Aba Priev

Hydration of polyethylene glycol-grafted liposomes.

This study aimed to characterize the effect of polyethylene glycol of 2000 molecular weight (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodynamic stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodynamic and hydrational characterization. Using a differential scanning calorimetry technique we showed that each molecule of PEG2000 binds 136 +/- 4 molecules of water. For PEG2000 covalently attached to the lipid molecules organized in micelles, the water binding increases to 210 +/- 6 water molecules. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concentrations over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calculated from ultrasound velocity and density) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concentration up to approximately 7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodynamic stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concentrations, compressibility and partial volume of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five additional water molecules per lipid molecule for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.

Formation of complement-activating particles in aqueous solutions of Taxol: possible role in hypersensitivity reactions

We reported earlier that the anticancer drug paclitaxel (Taxol) activated the complement (C) system in human serum in vitro, raising the possibility that C activation might play a role in the ill-understood hypersensitivity reactions (HSRs) to this drug [J. Natl. Cancer Inst. 90 (1998) 300]. In pursuing the mechanism of C activation by Taxol, the present study provided evidence that dilution of the injection concentrate in aqueous solvents led to the formation of micelles and needle-like structures, both of which caused C activation in vitro. Micelles were formed mainly from Cremophor EL (CrEL), the nonionic emulsifier vehicle of paclitaxel, whose level in Taxol infusion exceeded its critical micelle concentration by at least 400-fold. CrEL micelles were shown by quasi-elastic light scattering and cryo-transmission electron microscopy (cryo-TEM) to be spherical with diameters in the 8-22 nm range; however, de novo formation of 50-300 nm microdroplets following incubation with human plasma suggested further fundamental structural transformation in blood. The needle-like structures extended to the multimicron range and were shown by electron diffraction to be crystalline paclitaxel. Taxol-induced C activation was manifested in varying rises of serum C3a-desarg, iC3b and SC5b-9. The causal role of CrEL micelles in C activation was demonstrated by the fact that filtration of aqueous solutions of Taxol or pure CrEL via 30-kDa cutoff filters eliminated, while the filter retentate restored C activation. C activation by Taxol was also inhibited by 10 mg/ml human immunoglobulin (IVIG). If proven clinically, HSRs to Taxol may represent a hitherto vaguely classified adverse drug reaction recently called C activation-related pseudoallergy (CARPA) [Circulation 99 (1999) 2302].

Liposomes act as effective biolubricants for friction reduction in human synovial joints.

Phospholipids (PL) form the matrix of biological membranes and of the lipoprotein envelope monolayer, and are responsible for many of the unique physicochemical, biochemical, and biological properties of these supermolecular bioassemblies. It was suggested that phospholipids present in the synovial fluid (SF) and on the surface of articular cartilage have major involvement in the low friction of cartilage, which is essential for proper mobility of synovial joints. In pathologies, such as impaired biolubrication (leading to common joint disorders such as osteoarthritis), the level of phospholipids in the SF is reduced. Using a human-sourced cartilage-on-cartilage setup, we studied to what extent and how phospholipids act as highly effective cartilage biolubricants. We found that large multilamellar vesicles (MLV), >800 nm in diameter, composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or of a mixture of DMPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) are superior lubricants in comparison to MLV composed of other phosphatidylcholines. Introducing cholesterol into liposomes resulted in less effective lubricants. DMPC-MLV was also superior to small unilamellar vesicles (SUV), <100 nm in diameter, composed of DMPC. MLV are superior to SUV due to MLV retention at and near (<200 microm below) the cartilage surface, while SUV penetrate deeper into the cartilage (450-730 microm). Superiority of specific PL compositions is explained by the thermotropic behavior (including compressibility) of the lipid bilayer. Correlating physicochemical properties of the MLV with the friction results suggests that MLV having lipid bilayers in the liquid-disordered phase and having a solid-ordered to liquid-disordered phase transition temperature slightly below physiological temperature are optimal for lubrication. High phospholipid headgroup hydration, high compressibility, and softness are the common denominators of all efficient PL compositions. The high efficiency of DMPC-MLV and DMPC/DPPC-MLV as cartilage lubricants combined with their resistance to degradation at 37 degrees C supports further evaluation of these MLV for treatment of joint impairments related to poor lubrication. This work also demonstrates the relevance of basic physicochemical properties of phospholipids to their activities in biological systems.

Correlation between drug release kinetics from proteineous matrices and protein folding: elasticity and compressibility study

Naproxen sodium (NS) release mechanism from proteineous matrices based on egg albumin (EA) and bovine serum albumin (BSA) was investigated by several physico-chemical methods. The gel strength, modulus of elasticity and erosion properties of the matrices were studied and correlated with drug release kinetics. The results revealed that NS release rate from EA and BSA matrices was markedly different, indicating the significant role of protein nature and conformation on matrix behavior. Unexpectedly it was found that incorporation of NS to EA matrix increased gel strength and modulus of elasticity and decreased matrix erosion. This effect was dependent on NS concentration in the matrix. In contrast to EA, BSA behaved as a non-gelling matrix and was unable to retard drug release because of its high solubility. The influence of NS on protein folding and compressibility in protein solutions was studied using densitometric and ultrasonic techniques. Adiabatic compressibility measurements revealed that NS caused unfolding of EA, an effect which led to a decrease in EA intrinsic compressibility and the exposure of atomic side groups buried in protein interior. Unfolding of EA led to an increase of modulus of elasticity in solution (measured by ultrasonic velocimetry technique) which is in correlation with the modulus of elasticity measurements of gelled tablets (measured by Instron). In concentrated EA solutions, the results showed a large increase in EA compressibility and ultrasonic absorption in the presence of NS indicating a strong aggregation of the denatured state of EA. Regarding BSA, the results suggested that NS affected the packing of the protein interior, transforming it to a molten globule intermediate state, an effect that led to an increase in BSA compressibility. At high BSA concentrations, aggregation of the molten globule state was observed as indicated by an increase of BSA compressibility and ultrasonic absorption values.

REDUCTION OF PROTEIN VOLUME AND COMPRESSIBILITY BY MACROMOLECULAR COSOLVENTS : DEPENDENCE ON THE COSOLVENT MOLECULAR WEIGHT

The partial specific volume (V) and adiabatic compressibility (beta) of myoglobin have been shown to be reduced by small cosolvents such as glycerol (A. Priev, A. Almagor, S. Yedgar, B. Gavish, Biochemistry 35 (1996) 2061-2066). To elucidate the effect of the cosolvent size on these protein properties, in the present study we determined V and beta of myoglobin in solutions containing a homologous cosolvent series from sucrose to dextran--500 (M.W. 500,000). It was found that in addition to the expected effect of the cosolvent concentration, V and beta decrease with increasing cosolvent M.W. This suggests that structural properties of the cosolvent contribute to its effect on the protein interior.

The Role of Hydration in Stabilization of Liposomes: Resistance to Oxidative Damage of PEG-Grafted Liposomes

Phospholipids are amphipathic compounds which form structures such as micelles and liposomes in aqueous media as a result of minimizing hydrophobic interaction. Thermodynamic stability of the lipid assemblies is due to a balance between the weak attractive van der Waals and hydrophobic forces, and repulsive electrostatic, steric, and hydration ones (Lasic and Martin, 1995). Lipid peroxidation (LPO) is a major damaging process in membranes and in liposomal dispersions. Oxidation processes in membranes are initiated by reactive oxygen species (ROS) formed in the aqueous phase and in the lipids, leading to chemical degradation, especially of polyunsaturated acyl chains of phospholipids.

Cylindrical standing wave resonator for liquid food quality control.

In this paper, an innovative technology based on the use of ultrasonic cylindrical standing waves for continuous monitoring of quality of various liquid food products, such as milk, juices, beer, wine, and drinking water is described. A proprietary unique feature of the developed ultrasonic analyzer is that it employs a combined mode of operation using both high‐intensity and low‐frequency (10 W/cm2, 1 MHz) waves for separation and concentration of the high‐molecular‐weight particles (fat globules or cells) and low‐intensity and high‐frequency (0.5 W/cm2, 10 MHz) waves for compositional analysis. High accuracy for ultrasound velocity measurements (up to 0.001%) and ultrasound attenuation (of about 1%) and rapid testing time (2–20 s) have been achieved. Comparative analyses of the ultrasonic method with standard reference techniques have produced linear calibration curves for major components with correlation coefficients higher than 0.95. It is thus possible to monitor total protein and fat content, and s...

Acoustic radiation force for rapid detection of particles in biological liquids

As known, ultrasonic standing waves can be used to concentrate particles and biological cells into separated bands. Acoustic separation in plane standing waves is limited to particles of few microns and larger. This presentation concerns using acoustic radiation force (RF) produced by cylindrical standing waves, for detection of high-density standing waves in pressure nodes and low-density particles (fat globules) in antinodes. Theoretical calculations show that in a cylindrical ultrasonic resonator RF near the central node can exceed the force at the periphery by about 20 times. In a cylindrical standing wave, RF can induce movement of bacteria with a speed of a few mm/s at frequency of 2 MHz and pressure amplitude of 100 kPa, whereas the speed of bacteria in a plane standing wave does not exceed 0.2 mm/s under the same conditions. The cylindrical standing wave system performance was tested for the E. coli bacteria in water and for a multi-component system containing fat globules and somatic cells in mil...

Ultrasound‐enhanced latex immunoassay of pathogens in water

Real‐time knowledge of contaminants in water is an essential component of any potable water security system. Latex immunoassay is the basic technique in rapid identification of pathogens. We used acoustic radiation to accelerate the latex immunoassay and to bring about separation between free and bound antigen (rotavirus SA‐11) in less than one minute. The ultrasonic cylindrical standing‐wave device of NDT Instruments, UltraAssay 101, creates areas of maximum and minimum potential energy (nodes). Acoustic radiation forces acting on the pathogens drive them directly to the central node. In this manner, antibody‐antigen complexes accumulate in the nodes in 40‐60 sec. The nonbound antibodies are washed out of the separation area by the buffer flow. The forces responsible for separation of particles in the UltraAssay 101 depend on the frequency of the standing‐wave resonator and on the density, compressibility and size of the immune complexes. Additionally, UltraAssay 101 is able to directly monitor water sal...

Acoustic radiation forces in monitoring of milk composition

High sensitivity of ultrasonic velocity and attenuation to composition and state of milk and other liquid food products is now well established. Unfortunately, existing devices include measurements of the acoustic properties of milk at different temperatures and therefore require waiting for temperature equilibration of the milk when the sample is heated or cooled and cannot be used for real‐time monitoring. In this paper ultrasonic particles analyzer of NDT Instruments, AcoMilk‐02, used for continuous monitoring of fat globules and somatic cells of raw milk in computerized milking station of cowsheds has been described. This device is based on high intensity standing wave for preliminary separation and concentration of the fat globules and somatic cells by the acoustic radiation forces and low intensity standing wave to measure their content. Testing of analyzer was carried out on 5 cows during one‐month period. It was found that milk production level, stage of lactation, and outside temperature have sig...