Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) o r 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) are widely used in gene t herapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures fo r DNA quantification from DNA complexes as well as their stability in the p resence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degreesC in the presence of DNase I and sera from the three species. Both high pH and heparin were able to r elease DNA from its complexes. Naked DNA formed aggregates with serum prote ins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA fro m digestion by serum DNase, and the DNA digesting ability of serum was: mou se>rat>human. The DNA from the complexes was resistant to degradation by DN ase I, although a low proportion of DNA from the complexes was partially di gested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DN A complexes were stable in the presence of all sera. Heparin and high pH re lease DNA from its complexes. The order of DNA degradation is: mouse>rat>hu man, but DOTAP and PEI avoid degradation of DNA by serum compounds. (C) 200 1 Elsevier Science B.V. All rights reserved.