In Vitro Targeting of Acoustically Reflective Immunoliposomes to Fibrin Under Various Flow Conditions

Abstract

We have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b). The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Flow conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2 (15 dynes/cm2) over 70% of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction 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 likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.

Intravascular ultrasound molecular imaging of atheroma components in vivo

“…We have performed previous experiments to demonstrate the link between Ab-labeled liposomes and fibrin using scanning electron microscopy (3). We have demonstrated in a flow chamber that 1) anti-fibrinogen conjugated liposomes retain their adherence to fibrin under physiologic sheer/flow (17) and 2) ELIPs retain their echogenic properties after 9 min of physiologic sheer (20). These studies provide evidence for ELIP targeting under physiologic conditions while retaining echogenic characteristics.…”

“…Binding was specific, as it was Ͼ90% inhibited by the addition of soluble fibrinogen during Ab-ELIP incubation. Avidity and K assoc have been reported (15,17,18). We have demonstrated that the ELIP particle size after conjugation remained Ͻ1 m and, in most cases, remained between 500 and 800 nm (3).…”

Improvement of drug safety by the use of lipid-based nanocarriers

“…Either the whole antibody or the variable Fab fragment of the mAb can be chemically conjugated to the surface of lipid nanocarriers to bring about increased cellular uptake at the targeted disease sites. We have previously conjugated antihuman fibrinogen and intercellular adhesion molecule (ICAM-1) antibodies onto acoustically reflective liposomes composed of phospholipids and cholesterol [110]. When injected into miniswine with plaque formation induced in the carotid and iliofemoral arteries, antifibrinogen conjugated liposomes were found to attach to thrombi and atheroma while anti-ICAM-1 conjugated liposomes attached to early atheroma, providing ultrasonic image enhancement of the targeted structures compared to unconjugated liposomes.…”

“…Monoclonal antibody (mAb) is another class of commonly used targeting moiety in drug delivery [23,109,110]. Either the whole antibody or the variable Fab fragment of the mAb can be chemically conjugated to the surface of lipid nanocarriers to bring about increased cellular uptake at the targeted disease sites.…”

Left Ventricular Thrombus Enhancement After Intravenous Injection of Echogenic Immunoliposomes

“…ELIPs are composed of 4 primary lipids: PC, MPB-PE, PG, and cholesterol, and are made by a lyophilization process. 7,8,15 We have been able to quantitate the ELIP intravascular ultrasound enhancement of fibrin in vitro 9,18,19 and qualitatively demonstrate acoustic image enhancement of atheroma with anti-fibrinogen ELIPs delivered locally and imaged by transvascular and intravascular ultrasound. 10 Tiukinhoy et al 14 showed comparable echogenicity per particle of ELIPs compared with Albunex and other liposomal preparations.…”