In Vitro Gene Delivery with Ultrasound-Triggered Polymer Microbubbles

In the work described here, gene delivery using polymer microbubbles triggered by ultrasound in vitro was investigated. The effects of pressure amplitude (0–2 MPa), center frequency (1–5 MHz), pulse length (3–12,000 μs), pulse repetition frequency (5–20,000 Hz) and exposure time (0–30 s) on transfection efficiency and cell viability were examined. The effects of radiation force, calcium ion concentration and timing of treatments were also examined. Cells were successfully transfected with pressure amplitudes as low as 250 kPa. Transfection was most efficient at lower frequencies and longer pulse lengths, with a transfection efficiency of 24.2 ± 2.0% achieved using a center frequency of 1 MHz, pressure amplitude of 1 MPa, pulse length of 12,000 μs and pulse repetition frequency of 5 Hz. Gene delivery was also affected by the extracellular calcium ion concentration and the timing of treatments.     

Analysis of in vitro Transfection by Sonoporation Using Cationic and Neutral Microbubbles

The objective of the study was to examine the role of acoustic power intensity and microbubble and plasmid concentrations on transfection efficiency in HEK-293 cells using a sonoporator with a 1-MHz transducer. A green fluorescent protein (GFP) reporter plasmid was delivered in as much as 80% of treated cells, and expression of the GFP protein was observed in as much as 75% of cells, using a power intensity of 2 W/cm² with a 25% duty cycle. In addition, the relative transfection abilities of a lipid noncationic and cationic microbubble platform were investigated. As a positive control, cells were transfected using Lipofectamine reagent. Cell survival and transfection efficiency were inversely proportional to acoustic power and microbubble concentration. Our results further demonstrated that high-efficiency transfection could be achieved, but at the expense of cell loss. Moreover, direct conjugation of plasmid to the microbubble did not appear to significantly enhance transfection efficiency under the examined conditions, although this strategy may be important for targeted transfection in vivo. (E-mail: mbl2a@virginia.edu)     

Enhancement of Microbubble Mediated Gene Delivery by Simultaneous Exposure to Ultrasonic and Magnetic Fields

It has been shown in previous studies that gene delivery can be enhanced by a variety of minimally-invasive techniques including: (1) exposure of cells to ultrasound in the presence of DNA and gas microbubbles and (2) exposure of cells to a magnetic field in the presence of DNA conjugated to magnetic nanoparticles. The aim of this work was to investigate whether it was possible to combine the advantages of both these techniques. It was found that transfection of Chinese hamster ovary cells by naked plasmid DNA was enhanced by combined exposure of the cells to ultrasound (10 s at 1 kHz pulse repetition frequency with 40 cycle 1 MHz sinusoidal pulses, 1 MPa peak to peak pressure) and a magnetic field (provided by five square cross-section N52 grade NdFeB magnets 25 × 10 × 10 mm with transversal magnetisation Br = 1.50 T arranged in a Halbach array), in the presence of one of two different microbubble/nanoparticle preparations. The first preparation consisted of phospholipid coated microbubbles mixed with micelles containing magnetic nanoparticles. The second consisted of microbubbles which were themselves magnetically active. These preparations were found to be more effective than either magnetic micelles or phospholipid coated microbubbles alone by a factor of 2.8 (total flux ∼4 versus 1.4 × 10⁶ photon/s) and the results were found to be statistically significant (p < 0.01). Two mechanisms are proposed to explain these observations: firstly, that the magnetic field facilitates close proximity between the cells and the microbubbles and hence increases the likelihood of transfection; second, that there is sensitisation of the cells, as a result of exposure to the magnetic field in the presence of the micelles, which increases their ability to be transfected upon exposure to ultrasound. Further work is in progress to determine which of these mechanisms is the most significant and the potential for other therapeutic applications. (E-mail: e_stride@meng.ucl.ac.uk)     

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)     

Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles

The aim of this study was to assess the relative efficacy and mechanism of gene transfection by ultrasound (US) destruction of plasmid-bearing microbubbles. Luciferase reporter plasmid was charge-coupled to cationic lipid microbubbles. Rat hindlimb skeletal muscle was exposed to intermittent high-power US during dose-adjusted intra-arterial (IA) or IV administration of plasmid-bearing microbubbles via the carotid artery or jugular vein, respectively. At 4 days, luciferase activity in US-exposed skeletal muscle was 200-fold greater with IA than with IV administration of plasmid-bearing microbubbles, and was similar to transfection achieved by IM injection of plasmid (positive control). No transfection occurred with US and IA injection of plasmid alone. Intravital microscopy of the cremaster muscle in mice following administration of microbubbles and US exposure demonstrated perivascular deposition of fluorescent plasmid, the extent of which was twofold greater for IA compared to IV injection. Electron microscopy demonstrated a greater extent of myocellular microporations in US-exposed muscle after IA injection of microbubbles. We conclude that muscle transfection by US destruction of plasmid-bearing cationic microbubbles is amplified by IA, rather than IV, injection of microbubbles due to greater extravascular deposition of plasmid and to greater extent of myocellular microporation. (E-mail: jlindner@virginia.edu)     

In Vitro Gene Transfer by Electrosonoporation

Among the nonviral methods for gene delivery in vitro, electroporation is simple, inexpensive and safe. To upregulate the expression level of transfected gene, we investigated the applicability of electrosonoporation. This approach consists of a combination of electric pulses and ultrasound assisted with gas microbubbles. Cells were first electroporated with plasmid DNA encoding–enhanced green fluorescent protein and then sonoporated in presence of contrast microbubbles. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Sonoporation can improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells. These results demonstrated that in vitro gene transfer by electrosonoporation could provide a new potent method for gene transfer. (E-mail: rols@ipbs.fr)     

Pre-Treatment with Either L-Carnitine or Piracetam Increases Ultrasound-Mediated Gene Transfection by Reducing Sonoporation-Associated Apoptosis

Sonoporation, the use of ultrasound to alter the permeability of cell membranes, is a non-viral technique used to facilitate gene delivery, possibly by opening transient pores in the cell membrane. However, sonoporation may have negative bio-effects on cells, such as causing apoptosis, which limits its efficacy in gene delivery. In this study, we investigated whether pre-treatment with either L-carnitine or piracetam could protect cells from undergoing apoptosis after sonoporation and the possible mechanisms. We found that either L-carnitine or piracetam can promote gene transfection without reducing cell viability, possibly by reducing cavitation-induced reactive oxygen species generation, reversing alterations of mitochondrial membrane potential, preventing caspase-3/7 activity and facilitating mitochondrial ATP production. In conclusion, pre-treatment with either L-carnitine or piracetam could protect cells from sonoporation-associated apoptosis by preserving mitochondrial function.     

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.     

靶向超声造影剂促进反义基因转染的体外研究

目的 探讨超声破坏造影剂微泡联合半乳糖化多聚赖氨酸(G-PLL)对基因治疗肝癌的靶向促进作用.方法 将合成的超声造影剂微泡、G-PLL及c-myc反义寡核苷酸(ASODN)耦联物,结合超声辐照作用于表达c-myc基因的人肝癌及人肺癌细胞株进行体外效应研究.观察细胞形态学改变及耦合物与细胞结合情况并检测c-myc基因的表达情况.结果 镜下显示肝癌细胞较肺癌细胞更易于与耦联物结合,多数细胞呈凋亡改变.经超声辐照,耦联物作用后的人肝癌及肺癌细胞c-myc基因表达量均有降低,而G-PLL对肝癌细胞c-myc基因表达有明显抑制作用.结论 超声辐照下,造影剂结合G-PLL及反义基因耦联物可靶向性促进反义基因转染肝癌细胞.     

超声辐照和SonoVue微泡在介导hAng-1基因体外转染时的作用和量效关系探讨

目的 探讨超声辐照和SonoVue微泡分别使用和联用在介导hAng-1基因体外转染过程中的作用以及辐照强度和微泡浓度对转染效率和细胞活性的影响.方法 实验分四组A组:单纯超声辐照+质粒组;B组:微泡+质粒组;C组:超声辐照+微泡+质粒组和空白对照组D组. C组内转染参数分别设置为超声照射强度0.5、1.0 、1.5和2.0 W/cm~2,微泡浓度5%、10%、20%、30%和40%.将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡对293T细胞进行转染,48 h后检测各组基因转染效率和细胞存活率. 结果转染48 h后C组转染效率最高,荧光阳性细胞数最多,强度最大;A组转染效率很低,见少量荧光表达;B、D组无明显基因转染发生.随着超声照射强度和微泡浓度的增加,基因转染效率会逐步升高,具有统计学意义.微泡浓度大于20%、超声照射强度超过1.5 W/cm~2后基因转染效率不再升高甚至降低,细胞死亡率显著增高(P<0.01).结论 SonoVue微泡介导外源基因转染必须联合超声辐照才能获得较好的转染效率.对于hAng-1基因和SonoVue微泡,选择声强1.5 W/cm~2,微泡浓度20%是相对最佳转染条件.     

超声辐照微泡声学造影剂增强脂质体介导肝细胞基因转染的体外实验研究

目的研究超声破坏微泡声学造影剂提高脂质体介导肝细胞生长因子质粒（plRES—EGFP—HGF）在肝细胞中转染率的可行性。 方法将培养的肝细胞分为4组：（1）单纯对照组，（2）脂质体转染组，（3）超声辐照脂质体转染组，（4）超声辐照微泡＋脂质体转染组。第（4）组按照每孔加入造影剂剂量又分为1）10μl组，2）20μl组，3）30μl组，4）40μl组4亚组。超声辐照微泡＋脂质体转染组在脂质体介导肝细胞生长因子转染肝细胞1h后，在24孔板中每孔加入微泡声学造影剂10，20，30或40μl，超声辐照60s。24h后用荧光显微镜观察绿色荧光蛋白表达情况、MTT法检测肝细胞增殖率和流式细胞仪检测基因转染率。 结果超声频率1MHz，功率0．5W／cm。辐照60S，每孔加入造影剂30μl时肝细胞基因转染效率最高，与脂质体转染组比较有显著性。 结论在一定条件下，超声辐照微泡声学造影剂可明显提高脂质体介导肝细胞基因转染效率，为基因治疗提供了新的思路。     

超声微泡介导EGFP质粒转染视网膜母细胞瘤细胞与传统转染方法效率对比

目的探讨超声微泡造影剂在一定能量的超声波辐照下，介导EGFP质粒转染视网膜母细胞瘤（RB）细胞的效率及可行性。 方法将RB细胞分为6组，1组以一定能量的超声波辐照，2组加适当剂量的微泡造影剂，3组加入质粒，4组加入质粒与微泡，5组加入质粒、微泡，并用一定能量的超声辐照，6组予脂质体与质粒。转染24-48h后观察EGFP表达，并用RT—PCR进行检测。同时对1、2组予以染色。 结果超声微泡介导的DNA质粒对RB细胞的转染效率，与脂质体介导的质粒转染效率相似，明显高于其他实验组。一定能量和时间的超声波辐照，及适当浓度的微泡，对RB细胞的活性无明显抑制。 结论利用低频率和一定能量的超声击碎携带EGFP质粒的超声微泡造影剂，能够有效地提高DNA质粒在RB细胞中的转染效率。     

腺病毒载体介导肝细胞生长因子基因对血管平滑肌细胞的作用

目的：研究腺病毒载体介导肝细胞生长因子（HGF）基因感染血管平滑肌细胞后在缺氧和正常供氧时的细胞凋亡和迁移情况。方法：以肝细胞中抽提的总RNA为模板，反转录cDNA，采用逆转录-聚合酶链反应技术（RT-PCR）获得HGF基因，并引入酶切位点，转染大肠杆菌，筛选阳性菌落，经酶切及测序，转染腺病毒，经三轮扩增，制备高效表达HGF基因腺病毒载体（Ad-HGF），再感染人血管平滑肌细胞（VSMC）为观察组（Ad-HGF组）；未感染为阴性对照组（DMEM组）；以含人工合成HGF为阳性对照组（HGF组），分别在正常供氧和缺氧情况下观察细胞的凋亡和迁移情况。结果：与DMEM组及HGF组比较，Ad-HGF组在缺氧和正常供氧时VSMC的迁移率及凋亡细胞数差异均无显著性意义。结论：HGF基因感染VSMC后，在缺氧情况下并不促进VSMC的迁移。亦不抑制其凋亡。     

Development of Localized Gene Delivery Using a Dual-Intensity Ultrasound System in the Bladder

A dual-intensity ultrasound system (DIUS) using nanobubbles offers opportunities for localized gene delivery. This system consists of low-/high-ultrasound intensities. The bladder is a balloon-shaped closed organ in which the behavior of nanobubbles can be controlled spatially and temporally by ultrasound exposure. We hypothesized that when a DIUS with nanobubbles was used, low-intensity ultrasound would direct nanobubbles to targeted cells in the bladder, whereas high-intensity ultrasound intensity would collapse nanobubbles and increase cell membrane permeability, facilitating entry of exogenous molecules into proximate cells. A high-frequency ultrasound imaging system characterized movement and fragmentation of nanobubbles in the bladder. Confocal microscopy revealed that fluorescent molecules were delivered in the localized bladder wall, whereas histochemical examination indicated that the molecular transfer efficiency depended on the acoustic energy. A bioluminescence imaging system showed luciferase plasmid DNA was actually transfected in the bladder wall and subsequent transfection depended on acoustic energy. These findings indicate that delivery of exogenous molecules in the bladder using this approach results in high localization of molecular delivery, facilitating gene therapy for bladder cancer. (E-mail: kodama@bme.tohoku.ac.jp)     

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.     

野生型p53基因转移对大鼠血管平滑肌细胞凋亡的影响

目的：血管平均滑肌细胞的凋亡在球囊损伤后３０分钟内即已经开始,用基因转移的方法加速这一经过是控制再狭窄的途径之一。选择野生型ｐ５３作为目的基因,观察其对血管平滑骨细胞生长及凋亡的影响。方法：构建了野生型ｐ５３基因重组反转录病毒载体ｐＬＸＳＮ－ｐ５３。重组载体转染培养的大鼠主动脉增滑肌细胞,测定转染效率和ｐ５３基因的表达,用细胞存活曲线、流式细胞仪和ＤＮＡ “ｌａｄｄｅｒ”法检测了细胞生长和凋亡状况     

腺病毒介导白细胞介素-10基因转染抑制血管平滑肌细胞的增殖及机制

目的：观察白细胞介素（interleukin，IL-10基因转染对血管平滑肌细胞增殖的影响，初步研究其相关机制。方法：不同感染复数（multiplicity of infection，MOI）的IL-10重组腺病毒（Ad／IL-10）转染原代培养的人脐带动脉平滑肌细胞（HUASMcs），用酶联免疫吸附试验检测培养上清中IL-10的表达，噻唑蓝法测定吸光度值（OD570值），绘制细胞生长曲线，流式细胞术分析细胞周期、细胞凋亡，实时荧光定量聚合酶链反应检测p21的表达。结果：MOI=100时培养上清中IL-10含量最高，为5．746ng／ml；转染72h、96h，MOI为100、150、200组与MOI为10、20组及空白对照组比较，显著抑制HUASMCs的增殖（P〈0．01）；与空白及空载体转染对照组比较，MOI100的Ad／IL-10转染组的Gn-1期细胞数、凋亡细胞的平均百分数均显著增加（P〈0．01），p21基因表达的拷贝数较低。结论：MOI为100的Ad／IL-10可有效抑制HUASMCs的增殖，此作用与细胞周期阻滞和凋亡有关，为ID-10基因转染治疗血管内膜增生提供了理论依据。     

The Application of Clinical Lithotripter Shock Waves to RNA Nucleotide Delivery to Cells

The delivery of genes into cells through the transfer of ribonucleic acids (RNAs) has been found to cause a change in the level of target protein expression. RNA-based transfection is conceptually more efficient than commonly delivered plasmid DNA because it does not require division or damage of the nuclear envelope, thereby increasing the chances of the cell remaining viable. Shock waves (SWs) have been found to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanying SW bio-effects include dose-dependent irreversible cell injury and cytotoxicity. Here, the effect of SWs generated by a clinical lithotripter on the viability and permeabilisation of three different cell lines in vitro was investigated. Comparison of RNA stability before and after SW exposure revealed no statistically significant difference. Optimal SW exposure parameters were identified to minimise cell death and maximise permeabilisation, and applied to enhanced green fluorescent protein (eGFP) messenger RNA (mRNA) or anti-eGFP small interfering RNA delivery. As a result, eGFP mRNA expression levels increased up to 52-fold in CT26 cells, whereas a 2-fold decrease in GFP expression was achieved after anti-eGFP small interfering RNA delivery to MCF-7/GFP cells. These results indicate that SW parameters can be employed to achieve effective nucleotide delivery, laying the foundation for non-invasive and high-tolerability RNA-based gene therapy.     

SM22α启动子靶向性调控血管平滑肌细胞过表达p27的研究

目的:构建由SM22α特异性启动子介导血管平滑肌细胞(VSMCs)靶向性过表达p27蛋白的慢病毒载体来研究对VSMCs细胞周期的影响。方法:构建重组慢病毒载体(Lenti-SM22α-p27-EGFP),感染原代培养的VSMCs及内皮细胞(VECs)。结果:SM22α-p27慢病毒能对VSMCs表现出高效且精确的抑制作用。SM22α启动子靶向标记的p27能在VSMCs内过表达,且通过G0/G1期阻滞来抑制细胞增殖。结论:由SM22α特异性启动子驱动的重组慢病毒载体能选择性感染VSMCs,有效地引起p27蛋白过表达并产生G0/G1期阻滞。