The ultrasonic degradation of biological macromolecules under conditions of stable cavitation. II. Degradation of deoxyribonucleic acid

Solutions of T7 bacteriophage or calf thymus DNA arc degraded in solution by ultrasonic fields of low intensity in the presence of vibrating air bubbles but are not degraded at these low intensities when such bubbles are absent. Evidence is presented for the hydrodynamic nature of the observed degradation and theoretical simulation of a plausible degradation mechanism is compared with experimental degradation studies. It is concluded that degradation of such linear macromolecules as DNA may occur as a result of stresses induced in the macromolecule; these stresses are the result of a relative movement of solvent molecules and the macromolecules in the time-independent flow of solvent near the vibrating bubbles.     

Free radical formation induced by ultrasound and its biological implications.

The chemical effects of ultrasound in aqueous solutions are due to acoustic cavitation, which refers to the formation, growth, and collapse of small gas bubbles in liquids. The very high temperatures (several thousand K) and pressures (several hundred atmospheres) of collapsing gas bubbles lead to the thermal dissociation of water vapor into .OH radicals and .H atoms. Their formation has been confirmed by electron spin resonance (ESR) and spin trapping. The sonochemistry of aqueous solutions of gases and of volatile and nonvolatile solutes is reviewed. The similarities and differences between sonochemistry and radiation chemistry of aqueous solutions are explained. Some unusual characteristics of aqueous sonochemistry can be understood by considering the properties of supercritical water. By the use of rare gases with different thermal conductivities, it is possible to distinguish between temperature-dependent processes such as redox reactions initiated by .OH radicals and .H atoms and pressure-dependent processes which lead to polymer degradation and cell lysis. The evidence for free radical formation in aqueous solutions by pulsed ultrasound is discussed. This subject is of interest because it is related to the possible deleterious effects of ultrasonic diagnostic devices. The role of free radicals and of mechanical effects induced by ultrasound in DNA degradation, inactivation of enzymes, lipid peroxidation, and cell killing is reviewed.     

The role of cavitation in liposome formation

Liposome size is a vital parameter of many quantitative biophysical studies. Sonication, or exposure to ultrasound, is used widely to manufacture artificial liposomes, yet little is known about the mechanism by which liposomes are affected by ultrasound. Cavitation, or the oscillation of small gas bubbles in a pressure-varying field, has been shown to be responsible for many biophysical effects of ultrasound on cells. In this study, we correlate the presence and type of cavitation with a decrease in liposome size. Aqueous lipid suspensions surrounding a hydrophone were exposed to various intensities of ultrasound and hydrostatic pressures before measuring their size distribution with dynamic light scattering. As expected, increasing ultrasound intensity at atmospheric pressure decreased the average liposome diameter. The presence of collapse cavitation was manifested in the acoustic spectrum at high ultrasonic intensities. Increasing hydrostatic pressure was shown to inhibit the presence of collapse cavitation. Collapse cavitation, however, did not correlate with decreases in liposome size, as changes in size still occurred when collapse cavitation was inhibited either by lowering ultrasound intensity or by increasing static pressure. We propose a mechanism whereby stable cavitation, another type of cavitation present in sound fields, causes fluid shearing of liposomes and reduction of liposome size. A mathematical model was developed based on the Rayleigh-Plesset equation of bubble dynamics and principles of acoustic microstreaming to estimate the shear field magnitude around an oscillating bubble. This model predicts the ultrasound intensities and pressures needed to create shear fields sufficient to cause liposome size change, and correlates well with our experimental data.     

Cavitation bubble- and gas bubble-induced fractional precipitation of paclitaxel from Taxus chinensis

In this study, a fractional precipitation technique of paclitaxel using ultrasonic cavitation bubbles and gas bubbles is presented. Precipitation efficiency has been dramatically improved, and the time required for precipitation has been reduced by 20–30 times compared to conventional methods. As a result of investigating the mechanism of fractional precipitation in which cavitation and gas bubbles were introduced, it was found that the bubble surface itself acts as a nucleation site, resulting in faster nucleation and thereby improving precipitation efficiency. In addition, compared to the conventional fractional precipitation, the particle size was reduced by 7.8–8.9 times and 4.7–4.9 times for cavitation bubbles and gas bubbles, respectively, and the diffusion coefficient was increased by 10.3–11.9 times (cavitation bubble) and 4.7–4.9 times (gas bubble).     

超声对胃蛋白酶，胰蛋白酶，过氧化氢酶作用的研究

以胃蛋白酶,胰蛋白酶,过氧化氢酶溶液在超声处理下的酶活变化为指标研究超声对蛋白质作用的机理和影响因素。结果表明超声对蛋白质的破坏程度随着功率的升高或处理时间的延长而增加;三种酶在超声作用下酶活变化形式和程度各不同;浓度是影响超声对酶作用效果的一个重要因素,可通过调整酶溶液浓度来减少酶所受到的破坏程度。自由基清除剂甘露醇和非离子表面活性剂吐温-80可以对酶活在超声作用下起到一定的保护作用,说明自由基和超声空穴是超声破坏酶结构的重要机理,研究结果同时表明对于不同的酶,超声的破坏作用可能有不同的发挥主导作用机理。     

Ultrasound-biophysics mechanisms

Ultrasonic biophysics is the study of mechanisms responsible for how ultrasound and biological materials interact. Ultrasound-induced bioeffect or risk studies focus on issues related to the effects of ultrasound on biological materials. On the other hand, when biological materials affect the ultrasonic wave, this can be viewed as the basis for diagnostic ultrasound. Thus, an understanding of the interaction of ultrasound with tissue provides the scientific basis for image production and risk assessment. Relative to the bioeffect or risk studies, that is, the biophysical mechanisms by which ultrasound affects biological materials, ultrasound-induced bioeffects are generally separated into thermal and non-thermal mechanisms. Ultrasonic dosimetry is concerned with the quantitative determination of ultrasonic energy interaction with biological materials.

Whenever ultrasonic energy is propagated into an attenuating material such as tissue, the amplitude of the wave decreases with distance. This attenuation is due to either absorption or scattering. Absorption is a mechanism that represents that portion of ultrasonic wave that is converted into heat, and scattering can be thought of as that portion of the wave, which changes direction. Because the medium can absorb energy to produce heat, a temperature rise may occur as long as the rate of heat production is greater than the rate of heat removal. Current interest with thermally mediated ultrasound-induced bioeffects has focused on the thermal isoeffect concept. The non-thermal mechanism that has received the most attention is acoustically generated cavitation wherein ultrasonic energy by cavitation bubbles is concentrated. Acoustic cavitation, in a broad sense, refers to ultrasonically induced bubble activity occurring in a biological material that contains pre-existing gaseous inclusions. Cavitation-related mechanisms include radiation force, microstreaming, shock waves, free radicals, microjets and strain. It is more challenging to deduce the causes of mechanical effects in tissues that do not contain gas bodies. These ultrasonic biophysics mechanisms will be discussed in the context of diagnostic ultrasound exposure risk concerns.     

Observation and quantification of gas bubble formation on a mechanical heart valve

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.     

Detection of ultrasonic cavitation based on low-frequency analysis of acoustic signal

The acoustic cavitation phenomenon constitutes a potential hazard in ultrasound diagnostics and therapy so that early and effective detection of cavitation is of great interest. However, cavitation might even bring a higher risk especially when an echocontrast agent based on microbubbles is used. The major goal of the present work was to develop a cavitation detection method based on increased level of cavitation noise in the range of low frequencies (about 1 Hz). This method was applied in vitro using a model of body fluid containing a model echocontrast agent, such as 5% solution of lyophilized egg albumin, which was sonicated by ultrasound disintegrator. Ultrasound signal evokes cavitation in microbubble suspension accompanied by a certain level of cavitation acoustic noise. The level of noise voltage increased in the frequency range of 0.1 to 2 Hz in the presence of cavitation. Hence, this method makes it possible to determine the value of cavitation threshold. In addition, we examined how the cavitation threshold is affected by temperature and viscosity. It was found that the cavitation threshold decreased with growing temperature while it increased with growing viscosity.     

Kinetics of ultrasonic degradation and polymerisation degree distribution of sonochemically degraded chitosans

The process of physical degradation by means of the ultrasonic action towards chitosans with mole fraction of 2-acetamido-2-deoxy-β- -glucopyranose units (the degree of N-acetylation, F_A) in the range of 0.10≤F_A≤0.28, and the weight average polymerisation degree in the range of has been investigated. The decrease of as well as changes in the distribution of the degree of polymerisation (P) has been determined as a function of time, F_A, temperature, concentration of chitosan solution and concentration of acetic acid in the solution. The use of low-power ultrasound emitter allowed to establish that in the case of chitosan (binary heteropolysaccharide) the general rate parameter (k) increased with F_A. This can be explained by the relatively stronger aggregation of macromolecules with higher F_A, which results in size increase of macromolecular individuals and hence in their higher susceptibility to ultrasonic action. It was also observed that k decreased with chitosan concentration and temperature. The value of limiting degree of polimerisation (x_e) was found to be influenced by structural parameters of chitosan chains (F_A, aggregation). The increase of acetic acid concentration caused the increase in the k value, what indicated accelerating effect of ultrasound towards acidic hydrolysis of chitosan. The shape of the P curve of sonochemically degraded chitosans are in good correlation with the mid-point breakage concept of degradation accepted in sonochemical degradation of polymers.     

High Speed Imaging of Cavitation around Dental Ultrasonic Scaler Tips

Cavitation occurs around dental ultrasonic scalers, which are used clinically for removing dental biofilm and calculus. However it is not known if this contributes to the cleaning process. Characterisation of the cavitation around ultrasonic scalers will assist in assessing its contribution and in developing new clinical devices for removing biofilm with cavitation. The aim is to use high speed camera imaging to quantify cavitation patterns around an ultrasonic scaler. A Satelec ultrasonic scaler operating at 29 kHz with three different shaped tips has been studied at medium and high operating power using high speed imaging at 15,000, 90,000 and 250,000 frames per second. The tip displacement has been recorded using scanning laser vibrometry. Cavitation occurs at the free end of the tip and increases with power while the area and width of the cavitation cloud varies for different shaped tips. The cavitation starts at the antinodes, with little or no cavitation at the node. High speed image sequences combined with scanning laser vibrometry show individual microbubbles imploding and bubble clouds lifting and moving away from the ultrasonic scaler tip, with larger tip displacement causing more cavitation.     

Mechanism of disintegration of biological cells in ultrasonic cavitation

On the basis of elastic waves released by imploding cavitation bubbles, a mechanism for biological cell disintegration in high intensity ultrasounds has been proposed. Comparison of this mechanism with the published results on yeast cells shows many points of agreement suggesting that yeast cell disintegration in ultrasonic cavitation occurs by shear stresses developed by viscous dissipative eddies arising from shock waves.     

Sea-trial verification of ultrasonic antifouling control

An ultrasonic antifouling treatment was applied to a 96,000 m³ class drill-ship to verify its feasibility through a sea-trial. Soon after the hull cleaning had been performed, six ultrasonic projectors were evenly deployed around the starboard shell plate. Driven by a 23 kHz sinusoidal ultrasound in an intermittent manner, the projectors emitted a high-intensity sound reaching 214 dB at the source level causing cavitation around the adjacent water and eventually deterring the settlement of marine fouling organisms. Underwater photographs acquired after four months showed fairly clean slabs on the starboard side, but heavy fouling on the port side. This experiment revealed that ultrasound treatment is a promising method for inhibiting fouling accumulation, even for large-scale ship applications.     

Tumor cytotoxicity in vivo and radical formation in vitro depend on the shock wave-induced cavitation dose

Local tumor therapy using focused ultrasonic waves may become an important treatment option. This technique exploits the ability of mechanical waves to induce thermal and nonthermal effects noninvasively. The cytotoxicity to cultured cells and biological tissues in vivo that results from exposure to ultrasonic shock waves is considered to be a nonthermal effect that is partly a consequence of ultrasound-induced cavitation. Cavitation is defined as the formation of bubbles during the negative wave cycle; their subsequent oscillation and/or violent implosion can affect surrounding structures. To investigate cavitational effects in cells and tissues, defined cavitation doses must be applied while ideally holding all other potential ultrasound parameters constant. The application of independent cavitation doses has been difficult and has yielded little knowledge about quantitative cavitation-tissue interactions. By using a special shock-wave pulse regimen and laser optical calibration in this study, we were able to control the cavitation dose independently of other physical parameters such as the pressure amplitudes, and averaged acoustic intensity. We treated Dunning prostate tumors (subline R3327-AT1) transplanted into Copenhagen rats with shock waves at three cavitation dose levels and then determined the tumor growth delay and the histopathological changes. All of the treated animals exhibited a significant tumor growth delay compared to the controls. Higher cavitation doses were associated with a greater delay in the growth of the tumor and more severe effects on tumor histopathology, such as hemorrhaging, tissue disruption, and necrosis. In vitro, the cavitation dose level correlated with the amount of radical formation. We concluded that the process of acoustic cavitation was responsible; higher cavitation doses caused greater effects in tumors both in vivo and in vitro. These findings may prove important in local tumor therapy and other applications of ultrasound such as ultrasound-mediated drug delivery.     

超声空化强度测量的研究进展

近几十年来,超声波已经广泛应用于生物学和医学领域,在医学领域中超声波可以作为信息载体用于探测人体的病变信息,并且可以用一定剂量的超声波作用于人体病变组织,并通过它对组织的作用达到一定的治疗目的。作为一种无创、非介入性外科技术,它的疗效和安全性越来越被人们所关注。超声波与人体组织的相互作用有三种,分别是机械机制,热学机制,和空化机制。对于机械机制和热机制人们比较熟悉,而对于空化机制则相对陌生。随着超声空化在医学中的应用越来越广泛,其安全性越来越受到人们的关心。要么是其强度迭不到治疗效果,要么是其强度过大损伤人体,因此其强度已成为人们关心的主要主题。本文主要介绍超声空化的主要探讨了几种测量方法及对超声空化有影响的几种参量,并对超声空化的发展进行了展望。     

Correlation between sonochemistry of surfactant solutions and human leukemia cell killing by ultrasound and porphyrins

The synergistic effect of ultrasound and drugs on cells is known as sonodynamic therapy. The use of sonodynamic therapy for the potential clinical treatment of certain tumors is promising, however, the mechanism of sonodynamic therapy could be due to either sonomechanical and/or sonochemical effects on the cells. The aim of the current study is to determine the importance of the sonochemical mechanism for sonodynamic therapy. Sonochemical effects arise from the formation of radical species following collapse of cavitation bubbles. The synergistic effect of ultrasound (47 kHz) and analogues of a gallium-porphyrin derivative (ATX-70) on cytolysis of Human leukemia cells (HL-525 and HL-60) suspended in a cell culture medium were studied. Organic surfactants preferentially accumulate and subsequently decompose at the gas/solution interface of cavitation bubbles, producing secondary radicals that can diffuse to the bulk solution. The gallium porphyrin analogues used in the current study possess two n-alkyl side chains (ATX-C(x), where x = number of carbon atoms, ranging from x = 2 to x = 12). By varying the n-alkyl chain length, thereby modifying the surfactant properties of the ATX-C(x) derivatives, cell killing in relation to the accumulation of ATX-C(x) derivatives at the gas/solution interface of cavitation bubbles was determined. Following sonolysis in the presence of ATX-C(x), a strong correlation for the yield of carbon-centered radicals and cell killing was observed. These results support the hypothesis that a sonochemical mechanism is responsible for the synergistic effect of ultrasound and ATX-C(x) on HL-525 and HL-60 cells.     

Interactions of inertial cavitation bubbles with stratum corneum lipid bilayers during low-frequency sonophoresis

Interactions of acoustic cavitation bubbles with biological tissues play an important role in biomedical applications of ultrasound. Acoustic cavitation plays a particularly important role in enhancing transdermal transport of macromolecules, thereby offering a noninvasive mode of drug delivery (sonophoresis). Ultrasound-enhanced transdermal transport is mediated by inertial cavitation, where collapses of cavitation bubbles microscopically disrupt the lipid bilayers of the stratum corneum. In this study, we describe a theoretical analysis of the interactions of cavitation bubbles with the stratum corneum lipid bilayers. Three modes of bubble-stratum corneum interactions including shock wave emission, microjet penetration into the stratum corneum, and impact of microjet on the stratum corneum are considered. By relating the mechanical effects of these events on the stratum corneum structure, the relationship between the number of cavitation events and collapse pressures with experimentally measured increase in skin permeability was established. Theoretical predictions were compared to experimentally measured parameters of cavitation events.     

Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizophyllum commune Fries

Schizophyllan, a water-soluble beta-D-glucan elaborated by Schizophyllum commune Fries, was partially depolymerized by ultrasonic irradiation to a low-molecular-weight polysaccharide, designated "sonic-degraded schizophyllan". Both native and degraded polysaccharides exhibited essentially the same antitumor activities against Sarcoma-180 ascites. Both glucans are comprised solely of D-glucose residues and have a main chain of (1 leads to 3)-beta-D-glucopyranosyl residues, one out of three glucose residues being attached as single, (1 leads to 6)-beta-D-glucopyranosyl groups. Although both glucans have similar structural features, significant differences are observed in such physical properties as molecular weight and intrinsic viscosity. End-group analysis by using radioisotope-labeled glucans suggests that ultrasonic degradation occurs mainly by cleavage of glycosidic bonds of the main chain of schizophyllan. The molecular weights of the native and sonic-degraded schizophyllan were shown to be 75% of those of corresponding, original schizophyllan preparations, suggesting that there is no anomalous linkage sensitive to periodate oxidation, and ultrasonic irradiation may cause random hydrolysis of (1 leads to 3)-beta-D-glucosidic linkages in the main chain.     

An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes

Application of ultrasound transiently permeabilizes cell membranes and offers a nonchemical, nonviral, and noninvasive method for cellular drug delivery. Although the ability of ultrasound to increase transmembrane transport has been well demonstrated, a systematic dependence of transport on ultrasound parameters is not known. This study examined cell viability and cellular uptake of calcein using 3T3 mouse cell suspension as a model system. Cells were exposed to varying acoustic energy doses at four different frequencies in the low frequency regime (20-100 kHz). At all frequencies, cell viability decreased with increasing acoustic energy dose, while the fraction of cells exhibiting uptake of calcein showed a maximum at an intermediate energy dose. Acoustic spectra under various ultrasound conditions were also collected and assessed for the magnitude of broadband noise and subharmonic peaks. While the cell viability and transport data did not show any correlation with subharmonic (f/2) emission, they correlated with the broadband noise, suggesting a dominant contribution of transient cavitation. A theoretical model was developed to relate reversible and irreversible membrane permeabilization to the number of transient cavitation events. The model showed that nearly every stage of transient cavitation, including bubble expansion, collapse, and subsequent shock waves may contribute to membrane permeabilization. For each mechanism, the volume around the bubble within which bubbles induce reversible and irreversible membrane permeabilization was determined. Predictions of the model are consistent with experimental data.     

Disintegration of nascent replication bubbles during thymine starvation triggers RecA- and RecBCD-dependent replication origin destruction

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA(+) cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction.