Ashwin Nagaraj

In vivo targeting of acoustically reflective liposomes for intravascular and transvascular ultrasonic enhancement.

OBJECTIVES The purpose of this study was to target acoustically reflective liposomes to atherosclerotic plaques in vivo for ultrasound image enhancement. BACKGROUND We have previously demonstrated the development of acoustically reflective liposomes that can be conjugated for site-specific acoustic enhancement. This study evaluates the ability of liposomes coupled to antibodies specific for different components of atherosclerotic plaques and thrombi to target and enhance ultrasonic images in vivo. METHODS Liposomes were prepared with phospholipids and cholesterol using a dehydration/ rehydration method. Antibodies were thiolated for liposome conjugation with N-succinimidyl 3-(2-pyridyldithio) propionate resulting in a thioether linkage between the protein and the phospholipid. Liposomes were conjugated to antifibrinogen or anti-intercellular adhesion molecule-1 (anti-ICAM-1). In a Yucatan miniswine model, atherosclerosis was developed by crush injury of one carotid and one femoral artery and ingestion of a hypercholesterolemic diet. After full plaque development the arteries were imaged (20-MHz intravascular ultrasound catheter and 7.5-MHz transvascular linear probe) after injection of saline, unconjugated liposomes and antibody conjugated liposomes. RESULTS Conjugated liposomes retained their acoustically reflective properties and provided ultrasonic image enhancement of their targeted structures. Liposomes conjugated to antifibrinogen attached to thrombi and fibrous portions of the atheroma, whereas liposomes conjugated to anti-ICAM-1 attached to early atheroma. CONCLUSIONS Our data demonstrate that this novel acoustic agent can provide varying targeting with different antibodies with retention of intravascular and transvascular acoustic properties.

Left Ventricular Thrombus Enhancement After Intravenous Injection of Echogenic Immunoliposomes Studies in a New Experimental Model

Background—Targeted echogenic immunoliposomes (ELIPs) for ultrasound enhancement of atheroma components have been developed. To date, ELIP delivery has been intra-arterial. To determine whether ELIPs can be given intravenously with enhancement of systemic structures, a left ventricular thrombus (LVT) model was developed. Methods and Results—In 6 animals plus 1 dose-ranging animal, the apical coronary arteries were ligated, and an LVT was produced by injecting Hemaseel fibrin adhesive through the apical myocardium. The thrombus was imaged epicardially and transthoracically at 0, 1, 5, and 10 minutes after anti-fibrinogen ELIP injections. The dose of ELIPs was varied. PBS and unconjugated ELIPs were controls. The apical thrombi were easily reproduced and clearly visible with epicardial and transthoracic ultrasound. Enhancement occurred with 2 mg anti-fibrinogen ELIPs and increased with dose. With 8 mg ELIPs, enhancement was different from control within 10 minutes (P <0.05). Rhodamine-labeled anti-fibrinogen ELIPs were seen with fluorescence microscopy of the LVT. Blinded viewing detected enhancement by 10 minutes in all animals after anti-fibrinogen ELIPs. Conclusions—We describe an easily reproducible LVT model. Anti-fibrinogen ELIPs delivered intravenously, as a single-step process, rapidly enhance the ultrasound image of a systemic target. This allows for future development of ELIPs as a targeted ultrasound contrast agent.

Physical correlates of the ultrasonic reflectivity of lipid dispersions suitable as diagnostic contrast agents

The objective of this study was to determine the physical basis of ultrasound (US) reflectivity of echogenic lipid dispersions. These dispersions were made using a process previously described involving sonication of the lipid in water, addition of mannitol, freezing, lyophilization and rehydration. The component lipids were egg phosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol and cholesterol in a molar ratio of 69:8:8:15. Ultrasound reflectivity, as assessed with a 20-MHz intravascular US catheter and analyzed using computer-assisted videodensitometry, was found to be sensitive to variations in ambient pressure; echogenicity was greatly reduced by exposure to 0.5 atm vacuum for 10 s or 1.5 atm pressure for 10 s. Pressure changes of the magnitude that obtain in the arterial circulation had little effect on echogenicity. Vacuum treatment resulted in the release of approximately 100 microL air from a standard preparation of 10 mg lipid in 1 mL. Maximum ultrasonic reflectivity required the presence of 0.1-0.2 mol/L mannitol during the lyophilization step; mere addition of mannitol to the lipid lyophilized in the absence of mannitol produced nonreflective dispersions. Inclusion of sodium phosphate or other electrolytes reduced echogenicity. High echogenicity was associated with the presence of large-volume freeze-dried cakes and fusion of liposomes (which led to a 10 times increase in liposome diameters) during freezing before lyophilization. Lyophilization from water led to liposome fusion, but the cakes were small and US reflectivity was weak. Lyophilization from solutions of cryoprotectants such as trehalose produced large cakes, but little liposome fusion and also led to weak US reflectivity. Filtration through defined pores revealed that approximately 50% of the echogenicity originated from particles smaller than 1 microm and about 2/3 from particles smaller than 3 microm. These results indicate that lyophilization from 0.2 mol/L mannitol solution generates a disrupted array of lipid bilayers that, upon rehydration, fuse and trap small amounts of air distributed among liposome-size particles.

Pulsatile flow simulation in arterial vascular segments with intravascular ultrasound images

Previous studies have indicated a correlation between local variation in wall shear stress in arterial blood flow and atheroma development. The purpose of this study was to analyze the hemodynamics in vascular segments from morphologically realistic three-dimensional (3D) reconstruction, and to compare the computed wall shear stress in a compliant vascular segment model and the corresponding rigid walled model. Cross-sectional images of the segments of femoral and carotid arteries in five Yucatan miniswine were obtained using intravascular ultrasound (IVUS) imaging and the segment geometry was reconstructed at different times in the cardiac cycle. The actual measured wall motion from the reconstruction was employed to specify the moving boundaries for simulation of physiological distensibility. Velocity profiles and wall shear stress were computed using unsteady computational fluid dynamics analysis. The computed results revealed that the maximum wall shear stress in the compliant model was approximately 4-17 percent less than that in the rigid model if the wall motion is larger than 10 percent. Our analysis demonstrates that inaccuracies due to inflow velocity profile can be minimized by the extension of the model upstream. The phase angle between the diameter change and wall shear is affected by the local changes in geometry of the arteries. These simulations can be potentially used to analyze the effect of regional wall motion changes in the presence of atherosclerotic lesions on the local fluid dynamics and to correlate the same with subsequent growth of the lesions.

Three-dimensional finite element analysis of residual stress in arteries.

Calculation of residual stress in arteries, using the analytical approach has been quite valuable in our understanding of its critical role in vascular mechanics. Stresses are calculated at the central section of an infinitely long tube by imposing a constant axial stretch while deforming the artery from the stress-free state to its unloaded state. However, segments used to perform opening-angle measurements have finite lengths. Further, the stress-free artery configuration is assumed to be circular. Experiments show that they are slightly noncircular. The numerical approach to residual stress calculation can allow us to study both these issues. Using 3D cylindrical geometries and an isotropic material model, we investigated how segment length can affect residual stress calculations and identified the appropriate segment length for experiments. Further, we recorded and used the true noncircular stress-free state of an artery segment, computed the residual stress distribution, and compared it to that from a similar, but circular segment. Our findings suggest that segment length must be ten times the wall thickness for it to be “long” enough. We also found that the circularity assumption may be a reasonable approximation for typical arteries.

Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery.

Tiukinhoy SD, Mahowald ME, Shively VP, et al. Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery. Invest Radiol 2000;35:732–738. RATIONALE AND OBJECTIVES.Echogenic antibody-conjugated anionic liposomes have been developed that allow directed tissue targeting and acoustic enhancement. These are not efficient for gene delivery. A cationic formulation that allows directed gene delivery while retaining acoustic properties may provide more efficient transfection. METHODS.Cationic liposomes were prepared and acoustic reflectivity was determined. Anti-fibrinogen–conjugated liposomes were laid on fibrin-coated slides and adherence was quantified using fluorescence techniques. Liposomes were combined with a reporter gene and plated on cell cultures. Human umbilical vein endothelial cells were stimulated to upregulate intercellular adhesion molecule-1 (ICAM-1) and were treated with anti–ICAM-1–conjugated liposomes, and gene expression was quantified. RESULTS.Cationic liposomes retained their acoustic reflectivity and demonstrated specific adherence to fibrin under flow conditions. Significant transfection of human umbilical vein endothelial cells was demonstrated, with higher gene expression seen with specific antibody-conjugated liposomes. CONCLUSIONS.Novel acoustic cationic liposomes have been developed that can be antibody conjugated for site-specific adherence and directed cell modification. This presents exciting potential for a vector that allows tissue enhancement and targeted gene delivery.

A method for in-vivo analysis for regional arterial wall material property alterations with atherosclerosis: preliminary results.

Atherosclerosis is a diffuse arterial disease developing over many years and resulting in a complicated three-dimensional arterial morphology. The arterial wall material properties have been demonstrated to show regional alterations with atheroma development and growth. We present a mechanical analysis of diseased arterial segments reconstructed from intravascular ultrasound images in order to quantitatively identify regional alterations in the elastic constants with atherosclerotic lesions. We employ a finite element and a displacement sensitivity analysis to divide the arterial segment into regions with different material properties and use an optimization algorithm to identify the elastic constants in these regions. The results with regional variations identified with this method correlated qualitatively with the extent and location of atherosclerotic lesions identified by visual inspection of the affected arteries. The optimized elastic modulus in regions affected by early atherosclerotic lesions ranged from 90.9 to 93.0 kPa where as the corresponding magnitudes in normal arterial segments ranged from 97.9 to 101.0 kPa. This method can be potentially employed to identify the extent and location of atherosclerotic lesions in a systematic analysis and may potentially be used for the early detection of lesion growth.

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

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.

A physiologic flow chamber model to define intravascular ultrasound enhancement of fibrin using echogenic liposomes.

Hamilton A, Rabbat M, Jain P, et al. A physiologic flow chamber model to define intravascular ultrasound enhancement of fibrin using echogenic liposomes. Invest Radiol 2002;37:215–221. rationale and objectives.Echogenic immunoliposomes (ELIP) for enhancement of vasoactive and pathologic components of endothelium and atherosclerosis have been developed. A physiologic flow chamber model has been developed to define intravascular ultrasound enhancement of a fibrin interface. methods. A IgG ELIP was used, which nonspecifically associated with fibrin, to demonstrate the suitability of this model. With varying doses of IgG ELIP, the fibrin wells were imaged at 1, 2, 4, 6, and 9 minutes. results.IgG ELIP enhanced fibrin versus saline (P < 0.005) was visible at 1 minute, lasted at least 9 minutes, and at 6 minutes the interface enhanced 27% ± 6.1%. Enhancement was caused by increases in interface thickness and brightness. Enhancement increased with dose up to 8 mg lipid (n = 4 per time point). conclusion.This model can quantitate the components of IVUS enhancement of an interface produced by ELIP. This model may allow for further development and understanding of ELIP and other targeted ultrasound contrast agents.

Gadofluorine-Enhanced Magnetic Resonance Imaging of Carotid Atherosclerosis in Yucatan Miniswine

Objective:The aim of this study was to determine whether gadofluorine, a paramagnetic magnetic resonance imaging (MRI) contrast agent, selectively enhances carotid atherosclerotic plaques in Yucatan miniswine. Methods:Atherosclerotic plaques were induced in the left carotid arteries (LCA) of Yucatan miniswine (n = 3) by balloon denudation and high cholesterol diet. T1-weighted MRI was performed before and 24 hours after gadofluorine injection (at a dose of 100 &mgr;mol/kg) to assess the enhancement of the balloon-injured LCA wall relative to healthy, uninjured right carotid artery (RCA) wall. Histopathology was performed to verify the presence and composition of the atherosclerotic plaques imaged with MRI. Results:Gadofluorine was found to enhance LCA atherosclerotic lesions relative to RCA wall by 21% (P < 0.025) 24 hours after contrast injection. Enhancement of healthy LCA wall relative to healthy RCA wall was not observed. Conclusion:Gadofluorine selectively enhances carotid atherosclerotic plaques in Yucatan miniswine. Gadofluorine appears to be a promising MR contrast agent for detection of atherosclerotic plaques in vivo.