P. P. Kamaev

Quantification of Optison bubble size and lifetime during sonication dominant role of secondary cavitation bubbles causing acoustic bioeffects

Acoustic cavitation has been shown to deliver molecules into viable cells, which is of interest for drug and gene delivery applications. To address mechanisms of these acoustic bioeffects, this work measured the lifetime of albumin-stabilized cavitation bubbles (Optison) and correlated it with desirable (intracellular uptake of molecules) and undesirable (loss of cell viability) bioeffects. Optison was exposed to 500 kHz ultrasound (acoustic pressures of 0.6-3.0 MPa and energy exposures of 0.2-200 J/cm2) either with or without the presence of DU145 prostate cancer cells (10(6) cells/ml) bathed in calcein, a cell-impermeant tracer molecule. Bubble lifetime was determined using a Coulter counter and flow cytometer, while bioeffects were evaluated by flow cytometry. The lifetime of Optison cavitation nuclei was found to decrease and bioeffects (molecular uptake and loss of cell viability) were found to increase with increasing acoustic energy exposure. These bioeffects correlated well with the disappearance of bubbles, suggesting that contrast agent destruction either directly or indirectly affected cells, probably involving unstabilized cavitation nuclei created upon the destruction of Optison. Because Optison solutions presonicated to destroy all detectable bubbles also caused significant bioeffects, the indirect mechanism involving secondary cavitation bubbles is more likely.

Water transport phenomena in ‘green’ and ‘petrochemical’ polymers. Differences and similarities

Abstract The paper is devoted to the description of specific differences of water transport between petroleum-based (‘petro’ polymers) and environmentally friendly polymers (‘green’ polymers). The differences in water transport mechanisms are presented for poly-R-(3-hydroxybutyrate) and its blends with low density polyethylene (68–100 wt%) on the chemical and crystalline levels. The water diffusion coefficients and permeabilities were obtained using vacuum quartz spring microbalance techniques and permeability cells at 25°C. Spectral characteristics were obtained with FTIR procedure (IFS-48 Brucker IR spectrometer). ESR spectra for Tempol spin probe (sensitive to polar sites in polymer) are presented using Radiopan spectrometer within the temperature range of 20–75°C. As a result of polymer manufacture process, the accumulation of hydrophilic embedded groups is observed in petropolymers (PELD, PP, synthetic rubbers, etc.) which results in the water sorption increase and the effective diffusivity decrease. The immobilization of water both on polar polymeric groups and on impurities leads to an essential decrease in water molecule mobility. Due to their soft natural origin, this situation is less typical for green polymers. The effect of hydrophilic groups on water permeability was demonstrated for PHB/LDPE blends. Narrow MW distribution, stereo regularity and rigid order of polymeric fragments make for a more perfect crystalline structure as compared with petro polymers. The crystalline structure perfection leads to the decrease of water diffusivities as was shown for PHB. ESR data elucidate the relationship between the isotropic/textured PHB crystalline structure and spin probe rotating mobility. The study of the transport features in petro and green polymers is the necessary stage of investigation of such basic processes as physical aging at high humidity and corrosive stability of polymer membranes. The results may promote the design of novel environmentally friendly membranes for desalination and separation processes.

Modification via preparation for poly(3‐hydroxybutyrate) films: Water‐transport phenomena and sorption

The effect of the preparation technique on the sorption-diffusion parameters of poly(3-hydroxybutyrate) (PHB) films was studied. The films formed by a single-stage technique have an axial texture of the crystalline phase, with the polymer chain oriented predominantly perpendicularly to the film plane. Moreover, the crystallites in PHB are preferably ordered in stacks in the course of diffusion; the alignment of the crystallites noticeably decreases the sites of PHB (polar groups) which are accessible to water molecules. As a result, the sorption capacity decreases but the rate of diffusion increases. On the contrary, PHB films prepared by a two-stage technique are characterized by a poor ordering of crystallites without texture organization. Here, the sorption/immobilization on the polar groups of PHB is increased, but diffusivities are decreased. Concentration dependencies of the water-diffusion coefficient are discussed. Additional information on the existence of protein impurities in the PHB samples obtained by the above two preparation techniques was obtained by FTIR spectroscopy and H-D exchange methods.

Transport water and molecular mobility in novel barrier membranes with different morphology features

Abstract Bacterial poly(3-hydroxybutyrate) (PHB) — a new biopolymer and its derivatives are being extensively applied in medicine, biotechnology and membrane technologies. The relationship between structure-transport parameters and molecular mobility of PHB was investigated by quartz McBains microbalance, X-ray diffraction and ESR method. Two types of the PHB membranes with different structural organisation-isotropic and textured-were studied. The specific features of the sorption and diffusion characteristics of PHB are related to the structural organisation. The polymer matrixes are characterised by significantly microstructural heterogeneity as it was shown by the spin probe (TEMPO/TEMPOL) measurements. A non-crystalline phase of PHB includes two kinds of microfields with different molecular mobility. Moisture is found to change both the velocity of molecular motion and the mole content of such microfields. The diffusion behaviour of the membranes has been explained in accordance with dynamic information based on the ESR method.

Immobilization influence on the water sorption and diffusion in poly(3-hydroxybutyrate)

The temperature dependency of water vapor sorption and diffusion in poly(3-hydroxybutyrate) (PHB) was studied for the first time. Equilibrium sorption and diffusion kinetics were determined by a quartz McBains vacuum microbalance technique in the temperature range of 303–333 K. A probability of water molecule interaction with the polymer matrix was analyzed for wet PHB films by FTIR spectroscopy technique. Sorption isotherms are interpreted as the solution of free water molecules estimated by the Flory–Huggins equation and the sorption of water molecules immobilized on the carbonyl groups of PHB. The immobilization effect was described by a Langmuir-type equation. The dependency of diffusivity on water concentration was described in the frames of Fujitas immobilization model in which the growing function Dw versus Cw characterized the filling degree of carbonyl groups as sites of immobilization in the polymer. Enthalpy of free water sorption (12 kJ/mol) and water immobilization (42 kJ/mol), as well as the activation energy of water diffusion coefficients (71 kJ/mol), in noncrystalline areas of PHB were determined.

Desorption of human serum albumin and human fibrinogen from the poly(3‐hydroxybutyrate) surface

The protein desorption of human serum albumin and human fibrinogen from the surface of poly(3-hydroxybutyrate) films was studied using ATR–FTIR spectroscopy. The diffusion model for reversible and irreversible sandwiched layers was confirmed. The reversible ratio (ratio of reversible adsorbed concentration to irreversible adsorbed concentration as a function of time allows for a suggestion as to a kinetic model of the initial stage of thromb formation. The parameters of adsorption/desorption for both proteins are compared. The reversible ratio of plasma protein adsorption is proposed as a quantitative criterion of thromb-resistance behavior for polymers in biomedicine; namely, controlled drug release vehicles, artificial vessels, magistrals, reservoirs for blood storage, and surgical threads, especially. The mechanism of interaction of protein molecules with poly(3hydroxybutyrate (PHB) macromolecules is discussed.

Desorption of Human Serum Albumin and Human Fibrinogen from the Poly(3-Hydroxybutyrate) Surface

Abstract The protein desorption of human serum albumin and human fibrinogen from the surface of poly(3-hydroxybutyrate) films was studied with ATR-FTIR spectroscopy. The diffusion model for reversible and irreversible sandwiched layers was confirmed. The reversible ratio (ratio of reversible adsorbed concentration to irreversible one) registrated as a function of time allow us to propose the kinetic model of the initial stage of thromb formation. The parameters of adsorption/desorption for both proteins are compared. The reversible ratio of plasma protein adsorption is proposed as a quantitative criterion of thromb-resistance behavior for polymer in biomedicine, namely, controlled drug release vehicles, artificial vessels, magistrals, reservoirs for blood storage, and surgical threads especially. The mechanism of interaction of protein molecules with PHB macro-molecules is discussed.

Measurement of cavitation bubble lifetime and size distribution and its correlation with bioeffects

Ultrasound‐induced disruption of cell membranes can be used to deliver drugs and genes into cells. Because ultrasound bioeffects are governed by cavitation, measurement and control of cavitation bubble lifetime and size distribution should afford greater control over ultrasound’s effects on cells. In this study, 500 kHz ultrasound was focused onto suspensions of albumin‐stabilized gas bubbles (Optison) either with or without DU145 prostate cancer cells bathed in calcein. Bubble size and lifetime were determined by Coulter counting, while uptake of calcein and cell viability (i.e., bioeffects) were quantified by flow cytometry. We found that the lifetime and size of Optison bubbles decreased with increasing acoustic energy exposure. Moreover, bioeffects were shown to correlate well with disappearance of bubbles. For example, cell viability remained above 90% until approximately 75% of bubbles were destroyed; viability then dropped dramatically as more bubbles disappeared. Additionally, Optison solutions presonicated to destroy all detectable bubbles also caused significant bioeffects. These observations suggest that bioeffects were caused by the cavitation dynamics of free, short‐lived, and/or very small daughter bubbles liberated from albumin‐stabilized Optison parent bubbles. Regulation of bubble size distribution, possibly by presonication of Optison solutions, could provide a means to optimize high uptake and cell viability.

Bubbles, membranes and molecules: sequence of events from sonication to intracellular delivery

To elucidate mechanisms and control bioeffects for ultrasound‐mediated drug and gene delivery, we carried out an experimental study to quantitatively measure the effects of ultrasound and other physical parameters on the sequence of events leading from sonication to drug and gene delivery. Using a Coulter counter, the number and size distribution of bubbles was measured as a function of ultrasound pressure and time, and found to decrease as a function of acoustic energy exposure. The effects of these bubbles on cells were measured by electron and confocal microscopy, which indicated that cavitation created cell membrane defects that could be actively repaired and permitted the intracellular transport of molecules. Using flow cytometry, levels of molecular uptake and cell viability were quantified over a broad range of conditions and correlated with acoustic energy, the ratio of cells‐to‐cavitation nuclei and the size distribution of bubbles. Finally, levels of gene expression were quantified as a function...