Microbubble-endothelial cell interactions as a basis for assessing endothelial function.

Clinical signs and symptoms of coronary artery disease are predated in decades by endothelial dysfunction, an aberration in the vascular lining that permits the development and propagation of atherosclerotic lesions and vasomotor dysfunction in the arterial circulation. These ultimately lead to acute and chronic coronary ischemic syndromes. Other pathophysiologic scenarios encountered in clinical cardiology practice, such as cardiac transplant rejection and the period following coronary angioplasty or cardiac surgery, also are associated with endothelial dysfunction.

Polymer surfaces derivatized with poly(vinyl-N-hexylpyridinium) kill airborne and waterborne bacteria.

A facile methodology has been developed for covalently derivatizing the surfaces of common materials with a designed antibacterial polycation, poly(vinyl-N-pyridinium bromide), wherein the first, key step involves surface coating with a nanolayer of silica. Various commercial synthetic polymers derivatized in this manner become bactericidal-they kill up to 99% of deposited, from either an aerosol or an aqueous suspension, Gram-positive and Gram-negative bacteria on contact.

Noninvasive imaging of myocardial reperfusion injury using leukocyte-targeted contrast echocardiography.

Background: We hypothesized that myocardial contrast echocardiography (MCE) with leukocyte-targeted microbubbles could temporally and spatially characterize the severity of postischemic myocardial inflammation.

Methods And Results: In 9 open-chest dogs, either the left anterior descending or left circumflex coronary artery was occluded for 90 minutes (n=6), while the remaining dogs served as non-ischemic controls. During occlusion, MCE was performed to determine the risk area (RA) and regions supplied by collateral flow.

Asymmetric sulfoxidations mediated by alpha-chymotrypsin.

The oxidation of aryl alkyl sulfides with H(2)O(2) in aqueous solution is a reasonably facile reaction producing racemic sulfoxides. We show that in the presence of the hydrolytic enzyme alpha-chymotrypsin such a sulfoxidation is accelerated and, more importantly, becomes stereoselective. With phenyl isobutyl sulfide as a model, the chymotrypsin-mediated, highly asymmetric oxidation is shown to occur in the hydrophobic binding pocket of the enzyme active site.

Binding of hydrophobic hydroxamic acids enhances peroxidase's stereoselectivity in nonaqueous sulfoxidations.

Horseradish peroxidase exhibits a meager stereoselectivity (E) in the sulfoxidation of thioanisole (1a) in 99.8% (v/v) methanol. The E value, however, is greatly enhanced when the enzyme forms a complex with benzohydroxamic acid (2a).

Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin.

Background: Routine methods capable of assessing tissue inflammation noninvasively are currently not available. We hypothesized that tissue retention of microbubbles targeted to the endothelial cell adhesion molecule P-selectin would provide a means to assess inflammation with ultrasound imaging.

Methods And Results: Phospholipid microbubbles targeted to P-selectin (MB(p)) were created by conjugating monoclonal antibodies against murine P-selectin to the lipid shell.

Inhalation delivery of proteins from ethanol suspensions.

To circumvent inherent problems associated with pulmonary administration of aqueous-solution and dry-powder protein drugs, inhalation delivery of proteins from their suspensions in absolute ethanol was explored both in vitro and in vivo. Protein suspensions in ethanol of up to 9% (wt/vol) were readily aerosolized with a commercial compressor nebulizer. Experiments with enzymic proteins revealed that nebulization caused no detectable loss of catalytic activity; furthermore, enzyme suspensions in anhydrous ethanol retained their full catalytic activity for at least 3 weeks at room temperature.

Designing surfaces that kill bacteria on contact.

Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e.

p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups.

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds.

Improving biomaterial properties of collagen films by chemical modification.

Films of bovine collagen were chemically modified with the goal of improving their biomaterial properties. The modified films were investigated with respect to their affinity to fibroblast and endothelial cells, as well as their antibacterial properties tested by adhesion of Staphylococcus aureus. Modifications that only change the net charge of collagen, such as acetylation, succinylation, and treatment with glutaraldehyde (all increase the negative charge), and amination with ethylenediamine (EDA), N,N-dimethyl-EDA (DMEDA), or butylamine (all increase the positive charge), did not dramatically alter the mammalian cell attachment to the film.

Improving enzymes by using them in organic solvents.

The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than their natural aqueous reaction media. Studies over the past 15 years have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments enzymes can catalyse reactions impossible in water, become more stable, and exhibit new behaviour such as 'molecular memory'. Of particular importance has been the discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, can be markedly affected, and sometimes even inverted, by the solvent.

Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes.

Background: Lipid microbubbles used for perfusion imaging with ultrasound are retained within inflamed tissue because of complement-mediated attachment to leukocytes within venules. We hypothesized that incorporation of phosphatidylserine (PS) into the microbubble shell may enhance these interactions by amplifying complement activation and thereby allow ultrasound imaging of inflammation.

Methods And Results: In 6 mice, intravital microscopy of tissue necrosis factor-alpha-treated cremaster muscle was performed to assess the microvascular behavior of fluorescein-labeled lipid microbubbles with and without PS in the shell.

Peroxidase-catalyzed asymmetric sulfoxidation in organic solvents versus in water.

Peroxidase-catalyzed asymmetric sulfoxidations, while synthetically attractive, suffer from relatively low reaction rates due to poor substrate solubilities in water and from appreciable spontaneous oxidation of substrates (especially aryl alkyl sulfides) with H(2)O(2). In this work, we found that both of these shortcomings could be alleviated by switching from aqueous solutions to certain nearly anhydrous (99.7%) organic solvents as sulfoxidation reaction media.

Broadband time-domain reflectometry measurement of attenuation and phase velocity in highly attenuating suspensions with application to the ultrasound contrast medium Albunex.

We describe a technique for broadband measurements of the attenuation coefficient and phase velocity of highly attenuating liquid suspensions. To validate the technique we apply it to the ultrasound contrast agent Albunex at concentrations ranging from 0.69 x 10(6) particles/mL to 364 x 10(6) particles/mL.

Improving lipase enantioselectivity in organic solvents by forming substrate salts with chiral agents.

We recently demonstrated (J Am Chem Soc 121:3334-3340, 1999) that enzymatic enantioselectivity in organic solvents can be markedly enhanced by temporarily enlarging the substrate via salt formation. In the present study, this approach was expanded by finding that, in addition to its size, the stereochemistry of the counterion can greatly affect the enantioselectivity enhancement. For example, the enantioselectivity [E = (k(cat)/K(M))(S)/(k(cat)/K(M))(R)] of crystalline Pseudomonas cepacia lipase in the propanolysis of phenylalanine methyl ester (PheOMe) in anhydrous acetonitrile was found to be 5.

Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging.

Gas-filled microbubbles, with the size of several micrometres, are strong scatterers of ultrasound waves used in diagnostic imaging. Application of these microbubbles as ultrasound contrast materials is discussed, in view of the design of materials capable of selectively targeting the diseased tissues/organs. Methods of preparation, mechanisms of action, biodistribution and stability in vitro and in vivo are reviewed.

Calorimetric evidence for a native-like conformation of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH (from pH 2.5 to 10.0) and then dissolved in water and in anhydrous glycerol, has been studied by high-sensitivity differential scanning microcalorimetry over the temperature range from 10 to 150 degrees C.

Structural stability of DNA in nonaqueous solvents.

One of the defining physicochemical features of DNA in aqueous solution is its ability to maintain a double-helical structure and for this structure to undergo a cooperative, heat-induced denaturation (melting). Herein we show that a 21-mer synthetic DNA can form and maintain such a duplex structure not only in water but even in 99% glycerol; moreover, this double-helical structure reversibly and cooperatively melts in that solvent, with a T(m) value of some 30 degrees lower than in water. Two much larger, natural DNAs, from calf thymus and salmon testes, exhibit similar behavior in glycerol.

Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres.

Purpose: In the past decade, biodegradable polymers have become the materials of choice for a variety of biomaterials applications. In particular, poly(lactic-co-glycolic acid) (PLGA) microspheres have been extensively studied for controlled-release drug delivery. However, degradation of the polymer generates acidic monomers, and acidification of the inner polymer environment is a central issue in the development of these devices for drug delivery.

Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes.

Background: Albumin microbubbles that are used for contrast echocardiography persist within the myocardial microcirculation after ischemia/reperfusion (I-R). The mechanism responsible for this phenomenon is unknown.

Methods And Results: Intravital microscopy of the microcirculation of exteriorized cremaster muscle was performed in 12 wild-type mice during intravenous injections of fluorescein-labeled microbubbles composed of albumin, anionic lipids, or cationic lipids.