Aqueous Two‐Phase System Patterning of Microbubbles: Localized Induction of Apoptosis in Sonoporated Cells

Ultrasound‐driven microbubbles produce mechanical forces that can disrupt cell membranes (sonoporation). However, it is difficult to control microbubble location with respect to cells. This lack of control leads to low sonoporation efficiencies and variable outcomes. In this study, aqueous two‐phase system (ATPS) droplets are used to localize microbubbles in select micro‐regions at the surface of living cells. This is achieved by stably partitioning microbubbles in dextran (DEX) droplets, deposited on living adherent cells in medium containing polyethylene glycol (PEG). The interfacial energy at the PEG‐DEX interface overcomes microbubble buoyancy and prevents microbubbles from floating away from the cells. Spreading of the small DEX droplets retains microbubbles at the cell surface in defined lateral positions without the need for antibody or cell‐binding ligand conjugation. The patterned microbubbles are activated on a cell monolayer exposed to a broadly applied ultrasound field (center frequency 1.25 MHz, active element diameter 0.6 cm, pulse duration 8 μs or 30 s). This system enables efficient testing of different ultrasound conditions for their effects on sonoporation‐mediated membrane disruption and cell viability. Regions of cells without patterned microbubbles show no injury or membrane disruption. In microbubble patterned regions, 8 μs ultrasound pulses (0.2‐0.6 MPa) produce cell death that is primarily apoptotic. Ultrasound‐induced apoptosis increases with higher extracellular calcium concentrations, with cells displaying all of the hallmarks of apoptosis including annexinV labeling, loss of mitochondrial membrane potential, caspase activation and changes in nuclear morphology.     

声致穿孔仪的研制

在超声波作用下所引起的振动能改变细胞膜表面结构，可使细胞膜通透性增加。利用这一原理，近十几年来新兴起了使用超声作为辅助手段的声致穿孔方法。可以借助超声作用将目标大分子(如药物、DNA等)定点传输到细胞内，起到精确治疗和基因治疗的作用，并借助超声增强治疗效果。但其相应的实验用超声设备尚未达到完美，有多处可作改进。现有设备每次实验时只能对一个样本进行操作，作者在已有的设计思想基础上进行改进，设计制造了新的声致穿孔仪，并从导纳特性、电声效率、声功率以及声压分布等多方面对仪器的性能进行了测试。结果表明，该仪器性能达到了设计要求，使用该仪器进行的声致穿孔实验也取得了较为满意的结果。     

Controlled release of Fe3O4 nanoparticles in encapsulated microbubbles to tumor cells via sonoporation and associated cellular bioeffects

Fe(3)O(4) nanoparticles embedded in the shells of encapsulated microbubbles could be used therapeutically as in situ drug-delivery vehicles. Bioeffects on liver tumor cells SMMC-7721 due to the excitation of Fe(3)O(4) nanoparticles attached to microbubbles generated by ultrasound (US) are studied in an in vitro setting. The corresponding release phenomenon of Fe(3)O(4) nanoparticles from the shells of the microbubbles into the cells via sonoporation and related phenomena, including nanoparticle delivery efficiency, cell trafficking, cell apoptosis, cell cycle, and disturbed flow of intracellular calcium ions during this process, are also studied. Experimental observations show that Fe(3)O(4) nanoparticles embedded in the shells of microbubbles can be delivered into the tumor cells; the delivery rate can be controlled by adjusting the acoustic intensity. The living status or behavior of Fe(3)O(4) -tagged tumor cells can then be noninvasively tracked by magnetic resonance imaging (MRI). It is further demonstrated that the concentration of intracellular Ca(2+) in situ increases as a result of sonoporation. The elevated Ca(2+) is found to respond to the disrupted site in the cell membrane generated by sonoporation for the purpose of cell self-resealing. However, the excessive Ca(2+) accumulation on the membrane results in disruption of cellular Ca(2+) cycling that may be one of the reasons for the death of the cells at the G1 phase. The results also show that the Fe(3)O(4) -nanoparticle-embedded microbubbles have a lower effect on cell bioeffects compared with the non-Fe(3)O(4) -nanoparticle-embedded microbubbles under the same US intensity, which is beneficial for the delivery of nanoparticles and simultaneously maintains the cellular viability.     

Single-site sonoporation disrupts actin cytoskeleton organization

Sonoporation is based upon an ultrasound–microbubble cavitation routine that physically punctures the plasma membrane on a transient basis. During such a process, the actin cytoskeleton may be disrupted in tandem because this network of subcellular filaments is physically interconnected with the plasma membrane. Here, by performing confocal fluorescence imaging of single-site sonoporation episodes induced by ultrasound-triggered collapse of a single targeted microbubble, we directly observed immediate rupturing of filamentary actin (F-actin) at the sonoporation site (cell type: ZR-75-30; ultrasound frequency: 1 MHz; peak negative pressure: 0.45 MPa; pulse duration: 30 cycles; bubble diameter: 2–4 µm). Also, through conducting a structure tensor analysis, we observed further disassembly of the F-actin network over the next 60 min after the onset of sonoporation. The extent of F-actin disruption was found to be more substantial in cells with higher uptake of sonoporation tracer. Commensurate with this process, cytoplasmic accumulation of globular actin (G-actin) was evident in sonoporated cells, and in turn the G-actin : F-actin ratio was increased in a trend similar to drug-induced (cytochalasin D) actin depolymerization. These results demonstrate that sonoporation is not solely a membrane-level phenomenon: organization of the actin cytoskeleton is concomitantly perturbed.     

超声法转基因及其应用前景

目前基因工程蓬勃发展，转基因的方法也越来越多，越声皮作为一种新兴的转基因方法开始被应用于科研工作，文章主要阐述了超声法转基因的机理、步骤、影响因素、存在问题及应用前景。     

Ultrasound Targeted Microbubble Destruction Stimulates Cellular Endocytosis in Facilitation of Adeno-Associated Virus Delivery

The generally accepted mechanism for ultrasound targeted microbubble destruction (UTMD) to enhance drug and gene delivery is through sonoporation. However, passive uptake of adeno-associated virus (AAV) into cells following sonoporation does not adequately explain observations of enhanced transduction by UTMD. This study investigated alternative mechanisms of UTMD enhancement in AAV delivery. UTMD significantly enhanced transduction efficiency of AAV in a dose-dependent manner. UTMD stimulated a persistent uptake of AAV into the cytoplasm and nucleus. This phenomenon occurred over several hours, suggesting that some viral particles are endocytosed by cells rather than exclusively passing through pores created by sonoporation. Additionally, UTMD enhanced clathrin expression and accumulation at the plasma membrane suggesting greater clathrin-mediated endocytosis following UTMD. Transmission electron microscopy (TEM) revealed that UTMD stimulated formation of clathrin-coated pits (CPs) and uncoated pits (nCPs). Furthermore, inhibition of clathrin-mediated endocytosis partially blocked the enhancement of AAV uptake following UTMD. The results of this study implicate endocytosis as a mechanism that contributes to UTMD-enhanced AAV delivery.     

Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles

Site-specific perforation of the plasma membrane can be achieved through ultrasound-triggered cavitation of a single microbubble positioned adjacent to the cell. However, for this perforation approach (sonoporation), the recovery manoeuvres invoked by the cell are unknown. Here, we report new findings on how membrane blebbing can be a recovery manoeuvre that may take place in sonoporation episodes whose pores are of micrometres in diameter. Each sonoporation site was created using a protocol involving single-shot ultrasound exposure (frequency: 1 MHz; pulse length: 30 cycles; peak negative pressure: 0.45 MPa) which triggered inertial cavitation of a single targeted microbubble (diameter: 1–5 µm). Over this process, live confocal microscopy was conducted in situ to monitor membrane dynamics, model drug uptake kinetics and cytoplasmic calcium ion (Ca²⁺) distribution. Results show that blebbing would occur at a recovering sonoporation site after its resealing, and it may emerge elsewhere along the membrane periphery. The bleb size was correlated with the pre-exposure microbubble diameter, and 99% of blebbing cases at sonoporation sites were inflicted by microbubbles larger than 1.5 µm diameter (analysed over 124 sonoporation episodes). Blebs were not observed at irreversible sonoporation sites or when sonoporation site repair was inhibited via extracellular Ca²⁺ chelation. Functionally, the bleb volume was found to serve as a buffer compartment to accommodate the cytoplasmic Ca²⁺ excess brought about by Ca²⁺ influx during sonoporation. These findings suggest that membrane blebbing would help sonoporated cells restore homeostasis.