Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine).
DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.
Parker
Alan L
AL
CRC Institute for Cancer Studies, University of Birmingham, B15 2TA, United Kingdom.
Oupicky
David
D
Dash
Philip R
PR
Seymour
Leonard W
LW
D004587:Electrophoresis, Agar Gel; D004996:Ethidium / Q000378:metabolism; D005453:Fluorescence; D036103:Nanotechnology; D010957:Plasmids / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry*
D011107:Polylysine; D004996:Ethidium