Guanli Wu

In vitro studies of a new thrombus-specific ultrasound contrast agent

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.

Liposomal mr contrast agents

AbstractLiposomes are spheres composed of relatively non-toxic and biodegradable lipids which are useful for entrapping a variety of drugs, decreasing drug toxicity and targeting. For a number of years we have evaluated the use of liposomes as MR contrast agents. We have prepared and tested contrast agents entrapped within the internal aqueous space of liposomes as well as liposomes incorporating lipophilic contrast agents in the lipid bilayer. When chelates such as Gd-DTPA are entrapped within the internal aqueous space of lipid vesicles, delivery is primarily to the Kupffer cells and clearance is slow. Manganese ions entrapped within lipid vesicles cause more enhancement per micromole of paramagnetic ion than gadolinium. Lipophilic derivatives of manganese EDTA chelates when incorporated into liposomes confer the greatest hepatic enhancement per micromole of metal ion and have favorable clearance kinetics. An apparently hepatocyte specific liposomal MR contrast agent has been prepared based upon a lipop...

Copolymers and their use as contrast agents in magnetic resonance imaging and in other applications

Novel compounds having use as contrast agents in magnetic resonance imaging, as well as other uses, are described. The compounds are comprised of a copolymer which comprises at least two of a first monomer of the formula X1 -(CHR2 CHR2 --Y)n --(CHR2)m --CHR2 CHR2 --X2, wherein X1 and X2 are, independently, OH, NH2, NHR1, COOH, COOR1, SH, or Z, Y is O, NH, NR1, S, or CO, n is 0-10,000, m is 0 or 1, each Z is, independently, Cl, Br, or I, each R1 is, independently, a C1 -C20 substituted alkyl or cycloalkyl, and each R2 is, independently, H or OH, and at least one of a second monomer which is a polynitrilo chelating agent having at least two COOH, COOR1, or COZ groups, the first and second monomers being bound to one another to form a copolymer through an ester, amide, or carboxylic thioester linkage of at least one of the OH, NH2, NHR1, COOH, COOR1, SH or Z groups of the first monomer, and at least one of the COOH, COOR1, or COZ groups of the second monomer. Optionally, the copolymer may also include at least one of a third monomer which is a targeting agent or a targeting agent ligand having an OH, NH2, NHR1, COOH, COOR1, SH, or COZ group, wherein R1 is described as above, and wherein the third monomer is also bound with the first and second monomers to form a copolymer through an ester, amide, or carboxylic thioester linkage. For magnetic resonance imaging, for example, the copolymer may further comprise a paramagnetic ion chelated to the chelating agent.

Manganese-based liposomes. Comparative approaches.

RATIONALE AND OBJECTIVES.The purpose of this study was to further develop and compare manganese-based liposomes prepared by two different approaches wherein a manganese ion was entrapped within the internal aqueous space of the vesicles or into the bilayer surface via membrane bound complexes. METHODS.Small unilamellar liposomes (SUVs) were prepared entrapping manganese chloride. Alkylated complexes of manganese were prepared and also incorporated into SUVs. The two different manganese-based liposomes were compared for in-vitro relaxivity, stability, toxicity, and in-vivo imaging in rats with liver tumors. RESULTS.Liposomes entrapping manganese had a concentration-dependent change in relaxivity that was maximal at a several-fold molar excess of phospholipid relative to manganese ion. Liposomes bearing membrane-bound complexes showed relaxivity inversely proportional to vesicle size. In-vivo imaging showed greater and more specific hepatic enhancement with manganese liposomes bearing alkylated complexes than those entrapping manganese ion. CONCLUSIONS.Correlation effects likely explain the increased relaxivity of manganese entrapped in phospholipid vesicles. Greater efficacy, however, is afforded by liposomes bearing alkylated complexes.

Gas-filled lipid bilayers as ultrasound contrast agents

Unger E, Shen D, Fritz T, Kulik B, Lund P, Wu G-L, Yel-lowhair D, Ramaswami R, Matsunaga T. Gas-filled lipid bilayers as ultrasound contrast agents. Invest Radiol 1994;29: S134–S136.

Gas-filled liposomes as echocardiographic contrast agents in rabbits with myocardial infarcts.

RATIONALE AND OBJECTIVES The authors evaluated gas-filled liposomes as echocardiographic contrast agents in rabbits with myocardial infarcts. METHODS Ten rabbits underwent ligation of the left anterior descending coronary artery. Five animals underwent echocardiography before and after production of myocardial infarct (MI) and four animals had post-MI imaging only. In either case, images were obtained before and after injection of a single dose of 1 mL of gas-filed liposomes. Three radiologists blinded to clinical information reviewed the pre- and postcontrast images and assessed endomyocardial border definition, wall motion, confidence levels for normal versus abnormal wall motion and visualization of papillary muscle and mitral valve. RESULTS Postcontrast scans showed significant improvement (P < .05) in endomyocardial border definition, visualization of wall motion, papillary muscle and mitral valve as well as increased reader confidence level. CONCLUSIONS These results are encouraging and suggest that gas-filled liposomes may be a useful contrast agent for echocardiography.

Gadolinium-containing copolymeric chelates—a new potential MR contrast agent

Rationale and objectives: To develop and partially characterize a new class of potential blood pool magnetic resonance (MR) contrast agents.Methods: Various copolymeric chelates of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) were prepared with differing molecular weights of polyethylene glycol (PEG) or polypropylene glycol (PPG) as linkers between the monomeric chelate units. Gadolinium content of the polymeric chelates was determined by atomic absorption spectra. Relaxivity of the polymeric chelates was measured at 1.5 Tesla and compared with Gadolinium-DTPA. MR angiography (MRA) was performed in rabbits comparing Gd-DTPA with Gd-copolymers.Results: The gadolinium content of the copolymeric chelates ranged from 2.95 to 22.2% on weight basis. The molecular weight of the PEG linkers in the copolymers ranged from about 150 to about 3400. Ther1 (1/T1, mM−1 s−1) for Gd-DTPA=4.1. Ther1 values for the different Gd-containing polymers ranged from 3.8 to 5.8, with the lowestr1 for the polymer prepared with the lowest-molecular-weight complex. The higher-molecular-weight complexes resulted in moderately higher relaxivity. MRA with Gd-copolymers, in rabbits, showed markedly greater vascular enhancement relative to an equivalent dose of Gd-DTPA. Vascular enhancement was much more sustained with the copolymeric agent and confined to vascular space; i.e. no appreciable background tissue enhancement—a reflection of distribution into extravascular fluid space—was observed.Conclusions: Relative to Gd-DTPA monomers, PEG-containing Gd-DTPA polymeric complexes provided moderate increases in relaxivity but markedly greater efficacy during in vivo MRA. In vitro relaxivity studies of Gd-copolymers showed only an approximately 50% increase inr1 relaxivity compared with Gd-DTPA. The PEG-containing complexs lack of rigidity may have diminished the effect of spin diffusion on relaxation, thereby accounting for this modest increase. The greater efficacy of Gd-copolymers during in vivo MRA may reflect compartmentalization within the vascular space and possibly enhanced relaxation of the macromolecular copolymers in the blood. Gd-copolymers are promising agents that merit additional study.

Gene activation and gene delivery with ultrasound

Transfection experiments were performed in vitro in cell culture and in vivo in fish and mice using cationic liposomes with several different reporter genes. The effect of continuous‐wave 1‐MHz ultrasound on cell viability, cell permeability, and gene expression was assessed. RT‐PCR was used to monitor gene expression of cell repair genes in insonated cells. Acoustically active halocarbon‐containing liposomes were prepared for gene delivery. Ultrasound increased cell permeability and gene expression following liposomal transfection. RT‐PCR showed upregulation of repair of a number of cell repair genes after ultrasound exposure. The acoustically active gene delivery vehicles provided highly efficient transfection both with and without ultrasound in vitro and in vivo. Following IV administration of halocarbon‐containing liposomes/gene complexes, preferential gene expression was observed in the insonated tissue. Ultrasound has potentially useful applications for targeting and enhancing gene therapy.

Gas‐filled liposomes as ultrasound contrast agents for blood pool, thrombus‐specific and therapeutic applications

Our group has developed technology for stabilizing microbubbles with phospholipid coatings (Aerosomes®‐ImaRx Pharmaceutical Corp.). The first agent (MRX‐115) is based upon lipid‐coated microspheres filled with perfluoropropane gas and is in phase III clinical trials (October, 1997) for radiology and cardiology applications. Myocardial perfusion studies show the potential for the agent to detect ischemia in patients with myocardial infarcts. Targeting ligands have been covalently bound to lipids and incorporated into the stabilizing shells on the microbubbles. The first targeted agent planned for clinical trials is MRX‐408 (perfluorobutane gas, linear hexapeptide‐RGD analog). In vitro studies show enhanced visualization of thrombi, and in vivo studies in dogs with arterial and venous thrombi show selective enhancement, even in animals injected with heparin. In vitro studies have been performed to test sonothrombolysis comparing MRX‐115 to MRX‐408 with and without urokinase. The targeted contrast agent MRX‐...