Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1beta (IL-1beta). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

We investigated the therapeutic effects of two different versions of A␤ 1-15 (16) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetrapalmitoylated amyloid 1-15 peptide (palmA␤ 1-15), or with amyloid 1-16 peptide (PEG-A␤ 1-16) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmA␤ 1-15 liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-A␤ 1-16 had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmA␤ 1-15, whereas those elicited by PEG-A␤ 1-16 were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly ␤-sheet conformation of palmA␤1-15 and random coil of PEG-A␤ 1-16. We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a ''conformational'' disease, implying that antibodies against amyloid sequences in the ␤-sheet conformation are preferred as potential therapeutic agents.Alzheimer's disease ͉ ␤-amyloid ͉ vaccine C linical manifestations of Alzheimer's disease (AD) include progressive memory loss, cognitive impairment, confusion, and personality changes. The major neuropathological changes in the brains of AD patients are senile plaques and neurofibrillar tangles causing progressive neuronal dysfunction. These pathological alterations are likely causally involved in eventual neuronal death, particularly in brain regions related to memory and cognition (1-4). Senile plaques are formed predominantly by the ␤-amyloid peptide A␤ 1-42 that is coiled and ␣-helical in its soluble form but, upon conformational transition, aggregates into ␤-sheeted multimers. The monomeric A␤ peptide is a physiological metabolite of the large amyloid precursor protein (APP, 695-770 aa) that undergoes processing by several sequential proteolytic steps (5). The A␤ 1-42 aggregates are proposed to play the key role in the pathogenesis of AD (6). In transgenic animals that overexpress mutant human APP, anti-A␤-specific antibodies decreased the A␤ burden and improved memory after either passive (7-11) or active (12-18) immunization.We previously demonstrated that i.p. inoculation of tetrapalmitoylated A␤1-16 reconstituted in liposomes to transgenic NORBA mice elicited significant titers of anti-A␤ antibodies, that solubilized amyloid fibers in vitro and pancreatic A␤ plaques in vivo (19). To circumvent T cell-mediated immune responses known to be causatively involved in the adverse events of meningoencephalitis of AD patients immunized with A␤1-42 (20-22), the A␤1-16 and 1-15 sequences were used for immunization of APPxPS1 double-transgenic mice (23) because strong T cell epitopes are located more toward the C-te...

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