Role of constituents of Optison in Optison-mediated gene transfection enhancement in skeletal muscle in vivo.

OBJECTIVES: The mechanism by which Optison (an albumin-shelled, octafluoropropane gas-filled microbubble contrast agent; Amersham Health, Amersham, England) enhances gene transfection in skeletal muscle in vivo with or without ultrasound (US) is unclear. The possible mechanisms were investigated by experimenting with different constituents, both with and without US. METHODS: Plasmid DNA (10 µg) encoding green fluorescent protein was mixed with Optison or its constituents dissolved in saline (in an equivalent concentration as in Optison) and injected into the tibialis anterior muscle of mice with or without adjunct US (1 MHz, 2 W/cm², 30 seconds, and 20% duty cycle). The efficiencies of green fluorescent protein transgene expression were determined under different experimental conditions: (1) plasmid plus saline as a negative control; (2) plasmid plus Optison as a positive control; (3) plasmid plus heat-treated Optison (without microbubbles); (4) plasmid plus human serum albumin; (5) plasmid plus N-acetyltryptophan; and (6) plasmid plus caprylic acid. Transfection efficiency was assessed by counting the maximum number of green fluorescent protein-positive fibers. Tissue damage was assessed by measuring the damaged area on serial sections. RESULTS: Heat-treated Optison with or without US and albumin with US showed similarly high levels of transgene expression as Optison in mouse muscle without substantially increased tissue damage. N-Acetyltryptophan and caprylic acid had no effect on the delivery of plasmid green fluorescent protein into mouse muscle but instead showed the potential to increase tissue damage. CONCLUSIONS: These data suggest that US and albumin separately potentiate transfection in this model. The combination of albumin and perfluoropropane is highly effective, which probably explains why Optison is so effective.

Targeted disruption of the blood-brain barrier with focused ultrasound: association with cavitation activity.

Acoustic emission was monitored during focused ultrasound exposures in conjunction with an ultrasound contrast agent (Optison) in order to determine if cavitation activity is associated with the induction of blood-brain barrier disruption (BBBD). Thirty-four locations were sonicated (frequency: 260 kHz) at targets 10 mm deep in rabbit brain (N = 9). The sonications were applied at peak pressure amplitudes ranging from 0.11 to 0.57 MPa (burst length: 10 ms; repetition frequency of 1 Hz; duration: 20 s). Acoustic emission was recorded with a focused passive cavitation detector. This emission was recorded at each location during sonications with and without Optison. Detectable wideband acoustic emission was observed only at 0.40 and 0.57 MPa. BBBD was observed in contrast MRI after sonication at 0.29-0.57 MPa. The appearance of small regions of extravasated erythrocytes appeared to be associated with this wideband emission signal. The results thus suggest that BBBD resulting from focused ultrasound pulses in the presence of Optison can occur without indicators for inertial cavitation in vivo, wideband emission and extravasation. If inertial cavitation is not responsible for the BBBD, other ultrasound/microbubble interactions are likely the source. A significant increase in the emission signal due to Optison at the second and third harmonics of the ultrasound driving frequency was found to correlate with BBBD and might be useful as an online method to indicate when the disruption occurs.

Ultrasonic contrast agent shell rupture detected by inertial cavitation and rebound signals.

Determining the rupture pressure threshold of ultrasound contrast agent microbubbles has significant applications for contrast imaging, development of therapeutic agents, and evaluation of potential bioeffects. Using a passive cavitation detector, this work evaluates rupture based on acoustic emissions from single, encapsulated, gas-filled microbubbles. Sinusoidal ultrasound pulses were transmitted into weak solutions of Optison at different center frequencies (0.9, 2.8, and 4.6 MHz), pulse durations (three, five, and seven cycles of the center frequencies), and peak rarefactional pressures (0.07 to 5.39 MPa). Pulse repetition frequency was 10 Hz. Signals detected with a 13-MHz, center-frequency transducer revealed postexcitation acoustic emissions (between 1 and 5 micros after excitation) with broadband spectral content. The observed acoustic emissions were consistent with the acoustic signature that would be anticipated from inertial collapse followed by "rebounds" when a microbubble ruptures and thus generates daughter/free bubbles that grow and collapse. The peak rarefactional pressure threshold for detection of these emissions increased with frequency (e.g., 0.53, 0.87, and 0.99 MPa for 0.9, 2.8, and 4.6 MHz, respectively; five-cycle pulse duration) and decreased with pulse duration. The emissions identified in this work were separated from the excitation in time and spectral content, and provide a novel determination of microbubble shell rupture.

Enhanced heat deposition using ultrasound contrast agent--modeling and experimental observations.

Ultrasound contrast agents (UCA), created originally for visualization and diagnostic purposes, recently have been suggested as efficient enhancers of ultrasonic power deposition in tissue. The ultrasonic energy absorption by the contrast agents, considered as problematic in diagnostic imaging, might have beneficial impact in therapeutic applications such as targeted hyperthermia-based or ablation treatments. Introduction of gas microbubbles into the tissue to be treated can improve the effectiveness of current treatments by limiting the temperature rise to the treated site and minimizing the damage to the surrounding healthy tissues. To this end, proper assessment of the governing parameters of energy absorption by ultrasonically induced stabilized bubbles is important for both diagnostic and therapeutic ultrasound applications. The current study was designed to predict theoretically and measure experimentally the dissipation and heating effects of encapsulated UCA in a well-controlled and calibrated environment. The ultrasonic effects of the microbubble concentration, transmitted intensity, and frequency on power dissipation and stability of the UCA have been studied. The maximal temperature elevation obtained during 300 s experiments was 21 degrees C, in a 10 ml volume target containing UCA, insonifled by unfocused 3.2 MHz continuous wave (CW) at spatial average intensity of 1.1 W/cm2 (182 kPa). The results also suggest that higher frequencies are more efficiently absorbed by commonly used UCA. In particular, for spatial average intensity of 1.1 W/cm2 and concentration of 5 x 10(6) microspheres/cm3, no significant reduction of UCA absorption was noticed during the first 150 s for insonation at 3.2 MHz and the first 100 s for insonation at 1 MHz. In addition, when lower average intensity of 0.5 W/cm2 (160 kPa) at 3.2 MHz was used, the UCA absorptivity sustained for almost 200 s. Thus, when properly activated, UCA may be suitable for localized hyperthermic therapies.

Radiosterilization of albuminated polyester prostheses.

The study reported here is concerned with the radio-sterilization of Dacron vascular prostheses coated with crosslinked albumin. gamma-Radiations have no effect on the mechanical properties of the polyester fibres or on their crystallinity, whether irradiated in a dry state or immersed in saline. Special attention has been paid to the release of the albumin, or protein fragments from the reticulum using 125I-labelled albumin as a radiotracer. The albumin leakage depends upon the type of Dacron fabrics considered but the values derived from radioactivity measurements are always greater than those directly measured, which indicates a radio-induced break of the bond between iodine and albumin; this has nothing to do with the break of the association between albumin and Dacron. Moreover no cytotoxicity of the irradiated immersion medium has been observed using a test based on organotypic culture in liquid medium. Thus radio-sterilization of an albuminated polyester vascular prosthesis immersed in saline appears to be a suitable procedure.

Dynamics of pulsed holmium:YAG laser photocoagulation of albumen.

The pulsed holmium:YAG laser (lambda = 2.12 microm, tau(p) = 250 micros) has been investigated as a method for inducing localized coagulation for medical procedures, yet the dynamics of this process are not well understood. In this study, photocoagulation of albumen (egg white) was analysed experimentally and results compared with optical-thermal simulations to investigate a rate process approach to thermal damage and the role of heat conduction and dynamic changes in absorption. The coagulation threshold was determined using probit analysis, and coagulum dynamics were documented with fast flash photography. The nonlinear computational model, which included a Beer's law optical component, a finite difference heat transfer component and an Arrhenius equation-based damage calculation, was verified against data from the literature. Moderate discrepancies between simulation results and our experimental data probably resulted from the use of a laser beam with an irregular spatial profile. This profile produced a lower than expected coagulation threshold and an irregular damage distribution within a millisecond after laser onset. After 1 ms, heat conduction led to smoothing of the coagulum. Simulations indicated that dynamic changes in absorption led to a reduction in surface temperatures. The Arrhenius equation was shown to be effective for simulating transient albumen coagulation during pulsed holmium:YAG laser irradiation. Greater understanding of pulsed laser-tissue interactions may lead to improved treatment outcome and optimization of laser parameters for a variety of medical procedures.

Photografting of albumin onto dimethyldichlorosilane-coated glass.

4-Azido-2-nitrophenyl albumin (ANP-albumin) was prepared by displacing the fluoro group of 4-fluoro-3-nitrophenyl azide (FNPA) by an amino group of albumin. Photolysis of phenyl azides of ANP-albumin was studied by Fourier-transform infrared (FTIR) spectroscopy. The band of phenyl azide disappeared completely after a 12-min exposure to long wave UV light (366 nm), and the photolysis was first-order. Albumin was grafted onto dimethyldichlorosilane-coated glass (DDS-glass) by photolysis of the azido groups of ANP-albumin without any premodification of the surface. The albumin-grafted DDS-glass was characterized by determining the relative amount of nitrogen resulting from the grafted albumin on the surface using electron spectroscopy for chemical analysis (ESCA). The amount of nitrogen increased when the concentration of ANP-albumin in the adsorption solution increased up to 0.1 mg/ml. As the solution concentration increased above this value, the amount of nitrogen decreased. The platelet resistance of the albumin-grafted surfaces was evaluated by measuring the number of adherent platelets and the extent of activation that was quantitated by the area of platelets spread on the surfaces. The maximum platelet-resistant effect was observed when the ANP-albumin was adsorbed for more than 50 min at the solution concentration ranging from 0.05 to 10 mg/ml.

[Supernarrow EPR spectrum of aqueous protein solutions, UV-irradiated at 77 K]. / Sverkhuzkii spektr EPR vodnykh rastvorov belkov, UF-obluchennykh pri 77 K.

The nature was studied of paramagnetic centres photoinduced in aqueous solutions of proteins, amino acids and peptides at 77 K, having a supernarrow ESR spectrum with the width 8 mkTl and g = 1.9988 +/- 0.0002. It has been shown that seized electrons localized in deep traps are responsible for this signal. It is suggested that these are metal ions whose nucleus magnetic moment equals zero.

Albumin acts like transforming growth factor ß1 in microbubble-based drug delivery.

Unlike lipid-shelled microbubbles (MBs), albumin-shelled microbubbles (MBs) have not been reported to be actively targeted to cells without the assistance of antibodies. Recent studies indicate that the albumin molecule is similar to transforming growth factor ß (TGF-ß) both structurally and functionally. The TGF-ß superfamily is important during early tumor outgrowth, with an elevated TGF-ß being tumor suppressive; at later stages, this switches to malignant conversion and progression, including breast cancer. TGF-ß receptors I and II play crucial roles in both the binding and endocytosis of albumin. However, until now, no specific albumin receptor has been found. On the basis of the above-mentioned information, we hypothesized that non-antibody-conjugated albumin-shelled MBs can be used to deliver drugs to breast cancer cells. We also studied the possible roles of TGF-ß1 and radiation force in the behavior of cells and albumin-shelled MBs. The results indicate that albumin-shelled MBs loaded with paclitaxel (PTX) induce breast cancer cell apoptosis without the specific targeting produced by an antibody. Applying either an acoustic radiation force or cavitation alone to cells with PTX-loaded albumin MBs increased the apoptosis rate to 23.2% and 26.3% (p < 0.05), respectively. We also found that albumin-shelled MBs can enter MDA-MB-231 breast cancer cells and remain there for at least 24 h, even in the presence of PTX loading. Confocal micrographs revealed that 70.5% of the breast cancer cells took up albumin-shelled MBs spontaneously after 1 d of incubation. Applying an acoustic radiation force further increased the percentage to 91.9% in our experiments. However, this process could be blocked by TGF-ß1, even with subsequent exposure to the radiation force. From these results, we conclude that TGF-ß1 receptors are involved in the endocytotic process by which albumin-shelled MBs enter breast cancer cells. The acoustic radiation force increases the contact rate between albumin-shelled MBs and tumor cells. Combining a radiation force and cavitation yields an apoptosis rate of 31.3%. This in vitro study found that non-antibody-conjugated albumin-shelled MBs provide a useful method of drug delivery. Further in vivo studies of the roles of albumin MBs and TGF-ß in different stages of cancer are necessary.

Quantitative guidelines for the prediction of ultrasound contrast agent destruction during injection.

Experiments and theory were undertaken on the destruction of ultrasound contrast agent microbubbles on needle injection, with the aim of predicting agent loss during in vivo studies. Agents were expelled through a variety of syringe and needle combinations, subjecting the microbubbles to a range of pressure drops. Imaging of the bubbles identified cases where bubbles were destroyed and the extent of destruction. Fluid-dynamic calculations determined the pressure drop for each syringe and needle combination. It was found that agent destruction occurred at a critical pressure drop that depended only on the type of microbubble. Protein-shelled microbubbles (sonicated bovine serum albumin) were virtually all destroyed above their critical pressure drop of 109 ± 7 kPa Two types of lipid-shelled microbubbles were found to have a pressure drop threshold above which more than 50% of the microbubbles were destroyed. The commercial lipid-shelled agent Definity was found to have a critical pressure drop for destruction of 230 ± 10 kPa; for a previously published lipid-shelled agent, this value was 150 ± 40 kPa. It is recommended that attention to the predictions of a simple formula could preclude unnecessary destruction of microbubble contrast agent during in vivo injections. This approach may also preclude undesirable release of drug or gene payloads in targeted microbubble therapies. Example values of appropriate injection rates for various agents and conditions are given.

New insights into the molecular pathology of radiation-induced pneumopathy.

BACKGROUND AND PURPOSE: Pneumonitis and fibrosis constitute dose-limiting side effects of thorax or total body irradiation. An improved understanding of the underlying mechanisms is a prerequisite for the development of effective radioprotective strategies. Here we characterized the behavior of resident and immune cells in a murine model of radiation-induced pneumopathy. MATERIALS AND METHODS: Wild type (WT) or RAG-2 deficient C57BL/6 mice received 15 Gray of (hemi)-thorax irradiation in a single dose. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected at defined time points post-irradiation for the determination of apoptosis, microvascular injury, and histological and immunohistochemical analyses. RESULTS: Higher albumin levels and increased apoptosis were detected in the BALF 21 days after irradiation, indicative for delayed damage to resident cells. Irradiation also induced time-dependent changes in the BALF cytokine profile, the recruitment of activated T-cells into the lung and the formation of lipid-loaded resident cells. Lung fibrosis occurred earlier in RAG-2(-/-) mice, which lack mature T and B cells, compared to WT mice. CONCLUSIONS: Thorax irradiation triggers a delayed disturbance of tissue integrity and lipid metabolism in the lung. Activated T-lymphocytes infiltrating the lung tissue upon thorax irradiation participate in the protection of the lung from radiation-induced fibrosis.

Stability of alteplase in presence of cavitation.

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9 mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1 mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2 MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5 MPa for a duration of 60 min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60 min of 2 MHz ultrasound at acoustic pressures ranging from 0.5 MPa to 3.5 MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.

Difference frequency and its harmonic emitted by microbubbles under dual frequency excitation.

Vibro-acoustography is an elasticity imaging method that uses two ultrasound beams of slightly different frequency to excite an object and detects the resulting acoustic emission (AE) at the difference frequency. This method is especially sensitive to bubbles due to their nonlinearity. This study explores the harmonic acoustic emission (HAE) at twice the difference frequency emitted from bubbles. A perturbation method based on the dynamic bubble equation is used to derive the AE and HAE from a single bubble excited by dual frequency waves. Simulation shows that HAE is generated only by microbubbles whose resonant frequencies match the incident ultrasound frequencies. In contrast, AE is more sensitive to resonance at the difference frequency, which is relevant to sub-millimeter bubbles. This finding was confirmed by experiments where HAE was produced from Optison microbubbles, but not from larger air bubbles which are off resonance at the incident ultrasound frequency. In conclusion, harmonic acoustic emission is present for microbubbles. It is very sensitive to the size of the bubble and may be used for selective detection of microbubbles.

[Effect of melanin on the free-radical states of gamma-irradiated proteins and lipids]. / Vliianie melanina na svobodno-radikal'nye sostoianiia gamma-obluchennykh belkov i lipidov.

It was shown that the concentration of paramagnetic centres of dihydroxyphenylalanine-melanin increased after gamma-irradiation (60Co) both at room temperature (an irreversible increase) and at 77 K (a reversible increase). The accumulation of paramagnetic centres in gamma-irradiated albumin at room temperature was found to slow down appreciably in the presence of melanin. This effect is thought to be associated with the antiradical activity of the pigment.

[Changes in the total protein and protein fractions in cow's milk irradiated with ultraviolet rays]. / Promeni v obshtiia beltuk i beltuchnite fraktsii v krave mliako, oblucheno s ultravioletovi luchi

Comparative experimental electrophoretic studies were carried out to establish the effect of ultraviolet treatment on cow's milk at various rates of irradiation (5, 10 and 15 min) with a view to raising and regulating the content of the total protein and the protein fractions in milk intended for the feeding of calves. Results showed that there were no substantial changes in the level of the total protein, the albumins having a rising trend that was manifested best at a 15-minute treatment. Most characteristic were the changes taking place in the immunoglobulin fraction which rose by 4.7 per cent. The beta-lactoglobulin fraction dropped by 4.5 per cent, on an average, at all rates of treatment, and the level of alfa-lactoglobulin at 5- and 10-minute irradiation underwent almost no changes, it rising by about 2.09 per cent, on an average, at 15-min treatment. Similar was the trend manifested by the proteoso-pepton fraction showing a rise as early as the first ten minutes of treatment.

A simple, dual tracer method for the measurement of transvascular flux of albumin into the lung.

A simple, first-order model of albumin kinetics in the rat lung is presented and validated with a more sophisticated model. The simple model assumes that intravascular concentration of tracer albumin is constant over 30 min after injection and transvascular flux of tracer albumin is unidirectional and proportional to the permeability-surface area product (PS). 125I-albumin is injected initially and 131I-albumin at 20 min. At 30 min the rat is sacrificed and plasma and tissue samples are obtained for gamma counting. Simultaneous equations are set up for the two tracers and solved for PS and plasma volume. The accuracy of this approach is examined with data generated from a more complete model. This model uses the concepts of hydraulic conductivity, solvent drag, reflection coefficients, hydrostatic and osmotic pressures, exclusion volumes, and lymph flow, as well as PS. Based on known PS and clearance rates from the complex model, the simple model estimates tracer albumin leakage rate with less than 5% error over the range of PS encountered in rat studies.