Sonothrombolysis: the contribution of stable and inertial cavitation to clot lysis.

Microbubble-mediated sonothrombolysis (STL) is a remarkable approach to vascular occlusion therapy. However, STL remains a complex process with multiple interactions between clot, ultrasound (US), microbubbles (MB) and thrombolytic drug. The aim of this study was to evaluate the ability of combining US and MB to degrade fibrin and, more specifically, to assess the roles of both stable (SC) and inertial (IC) cavitation. Human blood clots containing radiolabeled fibrin were exposed to different combinations of recombinant tissue plasminogen activator (rtPA), US (1 MHz) and phospholipid MB. Three acoustic pressures were tested: 200, 350 and 1,300 kPa (peak-negative pressure). Clot lysis was assessed by diameter loss and release of radioactive fibrin degradation products. The combination rtPA + US + MB clearly revealed that IC (1,300 kPa) was able to enhance fibrin degradation significantly (66.3 ± 1.8%) compared with rtPA alone (51.7 ± 2.0%, p < 0.001). However, SC failed to enhance fibrin degradation at an acoustic pressure of 200 kPa. At 350 kPa, a synergistic effect between rtPA and US + MB was observed with an absolute increase of 6% compared to rtPA alone (p < 0.001). Conversely, without rtPA, the combination of US + MB was unable to degrade the fibrin network (0.3 ± 0.1%, p > 0.05 vs. control), but induced a distinct loss of red blood cells throughout the entire thickness of the clot, implying that MB were able to penetrate and cavitate inside the clot.

Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa - characterization and in vitro testing.

Abciximab-grafted ultrasound sensitive microbubbles were developed for the diagnosis of stroke. The antibody fragment abciximab, which binds to the GP IIb/IIIa and alpha v beta 3 receptors expressed by activated platelets, was chosen because most ischemic strokes are due to arterial thrombi that are mainly composed of platelets. The abciximab antibody fragment was activated by reduction of the disulfide bond for grafting on the microbubbles. The suspension was freeze-dried after the grafting was performed directly on the formed microbubbles. Quantification of the amounts of abciximab present on the surface of the microbubbles was assessed semi-quantitatively by flow cytometry, and quantitatively using radio-labeled abciximab. A protocol for human and rat platelets deposition and fixation was implemented and the expression of the GP IIb/IIIa receptor was validated by immunostaining. The abciximab-grafted microbubbles showed high static and dynamic binding to fixed platelets. Detection by ultrasonography of microbubbles bound on white and red clots gave higher signals compared to Sono Vue microbubbles.

Gray-scale liver enhancement in VX2 tumor-bearing rabbits using BR14, a new ultrasonographic contrast agent.

RATIONALE AND OBJECTIVES: We evaluated the potential of BR14, a novel gas-based echo contrast agent, for gray-scale liver imaging and VX2 tumor detection/delineation in the conventional and harmonic imaging modes. METHODS: A single dose (0.2 mL/kg) of BR14 was administered to eight VX2 tumor-bearing rabbits. Imaging was performed with an ATL-HDI 3000. The B-mode gray levels were analyzed by videodensitometry in areas selected in the liver tissue, tumors, aorta, and the portal vein from frames recorded up to 1 hour after injection. The tumors were further assessed using subjective scores just before and after contrast injection, as well as 15 minutes later in both conventional gray-scale and harmonic imaging modes. RESULTS: Gray-scale enhancement of the liver tissue was detectable soon after BR14 injection and remained at a high level even after clearance of the agent from the blood stream. On precontrast, of 42 observed tumors, 4 were perfectly detected (4/42). After injection of BR14, the number rose to 10/20 in the vascular phase and 12/20 in the delayed phase. In harmonic imaging, the corresponding numbers were even higher, 17/22 in the vascular phase and 18/22 in the delayed phase. The number of tumors perfectly delineated increased after BR14 from 1/42 to 3/20 in the vascular phase and 5/20 in the delayed phase. In the harmonic mode, 9 of 22 tumors were perfectly delineated in the vascular phase and 10 of 22 in the delayed phase. CONCLUSIONS: BR14 is a promising new agent for the study of tissue perfusion and in particular for liver imaging. It shows not only strong, but also persistent gray scale enhancement of the parenchyma but not of the tumors. The increased conspicuousness allows considerable improvements in tumor detection and tumor delineation in conventional imaging and even more so in harmonic imaging.

Polymeric microballoons as ultrasound contrast agents. Physical and ultrasonic properties compared with sonicated albumin.

Air-filled polymeric microballoons were prepared with number-mean diameters of approximately 3 microns, volume-mean diameters of approximately 5 microns, and narrow particle-size distributions (standard deviation [SD] = 1.2 microns in number and SD = 2.0 microns in volume). More than 99% of the particles were below 8 microns. These particles were found to be highly echogenic for ultrasound, showing backscatter coefficients at 7.5 MHz, similar to the ones obtained with sonicated albumin microspheres. However, at 2.25 MHz, microballoons were less echogenic than albumin microspheres. These results are consistent with ultrasound attenuation measurements, which showed a maximum at 8 to 9 MHz for the microballoons compared with a reported value of 3.5 to 4.5 MHz for albumin microbubbles. Polymeric microballoons were found to be stable in plasma or under applied pressure as evidenced by unchanged particle concentration and echogenicity. Albumin microspheres were particularly unstable to applied pressure (150 mm Hg) and showed a rapid decrease in both particle counts and echogenicity.

Novel solid-phase synthesis of thiol-terminated-poly(alpha-amino acid)-drug conjugate.

A new method using a controlled pore glass solid support for the preparation of a thiol-terminated-polymerdrug, notably poly-L-glutamate-daunomycin having a terminal thiol group, is described. The method consists of first polymerizing an ester-protected glutamic acid onto an amino-disulfide functionalized controlled pore glass support. The ester protecting group is then removed, freeing the gamma-carboxyl groups of the grafted polymer which then allows it to react with daunomycin. Finally, the disulfide bond linking the conjugated polymer-drug to the solid support is broken by thiolysis, thus releasing the desired product. The final product consists of only polymer-drug conjugates with terminal thiol groups (global yield 26%). This novel method is much simpler and more elegant than more conventional preparation methods requiring solution phase techniques.