ANALYTICAL METHODS FOR THE CHARACTERIZATION OF CATIONIC LIPID NUCLEIC-ACID COMPLEXES

Five analytical assays are described that provide a platform for syste matically evaluating the effect of formulation variables on the physic al properties of cationic lipid-DNA complexes (lipoplexes). The assays are for (i) lipid recovery, (ii) total DNA, (iii) free DNA, (iv) nucl ease sensitivity, and (v) physical stability by filtration, Lipid reco very was determined by measuring lipid primary amino groups labeled wi th the fluorescamine reagent in the presence of the detergent Zwitterg ent., Zwittergent was effective at disrupting lipoplexes, making the p rimary amine accessible to the fluorescamine reagent, Total DNA was de termined with the PicoGreen reagent, also in the presence of Zwitterge nt, The PicoGreen assay in the absence of Zwittergent gave the percent age of the total DNA that was not complexed with cationic lipid, The r esults of this assay for free DNA agreed well with the amount of DNA t hat could be separated from complexes by centrifugation as well as wit h the amount of DNA that was accessible to DNase I digestion. Monitori ng the lipid and DNA recoveries after filtration through polycarbonate membranes provided a quantitative method for assessing changes in lip oplex physical characteristics. Together, these assays provide a conve nient high-throughput approach to assess physical properties of lipopl exes, allowing systematic evaluation of different formulations.