IN-VITRO TARGETING OF ACOUSTICALLY REFLECTIVE IMMUNOLIPOSOMES TO FIBRIN UNDER VARIOUS FLOW CONDITIONS

We have previously demonstrated the development of acoustically reflec tive liposomes as a novel ultrasound contrast agent, that can be conju gated to antibodies for site specific acoustic enhancement of patholog ically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less th an one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-f ibrinogen antibodies, the liposomes have the ability to attach to fibr in coated surfaces and thrombi in vitro as demonstrated by scanning el ectron microscopy and ultrasound imaging (Demos et al,, 1997a), Anti-f ibrinogen liposomes were shown to attach to fibrous atheroma and throm bi in a Yucatan miniswine model of induced atherosclerosis whereas lip osomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM -1) were demonstrated to target early stage atherosclerotic plaques (D emos et al., 1997b). The purpose of this study is to evaluate the bind ing characteristics of anti-fibrinogen liposomes in vitro under a vari ety of flow conditions in order to optimize the targeting ability of t he immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applie d to fibrin coated filter paper and placed in a flow circuit under con trolled flow conditions. Flow conditions were altered to study the eff ects of different shear stresses, temperature, plasma flow and pulsati le flow on the retention of liposomes to fibrin after set time periods . The retention of liposomes conjugated to polyclonal and monoclonal a ntibodies as well as Fab fragments made from monoclonal antibodies wer e compared. The binding characteristics of liposomes conjugated to dif ferent quantities of polyclonal antibodies were analyzed. At physiolog ical shear stress of 1.5 N/m(2) (15 dynes/cm(2)) over 70% of the lipos omes remained attached to fibrin after two hours. A smaller and greate r portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effec t on liposomal retention whereas pulsatile flow resulted in a slight r eduction in binding. Monoclonal antibodies showed a slight trend of re duced retention to fibrin over time as compared with polyclonal antibo dies and Fab fragments. The quantity of antibody conjugated to the lip osomes plays a role in liposome retention as demonstrated by the reduc tion in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions like ly to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after inj ection.