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

Autores de IIS La Fe

• Inés Moret Tatay

  Autor

• Marta Benet Giménez

  Autor

• Francisco José Dasí Fernández

  Autor

• Salvador F Aliño Pellicer

  Autor

Participantes ajenos a IIS La Fe

• Peris, JE
• Guillem, VM
• Revert, F
• Crespo, A

Abstract

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence 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 release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds. (C) 2001 Elsevier Science B.V. All rights reserved.

Keywords

• polyplex; lipoplex; DNA complex; DNA digestion; DNA stability