Analytical methods for the characterization of cationic lipid-nucleic acid complexes.
Five analytical assays are described that provide a platform for systematically evaluating the effect of formulation variables on the physical properties of cationic lipid-DNA complexes (lipoplexes). The assays are for (i) lipid recovery, (ii) total DNA, (iii) free DNA, (iv) nuclease sensitivity, and (v) physical stability by filtration. Lipid recovery was determined by measuring lipid primary amino groups labeled with the fluorescamine reagent in the presence of the detergent Zwittergent. Zwittergent was effective at disrupting lipoplexes, making the primary amine accessible to the fluorescamine reagent. Total DNA was determined with the PicoGreen reagent, also in the presence of Zwittergent. The PicoGreen assay in the absence of Zwittergent gave the percentage of the total DNA that was not complexed with cationic lipid. The results of this assay for free DNA agreed well with the amount of DNA that could be separated from complexes by centrifugation as well as with the amount of DNA that was accessible to DNase I digestion. Monitoring the lipid and DNA recoveries after filtration through polycarbonate membranes provided a quantitative method for assessing changes in lipoplex physical characteristics. Together, these assays provide a convenient high-throughput approach to assess physical properties of lipoplexes, allowing systematic evaluation of different formulations.
Ferrari
M E
ME
Vical Inc., San Diego, CA 92121, USA.
Nguyen
C M
CM
Zelphati
O
O
Tsai
Y
Y
Felgner
P L
PL
D002412:Cations; D004247:DNA / Q000032:analysis*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004337:Drug Carriers; D005450:Fluorescamine; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D008055:Lipids; D008081:Liposomes; D008567:Membranes, Artificial; D009930:Organic Chemicals; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics
D002412:Cations; D004337:Drug Carriers; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D008567:Membranes, Artificial; D009930:Organic Chemicals; C099476:PicoGreen; D005450:Fluorescamine; D004247:DNA; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase