Vectors based on reducible polycations facilitate intracellular release of nucleic acids

Martin L. Read, K. Helen Bremner, David Oupicky, Nicola K. Green, Peter F. Searle, Leonard W. Seymour

Research output: Contribution to journalArticle

137 Citations (Scopus)

Abstract

Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L-lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys-Lys10-Cys and used to condense nucleic acids. The stability of RPC-based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC-3 and B16-F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride) (DOTAP). A CMV-driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC-based delivery vectors. Results: A 187-fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non-reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9%), LNCaP (55.2 ± 6.7%) and PC-3 (66.1 ± 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20-75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). Conclusions: These results demonstrate that lipid-mediated activation of RPC-based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity.

Original languageEnglish (US)
Pages (from-to)232-245
Number of pages14
JournalJournal of Gene Medicine
Volume5
Issue number3
DOIs
StatePublished - Mar 1 2003

Fingerprint

Nucleic Acids
Nitroreductases
Genes
Transfection
Lipids
DNA
Chloroquine
Prodrugs
Green Fluorescent Proteins
polycations
Luciferases
Reporter Genes
Inhibitory Concentration 50
Lysine
Electrophoresis
Chlorides
Flow Cytometry
Plasmids
Therapeutics
Molecular Weight

Keywords

  • Cancer
  • DNA
  • Gene therapy
  • Lipid
  • Nitroreductase
  • Poly(L-lysine)

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

Cite this

Vectors based on reducible polycations facilitate intracellular release of nucleic acids. / Read, Martin L.; Bremner, K. Helen; Oupicky, David; Green, Nicola K.; Searle, Peter F.; Seymour, Leonard W.

In: Journal of Gene Medicine, Vol. 5, No. 3, 01.03.2003, p. 232-245.

Research output: Contribution to journalArticle

Read, Martin L. ; Bremner, K. Helen ; Oupicky, David ; Green, Nicola K. ; Searle, Peter F. ; Seymour, Leonard W. / Vectors based on reducible polycations facilitate intracellular release of nucleic acids. In: Journal of Gene Medicine. 2003 ; Vol. 5, No. 3. pp. 232-245.
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abstract = "Background: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L-lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. Methods: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys-Lys10-Cys and used to condense nucleic acids. The stability of RPC-based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC-3 and B16-F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride) (DOTAP). A CMV-driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC-based delivery vectors. Results: A 187-fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non-reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 ± 7.9{\%}), LNCaP (55.2 ± 6.7{\%}) and PC-3 (66.1 ± 3.7{\%}) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC50 values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20-75{\%} of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5{\%} of cells). Conclusions: These results demonstrate that lipid-mediated activation of RPC-based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity.",
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