Biophysical characterization of an integrin-targeted non-viral vector.
BACKGROUND
The formulation of polycationic complexes containing plasmid DNA for optimal transfection in vitro and in vivo for DNA vaccination, gene therapy and other applications continues to be a major research goal. Here we present new data on the biophysical properties of an integrin-targeted plasmid DNA (LID) formulation.


METHODS
Two plasmids (D), pEGFP (4.7 kb) and pCI-luc (5.7 kb), were mixed with a synthetic a5b1 integrin-targeted peptide (I), [K]16 GACRRETAWACG, in the presence of a cationic liposome (L), Lipofectin, composed of DOTMA and DOPE to form LID complexes. The physical properties of the complexes were measured using a variety of techniques including dynamic light scattering and fluorescence methods. The in vitro gene delivery to neuroblastoma cells with LID complexes was also assessed.


RESULTS
We demonstrate the effects of complex size and charge ratio on in vitro transfection of mouse (Neuro-2A) and human (IMR-32) neuroblastoma cells. We report a significant increase in the level of luciferase and green fluorescent protein expression when transfection is performed in buffers of physiological ionic strength and hypothesise that the enhancement in transfection is caused by an increase in the size of the complexes observed during mixing and maturation.


CONCLUSIONS
Cell transfection is also shown to be dependent on complex size and charge ratio, with large complexes prepared at charge ratios above 4.0 demonstrating efficient transfection.
Lee
Li K
LK
Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, London, England.
Siapati
Elena K
EK
Jenkins
R Gisli
RG
McAnulty
Robin J
RJ
Hart
Stephen L
SL
Shamlou
Parviz A
PA
D000818:Animals; D001703:Biophysics / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D016023:Integrins / Q000235:genetics*; D008027:Light; D051379:Mice; D009930:Organic Chemicals; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D012542:Scattering, Radiation; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D016023:Integrins; D009930:Organic Chemicals; D010714:Phosphatidylethanolamines; C099476:PicoGreen; C020888:1,2-dielaidoylphosphatidylethanolamine