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Echocardiographic evaluation for the recognition of intravascular and left atrial appendage thrombus remains a difficult problem. A thrombus-specific ultrasonographic contrast agent has the potential for an alternative approach for their delineation. The aim of this study was to investigate the usefulness of thrombus-specific contrast agent MRX-408A1 for the detection of acute experimentally created intravascular and intracardiac thrombus. In the first study, we created inferior vena cava thrombus in 9 dogs. With the use of fundamental 2-dimensional echocardiography imaging, we recorded images of the inferior vena cava thrombus at baseline (n = 9), with the thrombus-specific contrast agent MRX-408A1 (n = 9), and with nonspecific contrast agent MRX-113 (n = 6). In the second study, we created a left atrial appendage thrombus in 8 dogs. We imaged left atrial appendage thrombus at baseline and during MRX-113 and MRX-408A1 infusion. Thrombus was successfully created in all dogs in study 1 and in 6 of 8 dogs in study 2. MRX-408A1 produced a visually apparent increase in ultrasonographic contrast enhancement of the thrombus in all cases in which thrombus was found on autopsy. In both studies, MRX-408A1 increased the videointensity of the thrombus significantly compared with baseline images and images obtained during MRX-113 infusion. The size of the visually detectable thrombus on the image was also significantly larger during MRX-408A1 infusion than at baseline and during MRX-113 infusion. These data provide in vivo demonstration of the efficacy of a thrombus-specific contrast agent, MRX-408A1, in the detection of acute intravascular and intracardiac thrombus. It has the potential to improve the diagnostic accuracy of ultrasonography for the detection of acute thrombi at various cardiovascular sites in the clinical setting.

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RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.

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RATIONALE AND OBJECTIVES: Paclitaxel-carrying lipospheres (MRX-552) were developed and evaluated as a new ultrasound contrast agent for chemotherapeutic drug delivery. METHODS: Paclitaxel was suspended in soybean oil and added to an aqueous suspension of phospholipids in vials. The headspace of the vials was replaced with perfluorobutane gas; the vials were sealed, and they were agitated at 4200 rpm on a shaking device. The resulting lipospheres containing paclitaxel were studied for concentration, size, acute toxicity in mice, and acoustic activity and drug release with ultrasound. Lipospheres containing sudan black dye were produced to demonstrate the acoustically active liposphere (AAL)-ultrasound release concept. RESULTS: Acoustically active lipospheres containing paclitaxel had a mean particle count of approximately 1 x 10(9) particles per mL and a mean size of 2.9 microns. Acute toxicity studies in mice showed a 10-fold reduction in toxicity for paclitaxel in AALs compared with free paclitaxel. The AALs reflected ultrasound as a contrast agent. Increasing amounts of ultrasound energy selectively ruptured the AALs and released the paclitaxel. CONCLUSIONS: Acoustically active lipospheres represent a new class of acoustically active drug delivery vehicles. Future studies will assess efficacy of AALs for ultrasound-mediated drug delivery.

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Ultrasound is used as a primary diagnostic technique for the detection of deep venous thrombosis. The purpose of this study is to describe the development of a new thrombus-specific ultrasound contrast agent: The linear hexapeptide (lysine-glutamine-alanine-glycine-aspartate-valine) was synthesized and coupled to a lipid moiety. The targeted lipid was then incorporated into the lipid blend for the contrast agent Aerosomes (ImaRx, Tucson, AZ, USA). The lipid blend was used to entrap perfluorobutane microbubbles. The microbubbles were sized and studied in vitro for acoustic stability, binding to blood clot, and ultrasound enhancement in vitro of blood clot. The results showed the mean size of the specific ultrasound contrast agent (MRX-408) was about 2.0 microm. The microbubbles appeared as smooth spherical structures. Microscopy showed that the targeted bubbles bound to blood clot whereas control, nontargeted bubbles did not bind to blood clot. In vitro acoustic study showed similar stability of the microbubbles compared with control microbubbles. The targeted microbubbles enhanced blood clot in vitro whereas nontargeted microbubbles did not enhance clot. Thus this promising new thrombus-specific ultrasound contrast agent could potentially improve detection of thrombosis by ultrasound and might be useful for distinguishing between new and old thrombosis. In vivo studies are in progress.

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RATIONALE AND OBJECTIVES: Cationic liposomes are under development as delivery agents for gene therapy. The authors studied the effect of ultrasound on gene expression in cell cultures during liposomal transfection experiments. METHODS: Cationic liposomes of dipalmitoylethylphosphocholine and dioleoylphosphatidylethanolamine were used to transfect cultured HeLa, NIH/3T3, and C127I cells with the chloramphenicol acetyl transferase (CAT) gene. A cell viability assay was performed on cultured HeLa cells that were exposed to varying durations (5 seconds or 30 seconds) and intensities of 1 MHz continuous-wave therapeutic ultrasound after transfection, and gene expression was measured 48 hours later. RESULTS: Cells survived 30 seconds or less at a power level of 0.5 watts/cm2 but died when exposed for 60 seconds or longer. Exposures of 5 seconds and 30 seconds of ultrasound resulted in significant increases in gene expression in all three cell types tested in this experiment. CONCLUSIONS: Relatively low levels of ultrasound energy can be used to enhance gene expression from liposomal transfection. Additional experiments are needed to optimize this process and clarify the mechanisms involved.

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The purpose of this study was to determine if the sonographic contrast agent MRX-115 could improve the accuracy of Doppler ultrasonographic diagnosis of testicular ischemia. Testicular duplex ultrasonography was performed on six dogs before and after surgical ligation of the testicular artery, and before and after intravenous injection of MRX-115. Six radiologists blinded to experimental conditions rated the testicular blood flow. Receiver operating characteristics curves for conditions with and without administration of contrast agent were compared using Student's t-test for paired observations. Statistically significant differences were found both for the tests viewed individually (P = 0.003) and for the testes in comparison to the contralateral side (P = 0.0001). MRX-115 therefore is found to improve duplex sonographic evaluation of testicular ischemia.

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Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.

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