Induction of luciferase activity under the control of an hsp70 promoter using high-intensity focused ultrasound: combination of bioluminescence and MRI imaging in three different tumour models.

The in vivo temporal changes of luciferase activity were investigated under the control of an hsp70 promoter in three tumour models after the application of different intensities of high-intensity focused ultrasound (HIFU). Three cell lines, SCCVII, NIH3T3 and M21 were stably transfected with a plasmid containing the hsp70 promoter and luciferase reporter gene, and tumours were subcutaneously initiated into mice. At a size of 1300 ± 234 mm(3), the tumours were exposed to five intensities of continuous HIFU (802-1401-2157-3067-4133 W/cm(2)) for 20 sec. Bioluminescence and MR imaging were performed to assess luciferase activity and signal intensity changes in the tissue. The MRI scan protocol was pre- and post-contrast T1-wt-SE, T2-wt-FSE, DCE-MRI, diffusion-wt STEAM sequence, T2 relaxation time determination obtained on a 1.5-T GE MRI scanner. The NIH3T3 tumours showed the highest luciferase activity of 328.1 ± 7.1 fold at 24 h at a HIFU intensity of 3067 W/cm(2), the M21 tumours of 3.2 ± 0.6 fold 8 hours and the SCCVII tumours 2.9 ± 0.9 fold 4 hours post-HIFU at 2157 W/cm(2). The greatest increase in T2 signal intensity and T2 relaxation time of 20.7 ± 3.4% was seen in the SCCVII tumours. The highest contrast medium uptake of 10.1 ± 1.1% was noted in the M21 tumours, and 14.8 ± 1.9% in the SCCVII tumours. In all tumours, a significant increase in the diffusion coefficient was seen with increased HIFU intensity, the highest of which was 40.3 ± 4.1% in the SCCVII tumours. The three tumour cell lines stably transfected with the hsp70/luciferase gene showed differential luciferase activity, which peaked at different times after the application of HIFU and was dependant on tumour type and HIFU energy deposition.

Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging.

Angiogenesis, the formation of new blood vessels, is a requirement for malignant tumor growth and metastasis. In the absence of angiogenesis, local tumor expansion is suppressed at a few millimeters and cells lack routes for distant hematogenous spread. Clinical studies have demonstrated that the degree of angiogenesis is correlated with the malignant potential of several cancers, including breast cancer and malignant melanoma. Moreover, the expression of a specific angiogenesis marker, the endothelial integrin alphaVbeta3, has been shown to correlate with tumor grade. However, studies of tumor angiogenesis such as these have generally relied on invasive procedures, adequate tissue sampling and meticulous estimation of histologic microvessel density. In the present report, we describe a novel approach to detecting angiogenesis in vivo using magnetic resonance imaging (MRI) and a paramagnetic contrast agent targeted to endothelial alphaVbeta3 via the LM609 monoclonal antibody. This approach provided enhanced and detailed imaging of rabbit carcinomas by directly targeting paramagnetic agents to the angiogenic vasculature. In addition, angiogenic 'hot spots' not seen by standard MRI were detected. Our strategy for MR imaging of alphaVbeta3 thus represents a non-invasive means to assess the growth and malignant phenotype of tumors.

In vivo target-specific delivery of macromolecular agents with MR-guided focused ultrasound.

Magnetic resonance (MR) images in rabbit muscle were used to select a target region for application of pulsed-focused ultrasound. Liposomes encapsulating gadopentetate dimeglumine, an MR-detectable model representing pharmaceutical agents, were then injected intravenously. Focal enhancement on T1-weighted images, representing gadolinium-liposome accumulation, occurred in the pulsed-focused-ultrasound focal zone. Therefore, MR-guided pulsed-focused ultrasound targeted the delivery of macromolecular pharmaceutical agents within tissues.

Paramagnetic polymerized liposomes as new recirculating MR contrast agents.

We describe a well-tolerated blood pool contrast agent with extended recirculatory half-life based on paramagnetic polymerized liposomes (PPLs). PPLs were constructed from a new type of polymerizable lipid molecule that has a derivative of gadopentetate dimeglumine as the hydrophilic head group and diacetylene groups in the hydrophobic acyl chains, which cross-link when irradiated with ultraviolet (UV) light. Biodistribution, blood pool half-life, and MR image enhancement were determined for PPLs composed of 10% of the gadopentetate dimeglumine lipid and 90% of ditricosadiynoyl tricosadiynayl phosphatidylcholine (DAPC) at a dose of 0.015 mmol Gd+3/kg in rats. In T1-weighed MR images (TR/TE = 400/18 msec), an average signal enhancement of 34% in the kidneys and 20% in the liver was observed, which persisted for at least 90 minutes after administration of the PPLs. Biodistribution studies using radiolabeled PPLs confirmed that 80% of the injected dose remained in the blood pool after 2 hours. The half-life of elimination from the blood pool was 19 hours. The preparation was well tolerated in rats and produced similar MR contrast enhancement of the blood pool as produced by other liposome contrast agents. However, the half-life of PPL elimination from the blood pool was prolonged relative to other liposome systems.

Direct colorimetric detection of a receptor-ligand interaction by a polymerized bilayer assembly.

Detection of receptor-ligand interactions is generally accomplished by indirect assays such as enzyme-linked immunosorbent assay. A direct colorimetric detection method based on a polydiacetylene bilayer assembled on glass microscope slides has been developed. The bilayer is composed of a self-assembled monolayer of octadecylsilane and a Langmuir-Blodgett monolayer of polydiacetylene. The polydiacetylene layer is functionalized with an analog of sialic acid, the receptor-specific ligand for the influenza virus hemagglutinin. The sialic acid ligand serves as a molecular recognition element and the conjugated polymer backbone signals binding at the surface by a chromatic transition. The color transition is readily visible to the naked eye as a blue to red color change and can be quantified by visible absorption spectroscopy. Direct colorimetric detection by polydiacetylene films offers new possibilities for diagnostic applications and screening for new drug candidates or binding ligands.

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study.

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.