Effect of ultrasound-activated microbubbles on the cell electrophysiological properties

New clinical applications of ultrasound contrast microbubbles extend beyond imaging and diagnosis toward therapeutic applications. Cell membrane permeability and the uptake of substances have been shown to be enhanced by microbubbles under ultrasound stimulation. However, the mechanisms of action of ultrasound-activated microbubbles are still unknown. The aim of our study was to examine how microbubbles and ultrasound interact with cells in an attempt to understand the sonoporation mechanism. The ruptured-patch-clamp whole-cell technique was used to measure membrane potential variations of a single cell. SonoVue microbubbles and mammary breast cancer cell line MDA-MB-231 were used. Ultrasound was applied using single-element transducers of 1 MHz. Microbubbles and cells were simultaneously video monitored during ultrasound exposure. Our results showed that, during sonoporation, a marked cell membrane hyperpolarization occurs (n = 6 cells) at negative pressures above 150 kPa, indicating the activation of specific ion channels while the cell and the microbubbles remain viable. The hyperpolarization was sustained for as long as the microbubbles are in a direct contact with the cell and the ultrasound waves are transmitted. Smaller acoustic amplitudes induced only mild hyperpolarization, whereas shutting off the ultrasound brings the cell membrane potential to its resting value. However, ultrasound alone did not affect the cell membrane potential. A similar hyperpolarization of the cell membrane was observed when a mechanical pressure was applied on the cell through a glass probe. In conclusion, the results demonstrate that microbubbles' oscillations under ultrasound activation entail modifications of the electrophysiologic cell activities by triggering the modulation of ionic transports through the plasmic cell membrane. However, only cells in direct contact with the microbubbles are impacted. The mechanisms involved are likely related to activation of specific channels sensitive to mechanical stresses (stretch-activated channels) and possibly nonspecific ion channels.     

国产超声造影剂在超声辐照下对肝细胞产生的生物效应

目的探讨国产超声造影剂全氟显在不同机械指数(MI)超声辐照下对正常肝细胞(HL-7702)的声孔效应及细胞损伤。方法在不同的MI(分别为0.15、0.61、1.2、1.9)下分别对各组加入全氟显的旋转的HL-7702细胞悬液进行超声辐照,频率2MHz,持续照射时间10min。对照组未经超声辐照。应用荧光显微镜观察大分子物质FD500进入细胞的情况,检测发生声孔效应的百分率,应用台盼蓝染色检测细胞活力,以Annexin Ⅴ-FITC/PI双染并应用流式细胞仪检测细胞凋亡。结果声孔效应与对照组比较其余各组声孔效应均有增加,有统计学意义(P<0.05)。随着MI的升高,声孔效应也随之增加,MI为1.2及1.9组与其他各组比较,差异有统计学意义(P<0.05)。细胞损伤随着MI的升高,细胞损伤(溶解 凋亡)虽有增加,但与对照组相比,仅当MI为1.9时细胞损伤增加的程度才有统计学意义(P<0.05)。结论应用于诊断的超声可使加入全氟显的HL-7702细胞悬液发生声孔效应。随着MI的升高声孔效应也随之增加;MI≤1.2时,全氟显不引起明显的细胞损伤,当MI=1.9时细胞损伤有统计学意义。     

Enhanced Intracellular Delivery of a Model Drug Using Microbubbles Produced by a Microfluidic Device

Focal drug delivery to a vessel wall facilitated by intravascular ultrasound and microbubbles holds promise as a potential therapy for atherosclerosis. Conventional methods of microbubble administration result in rapid clearance from the bloodstream and significant drug loss. To address these limitations, we evaluated whether drug delivery could be achieved with transiently stable microbubbles produced in real time and in close proximity to the therapeutic site. Rat aortic smooth muscle cells were placed in a flow chamber designed to simulate physiological flow conditions. A flow-focusing microfluidic device produced 8 μm diameter monodisperse microbubbles within the flow chamber, and ultrasound was applied to enhance uptake of a surrogate drug (calcein). Acoustic pressures up to 300 kPa and flow rates up to 18 mL/s were investigated. Microbubbles generated by the flow-focusing microfluidic device were stabilized with a polyethylene glycol-40 stearate shell and had either a perfluorobutane (PFB) or nitrogen gas core. The gas core composition affected stability, with PFB and nitrogen microbubbles exhibiting half-lives of 40.7 and 18.2 s, respectively. Calcein uptake was observed at lower acoustic pressures with nitrogen microbubbles (100 kPa) than with PFB microbubbles (200 kPa) (p < 0.05, n > 3). In addition, delivery was observed at all flow rates, with maximal delivery (>70% of cells) occurring at a flow rate of 9 mL/s. These results demonstrate the potential of transiently stable microbubbles produced in real time and in close proximity to the intended therapeutic site for enhancing localized drug delivery.     

In vivo gene transfer into the ocular ciliary muscle mediated by ultrasound and microbubbles

This study aimed to assess application of ultrasound (US) combined with microbubbles (MB) to transfect the ciliary muscle of rat eyes. Reporter DNA plasmids encoding for Gaussia luciferase, β-galactosidase or the green fluorescent protein (GFP), alone or mixed with 50% Artison MB, were injected into the ciliary muscle, with or without US exposure (US set at 1 MHz, 2 W/cm², 50% duty cycle for 2 min). Luciferase activity was measured in ocular fluids at 7 and 30 days after sonoporation. At 1 week, the US+MB treatment showed a significant increase in luminescence compared with control eyes, injected with plasmid only, with or without MB (×2.6), and, reporter proteins were localized in the ciliary muscle by histochemical analysis. At 1 month, a significant decrease in luciferase activity was observed in all groups. A rise in lens and ciliary muscle temperature was measured during the procedure but did not result in any observable or microscopic damages at 1 and 8 days. The feasibility to transfer gene into the ciliary muscle by US and MB suggests that sonoporation may allow intraocular production of proteins for the treatment of inflammatory, angiogenic and/or degenerative retinal diseases.     

Sonoporation induces apoptosis and cell cycle arrest in human promyelocytic leukemia cells

Despite being a transient biophysical phenomenon, sonoporation is known to disturb the homeostasis of living cells. This work presents new evidence on how sonoporation may lead to antiproliferation effects including cell-cycle arrest and apoptosis through disrupting various cell signaling pathways. Our findings were obtained from sonoporation experiments conducted on HL-60 human promyelocytic leukemia cells (with 1% v/v microbubbles; 1 MHz ultrasound; 0.3 or 0.5MPa peak negative pressure; 10% duty cycle; 1 kHz pulse repetition frequency; 1 min exposure period). Membrane resealing in these sonoporated cells was first verified using scanning electron microscopy. Time-lapse flow cytometry analysis of cellular deoxyribonucleic acid (DNA) contents was then performed at four post-sonoporation time points (4 h, 8 h, 12 h and 24 h). Results indicate that an increasing trend in the apoptotic cell population can be observed for at least 12 h after sonoporation, whilst viable sonoporated cells are found to temporarily accumulate in the G₂/M (gap-2/mitosis) phase of the cell cycle. Further analysis using western blotting reveals that sonoporation-induced apoptosis involves cleavage of poly adenosine diphosphate ribose polymerase (PARP) proteins: a pro-apoptotic hallmark related to loss of DNA repair functionality. Also, mitochondrial signaling seems to have taken part in triggering this cellular event as the expression of two complementary regulators for mitochondrial release of pro-apoptotic molecules, Bcl-2 (B-cell lymphoma 2) and Bax (Bcl-2-associated X), are seen to be imbalanced in sonoporated cells. Furthermore, sonoporation is found to induce cell-cycle arrest through perturbing the expression of various cyclin and Cdk (cyclin-dependent kinase) checkpoint proteins that play an enabling role in cell-cycle progression. These bioeffects should be taken into account when using sonoporation for therapeutic purposes.     

Recovery of Skin Barrier Properties after Sonication in Human Subjects

The SonoPrep® ultrasonic skin permeation system is used clinically to increase skin permeability for rapid, noninvasive delivery of local anaesthetics. This study tested the hypothesis that sonication can generate a long-lived increase of skin permeability for continuous transdermal drug delivery and diagnostic metabolite extraction. To accomplish this, the volar forearm skin of ten healthy adult subjects was sonicated. As a surrogate measure of skin permeability, skin electrical impedance was measured at occluded and nonoccluded sites every hour over a period of 48 h. Sonication dramatically increased skin permeability, as demonstrated by a large drop in skin impedance. Under occlusion, sonicated skin remained highly permeable during the entire 42-h period of occlusion, which was followed by an immediate decrease in permeability upon removal of occlusion. Without occlusion, sonicated skin retained elevated permeability throughout the 48-h experiment, but regained its barrier function more quickly. Therefore, sonication can increase skin permeability for prolonged periods of time, especially under the effect of occlusion, and has potential to facilitate continuous transdermal drug delivery and diagnostic metabolite extraction. (E-mail: prausnitz@gatech.edu)     

Calibration of the 1-MHz Sonitron Ultrasound System

Successful drug and gene delivery across cellular membranes can lead to improved therapeutic outcomes. Recent studies have suggested that sonoporation may enhance drug and gene delivery across cellular membranes. The enhancement may be a result of transient permeation of the membrane from cavitation or microstreaming effects of microbubbles exposed to ultrasound. Given limited acoustic pressure calibration and beam profile characterization of the Sonitron ultrasound systems in cellular bioeffects studies previously published, the objective of this work was to calibrate the acoustic output and explore the potential for standing waves in a cell-well plate. In this study, three 1-MHz transducers driven by Sonitron ultrasound systems, which have been used in a number of sonoporation studies, were calibrated. Transducers with 10-mm, 6-mm and 20-mm-diameter apertures (Sonitron 1000 and 2000, Rich-Mar, Inola, OK, USA) were calibrated using polyvinylidene fluoride (PVDF) needle hydrophones. Axial and transverse beam profiles were obtained, and the pressures were measured as a function of Sonitron intensity dial setting and duty cycle. The acoustic intensity was calculated and compared with the Sonitron intensity dial setting for duty cycles from 10–100%. Standing waves caused by reflections from the hydrophone holder were detected for each transducer. This observation may also have implications for in vitro sonoporation studies. Acoustic field characterization is an important first step in understanding the mechanisms of sonoporation and drug delivery across biomembranes. (E-mail: kopechja@uc.edu)     

Evaluation of Transfection Efficiency in Skeletal Muscle Using Nano/Microbubbles and Ultrasound

Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)—human albumin shell bubbles, lipid bubbles and acoustic liposomes—to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection. (E-mail: kodama@bme.tohoku.ac.jp)     

The Combination of Intralymphatic Chemotherapy with Ultrasound and Nano-/Microbubbles Is Efficient in the Treatment of Experimental Tumors in Mouse Lymph Nodes

Intravenous chemotherapy is a therapeutic option for the treatment of lymph node metastasis, but the drugs often have difficulty accessing the lymphatic system. The aim of this study was to determine whether the combination of intralymphatic chemotherapy with ultrasound and nano-/microbubbles is active against tumors in mouse lymph nodes. Intralymphatic chemotherapy in mice with lymph nodes containing tumors was found to have a marked anti-tumor effect, compared with intravenous administration, and the addition of ultrasound combined with nano-/microbubbles enhanced the effect of the anti-cancer drug, but only when the drug was administered intralymphatically. Furthermore, decreases in the volumes and blood vessel densities of tumor-bearing lymph nodes are reliable measures of therapeutic effect, confirmed by histopathological evaluation. The main conclusion is that combining ultrasound with nano-/microbubbles and intralymphatic chemotherapy improves drug delivery to the lymphatic system and has a more potent anti-tumor effect.     

Analysis of ultrasound fields in cell culture wells for in vitro ultrasound therapy experiments

Ultrasound is an established therapy method for bone fracture healing, hyperthermia and the ablation of solid tumors. In this new emerging field, ultrasound is further used for microbubble-enhanced drug delivery, gene therapy, sonoporation and thrombolysis. To study selected therapeutic effects in defined experimental conditions, in vitro setups are designed for cell and tissue therapy. However, in vitro studies often lack reproducibility and the successful transfer to other experimental conditions. This is partly because of the uncertainty of the experimental conditions in vitro. In this paper, the ultrasound wave propagation in the most common in vitro ultrasound therapy setups for cell culture wells is analyzed in simulations and verified by hydrophone measurements. The acoustic parameters of the materials used for culture plates and growth media are determined. The appearance and origin of standing waves and ring interference patterns caused by reflections at interfaces is revealed in simulations and measurements. This causes a local maximal pressure amplitude increase by up to the factor of 5. Minor variations of quantities (e.g., growth medium volume variation of 2.56%) increase or decrease the peak rarefaction pressure at a cell layer by the factor of 2. These pressure variations can affect cell therapy results to a large extent. A sealed cell culture well submersed in a water bath provides the best reproducibility and therefore promises transferable therapy results.