. 19, No. 12. pp. 2499-2512.
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abstract = "A series of pyridinium lipids containing a heterocyclic ring and a nitrogen atom were synthesized to determine the structure-activity relationship for gene delivery. Pyrylium chloroaluminate was synthesized by monoacylation of mesityl oxide and converted into pyrylium hexafluorophosphate, which was used as the key intermediate for reaction with different primary amines, to yield hydroxyethylpyridinium hexafluorophosphate and aminoethylpyridinium hexafluorophosphate. Acylation of these pyridinium salts with different types of fatty acid chlorides afforded the final pyridinium lipids, which were mixed with a co-lipid, such as L-α-dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) to prepare cationic liposomes by sonication. These liposomes were mixed with plasmid DNA encoding enhanced green fluorescent protein (pCMS-EGFP) or luciferase (pcDNA3-Luc) and transfected into Chinese hamster ovary (CHO) cells. Several factors including hydrophobic anchor chain length, anchor chain type, configuration of double bond, linker type, co-lipid type, cationic lipid/co-lipid molar ratio, charge ratio (N/P), and cell type had significant influence on transfection efficiency and cytotoxicity. Pyridinium lipids with amide linker showed significantly higher transfection efficiency compared to their ester counterparts. Liposomes prepared at a 1:1 molar ratio of pyridinium lipid and co-lipid showed higher transfection efficiency when either DOPE or cholesterol was used as a co-lipid to prepare cationic liposomes for complex formation with plasmid DNA at 3:1(+/-) charge ratio. Pyridinium liposomes based on a hydrophobic anchor chain length of 16 showed higher transfection efficiency and lower cytotoxicity. The pyridinium lipid with a trans-configuration of the double bond in the fatty acid chain showed higher transfection efficiency than its counterpart with cis-configuration at the same fatty acid chain length. In the presence of serum, C16:0 and Lipofectamine significantly decreased their transfection efficiencies, which were completely lost at a serum concentration of 30{\%} and higher, while C16:1 trans-isomer still had high transfection efficiency under these conditions. In conclusion, pyridinium lipids showed high transfection efficiency and have the potential to be used as transfection reagents in vitro and in vivo.",
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N2 - A series of pyridinium lipids containing a heterocyclic ring and a nitrogen atom were synthesized to determine the structure-activity relationship for gene delivery. Pyrylium chloroaluminate was synthesized by monoacylation of mesityl oxide and converted into pyrylium hexafluorophosphate, which was used as the key intermediate for reaction with different primary amines, to yield hydroxyethylpyridinium hexafluorophosphate and aminoethylpyridinium hexafluorophosphate. Acylation of these pyridinium salts with different types of fatty acid chlorides afforded the final pyridinium lipids, which were mixed with a co-lipid, such as L-α-dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) to prepare cationic liposomes by sonication. These liposomes were mixed with plasmid DNA encoding enhanced green fluorescent protein (pCMS-EGFP) or luciferase (pcDNA3-Luc) and transfected into Chinese hamster ovary (CHO) cells. Several factors including hydrophobic anchor chain length, anchor chain type, configuration of double bond, linker type, co-lipid type, cationic lipid/co-lipid molar ratio, charge ratio (N/P), and cell type had significant influence on transfection efficiency and cytotoxicity. Pyridinium lipids with amide linker showed significantly higher transfection efficiency compared to their ester counterparts. Liposomes prepared at a 1:1 molar ratio of pyridinium lipid and co-lipid showed higher transfection efficiency when either DOPE or cholesterol was used as a co-lipid to prepare cationic liposomes for complex formation with plasmid DNA at 3:1(+/-) charge ratio. Pyridinium liposomes based on a hydrophobic anchor chain length of 16 showed higher transfection efficiency and lower cytotoxicity. The pyridinium lipid with a trans-configuration of the double bond in the fatty acid chain showed higher transfection efficiency than its counterpart with cis-configuration at the same fatty acid chain length. In the presence of serum, C16:0 and Lipofectamine significantly decreased their transfection efficiencies, which were completely lost at a serum concentration of 30% and higher, while C16:1 trans-isomer still had high transfection efficiency under these conditions. In conclusion, pyridinium lipids showed high transfection efficiency and have the potential to be used as transfection reagents in vitro and in vivo.

AB - A series of pyridinium lipids containing a heterocyclic ring and a nitrogen atom were synthesized to determine the structure-activity relationship for gene delivery. Pyrylium chloroaluminate was synthesized by monoacylation of mesityl oxide and converted into pyrylium hexafluorophosphate, which was used as the key intermediate for reaction with different primary amines, to yield hydroxyethylpyridinium hexafluorophosphate and aminoethylpyridinium hexafluorophosphate. Acylation of these pyridinium salts with different types of fatty acid chlorides afforded the final pyridinium lipids, which were mixed with a co-lipid, such as L-α-dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) to prepare cationic liposomes by sonication. These liposomes were mixed with plasmid DNA encoding enhanced green fluorescent protein (pCMS-EGFP) or luciferase (pcDNA3-Luc) and transfected into Chinese hamster ovary (CHO) cells. Several factors including hydrophobic anchor chain length, anchor chain type, configuration of double bond, linker type, co-lipid type, cationic lipid/co-lipid molar ratio, charge ratio (N/P), and cell type had significant influence on transfection efficiency and cytotoxicity. Pyridinium lipids with amide linker showed significantly higher transfection efficiency compared to their ester counterparts. Liposomes prepared at a 1:1 molar ratio of pyridinium lipid and co-lipid showed higher transfection efficiency when either DOPE or cholesterol was used as a co-lipid to prepare cationic liposomes for complex formation with plasmid DNA at 3:1(+/-) charge ratio. Pyridinium liposomes based on a hydrophobic anchor chain length of 16 showed higher transfection efficiency and lower cytotoxicity. The pyridinium lipid with a trans-configuration of the double bond in the fatty acid chain showed higher transfection efficiency than its counterpart with cis-configuration at the same fatty acid chain length. In the presence of serum, C16:0 and Lipofectamine significantly decreased their transfection efficiencies, which were completely lost at a serum concentration of 30% and higher, while C16:1 trans-isomer still had high transfection efficiency under these conditions. In conclusion, pyridinium lipids showed high transfection efficiency and have the potential to be used as transfection reagents in vitro and in vivo.

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