EPR Spin Trapping Study of the Decomposition of Azo Compounds in Aqueous Solutions by Ultrasound: Potential for Use as Sonodynamic Sensitizers for Cell Killing

Sonodynamic therapy, a promising new approach to cancer treatment, is based on synergistic cell killing by combination of certain drugs (sonosensitizers) and ultrasound. Although the mechanism of sonodynamic action is not understood, the role of free radicals produced from sonosensitizers by ultrasound is implicated. In this work, we studied formation of free radicals during the decomposition of several water-soluble azo compounds by 50 kHz ultrasound in aqueous solutions. Using the spin trap 3, 5-dibromo-4-nitrosobenzene sulfonate (DBNBS) tertiary carbon-centered radicals from 2, 2'-azobis (N,N'-dimethyl-eneisobutyramidine) dihydrochloride (VA-044), 2-(carbamoylazo)-isobutyronitrile (V-30), and 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and CH3 radicals from 1, 1'-azobis (N,N'-dimethylformamide) (ADMF) were detected in argonsaturated solutions and the corresponding oxygen-centered radicals (alkoxyl and peroxyl) from VA-044, V-30, and AAPH were identified using the spin trap 5, 5'-dimethyl-l-pyrroline-N-oxide (DMPO) in aerated sonicated solutions. No free radicals from 4, 4'-dihydroxyazobenzene-3, 3'-dicarboxylic acid, disodium salt (DHAB) could be found in either system. While VA-044 and AAPH could also be readily decomposed by heat (42.5°C and 80°C), V-30 decomposition only occurred in the ultrasound-exposed solutions. The most likely mechanism of decomposition of azo compounds by ultrasound is their thermolysis in the heated shell of the liquid surrounding ca vita ting bubbles driven by ultrasound and/or by pyrolysis inside these bubbles. Experiments using scavengers of ·OH and ·H, which are produced by sonolysis in aqueous solutions, demonstrated that these radicals are not involved in the ultrasound-mediated radical production from the azo compounds. Due to the known cytotoxic potential of free radicals produced from azo compounds, the use of these compounds as ultrasound sensitizers appears to be a promising approach for sonodynamic cell killing.     

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.     

Mechanism of porphyrin-induced sonodynamic effect: possible role of hyperthermia

The biological effects of ultrasound have been investigated vigorously for various applications including the thermal coagulation of tissues, the opening of tight junctions, and localized gene or drug introduction. The synergistic cell killing effect of ultrasound and porphyrin derivatives, the so-called sonodynamic effect, holds promise for cancer treatment. Although several models to explain the sonodynamic effect have been proposed, its exact mechanism, especially in vivo, remains unknown. We examined the effect of a porphyrin derivative, protoporphyrin IX, on ultrasound-induced killing of HeLa cells. In some experiments, the intracellular protoporphyrin IX concentration was increased by 5-aminolevulinic acid treatment of the cells. Although extracellular protoporphyrin IX showed an enhanced cell killing effect by microbubble-enhanced ultrasound, intracellular protoporphyrin IX did not. On the other hand, intracellular protoporphyrin IX enhanced the cell killing effect of hyperthermia, which can be produced by ultrasound exposure, in a moderately acidic environment (pH 6.6). Because porphyrin derivatives are generally imported into the intracellular component in vivo, our results suggest that hyperthermia caused by ultrasound may play an important role in the sonodynamic effect induced by porphyrin derivatives.     

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.     

Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 W x cm(-2) in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were--1 s drop time, 5 mV x s(-1) voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag/AgCl/3 M KCl as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy.     

Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

Sonodynamic therapy (SDT) is one of antitumor strategies that kill tumor cells through the synergistic effects caused by the combined use of HpD and US[1]. SDT is based on the following principle. Ultrasound used in SDT can penetrate the deep tissue and activate HpD, which accu- mulated in tumor cell, and produce highly active oxygen species[2] such as singlet oxygen (1O2), which can destroy the structure of tumor cells. So far the studies on the SDT have focused mainly on the mechan…     

Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

The inhibition of ascitic S180 and induced sarcoma 180 in vivo was studied with the combination of hematoporphyrin derivatives (HpD) and ultrasound (US) at the frequency of 1.1 MHz and different intensities by light microscopy observation, electronic microscopy observation, cytochemical analysis and fluorescence labeling. The present study indicated that the injury of ascitic S180 increased as time passed and the inhibitory effect was stronger in US plus HpD group than that in other groups. Our results also indicated that the changes in cell structure, cytochrome C oxidase activity, the degradation and missing of DNA were the important factors that inhibited the tumor cell growth and even induced celldeath. The phenomenon of apoptosis of tumor cells indicated that cell death andinduced apoptosis exist in the treatment of sonodynamic therapy (SDT). Our study investigated the mechanism underlying the killing effect of S180 induced by USactivating HpD by the observation of cell morphology and dynamic changes from seminal injury to succeeded injury even to death. It would provide rich referencefor the study of SDT.     

Absence of synergistic enhancement of non-thermal effects of ultrasound on cell killing induced by ionizing radiation

The present study was performed to elucidate the role of non-thermal effects (cavitation and direct effects) of ultrasound, in simultaneous combination with X-irradiation on the cytotoxicity of mouse L cells. Firstly, mouse L cells were exposed to X-rays and ultrasound (1 MHz continuous wave, spatial peak temporal average intensity; 3.7 W/cm2) simultaneously at 37 degrees C under O2 or Ar saturated conditions to examine the cavitational effect of ultrasound. Secondly, cells were exposed to X-rays and ultrasound at 37 degrees C under N2O saturated conditions, which suppresses the cavitation, to examine the direct effects of ultrasound. The cavitational effect under O2 and Ar saturated conditions induced an exponential decrease in cell survival, and resulted in an additive effect on cell killing with the combination of X-rays and ultrasound. The direct effect in the N2O conditions induced no cell killing and did not modify the cell killing induced by X-rays. These results suggested that the non-thermal effects of ultrasound did not interact synergistically with X-rays for cell killing.     

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.     

Effect of non-volatile scavengers of hydroxyl radicals on thymine radical formation induced by gamma-rays and ultrasound

In order to investigate the mechanism of sonolysis of nucleic acid constituents, the yield of thymine radicals generated by 50 kHz ultrasound in Ar-saturated aqueous solution was compared with that formed by gamma-radiolysis in N2O-saturated solutions in the presence of various non-volatile scavengers, which cannot act in the gas phase of the cavitation bubbles. For comparison of thymine radical yields by sonolysis and gamma radiolysis, the method of spin trapping with 3,5-dibromo-4-nitrosobenzenesulphonate (a water-soluble, non-volatile, aromatic nitroso spin trap) combined with ESR was used. The efficiency of OH radical scavenging is expressed by the reciprocal value of C1/2, the scavenger concentration at which the thymine radical yield is decreased by 50 per cent. In gamma radiolysis the scavenging efficiencies of the solutes depend on their rate constants with OH radicals. For sonolysis the C1/2 values were similar to those obtained for gamma radiolysis except for the hydrophobic 5,5-dimethyl-1-pyrroline-N-oxide. These results suggest that thymine radicals induced by ultrasound are produced in the bulk of the solution as well as in the interfacial region.     

超声空化效应和超声微泡在生物医学中的应用

近年来,随着超声技术在医疗领域的广泛应用及超声造影剂研制的进展,空化效应和超声造影剂协同运用作为一种高效、安全、操作简单,且具有一定靶向性的无创治疗手段,在基因治疗、药物输送、溶栓和治栓,以及炎症与肿瘤的靶向诊断与治疗方面显示了巨大的运用潜力。简要综述了超声空化效应和超声微泡的治疗机制及应用。     

EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media

Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic. The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out. In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids. Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect. In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease. These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.     

Involvement of protein kinase C-beta and ceramide in tumor necrosis factor-alpha-induced but not Fas-induced apoptosis of human myeloid leukemia cells.

The role of protein kinase C-beta (PKC-beta) in apoptosis induced by tumor necrosis factor (TNF)-alpha and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-beta. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-alpha-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-beta reestablished their susceptibility to TNF-alpha-induced apoptosis. The apoptotic effect of TNF-alpha in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-beta deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-alpha-induced apoptosis involves PKC-beta and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.     

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.     

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.     

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.     

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.     

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.