Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin.

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF-SPLPs and untargeted SPLPs labeled with [3H]cholesteryloleyl-ether were injected into rats. About 87% of the LF-SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h. Fifty-two percent of the LF-SPLPs were taken up by hepatocytes, while non-parenchymal liver cells accounted for 16% of the uptake. Despite the efficient targeting of LF-SPLPs to hepatocytes and their capacity to transfect HepG2 and COS-7 cells in vitro, expression of a reporter gene was not detected in vivo. Overall, covalent coupling of LF to SPLPs leads to massive delivery in hepatocytes after systemic administration. However, these LF-SPLPs are not able to transfect these cells in vivo.

Cell-specific targeting of lipid-based carriers for ODN and DNA.

It is well recognized that there is an urgent need for non-toxic systemically applicable vectors for biologically active nucleotides to fully exploit the current potential of molecular medicine in gene therapy. Cell-specific targeting of non-viral lipid-based carriers for ODN and DNA is a prerequisite to attain the concentration of nucleic acids required for therapeutic efficacy in the target tissue. In this review we will address the most promising approaches to selective targeting of liposomal nucleic acid carriers in vivo. In addition, the routes of entry and intracellular processing of these carrier systems are discussed as well as physiological factors potentially interfering with the biological and/or therapeutic activity of their nucleotide pay-load.

Optimized targeting of polyethylene glycol-stabilized anti-intercellular adhesion molecule 1 oligonucleotide/lipid particles to liver sinusoidal endothelial cells.

We prepared polyethylene glycol (PEG)-stabilized antisense oligonucleotide (ODN)/lipid particles from a lipid mixture including the positively charged amphiphile 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and anti-intercellular adhesion molecule 1 (ICAM-1) antisense ODN by an extrusion method in the presence of 40% ethanol. These particles were targeted to scavenger receptors on liver endothelial cells by means of covalently coupled polyanionized albumin. Two types of such targeted particles were prepared, one with the albumin coupled to a maleimide group attached to the particle's lipid bilayer and the other with the protein coupled to a maleimide group attached at the distal end of added bilayer-anchored PEG chains. Upon intravenous injection, the ODN particles with bilayer-coupled albumin were cleared from the blood circulation at the same low rate as untargeted particles (<5% in 30 min). By contrast, the distal-end coupled particles were very rapidly cleared from the blood and preferentially taken up by the endothelial cells of the hepatic sinusoid (55% of injected dose after 30 min). Despite this substantial endothelial targeting, no consistent inhibition of ICAM-1 expression could be demonstrated in this cell type, either in vivo or in vitro. However, in J774 cells that also express scavenger receptors and ICAM-1, significant down-regulation of ICAM-1 mRNA was achieved with distal-end targeted lipid particles, as determined with real-time RT-PCR. It is concluded that massive delivery of ODN to cell types that express scavenger receptors can be achieved if lipid particles are provided with negatively charged albumin distally attached to bilayer anchored PEG chains.

Stabilized lipid coated lipoplexes for the delivery of antisense oligonucleotides to liver endothelial cells in vitro and in vivo.

We report on the preparation and in vivo/in vitro disposition of antisense ODN encapsulating coated cationic lipoplexes (CCLs), prepared by a procedure essentially developed by Stuart and Allen (Stuart, D.D. and Allen, T.M. (2000) "A new liposomal formulation for antisense oligodeoxynucleotides with small size, high incorporation efficiency and good stability", Biochim. Biophys. Acta 1463, pp. 219-229). The behavior of untargeted CCLs was compared with CCLs that were targeted to scavenger receptors on liver endothelial cells by covalent coupling of the poly-anion aconitylated human serum albumin (Aco-HSA) to the particle surface. By means of cryo transmission electron microscopy (cryo-TEM) particles of high electron density could be distinguished from electron-translucent particles, representing high and low ODN encapsulation, respectively. The two populations were separated by sucrose density gradient centrifugation. Upon injection into rats, the untargeted particles showed long circulating properties with a half-life of >10 h. These untargeted CCLs barely bound to liver endothelial cells in vitro while Aco-HSA CCLs massively and specifically interacted with scavenger receptors on these cells. With J774 cells, a macrophage cell line expressing scavenger receptors, downregulation of ICAM-1 mRNA levels was achieved when the ODN was specifically delivered by Aco-HSA targeted CCLs.

Massive and selective delivery of lipid-coated cationic lipoplexes of oligonucleotides targeted in vivo to hepatic endothelial cells.

PURPOSE: Previously we reported on massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (AcoHSA) by liver sinusoidal endothelial cells (EC) in vivo. In the present work we applied this principle for the in vivo delivery of antisense oligonucleotides (ODN) to these cells. METHODS: Anti ICAM-1 ODN was complexed with the cationic lipid DOTAP and the complex was coated by an excess of neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was coupled to the coated cationic lipoplexes (CCLs). Plasma disappearance, organ and intrahepatic distribution of Aco-HSA modified CCLs were determined in rats, using [3H]-cholesteryl oleyl ether and 32P-labeled ODN as markers. RESULTS: The Aco-HSA coupled CCLs were <160 nm in size, contained 1.03+/-0.35 nmol ODN and 54+/-18 microg Aco-HSA per micromol total lipid. These CCLs were rapidly eliminated from plasma, about 60% the injected dose of 3H- or 32P-label being recovered in the liver after 30 min. Within the liver, the EC accounted for two thirds of total liver uptake. Control non-targeted CCLs were eliminated very slowly: after 30 min still >90% of the particles was in the blood. CONCLUSIONS: Our results demonstrate efficient targeting of antisense ODN to EC in vivo, employing plasma-stable coated cationic lipoplexes, surface modified with negatively charged albumin. 40% of the injected ODN was delivered to the target cells within 30 min.