title: Lipopolyplex ternary delivery systems incorporating C14 glycerol-based lipids.
abstract: The structure, biophysical properties and biological behavior of lipopolyplex ternary gene delivery vectors incorporating novel C14 glycerol based lipids of varying alkyl chain geometry (containing cis, trans or alkyne double bonds) have been studied in the presence and absence of a bifunctional targeting peptide designed to both condense DNA and confer integrin-specific targeting. In vitro transfection studies in breast cancer MDA-MB-231 cells revealed that ternary formulations of lipid:peptide:DNA (LPD) complexes prepared using the aforementioned lipids possessed highly synergistic transfection activity up to 2500-fold higher than their respective lipid:DNA (LD) or peptide:DNA (PD) counterparts. Furthermore, the small structural differences in the lipid alkyl chain geometries also resulted in pronounced differences in transfection within each type of formulation, whereby the trans lipids showed best activity when formulated as LD complexes, whereas the cis lipids were superior in LPD formulations. Confocal fluorescence internalization studies using labeled components of the formulations showed both the lipid and the DNA of LD complexes to be trapped in endocytic compartments, whereas in the case of LPD complexes, the DNA was clearly released from the endosomal compartments and, together with the peptide, internalized within the cell nucleus. Physicochemical characterization of the formulations carried out by light and neutron scattering, zeta potential measurement, and negative staining electron microscopy detected major structural differences between LD and LPD complexes. Gel electrophoresis assays additionally showed differences between the individual lipids tested in each type of formulation. In conclusion, the superior transfection of the trans lipids in the LD complexes was thought to be attributed to superior DNA binding caused by a more closely matched charge distribution of the more rigid, trans lipids with the DNA. In the case of the LPD complexes, the DNA was thought to be predominantly condensed by the cationic portion of the peptide forming a central core surrounded by a lipid bilayer from which the targeting sequence partially protrudes. The more fluid, cis lipids were thought to confer better activity in this formulation due to allowing more of the targeting peptide sequence to protrude.
authors.affiliation: Institute of Pharmaceutical Science, King's College London, Franklin-Wilkins Building, 150 Stamford Street, Waterloo Campus, London SE1 9NH, UK. Laila.kudsiova@kcl.ac.uk
authors.forename: Laila
authors.initials: L
authors.lastname: Kudsiova
authors.forename: Barbara
authors.initials: B
authors.lastname: Fridrich
authors.forename: Jimmy
authors.initials: J
authors.lastname: Ho
authors.forename: M Firouz Mohd
authors.initials: MF
authors.lastname: Mustapa
authors.forename: Frederick
authors.initials: F
authors.lastname: Campbell
authors.forename: Katharina
authors.initials: K
authors.lastname: Welser
authors.forename: Melanie
authors.initials: M
authors.lastname: Keppler
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
authors.forename: David J
authors.initials: DJ
authors.lastname: Barlow
authors.forename: Alethea B
authors.initials: AB
authors.lastname: Tabor
authors.forename: Helen C
authors.initials: HC
authors.lastname: Hailes
authors.forename: M Jayne
authors.initials: MJ
authors.lastname: Lawrence
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism / Q000473:pathology; D055598:Chemical Phenomena; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism / Q000473:pathology; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D018014:Gene Transfer Techniques; D005995:Glyceryl Ethers / Q000737:chemistry; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D008024:Ligands; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008560:Membrane Fluidity; D008968:Molecular Conformation; D009363:Neoplasm Proteins / Q000378:metabolism*; D010316:Particle Size; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: D005456:Fluorescent Dyes; D005995:Glyceryl Ethers; D039081:Integrin alpha5beta1; D008024:Ligands; D008055:Lipids; D009363:Neoplasm Proteins; D010455:Peptides; D004247:DNA