Examination of the biophysical interaction between plasmid DNA and the polycations, polylysine and polyornithine, as a basis for their differential gene transfection in-vitro.
The impetus to develop non-viral gene delivery vectors has led to examination of synthetic polycationic polymers as plasmid DNA (pDNA) condensing agents. Previous reports have highlighted superiority (up to x 10-fold) in the in-vitro transfection of pDNA complexes formed by poly-(L)-ornithine (PLO) compared to those formed with poly-(L)-lysine (PLL). The apparent basis for this consistent superiority of PLO complexes remains to be established. This comparative study investigates whether physico chemical differences in the supramolecular properties of polycation:pDNA complexes provide a basis for their observed differential gene transfection. Specifically, particle size distribution and zeta potential of the above complexes formulated over a wide range of polycation:pDNA ratios were found to be consistent with a condensed (150-200 nm) cationic ( + 30-40 mV) system but not influenced by the type of cationic polymer used. A spectrofluorimetric EtBr exclusion assay showed that polycation:pDNA complexes display different pDNA condensation behaviour, with PLO able to condense pDNA at a lower polycation mass compared to both polylysine isomers, and form complexes that were more resistant to disruption following challenge with anionic counter species, i.e. poly-(L)-aspartic acid and the glycosaminoglycan molecule. heparin. We conclude that particle size and surface potential as gross supramolecular properties of these complexes do not represent, at least in a non-biological system, the basis for the differential transfection behaviour observed between these condensing polymers. However, differences in the ability of the polylysine and polyornithine polymers to interact with pDNA and to stabilise the polymer-pDNA assembly could have profound effects upon the cellular and sub-cellular biological processing of pDNA molecules and contribute to the disparity in cell transfection efficiency observed between these complexes.
Ramsay
E
E
Pharmaceutical Cell Biology, Welsh School of Pharmacy, Cardiff University, UK.
Hadgraft
J
J
Birchall
J
J
Gumbleton
M
M
D000230:Adenocarcinoma / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D019556:COS Cells; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium; D005456:Fluorescent Dyes; D006801:Humans; D010316:Particle Size; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry*; D014162:Transfection; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D010455:Peptides; C008973:polyornithine; D011107:Polylysine; D004247:DNA; D004996:Ethidium