(600f) Immuno-Targeting of Non Ionic Surfantant Vesicles

Normal administration of drugs or therapeutic agents does not allow for concentrated accumulation of drug at diseased sites due to an essentially uniform distribution of drug throughout the blood circulation. In order to adequately treat affected sites using traditional systemic administration high dosages of drug must be delivered. This not only increases costs, but in some instances creates toxic side effects as normal tissues and organs are exposed to unneeded pharmaceuticals. Myriad drug delivery systems have been developed to maintain greater control of therapeutic levels and focused concentration of drugs at target locations through either passive or active delivery. Vesicular drug delivery has been studied widely as a means to increase efficacy and reduce systemic toxicity in tumor targeting and cancer therapies, and in cardiovascular imaging and plaque targeting. Niosomes are self assembled vesicles composed primarily of synthetic surfactants and cholesterol. They are analogous in structure to the more widely studied liposomes formed from biologically derived phospholipids. Drug delivery specifically targeted to vascular inflammation has numerous therapeutic cardiovascular applications because inflammation has been shown to initiate and propagate pathologies such as atherosclerotic plaques and coronary artery disease. Using vesicular delivery targeted to specific actors in the disease process, for example inflamed endothelial cells or monocytes taken up in plaque progression could interrupt disease progression while also providing drug therapy. Cellular adhesion molecules (CAMs) are glycoproteins expressed by inflamed endothelium, initiating the cellular response to injury, which provide a potential therapeutic target. Interruption of the inflammatory process has been studied using the cell surface adhesion molecule CD44 blocked by antibody IM7. Dramatic suppression of inflammation was achieved through the highly selective binding of the antibody-antigen couple. We have synthesized niosomes by thin-film hydration methods using a mixture of biocompatible sorbitan ester surfactants and cholesterol. We conjugated them to IM7 antibodies through a novel polyoxyethylene sorbitan monostearate (Tween 61)-cyanuric chloride (CC) linker incorporated in the vesicle membrane. Tween 61 was functionalized prior to niosome synthesis by activation the hydroxyl groups on the ends of the polyethylene oxide (PEO) chains. In the presence of diisopropyl ethyl amine (DIPEA), Tween 61 and cyanuric chloride are incubated in a nitrogen environment. The cyanuric chloride undergoes nucleophilic substitution binding to the terminal hydroxyl group of a PEO chain on the Tween 61 molecule. The molar ratio of Tween:CC:DIPEA was 1:0.8:2. The resulting functionalized Tween-CC solution added to the surfactants and lipids in chloroform prior to forming a thin film. Vesicles are composed of a 1.0:1.0:0.1 molar ratio of surfactant:cholesterol:DCP at a concentrations between 0.0144 and 0.144 M. Niosomes were separated from unencapsulated dye and unformed lipids suing gel exclusion chromatography by passing vesicles through a Sephadex G50 column. Entrapment of fluorescent dye used as a drug model was measured using both UV absorption during the purification step, and fluorescence intensity of disrupted vesicle suspensions relative to a standard curve. Formation of vesicles was assessed by light and fluorescent microscopy. Mean particle size and distribution of formed vesicles was determined by light scattering and obscuration techniques. Once formed, purified niosome solutions were adjusted to pH 8.8 and were incubated with monoclonal anti-CD44 IM7 antibodies. At pH 8.8 the binding of a terminal carboxyl group on the antibody is preferred over that of an amino group at the antigen binding terminus. The resulting ?immuno-niosomes' bind selectively and specifically to CD44 antigen targets on synoviocytes, our initial cell model, at antibody concentrations far lower than advocated by traditional immuno-liposome literature. Bovine aortic endothelial cell models express CD44 and show enhanced binding of immuno-niosomes when activated with pro-inflammatory cytokines such as interleukin 1 beta. A contrast and fluorescence overlay micrograph of IM7 conjugated immuno-niosomes has shown adherence to the surfaces of CD44 expressed cells.