Delivery of negatively charged liposomes into the atherosclerotic plaque of apolipoprotein E-deficient mouse aortic tissue.

Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE(-/-)) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE(-)(/)(-) mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.

Delivery of negatively charged liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits.

Liposomes have been used as imaging and therapeutic agents in various tissues but only infrequently in the cardiovascular system. We prepared a liposome to target atheromas in a Watanabe heritable hyperlipidemic (WHHL) rabbit model. Liposomes labeled with rhodamine and nanogold were injected intra-arterially into the descending thoracic aortas of WHHL rabbits. The arterial segments of interest were perfusion-fixed and evaluated with immunohistochemistry, light microscopy, and electron microscopy. Deconvolution microscopy showed that rhodamine label was concentrated in the plaque shoulder regions of advanced-stage atheromas; however, rhodamine label was not found in adjacent, non-atherosclerotic aorta. Transmission electron microscopy revealed liposome remnants and the highest concentration of nanogold label in lipid-laden areas of atheromas. Liposomes were concentrated in areas of lipoprotein-associated phospholipase A(2) expression. We conclude that modified liposomes can be delivered to the shoulder regions of advanced atheromas in WHHL rabbits and may be useful therapeutically for targeting metabolically active plaque.