Vol. 297, Issue 3, 1129-1136, June 2001

In Vivo Delivery of Antisense Oligonucleotides in pH-Sensitive Liposomes Inhibits Lipopolysaccharide-Induced Production of Tumor Necrosis Factor- in Rats

Biddanda C. Ponnappa, Indranil Dey, Guang-chou Tu, Feng Zhou, Maria Aini, Qing-na Cao and Yedy Israel

Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (B.C.P., I.D., G-c.T., F.Z., M.A., Q-n.C., Y.I.); and Millenium Institute, University of Chile, Santiago, Chile (Y.I.)

Kupffer cells play an important role in the pathogenesis of liver diseases. During endotoxemia and alcohol-induced liver disease, tissue injury is preceded by an excessive release of cytokines by these macrophages. Tumor necrosis factor- (TNF-) is one of the key cytokines associated with liver injury. Pre-exposure of animals to TNF- antibodies has been shown to prevent macrophage-mediated liver injury in experimental animals. In this article, we describe a method to encapsulate in pH-sensitive liposomes and to deliver an antisense phosphorothioate oligonucleotide (TJU-2755) against TNF-. We describe the efficacy of this formulation in inhibiting endotoxin-mediated production of TNF-. The liposomes prepared were stable for over 4 weeks at pH 7.4, but readily released their contents when exposed to an acidic environment below pH 6, similar to the pH that exists in early endosomes. Male Sprague-Dawley rats were administered (i.v.) liposome-encapsulated TJU-2755 (1-2 mg/kg body wt.). Empty liposomes served as controls. Forty-eight hours postinjection, the animals were administered a single dose of lipopolysaccharide (50 µg/kg body wt.) and were sacrificed 90 min later. The TNF- produced by excised liver incubated ex vivo and the levels of plasma TNF- were determined. After a single administration of liposome-encapsulated antisense TJU-2755, a 30% reduction in TNF- produced by liver slices was observed. Two daily doses of the antisense oligonucleotide inhibited TNF- production by 50%. This was associated with a 65 to 70% reduction in plasma levels of TNF-, compared with controls. These results indicate that oligonucleotide TJU-2755 encapsulated in pH-sensitive liposomes can be used to effectively reduce endotoxin-mediated production of TNF- in macrophages in vivo and thus may be of value in attenuating or preventing macrophage-mediated liver injury.