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.     

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.     

Use of chemical dosimetry for comparison of ultrasound and ionizing radiation effects on cavitation

A comparison of the effects of ultrasound produced by low- and high-frequency ultrasonic apparatuses upon biological systems is one of the basic problems when studying ultrasound cavitation effects. One possibility for how to compare these effects is the indirect method which uses well-known physical quantities characterizing the interaction of ionizing radiation with matter and which also converts these quantities to one common physical quantity. The comparison was performed with two methods applied to the chemical dosimetry of ionizing radiation. The first method employed a two-component dosimeter which is composed of 50 % chloroform and 50 % re-distilled water (i.e. Taplin dosimeter). The other method used a modified iodide dosimeter prepared from a 0.5 M potassium iodide solution. After irradiation or ultrasound exposure, measurable chemical changes occurred in both dosimeters. The longer the exposure, the greater the chemical changes. These effects are described by the relationship of these changes versus the exposure times in both dosimeters. The UZD 21 ultrasonic disintegrator (with a frequency of 20 kHz, 50 % power output) was used as a low-frequency ultrasound source, and the BTL-07 therapeutic instrument (with a frequency of 1 MHz and intensity of 2 W/cm2) was used as a high-frequency cavitation ultrasound source. For comparison, a 60 Co gamma source was applied (60 Co, gamma energies of 1.17 and 1.33 MeV, activity of 14 PBq). Results of this study have demonstrated that the sonochemical products are generated during exposure in the exposed samples of both dosimeters for all apparatuses used. The amount of these products depends linearly upon the exposure time. The resulting cavitation effects were recalculated to a gray-equivalent dose (the proposed unit is cavitation gray [cavitGy]) based on the sonochemical effects compared to the effects of ionizing radiation from the 60 Co source.     

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.     

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.     

Monitoring and control of inertial cavitation activity for enhancing ultrasound transfection: The SonInCaRe project

Sonoporation process, at the origin of ultrasound cell transfection, is ruled by the interaction between cells and cavitating bubbles. Due to the stochastic behavior of acoustic cavitation, there exists a need in ensuring a stable state of cavitation within a medium during cell transfection to enhance transfection efficiency and control mortality. The goal of the SonInCaRe project is thus to define a controlled-cavitation device in order to monitor, control and stabilize inertial cavitation activity during cell sonication in real-time. This device, based on a feedback loop acting in real-time, allows ensuring fixed cavitation conditions during a pulsed sonication. Its application to suspended and adherent cells transfection shows better reproducibility compared to the fixed acoustic intensity sonication. The regulation device thus provides a better control of cavitation activity and its bioeffects which are of crucial importance for clinical applications of ultrasound-mediated gene transfection.     

Free Radical Formation by Ultrasound in Aqueous Solutions. A Spin Trapping Study

Our recent spin trapping studies of free radical generation by ultrasound in aqueous solutions are reviewed. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions exposed to ultrasound lead to the thermal dissociation of water vapor into H atoms and OH radicals. Their formation has been confirmed by spin trapping. Sonochemical reactions occur in the gas phase (pyrolysis reactions), in the gas-liquid interfacial region, and in the bulk of the solution (radiation-chemistry reactions). The high temperature gradients in the interfacial regions lead to pyrolysis products from non-volatile solutes present at sufficiently high concentrations. The sonochemically generated radicals from carboxylic acids, amino acids, dipeptides. sugars, pyrimidine bases. nucleosides and nucleo-tides were identified by spin trapping with the non-volatile spin trap 3.5-dibromo-2.6-dideuterio-4-nitrosobenzenesulfonate. At low concentrations of the non-volatile solutes. the spin-trapped radicals produced by sonolysis are due to H atom and OH radical reactions. At higher concentrations of these non-volatile solutes, sonolysis leads to the formation of additional radicals due to pyrolysis processes (typically methyl radicals). A preferred localization of non-volatile surfactants (compared to analogous non-surfactant solutes) was demonstrated by the detection of pyrolysis radicals at 500-fold lower concentrations. Pyrolysis radicals were also found in the sonolysis of aqueous solutions containing only certain nitrone spin traps. The more hydrophobic the spin trap, the lower the concentration at which the pyrolysis radicals can be observed. The effect of varying the temperature of collapsing transient cavities in aqueous solutions of different rare gases and of N₂O on radical yields and on cell lysis of mammalian cells was investigated.     

Sonochemistry of nitrone spin traps in aqueous solutions. Evidence for pyrolysis radicals from spin traps

When argon-saturated aqueous solutions of alpha-phenyl-N-tert-butylnitrone (PBN) were sonicated, the spin adducts PBN-Phenyl (Ph), PBN-X, and PBN-H were observed. It can be inferred that PBN-Ph and -X arise from spin adducts of thermal decomposition products of PBN induced by the high temperature due to ultrasonic cavitation. The ESR signal of PBN-H was observed at a lower PBN concentration than those of PBN-Ph and PBN-X. The ratios of ESR intensity of PBN-H to those of PBN-Ph and PBN-X increased with the final temperatures of the cavitation bubbles created by different rare gases. The spin adducts of methyl and tert-butyl radicals from the pyrolysis of PBN, induced by the high temperatures due to cavitation, were found from spin trapping experiments in which 3,5-dibromo-2,6-dideuterio-4-nitrosobenzene sulfonate was used as a spin trap. Similar spin adducts induced by pyrolysis were also observed in sonicated aqueous solutions of other nitrone spin traps, such as alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and alpha-(4-nitrophenol) N-tert-butylnitrone. The greater the hydrophobicity of the spin traps, as measured by the 2-octanol/water partition coefficients, the lower the concentration of spin trap at which methyl radicals generated by thermal decomposition of the spin trap can be observed. The present results indicate that the nonvolatile, highly hydrophobic spin traps accumulate preferentially in the interfacial region of cavitation bubbles where they undergo thermal decomposition during cavitation to produce the radicals.     

Submicron-Bubble-Enhanced Focused Ultrasound for Blood–Brain Barrier Disruption and Improved CNS Drug Delivery

The use of focused ultrasound (FUS) with microbubbles has been proven to induce transient blood–brain barrier opening (BBB-opening). However, FUS-induced inertial cavitation of microbubbles can also result in erythrocyte extravasations. Here we investigated whether induction of submicron bubbles to oscillate at their resonant frequency would reduce inertial cavitation during BBB-opening and thereby eliminate erythrocyte extravasations in a rat brain model. FUS was delivered with acoustic pressures of 0.1–4.5 MPa using either in-house manufactured submicron bubbles or standard SonoVue microbubbles. Wideband and subharmonic emissions from bubbles were used to quantify inertial and stable cavitation, respectively. Erythrocyte extravasations were evaluated by in vivo post-treatment magnetic resonance susceptibility-weighted imaging, and finally by histological confirmation. We found that excitation of submicron bubbles with resonant frequency-matched FUS (10 MHz) can greatly limit inertial cavitation while enhancing stable cavitation. The BBB-opening was mainly caused by stable cavitation, whereas the erythrocyte extravasation was closely correlated with inertial cavitation. Our technique allows extensive reduction of inertial cavitation to induce safe BBB-opening. Furthermore, the safety issue of BBB-opening was not compromised by prolonging FUS exposure time, and the local drug concentrations in the brain tissues were significantly improved to 60 times (BCNU; 18.6 µg versus 0.3 µg) by using chemotherapeutic agent-loaded submicron bubbles with FUS. This study provides important information towards the goal of successfully translating FUS brain drug delivery into clinical use.     

Acoustic emission analysis and experiments with physical model systems reveal a peculiar nature of the xylem tension

Advanced acoustic emission analysis, special microscopic examinations and experiments with physical model systems give reasons for the assumption that the tension in the water conducting system of vascular plants is caused by countless minute gas bubbles strongly adhering to the hydrophobic lignin domains of the xylem vessel walls. We ascertained these bubbles for several species of temperate deciduous trees and conifers. It is our hypothesis that the coherent bubble system of the xylem conduits operates as a force-transmitting medium that is capable of transporting water in traveling peristaltic waves. By virtue of the high elasticity of the gas bubbles, the hydro-pneumatic bubble system is capable of cyclic storing and releasing of energy. We consider the abrupt regrouping of the wall adherent bubble system to be the origin of acoustic emissions from plants. For Ulmus glabra, we recorded violent acoustic activity during both transpiration and re-hydration. The frequency spectrum and the waveforms of the detected acoustic emissions contradict traditional assumptions according to which acoustic emissions are caused by cavitation disruption of the stressed water column. We consider negative pressure in terms of the cohesion theory to be mimicked by the tension of the wall adherent bubble system.     

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。     

Dynamics of sonoporation correlated with acoustic cavitation activities

Sonoporation has been exploited as a promising nonviral strategy for intracellular delivery of drugs and genes. The technique utilizes ultrasound application, often facilitated by the presence of microbubbles, to generate transient, nonspecific pores on the cell membrane. However, due to the complexity and transient nature of ultrasound-mediated bubble interaction with cells, no direct correlation of sonoporation with bubble activities such as acoustic cavitation, i.e., the ultrasound-driven growth and violent collapse of bubbles, has been obtained. Using Xenopus oocytes as a model system, this study investigated sonoporation in a single cell affected by colocalized cavitation in real time. A confocally and collinearly-aligned dual-frequency ultrasound transducer assembly was used to generate focused ultrasound pulses (1.5 MHz) to induce focal sonoporation while detecting the broadband cavitation acoustic emission within the same focal zone. Dynamic sonoporation of the single cell was monitored via the transmembrane current of the cell under voltage-clamp. Our results demonstrate for the first time, to our knowledge, the spatiotemporal correlation of sonoporation with cavitation at the single-cell level.     

Ultrasound: mechanical gene transfer into plant cells by sonoporation

Development of nonviral gene transfer methods would be a valuable alternative of gene therapy or transformation. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation. Cavitation bubbles can induce cell death or transient membrane permeabilization (sonoporation) on cells. Application of sonoporation for gene transfer into cells or tissues develops quickly in recent years. Many studies have been performed in vitro exposure systems to a variety of cell lines transfected successfully. In vivo, cavitation initiation and control are more difficult, but can be enhanced by ultrasound contrast agents (microbubbles). The use of ultrasound for nonviral gene delivery has been applied for mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine. In this paper, ultrasound applied to plant cell transformation or gene transfer is reviewed. Recently, most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues. Microbubbles are also proposed to apply to gene transfer in plant cells and tissues.     

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.     

n-Alkyl glucopyranosides completely inhibit ultrasound-induced cytolysis

The mechanism(s) responsible for sudden cytolysis observed when cells are exposed to ultrasound could be mechanical and/or free radical in nature. Free radical reactions are initiated in the core and in the interfacial regions of collapsing acoustic cavitation bubbles. Because cyclic sugars are known to inhibit free radical chain reactions, we investigated the effects of n-alkyl-β-d-glucopyranosides of varying hydrophobicity on ultrasound (1.057 MHz)-induced cytolysis of HL-60 cells in vitro. n-Alkyl glucopyranosides with hexyl- (5 mM), heptyl- (3 mM), or octyl- (2 mM) n-alkyl chains protected 100% of the cell population from ultrasound-induced cytolysis under a range of conditions that resulted in 35 to 100% cytolysis in the absence of glucopyranosides. The protected cell populations also possessed long-term reproductive viability. However, the hydrophilic methyl-β-d-glucopyranoside could not protect cells, even up to a concentration of 30 mM. Furthermore, none of the glucopyranosides could prevent cytolysis of cells from a mechanically induced shear stress. Spin trapping and electron spin resonance experiments confirmed the presence of inertial cavitation in cell suspensions both in the presence and in the absence of the surfactants. It is concluded that surface-active glucopyranosides efficiently quench cytotoxic radicals and/or their precursors at the gas/solution interface of collapsing cavitation bubbles.     

n-Alkyl glucopyranosides completely inhibit ultrasound-induced cytolysis

The mechanism(s) responsible for sudden cytolysis observed when cells are exposed to ultrasound could be mechanical and/or free radical in nature. Free radical reactions are initiated in the core and in the interfacial regions of collapsing acoustic cavitation bubbles. Because cyclic sugars are known to inhibit free radical chain reactions, we investigated the effects of n-alkyl-β-d-glucopyranosides of varying hydrophobicity on ultrasound (1.057 MHz)-induced cytolysis of HL-60 cells in vitro. n-Alkyl glucopyranosides with hexyl- (5 mM), heptyl- (3 mM), or octyl- (2 mM) n-alkyl chains protected 100% of the cell population from ultrasound-induced cytolysis under a range of conditions that resulted in 35 to 100% cytolysis in the absence of glucopyranosides. The protected cell populations also possessed long-term reproductive viability. However, the hydrophilic methyl-β-d-glucopyranoside could not protect cells, even up to a concentration of 30 mM. Furthermore, none of the glucopyranosides could prevent cytolysis of cells from a mechanically induced shear stress. Spin trapping and electron spin resonance experiments confirmed the presence of inertial cavitation in cell suspensions both in the presence and in the absence of the surfactants. It is concluded that surface-active glucopyranosides efficiently quench cytotoxic radicals and/or their precursors at the gas/solution interface of collapsing cavitation bubbles.     

Sonolysis, radiolysis, and hydrogen peroxide photolysis of pyrimidine derivatives in aqueous solutions: a spin-trapping study

The sonolysis of aqueous solutions of various dihydropyrimidines and substituted pyrimidines was investigated by ESR and spin trapping with the nonvolatile, water soluble spin trap, 3,5-dibromonitrosobenzene sulfonate (DBNBS) and its deuterated analog to examine the possibility of detecting new radicals specifically generated in the high temperature zones produced by collapsing cavitation bubbles. Similar ESR spectra were obtained from sonolysis of argon-saturated aqueous solutions, from uv photolysis of aqueous solutions containing H2O2, and from gamma radiolysis of nitrous oxide saturated solutions, although sonolysis of aqueous solutions leads to the formation of pyrimidine radicals by H atom as well as OH radical addition to the 5,6 double bond of pyrimidines. No evidence for specific new radicals formed in the high temperature regions induced by cavitation could be found. For the reactions of dihydropyrimidines with hydroxyl radicals additional spin adducts could be detected and identified with the spin trap DBNBS compared to 2-methyl-2-nitrosopropane which was used in previous studies; however, for alkylpyrimidines fewer spin adducts were observed. The use of the deuterated analog of DBNBS is helpful for unambiguous radical structure assignment.     

The ultrasonic degradation of biological macromolecules under conditions of stable cavitation. I. Theory,methods, and application to deoxyribonucleic acid

Solutions of calf thymus DNA have been degraded in the presence of vibrating air bubbles in ultrasonic fields of low power which would not normally induce ultrasonic cavitation. The DNA was degraded to a limiting intrinsic viscosity, after which further irradiation by ultrasound had little or no effect. This limiting intrinsic viscosity decreased with increase in the ultrasonic intensity. Previously developed theories have-been adapted to calculate the maximum velocity gradient associated with the streaming of the solution around such vibrating air bubbles. The tensile force which is induced and which acts on the DNA has been calculated on the basis of current theories of degradation by hydrodynamic shear. These calculations indicate that the degradation of the DNA by ultrasound under conditions of “stable cavitation” is mainly the result of the shearing forces engendered in the solution around the oscillating bubbles.     

Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H₂, H₂O₂, and HO₂. The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.     

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.