Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids: J Control Release

C. W. Pouton, P. Lucas, B. J. Thomas, A. N. Uduehi, D. A. Milroy, S. H. Moss

Research output: Contribution to journalArticleResearchpeer-review

Abstract

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.
Original languageEnglish
Pages (from-to)289-299
Number of pages11
JournalJournal of Controlled Release
Volume53
Publication statusPublished - 1998

Keywords

  • Animals COS Cells Cell Line, Transformed DNA/*administration & dosage/chemistry Genetic Therapy Humans Lipids/administration & dosage/chemistry/*pharmacology Mice Peptides/administration & dosage/*pharmacology Plasmids Polyamines/*chemistry *Transfection Tumor Cells, Cultured

Cite this

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title = "Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids: J Control Release",
abstract = "DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.",
keywords = "Animals COS Cells Cell Line, Transformed DNA/*administration & dosage/chemistry Genetic Therapy Humans Lipids/administration & dosage/chemistry/*pharmacology Mice Peptides/administration & dosage/*pharmacology Plasmids Polyamines/*chemistry *Transfection Tumor Cells, Cultured",
author = "Pouton, {C. W.} and P. Lucas and Thomas, {B. J.} and Uduehi, {A. N.} and Milroy, {D. A.} and Moss, {S. H.}",
note = "M1 - 1-3 Pouton, C W Lucas, P Thomas, B J Uduehi, A N Milroy, D A Moss, S H eng Comparative Study NETHERLANDS 1998/09/19 J Control Release. 1998 Apr 30;53(1-3):289-99.",
year = "1998",
language = "English",
volume = "53",
pages = "289--299",
journal = "Journal of Controlled Release",
issn = "0168-3659",
publisher = "Elsevier",

}

Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids : J Control Release. / Pouton, C. W.; Lucas, P.; Thomas, B. J.; Uduehi, A. N.; Milroy, D. A.; Moss, S. H.

In: Journal of Controlled Release, Vol. 53, 1998, p. 289-299.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids

T2 - J Control Release

AU - Pouton, C. W.

AU - Lucas, P.

AU - Thomas, B. J.

AU - Uduehi, A. N.

AU - Milroy, D. A.

AU - Moss, S. H.

N1 - M1 - 1-3 Pouton, C W Lucas, P Thomas, B J Uduehi, A N Milroy, D A Moss, S H eng Comparative Study NETHERLANDS 1998/09/19 J Control Release. 1998 Apr 30;53(1-3):289-99.

PY - 1998

Y1 - 1998

N2 - DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.

AB - DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.

KW - Animals COS Cells Cell Line, Transformed DNA/administration & dosage/chemistry Genetic Therapy Humans Lipids/administration & dosage/chemistry/pharmacology Mice Peptides/administration & dosage/pharmacology Plasmids Polyamines/chemistry Transfection Tumor

M3 - Article

VL - 53

SP - 289

EP - 299

JO - Journal of Controlled Release

JF - Journal of Controlled Release

SN - 0168-3659

ER -