Mechanistic Insight into Sonoporation with Ultrasound-Stimulated Polymer Microbubbles

Sonoporation is emerging as a feasible, non-viral gene delivery platform for the treatment of cardiovascular disease and cancer. Despite promising results, this approach remains less efficient than viral methods. The objective of this work is to help substantiate the merit of polymeric microbubble sonoporation as a non-viral, localized cell permeation and payload delivery strategy by taking a ground-up approach to elucidating the fundamental mechanisms at play. In this study, we apply simultaneous microscopy of polymeric microbubble sonoporation over its intrinsic biophysical timescales–with sub-microsecond resolution to examine microbubble cavitation and millisecond resolution over several minutes to examine local macromolecule uptake through enhanced endothelial cell membrane permeability–bridging over six orders of magnitude in time. We quantified microbubble behavior and resulting sonoporation thresholds at transmit frequencies of 0.5, 1 and 2 MHz, and determined that sonic cracking is a necessary but insufficient condition to induce sonoporation. Further, sonoporation propensity increases with the extent of sonic cracking, namely, from partial to complete gas escape from the polymeric encapsulation. For the subset that exhibited complete gas escape from sonic cracking, a proportional relationship between the maximum projected gas area and resulting macromolecule uptake was observed. These results have revealed one aspect of polymeric bubble activity on the microsecond time scale that is associated with eliciting sonoporation in adjacent endothelial cells, and contributes toward an understanding of the physical rationale for sonoporation with polymer-encapsulated microbubble contrast agents.     

Interactions between Individual Ultrasound-Stimulated Microbubbles and Fibrin Clots

The use of ultrasound-stimulated microbubbles (USMBs) to promote thrombolysis is well established, but there remains considerable uncertainty about the mechanisms of this process. Here we examine the microscale interactions between individual USMBs and fibrin clots as a function of bubble size, exposure conditions and clot type. Microbubbles (n = 185) were placed adjacent to clot boundaries (“coarse” or “fine”) using optical tweezers and exposed to 1-MHz ultrasound as a function of pressure (0.1–0.39 MPa). High-speed (10 kfps) imaging was employed, and clots were subsequently assessed with 2-photon microscopy. For fine clots, 46% of bubbles “embedded” within 10 μm of the clot boundary at pressures of 0.1 and 0.2 MPa, whereas at 0.39 MPa, 53% of bubbles penetrated and transited into the clots with an incidence inversely related to their diameter. A substantial fraction of penetrating bubbles induced fibrin network damage and promoted the uptake of nanobeads. In coarse clots, penetration occurred more readily and at lower pressures than in fine clots. The results therefore provide direct evidence of therapeutically relevant effects of USMBs and indicate their dependence on size, exposure conditions and clot properties.     

Lipid Microbubbles as Ultrasound-Stimulated Oxygen Carriers for Controllable Oxygen Release for Tumor Reoxygenation

Microbubbles are proposed as a potentially novel method for oxygen delivery in vivo in initial studies. The lack of commercial microbubbles for oxygen delivery in preclinical research prompted us to fabricate an oxygen-loaded lipid microbubble. We aimed to extend the innovative strategy to modulate the tumor hypoxic microenvironment, using microbubbles intravenously as an oxygen carrier for the controllable tumor-specific delivery of oxygen by ultrasound (US). In our experiment, an oxygen-loaded lipid-coated microbubble (OLM) with mixed gas (O₂/C₃ F₈, 5:1 v/v) was fabricated and exhibited a higher rate of oxygen release to a desaturated solution through burst by US than that in the absence of US. Although in in vivo studies, OLMs could be imaged and triggered by US to elevate the pO₂ level in the breast VX2 tumor dramatically within a matter of minutes. The added presence of US-activated OLMs elicited a nearly six-fold increase in pO₂ levels within 1?min compared with that of the pre-injection. Owing to the high oxygen payload, great acoustic stability and acoustic properties, OLMs may be proposed as an ideal radio-sensitizer. We conclude that oxygen release mediated by ultrasound-targeted microbubble destruction is feasible and shows potential in image-guided, site-specific cancer radiotherapy.     

Effects of Biophysical Parameters in Enhancing Radiation Responses of Prostate Tumors with Ultrasound-Stimulated Microbubbles

We show here that ultrasound-stimulated microbubbles can enhance cell death within tumors when combined with radiation. The aim of this study was to investigate how different ultrasound parameters, different microbubble concentrations and different radiation doses interact to enhance cell death. Prostate xenograft tumors (PC-3) in severe combined immunodeficiency mice were subjected to ultrasound treatment at various peak negative pressures (250, 570 and 750 kPa) at a center frequency of 500 kHz, different microbubble concentrations (8, 80 and 1000 μL/kg) and different radiation doses (0, 2 and 8 Gy). Twenty-four hours after treatment, tumors were excised and assessed for cell death. Histologic analyses revealed that increases in radiation dose, microbubble concentration and ultrasound pressure promoted apoptotic cell death and disruption within tumors by as much as 21%, 30% and 43%, respectively. Comparable increases in ceramide, a cell death mediator, were identified using immunohistochemistry. We also show here that even clinically used microbubble concentrations combined with ultrasound can induce significant enhancement of cell death.     

Bioeffects of Ultrasound-Stimulated Microbubbles on Endothelial Cells: Gene Expression Changes Associated with Radiation Enhancement In Vitro

Ultrasound can be used to target endothelial cells in cancer therapy where the destruction of vasculature leads to tumor cell death. Here, we demonstrate ultrasound bioeffects in which the levels of genes in endothelial cells can be significantly altered by ultrasound-stimulated microbubble exposure. These were compared with established effects of radiation on endothelial cells at a gene level. Human-endothelial cells were exposed to ultrasound and microbubbles, radiation or combinations of ultrasound, microbubbles and radiation. Gene expression analyses revealed an up-regulation of genes known to be involved in apoptosis and ceramide-induced apoptotic pathways, including SMPD2, UGT8, COX6B1, Caspase 9 and MAP2K1 with ultrasound-stimulated microbubble exposure but not SMPD1. This was supported by immunohistochemistry and morphologic changes examined with cell microscopy, which showed changes in SMPD1 gene product in cells with microbubble exposure. This supports the hypothesis that ultrasound-stimulated microbubbles can induce significant bioeffect-related changes in gene expression and can affect ceramide signaling pathways in endothelial cells, leading to apoptosis.     

干酪乳杆菌对免疫效应细胞杀伤作用影响的研究

目的研究干酪乳杆菌对自然杀伤细胞杀伤肿瘤细胞活性的影响。方法干酪乳杆菌与结肠癌细胞系HT-29细胞37℃5%CO2环境中共同孵育12h,应用MTT法检测NK细胞对HT-29细胞的杀伤活性。结果干酪乳杆菌作用HT-29细胞明显增强NK对其杀伤活性。结论该研究为益生菌抗肿瘤作用的研究提供了新的依据。     

超声辐照LHRHa靶向微泡增强顺铂对卵巢癌移植瘤的抑制作用

目的 探讨超声辐照携带促黄体生成素释放激素类似物(LHRHa)的靶向微泡能否增强顺铂(DDP )对人卵巢癌裸鼠腹腔移植瘤的抑制作用.方法 利用LHRH受体阳性的人卵巢癌耐药细胞A2780/DDP建立裸鼠腹腔移植瘤模型.将30只荷瘤鼠随机分成6组,即DDP+靶向微泡+超声组(Ⅰ),DDP+普通微泡+超声组(Ⅱ),DDP组(Ⅲ ),靶向微泡+超声组(Ⅳ),靶向微泡组(Ⅴ)和生理盐水对照组,Ⅰ～V组为实验组.处理结束后记录肿瘤个数,剖取瘤块称湿质量,计算抑瘤率;对肿瘤及主要脏器进行病理组织学检查;免疫组化SP法检测肿瘤组织中基质金属蛋白酶-9 (MMP-9)的表达.结果 各组的肿瘤个数、质量和MMP-9表达均有差异(P<0.01),其中,工组的抑瘤率最大,其肿瘤个数、质量和MMP-9表达均显著低于其他4个实验组(P<0.05)和对照组(P<0.01).结论 超声辐照LHRHa靶向微泡能显著增强DDP对卵巢癌移植瘤的抑制作用.     

Plasma Membrane Blebbing Dynamics Involved in the Reversibly Perforated Cell by Ultrasound-Driven Microbubbles

The perforation of plasma membrane by ultrasound-driven microbubbles (i.e., sonoporation) provides a temporary window for transporting macromolecules into the cytoplasm that is promising with respect to drug delivery and gene therapy. To improve the efficacy of delivery while ensuring biosafety, membrane resealing and cell recovery are required to help sonoporated cells defy membrane injury and regain their normal function. Blebs are found to accompany the recovery of sonoporated cells. However, the spatiotemporal characteristics of blebs and the underlying mechanisms remain unclear. With a customized platform for ultrasound exposure and 2-D/3-D live single-cell imaging, localized membrane perforation was induced with ultrasound-driven microbubbles, and the cellular responses were monitored using multiple fluorescent probes. The results indicated that localized blebs undergoing four phases (nucleation, expansion, pausing and retraction) on a time scale of tens of seconds to minutes were specifically involved in the reversibly sonoporated cells. The blebs spatially correlated with the membrane perforation site and temporally lagged (about tens of seconds to minutes) the resealing of perforated membrane. Their diameter (about several microns) and lifetime (about tens of seconds to minutes) positively correlated with the degree of sonoporation. Further studies revealed that intracellular calcium transients might be an upstream signal for triggering blebbing nucleation; exocytotic lysosomes not only contributed to resealing of the perforated membrane, but also to the increasing bleb volume during expansion; and actin components accumulation facilitated bleb retraction. These results provide new insight into the short-term strategies that the sonoporated cell employs to recover on membrane perforation and to remodel membrane structure and a biophysical foundation for sonoporation-based therapy.     

Modulation of Gyrase-Mediated DNA Cleavage and Cell Killing by ATP

An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and an aconitase inhibitor, fluoroacetic acid, both of which are known to lower the cellular ATP pool, protected Escherichia coli cells from the bactericidal actions of gyrase poisons including quinolone antibiotics, nalidixic acid and ciprofloxacin, and the epipodophyllotoxins VP-16 and VM-26. Using purified E. coli DNA gyrase, we examined the effect of ATP on gyrase-mediated DNA cleavage in the presence of these gyrase poisons. ATP was shown to stimulate gyrase-mediated DNA cleavage from 10- to more than 100-fold in the presence of these gyrase poisons. ADP antagonized the stimulatory effect of ATP. Consequently, gyrase-mediated DNA cleavage induced by gyrase poisons is modulated by the ATP concentration/ADP concentration ([ATP]/[ADP]) ratio. Coumermycin A1, an inhibitor of the ATPase subunit of DNA gyrase, like ADP, also effectively antagonized the stimulatory effect of ATP on gyrase-mediated DNA cleavage induced by gyrase poisons. Furthermore, coumermycin A1, like DNP and fluoroacetic acid, also protected cells from the bactericidal action of gyrase poisons. In the aggregate, our results are consistent with the notion that the [ATP]/[ADP] ratio, through its modulatory effect on the gyrase-mediated DNA cleavage, is an important determinant of cellular susceptibility to gyrase poisons.     

Cell Killing by Simian Virus 40: Protective Effect of Chloroquine

Treatment of CV-1 cells with chloroquine before infection by simian virus 40 resulted in the accumulation of fewer nonviable, trypan blue-stainable cells at 72 h. The drug did not affect the fraction of infected T-antigen-producing cells or the viral yields. It did diminish the apparent redistribution of lysosomal N-acetyl-beta-glucosaminidase from a particulate to a soluble cell fraction, and it caused an increase in the size and number of lysosomes.     

CD3AK细胞对白血病细胞杀伤活性的实验研究

目的应用CD3单克隆抗体(CD3McAb)和重组人白细胞介素-2(rhIL-2)共同诱导外周血单个核细胞制备CD3AK细胞,研究其对白血病细胞的杀伤作用。方法用台盼蓝活细胞计数法计算细胞扩增倍数,MTT法检测CD3AK细胞杀伤活性。结果正常人CD3AK细胞对各型急性白血病细胞及K562细胞均有明显的杀伤活性,且无显著性差异(P>0.05);急性白血病完全缓解期CD3AK细胞与正常人CD3AK细胞对白血病细胞的杀伤活性,亦无显著性差异(P>0.05);急性白血病未缓解期CD3AK对白血病细胞杀伤活性明显减弱,与正常人CD3AK细胞比较,差异非常显著(P<0.01)。结论正常人与急性白血病完全缓解期CD3AK细胞具有明显的抗白血病作用,急性白血病未缓解期CD3AK细胞杀伤活性明显减弱。     

T Cell Receptor Signals Enhance Susceptibility to Fas-mediated Apoptosis

Fas(CD95) and its ligand (FasL) interaction plays a pivotal role in T cell receptor (TCR)-mediated apoptosis. However, the susceptibility of T cells to Fas-mediated apoptosis is tightly regulated during immune responses, a regulation which is thought to maintain the antigen-specificity of T cell apoptosis. Here we show that TCR stimulation enhances the induction of Fas-mediated apoptosis. In addition, using a mutant T cell hybridoma with impaired FasL expression, we show that the synergy provided by TCR stimulation can be mimicked by activators of PKC but not calcium influx. This effect cannot be inhibited by actinomycin D, suggesting that TCR stimulation leads to the alteration in preexisting signaling molecules to enhance Fas-mediated apoptosis. Our results therefore provide a mechanism of how Fas-FasL interactions lead to T cell death in an antigen-specific manner via repetitive antigen stimulation.     

Amalgamating Oncolytic Viruses to Enhance Their Safety,Consolidate Their Killing Mechanisms,and Accelerate Their Spread

Oncolytic viruses are structurally and biologically diverse, spreading through tumors and killing them by various mechanisms and with different kinetics. Here, we created a hybrid vesicular stomatitis/measles virus (VSV/MV) that harnesses the safety of oncolytic MV, the speed of VSV, and the tumor killing mechanisms of both viruses. Oncolytic MV targets CD46 and kills by forcing infected cells to fuse with uninfected neighbors, but propagates slowly. VSV spreads rapidly, directly lysing tumor cells, but is neurotoxic and loses oncolytic potency when neuroattenuated by conventional approaches. The hybrid VSV/MV lacks neurotoxicity, replicates rapidly with VSV kinetics, and selectively targets CD46 on tumor cells. Its in vivo performance in a myeloma xenograft model was substantially superior to either MV or widely used recombinant oncolytic VSV-M51.     

自制靶向脂质微泡与普通脂质微泡超声显影的对比实验研究

目的探讨自制的靶向脂质微泡超声造影剂在正常兔肝脏与VX2兔肿瘤模型的超声成像特点,对比研究其与普通脂质做泡超声造影剂超声成像的异同。方法分别从正常兔及VX2肿瘤兔耳缘静脉团注普通脂质微泡超声造影剂与自制靶向脂质微泡超声造影剂,使用飞利浦iU22超声诊断仪的造影模式,实时监控2种不同造影剂各自的超声显影特点。DFY定量分析诊断仪分析图像的达峰时间、峰值回声强度、清除时间并对各组参数进行比较。结果在正常兔肝实质,普通脂质微泡超声造影剂(MB)达峰时间明显早于自制的靶向脂质微泡超声造影剂(MB’)达峰时间,峰值回声强度前者高于后者,清除时间前者显著短于后者。在VX2肿瘤区,MB达峰时间明显早于MB’,峰值回声强度前者明显高于后者,清除时间前者显著短于后者。结论自制靶向脂质微泡超声造影剂有其自身独特的超声显像过程和特点,呈"负向显影"模式。     

Ex Vivo Imaging of Ultrasound-Stimulated Metabolic Activity in Rat Pancreatic Slices

Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 and 633 nm to image lipodamine dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence, respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm² at both 800 kHz and 1 MHz. Additionally, 800 kHz at 1 W/cm² was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.