Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides

Antisense therapy for the treatment of bacterial infections is a very attractive alternative to overcome drug resistance problems. However, the penetration of antisense oligonucleotides into bacterial cells is a major huddle that has delayed research and application in this field. In the first part of this study, we defined efficient conditions to encapsulate plasmid DNA and antisense oligonucleotides in a fluid negatively charged liposome. Subsequently, we evaluated the potential of liposome-encapsulated antisense oligonucleotides to penetrate the bacterial outer membrane and to inhibit gene expression in bacteria. It was found that 48.9 +/- 12% and 43.5 +/- 4% of the purified plasmid DNA and antisense oligonucleotides were respectively encapsulated in the liposomes. Using fluorescence-activated cell sorting analysis, it was shown, after subtraction of the fluorescence values due to the aggregation phenomenon measured at 4 degreesC, that about 57% of bacterial cells had integrated the encapsulated antisense oligonucleotides whereas values for free antisenses were negligible. The uptake of the encapsulated anti-lacZ antisense oligonucleotides resulted in a 42% reduction of beta -galactosidase compared to 9% and 6% for the encapsulated mismatch antisense oligonucleotides and the free antisense oligonucleotides respectively. This work shows that it is possible to encapsulate relatively large quantities of negatively charged molecules in negative fluid liposomes and suggests that fluid liposomes could be used to deliver nucleic acids in bacteria to inhibit essential bacterial genes. (C) 2001 Elsevier Science B.V. All rights reserved.