Radiolysis, Photolysis, Sonolysis and Sonoprotection of Cells

工作概述：声动力疗法(8)是一种很有前景的癌症治疗新方式，基于声敏剂和超声联合杀伤细胞的协同效应。超声波可以深入组织，并可以聚焦在肿瘤的一个小区域，以激活无毒分子（如卟啉），从而最大限度地减少不良的副作用。实验证据表明，声敏化是由于在热坍缩空化泡内部或附近的声敏化剂被化学激活，通过在水气界面直接热解敏化剂或通过水的热解形成氢原子和羟基自由基的反应而形成敏化剂衍生自由基。来自声敏剂的自由基（主要以碳为中心）与氧反应形成过氧基和烷氧基自由基。与在空化泡内通过热解形成的OH自由基和H原子不同，烷氧基和过氧基自由基在生物介质中与有机化合物的反应活性要低得多，因此它们到达细胞关键部位的可能性更高。我们最近的研究表明，长链（C5-C8） n-烷基葡萄糖吡喃苷完全抑制超声诱导的细胞溶解(5)。这种保护作用可能应用于HIFU（高强度聚焦超声）的肿瘤治疗和超声辅助药物传递和基因治疗。含有己基（5mM）、庚基（3mM）、辛基（2mM）正烷基链的正烷基吡喃葡萄糖苷在1.057 MHz超声诱导的细胞溶解条件下保护了100%的HL-60细胞，在不含吡喃葡萄糖苷的情况下细胞溶解率为35%至100%。然而，亲水性的甲基- β - d -葡萄糖吡喃苷对细胞没有保护作用。表面活性的n-烷基糖苷在空化气泡的气液界面处聚集。坍塌空化泡中形成的OH自由基和H原子通过从n-烷基葡萄糖苷的正烷基链或葡萄糖部分提取H原子发生反应。由于空化气泡气液界面处长链表面活性剂的高浓度，烷基链上最初形成的碳自由基转移到葡萄糖部分生成自由基，并与氧反应生成过氧化氢。我们最近测量的(2)在614 kHz和1.057 MHz下，长链（己基、辛基）葡萄糖苷与甲基- β - d -葡萄糖苷在氧饱和溶液中过氧化氢产率的比较结果与提出的声保护机制一致。这一系列事件通过过氧化和/或烷氧基自由基在细胞膜上引发脂质过氧化链式反应来防止声动力细胞的杀伤。研究了超声频率（47 kHz至1 MHz）对同源系列n-烷基葡萄糖吡喃苷保护细胞免受超声诱导的细胞溶解能力的影响。将这一系列的n-烷基吡喃葡萄糖苷的保护能力与我们之前研究的它们在气穴气泡的气/液界面上的积累进行比较，表明这些表面活性剂在气/液界面上的积累能力是由这些表面活性剂的动态吸收特性而不是这些表面活性剂的平衡吸收特性决定的。综述了超声在药物激活、细胞凋亡诱导、基因转移和基因表达改变等方面的治疗应用(3)。金纳米颗粒用于药物输送的医学应用最近得到了证实。我们发现，柠檬酸盐覆盖的直径大于15纳米的金纳米颗粒（36微摩尔或更小）不会影响人类细胞的活力。5-氨基乙酰丙酸盐酸盐（5-ALA）或5-氨基乙酰丙酸甲酯盐酸盐（5-ALA- me）在无光照条件下诱导氧化应激导致坏死细胞死亡。17 nm大小的金纳米颗粒与5-ALA衍生物联合使用对多种人类细胞系（乳腺癌细胞（MCF-7）、髓性白血病细胞（HL-60）、肝细胞肝癌细胞（HepG2）和正常人成纤维细胞（1522））的杀伤效果优于单独使用5-ALA衍生物。超氧化物歧化酶和过氧化氢酶对其破坏作用起保护作用。电镜形貌分析表明，5-ALA与金纳米颗粒复合后的损伤与单独使用5-ALA衍生物所造成的损伤基本相同。在这两种情况下，都显示了金纳米颗粒进入细胞的吸收。研究了不同脉冲重复频率对体外低强度超声诱导的化学和生物效应的影响。（与T. Kondo等人合作）李建军，李建军，李建军，等。正烷基葡萄糖吡喃苷的动态吸附性能及其对声空化介导的细胞溶解的抑制作用。物理化学学士B（已出版）刘建军，李建军，李建军，李建军，等。超声诱导肿瘤细胞溶解的研究进展。2 .超声与超声化学学报，2003,22(4):481 - 481。吉田,T。， Kondo， T., Ogawa， R., Zhao， Q., Hassan， M., Watanabe， A., Takasaki， I., Tabuchi， Y., Shoji， M., Kudo， N., Feril， L.，立花，K., Buldakov， M., Honda， T., Tsukada， K.& Riesz， P.，超声分子治疗。药物活化、细胞凋亡诱导、基因转移及基因表达变化的机制。热医学（日本），23,113-122,2007。Cheng， J.Y. & Riesz， P.，长链n-烷基糖苷对超声诱导HL-60细胞溶解的保护作用机制。5.中国生物医学工程学报，2007,32(4):558 - 558。Sostaric， J.Z, Miyoshi， N., Riesz， P., De Graff， W.G.和Mitchell， J.B.， N -烷基葡萄糖吡喃苷完全抑制超声诱导的细胞溶解。6.中国生物医学工程学报，2009,32(5):481 - 481。Feril, l.l.b, Tsuda， Y., Kondo， T., Zhao， Q.L, Ogawa， R.，崔志刚，Tsukada， K., Riesz P.，超声波诱导单核细胞U937细胞凋亡的研究。中国医学杂志，2004,32(5):481 - 481。7. 李建平，李建平，李建平，李建平。超声诱导细胞凋亡和细胞溶解的研究进展。国际放射生物学杂志80,165-175（2004）。8. Rosenthal， I., Sostaric， J. & Riesz， P.，声动力疗法——药物和超声协同作用的综述。超声化学11,349-363（2004）。9. Rosenthal， I., Sostaric， J. & Riesz， P.，开明的声化学。化工中间体研究30,685-701（2004）。

Summary of work: Sonodynamic therapy (8)is a promising new modality for cancer treatment based on the synergistic effects of cell killing by a combination of sonosensitizer and ultrasound. Ultrasound can penetrate deeply into tissue and can be focused in a small region of tumor to activate non-toxic molecules (e.g. porphyrins ) thus minimizing undesirable side effects. The experimental evidence suggests that sonosensitization is due to the chemical activation of sonosensitizers inside or in close vicinity of hot collapsing cavitation bubbles to form sensitizer-derived radicals either by direct pyrolysis of the sensitizer at the water-gas interface or due to the reactions of hydrogen atoms and hydroxyl radicals formed by the pyrolysis of water. The free radicals derived from the sonosensitizer (mostly carbon-centered) react with oxygen to form peroxyl and alkoxyl radicals. Unlike OH radicals and H atoms which are formed by pyrolysis inside cavitation bubbles, the reactivity of alkoxyl and peroxyl radicals with organic compounds in biological media is much lower and hence they have a higher probability of reaching critical cellular sites. Our recent studies have shown that the long chain ( C5-C8 ) n-alkyl glucopyranosides completely inhibit ultrasound induced cytolysis (5). This protective effect has possible applications in HIFU ( High intensity focused ultrasound ) for tumor treatment and in ultrasound assisted drug delivery and gene therapy. n-Alkyl glucopyranosides with hexyl ( 5mM ), heptyl ( 3mM ), octyl ( 2mM ) n-alkyl chains protected 100 % of HL-60 cells in vitro from 1.057 MHz ultrasound induced cytolysis under a range of conditions which resulted in 35% to 100% cytolysis in the absence of glucopyranosides. However the hydrophilic methyl-beta-D-glucopyranoside did not protect cells. The surface active n-alkyl glucopyranosides accumulate at the gas-liquid interface of cavitation bubbles. The OH radicals and H atoms formed in collapsing cavitation bubbles react by H-atom abstraction from either the n-alkyl chain or the glucose moiety of the n-alkylglucopyranosides. Owing to the high concentration of the long chain surfactants at the gas-liquid interface of cavitation bubbles , the initially formed carbon radicals on the alkyl chains are transferred to the glucose moieties to yield radicals which react with oxygen leading to the formation of hydrogen peroxide. Our recent measurements (2) of the hydrogen peroxide yields at 614 kHz and 1.057 MHz from oxygen-saturated solutions of long chain ( hexyl , octyl ) glucopyranosides compared with methyl-beta-D-glucopyranoside are consistent with the proposed mechanism of sonoprotection. This sequence of events prevents sonodynamic cell killing by initiation of lipid peroxidation chain reactions in cellular membranes by peroxyl and/or alkoxyl radicals. The effect of ultrasound frequency (from 47 kHz to 1 MHz ) on the ability of a homologous series of n-alkylglucopyranosides to protect cells from ultrasound-induced cytolysis was investigated. Comparisons of the protective ability of this series of n-alkylglucopyranosides with our earlier studies of their accumulation at the gas/solution interface of cavitation bubbles show that the ability of these surfactants to accumulate at this gas/solution interface is governed by the dynamic absorption properties and not the equilibrium absorption properties of these surfactants. Therapeutic applications of ultrasound to drug activation, apoptosis induction, gene transfer and changes of gene expression were reviewed (3). The medical applications of gold nanoparticles for drug delivery have recently been demonstrated. We have found that citrate capped gold nanoparticles with a size of more than 15 nm diameter ( at 36 micromol or less ) did not affect the viability of human cells. The oxidative stress induced by 5-aminolevulinic acid hydrochloride ( 5-ALA ) or aminolevulinic acid methyl ester hydrochloride (5-ALA-Me) in the absence of light cause necrotic cell killing. The combination of 17 nm sized gold nanoparticles and the 5-ALA derivatives were more effective in cell killing than the 5-ALA derivatives alone for several human cell lines [breast cancer cells (MCF-7),myeloid leukemia cells (HL-60), hepatocellular liver carcinoma cells (HepG2) and normal human fibroblast cells (1522). The damaging effects were protected by superoxide dismutase and catalase.The morphological appearance analysed by electron microscopy showed that the damage by the combination of 5-ALA and gold nanoparticles was almost the same as for the damage induced by 5-ALA derivatives alone. In both cases the uptake of the gold nanoparticles into the cells was shown. The influence of changing Pulse Repetition Frequency on the Chemical and Biological Effects induced by Low Intensity Ultrasound in-vitro was studied. (collaboration with T. Kondo et al.) 1. Sostaric J., Miyoshi N., Cheng J.Y., Riesz P. Dynamic adsorption properties of n-alkyl glucopyranosides determine their ability to inhibit cytolysis mediated by acoustic cavitation. J.of Physical Chemistry B (in press) 2. Miyoshi N., Tuziuti, T. Yasui K., Iida Y., Shimizu N., Riesz P., & Sostaric J.Z.Ultrasound-induced cytolysis of cancer cells is enhanced in the presence of micron-sized alumina particles. Ultrasonics Sonochemistry 15, 881-890, 2008 3. Yoshida,T., Kondo, T., Ogawa, R., Zhao, Q., Hassan, M., Watanabe, A., Takasaki, I., Tabuchi, Y., Shoji, M., Kudo, N., Feril, L., Tachibana, K., Buldakov, M., Honda, T., Tsukada, K.& Riesz, P., Molecular therapy by ultrasound. The mechanism of drug activation, apoptosis induction, gene transfer, and change of gene expressions. Thermal Medicine ( Japan), 23,113-122, 2007 4. Cheng, J.Y. & Riesz, P., Mechanism of the protective effects of long chain n-alkyl glucopyranosides against ultrasound-induced cytolysis of HL-60 cells. Ultrasonics Sonochemistry 14, 667-671 (2007) 5. Sostaric, J.Z., Miyoshi, N., Riesz, P., De Graff, W.G. & Mitchell, J.B., n-Alkyl glucopyranosides completely inhibit ultrasound-induced cytolysis. Free Radical Biology & Medicine 39, 1539-1548, (2005) 6. Feril, L.B., Tsuda, Y., Kondo, T., Zhao, Q.L., Ogawa, R., Cui, Z.G., Tsukada, K. & Riesz P., Ultrasound-induced killing of monocytic U937 cells enhanced by 2,2'-azobis(2-amidinopropane) dihydrochloride. Cancer Science 95, 181-185 (2004). 7. Feril, L., Kondo, T., Takaya, K. & Riesz, P., Enhanced ultrasound-induced apoptosis and cell lysis by a hypotonic medium. International Journal of Radiation Biology 80, 165-175 (2004). 8. Rosenthal, I., Sostaric, J. & Riesz, P., Sonodynamic therapy - a review of the synergistic effects of drugs and ultrasound. Ultrasonics Sonochemistry 11, 349-363 (2004). 9. Rosenthal, I., Sostaric, J. & Riesz, P., Enlightened sonochemistry. Research on Chemical Intermediates 30, 685-701 (2004).